Effects of 1,2,3,4-Tetrahydroimidazo[4,5-c]-Pyridine Derivatives on Ethanol Oxidation by Alcohol Dehydrogenase Isoforms from Human Liver

Abstract

We studied in vitro effects of four 1,2,3,4-tetrahydroimidazo[4,5-c]-pyridine derivatives formed in the reaction of the corresponding aldehydes with histidine on the rate of ethanol oxidation by alcohol dehydrogenase isoforms from human liver. None of test compounds inhibited ethanol oxidation by these enzymes. Some of them increased alcohol dehydrogenase activity to 220-240% of the initial level. Only one test compound accelerated ethanol oxidation by b1b2-alcohol dehydrogenase (150% of the control). The molecular mechanism underlying these effects of 1,2,3,4-tetrahydroimidazo[4,5-c]-pyridine derivatives on ethanol oxidation by alcohol dehydrogenase isoforms from human liver is discussed.

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541 0007-4888/01/1316-0541$25.00 (C) 2001 Plenum Publishing Corporation PHARMACOLOGY AND TOXICOLOGY Effectsof1,2,3,4-Tetrahydroimidazo[4,5-c]-PyridineDerivativesonEthanolOxidationbyAlcohol DehydrogenaseIsoformsfromHumanLiver Yu.G.Plyashkevich,V.M.Shcherbakov,E.N.Korneva,N.I.Kiseleva,andN.A.Plyashkevich

Translatedfrom ByulletenS^EksperimentalS^noiBiologiiiMeditsiny ,Vol.131,No.6,pp.640-643,June,2001OriginalarticlesubmittedMarch16,2001

Westudied invitro effectsoffour1,2,3,4-tetrahydroimidazo[4,5-c]-pyridinederivativesformedinthereactionofthecorrespondingaldehydeswithhistidineontherateofethanol oxidationbyalcoholdehydrogenaseisoformsfromhumanliver.Noneoftestcompoundsinhibitedethanoloxidationbytheseenzymes.Someofthemincreasedalcoholdehydrogena- seactivityto220-240%oftheinitiallevel.Onlyonetestcompoundacceleratedethanolox-idationby b

1b2-alcoholdehydrogenase(150%ofthecontrol).Themolecularmechanismun-derlyingtheseeffectsof1,2,3,4-tetrahydroimidazo[4,5-c]-pyridinederivativesonethanol

oxidationbyalcoholdehydrogenaseisoformsfromhumanliverisdiscussed. KeyWords:1,2,3,4-tetrahydroimidazo[4,5-c]-pyridine;derivatives;alcoholdehydrogenase;isoforms;ethanol

Department of Toxicology and Drug Testing, Center for Molecular Diagnostics and Therapy, Moscow

Alcoholdehydrogenase(ADH,EC1.1.1.1)isthemainenzymeinvolvedinprimaryalcoholbiotransformation intheliver.Thisenzymeisadimerpresentedbyvari-ousisoforms[2,6],whicharedividedinto3classesde- pendingontheirstructuralandfunctionalproperties[5].ClassIi soenzymesco ntaina-,b,and g-subunits[6].

TypicalADHdisplaysmaximumactivityatpH10.5andcontains b

1b2-subunits,whileatypicalADHpossessesmaximumactivityatpH8.8andconsistsof

b2b2-subunits[1].ADHisoformshavedifferentcatalyticactivities[3].

Theregulationofenzymeactivitiesmodulatesalcoholmetabolisminvariousethnicgroups.Thioureaspecifi- callyaffectsADHisoformsfromhumanliver.Thiscom-poundactivatesethanoloxidationby b

1b2-ADH,butinhibits b 2b2-ADH[9].However,thiseffectisob-

servedonlyatmillimolarconcentrationsofthemodu-lator.Thesearchfornewmodulatorsofethanolmeta- bolismbyliverADHisoformsisofconsiderableim-portanceforscientificpurposesandmedicalpractice.

Westudiedtheeffectsof1,2,3,4-tetrahydroimida-zo[4,5-c]-pyridinederivativesformedinthereaction ofaldehydes(productsofalcoholbiotransformation)withhistidineoncatalyticactivityofADHisoforms.

MATERIALS AND METHODS 1,2,3,4-Tetrahydroimidazo[4,5-c]-pyridine-3-carbonicacidhydrochlorides(S1-H)and1-methyl-1,2,3,4-te-

trahydroimidazo[4,5-c]-pyridine-3-carbonicacid(S1-M)weresynthesizedfromL-histidine[10].High-per- formanceliquidchromatographyshowedthatthepuri-tyofpreparationswas99%.Physicochemicalchar- acteristicsofS1-Hcorrespondedtopublisheddata[10].S1-Mwasamixtureofcis-andtrans-isomers,

Bulletin of Experimental Biology and Medicine, Vol. 131, No. 6, June, 2001542 Fig.1.Ethanoloxidationbyb1b2-(a)andb2b2-ADHfromhumanliver(b)inthepresenceofvariousconcentrationsof1-methyl-3-carboxy-(1), trans-1-phenyl-3-carboxy-(2),3-carboxy-(3),andcis-1-phenyl-3-carboxy-1,2,3,4-tetrahydroimidazo[4,5-c]-pyridine(4).

whichcannotbefractionatedbyextractionandcrys-tallization.Cis-andtrans-1-phenyl-1,2,3,4-tetrahy- droimidazo[4,5-c]-pyridine-3-carbonicacids(S1-PCandS1-PT)weresynthesizedbythefollowingmethod. L-Histidinehydrochloridemonohydrate(5g,0.12M)wasdissolvedin60mldistilledwater.KOH(13.4g) in60mldistilledwaterandbenzaldehyde(19.1g)in90mlethanolwereadded.Thereactionmixturewas boiledundervigorousstirringfor2h,cooled,andvac-uumevaporatedtoafinalvolumeof120ml.Benzal- dehydeexcesswasextractedwithether(2*30ml).TheaqueousfractionwasadjustedtopH6.0with0.2M H2SO4andcooled.Crystalswerefiltered,washed,andvacuumdried.Theprocedureyielded12.38g(4 2%)S1- PTtrans-isomerwithameltingpointof259 oC(255-257

oCasreportedpreviously[10]).Itschromatogra-

phicmobilityonSilufolplatesinanethanol:aceticacid:watersystem(6.5:1:3.5)was0.28(yellowspot

afterdevelopmentwithninhydrin). 1H-Nuclearmag-neticresonance(

1H-NMR,D2O,\Delta ppm):3.06-3.16(1H,

2d,CH 2),3.32-3.42(1H,2d,CH 2),4.06-4.12(1H,2d,CH),5.79(1H,s,CH),7.3-7.5(5H,m,Ph),and7.79

(1H,s,CHim).Chemicalshiftsweremeasuredin rela-tiontotheresidualwatersignalat40

oC(4.68ppm).

Afterseparationofthetrans-isomer,thestockso-lutionwascooledto0

oC.Whitecrystalswerefiltered,

washed,andvacuumdried.Theprocedureyielded7.59g(26%)S1-PCcis-isomerwithameltingpoint

of208oC(212-213oCasreportedpreviously[10]). 1H-NMR(D

2O, \Delta ppm):3.04-3.16(1H,2d,CH 2),3.30-3.38(1H,2d,CH

2),4.16-4.22(1H,2d,CH),5.57(1H,s,CH),7.37-7.50(5H,m,Ph),and7.71(1H,s,CHim).

ADHisoformswereisolatedfromhumanliver.Theliver(300g)wasmincedandhomogenizedindis-

tilledwater(1:3w/v).Thehomogenatewascentri-

fugedat30,000gfor10minona12-21Beckmancen-trifugeandrecentrifugedat105,000 gfor60minona Beckman18-80Mcentrifuge.Thesupernatant(~1li-ter)wasconcentratedto100ml,anditsvolumewas adjustedto200mlwith20mMTris-HClbuffer(pH8.2).ThecolumnpackedwithDEAE-52(5.3 *50cm) wasbalancedwiththesamebuffer.ADHwaspresentinafreevolume.Opticaldensitywasmeasuredat280 nm.FractionswithADHactivitywerecollectedandconcentratedtoafinalvolumeof100-200ml(protein contentnotlessthan1-2mg/ml).ADHphenotypewasdeterminedbyoptimumpHforethanoloxidationre- actionandthioureainhibition[9].ForevaluationofADHactivity,thetestcom- poundsin20t,ldistilledwaterwereaddedto3mlreactionmixturecontainingADH(0.5mgprotein/ml) and 10 mM NADH+in50mMpyrophosphatebuffer(pH9.6).Thereactionwasinitiatedbyaddingetha- nol(finalconcentration40mM)at37 oC.Thereactionratewasestimatedbychangesinopticaldensityat340 nm(NADHreductionaccompanyingADH-catalyzedethanoloxidation).Opticaldensitywasmeasuredon aHitachi-557spectrophotometerinasingle-beamreg-imen.NADHmolarextinctioncoefficientis0.0062 MU*1*cmU*1.Thereactionrate(V)wascalculatedbytangentofthelinearsegmentofthekineticcurve.The percentofactivationwascalculatedbytheformula:(Vexperiment/Vcontrol) *100%.

RESULTS PreliminaryexperimentsshowedthatADHdoesnotcausebiotransformationof1,2,3,4-tetrahydroimida-

zo[4,5-c]-pyridinederivatives.S1-Mdose-dependent-lyacceleratedethanoloxidationby b

1b2-ADH,which

Bulletin of Experimental Biology and Medicine, No. 6, 2001 PHARMACOLOGY AND TOXICOLOGY543 wasmaximum(150%)at400t,Mandthenreachedtheplateau.S1-PTinsignificantlyactivatedethanoloxida- tion(120%ofthecontrol)onlyinaconcentrationof200t,M.S1-PCandS1-Hhadnoeffectonthereac- tionrate(Fig.1, a).Theeffectsof1,2,3,4-tetrahydroimidazo[4,5-c]- pyridinederivativeson b2b2-ADHweremoreprono-unced(Fig.1, b).Thedependenceoftheactivating effectsofS1-PTonitsconcentrationwasde scribedbyabell-shapedcurvewithamaximum(240%)at 200-300 t,M.S1-Mcausedmaximumactivation(220%)inaconcentrationof300t,M.S1-PCinaconcentrationof 20t,Minsignificantlyactivatedthereaction(150%ofthecontrol).S1-Hwasineffective(similarlytoexper- imentswith b1b2-ADH).Thus,S1-MandS1-PT2-foldacceleratedethanol oxidationbyhumanliver b2b2-ADH.Itshouldbeemphasizedthatthepresenceofsub- stituentsinposition1andtheirtrans-orientationinrelationtothecarboxylicgroupplaythemajorrolein imidazopyridine-inducedactivationofADH.Thevol-umeofthissubstituentcanalsobeimportant,since S1-Mproducesactivatingeffectsinlowerconcentra-tionscomparedtoS1-PT.Thelesspronouncedacti- vatingeffectofS1-Monethanoloxidationanditsdependceonactivatorconcentrationprobablyattested tothepresenceofbothcis-andtrans-isomersinthetestsample.Itcanbeassumedthatonlytrans-isomers areactive(similarlyto1-phenylderivatives).AtypicalADHdiffersfromtypicalADHbysub- stitutionofarginineinposition47tohistidine[7].Pre-viousstudiesshowedthatthissubstitutiondrastically changescatalyticpropertiesofADH[4],sincethe

aminoacidresidueinposition47isinvolvedinNADH +coordinationintheactivecenterofADH.Degradation oftheADH-NADH-productcomplexandthereleaseofaldehydeandNADHistherate-limitingstagein oxidationofprimaryalcoholsbyADH[3].Thesedataandstereospecificeffectsofcompounds suggestthatS1-MandS1-PTbindtotheactivecen-terofADHandchangeNADH

+coordination.Themo-

lecularmechanismsofthisprocessrequirefurtherin-vestigations.Ourfindingsindicatethatnewselective

ADHactivatorsbelongingto1,2,3,4-tetrahydroimida-zo[4,5-c]-pyridinederivativesholdmuchpromise.

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Yu. G. Plyashkevich, V. M. Shcherbakov, et al.