73
BIOLOGICAL TESTING SPECTRQSCQPIC ANALYSIS ,.
BIOLOGICAL TESTING ~ SPECTRQSCQPIC ANALYSIS
,.
~~li.tl.l2.lll
BIOLOGICAL TESTING
247
TESTING ~ ~ITUBERCULAR ~ ANTIBACTERIAL ACTIVIT!
eL~ COMPOUNDS SYNTHESISED l! PARTS I.!! L 111
IN!RQPUCTIQN319.3B5
In 192B. a German scientiat. C.E. Ehrenberg uasd
the tsr. 'bacteriu.'. The bacteria ere a.ell .icroacopic
orgenia.e with reletively eimple end pri.itive farm of
the cellular orgenieation known ee 'Procaryotic'. The
staining reactione of becteria ere of greatsr importence
in their differentietion and identificetion. In 1SS.,
Daniah phySician Gram diacovered the strain. known es
Gram-stain. neDaly Gram positive and Gram negative.
The gram poaitive bacteria reaiet decolourieation with
acetone. alcohol and r •• eins .tained with methyl
violet in dark purple colour. In tha ea.e of Grem
negative bacterie. decolourisetion takes pleee.
Bacteria een be clasaified eceording to their
morphological charecterietica ae lower and higher.
The lower bacterie ere generally unicelluler etructure
and never in the form of a mycelium or sheethad
fila.ente e.g. COCCi, bscilli. bivrioue. Ipirille and
apirohatee. The higher becterie are fila.entoue
oroanilme. lome beino sheathed ond growing with
•
branching to form a myc.lium, having c.rtain cella
.pecialised for reproduction. The microorgania.a,
capable of producing di ••••• in anim.l or hum.n b.inV,
aro known a. pathogenic. Moat of the .icro-organi •• a
pr ••• nt on the akin and mucous .embranae ara non
pathogenic •.
B.ct.riology i. the acience thet deel. with the
study of bacteria. aacteria ia the microecopic
organi •• of plant kingdom, d.void of chlorophyll.
The th.r.p.utic. known b.for. the ti .. 0' E~lich .as
cinchona for m.leria, ip.ach fore.oabic dye.nt.ry and
mercury for treating eyphilie have ba.n ueed. Th.
di •••••• of protozoal end spiroch •• tel origin have bean
m.d. to re.pond to .ynth.tic ch •• Qth.r.p.utic .vente
during the fir.t two dec.d •• of '9th century. The
microbiologiet and clinical personnel ov.rlooked the
po •• ibl1ity th.t the bacterioetatic compounds would
inhibit rapid r.production of pathog.nic bacteria .nd
enable the leucocyt.a and other defenca mecheni •• of
the ho.t to cope with f.w static invadera.
Peul Ehrlich, father of chemotherapy u.ed the
term chBllotharapy to d •• crib. the cur a of an infectious
di.eae. without injury to the hoat known .a ch.moth.rap.utic
agsnt. and clasaified ecco~dlng to di ••••••• nd the
inf.ction., .uch as entibact.ri.l, .ntiprotozoal,
.ntivi~.l, .ntin.opl •• tic, .ntitubercular and antifungal
ag.nt ••
In this •• ction .. thod. u •• d for tin vitrot
•••••••• nt of antib.cteri.l agent. hes b.en de.cribad.
Antibacterial subatanc •• ~nd preparations ere cla •• ified
as disinfectants, antis.ptica and chemotherepeutic .gent ••
The t.rm disinfactant i. u.ed to elimineto o~ d.stroy
infection .nd should b. capable of killing e wid • •
range of bacteria. An antieeptic ia u •• d to control
or .liminato bact.riel infection. A ch.rr.otherap:!utic
agent i. an antibactarial .ubstanc. administered
.y.te.etically fo~ the tr.atment of inf.ction, .ey
be either bacteriostatic or bacteriocidal in it. action,
its main function ie to prevent the multiplication
Antibact.rial egents
They are an. type of chemother.peutic egent.
u.ed egeinot tha bacterial dise.see end divided into
two typ.e according to thair action on bacteria nom.ly
becterioetatic end bect.ricidel agente. An agant i.
I
l
I) r: () .... d
conaidered 'bactarioatatic' when it inhibite furthe~
growth or Multiplication of becteria and cleaaed ae
'bactaricidal' when it kille the becteri •• AntiMiarobi.l
egents ars the chemotherapeutic subatenc.e thet dostro¥
or inhibit the growth of Microorganieme in the living
tiseus. Antibiotics ere aubetencee produced b~ living
orgeni.M end ere .ufficient1~ noftootoxic to b. u •• d a.
anti.icrobiel .g.nt ••
EVALUATION gt ANTIDACTERIAl ACTIVITY
Varieties of '~vivo' .creening method. hee
been ueed to evaluete the entibacteriel ectivit,.
Testing in lIic8 hea baco," Btendard. the ."n.Uivit,
of bacteria to antimicrDbiel agenta ie tea ted b, the
B811e methods 8S in other form of
principIa. of which ere Bhown in
microbiological 386 figure •
I N C U I A T I o N
Result.
No v1eible org.nism
No visible growth
... , " . '
. ~ ,
.. ' . . _. . .. . - " ..
lilt
Via.t.bla growth
Bactericidal concantration
Bacteriostatic concentration
In vitro !!thods
'In vitro' teating is u.efUl for entibect.:ri.l
epect:rum deter_in.tion of • coapound end comparinll it
with other Silent.. Several type. of procedure. are in
use for a.aaying·th. potency of antibiotic prep.ration
fo:r ther.p.utic purpo... The _.thod. h.ve b.en
_edified and used for e.nsitivity tos' of unknown
org.nialll.
i) Serial dilution 1n broth3B1 -Seri.l dilutions of the drug being •••• y.d .1'.
made in unifo~m emounts of stenderd b~oth in cultu~e
tub.s. These era inoculat.d with e unifo~m nu.be~ of
cells to test organis.. After incubation, turbidity
(or .bs.nc.) is •• eBured by turbidimeter and
turbiditi.s (e.ount of growth) ar. comp.red with a
dilution ss~ies made in the aame wey but with entibiotic
r.fe.ence et.nd.~d of •••• ured potency.
ii) Str.ak enesy in .gor (lac. cit.,381
Gr.ded dilutione of the subet.nce to b. t.st.d
e~e plsced in e eeriee of petri diehe. in which i.
pou~.d .bout 10 al of melted end cooled eoer, contents
mi •• d with d~ug dilution. Aft.r ege~ he. herdened,
the pl.tes me~ked into savere1 sectore, eech of which
ie stre.k.d with different test oroenism.
iii) Diffueion tsete
Diffusion teete on solid medie have been adopted
by most of the lebor.to~ies wherein the entimic~obial
egent is held in e reservoir from which it diffuse.
t~ouOh egar medium to form diffusion gradient to
which tha microo~geni •• s, growin, in Or on the oger,
ere e.poeed. The eize of inhibition zone depends
upon tha factor that influanca tha diffuaion of the
antimicrobial agent as well ea the rata of growth of
tha ol'gania ...
a) Agar atrip diffusion!!!! !2I sensitivity
This is a siMpls technique which has been
originally usad by rleming. A strip of agar ia cut
frOM tha cantre at e place of suitab1s cultura madiuM.
Appropriata a .. aunt of sntiMicrabial agant ia addad to
lIoltan agar and pipattad into the guttar in the ~dium
and the DUl'feca of the agcr is inocUlated by etr@~king
Clulture,a to ba teeted.
b) Replica platB method to ~ bacteriostatic
~ bectaricida1 action 3BB
A zane of inhibition of growth around a diek
.ay indicata that tha antimicrobial agant is aither
bactariaCidal 01' bactariostatic. Tho presence 01'
abesnca of living o~ganisme within tha zone of
apparent caMp1ata inhibition of growth an diffusion
p1atas hava baan ahown by replica plate method.
c) Diffusion t.sta ~ filte~ paper diake !2! d.t.~Mining .ansitivity 319
This conatitute e i.pla and ~elieble technique
i.p~egnating •• all disk of standard filte~ peper with
given amount of antibiotic placing the. on plates of
cultu~ Medium inoculatad with the organia. to be
teated. Afte~ inoculation the degree of eeneitivit~
by maasuring the aasily visible era._ of inhibition of
growth which hae bean p~oduced b~ the diffusion of
entibiotic frOM tha disk into the surrounding is
DISCUSSIQN
W. have undertaken ths apaciee StaphYlococcua
eureu. and ~E~e_c_h_e_~~i_c_h_i_a coli for the antibacterial
ecreening of the cOMpounde synthesieed in Parta If
II L III.
1, Stephylococcue eureus - re.ily I MicFococceceee
In 1811, Koch obee~ved .tcrococcua like
organiam in pUSS Poeteur (1880) cultivated theee cocci
in liquid ~edia. ogaoton (1881) found it prasent
in pus of acute chromic absceeses end found it
pathoganic fo~ .ica and Quinaa piga.
They ere &~am (+) cocci, ovoid or spheroidal,
nan-matila. arranged in group of cluate~a. grow on
nutriant agar end produce colonies, which ara goldan
yellow. white or lemon yellow in colour, aerobaa or
feculetive aneerobee, biochemical activities end
hae~lytic power are varieble. pathogsnic at~aina
'produca coagula.a, far.ant glucoaa, lecto.a, mannitol
with production of ecid. liquefy gelation and produce
pus in the leeion.
Genua I Staphylococcus
Staphylococcus i. diffarentiatad fro •• icro
coccus, enother genus of the .ame family, by the
ability to utilise glucosa, .annitol a~ pyruvate
anacrcbically. Staphylococci ara found on the akin
or mucous membranee of tha animal body, especially of
tha no •• and mouth. where they often occur in largs
numbera even under nor •• l conditions.
Speciee I StaphylocoCCUs aureue
The individual cells ere 0.8 to 0.9 U in
die.eter. Thel ere sphericel, non-.. tlle, non
capsuleted, non-sporing, stein with ordinarl aniline
dyes and Gram (+)ve, typically arranged in groups.
The.e ere e.robe. or facultative enaerobes end grow
eoeilyon nutrient agar. The optimum te.perature for
the growth is 37- but the range of temperature veries
frOM '0- to .0- optiMUM pH is 7 •• to 7.6.
2) Eecherichia coli - ramily I Enterobacteriaceee
They ere Gre. (-)ve rode, motle with peritrichate
flegelle, or non-motile. They do not for. sporee end
are primerily environmantel seprophytas end ecevengers,
found in the inteetinal trect of man or lower animels.
GenUE I Eecherichia
This genue comprisee Eacherichia ~ and
eeverel variente, and ie af particular interoat aince
they occur comMonll in the normal intestinal tract of
man and animals. Eecherichia coli i. the ... t -di.tinctively fecal opecie ••
') r:: 7 .... \)
Speciee • Escherichie E2!!
Eecherichie in 1885 diecoversd E.cherichi.
coli from the f •• ce. of the new born who ehowed the -org.nisms in the intestins within,3 d.ys .fter birth.
The ••• re Gr.~ -(-lve rode 2 to • ~, commonly eean in
coccob.cillery forM .nd r.re1y in fil.mentoue form ••
E.coli ere gener.lly non-pathog.nic .nd incriMin.t.d
•• p.thogen., becsu.e eometime •• trains have been found
to bladder, .ppendix, .eningiti., pneoMOnie and other
infectione end this species ie a recognieed p.thogene
in the vet.rinary field.
IN VITRQ TESTING
Becterioet.tic .ctivity c.n be d.termined on
eolid or liquid medi., e.ch depende on •••••• in9 by
.0 •• meen. the extont of inhibition of growth. We
heve adopted 'The diec or Teblet method' for the
sen.itivity teeting.
After the report of the 'Internation.l 390 collaborative atudy which we. involved with
258
investig.ting 'the dilc test', thl method recommended
hal be.n adopt.d in Sweed.n. In U.S.A. the modified
KirbY_B.u.r3,t t.chnique he. been edopted a. the
officiel .ethod by the food .nd druge ad.inietretion.
The main etimulu. for atand.rdi •• tion in U.K. h •• co.e
from r.commendetion of the ule of the controlled
.ingle diec method3'2.
In thie method nutri.nt .gar of appropri.t.
compoeition i. h •• vily inoculated with the de.ired
org.ni.m .11 ov.r the eurf.c. of the eolidifi.d .gar
or mix.d with .g.r, while .till fluid, b.fore pouring
the pl.te. If an antibiotic .olution of unknown potancy
i. b.ing •••• y.d. the organism us.d i. atock .train of
known •• n.itivity to .t.nd.rd cl ••••• of th.t .ntibiotic.
Me •• ur.d .tr.ngth. of the .ntibiotic eolution to be
teet.d ere .pplied to tha inoculated .gar in diec or
uniform thickn.s. of sterile filter peper. end
pleced on the surface of the plet. b.fore incubating.
Saveral d.vic •• er. evaileble for epplic.tion of the
di.cs. The .ntibiotic diffue •• outward from eech
diec end inhibit. growth in the .gar eround it. The
width of the zan. indicat.e, the eeneitivity of the
organism to the agent or .gente being t.et.d through
the pres.nc. or ab.enc. of a zone and i. of gr.eter
.ignificence.
() f:)'9 ._l
As bacteriostatica ia a dynamic aituation
dependant not on tha sgent concerned but also a
number of other fectore particularly tim. of contact,
te.perature and nutritional environment in which the
organisma find the.selves as well aa tha typa and
numbar of celle involvad.
factors ~uencinq inhibition ~ si •• s
i) Ingrsdisnta s! culture madia
Many substances era pre.ent in culture madia
which may affect tha zone of inhibition, common
ingrediant •• uch a. peptone, tryptona, ye •• t eKtract
and agar may vary in mineral content and .. y influence
tha ectivity of ao.a antibiotic. It is well known
that Ca, Mg and fe affact aenaitivity zonee produced
by tetracycline a and gantamycin.
11) ~C~h;o:i:ce: g! medium
Coneiatent end reproducible re.ulta are obtainad
in media preparad e.pacially for aenaitivity t.ating.
The plata. mu.t be poured flat with an even depth,
very thin plate. ara unaatisfectory.
iii) Effect s! 2H
The activit~ of eminogl~coside& i& enhanced
in alkaline media. reduced in acidic madie. the
reveree i8 .hown by t.trecyclin.
h) Size of inoculum --Although many entibiotics sre not msrkedly
effect.d by lerge number orgeni... ell inhibition
zone. are diminished by heav~ inocula. The ideal
inoculum ie the ona which givas an even dense growth
without being confluent. Overnight broth culture. of
organiame end Buitebl. euspeneions from Golid medie
can be diluted accurately to give optimum inoculator
for eensitivity teeting. In prectice ••• ti.f.ctor~
results can be echieved by teking a loopful of a well
grown culture or a euitably made auepen.lon of
organism and spreading It with dry ateriie eweb393•
The performence s! diffueion technique
i) Composition e! nutrient eger
Peptone 2.0 9' NeCl 2.0 gJ Meat extract 3.2 la
281
Agar-agar powder B.O gl PH 7 •• , distillad water 1000 .1.
ii) Strangth 2! .ntibiotic
Until very r.cently, there hea b.an little or
no agreement reg.rding the strength of antibiotic
diece for uee 'in vitro' e.neitivity te.ta.
iii) Storege ~ di.c.
Di.cs should alwsys be kept cool and dry and
wh.n .pplied to the .. diu •• hould b. pr •••• d firMly
to eneure proper cont.ct and .ven diffu.ion diec mey
be epplied to culture madi. v.ry conveni.nt with fino
point.d forc.p, di •• ecting nesdl •• or hypod.rmic
needls. of conv.nient .ize.
iv) Incub.tion !!!!
It should ideally be the miniMum required for
the normal growth of the organism. Prolonged incubation
which heve been pr.vented frOM reproducing but which
are not killed. It ie claar thet ouoh methode ara
euiteble only for organism that reproduce repidly,
282
and anything in the ~ediu. or leck of any optimum
phyeical condition reducaD the growth, will produce
artificelly large zonee of inhibition.
v) Controle
ror the correct interpretetion of resulte and
recognition of eny eource of error in disc diffueion
eensitivity teeta the correct uee of control organie.
is eseentiel. If ~ultiple disce, neceeeitating tho
u.a of whale platee era ueed, cant~ol pletee ahould
be eet up for avery drug and medium used. ror
routing daily uee, the 0~g8ni.ms are most conveniently
kept in a refrigeretor et ,. on eterile throet ewebe,
e jar full of such awebe can be impregneted at one
ti .. ee thay keep well et leeet e weak.
TESTING 2L ANTITUEERCULAA ACTIVITY CHE~THERAPY Dt
TUBERCULDSIS394- 4'.
Although chemotherepy of tuberc~lo&is hes be.n
precti •• d in aome for~ or the other, for over two
thoueand yeare, it hea b.en a clinicel re.lity for
the last three decades only. Primativa treetment
fo~ tuberculoaia such ee injection of tannin or
263
.nim.1 charco.1, b1.ck mud batho, 1argD doi1r
consumption of .pDen and •• eBiv. gold •• 1t injection.
parBiated for manr ¥eer •• all th ••• war. r.ported br
Koch •• inf.ctiva ag.in.t guin •• pig tub.rcu10.is.
Th.re .r. manr ob.t.c1e. that confront the ehemoth.ropr
of tubarcu10aia. Some of them era -
i) Tubercle beci11uB eurvivaB for a number of rBere
in • hoBt without inducing .nd provoc.tion.
ii) It is • slow growing org.niB. which doeo not
81icit a .harp and maaeive reaction from the
hoet.
iii) Tubercula_is iu a communic.b1e disa.a. and h ••
• xiuted aa • conutent streaB on human surviv.1.
iv) ThB di.eas. c.uu. axtanuiv. ti.sue de.truction
.nd live viru1ant bacilli g.t protection in
the c.vitiee, debri •• nd n.crotic ti.au •• ,
where ther remein protected to che.other.peutic
att.ck.
It w.e therefore con.id.red naceo •• rr to .volve
.n ideal, more effective and e.fer che.otherepeutic
drug treetment for the d •• dlr infectiou. die •••••
284
The drug ao evolved ehould be alow in producing the
raaietant atrain and relativaly fraa fro~ undeairable
aide effacta.
Tubercla bacilli known aa Bacillua Cal.ette
&uarin or ECG4BO has baan daveloped. [ven though
to data there ia no conclueive evidence thet vaccinotion
with BCG cDnfere ecquired reaietance on hu •• n being,
but it ia conaidered p.obable that it ia of aome
banefit.
Theaearch for aubatance to entagonia. the
reepiratory stimulant. of M. tuberculoaia by aalicylic 415 acid led to the diacove:ry of P.A.S. • Howaver, the
letter does not .ffact tha :raapiration or antagoniaa
Dalicylic acid, which is fact ia not :raapiratory
aubatrata in the Mycobacterium. Tha retional parauit
of acraaning haa ao •• time bean faithful aa a reeult of
common practice of blind acreening of chemical
inter.edi.tea.
265
Metho~ !! teetinq !! tub.~culoetetic drugs
e) In vitro !£!!
The in vitro t~et principelly ccnsiets 01
se.ding tubercle becilli into e eynthetio culture
~edium. Thie conoi.ts of gr2ded concentration of the
subetance to be tB.ted, renging fro~ zero concentration.
for contzvl purpo.e. to conc.ntration approaching the
toxic dose.
b) In vivo t.st
Mice, guina. pig or rabbits ere inoculated with
bovine tubercle bacilli. Some of the infected eni .. 1e
treated orally or par.ntorally at various dose lev.le
with the aubetence to be t.st.d while othars untreated
animale ere maintained ae controle. A widely used
technique following pathology of the di ••••• pzvc •••
by .acrificing traated and untreeted infected eniDal.
in which the infection epread to various organs. A
series of druga affactive egein.t axp.rimentel and
clinical tuberculo.i. hes been dav.loped durin, the
last thirty yeers beginning with 4,4'-diomino diphenyl
eUlphone. atreptomycin and ethambutol. Exheuetive
reviewa by different authore heve boon publiehad on 4"_41' thie topic of cheaotherapy of tuberculoeia •
The -lIIDdern work could be considered to have
started with the obeervation of Riet, Rich end rallis,
their finding that the larga dose of sulph.nilamidea
produced a beneficiel effect on ths davelopmant of
experimental tuberculoeie, we confirmed by Suttle
and Parieh42D-422.
In this aection the perticulare of in vitro
testing of the com,ounds deacribed 1n Parte I to III
for their antitubercular a'nd entibacterial ectivitJ,f have
baen recorded in tablae - 15 to 25.
A~!TlMICRD1HAL SUSCEPTIBJLIU TESIINi
The stud1 hss been carried out according to the
mathod adoptsd by aauar-Kirby at al. 73 Discs praparsd
trom whatman til tar papel' no. 1 with 4 •• diamatel'
which heve been sterilized by heat (160 1 tor 30 min).
Tha concantration of compound in diac is 200 ~g/diGks.
which havs baen prapal'ad using acatona/DMr aa a aolvant.
Nutriant agar has been uaad tor growth of Staphylococcua
auraua and E.coli. Tha auapenaion af the organiam
hac baan apread on nutl'ient agar plata b1 stel'ililad
cotton swab and discs put by sterilized 'orcap.
InCUbation has baan carriad out at 37- for 24 hl'a. tho
diamatel' at gl'owth inhibition Zona noted. S. 8Ul'8US
rspreeenta graM poaitiva organiam and r.coli reprosenta
gram nagativs organia ••
Antitubarculoais ausceptibility tasting
The compound tested against standard strain
of Mycobacterium tuberculosis H3yRv' The compounda
diSSolved in D~F/acetona and added in Lowanstein
Janao'a r.edium so thet final concentration of tha
compound in 10 pg/ml and 100 pg/ml of medium.
2G8
The compounda .dded before inepiae.tion, the
chemicel containing medium is distributed in 7 ml
amount ia eere. capped McCortueyla tubes. All the
tubes inspisaated in elopping poaition at ao' for .5 minutea. The medium io inoculeted according to the
423 reco •• endation of W.H.O.
The inoeulu~ for the susceptibility teeta hee
been prepered by edding approximately 2.0 mg of growth
from tho prim.ry cultUre on B loop to 5 .1 eterile
diatilled water in a 7.0 ml screw cepped tuba together
with 6x3 am gl.aa beede. The tubea ahoken mechanically
for one minute end e full 3 .m loopful of auepeneion
inoculated onto each slope. Duplicete alopee fro.
e.ch compound inoculated end the drug free control
slope aleo aat up with each teat. The tubea incubated
at 37' and tho rasulte of the teeta raad after 6 weeke.
We have etudiod the activity at two different concen
tratione, t.e. 10 ~g/.l and 100 ~g/ml.
269
TESTING ~ ANTIBACTERIAL AND ANTITUBERCULAR ACTIVIT!
~ ANTIBACTERIAL ACTIVIT! ~ COMPOUNPS
+ Indicetea inhibition aona occurs
++ Indicates aon. diemeter mora than 15 mm.
_ Indicates no inhibitory aona around the diRe.
tQa ANTITUBERCULAR ACTIVITY ~ COMPOUNPS
i) -ii) +
Indicata. no inhibitory .ff.ct, i.e. co.pound
ie not affactive.
Indicates inhibitory effect, i.a. compound
is affactive.
iil) Solvent control end plein control indicatee tha
growth of bacterie end has not bean recorded in
the 'tables.
TABLE I II ANIIPACIERIAL llN1 ANTITUBERCULAR ACTIVIT! or THE CQMPOUNDS
Q!. l!!l TyPE (1)
I
HO
(I)
------------.---------.--------------------------------------Sr. f~o •
R
Antibacterial . !::!~!!I----__ -S.eureue E.coli
Antitubercular acti-~1_i!2~f~~~a!!!1_
to 100
-------------------------------------------------------------1. -c 6HS - .. - -Z. -CH2C6"5 - - - +
3. -J.O.e,H5 .. + + +
•• -4.Cl.C,H. + + - +
5. -2.e1.C,H4 - - - -6. -2.0CH3·C,H .. - - - -T. -J.OCH3·C,H. + - - -B. -"~OCH3·C6H .. + - - -,. -Z.CH3·e,H. - - + +
to. -4.CH3·',H .. - + - +
H. -2,0'2"S·C,H .. - .. .. -12. -4.0C 2HS·C,H4 - + .. -13. -",COO'2 H5.1: 6H• - - - .. ---------------------.--.--.---------------------------------
~271
TABLE • .!.! ANTIBACTERIAL AWl. AtJTIIl/BERCULeR ACTIVITy QL I!!t COMPOUNpS
gr !,!:!!;. ll!l (11) •
Br ~I
R
Cl o S I
R (II)
-------------------------------------------------------------Antibactorial Antitubuculu Sir. R R' activity activity No. iconcn • .1.18/mu
~-------r--DIi --rn---T n-.aureua .CD -------------------------------------------------------------, . -CH-CH-C ,H5 H - - - -2. -2.0H.C,H4 H - - + +
3. -'.N0 2·C,H4 H + - - -•• -l.N02 ·C,H • H - - - -s. -'.OCHl·C,H. H + + - -Ii. -3,4-CH2(0)2C,H3 H - - - -T. -'.a1'.'.20H.C'"3 H ++ + - +
8. -'.Cl.C ,H. H + + - -9. -2.0H.C,". eHt=OOH - - - -10. -'.OH.C,H4 eH2COOH - - - -1.1. -'.0 H. 30e H3C ,H3 CH2CODH - - - -12. -'.N0 2·C,H. C"2COOH - + - -13. -3.N02·C,". C"2COOH - - - -1 •• -'.DCH3oC,H. CH2CODH - - - + 15. -3,4-CH2(0)2C,H3 CH2eOOH + - - --------------------------------------------------------------
') .... 2· ..... ( ....
TABLE I 11 ANTIBACTERIAL !!l ANTITUBERCULAR ACTIVITY Qt l]t Cp~POUNDS
Q!. THE ~ (1ll)
(II I)
-------------------------------------------------------------51'. No.
Antibactel'ia1 AntitubeZ'Cu1al' actl-!;!!!~l_______ ~~I_l~~~~J~!l S.eureu3 E.co1i . 10 100
-------------------------------------------------------------1. • •• CH3·C,". + + + +
2. -C'"5 + - - +
3. -tH-tH.C,"S - - - +
•• - •• OH.C,". ++ + + +
5. • •• N0 2 ·C,H4 + - - +
6. -3.N0 2·C,". + - - -1. -2.0".C,". + + + +
I. -3'''I:H2 (0)21: 6H3 • - - -9. -"0",301:"3·1: 6"3 - - + + 1 O. -3 ,.-di-OC"3'C ,H3 • - • +
11. -5-II',2-0H.1: 6"3 ++ + + +
12. -5-11'-3, .. dl-0I:H3 ++ + - +
13. -2-C1.C,H. - - + --------------------------------------------------------------
TIl IlL E I !.!!. ANTI!1ACTERtAI. ACTIVITY Qf..!.!:!.l COMPOUNDS Q!. .I!!.t Ilfl (a)
<O~Br O~CHNH-R
I CN
(Iv)
---------------------------------------------------------s~. No. R
__ ~~!!~~;!!!!~!_~!l!!!~l ___ S.eureuD E.eoll
---------------------------------------------------------1 • -2-0CH3·C,". - -2. .4-oC~.C,H. + + "
3. -2-CH 3·C,H. - -•• -4-CH3oC,". + -s. .2-OC2 "5. C 6H• - •
5. .4-C1.C 6". + +
1. -C"2· C,HS - •
6. • •• OC2 H5·C 6H4 - + ,. -e,"S - -10. -3-C"3 oC ,H. - -11. -2-C1.C 6H4 - -12. -3-oCH3 ·C,H. + -13. -3-C1.C 6H. - +
---------------------------------------------------------
274
TABLE • 11 ANTIBACTERI6~ 6CTIVITY Q( !ttt COMPOUNDS Q! ~ TYPF. (~)
Br
HO
Br
(V)
CONHNHCH-R I CN
--------.-------------------------------------------------51'. No. R
~~!!~!~!~!!!_!=!:~!!r ____ _ 5.aureua E.cali
--------------------------------------------------.-------1 • -C 6H5 - -2. -t H-CH-t 6HS + -3. -4-oCH3 ·C,H4 - .+
4. -4.0HoC,H4 + . ++
5. -2.0H.C 6H4 - -6. -4.Cl.C,H. + .+
1. -2.Cl.C6H. - -o. -4.N~ .C,H4 + +
9. -3.N0 2·C6H. - -10. -3.4-CH2(0)2oC 6H4 - -11. -4.0H. 3OCH3 .C ,H3 - +
12. -3.4-di-OCH3 oC 6H3 - -13. -3. 5-dlBr.2.0H.C ,HZ + +
--------.-------------------------------------------------
TABLE t !!!. ANTIBACTERIAL ACTIVITY ~ ~ COMPOUNDS ~ ~ Ilfl (Xl)
(VI)
-------------------------------------------------------s~. No. R
~!!~!:!!:!!!_~:!!~!!l ____ _ S.eureus E.coli
-------------------------------------------------1. -C,HS - -2. -CHtoCH.C 6HS - -3. -4.0CH3 ·C,HA - +
4. -4.0 H. C ,HA - +
5. -2.0H.C 6HA + + ,. -4.Cl.C 6HA - -T. -2.Cl.C,HA + -B. -A.N0 2·C,HA - +
9. -3.1\'02· C 6H4 - -10. -3.4-CHZ(O)2C,H3 - -11. -4-PH.30CH3·C 6H3 + + 12. -3, 4-d1-PC H3 • C ,H3 - -13. -S-Br.3,4-di-OCH3·C,H2 - -1A. -S-B~. 2-0H.C ,H3 - ---------------------------------------.------------------
T ABLE I II ANTIBACTERIAL ACTIVITY ~ ~ COMPpUNDS ~ ~ lIe! (~
I
HO CHNH -R I CN
(VII) .
--------------------------------------------------------Sr. No. R
~~!!~!!~!!!!!_~2!!!!!1._ S.aurau8 E.coli
-------------------------------------------------, . -C,H!! - -2. -2.0CH3·t,H4 - -3. -3.0CH3·C,H. + -•• -4 .OCH 3• C ,H4 ++ +
5. -2.CH3·C,H4 - -15. -3.C H3• C6H. - +
7. -4.CH 3·C 15H. + -8. -2.0C2 HS·C I5H• - -II. • •• OCZH,.C,H. + + , O. _z. Cl.t iSH. - -, , . .l.Cl.C,H. - +
'2. - •• Br.C ,H. - +
'3. -CHZ·C,HS - -'4. - •• Cl.C,H. ++ ++
--------------------_._----------------------------------
TABLE • 22 -ANTIBACTERIAL AND ANTITUBERCULAR ACTIVITY ~ ~ COMPOUNDS
ru:. m. !l!!I. (illl)
N-N
I OJlSCH2CONH~R
(YIII)
----------------------------------------------.-------------. S". No. R
Ant1bacta,,1al Ant1tubal'cula" activit~ activity _________________ t22~~2~_~2~!!l. __ S.au"aua f.coli 10 100
-------------------------------------------------------------1 • -C,HS - + + +
2. -2-Cl.C,H. + - + +
3. -3-Cl.C,H. + +. - +
4. .4-Cl.C,H. + + + +
5. -2.CH3 ·C,H. • • • -,. -3.C H3 .C ,H. + + - +
7. - •• CH3 ·C,H. + + + +
S. • •• OCH 3 ,C(jH. + + + +
9. -2.0C 2HS·C,H. • - - -10. -3.0CH3·C,H. + + • +
--------------------------------------------------------------
')-8 .~ , TABLE I II
ANTIBACTERIAL Ml! ANTITUBERCULAR eCTIVITY J2!.. THE COMPOUNDS
n!. THE ~ (a)
N-N s N~"\ I II II
~NHNHCNH-R - 0
(IX)
.-----------------.------------------------------------------Antibacterial Antituberculer Sr. R activitr activity No. ________________ i!2~!~_~~!!!1 ___
5.eureue E.coli 10 1 DO
-------------------------------------------------------------1 • -2.CH3·C 6H4 + - • -2. -3.CH3·C 6H4 - - - -3. -4.C "3. C 6"4 - + - -4. -2.Cl.C 6H4 + ++ + +
5. -3.C1.C 6"4 + + • +
I. -4.Cl.C 6". ++ + + +
T. -2.0CH3·C 6"4 + • - -8. -3 .OC"3. C,H. - - - -t. -4.0C"3· C,H. + + - -10. .4.0C2 H5·C 6H. + + - -11. -CHz·C,Hs + + - -12. -C 15"5 - - - --------------------------------------------------------------
TABLE • !! ANTIBACTERIAL AND ANTITUBERCULAR ACTIVITY gr THE COMPOUNDS
L1!. l.!1t I.l.el. (IS'>
N-N S I II II
O/"-SCH2CONHNHCNH - R
(X)
--------------------------------------------------------------Antibllctllrial Antitubarcular Sr. R activity . activity No. .________________ i~~~~~~~2!!!l ____
5.eureue f.col! 10 100
-------------------.-----------------------------------------, . -t,"S - - - -2. -4.tH 3·C 6H. - + - -3. -3.CH3·C,". + - + +
4. -2.CH3 ·C,H. - - - -5. -4.Cl.C,H. ++ + + +
6. -3.Cl.C6". + + - +
7. -2.Cl.C6H• + - - -8. -2.0C"3· C'"4 - + - -,. -3.0CH3 ·C,H4 + - - -10. -4.0C H3.t,H4 ++ + + +
11. -4.0C2"S·C,H. ++ + - + , 2. -tH2·t,H. - - - --------------------------------------------------------------
•
::HO
TABLE I II ANTIBACTERIAL ~ A~~tTUBERCULAR ACTIVIII g[ THE COMPOUNDS
Q!. l.!K n!!. (U,)
(XI)
--------------------------------------------------------------Antibacterial Anti tube1'cu1 8ZO Sr. R, R2
IIctivity IIctivity No. l~2~!!~i!--!1 ___ .-------------
S.eU1'IlU8 E.coli 10 tOO ----------.---------------------------------------------------1. tH3 tH3 + + + +
2. t2 H5 C2HS - + + +
3. t ,H/5 CH, + + + +
4. t,H6 C6H, + + - +
5. CH 2CH2OH CH2tH2OH - - - -6. t,H6 C2H5 - - + +
T. Ind010 + + - +
B. JI'Io1'pho1ino - - - -,. Piperidino + - + +
10. P),1'1'010 - + + +
11. P he nothillzino + + - + 12. Tet1'IIch101'ophenothiazino + + + +
--------------------------------------------------------------
:~ 8.1
DISCUSSION Q( !]! BIOLOGICAL TESTING RESULTS
Resulte !! table. 1!
The biologicel atudi.e of 2-(4-hydroMy.Seiodo
.3.methoMY phenyl).3.eryl.4.thiazolidinone reveeled
tho compounds did not ahow any noteworthy entibacterial
activity Bince Moat of the compound a ere inactiva
with two or thraa aMcaptiona which ahow alight ectivity.
The aimilar typa of concluaion .ay ba drawn f:lO •.
their antitubercular activity r.aults. Tha co.pounda
with chloro and mathyl groupe Gubetitution to 3-phenyl
ring hava good activity_
Resulta of tsbla - '6 .. -
The activity date of 2-eryl-3-(4-broQO.l.
chloro phenyl)-S.aubatituted.4-thielolidinone show
that mora compounda ara active ageinet S.aureue than
E.coli but thay aro modarately active. Moat of tha
co.pounda hava been found inactive ageinst ~7Rv at.ain
at low concantration and at higher concentration, aoma
compounde inhibit the growth of the bacillua.
Raaulta 2! table. 11
The antibacterial and antitubarcular activit~
of 2.eryl-3-C4-chloro phano.~ acat8mido)-5-carbD.~
.ath~l-"thiazolidinone hava baen recordad in thin
tabl.. They found to poasaae antibectarial activity
against S.auraua and E.coli to ao.a a.tant ahowing
,,' u ') .... () ......
varr •• all inhibition zonee. Some compoundo hava baan
found to show antitubercular activit~ against H3TRv
atrain of M~ tubarculo.h, lID.tl~ at highar concantration.
~R_o_a_u_l_t_s 2! table • 11
Thia tabla includ.a the activit~ reaults of
=-(aubatituted or~l em1no).2-bromo~4,5-.eth~lenedio.~
phen~l ecetonitrilea. So .. of the cOMpounds show
antibactarial activit~ against S.eureua and E.coli.
The compounds with ortho and mota, substitution found
more active.
Raaulta !!2! tebla - !1
This tabla indicataa tha biologicel scraaning
rasults of =-(3,S-dibromo-4-hydro.~ benzorl hydrazino)
Dubstituted eryl acetonitrilas. Tha m.n~ compounds
are farelv active againat E.coli. Soma of them ara
also active again.t S.aureu.. Tha compound. having
pera substitution to aryl ring shows good activity.
Roeult. 2! table - 19
oc-(4-Mathyl ph.noxy ecetyl hydrazino).aryl
acetonitrilea ar. t.st.d for their bactaricidal
property. Moat of the compounds found inactiv. aoain.t
S.eureua end E.coli. They hove baen elso testad for
antitub.rcular activity but the raaulta era noteworthy
becausa almoat all tha compounds ara inactiva.
Reaulta of tabla - 21 - -The a.riaa of this tabla ia of inter.st becausa
of the amino nitrila. group having hydroxy and broMO
group in ph~nyl zing. Moot of tha compound. era
equ.lly la.a or mora activa againat both otrsin.
Compounds having 4-.ethoxYt 4-chlolo t 4-.ethyl moiaty
ahow oignificant activity.
Rosulta of tabla - 22 - -'10m ths expari •• ntal data 1t waa ob.erved that
284
all the compounds ahow hiQh activity againat bacteria
S,"auroue and E.coli. Antitubercular activity of
compounds heD been determined uaing HlyRy atrain of
Mycobacterium tubarculaaia. All the compounds ehow a
little to atrong entitubarcu1ar activity. It haa be.n
observed that aubatitution is para and mata poeition
in tha phenyl ring ia more potent than tha ortho
aubaUtution.
Reaulte of table - 23 - -All the cDmpDunda ohow activity Dgainat S.sureu ••
out of tham. o-aniayl. benzyl and p-chlorophenyl
derivDtiva. are more active againat S.aureua, whila
.-chlorophenyl and benzyl darivativea ara mora active
egeinet t.coli e. comparad ta athar compounda. Only
chloro Gubatituted compound ahow antitubarcular activity.
Beeulte 2! !2! teble - ~
Moat of the compoundu wera found to axhibit
activity againet 5.auraua and E.coli. Organia •• para
position of phenyl ia MOra activa than ortho and meta
poaition. It haa becn alao observed that titla compounde
ara mora active againet S.eureue .then E.coli. The
ecra.ning resulte of the 'in vitro' antitubercular
activity of compounde shaw that all compounds .re
inactiv. at low concentration. but sarno active at
highar concentration (100 ug/.l).
It haa been obeerved that .aet of the compounds
ehow aignificant activity against 5.eureus and E.cali.
The compounds found more active cgainst the etrain
of M. tuberculoeie. Compounde heving indolyl. methyl
ani11nyl and phonathiezinyl ~~i.ty shaw higheat
activity againet H37Rv .train.
Generel diacuseion SD ~ctivitY2! 4-thiezolidinonee
The 4-thiazolidinanae. etudiad have found to
poeaess antibacterial activity but most of them are
inactive to moderately active. Some compounds shaw
goad antitubercular activity. No atructure activity
correlation cen be aatobliehad fram the resulte. The
aryl or aryloxy acetyl hydrazino derivatives also have
ne influence an the activity. in the etudy of the
Gene~el diecuseion ectivity £! ~-eminonitrilee ~ releted compounds
The =-aminonitrilea derivatives have got no
interesting antibecteriel activitN except few of
them. The derivativea from 5-iodovenilline have more
number of compounde which are Bctive. No much effect
of cyeno group ha. been found the ectivitN.
Generel diecu8sion!n ~ivity!! 1,3,4-oxBd!ezole
derivetives
The 1,3,4-oxBdiezole derivative. studied ehowed
feirlN good antituberculer activitN. Compounds show
a little to strong antibacterial activity. PyridNl
ring increaeee the becteriel ectivitN. nut no cleer
etructure activity reletion h •• been concluded. It
ie cleer thet 1,3,4-oxsdiezoles have interesting
entibacterial Dnd antitubercular activity.
SECTIONI2 • • • • • •• •
SPECTRQSCQPIC AVALY5I5
287
SPECTRQSCOPIC ANALYSIS
Infr.red (ir), nuclear magnetic re.onance (nmr) •
and ultraviolet (uv) apactra of aever.l rapr.a.ntativ.a
from the aynthaaised compounda h.v. be.n racordad.
Tha spectroacopic snalysis of a compound reva.ls much
about ita structura. The infrarad apectra giva infor
matiDn about function.l grDupe in tha compound. The
nucle.r magnatic reaon.nca spectra show situ.tion
of protona and tha ultr.violat atudy indic.ting
elactronic aituation of the molecula. The epectr. of
a.var.l compounda, recorded .s 'raphe 1 to 19.
pISCUSSION ~ l! SPECTRA
Tho ir apectre of aevarel eyntheai.ed compound.
h.ve been meesured in KBr pallets. The spectra h.v.
bean taken on lackman. Acculab-10 sp.ctrophotometer
.t C.ntr.l S.lt .nd M.rin. Chamic.l. R •••• rch Inatitut.,
Bh.vn.g.r, between the r.ng. 2.5 _ 16.5 ~ (4000-600 em-')
end .hown ea Graphs 1 - ,. Th. d.ta has be.n
interpret ad .nd di.cua.ed.
Graph I ~
The infr.red epectr. of 2-(4-hydroxy-5_iodo_3_
:288
.methoxy phenyl) -3- (3-methoxyphenyl) -4-thiozoU.dinone
ehowa a weak absorption band et 3300 ca- ' auggesting
the presence of stretching vibration of -DH group. A -1 .
bsnd at 1650 em may indicate the prsoence of C-O
group in thiazolidinone. The peculier band at .1 -1 1210 cm end 1040 cm for C-O stretohing of erylkyl
group. Ths ather bsnda may bs aesigned to C-H, C-C
stretching end deformation vibratione.
iraph ! 2
The ir spsctra of 2-(4.nitrophBnyl)-3-(4-bro~o-
.3-chloro phsnyl).S-cerboxy mBthyl-4.thiszolidinona
1 _1
ehow the characteristic absorption bsnd at 680 cm
1 _1
aey indicate the pressncs of -C-O group, at 1 40 em -1 in the range of 1200.1025 ca for thiezolidinone
ring syetam, bands at 1525 and 1325 em-' indieataa .1 vibration in sryl nitro compounds at 2845 cm for
-1 C-H (stretching) in CH 2 and at 810 cm for C-N
NO 2
stretching. Absorption for nuJol (as mulling agent)
hava baen obssrved at 300-2900, 1460-'460 and 1380 cm-1•
Tha other benda may ba assignsd to C-C and C-H
stretching snd deformation vibrations. The halogena
e -1 ahow bands bstwean 00 - 600 em •
Graph I 3
(4-chlorophenoxy Bcetemido)-5-carboxymethyl-4-
thiezolidinone exhibit the following characterietic -1 -1 bands in Clll at 2900 end 2830 cm for N-H stretching
of -CONH-, ot -1 1680 and 1640 CIII for C.O etretching -1 in amide and thiazolid!none at 1125 cm for C-o(atretch)
, -1 in ether linkage et 1600 CIII for -N-C-S grouping, at
1020 c.- ' fo'r C-Cl etretching. The other banda may
be a.aignad to C-C and C-H atretching and daforMation
vibrationa.
Graph. !
Tha iJ: apectre 0' a: -( J ,5-dibJ:OIII0-4-h~roxy
benzoyl hydrazino)-4-.athoxy phe.yl acetonitrile ahow
the characteristic fJ:equenciea of various functional
groupe ee under. A week band at 3650 cIII- ' for -OH
group at 2'00 and 2830 cm- ' 'oJ: N-H atretching of
-CONH, et 1680 c.-, fOJ: C-o (atretching) in acid
1 -t -1 hydrazide, at 450 cm C-H (daf.) in methyl, "5 CIII
-1 for C-O (etr.) in ether linkege between BOD - 600 em
fOJ: C-BJ: groupa. The otheJ: bende cen b. aaeigne,d to
C-H and C.C stretchln; and deformation vibrations.
Tho absorption for C~N cannot be observed due to 73 adjacent hetero atom •
Graph I l
The infrared epectra of a: -( 4 •• ethyl phenoxy
acatyl hydrazino)-3.4-methylenedlOlCY phenyl aceto
nitrile ahow the cheroctariotic froquanciee of various
functional ;roupa ae under. At 3060 end around
2900 cm-' for N-H(etr.) in hydrazide, et 1665 em-' for -1 C.O stretch. at 1600 c. for aromatic ring. at 1240 .,
and 1030 em for t-o-c etrotching for acymrnetriesl ., and ay •• atrical reapectively. at 1440 em (atr) in
methyl. at 1600 (w) for aromatic rino, at 1250 (m)
and 1110(m), 1030 (m) for ethar linkage of 3,4 •
• ethylenediaxy phenyl gnup. In cc-arnino nitrilu the
band for C~N ia elmoat abeent due to preeence of
vicinaa nitrooen atom.
Graph I !
Tho ir spectra of 2.'-N-(4-methoxyphenyl).
carboxamido methyl thisr.5-(4-mathylphenyl).1,3,4_
oxediazole ahow the charectarietic frequancias of
varioue functionel under. At 3240 -1 groups ae o.
for N-H stretching in amide, at 1660 -1 for C-o CID
etretching, between -1 1460 to 1 UO cm for C-H etretch-
ing in C"3' at 1220 1 _1 and 025 c. for C-O-C ethersl -1 linkage, at 1590 cm for C.N in oMadiazole ring.
Tha othar banda .ay be easignad to C-H, C-C, N-H stretch
ing or daformetion vibrations.
Graph , 7
The ir apectre of 2-("phenyl thioa.m!carbazido)-
1 ,3,'-oxediezols ahow tha charecteristic frequenciea
of various functional groupa oa under. At 3460 e.- ' for N-H atratching, at 1570 e.- ' for C.N etretching in
1 -1 conjugated, et 13 0 em for C.S stretching -1 end 1010 em for t-o-t in oxadiazola ring. The
other banda .ay be a.eigned to C-H, t-t, N-H etretch
ing or defor.etion vibrationa.
Greph I 8
Tha ir apectra of 4-(2-mathylphenyl)-~(5-phenyl_
1,3,4-oMadiazol-2-yl-thio)acetyJ7-thioae.icarbazida
ahow- the characteriatic frequencies of verioua
functional groupe as under. At 3240 cm- ' for N-H -1 stretching in e.ide. at 1650 em for C-o etrAtching.
et 1440 and 1310 ca- ' for C.S linkege. et '.10 c.- t
-t for t-H stretching in CH3 at 1600 cm for C.N in
oxediazole ring.
Graph' ,
The ir epectra 0' Mannich baees show the
cheracteristic frequenciee of varioue functional
groups as under. At 1600 cm- t for C.N group in oxediazole
1 1 -1 ring. between 0'0 to 050 cm for Cas otrotching
1 _1 f h vibration. at 010 cm or C-O-C at erel linkage in
oxediazolo ring. The other bande me~ be aesignad to
C-H. C-C etretching or deformation vibratione.
PISCUSSION ~ ~ SPECTRA
Ths UV epectre of eeverel e~nthesieed compound
were meeeured in ebooluta alcohol ee a solvent. The
epectra were taken on Toehnl~ol Spectrophotometer.
model No. RLD-2. The correeponding nil VS 00
curves ere drswn e. graphs (10 and 1t). The region
etudied is from 200 nm to .00 nm.
;)()3 '. ,
Gnph , t,o
Tha UY &pect~a of m-a.inonitrila of broaa
piperonal type (~) and 5-iodo vanillin (II) ahowad
threa ebaorption maxima whieh a~a charactaristic of
thia typa of coapounda (l) DC -( 4_mathoxyphe nyl Elmino)
.2_bromo-4,5-methylenadioxy phenyl acatonitrile gava
abaorption .axia. at 224 nm, 234 nm and 217 nm.
(II) CC -(3-Anisidino).4-hydroxy-5-ioda-3-methoxy
phenyl acetonitrila gava abaorption aaxi .. et 223 na,
232 na and 264 nm.
Graph I tt
The UY apeetra af 2_(2 .. hydroxy phanyl)-3-(4_
chloro phanoxy acataaido)_5-cerbaxy mathyl-'
thiezolidinona show three absorption aaxi .. at 2tS na,
232 nm and 295 nm which era characteristics of the
type of coapounda.
DISCUSSION Il!. Nm SPECTRA
The nuclaa~ megnatic reaonance (NMR) spectroscopy
of aelsctad compounda has besn studied. Tha apactra
294
have bean taken ueing appropriete deutrated aolvent
by Regional Sophieticated Instrumentetion Centre. lIT.
Bombey.
Graph No. 12
2- (4-Chlorophany1) -3-( 4-bromo-3-ch1 oropheny1)
-4-thie.o1idinone in ecetone d, ee eo1vent ehow
absorption between 2.22 to 2.8-r'(lIu1tip1~te) for
arDmatic protonc and at about 6.07 for CH2 group of
thie.o1idinone ring. The peak at 3.4 'I .ay be for
t-H reeonanca of the ring.
Graph I 13
Acetone d6 ha. been u.ed a. a aolvant for
p.r atudy of 2.(2-hydroxy pheny1)-3-(4-ch10rophenoxy
acetamido)-5-cerboxYllethyl-4-thiazolidinona. It
give. the following abSorption bandl!l 0.95 T (singlet)
for -COOH proton. betw.an 2.25 to 2.6 7 ([email protected])
for 2-hydroxyphenyl protene. between 2.85 to 03.05 T (lIu1tiplata) for 4-ch1orophenoxy proton. and at
1.05 T (8ing1et) for -CH2 of cerboxy .. thylane group.
.) ()~ ,..,l V
Graph I 14
The NMR epectre of 1-(4-methylphenyl amino)
1-(6-bro~,3,4-.ethylenedioxy phenyl)-acetonitrila
(in acetona d, as a aolvant) ahow absorption betwean
2.67 to 3.25 'T' (mUltiPlete) for BrOllllltiC protona, at
3.B4 T' (singlet) for CH2 of -o-C"2-0- group, and et
7.6' 7' (ainglat) for C"3 group.
Graph , 15
m-(3,S-Dibromo-4-hydroxy benzoyl hydrezino)_
-4-methoxy phanyl acetonitrila in acetone d, ee a eolvant
sholt absorption betwsen 1.13 to 2.16 7' (multipl!to)
and about 2.75 to 2.95 ~ (multipl!te) for aromatic
protona end et 6.0' 'T' (8inglet) for -oCH3 protons.
Graph I 16
CDCl3 hes bean ueed ea a solvent for pmr etudy
of =-(4-methylphenoxy acatyl hydrezino)-3.4-
.athylenedioxy phenyl acetonitrile. It ,ivea the
following ebsorption bands.between 2.46 to 3.2 T
(mulU.p1e.te) for erolletic protone. ot 3.,67' (d.nght)
fu CH2
of -o-CH2-O- group end at 5.35'7' (singht)
for CHZ protona of -oCH2CO- group.
Graph , 11
Acetone d, he. b •• n ua.d .e • aolvent for pmr
a tudyof a: - (4-c:hlor D 8n111no) - 4- h,vdrolCy-5-iodo ... 3-
m.thoxy phenyl acetonitrile. It givos the followin,
abaorption bands, batw.en 2.5 to 2.6!) 'I (multipltte)
for Dramatic: protons at 6.2 rr (ein,l.t) for CH3 proton of -oCH3 group.
Graph I 18
NMR apectra of 2_~N_(4_matho.yph.nyl) ...
carboxamido~ethyl thiSi-S-(4-.ethylphanyl)_1.3,4-
olCldlelole in scatone d, es Bolvent show absorption
bands b.tween 1.9 to 3.0 ~ (multlpl~te), 2.33 to
2.45 'T' (multlPltte) and 3.0 to 3.05 7' (lnult1pl!te) fa.
aro •• Uc: protons. And at 5.U '7' (Singlet) of CHZ protons,
at 6.2 'T (Singlet) for CH3 p.l'otDn of -OCH3 group and
at 7.15 7' (linglet) for CH3 group.
297
Graph I 1,
Acetone d, ha. b •• n u •• d •• a eolvent for
pmr .tudy of "(3-•• thylph.n~l)-L-(5-phenyl-1.3.4-
oxediezol-2-yl-thio)-ac.ty17-thio.emicorbazida.. It
giv •• the following absorption bende, betw.en 2.25 to
2 •• 0 rr' (.ultip1et.) about 2.6 to 2.15 '7' (.ultipl~t.)
end 2.9 to 3.0 'I' (.ul tiplet.) for .romatic protone.
And about 7.2 '7' (elnglet) for CH3 protons.
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