Analytical Profiles of Drug Substances [Vol 16] - K. Florey (AP, 1987) WW

Analytical Profiles

of

Drug Substances

Volume 16

EDITORIAL BOARD

Abdullah A. Al-Badr Steven A. Benezra Eugene Inman Gerald S. Brenner Joseph Mollica Glenn A. Brewer, Jr. Nicholas DeAngelis John Zarembo

Lee T. Grady

Milton D. Yudis

Analytical Profiles of

Drug Substances Volume 16

Edited by

Klaus Florey The Squibb Institute for Medical Research

New Brunswick, New Jersey

Contributing Editors

Abdullah A. Al-Badr Gerald S. Brenner

1987

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando San Diego New York Ailstin Boston London Sydney Tokyo Toronto

Academic Press Rapid Manuscript Reproduction

COPYRIGHT 0 1987 BY ACADEMIC PRESS. INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. Orlando, Florida 32887

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. 24-28 Oval Road, London NW I 7DX

Library of Congress Cataloging in Publication Data (Revised for volume 16)

Analytical profiles o f drug substances.

Compiled under the auspices o f the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences.

Includes bibliographical references and indexes. 1. Drugs-Analysis-Collected works. 2. Chemistry,

Pharmaceutical-Collected works. I . Florey, Klaus, 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis Control Section. [DNLM: 1. Drugs-Analysis-Yearbooks. QV740 AA1 ASS]

ISBN 0-12-260816-X (v. 16 : alk. paper) RS 189.A58 6 lSI .1 70-187259

PRINTED IN THE UNITED STATES OF AMERICA

8 7 8 8 8 9 9 0 9 8 7 6 5 4 3 2 1

CONTENTS

Affiliations of Editors and Contributors Preface

Bromazepam Mahmoud M . A. Hassan and Mohammad A. Abounassif

Busulphan Mohammad Tariq and Abdullah A. Al-Badr

C hlo ram bucil Mohammad Tariq and Abdutlah A. Al-Badr

Chlorzoxazone James T . Stewart and Casimir A. Janicki

C yclosporine Mahmoud M. A. Hassan and Mohammed A. Al-Yahya

Enalapril Maleate Dominic P . fp and Gerald S. Brenner

Homatropine Hydrobromide Farid J . Muhtadi, Mahmoud M . A. Hassan, and Abdul Fattah A. Afify

Mebendazole A. A. Al-Badr and M . Tariq

uii ir

1

53

85

119

145

207

245

29 1

V

vi CONTENTS

Metoclopramide Hydrochloride Davide Pitre and Riccardo Strudi

Mitomycin C Jos H . Beijnen, Auke Bult, and Willy J . M .

Neostigmine A. A. Al-Badr and M . Turiy

Jnderberg

Pirenzepine Dihydrochloride Humeida A. El-Obeid, Salim A. Babhair, and Abdullah A. Al-Badr

Streptomycin faber S . Mossa, Abdut Hameed U . Kader Taragan, and Mahmoud M . A. Hassan

Thiabendazole Vijuy K . Kapoor

Timolol Maleate David J. Mazzo and Alice E , Loper

Trazodone Hydrochloride Dennis K . G. Gorecki and Roger K . Verbeeck

Yohimbine A. G. Mekkawi and A. A. Al-Badr

Cumulative Index

327

36 1

403

445

507

61 1

64 1

693

73 1

769

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Mohammad A . Abounassif, College of Pharmacy, King Saud University,

Abdul Fattah A . Afify, College of Pharmacy, King Saud University,

Abdullah A . Al-Budr, King Saud University, Riyadh, Saudi Arabia Mohammed A. Al-Yahya, College of Pharmacy, King Saud University,

Salim A . Babhair, College of Pharmacy, King Saud University, Riyadh,

Jos H . Beijnen, Slotervaart Hospital, Amsterdam, The Netherlands Steven A. Benezra, Wellcome Research Laboratories, Research Triangle

Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West

Glenn A. Brewer, Jr., The Squibb Institute for Medical Research, New

Auke Bult, Faculty of Pharmacy, State University of Utrecht, Utrecht,

Nicholas J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania Humeida A. El-Obeid, King Saud University, Riyadh, Saudi Arabia Klaus Florey, The Squibb Institute for Medical Research, New Bruns-

Dennis K . G . Gorecki, College of Pharmacy, University of Saskatchewan,

Lee T . Grady, The United States Pharmacopeia, Rockville, Maryland Mahmoud M . A . Hassan, King Saud University, Riyadh, Saudi Arabia Eugene L. Inman, Lilly Research Laboratories, Indianapolis, Indiana

Riyadh, Saudi Arabia



Saudi Arabia

Park, North Carolina

Point, Pennsylvania

Brunswick, New Jersey

The Netherlands

wick, New Jersey

Saskatoon, Saskatchewan, Canada

vii

AFFILIATIONS ...

Vll l

Dominic P . I p , Merck Sharp & Dohme Research Laboratories, West

Casimir A. Janicki, McNeil Pharmaceutical, Spring House, Pennsyl-

Vijay K . Kapoor, Department of Pharmaceutical Sciences, Panjab Uni-

Alice E . Loper, Merck Sharp & Dohme Research Laboratories, West

David J . Mazzo, Travenol Laboratories, Morton Grove, Illinois A. G. Mekkawi, College of Pharmacy, King Saud University, Riyadh,

Joseph A. Mollica, Pharmaceuticals, Du Pont, Wilmington, Delaware Jaber S. Mossa, College of Pharmacy, King Saud University, Riyadh,

Farid J . Muhtadi, King Saud University, Riyadh, Saudi Arabia Davide Pitrc?, Faculty of Pharmacy, University of Milan, Milan, Italy James T. Stewart, College of Pharmacy, University of Georgia, Athens,

Riccardo Stradi, Faculty of Pharmacy, University of Milan, Milan, Italy Abdul Hameed U . Kader Taragan, College of Pharmacy, King Saud Uni-

Mohammad Tariq, College of Pharmacy, King Saud University, Riyadh,

Willy J . M . Underberg, Faculty of Pharmacy, State University of Utrecht,

Roger K . Verbeeck, College of Pharmacy, University of Saskatchewan,

Milton D. Yudis, Schering-Plough, Kenilworth, New Jersey John E . Zarembo, W. H. Rorer, Inc., Fort Washington, Pennsylvania

Point, Pennsylvania

vania

versity, Chandigarh, India

Point, Pennsylvania

Saudi Arabia

Saudi Arabia

Georgia

versity, Riyadh, Saudi Arabia

Saudi Arabia

Utrecht, The Netherlands

Saskatoon, Saskatchewan, Canada

PREFACE

Although the official compendia define a drug substance as to identity, purity, strength, and quality, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. Such information is scattered through the scientific literature and the files of pharmaceutical laboratories.

I perceived a need to supplement the official compendial standards of drug substances with a comprehensive review of such infoimation, and sixteen years ago the first volume of Anulyticul Profiles of Drug Sub- stances was published under the auspices of the Pharmaceutical Analy- sis and Control Section of the APhA Academy of Phaimaceutical Sci- ences. That we were able to publish one volume per year is a tribute to the diligence of the editors to solicit articles and even more so to the enthusiastic response of our authors, an international group associated with pharmaceutical firms, academic institutions, and compendial au- thorities. I would like to express my sincere gratitude to them for making this venture possible.

Over the years, we have had queries concerning our publication pol- icy. Our goal is to cover all divg substances of medical value and, therefore, we have welcomed any articles of interest to an individual contrib- utor. We also have endeavored to solicit profiles of the most useful and used medicines, but many in this category still need to be profiled.

Klaus Florey

ix

This Page Intentionally Left Blank

BROMAZEPAM

Mahmoud M.A. H a h a n , PhO6eAhOh 06 PhahmaceuLLcd C h m h L t y , Depah;tment od PhahmaceuLLcd C h m h L t y , CoMege 06 Phanmacy, King Saud U n L v m L t q , Riyadh, Saudi Axabia.

Mohammad A. Abuumnid, AnbhXant Phodeddon 04 PhamaceuLi- c d C h m h i x y , Heud 04 Xhe DepdmenX 06 P h a m a c e d i c d C h m h L t y , College 06 Phahmacy, King Saud Un ivmi , t y , Riyadh, Saudi Arrabia.

1. His tory and Therapeut ic Category

2 . Descr ip t ion

2.1 Nomenclature

2 . 2 Formulae

2.3 Molecular Weight

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved. 1

2 MAHMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

.3. Physical P rope r t i e s

3.1 Appearance, Color, Odor and Taste

3.2 Melting Range

3.3 Dissoc ia t ion Constants

3.4 Crys ta l S t ruc tu re

4 , Spec t r a l P rope r t i e s

4.1 U l t r a v i o l e t Spectrum

4.2 In f r a red Spectrum

4.3 Nuclear Magnetic Resonance Spec t ra

4.4 Mass Spectrum

5 . Synthes is

6. Metabolism

7. Pharmacokinetic S tudies

8. Methods of Analysis

8.1 Elemental Analysis

8.2 I d e n t i f i c a t i o n Tests

8 .3 T i t r i m e t r i c

8.4 Spectrophotometric

8.5 Polarographic

8.6 Atomic Absorption

8.7 Radioimunoassays

8.8 Chromatographic

9. References

B R 0 MAZE P AM 3

Analytical P r o f i l e of Bromazepam

1. History and Therapeutic Category

The las t quarter century, and i n p a r t i c u l a r t h e decade 1952-1962, has witnessed a break-through i n t h e treatment and outlook of t h e mentally ill. Two groups of drugs were developed namely psychotic and psychotropic agents. Among t h e last named psychotropic agents , t h e benzodiazepines. These drugs such as diazepam, chlordiazepoxide and oxazepam have been shown t o exert anx io ly t i c , s l eep inducing, muscle relaxant and anticonvulsant e f f e c t s i n g rea t e r or l e s s e r degree. Search continued f o r making more de r iva t ives t o produce s u i t a b l e drug f o r spec i f i c cases or symptoms. In t h i s way nitrazepam and more r ecen t ly flurazepam have been found t o be spec ia l ly effec- t i v e s l eep inducers and clonazepam exe r t s even more potent ly t h e an t i conwlsan t a c t i v i t y known already f o r diazepam.

Search has continued and i n 1974 bromazepam w a s marketed as a new psychotropic drug of t h e benzodiazepine s e r i e s but belonging t o a new c l a s s of pyridyl-benzodiazepines. It possesses c e r t a i n biochemical f ea tu re s which d i s t i n - guish it from other benzodiazepines. It has been shown t o exer t a more profound anx io ly t i c e f f e c t t han other similar substances.

2. Description

2.1 Nomenclature

2.1.1 Chemical Names

7-Brom0-1,3-dihydro-5-(~2-pyridyl)-2H-l,~- benzodiazepine-2-one.

2.1.2 Generic Name

Bromazepam.

2.1.3 Trade Names

Lectopam; Lexomil; Lexotan; Lexotanil.


2.2 Formulae

2 .2 .1 Ehp i r i ca l

C14H10BrN 0 3

2.2.2 S t r u c t u r a l

H

2.2.3 Research Number (1)

Ro 5-3350.

2.2.4 Chemical Abs t r ac t s Reg i s t ry Number

[ 1812-30-21


316.16

3. Phys ica l P r o p e r t i e s

3 .1 Appearance, Color, Odor and Taste

A p a l e yellow odor l e s s c r y s t a l l i n e s o l i d ( 2 ) .

3.2 Melting Range

BROMAZEPAM 5

3.3 Dissoc ia t ion Constants

Bromazepam has t h r e e pKa va lues o f 2.5, 5.2 and 11.8 corresponding t o pro tona t ion at t h e azomethine and pyr id ine n i t rogen atoms, and deprotonat ion at t h e n i t rogen atom i n p o s i t i o n 1, respec t ive ly . These pKa values were determined by s p e c t r a l and polarographic a n a l y s i s ( 3 ) .

3.4 Crys t a l S t r u c t u r e ( 4 )

The c r y s t a l s t r u c t u r e of bromazepam w a s determined us ing c r y s t a l s from amyl ace ta te -e thanol obtained by slow evaporat ion. The i n t e n s i t y d a t a w e r e c o l l e c t e d on an Enraf-Nonius CAD-4 d i f f r ac tomete r , c r y s t a l s i z e 0.3 x 0.25 x 0.2 mm, c e l l dimentions from s e t t i n g angles of 25 r e f l e c t i o n s and g r a p h i t e monochromated MoKa r a d i a t i o n . Bromazepam i s mono- c l i n i c wi th space groups P2l /c (non-centrosymetric) . The c e l l dimentions are a = 10.304 ( 4 ) , b = 15.897 ( 5 ) , c = 8.122 ( 3 ) A', 6 = 106.8 (3)', U = 1273.6 AO,

Z = 4 , Dx = 1.649 Mg m-3, p(MoKa, A = 0.71069 A') = 3.13 mm-1, F(000) = 632, room temperature , R = 0.040 f o r 1470 observed r e f l e c t i o n s .

I n bromazepam t h e 7-membered r i n g i s i n a boat conformat ion. o f t h e 1,4-benzodiazepine system and t h e he te ro- c y c l i c r i n g system i n t h e ?-posi t ion i s 60.3 ( 6 ) ' . Fina l atomic parameters f o r bromazepam are l i s t e d i n Table 1; bond l eng ths , bond angles and s e l e c t e d t o r s i o n angles are l i s t e d i n Table 2. The atomic numbering scheme i s i l l u s t r a t e d i n Fig. 1. Bond l eng ths and angles gene ra l ly agree w e l l wi th values found i n analogous molecules ( 5 - 7 ) . The N(l)-C(2) formal s i n g l e bond i s shortened t o 1.351 ( 5 ) A' i n bromazepam and t h e d i s p o s i t i o n of bonds at N ( 1 ) i s near p lanar s o t h a t t h e geometry of t h e bond resembles t h a t of a normal double bond ( c f . t o r s i o n angles i n Table 2 ) ( 6 ) . The N ( 4 ) - C ( 5 ) l eng th i n bromazepam corresponds t o t h a t o f a C = N bond and t h e C ( 5 ) - C ( l ' ) and C ( 5 ) - C ( l l ) l eng ths are wi th in t h e accepted range (1.48-1.50 A') for a C(sp2)-C(sp2) s i n g l e bond. There i s , t h e r e f o r e , no evidence f o r any e l e c t r o n de loca l i za t ion between t h e 5-pyridyl r i n g i n bromazepam and t h e 1,4-benzodiazepine system.

The angle between t h e benzo-moeity

a, c

w

0

c,

F

(ud

BROMAZEPAM 7

The seven-membered r i n g i s i n a cyc lohepta t r iene- l i k e boat conformation, C(10) and C ( 1 1 ) forming t h e ' s t e r n ' and C(3) t h e 'bow'. The bow ang les , 60.3 ( 8O) i n bromazepam and t h e s t e r n angle 30.8 (8' ) .

4 Table 1. Frac t iona l atomic coord ina tes ( x 1 0 ) with e . s . d . ' s

i n parentheses and equivalent i s o t r o p i c temperature f a c t o r s ( ~ 2 x 103)

Z -

X Y - -

617 (1) -1404 ( 3 )

-2169 ( 2 ) -1209 ( 2 )

-528 ( 2 )

-1298 ( 3 ) -2004 ( 3 ) -1626 ( 5 )

-520 ( 3 ) 237 ( 3 ) 726 ( 3 ) 449 ( 3 )

-324 ( 3 ) -821 ( 2 )

-1841 ( 3 ) -1400 ( 3 ) -2190 ( 3 ) -2820 ( 4 ) -2650 ( 3 )

U eq

184 177

94 102

95 110 163

86 1 0 1 1 0 4 108 119

78 75 79

1 1 0 126 125

99

The major conformational d i f f e rence between t h i s compound and o t h e r known 5-phenyl-1,4-benzodiaze- p ines i s i n t h e o r i e n t a t i o n of t h e 5-aryl r i n g . The angle between t h e mean plane of t h e 5-aryl r i n g and t h e 'benzo' plane i s 60.3 (6') i n bromazepam and 75.5 (9') i n o the r benzodiazepines ( 8 ) .


Table 2. Molecular dimensions

(b) Bond angles ( " )

1.351 1 .214 1.453 1.278 1.483 1.394

1.376 1.362

1 - 371

1 397 1 * 395 1.402 1.892

- - - -

1.378 - ( 6 ) C ( 11 ) -C( 10) -N( 1 ) C( 10) -c( 11) -c( 6) c( 1O)-C( ll)-C( 5) C( 6 ) -c( 11 ) -c( 5 )

c(5 -C(l')-C(2') c(5 -c(if)-c(6I) c(2 )-c( 1' 1-c( 6' )

C( 5 ) -C( 1 ' ) -N( 2 ' )

N ( 2 )-c(it)-c(6') C( 2 )-C( 2' )-C( 3' ) C( 1 ) -C( 2' )-C( 3' ) C( 1 ) - C ( 2')-F C( 3 ' ) -C( 2 ' ) -F c( 2 ' )-C(3')-C( 4' N( 2' ) -C( 3' )-C( 4 ' ) c( 3' )-c( 4 ' )-c( 5 ' ) C( 4 ' )-C( 5')-~(6') C( 5' 1-C( 6 ' 1-C( 1' )

- -

119 (31 113 (3)

127.1 (4)

114.6 (4) 123.3 ( 4 ) 111.3 (4) 117 .4 ( 4 )

122.1 (5)

126.1 ( 4 ) 116.3 (4) 117.6 (3) 121.3 (4) 120.3 ( 4 ) 119.8 ( 4 ) 119.8 (3) -

- L

- -

119.5 ( 4 )

119.5 ( 4 )

123.5 ( 4 )

112.8 (4) 119.0 ( 4 ) 117.9 ( 4 )

120.7 ( 4 )

122.3 (4)

121.2 (4)

116.9 (4)

118.1 ( 4 )

-

-

116.8 ( 4 ) - - - -

124.4 (5)

119.2 ( 5 ) 119 .4 ( 5 )

117.8 (5)

BROMAZEPAM 9

( c ) Se lec ted t o r s i o n angels ( O ) ; e . s . d . ' s ca 0.6'

C ( l O ) - N ( 1 ) - C ( 2 ) - C ( 3) -1.1 C(l l ) -C(lO)-N(l)-C(2) 39.8 N ( l ) - C ( p ) - C ( 3)-N(4) -70.3 N ( 4 ) - C ( 5 ) -C( 1' )-N( 2 ' ) 141.6

C ( 3 ) - N ( 4 ) -C( 5 ) -C( 11) N ( 4 ) - C ( 5 ) -C( ll)-C( 10)

c(2)-C(3)-N(b)-C( 5 )

C ( 5 ) - C ( 11) -C( 10)-N( 1)

72.3 -2.7

-1.2

N f 4 ) -C( 5 ) -C( 1 ' ) -C( 2 ' ) - C( 11) -C( 5 ) - C ( 1' ) -N( 2 ' ) -39.8

-37.6 C( I l ) - C ( 5 ) - C ( 1 ' ) - C ( 2 ' ) -

4 . Spec t r a l P rope r t i e s

4 . 1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum o f bromazepam i n methanol i s shown i n F ig . 2. The spec t r a of bromazepam i n 1 N NaOH and 1N H C 1 are presented i n F ig . 3 and Fig. 4 , r e spec t ive ly . The q% and maximum wavelengths

cm are given i n Table 3 ( 9 ) . These values agrees wi th t h e publ ished da ta (10-12). Lev i l l a in (13 ) , has s tud ied t h e r e l a t i o n s h i p of s t r u c t u r e and t h e W absorp t ion c h a r a c t e r i s t i c s of a series of 1,4-benzo- d iazepines , inc luding bromazepam, consider ing t h e e l e c t r o n i c d i s t r i b u t i o n o f t h e var ious subs t i t uen t s and t h e s tereochemistry. The spectrum of bromazepam i s cons i s t en t w i th t h a t of o ther benzodiazepines wi th s i m i l a r s t r u c t u r e . Also bromazepam was i d e n t i f i e d i n s o l i d dosage forms by W absorpt ion spectrometry ( 1 4 ) .

Table 3. UV S p e c t r a l c h a r a c t e r i s t i c s

Solvent ( n m ) max 1%

cm

Methanol 320 54.54 258 398.08 233 1004.78

1 N NaOH 350 59.33 268 493.77 238 794.25

270 386.60 1N H C 1 348 39 * 23

238 552.15

The above va lues agrees wi th t h a t of bromazepam.

90

80

70

60

M

40

3a

2G

10

z

z

4

C

.- 3 n N

h P 0

E

1cI

e CI

V

a

5 3

0, c

w m

M

i*

.r(

11

91

81

7(

6t

5(

4f

200 250

Fi:;. 4 . The UV spectrum of bromazeparn i n IN H C I .

A

4 . 2 In f r a red Spectrum

An i n f r a r e d absorp t ion spectrum o f potassium bromide d i spe r s ion of bromazepam (Roche - Reference Standard Lot 0401055) i s shown i n F ig . 5. The s p e c t r a l band assignments (9 ,16,17) l i s t e d i n Table 4 .

Table 4. In f r a red s p e c t r a l assignments o f bromazepam

Wave number (em-') Assignment (Vibra t ion m o M

3220,3150 , 3080 N-H s t r e t c h

3000,2940,2860 C-H s t r e t c h

1695 C = 0 s t r e t c h

161 5 C = N s t r e t c h of both benzodiazepine and pyr id ine .

1600,1590,1570,14~0 Aromatic C = C s t r e t c h

81 5 Out o f p lane CH-deforma- t i o n o f 1 ,2 ,4 - t r i - s u b s t i t u t e d aromatic

Out o f p lane CH deforma- t i o n of or tho-d isubs t i - t u t e d aromatic

1 , 2 ,h-Trisubst i t u t e d aromatic and o-d isubs t i - t u t e d aromatic . A l s o 2 -subs t i tu ted pyr id ine .

Other c h a r a c t e r i s t i c 1320, 1230, 1200, 1090, 1040, 1000, 990, 890, and 790 em-1, In f r a red has been used f o r i d e n t i f i c a t i o n of bromazepam with o t h e r psychotropic drugs (18) . Bromazepam was i d e n t i f i e d i n s o l i d dosage forms by I R absorp t ion spectrometry (14).

bands a r e 1440, 1385, 1340,

BROMAZEPAM 15

4.3 Nuclear Magnetic Resonance Spectra

4.3.1 'H-NMR Spectrum

A t y p i c a l 'H-NMR spectrum o f bromazepam i s shown i n Fig. 6. The sample was dissolved i n DMSO d6 and t h e spectrum was recorded on a 400 MHz NMR spectrometer, using TMS as an i n t e r n a l reference standard. NMR spectrum recorded on a Jeol-FX-100 90 MHz spectroxeter i s a l s o shown i n Fig 6a. ments are l i s t e d i n Table 5. The spectrum a f t e r addi t ion of D20 i s shown i n F i g . 7 .

The proton assign-

H Table 5. PMR c h a r a c t e r i s t i c s of bromazepam

Proton Chemical s h i f t ppm p o s i t ion Group and split?;irig

c-3 -CH2 4.22 ( s ) c-9 -9H (Benzenoid) 7.23 ( d ) C-6 -6H ( " " ) 7.44 ( s ) c-5' -5'H( F'yridine ) 7.49 (m) C-8 -8H (Benzenoid) 7.41 (m) c-4' -4'H( Fyridine) 7.95 ( m ) c-3' -3'H( Fyridine) 8.86 ( d ) c-6' -6'H( Pyridine) 8.57 ( d ) N-1 - l H (Diazepine) 10.66 (s)

(disappeared a f t e r D20 exchange )

s = s ing le t d = doublet rn = mul t ip l e t

BROMAZEPAM 17

F i g . 6s. 'H-NYR spectrum of bromazepam i n DMSO d6 and TMS.

Fig. 7 . 'H-NMR spectrum of bromazepam i n DMSO d6 and TYS after DzO t=xchsngr.


4.3.2 13C-NMR Spec t ra

13C-NMR noise-decoupled and off-resonance spec t r a of bromazepam are shown i n F ig . 8, and Fig. 9 , r e spec t ive ly . Both were recorded over 5000 Hz range i n deutera ted dimethyl su l foxide (DMSO d6) , (cane. 100 mg/ 1 m l ) , on J e o l FX-100 90 MHz inst rument . Sample tube 1 0 mm and t e t r ame thy l s i l ane as an i n t e r n a l re ference s tandard a t 2OoC were used. The carbon chemical s h i f t s are assigned on t h e b a s i s of t h e chemical s h i f t theory and t h e off-resonance s p l i t t i n g p a t t e r n (Table 6 ) .

9 0

5' Table 6. Carbon chemical s h i f t s of bromazepam

Chemical s h i f t Carbon No. Chemical s h i f t 6 (ppm) 6 (ppm)

Carbon No.

c-2 169.72 ( s ) C-8 133.79 ( d ) c-3 56.89 ( t ) c-9 123.34 ( d ) c-5 167.55 ( s ) C-la 138.49 ( s ) C-5a 127.28 ( s ) c-2' 155.63 ( s ) C-6 133.50 ( d ) c-6' 148.24 ( d ) c-7 113.94 ( s f c-5: 122.87 ( a )

c-4 136.96 ( d ) c-3' 124.81 ( s )

s = s i n g l e t d = doublet t = t r i p l e t

BROMAZEPAM 19

. Fig. 8 . “C-NMR noise-decoupled Spectrum o f bmmazepam i n OMS0 and TUS.

Pig. 9 . 13C-NMR off-resonance spectrum of bromazepam in DUSO and TUS.


4.4 Mass Spectrum

The E l mass spectrum of bromazepam (Roche Reference Standard, Lot 0401055) was obtained on a Finigan 1020 quadrupole mass spectrometer and shown i n Fig. 10. I ts GC t r a c e i s shown i n Fig. 11. The ionizing electron beam energy w a s at 70 eV. It shows a molecular ion !d+ at m/e 316 ( r e l a t i v e i n t e n s i t y 34.87) and M+ + 1 at m / e 317 ( r e l a t i v e i n t e n s i t y 63.88). a r e given i n Table 7 (9).

Some of t h e most prominent ions

Table 7. The most prominent fragments of bromaz epam

m/e Relative i n t e n s i t y m/e Relative i n t e n s i t y

317 63.88 236 100.00 (base

31 6 34.87 208 43.56 Peak)

31 5 60.70 207 30 - 37 314 26.84 206 23.33

289 33.24 179 30.89

288 59.95 104 21.09

287 35.95 90 35.54

286 52.84 79 19.69

261 19.28 78 31 - 90 259 19.36 77 24.08

51 25.67

F i g . 10. EI-mass spectrum of bromazeparn.


5. Synthesis

Di f fe ren t rou te s f o r t h e synthes is of bromazepam were reported (19-21).

Route I

Br [11

CH NH COOC2H5.HC1

pyridine /ref lux

2 2

H I 0

BROMAZEPAM 23

Br2

2-( 2-Amino-5-bromobenzoylpyridine [ 41 was prepared from 2-phenacylpyridine p-bromophenyl hydrazone [ 11 through cyclization with concentrated hydrochloric acid to give 5-bromo-2-phenyl-3-( 2-pyridy1)indole [ 21. This compound was oxidized with chromium trioxide to give 2-(2-benza- mido-5-bromobenzoy1)pyridine [3] ( 2 9 , 2 2 ) which on hydrolysis with concentrated hydrochloric acid afforded the desired compound 2( 2-amino-5-bromobenzoy1)pyridine [4] . This compound is considered to be the starting material for the synthesis of 1,4-benzodiazepines. A mixture of [4], glycine ethyl ester hydrochloride and pyridine was refluxed to give bromazepam [ 5 ] .

in glacial acetic acid

H \ / I I 0 V

[5 1 [41

Alternatively, the key intermediate compound [4] was prepared by bromination of 2-(2-aminobenzoyl)pyridine [6] with bromine and glacial acetic acid (23,24).


Route 3

H O

BrCOCH2Br

H

B r

[ 51

B r o m a z e p a m

-6 Heat

In t h i s route 2-(2-aminobenzoyl)pyridine [ 41 w a s t r e a t e d with bromoacetyl bromide in g l a c i a l a c e t i c a c i d . The crude bromoacetyl de r iva t ive [ 71 w a s converted d i r e c t l y t o bromazepam [ 5 ] by react ion with ammonia through t h e intermediate aminoacetamidobenzoyl pyridine [a] (20).

BROMAZEPAM

Route 4

25

[41

H

CH2 \ o -/ \\

H-N IC - 0- c

Q I Br

B r

' - 4 - a I CH3COOH L [ 51

An a l t e r n a t i v e method (20) f o r t h e syn thes i s of bromazepam proceeded v i a t h e carbobenzoxyglyclyl d e r i v a t i v e [ 9 ] . This compound i s prepared according t o t h e method of Sheehan and Hess devised f o r t h e syn thes i s of pep t ides ( 2 5 , 2 6 ) . dine and carbobenzoxyglycine i n methylene c h l o r i d e w a s added. N,N'-dicyclohexylurea w a s concentrated i n vacuo a t room temperature . chromatographed t o g ive [ g ] . Cleavage of t h e carbobenzoxy group wi th hydrogen bromide i n g l a c i a l a c e t i c a c i d (27) gave bromazepam [ 5 ] d i r e c t l y without i s o l a t i o n of t h e in te rmedia te , 2-( 2-aminoacetamido-5-bromobenzoy1)pyridine.

A mixture of t h e 2-(2-amino-5-bromobenzoyl)pyri-

The methylene c h l o r i d e s o l u t i o n a f t e r removal of

The r e s idue w a s d i sso lved i n benzene and


Route 5

Clarke e t a1 (1980) (28) have described i n d e t a i l s t h e preparation of bromazepam [ 5 ] from t h e corresponding benzophenone by t h e use of hexamine and hydrochloric ac id as outlined below:

H

Br

/ t -

+ 3MeC6H12N4C1

t NH4C1

t 3HC02Me

BROMAZEPAM 27

Three main by-product were i d e n t i f i e d : two imidazol idi- nones [lo], [ll] and an aminobenzodiazepine ( 1 2 ) .


6. Metabolism

The f irst reported metabol ic work on bromazepam w a s t h a t published by Sawada i n 1972 (29 ,30) . This s tudy w a s c a r r i e d out both i n dogs, r a b b i t s and rats. The i s o l a t e d u r ina ry metabol i te was assumed t o be a 3-hydroxylated de r iva t ive of 2-amino-5-bromobenzoylpyridine [15] . The s t r u c t u r a l e luc ida t ion w a s based on elemental a n a l y s i s , mass spectrometry and nuclear magnetic resonance s tud ie s .

OH

The biotransformation of bromazepam, was s tudied i n four spec ies namely human, dog, rat and mouse (31) . Four metabol i tes w e r e i d e n t i f i e d and t h e i r excre t ion w a s q u a n t i t a t i v e l y determined. excreted by t h r e e humans given s i n g l e 1 2 mg o r a l doses of bromazepam-5- 1 4 C were t h e conjugated forms of 3- hydroxy bromazepam [13] accounting f o r 13-30% of t h e dose, and 2-( 2-amino-5-bromo-3-hydroxybenzoyl)pyridine [15] accounting f o r 4-25% of t h e dose. Conjugated [131 w a s a l s o t h e major metabol i te i n dogs and mice adminis tered 14C-bromazepam, but i t s excre t ion by rats was very l imi t ed . amino-5-bromobenzoy1)pyridine [4] were de tec ted i n t h e excre ta of t h e human, dog, rat and mouse. Although t h e pyridyl N-oxide [I81 d e r i v a t i v e o f bromazepam w a s not de tec ted as a metabol i te i n any spe ices , t h e Nq-oxide [14] was a minor metabol i te i n dog u r ine . This i s t h e f i r s t reported ins tance of N4-oxidation of a 1,4-benzodiazepine-2-one. This s tudy has shown t h a t t h e metabolism of bromazepm i n human, dogs but not i n rats f i t s t h e same p a t t e r n of o the r lY4-benzodiazepine-2-one i n a f fo rd ing conjugated 3-hydroxylated d e r i v a t i v e (32). The i d e n t i - f i c a t i o n and q u a n t i t a t i o n of t h e d i f f e r e n t metabol i tes were c a r r i e d out by t h i n l a y e r chromatography us ing seve ra l so lvent systems.

The major u r i n a r y me tabo l i t e s

Together wi th [13] , metabol i te [15] and 2-( 2-

BROMAZEPAM 29

de Silva et al, 1974 (33) have reported a sensitive and specific electron-capture GLC assay for bromazepm and its major metabolites (see under methods of analysis). Table 8 shows the chemical names and physical properties of bromazepam and its major metabolites.

Table 8. Chemical names and physical properties of bromazepam and metabolites

Chemical name No. Molecular Melting weight point

5

13

14

4

15

16

17 18

7-Bromo-1,3-dihydro-5-(2- pyridyl) -2H-1,4-benzodiazepin-2-one (bromazepam)

7-Bromo-1,3-dihydro-5-(2- pyridyl) -2H-1,h-benzodiazepin-2-one 4-oxide.

2-Amino-5-bromobenzoylpyridine.

2-Amino-5-brom0-3-hydroxy- benzoylpyridine.

Bromazepam mercapturic acid methylester( 7-bromo-1,3- dihydro- 5- [ 2 ' - ( 6 ' -N-ac etyl- L-cyst ein-S-y1)-pyridyl] -2H- 1,4-benzodiazepin-2-one.

Met hylt hiobromaz epam Pyridyl N-oxide of bromazepam

316.16

332 - 2

332.2

277.12

293.12

228

- -

237438.50 dec .

198-2ooo

263O dec.

97 * 5-99O

190-196O

-

- -

Taleishi and Shimizu 1976 (34) has recently reported the isolation of a methylthio-containing metabolite which was identified as 7-bromo-l,3-dihydro-5-[2'-( 6I-methylthio)pyridyl]-2H-1,4-benzodiazepin - 2-one (methylthiobromazepam) [17]. Also they have shown that the methionine donor theory (34) format ion of methylthiobromazepam.

is very unlikely in the case of the Isolation of the mer-

I

0

01

e Y

Y M

E

BROMAZEPAM 31

capturic acid conjugate of bromazepam from bile of rat given the drug, its identification and subsequent conversion of the mercapturic acid to methylthiobromazepam in rat liver preparation was reported (35). Scheme 1 shows a proposed pathway for the biotransformation of bromazepam to methylthiobromazepam in the rat.

H

[51

H O

CH3 I II

H ' I COOH

Bromazepam mercapturic acid H I

14 [Me- C -SAM] Y

Br

[I71 @-S: 14CH3

Scheme I. A proposed pathway f o r the biotransformation of bromazepam to methylthiobromazepam in the rat.

14 In this study non-radioactive bromazepam, [5- C] bromazepam and bromazepam-N'-oxide ('j'-bromo-1,3-dihydro-5-[2'- (1 ' -ox0 ) -pyridyl 3 -2H-1 ,h-benzodiazepin - 2-one were employed. 7-Bromo-l,3-dihydro-5-[ 2 I - ( 6'-methyl ,N-acetyl- L-cysteinate-5-yl)-pyridyl]-2H-1,4-benzodiazepin-2-one (bromazepam rnercapturate methylester) was synthesised from bromazepam "-oxide and methyl N-acetyl-L-cysteinate in acetic anhydride according to the pyridine-N-oxide


nucleophi l ic s u b s t i t u t i o n descr ibed by Bauer and Dickerhofe ( 3 6 ) . thiobromazepam i s o l a t e d previously i n t h e r a t u r i n e i s formed a t l e a s t i n p a r t v i a t h e mercaptur ic ac id and t h a t rat l iver conta ins enzyme(s) capable of ca t a lys ing t h e conversion from t h e mercapturic a c i d t o methylthiobromazepam. Chemical names and phys ica l p r o p e r t i e s of bromazepam and i t s metabol i tes are l i s t e d i n Table 8. r eac t ions of bromazepam and i t s known metabol i tes are shown i n Scheme 2.

The r e s u l t s i nd ica t ed t h a t 6'-methyl-

Chemical

H I 0 OH

""3 I1 I

COOH

[ I 7 1 Bromazepam mercapturic acid

Scheme 2. Chemical r eac t ions of bromazepam and i t s known m e t a - b o l i t e s .

BROMAZEPAM 33

Pharmacokinetic S tudies

Di s t r ibu t ion and e l imina t ion of bromazepam has been s tudied i n 4 human sub jec t s fol lowing s i n g l e o r a l and intravenous doses o f 6 mg 14C-bromazepam ( 3 7 ) . t i o n and d i f f u s i o n o f t h e substance t a k e s p l ace r a p i d l y , peak plasma l e v e l s being reached about an hour a f t e r o r a l adminis t ra t ion . Bromazepam i s mainly e l imina ted i n t h e u r i n e , and, un l ike most o t h e r benzodiazepines, excre- t i o n fol lows a regu la r , r e c t i l i n e a r p a t t e r n which i s p r a c t i c a l l y completed a f te r 120 hours. metabol i tes c l o s e l y p a r a l l e l s t h a t of t h e unchanged substance. H a l f - l i f e f o r t h e e l imina t ion of bromazepam from t h e plasma is 20.1 hours following intravenous admin i s t r a t ion , compared wi th 22.5 hours f o r t o t a l r a t i o - a c t i v i t y (unchanged bromazepam p lus me tabo l i t e s ) . 1 2 and Fig. 13 f o r intravenous and o r a l admin i s t r a t ion are p r a c t i c a l l y i d e n t i c a l . plasma p r o t e i n s w a s s tud ied by e q u i l i b r a t i o n d i a l y s i s , which showed t h a t about 70% of t h e substance present i s bound. This i s cons iderably less than f o r diazepam and most o t h e r benzodiazepines, and i s a t t r i b u t e d t o t h e increased p o l a r i t y of t h e molecule due t o t h e presence of t h e pyr idyl r a d i c a l (38) .

Absorp-

Excret ion o f t h e

F ig .

Binding of bromazepam by

Another work descr ibed t h e app l i ca t ion of gas l i q u i d chromatography fo r pharmacokinetic s t u d i e s i n man ( 3 9 ) . The a s say can be used a l s o for r o u t i n e plasma l e v e l monitoring. The important pharmacokinetic parameters have been ca l cu la t ed from t h e plasma concent ra t ion .

- 1 2 T - ( B ) = e l imina t ion h a l f - l i f e = 18.5 hours, C1 = t o t a l ~

body plasma c l ea rance = 347 m l / m i n and VdR = apparent volume of d i s t r i b u t i o n = O.gl/kgm following s i n g l e o r a l dose of 6 mg/day.

1 3 mg/day, it w a s found t h a t T 2 ( B ) = 19.5 hours =

243 ml/min and Vdg = 071/kg. Kaplan e t a l , 1976 (40) have repor ted plasma concent ra t ions and t i m e p r o f i l e s which i n agreement wi th t h e above mentioned work.

However, us ing mul t ip l e dosing wi th


Fig. 12 Fig. 13

- Bromazepam . . . . . ,

Bromazepam + metabolites

Fig. 12. 14C-Bromazepam and total radioactivity (ug/ml) in plasma after I.V. injection o f 6.0 mg.

Fig. 13. 14C-Bromazepam in plasma (pg/ml) after oral administration of 6.0 mg.

BROMAZEPAM

React ion with 1,3-

Diazo- dinitroben- I n In coupling zene 1 M N a O H 2 M HC1

(CH3)bN OH

Bromaze- Orange Red v i o l e t Yellow Yellow red Pam

i


8.1 Elemental h a l y s i s

The elemental composition of bromazepam is

Element % Theoret ical

C 53.18

H 3-19

B r 25.28

N 13 * 29

0 5.06

8.2 I d e n t i f i c a t i o n Tests

I n DMSO

Orange brown

8.2.1 Color Reactions

Reported color react ions for bromazepam a r e as follows ( 4 1 ) :

8.2.2 Thin-Layer Chromatogram (42)

Layer : S i l i c a g e l F254, MERCK pre-

coated p l a t e s 0.25 mm.

Mobile phase : Ethyl a c e t a t e - conc. ammonia (100 + 1) (prepare f r e sh ly ) without s a tu ra t ion of t h e chamber atm0sphel.e.

36 MABMOUD M. A. HASSAN AND MOHAMMAD A. ABOUNASSIF

Application : 10 pl of each solut ion (sample and comparison)

Sample solut ion: Shake 400 mg of t a b l e t powder with 5 ml of chloroform for approx. 3 min and f i l t e r .

Bromazepam comparison solu- t i o n : Dissolve 60 mg of bromazepam i n 25 m l of chloroform.

Front dis tance : 12 cm

Detect ion : 1. Observe under short-wave W-l ight : decrease of t h e fluorescence through bromazepam.

2. Spray t h e dr ied p l a t e with a f r e s h l y prepared solut ion of f e r rous s u l f a t e , dry for a short moment and spray then with an ammonia (1%): Bromazepam appears as a v i o l e t spot.

Rf-value : Bromazepam approx. 0.6

Ferrous s u l f a t e solut ion : Dissolve 1 g of FeS04. 7H20

i n 100 m l of d i s t . water.

8.2.3 Microcrystal Tests

Bromazepam w a s i den t i f i ed i n solut ion by microcrystal t e s t s i n microgram quan t i t i e s (43) *

A micro drop of t h e t e s t solut ion ( i n 50% ethanol) w a s placed on t h e g l a s s cover s l i p and a micro drop of t h e reagent was added. The drop being s t i r r e d with aslight scratching of t h e g l a s s t o promote t h e formation of c r y s t a l s . The appearance of t h e c r y s t a l s w a s recorded as follows:

1. With potassium cadmium iodide, c r y s t a l s i n t h e form of s m a l l t a b l e t s were obtained a f t e r one hour (Fig. 14a).

BROMAZEPAM 37

2. With potassium mercuric iodide, c r y s t a l s i n t h e form of round t r i f e r c a t e blackish s t ruc tu res , white on black back ground were formed a f t e r one hour (Fig. 14b) .

3. With gold bromide hydrochloride, c r y s t a l s i n t h e form of flowers of dendri tes were obtained i n s t a n t l y (Fig. 14c ) .

The s e n s i t i v i t i e s of t h e t e s t s were 0 .2 , 0.5 and 0 .1 microgram respect ively.

8.3 T i t r ime t r i c

8.3.1 Non-aqueous Ti t ra t ion

Among several 1,h-benzodiazepine de r iva t ives , bromazepam w a s t i t r a t e d with perchlor ic acid i n dioxane or tetrabutylammonium hydroxide i n benzene-methanol ( 4 3 ) .

8.4 Spectrophotometric

Unlike other 1 ,b-benzodiazepines, t h e exis tence of t h e a, a ' -dipyridyl type moiety enables bromazepam t o form complexes with divalent metal ions. Sabatino -- e t a1 (45 ) reported t h a t ferrous ions form purple complexes with pyridyl benzodiazepin-2-ones; compounds having a basic dipyridyl type bond s t r u c t u r e which has excellent metal complexing propert ies .

HN- -N

c - C \\

The method has been applied ( 4 6 ) t o t h e determination o f bromazepam i n biological mater ia ls by adding methanol solut ion of ferrous chlor ide t o t h e bromazepam obtained a f t e r extract ion from blood o r u r ine and measuring t h e absorbance of t h e solut ion a t 580 nm. The method proved t o be spec i f i c for bromazepam with no interference from 2-amino-5-bromobenzoylpyridine, one of i t s p r inc ipa l metabolites.

The procedure w a s a l s o applied t o t h e determination of bromazepam i n r a t blood ( 4 7 ) .

Fig. 14a Fig. 14b Fig. 14c

Fig. 14a. Microcrystals of bromazepam with potassium cadmium iodide.

Fig. 14b. Microcrystals of bromazepam with potassium mercuric iodide.

Fig. 14c. Microcrystals of bromazepam with gold bromide hydrochloride.

BROMAZEPAM 39

Smyth e t a1 (48) s tudied t h e che la t ion of bromazepam wi th F e ( I I ) , C u ( I I ) , and Co(I1) us ing spectroscopic techniques such as W - v i s . spectrophotometry.

Color imetr ic determinat ion of bromazepam, as w e l l as some o t h e r benzodiazepines, w a s a l s o performed a f t e r r e a c t i o n wi th a modified Marquis's reagent ( 4 9 ) . pKa values and mechanism of hydro lys is were a l s o repor ted using W spectrophotometry ( 3 ) .

Another W-spectrophotometric method used by F. Hoffnann - La Roche & Co Limited i s as fol lows: (50)

Assay: The determinat ion should be c a r r i e d out w i th in one hour under subdued day-l ight .

Pulver ize 20 tab le t s and accu re t e ly weigh approxima- t e l y 1000 mg of t a b l e t powder i n t o a 250 ml-volumetric f l a s k . Add about 200 m l of 0.1 N methanolic s u l f u r i c a c i d and shake f o r 1 5 min o r t r e a t wi th u l t r a s o n i c s . D i l u t e t o volume with 0 .1 N methanolic s u l f u r i c ac id . F i l t e r a p a r t o f t h i s so lu t ion ( d i s c a r d t h e f i r s t 1 5 m l ) and d i l u t e 10.0 m l of t h e c l e a r f i l t r a t e t o 100.0 m l wi th 0.1 N methanolic s u l f u r i c a c i d ( = t e s t s o l u t i o n ) .

Method. of Analysis: t h e ex t inc t ion o f t h e t e s t s o l u t i o n at 285 nm (max.) aga ins t 0 .1 N methanolic s u l f u r i c a c i d as a blank us ing 1 em-quartz c e l l s .

With a spectrophotometer measure

Calcu la t ion :

mg of bromazepam tab le t . =

E = ex t inc t ion o f t h e sample so lu t ion

E. 25000 . A 300 . B

A = average weight (mg)

B = weight of sample (mg)

300 = E ( 1 % , 1 cm)-value of bromazepam a t 285 nm (maximum) i n 0.1 N methanolic s u l f u r i c ac id .

Methanolic s u l f u r i c ac id : D i l u t e 5.0 g o f 98% s u l f u r i c a c i d t o 1000 m l with methanol. Prepare t h i s s o l u t i o n a t l e a s t 24 hours before use.


8.5 Polarographic

The ease of reduct ion of t h e azomethine ( > C

and t h e ( > C = 0) carbonyl group of i t s benzophenone metabol i tes , promotes t h e i r q u a n t i t a t i o n i n t h e subnanogram range by d i f f e r e n t i a l pu l se polarography.

= N4) group of bromazepam and i t s 3-hydroxy metabo 7 i t e ,

D e S i l v a et a1 (33 ) descr ibed a d i f f e r e n t i a l pu l se polarographic method f o r t h e determinat ion of bromazepam and i t s metabol i tes , 3-hydroxy-bromazepam, 2-amino-3-hydroxy-5-bromobenzoylpyridine and 2-amino- 5-bromobenzoylpyridine i n u r i n e .

Polarography w a s c a r r i e d out us ing phosphate b u f f e r as t h e support ing e l e c t r o l y t e wi th pH of 5.5. The peak p o t e n t i a l , Ep, due t o t h e reduct ion o f t h e azomethine group of bromazepam and i t s 3-hydroxy- metabol i te occured at -0.535 and -0.555 V ve r s calomel e l ec t rode r e spec t ive ly , whereas t h e P e z due due t o t h e reduct ion of t h e carbonyl group of t h e metabol i tes 2-amino-5-bromobenzoylpyridine and 2-amino-3-hydroxy-5-bromobenzoylpyridine occured at -0.630 and -0.635 V versus calomel e l e c t - rode, r e spec t ive ly (F ig . 15). S e n s i t i v i t y l i m i t s ranged between 50 and 100 ng/5 ml u r ine .

Trace levels of bromazepam i n blood w a s determined by d i f f e r e n t i a l pu lse polarography ( 5 1 ) . cur ren t r e s u l t i n g from reduct ion o f t h e 4,5-azome- t h i n e bond of bromazepam w a s measured at -0.610 V versus s i l v e r ch lo r ide e l ec t rode . The method has a recovery of 62.1% * 7.8 i n t h e range of 10-1000 ng bromazepam/l ml of blood.

The

Polarography w a s a l s o used t o s tudy acid-base and complexing behaviour of bromazepam (3), and f o r t h e i d e n t i f i c a t i o n of any one o r more of several 1 ,4 - benzodiazepines inc luding bromazepam ( 5 2 ) .

8.6 Atomic Absorption

Gonzales-Perez et a1 (53 ) descr ibed a method f o r t h e determinat ion of bromazepam by atomic absorp t ion spectrophotometry. ex t r ac t ion of t h e ion-pair formed by t h e bromazepam- n i cke l (11) c a t i o n i c che la t e and pe rch lo ra t e from aqueous t o methyl isobutylketone (MIBK) so lu t ions .

The method i s based on t h e

BROMAZEPAM 41

I

POTENrl AL VOLTS VERSUSSATURATED

I I - 0.350 -0550 -0.750

CAZOMEL ELECTROOE Cb)

Fig. 15. Differential pulse polarograms of: (a) I and I1 (b) I 1 1 and IV in 1.0 M pH 5.5 phosphate buffer as the supporting electrolyte. Key: A, control urine blank; B, authentic standard mixture; and C, authentic recovered from urine.

I. Bromazepam 11. 2-Amino-5-bromobenzoylpyridine 111. 3-Hydroxymetabolite of bromazepm IV. 2-Amino-3-hydroxy-5-brombenzoylpyridine


The absorbance of n i c k e l i n t h e organic phase w a s determined at t h e n i cke l resonance a 232.0 nm l i n e . The method t h a t showed a de tec t ion l i m i t o f 2 X 10-6M, was app l i ed t o t h e determinat ion of bromazepam i n drugs.

8.7 Radioimunoassay

Robinson e t a1 (54 ) descr ibed fou r radioimmunoassays f o r t h e determinat ion of bromazepam and o the r benzodiazepines. I n t h e most s e n s i t i v e method, t h e antiserum from t h e Finit-tox serum benzodiazepine

a assay k i t w a s used wi th [-H]flunitrazepam and proved t o be s u i t a b l e f o r de t ec t ing sub-therapeut ic l e v e l s of a l l benzodiazepines t e s t e d except one. The assay i s p a r t i c u l a r l y app l i cab le t o blood samples of fo rens i c i n t e r e s t which may be hemolyzed or decom- posing, and r equ i r e only 75 m l o f blood.

A rad ioreceptor assay f o r benzodiazepines inc luding bromazepam i n human blood, plasma, s a l i v a and u r i n e w a s developed ( 5 5 ) . tit ion between [ 3H]flunitrzepam and b i o l o g i c a l l y a c t i v e benzodiazepines i n b i o l o g i c a l f l u i d s f o r bra in-spec i f ic r ecep to r s , prepared i n a s t a b l e , d ry form and easy t o handle. The method i s s p e c i f i c f o r b i o l o g i c a l l y a c t i v e benzodiazepines. Other method, descr ibed t h e determinat ion of benzodiazepines i n serum us ing t h e enzyme mul t ip l i ed immuno assay-single t e s t (EMIT-ot) drug de tec t ion system ( 5 6 ) . method i s use fu l t o c l i n i c a l tox ico logy p r a c t i c e .

The method is based upon compe-

The

8.8 Chromatographic

8.8.1 Paper Chromatography

Bromazepam w a s separa ted from o t h e r benzodiazepines by using a t h i n l a y e r system: Merck aluminium oxide F254 and CHC13 - PhMe - E t O H (40:60:2) complemented by a Whatman S.G. 81 s i l i c a g e l loaded paper system developed i n CHCl3-EtOH (49:l). shor t W wavelength and a c i d i f i e d potassium iodoplat i n a t e ( 57 ) .

The spo t s were loca ted by

BROMAZEPAM 43

8.8.2 Thin-Layer Chromatography

Haefe l f inger (58 ) has repor ted a s p e c i f i c and s e n s i t i v e method for t h e determinat ion of bromazepam i n plasma by q u a n t i t a t i v e t h i n - l a y e r chromatography. S i l i c a g e l p l a t e s were used wi th methylacetate-ammonia 33% (1OO:l) as a so lvent system. The q u a n t i t a t i o n of t h e spo t s w a s performed e i t h e r by measuring t h e W r e f l e c t a n c e of t h e p l a t e o r by t h e hydroly- s i s of bromazepam t o 2-amino-5-bromobenzoyl- py r id ine on t h e p l a t e , d i a z o t i z a t i o n and coupl ing with N-( 1-naphthy1)ethylenediamine t o form an azo-dye, which was then evaluated dens i tome t r i ca l ly . The recovery from plasma was found t o be over 90%. Bromazepam has an Rf value of 0.6-0.65.

Thin-layer chromatographic systems and detec- t i o n methods descr ibed f o r t h e i d e n t i f i c a t i o n and determinat ion of bromazepam are summarized i n Table 9 .

Table 9 . Solvent systems and de tec t ion methods fo r br oma z epam

Support

S i l i c a AR 7GF-5 (twc dimen- t i o n a l

TLC )

S i l i c a ge l G

S i l i c a ge l

6o *254

Solvent system

Hept ane /e t hyl ace t a t e/ethanol, concentrated ammonia ( 50 : 50 : 3) and : Heptane/ chloroform/ ethanol/conc en t r a - t e d ammonia (50:50:20:1)

Chloroform/acetone (gO:lO), benzene/isopropanol/ammonia ( 85 : 15 : 10)

Diisopropylether-toluene- e t h y l a c e t a t e methanol - di - ethylamine (70:15:10:5:2)

Detect ion

W- l igh t

Bratton-Marshall, dragendorff and iodopla t i n a t e reagents . W-l igh t (254 nm: 10% w / v ~ $ 0 4 heat a t 120' for 1 0 minutes chamber wi th n i t r o u s fumes Spray wi th a- napht h y l e t hylme- diamine R.

- Ref.

59 -

60

50


Thin l a y e r chromatographic de t ec t ion and i d e n t i f i c a t i o n of bromazepam among o the r lY4-benzodiazepines has a l s o been repor ted (61-64).

8.8.3 Gas Chromatography

Numerous methods f o r t h e gas chromatographic ana lys i s o f bromazepam i n b io log ica l f l u i d s have been repor ted . Most of t h e s e methods descr ibed t h e determinat ion o f t h e compound e i t h e r as t h e i n t a c t 1 ,b-benzodiazepine-2-0ne o r as t h e hydrolyzed 2-amino-5-bromobenzoyl- pyr id ine (ABBP) (33, 65-69). Bromazepam has a l s o been assayed as t h e "-methyl d e r i v a t i v e ( 7 0 ) .

The presence of e l ec t ronega t ive func t iona l groups ( t h e halogen i n t h e 7-posi t ion and t h e 2-carbonyl) renders bromazepam amenable t o s e n s i t i v e determinat ion by electron-capture gas- l iquid chromatography (EC-GLC); t h e r e f o r e a l l (except one) t h e above mentioned methods appl ied (EC-GLC) t o t h e determinat ion o f t h e drug. I n p r i n c i p l e , t h e s e methods are capable of de tec t ing nanogram amounts o f bromazepam i n b i o l o g i c a l f l u i d s where they compete wi th radioimmunoassays. Recently, it has been coupled with mass spectrometry (71). The experimental condi t ions used f o r t h e a n a l y s i s of t h e drug i n b i o l o g i c a l f l u i d s are summarized i n Table ( 1 0 ) .

Table 10. Gas liquid chromatographic conditions f o r the determination of bromazepam and its metabolites

Drug o r metabolite

2-Amino-5-bromo-benzoylpyridine (ABBP)

2-Amino-5-bromo-benzoylpyridine (ABBP)

Bromazepam

Bromazepam

Bromazepam

N'-Methylbromazepam

Column packing

2% Carbowax 20M-TPA on silanized Gas ChromP (100-120 mesh)

3% OV 225 (methyl-phenylcyanopropyl. silicone) on Gas Chrom Q ( 6 0 / 8 0 Rest

3% OV-17 on Gas Chrom Q (60/80mesh)

3% OV-17 on Gas Chrom Q (60/80mesh)

10% UCC-W-98 on Chromosorb W AW DMC: (80/100 mesh)

0.5% OV-17 Chromosorb G HP (100/200 mesh)

4% SE-52 silica capillary column

20% UCC-W on Chromosorb W AW DMCS (80/100 mesh)

Carrier

N2

Ar-cH4 (9O:lO)

Ar-CH4 (90:lO)

Ar-CH4 ( 90-10 1

N2

N2

He

He

Column temp .

220 * 20

214O

24 5 O

240°

265"

270°

Program- med

130-260'

Program- med

100-310°

RT mi ns -

6-7

12.5

6

9.6

4

3-4

3-4

-

)et ect or 5

EC

EC

EC

EC

FI

EC

EC

Mass spect rometer

-

2e:

-

65

66

33

67

68

70

69

71

- Ar = Air


8.8.4 High Pressure Liquid Chromatography

A high pressure l i q u i d chromatographic procedure f o r t h e determinat ion of bromazepam i n human plasma has been descr ibed ( 7 2 ) . A f t e r ex t r ac t ion from plasma, t h e e x t r a c t w a s evaporate& and t h e res idue w a s suspended i n acetonitrile/tetrabutylammonium hydroxide (60:40, v/v) before i n j e c t i o n i n t o t h e chromatograph. Analysis w a s performed on a 1-1 Bondpack c18 column with mobile phase made

from 20 m l MeOH, 30 m l MeCN, and T O O ml of so lu t ion conta in ing 20 ml 10% t e t r a b u t y l - ammonium hydroxide i n 1 L water. The i n t e r - n a l s tandard w a s carbamazepine and de tec t ion w a s by W spectroscopy.

In order t o determine t h e lower the rapeu t i c range of drug l e v e l s i n serum (73 ) , bromazepam, among o the r bas i c drug, w a s chromatographed on an ~ ~ 1 8 Micropack MCH 10 column us ing UV de tec to r and perchlorate/MeCN so lu t ion as e luent .

Hochmuth -- e t a1 (74) descr ibed a simple and r ap id method f o r t h e tox ico log ica l a n a l y s i s of some benzodiazepines and an t idep res san t s including bromazepam. A f t e r solvent extrac- t i o n at pH 9 , t h e a n a l y s i s w a s performed by HPLC on a reverse-phase column with MeCN 0.6% KH PO (1:l) adjus ted t o pH 3.

Heizmann -- e t a1 (75 ) repor ted a HPLC method f o r t h e determinat ion of bromazepam i n plasma and of i t s main metabol i tes i n u r ine . The unchanged drug was ex t r ac t ed from plasma wi th dichloromethane, and t h e r e s idue was subsequent ly analysed by reversed phase HPLC wi th W de tec t ion (230 nm) . For t h e determinat ion of t h e metabol i tes , t h e u r i n e samples were incubated t o e f f e c t enzymatic deconjugat i on before ex t r ac t ion with dichloromethane. The column used w a s a 15 cm Supelcos i l LC18 and t h e mobile phase cons i s t ed o f a mixture o f methanol/phosphate b u f f e r , pH 7 .5 , a t a flow r a t e of 1 ml/min. from 6 t o 20 minutes.

2 4

The r e t e n t i o n times ranging

BROMAZEPAM 41

9. References

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2. Mart indale , The Extra Pharmacopoeia, J.E.F. Reynolds and A.B. Prasad, 28th Ed., The Pharmaceutical P res s , London, 1982, p. 1505.

3. M.R. Smyth; T.S. Beng and W.F. Smyth, Anal. Chem. Acta, - 92, 129 (1977).

4 . H . Butcher, T.A. Hamor and I . L . Martin, Acta Crys t . , Sect C : Cryst . S t r u c t . Commun. , -, 1469 (1983).

5. P. Chonanont , T.A. Hamor and I .L. Mar t in , Cryst . S t r u c t . Comun. , c, 393 (1979).

6.

7. G. G i l l i , V . B e r t o l a s i , M. Sacerdot i and B.A. Borea,

I b i d , Acta Cryst . , my 1371 (1981).

Acta Cryst . my 2664 (1977).

8. P. Chananont, T.A. Hamor and I . L . Martin, Acta Crys t . , - B36, 1690 (1980).

9. M.A. Abounassif and M.M.A. Hassan, unpublished d a t a .

10. R.W.T. S e i t z i n g e r , Pharm. Weekbl., 110, 1109 (1975).

11. M. Kahnert-Brandstaet ter , A. Kofler and G. F r iedr ich- Sander, S c i . Pharm. , 9, 234 (1974) .

12. J. Bavre t t , W.F. Smyth and I . E . Pavidson, J. Pharm. Pharmacol. , 25, 387 (1973).

13. P. Lev i l l a in , M. Bertucat and B. Pe r ro t , Eur. J. Med. Chem. Therapeut ica , 10, 433 (1975).

1 4 . 0. Oturn, T. Takhashi, T. Tomiyama, Kagaku Keisa tsu Kenkyusho Hokoku, Hakagaku Hen, 36, 98 (1983).

15. J . G . Ruters and C.U. Shearer , Analyt. Prof. of Drug. Subst. Edi ted by Klaus Florey, Academic Press , London, Vol. 9 (1980) p. 404.

16. "The I n f r a r e d Spec t ra of Complex Molecules" , L. J. Bellamy , 2nd Ed. , John Wiley & Sons , Inc . , New York (1964).


17. In f r a red Absorption Spectroscopy, K. Nakanishi and P.H. Salomon, Second Edi t ion , Holden-Day, Inc . , San Francisco, U.S.A. (1977).

18. F. Pa la , G. D e Cosmo, A.F. Sabato and A. Bondoli, Resusc i ta t ion , 9, 125 (1981).

19. The Benzodiazepin S tory , L.H. Sternbach, Edi t ions (Roche), Basle , Switzer land, Progress i n Drug Research, Vol. 22, B a s 1 1 : Burkhauser, (1980) p. 27.

20. R . I . F ryer , R . A . Schmidt and L.H. Sternbach, J . Pharm. S c i . , 53, 264 (1964) and U . S . Pa t . 3,100, 7 7 0 , (Aug. 13, 1963).

21.

22.

25 *

26.

27 *

28.

31

32

S. Inaba, K. Ishizumi, T. Okomoto and H . Yamamoto, Chem. Pharm. B u l l , 23, 3279 (1975) .

D.W. Ockenden and K. Schof ie ld , J. Chem. Soc., 3440 (1953) - A . J . Nunn and K. Schof ie ld , I b i d , 583 (1952) .

S. Raynolds and R . Levine, J . Am. Chem. SOC., 82, 472 ( 1960) . J . C . Sheehan and G.P. Hess, I b i d , 77, 1067 (1955).

A. Stempel and F. Handgraf, J. Org. Chem., 3, 4675 ( 1962 1 * D. Ben-Ishai and A. Berger, I b i d , 17, 1564 (1952) .

G.M. Clarke, J . B . Lee, F . J . Swinbourne and B. Williamson, J. Chem. Research ( 5 1 , 399 (1980) .

H. Sawada, Ekper ien t ia , 28, 393 (1972) .

H. Sawada, H. Yano and A. Kido, J . Pharm. SOC. Jap . , - 92, 1237 (1972).

M.A. Schwartz, E. Postma, S.J . Kol i s and A.S. Leon, J . Pharm. S c i . , 62, 1776 (1973).

M.A. Schwartz, i n "Benzodiazepines", S. G a r a t t i m i , E. Mussini, and L.O. Randal l , Eds . , Raven Press , New York, N . Y . , 1973, PP. 53-74.

BROMAZEPAM 49

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

J .F. D e S i l v a , I. Bekersky, M.A. Brooks, R.E. Weinfield, W . Glover and C.V. P u g l i s i , J . Pharm. S c i , 63, 1440 ( 1974 ) .

M. T a l e i s h i and H. Shimizu, Xenobiotica, 6, 431 (1976).

M. T a l e i s h i , S. Suzuki and H. Shimiza, Biochemical Pharmacology, 2'J, 809 (1978).

L. Bauer and T.E. Dikkerhofe, J . Org. Chem., 3, 2183 (1964 1 . Raaflaub and J .S . Pe i se r - Courvois ier , Arzneimit te l - Forsch. , - 24, 1841 (1974).

W. Muller and U. Woller t , Naunya-Schmiedeberg's, Arch. Pharmakol, 278, 301 (1973).

U. Klotz , J. Chromatogr., 222, 501 (0981).

S.A. Kaplan, K.L. Jack , R.E. Weinfield, W . Glover, L. Weissmann and S. Cu t l e r , J . Pharmacokin. Biopharm., 4, 1 (1976).

(1983). K.A. Kovar and D. Linden, Pharm. Acta Helv., 58, 66

F. Hoffmann - La Roche & Co. Ltd., Control Department, Basle , Switzer land ( P r i v a t e Communication) (1986).

S . N . T e w a r i and S.K. Shukla, Pharmazie, 33, 372 (1978).

F. Barbato, C . Grieco, C . C i l i po and A. V i t t o r i a , Farmaco, Ed. P ra t ., &, 187 (1979).

J.D. Sabat ino, O.W. Weber, G.R . Padmanobhan, and B.Z. Senkowski, Anal. Chem., 41, 905 (1969). N. Gallo, V.D. Bianco and S. Doronzo, I L Farmaco, 35, 497 (1980).

M. Fukumoto, Chem. Pharm. Bu l l , 3, 3678 (1980).

W.F. Smyth, R. Scannel l , T.K. Goggin D. Lucas-Hernandez, Analyt ica Chimica Acta, 141, 321 (1982). H.M. Stevens, J. Fores ic S c i . Soc., 18, 69 (1978).


50. Spec i f i ca t ions for Lexotan "Roche" , Control Department , Hoffhann - La Roche & Co Ltd. , Basle, Switzer land (1975) -

51. M.A. Brooks and M.R. Hackman, Anal. Chem., 47, 2059 (1975).

52. W.F. Smyth, M.R. Smyth, J.A. Groves and S.B. Tan, Analyst , 103, 497 (1978).

53. C. Congalez-Perez, J. Hernandez-Mendez and M . I . Gonzalez- Martin, I L Farmaco, Ed. P r a t . , 3, 383 (1983) .

54. K. Robinson, M. Ru t t e r fo rd , R.N. Smith, J. Pharm. Pharmacol. , 32, 773 (1980) .

J. Lund, Scand. J. Clin. Lab. Inves t . , 41, 275 (1981).

Clin. Toxicol. , 9, 303 (1982).

I 11, 183 (1971).

P. Haefe l f inger , Chromatographia, 11, 1 0 (1978).

55.

56. R.H. Drost , T.A. Plomp and R.A.A. Maes, J. Toxicol.

57. H.M. Stevens and R.W. Jenkins , Forens ic , S c i . SOC., J.

58.

59. M.A. Schwartz, W.R. Pool D.L. Hane and E. Postna, Drug Metabolism and Dispos i t ion , - 2 , 31 (1974).

H. Schuetz, Fresenius ' Z. ana l . Chem., 294, 135 (1979). 60.

61. L. La r in i , P.E.T. Salgado, T.C. Ralpho and M.G. Vilela, An. Farm. Quim. Sao Paulo, 17, 37 (1977).

62. P. F i c a r r a , M . G . V igor i t a and A. Tomanapini, Bol l . Chim. Pharm. , 116, 720 (1977).

63. D. Dekker and P.M.C. P a r e l , Pharm. Weekbl. , 118, 465 ( 1978 1 .

64. A . Hulshoff and J.H. P e r r i n , J . Chromatogr., 129, 263 ( 1976 1.

65. J.A. D e S i l v a and J. Kaplan, J. Pharm. S c i , 55, 1278 (1966).

66. J.P. Cano, A.M. Baile and A . Viala, Arzneim. Forsch, 25 1012 (1975).

BROMAZEP AM 51

67. J . A . De S i l v a , I. Bekersky, V.C. P u g l i s i , M.A. Brooks and R.E. Weinfield, Anal. Chem. , 48, 1 0 (1976).

68. T. Kaniewska and W. Wejman, J . Chromatogr., 182, 81 ( 1980 1 . J.M.F. Douse, J. Chromatogr., 301, 137 (1984).

U. K l o t z , J . Chromatogr., 222, 501 (1981).

H. Maurer and K. P f leger , J. Chromatogr., 222, 409

69.

70.

71. (1981).

72. H. Hirayama, Y . Kasuya and T. Suga, J. Chromatogr., 277 414 (1983).

73. H. Kaefers te in and G. S t i c h t , B e i t r . Ge r i ch t l . Med., 41, 95 (1983).

74. P. Hochmuth, J.L. Robert , H . Schreck and R. Wennig, Anal. Chem. Syrup. Se r . , 20, 143 (1984).

75. P. Heizmann, R . Geschke and K. Zinapold, J. Chromatogr., - 310, 129 (1984).

Acknowledgement

The au thors would l i k e t o thank M r . Tanvir A. Bu t t , College of Pharmacy, King Saud Univers i ty , for typ ing of t h i s manuscript .

BUSULPHAN

Muhammad T d q " and AbduReah. A . Al-Ba&**

*U~pumincnX 06 P h a c o L o g y and "Vepahtment 06 PhmacclLticd CCZmhfiy, CoReegc 06 Phmacy , King Saud UniuemLty, Riyadh, Saudi hub&.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 53

Copyright 0 1987 by Academic Press, Inc. A11 rights of reproduction in any form reserved.

54

1.

2.

3.

4 .

5.

6.

7.

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

Foreword

Description

2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2 . 4 Elemental Composition 2.5 Appearance, Color and Odor

Physical Properties

3.1 Melting Point 3.2 Solubility 3.3 Storage 3.4 Spectral Properties

Synthesis

Pharmacokinetics

Toxicology

Methods of Analysis

7.1 Identification 7.2 Titrimetric Methods 7.3 Chromatographic Methods 7.4 Nuclear Magnetic Resonance Methods

References

BUSULPHAN 55

1. Foreword

Busulphanis a chemotherapeutic agent, which has been widely used as antineoplastic drug since its discovery in 1953 (1). This alkylating agent belongs to the series of alkanesulfonic acid esters, possessing significant cytotoxic activity and is the drug of preference in the treatment of chronic myelocytic or granulocytic leukaemia. It has recently been proposed as a drug of choice in polycythaemia and thrombocythemia (2-13). Busulphanhas also been used for the treatment of bronchial carcinoma (18-19) , myasthenia gravis (20) and chronic granulomatous disease in children (21). The drug is also used as reproductive sterilant for insect (22-23) and as a general immunosuppressant for organ transplant in the treatment of autoimmune disease (24-26).

2. Description

2.1 Nomenclature

2.1.1

2.1.2

2.1.3

Chemical Names

1,4-Butanediol dimethanesulfonate; 1,4-Bis (methanesulf 0noxy)butane; 1,4-di(methanesulfonyloxy)butane; 1,4-di(methylsulfonoxy)butane; 1,4-Butanediylbismethane sulphonate; Methanesulfonic acid tetramethylene ester; Te t rame thylene bis (me thanesul f onat e) (2 7-2 9) . Generic Name

Busulphan, CB 2041, GT 41, NSC 750, WR 19508 Busulfanum (29).

Trade Names

Myleran, Misulban, Mitosan, Myelosan, Myeleukon, Myeloleukon, Sulfabutin, Mielucin (27).

56 MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

2.2.2 Structural

0 0 I1 I I

H C-S-O-CH2-CH2-CH2-CH2-O-S-CH3 3 I t II

0 0

2.2.3 CAS No.

[55-98-17 (27)


246.31 (31)

2.4 Elemental Composition

C, 29.26%; H, 5.73%; 0 , 38.98%; S, 26.03% (27).

2.5 Appearance, Color and Odor

A white almost odorless crystalline powder (29, 32).

3. Physical Properties

3.1 Melting Point

114-118O, 119O, 116'. (27).

3.2 Solubility

Soluble, at 20°, in 750 parts of water (in which it is slowly hydrolyzed) and in 25 parts of acetone; very slightly soluble in alcohol (27, 32).

3.3 Storage

Busulphan should be kept in a well-closed container, protect from light (30).

BUSULPHAN

3.4 Spectral Properties

3.4.1 Ultraviolet Spectrum

The ultraviolet spectrum of busulphan in methanol, ethanol and in water was scanned from 200 to 400 nm using Varian DMS 90 Spectrophotometer. No absorbance has been noticed .

3.4.2 Infrared Spectrum

The infrared spectrum of busulphan as KBr disc is shown in Figure 1. It was recorded on a Pye-Unicam SP 1025 infrared spectrophotometer. The assignments of the characteristic bands in the infrared spectrum is shown below:

-1 Frequency cm Assignments

2960-3060 CH stretch

1430-1415 -CH2-0 vibration

1352 S ( = 0>2 stretch (Assymm.)

1180 S ( = 0)2 stretch (Symm.)

1000-770 S-0-C stretch

1 3.4.3 Proton Nuclear Magnetic Resonance ( H NMR)

Spectrum

1 The H NMR spectrum of Busulphan is shown in Figure 2. and its spectrum determined on a Varian - T60 A NMR spectrometer using TMS as the internal standard.

The drug is dissolved in DMSO-db

Assignment of the chemical shifts to the different protons is shown below:

Wavelenqth urn

Jo 3500 3ooo 2500 zoo0 1800 1600 1400 1200 loo0 800 625

Wavenumber Figure 1 : Infrared spectrum of Busulphan, KBr disc.

ch W

I I " ' ' I ' * I ' I ' . " 1 . " " " " ' I

500 400 300

6 ! 5 4 3 2 9 1

0 i; CH3-~-O-CH2-CHj-CH~CH,-0 -S- CH3

1

0

TM:

- -

, a . . . a . . . . . . . . 8.0 7.0 6 .O

Figure 2 : Proton nuclear magnetic Resonance spectrum of Busulphan in

DMSO -dg using TMS as reference standard


Chemical Proton shift (6) Multiplicity assignment*

1.68-1.90 Multiplet 3 and 4 (Cg2)

3.10 Sing 1 e t 1 and 6 (Cg3)

4.1-4.40 Mu1 t iple t 2 and 5 (Cl12)

*Please refer to Figure 2.

3.4.4 Carbon-13 Nuclear Magnetic Resonance Spectra

(C-13 NMR)

The carbon-13 NMR spectra of busulphan in DMSO-d6 using TMS as an internal reference are obtained using a Jeol FX 100 MHz spectrometer at an ambient temperature. Figure 3 and 4 represent the proton-decoupled and off-resonance spectra respectively.

0 0 6 11 5 4 3 2 II 1

iI 2 2 2 2 11 CH3-S-0-CH CH CH CH -0-S - CH3

0 0

The carbon chemical shifts are assigned on the basis of the chemical shift theory and the off-resonance splitting pattern and are shown below:

Chemical Carbon shift (6) Multiplicity assignments

24.70 Triplet 3 and 4

36.52 Quartet 1 and 6

69.40 Triplet 2 and 5

Figure 3 : Proton - decoupled carbon - I3 nuclear magnetic resonance spectrum of Busulphan in DMSO- dgusing TMS as reference standard.

Ill I I

Figure 4 : Off- resonance carbon-13 nuclear magnetic resonance spectrum of Busulphan in DMSO-dG using TMS as reference standard.

BUSULPHAN 63

3.4.5 Mass Spectrum

The electron impact (EI) mass spectrum of busulphan at 70 eV recorded on Varian Mat 311 mass spectrometer and the direct chemical ionization (DCI) mass spectrum obtained with Finnigan 4000 mass spectrometer are shown in figures 5 and 6 respectively. The (EI) spectrum (Figure 5) shows a base peak at m/e 7 1 , the molecularion peak was not detected. Other major fragment are at m/e 175, 122, 111, 109, 97, 79, 55 and 42. A proposed mechanism of fragmentation of the EI fragments is shown in scheme 1.

The CI spectrum (Figure 6) is simple and shows a base peak at m/e 151 and at molecular ion peak at 247 corresponding to (M + 1) and minor peak at m/e 125.

4. Synthesis

By esterifying 1,4-butanediol [HOCH CH CH CH OH] with methanesulfonyl chloride [CH SO2C1] in the presence of pyridine (31 , 33) as shown below:

2 2 2 2 3

0 I I P y r i dine c

II 0

CH3-S-C1 + OH-CH2CH2CH2CH2-OH r

0 I I

0 II

CH3-S-O-CH2CH2CH2CH2-O-E-CH3 1 1 0 0


175

-29568

-

100 150 200 2 50 300 Figure 5 : Electron impact (El) mass spectrum of Busulphan.

100 150 200 250 300 350 Figure 6:Ckmical imitation(CI 1 mass spectrum of Busulphan.

+O

X

m

5 I 1

X 'c:

+o=(.=

0

I re: 0

..

U

0

I 0

I 0 =--a I

o=

m=

o

I Xm

U

co m

I m

8

t m

X

V

w 0

PO

1 .rl

m

X

U

0

I + c o=

m=

0

-5

1

+o- I -

,m-0

c'

i Y

\ 0 0

m/e 246

+ OH

0 CH3

/ ‘ 0 + OH

+;,

(4*7”J;-m3 iI

0

0 OH

0 0

m / e 175

+

m / e 7 1

Scheme 1 : Continued.

m

T;

0

0

1. I

+O

=T

=O

I II 51 0 -m

=o

P

0 --m=

0

I T; m

m

+O

P

.. 4

m

E

67


5. Pharmacokinetics

Absorption, Metabolism and Excre t ion

Busulphan i s r e a d i l y absorbed from t h e gas t ro- i n t e s t i n a l t rac t and r a p i d l y d isappears from t h e blood. I t i s l a r g e l y exc re t ed i n t h e u r i n e as su lphur - conta in ing me tabo l i t e (29) . Due t o i t s a l k y l a t i n g a c t i v i t y , t h e compound might be bound both r e v e r s i b l y and i r r e v e r s i b l y (covalen t ly) t o blood component (34). The metabolism t akes p l a c e mainly i n t h e l i v e r . The c l i n i c a l response u s u a l l y begins wi th in 1 t o 2 weeks a f t e r i n i t i a t i o n of therapy. It appears t o be p r a c t i c a l l y completely excre ted i n t h e u r i n e as methane-sulphonic a c i d (35)

Nadkarni -- e t a1 ( 4 ) us ing 14C and 35S-labeled busulphan found t h a t , a f t e r in t ravenous dosing, t h e r a d i o a c t i v i t y i n t h e blood r a p i d l y decreased, and between 20% and 95% was removed wi th in 3 t o 5 minutes. Af t e r o r a l dosing an i n i t i a l l o g phase of about 0.5 t o 2 hours was observed be fo re measureable blood l e v e l s were achieved. Af t e r o r a l 3H-labeled busulphan, Vodopick -- e t a l , (36) found a prompt r i s e i n t h e plasma l e v e l of r a d i o a c t i v i t y , which i s peaked wi th in t h e f i r s t hour , followed by a rap id f a l l and then a gradual rise i n r a d i o a c t i v i t y . Busulphan i s e l imina ted wi th t$ of about 2.5 hour , f o r t h e i n i t i a l phase of r a d i o l abe led drug i n plasma. Only about 1% of a dose of busulphan i s exc re t ed as unchanged drug wi th in 2 4 hours . busulphan i s probably e l imina ted by enzymatic a c t i v i t y r a t h e r than pure chemical degrada t ion

The major p o r t i o n of

(36).

Ehrsson e t a1 (37) have s tud ied t h e busulphan k i n e t i c s and r epor t ed t h a t k i n e t i c s were followed i n p a t i e n t wi th chronic myelocyt ic leukemia a f t e r o r a l dose of 2.4 t o 6 mg. The plasma concent ra t ion- t i m e d a t a could be f i t t e d t o a zero-order abso rp t ion one-component open model. The e l imina t ion rate cons tan t averaged 0.27 ? 0.05 hr-1 (SD). The plasma AUC w a s l i n e a r l y r e l a t e d t o t h e dose. t i m e f o r t h e s tar t of abso rp t ion , t h e t i m e absorp- t i o n ends, and t h e absorp t ion rate cons t an t showed

The l o g

BUSULPHAN 69

some inter-individual variation. About 1% of the drug is excreted unchanged in urine over 24 hours.

The fate of the drug in man has been studied after the administration of radiolabelled drug followed by measurement of total radioactivity in plasma and urine (4, 36, 37).

The drug is rapidly eleminated from the plasma and is reported to be extensively metabolized; twelve metabolites have been isolated, including methane-sulphonic acid and 3-hydroxytetrahydro- thiophene-1,l-dioxide, most of the metabolites have not been identified (38).

Wiygul et a1 (39) have studied metabolism of busulphan in the boll weevil. When one-day male boll weevils were force-fed 3H- and Ik-labelled busulphan, 34.34% of total radioactivity was recovered from the feces, about 1.09% as busulphan, at 72 hours after injection. Most metabolism of busulphan took place within 24 hours postingestion. 1,4-Butanediol, 2,3-butanediol, sulfolanes, malic acid, malonic acid, succinic acid, aminoacids, and methanessulphonic acid were identified as principal metabolites.

Jones and Campbell (40) have studied the metabolism, reactions and biological activities of some cyclic dimethanesulphonates. Relevance to the mechanism of action of busulphan (Myleran). The results obtained suggest that cycloalkylation of thiol groups by busulphan may represent a normal detoxification route rather than a mechanism of act ion.

Other studies on the metabolism of busulphan in b o l l weevils, are also reported (41, 42, 43).

6. Toxicity

Recently Bishop and Wassom (44) have published a detailed review article on the toxicity of busulphan incorporating around 290 references. The most important side effect of busulphan in high doses are thrombocytopenia and damage to haemotological tissues (45,53, 29) .


I n t e r s t e t i a l pulmonary f i b r o s i s , known as "Busulphan lung", i s as w e l l r e c o p i s e d s i d e e f f e c t busulphan therapy (54-64). A wast ing o r Addison - l i k e syndrome has occurred i n some p a t i e n t s r ece iv ing long term therapy wi th Busulphan (59, 65, 66) . Other adverse e f f e c t s inc lude endocardia1 f i b r o s i s (67) , c a t a r a c t s (68) ) c e l l u l a r dysp la s i a of pancreas ( 6 9 ) , adrena l i n s u f f i c i e n c y (70) and bone marrow damage (71-83) . Laboratory s t u d i e s i n animals have shown s i g n i f i c a n t immunosuppressive ( 4 8 , 81,) 85, 82, 86) and reproduct ive t o x i c i t y of busulphan (87 - 100) .

Busulphan, adminis tered dur ing t h e t h i r d trimester of pregnancy, can adverse ly a f f e c t f e t a l growth (101). Teratogenic e f f e c t of t h i s drug i n animals has been repor ted by s e v e r a l workers (102 - 106) . Busulphan i s p o t e n t i a l l y mutagenic (104 - 114) and carc inogenic i n man (115 - 123).

7 . Methods of Analysis

7 . 1 I d e n t i f i c a t i o n

The USP XX (1980) ( 3 2 ) desc r ibes t h e fo l lowing i d e n t i f i c a t i o n t e s t s f o r busulphan.

A) Fuse about 100 mg with about 100 mg of potassium n i t r a t e and a p e l l e t of potassium hydroxide weighing approximately 250 mg. Cool, d i s s o l v e t h e r e s idue i n water, a c i d i f y wi th d i l u t e d hydrochlor ic a c i d , and add a few drons of barium c h l o r i d e TS; a whi te p r e c i p i t a t e i s formed.

B) To 100 mg add 10 m l of water and 5 m l of 1 N sodium hydroxide. Heat u n t i l a clear s o l u t i o n i s obta ined; an odor c h a r a c t e r i s t i c of methanesulfonic ac id i s p e r c e p t i b l e .

C) Cool t he s o l u t i o n obtained i n I d e n t i f i c a t i o n Test B , and d i v i d e i t i n t o two equal po r t ions . To one p o r t i o n add 1 drop of potassium permanganate TS; t h e purp le c o l o r changes t o v i o l e t , then t o b lue , and f i n a l l y t o emerald- green. Acid i fy the second por t ion of t h e solu- t i o n wi th d i l u t e d s u l f u r i c a c i d , and add 1 drop

BUSULPHAN 71

of potassium permanganate TS: t h e permanganate i s no t discharged.

B r i t i s h Pharmacopoeia (1980) (30) has a l s o repor ted similar procedures f o r t h e i d e n t i f i c a t i o n of busulphan.

t he c o l o r of

7.2 Ti t r imet r ic Methods

7.2.1 Aqueous

B r i t i s h Pharmacopoeia (1980) (30) descr ibed an aqueous t i t r a t i o n procedure f o r t h e assay of busulphan a s fol lows:

To 0.25 g of t h e drug add 20 m l of water and b o i l g e n t l y under a r e f l u x condenser f o r t h i r t y minutes. Wash t h e condenser wi th a small q u a n t i t y of water, cool and t i t r a t e w i t h 0.1 M sodium hydroxide VS, us ing phenolphthalein s o l u t i o n as i n d i c a t o r . Each m l of 0.1 M sodium hydroxide VS i s equ iva len t t o 0.01232 g of C6H1406S2.

7.2.2 Gravimetr ic

Busulphan t a b l e t s are sugar coated and con ta in only mi l l ig ram q u a n t i t i e s , hence t h e f l a s k combustion method i s no t app l i cab le becuase of t h e l a r g e amount of organic matter. It i s necessary t o e x t r a c t t h e busulphan wi th acetone and decompose i t i n a s ea l ed tube with n i t r i c ac id a s fol lows:

T r i t u r a t e an accu ra t e ly weighed q u a n t i t y of powdered t a b l e t s equiva len t t o about 2 mg of busulphan wi th 10 m l of ho t acetone, decant t h e l i q u i d through a f i l t e r i n t o a Carius tube and evaporate t o small volume under a j e t of warm a i r . Ex t r ac t i n t h e same way wi th two f u r t h e r 10-ml q u a n t i t i e s of acetone, decant ing and evapora t ing a f t e r each e x t r a c t i o n and evapora t ing t o dryness a f t e r t h e t h i r d e x t r a c t i o n . Add 0.5 m l of fuming n i t r i c a c i d , s e a l t h e tube and h e a t a t 300° f o r two hours. Af t e r opening t h e


Carius tube transfer its contents to a small dish with 15 ml of water, evaporate to dryness, dissolve the residue in water and acidify with concentrated hydrochloric acid. Add an excess of 10 per cent barium chloride solution, heat on a water-bath for three hours, filter through a platinum filtering crucible, ignite at 800° for one hour, cool and weigh. Each mg of residue = 0.5276 mg of busulphan (124).

7.3 Chromatographic Methods

7.3.1 Gas Chromatographic Method

Hassan and Ehrrson (125) developed a gas chromatographic method for the determination of busulphan in plasma with electron capture detection. derivatization technique for extraction of busulphan from plasma in a single step combined with high separation efficiency and sensitivity by using capillary column in combination with electron capture detection. The derivatization of busulphan was done directly in plasma by adding sodium iodide in acetone. Plasma sample (1.0 ml) was mixed with 0.1 ml of 1.0 ug/ml of 1,5-bis(methanesulfoxy)pentane in acetone and sodium iodide in water (1 ml acetone and 8 M Na' iodide). 0.4 ml n-heptane the reaction was carried out at 7OoC for 70 minutes under stirring. The organic phase was separated for analysis on gas chromatography. 1-2 pl of solution was injected into Varian 3700 GC equipped with a constant current 63Ni lectron- capture detector. An OV-I fused silica cappillary column (25 m x 0.3 mm i.d.) with a film thickness of 0.52 um (Hewlett- Packard) was used.

The method comprise a new

After addition of

The instrument was operated isothermally with the oven, detector and injector port temperatures at 145OC, 25OoC and 2OO0C respectively. The helium was used as carrier gaswith a flow rate of 2 ml/min,the inlet pressure was

BUSULPHAN 73

0.4 bar . Nitrogen was used a s make up gas and added through t h e hydrogen i n l e t a t a rate of 30 ml/min i n the d e t e c t o r base. A s p l i t r a t i o of 1 : 10 w a s used. A high sepa ra t ion e f f i c i e n c y and s e n s i t i v i t y w a s obtained us ing t h i s method, t he r e t e n t i o n t i m e w a s 8 minutes. The s tandard curve us ing plasma w a s l i n e a r wi th in t h e range of 5-300 ng/ml. t 3.9% (c.v.) a t the 10 ng/ml l e v e l (n = 5) and ? 2 . 3 % (c.v.) a t t h e 100 ng/ml (n = 5) .

The p rec i s ion of t h i s method

7.3.2 Gas Chromatography - Mass Spectrometry

(GC-MS)

Ehrsson and Hassan (126) developed a chromatographic method f o r t h e determina- t i o n of busulphan i n plasma by GC-MS wi th se l ec t ed ion monitoring. Busulphan ex t r ac t ed from plasma with methylene ch lo r ide and converted t o 1,4-diiodobutane. 1.0 M 1 of plasma was mixed wi th 0.1 m l of i n t e r n a l s tandard (1,5-pentanediol dimethanesulfonate 1.0 pg/ml) i n acetone and ex t r ac t ed with 4 m l of methylene ch lo r ide using a mechanical shaker (100 s t rokes/min) . The organic phase w a s separa ted and evaporated t o dryness , d e r i v a t i z a t i o n was done by adding 0.1 m l of 1 M sodium iod ide i n acetone wi th a r eac t ion t i m e of 20 minutes a t 7OoC. Af t e r addi t ion 0.05 m l of n-hexane and 0.1 m l of water t h e mixture was vigorously shaken. The a l iquo t (1-2 pg of n-hexane) w a s used f o r the a n a l y s i s by GC-MS;LKB 2091 wi th an ion iz ing energy of 70 e V was used. The column(1.5 m x 2 mm i . d . ) w a s packed wi th 10% SP-2401 on 100-120 Supelcoport and w a s operated a t 140°using a helium flow rate of 20 ml/min. The i n j e c t o r and the i o n source temperature w e r e 230° and 27OoC, respec- t i v e l y .

The mininam! de tec t ab le concent ra t ion was 5.7 x 10-16 mole/sec. The s tandard curve w a s l i n e a r over a range of 10-400 ng/ml. The r e l a t i v e S.D. was 2.6% a t 100 ng/ml and ? 4.3% a t 10 ng/ml (n = 5) .


7.4 NMR Spect rometr ic Methods

a ) Truczan and Lau-Cm(l27) desc r ibed a s imple

20 G r a m of busulphan NMR spec t roscopic method f o r t h e a s say of busulphan i n t a b l e t s . t a b l e t s are f i n e l y powdered, an a c c u r a t e l y weighed q u a n t i t y of powder equ iva len t t o 6 mg of busulphan i s t r a n s f e r r e d t o a 15 m l v i a l . To t h i s 1 m l of i n t e r n a l s tandard s o l u t i o n (11.68 mg of methenamine i n 10 m l of chloroform) was added, t h e v i a l was c losed wi th a septum and crimper sea l ed wi th an aluminium seal. 2 M I of chloroform w a s added us ing a sy r inge by in t roduc ing t h e needle through septum. The conten ts were shaked v igorous ly . The i n s o l u b l e matter i s allowed t o se t t le and 0.5 t o 1 m l of upper l a y e r i s t r a n s f e r r e d t o NMR tube. 1 Drop of r e fe rence s tandard (50 mg te t ramethyl - s i l a n e (TMS) i n 10 m l chloroform) was added, caped and shaken. The NMR s p e c t r a were recorded wi th a 200 MHz, Wise bore , Four i e r t ransform spectrometer equipped wi th 5 mm proton probe and computer wi th 24 K memory. The spectrometry cond i t ions were: ambient temperature = 30OC, frequency 1 h = 200.06 MHz, Observation frequency range = 1600 H2.

Pu lse width = 9.13 sec. Pulse r e p e t i t i o n t i m e = 14 sec. FTD d a t a p o i n t s 3 8 Observation t i m e = 2.2 minutes .

I n t e g r a t e t h e broad t r i p l e t a t about 4.3 ppm due t o t h e 4-methylene protons (-CH2-O-S02-) of busulphan and t h e s i n g l e t s and about 4.7 ppm due t o t h e 12 methylene pro tons of t h e i n t e r n a l s tandard. obtained from:-

The q u a n t i t y of busulphan i n mg i s

(AU/AS) x (EV./ES) x c , where

Au = I n t e g r a l va lue of signal r ep resen t ing

A s = I n t e g r a l va lue of signal r ep resen t

Ev = Formula weight of busulphan 14, 61.58. E s = Formula wieght of methenamine/l2, 11.68.

bulsulphan.

methanamine.

C = Weight i n mg of methenmine taken f o r t h e a n a l y s i s .

BUSULPHAN 15

b) Feit and Rastrup-Andersen (128) have investigated the hydrolysis of busulphan by means of NMR spectroscopy. The final product was found to be tetrahydrofuran (THF). The rather unstable intermediate, 4-methane- sulphonybutanol , was synthesized and proved to undergo cyclization to THF by intramolecular alkylation. reaction in aqueous solution at 3 7 O was determined to be approximately 12 minutes at pH 3 as well as at pH 7.4. From the data obtained, it is concluded that 4-methansulphony- loxybutanol is unlikely to be responsible for the biological action of busulphan.

The half-life of this first-order

Acknowledgements

The authors would like to thank Mr. Syed Rafatullah, Assistant Researcher, College of Pharmacy, King Saud Univeristy, for his technical assistance and to Mr. Tanvir A. Butt, secretary to the Pharmaceutical Chemistry Dept. for his secretarial services.


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2.

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17.

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67. A. Weinberger, J. Pinkhas, U. Sandbank, M. Shaklai, A. de Vries J. Am. Med. Assoc. 231, 495 (1975).

68. M.P. Ravindranathan, V.J. Paul and E.T. Kuricose, Br. Med. J. 1, 218-219 (1972). ---

69. R.H. Kirschner and J.R. Esterly, Cancer 27(5) 1074- 1080 (1971).

70. A. Rodriques Cuartero, E.D. Cara and J.P. Redondo, Rev. Clin. Esp. 131, 73-78 (1973). --

71. AMA Drug Evaluation, 4th Ed., American Medical Association, Chicago, Illinois, page 1165 (1980).

72. L.A. Elson, Br. J. Haematol 1, 104-116 (1955).

73. L.A. Elson, Biochem. Pharmacol., 1, 39-47 (1958).

74. L.A. Elson, Ann. N.Y. Acad. Sci., 68, 826-833 (1958).

75. S.S. Esterberg, F.S. Philip and J. Scholler, Ann. N.Y.

--

Acad. Sci. 68, 811-825 (1958). - 76. L.A. Elson, D.A.G. Galton and M. Till, Brit. J. --

Haematol. - 4, 355-374 (1958).

77. M. Asano, T.T. Odell, T.P. McDonald and A.C. Upton, Arch. Pathol. - 75, 250-263 (1963).

78. E. Niskanen, Acta. Pathol. Microbiol. Scand. 70 Suppl. 190.1 (1967).

79. A.A. Wardhaugh. Mutation Res. 88, 191-196 (1981).

80. O.P. Ganda and A. Mangalik, J. Assoc. Physcns. India, 21(6), 511-516 (1973).

81. A. Dunjic and A.M. Cuvelier. Exp. Hematol., L(1), 11- 12, (1973).

82. C.A. Pugsley, I.J. Forbes and Morley, Blood 51, 601- 610 (1978).

BUSULPHAN 81

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

T. Tange, Acta. Pathol. Jpn., 24(1), 93-117 (1974).

K.M. Baisakh, D.S. Agarwal, B. Iyengar and V.N. Bhatia, Indian J. Med. Res. 63(12), 1716-1731 (1975).

H.I. Mamura, K. Ikegani and Takeuchi , J. Pharmacol. SOC. Jpn. 95, 453-459 (1975).

-----

M.C. Berenbaum. Immunology 36, 355-365 (1979).

W. Bollag, Experentia, 9, 268 (1953).

W. Bollag, Schweiz Med. Wschr., 84, 393-395 (1954).

A.W. Carig, B.W. Fox and H. Jackson, Nature, 181, 353- 354 (1958).

H. Jackson, B.W. Fox and A.W. Craig, Br. J. Pharmacol. - 14, 149-157 (1959).

H. Jackson, M. Partington and B.W. Fox, Nature, 194, 1184-1185 (1962).

H. Jackson, B.W. Fox and A . W . Craig, J. Reprod. Fertil., 2, 447-465 (1961).

J.F. Monoyer, C.R. SOC. Biol. (Paris), 156, 968-970 (1962).

B.W. Fox, W.H. Jackson, A.W. Craig and T.D. Glover, J. Reprod. Fertil.,5, 13-22 (1963).

A.B. Kar, V.P. Kamboj and H. Chandra, J. Repr. Fertil. - 16, 165-170 (1968).

P. Jones and H. Jackson, J. Reprod. Fertil. 2, 322 (1972).

W.M. Generoso, S.W. Huff and S.K. Stout, Mutation - 11, 411-420 (1971).

319-

Res. -

R.F. Moore and H.M. Taft, J. Econ. Entomol. 66(1) 96- 98 (1975).

H.M. Flint, N. Earle, J. E8tOn and W. Klassen, J. Econ. Enatomol. 66(1), 47-53 (1973).


100. E.B. Mitchell, J.W. Haynes, P.M. Huddleston and D.D. Hardee, -- J. Econ. Entomol. 68, 610-612 (1975).

101. S.J. Bros and J.W. Reynolds, Am. J. Obstet. Gynecol., - 129, 1, 111-112 (1977).

102. J. Pinto Machado, Acts Gynaecol. Obstet. Hisp-Lusit - 15, 201-212 (1966).

103. J. Pinto Machado, Arch. Portug. Sci. Biol. 16, 5-31 (1968).

104. G.F. Somers, Proc. Eur. SOC. Study Drug Toxic., lo, 227-234 (1969).

105. P.L. Weingarten, J.R. Ream Jr., and A.M. Pappas, Clin. Orthop. Relat. Res. 3, 236-247 (1971).

106. W.J. Swartz, Teratology 1-8 (1980).

107. J.Y. Richmond and B.N. Kaufmann, Exp. Cell. Res. 54, 37-380 (1969).

180. T. Fukishima and L.J. Zeldis, Acts Haematol. Jpn., 33, 312-320 (1970).

109. T. Fukishima and L.J. Zeldis, Acts Haematol. Jpn., 33, 321-328 (1970). -

110. J.R. Honeycombe, Mutation Res. 57, 35-49 (1978).

111. E. Gebhart, G. Schwanitz and G. Hartwich, Dtsch. Med. Wschr. 99, 52-56 (1974). --

112. A. Schinzel and W. Schmid, Mutation Res. 40, 139-166 (1976).

113. J.R. Honeycombe, Mutation Res. 84, 399-407 (1981).

114. J.R. Honeycombe, Mutation Res. 81. 81-102 (1981).

115. H. Waller, Pathol. Microbial - 23, 283 (1960).

116. N. Gureli, S.W. Denham and S.W. Root, Obstet. Gynecol. - 21, 466-470 (1963).

117. B.M. Nelson and G.A. Andrews, Am. J. Clin. Pathol. 42, - 37-44 (1964).

BUSULP H AN 83

118. M.L. Feingold and L.G. KOSS, Arch. Intern. Med., - 124, 66-71 (1969).

119. S. Dahlgren, G. Holm, N. Svanborg and R. Watz, Acts Med. Scand. 192(1), 129-135 (1972). ---

120. I. Diamond, M.M. Anderson and S . R . McCreadie, Pediatrics, 25, 85-90 ( 1960).

121. J.F. Pezzimenti, H.C. Kim and .T. Lindenbaum, Cancer - 38, 2242-2246 (1976).

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F.H. Sobles, Pharm. Weckble. - 107, 365 (1972). 123.

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CHLORAMBUCIL

Mohammad T L q * and AbduReah A . ke-Bad&**

*UepcuLtment 06 P h m a c o R o g y , **UepahGncn;t 06 Phacu~laceuLicaI Chern&in.fhy, College 06 P h m a c y , K i m j Saud LlniLImiXy, P.O. Box 2 4 5 7 , Riyadh-17451, S a u d i RtLabia.


Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form resewed. 85


1. History

2. Description

2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Color and Odor 2.6 Crystal Properties


3.1 Melting Point 3.2 Extraction 3.3 Solubility 3.4 Moisture Content and Hygroscopicity 3.5 Dissociation Constant 3.6 Storage 3.7 Spectral Properties

4. Synthesis

5. Pharmacokinetics

6. Therapeutic Uses

7. Toxicity


8.1 Identification 8.2 Titrimetric Methods 8.3 Spectrophotometric Methods 8.4 Chromatographic Methods

References

CHLORAMBUCIL 87

1. History

This aromatic derivative of mechlorethamine was synthesized first at the Chester Beatty Research Institute in England. It was synthesized among other aromatic derivatives of the chloroethyl- mines by the British chemists, Everett, Roberts and Ross (1, 2). of human malignancies (3). These include chronic lymphocytic leukemia (4,5). Hodgkin's and non-Hodgkin's lymphoma ( 6 ) , choriocarcinoma, ovarian carcinoma, and breast carcinoma (7). It is most often employed in the long-term maintenance management of chronic lymphocytic leukemia. rate of 19% was achieved in 52 advanced breast cancer patients.

Chlorambucil has shown activity in a variety

Moore -- et a1 (8) found that a response

2. Description

2.1 Nomenclature

2.1.1

2.1.2

2.1.3

Chemical Names

4-[Bis( 2-chloroethyl)amino]benzenebutanoic acid; 4- [ p- [ Bis ( 2- chloroethyl ) amino] phenyl ] butyric acid; N,N-di-2-chloroethyl-y-p-aminophenylbutyric acid; ~-[p-Di(2-chloroethyl)aminophenyl]butyric acid; 4- [ 4-Di-( 2-c hloroet hyl ) aminophenyl ] butyric acid; 4-[4-(Bis(2-chloroethyl)amino)phenyl]butanoic acid; p-Di(2-chloroethyl)aminophenyl butyric acid (9 -12).

Generic Names

Chlorambucil, C.B. 1348. NSC 3088 Chlorbuti- num (10-12).

Trade Names

Chlorambucil , Leukeran, Chloroambucil ,

Amboclorin; Chloraminophene; Linfolysin (11) - (10-12) .


2.1.4 CAS Regis t ry No.

( 305-03-3) . 2.1.5 Wiswesser Line Notat ion

QV3R DN2G2G (13)

2.2 Formulae

2.2.1 Empirical

C14H19C12N02 (14 J 5 ) .


C 1 CH 2CH2

\ N

/ C1CH2CH2

CH CH CH COOH

2 .3 Molecular Weight

304.20 (10,15) . 2.4 Elemental Composition

C 55.27%; H 6.30%, C1 23.31%, N 4.60%, 0 10.52% ( 1 0 ) .

2.5 Appearance, Color and Odor

A white or off-white c r y s t a l l i n e or granular powder with a s l i g h t odor (11).

2.6 Crys t a l P rope r t i e s

F la t tened needles from petroleum e t h e r ( 1 0 ) .

3. Physical. P rope r t i e s

3.1 Melting Point

Melts between 64' and 69' ( 1 2 ) .

CHLORAMBUCIL 89

3.2

3.3

3.4

3.5

3.6

3.7

Ext rac t ion

Chlorambucil i s ex t r ac t ed by organic so lven t s from aqueous a c i d s o l u t i o n s ( 1 2 ) .

S o l u b i l i t y

P r a c t i c a l l y in so lub le i n water (14). 2OoC i n 1 .5 p a r t s of a l coho l , i n 2.5 p a r t s of chloroform, and i n 2 p a r t s of acetone, so lub le i n e t h e r ( 1 0 ) and i n d i l u t e so lu t ion of a l k a l i hydroxides (11).

Soluble at

Moisture Content and Hygroscopicity

Not more than O . 5 % , determined by Fischer t i t r a t i o n

(14).

Dissoc ia t ion Constant

pKa va lue 5.8 (16) .

Storage

It should be s to red i n a cool p lace i n a i r t i g h t , l i g h t r e s i s t a n t con ta ine r s (11).

Spec t r a l P rope r t i e s

3.7.1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t spectrum of chlorambucil i n methanol e x h i b i t s maxima a t 258 nm ( E 1%, 1 cm 650) and 302 nm ( E 1%, 1 cm 85) ; and minima at 225 nm and 281 nm ( 1 2 ) .

The W spectrum o f chlorambucil i n w a t e r and i n methanol which was scanned from 200 t o 400 nm us ing DMS 90 Varian spectrophotometer. It e x h i b i t s absorp t ion maxima at 257 nm and a t 302 nm and minima a t 224 and 280 nm. The W spec t r a i n w a t e r and i n methanol a r e shown i n Figs [l] and [ 2 ] , r e spec t ive ly .

MOHAMMAD TARIQ AND ABDULLAH A. AL-BADR

so.

8 0

2

90

- ' 80

I50 2 00 I S 0 W a r d e p t h 300

Figure 2 : U l t r a v i o l e t Spectrum o f Chlorambucil i n Methanol.

.

.

.

4 0 . .

.

10 .

A 150 2 00 250 W a v e l e n p l h 300

70

so

50

LO

. l o

20

.10

CHLORAMBUCIL 91

3.7.2 I n f r a r e d Spectrum

The i n f r a r e d spectrum of chlorambucil i s shown i n Fig. [ 3 ] . The spectrum w a s obtained a s KBr d i s c and i s recorded i n Pye-Unicam SP 1025 i n f r a r e d spectrophotometer. The frequencies and t h e i r s t r u c t u r a l assignments are as follows:-

Frequency (em-') Assignment

3100-29'70

1720

O H s t r e t c h and CH s t r e t c h C = 0 a c i d

1622 C = C aromatic

1450 CH deformation

730 C-C1 s t r e t c h

Other f i n g e r p r i n t bands a r e 1530, 1465 and 830 cm-1. Clarke ( 1 2 ) repor ted t h e p r i n c i p a l peak a r e 1695, 1520 and 1229 cm-l.

3 .7 .3 Carbon-13 Nuclear Magnetic Resonance Spec t ra ( C - 1 3 NMR)

The C - 1 3 NMR no i se decoupled and o f f - resonance s p e c t r a are shown i n Figs. [ 4 ] and 151, r e spec t ive ly . Both were recorded i n deu te ra t ed chloroform on a Jeol-XL-100 MHz NMR spectrometer us ing t e t r ame thy l s i l ane as a r e fe rence s tandard. The s p e c t r a l assignments are l i s t e d below:-

10 9 c i C H ~ C H~

4 3 2 1 / &@ CH2-CH2-CH2-COOH

C1CH2CH2 7' 6'

10' 9'

L

92

Wavenumber Figure 3 : Infrared Spectrum of Chlorambucil as KBr Disc.

,

93

W

v)

.r

0 L

Y

E

.r

c

Y-

O .. d

Figure 4 : "C-NMR Spectrum o f Chlorambucil in CDC13 (Noise Decoupled).

\ a

I a

a

Figure 5 : 13C-NMR Spectrum o f Chlorambucil i n CDC13 (Off-Resonance).

CHLORAMBUCIL

Carbon No.

1

2

3

4

5

6 and 6'

7 and 7'

8

9 and 9'

10 and 10'

Chemical s h i f t ( PPm 1

180,3

33.8"

33.4"

26.4

130.2

129.6

112.1

144.3

53.5

40.4

95

Mult i p l i c it y

S

t

t

t

S

d

d

S

t

t

s = s i n g l e t d = doublet t = t r i p l e t

I 3.7.4 Proton Magnetic Resonance Spec t ra ( H-NMR)

1 The 60 MHz i n deutera ted chloroform is shown i n Fig. [ 61. The spectrum w a s recorded on Varian T ~ O - A NMR spectrometer us ing t e t r ame thy l s i l ane (TMS) as t h e i n t e r n a l s tandard . The fol lowing are t h e s t r u c t u r a l assignments:

H-NME3 spectrum of chlorambucil

Chemical s h i f t (ppm) Assignments

Mul t ip le t at 1.8-2.8 -(CH ) -CO pro tons

S ing le t a t 3.60 -N-CH2CH2 protons

Mul t ip l e t at 6.6-7.08 Aromatic protons

S ing le t at 11.5 O H ( a c i d i c ) exchange-

2 3

ab le with D20, Fig . [ T I .

4 . - . . . . . . , . . . . . . . " . . , " . , , ' , . . - - , - l - - - . v 300 200 100 0 500 400

T Y I

[ . . . . I . . . . 1 I . . . I . . . . I . . . . I . . . . I . . . . 1 . . . . Qo 7-0 6-0 5-0 6 4-0 3-0 2.0 1 4 0

Figure 6 : 'H-NMR Spectrum o f Chlorambucil i n Deuterated Chloroform.

200

L'

100 0

Figure 7 : 'H-NMR Spectrum o f Chlorambucil in Deuterated Chloroform + D20.


3.7.5 Mass Spectrum

The e l e c t r o n impact ( E I ) mass spectrum of chlorambucil a t 70 e V , recorded on Varian Mat 311 mass spectrometer us ing ion source p re s su re of 10-6 Tor r , ion source temperature of 1 8 0 O c and an emission cu r ren t of 300 uA. The spectrum i s shown i n F ig . [ a ] . The spectrum is dominated by m / e 254 ion (base peak) r e s u l t i n g from t h e l o s s of CH2C1 and H . A proposed mechanism of f ragmentat ion and t h e m / e o f t h e major fragments i s given i n Scheme 1.

The chemical i o n i s a t i o n ( C I ) spectrum (Fig . [ 9 ] ) is obtained on a Finnigan 4000 mass spectrometer wi th ion e l e c t r o n energy of 100 e V , i on source p re s su re o f 0.13 Torr, ion source temperature of 1 5 0 O c and emission cu r ren t of 300 PA. The spectrum shows a pronounced peak r e s u l t i n g from t h e loss of HC1 (& - 36) c o n s t i t u t e t h e base peak at m / e 268 and a molecular ion peak at m / e 304. A proposed fragmentat ion pathway for t h e fragment i n t h e E I spectrum i s shown i n Schemes l a and lb. The mass s p e c t r a l assignments of t h e prominant ions under C I condi t ions a r e given i n t h e fol lowing

m/e

306

305

Fragment

[ M + 2H]+

MH+

304 M+

303

286

268

270

254

232

[MH - [M - H20]+

[ M - H C l ] '

[ M + 2 H - H20]

[MH - (H2 + C H 2 C l ] +

[ M - 2 H C 1 ] +

+

1oo.c

50.0

WIE 50 1 0 0 1 so ZOO 250 300

Figure 8 : Mass Spectrum of Chlorambucil ( E I ) .

1oo.c

5O.C

I

'r M I € 200 220 240 260

I

3

'i

2a 300 320 340 I 0 310 100

Figure 9 : Mass Spectrum of Chlorambucil (CI).

z

0

8

6 LW N

N

-N

M

0

M

a, \

E

+z

I' Y z

-z

u

W

4

4

a, \

E

4

00

4

a, \

E

+z

Ill

db ..

r. 4

4

0

m

w 0

E .rl

+J

rd t,

k rd k

w

w 0

a 8 s .d

a a,

m 0

PI 0

k a

0

m/e 254

H

Scheme l b : Proposed mechanism of fragmentation of chlorambucil.

CHLORAMBUCIL 103

4. Synthesis

Evere t t et g (1) have synthes ized chlorambucil and some o t h e r n i t rogen mustard. The syn thes i s have been repor ted (17,18) as f o l l o w s :

CH2CH2CH2COOH 0 N i t r a t ion L

Esterification 02N a CH2CH2CH2COOH - Reduction Pd/CaCO,

3

02N -(Ot CH2CH2CH2COOCH3 - Ethylene oxide

HzN a CH2CH2CH2COOCH3 CH3COOH

HOCH2CH2 POC 1 4 0 CH2CH2CH2COOCH3 -

/ I

HOCH,CH, C ~ C H ~ C H ~

Hydrolysis 2J a- CH2CH2CH2COOCH3 P /

C1CH2CH2 - ClCH CH

\N CH2CH2CH2COOH I

ClCH ,CH, L L


5. Pharmacokinetics

5 .1 Absorption, Distr ibut ion, Metabolism and Excretion

Oral absorption of chlorambucil i s adequate and r e l i a b l e . The drug has a h a l f - l i f e i n plasma of approximately 90 minutes, and it i s almost completely metabolized ( 1 9 ) . Although t h e i n t r a - venous route produced higher concentrations, absorp- t i o n from t h e gas t ro - in t e s t ina l t r a c t w a s consis- t e n t l y rapid and appeared t o be complete; peak- plasma concentrations were achieved i n 40 t o 70 minutes. A n hour a f t e r administration by e i t h e r route the r a t e of metabolism w a s s i m i l a r and su f f i c i en t t o make a contr ibut ion t o the a c t i v i t y of the drug (20 ) .

There appears t o be extensive metabolic degradation of t h e drug and a major metabol i te , an aminophenyl- ace t i c acid der ivat ive which has a h a l f - l i f e of about 2.5 h r i n man, has been iden t i f i ed . The ul t imate metabolite( s ) of chlorambucil i n man has yet t o be defined and fu r the r work i s required t o confirm these i n i t i a l pharmacokinetic observations (21).

McLean e t a1 (20) reported two metabolites one w a s i den t i f i ed as t h e @-oxidation yroduct of chlorambuci l ; 2[ k-N,N-bis( 2-chloroethyl)aminophenyl] a c e t i c acid, a l s o ca l l ed phenylacetic mustard. This metabolite i s known t o have an alkylat ing ac t ion i n animals.

Dorr and W i l l i a m ( 2 1 ) extensively reviewed the disposi t ion of chlorambucil i n human being and experimental animals. In rats, radioact ive drug appear t o be cleared from t h e blood rapidly, and at one hr t he highest t i s s u e concentration i s found i n the l i v e r . However, f a i r l y homogeneous t i s s u e d i s t r i b u t i o n w a s noted. The drug h a s a l s o been shown t o d i s t r i b u t e well i n t o a s c i t i c f l u i d a f t e r subcutaneous administ-ation. Sixty percent of drug r ad ioac t iv i ty w a s excreted i n t o the ur ine by 24 h r , with the majority of t he remainder ex i s t ing as t i s s u e as tissue-bound drug (22 ) . Signif icant drug binding t o gamma globulin p ro te ins has been observed. Apparently no drug is excreted i n t h e feces . Because a portion of the drug molecule has l i p o p h i l i c

CHLORAMBUCIL 105

prope r t i e s , some f a t (depot) drug s torage may occur. This could explain the prolonged c l i n i c a l e f f e c t of chlorambucil occasionally observed i n man.

6. Therapeutic Uses

C l in i ca l t r i a l s of ch lo rmbuc i l were i n s t i t u t e d i n 1954. During t h e subsequent 5 years t h e substance w a s s tudied i n qu i t e some d e t a i l . The therapeut ic uses of chlorambucil have been discussed by Larionov (23) . The drug i s given o r a l l y i n t a b l e t form i n doses from 0 .1 t o 0.4 mg/kg, i . e . 6 t o 25 mg da i ly . The course of treatment usual ly las ts 3-9 weeks. The t o t a l dose on average i s about 400 mg per course but i n individual pa t i en t s it v a r i e s g rea t ly with t h e form of t h e disease, t h e s tage, t h e condition of t h e organism, previous treatment, e t c . The therapeut ic e f f e c t does not come a t once; but usual ly i n the 2nd t o 3rd week a f t e r starting the drug. A t these doses s ide e f f e c t s i n t h e gastro- i n t e s t i n a l t r a c t a r e r a r e l y observed. Towards t h e end of t he course , neutropenia and thrombocytopenia may appear, thus necessi ta t ing withdrawal of t h e preparation. The maintenance dose of t h e compound i s from 0.03 t o 0 . 1 mg/kg, i . e . 2-6 mg da i ly .

Chlorambucil gives t h e best e f f e c t i n chronic lymphoid leumaemia both i n the leukaemic and aleukaemic form and, i n p a r t i c u l a r , i n t h a t var iant known as f o l l i c u l a r lymphoma Galton and T i l l (24 ) . Altman e t a1 (25) , I s r a e l s _ - et a1 (261 Doan e t a1 ( 2 7 ) . I n chronic lymphoid leukaemias, chlorambucil i s given i n doses of 0 .1 - 0.2 mg/kg, i . e . 6-12 mg d a i l y f o r 4-8 weeks. The e f f e c t cons i s t s remission expressed, i n p a r t i c u l a r , i n reduction of t h e lymph nodes, spleen and l i v e r , increased haemoglobin, f a l l i n lymphocytosis and sometimes increase i n neutrophils i n t h e blood. The remission las t s from 3 months t o 1-3 years. After t he onset of a relapse, remission can again be obtained i n some p a t i e n t s by t h e same drug ( 2 3 ) .

Moore et a1 (8 ) used a continuous da i ly dose of 0 .1 - 0.2 mg/kg. intermit tent bas i s (0 .4 mg/kg every four weeks (28 ) . Both regimens commonly include prednisone. On t h e i n t e r - mit tent schedule, monthly pulse doses as high as 1 . 5 - 2.0 mg/kg were w e l l t o l e r a t e d and did not produce undue marrow t o x i c i t y . A n e f f e c t i v e biweekly dosing schedule

The drug may a l s o be administered on an


with lessened hematologic t o x i c i t y i s a l s o repor ted : 0.4 mg/kg every 2 weeks, increas ing by 0 . 1 mg/kg increments t o t o x i c i t y o r remission ( 5 ) .

7 . Toxic i ty

According t o Dorr and W i l l i a m ( 2 1 ) chlorambucil i s one of t h e bes t t o l e r a t e d o r a l a l k y l a t i n g agents . Bone marrow depression, including neutropenia , thrombocytopenia , and lymphopenia, occurs wi th prolonged use. I r r e v e r s i b l e bone marrow damage, however, may occur . Thus myelosuppression i s t h e common dose- l imit ing t o x i c i t y with chlorambucil. G a s t r o i n t e s t i n a l d i s t r e s s wi th l a r g e r doses occurs but i s u s u a l l y not s e r i o u s l Rare h e p a t i t i s and dis turbances o f l iver func t ion have a l s o been repor ted .

Cent ra l nervous system s t imula t ion has occurred wi th chlorambucil but i s uncommon un le s s l a r g e doses are used. Inadvertent t o x i c inges t ions i n ch i ld ren of 1 . 5 - 5 mg/kg have occurred without fa ta l i t ies but wi th moderate t o x i c i t y cha rac t e r i zed by vomiting, a g i t a t i o n , i r r i t a b i l i t y , and hype rac t iv i ty followed by l e t h a r g y and eventua l mild pancytopenia (29 ,30) . Chromosomal damage i s w e l l documented f o r t h i s agent although t h e exact mechanism i s not w e l l known ( 3 1 ) . Lerner (32 ) has f u r t h e r assoc ia ted chronic chlorambucil therapy wi th a high incidence of secondary acu te myelogenous leukemia.

S imi la r t o some o t h e r a l k y l a t i n g agents , chlorambucil can a l s o cause a l v e o l a r dysplas ia and pulmonary f i b r o s i s wi th long-term use ( 3 3 ) . A good c l i n i c a l response t o drug discont inuance and c o r t i c o s t e r o i d s w a s noted i n t h i s case . Spermatogenesis i s a l s o depressed while t h e p a t i e n t i s on chlorambucil therapy ( 3 4 ) . i n a s i n g l e adminis t ra t ion i s 21-23 mg/kg ( 3 5 ) .

LD 0 i n rats


8.1 I d e n t i f i c a t i o n

B r i t i s h Pharmacopoeia 1980 ( 9 ) descr ibes the following i d e n t i f i c a t i o n tes t s :

1) Mix 0 .4 g of chlorambucil with 1 0 m l of 2 M hydrochloric ac id and a l low t o s tand f o r t h i r t y minutes, shaking occas iona l ly . F i l t e r , wash t h e

CHLORAMBUCIL 107

res idue with two q u a n t i t i e s , each of 1 0 m l , of water , and r e se rve t h e mixed f i l t ra tes and washings for t e s t f o r i d e n t i f i c a t i o n 2. Melting poin t of t h e r e s idue , a f t e r drying over phosphorous pentoxide a t a pressure not exceeding 0.7 KPa (about 5 t o r r ) f o r t h r e e hours , about 1 4 6 O .

2 ) To 10 ml of t h e mixed f i l t r a t e and washings reserved i n t e s t f o r i d e n t i f i c a t i o n , add 0 . 5 m l of potassium mercuri iodide so lu t ion ; a buf f - colored p r e c i p i t a t e i s produced. To a f u r t h e r 10 m l add 0.5 m l of potassium permanganate so lu t ion t h e purple co lo r i s discharged.

3 ) Mix 0.6 g with 0.2 m l of phenylhydrazine, hea t i n a water ba th f o r t e n minutes, s t i r r i n g occas iona l ly , add 2 m l of absolu te e thano l , hea t f o r twenty seconds, f i l t e r immediately, and a l low t o stand f o r t h i r t y minutes. Crys t a l s of phenylhydrazinium ch lo r ide are formed, which, a f te r washing with two q u a n t i t i e s , each of 1 m l , o f absolu te e thanol and drying a t 105O, have a mel t ing poin t of about 245' , with decomposition.

U.S. Pharmacopeia XX ( 1 4 ) descr ibe t h e fol lowing tes ts f o r t h e i d e n t i f i c a t i o n o f chlorambucil:

1) The i n f r a r e d absorp t ion spectrum of a 1 i n 125 so lu t ion i n carbon d i s u l f i d e , i n a 1 mm c e l l , e x h i b i t s maxima only a t t h e same wavelength as t h a t of a s i m i l a r s o l u t i o n of USP Chlorambucil Reference Standard.

2 ) Dissolve 50 mg i n 5 ml of ace tone , and d i l u t e with water t o 1 0 m l . Add 1 drop of 2 N s u l f u r i c a c i d , t hen add 4 drops of s i l ve r n i t r a t e ( t e s t s o l u t i o n ) : no opalescence is observed immediately (absence of ch lo r ide i o n ) . Warm t h e s o l u t i o n on a steam bath : opalescence develops ( presecne o f i on i sab le ch lo r ine ) .

8.2 T i t r i m e t r i c Methods

U . S . Pharmacopeia XX ( 1 4 ) descr ibes t h e following method:

Dissolve about 200 mg of chlorambuci l , accura te ly weighed, i n 1 0 ml of ace tone , add 10 m l of water,


and t i t r a t e with 0 .1 N sodium hydroxide VS, us ing phenolphthalein TS as t h e i n d i c a t o r . Each m l of 0 . 1 N sodium hydroxide i s equivalent t o 30.42 mg of C14HlgC12N02.

B r i t i s h Pharmacopoeia 1980 ( 9 ) descr lbes t h e fol lowing method:

To 0.5 g of chlorambucil, add 20 m l of M sodium hydroxide and b o i l under a r e f l u x condenser f o r one hour. Cool, t r a n s f e r t h e mixture t o a 100-ml graduated f l a s k with t h e a i d of water , add 50 m l of 0 .1 M s i l v e r n i t r a t e VS and 5 m l of n i t r i c a c i d , mix, and add s u f f i c i e n t water t o produce 100 m l . F i l t e r and t i t ra te t h e excess of s i l v e r n i t r a t e i n 50 m l of t h e f i l t r a t e wi th 0 .1 M ammonium th iocyanate VS, using 3 m l o f ammonium i r o n (111) s u l f a t e so lu t ion as ind ica to r . Each m l of 0.1 M s i l v e r n i t r a t e VS i s equivalent t o 0.01521 g of

C14H19C12N02 *

8.3 Spectrophotometric Methods

8 .3 .1 Colorimetr ic Method

Pe ter ing and Van Giessen (36 ) descr ibed a co lo r ime t r i c procedure f o r determinat ion of n i t rogen mustards inc luding Chlorambucil , Dopan, Urac i l Mustard and Mustargen i n plasma. d i sso lved i n a 5% s o l u t i o n of dimethyl- acetamide i n e i t h e r e thanol o r 0.9% N a C l s o l u t i o n , with ph tha la t e bu f fe r s o l u t i o n (pH 4.0) (1 m l ) , 5% b (p -n i t robenzy1) - pyr id ine so lu t ion i n acetone (1 m l ) and 0.9% N a C l so lu t ion (1 m l ) , and d i l u t e with e thanol t o 4 m l . cool i n i c e , add N-KOH i n 90% e thanol (0 .1 nil), d i l u t e wi th e thanol a t 600 mp wi th in 2 o r 3 min. Carry out a blank determinat ion.

Mix t h e drug (10 t o 70 ug) ,

Heat at 80' f o r 20 min. ,

8.3.2 In f r a red Spectrometr ic Method

Kozlov and Sbezhneva (37) repor ted an i n f r a r e d spectroscopic ana lys i s of chloram- b u c i l i n pharmaceutical p repara t ions . Spec ia l ly t o determine t h e amount of a c t i v e

CHLORAMBUCIL 109

i ng red ien t s i n c a r c i n o s t a t i c prepara t ions

t h e prepara t ion i s ex t r ac t ed i n 100 m l of acetone and t h e absorbance o f a l iquo t i s measured at 770 cm-1. The e r r o r does not exceed f 1%.

11 conta in ing chlorambucil. 0 . 1 g of sample of

8 .3 .3 U l t r a v i o l e t Spectrometric )Jethod

El-Tarras e t a 1 (38) developed a simple method f o r t h e determinat ion of chlorambucil i n pharmaceutical p repara t ions . The powdered t a b l e t s are e x t r a c t e d i n t o e thano l (4 X 1 0 ml). The f i l t e r e d e x t r a c t w a s d i l u t e d t o 50 m l wi th e thanol . A po r t ion of t h i s so lu t ion w a s f u r t h e r d i l u t e d as requi red wi th e thanol and t h e absorbance measured at 250 nm vs e thano l . The concent ra t ion o f t h e drug i s ca l cu la t ed using a c a l i b r a t i o n curve.

8 .3 .4 Mass Spectrometr ic Methods

Chang _ - e t a1 (39) developed a s e n s i t i v e and s p e c i f i c method f o r t h e determinat ion of chlorambucil and i t s metabol i tes i n bio- l o g i c a l samples. The method i s based on se l ec t ed ion monitoring de tec t ion fol lowing simple e x t r a c t i o n of t h e parent compound, i t s metabol i te and i n t e r n a l s tandard (Chlorambucil-d8) from plasma and u r i n e samples. To determine chlorambucil and b [ b i s - ( 2 chloroethyl)ar,ino]phenyl a c e t i c a c i d i n 0.5 m l plasma or u r i n e add 0.5 m l of 4% HCl04 ( p e r c h l o r i c a c i d ) and [2H8] chlorambucil as i n t e r n a l s tandard. The mixture is ex t r ac t ed with e t h y l a c e t a t e and hexme (1:l). The organic l a y e r w a s d r i e d with p u r i f i e d N2 a t room temperature and s e v e r a l drops of methylene ch lo r ide were added t o dry res idue t o form t r ime thy l s i l y l de r iva t ive . This i s submitted t o GLC column ( 5 0 cm X 2 mm) of 3% of OV-17 on gas Chrom Q (100 t o 120 mesh) a f te r 40 seconds at 190°C t h e column temperature i s r a p i d l y r a i s e d t o 31OoC; 70 eV-m.s de t ec t ion i s used. T r ime thy l s i ly l a t ed chlorambucil i s


monitored a t m / e 3?6 and 328, t h e d e r i v a t i v e of t h e metabol i te of chlorambucil a t m / e 298, and t h a t of t h e i n t e r n a l s tandard a t m / e 383 and 385. s tandard devia t ion were 94.3 * 1.3%.

The mean recovery and

Jakhammer e t a1 (40) descr ibed an a n a l y t i c a l procedure f o r simultaneous determinat ion of chlorambucil and prednimustine i n plasma. The method involves e x t r a c t i o n and separa- t i o n followed by d e r i v a t i z a t i o n t o chloram- b u c i l methyl es ter and mass fragmentography. The drugs are ex t r ac t ed i n t o chloroform, hexane (3 :7 ) and sepa ra t ion i s done by p a r t i t i o n af ter adding 0.1 M phosphate - 0.1 M bora te buf fer (pH 9 . 0 ) . Chlorambucil i n t h e aqueous phase add predenimustine i n organic phase are each t r a n s e s t e r i f i e d wi th methanol-3F3 d i e t h y l e t h e r a t e ( 4 : l ) . The compounds are determined us ing fragmentography. A Varian MAT mass spectrometer i n combination with Varian Aerograph 1400 gas chromatograph with a column of 0.5% o f Carbowax 20 M on Chromosorb W-HP, are used. Analogues, prepared by introducing e igh t 2H atoms i n t o t h e b is - ( 2-chloroethy1)amino group of both compounds are used as i n t e r n a l s tandards and c a r r i e r s . The de tec t ion l i m i t i s about 8 ng/ml f o r both determinat ions and t h e r e l a t i v e s tandard devia t ion 3% f o r chlorambucil and 5% prednimustine a t 30-60 ng/rnl l e v e l .

--


8 .4 .1 Thin Layer Chromatograph (TLC)

Norpoth e t a1 ( 4 1 ) descr ibed a TLC method for t h e determinat ion of a l k y l a t i n g cyto- s t a t i c agents on a t h i n l a y e r p l a t e wi th isonicot inaldehyde benzothiazol-2-yl- hydrazone. The drug samples are d i s so lved i n methanol. The spot i s appl ied on a shee t o r precoated p l a t e s . After developing and drying t h e chromatogram, it i s sprayed wi th 3% so lu t ion of reagent i n acetophenone - dimethylformamide (7 :3 ) conta in ing 0.01 m l of concent ra t ion H C 1 per 100 m l . It i s then

CHLORAMBUCIL 111

placed over a ba th o f acetophenone and ba th and p l a t e are heated at 2OO0C for 20 minutes; t h e cooled p l a t e i s then sprayed wi th t r i e thy lamine . Chlorambucil produces red spo t . For q u a n t i t a t i v e determinat ion t h e co lo r i n t e n s i t y of t h e spot i s measured using chromatogram spectrophotometer .

8.4.2 Gas Liquid Chromatography ( G L C )

Ehrsson _ _ e t a1 (42) descr ibed a GLC technique f o r determinat ion of chlorambucil i n plasma by gas- l iqu id chromatography with s e l e c t e d ion-monitoring. 2 m l of plasma sample conta in ing 8 l.~g of chlorambucil pe r m l w a s mixed with hydrochlor ic a c i d , phosphoric a c i d , or phosphate b u f f e r ( 0 . 2 m l ) . The mixture w a s e x t r a c t e d with methylene ch lor ide ( 2 m l ) f o r 30 minutes. An a l i q u o t o f organic phase was separated and evaporated t o dryness with n i t rogen gas . The res idue w a s d i sso lved i n methanol-water 0 .1 M , a c e t i c a c i d (75:15:10) and analysed on gas chromatography. A l t e rna t ive ly t h e plasma samples w e r e ad jus t ed t o pH 3 with 1 M phosphoric ac id and ex t r ac t ed with e thylene d i ch lo r ide f o r 30 minutes. The organic phase w a s separa ted and e x t r a c t e d with 0 .5 m l of 0 . 1 M sod. sulphide f o r 5 minutes. The aqueous phase w a s separa ted and heated f o r 1 5 minutes a t 80°C t o convert chlorambucil i n t o t e t r ahydro th i az ine d e r i v a t i v e , then concentrated phosphoric a c i d (0.91 m l ) was added, and H2S removed with N2 f o r 5 minutes. To t h i s O . 0 5 m l of 12 M NaOH, 0 .2 m l of 0.5 M t e t r a b u t y l ammonium, 0.2 ml methylene ch lo r ide and 0.05 m l a l lylbromide were added and t h e mixture w a s shaken f o r 30 min at 2 5 O C . The organic phase (1-2 mcg) w a s i n j e c t e d i n GLC (a t Z50°C) on a column of 5% of OV-17 on gas-Chrome-Q operated with 70 eV.m. s . de t ec t ion . The i n t e r n a l s tandard used w a s [%8] chlorambucil. at m / e 305 and 313 f o r chlorambucil and

The peaks were monitored


2 [ H8] chlorambucil r e spec t ive ly . The c o e f f i c i e n t of v a r i a t i o n f o r 10 ng/ml o f chlorambucil w a s 5%.

8 .4 .3 High-pressure Liquid Chromatography (HPLC)

N e w e l 1 -- e t a1 (43 ) developed a HPLC method f o r es t imat ion of chlorambuci l , phenyl- a c e t i c mustard and predenimustine i n human plasma. One m l plasma w a s ex t r ac t ed wi th 2 ml of e t h y l a c e t a t e , t h e emulsion formed by vigorous a g i t a t i o n on a Whirlimixer was frozen by immersing t h e tubes i n a methanol carbondioxide ba th a t - 6 8 O C . The tubes w e r e cen t r i fuged a t 600 pg f o r 10 minutes at 4 O C unti l t h e thawed aqueous phase could be separa ted . The procedure was repea ted with f u r t h e r 2 m l of e t h y l a c e t a t e , t h e pooled e t h y l a c e t a t e e x t r a c t s were d r i e d over anhydrous sodium su lpha te f o r 1 hour a t room temperature . 2 m l of d r i ed e t h y l a c e t a t e e x t r a c t was evaporated t o dryness i n a stream of n i t rogen at 4 5 O C . The res idue w a s d i sso lved i n 100 m l of e t h y l a c e t a t e f o r chromatographic a n a l y s i s . 50 1.11 of t h e e x t r a c t w a s i n j e c t e d on a 1.1 Bondapak c18 column. Linear grad ien t o r e l u t i o n w a s done at 2 m l pe r minute with aqueous 0.175 M - a c e t i c a c i d (from 40 t o 0%) i n methanol during 1 0 minutes followed by i s o c r a t i c e l u t i o n wi th methanol. This method gave a good sepa ra t ion of chloram- b u c i l i n less than 12 minutes . The absorbance of e l u t e w a s monitored a t 254, and 280 nm simultaneously. Detector response w a s r e c t i l i n e a r over a range of 5 t o 1000 ng of t h e compound. The recovery of chlorambucil w a s constant over t h e range of 0.05 t o 10 m.

Leff and Bardsley ( 4 4 ) have repor ted pharmacokinetic of chlorambucil i n ovarian carcinoma using a new HPLC assay. The apparatus cons is ted of a Waters Assoc ia tes ~ 6 0 0 0 pump, a 10 x 0.46 cm s tee l column packed with S 5 ODs, a Waters Associates Model U6K i n j e c t o r , a me-Unicam LC3 v a r i a b l e

CHLORAMBUCIL 113

wavelength W d e t e c t o r opera t ing at 254 nm. The mobile phase was water: methanol 20:80 ( v / v ) with 0.1% ammonium a c e t a t e (w/v ) . The flow rate used w a s 1 ml min-1 ( = 500 p s i ) .

Zakaria and Brown ( 4 5 ) repor ted a r ap id assay f o r plasma chlorambucil and i t s metabol i te ( phenylacet ic mustard) using reversed-phase l i q u i d chromatography. Plasma (20 o r 30 ~ 1 ) i s i n j e c t e d d i r e c t l y i n t h e chromatographic system, which incorpora tes a 5 cm Co: Pe l1 ODS pre-column for sample c l ean up. A p a r t i s i l PXS-10/25 ODS column ( 2 5 cm X 4 .6 mm) o r , f o r f a s t e r e l u t i o n , a chromegabond MC-18 column ( 1 5 cm X 4.6 mm) i s used for t h e a n a l y s i s . The mobile phase being methanol - 0.02 M - KH2P04 (1:l o r 11:9, r e spec t ive ly ) at 1 . 5 o r 1 . 0 m l min-l, r e spec t ive ly . graphs are r e c t i l i n e a r over t h e range of i n t e r e s t . Simultaneous de t ec t ion a t 280 and 254 nm al lows picomole amounts t o be determined.

Ca l ib ra t ion

C h a t t e r j i _ _ e t a1 ( 4 6 ) have s tud ied k i n e t i c s of chlorambucil hydro lys is us ing high- pressure l i q u i d chromatography. The high- pressure l i q u i d chromatograph w a s equipped with a fixed-volume loop i n j e c t o r and a f ixed wavelength de t ec to r . A 250 X 4.6 mm i . d . reversed-phase column w a s used. Separat ion was performed on a reversed-phase column (Zorbax C-8, 6 pm average p a r t i c l e s i z e , Dupont Instruments , Wi l l ing ton , D e l . ) and methanol-acetoni t r i le-0.01 M a c e t a t e b u f f e r , pH 4.5 (65 :5 :30 ) , a t a flow rate of 1 . 6 ml/min was used as t h e mobile phase. A 1 0 p1 full loop volume w a s q u a n t i t a t i v e l y i n j e c t e d , and t h e recorder w a s set a t 0.32 au f s (254 nm d e t e c t o r ) .

Ekrsson e t a1 (47 ) descr ibed a reverse phase HPLC method t o s tudy degradat ion of chlorambucil i n aqueous so lu t ion . Chloram- b u c i l and i t s degradat ion products;4-( 4-( 2- chloroethyl-2-hydroxyethylamino)-phenyl) bu ty r i c acid, are determined i n aqueous methanol. The chlorambucil s o l u t i o n i s

114 MOHAMMAD TARlQ AND ABDULLAH A. AL-BADR

prepared by adding 1 0 mg of compound i n 0.5 m l methanol and t h e volume i s made upto 100 m l with d i s t i l l e d water. A t d i f f e r e n t t i m e i n t e r v a l s e x t r a c t i o n i s done with e t h y l a c e t a t e , t h e so lvent i s evaporated t o 0.2 m l under a n i t rogen atmosphere. Reversed-phase HPLC ana lys i s i s done on a column (15 cm X 4 mm) of Li-Chrosorb RP-18 ( 5 urn). Methanollwater conta in ing 0.01 M phosphoric ac id i s used as mobile phase. A W d e t e c t o r ad jus t ed a t 253.7 nm w a s used f o r determinat ion of t h e concent ra t ion .

Ahmed _ _ e t a1 (48) descr ibed a q u a n t i t a t i v e method for determinat ion of chlorambucil i n plasma by rever sed-phas e h i gh-pe r f ormanc e l i q u i d chromatography. The plasma samples a re depro te in ised by adding 4 volumes of a c e t o n i t r i l e a t pH 3 . The tubes w e r e c e n t r i - fuged f o r 2 minutes and t h e supernatent w a s r ap id ly frozen i n s o l i d carbondioxide-acetone t o complete dep ro te in i sa t ion . The tubes a r e cent r i fuged for 2 minutes . The a l i q u o t so obtained w a s analysed on Water's Radial-PAK c18 (10 u m ) c a r t r i d g e , with a c e t o n i t r i l e - 0.2% a c e t i c ac id (13:7) as mobile phase. Absorbance was monitored at 263 nm and recorded on a Data Module. A s tandard curve was developed using a c e t o n i t r i l e s o l u t i o n of chlorambucil s tandard .

Acknowledgement

The au thors would l i k e t o thank Mr. Syed A l i Jawad f o r t h e t e c h n i c a l a s s i s t a n c e and Mr. Tanvir A . But t f o r t h e s e c r e t a r i a l a s s i s t ance i n typ ing t h e manuscript.

CHLORAMBUCIL

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30.

31.

32.

33.

34.

35.

36.

37 -

38.

39.

40.

41.

42.

43.

44.

A. Sawitsky; K.R . R a i ; 0. Glidewell and R.T. S i l v e r , Blood, 50, 1049 (1977).

A.A. Green; and J . L . Naiman, Am. J. D i s . Chi ld . , 116, 190 (1968).

S. Wolfson and M.B. Olney, -- JAMA, 165, 239 (1957).

B.R. Reeves, Br. Med. J . , 4, 22 (1975). H.F. Lerner , Cancer Treat. Rep. , 62, 1135 (1978). S.R. Cole; J.J. Myers and A.U. Klatsky, Cancer, 41, 455, _ _ _ - (1978).

P. Richter ; C.J. Calamers and M.C. Morganfeld, Cancer, - 25, 1026 (1970).

Y

~ 5 9 (1963).

N.E. Kozlov and V.G. - 29, 33 (1980).

M.F. E l - T a r r a s ; M.M. Pharma Fenn. , 2, 31

L.A. Elson, Ann. N . Y . Acad. S c i . , - 68, 826 (1958).

H.G. Pe t e r ing and G . J . Van Giessen, J. Pharm. S c i . , 52,

Sbezhneva, Farmatsiya (Moscow),

E l l a i t h y and N.B. Tadros, 9 (1983).

S.Y. Chang; B.J. Larcom; D.S. Alber t s ; B. Larsen; P.D. Walson and I . G . S ipes , J . Pharm. S c i . , 69, 80 (1980).

T. Jakhammer; A. Olsson and L. Svensson, Acta Pharma. Suec. , 14, 485 (1977). K. Norpoth, H.W. Addicks and U. Wit t ing , Arzne imi t te l - Forsch. , 23, 1529 (1973).

H. Ehrsson; S. Eksborg; I . Wallin; Y . Marde and B . Joansson, J. Pharm. S c i . , 6 9 ( 6 ) , 710 (1980).

D . R . N e w e l l ; L . I . Heart and K.R . Harrap., J. Chromatogr 164, Biomed. Appl., 6, 114 (1979).

P. Leff and W.G. Bardsley, Biochem. Pharmacol., 28, 1289 (1979).


45. M. Zakaria and P.R. Brown, J. Chromatogr., 230, Biomed. Appl. , 19, 381 (1982).

46. D.C. Chatterji, R.L. Yeager and J.F. Gallelli; J. Pharm. .) Sci - 71, 50 (1982).

H. Ehrsson; S. Eksborg; Inger Wallin and S.G. Nilsson, J. Pharm. Sci., @, 1091 (1980).

A.E. Ahmed; M. Moenig; and H.H.J. Farrish, J. Chromatogr. , 233, (1982) , Biomed. Appl. , 22, 392 (1982) .

47.

48.

CHLORZOXAZONE

James T. Stewart Department of Medicinal Chemistry and Pharmacognosy

College of Pharmacy University of Georgia

Athens, Georgia

Casimir A. Janicki Director, Analytical Control

McNeil Pharmaceutical Spring House, Pennsylvania

1. Foreword, History, Therapeutic Category 2 . Description

2.1 Names, Formula, Molecular Weight 2.2 Appearance, Color, Odor, Taste

3. Synthesis 4 . Physical Properties

4 . 1 Infrared Spectrum 4.2 Ultraviolet Absorption Spectrum 4 . 3 Nuclear Magnetic Resonance Spectra 4 . 4 Mass Spectra 4.5 X-Ray Diffraction Pattern 4 . 6 Thermal Analysis 4 .7 Melting Range 4 .8 Solubility 4 . 9 pKa

5 . Methods of Analysis 5.1 Fluorescence Spectrophotometry 5.2 Ultraviolet Spectrophotometry 5 .3 Iodometric Titrimetry 5 . 4 Gas Chromatography 5 . 5 Thin-Layer Chromatography 5 . 6 Paper Chromatography 5.7 High Performance Liquid Chromatography 5 . 8 Non-Aqueous Titrimetry 5 . 9 Colorimetry 5 .10 Color Test 5 . 1 1 Polarizing Microscopy 5 .12 Potentiometry

6 . Stability 7. Drug Metabolic Products, Pharmacokinetics, and Bioavail-

ab i 1 i ty Copyright 0 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved. ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 119

120 JAMES T. STEWART AND CASIMIR A. JANICKI

7.1 Drug Metabolic Products 7.11 Urine Metabolism 7.12 In-vitro Metabolism

7.2 Pharmacokinetics and Bioavailability Identification and Determination in Body Fluids and Tissues

8.

8.1 Ultraviolet Spectrophotometry 8.2 Fluorescence Spectrophotometry 8.3 High Performance Liquid Chromatography 8.4 Gas Chromatography

9.1 Colorimetry 9.2 Ultraviolet Spectrophotometry 9.3 Fluorescence Spectrophotometry 9.4 Potentiometry 9.5 High Performance Liquid Chromatography

9. Identification and Determination in Pharmaceuticals

10. References

CHLORZOXAZOIW 121

1. Foreword, History, Therapeutic Category Chlorzoxazone is a centrally active muscle relaxant for the treatment of painful muscle spasms associated with muscloskeletal disorders, such as fibrositis, bursitis, myositis, spondylitis, sprains, and muscle strains. The skeletal muscle relaxant properties were first discovered by Marsh at McNeil Laboratories In the late 1950s (1). Onset of therapeutic activity is observed within one hour and the duration usually lasts up to 6 hours. The drug was reported to be useful in the treatment of painful spasm of skeletal motor muscles by acting at spinal levels of the central nervous system (2). The drug exhibits minimal adverse effects and almost no gastrointestinal irritation. Chlorzoxazone was ranked among the top 100 most widely prescribed drugs for many years ( 3 ) .

2. Description 2.1 Names, formula, molecular weight

Generic name - Chlorzoxazone Trade name - Marketed by McNeil Pharmaceutical under the trade name, Paraflex. Chemical names - 5 Chloro-2-(3H)-benzoxazolone, 5-Chloro-2-benzoxazolol, 5-Chloro-2- hydroxybenzoxazole, 2-Hydroxy-5-Chlorobenzoxazole, 5-Chlorbenzoxazolin-2-one, 5-Chlorobenzoxazolidone. Chemical Abstracts Registry No. 95-25-0

-__.

-

C7H4C1N02 Mol. Wt. 169.58

2.2 Appearance, Color, Odor, Taste - Odorless colorless crystals or a white to creamy white crystalline powder with a bitter taste.


3. Synthesis Chlorzoxazone is synthesized by either the acid hydrolysis of 2-amino-5-chlorobenzoxazole (4) or by treating 2-amino-4-chlorophenol with phosgene in ethyl acetate (5). sodium ethoxide with 2-ethoxycarbonylamino- 4-chlorophenol in tetralin (6).

It can also be prepared from the reaction of

C l C O C l t

4 . Physical Properties 4.1 Infrared SDectrum - An FTIR sDectrum of chlorzoxa- I

zone is shown in Figure 1.

-1 cm 3470 3155 3117 3087 3057 2978 2282 1924 1885 1772 1621

1463 1482

% T 72.6 36.3 39.7 37.3 35.2 46.4 69.1 67.4 70.2 3.2 30.5 13.5 34.3

% T 1367 43.8 1330 53.6 1300 27.3 1262 26.2 1153 21.1

1064 54.8 964 16.4 923 33.1 867 46.1 845 23.7 805 19.2

-1 cm --

1101 58.7

% T cm 732 52.8 713 32.2 600 45.8 591 41.8 552 49.0 417 67.6 394 70.2 382 79.1 352 66.1

216 5.0 210 6.7

-1 --

234 8.8

.....

.... -

I I I i

I I I

r I I I

i I r

r i

I

r i

I

I

11 i

Fig. 1 FTIR spectrum of chlorzoxazone


4.2 Ultraviolet Absorption Spectrum - A solution of the drug in absolute methanol shows a maximum at 282 ? 2 nm and a m i n i m u m a i 248 _t 2 nm. There is an inflection at about 228 GII! ( see Figure 2).

4.3. Nuclear lvlagnetic Resenance hpectra - 13C and proton nuclear magnetic resonance spectra are shown in Figures 3 and 4.

-- - -- --

b

C

13C NMR Carbon 6 (ppm)

a 39.4 DMSO-dc b 109.8 C 110.8 d 121.4 e 127.7 f 131.7 g 142.1 h 154.2

J

C

NH f

d Proton NMR

Proton f i (DDm) Y .r r --I ~~ ~

a 2.49 DMSO-d, b 3.36 HDO C 7.10 d 7.13 e 7.28 f 11.8

CHLORZOXAZONE

220

WAVELENGTH (nm)

350

Fig. 2 W spectrum of chlorzoxazone in methanol

I

Fig. 3 13C-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide

127

"-2.1 1d.r 77 8.1 7.1 6.1 5.0 4 .1 3.1 2.1 1.0 - ...... I ..-.--I---* . I

PPM

Fig. 4 'H-NMR spectrum of chlorzoxazone in deuterated dimethylsulfoxide


4.4

4.5

4.6

4.7

4.8

4.9

ME Spectra - Electron impact (EI) and chemical ionization (CI) mass spectra are shown in Figures 5 and 6.

X-Ray Diffraction - Figure 7 shows an X-ray diffraction pattern for chlorzoxazone powder.

Thermal Analysis - A DSC thermogram of chlorzoxazone is shown in Figure 8.

Melting Range - The melting point range is 189-194°C.

Solubility - Chlorzoxazone is very slightly soluble in water (0.2 - 0.3 mg/ml), sparingly soluble in ethanol and methanol, soluble in chloroform (1 g in 250 ml) and ether (1 g in 60 ml). It is freely soluble in aqueous solutions of ammonia and alkali hydroxides.

pKa - The pKa of chlorzoxazone in 8.3.

5. Methods of Analysis 5.1 Fluorescence Spectrophotometry - Stewart and Chan

have studied the reaction of chlorzoxazone with dansyl chloride, fluorescamine, 2,4-dihydroxy- benzaldehyde, and salicylaldehyde to form fluoro- phores (7). Fluorescamine gave the highest intensity product and was used to analyze chlorzoxazone in the 0.27 - 3.4 pg/ml range. The same authors also reported that chlorzoxazone exhibits native fluorescence in chloroform using excitation and emission wavelengths of 286 and 310 nm, respectively (8).

5.2

5.3

5.4

Ultraviolet Spectrophotometry - Conney -- et a1 utilized a back-extraction approach to quantitate chlorzoxazone at 289 nm (9). The drug can also be directly determined at 282 nm in absolute methanol (10) *

Iodometric Titrimetr - Beral et a1 titrated c h l o r z o x a z o d t r e a t m e n t with boiling 10% sulfuric acid, potassium bromide and potassium bromate) with 0.1 N sodium thiosulfate after potassium iodide was added to a cooled solution (11).

--

- Gas Chromatography - Kaempe has chromatographed chlorzoxazone on a packed 15% Dexsil 300 on HP

90 - 80 - 70 - 60 - 50 -

I

11

113

51 63

30 - 20 d

I d. I,,, .. . .. , 40 60 30 100 120 140 160

I I

9

I; . . , [ A S S 130 200

Fig. 5 Electron impact (EI) mass spectrum of chlorzoxazone

MH+

'0°1

80 -

60 -

%I

a !!

50 90 I30 I 70 210 250 m / e

Fig. 6 Chemical ionization (CI) mass spectrum of chlorzoxazone

90

80

70

60

50

e

e W

-

-

-

-

-

1

Fig. 7 X-Ray diffraction pattern of chlorzoxazone

5.00 7

CHLORZOXAZONE. L O T 2933 WT: 1.00 mg

SCAN RATE: 5.00 deg/rnxn

*. MI 380. M 4M.M 42O.M bu). MI 4 h no 0.00 ' TEMPERATURE (K)

460.00 5th 01

DSC

Fig. 8 DSC thermogram of chlorzoxazone

CHLORZOXAZONE 133

Chromosorb W 801100 mesh glass column (6 ft x 6.4 m i.d.). The retention time was 0.62 relative to caffeine using a column temperature of 220°C (12). Parker -- et a1 attempted to chromatograph the drug on SE-30 (0.05% on glass microbeads 60/80 mesh) at 150 and 165°C column temperatures with no success (13). Desiraju et a1 chromatographed chlorzoxazone on a 6 ft x 4 mm i.d. glass column packed with 3% OV-1 on 60/80 mesh Gas Chrom Q (14). was 130°C and retention time was 3.25 minutes using a column flow rate of 30 ml/minute.

Column temperature

Beckett et a1 reported that chlorzoxazone can be chromatographed on a 2.5% SE-30 on 80/100 mesh Chromosorb W acid-washed HMDS treated 5 ft x 4 mm i.d. glass column at a column temperature of 225°C (15). Retention time was 0.69 relative to diphenhydramine. Nitrogen was used as carrier gas at 50 ml/min a.nd FID detection was utilized. Ardrey and Moffat also reported that a 2 m x 4 mm i.d. glass column containing 2.5% SE-30 on 80/100 mesh Chromosorb G acid-washed HMOs treated will separate chlorzoxazone using a column temperature of 173°C (16). Retention time is given relative to n-alkanes.

Pedroso and Moraes have chromatographed chlorzoxazone on 2.5% SE-30 and 3% OV-17 at 200 and 220°C column temperatures, respectively (17). They used the retention data to perform qualitative analysis of the drug based on Kovat's Retention Indices.

--

-

5.5 Thin-Layer Chromatography

Stationary Phase Mobile Phase

18 Silica Gel HF254 Chloroform-methanol 19:l 0.32

Chloroform-methanol 9:l 0.58 Chloroform-methanol 4:l 0.87 Chloroform-methanol 7:3 1.00 Chloroform-methanol 3:2 1.00

Kieselgel 60F254 0.69 Toluene - ethylacetate - 85% formic acid (50:45:5)

19

134

Kieselgel 60F254

Kieselgel 60F254

Silica Gel 60F254

Silica Gel 60F254

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

JAMES T. STEWART AND CASIMIR A. JANICKI

19 0.68 Toluene - Isopropanol - concd. ammonium hydroxide (70 : 29 : 1)

Toluene-dioxane - methanol - ammonium hydroxide (20:50:20:10)

Toluene - Acetone - 2N Acetic Acid - (30:65:5) -

Toluene - Isopropanol - Ethyl Acetate - 2N Acetic Acid (10:35:35:20)

Methanol - 0.5% Phosphoric Acid 3% Sodium Chloride (60:10:30)

Isopropanol - 0.5% Phosphoric Acid - 3% Sodium Chloride ( 4 0 : 10:50)

Isopropanol - Methanol - 0.5% Phosphoric Acid - 3% Sodium Chloride (23:23:10:44)

Tetrahydrofuran - Methanol - 0.5% Phosphoric Acid - 3% Sodium Chloride (28:28:10:34)

Methanol - 2 N Ammonia - 3% Sodium Chloride (60:10:30)

19 0.55

0. 8320

20 0.95

0. 3220

20 0.35

20 0.33

20 0.32

20 0.60

CHLORZOXAZONE

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Bonded C18F

Isopropanol - 2N Ammonia 3% Sodium Chloride (40:10:50)

Isopropanol - Methanol - 2N Ammonia - 3% Sodium Chloride (23:23:10:44)

Tetrahydrofuran - Methanol - 2N Ammonia - 3% Sodium Chloride - (28:28:10:34)

135

20 0.47

0. 4320

0. 5520

20 Acetonitrile - 0.46 3% Sodium Chloride (50: 50)

Methanol - 0.32 3% Sodium Chloride (60 : 40)

Ace tone 0.25 3% Sodium Chloride (50 : 50)

Tetrahydrofuran - 0.18 3% Sodium Chloride (43:57)

Isopropanol - 0.35 3% Sodium Chloride (40:60)

Methanol - 0.36 Isopropanol - Tetrahydrofuran - 3% Sodium Chloride (17:17:17:49)

20

20

20

20

20

5.6 Paper Chromatography - Clarke reported that chlorzoxazone gave an R 87 : 13 - n-butanol-waEer containing 0.48% citric acid

of 0.93 using a mobile phase of

(21) *


6

5.7 High Performance Liquid Chromatography - Stewart a1 have chromatographed chlorzoxazone on an octadecylsilane column using varying proportions of absolute methanol-distilled water (22). Chlorzoxa- zone was effectively separated from acetaminophen in a mixture with 50:50 methanol-water at a 2.0 ml/min flow rate using refractive index detection. The drug has also been chromatographed on an octadecylsilane column using 60:40 water- methanol (23), and a 1:l acetonitrile-water mixture (10). These methods utilized ultraviolet detection at 280 nm.

-

5.8 Non-Aqueous Titrimetry - Chlorzoxazone is titrated with 0.1 N sodium methoxide in dimethvlformamide using thymol blue as indicator ( 2 4 ) . The drug can also be titrated in dimethylsulfoxide using O.1N propanolic potassium hydroxide as titrant and metanil yellow as visual indicator (25).

5.9 Colorimetry - Sanghoi reported a colorimetric procedure for chlorzoxazone based on base hydrolysis of the drug followed by diazotization of the product with nitrous acid formed in situ (26). color measured at 405 nm is stable for 60 minutes and obeys Beer's Law in the 4-32 pg/ml range.

The --

5.10 Color Test - The Koppanyi-Zwikker test gives a -- violet color (27).

5.11 Polarizing Microscopy - Watanabe -- et al., reported that polarizing microscopy can be used as a qualitative identification tool for chlorzoxazone (28).

5.12 Potentiometry - A potentiometric method for the determination of chlorzoxazone, based on the use of a carbon dioxide gas-sensing electrode, is described by Tagami and Muramoto (29). Upon refluxing the drug with 3N sodium hydroxide, aminophenol and sodium carbonate are formed.

Acidification of the reaction mixture yields carbon dioxide which is sensed by the gas-permeable membrane electrode. A line r calibrati n plot was obtained within the 3 x 10 M - range of chlorzoxazone.

-8 -3 to 5 x 10

Stability Chlorzoxazone is stable in the solid state for up to 5 years at room temperature. Storage at temperatures for

CHLORZOXAZONE 137

up to 80°C and in artificial sunlight conditions (1000 foot candles) for up to 4 weeks did not cause significant degradation.

Tablets of chlorzoxazone have also been found to be stable for up to 5 years at room temperature. Tempera- tures for up to 60"C, humidity conditions of 40"C/80% relative humidity and artificial sunlight (1000 foot candles) €or up to 4 weeks have not caused degradation of the product.

An aqueous suspension of the drug in distilled water or acidic media is generally stable but in solutions with a pH greater than 7, the solution is generally not stable. Chlorzoxazone yields 2-amino-4-chlorophenol as its major degradation product.

Metabolic Products

7. Drug Metabolic Products, Pharmacokinetics, and Bioavail- ab i 1 it y 7.1 Drug

7.11

7.12

Urine metabolism - The metabolic product of chlorzoxazone was isolated from urine of human subjects administered an oral dose of the drug. Ether extraction of urine yielded a residue which was identified as 6-hydroxychlorzoxazone (9). Studies showed that the metabolite is eliminated as the glucuronide conjugate to the extent of 74% of the dose. 6-Hydroxychlorzoxazone had little or no muscle relaxant activity when tested in mice and rats. In 1982, Twele and Spiteller identified two additional chlorzoxazone metabolites from human urine, a 5-chloro-2,4-dihydroxy- acetanilid produced from 6-hydroxychlorzoxazone by ring cleavage and acetylation of the amino moiety, and 6-hydroxybenzoxazolone, produced by substitution of hydrogen for chlorine and hydroxylation of a neighbor ring-carbon atom (30).

In-vitro metabolism - Chlorzoxazone was incubated with fortified homogenates of mouse and rat liver. The reaction was stopped by addition of 3N hydrochloric acid. Spectro- photometric assay of a sample preparation identified 6-hydroxy-chlorzoxazone as the major metabolite (31). It has been established that the liver is the principal site of metabolism.


7 . 2 Pharmacokinetics and Bioavailability - The following pharmacokinetic data were obtained following a 750 mg oral dose of chlorzoxazone to human subjects ( 1 4 ) :

Peak plasma concentration Peak time AUC (0-10 hr) Apparent Volume of

Elimination Rate Constant (K) Absorption Rate Constant ( ka) Elimination Half Life (t ) Apparent Clearance (KV/F)'

Distribution (V/F)

36.3 f 2.3 pg/ml 38 f 3.3 minutes 4084 f 284 pg min/ml 13.70 f 5.07 liters

0.73 2 0.29 hrI: 2.28 2 2.08 hr 1.12 f 0.48 hr 148.0 f 39.9 ml/min

There is rapid absorption and elimination of chlorzoxazone. Chlorzoxazone is widely distributed in plasma and tissues. Less than 1% of the drug is excreted unchanged in urine. The drug concentration in fat is twice the plasma concentration, while liver, muscle, brain and kidney concentrations are one-half or less that found in plasma. The apparent volume of distribution of approximately 14 liters suggests that the drug is not widely distributed and that it would be confined to the circulatory system and possibly the extracellular fluid .

8 . Identification and Determination in Body Fluids and Tissues 8.1 Ultraviolet Spectrophotometry - Conney et a1 --

reported the extraction and estimation of chlorzoxazone in urine ( 9 ) . containing 1.5% isoamyl alcohol was shaken with the urine sample for 60 minutes. The organic phase was separated and the drug was back extracted into 0.5 N sodium hydroxide. Absorbance read at 289 nm followed Beers Law. was 93 f 2%.

Petroleum ether

Recovery of drug from urine

8.2 Fluorescence Spectrophotometry - Stewart and Chan reported the quantitation of chlorzoxazone from plasma and/or -urine samples ( 8 ) . Chlorzoxazone was extracted from the biological sample at pH 2.5 with petroleum ether containing 1.5% isoamyl alcohol. The organic phase was re-extracted with strong base, the pH adjusted to pH 6 .8 with hydrochloric acid, and the drug extracted into chloroform for the measurement step. Excitation and emission wavelengths were 286 and 310 nm, respectively.

CHLORZOXAZONE 139

Drug concentration arid fluorescence were linear from 0.06 - 3.2 pg/ml and 0.13 - 3.0 vg/ml for plasma and urine, respectively. Limits of detection were 60 and 130 ng/ml for plasna and urine, respectively. Percent recoveries of chlorzoxazone from plasma and urine were 86.63 2 1.66 and 95.37 2 2.42%, respectively.

Stewart and Chan also reported a procedure for the analysis of chlorzoxazone based upon solvent extraction of the drug from human plasma or urine followed by chemical derivatization with fluorescamine (7). Linear detection range was 2-10 pg/ml using excitation and emission wavelengths of 370 and 505 nm, respectively.

8.3 High Performance Liquid Chromatography - Honigberg, Stewart, and Coldren reported an HPLC assay for chlorzoxazone and 6-hydroxy-chlorzoxazone in plasma based on ether extraction of the compounds from acidic plasma (22). The separation was achieved on an octadecylsilane column (30 cm x 3.9 mm, i.d.) at a flow rate of 2 ml/min with UV detection at 280 nm and a mobile phase of 40:60 absolute methanol- distilled water. Retention times for the hydroxy compound and drug were approximately 4 and 8 minutes, respectively. The limit of detection for each compound was calculated to be 80 ng at signallnoise = 2.

Another HPLC assay for chlorzoxazone in plasma reported by Ng at McNeil Pharmaceutical (32) used at 1:l acetonitrile-distilled water mobile phase, an octadecylsilane column (25 cm x 4..6 mm, i.d.), a 2.0 ml/min flow rate, and UV detection at 280 nm. Retention time for the drug is 2.9 minutes. Ethyl 5-chloro-2-hydroxycarbanilate was used as internal standard (retention time of 4.5 minutes). A plasma sample was made acidic with hydrochloric acid and extracted once with ethyl acetate. The ethyl acetate was evaporated to dryness and the residue dissolved in methanol prior to injection into the HPLC . Stewart and Carter reported modifications in the above mentioned HPLC assay by Honigberg, Stewart and Coldren in which an octadecylsilane solid-phase extraction column was used t o separate chlorzoxazone and 6-hydroxychlorzoxazone from human serum (33). Percent recoveries were 90.24 2 4.28 and


86.06 It: 3.58% for drug and metabolite, respectively. The separation was achieved on an octadecylsilane column using a mobile phase of 50:50 acetonitrile-aqueous 0 .05 M sodium dihydrogen phosphate, pH 4 .5 with phenacetin as internal standard. The two compounds were detected at the glassy carbon electrode using a cell potential of + 1300 mV. These modifications halved the original HPLC analysis time and increased detection for each compound to 2.5 ng, 30 times the previous limit of detection using 280 nm.

8.4 - Gas Chromatography - Desiraju -- et a1 reported a packed column method for the quantitation of chlorzoxazone in plasma samples ( 1 4 ) . The drug is extracted from acidified plasma with ethyl acetate containing the internal standard, n-hexadecane. The ethyl acetate is separated andevaporated to dryness. Pyridine and acetic anhydride are added to the residue, the tube is capped, and the reaction allowed to proceed at 42°C for 20 minutes. An aliquot of the mixture is injected into the gas chromatograph.

The GC parameters were flame ionization detection (30 and 300 ml/min flow rates for hydrogen and air, respectively), carrier gas (nitrogen at 30 ml/min), 6 ft x 4 mm i.d. glass column packed with 3% OV-1 on 6 0 / 8 0 mesh Gas Chrom Q, and column temperature of 130°C. The retention times of chlorzoxazone and internal standard were 3.25 and 4.5 minutes, respectively. The limit of detection was 0.5 ug/ml using 1 ml of plasma. The calibration curve was linear in the 0.5 - 25 ug/ml range. efficiency for chlorzoxazone was 88 It: 8% (n = 6 ) .

Extraction

9 . Identification and Determination in Pharmaceuticals Colorimetry - Twenty tablets were weighed and powdered in a dry mortar and pestle. the powder equivalent to 40-60 mg of chlorzoxazone was hydrolyzed with 20% sodium hydroxide solution, cooled, and reacted with nitrous acid to yield the diazonium salt, whose absorbance measured at 405 nm obeyed Beer's Law in the 4-32 ug/ml range ( 2 6 ) . Acetaminophen and indomethacin were shown to interfere with the method. zone in bulk powder and tablet samples was in the 98.3 - 99.9% range utilizing the assay procedure.

9.1 An aliquot of

Recovery of chlorzoxa-

CHLORZOXAZONE 141

9 . 2 Ultraviolet Spectrophotometry - Kirschner of McNeil Pharmaceutical reported an assay for the drug in tablets as follows (10):

Standard Preparation: Dissolve a suitable quantity of USP Chlorzoxazone KS, accurately weighed, in methanol, and dilute quantitatively and stepwise with methanol t o obtain a solution having a known concentration of about 20 pg/mL.

Assay Preparation: Weigh and finely powder not less than 20 Chlorzoxazone tablets. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of chlorzoxazone, to a 100-ml volumetric flask. Add about 80 ml of warm methanol, and shake by mechanical means for about 15 minutes. Dilute with methanol to volume, and mix. Filter, discarding the first 20 mL of the filtrate, and pipet 2 ml of the filtrate into a 100-ml volumetric flask. Dilute with methanol to volume, and mix.

Procedure: Concomitantly determine the absorbances of the Assay and Standard Preparations at 282 nm, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity, in mg, of chlorzoxazone in the portion of tablets taken by the formula 5C (Au/As), in which C is the concentration, in pg per ml, of USP chlorzoxazone RS in the Standard Preparation and Au and As are the absorbances of the Assay and Standard Prepa- rations, respectively.

9.3 Fluorescence Spectrophotometry - Stewart and Chan used the intrinsic fluorescence of chlorzoxazone to assay for the drug in commercial tablets containing acetaminophen ( 8 ) . Recovery of drug was in the 98-101% range. These same authors also demonstrated that chlorzoxazone could be successfully analyzed in a tablet dosage form chemical derivatization with fluorescamine to form a fluorophore (7).

9 . 4 Potentiometry - Tagami and Muramoto described a method based on the use of a carbon dioxide gas-sensing electrode ( 2 9 ) . Twenty tablets were powdered and a portion equivalent to about 424 mg of drug was weighed and extracted with 20 ml of acetone. After stirring and centrifugation, the supernatant acetone solution was removed and


diluted with additional acetone. This extraction procedure was performed four times. The collected acetone fractions were evaporated to dryness. The residue was refluxed for 2 hr with 50 ml of 3N sodium hydroxide and after acidification, the carbon dioxide was determined using a gas permeable electrode. Mean recovery of chlorzoxazone was 99.6% with a standard deviation of 0.24.

9.5 High Performance Liquid Chromatography - The USP XXI method for Chlorzoxazone with Acetaminophen tablets is a gradient HPLC method (34). In the method, m-chloroaniline, p-aminophenol (a degradation prozuct of acetaminophen), p-chlorophenol and 2-amino-4-chlorophenol are separated from chlorzoxazone, acetaminophen and the internal standard phenacetin.

An example of the separation is as follows:

Component Acetaminophen

Retention time, minutes 2.3

2-Amino-4-chlorophenol 6.0 p-Aminophenol 7.1

Internal Standard (phenacetin) 8.3 p-Chlorophenol 9.2 Chlorzoxazone 10.1

m-Chloroaniline - 7.5

10. References

1. Current Therapeutic Research (1975) 17, p 123, Litera- ture Services Section, McNeil Laboratories, Inc., Fort Washington, PA. A.P. Roszkowski, J. Pharmacol. Exptl. Therap., 129 (1960) p 75.

2.

3. Pharmacy Times, 48 (1982) 23-32. 4. D. Marsh, U S Patent 2, 895, 877 (1959). 5. J. Sam and J . N . Plampin, J.Pharm. Sci., 53, (1964)

6. K. Harsanyi, F. Toffler, KD. Korbonitis and G. 538-544.

Leszkovszky, Hung. Patent 150, 577 (1961); thru Chem. Abstr., 60, 5507b (1964).

7. J.T. Stewart and C.W. Chan, Anal. Letters, Bl1 (9),

8. J.T. Stewart and C.W. Chan, J. Pharm. Sci., 68 (1979) (1978) 667-680.

9 10-912. -

9. A.H. Conney, N. Trousof and J.J. Burns, J. Pharmacol. Exptl. Therap., 128 (1960) 333.

CHLORZOXAZONE 143

10.

11.

12. 13.

14.

15.

16.

1 7 .

18.

19.

20.

21.

22 *

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

E.L. Kirschner, McNeil Pharmaceutical, Spring House, PA, (1986), Personal communication. H. Reral, V. Gamentzy and I. Bucur, Rev. Chim. (Bucharest) 16 (1965) 322. B. Kaempe, Arch. Pharm. Chemi., g. Ed., 1. (1974), 145. K.D. Parker, C.R. Fontan and P.L. Kirk, Anal. Chem., 34 (1962) 757. R.K. Desiraju, N.L. Renzi, R.K. Nayak and K. Ng, J, Pharm. Sci. 72 (1983) 991-994. A.H. Beckett,G.T. Tucker and A.C. Moffat, J. Pharm. Pharmac., 19 (1967) 273. "Clarke's Isolation and Identification of Drugs," 2nd Edition, A.C. Moffat, Ed., The Pharmaceutical Press, London, 1986, p 193. R.C. Pedroso and E.D.C.F. Moraes, Rev. Farm. Rioqulm. Univ. S. Paulo, 18 (1982) 183-195. I. Ullah, D.E. Cadwallader, and I.L. Honigberg, - J. Chromatogr., 46 (1970) 211-216. R.A. Egli andS. Tanner, Fresenius 2. Anal. Chem. 295 (1979) 398-401. R.A. Egli and S. Keller, - J. Chromatogr., 2 (1984) 249-256. E.G.C. Clarke, "Isolation and Identification of Drugs," Volume 1, The Pharmaceutical Press, London (1969), p 34. J.T. Stewart, I.L. Honigberg, and J.W. Coldren, - J. Pharm. Sci., 68, (1979) 32-36. I.L. HonigberE J.T. Stewart, and J.W. Coldren, - J. Pharm. Sci., 68, (1979) 253-255. Japan Pharmacopeia, 9th Ed., Society of Japanese Pharmacopeia, Japan, p 157. V.J. Schnekenburger and M. Quade-Henkel, Dtsch.

N.M. Sanghoi, N.G. Joshi, and K.S. Dubal, Indian Drugs,

Tlarke's Isolation and Identification of Drugs," 2nd Edition, A.C. Moffat, Ed., The Pharmaceutical Press, London, 1986, p 465. A . Watanabe. Y. Yamaoka. K. Kuroda, T. Yokoyama and T.

Ap0th.-Ztg., 124 (1984) 1167-1170.

18 (1981) 299-302.

Umeda, Yakugaku Zasshi,-l05 (1985) -481-490.- S. Tagami and Y. MuramotFChem. Pharm. Bull, 32 (1984) 1018-1024. V.R. Twele and G. Spiteller, Arzneim. Forsch., - 32 (1982)

A.H. Conney and J.J. Burns, J. Pharmacol. Exptl. Ther., 128 (1960) 340. m. Ng, McNeil Pharmaceutical, Spring House, PA (1986), Personal Communication. J.T. Stewart and H.K. Carter, J. Chromatogr., Biomed.

759-763.

Appl., 380 (1986) 177-183.


3 4 . Second Supplement, United States Pharnacopeia , 2 1 s t - E d i t i o n , United S t a t e s Pharmacopeial Convention, Bethesda, MD, (1985), p. 1827.

L i t e r a t u r e surveyed through December, 1986.

CYCLOSPORINE

*Mahmaud M.A. H c u a n and **Mohammed A . AL-Yahya

*Pho6ehboh 0 6 PhcuunacWcd Chmbin.thy, Vep&evLt 0 6 P h m a c e d c d Che.mhin.thy, CoUege 04 Phmacy , King Saud U n i w m L t y , Riyadh, Saudi Atrabiu.

**kshoch~& PhOdeAhotr 0 6 Phahmacognob y , Ve-ed 0 6 Phahmacognon y. VhecZoh 06 ,the MediciMae Akomatic and Pohonoun Plan& Runahch Cenhn, CoLtege 06 Phahmacy, King Saud U n i w m L t y , Riyadh, Saudi k a b i a .



146 MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

1.

2 .

3.

4.

5 .

6.

7 .

8.

9.

10.

11.

12.

13.

History and Therapeutic Category

Description

2.1 Nomenclature 2.2 Formulae

2.2.3 Degradation and Structural Elucidation 2.2.4 Conformation

2.3 Molecular Weight 2.4 Appearance, Color, Taste, Odor

Physical Properties

3.1 Crystal Properties 3.2 Melting Range 3.3 Solubility 3.4 Optical Rotation 3.5 Spectral Properties

I so lat i on

Biosynthesis

S y n thes is

6.1 Cyclosporine 6.2 Cyclosporine Analogs 6.3 Structure-Activity Relationship

Met abo 1 ism

Pharmacokinetics

Pharmacological Activity and Clinical Applications

Pharmaceutical Forms

Methods of Analysis

11.1 Elemental Composition 11.2 Introduction to Analysis 11.3 Radioimmunoassay 11.4 High Performance Liquid Chromatography

References

Acknowledgement

CYCLOSPORINE

Cyclosporine

147

1. His tory and Therapeut ic Category

It w a s from Hardanger Vidda, a b leak treeless p l a t eau i n t h e South of Norway, t h a t a s o i l sample w a s ob ta ined i n t h e e a r l y seven t i e s . The fungus i s o l a t e d from t h i s sample was c a l l e d Tolypocladium i n f l a t m Gams (1) , formerly ( 2 ) designated as Trichoderma po lysporm (Link ex Pe r s . ) ( 3 ) . Severa l metabol i tes o f t h i s fungus have been i s o l a t e d of which cyc lospor ins A , C and G t o have so far been shown t o possess s t rong immunosuppressive a c t i v i t y . The main component i n normal fermentat ion b ro ths w a s named cyc lospor in A ( A ) , now known as cyclosporine. A s repor ted by Bore1 e t a1 (5 -9 ) , cyclosporine i s a s e l e c t i v e immunosuppressive drug with an t i funga l and ant i inf lammatory p r o p e r t i e s .

R i f a i or Cylindrocarpon luciderm Booth

Today important r ecen t innovat ion i n t h e f i e l d of organ t r a n s - p l a n t a t i o n .

cyclosporine can be regarded as t h e m o s t

2. Descr ip t ion

2.1 Nomenclature

2 . 1 . 1 Chemical Names

Cyclosporin A; Cyclosporine.

2.1.2 Generic Names

Cyclosporine (USAN) and i s adopted f o r t h e bas i c s t r u c t u r e , meanwhile t h e fol lowing names has been accepted i n o t h e r coun t r i e s . England, cyc lospor in (BAN name); France, c icy lospor ine ; Switzer land and o t h e r s , c i c l o s p o r i n (INN name, WHO).

2.1.3 Trade Names

Sandimmune , Sandimmun


2.2 Formulae

2.2.1 E h p i r i c d

N O C62Hl l l 11 1 2


Cyclosporine (F ig . 1) is a n e u t r a l , hydrophobic c y c l i c pept ide composed o f 11 amino ac id r e s idues , a l l having t h e S-configura- t i o n of t h e n a t u r a l L-amino a c i d s , except f o r t h e D-Ala i n p o s i t i o n 8, which has t h e R- conf igura t ion , and Sa r i n p o s i t i o n 3. Seven ac ids a r e N-methylated. Ten are known a c i d s , t hey are a-Abu i n p o s i t i o n 2 , Sar-in p o s i t i o n 3, N-MeLeu i n pos i t i on 4,6,9 and 1 0 , V a l i n p o s i t i o n 5 , Ala i n p o s i t i o n 7 , D-Ala i n posi- t i o n 8 , N - M e V a l i n p o s i t i o n 11 and MeBmt i n pos i t i on 1.

2.2.3 Degradation and S t r u c t u r a l E luc ida t ion

The s t r u c t u r e o f cyclosporine [l] (F ig . 1) was determined by chemical degradat ion (4) and x-ray c rys t a l log raph ic ana lys i s (10,ll). Also with an x-ray c rys ta lographic a n a l y s i s of t h e iodo d e r i v a t i v e [ l a ] . It has t h e molecular formula C62HlllN11012 as deduced by NMR and mass spec t r a . Hydrolytic cleavage of cyclosporine furn ished 11 amino ac ids as fragments, among them an a r t i f a c t of a new C9-amino ac id . The amino a c i d sequence w a s determined by Edman degradat ion of i socyc lospor in , a bas i c rearrangement product formed from cyclosporine by N,O- a cy l migra t ion . On t h e b a s i s of t h e chemical, spec t roscopic and c rys t a l log raph ic evidence, t h e s t r u c t u r e of cyclosporine w a s ,

e luc ida ted as t h e n e u t r a l c y c l i c o l igopep t ide It i s composed of e leven amino ac id r e s idues , a l l having t h e L-configuration of t h e n a t u r a l amino a c i d s , except f o r t h e D-alanine i n p o s i t i o n 8 and t h e non-chiral sa rcos ine (N-methylglycine) i n p o s i t i o n 3. Seven amino a c i d s - i n p o s i t i o n s 1, 3, 4, 6, 9 , 1 0 and 11- are N-methylated. Ten o f t h e

A

u Fig. I . Struciure of cyclosporine 1 (schematic). McBmt - (4R)-4-[(€)-2-bute-

nyl]-4,N-dimcthyl-~-threoninc (see text).


Ib J

cyclic derivotivool the Nmdhyl-C9-amino ocid

cyclorporine not obtoincd

1

cyclosporlnc IS OCYCLOSWRINE onhydro- mrthylthio

de rivot ive 1 Ic hydontoin

Edman chosen mild conditions

pept ide hydrolysis with 6N HCI followed by ion erchange chroma tog raphy.

degradat ion with CHI-NCS under carelul ly

T l O A c l I t -+ t--- Zn I HOAc

n 'F,. I

n

cy cloiporlnc

1

CYCLOSPORINE 151

eleven ring members are derivatives of known aliphatic amino acids: a-aminobutyric acid (Abu) in position 2, Sarcosine (S.ar) in position 3, N-methylleucine (MeLeu) in positions 4, 6, 9 and 10, Valine (Val) in position 5, Alanine (Ala) in position 7, D-Alanine (D-Ala) in position 8, and N-methylvaline (Me Val) in position 11. The amino acids were readily characterised following acid hydrolysis of cyclosporine.

The novel amino acid was found to have the composition ( 2S, 3R, 4R, 6E) -3-hydroxy-b- methyl-2-( methylamino) -6-octenoic acid, and in accord with amino acid nomenclature, is now designated as (4R)-4-[(E )-2-butenyl]- 4-N-dimethyl-L-threonine and abbreviated MeBmt . Thus the novel amino acid has the polar features of an N-methyl-L-threonine which is substituted at the end of the carbon chain by butenyl and methyl groups. This amino acid was hitherto not known in the free form, since only antifacts and derivatives were obtained from degradation experiments on cyclosporine(4).

Hydrolysis of cyclosporine followed by ion exchange chromatography afforded a cyclic derivative [lb] of the MeBmt minoacid. Acidic treatment of cyclosporin [l] in the absence of water effects an N, O-acyl- migration of the methylvalyl furnishes isocyclosporine [lc]. A modified Edman degradation of isocyclosporine [1] using methyl isothiocyanate produced an anhydrothiohydantoin-derivative [l] and thus established MeBmt as the first amino acid in the peptide sequence. The complete sequence was established by repetitive Edman degradation.

moiety and

2.2.4 Conformation of Cyclosporine

The conformation of cyclosporine in the crystal and in solution was reported (11). The


conformational ana lys i s was based on X-ray c rys ta l lography i n t h e c r y s t a l and on NMR spectroscopy i n so lu t ion us ing d i f f e r e n t l i p o p h i l i c so lvents . Cyclosporine molecule i s h ighly l i p o p h i l i c , t h e r e f o r e t h e s o l i d s t a t e conformation and t h a t deduced from NMR s t u d i e s i n apolar so lven t s a r e c l o s e l y similar. It assumes a r a t h e r r i g i d conformation, both i n t h e c r y s t a l l i n e s ta te and i n so lu t ion ( F i g . 2 ) . The only d i f f e rences a re t h e side-chain conformation o f MeBmt ( a major change between t h e chain fo lded i n upon t h e molecule i n t h e solid s ta te , and po in t ing out i n t o so lvent i n t h e NMR s tudy) , t h e side-chain conformation of MeLeu-10, and t h e d e t a i l e d backbone conformation i n t h e region of D- A l a - 8 ( F i g . 3 ) . Here t h e X-ray s t r u c t u r e c l e a r l y shows one H-bond from D-Ala-8 ( N H ) t o MeLeu-6 ( C O ) , whereas i n so lu t ion a b i fu rca t ed H-bond t o both t h e carbonyl 0-atom of MeLeu-6 and t o t h e carbonyl 0-atom of D-&a-8 i t s e l f . The conformation change needed t o convert t h e X-ray backbone i n t o t h e NMR backbone i s small, and can be brought about by r e l a t i v e l y s m a l l r o t a t i o n i n t h e backbone t o r s i o n angles i n t h e reg ion MeLeu-6 t o MeLeu-9, p r i n c i p a l l y around t h e 7- and 8- res idues .

The side-chain conformations of M e V a l and V a l and t h r e e of t h e f o u r MeLeu-residues (MeLeu -4 , -6 and -9) are i d e n t i c a l i n c r y s t a l and i n so lu t ion . Only t h a t o f MeLeu-10 i s r o t a t e d by 120°. The side-chain conformation of MeBmt i n so lu t ion can be der ived from t h e X-ray conformation simply by means of 120' r o t a t i o n about t h e cl,B-bond. This r o t a t i o n which i s e n e r g e t i c a l l y allowed, could be t h e r e s u l t from t h e formation of an in t ramolecular H-bond between t h e B-OH and t h e carbonyl 0-atom of MeBmt rep lac ing t h e in te rmolecular H- bond found i n t h e c r y s t a l . A l a r g e po r t ion of t h e backbone ( r e s idues 1-6) adopts an a n t i p a r a l l e l &pleated shee t conformation which conta ins t h r e e t ransannular H-bonds

CYCLOSPORINE 153

Fig. 2. Stereoview of cyclosporine. a) Solid-state conformation, b) Computer-generated conformation in apolar solvents (in solution the distal atoms of Abu-2 and YeBmt-1 have a high flexibility).

Fig. 3. Superposition of the solid-state conformation (thin line;) and the computer-generated conformation of cyclosporine in apolar solvent (thick lines).

CY CLOSPORINE 155

and is markedly twisted. The sarcosine in position 3 and N-methylleucine in position 4 participate in a type 11B-turn (12-14). This means that, in the orienta- tions chosen in Figures l and 2, the CC group of Abu-2 and the N-CH? group of MeLeu-4 are up and the CC gEoup of Sar-3 and NH group of Val-5 are down - pro -S proton of the methylene group of Sar 3 is axial, and the side-chain of MeLeu-4 is requatorial. The remaining residues 7-11 form an open loop. only cis- amide linkgage in the molecule between the two adjacent N-methylleucine residues 9 and 10. The remaining H-bond of a y-type and serves to hold the backbone in a folded L-shaped.

This loop contains the

As a consequence of this rather rigid conformation of the cyclosporine skeleton, six amino acids have their side-chains directed quasi-perpendicular to the plane of the peptide ring; in Abu-2, Val-5 and MeLeu-6 they are pointing upwards, in MeBmt-1 and ~ e ~ e u - 6 they are pointing downwards. The side-chains of the remaining aminoacids lie more or less in the plane of the peptide ring.

The resulting overall molecular shape in apolar solution is that of the previously postulated "butterfly" (10) , in which the MeE3mt side-chain, known to be important for immunosuppressive activity, stands out, probiscis - like, in a manner suggestive of special function (Fig. 4).

2.2.5 CAS Registery Number

Cyclosporine [79217-60-0] Cyclosporine A [ 59865-13-31

2.2.6 Clinical Code

CyAIOL 27-400


Fig. 4. Two roughly orthogonal views of a model of native cyclosporine in the conformation suggested as probable. The backbone conformation corresponds to that of iodocyclosporine.

CY CLOSPORINE 157

2 . 3 Molecular Weight

1202.64

3.

2.4 Appearance, Color , Taste, Odor

White p r i sma t i c c r y s t a l s .

Phys ica l P r o p e r t i e s

3 .1 Crys t a l P r o p e r t i e s

3.1.1 X-ray D i f f r a c t i o n

Crys t a l Data

X-ray c r y s t a l s t r u c t u r e s of cyc lospor ine and i t s iodode r iva t ive [ I a ] were determined (10,ll). A summary o f t h e c r y s t a l d a t a and re f rac tomet ry for cyclospor ine i s given i n Table 1 (11).

Table 1. Crys t a l and D i f f r a c t i o n Data

Molecular for - mula

Molecular weight

C r y s t a l l i s a - t i o n

Crys t a l form

Crys ta l s i z e

Space group

Cell dimen-. s i o n s

Crys t a l den- s i t y ( c a l c . )

N O C 6 2 H l l l 11 12

1202.5

from acetone

c o l o u r l e s s , p r i s m a t i c

ca . 0 .2 x 0 .2 x 0.3 mm

c = 41.242(33 A; V = 7896A

dc = 1.042 g. cm-3

Molecules per c e l l

D i f f r ac to - meter

Radiat ion

I n t w s i t y scans

Sphere of r e f l ex ion

I n t e n s i t ie: measured I n t e n s i t i e s s ign i f i can t

z = 4

CAD4 (Enraf - Nonius)

C a a ( Graphi te monochromat o r )

Ow = 1.0°+0.3 w/2e = 1.0;

t a n 0

a(T) / I = 0.02 = 120s) ( tmax

sine/X 4 0.51 ( 36508 r e f l ex ion r )

5057 (unique)

4272(I 2 .50(1) ;


The c r y s t a l da t a for iodocyclosporine (10) , N 0 + , M = 1327, c o l o r l e s s

'62'110 11 1 2 - prisms from n-heptene, 2 . 5 : l monoclinic, a = (10 .475(5) , b = 1 9 . 6 0 ( 1 ) , c = 21.05(1)A0, f3 = 99.35 (2'1, U = 4264 A o 3 , Dc = 1.03, z = 2, space group ~2~ , ( c;, NO. 4). r a d i a t i o n , A = 1.54187 A o , g r aph i t e monochromator. A c r y s t a l o f approximate dimensions 0.3 x 0.7 x 0.4 mm w a s used on an Enraf-Nonius CAD4-F automatic d i f f r a c t o - meter.

CUK ci

A s tereoview o f iodocyclosporine molecule i s shown i n F ig . 5a, and t h a t of cyclosporine i n Fig. 5b.

The iodocyclosporine showed t h e a n t i p a r e l l e l &pleated shee t conformation of r e s idues 1-6, t h e open loop of r e s idues 7-11, and t h e r a t h e r l a r g e thermal v i b r a t i o n s of some of t h e side-chain atoms , p a r t i c u l a r l y those of MeLeu-9 are apparent . The abso lu te conf igura t ion w a s not determined as p a r t of t h e s t r u c t u r e a n a l y s i s , s ince s u f f i c i e n t of t h e hydro lys is products could r e l i a b l y be i d e n t i f i e d as L-amino-acids. It became apparent during t h e a n a l y s i s , however , t h a t A l a - 8 has t h e D-configuration (15).

The conformation of cyclosporine observed i n t h e c r y s t a l (F ig . 5b) wi th 50% p r o b a b i l i t y v i b r a t i o n a l e l l i p s o i d s of t h e atomic thermal motion (16) which d i sp lay t h e r e l a t i v e l y s m a l l , i so top ic v i b r a t i o n s of t h e backbone atoms - i nd ica t ing conforma- t i o n a l r i g i d i t y - i n c o n t r a s t t o t h e more f l e x i b l e side-chain atoms whose v i b r a t i o n a l amplitudes increase with t h e d i s t ance from (a).

Fig. 6a shows t h e backbone of t h e c y c l i c pept ide with t h e 4-, I)-, and w- angles and some s t r u c t u r a l f e a t u r e s . Three H-bonds br idge t h e two shor t @ - s t r ands adding t o t h e s t a b i l i t y of @-fragment.

/!

Fig. 5a. A stereoscopic view of the iodocyclosporine molecule showing 50% probability ellipsoids of thermal vibration. The Cg amino acid is at centre left on the forward side of the molecule and the conventional sequence then extends upwards at the front through a-aminobutyric acid, glycine etc. of the 8-pleated sheet is indicated (dotted) in the centre and the interesting hydrogen bond Ala-8 4 Meleu-6 appears at centre right.

One of the hydrogen bonds

Fig. 5b. ORTEP drawing of the crystal structure of cyclosporine. Anisotropic atomic vibrations are represented by 50% - probability ellipsoids. The numbers shown are residue numbers.

Fig. 6a.

\ bet a loop

Backbone conformation of crystalline cyclosporine with indication of the loop and 6-fragment. The torsion angles 6, $I, and w and in brackets the computer- modeled values for the NMR conformation are given. The dashed lines indicate B-bonds, the dotted lines close contacts from CH3N of MeVal to different atoars (0 11:3.04 Ao; C 7:3.36 Ao; 0 7:3.26 AO; C 8:3.41 AO; N 9:3.54 AO; N 10:3.12 Ao).

F i g . 6b. Stereoview of a space-filling model cyclosporine showing the side chain

of of

between MeLeu-6 and-Meleu-4 into the ply of the &fragment

CYCLOSPORINE 163

The s i d e cha ins of t h e Abu, V a l , and MeLeu r e s idues are i n t h e s taggered conformation, t h e MeLeu s i d e cha ins being extended. Rather unexpected i s t h e conformation of t h e Cg-alkylene s i d e chain of MeBmt-1; it i s n e a t l y fo lded i n t o t h e p ly of t h e 6-pleated shee t which al lows t h e molecule t o adopt a g lobular compact shape (F ig . 6b ) .

3.2 Melting Range

White pr i smat ic needles from acetone at - 15', m.p. 148-151' (17). The syn the t i c cyclosporine m.p. 149-150° (18) .

3.3 S o l u b i l i t y

The compound i s n e u t r a l , r i c h i n hydrophobic amino a c i d s , i n so lub le i n water and n-hexane, but very so lub le i n a l l o the r organic so lvents . It i s very so lub le i n methanol, e thano l , acetone, e t h e r and chloroform.

3.4 Opt ica l Rotat ion (17)

- 244' ( C = 0.6 i n chloroform).

[a]:' - 1.89' ( C = 0 .5 i n methanol) .

3 .5 S p e c t r a l P rope r t i e s


The W spectrum of cyclosporine i n methanol w a s recorded on a Beck~nann-DK2 spectrophotometer shows only an end absorp t ion a t 200 nm ( 4 )

3.5.2 In f r a red Spectrum

The i n f r a r e d c h a r a c t e r i s t i c s i n methylene- ch lo r ide ( C H 2 C 1 2 ) and i n ca rbon te t r ach lo r ide ( C C l 4 ) were repor ted ( 4 , l l ) . spectrum i n methylene ch lo r ide recorded on a Perkin E l m e r 21.IR spectrophotometer i s shown i n Fig. 7. It should be noted t h a t so lu t ion s t u d i e s , i nd ica t ed t h e NH groups

The I R

Fig. 7. IR-spectrum of cyclosporine in CH2C12.

CYCLOSPORINE 165

are involved i n H-bonding. Thei r absorp t ion bonds ( 3330, 3290 cm-l i n C C l 4 s o l u t i o n and 3400-3330 cm-1 i n CH2C12 s o l u t i o n ) are independent o f t h e concent ra t ion i n t h e range of 4.10-2 t o 4 . 1 O q 4 M i n C C 1 4 so lu t ion . The spectrum shows a l s o an in t ens ive amidecarbonyl bond at 1630-1670 cm-1.

3.5.3 Nuclear Magnetic Resonance Spec t ra

1 'H-NMR, 13C-NMR and H-NMR-NOE-difference s p e c t r a of cyclosporine i n deuterochloroform and deuterobenzene were repor ted ( 4 ~ 8 , 11). However, t h e complete assignment of lH-NMR, 13C-NMR and 15N-NMR s p e c t r a i n deuterochloroform and deuterobenzene by a combination of homonuclear and he tero- nuclear two dimentional techniques were descr ibed by Kessler e t a1 (19 ) .

3.5.3.1 'H-NMR Spec t ra

1 The H-NMR spectrum of cyc lospor ine (F ig . recorded on Bruker HX-gO-E, 90 MHz instrument ( 4 ) . A p a r t of 1~-NMR spectrum o f cyclosporine i n ~ 6 ~ 6 (F ig . 9 ) w a s recorded on Bruker HX-360, 360 MHz instrument . This has proved t h e E-configura- t i o n o f t h e double bond i n cyc lospor ine by us ing t h e double resonance technique (JE, 5 = 16 Hz) ( 4 ) . From t h e s e s p e c t r a , i d e n t i - f i c a t i o n o f sp in systems were obtained. It i s evident from t h e s p e c t r a t h a t one conformation s t rong ly dominates. Some minor peaks s p e c i a l l y i n t h e N-methyl reg ion (2.5-3.5 ppm) are v i s i b l e i n d i c a t i n g t h e presence o f another conformation i n s l o w exchange. A completely d i f f e r e n t s i t u a t i o n i s observed i n DMSO d6 s o l u t i o n , where a mixture of at l ea s t seven conformations l e a d s t o a spectrum of h igh complexity. Roughly

8 ) i n CDC13 and TMS w a s

8 * 0 0 T 6 5 I 3 2 I 0 ??a

F i g . 8 . 'H-NMR-spectrum of cyclosporine by 90 !Mz in CDcl3, TMS -- 0 ppm.

CYCLOSPORINE 167

F i g .

C

b

0

9.

speaking, f i v e more o r less separated spec t r a l regions are dist inguishable:

NH protons 7 - 8.3 P P ~

a-protons as well as 4.5 - 6 pprn the v iny l i c protons

N-methyl groups 2.6 - 3.8 ppm

C-methyl groups 0.6 - 1.8 ppm

Aliphatic f3-, y- 1.1 - 2.7 PPRI and 6 protons

The proton chemical shifts for cyclosporine i n CDC13 and C6D6 are l i s t e d i n Table 2 .

h

H-c c

h

1 t

Part of the a) Undecoupled spectrum. b) Decoupled snectrm keeping lock on H-C(n) of Cg As c ) Decoupled spectrum keeping lock on H-C(6) of Q As

H-NMR-spectrum of cyclosporlne by 360 llHz in CsDs.

Table 2. h- and 13C-NMR chemical shifts of cyclosporine [I] in C D C l and C G D ~ ~ ) 3

Resi- Amino-acid ~~ l3C-NMR Group

CDC13 C6D6 CDC13 C6D6 due residue lH-NMR

1 MeBmt: CH3N 3-52 3.51 3.73 33.97 9 34.33

co - - - - 169.65 s U ’ O * 73

H-C(a ) 5.45 5.47 5.7 5.72 58.75 d 59.96

H-C(B) 3.82 4.21 4.20 74.74 d 74.95

OH 3.87 3.5

1.63 2.07 35.99 d 36.30

- -

2 Abu NH

co

2.41 2.65

1 - 7 3 2.13

0.72 0.71 1.15

5.36 5.64

5.35 5-52

1 .62 1 - 7 5

35.63 t 36.04

16.76 q 18.30

126.32 d 126.76

17.96 q 18.69

129.68 d 131.38

- 7.93 7.96 8.26 - - - - - 173.04 s 174.29

Table 2. (Cont inued)

Resi- Amino-acid Group H-NMR 13C-NMR

3 C6D6 C D C l C6D6 due residue C D C l

2 K 3 2 K 2 K 3 2 K No.

H-C( a) 5-03 5.12 5.12 48.86 d 49.54

H-C( 6) 1.6-1.74 1.79 25.06 t 26.11

3.40 3.08 39.40 q 39.58 - - - - 170.50 s 171.80

H-C( Y) 0.87 0.89 9.93 9 10.73

m3N 3 Sar

co H-C( a ) 4.76 4.74 4.01 50 - 37 t. 50.10

4 MeLeu CH N 3.11 2.59 31.32 q 31.39

H-C( ~ 1 ) 5.34 55.51 a 56.20

3 co 169.35 s 170.21

H1-C( B ) 2.00 35.99 t 37 * 01

5 V a l

5.60

1 .58

0.98

0.91

2.27

1.42

7.46

24.90 d 25.79

23.49 q 24.28

21.18 q 22. TOb)

W

RW

V M

d

V

e

4

0

wln

mr

J&

w

c-

or

i

0

d

ri I

W

1-

3

?

r-

dW

0

WA

OO

\

*.

..

I

A(

ur

l0

M

(u

m

? 0‘

m

Ln

d

M

0

cu m

Ln

cu M

I

ad

d

I

co

t- ?

ul

170

co

..

Ln

dL

nL

n

co gt-

59

?

??

?

Ln

Ln

m

ti

co

o

cu

r-

JJ

cucu

ti

LP

U

J

m

cu

M

cu ?

cu r-

M

t- co

IL

n

0

IL

n

cs

m

171

Table 2 . ( Continued)

Resi- Amino-acid Group lH-NMR 13C-NMR

3 C6D6 CDC13 C6D6 due residue C D C l

2 K 3 2K 2 K 32K No.

CH3(62) 0.89 0.84 21.86 9 24.81

10 MeLeu CH 3N 2.70 2.85 29.83 q 30.47

co - - - - 169.41 s 170.98

H-C(CX) 5.10 5.35 57.54 d 58.36

H1-C( 6 ) 2.13 2.42 2.42 40.73 t 42.06

11 M e V a l

1.24 1.29

H-C(y) 1 .49 1 .79 CH3(61) 0.98 1 .17

CH3( 6 0.98 1.16

24.55 d 25.62

23.85 q 24.34

23.38 q 22. 6bb)

2.71 2.98 29.81 q 30.96

174.61 - - - - 172.85 s co H-C(CX) 5 -15 5.14 5-27 57.93 d 58.84

m3N

H-C( 6 ) 2.17 2.27 29.05 t 29 - 99

Pi

3

0

k

0

WD

V)

am

0

0

..

ri

m

oc

o

do

..

wl

kk

G

ik

n

Ld

173

Table 2. ( Continued)

e

4 W

Resi- Amino-acid Group 1H-NMR 13c-NMR due residue CDC1,

0.96

0.66

18.75 q 19.38

20.26 q 20.59

a 1 ) At 296 K in pprn from internal TMS. The H-NMR data were obtained from a conventional

I 32K spectrum or from the cross-sections of a H,$-COSY with 2K data points in f2 at

300 MHz

b, The signals of MeLeu-4(6 ) and MeLe~-9(6~) as well as those of MeLeu-6(6 ) and MeLeu-lO(6 )

in C D 2 1 2

may be exchanged). 6 6


3.5.3.2 13C-NMR Spec t ra

I n t h e broadband-decoupled 13C-NMR s p e c t r a i n CDC13 or C6D6 were repor ted ( 1 9 ) . Almost a l l 62 C- s i g n a l s a r e reso lved and only few s i g n a l s over lap , bu t t hese s p l i t when t h e so lvent i s changed. A l l carbonyl C-atoms resonate i n t h e range between 169 and 175 ppm. Pa r t of t h e l 3 C - m spectrum ( t h e carbonyl reg ion) of cyclosporine i n CDC13 measured on a Bruker-WH-360, 360 MHz instrument i s shown i n Fig. 1 0 (11). The two o l e f i n i c C-atoms of MeBmt are a l s o i n a t y p i c a l range 126-132 ppm, whereas 49 C-signals a r e i n t h e a l i p h a t i c region. The number of a t t ached protons were obtained v i a t h e usua l DEPT technique (20 ,21 ) . A he te ronuclear 2D-J, 6 spectrum (22-26) w a s a l s o performed i n CDC13 The chemical s h i f t f o r a l l carbons and m u t l i p l i c i t i e s i n C X 1 3 and i n C6D6 are given i n Table 2 .

3.5.3.3 15N-NMR Spec t ra

AH-decoupled 15N-NMR spectrum shows eleven N-signals (Table 3 ) .

Table 3. 15N-NMR chemical s h i f t s of 1. 6 ppm from N H 4 l 5 N O 3 i n an e x t e r n a l c a p i l l a r y .

i n CDC13

- 248.8 A7

- 255.5 Abu

- 256.9

- 257.3 A8

- 258.0

- 258.6 Val

i n C6D6

- 248.1 A7

- 255.1 Abu

- 256.7

- 256.9 ~8

- 258.3

- 259.2

~~

i n C6D6 3

i n C D C l

- 259.2 - 260.4 V a l

- 260.1 - 261.8

- 261.8 - 263.1

- 264.2 - 263.9

- 265.2 - 265.1

F i g .

1 1 1

I73 m m 170

-6 [PpnJ 10. ''C-NNFI spectrum of cyclosporlne: CO region.

shifted downfield are marked In black. lowest from those attached to NE groups (residues 1 , 4 , 6 , and 7).

Those CO groups whose signals The CO sign81 of residue 8 (shaded)


3.6 Mass Spectrum

3.6.1 EI Spectrum

The e l e c t r o n impact spectrum of cyclosporine was measured on a CEC-mass spectrometer 21-llOB ( 4 ) . The e l e c t r o n energy w a s 70 e V , t h e acce le ra t ion rate 4.6-8 KV, t h e tempera- t u r e of t h e source 280-300°C and a pressure of 10-5 - 10-6 Tom. The molecules w a s uns tab le g iv ing a molecular ion peak a t m/e 1201 which vanishes quickly. vanishes a peak at m / e 1183 inc reases i n i n t e n s i t y and t h e spectrum remains s t a b l e . The peak a t m/e 1183 i s formed by elimina- t i o n of a molecule of water. The exac t mass measurement were made at 1183.831 i 0.003 and 1201.842 f 0.003. The molecular fo r - m u l a obtained by t h i s measurement w a s C62HlllN11012 (phys ica l molecular mass

1201.841). This w a s i n agreement wi th t h a t deduced from NMR and o t h e r f ind ings . Other mass s p e c t r a l da t a w i l l be presented under metabolism.

A s t h e M+

3.6.2 FD Spectrum

The f i e l d desorp t ion mass spectrum w a s measured on a MAT 711 mass spectrometer ( 4 ) . The acce le ra t ion energy rate 6 KV, t h e temperature of t h e ion source goo and t h e power f a c t o r f o r m u l t i p l i e r 106. A mole- c u l a r ion peak M+ of m / e 1201.8 w a s obtained and m / e 1203.6 f o r t h e dihydrocyclosporine. Another FD spectrum w a s repor ted (18). A molecular ion peak Mc a t m / e 1202, NH' a t m / e 1203 and ( M + N a + ) a t m / e 1225.

4 . I s o l a t i o n of Cyclosporine

Dreyfuss e t a 1 ( 2 ) and o t h e r s ( 4 , 27-30) have repor ted t h e i s o l a t i o n of cyclosporine from o t h e r fungal m e t a - b o l i t e s of t h e fungi Tolypocladium inf la tum Gams. This fungus w a s previously known as Trichoderma polysporum (Link ex P res . R i f a i ( 2 ) .

CYCLOSPORINE 177

The s t r a i n used i s Tolypocladium inf la tum designated No. N RRL 8044. This was grown i n an aerobic submerged c u l t u r e .

A repor ted method of i s o l a t i o n of cyclosporine from fermentat ion media w a s as fol lows ( 4 ) : The fermentat ion medium w a s ex t r ac t ed wi th n-butylacetate. Then a f te r t h e separa t ion of t h e organic l a y e r it w a s concentrated under vacuum. The crude res idue obtained w a s d e f a t t e d with 90% methanol and petroleum e t h e r . The product obtained was d isso lved i n chloroform and appl ied t o a column chromatography of s i l i c a g e l . The p o l a r i t y of t h e e l u t i n g so lvent w a s g radual ly increased by adding methanol t o chloroform. The f r a c t i o n s obtained by chloroform-methanol ( 98 : 5 : 1 . 5 ) contained cyc lospor in A . F rac t ions contained cyclosporin -C w a s obtained with chloroform-methanol (97:3). The f r a c t i o n s conta in ing cyclosporin A were subjec ted t o g e l f i l t r a t i o n with Sephadex LH-20 with methanol. The t o p f r a c t i o n s w e r e d i sso lved i n to luene and appl ied t o column chromatography o f aluminium oxide us ing to luene-e thylace ta te as t h e e luen t . The f r a c t i o n s obtained were evaporated and d isso lved i n a lcohol , t hen t r e a t e d with charcoa l . The f i l t r a t e was evaporated t o g ive cyclosporin A as an amorphous powder.

Another repor ted procedure ( 2 7 ) w a s as fol lows:

The fermentat ion bro th w a s separa ted i n t o t h e mycel ia l cake and t h e c u l t u r e f i l t r a t e . The former w a s homoge- nized t h r e e t i m e s wi th methanol-water (9:l) and t h e combined f i l t ra tes were concentrated under vacuum t o remove t h e methanol. The water so lu t ion w a s e x t r a c t e d t h r e e times wi th l ,2-dichloroethane and t h e organic l a y e r s were evaporated t o dryness . c u l t u r e f i l t r a t e by e x t r a c t i o n wi th 1,2-dichloroethane r e s u l t e d i n an add i t iona l por t ion of crude material. The combined ex t r ac t ed were submitted t o g e l f i l t r a t i o n on Sephadex LH-20 wi th methanol. Those f r a c t i o n s which conta in t h e cyclosporine (as a mixture) were pooled and then separa ted i n t o t h e components by s i l i c a g e l column chromatography (Merck , 0.063-0.2 mm) wi th e t h y l a c e t a t e s a t u r a t e d wi th water. I n accordance t o t h e i r p o l a r i t y , t h e cyc lospor ines w e r e e l u t e d i n t h e fol lowing order : D , G , A , B and C . The crude cyc lospor ines were f u r t h e r p u r i f i e d by c r y s t a l l i z a t i o n (cyc lospor ine G from ether /petroleum e t h e r a t room temperature; cyclosporines A, C and D f r o m acetone a t - 15OC).

Working-up of t h e

The


r e s u l t i n g pure cyc lospor ines A, B, C, D and G w e r e charac te r ized by a n a l y t i c a l methods ( t h i n l a y e r chromatography, mel t ing po in t , o p t i c a l r o t a t i o n ) and s p e c t r a l da t a ( U , I R , 'H- and l3C-NMR, mass spectrum). were found t o be i d e n t i c a l i n a l l r e s p e c t s with au then t i c samples i s o l a t e d from fermentat ion i n complex n u t r i e n t media as publ ished by Dreyfuss e t a1 ( 2 ) , Ruegger _ _ e t a1 ( 4 ) and Traber e t a1 (28 ,30 ) .

They

5. Biosynthesis of Cyclosporine

Kobel _ - e t a1 (31) and o t h e r s (27,32,33) have repor ted t h e b iosynthes is of cyclosporine and o the r fungal meta- b o l i t e s . I n t h e i n i t i a l b iosyn the t i c s t u d i e s of Kobel _ _ et a1 (31) , 3H- and 13C-labeled precursors w e r e fed t o t h e c u l t u r e and t h e p o s i t i o n of incorpora t ion of t h e l a b e l i n cyclosporine w a s determined by NMR spectroscopy. It w a s demonstrated t h a t t h e N-methyl groups of t h e mole- c u l e and t h e methyl group i n t h e y-posi t ion o f t h e unsa tura ted amino a c i d MeBmt a r e introduced as i n t a c t methyl groups from methionine and t h a t t h e remaining carbons of t h e MeBmt moiety a r e der ived from t h e head- t o - t a i l condensation of fou r a c e t a t e u n i t s . spectrum of the enriched cyclosporine der ived from [ l-13C]acetate showed four enhanced s i g n a l s corresponding t o t h e carbon atoms 1, 3, 5 and 7 of t h e MeBmt u n i t and no '3C incorpora t ion i n t o any o t h e r amino ac id .

The l3C-NMR

I n enn ia t in , an analogous example of non-ribosomal c y c l i c pept ide syn thes i s by fungi , Zocher and Kleinkauf ( 32) demonstrated t h a t a mul t i func t iona l enzyme c a r r i e s ou t t h e N-methylation of cons t i t uen t amino ac ids following an a c t i v a t i o n s t e p when t h e a c i d s are bound t o t h e enzyme as t h i o e s t e r s . Supporting evidence f o r a similar mechanism i n cyclosporine b iosynthes is w a s provided by t h e observat ion of Zocher e t a1 (33) t h a t [14C] sarcos ine w a s not 'ncorporated and t h a t t h e r a d i o a c t i v i t y from [Me-16C]methionine incorporated i n t o each N-methylated amino a c i d w a s d i r e c t l y propor- t i o n a l t o t h e number of t h e corresponding amin a c i d res idues i n cyclosporine, thus i n d i c a t i n g t h a t N- methylat ion of t h e amino ac ids occurred s imultaneously.

From t h e r e s u l t s o f Kobel e t a1 (31 ) t h e s i t e of b iosynthes is of t h e Bmt u n i t remains unc lear . However, t h e results obtained by t h e Zocher group (33) wi th short-term feeding experiments using l abe led a c e t a t e and methionine, i n which probably only incorpora t ion of

CY CLOSPORINE 179

label in the N-methyl group occurred, suggest that the amino acid Bmt is not biosynthesized on the enzyme matrix but at some other site before incorporation into cyclosporine.

6. Synthesis

6.1 Cyclosporine

Winger (18, 34-38) has reported the total synthesis of cyclosporine and analogues. The strategy of the total synthesis of cyclosporine has involved the following steps:-

I.

11.

111.

TV.

V.

VI .

VII.

VIII.

The synthesis of the N-methyl-C-9-amino acid (Scheme 1) (36, 39-44).

Building of the tetrapeptide BOC-D-Ala-MeLeu- MeLeu-MeVal-benzylester (37).

Preparation of the dipeptide BOC-Abu-Sar- benzylester (18,37,46).

Building of the tetrapeptide BOC-MeLeu-Val- MeLeu-Ala-benzylester (18).

The synthesis of the hexapeptide BOC-Abu- Sar-MeLeu-Val-MeLeu-Ala-benzylester (18).

Synthesis of the heptapeptide H-Me-C9-Abu- Sar-MeLeu-Val-MeLeu-Ala-benzylester (18,47) . Formation of the undecapeptide BOC-Ala-MeLeu- MeLeu-MeVal-Me-Cg-Abu-Sar-MeLeu-Val-MeLeu- Ala-benzylester (18,48).

Cyclization of the undecapeptide with a free amino group and free carboxyl group to produce cyclosporine (48,50,51).


7 Steps tio- F:, b

COOEI

(ZR,3R)-3-methyl-l,2,4,-butane- trio1

6 Steps

(2R,3R,SE)-3-rnethyl-S-heptene- 1,2-diol

"II_ c y k c y

(2R, 3R, 5E) -2-hydroxy-3-methyl- 5-heptenal

(2S, 3R, 4R, 6E) -3-hydroxy-4- methyl-2-(methylamino)-6-octenoic

acid (MeBmt)

Scheme 1. Synthesis of MeBmt

CYCLOSPORINE 181

V I I I . Total Synthesis of Cyclosporine (18, 34, 35, 37 1

Strategy Used f o r t h e Synthesis of Cyclosporine

For t h e synthesis of cyclosporine [l] (Fig. 1) t h e peptide bond between t h e L-alanine i n posi t ion 7 and t h e D-alanine i n pos i t i on 8 was chosen f o r t he cycl izat ion s tep. There were two main reasons f o r choosing t h i s s t r a t egy : 1. The intramolecular hydrogen bonds between t h e amide groups of t h i s l i n e a r peptide could operate so as t o s t a b i l i z e t h e open chain i n folded conformations approximating the cycl ic s t r u c t u r e of cyclosporine and thus assist cyc l i za t ion . 2. The bond formation between N-methylated amino acids i s more d i f f i c u l t than between non-methylated amino acids (37,45) ; there- f o r e , bond formation between the only consecutive p a i r of non-methylated amino ac ids i n cyclosporine appeared t h e l o g i c a l choice f o r t h e cycl izat ion s tep.

For the synthesis of t h e undecapeptide, a fragment-condensation technique introducing t h e amino acid MeBmt a t t h e end of t h e synthesis w a s used. I n t h i s way, t h e number of s t eps a f t e r t h e introduct ion of t h i s amino ac id w a s minimized. Scheme 5 shows t h e s t ep sequence used f o r joining t h e (protected) peptide fragments. The carboxy groups were general ly ac t iva t ed using a va r i a t ion of t he mixed p i v a l i c anhydride method reported by Zaoral (46) and adapted f o r t h e N-methylamino ac id de r iva t ives ( 3 7 ) . T h i s s t r a t egy allowed slow anhydride formation i n chloroform at - 2OoC with pivaloyl chlor ide i n t h e presence of 2 equivalents of a t e r t i a r y base such as N-methylmorpholine before adding t h e amino acid o r peptide e s t e r s t o be coupled i n t h e form of t h e i r f r e e bases. The f r e e bases were obtained by removal of t h e Boc-protecting groups with t r i f l u o r o - ace t i c acid at - 2OoC and subsequent


neu t ra l i za t ion with NaHC03; t h e benzyloxy- protect ing groups of t h e peptide i n t e r - mediates were removed hydrogenolytically with palladium/carbon i n ethanol.

The t e t rapept ide Boc-D-Ala-MeLeu-MeLeu- MeVal-OBzl w a s synthesized from t h e l e f t t o t h e r i g h t by forming bonds 1, 2 and 3 (Scheme 2 ) . Bond 4 was formed on syn- t h e s i s of t h e dipeptide Boc-Abu-Sar-OBzl. The t e t r apep t ide Boc-MeLeu-Val-MeLeu-Ala- OBzl w a s synthesized from t h e r i g h t t o t h e l e f t by forming bonds 5 , 6 and 7 i n t h a t order. Subsequent coupling (bond 8) with the previously synthesized dipeptide then furnished t h e hexapeptide Boc-Abu-Sar- MeLeu-Val-MeLeu-Ala-OBzl. During incorpora- t i o n of t h e amino acid MeBmt, t h e hydroxy- and N-methylamino functions were protected i n t h e form of a dimethyloxazolidine deriva- t i v e . This isopropylidene-protecting group was r ead i ly introduced by ref luxing t h e amino acid i n acetone and has t h e advantage of avoiding epimerization of t h e amino a c i d MeBmt during peptide-bond formation. This means t h a t t h e oxazolidine r ing r e t a i n s the thermodynamically more s t a b l e t rans- configuration of subst i t uen t s during carboxy ac t iva t ion and peptide formation. On preparation, but before ac t iva t ion , t h e protected MeBmt amino ac id w a s s t a b i l i z e d by addi t ion of N-methylmorpholine (1 equiv.) . To prepare the protected heptapeptide from t h e protected MeBmt amino acid and the hexapeptide, t he dicyclohexylcarbodiimide ( D C C I ) coupling method w a s used i n presence of N-hydroxybenzotriazole ( 4 7 ) . After removal of t h e isopropylidene-protecting group (1 equiv. of 1 N HCl/MeOH), t h e f i n a l amide l inkage 1 0 w a s made by coupling Boc-D-Ala-MeLeu-MeLeu-MeVal-OH with t h e heptapeptide i n presence of N-methylmorpho- l i n e i n CH2C12 at room temperature using t h e reagent (1H-1,2,3-benzotriazol-l-yloxy)tris (dimethylamino)-phosphonium hexafluoro- phosphate (BtOP( NMe2) 3' PFZ) developed by Castro -- e t a1 (48) . After hydrolysis of t h e

CYCLOSPORINE 183

step sequence for the peptide bond formation

1 - 4 -

1 i i i T i i i i i i 0 - A l o HeLeu HeLeu HeVol H e 6 m t Abu Sor MeLeu Vol HeLeu A l o

8 9 10 1 1 1

H e L c u - c H c V o l ~ H e B ~ t - c A b u ~ S a r

HeLeu

0- A lo --A lo t- H rLeu-Vol --HeLeu

t

t

Scheme 2.Syniherir olcyclosporinc 1 . Bold arrows: slralrgy lor the synthesis o l the pcptidcs. For delailr see teat. Boc-rrrr- buloxycarbonyl. BzI -bcnzyl. >-OH - isopropylidcne.proleacd MeBml.


e s t e r group (NaOH a t O°C) and removal of the Boc group (CF3COOH a t - 2OoC) , t h e unprotected undecapeptide w a s cyclized at room temperature t o cyclosporine 1 (0.0002 M CH2C12, 1 equiv. BtOP(NMe); PFZ

(48) , N-methylmorpholine). The y i e l d of c r y s t a l l i n e cyclosporine w a s 62%. the mixed phosphonic anhydride method described by Wissmann and Kle iner ( 4 9 ) o r the pentafluorophenol-DCCI complex of Kovacs (50 ) t o e f f e c t cycl izat ion of t he unprotected undecapeptide , t h e y i e l d of cyclosporine could be increased t o 65%. Using t h e fragment-condensation technique described here it is now possible t o synthesize cyclosporine very e f f i c i e n t l y i n 27.5% y i e l d with respect t o t h e amino acid MeBmt. Thus, 1 . 6 g of cyclosporine can be produced s t a r t i n g from 1 g of t he amino acid MeBmt .

By using

7. Metabolism of Cyclosporine

Maurer e t a1 (52) have reported t h e disposi t ion of cyclosporine i n man and several animal species and a l s o s t r u c t u r a l e lucidat ion of i t s metabolites. The metabo- l i t e s were i so l a t ed from dog and human ur ine and from rat b i l e and faeces. The s t ruc tu res of t h e i d e n t i f i e d metabolites were used t o e s t a b l i s h t h e biotransformation processes. 3H-labeled cyclosporine with spec i f i c a c t i v i t i e s ranging from 25 t o 80 uCi/mg w a s prepared biosynthet ical ly by feeding submerged cu l tu re s of a selected s t ra in of t h e fungus r. inflatum G a m s with [methyl-H3] methionine as labeled precursor ( 3 1 ) . The radiochemical pu r i ty of t he labeled substance w a s b e t t e r than 95%. incorporated tritium t o be located i n t h e seven N- methyl groups and i n t h e methyl group at posi t ion 4 (C,) of amino acid (Fig. 11).

The separation of metabolites was performed by HPLC. Nine ether-extractables of cyclosporine were i s o l a t e d from urine of dog and man and from rat b i l e and faeces (Fig. 12).

3H-NMR analysis revealed t h e

H

c 0

r)

.. 1)

F i g . 11. Site and type of biotransformation in cyclosporine


Fig . 12. Structure of cyclosDorine and i t s metabolites .

co 1.u 0 . . I & A 8 0 b A I

h - R 2 I I

I 6 .., $ ... R - - - C t i z - : t i * . *

cl'I CkI,-N , ,, II . . I A I ... f

O C . CH-N- CO - C H - N - C ... C H - N - i - CH - N - C O - CH I I I b I I 1 I

I C H j 1.i CH, 0 CI.C, CII, ,C!l CHI

C n , C l i , . I ......................

CI(,/: 'cn, CH+ CH,

*3

----- - V e t ~ b o ; i t e R R l R2 R l R 4 Other modi- Ho I ccul a r h o . f i r r t i on ueiqht

Cyclosporinc n tj CH, n M 1202 . C 4

--

- I 011 t i cti, h H I 2 I 0 . 6 ~

1! On OH CH, n n 9 081 n n n or.

- 10 011 It CH] OH n 16 on n CH) n on

1214 .64

1220.62 1234.64

1214.64 I 1 H of( Cn, n n 1218.64

/9

z c r .

- - I n n oti ciil n II CH ch-cnZ A ~ I 1218.64

?! H I I n II t i 1188.61

CYCLOSPORINE 187

Despite t h e complex s t r u c t u r e of cyc lospor ine , t h e r eac t ions involved i n t h e biodegradat ion of t h e molecule are l i m i t e d and produce only a r e l a t i v e l y small number of metabol i tes . The r e s u l t i n g products a r e l i p o p h i l i c as ind ica t ed by t h e i r occurrence i n t h e e t h e r phase o f t h e l i qu id - l iqu id e x t r a c t i o n .

S i t e and type of b io t ransformat ion i n cyclosporine are summarized i n Fig. 11. The c y c l i c o l igopept ide s t r u c t u r e of cyclosporine i s preserved i n a l l i d e n t i f i e d meta- b o l i t e s . Metabolism mainly c o n s i s t s o f ox ida t ive processes (phase I r e a c t i o n s ) . Typical phase I1 r eac t ions l i k e conjugat ion with s u l f u r i c a c i d or glu- curonic ac id could not be de t ec t ed . Only a l i m i t e d number o f t h e cyclosporine subuni t s , namely fou r of 11 amino a c i d s , undergo r eg iospec i f i c ox ida t ive modi- f i c a t i o n s . Hydroxylation r e a c t i o n s appear t o be r e s t r i c t e d t o t h e rl-position of amino ac id 1 (Cg-amino a c i d ) and t h e y-posi t ion o f t h e N-methylleucines 4, 6 , and 9. Oxidative N-demethylation, so f a r , seems t o occur only on t h e N-methylleucine 4. The N-methylleu- c i n e s 6 and 9 a r e sub jec t of a s i n g l e r e a c t i o n (hydroxyla t ion) whereas amino a c i d 1 and t h e N-methyl- l euc ine 4 are s u b s t r a t e s f o r two types of transforma- t i o n : hydroxylat ion and in t ramolecular e t h e r formation o r hydroxylat ion and N-dernethylation.

The proposed pathways f o r t h e b io t ransformat ion of cyc lospor ine (F ig . 13) are based on t h e s t r u c t u r e o f t h e i s o l a t e d me tabo l i t e s . The regioisomeric mono- hydroxylated cyclosporines 1 and 17 and t h e N-demethy- l a t e d cyclosporine 2 1 are primary metabol i tes . Metabol i tes 1 and 17 r e s u l t from hydroxylat ion of e i t h e r t h e N-methylleucine 9 at t h e y-posi t ion or t h e amino a c i d 1 a t t h e q-pos i t ion . Metabol i te 21 r ep resen t s the product o f N-demethylation on t h e N-methylleucine 4 . b o l i t e l on e i t h e r one of t h e N-methylleucines 4 and 6 o r on t h e Cg-amino ac id 1, and o f metabol i te 17 on t h e N-methylleucine 9 genera tes dihydroxylated d e r i v a t i v e s of cyclosporine (me tabo l i t e s 8 , 1 0 , and 1 6 ) . i d e n t i f i c a t i o n of a dihydroxylated and N-demethylated d e r i v a t i v e of cyclosporine (metabol i te 9 ) i n d i c a t e s f u r t h e r ox ida t ion t o occur on e i t h e r t h e dihydroxylated metabol i te 16 by N-demethylation of t h e N-methylleucine 4 o r t h e primary N-demethylcyclosporine 2 1 by hydroxy- l a t i o n of bo th N-methylleucines 6 and 9.

Fur ther ox ida t ion o f meta-

The

Fig.

8

t

10

'En,

I

t

21

13. Proposed pathways for the biotransformation of cyclosporine.

CYCLOSPORINE 189

Metabolite 18 , containing a cyclic ether moiety (tetrahydrofuran), may be derived from 17 (monohydroxycyclo- sporine) by intramolecular addition of the 6-hydroxyl group to the double bond of the C9-amino acid 1. This cyclization is suggested to proceed by a nonenzymatic mechanism. An artifact with a similar cyclic ether structure has previsouly been observed among the acid hydrolysis products of cyclosporine.

Three other metabolites possessed the basic cyclic structure of the parent drug. The spectroscopic data of two of them were compatible with hydroxylated and N-demethylated derivatives of cyclosporine, whereas those of the third indicated a dihydroxylated derivative. Their structures remain incompletely determined.

It should be noted that metabolites 10 and 16 were not found in man (53).

8. Pharmacokinetics of cyclosporine

The absorption, distribution, metabolism and elimination of cyclosporine have been investigated by many authors ( 52-57) - The pharmacokinetic data on cyclosporine in man as follows :

Absorption

Ab solute or a1 bi oavai labi 1 it y

Plasma peak concentration time

Di st ri but ion

Binding to plasma proteins

Apparent volume of distribution

Metabolism

Extent of metabolism

Number of components

20-50% (mean 34%)

1-6 hours

go% (20OC)

3.5 1/Kg

Ca. 99%

10-12(9 of which have been characterised as metabolites).

MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

Eliminat ion

Terminal e l imina t ion h a l f - l i f e 14-26 hours

Tota l body c learance 0.27-0.47 l/h/Kg

because of i t s very low wa te r - so lub i l i t y , t h e o r a l dose form of cyclosporine i n an ol ive-oi l -based so lu t ion . The absolu te o r a l b i o a v a i l a b i l i t y from t h i s s o l u t i o n ( i n s teady s t a t e ) i s 20-50% (mean 34%).

I n whole blood, about 50% of cyclosporine i s taken up by ery throcytes and 10-20% by leucocytes . amount of drug i n t h e plasma, approximately 90% i s protein-bound, mostly t o l i p o p r o t e i n s .

I n animal experiments using3H-cyclosporine, t h e h ighes t t i s s u e concent ra t ions of r a d i o a c t i v i t y were found i n l i v e r , kidney, ad rena l s , pancreas , thymus, t hy ro id and r e n a l f a t . From blood concentration-time da ta fol lowing intravenous adminis t ra t ion i n man, t h e drug was found t o be d i s t r i b u t e d according t o a three-compartment d i s t r i b u t i o n model. The t o t a l apparent volume of d i s t r i b u t i o n was 3.51/Kgm.

O f t h e t o t a l

Cyclosporine i s slowly but ex tens ive ly metabolised by l i v e r . I n addi t ion t o t h e parent drug, 10-12 meta- b o l i t e s have been de tec t ed i n human plasma. Nine meta- b o l i t e s have been i s o l a t e d and i d e n t i f i e d ; a l l contained t h e i n t a c t cyc l i c o l igopept ide s t r u c t u r e of t h e parent molecule. S t r u c t u r a l modif icat ions cons i s t ed of mono- and di-hydroxylation and N-demethylation at var ious pos i t i ons on t h e cyclosporine.

Terminal e l imina t ion ha l f - l i ves from blood o r plasma of 14-26 hours have been reported.

The drug and i t s metabol i tes are excre ted mainly via t h e l i v e r and only t o a s l i g h t ex ten t v i a t h e kidneys. I n man t h e t o t a l u r ina ry excre t ion of r a d i o a c t i v i t y i n t h e per iod up t o 96 hours fol lowing a s i n g l e dose of 3H-cyclosporine w a s 6% of t h e dose; unchanged cyclospor ine accounted fo r on ly 0.1% o f t h e or& dose,

CYCLOSPORINE 191

9 . Pharmacological Activity and Clinical Application

As reported by Bore1 et a1 (5-9), cyclosporine is a selective immunosuppressive drug with antifungal and antiinflammatory properties. It has been shown to strongly suppress the production of haemagglutinating antibodies against sheep erythrocytes in mice and to be effective in inhibiting both humoral and cell- mediated immune responses. Its effect is reversible, and specific for T-lymphocytes. The hemopoietic tissues in immunosuppressed animals are not affected. Bueding _ _ et a1 (58) demonstrated an antishistosomal activity and Thommen-Scott ( 59) an antimalarial effect of cyclosporine, both antiparasitic activities being independent of immunosuppression. The first trans- plantation in humans with the aid of cyclosporine were reported in 1978 by Calne et a1 (60) f o r kidney and by F’owles et a1 (61) f o r bone marrow. Today cyclosporine is used successfully to prevent graft rejection following bone marrow and organ transplantations.

Other possible applications, such as in the treatment of autoirnune and other diseases, are currently under investigations.

10. Pharmaceutical Forms

Cyclosporine ( Sandimmun) is available as :

a) Drinking Solution: 100 mg/ml cyclosporine dissolved in olive oil and labrafil.

b) Intravenous Preparation: Ampoules of 5 m l (250 mg cyclosporine) and 1 ml (50 mg cyclosporine).

11.Methods of Analysis


Cyclosporine (C H N 0 ) : 62 111 11 12

C H N 0

61.90 9-30 12.80 16.00


11.2 Introduction to Analysis

Several methods have been developed for the analysis of cyclosporine in plasma and or blood. The first method reported was the radioimmunoassay (RIA) of Donatsch (62) which is now available as a kit and has been used extensively during the clinical development for cyclosporine. The method was initially developed for plasma, and as shown later, is also applicable to blood samples. The main criticism of the RIA method has been the cross-reactivity of the antisera with some of the circulatinc metabolites of cyclosporine. Other RIA procedures were reported ( 54,55, 63-67).

Subsequent to the introduction of the RIA method, Niederberger et a1 (68) developed a high pressure liquid chromatogri:phy ( HPLC) method based on gradient elution. The technical complexity of this method encouraged several investigators , (69-75) to reinvestigate the method. The results of these activities are shown in Table 4.

11.3 Radioimmunoassay ( RIA)

Several radioimmunoassay procedures have been developed for both the analysis and immunopharmacological monitoring of cyclosporine ( 54,55, 63-67) - One method described recently by Randall et a1 (64) is as follows:

Sample Preparation: Venous blood was collected in heparinized tubes from 38 post-transplant patients receiving cyclosporine, just before their next dose and 12 h after their last.

Non-temperature-standardized protocol: About 0.5 to 3 h after collection, an aliquot of each sample f o r whole-blood analysis was set aside and centrifuged the remaining specimen at room temperature to remove cells. The plasma and whole-blood specimens were both then stored at - 20%.

CYCLOSPORINE 193

Temperature-standardized pro tocol : After co l l ec - t i o n , samples were r e f r i g e r a t e d for no longer than 4 h, r e -equ i l ib ra t ed t o 37OC by incubat ion i n a water ba th f o r 30 min, then cen t r i fuged without delay (1100 X g , 10 min, room temperature) The r e s u l t i n g plasma w a s s to red frozen a t -2OOC u n t i l a n a l y s i s .

Time and temperature experiments: Aliquots of whole blood from p a t i e n t s r ece iv ing cyc lospor ine

were incubated for 0.25, 0.5, 1, 2, 4 , 6, and 24 h at 4'12, room temperature (about 22OC), and 37OC. A t t h e s e s p e c i f i e d t i m e s , samples s t o r e d a t room temperature or 4°C were cen t r i fuged a t room temperature; t hose s to red a t 3 7 O C were cent r i fuged a t 37OC and t h e plasma was decanted and frozen.

I n add i t ion , t h e t r e a t e d a l i q u o t s of samples s t o r e d a t room temperature and 4 O C f o r t h e above t i m e s by r e -equ i l ib ra t ing t o 3 7 O C by incubat ing for 1 5 min i n a 37OC w a t e r ba th , followed by c e n t r i f u g a t i o n at 37OC and prompt removal of t h e plasma.

Equ i l ib ra t ion time experiment: Al iquots o f whole blood from p a t i e n t s r ece iv ing cyc lospor ine

w e r e maintained a t 4 O C f o r 4 h af ter co l lec- t i o n , t hen e q u i l i b r a t e d i n a 37OC water ba th for 0, 10, 1 5 , 30, 60, 120, and 180 min. All specimens were cent r i fuged at 3 7 O C , and t h e plasma was immediately decanted and stored at -2OOC u n t i l ana lys i s .

Measurement of cyclosporine: Cyclosporine w a s measured by radioimmunoassay, using t r i t i a t e d cyc lospor ine as t r a c e r and an ant ibody i n a k i t suppl ied by Sandoz Ltd . , Bas le , Switzer land. Dupl ica te 5 pL p a t i e n t ' s samples were mixed wi th 100 pL of t r a c e r , T O O 1J.L o f T r i s bu f fe r ( 0 . 5 mol/L, p H 8 . 5 ) , and 100 UL o f sheep ant iserum t o cyc lospor ine , then incubat ing for 2 h a t room temperature . Free and bound f r a c t i o n s were separa ted by e x t r a c t i o n wi th charcoa l for min at 4 O C . The r a d i o a c t i v i t y was measured i n each specimen wi th a l i q u i d s c i n t i l l a t i o n

1 94 MAHMOUD M. A. HASSAN AND MOHAMMED A. AL-YAHYA

counter , us ing a quench-corrected count ing program. i n plasma and whole blood supplemented wi th t h e drug w a s about 75%.

The a n a l y t i c a l recovery of cyclosporine

Correct ion f o r hematocri t : Hematocrits of samples f o r cyclosporine a n a l y s i s were measured as p a r t o f t h e rou t ine blood count on a Coul ter S P lus (Coul te r Instruments , Hialeah, FL) . The plasma cyclosporine r e s u l t s were co r rec t ed f o r hematocrit by mul t ip ly ing t h e uncorrected plasma cyclosporine r e s u l t s by t h e pa t i en t ' s hematocrit / O .39, t h e n o m a l va lue f o r t h e hematocri t .

1 1 . 4 High Performance Liquid Chromatography

Several HPLC procedures were repor ted for t h e es t imat ion of cyclosporine i n blood and o r plasma a s w e l l as immunopharmacological moni- t o r i n g of cyclosporine ( 5 3 , 5 4 , 67, 69-75). These methods were developed t o overcome t h e drawbacks of t h e R I A methods p a r t i c u l a r l y t h e overes t imat ion of cyclosporine i n b i o l o g i c a l f l u i d s . This i s because, t h e an t ibod ie s c ros s r e a c t with t h e metabol i tes . The HPLC condi t ions used f o r t h e ana lys i s of cyclosporine a r e l i s t e d i n Table 5. A r ecen t HPLC method w a s repor ted by Kates and L a t i n i (75 ) and i s as fol lows :

Chemicals and reagents : Cyclosporine and t h e i n t e r n a l s tandard , cyclosporin D ( F i g . 14). are obtained from Sandoz (Basle, Swi tzer land) . G las s -d i s t i l l ed d i e t h y l e t h e r , a c e t o n i t r i l e , q-hexane, and methanol are obtained from Burdick and Jackson Labs. U.S.A.). Tris(hydroxymethy1)ainomethane is purhcased from Aldrich (Milwaukee, W I , U.S.A.)

(Muskegon, M I ,

Ins t rumentat ion and chromatograghic condi t ions : A Waters Instrument (Mi l ford , MA, U.S.A.) Model 6 0 0 0 ~ HPLC pump, Model 480 var iab le- wavelength UV d e t e c t o r , Model 7lOB sample i n j e c t o r and Model 730 d a t a module a r e employed. The column used i s a Brown-Lee Labs. (San ta Clara, CA, U.S.A.) RP-8 MLPC a n a l y t i c a l c a r t r i d g e

CY CLOSPORINE 195

(10 ern X 4.6 mm I.D.; p a r t i c l e s i z e 10 m). A 3-em RP-8 guard c a r t r i d g e i s pos i t i oned a t t h e head of t h e column. The column i s maintained a t 7 O o C with an Eldex (Menlo Park, CAY U . S . A . ) column hea te r . The h e a t e r i s l e f t on a t a l l t i m e s and a cons tan t flow is maintained through t h e column. The flow-rate of t h e mobile phase i s 0.6 ml/min which produces a precolumn pressure of about 34 bars ( 500 p. s . i. ) The e f f l u e n t from t h e column i s monitored a t a wavelength of 215 nm.

Mobile phase: The mobile phase c o n s i s t s of a simple mixture of ace ton i t r i l e -wa te r ( 72:28). This mixture i s f i l t e r e d and degassed by vacuum and son ica t ion . The mobile phase i s cont inuously recyc lsed and rep laced about every two weeks.

Prepara t ion o f e x t r a c t i o n columns: The ex t rac- t i o n o f cyclosporine involves t h e use o f a Baker-10 SPE e x t r a c t i o n system ( J . T . Baker, Ph i l l i p sburg , N J , U.S.A.). The columns used are t h e 3 ml cyano d isposable e x t r a c t i o n columns. These columns a r e prepared by washing wi th 6 ml of methanol and then 6 ml of water under vacuum. They a r e l e f t approximately one h a l f t o t h r e e q u a r t e r s f u l l of w a t e r u n t i l t h e sample i s added.

Ext rac t ion procedure: One ml of whole blood or serum i s added t o PTFE-lined, screw-capped tubes . To t h e blood or serum i s added 450 ng of i n t e r n a l s tandard , 3 ml of 0 . 1 M T r i s b u f f e r (pH 9 .8) and 10 m l o f d i e t h y l e t h e r . They are rocked f o r 20 min on a labquake rocker (Lab. I n d u s t r i e s , Berkeley, CA, U.S.A.) and c e n t r i - fuged for 20 min t o sepa ra t e t h e e t h e r and aqueous l a y e r s . The d i e t h y l e t h e r i s p i p e t t e d i n t o a c lean tube and t h e blood or serum residue i s d iscarded . To t h e d i e t h y l e t h e r i s added 200 p1 of 75% methanol i n water . The d i e t h y l e t h e r i s then evaporated at room tempera- t u r e wi th a g e n t l e stream of n i t rogen . Only t h e d i e t h y l e t h e r i s evaporated, l eav ing 150- 200 p1 of t h e methanol-water remaining. To t h i s are added another 100 u1 of methanol. The


methanol-water i s then t r a n s f e r r e d t o a 3 m l Baker ex t r ac t ion column, The r e s idue i s e l u t e d onto t h e column wi th water. The column is then washed wi th 3 ml of 25% a c e t o n i t r i l e i n water and 6 m l of n-hexane. The columns a r e then d r i e d by drawing through a i r for 4 min. Cyclosporine and cyclosporin D are then d lu t ed off t h e column wi th t h r e e washings o f 200 p l of methanol. The methanol i s c o l l e c t e d i n t o disposable tubes and evaporated t o dryness wi th a stream of ni t rogen a t 5OoC. The r e s u l t i n g r e s idue i s d isso lved i n 200 p 1 of t h e mobile phase. An a l iquo t of 100 p 1 i s then i n j e c t e d onto t h e column ( F i g . 1 4 ) .

Prepara t ion of c a l i b r a t i o n s tandards : Stock s o l u t i o n s of cyclosporine and cyclosporin D are prepared i n methanol and s to red i n amber b o t t l e s at room temperature . A s tock s o l u t i o n of cyclosporin D i s prepared i n a concent ra t ion of 30 ng/ul . The s tock s o l u t i o n of cyclospor ine i s prepared i n a concent ra t ion of 10 ng/pl . This so lu t ion i s used t o prepare s tandards for t h e c a l i b r a t i o n curve. The c a l i b r a t i o n curve samples are prepared by adding amounts of cyclospor ine (50-800 ng) t o whole blood or serum from a normal volunteer . These are then t r e a t e d as descr ibed i n t h e e x t r a c t i o n procedure. This procedure has a lower l i m i t of s e n s i t i v i t y below 50 ng/ml.

d I n 0

C

71 I

11

\ L Fig . 14. Chromatograms of extracted whole blood samples. ( A ) Blood from normal healthy

volunteer, not taking cyclosporine; (B) blood to which has been added 200 ng/rnl of cyclosporine and the internal standard; (C) blood from a patient who was taking cyclosporine ( the concentration in this patient sample is 304 nglml). Peaks: I = cyclosporine; I1 = cyclosporin D, internal standard.

Table 4. Comparison o f publ ished methods o f a n a l y s i s o f cyclosporine

* %

ana lys i s

HPLC-Isocr . HPLC-Isocr.

HPLC-Col . Swit

HPLC-Isocr . HPLC-Isocr.

HPLC-Grad.

HPLC-Isocr.

HPLC-Col .Swit

HPLC-Isocr .

RIA**

HPLC-Grad.

Sample type

Blood/plasma

Plasma

Serum

Blood/plasma

Blood/plasma

P l a s m a

Blood/plasma

Serum

Plasma

3loodlplasma

Sample* prepara t ion

Long

Long

Very long

Short

Long

Long

Long

Long

ihort s emiaut oma-

t e d )

Long

Detect ion l i m i t

p g / l i t r e )

1-20

20

100

25

20

100

100

30-50

31

8

50

S p e c i f i c i t y

Metabol i te x-react i v i t y

High

-

High

High

High

High

High

High

High

High

S t an dar- d i z a t i on

~

Ext . I n t . Ext . I n t . I n t . I n t . I n t . I n t . I n t . Ext .

I n t .

Chromat o-

Time( min)

-

30

5 1 0

30

20

45

1 5

30

1 5

19

R e f .

54955, 63-67

68

69

70

71

72

63

73

74

67

75

"Short: p r o t e i n p r e c i p i t a t i o n and i n j e c t i o n ; Long: e x t r a c t i o n & evaporat ion; Very Long: a d d i t i o n a l sample manipulat ion.

**RIA: Radioimmunoassay. Ex:. = .Externa l ; I n t . = I n t e r n a l ; Grad. = Gradient ; I s o c r . = I s o c r a t i c ; Col-Swit . = Column switching.

Table 5 . HPLC condi t ions used f o r t h e a n a l y s i s of cyclosporine

Sample Instrument

Blood/snrum Waters Model 6 0 0 0 ~

,Blood/ 1 Technicon

RP-8 YLPC a n a l y t i - c a l c a r t r i d g e , 10 cm x 4.6 mm I . D . 3-em RP-8

b lood/

~

plasma

c

A c e t o n i t r i l e l water 72:28

Altex pumps

UV de tec to r LCD 725 Uvi- kon . Recorder Tarkan 600 W+W . Altex m i croproc es- sor 420.

AX-110.

1

Column Mobile phase

I

guard c a r t r i d g e , at 7OoC.

Flow ra te

m l /min

0.6

LC-8 Supe lcos i l , 5 urn, supelco, Inc . , Be l l e fon te , PA., a t 7 5 O c .

Acet o n i t r i l e / water 55:45 v/v

3.0

I I

Column I; Altex pe r i so rb RP-8 , 40 X 4.6 mm I . D . , 30-40 urn. Column 11; Lichro- sorb RP-18, 150 X 4.6 mm I . D . , 5 urn, a t 70Oc. Al te rna t ive column; Beckman Ul t rasphere O D s , 150 X 4.6 mm I . D . , Guard column; Altex pe r i so rb RP-2 40 X 3.2 mm I . D . , 30-40 urn.

Cons is t s of 6 solvent s. 1. A c e t o n i t r i l e 1

methanollwat er 35 : 20 : 45 v/v/v

water 55:45 v/v

water 72:28 vlv

2 . A c e t o n i t r i l e 1

3. A c e t o n i t r i l e l

4. Tetrahydrofu-

5 - A c e t o n i t r i l e /

6. Methanol

r an

water 90110 v/v

1 - 7

Tetent ior time

(mins)

9.2

Detect ion

UV a t 215 nm

UV at 202 nm

W a t 210 nm

Table 5.

Sample

Serum

Serum

( Continued)

1 I Instrument

V a r i a n V i s - t a HPLC system.

Beckman ul t rasphere ODs, 25 X 0.46 cm, 5 um. Precolumn Vydac C18, 4 X 0.4 cm. Both columns a t 7OoC.

I Waters sys- Beckman ultrasphere- t e m con- t r o l l e d with mm, M-660 so l - 5 pm. vent pro- Maintained a t 72Oc. grammer .

oc ty l , 25 cm X 4.6

Flow Mobile phase r a t e

(ml/min

S t a r t i n g with 1 tri fluoroacet i c a c i d l a c e t o n i t r i l e 35:65 and increasing proport ions u n t i l 5:95 a t 1 5 mins.

Ace ton i t r i l e / 1 - 5 methanol / w a t er 47 : 20 : 33 by Vol .

Retention

(mins ) time Detect ion

1 4 . 1 IUV at

20.46 UV a t and 210 nm

3ef

73

-

74

CYCLOSPORINE 201

12.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

References

W. G a m s , Persoonia,6, - 185 (1971).

M. Dreyf'uss, E. Harri, H. Hofhan, H . Kopel, W. Pache, H. Tscherter , Eur. J. Appl. Microbiol., 3, 125 (1976).

J .F. Borel: I n White D J G ( e d ) : Cyclosporin A. New York, Elsevier Biomedical, 1982, p. 5 .

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-

Actions, 6, 468 (1976) .

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17

18.

19 *

20.

21.

22.

23.

24.

25 *

26.

27 *

28.

29 -

30.

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M.R. Bendal l , W.E. Hu l l , 'DEPT', brochure by Bruker Analyt ic , Karlsruhe , 1982.

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CYCLOSPORINE 203

32.

33.

34.

35.

36.

37.

38.

39.

40.

41 .

42.

43.

44 .

45.

46.

47

48.

R . Zocher, H . Kleinkauf , Biochem. Biophys. Res. Commun., - 81, 1162 (1978).

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R . Wenger, Transplan ta t ion Proceedings - 1 5 (4-Suppl. 1) , 2230 (1983).

R . Wenger, Helv. Chim. Acta, - 66, 2308

R. Wenger, Helv. Chim. Acta 66, 2672

R . Wenger, Angew. Chem. I n t . Ed. Engl (1985 .

(1983).

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R.S. Cahn, C . Ingold, V. P re log , Angew. Chem. - 78, 413 ( 1966 1 ,

D. Seebach, E . Hungerbuhler i n R . Scheffold, Modern Synthe t ic Methods, S a l l e & Sauerlander , Frankfur t / Aarau 1980, p. 93.

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49.

50 -

51.

52 *

53.

54.

55 *

56.

57.

58.

59.

60.

61.

H. Wissmann, H . J . Kle iner , Angew. Chem., 92, 129 (1980); Angew. Chem. I n t . Ed. Engl . , 9, 133 (1980) .

J. Kovacs "Rcemization and Coupling N -pro tec ted Amino Acid and Pept ide Active Es t e r s " , i n t h e 'Pept ides ' eds. E. Gross and J Meinenhofer, Vol. 2 , Academic P res s , New York-London, 1980, p. 48s.

a

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CYCLOSPORINE 205

62,

63.

64.

65.

66.

67.

68.

69.

70 .

71 *

72.

73.

74.

75 .

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13. Acknowledgement

The authors would like to express their sincere gratitude to Mr. Abdalla I. Babikir of the Medicinal Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia for his continuous and indispensable assistance throughout the preparation of this profile.

ENALAPRIL MALEATE

Dominic P. Ip and Gerald S . Brenner

1. History and Therapeutic Properties

2. Description

2 - 1 Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 CAS Registry Number

2.2 Formula and Molecular Weight 2.3 Appearance, Color, Odor

3 . Synthesis


4.1 infrared Spectrum

::; 1% - Nuclear Magnetic Resonance Spectrum 4 - 4 Ultraviolet Spectrum 4.5 Mass Spectrum 4.6 Optical Rotation 4.7 Thermal Behavior 4.8 Solubility 4.9 Crystal Properties 4.10 Dissociation Constants 4.11 Partition Behavior 4.12 Confocmational Isomers

- Nuclear Magnetic Resonance Spectrum



208 DOMINIC P. IP AND GERALD S. BRENNER


5.1 Elemental Analysis 5.2 Colorimetric/Spectrophotometric 5.3 Chromatographic

5.3.1 Thin-Layer Chromatography 5.3.2 High Performance Liquid Chromatography

5.4 Identification Tests 5.5 Titration

6. Stability and Degradation

6.1 Solid State Stability 6.2 Solution Stability

7. Pharmacokinetics and Metabolism

8. Determination in Biological Fluids

9. References

ENALAPRIL MALEATE 209

1. History and Therapeutic Properties

Enalapril is the ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. Enala- pril maleate, discovered and developed by the Merck Sharp and Dohme Research Laboratories (l), is indicated for the treatment of essential and renovascular hypertension. Enalapril is a pro-drug; following oral administration, it is bioactivated by hydrolysis of the ethyl ester to enalaprilat, which is the active angiotensin converting enzyme inhibitor.

CH3

COOH

I e C H 2 C H 2 y H N H C H -CO -N

COOCHPCH3

Enalapril

I CH3

COOH

I CHZCHzCHNHCH - CO - N

COOH

Enalaprilat

Since the oral absorption of enalapril is superior to that of enalaprilat, it is the compound that is used in oral dosage forms. Enalaprilat, the deesterified parent diacid of enalapril is the form of the drug that is administered intravenously. Several review articles give a detailed account of the history, design, chernis- try and pharmacology of the drug. (2-6).

2. Description

2.1 Nomenclature

2.1.1 Chemical Name

( S ) -1- [N- [ 1- ( ethoxycarbonyl ) -3- phenylpropyll -Galany11 ->proline, (2)-2- ixltenedioate(1:l)salt


2.1.2

2.1.3

2.1.4

2.1.5

Generic Name

Enalapril Maleate

Laboratory Codes

G154,739-001D, MK-0421

Trade Names

Vasotec, Renitec, Tnnovace, Pres, Xanef

CAS Registry Number

76095-16-4

2.2 Formula and Molecular Weight

mol wt 492.53

2.3 Appearance, Color, Odor

Enalapril maleate is a white to off-white, crystalline, odorless powder

3. Synthesis

Enalapril maleate has been prepared by the scheme outlined in Figure 1 (7 ) . Ethyl 2-0x0-4-phenylbutanoate - (1) is condensed with L-alanyl-L-proline (2) to give Schiff base 3, as a mixture of syn and anti isomers. Reduction of the imine bond in 3 results in formation of the diastereomeric mixture 4 (SSS and RSS). The more biologically active diastereomer (SSS) is isolated

z

t T

z I CU

+

o=

0,

0, I

I

a

f

a A"

0" 5

3'

-l-

0" 5

aJ c1 m

aJ rl m

c

I4

.A

I4

m e w

w

0

tn aJ c U

C

h

v)


by fractional crystallization of the maleate salts to yield enalapril maleate (5) of greater than 99% purity in 32 - 34% overall yield. tic route, others have also been described in the literature (1, 8, 9).

In addition to this synthe-


4.1 Infrared Spectrum

The infrared spectrum is shown in Figure 2 (10). The spectrum was obtained as a potassium bromide pellet using a Perkin-Elmer Model 281B spectrophotometer. Assignments for the characteristic bands in the spectrum are listed in Table I.

Table I

Wavenumber, an-’

3100-3010 2970 and 2920

2800-2200

1750 1727 1645 1570

(Broad, structured)

1500-1390 1375 1355 1187 750

695

Assignment

Aromatic C-H Stretch Methyl and methylene C-H asymmetric stretch / \NH$ Stretch

Acid carbonyl stretch Ester carbonyl stretch Amide carbonyl stretch Monohydrogen maleate

Mainly methylene bending Methyl symnetric bending Methylene bending Ester C-0 stretch In-phase, out-of-plane hydrogen bending (5- adjacent aromatic hydrogens)

bending

carboxyl stretch

Phenyl out-of-plane ring

4.2 ’H-Nuclear Magnetic Resonance Spectrum

The proton magnetic resonance spectrum is shown in Figure 3 (11). Varian Associates Model SC300 spectrometer at a concentration of 0.55% w/v in deuterium oxide (D20).

The spectrum was obtained using a

Active protons exchange with D20 in

10 N

E

B b

0

8 L2 s 0

0

0

0

Li 0

0

9 ? F E 5: 0 0

0 0

0 0

0

N

0 0 0

rr)

0

lo 5:

-m

-m

F i g u r e 3 . The Pro ton Magnetic Resonance Spectrum of E n a l a p r i l Maleate


solution and appear in the HOD signal at approximately 4.8-4.9 ppm. expanded scale spectrum which mre clearly illustrates the signals pattern.

Figure 4 shows an

For several protons in the molecule, slow rotation about the proline amide C-N bond gives rise to paired signals. The phenomena has been attributed to the existence of rotational conformers as in the case of captopril in aqueous solution (12 ) . A tabulation of the chemical shifts and assignments for the numbered structure below is shown in Table 11. The notations 'major' and 'minor' denote the relative abundance of the two rotamers relative to each other in the spectrum.

H\ /COOH C I I

m o H/'\COOH

Table I1

hemical Shift '€I, 6 (ppm) Multiplicity

1.30 t 1.54 d

1.59 d

1.75 m

2.02 m

2.02 m

2.33 m

2.33 2.80 3.60 3.95 3.97 4.27 4.09

Assignment

CE3CH 0- Alanyl C% (minor )

Alanyl C%

Proline C-4 proton (minor )

Proline C-4 proton (major )

Proline C-3 protons (minor )

Proline C-3 protons (major )

C-3 ' protons C-4' protons Proline C-5 protons C-2' proton (minor) C-2' proton (major)

'a proton (minor)

(major 1

CH3CZ20-

--csr I

6.0

3.0 PPM 2.0 1.2 4.0

Figure 4 . The Proton Magnetic Resonance Expanded Spectrum of Enalapril Maleate


hemical Shift 'H, 6 (ppm) Multiplicity Ass i gnmen t

9 'a proton (major) 4 . 3 3

6 . 3 3

7 .37 7 .41

S - H @ L'C02H

Aryl protons - o & p m Aryl protons - m m

4 . 3 13C Nuclear Magnetic Resonance Spectrum

The l3C magnetic resonance spectrum shown in Figure 5 was run using a Varian Associate Model XL- 200 spectrometer at a concentration of 8 .4% w/v in deuterated methanol (13). The internal standard was tetramethylsilane. acquisition time was 0.9 s.

The pulse width was 4 ps,

The assignments for the numbered structure below are shown in Table 111. As discussed above, the two rotamers are designated 'major' and 'minor' in order of their relative abundances.

v

160 PPM

50

Figure 5. The Carbon-13 Magnetic Resonance Spectrum of Enalapril Maleate


Table 111

Chemical 13c, 6 (ppm)

16.2 17.4 18.1

24 -9 27.7 31.8 33.7 35.0 35.2

49.8

57.9 58.4 61.7 61.8 62.3 62.4 65.6 65.7

129.3

131.2 131.4

138.0 142.9

170.5 171.2 171.6 172.0 172.4 176.5 176.7

Assignment

iE:$2H3 (major) Alanyl ?[I3 - (minor)

The signals at 24.9-35.2 p p m arise from the four carbons - C-3', C-4' and C-4, C-3 proline including signals corresponding to rotamers. An exact assignment of the signals in this region was not made.

Proline C5

The signals at 57.9-65.7 ppm arise from the four carbons - Proline C-2', Alanyl C-a, C-2' and -gH2CH3 including s igna Is attributable to rotamers. An exact assignmnt of the signals in this region was not made.

Phenyl C-4"

Phenyl C-2" , C-6" , and phenyl c-3" , c-5"

Naleate -HcSH- Phenyl C-1"

The signals at 170.5-176.7 arise from alanyl, proline, ester and maleate carbonyl carbons. However, defini- tive assignments were not made for the carbonyl carbons.

Carbon-13 NMR may also distinguish enalapril mleate and 'ts RSS diastereomer. Major differences between between 31.8 and 34.0 ppm, between 54.7 and 60.8

spectra of the two compounds occur


ppm and between 168.6 and 170.7 ppm. this technique is not preferred for accurate measurement of small quantities of either compound in the other.

However,

4.4 Ultraviolet Spectrum

Enalapril maleate is characterized by inflections or "shoulders" at about 251 and 263 y%and barely discernible maxima at abut 21J nm (A1 cm, about 15) and abut 267 nm (A1 cm, about 8.6). spectra of enalapril maleate in N/10 methanolic hydrochloric acid at concentrations of 0.991 mg/ml and 0.0991 mg/ml.

The low intensity and lack of well-defined maxima in the ultraviolet absorption spectrum of enalapril maleate, typical of an unconjugated phenyl moiety, does not make it useful for characteriza- tion or quantitation of the compound.

Figure 6 shows the ultraviolet absorption

4.5 Mass Spectrum

The direct probe electron impact (EI) mass spectrum of enalapril maleate as shown in Figure 7 was obtained on the VG MM7035'mass spectrometer employing 70 eV electron energy and a probe temperature of 16OoC (14).

Under the condition of analysis the maleic acid moiety is pumped off before the peptide derivative is vaporized. Molecular ion information for enalapril base is also absent in the spectrum obtained by direct probe EI at 70 eV and 20 eV, respectively. Such information is only possible when a gross sample overload is employed. Under field desorption (FD)/MS however, good molecular ion and pseudomolecular ion [M+H]+ formation (376, 377) is observed (Figure 8 ) .

The low resolution EI 70 eV spectrum (Figure 7) shows a base peak at m/e at 91 (benzylic ion), [M- H20] atm/e 358 and other major fragment ions. Accurate mass measurements (high resolution, 70 eV, 16OOC) of the diagnostic fragment ions at m/e 358, 313, 285, 257, 254, 234, 208,

c

2

1

al 0

0

c a u1 *

1

C

\ 220 240 260 280 300 320 340 220 240 260 280 300 320 340

Wavelength (nm I

F igure 6. The U l t r a v i o l e t Absorption Spectrum of E n a l a p r i l Maleate i n (a ) N / 1 0 Methanolic Hydrochlor ic Acid; Concentrat ion: 0.991 mg/ml and (b) N i l 0 Methanolie Hydrochlor ic Acid; Concentrat ion: 0 . ~ 9 9 1 mg/ml

80

)r

u) c 0)

U

x 0 0)

c .-

60-

a

k

-

50

'T' 150 250

Mass

350

Figure 7. The Low Resolu t ion (EI) Mass Spectrum of Enalapril Maleate

223

F i g u r e 8 . The F i e l d D e s o r p t i o n (FD) Mass Spectrum of E n a l a p r i l Maleate


169, 168, 160, 126, 9 1 and 70 facilitated a proposed fragmention pattern as shown in Figure 9.

4.6 Optical Rotation

The optical rotation, of enalapril maleate (three asymnetric centers) in methanol is, -42.3' (c=l%).

4.7 Thermal Behavior

Enalapril maleate exhibits a single melting endotherm by differential thermal analysis (DTA) under a stream of nitrogen. The peak temperature of the endotherm varies with heating rate, indicating accompanying decompostion during melting of the conpound (- 14OOC at 2OC/min. and - 149OC at 2OoC/ min.) on a DuPont 1090 equipped with a DTA head is given in Figure 10.

A DTA curve for enalapril maleate obtained

By capillary melting point determination (USP Class Ia method) enalapril maleate melts at - 143- 144OC (15).

4.8 Solubility

The following approximate solubility data (Table IV) have been obtained at ambient temperature.

Table IV

Solvent

Water Methanol Ethanol Isopropanol Ace tone Acetonitrile Dime thylf ormamide Chloroform Diethyl ether Hexane Polyethylene glycol

Solubility (mg/ml)

25 200

80 1.4 9.1 3.3

0.6 < 0.1 < 0.1

300 < 0.1

>400

t

4

\" 3

+5 I

+*

r

Figure 9. A Proposed Fragmentation Pattern to Explain the Mass Spectrum of Enalapril Maleate

-O

r N

r

od

-r

r,

(D

"" 0

(0

N

0

d- N

0

N

N

0

0

E

2

5 2

?

0

0

0

0

0

(D

0

t

ar U

m

ar 4

2 4 'rl k

a

m 4

m

G

w

u-i 0 al > k

3

u 4

m

fi k

al c

b

-4

b

n

0

ar k 3

M

'rl FL(

.-(


The solubility of enalapril maleate increases with pH. A pH versus solubility profile constructed to compare theoretical points to actual data is shown in Figure 11 (16).

4.9 Crystal Properties

Enalapril maleate is a white to off-white crystalline corrpound. The compound is polymorphic. TWO nonsolvated polylmrphs have been detected and characterized by high resolution spec oscopic techniques ( F T I R , Raman, solid state % NMR) and solution calorimetry ( 1 7 ) . However, these plymrphs, referred to as Form I and Form I1 are very similar in energy, differing only by 0.6 Kcal/ml. The X-ray powder diffraction spectra for Form I and Form I1 of enalapril maleate are shown in Figures 12 and 13, respectively. The spectra were obtained using a Philips Electronics X-ray diffractometer using CuKa irradiation.

4.10 Dissociation Constants

Aqueous acidicfiasic potentiometric titration yielded pKa values of 2.97 and 5.35 at 25OC for enalapril (18).

4.11 Partition Behavior

At room temperature, enalapril maleate does not partition out of the the aqueous media listed below into mineral oil or n-octanol (19).

Solvent Pairs n-Octanol/pH 7 buffer mineral oil/pH 3.5 buffer mineral oil/pH 4.5 buffer mineral oil/pH 5.5 buffer

4.12 Conformational Isomers

Proton NMR and carbon-13 NMR spectra of enalapril maleate are characterized by several paired signals attributed to slow rotation about the proline amide bond, resulting in a slow equilibrium between two rotational conformers (cis and trans). The two rotamers are distinguishable on the NMR

228

1000000

100000

DOMINIC P. IP AND GERALD S. BRENNER

100

10

PH

-D- Calculated Pts o Experimental Pts

Figure 1 1 . pH - Solubility Profile of Enalapril Maleate

F i g u r e 12. Powder X-Ray D i f f r a c t i o n P a t t e r n of E n a l a p r i l Maleate, Form I

In

P 9

R In

(u

In

m 0

O*

H

H

E

N

0

Fa d

.d

N a

fd rl fd c w

w 0

E al

a::

Nf

d

PI

ENALAPRIL MALEATE 23 1

time scale. In addition, HPLC has provided firm evidence for their existence by demonstrating the chromatographic behavior of enalapril maleate at various temperatures (20). Figure 14 shows chrom- tograms obtained by reverse-phase HPLC at column oven temperatures of 10°C and 80°C. At 10°C, in addition to the early eluting maleic acid peak ( - 1.2 minute), the enalapril base peak is resolved into two distinct rotamer peaks. At 8OoC, the rotamer peaks have coalesced to yield a single symme- trical peak.



Analysis of Merck Sharp & Dohme reference l o t , L-154,739-001D22 for carbon, hydrogen and nitrogen was reported as follows:

Element % Calculated % Found

C H N

58.53 58.53 6.55 6.75 5.68 5.82

5.2 Colorimetric/Spectrophotometric

Due to the lack of strong W absorption maxima, mre sensitive ways for the analysis of enalapril maleate are needed. analyzer colorimetric assay procedure (21). In this approach enalapril maleate forms a yellow dye complex with bromothymol blue in acidic aqueous, pH 2/CHC13 mixture and is then assayed spectro- photometrically at 415 nm after extraction with chloroform. This method is suitable for content uniformity and dissolution assays of enalapril maleate capsules and tablets.

One approach has been an auto-

Analysis of enalapril in pharmaceutical dosage forms via flow injection analysis has recently been reported (22). Enalapril is determined by extraction as an ion-pair with bromthyml blue in an unsegmented flow system. The procedure is stability indicating: the degradation products of enalapril and excipients in the dosage forms do not interfere.

232

10°C

c

Time (Minutes 1

DOMINIC P. IP AND GERALD S. BRENNER

80" C

a0 N : - r

Time (Minutes

Figure 14. HPLC Chromatograms of Enalapril Maleate at 10" and 80°C


5 .3 Chromatographic

5.3.1 Thin-Layer Chromatography

Three solvent systems (23) for the thin- layer chromatography of enalapril maleate are given below. enalapril base from its salt forming acid (maleic acid) and its hydrolysis product (enalaprilat - see Section 6.1)

The systems separate

Solvent System Rf (Approximate )

A (chloroform/methanol/ 0.6 (enalapril acetic acid base ) 90 :10 :1) 0.2 (maleic acid)

0 (enalaprilat)

B

acetic acid 3:l:l) (n-Butanol/water/ 0.7 (enalapril

base and maleic acid)

0.35 (enalaprilat)

Solvent System Rf (Approximate)

C (n-Butanol/toluene/ 0.55 (enalapril water/acetone/acetic base ) acid 1:l:l:l:l) 0.45 (maleic acid

and enalaprilat)

These solvent systems use a silica gel adsorbent containing an ultraviolet-fluorescence quench indicator (Analtech GF or What- man K1F or E Merck Silica Gel G60 type plates). Detection of components is accomplished by visualization of dried plates under short wavelength (254 nm) ultraviolet light. spot for the enalapril base but does not visualize the maleic acid.

Iodine vapor yields a mre intense

In the systems reported above, the low sensitivity of the method (detection limit 1 to 2%) renders it unsuitable for the


detection and quantification of low levels of impurities in enalapril maleate bulk substance.

5.3.2 High Performance Liquid Chromatography (HpLC)

Several HPLC methods have been developed which are suitable for: (a) quantification of enalapril maleate and process impurities in the bulk substance (24 ) , (b) analysis of enalapril mleate and degradates in tablets (25) , (c) content uniformity assays of enalapril maleate in tablets (26) and (d) dissolution of enalapril maleate in tablets (27) . Reverse-phase HPLC conditions are employed. Detection is by uv at 210 or 215 nm. Flow rate is normally 1.8 or 2.0 ml/min. phate buffer and organic modifier (acetonitrile or a mixture of acetonitrile and methanol) and an oven temperature of 7OoC or 80° provide the selectivity and sensitivity required for satisfactory chromato-

The significance of specifying a high column temperature has been discussed in section 4.12. tems is given in Table V.

A mobile phase of aqueous phos-

graphy.

A summary of these HPLC sys-

5.4 Identification Tests

Identification of enalapril maleate can be carried out by infrared absorption (section 4.1), HPLC (section 5.3.2) and thin-layer chromatography (section 5.3.1).

Supportive evidence for identification can be obtained by differential thermal analysis (section 4 .7 ) and complexation with bromothyml blue (section 5.2).

5.5 Titration

Enalapril maleate can be determined by potentiometric titration with aqueous sodium hydroxide and perchloric acid. With respect to the sodium

V 0

OD

u 0

a3

0

0

m

c-

v-l

JJ c

F! 2

- 0 9

cv 9

w

v

X 0, Y

d N z

ax

4:; obl

x a .

.

v

.. obl

mR

J

a, oc

4J .d

& 0

ru

%

In

'9 v

X

W

a

w

W

X

X

W

w X 0

In

0

0 0-

. 07 u

236 DOMINIC P. IP AND GERALD S. BREWER

hydroxide titration, 0.1N sodium hydroxide is the titrant and a combination pH electrode is employed as the electrode system. The perchloric acid titration employs 0.1N perchloric acid in glacial acetic acid as the titrant and an anhydrous electrode system, e.g. Metrohm #EA 441/5 silver- silver chloride electrode filled with 0.1N lithium perchlorate in glacial acetic acid (or acetic anhydride), versus a glass electrode is used.


6.1 Solid State Stability

There are two major potential modes of degradation for enalapril maleate: (a) hydrolysis of the ethyl ester to 11, enalaprilat, the free acid and (b) cyclization to a diketopiperazine derivative 111. The structure of these compounds are shown in Figure 15.

Crystalline enalapril maleate is a very stable solid. (amber glass) for four years shows no evidence of degradation by HPLC analysis. degradation can be induced by storage at 8OoC for 3 weeks (in screw-cap vials) and < 1% of decomposition at 4OoC and 75% RH (open vial) for 16 weeks.

The compound stored at room temperature

Less than 2% of

Enalapril maleate tablets are stable when protected from elevated temperatures and high humidity. Degradation can be induced, however, if tablets are stored at 4OoC and 75% RH in open containers, resulting in enalapril maleate loss of about 10% or greater for three months. The major degradation product is 11. loss OF enalapril maleate and formation of I1 and I11 has been demonstrated.

The mass balance between

6.2 Solution Stability

The stability of enalapril maleate in aqueous buffer solutions has been studied as a function of pH in the range of 2-7 (28). Maximum solution stability occurred around pH 3 . loss and the mode of degradation are dependent upon the solution pH.

The rate of enalapril

At room temperature, tgO

I N

I

V I In

I, 0

h

v)

m

v)

U

C 3

0 a

u

U

m

rl aJ crr: U

C

m

ri

.d

Ll a m

'd C w

W

0


7.

(time required for 10% loss of starting material) values are 262 days and 114 days €or 0.5 W/ml at pH 2 and 5, respectively. For up to 20% loss at pH 2, I11 was the major degradation product while I1 represented only a minor fraction of the loss. A t pH 3 , I11 was the major degradate but the fraction of I1 was increased. accounted for the bulk of enalapril loss with I11 present only in trace quantities.

At pH 5 and ahve, I1

Pharmacokinetics and Metabolism

The pharmacokinetics and metabolism of enalapril maleate follming oral administration have been the subject of a number of investigations and reviews (4, 29-32).

Following oral administration of enalapril maleate to man (33) peak serum concentrations of enalapril and enalaprilat occur within 0.5 to 1.5 and 3 to 4 hours respectively. Based on urinary recovery of the total drug (enalapril plus its active metabolite, enalaprilat) absorption is at least 61%. The primary route of excretion of the drug is renal. Approximtely 94% of the dose is recovered in the urine and feces as enalaprilat or enalapril.

Enalapril, when administered orally, is rapidly absorbed and bioactivated extensively to enalaprilat. Bioactivation appears to be largely post-absorptive and there is no evidence for metabolism of enalapril beyond bioactivation to enalaprilat in man. The in vitro conversion of enalapril to enalaprilat in human autopsy tissues has been examined (34). Human cadaver liver was the only tissue in which significant conversion was demonst rated.

Enalapril absorption is not influenced by the presence of food in the gastrointestinal tract (35). was administered to normal volunteers both in the fasting state and with a normal meal. serum concentration-time curves for enalaprilat and urinary recoveries for enalaprilat and the total drug did not differ significantly between the fed and fasted conditions.

The drug

The area under the

The pharmacokinetics of enalapril maleate was determined in normal volunteers after repeated single daily


doses for eight days (36). Serum profiles show little accumulation of enalprilat over the course of the study. tion of approximately 11 hours was calculated from urine data. prilat is achieved by the third or fourth dose. serum concentration profile of enalaprilat is characterized by a prolonged terminal phase, apparently representing a small fraction of the administered dose that has been bound to angiotensin converting enzyme. The amount bound does not increase with dose indicating a saturable site of binding.

An average effective half-life for accumula-

An average steady state recovery of enala- The

8. Determination in Biological Fluids

A number of procedures have been developed for the analysis of enalapril and enalaprilat in biological Eluids. enzyrw inhibition and radioimuno-methods.

Those of primary utility include radiometric,

The physiological disposition and metabolism of enalapril maleate in laboratory animals has been studied with both radiolabeled drug and an enzyme-inhibition assay (37). Following administration of the radioactive drug; urine, feces or plasm samples may be counted by liquid scintillation techniques for total radioactivity. Urinary radioactivity may be separated into enalapril and enalaprilat by thin-layer chromatography *

Enalaprilat can also be determined by its in vitro inhibition of angiotensin converting enzyme (37). Plasm, urine and homogenates of feces are placed on xAD.4 columns, washed and then eluted with methanol to yield enalapril and enalaprilat. component of the mixture is determined by the enzyme assay which uses as a substrate, the tripeptide carbo- benzoxycarbonyl-phenylalanyl-histidyl leucine and converting e n z w isolated from porcine plasm. The product, histidyl-leucine is then quantified by fluorescence after reaction with o-phthalaldehydc ( 3 8 ) . For the determination of the total drug (enalapril plus enalaprilat) samples are hydrolyzed at high pH and then reassayed for enalaprilat. be calculated as the difference between the total drug and enalaprilat prior to hydrolysis. tion assay for enalaprilat has also been developed

The enalaprilat

Enalapril levels may then

An enzyme inhibi-


which uses radiolabeled substrate (39 ) .thereby elimi- nating derivatization with o-phthalaldehyde and fluorescence detection of the enzyme roducts. The radiolabeled substrate employed is [ HI hippuryl-kglycyl-

3 L-glycine. The product, [ HI hippuric acid is extracted into a water-immiscible scintillation cocktail and then quantified by counting in a liquid scintillation spectrometer.

fl

Enalaprilat has also been determined in serum and urine samples by radioimmunassay procedures. (40, 41). In one such procedure (40 ) antibodies for the radioimunoassay are raised in rabbits using an immunogen prepared by coupling the lysine analog of enalaprilat (lisinopril) to albumin with difluoro-dinitrobenzene. The radiolabeled component is obtained by iodination of the

CH2CH2 -c -N A COOH

- c -c I

-N I 51

*’ COOH

NH2 Lisinopril

amidine derived from the reaction of lisinopril and methyl p-hydroxybenzimidate. measured as enalaprilat after sample hydrolysis by rat liver homogenate. Enalapril can then be calculated as the difference between enalaprilat before and after hydrolysis.

The total drug is

Acknowledgements

The authors wish to thank Mrs. Betty Moyer €or typing the manuscript and Ms. Florence Berg for performing the literature search.

ENALAPRIL MALEATE 24 I

9. References

1. A. A. Patchett, E. Harris, E. W. Tristram, M. J. Wyvratt, M. T. Wu, D. Taub, E. R. Peterson, T. J. Ikeler, J. temroeke, L. G. Payne, D. L. Ondeyka, E. D. Thorsett, W. J. Greenlee, N. S. Lohr, R. D. Hoffsomer, H. Joshua, W. V. Ruyle, J. W. Rothrock, S. D. Aster, A. L. Maycock, F. M. Robinson, R. Hirschmann, C. S. Sweet, E. H. Ulm, D. M. Gross, T. C. Vassal and C. A. Stone, Nature 288, 280 (1980).

2. A. A. Patchett in "Hypertension and the Angiotensin System: Therapeutic Approaches", A. E. Doyle and A. G. Bearn Eds., Raven Press, New York, N.Y., 1984.

3. A. A. Patchett, Br. J. Clin. Pharmacol. - 18, 201s (1984).

4. P. H. Vlasses, G. E. Larijani, D. P. Conner and R. K. Ferguson, Clin. Pharrn. - 4 , 27 (1985).

5. C. S. Sweet, Fed. Proc. - 4 2 , 167 (1983).

6. D. M. Gross, C. S. Sweet, E. H. Urn, E. P. Backlund, A. A. Morris, D. Weitz, D. L. Bohn, H. C. Wenger, T. C. Vassil and C. A. Stone, J. Pharmac. Exp. Ther 216, 552 (1981).

7. M. J. Wyvratt, E. W. Tristram, T. J. Ikeler, N. S. Lohr, H. Joshua, J. P. Springer, B. H. Arison and A. A. Patchett, J. Org. Chem. - 49, 2816 (1984).

8. A. A. Patchett, E. E. Harris, M. J. Wyvratt and E. W. Tristram (Merck and Co., Inc.) Eur. Pat. Appl. 12,401.

9. E. E. Harris, A. A. Patchett, E. W. Tristram, and M. J. Wyvratt (Merck and Co., Inc.) US 4,374,829.

10. Merck Sharp and Dohme Research Laboratories, unpublished data.


12. D. L. Rabenstein and A. A. Isab, Anal. Chem., 2, 526 (1982).


13. Merck Sharp and Dohrne Research Laboratories, unpublished data.

14. H. G. Ramjit, Merck Sharp and Dohme Research Laboratories, personal comunication.

15. Nerck Sharp and Dohme Research Laboratories, unpublished data.

16. R. G. Bergstcorn, Merck Sharp and Dohme Research Laboratories, personal comunication.

17. D. P. Ip, G. 5. Brenner, J. M. Stevenson? S. Lindenbaum, A. W. Douglas, S. D. Klein and J. A. McCauley, Int. J. of Pharm., 28, 183 (1986).

Laboratories, personal comunication. 18. J. A. McCauley, Merck Sharp and Dohme Research

19. J. P. Draper, Merck Sharp and Dohme Research Laboratories, personal comunication.

20. R. Roman, formerly of Merck Sharp and Dohme Research Laboratories, unpublished data.

21. J. M. Konieczny, Merck Sharp and Dohme Research Laboratories, personal comnunication.

22. T. Kato, Anal Chim. Acta, 175, 339 (1985).

23. Nerck Sharp and Dohme Research Laboratories, unpublished data.


25. J. DeMarcO, Merck Sharp and D o h Research Laboratories, personal comnunication.

26. R. Roman, formerly of Merck Sharp and Dohme Research Laboratories and C. Bell, Merck Sharp and Dohm Research Laboratories, personal comunication.

27. D. J. Mazzo, formerly of Merck Sharp and Dohme Research Laboratories, unpublished data.


28. L. L. Ng, Merck Sharp and Dohme Research Laboratories, personal communication.

29. E. H. U l m , Drug. Metab. Rev., 14 , 99 (1983).

30. P. A. Todd and R. C. Heel, Drugs - 31, 198 (1986).

31. R. S. Perry, M e d . A c t u a l . , - 21, 31 (1985).

32. A. F. Lant, R. W. McNabb and F. H. Noormohamed, J. Hypertens., 2, 37 (1984).

Hand, T. C. Vassi l , J. Biollaz, H. R. Brunner and J. L. Schnelling, B r . J. Clin. Pharmacol., - 14, 357 (1982).

Br . J. Clin. Pharmacol., 19, 701 (1985).

Bergquis t , A. E. T i l l , J. D. I r v i n and K. Harris, J. Pharm. Sci. - 73, 1655 (1984).

W. R. McNabb, B. A. Brooks , F. Noormhamed and A. F. Lant, Biopharm. Drug Dispos., 2, 273 (1984).

33. E. H. U l m , M. Hichens, H. J. Gomez, A. E. T i l l , E.

34. I. Larmour, B. Jackson, R. Cubelal and C. I. Johnston,

35. B. N. Swanson, P. H. Vlasses, R. K. Ferguson, P. A.

36. A. E. T i l l , H. J. Gomez, M. Hichens, J. A. Bolognese,

37. D. J. TOCCO, F. A. deLuna, A. E. W. Duncan, T. C. Vassil and E. H. U l m , Drug Metab. Dispos. - 10, 15 (1982).

38. Y. Piquillaud, A. Reinharz and M. Roth, Biochem. Biophys. Acta, 206, 136 (1970).

39. B. N. Swanson, K. L. Stauber, W. C. Alpaugh and S. H. Weinstein, Anal. Biochem. - 148, 401 (1985).

40. M. Hirhens, E. L. Hand and W. A. Mulcahy, Ligand Q. - 4 , 43 (1981).

41. P. J. Worland and B. J a r r o t t , J. Pharm Sci. - 75, 512 (1986).

The l i t e r a t u r e w a s reviewed to Ju ly , 1986.

HOMATROPINE HYDROBROMIDE

F a i d J. Muktadi, Mahmoud M.A. H u b a n , and

AbduR FaMah A . A d i h y

C o U e g e 06 Phanmacy, King Saud U n i v m L t y , Riyadh, Saudi A h a b i a .

F.J. Muktadi : Uepan;lmeMA: od Phatunacognony.

M.M.A. Hanun : M e d i c i d , AmmaZic and P o h a n o u n P&m% R e n u c h Cevtta.

A.A. A d i d y : Qep'ahtment 06 Phatunacagnony.


Copylight 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved. 245

246 FARID J . MUHTADI ETAL.

1. Description

1.1 Nomenclature

1.2 Formulae



1.5

1.6 Dissociation Constant

1.7 pH Range

Appearance, Color, Odor and Taste


2.1 Melting Point

2.2 Solubility

2.3 Crystal Structure

2.4 X-Ray Powder Diffraction

2.5 Spectral Properties

2.5.1 W Spectrum

2.5.2 IR Spectrum

2.5.3 Nuclear Magnetic Resonance Spectra

2.5.4 Mass Spectrum

3. Preparation of Homatropine Hydrobromide

4. Total Synthesis

4.1 Total Synthesis of Tropine

4.2 Total Synthesis of Mandelic Acid

4.3 Total Synthesis of Homatropine

5. Therapeutic Uses and Pharmacology

6 . Drug Stability



7.1 Tests of Identification

7.2 Microcrystal Formation

7.3 Titrimetric Methods

7.4 Complexornetry

7.5 Polarographic Methods



Acknowledgement

247

References

248 FARID J. MUHTADI ETAL.

1. Descr ip t ion

1.1 Nomenclature


- a- Hydroxybenzene a c e t i c a c i d 8-methyl-8- azabicyclo [3.2.1] oct-3-yl e s t e r , hydrobromide.

- l a H , 5aH-tropane-3a-01 mandelate (es ter) hydrobromide.

- Benzeneacetic a c i d , a-hydroxy-, 8-methyl-8- azabicyclo [ 3.2.17 oct-3 y l ester hydrobromide , endo-( 2 ) - .

1 . 1 . 2 Generic Names

Homatropine Hydrobromide, DL-Hornatropine Hydrobromide, The hydrobromide of t r o p i n e mandelate , Tropine mandelate Hydrobromide , Mandelyltropine Hydrobromide.

1.1.3 Trade Names

Homatrisol, Bufopto, Homatrocel.

1 . 2 Formulae

1.2.1 Empirical

CIGH2103N. HBr

1 .2 .2 S t r u c t u r a l

7 HO

I I OH

I 0- C-CH

HOMATROPINE HYDROBROMIDE 249

1 .2 .3 CAS Regis t ry No.

[Sl-56-91 C16H2103N. H B r

1 . 2 . 4 Wiswesser Line Notation

T56 A ANTJ A GOVYGR 6 EH (1) -

1.2.5 Stereochemistry

Baminat ion of the M spec t r a of some t ropane deuterohal ides has shown t h a t t he N-substitu- en t i n t ropanes i s predominantly equa to r i a l (2 1. X-ray ana lys i s of t rop ine hydrobromide has shown t h e presence of c h a i r conformation (3) . Study of the dipole-moment and Kerr-constant measurements of a number of t ropane d e r i v a t i - ves has shown t h a t t h e p ipe r id ine r i n g i s i n t he c h a i r form wi th the N-methyl equa to r i a l ( 4 ) . NMR spec t r a o f some t ropane de r iva t ives have confirmed t h a t t h e p ipe r id ine r i n g i s i n t h e chair conformation wi th t h e N-methyl group predominantly equa to r i a l ( 5 ) . I n t rop ine , however, t h e predominant conformation i s t h e p iper id ine r i n g i n a deformed c h a i r form to - gether wi th a minor amount i n t h e boat form

Another study of t h e dipole-moments and

( 6 I *

HO

Tropine

I n a t rop ine , t h e a-3-subst i tuent i s of gree- ter bulk than t h e hydroxyl, and t h e boat form may w i l l be favored because of t h e increased i n t e r a c t i o n s involv ing the dimethylene br idge i n the c h a i r conformation ( 7 ). Hornatropine would be the same as a t rop ine .


I I 011 e /

N -CH3 0-c-CH

‘‘6HS

A d e t a i l e d review i s ava i l ab le f o r t he boat o r c h a i r conformation i n t rop ines ( 8 ) .

Other PMR s tudy suggested a preference f o r t he boat conformation i n seve ra l t ropane d e r i v a t i v e s . This s tudy showed s t rong c ross - r ing in t ramolecular i n t e r - a c t i o n s of t h e type N---C---O and N---H-0 were i n d i - ca ted by the broadening of t h e proton s i g n a l due t o t h e coupl ing between 1(5)-H and 2(4)-H protons i n the boat conformer compared with t h e c h a i r . This broadening a r i s e s a s a consequence of e c l i p s i n g of t hese protons i n t he boat conformer ( 9 ) .

1 Applicat ion of and coba l t d i ace ty l ace tona te s , t o t r o p i n e s has demonstrated t h a t t h e six-membered r i n g of t r o p i n e s is i n a near ly semiplaner form. I t i s suggested t h a t t he p i p e r i d i n e r i n g of t ropanes is somewhat d i f f e r e n t i n conformation from t h e normal c h a i r form (10) .

Carbon-13 magnetic resonance s tudy has a l s o sugges-

H-NMR con tac t s h i f t s us ing n i c k e l

t ed a non-chair t i v e s (11).

conformations i n t ropane d e r i .va-

Me


275.33 (Homatropine) 356.3 (Homat ropine HBr)

HOMATROPINE HYDROBROMIDE 25 1

2 .

1 . 4

1 .5

1 .6

1.7

Elemental Cormosition

C , 69.79%; H, 7.69%; N , 5 .09%; 0 , 17.43% Il-lomatropine) . C , 53.94%; H , 6 .22%; B r , 2 2 . 4 4 % ; N , 3.93%; 0, 13.47% (Homatropine HBr).

Appearance, Color , Odor and Tas te

Co lo r l e s s c r y s t a l s o r a w h i t e c r y s t a l l i n e powder, odor l e s s , with a b i t t e r t as te (12) .

Orthorhombic, bipyramidal pr isms (from wa te r ) , odor l e s s (13) .

D i s soc ia t ion Constant

pKa 9.9(2O0) (14)

pH Range --

A 2% s o l u t i o n i n water has a pH 5.5 t o 7 (12 ) , a 5.67% s o l u t i o n i n water is iso-osmotic w i t h serum. pH (1% aqueous s o l u t i o n ) : 5.4 (13).

i'hysical Proper t i.es

2 . 1 Melting Poin t

214 - 217O (with decomposition) ( 1 2 ) Other mel t ing p o i n t s were a l s o r epor t ed : 215 (with decomposition) f 1 5 ) 217-218 (with decomposition) ( 1 )

2 . 2 S o l u b i l i t y

One gram d i s s o l v e s i n 6 ml water, 40 m l a l coho l , 1 2 m l a lcohol a t 60°, 420 m l chlornform. Inso luble i n e t h e r . The s o l u b i l i t y inc reases r a p i d l y as t h e t eEpera tu re rises (12 ) .

2 . 3 Crys t a l S t r u c t u r e

The c r y s t a l s t r u c t u r e of both homatropine and homa- t r o p i n e hydrobromide were r epor t ed (16). C r y s t a l s of homatropine were obta ined by r e c r y s t a l l i z a t i o n from e thano l , whereas the c r y s t a l s of homatropine hydrobromide (C16H21N03. H R r ) were obta ined by r e c r y s t a l -

252 FARID J . MUHTADIETAL.

l i z a t i o n from water. O s c i l l a t i o n and Weissenberg photographs were used t o determine the space group (Table 1). In both homatropine and homatropine hydrobromide t h e n i t rogen atom may be o p t i c a l l y a c t i v e , s i n c e the space groups a r e centrosymmetric, t h e u n i t c e l l s must be assumed t o conta in equal numbers of L - a d D-molecules (16) .

Table 1. The Crys t a l S t r u c t u r e o f Homatro- p ine and of Homatropine H B r .

Compound a(A") b(A") c(A") Density (g.cmR3) Z Space observed c a l c u l a t e d group

Homatropine 14.5 15.1 6.97 1.21 1.22 4 P211/c

Homatropine 10.3 16.4 19.25 1.48 1.46 8 Pcab H B r

2 . 4 X-ray Powder D i f f r a c t i o n

The X-ray d i f f r a c t i o n p a t t e r n of homatropine hydrobromide was determined with a P h i l i p s X-ray d i f f r a c - t i o n spectrogoniometer equipped wi th PW 1730 genera t o r . X-ray r a d i a t i o n was provided by a copper t a r g e t (Cu- anode 2000 W ) . High i n t e n s i t y X-ray tube opera ted a t 40 KV and 20 mA. The monochromator was a s i n g l e curved c r y s t a l ( y = l .5918A0) The u n i t was equipped with P h i l i p s PM 821.0 p r i n t i n g recorded d i g i t a l p r i n - ter . The scanning speed of t he goniometer used was 0.02 2e /sec . , r eco rde r f u l l s c a l e was 10,000 counts a t r eco rde r speed of 4 mm/28. The lower level and t h e upper l eve l of t h e s i g n a l c o n t r o l waz 35 E 75 r e s - pec t ive ly . The u n i t was a l igned and examined, us ing a Sol icon s tandard , t o a t t a i n maximum ope ra t ion cond i t ions . The X-ray p a t t e r n of homatropine hydrobromide i s presented i n Fig. 1. In t e rp l anna r d i s t a n c e and r e l a t i v e i n t e n s i t y are t a b u l a t e d i n t a b l e 2 .

Divergance s l i t and t h e r ece iv ing s l i t were 1".

HOMATROPINE HY DROBROMIDE

Table 2 : X-Ray Powder D i f f r a c t i o n P a t t e r n of Homatropine Hydrobromide

253

- ~ ~~

6.98 17.8% 6 .41 17.3% 6.00 22 .1% 5.15 15.7% 5.04 21.1% 4.84 73.5% 4.64 12.6% 4.48 14.4% 4.35 39.0% 4.28 85.9% 4 . 1 9 100.0% 3.96 36.3% 3.57 48.7% 3.50 46.1% 3.43 1 1 . 2 % 3.30 38.5% 3.23 21.3% 3.17 20.2% 3.12 22.7% 3.07 20.7% 3.00 20.8% 2.97 17.8%

2.87 7.83 2.72 2.64 2.61 2.58 2.52 2.50 2.47 2.35 2.32 2.29 2.26 2.15 2.07 2.03 1.98 1 .93 1.87 1.80 1.77

11 ,7% 24.1% 19.5% 10.7% 14.2% 20.0% 22.9% 1 2 . 1 % 22.0% 13.5% 25.9% 13.7% 13.1% 11.9% 11.3% 16.4% 10.7% 13.0% 11.2% 11.0% 13.5%

~ ~~

d = i n t e r p l a n a r d i s t a n c e , I / Io = re la - t i v e i n t e n s i t y (based on t h e h ighes t i n t e n s i t y of 100).

J 1 lY 1 X-RAY DIFFRACTION PATTEW OF HCMATROPINE HYDROIIRO\IIDL.


2.5 Spec t r a l P rope r t i e s


The UV spectrum of homatropine hydrobromide i n methanol (Fig. 2 ) was scanned from 200 t o 350 nm us ing DMS 90 Varian Spectrophoto- meter. I t e x h i b i t s t h e fol lowing UV d a t a (Table 3 ) .

Table 3 . UV C h a r a c t e r i s t i c s of Homatropine

Xmax. a t nm E (A 1%, lcm)

252 224.28 258 26 7 264.5 228.5 273 78.32

6.3 7.5 6.42 2 . 2

Other r epor t ed UV s p e c t r a l d a t a €or homatro- p ine i n 0 . 1 N s u l f u r i c ac id ( 1 7 ) .

Xmax. a t 252 m u ( E 1%, 1 cm 4 .8 ) , 258 mu ( E 1%, 1 cm 6.1) and 264 m u ( E 1%, 1 cm 5 ) .

Amax. i n 0 .1 N hydrochlor ic ac id a t 254 nm ( E 1%, 1 cm = 3 ) ; a t 259 nm ( E 1%, 1 cm =5 .2 ) and 266 nm. (18) .

2.5.2 In f r a red Spectrum

The I R spectrum of homatropine hydrobromide as KBr-disc was recorded on a Perkin E l m e r 580 B I n f r a r e d Spectrometer t o which i n f r a r e d d a t a s t a t i o n i s a t t ached (Fig. 3 ) . The s t r u c t u r a l assignment have been c o r r e l a - t e d with t h e fol lowing f requencies (Table 4 ) .

Table 4. I R C h a r a c t e r i s t i c of Homatropine

Frequency cm-’

Base H B r

Ass i gnment

3350 3270 OH ( s t r e t c h ) 2940,3050 2965 CH ( s t r e t c h )

+ - 2 80 0 - 2.58 5 NH

R 1735 1755 -0 -C- ( e s t e r )


20 0 250 30 0 350

Fig. 2 : ‘THE W SPECTRUM OF HOMATROPINE IIYDROl3KOMIDE IN PE’MANOL

1 I I I I I 19

I I I 1 I 1 1 1 1 1

3500 3000 2500 2000 1800 1600 1400 1200 1000 800 600

Fig. 3 : ‘IHE IR SPECTRLM OF HOMATRDPINE HYDROBRDMIDE AS KBr DISC.

HOMATROPINE HY DROBROMIDE 257

-1

Ease HBr

Frequency cm Assignment

1600 1600 C=C (aromatic) 145 0,1500 1475 C=C (aromatic) 1100,1065 1170,1070 C-0-C (ether) 755 , 700 735,700 5 H (monosubstituted

aromatic)

Other characteristic bands are 2750, 2710, 1450, 1420, 1395, 1378, 1328, 1288, 1265, 1195, 1105, 1025, 960, 885, 820 and 615 cm-l. Other IR data for homatropine and the hydrobromide salt have been reported (1,14,17).

2.5.3 Nuclear Magnetic Resonance

2.5.3.1 'H-NMR Spectra

The 'H-NMR spectra of homatropine hydrobromide in DMSO-D6 and in D20

were recorded on a Varian T-60A, 60 MHz NMR spectrometer iising TMS (tetramethylsilane) as an internal reference. These are shown in F i g . 4 and 5 respectively. The following structural assignment have been made (Table 5). 8

A H 3

7 6 >&$H0 " OH ' Oi4 12 13

0- C-CH 9 10

1 '6 Table 5. The H-NMR Characterjstics of Homatropine

Group Chemical Shift (pmm)

D2° DMSO- d6

5 aromatic protons 7.33 (s) 7,4 (s) (12,13,14,15,16 H) 10 H (OH) 6.03 (d)

10 H 5.13 (d) 5.33 ( s )

J . I , . , , 1 , , . , , , I , , , . 1 , . L . 1 . . . . I . . . . l . . . . l . . . . I . . . . I . . * . I . L

I n I n 6 0 s o P P W I I I a n . 1 0 Y O 1 0

Fig. 4 : THE 'H-NMR SPECTRUM HOMATROPINE HYDROBROMIDE IN D I W - L ) ~

Fig. 5 : THE 'H-NMR SPECTRUM OF HOMATROPINE HYDROBROMIDE IN D20.

260 FARID J. MUWTADIETAL.

Group Chemical S h i f t (pmm)

D2° DMSO- d6

3H 4.70 ( t ) 5.03 ( t )

1 H and 5 H 3.71 (bs) 3.66 (bs)

8-N-Me 2.63 (s) 2.67 (s)

2,4,6,7 H 2.4-1.27 (m) 2.4-1.8 (m)

s = s i n g l e t , d=doublet , t = t r i p l e t , bs=broad s i n g l e t 1

Other H-NMR da t a f o r homatropine hydrobromide was repor t ed ( 1 ). Ohashi e t a1 (10 ) have r epor t ed 'H nuc lear magnetic resonance con tac t s h i f t s o f some t ropanes inc luding homatropine i n CDC13 a t 220 MHz. The contac t s h i f t s used were n i c k e l and c o b a l t d i a - ce ty l ace tona te s .

2.5.3.2 13C-NMR

The I3C -NMR complete l y decoupled and o f f resonance s p e c t r a of homa- t rop ine hydrobromide a r e presented i n Fig.6 and Fig. 7 r e spec t ive ly . Both were recorded over 5000 Hz range i n a mixture o f DMSO-D6 and CDC13 (conc. 100 mg/ml), on a J o e l FX-100-90 MHz spectrometer . A sam- p l e t u b e ' l 0 mm and TMS a s i n t e r n a l s tandard a t 20' were used. The carbon chemical s h i f t s are assigned on t h e b a s i s o f t h e chemical s h i f t theory and t h e off-resonance s p l i t - t i n g p a t t e r n (Table 6 ).

8

I I I

7

0- C-CH 9 10

12 13

Fig. 6 : 'IHE l 3 C - I W W L E T E L Y DECOUPLED SPECTRUM OF HOMATROPINE HYDROBNNIDE.

Fig. : THE ' 3 C - W OFF-RESONANCE S P E m M OF Wl'p.OPINE HWWBROFlIDE.


Table 6 . Carbon Chemical S h i f t s of Homatropine

Carbon Chemical S h i f t Carbon Chemical S h i f t No. [PPml No. [PPml

‘1

c2

c3

‘4

c5

c7

169.38 (s)

62.76 (d)

137.38 (s)

126.34 (d)

124.76 (d)

125.99 (d)

124.76 (d)

126.34 (d)

‘ 9

5 0

c l l

5 2

59.18 (d)

32.11 ( t )

70.74 (d)

32.11 ( t )

59.18 (d)

21.19 ( t )

21.19 ( t )

40.21 ( 9 )

‘13

14 ‘6

5 5

‘16 ‘8

s = s i n g l e t , d = d o u b l e t , t = t r i p l e t , q = q u a r t e t .

2.5.4 Mass Spectrum

The mass spectrum of homatropine o b t a i n e d by e l e c t r o n impact i o n i z a t i o n a t 70 eV, was recorded on a Finigan-Mat 5100 Mass S p e c t r o - meter. The spectrum (Fig . 8) shows a mole- c u l a r i o n peak M+ at m/e 275. The base peak i s a t m/e 124. The most prominent f ragments , t h e i r r e l a t i v e i n t e n s i t i e s and some proposed ion fragments are shown i n table 7.

Table 7 . Mass Fragements of Homatropine

- m/e R e l a t i v e I n t e n s i t y % I o n s - 275 4.4 M+

234

193

165

142

140

125

1 . 2

1 . 2

1 . 3

6 .4

4.6

10 .5

- +

[IT)]’

I- Fig. 8 : 'THE MASS SF'ECIRLN OF ~ l R O P I t E


m/e R e l a t i v e I n t e n s i t y %

124 100

1 2 2 1 .6

107 7 .5

106 3.8

105 6.1

-

96 14

95 9 . 3

94 21.5

83 20.3

8 2 29.2

79 14 .3

77 19.2

68 5.9

6 7 19.1

55 6 .7

Ions - 125-H

96 -H

95-H +

-

68 -H +

[CH 2 =N=CH-CH2]

Other mass s p e c t r a l data of homatropine have been r e p o r t e d (19,20) .


3 . Preparat ion of Homatropine Hydrobromide

Atropine i s hydrolyzed wi th barium hydroxide s o l u t i o n t o g ive t r o p i n e and t r o p i c ac id . The t rop ine i s hea ted with mandelic ac id i n t h e presence of hydrogen ch lo r ide . Ammonia i s added, and t h e l i b e r a - t e d homatropine i s e x t r a c t e d with chloroform. The chloroform so lu t ion i s evaporated t o dryness . The r e s idue i s t r e a t e d with hydrobromic ac id and t h e homatropine hydrobromide obtained is p u r i f i e d by r e c r y s t a l l i z a t i o n (21, 2 2 ) . The p repa ra t ion i s ou t l ined i n Scheme I .

Scheme I . H f) / CH20H

0-C-CH \ C 6 H

Hydrolysis ’

HOOC -CH-CHZ OH eoi 6 HOOC\ +

Ho’cH@ 1 0 OH

0-E-cii’ ‘‘6HS

Homatropine


An alternative method of hydrolyzing atropine was achieved microbically by the action of Arthrobacter atropini (23).

4. Total Synthesis

Since Homatropine is the tropine ester of mandelic acid, schemes for the total synthesis of tropine and of mandelic acid were reported.

4.1 Total Synthesis of Tropine

Four schemes for the total synthesis of tropine are known. Scheme I1 was also modified to give a much better yield. Scheme 11: Willstatter's total synthesis of tropine

Suberone (cycloheptanone) [l] is reduced to suberol which is treated with hydrogen iodide to give suberyl iodide [2]. This is treated with potassium hydroxide in ethanol to give cycloheptene [ 3 ] . Cycloheptene is brominated to give 1,Z-dibromocycloheptane [4] which is treated with dimethylamine to yield dimethy- laminocyclohept-2-ene [5]. The latter is converted to cyclohepta-1,3-diene [6] by exhaustive methylation. [6] is brnminated at 1,4-positions to give 1,4-dibro- mocyclohept-2-ene [7]. Elimination of two moles of the hydrogen bromide of [7] is effected by quinaline to give cycloheptatriene [ 8 ] .

Substance [8] is treated with hydrogen bromide to give bromocyclohepta-3,5-diene [9 ] which is reacted with dimethylamine to give dimethyl aminocyclohepta- 2,4-diene [ l o ] . The latter is treated with sodium in ethanol followed by bromination to give 1,2-dibromo- 5-dimethylamino-cycloheptane [ll]. This is warmed in ether when intramolecular alkylation occurs to give 2-bromotropane methobromide [12]. Hydrogen bromide is eliminated from [12] by the action of alkali to yield tropidine methobromide [ 1 3 ] . This is transformed to tropidine methochloride [14] by the action of potassium iodide followed by the action of silver chloride. Sub-rtance [14] is pyrolized to give tropidine [15].

Hydrogen bromide is added to an acetic acid solution of tropidine [15] to yield 3-bromotropane [16! which is hydrolysed with 10% sulfuric acid at 2@0-21@0 to give pseudotropine [ 171. JI-tropine [ 171 is oxidized with chromium trioxide to give tropinone [18]. This ketone is reduced with zinc and hydriodic acid to tropine [ 191.

(24) *


Scheme 11 : W i l l s t a t t e r ' s t o t a l synthes is o f a t ropine

exhaust

methyln L

Q- B r

B r [ 4 I

B r

quinol i ne

1 5 0 0 ~ *

w a r m

i n Et20 B r

[ I 2 1 Br [111

f


Scheme T I l : Robinson's t o t t i l s y n t h e s i s of atropine

C H 3 + \

CH-OH

N -CH 3

/"=" CH3 G [ 4 1

CH3NH2 cond.

121 CH-OH

I 3 1

[ G I I 5 1


Scheme 111: Robinson's synthes is (25)

Succindialdehyde [ 11 i s condensed with methylamine [ 2 ] t o give t h e condensate b iscarb in- olamine [3]. This i n t u r n condensed wi th acetone [ 4 ] t o give t ropinone [ 5 ] ( T h i s mixture i s allowed t o s tand i n water a t ordinary t e m - pera ture f o r ha l f an hour) . Tropinone [ 5 ] i s reduced with zinc and hydr- i od ic ac id t o t rop ine [6 ] .

The y i e l d can be improved by s u b s t i t u t i o n of t h e more r eac t ive acetone dicarboxylate o r i t s ester f o r acetone. Succindialdehyde [l] i s condensed with mthyla- mine [21 t o give b i sca rb ino lmine [31. I31 i s condensed with calcium acetonedicarboxy- l a t e [4] t o af1'31-d t h e condensate [51. This i s warmed with hydrochloric a c i d t o give t ro - pinone [61. z inc and hydriodic a c i d t o t rop ine [71. Scheme IV-:

Succinyldiacet ic e s t e r [ 11 i s condensed with methylamine [ 2 ] t o give diethyl-N-methylpyr- ro l ed iace t a t e [ 31. This i s reduced (H2+Pt) t o a f ford diethyl-N-methylpyrrolidinediace- t a t e [41. The c i s form of [41 is cycl ized i n t he presence of Na and p-cymene t o give e thy l - tropinone-2-carboxylate [ 51. Hydrolysis of [5] with 10% s u l f u r i c a c i d gives e thy l t rop i - none-2-carboxylic ac id [ 61. heated t o y i e l d tropinone [TI which i s reduced wi th zinc and hydriodic a c i d t o t rop ine [a]. Scheme V:

Tropinone can a l s o be synthesized ( 2 7 ) us ing methylamine hydrochloride acetondicarboxyl ic ac id and generat ing succindialdehyde i n s i t u by t h e ac t ion of ac id on 2,5-dimethoxy t e t r a - hydrofuran as follows :

Tropinone [61 i s reduced wi th

Willstatter I s second synthesis(26)

The l a t t e r i s

H+ CH3NH2HC.l

CO( CH2COOH)2

CHO

"0

HOMATROPINE HYDROBROMIDE 27 1

Scheme 111 : Robinson's s y n t h e s i s ( y i e l d improvement)

CH-OH CH2COOCa

c=o \

/ cond

CH2COOCa

[ 4 1 - ';-... +

f NHeCH3 :li 121 CH-OIf

[ 3 1 \ I 1 1

COOCa 4

Scheme I& Willstatter ' s second s y n t h e s i s

CH= Cli- CH2-COOC H 2 5

1 1 1

H I

CH2- C- CH2-COOC H

I CH -C-CH2-COOC H

2 5 I 1

H3CT c=o

2 1 2 5 H

\ 'A (51

CH= C- CH,-COOC H I I - 2 5

+ H2NCH3

CH= C- CH2-COOC H [21 2 5

H I I I 3 ] CH -C- CH2-COOC I I 1 2 I 2 5 I II- Cli3

.(-------------- I CR -C- CH2-COOC H

2 1 2 5

H I

H

[ 4 1

CH - c- CH-COOH i 2 i I

* 1 II CN c=o 3 1 I

CH2- c- &I2 I H

(61


CH2 - C H - C H 2

\ I I Me-N C = O I / I CH2 - CH - CH2

[71

Zn

HI L

2 CH2 - CH - C H

[81

4 .2 To ta l Synthes is of Mandelic Acid

Severa l schemes f o r t h e t o t a l s y n t h e s i s a r e known (28-30) .

Scheme I Benzaldehyde [ l ] i s t r e a t e d with a mixture of a s a t u r a t e d s o l u t i o n of sodium hydrogen s u l f i t e and aqueous sodium cyanide t o g ive mande lon i t r i l e [ 2 ] . [ 2 ] i s hydrolyzed with co ld concent ra ted hydro- c h l o r i c a c i d t o y i e l d mandelic a c i d [3] (28).

Scheme I1 Acetophenone [ l ] i s converted i n t o d ich loroace to- phenone [ 2 ] by t h e ac t ion of c h l o r i n e i n a c e t i c a c i d . [ 2 ] i s t r e a t e d with sodium hydroxide t o a f f o r d t h e sodium s a l t o f mandelic a c i d , which i s t r e a t e d wi th hydrochlor ic ac id t o g ive mandelic ac id [ 3 ] ( 2 9 ) .

Scheme I11 Amygdalin [ 13 is t r e a t e d wi th fuming hydrochlor ic ac id t o produce mande lon i t r i l e [2 ] which i s then hydrolyzed with hydrochlor ic a c i d t o g ive mandelic a c i d [ 3 ] (30).

4 .3 To ta l Synthes is of Homatropine

Tropine is then e s t e r i f i e d with (+) -mandelic a c i d i n t h e presence of hydrogen c h l o r i d e (F ischer - Spe ie r e s t e r f i c a t i o n ) t o g ive ( 2 ) -homatropine.


Total Synthesis of Mandelic Acid

Scheme I

NaCN NaHSO

Scheme I1

CHC 1 2

c12 @Lo CH3COOH b

/ [21 / i) NaOH

ii) HCI

Scheme I11

HO

HO

213


5. Therapel.itic Uses and Pharmacological E f fec t s

Homatropine i s used a s a mydriat ic and cyc lopleg ic drug i n ophthalmology. I t i s p re fe r r ed t o a t rop ine f o r d iag- n o s t i c purposes because i t s ac t ion i s more r ap id , less prolonged and i s zore r e a d i l y c o n t r o l l e d by physos t igmine (12) . The e f f e c t of homatropine i s exer ted i n 15 t o 30 minutes and passes o f f i n 1 2 t o 24 hours . I t j s sorne- times used with cocaine which enhances i t s mydr ia t ic ac t ion ( 1 2 ) . Homatropine has a l s o l e s s tendency than a t rop ine t o i n c r - ease t h e in t r a -ocu la r pressure (18), bu t it produces a l e s s s a t i s f a c t o r y mydriasis i n ch i ld ren (18). I t i s seldom used i n t e r n a l l y (12). Homatropine is an a n t i c h o l i n e r g i c drug with e f f e c t s s i m i - l a r t o hu t much weaker (about one-tenth) than those of a t rop ine (12,31l , gene ra l ly t h e bel ladonna a l k a l o i d s a r e absorbed r a p i d l y from t h e g a s t r o i n t e s t i n a l t r a c t . a l s o e n t e r t h e c i r c u l a t i o n when appl ied l o c a l l y t o t h e mucosal su r f aces o f t h e body (31). Some of these compounds (a t ropine and homatropine) when appl ied l o c a l l y t o t h e eye can cause mydriasis and cyc lopleg ia (31). Homatropine i s used a s a 2% s o l u t i o n i n c a s t o r o i l , but more o f t e n i n form of 2 t o 5% hornatropine hydrobromide ophthalmic so lu t ions (12,31). I t has been repor ted (32) t h a t hornatropine i n doses of 0 .5 t o 4.0 mg, induces bradycardia i n man, when it i s adminis tered subcutaneously

They

6. Drug S t a b i l i t y

Homatropine hydrobromide i n t h e dry s t a t e , i n a i r t i g h t con ta ine r a t room temperature and p ro tec t ed from l i g h t , showed no decomposition a f t e r f i v e yea r s when examined according t o t h e B.P. (15) . In aqueous s o l u t i o n , homatropine hydrolyses t o t r o p i n e andmandelic ac id . Hydrolysis i s ca ta lyzed by hydrogen ions and hydroxide ions (18). A t 25", t h e r a t e of hydro- l y s i s i s a minimum a t pH 3 . 7 . A so lu t ion conta in ing homatropine hydrobromide 1% and b o r i c ac id 1 . 5 5 % , l o s t 2% of i t s homatropine conten t a f te r au toc lav ing f o r 20 minutes a t 120" (12). The pH of t h i s s o l u t i o n f e l l from 4 . 5 t o 3.8 (33,34).



7 .1 I d e n t i f i c a t i o n Tests

The fol lowing i d e n t i f i c a t i o n t e s t s a r e mentioned i n t h e B.P. (15) .

- Dissolve 10 mg of homatropine hydrobromide i n 1 m l of water, add a s l i g h t excess of 10M ammonia and shake with 5 m l of chloroform. Evaporate t h e ch loro- form l a y e r t o dryness on a water ba th and add 1 . 5 m l of a 2 pe r cent w/v s o l u t i o n of mercury (11) ch lo r ide in e thanol (60%); a yellow c o l o r develops which t u r n t o r e d on warming.

- A 2 p e r c e n t w/v s o l u t i o n y i e l d s t h e r e a c t i o n charac- t e r i s t i c of a l k a l o i d s and t h e r eac t ions c h a r a c t e r i s - t i c of bromides.

- The fol lowing t e s t i s mentioned i n t h e U.S.P. (35) . The i n f r a r e d absorp t ion spectrum of a potassium bromide d i spe r s ion of i t , e x h i b i t s maxima only a t t h e same wavelengths a s t h a t of a s i m i l a r prepara- t i o n of USP Homatropine Hydrobromide Reference Standard .

- A s imple, r ap id and s e n s i t i v e t e s t €or t h e d e t e c t i o n of homatropine and o t h e r a l k a l o i d s i n phys io log ica l f l u i d s such a s s a l i v a and u r i n e i s r epor t ed (36) To the so lu t ion of a l k a l o i d s , a methanolic bromo- phenol b lue i s added t o g ive b lue s t a b l e c o l o r .

- Another c o l o r t e s t was repor ted a s fol lows (37): Homatropine i s evaporated t o dryness with a mixture of fuming n i t r i c a c i d - a c e t i c anhydride (4 : 3 ) and 5 % met hano 1 i c t e t rame thy1 ammonium hydroxide so l u t ion i s added t o a s o l u t i o n of t h e r e s idue i n acetone, t h e co lo r develops and remains cons tan t f o r 6 t o 8 minutes. I t i s poss ib l e t o determine t h e a l k a l o i d q u a n t i t a t i v e l y by measuring t h e c o l o r a t 570 ni.1.

- Other i d e n t i f i c a t i o n tes t s have been a l s o mentioned (38) *

7 . 2 Microcrvstal Tests

Homatropine hydrobromide was d isso lved i n water (10 mg i n 10 m l ) , 1 t o 2 drops of t h i s s o l u t i o n was t r e a t e d with the reagent on a microscopical g l a s s s l i d e . After s p e c i f i c time, t h e c r y s t a l s were microscopica l ly examined (39) .


7.2.1 Wagner's reagent was added t o the t e s t so lu - t i o n , minute irregular brown b lades were formed a f te r 2-3 minutes (F ig . 9 ) .

7.2.2 P i c r i c a c i d was added t o the t e s t s o l u t i o n , r a d i a t i n g rods and i r r e g u l a r b lades were formed a f t e r 10 minutes (F ig . 10 ) .

7.2.3 Dragendroff ' s reagent was added t o t h e t e s t s o l u t i o n , minute i r r e g u l a r orange c r y s t a l s were formed a f t e r 1-3 minutes (Fig. 11 ) .

Other microcrys ta l t e s t s have a l s o been r epor t ed (17,40,41).

7.3 T i t r i m e t r i r Determinations

7.3.1 Aqueous

The fo l lowing assay i s mentioned i n U.S.P (35)

mide, accu ra t e ly weighed, i n water t o make 50 m l and mix. Transfer 10 m l of t h i s s o l u t i o n t o a beaker , add 5 m l of sodium hydroxide TS, and hea t t he s o l u t i o n j u s t t o b o i l i n g . Add 10 m l of d i l u t e n i t r i c a c i d (1 i n 15) , add water t o make 50 m l , and cool i n an ice ba th . Concomitantly add 5 m l o f d i l u t e n i t r i c a c i d (1 i n 15) t o a second 10 m l po r t ion of t h e s o l u t i o n of Homa- t r o p i n e Hydrobromide, add water t o make 50 m l , and cool i n an i c e ba th . Add 1 drop of n i t r o - phenanthrol ine TS t o each s o l u t i o n and while keeping the s o l u t i o n s co ld , t i t r a t e wi th O.OSN c e r i c ammonium n i t r a t e u n t i l t h e pink c o l o r is d ischarged . Each m l o f t h e d i f f e r e n c e i n volumes of 0.05N c e r i c ammonium n i t r a t e r equ i r ed i s equ iva len t t o 8.907 mg of CI6HZ1NO3.HBr .

aqueous 0.01 M-tetraphenylborate , with t e t r a - bromophenolphthalein e t h y l e s t e r as an ind ica - t o r i n 1 , 2-dichloroethane (42).

hydrobromide i n g a l e n i c a l p repa ra t ions wi th sodium tetraphenylboron was descr ibed (43) .

- Dissolve about 400 of Homatropine Hydrohro-

- Homatropine was determined by t i t r a t i o n wi th

- A po ten t r iome t r i c t i t r a t i o n of homatropine


Fig, 9. Hornatropine HBr with Wagner's reagent

~~

Fig. 10. Hornatropine HBr with picric acid reagent

a-

Fig. 11 Hornatropine HBr with Dragendorff's reagent


- Homatropine hydrobromide was t i t r a t e d i n dimethyl sulphoxide medium with 0.1 M AgN03. End-point was determined by conductometric, po ten t iomet r ic and po la r ime t r i c techniques , and t h e r e s u l t s were s a t i s f a c t o r y by a l l t h r e e techniques. Amounts of 0 .1 mmol (44) could no t be determined.

7.3.2 Non-aqueous

- The fol lowing assay i s mentioned i n t h e B.P. (15) * Dissolve 0.3 gm in 20 m l of anhydrous g l a c i a l a c e t i c ac id , add 10 m l o f mercury (IT) a c e t a t e so lu t ion and ca r ry out Method I f o r non-aqueous t i t r a t i o n , Appendix VIII A, determining t h e end-point po ten t iome t r i ca l ly . Each m l of 0 .1 M p e r c h l o r i c ac id VS i s equiva len t t o 0.03563 gm of C16H21N03.HBr.

- An a l t e r n a t i v e method involves d i r e c t t i t r a - t i o n of homatropine hydrobromide wi th perchlo- r i c ac id i n a c e t i c anhydride medium us ing naphthalbenzein o r Sudan red B as i n d i c a t o r (45) - Other non-aqueous t i t r a t i o n has been a l s o repor ted (46).

-

7.4 Complexometry

- P r e c i p i t a t i o n of homatropine a s a thal ium complex us ing one p a r t 0 .1 N t ha l l i um s u l f a t e and 3 p a r t s 0 .1 N iod ine i n 0.15 M potassium iodide so lu t ion . The p r e c i p i t a t i o n i n a n e u t r a l medium i s most s e n s i - t i v e . The use of t h i s p r e c i p i t a t i o n reagent f o r an i n d i r e c t de te rmina t ion of small amounts of homatro- p ine i s based on t h e determinat ion of t ha l l i um i n t h e p r e c i p i t a t e (47) .

Another method u t i l i z i n g merceuric-potassium iod ide reagent i n 0 .3 sodium hydroxide so lu t ion was repor- t e d (48 ) . The determinat ion i s based on t h e ex t r ac - t i o n of t he p r e c i p i t a t e formed with chloroform. The combined e x t r a c t s a r e evaporated and water and 0.1M- s i l v e r n i t r a t e a r e added. Methylthymolblue i s used a s i n d i c a t o r and s u f f i c i e n t 20% hexamethylenetetra- mine so lu t ion i s added t o g ive a b lue co lo r . The mercury i n t h e so lu t ion i s then t i t r a t e d with 0.05M EDTA t o a yellow end poin t .

-


7 . 5 Polarographic

- The osc i l l opo la rograph ic behaviour of homatropine and o the r a l k a l o i d s was repor t ed A dropping mercury e l ec t rode served a s a p o l a r i z a b l e e l e c t r o d e , and a g raph i t e one a s a re ference . The depo la r i z ing p o t e n t i a l s were r e f e r r e d t o t h e p o t e n t i a l s of T1 ions . Semiquant i ta t ive determina- t i o n from t h e depth of the c h a r a c t e r i s t i c Cuts- in on t h e curve is poss ib l e i n a l k a l i n e s o l u t i o n s .

Another o sc i l l opo la rograph ic method f o r i d e n t i f i c a - t ion of homatropine hydrobromide i n a l k a l o i d a l mixtures was a l s o descr ibed (50).

( 4 9 ) .

-

7.6 Spectrophotometric methods

7.6.1 Colorimetry

- A co lo r ime t r i c method f o r t h e de te rmina t ion of homatropine and o t h e r r e l a t e d a l k a l o i d s was r epor t ed (51) . The method i s based on t h e n i t r a t i o n of t h e drug with a s o l u t i o n of 20% potassium n i t r a t e i n concentrated s u l f u r i c ac id a t 50-60' f o r 30 minutes. The product is made a l k a l i n e with hot 20% sodium hydroxide and t h e c o l o r which develops i s measured.

t ion of homatropine hydrobromide and methobromide and t h e i r degradat ion products i n ga l en i - c a l p repa ra t ions was r epor t ed ( 5 2 ) . The method i s a s fol lows:- To 5 ml of prepared so lu t ion (z = 3 mg of a lka lo id ) i n a 5Q m l f l a s k placed i n an i c e - water ba th , add s a t u r a t e d aqueous hydroxylammonium ch lo r ide s o l u t i o n (1 ml) and 10 .5 M potasium hydroxide (1 ml) , mix and se t a s i d e f o r 1 h r . Add 4 M hydrochlor ic a c i d ( 2 ml) t o give pH 1 . 2 t o 1 . 4 , m i x , add 0 . 3 7 M F e r r i c ch lo r ide s o l u t i o n i n 0 .1 M hydrochlor ic ac id (1 ml) and mix again. Remove t h e f l a s k from t h e ba th , a l low t h e evolu t ion of gas t o subside and measure t h e e x t i n c t i o n o f t h e s o l u t i o n a t 540 nm.

Absorpt iomet r ic de te rmina t ion of homatropine hydrobromide i n eye drops (53) ; D i lu t e 2 . 5 m l of sample t o 25 m l and t o 1 m l of t he r e s u l t i n g s o l u t i o n [0 .25 t o 2 mg of

- A s t a b i l i t y i n d i c a t i n g assay f o r t h e determina-

-


homatropine hydrobromide] add 2.5 m l o f water, 3 m l of 5% sodium hydroxide s o l u - t i o n and a f t e r 5 minutes, 3 .5 m l of Fol in reagent . Af te r 10 minutes , measure t h e e x t i n c t i o n of t h e b lue s o l u t i o n wi th use of a r e d f i l t e r . The s e n s i t i v i t y of t h e method i s 0.25 mg i n t h e f i n a l s o l u t i o n t h e mean e r r o r i s + 3 % .

homatropine hydrobromide has been a l s o r epor t ed (54). Dissolve 1 mg of sample i n 100 m l of water and t o 1 m l of t he s o l u t i o n add 3 m l o f mM-bromocresol pu rp le and 2 m l o f b u f f e r s o l u t i o n (pH 4 ) . Ex t r ac t t he co lo red 1:l product i n t o chloroform (2x5 ml) ; d i l u t e t h e combined e x t r a c t s t o 10 m l wi th ch loro- form and measure t h e absorbance a t 405 t o 410 nm. Hydrolysis products of t he drug does n o t i n t e r f e r e u n l e s s present i n t h r e e f o l d amounts. The r e l a t i v e e r r o r of t h e method does not exceed 2 1%.

t r o p i n e by c i s - a c o n i t i c anhydride has been descr ibed (55,56). I t i s as fo l lows: The sample (0.05 gm) was d isso lved i n 50 m l t o luene and from t h i s s o l u t i o n , t h e s t a n - dards a t 20,50 and 100 y / m l were prepared. Each s tandard ( 2 m l ) was d i l u t e d with 2 m l to luene and 1 m l r eagent prepared by d i s - so lv ing 0.25 gm c i s - a c o n i t i c anhydride i n 100 m l r e d i s t i l l e d a c e t i c anhydride. The s o l u t i o n s were hea ted f o r 45 minutes on a steam ba th and cooled f o r 15 minutes i n t h e dark. Then were d i l u t e d t o 10 m l . wi th 20: 80 a c e t i c anhydride- toluene mixture and absorbancy measured a t 535 nm. Aqueous s o l u t i o n s of t h e s a l t was b a s i f i e d wi th ammonia and e x t r a c t e d with to luene .

- Homatropine was determined us ing t e t r a - bromophenolphthalein e t h y l e s t e r a t pH 8-10.5 and the a d d i t i o n compound formed was e x t r a c t e d wi th 1,2-dichloroethane and e x t i n c t i o n i s measured a t 560 nm (57).

- Color imet r ic de te rmina t ion of t h e tha l ium complex of homatropine hydrobromide us ing c r y s t a l v i o l e t was a l s o r epor t ed (58) .

- Another c o l o r i m e t r i c de te rmina t ion of

The method i s as fo l lows:

- Other co lo r ime t r i c de te rmina t ion of homa-

7.7 A

7 . 7 . 1 Paper chromatography

Homatropine can be de t ec t ed by paper chromatography. chromatographic condi t ions used €or homatropine.

Table 8 inc ludes paper

Table 8. Paper Chromatography of Hornatropine

Solvent system

s a t u r a t e d with octanol - 5% aqueous ammonia

- 4.8 gm of c i t r i c a c i d i n a mixture of 130 water and 870 of n- But an o 1

(pH 4.58) - a c e t a t e bu f fe r

- BuOH- AC OH- H 2O (40 : 10 : 50) BUOH - HCO 2H- H2 0

BuOH-C2H5C 02H-H20 (10: 1 : 10)

(10: 1: 10)

Condit ions Detect ing reagent R va lue Reference -€ - Whatman No. 1 paper impreg- - (59)

na ted wi th 7 % methanol ic oc tanol . Descending t echn i - que. Drying t h e paper i n a i r f o r 5 t o 10 minutes.

Paper Whatman No.1 dipped U . V . o r Iodop la t ina t e 0 . 3 C l a r k i n 5% so lu t ion of sodium (17) dihydrogen c i t r a t e and d r i e d

Whatman No.1 o r No.3 impre- Location under U . V . 0.96 Clark gnated by dipping i n 10% Light (254 mu) o r (17) s o l u t i o n of T r ibu ty r in i n Iodop la t ina t e spray acetone and dry ing i n a i r .

Descending technique 0.2% Iodine so lu - t i o n spray

(60)

Other paper chromatography has a l s o been repor ted (61).

7.7.2 Thin l aye r chromatography (TLC)

Homatropine can be de t ec t ed by t h i n l a y e r chromatography. Table 9 i nc ludes t h i n l a y e r chromatographic cond i t ions used f o r homatropine.

Table 9. Thin l a y e r chromatography of Eomatropine

Solvent system Condit ions Detec t ing r eagen t R va lue Reference f-

Strong ammonia s o l u t i o n : s i l i c a ge l G a c i d i f i e d i o d o p l a t i n a t e 0.15 Clark methanol (1.5 : 100) o r under U . V . (254 nm) ( 17)

Ethanol-pyridine-water alumina i o d o p l a t i n a t e spray 0.87 (65) (1 : 6 : 4)

Chloroform-acetone-diethylamine Kiese lge l GF Dragendorf f spray ( 5 : 4 : 1 ) 254 Acetone - wat e r - 2 5 % ammonia Kiese lge l G Dragendorff e t h y l a c e t a t e - (64)

( 9 0 : 4 : 6 )

The upper phase of But ano 1 - a c e t i c ac id-wat e r Ce 1 l u l ose Dragendorff spray - (65) ( 10 : 1 : 5)

s o l u t i o n reagent

Other T.L.C. f o r homatropine has been a l s o r epor t ed (66) .


7.7 .3 E lec t rophores i s

- Elec t rophores i s of homatropine was performed on Whatman No.1 paper (18x46 cm) a t 8 v./cm f o r 3 h r s . i n t h e presence of un ive r sa l buf- f e r . The r e l a t i v e displacements a t pH 2 . 3 , 4 .3 , 6 .4 , 8 . 2 , 10.5 and 11.4 were 63,54, 104,105,100,16 r e s p e c t i v e l y (67,68) .

have been repor ted (69) . - Other e l e c t r o p h o r e s i s f o r s e v e r a l a l k a l o i d s

7.7.4 Column chromatography

Chromatographic a s say of homatropine hydrobromide and o t h e r r e l a t e d a l k a l o i d s a r e quan- t i t a t i v e l y e l u t e d wi th 95% acetone from a column packed with b a s i c alumina (70).

The p u r i t y of homatropine hydrobromide can be determined on alumina column as fo l lows

Homatropine hydrobromide (0.1 gm) was e l u t e d over a column packed with b a s i c alumina (5 gm) by us ing aqueous acetone as so lven t . After e l u t i o n , t h e e l u a t e was d i l u t e d wi th water and homatropine hydrobromide i s t i t r a - t e d wi th 0.1 N hydrochlor ic ac id us ing brom- ophenol b lue as an i n d i c a t o r .

(71) '

7.7.5 Gas chromatography ( G C )

The gas chromatography f o r homatropine a r e l i s t e d i n t a b l e (lo).

Table 10. The GC of Homatropine

Retent ion time __ Column condi t ion C a r r i e r gas Detector

2.5% SE-30 on 80-100 mesh chromosorb W (5 F t x 4mm i n t e r n a l d iameter ) . Column temperature 225'.

3% XE-60 s i l i c o n e n i t r i l e polymer on 100-120 mesh Chromosorb W . Column t enpe ra tu re 225".

5% SE-30 on 60-80 mesh Chromosorb W AW (5 f t x 1/8 diameter) Column temperature 230'.

3% J X R on s i l a n i s e d G Chrom P on 100-120 mesh (1.8m x 2mm) Column temperature 250".

5% SE-30 on 60-80 mesh ac id washed Chromosorb W a t 190,210,230, 250-270°

15 % QF-1 on 60-80 mesh, Gas Chrom(R)Q. , (1.5m x 3 . 2 mm) column temperature 160".

Nitrogen (SO ml/min.)

Nitrogen (50 ml/min.)

Nitrogen (30.7 ml/min.)

Nitrogen (50 ml/min.)

Nitrogen (50 ml/min)

F . I .D . hydrogen 0.36 r e l a t i v e (50 ml/min.) t o codeine a i r (300 ml/min.)

F . I . D . hydrogen 0.39 r e l a t i v e (50 ml/min.) t o codeine a i r (300 ml/min.)

F.I.D. hydrogen 0.44 r e l a t i v e (22 ml/min.) t o codeine

F . I . D . -

F . I . D . -

F.I.D. 4 . 2 (min)

Ref.

C l a r k (17)

-

Clark (17)

Clark (17)

(73)

Other gas chromatographic a n a l y s i s have been r epor t ed (75-78)


7.7.6 High Pressure Liquid Chromatography (HPLC)

F e l l _ _ e t a l . (73) have r epor t ed an a n a l y s i s of homatropine hydrobromide by reversed-phase h igh-pressure l i q u i d chromatography. Homatro- p ine hydrobromide was determined on a column of Hypersi l ODS ( 5 um) with 50 mM. Sodium a c e t a t e i n 10 mM-tetrabutylammonium s u l f a t e (pH 5 . 5 ) - a c e t o n i t r i l e ( 3 : l ) as t h e mobile phase and d e t e c t i o n a t 254 nm. The i n t e r n a l s tandard was p - t o l u i c a c i d . R e c t i l i n e a r c a l i b r a t i o n graph was obta ined f o r homatropine hydrobromide ( i n eye drops) i n the range 0 t o 4 mg ml-1.

S t u t z and Sass (80) have descr ibed a high-speed, high p res su re l i q u i d chromatography of t ropane a l k a l o i d s inc lud ing homatropine. The compound was separa ted on a s t a i n l e s s - s t e e l column (1 meter x 4 . 6 mm) packed with sil-X absorbent with 28% aqueous ammonia te t rahydrofuran (1: 100) as t h e so lven t and wi th a column i n l e t p re s su re of 500 l b pe r sq. inch . A d i f f e r e n t i a l r e f r a c t i v e index d e t e c t o r and a UV d e t e c t o r ope ra t ing a t 254 nm were used t o monitor t h e e l u a t e .

ACKNOWLEDGEMENT

The au tho r s would l i k e t o thank Mr. Uday C. Sharma of Department of Pharmdcognosy, c o l l e g e of Pharmacy, King Saud Univers i ty €or h i s s e c r e t a r i a l assistance i n t h e reproduct ion of t h e manuscr ipt .

286

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2 .

3.

4 .

5.

6.

7.

8.

9.

FARID J. MUHTADI ETAL.

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D . N . Gore and J . M . Adshead, J . Pharm. and Pharmacol. - 4, 803 (1952) .

K. Macek, J . Hacaperkova, and B. Kakac, Pharmazie - 11, 533 (1956).

K . C . Guven and A. Hincal . Eczac. Fak. Mecm. ( I s t a n b u l ) , - 1 ( 2 ) , 153 (1965) .

S . Ebel, W . D . Mikul la , and K . H . Weisel , D t . Apoth. Z tg . , 111 ( 2 5 ) , 931 (1971).

J. Vamos, L . Ambrus, S.G. I s tvan ; and A. Brantner , Gyogyszereszet, (Budapest) 21 ( 6 ) , 206 (1977).

L. Ambrus, J. Vamos, G. Szasz and A. Brantner? Gyogysze- r e s z e t (Budapest) , - 22 (12 ) , 467 (1978).

-

-

I . C . Di jkhuis Pharm. Weekbl. (Ned.), 104 (421, 1317 (1969) . G . B . Marini - Bet to lo and J . A . Coch Frugoni. Rend. 1st. super . S a n i t a (Uruguay) - 21, 319 (1958).

G . B . Marini - Bet to lo and Juan A. Coch Frugoni, Gazz . Chim. ( I t a l . ) - 86 , 1324 (1956).


69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

C. Apreotesei and M. Teodosiu, Farmacia (Bucharest) - 10, 321 (1962).

13. Bohme, G . Berg, H. Stamm, and E. Tauber, Arch. Pharm., - 293, 342 (1960); - 294, 315 (1961).

H. Bohme, G. Berg, H. Stamm and E. 'Tauber, Arch. Pharm., - 294 (7 ) , 447 (1961).

V . Quercia, C. Cardini and B. Tucci, Boll. Chim. farm. (Ital.), - 107 (12), 737 (1968).

K.D. Parker, C.R. Fontan, and P.L. Kirk, Anal. Chem., - 35, 356 (1963).

B.F. Gorbowski, B.J. Softly, B.L. Chang, and W.G. Haney, J. Pharm. Sci., - 62, 806 (1973).

E. Brochmann - Hanssen and C.R. Fontan, J. Chromatog.,, - 19 (2) , 296 (1965).

E. Brochmann-Hanssen and A.B. Svendsen, J. Pharm. Sci. - 51, 1095 (1962).

V. Quercia, Farmaco, Ed. prat., - 25 ( 2 ) , 122 (1970).

E. Shami, J. Dudzinski, J. Lachman., and J. Tingstand, J. Pharm. Sci., - 62 (81, 1283 (1973).

A.F. Fell, S.J.K. Johnstone, and G. Smith, J. Pharm. Pharmacol., 31 (Suppl.) 20 (1979).

M.H. St.utz, and S. Sass; Analyt. Chem., - 45 (12), 2134, (1973).

MEBENDAZOLE

A . A . AL-Budrz* und M . Td/ t iq* *

*Vepu,ahtmeMA: 0 6 PhatunuzceuLlcal C h m i ~ f i y and VcpahXment** 0 6 Phahmacology, Coflecje ozj Phahmucy, King Saud UnivehhLty, P.O. Box 2457, Riyudh-11451, Saudi Ahabia.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 29 1

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

292 A. A. AL-BADR AND M. TARIQ

1.

2.

3 .

4.

5.

6.

7 .

8.

Forward, History

Description

2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Color and Taste

Physical properties

Spectral Properties

Synthesis

Pharmacokinetics

Toxicity

Methods of Analysis

8.1 Identification 8.2 Titrimetric Methods 8 .3 Spectrophotometric Methods 8.4 Chromatographic Methods 8.5 Polarographic Method 8.6 Radioimmunoassay Method

References

MEBENDAZOLE 293

Foreword, His tory

Mebendazole i s a broad spectrum an the lmin t i c agent synthesized and developed by Janssen Pharmaceutica, Research Laboratody, Beerse, Belgium. Af t e r i t s in t roduc t ion t h e r e i n 1972, t h e drug became a v a i l a b l e i n numerous coun t r i e s around t h e world, inc luding t h e United S t a t e s and Canada (1). I t produces high cure rates i n i n f e s t a t i o n s by Ascar i s , threadworms (Enterobius) , hookworms (Ancylostoma and Necator Spp.) , and Whipworms (Tr i chur i s ) . I t is a l s o a c t i v e i n some (about 40%) Dwarf tapeworm (Hymenolepis) i n f e s t a t i o n s (2-5).

2 . Descr ip t ion

2 . 1 Nomenclature


(5-Benzoyl-1H-benzimidazol-2-yl)-carbamic ac id methyl ester. 5-Benzoyl-2-benzimidazolecarbamic a c i d methyl es ter . Methyl 5-benzoyl-2-benzimidozolecarbamate. Methyl-5-benzoylbenzimidazol-2-ylcarbamate. Methyl-(5-benzoyl-1H-benzimidazol-2-yl) carbamat e. Carbamic acid,5-benzoyl-1H-benzimidzol-2-yl, methyl e s t e r . (6)

CAS Regis t ry Number

[31431-29-71 (6)

2.1.2 Generic N a m e

Mebendazole; R 17,635 (7) .

2 .1 .3 Trade Names

Propre ta ry Names

Bendrax, Besantin, Esadi rase , Equivuarm p l u s Forverm, Gendal, Granverm, Lomper, Mebendacin, Mebendozol M K , Mebendazole, Mebenix, Mebutar, Nemasole, Oxiben, Pantelmin, Parelmin, S i rben , Telmin, T r o t i l , Vermirax, Vermox, and Zakor (1,6, 8-11).


2 . 2 Formulae

2 . 2 . 1 Empirical

2 . 2 . 2 Structural

H I 0

2 .3 Molecular Weight

295.30 and 295 ( 7 E 9 ) ,

2 .4 Elemental Composition

C , 65.08%, H , 4.44%, N , 14.23%; 0, 16.25%

2.5 Appearance, Color and Taste

Off-white to slightly yellow amorphous powder and not unpleasant to taste. (10-12)

3 . Physical Properties

3 .1 Melting Point

Melts at about 290°, 288.5', above 280' with decomposition (6-8 6 l o ) .

3.2 Solubility

Almost insoluble in water, ethanol, ether, chloroform, and dilute mineral acids; readily soluble in formic acid (9 E 11).

3 . 3 Hygroscopicity

Mebendazole is not hygroscopic and it is stable in air.

MEBENDAZOLE 295

3 .4 Ex t rac t ion

Mebendazole i s e x t r a c t e d by chloroform from aqueous a l k a l i n e s o l u t i o n s (11) .

4 . S p e c t r a l P r o p e r t i e s

4 . 1 U l t r a v i o l e t Spectrum

The u l t r a v i o l e t abso rp t ion spectrum o f mebendazole obtained from a s o l u t i o n i n n e u t r a l methanol i n t h e reg ion of 200 t o 400 nm using DMS 90 spec t rophoto- meter is shown i n F igure 1. maxima a t about 210, 247 and 310 nm and two minima a t 228 and 273 nm were observed.

Three abso rp t ion

Clarke (11) r epor t ed t h e fol lowing:

Mebendazole i n 0.05% formic a c i d i n isopropanol; maxima a t 248 nm ( E l % , 1 cm 1005) and 313 nm ( E l % , 1 cm 518) , minima a t 230 nm and 275 nm.

Mebendazole i n 0.01 N sodium hydroxide i n isopropa- no l , maxima a t 270 nm ( E l % , 1 cm 802) and 355 nm ( E l % , 1 cm 653), minima a t 245 nm and 302 nm. Mebendazole i n 0 . 1 N hydrochlor ic a c i d i n i sopro- panol , maxima a t 234 nm (El%, 1 cm 1000) and 288 nm (El%, 1 cm 524) , minima a t 215 nm and 266 nm.

4 . 2 I n f r a r e d Spectrum

The i n f r a r e d spectrum of mebendazole i s presented ; i n F igure 2. The spectrum was obta ined from K B r d i s c us ing a Pye Unicam SP 1025 i n f r a r e d spectrophotometer . The s p e c t r a l ass ignments are presented i n t h e fo l lowing t a b l e .

2% A. A. AL-BADR AND M. TARIQ

200 250 300Wavelength 350 400

(nm)

FIGURE I : ULTRAVIOLET SPECTRUM OF MEEENDAZOLE IN METHANOL

C 0 e - .- E 20. m C

;" 0;

FIGURE 2 . I N F R A R E D S P E C T R U M OF M E B E N D A Z O L E ( KBr d i s c )

*20

0


-1 Frequency cm

34 15

2500-2950

1720

1650

1530 1600 I 141 0-1460

700- 765

1 878

900)

Assignment

NH s t r e t c h

CH s t r e t c h

C = 0 (amide) s t r e t c h

C = 0 s t r e t c h

C = C s t r e t c h

CH3-0 (C-0) s t r e t c h

Monosubsti tuted benzene

1 , 2 , 4 T r i s u b s t i t u t e d benzene

Clarke (11) r epor t ed t h e p r i n c i p a l peaks (KBr d i s c ) a r e 705, 1260, 1590, 1635 and 1730 cm-1.

1 4 . 3 'H Nuclear Magnetic Resonance ( H NMR) Spectrum

1 The H NMR spectrum of mebendazole i s shown i n F igure 3. The drug is d i s so lved i n T r i f l u o r o - a c e t i c ac id (TFA) and i ts spectrum determined on a Varian T60 A NMR spec t rometer us ing TMS ( t e t r a - methyls i lane) as t h e i n t e r n a l s tandard . Assignment of t h e chemical s h i f t s t o t h e d i f f e r e n t p ro tons is shown be low:

Chemical S h i f t M u l t i p l i c i t y Proton Assignment PPM (6)

7 .5 - 8 . 3 mu 1 t i p 1 e t Aromatic

4.08 s i n g l e t methyl

FIGURE 3: PROTON MAGNETIC RESONANCE SPECTRUM OF MEBENDAZOLE IN TRIFLOUROACE T I C

A C I D ( T F A I


4.4 I 3 C Nuclear Magnetic Resonance (13C NMR) Spectrum

The I3C NMR s p e c t r a of mebendazole i n formic a c i d us ing TMS ( t e t r ame thy l s i l ane ) as an i n t e r n a l s tandard are obta ined us ing a Joel FX 100 MHz spectrometer a t an ambient tempera ture . F igu res 4 and 5 r ep resen t t h e 1H-decoupled and of f - resonance s p e c t r a r e spec t ive ly .

/ 0 .,

The carbon assignments a r e based on chemical s h i f t va lues and by comparison with model compounds and are shown below:

Chemical S h i f t PPM (6)

57.5

155.8

148.4

139.2

134.4

118.2

130.5

130.9

115.8

200.5

136.7

132.9

131.3

136.3

M u l t i p l i c i t y C a r bon Ass i gnm en t

q u a r t e t

s i n g l e t

s i n g l e t

s ing 1 e t

s i n g l e t

double t

s i n g l e t

doublet

double t

s i n g l e t

s i n g l e t

double t

double t

double t

c1

c2

c3

c4

c 5

c 7

c9

c l o 5 1

‘6

‘12 and ‘16

‘13 and ‘15

‘14

MEBENDAZOLE 301

FIGURE L : PROTON OECOUPLEO CARBON-13 NMR SPECTRUM OF MEBENDAZOLE IN FORMIC ACID

FIGURE 5 : OF-RESONANCE CARB3N-I3 NMR SPECTRUM OF MEBENDAZOLE IN FORMIC ACID


4 . 5 Mass Spectrum and Fragmentometry

F igure 6 shows t h e 70 eV e l e c t r o n impact ( E I ) mass spectrum obta ined on Varian MAT 311 mass spec t ro - meter using ion source p re s su re of 10-6 Torr , ion source temperature of 18OoC and an emission c u r r e n t of 300 PA. The spectrum is dominated by m/e 186 ion (base peak) r e s u l t i n g from t h e l o s s of CH30H and C6H5 fragments. f ragmentat ion and t h e mass/charge r a t i o s of t h e major fragments i s given i n Scheme 1 (a ar,d b ) .

A proposed mechanism of

Chemical i o n i s a t i o n (CI) spectrum i s presented i n F igure 7 and i s obtained on a Finnigan 4000 mass spectrometer with ion e l e c t r o n energy of 100 eV, ion source p re s su re o f 0 . 3 Torr , ion source temperature of 15OoC and emission c u r r e n t of 300 PA.

The mass s p e c t r a l assignment of t h e prominent i ons under C I cond i t ions i s given below:

m/e Species

296 M+ + 1

2 95 M+

218 M+ - C6H5

MEBENDAZOLE

J

303

324.2 2180

lBO(

50.C

208 \

2

336.2 235.8 251 6 2Ul

1

,330

50.0

M / E

I05

I86

263

218 2 95

32832.

FIGURE 7 MASS SPECTRUM OF MEBENDAZOLE (c l )

b .+

G a +. H

&@- N?-NH-c-o -cH3

m/e 295 '\ O+ - co

7 0-C' ____)

m/e 77

E [@!$==y m/e 105 Ill O 1

-+ 0 H NH - \\ C - OCH3 -CH30H [ ,i,@-yNH-C -A* I -co m/e 158 L -

m/186 /4 m/e 218 m/e 130

Scheme l a : Proposed mechanism of fragmentation of mebendazole.

m/e 295 m/e 295

OI +

t N7rNH-C = O -co W -CH30H s 0 '& m/e 263 4 m/e 235

Figure lb : Proposed mechanism of f ragmenta t ion of mebendazole.

A. A. AL-BADR AND M. TARIQ

5. Synthesis

1- Mebendazole was prepared (13) from 3,4-

according to the following scheme. (NH2)2C,H,CoC6H5 and NHz(CH3S)C = N - COOCH3

0

I I C

0

0

U

NH3 - HN 0 v II

c' C-NH-C-0-CH3

3

.CH3 11 ,C = N-C-0-CH3

H2N, 11

s, 0 CH3

NH CH3

-c-0- II 0

MEBENDAZOLE 307

2- Mebendazole was also prepared in 52% yield by decomposition of methyl 7-benzoyl-lH-2,1,4- benzothiadiazine-3-y1 carbamate in methanol with 2 N HC1. The latter compound is prepared in two stages from 4-benzoyl-2-ni t roani l ine (14) .

Two steps *

0 CH 30H I I - CH3 2N HC1

H 0

6. Pharmacokinetics

6.1 Absorption, Distribution, Metbolism and Extraction

Mebendazole is poorly absorbed by gastrointestinal tract after oral administration (10). Only 5-10% of the orally administered frug is found in the blood (1). The poor intestinal absorption of mebendazole is of therapeutic advantage in the treatment of intestinal helminthis. However, for tissue dwelling organism high oral doses are required (15) .

The absorption of drug in man is markedly enhanced when the drug is used together with the meals, as concomitent fat ingestion ehances its intestinal absorption. Following oral administration peak plasma level reach in 2-4 hours (16-18).

Munst -- et a1 (19) reported a significant variation in plasma mebendazole level in human subjects following oral ingestion. According to this study the plasma half-life was ranging between 1.5 and


5.5 hours . adminis t ra t ion were de tec ted a t 30 minutes t o 2 hours of t he dose. Mebendazole is metabolized pr imar i ly i n l i v e r by decarboxylat ion (20) , t h e major metabol i te was 2 -amino-5 (6-benzimidazolyl phenylketone. Brai thwaite -- e t a1 (21) repor ted t h a t mebendazole is more l i p o p h i l i c than any of i t s metabol i tes . The d i s t r i b u t i o n s tudy by same author showed t h a t 63.3% of mebendazole was d i s t r i b u t e d i n plasma and 36.7% i n c e l l u l a r f r a c t i o n . of t h e drug i n plasma (95%) was present i n t h e p r o t e i n bound form. i n l i v e r was considerably h igher as compared t o the f a t t y t i s s u e . G i lba ld i and Perrier (22) repor ted t h a t s teady s t a t e AUC of mebendazole is independent of absorpt ion r a t e i n human s u b j e c t s . 90% o f t h e o r a l l y adminis tered dose of mebendazole i s el iminated pr imar i ly a s unchanged drug i n faeces. About 5-10% of t h e adminis tered drug may be recovered i n t h e form o f i t s conjugates , and metabol i tes i n t h e u r ine (11) .

In r a t s about 85% of an I . V . dose was e l imina ted with the b i l e and remainder with t h e u r ine . The major i ty o f t h e dose was recovered a s conjugated metabol i tes (23-24). In another s tudy (25), t h r e e metabol i tes of mebendazole were i s o l a t e d from t h e b i l e of r a t s and i d e n t i f i e d as : methyl-5- (a-hydroxybenzyl)-2-benzimidazole carbamate; 2- amino-5- (a-hydroxybenzyl) benzimidazole and 2-amino- 5-benzoylbenzimidazole.

The peak concent ra t ion following o r a l

Most

The concent ra t ion of mebendazole

Approximately

7. Toxic i ty

Gas t ro in t e s t ina l symptoms including cons t ipa t ion have been repor ted in f r equen t ly fol lowing use of mebendazole (26). High doses may on occas io lsbe assoc ia ted with mild c o l i c pain o r d ia r rhoea as repor ted by Chavarr ia ( 2 7 ) using doses of upto 300 mg B I D i n t h e t reatment of t aenias i s . Acute and chronic t o x i c i t y s t u d i e s i n animals i n d i c a t e a wide range between the rapeu t i c and t o x i c doses . The LD50 (mg/kg o r a l l y ) was more than 640 mg/kg in r a b b i t s and dogs and more than 1280 mg/kg i n mice and rats (11). S ign i f i can t haematologic, biochemical, o r patho- l o g i c abnormali t ies were not found i n animals given 40 mg/kg d a i l y f o r 13 weeks but were noted i n r a t s

MEBENDAZOLE 309

given a d a i l y dosage of 130 mg/kg. con t r a ind ica t ed i n pregnant women because i t has shown embryotoxic and t e r a t o g e n i c a c t i v i t y i n s i n g l e p e r o r a l dose a s low as 10 mg/kg (18, 28 , 29 ) . Rare cases o f leukopenia i n human s u b j e c t s fol lowing t h e admin i s t r a t ion of mebendazole was a l s o reporqed ( 3 0 ) . In high doses 40-50 mg/kg/day, t h e drug has been a s soc ia t ed wi th severe i r r e v e r s i b l e neut ropenia . No s i g n i f i c a n t CNS e f f e c t s i n t h e p a t i e n t s t ak ing mebendazole therapy has been repor ted except s l i g h t headache and d i zz iness (31) .

Mebendazole i s


8 . 1 I d e n t i f i c a t i o n

In f r a red Spectrum

The i n f r a r e d absorp t ion spectrum of a potassium bromide d i spe r s ion o f i t , p rev ious ly d r i ed , e x h i b i t maxima only a t t h e same wavelength as t h a t of a s i m i l a r p repa ra t ion of USP mebendazole RS ( 3 2 ) .

X-ray Di f f r ac t ion

The X-ray powder d i f f r a c t i o n d a t a f o r i d e n t i - fy ing mebendazole and o t h e r an the lmin t i c s have been obtained by d i f f r ac tomete r and Debye - Scherrer camera techniques (33) . The d a t a were t abu la t ed i n terms of t h e l a t t i c e spacings and t h e r e l a t i v e i n t e n s i t i e s o f t h e l i n e s . Pa t t e rns us ing t h r e e d i f f e r e n t X-ray wavelengths with t h e camera method a r e compared with each o t h e r and wi th t h e d i f f r ac tomete r p a t t e r n .

8.2 Ti t r imetr ic Method

USP XX (1980) (32) descr ibed t h e fol lowing t i t r i m e t r i c method f o r t h e assay o f mebendazole:

Dissolve about 225 mg of mebendazole, a c c u r a t e l y weighed, i n 30 m l o f g l a c i a l a c e t i c a c i d . T i t ra te with 0 .1 N p e r c h l o r i c ac id VS, determining t h e end-point po ten t iome t r i ca l ly , us ing a calomel- g l a s s e l e c t r o d e system. Perform a blank determina- t i o n , and make any necessary co r rec t ion . Each ml

3 10 A. A. AL-BADR AND M. TARIQ

of 0 .1 N p e r c h l o r i c ac id i s equiva len t t o 29.53 mg

Of C16H13N303'

Wahbi and Onsy, (34) developed t h e two t i t r imet r ic methods t o determine t h e mebendazole i n pharma- c e u t i c a l p repa ra t ions . t i t r a t i o n methods are mentioned a s fol lows:

Di rec t t i t r a t i o n and back

a) Di rec t T i t r a t i o n Method

200 Mg o f mebendazole was taken i n dry con ica l f l a s k and d i s so lved i n 2 m l o f 99% formic a c i d followed by t h e add i t ion of 60 m l o f g l a c i a l a c e t i c a c i d . This s o l u t i o n was t i t r a t e d wi th 0 . 1 N p e r c h l o r i c ac id i n g l a c i a l a c e t i c a c i d using c r y s t a l v i o l e t a s i n d i c a t o r , development o f a p e r s i s t e n t green c o l o r was designated a s end p o i n t . Blank determinat ion was performed and the necessary co r rec t ions were made. The c a l c u l a t i o n was done by us ing t h e equivalence f a c t o r which is 1 m l o f 0 .1 N p e r c h l o r i c a c i d i s equal t o 29.53 mg of mebendazole.

b) Back-Titrat ion Method

200 Mg of mebendazole was taken i n dry con ica l f l a s k , t h e powder was d i s so lved in 20 m l o f 1 M p e r c h l o r i c ac id i n g l a c i a l acetic a c i d . This so lu t ion was t i t r a t e d with 0 .1 N sodium a c e t a t e i n g l a c i a l a c e t i c a c i d , c r y s t a l v i o l e t was used a s i n d i c a t o r , development of b l u i s h green a s i n d i c a t o r . reproducible wi th s tandard dev ia t ion o f 0 .3%.

Mebendazole ( i n amounts upto 250 mg) i n anhydrous a c e t i c ac id medium can be t i t r a t e d with 0 . 1 M - perch lo r i c ac id ( a l s o i n a c e t i c ac id) us ing 0.5% c r y s t a l v i o l e t s o l u t i o n a s i n d i c a t o r . Resul t s agree with those obtained by spectrophotometry a t 313 nm (35) .

These methods a r e

8 . 3 Spectrophotometric Methods

8 .3 .1 U l t r a v i o l e t Spectrometr ic Methods

Pate1 e t a1 (36) repor ted two spectrophotometric methods f o r t h e e s t ima t ion o f

--

MEBENDAZOLE 311

mebendazole i n raw m a t e r i a l . A s o l u t i o n of t h e sample i n formic a c i d was d i l u t e d with a mixture of chloroform : methanol (17:3). Thin-layer chromatography was run on s i l i c a g e l i n chloroform : e t h y l a c e t a t e ; acetone : formic ac id (70:80:9.5:0.5), developed f o r 15 cm and d r i e d f o r 30 minutes at 115O. The drug i s then e x t r a c t e d i n t o chloroform and measured a t 313 nm.

In another methods, a so lu t ion o f t h e drug i n formic ac id i s d i l u t e d with isopropanol and t r e a t e d with MeOH and 30% potassium hydroxide. measured a t 420 nm.

After 30 min. t h e absorbance was

Wahbi and Osny (34) repor ted a spectrophotometric method o f a n a l y s i s of mebendazole i n t a b l e t s with a s tandard dev ia t ion of 1 .4%. of mebendazole were taken i n t o 50 m l s tandard f l a s k followed by 10 m l o f 70% p e r c h l o r i c a c i d . 10 minutes and f i l t e r e d through a d ry f i l t e r paper . 100 m l f lask and d i l u t e d t o volume wi th d i s t i l l e d water . The absorbance of t h i s so lu t ion was measured i n a 1 cm ce l l a t 288 nm wavelength us ing a spectrophotometer. 0 .2 M 1 o f 70% p e r c h l o r i c ac id d i l u t e d wi th 100 m l of d i s t i l l e d water was used a s b lank . Ca lcu la t ion of concent ra t ion was done by t ak ing 566 a s t h e (E 1%, 1 cm) value a t 288 nm o r by us ing a s u i t a b l e c a l i b r a t i o n curve.

Powdered t a b l e t s equiva len t t o 50 mg

The mixture was shaken f o r

1 M 1 o f f i l t r a t e taken t o a

8.3 .2 Phosphorescence Method

Mebendazole has been repor ted t o show good phosphorescence a t room temperature when determined on Whatman 42 f i l t e r paper us ing Pb ( I V ) and T1 ( I ) a s phosphorescence enhancing agents . In t a b l e t s , t h i s method gave a recovery o f 97.6% of mebendazole (37) *


8 . 3 . 3 F luo r ime t r i c Method

Abdel Fa t t ah -- e t a1 (38) have r epor t ed a f l u o r i m e t r i c de te rmina t ion method of mebendazole i n pharmaceut ical dosage forms a f t e r a l k a l i n e hydro lys i s . In t h i s method 2 t o 5 mg of mebendazole is d i s so lved i n 10 m l of 1 M sodium hydroxide and hea ted f o r 1 h r on a b o i l i n g water ba th . After d i l u t i o n with methanol, 1 M sodium hydroxide (49:1) , t h e s o l u t i o n was spo t t ed on t o Whatman 42 f i l t e r paper and d r i e d a t 65 f o r 5 t o 10 minutes . The f luorescence was measured a t 460 nm ( e x c i t a t i o n a t 365 nm). The l i m i t of d e t e c t i o n was 0 .1 f o r mebendazole with r e c t i l i n e a r c a l i b r a t i o n graphs upto 10 and 50 ppm ( i n t h e f i n a l s o l u t i o n ) . For 8 ng o f mebendazole p e r spo t , t h e c o e f f i c i e n t o f v a r i a t i o n was 9.5% (n = 19) . For pharmaceut ical formula t ions , recovery o f mebendazole ranged from 99.04 t o 100.56%, with c o e f f i c i e n t o f v a r i a t i o n of 2.27 t o 2.97%.

8.3.4 Color imet r ic Method

Rana et& (39) have desc r ibed a method f o r c o l o r i m e t r i c de te rmina t ion of mebendazole i n t a b l e t s and suspension by adding hydroxyl amine hydrochlor ide , dicyclohexylcarbodiimide and fe r r ic ch lo r ide t o a s o l u t i o n of t a b l e t s (or a d i l u t i o n o f a suspension) i n isopropanol and formic a c i d and measuring t h e absorbance a t 520 nm. The c a l i b r a t i o n graph was l i n e a r f o r 0 .4 t o 2 . 0 pg/ml of mebendazole .

8 .4 Chromatographic Methods

8 .4 .1 Thin-Layer Chromatography (TLC)

Clarke (11) has descr ibed t h e fo l lowing TLC system f o r t h e i d e n t i f i c a t i o n of mebendazole.

Solvent system: Chloroform : methanol : formic ac id (90:5:5).

Absorbent: S i l i c a g e l HF 254 (Merck) .

MEBENDAZOLE 313

Visua l i z ing agent : Iodine vapour; d ragendorf f sp ray (Location under u l t r a v i o l e t l i g h t ) . R f : 0.53.

United S t a t e s Pharmacopeia "USPXX 1980 (32) have used TLC t o determine t h e p u r i t y o f mebendazole. The method i s descr ibed as fol lows:

Dissolve 50 mg i n 1 . 0 m l o f 98 percent formic a c i d i n a 10-ml volumetr ic f lask , add chloroform t o volume, and mix. S i m i l a r l y prepare a s o l u t i o n of USP Mebendazole RS i n the same medium having a concen t r a t ion of 5 mg pe r m l . S tandard s o l u t i o n t o a 200-ml volumetr ic f l a s k , add a mixture of chloroform and 98 percent formic a c i d (9 : l ) t o volume, and mix ( d i l u t e d Standard s o l u t i o n ) . On a s u i t a b l e t h i n - l a y e r chromatographic p l a t e , coated wi th a 0.25-mm l a y e r of chromatographic s i l i c a ge l mixture , spot 10-1-11 p o r t i o n s of t h e t e s t s o l u t i o n , t h e Standard s o l u t i o n , and t h e d i l u t e d Standard s o l u t i o n . Allow t h e s p o t s t o dry , and develop t h e chromatogram i n a so lven t system c o n s i s t i n g o f chloroform, methanol, and 98 percent formic a c i d (90:5:5) u n t i l t h e so lven t f r o n t has moved about t h ree - fou r ths o f t h e l eng th o f t h e p l a t e . developing chamber, mark t h e so lven t f r o n t , a l low t h e so lven t t o evapora te , and examine t h e p l a t e under short-wavelength u l t r a v i o l e t l i g h t . The R f value of t h e main spo t ob ta ined from t h e t e s t s o l u t i o n corresponds t o t h a t ob ta ined from t h e Standard s o l u t i o n , and no s p o t , o t h e r than t h e main s p o t , i n t h e chromatogram o f t h e t e s t s o l u t i o n is l a r g e r o r more i n t e n s e than the main spo t ob ta ined from t h e d i l u t e d Standard s o l u t i o n .

T r a n s f e r 1 .0 m l o f t h i s

Remove t h e p l a t e from t h e

8 . 4 . 2 High-pressure Liquid Chromatography (HPLC)

A s e n s i t i v e method f o r s epa ra t ion and s imultaneous de te rmina t ion o f mebendazole and hydroxymebendazole i n human plasma o r c y s t l i q u i d sample us ing f lubendazole as


i n t e r n a l s tandard by high-performance l i q u i d chromatography with electrochemical d e t e c t o r i s descr ibed (40) .

The chromatographic condi t ions : The column (4.6 mm X 5 cm) was packed with Spherisorb 5 ).rm ODS (Phase Sep. Queensferry Clwyd, U.K.). The mobile phase cons i s t ed of 20% 2-propanol i n a pH 7 . 0 phosphate b u f f e r . The e luen t was de l ive red by Kipp 9208 high-pressure l i q u i d chromatography pump, a t a flow r a t e of 2.5 ml/min. The electrochemical d e t e c t o r equipped with a r o t a t i n g d i s c working e l e c t r o d e was used. The l i m i t of de t ec t ion f o r mebendazole and hydroxymebendazole was - 5 and 2.5 ng/ml r e spec t ive 1 y . Allan _ - e t a1 (41) repor ted two high- performance l i q u i d chromatographic methods f o r determinat ion o f mebendazole and i t s metabol i tes i n human plasma using a r a p i d Sep Pak C18 e x t r a c t i o n . na t e t h e need f o r so lven t e x t r a c t i o n a s such. One method inc ludes i s o c r a t i c e l u t i o n and o t h e r , g rad ien t e l u t i o n . The g rad ien t e l u t i o n system provides s u p e r i o r r e s o l u t i o n s

Plasma samples ( 5 ml) was sp ike wi th major metabol i tes o f mebendazole, methyl 5- fa-hydroxybenzyl) -2-benzimidazole carbamate, 2-amino-5-benzoyl-benzimidazole and 2- amino-5- (a-hydroxybenzyl) -benzimidazole and with 5 . 0 pg of ethyl-5-benzoylbenzimidazole carbamate as i n t e r n a l s tandard i n t h e range of 10 ng - 30 pg. Samples were ad jus ted t o pH 6 with d i l u t e hydrochlor ic ac id o r sodium carbonate so lu t ion and ex t r ac t ed by pass ing through a Sep Pak C18 c a r t r i d g e f i t t e d t o a l u e r lock g l a s s syr inge . Af t e r t h e spikedplasma was passed through t h e car t r idge , it was washed with 20 m l o f d i s t i l l e d water , 0 . 5 m l o f 40% methanol i n water and 0.4 m l of methanol. The next 1 . 6 m l of methanol e l u t e d t h e f i v e compounds from c a r t r i d g e , t h i s 1 .6 m l o f methanol was evaporated t o dryness i n

The methods elimi-

MEBENDAZOLE 315

poin ted and t h e r e s idue was d isso lved i n 100 1-11 DMSO. i n j e c t e d i n t o HPLC system. The instrument used was Altex Model 322 MP HPLC (Altex S c i e n t i f i c , Berkeley, CA, USA) equipped with f ixed wavelength d e t e c t o r (254 nm, 8-ul flow c e l l ) , a Rheodyne (Berkeley, CA, USA) Model 7120 i n j e c t o r and Hewlett-Packard (Avondale, PA. USA) Model 3380 A i n t e g r a t o r r eco rde r . For mebendazole t h e determinat ion limits a r e 20 ng/ml ( i s o c r a t i c system) and 10 ng/ml (grad ien t system).

Aliquotes of 20 1~1 were

Chromatographic Condit ions:

Column

Mobile phase :

Flow r a t e :

Pressure

(250 x 4 .6 mm I.D.) LiChrosorb, RP-8 10-ym reversed-phase packing. A precolumn (35 x 3 .2 mm I.D.) dry packed with Coras i l C18 was a l s o used.

Methanol-water (55:45 pump A) and methanol-aqueous ammonium phosphate 0.05 M pH 5 . 5 (55:45 pump B) . 1 . 7 ml/min.

1200-1300 p . s . i .

Karlaganis e t a1 (42) developed an HPLC method f o r t h e determinat ion of mebendazole i n b io log ica l samples. 2 M 1 plasma of t h e p a t i e n t s was a l k a l i n i z e d wi th 8 ml of sodium carbonate b u f f e r (0.05 M , pH 11.3) and was e x t r a c t e d with 10 m l o f chloroform by shaking i n a g l a s s tube f o r 15 minutes . The conten ts were cen t r i fuged and aqueous phase was a s p i r a t e d and t h e chloroform l a y e r was evaporated under reduced p res su re . The r e s idue was d isso lved i n 200 1~1 mobile phase, 150 1-11 were i n j e c t e d . Analysis us ing Waters Model M6000A High-pressure Liquid Chromatograph equipped with Wisp Model 710 i n j e c t o r and a Perkin-Elmer W d e t e c t o r Model LC55.

--



Column : 25 cm x 3 mm ( i . d . ) LiChrosorb S1 60 5 m .

Mobile phase : Acetonitrile/water-saturated ch 1 orof orm/2 5% (weight / volume) ammonia i n water (75/92.5/0.1, by volume) , pH 6 . 5 .

Flow r a t e : 0.8 ml/min

Pressure : 400 p s i

Column temperature : Ambient

Detector wavelength : 307 nm

Recorder : 5 mV (0.02 absorp t ion u n i t s f u l l s c a l e ) .

C a l i b r a t i o n curve was prepared us ing 40 , 80, 200 and 400 ng of mebendazole. 1 Mg of c ic lobendazole was used as a i n t e r n a l s tandard , de t ec t ion r a t e i n t h e range of 6.117 ng/ml.

Alton e t a1 (43) developed a s imple, r a p i d and s p e c i f i c HPLC method €or q u a n t i t a t i v e de te rmina t ion of mebendazole i n plasma. 2 M 1 o f plasma was d i l u t e d wi th 2 n i l o f 0.05 M potassium hydrogen phosphate b u f f e r (pH 7.0) and e x t r a c t e d wi th 7 m l of e t h y l a c e t a t e . The organic l a y e r was removed and d r i e d , t h e r e s idue was d isso lved i n 0.001 N HC1 and r e -ex t r ac t ed wi th 6 m l o f petroleum e t h e r and cen t r i fuged , supe rna ten t was d iscarded t h e aqueous f r a c t i o n was a l k a l i n i z e d wi th 2 m l 0 .01 N NaOH and e x t r a c t e d twice with e t h y l acetate. The so lvent was d r i e d and d i s so lved i n 60 ~1 of e t h y l acetate and 20 p1 was i n j e c t e d i n chromatograph (Model ALC 202/204 Waters Assoc ia tes ) f o r a n a l y s i s .

--

MEBENDAZOLE 317


Column : u Bondapak C-18 300 x 3.0 mm ( i . d . ) reversed-phase .

Mobile phase : 0.05 M KH2P04-NaOH b u f f e r (pH 6 . 0 ) - a c e t o n i t r i l e ( 7 3 : 2 7 v/v) .

Flow r a t e : 2 . 5 ml/min

Pressure : 2500 p s i

Column temperature : Ambient

Detec tor wavelength : UV (313 nm).

Flubendazole was used as i n t e r n a l s t a n d a r d . i n t h e q u a n t i t y of 0.15 ug/ml f o r each sample. This method can be used t o measure plasma mebendazole l e v e l as low as 10 ng/ml.

A simple and r ap id method t o d e t e c t the presence of d i f f e r e n t benzimidazole compounds i n drug p repa ra t ions have been repor ted (44 ) . The procedure involves two d i f f e r e n t chromatography systems:

i- Reversed-phase g r a d i e n t .

ii- Normal p a r t i t i o n chromatography wi th i s o c r a t i c e l u t i o n .

Three d i f f e r e n t mixtures A , B , and C were used. The benzimidazoles were d i s so lved i n pure formic a c i d (5 ml) then i n concent ra ted hydrochlor ic a c i d (5 ml) and i n 50% (%) aqueous e thano l (90 m l ) . Mixtures A and C con ta in mebendazole. The s o l u t i o n s A and B were sepa ra t ed by reversed-phase g rad ien t e l u t i o n wi th t h e so lven t conta in ing ace to- n i t r i l e - aqueous 1% HzS04 with t h e programme: 10% a c e t o n i t r i l e f o r 2 minutes then from 10% a c e t o n i t r i l e t o 30% a t a rate of 5% p e r minute. Mixture C was e l u t e d


i s o c r a t i c a l l y on a amino phase chemical ly bonded t o s i l i c a g e l (Type NH2) wi th t h e so lven t : methylene c h l o r i d e + i sopropyl a lcohol (90 + 10) - a c e t o n i t r i l e (50:50) . I n i t i a l l y f o r de te rmina t ion of r e t e n t i o n t ime each of t h e benzimidazoleswas s e p a r a t e l y chromatographed.


Chromatograph :

Column

Mobile phase :

Flow rate :

Detector s e n s i t i v i t y :

Column p res s ure

Detector wavelength :

Varian LC 8500 HPLC wi th UV d e t e c t o r .

LiChrosorb RP 8 - micro- p a r t i c u l a t e (15 cm 4 . 7 mm i . d . ) LiChrosorb NH2 (Merck) (15 cm x 4.7 mm i . d . )

Methylene ch lo r ide + i s o - propyl a l coho l (90 + 10) - a c e t o n i t r i l e (5O:SO).

80 m l h - l

0 . 5 AUFS

500 p s i

(UV) 254 nm

This method would be u s e f u l i n t h e q u a l i t y con t ro l of raw m a t e r i a l s i n the q u a n t i f i c a - t i o n o f benzimidazoles i n foods tu f f s of animal o r i g i n , and i n t h e de t ec t ion of r e s idues .

A simple , accura t e and reproducib le HPLC method has been descr ibed (45) f o r determina- t i o n of mebendazole i n t a b l e t s . 20 Tab le t s were taken and ground i n f i n e powder, equiva len t t o about 10 - 20 mg of mebendazole was a c c u r a t e l y measured ou t €or each de termina t ion . The powder was e x t r a c t e d with 5 m l o f formic a c i d . 5 M 1 of s a l i c y l a - mide s o l u t i o n (8.5 mg/ml) was added t o each

MEBENDAZOLE 319

e x t r a c t as i n t e r n a l s t anda rd . The mixture was cen t r i fuged and t h e superna ten t was used f o r HPLC de te rmina t ion .


Chromatograph: Waters Assoc ia tes Model 440

Column : u Bondapak C-18

Mobile phase : Tetrahydrofuran-0.5% formic ac id (30:60) .

Flow rate : 1 .9 ml/min.

Temperature : 25-28OC.

Pressure : 1500 p s i .

Detect o r wavelength : (UV) 254 nm

The recovery was between 99.58 - 100.15%.

An HPLC method f o r t h e e s t ima t ion mebendazole, i t s prodrug, 4-arnin0-3-(3~-methoxycarbonyl 2 -thioureido)benzophenone , and t h e i r known o r expected me tabo l i t e s and degrada t ion products i n aqueous media and r a t blood was developed (46) . After o r a l adminis t ra - t i o n of t h e prodrug, t h e prodrug was r a p i d l y converted t o mebendazole and t h e area under t h e blood level vs time curve o f mebendazole, i n rats dosed wi th prodrug, was more than twice t h a t ob ta ined a f t e r g iv ing rats an equimolar amount of mebendazole. Only t h e prodrug, mebendazole and known me tabo l i t e s of mebendazole were de tec t ed i n ra t s given t h e prodrug.

Behm e t a1 (47) have measured t h e concent ra - t i o n of mebendazole and i ts major me tabo l i t e s by HPLC i n sheep plasma a t 12.5, 25, 50 o r 100 mg/kg. A t 12.5 mg/kg t h e peak plasma concen t r a t ions occured between n ine and 24 hours f o r a l l dose rates and d e c l i n e r a p i d l y . Two major me tabo l i t e s were d e t e c t e d ; t h e i r

--


concent ra t ions exceeded t h a t o f mebendazole at a l l dose r a t e s .

8 .5 Polarographic Method

P inzau t i e t a1 (48) descr ibed a d i r e c t - c u r r e n t polarographic reduct ion method of mebendazole a t t h e dropping-mercury e l ec t rode , and e s t a b l i s h e d t h e optimum condi t ions f o r t h e determinat ion o f t h i s drug i n dosage form. S ix t a b l e t s of mebendazole (about 0 . 3 g) were ground. A q u a n t i t y o f t h e powder equiva len t t o 50 mg o f mebendazole was t r a n s f e r r e d t o 100 m l volumetr ic f l a s k . To t h i s powder 8.6 m l o f 70% p e r c h l o r i c ac id was added and t h e mixture s t i r r e d f o r 10 minutes and d i l u t e d t o volume wi th water. The suspension was f i l t e r e d under suc t ion through a f i n e p o r s i t y g l a s s c r u c i b l e . po r t ion of t h e f i l t r a t e was t r a n s f e r r e d t o a 50 m l volumetric f l a s k , 3 m l o f 1 M pe rch lo r i c a c i d was added and t h e s o l u t i o n d i l u t e d t o volume with t h e McIlvaine b u f f e r pH 2.6 (2.18 v o l . 0.2 M sodium phosphate with 17.82 vol . 0 . 1 M c i t r i c a c i d ) .

A 20 m l of t h i s so lu t ion (corresponding t o 0 .4 mg o f mebendazole) was t r a n s f e r r e d i n t o polarographic ce l l (Metrohm E506 recording polarograph equipped with polarography s t and) .

--

2 M1

Deaeration and polarography were performed on us ing Metrohm EA 290 hanging mercury drop system. number of e l e c t r o n s involved i n t h e reduct ion was ca l cu la t ed a t a mebendazole concent ra t ion of 1 x 10-4 M us ing a s o l i d s ta te c o n t r o l p o t e n t i a l coulometr ic appara tus . A c a l i b r a t i o n curve was prepared using 5 t o 50 pg/ml mebendazole s o l u t i o n prepared i n McIlvaine b u f f e r , us ing t h i s method mebendazole a n a l y s i s c o u l d - b e performed with a l i m i t of de t ec t ion 100 ng/ml (48) .

The

8.6 Radioimmunoassay Method

Michiels -- e t a1 (49) developed an extremely h igh ly s p e c i f i c radioimmunoassay procedure f o r t h e determinat ion o f mebendazole i n plasma. was converted t o methyl [5-[4-(2-aminoethyl) ben zoy 1 1- 1H-ben zimidazol - 2- y l ] carbamate . hapten was coupled t o bovine serum albumin us ing a water so luble carbodiimide as fol lows:

Mebendazole

This

MEBEND AZOLE 321

0 II

CH3-C-NH-CH2CH2

0 I I ~ T N H - C H -0- C H ~

N

HC1, 1 N

0 I t

H

H 2 NCH2CH2 a ! a 7 NH-C-0-CH3 1. DMF/MeOH

c arbodi imide /HC 1

2 . Bovine Serum Albumin (BSA).

0 H I .

0 II

BSA-C-HN-CH2-CH2

Synthesis of the hapten and the mebendazole-protein conjugate used f o r the immunization of the rabbits (49).


The r e s u l t i n g conjcgate was used t o immunize r a b b i t s according t o convent ional procedures . serum e l i c i t e d i n t h i s way was t e s t e d f o r i ts a b i l i t y t o bind s p e c i f i c a l l y mebendazole. was c o l l e c t e d on EDTA and plasma was obtained a f t e r cen t r i fuga t ion of t h e blood a t 2200 g f o r 15 minutes. Unmetabolized mebendazole was measured by d i r e c t radioimmunoassay using an t i -bodies . Under t h e s t anda rd ized assay cond i t ion , t h e r a b b i t serum bound s p e c i f i c a l l y n e a r l y 50% of added 0 . 2 ng H3-Mebendazole at 1/100 serum d i l u t i o n . Unspecif ic adsorpt ion t o plasma c o n s t i t u e n t s d id not exceed 2 % . The method is q u i t e s e n s i t i v e t o a s say mebendazole i n plasma a t concent ra t ion as low a s 100 picogram/ml. with t h e binding of parent drug t o an t ibod ie s .

The a n t i -

Blood

Metabol i tes d i d no t i n t e r f e r e

Acknowledeements

The au thors would l i k e t o Thank M r . Syed Rafa tu l lah , Ass i s t an t Researcher, f o r h i s t echn ica l a s s i s t a n c e and Mr. Tanvi r A . Butt f o r h i s s e c r e t a r i a l s e r v i c e s .

MEBENDAZOLE

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MEBENDAZOLE 325

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

United S t a t e Pharmacopeia USP XX, page 465 (1980).

J .T.R. Owen, B.M. Hashim and F.A. Underwood, J . Assoc. O f f . Anal. Chem., - 66, 161 (1983).

A.A.M. Wahbi and S . Onsy, T a l a n t a , - 25, 716 (1978).

A . A . Patel, T.P. Gandhi, P .R . P a t e l and V . C . P a t e l , Ind ian J. Pharm., - 40, 20 (1978).

A . A . P a t e l , T.P. Gandhi, P.R. P a t e l and V . C . Patel , Indian J . Pharm. S c i . , - 40, 76 (1978).

F. Abdel-Fat tah, W . Baeyens and P . De Moerloose “Biolumin Chemilumin . , [ I n t . Symp. Anal. Appl . Biolumin. Chemilumin] . Page 335 (1981).

F. Abdel F a t t a h , W . Baeyens, P . De Moerloose, Anal. Chim. Acta, - 154, 351 (1983).

N . G . Rana, R.V. Dave and M.R. P a t e l , Ind ian Drugs, - 18 333 (1981).

B . Oos terhuis , J . C . F.M. Wetsteyn and C . J . V a n Boxte l , Therap. Drug Monitor . , - 6 , 215 (1984).

R . J . Al lan, H.T. Goodman and T.R. Watson, J o u r n a l of Chromatography, - 183, 311 (1980).

G . Kar laganis , G . J . Munst and J . B i r c h e r , J . High Resolu. Chromatogr. and Chromatogr. Commun. - 2 , 141 (1979).

K . B . Al ton, J . E . P a t r i c k and J . L . McGuire, J . Pharm. S c i , 68, 880 (1979).

D . Mourot, J . Boisseau and G . Gayot, Anal. Chim. Acta, - 99, 371 (1978).

Da-Peng Wang and Hsin-Shang, Lo, Analys t , - 109, 669 (1984).

- -

M . Dawson, T.R. Watson, Eur . J . Drug Metab. Pharmacokinet. - 8, 329 (1983).

C . A . Behm, R.A. Cornish and C . Bryant, Res. V e t . S c i . , - 34, 37 (1983).


48. S . Pinzaut i , E . La Por t a and G . Papeschi, J . Pharmaceuti. Biomed. Anal., 1, 223 (1983) .--

49. M. Michiels , R . Hendriks, J. Heykants and H.V.D. Bossche, Arch. Int. Pharmacodyn., 256, 180 (1982).

METOCLOPRAMIDE HYDROCHLORIDE

Davide P i t r g and Riccardo S t r a d i

F a c u l t y o f Pharmacy, U n i v e r s i t y of Milan

1. D e s c r i p t i o n 1.1 Nomenclature

1 .2 Chemical names 1.3 Generic names 1.4 Trade names 1.5 Formula and Molecular Weight 1.6 Appearance, Color, Odor and Taste

2. P h y s i c a l P r o p e r t i e s 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance S p e c t r a



328 DAVIDE PITRe AND RICCARDO STRADI

3.

4.

5.

2.2.1

2.2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12

'H-NMR

13

Ultraviolet Spectrum Mass Spectrum Fluorescence Spectrum X-Ray, Powder Diffraction Crystal Structure Melting Range Solubility

PKa PH Partition Coefficient

C-NMR

Manufacturing Procedures 3.1 Synthesis

Stability

Metabolism and Pharmacokinetics 5.1 Metabolism 5.2 Pharmacokinetics 5.3 Acute Toxicity

6. Methods of Analysis 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11 6.12

Element a1 Identification Tests Nonaqueous Titration Complexometric Analysis Colorimetric Analysis Ultraviolet Spectrophotometric Analysis Fluorimetric Analysis Paper Chromatography Thin-Layer Chromatography High Performance Thin-Layer Chromatography Gas Liquid Chromatography High Performance Liquid Chromatography

7.

8.

Determination in Body Fluids and Tissues

References

METOCLOPRAMIDE HYDROCHLORIDE 329

1. Description

1.1 Nomenclature

1.2 Chemical Names

Benzamide, 4-amino-5-chloro-N-[2-(diethylamino) ethyl 3-2-methoxy, monohydrochloride, rnonohydrate CAS 54143-57-6

4-Amino-5-chloro-N-[2- (diethy lamino) ethyllo-anisa- samide

anhydrous CAS 7831-21-5

1.3 Generic Names

Metoclopramide Hydrochloride USAN-BP 1980-F.U.IX Metoclopramidhydrochloride DAC 1979 Metoclopramidum hydrochloricum 2. AB-DDR

1.4 Trade Names

Cerucal (Arzneimittelwerk-Dresden) Elieten (Nippon Kayaku, Tokyo-Japan) Emperal (Neofarma-Helsinki ) Maxeran ( Delagrange-Paris) Plasil ( Lepetit-Milan) Peraprin (Taiyo, Gifuken-Japan) Primperan (Delagrange-Paris) Reglan (Robins, Richmond, USA)

330 DAVIDE PITRh AND RICCARDO STRADI

1.5 Formula and Molecular Weight

CI

C 0 N H - C H 2- I

OCH3

.HCI

CH2-N \

N H p

C H C1N 0 . H C l . H 0 14 22 3 2 2

1.6 Appearance, Co lo r , Odor, Taste

c2 * H20

Mol.Wt. = 354.3

A wh i t e o r a lmos t white c r y s t a l l i n e powder, odorless (1)

METOCLOPRAMIDE HYDROCHLORIDE 33 1

2. Phys ica l P r o p e r t i e s

2.1 In f r a red Spectrum

The i n f r a r e d spectrum of rnetocloprarnide hydrochlo- r i d e , is presented i n Fig. 1. The spectrum was recorded with a s o l i d sample d i s c composed of 1 m g of compound/200 m g KBr on a P e r k i n E l m e r Model 1310.

The fo l lowing bands (cm ) were assigned and a r e r epor t ed i n Table 1.

-1

Table 1

Wave Number

3200, 3300, 3340, 3400, 3460

2860, 2950, 2980

3030

2100, 2500, 2660, 2710

1600

1540

1270

700

Assignments

V N H , V O H

V C H SP3

SP2 V C H

v N+H

v c=o

6 N H (Amide)

v C-0-C (Asymmetric)

v c-c1

F i g . 1 - Infrared Spectrum of Metocloprarnide Hydrochloride


2.2 Nuclear Magnetic Resonance Spec t ra

1 2.2.1 H-NMR

1 H-NMR spectrum o f metoclopramide hydrochlor ide,

shown i n Fig. 2 , was obtained i n DMSO-d with a Varian spectrometer EM-390 opera t ing a t 90 MHz. The chemical s h i f t s a r e l i s t e d i n t h e Table 2.

6

Table 2

1.33

3.0-3.2

3.72

3.95

6.03

6.60

7.72

8.44

10.85

Moltepli c i t y

t

m

9

S

broad s

s

S

t

broad s

Number of protons

~

6

6

2

3

2 ,exch.

1

1

1, exch.

1, exch.

Assignments

N-(CH2-CH )

CH -N-(CH -CH ) -2 -2 3 2

-3 2

NH-CH -2

3 OCH

NH 2

H~ (arom.)

H (arorn.) 6

CONH

h H

1

hydrochlor ide i n DMSO-d

Fig. 2 - H-NMR (90 MHz) Spectrum of Metocloprarnide

6


13 2.2.2 C-NMR

13 The C-NMR spectrum o f metoclopramide hydrochlo-

r i d e i s shown i n Fig. 3. The spectrum was recorded i n D 0

with Varian XL.200 spectrometer ope ra t ing a t 50.391 MHz. The

chemical s h i f t s are l i s t e d i n Table 3.

2

T a b l e 3

Line

N.

1

2

3

4

5

6

7

8

9

10

11

12

2.3

(p.p.m)

8.38

35.15

48.27

51.40

55.89

98.00

109.18

110.61

131.43

148.57

158.07

167.46

Mu1 t i p 1 i c i t y

q

t

t

t

q

d

S

S

d

S

S

S

U l t r a v i o l e t Spectrum

Assignments

N(CH2.CH )

CH -N(C2H512 -2

N(CH -CH ) -2 3 2

-3 2

NH.CH -2

3 OCH

c (a romat ic )

C o r C

C o r C

‘6

C

3

1 5

5 1

4

c2

c=o

The u l t r a v i o l e t spectrum of metoclopramide hydro- -5

ch lo r ide i n water ( c = 5.763 10 m/l) was obtained (60)

with a Beckman Mod. 24 spectrophotometer and i t is shown i n

Fig. 4

1 8 :

13

hydrochloride in DMSO-d Fig. 3 - H-NMR (90 MHz) Spectrum of Metoclopramide

6

i I I

- . . ' jl

I

!

i

i

h - ' i


i ! ,

Fig. 4 - Ultraviolet Spectrum of Metoclopramide hydrochloride in Water

DAVIDE PITRG AND RICCARDO STRADI 338

2.4 Mass Spectrum

The mass spectrum of metoclopramide hydrochlor ide was recorded (Fig.5) on Finnigan 1020 spectrometer by

cowen t iona l e l e c t r o n impact i o n i s a t i o n a t 70 eV. Prominent fragments and t h e i r re la t ive i n t e n s i t y a r e shown i n Table 4.

m/e

~

229

227

201

181

169

141

99

86

Table 4

I n t e n s i t y

0.23

0.90

7.60

6.43

0.66

1.78

27.96

100

At t r ibu t ion

+

+ c1 QCH3 NH2

4fCH3 NH2 II c 1 0

c1 J$ro NH2

c 1 -0"'

+/CZHS CH -N

2- \C2H5


1W.Q

50.1

WE

188.1

se. I

I

M

DulR: STRW16 (72 Ell% N.E: 96 RlCi 367616. '108 t 2128

n.w.02.cL. 173712.

10.

99

M n : SIFWI6 172 B P X M'E: P6 PIC: 167616.

mss S P E C T M Wrn.'86 9zs7;oo t 2128 WREi Cll.H22.tI3.02.cL.

270

272 i' 281

2Qe""'"- 285 ~ " " " ' 2 6 29s m

F i g . 5 - Electron impart Mass Spectrum of Mono- cloprarnide hydrochloride

340 DAVIDE PITRB AND RICCARDO STRADI

2.5 Fluorescence Spectrum

A solution of metoclopramide hydrochloride in water exhibits fluorescence when excited with ultraviolet light. When excited at 353 nm, it shows a sharp peak at 353 nm and a second peak at about 273-276 nm. The emission spectrum of the compound consists of a single peak at 355 nm. The spectra obtained in water (5 mcg/ml), on a spectrophotometer Perkin-Elmer MPE 44A,are given in Fig.6.

2.6 X-Ray Powder Diffraction

The X-ray powder diffraction data of metoclopramide hydrochloride was determined (2) by a Philips Powder Diffractometer PW 1710 with nichel-filtered copper radiation (1.54051 A) with the following instrumental conditions:

TUBE: BF type, CU/Ni, 40 KV, 40 mA; SLITS: lo-0.1 mm-lo; DETE TOR:PW1711 proportional counter + discriminator; SCALE: 2x10 cps; TIME CONSTANT: 1"; SCANNING SPEED: 0.008°xl'1; PAPER SPEED 1 cmxlo; SPECIMEN HOLDER: Niskanen. The X-ray diffraction data are given in Table 5.

2


Fig. 6 - Fluorescence Spectrum of Metoclopramide

hydrochlor ide i n water


Table 5

N.

1 2 3 4 5

6 7

8

9

10 11 12

13 14 15

16

17 18

19 20

21 22

23 24

25 26

d(A)*

9.23 8.46 7.64

6.98 6.64

6.28

6.02

5.26 5.18

4.87

4.71 4.60

4.48

4.27

4.23

4.03

3.95

3.82 3.78 3.71

362 3.50 3.47

3.41

3.36 3.32

I&**

5 25 20

22

13 65

8 20

10

38

45 30 31 10 20

20

6 47

35 17

10 58 30

75 100

11

N.

27 28

29

30 31

32 33

34 35

36 37

38

39 40

41

42

43 44

45 46

47 48 49

50

50 51 52

3.15 3.07 3.04 2.99

2.95

2.92

2.86 2.82 2.78

2.74

2.69 2.59

2.53 2.51 2.43

2.33

2.31 2.29

2.26 2.16

2.14

2.05 2.01

1.81 1.81

1.71 1.63

PLUS OTHER LINES c5

Interplanar distances = 1.54051/2sin diam.

I&*+

12 8

6 11

12 10

20

13

8 21

11

10 9

13

18 10

8 10

11

11 10 8

9

8 8

7 9

** Relative intensities are based on highest intensity of 100


Solvent

Water Ethanol 95 Ethanol Benzene Chloroform

2.7 Crystal Structure

~~

Base

0.02

2.90 1.90 0.10 6.60

The metoclopramide hydrochloride monohydrate is monoclinic, space group P2 /n with a = 12.138(3), b = 8.553(2), c = 17.120(4) A and p= 98.06(7) Z = 4. The structure was solved by direct methods and refined to R = 0.055

The metoclopramide free base (CAS 364-62-5) is triclinic, space group P1 with a = 13.310 b = 8.728, c=7.477 A , a = 114.20, p = 81.13 and Y = 101.23O, Z = 2. A study of structure-activity relations shows an analogy between metoclopramide and apomorphine (4) .

1

(3).

2.8 Melting Range

Employing the USP method the metoclopramide monohydrochloride monohydrate melts at 182-185O(5) and with ter- ma1 microscopy the melting begins at 181O and completes at 183O(6). The dihydrochloride monohydrate melts in the range of 148O with decomposition. The melting points f o r metoclopramide free base are about 148O(7) or m.p. 146-148O(8). For dihydrochloride is also reported an eutectic melting point of 160° with phenacetin and of 117O with benzanilide(7).

2.9 Solubility

At 25O metoclopramide monohydrochloride is soluble in 0.7 g of water, 3 g of ethanol (96 per cent) and 55 g of chloroform, whereas it is practically insoluble in ether (10). Solubility of the base and of dihydrochloride are reported in Table (6). (11).

Table Solubility of metocloprarnide g/100 ml at 25O

Dihydrochloride

48 9 6

0.10 0.10

48 9 6

0.10 0.10

344 DAVIDE PITRfi AND RICCARDO STRADI

2.10 €5

Metoclopramide hydrochloride shows two ionisation costants; pic = 9.71 and pK = 0.42. The determination was carried out spectrometricalfy in aqueous solution and the values are a mean of 16 determinations with a standard deviation of 0.03 and 0.02 (12).

1

2.11 pH Range

The pH of a 10% water solution must be between 4.6 and 6.5 according to BP 1980.

2.12 Parti tion Coefficient

The partition coefficient in octanol/water has been determined(l3) by reversed-phase HPLC at 20°. The hydrochloride gives

Experimental LogP = 2,667

Calculated from R.A. Deckker (14) LogP = 2.76


3. Manufacturing Procedures

3.2 Synthesis

Among the synthetic methods leading to metoclopramide (I) it seems suitable to report only the ones of greatest interest that are those using p-aminosalicilic acid (II), as starting product, a compound whose properties are economically excellent. The two main processes are listed in Fig 7. In Scheme A, referring to the first invention(l5), the acid (1I)is acetylated to (111) and then treated with methyl sulfate to give the ether-ester (IV). This compound is reacted with (C H ) N-CH CH -NH to the corresponding amide. The subsequent Zesacetylation leads to metoclopramide ( 1). In scheme B the first step in the synthesis is the ether- ester (VI). The protection of the amino aromatic group was not considered necessary (16). The direct chlorination (18) of (VI) with sodium ipochloride gives (VII) which is

transformed in excellent yields into (I).

2 5 2 2 2

4 . S tabi 1 i ty

Aqueous solution

The maximum stability of metoclopramide, investigated at different pH values, was found at pH = 7.6 while the maximum degradation was found at pH = 2. One of the degradation products was identified as N,N-diethylethylen- diamine(l7).

S c h e m e A

COOCH3 COOCH3

COOH I

60 b

FOOH FOOCH3 FOOCH3

Fig. 7 - Synthesis of Metoclopramide


5. Metabolism and Pharmacokinetics

5.1 Metabolism

The metabolites identified in the in vitro and in

vivo studies are listed in Table 7, in which metoclopramide

is indicated as (I).

Table 7 Non conjugated metabolites found

CI @OR2

NH2

N.

I

I1

I11

IV

V

VI

VII

VIII

IX

X

R 1

-NHCH CH N(C H ) 2 2 2 5 2

-NHCH CH NHC H 2 2 2 5

-NHCH CH NH 2 2 2

-OH

-NHCH CH N(C H ) 2 2 2 5 2

-NHCH CH N(C2H512 2 2

-NH 2

-NHCH COOH 2

-NHCH = CH 2

-NHCH CH NHCOCH 2 2 3

R 2

3 -C H

3

-C Hg

-C H

-CH3

-H

-H

3 -CH

-C H 3

3 -CH

-CH 3

REFERENCES

348 DAVIDE PITRe AND RICCARDO STRADI

Dosage p.0. 100 20 10

m g k Rats Dogs humans

U F U F U F

0-24 71.9 5.7 65.3 ---- 77.8 20

24-48 8.5 4.4 6.9 18.3 8.7 1.0 48-72 1.0 1.7 1 .o 0.8 0.8 1.6

hours

r

By studies of the in vitro metabolism of (I) using hepatic fractions from several animal species (1) (2) five metabolites (11) (111) (IV) (V) (VI) were detected. Identification studies of the metabolites in urinary ex- cretes of rat and dog (20) after enzimatic hydrolysis evi- denced some compounds already known from the in vitro studies and some new ones corresponding to (VII) (VIII) IX) (X). In particular in rat urine, were found metabolites (11) (IV) (V) (VII) (VIII), while in dog urine were present metabolites (V) (VI) (VIII) (IX) (X). In human urine the excretion was found to be 60% in the first 24 hours. After enzimatic hydrolisis was identified metabolite (VIII)(CAS 52702.04.2),

but the main metabolite was found to be unchanged metoclopramide originated from the sulfate and glucuronide. Meta- bolic products were quantified in rabbit urines (21) (Ii) and (IV) but also unchanged (I),as N -glucoronide and N - sulfonate were identified. Another product of N-oxidation of (I) was also found. Metoclopramide N -sulfonate (CAS 27260-

42.0) is the main metabolite in human urine.

4

4

5.2 Pharmacokinetic

U = urinary excr. F = fecal excr. % of the administered dose

14 The studies of pharmacokinetics of C-metoclopra-

mide in rats, dogs and humans show that the drug is distributed within a few minutes after oral administration and it

is eliminated mainly in the urine of the first 24 hrs. (20). Table 8

14 Excretion of C-metoclopramide in rats, dogs, and humans


The pharri iacokinetic of metoclopramide H C 1 was s t u d i e d i n

normal male v o l u n t e e r s a f t e r i . v . dosage o f 10 mg. The shape o f t h e plasma l e v e l cu rve i n t h e s e s u b j e c t s f i ts a two-compartment model w i th a d i s t r i b u t i o n o f T 1 a = 4.6

min. and an e l i m i n a t i o n o f T p = 165.7 min./2The body plasma c l e a r a n c e was 10.9 ml/min g ( 2 2 ) ( 2 3 ) . G. Be rne r and co-workers ( 2 4 ) r e p o r t e d t h e s t u d y of b i o a v a i l a b i l i t y of commercial f o r m u l a t i o n s o f metoclopramide i n male h e a l t h y v o l u n t e e r s . The f o l l o w i n g r e s u l t s were o b t a i n e d : f o r t a b l e t s 57.6-80.3%, f o r s o l u t i o n s 49.7-76.7% and fo r c a p s u l e s 54.8%.

.I/?

5.3 Acute T o x i c i t y

The a c u t e t o x i c i t y i . v . (LD ) o f t h e metoclopramide d i h y d r o c h l o r i d e is 85.7 mg/kg f o r t h e r a t and 71 mg/kg

f o r t h e mice ( 2 5 ) .

50

6. Methods of Ana lys i s

6 . 1 Elemental Composition

C, 47.4; H , 7.12; C 1 , 20.0; N , 11.9; H 0, 5.1 fo r t h e

C , 50.01; H , 6 .89; C 1 , 21.08; N, 12 .5 ; 0, 9.50 f o r t h e

C, 56.9; H , 7.40; C 1 , 11.83; N , 14.02; 0, 10.67 f o r t h e

2 monohydrate

anhydrous form

base .

6 .2 I d e n t i f i c a t i o n Tests

a) I n f r a r e d S p e c t r o s c o p i c Test

BP 1980 (10) c i tes t h e u s e of t h e i n f r a r e d ab-

s o r p t i o n spectrum o f a potassium c h l o r i d e d i s p e r s - i o n o f t h e rnetoclopramide h y d r o c h l o r i d e i n accordance w i t h t h e r e f e r e n c e spectrum reproduced i n t h e appendix.

350 DAVIDE PITRk AND RICCARDO STRADI

b)

C)

d)

6.3

Ultraviolet Spectroscopic Test

The light absorption, in the range 230 to 350 nm of a 2-cm layer of a 0.001 per cent w/v solution in 0.01M hydrochloric acid exhibits two maxima, at 273 nm and 303 nm; absorbance at 273 nm about 0.79 and at 309 nm about 0.69. This is another identification test recommended by B.P.1980 (10).

Color Tests

With a solution of 4-dimethylaminobenzaldehyde - yellow-orange(1); ammonium vanadate test - light brown (sensitivity : 1.0 ug), (7). Vitali's test: pale-yellow/light brown (sensitivi-

/

ty : 1.0 ug) (7). /

Crystal Tests

Gold Cyanide solution - needles, sometimes in rosettes (sensitivity: 1 in 1000).

Lead iodide solution - feathery rosettes (sensitivity: 1 in 1000) (7).

Nonaqueous Ti tration

The metocloropramide hydrochloride may be titrated (10) potentiometrically in glacial acetic acid containing mercuric acetate with perchloric acid in glacial acetic acid as titrant.

6.4 Complessometric Analysis

The method is ba ed on the use of a column of amberlite IR 120 in the Zn form. The released quantity, corresponding to the absorbed metoclopramide, is titrated with EDTA using Eriochromo Black T as indicator (26).

5+

METOCLOPRAMIDE HYDROCHLORIDE 35 1

6 .5 Colorimetric Analyses

The colorimetric assays have been the most widely used especially to determine metoclopramide in pharmaceutical preparations. They are listed in the following table 9.

Table 9 Colorimetric assays of Metoclopramide

N.

1

2

3

4

5

6

7

8

9

10

Citric acid in Ac 0 2

NH4SCN t Co (N03)2

Z a, P-dinitrostilbene

DiazotizationtN(1-naphthyl)

ethylendiamine

HNO to form the nitrous

derivative

Diazotization + a-Naphthyl- amine

Diazotization + R-Salt Dragendorff's

Diazotization + NH Sulfamate

Diazotization + 2,4-dihydroxybenzoic acid

2

4

max

-- 600

620

390

525

3 75

510

500

475

495

440

Bear's law ,ug/ml

2-14

--

- -

0.4-4

10-60

0.1-11

1-14

20-120

1-11

0.8

Ref.

27

28

29

30-11

31

32

33

34

35

36


6.6 Ultraviolet Spectrophotometric Analysis

B.P 1980 (10) has described a spectrophotometric assay for the quantitative determination of metoclopramide tablets. After the extraction of the base in chloroform, the metoclopramide can be determined by measurement of absorbance at 305 nm. E(1%, lcm) = 265.

6.7 Fluorimetric Analysis

Fluorimetric measurements are carried out in pH=2 buffer solution on the basis of intense emission at 360 nm that occurs when the sample is exci ed at 310 nm. The method, with detection limit of 3x10 ,ug ml , was successfully applied to analyses of pharmaceutical formulations with a recovery of 100.8-101.8% and a coefficient of variation of 0.9-1.9% (37).

-8 -1

Chromatography

6.8 Paper Chromatography

Several chromatographic assays are summarized in the following table 10.

Table 10

Solvent system

4.6 g of citric acid in 130 ml H 0 and 870 ml BuOH

Acetate buffer pH = 4.58

Phosphate buffer

2

pH = 7.4

Toluene : Methanol : conc. NH40H

Paper Detection

U.V. Iodoplatinate

spray

II

II

1% of chlora- nyl in Bz

RfxlOO

45

77

39

-

Ref.


Paper: A - Whatman N 1, shee t 11 x 6 cm buffered by dipping i n a 5%

solut ion of sodium c i t r a t e , b l o t t i n g and drying a t 25O f o r 1 hour.

B - Whatman N 1 N 3, sheet 17x 19 cm impregnated by dipping i n a 10% solut ion of t r i b u t y r i n i n acetone and drying i n a i r .

C - Whatman N 1

6.9 Thin-Layer Chromatographx

The methods f o r separation and detect ion of metoclopramide a r e summirized i n t ab le 11.

Table 11

Solvent system

I I1 III IV V VI VII

VIII IX X XI

XI1

Plate

A,B C C

C C

C C F C C F F

Rf x 100

40 10 46

16 54 29 55 -

98 68 47 13

Reference

354

Solvent

I I1 I11

IV V

VI VII VIII IX X

XI XI1

DAVIDE PITRfi AND RICCARDO STRADI

Sytem

Strong ammonia solution: methanol (1,5:100) Methanol-chloroform (1:4) 1,2-Dichloroethane-ethanol-ammonia solution (sp.gr.0.88) (70: 15:2) n-Butanol-acetic acid-water (4:l:l) Isopropanol-ammonia solution (sp .gr. 0.88) ( 80 : 4 : 5)

Butanol-acetic acid-water (4:l:l) Isopropanol-NH OH-water (80:4:5) Dioxan-NH OH-benzene (8:l:l) Chloroform-methanol-28% ammonia (10:4:1) Chloroform-methanol-dioxane-28% ammonia (90:14:10:3) Methanol-ammonia (100:1.5) Acetone

4 4

Detection

- Acidified iodoplatinate spray - Spray solution: 1% solution sodium nitrite in HC1 N and then with 0.4% of N-(1-naphthy1)-ethylaminediammonium di- chloride as coupling agent in methanol.

- Spray solution: 0.2% solution of chloranil in acetonitrile followed by heating the plate at 100-105° for 2 min.

- Spray solution: 2 g of iodic acid dissolved in 10 m l H 0 2

and made up to volume of 100 m l with 90% (w/v) sulfuric acid (5).

- Ultraviolet light (250 nm).

Plate

A. Glassplate, 20 x 20 cm coated with silica gel B. Pre-coated silica gel G (Merck) C. Silica1 gel 60 F (Merck) D.8 GF Plate. Analtech (6) E. Kodak Silice Fluorescente K301 F. Silica dipped in 0.1N KOH and dried

2 54


6.10 High Performance Thin-Layer Chromatography

HPTLC was performed on metoclopramide base using HPTLC Fertiplatten Kiegelgel 6OF(Merck)and Me0H:CHCl NH OH 25% (80:85:0.2) as solvent. The compound may be quantified by means of excitation fluorimetry at 312 nm and emission at 365 nm integrating the peak area with a videointegrator. The results are linear from 160 ug to 20 ug with a standard deviation of 2-5% (43).

3: 4

/ /

6.11 Gas Liquid Chromatography

T. Daldrup and co-workers (44) described a GLC assay method on a glass-column (6 ft x 2 inc.) packed with 3% OV-1 on Chromosorb W-HP (100 to 120 mesh) and operated at 15Oo-25O0 (lO°C/min), with N as carrier gas (50 ml min )

2 and a flame ionization detector. Internal standard: 2-amino-5-chlorobenzophenone. Detector and injection temperature were 350° and 250OC. Retention time: 1.87. GLC has been used as the method for the determination of the drug and its 15 metabolites in biological fluids (45) (46) (47) (48) and Y.K. Tamond, J . E . Axelson (49) have been determined the mctoclopramide in picogram quantities in plasma with a Ni-electron capture detector. The procedure involved the extraction of the drug from an alkalinized aqueous solution in benzene and derivatization with heptafluorobu- tirric anhydride. The determination of the derivative was done using diazepam as internal standard. Working conditions: glass column (1.2 m x 2 inc. packed 3% OV-17, coated with 80-160 mesh, chromosorb W. Injection 2 5 8 O ; oven 250 and detection 350. A mixture of argon-methane (95:5) was employed with a flow rate of 40 ml/min . The retention times were 3.76 min for the metoclopramide and 9.1 min for diazepam.

-1

63

-1


6.12 High-pressure Liquid Chromatography

Teng et al.(l) used a column 15 cm x .5 mm packed with silica gel M 171; flow rate 2.0 ml/min and CH OH- -CHC1 -NH OH conc. (30:70:0.5) at room temperature. Detect- ion 280 nm. The retention times of metoclopramide is 2.8 min and 4.5 min for the internal standard (4-amino-5-chloro-N- D- ( prop yl am ino e t hyl]-Z-me t hoxyben z m i de ) . W . Block et a1 ( 5 0 ) determined metoclopramide using reverse- -phase HPLC with an RP-18 column under isocratic conditi n (CH 0H:H 0:NH OH conc.,75:24.9:0.1). Flow rate 1.2 ml/min Detection at 308 nm. Elution after 5 min.

-? 3

4

-4. 3 2 4

7. Determination of metoclopramide in Body Fluids and Tissues

The determination of metoclopramide in biological fluids of man and of various animal species is the object of many communications as reported below:

Blood (Plasma, Serum)

Colorimetry: (11) TLC : ( 3 ) (41) (40) (52) HPLC: (24) GC : (45) (46) (47) (53) (48) HPLC : (54) (55) (20) (50) (53) (56) (57) GC-Mass Spectrometry: (15)

Urine

Colorimetry : (21) (52) TLC : (21) (40) (39) HPCL : (20) (56)

GLC-Mass Spectrometry: (58) GC : (54) (19)

Saliva HPLC : ( 59)

METOCLOPRAMIDE HYDROCHLORIDE

B i l e Colorimetry : (21)

Feces Radioactivity with 14C-metoclopramide : (20)

Microsomal fraction of liver homogenate HPLC : (39)

357

DAVIDE PITRE AND RICCARDO STRADI

REFERENCES

F.U. IX G. Liborio, Liiversity of Milan, Ins gy, Personal Communication.

itute of Mineralo-

N.M. Blaton, O.M.Peeters, C.J. Be Ranten Cryst. Struct.Comm. 9 , 857 (1980) M. Cesario, C. Pascard, M.E1 Moukhtari, L. Jung Eur.J.Med.Chem.-Chim.Ther. Is, 13 (1981) The Merck Index, IX Ed., 6109 J. Topard, M. Hanocq, Van Damme, L. Molle J.Pharm,Belg. 2, 633 (1973) E.G.L. Clarke, Isolation and Identification of Drugs The Pharmaceutical Press, London 1969 R. Pakula, K. Butkiewics, 2. Trojanowska, J. Ruszczak Arch.Pharm. (Weinheim) - 313, 297 (1980) M. Kuhnert-Brandstatter, L. Bosch, G. Eckstein Sci.Pharm. 461, 54 (1978) B.P.1980. Vol. 1, pag. 273 G.P.Pite1, Th.Luce - Ann.Pharm. Fr. 23, 673 (1965) M. Manocq, J. Topart, M. Van Damme, L. Molle J.Pharm.Belg. 28, 649 (1975) N. Verbiese-Genard, M. Hanocq, M. VanDarnme, L. Molle Int. J. Pharm. - 9, 265 (1981) R.F. Reckker, H.M. d e Kort Eur. J. Med.Chem.-Chim.Ther. - 14, 479 ( 1979) SOC. d'Etudes Scientifique Industrielle de 1'Ile de France - Fr. 1.407.055 M. Murakami, N. Inukai, A. Koda, N. Nakano Chem.Pharm.Bul1 (Tokyo) 19, 1696 (1971) T. Kaniewska, W. Wejman. Farm.Po1. 32, 927 (1976) A.H.Beckett, G. Huing J.Pharm.Pharmaco1. 27, Suppl. 24P (1975) D.A.Cowan, G. Huing, A.H.Beckett Xenobiotica - 6, 605 (1976) L. Teng, R.B. Bruce, L.K. Dumming J.Pharm.Sci. 66, 1615 (1977) T. Arita, R. Hori, K. Ito, K. Ichikawa, T. Uesugi Chem.Pharm. Bull. (Tokyo), g, 1663 (1970)


D.N. Bateman, D.S. Davies, C. Kahn, K. Maskiter Br. J. Clin.Pharmaco1. 4, 6508 (1977) D.N. Bateman, C. Kahn, K. Mashiter, D.S. Davies Br. J. Clin.Pharmaco1. 5 , 401 (1978) G. Beiner, B.Emschermann, E. Glaottke, W. Haase, F. Leuschner, V. Vogtle-Junkert, H.H. Wagener

C. Agralo, D. Bulgach, H. Araldi, Jorndas Argent Toxicol. Anal. Actas 1st. 194

J. Jarzebinski, Z. Swajber, G. Klos Pharm. Pol. 34, 237 (1978) J. Emmanuel, T.V. Yogyanaranan East Pharm. 24, 133 (1981) CA 95-1758 8 9 k J. Dobrecky, R.J. Calleja Rev.Farm. (Buenos Aires), 115, 12 (1975) CA 80-100249k P. Dubois, J. Lacroix, P. Levillan, C. Vie1 J.Pharm. Belg. - 36, 203 (19811, CA 95-121218~ D.M. Skingbal, K.V. Sawant Indian Drugs 19, 239 (1982) CA 96-223389s D.M. Shingbal, S.D. Naik Indian Durgs 18, 441 (1981) CA 96-24856s O.S.Kamalapurttar, R.S. Priolkar Indian Drugs 20, 108 (1982) CA 98-221907b R.T. Sane, A.Y. Okamantar, U.R. Pandit, A.B. Ambardaker Indian Drugs 20, 73 (1982) CA 98-78232v R.G. Bhatkar, K.V. Nagvenkar East Pharm. 26, 205 (1983) CA 99-164094a O.S. Kamalapurkar, J.J. Chudasama Indian Drugs 3, 298 (1983) CA 99-10952~ J. Emmanuel, S.D. Naik, N. Pradego Indian Drugs 20, 387 (1983) CA 99-181559q W. Balyens, P.De Moerloox Analist 103, 1356 (1978) A.M. Taka, M.A. Abd El-Kader J. Chromatog. 177, 405 (1979) G. Huinzing, A. Beckett, J. Segura J. Chromatogr. 172, 227 (1979) O.M. Bakke, J. Segura J.Pharm.Pharmaco1. 28, 32 (1976) D. Schuppan, I. Schmidt, M. Heller Arzneim.Forsch. 9, 157 (1979)

Arzneim. Forsch. 32, 169 (1982)

(Pub. 1972) 1, 214 - CA 79-73800

-

-

DAVIDE PITRE AND RICCARDO STRADI 360

42

43

44 1

45)

46

A.M. Stead, R. Gill, T. Wright, J.P. Gibbs, A.C.Moffat Analyst 107, 1106 (1982) M. Prosek, M.Watre, I. Verbic Comput. suppl. Lab. - 1, 223 (1986) T. Daldrup, F. Susanto, P. Michalke Fresenius 2. Anal.Chem. 308, 413 (1981) L.M. Ross-Lee, M.J. Eddie, F. Barier, W.D. Hooper, J.H. Tyrer J. Chromatogr. 183, 173 (1980) K.W. Riggs, J.E. Skelson, D.W. Rurak, D.I. Hasman, B.McErlane, M. Bylsma-Havell, G.H. MacMorland, R.Dugley, J.D.E. Price J.Chromatogr. 276, 319 (1983) Y.K.Tam, J.E. Axelson, R. Ongley J.Pharm.Sci. 68, 1254 (1979) M. Stanley, D. Wazer Neuropsychobiology lo, 108 (1984) Y.K. Tam, J.E. Axelson J.Pharm. Sci. 67, 1073 (1978) W. Block, A . Pingoud, M. Khan, P. Kjeldrap Arzneim.Forsch. 2, 1041 (1981) G. Pitel, Th. Luce Ann.Pharm.Fr. 28, 595 (1970) T. Arita, R. Hori, K.Ito, M. Sckikawa Chem. Pharm. Bull. (Tokyo), 18, 1675 (1970) J.P. Leppard, E. Reid Methodol . Surv . Biochem. 3, 315 ( 19781, CA 90-66423~ W. Block, B. Pingoud Fresenius Z.Ana1. Chem. - 301, 109 (1980) G.B. Bishop-Frendling, H. Vergin J.Chromatogr. 273, 453 (1983) J. Popovic Ther.Drug Monit. 5 , 77 (1984) J.L. Choen, G.M. Hisayasu, E. J. McDermed, S.B. Stram Pharm. Res. (1984) Y.K. Tam, J.E. Axelson J .Pharm.Sci. 67, 1073 (1978) R.P. Kapil, J.E. Axelson, R. Ongley, J.D.E. Price J.Pharm.Sci. 2, 215 (1983) C. Bugatti. Faculty of Pharmacy, University of Milan, Personal Communication

MITOMYCIN C

J o s H. Beijnen Slotervaart HospitalfNetherlands Cancer Institute

Amsterdam, The Netherlands

Auke Bult and Willy J.M. Underberg Faculty of Pharmacy, Department of Pharmaceutical Analysis

State University of Utrecht Utrecht, The Netherlands

1. Historv 2 . Description

2.1 Name, Formula, Molecular Weight 2.2 Appearance, Colour, Odour

3 . Biosynthesis, Chemical Synthesis 4 . Physical Properties

4.1 Ultraviolet-Visible Spectrum 4.2 Infrared Spectrum 4.3 Nuclear Magnetic Resonance (NMR) Spectrum 4.4 Mass Spectrum 4.5 Circular Dichroism (CD) Spectrum 4.6 Optical Rotation 4.7 Melting Point 4.8 Differential Scanning Calorimetry 4 . 9 Solubility 4.10 Partition Coefficient 4.11 Dissociation Constants 4.12 Electrochemistry

5 .1 Elemental Analysis 5.2 Ultraviolet Spectrophotometry 5.3 5 . 4 Gel Filtration Chromatography 5.5 High Performance Liquid Chromatography 5.6 Identification and Purity Tests in Official


Thin Layer and Paper Chromatography

Compendia 6. Stability, Degradation

6.1 Stability in Aqueous Solutions 6.2 Stability in Pharmaceutical Formulations 6 . 3 Stability in Biological Media

7.1 Mechanism of Action 7 . 2 Clinical Antiturnour Activity 7.3 Cljnical Toxicity 7 . 4 Pharmacokinetics

8 . Determination in Body Fluids References

7. Pharmacology



362 JOSH. BEIJNEN ETAL.

1. HISTORY

In 1956 Hata and coworkers (1) first reported about the isolation of a new group of antitumour antibiotics which they named mitomycins. These pigmented compounds were obtained by extraction and chromatographic purification of the fermentation broth filtrate of Streptomyces caespitosus. In 1958 another Japanese group, led by Wakaki, succeeded in the isolation of a mitomycin derivative, called mitomycin C, from the same actinomyces strain (2). Several mitomycin derivatives have since been isolated from Streptomyces species (3-8). Ex- tensive chemical degradation studies, X-ray diffraction crys- tallography, chromatographic and spectroscopic analysis and biosynthesis studies have contributed to the elucidation of the structure and absolute stereochemistry of mitomycin C and related mitomycins (9-23a). Mitomycin C was selected for further development after it had been established that mitomycin C possessed profound antineoplastic activity and that the therapeutic index was more favourable than that of related mitomycin congeners. The drug has activity against an array of solid tumours such as adenocarcinomas of the gastrointestinal tract. Unfortunately, clinical experience with mitomycin C has shown that it causes severe toxicity in particular a delayed, cumulative bone marrow suppression, Mitomycin C is rarely used as a single agent except for the treatment of superficial cancer of the urinary bladder, intravesically administered. This administration mode has been shown to be very effective and devoid of serious haematological toxicities. For more specific information about the clinical usefulness of mitomycin C combinations in lung, breast and stomach cancer the reader is referred to review articles and books (24-31) .

2 . DESCRIPTION

2.1 Name, Formula, Molecular Weight The generic name is mitomycin C (50-07-7). The drug is

marketed under the trade names of MutamycinB, Mitomycin-C Kyowa@ and Ametycine@ . The molecular formula for mitomycin C is CI5Hl8N4O5, The molecular weight is 334.13.

MITOMYCIN C 363

2.2 Appearance, Colour, Odour Blue-vi.olet crystalline powder which is odour less .

3. BIOSYNTHESIS, CHEMICAL SYNTHESIS

By using 13C, 14C, 3H and 15N-labeled precursors in feeding experiments, investigators have studied the biosynthetic pathways of mitomycins (20, 21, 32-35). Although considerable progress has been made in elucidating the biosynthesis routes the exact pathway remains to be established. Feeding studies have revealed that D-glucosamine, L-methionine, L-citrulline, L-arginine, pyruvate and D-erythrose are candidates as biosynthetic precursors of the mitomycines (21). The total chemical synthesis of mitomycin C has been accomplished from 2,6-dimethoxytoluene as starting material by Kishi and collaborators (36). The synthesis route is lengthy (47 steps) and complex.

4. PHYSICAL PROPERTIES

4.1 Ultraviolet-Visible Spectrum The ultraviolet spectrum of mitomycin C ( ~ x ~ O - ~ M ) in

water is depicted in Figure 1. The spectrum was recorded by using a Shimadzu UV-200 double beam spectrophotometer in 1 cm quartz cell. In the visible region a broad band of low intensity is present. At pH values over 10 the absorption maximum at 360 nm shifts to 295 nm due to keto-enol tautomerization and deprotonation of the 7-amino quinone part of the mitomyzin C molecule (Figure 2)(37). Molar absorptivities (E)

and A1 cm values are reported in Table I (9).

Table I UV-VIS Spectral Data for Mitomycin C in Methanol(9)

360 689 23,000 555 6.3 209

364 JOS H. BEIJNEN ETAL.

01 absorbance I

400 350 300 250 200 wavelength I nrn)

MMC MMC-

Figure 2. Keto-enol tautomerization and deprotonation of mitomycin C (MMC)

365 MITOMYCIN C

0

4.2 Infrared Spectrum The infrared spectrum of mitomycin C in a potassium bro-

mide pellet is presented in Figure 3 . The spectrum was recorded with a Jouan-Jasco IRA-grating infrared spectrophotometer. Assignments o f a number of prominent bands is listed in Table 11.

, , , , , , , 1 , , , , , , , , , , , , , , 1 1

Table I1 Infrared Assignments for Mitomycin C

-1 wavenumber (cm ) assignment

346OS, 331OS, 3260' V(NH) and V (NH2) of aziridine,

carbarnate and 7C-NH 2 1735' V(C=O) of carbamate function

16OOs, 1558' V(C=O) of quinone

'"1

Figure 3 . Infrared spectrum of mitomycin C


4.3 Nuclear Magnetic Resonance fNMR) Spectrum The proton NMR spectrum was recorded in pyridine-d con-

taining tetramethylsilane as internal reference and wi ?h the use of a Bruker WM-300 spectrometer at frequency 300.13 MHz (37a). The spectrum (detail) is presented in Figure 4 and re- fers to the situation after proton exchange. The spectral assignments are presented in Table 111 (in pyridine-d ) and are derived from Low and Begleiter ( 3 8 ) . Irrigularitges in intensity comparison of the signals make the assignments tentative. Proton exchange with deuterium oxide results in disappearance of NH, NH and H-1 signals, while the H-3, H-9, H-10 and H-10' signals shift 0.10 to 0.20 to lower ppm values (spectrum Fig . 4 ) .

Table I11

2

'H NMR Assignments for Mitomycin C in pyridine-d5

~ ~~~~

Chemical shift Multiplicity Number of Assignment 6 (ppm) atoms

2.00

2.10

2.72

3.13

3.19

3.57

4.02

4.53

5.11

5.43

7.6

singlet

triple t

quartet

singlet

doublet

quartet

doublet

triplet

quartet

broad

3

1

1

1

3

1

1

1

1

1

4

CH3 (C-6)

NH (Cl-C2)

H-2

H- 1

OCH3 ( C - 9 )

H-3 ' H-9

H- 3

H-10

H-10'

NH2(C-7)

and CONH2

The natural abundance carbon 13 NMR spectrum was recorded under the same experimental conditions except that the frequency was 75.46 MHz (37a). The proton-noise decoupled spectrum is presented in Figure 5 and the spectral assignments are summarized in Table IV. The assignments of Keller and Hornemann are followed ( 3 9 ) .

MITOMYCIN C 367

1 , . . I . . , . 1 . . . . 1 . . , . 1 . . . . 1 . . . . , . . . . , . . . . 5.0 4 .O 3 .O 2.0

chemical shift (ppm)

Figure 4 . Proton NMR spectrum of mitomycin C (detail)


180 160 140 120 100 80 60 40 20 0

chemlcal shift (ppm)

Figure 5.

I3c NMR spectrum of mitomycin c

MITOMYCIN C 369

5 Table IV

Chemical shift 6(ppm) Ass-ignment

13C NMR Assignments for Mitomycin C in pyridine-d

~ ~~

8.8

32.6

36.7

44.2

49.5

50.6

62.5

104.3

106.8

110.7

149. 6a

156.1

158.1

176.7

178.3

CH3 (C-6)

c- 2 c-1

c-9

OCH3 (C-9a)

c-3

c-10

C-6

C-9a

C-8a

c-7

C-5a

C-lOa

C-8

c-5 ~~~ ~~ ~

a obscured by solvent

4.4 Mass Spectrum Electron impact (EI) mass spectroscopy of mitomycins,

including mitomycin C, has been investigated in detail by Van Lear (40). His observations were confirmed in later studies (41, 42). For this analytical profile electron impact (EI), chemical ionization (CI) , field desorption (FD) and fast atom bombardment (FAB) in the positive and negative ion mode, spectra of mitomycin C (lot nr. 010783) were recorded. EI mass spectrometry was performed by direct probe analysis on a Kratos MS-80 mass spectrometer. Source conditions were: 200 OC source temperature, 70 eV electron energy and 100 PA ionising current. Direct insertion probe CI mass spectra were obtained by using a Finnigan 3200 quadrupole mass spectrometer combined with a Finnigan 6000 data system. Methane was used as rhe ionising gas. The emission current was 0.20 mA and the multiplier voltage 1800 V. FD mass spectra were obtained with a Varian MAT 711 double focussing mass spectrometer equipped with a MAT 100 data acquisition unit. 10 pm tungsten wire FD emitters containing carbon microneedles


Figure 6. EI mass spectrum of mitomycin C

.360 x 4 242

292 I

3ab

Figure 7. CI mass spectrum of mitomycin C

MITOMYCIN C 371

with an average length of 30 pm were used. The mitomycin C sample was dissolved in methanol and then loaded onto the emitters by the dipping technique. An emission current of 12 mA was used to desorb the sample. The ion source temperature was 70 'C. FAB mass spectrometry was carried out using a V.G. micromass ZAB-2F mass spectrometer, an instrument with reverse geometry and fitted with a high field magnet and coupled to a V.G. 11-250 data system. The sample was loaded in thioglycerol solution onto a stainless steel probe and bombarded with Xenon atoms having a 8 keV energy. The recorded spectra are depicted in Figures 6, 7, 8, 9 and 10 and the major ion assignments are given in Table V. A high resolution mass spectrum exhibited the molecular ion at m/z 334.1280 with composition C15H18N405 (calculated mass 334.1277).

4

Figure 8. FD mass spectrum of mitomycin C

3 72

98.

88

18.

68.

58.

18.

38.

JOSH. BEIJNEN ETAL.

28

18

e

3

388

2r

7

L 488.

21s

I88 288 388 480

Figure 9/10. FAB(+) mass spectrum (upper spectrum) and FAB(-) mass spectrum (lower spectrum) of mitomycin C

MITOMYCIN C 373

Table V Mass Spectral Data for Mitomycin C

m/z assignment

- FD 334 M+*

335 (M+H)+

FAB (+) - 358

35 7

336

335

(M+H+Na) +

(M+Na ) +

(M+2 H) +

(M+H) +

274 ( (M+H)-02CNH3) + 242 ( ( (M+H) -H~COH) -O~CNH~) +

m/z 57, 91 and 215 signals come from the thioglycerol matrix

- FAB (-) 334

333

29 1

M-'

(M-H)-

(M-CONH) - m/z 107, 215 and 321 signals come from the thioglycerol matrix m/z 139 and 265 signals cannot be assigned with certainty

CI 363

335

302

274

2 42

-

EI - 334

302

29 1

(M-H3COH) +

( (M-H3COH) -02CNH2) + +

(M-02 CNH2)

M+

(M-H~COH)+

(M-OCWH) +

+ 273 (M-0 CNH 3) 259 ( (M-02CNH2)-CH3) + 242 ( (M-H3COH) -02CNH2) +


4.5 Circular Dichroism (CD) Spectrum The chiral centers at C1, C2, C9a and C9 in the mitomy-

cin C molecule are responsible for the CD Cotton effects at 355 nm and 395 nm. The CD spectrum of mitomycin C in a methanolic solution (c=O.OOl%), determined by using a Jobin Yvon Dichrograph 111, is shown in Figure 11.

+A c

Figure 11. CD spectrum of mitomycin C

MITOMYCIN C 375

4 .6 Optical Rotation The optical rotation of mitomycin C in methanol

(c-0.1%) was determined to be +232" . This analysis was performed with a Thorn NPL Automatic Polarimeter Type 243 at 589 nm. The optical rotatory dispersion (Om) spectrum of mitomycin C has been reported by Hornemann et al. ( 4 3 ) .

4.7 Melting Point In the literature no melting point for mitomycin C is

mentioned except for the statement that it should be over 300 "C (11).

4.8 Differential Scanning Calorimetry In nitrogen atmosphere no transitions were recorded up

to 330 OC in a closed (air tied) sample holder. In an open sample holder an undefined large exothermic decomposition takes place from 215 "C. The applied apparatus was a Feteram 111, the heating rate was 5 K/min and the sample size was 3 mg ( 4 4 ) .

4.9 Solubility Mitomycin C is readily soluble in methanol, acetonitrile

normale saline and water, but is nearly insoluble in hydro- carbon solutions, The following solubility data have been reported ( 4 5 ) , determined by suspending an excess amount of mitomycin C in a solvent, followed by filtration and HPLC analysis.

Table VI Solubilities of Mitomycin C

solvent solubility (mM) temperature ("C)

water 2.73 sesame oil 0.0180 isopropylmyristate 0.0193 hexane 0 . 0 2 3 4 ~ 1 0 - ~

~

25 25 37 25


4.10 Partition Coefficient The partition coefficient for mitomycin C has been mea-

sured in various organic phaseldistilled water systems. The results have been combined in Table VII.

Table V I I Partition Coefficients for Mitomycin C

solvent partition coefficient

chloroform 0.26; 0.27, n-octanol 0.41; 0.42 ethyl acetate 0.51 1-pent an 1 2.32 chloroform/2-propano1 (90+10) 0.50 chloroform/2-propanol (50+50) 1.52

chloroform/l -pentan01 (80+20) 1.85 dichloromethane/2-propanol (50+50) 1.04

chloroform/l-pentanol (90+10) 1.12

reference

a the aqueous phase was 0.07M phosphate buffer pH = 7.4

The organic solvents chloroform (1,2) and ethyl acetate (7, 48) have been used with success €or the isolation of mitomycins from the aqueous fermentation broths of Streptomyces species.

4.11 Dissociation Constants The mitomycin C molecule contains several prototropic

functions. Basic groups are the 7-amino group, the N-4 nitrogen and the aziridine nitrogen. Due to rapid degradation the pKa values of the conjugated acid concerning the 7-amino group and N-4 nitrogen can not be deduced from titration experiments with intact mitomycin C. Therefore the acid dissociation constants for these €unctions were derived from titrations with stable analogs (37). Results are listed in Table VIIT. The pKa of the aziridine nitrogen has been determined titrimetrically (11) and kinetically from the pH-rate re1.ationship.s in degradation studies (49-51) although the titrimetrical determination also has been influenced by degradation. Mitomycin C also has acidic properties with an (apparent) pKa 12.44 at room temperature. These acidic properties emerge after keto-enol tautomerization of the 7-amino quinoid moiety which precedes deprotonation in alkaline medium (Figure 2)(37).

MITOMYCIN C 377

Table V I I I Prototropic Functions of Mitomycin C

function determination method

N-4 spectrophotometry 7-amino spectrophotometry aziridine potentiometric titration

via pH-rate profile via pH-rate profile via pH-rate profile via pH-rate profile

7-amino spectrophotometry

pKa(25"C) reference

-1.2 -1.3

3 .2 2 .8 2 2.74 2 . 5 0 ( 4 9

12 .44

4 . 1 2 Electrochemistry Due to the presence of the quinone ring of the mitomycin

C molecule the compound can be reduced and transformed into its hydroquinone form. This conversion triggers a complicated pattern of chemical consecutive reactions. Although much progress has been made in the elucidation of the processes occurring during and following electrochemical reduction of mitomycin C the exact complicated mechanisms have not yet been unravelled (50). This is due to: - rapid degradation of mitomycin C at pH<3 and pH>12; - the extremely high reactivity of the hydroquinone form

of mitomycin C; - the fact that chemical degradation products as well as

electrochemically generated degradation products of mitomycin C can be reduced within the region of reduction potentials of mitomycin C itself ( 5 0 ) .

Electrochemistry of mitomycin C has been studied in aqueous solutions (50, 52-54) and aprotic solvents ( 5 5 ) , by using polarography ( 5 0 , 52-54) and cyclic voltammetry ( 5 0 , 5 2 , 5 3 , 5 5 ) . Direct current polarographic curves for mitompcin C are shown in Figure 1 2 . Rapid degradation of mltomycin C within the time scale of recording a direct current polarographic curve at pH< 3 and pH > 1 2 strongly influences the shape and heights of these curves ( 5 0 ) .

378 JOS H. BEIJNENETAL.

I

I I

PH = S O 5

I I I I

0 -500 -1000 -1500

Figure 12.

potential (mV)

-4 Direct current polarographic curves of mitomycin C (10 M)

MITOMYCIN C 379

5. METHODS OF ANALYSIS

5.1 Elemental Analysis The elemental analysis of a purified mitomycin C sample

for C, H, N and 0 was reported to be as follows:

Table IX El-emental Analysis of Mltomycin C (11)

element % theory % found

C 53.83 53.55 H 5.43 5.57 N 16.76 16.86 0 23.98 24.13

5.2 Ultraviolet Spectrophotometry Mitomycin C has been determined by using UV spectropho-

tometry at 360 nm &n methan-qjl solution. This assay is linear in the range 2x10- to 4x10 M y with a precision of ? 1%.

5.3 Thin Layer and Paper Chromatography A diverse array of thin layer chromatographic systems

have been described in the literature for analysing mitomycin C and derivatives (55). Some representative systems have been summarized in Table X. The mitomycin C spot can be located by: - irradiation with UV light of 254 nm; - examination under daylight (mitomycin C has a blue-vio-

let colour) ; - spraying with a 1 in 100 solution of ninhydrin in alco-

hol, followed by heating the plate at 110°C for 15 minutes by which mitomycin C appears as a pink spot (56).

Table X Thin Layer and Paper Chromatography of Mitomycin C ~ ~ ~ _ _ _ _ _ _ _ _ _

plate solvent (v/v) Rf reference

silica- butanol (sat. with water) 0.57 (12) gel ethyl acetate (sat. with water) 0.56 (12)

methanol-benzene-water (10:5:5) 0.02 (12) isopropylalcohol-1ZNH OH (2:l) 0.88 (57)

0.80 (57) met hano 1 1-octanol-acetone-ligroin (9O-115°C) (2:5:5) 0.11 (58) chloroform-methanol (9: 1) 0.12 (59)

paper butanol-water (84:16) 0.57 (60) methanol-benzene-water (1:1:2) 0.02 (60) ethyl acetate (sat. with water) 0.56 (60)

4

cellulose isobutyric acid-0.5M NH40H (10:6) 0.75 (57)


5.4 Cel Filtration Chromatography For the separation of mitomycin C from its degradation

products and alkylation adducts Tomasz et a1 (57, 61-64) utilized pel filtration chromatography on Sephadex G-25 columns and 0 . 0 2 M NH HCO solution as eluent. By using a 1 . 5 ~ 41.5 cm Sephadex G-25 (fine) column the elution volume of mitomycin C is 96 ml ( 5 7 ) .

4 3

5.5 High Performance Liquid Chromatography HPLC procedures reported in the literature have been de-

veloped mainly for the analysis of mitomycin C and derivatives in decompositinn mixtures ( 5 1 , 5 9 , 65-71) in biological matrices (plasma, urine, bile, tissues) ( 4 6 , 72-86) and other systems such as (enzyme containing) culture media ( 6 2 , 6 9 , 87-90). Detection by W absorbance measurements at 365 nm is usually preferred ( 5 5 , 7 5 , 7 6 ) because of the high specifi- city and sensitivity, but detection at 254 nm, which is less sensitive, is also reported ( 6 6 , 91, 92). The polarographic electrochemical detection mode is an elegant alternative for the detection of mitomycin C ( 5 0 , 75, 93), but demands extensive experimental precautions to be taken, such as removal of oxvgen from the mobile phase and damping of pulsating flow of the solvent delivery system ( 9 3 ) . Important HPLC systems have been enumerated in Table XI,

Table XI High Performance Liquid Chromatography for Mitomycin C ~~ ~~ ~~

column mobile phase reference

Reversed Phase HPLC p-Bondapak C18 (10 ,pm) methanol-water (35: 65) (77-80) p-Bondapak C18 (10 pm) (46) Lichrosorb RP18 (10 pm) methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0 (67) Lichrosorh RP8 (5 p'm) methanol-water-phosphate buffer pH 7.0 (20:70:10) (82) Ultrasphere ODX acetonitrile-0.03M potassium phosphate buffer (12.5:87.5) (91)

(94)

methanol-0.01 M phosphate buffer (pH 6,0)(30:7O;w/w)

Hypersil-MOS (5 pm) acetonitrile-0.05M phosphate buffer pH 7 (10:90) (75) Hewlett-Packard C8 (10 pm) acetonitrile-water; gradient from 5% to 41% acetonitrile Radial Pak C18 (10 pm) methanol-0.01M phosphate buffer pH 7; gradient from 0% to 50% methanol ( 4 2 )

Ion Pair HPLC Lichrosorb RP18 (10 pm) Lichrosorb RP18 ( l o p ) methanol-water (30:70;w/w) + 0.5% (v/w) phosphate buffer pH 7.0

+ 0.5% (v/w) tetrabutylammoniumbromide solution (20%, w/w) (59) Hypersil ODS (5 pm) acetonitrile-water (25:75) + 0.2% sodium laurylsulphate + 0.025M

sodium acetate (66) Hypersil ODS (5 pm) acetonitrile-water (20:80) + 0.2% cetrimide + 0.0125M sodium acetate (66) Resolve C18 (5 pm) methanol-1 mM octanesulfonicacid sodium salt solution (30:7O;v/v)

methanol-water (40:63;w/w) + 0.5X (v/w) glacial acetic acid (~H*=4.3)~ (65)

(pH* 4.5)a (85)

Porasil P (10 pm) Normal Phase HPLC

tetrahydrofuran-methanol-isooctane-dichloro~t~ane (3:7:15:75) Porasil (10 pm) chloroform-methanol (9: 1)

ethyl acetate-methanol (95:l) Silica Si-60 ethyl acetate-methanol-water-dichloromethane (97:2:1:1)

a pH values of organic modifier-water mixtures


5.6 Identification and Purity Tests in Official Com-

In the United States Pharmacopeia XXI (56) the monographs "Mitomycin" and "Mitomycin for Injection" have been included. In these monographs the identification of mitomycin C is based on IR and UV spectroscopy and chromatography by which the sample should exhibit the same spectroscopic and chromatographic properties as a similar preparation of a USP Mitamycin Reference Standard. The pH of a solution containing 5 mg/ml should be between 6.0 and 8.0. The water content of the raw material may not exceed 5%. Furthermore Mltomycin and Mitomycin for Injection (a dry mixture of mitomycin C and mannitol) must meet the requirements of specific tests described in the USP XXI such as: Safety Tests, Depressor Sub- stances Tests, Pyrogen Tests and Sterility Tests (56).

pendia

6. STABILITY, DEGRADATION

6.1 Stability in Aqueous Solutions Mitomycin C and related mitomycins are not stable when

stored as aqueous solutions (11-13, 51, 55, 57, 59, 65-68, 82, 96-108). Degradation is accelerated by acid, alkali, (buffer) ions and high temperatures (108). At pH<7 mitomycin C is converted into l-hydroxy-2,7-diaminomitosenes and at pH > 7 it is hydrolysed into 7-hydroxymitosane. The overall degradation scheme is given in Figure 13. In the mitosene compounds the C9a methoxy group has been cleaved, a double bond between C 9 and C9a has been introduced and the aziridine moiety has been opened. Aziridine ring opening occurs with configurational retention at C2 and water attachment at C1 with inversion as well. as retention of the C1 stereochemistry yielding 1,2-trans- and 1,2-cis-l-hgdroxy-2,7-diaminomitosenes, respectively. Under drastic acidic conditions progres- sive degradation reactions lead to hydrolysis of the 7-amino group and the C10 carbamate side chain (9-11, 1 3 ) . The mechanism of the initial degradation step of mitomycin C in acid is shown in Figure 14 (107, 108). In the cascade of reactions an intermediate is proposed, having a positive charge at C1 (49, 107-110). This electrophilic center serves as target for attacking (nucleophil ic) water molecules resulting in the formation o f l-hydroxv-2,7-diaminomitosenes. The degree of protonation of the 2-amino function in the proposed intermediate species (pKa:2.8(65)) determines the C1 stereochemistry of the resulting mitosenes. A protonated 2-amino function may direct incoming water molecules to a cis configuration. When- ever the amino function in the intermediate is not protonated the directing electrostatic power is absent and a reacting water molecule may approach the C1 carbonium ion from both

383 MITOMYCIN C

sides with roughly equal chances. This explains the remark- able influence of pH on the C 1 stereochemistry of the resulting mitosenes. At pH=l the cisltrans ratio is about 4 while at pH=6 the ratio approaches 1 (65). When mitomycin C degrades in acidic medium in the presence of nucleophiles such as acetate, phosphate or ever! DNA parts, l-nucleophile-2,7-diaminomitosenes are generated (57, 103, 111-114). These are important observations which lend credibility to the hypothesis that acid activation may play a role in the in vivo mitomycin C DNA alkylation.

In alkaline solution (pH 7 to 13) the 7-amino group of the mitomycin C molecule is substituted by a hydroxy function (59, 100-102). Under prolonged severe alkaline treatment the quinone chromophore is destroyed (59) .

In huffered media the degradation kinetics o f mitomycin C can be described adequately by (pseudo) first order kinetics. Some kinetic data are summarized in Table X I I . For further detailed kinetic information is referred to the literature (49, 51, 55, 67, 107, 108).

mitomycin C \ OH-

cis- mitosene

7 - hydroxymitosane

+ i \ i, NH,

trans -mitosene

Figure 13. Overall degradation scheme of mitomvcin C


.." L

c

0 II

0 1

+ CH.OH N-H

Figure 14. Acid degradation mechanism of rnitomycin C

MITOMYCIN C 385

Table XI1 Degradation Kinetics for Mitomycin C

conditions/method kobs (s-l) reference

pH 0.87; perchloric acid solution;

pH 2.86; 0.001M acetate buffer;

pH 3.0; 1.OM phosphate buffer;

pH 4.64; 0.1M phosphate buffer;




2OoC/ HPLC 1 . M O - ~ (65)

20°C/ HPLC 4. Ox (65)

25'C/ HPLC 1.2~10-~ (114)

40°C/ spectrophotometry 1.4~10-~ (107)

37'C/ HPLC 4.1~1O-~ (105)

37"C/ spectrophotometry 1.8~10-~ (104)

25OC/ HPLC 5.3~ (67)

6.2 Stability in Pharmaceutical Formulations Mitomycin C is commercially available in a lyophilized

form containing mannitol (Mutamycin@ : 5 mg mitomycin C + 10 mg Mannitol) or sodium chloride (Mitomycin C-Kyowam :10 mg mitomycin C + 240 mg sodium chloride; Ametycine lo@ : 10 mg mitomycin C + 240 mg sodium chloride) as excipients. In this state the drug is stable for at least two years if kept dry and in well closed containers at room temperature in the dark (115). After reconstitution the drug has only limited stability, depending strongly on the pH of the final solution (68). An overview of stability data of mitomycin C in infusion fluids is given in Table XIII. More specific and extended data are described in the references concerned (68, 70, 71, 116-120).

Table XI11 Stability of Mitomycin C in Infusion Fluids at Room Temperature and - 3 O O C

infusion fluid concentration stability reference ~~

5% dextrose

0.9% sodium chloride

sterile water

5% dextrose


phosphate buffer pH 7.75

5% dextrose



50 pglml

50 pg/ml

2 mg/ml

50 pg/ml

50 pglml

50 pg/ml

0.4 mg/ml

0 .4 mg/ml

0.6 mg/ml

tgO = 3 . 0 h; t50 = 7 8 h

93% remained after 5 days

stable for 7 days

26% remained after 12 hours

t50 = 12 h

tgO = 15 days

tgO = 1 h

t = 2 4 h

99% remained after 4 weeks storage at -30°C 90

MITOMYCIN C 387

Further research is required to resolve the problem of conflicting stability data on mitomycin C in 0.9% sodium chloride solution (68, 70, 116, 119, 120).

6.3 Stability in Biological Media Chemical instability of mitomycin C in biological fluids

and tissues may be a complicating factor in the bioanalysis of the drug. Ignorance of the chemical stability of the drug during sampling and sample pre-treatment procedures may lead the researcher to misinterpretations. Recently, den Hartigh et al. (86) published an extehsive study concerning the hand- ling of mitomycin C biological samples. The following recom- mendations and guidelines were given by these authors (86): - whole blood samples should be placed in ice immediately

after sampling; separation of plasma and red blood cells should take place within half an hour after collection of the samples;

- mitomycin C samples should be protected from long-term exposure to daylight in order to prevent analyte degradation;

- the pH of urine samples should always be checked and, if necessary, adjusted to neutral values, as a low pH will induce mitomycin C decomposition and the formation of precipitates upon which mitomycin C may be adsorbed:

- plasma and urine samples need not necessarily be placed in the refrigerator or freezer immediately after preparation, provided analysis is carried out within a period of 2 h;

- plasma and urine samples may be stored in a freezer at -2OOC; however, analysis should take place within 3 weeks as longer storage at this temperature may induce considerable reduction of the mitomycin C concentration;

- repeated freezing and thawing of plasma and urine samples does not influence the analytical results of the mitomycin C assay;

- extracts of biological samples may be kept for 24 h, in the dark, either dry or dissolved in at 4 ° C or 20°C prior to analysis.

The in vitro half life of mitomycin C was determined > 14 days under conditions of the clonogenic assay (

at least methanol

to be 21). Mi-

tomycin C is fairly stable (70-802 of the drug remained) when incubated for 5 days at 37°C in Medium E, RPMI, Fischer's or MEM culture media, supplemented with serum (69). In Mark's M-20 culture medium with fetal calf serum, Proctor and Gaul- den (90) observed that the amount of mitomycin C was reduced by 29% after 30 minutes incubation.


7. PHARMACOLOGY

7.1 Mechanism of Action Mitomycin C induced cytotoxicity is believed to be medi-

ated by DNA binding. The drug as such, in its oxidized form, is not cytotoxic although some interaction with nucleic acids may exist (122). Mitomycin C must be activated before it is capable of alkylating DNA (123-128). The alkylating capacity is unmasked by reductive or acidic activation. An outline of the reductive activation pathway is given in Figure 15. Re- duction can be mediated by enzyme systems (87-89, 129-132) or by chemical reducing agents (58, 126). The conversion of mitomycin C into its reduced form destabilizes the compound drastically and almost instantaneously the C9a methoxy group is lost (Figure 15). A subsequent rearrangement in species C leads to the formation of the quinone methide D which acts as target for nucleophilic nucleic acid attack, by which a mono- fu ctional alkylation adduct is formed. An intramolecular SN -displacement of the carbamate moiety leads to the bifunc- tional cross-linked adduct F (Figure 15) (128). The interaction of mitomycin C with DNA is more than 9OX of the mono- functional type. Chemical structures of monofunctionally linked mitomycin C adducts with DNA, nucleotides and nucleosides have been reported (62-64, 133-136). Reduction of mitomycin C may follow either one or two electron reduction steps. The semiquinone radical is also believed to be capable of producing interstrand cross-links between complimentary strands of DNA (87, 137). Under aerobic conditions, the semiquinone radical formed after a one electron reduction can transfer its electron to oxygen to generate superoxide anion under regeneration of the oxidized quinone (138-142). Reactive oxygen species can cause DNA strand scission and membrane lipid peroxidation, leading to cell damage and can contribute in this manner to mitomycin C induced cytotoxicity. In the absence of any reducing agent or enzymes mitomycin C is also capable of coupling covalently with DNA or nucleic acid parts, but then, activation by acid is a requisite (57, 103, 111-113, 143, 144). Acid catalyzed degradation of mitomycin C (Figure 14) occurs through reaction intermediates possessing an electrophilic C1 center which is the target for nucleophilic nucleic acid attack.

More studies are necessary in order to elucidate which of the proposed mechanisms or combination is actually responsible for mitomycin C induced cytotoxicity in vivo.

9

MITOMYCIN C 389

OH OH

Figure 15. Reductive activation mechanism of mitomycin C


7.2 Clinical Antitumour Activity Single agent activity of mitomycin C has been demonstra-

ted against various solid neoplasms in humans such as breast cancer (29, 145) , stomach cancer (146), pancreatic cancer (147), colorectal cancer (29), non-small cell lung cancer (148) , squamous cell carcinoma of the uterine cervix (28) and head and neck cancer (28). Encouraging results have been obtained on the treatment of superficial transitional cell carcinoma of the bladder when mitomycin C is administered intravesically (28-30, ,l49). Mitomycin C is usually used in combination with other cytostatics (28-30).

7.3 Clinical Toxicity Delayed cumulative myelosuppression is the most impor-

tant toxicity. Others include nausea, vomiting, anorexia, diarrhea, stomatits, skin rashes and alopecia (24, 28, 150). Extravasation of mitomycin C causes severe cellulitis accom- panied with chronic pain and ulceration (151). Interstitial pneumonitis, pulmonary fibrosis, hemolytic-uremic syndrome and renal failure have occasionally also been documented as due to the drug. Following topical intravesical mitomycin C therapy for bladder cancer dermatological toxicity has been encoutered in some cases (154).

7.4 Pharmacokinetics Following intravenous bolus injection of mitomycin C the

plasma concentration-time curves show a bi-phasic, exponential decline (72, 155). Mitomycin C pharmacokinetics (at clinically relevant doses) is not dose dependent (72, 155). Ma- j o r routes of elimination are liver metabolism and urinary excretion. Metabolites have never been identified. Absorption after oral administration is very irregular(l56). After intravesical instillation, limited absorption of mitomycin C into the systemic circulation occurs (30, 157). Pharmacoki- netic data after intra-arterial administration are available (73).

391 MITOMYCIN C

8. DETERMINATION IN BODY FLUIDS

The analysis of mitomycin C in biological samples is hampered by its instability, low concentrations and the polar nature of the mitomycin C molecule (55, 7 6 ) . The following techniques have been used: microbiological assays ( 7 4 , 9 5 , 1 0 4 , 158-161, 1 6 6 ) , bioassay by using a repair deficient mu- tant of Chinese hamster ovary cells ( 1 6 2 ) , immunological assay ( 1 6 3 , 1 6 4 ) , high-performance differential pulse polarography ( 5 4 ) and HPLC assays ( 4 6 , 72-86 , 93-96, 1 5 5 ) . The HPLC assay designed by Den Hartigh et al. ( 4 6 , 7 2 ) has shown to be very suitable for pharmacokinetic studies ( 7 2 - 7 4 ) . The following procedure is followed for plasma samples: 0 . 2 , 0.5 or 1.0 ml samples is mixed with the internal standard porfiromycin (about 500 ng) and with 2 . 0 , 5 . 0 or 10.0 ml, respectively, of chloroform-2-propanol ( l+l , w/w). After shaking for 1 minute and centrifugating for 5 minutes ( 2 5 0 0 g), the clear supernatant is transferred into a conical tube and evaporated to dryness at 30-40°C under nitrogen. The residue is dissolved in 50-100 pl of methanol with the aid of a vortex- type mixer. Aliquots of 10-20 PI are injected into the chromatograph. HPLC: column: p Bondapak C18 ( 3 0 cm x 3 . 9 mm i.d., particle size 10 pm); mobile phase: 0.01 M phosphate buffer pH 6.0-methanol (70+30, w/w); flow rate 1.0 ml/min; detection: 365 nm.

An overview of published determination methods of mitomycin C in body fluids is given in Table X I V .

Table X I V Determination Methods for Mitomycin C in Body Fluids

matrix sample pretreatment determination limit(ng/ml) - test organism reference a b

microbiological assays

blood, tissues, urine, bile plasma, urine plasma, urine

W tJ serum, urine

no 2 ~~~

E.coli R

no no no

10 500

60

E.coli B B.subtilis E. coli

bioassay with hamster ovary cells

plasma no 1 (162)

immunological assays

serum, urine no 4 (163)

polarography

plasma, urine lI0 200 (54) plasma, urine 1.s 25 (54)

Table XIV (continued)

matrix high performance liquid chromatography

chrom to ra hic reference mode - g g p sample pre- internal determination

treatment - standard - limit(ng/rnl) - a C

serum, urine, ascites plasma plasma plasma, urine, bile, asc i t e s serum, plasma, urine

1.1. 1.1. 1.1.

1.1. 1.S.

plasma, urine plasma, urine

plasma, urine w

plasma, urine serum serum

plasma, urine plasma, urine

1.s. 1.s.

1.1.

1.1. (plasma) 1.S. deproteination with methanol 1.S. 1.1.

no no no

Yes Yes

yes no

Yes

no no no

Yes Yes

40 (serum) 1 1

1 5

4 (plasma) 2 (plasma)

1 (plasma)

1 (plasma)

50 (urine)

500 (urine)

200 (urine)

10 10

25 10

RP RP RP

RP RP

RP (gradient) RP

NP

RP RP RP

RP RP

a - 1.1.: sample pre-treatment by liquid-liquid extraction; 1.s.: sample pre-treatment by liquid-solid

b - determination limits for 25 p1 ( 8 0 ) , 50 pl ( 8 5 , 1 6 3 ) , 100 pl ( 8 4 ) , 0.2 ml (160), 1.0 ml ( 4 6 , 72-74,

- the internal standard is porfiromycin d - RP: reversed phase chromatography; NP: normal phase chromatography.

extraction.

77, 7 8 , 82, 162), 2.0 ml (54, 7 5 , 9 5 ) and 2.5 ml ( 9 4 ) samples C


Acknowledgements The authors wish t o thank Ms C. de Graaf €or typing the manuscript and Mr P.W.Y. Chan for preparing the figures.

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MITOMYCIN C 395

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MITOMYCIN C 391

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MITOMYCIN C 401

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-

NEOSTIGMINE

A . A . Al-Ba&* and Ad. lahiq"

*DepahAnent 06 Phanmacaficak? Chemhin.thy and **Vep&men;t 06 Phatrmacotogy, College 06 P h m a c y , King Saud UVLive.&Lty, P.O. Box 2457, Riyadh-77451, (Saudi A m b h l .


Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved. 403


1. History

2. Description

2.1 Nomenclature

2.2 Formulae





4. Synthesis

5. Pharmacokinetics

6 . Therapeutic and Other Uses

7. Toxicity


8.1 Identification


8 . 3 Biological Method



8.6 Ion-Selective Electrodes Method

8.7 Polarographic Method

Refer en c es

NEOSTIGMINE 405

1. His torv

As a r e s u l t o f t h e b a s i c r e sea rch o f Stedman and a s s o c i a t e s (1 ,2 ,3) i n e l u c i d a t i n g t h e chemical b a s i s o f t h e a c t i v i t y of physostigmine, Aeschlimann and Reiner t (4) sys t ema t i ca l ly i n v e s t i g a t e d a s e r i e s of s u b s t i t u t e d phenyl esters o f a l ly lcarbamic a c i d s . Neostigmine (Prost igmin) , a most promising member o f t h i s series, was introduced i n t o the rapeu t i c s i n 1931 f o r i t s s t imulant a c t i o n on t h e i n t e s t i n a l t r a c t . I t was repor t ed independent ly by Remen (5) and Walker (6) t o be e f f e c t i v e i n t h e symptomatic therapy of myastheniagravis which is due t o d e f e c t i n synap t i c t ransmiss ion at neuromuscular j unc t ion . When t h e patient i s given an appropr i a t e dose o f neos t igmine, t h e response t o t i t a n i c s t imu la t ion i s improved along with symptomatic improvement i n muscle s t r e n g t h ( 7 ) . Neostigmine was a l s o found usefu l i n p a r a l y t i c i l l e u s , a tony o f u r ina ry b ladder and i n glaucoma (8 ,9) .

2 . Descr ip t ion

2 .1 Nomenclature


3- (Dimethylcarbamoyloxy) -N , N ,N-tr imethyl- an i l in ium bromide. 3-(Dimethylcarbamoyloxyphenyl)trimethylammonium bromide. Benzenaminium, 3- [ [ (dimethylamino)carbonyl] oxy] -N ,N,N-trimethyl bromide. (m-Hydroxypheny1)timethylammonium bromide dimethyl carbamate. 3- [ [ (Dimethylamino)carbonyl]oxy] - N , N , N , trimethylbenzenaminium bromide 3- [ [ (Dimethylamino) carbonyl loxy] -N ,N ,N- trimethylbenzaminium methyl su lpha te . (m-Hydroxyphenyl) t r ime thy l ammonium methyl su lpha te dimethylcarbamate. (3-Dimethylcarbamoxyphenyl)trimethylamonium methyl su lpha te (10-13).


2.1.2 Generic Names

Neostigmine bromide; Neostigmine DCF; NFN; Neoeserine; Proser ine ; Synstigmine; Eus t igmine ; Phi 10s t igmine ; Neos t igmin i bromidum; Neost igmini i bromidum; Neostigmium bromatum (13,14).

2 .1 .3 Trade Names

Neost i pmine bromide

Prost igmin; Juvast igmine; Konstigmin; Metastigmin; Normastigmin; Prost igmin; Leostigmin (13,14) .

Neostigmine methyl su lpha te

In t r a s t igmina ; Juvast igmin; Mestastigmin; Neoesser in; Normastigmin; Prost igmin; Hodostin; S t i g l y n ; Stigmosan (13,14).

2 .1 .4 CAS Regis t ry Number

59-99-4 Neostigmine

51-60-5 Neostigmine methyl su lpha te (14) 114-80-7 Neostigmine bromide

2 .1 .5 Wiswesser Line Notat ion

1 N 1 & VOR CK-& E (Neostigmine bromide) (15)

2 . 2 Formulae

2 . 2 . 1 Empir ical

C H BrN202 Neostigmine bromide

C13H22N206S Neostigmine methyl su lpha te

1 2 19

NEOSTIGMINE 401

2 . 2 . 2 S t r u c t u r a l

CH ‘I

0

’ - c II

x -

,CH3 -N

‘CH3

X = B r - Neostigmine bromide

X = CH3SOi Neostigmine methyl su lpha te

2 . 3 Molecular Weipht

Neostigmine bromide 303.2 Neostigmine methyl su lpha te 334.4


C 47.53%, H 6.32%, N 9.24%, B r 26.36%, 0 10.55% (bromide). C 46.69%, H 6 .63%; N 8 .38%, S 9.59%, 0 28.71% (methyl su lpha te) .


Odorless , c o l o r l e s s c r y s t a l o r a white c r y s t a l l i n e , s l i g h t l y hygroscopic powder with a b i t t e r t a s t e (14) *

3. Physical P r o p e r t i e s

3 .1 Melting Point

Neost igmine bromide

Crys t a l s from a lcohol and e t h e r melt a t 167’ wi th decomposition (13) .

Neostigmine methyl su lpha te

Crys t a l s from alcohol melt between 142OC and 145OC (13) *

408 A. A, AL-BADR AND M. TARIQ

3.2

3 .3

3.4

3 . 5

3.6

3.7

Ext rac t ion

Neostigmine s a l t s a r e qua ternary ammonium compounds and a r e so lub le i n water. i s a c i d i f i e d with d i l u t e a c e t i c a c i d , evaporated t o dryness and ex t r ac t ed with methanol. The methanol e x t r a c t w i l l conta in most o f t h e qua ternary ammonium compound and may be p u r i f i e d by paper chromatography (16) .

The aqueous s o l u t i o n

S o l u b i l i t y

Neostigmine s a l t s a r e f r e e l y s o l u b l e i n water . Moderately so lub le i n chloroform and e thanol . Inso luble i n e t h e r (11).

Acid i ty

Dissolve 0 .20 g i n 20 m l carbon d iox ide - f r ee water and t i t r a t e a t pH 7 .0 with 0.02 N sodium hydroxide (carbonate- f ree) no t more than 0.2 m l i s requi red (17).

Moisture Content and Hygroscopici ty

Not more than I%, determined by drying a t 1 0 5 O C (10) - Storage

I t should be s t o r e d i n a i r t i g h t con ta ine r s p ro tec t ed from l i g h t (10 ) .

Spec t r a l P r o p e r t i e s

3 . 7 . 1 U l t r a v i o l e t SDectrum

The UV spectrum of neostigmine bromide i n e thanol i s given i n Figure 1. I t shows two maxima a t 260 nm and 266 nm. The spectrum was recorded on Varian spectrophotometer model DMS 90.

Clarke (11) repor ted t h e following: Neostigmine methyl s u l f a t e i n 1 N H2SO4; maxima a t 260 nm (E 1%, 1 cm 20) and 266 nm (E 1%, 1 cm 18) .

NEOSTIGMINE

lot

2 00 2SOWavelength 300 (nm) 350 400

Figure I: Ultraviolet spectrum of neostigmine bromide in methanol.


3.7.2 In f r a red Spectrum - (IR)

The IR spectrum of neost igmine bromide i n K B r d i s c is presented i n Figure 2 , and was recorded on Pye-Unicam I R spectrophotometer model SP 1025. t h e s t r u c t u r a l assignments a s shown below:

Frequency cm-l

The f requencies and

As s i gnmen t

CH s t r e t c h 1 3020-

2900 .)

1730 C = 0 (amide) s t r e t c h

1 1610 C = C s t r e t c h (aromatic)

1595)

1490-1450 CH bending v i b r a t i o n s

1 1030

1070) C - N v ib ra t ion ( a l i p h a t i c )

780 and 895 CH (aromatic) bending v i b r a t i o n s

Clarke (11) repor ted t h e fol lowing:

Neostigmine bromide, potassium bromide d i s c t h e p r i n c i p a l peaks a r e 1711, 1215 and 1154 cm-1.

1 3.7 .3 Proton Nuclear Magnetic Resonance ( H NMR)

Spectrum

1 The H NMR spectrum of neostigmine methyl su lpha te is shown i n Figure 3. d i sso lved in deuterium oxide (DzO) and i t s spectrum was determined on a Varian - T60 A NMR spectrometer us ing sodium 2,2-dimethyl 2 - s i l apen t ane- 5-sulphonate (DSS) a s t h e i n t e r n a l s tandard .

The drug was

I - -

I

f

8.0 7.0 6.0 S.OPpm( 6k.O 3 -0 2 .o I .o 0

Figure 3: Proton NMR spectrum of neostigmine methyl sulphate in D20 with

TMS as internal reference.

NEOSTIGMINE 413

Assignment of t h e chemical s h i f t s t o t h e d i f f e r e n t protons i s shown below:

Chemical s h i f t Mult i - (ppm) p l i c i t y Proton assignment

0 3.04) II

s i n g l e t s -C-N(CH ) 1 -3 2 3.12

3. 72 s i n g l e t 4- ( g 3 )

3 .80 s in g 1 e t CH, - S O4

7.15-7.60 mul t ip l e t s Aromatic pro tons 1 7.66-8.30

3.7.4 Carbon-13 Nuclear Magnetic Resonance

(C-13 NMR) Spectrum

The C-13 NMR s p e c t r a of neostigmine methyl su lpha te i n deuterium oxide (D2O) us ing DSS (sodium 2,2-dimethyl 2-s i lapentane- 5-sulphonate) as an i n t e r n a l r e fe rence were obtained us ing a J e o l FX 100 MHz spec t rometer a t an ambient temperature . Figures 4 and 5 r ep resen t t h e proton-decoupled and o f f - resonance s p e c t r a r e spec t ive ly .

CH3

1 2 I +

5

13 CH3 - S O i

1 C H

.CH3

3 0 - C -N

" 0 2

The carbon chemical s h i f t s were assigned on t h e b a s i s of t h e chemical s h i f t theory and t h e off-resonance s p l i t t i n g p a t t e r n and a r e shown below:


Figure 4 : Proton -decoupled carbon-I3 NMR spectrum Of n-stigmine methyl wlphate

in D20 with DSS as internal reference.

Figure 5 : Off -Resonance carbon -13 NMR spectrum of neostigmine methyl sulphate

in 9 0 with DSS a s internal reference.

NEOSTIGMINE 415

Chemical s h i f t Mul t i - (ppm) p l i c i t y Carbon assignment

38.9 q u a r t e t 1

38.8 q u a r t e t 2

158.3 s i n g l e t 3

149.8 s i n g l e t 4

126.9 double t 5

134.0 doub 1 e t 6

119.8 doublet 7

154.5 s i n g l e t 8

117.1 doublet 9

59.6 q u a r t e t 10, 11 and 12

57.9 q u a r t e t 13

3 . 7 . 5 Mass Spectrum

The e l e c t r o n impact ( E I ) mass spectrum a t 70 e V was recorded on Varian MAT 311 mass spec t rometer i s shown i n F igure 6 . spectrum an ion a t m/e 428 was observed which most probably a r i s e s from a recombination o f fragments. The spectrum shows a base peak a t m/e 72. The most prominent i ons are shown below:

In the

Figure 6: Electron impld (El)mass spectrum Of neostigmine methyl sulphate.

NEOSTIGMINE 417

72

208

356

42 8

Fragment

CON ( c H ~ ) ~ +

0-CON ( c H ~ $ + @- 428 - CON (CH,),I+

L-. OCON ( CH3)

1'

1 CH2

The chemical i o n i s a t i o n ( C I ) mass spectrum was obtained on Finnigan 4000 mass spectrometer and is shown i n Figure 7 . The spectrum shows ions at m/e 127 ( t h e base peak) and a t m/e 303 and both a r e probably a r i s e d from a recombination o f fragments. The most prominent ions a r e shown below:

Figure 7: Chemical ionization ( C I mass Spectrum Of neostigmine methyl sulphate.

NEOSTIGMINE 419

m/e

127

Fragment

l+ 0 II

CH30-S-O-CH3 .t H II 0

209

303 l+

4. Synthes is

Neostigmine bromide can be prepared from 3-hydroxydi- methylan i l ine , which is made from 3 - n i t r o a n i l i n e by o rd ina ry s y n t h e t i c methods. The 3-hydroxydimethyl- a n i l i n e i s d isso lved i n an a l coho l i c s o l u t i o n of t h e c a l c u l a t e d amount of potassium hydroxide, and t h e s o l u t i o n of t h e potassium hydroxide, and t h e s o l u t i o n o f t h e potassium d e r i v a t i v e so formed is t r e a t e d wi th dimethylcarbamoyl c h l o r i d e , Me2N.COC1 (made from dimethylamine and carbonyl c h l o r i d e ) . This g ives a t e r t i a r y base which, by combination with methyl bromide, y i e l d s t h e requi red qua ternary s a l t - i . e . t h e dimethylcarbamic ester o f 3-hydroxy-NNN-trimethylani- l inium bromide: (18, 19) .


OCON (CH3)

OCON (CH3)

Neostigmine methylsulphate can be made i n the same way, except t h a t i n t h e l a s t s tage the secondary amine is combined with methyl sulphate t o form the sal t

[ (R-AMe3)MeSOi] (18).

OK OCON (CH3)

0-C-N, ” 0 CH3

C H 3 S 0 i

NEOSTIGMINE 42 1

A modif ica t ion of t h i s r o u t e is t o convert t h e hydroxydimethylani l i n e i n t o t h e qua r t e rnary s a l t (by combination wi th methyl bromide o r methyl su lpha te) , and then t o t rea t t h e s a l t with dimethylcarbamoyl c h l o r i d e (18).

9 N(CH3)2

OK C l C O N (CH3)

P

+ -

Neostigmine can be synthesized e i t h e r d i r e c t l y from 3-hydroxydimethylaniline with phosgene t o g ive t h e carbonyl ch lo r ide and then with dimethylamine t o g ive 3-dimethylaminophenyldimethylurethane or t h e l a t t e r may be prepared from t h e sodium s a l t of t h e s t a r t i n g ma te r i a l and dimethylcarbamic ch lo r ide . case t h e product i s converted t o t h e methylsulphate by t reatment with dimethyl su lpha te (20).

In e i t h e r

Y ON a OCON (CH31

OH

COC 12 N 2-

OCOCl . CH3S0i

+

OCON (CH3)


5. Pharmacokinetics

Neostigmine is absorbed poor ly a f te r o r a l admin i s t r a t ion , such t h a t much l a r g e r doses a r e needed than by t h e p a r e n t e r a l rou te . Whereas t h e e f f e c t i v e p a r e n t e r a l dose of neostigmine i n man i s 0 .5 t o 2.0 mg, t h e equiva len t o r a l dose may be 30 mg o r more. Large o r a l doses may prove t o x i c i f i n t e s t i n a l absorp t ion is enhanced f o r any reason and t h e qua ternary a lcohol and parent compound are excre ted i n t h e u r i n e . Py r idos t ig - mine and i t s quaternary a lcohol are a l s o t h e predominant e n t i t i e s found i n u r i n e a f t e r admin i s t r a t ion o f t h i s drug t o man (21).

Neostigmine was r a p i d l y e l imina ted from t h e plasma o f 5 p a t i e n t s t o whom 5 mg of t h e methylsulphate had been given t o an tagonise r e s i d u a l neuromuscular b lock . The plasma concent ra t ion o f neost igmine dec l ined t o about 8% o f i t s i n i t i a l va lue a f te r 5 minutes with a d i s t r i - bu t ion h a l f - l i f e of less than one minute. E l imina t ion h a l f - l i f e ranged from about 15 t o 30 minutes. amounts o f neost igmine could be de t ec t ed i n t h e plasma after one hour ( 1 4 ) .

Trace

O f neost igmine t h a t reaches t h e l i v e r , 98 pe r cen t is metabol ized i n 10 minutes. ( 2 2 ) I ts t r a n s f e r from plasma t o l i v e r cells and then t o b i l e is probably pass ive i n cha rac t e r . Since c e l l u l a r membranes permit t h e passage o f plasma p r o t e i n s synthes ized i n l i v e r i n t o t h e blood stream through c a p i l l a r y walls o r lymphatic v e s s e l s , t hey may no t p re sen t a b a r r i e r t o t h e d i f fus ion of qua ternary amines such as n e o s t i g - mine. Poss ib ly t h e r ap id hepa t i c metabolism of neost igmine provides a downhill g rad ien t f o r t h e con t inua l d i f f u s i o n of t h i s compound ( 2 3 ) . A c e r t a i n amount may be hydrolyzed s lowly by plasma chol ines- t e r a s e . p a t i e n t s with myasthenia g rav i s less than 5% was found unchanged i n t h e u r i n e (14 ) . Following in t ramuscular admin i s t r a t ion about 65% o f a dose i.s excre ted i n t h e u r i n e unchanged (10) . 20% of an o r a l dose is excre ted i n t h e u r i n e and 50% i n t h e faeces; less than 5% of t h e o r a l dose i s excre ted i n t h e u r i n e unchanged.

When neost igmine was given by mouth t o 2

NEOSTIGMINE 423

6. Theraneut ic and Other Uses

Neostigmine is a qua ternary ammonium a n t i c h o l i n e s t e r a s e . I t acts a t t h e e s t e r a t i c s i t e of t h e enzyme t o form t h e i n a c t i v e dimethylcarbamoyl enzyme. Neostigmine has widespread a c t i o n s , bu t f o r t u n a t e l y i t s effects are more prominent on c e r t a i n s t r u c t u r e s than on o t h e r s , being p a r t i c u l a r l y e f f e c t i v e on t h e bowel, u r i n a r y b l adde r , and s k e l e t a l muscle, t h e p u p i l , t h e h e a r t , blood p res su re , and s e c r e t i o n s be ing a f f e c t e d t o a much lesser e x t e n t i n doses t h a t are o r d i n a r i l y e f f e c t i v e on r e l a t e d s t r u c t u r e s (19) .

The drug i n h i b i t s c h o l i n e s t e r a s e a c t i v i t y and pro longs and i n t e n s i f i e s t h e muscar in ic and n i c o t i n i c effects of ace ty l cho l ine . I t probably a l s o has d i r e c t effects on skeletal muscle f i b r e s . The a n t i c h o l i n e s t e r a s e a c t i o n s o f neost igmine are r e v e r s i b l e . I t is used mainly f o r i t s a c t i o n on s k e l e t a l muscle, and less f r equen t ly t o i n c r e a s e a c t i v i t y of smooth muscle (14 ) .

Neostigmine methylsulphate i s used i n t h e t r ea tmen t o f myasthenia g r a v i s i n usua l doses of 1 t o 2 .5 mg d a i l y given i n d iv ided doses by subcutaneous, in t ramuscular , o r in t ravenous i n j e c t i o n according t o t h e s e v e r i t y o f t h e condi t ion (14) .

Neostigmine i s used i n cond i t ions o f u r i n a r y b l adde r a tony due t o p o s t a n e s t h e t i c depress ion o r t o neu ro log ica l d i s o r d e r s (19) . I t is a l s o used i n t h e t rea tment o f p a r a l y t i c i l e u s pos tope ra t ive u r i n a r y r e t e n t i o n , i n doses o f 0 .5 t o 1 mg. For e x p e l l i n g i n t e s t i n a l f l a t u s p r i o r t o radiography o f t h e ga l l -b l adde r , kidneys , o r u r e t e r s , a s i n g l e dose o f 500 ug has sometimes been used (14) .

Neostigmine can be employed as a d i a g n o s t i c t e s t , e s p e c i a l l y a f t e r symptoms have been purposely accentua ted by t h e admin i s t r a t ion o f qu in ine . mine is used as d i a g n o s t i c t e s t agent i n myotonia congeni ta , i n which cond i t ion neost igmine aggrava tes t h e symptoms. f o r e a r l y pregnancy o r t o t rea t delayed menstruat ion: given in t r amuscu la r ly on t h r e e success ive days i t w i l l induce menstruat ion wi th in 72 hours a f t e r t h e last dose un le s s t h e p a t i e n t i s pregnant . t o p i c a l l y t o treat primary open-angle glaucoma and i n

Neost ig-

I t may be used as a d i a g n o s t i c agent

Neostigmine i s used

424 A. A. AL-BADR AND M. TARlQ

t h e emergency t reatment of primary acute-angle glaucoma. Miosis lasts 1 2 t o 36 hours . Longer-acting a n t i c h o l i - n e s t e r a s e s a r e p re fe r r ed f o r t h e t rea tment o f accommo- d a t i v e e so t rop ia (19).

7 . Toxic i ty and Side E f f e c t s

Adverse and s i d e e f f e c t s o f neostigmine inc lude s a l i v a t i o n , anorexia , nausea and vomiting , abdominal cramps and d ia r rhoea . sweating, lachrymation, watery n a s a l d i scharge , e r u c t a t i o n , involuntary defaeca t ion and u r ina t ion f lu sh ing , miosis , conjunct iva l congest ion, c i l i a spasm, brow ache, nystagmus, r e s t l e s s n e s s , a g i t i o n , fear , excess ive dreaming, increased b roch ia l s e c r e t i o n combined with bronchocons t r ic t ion , bradycardia and hypotension, muscle cramps, s c a t t e r e d f a s i c u l a t i o n s and eventua l severe weakness and p a r a l y s i s , convuls ions, and coma. e f f e c t could occur due t o i n t e r a c t i o n between n i c o t i n i c and muscarinic ac t ions ; a consequence of t h i s could be some evidence o f an acce le ra t ion pu l se - r a t e and e l eva t ion of blood pressure . Death may fol low due t o ca rd iac a r r e s t o r c e n t r a l r e s p i r a t o r y p a r a l y s i s and pulmonary oedema. i n myasthena g r a v i s i s increased muscular weakness (14) .

I t is con t r a ind ica t ed i n as thmat ic p a t i e n t s . I t should not be employed along with cho l ine esters except f o r ophthalmologic use. o f neostigmine (19) .

Symptoms of overdosage inc lude

I t has a l s o been s t a t e d t h a t paradoxical

The major symptom of overdosage

Quinidine i n t e r f e r e s wi th t h e ac t ion

Neostigmine methyl su lpha te , by inc reas ing i n t e s t i n a l m o t i l i t y , may cause d i s rup t ion o f i n t e s t i n a l s u t u r e l i n e s (10 ) .

8. Methods o f Analysis

8 .1 I d e n t i f i c a t i o n

a ) To 0 .1 m l o f a 1 pe r cent w/v s o l u t i o n , add 0.5 m l of sodium hydroxide s o l u t i o n and evaporate t o dryness on a water-bath. o i l -ba th t o about 250' and maintain a t t h i s temperature f o r about t h i r t y seconds. d i s so lve t h e r e s idue in 1 m l o f water, cool i n i c e water, and add 1 m l o f diazoaminobenzene-

Heat qu ick ly on an

Cool,

NEOSTIGMINE 425

su lphonic ac id ; a cher ry- red co lour i s produced (15) .

Crys t a l tes ts . Micro: l ead iod ide s o l u t i o n - branching needles ( s e n s i t i v i t y : 1 i n 400) ; p l a t i n i c i od ide s o l u t i o n - bunches of curved ( s e n s i t i v i t y : 1 i n 400) (11).

The i n f r a r e d absorp t ion spectrum of a potassium bromide d i spe r s ion of i t , previous ly d r i e d a t 1 0 5 O f o r 3 hours , e x h i b i t s maxima only a t t h e same wavelengths as t h a t of a s i m i l a r prepara t ion o f USP Neostigmine Bromide Reference Standard (12).

A s o l u t i o n (1 i n 50) responds t o t h e t e s t o f bromide (neostigmine bromide) (12).

8 .2 T i t r i m e t r i c Methods

8 .2 .1 Aqueous T i t r a t i o n

B r i t i s h Pharmacopoeia (1973) (17) r epor t ed t h e fol lowing procedure f o r t h e assay of neostigmine methyl su lpha te :

Dissolve 0.15 g i n 20 m l of water , t r a n s f e r t o a semimicro ammonia d i s t i l l a t i o n appara tus , and add 20 m l of a 50 p e r cent w/v s o l u t i o n o f sodium hydroxide. Pass a cu r ren t o f steam through t h e mixture , c o l l e c t t h e d i s t i l l a t e i n 50 m l o f 0 .1 N su lphur i c ac id u n t i l t h e t o t a l volume reaches about 200 m l , and t i t r a t e t h e excess of ac id wi th 0.02 N NaOH us ing methyl red s o l u t i o n as i n d i c a t o r . Repeat t h e ope ra t ion without t h e substance being examined; t h e d i f f e r e n c e between t h e t i t r a t i o n s represents t h e amount of a c i d requi red t o n e u t r a l i s e t h e dimethylamine formed from t h e neost igmine. Each m l o f 0.02 N su lphur i c a c i d i s equiva- l e n t t o 0.006688 g of Cl3HZ2N2O6S.

Huang -- e t a1 (24) descr ibed a s imple, r a p i d and accurate method us ing t h e app l i ca t ion of a l t e r n a t i n g - c u r r e n t o sc i l l opo la rograph ic t i t r a t i o n i n pharmaceutical a n a l y s i s . In


t h i s method neostigmine methyl su lpha te i n i n j e c t i o n s was t i t r a t e d wi th sodium t e t r apheny l bo ra t e . To t h e i n j e c t i o n s o l u t i o n (20 ml) conta in ing 10 mg of neostigmine methyl su lpha te were added 10 m l of 0 .01 N sodium t e t r apheny l bo ra t e and 10 m l o f a c e t a t e b u f f e r so lu t ion (pH 5 . 3 ) . The s o l u t i o n was d i l u t e d t o 50 m l wi th water , a f te r 5 minutes t h e p r e c i p i t a t e was formed, t h e con ten t s were f i l t e r e d . A 25 m l po r t ion of f i l t r a t e was t r e a t e d wi th 8 drops o f 40% sodium hydroxide s o l u t i o n and t i t r a t e d with 0.01 N TI2S04, t h e d i s - appearance o f t h e i n c i s i o n o f sodium te t raphenyl bo ra t e on t h e curve of d/E/dt vs E being used t o i n d i c a t e t h e end p o i n t . The r e s u l t s were co r rec t ed f o r a blank value. The recovery range from 99.3 - 100.2% and t h e c o e f f i c i e n t of v a r i a t i o n was 0.42%.

Tsubouchi e t a1 (25) r epor t ed a method o f a n a l y s i s o f neostigmine us ing t h e app l i ca t ion o f one-phase end-point change system i n two phase t i t r a t i o n t o amine drug a n a l y s i s . Neostigmine i n aqueous s o l u t i o n ( 5 o r 10 mM) was determined by t i t r a t i o n wi th aqueous 0.01 M t e t r apheny l bo ra t e with tetrabromo phenolphthal ien e thy l ester a s an i n d i c a t o r i n t h e organic phase (1,2-dichloroethane) , i n b o r a t e phosphate b u f f e r medium (pH 5.5.-7.5) . The end-point was de tec t ed by means o f c o l o r change of t h e i n d i c a t o r i n t h e organic phase without t r a n s f e r of i n d i c a t o r between phases. Various common ions ( inc luding carbonate , a c e t a t e , c i t r a t e , and tannate) d id not i n t e r f e r e bu t thiamine , papaverine , dodecyle su lpha te and mercury caused i n t e r f e r e n c e .

--

Diamandis and Chris topoulos ( 2 6 ) developed a poten t iomet r ic t i t r a t i o n of neostigmine bromide i n pharmaceutical compounds by t i t r a t i n g them with sodium t e t r apheny l bo ra t e . 25 M 1 o f aqueous so lu t ion (0.2 - 1 mM) of neostigmine bromide and 5 m l o f t h e appropr i a t e b u f f e r s o l u t i o n was added.

NEOSTIGMINE 421

The s o l u t i o n was t i t r a t e d po ten t iome t r i ca l ly a t 0.36 m l per minute with 0.01 M sodium te t raphenyl bo ra t e a t 2 2 O 5 20 with continuous s t i r r i n g . The end-point was de t ec t ed by a t e t r apheny lbora t e s e l e c t i v e e l ec t rode . This method was adopted f o r determinat ion o f neostigmine i n powders, i n j e c t i o n s , drops and syrups.

United S t a t e s Pharmacopeia X I X (1980) (12) descr ibed t h e fol lowing procedure f o r t h e assay o f neostigmine methyl su lpha te :

Place about 100 mg o f neostigmine methyl su lpha te , accu ra t e ly weighed, i n a 500 m l Kjeldahl f l a s k , d i s so lve in 150 m l o f water , and add 40 m l of sodium hydroxide s o l u t i o n (1 i n 10) . Connect t h e f l a s k by means of a d i s t i l l a t i o n t r a p t o a well-cooled condenser t h a t d i p s i n t o 25 m l o f b o r i c a c i d so lu t ion (1 i n 25) , d i s t i l about 150 m l o f t h e con ten t s of t h e f l a s k , add methyl purp le TS t o t h e so lu t ion i n t h e r e c e i v e r , and t i t r a t e with 0.02 N su lphur i c ac id . Perform a blank de termina t ion , and make t h e necessary co r rec t ion . su lphur i c ac id is equiva len t t o 6.688 mg of

C13H22N206S'

B r i t i s h Pharmacopoeia (1973) (17) r epor t ed t h e fol lowing procedure f o r t h e a s say of neos t igmine t a b l e t s :

Weigh and powder 2 t a b l e t s . T rans fe r a q u a n t i t y of t h e powder, equivalent t o 0.15 of neostigmine bromide, t o a semi-micro ammonia d i s t i l l a t i o n appara tus , add 20 m l o f a 50% w/v s o l u t i o n of sodium hydroxide and 0.5 m l o f a 2% s o l u t i o n o f octan-2-01 i n l i q u i d p a r a f f i n and complete t h e assay descr ibed under neostigmine methyl su lpha te . (above) begining a t t h e words "Pass a c u r r e n t of steam . . . . I ! . Each m l o f 0.02 N su lphur i c a c i d is equiva len t t o 0.006064 g of C12H19BrN202.

Each m l of 0 .02 N


8.2.2 Ti t r imetr ic Methods

Non-aqueous T i t r a t i o n

United S t a t e Pharmacopeia X I X (1980) (12) descr ibed t h e fol lowing procedure f o r t h e assay of neostigmine bromide:

Dissolve about 750 mg of neostigmine bromide accu ra t e ly weighed, i n a mixture o f 70 m l of g l a c i a l a c e t i c a c i d and 20 m l o f mercuric a c e t a t e TS, add 4 drops of c r y s t a l v i o l e t TS, and t i t r a t e with 0 .1 N p e r c k l o r i c a c i d t o a blue end-point . Perform a blank determina- t i o n , and make any necessary co r rec t ion . Each m l of 0 .1 N p e r c h l o r i c ac id is equiva len t t o 30.32 mg of C12H19BrN202 (12) .

Bayer and Posgay (27) suggested a p e r c h l o r i c ac id t i t r a t i o n method f o r t h e determinat ion of neostigmine i n pharmaceutical p repa ra t ions . The neostigmine so lu t ion was t i t r a t e d with 0 .1 N p e r c h l o r i c a c i d which has been s tandard ized with anhydrous potassium carbonate wi th 0.1% gent ian v i o l e t i n a c e t i c ac id conta in ing 3% mercuric a c e t a t e a s i n d i c a t o r .

Surmann e t a1 (28) r epor t ed a t i t r a t i o n method f o r t h e pharmaceutical hydrochlor ides and hydrobromides i n non-aqueous media. This method involve d i r e c t t i t r a t i o n of drug wi th pe rch lo r i c a c i d i n a c e t i c anhydride medium, with naphthol benzein o r Sudan Red B an i n d i c a t o r f o r hydro- ch lo r ides f o r hydrobromides r e s p e c t i v e l y .

Kracmarova and Kracmar (29) repor ted t h e fol lowing non-aqueous tit r a t i o n :

Evaporate t h e sample o f t h e eye drops (5 ml) on a water ba th t o dryness , d i s so lve t h e res idue i n anhydrous a c e t i c ac id (2 x 20 ml) , add c r y s t a l v i o l e t (0.2% i n anhydrous a c e t i c ac id ) (2 drops) a s i n d i c a t o r , and t i t r a t e with 0 .1 N p e r c h l o r i c ac id t o a blue end-point , add 5% HgII a c e t a t e s o l u t i o n

--

NEOSTIGMINE 429

(5 ml), mix and t i t r a t e again t o a b lue end- po in t ; t h e d i f f e rence between t h e t i t r e s corresponds t o neostigmine bromide.

8 .2 .3 Iodimet r ic Methods

Koka (30) developed an iod ime t r i c determina- t i o n o f p rose r ine (neostigmine methyl su lpha te) i n drug formula t ions . The drug sample is d isso lved i n water and t h e so lu t ion is t r e a t e d wi th 2 -3 m l o f d i l u t e su lphur i c a c i d , 2-3 m l o f 10% potassium iod ide so lu t ion and 5 m l of 0.1 N i od ine , d i l u t e d t o 25 m l with water and f i l t e r e d ; 10 m l o f f i l t r a t e i s t i t r a t e d with 0 . 1 N sodium th iosu lpha te .

Mitchenko and Kirichenko (31) repor ted t h e use of i od ime t r i c method f o r t h e determina- t i o n of neostigmine methyl su lpha te and p i loca rp ine HC1 i n eye-drops. Neostigmine methyl su lpha te and p i loca rp ine HC1 a r e determined by r e a c t i o n with iod ine s o l u t i o n i n dark, f i l t e r i n g t h e mixture and t i t r a t i n g unreacted iod ine wi th sodium th iosu lpha te s o l u t i o n . When both a r e present t h e sum o f t h e two i s determined by above method and only p i loca rp ine is determined a lka l i - m e t r i c a l l y , a r g e n t i m e t r i c a l l y o r mercuri- m e t r i c a l l y . Neostigmine methyl su lpha te is determined by t h e d i f f e rence . The re la t ive e r r o r i n determining the two drugs i n eye-drops is 3.56 and 2.85 r e s p e c t i v e l y .

8 .3 Bio logica l Method

Buckles and Bullock (32) repor ted t h e a p p l i c a t i o n of enzyme i n h i b i t i o n t o t h e es t imat ion of small q u a n t i t i e s o f drugs possess ing a n t i c h o l i n e s t r a s e a c t i v i t y . The method is based on t h e i n h i b i t i o n of t h e pseudochol ines te rase a c t i v i t y o f horse serum. neostigmine is mixed wi th 1 m l o f horse serum, 1 m l o f 0.2% cresol red s o l u t i o n and 37 m l o f water . ad jus ted t o 7.9. After 15 minutes add 5 m l o f 3% ace ty l cho l ine pe rch lo ra t e s o l u t i o n and r ead jus t

5 M 1 o f sample conta in ing 5 ug of

The mixture i s hea ted t o 4OoC and pH


t h e pH t o 7 .9 . 7 .8 and 8.0 dur ing 15 minutes by dropwise add i t ion o f 0.025 N sodium hydroxide and record t h e volume of a l k a l i used. Correc t f o r non- enzymic hydro lys i s by conduct ing an experiment with 1 m l b u f f e r i n s t ead of ho r se serum and c a r r y out a blank with 5 m l water i n s t e a d o f sample. The percentage i n h i b i t i o n i s a l i n e a r func t ion of t h e log concent ra t ion of neost igmine methyl su lpha te .

Maintain t h e pH between

8 .4 Spectrophotometr ic Methods

8 .4 .1 Color imet r ic Methods

Belal et a l , (33) descr ibed a spec t ro - photometr ic de te rmina t ion o f neost igmine i n t a b l e t s and ampoules. Table t p repa ra t ions conta in ing neost igmine methyl su lpha te were powdered and d i s so lved i n 50 m l o f water and 1 m l of s o l u t i o n was mixed wi th 3 m l o f c i t r a t e -phospha te b u f f e r s o l u t i o n (pH 7.8) and 2 m l o f a 0.1% s o l u t i o n of bromothymol b lue i n 0 .01 N sodium hydroxide. mixture was e x t r a c t e d wi th chloroform be fo re measurement o f i t s absorbance a t 416 nm vs a reagent blank. of chloroform e x t r a c t was t r e a t e d wi th 10 ml of 0 . 0 1 N sodium hydroxide and t h e aqueous l a y e r was separa ted and made up t o 25 m l with water, i t s absorbance being measured a t 615 nm as a reagent b lank . The c a l i b r a - t i o n graph were r e c t i l i n e a r f o r 0 .2-1.6 mg. The recovery by t h i s method was 100.5%.

Belikov e t a1 (34) have used sulphoneph- t h a l e i n dyes as e x t r a c t i o n reagents f o r t he e x t r a c t i o n of qua te rna ry ammonium compounds. value, Xmax, d i s t r i b u t i o n c o e f f i c i e n t and s e n s i t i v i t y of r e a c t i o n s f o r t h e ion a s soc ia t ion complexes of neost igmine methyl su lpha te . determining t h e qua te rna ry ammonium compounds, 0 . 5 m l o f 0.01% s o l u t i o n of compound is added t o 5 m l o f a b u f f e r s o l u - t i o n o f appropr i a t e pH, 0 .5 m l of 0.1% dye

--

The

A l t e r n a t i v e l y 10 m l

--

They r epor t ed t h e optimum pH

In a genera l procedure f o r

NEOSTIGMINE 43 I

s o l u t i o n i s added, t h e complex is e x t r a c t e d i n t o 5 m l o f chloroform and absorbance o f o rgan ic phase i s measured a t 400-434 nm. Relative e r r o r i s less than 2%.

Tsubouchi (35) r epor t ed a spec t rophotometr ic de te rmina t ion of o rgan ic c a t i o n s by so lven t e x t r a c t i o n wi th tetrabromophenol- ph tha l i en e thy l e s t e r . 5 1.11 of t h e sample con ta in ing less than 0.01 WM of neost igmine was e x t r a c t e d wi th 2 m l o f 0.07% e t h a n o l i c tetrabromophenolphthalien e t h y l es ter (potassium s a l t ) and 5 m l o f b o r a t e - phosphate b u f f e r (pH l o ) , d i l u t e t o 25 ml with water and shake f o r 2 minutes wi th 1 0 m l o f 1 ,2 ,d ich loroe thane . Separa te and f i l t e r t h e organic phase and measure i t s absorbance a t 615 nm a g a i n s t reagent b lank . The c o e f f i c i e n t of v a r i a t i o n was 1.11%.

8.4.2 U l t r a v i o l e t SDectronhotometric Method

Rita (36) developed a u l t r a v i o l e t spec t rophotometr ic a s say o f neost igmine methyl su lpha te as t h e a l k a l i n e hydro lys i s product , 3- (dimethy1amino)phenol. An a l i q u o t of i n j e c t i o n p repa ra t ion equ iva len t t o 5 mg o f neost igmine methyl su lpha te was a c i d i f i e d and any phenol o r p-hydroxy benzoate p r e s e r v a t i v e p re sen t was e x t r a c t e d with chloroform. The r e s i d u a l aqueous s o l u t i o n was made a l k a l i n e and hea ted on steam ba th f o r 30 minutes and t h e 3- (dimethylamino) phenol formed is determined by UV-spectrophotometry.

Kracmarova and Kracmar (29) descr ibed a spec t rophotometr ic method f o r eva lua t ion of neost igmine bromide and neost igmine methyl su lpha te wi th regards t o t h e degrada t ion products . 2 m l o f 0 .05 N H2SO4 was added. was d i l u t e d t o 10 m l wi th water. The absorbance was recorded at 261 nm f o r neost igmine bromide o r a t 260 nm f o r neost igmine methyl su lpha te o r a t 266 nm f o r both o f t h e compounds.

To 5 ml of i n j e c t i o n s o l u t i o n , The mixture


Mytchenko and Kyrychenko (37) r epor t ed a spectrophotometr ic de te rmina t ion o f p rose r ine (neost igmine methyl su lpha te) i n medicinal formula t ions . The t a b l e t s are gr inded i n water and shaked. s o l u t i o n i s f i l t e r e d and t h e absorbance of f i l t r a t e i s d i r e c t l y recorded a t 260 nm. This method can a l s o be used f o r a n a l y s i s of neost igmine i n eye drops (conta in ing a l s o p i l o c a r p i n e HC1) a f t e r d i l u t i o n . The accuracy of method i s wi th in ? 0.66%.

0.5%

8 .4 .3 In f r a red Spectrophotometr ic Methods - I R

Mynka -- e t a1 (38) r epor t ed t h e i n f r a r e d spectroscopy of drugs o f t h e amide class. The I R spectrum of neost igmine methyl su lpha te was determined and i n t e r p r e t e d by measurement a t 1732 cm-l, be done wi th a maximum e r r o r o f 1.66%.

Analysis can

Kracmarova and Kracmar (29) r epor t ed spectrophotometr ic a n a l y s i s of neost igmine i n i n f r a r e d reg ion . wi th l i q u i d p a r a f f i n (12 drops) and record t h e spectrum i n a sodium ch lo r ide ce l l i n the range 3-15 1-1. C h a r a c t e r i s t i c band f o r neostigmine bromide and neost igmine methyl su lpha te a r e observed.

Grind t h e sample

8 .4 .4 Photometric Method

Kracmarova and Kracmar (29) r epor t ed a photometr ic method €or t h e eva lua t ion o f neost igmine bromide and neost igmine methyl su lpha te as fo l lows:

To t h e i n j e c t i o n s o l u t i o n (1.5 ml) add b u f f e r s o l u t i o n (pH 4.6) (5 ml) , bromo- c r e s o l purp le s o l u t i o n (2 g i n 15 m l o f water and 3 . 2 m l o f 0 . 1 N - potassium hydroxide d i l u t e d t o 250 m l wi th water) (S ml) and chloroform (25 ml) . Shake f o r 1 minute i n a s e p a r a t i n g funnel , and f i l t e r t h e chloroform l aye r ; r epea t t h e e x t r a c t i o n with 2 m l of chloroform and d i l u t e t h e combined e x t r a c t s wi th chloroform t o 50 m l .

NEOSTIGMINE 433

8.5

To a 25 m l po r t ion add 0 .1 N potassium hydroxide (20 m l ) , shake f o r 30 seconds, f i l t e r t h e aqueous l a y e r , d i l u t e t o 100 m l wi th water and measure t h e e x t i n c t i o n a t 570 nm.

Chromatographic Methods

8 . 5 . 1 Paper Chromatographic Method

Giebelmann (39) developed a paper chromatographic d e t e c t i o n f o r phenoxy compounds having a phenyl es ter func t iona l group us ing t h e fol lowing s o l v e n t s - butanol-20% formic a c i d (4 :1) , methanol acetone- t r ie thanolamine (100: 100: 3) and butanol - water ( 6 : l ) . As l i t t l e as 2.5-10 1-16 of neost igmine can be de t ec t ed wi th Mi l lon ' s r eagen t .

Sco t t -- e t a l , (40) repor ted an i n v e s t i g a t i o n on t h e metabolism of neost igmine i n p a t i e n t s with myasthenia g r a v i s . Neost ig- m i n e an d m - h y d r oxyphen y 1 t r i m e t h y 1 ammon i urn bromide are p r e c i p i t a t e d from u r i n e with aqueous bromine. The p r e c i p i t a t e i s e x t r a c t e d with 50% methanol. This e x t r a c t is app l i ed t o Amberli te CG-50 r e s i n a t pH 6.86. Ten bases are e l u t e d wi th 0 . 2 N hydrochlor ic a c i d and submit ted t o chromatography on Whatman wi th butanol-ethanol-water-acetic a c i d (32: 8 : 1 2 : 1) as s o l v e n t . The Rf va lue of neost igmine was between 0.5 - 0.3 .

NO 541 paper

Kracmarova and Kracmar (29) descr ibed procedures f o r t h e de te rmina t ion o f neost igmine bromide and neos t igmine methyl su lpha te i n var ious pharmaceut ical p repara- t i o n s , a s wel l as f o r t h e chromatographic c o n t r o l o f t h e degrada t ion products ; 3 dimethylaminophenol and (3-hydroxyphenyl) trimethylammonium sa l t s , with t h e use of Whatman No. 1 paper , butanol-conc-aqueous ammonia-water (3: 1 : 4) as so lven t , and Dragendorff reagent as t h e d e t e c t i n g agent .


Clarke (11) descr ibed t h e fol lowing:

(1) Paper: Whatman No. 1, shee t 14 x 6 i n , buf fered by dipping i n a 5% so lu t ion o f sodium hydrogen c i t r a t e , b l o t t i n g , and drying a t 250 f o r 1 hour. s to red immediately.

Sample: 2 .5 1-11 of a 1% s o l u t i o n ; i n 2 N a c e t i c a c i d i f poss ib l e , otherwise i n 2 N hydrochlor ic ac id , 2 N sodium hydroxide, o r e thanol .

Solvent : 4 .8 g of c i t r i c a c i d i n a mixture of 130 m l o f water and 870 m l o f n-butanol . This may be used f o r s e v e r a l weeks i f water i s added from time t o time t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o 0.844.

Development: Ascending, i n tank 8 x 11 x 15% i n ; 4 s h e e t s being run a t a time. Time of run, 5 hours .

Location under u l t r a v i o l e t l i g h t , absorp t ion ; l oca t ion reagents : weak r eac t ion ; bromocresol green sp ray , weak r eac t ion . Rf 0.20.

I t can be

I odoplat i n a t e spray ;

( 2 ) Paper: Whatman No. 1 o r No. 3, shee t 17 x 19 cm, implegnated by dipping i n a 10% s o l u t i o n of t r i b u t y r i n i n acetone and drying i n a i r .

Sample: e thanol o r chloroform.

Solvent : Acetate b u f f e r (pH 4.58) .

Equ i l ib ra t ion : The beaker conta in ing t h e so lvent i s e q u i l i b r a t e d i n a t h e r m o s t a t i c a l l y con t ro l l ed over at 95O f o r about 15 minutes.

Development: Ascending, t h e paper is folded i n t o a cy l inde r and c l ipped , t h e cy l inde r is i n s e r t e d i n t h e beaker conta in ing t h e so lvent which i s not removed from t h e oven. A p l a t e - g l a s s d i sk t h i c k l y smeared with s i l i c o n e grease may se rve as a cover. Time of run, 15 t o 20 minutes.

5 p1 of 1 t o 5% s o l u t i o n s in

NEOSTIGMINE 435

Location: Under u l t r a v i o l e t l i g h t , absorp t ion , R f 1.00.

(3) Paper and sample a s i n 2 above. Solvent : Phosphate b u f f e r (pH 7 .4 ) . Equ i l ib ra t ion : The peak conta in ing t h e so lvent is e q u i l i b r a t e d i n a t he rmos ta t i - c a l l y c o n t r o l l e d oven a t 86' f o r about 15 minutes.

Development: as i n 2 above.

Location: Under u l t r a v i o l e t l i g h t , absorp t ion , Rf 0.66.

8 .5 .2 Thin-Layer Chromatography (TLC)

Anand (41) r epor t ed a th in- l a y e r chromatographic and spectrophotometr ic i n v e s t i g a - t i o n on t h e i d e n t i f i c a t i o n o f a phenol ic impuri ty i n neost igmine. The samples o f neostigmine were app l i ed on TLC p l a t e s coated wi th alumina-D and were developed f o r 30 minutes with chloroform-benzene- methanol (5:4:1) t o g ive a good s e p a r a t i o n .

Neostigmine was de t ec t ed with iod ine vapours o r with Dragendorff ' s r eagen t . u l t r a v i o l e t spectrophotometr ic technique is a l s o used f o r q u a n t i t a t i v e e s t ima t ion . Neostigmine has a maxima a t 261 nm.

Por s t and Kny (42) desc r ibed a n a l y s i s o f neostigmine eye drops us ing TLC system and absorp t ion spectroscopy. The drug is de tec t ed and determined by TLC on c e l l u l o s e and u l t r a v i o l e t o r v i s i b l e spectrophotometry is done a f t e r formation o f co lored compound with 3,S-dichloro-p- benzoquinonechlorimine o r sodium n i t r i t e o r 4-dimethylaminobenzaldehyde.

The

Clarke (11) descr ibed t h e fol lowing:

Plate: Glass p l a t e , 20 x 20 cm, coated wi th a s l u r r y c o n s i s t i n g o f 30 g o f s i l i c a ge l G i n 60 m l o f water t o g i v e - a l a y e r 0.25 mm l i k e and d r i e d a t 110 f o r 1 hour.


Sample: ac id .

Solvent : Strong ammonia so lu t ion : methanol (1.5 : 100). I t should be changed a f te r two runs.

Equ i l ib ra t ion : The so lven t i s allowed t o s tand i n t h e tank f o r 1 hour .

Development: Ascending, i n a tank , 2 1 x 21 x 10 cm, t h e end of t h e tank being covered wi th f i l t e r paper t o assist evapora t ion . Time o f run, 30 minutes.

Location reagent : Ac id i f i ed i o d o p l a t i n a t e spray , p o s i t i v e r e a c t i o n , R f 0.02,

8.5.3 Gas Liquid Chromatographic Methods

1 u1 of a 1% s o l u t i o n i n 2 N a c e t i c

Chan e t a1 (43) developed a s e n s i t i v e and s e l e c t i v e a n a l y t i c a l method f o r t h e measurement of neostigmine i n human plasma using gas chromatography. Plasma conta in ing neostigmine i s washed wi th e thy l e t h e r and buf fered wi th potassium iodide-g lyc ine s o l u t i o n , then iodide- glycine-drug complexes are e x t r a c t e d i n t o dichloromethane. The e x t r a c t i s evaporated and t h e r e s idue is d i s so lved in methanol. 2 - 5 1-11 of methanol ic s o l u t i o n was i n j e c t e d i n t o gas chromatographic g l a s s column (2 m x 0 .25 inches O.D.) packed wi th Diatomite CQ coated with 3% of OV-1 , 3% of OV-17 o r 3 . 8 % of SE-30 and s i l a n i s e d ; a n i t rogen s e l e c t i v e d e t e c t o r was used. Pyridostigmine bromide was used a s i n t e r n a l s tandard . The temperature was maintained a t 205OC and t h e flow rate o f n i togen was 30 m l p e r minute. The de tec t ion l i m i t was 5 ng/ml and t h e r e was no i n t e r f e r e n c e with o t h e r b a s i c drugs.

--

Ward e t a1 (44) descr ibed a simple GLC assay of neostigmine us ing d i e t h y l analog of neostigmine a s i n t e r n a l s t anda rd . Neostigmine and i ts analog were d i s so lved in 25 1.11 of chloroform. 2 1.r1 o f chloroform s o l u t i o n was i n j e c t e d i n t o a

--

NEOSTIGMINE 437

gas chromatograph equiped with a flame i o n i s a t i o n d e t e c t o r and 1 . 2 m x 2 mm I . D . g l a s s column packed with 5% O V - 1 7 on Gas-Chrom Q is used a t 210' with helium (40 ml/minute) a s a carrier gas and flame ion iza t ion d e t e c t o r . For de te rmina t ion o f neostigmine i n b i o l o g i c a l f l u i d s , t h e drug is ex t r ac t ed i n t o chloroform with added 3-diethylcarbamoyloxy-"N-trimethylanil inium iodide as t h e i n t e r n a l s tandard . The r e l a t i v e r e t e n t i o n time of t h e drug and t h e s tandard is 1 t o 1 .35.

De Ruyter -- e t a1 (45) developed a reversed- phase, i on -pa i r l i q u i d chromatography o f qua r t e rna ry ammonium compounds €or t h e determinat ion o f pyridost igmine, neost igmine, and edrophonium i n b i o l o g i c a l f l u i d s . The method i s based on e x t r a c t i o n of neostigmine i n t o wa te r - sa tu ra t ed dichloromethane, then back-ext rac t ion is done wi th t e t r a b u t y l ammonium hydrogen su lpha te t o enhance t h e recovery t o more than 86%. The e x t r a c t s a r e submit ted t o chromatography on a v a r i e t y o f reversed- phased columns f i t t e d with 214 nm d e t e c t o r s . For b i o l o g i c a l e x t r a c t s a column (15 cm x 4.6 mm) o f Ul t rasphere o c t y l ( 5 pm) provided with an RP-2 pre-column was used. For neostigmine e l u t i o n was c a r r i e d out with a s o l u t i o n conta in ing sodium heptanesulphonate (0.01 M ) , sodium hydrogen phosphate (0.01 M) and te t ramethyl ammonium ch lo r ide (2.5 mM) i n aqueous 20% a c e t o n i t r i l e . The mobile phase (2 m l p e r minute) was ad jus t ed t o pH 3 with su lphur i c ac id , edrophonium was used a s an i n t e r n a l s tandard . The c a l i b r a t i o n graphs were r e c t i l i n e a r f o r upto 400 ng per m l . The l i m i t of de t ec t ion was 5 ng p e r m l . o f v a r i a t i o n was 1 .5%.

The c o e f f i c i e n t

Davison e t a1 (46) descr ibed a method f o r simultaneous monitoring of plasma l eve l of neostigmine and pyridost igmine i n man. The assay involve pre l iminary ion p a i r


e x t r a c t i o n o f t h e drugs and i n t e r n a l s tandards (3-diproypl carbamoyloxy) 1-methyl pyridinium bromide) wi th t h e use o f potassium iodide and of g lyc ine b u f f e r . i s analysed by GLC (10% of OV-17 on Chromosorb W-AW (100-120 mesh) with a n i t rogen- sens i t i ve d e t e c t o r . The cali- b ra t ion graphs a r e r e c t i l i n e a r and reproduced over t h e range 5-100 ng p e r m l o f e i t h e r drugs i n 3 m l samples.

The e x t r a c t

Pohlmann and Cohan (47) developed a s impl i - f i e d de t ec t ion of qua r t e rna ry ammonium compounds by gas chromatography. The method has been appl ied t o pyridost igmine, neostigmine and ace ty l chol ine . The qua r t e rna ry salts i n t h e serum o r u r i n e a r e first converted wi th potassium t r i i o d i d e (K13) i n t o t h e i r i o d i e s which a r e e x t r a c t e d i n t o chloroform. The e x t r a c t i s evaporated i n vacuum and t h e res idue is d isso lved i n water o r hexane. The s o l u t i o n is i n j e c t e d on t o a column (1 .8 m x 2 mm) of Porapak Q (800-100 mesh), Chromosorb 101 (80-100 mesh) o r Chromosorb 105 (80-100 mesh), opera ted a t 14SoC-17O0C with helium o r n i t rogen a s c a r r i e r gas (20 m l pe r ml). quar te rnary ammonium iod ides a r e thermal ly decomposed, and t h e methyl iod ide r e l eased is measured with a 63Ni e l ec t ron -cap tu re de t ec to r . Ext rac t ion y i e l d s a r e a t l e a s t 95% and down t o 1 0 f-mol of qua r t e rna ry ammonium compound can be de t ec t ed wi th good r e p r o d u c i b i l i t y .

On t h e column t h e

Chan and Dehghan (48) descr ibed a GLC method f o r i s o l a t i o n and determinat ion o f neostigmine, pyridost igmine and t h e i r metabol i tes i n human b i o l o g i c a l f l u i d s . The method involves a pre l iminary , s e l e c t i v e ion -pa i r e x t r a c t i o n of t h e drugs and t h e i r metabol i tes i n t o dichloromethane and i n (dichloromethane-acetone) , r e spec t ive ly . The a n a l y s i s of e x t r a c t was done by GLC on a column o f 3% (drug) o r 10% (metabol i te) of OV-17 on Chromosorb W AW,

NEOSTIGMINE 439

with a n i t rogen- sens i t i ve de t ec to r . For t h e determinat ion o f 3-hydroxytrimethyl an i l in ium ion; a major metabol i te o f neostigmine, 3-hydroxy-N-methylpyridinium ion was used a s a i n t e r n a l s tandard. As l i t t l e as 3 ng/ml of neostigmine and upto 50 ng/ml of i t s metabol i te can be determined i n b io log ica l samples.

8 .6 Ion-Select ive Elec t rodes Method

Kina -- e t a1 (49) descr ibed a method of neostigmine a n a l y s i s based on ion - se l ec t ive e l ec t rodes s e n s i t i v e t o some organic compounds used a s drugs. Liquid membrane e l ec t rodes s e n s i t i v e t o neos t ig - mine have been made by t h e ion-assoc ia t ion e x t r a c t i o n method. The exchange components used were c r y s t a l v i o l e t , d ipicrylamine, sodium t e t r a p e n t y l bo ra t e , l , lO-phenanthrol ine, 4,7- diphenyl- 1,lO-phenanthroline . f o r t hese compounds were 1,Z-dichloroethane and ni t robenzene. The choice of so lven t s a f f e c t e d with s e l e c t i v i t i e s o f t h e membranes but t h e choice of ion-exchange components d id no t . s e l e c t i v i t i e s r e l a t i v e t o univa len t c a t i o n s were mostly b e t t e r than 10-4. range o f e l ec t rodes was 10 VM t o 0 . 1 M wi th a response o f 59 mV per decade change i n t h e concent ra t ion o f t h e drug. be used i n ana lys i s of mixed pharmaceutical p repara t ions .

Diamandis and Chris topoulos (26) repor ted a poten t iomet r ic t i t r a t i o n o f neostigmine and o t h e r pharmaceutical compound i n pharmaceutical formu- l a t i o n with sodium te t rahydrobora te . (See Sect ion 8 .2 .1 . ) . tetraphenylborate-selective e lec t rode .

The so lven t s used

The

The use fu l concent ra t ion

The e l ec t rodes could

The end poin t was de tec ted by a

8 .7 Polarographic Method

Novotny (50) devised a polarographic method f o r t h e determinat ion of neostigmine i n pharmaceutical p repa ra t ions having a concent ra t ion o f 0 .5 mg/ml of t h e drug. hydro lys is of neostigmine and n i t r a t i o n o f r e s u l t i n g phenol, followed by polarography o f

This method is based on t h e


r e s u l t i n g d e r i v a t i v e . The neost igmine s o l u t i o n ( 0 . 2 t o 0 . 6 m l of 0 .1%) i s evaporated t o dryness i n a 50 m l beaker on a water ba th . 1 bll o f KOH s o l u t i o n (20%) i s added and warmed on t h e ba th f o r 20 minutes and again evaporated t o dryness . 0 . 5 M 1 of water and 2 m l o f conc. HN03 (65%) is added and warmed f o r 20 minutes and cooled. 10 M 1 of KOH s o l u t i o n (20%) is added and t h e volume made upto 14.5 m l wi th water. The e s t ima t ion i s c a r r i e d out by polarography, wi th a galvanometer s e n s i t i v e t o 10-9 amp. concent ra t ion o f neostigmine i n an unknown s o l u t i o n , s o l u t i o n of t h e unknown concent ra t ion o f neostigmine is t r e a t e d as descr ibed above, both with and without a known amount of neost igmine.

To determine t h e

Acknowledgements

The au thors would l i k e t o thank Mr. Syed A l i Jawad, Col lege of Pharmacy, King Saud Univers i ty f o r h i s t e c h n i c a l assistance and Mr. Tanvir A. Butt €or h i s s e c r e t a r i a l a s s i s t a n c e i n typ ing t h e manuscript .

NEOSTIGMINE 441

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NEOSTIGMINE 443

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A. A. AL-BADR AND M. TARIQ 444

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PIRENZEPINE DIHYDROCHLORIDE

f fumei ida A . ER-Ob&id*, S a L h A . Rubha,&**, and A b d u U a h A . M-Badrr*



446 HUMEIDA A. EL-OBEID ETAL.

Contents

1. Description

1.1 Nomenclature 1.2 Formulae 1 . 3 Molecular Weight 1 . 4 Elemental Composition 1.5 Appearance

2. Physico-chemical Properties

2.1 Melting Range 2.2 Solubility 2.3 Distribution Ratio 2.4 Spectral Properties 2.5 Crystal Structure

3. Synthesis

4. Stability 5. Methods of Analysis

5.1 Spectrophotometric Methods 5.2 Chromatographic Methods 5 . 3 Radioimmunoassays

6. Pharmacokinetics

6.1 Absorption 6.2 Distribution 6 . 3 Metabolism 6 . 4 Excretion

7. Pharmacology

7 . 1 Antisecretory Effects 7 . 2 Efficacy 7 .3 Adverse React ions 7.4 Tissue Selectivity 7 . 5 Mechanism of Action

Acknowledgement

Re fer en c e s

PIRENZEPINE DIHYDROCHLORIDE 447

1. Descrivt ion

1.1 Nomenclature


5,11-Dihydro-ll-[(4-methyl-l-piperazinyl) a c e t y l ] -6H-pyrido[ 2,3-b] [ 1 , 4 ] benzodiazepin- 6-one d i h y d r o c h l o r i d e monohydrate.

11- [ (4-Methyl-1-piperazinlyl) a c e t y l ] - p y r i d o [ 2 , 3-b] [ 1 , 4 ] benzodiazepin-6 (5 H) -one d i h y d r o c h l o r i d e monohydrate.

1 . 1 . 2 Gener ic Names

L-S 519, P i renzepine

1 . 1 . 3 Trade Names

B i s v a n i l , Gasteri l , Gas t rozepin , Leblon, Tabe, Ulcosan.

1 . 2 Formulae

1 . 2 . 1 Empir ica l

C 9H2 3C 1 2N502 (dihydrochl o r i d e )

C19H21N502 (base)

1 . 2 . 2 S t r u c t u r a l

.2HC1, H 2 0 I

CH2- NwN - CH3


1.2.3 CAS No.

2.

[ 29868-97-11 as dihydrochloride salt.

[28797-61-71 as free base.


424.33 as dihydrochloride salt

351.42 as free base.


Dihydrochloride salt: 0 7.52%, C1 16.71% (1).

C 53.78%, H 5.46%, N 16.50%,

Free base: C 64.94%, H 6.02%, N 19.93%, 0 9.11% (2).

1.5 Appearance

Pirenzepine dihydrochloride is a white crystalline substance (2).

Physico-chemical Properties

2.1 Melting Range

Pirenzepine dihydrochloride melts in the range 250-252OC (corrected) with decomposition (I).

2.2 Solubility

Pirenzepine dihydrochloride is readily soluble in water, slightly soluble in methanol and practically insoluble in ether (1).

2.3 Distribution Ratio

Pirenzepine is a tricyclic compound, but its physioco-chemical properties differ considerably from those of other tricyclic compounds which are used as centrally-acting drugs. Pirenzepine is unusual because of its pronounced hydrophilic properties. Comparison of the distribution coefficient of pirenzepine with those of other


t r i c y c l i c drugs a t pH 7.4, has been ca r r i ed out (3). The r e s u l t s depicted i n Table 1 show t h a t pirenzepine i s 200-13000 l e s s l i p i d soluble ,

Table 1 : Distr ibut ion coe f f i c i en t of pirenzepine and other t r i c y c l i c drugs.

- H 7 .4 CNS e f f e c t s Drug $Ppe-- - -

Chlorpromazine 3160 +

Cyprohept adine 1600 +

Imipramin 250 +

Dibenzepine 51 +

Pirenzepine 0.23 -

Eberlein -- e t a1 (3) analysed t h e pirenzepine molecule i n t o f i v e s t r u c t u r a l elements. Each o f t h e f i v e elements can be al located a hydrophobic subs t i t uen t consant (TX) and compared with those assigned f o r corresponding s t r u c t u r a l elements i n imipramine molecule (Table 2 ) . A pos i t i ve T X

value indicates contribution t o t h e l i p o p h i l i c character of t h e molecule and a negative TX

value indicates contribution t o hydrophi l ic i ty of t h e molecules.


Table 2 : C o n t r i b u t i o n o f t h e d i f f e r e n t f ragments of p i r e n z e p i n e and imipramine molecules t o t h e i r d i s t r i b u t i o n c o e f f i c i e n t s .

' CH,

, CH, CH2CH2CH2 f N I

1

5 4 -

-

I m i p r am i n e

Fragment Fragment No. s t r u c t u r e TX

-CH2-CH2- +10.02

+ 1 . 9 6

+ 1.96

\

/ N-CH2CH2CH2- + 0.50

~ ~

LogBase ( c a l c u l a t e d ) + 5.12

LogBase(experimental) + 4.80

H n

P i r e n z e p i n e ~~~~

Fragment s t r u c t u r e nx

\ / N-COCH2-

n

-1 .49

+ O . 65

+l . 96

-0.97

( c a l c u l a t e d ) +O. 010 LogBase

LogBase (experimen- -0.64 t a l pH 7 .4)

PIRENZEPINE DIHYDROCHLORIDE 45 1

2.4

I t can be shown t h a t t h e s t r u c t u r a l elements 1 and 4, i n p a r t i c u l a r , c o n t r i b u t e l a r g e l y t o t h e h y d r o p h i l i c i t y of p i renzepine . Fragments 1 and 4 i n p i renzepine c o n s i s t o f amide groupings each with two d ipo le s which can i n t e r a c t s t r o n g l y with water molecules. The py r idy l group of fragment 2 i n p i renzepine i s a l s o expected t o c o n t r i b u t e t o t h e h y d r o p h i l i c i t y of t h e molecule. i nd iv idua l hydrophobic cons t an t s f o r p i renzepine molecular fragments a log P of 0.01 i s obta ined for t h e whole molecule, which i n d i c a t e s t h a t p i r e n - zepine a s t h e uncharged free base w i l l be d i s t r i - buted uniformly between t h e oc tanol and water . A t phys io logica l pH, however, t h e r a t i o s h i f t s even more towards water s o l u b i l i t y due i o n i z a t i o n o f t h e molecule. (pH 7.4) t h e experimental ly determined log P is - 0.64 (Table 2 ) , i n d i c a t i n g t h a t about 5 times more o f t h e compound i s d i s t r i b u t e d i n t h e aqueous phase than i n oc t ano l . Under t h e same cond i t ions imipramine accumulates much more s t r o n g l y i n t h e organic phase. As w i l l be shown l a t e r , t h e pronounced hydroph i l i c p r o p e r t i e s o f p i renzepine expla in many of t h e s p e c i f i c pharma- c o k i n e t i c c h a r a c t e r i s t i c s of t h e drug.

Adding up t h e

Under phys io logica l cond i t ions

Spec t r a l P r o p e r t i e s

2 . 4 . 1 U l t r a v i o l e t Suectrum

The u l t r a v i o l e t absorp t ion spectrum o f p i r en z ep i n e i n met h an o 1 /HC 1 was ob t a i n ed on a Cary 219 spectrophotometer . spectrum, shown i n Figure 1, is cha rac t e r - i zed by a maximum a t 283 nm and a minimum a t 264 nm. repor ted i n t h e l i t e r a t u r e (4, 5 ) . I t is a l s o r epor t ed t h a t p i renzepine i n 0 . 1 N sodium hydroxide s o l u t i o n shows absorp t ion maximum a t 295 nm (4 ) .

The

The r e s u l t s ag ree wi th those

2 . 4 . 2 In f r a red Spectrum

The i n f r a r e d absorp t ion spectrum o f p i renzepine obtained from a potassium bromide d i spe r s ion was recorded on a Pye

452 HUMEIDA A. EL-OBEIDETAL.

Figure 1 : ULTRAVIOLET SPECTRUM OF PIRENZEPINE DItiYDRDQiLORIDE IN METHANOLIC HCl.


Unicam SP 1025 spectrometer and i s shown i n Figure 2 . The c h a r a c t e r i s t i c bands of t h e spectrum with t h e assignments a r e l i s t e d below:

Frequency (cm-l)

3520, 3463

3240

3020, 2990

2700 - 2400

1717, 1678

1605, 1585, 1502,) 1479, 1432 1

1370

800, 785, 760

Assignment

Amide N-H s t r e t c h

Water molecule

Aromatic C-H s t r e t c h

t -Amine sa l t N-H s t r e t c h

Amide C = 0

Aromatic C - C , C *- N s t r e t c h .

Aryl C - N s t r e t c h

Aromatic C - H out- of-plane bending.

2.4,3 'H Nuclear Magnetic Resonance ('H NMR)

SDectrum

Pirenzepine solut ion i n D 2 0 was used t o obtain t h e proton magnetic spectrum on a Varian-T60 NMR spectrometer with TMS a s i n t e r n a l standard. i n Figure 3, and assignment of t h e chemical s h i f t s t o the d i f f e r e n t protons is summarized below:

The spectrum i s shown

Microns

P VI P

4000 3000 2500 2000 1800 1600 1400 1000 800 600 400 200 Wavenumber

Figure 2 : INFRARED SPECTRUM OF PIRENZEPINE DIHYDROCHLORIDE, KBr DISC.

R ul

I I

400 3 200 100 0

I7 16

l . . . . l . . . . l . . . . 1 . . . . l . . . . l . . . . 1 . . . . 1 . . . . 8.0 7.0 60(ppm)Ei.O ( 6 ) 4-0 3-0 2-0 1.0 0

I

Figure 3 : 'H SIR SPECIRUM CF PIRENZEPINE DIHYDROCMRIDE IN DZO WITH ?Hs As IkTERUL REFERENCE.


I 0 = c 1 2 1 4 1 5

1 n 1 8 C H ~ - N N -CH3

1 3 I U 6

Chemical s h i f t Proton No. of (6) Mul t ip l i c i ty Assignment protons

3.05 Sing 1 e t -N-CH -3 3

-CH -(piperazine) 8 -2 3.55 Mu I t i p 1 et

0 It

4.15 Mult i p l e t - 3 - C - 2

7 . 7 Mu 1 t i p l e t Aromat ic-H 6

8.4 Mu 1 t i p let Aromatic-H 1

2.4.4 13C Nuclear Magnetic Resonance ( 13 C NMR)

Spectrum

The 13C NMR spectra of pirenzepine i n a mixture of D20 and formic acid using dioxane as in t e rna l reference a r e obtained using a J e o l FX 100 90 MHz spectrometer a t ambient temperature. t he IH-decoupled and off-resonance spectra , respect ively. The carbon chemical s h i f t s , assigned on bas i s of t he off-resonance s p l i t t i n g pat tern and the theories of chemical s h i f t s , are presented below:

Figure 4 and 5 show


Dioxan 67.4

Figure 4 : 'ti DEaWPLED 13C tW SPEclRuM OF PIRENZEPINE DIHYDROMLORIDE I N DzO/ RIRMIC ACID FlIXlURE W I ' M DIOXANE AS INNWU. REFERNECE.

Figure

- 5 :

a'


Chemical S h i f t (6 1

44.83

50.72

51.87

57.77

122.41

128.22

128.88

130.57

131.57

132.15

132.74

135.24

139.43

143.61

146.77

165.21

165.31

I Y 10 1 2 1

0-c I

CH2- NWN l3 1 7 16

M u l t i p l i c i t y

Quartet

T r i p l e t

T r i p l e t

T r i p l e t

Doublet

Doub 1 e t

Doub 1 e t

Doublet

S ing le t

Doublet

Doub 1 et

S ing le t

Doublet

S ing le t

S ing le t

S ing le t

Sing 1 e t

18

CH3

Carbon Assignment

‘18

‘15’ ‘16

‘14’ ‘17

‘13

c lo

c7

‘3

c9

c4

c7’

c2

c31

c 2 1

‘12

‘8

‘6’

‘6


2.4.5 Mass SDectra

The 70 eV electron impact mass spectrum of pirenzepine, presented i n Figure 6 , was obtained on Varian MAT 311 mass spectrometer using ion source pressure of Torr, ion source temperature of 18OoC and an emission current of 300 PA. The molecular ion i s detectable at m/e 351 and the base peak a t m/e 113. pat tern and the mass/charge r a t i o s of t h e major fragments i s shown i n Scheme 1. The d i r e c t chemical ionizat ion (DCI) spectrum (Figure 7) was obtained on a Finnigan 4000 mass spectrometer using methane gas a reagent with ion electron energy of 100 eV, ion source pressure o f 0 . 3 Torr, ion source temperature of 150°C and emission current o f 300 PA. by a quasi-molecular ion (M + 1 ) . Two peaks appearing a t m/e 380 and m/e 392 a r e a t t r i b u t e d t o t h e t r a n s f e r of carbocations from the c a r r i e r gas. assignments of the prominent ions under DCI conditions a r e shown in Table 3.

A proposed fragmentation

The spectrum is dominated

The mass spec t r a l

Table 3. Mass spec t r a l assignments of pirenzepine using DCI with methane as reagnet gas.

m/e Species .__

392 [M + C ~ H ~ I +

352 MH+

35 1 M+

380 [M + CZH51+

2 . 5 Crystal Structure

The c r y s t a l s t r u c t u r e o f pirenzepine monohydrate f r e e base was s tudied by Ruzic-Toros and Kojec- Prodic (6). Pirenzepine monohydrate was t o be monoclinic, P2 a = 13.160 [7], b = 12.766 [Sly

1/c


Figure 6 : ELECTRON IMPACT W S SPECTHW! OF PSWZEPINE.

Figure 7 : DIRECT WICAL IONIZATION W S SPECIIW OF PIRENZEPINE.

H

@% N -238 dh N +p -CH -43 3 -N = CH2

0 =c I I ____t H2C = 'd -cH3- =CH2 0 =c +

m / e 70 %lac. D m/e 113 H,C - N N -CH, L u 5

-42

2

m/e 351

2 =N=CHC

m/e 113

- 140 - tH 1 ~ ~ 1 A N - CHJ 0 = C-CH-N w H

m/e 211

Scheme 1. Proposed f r a p e n t a t i o n of pirenzepine.

@ 3 N + ~ c H

m/e 71

1 - CH3

m/e 56


c = 12.439 171 A’, B = 113.2 [4]O, U = 1920.8 Ao3,

Z = 4, Dc = 1 . 2 7 7 Mgm-3, p(Mo Ka) = 0.128 mm . Fina l R = 0.045 f o r 2681 observed r e f l ex ions .

-1

Bond d i s t ances and angles (Table 4) i n t h e fused r i n g s , benzene, diazepine and py r id ine , a r e a f f e c t e d by conjugat ion. expected €or t h e given atom type and hybr id i za t ion could not be explained by t h e thermal v ib ra t ions of t h e atoms (Table 5 ) . The shortening o f C[7] - C[8] , 1.372[4]Ao, i n t h e benzene r i n g could be due t o in te rmolecular con tac t s , involving these atoms, with N[1][3.352[3]; 3.470[3]Ao.

Deviation from t h e values

The seven-membered diazepine r i n g is folded a t “51 - C[6] and “111 t o form a boat conformations. The pyr id ine and benzene r i n g s a r e p lanar while t h e p iperaz ine r i n g i n a c h a i r conformation. A value o f 60.9[41° was obtained f o r t h e d ihedra l angle between t h e benzene and t h e pyr id ine r ings .

The water molecule is involved i n hydrogen bonds with t h e amide group o f t h e diazepine r ing , “51 - H[5] .. O[W]I2.800[3])and O(W)-H(W)2 ... O[ 61 c2.907[ 31 Ao) . is hydrogen-bonded t o the water molecule by O(W)- H(W)1 . . . N[4’](2.821[3]A0). Molecular packing i s a l s o inf luenced by van der Wall i n t e r a c t i o n s . The r e l a t i v e o r i e n t a t i o n o f t h e py r id ine and p iperaz ine r ing , with a d ihedra l angle o f 22 .2 [ 41°, enables a s h o r t intramolecular contac t “11 ... “1’1 of 3.087[2]Ao. The s t r u c t u r a l formula and atomic numbering o f pirenzepine is given i n Figure 8. The confonna- t i o n o f t he molecule is shown i n Figure 9 and Table 6.

N [ 4 ’ ] of t h e p iperaz ine r i n g

Trummlitz -- e t a l . ( 7 ) , using X-ray ana lys i s , determined t h e c r y s t a l s t r u c t u r e s of p i renzepine dihydrochlor ide and i ts monoprotonated form, pirenzepine monohydrochloride (Figure 10) . Molecular mechanics (MMPI) and semiempirical quantum chemical (MNDO) c a l c u l a t i o n s showed t h a t t h e ca l cu la t ed minimum energy conformations o f t h e t r i c y c l e and of t h e exocycl ic amide group are


Table 4. Bond d i s t a n c e s (A) and a n g l e s (O)

1.348 (3) 1.315 (3) 1.364 (4)

1 .375 (3) 0.97 (2)

0 .97 (2) 1.362 (3) 1 .403 (3) 0.86 (2) 1 .231 (3) 1 .484 (4) 1 .397 (4) 0.97 (2) 1.372 (4) 1.377 (4)

1 . 3 8 3 (4) 0.92 (2)

0 .99 (2)

1 .395 (3)

1 .00 (3)

C(lO)-C(15) 1 .381 (3) C(lO)-H(lO) 0.94 (2) N(l1)-C(12) 1 .438 (3) N(l1)-C(15) 1 .433 (3) N(l1)-C(16) 1.362 (2) C(12)-C(13) 1 .387 (3)

C (14) -C (15) C(16)-O(16) C (16) -C ( 1 7) C (17) -N (1 ' ) C(17)-H(17)1 C ( 1 7) -H (1 7) 2 N ( l ' ) - C ( Z ' ) N ( 1 ' ) -C (6 ' ) C (2 ' ) -C ( 3 ' ) C (2 ' ) -H(2 ' ) 1 C (2 ' ) -H (2 ' ) 2 C ( 3 ' ) -N (4 ' ) C(3 ' ) -H(3 ' )1 C ( 3 ' ) -H ( 3 ' ) 2 N (4 ' ) -C (5 ' ) N ( 4 ' ) -C ( 7 ' ) C (5 ' ) -C (6 ' ) C (5 ' ) -1-1 (5 ' ) 1 C (5 ' ) -H ( 5 ' ) 2 C (6 ' ) -H(6 ' ) 1 C (6 ' ) -H (6 ' ) 2 C (7 ' ) -H (7 ' ) C ( 7 ' ) -H( 7 ' ) 2 C (7 I ) -H ( 7 ' ) 3 O(W)-H(W)1 O(W)-H(W)2

1.388 (3) 1.229 (3) 1 .513 (4) 1.452 (3)

1 .05 (2) 1 .460 (3) 1.454 (3) 1 .505 (4) 1 .04 (2) 1 .04 (2) 1 .465 (3) 1.05 (2) 1 . 0 1 (3) 1.462 (4) 1.468 (5) 1.502 (4) 1 .03 ( 2 )

1 .04 (2) 1 . 0 3 (3)

0.97 (4) 1 . 0 5 (3) 0 .93 (3) 0 .78 (5)

1 . 0 1 (2)

1 . 0 1 (2)

1 .09 (3)

C(Z)-N(l)-C(12)

N (1) -C ( 2 ) -H (2) C(3)-C(Z)-H(2)

c (1) -c (2) -c (3)

c (2) -c (3) -c (4)

c (3) -c (4) -c (13)

C (2) -C (3) -H( 3) C (4) -C (3) -H (3)

C(3)-C (4)-H(4) C (1 3) -C (4) -H (4) C(6)-N(S)-C(13) C(6)-N(S)-H(5) C (13) -N (5) -H (5) N (5) -C (6) -0 (6) N (5) -C (6) -C (14) O(6) -C (6) -C (14) C(8) -C (7) -C (14) C (8) -C (7) -H (7)

116.7 (2) 122.9 (2) 115 (1)

119.6 (2) 1 2 1 (1)

120 (1) 120 (1) 118.7 (2) 123 (1) 118 (1) 129.8 (2) 114 (1) 115 (1) 119.3 (2) 119.3 (2)

120.5 (2) 121.2 (2)

1 2 1 (2)

C (12) -N (1 1 ) -C (16) C (15) -N (11) -C(16) N ( 1) -C (1 2 ) -N (1 1) N ( l ) -C (12) -C (13) N (11) -C (12) -C(13) C(4) -C (13) -N (5) C (4) -C (13) -C (12) ~ ( s j - c ( 1 3 j -c (12)

c (7) -c (14) -c (15)

C(10) -c (15) -c (14)

C (6) -C (14) -C (7) C(6) -C (14) -C (15)

C (1 0) -C (15) -N (1 1)

N (11) -C (15) -C (14) N (11) -C (16) -0 (16) N (11) -C (16) -C (17) 0 (16) -C (16) -C (17) C (16) -C (17) -N (1 ' )

124.9 120 .6 117.0 125 .0 118 .0 119 .8 116 .8 123.1 118.1 123.2 118.6 120.5 120.9 118.5 120.8 117.9 121.3 113.2

463


Table 4. Contd . . . . . C (14) -C (7) -H (7) C (1 7) -N ( 1 ' ) -C (2 ' ) C (7) -C (8) -C (9) 120.2 (3) C ( 1 7 ) - N ( l f ) - C ( 6 ' ) C (7) -C (8) -H (8) C (2 ' ) -N ( 1 ' ) -C (6 ' ) C (9) -C (8) -H (8) N (1 ' ) -C (2 ' ) -C ( 3 ' ) C (8) -C (9) -C (10) 120.4 (2) C(2 ' ) -C(3 ' ) -N(4 ' ) C (8) -C (9) -H(9) 123 (1) C(3' ) -N ( 4 ' ) -C(S') C(l0)-C(9) -H(9) 117 (1) C (3 ' ) -N ( 4 ' ) -C ( 7 ' ) C (9)-C (10) -C (15) 119 .4 (2) C(5 ) -N(4' ) -C (7 I )

C(9)-C(lO)-H(lO) 1 2 1 (1) N (4 ') -C ( 5 ') -C (6 ' ) C(lS)-C(lO)-H(lO) 119 (1) N ( 1 ' ) -C(6 ' ) -C(S') C( 12) -N(11) -C(15) 114 .3 (1) H(W) l-O(W) -H(W) 2

118 (2)

120 (2) 120 (2)

111.0 (2) 111.7 (2) 110 .6 (2) 109.9 (2) 110.8 (2) 109 .3 (2) 1 1 0 . 9 (2) 110.9 (2) 1 1 0 . 1 (2)

110 (4) 110 .6 (2)

Table 5. Final atomic coordinates (x l o 4 f o r C , N and 0; 5 x 10 for H) and i so t rop ic thermal parameters

(x l o2 )

U

U = - C . C . U..a* a* . a* . a . . a

i s derived from the anisotropic thermal parameters by eq 1 eq 3 1 J 1 J J 3 1 j

X

6263 (1) 6199 (1) 6380 (1) 6615 (1) 7023 (1) 6726 (1) 7131 (1) 5060 (1) 4228 (2) 4186 (1) 4978 (1) 6635 (1) 6537 (1) 6695 (1) 5866 (1) 5818 (1) 7373 (1) 7400 (1) 8146 (1) 8712 (1)

Y

1423 (1) 1306 (1)

372 (2) -503 (1)

-1266 (1) -1540 (1) -2330 (1) -1465 (1)

-932 (2) 143 (2) 698 (1) 716 (1) 588 (1)

-404 (1) -922 (1)

162 (1) 1361 (1) 1426 (1) 1997 (1) 1375 (1)

z

U o r

ueq('42)

6087 (1) 4 .2 (2) 4985 (1) 4 . 6 (2) 4557 (1) 4 . 7 (2) 5255 (1) 4 . 3 (2) 7162 (1) 4 . 3 (2) 8057 (1) 7.2 (2) 8634 (1) 6 . 4 (2) 8510 (1) 4 . 8 (3) 8677 (1) 5.1 (3) 8615 (1) 5 . 1 (3) 8379 (1) 4 .5 (2) 7947 (1) 3.7 (2) 6761 (1) 3.4 (2) 6403 (1) 3.5 (2) 8266 (1) 3 .9 (2) 8216 (1) 3 .7 (2) 8752 (I) 4.2 (2) 9729 (1) 6 .2 (2) 8376 (2) 4 . 6 (3) 7805 (1) 3.8 (2)


Table 5 . Contd. . . .

465

X

9214 (1) 9766 (2)

10604 (1) 10082 (2)

9536 (2) 11162 (3) 2165 (2)

603 (2) 632 (2) 676 ( 2 )

509 (2) 366 (2) 362 (2) 496 (2) 868 (2) 768 (2) 857 (2) 978 (2) 918 ( 2 )

1017 (2) 951 (2)

1068 ( 2 )

917 (2) 1175 (3) 1152 (3) 1057 (3)

162 (3) 223 (4)

737 (2)

1012 (2)

Y

2042 (1) 1377 (2)

696 (1)

688 (2)

2153 (1) 195 (2)

28 (1)

6 3 (3)

32 (2)

-222 (2)

5 3 (2 )

-118 (2) -175 (2)

-133 (2)

143 (2) 237 (2) 259 (2) 248 (2) 255 (2)

184 (2) 9 1 (2)

-44 (2) 1 1 2 (2)

21 (2) -45 (3)

55 (3)

-46 (2)

-38 (3) 167 (3) 217 (4)

Z

7195 (2) 6583 (2) 7417 (1) 8008 (2) 8627 (2) 6827 (3) 8845 (2)

451 (2) 375 (2) 499 (2) 696 (2) 854 (2) 886 (2) 871 (2) 834 (2)

780 (2) 661 (2)

909 (2)

777 (2) 594 (2) 621 (2) 741 (2 ) 857 (2) 929 (2) 904 (2 )

648 (3) 615 (3) 843 (3) 950 (4)

747 (3)

U o r

Ue9(A2)

4 . 9 (3)

5.4 (3) 4 . 7 (2)

7.9 (2)

5 . 8 (3) 5 . 0 (2)

8 .4 (4)

6 . 1 (7) 5 .2 ( 6 ) 3.9 (6) 4 . 3 (6) 5 . 7 (7) 7 .1 (8) 5 .2 (6) 4.2 (6) 6 . 3 (7) 5 .7 ( 7 ) 6 . 0 (7) 6 . 6 (8) 6 . 6 (9) 8 . 3 (9) 6 .6 (7) 7 .0 (8) 5 .1 (6) 7.5 (8)

11 (1) 9 (1)

1 0 (1) 1 0 (1) 1 4 (2)


Figure 8 : STRUCTURAL FORMULA OF PIRENZEPINE WITH ATOMIC NUMBERING (6).

Figure 9 : A VIEW OF THE CRYSTAL STRUCTURE SHOWING THE PACKING, HYDROGEN BONDS AND CONFOR-

-MATION OF THE BENZODIAZEPINE AND

PIPERAZINE R I N G S ( 6 ) .

Table 6. Torsion a n g l e s (O)

C (12) -N (1) -C (2) -C (3) N ( l ) -C(2) -C(3) -C (4) C (2) -C (3) -C (4) -C (13) C(3) -C (4) -C(13)-C (12) C (3) -C (4) -C (13) -N (5) C (4) -C (13) -C(12) -N (1) C(4) -C(13)-C (12) -N(11) C(13)-C (12) -N(1) -C(2) C (7) -C (8) -C (9) -C (10) C(8)-C(S)-C(lO)-C(15) C (9) -C (10) -C (15) -C (14) C (9)-C(lO)-C(15) -N(11) C (10) -C (15) -C (14) -C (7) C(10) -C(15) -C(14) -C(6) C(15) -C (14) -C (7) -C (8) C (14) -C (7) -C(8) -C (9) N(S)-C(6)-C(14)-C(15) C(6) -C (14) -C (15)-N (11)

1 . 4 (3) 1 . 5 (4)

-2 .0 (3) -0 .4 (3)

-174.7 (2)

-178.9 (2) 3 .6 (3)

- 4 . 1 (3)

-0 .5 (3) 1.1 (3)

-1 .4 (3)

1.1 (3)

0 .2 (3)

0 . 3 (3)

178.7 (2)

177.8 (2)

-0.6 (3) -42.4 (3)

C (14) -C (15) -N (11) -C (12) N (1 1 ) -C (12) -C (13) -N (5) C ( 12) -C ( 13) -N (5) -C (6) C (1 3) -N (5) -C (6) -C (14) C (15) -N (11) -C (12) -C (13) C (13) -N (5) -C (6) -0(6) C(12) -N(11) -C(16) -0(16) C(12) -N(11) -C(16) -C(17) N (11) -C (16) -C (17) -N ( 1 ' ) C (16) -C (17) -N (1 ' ) -C (2 I )

C (17) -N (I ) -C (2 ' ) -C (3 ' ) N ( 1 ' ) -C (2 ' ) -C ( 3 ' ) -N ( 4 ' ) C (2 ) C (2 I ) -C ( 3 ) -N (4 ' ) -C ( 7 ' ) C (3 ' ) -N (4 I ) -C (5 I ) -C (6 ' ) N (4 ' ) -C ( 5 I ) -C (6' ) -N (1 I )

C(S')-C(6') -N(1 I ) - C ( 2 ' )

) -C ( 3 ) -N (4 ' ) -C ( 5

69.5 (2)

38.3 (3) -4 .7 (3)

5 . 3 (3) -64.5 (2)

-178.8 (2) 179.8 (2)

0 .2 (3) -50.0 (2)

-57.9 (2)

165.2 (1) -178.4 (1)

58.7 (3) -178.7 (2)

-58.6 (2) 58.6 (2)

-57.7 (2)


Figure 10: STEREO-PAIRS OF DRAWINGS FOR THE MOLECULAR STRUCTURES OF PIRENZEPINE DIHYDROCHLORIDE(a)AND

PI RENfEPlNE MONOHYDROCHLORIDE (b) OBTAINED

FROM X-RAY ANALYSIS ( 7 ) .

1.491 (3) 1.503(6) a-

Figure l l : ATOM NUMBERING SCHEME AND CRYSTALLOGRAPHIC BOND

ANGLES (A, STANDARD DEVIATONS IN PARENTHESES)

FOR PIRENZEFINE DIWDROCHLORIDE ( FIRST VALUE) AND

PIRENZEPINE MONOHYDROCHLORIDE (SECOND VALUE I ( 7).


i n agreement with t h e c r y s t a l s t r u c t u r e s . The t r i c y c l i c r i n g system c o n s i s t s of t h e p l ana r r i n g s A and C and nonplanar r i n g B (Figure 11 ) . For both compounds t h e seven-membered r i n g B has a d i s t a r t e d boat shape. This r i n g i s folded along a fo ld ing l i n e pass ing through N 1 and t h e middle o f t h e bond N 8 - C9. This s i m i l a r i t y i n t h e geometry o f t h e t r i c y c l i c systems o f both compounds does no t ho ld f o r t h e s i d e cha ins which have t o t a l l y d i f f e r e n t arrangements with r e spec t t o t h e t r i c y c l i c system i n both compounds (Figures 10 and 12) . The s t e r i c s i t u a t i o n is q u i t e s i m i l a r along t h e bond N 1 - C16, i n t h a t t h e carbonyl groups are almost i n ec l ip sed p o s i t i o n s t o N 1 - C15. S i g n i f i c a n t d i f f e r e n c e s are observed, however, a long t h e C16 - C 1 7 bond. For t h e d ihyrochlor ide N18 is trans t o N 1 (T N18-Cl7-Cl6-Nl = 171.3') whereas i n t h e monohydrochloride N18 and N 1 a r e i n gauche pos i t i ons (T N18-Cl7-Cl6-Nl = 64.1'). In t h i s arrangement t h e p iperaz ine r i n g w i l l be s i t u a t e d below t h e t r i c y c l i c r i n g system i n t h e c r y s t a l s t r u c t u r e o f p i renzepine monohydrochloride. The c r y s t a l s t r u c t u r e s of both s a l t s showed s t rong N-H . . . C 1 - hydrogen bonds f o r t h e pro tona t ed n i t rogens of t h e p iperaz ine r ings . A r e l a t i v e l y weak 0 . . . C 1 - hydrogen bonds a l s o e x i s t f o r t h e d i s t o r t e d water molecule i n t h e c r y s t a l l a t t i c e i n t h e dihydrochlor ide sal ts . The amide n i t rogen N8 o f t h e monohydrochloride sal t shows a f u r t h e r N-H ... C 1 - hydrogen bond which is not observed i n t h e dihydrochlor ide s t r u c t u r e .

3. Synthes is

P i r enzep ine , toge the r with o t h e r 11 - subs t i t u t ed 11H- pyrido[ 2 , 3-b] [ 1,5] benzodiazepin-5 (6H) -ones , had been prepared by D r . Karl Thomae (8) . The compounds are prepared by t reatment of an 11H-pyrido[ 2 , 3-b] [ 1 , 51 benzodiazepin-5 (6H) -one with a dihalogen compound, XCH2COX' i n which X and X I may be a l i k e o r d i f f e r e n t and des igna te an atom of C 1 , B r and I . ha loace ty l in te rmedia te is then t r e a t e d with t h e appropr ia te amine. Thus, a b o i l i n g s o l u t i o n o f 2 1 g 11H-pyrido[ 2 , 3-b] [ 1 , 51 benzodiazepin-5 (6H) -one) i n 300 m l

The 11-


C16 -NI C 17- C I6 N 18-C I7

HI71 HI8

HI72

Figure 12 : NEWMAN PROJECTIONS DOWN THE BONDS ClbNl, c r C 1 6 &

v8-Ci7 FOR PIRENZEPINE DIHYDROCHLORIDE ( ABOVE ) AND THE

MONOHYDROCHLORIDE (BELOW) ( 7 1.


abso lu te dioxane t r e a t e d dropwise s imultaneously i n 30 min wi th 15.8 g ClCH2COC1 i n 40 m l abso lu t e dioxane and 1 4 . 4 g E t 3 N i n 30 m l abso lu t e dioxane, t h e mixture r e f luxed f o r 8 h r and t h e hot f i l t r a t e evaporated i n vacuo gave ll-chloroacetyl-11H-pyrido[2,3-b][1,5] benzodiazepin-5 (6H) -one (Scheme 2 ) . The ch lo roace ty l d e r i v a t i v e and N-methylpiperazine i n abso lu te a lcohol re f luxed f o r 18 h r f i l t e r e d and t h e hot f i l t ra te evaporated i n vacuo t o g ive p i renzepine .

C 1 CH, COC 1 %

N H

I 0 =c

I C H 2 C 1

H 0

H2C -N wN- CH3

Scheme 2 . Synthes is of p i renzepine


4. S t a b i l i t y

S t a b i l i t y of p i renzepine depends on bo th moisture conten ts and pH and t h e e f f e c t o f t h e moisture con ten t s could be minimized by ad jus t ing t o optimum pH ( 9 ) . Pirenzepine d ihydrochlor ide i n t a b l e t was shown s t a b l e t o exposure No change i n co lo r , smell, t a s t e o r genera l appearance o r presence of degradat ion products was observed (10).

t o l i g h t a t 50 - 5 5 O .


5 . 1 Spectrophotometr ic Methods

The determinat ion of p i renzepine d ihydrochlor ide spec t rophotometr ica l ly using AA method have r e c e n t l y been repor ted ( 4 ) . The mean percentage recovery f o r t a b l e t s , each l a b e l l e d t o conta in 25 mg,was 100.2 -+ 1.15. The drug shows maximum absorpt ion a t 280 nm i n hydrochlor ic ac id and a t 295 nm in 0 . 1 N sodium hydroxide due t o keto-enol tautomerism. AA at 300 nm was found t o be l i n e a r l y r e l a t e d t o t h e drug concent ra t ion over a range of 10-80 Ug/ml with a r e l a t i v e s tandard dev ia t ion of 0.4%.

A r ap id and d i r e c t method f o r t h e determinat ion of p i renzepine d ihydrochlor ide i n t h e presence o f degradat ion products us ing f i rs t d e r i v a t i v e spectrophotometry has a l s o been repor ted ( 5 ) . The method was appl ied us ing s y n t h e t i c mixtures of t h e i n t a c t drug and degradat ion products . procedure i s s u i t a b l e f o r monitor ing t h e drug s t a b i 1 i t y .

The

5 .2 Chromatographic Eilethods

5 . 2 . 1 Thin-Layer Chromatographic (TLC) Methods

Thin l a y e r chromatography have been used f o r t h e a n a l y s i s o f p i renzepine (10) . Precoated TLC p l a t e s with 0 .2 mm s i l i c a g e l FZs4 were used.


5.2.2 High-pressure Liquid Chromatography (HPLC)

Daldrup -- e t a1 (11) have used reversed-phase l i q u i d chromatography a s a t e s t f o r sc reening pharmaceuticals , drugs and i n s e c t i c i d e s .

High performance r e v e r s e - p h a s e l i q u i d chromatography r e t e n t i o n da ta f o r p i renzepine and o the r compounds (560 compounds) a r e given. The r e l a t i v e r e t e n t i o n times were ca l cu la t ed a s t h e r a t i o s of r e t n t i o n times of compound and r e fe rence compound, 5- (p-methylphenyl) -5-phenyl hydantoin. The UV d e t e c t o r wavelength was 220 nm, where most of t h e compounds gave response. prepacked column C - 1 8 f o r t h e ana lys i s .

Two so lvent programs and a SIL-X-10 were used

Babhair (12) repor ted a s imple and s e n s i t i v e method for t h e determinat ion of p i renzepine , i n dosage forms and i n b io log ica l f l u i d s , us i n g high-per formance 1 i q u i d chromatography wi th U . V . de tec to r . A t a b l e t of 25 mg drug was ground, suspended i n 10 m l o f water , shaken and then f i l t e r e d . A known volume of t h e f i l t r a t e is ad jus ted t o appropr ia te concent ra t ion . Twenty 1.11 of t h i s s o l u t i o n were in jec ted . Plasma o r u r i n e samples were made a l k a l i n e with ammonia before e x t r a c t ion with chloroform which was evaporated and t h e res idue was d isso lved i n t h e mobile phase, 20 1-1 of t h i s so lu t ion were in j ec t ed . The determinat ion l i m i t f o r q u a n t i t a t i o n was about 1 pg/ml o f p i renzepine . Complete separa t ion of t h e drug was achieved i n about 5 .4 minutes under the present chromatographic condi t ions . A 30 X 3.9 cm i. d. commercially a v a i l a b l e s t a i n l e s s s tee l C-18 column was used. Mobile phase cons i s t ed of a c e t o n i t r i l e , methanol and 5% a c e t i c ac id (70:40:15).

High-performance l i q u i d chromatographic determinat ion o f p i renzepine dihydrochlor ide i n i t s pharmaceutical formulat ion have a l s o


been repor ted (13). Normal phase l i q u i d chromatography has been performed on a Micropack Si-10 column us ing ammonium hydroxide (28-30% NH3) i n methanol (0.75 : 99.25% v/v) mobile phase and a flow r a t e of 2 ml/min. Clobazam has been used a s i n t e r n a l s tandard with r e t e n t i o n times of 1 . 9 and 2.8 minutes f o r clobazam and p i renzepine d ihydrochlor ide , r e spec t ive ly a t 254 nm. t o contain 25 mg p i renzepine d ihydrochlor ide gave mean percentage result o f 99.98 2 0.4. The method have a l s o been repor ted t o be a s t a b i l i t y i n d i c a t i n g method.

Tab le t s each l a b e l l e d

5 .3 Radioimmunoassay

A radioimmunoassay system developed f o r p i renzepine was used t o spec i fy t h e requirements f o r us ing such system i n pharmacokinetics and t o i l l u s t r a t e t h e genera l s t r a t e g y i n e s t a b l i s h i n g s p e c i f i c i t y , s e n s i - t i v i t y and r e l i a b i l i t y (14 ) . The type of e r r o r i n t h e f i n a l d a t a was considered. I t is a widespread assumption t h a t weak c ross - r eac t ions i n me tabo l i t e s r e s u l t i n a r e l a t i v e e r r o r and, t h e r e f o r e , have no relevance t o t h e de t ec t ion o f t h e parent drug. In c o n t r a s t t o t h i s assupt ion , it was shown t h a t such weak c r o s s - r e a c t i v i t y in b i o l o g i c a l samples g ives r ise t o an abso lu te e r r o r which i s independent o f t h e concent ra t ion and analogous t o t h a t caused by a blank value i n chemical ana lys i s .

Tanswell and Zahn (15) appl ied monoclonal assays f o r s tudying t h e pharmacokinetics o f p i renzepine .

6. Pharmacokinetics

The marked physicochemical p r o p e r t i e s o f p i renzepine r e s u l t i n t h e drug being h ighly hydroph i l i c a t phys io logi - cal pH. This proper ty determines t h e fa te o f p i renzepine i n t h e organism due t o t h e l imi t ed a b i l i t y of t h e drug t o pene t r a t e l i p i d membranes. This l imi t ed pene t r a t ion i s r e f l e c t e d in i t s absorp t ion , d i s t r i b u t i o n , biotransforma- t i o n and exc re t ion .

6 .1 Absorption

Ora l ly adminis tered p i renzepine was almost completely absorbed by r a b b i t s and dogs bu t on ly about 11% absorbed by r a t s and t h e blood l e v e l maxima reached a f t e r 3 h r (16).


After o r a l adminis t ra t ion of aqueous so lu t ions o r t a b l e t s 20-30% of t h e dose is absorbed ( 1 7 ) . In an i n t e r n a t i o n a l pharmacokinetic s t u d i e s 20% o f t h e o r a l dose adminis tered t o t h e panel of 87 volunteers was absorbed (18). Uniform plasma l e v e l s were observed i n t h i s la rge panel from 10 d i f f e r e n t coun t r i e s a f t e r a s i n g l e o r a l dose of 50 mg, with minimal i n t e r p a t i e n t v a r i a t i o n . Peak serum concent ra t ion were approximately 50 ng/ml occurr ing wi th in 2 h r s . After o r a l dose o f 20 mg/kg, t h e drug appeared i n t h e blood a t a concent ra t ion of 0 .17 ug/ml only 15 min a f t e r admin i s t r a t ion . Afterwards, however, i nc rease i n t h e concent ra t ion was shown reaching t h e maximum of 0 . 4 2 ug/ml approximately 3 h r a f t e r adminis t ra t ion (19) . The r e s u l t s a r e comparable t o those of Hammer e t a1 (16) who repor ted t h a t t h e maximum blood concent ra t ion was a t t a i n e d 3 h r a f t e r adminis t ra t ion at t h e l e v e l of 0.55 ug/ml.

- _

After o r a l adminis t ra t ion of 25 mg of t h e drug, t he maximum blood l eve l occurred in 2 - 3 h r and was about 24 ng/ml. Computer s imula t ion was used t o de r ive a s i n g l e dosage scheme f o r p i renzepine cons i s t ing of an i n i t i a l dose o f 50 mg and maintenance dose of 25 mg a t 12 h r - i n t e r v a l s . This schedule maintained a maximum plasma l eve l o f - > 24.1 ng/ml i n normal s u b j e c t s (17) .

The maximum plasma l eve l appeared 2 h r a f t e r t h e adminis t ra t ion o f 1 2 . 5 - 150 mg pirenzepine o r a l l y t o volunteers (20) . Af t e r mu l t ip l e adminis t ra t ion (25 - 50 mg a day) t h e plasma l eve l was increased f o r t h e f i rs t 3 days o f adminis t ra t ion and remained constant t h e r e a f t e r .

Simulated plasma l e v e l s of p i renzepine a f t e r an o r a l t h e r a p e u t i c dosage regimen (50 mg, twice d a i l y ) on t h e b a s i s of s i n g l e adminis t ra t ion d a t a , showed s teady s ta te within 2 days of t reatment (18). Pirenzepine adminis tered i . m . was completely absorbed i n r a b b i t s and dogs. Rats t h a t poorly absorb o r a l p i renzepine showed complete absorp t ion of i . m . dose of t h e drug.

Using monoclonal assays (15), plasma p i renzepine l e v e l s and u r ina ry excre t ion were measured over time


i n 10 hea l thy male sub jec t s a f te r 10 mg i . v . or i . m . dose, in randomized crossover s tudy. The da ta were f i t t e d t o a 3-compartment open model. Absorption a f t e r i . m . i n j e c t i o n was rap id and complete (mean b i o a v a i l a b i l i t y 100 .3%) .

After i . v . bolus i n j e c t i o n high plasma l e v e l peaks are reached which last only f o r a sho r t time per iod s ince due t o t h e d i s t r i b u t i o n of p i renzepine i n t o body t i s s u e s t h e plasma l e v e l dec l ines r a p i d l y with an i n i t i a l h a l f - l i f e of about 5 min. (17, 2 1 ) . The peak concentrat ion a f t e r i . v . adminis t ra t ion (22) was much h igher than t h e the rapeu t i c l e v e l a f te r o r a l adminis t ra t ion . In t h e f i r s t minutes a f t e r i . v . i n j e c t i o n of 10 mg pirenzepine (0.15 mg/kg) , t h e plasma concent ra t ions were almost 10-fold h igher than t h e s teady s t a t e l eve l reached a f t e r d a i l y doses o f o r a l 100 mg. In less than one hour, however, t h e plasma l e v e l o f t h e i . v . dose i s comparable t o the s teady s t a t e concentra- t i o n s after o r a l adminis t ra t ion (18).

The absorpt ion of S . C . dose of p i renzepine i s very swift and comparable t o t h a t of i . v . (4) and unl ike t h a t o f i . m . r epor ted by Hammer -- et a1 (16).

6 .2 D i s t r ibu t ion

Informations provided by whole-animal autoradiography of r a t 10 minutes a f t e r intravenous i n j e c t i o n o f 2 mg/kg o f r ad ioac t ive p i renzepine (15, 2 1 ) , showed t h a t t h e drug is widely d i s t r i b u t e d i n t h e body. i n t e r n a l organs and s k e l e t a l muscles. No rad io- a c t i v i t y was de tec t ed i n t h e b r a i n o f t h e r a t o r t h e f e tuses of g e s t a t i n g mouse suggest ing t h a t t h e blood-brain b a r r i e r r ep resen t s a genuine b a r r i e r t o p i renzepine and t h a t t h e drug does not c ros s t h e p l acen ta l b a r r i e r . Continuous intravenous infus ion of p i renzepine over a per iod o f 2 days a t a r a t e of 2 mg/hr/8-9 kg baboon (10 times t h e human infus ion dose) , showed t h a t t h e h ighes t l e v e l s a r e i n t h e organs respons ib le f o r t h e e l imina t ion of t h e drug i . e . l i v e r and kidneys. The o the r i n t e r n a l organs, t h e sk in and s k e l e t a l muscles, however, showed a h igher pirenzepine concentrat ion p e r g of t i s s u e than t h e plasma.

The r ad ioac t ive drug i s found in a l l


Extremely low drug concent ra t ion was found i n t h e b r a i n .

The d i s t r i b u t i o n o f p i renzepine i n t h e t i s s u e s a f t e r one-time o r a l admin i s t r a t ion was i n v e s t i g a t e d by Kobayashi e t a1 (19) a f t e r i s o l a t i o n o f t i s s u e s and whole-body autoradiography technique. After 3 h r , t h e time o f maximum blood concen t r a t ion , h igh concent ra t ion of p i renzepine was observed i n t i s s u e s l i k e l i v e r , kidney, pancreas , lung, p i t u i t a r y gland, s a l i v a r y gland, adrena l gland e t c . These resu l t s , t h e r e f o r e , agree wi th those of Hammer and Koss (21) i n t h a t r ad ioac t ive p i renzepine was found d i s t r i b u t e d i n a l l t i s s u e s except t h e c e n t r a l nervous system. The r a d i o a c t i v i t y d i s t r i b u t e d in t h i s way decreased as time passed. Twenty-four hours a f t e r adminis t ra t ion , t h e concent ra t ion was s l i g h t l y hgih only i n t h e g a s t r o i n t e s t i n a l t r a c t and l i v e r . Rad ioac t iv i ty i n t h e t es t i s showed a tendency t o decrease g radua l ly dur ing 72 h r . Kaubisch e t a1 (23) observed s t rong r a d i o a c t i v i t y d i s t r i b u t e d i n t h e g a s t r o i n t e s t i n a l t r a c t , kidney and s a l i v a r y glands i n a whole-body autoradiography performed a f t e r i . v. adminis t ra t ion of 14C-pirenzepine i n a dose of 2 mg/kg. g a s t r o i n t e s t i n a l t r a c t was considered t o r e s u l t from t h e exc re t ion of t h e r a d i o a c t i v i t y i n t o t h e i n s i d e o f t h e i n t e s t i n a l t r a c t v i a t h e blood v e s s e l s and i n t e s t i n a l wal l . This is supported by t h e f ind ing o f Kobayashi e t a1 (19) who observed c lean p i c t u r e s o f r a d i o a c t i v i t y d i s t r i b u t i o n a t t h e wall o r mucus o f t h e stomach and i n t e s t i n e s i n autoradiography 3 h r and 9 h r after admin i s t r a t ion and considered t h a t t h e r a d i o a c t i v i t y was s e c r e t e d from t h e g a s t r o i n t e s t i n a l wall o r was depos i ted t h e r e .

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The presence of r a d i o a c t i v i t y i n t h e

L-

After mul t ip l e o r a l dose, Kobayashi e t a1 (19) , observed a s l i g h t tendency towards accumulation i n t h e l i v e r , kidney and t e s t i s whereas r ad io - a c t i v i t y decreased s lowly i n one-day admin i s t r a t ion . However, t h e r a d i o a c t i v e concent ra t ion i n each t i s s u e d i d not i nc rease i n propor t ion t o t h e admin i s t r a t ion times bu t decreased as time passed and le f t only t r a c e s one week a f t e r t h e te rmina t ion of admin i s t r a t ion . In mul t ip l e admin i s t r a t ion

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HUMEIDA A. EL-OBEID ETAL.

almost a l l of t h e r ad ioac t iv i ty was found excreted i n t o ur ine and feces 24 h r a f t e r each administration. I t i s , therefore , considered t h a t t h e accumulation of t he drug i n the body is small. Jaup and Blomstrand (24) measured serum and cerebrospinal concentrations of pirenzepine i n healthy volunteers a f t e r therapeut ic dosage over 2-5 days. The cerebrospinal f l u i d - to-serum r a t i o was 0.095 t o 1, indicat ing t h a t pirenzepine passed the blood-brain b a r r i e r , but only t o a small extent . i n t h e cerebrospinal f l u i d was about 10% those of serum.

Pirenzepine concentrations

That the penetration of pirenzepine through the blood-brain b a r r i e r i s very l imited was a l s o demonstrated by o the r s tudies (18, 25-27).

6 . 3 Metabolism

Pirenzepine undergoes only s l i g h t metabolism i r r e spec t ive of the route of administration and the re i s no s ign i f i can t f i r s t - p a s s e f f e c t . Only small amounts of metabolites have been detected (16-18, 21), of which t h e desmethyl der ivat ive accounted f o r s i g n i f i c a n t l y less t h a n 10% of t h e dose. e f f e c t . The t r i c y c l i c moiety, despiperazinly pirenzepine was a l so detected as a metabolite i n negl igible amounts ( 1 7 ) . Unlike cimetidine, pirenzepine does not i n h i b i t t h e hepat ic mixed function oxidase system (28).

The desmethyl compound has no pharmacological

6.4 Excretion

In man as well as i n other animals, pirenzepine is excreted unchanged in ur ine and feces (17 , 18, 21). Due t o t h e special physicochemical propert ies of pirenzepine only about 10% of t h e dose is bound t o plasma proteins (17, 20) and hence it is almost completely f i l t e r e d i n t o t h e kidneys. of d i s t r ibu t ion of pirenzepine is iden t i ca l t o the e x t r a c e l l u l a r space which indicates t h a t pirenzepine rapidly equ i l ib ra t e s between plasma and the ex t r ace l lu l a r f l u i d s of t h e peripheral t i s s u e s . The t o t a l plasma clearance of pirenzepine is 255 ml/min. Because of i t s l imited

The volume


l i p i d s o l u b i l i t y , the drug cannot be reabsorbed from the renal tubules s o t h a t t h e renal clearance of 129 ml/min corresponds t o the glumerular f i l t r a t i o n r a t e . Hammer e t a1 (16) reported t h a t approximately equal amounts of pirenzepine were excreted via t h e b i l e and ur ine. clearance, mostly b i l i a r y excretion i s about 125 ml/min (17).

-- The hepat ic

Following o ra l administration of a therapeut ic dose of labeled drug, 82% of the substance excreted i n ur ine is unchanged, as is 92% of t h a t i n the feces. The drug i s eliminated from the organism f u l l y and almost equally i n ur ine and feces , elimination being almost complete a f t e r 4 days (17). Kobayashi -- e t a1 (19) reported t h a t i n s i n g l e a s well as i n mult iple administration, almost a l l of t h e dose was excreted in u r ine and feces. In i . v . administration, 44% and 50% of t h e administered dose were excreted i n t o ur ine and feces , respect ively 24 h r a f t e r administration and 46% and 53% respect ively, 4 days a f t e r administ- r a t ion . Similar r e s u l t s were obtained with subcutaneous administration. Hammer -- e t a1 (16) reported t h a t 36% and 56% of the administered dose were excreted i n u r ine and feces , respect ively, following i . v . administration. 42% of a 1 0 mg dose was a l s o reported (15) eliminated r ena l ly with t o t a l clearance o f 253 ml/min and renal clearance of 107 ml/min. The mean t r a n s i t time was 9 . 3 h r . A urinary excretion r a t e of 7-12% was reported by Ohashi e t a1 (20) within 4 days a f t e r multiple administration of pirenzepine (25-50 mg, 3 times a day f o r 4 days). - a1 (16) reported approximately 4% of the adming- t e r e d dose was excreted i n ur ine a f t e r o r a l administration. According t o Kobayashi -- e t a1 (19) about 8% was excreted i n ur ine a f t e r s i n g l e o r a l administration and about 4.5% i n multiple administration. The difference i n t h e value was a t t r i b u t e d t o the difference i n absorption r a t e s between f a s t ed and nonfasted animals.

-- Hammer e t

Elimination h a l f - l i f e of 10 h r was reported (21) following i . v . dose of 8 mg pirenzepine t o humans. Oral administration of 25 mg t a b l e t s t o volunteers r e su l t ed i n elimination h a l f - l i f e of


7 .

11 h r (21). Ohashi e t a1 (20) reported an elimination h a l f - l i f e of 13 h r . The long elimina- t i o n h a l f - l i f e was a t t r i b u t e d t o slow red i s t r ibu t ion of pirenzepine from the t i s s u e s r a t h e r than t o slow excretion (21).

Intravenous administration of 14C-pirenzepine t o dams i n a dose of 2 mg/kg caused t h e appearance of r ad ioac t iv i ty i n m i l k almost equivalent t o t h a t i n blood (19).

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Pretreatment of healthy volunteers with therapeut ic doses of pirenzepine over 5 days showed no s ign i f i can t e f f e c t on t h e absorption, d i s t r i b u t i o n o r elimination of ant ipyrine (28).

Pharmaco 1 ogy

7.1 Antisecretory Effects

Secretory s tud ie s with pirenzepine c l e a r l y demonstrate t h a t pirenzepine suppresses both basal and stimulated acid and pepsin secret ion (30-52). Intravenous pirenzepine reduces basal acid secre- t i o n by 90% and pentagastrin-stimulated secret ion by approximately 50% ( 3 0 ) . Fr i t s ch -- e t a1 (31) reported t h a t o r a l 50 mg of pirenzepine, i n pa t i en t s with duodenal u l c e r , inhibi ted the basal acid secret ion by 39.9% and peptone-induced acid output by 21.3%. near ly doubled i f t he same dose is given a f t e r a treatment with 50 mg pirenzepine twice d a i l y f o r 3 t o 7 days. l eve l s can be demonstrated by a s i n g l e dose of pirenzepine. After pretreatment only small increase of basal gas t r in l eve l s i s observed, serum gas t r in does not change during stimulation. Oral pirenzepine (25 mg) reduces acid output by 50% and pentagastrin-stimulated secret ion by 30-40%. Doubling the dose increases the degree of inhibi t ion of st imulated secret ion by (30). Jaup -- e t a1 (32) reported t h a t pirenzepine 50 mg PO b . i . d . reduced basal g a s t r i c acid secret ion by 54% a t two hours following t h e last dose. Pentagastrin-stimulated secret ion of hydrochloric acid (1 mcg/kg/hr) by continuous i . v . infusion was reduced by 31%. Secretory s t u d i e s by

The percentage inh ib i t i on

No acute e f f e c t on serum gas t r in

10%


Jaup -- e t a1 (33) c l e a r l y demonstrated t h a t p i renzepine suppresses b a s a l , pentagas t r in-s t imula ted and insu l in-s t imula ted ac id sec re t ion i n a dose- re la ted manner. In a double-bl ind, placebo con t ro l l ed , randomized s t u d i e s on p a t i e n t s with dyspepsia, Bianchi Porro e t a1 (34) repor ted a 48% decrease of g a s t r i c s e c r e t i o n i n t h e first hour and 30% i n t h e second hour a f t e r 50 mg o r a l dose o f pirenzepine. Pirenzepine caused a reduct ion t o 20% of t h e concent- r a t i o n o f t h e ac id produced by vagal s t imu la t ion (35). The drug i n h i b i t e d t h e vaga l ly t r ansmi t t ed ac id sec re t ion by about 45% even when t h e vaga l ly s t imula ted ac id sec re t ion amounts t o more than 50% of t h e pentagas t r in-s t imula ted sec re t ion . These e f f e c t s o f p i renzepine a r e s i m i l a r t o those of a t r o - p ine but d i f f e r e n t from the e f f e c t s of c imet idine. Eugenides -- e t a1 (36) repor ted t h a t histamine- s t imula ted ac id output p e r two hours i n hea l thy volunteers was reduced with 25 mg p i renzepine b .d . by 12.5% and with 50 mg t . d . s . by 24%. Stockbrugger e t a1 (37) s tud ied t h e e f f e c t of t h r e e d i f f e r e n t doses o f p i renzepine on ac id sec re t ion s t imula ted by modified shamfeeding (MSF) on hea l thy volunteers . Oral p i renzepine 25 mg b . i . d . , 50 mg b . i . d . o r 50 mg t . i . d . reduced basa l output (0-30 min) by 48%, 59% and 66% r e spec t ive ly , i n s t imula ted ac id output (0 - 120 min) by 45%, 58% and 48% respec t ive ly . When t h e a c i d sec re t ion was c a l c u l a t e d a s volume co r rec t ed f o r duodenal-gastr ic r e f l u x and p y r o l i c l o s s e s , t h e corresponding f i g u r e s were 33.7%, 36 .3% and 42.6%. Mignon e t a1 (38) repor ted a dose- re la ted reduct ion of meal-stimulated ac id response with inc reas ing doses o f i . m . p i renzepine. Resul t s with 0 .5 mg/kg o f pirenzepine showed 53% reduct ion i n ac id secre- t i o n . p a t i e n t s 45 min and again 10 min before t h e start of MSF blocked 48% of t h e response (39).

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Pirenzepine 10 mg given t o duodenal u l c e r

Pirenzepine (25 pg/kg) adminis tered i . m . t o dogs 10 min before infus ion of bombesin, s i g n i f i c a n t l y i n h i b i t e d t h e hypersecret ion induced by t h e pept ide i n t h e main stomach and i n a dose o f 100 ug/kg pirenzepine v e r t u a l l y abbol ished t h e a c i d response t o bombesin (40 ) . In Heidenhain pouch both doses f u l l y prevented ac id sec re t ion . Pirenzepine i n j e c t e d during t h e sec re to ry p l a t eau


induced by bombesin, produced a mean peak inh ib i t i on of acid output from the main stomach of 76.5% and 80% with doses of 25 and 100 g/kg respectively. Pirenzepine (25 and 100 ug/kg i . v . , 10 min before a meal t e s t ) f u l l y prevented acid secret ion f o r Hiedenhain pouch. no e f f e c t on bombesin - or meal-stimulated g a s t r i n re lease. Acid and pepsinogen secret ion induced by bethanechol i n i so l a t ed perfused mouse stomach a r e inhibi ted by pirenzepine (41).

The drug showed

7 . 2 Efficacy

Pirenzepine i s e f f ec t ive i n t h e treatment of g a s t r i c and duodenal ulcers . I t is a l s o e f f ec t ive in non- u l ce r dyspepsia and i n preventing u l c e r recurrence. Pirenzepine i n combination therapy with cimetidine can be used successful ly f o r t he treatment o f c imet idine-resis tant conditions such as duodenal ulcer and Zollinger-Ellison syndrome. The l i t e r a t u r e reports a number of s tud ie s on t h e e f f i cacy of pirenzepine in t h e treatment of g a s t r i c and duodenal u l ce r s (43, 53-130). The published world l i t e r a t u r e (from 1977 t o 1981) on the e f f i cacy of pirenzepine had been the subject of a review and commentary by Bianchi Porro and P e t r i l l o (53). The review examined t h e data published i n Europe which deal with pirenzepine i n the treatment of g a s t r i c and duodenal u l ce r s , taking i n t o account only those r e s u l t s obtained from control led endoscopic t r ia ls . placebo-controlled s tud ie s pirenzepine was administered t o 475 duodenal u l ce r pa t i en t s with incidence of endoscopically proven healing of 74% when the p a t i e n t s received d a i l y dosage of 100 o r 150 mg pirenzepine, but only 55% i f t h e dosage of t h e drug was 75 mg o r less. Similar healing rates were observed (72% when da i ly dose of pirenzepine was 100 o r 150 mg, 55% when the dose was 75 mg o r l e s s ) i n 4 randomized double- b l ind placebo -controlled s tud ie s i n which 84 pa t i en t s with g a s t r i c u l ce r were admitted.

In another review, Texter and Re i l ly (83) evaluated the c l i n i c a l evidence concerning t h e healing e f f ec t of pirenzepine i n g a s t r i c and duodenal u l ce r s .

In 12 prospective randomized double-blind

The reviewers subjected t h e


results of t h e previous double-blind, therapeut ic s tud ie s on u l ce r healing t o mathematical treatments. The therapeut ic s tud ie s reviewed included a l a rge number o f p a t i e n t s i n Europe and Japan. response curve f o r duodenal u l ce r incorporating 16 data points from 8 t r ia l s i n 530 p a t i e n t s , 343 of whom received pirenzepine, r e su l t ed i n a co r re l a t ion coe f f i c i en t r = 0.887 with P < 0.005. Although the a n a l s i s is not s t r i c t l y mathematically co r rec t , it suggests t h a t pirenzepine heals duodenal u l ce r compared t o placebo with a pos i t i ve r e l a t ionsh ip between dose and e f f e c t . d i c t ed healing r a t e a t 150 mg was 79%. The analysis a l so shows t h a t endoscopic healing from g a s t r i c and duodenal ulcers is l i n e a r with respect t o time upto 6 weeks ( r = 0.75, P < 0.001) and suggests t h a t healing r a t e s improve with time on therapy. This observation on time course is strengthened by the r e s u l t s of a l a rge Japanese mult icentre t r i a l on pirenzepine. Therapeutic s tud ie s on g a s t r i c u l c e r , showed endoscopic healing r a t e o f 66.7%, using 75 mg/day pirenzepine f o r 8 weeks, which increased t o 72% with 150 mg dose f o r 8 weeks.

The dose-

The pre-

Brunner (94) reported t h a t pirenzepine is equally o r a t least not e s s e n t i a l l y l e s s e f f e c t i v e than cimetidine i n t he treatment of duodenal u l ce r . The e f f e c t of pirenzepine and cimetidine on healing, symptoms and relapse r a t e of duodenal u l c e r was s tudied by Eichenberger -- e t a1 (95). No s i g n i f i c a n t differences i n u l ce r healing between the eff icacy of t he drugs when 1 gm/day of cimetidine and 75 mg/day pirenzepine were used. re lapse r a t e a f t e r treatment with pirenzepine was lower than a f t e r treatment with cimetidine.

The

Do -- e t a1 (96) reported the e f f i cacy of pirenzepine, 100 mg daily, i n t he treatment of duodenal u l ce r s i n a placebo-controlled study, Pirenzepine produced healing i n 14 out of 18 p a t i e n t s (78%) following 6 weeks o f treatment as compared t o 7 of 20 p a t i e n t s (35%) receiving placebo. Relief of daytime and nighttime pain was g rea t e r i n pirenzepine-treated p a t i e n t s , as well as lower consumption of antacid.


Cheli -- e t a1 (97) reported t h a t pirenzepine i n 100 mg o r a l l y da i ly f o r 28 days r e su l t ed i n c l i n i c a l cure i n 34 of 44 duodenal u l ce r pa t i en t s . p a t i e n t s completing treatment, were given pirenzepine 50 mg da i ly f o r 6 months, r e su l t ed i n u l ce r re lapse i n 18.7%. In 16 other p a t i e n t s , who interrupted treatment completely without pirenzepine, c l i n i c a l recurrence of duodenal u l c e r occured i n 68.7%, with endoscopic recurrence i n 62%, i n the following 6 months.

Sixteen

Pirenzepine was shown by Dal Monte e t a1 (98) t o be e f f ec t ive i n the maintenance treatment of duodenal u l ce r s t o prevent u l ce r recurrence when given i n o r a l doses of 100 mg da i ly f o r one year. Pirenzepine has a l s o been e f f e c t i v e i n doses of 50 mg da i ly f o r one year (100, 104, 107). However, other invest igators have reported no s i g n i f i c a n t difference between pirenzepine 25 mg o r a l l y b . i . d . and placebo i n the prevention of duodenal u l ce r re lapse i n a one-year study (102). Hoffenberg (103) reported pirenzepine i n o r a l dose of 50 mg b . i . d . f o r 4 weeks, i n i t i a l l y , followed by 50 mg b . i . d . f o r 4 weeks, was no more e f f ec t ive than placebo i n the treatment of duodenal u l ce r (48% versus 40% complete healing). These data suggest t h a t longer courses of therapy (6-8 weeks) may be indicated.

--

Pirenzepine in o r a l da i ly doses of 100 - 150 mg has been e f f e c t i v e i n producing healing of 74% of pa t i en t s with duodenal u l ce r s i n control led c l i n i c a l t r ia l s (range 52 - 90%) (53, 55, 57, 60, 74, 76, 104, 105), whereas lower doses of 75 mg da i ly o r l e s s have produced lower response r a t e s ( z 55%) (53, 54, 106). These results a r e supported by more recent c l i n i c a l s tud ie s with pirenzepine i n o r a l da i ly doses of 100 - 150 mg and t h i s appears t o be the optimal dose (107 - 113).

Available c l i n i c a l t r i a l s on g a s t r i c u l ce r suggest t h a t o ra l doses of 100-150 mg da i ly can produce healing in approximately 72% of pa t i en t s with gas- t r i c u l ce r , s imi l a r t o duodenal u l ce r , doses of 75 mg da i ly o r l e s s produce lower healing rates (53, 89, 114, 115).


Oral 50 mg d a i l y dose of pirenzepine was reported e f f e c t i v e i n t he treatment of non-ulcer dyspepsia i n a double-blind comparative t r i a l i n 59 p a t i e n t s f o r a period of 4 weeks (116). A da i ly dose of 100 mg was a l so shown t o be e f f ec t ive (117). Short-term pirenzepine therapy (100 mg d a i l y f o r 4 weeks) was reported e f f e c t i v e i n improving t h e endoscopic appearance of acute conjest ive and erosive g a s t r i t i s and non-ulcer-associated severe duodenitis (118). The drug was s i m i l a r l y e f f e c t i v e as cimetidine 1 gm da i ly f o r 4 weeks.

Comparative e f f i cacy of pirenzepine 100 mg PO d a i l y and cimetidine 1 gm PO da i ly i n 6-week treatment o f duodenal u l ce r i n ou tpa t i en t s has been indicated by several control led c l i n i c a l t r i a l s (67, 68, 81, 107, 108). have a l so reported t h a t pirenzepine 150 mg PO d a i l y was comparable t o cimetidine 1 gm d a i l y i n the treatment o f a c t i v e duodenal u l c e r i n 90 pa t i en t s t r e a t e d f o r a period of 4 weeks (72% versus 75% respect ively) . t he eff icacy of both drugs was approximately 70%. Both drugs have been equally e f f ec t ive i n preventing duodenal u l c e r recurrence following healing with conventional treatment with e i t h e r drug (99, 101, 120, 121). Combination therapy with cimetidine has been e f f ec t ive i n Zollinger-Ellison syndrome r e s i s t a n t t o cimetidine alone (121).

A s i n g l e 400 mg o ra l dose of cimetidine demonstrated approximately twice the an t i s ec re to ry a c t i v i t y of 50 mg pirenzepine s i n g l e o r a l dose i n both basal and stimulated g a s t r i c acid sec re t ion following pentagastrin st imulation. With pirenzepine i n doses of 50 mg, i nh ib i t i on of pentagastr inst imulated acid sec re t ion was similar t o t h a t o f an o r a l 200 mg cimetidine, t h i s cimetidine dose, however, had a g rea t e r i nh ib i to ry e f f e c t on g a s t r i c basal acid secret ion (122). The authors ind ica t e t h a t high doses of pirenzepine should be avoided due t o antimuscarinic e f f e c t s such as dry mouth and pa lp i t a t ions and t h a t cimetidine is indicated as agent of choice f o r reduction of g a s t r i c acid secret ion. Pa lp i t a t ions occurred i n several pa t i en t s i n t h i s study with doses of 50 mg. In a s ingle-bl ind multicenter study Brunner -- et a1 (108) evaluated t h e e f f i cacy of pirenzepine 100 mg d a i l y

Porro -- et a1 (109)

In most c l i n i c a l s tud ie s ,


(50 mg before breakfast and dinner) compared with t h a t of cimetidine 1 gm da i ly (200 mg t . i . d . with meals and 400 mg h . s . ) in t he treatment of endoscopically-proven duodenal u l ce r i n 254 pa t i en t s . Endoscopy was repeated a f t e r 4 weeks of treatment with both regimens; t h e endoscopist was blinded as t o treatment regimens. 73% occurred with pirenzepine and cimetidine, respect ively, which was not s t a t i s t i c a l l y s i g n i f i c a n t . with both drugs and t h e incidence of s ide e f f e c t s was s imilar .

Healing r a t e s of 64% and

Pain r e l i e f was achieved rapidly

Pirenzepine i n an o r a l dose of 50 mg b . i . d . was reported s imi l a r ly e f f ec t ive as cimetidine 400 mg b . i . d . o r a l dose (low-dose therapy) i n the treatment of duodenal ulcers i n multicenter t r i a l (125). occured in 27 of 37 pirenzepine-treated p a t i e n t s (73%) and 29 of 38 cimetidine-treated p a t i e n t s (76%). Side e f f e c t s were s imi l a r i n each group, except f o r more frequently reported antimuscarinic e f f e c t s i n pirenzepine-treated pa t i en t s . No adverse e f f e c t s on i n t r a g a s t r i c milieu were reported. I n t r a g a s t r i c microbial concentrations were increased s i g n i f i c a n t l y with both drugs but remained within normal limits; n i t r i l e concentra- t ion p r i o r t o o r following treatment did not exceed t h e normal range. Oral 300 mg da i ly dose r an i t i d ine was reported (104) equally e f f e c t i v e as o r a l 100 mg da i ly dose of pirenzepine i n the treatment of duodenal u l ce r i n one control led study (response rates of 87% i n each group). Pirenzepine was equally e f f e c t i v e as atropine as g a s t r i c an t i s ec re to ry agent, but unlike atropine, pirenzepine had no e f f e c t s on CNS, increase i n hear t r a t e , mydriasis, urinary bladder contract ion o r motor function of gastroenter ic t r a c t (124).

Following 4 weeks of therapy, healing

Londong e t a1 (125) reported t h a t concomitant pirenzepine and cimetidine therapy r e s u l t i n s i g n i f i c a n t l y greater suppression of st imulated acid secret ion than e i t h e r drug alone. suggested t h a t combination therapy may exhibi t syne rg i s t i c e f f e c t s on p a r i e t a l c e l l function and could be advantageous in p a t i e n t s w i t h gastrinoma,

--

I t i s


u l c e r s and hypersecre t ion r e s i s t a n t t o s i n g l e drug therapy, and i n prophylaxis of stress u l c e r bleeding i n c r i t i c a l l y - i l l p a t i e n t s .

Weberg e t a1 (126) has demonstrated t h a t p i renzepine enhances and prolongs t h e effects o f an tac ids on i n t r a g a s t r i c a c i d i t y . Addition o f p i renzepine t o an tac id therapy r e s u l t e d i n a more sus ta ined pH e l eva t ion than t h a t observed with twice t h e amount of an tac id adminis tered alone.

--

Combination therapy with H2-receptor an tagon i s t s has proven more e f f e c t i v e than H2-antagonist therapy alone (118, 127) . The combination of p i renzepine 50 mg h . s . and r a n i t i d i n e 150 mg b . i . d . given f o r a per iod o f 6-12 weeks was e f f e c t i v e i n a l l o f 7 p a t i e n t s with a c t i v e r ecu r ren t duodenal u l c e r . Continuous maintenance therapy with r a n i t i d i n e 150 mg d a i l y and p i renzepine 50 mg d a i l y r e s u l t e d i n prevent ion of u l c e r r e l apse i n 5 p a t i e n t s who, i n t h e previous year , con t inua l ly re lapsed while rece iv ing s ingle-agent (c imet id ine o r r a n i t i d i n e ) therapy. These r e s u l t s suggest t h e e f f i c a c y of combined H2-antagonist and p i renzepine therapy i n h ighly r ecu r ren t o r r e s i s t a n t duodenal u l c e r s (127). Mignon -L e t a1 (121) repor ted t h e e f f ec t iveness o f combination therapy of p i renzepine and c imet id ine i n 3 of 4 p a t i e n t s with Zol l inger - E l l i son syndrome who were unresponsive t o cimet i d i n e a lone .

Unlike c imet id ine , p i renzepine appears t o lack i n h i b i t o r y effects on t h e hepa t i c mixed func t ion oxidase system and it should be safe t o adminis te r concomitantly with drugs t h a t a r e metabolized by t h i s system (128).

The a v a i l a b l e informations suggest t h a t p i renzepine possesses s e l e c t i v e an t imuscar in ic effects and minimal CNS effects. S tud ie s presented so f a r do not show t h a t t h e drug i s more e f f e c t i v e than c imet id ine o r r a n i t i d i n e and no s i g n i f i c a n t d i f f e rences i n t o x i c i t y have been observed. Pirenzepine i n combination therapy with H2-receptor an tagon i s t s , however, suggest t h a t p i renzepine may be very use fu l i n t h e t reatment of r e s i s t a n t duode- n a l u l c e r and Zol l inger -El l i son syndrome.


7.3 Adverse Reactions

The major s ide e f f e c t s of pirenzepine a re due t o i ts ant ichol inergic a c t i v i t y which indicates t h a t t h e s e l e c t i v i t y of the drug is not absolute and the ant ichol inergic e f f e c t s can occur which appear t o be dose-dependents.

Dry mouth is the most common s i d e e f f ec t of pirenzepine. Sal ivary secret ion e f f e c t s have been noted i n many therapeut ic t r ia l s . In the pharmacological s tud ie s i n man, pirenzepine i n a dose of 50 mg b . i . d . inhibi ted sa l iva t ion compared t o placebo (131) but not as much as R-hyoscyamine i n a dose of 0.6 mg b . i . d . which was approximately equipotent i n suppression of g a s t r i c acid. Ten of 18 pa t i en t s i n the t r i a l s reported dry mouth with pirenzepine and 17 of 18 on R-hyoscyamine. Dry mouth i s reported t o occur i n 13.5% of pa t i en t s receiving pirenzepine in doses o f 150 mg dai ly . The incidence is reduced t o 4% with doses of 100 mg d a i l y and t o 2.5% with doses of 75 mg da i ly o r l e s s (54). Dry mouth does not appear t o be severe enough t o warrant withdrawal of treatment in most cases (106).

Pirenzepine i n high doses i n h i b i t s the c o n t r a c t i l e (132) a c t i v i t y of the stomach and colon (132). Bianchi Porro -I e t a1 (34), however, reported pirenzepine (50 mg o ra l ly ) showed no e f f e c t s on g a s t r i c emptying. Similar r e s u l t s were reported by Stacher -- e t a1 (133) when healthy men received e i t h e r 50 mg pirenzepine twice d a i l y o r placebo. Despite t he considerable e f f e c t s of pirenzepine on an t r a l mo t i l i t y , its delaying e f f e c t on g a s t r i c emptying was ins ign i f i can t . Constipation has occurred with pirenzepine i n approximately 2.6% of pa t i en t s receiving doses of 150 mg o r a l da i ly dose (54). Diarrhea has a l s o occurred with pirenzepine therapy, however, t he incidence appears t o be l e s s than t h a t of constipation (23, 106, 134). Jaup (135) measured esophageal p e r i s t a l t i c a c t i v i t y a f t e r pirenzepine, 10 mg i . v . o r 50 mg b . i . d . (oral ly) i n comparison with atropine 0.5 mg i . v . o r R-hyoscyamine 0.6 mg b . i . d . The l a t t e r two agents reduced esophageal p e r i s t a l t i c pressure compared t o placebo while pirenzepine showed


marked e f f e c t s only sho r t ly a f t e r i . v . dosing, when plasma l eve l s were high. s l i g h t l y decreased (- 15%) p e r i s t a l t i c amplitude compared t o placebo while R-hyoscyamine decreased t h i s parameter over 50%. Other s tud ie s (136, 137) have shown decreased amplitude of esophageal contractions as well as inh ib i t i on of lower esophageal spincter pressure with parenteral administration of pirenzepine. reduced by 0.3 mg/kg i . m . (138) and g a s t r i c smooth muscle e f f e c t s a r e s l i g h t a t 0 . 2 mg/kg i . m . (139) o r 50 mg b . i . d . (131). Despite pirenzepine e f f e c t s i n a l t e r i n g esophageal and colonic mot i l i t y , t he ava i l ab le data does not suggest t h e drug w i l l increase the r i s k of pep t i c gastro- esophageal r e f lux (140).

Oral pirenzepine

Colonic m o t i l i t y is

The e f f e c t s of intravenous and o r a l pirenzepine on various g a s t r o i n t e s t i n a l hormones and meta- b o l i c parameters a r e s imi l a r t o those observed f o r antimuscarinic agents except f o r the lack of depression of pancreatic bicarbonate secret ion (141, 142) and possibly lack of g a s t r i n (143-145). Pirenzepine produced no changes i n g a s t r i c i nh ib i - t o r y polypeptide, i n su l in , glucagon, neurotensin, vasoactive i n t e s t i n a l polypeptide, somatostatin, s e c r e t i n , f r ee f a t t y acids , l a c t a t e , pyruvate, f3-hydroxybutyrate, acetoacetate , p ro l ac t in and growth hormone. Pancreatic secret ion volume, amylase, t ryps in , chymotrypsin,. l i pase , pancreat ic polypeptide, enteroglucagon, pepsin and g a s t r i c acid sec re t ions a r e decreased. One study (146) noted a s l i g h t prolongation of gas t r in post- prandial ly , while o the r s (143, 145) observed decreases i n contrast t o a t ropine and another t r ia l (144) showed no change. Masala e t a1 (147) reported t h a t pirenzepine i n s ing le doses of 75 mg reduced p ro lac t in l eve l s i n females but produced only minimal e f f e c t s on p ro lac t in in males. reported by Alagna -- e t a1 (148) t o produce no d i r e c t e f f e c t upon the adrenal glands. responsiveness o f the adrenal glands t o exogenous ACTH was unchanged, However, a reduction i n basal c o r t i s o l l eve l s occurred with pirenzepine, suggesting a reduction i n ACTH secret ion. drug has not produced adrenal insuff ic iency.

--

Single doses o f 75 mg pirenzepine were

The

The


Visual disturbances have been reported i n 42% of subjects (149). Brunner (94) however, reported t h a t only small number of p a t i e n t s t r e a t e d with pirenzepine complained about impairment of vis ion. Blurred vision and double vision have occurred during p i renz ep i n e t r e a t men t i n approximate 1 y 6.3% of pa t i en t s receiving o r a l d a i l y doses of 150 mg (75, 83). Hoffenberg (103) described amblyopia i n 24% of pa t i en t s receiving dose of 100 mg dai ly .

Urinary retent ion has been reported i n 0.3% of pa t i en t s receiving pirenzepine i n doses of 100 mg da i ly ( 8 3 ) . Kuhn -- e t a1 (150) reported t h a t pirenzepine (8 mg i.m.) decreased t h e pulse r a t e and s y s t o l i c and d i a s t o l i c blood pressure. Tachycardia has been observed during pirenzepine treatment (103). In most s tud ie s , however, no e f f e c t s on hea r t r a t e were observed (83). Pirenzepine i n dose of 50 mg da i ly caused pa lp i t a t ions (122). CNS t o x i c i t y has been minimal i n most s tud ie s with pirenzepine (83, 151). This can be explained on the bases of pirenzepine s e l e c t i v i t y and a l s o t o hydrophilic c h a r a c t e r i s t i c s . Dizziness, mental confusion, as thenia and headache have been reported i n a few p a t i e n t s (152, 153). Studies i n healthy volanteers showed t h a t no psychotropic e f f e c t s occurred t h a t a r e commonly encountered with t r i c y c l i c ant idepressants , indicat ing t h a t pirenzepine lack appreciable CNS t o x i c i t y (154). Fink and Erwin (149) a l so reported lack of CNS e f f e c t s by pirenzepine with doses upto 150 mg.

7.4 Tissue Se lec t iv i ty

Pirenzepine blocks the acetylcholine receptors of the p a r i e t a l c e l l s o f the stomach and i s thereby a marked i n h i b i t o r of g a s t r i c acid secret ion both i n animals and men. Pharmacological and c l i n i c a l s tud ie s have shown t h i s act ion of pirenzepine t o be se l ec t ive , s ince i n doses which s i g n i f i c a n t l y i n h i b i t g a s t r i c secret ion, s i d e e f f e c t s t yp ica l of ant ichol inergic agents do not occur o r minimal (30, 33, 45) and much higher doses a re needed t o i n h i b i t s a l iva t ion (132) and contraction of stomach and colon (138) o r t o induce tachycardia (52, 132).


Engelhorn (155) has evaluated t h e s e l e c t i v i t y o f p i renzepine and a t ropine and h i s experiments i nd ica t ed s u b s t a n t i a l d i f f e rences between t h e inf luence of e i t h e r ant imuscarinic agent on g a s t r i c u l c e r and g a s t r i c s ec re t ion on one hand and on smooth muscle organs on t h e o the r . a n t i - u l c e r and a n t i s e c r e t o r y effects of p i renzepine approach those o f a t rop ine , s i g n i f i c a n t d i f f e rences a r e demonstrated i n i s o l a t e d muscle prepara t ions a f t e r ace ty lchol ine s t imu la t ion a s wel l a s af ter t ransmural s t imu la t ion . was 16.5 times weaker than a t rop ine i n reducing s a l i v a t i o n i n r a b b i t and is 100 times less e f f e c t i v e than a t rop ine i n doubling p a p i l l a r y diameter. Atropine caused a dose-dependent i n h i b i t i o n of t h e g a s t r o i n t e s t i n a l t r a n s p o r t while p i renzepine , even a t h igher doses , d id not cause a decrease but r a t h e r an increase . caused a dose-dependent i nc rease i n h e a r t r a t e which, however, could only be obtained with p i renzepine i n doses 10 t o 100 times g r e a t e r than with a t rop ine . Jaup -- e t a1 (131, 156) compared t h e effects of p i renzepine and R-hyoscyamine when taken i n equipotent doses i n i n h i b i t i n g g a s t r i c s e c r e t i o n . s e c r e t i o n but t h e i n h i b i t i o n by pirenzepine was s i g n i f i c a n t l y less than t h a t by R-hyoscyamine. Gastric emptying was s i g n i f i c a n t l y delayed by R-hyoscyamine compared t o pirenzepine. Swallowing induced eosphageal p e r i s t a l s i s was s i g n i f i c a n t l y i n h i b i t e d i n 51%. Pupi l s i z e and nea r po in t d i s t ance were s i g n i f i c a n t l y a f f e c t e d by R-hyoscyamine but not by pirenzepine. The minimal effects o f p i renzepine on t h e CNS have been a t t r i b u t e d t o t h e drug s e l e c t i v i t y and hydrophi l i - c i t y .

Although t h e

Pirenzepine

Atropine

Both drugs a f f e c t e d t h e s a l i v a r y

7.5 Mechanism of Action

I t i s gene ra l ly accepted t h a t p i renzepine is an an t imuscar in ic drug. Among o t h e r examples demonstrating t h i s , is the competi t ive i n h i b i t i o n by t h e drug of t h e carbachol effect on t h e spontaneously bea t ing guinea p ig atrium ( 1 5 7 ) , t h e s e l e c t i v e antagonism o f carbachol-s t imulated ac id s e c r e t i o n i n t h e perfused rat stomach while no such antagonism was no t i ced when his tamine o r


pen tagas t r in was used as a s t imulus (48), as well a s t h e s e l e c t i v e antagonism, by p i renzepine , o f t h e muscarinic response t o bethanechol on conscious c a t s with g a s t r i c f i s t u l a (157) and t h e f ind ing t h a t p i renzepine , i n g r e a t e r amount t h a t a t ropine , blocked only t h e muscarinic e f f e c t s o f ace ty l cho l ine without a l t e r i n g t h e n i c o t i n i c component (151). S imi l a r t o o t h e r an t imuscar in ic agents , pirenzepine a f f e c t s only t h e volume and a c i d output , and no t g a s t r i c pH (83). (158) have demonstrated t h a t p i renzepine (0.15 mg/kg, i . m . ) i n h i b i t s t h e motor response i n t h e human colon

S tud ie s on r ecep to r binding, pharmacological i n v e s t i g a t i o n on i s o l a t e d t i s s u e s and s t u d i e s i n man have shown t h a t p i renzepine d i s t i n g u i s h e s between d i f f e r e n t subc lasses of muscarinic r ecep to r s (83, 150, 159-170). Using p i renzepine a s novel t o o l t h a t r evea l s he te rogenei ty of muscarinic r ecep to r s , evidences have been provided (164-166) o f t h e ex i s t ence o f t h r e e d i s t i n c t i v e types of muscarinic r ecep to r s c l a s s i f i e d a s types A , B and C. With p i renzepine t h e log a f f i n i t y cons tan t f o r t h e A-type is about 7.7, f o r t h e B- type about 6.6 (10-fold weaker) and 4- fo ld f u r t h e r weaker f o r t he C-type. t h e log a f f i n i t y f o r a t rop ine i s about 8.5 t o 9 , i n d i c a t i n g t h a t t h e pharmacological response o f p i renzepine as compared t o t h a t o f a t r o p i n e w i l l be about 10-fold weaker a t t h e A-type, 100-fold weaker at t h e B-type and 400-fold weaker a t t h e C-type. potency of p i renzepine as well a s d a t a from binding s t u d i e s suggest t h a t i n t h e calf sympathet ic gangl ia t h e r e seems t o be predominantly A- r ecep to r s and i n t h e r a t sympathet ic gang l i a more of B-receptors . C-receptors , t h e conduction t i s s u e s i n t h e h e a r t conta in B-type r ecep to r s while t h e ileum seems t o have both type B and C r ecep to r s and t h e CNS both type A and B r ecep to r s . t h e r e i s good q u a n t i t a t i v e agreement between t h e binding ana lys i s and t h e pharmacological r e s u l t s (132, 155). Evidence f o r t h e he t e rogene i ty o f muscarinic r ecep to r s is a l s o presented (171-173) us ing t h e s e l e c t i v e gangl ionic r ecep to r agonis t

Baldi -- e t a1

t o a cho l ine rg ic s t imulus wi th prost igmine.

In t h e same system

The pharmacological d a t a a v a i l a b l e on t h e

The myocardium have predominantly

In most of t h e cases


McN-A-343 [ 4- (m-chlorophenylcarbamoyloxy) -2- butenyltrimethylammonium chlor ide] . The compound s e l e c t i v e l y st imulates the muscarinic receptors on ganglionic receptors with minimal influence on the muscarinic receptors i n the hear t and smooth muscle. Those receptors st imulated s e l e c t i v e l y by McN-A-343 have been named as M1-receptors and those st imulated by conventional muscarinic drugs are ca l l ed M2-receptors. gations a r e needed on pirenzepine and McN-A-343 t o determine which nomenclature t o follow. I t appears t h a t M I and M2 receptors correspond t o t h e A-type and C-type receptors respect ively. Pirenzepine may a l so have a cytoprotective e f f e c t i n addition t o i t s an t i s ec re to ry e f f e c t s (174, 175). Other mechanism f o r pirenzepine ulcer-heating e f f e c t s were a l s o reported (176).

Further i nves t i -

Acknowledgement

The authors would l i k e t o thank M r . Tanvir A. B u t t for typing t h i s manuscript.

494

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151. E . Kuhn, R. Honzak, A . Hnidkova, V . Brodan and M . Andel, Therapiewoche, - 27(9) , 1608, 1611, 1614, 1617, 1619 (1977).

152. G.P. Viscont i , D . Spo t t e , G . A . Grasso et a l , Curr . -- - Ther. Res., - 34, 853 (1983).

153. F. Krempler, F. Sandofer and A . Kalk , Wien Med. Wschr., 132, 427 (1982). -

154. E . Kuhn and R . Hanzak, Scand. J. Gas t roen t . , - 15(66) , 47 (1980).

155. R. Engelhorn, Scand. J . Gas t roen t . , - 17(72) , 49 (1982).

156. B .H. Jaup, R.W. Stockbrugger and G . Doteva l l , - Scand. J. Gas t roent . , - 17(72) , 119 (1982).

157. K.F. Sewing, Scand. J . Gas t roen t . , - 17(72) , 265 (1982)

158. F. Baldi , F . F e r r a r i n i , M . Cassan -- e t a l , Curr. Ther. Res., 33, 802 (1983). - -


159,

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

170.

171.

172 .

173.

N . J . M . B i r d s a l l , A.S.V. Burgen, R . Hammer e t a l , Stand. J. Gas t roent . , - 15(66), 2 7 (1980).

N . J . M . B i r d s a l l , A . S. V. Burgen, R . Hammer, E . C .

--

Hulme and J . Stockton, - Scani . J . Gastroent . , - 15(66) , 1 (1980).

R . Hammer, C .P . Ber r ie , N . J . M . B i r d s a l l , A.S.V. Burgen and E.C. Hulme, Nature, 283 , 90 (1980). -- E.C. Hulme, N . J . M . B i r d s a l l , A.S.V. Burgen and P . Mehta, Molec. Pharmac., - 1 4 , 737 (1977).

R. Hammer, Scand. J. Gas t roent . , - 15(66) , 5 (1980).

R . Hammer, Scand. J. Gas t roent . , - 17(72) , 59 (1982).

J . Stockton, N . J . M . B i r d s a l l , A.S.V. Burgen and E.C. Hulme, Scand. J. Gas toent . , - 17(72), 43 and 268 (1982).

_I_II_

J. Stockton, N . J . M . B i r d s a l l and R . Hammer, Scand. J. Gas t roent . , - 17(72) , 268 and 270 (1982).

D.A. Brown, A. Forward and S . Marsch, B r . J . Pharmac., 71, 362 (1980). - R. Hammer and F.W. KOSS, Scand. J. Gastoent . , 14 (57) , (1979).

S. Ber togl io , A . Anfossi, G . Arnulfo, P . Va len t in i and - - E . Berti R ibo l i , Scand. J . Gast roent . , g ( 7 2 ) , 179 (1982).

L. Bustos-Fernandez, J . A . Kofoed, E . Gonzalez and M. I . Ledesma-Paolo, Scand. J. Gas t roent . , z ( 7 2 ) , 95 (1982).

R .K. Goyal and S . J . Rat tan, J . C l in . Inves t . , L 55, 1119 (1975).

R . K . Goyal and S . J . Rat tan, Gastroenterology, - 74, 598 (1978).

H. Abrahamsson, Scand. J . Gas t roent . , c ( 7 2 ) , 7 (1982) .


174. S.J. Konturek, T. Brzoozowski, T. Radecki and I . P i a s tuck i , Scand. J . Gas t roen t . , - 17(72) , 225 (1982).

175. V. Rovat i , D. Foschi, F. F e r r a n t e , P . Del Solda to , S. D a n i o t t i and L. Varin, Scand. J . Gas t roen t . , - 17(72) , 261 (1982).

176. H. Kitagawa, K . Kurahashi, M. Fujwara and K . Kohe, Arzneim. Forsch. , - 28, 2 1 2 2 (1978).

STREPTOMYCIN

J a b e r S . Mossa, Abdul Hameed U . Kader Taragan,

and Mahmoud M . A . Hassan,

Jabe r S . Mossa, Assoc ia te P ro fes so r o f Pharmacognosy, Col lege of Pharmacy, King Saud Unive r s i ty , Riyadh, Saudi Arabia .

Department of Pharmacognosy, Col lege of Pharmacy, King Saud Unive r s i ty , Riyadh, Saudi Arabia .

Mahmoud M.A. Hassan, Professor of Medicinal Chemistry, Department of Pharm. Chem., Col lege of Pharmacy, King Saud UNiversi ty , Riyadh, Saudi Arabia.

Abdul Hameed U . Kader Taragan, Lec turer of Pharmacognosy,



508

1. History

JABER S . MOSSA ETAL.

2. Description

2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2.4 Appearance, Color, Odor and Taste 2.5 pH Range.


3.1 Melting Range 3.2 Solubility 3.3 Loss on hying 3.4 Optical Rotation 3.5 Crystal Structure 3.6 Spectral Properties

4. Production of Streptomycin

5. Synthesis of Streptomycin

6. Biosynthesis of Streptomycin

7. Pharmacology and Therapeutic Category

8. Pharmacokinetics

8.1 Absorption 8.2 Distribution 8.3 Excretion 8.4 Half -life

9. Toxicity

10. Dosage

11. Pharmaceutical Forms

12. Mechanism of Action


STREPTOMYCIN 509

13.1 Elemental Composition 13.2 Identification 13.3 Titrimetric Methods 13.4 Gasometric 13.5 Electrometric 13.6 Spectroscopic Methods 13.7 Chromatographic Methods 13.8 Bio-Assay Methods. 13.9 Biochemical Methods.

Acknowledgement

References

510 JABER S . MOSSA ETAL.

1. HISTORY

Streptomycin is a water soluble organic base biosynthesised by appropriate strain of the actinomycetes , SXkepXornqcU g h i n m . Prior to the development of Penicillin Waksman and his collaborators began to study the production of antibiotic substances in the department of microbiology of New Jersey Agricultural Experiment Station, Rutgers University in 1939 (1). In the succeeding years they examined some thousands of actinomycetes, hundreds of fungi and many bacteria and extracted from their culture media a number of antibacterial substances. Particular attention was devo- ted to obtaining substances which acted prominently on gram negative bacteria. As a result of this work, the organism producing streptomycin was isolated from heavily manured soil and from a chicken's throat (2,3). The first publication on streptomycin was made by Schatz, Bugie and Waksman (3) in 1944. mycin from 1944-1952 was recorded by Waksman in "The Litera- ture on Streptomycin" (4). The cultural descriptions and the nutritional requirement of ShepXornqcu g h i n w are also discussed by Waksman ( 5 , 6 ) . The early promising clinical reports of 1944 based on crude Streptomycin prepared in the Merck Research Laboratories showed the importance of the crude drug and started an intensive development program for its large-scale production. The general line of development of the work on streptomycin have closely followed those of the work on penicillin. Rapid progress in the study of streptomycin was greatly helped by the experience gained with penicillin and by the fact that there already existed organizations for large-scale manufacture and exploitation of the latter.

A complete bibliography of the work on strepto-

2. DESCRIPTION

2.1 Nomenclature

2 .11 Chemical Names

a) O-2-Deoxy-2-(methylamino)-a-L-glycopyranosyl- (1 + 2)-0-5-deoxy-3-C-formyl-a-L-lyxofuranosyl- (1 + 4) - N , N ' -bis (aminoiminomethyl) -D-strepta- mine ( 7 ) .

b) 4-0-(2-0-(2-Deoxy-2-methylamino- a -L-glycopyranosyl)-5-deoxy-3-C-formyl-a-L-lyxofuranosyl)-N N' -diamidino -D -streptamhe (8).

STREPTOMYCIN 511

c) 2,4-Diguanidino-3,5,6-trihydroxycyclohexyl- 5-deoxy-2-0-(2-deoxy-2-methylamino-a -L-glucopyranosyl ) -3 -formy1 -a - L 1 yxopent anofurano - side (9).

d) N-Methyl-L-glucosamidinostreptosidostrep- tidine (10).

e) 2 - (N-methyl -a-L-glucopyrano saminido) - 3-C- forinyl-5-desoxy-L-aldopentofuranose (11) .

2.12 Generic Name

Streptomycin A. (12)

2.13 Trade Names

a) Estreptomicina b) Streptomycinum c) Streptomyseen d) Streptomycini e) Streptovex f) Stryzolin g) Crystamycin h) Dimycin i) Oroject NS j) Streptopen

2.2 Formulae

2.21 Empirical

a) Streptomycin = C H N 0 21 39 7 12 b) Streptomycin sulphate = (C21H39N,012)23H2S04

c) Streptomycin Hydrochloride =

C21H39N7012.3HC1.

d) Streptomycin calcium chloride =

(C21H39N7012. 3HC1) CaC12

512 JABER S. MOSSA ETAL.

2 . 2 2 Structural

MH NH- C-NH2

0

H3C NH

Dl HO

Streptomycin i s an o p t i c a l l y ac t ive , t r i a c i d i c base, C21H39N7012; possessing an aldehydic carbonyl group. The high proportion of oxygen molecule is c h a r a c t e r i s t i c of a carbohydrate. Streptomycin [ l ] is made up o f t h ree components : St rep t id ine [ Z ] , Streptose [ 31, and N-methyl- L-glucosamine [4 ] , linked together by glycosidic bonds (16).

STREPTOMYCIN

I NH

513

Streptomycin

P I

5 14 JABER S. MOSSA ETAL.

2.23 Structural elucidation

The difficult task of elucidating the structure of streptomycin was carried out principally by four groups of workers led by K. Folkers, 0. Wintersteiner, H.E. Carter and M.L. Wolfrom. Their work has excellently reviewed for several times (11, 17-21). The structural elucidation of streptomycin is based on studies o f its various degradation products.

1) On acid hydrolysis, the weaker glycosidic bond in streptomycin between streptidine and streptose splits to give streptidine [21 and streptobiosamine 151 (16,22-26). 2) Mild methanolysis o f streptomycin [ 11 with methanolic hydrogen chloride gives streptidine [ 21 and methyl streptobiosaminide dimethyl acetal hydrochloride [61. captan and hydrogen chloride gives ethyl thio- streptobiosaminide diethyl mercaptal hydrochloride [ 7 1 which is also obtained by the action of ethyl mercaptan and hydrogen chloride on streptomycin. By Raney-nickel and subsequent acid hydrolysis of [71 , it gives N-methyl-L-glucosamine [4] and bisdeoxy-streptose 181 (27-28). 3) Hydrolysis of streptomycin xith N1 sodium hydr- oxideofor three minutes at 100 at 40 yields a weakly acid substance, which has been characterized as maltol [9! (29).

Treating [6] with ethyl mer-

or eighteen hours

qCH3 OH

0

The degradation reactions of streptomycin is well summarized in Scheme I.


Streptidine

Streptidine 121, ~8~18~604, is a meso Form of 1,3- diguanido-2,4,5,6-tetrahydrocyclohexane (24,25, 30, 31). confirmed by synthesis from D-glucosamine (32,33).

The structure of streptidine [21 was

Streptobioasamine

The structure of Nitrogen containing disaccharide, streptobiosamine [51 was determined by a study of hydrolysis products of the methyl ether and the ethyl thio-ether formed by the action of hydrogen chloride in methanol and ethyl mercaptan respectively (27, 29, 34, 35). The hydrolysis of methyl-streptobiosaminide dimethyl acetal gave N-methylglucosamine 141 (27,28). The structure of N-methyl-L-glucosamine has been established by synthesis from L-arabinose (36). Because of its instability, it is very difficult to isolate streptose [ 31 , the second component of streptobiosamine [51. The molecular formula CgH1005 was calculated from the known formulae of streptomycin 111 , Streptidine [ 2 ] and N-methyl-L-glucosamine [41 and its structure was deduced by identifying oxidation and reduction products. shown to be 3-C-formyl-5-deoxy-L-lyxose, a pentose containing the reactive aldehyde group associated with streptomycin molecule (29,37-40). The structure of streptomycin [I] was finally confirmed by total synthesis (Scheme 11).

It was finally

2.24 CAS Registry Number

1) Streptomycin (57-92-1) 2) Streptomycin sulphate (3810 - 74-0) 3) Streptomycin Hydrochloride (6160-32-3)

2.25 Wiswesser Line Notation

Streptomycin sulphate :

T60TJ BlQ CQ -DQ EM1 FO- DT50TJ B CV H CQ EO - AL6T J BQ CQ DMYZ UM EQ EMYZU M WSQQ 3. (41)

STREPTOMYCIN 517

2.26 Stereochemistry and Absolute Configuration

The chemistry of the component fragments of streptomycin was thoroughly studied and their structures were established (23-40). The absolute configuration of streptomycin has been elucidated by chemical (applying Reeve's Copper complexing method to various degradation products of streptomycin, dihydrostreptomycin and streptobiosamine) (42,43) and optical correlations (44). The absolute stermch emistry of streptose was established to be (R) at C-4 and (S) at C-2 (37,40,45). The streptidine was shown to possess all mrn configuration (46). form, the asymmetric attachment of streptobiosamine to it causes each of the ring carbon of streptidine in streptomycin to be asymmetric. The asymmetry of streptidine ring in streptomycin is 1(R), 2(R), 3(S), 4(R), 5(R), 61s). The streptobiosamine moiety of streptomycin has been shown to be attached to position 4 of streptidine in either R or S absolute configuration (46-48). The three units of the streptomycin are attached together by glycosidal bonds which were shown to be formed by condensations between : (i) the C-2 hydroxyl of streptose and C-1 hydroxyl

of N-methyl-L-glucosamine (28,38,39,49-51). (ii) C-1 hydroxyl of streptose and a hydroxl

group of streptidine that is adjacent to only one guanidino group (i.e., position 4)

Although streptidine is a meso

(22,38,39,52-54) . All glycosidic linkages have an a-S configuration which was confirmed by NMR and crystallographic studies (44, 55-57). By using the configurational assignment, the structure of streptomycin in complete stereochemical details may be written as indicated below :

518 JABER S. MOSSAETAL.

YH2 C=NH I N-H

HOCH2

- \, YH2

HO H NH

- 11- L - I

I I I OH


a) Streptomycin = 581.6 b) Streptomycin sulphate = 1457.4 c) Streptomycin hydrochloride = 691.0 d) Streptomycin calcium chloride = 1492.9 (8)

2 . 4 Appearance, Color, Odor and Taste

Streptomycin occurs as white to slightly pink o r pale brownish powder or granules; odor: odorless o r with a slight cdor; taste : slightly bitter. Streptomycin is hygroscopic and may deliquensce on exposure to air but not affected by air or light ( 1 3 ) .

2 . 5 PH Range

A 25 per cent w/v solution of streptomycin has a pH between 5 and 7.

STREPTOMYCIN

3 . PHYSICAL PROPERTIES

519

3 . 1

3 .2

3.3

3.4

Melting Range

I n d e f i n i t e (12).

So 1 ub i 1 it y

1) Streptomycin : I t i s very so lub le i n water ; almost in so lub le i n a lcohol ( 9 5 % ) , chloroform, e t h e r and petroleum e t h e r ( 1 3 ) .

2) Streptomycin su lpha te : I t i s very s o l u b l e i n water; p r a c t i c a l l y in so lub le i n a lcohol acetone, chloroform, e t h e r and petroleum e t h e r (8) .

3) Streptomycin hydrochlor ide : I t is very so lub le i n water, p r a c t i c a l l y i n s o l u b l e i n a lcohol , chloroform and e t h e r (8) .

4) Streptomycin calcium ch lo r ide : I t is very s o l u b l e i n water, p r a c t i c a l l y in so lub le i n a l coho l , chloroform and e t h e r (8).

Loss on Drying

Strephomycin l o s e s when d r i e d over phosphorus pentoxide a t 60 C and a t a p r e s s u r e no t exceeding 5 mm. o f mercury, f o r 3 hours, no t more than 5 p e r cent (13).

O m i c a l Rotat ion

Streptomycin is o p t i c a l l y a c t i v e with lev0 r o t a t i o n . The fol lowing o p t i c a l r o t a t i o n s were repor ted f o r t h e var ious s a l t s o f s t reptomycin ( 7 , 1 2 ) :

Solvent 25 [a1 D

1. Streptomycin hydrochlor ide . -84: Water (C = l . O ) 2 . Streptomycin calcium chlor ide . -76 Water (C -1.0)

The s p e c i f i c r o t a t i o n €or s t reptomycin su lpha te was determined i n our l abora to ry on a Perkin E l m e r Pola- r ime te r , Model 241 MC and found t o be [a125 - 75' i n water (C = 1 . O ) . D


3.5 Crys ta l S t ruc tu re

Neidle, e t a1 (57) have repor ted t h e X-ray c r y s t a l l o - graphic a n a l y s i s of s t reptomycin oxime s e l e n a t e . S t r ep - mycin oxime selenate', C21H40Ng012 [ 1%H2Se04], 4H20

c r y s t a l l i z e s from aqueous methanol as monoclinic needles , s ace groupoC2, a = 17.10, b = 14.36, c = 16.13 B, B = 108 , D = 1.54 g . ~ m - ~ , Dc = 1.55 g . cm-3 f o r four formula u n f t s pe r c e l l . The [OOl] pro- j e c t i o n of one streptomycin oxime ion i n selenate c r y s t a l i s shown i n Fig. 1.

3 .6 Spec t r a l P rope r t i e s

3 .61 U l t r a v i o l e t spectrum

The UV spectrum of s t reptomycin su lpha te i n water was scanned from 200 t o 400 nm on DMS 100 Varian AG Spectrophotometer (Fig. 2 ) . Three maxima were observed a t 241.6, 270 and 328.2 nm. Streptomy- c i n i n 0.2N su;phuric a c i d , showed a maxima a t 280 nm wi th E l 4 = 0.2 (9) .

1 cm 3.62 In f r a red spectrum

The i n f r a r e d spectrum of s t reptomycin su lpha te a s K B r d i s c recorded on a Pye Unicam i n f r a r e d spectrophotometer is shown i n F ig . 3.

The s t r u c t u r a l assignments have been c o r r e l a t e d with t h e fol lowing f requencies (Table 1) :

Table 1 : I R C h a r a c t e r i s t i c s of Streptomycin Sulphate .

Assignment -1 Frequency cm

3100-3500 NH and OH s t r e t c h (vb).

1630- 171 0 Y I

-C=O and -C=NH s t r e t c h (vb) . 1480 (vb) * 1100-1200 C-0-C-stretch (vb) *

vb = Very broad.

Other r epor t ed I R d a t a 41) a r e 3330, 1670, 1470, 1390, 1110 and 1040 cm- 5 .

STREPTOMYCIN 521

Fig. 1 : [OOlI Pro jec t i on o f one Streptomycin oxime i o n i n t h e Selenate Crys ta l .

Fig. 2 : UV Spectrum of Streptomycin Sulphate in Water.

80 t

1 I 1 1

2000 1800 1600 1400 1200 1000 800 I

Fig. 3 : IR S p e c m ~ a of Streptomycin Sdphrte as K k diw.

STREPTOMYCIN 523

3 . 6 3 Nuclear Magnetic Resonance Spectra

1 3 .631 H-NMR Spectra

1 The (F ig . 4) were recorded on a T60A MHz N M i spectrometer with TMS as in t e rna l reference. The proton chemical s h i f t s a r e shown i n

H-NMR spectra of streptomycin i n D 0

Table 2 . NH

8

1 I

CH OH 7* 3

Table 2 : PblR Character is t ics Sulphate

o f Stremomycin

Chemical Group. s h i f t .

D20 H-1 N-methylglucosamjne 5 . 5 3

H - 1 Streptose 5 . 3 0

3 -ti Formyl s t r ep tose 5 . 0 6

3 . 8 3

3 . 5 6

2 - N Me of N-methyl glucosamine. 2 . 8 6

4 - Sec-Me of Streptose. 1.23

1 1 ' , I . I . . . . I . I I . I . . . , I . . . . 1 . . . . 1 . . . , 1 .

, I I 1 I . I I 5.0 m (4) 4.0 3.0 Z.0 1.e 0

1 . . . . 1 , 8.0 r.0 6.0

I Fig. 4 : H-NMW Spectrum of Streptomycin Sulphatr iii I)$) and TMS.

STREPTOMYCIN 525

Table

Carbon No.

3.632 "C-NMR Spec t r a

The n a t u r a l abundance C-13 NMR noise-decoupled and s i n g l e frequency off-resonance decoupled (SFORD) s p e c t r a (Fig. 5 and Fig.6) i n deuterium oxide were obtained on a J o e l FX 90 - 90-MHz inst rument . The carbon chemical s h i f t s were assigned on t h e b a s i s o f t h e theo ry of chemical s h i f t and SFORD s p l i t t i n g p a t t e r n (Table 3 ) .

1 2 3 4 5 6 7 8 1' 2 ' 3 ' 4 ' 5 ' 6 ' 1" 2'7 3" 4" 5" 6" 7''

3 : Carbon Chemical S h i f t s of Streptomycin Sulphate .

Chemical S h i f t 6 ppm M u l t i p l i c i t y

57.06 87.13 60.82

63.11 61.52

160.33 160.80 108.61 o r 97.11

71.92 84.89 80.67 34.81

186.31 108.61 o r 97.11

64.50 73.33 74.21 80.14 75.97 15.20

108.61 o r 97.11

d d d d d d S

S

d d

d 4 d d d d d d t 4

S

Fig. 5 : 13C-NMR Noisc-decoupkd Spectrum of Streptomycin SuIphate in D20 and TMS.

hl i 1

Fig. 6 : 13C- NMR Off-Resonance Spectrum of Streptomycin Sulphate in D 2 0 and TMS.


160.33 (s)

57.06 (d)

87.13(d)

160 .8 (5)

63. i 1 (d) 108.61 o r

97.11 (d)

97.11 (d)

H2N - C - HN 0

60.82(d) H

0

80.14 (d) 0

64.05 (d)

74.21 (d)

73.33 (d)

STREPTOMYCIN 529

4 . PRODUCTION OF STREPTOMYCIN

Streptomycin i s produced by t h e micro-organism b;trreptomyca g h h w . I t i s a l s o produced by o t h e r s t r a i n s of organisms l i k e Acfinomyca cj1obhppohu.b (58) , pink v a r i a n t of bRtreptu- mqca g h i n e u n (591, bAh.epXumycu g d b u (60). However, f o r l a r g e s c a l e product ion , appropr i a t e s t r a i n of baheptomyca g&mb i s used.

SXtepXomycu g & e u can b iosynthes ize s t reptomycin i n a medium conta in ing glucose, sodium c i t r a t e and ino rgan ic salts , but much higher y i e l d s are obta ined by us ing t h e media conta in ing a l s o an organic source of n i t rogen such a s meat e x t r a c t , yeas t au to lysa t e , Soybean f l o u r , corn s t e e p l i q u o r o r fermentat ion so lub le s (61-88). One o r more of t hese a r e used f o r i n d u s t r i a l b iosyn thes i s . Tre- mendous amount of work has been done on t h e product ion of s t reptomycin (64 , 89-103) .

The l a rge - sca l e product ion has been accomplished by e i t h e r su r face c u l t u r e o r deep c u l t u r e process (64,65) . The deep c u l t u r e process i s more o f t e n used f o r l a rge - sca l e pro- duc t ion o f s t reptomycin. o f s t reptomycin i s well summarised below (104):

A b r i e f o u t l i n e of t h e product ion

a) Product ion of Spore Suspension

The spores of h%%eptumqcu g h i n ~ . ~ ~ requi red f o r inocula- t i o n i s obtained from r a p i d l y growing c u l t u r e on s o l i d medium (64, 105) . The spores are e i t h e r suspended i n s t e r i l e water o r i n skimmed m i l k which i s inocula ted w i t h t h e medium i n t h e shake f l a s k s .

b) Prepara t ion of Medium

A l l t h e s o l i d c o n s t i t u e n t s of t h e c u l t u r e medium a r e placed i n t h e ferinenter through t h e charge hole and water i s added t o make up t h e volume. The medium i s s t e r i l i z e d by t h e d i r e c t i n t roduc t ion of steam. S t e r i l i z a t i o n i s c a r r i e d o u t a t 15 l b p re s su re f o r 2 5 minutes. Af te r shu t - t i n g o f f t h e steam, water i s passed through t h e j a c k e t and an a i r pressure o f 10 l b i s The sitrrer is working dur ing s t e r i l i z a t i o n , cool ing and ferment a t ion .

maintained i n t h e fe rmenter .

c) Prepara t ion o f t h e inoculum

The s t e r i l i z e d medium i n t h e seed tank (115 L . c apac i ty , provided with st irrer and inoculum measuring tank) is


Flow Sheet 1 : The production of Streptomycin.

STREPTOMYCIN 53 1

5.

inocula ted wi th t h e conten t of shake f l a s k . After t h e inocu la t ion , t h e niycelium i s grown f o r 36 hours wi th an a e r a t i o n r a t e of 100 L p e r minute. After 36 hours , a heavy growth of mycelium is obta ined . o f t h i s mycelium suspension is blown i n t o t h e measuring tank and f i n a l l y t o t h e fermenters through p i p e - l i n e .

d) Fermentation

The fermenters a r e made up of s t a i n l e s s s tee l , having t h e capac i ty of 15000 ga l lons and provided with s i t r r e r , a n t i - foam nozzle , a i r sparger and a i r o u t l e t . After t h e inoculum from t h e seed tank has been added t o t h e fermen- t e r , a e r a t i o n i s s t a r t e d a t once. Then t h e fe rmenta t ion i s c a r r i e d ou t a t an optimum temperature of 25 -30 C . Foaming dur ing fermentat ion is con t ro l l ed by t h e add i t ion of ant i foaming agents . The f i n a l concent ra t ion of s t r e p - tomycin (50,000 u n i t s p e r l i t r e ) w i l l be reached i n 72 hours . shee t I .

About twenty l i t e rs

0 0

The product ion of s t reptomycin is given i n flow

e) I s o l a t i o n and P u r i f i c a t i o n

The s t reptomycin from t h e c u l t u r e f l u i d i s separa ted by adsorp t ion on t h e charcoal and by subsequent e l u t i o n wi th methanol ic hydrochlor ic ac id o r o t h e r s u i t a b l e s o l v e n t s (106-117).

Fur ther p u r i f i c a t i o n i s c a r r i e d o u t by chromatography on alumina (118-122) o r ion exchange resins (123-164) and by p r e c i p i t a t i o n wi th var ious base p r e c i p i t a n t s (165-179). Solvent e x t r a c t i o n techniques (180-188), e l e c t r o d i a l y s i s (189) and e lec t roosmot ic (190) procedures a r e a l s o employed.

SYNTHESIS OF STREPTOMYCIN

Although s t reptomycin was t h e f i rs t aminoglycoside a n t i - b i o t i c t o be d iscovered , i t was not synthes ised u n t i l some 30 years l a t e r . The t o t a l s y n t h e s i s of s t reptomycin was achieved by Sumio Umezawa e t a l , i n 1973 (191,192). The r e a c t i o n s involved a r e i n shown i n Scheme I 1 €j summarized below:

Condensation of blocked d e r i v a t i v e o f d ihydro-s t rep tose (benzyl a-L-dihydro-streptose)[lO] with t h e glucosamine d e r i v a t i v e (L-glucopyranosyl b romide ) [ l l ] i n benzene i n t h e presence of mercuric cyanide, followed by hydro- l y s i s of t h e S c h i f f base w i t h 50% a c e t i c ac id and

532

SCHEME I1

JABER S . MOSSA ETAL.

STREPTOMYCIN 533

1111

PhCOO phcg-J 0

0

1191

PhCOO


PhCOO phcol&j 1221

NH

NHCNH2 I1 0 NHCNHz tlH

I OH

STREPTOMYCIN 535

reaction with methyl chloroformate (Na2Cog, aq. ace-

tone) gave the protected streptobiosaminide[ 121. N- Methvlation of [121 gave [131 Deacetylation followed by deisopropylidenation (IN HC1, Aqueous MeOH) gave [ 141 which was identical with benzyl a-S-dihydrostreptobio- asminide. Hydrolysis of 1141 with 10% Ba(OH)2 affored benzyl a-L-dihydro-Streptobiosaminide [ 151 . Treatment of [ 151 with 2,2-dimethoxypropane (p-toluenesulfonic acid) gave a diisopropylidene derivative and further blocking by use of p-ni t rophenoxycarbonylchlor ide (NaOH, aqueous acetone) gave [ 161. Partial hydrolysis of [161 (25% AcOH in MeOH) gave the diol 1171, This was converted into the dibenzoyl derivative [18]. Partial hydrolysis of [181(75% Acetic acid) removed the isopropylidene group giving a diol which was transformed (p-N02C6H ococ 1, [19] : Hydrogenofysis of the glycoside linkages (Palladium Black-dioxane) gave free sugar 1201. The reaction of [201 with thionyl chloride at room temperature offered the a-glycosyl chloride [211. Direct treatment of [ 151 with p-nitrophenoxycarbonyl chloride o r phosgene followed by benzolyation also gave [191. But the yield is very poor.

Finally condensation of [211 with di-N-acety1-di-N- benzyloxycarbonyl -0-cyc lohexyl idenes t rep t id ine gave the 4-0-glycoside [22]. Hydrolysis of 1221 with 0.05 N Ba(OH2) and dioxane successively removed the carbamate, carbonate, benzoyl and acetyl groups. Removal of the remaining protecting groups with 50% acetic acid followed by hydrogenolysis with Palladium black gave dihydrostreptomycin [23] . Subsequent mild oxidation of [23] afforded streptomycin[l] .

pyridine) into the carbonate

6. BIOSYNTHESIS OF STREPTOMYCIN

Biosynthesis of streptomycin has been quite extensively studied. Streptomycin is formed from glucose as a primary precursor (193). Biosynthesis of the components of streptomycin, streptidine, streptose and N-methyl-glucosamine are well summarised by Horner (193) E Snell (194). General indication of the metabolic relationships of glucose L241 to the various components of streptomycin is shown in Scheme 111.


SCHEME I11

Tranunnular

O<QH Icarranttemcnt. - H,yo3n on on O H

HO

Mvoinotitol

STREPTOMYCIN 537

Experiments with labe led compounds demonstrated t h a t t h e s p e c i f i c a l l y l abe led glucose [ 2 4 ] i s converted i n t o s t r e p - tomycin [ l ] i n which each of t h e sub-uni t s i s labe led a t t h e corresponding carbon (Scheme IV). This was e s t a b l i s h e d by seve ra l groups (195-203). Some of t h e in t e rmed ia t e s i n t h e b i o s y n t h e t i c pathway of s t reptomycin have been i s o l a - ted ,but some o f t h e key in t e rmed ia t e s a r e not ye t de t ec t ed . Recent s t u d i e s e s t ab l i shed t h e presence o f s t r e p t i d i n e - 6 - phosphate and dihydro-streptose-dTDP, i n t h e s t reptomycin b iosyn the t i c pathway (204). Some in t e rmed ia t e s between glucose and d ihydros t r ep tose a r e known as a r e s u l t o f t h e work of Grisebach (205) who demonstrated t h a t [14C-] - glucose-dTDP was conver t ed to 4-Keto4,6-dideoxyglucose-dTDP which i n t u r n was converted t o dihydrostreptose-dTDP.

Nothing i s known ye t regard ing t h e in te rmedia te between glucose and N-methylglucosamine. Recently Kniep and Grisebach (206) e s t a b l i s h e d t h e r o l e of d ihydros t rep tosy l - s t r e p t i d i n e 6-phosphate [25] i n t h e b iosyn thes i s of s t r e p - tomycin. They concluded t h a t t he nucleotide-bound N-methyl- 4-glucosamine moiety i n S. g h i n w was c a t a l i z e d and t r a n s - f e r r e d t o d ihydros t rep tosy l s t r e p t i d i n e 6-phosphate [25] which i n t u r n s was converted t o dihydrostreptomycin 6- phosphate [26] . Fur ther ox ida t ion of [26] gave s t r e p t o - mycin-6-phosphate[27] (207) hid hydro lys i s y i e l d s t r e p t o - mycin [ l ] (208). The r e a c t i o n i s summarized i n Scheme V .

7 . PHARMACOLOGY A N D THERAPEUTIC CATEGORY

Streptomycin i s an aminoglycoside a n t i b i o t i c which is a c t i v e aga ins t a wide range of b a c t e r i a , both Gram p o s i t i v e and Gram negat ive (209). I t i s a va luable t h e r a p e u t i c agent f o r a l l forms of t u b e r c u l o s i s (210-252). S t r ep to - mycin i s e f f e c t i v e aga ins t Tularemia (253,254). I t i s h igh ly s p e c i f i c and one of t h e most e f f e c t i v e agent f o r t h e t reatment o f a l l forms of plauge (255). I n combina- t i o n wi th Penici l l in G , it i s considered t o be t h e drug of choice i n i n f e c t i o n s wi th SfiepXococcU vh idavm and EvLtmococcu (256) . I t i s a l so e f f e c t i v e aga ins t h&phy- Lococcud uLbw (257).

Streptomycin i s a drug of second choice i n i n f e c t i o n s due t o suscep t ib l e s t r a i n s of anaerobic bfiCLp,&jCOC&, bactero- i d e s , AmobacXm aehogena, A . d e c a l h , S h i g e l L a , BhuceRea and Vibh,Lo carnnin and i n combination wi th t e t r a c y c l i n e , i n P o t l ; t e ~ i n f e c t i o n s and i n b r u c e l l o s i s (256,258-264). Since s t reptomycin i s e f f e c t i v e aga ins t H. i n d h e n z a e , E. CULL, K&bbLelLa pneurnanhe and S&andl?a, i n combination wi th

SCHEME IV NH

NHCNH I I NH

II

*

STREPTIDINE

I 0

HO i-koH 0 - . H3C #q* OH STREPTOSE

OH

CH20H N-METHYL-

# CH3NH * L-GLUCOSAMINE HQ ~241

OH [ 1 1

STREPTOBIO- SAMINE

STREPTOMYCIN

SCHEME V

539

1251

H $ N M nvo O M 1261

OH I l l


o t h e r a n t i b i o t i c s it i s use fu l i n t h e t reatment o f r e s p i - r a t o r y t r a c t i n f e c t i o n s , b a c t e r i a l men ing i t i s and u r i n a r y t r a c t i n f e c t i o n s (256,265-273). Streptomycin i s a l s o e f f e c t i v e f o r typhoid f eve r (274-277) , whooping cough (278) , d i p h t h e r i a (279) and i n murine leprosy (280-283).

8 . PHARMACOKINETICS

8 .1 Absorption

Streptomycin is absorbed poorly and i r r e g u l a r l y from t h e g a s t r o i n t e s t i n a l t r a c t (255,284). Therefore , t h e o r a l r o u t e i s not s u i t a b l e f o r admin i s t r a t ion of s t r e p - tomycin f o r systemic a c t i o n . However, t h e o r a l r o u t e has been recommended i n t h e t rea tment of i n t e s t i n a l d i s o r d e r s due t o s t reptomycin s e n s i t i v e s t r a i n s of b a c t e r i a o r t o reduce t h e b a c t e r i a l f l o r a of t h e i n t e s t i n e p r i o r t o opera t ion on i n t e s t i n a l t rac t . Streptomycin i s r ap id ly absorbed when adminis tered by subcutaneous, in t ramuscular o r in t ravenous r o u t e s (255,256,285). Streptomycin can a l s o be absorbed through rectum when adminis tered as cocoa b u t t e r o r carbowax mixture (286,287). P a r e n t r a l admin i s t r a t ion must be employed t o ensure t h e r a p e u t i c a l l y adequate plasma and t i s s u e l e v e l s of s t reptomycin. After an in t ramuscular dose of 1 g of streptomycinipeak serum concent ra t ions of 25 t o 50 mg/ml a r e a t t a i n e d i n 0.5- 2 hours , f a l l i n g t o 5 t o 10 mg/ml a t 12 hours (9, 15 ) . The e f f e c t i v e minimum serum concent ra t ion i s about 7 mg/ml (15) .

8 .2 Di s t r ibu t ion

No metabol i te of streptomycin has so far been de tec t ed . About one t h i r d t h e c i r c u l a t i n g st-reptomycin i s bound t o serum pro te in . Streptomycin i s widely d i s t r i b u t e d t o a l l organs except b ra in .

The drug pene t r a t e s r a p i d l y i n t o t h e p e r i t o n e a l , p e r i - c a r d i a l , p l e u r a l and synovial f l u i d s . enters i n t o cerebrospina l f l u i d except i n meningi t i s (256,285,288,289) ,

Very l i t t l e

8.3 Excret ion

About 50-80% of s t reptomycin i n j e c t e d i s excre ted i n u r i n e unchanged i n 24 hours (15,285,290,291). Most of t h e o r a l dose i s e l imina ted i n t h e f aeces (15,256). About 0.5% is excre ted i n t h e b r e a s t m i l k i n 24 hours and 1% is excre ted i n b i l e (15,286).

STREPTOMYCIN 541

8 . 4 H a l f - l i f e

Serum h a l f - l i f e o f s t reptomycin i s about 2 .5 hours i n young a d u l t s . This w i l l be increased i n t h e new born and i n a d u l t s above 40 yea r s . In r ena l func t ion impairment, t h e h a l f - l i f e may be increased t o 2-5 days (15) *

9 . TOXICITY

Al l e rg i c r e a c t i o n s a r e r e l a t i v e l y very common with t h e admin i s t r a t ion of s t reptomycin. toms a r e eos inoph i l i a , fever and sk in r a shes . The drug a l s o causes headache, nausea, a r t h r a l g i a , b l u r r i n g of v i s i o n , v e r t i g o , r ena l i r r i t a t i o n and neuro tox ic i ty . The most s e r i o u s t o x i c effect i s damage t o t h e e igh th c r a n i a l nerve, causing deafness which i s permanent. Damage t o r ena l t ubu le s , bone marrow and l i v e r r a r e l y occurs a t t h e usual t h e r a p e u t i c t i s s u e l e v e l . Applicat ion of s t r e p t o - mycin d i r e c t l y t o t h e peritoneum o r i t s in t ravenous i n j e c - t i o n i n l a r g e doses may cause dea th by p a r a l y s i s of t h e r e s p i r a t i o n (285,286,288, 292-303).

The usua l a l l e r g i c symp-

10 . DOSAGE

About 0 .5 t o 1 gram by in t ramuscular i n j e c t i o n d a i l y o r a t long i n t e r v a l s . A s an i n t e r n a l a n t i s e p t i c , 500 mil l igrams by mouth every e i g h t hours (304).

11. PHARMACEUTICAL FORMS

1) Streptomycin su lpha te U.S.P. (305). 2) Streptomycin su lpha te i n j e c t i o n B .P . (304) . 3) Streptomycin calcium ch lo r ide B.P . (8) *

4) Streptomycin hydrochlor ide B . P . (8) - 1 2 . MECHANISM OF ACTION

The a n t i b a c t e r i a l a c t i o n of s t reptomycin i s a consequence o f i t s combination wi th t h e 30s ribosomes of s u s c e p t i b l e organisms, thereby d i s t o r t i n g t h e t r a n s l a t i o n of t h e gene t i c code. As a r e s u l t o f t h i s condi t ion t h e wrong amino a c i d s a r e incorpora ted i n t o t h e nascent po r t ion of t h e p r o t e i n molecule, t h e p r o t e i n syn thes i s i s d i s rup ted and t h e c e l l d i e s (256, 306, 307).

542

13. METHODS OF ANALYSIS

JABER S. MOSSA ETAL.

13.1

13.2

Elemental Composition

c = 43.7% N = 16.86% H = 6.76% 0 = 33.01% ( 7 ) .

I d e n t i f i c a t i o n

13.21 Pharmacopeial Tes ts

The fol lowing t e s t s have been descr ibed i n B r i t i s h Pharmacopoeia f o r t h e i d e n t i f i c a t i o n of s t reptomycin (304).

a ) Dissolve about 0 .2 g o f s t reptomycin i n a mixture o f 2 m l o f methyl a lcohol and 0.1 m l o f su lphur i c ac id , f i l t e r i f necessary and allow t o s tand a t about 250; c r y s t a l s of s t r e p t i d i n e su lpha te sepa ra t e i n t h e course of 2 o r 3 days. Dissolve t h e c r y s t a l s i n 10 ml of ho t water conta in ing 0.1 g o f t r i - n i t rophenol and coo l . The melt ing po in t o f t h e p r e c i p i t a t e a f t e r r e c r y s t a l l i s a t i o n from hot water i s about 283%.

b) Boil a small quan t i ty of s t reptomycin wi th 1 N sodium hydroxide f o r a few minutes and add a s l i g h t excess of hydrochlor ic ac id and a few drops o f f e r r i c ch lo r ide t e s t s o l u t i o n ; a b r i l l i a n t v i o l e t c o l o r i s produced.

13.22 Color Tes t s

The fol lowing co lo r tes ts have been descr ibed f o r t h e i d e n t i f i c a t i o n o f s t reptomycin :

a) Glucosamine Test

i ) A 10 mg of s t reptomycin i s d isso lved i n 1 m l o f water, 2 m l o f 1 N hydrochlor ic a c i d is added and t h e s o l u t i o n is heated on a steam ba th f o r 10 minutes. Then 2 m l of 2 N sodium hydroxide and 1 m l o f 20% aqueous ace ty lace tone a r e added. The s o l u t i o n i s again heated f o r 5 minutes and cooled. About 1 m l o f e thanol and 25 m l o f concentrated hydrochlor ic a c i d

STREPTOMYCIN 543

a r e then added. produced (308).

i i ) To about 2 m l o f an aqueous s o l u t i o n of s t reptomycin, 1 m l of a 2% s o l u t i o n o f ace ty lace tone i n water and 1 m l o f 1 N sodium hydroxide a r e added. i s heated f o r 10 minutes i n a b o i l i n g water ba th and then cooled. i s developed on add i t ion o f 2 m l o f a s o l u t i o n o f E h r l i c h ' s aldehyde (309).

A cher ry r ed c o l o r i s

The mixture

A pink co lo r

b) Guanido Test

i ) A 10 mg o f s t reptomycin i s d i s so lved i n 1 m l o f water and 1 m l of ox id ized n i t r o - pn r s s ide reagent i s added ( t h e reagent i s prepared by mixing equal volumes of 10% sodium n i t r o p r u s s i d e , 10% potassium f e r r i c y a n i d e and 10% sodium hydroxide, i n t h a t o rde r ; a f t e r t h e c o l o r becomes b r i g h t green-yellow, 1 m l i s d i l u t e d t o 100 m l wi th wa te r ) , a r ed c o l o r develops (310).

i i ) Aqueous s o l u t i o n of s t reptomycin when t r e a t e d wi th d i a c e t y l i n t h e presence of oxygen g ives a pink co lo r which i s i n t e n s i f i e d by t h e add i t ion o f l-naph- t h o l (311) .

i i i ) When s t reptomycin i s t r e a t e d with l-naph- t h o l and sodium hypobromite, a red co lo r i s produced (312,313).

i v ) When s t reptomycin i s t r e a t e d with sodium hypobromite i n s t rong a l k a l i n e s o l u t i o n , a v o l a t i l e bromo amine i s produced which t u r n s a c i d i f i e d s t a r c h iod ide s o l u t i o n s t o dark b lue c o l o r (314).

c) Other c o l o r T e s t s

i ) An a l c o h o l i c s o l u t i o n o f s t reptomycin g ives a pink c o l o r wi th su lphur i c a c i d (315).


ii) An alcoholic solution of streptomycin produces a yellowish-red color with phosphoric acid (315).

iii) With Sakaguchi solution (100 mg o f boric acid in 10 ml of concentrated sulphuric acid) streptomycin gives pale-green after 3-5 minutes (315).

13.23 Microbiological Tests

a) Test with Streptococcus lactis

Streptomycin when treated with milk inoculated with SRhepAococcud ,f!ucLh, prevents the coagulation of the milk in the usual way (316) .

b) Triphenyltetrazolium Chloride (TTC) Test

When streptomycin is treated with a culture of SR/LepAocaccw t h m o p k i e e u d on a sterile milk medium containing triphenyltetrazolium chloride, prevents the usual color change from leucoform to red (317,318).

c) Water-blue Test

A dilute solution of streptomycin prevents the formation of the blue color when treated with a culture of KLeebtA.eRea pfiewnoniae containing water blue dye (319).


13.31 Aqueous

Delaby and Stephan (320) have reported an iodimetric method for the determination o f streptomycin. The method is as follows :

Dissolve about 0.7 to 1 g of Streptomycin in 15 ml of water, add 15 ml of Nessler's reagent and 1 ml of 10% potassium iodide. 20 ml of water, neutralize with 2N hydrochloric acid and add 1 ml in excess. Add 15 ml of 0.1N iodine solution. Shake and titrate the excess of iodine with 0.05N sodium thiosulphate solution.

Then add

STREPTOMYCIN 545

13

An iodometr ic method involv ing t h e r e a c t i o n of s t reptomycin with sodium hyphobromite has been a l s o repor ted (321). The method i s a s fol lows :

The sample conta in ing s t reptomycin su lpha te i s added t o 5-10 m l b a s i c sodium hyphobro- mi te s o l u t i o n and a i r is swept success ive ly through t h e sample and 2 U-tubes each con- t a i n i n g 5 m l o f 0.1N su lphur i c a c i d and 0.1-0.2 g potassium iod ide . Af t e r sweeping a i r (10-15 bubbles/sec) through t h e system €or about 15 minutes , t h e l i b e r a t e d iod ine i s t i t r a t e d with 0.005N sodium th iosu lpha te . Analysis o f 4-8 mg por t ions of 90.2% pure s t reptomycin su lpha te showed va lues of 89.2, 88.7 and 88.4%.

Huttenrauch and Keiner (322) have descr ibed a t i t r imet r ic method f o r t h e determinat ion o f s t reptomycin su lpha te . fo l lows :

The method i s a s

The sample conta in ing about 50 to 150 mg o f s t reptomycin su lpha te is d isso lved i n 25 m l o f water ; 2 m l o f 1 N ,sodium hydroxide i s added and t h e mixture is heated a t l ooo f o r 5 minutes. Af t e r cool ing, it is a c i d i - f i e d wi th 1 N su lphur i c a c i d , then d i l u t e d t o 100 m l wi th water and about 0 . 1 m l o f 2% f e r r i c c h l o r i d e s o l u t i o n is added. The s o l u t i o n i s set a s i d e f o r 7 minutes and then t i t r a t e d with 0.01N c e r i c su lpha te u n t i l t h e c h a r a c t e r i s t i c red co lour i s discharged. This method can a l s o be employed f o r t h e de te rmina t ion of s t reptomycin i n c u l t u r e media (323).

Modif icat ion of Huttenrauch and Keiner method by r ep lac ing c e r i c su lpha te s o l u t i o n with po t - assium permanganate s o l u t i o n , was r epor t ed by Chandra e t a1 (324). This modified method g ives r e s u l t s wi th sharper end p o i n t .

32 Non-aaueous

Penau e t a1 (325) have descr ibed a nonaquous method of de te rmina t ion o f a n t i b i o t i c s inc luding


st reptomycin. tomycin su lpha te is d isso lved i n p e r c h l o r i c a c i d and 2 m l of e thyleneglycol . Then about 0.05M benzidine so lu t ion i n acet ic a c i d i s added. After 10 minutes, 2 drops of i n d i c a t o r (1% thymolphthalein i n e thanol o r 0 .5% t h i a z o l e yellow i n e thanol ) is added and t h e excess of pe rch lo r i c ac id i s t i t r a t e d with ac id potassium p h t h a l a t e s o l u t i o n . l m l o f 0.1N H C 1 0 4 ac id i s equiva len t t o 581.6/30 mg of s t reptomycin base.

The s p e c i f i e d amount of S t r ep -

Gaut ie r and Pe l le r in (326) have repor ted a non- aqueous method of es t imat ion o f su lpha te of t h e organic bases l i k e s t reptomycin, dihydro- s t reptomycin, and o t h e r s by d i r e c t t i t r a t i o n of p e r c h l o r i c ac id i n g l a c i a l a c e t i c ac id . procedure i s a s fol lows :

To a 0.1N s o l u t i o n of t h e sample i n g l a c i a l a c e t i c ac id , add s u f f i c i e n t benzidine (as 0.05M s o l u t i o n i n g l a c i a l acet ic ac id ) t o p r e c i p i t a t e 95 % of t h e su lpha te ; a l low t h e p r e c i p i t a t e t o s e t t l e and t i t r a t e t h e superna tan t l i q u i d with 0.1 N p e r c h l o r i c ac id i n g l a c i a l a c e t i c a c i d .

The

13.4 Gasometric

Gasometric de te rmina t ion of s t reptomycin was r epor t ed by Yamagishi and Yokoo ( 3 2 7 ) and Viala (328). Pure s t reptomycin i n s o l u t i o n o r i n complex p repa ra t ions o r i n a s soc ia t ion wi th p e n i c i l l i n s , are determined by measuring t h e volume of n i t rogen evolved by s t r e p t i - d ine f r a c t i o n c.f tlie molecule under t h e a c t i o n o f sodium hypochlor i te o r sodium hypobromite and compar- i ng it with t h a t f r eed by r e fe rence s o l u t i o n s of guanidine hydrochlor ide o r guanidine a c e t a t e .

13.5 Elec t romet r ic

13.51 Polarographic

Streptomycin is r educ ib le a t t h e dropping mercury e l ec t rode . A po larographic a n a l y s i s has been developed (329) . The concent ra t ion - d i f f u s i o n current curve i s l i n e a r f o r concen- t r a t i o n s from 0 .1 t o 1 .0 mg/ml. The s o l u t i o n o f s t reptomycin i n 3% t e t ramethyl ammonium

STREPTOMYCIN 541

hydroxide is polarographed at 13.6O; the Eh is 1.45 volts vs a mercury electrode. The rate of decomposition o f streptomycin in alkaline solution was polarographically studied by Bricker and Vail ( 3 3 0 ) .

Goodey et a1 (331) has described a polarographic method for the assay of streptomycin in fermenter broth and associated recovery stages. A Tinsley Mark 19 pen recording polarograph is used, with mercury capillaries of drop times between 2 and 3 seconds at 50 cm height. Lithium hydroxide is used as base electrolyte.

Doan and Riedel (332) have reported a polarographic method for the estimation of streptomycin sulphate.Solutions containing 100-400 y /ml were used for the determination. The pH of the solution was adjusted to 1 2 . 4 by adding 2 ml of 1 N sodium hydroxide. oxygen was removed by bubbling nitrogen through the sample f o r 5 minutes after that the surface was flushed with nitrogen. A mercury drop rate of 4-5 seconds was maintained. For all concentrations the half wave potential (E%) was 1 . 2 8 - 1 . 2 9 volts. Plotting the diffusion current against concentration resulted in a straight 1 ine .

The dissolved

Cunha (333) has published a similar polarographic method f o r determination of streptomycin sulphate. The limit o f identification of streptomycin sulphate was 0 . 5 8 - 7 2 . 8 7 y/ml.

Caplis et a1 (334) have reported a method f o r determination o f antibiotics including streptomycin by alternating-current polarography.

A survey of the applications of polarographic methods in the production control of antibiotics including streptomycin was described by Weissbuch and Unterman (335 ,336) .

1 3 . 5 2 Amperometric

Streptomycin forms water insoluble salt with various anionic dyes. amperometric estimation of streptomycin (337) .

This can be used €or


The method is based on the precipitation of streptomycin with a trivalent dye which is synthesised by coupling diazotized para rosaniline with 1-naphthol-4-sulphonic acid. About 1-3 mg of streptomycin is dissolved in 5 to 10 ml of 0.02M triethylamine citrate buffer, pH 2.8 and is titrated with standard solution of the dye. The potential is maintained at -0.08 volts.

Konopik (338) has described the application of metal salt solutions to amperometric determination of organic compounds including streptomycin.

13.53 Potentiometric

Machek (339) has carried out a potentiometric titration for the determination of streptomycin in mixed preparations. follows :

The method is as

Mix the sample with 15 ml of water and 20 ml of butyl acetate and adjust to pH 2.1 by addition of 2N hydrochloric acid and shake for five minutes. Extract a 1 ml aliquot of the filtered upper layer with 10 ml of phosphate buffer solution (pH 7.2) and determine the penicillin in the extract iodime- trically or microbiologically. Mix the filtered lower layer with 20 ml of butyl acetate, adjust to pH 9.5 by addition of 2N sodium hydroxide, add Barium chloride (2ml) and shake for 1 minute; centrifuge, separate and dilute the aqueous phase to 50 ml, adjust the pH of a 20 ml aliquot to 5.8 by addition of 2N hydrochloric acid and titrate the streptomycin potentiometrically with 0.1N NaOH to pH 9.6.

A potentiometric microbiological assay for the antibiotic substances including streptomycin was proposed (340). The method for the determination of tetracycline hydrochloride with suspension of E b C h d C k i d cof i and a potentiometric C02 sensor (HNC Model 10-22-00 or an Orion Model 95-02) has been extended to the determination of streptomycin. Sample solutions containing 2-4 pg ml-1 of streptomycin

STREPTOMYCIN 549

was used. wi th range 0.33 t o 16.67 pg m l - 1 .

The c a l i b r a t i o n graph was rect i l inear

13.6 Spec t roscopic Methods

13.61 Color imet r ic Methods

13.611 The maltol Method

When s t reptomycin is heated i n d i l u t e a l k a l i n e s o l u t i o n it forms mal to l (29). Maltol r e a c t s with ferr ic ions t o g ive a pu rp le co lo r (304) and with t h e Fol in-Cioca l teu phenol reagent it produces a b lue co lo r (341). This f a c t may be used f o r t h e assay o f streptomycin.

A co lo r ime t r i c assay involv ing t h e mal to l formation, f o r t h e es t imat ion o f s t r e p - tomycin i s descr ibed i n t h e European Pharmacopoeia (342). The procedure i s as fol lows :

Dissolve 0 . l g o f s t reptomycin i n water and d i l u t e t o 100.0 m l with t h e same so lven t . To 5 . 0 m l o f t h i s s o l u t i o n add 5.0 m l o f 0.2N sodium hydroxide and hea t f o r e x a c t l y 10 minutes i n a water ba th . Cool i n ice f o r exac t ly 5 minutes, add 3 m l of a 1 .5 per cent w/v s o l u t i o n of fe r r ic ammonium su lpha te i n 0.5N s u l - phur ic a c i d and s u f f i c i e n t water t o produce 25 m l and mix. minutes a f t e r t h e add i t ion of t h e f e r r i c ammonium sulphate , measure t h e e x t i n c t i o n of a 2 c m l a y e r a t about 525 nm, us ing a s t h e compensation l i q u i d a s o l u t i o n prepared i n t h e same manner, omi t t i ng t h e substance t o be examined. The e x t i n c t i o n i s not less than 90.0 pe r cen t o f t h a t ob ta ined by ca r ry ing ou t t h e ope ra t ions s imultaneously, us ing s t reptomycin su lpha te CRS ins t ead o f t h e substance t o be examined. Ca lcu la t e t h e percentage with r e fe rence t o t h e subs- t ance t o be examined and t h e streptomy- c i n su lpha te CRS, both d r i e d f o r 4 hours

Exact ly 20


at 60' over phosphorus pentoxide a t a p re s su re not exceeding 5 Torr .

Pharmacopoeia of United S t a t e s , 1955 (343) descr ibed a similar mal to l c o l o r i - metric assay f o r de te rmina t ion o f s t r e p - tomycin i n s t rep toduocin €or I n j e c t i o n , U.S.P.

Boxer e t a1 (344) have repor ted a s i m - p l e and r ap id co lo r ime t r i c assay f o r streptomycin i n c l i n i c a l p repa ra t ions , u r i n e and broth. The method is based on t h e formation of mal to l from s t r e p t o - mycin, s epa ra t ion of t h e maltol from t h e i n t e r f e r i n g subs tances by ex- t r a c t i o n with chloroform and subsequent determinat ion by e i t h e r phenol reagent o r ac id f e r r i c ammonium su lpha te .

Eisenman and Bricker (345) have d e s c r i - bed a co lo r ime t r i c method €or t h e e s t i - mation of s t reptomycin i n which t h e mal to l i s separa ted from t h e r e a c t i o n mixture by steam d i s t i l l a t i o n . The method i s success fu l ly appl ied f o r t h e determinat ion of s t reptomycin i n fermen- t a t i o n bro ths , concent ra tes and c l i n i c a l samples. streptomycin a r e p re sen t t h e colorime- t r i c r eac t ion wi th ferr ic ion is employed and t h e c o l o r is read a t 550 nm. I f less thean 500 u n i t s a r e p re sen t , t h e phenol reagent i s used and t h e co lor is read a t 775 nm. The s tandard curves prepared from pure s t reptomycin are l i n e a r .

I f more than 500 u n i t s of

Bagdasarian and Michalska (346) have r epor t ed the maltol method f o r determina t ion of s t reptomycin i n u r i n e without e x t r a c t i n g t h e s t reptomycin p r i o r t o t h e assay, provided t h a t dosage was mode- r a t e . This method is recommended f o r r o u t i n e use, but cannot be appl ied t o determining s t reptomycin i n concentra- t i o n below 100 y/ml.

STREPTOMYCIN 551

Doery and Mason (347) and Kartseva e t a1 (348) separa ted s t reptomycin from o t h e r fe rmenta t ion products by ion exchange chromatography on c a t i o n exchange r e s i n before conver t ion i n t o ma l to l . F ina l de te rmina t ion of s t reptomycin was done by Maltol method.

Bruns e t a1 (349) , have repor ted a colorimetric method f o r t h e de te rmina t ion of s t reptomycin from t h e bro th . The a n t i - b i o t i c s are c o l l e c t e d from t h e s o l u t i o n a t pH 8 . 3 - 8 . 5 on s i l ica ge l and extrac- t e d i n su lphur i c a c i d . determined by mal to l method.

The conten t i s

Desai e t a1 (350) have descr ibed a d i r e c t method f o r e s t ima t ion of s t r e p - tomycin i n fermenter b ro th sample. b ro th sample was depro te in ized by t h e add i t ion o f 4 m l of t r i c h l o r o a c e t i c a c i d , f i l t e r e d cent r i fuged and d i l u t e d . After t h e add i t ion of 2 m l of 2N sodium hydroxide t o 5 m l o f t h e b ro th , t h e mixture was heated i n a b o i l i n g water b a t h f o r t h r e e minutes and cooled. The absorbance was read af ter add i t ion of f e r r i c ammonium su lpha te . run concurren t ly , cons i s t ing of t h e same sample without hea t .

F e r r a r i e t a1 (351) have adapted t h e mal to l method f o r automated a n a l y s i s of s t reptomycin i n fermentat ion media us ing d i a l y s i s t o sepa ra t e mal to l from t h e i n t e r f e r i n g subs tances . The ma l to l t h u s separa ted was automat ica l ly t r e a t e d wi th stream of fe r r ic ch lo r ide i n 2.2N hydrochlor ic ac id and t h e e x t i n c t i o n of t h e r e a c t i o n product at 530 nm was auto- m a t i c a l l y recorded. E r ro r s were claimed t o be less than with convent ional manual methods.

The

A blank was

Fur ther development of t h e au to-ana lyzer method f o r eva lua t ion of s t reptomycin were descr ibed by Shaw and Fortune (352) . They claimed t h a t high p rec i s ion could


be maintained even wi th cons iderable s i m p l i f i c a t i o n t o a l low f o r h igh through- put of samples.

Iodimetric-absorptiometric method was adopted by Sauciuc and Ionescu (353) f o r micro-determination o f s t reptomycin i n fermentat ion l i q u i d . The method i s a s fol lows :

The sample conta in ing 25-150 Ug of s t reptomycin is heated with 0.1N sodium hydroxide f o r 4 minutes. i s then ad jus ted t o pH 7.5 by adding 0.055N phosphoric ac id then t r e a t e d wi th f r e s h l y d i l u t e d 0.001N iod ine i n potassium iod ide s o l u t i o n . After 5 t o 10 minutes 5 m l of reagent A (300 m l water, 200 m l acetic ac id ,6g potassium bromide and l m l hydrochlor ic ac id ) is added followed by a s t rong j e t of 1 m l of reagent B (100 m l water, 0.72g Sulpha- ni lamide 0.02 g N-naphthyl-ethylenediamine d ihydrochlor ide and 5g hydroxylammonium c h l o r i d e ) . After 10 minutes t h e e x t i n c t i o n of t h e s o l u t i o n is measured aga ins t t h e blank s o l u t i o n which is s i m i l a r l y t r e a t e d .

The s o l u t i o n

Krystyna (354) has repor ted t h e mal to l co lo r ime t r i c method €or t h e determina- t i o n of s t reptomycin r e s idues i n milk. A n t i b i o t i c - f o r t i f i e d milk samples were cooled, d e f a t t e d by c e n t r i f u g a t i o n , depro te in ized with z inc su lpha te i n t h e presence of barium hydroxide, concen- t r a t e d and aga in d e f a t t e d wi th l i g h t petroleum. Then t h e sample was t r e a t e d with sodium hydroxide and heated on t h e water ba th . The mal to l formed was ex t r ac t ed i n t o chloroform from t h e a c i d i c medium and back-extracted i n t o water a t a l k a l i n e pH. The aqueous e x t r a c t was mixed wi th Fol in Cioca l teu phenol reagent , a f t e r 10 minutes t h e absorbance was measured a t 660 nm and r e f e r r e d t o a c a l i b r a t i o n graph.

STREPTOMYCIN 553

The maltol method has been extensively used in studies of stability (355) and in the analysis of streptomycin in pharmaceutical preparations (356), feeds (357) and urine (358). Minor modifications have been suggested by Borowiecka (359), Kartseva and Bruns (360) and Wahbi et a1 (361).

13.6 12 Guanido reaction Method

Both streptomycin and dihydrostreptomycin form orange-red color with oxidized nitroprusside reagent (310,362). Based on this principle Valedinskaya (363) has described a colorimetric method for estimation o f streptomycin. The procedure is as follows :

10 ml of oxidized nitroprusside reagent is added to 10 ml of aqueous sample solution containing approximately 0.1 mg of streptomycin o r dihydrostreptomycin. After 3 minutes the absorbance is determined at 490 nm against a reagent blank prepared by substituting 10 ml of water for the sample aliquot. is prepared with the appropriate compound following the same procedure. The color is stable for about 4 hour.

A working curve

Vieira and Pereira (364) have adapted the nitroprusside method for the determination of streptomycin in urine. The urine sample is treated with acetone and centrifuged after 30 minutes. Then the precipitate is stirred with water and filtered o r centrifuged. To an aliquot of the clear solution,water is added to bring to 2.5 ml, then treated with nitroprusside reagent an3 the absorbance is measured.

Halliday (311) described the application of the modified Voges-Proskauer reaction to streptomycin, in which a pink color is generated by the reaction of the guanidine moiety with 3iacetyl and


1-napthol. Quantitative analytical procedure was developed by Szafir and Bennett (365), Ashton et a1 (366) and Banerjee (367). The method is as follows :

Transfer 2 ml of the sample solution to a test tube, add 15 ml of water and then 1 ml of 0.4 per cent diacetyl solution, 1 ml of 20% potassium hydroxide solution and 1 ml of 10% solution of 1-naphthol in anhydrous ethanol, in that order. inversion after each addition. later determine the extinction at 525 MI in a 1 cm cell against water. graph from suitable dilutions of a standard streptomycin solution, using the same procedure for color development as described for the sample. Determine the potency of the diluted sample solution from the standard graph. A standard graph should be prepared for each determination to allow for differences in room temperature, reagents, and so on.

Mix the contents of the tube by Forty minutes

Prepare a standard

Schulz and Posada (368) have developed a colorimetric method involving guanidino group reaction for determination of streptomycin. The aqueous sample solution is treated with an ethanolic reagent containing diacetyl and naphthalene-2,7-diol; ethanolic sodium hydroxide is added and the mixture is set aside at room temperature for 45 minutes. The extinction is then measured at 555 nm and the streptomycin content is calculated with reference to a standard solution containing 50 1.18 of streptomycin sulphate per ml.

The Sakaguchi reaction (312,313) has been adapted by Buck et a1 (369) to the analysis of streptomycin and dihydrostreptomycin, using spectrophotometric determination of the red color formed by the reaction of 1-naphthol and sodium hypobromite with the antibiotics.

Natarajan and Tayal (370) have reported a rapid method of colorimetric estimation of streptomycin, based on the Sakaguchi reaction. The procedure i s as follows :

STREPTOMYCIN 555

Mix an aqueous s o l u t i o n of t h e sample wi th 1 m l o f 10% sodium hydroxide, and 2 m l o f 0.02% e t h a n o l i c 1-naphthol. After 15 minutes , add 1 m l of sodium hypobramite reagent and 10 m l o f c a r b o n t e t r a c h l o r i d e . Shake well, and add 5 m l o f abso lu t e e thano l , shake aga in and measure t h e e x t i n c t i o n of t h e aqueous phase a t 530 nm i n a 1 cm cuve t t e with a reagent blank. from microbio logica l method.

The r e s u l t s were comparable wi th those

The a p p l i c a t i o n of t h e Sakaguchi r e a c t i o n f o r t h e co lo r ime t r i c de te rmina t ion o f streptomy- c i n was a l s o repor ted by Fontes (371) .

Modif icat ion of t h e above method by us ing 8- hydroxyquinoline was descr ibed by T i b a l d i (372).

S z i l a g y i and Szabo (373) have repor ted a simi- la r co lo r ime t r i c method f o r t h e a n a l y s i s o f Sakaguchi-posi t ive a n t i b i o t i c s , us ing N-bromo- succinimide. The method i s as fol lows :

5 m l O f t h e s o l u t i o n o f t h e sample is t r e a t e d wi th 2 m l o f 1-naphthol reagent . shaken and set a s i d e f o r 3 minutes, t h e mix- t u r e is r a p i d l y poured i n t o 0.5 m l o f N-bromo- succinimide s o l u t i o n , t h e mixture i s shaken v igorous ly and 1 m l o f 40% urea s o l u t i o n is added i n 15 second. A v iv id red co lour deve- l ops , t h e e x t i n c t i o n o f which is read i n 5 minutes a g a i n s t blanks, with a b lue f i l t e r , r e s u l t s being r e f e r r e d t o s tandard curves. The r e l a t i v e e r r o r of t h e method i s f 4%.

After being

13.613 Glucosamine r e a c t i o n Method

The glucosamine t e s t f o r t h e i d e n t i f i c a t i o n of s t reptomycin us ing a c e t y l acetone and E h r l i c h ' s reagent , can be used q u a n t i t a t i v e l y (309). Based on t h i s p r i n c i p l e , Kamata e t a1 (374) have descr ibed a co lo r ime t r i c assay f o r s t r e p - tomycin. The procedure of t h e method is as fol lows :


To 4 m l of s t reptomycin s o l u t i o n , add 1 m l o f 2.5N su lphur i c ac id . Boil f o r 1 hour, cool , n e u t r a l i z e and d i l u t e t o 10 m l . To about 2 m l o f t h i s s o l u t i o n add 5.5 m l o f reagent A (2.225 m l ace ty lace tone , 113.75 m l 2 N sodium carbonate 34.1 m l 1N hydrochlor ic a c i d and water) , s toppe r , b o i l f o r 20 minutes, cool , add 10 m l . o f e thanol , f i n a l l y add reagent B (0 .8 o f Ehrlich'saldehyde, 30 m l o f concen- t r a t e d hydrochlor ic a c i d and abso lu te e thano l ) . Measure t h e i n d e n t s i t y o f t h e co lo r a t 532 nm. The method is r e l i a b l e only f o r samples o f high p u r i t y .

The a c e t y l acetone-Ehrlich's aldehyde method has been adapted by Masquelier (375) f o r t h e d e t e r - minat ion o f s t reptomycin i n u r ine . The u r i n e sample conta in ing s t reptomycin i s t r e a t e d wi th lead a c e t a t e , t h e excess o f lead is removed by sodium su lpha te and f i l t e r e d . To about 1 m l o f t h e f i l t r a t e 1 m l o f a c e t y l acetone reagent and 2 drops of sodium hydroxide a r e added. The s o l u t i o n i s heated i n a water ha th f o r 10 min- u t e s and cooled. 2 m l o f theEhrl ich 's aldehyde reagent is then added and t h e red co lo r developed i s compared wi th a s tandard s o l u t i o n of s t reptomycin and with a blank of u r i n e sample.

13.614 Other Methods

The aldehyde group of s t reptomycin was exp- l o i t e d by Marshall e t a1 (358) who developed a q u a n t i t a t i v e procedure based on t h e forma- t i o n of a colored semicarbazone with 4-[4- (p-chlorophenazo) -1 -naphthyl ] semicarbazide. The excess of t h e reagent i s ex t r ac t ed wi th chloroform and hydrochlor ic a c i d i s added. The c o l o r developed i s measured a t 580 nm.

Savi tskaya and Kartseva (376) used 2 ,4-d in i - t ropheny lhydraz ine which reacts wi th s t r e p - tomycin t o form a yellow hydrazone. excess reagent is e x t r a c t e d wi th buty l acetate and t h e concent ra t ion o f t h e s t reptomycin is determined from c a l i b r a t i o n curve prepared with pure s t reptomycin.

Mattick (377) has repor ted a co lo r ime t r i c method f o r t h e de te rmina t ion o f a n t i b i o t i c s

The

STREPTOMYCIN 557

i nc lud ing s t reptomycin i n m i l k which is based on t h e i n h i b i t i o n of t h e reduct ion of n i t r a t e t o n i t r o u s oxide by Miwiacoccus pqkogcnu. The n i t r o u s oxide produced i s determined colo- r i m e t r i c a l l y a f te r d i a z o t i s a t i o n wi th sulpha- n i l i c a c i d and coupl ing with l-naphthylamine.

B a r i l a r i and Katz (378) have repor ted a method us ing copper t a r t r a t e and phosphomolybdic ac id reagents f o r co lo r ime t r i c de te rmina t ion o f s t reptomycin. The f r e s h l y prepared s o l u t i o n o f streptomycin su lpha te is heated f o r 8 min- u t e s wi th 2 m l of water and 2 m l of copper t a r t r a t e so lu t ion . molybdic ac id i s added and t h e b lue c o l o r developed is read pho toco lo r ime t r i ca l ly .

A photocolor imet r ic method f o r t h e determina- t i o n of s t reptomycin wi th r e inecka te sa l t was descr ibed by B a r i l a r i et a1 ( 3 7 9 ) . St rep to - mycin i s heated with water a t 40° and equal volume o f ammonium re inecka te s o l u t i o n is added. The p r e c i p i t a t e t hus formed is sepa- r a t e d , washed with ethanol and d isso lved i n acetone water mixture . Then acetone is evapora ted . The s o l u t i o n i s then t r e a t e d with sodium hydroxide, d i l u t e d wi th water and neu- t r a l i z e d with n i t r i c a c i d . 5 m l o f t h i s so lu - t i o n i s t r e a t e d with 5 m l of ferric n i t r a t e and t h e concent ra t ion of s t reptomycin i s d e t e r - mined c o l o r i m e t r i c a l l y . The a p p l i c a t i o n o f t h i s method i n t h e pharmaceutical a n a l y s i s was repor ted by Basu and Dutta (380).

After cool ing t h e phospho-

Color imet r ic de te rmina t ion o f aldehyde and ketones with o x a l i c a c i d dihydrazide was ada- p ted by Bartos and Bur t in (381) f o r determina t ion o f s t reptomycin. About 1 m l of t h e sample i s t r e a t e d with 2 m l o f phosphate-c i t - r a t e - b o r a t e b u f f e r and 2 m l of o x a l i c ac id d ihydraz ide reagent . The mixture i s allowed t o s t and f o r 4 hours . The b lue o r v i o l e t c o l o r r e s u l t i n g i s read c o l o r i m e t r i c a l l y .

Using p i c r i c a c i d reagent , Agrawal and Dutta (382) have developed a r ap id co lo r ime t r i c a s say method f o r s t reptomycin i n mixture wi th d ihydros t r ep tomyc in . The test sample and


the blank are treated with 3 ml of 0.1% picric acid,2 ml of 5% sodium hydroxide; heated for 3 minutes, cooled diluted to 10 ml and the extinction is measured at 500 nm. Dihyd- rostreptomycin does not give this color reaction, but can be determined after being converted into streptomycin.

Yavors'kli (383) has reported the application of dinitrobenzene and their derivatives as reagent in colorimetric analysis of organic medicinal substances including streptomycin.

A new spectrophotometric method for the assay of streptomycin, by salt formation with bromothymol blue was reported by Nour El-Deen et a1 (384).

Barilari et a1 (385) have reported a new photo- colorimetric method for the determination of streptomycin. Streptomycin solution is treated with potassium iodate solution and sodium arsenite solution. After the disappearance of the yellow color, 0 .03 ml of phenol and 4 ml of sulphuric acid are added, boiled, cooled and the absorbance is read at 490 nm.

A spectrophotometric method for the estimation of streptomycin by salt formation with tropaeolin 000, was reported by Nour El-Deen et a1 (384) and Beltagy et a1 (386). The method is as follows :

To about 3 ml of sample in water add about 1 ml of cold, dilute sulphuric acid, and 3 ml of cold aqueous solution Tropaeolin 000. Mix and cool in ice for one hour, centrifuge, decant and wash the precipitate with 10 n i l of cold dilute sulphuric acid. Dissolve the residue in 2 ml of sodium hydroxide, adjust to 100 ml with water. Dilute a mixture of 5 ml of the solution and 1 ml of dilute sulphuric acid to 50 ml and measure the extinction at 485 nm.

A new sensitive colorimetric determination of streptomycin by thiobarbituric acid method was proposed by Erno (387). The method is

STREPTOMYCIN 559

s p e c i f i c and s e n s i t i v e . fol lows :

The procedure is a s

The s o l u t i o n conta in ing s t reptomycin i s mixed wi th 0.1M potassium i o d a t e i n 0.05 m l o f 0.5M su lphur i c a c i d , a f t e r 15 minutes, a 2% s o l u t i o n o f sodium a r s e n i t e i n 5 m l hydrochlor ic ac id i s added. When t h e brown co lo r i s disappeared, 1 m l o f t h i o b a r b i t u r a t e reagent is added. The mixture i s heated f o r 10 minutes and then t h e e x t i n c t i o n of t h e s o l u t i o n i s measured a t 415 nm a g a i n s t a blank.

The modif icat ion of t h i o b a r b i t u r i c a c i d method was repor ted by Erno e t a l . (388) and Fukumoto Hisako (389).

Alykov (390) has descr ibed an absorp t iomet r ic method f o r t h e de te rmina t ion of amino g lycos ide a n t i b i o t i c s i n b i o l o g i c a l f l u i d s . Streptomycin and o t h e r amino g lycos ides form a complex with s t i lbazochrome P i n a c e t a t e o r c i t r a t e b u f f e r medium. The blood sample is t r e a t e d with 1 m l o f water and 0 . 5 m l o f t r i c h l o r o a c e t i c a c i d s o l u t i o n and cen t r i fuged . l i q u i d 1 m l of s t i lbazochrome P reagent i n c i t r a t e bu f fe r s o l u t i o n , 1 m l o f 1 mM PrC13 and 6 ml of e thanol added. After 20 minutes t h e mixture i s cen t r i fuged and t h e absorbance o f t h e superna tan t l i q u i d is measured a t 560 nm.

To t h e superna tan t

A new and s e l e c t i v e spectrophotometr ic d e t e r - mination o f s t reptomycin using O-hydroxyhydro- quinonephthalein [2',3',6',7'-tetrahydroxyspiro (isobenzofuran; 1 ( 3 H ) , 9 ' - (9H) xanthen) -3-one) ] and manganese, was repor ted (391). The method

involves formation of a t e r n a r y complex i n a medium conta in ing 40% of methanol and 1% of polyvinylpyrro l id inone . Measurements a r e made a t 570 nm a t pH 10 t o 1 0 . 3 .

A photometr ic method f o r t h e de te rmina t ion of s t reptomycin and o t h e r aminoglycoside a n t i b i o - t i c s was proposed by Alykov (392). The method involves t h e measurement of t h e decrease i n t h e absorbance a t 575 nm, due t o Acid b lack S caused by p r e c i p i t a t i o n o f a complex of C . 1 Acid Black 24 with t h e a n t i b i o t i c s .


13

Eriochrome the photometric determination of streptomycin and other antibiotics in biological fluids (393).

Slack T was used as a precipitant in

Ninhydrin spectrophotometric method has been adapted f o r the determination of aminoglycoside antibiotics in pharmaceutical preparations (394). About 10 l.11 portions of the solutions of streptomycin in water were applied to precoated Silica gel plate; the chromatogram was developed in a solution of l g of ninhydrin in 50 ml of 95% ethanol-anhydrous acetic acid (5:l). transferred to test tubes and shaken with 5 ml of propan-2-01 containing 5 drops of ammonium hydroxide and the tubes were centrifuged. The absorbance spectrum of supernatant solution was recorded in the range 400 to 800 nm against water.

After drying the colored bands were

Divakar et a1 (395) have reported a spectrophotometric method for the determination of streptomycin, tetracycline and chloramphenicol using brucine and sodium periodate. About 10 ml of streptomycin solution was boiled under reflux f o r 45 minutes with 10 ml of 2M hydrochloric acid and the excess of the acid was removed in vacuo. The residue was dissolved in 20 ml of warm water and diluted to 50 ml. About 0.5 to 3.0 ml of the solution was treated with 3 ml of SmM-brucine, 1.5 ml of 5m.M.sodium metaperiodate and 2 ml of 2.3M.-sulphuric acid. solution is then diluted, boiled for 15 minutes, cooled and the absorbance was measured at 430-450 nm.

The

62 Ultraviolet Methods

When streptomycin is heated with 0.1N sodium hydroxide, it produce maltol (L9), which exhibits an absorption maximum at 322 nm in alkaline solution. Therefore the streptomycin can be assayed by measuring the absorbance at 322 nm before and after hydrolysis in sodium hydroxide solution (396).

Katz (397) has reported an ultraviolet spectroscopic method for the determination of

STREPTOMYCIN 561

st reptomycin i n feeds . feeds was ex t r ac t ed with d i l u t e su lphur i c ac id , separa ted , n e u t r a l i z e d , f u r t h e r separa ted , concen t r a t ed and p u r i f i e d by ion exchange. s t r e p t o s e moiety was converted i n t o mal to l and q u a n t i t a t i v e l y determined by measuring i t s u l t r a v i o l e t absorbance.

The s t reptomycin i n

The

13.63 F luo r ime t r i c Methods

Boxer and J e l i n e k (398) have repor ted a f l u o r i - metric method f o r t h e determinat ion o f s t r e p - tomycin i n blood and sp ina l f l u i d . The b a s i s o f t h i s method i s t h e formation o f hydrazone o f s t reptomycin with f luo rescen t 9-hydrazino- a c r i d i n e hydrochlor ide. reagent t oge the r wi th hydrazones of a c i d i c , n e u t r a l and b a s i c compounds are separa ted from t h e s t r o n g l y b a s i c s t reptomycin hydrazone by e x t r a c t i o n from t h e so lu t ion with benzyl a l co - ho l . The aqueous base conta in ing s t reptomycin hydrazone i s cen t r i fuged and t h e i n t e n - s i t y of t h e f luorescence of t h e clear s o l u t i o n is measured. The method has been extended t o determine s t reptomycin i n u r i n e and t i s s u e s

The excess of t h e

(399).

Streptomycin and dihydrostreptomycin i n a lka - l i n e so lu t ion , produce a s t rong f luorescence wi th sodium 1,2-naphthaquinone-4-suIphonate (400). Based on t h i s p r i n c i p l e , Faure and Blanquet (401) have developed an a n a l y t i c a l procedure f o r t h e de te rmina t ion of s t reptomycin. Aqueous s o l u t i o n o f s t reptomycin was t r e a t e d wi th sodium hydroxide and sodium 1,2-naphthaquinone-4-sulphonate s o l u t i o n . Then t h e so lu - t i o n was set a s i d e i n t h e dark €or 1 hour a t 20° and then t h e f luorescence was measured. The d e t a i l e d s t u d i e s of f luorescence made i n subsequent pub l i ca t ions (402,403). Appl ica t ion o f t h e method f o r t h e de te rmina t ion o f s t reptomycin i n blood serum o r plasma was a l s o explored (404-406).

A continuous-flow Auto-Analyzer f l u o r i m e t r i c procedure f o r t h e de te rmina t ion of l ac t ic a c i d i n m i l k was appl ied t o t h e d e t e c t i o n o f a n t i - b i o t i c s (407) . The procedure was based on t h e

s p e c t r a were


i n h i b i t i o n by a n t i b i o t i c s of t h e a b i l i t y of s t a r t e r c u l t u r e s t o produce l ac t i c a c i d .

Alykov (408) has descr ibed a new f l u o r i m e t r i c method f o r t h e determinat ion o f aminoglycoside a n t i b i o t i c s . The method involves e x t r a c t i o n of f luo resce in complexan-Pr3+ aminoglycoside spec ie s from an aqueous medium of pH 5.8 t o 6 .2 i n t o isoamyl alcohol-benzene (1 :1) r e e x t - r a c t i o n o f f luo resce in complexan a s soc ia t ed wi th t h e aminoglycoside i n t o aqueous 0.1M sodium f l u o r i d e and Fluor2metr ic de te rmina t ion o f f luo resce in complexan i n t h i s aqueous s o l u t i o n a t 533 nm. The method i s s u i t a b l e f o r t h e de te rmina t ion o f s t reptomycin and t h e r e l a t e d a n t i b i o t i c s i n blood, milk and u r ine . Pro- t e i n s a r e removed from t h e blood samples by p r e c i p i t a t i o n with t r i c h l o r o a c e t i c a c i d .

13. 7 Chromatographic Methods

13.71 Column Chromatography

Column chromatographic (CC) method has been used ex tens ive ly f o r t h e sepa ra t ion and p u r i - f i c a t i o n of s t reptomycin. Severa l r e p o r t s have been publ ished regard ing t h e a p p l i c a t i o n o f charcoal ( lQ6-117), a c i d washed alumina (118-122) and ion exchange r e s i n s (123-164) f o r t h e sepa ra t ion and p u r i f i c a t i o n o f s t r e p t o - mycin.

Okumura e t a1 (409) have used columns o f a magnesium a lumino- s i l i ca t e f o r p u r i f i c a t i o n o f b a s i c a n t i b i o t i c s . Resolut ions such a s s t r e p - tomycin and kanamycin from oleandomycin were descr ibed . The method appears t o have app l i ca - b i l i t y i n t h e i s o l a t i o n o f t h e a n t i b i o t i c s from b i o l o g i c a l f l u i d s such a s c u l t u r e media, blood and u r i n e .

S t . John e t a1 (410) used column of ca t ion exchange r e s i n . Amberli te IRC-50 t o s e p a r a t e streptomycin and s t reptomycin B from fermenta- t i o n b ro ths , a n t i b i o t i c being determined co lo- r i m e t r i c a l l y [maltol method), with good agreement wi th microbio logica l procedure. Applica- t i o n o f t h e above method f o r determinat ion of

STREPTOMYCIN 563

st reptomycin i n animal feed (397) and i n formu- l a t i o n s (411) were r epor t ed . h b e r l i t e CG 120 ion exchange r e s i n was u t i l i z e d by Conca and Pazdera (412) f o r s epa ra t ion of s t r e p t i d i n e i n s t reptomycin .

Comparative s tudy o f carboxyl ic c a t i o n exch- angersin r e s p e c t s o f t h e so rp t ion and desorp t ion o f s t reptomycin was repor t ed (413).

Other r e p o r t s have descr ibed i n d e t a i l s t h e u s e o f permut i t (414), phenoxy-acetic a c i d c a t i o n exchanger and phenol ic r e s i n s (415), t h e reso- l u t i o n of components of s t reptomycin complex (416), t h e sepa ra t ion o f s t reptomycin from fermenta t ion b ro ths us ing continuous process (417) and eva lua t ion o f var ious exchangers f o r s epa ra t ing co lored impur i t i e s (418). The chromatographic behaviour of s t reptomycin on Sephadex G-10 has been descr ibed by S t o r l (419).

13.72 Paper Chromatography

Horne and Pol lard(420) have descr ibed a method f o r i d e n t i f i c a t i o n of s t reptomycin by us ing p a p e r - s t r i p chromatogram. The p o s i t i o n o f t h e a n t i b i o t i c were de tec t ed by means o f t h e Sakaguchi r eagen t . 3% ammonium c h l o r i d e was found t o be necessary f o r good sepa ra t ion .

Wj-nsten and Eigen (421) were success fu l i n r e so lv ing mixtures of s t reptomycin and c l o s e l y r e l a t e d subs tances . Butanol-piper idine- to luene sulphonic a c i d mixture was used as a developing agen t . The p o s i t i o n o f t h e a n t i - b i o t i c s were revea led by bioautography on aga r .

Solomons and Regna (422), S t o d o l l a e t a1 (423) Kowezyk (424) have appl ied similar method f o r t h e sepa ra t ion o f s t reptomycin. Pe terson and Reineke (425) descr ibed a s i m i l a r method f o r t h e de te rmina t ion of s t reptomycin i n salt-free prepara t ions and a p p l i c a t i o n s o f t h e method t o s t u d i e s o f b iosyn thes i s were publ ished by Hunter e t a1 (199). E f f e c t s of s a l t s on t h e movement and zone shape of s t reptomycin i n paper chromatography was d iscussed by Sokolski and Lummis (426).


A d e t a i l e d s tudy by Heding (427,428) produced a system capable of c l e a r l y r e so lv ing s t r e p t o - mycin, and i t s r e l a t e d compounds wi th a development time o f 8 hours. was prepared by mixing equal volume of amyl a lcohol conta in ing 1% of b is - (2-e thylhexyl ) phosphate and 0.5% sodium c h l o r i d e s o l u t i o n i n bo ra t e b u f f e r . The s p o t s were de tec t ed e i t h e r by us ing a spray o f 1-naphthol -d iace ty l o r by bioautogrsphy on a n u t r i e n t agar p l a t e seeded with spores of B a W bub-.

The developer used

Other r e p o r t s o f r e s o l u t i o n have been p u b l i - shed by Makisumi (429), Kondo e t a1 (430) and Pant and Agrawal (431). The former used buta- n o l , acet ic a c i d , py r id ine . water (4: 1 : 1 :2) and butanol : 6N hydrochlor ic ac id ( 7 : 3 ) as developing reagent and Sakaguchi 's reagent and J a f f e ' s reagent f o r d e t e c t i o n . The Rf va lues of s t reptomycin i n t h e above so lven t systems were 0.17 and 0 .08 r e s p e c t i v e l y .

Albu-Budai and Unterman (432) repor ted a quan- t i t a t i v e procedure based on paper chromatograph ic r e s o l u t i o n , e l u t i o n and spectrophotometr ic de te rmina t ion after co lo r development wi th 1-naphthol -d iace ty l . MN 261 paper s t r i p s prev ious ly impregnated with phosphate b u f f e r of pH 7 were used. pentachlorophenol-butanol-sodium hydroxide-water.

The developing so lvent were

Numerous spray reagents have been used i n t h e d e t e c t i o n of s t reptomycin based l a r g e l y on colo- r imet ry . This inc ludes potassium permanganate (433), iod ine (434), sodium pe r ioda te wi th p i c r i c a c i d (435), t h e a n i l i n e , l ac t ic a c i d , p i c r i c a c i d mixture (436) and bromine-starch- potassium iod ide , reagent (9).

Couling and Goodey (437) have descr ibed a new method o f s t reptomycin chromatography and i t s u s e i n t h e examination of t h e r e a c t i o n between s t reptomycin and ammonia, Paper p rev ious ly impregnated wi th phosphate bu f fe r at pH 7 and sodium hydroxide-pentachlorophenol-butanol as developing so lven t were U s e d . To d e t e c t t h e compounds, d i p t h e chromatogram i n p e r i o d a t e acetone and then i n benz id ine - acetone t o

STREPTOMYCIN 565

g ive yellow o r white spot i n a b lue background; o r d i p i n a mixture o f d i ace ty l and 1-naphthol t o give red spot on a white background.

1 3 . 7 3 Thin-Layer Chromatography

Thin l aye r chromatographic technique was ex- t e n s i v e l y used €or t h e sepa ra t ion and i d e n t i - f i c a t i o n of s t reptomycin and i t s r e l a t e d compounds by Kondo (430) and Borowiecka (438). The former used p l a t e s coated with a c t i v a t e d carbon whereas t h e latter used p l a t e s coated e i t h e r with s i l i ca ge l G o r Kieselguhr G. VonSchantz e t a1 (439) s tud ied t h e app l i ca t ion of metal p l a t e s f o r t h i n l aye r de t ec t ion of pharmaceutical compounds.

Several r e p o r t s have appeared on t h e app l i ca - t i o n o f t h i n l a y e r chromatography i n i d e n t i - fy ing aminoglycoside a n t i b i o t i c s . The s o l - vent system and t h e Rf values are repor ted i n t a b l e . 4.

Voigt and Bared (443) and Katayama and Ibeda (444) have r epor t ed two-dimensional TLC method f o r separa t ion and i d e n t i f i c a t i o n of s t r e p t o - mycin and i t s r e l a t e d compounds.

Sa t0 and Ikeda (445) were a b l e t o r e so lve streptomycin and i t s dihydro-and dihydrodeoxy d e r i v a t i v e s by ascending TLC procedure. Simi- l a r claims were made by Katayama and Ikeda (446).

Thin layer-bioautographic method has been employed f o r t h e sepa ra t ion and i d e n t i f i c a t i o n o f streptomycin (447,448) . The spo t s obtained from streptomycin by TLC procedure have been e l u t e d wi th phosphate buf fer and examined microbio logica l ly with B a W bubLLLi.6 spores A s i m i l a r procedure has been adapted by Zuidweg e t a1 (449) and Hamann e t a1 (450) t o r e so lve streptomycin and o t h e r a n t i b i o t i c s . The former used t h e p l a t e s coated with sephadex and t h e l a t t e r used t h e c e l l u l o s e l aye r .

Applicat ion 4-Dimethylaminobenzaldehyde-anti- mony t r i c h l o r i d e a s a de t ec t ing agent f o r a n t i - b i o t i c i n TLC was repor ted by Mathis and

Table 4 : TLC of Streptomycin

Adsorbent Solvent System. Spray Reagent Rf value Reference

1. Silica gel G / Propanol- Aluminium Ethyl acetate-water- Ninhydrin. 0.62-07 44 0 oxide. Ammonium hydroxide

(50:10:30:10).

2. Silica gel G Butanol-water- Sodium Nitroprus- methanol-tolune side, potassium 0.4 -p-sulphonic acid permanganate and (40 :20: 10: 1) sodium hydroxide

mixture.

441

3 . Silica gel G 1) Acetic Acid : 10% aqueous solution 0.32

2) Benzene-acetone with 2% ammonia. 0.32 butanol (7 : 2) Copper sulphate 44 2

acetic acid 2:2:1. > J

STREPTOMYCIN 567

13.74

Schmitt (451). Paunez (452) described a TLC method utalizing the layer o f ion-exchange resin.

More extensive examination of streptomycin group was reported by Heding (453) and More recently Borowiecka (454) . Forty seven solvent systems were investigated in deciding on optimum conditions for resolution of streptomycin and related compounds. Visualization was made by a spray reagent of sodium nitroprusside- potassium permanganate.

High Performance Liquid Chromatography

Bagon (455) has developed an assay of antibiotics in pharmaceutical preparations using reversed-phase high-pressure liquid chromatography. Adapting this method, antibiotics like streptomycin were determined by HPLC on a column packed with Spherisorb SS-ODs. Aqueous methanol at a flow rates of upto 1 ml/min. was used as an eluent. The elutes were monitored by spectrophotometric detection.

Whall (456) has reported a HPLC method for the determination of streptomycin and dihydrostreptomycin sulphate. follows:

The method is as

Samples were dissolved in water and diluted to 50 ml., 3 ml of each solution is then diluted to 50 ml with mobile phase containing 0.02 M sodium hexanesulphonate - 0.025 M sodium phosphate in aqueous acetonitrile, adjusted to pH 6 . on a column packed with LI Bondapak C18. column was fitted with a reversed-phase guard column and elution was carried out at 1 mljmin. at 25O. Solutes being detected at 195 nm.

A 25 pl portion was subjected to HPLC The

HPLC assay of pyrazinoic acid in human plasma in the presence o f antituberculosis drugs using an automatic sampler was proposed by Ratti et a1 (457) . This method could also be used to determine streptomycin. The chromatographic conditions are : Mobile phase : 0.01M ammonium dihydrogen phosphate - acetonitrile ( 3 : 7 ) .


Column condition : Column of 25 cm length 4.6 mm width packed with Lichrosorb-NH2. at 2 rnl/min. The elutes are detected at 254 nm.

Flow rate

Inchauspe and Samain (458) have described a ion-pair reversed-phase high performance liquid chromatographic method for separating aminoglycoside antibiotics using perfluorinated carboxylic acids. The aminoglycoside antibiotics were separated at ambient temperature on a column (25 cm x 4.6 mm) packed with ultrasphere c18 (5 pm) with methanol - 50 mM-pentafluoropropio- nic acid (21:29), methanol - 50 mM-heptafluoro- butyric acid (3:2), methanol - 50 mM - camphorsulphonic acid (29:21) o r 0.2 M trifluoroace- tic acid as mobile phase and refractive index detection.

13.75 Electrophoresis

Foster and Ashton (459) described a method for separation of streptomycin and related compounds by paper electrophoresis. were applied to the paper which is then wetted with a buffer solution of pH 5. After passage of current for 16 hours the paper was removed and dried. The spots were revealed by one of following reagents : 1) diacetyle; 2) naphtha- resorcinol spray; 3) Elson Morgans reagent. Alternatively, the potency was determined microbiologically by laid down the papers on agar plates seeded with suitable organisms.

The test solutions

Takahashi and Amano (460) employed the paper electrophoresis method for the identification of various antibiotics.

Philippe et a1 (461) have reported a similar method of electrophoresis f o r the separation of streptomycin and dihydrostreptomycin. Whatman 3MM paper in borate buffer at pH 9 were used. The electropherogram was run €or 3 hours at 500 V and the spots were developed with Monastero's alkaline nitroprusside-ferric cyanide reagent.

Other reports of resolution of streptomycin and other antibiotics have been published by Paris

STREPTOMYCIN 569

13.76

and Theallet (462) Lightbrown and de Rossi (463). The application of electrophoresis for separation and identification of the antibiotics in Pharmaceutical mixtures was reported by Apreotesei and Teodosiu (464).

Katayama and Ikeda (465) achieved a similar resolution using electrophoresis layers of silica gel. sional technique using a combination of electrophoresis and chromatography on silica gel (466).

Gorge and Monteoliva (467) used two dimensional techniques on paper (chromatography in one direction and electrophoresis in the second) €or identification of many guanidino compounds including streptomycin. Application of electr- phoresis to the identification of several aminoglycoside antibiotics has been reported by Garber and Dobrecky (468) and Ochab (469).

They also described a two dimen-

Langner et a1 (470) have described a method of determination of antibiotics including streptomycin in mixtures at ultra-micro levels by rapid low-voltage electrophoresis. The antibiotics could be separated on microscopic slides coated with agarose gel. phoresis at 50 V per cm. for 5 to 30 minutes, the antibiotics were located by Bioautography on agar seeded with BaCieeUn c5ub;tieid.

After electro-

Counter-Current Distribution

Titus and Fried (471) have reported the use of counter-current distribution technique f o r the analysis of streptomycin preparation. The Craig technique of counter-current distribution was employed to show the presence of a related substance in crude streptomycin by distribution between butanol and water.

Craig countercurrent distribution technique has been extensively employed for the separation of streptomycin from mannosidostreptomycin by Plaut and McCormack (472) and O'Keeffe et a1 (473). The former obtained a better resolution by using the solvent systems of an aqueous phase containing 0.5% sodium bicarbonate and 1% sodium chloride and solvent phase


(Pentasol-amyl a lcohol ) con ta in ing 5% s t e a r i c ac id and t h e latter employed a system composed o f l a u r i c a c i d i n amyl a l coho l and aqueous phosphate-borate b u f f e r .

Meyer (474) has d iscussed i n d e t a i l s t h e d i f f e r - en t methods of counter -cur ren t d i s t r i b u t i o n techniques employed f o r t h e a n a l y s i s of va r ious compounds inc luding s t rep tomycin .

13.8 Bio-Assay Methods

The p r i n c i p l e o f t h e b i o l o g i c a l a s say i s based on t h e comparison of t h e i n h i b i t i o n of t h e growth of b a c t e r i a produced by known concent ra t ion of t h e s t anda rd pre- p a r a t i o n with t h a t produced by measured concen t r a t ion of t h e sample being t e s t e d .

13.81 Agar-Diffusion Method on S o l i d Media

Many aga r -d i f fus ion methods wi th t h e s u i t a b l e inoculum of t h e fol lowing test organisms have been r epor t ed :

Enchenicki cakX (475-477), BacAYw n u b U (478-483), B a W cd~cf.&ns (484), B a r n f icheni~onmn (485), B a W cetrew v a r . mgcoidu (486-487), SXaphyLocaccw awrew (488- 490) , S.thepRococcub h c ; t i n (491) K L e b n i d h pneumonkw (492) Lac tobac iUu b&atLicw (493) acid r e s i s t a n t microbacterium V-5(494). The method has been ex tens ive ly used i n determi- n a t i o n of s t reptomycin i n b i o l o g i c a l body f l u i d (495-499), u r i n e (500). food and animal feed (501-505) and pharmaceut ical p r e p a r a t i o n s (506).

Modi f ica t ions of agar d i f f u s i o n method have been r epor t ed . Paper d i s k method has been adapted f o r t h e de te rmina t ion of s t reptomycin by Loo et a1 (507) , Koelzer and Giesen (508) , R e i l l y and Sobers (509) , and Kanazawa & Kuramata (510). Resazurin d i s c has been employed by Shahani and Badami (511) .

Waksman and R e i l l y (512) app l i ed Agar-s t reak method €or assaying s t reptomycin and o t h e r a n t i - b i o t i c subs t ances : b i o t i c s p repa ra t ions can be determined by

The potency of t h e a n t i -

STREPTOMYCIN 571

incorpora t ing graded d i l u t i o n s i n s tandard nut - r i e n t agar media i n p e t r i p l a t e s and s t r e a k i n g t h e agar su r face with s e l e c t e d t e s t b a c t e r i a . After overn ight incubat ion , t h e potency is read and expressed as t h e r ec ip roca l of t h e h ighes t d i l u t i o n i n h i b i t i n g t h e d i f f e r e n t tes t organisms.

McGuire e t a1 (513) has suggested a new l i n e a r d i f f u s i o n method for microbio logica l assay of s t reptomycin. S l i d e - c e l l method (514) and modified A comparative s tudy of cup, paper-disk, pulp d i s k and superpos i t ion assay mthods was repor- t e d by I sh ida e t a1 (516) .

d rop-p la te method were a l s o r epor t ed .

Dewart e t a1 (517) and Light Brown e t a1 (518) have descr ibed a d i f f u s i o n assay f o r a n t i b i o t i c s by an automated procedure. fol lows :

The method i s as

Disposable p l a s t i c P e t r i d i shes loaded with agar prepared f o r assay are c a r r i e d au tomat ica l ly t o a dev ice t h a t punches ho le s i n t h e agar , t hen t o a d i spens ing u n i t t h a t d i spenses measured volumes (e .g . , 50 o r 70 1.11) of t e s t o r s tandard s o l u t i o n i n t o t h e holes ; a f t e r incubat ion , t h e a rea of zones of i n h i b i t i o n a r e measured.

Continuous automatic microbio logica l assay o f a n t i b i o t i c s was r epor t ed by Shaw and Duncombe (519). of r e s p i r a t o r y carbon d ioxide .

Improvements of Agar d i f f u s i o n method f o r assaying s t reptomycin were a l s o repor ted (520,521).

B r i t i s h Pharmacopoeia 1958 (304) and Egyptian Pharmacopoeia 1953 (13) desc r ibe a microbiolo- g i c a l Assay method f o r de te rmina t ion of s t reptomycin.

The method was based on t h e measurement

Assay Procedure :

F i l l i n uniform th i ckness p e t r i - d i s h e s , o r r ec - t angu la r t r a y s , t o a depth o f 3 t o 4 mm. with agar c u l t u r e medium which has been previous ly inocula ted wi th a s u i t a b l e quan t i ty o f a suspension o f spores of a s u i t a b l e s t r a i n of BacLUuh h u b U .


Allow the inoculated p l a t e s t o dry a t room temperature and keep a t 5OC., u n t i l used. Place on the surface of the inoculated medium, small s t e r i l e cylinders of uniform s i z e approximately 10 mm high or having an in t e rna l diameter of approximately 5 mm. , made of glass, porcelain o r aluminium, and keep a t about 150OC. Five, s i x , e ight cyl inders , or any other numbers which can be conveniently adjusted t o the s i z e of t he agar p l a t e , may be placed on each. Bore i n t h e medium, i n place of cyl inders , holes, 5 t o 8 mm i n diameter, by means of a s t e r i l e borer.

Prepare i n s t e r i l e phosphate buffer solut ion, pH 8 .0 , solut ions of t h e standard preparation of streptomycin, of known concentrations, as f o r example 0.5 t o 2 u n i t s per m l and solut ions of the sample t o be t e s t e d presumed t o be o f t he same order of concentration. F i l l i n t o t h e cylinders o r holes, t he solut ions of t he s tandard preparation and of t h e sample t o be t e s t e d i n a l t e r n a t e order by means of a p i p e t t e which de l ive r s a standard amount o f solut ion s u f f i - c ient almost t o f i l l t he holes when they a r e used,

Place the p l a t e s i n a r e f r i g e r a t o r f o r about 2 hours, and then incubate a t about 30° t o 37OC f o r about 16 hours. Measure, with the g rea t e s t possible accuracy, t he diameters of t he inh ib i - t i o n zones produced by the varied concentrations of t h e standard preparation of Streptomycin and of t he sample t o be t e s t ed , and from the r e s u l t s t h e potency of t he l a t t e r is calculated.

L i m i t of Error: The limits of e r r o r (P=O.99) t h a t can be obtained with t h i s method are 79 and 121 per cen t . , where 10 cylinders o r holes a r e used; 85Oand l l S O , where 20 cylinders o r holes a r e used; 89.50 and 110.50 where 40 cyl inde r s o r holes are used; and 92.50 and 107.50 where 80 cylinders o r holes a re used f o r t he standard preparation of streptomycin and f o r t he sample t o be t e s t ed , respect ively.

STREPTOMYCIN 513

13.82 Liquid Cul ture Method

13.821 Turbid imet r ic Method

Oswald and Knudsen (522) have repor ted a t u r b i d i m e t r i c method f o r assay of s t r e p - tomycin. Two series of tubes , one wi th graded amounts o f s tandard s t reptomycin and o t h e r with t h e unknown are inocula ted with t h e same amount o f a s tandard suspension o f t h e t e s t organism, incubated a t 37O f o r 4 hours . t ransmiss ion read i n a p h o t o e l e c t r i c co lor imeter . t e s t can be read d i r e c t l y from t h e s t an - dard curve .

The percentage o f

Potency of t h e sample under

Appl ica t ions of t h e t u r b i d i m e t r i c method €or t h e a n a l y s i s of s t reptomycin i n u r i n e , serum and cerebrospina l f l u i d were repor- t e d by Gibb (523) and Whitlock e t a l (524) .

13.822 Other Methods

Bonifas and Chesni (525) have developed t h e d i l u t i o n method f o r t h e de te rmina t ion of s t reptomycin : K ~ e b b ~ ~ p n m o n i a e was allowed t o grow a t pH 9-9.5 i n t h e s y n t h e t i c medium of Monod t o which sugar and i n d i c a t o r c r e s o l red were added. The r e a c t i o n s h i f t e d towards t h e ac id s i d e upon s p l i t t i n g t h e sugar . Decreasing amounts o f s t reptomycin were added t o t h e medium. tomycin was determined by no t ing t h e d i l u - t i o n i n t h e tube immediately preceding t h a t i n which these was t h e f irst change o f c o l o r of t h e i n d i c a t o r .

The i n h i b i t i n g amount of s t r e p -

Dilution-method was extended t o determine s t reptomycin i n u r i n e and body f l u i d s (526).

A microbio logica l assay of a n t i b i o t i c s based on i n h i b i t i o n of ammonia product ion was repor ted by Kobos (527). The test s o l u t i o n was incubated f o r 2 hours a t 37O with a suspension of E . cofi i n 6% peptone medium., t h e pH o f t h e mixture was


13.9

ad jus ted w i t h sodium hydroxide and t h e concent ra t ion of ammonia was determined with use of an Orion 95-10 ammonia sensor .

Biochemical Methods

A radioimmuno assay (528,529) was developed f o r d e t e r - mination of s t reptomycin i n plasma and u r ine . The method involves enzyme-catalysed in t roduc t ion o f a l a b e l l e d group i n t o t h e molecule and sepa ra t ion o f t h e l a b e l l e d product by paper chromatography before count ing . Schwenzer and Anhalt (530) have r epor t ed an automated f luorescence p o l a r i z a t i o n immunoassay f o r s t reptomycin i n blood serum. I n t h e assay descr ibed f luo resce in - l a b e l l e d streptomycin was used a s t h e tracer and a n t i - serum aga ins t streptomycin was r a i s e d i n r a b b i t s . On mixing of t r a c e r , sample and ant ibody, t h e p o l a r i z a - t i o n o f t r a c e r f luorescence was measured i n an Abbott TDX f luo r ime te r .

Acknwo 1 edgement

The au thors s i n c e r e l y thank Mr. Al ta f Hussain Naqvi and M r . Uday C . Sharm €or t h e i r s e c r e t a r i a l he lp .

STREPTOMYCIN

References

575

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STREPTOMYCIN 511

31.

32.

33.

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36.

37.

38.

39.

40.

41.

4 2 .

43.

4 4 .

45.

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M . L . Wolfrom and W.J. Polg lase ; J. An. Chem. SOC., 70, 1672 (1948).

M . L . Wolfrom, S.M. Olin and W . J . Po lg lase ; J. Am. Chem. SOC. - 72, 1724 (1950).

J. Fr ied and 0. Win te r s t ine r , J . Am. Chem. SOC; 69, 79 (1947); C .A. ; - 41 ; 1620 (1947).

F . A . Kuehl, Jr. , E.H. Flynn, N . G . Brink and K . Folkers ; J . Am. Chem. SOC., 68, 2679 (1946); C . A . ; 41, 21004 (1947).

F.A. Kuehl, Jr. , E.H. Flynn, F.W. Holly, R . Mozingo and K. Folkers ; J. h. Chem. SOC., 69, 3032 (1947).

J. F r i ed , D.E. Walz and 0. Win te r s t e ine r , J . Am. Chem. SOC; - 68, 2746 (19461.

N . G . B r i n k , F.A. Kuehl, Jr. , E .H. Flynn and K . Folk- erS;J. Am. Chem. SOC. , 68, 2405 (1946).

N.G. Brink, F.A. Kuehl, Jr. , E . H . Flynn and K . Folk- crs;J. Am. Chem. SOC., 70, 2085 (1948).

John R . Dyer, W.E. PlcGonigal and K.C. Rice, J. Am. Chein. SOC. , 87, 654 (1965).

J . C . C r a s s e l l i and W . M . Ri tchey, "Atlas of S p e c t r a l Data and Phys ica l Cons tan ts f o r Organic Compounds" , 2nd ed . Vol. IT, CRC Press, Inc. , Cleveland, Ohio (1975).

-

-

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-

S. 'Tatsuka and S. Hor r i , Proc. Jap . Acad., - 39, 314 (1963).

J. R. Dyer and A.W. Todd, J . Am. Chem. SOC., 85, 3896 (1963).

M.L. IVolfrom, M..J. Cron, C.W. DclValt and R . N . I lus- band; J . Am. Chcm. SOC., 76, 3675 (1954); C . A . , - 49, 10194 (1955).

M . L . lVolfroiii and C . N . I)cl\ralt; J . Am. Chem. SOC; - 70, 3148 (1948).

0. Win te r s t e ine r and A. Kingsbergs , J . Am. Chcm. SOC. , - 73, 2917 (1951).

JABER S. MOSSA ETAL. 578

47.

48.

49.

50.

51.

52.

53.

54 *

5 5 .

56.

57.

58.

59.

60.

61.

K.L. I l inehart , Jr. , M. Iiichens, A . D . Argoudel is , W.S. Chi l ton , H.E. Carter, M.P. Georgiadis , C . P . Schasf fner and T.R. S c h i l l i n g s ; J. Am. Chem. SOC. , - 84, 3218 (1962).

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R.U. Lemieux, C.W. DeWalt and M.L. Wolfrom, J . Chem. S O C . - 69, 1838 (1947); C.A. , - 41 , 6207 (1947).

F.A. Kuehl, Jr. , R . L . Peck , C . E . Hoffhine, Jr. , E.W. Peel and K. Fo lkers ; J . Am. Chem. SOC. 69, 1234 (1947).

R . L . ,Peck, F.A. Kuehl, Jr. , C . E . t loffheine, Jr . , E.W. Peel and K. Folkers ; J . Am. Chem. SOC. , 70, 2321 (1948).

F.A. Kuehl , Jr. , R.L. Peck, C. E. Hoffhine, Jr. , and K. Fo lkers ; J. Am. Chem. SOC. , 70, 2325 (1948).

F.A. Kuehl, Jr. , M . N . Bishop, E.H. Flynn and K. Fo lkers ;

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J . Am. Chem. SOC. , 70, 2613 (1948).

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H. Umezawa; Y. Ogata, T. Takeuchi and T. Tabato; Japan. Med. J . ; 1, 397 (1948); C .A. , - 45, 5757 (1951).

Masao Murase , Tomohisa Tak i t a ; Kofumi o h i , Hiroko Kondo , Yoshiro okzmi and Hamio Umezawa; J . A n t i b i o t i c s , 1 2 , 126, 257 (1959).

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S . A . Waksman, A . Schatz and H.C. R e i l l y ; 7353 (1946).

J . Bact; 51,

62. C.IV. Rake.and R. Donovick; J . Bact. 52, 223 (1946).

STREPTOMYCIN 579

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

K . R . Rao,, S . S . Rao and P.R. Venkataraman; Nature - 158, 23 (1346).

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A.C . Thaysen and M. Morr i s ; Nature, 159, 100 (1947).

H.B. Woodruff; J. Bact., 54, 42 (1947).

C.V. Hubbard and H.H. Thornberry; Acad. Sc . , 39, 57 (1947); Biol . Abst . , 22, 22933 (1947).

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H.H. Thornberry and H.W. Anderson; Arch. Biochem; 16, 389 (1948); U.S. - 2 , 621, 146 (1952).

J . F . S p i l s b u r y ; Trans. Brit. Mycol. SOC., 31, 210 (1948).

H.M. Eiser and W.D. McFarlane; Canad. J. Res., 26, 164 (1948).

Eugene L. Dulaney., J. Bact; - 56, 305 (1948). C .A. , p3, 271 (1949).

Ayerst McKenna and H a r r i s o n Ltd. , B r i t , 616, 331 (1949); C.A. , - 43, 4434 (1949).

Abraham L. Baron, B r i t . , 639, 863 (1950); C.A. - 44, 10270 (1950).

Paul C. T r u s s e l l ; U.S. - 2 , 541, 726 (1951); C . A . , - 45, 4006 (1951).

L . E . McDaniel and David I-Ie!dlin.; U.S. - 2 , 538, 943 (1951); C . A . ; 45, 2637 (1951).

Merck and Co, Inc . ; B r i t . , 650, 087 (1951); C . A . , - 45, 9814 (1951).

C h r i s t o p h e r J. Jackson , P h i l l i p s D. Coppock and Brendan K . Kelly. B r i t . 660, 203 (1951); C . A . - 46. , 4610 (1952).

Merdic E Co. I n c . , Rrit., 305 (1953) , C . A . , - 48, 335 (1954).

R . Raghunandana Rao; C u r r e n t S c i . , 22, 203 (1953) , C . A . , 48. 4043 (1954) .

j -


81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

Robert D. Muir and Alden B. Hatch; U.S. 2, 682, 493 (1954), C.A. , - 48, 11738 (1954).

Kyoichi Kawasaki; Japan; 3750 (56) (1956); C.A. , - 51 11666 (1957).

E . I . Sur ikora , G.F. Zavi le i skaya , N.T. Dygern and G.D. Pesterova; A n t i b i o t i k i , 4, 12(1959) , C.A. , 53, 22744 (1959).

V.A. Severina; S.V. Gorskaya and I . V . Gracheva., Doklady Akad. Nauk. SS.S.R. - 126, 110311959)C.A. - 54, 3590 (1960).

Hiromi Nakayama, Sh i ro S h i r a t s u c h i and Sh in ich i ro Esumi; Japan 7969 (1958). , C.A.; 54, 3869 (1960).

Hi rosh i ogawa, Yasuji Sekisawa, Masao ogasawara, Sh in ich i Kondo and Masako Aoki; Japan, 9 , 0 9 9 (1961) , C.A. , 57, 2681 (1962).

Sh i ro Sh i r a to , Hi rosh i Motoyama and Hiroko Ueda; Hakko Kyokaishi, 2, 416 (1961); C . A . , - 59, 15919 (1963).

Hideo Kubo, Hiromi Nakayama Sh i ro Sh i r a t such i Hi roshi Motoyama and Noriko Yanagita, Japan,11,598 - (1960); C.A., 57, 13019 (1962).

C.R. Addinal l ; Drug Cosmetic Ind., 57, 628, 710 (1945); C . A . , - 40, 1279 (1946).

Herbert S i lcox; Chem. Eng. N e w s , 24, 2762 (1946); C . A . , - 41, 252 (1947).

91. Richard W. Po r t e r ; Chem. Eng. 53, 94 (1946); C.A. , 41, 565 (1947).

92. P h i l i p D. Coppock; U.S. - 2 523, 245 (1950), C . A . , 4 5 , 715 (1951).

93. Hamao umezawa, Yasusuke osa to , Ryozo Utahar, Koki Yagish i ta and Yoshiro okami; J. A n t i b i o t i c s - 4 , 23 (1951), C .A. , 45, 9223 (1951).

94. Eugene L. Dulaney; U.S. __ 2, 571, 693 (1951); C.A. , - 47, 694 (1952) *

95. Hiromi Nakayama, Yonosuke Ikeda and Makoto Kawasaki, Repts. Sc i . Research I n s t . , 28, 387 (1952). C.A. , 47, 8833 (1953).

STREPTOMYCIN 581

96. Merck 4 Co; Inc.; Brit. 698, 113 (1953); C . A . , 48, - 4186 (1954).

97. I d e m . , Ib id 667, 423 (1952); C.A. , 48 6082 (1954).

98. A. Kramli, A. Kovacs, B. Matkovics, M. Natonek, G. Pulay and P. Turay; Acta Biol. Acad Sc i . Hung., - 5, 79 (1954); C . A . , - 49, 4938 (1955).

A. Waheed Khan and I . D . Chughtai; Pakis tan J. Sc i , - 7 , 15 (1955); C . A . - 50, 531 (1956).

-

99.

100. S.V. Trachuk and S.G. Chekaida, Med. Prom. SSSR - 16, l l (1962) ; C . A . , 57, 12639 (1962).

101. Shi ro Sh i r a to and Hiroshi Motoyama; Hakko Kyokaishi; - 19, 549 (1961); C.A. , 59 , 15919 (1963).

102. Henrik Hedding, Dan. K e m i , - 48, 85 (1967), C.A. , - 67, 10736-.2e (1967).

103. L. A. Voroshilova, E.S. Bylinkina and S.V. Gorskaya; Khim - Farm. Zh., 3, 41 (1969); C . A . , - 71, 29266t (1969).

104. H.W. Flory, E . Chain, N . G . Heatly, M.A. Jennings A. G. Sanders; E.P. Abraham and M.E. Florey "Antibiot ics" Vol. 11, Oxford Univers i ty Press, London (1949).

105. F. Carva ja l ; Mycologia, 38, 596 (1946).

106. T.J. Woodthrope and D.M. I r e l and . , J. Gen. Microbiol., - I , 344 (1948); C .A. , - 4 2 , 5944 (1948).

107. Merck Fr Co., Inc. , B r i t , - 607, 186 (1948); C.A., - 43, 1158 (1949).

108. Idem. , I b i d 625, 820 (1949); C.A. , 44, 1235 (1950). - -

109. P h i l i p D. Coppock, L i ly L. Mulligan and Alan G. White; B r i t , - 622, 682 (1949); C .A. - 44, 1656 (1950).

110. HarveyE. Alburn and Eric G. Snyder; U.S., - 2 , 505, 318 (1950); C . A . , - 44, 6087 (1950).

111. Robert D. Babson and Max T i sh le r ; U.S., - 2 , 521, 770 (1950); C.A. , - 44, 11041 (1950).


112 . Robert L. Peck, U.S. - 2 , 540, 284 (1957); C.A. 43, 4894 (1951).

113. Wm. A. Bi t tenbender and Robert D. Babson; U.S. 2, 540, 238 (1951); C.A., - 45, 4414 (1951).

114. Frank J. Wolf; U.S., - 2, 538, 140 (1951); C.A., - 45, 3131 (195 1) .

115. S.A. Waksman, J. H i s t . Med. A l l i e d Sc i . , - 6, 318 (1951), C.A. - 46, 1220 (1952).

116. David L. Johnson; U.S., - 2 , 643, 997 (1953), C.A. - 47, 8970 (1953).

117. Arne N. Wick and Mil ton, J. Vander Brook; U.S. , 2, 701, 795 (1955); C . A . , - 49, 6550 (1955).

118. H.E. Carter; R.K. C lark , Jr.; S.R. Dickman, Y.H. Loo, P.S. Ske l l and W.A. Strong; J. Biol . Chem; - 160, 337 (1945) ; C.A. - 40, 3485 (1946).

119. F.A. Kuehl, Jr., R . L . Peck, C.E. Hoffhine, Jr., R.P. Graber and K. Folkers ; J. Am. Chem. SOC., 68, 1460 (1946); C .A. ; - 40, 7211 (1946).

120. M . J . Vander Brook, A.N. Wick, Wm. H. DeVries, R. Harris and G.F. Car t land; J. Biol . Chem., 165, 463 (1946); C . A . , - 41, 1276 (1947).

121. George P. Mueller; J. Am. Chem. SOC. - 69; 195 (1947); C .A. , - 41, 3927 (1947).

1 2 2 . Herman Sokol and Robert P. Popino; U.S. - 2, 653, 151 (1953); C .A. , - 48, 959 (1954).

123. Roy J. Taylor; U.S. - 2 , 528, 188 (1950); C .A. 45, 2158 (1951).

124. Eugene E. Howe and I r v i n g P u t t e r ; U.S. - 2; 541, 420 (1951); C.A. - 45; 8726 (1951).

125. Char les H. McBurney; U.S., 2, 613, 200 (1952); C.A. , - 47, 798 (1953).

126. Hideo Kubota;Japan, 5248 (1954); C . A . , - 49, 13605 (1955).

127. Sumio Umezawa and Tetsuo Suami; Japan; 4296 (1954); C.A. - 49, 10589 (1955).

STREPTOMYCIN 583

128. Kaneyuki Takagi; Japan; 5649 (1954); C . A . , - 49, 14278 (1955).

129. Josef Hoffman and J i r i n a Capkova; Czech, - 84, 779 (1955); C.A. , - 50, 10350 (1956).

130. Josef Hoffman, J i r i n a Capkova, Miroslav Hermansky and Zdenek Severa; Czech; - 85, 067 (1955); C .A. - 50, 10351 (1956).

131. O . B . Fardig and I . R . Hooper; U.S. - 2 , 717, 892 (1955); C . A . , S J , 1272 (1956).

132. O.B. Fardig and M.A. Kaplan; U.S. - 2 , 754, 295 (1956); C.A. , S J , 15031 (1956).

133. C.R. Bartels; B. Berk and W.L. Bryan ; U.S. - 2 , 765, 302 (1956); C . A . , - 51, 3939 (1957).

134. G.V. Samsonov and S.E. Bresler; Kolloid. Zhur., - 18, 337 (1956) , C . A . , - 51, 799 (1957).

135. C.R. Bartels; G . Kleiman, D . R . I r i s h and J . N . Korzun, U.S., 2, 786, 831 (1957), C.A. - 51, 8385 (1957).

136. Zbigniew Kotula, Acta Polon. Pharm. , 2, 133 (1957); C.A. , 51, 14206 (1957). -

137. Josef Hoffman. Miroslav Hermansky. Helena Parizkova and Jiri Doskocil; Chem. Tech. , - 9, l k l (1957); C.A. , - 51, 17093 (1957).

138. C.R. Ba r t e l s , W.L. Bryan and B. Bark; U.S. , - 2 , 868, 779 (1959); C . A . , - 53, 6542 (1959).

139. S . I . Kaplan, Med. Prom. S.S.S.R., 12, 24 (1958); C.A. , - 53, 11762 (1959).

140. S.M. Mamiofe, E.M. Savi tskaya, B.P. Burns, Z.T. S in i tzyna and N . N . Shel lenberg; Med. Prom. S.S.S.R., - 1 2 , 39 (1958); C . A . , - 53, 11762 (1959).

141. V.A. Zhelobenko, F.M. Dinulov; V.M. Vishnevski l , P.S. Romanenko, V.N. Papchinski, G.T. Godunov, R.A. Myrsina, L.V. Fedorovskaya, I . T . Kondokova, I.S. Basenko and I .R. Grekova; U.S.S.R., - 114, 385 (1958); C . A . , - 53, 11771 (1959).


142. S.E. Bresler, A n t i b i o t i k i , - 5, 33 (1960), C.A. , 54; 15840 (1960) .

143. G.V. Samsonov, Yu. B. Bol taks , N.P. Kuznetsova, A.P. Bashkovich and R.B. Ponomareva; Kol lo id , Zhur. - 21, 471 (1959). C . A . , 54, 7286 (1960).

144. S.I. Kaplan; Med. Prom. S.S.S.R., 13, 16 (1959). C .A. - 54, 16739 (1960).

145. G.V. Samsonov and Yu. B. Bol taks; Trudy Leningrad Khim - Farm. In s t . , 1, 35 (1959); C.A. , 55, 5085 (1961).

146. J u s t i n J. Murtaugh and I s i d o r o Caldas; Jr., U.S. - 2 , 970, 138 (1961); C.A. 55, 12782 (1961).

147. L.F . Yakhontova;E.M.Savitskaya and B.P. Bruns.; Khroma- tog- ee Teoriya Primenen; Akad. Nauk. S.S.S.R.; Trudy Vsesoyuz. Scveshchaniya Moscow; 100 (1960). C.A., 55, 19135 (1961).

148. L.F. Yakhontova, B.P. Bruns; Yu. S. Chekulaeva, N . N . She l lenberg , N.A. Vakulenko and S.N. Kovardykova., Med. Prom. S.S.S.R., 15, 26 (1961); C.A. - 55, 27776 (1961).

149. Wolfgang Sonnenkalb and Horst Henkel. Ger. (Eas t ) ; - 18, 098 (1960); C.A. - 55, 26380 (1961).

150. L . F.Yakontova, B. P. Bruns, E. S .Byl inkina, A.M.Zisserman,V.V. Matveev, A . B . Pashkov, E.M. Savi t skaya , K.M. Saldadze, M.A. Slobodnik, N.N. She l lenberg , Yu. S. Chekulaeva and A.A. Khints, U.S.S.R., 139, 052-(1960) ; C .A. - 56, 6100 (1962).

151. Shigeo F u j i t a , Hi rosh i Kawabe and Masaya Yanagi ta ; Rika- gaku Kenkyusho Hokoku; - 36, 595 (1960); C.A. - 56, 10707 (1962).

152. L.E. Yakhontova, B.P. Bruns, Yu. S. Chekulaeva; N . N . Shel lenberg, N.A. Vakalenko and S.N. Kovardykova; Ionoob- men Sorbenty, v Prom. Akad. Nauk S.S.S.R., I n s t . F i z . Khim. 203 (1963); C . A . , - 60, 5281 (1964).

153. S.G. Chekaida, R. Ya. Ladiev-and S.V. Trachuk, - 152, 544 (1964); C . A . , - 60, 16468 (1964).

154. All-Union Sc ien t i f ic -Research I n s t i t u t e of a n t i b i o t i c s ;

U.S.S.R.,

Fr. - 1. 402, 160 (1964). C .A. , 63, 11263 (1964).

STREPTOMYCIN 585

155. Zbigniew Kotula and Teresa Czolowska; Po l . - 49, 876 (1965); C . A . , - 65, 576 (1966).

156. E.S. Vaisberg, L . F . Yakhontova and B.P. Bruns, Zh. F iz . Khim., - 40, 1884 (1966); C.A. - 66, 14337Q (1967).

157. Gheorghe Dragan, Mixcea Rusan and Mircea Toma; F r . 1, 444, 738 (1966). C . A . - 66, 27706j (1967).

- 207, 4.3 (1966). , C . A . , - 66, 31942a (1967).

159. S t an ley W. Ensminger, U.S. - 3, 313, 693 (1967), C . A . , - 67, 52740u (1967) .

158. V.A. Ezhov and V . I . Di tyantseva ; Med. Prom. S.S.S.R.,

160. Ionescu, Sabin; J a luba Margareta; Danet , Rada; Ionescu Lig ia ; and Dumitrescu, Mihalache; Rom - 9- 51, 161 (1968); C . A . - 70, 66804s (1969).

161. Urs F. Nager and D u rsch F r i e d r i c h , U.S.. 3, 451, 992 (1969); C . A . , - 71, 59638 h (1969).

162. P f i z e r , Chas., and Go. Inc. F r . - 1, 518, 916 (1968); C . A . , 71, 6525n (1969).

163. N . I . Ge l ' pe r in , L.M. Klyueva and L.L. Stremovski i Khim.-Farm. Zh., - 4, 23(1970)., L .A . - 72, 1 3 6 4 6 3 ~ (1970).

164. Kotula Zbigniew, Kunstharz - Ion enaus tauscher P lenar - Diskuss ionsvor t r . Symp. 415(1968); C . A . , - 74, 45569 m (1971).

165. P e t e r P. Regna, I s a i a h A. Solomons, I11 and Richard Pasternack; U.S. - 2 , 555, 762, 763 (1951), C . A . , - 45, 7754 (1951).

166. Idem, i b i d . , U.S. 2 , 555, 760, 761 (1951); C.A. - 45,7754 (1951).

167. Idem, I b i d , - 2, 560, 890 (1951) , C . A . , 45, 9228(1951).

168. Wm. Braker, Wm. A. Lett and Andrew E.O'keefe; U.S. , 2, 631, 143 (1953), C . A . , - 47, 7742 (1953).

169. Leon J. Heuser, Morris A. Dol l ive r and Eric T. S t i l l e r ; J. Am. Chem. SOC. , 75, 4013 (1953); C.A. , 48, 10624 (1954).


170. Wm. A. Lott, Jack Bernstein and Leon J. Heuser, U.S. - 2, 669, 419 (1953), C . A . , - 48, 3642 (1954).

171. Robert B. McCormack, Asger F. LagLykke and David Perlman; U.S. - 2 , 656, 300 (1953), C.A., - 48, 3643 (1954).

172. Hiroshi Ikeda,Hatsuko Ikeda, I t s u o Fujimaki, Kenji Shiroyanagi, Mitsuhiko Katayama and Junichi Sugoyama, J. Sci. Research IASt., - 48, 216 (1954); C.A. 49, 4239(1955).

173. Sadajiro Yabuta and Hiroshi Ikeda; Japan 4297 (1954); C .A. , - 49, 10589 (1955).

174. David A. Johnson; U.S. - 2 , 717, 893 (1955), C.A. 50, 12407 (1956).

175. Sadajiro Yabuta and Hiroshi Ikeda; Japan, 98 (1956); C.A. - 51, 3938 (1957).

176. F r i t z Ziegler; U.S. - 2 , 857, 376 (1958), C . A . - 53, 3611 (1959).

177. T e i j i r o Yabutta, Hiroshi Ikeda and J o Tanaka; Japan 8295 (1955), C.A. - 51, 18494 (1957).

178. Virgi l V. Bogert; U.S. - 2, 872, 442 (1959); C.A. - 53, 13518 (1959).

179. Leon J. Heuser; U.S. - 2 , 2921, 062 (1960); C . A . , - 54, 10245 (1960).

180. Minoru Tanaka; Hiroshi Yonehara, Takuro Soda and Yusuke Sumiki; J. Antibiot ics ; - 4, 20(1951), C.A.,%, 1720 (1952).

181. Hiroshi Ikeda and Hatsuko Ikeda; J. Sci. Research Inst . , - 45, 161 (1951); C . A . , - 46, 4739 (1952).

182. Walter A. Winsten; U.S. - 2 , 609, 369 (1952), C . A . , - 47, 11670 (1953).

183. E . J . Goett and R . J . Taylor; U.S. - 2, 676, 960 (1954), C . A . , - 4R, 9023 (1954).

184. Adilia Seixas Antao; Congr. Luso-Espan farm, 277 (1952) ; C . A . , 48, 13784 (1954).

185. Hiroshi Ikeda; Kenji Shiroyanagi, Hatsuko Ikeda, I tsuo Fujimaki, Mitsuhiko Katayama, Saichi Yamagiwa and Teruo Motokawa; J. Sci. Research Ins t . , - 48, 2 1 1 (1954); C .A. , - 49, 4238 (1955).

STREPTOMYCIN 587

186. S.I. Kaplan; N . G . Semizhen, E.T. Tuf f l in , N.P. Krivosheev, A . I . Volakhanovich and V.S. Gin'ko, U.S.S.R., 117, 630 (1959), C . A . , 53, 17438 (1959).

~

-

187. Egon S. Andersen and Henrik Heding; Dan. , 106, 272 (1967); C.A. , 67, 1 4 8 3 3 ~ (1967).

188. Heding, Henrik, Joergensen, J. Hartvig and Andersen Egon Steen; J. Ant ib io t . , - 21, 361 (1968); C . A . - 70, 27601d [ 1969).

189. Henry Fischbach; J. Am. Pharm. Assoc. Sc i . Ed. - 37, 470 (1948); C . A . , - 43, 7195 (1949).

190. Jack D. Sa r t akof f , U.S. - 2 , 547, 231 (1951), C . A . , - 45, 6352 (1951).

191. Sumio Umezawa, Tsutomu Tsuchiya; Testsuro Yamasaki, Hi rosh i Sano and Yoshikazu Takahashi; J. Am. Chem. SOC. - 96, 920 (1974).

192. P.G. Sammes, "Topics i n A n t i b i o t i c Chemistry", Vol. I, E l l i s Horwood Limited, England (1977).

193. W.H. Horner, "Streptomycin", i n "Ant ib io t ic II-Biosyn- thes i s " Ed. Go t t l i eb , D. and Shaw, P .D . , Springer- Verlag, N e w York (1967).

194. J . F . S n e l l ; IIBiosynthesis of a n t i b i o t i c s " , Vol. I , Academic Press, New York (1966).

195, D . J . Candy, N . L . Blumson and J. Baddiley; Biochem. J . , 91, 31 (1964).

196. D.J . Candy and J. Baddiley; Biochem. J . , 96, 526 (1965), C.A. - 63, 7248 (1965).

197, M. Silverman and S.V. Rieder; J. Biol. Chem. 235, 1251 - (1965).

198. J. Burton, W.H. Horner and G.A. Russ; J. Biol . Chem. , L42, 813 (1967). -

199. G . D . Hunter and D.J .D. Hockenhull; Bio Chem. J . ; 59, - 249 (1955).

200. R.M. Bruce, H.S. Raghib and H. Weiner; Biochem. Bio- phys. Acta., 158, 499 (1968).


201. W. H. Horner and G . A . Russ; Biochem. Biophys. Acta; 237, 123 (1971). -

202. M.H.G. Munro, M. Taniguchi, K . L . R inehar t , Jr . , D. G o t t l i e b , T.H. Stoudt and T.O. Rogers; J. Am. Chem. SOC., 97, 4782 (1975).

203. W.H. Horner; J. Biol . Chem., 239, 578 (1964); C . A . , - 60, 5777 (lY64).

204. K.L. R inehar t , Jr., and T. Suami; "Aminocycletol A n t i - b i o t i c s " , American Chem. SOC., Washington (1980).

205 . H. Grisebach; Adv. Carbohydr. Chem. Biochem., 34, 81 - (19 78) .

206. B. Kniep and H. G r i s e b x h ;

207. S. Maier and H. Grisebach; Biochem. Biophys. Acta,

J. An t ib io t . , 23, 416(1980).

586, 231 (1979). -

209. S.A. Waksman and H. C h r i s t i n e K e i l l y ; J . i n f e c t i o u s Diseases; - 75, 150 (1944) , C.A. , - 39, 2089 (1945).

210. W.H. Feldman and H.C. Hinshaw; Proc. S t a f f Meeting Mayo C l i n i c , 2, 593 (1944), C . A . , - 39, 4677 (1945).

211. M.I. Smith, and W . ' l . McClosky, U.S. Pub. Heal th Reports , - 60, 1129 (1945); C.A. , - 39, 359 (1945).

212. A. Schatz and S.A. Waksman. , Proc. SOC. Expt l . Biol . Med., - 57, 244 (1944); C .A. , - 39, 1193 (1945).

213. Guy P. Youmans and John C. McCarter; Am. Rev. Tuberc. , 52 , 432 (1945). C.A. 40, 1226 (1946). - -

214. J . D . Wassersug; N e w Engl. J. Med.; 235, 220 (1946); C.A; - 40, 5832 (1946).

215. Guy P. Youmans and John C. McCarter; Quart . Bul l . Northwestern Univ. Med. School. , 19, ZlO(1945); C.A. , - 40, 6680 (1946).

216. Tom F. Paine, Koderick Murray and Maxwell Finland; N e w s Engl. J. Med. - 236, 748(1947); C . A ; - 41, 5262(1947).

STREPTOMYCIN 589

217. Joseph D. Wassersug; New Engl. J. Med., 237, 2211947); C.A. - 41, 5981 (1947).

218. H. Corwin Hinshaw and Wm. H. Feldman; Ann. N . Y . Acad. Sc i . , - 48, 175 (1946); C.A. , - 41, 810 (1947).

219. F r i t z T. Callomon, John A. Kolmer, Anna M. Rule and Alber t J. Paul ; Proc. SOC. Expt l . Biol . Med., - 63, 237 (1946); C .A. , - 41, 1323 (1947).

22U. Wm. H. Feldman; Trans & Stud ies Col l . Phys ic ians P h i l a - 14, 81(1946); C . A . , - 41 , 2168 (1947j.

221. C. Levad i t i and A. Vaisman, Bul l . Acad. Nat iona le Med; - 131, 173(1947); C . A . , - 42, 2353 (1948).

222. Geo Brownlee and C.R . Kennedy; B r i t . J . Pharmacol, - 3, 37(1948), C . A . , - 43, 3100 (1949).

223. M . I . Smith, E.W. Emmart and W.T. McClosky; Am. Rev. Tubere; - 58, 112(1948); C . A . , - 43, 3878(1949).

224. Antonio t ir ignolo; Minerva Med., - 39, 1, 16 (1949).

225. Morr is Solotorovsky, Henry S i e g e l , E l i zabe th J. Bugie and Franc is J. Gregory; Am. Kev. Tuberc; - 60, 366 (1949).

226. F. Gongalez and J. Q u i n t a n i l l a ; eo l . SOC. b i o l . San t i ago Ch i l e - 6 , 79(1949); C . A . , - 44, 4151 (1950).

227. M.1. Smith, E . L . Jackson and H. Bauer; Ann. N.Y. Acad. Sci . , 52, 704(1949); C . A . , - 44, 5011(1950).

228. R.J .W. Rees and J . M . Robson; B r i t . J. Pharmacol, - 5, 77(1950); C . A . , - 44, 6032 (1950).

229. E. Hoggarth and A.R. Martin, B r i t . J. Pharmacol., - 5 , 188(1950); C.A. , - 44; 8534 (1950).

230. Gerhard Domagk, Beitr Klin. ‘Tuberk - 102, 603(1950); C.A. , 44, 10144(1950).

231. H. Corwin Hinshaw; Am. J. Med., - 9, 654 (1950), C . A . , - 45, 1734 (1951).

232. P. Huebschmann, F . J . Pothmann and R. Schaukowski Z . Tuberk, - 96, 14(1950), C . A . , L 45, 2083 (1951).

233. S.A. Waksman, Minerva Med., - 41, 157(1950); C . A . , - 45, 3502 (1951).


234. Donald S. King; New Engl. J. Med. - 243, 530(1950); C.A. , - 45, 5316 (1951).

235. Alfred G. Karlson; Karl H. Pfue tze , Uavid T Carr., W i l l i a m H. Feldman and H. Corwin Hinshaw; Proc. staff Meetings Mayo C l i n i c , - 24, 85 (19491, C.A. , - 45, 5322 (1951).

236. Lynn A. James, Leroy J. S ides , Wm. E. Dye and Vera F. Dyke., Am. Rev. Tuberc. , - 63, 275 (1951); C.A. , - 46, 3648 (1952).

237. Roger Linz; Ann. SOC. roy Sc i . Med. e t n a t . Bruxel les , - 4 , 142 (1951); C . A . , - 46, 1089 (1952).

238. Alber t0 Tesi and Domenico Pisani; Sperimemtale, 102, 298(1952); C . A . , - 47, 2881 (1953).

World Med., - 6, 245 (1949); C x - 47, 6555 (1953).

240. E. Chauvin, A. Orsoni and H.F. Chauvin, J. Urol. Med.

239. G . F r o n t a l i , Ann. Paed ia t . , 171, 286 (1949); Abs t r ac t s

Chi r , - 56, 239(1950), C . A . , - 47, 7674(1953).

241. G. M i s s i r o l i ; Bol l . O c u l i s t , - 29, 317(1950); C.A. , - 47, 8264(1953).

242. Alan C . Woods, Ronald M. Wood and Horward A. Naquin; Arch. Ophthalmol-, - 43, 834(1958); C.A. , - 47, 10065(1953).

243. E. Benhamm and F. Destaing; Bul l . Acad. Natl. Med. - 133, 301(1949); Excerpta Med. Sec t . - 3 , 39(1950), C.A. , - 47, 10739(1953).

244. C. Bazzicalupo, A. P o r t e l l a and M. C o n t i e r i , I1 Farmaco Ed. S c i . , - 8, 538 (1953), C . A . , - 48, 879(1954).

245. Sumner S . Cohen. Lynn Johsen. M.R. L i ch tens t e in and Wm. J. Lynch, Am'. Rev. Tuberc:, - 68, 229(1953), C .A. , - 48, 6586 (1954).

246. Ryosuke Katayama, Yasuto Itami, Kauji oya, J u n j i Tanaka and E i j i Maruno; Ann. Tuberc., 2, 13(1954); C.A. , - 49, 5664 (1955).

247. Bet ty Croshaw, Proc. SOC. Appl. Bac te r io l . - 14, 131 (1951); C . A . , - 49, 5680(1955).

STREPTOMYCIN 591

248. P. Mutschler and G . Hasche-Klunder., Beitr - k l i n . Tuberk., 114, 406 (1955). C.A. - 50, 2844 (1956).

249. Kimifusa Mizunoe, Japan, J. B a c t e r i a l , - 7, 293 (1952), C . A . , - 50, 2860 (1956).

250. Roger S. Mi tche l l and J. C a r r o l l Bel l . ; A n t i b i o t i c s Monographs, 11, 1 (195s) ; C . A . , 2, 22520 (1959).

251. Arata Fukui, Kekkaku, - 34, 793 (1959); C . A . , - 54, 17709 (1960).

252. Hi rosh i Takahashi; Kekkaku, - 34, 607 (1959); C . A . , 54, 21499 (1960).

253. F . R . Heliman, Proc. S t a f f . Meetings Mayo C l in i c , - 19, 553 (1944), C . A . , - 39, 4387 (1945).

254. P. Nelis, A. Lafontaine, H. Massa and S . de Maeyer Cleempoel; Rev. Immunol.; - 16, 305 (1952); C . A . , - 47, 1230 (1953).

255. "The United S t a t e s Dispensatory", 27th Ed. J. B. Lippin- g o t t Company, Lippingot t , Toronto (1973).

256. A. Grollman and E . F . Grollman, "Pharmacology and Therapeut ics" , 7th ed . , Lea and Febiger , Ph i l ade lph ia (1970) .

257. C . A . Elias, M. Miranda, M.A. Salgado and L . R . Caldas , Mammalian Cytogenet. Related Probl . Radiobio l ; Proc. Symp. Sao Paul0 Rio de J a n e i r o . , 335 (1962); C . A . , - 63, 4698 (1965).

258. D. Jones, H . J . Metzger; A. Schatz and S.A. Waksman; Science 100, 103 (1944). -

259. Car los Fernandez; Bol. Of i c ina S a n i t . panamer 29, 400 (19SO), C . A . , 44, 6970 (1950).

260. Abraham I . Brandcand Wesley W. Spink. J. Immunol., 65, 185 (1950); C . A . , - 44, 10172 (1950).

261. Char les A. Werner and Vernon Knight, J. Immunol, - 65, SO9 (1950); C .A; - 45, 1251 (1951).

262. L. O l i t z k i , H. Farkas m d D. Friedman; In t e rn . Congr. Microbiol. Abstrs . o f Papers , 5 t h Congr. Rioa de J a n e i r o , 88 (1950); C . A . , - 48, 1480 (1954).


263. Jozef Parnas and Stefan Stepkowski., Ann. Univ. Mariae Curie-Sklodowska, Lublin Polonia; - 5, 439 (1950); C.A. - 48, 4118 (1954).

264. Zof ia Kuranocowa; Ann. Univ. Mariae Cur ie Sklodowska, Lublin-Polonia, 2, 145 (1953/54). C.A. , - 49, 6464 (1955).

265. W.W. U m b r e i t . , J . Biol . Chem. - 177, 703 (1949); C . A . , - 43, 4334 (1949).

266. E . L . oginsky, P.H. Smith, and W.W. Umbreit, J. Bact., - 58,761 (1949); C.A.,44, - 2078 (1950).

267. J. Douglas Reid, Muriel M. Jones and Evelyn C. Bryce; A n t i b i o t i c s and Chemotherapy - 2, 351 (1Y52), C .A. , - 47, 3920 (1953).

268. Eugene A. Gorzynski and Erwin Neter, A n t i b i o t i c s and Chemotherapy, - 3, 798 (1953), C.A., - 48, 7686 (1954).

269. H. Leiguarda and A. Peso, Arch. farm. Y . bioquim. Tucuman; 7, 45 (1955); C . A . , - 50, 14856 (1956). - -

270. J. Michel, M. Cluzel-Nigay, R. Vaurs and R. Cluzel. , Compt. Rend. SOC. Bio l . , - 157, 548 (1963). C . A . , 59, 13133 (1963).

271. G.E. P lunket t ; J. Gen. Microbiol . , - 38, 189 (1965). C.A., 63, 2150 (1965).

272. Ronald Gwatkin, Can. J. Comp. Med. Vet. S c i . , - 14, 157 (1950), C . A . , - 45, 256 (1951).

273. Kuan-How Chang and Henrik J. S t a f s e t h , Poul t ry S c i . , - 29, 130 (1950).

274. H.A. Reiman, W.F. Elias and A.H. Price, J. Am. Med. Assoc. - 128, 175 (1945).

275. Henry Welch, Robert J. Reedy and S tan ley W . Wolfson. J. Lab. Cl in . Med., - 35, 663 (1950); C.A. , - 44, 9000 (1950).

276. I p p e i Nagao, Tohoku J. Expt l . Med. - 58, 169 (1953); C .A. , 48, 6595 (1954).

277. H.K. Robinson, Dorothy G. Smith and O.E. Graessle, Proc. SOC. Expt l . Biol . Med., - 57, 226 (1944).

STREPTOMYCIN 593

278. I . V . Dyadyunova, L. 1. Gerasimenko and A.A. Yusufcwa, Ped ia t r iya , - 5 , 26 [1955). C . A . - 5 0 , 5915 (1956).

279. L . F . H e w i t t , B r i t . J . Expt l . pa th . , 31, 597 (1951). C . A . , 45, 8077 (1951).

280. Y . ' l ' . Chang; In te rn . J. Leprosy, 22, 331 (1954). C . A . , 49, 10511 (1955). -

281. E. T o r e l l a G i l . , F o n t i l l e s , 3, 2 1 (1952); Excerpta Med., - 8, 69 (1954)., C.A. - 49, 1351-(1955).

282. Y.T. Chang; In t e rn . J. Leprosy, 24, 307 (1956). C . A . , - 53, 8981 (1957).

283. Antonio Carlos Mauri, Rev. I n s t . Med. t r o p . Sao Paulo, - 2 , 5 (1960); C . A . , - 5 5 , 26237 (1961).

284. Haruo Nishimura and Masafumi Shimohira, Ann. Rept. Shionogi Research Lab - 6 , 273 (1956), C . A . , - 51, 4563 (1957).

285. U.H. Heilman; F.R. Heilman, H.C. Hinshaw; D . R . Nichols and W.E. Herrell., Am. J. Med. S c i ; - 210, 576 (1945), C.A. , - 40, 1591 (1946).

286. L. Nassi and M. De t to r i , Boll. SOC. i t a l . b i o l . spe r . , - 30, 327 (1954); C . A . , - 48, 13066(1954).

287. P. Gugl ie lmet t i and F. Z in i , Rass. p a t o l . apparato r e s p i - r a t . - 5 , 547 (1955), C . A . , - 50, 132Y9 (1956).

288. Louis S. Goodman and Alfred G . Gilman "The Pharmacologi- c a l Basis of Therapeutics", 5 t h ed . , Macmillan Publ ishing Co., Inc. , New York (1975).

289. T. Honda, Repts. Research I n s t . Tuberc and Leprosy, Sc i . Repts. Research I n s t s . Tohoku Univ., - 3, 349 11952); C .A. , - 49, 14096 (1955).

290. R . B . S tebbins , O . E . Grassle and H . J . Robinson; Proc. SOC. Exptl. Biol . Med., - 60, 68 (1945)., C.A. - 40, 403 (1946).

291. D.D. Ruts te in , R .B . S tebbins , R.T. Cathcar t and R.M. Harvey; J. Cl in . Inves t iga t ion - 24, 898 (1945); Vol. - 40, 2529 (1946).


292. Hans Moli tor , Ot to E. Graessle , Samuel Kuna, Charles W. Mushett and Robert H. S i l b e r t ; J. Pharmacol., - 86, 151 (1946); C.A. - 40, 3186 11946).

293. H. Vanderhaeghe; Arch. i n t e rn . pharmacodynamie, - 81, 500 (1950); L . A . 44, 7430 (1950).

294. krna Chris tensen, Helge Hertz, Niels Riskaer and Gustav Vraa - Jensen, Acta Pharmacol. l o x i c o l , - 6, 201 (1950); U . A . , - 45, 260 (1951).

295. Klaus Wechselberg and Edelgard Weidenbusch; Ergeb. inn. Med. U. Kinderhei lk . , - 2 , 713(1951) ; C . A . , - 46, 3164(1952).

777(1952); C.A., - 47, 8903 (1953). 296. J . M . Robson and Roy Goulding; Proc. Roy. SOC. Med. - 45,

297. N.V. Svinkina; Trudy Akad. Med. Nauk S.S.S.R. 5, Voprosy Khimioterap-Bakterial. I n f e k i t s i i , - 1 , 130(1950), C . A . , - 49, 11882 (1955).

298. K.A. Veis ; Trudy Vsesoyuz. Obshchestva Fiziologov Biokh- imikov i Farmakologov Akad-Nauk S.S.S.K., 2 , - 198(1954); C.A. - 49, 8562(1955).

299. T. Manford McGee; l 'rans. Am. Acad. opthalmol. o to l a ry - ngol . , - 65, 222(1961). , C.A. , - 55, 1469911961).

300. Olavo Ehmke; Rev. P a u l i s t a Med., - 6 2 , 109 (1963); C . A . , - 59, 6883 (1963) .

301. R. Amann., Arzneimit te l - Forsch; - 1 2 , 887(1962); C . A . , - 58, 8331 (1963).

302. A.M. Kharitonova; An t ib io t ik i ; 1 2 , 830(1967); C . A . , 67, - - 115566Q(1967).

303. Kobert H. Dreisbach, "Hand Book of Poisoning" 5 th Ed., Blackwell S c i e n t i f i c Publ ica t ions , Oxford (1966).

304. "Br i t i sh Pharmacopoeia", Pharmaceutical Press, London (1958).

305. "l'he United States Pharmacopeia XIX"; United S t a t e s Pharmacopeial Convention, Inc . , Rockvi l le , Md. 20852, U.S.A. (1975).

306. Michael Barza and Richard T. Cheife; Am. J. Hosp. Pharm., - 34, 723 (1977).

STREPTOMYCIN 595

307. W.W. Umbreit; 2nd Congr. In t e rn . biochim; chim. b i o l . V I , Symposium. Sur l e mode d ’ac t ion des a n t i b i o t i q u e s , 63(1952); C . A . , - 47 , 4955(1953).

c e u t i c a l ana lys i s” , l n t e r sc i ence publ i shers , New York (1961).

308. Takeru Higuchi and Einar Brochmann - Hanssen. ,“Pharma-

309. John V. Scudi, Geo E. Boxer and Viola C. J e l inek . , Science, - 104, 486 (1946)., C . A . , - 41, 1014 (1947).

310. Albert Roax; Ann. Pharm. Franc., 6, 567 (1948). C . A . , - 46, 3595 11952).

311. W.J. Hall iday, Nature, - 169, 335(1952), C.A. - 46, 7171(1952).

312. S. sakaguchi; J. Biochem; - 5, 2 s ( 1 ~ 2 5 ) ; C . A . , - 19, 3506 (1925).

313. C . J . Weber, J. Biol. Chem. , 86, 217 (1930); C . A . , - 24, 2485 (1Y30).

314. H.Kiryakov; Compt. Rend. Acad. Bulgare Sci . 16, 43 (1963) , C . A . , - 60, 2723(1964) , Anal. Abst. - 12,1359(1965).

315. K . C . Wornick and G.O. Kuhn, Proc. Ann. Meeting Am. Assoc. Feed Microscopist 6 , - - 33(1958); C.A. , - 53, 15420 (1959).

316. Maurice Lemoigne; George Sanchez and Henry Girard; La i t , - 33, lS(1953); C . A . , - 47, 12674 (1953).

317. C . E . Neal and H.C . Ca lber t , J. Dairy Sc. - 38, 629(1955); C .A. , 49, 11197 (1955).

318. Takeo Matsui, Keiko Sano; Yoriyuki Akao and Mitsuo Kambayashi; Shokuhin Eiseigaku Zasshi , - 2 , 34(1961); C.A. , - 56, 10645 (1962).

319. O t to V. Viet inghoff - Scheel and Erich Hagendorff, Klin. Wochschr, - 29, 703(1951); C .A. , - 4 7 , 4939(1953).

320. Raymond Delaby and Fouad Stephan. , Ann. Pharm. Franc., - 8, 513 (1950) . , C .A. , - 45, 2147 (1951).

321. H. Kiryakov; Compt. Rend. Acad. Bulgare Sc i . , - - 16, 385 (1963) , C.A. , - 60, 5281 (1964).

322. K. Huttenrauch and J. Keiner, Pharm. Zentralh. 102, 615 (1963); Anal. Abst. , 1 2 , 308(1965) , C.A. , - 60, 7874(1964).


323. Idem.; Ger. , 34, 175 (19641, C . A . , 63, 6798(1965).

324. Umesh Chandra, J . N . 'I'ayal and Purna Nand; Z . Analyst Chem. , 238, 457(1968)., Anal. Abst. , 17, 2362(1969).

325. H. Penau, E. Sa ias and J. Ferdet ; Ann. Pharm. Franc; - 11, 740(1953); C .A. , - 48, 14118[1954).

505(1954); Anal. A b s t ; - 2, 1320(195S). 326. J . A . Gaut ier and F. P e l l e r i n ; Ann. Pharm. Franc., - 1 2 ,

327. M. Yamagishi and M. Yokoo; J. Pharm. SOC. Japan; 74 , 283 (1954). -

328. A. Viala, Trav. SOC. pharm. Montpe l l ie r ; 18, 10411958); C.A. , 53, 853411959).

329. Gabor B. Levy, P h i l i p Schwed and J. Warren Sacket t , J. Am. Chem. SOC. - 68, 528 (1946), C.A. , - 40, 3494 (1946).

330. C l a r k E. Bricker and W . Aubrey Vai l ; J. Am. Chem. SOC. L3, 585 (1951).

331. R. Goodey, T.E. Pharmacol. , 14 , C . A . , - 58, 7787

332. L. Doan and B . E - 96, 109 (1963);

Couling and J . E . Hart; J. Pharm. 122T (1962), Anal. Abst; - 10, 4359 (1963). 1963).

Riedel ; Can. Pharm. J . , Sci . Sec t . , C.A. 59, 6198 (1963). -

333. Antonio Proenca Mario Augusto da cunha; Biol. Escola Farm., Univ. Coimbra, Ed. c i e n t ; 23, 1 (1963). C . A . , - 62, 1515 (1965).

334. Michael E. Capl i s , Hussein S. Ragheb and Elwyn D. S c h a l l . , J. Pharm. Sci . , - 54, 694 (1965); C.A. , - 63, 1656 (1965).

335. S. Weissbuch and H.W. Unterman; s t u d i i ce rc . Chim., 14, 793 (1966), Anal. A b s t . , - 15, 1003 (1968). -

336. H.W. Unterman and S. Weissbuch, Pharmazie; 29, 752(1974); Anal. Abst. , - 29, 1 E 2 1 (1975).

337. John B. Conn and Sara L. Norman.; J . Cl in . i n v e s t , 28, - 837 (1949). , C.A. - 44. , 11024 (1950).

338. N . Konopik; O s t . Chemztg. , 55, 1 2 7 (1954), Anal. Abst. , - 2 , 3411 (1Y55 ) .

STREPTOMYCIN 597

339. G. Machek; Sc i . Pharm., 28, 97 (1960j, Anal. Abst. - 8 , 778 (1961).

-

340. D. L. Simpson, R . K . Kobcs; Anal. Chem.; 55, 1974 (1983); - Anal. Abst. - 46, 5E22 (1984).

341. 0. Folin and V. Cioca l t eu ; J. Biol. Chem. 73, 627 (1927).

342. "European Pharmacopoeia" Volume I I . , Council of Europe, Maisonneuve S.A., France (1971).

343. "Pharrnacopeia of t h e United S ta t e s " , 15 th Revis ion; Mack, Easton, Pa (1955).

344. George E. Boxer; Viola C. J e l i n e k and P a t r i c i a M. Leghorn; J. Biol. Chem., - 169, 153 (1947) . , - 41, 6594 (1947).

345. Wm. Eisenman and Clark E. Bricker; Anal. Chem. 2 1 , 1507 - (1949); C . A . , 44, 2703 (1950).

346. Grzegorz Bagdasarian and Krystyna Michalska, Gruz l ica , - 24, 1165 (1956); C . A . , - 53, 3351(1959).

347. 11. M . Doery and E . C . Mason; Anal. Chem. - 2 2 , 1038 (1950).

348. V . D . Kartseva, Yu. S. Checkulaeva, V.B . Korchagin and B.P. Burns, A n t i b i o t i k i , - 5 ( 4 ) , 50(1960]. Anal. Abst. , - 18, 1202(1961).

Korobitskaya; Zhur. Anal. K i m . , - 12(2) , 262 (1957) ; A n a l . Abst . , 2, 684 (1952).

Current Science 33(93 ; 272 (1964) ; C . A . , 61, 2435(1964).

351. A . F e r r a r i ; F.M. Russo-Alesi and J.M. Kelly; Anal. Chem.

349. B . P . Bruns, V . D . Kartseva; E.M. Savitskaya and A . A .

350. K . M . Desai, N. Narasimhachari and G. Ramana Rae,

L -

31(10) , 1710 (1959); Anal. Abst. , 7 , 2431 (1960). - -

352. W.H.C. Shaw and I . Fortune; Analyst , 87, 187(1962); C . A . , 57, 7735 (1962); Anal. Abst . , - 9 r 4 4 6 0 (1962).

353. A. Sauciuc and L. Ionescu; Revta Chem. , 17 , 47(1966), C . A . , 64, 19319 (1966); Anal. Abst. 1 4 ; 3510 (1967). - -

354. Rybinska Krystyna; Rocz. Pnns tw. Zakl. Ilig. 29(3) 287 (1978); Anal. A b s t ; 36, 4 F 4 2 ; - 3 7 , 2 F36 (1947). -

355. Ikijiwnra, 1 1 . ; Ann. Rep. I'akeda. Lab. - 14, 23(1955); C . A . , S O , 5821 (1956).


356. Vivina 0. Angeles 0 . ; Bol. SOC. Quim: Peru; 21, 189(1955); C . A . , 50, 8970 (1956); Anal, Abst. - 3, 3446 (1956).

3 5 7 . Stanley E. Katz ; J. Agr. Food Chem., - 10, 39(1962), C . A . , - 57, 11620 (1962).

358. Marshal l , E .K. , Jr . , Blanchard, K . C . , and Buhle, E . L . , J . Pharmacol Exptl . Therap -- 90, 367 (1947); C.A; - 42, 630 (1948).

359. B. Borowiecka; Med. Doswiadczalna i Mikrobiol. 9, 23 - (1957); C . A . , - 52,8'277e(1958).

(1958); C .A. , - 53, 2336(1959) , Anal. Abst. - 6 7 1503(1959). 360. V.D. Kartseva and B.P. Bruns; A n t i b i o t i k i , 3 (5 ) , 39

361. A.M. Wahbi, H. Abdine and S.A. Rafik; Pharmazie, - 32 (11) Y 690(1977); Anal. Abst. - 34, 5E26 (1978).

362. F. Monastero; J. Am. Pharm. Assoc. Sc i . Ed., 41, 322 - (1952).

363. L.K. Valedinskaya; A n t i b i o t i k i i ikh Primenenie, - 22(1) , 82(1952); C . A . , - 50, 3711 (1956).

263 (1955); C . A . , - 50, 10843 (1956). 364. J . A . K . V ie i r a and N . A . Pere i r a ; Rev. b r a s i l . farm. 36, -

365. J.J. S z a f i r and E.O. Bennett . , Science, 117, 717(1953).

366. G.C. Ashton, M.C. Fos te r and M. Fa ther ley ; Analyst , - 78, 581 (1953).

367. H. K . Banerjee; Indian Pharmacist , 10, 301 (1955). -

368. E.P. Schulz and A. Posada R. , Revta. SOC. Quim. Mex., l l ( 4 ) 114 (1967), Anal. Abst, - 15, 6258 (1968).

369. R . R . Buck; W . J . Mader and H.A. F red ian i , Bol. Col. Quim. Puerto Rico,:, 6 , 9(1949) ; C . A . , 44, 282(1950).

370. R. Natarajan and J . N . Tayal. J. Pharm. and Pharmacol

-

- 9 326 (1957); C . A . , - 51, 15889 (1957); Anal, Abst, - 4, 3423 (1957).

371. Dulce Mello Fontes; Anais Fac. Farm. Univ. Recife 3, 33 (1960), C.A. , 55, 15835 (1961).

STREPTOMYCIN 599

372.

373.

374.

375 *

376.

377.

378.

379.

380.

381.

382.

383.

384.

385.

386.

E.Tibaldi , 11 Farmaco-Ed. S c i . , 8 , 160 (1953).

I. Sz i l agy i and I. Szabo, Nature, 181, 52 (1958) Anal. Chem. - 6, 3122 (1959).

1. Hideo Kamata, Hirobumi I r i s a , Yasuo Fujimoto and Kazukogoto; J. An t ib io t i c s - 6 , 231 (lY53) ; C . A . , - 53, 6333(1959).

J. Masquelier; Bulb-trav. SOC. pharm, Bordeaux - 87, 53 (1949); C.A. 46, 2114 (1952).

E.M. Savi tskaya and V . D . Kartseva; Zhur. Anal. Khim. - 8, 46 (1953); C . A . , - 47, 5070 (1953).

L . R . Mattick; Dissert. Abstr. 15, 1 (1955); Anal. Abst., 2 , 2223 (1955).

Eduardo M. B a r i l a r i and Miguel Katz; Anales asoc. quim.

-

arg. 46, 310 (1958); C . A . - 54, 16741 (1960); Anal. Abst. - 7 , 710(1960) .

Eduardo M. Barilari , Eduardo Ocaranza and Arturo Teran Navarro; Publ. Inst . Inves t . Microquim. Univ. Natl . L i t o r a l ; 25, 23, 10 (1959-60); C . A ; 57, 7388 (1962).

- -

K. Basu and B . N . Dutta; J. Proc. I n s t . Chemist, - 34, 142 (1962); C . A . , - 58, 4378 (1968).

J a ros l av Bartos and Jean Francois Burt in; Ann. Pharm. Franc. - 19, 769 (1961); C.A. - 57, 957 (1962).

K.C. Agrawal and B . N . Dutta; Sc i . and Cul ture , 27, 450 (1961); C . A . - 56, 14401 (1962); Anal. Abst., - 1 0 , 5 2 5 (1963).

M.P. Yavors 'k l i ; c 34, 1E12 (1978).

ti. Nour El-Deen, N.M. Dessouky .and M. El-Kirdani ; Bul l . Fac. Pharm. Cairo Univ. - 9, 267 (1970).

E.M. B a r i l a r i , M. Cobos and A. ch. D e Gepner; Arch. Bioquim.,Quim. Farm; 89 (1969); C . A . , - 47, 146455 b (1971).

Farm. Zh., - 5, 26 (1976), Anal. Abst.

Y . A. Beltagy, A. I s s a and S.M. Rida; Pharmazie, 31(4) 258 (1976) ; Anal. Abst. , - 31, 5E26 (1976).


387. Duda Erno; Analyst Biochem. SJ ( 2 ) , 651 (1973) Anal. Abst. 25, I 2561 (1973).

388. Duda Emo, L. Marton and G. Kiss; Biochem. Med., - 15, 330 (1976); Anal. Abst. 32, - 5D42 (1977).

Anal. Abst, - 33, 1D38 (1977). 389. Fukumoto Hisako; Eisei Kagaku, - 22, 380 (1976)

390. M . M . Alykov; A n t i b i o t i k i , - 25, 831 [1980), Anal. Abst, - 41, 1D41 11981).

391. Y. F u j i t a , I . Mori and S. Kitano; Chem. Pharm. Bul l . , - 31, 1289 (1983); Anal. Abst. - 46, 1E15 (1984).

392. N.M. Alykov; A n t i b i o t i k i , - 29, 336 (1984); Anal. Abst. , - 46, 12D69 (1984).

393. N.M. Alykov, N . A . Fek l i s tova , T.V. Alykova and T.V. Kamzel; Zh. Anal. Khim., 38, 1856 (19831, Anal. Abst. - 46, 8E37 (1984).

394. R.C. Evangel i s ta and E.E.S. Schapoval; Rev. Cienc. Farm., 2, 29 [1983); Anal. Abst., 47, I 6E37 (1985).

395. T.E. Divakar, M.K. Tummuru and C.S.P. S a s t r y ; Indian Drugs, z ( 1 ) , 28(1984); Anal. Abst. 4 7 , 7E40 (1985).

396. Elwood T i t u s and Jose f Fr ied ; J. Biol . Chem. 174, 57 - [ 1948).

397. S t an ley E. K a t z ; J. Agr. Food Chem., 8, 501 (1960). C . A . , 55, 19063 (1961)., Anal. Abst.-8, - 2669 (1961).

398. George E. Boxer and Viola C. Je l inek; J. Biol . Chem.,

I

- 170, 491 (1947).

399. Viola C. J e l i n e k and George E. Boxer., J. Biol . Chem. 175, ,367 (1948); C . A . 43, 710 (1949). Y -

400. F. Faure and P. Blanquet; Bul l . SOC. Pharm. Bordeaux, 100, 105 (19611, C . A . 56, 1731 (1962). - -

401. Idem., Ib id 101, 7 (1962) , C . A . , 57, 8661 (1962), Anal. - Abst. , - 10, 3858 (1963).

402. Idem., l b i d , 102, 79 (1963); C . A . , 59, 13484 (1963). - -

STREPTOMYCIN 601

403. Idem.,Clin Chim. Acta 9 , 292(1964); C . A . , - 61, 535(1964) Anal. Abst , - 1 2 , 3491 (1965).

404. Idem. , Bull . SOC. Pharm. Borcleaux, 102, 239 (1963); C . A . , - 65, 3669 (1966).

405. Idem., I b i d ; - 104, 89(1965); C . A . , - 64, 7028 (1966).

406. Idem; Pharm. b i o l . , - 4 , 433 (1966) , C . A . , - 66, 44189 (1967).

407. J. Harnann, W. Heeschen, A. T o l l e and G. Milchwissen- s c h a f t H a h n ; s , 139 (1975). Anal. Abst. - 29 (1979).

408. N.M. Alyokov; Zh. Anal. Khim. - 36, 1387 (1981), Anal. Abs t . , - 42, 3D52 (1982).

409. F. Ukumura, T. N o j i r i and D. Tquri i ; J . Agr. Chem. S O C . , - 48, 397 (1974).

410. C.V. S t . John, D.E. F l i c k and J . R . Tepe; Anal. Chem. - 23, 1289 (1951).

411. C . K . Kohli and L.M. V i j a y Prasad; Indian J. Pharm. Sc . ,43 - 112 (1981), Anal. Abst. - 4 2 , 5E29 (1982) .

412. N . Conca and H . J . Pazdera , Acad. S c i . , E , 596 (1965) Anal. Abs. I 1 4 , 2200 (1971) .

413. t l . G a l i n a , B.N. Kolarz and K . Dzieg ie lewski ; J . Chroma- t o g . 179, 173 (1979). Anal. Abst . , - 39, 1E19 (1980).

414. S.E. Bresler and G . V . Samsonov; Kol lo id Zhur. 18, 155 - (19561, Anal . , Abs t . , 4 - 3193 (1957).

Kenkyusho Hokoku - 36, 595, 604, 612 (1960) , C . A . , - 5 6 , 10707 d , f (1962).

415. S. F u j i t a , 11. Kawabe, and M. Yanagi ta ; Rikagaku

416. J . N . P e r e i r a ; J . Biochem. Microbiol Technol. Engng; 3, 79 (1961); Anal. A b s t . , - 9 , 2895 (1962).

417. M . Uatui-ner; 'IY. Lellingr. Khim-Farmatser. I n s t . 149 (1962) Chem. A b s t . - 6 0 , 110981 f (1964).

418. 2. k o t u l a ; Kuns tha rz - Ionenaus tausche r , Plenar - D i s k u s s - i o n s v o r t r . Symp. 415 (1970) , C . A . , z, 45569m 11971).

419. 1 1 . . J . S t c l ' 1; J . Chromat , - 4 0 , 71 (1969); Anal. Abst , - 19, 639 (1970).


420. R .E . Horne and A.L. P o l l a r d ; J. Bact. - 55, 231 (1948).

421. Walter A. Winsten and Edward Eigen; J. Am. Chem. SOC. - 70, 3333 (19481, C . A . , - 43, 820 (1949).

422 . I . A . Solomons and P .P . Regna; J. Am. Chem. SOC. 72, 2974 (1950).

423. F.H. S t o d o l l a , O.L. Shotwel l ; A.M. Borud, R.G. Benedict and A.C . R i ley , Jr. , J. Am. Chem. SOC. - 73, 2290 (1951).

424. Zuzanna Kowezyk., Med. Deswiadczalna i Mikrobiol . , - 6 , 397 (1954) ; C.A. - 49 , 4944C(1955).

425. D.H. Peterson and L.M. Reineke; J. Am. Chem. SOC., - 72 3598 (1950).

426. W.T. Sokolski and N . E . Lummis, An t ib io t . 6 Chemother; 11, 271 (1961); Anal. Abst. - 9, 335 (1962). -

427. H. Heding; Acta Chem. Scand. 20, 1743 (1966) , Anal. Abst. - 14, 7105 (1966).

428. Idem, Chromatogr. E lec t rophor , Symp. I n t . 4 th , 569

429. Satoru Makisumi, J . Chem. SOC. Japan, 3, 737 (1952);

430. S. Kondo, M. sezaki and M. Shimura; J. A n t i b i o t i c s , 17,

(1968) , C . A . 72, 59120 (1970).

C.A. , 47, 5848 (1953).

1 (1964), Anal. Abst. - 1 2 , 2996 (1965).

- -

431. Radha Pant and Harish C . Agrawal; 2 . Physiol . Chem. - 335, 203 (1964) C . A . , - 61, 2164 (1964).

432. M. Albu-Budai and H.W. Unterman; Revta. Chim., 18, 173 - (1967).

433. E. Pacsu; T.P. Mora, and P.W. Kent, Sc ience , - 110, 446 (1949); C .A. - 44 , 2417 (1950).

434. R.M. Greenway, P.W. Kent and M . W . Whitehouse; Research (London) - 6 , Suppl. No.1 6s(1953) , C .A. , - 48, 1203i (1954).

Analyst , 86, 132(1961) , 435.R.A. Cadenas and J.0. D e f e r r a r i ; - C . A . , - 55, 21984 (1961).

436. P. Castel, R. Mus and J. Storck; Ann. Pharm. f r a n c e 17, - 63 (1959) ; C . A . , - 5 3 , 17425 (1959).

STREPTOMYCIN 603

437. T.E. Coulingand H. Goodey; J. Pharm. Pharmacol. , - 1 5 ,

438. E . S o r o u i e c k a d i s s n e s pharm. pharmac. 2 2 ( 5 ) ,

295T (1963); Anal. Abs t . , - 1 2 , 1952 (1965) .

345 (1970) ; C . A . , 74, 79734 (1971) . 1803 (1972).

Anal. A E t . , 22,

439. M. von Schantz , I . I v a r s and K . Ruoppi-Viholainen, Farm. A i k a k , - 7 7 ( 9 ) , 173 (1968) ; Anal. Abst. - 18, 493 (1970) .

Anal. Abst. - 1 2 , 48U2 (1965); C . A . - 61, 2903 (1964).

441. P .A. Nussbaumer and M. S c h o r d e r e t , Pharm. Acta. Helv . ,

440. R . Hut tenrauch and J. Schulze ; Pharmazine, 1 9 , 334(1964);

- 40, 205 (1965); C . A . , I 6 3 , 432 (1965) . , Anal. Abst. l-3, 4389 (1966).

442. K.C. Guven and G. o z s a r i , E c z a c i l i k Bul . , 9 ( 2 ) 19(1967) ; C . A . - 67, 71008 (1967~. , Anal. Abst., 15, 2902-(1968).

443. R. Voigt and A.G. Maa Bared; J. Chromat., 36, 120 (1968); Anal. Abst. 17 , 3750 (1969) . -

444. T , Katayama and H. l k e d a ; S c i . Pap. I n s t . Phys. Chem. Res., 63, 49 (1969)., C .A. - 71, 128786U(1969).

445. ‘lomonori S a t 0 and H i r o s h i Ikeda; I b i d . , 59, 159 (1965); - C . A . , - 64, 12400 (1966).

446. T. Katayama and H. Ikeda; I b i d 60, 85 (1966); C . A . , - 66, - 58853n (1967).

447. R.M. Kl ine and T. Golab; J . Chromatog. - 18, 409 (1965)., C . A . , - 63, 11977 (1965).

448. P.A. Nussbaumer and M . S c h o r d e r e t , Pham. Acta Helv. , - 40, 477 (1965)., Anal. Abst. - 13, 7080 (1966).

449. M. ti . J . Zuidweg, J . C. Oostendorp and C. J . K . Bos; J. Chromatogr., - 4 2 , 552 (1969); C . A . - 71, 74072n (1969).

450. J. Iiamann, W. Heeschen and A. ‘Tolle; M i l c h w i s s e n s c h a l f t , 34, 479 (1979) ; Anal. Abs t . - 39 , 6F45 (1980). -

451. C. Mathis and J . P . Schmi t t ; Annls. pharm. f r . , 26, 727 (1968); Anal. Abst. 18 , 1950 (1970). - -

452. J.K. Pnunez; .I. A n t l b i o t i c s , 25, 677 (1972) .

JABER S . MOSSA ETAL. 604

453. H. Heding; Acta. Chem. Scand., 24, 3086 (1970).

454. i3 . B O r O W i e C i c a , J. Phamac. Pharm. - 25, 99 (1973), Anal. Abst. - 26, 2854 (1974).

455. Kay R. Bagon, J. High Kesolut. Chromatogr. Chromatogr. Commun., 2, 211 (197Y), Anal. Abst. - 38, 1E20 (1980).

456. T . J . Whall; J. Chromatogr., - 219, 89 (1981); Anal. Abs t . , - 42, 6E28 (1982).

457. B . Ra t t i , A. T o s e l l i , E . Be re t t a and A. Bernareggi; Farmaco. Ed. Prat . , 37, 226 (1982)., Anal. Abst. , - 44, 3D90 (1983).

458. G. Inchauspe, D. Samain; J. Chromatogr., 303, 27711984); Anal. Abst. ; - 47; 6E34 (1985).

459. M.C. Fos te r and G . C . Ashton; Nature , 172, 958 (1953) Anal. Abst; - 1, 561 (1954).

(1954); C . A . , - 53, 9335 (1959). 460. B i n j i Takahashi and Yasuj i Amano; cJ. A n t i b i o t i c s - 7, 81

461. J. Ph i l ippe ; Y. Jo l ch ine and 0. Alcaraz, Ann. Pharm. France, 15, 546 (1957); Anal. Abst. - 6 , 3123 (1959).

462. K . R . Paris and J . P . T h e a l l e t , Ann. I b i d . France. , 3, 436 (1962), C . A . , - 57, 16746 (1962).

463. J.W. Lightbrown and P. de Rossi;

464. C . Apreotesei and M. Teodosiu; Farmacia; - 10, 321(1962)

Analyst , - 90, 89(1965).

C . A . , E, 4375 (1965).

465. T. Katayama and H. lkeda; Sc i . Pap. I n s t . Phys. Chem. Res., - 62, 130 (1968).

466. Idem. I b i d , - _ 63, 49, (1969).

467. J u l i o Lopez Gorge and Miguel Monteoliva; J . Chromatogr., - 29, 300 (1967); C .A. - 67, 96642t (1967).

468. C. Garber and J. Dobrecky; Rev. Assoc. Bioquim. Argent. , 33, 180 (1963); C . A . , 7 2 , 35816j (1970).

469. S . Ochab , Pol . J. Pharmac. Pharm. - 25, 10511973); Anal. Abst ; 26, 2855 (1974).

STREPTOMYCIN 605

470. H.J.Langner, U.Teuf'e1, M . Siegert , Y. Fro:cmhold a n d Y. S e i d l e r , I : l e i s c h w i r t s c h a f t , - 5 3 , 557 (1973) .

471. Elwood T i t u s and J o s e f F r i e d ; J . B io l . Ohem., - 168, 392 (1947); C . A . , - 41, 4190 (1947) .

472. G.W.E. P l a u t and R . B . MeCorrnack, J. Am. Chem. SOC., 2, 2264 11949); C . A . , - 43, 7192 (1949) .

473. A . E . O'Keeffe; M.A. D o l l i v e r and E.T. S t i l l e r , J. Am. Chem. SOC., 71, 2452 (1949) .

474. C . Meyer, Chem. Tech. B e r l i n , - 7 , 281 (1955)., Anal. Abst., 2 , 2615 (1955) . -

475. S.A. Waksman; S c i e n c e , - 102, 40 (1945) , C . A . , - 39 , 4193 (1945).

476. David Prarner and Robert L. S t a r k e y ; S o i l . Sci., - 94, 48 (1962); C . A . , - 59, 15884 (1963) .

477. Sands, D.C. and S c h r o t h , M . N . , Phytopathology, - 60, 567 (1970); C . A . , - 73, 28072 (1970).

478. Andrew D. Hunt, Jr. and Mary B . Fe l l , J. Lab. C l i n . Med., 33, 886 (1948); C . A . , - 42 , 7361 (1948).

479. Reginald H. H a l l and Donald P. Young; Brit., 846, 168 (1960); C . A . , - 55, 4894 (1961) .

480. J i r o Tsukada and S o s h i c h i Kamegaya; Meiji Sh ika Kenkyu Nempo, 4 (1960); C . A . , - 57, 2530 (1962) .

481. E .N. Druzhinina and D . V . Suvork ina , A n t i b i o t i k i , - 7 , 825 (1Y42); C . A . , - 61, 2908 (19641.

482. Antao, A. S e i x a s ; Simoes, M.H. Ca r reco ; Rev. P o r t . Farm., - 18, d9 (1968); C . A . , - 70, 71103s (1969) .

483. L.D. S a b a t h , J . I . Zasey, P. ii.Ruch, L . L . S t a m p f , and F in land , M. ; Antirnici.nb. Ag. Chemolter; 8 3 (1970) , C . A . , - 75, 107921b (1971) .

484. C.W. Price, J .K .Nic l sen and H . Welch; S c i e n c e , - 103, 56 (1946); C . A . , - 40, 2186 (1946) .

485. F. Fcrnandez; Mic rob io l . Expan., 17, 209 (1964); C . A . , - 6 3 , 18639 (1965).


486. Vakulenko, N . A . , A n t i b i o t i k i , - 13, 1010 (1968); C.A. , - 70, 31701d (1969).

487. J. Jacquet and L. S teeg; Ann. f a l s . e t . f raudes ; - 46; 5 (1953); C . A . , - 47, 5996 (1953).

11947); C . A . , - 41, 3169 (1947).

184 (1949); C . A . , - 44, 3550 (1950).

53 (1952); C . A . , - 50,9484 (1956).

488. E . E . Osgood and S.M. Graham, Am. J. C l in Path. - 17, 93

489. D.A. Mitchison and C . C . Sp icer , J. Gen. Microbiol . , - 3,

490. Hector Enrique Cammarota; Rec. fac. cienc. quim. - 27,

491. L.K. Rubksova, Trudy Akad. Med. Nauk. S.S.S.R. Antibio- t i k i i ikh Primenenie, - 2 2 , 88 (1952), C . A . , - 50, 37116 (1956).

492. Erna Al tu re - Werber and Leo Loewe., Proc. SOC. Exptl . Biol . Med., - 63, 277(1946); C . A . , - 41, 1268 (1947).

493. Georges Sanchez and Andre Lamensans; Compt. rend . , 224, 1189 (1947), C . A . , 43, 7076 (1949). -

494. Yu. K. Veisfeiler and T.N. Zykova; U.S.S.R., 3, 702 (1962), C.A. - 58, 5979 (1963).

495. D.H. Heilman and Bet.ty McCray; Proc. S t a f f Meetings Mayo C l i n i c ; - 20, 145 (1945); C.A., - 39, 4640 (1945).

496. R.B. S tebbins and H . J . Robinson; Proc. SOC. Ep t l . Biol . Med., - 59, 255 (1945); C . A . , - 39, 4341 (1945).

497. Baudolino Mussa; Minerva p e d i a t , - 1, 372 (1949 - 45, 9594 (1951).

498. Gosta Wallmark, Acta Path. Microbiol. Scand., 397 (1951); C . A . , - 46, 3120 (1952).

- 29 ,

499. G. Ya Kivman and I . Ya Geitman; (1971); C . A . , - 75, 1158t (1971).

A n t i b i o t i k i ; - 16, 331

500. A . L . O l i t z k i and Z . O l i t z k i ; A n t i b i o t i c Med., 2 , 317 - (1956); C . A . , - 50, 14854 (1956).

STREPTOMYCIN 607

5U1. Murakami. Hajime; Kanzaki. Masako; Fujimoto- Chizuru and Haruta. Misao; Shokuhin Eiseigaku Zassh i , 12, 86 (1971); C . A . , - 75, 117193e (1971).

502. J.J. Mayernik, J. Ass. O f f . Agric. Chem., 44, 33(1961); Anal. Abst. , - 8, 3973 (1961).

Abst. , - 9, 2550 (1962). 5U3. S.E. Katz and J. Spock; Ib id . 44, 742 (1961); Anal. -

504. E.N. Druzhinina and D . V . Suvorkina; A n t i b i o t i k i , - 7,

505. J . J..Mayernik, J. Ass. Offic. Anal. Chem. - 52, 679

825 (1962)., Anal. Abs t . , - 10, 2877 (1963).

(1969); C.A. , - 71, 59682t (1969); Anal. Abst . , - 19, 2678 (1970).

506. A. E . Tebyakina and E.N. Druzhinina; Epidemiol , Mikro- b i o l . Infek. B o l e s t i , - - 8 , 48 (1971).) C . A . , 75, 40496w (1971).

507. Y.H. Loo, B.S. Skell, H.H. Thornbery, John Ehr l i ch , J.M. McGuire, G.M. Savage and J . C . Sy lves t e r ; J . Bact. - 50, 7Cl (1945); C . A . , - 40, 2495 (1946).

508. P. Koelzer and J . Giesen; Med. K l i n ; 44, 1442 (1949); C . A . , - 44, 3064 (1950).

509. H. C h r i s t i n e R e i l l y and Hazel overby Sobers , A n t i b i o t i c and Chemotherapy, - 2 , 469 (1952); C . A . , - 47, 7160(1953).

51u. Y.Ksnazawa and T.Kurarnata , ~ Nippon Kagaku Ryohogakukai Zassh i , - 19, 159 (1971); C . A . , - 75, 115799h [ 1971).

(1958); Anal. Abst. , - 6, 3196 (1959). 511. K . M . Shahani and M.C. Badami; J. Diary S c i . , 4 1 , 1510 -

512. S.A. Waksman and H. C h r i s t i n e K e i l l y ; Ind. Eng. Chem. Anal. Ed., - 17, 556 (1945); C.A. - 39, 4908 (1945).

513. J . M . McGuire, W. W. Davis , T.V. P a r k e and W.A. Dai ly; J. C l i n . I nves t . , 28, 840 (1949); C.A. , - 44, 10029(1950).

514. Yves Chabberc and Bernard Sureau; Ann. I n s t . Pas t eu r , - 73, 1130 (1947); C . A . , - 4 4 , 10027 (1950).


515. George V. Tap l in , Fred A. Bryan, Jack Presberg; Louis Jensen, Carl Hornberger, Edward Roche, Mary LouiseGautschi and Helene Drusch; S tanford Med. Bu l l . , - 8, 97(1950); C . A . , - 45, 3035 (1951).

516. Nakao I sh ida , Ken Ka tag i r i and Reiko Chida, Tohoku; J. Expt l . Med., - 54, 188(1951); C . A . , - 46, 3596 (1952).

517. R. Dewart, F. Naudts and W. Lhoest; Ind. Chim. Belge, I 32 , 272 (1967); C.A. , - 70, 60865 [1969).

Sharpe and A. J0nes;Analys.t; - 104, 201 [1979); Anal. Abst. , 37, 4E34 (1979).

518. J. W. Light-brown, R.A. Broadbridge, P. Isaacson, J . E .

- 519. W.H.O. Shaw and K.E . Duncombe; Analyst - 8 , 694(1963);

C.A. , 59, 15595 (1963).

520. N . D . I e r u s a l i m s k i i , I . V . Konova and N . M . Neronova; Mikrobiologiya, 28, 433 (1959); Anal. Abst. , - 8, 1729 (1961), C . A . , - 53722252 (1959).

521. M.A. El-Nakeep and R.T. Yousef; Arzneim-Forsch, - 20, 103 (1970); C . A . , - 72, 829Y7d (1970).

522. El izabe th J. Oswald and L i l a F. Knudsen; J. Am. Pharm. ASSOC., 39, 61 (1950); C . A . , - 44, 4200 (1950).

523. Mary C. Gibb; Am. J. Med. Technol. , - 17, 14 (1951), C.A., - 45,9610 (1951).

524. C.M. Whitlock, Jr., A.D. Hunt, Jr., and S.G. Tashman; J. Lab. Cl in . Med., 37, 155 (1951); C.A. , 45, 5755(1951). - -

525. V. Bonifas and Y. Chesni; Expe r i en t i a , - 4 , 355(1948); C . A . , - 43 , 1825 (1949).

526. Georg Hennberg and Wolfgang Waterstraat; Zentr . Bakt. Paras i tenk I Abt. Orig. , -., 258(1950); C . A . , 45, 3448 - [ 1951).

527. D. L. Simpson, R.K. Kobos; Anal. Chin!. Acta, 164, 273 c

(1984); Anal. Abst. , - 47, 9E25 (1985).

528. Arden W. Forrey, Andrew D. Blair, Michelle A. O ’ N e i l and Ralph E - Cutlex;. Methodol. Dev. Biochem., - 5, 235 (1976); Anal. Abst., - 34, 1D49 (1978).

STREPTOMYCIN 609

529. T. Ya Goncharskaya and V . G . Koroleva; A n t i b i o t i k i , - 24, 859 (1979); Anal. Abst., - 38, 6 D 68 (1980).

530. K.S. Schwenzer and J . P . Anhalt; Antimicrob Agents Chemother, - 23, 683 (1983) ; Anal. Abst. - 46, 5D54 (1984).

THIABENDAZOLE

Vijay X. Kapoor

1. Description

1.1 Name, Formula and Molecular Weight 1.2 Appearance, Color, Odor 1.3 Salts

2.

3.

4.

5.

6.

7.

8.

Physical Properties

2.1 Infrared Spectrum 2.2 Ultraviolet Spectrum 2.3 Optical Rotation 2.4 Melting Range 2.5 Solubility 2.6 Crystal Properties

Synthesis

Stability and Degradation

Metabolism and Phannacokinetics

Methods of Analysis

6.1 Elemental Analysis 6.2 Identification Color Test 6.3 Titrimetric Analysis 6.4 Bioassay 6.5 Polarographic Analysis 6.6 Colorimetric Analysis 6.7 Spectrophotometric Analysis

6.71 Ultraviolet 6.72 Infrared

6.8 Spectrofluorometric Analysis 6.9 Chromatographic Analysis

6.91 Paper Chromatography 6.92 Thin-Layer Chromatography 6.93 Gas Chromatography 6.94 High Performance Liquid

Chromatography 6.10 Miscellaneous

Limits of Tolerance

References


Copyright 0 1987 by Academic Press, Inc. AU riehts of reproduction in any form reserved. 61 1

612 VIJAY K. KAPOOR

1 Description

1.1 Name, Formula and Molecular Weight

Thiabendazole, an anthelmintic and antifungal agent, was developed by scientists of Merck laboratories1 in 1961. Thiabendazole is 2-(4-thiazolyl)-1H_-benzimidazole. The CAS Registry number is 148-79-8. The most common proprietory name is Mintezol .

Molecular Weight: 201-25 10H7N 3'


White to cream-colored, odorless or almost odorless powder.

1.3 Salts

Thiabendazole is a weak base. The hydrochloride (sublimed at 265O) and sulfate (melted at 262-6O) have been prepared.2 Water- soluble lactate of thiabendazole has also been prepared along with the citrate, fumarate and salicylate which had limited solubility in water.



The infrared spectrum4 (KBr disc) of thiabendazole is presented in Figure 1. The principal peaks are at 1408, 906 and 74OCm-1.


The light absorption, in the range 230 to 350 tun, of a 2-cm layer of a 0.0004 per cent w/v solution in 0.1N hydrochloric acid exhibits two maxima, at 243 ?h~ and at 302 nm; absorbance at 243 nm, about 0.47 and at 302 nm, about 0.98.5

THIABENDAZOLE

Wavelength microns 6 7 8 9 10 11 12 131415

613

90

80

70

60

50

40

30

20

90

80

70

60

50

40

30

20

10

I I I I I I I I 2000 1800 1600 1400 1200 1000 800 650

l o

2000 1800 1600 1400 1200 1000 800 650

Wavenum ber

Figure 1. Infrared Spectrum of Thiabendazole


Thiabendazole exhibits no optical activity.

The melting range6 of thiabendazole is

2.4 Melting Range

between 296O and 303O.

2.5 Solubility 7 The solubility of thiabendazole at

ordinary room temperature is as follows:

Solvent Solubility, mg/ml

Water

A l c o h o l

Chlorofonn

practically insoluble

6.66

3.33

Ether 0.5

A c e t o n e slightly soluble

Dilute mineral acids soluble

614 VIJAY K. KAPOOR


Crystal structure of thiabendazole is described.* Crystals of thiabendazole are orthorhombic, space group p s, with a = 17.052(7), b = 10.998(4) , and = 10.030?8) A. There are erght formula units per cell; observed -3 and calculated densities are 1.414 and 1.421 g cm . Intensity data were collected on an automatic diffractometert the structural parameters were refined by full-matrix least-squares to an index of 0,066 fo r 1805 reflections, The two ring systems are approximately planar, but are twisted by loo with respect to each other. The C-C bond connecting the two ring systems is 1.442(10) %, long. H(l)---N(14) hydrogen bonds to form chains parallel to the axis,

X-ray powder diffraction data for thiabendazole is given in Table 1.9 diffractometer and Debye-Scherrer camera techniques, and are tabulated in terms of the lattice spacings and the relative intensities of the line. Patterns using three different X-ray wavelengths w i t h the camera method are given.

Molecules are linked together by N ( 1 ) -

The data were obtained by

3. Synthesis

reported the synthesis (Scheme I) of thiabendazole by the reaction of 4-thiazolecarboxamide with - O-phenylenediamine in polyphos horic acid at 250° for three hours. thiabendazole is reported through another route (Scheme 11). O-Nitroaniline was condensed with lactic acid or-the acid halide to give g-lactoyl- 2-nitroacetanilide which on oxidation followed by bromination gave ~-(bromopyruvoyl)-2-nitroanilide. The latter on refluxing overnight with thiofomamide hydrate followed by zinc dust reduction gave thiabendazole.

described.11812 thiabendazole i s also described.13

Brown et a1.l from Merck laboratories first

In a patent18 the synthesis of

Alternate methods of synthesis havs also been Synthesis of 14C- or 3 S-labeled

Table 1

X-Ray Diffraction Data f o r Thiabendazole

Diffractometer Camera CuKa CuKa corn CrKa.

d ( f t ) 1/11 d (1) 1/11 d <A) 1/11 d ( A ) 1/11

6.81 8 6.76 17 6.76 17 6076 13 5.64 4 - I - - - - 5056 7 5057 18 5.57 14 5057 11 5 007" 43* 5oO3* 68* 5 04" 75* 5 o O 3 * 63* 4r23* loo* 4 0 2 1 ~ loo* 4.21* loof 4021* loo* - - 4.02 3 4001 5 (c. - 3.72 14 3.71 26 3071 27 3.70 20 3 39* 4 3* 3 0 38* 51* 3 38* 5 1* 3 0 38* 43* 3027 2 3026 5 3.26 7 3025 7 - - 3011 3 30 10 4 I I

2,814 10 2 0803 19 20806 21 2.808 14 20695 2 2.681 6 2 0 685 7 - -

7 - ... 2,525 3 2,517 12 2.515 12 2.519 8 - - 2.419 1 20418 17 - I

2 327 3 2.321 9 2.316 21 20311 7 - - 2,253 4 2.252 5 - - 20137 5 2.132 4 2,062 2 2,055 7 2.056 7 20025 1 20022 4 2 0 000 5

20629 3 20630 - -

*Most intense lines

616 VIJAY K. KAPOOR

$7 PolYphosphoric acid , NHpCO N 250°/ 3 hr

Scheme I. Synthesis of thiabcndazole

+ CHaCHOHCOR UNo2 N - H

c=o I

I I CH3

R =OH, C I

H COH

aNo2 N - H

I I - I

c=o 0r2/0O0

c =o

CHI Br

1

N-H

c=o

c = o

C H I

I I I

'NH2 HC

II aNo2 i i y a Z n / H C l

NH-C N

Scheme n. Alternate synthetic pathway t o thiabendazole

THIABENDAZOLE 617


In general thiabendazole is a stable compound. The USP XXI NF XVI directs that it should be packed and stored in well-closed containers .6 zole, however, has been reported14-17 to be slightly uv (sunlight) sensitive. Photolytic degradation products of thiabendazole have been identified as benzimidazole, benzimidazole-2- carboxamide, triazole-4-carboxamide, thiazol-4- ylamidine and methyl thiazol-4-carboxylate .14.16 A recent study involving photooxidation of thiabendazole in methanol in presence and absence of a photosensitizer, methyleneblue also identified dimethyl oxalate, thiazole-4-(pcarbomethoxy)- carboxamlde, methyl benzimidazole-2-carboxylate, benzimidazole-2-carboxamide and benzimidazole as the main products of photolysis.19 structures of the main degradative products,

Thiabenda-

Figure 2 gives the

Benzi midosole Benzimidozole-2-corboxomide

c;sa CH3OOCNHCO

Methyl benrim~dozole - 2 -corboxylote 1 hiozote -4 - f N-car bomethoxy) - carboxamide

Figure 2. Moin products of photolysis of thiabendazole

A recent review discusses the different breakdown products due to environmental factors of various fungicides including thiabendazole . 2o

5 , Metabolism and Pharmacokinetics

hydroxythiabendazole which is excreted in the urine conjugated either B S glucuronide or as the sulfate (Figure 3) . I 3 absorbed from the gastrointestinal tract and peak plasma levels of 13 to 18 pg/ml are found within an hour of ingestion. The drug and its metabolites are excreted rapidly, 87% in the urine and 5% in

Thiabendazole is metabolized chiefly to 5-

Thiabendazole is rapidly

618 VIJAY K. KAPOOR

Thiabendazole 5 -Hydroxythiabendazole

Hp N

mN Sulfate conjugate Glucuronide conjugate

Figure 3 . Metabolism o f thiabendazole

the faeces within 48 hours. 21 both unchanged thiabendazole and free 5-hydroxythiabendazole may be found in both the plasma and urine. Gastrointestinal absorption and secretion of thiabendazole has been studied.22 Wilson & - a1.23 have reported that 5-hydroxylation of thiabendazole in rat liver microsomal preparation is dependent on cytochrome P-450 and has a Km of 3.92 x 10-431 and Vmax of 0.484pole of 5- hydroxythiabendazole produced/hr/mg of protein. 5-Hydroxythiabendazole gave a type If binding spectrum w i t h cytochrome P-450, Desmethylimipramine and ethoxyquin are reported to inhibit the hepatic microsomal hydroxylation of thiabendazole.24 A single oral dose of desmethylimipramine (80 mg/kg) administered to rats inhibited the hydroxylation of thiabendazole by 45% in vitro at 5 hr after dosage but did not decrease cytochrome P-450. A single oral dose of ethoxyquin (200 mg/kg) to rats inhibited the hydroxylation by 65% in vitro at 1 hr after dosage, inhibition was less at 5 hr. Oral ethoxyquin (400 mg/kg) or desmethylimipramine (80 rng/kg) administered 30 minutes before oral thiabendazole (100 and 200 mg/kg, respectively) delayed absorption of thiabendazole and resulted in its decreased plasma concentrations. Bauer

small amounts of

THIABENDAZOLE 619

-- et a1.25 have studied the phamacokinetics of thiabendazole and its metabolites in an anephric patient undergoing hernodialysis and hemoperfusion. The half-life, volume of distribution, and clearance of thiabendazole were 1.17 hr, 2.76 l/kg, and 27.2 ml/rnin/kg, respectively. While thiabendazole and the 5-hydro= metabolite did not accumulate during multiple dosing, the glucuronide and sulfate conjugates accumulated extensively despite hemo- dialysis and hemoperfusion.

studied in cattle, 268 27 sheep, 28 and avian29 species. The findings are consistent with the literature data that metabolism and excretion of thiabendazole are more extensive in cattle than in sheep.

Pharmacokinetics of thiabendazole has been



The elemental analysis of thiabendazole is as follows:

11 E 1 emen t % Calcd. % Found

C 59.68 59 074

H 3.51 3.62

N 20.88 20.57

S 15.93 15.99

6.2 Identification Color Test

to identify a sample of thiabendazole.586 About 5 mg of the sample are dissolved in 5 ml of 0.12 hydrochloric acid and 3 mg of E-phenylenedimine dihydrochloride are added f 01 lowed by shaking to dissolve the contents. About 0.1 g of zinc dust is then mixed and the mixture allowed to stand f o r 2 minutes. 5 ml of a solution prepared by dissolving 20 g of ferric ammonium sulphate in 7 5 rnl of water, and 10 ml of 1E sulphuric acid are then added. When diluted with water to 100 ml a blue or blue-violet color is developed-

The following color reaction can be used

6.3 Titrimetric Analysis

The titration with perchloric acid is the method of choice to assay thiabendazole.586

620 VIJAY K. KAPOOR

The sample is dissolved i n glsclal acetic acid. After speciEic quantities of acetic snhydride, mercuric aceta%e and two drops of crystal violet soluticn are added, the titration with 0.1E perchloric acid to a hlue-green end point is carried out. A blank determination is performed and any necessary correction is made. Each r n l of 0.1N perchloric acid is equivalent to 20.13 mg of CIOH7N3S.

A titration method utilizing the complex formed by thiabendazole with silver Ions is also described. 30

6.4 Bioassay

Several microbiological assay methods using sensitive fungi for the quantitative determination of thiabendazole have been reported. 31-38 methods have been employed for determining the residue of the fungicide on plant materials or in soil. A simple and rapid bioassay €or the direct determination of thiabendazole residue in soil involves placing pellets composed of mixtures of soil (200-500 my> and agar on an agar medium pre- inoculated with the test organism Penicillium digitatum.37 incubatlon at 2 7 O , the size of the inhibition zone is measured (Figure 4 ) . The lowest de%ectable concentration of thiabendazole in a sand soil was

from citrus fruit and bananas were applied as spots to silica gel in P e t r i dishes which were then uniformly inoculated with Penicillium cyclopium spores. Fungicide concentration was detennined from comparison of inhibition zones with those of a standard 48-hr incubation. Detection threshold was 0.5 p g thiabendazole.

Other microorganisms employed in the bioassa studies are Penicillium exansum,32 Graphium ulmf3 and Sporobolomyces roseus.38

These

After cold pre-incubation followed by

found to be 1.0 ) i g / g . In another method Y 5 extracts

6.5 Polaroqraphic Analysis

A polarographic method for determining thiabendazole is described. 39 behavior of thfabendazole was investigated by sampled doc. polarography and differential pulse polarography. Thiabendazole showed one wave peak in the polarogram when a short drop time (0.4 s ) was used. Detection was most sensitive at pH 8.

The electrochemical

THIABENDAZOLE 621

c

“E 250 c Y

C 0 .- -2 200 .- c C .-

t

2 4 6 8 10 12 Thiabendazole concentration in soil (pg/g)

Figure 4. Effect of thiabendazole concentration in-soil on growth inhibition of Penicfllium di-itatum using the soil-agar pellet technique. Grow + inhibition is expressed a s the square

The current measured was proportional to the concentration. The detection limit was 0.5 ppm. Differential pulse polarography has been used for quantitative determination of thiabendazole In citrus fruit peels.

6.6 Colarimetric Analysis

Thiabendazole can be analyzed colori- The method involves: metrically in feeds.40-43

(a) complete extraction of thiabendazole from Eeed w i ’ 3 1 C.2N HC1 in SO% methanol; (h) separation from other suEstences by ext.raction with chlorof o m from a l k a l i n e solution; (c) further purif ication by reextraction into dil.vte H C l ; (d) reduction with

622 VIJAY K. KAPOOR

zinc dust in the presence of pphenylenediamine to form a complex; (el subsequent oxidation with a ferric solution to form a thiazine dye; and (f) extraction of the dye into butanol and measurement at 605 nm against reference standard thiabendazole. The method gives most accurate and reproducible results. he method described above was automated by !~;hite.~~ In the automated system the solution was reduced with 0.3% zinc dust in glycerol, and the products were coupled with 0.17% pphenylenediamine in 2N H2SO4 and 15% Fe NH4 (SOg) 2 in 0.1g H2SO4. The glue complex was measured at 610 nm. The relative standard deviation was 1.2%, and the recovery was 95-100% from feed containing 1% thiabendazole. A modified method was used to determine the residues of thiabendazole in citrus fruits -45

A method for estimating thiabendazole in the milligram and submilligram ranges (0.1-2.0 mg/ml) has been descrlbed which involves measuring of absorbance of a copper-thiabendazole-tetramethyl- guanidine complex at 350-400 nm.46 A modified method in which absorbance of the complex was measured at 311 nm permitted estimation of 2 pg/ml thiabendazole. An improved procedure involves shaking an alkaline aqueous suspension containing thiabendazole with solution of cupric acetate and 1-dimethylamino-2-propanol in chloroform to produce a green color in the chloroform phase.47 analysis is completed by spectrophotometric measurement. Thiabendazole in concentration range of 0.1-1.0 mg/ml can be estimated by this method.

reaction of thiabendazole in ch1.oroform with alkaline buffered bromocresol green is also 6escribed.48

6.7 Spectrophotometric Analysis

The

A simple colorimetric method involving the

6.71 Ultraviolet

Thiabendazole is estimated in various fruits as a residual fungicide by ultraviolet spectrophotometry.49-60 The method in general involves extraction with ethyl acetate-ammonium hydroxide, purification by acid-base procedure, and measuring the absorbance of the acid solution at 302-3 nm. In another purification procedure the

THIABENDAZOLE 623

f r u i t p e e l s o r pulps are extracted with chloroEorm, the e x t r a c t i s acidif ied and c h l o r o f o m evaporated. The a c i d i c concent ra te i s cleaned up with chloroform, and thiabendazole e x t r a c t e d a t pH 9.5. Using t h i s method thiabendazole w a s determined spectrophoto- m e t r i c a l l y a t 302-3 nm a t concent ra t ions of 0.1 pprn i n 100 g peel or pulp, o r 0.03 ppm i n whole c i t r u s f r u i t . Aspect of p ro te in i n t e r f e r e n c e i n thiabendazole determinat ion i s discussed.61

6.72 I n f r a r e d

Thiabendazole can a l s o be analyzed by i n f r a r e d spectrophotometry.62 The method involves d i s so lv ing 0.12 g of thiabendazole sample i n 5 m l of l?,~-dimethylformamide and adding 1 g of anhydrous sodium sulphate . A por t ion of t h e superna tan t i s used for i n f r a r e d spectrophotometry, scanning between 2.5 and 16.7 )un. 11.09 )xn is used f o r quan t i t a t ion . The s t a t i s t i ca l a n a l y s i s of mul t ip l e determinat ion by t h i s method gave a r e l a t i v e e r r o r of \ (2% and a c o e f f i c i e n t of v a r i a t i o n 4 1%.

Absorbance a t

6.8 Spectrof luorometr ic A n a l y s i s

Thiabendazole is anal zed by spectro- f luorometr ic method also.54,63-%8 can be detected i n crops by acetone e x t r a c t i o n followed by p a r t i t i o n i n g i n acetone-water and e t h y l a c e t a t e and clean-up on magnesium oxide- cel i te-alunfnium oxide column. The cornpound i s then q u a n t i t a t i v e l y measured by direct fluorometry. The e x c i t a t i o n and emission wavelengths are 305 and 345 nm, r e spec t ive ly f o r thiabendazole. The method i s reported t o be s e n s i t i v e t o 0.02 ppm thl sbendazole. 63 An automated f luorometr ic method f o r t he determinat ion of thiabendazole i s a l s o descr ibed which gives the minirnun l e v e l of de t ec t ion of t h e fungic ide asw0.05 pg/ml.6* A s e n s i t i v e f luorometr ic method for determinat ion of t h i a z o l e conta in ing compound i s r e c e n t l y described. 68

The fungic ide

6.9 Chromatographic A n a l y s i s

6.91 Paper Chromatoqraphy

4 The fol lowing system has been descr ibed.

624 VIJAY K. KAPOOR

Paper:

Solvent :

Khatrnan Noel, sheet 14 x 6 i n , buffered by dipping i n a 5% s o l u t i o n of sodium d ihydr ogen c i tre t e , b l o t t i n g , and drying a t 2 5 O for 1 hour.

4.8 CJ of c i t r i c a c i d in a mixture of 330 ml of water and 870 ml of rpbutanol .

Equi l ibra t ion : None Development: Ascending. Time of run,

Location: a. Examination under u l t r a v i o l e t l i g h t

5 hours . b. Bromocresol green spray

C. Fotassium permanganate spray

Rft 0.06

An a l t e r n a t e system employs acetone-water (l:6) i n ascending mode which g i v e s Rf va lue of thiabendazole as 0.49.69 allowing t h e paper t o react with i o d i n e vapor for 1 minute.

Location is done by

6.92 Thin-Layer Chromatography

graphic sys t ems using s i l ica g e l or si"l1ca g e l GF254 p l a t e s f o r i d e n t i f i c a t i o n and eva lua t ion of thiabend- azo le have been reported. These are t abu la t ed below:

Solvent System Reference

A v a r i e t y of t h in - l aye r chromato-

Benzene-acetic acid-acetone-water 70 (10:4: 1:0.4)

Benzene-acetic acid-acetone-water (70: 20: 10: 2)

71,72

Ethyl ace ta te -e thyl methyl ketone- 65,67 formic acid-water (50: 30: 10: 10)

Ethyl acetate-benzene (50:50) 73

Diethyl e the r -g l ac i a l acetic acid- 74 methanol (100:5:2)

Acetone 74

THIABENDAZOLE 625

Solvent. System Reference

Light petroleum (60-80°)-acetone 74 (30:lO)

Chloroform-acetone (80: 20) 75

Using aluminium oxide F254 n e u t r a l p l a t e 74 d i e t h y l ether-methanol i s t h e s y s tern employed.

Cole e t has given t h e Rf values of t h i a b e n d a z x e i n d i f f e r e n t so lven t systems. These are given below:

Solvent System R f Value x 100

E t h y l acetate

Ethyl a c e t a t e s a t u r a t e d wit.h ammonia

E t h y l acetate-toluene-anunonia (60:40:2)

Ethyl acetate-toluene-ammonia (40: 60: 2)

Ethyl acetate-toluene-anunonia (20:80:2)

Toluene-ethanol-ammonia (80:20:2)

Toluene-ethyl acetate-ethanol- ammonia ( 60 : 10 : 30 : 2 1 To 1 uene- e t k i y 1 ace t a t e- e t h an o 1 - ammonia (70: 15: 15:2)

Toluene-ethyl acetate-ethanol- arnmonia (60:10:10:2)

55

60

41

31

2 1

43

58

52

38

Detect ion of spo t of thiabendazole on t h e p l a t e has been c a r r i e d ou t by d i f f e r e n t methods such as v i s u a l i z a t i o n under u l t r a v i o l e t l igh t ,74876#77 by spraying with potassium iodobismuthate solution followed by exposure t o bromine A b i o l o g i c a l method f o r d e t e c t i o n and eva lua t ion of thiabendazole has a l s o been descr ibedO73 I t i s done by spraying t h e p l a t e s with spores of any of t h e th i r ty two fungal. species followed by incubat ion. Guan t i t a t ive relations were es t ab l i shed be tween spot su r face a r e a and t h e amount of fungicide. An enzyme i n h i b i t i o n

vaposs74 o r by exposure t o iod ine vapors. 76

626 VIJAY K. KAPOOR

75 technique i s a l so reported.

determination of thiabendazole has been ca r r i ed oiit spectrophotometricall 70,71,72,78,79 or fluorometrically65,67,8~,~1. Otteneder and FIezel65 have reported a method by which thiabendazole can be Getermined d i r e c t l y on the chromatogram by reflectance-absorption photometry with measurement at 302 nrn o r f luorometr ical ly a t 355 nm, with exc i t a t ion a t 313 nm. Compared with t h e e lu t ion and subsequent photometry, direct evaluation on the chromatogram has the advantage t h a t it saves t i m e and requires less substance pe r spot.

using t h i n - 1 aye r chsornat oy r aphy want i t a t i ve

6.93 G a s Chroniatoqraphy

been used t o determine residue of thiabenda- ,o1e050,53,67,82-94 the fungicide i n whole c i t rus f r u i t and i n c i t r u s and bansna pulp is described by Mestres e t al.50 Samples were made a lka l ine with ammonia so lu t ion and macerated w i t h e thy l ace t a t e and filtered. The f i l t r a t e was cleaned up by ex t rac t ion i n t o 0.1g HC1, and making the aqueous phase a lka l ine and reext rac t ing back i n t o e t h y l ace ta te . The concentrated e x t r a c t was chromatographed on a lo”/, DC-200 s t a t iona ry phase using FPD set i n t h e mode f o r determining su l fur . de tec t ion was of the order of 0.1 pj. Hey used SE-30 a s s t a t iona ry phase with a ni t rogen-sensi t ive de tec t ion . thiabendazole a s i ts methyl der ivat ive with flame ioniza t ion de tec t ion and a column of lo”/, Dc-200 on Gas-Chrom Q a t 240O. It permits de t emlna t ion i n concentrations down t o 0,l ppm,

Thiabendazole w a s determined by Nose e t al. a s g-pentafluorobenzoyl de r iva t ive by electron- capture gas chromatography following react ion of thiabendazole with pentafluorobenzoyl chlor ide. The m i n i m u m amount of thiabendazole detectable by t h i s method was 0.01 ppm. Pr inc ipa l operat ing condi t ions were: 1.5 m x 3 mm I.D. g l a s s column packed with 5% OV-101 on Gas Chrom G (HP) (80-100 mesh), i n j e c t i o n po r t and de tec to r temperature 270°, column temperature 230O. Nitrogen was used as a c a r r i e r gas a t a flow-rate of

Gas chromatographic methods have

An e a r l i e r method t o determine

The l i m i t ofs3

Tanaka and Fujimoto83 determined

84

THIABEND AZOLE 627

40 ml/min. Combined gas chromatography-mass spectrometry was operated with column temperature 2 3 0 ° , separator temperature 260°, ion source temperature 290° and flash heater temperature 270'. The carrier gas was helium at a flow-rate of 30 ml/min. Recently, Bardalaye and Wheeler85 employed this technique to determine thiabendazole in yams with operating conditions as: 1.83 m x 4 mm I . D . glass column packed with 5% OV-17 on 80-100 mesh Gas-Chrom 0, argon-methane carrier gas (95 + 5) at a flow-rate of 60 ml/min. Temperatures

66 of oven, detector and injectors used were 240 300 and 200°, respectively. Tjan and Jansen have used pentafluorobenzoyl bromide f o r derivatization of thiabendazole.

A combined gas Chromatography - mass spectrometry confirniatory assay for thiabendazole and 5-hydroxythiabendazole at 0.1 p m in animal

methylation converts these compounds to their E- methyl and E,g-dimethyl derivatives, respectively. Identification and quantitation are achj eved by selective ion monitoring of M-1, M and Etl ions from E-methylthiabendazole and the I4 and M-15 i ons from IJ,g-dimethyl derivative of fj-hydroxy= thiabendazole.

tissue isolates has been developed. B 5 On-column

6.94 High Performance Liquid Chroma toqraphy

In recent years high performance liquid chromatography (HPLC) has become one of the major analytical tools to determine the residual thiabendazole alone96-100 or simultaneously with other addit.iveslOl-lll in fruits. Isshiki -- et a l o g 6 have described a simple HPLC method for eetermining thiabendazole in fruits. Thiabendazole is extracted with methanol and the extract is washed with phexane saturated with methanol. The HPLC determination is carried out us ing LiChrosorb RP-8 as stationary phase and methanol-2.8"A ammonia wzter (60:40) as mobile phase; 2-methylindole is added as an internal standard. The detection is carried out fluorometrically and the limit of detection is 0.1 pg thiabendazole/ . Recoveries are 92%. determined thiabendazole in curds (artificial marmalades) and orange and lemon marmalades

Collinge and Nairfalisegg have

628 VIJAY K. KAPOOR

by using aqueous solution containing 0,PA of ammonium nitrate and 0.7% of ammonia (buffered solution) -methanol (1 : 1) as mobile phase for curds , and buffered solution-methanol ( 3 : 2) for marmalades. The flow-rate in column was 1.0 ml/min, the column temperature was 22-26O and the detection wavelength was 305 nm. have described a detailed procedure for the separation of thiabendazole in fruits in which the uv-absorbing Contaminants are removed by fractional separation with an aqueous-ethyl acetate system. The HPLC conditions for thiabendazole determination reported aret column, Unisil C-18 10 p 4.6 mm x 300 mmt eluant, methanol- 0.16 M KH2PO4 water ( 5 0 : 5 0 , v/v % ) I flow rate, 1.0 ml/min; pressure, 60 kg/cm2; detector uv (298 nm); range 0.01 AUF; and sample size 5 pl. The detection limit for this method was found to be 0.08 ppm, and it was shown that 1 ppm of thiabendazole in the fruits could be estimated within 10%. An iongg air HPLC has been described by Belinky.

A simultaneous HPLC determination of thiabendazole, 0-phenylphenol and diphenyl residues in cityus fruits without prior clean up has been described by Kitada et a1.1°6

A rapid, sensitive and precise HPLC method using fluorescence detection has been developed by Watts et a1,112 determination of thiabendazole and unconjugated 5-hydroxythiabendazole in human serum. Sample pretreatment consists only of protein precipitation with acetonitrile containing the internal standard, 2-methylindole. Detection limits were found to be 0.1 pg/ml serum for thiabendazole and 0.4 pg/ml serum for 5-hydroxythiabendazole. A microenzymatic method for the conversion of the glucuronide and sulfate esters of 5-hydroxythiabendazole was also developed using P-glucuronidase and sulfatase, respectively. Thus, quantitation of the separate metabolites was possible. A special adaptation of the chromatographic procedure was utilized for the determination of 5-hydroxythiabendazole metabolites in the sera of uremic patients, which can contain large mounts of interfering fluorescent substances. The method should be of particular use for monitoring thiabendazole therapy in

Yamada et &.lo0

for the simultaneous

THIABENDAZOLE 629

patients to eliminate the potentially toxic metabolites.

HPIC methods for determining thiabendazole in waste waters1 138 114 and in textile productsll5-117 are also described.

6.10 Miscellaneous

A radioactive indicator method for the determination of thiabendazole residues has been described.118

7 . Limits of Tolerance

Tolerances in ppm for the residues of the fungicide thiabendazole in or on various raw agricultural commodities established under the Federal Food, Drug, and Cosmetic Act fixed by the United States Environmental Protection Agency are: 0.02 for sweet otatoes;llg 0.1 for dried beans,l20 eggs, 121 meat; 151 and soyabeanso122 0.4 for banana pulp119 and rnilki123 1 for hubbard squashr124 3 for bananas119 and rough rice1125 5 f o r strawberriesol26 6 €or su ar beetsol21 8 for rice

fruits 130 grapes, P93 pears 128 potatoesl31, rice straw, $25 sugar beet tops135 and wheat grains; 131 15 for cantaloupes; 126 and 40 for rnushro0m.13~

hullst127 10 for a les,l 8 8 carrots,l29 citrus

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VIJAY K. KAPOOR 634

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2. Hamadi, M. Pierson and P. Chambon, Bull. T r a v . SOC. Pharm. Lyon, 22, 3 (1978); C.A. - 93, 44048~ (1980).

F. Chiti and G. Modi, Boll. Chim. Unione Ital. Lab. PTOV., Parte Sci., 2, 693 (1979); C .A*, 92, 196470~ (1980)

J. Tancogne and R. Delon, Ann. Tab. Sect.2, - 16, 67 (1980) : C.A., 95, 19523f (1981) . P. B. Baker, J. E. Farrow and R. A. Hoodless, J. Chromatogr., 8 1 8 174 (197310

G. H. T j a n and L. J. Burgers, J. Assoc. Off. Anal. Chert., 56, 223 (1973).

THIABENDAZOLE 635

76. E. R , C o l e , 2 . Crank and A. Sheikh, J. Chromztoyr., 78, 323 (1973).

Lebensm., 2, 57 (1973); C.A., 79, 124782g 77. C. Reinhard , Cheni., Mikrobiol., Technol.

(1973) . 78, E , Kmeller, Deut. Lsbensrn.-Rundsch., 67,

229 (1971).

79, F. Mail.ath, F. Medve and 14. J o z s e f , A c t a Pharrn. Hung., 44, 71 (1974); C . A . , 3, 1157132 ( 1 9 7 4 1 7

80, S. M. Norman, D. C. Fouse and C . C. C r a f t , J. Assoc. Off. Anal. Chem., 55, 1239 (1972).

Rundsch., 2, 133 (1974).

and R. Cornet, Ann. F a l s i f . Expert . Chim., 67, 585 (1974) ; C.A., 83, 56783p (1975) . 117, 149 (1976).

81, S, Ebel and G. Herold, Deut. Lebensm.-

82. R. Mestres, J. Toiirte, V, Campo, S. Il les

- 83. A. Tanaka and Y. Fujimoto, J. Chromatogr., -

84. N , Nose, S. Kobayashi, A, Tanaka, A. Hirose and A. Katanabe, J. Chromstogr., 130, 410 (1977) .

85. P. C. Bardalaye and W. B. Wheeler, J. Assoc.

86. G. H. T j a n and J. T. A . Jansen, J. Assoc.

87. T. Chikamoto and S. Nagata, Kyoto-fu E i s e i

O f f . Anal. Chem., 69, 114 (1986).

Off . Anal. Chem., 62, 769 (1979).

Kogai Kenkyusho Nenpo, 24, 48 (1979); C.A., - 95, 167278r (1981).

Nippon Nogel Kagaku Kaishi, 5 5 8 695 (1981); C.A., 95, 2021392 (1981).

Nippon Nogei Kagaku Kaishi, 2, 1045 (1980)t C.A., 94, 137876a (1981) .

88. K. I s s h i k i , S. Tsumura and T, Watanabe,

89. K. I s s h i k i , K. Tsumura and T, Watanabe,

636 VIJAY K. KAPOOR

94. Y. Toiioc,r?i, H. Sano, Y. It0 and 14. IwA~~s, Shokuhln Eiseigakv Zasshi , 16, 397 (1975) J C.A.8 84, 1470922 (1976)

95. H. Vanden, J . A . William 1x1, J. S. hood, M. EiGiovanni and R. ;i. i ialker, ,J. A S r l c . Food Chem., 25, 386 (1977); C.A., c 86, 119342d(1977).

J. Asscc. Off. Anal. Chern., s, 747 (1980). 96. K. I s s h i k i , S , Tsumura and T. ha tambe,

97. F. T a f u r i , C. DIarrucchin1, M. Pdtumi and M. R u s i n e l l i , J. A g r i c . Food Cheni. , 2, 1150 (1980) .

98. B. R. Relinky, J. Chrornatogr., 238, 506 (1982).

99. A. C a l l i n g e and A. No i r f a l i s e , J. C h r o m a t . o y r . , - 257, 416 (1983).

Chem. , 48, 1883 (1984). 100. T. Yamada, H. Aokl. and M. A k a F i , Agric . Biol.

101. 14. llaeda and A. Tsuji, J. Chrornatogr., 120, 449 (1976).

1 0 2 . D. J. A u s t i n , K. A . Lord and I. H. Williams, Fest ic . Sci., 2 , 211 (1976); C.A., 85, Y8362h (1976).

103. J. E. Farrow, R. A . Hoodless , M. Sargent and J. A . S i r l w e l l , Analyst, 102, 752 (1977).

THI ABENDAZOLE 637

104.

105

106.

107

108.

109 . 110.

111.

112.

113.

114.

115.

116.

r). Mourot, J. B o i s s e a u and (3. Gayot , Anal. C h i m . A c t a , 9 9 8 371 (1978).

F. Burzi F i o r i , A. Berri and M. Zacchet-ti, R i v . SOC. I t a l . sci. A l i m e n t . , 10, 19 (1981):

Y. Kitada, PI. Sasaki and K. Tanigawa, J. ASSOC. O f f . Anal . Chem. , 65, 1302 (1982) . Y. Kitada, K. Tamase, M. Inoue, M. Sasaki and K. Tanigawa, Shokuhin Eiseigaku Zasshi , - 23, 21 (1982)~ C.A.0 97, 22209e (1982).

I. Takahashi, A. Tomioka, T. I i t s u k a and A. U j i e , Gunmaken E i s e i Kogai Kenkyusho Nenpo, - 13, 58 (1981); C.A., 97, 125775r (1982).

G. S t r i n g a r i , I n f . F i topa to l . , 35, 48 (1985).

C.A.8 95, 78566~ (1981)

B. Ohlin, V& Foda, 38(supp1.2)# 111 (1986).

U. Gieger, Lebensmittelchem. Ger i ch t l . Chem. , - 40, 25 (1986) . M. T. Watts, V. A. Raisys and L. A. Bauer, J. Chromatogr. 8 230, 79 (1982).

D. M. Victor , R. E. H a l l , J. D. Shamis and S. A. Whitlock, J. Chromatogr., 283, 383 (1984).

R. C. C. Wegman and H. H. Van den Broek, Meded, Fac. Landbouwwet. Rijksuniv. Gen t , - 47, 361 (1982); C.A., 98, 48523y (1983).

A. Kanetoshi, H. Ogawa, M. Anetai , E. Katsura and H. Kaneshima, E i s e i Kagaku, - 31, 245 (1985); C.A., 104, 226211~ (1986).

T. Amemiya, M. Sakai, K. Mori, S. Suzuki and M. Kazama, Kenkyu Nenpo-Tokyo-toritsu E i s e i

88997 j (198g. Kenkyusho, 35, 133 (1984); C.A.8 103,

638 VIJAY K. KAPOOR

117.

118.

119.

120.

121.

122.

1230

124.

125

126.

127

1 2 8 0

129

130.

131,

T. Amemiya, M. Sakai , K. Ikeda, K. Mori, S . Siizuki and Y. Watanabe, Kenkyu Nenpo- Tokyo-toritsu Eisel Kenkyusha, 36, 123 (1985); C.A., I&, 1 3 5 3 3 5 ~ ( 1 9 8 n .

C. P.osenblum anc? H. T , Meriwether, J. Radioanal. Chern., 58 379 (1970); C . A . 8 - 74, 98714m (1971).

Fed. Regist,., 17 Nav 1983, 48 (223), 52304; C.A., 100, 4922h (1984).

Fed. Regist . , 3 A p r 1985, 50 (641, 13195r c.A., 1 0 2 8 183870a (1985).

Fed. Regist . , 22 Jan 1980, 45 (141, 3907; C.A.8 92, 145132d (1.980)

Fed. Regist . , 2 8 Jun 19778 42 (1241, 32782; C.A., 87, 116494q (1977).

Fed. Regist . , 8 Sept 1982, 47 (1’741, 39488; C.A., 97, 161179h (1982).

Fed. Regis t . , 26 May 1972, 37 (103) , 10670; C.A.8 2 0 46820k (1972)

Fed. Regist., 9 J u l 1980, 4S (1331, 46073; C.A*, 93, 130751b (1980).

Fed. Regist., 19 Mar 1986, 51 (531, 9448;

Fed. Regist., 9 July 1980, 45 (1331, 46067;

Fed, Regist . , 7 O c t 1972, 37 (19618 212787

C.A.8 104, 3 6 7 0 4 4 ~ (19$6)

C.A., 93, 130752~ (1980)

C.A.8 7 8 0 2 8 7 4 ~ (1973)

Fed, Regi s t . , 2 8 NoV 19808 45 (2311, 79068; C.A., 94, 63918s (1981).

Fed. Regis t . , 30 Oct 1974, 39 (2lO)#

Fed. Regist., 10 A p r 7985, 50 (691, 14106;

38229; C.A.0 82, 29789t (1975).

C .Am, 102, 2 0 2 7 2 9 ~ (1985)

THIABENDAZOLE 639

132. Fed. Regist. , 25 Jun 1973, 38 (121), 16644;

133. Fed. Regist. , 10 hpr 1385, 50 (69), 14104;

C.A. , 2, 64669~ (1073)

C . A . , 102, 2 0 2 7 2 7 ~ (1985).

TIMOLOL MALEATE

DAVID J. MAZZO* AND ALICE E. LOPER#

* MANAGER, PARENTERALS RESEARCH AND DEVELOP- MENT, TRAVENOL LABORATORIES, MORTON GROVE, ILL I NO1 S

#RESEARCH FELLOW, PHARMACEUTICAL RESEARCH AND DEVELOPMENT, MERCK SHARP AND DOHME RESEARCH

LABORATORIES, WEST POINT, PENNSYLVANIA


Copyright 0 1987 by Academic Press, Inc. AU rights of reproduction in any form reserved. 641

642 DAVID J. MAZZO AND ALICE E. LOPER

1. Introduction

1.1 Therapeutic Category

1.2 History

2. Description

2.1 Chemical ..me, Formu Weight

a, Molecular

2.2 Definition


3. Synthesis



4.2 Nuclear Magnetic Resonance Spectrum

4.2.1 Proton NMR

4.2.2 Carbon-13 NMR


4.4 Mass Spectrum


4.6 Electroanalytical Behavior

4.7 Thermoanalytical Behavior

4.7.1 Melting Point

4.7.2 Differential Thermal Analysis Behavior

TIMOLOL MALEATE 643

4.7.3 Thermogravimetric Analysis Behavior

4.8 Solubilities


4.10 Hygroscopicity

4.11 Dissociation Constant



5.1.1 Ultraviolet Spectrophotometry

5.1.2 Infrared Spectroscopy

5.1.3 Elemental Analysis

5.2 Titrimetric Analysis


5.3.1 Direct Ultraviolet Spectro- photometry

5.3.2 Ultraviolet Spectrophotometry via Flow Injection Analysis

5.3.3 Specific Rotation

5.4 Chromatographic Analysis

5.4.1 Thin-layer Chromatography

5.4.2 High Performance Liquid Chromatography

6. Stability - Degradation 6.1 Solid State Stability

6.2 Solution Stability


7. Biopharmaceutics and Metabolism

7.1 Absorption and Bioavailability

7.2 Metabolism

7.3 Pharmacokinetics

8. Determination in Biological Matrices

9. Determination in Pharmaceuticals

9.1 Dissolution Testing

9.2 Potency

9.2.1 Assay

9.2.2 Dosage Uniformity

9.2.3 Stability Testing

10. References

TIMOLOL MALEATE

1. Introduction

1.1 Therapeutic Category (1)

645

Timolol maleate is a beta adrenergic blocker which is non-selective between beta and beta adrenergic receptors. It dhes not ha6e significant intrinsic sympathomimetic, direct myocardial depressant or local anesthetic (membrane-stabilizing) activity. Timolol maleate is effective in lowering intraocular pressure and is widely used in patients with open- angle glaucoma and aphakic glaucoma. Timolol maleate is also indicated both for the treatment of hypertension (alone or in combination with other anti-hypertensive agents, especially thiazide-type diuretics) and to reduce cardiovascular mortality and the risk of reinfarction in patients who have survived the acute phase of myocardial infarction and who are clinically stable. Timolol maleate, available for oral dosing as tablets and for injection and ophthalmic dosing as distinct sterile aqueous solutions, is usually well tolerated with most adverse effects being mild and transient. However, a number of adverse effects have been reported with other beta- adrenergic blocking agents and should be considered potential adverse effects of timolol maleate. A number of adverse reactions considered to be causally related to timolol maleate have been reported. For example, severe respiratory reactions and cardiac reactions, including death due to bronchospasm in patients with asthma and rarely death in association with cardiac failure, have been reported (1)


1.2 History

Timolol maleate, belonging to the thiadiazole class of compounds, was first synthesized in the Merck-Frosst Laboratories in Montreal, Canada. The first non-patent literature reference to timolol maleate appeared in 1972 (2). Timolol maleate, since its introduction in pharmaceutical formulations, has gained a wide acceptance as an anti-hypertensive and anti-glaucoma agent. A search of Chemical Abstracts (1966 - 1986) and Pharmaceutical Abstracts (1974 - 1986) produced over 210 unique bibliographic citations for works dealing with timolol maleate.

2. Description

2.1 Chemical Name, Formula, Molecular Weight

The accepted chemical name for timolol maleate (MK-950) is: (S)-l-[(l,l- dimethylethyl)-aminol-3-[[4-(4- morpholinyl)-1,2,5-thiadiazol-3- ylloxyl-2-propanol, (2)-butenedioate (1:l) salt. The CAS registry number is 26921-17-5.

Other names which have been used for timolol maleate include the maleate salts of (s)-(-)-3-(3-tert-butyl-amino- 2-hydroxypropoxy)-4-morpholino-l,2,5- thiazide, (-)-3-morpholino-4-(3-tert- butylamino-2-hydroxypropoxy)-1,2,5- thiadiazole and (-)-1-(tert- butylamino)-3-[(4-morpholino-1,2,5- thiadiazol-3-yl)oxy]-2-propanol.

TIMOLOL MALEATE

2 . 2 Definition

647

Timolol maleate possesses an asymmetric carbon atom in its chemical structure and is provided as the levoisomer. It has tradenames of BLOCADRENs and TIMOPTICs. Timolol, when referred to, indicates the free base (CAS Registry Number = 26839-75-8).

2 . 3 Appearance, Color, Odor

Timolol maleate is a white, odorless, crystalline powder.

3 . Synthesis

Timolol maleate has been prepared through a series of synthetic steps beginning with D-mannitol and acetone (3). The synthetic route is presented in Figure 1. The starting material, I, is reacted with acetone and the product (11) is washed with benzene and crystallized from hexane. This substituted mannitol (I11 is then oxidized in tetrahydrofuran with lead tetraacetate to the substituted glyceraldehyde (1111, hydrogenated with t-butylamine (IV) and treated with benzaldehyde to form a substituted phenyloxazolidine (V). In a separate reaction sequence,

C13H24N403S-C4H404 Molecular Weight 432.49 g/mole

HO - CHe H3C..c/0-?H2 CHO H3C' '0-C-H I

HO-C-H Pb(0Ach , [""' H,C/~'O-CH /o-i:6] (CH3)aCNH2 HOCHzCHCHzNHC(CH3)3 + 6 - o&N\C(CH3)3

I

I (CH3)zCO

I I H-C-OH

I H-C-0, /CH3

I C HeC-O/ 'CH3

HO-C-H

HO-C-H \

I / OH

H-C-OH I

H-C-OH I

C H D H

m Ip Y I II

H

rn

N, ,N S

Y m Figure 1.

Ix X

Synthetic route to timolol maleate.

TIMOLOL MALEATE 649

aminoacetonitrile hydrochloride (VI) is reacted in the presence of chlorine with sulfur monochloride to form a chlorinated thiadiazole (VII), which, in turn, is reacted with morpholine to form VIII. Compounds VI and VIII are reacted to timolol free base (1x1 which is then converted to timolol maleate by refluxing with maleic acid in acetone ( X ) .



The infrared spectrum of timolol maleate taken in a KBr pellet is shown in Figure 2 (4). A Digilab Model FTS-15C Fourier transform infrared spectrophotometer was used to acquire the spectrum. Frequency assignments for some of the characteristic bands are listed in Table I.

Table I

Infrared Spectral Assignments for Timolol Maleate

Frequency ( cm’l) Ass i qnmen t

3250 3000 1700 1200 1100

0-H stretch N-H stretch C=N stretch c-o(c-OH) stretch c-0-c stretch

The infrared spectrum of timolol maleate taken in a mineral oil mull is shown in Figure 3 (4). Both of these spectra are consistent with the structure of timolol maleate.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l l l l l l l I I I I

i V

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l l l l l l l l l l l I I I I

4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 900800 700 600500

Wavenumbers Figure 2. Infrared spectrum of timolol maleate taken in a KBr pellet.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

”;

1 1 1 1 1 1 1

3700 3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 800700600

Wavenumbers

Figure 3. Infrared spectrum of timolol maleate taken in a mineral oil mull. Mineral oil contributes to the spectrum at ca. 1350-1450, 1500- 1750 and 2875-3000 wavenumbers.


4.2 Nuclear Magnetic Resonance Spectrum

4.2.1 Proton NMR Spectrum

The proton magnetic resonance spectrum of timolol maleate was obtained using a Nicolet NT 360 spectrometer. The spectrum was acquired from an approximately 0.02 M solution using d - DMSO as the solvent. shown in Figure 4 and the spectral assignments are listed in Table I1 ( 4 , s ) .

The spgctrum is

A - 20 1

LL / " ' I " '

1 0 8 b

r I

ill 7 4

Figure 4. Proton NMR spectrum of timolol maleate.

654 DAVID J . MAZZO AND ALICE E. LOPER

2*> 3.

3.35

3.47

3.72

4.2

4.4

5.97

6.03

8.38

20.1

Table I1

Re la t ive No. Protons Assignment

d - DMSO sglvent effect

2 - CH- Cg2 -N

- residual H20/HOD

4

4

CH - C H h / -2

‘CH2 - CHf

-N 0 ‘CH - Cgf

0 -N

,CHpE&

1 - CH- I 0

2 0-C€12-

1 -0-g

2

2

2

I I - HC=Cg

H - I +

-N-C(CH3)3 I

I - c02

H - I

-C=C-COOH

TIMOLOL MALEATE 655

4.2.2 CI3 NMR Spectrum

The CI3 NMR spectrum shown in Figure 5 was obtained on a Varian CFT-20 spectrometer using a 0.5 M timolol maleate fglution in d - DMSO (5). The C spectral assignments are listed in Table 111.

Table I11

CI3 NMR Spectral Assignments for

Timolol Maleate

6 (ppm) Assignment

167.4 C02H/C02-

153.3

149.8

135.9 HC=CH

c3

c4 I I

72 .0 -0cH2-(Ca)

65.6 O-(CH2-l2 (a)

65.0 -CHOH-(C@) -

56.5 -NH-C-

43.7 -NHCH2-(Cy)

24.9 C(CH313

I I I I I I I I I I 1 I I I I I I I I 1

180 170 160 150 145 130 120 110 100 90 80 70 60 50 40 30 20 10 0

(PPm)

Figure 5. CI3 NMR spectrum of timolol maleate.

TIMOLOL MALEATE 657

a)

b )

4.3

4.4

1 t

The C and C carbons are equivalent; theregore, onfy a single line is observed for these two carbons.

The C and C carbons are equivalent; there$ore, on?y a single line is observed for these two carbons.

Both the proton and CI3 NMR spectra are consistent with the timolol maleate structure.

1 I

Ultraviolet Spectrum

The ultraviolet absorption spectrum of timolol maleate in 0.1 N aqueous hydrochloric acid is characterized by maxima at approximately 210 nm and 294 nm.

The absortivity in absorbance units drug solution in a 1 cm

is 200 at 294 nm. The W in Figure 6 was obtained

using a Hewlett-Packard Model HP8451A diode array spectrophotometer.

Mass Spectrum

A mass spectrum of timolol is shown in Figure 7. This spectrum was obtained using an LKB model 9000 mass spectrometer operated at 70 eV in the direct probe-electron impact mode. The fragmentation pattern observed is consistent with the chemical structure of timolol.

The molecular ion is noted at m/e = 316. A strong M-minus-methyl peak is observed at m/e = 301. The loss of CIHION leads to m/e = 244. Loss of C H produces a fragment at m/e = 259 wheh lacks the intensity to be seen in a high resolution spectrum. The loss of C H N(H)-CH gives rise to m/e = 230 with4t8e peak it m/e = 86 representing this lost group. Principle fragmenta- tions of the morpholine ring are seen

f 0

8

w 0

658

100

144 154 187 188 229 230 286 3 - I l A - A l

80

316

60

Relative Intensity

40

20

0

I

57 130

50 100 150 200 250 300

(mle) Figure 7. Low resolution mass spectrum of timolol maleate taken

in the electron impact mode.

DAVID J. MAZZO AND ALICE E. LOPER

at m/e = 286 and m/e = 272. A MacClafferty rearrangement leads to m/e = 187 and two ions at m/e = 130 (see Figure 8). Fragmentation of the heter- ocyclic nucleus produces ions at m/e = 144 and possible M-144 at m/e = 172. The peak at m/e = 243 is due to the loss of C H 0 from the molecular ion. This fraAe8t also appears as a characteristic doubly charged ion at m/e = 121.5 (due to the same decomposition). The origin of this peak is apparent from the metastable peak at m/e = 196 which corresponds to the transition from m/e = 301 to m/e = 243.


Timolol maleate, having one chiral carbon, is optically active. The levorotatory enantiomer is the biologically active form. The optical rotation of a 5% (w/v) solution of timolol maleate in i50 N aqueous HCt5at 25 "C and 405 nm is -12.2" (AD = -4.2 " ) (6).

r f7 0 UN

L

mle 187,0407 (1 87.041 5) (1 30.1 232)

-(CH,-0-CH-CH,)

0 /

/ 154.061 6 (1 54.0625)

CH, = N H r f

@ N\s, NH

mle 130.0057 (130,0075) (minor)

(major)

I -H,O

@ CH3 I I CH,

CH,=CH-CH = NKC-CH,

mle112

Figure 8. Schematic representation of the mass spectral fragmentation of timolol maleate.


4.6 Electroanalytical Behavior

Timolol maleate is electrochemically active and it undergoes a single irreversible electro-oxidation. The E of an approximately 40 vg/mL skliition of timolol maleate in 0.01 M aqueous KC1 is +730 mV (vs Ag/AgCl). Figure 9 shows the voltammogram of this solution obtained using a Princeton Applied Research Model 174A Polarographic Analyzer with a glassy carbon working electrode, a silver/silver chloride reference electrode and a platinum auxillary electrode.

4.7 Thermoanalytical Behavior

4.7.1 Melting Point

The melting point of timolol maleate varies between 201.5 'C and 202.5 'C as determined by the USP class Ia capillary method (6).

4.7.2 Differential Thermal Analysis Behavior

The differential thermal analysis (DSC) behavior of timolol maleate under nitrogen is shown in Figure 10. This curve was obtained using a Perkin-Elmer Series 7 DSC scanning from 40 "C to 250 "C at 1 0 "C/minute. A primary endotherm corresponding to melting is observed with an actual peak temperature of 205.8 "C. An additional endotherm with a peak temperature of ca. 215 'C is noted corresponding to compound decomposition.

I I I I I I 1 I I I I

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

Potential (Volts vs. AgIAgCI)

Figure 9. Scanning voltammogram of timolol maleate in 0.1 M K C 1 (aq).

100.0

90.0 -

80.0 -

70.0 - n

60.0 - 3 = 50.0 - - U

m c

40.0 -

30.0 -

20.0 -

10.0 -p

0.0 ' I I I I 1 I I

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0

Temperature ("C)

Figure 10. Differential scanning calorimetry behavior of timolol maleate.

TIMOLOL MALEATE 665

4.7.3 Thermogravimetric Analysis Behavior

Thermogravimetric analysis of timolol maleate indicates no weight loss until 205.8 "C. From 205.8 " C to ca. 350 " C there is an approximately 80% weight loss due to decomposition/vaporization. A typical TGA curve for timolol maleate is shown in Figure 11. The curve was obtained using a Perkin- Elmer Series 7 TGA scanning from 30 "C to 400 "C at 1 0 "C/minute. A sample of 3.523 mg of timolol maleate was used.

4.8 Solubilities

The solubilities of timolol maleate in a variety of solvents at room temperature ( ~ 2 5 "C) is presented in Table IV (6). Note that these solubilities are stated in terms of the current U.S.P. definitions ( 7 ) .

Table IV

Solubility of Timolol Maleate at

Room Temperature

Solvent Solubility

Water Methanol Ethanol Chloroform Propylene Glycol Ether Cyc lohexane Isooctane

Soluble Soluble Soluble Sparingly Soluble Sparingly Soluble Practically Insoluble Practically Insoluble Practically Insoluble

I I

I I

I I

I

8 m 0

8 s: 8 rr, cu

8 0 cv 8 2 8 ?

9

0

m

TIMOLOL MALEATE 667

4 . 9 crystal Properties ( 8 )

Timolol maleate exists as a crystalline powder. No polymorphs of timolol maleate have been reported. Figure 1 2 shows the x-ray powder diffraction pattern of timolol maleate obtained with a Phillips Electronics model APD3720 powder diffractometer using CuKa radiation. Table V lists the interplanar distances and the relative intensities of the major lines in the x-ray powder diffraction pattern of timolol maleate.

Table V X-Ray Powder Diffraction of Timolol Maleate

Peak Angle ( 2 0 " ) D Spacinq (I/Imax)(%)

7 . 1 9 . 9

1 1 . 4 1 4 . 2 1 4 . 8 1 5 . 6 1 6 . 3 18.1 1 9 . 0 1 9 . 5 1 9 . 8 2 0 . 5 2 0 . 6 2 0 . 9 2 1 . 4 2 2 . 4 2 3 . 0 2 3 . 8 2 4 . 8 2 6 . 0 2 6 . 5 2 7 . 1 2 8 . 3 2 9 . 1 2 9 . 5 3 1 . 6 3 4 . 0 3 8 . 8 3 9 . 3

1 2 . 4 9 8 . 9 5 7 . 7 6 6 . 2 3 5 . 9 6 5 . 6 8 5 . 4 4 4 .90 4 . 6 7 4 . 5 6 4 . 4 7 4 . 3 3 4 . 3 1 4 . 2 4 4 . 1 4 3 . 9 7 3 . 8 6 3 . 7 4 3 . 5 8 3 . 4 2 3 . 3 6 3 . 2 9 3 . 1 6 3 . 0 7 3 . 0 3 2 . 8 2 2 . 6 3 2 . 3 2 2 . 2 9

1 3 1 3

6 1 0 0

27 1 2 3 0 43 6 2 2 4 1 8 5 2 5 4 6 7 7 5 3 5

9 50 4 5

7 1 6 60 3 2 1 3

5 26 1 2 1 9 1 4

TIMOLOL MALEATE

4.10 Hygroscopicity (9)

669

The maleate salt of timolol is a stable anhydrous compound. No hydrates of timolol maleate have been reported. The free base of timolol, however, exists both as a stable crystalline hemihydrate and anhydrous compound. Anhydrous timolol maleate is non- hygroscopic when stored under ambient temperature conditions (-25 " C ) and relative humidity conditions ranging from 33% to 76%.

4.11 Dissociation Constant ( 9 )

The pKa of timolol base obtained by potentiometric titration in water at 25 " C is approximately 9.2. The native pH of a saturated aqueous solution of timolol maleate is 2 4.



5.1.1 Ultraviolet Spectrophotometry (10)

Ultraviolet spectrophotometry is used to identify timolol maleate. A solution in 0.1 N HC1 (aq) scanned from 200 nm to 400 nm qualitatively exhibits the same absorbance characteristics at identical wavelengths as does a similarly prepared and concomitantly measured solution of a timolol maleate standard. Quantitatively, equimolar sample and standard solutions will exhibit absorbances at 2 294 nm (Xmax) whi Q differ by no more than 3% (AX&= 2 0 0 ) .

5.1.2 Infrared Spectroscopy

Infrared spectroscopy may also


be used to identify timolol maleate. The infrared spectrum prepared as a mineral oil dispersion compares qualitatively (with maxima only at the same frequencies) to the spectrum of a similarly prepared timolol maleate standard.

5.1.3 Elemental Analysis

Timolol maleate may be identified through the use of elemental analysis. The results of an elemental weight percent determination of carbon, nitrogen, sulfur, hydrogen and oxygen are compared to the respective theoretical values of 49.35%, 17.71%, 10.15%, 7.65% and 15.17%.

5 . 2 Titrimetric Analysis (10)

Timolol maleate may be determined by perchloric acid titration to a visual or potentiometric endpoint. An accurately weighed sample ( = 800 mg) is dissolved in = 90 mL glacial acetic acid and titrated with 0.1 N perchloric acid in dioxane to a green endpoint. Alternatively, the endpoint may be determined potentiometrically as that point at which the greatest change in potential per unit volume change of titrant is noted. For potentiometric titrations, the electrode system should consist of an indicating electrode prepared by emptying a sleeve type calomel electrode, drying it and filling with 0.1 N LiClO in acetic anhydride. A platinum rfng reference electrode is to be used.


5.3.1 Direct Ultraviolet Spectrophotometry

TIMOLOL MALEATE 67 1

Timolol maleate exhibits an ultraviolet absorption band near 294 nm attributed to the thiadiazole core structure. This absorption is the basis for the quantitative determination of the drug. Assay of the compound is performed by comparing the net absorbance at 294 nm of a sample dissolved in 0.1 N HC1 (as) with the net absorbance of a standard in 0.1 N HC1 (as) of known concentration. The net absorbance is calculated by subtracting the drug free matrix contribution to absorbance at the wavelength of determination from the absorbance of the drug solution at the same wavelength.

where :

= Concentration of the sample

= Concentration of the standard

= Absorbance at 294 nm of the

solution

‘std solution

Asam sample solution = Absbrbance at 294 nm of the standard solution

= Absorbance at 294 nm of the drug free solution matrix Ab

5.3.2 Ultraviolet Spectrophotometry via Flow Injection Analysis

Ultraviolet absorbance is also used as the detection mode for timolol maleate determinations by flow injection analysis (11). In the case of the flow injection technique assay, timolol maleate sample and


standard solutions are periodically injected into a flowing stream. The resulting changes in UV absorbance at 294 nm of the stream are measured relative to the analyte-free stream. Calculation of the concentration of timolol is made in an identical fashion as direct UV spectrophotometry.

5.3.3 Specific Rotation

The specific rotation of timolol maleate is determined in 0.1 N HC1 (aq). Using a solution of = 50 mg/mL, the angle of rotation at 405 nm, 25 "C, in a 10 cm cell is determined with a suitable photoelectric polarimeter. Specific rotation, calculated on the dried basis, is between -11.7" and -12.5'.

5.4 Chromatographic Analysis

5.4.1 Thin Layer Chromatography (10)

Normal-phase thin layer chromatography on silica gel has been employed for timolol maleate. A developing solvent system consisting of chloroform/methanol/(28% ammonia water) [80/20/1 (v/v)l is used to develop a spot resulting from 10 pL of an approximately 5 mg/mL solution of the drug in methanol. A reference spot for impurity purposes applied as 10 pL of = 20 pg/mL solution of timolol maleate in methanol is also employed. R for timolol maleate in this &stem is = 0.7. Detection is made under visible light after the developed and air-dried plate has been exposed

TIMOLOL MALEATE 613

to iodine vapor for 2 hours. Sensitivity is estimated to be within an order of magnitude of the amount of timolol maleate in the reference spot solution ( = 200 ng drug).

5 . 4 . 2 High Performance Liquid Chromatography (12)

High performance liquid chromatography may be employed to analyze timolol maleate. A chromatographic system consisting of a column (300 mm x 4.6 mm i.d.) packing with p-phenyl stationary phase (10 pm particle size) has been used with a mobile phase consisting of 1 part methanol and 2 parts 0.005 M aqueous hexane sulfonic acid (pH adjusted to 1 with acetic acid). A column temperature of 30 'C and a flow rate of 2.00 mL/min are used. Detection is by UV absorbance at 280 nm. Typically, 20 pL injections of a timolol maleate solution are made. Under these conditions, timolol is separated from its known potential process impurities.

6 . Stability - Degradation

6.1 Solid State Stability (13)

Timolol maleate is an extremely stable compound at room temperature in the solid state. In fact, prolonged exposure to extremes of temperature and humidity caused minimal degradation of the solid compound. For example, timolol maleate stored for = 2 months at 105 O C in an air atmosphere (ambient relative humidity) showed no difference in potency by TLC chromatographic


profile when compared to a sample of unstressed solid. Only heating timolol maleate to above its melting point and keeping it molten for 10 minutes produced discoloration and an approximate 5% loss in potency. (TLC of this sample did detect small quantities of several unknown decomposition products.) Timolol maleate, when heated at 95 O C for = 3 weeks in an air atmosphere (100% relative humidity), also exhibited a loss in potency of 25% (by W assay). Two degradates were isolated from this sample and were identified as the cis/trans isomer pair of timolol 0-fumarate ester and timolol 0-maleate ester (Figure 13).

Timolol maleate exhibited surface discoloration when exposed in an open dish to intense UV light ( z 20 times normal sunlight). This sample did not, however, show any loss of potency by UV assay nor were any decomposition products detected by TLC.

6.2 Solution Stability (13)

Timolol maleate is extremely stable in aqueous solutions (protected from light) with no decomposition being detected in injectable formulations stored at room temperature for = 5 years. Timolol maleate can be degraded in solution if exposed to elevated temperatures or intense ultraviolet radiation. The solution decomposition of the drug is pH dependent with maximum stability occurring near pH = 4. Solutions of timolol maleate prepared in each of 0.1 N HC1 (aq) (pH = 1) and phosphate buffer (pH = 7 .2 ) and then stored in sealed glass vials for = 2 months at 105 OC yielded considerable amounts of degradation products. A solution prepared in distilled water and stored identically

n 0 OCH,CHCH,NHb-CH,

1 CH3

I 0 I

I

N \ /

N

S c=o

HC = CHCOOH

cis and trans

Figure 13. Potential solid state degradates of timolol maleate exposed to 105 " C / l O O % relative humidity.


was found to be virtually the same as unstressed control solution. Studies of timolol aqueous solutions (pH - 5 ) under autoclave conditions indicated that timolol maleate degrades at the rate of approximately l%/hour at 120 O C . Three decomposition products were isolated from solutions which were autoclave degraded. Figure 14 depicts the proposed degradation mechanism and indicates the three modes of thermally induced solution degradation; (1) rearrangement to isotimolol; (2) ether cleavage to form 4-hydroxy-3- morpholino-1,2,5-thiadiazole; and (3) oxidation followed by ether cleavage to form 4-hydroxy-3-morpholino-l,2,5- thiadiazole 1-oxide.

Aqueous solutions of timolol maleate (pH 5 ) have also been shown to be susceptible to degradation when exposed to intense ultraviolet radiation. Exposure for 2 hours to U V light equivalent to = 20 times normal sunlight produced approximately 2% loss in potency. Evidence of the three thermal degradation products mentioned above as well as several unidentified degradation products was found in this solution. Because of the potential for light-induced degradation, it is recommended that aqueous timolol maleate solutions be stored protected from light.

7. Biopharmaceutics and Metabolism

7.1 Absorption and Bioavailability

Timolol maleate is rapidly and completely absorbed after oral administration (14). Maximum blood plasma concentrations ranging from 10 ng/mL to 100 ng/mL are attained within 1 to 2.4 hours after either acute or

OH CH,

"\_/", n , IIOCH,CHCH,NHC-CH, I I I

N N\ / S

CH,

Timolol

CH,OH n I

o=N'rrr(opH FH3

Y Y S

lsotimolol

Figure 14.

CH~NHC-CH, I CH,

I

'OH

n oWNllfoH N N

' S' I 0

4-hydroxy-3-morpholino- 1,2,5-thiadiazole l-oxide

4-hydroxy-3-morpholino- 1,2,5-thiadiazole

Proposed aqueous solution degradation mechanism for timolol maleate in solutions exposed to autoclave conditions.


7.2

chronic administration of 2.5 mg to 20 mg of timolol maleate twice daily (15-16). The bioavailability of oral timolol is reported to be 61% to 75% of a reference intravenous dose (17-18). Bioavailability of less than 100% is attributed to first-pass metabolic extraction by the liver after oral administration rather than to incomplete gastrointestinal absorption (14). The effect of food on the rate and extent of oral absorption of timolol maleate is not significant (19)

Timolol maleate administered directly to the eye as an ophthalmic drop appears to be rapidly absorbed, producing a decrease in intraocular pressure within three hours (20). Systemic absorption of timolol also occurs after ocular administration, producing peak blood plasma levels of no more than 2 to 5 ng/mL at 0.5 hours to 1.5 hours after instillation of 2 drops of 0.5% timolol maleate ophthalmic solution in each eye (21). Some small but statistically significant cardiovascular effects have been reported after ophthalmic administration (20,221. Supporting studies in rabbits indicate that timolol reaches peak levels in most tissues of the eye at ten minutes after ocular administration with blood serum concentrations peaking at 30 to 60 minutes (23).

Timolol free base is rapidly absorbed through intact skin (24).

Metabolism

After orf4 administration of a 4 mg dose of C- timolol maleate to man, 72% of the I4C label is excreted within 84 hours with 66% in the urine and 6% in the feces. Only 20% of the timolol

TIMOLOL MALEATE 679

dose is recovered unchanged from the urine. Over 80% of the blood plasma radioactivity represents timolol metabolites (14).

Oxidation and hydrolytic cleavage of !be morpholine ring of an oral dose of

urinary metabolites in man (25). C- timolol maleate produces two major

HO-CH2 - CH 0-CH -CH-CH2-NH-C(CH3)3 * bH

HOOC-CH< \ / S

Metabolite I

N-{{4-[3-(l,l-dimethylethyl)aminol-2-hydroxypropoxy}-1,2,5- thiadiazol-3-yl}-N-(2-hydroxyethyl) glycine.

H O - C H ~ - C H ~ - N H - ~ O - C H -CH-CH~-NH-C(CH~)~ N N bH ‘S’

Metabolite I1

l-(l,l-dimethylethylamino)-3-{[4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-ylloxy}-2-propanol.

Metabolite I represents approximately 30% of the total urinary radioactivity while Metabolite I1 accounts for 10% (14,251.

Oxidation of the basic oxypropanolamine side chain of orally administered timolol maleate results in two other minor urinary metabolites in man. Metabolites I11 and IV represent 6% and 3 % , respectively, of administered radioactivity (26).


.. N\s/ N OH

Metabolite I11

2-hydroxy-3-[{4-(4-morpholinyl)-l,2,5-thiadiazol-3-yl}oxy]propanoic acid

H-CH2NH-C-CH2-OH I

O w N - \ / CH3

S

Metabolite IV

l-~[(1,1-dimethyl-2-hydroxy)-ethyllamino}-3-{[4- (4-morpholinyl)-1,2,5-thiadiazol-3-yl]oxy}-2- propanol.

Biological testing of synthetic samples of Metabolites I, I1 and I11 shows only Metabolite I1 to have significant 6-adrenergic blocking activity. However, activity was only one-seventh that of timolol maleate in anesthetized dogs (26).

These four metabolites are also produced by other species. Metabolite I11 represents the major metabolic pathway for timolol maleate in the dog. Other metabolites in rodents result from hydroxylation and oxidation of either the morpholino ring or the oxypropanolamine side chain (25).

7.3 Pharmacokinetics

Oral administration of timolol maleate in doses ranging from 5 mg to 20 mg

TIMOLOL MALEATE 681

results in peak plasma concentrations of timolol maleate within one to two hours, followed by an exponential decline of plasma concentrations with time ( 1 5 ) . The mean apparent half-life of elimination of unmetabolized drug from plasma is approximately 2.5 hours (15 ,171, although plasma half-lives as long as five hours have been reported in normal volunteers ( 2 7 ) . Half-life is independent of the administered dose. Timolol maleate appears to follow simple linear kinetics over the dosage range studied; area under the plasma concentration versus time curve is proportional to the administered dose ( 1 5 ) . The mean renal clearance of unchanged timolol maleate in normal volunteers was reported as 69.6 mL/minute ( 2 7 ) . Pharmacokinetic parameters do not change upon one week chronic administration of oral timolol maleate ( 1 5 ) .

Following intravenous administration, plasma levels decrease biexponentially with time ( 2 8 ) . Reported half-lives for timolol maleate elimination from plasma, 3.3 to 4 . 1 hours, are comparable to the oral half-life.

The pharmacodynamics of oral timolol maleate have been well characterized (15 ,161 . Maximal suppression of exercise-induced tachycardia, a measure of @-blockade, occurs when timolol maleate plasma levels reach 27 to 30 ng/mL. Fifty percent of maximum inhibition of exercise-induced tachycardia is produced by timolol maleate plasma concentrations of 3 to 4 ng/mL.

8. Determination in Biological Matrices

Timolol maleate may be determined in blood plasma and urine by isolation and


derivatization followed by gas-liquid chromatography with electron capture detection (29). Timolol and the internal standard, desmethyltimolol, are extracted from either 1.0 mL of alkalinized plasma or 0.1 mL of alkalinized urine into 4% isoamyl alcohol in heptane. The compounds are then back-extracted into 0.1 N hydrochloric acid, the acidic phase alkalinized, and the compounds extracted into methylene chloride. After evaporation of the methylene chloride, timolol maleate and the internal standard are derivatized by addition of 0.1 mL of heptaflurobutyrylimidazole-ethyl acetate reagent and immersion in a boiling water bath for one hour. The diheptafluorobutyryl derivatives are recovered in n-heptane, which is evaporated at 60 "C under a stream of nitrogen to concentrate the sample. Typically, 1 to 5 p 1 of sample is chromatographed on a 1.83 m X 0.64 cm column packed with 1% OV-17 on 80-100 mesh Gas Chrom Q. The chromatograph is operated isothermally with injection port, oven and detector temperatures of 250 OC, 185 "C and 300 "C, respectively. Helium, the carrier gas, and 10% methane in argon, the purge gas,63re maintained at 75 mL per minute. The Ni electron-capture detector was operated in the pulse mode of voltage with a pulse interval of 50 microseconds. Retention times of derivatized timolol maleate and internal standard are 7.6 and 6.0 minutes, respectively. Limits of detection are 2 ng/mL in plasma and 20 ng/mL in urine.

A capillary column gas-liquid chromatographic-mass spectrometric assay with selectefiion monitoring for timolol maleate and C-timolol maleate in blood plasma is reported to have a sensitivity of 0.5 ng/mL (30). This method provides the capability to examine administration of drug and its stable isotope-labeled counterpart simultaneously for comparison of two different routes of administration. The

TIMOLOL MALEATE 683

isolation procedure prior to assay consists of passing 9.5 to 1.5 mL of plasma, containing H-timolol maleate as an internal standard, through a cartridge containing an octadecyl-silica bonded reversed-phase packing. The cartridge is flushed and the compounds are then eluted with acetonitrile- 0.05 N hydrochloric acid, which is subsequently alkalinized. Back extraction from cyclohexane-toluene into an aqueous phase followed by extraction into methylene chloride precedes evaporation and derivatization with bis(trimethylsily1)-trifluoroa- cetamidepyridine. One to two ~ J L are injected onto a 16 m X 0.25 mm capillary column coated with SE-30. The splitless injection port temperature is 260 "C and oven temperature is 225 " C . Carrier gas (helium) velocity is 30 cm/second.

The mass spectrometer is operated in the electron-impact ionization mode at an ionizing potential of 70 eV, a filament emission current of 0.8 mamp and electron multiplier setting of 1900 V. Selected ion monitoring at m/e 86, 89 and 95, base peaks corresponqing to the side chains ff timolol maleate, C-timolol maleate and H-timolol maleate, respectively, allows construction of a standard curve from the ratios ~

Intensity86/Intensity95 and Intensitygg/ Intensityg5.

Other gas-liquid chromatographic methods employing mass fragmentographic detection (31) or nitrogen-selective flame ionization detection (18) have been reported.

High performance liquid chromatography with electrochemical detection may also be employed for analysis of timolol maleate in human blood plasma and breast milk (32). A 25 cm X 4.6 m i.d. column packed with PXS 5/25 Partisil ODS 3 (5 pm) is used with a mobile phase of methanol :0.2 M sodium dihydrogen phosphate :88% orthophosphoric acid :water (500:200:3:297) pumped at a flow


rate of 1.0 mL per minute. The applied potential for oxidative electrochemical detection is +1.2 V for maximum sensitivity. A glassy carbon working electrode is employed with a silver/silver chloride reference electrode and a platinum auxillary electrode. Timolol maleate and the internal standard, propranolol for plasma or serum, pindolol for breast milk, are extracted from biological matrices with diethyl ether. Compounds are extracted from the organic phase into dilute orthophosphoric acid. For plasma or serum, a hexane wash of the organic phase is sufficient to prepare the acidic aqueous phase for injection of 100 pL into the chromatographic column. For breast milk, the acidic aqueous phase must be alkalinized and the compounds back-extracted into ether. Timolol maleate and internal standard are once again extracted into dilute orthophosphoric acid and the hexane wash and assay are performed as for plasma. Calibration curves are linear from the 2 ng/mL limit of detection up to 100 ng/mL.

9. Determination in Pharmaceuticals

9.1 Dissolution Testing

The dissolution of timolol maleate tablets is evaluated based on methodology and apparatus described in USP XXI(33). The acidic dissolution medium (500 mL of 0.1 N hydrochloric acid) is maintained at 37 5 0.5 "C and basket speed is 100 rpm. After introduction of the tablet, filtered aliquots of the dissolution medium are withdrawn for assay for timolol maleate content against appropriate standards. The official assay is a high performance liquid chromatographic method developed for a system with an ultraviolet detector. The wavelength for detection is 295 nm. A mobile phase consisting of pH 2.8 phosphate

TIMOLOL MALEATE 685

buffer: methanol (3:2) is pumped at a rate of 1.8 mL/minute through a 30 cm X 3.9 mm column packed with octadecyl- bonded silica. About 15 p 1 of dissolution sample is injected onto the column. Not less than 80% of the labeled amount of timolol maleate is dissolved in 20 minutes. Alternative assay methods, as outlined in the previous sections (direct ultraviolet spectrophotometry, ultraviolet spectrophotometry via flow injection analysis and high performance liquid chromatography), can be used if an official cornpendial method is not required.

9.2 Potency Testing

9.2.1 Assay (33)

Official compendial methods are available for both timolol maleate tablets and ophthalmic solution. Tablets are prepared for assay by weighing and finely powdering not less than twenty tablets. An accurately weighed portion of powder is dispersed in 0.05 M monobasic sodium phosphate and sonicated for five minutes. Acetonitrile is added followed by an additional five minutes of sonication. Water is then added and the dispersion is shaken for ten minutes. After dilution to volume with water, the sample may be centrifuged to remove dispersed solids from the timolol maleate solution. The supernatant liquid may be assayed with appropriate standards with the chromatographic system outlined above (Dissolution, Section 9.1) or by an alternative assay if an official cornpendial method is


not required. Tablets must contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of timolol maleate.

The official compendia1 assay for timolol maleate opthalmic solution requires that an aliquot of the solution be diluted to volume with water to produce a 0.5 mg per mL timolol maleate solution. Five mL each of this solution and reference standard solutions are transferred to separators and made basic with pH 9.7 carbonate buffer. Timolol is extracted into toluene from the alkaline aqueous phase. The aqueous phase in each separator is then transferred to a second set of separators for an additional toluene extraction. The aqueous phase in the second set of separators is then discarded. Additional pH 9.7 buffer is added to the toluene remaining in the first set of separators and, after extraction, the aqueous phase is transferred to the second set of separators. After shaking, the aqueous phase is discarded and the toluene from both sets of separators is combined. Timolol in the combined toluene solutions is extracted into four portions of 0.1 N sulfuric acid. The sulfuric acid extracts are diluted to volume with additional acid solution. The extracts are assayed against a 0.1 N sulfuric acid blank at 294 nm with an ultraviolet spectrophotometer. The timolol maleate ophthalmic solution must

TIMOLOL MALEATE 687

9.2.2

contain not less than 90.0 percent nor more than 110.0 percent of label claim.

Dosage Uniformity ( 3 3 )

Tablets must also meet official compendia1 standards for uniformity of dosage units. Ten tablets, assayed individually must exhibit 85.0 to 115.0 percent of the label claim and have a relative standard deviation less than or equal to 6.0 percent. If one unit is outside the label claim range or the relative standard deviation is greater than 6.0 percent, or both, an additional twenty tablets must be tested. Of the thirty tested, not more than one may be outside the 85.0 to 115.0 percent range, and that must be within the 75 .0 to 125.0 percent of label claim range ( 3 4 ) .

Direct ultraviolet spectrophotometry, ultraviolet spectrophotometry via flow injection analysis and reversed- phase high performance liquid chromatography with ultraviolet detection, all as previously described, are acceptable techniques for uniformity determinations. Typically sample preparation involves the disintegration and dissolution of single tablets in solvents identical to those used for assay. A set of extractions and other sample preparation steps identical to those described in the potency assay ( 9 . 2 . 1 ) are then employed.


9.2.3 Stability Testing (33)

Timolol maleate is tested for stability using the high performance liquid chromatographic method described in the USP XXI. Based upon data generated in accelerated and room temperature stability studies, timolol maleate tablets carry a five year expiration dating.

TIMOLOL MALEATE

References

689

4)

6)

9)

Physicians Desk Reference, 41st edition, Edward R. Barnhart, Publisher, Medical Economics Company, Inc., Oradell, NJ, U.S.A., 1987, pp. 1249-1251, 1340-1342.

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E. Oberholtzer, and Joseph V. Bondi; Department of Pharmaceutical Research and Development, Merck Sharp and Dohme Research Laboratories, West Point, PA, U.S.A., personal communication.


10 1

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18

19 1

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The United States Pharmacopeia, 21st revision, Mack Publishing Company, Easton, PA, U.S.A., 1985, pp. 1063-1064.

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TIMOLOL MALEATE 69 1

2 1 1

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30

3 1

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34) Ibid, pp. 1277-1278.

TRAZODONE HYDROCHLORIDE

DeMiS K.J. Gorecki and Roger K. Verbeeck College of Pharmacy

University of Saskatchewan Saskatoon, Saskatchewan, Canada

1. Introduction

1.1. History 1.2. Therapeutic Category

2. Description

2.1. Nomenclature 2.1.1. Chemical Name 2.1.2. Generic Name 2.1.3. Proprietary Names

2.2.1. Empirical 2.2.2. Structural 2.2.3. Registry Numbers

2.3. Molecular weight 2.4. Elemental Composition 2.5. Appearance, Color and Odor 2.6. Patent Information

2.2. Formulae

3. Phvsico-Chemical ProDerties

3.1. Melting Range 3.2. Solubility 3.3. Stability and Storage 3.4. Dissociation Constant 3.5. Crystal structure 3.6. Differential Scanning Calorimetry 3.7. Spectral Properties

3.7.1. Ultraviolet Spectrum 3.7.2. Fluorescence Spectrophotometry 3.7.3. Infrared Spectrum 3.7.4. Nuclear Magnetic Resonance (NMR) Spectra

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 16 693

694 DENNIS K. G. GORECKI AND ROGER K. VERBEECK

3.7.4.1. Proton Magnetic Resonance (PMR)

3.7.4.2. Carbon-13 Nuclear Magnetic Resonance

3.7.5.1. Electron Impact (EI) 3.7.5.2. Chemical Ionization (CI)

Spectra

Spectrum (~%-NMEX) 3.7.5. Mass Spectrometry

4. Synthesis

5. Pharmacokinet i cs

5.1. Absorption 5.2. Distribution 5.3. Elimination


6.1. Identification 6.2. Chromatographic Analysis

6.2.1. Thin Layer Chromatography (TLC) 6.2.2. Gas-Liquid Chromatography (GLC) 6.2.3. High Performance Liquid Chromatography (HPLC)

7. References

TRAZODONE HYDROCHLORIDE 695

1. Introduction

1.1. History

in 1966 by Palazzo and Silvestrini (1) in the chemical laboratories of the firm Francesco Angelini. molecule consists of a triazolopyridine ring and a phenyl piperazinylalkyl moiety. It is the first triazolopyri -dine derivative to be used clinically, and therefore, represents a new chemical class of antidepressant drugs. Its development was a result of an organized approach rather than fortuitous discovery and was based on the hypothesis that the mechanisms responsible for physical and mental pain are the same (2,3).

The history, pharmacology and clinical data on trazbdone hydrochloride is well documented in a number of review articles and monographs ( 3 - 8 ) . In addition, an analytical profile has been described (9).

1.2. Therapeutic Category

indicated in the symptomatic treatment of moderate to severe depression. incidence of anticholinergic and cardiovascular side effects along with minimal stimulatory effects upon dopamine and norepinephrine receptors and is, therefore, considered particularly useful in the geriatric population (5).

Trazodone hydrochloride was originally synthesized

The

Trazodone hydrochloride is an antidepressant drug

Its major advantages include a low

2. Description

2.1. Nomenclature

2.1.1. Chemical Name

2-{3-[4-(3-chlorophenyl)-l-piperazinyll propyl)-1,2,4-triazolo-[4,3-a]pyridin-3(2H)-one - - monohydrochloride


2.1.2. Generic Name

Trazodone hydrochloride

2.1.3. Proprietary Names

Thombran, Trittico Desyrel, Manegan, Molipaxin, Pragmazone,

2.2. Formulae

2.2.1. Empirical

C19H22C1N50 HC1

2.2.2. Structural

2.2.3. Registry Numbers

Chemical abstracts; Trazodone:19794-93-5 Trazodone hydrochloride:25332-39-2

2.3. Molecular Weight

408.33

2.4. Elemental Composition

C, 55.89%; HI 5.68%; C1 17.36%; N, 17.15%; 0, 3.92%

2.5. Appearance, Color and Wor

taste. White, odorless crystals (plates) with a bitter

2.6. Patent Information

U.S. Pat. 3,381,009 (1968 to Angelini Francesco)(l). Brit. Pat. 1,117,068 (11).


3 . Physico-Chemical Properties

3.1. Melting Range

(9). the range 222-228 C (grlOr1l). Under vacuum decomposihion does not occur and a melting range of 231-232.5 C is reported (9).

The melting point for trazodone free base is 96OC The hydrochboride salt melts with decomposition in

3.2. Solubility

Trazodone hydrochloride is soluble in chloroform, sparingly soluble in water, ethanol and methanol (10) and insoluble in common organic solvents (9).

3.3. Stability and Storage

room temperature in tight, light resistant gontainers and protected from temperatures in excess of 40 C (13).

3.4. Dissociation Constant

Trazodone hydrochloride tablets should be stored at

The reported pKa for trazodone, in 50% ethanol, is 6.14 (9). This value was obtained potentiometrically using a glass-calomel electrode.

3.5. Crystal Structure

hydrochloride has been determined (14). diffraction data were obtained using an Oak Ridge diffractometer employing molybdenum Kolradiation with a niobium filter.

Trazodone hydrochloride, C H C1N 0-HC1, crystals are monoclinic with space group182?3c a8d with cell dimension8 a=16.866[31, b..1O.66[2lr c=11.230[21, A, =93.04(1) and Z=4 with the final R factor of 0.042 for 3017 reflections. are 1.344 and 1.345 mg m-

structure of trazodone hydrochloride (58). and angles are tabulated in Tables 1 and 2.

The crystal and molecular structure for trazodone X-ray

The megsured and calculated densities respectively for Z=4.

Bond lengths Figure 1 depicts the 3-dimensional, X-ray derived,

Figure 1. Three-Dimensional, X-Ray Derived, Structure of Trazodone Hydrochloride.

0

Table 1. Intramolecular Bond Lengths ( A ) of Trazodone Hydrochloride

0

A - D i s t anc e s

1.222 N(2)-C(10) 1.349 C( lO)-C(ll) 1.386 C(ll)-C(12) 1.308 C( 12) -N( 13) 1.384 N( 13)-H(13) 1.419 N( 13)-C1(1) 1.344 H( 13)-C1(1) 1.421 N( 13) -C( 14) 1.337 C (14) -C ( 15) 1.384 C (15) -N( 16) 1.394 N(16)-C(17)

0

A - Distances 0

A -

1.451 C(17)-C(18) 1.506 1.518 C(18)-N(13) 1.495 1.512 N(16)-C(19) 1.407 1.498 C(19)-C(20) 1.394 0.872 C(20)-C(21) 1.378 3.052 C(21)-C(22) 1.380 2.180 C(22)-C(23) 1.363 1.491 C(23)-C(24) 1.383 1.497 C(24)-C(19) 1.391 1.455 C(22)-C1(2) 1.747 1.463

Table 2. Bond Angles ( " ) of Trazodone Hydrochloride

Angles

0 (1) -C (1) -N(l) 0 (1)-C( 1)-N(8) C(l)-N(2)-C(lO) C (l)-N( 2)-N( 3) C ( 1) -N ( 8 ) -C ( 9) N(2)-C(l)-N(8) C( 10)-N( 2)-N(3) N( 2)-N( 3)-C(9) N(3)-C(9)-C(4) N (3) -C (9) -N (8) C(9) -C(4) -c(5) C(4)-C(5)-C(6) C (5) -C( 6)-C ( 7 ) C(6)-C(7)-N(8)

(">

130 128 125 114 108 103 121 104 130 112 119 122 121 118

Angles (")

C(7)-N(8)-C(9) 123 N(8)-C(g)-C(4) 118 C(l)-N(8)-C(7) 129 N(2)-C(lO)-C(11) 113

C(ll)-C(l2)-N(13) 113 C(12)-N(13)-C(14) 109 C(12)-N(13)-H(13) 114 N (13) -H (13)-C1(1) 172 N (13) -C (14)-C( 15) 112 C ( 14) -C ( 15) -N (16) 111 C(15)-N(16)-C(17) 111 N(16)-C(17)-C(18) 111 C(17)-C(18)-N(13) 111

C( lO)-C(ll)-C( 12) 112

Angles

C( 18)-N( 13) -C ( 1 4 ) C(17)-N(16)-C(19) C(15)-N(16)-C(19) N( 16) -C (19) -C(20) N(16)-C(19)-C(24) C(19)-C(20)-C(21) c (20) -c (21 1 -c (22 1 C(21)-C(22)-C(23) C(22)-C(23)-C(24) C( 23)-c(24)-c( 19) c (24) -c (19) -c (20) Cl(2) -C( 23) -C(22) C1(2)-C(23)-C(24)

108 118 117 121 121 120 122 117 118 120 118 119 118

TRAZODONE HYDROCHLORIDE 70 1

This study revealed a number of interesting features about trazodone which may be useful in proposing structural requirements for neuroleptic activity. The piperazine ring is in the normal chair conformation with “13) extending upward and N(16) down. The bond lengths and angles about N(13) resemble that of a protonated2 mine and those associated with N(16) demonstrate sp character due to the attached chlorophEny1 ring. Torsiogal angles about N(2)-C(10)(93.2 1 C(ll)-C(12) (178.2 ) suggest that the propylene side chain is fully extended. In addition, the triazolopyridine nucleus (including C(10)) and the chlorophenyl moiety are planar groups . 3.6. Differential Scanning Calorimetry

A differential scanning calorimetry (DSC) curve for trazodone hydrochloride has been reported (9). obtained using a Perkin Elmer DSC 1B instrument was used as a purity index for trazodone.

Data

3.7. Spectral Properties

3.7.1. Ultraviolet Spectrum

The ultraviolet spectrum of trazodone hydrochloride was scanned from 200 to 360 nm with a Gilford Response spectrophotometer at a concentration of 30 and 75!~moles/L in methanol and 30 Umoles/L water (Figure 2). Each spectrum is characterized by two well defined and two less prominent peaks. The spectra in methanol show maxima at 213, 256, 278 and 312 nm while that in water displays maxima at 211, 246, 275 and 312 nm. The latter are in agreement in previously reported data (9,101.

The reported A (l%, 1 cm) and molar absorptivity values for trazodone hydrochloride in water are smrized in Table 3 (9).

Table 3. Ultraviolet Characteristics of Trazodone Hydrochloride

max (m) A( 106, lcm) E

211 246 274 312

1225 50,100 287 11 , 730 94 3,040 94 3 , 840

A10

Figure 2 . Ultraviolet Spectra of Trazodone Hydrochloride (-, methanol; ---- , water).


3.7.2. Fluorescence Spectrophotometry

2-alkyl-substituted-l,2,4-triazolo-[4,3-al pyridin-3 ( 2H)-ones , shows an intense fluorescence-when irradiated with ultraviolet light. The excitation and emission wavelengths for trazodone are 317 and 455 nm respectively (9).

3.7.3. Infrared Spectrum

hydrochloride is shown in Figure 3. was obtained with a Beckman AccuLab 4 infrared spectrophotometer from a compressed KBr disc. Structural assignments of some of the characteristic absorption bands are presented in Table 4.

Table 4. Infrared Characteristics of Trazodone

Frequency (cm-' ) Intensity Assignment

Trazodone hydrochloride, like most

The infrared spectrum of trazodone The spectrum

Hydrochloride

2975-2840 2535,2460 1705 1640 1595

W Aliphatic CH M +N-H stretching S C=O stretching M triazoline C=N stretch S aromatic C=C stretch

M=Medium; S=Strong; W=Weak

Other characteristic bands appe r at 1540, 1491, 1447, 1350, 1273, 943 and 745 cm- 'i . Principle peaks, wavenumbers and structural assignments are in agreement with previously published results (9,lO).

3.7.4. Nuclear Magnetic Resonance (NMR) Spectra

3.7.4.1. Proton Magnetic Resonance (PMR) Spectra

The PMR spectra of trazodone base and trazodone hydrochloride were recorded on a Bruker AM 300 NMR spectrometer employing a frequency of 300.13 MHz. Spectral assignments were confirmed by homodecoup- ling, COSY and heteronuclear correlation experiments.

3 4 5 6 7 8 9 10 11 12 14 16 MICRONS

1 I I I I I I I I I I 1 I 1 I 1

10 okooo 3000 2000 I800 1600 1400 1200 1000 800 600

WAVENUMBER CM

Figure 3. Infrared Spectrum of Trazodone Hydrochloride-KBr Disc.


The spectrum of trazodone base obtained in CDCl Scale expansion $n the region 6.4 to 7.8 ppm is illustrated in Figure 5. shifts, multiplicities and assignments are compiled in Table 5. consistent with the results reported using a 60 MHz instrument ( 9 ) .

Table 5. Proton Magnetic Resonance

is presented in Figure 4.

The chemical

These data are

Assignments for Trazodone Base

12 13 5 6

3 /y\ 9 10 11 /-7 * \ i

U 1

Chemical Numbe r Shift of 6 (ppm) Multiplicities Protons Assignment

7.75 d 1 1 7.11 m 3 314,6 6.84 m 1 8 6.76 m 2 517 6.46 m 1 2 4.09 t 2 9 3.13 t 4 13 2.56 t 4 12 2.49 t 2 11 2.05 m 1 10

d=doublet; t=triplet; m=multiplet

The PMR spectrum of trazodone hydrochloride in DMSO-d 6. Scale expansion in the region 6.5 to 8.0 ppm is depicted in Figure 7. Chemical shifts and assignments are given in Table 6. It can be seen that protonation of the piperzine ring nitrogen nearest the propyl chain affects the chemical shift and splitting pattern of adjacent protons in the piperazine ring.

is shown in Figure

UCJ LL I " " I " " I ~ " ' " ' 1 " ' " " ' ' " I ' " ' I " " I ' " ' I ' ' ~ ' I " " I ' ' "

8.0 7 . 5 7.0 6.5 6.0 5 .5 5.0 4.5 4.0 3.5 3.0 2.5 2 . 0 PPM

Figure 4 . Proton Magnetic Resonance Spectrum of Trazodone Base i n CDC13.

r I I 1 - ' I I I

7 . 8 7 . 6 7 . 4 7 . 2 7 . 0 PPM

I

6.6 I I

6 . 6 6 . 4

Figure 5. Proton Magnetic Resonance Spectrum o f Trazodone Base in CDCl with Scale 3 Expansion (6.4-7.8 ppm).

I " " " " ' I I 1 1 1 1 ' 1 I I 1 1 1 " [ ' I 1 1 I l " l 1 ' I 1 1 1 ' 1 I I 1 1 1 1 ~ I I I I I ~ 1 l 1 1 ~ 1 1 1 1 ] 1 1 1

8.0 7 .5 7 .0 6.5 6.0 5 . 5 5 . 0 4.5 4.0 3.5 3.0 2 . 5 2.0 PPM

Figure 6. Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d6.

r I I 1 I I I I I I I I I I I 7 . 9 7.8 7.7 7 . 6 7.5 7.4 7.3 7 . 2 7.1 7.0 6.9 6.6 6.7 -6 .6

PPM

Figure 7. Proton Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d with 6 Scale Expansion (6.5-8.0 ppm).


Table 6. Proton Magnetic Resonance Assign- ments for Trazodone Hydrochloride

Chemical Number Shift of

6 (ppm) Multiplicities Protons Assignment

7.87 7.25 7.04 6.95 6.86 6.64 4.01 3.86 3.53 3.15 2.25

m m m dd dd m t d d m m

1 1 3 31416 1 8 1 5 1 7 1 2 2 9 2 12 , 13 2 12,13 6 11,14,15 2 10

d=doublet; dd=doublet,doublet; t-triplet m=multiplet

3.7.4.2. Carbon-13 Nuclear Magnetic Resonance Spectrum (IjC-NMR)

The 13C-NMR spectrum of trazodone hydrochloride base obtained in DMSO-d6 is given in Figure 8. obtained at ambient temperature on a Bruker AM 300 NMR spectrometer at 75.47 MHz with broad band proton decoupling. Chemical shifts, and assignments are outlined in Table 7. Spectral assignments were confirmed by C-13m-1 heteronuclear correlation experiments and published chemical shift data.

The spectrum was

d

1 ' ~ ' l ' ~ ' l ' , ' l ' ~ ' , ' , ' , ' , ' l ' , ' " , ' l '

1 6 0 1 4 0 1 2 0 1 0 0 80 60 40 2 0 PPM

Figure 8. Carbon-13 Nuclear Magnetic Resonance Spectrum of Trazodone Hydrochloride in DMSO-d 6'


Table 7. Carbon-13 Nuclear Mametic _. .~~

Resonance Assignments for- Trazodone Hydrochloride

Chemical Shift 6 (ppm)

150.69 147.88 141.06 133.84 130.51 130.47 123.82 119.03 115.12

Assignment

6 1 7 11

4 , s or 9 4,5 or 9

2 10 12

Chemical Shift

6 (ppm) Assignment

114.92 113.99 110.76 52.79 50.23 44.66 42.54 22.78

4,s or 9 8 3 13 17 16 15 14

3.7.5. Mass Spectrometry

3.7.5.1. Electron Impact (EI)

An electron impact mass spectrum of trazodone hydrochloride is presented in Figure 9. This was obtained by direct solid insertion probe at an electron eneljgy of 70 eV and a source temperature of 180 C using a VG Analytical MM 16F single focussing mass spectrometer linked to a VG 2035 data system+ The spectrum shows a molecular ion peak M at a mass/charge (m/z) ratio of 371 with a relative intensity of 13.2% and a base peak at 205. are observed at m/z 70, 136, 166, 176, 209, 231 and 278. The fragmentation pattern leading to the various ions is outlined below.

Prominent diagnostic ions


20:91J 231

Mass spectra of trazodone and other related compounds have been reported elsewhere (9,15,16,17,18).

3.7.5.2. Chemical Ionization (CI)

The chemical ionization spectrum of trazodone hydrochloride is shown in Figure 10. determined on a VG Analytical MM 16F mass spectrometer, however, using ammonia as a reagent gas. The spectrum shows a pseudo molecular ion at a mass to charge ratio of 372.

This spectrum was also

4. Synthesis

the synthesis of trazodone hydrochloride (1,11,19,20). All of the reported routes employ 1,2,4-triazo-[4,3-a]-pyridin- 3( 2H)-one (I) or 2-substituted derivatives as stzrting matFria1. 1.

The original synthesis reported by Palazzo and Silves- trini (1) consisted of the condensation of l-(3-chloro- pheny1)-4-(3-~hloropropyl)-piperazine with the sodium salt of I. Other related patented methods were simultaneously developed in the same laboratories (11).

More recently, modications of existing pathways were reported (19). One method involves the condensation of the usual triazopyridine (I) with bromochloropropane to yield the 2-(3-~hloropropyl)-derivative (11). Subsequent treatment with N-(3-chlorophenyl)piperazine gives trazodone. Alternatively, I is alkylated with bromochloropropane and treated with diethanolamine to form the di-alcohol (111). This product is then chlorinated with thionyl chloride and subsequently reacted with 3-chloroaniline to yield trazodone.

A number of synthetic pathways have been described for

The more important methods are outlined in Scheme

714

100-

80 -

> c cn

c z

W >

- H

6 60 - - -

-

DENNIS K. G. GORECKI AND ROGER K. VERBEECK

I 1s 231

I 1 1

375

0

m

50 100 1-50 200 250 300 350

MASSKHARGE RAT I0

Figure 9. Mass Spectrum of Trazodone Hydrochloride Electron Impact.

Figure 10. Mass Spectrum of Trazodone Hydrochloride- Chemical Ionization.

I1

C l - ( C H ) -N 2 3 w

c 1

HC1

I 0 TRAZODONE c1

HYDROCHLORIDE

.HC1

CH2 CH20H

CH2CH 20H

Scheme 1. Synthesis of trazodone hydrochloride

716 DENNIS K. G . GORECKI AND ROGER K. VERBEECK

The synthesis of 14-C-labelled trazodone hydrochloride has also been published (20) . This procedure also employs the classical condensation reaction described in the original patent.

5. Pharmacokinetics

5.1. Absorption

rapidly absorbed. concentrations typically varies from 0.5 to 4.0 hours in fasting subjects (21-26). Peak plasma concentrations following oral administration of a single 50 mg dose range from 0.5 to 1.0 pg/mL (21,24-26). Following oral ingestion of a single 100 mg trazodone hydrochloride dose average maxim plasma concentrations of 1.61 Pg/mt and 1.66 ug/mL were reported for a capsule and liquid formulation respectively ( 2 2 ) .

is almost complete as shown by the virtually identical plasma concentration-time profiles obtained following oral intramuscular and intravenous administration to healthy volunteers ( 2 3 ) . Ingestion of food delays the absorption of trazodone from the gastrointestinal tract but does not seem to affect its bioavailability (23,27). Studies in rats and rabbits have shown that food delays the oral absorption by decreasing the transfer rate of drug from the stomach to the duodenum (23) .

5.2. Distribution

Following oral administration to man, trazodone is The time to reach maxim plasma

Oral and intramuscular bioavailability of trazodone

Trazodone is a weak base with a pKa of 6.14 (91, and therefore, exists mostly in the unionized form at physiological pH. The drug distributes in a relatively large volume: Vd values reported in the literature or calculated from published data range approximately from 1-2 L/kg for healthy volunteers (21,22,24,25,28). Trazodone is highly bound to plasma proteins. In vitro protein binding studies with human plasma have shown that trazodone is 96, 98 and 97% bound at concentrations of 1.0, 0.1 and O.Olvg/mL respectively (29) .

Studies in laboratory animals show that trazodone readily crosses the blood brain barrier but is minimally transferred across the placenta (30,311. information is available in man concerning its distribution in organs and body fluids. trazodone in human breast milk has been studied and found to be very small (32) . The average milk/plasma ratio was 0.14, therefore, exposure to babies via breast milk would be minimal.

Little

Excretion of


5.3. Elimination

In general, plasma concentrations of trazodone in man decline in a biphasic manner with a mean half-life of approximately 4 hours during the period 3-10 hours following dosing and a terminal elimination half-life of 6-12 hours (21,22,25,28,32). Total body clearance (assuming an oral bioavailability of 100%) ranges from 150 mL/min. to 400 mL/min. (21,24,32). Patients treated with trazodone on a twice daily dosage schedule show significantly higher plasma levels in the morning as compared to the levels at night (33). variation in clearance may be responsible.

hydroxylation, N-oxidation and hydrolysis (Scheme 2) (34). (4-hydroxyphenyl metabolite, I) and in the triazolopyridine ring (dihydrodiol, 11) while N-oxidation occurs on a piperazine nitrogen (N-oxide, 111). A hydrolytic reaction results in the formation of 3-0~0-1,2,4-triazo10[4,3-a]-pyridin-2-yl propionic acid ( IV) and 1-m-chlorophenylpiperazine (V) , a pharmacologTcally active metabolite ( 35,36 1. concentrations of 1-m-chlorophenylpiperazine, approximately 1% of 'frazodone plasma concentrations, have been reported in man (37). Whether these small concentrations contribute to the overall effects of trazodone remains unclear. Approximately 70-75% of an oral dose of the drug is excreted in urine within 72 hours of administration, most as metabolites. Only less than 1% of the dose is recovered in urine as unchanged drug (26,34). The remainder of an oral dose is excreted in feces, involving biliary excretion, mainly as trazodone metabolites (23,271.

does not induce its own metabolites (38).

A possible diurnal

Trazodone is extensively metabolized via

Hydroxylation occurs in the benzene ring

Small

Results from animal studies indicate that trazodone


6.1. Identification

Trazodone hydrochloride reacts with certain reagents to produce characteristic colors which may be useful for its identificahion. reagent at 100 C (sodium nitrite-sulphuric acid) trazodone gives a transient violet color whereas with Mandelin's reagent (ammonium vanadate-sulphuric acid) a grey color develops which later changes to violet (10).

When treated with Liebermanns Test

0 / QOH

GLUCURONIDE OH

C l TRAZODONE \

0 IV

I GLUCURONIDE

Scheme 2. Biotransformation pathways of trazodone in man


An infrared spectroscopic method is described which is useful for the rapid emergency identification of certain psychtropic drugs including trazodone (39). The method employs chloroform extraction of patient's urine at different pH's and utilizes the infrared finger print region and spectra of authentic standards to confirm identity. Infrared spectra are determined as compressed potassium bromide discs from dried chloroform extracts.

6.2. Chromatographic Analysis

6.2.1. Thin-Layer Chromatography ( T L C )

A number of thin-layer chromatographic systems have been developed for the determination of trazodone. These systems have been employed for the identification (9,10,40,41,42) and quantitation (43,44,45) of the drug both as pure drug substance as well as in biological fluids and tissues.

used and most systems use silica gel plates as absorbent. methods is given below.

A variety of detection methods have been

A summary of some of the reported TLC

System I ( 9 ) Absorbent: Merck Kieselgel-0.25 mm Mobile Phase: i) cyclohexane-benzene-diethyl-

amine (5:4:1) ii) chloroform-methanol-isopro-

panol-ether-NH (60:25:25:2) i i i ) n-butanol : wa t e? : NH iv) abs. ethano1:NH (35:5) Detection: ultra-viol& light (366 MI), ninhydrin, rhodanine, ammonium sulphocyanide and cobaltous chloride or potassium dichromate, sulphuric acid with glacial acetic acid.

( 20 : 10 : 2 1

Absorbent: Silica gel G, 0.25 mm treated with potassium hydroxide in methanol. Mobile Phase: i) methano1:NH (100:1.5)

amine (75:15:10) ii) cyclohexane.toluene:diethyl- 3

Detection: ultra-violet light, Dragendorff's reagent or acidified iodoplatinate solution.

System I1 (10)


System 111 (40) Absorbent: Silica gel, F254, 0.25 mm Mobile Phase: i) methano1:NH (100:1.5)

amine (75:15:10) ii) cyclohexane.to1uene:diethyl- 3

iii) ch1oroform:methanol (9:l) iv) acetone Detection: Dragendorff's reagent

System IV (42) Absorbent: Silica gel 60, F254, 0.25 nun Mobile Phase: i) ethylacetate:methanol:30% NH3

(85: 10:5) ii) cyc1ohexane:toluene:diethyl-

amine (65:25:10) iii) ethy1acetate:chloroform

(50:50) iv) acetone Detection: 10% sulphuric acid followed by Dragendorff's reagent and acidified iodoplatinate solution

Absorbent: Silica gel, 0.25 nun Mobile Phase: i) methano1:ammonia (100:l.S) ii) ethy1acetate:methanol:amonia

Detection: 5% sulphuric acid, iodoplatinate solution followed by Dragendorff's reagent

( 85: 10: 5)

System V ( 4 5 )

6.2.2. Gas-Liquid Chromatography (GLC)

A number of GLC procedures have been published for the determination of trazodone. technique has been used for routine detection of the drug (9,10,17), biopharmaceutics and pharmacokinetic studies (16,19,28,47), as well as in therapeutic and overdose stiuations.(45,46) Chromatography of trazodone is readily accomplished without derivatization using a variety of stationary phases in packed or capillary columns. Detectors employed include flame ionization (FID), nitrogen-phosphorous (NPD) and mass spectrometry (MS). The conditions for the reported GLC methods are as follows:

The

TRAZODONE HYDROCHLORIDE 72 1

System I (9)

System I1 (10)

System 111 (16)

System Iv (45)

Type of sample: drug substance Column: 6 ft x 1/4" I . D . glass column packed with 1.5% OV-17 on Chromosorb W HP

Carrier gas: Nitrogen, &3 lb/inch Temperature: column 268 C, injector 32OoC Retention time: approximately 3.5 min. Detector: not stated, presumably F I D

Type of sample: general Column: 2 m x 4 mm I .D . glass packed with 2.5% SE-30 on chromosorb G 80-100 Carrier gas: nitrogen, 45 mt/min. Temperature: column, retention index - 10 Retention index: 3250

2 80-100

Detector: F I D or NPD

Type of sample: plasma ( rat) Column: 80 cm x 4 mm I . D . glass column packed with 1% OV-1 on Chromosorb Q 100-120 Carrier gas: nitrogen, 33 mL/min. Tevrature: column, 240 C; injector, 270 C Retention time: 2 min. Detector: F I D and MS

Type of sample: biological fluids and tissue

fused-silica capillary (0.25wm film) Carrier gas: hydrogen, 48 cwsec. Temperature: column, 265 C Retention time: 2.87 min. Detector: F I D (or NPD) In addition: i) Column: 1.8 m column with 3%

Column: 4 m x 0.25 mm I .D. DB-1

SP 2250 on Supegcoport 100-120 Temperape: 10 C/min. from 140-300 C, hold 5 min. Retention time: 3.89 min. Detector: NJ?D (MS)

SP 2100 on Supegcoport 100-120 Temperape: 13 C/min from 160-320 C, hold 7 min. Retention time: 3.10 min. Detector: NPD (MS)

ii) Column: 1.8 m column with 3%

722

System V (28)

System VI (47)

System V I I (17)

System VIII (18)

DENNIS K. G . GORECKI AND ROGER K. VERBEECK

"ype of sample: plasma Column: 1.23 m x 2 mm I.D. glass column packed with 3% OV-101 on Chromosorb W HP

Carrier gas: helium, 30 p/min Tevrature: column, 260 C, injector, 310 C; detector, 275OC Retention time: 5.1 min. Detector: NPD

80-100

Type of sample: plasma, brain tissue (rat) Column: 1 m x 3 mm I.D. glass column packed with 3% OV-1 on Gas Chrom Q 80-100 Carrier gas: nithogen, 30 ml/min. Temperature: 270 C Retention time: 4.2 min. Detector: NPD (MS)

Type of sample: urine (rat) Column: 60 cm x 1 mm I.D. nickel capillary column packed with 20% UCC-W (Hewlett Packard) on Chromosorb W AW DMCS

Carrier gas: helium, 7 p/min. Temperahure: column, 20 $pin. from 100-300 C; injector, 270 C Detector: MS

80-100

'rype of sample: plasma Column: 4 m x 0.25 mm I.D. DB-1 fused-silica capillary (0.25 pm film) Carrier gas: helium, 2000cm/sec. Teperature: colp, 170 C for 1 gin., 15 C/min. to 255 CA injector, 250 C; separator oven 255 C Retention time: 3 min. Detector: MS

6.2.3. High Performance Liquid Chromatography 3 HPLC )

Several HPLC methods have been developed for the determination of trazodone; most of which have been employed in analysis of biological fluids and tissues for unchanged drug and metabolites. The majority of the procedures utilize reverse-phase chromatography with mobile phases comprised of an aqueous buffer with organic solvent modifiers (21,32,43,48,50-52,54). Others include


reversed-phase with ion-pairing agents (49,56,57) and normal-phase systems (53,551. Detector systems include ultra-violet (w) at various wavelengths, fluorescence and electrochemical oxidation. A summary of the reported HPLC systems is presented below.

System I (48) Type of sample: serum or plasma Coluum: 150 x 4.6 mm Supelcosil LC-8 DB Mobile phase: phosphate buffer 0.01M (pH 4.6): methanol: acetonitrile (47:37:16) Temperature: ambient Flow rate: 2.1 mL/min. Retention time: 3.2 min. Detection: UV at 206 nm

System I1 (32) Type of sample: breast milk Column: 150 x 3.9 mm Nova-Pak C18 (4 p m ) Mobile phase: methano1:phosphate buffer 0.082 M (PH 6.5): acetonitrile (39:47:14) Temperature: ambient Flow rate: 1.5 mL/min. Retention time: 5 min. Detection: W at 211 mm

System I11 (49) Type of sample: plasma and tissue Column: 250 x 4.6 mm trimethyl silyl

Mobile phase: acetonitri1e:phosphate buffer (pH 3.0) (27:73) with 0.02 M heptane sulfonic acid and 0.04 M t r iethylamine Temperature: ambient Flow rate: 1.5 mL/min. Retention time: 12.8 min. Detection: W at 214 nm

(5 urn)

System Iv (50) Type of sample: plasma Column: 300 x 4.6 mm Bondapak C18 (10 VM) Mobile phase: phosphate buffer 0.05 M (pH 4.7): acetonitrhle (72:8) Temperature: 60 C Flow rate: 2.5 mL,/min. Retention time: 8 min. Detection: W at 214 nm

724

System V (51)

DENNIS K. 0. GORECKI AND ROGER K. VERBEECK

Type of sample: serum Column: 250 x 4.6 mm LiChrosorb l o w 8 Mobile phase: acetonitrile: phosphate buffer, 0.6%; pH 3.0 (1:1) Temperature: ambient Flow rate: 2 mL/min. Retention time: 3 . 8 min. Detection: W at 240 nm

System VI ( 5 2 ) Type of sample: biological fluids Column: 250 x 4.6 mm Ultrasphere ODs (octadecylsilyl) 5 p m) Mobile phase: i) phosphate buffer, 0.1 M (pH

7.0): acetgnitrile (10:3.8); 3.0 ml/min; 65 C; retention time (10 :3 .8 ) ; 8 min. phosphate buffer 0.1 M (pH 3.0): acgtonitrile (10:3); 1.5 mL/min.; 60 C; retention time, 1.25 min.

ii)

Detection: W at 242 nm

System VII (43) Type of sample: serum and urine Column: ODs (C18) Mobile phase: methanol: 0.011 M phosphate buffer (pH 7.5): acetonitrile (60:38:2) Detection: W at 250 nm

System VIII (21) "ype of sample: plasma Column: 250 x 4.6 rmn Spherisorb S5 ODs Mobile phase: acetonitrile: 0.05 M sulphuric acid ( 1 8 : l ) Temperature: ambient Flow rate: 2 mL/min. Retention tinre: 6 . 8 min. Detection: Uv at 254 nm

System IX (53) Type of sample: drug substance Column: 125 mm Spherisorb S5W Silica Mobile phase: methanolic ammonium perchlorate, 10 mM, pH 6 . 7 Temperature: ambient Flow rate: 2 mL/min. Relative retention time: 0.31 Detection: W at 254 nm, electrochemical, -I- 1 . 2 v


System X ( 54) Type of sample: plasma Columns: 250 x 4.6 mm MC-18 with 20% carbon load (5 pm), 300 x 4.6 mm Bond- apak C18 (10 um) Mobile phase: phosphate buffer, 0.2 M (pH 4.7): tetrahydrofuran: acetonitrile (90:5:5) Temperature: 45'~ Flow rate: 1.5 !nL/min. Retention time: 39 min. Detection: W at 254 nm

System XI (55) Type of sample: biological fluids Column: 125 x 5 mm Spherisorb S5W Silica

Mobile phase: methanol with 0.02% perchloric acid Temperature: ambient Flow rate: 2.0 mL/min. Retention time: 3.5 min. Detection: Fluorescence, A ex=200nm

Column: 150 x 4.6 mm Ultrasphere octyl (5 urn) Mobile phase: acetonitrile: water (50:50) with 0.05% of each of tetramethylammmonium hydroxide and perchloric acid (70%) Temperature: ambient Flow rate: l.O/min. Retention time: 7.7 min. Detection: Fluorescence, Xex=320 nm; Aem=440 nm

(5 pm)

System XI1 (56) Type of sample: plasma

System XI11 (57) Type of sample: plasma Column: 250 x 4.6 mm LC-1 (trimethysilyl) ( 5 urn) Mobile phase: phosphate buffer (0.05 M I pH 3.0): acetonitrile (9O:lO) with 0.005 M of each of sodium heptane sulfonate and - n-nonylamine Temperature: ambient Flow rate: 2.2 mL/min. Retention time: 13 min. Detection: electrochemical, +1.15 V


7.

1.

2.

3.

4 .

5.

6 .

7.

8.

9.

10.

11.

12.

13.

14.

References

G. Palazzo and B. Silvestrini, U.S. Patent No. 3,381 009. CA:69, - 52146W (1968).

B. Silvestrini, V. Cioli, S. Burberi and B. Catanese, Int. J. Neuropharmacol., 1, 587 (1968). B. Silvestrini. Trazodone. A new approach to the therapy of depression. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy", Eds., G. MOrOZOV, J. Saarma and B. Silvestrini, Edizioni Luigi Pozzi, Roma, 1978, pp. 1-10.

B. Silvestrini, R. Lisciani and M. De Greqorio, in Pharmacological and Biochemical Properties of Drug Substances, Vol. 3, Ed. M.E. Goldberg, Am. Pharm. Association, Washington, DC, 1981, pp. 94-119.

R.N. Brogden, R.C. Heel, T.M. Speight and G.S. Avery, Drugs, 2, 401 (1981). M.M. Al-Yassiri, S.I. Ankier and R.K. Bridges, - Life Sciences, 28, 2449 (1981). T.A. Ban and B. Silvestrini, Eds., Trazodone in Modern Problems of Pharmacopsychiatry, Vol . 9, S. Karger, 1974.

S. Gershon, K. Rickels and 8. Silvestrini, Eds., Trazodone: A New Broad Spectrum Antidepressant, Amsterdam: Excerpta Medica, 1980.

L. Baiocchi, A. Chiari, A. Frigerio and P. Ridolfi, Arzneim-Forsch., 23, 400 (1973).

A.C. Moffat, Ed., Clarke's Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1986, pp. 1035-1036.

Brit. Patent No. 1,117,068. CA:69, - 67429 q (1968). The Merck Index, M. Windholz, Ed., 10th edition, Merck and Co., Inc. Rahway, N.J., U.S.A., 1983 p. 1370.

Desyrel (Trazodone hydrochloride) Product Monograph, Mead Johnson and Co., Evansville, Indiana.

J.P. Fillers and S.W. Hawkinson, Acta Cryst., - B35, 498 (1979).

727 TRAZODONE HYDROCHLORIDE

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G. Belvedere, A. Frigerio and C. Pantarotto, - J. Chromatogr., - 112, 631 (1975).

H. Maurer and K. Pfleger, J. Chromatogr., - 305, 309 ( 1984 1.

R.E. Gammans, E.H. Kerns, W.W. Bullen, R.R. Covington and J.W. Russell, J. Chromatogr., - 339, 303 (1985).

L. Baiocchi and M. Giannangeli, Boll. Chim. Farm., 113, 152 (1981).

K. Sugiyama, S. Kono, C. Yamato and T. Fujita, J. Lab. Compd., 9, 723 (1973). S.I. Ankier, B.K. Martin, M.S. Rogers, P.K. Carpenter and C. Graham, Br. J. Clin. Pharmacol., - 11, 505 (1981).

A.W. Harcus, A.E. Ward, S.I. Ankier and G.R. Kimber, - J. Clin. Hosp. Pharm., - 8, 125 (1983).

R. Javch, Z. Zopitar, A. Prox and A. Zimmer, Arzneim-Forsch., 26, 2084 (1976).

R.E. Gammans, A.V. Mackenthun and J.W. Russell, Br. J. Clin. Pharmacol., - 18, 431 (1984).

A.J. Bayer, M.S.J. Pathy and S.I. Ankier, Br. J. Clin. Pharmacol., - 16, 371 (1983).

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F.W. Koss and U. Bosch, Pharmacokinetics and metabolism of trazodone in different species. In Proceedings of "Depression and the Role of Trazodone in Antidepressant Therapy", as., G. Morozov, J. Saarma and 8. Silvestrini, Edizioni Luigi Pozzi, Roma, 1978, pp. 11-20.

D.R. Abernethy, D.J. Greenblatt and R.I. Shader, Pharmacology, 28, 42 (1984). R.E. Gammans, Personal communication, 1984.

F.W. Koss and U. Bosch: Trazodone as an example-drug levels in blood and brain. In "Trazodone--A New Borad-spectrum Antidepressant". Eds. S. Gershon, K. Rickels and B. Silvestrini, Excerpta Medica, Amsterdam, 1980.

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Y. Suzuki: Teratogenicity and placental transfer of trazodone. Vol. 9", Eds. T. Ban and B. Silvestrini, Karger, Basel, 1974.

In "Modern Problems of Pharmacopsychiatry,

R.K. Verbeeck, S.G. Ross and E.A. McKenna, Br. J. Clin. Pharmacol., - 22, 367 (1986).

L.R. Baxter, J.N. Wilkins and G.B. Smith, J. Clin. Psychopharmacol., - 6, 223 (1986).

L. Baiocchi, A. Frigerio, M. Giannangeli and G. Palazzo, Arzneim.-Forsch., - 24, 1699 (1974).

A. Adamus, M. Sansone, M. Melzacka and J. Vetulani, - J. Pharm. Pharmacol., - 37, 504 (1985).

S. Caccia, M. Ballabio, R. Samanin, M.G. Zanini and S. Garattini, J. Pharm. Pharmacol., - 33, 477 (1981).

S. Caccia, M.H. Fong, S. Garattini and M.G. Zanini, - J. Pharm. Pharmacol., - 34, 605 (1982).

C. Yamato, T. Takahashi and T. Fujita, Xenobiotica, - 4, 313 (1974).

F. Pala, G. DeCosmo, A.F. Sabato and A. Bondoli, Resuscitation, - 9, 125 (1981).

G. Musumarra, G. Scarlata, G. S. Wold, J. Chromatoyr. Sci.,

T. Daldrup, F. Susanto and P. Anal. Chem., - 308, 413 (1981).

G. Musumarra. G. Scarlata. G.

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Michalke, Fresenius Z. CA:96, 29498m (1982).

Cirma, G. Romano, S.

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Palazzo, S. Clementi and G . Giulietti, J. Chromatogr. , - 350, 151 (1985).

P. Giovanni, C. DiNunzio, A. Scotto di Tella, P. Schiano di Zenise and P. Ricci, Boll. SOC. Nat. Napoli, - 93, 41 (1984). CA: - 608, 126695k (1986).

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W.H. Anderson and M.M. Archuleta, J. Anal. Toxicol., - 8, 217 (1984).


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58.

B. Munday, M.J. Kendal, M. Mitchard and T.A. Betts, Br. J. Clin. Pharmacol., - 2, 19 (1975).

S. Caccia, M. Ballabio, R. Fanelli, G. Guiso and M.G. Zanini, J. Chromatogr., 210, 311 (1981).

B. Gerson, S. Chan, F. Bell and K.M. Pappalardo, - J. Psychiat. Res., - 20, 69 (1986).

R.L. Miller and C.L. Devane, J. Chromatogr., - 374, 388 ( 1986 1.

-

S.H.Y. Wong, S.W. Waugh, M. Draz and N. Jain, Clin. Chem. , 30, 230 (1984). P. Hochmuth, J.L. Robert, H. Schreck and R. Wennig Simultaneous determination of benzodiazepines and antidepressants in emergency toxicology by HPLC. "Topics in Forensic and Analytical Toxicology", Ed. R.A.A. Mais, Elsevir, Amsterdam, 1984, pp. 143-149.

I. Root and G.B. Ohlson, J. Anal. Toxicol., - 8, 91 (1984).

- -

In

I. Jane, A. McKiMon and R.J. Flanagan, J. Chromatogr., - 323, 191 (1985).

S.H.Y. Wong and N. Marzouk, J. Liquid Chromatogr., - 8, 1379 (1985).

R. Caldwell and R.J. Flanagan, J. Chromatogr., 345, 187 (1985).

R.N. Gupta and M. Lew, J. Chromatogr., - 342, 442 (1985).

R.F. Suckow, J. Liquid Chromatogr., - 6, 2195 (1983).

J.M. Stewart and S.R. Hall. In "The XTAL system of crystallographic programs, Computer Science Centre, University of Maryland, College Park, M.D., 1983.


8. Acknowledgements

The authors would like to express sincere appreciation to Mr. Peter Pavlakidis for performing the literature search and providing some spectral data, to Mr. D. Leek for determining and Dr. M. Sardessai, Dr. S . Reid and Dr. J.R. Dimmock for assistance in analyzing the nuclear magnetic resonance spectra, to Dr. G. McKay for determining the mass spectra and to Dr. L. Delbaere, Dr. W. Quail and Mr. G. Loewen for interpretation of X-ray diffraction data and providing the three-dimensional structure of trazodone hydrochloride. Special thanks are extended to Mrs. C. Shuttleworth for typing this manuscript.

YOHIMBINE

A . G . Mehhawi* and A . A . Al-Ua&**

*vep&eitt 0 6 Phahmacagnany

**'Depcmhen.t 0 6 Humnaceu;ticcd ~ h m h h y Col lege 0 6 P h m a c y , King Saud UVLivmLty, P.U. Box 2 4 5 7 , Riyadh-] 1451 (Saudi Ahab,i.ul


Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form IeSeNed. 73 1

732 A. G . MEKKAWI AND A, A. AL-BADR

1. Description

1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition


3. Spectral Properties

4 . Natural Sources and Isolation

5. Biosynthesis of Yohimbine

6. Total Synthesis of Yohimbine

7. Pharmacology and Medicinal Uses


8.1 Qualitative Analysis 8.2 Quantitative Analysis

8.2.1 Gravimetric Methods 8.2.2 Titrimetric Methods 8.2.3 Spectrophotometric Methods 8.2.4 Spectrofluorimetric Methods 8.2.5 Chromatographic Methods

Acknowledgements

References

YOHIMBINE 133

From the h i s t o r i c a l po in t of view yohimbine i s t h e most important of t h e complex indo le a l k a l o i d types . I t was known s i n c e anc ien t t i m e s , i n t he curde drug bark o r t h e impure form, as an aphrod i s i ac by t h e West Afr ican n a t i v e s . It occurs i n va r ious t r o p i c a l trees such P a u s i n y s t a l i a yohimbe Pierre and r e l a t e d spe ices , Aspidosperma quebrachoblanco Schlecht and as a minor a l k a l o i d i n some Rawaolfia -. The f i r s t i s o l a t i o n of t h e drug w a s r epor t ed by Spiege l i n 1896 (1) and Witkop (2) i n 1943 suggested its c o r r e c t c o n s t i t u t i o n .

1. Descr ip t ion

1.1 Nomenclature

1.1.1 Chemical Name

17a-Hydroxyyohimban-16a-carboxylic ac id methyl ester ( 3 , 4 ) .

1.1.2 Generic Names

Quebrachine, Corynine, Aphrodine ( 3 , 4 , 5 , 6 ) .

1.1.3 Wiswesser Line Notat ion

a) Yohimbine base: T F6 D5 C666 E M ONbTTTTJ T Q UVOl ......... ( 4 ) .

b) Yohimbine hydrochlor ide: T F6 D5 C666 E . -- M ON&TTTTJ T Q UVOl &GH . . ( 4 ) .

1 . 2 Formulae

1 .2 .1 Empir ical

‘2 lH2 6°3N2

134 A. G . MEKKAWI AND A. A. AL-BADR

1.2.2 Structural

1 0

1 1

d 0 I

OH


354.45


C 71.16%, H 7.39%, N 7.90%, 0 13.54%.


2.1 Color, Odour and Taste

Colorless needles when crystallized from ethanol, colorless or white in bulk. Taste is bitter, odourless ( 4 , 6 , 7 ) .

2.2 Melting Point

240' - 241'.

2.3 Sublimation

Under vacuum the base sublimes at 159°/0.01 mm (7).

2.4 Solubility

Almost insoluble in water, freely soluble in ethanol, chloroform and sparingly soluble in diethyl ether ( 4 , 6 ) .

YOHIMBINE 735

2 . 5 Salts and Their Melting Point

The salts are well crystalline and the hydrochloride is colorless plates m.p. 3oOo - 302OC , the nitrate m.p. 269-270°C, the tarkarate hexahydrate m.p. 213OC, followed by solidification and remelting at 278OC. The thiocyanate is of rectangular crystals (m.p. 233' - 234OC) and the colorless methiodide plates from acetone have the m.p. 249 - 250OC. The melting point for the 0-acetate is 1 3 3 O C and the 0,N-diacetyl derivative melting point is 1 8 3 O C (7 , 8 ) .

2.6 Specific Rotations of Base and Salts

Various specific rotations have been recorded for

the base. [ a ] i O + 107.9' in pyridine; + 56 in

EtOH ( 9 ) ; + 62.2' in EtOH (7 ) ; the hydrochloride

+ 103.3 (H20) ( 7 ) .

3. Spectral Properties


The U.V. spectra of yohimbine in methanol and in 0.1 N H2SO4 were scanned from 200 to 400 nm using Varian DMS 90 spectrophotometer and the following maxima and (E 1%, 1 cm) values were found:

In methanol: 3 maxima at 289, 282 and 225 nm were recorded. The (E 1%, 1 cdfound were : 210

at 289 nm, 247 at 282 nm and 1079 at 225 nm. In the acidic solution a shift to lower wavelengths with some hyperchromic effect was observed (E 1%, 1 an) at 280 nm was 225, at 272 nm was 233

and at 221 nm was 1110. Previous reports in 0.1 N H2S04 (6) were as follows:

(E 1%, 1 an) at 278 nm 204; at 272 nm 208 and 220 nm

1064. Figure 1 illustrates the UV spectrum behaviour for both the methanolic and the acidic solutions of yohimbine.

YOHIMBINE

3.2 I n f r a r e d Spectrum

737

The i n f r a r e d spectrum of yohimbine hydrochlor ide i s presented i n F igure 2. The spectrum i s obta ined from K B r d i s c on a Perkin-Elmer I n f r a r e d Spectrophotometer model 580 B. It shows t h e fo l lowing :

-1 Frequency (cm ) Assignments

3540 OH - s t r e t c h ( f r e e )

3190 NH - s t r e t c h

2900)

2960) CH - s t r e t c h

1730 C = 0 - s t r e t c h

1460)

C = C - aromatic 1472) )

1448)

Other c h a r a c t e r i s t i c abso rp t ion bands are :

1172, 1150, 1025, 1000, 970, 750 and 702 cm-’.

I R d a t a of yohimbine hydrochlor ide have a l so been r epor t ed (3, 5 , 6 ) .

Clarke (6) r epor t ed t h e p r i n c i p l a l peaks at 1705, 741, 1160 o r 1197 o r 1436 cm-1.

3.3 Nuclear Mangetic Reonance Spec t ra (NMR)

3.3.1 Proton Nuclear Magnetic Resonance (PMR)

Spec t ra

The PMR spectrum of yohimbine hydrochlor ide i n DMSO-db and i n DMSO-d6 p lus D20 were shown i n F igures 3a and 3b r e s p e c t i v e l y . The s p e c t r a were recorded on a Varian T60 A NMR spec t rometer us ing t e t r a m e t h y l s i l a n e

Wavelength urn 3 4 5 6 7 8 9 10 11 12 13 1461qm

100. * 90

90 * 80- * 80

70,

* 20

Fac 10. -10 r-’ 0, 0

38003500 3ooo 2500 2000 1800 1600 1400 1200 1000 800 625 Wavenumber

Figure 2 : Infrared spectrum of yohimbine, HBr disc.

YOHIMBINE 139

., . . . . , . . - I ' I . ' . - I - - 1

400 300 1500

80 7.0 6.0 S.OPpM(r)L.O 3 0 2.0 1.0 0 Figure 3a: Proton magnetic resonance spectrum of yohimbine hydrochloride

in D M s 0 - d ~ using TMS as reference standard.

. . . I . . I . . . . , . . . I .

'7 O

200

8.0 7.0 60 WPPM(~)L.O 30 2.0 I0 0 Pigurc 3b: Proton magnetic resonance spectrum of yohimbinc hydrochloride

in DMs0-d~ PIUS @ o using TMS a5 reference standard.


(TMS) as reference standard. The structrual assignments as follows:

Chemical shift (PPMJ Assignments

Multiplets at 0.7-4.60

Singlet at 3.60 CH3 protons

Multiplet at 6.7-7.4 Aromatic protons

Singlet at 4.05 NH (exchangable

CH2 and CH protons

with D20).

with D 2 0 ) . Singlet at 10.65 OH (exchangable

Mills et a1 (3) have reported the proton NMR spectrum of yohimbine.

3.4 Carbon-13 Nuclear Magnetic Resonance Spectrum

(C-13 NMR)

The proton-decoupled and off-resonance carbon-13 NMR spectrum of yohimbine have been determined on Jeol FX 100 MHz spectrometer at an ambient temperature and are presented in Figures 4 and 5 respectively. The sample was dissolved in deuterated chloroform and DMSO-dC; using TMS as reference standard. The carbon assignments are based on chemical shift values and off-resonance spectrum and are listed below:

0 I

OH

(OHIMBINE 741

u

TMS

sil Figure4: Proton- decoupled carbon -13 NMR spectrum of yohimbine in

CDCI3 and D M s 0 - d ~ using TMS as reference standard.

TMS 0

Figure 5 : Off- resonance carbon-13NMR spectrum of yohimbin inCDCi3 and DMSO-ds using TMS a reference standard.

142 A. 0. MEKKAWI AND A. A. AL-BADR

Chemical s h i f t ( 6 )

135.1

70.2

52.6

21.7

106.9

127.0

117.6

118.5

120.5

111.0

136.1

34.7

36.5

52.4

66.8

31.9

23.2

39.8

61.3

174.8

51.6

M u l t i p l i c i t y

S i n g l e t

Doublet

T r i p l e t

T r i p l e t

S i n g l e t

S i n g l e t

Doublet

Doublet

Doublet

Doublet

S ing 1 e t

T r i p l e t

Doublet

Doublet

Doublet

T r i p l e t

T r i p l e t

Doublet

T r i p l e t

Sing 1 e t

Q u a r t e t

Assignment

c2

c3

‘5

c7

‘9

c l o

c1 1

c12

‘6

‘8

‘13

‘14

‘15

‘16

‘17

‘18

‘19

‘2 0

c2 1

c22

‘2 3

YOHIMBINE

3.5 Mass Spec t ra

143

The e l e c t r o n impact ( E I ) m a s s spectrum a t 70 e V recorded on Varian Mat 311 mass spec t rometer and t h e chemical i o n i s a t i o n (CI) mass spectrum obta ined wi th Finnigan 4000 mass spec t rometer are shown i n F igures 6 and 7 r e s p e c t i v e l y . The E I spectrum (Fig. 6) shows a base peak a t m / e 353 and another peak a t m/e 354 (&) of almost equal abundance (98%), o t h e r major fragments are a t m / e 184, m / e 169 and a t m / e 156. The C I spectrum (Fig. 7) shows a base peak a t m / e 355 (M+ + 1) . Other fragments are a t m / e 337 and at m / e 323.

Other m a s s spectrum d a t a has a l s o been r epor t ed (3) *

4. Natura l Sources and I s o l a t i o n

Yohimbine i s t h e major a l k a l o i d of t h e bark of P a u s i n y s t a l i a yohimba (Syn. Corynanthe yohimbe) and a l s o p re sen t i n the r e l a t e d spec ie s p. macroceras , - P. p a n i c u l a t a and 2. t r i l l e s i i ( f . Rubiaceae) (7 ,4 ,8 ) . Several Rauwolfia spec ie s w e r e r epor t ed t o con ta in yohimbine (e .g . &. se rpen t ina ) (4 , lO). &. c a f f r a ( l l ) , - R. t e t r a p h y l l a (12) and &. v o m i t o r i a ( l 3 ) , ( f . Apocynaceae).

The i s o l a t i o n of yohimbine from t h e bark of spec ie s conta in ing i t as t h e major a l k a l o i d can be done by t h e method of Le H i r -- e t a1 (8 ) , where yohimbine i n a mixture wi th the o t h e r isomers (a-yohimbine) w a s bo i l ed wi th d - t a r t a r i c a c i d , t h e yohimbine ta r ta ra te c r y s t a l l i z e d immediately while a-yohimbine ta r ta ra te remained i n s o l u t i o n and c r y s t a l l i z e d later. The e x t r a c t i o n of t he a l k a l o i d s from t h e i r n a t u r a l sources i s g e n e r a l l y c a r r i e d out by t h e convent iona l method. The drug powder i s t r e a t e d wi th Na2C03, d r i e d and e x t r a c t e d with a s u i t a b l e o rgan ic so lven t e.g. benzene o r chloroform (14,15). Le H i r -- e t a1 (8) a l s o used column chromatographic sepa ra t ion . Court and h i s s tuden t s (16-ZO), working on Rauwolfia spec ie s ,used p repa ra t ive th in- layer chromatography as a r o u t i n e technique f o r t he i s o l a t i o n and c h a r a c t e r i s a t i o n of i ndo le a l k a l o i d s . Although p repa ra t ive HPLC w a s n o t used i n t h e r o u t i n e i s o l a t i o n of such compounds but t h e technique might be of r o u t i n e use i n t h e f u t u r e .

744 A. G. MEKKAWI AND A. A. AL-BADR

100.0-

50.0-

169

-9904.

100.

504

4 67 '

337 I

100.0-

50.0-

323

383

18912.

M E 300 320 340 360 380 400 420 440

Figure7 : Chemical ionization (CI )mass spectrum of yohimbine

YOHIMBINE 745

5. Biosynthesis of Yohimbine

The major features of the probable biosynthetic pathway to yohimbine were summarized by Atta-ur-Rahman and Basha (21). Scheme 1 illustrates how yohimbine is biosynthesized from tryptamine and secologanin. The condensation of tryptamine [l] with secologanin [2] is catalysed by the enzyme strictosidine synthetase which controls the C-ring closure mechanism, and results in the formation of strictosidine [3]. A second glycosidase enzyme converts strictosidine via a series of reactive intermediates [4]-[6] to 4,21-dehydrocorynantheine aldehyde [7] by D-ring closure. This aldehyde can then isomerize to [8] which cyclizes to yohimbine.

6. Total Synthesis of Yohimbine

The following total synthesis of yohimbine was described by Van Tamelen et a1 (22). --

p-Benzophenone [l] was condensed with lY4-butadiene to give the adduct 1,4,5,8,9,1O-cis-hexahydronaphthalene- 1,4-dione [2]. Selective reduction by zinc metal and acetic acid gives the octahydronaphthalenedione [3] which was converted to glycidic acid ester [4], subsequent hydrolysis to the glycidic acid [5] and then final decarboxylation to the ketoaldehyde [ 6 ] . The ketoaldehyde [6] was oxidized to the corresponding keto- acid [7] using silver oxide in the presence of alkali. The keto acid [7] was then converted to its acid chloride [8] and the latter was reacted with tryptamine [9] in the presence of pyridine yielding tryptamide [lo]. The tryptamide [lo] is then converted to the cis-diol [ll] using solution of osmium tetraoxide in tetrahydrofuran added to a solution of amide [lo] in pyridine- THF and cooled at dry-ice-acetone temperature. Hydro- genation of the keto-diol [ll] over Adams' catalyst in ethanol gave a good yield of the desired triol [12]. Intermediate [12] was converted to the dialdehyde [13] by cleavage of the triol amide [12] in aqueous acetone and then was cyclized -- in situ using phosphoric acid and heating at 70°, where intermediate [14] was obtained. The lactol [14] was then transformed into the lactol- lactam methyl ether [15] by means of p-toluenesulfonic acid catalyzed methanolysis. Lithium aluminium hydride reduction of [15] in THF afforded the lactol ether base [16]. Hydrolysis of [16] by means of aqueous hydrochloric


,O Glu. + H20 - glucose -

c-0-cs3 [ 2 1 11

0

1 L

+ H 2 0 r -Glucose

CH3-0-C II

CB oc 3 \ \

I 1 + H 2 0 ' I

L -Glucose

CH3-0-C II

CB oc 3 \ \

OH U

-

Scheme 1: Biosynthesis

CH30-C *v , II I

OH of Yohimbine.

YOHIMBINE 141

111 0

t Diels-Alder

--. Condn. -- -@ 0

[21 COOEt

Zn-AcOH - Aqueous NaOH ( h o t ) under

n i t r o g e n

E t h y l c h l o r o a c e t a t e

K t -butoxide/benzene

H O

[31 [41

COOH

Silver

o x i d e / a l k a l i

(1) Cu powder i n d i e t h y l - g l y c o l a t e 155 - 160'

H O H O

[51 [61

Oxalyl c h l o r i d e HZ ~

.)

i n p y r i d i n e

[91 0 0

Scheme 2: T o t a l S y n t h e s i s of Yohimbine.


I H

OH OH H

H HL=O

- LAH

i n THF

OCH3 OH

[I61 [ I 7 1 Scheme 2: Continued.

YOHIMBINE 749

[ 181 OCOCH3

-

285 - 295'

[ 191

Metaperiodate

Cleavage

'OCHO o = c H

Chromic anhydride at Oo/H2 SO4 [211

* Scheme 2: Continued.


i- Chromatography ii- L-camphorsulphonic

acid -

' H \\

CH3-O-C 'OH

0 [ 23 1

Scheme 2. Continued.

YOHIMBINE 75 1

a c i d gave t h e l a c t o l base 1171. The l a t t e r w a s then converted t o t h e l a c t o l acetate [18 ] ( a s acetate s a l t ) by t rea tment wi th a c e t i c anhydride-pyridine. a c e t a t e s a l t [18] w a s sub jec t ed t o py ro lys i s c o n d i t i o n s , where a l i m i t e d amount of acetate s a l t under 0 .01 mm p res su re i n a subl imer was exposed t o a metal ba th p rev ious ly heated t o 285-295', where t h e enol e t h e r [19] have been formed, a s t he acetate s a l t . The' o l i f i n i c bond i n the c y c l i c enol e t h e r system w a s ox id ized us ing osmium te t raoxide-per ioda te . The ozmylation w a s c a r r i e d i n t h e eno l e t h e r a t - 7 8 O i n THF-pyridine, where t h e d i o l [20] was obta ined . Metaperiodate c leavage of 1,2- glycol [20] gave the 0-formate of pseudoyohimbaldehyde [21] . acetone and t r e a t e d a t Oo with chromic anhydride (2.0-3.5 equiv . ) i n s u l f u r i c a c i d f o r 30 minutes . A f t e r removal of t h e forrnyl group, pseudoyohimbine [22 ] was obta ined (low y i e l d ) us ing alumina chromatography. The r e s o l u t i o n of t he s y n t h e t i c base was accomplished by means of l-camphor su lphonic a c i d where pseudoyohimbine ep imie r i zes t o yohimbine[23]. Scheme 2 i l l u s t r a t e s t h e sequence of s y n t h e s i s descr ibed above.

The

The crude pe r ioda te was d i s so lved i n methanol-

7. Pharmacology and Medicinal Uses

Yohimbine produces a compet i t ive a-adrenergic blockade of l i m i t e d du ra t ion and i t i s r e l a t i v e l y s e l e c t i v e f o r t h e p re synap t i c a2-receptor . blocks pos t synap t i c a-adrenergic r ecep to r s . It a l s o blocks 5-HT r e c e p t o r s . It has a l i t t l e e f f e c t on smooth muscle and r e a d i l y p e n e t r a t e s t h e c e n t r a l nervous system and produces a complex p a t t e r n of responses i n doses lower than the r equ i r ed t o produce p e r i p h e r a l a- adrene rg ic blockade. These inc lude vasopress in release and hence mani fes t s an a n t i d i u r e t i c e f f e c t . General CNS e x c i t a t i o n inc lud ing e l eva ted blood p r e s s u r e , increased h e a r t r a t e , i n c r e a s e d motor a c t i v i t y , i r r i t - a b i l i t y and t remors a l s o occured. P a r e n t r a l adminis t ra - t i o n may induce sweat ing,nausea and vomiting (23 - 33) . Lecrubier e t a1 (29) i n v e s t i g a t e d t h e p o s s i b l e use of yohimbine t o improve the cond i t ion of o r t h o s t a t i c hypotension induced by clomipramine. The c o n t r o v e r s i a l aph rod i s i ac p rope r ty of yohimbine was r e c e n t l y i n v e s t i g a t e d by many au thors . Morales e t a1 (30) r epor t ed t h e a b i l i t y of 6 mg of yohimbine, given 3 times a day, t o improve p a r a t h e s i a of t h e lower l imbs i n 4 p a t i e n t s ou t of 6 d i a b e t i c p a t i e n t s . S t e e r s e t a1 (31)

A t h igh doses i t a l s o


s tud ied t h e pharmacological e f f e c t s of yohimbine i n -- i n - v i t r o p repa ra t ions of human and r a b b i t penis and i n v ivo on r a b b i t s and concluded t h a t , i n t he p e n i s , -- yohimbine e x h i b i t s a-adrenergic b locking p r o p e r t i e s and does no t a f f e c t catecholamine l e v e l i n t h i s t i s s u e . Condra e t a1 (32) s tud ied the e f f e c t i v e n e s s of yohimbine i n t h e t reatment of impotence and Nakatau e t a1 ( 3 3 ) have s tud ied t h e pharmacokinetics of yohimbine i n man. However, t he poss ib l e e f f e c t i v e n e s s of yohimbine as an aphrod i s i ac , a t l e a s t i n ra t s , has now rece ived support from a s tudy c a r r i e d out by Clark e t a1 ( 3 4 ) . teams of Condra and Nakatau ( 3 2 , 3 3 ) found t h a t i n p a t i e n t s wi th c l e a r o rgan ic impotence, t rea tment wi th yohimbine hydrochlor ide exh ib i t ed a p o s i t i v e response r a t e of approximately 30% and pos tu l a t ed t h a t t h e f a i l u r e t o improve the condi t ions of t he o t h e r two t h i r d s of p a t i e n t s may be due t o i n t e r i n d i v i d u a l d i f f e r e n c e s i n t h e pharmacokinetics of t h e drug which appears t o be c l ea red i n ind iv idua l s by metabolism.

The


8.1 Q u a l i t a t i v e Analysis

8.1.1 Color Tests

Yohimbine when r eac t ed upon by ammonium molybdate g ives a b lue c o l o r t h a t changes slowly t o green ( s e n s i t i v i t y 0.025 pg); wi th ammonium vanadate t e s t i t g ives a b lue changing t o preen and fad ing away ( s e n s i t i v i t y i s 0.1 pg). With V i t a l i ' s t es t t h e co lour sequence i s ye l low/ye l low/vio le t (6) . Other non-spec i f ic t es t s a r e t h e xanthydrol reagent and 4-dimethylamino benzaldehyde i n methanol ic HC1.

8.1.2 Microcrys ta l Tests

Wachsmuth (35) determined the p r e c i p i t a t i n g p r o p e r t i e s of f l a v i n i c a c i d ( i n e thanol - d i e t h y l e t h e r s o l u t i o n ) on s e v e r a l a l k a l o i d a l and o the r n i t rogen conta in ing subs tances and r epor t ed t h a t abundant p r e c i p i t a t e s were obtained wi th yohimbine ( s e n s i t i v i t y 1:lOO). Habib and Aziz ( 3 6 ) found t h a t yohimbine g ives i r r e g u l a r l a r g e r o s e t t e s of curved h a i r s wi th 0.5% aqueous ammonium r e i n e c k a t e

YOHIMBINE 153

which can be i d e n t i f i e d microscopica l ly . With potassium cyanide solut ion,yohimbine g ives r o s e t t e s of rods and wi th sodium carbonate s o l u t i o n i t g ives r o s e t t e s of rods o r dense r o s e t t e s ( s e n s i t i v i t y i n both reagents 1: 1500) ( 4 ) . C h a r a c t e r i s t i c c r y s t a l s wi th d i f f e r e n t reagents ob ta ined i n our l abora to ry , are i l l u s t r a t e d i n F igures 8-17.

8 . 2 Q u a n t i t a t i v e Analysis

8 .2 .1 Gravimetr ic Methods

Raymond (11) reviewed methods used a t h i s t i m e i n g rav ime t r i c a n a l y s i s . H e e x t r a c t e d powdered bark of yohimbehe wi th e t h e r : chloroform ( 4 : l ) mixture us ing 10 m l of so lvent f o r each gram of bark m a t e r i a l Feldhoff (15) modif ied t h e techniques used be fo re him by l i b e r a t i n g t h e bases i n t h e bark of Yohimbe and e x t r a c t i n g t h e drug i n a soxh le t w i th d i -o r - t r i ch lo roe thy lene mixed i n t h e r e c e i v e r wi th aqueous o x a l i c ac id s o l u t i o n . The o rgan ic so lven t was evapora ted , t h e aqueous a c i d s o l u t i o n w a s t r e a t e d wi th NH40H s o l u t i o n t o l i b e r a t e t h e a l k a l o i d s which were then e x t r a c t e d wi th e t h e r . The a lka- l o i d s were p r e c i p i t a t e d wi th an a l c o h o l i c s o l u t i o n of H C 1 i n a beaker and t h e aqueous a l c o h o l i c p a r t was washed twice wi th e t h e r and methyl acetate. The secondary a l k a l o i d s remained i n s o l u t i o n a f t e r c r y s t a l l i s a t i o n of yohimbine HC1. Af t e r be ing l e f t 24 hours i n a r e f r i g e r a t o r , t h e c r y s t a l l i n e yohimbine- H C l was f i l t e r e d on a weighed f i l t e r paper , washed and d r i e d a t 102% t o cons t an t weight .

wi th Na2C03 t rea tment

8.2.2 T i t r i m e t r i c Methods

P r e c i p i t a t i o n T i t r a t i o n

Vytras and Riha (37) c a r r i e d ou t p r e c i p i t a - t i o n t i t r i m e t r y f o r 9 a l k a l o i d s inc lud ing yohimbine us ing sodium t e t r apheny lbora t e reagent and a Crytur valinomycin ion- s e l e c t i v e e l e c t r o d e .


Figure8: Rosettes of circular plates of yohimbine with picric

acide reagent (a f ter 3 minutes 1.

Figure91 Radiating pointing plates of phimbine with mercuric

chloride reagent (a f ter 14minutes).

Figure 10 : Orange coloured radiating relatively wide crystal

plates of yohimbine with potassium chromate reagent

(after 9 minutes ).

YOHIMBINE 155

Figurell : Tagged plates of crystals of yohirnbine with

Dragendorf f's reagent ( after 5 minutes ),

Figure12. White crystalline minute rods of yohimbine after

reaction w i th lead iodide reagent (immediately

produced )

Figure 13: Crystal of tetragonal or hexagonal structures

af ter addition of Marme's reagent (after 5 minutes).


Figure 14 Radiating rod clusters of yohlrnblne and Naf03

solution (after 10 minutes)

Figure IS : Crystal plates, characteristic, to reaction of

yohimbine with NH4S CN aqueous solution

( a f t e r 5 - 7 minutes),

Figure 16: immediate sandy crystals w i t h Wagner's reagent.

Figure 17. Fine clusters of rods with KCN solution (after 15minutes)

YOHIMBINE

b ) M i cr 0- he te r ome t r i c T i t r a t ion

I n t h e i r t r i a l t o analyze l a r g e o rgan ic n i t r o g e n - con ta in ing compounds, Bobtelsky and Braz i ly (38) s t u d i e d t h e behaviour of micro amounts of yohimbine t o g e t h e r w i th n i t r o n , quinone , s t rychn ine , papaverine , n i c o t i n e , a t r o p i n e , morphine o r phenanthro- l i n e when t i t r a t e d h e t e r o m e t r i c a l l y wi th t u n g s t o s i l i c i c , tungstophosphoric o r molybdophosphoric a c i d a t pH 1 o r 7. They found t h a t t h e in f luence of t h e a c i d i t y of t h e s o l u t i o n was d i f f e r e n t i f t he o rgan ic molecule contained 1 o r 2 h e t e r o c y c l i c N atoms.

8.2.3 Spectrophotometr ic Methods

a ) Color imetry

Color imet r ic a s says of t he weakly b a s i c t e r t i a r y a l k a l o i d w e r e f i r s t done on c o l o r r e a c t i o n s c h a r a c t e r i s t i c of t h e i n d o l e group (8, 29). I n presence of o t h e r i n d o l e conta in ing compounds, such as those n a t u r a l l y p re sen t i n t h e n a t u r a l source,such a method i s of l i t t l e va lue . However, Kolsck (39) used t h e co lour r e a c t i o n of yohimbine i n d o l e nuc leus wi th p-dimethylaminobenzaldehyde and measured t h e co lour produced photometr ica l ly with f i l t e r S 39 o r S 49. The method i s summarized a s fo l lows:

Yohimbine-HC1 w a s d i s so lved i n water, i f d e s i r e d wi th a d d i t i o n of l i t t l e e thano l . In a 10 m l vo lumetr ic f l a s k t o 1 m l of t h e s o l u t i o n con ta in ing 0.05 - 3 mp, of t h e allcaloid sal t , 2 m l of p-dimethylylamino- benzaldehyde w e r e added and the mixture w a s l e f t t o cool f o r 10 minutes and 0.05 m l of a 5% H202 (o r 1% NaN02) s o l u t i o n w a s added, shaken, and t h e c o l o r w a s al lowed t o develop. To t h i s , d i s t i l l e d water was added t o make 10 m l of mixture and d e n s i t y was measured photometr ica l ly us ing a f i l t e r S 39 o r S 49. The mechanism of t h i s r e a c t i o n w a s s tud ied by Pindur (40).


Jung assayed reserpine and yohimbine (41 ,42) photometrically with xanthydrol reagent in presence of other nitrogen containing compounds. The indole nucleus of the two compounds forms a colored compound with xanthydrol and the extinction is measured at 515 mu. In this method a 50-150 pg of yohimbine were dissolved in 5 ml of a freshly prepared solution of 0.4% xanthydrol in glacial acetic acid Containing 1% v/v HC1 heated on a boiling water bath for 15 minutes, cooled and the extinction wasmeasured at 515 mu. The color is stable for 5 hr. The procedure can be used for tablets, injections or mixtures after extracting the alkaloid by a suitable method The method was also used by Budavaris - a1 ( 4 3 ) .

The yohimbine-methyl orange complex color as a quantitative method was utilized by Mohamed and Elsayed ( 4 4 ) for assaying the base in tablet formulations. A solution of the powdered tablets ( e 5 0 mg of yohimbine hydrochloride) in 50 ml of H20 was prepared A measured volume containing about 300 mg of tablet base was diluted to 50 ml. A 2 m portion of this solution was transferred to a separating-funnel containing 2 ml of buffer (pH 4.0) and 1 ml of 0.1% methyl orange solution in 20% ethanol. The yohimbine-methyl orange complex was extracted successively with chloroform, diluted to 25 ml with chloroform and the extinction was measured at 421 nm. For 5 determinations the recovery was 97.6 - 102.5%.

8 .2 .4 Spectrofluorimetric Methods

In their spectrophotofluorimetric study of a vast number of organic compounds of pharmaceutical interest, Underfried et a1 ( 4 5 ) using a Xe arc - source emitting a continuum from 200-800 mu in their spectro- fluorometer,reported that yohimbine at pH 7 had its maximum activation at 270 mu and its maximum emission of fluorescence at 360 mu.

YOHIMBINE 759

Chiang and Chen (46) assayed yohimbine after increasing its fluorescence in solutions by subjecting its heated solution in presence of H202 to ultraviolet light. Auterhoff and Schroeder ( 4 7 ) carried a similar assay and evaluated yohimbine in Yohimbe bark. They extracted 1 g of the bark with ethyl ether after alkalinisation with 10% aqueous ammonia. The ethereal extract was re-extracted with 0.1 N H2SO4 solution and the acid solution was basified with NH40H, extracted with chloroform, concentrated and diluted to 10 ml with chloroform. The chloroformic extract was chromatographed on a MN 300 Polygram cellulose plate impregnated with formamide : dimethylformamide : acetone (4:3:12) and developed with heptane : ethylmethyl ketone : 0.25% aqueous ammonia (4:2:1) for 15 cm and the zone of Rf 0.2, yohimbine, was extracted with CHC13 : CH3OH (100 ml). The extract was filtered, the solvent removed under reduced pressure at 35OC and the residue dissolved in 10 ml of 30% acetic acid. To 1 ml of this solution, 1 ml of 3% H202 and 4 ml of 30% acetic acid were added. A similar solution of standard yohimbine was prepared. Both solutions were heated for 2 hr at 80°C, cooled and the volume was adjusted to 10 ml with 30% acetic acid. After excitation at 365 nm the fluorescence was measured at 496 nm.

correspondin? to

Clarke ( 6 ) reported the quantitative estimation of yohimbine as follows:

The drug when in a mixture consisting of reserpine, ajmaline and yohimbine may be separated by thin-layer chromatography and determined by spectrophotometry or spectrofluorometry as described previously for indole alkaloids by some authors (48,49)


8.2.5 Chromatographic Methods

8.2.5.1 Paper Chromatography (P.L.C.)

Za f fa ron i ' s system (50) w a s t r i e d by Machovicova ( 51 ) on paper chromatography and was found t o be s u i t a b l e f o r t h e sepa ra t ion of r e se rp ine , r e s e r p i c a c i d and yohimbine. Quan t i t a t ion of yohimbine us ing P.L.C. by Auterhoff and Schroeder (47) i s descr ibed i n s e c t i o n 8.2.4 above

Clarke (6) repor ted t h e fol lowing paper chromatography systems f o r t h e sepa ra t ion of yohimbine:

a ) Paper: Whatman No. 1 , shee t 16 x 4 inch , buf fered by d r ipp ing i n a 5% s o l u t i o n of sodium dihydrogen c i t r a t e , b l o t t i n g , and dry ing a t 25' f o r 1 hour. be s t o r e d i n d e f i n i t e l y ) .

( I t can

Sample: 2 .5 ~1 of a 1X s o l u t i o n ; i n 2 N a c e t i c ac id i f p o s s i b l e , o therwise i n 2 N hydrochlor ic , 2 N sodium hydroxide, o r e thano l .

Solvent : 4.8 of c i t r i c a c i d i n a mixture of 130 m l of water and 870 m l of n-butanol. This may be used f o r s e v e r a l weeks i f water added from t i m e t o t ime t o keep t h e s p e c i f i c g r a v i t y a t 0.843 t o 0.844.

Development: Ascending, i n a tank 8 x 11 x 15% inch , 4 s h e e t s be ing run a t a t i m e . Time of run, 5 hour.

Location: Under u l t r a v i o l e t l i g h t , pa l e b lue f luorescence ; l o c a t i o n reagent : i o d o p l a t i n a t e spray , week r e a c t i o n ; bromocresol green spray; weak r e a c t i o n .

b) Paper: Whatman No. 1 o r No. 3 , shee t 1 7 x 19 cm, impregnated by d ipping i n a 10% s o l u t i o n of t r i b u t y r i n i n acetone and dry ing i n a i r .

YOHIMBINE 76 1

Sample: 5 1-11 of 1 t o 5% s o l u t i o n i s e thano l o r chloroform.

Solvent : Acetate b u f f e r (pH 4.58).

E q u i l i b r a t i o n : The beaker con ta in ing t h e so lven t i s e q u i l i b r a t e d i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 95' f o r about 15 minutes .

Development: Ascending. The paper i s fo lded i n t o a c y l i n d e r and c l ipped , t he c y l i n d e r i s i n s e r t e d i n t h e beaker con ta in ing t h e so lven t which i s n o t removed from t h e oven. A p la t e -g l a s s d i s k t h i c k l y smeared wi th s i l i c o n e g rease may se rve a s a cover. T i m e of run , 15 t o 20 minutes .

Locat ion: Under u l t r a v i o l e t l i g h t , b lue f luorescence . Rf va lue 0 .64 .

c) Paper and Sample: A s above i n (b) . Solvent: Phosphate b u f f e r (pH 7 . 4 ) .

E q u i l i b r a t i o n : The beaker con ta in ing t h e so lven t i s e q u i l i b r a t e d i n a t h e r m o s t a t i c a l l y c o n t r o l l e d oven a t 86' f o r about 15 minutes .

Development: A s above i n (b) . Location: A s above i n (b) . Rf va lue 0.04.

8.2.5.2 Thin-Layer Chromatography (T.L.C.)

S i l i c a g e l G. p l a t e s preapred wi th 0.1 M NaOH o r 0.1 M KHSO4 developed by 5 so lven t systems were used by Fike (52) i n t h e s tudy of t h e behaviour of 140 b a s i c drugs inc lud ing yohimbine and t h e Rf va lues of those drugs w e r e recorded and success fu l s e p a r a t i o n w a s achieved bu t none of t he i n d o l e a l k a l o i d s r e l a t e d t o yohimbine w e r e inc luded i n t h e s tudy. However, Court and

162 A. 0. MEKKAWI AND A. A, AL-BADR

and Habib (16) and Court and Timmins (17 ) , i n t h e i r s t u d i e s on Rauwolfia a l k a l o i d s and t h e i r behaviour on T.L.C. have piven va luab le informat ions on t h e co lour r e a c t i o n s and R f va lues of such r e l a t e d a l k a l o i d s on s i l i c a g e l l a y e r s employing 10 so lven t systems and emphasis can be concent ra ted on t h e r e l a t e d a l k a l o i d s :

Yohimbine, a-yohimbine, s e r p e n t i n e , sa rpagine and a jma l i c ine which come i n t h e s t r o n g l y b a s i c f r a c t i o n of Rauwolfia r o o t e x t r a c t .

Quan t i t a t ion photodens i tomet r ica l ly on s i l i c a g e l l a y e r s of a l k a l o i d s of p l a n t sources w a s c a r r i e d ou t by Massa e t a1 (53) who found t h a t f l uo r scence r e f l e c t a n c e readings of unsprayed s p o t s of f l u o r e s c e n t a l k a l o i d s were favourable i n a c t i v a t e d s i l i c a g e l p l a t e s . photodensi tometer wi th r eco rde r and i n t e g r a t o r func t ion ing i n t h e v i s i b l e and u l t r a v i o l e t l i g h t s . Nine so lven t s systems were used and s u i t a b l e concen t r a t ions of s tandards were run and the s tudy w a s done on 25 a l k a l o i d s of which 14 a l k a l o i d s sprayed wi th Dragendorff-Schuette r eagen t , were de t ec t ed a t 400 nm and 11 o t h e r s were determined by i n h i b i t i o n of f luorescence a t 528 nm and 13 a l k a l o i d s es t imated by t h e i r n a t u r a l f luorescence o r induced by a s u i t a b l e reagent such as KMnO4, Iodine o r d i c h l o r o a c e t i c ac id and h e a t , and e x c i t e d a t 358 nm. However, Court and Habib (16) q u a n t i t a t e d 10 Rauwolfia a l k a l o i d s by combined p repa ra t ive T.L.C. and co lo r ime t ry and found t h a t t h e i r method w a s b e s t s u i t a b l e f o r B-carboline a l k a l o i d s .

They used a double-beam

Clarke (6) descr ibed t h e fol lowing TLC system f o r t h e i s o l a t i o n of yohimbine:

P l a t e : Glass p l a t e s 20 x 20 c m , coated w i t h a s l u r r y c o n s i s t i n g of 30 g of s i l i c a p,el G i n 60 m l of water t o g ive a l a y e r 0.25 mm t h i c k and d r i e d a t l l O o f o r 1 hour .

YOHIMBINE 163

Sample: 1 p1 of a 1% s o l u t i o n i n 2 N a c e t i c ac id .

Solvent : S t rong ammonia s o l u t i o n : methanol (1.5 : 1 0 0 ) . It should be changed a f t e r two runs.

E q u i l i b r a t i o n : The so lven t i s allowed t o s t and i n the tank f o r 1 hour.

Development: Ascending, i n a tank 2 1 x 2 1 x 10 cm, t h e end of t h e tank being covered wi th f i l t e r paper t o a s s i s t evapora t ion . Time of run: 30 minutes .

Locat ion reagent : Ac id i f i ed i d o p l a t i n a t e spray , p o s i t i v e r eac t ion . Rf va lue 0.55.

8.2.5.3 (G.L.C.)

Dusci and Hackett (54) descr ibed a d i r e c t procedure f o r a n a l y s i s of yohimbine and o t h e r n e u t r a l drugs i n t i s s u e s . They used Hewlett-Packard gas chromatograph model 5700A equipped with a f l ame i o n i z a t i o n d e t e c t o r . The column w a s a 4 f t x 4 mm i . d . g l a s s column pakced with 3% OV-17 on Gas Chrom Q , 80-100 mesh. The ins t rument s e t t i n g s were a s fo l lows:

I n j e c t i o n po r t temperature , 25OoC; d e t e c t o r temperature , 30OoC; n i t rogen c a r r i e r gas flow ra te , 60 ml/min. Oven temperature programmed as 15OOC he ld f o r 2 minutes , i nc reased a t 8OC/min t o 29OoC and t h e f i n a l temperature was he ld f o r 4 minutes . Using c h o l e s t e r o l a s a s t anda rd , t he r e l a t i v e r e t e n t i o n t i m e (RRT) of yohimbine was 0.89.

Clarke (6 ) r epor t ed t h e fo l lowing GC system f o r t h e sepa ra t ion of yohimbine.

Column: 5% SE-30 on 60-80 mesh Chromosorb W AW, 5 f t x 118 inch i n t e r n a l d iameter s t a i n l e s s steel column.

Column temperature : 23OoC.

Carrier Gas: Nitrogen.


Gas Flow: 30.7 m l per minute.

Detector : Flame i o n i z a t i o n d e t e c t o r , hydrogen 22 m l per minute.

Retent ion t i m e : 0.38 r e l a t i v e t o codeine.

8.2.5.4 High-speed Liquid Chromatography(HPLC)

The advent of HPLC i n t h e f i e l d o f s e p a r a t i o n and q u a n t i t a t i o n of almost a l l types of chemical mix tures made t h e t a s k s of t h e a n a l y t i c a l chemists f a r easier than two decades ago. HPLC on a l k a l o i d s us ing va r ious types of chromatographic sepa ra t ion modes w a s c a r r i e d out by va r ious au tho r s . Yohimbine sepa ra t ion on adosrp t ion HPLC w a s success fu l and Verpoorte and Svendsen ( 5 5 ) were a b l e t o s e p a r a t e i t from r e s e r p i n e on Mercksorb S 1 70 ( 5 pm) column us ing 6 systems of e luen t and a Packard Model 8200 l i q u i d chromatograph equipped with a 254 nm and 280 nm UV d e t e c t o r w a s used. The column w a s a s t a i n l e s s s t e e l tube (30 cm x 2 mm i .d.) . The column temperature w a s maintained a t 2OoC and p res su res of 50-250 kg/cm2 were employed t o c o n t r o l f low rates. However, t h e r e l a t i v e l y high[E 1%, 1 cm]of

yohimbine i n U.V. l i g h t a t 226 nm makes t h e d e t e c t i o n of minute q u a n t i t i e s p o s s i b l e i f t he ins t rument U.V. wavelength can be va r i ed t o t h e optimum. Rodgers ( 5 6 ) w a s ab le t o ana lyse s e v e r a l classes of t r an - q u i l i z e r drugs inc lud ing Rauwolfia a l k a l o i d s by t h i s technique us ing adsorp t ion columns of s i l i c a g e l and c a t i o n exchangers.

Acknowledgements

The au thor would l i k e t o thank M r . Amir Shehata (B. Pharm.) of Pharmacognosy Dept. f o r performing t h e mic roc rys t a l t es t s and t o Tanvir A. Bu t t , Sec re t a ry t o Pharmaceut ical Chemistry Dept. f o r typing t h e manuscr ipt .

YOHIMBINE 765

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CUMULATIVE INDEX

Bold numerals refer to volume numbers

Acetaminophen, 3, I ; 14, 551 Acetohexamide, 1, I ; 2, 573 Allopurinol, 7, I Alpha-tocopheryl acetate, 3, 1 1 1 Amantadine, 12, 1 Amikacin sulfate, 12, 37 Amiloride hydrochloride, 15, I Arninoglutethimide. 15, 35 Aminophylline. 11, I Aminosalicylic acid, 10, I Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, 1: 7, 502 Ampicillin, 2, I ; 4, 518 Ascorbic acid, 11, 45 Aspirin, 8, I Atenolol. 13, 1 Atropine, 14, 32 Azathioprine, 10, 29 Bacitracin, 9, 1 Baclofen, 14, 527 Bendroflumethiazide, 5, 1; 6, 597 Benperidol 14, 245 Benzocaine, 12, 73 Benzyl benzoate, 10, 55 Betamethasone dipropionate, 6, 43 Bretylium tosylate, 9, 71 Bromazepam, 16, I Bromocriptine methanesulfonate, 8, 47 Busulphan, 16, 53 Caffeine, 15, 71 Calcitriol, 8, 83 Camphor, 13, 27 Captopril, 11, 79 Carbamazepine. 9, 87 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10, 729 Cefazolin, 4, 1 Cefotaxime, 11, 139 Cefoxitin. sodium, 11, 169 Cephalexin, 4, 21 Cephalothin sodium, 1, 319

Cephradine, 5, 21 Chloral hydrate, 2, 85 Chlorambucil, 16, 85 Chloramphenicol, 4,47, 518; 15, 701 Chlordiazepoxide, I, 15 Chlordiazepoxide hydrochloride, 1, 39; 4, 518 Chloroquine, 13, 95 Chloroquine phosphate, 5,61 Chlorpheniramine maleate, 7, 43 Chlorprothixene, 2, 63 Chlortetracycline hydrochloride. 8, 101 Chlorthalidone, 14, I Chlorzoxazone, 16, 119 Cholecalciferol. see Vitamin D3 Cimetidine, 13, 127 Cisplatin. 14, 77 : 15, 7% Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10, 75 Clofibrate, 11, 197 Clonazepam, 6,61 Clorazepate dipotassiurn, 4, 91 Clotrimazole, 11, 225 Cloxacillin sodium, 4, 113 Cocaine hydrochloride, 15, 15 I Codeine phosphate, 10,93 Colchicine, 10, 139 Cyanocobalamin, 10, 183 Cyclizine, 6, 83; 7, 502 Cycloserine, 1,53 Cyclosporine, 16, 145 Cyclothiazide.1, 66 Cypropheptadine, 9, 155 Dapsone, 5, 87 Dexamethasone, 2, 163; 4, 519 Diatrizoic acid, 4, 137; 5, 556 Diazepam, 1,79: 4, 518 Dibenzepin hydrochloride, 9, 181 Dibucaine and dibucaine hydrochloride, 12, 105 Diflunisal, 14, 491 Digitoxin, 3, 149 Digoxin, 9, 207 Dihydroergotoxine methanesulfonate, 7, 81

769

770 CUMULATIVE INDEX

Dioctyl sodium sulfosuccinate, 2, 199: 12, 713 Diperodon, 6 ,99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disopyramide phosphate, 13, 183 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride. 11, 257 Doxorubicine. 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10, 289 Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, I13 Erythromycin. 8, 159 Erythromycin estolate, 1, 101; 2, 573 Estradiol. 15, 283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Etomidate. 12, 191 Fenoprofen calcium 6, 161 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Flufenamic acid, 11, 313 Fluorouracil, 2, 221 Fluoxymesterone, 7, 25 I Fluphenazine decanoate. 9, 275; 10, 730 Fluphenazine enanthate, 2, 245; 4, 524 Fluphenazine hydrochloride, 2, 263; 4, 519 Flurazepam hydrochloride, 3, 307 Gentamicin sulfate, 9, 295; 10, 731 Glibenclamide, 10, 337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219; 9, 583 Guanabenz acetate. 15, 319 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, 1, 119; 2, 573; 14, 597 Heparin sodium, 12, 215 Heroin, 10, 357 Hexestrol, 11, 347 Hexetidine, 7, 277 Homatropine hydrobromide, 16, 245 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10, 405 Hydrocortisone, 12, 277 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate. 4, 209

Hydroxyzine dihydrochloride, 7, 319 Imipramine hydrochloride, 14, 37 Indomethacin, 13, 21 1 loddmide. 15, 337 lodipamide. 2, 333 lopanoic acid, 14, 181 Isocarboxazid. 2, 295 Isoniazide. 6, 183 Isopropamide. 2, 315; 12, 721 Isoproterenol, 14, 391 lsosorbide dinitrate, 4, 225; 5, 556 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10, 443 Ketotifen. 13, 239 Khellin. 9, 371 Leucovorin calcium, 8, 315 Levallorphan tartrate, 2, 339 Levarterenol bitartrate, 1, 49; 2, 573; 11, 555 Levodopa, 5, 189 Levothyroxine sodium, 5 , 225 Lidocaine base and hydrochloride, 14, 207; 15, 761 Lithium carbonate. 15, 367 Lorazepam, 9, 397 Maprotiline hydrochloride, 15, 393 Mebendazole, 16, 291 Mefloquine hydrochloride, 14, 157 Melphalan, 13, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 209; 4, 520; 11, 587 6-Mercaptopurine, 7, 343 Mestranol. 11, 375 Methadone hydrochloride, 3, 365; 4, 520; 9, 601 Methaqualone. 4, 245, 520 Methimazole, 8, 351 Methotrexate, 5 , 283 Methoxsalen. 9, 427 Methyclothiazide, 5 , 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metocloprarnide hydrochloride, 16, 327 Metoprolol tartrate, 12, 325 Metronidazole, 5, 327 Minocycline. 6, 323 Mitomycin C, 16, 361 Moxalactam disodium, 13, 305 Nabilone, 10, 499 Nadolol, 9, 455; 10, 732 Nalidixic acid, 8, 371 Naloxone hydrochloride, 14, 453 Natamycin, 10, 513 Neornycin, 8, 399 Neostigrnine, 16, 403 Nitrazepam, 9, 487

CUMULATIVE INDEX 77 1

Nitrofurantoin. 5, 345 Nitroglycerin. 9, 519 Norethindrone, 4, 268 Norgestrel, 4, 294 Nonriptyline hydrochloride. I , 233; 2, 573 Noscapine, 11, 407 Nystatin. 6, 341 Oxazepam. 3,441 Oxyphenbutazone. 13, 333 Oxytocin, 10, 563 Penicillamine. 10, 601 Penicillin-G benzothine, 11, 463 Penicillin C. potassium, IS, 427 Penicillin-V. 1. 249 Pentazocine, 13, 361 Phenazopyridine hydrochloride, 3,465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5 , 429 Phenobarbital. 7, 359 Phenoxymethyl penicillin potassium. I , 249 Phenylhutazone, 11,483 Phenylephrine hydrochloride. 3,483 Phenylpropanolamine hydrochloride, 12, 357; 13,

Phenytoin, 13, 417 Pilocarpine, 12, 385 Piperazine estrone sulfate. 5 , 375 Pirenzepine dih ydrochloride, 16, 445 Piroxicam. 15, SO9 Primidone, 2,409 Probenecid. 10, 639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride, 5, 403 Promethazine hydrochloride, 5 , 429 Proparacaine hydrochloride, 6, 423 Propiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, I , 301; 4, 520; 6,

Propylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8 ,489 Pyrazinamide, 12, 433 Pyridoxine hydrochloride, 13,447 Pyrimethamine, 12, 463

Quinidine sulfate, 12, 483 Quinine hydrochloride, 12, 547 Ranitidine. 15, 533 Reserpine, 4, 384; 5 , 557; 13, 737 Rifampin. 5, 467 Rutin. 12, 623 Saccharin, 13, 487 Salbutamol, 10, 665 Salicylamide. 13, 521

771

598

Secobarbital sodium, 1, 343 Silver sulfadiazine, 13, 553 Sodium nitroprusside, 6, 487; 15, 781 Spironolactone, 4,431 Streptomycin, 16, 507 Strychnine. 15, 563 Succinycholine chloride, 10, 691 Sulfadiazine, 11, 523 Sulfamethazine. 7, 401 Sulfamethoxazole, 2, 467; 4, 521 Sulfasalazine, 5, 515 Sulfisoxazole, 2, 487 Sulindac. 13, 573 Sulphamerazine. 6, 515 Terpin hydrate, 14, 273 Testolactone, 5, 533 Testosterone enanthate. 4, 452 Tetracycline hydrochloride, 13, 597 Theophylline. 4, 466 Thiabendazole, 16, 61 I Thiosrrepton, 7, 423 Timolol maleate, 16, 641 Tolbutamide, 3, 513; 5, 557; 13, 719 Trazodone hydrochloride, 16, 693 Triamcinolone, 1, 367; 2, 571; 4, 521. 524: 11,

Triamcinolone acetonide, 1, 397, 416; 2, 571; 4,

Triamcinolone diacetate. 1, 423: 11, 651 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2 , 507 Trifluoperazine hydrochloride, 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 521; 5,

Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprim, 7, 445 Trimipramine maleate. 12, 683 Trioxsalen, 10, 705 Tripelennamine hydrochloride. 14, 107 Triprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybamate, 4,494 Valproate sodium and valproic acid, 8, 529 Vidarabine. IS, 647 Vinblastine sulfate, 1, 443 Vincristine sulfate. 1, 463 Vitamin Dlr 13, 655 Warfarin, 14, 243 Xylometazoline hydrochloride, 14, 135 Yohimbine, 16, 731 Zomepirac sodium, 15, 673

593

521; 7, 501; 11, 615

557

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