SYNTHESISANDBIOLOGICALEVALUATIONOFSOME5[(SUBSTITUTED)PHENYL]3(4 CHLOROPHENYL)4,5DIHYDRO1HPYRAZOLE1CARBATHIOMIDEDERIVATIVES

VidhiR.Patel,C.N.PatelandDhruboJyotiSen
DepartmentofPharmaceuticalChemistry,ShriSarvajanikPharmacyCollege,GujaratTechnologicalUniversity, ArvindBaug,Mehsana384001,Gujarat,India,Email:vidhipatel708@yahoo.in

________________________________________________________________________________________________ Abstract Inthepresentinvestigation,aseriesofsomesynthesisandbiologicalevaluationofsome5[(substituted)phenyl]3(4 chlorophenyl)4,5dihydro1Hpyrazole1carbathiomide derivatives were synthesized and were tested for their antimycobacterialactivityinvitroagainstisoniazideresistantMycobacteriumtuberculosis.Compound3cwasfoundto bethemostpotentderivativesoftheserieswithanMICvalueof100g/ml. Keywords:Pyrazoline,MIC,INH ________________________________________________________________________________________________ Introduction Tuberculosisisthemostprevalentcommunicableinfectiousdiseaseonearthandremainsoutofcontrolinmany developingnations.RecentlyavailabletherapytocontrolTBissomewhatchalangingincaseofresistantstrain0f tuberculosis.Becauseoflongerdurationoftherapy,differentunwantedsideeffectofdrugsandhighcostfactor thereisneedofnewchemicalentityhavingmorepotencyandfewersideeffectstotacklethecurrentsituation. Inrecentscenarioheterocycleplaysamajorruleindrugsynthesis.Inthatrespectpyrazoleplaysasignificantrule among other heterocycles. From the literature survey, in recent years pyrazole derivatives have attracted considerable interest because of their therapeutic and pharmacological properties. Several of them have been foundtoexhibita widespectrum of biologicalactionslikeantiinflammatory, ulcerogenic,antibacterial, diuretic, analgesic,antiviral,antifungal,antimycobacterialactivity.Soitwasplanedtosynthesizeanovelseriesofsome5 [(substituted)phenyl]3(4chlorophenyl)4,5dihydro1Hpyrazole1carbathiomide derivatives and to check their activityasantimycobacterialactivity.14 Experimental TheentirechemicalsweresuppliedbyS.D.Finechem.(Mumbai),finarchem.Ltd(Ahmedabad)andLobaChemie. Pvt.Ltd.(Mumbai).Meltingpointsweredeterminedbyopentubecapillarymethodandareuncorrected.Purityof compoundswascheckedbythinlayerchromatography(TLC)onsilicagelGinsolventsystemhexaneethylacetae (3.5:1.5),thespotswerelocatedunderiodinevapoursorUVlight.IRspectraofallcompoundswererecordedon FTIR8400SShimadzuspectrophotometerusingKBr.Massspectrawereobtainedusing2010EVLCMSShimadzu instrument. GeneralprocedureforPreparationof1(4chlorophenyl)3(4substitutedphenyl)prop2en1one(1a6a)1 A mixture of 4Chloro acetophenone (12gm, 0.1mol), substituted benzaldehyde (0.1mol) in ethanol and sodium hydroxide(30%w/v,5ml)inpresenceof10mlofpetroleumetherwasstirredunderroomtemperaturefor8h.The resultingsolutionwasallowedtostandovernightandpouredintoicecoldwaterandthenitwasneutralizedwith hydrochloricacid.Thesolidsoobtainedwasfiltered,driedandcrystallizedfromethanol. GeneralprocedureforPreparationof5substituted3(4chlorophenyl)4,5dihydro1Hpyrazole(1b6b)1 Asolutionofchalconeinethanol,hydrazinehydrate(99%)wasaddeddropwise.Thereactionmixturewasheated underrefluxfor10handthencooledandpouredontocrushedice.Thesolidpyrazolineproductwasfilteredand recrystallizedfromethanol. General procedure for Preparation of 5[(substituted)phenyl]N,3bis(4chlorophenyl)4,5dihydro1Hpyrazole1 carbothioamide(1c6c)1 1chloro4isothiocyano benzene (0.1mol) was added to a solution of pyrazoline (0.1mol) in ethanol (20 ml). The reactionmixturewasrefluxedfor10handaftercoolingitwaspouredontocrushedice.Then,theseparatedsolid masswasfiltered,washedwithwaterandcrystallizedfromethanol. SchemeofSynthesis:

CH3 R O
1

Cl

CH

NaOH C2H5OH O 1-(4-chlorophenyl)-3-(4-substitutedphenyl)prop-2-en-1-one (1a-6a)

O Aldehydes Cl 1-(4-chlorophenyl)ethanone

C2H5OH Cl NH2NH2 H2O

Cl N NH Cl S N C2H5OH R
1

Cl

NCS HN

5-[(substituted)phenyl]-N,3-bis(4-chloro phenyl)-4,5-dihydro-1H-pyrazole-1-carb othioamide (1c-6c)

R 5-substituted-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole (1b-6b)

Table1:PhysicalCharacteristicsofSynthesizedCompounds Compound Molecular R1 Molecular Code Formula Weight (g/mol) 1c 2c 3c 4c 5c 6c C22H17Cl2N3OS 4OHphenyl 442.36 471.35 460.80 460.80 426.26 456.38

Melting Point (oC) 7680 8690 6266 6870 7173 7780

Yield (%w/w)

R f Value

40 30 46 45 62 35

0.68 0.68 0.71 0.78 0.61 0.65

C22H16Cl2N4O2S 4NO2phenyl C22H16Cl3N3S C22H16Cl3N3S C22H17Cl2N3S C22H17Cl2N3S 4Clphenyl 2Chlorophenyl Phenyl 4OCH3phenyl

Mobilephase:ethylacetate:hexane(1:4) Table2:Spectraldataofsynthesizedcompounds Compond UV IR(v,cm1) code (max,nm) 1c 278 OH(3200), CCl(821), NH(~3100), C=S(1207) 2c 279 CCl(825), NH(3240), C=S(1215),

Mass (m/z) 442(M+)

NMR

471(M+)

7.187.97(m,12H,ArH) 2.56(s,1H,NH) 1.24(d,2H,CH2)

3c

274

4c

261

5c

279

6c

236

NO2(1461,1492) CCl(821,1010), NH(3240), C=S(1207), C=N(1596) CCl(829,1010), C=S(1207), CCl(756,829,1010) CCl(825,1010), NH(~3100), C=S(1215), C=N(1593) CCl(829,1010), NH(3352), C=S(1245), NH(3352)

461(M )

1.40(t,1H,CH)

459.6(M+) 425.9(M+)

455.7(M+)

7.018.03(m,12H,ArH) 2.53(s,1H,NH) 1.24(d,2H,CH2) 1.43(t,1H,CH) 3.83(s,3H,CH3)

Antitubercularactivity67 Determination0fminimalinhibitoryconcentrationbyLowensteinJensenmethod Here LJ medium without potato starch with drug incorporation before inspissations was used. Screw cappedtubes17mmindiameter,containing7mlofmediumwereinspissatedat85Cfor4045minutes. The drug susceptibility test was carried out from a primary isolation or a subculture on LJ medium. A representativeportionoftheculturewasobtainedbysamplingasmanycoloniesaspossiblewithin1or2weeks afterappearanceofgrowth. Thesamplewashomogenizedinasterilescrewcappedbottle(e.g.14mlMcCartneybottleor5mlBijoux bottle)containing1mlofsteriledistillwaterand10glassbeads3mmindiameter. Themixturewashomogenizedinavortexmixtureforaminuteandifnecessarytheopacitywasadjusted byaddingsteriledistillswater,downtothatofastandardsuspensionof1mg/mlofBCG. Thesuspensionislefttosettleforabout30minutes. CompositionofmodifiedLJmedia Potassiumdihydrogenphosphate: 1.2g Magnesiumsulphate: 0.12g Magnesiumcitrate: 0.3g Lasparagine: 1.8g Glycerol/Sodiumpyruvate: 6.0ml/3.6g Distilledwater: 300ml Malachitegreen(2%): 16ml Egghomogenate: 500ml Benzylpenicillin: (1,000,000IU/ml)1ml Formediuminoculation,dilutionof103mg/mlofthestandardsuspension(1mg/ml)wasprepared.0.2ml oftheinoculumwasincorporatedtoeachtube. After inoculation the tubers were incubated at 37C in a slanted position with the screw caps slightly loosenedtoallowevaporationoftheinoculum. After2448hoursscrewcapsweretightenedandwerefurtherincubated.Thereadingofresultwascarried outatthe28thand40thday. Ifatthe28thdayreadinggrowthwasoptimalthannoneedtogoforthe40thday. The reading of the results consists of counting of growth on the different slants, calculation of the proportion of bacilli by comparing counts on drug free and drug containing LJ media and finally matching the calculatedproportionwiththecriticalproportion. RESULTSANDDISCUSSION

5[(substituted)phenyl]3(4chlorophenyl)4,5dihydro1Hpyrazole1carbathiomide,(1c6c)wereshowninTables 1and2andreactionsequencesforthepreparationisoutlinedinscheme.Thechalconeswerepreparedbyreaction of 4chloro acetophenone with appropriate aldehyde in a presence of base by conventional Claisen Schmidt condensation.Thereactionbetweennewlysynthesizedchalconswithhydrazinehydrateinethanolledtosynthesis of novel pyrazolines (1b6b), which on treatment with 4chlorophenylthiocyanates (1c6c) with 4050% yield. The purity of the compounds was checked by TLC and elemental analysis. Spectral data (IR,MS and 1HNMR) fully confirmedthestructures.1 Antimycobacterialactivity Amongtheringsubstitutedpyrazolinederivatives(1c6c)weretestedfortheirantimycobacterialactivityinvitro against INH resistant Mycobacterium tuberculosis, using the LJ slope method. The results are shown in table 3.Compound 3c was found to be most potent among the series having MIC 100g/ml. Others synthesised compounds were found to be active (having MIC 1c : 500g/ml, 2c : 500g/ml, 4c : 250 g/ml,5c :1000 g/ml, 6c:500g/ml).noneofthecompoundwasfoundtobeequipotentwithstandardisoniazid. ANTITUBERCULARACTIVITYOFSYNTHESISEDCOMPOUNDS Table3:Minimuminhibitoryconcentration(MIC)ofsynthesizedcompounds COMPOUNDCODE M.TUBERCULOSIS[g/ml] 1c 250 2c 500 3c 100 4c 250 5c 1000 6c 500 Isoniazid 0.2 h HistogramofMIC

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