Synthesisandtype-IIanti-diabeticactivityofPyrrolidine-2-carbonitrilederivedLigandsinSHR-STZAnimalmodel

 

BabasoV.Udugade^1*,ShivajiP.Gawade²

¹DepartmentofMedicinalChemistry,SataraCollegeofPharmacy,Degaon,Satara-415004MaharashtraIndia

²DepartmentofPharmacology,SahyadriCollegeofPharmacy,Methawade,Sangola-413307,Maharashtra,India

*CorrespondingAuthorE-mail:swarajudugade@gmail.com

Wereportedthesynthesisandtype-IIanti-diabeticactivityofpyrrolidine-2-carbonitrilederivedligandsinSHR-STZanimalmodels.3DimensionalQuantitativeStructuralactivity,pharmacophore,dockingstudiesusedfordesignofmolecules,studiesreveledthatnovelsubstitutedcyanopyrrolidineshadbeenpromisingcandidateforfurtherresearch.Moleculeshadbeensynthesizedandevaluatedforanti-diabeticactivitythroughtheuseofSpontaneouslyHypertensiveRat-Streptozotocinanimalmodel.Amongstallofthesynthesizedcompounds1-(2-(5-methylisoxazol-3-ylamino)acetyl)pyrrolidine-2-carbonitrile,1-(2-(5-methyl-1,2,4-oxadiazol-3-ylamino)acetyl)pyrrolidine-2-carbonitrileand1-(2-(1,2,4-thiadiazol-5-ylamino)acetyl)pyrrolidine-2-carbonitrilehadbeenfoundtobestronganti-diabeticactivity.

 

 

 

INTRODUCTION:

TypeIIDMisadisordercharacterisedbyimpairedmanagementofbloodsugarlevel,isprevailingworldwidemovingnearly6%ofthepopulation.¹It'soneamongstthequickestgrowinghealthissuesworldwideandwillhaveaneffecton366millionindividualswithinthenextthirtyyearsifcorrectpreventivemeasuresdon'tseemtobeenforcedwithintheimmediatefuture.²Indiaturningintohubofdiabetics.^3,4ThepresentoraltreatmentchoicesfortypeIIDMincludemetformin,sulfonylureaandthiazolidinedionederivatives,glycosidaseinhibitorsandthereforetherecentlyintroduceddipeptidylpeptidase-4(DPP-4)inhibitors.^5,6

 

DPP-4inhibitorsinhibittheenzymeDPP-4,aserineproteasethatdegradestheincretinhormone,glucagon-likepeptide-1(GLP-1),quicklytoitsinactiveform.GLP-1isfreewithinthegutinresponsetotheingestionoffoodandstimulatesinsulinbiogenesisandsecretion,whereasinhibitingthedischargeofglucagonwiththeexceptionofmanydifferenthelpfuleffects;GLP-1regulatesinsulininastrictlyglucose-dependentmanner.Thus,inhibitionofDPP-4hasbeenshowntoextendthehalf-lifeofGLP-1andtoprolongtheusefuleffectsofthisincretinhormone.⁷Moreover,DPP-4inhibitorsdidn'tshowtheundesirableaspecteffects,likeweightgainandhypoglycaemiathatareobservedwiththeutilizationofalternativeanti-diabeticagents.⁸Intenseresearchactivitiesduringthisareahaveresultedinthelaunchofsitagliptinandvildagliptinandtheadvancementofacoupleofalternativepotentialmedicationintopreregistration/phase3,e.g.,saxagliptinandalogliptin.^9,10VarietyofreviewarticlesarecurrentlyavailablethatcovernumerousaspectsofDPP-4inhibitorsextensively.^11-19ClinicalinformationrevealsthattherecentDPP-IVinhibitorsofferseveralprospectivebenefits,investigatingnoorlessweightgainandnoriskofhypoglycaemia.Still,somesideeffectsarewiththem,togetherwithsorethroat,gastrointestinalissueslikeloosenessofthebowelsanduppertractinfectionwiththeexceptionofthesesideeffects;reportedcompoundsareoflesspotent.Thus,there'sanecessitytoidentifynewpharmacologicalcompoundsthatmaynotonlytreathyperglycemiabutmayalsocorrectimpairedglucosephysiologicalconditionandpreserveendogenousβ-cellfunction,becausepatientswithnewdiagnoseddiabeteshaveonlyabout50%normalβ-cellfunction,withfurtherprogressivelossovertime.Aboveobservationsindicatesneedofpresentinvestigationondesignandsynthesisofnovelcyanopyrrolidinederivativesasantidiabeticagentswhichcaninhibittheactionofdipeptidylpeptidase-IVonGLP-1toattainmanagementonhyperglycemiawhilenotorwithnegligibleadverseeffects.WearegoingtoplacerigidconformationonP2siteofDPP-IVinhibitorspharmacophorethenefficiencyofcynopyrrolidinecontainingDDP-IVinhibitorswillincrease.Withthismotivation,wehereindiscloseddesign,synthesis,andevaluationofnovelcyanopyrrolidinecontainingDDP-IVinhibitors

 

MATERIALANDMETHODS:

Synthesis:

ChemicalsandsolventswereprocuredfromAldrichIndiaLtd.,andE.MerckIndiaLtd.ThesesolventsandreagentswereofLRgradeandifnecessaryrefinedbeforeuse.Meltingpointsweredeterminedwithlaboratorymeltingpointequipmentbyanopencapillarytechniqueandareuncorrected.TheIRspectrawererecordedonAT-FT-IRspectrophotometer(Bruker).ProtonmagneticresonancespectrawererecordedusingDMSO-d6assolventandTMSasaninternalstandardonBrukerNMRspectrometer.Chemicalshiftvalues(δscale)giveninppm.ThemassspectrawererecordedonaMSlowresolutionmassspectrometeroperatingat70ev.Thepurityofthecompoundswascheckedbythin-layerchromatography(TLC)onsilicagelplatewithvisualisationofcomponentsbyultravioletillumination(254nm).Alltestedcompounds’puritywaslargerthan95%.

 

Synthesisof1-(2-chloroacetyl)pyrrolidine-2-carbonitrile:

Wesynthesizedkeyintermediates,1-(2-chloroacetyl)pyrrolidine-2-carbonitrile(4),fromLproline(1)asperliteratureprotocol(scheme-1)²⁰Thepurecompoundwasobtainedasabrownishwhitesolid,Yield=55%;mp=50-560C;Rf=0.6(Methanol:Chloroform=8:2);ATR-FT-IR(cm-1):(C-C-H)2910,(C=C-H)3052,(C≡N)2219,(C=O)1660,(C-N)1253,1294,(C-O)1149,(Cl)767.1HNMR(300MHz,CDCl3):δ(ppm)1.700.1–2.101(s,2.17H,CH2),2.600δ(s,2.27H,CH2)3.520(s,2.28H,CH2)3.477-4.503(m,3.21H,Cl-CH2).LC-MSm/z:M+172(100%)

Generalprocedureforthesynthesisof1-(2-chloroacetyl)pyrrolidine-2-carbonitrilederivatives:(CYP-1toCYP-8)

Reportedmethod²¹wasusedforsynthesisof1-(2-chloroacetyl)pyrrolidine-2-carbonitrilederivatives(CYP-1toCYP-8).A250mlreactorwiththermometer,condenserandmagneticstirrerwaschargedwithTHF(50ml),powderedK2CO30.08mol,0.05mol,1-chloroacetyl-2-cyanopyrrolidine0.02molandKI0.001mol.TheresultingslurrywasheatedtorefluxuntilcompleteconversionbyTLC(approx.2h).Thewarmsuspensionwasfilteredandthesolidswashedwith20mlofTHF.Solventsweredistilledofftoobtainasolid.Thissolidwassuspendedin30mlofMEKandheatedtoreflux.Theresultingclearsolutionwasallowedtocoolandtheproductcrystallizedasawhitesolid.Theslurrywasstirredat0°C.for1hour,filtered,washeddriedundervacuumtoobtainfinalproduct.

 

 

Figure1:Synthesisof1-(2-chloroacetyl)pyrrolidine-2-carbonitrilederivatives.ReagentsandConditions:(i)Chloroacetylchloride,THF,reflux,2h.;(ii)1:DCC,DCM,stir,RT,2h.;2:NH₄HCO₃,stir,RT,1h.;(iii)1:TFA,THF,15^OC-RT,1h.;2:NH₄HCO₃,C₆H₅CH₃,RT,1h.;(iv)K₂CO_3,KI,THF,reflux,2h.

 

 

Synthesisof1-(2-(5-methylisoxazol-3-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-1):

Thepurecompoundwasobtainedasawhitesolid,Thepurecompoundwasobtainedasawhitesolid,Yield=59%;mp=176-1780C;Rf=0.8(Hexane:Ethylacetate(9:1);ATR-FT-IR(cm-1):(C-C-H)2911,(C=C-H)2983,(NH)3357,(C≡N)2360,(C=O)1662.1HNMR(300MHz,CDCl3):δ(ppm)1.400.1δ(s,2.12H,CH2),2.120-2.270(d,2.02H,CH2)2.779δ(s,3.10H,CH3)3.635-3.662(d,2.11H,CH2),4.116-4.140(s,2.87H,C-CH2-NH).LCMSm/z:234(100%),235(12%),234(2%),233(8%)

Synthesisof1-(2-(isoxazol-4-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-2):Thepurecompoundwasobtainedasawhitesolid,Yield=68%;mp=130-1360C;Rf=0.7(Hexane:Ethylacetate(9:1);ATR-FT-IR(cm-1):(C-C-H)2907,(C=C-H)2999,(NH)3280,(C≡N)2212,(C=O)1654.1HNMR(300MHz,CDCl3):δ(ppm)1.920(s,2.01H,CH2),2.620-2.650(s,2.11H,CH2)3.620-3.629(m,4.17H,CH2)4.309(s,0.71H,-NH-),4.915-5.062(d,3.10H2,CH).LCMSm/z:220(100%),221(10.5%),222(7%)219(5%),218(1%)

 

Synthesisof1-(2-(5-methyl-1,2,4-oxadiazol-3-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-3):

Thepurecompoundwasobtainedasawhitesolid,Yield=58%;mp=118-1200C;Rf=0.7(Hexane:Ethylacetate(8:2);ATR-FT-IR(cm-1):(C-C-H)2907,(C=CH)2999,(NH)3285(C≡N)2227,(C=O)1654.1HNMR(300MHz,CDCl3):δ(ppm)1.420-1.510(d,2.00H,CH2),2.120(d,2.82HH,CH3)2.410(s,2.13H,CH2)3.540(t,3.83H,CCH2-NH-).4.413-4.439(d,0.83H,-NH-),4.820-4.917(m,1.30H,CH2),LCMSm/z:m/e:235(100.0%),236(11.0%),237.11(2%)

 

Synthesisof1-(2-(4H-1,2,4-triazol-3-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-4):

Thepurecompoundwasobtainedasawhitesolid,Yield=59%;mp=120-1210C;Rf=0.7(Hexane:Ethylacetate(8:2);ATR-FT-IR(cm-1):(C-C-H)2883,(C=C-H)3003,(NH)3279,(C≡N)2245,(C=O)1666.1HNMR(300MHz,CDCl3):δ(ppm)2.441-2.632(d,3.82H,CH2),3.672(d,1.83H,CH2)4.021(d,1.10H,NH2)4.141(s,2.02H,C-CH2-NH-),4.404(s,0.77H,-NH-),4.844(s,1.23H,-CH-).7.646(s,0.71H,-CH-).LCMSm/z:m/e:235(100.0%),236(11.0%),237.11(2%)

 

Synthesisof1-(2-((tetrahydro-2H-pyran-4-yl)methylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-5):

Thepurecompoundwasobtainedasawhitesolid,Yield=80%;mp=198-2020C;Rf=0.8(Hexane:Ethylacetate(9:1);ATR-FT-IR(cm-1):(C-C-H)2926,,(C=CH)2991,(NH)3325,(C≡N)2204,(C=O)1660,1HNMR(300MHz,CDCl3):δ(ppm)1.308.1–1.317(m,4.97H,CH2),2.120-2.341(m,7.02H,-NH-,CH2)3.287-3.341(d,2.29H,CH2)3.513(s,1.12H,-CH2-NH-),3.598(d,3.74H,CH2),4.607(s,1.03H,CH2).LCMSm/z:220(100%),221(10%),222(9%)223(2%)

 

Synthesisof1-(2-(1,2,4-thiadiazol-5-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-6):

Thepurecompoundwasobtainedasawhitesolid,Yield=90%;mp=130-1340C;Rf=0.6(Hexane:Ethylacetate(9:1);ATR-FT-IR(cm-1):(C-C-H)2919,(C=C-H)3068,(NH)3295(C≡N)2218,(C=O)1672,1HNMR(300MHz,CDCl3):δ(ppm)2.371–2.350(d,3.42H,CH2),3.550(m,4.27H,CH2)4.367-4.394(d,1.20H,-NH-)4.474-4.999(m,1.17H,CH2),7.169(m,1.03H,CH)LCMSm/z:251.16(100%),252(14%),253(3%),254(1%)

 

Synthesisof1-(2-(1H-pyrrol-1-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-7):Thepurecompoundwasobtainedasawhitesolid,Yield=88%;mp=110-1120C;Rf=0.7(Hexane:Ethylacetate(9:1);ATR-FT-IR(cm-1):(C-C-H)2888,(C=C-H)3090,(NH)3696(C≡N)2261,(C=O)1661.1HNMR(300MHz,CDCl3):δ(ppm)1.432–1.533(d,1.39H,-NH-),2.050(s,1.91H,CH2)2.869-3.059(s,2.21H,CH2)3.222-3.572(d,4.14H,CH2).4.150(s,1.07H,CH2)6.352(s,4.13H,CH),LCMSm/z:237(100%),238(10.5%),239(4%)

 

Synthesisof1-(2-(2-ethyl-2H-tetrazol-5-ylamino)acetyl)pyrrolidine-2-carbonitrile(CYP-8):

Thepurecompoundwasobtainedasawhitesolid,Yield=86%;mp=150-1520C;Rf=0.8(Hexane:Ethylacetate(8:2);ATR-FT-IR(cm-1):(C-C-H)2871,(C=C-H)3055,(NH)3301,(C≡N)2267,(C=O)1655.1HNMR(300MHz,CDCl3):δ(ppm)1.210(s,3.12H,CH3),2.050(m,3.53H,CH2)3.151(s,2.10H,CH2)3.567-3.797(m,3.89H,Cl-CH2),4.110(s,0.97H,NH),4.472-4.511(s,1.09H,CH2).LCMSm/z:218(100%),219(12%),220(2%),221(1%)

 

PharmacologicalEvaluation:

ProtocolforanimalstudieswasapprovedbytheInstituteAnimalEthicsCommittee(IAEC),SataraCollegeofpharmacy,Satara,Maharashtra,India(Refno.SCOP/IEAC/43/14-15)andCommitteeforthePurposeofControlandSupervisionofExperimentsonAnimals(CPCSEA)guidelineswerefollowedforthemaintenanceofexperimentalanimals.

 

TypeIIAnti-diabeticactivitybyusingSHR-STZanimalmodel:

Fructoseinducedhypertensioninrats:

Increasesindietarycarbohydrateintakecanraisebloodpressureinexperimentalanimals.Theincreasedintakeofeithersucroseorglucosewasshowntoenhancethedevelopmentofeitherspontaneoushypertensionorsalthypertensioninrats.Groupsof40maleWistarratsweighing210–250gareused.Theywerehousedona12-hlight12-hdarkcycleandareallowedfreeaccesstostandardlaboratorydiet(Purinaratchow)anddrinkingfluid.Drinkingfluidconsistof10%-fructoseaddedwith4%NaClsolution.Usingthetail-cuffmethod,systolicbloodpressureandheartrateismeasuredbeforeandeverymonthduringtreatment.Outof40animals1animalomittedfromthestudybecauseofmildhypertension.

 

InductionofDiabetes:

Diabeteswasinducedin33ratesoutof39HypertensiveratesbyintravenouslyinjectingStreptozotocin(55mg/kgin0.9%NaCl).Controlrats(n=6)wereinjectedwithvehiclealone.Diabeteswasverified72hlaterbyevaluatingbloodglucoselevelswiththeuseofGlucometer.Ratshavingbloodglucoselevelof300mg/dlorgreaterwereconsideredtobediabetic.Outof33animals2diedandonewasomittedfromthestudybecauseofmildhyperglycemia.Remaining30diabeticanimalsweredividedinto5groupseachhaving6rats.Group1:VehicleControlrats,fednormalpelletdiet,receivedonlysinglei.p.injectionofcitratebuffer(1ml/kg)andservedasVehiclecontrolgroup.Group2:diabeticratsreceivedVildagliptinasstandarddrug.Group3:Diabeticcontrolratsreceivedonlyvehicleandservedasdiabeticcontrolgroup.Group4(MTD):diabeticratsreceivedMaximumTherapeuticDoseofcyanopyrrolidinederivatives.Group5(MTD/2):diabeticratsreceivedhalfofMaximumTherapeuticDose.Group6(MTD*2):diabeticratsreceiveddoubleofMaximumTherapeuticDose.^22,23

 

BloodpressureandheartratemeasuredbyTail-cuffmethod:

AllbloodpressuremeasurementsweredoneonratsusedinSHR-STZdiabetesmodelusingCODANon-InvasiveBloodPressureRecorderusingrattail-cuffmethod(KentScientificCorporation,Torrington,Connecticut,USA).Beforeyoubeginofexperimenttrainingwaregiventoanimalbykeepingtheminholderforfifteenminutesessions.Animalwarmingplatformweresetonlevel3.Animalwasplacedintotheholderandthebaseoftheanimal’stailinsertedintotheOcclusionCufffollowedbyVPRcuffandrunthesoftwareandbloodpressureandheartratewererecorded.²⁴

 

Lipidprofile:

BloodLipidprofileconsistingTotalcholesterol(TC),Triglycerides(TG),HighDensityLipoproteins(HDL),LowDensityLipoproteins(LDL)andVeryLowDensityLipoproteins(VLDL)establishedonratsusedinSHR-STZdiabetesmodel.Bloodsamples(2–3ml)forassayoftheserumlipidprofilewerecollectedfromtheratsbytheretro-orbitalpuncturemethodundergentleetheranaesthesia,aftera12-hovernightfast.Thebloodsamplesweredispensedintocleanplainglasstesttubesandallowedtostandfor30minatroomtemperature.Serumfortheassayswasthereafterseparatedfromtheclotbycentrifugationto3000rpmfor20min.Allthebiochemicaldeterminationswerecarriedoutimmediatelyafterseparationoftheserumfromtheclot.Totalcholesterol(TC),Triglycerides(TG),HighDensityLipoproteins(HDL),LowDensityLipoproteins(LDL)andVeryLowDensityLipoproteins(VLDL)determinedbyusingprietestTMclinicalchemistryreagentskitsmanufacturedbyROBONIK,Mumbai.²⁵

 

RESULTSANDDISCUSSION:

Table1:Selectedcompoundsforsynthesiswithcode,formulaandchemicalname

AllabovecompoundswereselectedforsynthesisbasedonSAR,QSAR,Pharmacophoreanddockingstudies.

 

 

Table2:Physiochemicalcharacterizationsofsynthesizedcompounds

Where;CN=1-(2-chloroacetyl)pyrrolidine-2-carbonitrileandCYP=Cyanopyrrolidines

 

 

Resultsofpharmacologicalevaluation:

TypeIIAnti-diabeticactivitybySHR-STZanimalmodel:

Table3:EffectofCYP-1,CYP-3andCYP-6onBodyweight,Bloodglucose,bloodpressureandheartrate

Allthedatawereexpressedasmean±S.E.M.(n=6).StatisticalsignificancewasdeterminedbyonewayANOVA(AnalysisofVariance)FollowedbyDunnetmultiplecomparisontestbyusingtheGraphpadPrismversion6.a=*P<0.05,b=**P<0.01andc=***P<0.001wereregardedassignificant

 

Lipidprofile:

Table4:EffectofCYP-1onlipidprofile

Allthedatawereexpressedasmean±S.E.M.(n=6).StatisticalsignificancewasdeterminedbyonewayANOVA(AnalysisofVariance)FollowedbyDunnetmultiplecomparisontestbyusingtheGraphpadPrismversion6.a=*P<0.05,b=**P<0.01andc=***P<0.001wereregardedassignificant

 

HistopathologyofPancreasandheartmuscles:

Histopathologicalchangesinpancreasandheartmuscleswereillustratedinfigure2and3respectively.InNormalcontrolacinarcellswereseentobenormal.Theisletpresentinadequateproportionbetacells.Therewasnoevidenceofinfiltrationorfibrosis.MTDx2demonstratessectionofregularpancreasofrat.Itexplainsnormalacinarpattern,isletcellsandnoevidenceofinfiltration.Numbersofisletsofβ-cellswerefoundtobeincreasedascomparedtotherapeuticdose.Mainlyregenerationofpancreaticβ-cellswasobserved.Theacinarcellsareseentobenormal.Heartmusclesfoundtobenormalexceptdiabeticgroup

 

Figure 2: Microphotographs of Histopathological study of pancreas

 

 

Figure 3: Microphotographs of Histopathological study of heart muscles

Where; MTD= Maximum Therapeutic Dose MTD/2= Half Maximum Therapeutic Dose and MTDx2= Double Maximum Therapeutic Dose

 

Conclusion:

EightpotentialnoveltypeIIantidiabeticagentsCYP-1toCYP-8CYP-1toCYP-8compoundswereselectedbydrugdesigntogetherwith3DQSAR,pharmacophoremodelinganddockingstudiesandsynthesized.Thepurityofthecompoundswascheckedbythin-layerchromatographyandmeltingpoints.StructuresofallwereconfirmedbyinterpretationofIR,1HmagneticresonanceandMassspectra.TypeIIanti-diabeticactivityofsynthesizedcompoundswasperformedinSHR-STZanimalmodelinwhichsynthesizeddrugstestedin3doselevelsMTD,MTD/2andMTDx2.CYP-1,CYP-3andCYP-6foundtobepromisingoneindecreasingenhancedbloodserumglucoselevelasstandardvildagliptin.Bloodpressureandheartbeatswerefoundtobefluctuatingoneascomparetostandardvildagliptin.lipidprofileofcontrolgroupwascomparedwithdiabetes.ithadbeenfoundtobethelevelsoftriglycerides,totalcholesterol,andldlwereenhancedconsiderably(P<0.01)whereasHDLlevelsweredecreased(P<0.05)group.whentreatmentwithVildagliptin,CYP-1CYP-3andCYP-6lipidprofilewerefoundtobeimprovedascomparedtothediabeticgroup.VildagliptinwasmoreeffectiveinreducingserumtriglyceridesvaluesthanCYP-1,CYP-2andCYP-6(P<0.01)AlltheseobservationandresultsitcanbeconcludedthatsynthesisofnoveltypeIIantidiabetic’sagentwerecarriedoutwithsuccesswithfruitfulresultsasCYP-1,CYP-3andCYP-6showingoutstandingresultsinanimalmodelstheycanbethemostpromisingcandidatesforhumanstudies.

 

Acknowledgement:

Thisresearchdidn'treceiveanyspecificgrantfromfundingagenciesinthepublic,commercial,ornot-for-profitsectors.AuthorsaregratefultoPrincipal,SataracollegeofPharmacy,Degaon,Satara,Maharashtra,Indiaforprovidingfacilitiestocarryoutthisresearchwork.

 

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Receivedon13.12.2017Modifiedon06.01.2018

Acceptedon24.01.2018©AJRCAllrightreserved