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.