jm034111v

FluorineSubstitutionCanBlockCYP3A4Metabolism-DependentInhibition:Identificationof(S)-N-[1-(4-Fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluorophenyl)acrylamideasanOrallyBioavailableKCNQ2OpenerDevoidofCYP3A4Metabolism-DependentInhibition

Yong-JinWu,*,†CarlD.Davis,‡StevenDworetzky,|WilliamC.Fitzpatrick,§DavidHarden,§HuanHe,†RonaldJ.Knox,§AmyE.Newton,|ThomasPhilip,‡CraigPolson,|DigavalliV.Sivarao,|Li-QiangSun,†SvetlanaTertyshnikova,|DavidWeaver,§

SureshYeola,‡MaryZoeckler,‡andMichaelW.Sinz‡

Bristol-MyersSquibbPharmaceuticalResearchInstitute,

5ResearchParkway,Wallingford,Connecticut06492

ReceivedMay23,2003

Abstract:TheformationofareactiveintermediatewasfoundtoberesponsibleforCYP3A4metabolism-dependentinhibition(MDI)observedwith(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide(1).Structure-3A4MDIrelationshipstud-iesculminatedinthediscoveryofadifluoroanalogue,(S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide(2),asanorallybioavailableKCNQ2openerfreeofCYP3A4MDI.

Chart1

Table1.HumanCytochromeP450InhibitoryPotentialof1withRecombinantHumanCYP450EnzymesCYP

IC50(µM)a

a

1A2782C95.32C19132D6>1003A4b

273A4c76

IC50valuesweredeterminedforinhibitionofdeethylationof3-cyano-7-ethoxycoumarin(CYP1A2andCYP2C19),dealkylationof7-methoxy-4-trifluoromethylcoumarin(CYP2C9)andforinhibi-tionofdemethylationof3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin(CYP2D6).TwodifferentsubstrateswereusedtodeterminetheIC50valuesforCYP3A4;theinhibitionofdealkylationofBzRes(benzoylresorufin)andofBFC(7-benzyloxy-4-trifluoromethylcoumarin).Thesevaluesarethemeanofduplicatedeterminations.bBFCusedasthesubstrate.cBzResasthesubstrate.

Table2.Time-DependentInhibitionChangesof1and2UsingrCYP3A4andBFC(IC50,µM)

compound12

troleandomycinketoconazole

5min9618610.016

15min6221330.013

30min3319200.017

45min2219160.022

CYP3A4isamajorCYP450isoformandaccountsfor30%oftotalCYPproteininhumanliver.CYP3A4hasabroadsubstratespecificityandisestimatedtobeinvolvedinthemetabolismofapproximately50%ofdrugsusedinhumans.CoadministeredCYP3A4inhibi-torssuchasketoconazoleandritonavirareamajorconcerninbothdrugdevelopmentandclinicalpracticebyvirtueofthisenzyme’sinvolvementintheclearanceofamajorityofdrugs.HenceitiscommonpracticetoscreenanewchemicalentityforitspotentialtoinhibitCYP3A4.1

Recently,wereportedthediscoveryof(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide(1,Chart1)asanovelKCNQ2potassiumchannelopenerwithexcellentoralbioavailabilityindogsandrats.2Thiscompounddemonstratedsignificantoralactivityinacorticalspreadingdepressionmodelofmigraine,sug-gestingthatKCNQ2openersmayhavepotentialforthetreatmentofsometypesofmigraineheadache.3Toassessthepotentiallikelihoodofdrug-druginterac-tions,weevaluatedthecytochromeP450inhibitorypotentialof1usingrecombinant(r)CYP1A2,CYP2C9,CYP2C19,CYP2D6,andCYP3A4(Table1).LowtomoderatelevelsofinhibitionwereobservedwithseveralmajorhumanP450enzymeswiththemostpotentinhibitionseenagainstCYP2C9(IC505.3µM).Insubsequentmedium-throughputtimedependentinhibi-*Correspondingauthor.Tel.:+1-203-677-7485;fax:+1-203-677-7702;e-mail:yong-jin.wu@bms.com.

†DepartmentofNeuroscienceChemistry.

‡DepartmentofPreclinicalCandidateOptimization.|DepartmentofNeuroscienceBiology.§DepartmentofNewLeadsBiology.

tion(TDI)studies,noTDIwasobservedwithrCYP2C9.However,whenrCYP3A4wasused,compound1re-sultedinanapproximate4-folddecreaseinIC50between5and45min,whilea3.8-folddecreaseanda1.4-foldincreasewereobservedforourreferencecompounds:troleandomycin(apotentmetabolismdependentCYP3A4inhibitor)andketoconazole(apotentreversible,non-metabolismdependentCYP3A4inhibitor),respectively(Table2).4Inourassay,compoundswith2-folddecreaseormoreinIC50between5and45minaredefinedastime-dependentinhibitors.ToconfirmtheCYP3A4time-dependentinhibitionobservationandfurtherchar-acterizetheinhibitionmechanismasmetabolismde-pendent,compound1(100µM)waspreincubatedwithhumanlivermicrosomesinthepresenceofNADPH.Microsomalaliquotsweretakenatvarioustimepointsanddiluted20-foldinasecondassaythatmeasuredtestosterone6󰀁-hydroxylationasanindicationofCYP3A4enzymeactivity(Figure1).Bothfluoxetineandtrole-andomycin,knownMDI’s,wereincludedaspositivecontrols.Thepreincubation-dilutionexperiment(de-scribedabove)wasalsocarriedoutinhumanlivermicrosomesatmultipleconcentrationsofcompound1.Figure2showsthattheCYP3A4-MDIwasobservedatallconcentrationstestedandespeciallysalientathigherconcentrations.Furthermore,theinhibitoryeffectonCYP3A4activitywasnoteliminatedafterdialysisoftheabovemicrosomalincubations,therebysignificantlyreducingthepossibilityofreversibleinhibitioncausedbyformationofaninhibitorymetabolite.Thus,theCYP3A4MDIcouldarisefromeithermechanism-basedinactivationorquasi-irreversibleinhibition.

Itiswell-knownthatcompoundswithdialkylaminefunctionalityundergosequentialdealkylation-oxidation

10.1021/jm034111vCCC:$25.00©2003AmericanChemicalSociety

PublishedonWeb07/26/2003

LettersFigure1.Metabolismandtime-dependentinhibitionof1,troleandomycin(TAO),andfluoxetineinhumanlivermi-crosomes(allcompoundsat100µM).

Figure2.Metabolismandtime-dependentinhibitionof1inhumanlivermicrosomesatvariousconcentrations.Testoster-one6󰀁-hydroxylationusedasanindicationofCYP3A4enzymeactivity.

reactionsleadingtoformationofanitrosointermediate.Thisreactiveintermediatecancoordinateinaquasi-irreversiblefashionwiththeprosthetichemeironofCYP3A4leadingtoformationofacatalyticallyinactivemetabolite-intermediatecomplex(MIC)withtheen-zyme.Macrolideantibioticssuchaserythromycin,clarithromycin,andtroleandomycinaretypicalex-amplesofdrugsthatformMIC’s.5However,noevidenceofaMICwasdemonstratedintherCYP3A4bindingspectrumof1,wheretroleandomycinandfluoxetinewereemployedaspositivecontrols(datanotshown).Wealsoinvestigatedtheeffectofpotentialmodifiers,suchasglutathioneandcatalase,ontheinactivationofCYP3A4whenincubatedwithhumanlivermicrosomesand1(100µM),andnosignificantdifferenceswereobservedintheirpresence(<15%differenceinthedegreeofenzymeinactivation),suggestingthattheprocesswaslikelyconfinedtotheCYP3A4activesiteanddidnotinvolveperoxidativereactionsoutsideoftheactivesite.Multipleenzymescanbeinvolvedinthemetabolismofasubstrateaswellasitsclearanceandtheformationofpotentialinhibitorymetabolites.Inthiscase,isoform-selectivechemicalinhibitionstudiesinhumanlivermicrosomes(Figure3)showedthatCYP3A4isamajorCYP450responsibleforthemetabolismof1asindicatedbythedecreaseintheextentofmetabolismbytheCYP3A4inhibitorketoconazole.

JournalofMedicinalChemistry,2003,Vol.46,No.183779

Figure3.EffectofCYPisoform-selectiveinhibitorsontheextentofmetabolismofcompound1inhumanlivermi-crosomesduringa60minincubation.Inhibitors:2µMketoconazolefor3A4;2µMquinidinefor2D6;20µMsul-faphenazolefor2C9;25µMfurafyllinefor1A2.

Scheme1

Takentogether,thesedatasuggeststhatcompound1ismetabolizedbyCYP3A4toformareactiveinter-mediate.ThisintermediateappearstoberestrictedtotheCYP3A4activesiteandcovalentlybindstotheCYP3A4enzyme,leadingtoirreversibleenzymeinac-tivation,alsocalledmechanism-basedinactivation.Thistypeofinhibitionisviewedasanundesirablecharac-teristicsincethedegreeofinhibitionextendsbeyondtheeliminationofthedrugandmayincreasewithmultipledosing,thusleadingtoseriousdrug-druginteractions.Moreover,compoundswithpotentirre-versibleinhibitioncharacteristicscanexhibitnonlinearpharmacokinetics.6Forthesereasons,wechosetodevelopanewanaloguewithKCNQ2openeractivityandapharmacokineticprofilecomparableto1,butdevoidof3A4MDI.Thisreportdescribesthediscoveryofacrylamide2(Chart1)thatappearstomeettheabovecriteria.

Thesynthesisofacrylamide2isshowninScheme1.Couplingof(S)-1-(4-fluoro-3-morpholin-4-ylphenyl)ethy-lamine(4)7with3-(4-fluorophenyl)acrylicacid(3)inthepresenceof1-(3-dimethylaminopropyl)-3-ethylcarbodi-imidehydrochloride(EDAC‚HCl),4-(dimethylamino)-pyridine(DMAP)andtriethylamineafforded2ingoodyield.

Thestructure-3A4MDIrelationshipstudieswerecarriedouttoidentifythepossiblesite(s)responsibleforthegenerationofthereactiveintermediatedescribedaboveforcompound1.ReductionoftheR,󰀁-unsaturateddoublebond(i.e.,5,Chart2)orfluorosubstitutionofthephenylontheleft(i.e.,6,7,and8)didnoteliminatetheCYP3A4MDI(usingtherCYP3A4-BFCassay),suggestingthatthereactiveintermediatemaybeformedontheN-phenylmorpholinemoietyof1.SinceCYP3A4MDIwasnotobservedwithcompound9,themorpholi-nylgroupmustplayaroleintheformationofthereactiveintermediateontheN-phenylmorpholinemoi-ety.Monohydroxylationofthismoietywasseeninthebiotransformationof1withbothhumanorratlivermicrosomes.Toblockthepotentialhydroxylationofthephenylringontheright,wesubstitutedthehydrogenattachedtoC4(seeChart2)withafluorine8asthispositionappearstobemostsusceptibletohydroxylationbasedonelectronicandstericconsiderations.Amonga

3780JournalofMedicinalChemistry,2003,Vol.46,No.18Chart2

seriesof4-fluoro-substitutedanaloguessynthesized,acrylamide2wasfoundtoexhibitcomparableKCNQ2openeractivityto1(videinfra)andyetnoCYP3A4MDI,asshowninTable2.ThelackofCYP3A4MDIobservedwiththedifluorocompound2isduetothefluorineadjacenttothemorpholinylgroup,notthefluorineattachedtothephenylontheleft(styrene),sincethemonofluoroanalogue7displayedCYP3A4MDI(3.6-folddecreaseinIC50between5and20min).Presumably,4-fluorosubstituentcanpreventhydroxy-lationatthisposition,thusblockingtheformationofareactivequinoneintermediatesuchas10or11.9Fur-therstudiesarerequiredtoestablishtheexactreactiveintermediate.

Theconcentrationresponseeffectsof2weredeter-minedonmKCNQ2channelsheterologouslyexpressedinHEK293cellsusingthewhole-cellpatch-clamprecordingtechniqueasdescribedpreviously.2Themem-branepotentialofindividualcellswasvoltage-clampedto-40mV,resultinginthegenerationofamaintained,steady-statebaselineKCNQ2currentof∼100pA.Next,increasingconcentrationsof2werelocallysuperfusedontothecells,causingalargeincreaseintheamountofKCNQ2currentmeasuredat-40mV.Themaximalsteady-statecurrentamplitudeproducedbyeachcon-centrationof2wasmeasuredandusedtoconstructtheconcentrationresponsecurveshowninFigure4.TheEC50,estimatedfromthesinglebindingsitemodellogisticfittothesedata,is1.2µM,comparabletothatobtainedwith1(about3µM).2

Theeffectsof2onmembranepotentialwerealsoinvestigatedusingthefluorescentmembranepotentialassayinSH-SY5Yhumanneuroblastomacells10ex-pressingnativeKCNQchannels.Therelativefluores-cenceintensitychangewasusedasanindicationofthechangeinmembranepotential.11AsshowninFigure5,applicationof2ledtoaconcentration-dependenthyperpolarizationofthecells.ThecalculatedEC50ofthiscompoundis1.55µM,againsimilartothatobtainedwith1(0.69µM).2

Compound2wascharacterizedinseveralpharma-cokineticstudies(Table3).Oralabsorptionwasrapidintheratwithapeakconcentrationoccurringwithin0.66h.Inivdosingstudiesthecompounddemonstratedamoderateplasmaclearanceandarelativelylongeliminationhalf-life.Theoralbioavailabilitymeasuredwas84%,when2wasadministeredasasuspension.Compound2wasalsoshowntobepresentinbrain

Letters

Figure4.Concentration-responseeffectsof1and2onsteady-statemKCNQ2currentproducedbyvoltage-clampingthecellmembranepotentialto-40mV.Datasetsarethemean(SEM,andtheyarefittedwithone-sitemodellogisticfunctions.

Figure5.Effectsofand1and2onrestingmembranepotentialinSH-SY5Yhumanneuroblastomacells(n)4).

Table3.PharmacokineticParametersof2intheRat

PKparameterrativa

dose(mg/kg)1t1/2(h)

3.5CL(mL/min/kg)17.8Vd(L/kg)

3.3po(suspension)b

dose(mg/kg)10tmax(h)0.66F(%)

84

a

CompounddosedtoSprague-DawleymaleratsasanivsolutioninPEG400(n)3).bdosedasanoralsuspensionin25%PEG400/0.5%Tween80/74.5%pH3water)(n)3).

tissuewhendosedorallyat8mg/kgin95%PEG400/5%DMSOinrats(∼1µMconcentrationof2).

Compound2wasexaminedforitsabilitytoreducethetotalnumberofcorticalspreadingdepressions12(CSD)followingintravenousadministration.CSDwasproducedbya10minapplicationofcrystallinepotas-siumchloridetoasmallregionintheratparietalcortex.Topicalapplicationofcrystallinepotassiumchlorideproducedacontinuoustrainofslownegativeexcursionsinthecorticalsurfacepotentialinallanimalstested.Thetotalnumberofdepolarizationsinthedistalleadofeachanimalwasusedtodeterminethemeannumberofdepolarizationsforeachgroup.Themeandepolar-izationsforthevehicle-treatedgroupwas26.4(0.9(n)8),whilethecorrespondinggroupmeanfor2treatedratswas19.8(1.9(n)8).Thisapproximately25%attenuationindepolarizationsinthedrug-treatedgroupwassignificant(p)0.009;two-tailedStudentt-test).

LettersFigure6.Effectofasingledoseof2(1mg/kg,iv)onKCl-inducedcorticaldepolarizations.Meandepolarizationsinthedistalleadfromindividualratsorganizedascolumnsbasedontreatment.Horizontalbarrepresentsthegroupmean(SEM.IntergroupcomparisonwasmadeusingtheStudent’stwo-tailedt-test;p<0.05wasdeemedsignificant.

Thus,acrylamide2evenat1mg/kg(iv)producedasignificantreductiononthetotalnumberofdepolar-izationevents.

Insummary,theCYP3A4MDIof1waseradicatedthroughspecificfluorinesubstitution,andthedifluoroacrylamide2wasidentifiedasanewKCNQ2opener13withcomparableactivityto1,butdevoidofCYP3A4MDI.Compound2showedthesameexcellentoralbioavailabilityinratsascompound1anddemonstratedsignificantinvivoactivityinacorticalspreadingdepressionmodelofmigraine.Thus,2mayhavepoten-tialforthetreatmentofsometypesofmigrainehead-achewithreducedlikelihoodofCYP3A4inhibitionrelateddrug-druginteractions.

Acknowledgment.WethanktheDiscoveryProfil-inggroupofBristol-MyersSquibbcompany(Walling-ford,CT)fortheP450inhibitiondata.

SupportingInformationAvailable:Experimentalde-tailsforthesynthesisof2anddescriptionsofthecorticalspreadingdepressionstudiesandCYP450enzymeinhibitionassays.ThismaterialisavailablefreeofchargeviatheInternetathttp:/pubs.acs.org.

References

(1)Kumar,G.N.;Surapaneni,S.RoleofDrugMetabolisminDrug

DiscoveryandDevelopment.Med.Res.Rev.2001,21,397-411.(2)Wu,Y.-J.;Boissard,C.G.;Greco,C.;Gribkoff,V.K.;Harden,D.

G.;He,H.;L’Heureux,A.;Kang,S.H.;Kinney,G.G.;Knox,R.J.;Natale,J.;Newton,A.E.;Lehtinen-Oboma,S.;Sinz,M.W.;Sivarao,D.V.;Starrett,J.E.,Jr.;Sun,L.;Tertyshnikova,S.;Thompson,M.W.;Weaver,D.;Wong,H.S.;Zhang,L.;andDworetzky,S.I.(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phen-yl-acrylamide:anOrallyBioavailableKCNQ2OpenerwithSignificantActivityinaCorticalSpreadingDepressionModelofMigraine.J.Med.Chem.2003,46,3197-3200.

(3)Forreviewsonpotassiumchannels,see:(a)Coghlan,M.J.;

Carroll,W.A.;Gopalakrishnan,M.RecentDevelopmentsinthe

JournalofMedicinalChemistry,2003,Vol.46,No.183781

BiologyandMedicinalChemistryofPotassiumChannelModu-lators:UpdatefromaDecadeofProgress.J.Med.Chem.2001,44,1627-1653.(b)Shieh,C.;Coghlan,M.;Sullivan,J.P.;Gopalakrishnan,Murali.PotassiumChannels:MolecularDe-fects,Diseases,andTherapeuticOpportunities.Pharmacol.Rev.2000,52,557-593.

(4)

Forreviewsonthemethodsandinterpretationofirreversibleinhibitors,see:(a)Yan,Z.;Rafferty,B.;Caldwell,G.W.;Masucci,J.A.RapidlyDistinguishingReversibleandIrrevers-ibleCYP450InhibitorsbyFluorometricKineticAnalyses.Eur.J.DrugMetab.Pharmacok.2002,27,281-287.(b)Guengerich,F.P.inHandbookofDrugMetabolism.InhibitionofDrugMetabolizingEnzymes:MolecularandBiochemicalAspects;Woolf,T.F.,Ed.;MarcelDekker:NewYork,1999;p203.(c)Jones,D.R.;Hall,S.D.inDrugDrugInteractions.Mechanism-BasedInhibitionofHumanCytochromeP450:InVitroKineticsandInVitro-InVivoCorrelations;Rodrigues,A.D.,Ed.;MarcelDekker:NewYork,2002;p387.

(5)

Pershing,L.K.;Franklin,M.R.CytochromeP-450Metabolic-IntermediateComplexFormationandInductionbyMacrolideAntibiotics;aNewClassofAgents.Xenobiotica1982,12,687-699.

(6)

Forreviewsontheimpactofirreversibleinhibitors:see:(a)Ito,K.;Iwatsubo,T.;Kanamitsu,S.;Ueda,K.;Suzuki,H.;Sugiyama,Y.PredictionofPharmacokineticAlterationsCausedbyDrug-DrugInteractions:MetabolicInteractionintheLiver.Pharmacol.Rev.1998,50,387-411.(b)Lin,J.;Lu,A.Y.H.RoleofPharmacokineticsandMetabolisminDrugDiscoveryandDevelopment.Pharmacol.Rev.1997,49,403-449.(c)Jang,G.;Harris,R.Z.;Lau,D.T.PharmacokineticsandItsRoleinSmallMoleculeDrugDiscoveryResearch.Med.Res.Rev.2001,21,382-396.

(7)Wu,Y.-J.;He,H.;Sun,L.Q.;Wu,D.D.;Gao,Q.;Li,H.Y.SynthesisofFluorinated1-(3-Morpholin-4-yl-phenyl)-ethy-lamines.Bioorg.Med.Chem.Lett.2003,13,1725-1728.

(8)

(a)Welch,J.T.;Eshwarakrishan,S.FluorineinBioorganicChemsitry;JohnWielyandSons:NewYork,1991.(b)Kirk,K.L.FluorineSubstitutionasaModulatorofBiologicalProcesses.InBiomedicalChemistry,ApplyingChemicalPrinciplestotheUnderstandingandTreatmentofDisease;Torrence,P.F.,Ed.;JohnWiley&Son:NewYork,2000;p247.

(9)

Forexamplesofreactivequinoneintermediates,see(a):Marques,M.M.;Beland,F.A.IdentificationofTamoxifen-DNAAdductsFormedby4-HydroxyltamoxifenQuinoneMethide.Carcinogen-esis1997,18,1949-1954.(b)Woolf,T.F.;Pool,W.F.;Chang,T.;Goel,O.P.;Purchase,C.F.,II;Schroeder,M.C.;Kunze,K.L.;Trager,W.F.BioactivationandIrreversibleBindingoftheCognitionActivatorTachrineUsingHumanandRatLiverMicrosomalPreparations:SpeciesDifference.DrugMetab.Dispos.1993,21,874-882.

(10)

Tosetti,P.;Taglietti,V.;Toselli,M.J.FunctionalChangesinPotassiumConductancesoftheHumanNeuroblastomaCellLineSH-SY5YduringInVitroDifferentiation.J.Neurophysiol.1998,79648-58.

(11)

Whiteaker,K.L.;Gopalakrishnan,S.M.;Groebe,D.;Shieh,C.C.;Warrior,U.;Burns,D.J.;Coghlan,M.J.;Scott,V.E.;Gopalakrishnan,M.ValidationofFLIPRMembranePotentialDyeforHighThroughputScreeningofPotassiumChannelModulator.J.Biomol.Screening2001,6,305-312.

(12)Foradiscussiononthecorticalspreadingdepressionanimalmodel,seeref2.

(13)

Preliminarystudiesusingthallium(I)influxassayshowedthat2,like1,2alsoactivatedothermembersoftheKCNQfamily.Althoughthesecompoundsactivatedthesechannels,furtherelectrophysiologicalcharacterizationisrequired.

JM034111V