N-Benzoyloxy-N-methyl-4-aminoazobenzene: its carcinogenic activity in the rat and its reactions with proteins and nucleic acids and their constituents in vitro.

LIONELA.POIRIER,-JAMESA.MILLER,ELIZABETHCMILLER,ANDKEISATOMcArdleLaboratoryforCancerResearch,MedicalCenter,UniversityofWisconsin,Madison,Wisconsin53706SUMMARYAT-Benzoyloxy-A'-methyl-4-aminoazobenzene(.V-benzoyloxy-MAB),anewderivativeofthehepatocarcinogenAr-methyl-4-aminoazobenzene(MAAŸ),wassynthesized.ThistoxicesterofA'-hydroxy-MABproducedsarcomasatthesiteofrepeateds.c.injectionsinrats;MABwasinactiveundertheseconditions.UnlikeMAB,Ar-benzoyloxy-MABreactedreadilyinvitroatpH7withprotein,RNA,andDXAtoformmacromolecular-bounddye.Fivenucleophiliccomponentsofthesemacro-molecules(methionine,cysteine,tryptophan,tyrosine,andguanosine)reactedwithAT-benzoyloxy-MABundersimilarconditionstoformpolardyes;othercommonaminoacidandnucleosidecomponentsdidnotreact.ThereactionsofN-benzoyloxy-MABwithproteins,nucleicacids,andtheircomponentsmaybeusefulprototypesinstudiesontheprotein-andnucleicacid-bounddyesformedinvivobytheaminoazodyes.ThebiologicandchemicalpropertiesofAr-benzoyloxy-MABparallelthoseofthecarcinogenicaromaticN-acyloxyamideAr-acetoxy-2-acetylaminofluoreneandlendfurthersupporttotheconceptthatthecarcinogenicaromaticaminesandamidesareactivatedinvivobyesterificationoftheirA^-hy-droxymetabolites.INTRODUCTIONAr-Hydroxylationhasbeendemonstratedasanimportantstepinthemetabolicactivationofcarcinogenicamides.Thus,AAF4(9,27)andits7-fluoroderivative(20),4-acetylamino-1Thisinvestigation wassupported byResearchTrainingGrantCRTY-5002andbyProgram-Project GrantCA-07175oftheNationalCancerInstitute, USPHS;byagrantfromTheJaneCoffinChildsMemorialFvmdforMedicalResearch;andbytheAlexanderandMargaretStewartTrustFund.2MontrealCancerInstitute,NotreDameHospital,Montreal,Quebec,Canada.3Department ofEducation, HirosakiUniversity, Hirosaki,Aomori-ken,Japan.4Thefollowingabbreviationsareused(theChemicalAbstractsnomenclatureisgiveninparentheses): AB,4-aminoazobenzene(p-phenylazoaniline); AAB,A7-acetyl-4-aminoazobenzene (p-phenylazoacetanilide); MAB, Ar-methyl-4-aminoazobenzeneOV-methyl-p-phenylazoaniline); DAB,JV,AT-dimethyl-4-aminoazobenzene(Ar,Ar-dimethyl-p-phenylazoaniline); AAF,2-acetyl-aminofluorene(A^-fluorenylacetamide).ReceivedMarch27,1967;acceptedMay6,1967.biphenyl(35),4-acetylaminostilbene(2,4),and2-acetyl-aminophenanthrene(20)areeachmetabolizedtoA^-hydroxyderivativesbyspeciesinwhichtheyarecarcinogenic.Furthermore,whenadministeredtorats,theseW-hydroxymetabolitesaremorecarcinogenicthantheparentcompoundsatsiteswheretheamidesareactiveandproducetumorsatsitesoflocalapplicationwheretheparentcompoundshavelittleornoactivity(2,5,20,23,24,28,35,39).TheisolationofN-hydroxy-AT-acetyl-4-aminoazobenzenefromtheurineofrats,mice,orhamstersadministeredABoritscarcinogenicA'-methylorA*,A-dimethylderivatives(MABorDAB)showedthattheaminoazodyesarelikewiseAr-hydroxylatedinvivo(42).However,neitherthismetabolitenorA^-hydroxy-ABwascarcinogeniconadministrationtorats(42).TheinactivityofthesecompoundsisconsistentwiththeapparentrequirementofaA'-methylgroupforstrongcarcinogenicityintheseriesofaminoazodyesstructurallyrelatedtoMABandDAB(3,6,29).Thesedatasuggestedthattheproximatecarcinogenicmetabolite(s)ofthesedyeswouldcontainbothaA^-methylgroupandaA"-hydroxygroup.WhenrepeatedattemptstosynthesizeA'-hydroxy-Af-methyl-4-aminoazobenzene(JV-hydroJSy-MAB)failed,itsO-benzoylderivative,A"-benzoyloxy-MAB,waspreparedonthepremisethatitmightreleasethepresumedcarcinogenicmetaboliteinvivo.Thisesterinducedahighincidenceofsarcomasinratsonrepeatedsubcutaneousinjection.Furthermore,A'-benzo-yloxy-MABreactednonenzymaticallyinneutralsolutionwithcertainaminoacidsorproteinsandwithguanosine,DXA,andRNA.Thesedata,whichformthesubjectofthisreport,andtherecentstudiesfromthislaboratoryonthesimilarreactivityandcarcinogenicityofAr-acetoxy-AAF(10,14-16,19)supporttheconcejilthatA"-hydroxymetabolitesofcarcinogenicaromaticaminesandamidesareactivatedforcarcinogenesisbyesterificationoftheAT-hydroxygroup(32).MATERIALSANDMETHODSPreparationofCompoundsSynthesisofN-Beiizoyloxy-MAB.A^-Benzoyloxy-MABwassynthesizedbyamodificationoftheprocedureusedbyHornerandSteppan(13)forthesynthesisofAr-benzoyloxy-A"-ethylaniline.Itwasfoundnecessarytoperformalloperationsbetween—2°Cand5°C,andallsolutionswerealsomaintainedwithinthistemperaturerange.MAB(20gm)dissolvedin70mlKXX) CANCERRESEARCHVOL.27

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