TESTING QI ANTIBACTERIAL. ANTITUBERCULAR ACTIVITY ~

PHYSICAL ~ (SPECTRA)

272

T~E~S~T~I~N~G ill: ANTIBACTERIAL ACTIVITY QL Ittt COMPOUNDS

SYNTHESISED !! PARTS 1 lQ !l!

INTRODUCTION33B-341

Bacteriology ie the science that deals with

the study of bacteria, which is the microscopic organism

of plant kingdom. The therapeutics known before the

times of Ehrlich were Cinchona for malaria, ipeac for

amoebic dysentery and mercury for treating syphiliS.

The disease of protozoal end 6pirochecatal origin

heve been made to respond to synthetic chemotherapeutic

agents during the first two decades of the 19th century.

Bactariostatic compounds would inhibit rapid reproduction

of pathogenic bactaria end enable the leucocytes and

other dafenca machanism of ths host to cope with few

static invaders.

paul Ehrlich the father of g~e~9therapy used

the term chemotherapy to describe the cure of an

infectious disease without injury to the hoet known

as chemotherepeutic agents and claseifi~d according

to diseasa and the infections, such as antibacterial,

antiprotozoel, antiviral, antineoplastic, anti­

tubarcular and entifungal agents.

273

This section describes method used for

'in vitro' assessment of antibacterial agents.

Antibacterial substancee and preparationa are classified

as disinfectants, antiseptics and chamotherapeutic

agents. The term disinfectant ia used to eliminate

or destroy infection and should be capable of killing

a ~ide range of bacteria. An antiseptic ia used to

control or eliminate bacterial infaction. A

chemotherapeutic agent is an antibacterial substance

administered systematically for the treatment of

infaction which may be either bscteriostetic or

bacteriocidal in its action.

Antibacterial agents

Chemotherapeutic agent uaed against the

bacterial diseaeea and divided into two types

according to their action on bacteria namely

bacteriostatic and bacteriocidal agents. An agent

ia conaiderad "bacteriostatic" when it inhibite further

growth or mutiplication of bacterie and classed as

-bactericidal" when it kills the bactaria. Antimicrobial

agents ara the chamotherapeutic aubstances that destroy

or inhibit the growth of micro-organisms in the living

tissue. Antibiotics are substances produced by living

organisms and are sufficiently non-toxic to be used as

antimicrobial sgsnte.

CLASSIFICATION Qf.. ANTIBACTERIAL AGENTS

Alcohols ~ related compoun&!

Various alcohols and alcohol darivatives have

baan used as sntiseptics e.g. athanol and propanol.

The antibscterial value of straight chain alcohols

increases with an increass in the molecular weight

and beyond Ca the activity begins to falloff. The

isomeric alcohols show a drop in activity from primary

to secondary to tertiary.

~ ~ _t_h~e:i~r derivatives

Salicylic acid has strong antiseptic and

germicidal properties being carboxylated phenol. The

presence of the carboxyl group eppeare to have -ve

sffect. Benzoic acid is used axternally es sn antiseptic

and employed in lotions and ointments.

274

Iodine containing compounds

Now many iodine containing compounds srs widely

used as antieeptics in the medicine todey. But iodine

ea a tincture or in aqueous solution is still widely

used as antiseptic.

Chlorine containing compounde

The bectericidal properties of hypochlorite

275

hes been firat studied by Robert Koch in 1881. N-chloro­

compounds are represented by amides, imidee and

amidinee in which one or more of the hydrogen ato.e

attached to nitrogen have been replacsd by chlorine

e.g. chloroamine - T.

oxidizing agente

Oxidizing agsnta are of value as antiaeptics

depending on oxygen like H20 2 , matal peroxides,

ursa peroxide etc.

Bacteriostatic dyes

Prior to advent of the Bulfonamides and the

antibiotics, the organic dyes have been ueed exten-

tively as antibacterial agents.

significance was first recognized

Their medical 342 by Churchman who

reported in 1912 on the inhibitory effect of crystal

violet on gram positivs organism. The yellow acridine

dyes have been discovered by Ehrlich for control of 343 trypanoeomal infections. Browing in 1913 discoversd

their entibacteriel propertiea which led to their

wide clinical use~

Antibacterial antibiotics

In the last twenty years since the diacovery

276

of arythromycin more than fifty antibacterial

antibiotics with a common chemical featUre - a macrolytic

lectona have been described. These mecrolide. ere of

great interest because of their antibacterial activity,

primarily against gram - positive bacteria and

mycoplasma species like mythymycin, erythromycin end

-carbomycin.

B-Hydroxy quinoline

B-Hydroxy quinoline or oxine ie unique among

tha isomeric hydroxy quinolines. It alone exhibits

antimicrobial activity attributed by ite ability to

chelate metals344 •

Antibecterial metal ione

Metals and their salts other than mercury and

eilver are lese important as practical antibacterial

agents~ Both organic and inorganic copper salts,

used mainly as industrial fungicides and preeervetives

which are strongly bacteriostatic but lack significant

bacteriocidal propartias. poe 345 studied the

entibacterial activity of a number of metallic salts

in solution and found them more effective.

Mode of ection and metabolism of antibacteriels -- - -

N-Chloro compounds

They slowly liberate hypochloroue acid in

contact with water.

R.CD.NH.CI + HDH---')~ RCDN'2 + HCID

The antiseptic property is greater at pH 7

and decreeses as the solution becomes more alkaline

27(

or acidic. Tha bactarial action of hypochlorite

reside in their ability to furnish HCIQ regerdlese

2,8

of the precursor. It ie known that HCIQ will chlorinate

a~ide nitrogen end eesums to ettach bacterial protein

by thia routB~

Protein

Bacteriostatic ~ intermediates

346 Steern end Stearn attributed bacteriostatic

activity of triphenyl methane dyes to their ability

and form unionised complexes with amphoteric

constituents of the cell thersby blocking important

microbiological processes. fiachsr and Munzo347

summarised the relationship between structure end

effectiveness of such dyes.

Chelation ~ drug action

A numbsr of drugs are metal-binding agents and

aD chelation may be considered as meane whereby a

drug can be removed from the circulation when chalate.

are in active form of a drug. It is moat likely that

they penetrate biological membranes in fully chelated

form and subsequently break down to an active form.

An effective demonetration is provided by classic

34B-349 work of Albert •

Study of sevan isomeric monohydroxy quinolines

suggest that only tha B-hydroxy isomer is active

in inhibiting the growth of micro-organisms and that

same isomer ie the only one to form metal chelates.

The site of action of oxine and its analogue has

been suggested inside bacterial cell. or on call

aurface350.351.

DETOXICATION OF ANTIBACTERIALS

p-Aminobenzoic acid is a growth factor for

certain microorganisms end competitively inhibits

the bacteriostatic action of sulfonamidee. The

metabolites identified in man are p-aminobenzoyl

glucoronide. p-emino-hippuric acid, p-acetyl amino

benzoyl glucoronide. p-acetyl amino hippuric acid

and p-acetyl amino benzoic acid.

278

280

Bactaria

In 1928 Germen scientist C.E. Ehrenberg used

the term 'bacterium'. The bacteria are smell

microscopic organism with a relatively simple and

primitive form of cellular organisation known as a

'Procaryotic'. In 1884 Danish physician Gram

discovsred tho stain known as Gram-stain. Staining

reaction has widest application which dividas all

bacterie into two cetagoriaa, namely Gram (+)ve and

Gram (-)va. The Gram (+)ve bacteria resist dacolourisa­

tion and remain stained as dark purple colour while

Gram (-)ve bacteria are decolourisad.

Bacteria can be classifiad according to thair

morphological characteristics as the form of mycelium

or shathed filements, e.g. cocci, bacilli, vibrios,

sprille and spirochetes. The microorganism capable

of producing diseese in animal or human being known

as ·pathogenic". Most of the microorganisms present

on the skin and mucous membranes ara non-pethogenic.

281

CLASSIFICATION QI IMPORTANT ORGANISMS

ClaSS I Schizomycetes

-----------------------------------------------------------Ordar Family Genus Species

-----------------------------------------------------------(1) (2) (3) (4) -----------------------------------------------------------Eubacterials Micrococea- StaphylococcU8 Staph.aureus

ceae Micrococcus M. tatragenua

Sarcina S.lutea

--------------------------------------------Lactobaci­llaceaa

Streptococcus

peptoetrepto­coccus

Diplo coccus

Lettobacillus

Str. pyoganas

Pep.putridi.

D.pneumoniae

L. ecidophilus _____________________ e .. _____________________ _

Neisseriace- Neisseria ae

N.gonorrhoeea

N. meningi tid is

N.catarrhalia

--------------------------------------------Coryncbecte- Corynebecte- C.diphtheriaa riaceae riu.

Listeria L.monocytogenes

Erysipelothrix E.rhusiopethiae

---------------------------------------------

282

-----------------------------------------------------.. ----(1) (2) (3) (4) --.------------------------------.-------------------------

Achromoba­cteriacaae

Alceligenee Alc.faccelie

--------------------------------------------Enterobe­cteril!lceae

Eecherichia

Kelbsiella

Citrobacter

Cloaca

Hafnia

Serrl!ltia

Salmonella

Shigella

Protous

Esch. coli

K.pneumoniae

K.serogenes

Cit.freundii

CI.cloacae

Haf.alvei

Ser.Ml!lrcescons

Salm.typhose

Sh.dysenteriae

Pr.vulgaris

--------------------------------------------Brucella­ceae

Pasteurlla

fancisella

Brucella

Haemophilus

P. peatie

P.psuedotuber­culosie

f.tulerensie

Br. lIIelitensie

Br. abortus

Br.suie

H.i"nfluenzae

H.duoreyi

283

---------------------------------------------.--------.---__ l!l _____________ l~l __________ l~l _____________ l~l _______ _

Bordetella Bord.pertussis

Moraxells M.lacunats

Actinobacillus A.mallie

A.ligniereaii

------------------------------.-------------Bactsrio­dacsse

Bacteroide Bact·. fragilis

Fusobacterium F. fusiforms

Streptobsci- St.moniliformie llus

Saphaerophorus Sph.necrophorus

--------------------------------------------Bacilla­ceae

Bacillus

Clostridium

B·.enthracis

B.subtilis

Cl. tatsni

Cl.l-!elchH

------------.----------------------------------------------P seudomone­dales

pseudomona- Pseudomonas daceae

ps.aeruginosa

--------------------------------------------Spirllaceae Vibrio V~cholerae

Spirillum Sp.minus

-------.---------------------------------------------------Mycoplasma­talea

Mycoplasma- Mycoplasma taceee

M.pneumoniee

M.mycoides

----------------------------.------------------------------

l""~ .~.IU I If. 11118 ..

~I .. of tt 'to , '---

284

------------------------------------------------------.-. 121 _______________ 1~1 __________ 1~1 _____________ 1~1 _____ --

Actinomy­cetatea

Mycobacteria- Mycobacterium ceae

Myco.tuber­culosis

Myco.laprae

-------------------------------------------Actinomyceta- Actinomyces caae

A.isrse1i

A.bovie

N. madurae

-------------------------------------------Streptomyce- Streptomyces tacaae

Strepto.griseus

-----------------------------------------------------------E. spirochae­tales

Spirochae­taceae

Spirochaeta Non pathogsnic

Saprospirs

-------------------------------------------Treponema­taceae

Borra1ia

Treponema

Leptospire

Bor.duttoni

Bor.recurentie

Bor.vincenti

Tr.pa11idum

Tr.pertenue

L.icterohae­morrhagies

----------------------------.------------------------------

We have undertaken species Eschericha coli

(Gram - negative bacterial strain) and Staphylococcus

aureus (Gram - po~itive bacterial strain) for testing the

antibacterial activity of these compound.

EVALUATION QE ANTIBACTERIAL ACTIVITY

Varieties of 'in vitro' screening methode has

been used to evaluate the antibacterial ectivity.

testing in mice has become standard, the sensitivity

of bacteria to antimicrobial agents is tested by the

eame methods as in othsr form of microbiological assey,

principles of which are shown in figure 352 •

Results

285

Reagjf Antibiotic Non visible organism

Non visible growth

Visible growth

Microorganisms

I n vitro methods

I N C U B A T I o N

Bactsrioci­dal concen­tration

.. . . . . ... :\;i~~ .... .... .. .. · , . · . ~ .. .... .., .

... ,.. ..' · . . K:~~{j~~~~~~ ... , t t ... .. -.' , . ' . , ~ . ',".. ..... . ,. , ... • , t . , . . . '

• · , · . .. .' . ... . .. ". .'. · , . · .. .. ". .. .... . .. . " ," .... , . . ~.::~ .. :. · ..... ...... .. " "I~~i · .'. : Ii · .. .. .

Bacteriostatic concantration

'In vitro' testing is useful for antibecterial

spectrum determination of e compound end comparing it

with other 8gent8~

Severel types of procedures are in use for

assaying the potency of antibiotic preparation for

therapeutic purpose. These methods have been modified

and used for sensitivity test of unknown organism.

353 Seriel dilution ~ Broth

Serial dilutions of ths drug being essayed ere

made in uniform amounts of standard broth in culture

tubes. These are inoculated with a uniform of cella

to test orgenism. After incubation, turbidity (or

286

its absence) is measured by turbidimeter and

turbidities (amount of growth) are compared with

dilution series mede in the same wey but with entibiotic

rsfsrencs standard of maasured potency.

streek assay ~ ager (loc. cit)353

Granded dilutions of the substances to bs tested

ere pleced in a series of petri dishes in which is

poured about 10 ml of melted and cooled egar, contents

mixed with drug dilution. After egar has hardened,

the plates marked into several sectors, each of which

is streaked with different test organisms.

Diffusion tssts

2B7

Diffusion tests on solid media have been adopted

by most of the laboratories wherein the antimicrobial

agent is held in reservoir from which it diffuses through

agar medium to form a diffusion gradient whersin the

micro organisms growing in or on ths agar are axposad.

The size of inhibition zone depands upon the factor

that influence the diffusion of the antimicrobial agsnt.

Agar strie diffueion 1!!i ~ sensitivity

This is a simple technique which has originelly

been used by Fleming. A strip of egar ia cut from the

centre at a placa of Buitable culture medium. Appro­

priate amount of antimicrobial agent ie added to molten

agar and pipet ted into gutter in the medium and the

surface of the agar ie inoculeted by etroking culturee

to be tested.

Replica plate

bacteriocidal

method ~ 354 action

show bacteriostatic and ...........

A zona of inhibition of growth around a dish

283

may indicate that the antimicrobial agent is aither

bactericidal or bacteriostatic. The presence or absence

of living orgenisms within the zones of eppersnt complete

inhibition of growth on diffusion plates have been

shown by replica plate method.

~iffusion tests with filter paper diske ~ determining 355 sensitivity

This constitutes a reliable and eimple technique

impregnating small disk of stsndard filter paper with

given amount of antibiotic placing them on plates of

cultura medium inoculated with organism to be tested.

After incubation the degraa of sansitivity by maaeuring

the easily visible areas of inhibition of growth hes

bean produced by diffusion of antibiotic from the disk

to the surrounding medium is determined.

DISCUSSION

We have undertaken the species Staphylococcus

aureUB and Escherichia coli for the antibecterial

screening of the compounds synthesised in Perts I to

III.

289

2taphylococcus aureus - Family I Micrococcaceee

In 1B7B, Koch observed micrococcus like

organiems in pUB. pasture (1BBO) CUltivated theee

cocci in liquid media. Ogaston (1BB1) found it present

in pus of acute chronic obscessed and found pathogenic

for mice and guinea pigs.

They sre Gram (+}ve cocci ovoid or epheroidel,

non-motile, arranged in group of culstersl grow on

nutrient agar and producecolonias, which are golden

yellow, white or lemon yellow in colour, aerobes or

faculative anaerobes, biochemical activities and

haemolytic power ara variable; pathogenic etraina

produce coagUlase, ferment glucoee, lactose, mannitol

with production of acid, liquify gelation and produce

pus in lesion.

290

~enus I Staphylococcus

Staphylococcus is differentiated from micrococcus,

another gsnus of the same family by ita ability to

utilize glucose, manitol and pyruvate anaerobically,

etaphylococci ere found on the skin or mucue membranas

of the animal body.

Species I Staphylococcus aureus

The individual cells srs 0.8 to 0.9 ~ in

diameter. They are ovoid or spherical, non-motile,

non capsulated, non-sporing, stein with ordinary aniline

dyes and Gram (+)ve, typicelly arrangad in groups.

These era aerobes or facultativs anaerobes and grow

sasily on nutrient - agar. The optimum temperature

for the growth is 370 but ths range of temperaturs

varies from 10. to 40., optimum pH is 7.4 to 7.6.

Escherichia coli - famil~ I Enterobecteriaceae

They are Gram (-)ve rods, metal with

peritrichate flagella, or non-motile. They do not

form epores end are primerily environmental eaprophytes

and scavengers, found in the intestinsl tract.

Genua I Escherichie

This genua comparisee Escherichia coli and

Beveral varients, and ia of particular interest einca

they occur commonly in the normal intestinal tract of

a man and animals. Escherichia coli ia tha most

diatinctively faacal speciea.

SpeCies I Escherichia coli

Escherichia in leeS discovered Eecherichia coli

from the faecea of the new born who showed the

organisms in inteatine within 3 daya aftar birth.

ThsBe ara Gram (-)ve rods. 2 to 4 ~. commonly sean

in cocca bacillary form and rarely filementous forms.

E.coli are generally non-pathogenic and incriminated

as pathogena because sometimes strains have been found

to produce septicemia, inflammations of liver gall

bladder. appandix. meningitis, pneumonia and other

infections.

291

TESTING Q[ ANTIBACTERIAL ACTIVITY Q[ SYNTHESISED

COMPOUNDS INCLUDED U! PARTS I TO III

In vitro testing

Bacteriostatic activity can b. determined on

eolid or liquid media, each depends on aeeeesing the

extent of inhibition of growth. We have adopted

disk or tablet method for the sensitivity testing.

l!!! ~ method

After the report of the "International 356 collaborative study" involved with investigation

by 'the disk test', the method recommended has

adopted in Sweden. In U.S.A. modified Kirby _

been 357

Bauer

teChnique has been adopted as an official method by

'Food and Drug Administretion'. The main stimulue

for standerdizetion in U.K. has come from the

recommendation of use of the controlled eingle disk

method3SB• Measured etreangths of the antibiotic

solution are applied to inoculatad agar in disks of

uniform thickness. of sterile filter paper, which are

placed on the surface of the agar plate before

292

incubating. The width of the zone indicates, the

sensitivity of the organism bsing tested through the

presence or absence of a zone and ia of greater

significsnce.

factors influencing inhibition !2n! sizes

Ingradients 2! culture media

Many substancss are prasented in culture media

which may affsct tha zone of inhibition, common

ingradients such as paptone, tryptone, yeast extract

293

and agar may vary in their mineral content may influence

the activity of soma antibiotic.

Choice of medium .....

Consistsnt and reproducible rsaults ere

obtainsd in media prepared eepecially for sensitivity

testing the plate must be poured fIst with an aven

depth. Very thin plates are unsatisfactory.

Effect s! EH-

The activity of aminoglycosides is enhsncsd

in alkaline media and reduced in acidic media, the

reverse is shown by tetrecycline.

Size 2! inoculum

Although many antibiotica ere not markedly

affected by large number of organisms all inhibition

zones ara diminiehed by haavy inocula. Overnight

broth cultures of organiems and suitable suspensions

from solid media can be diluted accurataly to give

optimum inocula for sensitivity testing. In practice

satisfactory results csn be s~hievsd by taking a

loopful of well grown culture. or a suitably mede

suspension of organisms and spreading it with dry

sterile swab359 •

!h! performance £! diffusion tachnique

Composition 2! nutrient agar

Peptone 2 g

NeCl 2 9

Meat extract 3.2 g

Agar agar powder 8 g

pH 7.4

Diatilled water 1000 ml

294

•

]trength of antibiotics

Until very recently, there has bsan little or

no agreament regarding the strength of antibiotic

disks for use 'in vitro' sensitivity tests.

Storage ~ disks

Disks should always be kept cool end dry when

applied to the medium should be pressed firmly to

ensure proper contact and even diffueion forceps,

dissecting needles.

Incubation time .........

It ehould idaally be the minimum requirement

for normal growth of the organism. Prolonged incu­

bation of a culture may result in inactivation of the

antibiotic end reeult in the eubsequent growth of

organism.

Controle

for the correct interpretation of results and

recognisation of any eource of error in disk diffusion

295

29G

sensitivity tssts, correct use of control organism

is essential. For routine the organism sre most

conveniently kept in s refrigeretor et 4-. On atarile

throet swabs a jer full of such swsbs cen be impragnated

at one time as they keep well at leaet a week.

The compounds described in Parts I to III hava

been screenad for entibacterial activity in tables 15

to 25 under the following heeds I

a) Thiazolidinonea

b) Cyanuric chloride

c) Acetoecetanilidea

d) ~.Aminonitriles

e) Thiourea

TESTING ill: ANTITUBERCULAR ACTIVITY g[ ~S_YN~T~H~E~S~I~S~E~D

COMPOUNDS INCLUDED l! PARTS! AND ill

Methods 2! testing 2! tuberculoststic drugs

I n vitro .!!!.!i

The in vitro test principally consiets of

seeding tubercle becilli into synthetic culture medium.

This consists of graded concentretions of the substences

to be tested, ranging from zero concentration for

control purpose to concentration approaching the toxic

dose.

In vivo .!!..!!

Mics, guinea pigs or rabbits era inoculated

with bovine tubercle becilli~ Soma of infected

animals orally or paranterally at various dose lavels

with the substances to be tested while other untreeted

enimals ere msintained as controls. A widaly used

techniqus following the pathology of the diseass

process by sacrificing treeted end untreated infected

animels in which the infection spread to varioua

297

organs. A series of drugs effective against experimental

end clinical tuberculoeis has been developad during

last thirty years begining with 4,4'-diamino-diphenyl

sulphone, streptomyc~n and ethambutol. Exhaustive

review by different authors have been published on

this topic360-364.

298

The modern work could be considsred to have

started with the observations of Rist, Rich and follis,

their findings that the large doseD of sulfanilamides

produce benificial effect on the

mental tuberculosie confirmed by

development experi-365-367 Buttle and Parish •

Sulphur containing compounds

SUlphonamides, sulphons, sulphanilamides ~ releted

compounds

Prior to 1935 no compounds have been known that

could alter the course of tuberculosis in enimals Dr

368-369 man • In 1938 sulphonamide has been reportad

to posaess a striking effect on the extent and

dietribution of M.tuberculoeis disease in guinea pigs370•

The sUlphonemideB elevate serum concentratione

of INH in man371 ,372 and supress emergence of

resistance of streptomycin in vitro. Rist and 373 feldman prepered sulphonee end reported them to be

more active in experimentel tuberculosie than

sulphsnliamides, over and above their epplication in

leprosy374.

o HN~···~~NH

2 -~_ II~_ 2 o (I)

4.4'-Diamino diphenyl eulphone (I) (DDS) and

its glucose bisulphite derivative have been the first

compounds to exhibit e good life extending effect

on tuberculosis animals. Replacement of one of the

amino group. any of the groupa euch aa hydroxy,

hydroxylamine or nitro resulta in a prectical loss

of activity375-376. The eulphons serias consist of

following I

PromHole (II)

o "-Clf)! R S~NH o 2

( II)

298

4-Nitro-4_formamido-diphenyl sulphone (III)

o o N ~- ~ ~NH-C=O

2 ~- 11\d-- I o H

(I II)

Treatment with the eulphones end derivatives

show aide reactions in central nervous system,

hemobutic anaemia cuanosis377 •

Thiosemicarbazone, thioureae and thioamides

Thiosemicarbazones

from the study of certain sulphathiazoles and

eulphathidiazoles which ara reported

antituberculous activity. Domagk et

to possess

HB el. showed

thst tuberculostatic activity of these compounds is

a function of thiazole ring. The specific activity,

although a function of the constituent sulphur group

is not limited to triazole ring compounds containing

300

301

open chein errangement of Nand 5 etoma which have baen

found to be more effective379 • Thioaemicarb8zone of

indole-3-aldehyda has been found to be sctive JBD subcutaneously • Thiosamicerbezone of 4-acetamido

benzaldahyde (thioacetazpna) (IV) is most affective.

s HF-OC-HN{ }CH=N-HN-~-NH2

whan

(Thioacetazone) (IV)

Activity of thioacetazone molecule is lost

>Ca5 moiety replaced by >CuD or >CcNH or

whan terminal amino group is replaced with -NHR,

-NH 2 or -5-CHJ , The ortho and meta isomers ara

successivaly less active than the para-isomer in

thioacetazone, while p-carboxy, p-hydroxy, p-acetoxy

end p-ethoxy derivatives of thioacetezone are reported

to poaseas high activityJB1,382.

Thioacetazons was activa orally against

experimental infections in animals being comparable to

383 streptomycin • In spite of theee dieedvantagas of

ite toxicity. thie drug is baing widely used to replace

PAS aa a companian drug to INH because it ie cheapar

384-386 and has greater patient acceptance •

Thioureaa

The taxonomic similarities between mycobacteria

and fungi prompted Meyer to test the effect of known

antifungal agente like sUlphur-containing compounds

against M.tuberculosia in vitr0387. In the attempte

302

to enhance the activity, extensive structural variation

are sought38B ,389. Mono-substituted thioureae usually

possess in vitro tuberculostatic activity. SUlphanilamide

thiouraa (V) displayed definite chemotherapeutic 39D activity in humen tuberculosis •

(V)

s x-{ }-HN-~ -NH-AIK:y1

(VI)

1.3-Diphenyl thiourea. and 1-(p-aceyl phenyl)­

thioureaa391 are more active amongst the thiourea.

known. p-Subetitution of both the phenyl rings with

electron donating group is necessary for the activity.

It is found that 1-phenyl-3-alkyl thiourees (IV)

bearing certain electronegative substituents in the

para position possess good activity. provided the

alkyl radical is short and unbranchad. The most

active among these compounde is 1_(p-acyl)-phenyl-3_

methyl thiourea (VII)

(VII)

The presence of -NH-CS-NH- group is essential

part of thioaemicarbazonBs and introduction of

303

CmO group between one phenyl and -NH group does not

deactivate the thiourees molecule whereas removal of

one -NH gr. inactivate the molecule in a group of new

thiourees namely the disubstituted thioureae.

Thioamidee

In vivo tuberculostatic ectivity has been

claimed for thio-isonicotinamide (VIII)392,393 teeted

earlier in vitro394.

(VIII) (IX) (X)

Many modifications have been made in attempts

to increase the activity. Increase in potency without

increase in toxicity has been observed in a series

304

of 2-alkyl (C 2 to C6 ) derivatives of thioisonicotinamide,

the most active being the ethyl (IX) end n-propyl (X)

derivatives395 ,396 which have besn found to be 4 timee

es active as thioisonicotinamide.

2-Ethyl thioisonicotinamide is about four times

as active as streptomycin. It is active against INH,

streptomycin as PAS resistant strains397 ,39 B has

approximately 1/10th activity and 1/5th the toxicity

of INH.

Mercaptans

Del Pianto observed that compounde capable of

releaaing ethanethiol (C2HSSH) are tuberculostatic.

In next few years the activity of other ethyl

mercaptan derivatives has baen studied and confirmed

their activity due to release of ethanethiol399 •

Among the compounds capable of releasing ethanethiol

in vivo, bis(ethylthio) isophthalate (XI) haa been

found to be more suitabla for human therapy. Ethyl­

thiogroup and its ~ -relationship to the carboxy group

i8 essential for the activity of sthyl thio-pyrazinoate

which is reported to possess apprecieble activity.

Y-COSC2H:

COSC2HS

(XI)

305

aUG

/3 -Ethyl thiopropionic acid (C 2H S.S.CH 2-CH 2-COOH)

a derivative of mercaptan is more active in vivo than

in vitro.

Higher activity and relatively low toxicity

of INH prompted many workers for synthesising a large

numbsr of structural analogues of INH, however, none

of tha derivative appeared to be superior to INH

as such. The active pthibazida (3-methoxy-4-hydroxy

benzylidene derivative of INH hydra zona (XII) has

been found to be more active and less toxic, Bxten-400 sively used clinically in U.S.S.R.

K( )-CO-HN-N==CH OH

(XII)

Ithambutol

Thomas and coworkers401 found that

3U7

N.N'-di-isopropyl ethylene diamine (XIII) is effective

in the treetment of tuberculoue infection in mice.

(XIII)

A number of congeners of this compound is

examined and one proved to be the most tuberculostatic

ie N.N'-bis-(1-hydroxy-2-butyl)-ethylene diamine

dihydrochloride (ethambutol) (XIV)

(XIV)

Activity

The etereospecifiCity of the antimycobacterial

activity of etha.butol ie reflected by the fact that

dextra-isomer is 12 times more active than higher

purified meso-form and 200-500 times more active than

402 the leavo isomer • About 75% of the strains of tha

human type are aensitive to 1 ~g/ml of ethsmbutol.

Ethambutol supreeees the growth of INH and straptomycin

resistance tubercle bacilli.

~bsorption, excretion ~ effects

75 to 80% of an administered dose of ethambutol

is absorbed from the gsetro-intestinal tract. Plasma

concentration ara maximum in the man 2 to 4 hours

after the drug is taken. It is partly excreted in the

form of an aldehyde and. dicarboxylic acid derivativee,

becauae of greater tuberculostetic activity, lower

incidence Qf toxic side effect and better pat~nt

403 acceptance •

P yrazi.,amide

Pyrazinamide (XV) exhibits tuberculostatic

activity in vitro only at a elightly acidic PH404 •

The growth of tubercle bacilli within monocutss in vitro

is completaly inhibited by the drug in concentration

of 12.5 ~g/ml.

(XV)

Pyrezinemide i8 more effective thsn PAS,

cycloserine or viomycin is experimental infections

with human strain of M. tuberculosis in mice in guinea

pigs. It is well absorbed from gsstro-intestinal tract

and it is widely distributed throughout the body, 40S sxcreted primarily through urine •

!:!.ydroxamic acid

Activity of PAS, prompted Urbanski to examine

aalicylicacid derivativas considering hydroxamic 406 ecid as a potential amino group • Salicyl hydroxamic

acid (XIV) (250 mg/kg against unexperimental infection

in mice) is found to be equivalent to PAS (500 mg/kg).

309

~CONHOH ~OH

CONHOH OH

(XVI) Br

(XVII)

310

Br /" ~ OH

~ CONHOH / OH

CONHOH

(XVIII) (XIX)

3- and 5-bromo derivatives (XVII) and (XVIII)

and 2-hydroxy naphthalan-3-hydroxsmic acid (XIX) have

been claimed to be active as streptomycin subcutaneously

in guinaa-pigs, but are found to bs inactive in the 407 mouse test •

Jl asic dyea

A good number of baaic dyes such as phenazines,

phenoxazines, etc. hava been tastad for thair

entibacteriel activity. Among the various groups

studied the azines ara outstanding and many azines

possess pronounced activity in vitro but only a few

of these are activa in vivo. In this section tha

particulars of in vitro testing of the compounds

deacribed in Parts I and III for their antitubercular

activity.

311

ANTIBACTERIAL ~ ANTITUBERCULAR ACTIVITY

Tables 15 to 2S

EQ! ANTIBACTERIAL ACTIVITY QL ~ COMPOUNDS

(+) - Indicatea inhibition zona occurs.

+ • Zone diamater less than 20 mm.

++ - Zone diamater more than 20 mm.

(-) - Indicate. no inhibitory zone around the disk.

fOR ANTITUBERCULAR ACTIVITY Of THE COMPOUNDS - --

(+) - Indicates no inhibitory effect,

i.e. compound is not effective.

(-) • Indicates inhibitory effect,

i.e~ compound i. active •

• (N.B.r Strein used for antitubercular screening is H

31R

v) •

•

312 T ABLE I 15 -

ANTIBACTERIAL AND ANTITUBERCULAR ACTIVITY Q! !tl[ COMPOUNDS

Q!. I.!:!S. TY P E (I)

CI-{ }CO~1j R R'

-------------------------------------------------------------A ntiblllcterillll Anti t ubercullllr

Sr. IIIctivhy activity. Con-

No. R R' I nterpretllltion centration of of zone of =~!!!f.:~i~~!.:. __ inhibition --------------- 5 30 5.lIIurllus E.coli

-._--.--------------------------------------------------------1 • -C 6HS -H + - + -2. -2-OO Z·C 6H4 -H - - + -3·. -4.N0 2 ·C 6H4 -H + + -4·. -4-0CH 3 ·C 6H4 -H - + + 5. -3,4-di-CH 3O.C 6H3 -H + - +

6. -C 6H5 -CH 2CO 2H + - +

7. -Z-NO 2. C 6H 4 -CHzC°2H - - + -B. -4-N02·C6H4 -CH 2C0 2 H + + -9. -4.0CH 3 ·C6H4 -CH ZC0 2H - - + -10. -3,4-di-OCH 3C6H3 -CH 2C0 2H ++ - + -11. -C6 HS -CH3 - - + -12. -2-N0 2 ·C 6H4 -CH3 - - + -1 3. -4-NO 2. C 6H4 -CH 3 ++ - + -1 4. -4.0CH 3·C 6H4 -CH3 - + + -1 5. -3.4-di-OCH 3C6H3 - CH3 + - + ---------------------------------------------------------------

313 T ABLE I .!.§.

A NTIBACTER I AL A.@. ANTIT UBERCULAR ACTIVITY m:. I..!:!!. COMPOUNDS

QL m TYPE (II)

-------------------------------------------------------.-.-Antibacterial Antitubercular

Sr. activity ectivity. Con-No. R R' Interpretation centration

of zone of of COlilp. ~g/ml inhibition - -------------------------- 5 30 S.auraus E.coli

-------------------------------------------------------~--

1 • -C 6HS -H + + -. 2. -3-N0 2 ·C 6H4 -H - - + -3. -4-N0 2 ·C 6H4 -H + - + -. 4. -4.0CH3.C6H4 .-H - - + -. s. -2-0H.C6H4 -H + + + -6. -3.4-di-OCH 3 ·C 6H3 -H - - + -. 7. -2-Cl.C 6H4 -CH 2C0 2H + + + -, 8. -3-Cl.C~4 -CH 2C02H ++ - + -9. -3. S-di-Br. 20H -CH 2C0 2H ++ + + --C 6H2

10. -3.4-CH20 2 ·C 6H3 -CH 2CO 2H - - + -11 • -4.0H.C ~4 -CHf0 2H - - + 1 2. -3-CH 3O.4-0H-C 6H3 -CH 2C0 2H - - + -----------------------_._-----------------------------------

314

T ABLE I 11 ANTIBACTERIAL ~ ANTITUBERCULAR ACTIVITY ~ ~ COMPOUNDS

Of" THE TYPE (III) ---

-----------------------------------------------------------Antibacterial Antitubercular

Sr. activity. activIty. No. R Interpretation of Concentration

zone of inhibition 2!_~2!!!e.!~~~!!!!.:._ -------------------S.Eluraua E..coli 5 30

-----------------------------------------------------------1 • -C 6HS - - + -2. -2-Cl.C 6H4 - - + -3. -3-Cl-C 6H4 . + - + -4. -2-HO.C 6H4 - - + -5. -4-HO.C 6H4 + - + -6. -4-oCH3·C 6H4 ++ + + -1. -4-0H ,-3-0CH 3-C 6H3 + .. + -8 •. -3,4-di-OCH 3·C 6H3 - .. + .. 51 •. -3-N0 2·C 6H4 .. .. + .. 10. -4-N0 2·C 6H4 - + + -11 • -CH-C6HS .. + .. 12. -C4H30 + .. + .. ----------------------------------------------------------

315 TABLE I .!!

ANTIBACTERIAL ACTIVITY QE COMPOUNDS Q[ ~ TYPE (IV)

N H NHCOAr

Ri<Z\\ A /l5«R ~_\ HN~J-NH--\d_

----------------------------------------------------------Sr. No. Ar

Antibacterial activity. R Intarpretation of zene

of inhibition ------------------------S.aureus E.cD1i

----------------------------------------------------------1 • -1:6HS -H -2.- -4-1:1-1: 6H4 -H -3. -I: SH4N -H + ... 4. -4-1:1-C 6H4 -2-C1 + -s. -4-0H-C 6H4 -2-1:1 - ... 6. -C SH4N -2-C1 + -7. -4-C1-C 6H4 -4-C1 - -8. -4-0H.C 6H4 -4-C1 ... -9.- -I: SH4N -4-1:1_ + -10. ...C~S -4.CH3 - -

• 1" -4-N0 2 ·C 6H4 -4-CH3 + -1 2. -c 5H 4N -4-I:H3 + -1 3.- -4-0H-C 6H4 -4-0C 2HS - -14. -4-N0 2 ·C 6H4 -4-0C 2HS -1 S. -C SH4N -4-oC 2HS + +

-----------------------------------------------------------

316

TABLE, .!!

ANTIBACTERIAL ACTIVITY.Q[ COMPOUNDS .QE ~ TYPE (V)

-----------------------------------------------------------Antibacterial activity

Sr. Ar R Interpretation of zonll No. of inhibition -----------------------

S.aureus E.coli -.---------------------------.-----------------_.-.-------1 • -C 6HS - CH 3 -2. -3-Cl.C 6H4 - CH 3 - -3. -4-Cl.C 6H4 - CH 3 + -4. -3-CH 3 ·C 6H4 - CH 3 - -5. -4-CH 3 ·C 6H4 - CH3 + +

6. -1-C 10H7 -

CH3 + -

7. -4-H SC 20 • C 6H4 - CH3 - +

B. -C 6H5 -CZHS - -9. -3-Cl.C.6H4 -CZHS - -10. -4-Cl.C ~4 -C 2HS + -11. -4-0CH 3 • C 6H 4 -C 2HS - +

12. -4-CH 3'C 6H4 -C2 HS + -1 3. -4-0C ZH 5' C 6HS -CZHS - -14. -4-H SCzO f·C 6H4 -CZHS + ---------------------------------------------------------.-

TABLE I lQ.

aNTIBACTERIAL ACTIVITY OF COMPOUNDS Q[ ~ ~ (VI)

------------------------------------------------------Sr. No. Ar

Antibactarial activity Interpretation of zona of inhibition -----------------------------

S.aureus Eo coli ------------------------------------------------------1 • -c 6H S -2. -2-CI-C 6 H4

3. -3~CI,C6H4 - -4. -4-CI-C 6H4 +

5. -2-H3CO.C 6H4 +

6. -4-H 3CO.C 6H4 +

7. -2-H 3C .C 6H4

8. -4-H3C.C 6H4 + -9. -4-H sC 2°2' C.C 6H4 +

1O. -3-N0 2·C 6H4 -11. -4-N0 2 ·C 6 H4 +

12. -4-H SC2O,C6 H4 +

------------------------------------------------------

317

TABLE I £1

ANTITUBERCULAR ACTIVITY QE COMPOUNDS QL ltlI TYPE (VII)

yOCH3

NH-CO-CH-CHTCO-NH-R /'" ~

"'-. /

-------------------------------------------------------Sr. No. R

Antitubercular activity Concantration Df cDmp. ~.9~!'!.:. ___________________ . __

5 30 ------------------------------------------------------1 • -C,HS +

2. -2-Cl-C,H4 + +

3. -3-Cl-C,H 4 +

4. -4-Cl-C,H 4 + -S. -3-CH 3·C,H 4 + +

6. -4-CH3 ·C 6H4 + -7. -4-0CH 3 ·C,H4 + +

B. -4-0C 2HS·C 6H4 + +

9~ -4-COOC 2 HS·C,H4 +

10. -1-C10H7 + -

------------------------------------------------------

318

318 TABLE I 22

ANTITUBERCULAR ACTIVITY QL COMPOUNDS .QL !!:!!. ~ (VIII)

COCH3 R"-NH-OO-CH2 ~H-CO-NH ~ }-COOR

--~---------------------------------------------------5r~

Antitubercular activity R' R Concentration of compo

No. ~3L~~~ _________________ 5 30

-~-----------------------------------------------------1 • -C 6HS -CH 3 +

2. -4-Cl-C 6H4 - CH3 +

3. -4-CH3 ·C 6H4 -CHJ + +

4. -4-0CH 3 ·C 6H4 - CH3 + +

S. -4-:-COO C 2H 5· C l'jH 4 - CH 3 + + ,. -3, 5-di-Br. 4- - CH 3 + -

-COOCH3-C 6H 2

7. -4-CH 3COO-C6H4 - CH3 + -B. -Cl'jHs -C 2HS +

9. -3-Cl.C 6H4 -C 2HS + +

1 D. -4-Cl-C 6H4 -C 2 HS +

11 • -3-CH3 ·C 6H4 -C 2HS + +

12. -4-CH 3-C 6H4 -C2HS + -1 3. -4-0CH r C6H4 -C 2HS + -14. -4-COOC 2HS-C 6H4 -C 2HS +

------------------------------------------------------

T ABLE I 23

ANTIBACTERIAL ACTIVITY Q[ COMPOUNDS [E !tlr ~ (IX)

HO{ }CO-NH-NH-~H-R CN

------------------------------------------------------Sr. No. R

Antibacterial activity Interpretation of zone of inhibition --------------------------

S.aureUB E.coli ------------------------------------------------------1 • -C 6H5 + +

2. -2-CI-C6H4 - -3. -3-CI,:"C 6H4 + -4. -4-0H-C 6H4 + +

5. -4-0CH 3·C 6H4 + +

, . -3-N02 ·C 6H4 -7. -4-N0 2·C 6H4 + -8. -3.4-di-OCH 3 ·C 6H3 +

9. -3-0H-4-0CH 3-C,H 3 +

10. -2-0H-C 6H4 + +

11 • -J.4-CH20 2C6HJ -

320

---------------------------------------------------------

TABLE I ~

ANTIBACTERIAL ACTIVITY Q( COMPOUNDS OF ~ ~ (Xl

Q-\ O-CH-CO-NH-NH-CH-R .- 2 I

/ CN R

------------------------------------------------------S l' ~ No. R R'

Antibacterial activity Interpretation of zone of inhibitio n -----------------------

S.aureus -------------------------------------------------------1 • -C 6HS 2-Cl -2. -2-CI-C 6 H4 2-C1 + +

3. -4-Cl-C 6 H4 2-Cl + +

4. -2-0H-C 6H4 2-C1 -5. -4-0CHJ .C 6H4 2-C1 + +

6 • -CH-C 6HS 2-Me - -7. -4-0H-C

6H

4 2-Ma -8. -4-0H-3-0CH3-C 6H 3 2-Me +

9. -3,4-di-OCH J-C 6H3 2-Me +

10. -3-NO 2° C f!i4 2-Me

11. -4-N0 2-C 15 H4 2-Me + +

------------------------------------------------------

321

322

TABLE I ~

ANTI BACTER IAL ~ ANT !TUBERCULAR ACTIVITY !!f.. COMPOUNDS

Q!. TYPE (XI)

Br S

R-HC= N-HN-OC / '\ NH - g - H N . ( )

Br

------------------------------------------------------------Sr. No. R

Antibacterial ectivity. Interpretation of zone of inhibition ------------------S.aurauB E.coli

Antitubercular activity. Concentration ~!_=~~E~_~2~!!~ __

5 30 --------.. --------------------------------------------------1 • -C 6HS +

2. -2-CI-C 6H4 - - + -3. -3-CI-C 6 H4 - - +

4. -3-N0 2 ·C 6H4 + - + -5. -4-NO 2-C 6H4 ++ - + -6. -4-0H-C 6H4 + - + -7. -4-0CH3 -C 6H4 + - +

8. -2-0H-C 6H4 + - +

9. -3,4-CH 20 2 ·C 6H3 - + +

10. -3,4.di-OCH 3 ·C 6H3 - - + -1 1 • -3,S-di-Br-2-0H-C 6H

2 + - + ------------------------------------------------------------

ANTIMICROBIAL SUSCEPTIBILITY TESTING

They study has been carried out according to 40B

the method adopted by Bauker - Kirby et sl.

Disks have been prepared from Whatman filtar paper

No.1 with 4 mm. diameter and sterilized by heat

(160- for 30 min). The discs concentration of 100 ~g/

disks of all the compounds have been prepared using

acetone as solvent. nutrient agar. has been used for

growth of staphylococcue aureue and Escherichia cali.

The suspension of organisms has baen spread on nutrient

agar plate by sterilized catton swab and the discs

put by sterilized forcsp. Incubation has been carried

out et 31 0 for 24 hours, the diameter of growth

inhibition zone noted. Staphylococcus aureus

represents Gram (+)ve organism while Escherichia cali

represents Gram (-)ve organism.

ANTITUBERCULOSIS SUSCEPTIBILITY TESTING

The compounds teeted against standard etrsin

of Mycobactsrium tuberculosis H37Rv. The compounds

dissolved in dimethyl formamide and added in

323

Lowenstein - Jensen's medium so that the finel con­

centration of the compound ia 100 ~g/ml of medium.

The compounds added before inepisstion snd the chsmical

containing medium ia distributed in 1 ml emount screw

cep Mc.Cartuay's tubes. All the tubes are inspissatad

in slopping position at 80' for 45 minutss.

Ths medium has been inoculated eccording to

the recommendetion of W.H.D. The inoculum for the

susceptibility tests hea been prepared by edding

approximately 2 mg of growth from the primery •

culture on a loap to 0.5 mI. Sterile distilled water

in 8 7 ml screw cap tube togsther with six 3 mm

glass beads. The tUbs shaksn mechanicelly for 1 min

and a full 3 mm. loapful of the suspension inoculated

ante each slope. A drug free control slope has been

set up with each test end tubes incubated at 37'.

The results of the teste read after 4 weeks incubetion.

Reeults ~ discussion ~ ~ tested compounds

mentioned !n teblee ~ !2 ~

All compounds show mors or less entibacterial

activity end good dsal of antitubercular activity of

the compounds recorded in tables 15 to 25.

324

Compounds 2! ~ table 11

It has been observed that compounds are mors

activa againet S.aureue and less against E.coli. It

has elso been obaerved that thiazolidinone compounds

having 4-chloro-benzamido group with diffarant

substituents increase the antibactarial activity.

Compounds ~ 1h! tabla ~

The aeries of this tabla ia of interest becausa

of the presence of 4-amino-3,5-dibromo banzamido ring.

It hes bean observed that thiazolidinones having

aromatic nuclaus at position 4 show more activity

and alao introduction of halo molacula such as

Compounds 2! 1h! table !L

The activity raaulta are of importance becausa

of 4-thiazolidinones having -C.C- at position 5.

Generally compounds are more active when substitution

takes place in para position of phenyl ring with

comparison to meta and ortho position a.g., 4-0HC 6H4

,

4-0CH 3C6H4•

325

326

Compounds E! the table 18

The compounda racorded in table - 18 have been

found to show good inhibitory effect against S.aureue

and poor inhibitory effect against E.coli. It can b.

SBan that the substitution in position 4 in the phenyl

ring is more potent than the other substitution

i.e., -4-N0 2-C 6H4, -4-0CH 3.C 6H4 , -4-CH 3-C 6H4 ,

4-Cl-C 6H4 are mora activs. Along with that ws can

aay that benzhydrazino and nicotinimido ring attachsd

via -NH groups show more activity when it is

isonicotinic acid hydrazide~ All the compounds are

more or lSaa potent egsinst S~eureus~

Compounds s! !h! table 11

The activity results srs of importance beceuse

it has been reported thet e-triazine nucleus is more

potent when eecond end third substitution is carried

out with p-aubatitutsd aromatic nucleus. The rssult

alao show that compounds having highast activity whan

substitution ia at p_position, i.e. -4.CH3.C 6H4 and

-4-0CH3 .C 6H4 • It is also active against E.coli.

Coppounds £! ~ table ~

The compounds tsetad heve baen found to show

inhibitory effect against S.aureus end less inhibitory

effect against E.coli spscies, The substitution in

para position in the phenyl ring i8 more potent than

other substitution, e.g., p-N0 2-C 6H4 , P-OCH3 .C 6H4 ,

p-COOC2 HSC6H4 , p-CI-C 6H4 • Substitution in aromatic

ring also increases the activity in gensral.

Compounds 2t table ~

Ths series of thia table ia of interest because

of tha amino-nitrile group, secondly it has benzsmido

nucleus and -OH gr. at para position in benzamido

nucleus. It can ba seen that the aUbstitution in para

position in the phenyl ring ie mora ,otant than the

meta sUbstitution. Generally activity increasee

together with introduction of the substitusnts, e.g.

p-OH-C6H4' P-OCH3 .C 6H4 and P-N02-C 6H4 show higheat

activity.

Compounda 2! table l!

Theae compounds show poor rasults against

E.coli activity. But in S.sureus epscies, ectivity

ie reported to be more becauae of eryloxy acethydrazide

moiety, ~-aminonitrile group and chlorine etom at

position 2, e.g. 2-CI-C 6H4 , 4-CI-C 6H4 , 4-0CH 3C6H4 , etc.

327

328

Compounds £! table 12

Antibacterial activity of compounda racorded

in this tabla is having much importance. Because

eromatic nucleus possess thioureido moiaty at position 4

and at position 1 there is hydrazino group in addition

to thie there are two bromine atoms et 3 and 5 position

which ars responsible for antibacterial activity.

Generally substitution et para position of aromatic

nucleus increases activity such as 4-N0 2-C 6H4,

4-0H-C 6H4, 4-0CH3-C 6H4•

Comperieon ~ antitubercular activity ~~ compounds

.!!!. ta bl e s .!.h .!!' .11, 1.1, II .!!..!l.2 12.

For antitubercular activity we have undertaken

following sedtion :

i) Thiazolidinones

ii) Acetoscetanilida derivatives

iii) Thiourea derivative

Litvinchuk (loc.cit)14 reported antitubercular

activity with low toxicity associated with 8 few

derivatives of 2-imino-4-thiazolidinones. Repeated

therapeutic doaes have been found to possess enti­

tubercular activity comparable to streptomycin

409 phthivazid. Kapustyark atudied structure

tubarculostatic activity relationship of eome

143 4-thiazolidinones. Fujkikawa et al. (loc. cit)

reported chemotherapeutic sffectivensss sgainst

Mycobscterium tuberculosis. A few derivstives have

been found to inhibit the growth of human tubercle

329

b ecilli. '3 7R v strain in s concentration of 12.5 ..ug/ml.

Antitubercular activity of above section,

we have used HJ7Rv strain of Mycobacterium tuber~ulosis.

The introduction of substitusnts into hydrazino group

has led to much more interasting compounds. Thus

2-aryl-3-(4-chloro benzamido}-S-substituted_4_

thiazolidinonss have been found highly active and less

toxic. It has been observed thst all compounds

possess -CH.N- linkage, with thiszolidinone ring

system, basically thiazelidinone show goed anti­

tubercular activity. So 2-sryl-3-(4-emino-3,S_

dibromo benzamide}-5-substituted-4-thiazelidinones

and S-erylidene-3-(4-carbethoxyphsnyl}_2_(J,4-dimethoxy

phenyl}-4-thiazolidinonas are found to be more active

against H37Rv strain.

On comparison of the experimental date on

Ii:3 7 Rv strains of Mycobacterium tubarculosis of

NI-(~-naphthYll-N2-(aubstituted phenyll-2-aceto

auccinamide and 1 2 N -(p-alkoxy phenyll-N -(substituted

phenyll-2-aceto succinamide, it has been concluded

thet the substances taeted have comparable anti-

tubercular activity with the data availabla regard­

ing the activity of other known tuberculostatic agents.

Furthermore, it hae been found that the para substi-

tution in the benzene nucleus activates the compounds

The retardation of the growth of Mycobacterium

tuberculosis H37Rv etrain has been studied in

loweinstein Jensen egg medium (4 mIl containing two

fold dilutions of the teet compounds namely 1-L-4-(phenyl thioureido-benzoyllT-2-substitutedbenzal

hydrazinas at 370. From experimental data, it has

been observed that thioureas possess antitubarcular

activity et higher concentration. It has been noted

that hydrazone of hydrazide gives entitubercular

ectivity. Secondly p-substitution and halo atoms

like bromine in aromatic nucleus increeses activity.

330

•

SEC T ION : 2 11: ••••• _ 1m

PHYSICAL DATA (SPECTRA)

1 QCI"'<t~ ~~ .. ~ j I "'".01 , til '" " 'I it. o

•

PHYSICAL ~ (SPECTRA)

The IR epectra and UV spectra of some

representatives of several synthesised compounds have

been recorded and data interpreted. The compounds

show charscteristic frequencies of verious functional

groups. The UV spectra of severel synthesised

compounds have been recorded on Toshniwsl Spsctropho-

tometer RL0 2 , 240 to 400 nm range using ethanol (99.~)

as the eolvent. The IR spectra of severel synthesised

compounds have been recorded on Spektromom 2000.

Infrered epectrophotometer in nujol and KBr phase -1 between 5000 to 700 cm range •

•

DISCUSSION

fig. !i2.:...1.

The IR spectra of the thiezolidinones of the

type (I) show the characteristic absorption bend at

3 5 -1 1 -1 about 4 0 - 2500 em • 200 - 1600 em • The

peculiar bend et 1220 - 1025 (S) cm-1 is common in

these compounds which may be the ebsorption band

331

for the thiezolidinones ring system. The absorption

bend for (1) L-2-(2-nitrophenyl17-J-(4-chloro benzsmidol-

332

-4-thiezclidincne at 2990. 2BOO (e). 1680. 1640 (a).

15BO(II). 1460(E). 1400. 1365(w). 1350. 12BO(s). 1200(w).

1160. 1125(s). 1100(m). 1085(s). 1040(111). 1020. 1005(s),

9S0. 90S(s), 840(s), Bl0(w), 740, 720(101) c .. -'. (2) 2_

(4-Hydroxy phanyl)-3-(4-smino-3,5-dibromo benzemido)­

-5-carboxy methyl-4-thiezolidinone et 3250. 2900(s).

2490. 1725. 1610(e). 1520. 14BO(a). 13BO(e), 1320(m).

1 1 3 11 -1 .

2BO. 2 5. 70(w). 940 cm •

Ths spectral data curvee of these compounds

heve been ahown in Fig. No.1.

Fig. No.2

The infrared spsctrs of several represontative

thiazolidinones of the type (11) show absorption bends

which are characteristic for such compounds. The

absorption bands for 5-(3-nitrobanzylidene)-3-

(4-carbethoxy phanyl)-2-(3.4-dimethoxy phenyl)-4-

thiazolidinona at 3500. 34BO, 3100, 2900, 2800, 2350,

2290. 16BO. 1590. 1520(s). 14BO. 1460. 1440. 1355(s).

1310. 1280. 1180. 1140. 1120. 1030(s). 900. 820, 7BO. -1 750 CIII •

fig. ~

The IR spectra of acetoacetanl1ida of type (III)

show absorption bands which ara characteristic of

such compounds. The absorption band for

(1) 1 2 N -(0: -naphthyl)-N -(m-chloro phenyl) -2-

aceto succinsmide at I 3500. 2800(s). 2500(w). 2350.

1880,1840, .1805,1735,1700,1640.1620,1600,1580,

1500, 1460(s), 1425 (m), 1400(s), 1360, 1320, 1270(s),

1240(s), 1210, 1160, 1160(s), 1120(s), 10BO(s).

1035, 920, B80, 800, 7BO, 760 cm-'.

( 2) 1 2 N _(p_carbethoxy phenyl)-N -(o:-naphthyl)-

-2-aceto-succinamida at 3300, 2950. 1700(a), 1610(s),

1550, 1460, 1420, 135m, 1320, 12BO, 1200. 1180. 1140,

1110, 10BO, 1045, 1020, 1000(s), 950(s), 860(s), 760,

730, 700 cm-1•

The infrared apectra of several rspresentative

0: -amino nitrilas of the type (IV) show characteristic

ebsorption band et 2000 - 2250(w) cm- ' which is common

for such compound msy be the absorption band for the

-c - N- stretch systelll. The banda observed as follows

333

for (1) 4-hydroxy benzoyl-1,1-(cc- cyeno-3,4-

dihydroxy-benzyl emino)-hydrazine at 3000, 2850, 1690,

1660,

1130,

1500(s), 1440, 1360(s), 1280(s), 1200, _1

1060, 1030, 960, 900, 885, 810 Clll •

1180,

334

( 2) N '-( 2-chloro phenyloxyacetyl) _N2 - (a: -eyanobenzyl)-

hydrazine at 2850, 2300, 1650(s), 1560, 1480(s), 1440,

1400,1340, 1270(s). 1250, 1230(s), 1100, 1070, 960, -1 900, 840, 760(e), 700 em •

The IR spectrs of some representative thioureaa

of the type (V) show characteristic bands at about

1180-1030 cm- ' which ia peculiar and common in these

compounds may be the absorption band for thiourea

type system. The abeorption banda for (1) '-L-4-(Phenyl

thioureido-3,5-dibromo benzoyl17-2-(4-hydroxy benzal)­

hydrazine at 2800, 1580(e), 1530,1500,1440,1370,

1300(s), 1220, 1150(s), 1050(e), 960, 890, 1340(e), -1 720 em •

(2) 1-L-4-(Phenyl thioureido-3,5-dibromo benzoYll(-

2-(3-chloro benzal)-hydrazine at 3200, 3100. 2895(s),

1620(s), 1545(e), 1470(e), 1360(e), 1285.1235,1140,

1060(s), 960, 900.880,780,720 em-1•

335

The UV spectra of thiazolidinones of the

type (VI) show absorption maxima which ara characteristic

for such compounds. The absorption msxims for

(1) 2-(4-mathoxy phenyl)-S-carboxy methyl-3-(4-chloro

benzamido) -4 .. thiazolidinone at 270 nm, 300, and 370 nm.

(2) 2-(3,4-Dimethoxy phenyl)-3-(4-chloro benzamido)-

-5-methyl-4-thiazolidinona at 260 nm, 2BO~., 300 nm

and 320 nm.

lli. No.7

The UV spectra of severel representatives

acetoBcetanilidee of the series (VII) hava been

studied. The absorption maxima for (1) N1-(p-carbethoxy_

phenyl l_N2_(3_methyl phanyl)-2-eceto-succinamids at

270 nm, 320 nm, 360 nm and 370 nm.

(2) . 1 . 2 . N -(p-Carbmethoxy phenyl)-N -(p-carbethoxy

phenyl).2-aceto-succinamide at 270 nm, 310 nm, 340 nm

and 3BO nm.

The UV apactra of some representatives

•

a: -aminonitrile and thiourea of the type (VIII) and

(IX) have been displayed. The absorption maxima for

(1) 4-hydroxy-1,1-L-a: -cyano-4-hydroxy benzyl amini!­

hydrazine at 250 nm, 310 nm end 380 nm.

(2) 1.L-4-(phenyl thioureido-3.5-dibromo benzoyl17-

-2-benzal hydrazina at 310 nm and 370 nm.

As a result of the UV and IR spectral data,

the characteri.stic groups in the compounds synthesised

could be ascertained and thus confirms the etructure

336

of the compounds. Some spectroscopic data of

thiazolinone dsrivativee give UV spectra at 2BO - 300 nm

end acatoacatanilida derivatives at 265 - 380 nm whereas

~-amino nitrile derivatives et 250 - 385 nm.

WAVELENGTH J N MICRONS

7 10 t2 14 ----"~t-_--"+. __L _____ ..L. __ ---L __ ~ _ _I____L. _ ____l..:._~

337

D

D

o

J A IPltCTRA WAVE JOoI9EA cJ , > ,. ( •• I, '.~ ,

338

....

£' ct ex

8'

~ t UJ Q.. Ill'

~

6' • , 0

,/ "I

U 0

3--

,...

.... -_£ ~

0 0 oS) '" • ...

WAVE LENGTH IN M ~CRGN<; 339 3 ~ ___ L __ -----'L§ __ ~ J _-....L._--,-V _-""O'----_-',~l_ _ _'tt:z..

WHEN R=]-CI --( I)

WHEN R= 4-cooePs ~ --- ---( 2)

80

!lO

10

..... ----um.-----..lMJ .... --~z.,.tDJ----... ,tiJ-----.'II!IXJ---.,""ll!I1Ir--~17:1ro::;:---,,'6~w..---eas~--,7I'X1I:I

F1tJ-l VI,""E NUMBER eM'

340 - WAVELENGTH IN MICRONES

~ ____________ i-____ ~ __ ~~ ________ ~ _______ ~ --~--~~~--~-~~----,

(1) (z)

HOOC~NHNH~--O-OH OH

)

IR SPEC! RA WAVENUM3ER

WAVE LENGTH IN MICRONS 341 -.L __ _ ? __ ~___ L_!l _____ 1:.::,2_..:;,!O;:...

WHEN R=4-0H ----(1)

WHEN R=3-CI ( 2) R. jj~ Sjj~ "©-CH=NNHOC-Q_ NH-~-NH-V

By

60

]0

so

40

l{)

FIG-S l'l SPECl RA WAVE NUMBER

342

h----------~ .. ----- .• --------------, FIG-6

·f4

o d ·10

'0,

•

2

! ., " UV SPECTRA WAVE LENGTH IN nl1l-~ ......

343

.L, '

Lt.' 0' ~OCf) 0 '\O{'''O'''O "-'"''''"-<' ~ ,.'

l lA'

Il.

"' Of(

."

o· . • t •

.... It. ••

UV SPECTRA WAVE LENGTH IN Jl"I>'L--~ .....

o d

fIG-7!II,

(,0'

}O'

b

UV SPEClRA

344

. , .. Jh "' .. WAVE LEIoI3TH IN 'ILY'I, --.... -

\.1'}

.c.<;,

o • .. uv Sf'EClkA

345

ii)

HU--{ / \ >-(ilNHNH -cH- ! \-()H ! 0 0 "=..I ~N-

::---- -lL

WAVE Llf'.l)TH IN l\. -m .. --- -o o

"'"