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J.Med.Chem.2004,47,1719-

N-(Cycloalkylamino)acyl-2-aminothiazoleInhibitorsofCyclin-Dependent2.N-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide(BMS-387032),aHighlyEfficaciousandSelectiveAntitumorAgentRajN.Misra,*Hai-yunXiao,KyoungS.Kim,SongfengLu,Wen-ChingHan,StephanieA.JohnT.Hunt,DavidB.Rawlins,?WeifangShan,SyedZ.Ahmed,?LigangQian,Bang-ChiChen,RulinZhao,MarkS.Bednarz,?KristenA.Kellar,?JanetG.Mulheron,?RobertaBatorsky,UrvashiRoongta,AmritaKamath,PunitMarathe,SunandaA.Ranadive,JohnS.Sack,JohnS.Tokarski,NikolaP.Pavletich,|FrancisY.F.Lee,KevinR.Webster,§andS.DavidKimball?Bristol-MyersSquibbPharmaceuticalResearchInstitute,POBox4000,Princeton,NewJersey08543-4000ReceivedNovember4,2003tobepotent,selectiveinhibitorsofCDK2/cycEwhichexhibitantitumoractivityinmice.Inparticular,compound21N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide,BMS-387032,hasbeenidentifiedasanATP-competitiveandCDK2-selectiveinhibitorwhichhasbeenselectedtoenterPhase1humanclinicaltrialsasanantitumoragent.Inacell-freeenzymeassay,21showedaCDK2/cycEIC50)48nMandwas10-and20-foldselectiveoverCDK1/cycBandCDK4/cycD,respectively.Itwasalsohighlyselectiveoverapanelof12unrelatedkinases.AntiproliferativeactivitywasestablishedinanA2780cellularcytotoxicityassayinwhich21showedanIC50)95nM.Metabolismandpharmacokineticstudiesshowedthat21exhibitedaplasmahalf-lifeof5-7hinthreespeciestomouse,rat,anddog,21showed100%,31%,and28%bioavailability,respectively.Asanantitumoragentinmice,21administeredatitsmaximum-tolerateddoseexhibitedaclearlysuperiorefficacyprofilewhencomparedtoflavopiridolinbothanip/ipP388murinetumormodelandinasc/ipA2780humanovariancarcinomaxenograftmodel.Cyclin-dependentkinases(CDKs)areafamilyofserine\threonineproteinkinaseswhichplaykeyrolesinthenormalgrowthandlifecycleofeukaryoticcells.1Specifically,CDKs,alongwiththeiractivatingsubunitcyclinsorinhibitorysubunitCDKi(e.g.,p16,p21,p27),areresponsibleforcellularresponsetoexternalstimuliorinsults.Togethertheyprovidefortheorderlycoor-dinationofcellulareventsthroughthecellcycleandensurecomplianceofthegeneticintegrityofnewcells.Becauseoftheirkeyroleasdriversofcellgrowthanddivision,theirmisregulationinanumberofcancers,2andthestrongpositivecorrelationbetweenCDK2activityandpoorclinicalprognosisincancerpatients,3oncologydrugdiscoveryprogramshavedirectedmajoreffortstowardtheidentificationofsmallmoleculeinhibitorsofCDKsaspotentialantitumortherapeutic

Figure1.StructuresofCDKinhibitorsflavopiridol(1)andaminothiazole2.abenchmarkbroad-spectrumATP-competitive advancedclinicaltrialsasanantitumoragent.5*Towhomcorrespondenceshouldbeaddressed.RajN.Misra,Ph.D.,12EatonPlace,Hopewell,NJ08525.E-mail:raj_n_misra@?Currentaddress:LexiconPharmaceuticals,350CarterRd.,Prin-ceton,NJ08540.?Currentaddress:FMCCorporation,POBox8,Princeton,NJ§Currentaddress:Astra-ZenecaR&DBoston,35GatehouseDr.,Waltham,MA02451.|Affiliation:MemorialSloan-KetteringCancerCenter,NewYork,

recentlydescribedN-phenylacetyl-2-aminothiazole2asactivitysuperiorto1.6Inthisdisclosurewedescribestructuralstudiesofaseriesrelatedto2,theN-(cy-cloalkylamino)acyl-2-aminothiazoles.7Itisfromthisseriesthatourclinicaldevelopmentcandidatehas10.1021/jm0305568CCC:$27.50 ?2004AmericanChemicalSocietyPublishedonWeb03/02/2004想了解更多內(nèi)容可以參考以下地址/////////1720JournalofMedicinalChemistry,2004,Vol.47,No. MisraetSchemeaReactionconditions.(a)NaN3,acetone,25°C,100%;(b)Pd/C,H2,aqHCl,MeOH,25°C,91%;(c)Et3N/CH2Cl2,-10°C,98%;(d)POCl3,105°C,80%;(e)Br2,KSCN,MeOH,26%;(f)NaBH4(2equiv),EtOH,25°Cfollowedbychloride6(1.1equiv),reflux,94%;(g)RCOOH,EDAC(2equiv),DMAP(1equiv),DMF,CH2Cl2,25°C.SchemeCH2Cl2,25°C,97%;(c)4NHClindioxane,CHCl3,45-55°C,83%.antitumoractivityofselectedanaloguesincludingourclinicaldevelopmentcandidateisdisclosed.SyntheticChemistryMethods.Aconvergentsyn-theticroutewhichinvolvedthecouplingofchlorom-loguesofgeneralstructure9(specificexamples12-27).Thus,asshowninScheme1,commerciallyavailableR-bromopinacolone3wasconvertedsmoothlytoR-ami-bycatalytichydrogenation.Acylationof4withR-chlo-roacetylchlorideaffordedketo-amide5whichwascyclizedtokeychloromethyloxazole6inrefluxingratedbytreatmentofcommerciallyavailable2-ami-nothiazolewithbromineandpotassiumthiocyanatetogive7inalowyieldbutmoderatelyscalableprocess.8Reductionof7byexposuretosodiumborohydrideinwithchloromethyloxazole6gavekey2-aminothiazoleintermediate8.Ingeneral,amine8wascoupledeitherdirectlytotheappropriatecarboxylicacidorwhennecessarytheN-t-Bocprotectedcarboxylicacidusing1-(3-dimethylami-presenceof4-(dimethylamino)pyridine(DMAP)and/orN-hydroxybenzotriazole,followedbyremovaloftheprotectinggrouptoaffordthedesiredacylated

analogues9.Aspecificexampleforthepreparation21isshowninScheme2.Compounds22-24werepreparedfrom21byelaborationoftheaminofunction-alityusingstandardknownsyntheticmethods.ResultsandInVitroBiologicalEvaluation.Compoundswereinitiallyevaluatedinacell-freeenzymeassayforawholecell72hcytotoxicityassay.Anovariancancercellline,A2780,wasutilizedinthecellularcytotoxitypreviouslyandareincludedintheExperimentalSec-tion.6TheresultsoftheseassaysareincludedinTable1aspartoftheSARstudyforthisseries.Structure-ActivityStudies.SummarizedinTable1istheSARofaseriesofN-acyl-2-aminothiazolesinwhichanonaromaticsidechaincontainingabasicaminehasbeenintroducedontheacylsubstituent.PreviousSARandstructuralstudiesinthisserieshadestablishedthattheacylsidechainextendstowardtheexterioroftheproteinandwasamenabletosignificantstructuralmodification.6ThisfindingwasexploitedtocontainsanaromaticacylsidechainwithabasicaminoInhibitorsofCyclin-DependentKinase JournalofMedicinalChemistry,2004,Vol.47,No.7Table1.Structure-ActivityRelationshipStudiesofAminothiazoles12-27aaSeeref6andorExperimentalSectionfordescriptionofassayconditions.bAssaywasperformedemploying25μM[ATP].toincreasesolubilityandreducebothproteinbindingandmetabolictransformation.Importantly,thismodi-ficationwasakeytoimprovinginvivoantitumorefficacy.Thisreportistheconclusionofthisworkinwhichwehavefocusedonexamininglowermolecularweightnonaromaticacylsidechainswithbasicaminesandhasledtotheidentificationofourclinicaldevelop-mentcandidate.Initialcomparisonofanalogues12and13indicatedthatintroductionofaprimaryamine

thesidechainaffordsapotentenzymeinhibitorwithonlya4-folddecreaseinCDK2inhibitoryactivitybutalarger10-foldlossofpotencyinthecellularantipro-ingtheaminegroupwithinorontoaringwewereabletorestorecellularantiproliferativeactivity(seeana-logues19-23,videinfra).Analoguesinwhichameth-ylenegroupispresentbetweenthecarbonylgroupandthecyclicsubstituent(14vs15,16vs17,20vs21)exhibiteda4-7-foldincreaseinenzymepotencyversusanaloguesinwhichamethylenespacerwasabsent.This,however,wasnotnecessarilytranslatedintoanincreaseincellularantiproliferativeactivity(e.g.,20vs21).Wespeculatethattheallowedorientationsofacyclicringattacheddirectlytothecarbonylgrouparesufficientlyrestrictedthatitdoesnotallowforsimul-taneousoptimalbindingofthesidechainandtheaminothiazolering.Introductionofamethylenespacerprovidesthenecessaryflexibilitytooptimizebindingofofphenylanalogues14and15withtheircyclohexylanalogues16and17indicatedthatthecarbocycleswere～15-foldlesspotentthantheirphenylcounterparts.Thereducedenzymeactivityof16and17relativetothephenylanaloguesmaybeduetoincreasedstericbulkofthecyclohexylgroupand/oritsincreasedlipohilicity.Comparisonofthecyclohexylanalogues16and17withtheirpiperidinylcounterparts20and21,however,revealeda5to6-foldincreaseinenzymeactivityforthepiperidinylanalogues.Amines20and21wereonly2to3-foldlesspotentthanphenylanalogues14and15.Thiscomparisonsupportsthehypothesisthatthereisnotastericissuewiththecyclohexylringsincethepiperidinylringisessentiallythesamesize.Thebasisforthediminishedactivityof16and17seemstobethehighlipophilicity(i.e.,lowisconsistentwiththesolid-statestructuraldatawhichindicatesthattheacylsidechainextendsintothehydrophilicenvironmentattheinterfaceoftheproteinandaqueousexternalenvironment.Highlylipophilicgroupswouldbeexpectedtobepoorlyaccommodatedinthisregion.Becauseoftheiracceptableenzymeandandreducedproteinbinding,wefocusedmorecloselyontheexplorationofanaloguescontaininganami-nocycleinthesidechainandemployed21asourbenchmarkinhibitor.Ringsizeandsubstitutionwereexploredthroughanalogues18and19.Thus,theracemicprolineanalogue18showedsimilarenzymeinhibitorypotencyto21althoughitwas16-foldlesspotentincells.Theracemic3-piperidinylanalogue19,however,wasonly2-foldlesspotentthan21inboththethesyntheticliabilityofachiralcenterwhichcouldnotintermediate.Theeffectofsubstitutiononthepiperidi-nylnitrogenwasexploredthroughanalogues22-25.N-Methyl,N-hydroxyethyl,andsulfonamideanalogues(22-24)showedsimilaractivityto21inbothenzymeandcellsassayswhiletheN-tert-butoxycarbonylana-logue25wassignificantlylesspotentinbothoftheseassays.Asnotedpreviouslywithcyclohexyl1722JournalofMedicinalChemistry,2004,Vol.47,No. MisraetTable2.MetabolismandPharmacokineticEvaluationofAminothiazoleAnaloguesinMicea2Figure2.Upperpanel:Solid-statestructureofpiperidinylTheinhibitorcarbonatomsareshowningreen,thenitrogenatomsareshowninblue,oxygenatomsareshowninred,andthesulfuratomsareshowninyellow.Theproteincarbonsareingray.Hydrogenbondsareshownbythemagentadottedlines.Lowerpanel:Relativebindingmodeofinhibitor21(carbonatomsinlightblue,nitrogenatomsindarkblue,oxygenatomsinredandsulfuratomsinyellow)boundtoinblue,oxygenatomsinred,andphosphorusatomsinmagenta)boundtoCDK2.16and17,thismaybetheresultofintroducingahighlylipophilicgroupintoahydrophilicspace.Finally,the7-foldmorepotentthantransisomer27intheenzymeassaybutonly2-foldmorepotentinthecellularanti-proliferationassay.Theoriginforthedifferencesinenzymeactivitybetweenthetwoisomerswerenotapparent.Inbothoftheassays,26comparedfavorablytoourbenchmarkinhibitor21.Solid-StateStructureStudy.Thethree-dimen-sionalsolid-statestructureof21incomplexwithCDK2wasdeterminedbyX-raycrystallography(seeSupport-ingInformationforsummaryofcrystallographicdata).Crystalswereobtainedbyincubatinginhibitor21(72h)withcrystallineproteinintheabsenceofcyclin.Thecrystalstructure,asshownintheupperpanelofFigure2,confirmedthat21bindsintheATP-bindingsiteandtheinhibitoradoptsthesameorientationand“folded”conformationpreviouslydescribedwiththisseries.6In

aSeeref6fordescriptionofassayconditions.bCompoundwasdosediv.towardthethiazolecoreandintotheribosepocketratherthanextendingtowardPhe-80.Noclearhydro-genbondinginteractionsareseenbetweentheoxazoleringandtheprotein.Importanthydrogenbondsbe-tweentheamidebackboneatomsofLeu-83andboththethiazolenitrogenandexocyclicamideprotonaretowardtheexterioroftheproteinconsistentwiththepositionoftheinhibitoristolerated.TheoriginsoftheCDK2selectivityofthisseriesarenotreadilyapparentfromthestructuraldata.Forcomparison,thelowerpanelofFigure2isanoverlaywhichshowstherelativeorientationofinhibitor21boundtoCDK2withthatofATPboundtoCDK2.9MetabolismandPharmacokinetics.Selectedana-logueswereassayedformouseserumproteinbindingusinganequilibriumdialysisprotocol(Table1)andformetabolicstabilitytowardmouselivermicrosomes(Table2).InhibitorswithsuitableinvitroCDK2inhibi-torypotencyinbothenzymeandcellassays(IC50<100nM)andlowtomoderateproteinbinding(65-85%)wereselected,andpharmacokinetic(PK)parametersweredetermined.Eachcompoundwasdosedintraperi-toneally(ip)atadoseof10mg/kginmice,withthedatashowninTable2.Althoughthecompoundswereallsimilarinstructure,cumulativedrugexposureover6hasmeasuredbyAUCvaried6-to7-fold.Inparticular,N-methylanalogue22showedsignificantlypoorerPKand,bothshorterhalf-life(T1/2)andmean-residencetime(MRT)while19,21,and26exhibitedrelativelyhighexposuresandlongerhalf-lives.WespeculatethatthepoorPKof22couldbeduetoeitheroxidationordemethylationofthetertiaryamine.InVivoAntitumorActivity.Onthebasisoftheirinvitropotencyandfavorableinvivoexposure,com-pounds19(homochiralisomer,seeExperimentalSec-tion),21,23,and26wereevaluatedforinvivoantitu-morefficacyinmiceusingP388murineleukemiatumorsandA2780humanovariancarcinomaxe-nografts.10IntheP388model,drugsweredosedintra-peritonially(ip)onceadayfor7days(qdx7)inimmu-nocompetentmiceimmediatelyafterthetumorswereimplantedip.Theantitumorefficacywasmeasuredasanincreaseinlifespan,andthedataareexpressedasaratiooflifespanofdrug-treatedgroup(T)versuswereimplantedsubcutaneously(sc)innudemice,andcompoundsweredosediponceadayfor8days(qdx8)startingwhentumorshadgrowntoamedianweightInhibitorsofCyclin-DependentKinase JournalofMedicinalChemistry,2004,Vol.47,No.7Table3.InVivoAntitumorActivityofAminothiazolesagainstP388andA2780TumorsinMiceaMTDddoseMTDddose10.6-2>3.6-aSeerefs6and10forexperimentalprotocols.bP388dosingschedule,ip/qdx7.cA2780dosingschedule,ip/qdx8.dMTD)maximumtolerateddose.eSeeref10fordefinitionofLCK.fHomochiralisomer,seeExperimentalSection.100mg.TheantitumorefficacywasmeasuredasaThedataaresummarizedinTable3.AllcompoundswereactiveintheP388modelshowing%T/Cvaluesbetween140and179,where%T/C>125wasdefinedasactive.IntheA2780xenograftmodel,3-piperidinylanalogue19and4-aminocyclohexylanalogue26pro-ducedagrowthdelayof1.6and1.7LCK,respectively.toflavopiridolbutsignificantlylessefficaciousthanaminothiazole2despitetheirsuperiorexposurenum-bers.4-Piperidinylanalogue21,whichhadshownintermediateexposure,however,producedagrowthdelaybetween3.6and5.0LCKandwassignificantlymoreefficaciousthaneitherflavopiridol(1)oramino-thiazole2.Theenhancedefficacyof21willbethesubjectoffuturereports.BiologySummaryfor21(BMS-387032).Onthebasisofitssuperiorantitumorefficacy,wefocusedourstudieson4-piperidinylanalogue21.Closerexamina-tionoftheCDKselectivity(Table4)confirmedthat21exhibitedasimilarCDK2-selectiveprofileasreportedically,21was10-foldselectiveforCDK2/cycErelativetoCDK1/cycBand20-foldselectiverelativetoCDK4/apanelofunrelatedkinases.ItexhibitedIC50>40μMforPKCR,PKC?,PKCγ,Chk1,IKK,EMT,andIGF-1R.Inbothmouseandhumanserum,21exposuretofreedrug.inmouse,rat,anddogareshowninTable5.Thecompoundexhibitsamoderatehalf-life(T1/2)ofbetween5and7hacrossthethreespecies.TheT1/2isconsistentwithinvitrostudieswhichindicatedalowrateofmetabolism(oxidationandglucuronidation)inlivermicrosomes.Inaddition,21waswelldistributedintotissuesasindicatedbyahighvolumeofdistribution(Vss).Examinationinthreespeciesindicated21isorallybioavailableinallthreespecies,althoughlesssoinratanddogthanmouse.Metabolismstudiesinratsindi-catedthatdosediv(8.9mg/kg)28%ofparentdrugisrecoveredunchangedinurineand11%inbile9hpostadministration.Minormetabolitesresultingfromcleavageoftheamidebond,oxidationofthethioetherlinker,andhydroxylationofthetert-butylgroupwere

acytochromeP450panelwhichincludedCyP1A2,CyP2C9,CyP2C19,CyP2D6,andCyP3A4.Theantitumorefficacyof21intheA2780humanovariancarcinomaxenograftmodelwasevaluatedingreaterdetailalongwithflavopiridolforcomparative(Figure3).Tumorswereimplantedscinnudemice21onceadayipfor8daysat18,36,and48mg/kgAminothiazole21wasfoundtohaveantitumoractivityatallthreedoses(i.e.,LCK>1.0).Atthelow18mg/kgdose,21showedminortumorregressionwithagrowthdelayof2.1LCK.Atthehigher36and48mg/kg21showedrapid,significanttumorregressionandgrowthdelaysof>5.6LCKand>6.5,respectively.Inaddition,afterextendedtimesatthetwohighdoses,cureswereseenin9of16animalsasdeterminedbynomeasurabletumoratday68.Atautopsythecuredanimalswerefoundtobefreeoftumorcells.Signifi-cantly,21wasfoundtobeactivenotonlyatitsMTDbutalsoatbothsub-MTDdoses(18and36mg/kg),Forcomparison,flavopiridolshowedagrowthdelay0.4LCK(inactiveinthismodel,LCK<1.0)withnoregressionorcuresatitsMTD(7.5mg/kg).SelectionofFinalForm.Becauseofthelowaque-oussolubilityoffreebase21(0.28mg/mL)andananticipatedclinicalplanwhichincludedanivdosingprotocol,anumberofmineralandorganicacidsaltsof21wereexaminedwiththegoalofincreasingaqueoussolubilityandidentifyingafinalformwithacceptable(acid:21)wasselectedasthefinalformduetoitsoverallfavorablephysicalpropertieswhichincludedexamina-tionofpolymorph,hydrate,solvateissues,ease,andstability,andaqueoussolubility(5.5mg/mL).Onthebasisofitsphysicalproperties,selectivity,pharmaco-the1/2-L-tartratesalt,wasselectedtoprogressintoclinicaldevelopmentasanantitumoragent.Aminothiazole21(BMS-387032,N-[5-[[[5-(1,1-dim-ridinecarboxamide)hasbeenidentifiedasaCDK2-selectiveinhibitorwhichhasbeenselectedtoenter21hasshownsuperiorantitumorefficacytobothflavopiridolandpreviouslyreportedanaloguesinthisseries.Thekeymodificationfromourpreviouslyre-portedaminothiazoleanalogueswasreplacementofasubstitutedaromaticacylsidechainwithanonaromaticaminoacylsidechain.Thissubstitutionreducedmo-lecularweight,proteinbinding,andinvitromeasuredmetabolisminlivermicrosomeswhileatthesametimeitincreasedaqueoussolubility.SARstudieswerecon-sistentwiththesolid-statestructureoftheinhibitorboundtoCDK2protein,whichindicatedthattheacylextraproteinspaceandwasamenabletomodification.Optimizationofpotency,pharmacokinetics,andevalu-ationinbothanip/ipP388murinetumormodel1724JournalofMedicinalChemistry,2004,Vol.47,No. MisraetTable4.SummaryofinVitroEvaluationofAminothiazoleA2780cellular%%IC50IC50IC50IC50bindingbindingTable5.PharmacokineticParametersforAminothiazole21inMouse,Rat,andDogdoseT1/2(h),MRT(h),AUCtot(μM?h),clearance(mL/min/kg),Vss(L/kg),Cmax(μM),Tmax(h),%oralaDosedin1:9EtOH/watervehicle.bDosedin1:19dextrose/water,podose2.4μmol/kg.Figure3.Comparativeantitumorefficacyof21(BMS-387032)andflavopiridol(1)inaA2780humanovariancarcinomaxenograftnudemousemodel.modelledtotheidentificationofthe4-piperidinylanalogue,21,asourclinicaldevelopmentcandidate.Detailedbiochemical,antitumorefficacy,andclinicalstudieswillbethesubjectoffuturedisclosures.ExperimentalAllair-andmoisture-sensitivereactionswereconductedunderanargonatmosphere.Allreagentsandstartingmateri-alswereobtainedfromcommercialsourcesandusedwithouttoN,N-dimethylforamide,TFAreferstotrifluoroaceticacid,DMAPrefersto4-(dimethylamino)pyridine,andEDACrefersto1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochlo-ride.1HNMR(400or500MHz)and13C(100or125MHz)spectrawererecordedonJEOLorBrukerspectrometersandusingaShimadzuHPLC10AsystemlinkedtoaMicromassMSdetectorinpositivechemicalionizationmode.Analyticalhighperformanceliquidchromatography(HPLC)wasper-formedonShimadzu10AsystemsfittedwithYMCS54.6×50mmcolumns,usingaUVdetectorat220or254

structures.Allnewcompoundswerehomogeneous(>98%)byHPLCanalysis.Allpreviouslyunreportedfinalanaloguesshowedacceptable(+0.4%)elementalanalysisand/orhigh-resolutionmassspectrum(HRMS).Meltingpoints(mp)areuncorrected.FlashchromatographywasperformedusingE.Mercksilicagel60undermediumnitrogenpressureelutingwiththesolventsystemsindicated.Concentrationinvacuoreferstotheuseofarotoraryevaporatorthenoilpumpvacuumforremovalofvolatilecomponents.Compounds2,6,8,12and15werepreparedpreviously.6Biologicalassays,metabolismandpharmacokineticstudies,andsolidstatestructuralstudieswereconductedutilizingpreviouslypub-lishedprotocols.6,10Preparationof2-Amino-5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]thiazole(8).Toa2Lthree-neckedR-bromopinacolone(134g,747mmol,1.0equiv),acetone(1.2L),andsodiumazide(63.2g,971mmol,1.3equiv).Thereactionmixturewasstirredatroom-temperatureovernightandthenfiltered,andthesolidswerewashedwithacetone×100mL).ThefiltratewasconcentratedinvacuotoprovideR-azidopinacolone(105.0g,100%)asanoil.1HNMR(CDCl3)δ1.17(s,9H),4.07(s,2H).Thecrudematerialwasusedinthenextstepwithoutfurtherpurification.Toa2Lthree-neckedround-bottomflaskfittedwithamechanicalstirrerwereaddedR-azidopinacolone(28.6g,203mmol,1.0equiv),methanol(1145mL),concentratedHCl(18mL),and10%Pd/C(3.5g,50%waterwet).Thereactionmixturewasstirredunderhydrogenat20psifor2h,themixturewasfilteredthroughapadofCelite,andtheresiduerinsedwithmethanol(2×50mL).Thefiltratewastratedunderreducedpressureatatemperaturebelow40Theresultingwetsolidwasazeotropedwith2-propanol(2whichformedwasstirredfor5min.Thesolidproductwascollectedbyfiltration,andthecakewaswashedwithdiethylether(2×30mL)andthendriedinvacuotogiveR-aminopi-nacolonehydrochloride4(28.0g,91%),mp205°C(lit.200°C).111HNMR(DMSO-d6)δ1.13(s,9H),4.06(s,2H),8.34(s,3H).Toa1Lthree-neckedround-bottomflaskfittedwithamechanicalstirrerwereaddedR-aminopinacolonehydrochlo-ride4(15.2g,100mmol,1.0equiv)andCH2Cl2(350mL).Theslurrywascooledto-5°C,andtriethylamine(35mL,250andcooledto-10°C.AsolutionofR-chloroacetylchloride(8.8mL,110mmol,1.1equiv)inCH2Cl2(20mL)wasaddeddropwiseover15minwhilekeepingthereactiontemperaturebelow-5°C.Thereactionwasstirredfor1handthenquenchedwith1NaqHCl(200mL).Thephaseswereseparated,andtheorganicphasewaswashedwith1NaqHClinvacuotoaffordR-N-2-(chloroacetylamino)pinacolone5(18.9g,98%)asawhitesolid,mp50-52°C.1HNMR(CDCl3)1.21(s,9H),4.09(s,2H),4.30(s,2H),7.35(s,1H).13C(CDCl3)δ210.13,166.62,45.23,43.51,42.79,27.45.Anal.(C8H14NO2Cl):C,H,N,Cl.Toa100mLroundedbottomflaskfittedwithamagneticstirrerwereadded5(18.9g,98.6mmol,1equiv)andPOCl3InhibitorsofCyclin-DependentKinase JournalofMedicinalChemistry,2004,Vol.47,No.7to105°Candstirredfor1h.Afterbeingcooledtoroomtemperature,thereactionmixturewaspouredcarefullyintoice(180g).Themixturewasextractedwithether(6×150mL).TheorganicextractswerecombinedandneutralizedtopH7-8withsaturatedsodiumbicarbonate(～700mL).Thesaturatedsodiumbicarbonate(100mL),water(100mL),andbrine(50mL),dried(MgSO4),andconcentratedinvacuo.crudematerialwasdistilledunderreducedpressuretogive5-tert-butyl-2-chloromethyloxazole,66(13.6g,80%),asacolorlessoil,bp38-40°C(0.25mm).1HNMR(CDCl3)δ1.32(s,9H),4.60(s,2H),6.70(s,1H).13CNMR(CDCl3)δ28.92,31.91,36.57,120.87,158.03,162.90.Toasolutionofthiocyanate78(10.0g,63.3mmol)inabsoluteEtOH(600mL)wasaddedNaBH4(4.8g,120mmol)portionwiseatroomtemperature.Themixturewasstirredfor1h,andthenacetone(300mL)wasslowlyintroduced.After1h,asolutionofoxazolechloride6(12.0g,69mmol)inEtOH(100mL)wasadded,andtheresultingdarkreactionmixtureheatedtorefluxfor1h.Theresultingmixturewascooled,concentratedinvacuo,andthenpartitionedbetweenEtOAcandbrine.Theorganicphasewasseparated,dried(MgSO4),andconcentratedinvacuotogiveacrudesolid.Thecrudematerialwastrituratedwithdiethylether/hexanetoprovideamine86(16.0g,94%)asapalered-brownsolid.PreparationofN-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]-methyl]thio]-2-thiazolyl]-2-aminoethanecarboxamideHy-drochloride(13).Toastirredsolutionofamine8(1.20g,4.45mmol)andN-tBoc-?-alanine(2.10g,11.1mmol)inanhydrousDMF(10mL)wasaddedEDACinseveralportions(2.55g,13.3mmol)over5minatroomtemperature.Themixturewasstirredfor1.5handthenpartitionedbetweenEtOAc(75mL)and1MaqHCl(75mL).Theorganicphase5%aqsodiumbicarbonate(75mL),andbrine(50mL),dried(Na2SO4)andconcentratedinvacuotogiveanoil.Thecrudematerialwaspurifiedbyflashchromatography(EtOAc)to(1.93g,98%)asasolidwhitefoam.1HNMR(CDCl3)δ11.9(s,1H),7.35(s,1H),6.62(s,1H),5.28(brs,1H),4.09(s,3.54(m,2H),2.72(m,2H),1.43(s,9H),1.27(s,9H).441[M+Toasolutionoftheabovet-Bocintermediate(1.82g,4.13mmol)inCH2Cl2(10mL)wasaddedTFA(5mL)rapidlyatroomtemperature.After1h,thesolutionwasconcentratedinvacuo.TheresiduewaspartitionedbetweenEtOAc(25mL)andwater(35mL)andthenneutralizedbyadditionofsolid(25mL),dried(Na2SO4),andconcentratedinvacuotogivethefreeamine(1.26g)asasolidfoam.Thefreeaminewassolubilizedwith1MaqHCl(3.7mL,1equiv)andwater(5mL)andthenlyophilizedtoaffordthehydrochloridesaltof13(1.37g,88%)asanoff-whiteamorphoussolidfoam.1HNMR(DMSO-d6)δ12.5(brs,1H),8.02(brs,2H),7.39(s,6.71(s,1H),4.06(s,2H),3.08(m,2H),2.84(t,J)7,(s,9H);LCMS:341[M+H]+.HRMSFAB[M+H]+calcdforC14H20N4O2S2340.1028;found341.1095.PreparationofN-[5-[[[5-(1,1-Dimethylethyl)-2-oxazolyl]-methyl]thio]-2-thiazolyl]phenylmethanecarboxamideTri-fluoroaceticAcidSalt(14).Toastirredmixtureofpheny-laceticacid(50mg,0.37mmol),amine8(100mg,0.37mmol),N-hydroxybenzotriazolehydrate(60mg,0.44mmol),andDMAP(45mg,0.37mmol)inDMF(1.4mL)wereaddeddiisopropylethylamine(0.16mL,0.89mmol)andEDAC(71mg,0.37mmol)atroomtemperature.ThereactionmixturetiveHPLC(YMCODSS520×100mmcolumn,30%to100%gradientover10minwith0.1%TFAinMeOH-watersolventsystem,20mL/min)togivetheTFAsaltof14(40mg,asawhitesolid.1HNMR(CDCl3)δ7.49-7.25(m,6H),6.54(s,1H),3.93(s,2H),3.79(s,2H),1.21(s,9H).LCMS:

[M+H]+.HRMSFAB[M+H]+calcdforC19H21N3O2S23