title: Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.
abstract: U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.
authors.affiliation: Drug Metabolism Research, Discovery Chemistry, Kalamazoo, Michigan 49001, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: K A
authors.initials: KA
authors.lastname: Koeplinger
authors.forename: G E
authors.initials: GE
authors.lastname: Padbury
authors.forename: M J
authors.initials: MJ
authors.lastname: Hauer
authors.forename: G L
authors.initials: GL
authors.lastname: Bundy
authors.forename: L S
authors.initials: LS
authors.lastname: Banitt
authors.forename: T M
authors.initials: TM
authors.lastname: Schwartz
authors.forename: D C
authors.initials: DC
authors.lastname: Zimmermann
authors.forename: P R
authors.initials: PR
authors.lastname: Harbach
authors.forename: J K
authors.initials: JK
authors.lastname: Mayo
authors.forename: C S
authors.initials: CS
authors.lastname: Aaron
mesh_terms: D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D004260:DNA Repair; D004285:Dogs; D016166:Free Radical Scavengers / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D008401:Gas Chromatography-Mass Spectrometry; D000882:Haplorhini; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D011485:Protein Binding; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity*; D051381:Rats; D011916:Rats, Inbred F344; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet
chemical_list: D016166:Free Radical Scavengers; D009153:Mutagens; D011743:Pyrimidines; D011758:Pyrroles; C097295:U 89843