Journal of Medicinal Chemistry, 2000 , vol. 43, _ 25 p. 4738 - 4746

EthyleneGlycolandAminoAcidDerivativesof5-AminolevulinicAcidasNewPhotosensitizingPrecursorsofProtoporphyrinIXinCells

YannBerger,†AlainGreppi,†OlivierSiri,†ReinhardNeier,*,†andLucienneJuillerat-Jeanneret*,‡

InstituteofChemistry,NeuchaˆtelUniversity,avenuedeBellevaux,P.O.Box2,CH-2007Neuchaˆtel,Switzerland,andUniversityInstituteofPathology,CHUV,Bugnon27,CH-1011Lausanne,SwitzerlandReceivedMay8,2000

ProtoporphyrinIX(PpIX)isusedasaphotosensitizingagentinphotodynamicdetectionandtherapy(PDT)ofcancerandissynthesizedintracellularlyfromaminolevulinicacid(ALA)precursors.ToevaluatemeanstospecificallytargetALAderivativestodefinedcells,wehavesynthesizedandcharacterizedethyleneglycolestersandaminoacidpseudodipeptidederivativesofALAaspotentialspecificsubstratesforcellularesterasesandaminopeptidases,respectively.ThePpIXformationinducedbytheseproductswasinvestigatedusingculturesofhumanandratcelllinesofcarcinomaandendothelialorigins.Thecytotoxicityofthesecompoundsintheabsenceoflightwasalsocontrolled.TheresultshaveshownthatethylenglycolesterscaninducehighlevelsofPpIXandareusefulatconcentrationsbelowtheircytotoxicitythreshold.FromtheALA-aminoacidderivativeswhichwereevaluated,thehighestPpIXproductionwasobtainedusingALAderivativesofneutralaminoacids,ascomparedtoacidicorbasicaminoacids.

Introduction

Photodynamicdetectionandtherapy(PDT)involvesthedetectionanddestructionofdiseasedtissuebyvisiblelightafterloadingofthetargettissuewithaphotosensitizer.AnapproachtoPDTofcancer,inpreclinicalandclinicalstudies,isbasedontheendog-enousaccumulationofprotoporphyrinIX(PpIX)follow-ingtopicalorsystemicadministrationof5-aminolevu-linicacid(ALA),usedasaprecursorphotosensitizer.1Thistherapeuticapproachisbecominganoptioninthetreatmentofdiseases,includingtumorsandpretumorallesionsorvascularproliferativedisorders,andhasrecentlybeenapprovedintheFoodandDrugAdmin-istrationintheUnitedStatesforthetreatmentofactinickeratosis.Cancertreatmentispresentlyat-temptedbytargetingthedestructionofthetumorcellsorthetumor-associatedvascularendothelialcells.WhereasALA-PDThasprovedverysuccessfulclinicallyforsuperficialapplicationsinskinandholloworganssuchasthebladder,problemshavearisenintreatingdeeperlesionsfollowingintravenousadministration,2,3,4resultinginanonspecificdistributionofALAderivativesincellsofanytype2andsensitizationtolight.Thus,tolimitunwantedsideeffectstonormaltissue,itwouldbevaluabletodesignALAderivativesdisplayingselec-tivityfortumor-associatedcellsorvascularendothelialcells,dependingontheparticulardiseasetobetreated.AlthoughtightlyregulatedPpIXformationexistsinnearlyallnucleatedcell,itpreferentiallyaccumulatesintissueswithahighcellularturnoversuchastumorcells.5,6BiologicalandchemicalapproacheshavebeenattemptedinordertoimprovetheselectivityofALAdeliveryoryieldofPpIXproductionfromALA.Using

*CorrespondencetoDr.L.Juillerat,Ph.D.,InstituteofPathology,Bugnon27,CH1011Lausanne,Switzerland.Phone:+41213147173.Fax+41213147175.E-mail:lucienne.juillerat@chuv.hospvd.ch.†NeuchaˆtelUniversity.

‡UniversityInstituteofPathology.

anadenoviralvector,aconstitutivelyactivecytoplasmicALA-synthase,thefirstregulatoryenzymeofthePpIXbiosyntheticpathway,wasexpressedinculturedtumorcells,inducingalargeincreaseinPpIXproductionandphotosensitivity.7AnotherapproachwastousemorelipophilicALAderivatives,suchasalkyl-esterssub-stratesforcellularesterases,8-11asALAisahydrophilicmoleculewithapoorpenetrationintocellularmem-branesandinterstitialspace.12However,theuseofALA-estersresultedinanonspecificdistributionofALAinallcelltypes,withacertainpreferencefortumorcells.Thisapproachrequired30-150-foldlowerconcentrationofALAderivatives,dependingontheparticularester,thanALAitselfforcomparableamountsofPpIXpro-duced.8,10,13

InanattempttoimprovecellselectivityofALAanditsderivatives,weinvestigatedPpIXformation,usingdirectfluorescencemeasurementincellsinculture,fromALAprecursors,eitherestersofthepolyethylenglycol(PEG)familyassubstratesforcellularesterases,orbasic,acidic,andneutralaminoacidderivativeswhichrepresentpotentialsubstratesforcell-surfaceandcy-toplasmicaminopeptidasesand/orligandforpeptideandaminoacidtransporters.Thesynthesis,characteriza-tion,andevaluationincellcultureofthesenewALAderivativesaredescribed.ALA-PEG-estersappearedtobeapotentiallybetterprecursorthanALA-alkylesters,leadingtohighlevelsofproductionofPpIXincellsanddisplayinganimprovedratioofPpIXproductionversuscytotoxicity.Oftheaminoacidderivativestested,ALA-pseudopeptidederivativesofneutralaminoacidsin-ducedthehighestPpIXproduction.Results

SynthesisofALADerivativePrecursorsofPpIX.1.SynthesisoftheEthyleneGlycolDerivatives.Theinitialfeaturewhichwasexaminedforitscapacitytoobtainderivativesof5-aminolevulinatecapableto

10.1021/jm000981qCCC:$19.00©2000AmericanChemicalSociety

PublishedonWeb11/22/2000

ALA-EstersandALA-AminoAcidsforPDTJournalofMedicinalChemistry,2000,Vol.43,No.2739

Scheme1a

a

Reagentsandconditions:(i)SOCl2,50°C,5h.

Table1.SynthesisofALAEthyleneGlycolEsterDerivativesacode3a3b3c3d

a

yield[%]908790

name

ALA-O-ethyleneglycolmonomethyletherALA-O-ethyleneglycolmonoethyletherALA-O-diethyleneglycolmonoethyletherALA-O-triethyleneglycolmonoethylether

Yieldofthesyntheses,andnumerationandnomenclatureofthederivativesusedincellcultures.

inducePpIXproductionincellswastheuseofethyleneglycolderivatives2a-dasalcoholsusedfortheesteri-fication.EarliertestswithsimplealkylestershadshownthatincreasingthechainlengthinducedhigherproductionofPpIX.9,13Amajorlimitationtothisap-proachwasthereducedsolubilityinwateroftheestersderivedfromlongchainalcohols.Ethyleneglycolshavetheinterestingpropertiestobeatthesametimehydro-andlipo-soluble.Theesters3a-dderived5-aminolevulinate(1)andthecorrespondingethyleneglycols2a-dwereobtainedinexcellentyieldsstartingfromtheinsitupreparedacidchlorideandthecorrespondingalcohols(Scheme1,Table1).Themajorproblemwas

Scheme2a

theremovalofthecorrespondingalcoholusedinexcess.Thecompoundswerefullycharacterizedspectroscopi-cally;however,theelementalanalysisindicatedthatsomeamountofthecorrespondingalcoholwasstillpresentinoursamples.Preliminarytestswereunder-takentodeterminetherelativerateoftheenzymatichydrolysisoftheseestersusingR-chymotrypsinandhogliveresterase.Therateofhydrolysisoftheseesters3a-dwascomparedwiththerateofhydrolysisofthen-hexyl5-aminolevulinate.Therateoftheenzymatichydrolysisofouresterswascomparableandinmostcasesevenhigherthantherateoftheenzymatichydrolysisofthen-hexyl5-aminolevulinate,acom-poundinducingtheproductionofhighlevelsofPpIXproductioninvitroandinvivo.

2.SynthesisoftheAminoAcidDerivatives.Thecondensationof5-aminolevulinate(1)toobtainthepep-tideanalogues6a-ewasstraightforward14(Scheme2).InourfirsttrialswehadusedFmoc-protectedaminoacidsforthecondensationreaction.However,itbecameclearthatthedeprotectionoftheFmoc-groupwasdifficult.Thepeptideanalogueswerenotstableunderbasicconditions.SoweturnedtotheBoc-protectedaminoacids4a-d.IncontrasttotheFmoc-protectedpeptideanalogues,mostoftheBoc-protectedpeptides6a-ewerenotcrystalline,whichrenderedthepurifica-tionlessconvenient.AfteroptimizationofthereactionconditionsstartingfromtheBoc-peptides6a-e,themono-deprotectedpeptides7a-ecouldbeobtainedingoodyieldastheirtrifluoroacetates(Scheme2,Table2).Totesttheimportanceoftheprotectinggroupsofthecarboxylateandtheaminofunctions,selectivemono-deprotectionofthecarboxylategroupwasachievedusingpigliveresterase(Scheme3).Compound8acould

a

Reagentsandconditions:(i)HOBt,EDC,Net3,roomtemperature,12h;(ii)TFA,roomtemperature,1h.

Table2.SynthesisofPseudopeptideAminoAcidALADerivativesa

code6a7a7b7c7d7e8a9a

a

R1b

methyl-methyl-methyl-benzyl--(CH2)4-NH3‚TFA-CH2-COOHmethyl-methyl

R2b-O-methyl-O-methyl-O-hexyl-O-methyl-O-methyl-O-methyl-H-H

yield[%]72717469728882

name

Boc-Ala-ALA-O-methylAla-ALA-O-methylAla-ALA-O-hexylPhe-ALA-O-methylLys-ALA-O-methylAsp-ALA-O-methylBoc-Ala-ALAAla-ALA

Yieldofthesyntheses,andnumerationandnomenclatureofthederivativesusedincellcultures.bR1andR2accordingtoScheme2.

4740JournalofMedicinalChemistry,2000,Vol.43,No.25Bergeretal.

Scheme3a

a

Reagentsandconditions:(i)PLE,phosphatebufferpH8.0,roomtemperature,5h;(ii)TFA,roomtemperature,1h.

Figure1.PpIXfluorescenceproductionfollowingexposureofhumanendothelialHCECandcarcinomaA9cellstoALA-ethyleneglycolesters.(A)EffectofthelengthoftheetherandglycolchaininHCECendothelialcells:[ALA-O-ethyleneglycolmonomethylether(3a)[87µM];9ALA-O-ethyleneglycolmonoethylether(3b)[82µM];2ALA-O-diethyleneglycolmonoethylether(3c)[100µM];OALA-O-triethyleneglycolmonoethylether(3d)[90µM].(B)EffectofthelengthoftheetherandglycolchaininA9carcinomacells:[ALA-O-ethyleneglycolmonomethylether(3a)[87µM];9ALA-O-ethyleneglycolmonoethylether(3b)[82µM];2ALA-O-diethyleneglycolmonoethylether(3c)[79µM];OALA-O-triethyleneglycolmonoethylether(3d)[85µM].

beobtainedingoodyield(Table2)andhighpurity.Thetotallydeprotectedpeptide9awasobtainedusingtrifluoroaceticacid.Allpeptideswhichcontainedanaminogrouphadtobestoredastheirtrifluoroacetatesat4°C.

EvaluationoftheProductionofPpIXfromSyn-theticPrecursorsinCellLines.1.ProductionofPpIXfromtheSyntheticALADerivativesofEth-ylenglycols.TheproductionofPpIXfromALA-estersoftheethylenglycolfamily3a-dwasevaluatedusinghumancellsincultureeitherfromendothelial(HCEC)(Figure1A)ortumor(A9)origin(Figure1B).First,theeffectofthechainlengthoftheethermoietywasdetermined.ProductionofPpIXfromtheALAO-methylethyleneglycolandO-ethylethyleneglycolesters3aand3bwerecomparedinendothelial(Figure1A)andtumor(Figure1B)cells.Inendothelialcells(Figure1A),

theO-ethyl-derivative3bproducedhigherlevelsofPpIXthantheO-methyl-derivative3a,whileintumorcells(Figure1B),thePpIXlevelwaslowerthaninendothe-lialcellsforbothderivatives.Theeffectofincreasingthechainlengthoftheethyleneglycolmoietywasevaluatednext.Inendothelialcells(Figure1A),nodifferenceswereobservedbetweenthemono-,di-,andtriethyleneglycolethylether3a,3c,and3d.Intumorcells(Figure1B),increasingtheethyleneglycolchainlengthslightlyincreasedthePpIXproduction.TheseresultssuggestthatsomeselectivityforendothelialcellstowardtumorcellscanbeobtainedusingALAestersoftheethyleneglycolfamilylike3a-d.Thecytotoxicityintheabsenceofaddedlightoftheseethyleneglycolderivatives3a-dwascontrolledbydeterminingtheefficacyindex,comparingPpIXfluorescenceat300minwithcellsurvival(MTTevaluation)after20hexposuretoincreasingconcentrationsofthesecompounds.NocytotoxicitywasobservedforHCECcells(Figure2A)orA9cells(Figure2B)exposedtolowconcentrationsofALA-ethyleneglycolethylether,whenthePpIXfluorescencewasalreadyhigh;whileincellsexposedtohigherconcentrationsoftheethyleneglycolderiva-tives,cytotoxicitywasobserved(Figure3A-D).Theresultsindicatedthatendothelialcellsaremoresensi-tivethantumorcellstothecytotoxiceffectofthesederivatives,parallelingthePpIXproduction.

2.ProductionofPpIXfromtheSyntheticALADerivativesofAminoAcids.Toevaluateadifferentapproachtocell-specifictargetingofALAderivatives,wepreparedtheaminoacidpseudopeptidederivativesofALA6a-e,7a-e,8a,and9awhicharepotentialsubstratesforcellularaminopeptidases.Aminoacid-ALAderivativesofacidic,basic,orneutralaminoacids,aspotentialsubstratesforaminopeptidaseA(APA),APB,orAPN/M,respectively,weresynthesizedandcharacterized(listedinTable2).Theexpressionoftheseaminopeptidaseactivitieswascontrolledinthecellslinesthatwereused(Table3).Aparticularaminopep-tidaseactivitywasexpressedathighlevelbyadefinedcelllineforeachoftheactivitiesevaluated:APAbyEC219cells,APBbyHCECcells,andAPN/MbyA9cells.A9cellsdidnotexpressmeasurableAPAactivity.Byfar,atequimolarconcentration,thebestprecursoristheneutralaromaticaminoacid-derivativesubstrate7cforAPN/M-likeaminopeptidasesinallcelllinestestedandwhateverthelevelofexpressionofthisenzymaticactivitybythedifferentcelllines(Figure4A-C).Theresultsshowthatbasicaminoacidderivativessuchas7darenotprecursorsforPpIXproductionwhateverthecellline,includingHCECcellswhichexpresshighlevelsofAPBactivity,andthesubstrate

ALA-EstersandALA-AminoAcidsforPDTFigure2.PpIXfluorescenceandMTTreductioninHCEC(A)andA9(B)cellsexposedtoincreasinglowconcentrationsofALA-O-ethyleneglycolmonoethylether(3b).[:PpIXfluorescence[a.u.];0:MTTreduction[A0nm].

concentrationused(Figure3C).Acidicderivativessuchas7earepoorprecursorsinthecellsexpressingAPA(Figure4A,B),EC219,andHCECcells,whilenoPpIXproductionwasobtainedinAPA-negativeA9cells(Figure4C).However,inEC219cellsexpressinghighAPAactivity,PpIXproductioncanbeobservedusinghighconcentrations,upto10mM,oftheprecursor(resultsnotshown).TotestaminopeptidaseAacces-sibilitytosubstrate,wedeterminedinthebrain-derivedendothelialEC219cellsandinbrain-derivedendothelialcellsofhumanglioblastomatheinsituactivityofAPAusingGlu-methoxy-naphthylamidesubstratewhichre-sultsinaredprecipitateofthemethoxy-naphthylaminewithdiazotatingreagentsfollowinghydrolysis(Figure5).TheresultsshowedthatAPAiseasilyaccessibleinEC219cellsaswellasinthevascularsystemofhumanglioblastoma.Thustargetingofcellularproteasesmaybeofuseinthetargetingofphotosentizerstodefinedcells.

Thespecificityofneutralaliphaticaminoacid-ALAderivativessuchas7aand7cforAPN/M-likeamino-peptidasewasfurtherevaluatedusingtheN-substitutedAla-ALAderivative6aand8a,whichwouldnotbesubstratesforsuchanenzyme,ortheAlaALA-methyland-hexylesters7aand7b,whichwouldmakethembettercell-permeantwithincreasinghydrophobicity.Theresultsshowedthat,asexpected,theN-substitutedproducts6a(notshown)and8a(Figure6A)arenotPpIXprecursors,validatingthenecessityforanAPN/M-likeactivitytoreleaseALAfromtheaminoacid

JournalofMedicinalChemistry,2000,Vol.43,No.2741

Figure3.EfficacyoftheALA-O-diethyleneglycolandALA-O-triethyleneglycolmonoethylethersinhumanendothelialandtumorcellsatincreasinghighconcentrationsofALA-ethyleneglycolderivatives.PpIXproduction(fluorescenceat300min)andcytotoxicityat20h(MTTassay,absorbanceat0nm)oftheALA-O-diethyleneglycolmonoethyl(3c)(A,C)andALA-O-triethyleneglycolmonoethyl(3d)(B,D)ethersinhumanendothelialHCEC(A,B)andcarcinomaA9(C,D)cellsweredetermined.Resultsaremeansofsextuplewells(SEM.Bars:PpIXfluorescence[a.u.];[:MTTreduction[A0nm].

Table3.ExpressionofAminopeptidase(AP)A,B,andN/MActivitiesintheEndothelialandCarcinomaCellLinesacelllineAPAb

APBb

APN/Mb

substratesrefGlu-,Asp-X

Lys-,Arg-X

Phe-,Ala-,Leu-X

A9c28-+++HCECd++++EC219d

27

++

+

+

a

Cellsweregrowntoconfluenceandenzymaticactivitiesweremeasured.bSymbols:-,nomeasurableactivity.+,lowenzymaticactivity;++,highenzymaticactivity.cA9cells:humanlungcarcinomacellline.dHCECandEC219cells:cerebralendothelialcellsofhumanandratorigin,respectively.

derivative,andthatincreasinghydrophobicity(Ala-ALA-methyl7aversusAla-ALA-hexyl7b)didresultinincreasedPpIXproduction.ComparisonofPpIXproduc-tionbetweenAla-ALA9aandAla-ALAhexylesterderivatives7b(Figure6A)withALAandALA-hexylester(Figure6B)demonstratedacomparablebehaviorwithincreasinglipophilicity,suggestingacommonmechanismforintracellularuptake.Discussion

Thetargeteddeliverytodefinedcellsofanticancertreatments,includingphotosensitizersinPDT,isanintensefieldofresearch.Precursorphotosensitizers,suchasALA,mustbeinternalizedbytargetcellsinordertobeprocessedtothephotosensitizerPpIXinmitochondria,dependingonthepresenceintargetcellsnotonlyofthenecessarytransportsystemsabletodeliverALAintothecytoplasm,butalsooftheprocess-ingenzymesabletoreleasefreeALAfromitspro-drug

4742JournalofMedicinalChemistry,2000,Vol.43,No.25Figure4.PpIXfluorescenceproductionfollowingexposureofhumanandratendothelialcellsandhumancarcinomaA9cellstoALA-aminoacidesters.(A)Acidic,basic,andneutralaminoacidderivativesaddedtohumanendothelialHCECcells:2Asp-ALA-O-methyl(7e)[1.57mM];9Lys-ALA-O-methyl(7d)[1.17mM];[Phe-ALA-O-methyl(7c)[1.43mM].(B)Acidic,basic,andneutralaminoacidderivativesaddedtoratendothelialEC219cells:2Asp-ALA-O-methyl(7e)[1.88mM];9Lys-ALA-O-methyl(7d)[1.61mM];[Phe-ALA-O-methyl(7c)[1.68mM].(C)Acidic,basic,andneutralaminoacidderivativesaddedtohumancarcinomaA9cells:2Asp-ALA-O-methyl(7e)[1.57mM];9Lys-ALA-O-methyl(7d)[1.17mM];[Phe-ALA-O-methyl(7c)[1.43mM].

andtoperformthebiosyntheticpathway.Thesespecificmetabolicprocessesmaybedifferentincellsofdifferentphenotypes.15OurpresentgoalwasthustodefinemeanstospecificallytargetALAdeliveryeithertotumor-associatedendothelialcellsortotumorcells.Toevaluatethepotentialoftargetingspecificcellularesterases,wecomparedPpIXproductionobtainedfol-lowingexposureofhumanendothelialcellsorhumancarcinomacellstoALA-estersoftheethyleneglycolfamily3a-dincomparisonwithinformationobtainedusingalkylestersofALA.8,13Inadifferentapproach,basedonthepreviousinformationthatdefinedacidic,basic,orneutralaminopeptidaseactivitiesarediffer-entlyexpressedintumorvasculaturewhencomparedtonormalvasculatureofthesameorgan,16weprepared

Bergeretal.

Figure5.InsituaminopeptidaseAactivity(histoenzymog-raphy)intumorvasculatureofhumanglioblastomaandintactEC219cells:(a)humanglioblastoma;(b)EC219cells.

Figure6.PpIXfluorescenceproductionfollowingexposureofhumancarcinomaA9cellstoN-andC-blockedAla-ALAesters.(A)[Boc-Ala-ALA(8a)[10.9mM];9Ala-ALA(9a)[11.1mM];2Ala-ALA-Me(7a)[11.4mM];0Ala-ALA-Hex(7b)[1.9mM];(B)[ALA[3.9mM];9ALA-hex[39.8µM].

aminoacid-ALAsubstratesforaminopeptidasesandcomparedPpIXproductionincelllinesofendothelialandcarcinomaphenotype,expressinghighorlowlevelsofthethreetypesofaminopeptidases.ALAestersoftheethyleneglycolfamily3a-ddemonstratedsomeselec-tivityforendothelialcellstowardtumorcells,andALAsubstratesforaminopeptidases,inparticularAPN/M,demonstratedthepotentialtoobtainPpIXfromsuchprodrugs.

IthasbeenpreviouslyshownthatALA-hexylesterisinternalizedmorerapidlyandefficientlythatALAorALA-methylester;8,9,13,17however,nodifferencesinthecellularlevelsofPpIXwereobservedbetweenendothelialandtumorcells(Juillerat,unpublishedresults).UsingALA-estersofethyleneglycols3a-d,weobtainedincreasedPpIXproductioninendothelialcells

ALA-EstersandALA-AminoAcidsforPDTcomparedtotumorcells,especiallyforlongchainethers.Inaddition,theethyleneglycolderivatives3a-dusedinthepresentexperimentsdisplayedabetterPpIXfluorescenceyieldversuscytotoxicityintheabsenceoflightthandidthealkylesters(Juillerat,unpublishedresults),suggestingthatreducedcytotoxicsideeffectsforbystandercellsmaybeobtainedusingthisclassofcompounds.However,cytotoxicityassociatedwithcellspecificitymayalsobeanadvantageincancertreat-ment.EvidenceexiststhatpartoftheefficiencyofPDTinvolvesvasculardamage.PDTmaycausetumorre-gressionandcelldeath(apoptosisornecrosis)18eitherbydirectdeathoftumorcellsthemselvesoralsobyinducingtumorhypoxiathroughmicrovascularshut-down.19,20Clinically,theselectivetargetingofdrugstotumor-associatedendothelialcellsaftersystemicinjec-tionisalsoanadvantagewhencomparedtotargetingthetumorcellsthemselves.

TheuseofaminoacidderivativesofALAhadnotbeenattemptedbefore,tothebestofourknowledge.Sub-stratesforbasicaminopeptidasessuchas7dwerenotprecursorsforPpIXproduction,evenincellsexpressingAPBactivity,whilesubstratesforacidicaminopepti-daseswereprecursorsonlyincellsexpressingAPAactivity.However,highconcentrationsofthepro-drughadtobeusedinordertoobtaintheintracellularPpIXconcentrationswhichcanberelevantforPDT.WedidnotattempttodeterminethekineticcharacteristicsofhydrolysisofLys-ALA7dorAsp-ALA7ebycellularaminopeptidasesorthemechanismsofcellularuptake.Thuswecannotwiththepresentexperimentsdeterminewhetherpoorkineticconstants,alowcellpenetration,ordeliveryoftheprodrugtoacellularcompartmentdifferentfromthecytoplasmwhereALAdehydrataseislocatedisresponsibleforthelowPpIXproduction.UsingsubstratesforAPN/Mactivity,Ala-ALA7aandPhe-ALA7c,weobtainedhighyieldsofPpIX.Theseenzymesarelocatedbothonthecellsurfaceandinthecytoplasm.PpIXproductionwasnotobservedusingtheN-terminalBoc-derivative6aor8a,whicharenotsubstratesforsuchanenzyme,demonstratingtheinvolvementofAPN/MactivityinthereleaseofALA.ProductionofPpIXfromAla-ALA,freeacid9a,andmethylorhexylesters7a,bwascomparabletoPpIXproductionobtainedfromALAfreeacidandmethylorhexylesters,suggestingthatthelipophilicityofaminoacidpro-drugsfacilitatestheircellpenetrationandthatbothaminoacid-ALAandALAderivativesuseacom-montransportsystem.However,ourapproachcannotformallydifferentiatebetweenthealternativethateither(i)Ala-ALAoritsestersarefirsthydrolyzedatthecellsurfaceandthenfreeALAoritsestersareinternalizedor(ii)thehydrolysisisperformedintrac-ellularlyfollowingtransportoftheaminoacid-ALAderivative.ThemechanismofentryofexternallyaddedALAanditsestersintocellshavenotbeendetermined.ALAuptakebycellsinculturehasbeenshowntobeinhibitedbysomeaminoacids,21peptides22ormodula-torsofion-cotransporters,17,22,23suggestingtheinvolve-mentofanaminoacid/peptide(co)transport.Inmam-maliancellstransfectedwithdipeptidetransporters,radioactiveALAwastranslocatedbysaturableandpH-dependenttransportmechanismsusingacation/ALAcotransport.17,22Di-andtripeptidesandserum,butnot

JournalofMedicinalChemistry,2000,Vol.43,No.2743

aminoacids,suchasGluorGABA,24competedfortransport,indicatingthattheaminoacidtransportsysteminvolveddoesnottransportacidicandbasicaminoacidanalogues.Thisinformationmayexplainthepoorefficiencyofacidic-andbasic-ALAderivativesasPpIXprecursors,suggestingthatthehydrolysisoftheaminoacid-ALAderivativeisperformedbyintracellularaminopeptidases,mainlyoftheneutraltype,followingentryofcompoundsintocells.AsAPAactivityhasbeenmainlydescribedasanectoenzyme,thismayalsoexplainthepoorefficiencyforthispro-druginPpIXproduction.

Inconclusion,wedemonstrateherethepotentialofanewapproachforthespecifictargetingoftumor-associatedendothelialcellsortumorcellsforPDTprotocols.Thecell-specificdeliveryofPpIXprecursorsmaybeobtainedusingcell-specificaddressesconsistingofestersofethyleneglycolsorpeptidederivativesofALAforcellularesterasesandpeptidases,respectively.ExperimentalSection

Synthesis.GeneralSyntheticMethods.MeltingpointsweredeterminedonaGallenkampMFB-595apparatusandareuncorrected.InfraredspectrawererecordedonaPerkin-ElmerFT-IR1720Xspectrometer.1HNMRand13CNMRspectrawererecordedonaBrukerAMX-400andarereportedinppmrelativetoTMSandreferencedtothesolventindicated.Massspectrawererecordedonthefollowinginstruments,usingthestatedionizationmethods:NermagRC30-20massspectrometer,electronimpactionizationmassspectra(EI),anddirectchemicalionizationmassspectra(DCI)usingNH3asreactant;FinniganLCQmassspectrometer,electrosprayionization(ESI);BrukerFTMS4.7TBioAPEXIImassspec-trometer,high-resolutionmassspectra(HRMS)measuredusingtheESItechnique,whichallowedtodeterminethemolecularformulaofthe[M+H]+orthe[M+Na]+peak.ElementalanalyseswereobtainedfromtheCHNanalyseslaboratoryofCibaSpecaltiesinMarlyandarewithin0.4%oftheoryunlessotherwisenoted.Reagentswereusedasacquiredfromcommercialsourceswithoutpurification.Anhydroussolventswereobtainedbydistillationoveranadequatedryingagent.25Solventwasremovedbyrotaryevaporationunderreducedpressure,andsilicagelchromatographywasper-formedusingMercksilicagel60withaparticlesizeof40-63µm.Thepurifiedcompoundswereanalyzedbythin-layerchromatography(silicagel60F20.2mm,Merck,solventCH2-Cl2/CH3OH/H2O70:30:5,detectionwithKMNO4).Tokeepthenumberingofthesynthesizedcompoundscoherent,thecarbonoftheesterfunctionorthecarbonofthecarboxylatefunctionwasalwaysattributedthenumber1.

GeneralProcedurefortheSynthesisofCompounds3a-d.Toasolutionof0.5mL(7mmol)ofthionylchloride(Fluka)in3mL(38mmol)ofmethoxyethanol(Fluka,2a)wasadded500mg(3mmol)of5-aminolevulinicacidhydrochloride(ALA,1),andthesolutionwasstirredat50°Cfor5hunderargonatmosphere.Theresultingsolutionwasdirectlyappliedtoasilicachromatographycolumn(50gofsilicagel(Merck),eluent:dichloromethane/methanol95/5)toprovidetheproduct(3a)asyellowishoil.

Compound3a:90%yield;1HNMR(CD3OD)δ4.30-4.28(m,2H,H2C(12)),4.13(s,2H,H2C(5)),3.70-3.67(m,2H,H2C-(13)),3.45(s,3H,H3C(14)),2.95-2.92(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.81-2.78(m,BB′ofAA′BB′-system,2H,H2C(2));13CNMR(CD3OD)δ203.4(C(4)),174.2(C(1)),71.7(C(13)),65.1(C(12)),59.4(C(14)),48.5(C(5)),35.6(C(3)),28.8(C(2));MS(EI)m/zrelativeintensity190(100,[M-Cl]+);HRMS(ESI)calcdforC11H22NO5+190.1073,obs.190.1071.Anal.calcdforC8H16NO4Cl:C,42.58;H,7.15;N,6.21.Found:C,38.97;H,6.75;N,5.71.

Compound3b:%yield;1HNMR(D2O)δ4.19-4.17(m,2H,H2C(12)),4.05(s,2H,H2C(5)),3.67-3.(m,2H,H2C(13)),

4744JournalofMedicinalChemistry,2000,Vol.43,No.253.53(q,3J(1,415))7.1,2H,H2C(14)),2.87-2.84(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.69-2.65(m,BB′ofaAA′BB′-system,2H,H2C(2)),1.11(t,3J(1,415))7.1,3H,H3C(15));13CNMR(D2O)δ206.5(C(4)),177.3(C(1)),70.3(C(13)),69.3(C(14)),66.8(C(12)),49.6(C(5)),36.8(C(3)),30.0(C(2)),16.6(C(15));MS(EI)m/zrelativeintensity204(100,[M-Cl]+),1(77),146(30),114(65);HRMS(ESI)calcdforC9H17-NO4+:204.2448,obs.204.2444.Anal.calcdforC9H18NO4Cl:C,45.10;H,7.57;N,5.84.Found:C,44.45;H,7.29;N,5.79.Compound3c:87%yield;mp25.0-30.0°C;1HNMR(400MHz,D2O)δ4.20-4.18(m,2H,H2C(12)),4.05(s,2H,H2C(5)),3.70-3.68(m,2H,H2C(13)),3.63-3.61,3.59-3.56(m,2H,m,2H,H2C(14),H2C(15)),3.51(q,3J(1,617))7.1,2H,H2C(16)),2.87-2.83(m,AA′ofAA′BB′-system,2H,H2C(3)),2.68-2.65(m,BB′ofAA′BB′-system,2H,H2C(2)),1.11(t,3J(1,617))7.1,3H,H3C(17));13CNMR(100MHz,D2O)δ206.6(C(4)),177.3(C(1)),72.2,71.5(C(14),(C(15)),71.0(C(13)),69.2(C(16)),66.7(C(12)),49.7(C(5)),36.9(C(3)),30.0(C(2)),16.7(C(17));MS(EI)m/zrelativeintensity248(46,[M-Cl]+),117(25),99(68),86(45),72(100);HRMS(ESI)calcdforC11H22NO5+:248.1491,obs.248.1495.Anal.calcdforC11H22NO5Cl:C,46.56;H,7.81;N,4.94.Found:C,44.93;H,7.18;N,4.93.

Compound3d:90%yield;1HNMR(400MHz,D2O)δ4.31-4.28(m,2H,H2C(12)),4.15(s,2H,H2C(5)),3.79-3.77(m,2H,H2C(13)),3.75-3.72,3.73,3.70-3.67(m,2H,′s′,4H,m,2H,H)7.0,2H,2C(17),HH2C(16),H2C(15),H2C(14)),3.(q,3J(1,819)2C(18)),2.95-2.92(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.82-2.79(m,BB′ofaAA′BB′-system,2H,H2C-(2)),1.28(t,3J(1,918))7.1,3H,H3C(19));13CNMR(100MHz,D2O)δ203.5(C(2)),174.0(C(1)),71.8,71.7,71.1(C(14)-C(17)),70.3(C(13)),67.8(C(18)),65.2(C(12)),48.6(C(5)),35.6(C(3)),29.0(C(2)),15.7(C(19));MS(ESI+)m/zrelativeintensity315(25,[M-HCl+Na]+),293(100,[M-Cl]+),201(27),196(56),179(99),149(37);HRMS(ESI)calcdforC13H26NO6+:292.1755,obs.292.1751.

GeneralProceduresforthePreparationofthePep-tideAnalogues7a-eviaCouplingof5-AminolevulinicAcidEsterswithBOC-ProtectedAminoAcids4a-dFollowedbyAcidInducedDeprotectionoftheBOC-Group.Coupling.Atotalof1mmolofBOC-aminoacids(4a-d)(Novabiochem)dissolvedin8mLofDMFwasactivatedduring45minbyadditionof0.82g(6.1mmol)ofN-hydroxybenzotriazole(HOBt,Novabiochem)and1.16g(6.1mmol)ofN-(3-dimethylaminoprpyl)-N′-ethylcarbodiimideHCl(EDC,Fluka).Asolutionof5.5mmolofALA-methyl(5a)orALA-hexyl(5b)estersin6mLofDMFwasaddedfor10min,then0.77mL(5.5mmol)oftriethylamine(NEt3,Fluka)wasadded.After12hstirringatroomtemperature,theDMFwasdistilledinaKugelrohroven(50°C/0.5mbar)andthecrudematerialwasextractedwith2×100mLofethylacetateandwashedsequentiallywith75mLof1Mcitricacid,NaHCO310%,andNaClsaturatedwater.Thesolutionwasappliedtoasilicachromatographycolumn(50gsilicagel(Fluka),eluent:ethylacetate:hexane,1:3to5:1).Thepureproductswereobtainedassolidsfollowingevaporationofthesolvent.

Compound6a:72%yield;1HNMR(400MHz,CDCl3)δ7.07(sbr,1H,NH-H2C(5)),5.30(sbr,1H,NH-HC(7)),4.19-4.16(m,1H,HC(7)),4.11(d,3J(5,NH))5.0,2H,H2C(5)),3.60(s,3H,H3C(12)),2.71-2.67(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.59-2.55(m,BB′ofaAA′BB′-system,2H,H2C(2)),1.37(s,9H,H3C(10a),H3C(10b),H3C(10c)),1.30(d,3J(7,72))7.1,3H,H3C(72));13CNMR(100MHz,CDCl3)δ203.8(C(4)),173.0,172.7(C(1),C(6)),155.3(C(8)),80(C(9)),51.7(C(12)),49.9(C(7)),48.9(C(5)),34.3(C(3)),28.1(C(10a),C(10b),C(10c)),27.4(C(2)),18.4(C(72));MS(EI)+)m/zrelativeintensity317(29,[M+H]+),261(53),217(16),145(31),144(100),140(26),115(68),88(80),87(20),70(20);HRMScalcdforC14H24N2O6-Na+339.1526,found339.1525.Anal.(C14H24N2O6)C,H,N.Compound6b:(65%yield);1HNMR(400MHz,CD3OD)δ4.22-4.13(m,3H,H2C(5),HC(7)),4.15(t,3J(12,13))6.7,2H,H2C(12)),2.87-2.84(m,AA′ofaAA′BB′,2H,H2C(3)),2.69-2.66(m,BB′ofaAA′BB′,2H,H2C(2)),1.71(quint.,3J(13,14))3J(13,12))6.7,2H,H2C(13)),1.53(s,9H,H3C(10a),H3C-(10b),H3C(10c)),1.47-1.37(m,6H,H2C(14),H2C(15),H2C(16)),

Bergeretal.

1.42(d,3J(72,7))6.9,3H,H3C(72)),1.32(t,3J(1,716))6.9,3H,H3C(17));13CNMR(100MHz,CD3OD)δ206.4(C(4)),176.5(C(1)),174.7(C(6)),157.9(C(8)),80.9(C(9)),66.1(C(12)),51.9(C(7)),50.0(C(5)),35.5(C(3)),32.9(C(15)),29.9(C(13)),29.0(C(2),C(10a),C(10b),C(10c)),26.9(C(14)),23.9(C(16)),18.7(C(72)),14.8(C(17));MS(DCI)+)m/zrelativeintensity387(18,[M]+•),331(40),313(25),288(40),287(51),270(55),269(100),201(13),144(25)114(12),100(29),99(20);HRMS(ESI)calcdforC19H34N2O6Na+409.2309,found409.2308.

Compound6c:75%yield;1HNMR(400MHz,CDCl3)δ7.30-7.17(m,5H,HC(74),HC(74′),HC(75),HC(75′),HC(76)),6.65(sbr,1H,NH-H2C(5)),5.00(sbr,1H,NH-HC(7)),4.41(sbr,1H,HC(7)),4.20-4.04(m,2H,H2C(5)),3.66(s,3H,H3C(12)),3.13-2.99(m,2H,H2C(72)),2.70-2.66(m,AA′ofanAA′BB′system,2H,H2C(3)),2.-2.59(m,BB′ofanAA′BB′system,2H,H2C(2)),1.38(s,9H,H3C(10a),H3C(10b),H3C(10c));13CNMR(100MHz,CDCl3)δ203.2(C(4)),172.7(C(1)),171.4(C(6)),155.3(C(8)),136.5(C(73)),129.2,128.6(C(7,474′,7,575′)),(C(7,474′)),126.9(C(76)),80.2(C(9)),55.6(C(7)),51.9(C(12)),49.0(C(5)),38.4(C(72)),34.4(C(3)),28.2(C(10a),C(10b),C(10c)),27.5(C(2));MS(APCI)+)m/zrelativeintensity394(23,[M+H]+),393(98,[M]+•),337(14),293(13),275,261,247,243,215;HRMScalcdforC20H28N2O6Na+415.1840,found415.1838.Anal.(C20H28N2O6)C,H,N.

Compound6d:71%yield;1HNMR(400MHz,CD3OD)δ4.23-4.09(m,3H,H2C(5),HC(7)),3.74(s,3H,H3C(12)),3.15-3.11(m,2H,H2C(75)),2.88-2.84(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.70-2.66(m,BB′ofaAA′BB′-system,2H,H2C-(2)),1.92-1.84(m,1H,HC(72a)),1.76-1.67(m,1H,HC(72b)),1.(s,18H,H3C(78a),H3C(78b),H3C(78c),H3C(10a),H3C(10b),H3C(10c)),1.-1.40(m,4H,H2C(73),H2C(74));13CNMR(100MHz,CD3OD)δ206.4(C(4)),175.9(C(1)),175.1(C(6)),158.8,158.2(C(76),C(8)),80.9,80.1(C(77),C(9)),56.3(C(7)),52.5(C(12)),49.9(C(5)),41.3(C(75)),35.5(C(3)),33.2(C(72)),30.9(s,C(74)),29.1(C(78a),C(78b),C(78c)orC(10a),C(10b),C(10c)),29.0(C(10a),C(10b),C(10c)orC(78a),C(78b),C(78c)),28.7(C(2)),24.4(C(73));MS(ESI+)m/zrelativeintensity475(23,[M+H]+),474(100,[M]+•),418(8),374(11),318(3),300;HRMS(ESI)calcdforC22H39N3O8Na+496.2629,found496.2631.Anal.calcdforC22H39N2O8:C,55.80;H,8.30;N,8.87.Found:C,55.27;H,8.40;N,8.56.

Compound6e:73%yield;1HNMR(400MHz,CDCl3)δ7.16(t,3J(5,NH))4.8,1H,NH-H2C(5)),5.68(d,3J(7,NH))8.2,1H,NH-HC(7)),4.46-4.43(m,1H,HC(7)),4.09(dxt,3J(5,NH))4.6,4J(3,5))2.2,2H,H2C(5)),3.59(s,3H,H3C(12)),2.79-2.73(m,1H,HC(72a)),2.69-2.65(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.61-2.(m,3H,H2C(2),HC(72b)),1.38(s,9H,H3C(75a),H3C(75b),H3C(75c)),1.36(s,9H,H3C(10a),H3C-(10b),H3C(10c));13CNMR(100MHz,CDCl3)δ203.2(C(4)),172.6(C(1)),171.0(C(6)),170.7(C(73)),155.3(C(8)),81.4(C(74)),80.1(C(9)),51.7(C(12)),50.6(C(7)),49.0(C(5)),37.2(C(72)),34.2(C(3)),28.1,27.8(C(75a),C(75b),C(75c)),C(10a),C(10b),C(10c)),27.4(C(2));MS(ESI)439(21,[M+Na]+),418(20,[M+H]+),417(100,[M]+•),416(11),361(16);HRMS(ESI)calcdforC19H32N2O8Na+439.2053,found439.2051.

ProcedurefortheDeprotectionoftheN-TerminalAminoAcidFunction.Atotalof0.3mmolofprotectedaminoacid-ALAderivativeswasdissolvedin3mL(39mmol)ofTFAunderargonatmosphere.Afterthesolutionwasstirredfor1hatroomtemperature,TFAwasevaporatedunderhighvacuum.Theproductswereobtainedingoodpurityandcharacterizedwithoutfurtherpurification.

Compound7a:98%yield;1HNMR(400MHz,DMSO-d6)δ8.81(t,3J(5,NH))5.6,1H,NH-H2C(5)),8.26(sbr,3H,H3N+),4.24(dxd,3J(5,NH))5.7,2J(5a,5b))12.7,1H,HC-(5a)),4.16(dxd,3J(5,NH))5.5,2J(5a,5b))12.9,1H,HC(5b)),4.07-4.00(m,1H,HC(7)),3.68(s,3H,H3C(12)),2.86-2.82(m,AA′ofaAA′BB′-system,2H,H2C(3)),2.62(m,BB′ofaAA′BB′-system,2H,H2C(2)),1.49(d,3J(7,72))7.0,3H,H3C(72));13CNMR(100MHz,DMSO-d6)δ204.9(C(4)),172.9(C(1)),170.2(C(6)),51.6(C(12)),48.5(C(7)),48.4(C(5)),34.2(C(3)),27.4(C(2)),17.5(C(72));MS(ESI)m/zrelativeintensity239(4,[M+Na]+),217(100,[M+H]+),199(38),171,167,146(18);HRMS(ESI)calcdfor[C9H17N2O4]+217.1183,found217.1191.

ALA-EstersandALA-AminoAcidsforPDT

Compound7b:99%yield;1HNMR(400MHz,D2O)δ3.66-3.65(AB-system,J2)7.3,1H,HC(7)),3.47(t,≈3J18.4,(12,12H,3))H2C(5)),3.63(q,3J(7,72)6.7,2H,H2C(12)),2.25-2.22(m,AA′ofaAA′BB′,2H,H2C(3)),2.04-2.01(m,BB′ofaAA′BB′,2H,H2C(2)),1.05-0.96(m,2H,H2C(13)),1.01(d,3J(72,7))7.1,3H,H3C(72)),0.80-0.73(m,6H,H2C(14),H2C(15),H2C(16)),0.32(t,3J(1,716))6.8,3H,H3C(17));13CNMR(100MHz,D2O)δ205.5(C(4)),173.8(C(1)),170.4(C(6)),.8(C(12)),48.6(C(7)),48.1(C(5)),33.5(C(3)),30.7(C(15)),27.6(C(13)),26.8(C(2)),24.7(C(14)),21.7(C(16)),16.0(C(72)),12.7(C(17));MS(ESI+)m/zrelativeintensity309(12,[M-HTFA+Na]+),287(100,[M-TFA]+),269(6),216(6).

Compound7c:99%yield;1HNMR(400MHz,D2O)δ7.19-7.07(m,5H,HC(74),HC(74′),HC(75),HC(75′),HC(76)),4.10(t3J(7,72))7.3,1H,HC(7)),3.91-3.90(AB-system,J2≈18.5,2H,J(7H2C(5)),3.44(s,3H,H3C(12)),3.02(dd,2J(72a,72b))14,32a,7))7.1,1H,HC(72a)),2.96(dd,2J(72b,72a))14,3J(72b,7))7.1,1H,HC(72b)),2.53-2.48(m,AA′ofaAA′BB′,2H,H2C(3)),2.39-2.36(m,BB′ofaAA′BB′,2H,H2C-(2));13CNMR(100MHz,D2O)δ207.5(C(4)),175.7(C(1)),169.4(C(6)),134.0(C(73)),129.7,129.4((C(7,474′,7,575′)),128.2(C(76)),.6(C(7)),52.7(C(12)),48.8(C(5)),37.1(C(72)),34.3(C(3)),27.6(C(2));MS(ESI+)m/zrelativeintensity315(43,[M-TFA]+),293(100,[M-HTFA+Na]+),275(15),120(8).Compound7d:97%yield;1HNMR(400MHz,D2O)δ4.01(d,2J(5a,5b))18.8,1H,HC(5a)),3.91(d,2J(5b,5a))18.8,1H,HC(5b)),3.79(t,3J(7,72))6.61H,HC(7)),3.38(s,3H,H3C-(12)),2.72(t,3J(7,574))7.6,2H,H2C(75)),2.58-2.55(m,AA′ofaAA′BB′,2H,H2C(3)),2.37-2.34(m,BB′ofaAA′BB′,2H,H2C(2)),1.68-1.62(m,2H,H2C(72),1.44(quin.,3J(7,475))(7,473))6.62H,H2C(74)),1.26-1.17(m,2H,H2C(73);13CNMR(100MHz,D2O)δ209.6(C(4)),177.9(C(1)),172.3(C(6)),55.4(C(7)),.7(C(12)),51.0(C(5)),41.4(C(75)),36.6(C(3)),32.7(C(72)),29.8(C(2)),28.7(C(74)),23.5(C(73));MS(ESI+)m/zrelativeintensity296(5,[M-2HTFA+Na]+),274(100,[M-2TFA-H]+),256,(34),239(9),211(3).

Compound7e:99%yield;1HNMR(400MHz,D2O)δ4.22(dd,3J(7,72a))5.1,3J(7,72b))7.5,1H,HC(7)),4.03-3.99(AB-system,J2≈18.3,2H,H2C(5)),3.44(s,3H,H3C(12)),2.(dd,2J(72a,72b))18.1,3J(72a,7))5.1,1H,HC(72a)),2.83(dd,2J(72b,72a))18.1,3J(72b,7))5.1,1H,HC(72b)),2.65-2.62(m,AA′ofaAA′BB′,2H,H2C(3)),2.44-2.41(m,BB′ofaAA′BB′,2H,H2C(2));13CNMR(100MHz,D2O)d207.6(C(4)),175.8(C(1)),172.7(C(6)),169.1(C(73)),52.6(C(12)),49.7(C(7)),49.0(C(5)),35.0(C(72)),34.4(C(3)),27.6(C(2));MS(ESI+)m/zrelativeintensity283(39,[M-HTFA+Na]+),261(100,[M-TFA]+),243(65),225(9),183(13),146(5).

ProcedurefortheEnzymaticDeprotectionoftheMethylEstertoFreeAcid8a.Atotalof1.58mmolofAla-ALA6awasdissolvedin50mLofphosphatebufferpH8.0,and0.2mLofpigliveresterrase(PLE)solution(Sigma,250units/mg,15mg/mgderivative)wasadded.ThepHwasmaintainedat8.0with0.2NNaOHfor48h,thenthepHwasloweredto1.5.Thesolutionwasextractedwith3×100mLofAcOEt,driedunderMgSO4,andpurifiedbycolumnchro-matography(55gsilicagel,eluent:dichloromethane/methanol5%).

Compound8a:88%yield;Rf(AcOEt/hexane/CH3COOH:100:20:1)0.67;1HNMR(400MHz,CD3OD)δ4.26-4.13(m,3H,H2C(5),HC(7)),2.80-2.75(m,AA′ofaAA′BB′,2H,H2C-(3)),2.-2.60(m,BB′ofaAA′BB′,2H,H2C(2)),1.53(s,9H,H3C(10a),H3C(10b),H3C(10c)),1.43(d,3J(7,72))7.2,3H,H3C-(72));13CNMR(100MHz,CD3OD)δ179.7(C(1)),176.6(C(6)),157.9(C(8)),81.0(C(9)),51.9(C(7)),50.2(C(5)),36.2(C(3)),31.0(C(2)),29.0(C(10a),C(10b),C(10c)),18.8(C(72));MS(APCI)m/zrelativeintensity304(17,[M+H]+),303(81,[M]+•),247(100),229(12),203(10).Anal.(C13H22N2O6)C,H,N.

Compound9a.UsingthestandarddeprotectionprocedurewithTFAstartingfrom8a(181mg,0.6mmol)thefullydeprotected9a(151mg,82%)wasisolated:Rf0.71;1HNMR(400MHz,D2O)δ3.02(s,2H,HC(5)),3.94(q,3J(7,72))7.1,1H,HC(7)),2.(t,3J(3,2))6.3,2H,HC(3)),2.44(t,3J(2,3))6.3,2H,HC(2)),1.34(d,3J(72,7))6.3,1H,HC(72));13CNMR(100MHz,D2O)δ207.9(C(4)),177.2(C(1)),172.8(C(6)),

JournalofMedicinalChemistry,2000,Vol.43,No.2749.3(C(7)),48.9(C(5)),34.4(C(3)),27.7(C(2)),16.7(C(72));MS(ESI+)m/zrelativeintensity225(53,[M+Na]+),203(100,[M+H]+),185(63),145(17),132(22);HRMS(ESI)calcdfor[C8H15N2O4]+203.1026,found203.1031.

CellLinesandCultureConditions.Humanlungcarci-nomaA9celllinewasfromATCC,humanmicrovascularendothelialcelllineHCEC26wasakindgiftofD.StaminirovicandA.Muruganandam,Ottawa,andtheratmicrovascularendothelialcelllineEC219hasbeendescribed.27CellsweregrowninDMEMmedium(Gibco)containingeither4.5g/L(A459andHCECcells)or1.1g/L(EC219cells)glucose,10%FCS,andpenicillin/streptomicin,at37°Cand6%CO2.Cellsweresubculturedin48-welldishes(Costar)48hpriortoexperimentsincompleteculturemedium.Aminopeptidaseactivitiesweredeterminedincellextractsessentiallyaspreviouslydescribed.27,28Experimentswererepeatedatleasttwiceandwereperformedinsextuplewells.Meansofresultswereused.

PpIXFluorescenceMeasurements.Forfluorescencemeasurements,confluentcelllayersin48-wellplateswereexposedtotheindicatedconcentrations(6wellsperconcentra-tion)ofthedifferentPPIXprecursorsin0.25mL/wellDMEMmedium,freeofphenolred(Gibco)andofFCS.Increaseinfluorescencewasmeasuredevery15-60mininathermostatedfluorescencemultiwellplatereaderat37°C(CytoFluorSeries4000,PerSeptiveBiosystems,MA)usingexcitationfilteratλ)409(20nmanddetectionfilteratλ)0(40nm.DeterminationofCellViability.Cellviabilitywasde-terminedafter20hexposuretoALAderivativesusingtheevaluationofmitochondrialfunctionsbytheMTTassay.Briefly,MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromid,Merck),finalconcentration250mg/mL,wasaddedtoeachwellandincubationwascontinuedat37°Cfor2h.Thesupernatantwasremoved,thecelllayerwasdissolvedin0.04NHCl/2-propanol,andabsorbanceat0nmwasquantitatedusinga96-wellELISAplatereader(iEMSReaderMF,Labsystems,Bioconcepts,Switzerland).

EvaluationofProductEfficacy(EfficacyIndex).Theratiooffluorescencemeasuredat300minafterinitialexposure/cellviability,asdefinedbyabsorbanceat0nm(MTTreduction)following20hexposuretothecompound,wasusedtodeterminethemoleculeproducingthemaximalPpIXfluorescencewiththeminimalcytotoxicity.Standarddevia-tions(SEM)werecalculated.

InSituEnzymaticActivities(Histoenzymography).EC219cellsgrownin6-wellplatesandwashedfromculturemediumwithoutfurtherfixationor5µmsectionsoffrozenbraintumorsampleswereexposedat37°CtoGlu-󰀁-meth-oxynaphthylamidesubstrate(Bachem,Switzerland)andFastBlueB(Sigma,Switzerland)inthesameconditionsasprevi-ouslydescribed.16Theywerecounterstainedwithlighthema-toxylinreagent.Enzymaticactivitywasvisualizedasaredprecipitate.

Acknowledgment.TheauthorsthankMrs.P.Des-sousL’EgliseMangeandRaymondeBoveyforexcellenttechnicalassistance.ThisworkwassupportedbytheSwissLeagueandResearchagainstCancer(GrantsSKL353-9-1996andKFS947-09-1999)andtheSwissNationalScienceFoundationforScientificResearch(Grants20-52490.97and3152-059219.99).

SupportingInformationAvailable:Additionalchemicalandspectralcharacteristicsofthesynthesizedcompounds.ThismaterialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.

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