Bioactivation of 2,3-Diaminopyridine-Containing Bradykinin B1 Receptor Antagonists: Irreversible Binding to Liver Microsomal Proteins and Formation of Glutathione Conjugates

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Abstract
The 2,3-diaminopyridine (DAP) moiety was found to represent a core structure essential for the potency of a new series of human bradykinin B(1) receptor antagonists. However, incubation of (14)C-labeled 2,3-DAP derivatives with rat and human liver microsomes resulted in substantial irreversible binding of radioactive material to macromolecules by a process that was NADPH-dependent. Trapping the reactive species with GSH led to significant reduction of the irreversible binding of radioactivity, with concomitant formation of abundant GSH adducts. One type of thiol adducts (detected in both human and rat liver microsomes), resulting from addition of 305 Da to the parent compound, was observed with all 2,3-DAP compounds. These adducts also were detected in rat hepatocyte incubates, as well as in rat bile, following intravenous administration of 2,3-DAPs. Formation of the conjugates appeared to involve modification of the DAP ring, based upon mass spectral analysis of a number of representative GSH adducts; this was corroborated by detailed LC NMR analysis of one compound. Formation of this type of GSH conjugate was markedly reduced when the 2-amino methyl group linking the 2,3-DAP and the biphenyl moiety was replaced with an ether oxygen atom. It is postulated, therefore, that oxidation of the 2-amino group serves as a key step leading to the formation of reactive species associated with the DAP core. In addition, this step appears to be mediated primarily by P450 3A, as evidenced by the marked decrease in both the irreversible binding of radioactivity and the formation of the GSH adducts in human liver microsomes following treatment with ketoconazole and monoclonal antibodies against P450 3A. A mechanism for the bioactivation of 2,3-DAP is proposed wherein oxidation (dehydrogenation or N-hydroxylation followed by dehydration) of the 2-amino group, catalyzed by P450 3A, results in the formation of a highly electrophilic species, pyridine-2,3-diimine.