BIOACTIVATION OF -2,4-DI-1-PYRROLIDINYL-7H-PYRROLO[2,3-D]PYRIMIDINE (U-89843) TO REACTIVE INTERMEDIATES THAT BIND COVALENTLY TO MACROMOLECULES AND PRODUCE GENOTOXICITY

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylac tic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in t he in vitro unscheduled DNA synthesis (UDS) assay, an assay which meas ures DNA repair following chemically-induced DNA damage in metabolical ly competent rat hepatocytes. Incubation of [C-14]U-89843 with liver m icrosomes resulted in covalent binding of radioactive material to macr omolecules by a process that was NADPH-dependent. U-89843 has been sho wn to undergo C-Ci methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. S ynthetical U-97924 is chemically reactive and undergoes dimerization i n aqueous solution. The dimerization of U-97924 was significantly inhi bited by addition of nucleophiles such as-methanol, glutathione, and N -acetylcysteine. Characterization of the corresponding methanal glutat hione, and N-acetylcysteine adducts of U-97924 supported the hypothesi s of a reaction pathway involving re active iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalen t binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced reduced glutat hione (GSH). A trifluoromethyl analog of U-89843 was prepared in-an ef fort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the ill vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C- 6 methyl group was eliminated. These observations suggest that the pos itive in vitro UDS results of U-89843 are mediated by the bioactivatio n of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.