Design, synthesis, DFT, docking studies and ADME prediction of some new coumarinyl linked

23 Pages·2017·6.68 MB·English

RESEARCHARTICLE
Design, synthesis, DFT, docking studies and
ADME prediction of some new coumarinyl
linked pyrazolylthiazoles: Potential standalone
or adjuvant antimicrobial agents
SunilKumar1,VikramSaini2,IndreshK.Maurya3,JayantSindhu4,MukeshKumari5,
RameshKataria1☯*,VinodKumar6☯¤*
1 DepartmentofChemistry&CentreofAdvanceStudiesinChemistry,PanjabUniversity,Chandigarhs,
India,2 DepartmentofBiotechnology,AIIMS-NewDelhi,Delhi,India,3 DepartmentofMicrobial
a1111111111
Biotechnology,PanjabUniversity,Chandigarh,India,4 K.M.GovernmentCollege,Narwana,India,
a1111111111
5 DepartmentofChemistry,KurukshetraUniversity,Kurukshetra,India,6 DepartmentofChemistry,M.M.
a1111111111
University,Mullana-Ambala,India
a1111111111
a1111111111 ☯Theseauthorscontributedequallytothiswork.
¤ Currentaddress:DepartmentofChemistry,MarkandaNationalCollege,Shahbad(M),India.
*[email protected](RK);[email protected](VK)
Abstract
OPENACCESS
Citation:KumarS,SainiV,MauryaIK,SindhuJ,
Thecontrolofantimicrobialresistance(AMR)seemstohavecometoadeadend.The
KumariM,KatariaR,etal.(2018)Design,
synthesis,DFT,dockingstudiesandADME majorconsequencesoftheuseandabuseofantibacterialdrugsarethedevelopmentof
predictionofsomenewcoumarinyllinked resistantstrainsduetogeneticmutabilityofbothpathogenicandnonpathogenicmicroor-
pyrazolylthiazoles:Potentialstandaloneoradjuvant
ganisms.We,herein,reportthesynthesis,characterizationandbiologicalactivitiesofcou-
antimicrobialagents.PLoSONE13(4):e0196016.
marin-thiazole-pyrazole(CTP)molecularhybridswithanefforttoexploreandovercomethe
https://doi.org/10.1371/journal.pone.0196016
increasingantimicrobialresistance.Thecompoundswerecharacterizedbyanalyzingtheir
Editor:MohammadShahid,AligarhMuslim
IR,Mass,1Hand13CNMRspectraldataandelementalanalysis.Theinvitroantimicrobial
University,INDIA
activityofthesynthesizedcompoundswasinvestigatedagainstvariouspathogenicstrains;
Received:December29,2017
theresultsobtainedwerefurtherexplainedwiththehelpofDFTandmolecularorbitalcalcu-
Accepted:April4,2018
lations.Compound1band1fdisplayedgoodantimicrobialactivityandsynergisticeffects
Published:April19,2018 whenusedwithkanamycinandamphotericinB.Furthermore,invitrocytotoxicityofcom-
Copyright:©2018Kumaretal.Thisisanopen pounds1band1fwerestudiedagainstHeLacells(cervicalcancercell)andHek-293cells.
accessarticledistributedunderthetermsofthe Theresultsofmoleculardockingstudywereusedtobetterrationalizetheactionandpredic-
CreativeCommonsAttributionLicense,which
tionofthebindingmodesofthesecompounds.
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
files. Introduction
Funding:Thisworkwasfinanciallysupportedby Overthepastfewdecades,globallyantibioticrepositoryhasbecomelesseffectiveduetoboth
theUniversityGrantCommissionintheformof
antibioticoveruseandanincreaseinantimicrobialdrugresistance.Evencommonbacterial
UGC-BSRresearchstartupgrant(F.30-86/2014
(e.g.Staphylococcusaureus,Staphylococcusenteric,Escherichiacoli)andfungal(e.g.Candida
(BSR)),NewDelhi,India.Therewasnoadditional
externalfundingreceivedforthisstudy.The albicans,Cryptococcusneoformans)pathogensaredevelopingresistanceagainstwidelypre-
fundershadnoroleinstudydesign,datacollection scribedantibacterialandantifungaldrugslikeamphotericinB(AmpB),fluconazole(FLC),
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
andanalysis,decisiontopublish,orpreparationof chloroamphenicolandpenicillin[1–3].Thisisparticularlydangerousconsideringthatthe
themanuscript. majorbacterialaswellasfungalpathogenssuchasCandidasp.accountfor~75%ofallinfec-
Competinginterests:Theauthorshavedeclared tionsandrepresentthe4thleadingcauseofnosocomialinfections[4].Thusmicrobialdrug
thatnocompetinginterestsexist. resistancehasemergedasamajorpublichealthchallengeacrosstheworld.Consequently,
strategiesareneededtodevelopnewtherapeuticcompoundsthatcouldeitheractasnew
drugsorcouldcomplementtheexistingdrugtherapymoreeffectivebyactingasanadjuvant
[5–7].
Inrecentpast,azoleshavegarneredconsiderableattentionespeciallyinthedesignandsyn-
thesisofcompoundswithsignificantbiologicalactivities[8–11].Inparticular,therearevari-
ousdrugsincorporatingthiazolenucleus(Fig1)whicharebeingusedastheantimicrobial
(sulfathiazole),antiinflammatory(fanetizole)andantifungal(abafungin)agents[12–17].Simi-
larly,compoundscontainingpyrazoleringhavebeenfoundtoexhibitavarietyofbiological
propertiesandmanyofthemsuchascelecoxib,pyrazofurinetc.(Fig1)areusedclinically
besidesusingforagriculturalpestmanagement[18–22].Especially,3,5-dimethyl-4-aryldiaze-
nylpyrazolederivativeshavebeenreportedtoexhibitcytotoxic[23],antioxidant[24],analgesic
[25]andsignificantantimicrobialpotential[26].Ontheotherside,compoundscontaining
coumarinmoiety(Fig1)havealsobeenprovedasapotentantimicrobial,antitumor,anticoag-
ulant,antiviral,andanti-inflammatoryagents[23,24,27–31].Ithasbeenwidelyacceptedthat
structuralpropertiesofthiazole,pyrazoleandcoumarinderivativesandtheirstabilityunderin
vivoconditionsaretheprimaryfactorsfortheirsuperiorpharmacologicalactivities[32–34].
Fig1.Somethiazole,pyrazole,4-aryldiazenylpyrazoleandcoumarincontainingbiologicalactiveheterocyclesandtargetcompounds.
https://doi.org/10.1371/journal.pone.0196016.g001
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
Adrugdiscoveryprograminvolvestheplanningofnovelchemicalentitiesusingmolecular
hybridizationapproachwheretwoactivepharmacophoricunitsofknowndrugswerecon-
nectedthroughacovalentbondinasinglematrix[35–37].Theselectionoftheactiveunitsin
thedualdrugisusuallyestablishedontheirobserved(oranticipated)synergisticoradditive
pharmacologicalactivities.Inprospectoftheabovediscussedfactsandincontinuationofour
researchtowardsthedevelopmentofnovelmolecularhybridsaspotentialbiologicallyactive
agents[38–42],wehavedecidedtobuildnewmolecularhybridsbasedoncoumarin-thiazole-
pyrazole(CTP).
Experimental
Chemistry
General. Meltingpointsweredeterminedbyopencapillarymethodandareuncorrected.
InfraredspectrumwasobtainedneatonaThermoScientificFischerspectrometer.Multinu-
clearNMR(1H,13C)spectrawererecordedonaBrukeravanceat300MHz.Chemicalshifts
arereportedinpartspermillion(ppm).Tetramethylsilaneservedasaninternalstandardin
13Cand1HNMR(δ0.00ppm).CHNSanalyseswereobtainedonaPerkinElmerModel2400
CHNSelementalanalyzerandMassspectroscopydataofsynthesizedcompoundwasrecorded
onwatersQQ-TOFmicroMassSpectrometer.Allthechemicalsusedforthesynthesisoftar-
getcompoundshavebeenpurchasedfromSigmaAldrichandwereusedasreceived.Allthe
compoundsgaveC,HandNanalysiswithin±0.5ofthetheoreticalvalues.ScannedSpectra
forallcompoundsareshowninS3–S19Figs.
Generalprocedureforthesynthesisof4-(coumarin-3-yl)-2-((3,5-dimethyl-4-phenyldia-
zenyl)pyrazol-1-yl)thiazoles(1a-1g). Anethanolicsolution(20mL)of3-bromoacetylcou-
marin(7,0.52g,2.00mmol),sodiumcarbonate(0.11g,1.00mmol)and3,5-dimethyl-4-(p-
chlorophenyl)diazenylpyrazole-1-thiocarboxamide(5a)(0.59g,2.00mmol)wasrefluxedona
waterbathfor6h.OncompletionofthereactionasobservedusingTLC(20:80,Ethylacetate:
Hexane(v/v)),thesolventwasevaporated.Thesolidthusobtainedwasdissolvedinchloro-
form(25mL)andwashedwithwater(50mL).Theorganiclayerwasseparatedandtheexcess
solventwasdistilledoff.Thesolidthusobtainedwascrystallizedwithethanoltoafford1ain
82%yield.Theprotocolwasfurtherextendedusingdifferentlysubstitutedaminesandcouma-
rinwhichresultedintotheformationofcompounds1b-1gingoodtoexcellentyield.
Spectraldata
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl)diazenyl)pyrazol-1-yl)thiazole(1a).
R:0.6;mp214–216˚C;Yield:82%;IR(cm-1):1719(C=O),1605(C=N);1HNMR(300
f
MHz,CDCl ,δppm):8.39(s,1H,coumarin4-H),8.06(s,1H,thiazole5-H),7.70(d,2H,
3
J=8.4Hz,p-chlorophenyl2,6-H),7.65(s,1H,coumarin5-H),7.60–7.52(m,1H,coumarin
7-H),7.45(d,2H,J=8.4Hz,p-chlorophenyl3,5-H),7.41–7.33(m,2H,coumarin6,8-H),2.86
(s,3H),2.31(s,3H);13CNMR(75MHz,CDCl ,δppm):164.33(C=O),159.22(C -thiazole),
3 2
154.38(C -coumarin),149.26(C -thiazole),147.14(C -coumarin),141.59(C-Ar),136.64
8’ 4 4
(C-Ar),133.14(C-Ar),129.23(C-Ar),128.82(C-Ar),125.07(C-Ar),123.35(C-Ar),120.87
(C-Ar),119.40(C-Ar),118.67(C-Ar),116.81(C-Ar),110.77(C-Ar),23.88(5-CH pyrazole),
3
14.70(3-CH pyrazole);MS(m/z)Found:463.1,461.0(3:1)[M+H]+;Calcd.:461.92;Anal.
3
Calcd.forC H ClN O S:C,59.80;H,3.49;N,15.16.Found:C,59.85;H,3.50;N,15.46.
23 16 5 2
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl)diazenyl)pyrazol-1-yl)
thiazole(1b).R:0.6;mp220–222˚C;Yield:88%;IR(cm-1):1719(C=O),1602(C=N);1H
f
NMR(300MHz,CDCl ,δ):8.30(s,1H,coumarin4-H),8.08(s,1H,thiazole5-H),7.65–7.61
3
(m,5H,coumarin5-H,Ph2,6,3,5-H),7.49(d,1H,J=8.7Hz,coumarin8-H),7.31(d,1H,
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
J=8.7Hz,coumarin7-H),2.74(s,3H,5-CH ),2.20(s,3H,3-CH );13CNMR(75MHz,
3 3
CDCl ,δppm):167.47(C=O),159.23(C -thiazole),154.49(C -coumarin),149.56(C -thia-
3 2 8’ 4
zole),147.15(C -coumarin),143.40(C-Ar),137.33(C-Ar),133.24(C-Ar),128.86(C-Ar),
4
125.07(C-Ar),124.26(C-Ar),124.16(C-Ar),122.74(C-Ar),119.53(C-Ar),118.64(C-Ar),
116.89(C-Ar),115.04(C-Ar),112.54(C-Ar),31.16(5-CH pyrazole),25.30(3-CH pyrazole);
3 3
MS(m/z)Found:542.1,540.1;Calcd.:540.82;Anal.Calcd.forC H BrClN O S:C,51.08;H,
23 15 5 2
2.80;N,12.95.Found:C,51.00;H,2.82;N,13.14.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl)diazenylpyrazol-1-yl)thiazole
(1c).R:0.6;mp240–241˚C;Yield:80%;IR(cm-1):1713(C=O),1601(C=N);1HNMR(300
f
MHz,CDCl ,δ):8.32(s,1H,coumarin4-H),8.02(s,1H,thiazole5-H),7.87(d,2H,J=8.7Hz,
3
Ph2,6-H),7.71(d,1H,J=8.4Hz,coumarin5-H),7.49(d,2H,J=8.4Hz,Ph3,5-H),7.62(t,
1H,J=7.5Hz,coumarin7-H),7.43–7.40(m,2H,coumarin6,8-H),2.74(s,3H,5-CH ),2.20
3
(s,3H,3-CH ),2.05(s,3H,p-CH );13CNMR(75MHz,CDCl ,δppm):162.73(C=O),
3 3 3
157.90(C -thiazole),147.90(C -coumarin),140.92(C -thiazole),140.62(C -coumarin),
2 8’ 4 4
139.09(C-Ar),134.47(C-Ar),133.02(C-Ar),132.82(C-Ar),130.27(C-Ar),128.85(C-Ar),
128.44(C-Ar),128.39(C-Ar),128.35(C-Ar),126.95(C-Ar),126.92(C-Ar),125.36(C-Ar),
123.28(C-Ar),118.07(C-Ar),117.99(C-Ar),113.57(C-Ar),35.10(5-CH pyrazole),29.61
3
(3-CH pyrazole),14.60(Ph-CH );MS(m/z)found:441.3;Calcd.441.50;Anal.Calcd.For
3 3
C H N O S:C,65.29;H,4.34;N,15.86;FoundC,65.69;H,4.26;N,15.85.
24 19 5 2
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-methylphenyl)diazenyl)pyrazol-1-yl)
thiazole(1d).R:0.6;mp255–256˚C;Yield:83%;IR(cm-1):1720(C=O),1601(C=N);1H
f
NMR(300MHz,CDCl ,δ):8.39(s,1H,coumarin4-H),8.00(s,1H,thiazole5-H),7.86(d,2H,
3
J=8.4Hz,Ph2,6-H),7.66(s,1H,coumarin5-H),7.63(d,2H,J=8.1Hz,Ph3,5-H),7.53(d,
1H,J=8.4Hz,coumarin7-H),7.35(d,1H,J=8.4Hz,coumarin8-H),2.69(s,3H,5-CH ),
3
2.19(s,3H,3-CH ),1.98(s,3H,p-CH );13CNMR(75MHz,CDCl ,δppm):164.37(C=O),
3 3 3
152.86(C -thiazole),151.56(C -coumarin),144.30(C -thiazole),143.71(C -coumarin),
2 8’ 4 4
139.30(C-Ar),138.86(C-Ar),131.69(C-Ar),131.60(C-Ar),129.69(C-Ar),128.69(C-Ar),
128.41(C-Ar),126.37(C-Ar),126.20(C-Ar),120.61(C-Ar),120.24(C-Ar),119.41(C-Ar),
116.44(C-Ar),113.44(C-Ar),20.94(CH -Ph),24.37(5-CH pyrazole),13.86(3-CH pyra-
3 3 3
zole);MS(m/z)Found:477.6,475.8;Calcd.475.95;Anal.Calcd.forC H ClN O S:C,60.56;
24 18 5 2
H,3.81;N,14.71;FoundC,60.55;H,3.71;N,14.63.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-(4-bromophenyl)diazenyl)pyrazol-1-yl)thiazole
(1e).R:0.6;mp210–212˚C;Yield:88%;IR(cm-1):1717(C=O),1600(C=N);1HNMR(300
f
MHz,CDCl ,δ):8.30(s,1H,coumarin4-H),8.08(s,1H,thiazole5-H),7.82(d,1H,J=8.1Hz,
3
coumarin5-H),7.71(d,2H,J=8.4Hz,Ph3,5-H),7.65–7.61(m,1H,coumarin7-H),7.47(d,
2H,J=8.4Hz,Ph2,6-H),7.28–7.24(m,2H,coumarin6,8-H),2.72(s,3H,5-CH ),2.32(s,3H,
3
3-CH );13CNMR(75MHz,CDCl ,δppm):164.81(C=O),159.22(C -thiazole),154.38(C -
3 3 2 8’
coumarin),149.26(C -thiazole),147.14(C -coumarin),141.59(C-Ar),136.64(C-Ar),133.14
4 4
(C-Ar),129.23(C-Ar),128.82(C-Ar),125.07(C-Ar),123.35(C-Ar),121.87(C-Ar),119.40
(C-Ar),118.67(C-Ar),116.81(C-Ar),110.77(C-Ar),25.08(5-CH pyrazole),14.71(3-CH
3 3
pyrazole);MS(m/z)found:506.1,504.1(3:1);Calcd.:506.37;Anal.Calcd.forC H BrN O S:
23 16 5 2
C,54.55;H,3.18;N,13.83.Found:C,54.48;H,3.12;N,13.82.
4-(6-Chlorocoumarin-3-yl)-2-(3,5-dimethyl-4-(4-chlorophenyl)diazenyl)pyrazol-1-yl)
thiazole(1f).R:0.6;mp235–236˚C;Yield:85%;IR(cm-1):1719(C=O),1605(C=N);1H
f
NMR(300MHz,CDCl ,δ):8.28(s,1H,coumarin4-H),8.07(s,1H,thiazole5-H),7.79(d,1H,
3
J=2.4Hz,coumarin5-H),7.65–7.61(m,5H,Ph4-Handcoumarin6-H),7.28(d,1H,J=8.1
Hz,coumarin8-H),2.84(s,3H,5-CH ),2.21(s,3H,3-CH );13CNMR(75MHz,CDCl ,
3 3 3
δppm167.44(C=O),159.23(C -thiazole),150.22(C -coumarin),147.40(C -thiazole),
2 8’ 4
142.56(C -coumarin),139.21(C-Ar),133.23(C-Ar),128.88(C-Ar),125.10(C-Ar),124.85
4
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
(C-Ar),124.80(C-Ar),122.43(C-Ar),119.34(C-Ar),118.64(C-Ar),116.81(C-Ar),113.49
(C-Ar),110.39(C-Ar),25.16(5-CH pyrazole),15.54(3-CH pyrazole);MS(m/z)found:
3 3
497.2,495.2(3:1);Calcd.:496.37;Anal.Calcd.forC H Cl N O S:C,55.65;H,3.05;N,14.11;
23 15 2 5 2
Found:C,55.64;H,3.10;N,14.17.
4-(Coumarin-3-yl)-2-(3,5-dimethyl-4-phenyldiazenyl)pyrazol-1-yl)thiazole(1g).R:0.6;
f
mp205–206˚C;Yield:82%;IR(cm-1):1715(C=O),1603(C=N);1HNMR(300MHz,
CDCl ,δ):8.34(s,1H,coumarin4-H),8.08(s,1H,thiazole5-H),7.82–7.78(m,2H,Ph2,6-H),
3
7.78(d,1H,J=8.4Hz,coumarin5-H),7.65–7.63(m,1H,coumarin7-H),7.62–7.57(m,4H,
Ph3,4,5-H,coumarin6-H),7.33(d,1H,J=8.7Hz,coumarin8-H),2.79(s,3H,5-CH ),2.24
3
(s,3H,3-CH );13CNMR(75MHz,CDCl ,δppm):167.79(C=O),159.35(C -coumarin),
3 3 8’
154.35(C -thiazole),148.40(C -coumarin),146.95(C -thiazole),142.99(C-Ar),138.95
2 4 4
(C-Ar),135.97(C-Ar),132.95(C-Ar),128.78(C-Ar),124.99(C-Ar),123.43(C-Ar),122.59
(C-Ar),120.05(C-Ar),119.79(C-Ar),118.76(C-Ar),116.75(C-Ar),110.52(C-Ar),24.83
(5-CH pyrazole),15.22(3-CH pyrazole);MS(m/z)found:427.3;Calcd.:427.28;Anal.Calcd.
3 3
forC H N O S:C,64.62;H,4.01;N,16.38;FoundC,64.55;H,4.05;N,16.45.
23 17 5 2
Antimicrobialassay
Themicrobialstrains(bacterialandfungal)usedinthestudywereobtainedfromtheInstitute
ofMicrobialTechnology(MTCC-IMTECH),ChandigarhandNationalCollectionofPatho-
genicFungi(NCPF),Post-GraduateInstituteofMedicalEducationandResearch(PGIMER),
Chandigarh.ThebacterialstrainsEscherichiacoliMTCC2961,StaphylococcusaureusMTCC
3160,EnterococcusfaecalisMTCC439,VibriocholeraMTCC3906,Streptococcuspyogenes
MTCC442wereculturedinMullarHintonbroth(MHB,HiMedia,India).Thefungalstrains
CandidaalbicansNCPF400034,CandidaglabrataMTCC3019,CandidakruseiNCPF44002,
CandidaparapsilosisNCPF450002,Candidakeyfer,NPCPF410004andCandidatropicalis
NPCPF420007wereculturedinyeastextract-peptone-dextrose(YEPDbroth,HiMedia,India)
andRPMI1640media(HiMedia,India).Foragarplates,2.5%(w/v)bacteriologicalagar
(HiMedia,India)wasaddedtothemedium.Thestrainswerestoredwith15%glycerolat
-80˚Casfrozenstocks.Thecellswerefreshlyrevivedonrespectiveagarplatesfromthestock
beforeeachexperiment[43,44].
Antibioticsusceptibilitytesting
Antibacterialactivity. Allthebacterialstrains(E.coli,S.aureus,E.faecalis,V.cholerae,S.
pyogenes)weregrownovernightandweredilutedinMueller-Hintonbroth(MHB)toacell
densityof105CFU/mL.100μLofthiscultureandcompounds1a-1g(250–0.112μM)dis-
solvedinDMSO,wereaddedintothe96-wellflatbottomedmicrotiterplate(Genexy-HiMe-
dia,India).Theplatewasincubatedat37˚Cwithoutshakingfor24h.Thevisualandoptical
densitywasmeasuredusingmicroplatereaderat600nm(Thermo,Model680).Theminimum
inhibitoryconcentration(MIC)wasdefinedastheconcentrationoftheantimicrobialagent
thatinhibit>99%growth.kanamycin(KAN),aknownantibacterialdrugisusedasapositive
control[45].
Antifungalactivity. Theantifungalactivitiesofcompounds1a-1gagainstfungalspecies
(C.albicans,C.glabrata,C.krusei,C.parapsilosis,C.keyfer,C.tropicalis)wereperformed
accordingtotheClinicalandLaboratoryStandardsInstitute(CLSI,formerlyNCCL)in
RPMI-1640mediumbybrothmicro-dilutionmethods[46].Theconcentrationsofcom-
pounds1a-1grangedbetween250μMand0.097μM.The96wellflatbottommicrotiterplates
wereincubatedwithoutshakingat30˚Cfor48h.Thevisualandopticaldensitywasmeasured
usingmicroplatereaderat492nm(Thermo,Model680)tostudytheinhibitioninfungal
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
growth.TheMICwasdefinedasthelowestconcentrationofantifungaldrugwhichresultedin
>99%inhibitionofgrowthcomparedtothatoftheuntreatedcontrol.amphotericinB,a
knownantifungaldrugisusedasapositivecontrol.
Timekillassay. S.aureusexponentialphasecells(~1x105CFU/mL)wasinoculatedin
MHBmediumcontainingcompound1f(at15.67μMconc.).TheS.aureuscellswereincu-
bated(37˚C;200rpm)and100μlaliquotswereremovedatpre-determinedtimepoints(0,4,
8,12,16,20and24h).Thealiquotswereseriallydiluted(10fold)insalineandplatedonthe
MHA(MullerHintonAgar)plates.Thenumbersofcolonieswerecountedafterincubating
theplatesat37˚Cfor24h.SimilarlyC.keyferexponentialphasecells(~1x104CFU/mL)were
inoculatedinRPMI1640mediumcontainingcompound1f(62.25μM).Thetubeswereincu-
bated(30˚C;200rpm),and100μLaliquotswereremovedatpre-determinedtimepoints(0,4,
8,12,16,and24h).Thealiquotswereseriallydiluted(10fold)insalinewaterandplatedon
YEPDagarplates.Thenumbersofcolonieswerecountedafterincubatingtheplatesat30˚C
for48h[44].
Checkerboardassayofcompound1band1f. Theinteractionofcompound1band1f
withwell-knownantimicrobialskanamycin,andamphotericinBandfluconazolewereevalu-
atedbythecheckerboardmethodasdescribedpreviouslyandrepresentedasthefractional
inhibitoryconcentration(FIC)index(FICI)i.e.sumoftheFICforeachcompounds.FIC
valueofthemosteffectivecombinationwasusedincalculatingtheFICI.FICI=FICofX
+FICofY=C comb/MIC alone+C comb/MIC alone,Where,MIC aloneandMIC alonearethe
X X X Y X Y
MICsofdrugXandYwhenactingaloneandC combandC combareconcentrationsofdrugs
X Y
XandYattheisoeffectivecombinations,respectively.Theinteractionwasdescribedassyner-
gisticwhentheFICIwas(cid:20)0.5,andwhen>4.0,itwasinterpretedasantagonistandanyvalue
inbetweenasindifferent[45].
Scanningelectronmicroscopy(SEM)of1ftreatedS.aureusandC.keyfercells. Thecell
suspensionsofS.aureusandC.keyferinexponentialphasewerepreparedinMHBandRPMI
1640medium(pH7),respectively.Compound1f(15.67μM)wasaddedtotheS.aureuscells
(~1x105CFU/mL),whereas1f(62.25μM)wasaddedtotheC.keyfer(~1x104CFU/mL)and
incubatedat37˚C(S.aureusfor12h)and30˚C(C.keyferfor12h).Aftertreatmentthesam-
plesforSEMstudywerepreparedasdescribedpreviouslyandobservedundertheSEM
(JEOL-6100F,JEOL,Japan)[47].
Mammaliancelltoxicity. Themammaliantoxicityofcompounds1a-1gagainstHek-293
(normalhumanembryonickidneycells)andHeLa(humancervicalcancercells)byMTT(3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide)protocol[48].Briefly,thecells
(5×104/well)wereculturedinRPMI1640mediumsupplementedwith10%FetalBovin
Serum(FBS)in96-wellmicrotiterplateat37˚Cforovernight.Thenextdaycompounds1a-1g
atMICconc.wereaddedtothecellsinseparatewellsandincubatedat37˚Cfor18h.Thecells
werefurtherincubatedat37˚Cfor3hto4hin20μLofMTTsolution(5mg/mL)inphos-
phatebuffersaline(PBS).Further,afterremovingsupernatant(120μL),Dimethylsulfoxide
(DMSO;100μL)wasadded,andthesuspensionwasmixedtodissolvetheformazancrystals.
ThepercentviabilityofcellswascalculatedbytheratioofO.D oftreatedcellstotheO.D
570 570
ofuntreatedcells.Untreatedcellsandnovobiocin(aminocoumarinantibiotic)weretakenas
negativeandpositivecontrol,respectively.
Computationalmethods
DFTstudies. Khon-Sham’sDFTmethodthatundergothegradient-correctedhybridden-
sityfunctionalB3LYPwereusedforalltheoreticalcalculations[49].TheBecke’sthreeparame-
tersfunctionalofLeeetal[50],isacombinationofnon-localexchangepotentialwiththenon-
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localcorrelationfunction.Afullgeometryoptimizationwasperformedforallstructures,
usingthisfunction[50]andthe6–31+g(d,p)basessetasimplementedbyGaussian03package
[51].AllgeometrieswerevisualizedusingAvogadro1.2softwarepackage.
Moleculardocking. Theprotein-ligandcrystalstructurehasbeenoptimizedtoitslower
energyconformationusingproteinpreparationwizardwhereallthewatermoleculeswere
removedduringpreprocessandmissingsidechainsofresidueshavebeenaddedusingprime.
Keepinginmindtheappropriateionizationstatesfortheacidicaswellasbasicaminoacids,
hydrogenatomswereaddedtotheproteinstructurecorrespondingtothephysiologicalpH
7.0.Finallyenergyminimizationwithroot-mean-squaredeviation(RMSD)valueof0.30Å
wascarriedoutusingoptimizedpotentialsforliquidsimulations(OPLS-2005)forcefieldafter
assigningchargeandprotonationstate.LigandpreparationwasdonebyusingtheSchrodinger
LigPreputility(Schrodinger,LLC,USA)whichgeneratesalowenergy3dstructures.
Theactivesiteofproteinwasdefinedbyaboundingbox(grid)thatwascenteredonthe
nativeligandinthecrystalcomplex.Extra-precisionglidedocking(GlideXP)whichdocks
ligandsflexiblywasusedtorankthedockingposesandtogauzethebindingaffinityofthese
ligandstowardtheprotein.
ADMEprediction. ADMEpropertieswerepredictedforsynthesizedcompounds1a–1g
usingcomputationalmethods.Inthisstudy,wehavecalculatedQpLogP /w,QPLogHERG,
o
QPPCaco,QpLogBB,QPPMDCK,QPlogKp,QpLogHSA,PercentageHumanoralabsorption
andLipinski’sruleoffiveusingQikProp.
Resultsanddiscussion
Chemistry
Inviewofouraimofbuildingcoumarin-thiazole-pyrazole(CTP)basedmolecularhybridsthe
coumarinanalogi.e.,6-substituted-3-acetylcoumarins(6)wassynthesisedfromareactionof
substitutedsalicylaldehydeswithethylacetoacetateinthepresenceofpiperidineascatalyst.
6-substituted-3-acetylcoumarins(6)thusformedwassubjectedtobrominationusingBr /
2
CHCl toyield6-substituted-3-bromoacetylcoumarins[52](7a-b)(Fig2).3,5-Dimethyl-
3
4-aryldiazenylpyrazole-1-thiocarboxamides(5a-d)requiredforthecondensationwith
6-substituted-3-bromoacetylcoumarins(7a-b)werepreparedbythereactionofthiosemicar-
bazidewith4a-daccordingtothepreviouslyreportedprocedures(Fig2)[53,54].Thecom-
pounds4a-dwerepreparedbycouplingofdiazotizedsubstitutedaniline(2)withacetyl
acetone(3)at0–5˚C(Fig2).
Inordertoachievethesynthesisof4-(coumarin-3-yl)-2-(3,5-dimethyl-4-aryldiazenylpyra-
zol-1-yl)thiazoles(1a-1g)exclusively,thecondensationof3,5-dimethyl-4-hydrazoarylpyra-
zole-1-thiocarboxamides(5)with6-substituted-3-bromoacetylcoumarins(7a-b)wascarried
outinrefluxingethanolunderalkalinecondition(Fig2).Thestructureofallthesynthesized
compoundswereestablishedbyanalyzingusingIR,NMR(1H,13C)andmass.
The1HNMRspectraofcompounds(1a-1g)revealedtwosingletsintherangeδ8.39–8.29
and8.00–8.10ppmcorrespondstocoumarin4-Handthiazole5-H,respectively,therebycon-
firmingtheformationof1.TwoSingletsintherangeδ2.19–2.32and2.69–2.86ppmappeared
dueto3-CH and5-CH groupattachedatpyrazolering.Ithasbeenwellestablishedthat
3 3
5-CH ismoredeshieldedcomparedto3-CH duetoalonepairofelectronspresentonthe
3 3
nitrogenatomofthethiazolenucleusandthereforeappearedataboutδ2.80ppm[41,55].
Moreover,theappearanceofothersignalsintheNMRspectracorrespondingtoaromatic
protonsandcarbonsfurhterestablishthestructureofthesynthesizedcompoundsandcom-
pleteassignmentof1HNMRalongwithotherspectraldatahasbeengiveninexperimental
section.
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CTPhybrids:Potentialstandaloneoradjuvantantimicrobialagents
Fig2.Synthesisof4-(coumarin-3-yl)-2-(3,5-dimethyl-4-hydrazoarylpyrazol-1-yl)thiazoles(1a-1g).
https://doi.org/10.1371/journal.pone.0196016.g002
Biologicalevaluation
Antimicrobialactivity. Theantimicrobialactivityofcompounds(1a-1g)wasevaluated
invitroaccordingtoClinicalandLaboratoryStandardInstitute(CLSI)guidelinesusingfive
clinicalbacterialstrains(Escherichiacoli,Staphylococcusaureus,Enterococcusfaecalis,Vibrio
choleraandStreptococcuspyogens)andsixfungalstrains(Candidaalbicans,Candidakrusei,
Candidaglabrata,Candidakeyfer,Candidatropicalis,Candidaparapsilosis).Theantimicrobial
activityofsynthesizedcompounds(1a-1g)rangedfrom250μMto15.67μMandresultshave
beensummarizedinTables1and2.Theactivitiesandefficaciesofthecompoundswerecom-
paredwithkanamycin,aroutinelyusedstandardantibiotic,andamphotericinB,awell-estab-
lishedantifungaldrug[56–58].Interestingly,thecompounds1band1fhavebeenfoundtobe
themostpotentantimicrobialagentsamongalltestedcompounds.Whenusedasastandalone
antibacterialagent,theMICof1bwaseithercomparable(S.aureus)oratleast50%reduced
(S.pyogens)comparedtoKAN.Incaseof1f,theMICis50%lesscomparedtothatofKAN
whentestedagainstS.aureus,E.faecalisandS.pyogens.
ThoughMICwasmuchhigherincaseofantifungalscreening,thecompounds1band1fas
standalonefoundtobemoreactiveincomparisontoothercandidateswhenAmpBwasused
asreferencedrug.Theresultsofantimicrobialactivitystudyrevealedthatincorporationofhal-
ogenatoms(ClandBr)oncoumarinylandphenylmoietieslinkedtopyrazolylthiazolesmay
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Table1. Minimuminhibitoryconcentration(MIC)ofthecompoundsagainstfivebacterialstrains.
Compound Minimuminhibitoryconcentrationvalues(cid:3)(μM)
S.aureus E.coli E.faecalis S.pyogens V.cholerae
1a >250 >250 >250 >250 >250
1b 31.25 >250 250 31.25 250
1c >250 >250 >250 >250 >250
1d >250 >250 >250 >250 >250
1e >250 >250 >250 >250 >250
1f 15.67 62.50 31.25 31.25 >250
1g >250 >250 >250 >250 >250
KAN# 31.25 03.90 62.50 62.50 62.50
(cid:3)MICvaluesofthecompoundswerecalculatedasCFUplatingmethodasdescribedinmaterialsandmethods
#KAN,afrequentlyusedantibacterialdrug,wasusedaspositivecontrolforthisstudy.
https://doi.org/10.1371/journal.pone.0196016.t001
beresponsibleforhigherpotentialof1band1f.Generally,substitutionofhydrogenbyhalo-
genincoumarinderivativescompoundsincreasesthelipophilicityandthusincreasestherate
ofcellpenetration,whichisanimportantfeatureofantimicrobialdrugefficiency[59].Substi-
tutionofchlorineatposition-6ofcoumarinringin1band1fsignificantlyincreasedactivity
againstthebacterialandfungalstrains.Theresultsdemonstratedthattheantimicrobialactiv-
ityoftheinvestigatedcompoundswasinfluencedbythephysicochemicalpropertiesofthe
typeandpositionofsubstituentonthearomaticring.
Furthermore,itisnoteworthyherethatS.aureusandcandidasp.areresponsibleformajor
opportunisticinfectionsinclinicalsettings.TherearegrowingconcernsabouttheabilityofS.
aureustoacquireresistance,eventowardsthenewestantibacterialavailableinthemarket[60].
Similarly,C.keyferinfectionsmostlyoccurinpatientswithhematologicmalignanciesorcarci-
nomaswhoarealreadyundergoinghighlycytotoxicchemotherapy[61].KANhasbeenused
asastandarddrugininfectionswithS.aureus[62].Likewise,AmpBisaoneoftheprimary
standarddrugsusedincaseofcandidainfections[61,63].Emergenceofresistanceagainst
thesefrontlinedrugshasbeenamajorpublichealthproblem.Animportantstrategytoreduce
theemergenceofdrugresistantstrainsisbyreducingtheeffectiveamountofdrugrequiredto
killtheinfectiousbacterialorfungalagent[64,65].Therefore,combinatorialtherapiesoruse
ofadjuvantisencouragedorusedtomodulatetheantimicrobialactivityandmitigatethepos-
sibilityofdevelopmentofdrugresistance[46].
Table2. Minimuminhibitoryconcentration(MIC)ofcompoundsagainstsixfungalstrains.
Compound Minimuminhibitoryconcentrationvalues(cid:3)(μMconc.)
C.albicans C.glabrata C.keyferkeyfer C.krusei C.tropicalis C.parapsilosis
1a >250 >250 >250 >250 >250 >250
1b 62.50 250 62.50 125 125 62.50
1c >250 >250 >250 >250 >250 >250
1d >250 >250 >250 >250 >250 >250
1e >250 >250 >250 >250 >250 >250
1f 62.50 250 62.50 62.50 125 62.50
1g >250 >250 >250 >250 >250 >250
AmpB# 0.78 0.78 12.50 0.78 0.78 0.78
#AmpB,wasusedaspositivecontrolforthisstudy
(cid:3)MICvaluesofthecompoundswerecalculatedasCFUplatingasdescribedinmaterialsandmethods
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Table3. Synergisticeffectofcompounds1band1fwithantibacterialandantifungaldrugsagainstS.aureusandC.keyfer.
Antibacterial/Antifungaldrug(μM) Compd.(μM) Conc.(μM)ofthedrugincombinationwith1bor1f Fractioninhibitoryconc.index(FICI)a Effect
4.0(KAN) 31.25(1b) 0.5(KAN)+7.81(1b) 0.500 Synergy
4.0(KAN) 15.67(1f) 0.5(KAN)+3.91(1f) 0.374 Synergy
12.50(AmpB) 62.50(1b) 3.12(AmpB)+7.81(1b) 0.364 Synergy
0.40(FLC) 62.50(1b) 0.08(FLC)+15.62(1b) 0.450 Synergy
12.50(AmpB) 62.50(1f) 1.56(AmpB)+6.25(1f) 0.240 Synergy
0.40(FLC) 62.50(1f) 0.05(FLC)+15.67(1f) 0.375 Synergy
aThefractioninhibitoryconcentrationindex(FICI)valueisdefinedasthecombinationoftwoactiveagentscanexertinhibitoryeffectsthataremorethanthesumof
theireffectalone.
https://doi.org/10.1371/journal.pone.0196016.t003
Synergisticeffect. Asdiscussedabove,wefurtherevaluatedwhetherthecompounds1b
or1fcanactasanadjuncttopotentiateareductioninMICofKANandAmpBagainstclinical
strainsS.aureusandC.Keyfer,respectively.Acheckerboarddilutionassayhasbeenusedto
determinethefractioninhibitoryconcentrationindex(FICI)forthecompounds1band1f,
whenusedincombinationwithKANandAmpBagainstS.aureusandC.Keyfer,respectively.
AsevidentfromTable3,weobservedanFICIof(cid:20)0.5inalldrugcombinationsreportedhere
indicatingasynergisticinteractionbetweenthecompoundsandknowndrugs.Therewasa
substantial8-foldreductionintheMICofKANagainstS.aureuswithcompounds1b,and
alsowith1f.Likewise,incaseofAmpB,thereisa5-foldreductioninMICwithcompound1b
and~8foldreductionwithcompound1fagainstfungalpathogenC.keyfer.
Cytotoxicevaluation. EvaluationofcytotoxicityusingHeLacells(cervicalcancercell)
andHek-293cells(normalembryonickidneycells)indicatedthatthecompounds1band1f
havecomparablecytotoxicity(>80%survival)atMICconcentrations(Fig3)andnegligible
cytotoxicity(>90%survival)attheconcentrationsusedincombination.Theircytotoxicity
wascomparablewithNovobiocin,acoumarinderivativeusedasapositivecontrol[48].Most
potentcompoundsemergingoutofourscreen1band1fhavecomparablecytotoxicityattheir
respectiveMICconcentrations.
Fig3.Evaluationofcytotoxicity.Celltoxicityofcompounds1a-1gintermofviabilityofHek-293andHeLacells.Datarepresent
resultsfromthreeindependentexperiments.Novobiocinwasusedasapositivecontrol.
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