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8-Amino-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline maleate (nomifensine), an antidepressant drug, was withdrawn from the market because of increased incidence of hemolytic anemia, as well as kidney and liver toxicity. Although the nature of the potentially reactive metabolites formed after nomifensine metabolism remains unknown and no glutathione (GSH) adducts of these nomifensine reactive metabolites have been reported, bioactivation has been postulated as a potential mechanism for the toxicity of nomifensine. This study was conducted to probe the potential bioactivation pathways of nomifensine in human and animal hepatocytes and in liver microsomes using GSH as a trapping agent. Two types of GSH conjugates were characterized by liquid chromatography/tandem mass spectrometry: 1) aniline oxidation followed by GSH conjugation leading to the formation of nomifensine-GSH sulfinamides (M1 and M2); and 2) arene oxidation followed by GSH conjugation yielding a range of arene C-linked GSH adducts (M3-M9). Nine GSH adducts (M1-M9) were identified in liver microsomes of humans, dogs, monkeys, and rats and in human and rat hepatocytes. In dog hepatocyte preparations, six GSH adducts (M1-M6) were identified. The GSH adducts in dog and rat liver microsomes were formed primarily through aniline and arene oxidation, respectively. Both pathways contributed significantly to the formation of the GSH adducts in human and monkey liver microsomes. The bioactivation pathways proposed here account for the formation of the observed GSH conjugates. These investigations have confirmed the aniline and the arene groups in nomifensine as potential toxicophores capable of generating reactive intermediates.

Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury. Its interest as a therapeutic target has led many to pursue the development of inhibitors. To date, only one series of nonpeptidic inhibitors have been described, and these have limited selectivity within the caspase family. Here we report the properties of a series of anilinoquinazolines (AQZs) as potent small molecule inhibitors of caspase-3. The AQZs inhibit human caspase-3 with Ki values in the 90 to 800 nM range. A subset of AQZs are equipotent against caspase-6, although most lack activity against this isoform and caspase-1, -2, -7, and -8. The AQZs inhibit endogenous caspase-3 activity toward a cell permeable, exogenously added substrate in staurosporine-treated SH-SY5Y cells. The AQZs reduce biochemical and cellular features of apoptosis that are thought to be a consequence of caspase-3 activation including DNA fragmentation, TUNEL staining, and the various morphological features that define the terminal stages of apoptotic cell death. Moreover, the AQZs also inhibit apoptosis induced by nerve growth factor withdrawal from differentiated PC12 cells. Thus, the AQZs represent a new and structurally novel class of inhibitors, some of which selectively inhibit caspase-3 and will thereby allow evaluation of the role of caspase-3 activity in various cellular models of apoptosis.