Indian Journal of Chemistry Vol. 42B, November 2003, pp. 2835-2845

Synthesis and antimicrobial activities of some quinoxalinonyl amino acid and peptide derivativest

A Y Ali , EzzEl-Din M Salem, J A Hasananen & M E Abdel-Fattah*

Department of Chemistry, Faculty of Science, Suez Canal University, Ismaili a, A.R. Egypt

Received 3 July 2002; accepted (revised) 22 April 2003

The sy nthesis and antimicrobial screening of some 3-methyl-2 ( I H)-quinoxalinon-I-yl acetyl amino ac id, peptide, dia­mine,thiazolyl and benzothiazolyl derivatives, in addition to three copper complexes, have been described. The sy nthesized compounds have screened against E.coli, P. aeroginosa, S.aureus and B.subtilis. It is noteworthy, to menti on that, the com­plex formati on enhanced considerably the antimicrobial action .

The polypeptide quinoxaline antibiotics 1 and 2 (Scheme I) are a group of bicyclic octadepsipeptides l

-5

.These antibiotics show activity against Gram-positive bacteria 6 and certain animal tumers.3.

7 However, they have limited clinical application due to their toxic ef­fects.s This prompted us to synthesize and test as an­timicrobial agents some relatively simple 3- methyl-2 (lH)-quinoxalinon-l-yl acetyl derivatives of amino acids and peptides in addition to other related deriva­ti ves. It is hoped that the synthesized compounds would possess the desirable biological qualities of qui­noxaline antibiotics without being toxic.

The design of these derivatives is based on the re­corded data on the importance of 3- methyl-2 (l H)­quinoxalinon-I-yl acetyl moeity for exhibiting antim­icrobial action against S.aureus. 9 It is believed that the suggested structures will facilitate the approach of these molecules to viral DNA and prevent DNA­directed RNA synthesis by virtue of its binding to CpG site on D A.I O This is in accordance with the conclusions derived by Waring which show the im­portance of the introduction of non polar groups into the drug structure for its development as DNA- tar­geted compounds. lo The sy nthesis of 3- methyl-2 ( I H)- quinoxalinon-I-yl acetyl amino ac ids esters 7a­k, Table I has been achieved by the azide method

+ This art icle has been partly presented in the form of abstract in the I" Internat ional Conference on "New Trends in Chemistry and Their Application", Chemi try Department, Faculty of Science at Beni - Sucf, Cairo Un ivers itry, Beni -Suef, A.R. Egypt (2-4 Feb., 2002). < Abbreviation accordi ng to IUPAC- IUP Commission, J Bioi Chem, 247 (977) 1972 are used throughout.Addi tional abbrevia­tions: DCC, dicyclohexylcarbodiimide; HOBt, 1- hydroxybenzo­triazole.

starting from the hydrazide 5 The used amino acids are in the optically active L- and D- forms. Some ra­cemic amino ac ids are used for comparison to a cer­tain the effect of optical purity and configuration on the antimicrobial activity.

The selection of the amino acids L- valine, L­

alanine and D- serine is based on the fact that they are major constituents of the quinoxaline antibiotics. 11

L-Methionine methyl ester derivative 7e has also been prepared by the DCC/HOBtt procedure . How­ever, in the absence of HOBt, the N- acy l urea deriva­tive was obtained, instead of the desired product. The structure of the by-product was confirmed by its syn­thes is via reaction of with dicyclohexyl-carbodiimide.

Further support of the proposed structure has been achieved by hydrazinolysis of the N-acyl urea derivative which afforded dicyclohexyl urea. Some other derivatives, bearing physiologically important functional groups such as hydrazides 9a-d, hydrazones 10 a,b amides lla-c,13 and the Mannich base 12 have been prepared by the con­ventional procedures (Scheme II).

Attempts to sy nthesize the free acid 3-methyl-2 (lH)- quinoxalinon-I-yl acetyl glycine by a lkaline hydrolysis of the corresponding ester 7a was unsuc­cessful. Accordingly, the synthes is of quinoxalinonyl dipeptide esters 14a,b by the DCC-procedure was abo li shed. Analogously , the azide procedure failed to give quinoxalinonyl dipeptide esters due to instabil ity of the quinoxalinonyl amino acid hydrazide 9c in the acid medium during its conversion to the correspond­ing azide. Therefore, the dipeptide ester derivatives 14a,b have been obtained by acylation of the partially

2836 INDIAN J. CHEM., SEC B, NOVEMBER 2003

CCN

I ~ CH3 CH3 # A I I

N CO-NH-CH-CO- NH - Cf-l- co -N -CH - co ~ I I -L~I CH2 " CH2 0 I ~ ___ I I

O I S ICH2 CH2 0

I r-I I I II '------llJ CO-CH- ~- CO- TH - NH - -CO- CH- NH- C'(ND

CH3 CH3 0 ~ I N #

x y

Triostin A Me Val Me Val

Triostin B Me Alloileu Me Alloileu

Triostin C DirreAUoileu DirreAlloileu

Triostins 1

yH3

CC"'" N~ CH3 CH3 H3C <;:H- CH3 /. A I I I I

/. N CONHCH-CONH-CH-CON-CH-CON-CH- co I r I CH? _..-/CH 0 I - s- I r o H c/ SCH3 CH2 I 2 1 I

0= C-CH-NCO-CH-NCO-CH- NH co- CH-NHCCA(N

D

I I I I r I '" H3C- CH CH3 CH3 CH3 ~

I N # CH3

Echinomycin 2

Scheme 1-Structures of quinoxaline antibiotics

protected dipeptide esters with the azide 6. The dipep­tide ester hydrochlorides were prepared by deprotec­tion of the corresponding Boc-/For- dipeptide methyl esters with 1M Hel in glacial acetic acid (Scheme III). The Boc-lFor-dipeptide esters have been synthesized by the mixed anhydried and car­bodiimide methods.

ester 4 with ethylene diamine, followed by acylation of the product obtained 15 with azide 6. Other amjdes 7 l-p of some aliphatic and aromatic amines have been also synthesized for comparison. On reflux, the azide 6 underwent Curtius rearrangement to yield the isocyanate 17 (Scheme IV) The urea derivative 18 which is structurally close to amide 7 b has been pre­pared from the reaction of the isocyanate with L­

phenylalanine methyl ester. Reaction of two equivalents of the azide 6 with one equivalent of the appropriate diarnine [H2N (CH2)n NH2; n= 2,7] in an inert solvent yielded readily the N,N'-polymethylene bisq uinoxaline carboxamides 16a,b. The ethylene diamine derivative 16a has been synthesized by alternative route via aminolysis of the

Importance of metal complexes has been estab­lished and some of them were found to possess higher antimicrobial activity than the parent ligands. 12 Ac­cordingly, in the present study some complexes of the hydrazide 5, acid 8, and amide 13 with copper ion

ALI el ai. : SYNTHESIS OF QUINOXALINONYL AMINO ACID & PEPTIDE DERIV ATIVES 2837

Table I- Phys ical and spectral data of 3- methyl- 2 (1 H)- quinox alinon- I-y l acetyl amino acid methyl esters/and some aliphati c and aromatic amides 7a- p

Compd

7a

7b

7c

7d

7e

7f

7g

7h

7 i

R

H

-CH!CHzSCH3 L

-CH] L

-CH2CH(CH3h L (elhyl eSler)

172-4

200- 1

198-9

151-3

142

18 1-3

185

128-30

123-4

Yie ld (%)

Molecul ar formula

(Mo l. wL)

Spectra l data Ca lcd. % (Found)

C H

62 C I. H ISN304 IR (KBr): 3293 (N H), 1754 (C=O.esler), 58. 13 5.19 14.53 (289) 1652 (C=O.Qx). IH_ IMR (CDCI3, 8): 7.9- (57 .90 5.60 1480)

7.3 (m, 4H, A r-H), 6.7 (5 , I H, NH), 5.0 (5,

2H, CHrCO), 4. 1 (d, 2H, HN-CHz). 3.7 (s. 3H, OCH3), 2.65 (5, 3H. CH3 Qx). M S (mlz) : 289 [Mr.

77.5 CZI I-I ZI N304 IR (KBr): 3289 (N I-I), 1752 (C=O.esler), 66.49 5.54 11.08 (379) 1652 (C=O,Qx). II-I -NMR (CDCI]. 8): 7.9- (66.60 5.3 1 11.00)

6.8 (m. 91-1, Ar- I-I), 5.0- 4.65 (d d , 21-1 . CHr CO). 4.85 (q, IH . I-I N-CHCOO), 3.75 (5 . 31-1 . OCH3) , 3.0 (d. 21-1 , C/-/zPh), 2.7 (5, 3H. CI-I .. Qx). MS (mlz) : 3791M r.

74 C21H21N304 IR (KBr): 3290 (N I-I), 1752 (C=O,esler), 66.49 5.54 11.0 (379) 1652 (C=O,Qx). II-I -NM R(CDC!". 8): 7.9- (66.64 5.6 1 11.05)

6.8 (m, 91-1 . Ar- I-I ), 5.0- 4.65 (d d, 21-1 , CH2-

CO), 4.85 (q, 11-1 , I-IN-CHCOO), 3.75 (5, 31-1. OCH3). 3.0 (d. 2H, CHzPh), 2.7 (5, 3H, CH3 Qx).

6 1 C17 HZIN304 IR (KBr): 33 10 ( H), 1746 (C=O.esler), 6 1.63 6.34 12.68 (33 1) 1652 (C=O. Qx). II-I -NM Rr (CDCI), 8) : 7.9- (6120 6.60 12.41 )

7.3 (m, 4H, A r-H), 4.9 (d d, 2H, CHz-CO). (I. I H4.45, IIN-CHCOO). 3.7 (5 , 3H, OCH3), 2.65 (s. 3H, CH3 Qx), 2. 15 (m. 11-1 , CH (CH)h), 0.9 (d. 6H. CH(CH)h). MS (mlz): 33 1 [Mr.

68 C 17HZ1 30 4S IR (K Br): 3302 (N H), 1749 (C=O,esler). 56.19 5.78 11 .57 (363) 1652 (C=O.Qx). (56.03 5.94 11.90)

55 Cl sH I7N304 IR (KBr) : 3303 (N H), 1753 (C=O,esler), 59.4 1 5.6 1 13.86 (303) 1652 (C=O.Qx). IH_ MR (CDCh. 8) : 7.93- 59.80 5.50 13.61

7.3 ( Ill , 41-1 , Ar-H), 4.95 (d d, 21-1 , CHrCO), 4.53 (m, 11-1, HN-HCOO), 3.7 (5, 3H, OCH3), 2.63 (5, 31-1 , C I-I3 Qx). 1.42 (d. 3H. CHCH»).

60 CISl-l 17N30 4 IR (KBr): 330 1 (N H). 1748 (C=O,esler). 59.4 1 5.6 1 13.86 (303) 1652 (C=O,Qx). II-I -NMR (CDC!", 8): 7.93- (59.4 1 5.66 13.81)

7.3 (m. 4H, Ar-H), 4.95 (d d, 2H, CI-I2-CO). 4.53 ( Ill , I H, HN-HCOO), 3.7 (5, 3H. OCHJ), 2.63 (5, 31-1. CH3 Qx). 1.42 (d. 3H. CHCH3).

60.5 Cl xH2.1N304 IR (KBr): 3328 (NI-I), 1745 (C=O,esler), 62.6 1 6.66 12.17 (345) 1652 (C=O,Qx). IH-N MR (CDCI3, 8): 7.86- (62.48 7.08 12.1 9)

7.35 (m, 41-1, Ar-H ), 4.95 (d of d, 21-1 , CHz-CO), 4.57 (q. I H, 1-1 -CHCOO), 3.64 (5, 3H. OCH3), 2.66 (s, 3H, CH) Qx). 1.65-1..J6 (m. 31-1. CH2CH- (CI-I»)z). 0.9 (d. 6H, CH (CH3lz).

55 CI91-1 2SN .. 0 4 IR (KBr): 3333 ( H), 1740 (C=O,ester). 63.5 1 6.42 11 .69 (359) 1652 (C=O,Qx). IH_ M R (CDCI). 8): 7.9- (63.44 7.23 11 .48)

7.23 (m, 4H , Ar-I-I), 4.95 (d d, 21-1, CH2-

CO), 4.57 (q, IH, I-I N-CHCOO). 4.1 5 (q, 2H. OCH2CH)). 2.66 (5, 3H, CI-I3 Qx), 1.65-1.46 (m. 3H. CH2CH- (CH3)2). 1.2 (I, 31-1 , OCHzCH3), 0.9 (d, 6H, C I-I (CH3h).

--Comel

2838 INDIAN J. CHEM .. SEC B, NOVEMBER 2003

Table I- Phys ica l and spectral data of 3- methy l- 2 ( I H)- quinoxa linon- I -y l acety l amino ac id methyl es ters/and some ali phatic and aromatic amides 7a- p--Collld

Compd

7j

7k

71

7m

711

70

7p

R

-CH201-1 L

-CI-I,OI-l D

Benzothiazolyl-2-yl

Thiazoly l-2-y l

n-propy l

ethyl

cyc lohexyl

168-70

169

297

309

203

266

24 1

Yield (%)

50

52

66

60

72

60

55

Molecular formula

(Mol.wt.)

C1s l-l 17N)Os (3 19)

C1 sH17N]Os (3 19)

Clx I-l I4NJO~S (350)

C 1JH I2 .02S (300)

C 14H 17N)0 2 (265)

CI.1 I-l lsN,02 (245)

C I7 H2I N)0 2 (299)

Spectral data

IR (K Br): 3466-346 1(OH), 3289 (N I-I), 1745 (C=O,ester), 1652 (C=O.Qx).

IR (K Br): 3466-346 I (OH), 3290 (N I-I ), 1750 (C=O.ester), 1652 (C=O,Qx).

IR (KBr): 3180 (1 H). 1706 (C=O. amide). 1652 (C=O.Qx).

1 R (KBr): 3 180 ( H), 1706 (C=O, amide). 1652 (C=O. Qx) .

IR (KBr): 3292 (N I-I), 1752 (C=O,amide) . 1652 (C=O,Qx) . IH_ M R (CDCk 8): 7.9-7.3 (m, 41-1 , Ar- H), 4.9 Cd of d, 21-1 . CH 2-

CO), 3.2 (t, 21-1, HN-CHzCH2CH», 2.66 (s, 31-1 . CI-I J Qxc). 1.5 (m. 2H. 1-1 -CI-I 2CH,CI-I ) , 0.85 (1. 31-1 , I-IN­CI-I2CI-I 2CHJ ).

IR (KBr): 3285 ( H). 1654 (C=O.amide), 1652 (C=O.Qx)

IR (KBr): 3290 ( 1-1 ).1 665 (C=O,amide) . 1652 (C=O,Qx).

Calcd. % (Found)

C 1-1

56.42 5.32 (56.29 5.32

56.42 5.32 (56.80 5.61

61.7 1 4.02 60.98 3.95

56.01 4.03 (56.3 1 3.86

63 .39 6.4 1 (63.40 6.70

63 .616. 12 (63.88 5.66

68.2 1 7.0 1 (67.68 6.76

N

13.1 6 13.40)

13.16 13.30)

16.0 1 15.89

18.6 1 18.29)

18. 10 18. 12)

17. 10 17 .2 1)

14.02 14.03)

* The compounds 17a- k, 11,01 were crystall ized from ethyl acetate- petroleum ether (b. p. 40- 60°). Compounds [71, III and pI were crys tallized from dimethylformamide-water.

were obtained when heated with cuppri c chloride in ethanol in equimolar rati os. The ligand -metal rati o of the complexes was fou nd to be I : I by titrati on aga inst EDT A. It is clear that the measured conductance va l­ues of the studi ed complexes are relatively low, whi ch indicate the nonel ectrolytic behaviour of these com­plexes. Conseq uentl y it could be conc luded that chlo­rine forms cova lent bond with the cen tral copper.

The synthesized compounds were sc reened against microorgani sms P.aeroginosa, Ecoli, St.au rel/ s, and B. subtilis. All the tes ted compounds were inacti ve at 10 ppm. However, at 100 ppm, out of 4 1 tested com­pounds 14, 30. and 13 co mpounds showed moderate activity against P.aerogillosa. Ecoli and S.al/reus respectively as shown in Table II

Some derivatives showed moderate acti vity onl y at 1000 ppm against P. aerog inosa, E co fi, S.aureus and B.subtilis respectively as shown in Table III.

From the results of the antimicrob ial action, the fo llowing conclusion can be drawn :

(i) Unexpectedly , the tested sy nthesized derivati ves were relati vely more ac tive against Gram-negati ve than Gram -positi ve bacteri a.

(ii ) Eco fi is the most sensit ive mi croorganism, whereas B.subtilis is the least one.

(iii ) Conjugat ion of the quinoxalinone moiety with amino acid esters improved the antimicrobial action .

(iv) The p-nitrobenza ldehyde hydrazone lOb is the more potent compound with the broadest spectrum among the studi ed series. This shows the necessity of testing other hydrazones substituted with different phys iologically acti ve functi onal groups such as CI, OR, NH2 and/or OH .

(v) Chelation with copper enhanced considerably the antimicrob ial ac ti on. These data add further sup­port to the previously published res ults in our labora­tory' 2, showing the importance of complex formati on for getting more potent antimicrob ial agents. This study also shows the need for preparation and testi ng other complexes with different cations and ligands from this seri es of compounds.

From the data of molecular modeling using Alkemy III program (Table IV) it is clear that the sy nthesized deri vatives are ac tuall y devo id of ac tivity towards Gram-positi ve bacteri a, whereas the qui noxa­line peptide antibioti cs Triostin A is hi ghl y act ive aga inst the same microorgani sms. 13 The high level of act ivity of the antibiot ic could be attributed to the hi gh non-polarity of Triostin A 1 by compari son with the synthesized quinoxaline derivatives . This observa­ti on is in accordance with the published data by War­ing et al14 on 0 ~A recogn ition by Quinoxaline antibi­otic, where they showed that binding of Triostin A to CpG sites is primarily due to hydrogen bonding inter

ALI el oJ.: SYNTHESIS OF QUINOXALINONYL AMINO ACID & PEPTIDE DERlVATIVES

H I

(X

NyO

~I ~ N CH)

[3]

CHoCO H1 I - -

(X

NyO

~I ~. N CH3

[1 3]

CHoCOOH CH2CON HCHCOOCHJ I - I I N (XN 0 CHP-l2SCH)

(X

I 1; 0 L-M et-OMe/DCC/HOBt ... : I :c: ~ N~rnl N rn)

/ [8] [7 e]

NaOH I LM"OM, /

18 a b

R CH2ph CH2pH D __ L

CH2CO- NHCI-ICONHCI-I, - N '\ I - J

(X

NyO

~ I ~ N CH3

[12J

Scheme II

c

H

CI-I,CO-NHCI-ICONHNI-I? I - \ -

(X~ I N;O R

N~CI-l 3 9 R

a CH2CH2SCH3

[9 a-dJ L b CH3 L

c CH2ph L

d CH2ph D

C I-1 2CO-NHCHCONHN=CH~ R'

(X~ro k '=T ~ I .-<

N CI-I 3 10 R R' [ 10 a. b] a -CH2ph H

L

b CH2ph L N02

2839

2840

7

R

o r

Boc-N H-CHCOOH I

RI +

CH2CON21-1 3 1

CCN:C:H3

[5]

l HNO,O"C

CH,CON 1 1 - -

CC~ N;O

# :::l... CH3

16J

~ R-N H2

CH,CO-NIIR 1 •

CCN:C:H3 [7 1-p]

I m

erN l}-. I '>--"" s s

INDlAN 1. CHEM., SEC B, NOVEMBER 2003

or

Boc-NHCHCO-N HCHCOOCH3

k, ~2 ~ 1M HCIIAcOH

HCI.HN-CHCO-NHCHCOOCH3 I I RI R2

14 RI R2 CH2CO-NHCHCO-NHCHCOOCH3 1 I I

a .CH2ph CH2C1-I (CH3)2

L

b H 1-1

CC:cO RI R2

N CH3

[ 14 a,bJ

Scheme III

[4] [ IS]

/

[171 11 8J

n 0 p

-CH2C1-1 2C1 1.1 -CH2CH3 6 Scheme IV

ALI et aJ.: SYNTHESIS OF QUINOXALINONYL AMINO ACID & PEPTIDE DERIVATIVES 2841

Table II-Active deriv3tives+ towards the tested microorganisms at 100 ppm"

P.aerogil1osa

4, 7a, 7 I, 7 m, 7 p, lOb, 12, IS, 16a, 16b, 18, Cu [5], Cu [8], Cu [13].

E.coli

5, 7 a, 7 b, 7 c, 7 d, 7 e, 7 g, 7h, 7 i, 7 I, 7 m, 7 n, 8, 9a, 9b, 9 c, 10 a, 10 b, 11 c,12, 13, 14a, 15, 16 a, 16 b, 17, 18, Cu [5], Cu [8], Cu [13].

S.aureLis

4, 5, 7 f, 7 j , 7 k, 7 m, 9 a, 9b, 10 b, lla, llb.14 a, 16a,

+ Compounds that showed inhibition zone 16-20 mm diameter at 100 ppm. '"Most of the tested compounds were actually inact ive towards B.subtiJis

Table IH- Active derivatives towards the tested microorganisms at 1000 ppm

P.aerogil1osa £.coli S.aureus B.subtiJis

5, 7b-f, 7h-k, 4, 5, 7f, 7a-e, 7h, 7i, 71, 4, 5, 7a, 71, 7n, 8, ge, 9d, 7k, 70, 12, 13, 14b, 15, 7m, 7p, ge, lOa, He, 13, 7p, 9d, 17, 18, Cu [5], lOb, lla, 14a, 14b, 17 lla, llb, Cu [8], Cu [13] Ilb, 16a, Cu

14b [5]

action between the highly non polar cyclic peptide of the antibiotic and the 2-amino group of guanine resi­due. In terms of drug design, this means that further development of DNA -targeted compounds could be achieved by judiciously introducing non polar groups into the drug structure . Therefore, it will be interest­ing to test other sy nthetic more nonpolar quinoxaline peptide derivatives, containing different N-alkyl amino ac ids and quinoline nucleus substituted with longer alkyl/ ary l side chains. These suggested struc­tures will facilitate the approach of these molecules to viral DNA and prevent DNA-directed RNA synthes i by virtue of its binding to DNA. 10

Experimental Section Melting points were uncorrected, and measured on

a MEL- TEMPII melting point apparatus. Microanaly­sis was performed by mi croanalytica l laboratory, Cairo University. lR spectra were recorded with a Perking Elmer 1430 ratio recording infrared spectro­photometer with CDS data station usi ng KBr Wafer technique, IH MR spectra on a Varian Gemini (300 MHz) spectrometer and mass spectra on a GC-MSQP 1000 EX Schimadzu (Cai ro University) . The purity of the synthesized compou nds was checked by TLC on glass coated plates in the laboratory with silica gel GF 254 type, 60 mesh, size 50-250 in the following sol-

Table IV-The antibacterial-sterric factor (Van der Waals forces) relationship of the synthesized quinoxaline derivatives

Compd

3

4

5

7e

7f

71

70

8

9b

9c

lOb

lla

13

IS

16a

Triostin A I

Antibacterial activity agai nst Gram +ve bacteria

S. aureus B. subtilis Concentration (ppm)

10 100 10 100

+ ++

++

+

++ +

++

++

++

+

+ ++

0.5 )!g

+

+

+

+

+

+

van der Waals force

1.398

1.803

3.107

-2.4 10

2.707

0.427

2.840

2.560

3.377

2.058

-1.252

0.137

2.003

3.415

4.180

-26

(++): moderatly active; (+): sli ghtly ac ti ve; (-): inactive

vent systems, S I: chloroform / methanol (95 : 5); S2: chloroform! acetic acid/ methanol (90 : 5 : 5). The spots on thin layer plates were detected by exposure to iodine vapour.

General procedure for the synthesis of 3-methyl-2(1H)-quinoxalinon-l-yl acetyl amino acid methyl esters and some aliphatic and aromatic amides by the azide method 7 a-po 3-Methyl-2(lH)-quino­xalinon-l-yl acetic acid hydraz ide 5 9 (0. 1856g, O. 8 mmole) was di ssolved in a mixture of acetic acid (6 mL), 5 N HCI (3 mL) and water (25 mL), afterwards the mixture was cooled to -soc. Sodium nitrite (0.07g, 1 mmole) in water (3 mL) was cooled to O°C and then added in one portion to the cooled hydrazide solution. After stirring the reaction mi xture at 0 DC for 15 min, the az ide formed as a yellow syrup, was taken up in cold ethyl acetate (30 mL). The ethyl acetate layer was kept cold while washing success ive ly with water, 3% NaHCO), water and briefly dried over anhyd . Na2S04. To the suspension of the amino acid methyl ester hy­drochloride (0.9 mmole) in ethyl acetate (20 mL) at O°C, triethyl amine (0 .2 mL) . was added, afterwords , the reaction mixture was stirred for 20 min . at O°c. The cold dried solution of 3-methyl-2(l H)­quinoxalinon-l-yl- acetyl azide was added to the cold

2842 INDIAN 1. CHEM., SEC B, NOVEMBER 2003

ethy l acetate solution of free amino ac id methyl es­ter/or the amine (0.9 mmole), then the react ion mix tu re was kept 12 hrs . in the refrigerator and for add itional 12 hrs. at room temperature. The ethyl acetate solution was washed with 0.5 N HCl, water, 3% NaHC03 and water then dried over anhyd . Na2S04. The so lvent w s evaporated in vacuo and the residue was crystallized from the suitable solvent to g ive the amino ac id es­ter/amide deri vatives 7 a-p, Table I.

Synthesis of 3-methyl-2(1H)-quinoxalinon-l-yl acetic acid 8. After stirring of ethy l- 3-methyl-2(lH)­guinoxalinon-l -yl acetate 4 (2.46g, 0.01 mole) and sodium hydroxide (0.4 g, 0.01 mole) in 95% ethanol (40mL) at room temperature for 3 hrs, the solut ion was fi ltered, cooled, then ac idified with dil. HCI. The solid formed after separation was crystallised from ethanol to give the con'esponding acid 8, (2.0 g, 92%), m.p. 22SOC, Rr 0.75 (SI)' Ana l. Calcd.for CIIHI2N402 (M.Wt. 218): C, 60.55; H, 4.58; N, 12.84. Found: C, 60.25; H, 4.6; N, 12.76%. IR (KBr): 3425 (OH), 1709 (C= 0, acid); IH NMR . (DMSO): 7.64- 7. 15 (m, 4H, Ar-H), 4.85 (s, 2H, CHzCO), 2.3 (s, 3H, CH3QXC): MS (mJz): 218 [Mr.

Synthesis of 3-methyl-2(1H)-quinoxalinon-1-yl acetyl-L-methionine methyl ester 7 e in the presence of DCCIHOBt.(IS) To a cold solution (-5°C) of the L­methionine methyl ester hydrochloride (0. 1995g, I mmole) in aceton itrile (6 mL) containing triethyl ami ne (0.14 mL, I mmole), 3-methyl-2( I H)-guinoxali non-I-yl acetic acid 8 (0.2 18 g, J mmole), J -hydroxy­benzotriazole (0. 135g, I mmole) and dicyclohexyl­carbodiimide (0.207 g, I mmole) were added succes­sively. The reaction mixture was sti lTed at O°C for 1 hr, at 5°C for additional hour, then for 8 hr, at room tem­perature. The reaction mixture was set side overnight. The precipitated dicyclohexylurea was fi ltered off and the filtrate was evaporated under reduced pressure. The residue was dissolved in ethyl acetate, filtered and the filtrate was washed with saturated sodium ch loride solu­tion, 5%NaHC03 solution, IN HCI and saturated solu­tion of sodium chloride and then dried over anhycl. Na2S04. After evaporation of the solvent, the remaining residue was crystall ized from ethyl acetate- pet. ether (b.p. 40- 60°C), to give yellow crystals (0.24 g, 68%) of the product 7e, m. p. 142°C, Rr 0.65 (SI)' Anal. Cald. for C17H21N30 4S (M.wt.363): C, 56.19; H, 5.78; N, 11.57. Found: C, 56.03; H, 5.94; N, 11.90%. IR (KBr): 3302 (NH), 1749 (C= 0, ester),1652 (C=O, Qx).IH­NMR, (CDCb): 7.9- 7.35 (m, 4H, Ar-H), 4.95 (d d, 2 , CH2CO), 4.7 (g, IH, - NHCH-COO), 3.7 (s, 3H,COOCH3), 2.7 (s, 3H, CH3 Qxc), 2.4 (t, 2H, CH-

CH2-CH2-SCH3), 2.1 (g, 2H, CH-CHr CH2-SCH3), 2.0 (s, 3H, SCH3); MS (mlz): 363 [Mr.

Synthesis of 3-metlhyl-2(1H)-quinoxalinon-1-yl acetyl amino acid hydrazides 9a-d. To the solution of 3-methyl-2( I H)-guinoxalinon-l-yl acetyl amino acid methyl esters 7a-k ( I mmole) in ethanol (20 mL), hydrazine hydrate (0.15 mL, 3 mmole) was added. The reaction mixture was refluxed for 4 hr, then kept aside to cool to room temperature. The formed precipitate was fi ltered off, washed with etha­nol and ether and then crystalli zed from the suitable solvent to give 9 3 - d .

3-methyl-2(1H)-quinoxalinon-1-yl acetyl L-

methionine hydrazide 9a: Yellow crystal S, 2.85g (78 %),m.p. 253°C (ag. ethanol), Rr 0.7 1 (S2) . Anal. Cald . for CI6H21Ns03S (M.Wt. 363): N, 19.28. Found: N, 19.21 %. IR (KBr): 3299 (NH2)' 3214 (NH), 1660 (C=O, amide), 1653 (C=O,Qx).

3-methyl-2(lH)-quinoxalinon-l-yl acetyl L-

alanine hydrazidle 9b: Yellow crystals, 2.48 g (82%), m.p. 277°C (DMF/water) , Rr 0.74 (Sz) . Anal. Cald. for CI4HI7NsOJ (M.Wt. 303): N, 23. 10. Found: 22 .60%. IR (KBr): 3389 (NH2), 3298 (NH), J 679 (C=O, amide), 1653 (C=O, Qx).

3-methyl-2(1H)-quinoxalinon-1-yl acetyl L-and D­

phenylalanine hydrazidle 9c,d: L-fonn: Yellow crys­tals, 3.41 g (90%),m.p. 259°C (DMF/water) , Rr 0.84 (S2). Anal. Cald . for C2oH21Ns03 (M .wt. 379): N, 18.46. Found: ,18.30%. IR (KBr) : 3365 (NH2), 3281 (NH), 1660 (C=O, amide), 1652 (C=O,Qx); D-form: Yellow crystals, 2.72 g (72%), m.p. 257°C (DMF/water), Rr 0.84 (S2). Anal. Cald . for C2oHzI Ns03 (M .Wt. 379): N, 18.46. Found : N, 18.33%. IR (KBr): the same as for L-fonn.

Condensation of hydrazide 9c with aldehydes; General procedllJre. A mixture of 3-methyl-2(lH)­guinoxalinon-l-yl acetyl L- phenylalanine hydrazide 9 c (3 .79 g, 10 mmole) , aldehyde (10 mmole) and two drops of conc. HCI in ethanol (30 mL) was heated under reflux for 6 hrs . The formed prec ipitate after cooling was filtered off and crystallized from the proper solvent to give 10 a, b .

[3-methyl-2(1H)-quinoxalinon-l-yl acetyl L-

phenylalanyl] benzal hydrazone lOa. Yellow crys­tal s 3.21 g (68.9 %), m. p. 284°C (DMF), Rr 0.7 (S2) . Ana l. Calcd . for C26H2S Ns0 3 (M.Wt. 467): C, 66.80; H, 5.35; N, 14.98. Found: C, 66.85; H , 5.61; N, 15 .19%. IR (KBr): 328 1 (NH), 1657 (C=O). MS : no molecular ion peak was found but showed peaks at 349,20 land 173 corresponding to [Mt -NHN=CHph, Qx-CH2CO and QX-CH2 respectively.

ALI et al.: SYNTHESIS OF QU INOXALINONYL AM INO ACID & PEPTIDE DERIVATIVES 2843

[3-methyl-2(1H)-quinoxalinon-1-yl acetyl L-phenyl alanyl] p- nitro benzal hydrazone lOb. Yel­low crystals 3.73 g (74.5 %), m.p. 273°C (DMF), Rr 0.8 (S2). Anal. Calcd. for C26H24 N60 S (M .Wt. 513): N, 16.42. Found: 16.41 %, lR (KBr): 3290 (NH), 1677 (C= 0 ).

Synthesis of 3-methyl-2(1H)-quinoxalinon-1-yl acetyl amino acid amides 11a-c. To the solution of 3-methyl-2( I H)- quinoxalinon-] -yl acetyl amino acid methyl ester 7 ( 10 mmole) in ethanol (20 mL), a satu­rated alcoholic ammonia solution (30 mL) was added . The reaction mixture was kept as ide at room tempera­ture for 2 days, afterwards the solution was concen­trated under vacuum. The solid formed after cooling, was fi ltered and crystall ized from ethanol to afford the amides 11a- c.

3-methyl-2(1H)-quinoxalinon-1-yl acetyl D-

phenylalanine amide 11a. Yellow crystals, 2.5 g (62%), m.p. 259°C, Rr 0 .68 (SI)' Anal.Cald . for C2oH20N40 3 (M.Wt. 364): C, 65 .93; H, 5.49; N, 15.38 Found: C, 65.89; H, 5.89 ; N, ]5.62%. IR (KBr): 3388 ( H2), 3297 (NH), 1660 (C=O, amide), 1652 (C=O, Qx). MS (mlz): 364 [Mr.

3-methyl-2(lH)-quinoxalinon-1-yl acetyl L-phenylalanine amide 11b. Yellow crystals, 3.4 g g (90%), m.p. 260°C, Rr 0.67 (SI) ' Anal.Cald. for C2oH20N40 3 (M. Wt. 364): N, 15.38 Found: N, 15.5 1 %. IR (KBr): 3389 (NH2), 3298 (NH), 1679 (C=O, am ide), 165 1 (C=O, Qx).

3-methyl-2(1H)-quinoxalinon-l-yl acetyl glycine amide 11c. Yellow crystals, 1.6 g (66%), m.p. 264°C, Rr 0.62 (SI). Anal.Ca ld. for C 13HI4N40 3 (M. Wt. 274): C, 56.93; H, 5. 10; N, 20.64 Found: C, 57.21; H, 5.32; N, 20.64%. lR (KBr): 3419 (NH2), 3288 (NH), 1679 (C=O, am­ide), 1653 (C=O, Qx). MS (mlz): 274 [Mr.

Synthesis of N-piperidino methyl-(3-mcthyl-2(1H)-quinoxa linon-1-yl acetyl)-L-phenylalanine amide 12. To a solution of am ide 11 a (0.364 g, I mmole) in ethanol (IS mL), 37 % formaline soluti on (0 .1 ml, I mmole) and piperidine (0.09 mL, 1 mmole) were added. After refluxing the reaction mixture for 6 hrs, the fo rmed solid was filtered off and crystalli zed from aq. ethanol to g ive 0.239 g (52%) of 12, m.p. 152°C, Rr 0.73 (SI)' IR (KBr): 3266 (NH), 1600 (C= 0 , amide), 1651 (C=O. Qx). IH MR (C DCl3): 7.9- 6.7 (m, 9H, Ar-H) , 5.1- 4.6 (d of d, 2H, CH2-

CO), 4.85 (q, I H, HN-CHCOO) , 3.7 (d, 2H, NHCH2N) , 3.2- 2.9 (d, 2H, CH2ph) , 2.6 (s, 3H, CH] Qx), 1.8- 1.5 (m, 10 H, piperidine ring).

Synthesis of 3-methyl-2(1H)-quinoxalinon-1-yl acetamide 13. The tit le compound was prepared from 3-methyl-2(1H)-quinoxalinon-l-yl acetate 4 (2.46 g, 10 mmole) and a saturated alcoholic ammonia solution (30 mL) using the same procedure described for the syn­thesis of compounds 11 a-c. After crystalization from ethanol white crystals of the amide 13 were obtained, 1.96 g (78%), m.p. 295°C, Rr 0.82 (SI)' IR (KEr): 3355 (NH2), 1680 (C=O, amide), 1651 (C=O, Qx).

Synthesis of 3-methyl-2(1H)-quinoxalinon-1-yl acetyl dipeptide methyl esters 14a,b. Boc-lFor­dipeptide esters were prepared by DCC and lor mixed anhydride methods as described in literature. 16.17 The NU-protecting group was cleaved by 1M HCI/HOAc to afford the dipeptide methy l ester hydrochlorides. 16

To the suspension of the dipeptide methyl ester hy­drochloride (0. 9 mmole) in co ld ethy l acetate (20 mL) at (O°C) , triethyl amine (0.2 mL) was added, and the reaction mixture was stirred for 20 min . at the same temperature. The cold dried solution of 3-methyl-2 (lH)-quinoxalinon-l -y l acetyl azide 6 (pre­pared from 0.185 g, 0.8 mmole. of the hydrazide) was added to the soluti on of dipeptide methyl ester and the reaction mixture was kept as ide for 12 hI', in the re­frigera tor and at room temperature for another 12 hI'S. The reaction mixture was washed with 0.5 N HCI, H20 , 3%NaHC03 and H20 , afterwards was dried over anhyd. Na2S04. After evaporation of the solvent under vacuum, the res idue was crys talli zed from ethyl ace­tate-pet.ether (b.p 40-60°C) to yield the dipeptide methyl ester derivatives 14a,b .

3-methyl-2(1H)-quinoxalinon-1-yl acetyl L-

phcnyla la nyl-L-Ieucine methyl ester 14 a. Yellow crystals, 0.26 g (66%), m.p. 188°C, Rr 0.74 (SI)' Anal.Cald . for C27H32N40 S (M. Wt. 492): C, 65.85; H, 6.5 1; N, 11.38. Found: C, 66 .1 I ; H, 6.72; N, 1l.60%. IR (KBr): 3304 (N H), 175 1 (C=O, ester), 165 1 (C=O, Qx).

3-methyl-2(lH)-quinoxa iinon-l-yl acetyl glycyl­glycine methyl ester 14 b . Yellow crystals, 0. 166 g (60%), m.p. 194°C, Rr 0.68 (S I)' Anal.Cald . for CI6HI SN40 S (M.Wt. 346): C, 55.49; H, 5 .21; N, 16.18. Found: C, 55.71; H, 5.6 1; N, 16.40%. IR (KBr): 3308 ( H), 1751 (C=O, ester), 165 1 (C=O, Qx).

Synthesis of N-(2-aminoethyl)-3-methyl-2(1H)­quinoxalinon-l-yl methylene-carboxamide 15. A mixture of ethyl-3- methyl-2( 1 H)-quinoxalinon-I -y l acetate 4 ( I g, ISO mmol e) and 1 ,2-di aminoethane (10 mL) was st irred at room temperature for 2 hr. The so lvent was evaporated under vacuum and the residue was triturated wi th diethyl ether (20 mL). The residue

2844 I DIAN 1. CHEM., SEC B, NOVEMBER 2003

formed was crystallized from ethanol 95% to give 0.739 g (70%) of the product 15, m.p. 203°C, Rr 0.64 (SI)' Anal.Calcd.for CI3HI6N402 (M.Wt. 260): C, 60.00; H , 6.10; N, 21.50. Found: C, 60.18; H, 6.09; N, 2 1.73%. IR (KBr): 3369 (NH2), 33 11 (NH), 1650 (C= 0, amide), 1643 (C=O, Qx).

Synthesis of N,N' -polymethylenebis-(3-methyl-2( IH)-q uinoxalinon -ly I )methy I-enecarboxamid e 16a,b: The diami ne ( I ,2-diaminoethanell ,7-diaminoheptane) (0. 9 mmole), was added dropewise to the dry cold ethyl acetate solution of 3-methyl-2 (lH)­quinoxalinon-l-yl acetyl azide 6 [prepared from 0 . 185 g. (0.8 mmole) of the hydrazide]. Immediately after the addition of the diamines a yellow precipitate began to separate and the reaction mixture was kept aside in re­frigerator for 8 hI's and at room temperature for 12 hrs. The precipitate formed after filtration was crystallized from DMF- water to afford yellow products of 16a, b;

N ,N' -dimethylenebis-(3-methy 1-2(1H)-q uinoxali­non-l-yl)methylenecarboxamide 16a: Yellow crys­ta ls, 0.26 g (71 %), m.p. 33 1°C, Rr 0.73 (S2)' Anal.Cald. for C24 H24 N60 4 (M.Wt. 460): C , 62.6 1; H, 5.21; N, 18.26. Found: C, 61.99; H, 5.49; N, 18.22%. lR (KBr): 329 1 ( H), 1659 (C=O, amide), 165 1 (C=O, Qx).

N ,N' -heptamethy lenebis-(3-meth y 1-2(IH)-q uino­xalinon-l-yl)methylenecarboxamide 16b: Yellow crysta ls, 0.27 g (64%), m.p. 250°C, Rr 0.69 (S2). Anal.Cald . for C29H3~N60~ (M.Wt. 530): C, 65 .32; H, 6.403; N, 15.84. Found: C, 65.32; H, 6.51; N, 16.13%. IR (KBr): 3292 (NH), 1663 (C=O, amide), 1651 (C=O, Qx) .

Synthesis of N,N' -dimethylenebis-3-methyl-2(1H)-quinoxalinon-l-yl methylene- carboxamide 16a. N-(2-aminoethyl)-3-methyl-2( 1 H)-gu inoxali non­l-yl methyle-necarboxamide 15 (0.9 mmole) was added to the cold dried solution of 3-methyl-2- ( IH)­guinoxalinon-I-yl acetyl azide 6 rprepared from (0.185 g, 0 .8 mmole) of the hydrazide], afterwards the mixture was kept in the refrigerator for 12 hrs . The precipitated product was filtered off and crysta llized from ag. DMF to give 1.2 g (68 %) of 16a, m.p. 332°C, Rr 0.72 (SI)' (The same product was prepared previously in this study from coupling of 3-methyl-2(JH)-quinoxalinon-l-yl acetyl azide 6 with ethyl­enediamine; m.p. 332°C, 65 % yield.

Synthesis of 3-methyl-2(1H)-quinoxalinon-l-yl methyl isocyanate 17. After heating a solution of azide 6 (prepared from 0.185 g, 0.8 mmole of the hydrazide) in ethyl acetate under refl ux for 3 hrs, it was cooled and the product was precipitated by the addition of pet.

ether (60- 80°C). After filtration, the isocyanate 17 was crystallized from ethyl acetate- pet. ether (60- 80°C) to give 13 (55%), m.p. 210°C, Rr 0.84 (SI)' IR (KBr): 1690 (C= 0, isocyanate); 1652 (C= 0, Qx); 1600 (C= N) .I H NMR (DMSO): 7.6- 6.8 (m, 4H, Ar- H), 4.7 (s, 2H, CH2N), 3.25 (s, 3H, CH3 Qx).

Synthesis of 3-methyl-2(1H)-quinoxalinon-l-yl methyl carbamyl-L-phenyl-alanine methyl ester 18. A mixture of isocyanate 17 (2.15 g, 10 mmole) and L­phenylalanine methyl ester (1.79 g, 10 mmole) in ethyl acetate was heated under reflu x for 3 hrs. The reaction mixture was washed with 0 .5 N HCI , H20, 3% NaHC03 and H20 then dried over an hyd. Na2S04. The solvent was evaporated under vacuum and the residue was crystallized from ethyl acetate- pet. ether (b.p 40-60°C) to give the L-phenylalanine methyl ester derivative 18 2.2 g (56%), m.p. 178°C, Rr 0.72 (SI)' lR (KBr): 3284 (NH), 1752 (C= 0, ester); 1652 (C= 0, Qx) , MS (m/z): 394 [Mr.

General method fOJ" the preparation of the cop­pel' complexes of the ligands 5, 8 and 13. To the solution of the ligand (3 mmole) in ethanol (15 mL), a so lution of cupric chloride (0.3 g, 3 mmole) in ethanol was added. The reaction mixture was refluxed for 3 hrs. The precipitated complex was separated by filtra­tion , wa 'hed with cold e thanol , and dried under vac­uum at room temperature.

The metal: li gand ratio was determined by titration aga inst EDT A using murexide as indicator after digestion with concentrated nitric ac id and hydrogen peroxide (30%)1 8 The conductance of the complexes was measured using conductiv ity meter Chemitrix by the procedure publi shed by Srivastava et al. 19

Copper complex of 5, brown precipitate, 0.579 g, (57%), m.p. 280°C. IR: 3450-3428 (H 20), 3 180 (NH), 1638 (C= 0 , hydrazide), 1636 (C=O, Qx), 598 (Cu-0), 540 (Cu- ). Molar conductance 1.4 x 10.7 mho cm2 mOrl .

Copper complex of 8, bl ack precipitate, 0.516 g, (53%), m.p. 340°C. lR: 3650-3426 (H20), 1740 (C=O, carboxyl) , 1659 (C=O, Qx), 610 (Cu-O). Molar conductance 1.8 x 10'7 mho cm2 mor l.

Copper complex of 13, brown precipitate, 0.524 g, (54%), m.p. 282°C. lR: 3448 (N H), 1681 (C=O, 'am­ide), 1653 (C=O, Qx), 511 (Cu-O), 571 (Cu-N). Molar conductance 1.5 x 10'7 mho cm2 mOrl.

(B) Antimicrobial activity The antimicrobial activity of the synthes ized com­

pounds has been screened in vitro against E.coli, P. aeroginosa, St.aureus and B.subtilis ?O

ALI et 01.: SYNTHESIS OF QUINOXALINONYL AMINO ACID & PEPTIDE DERIVATIVES 2845

The diameters of the clearing zones (in mm) have been taken as a parameter to express the antimicrobi al activity for each compound at concentrations 10, 100 and 1000 ppm. The results of the antimicrobial activ­ity are shown in Tables (II and III) .

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