Electrophilicity of Pyridazine-3-carbonitrile, Pyrimidine-2-carbonitrile, and Pyridine-carbonitrile Derivatives: A Chemical Model To Describe the Formation of Thiazoline Derivatives in Human Liver Microsomes

Certain aromatic nitriles are well-known inhibitors of cysteine proteases. The mode of action of these compounds involves the formation of a reversible or irreversible covalent bond between the nitrile and a thiol group in the active site of the enzyme. However, the reactivity of these aromatic nitrile-substituted heterocycles may lead inadvertently to nonspecific interactions with DNA, protein, glutathione, and other endogenous components, resulting in toxicity and complicating the use of these compounds as therapeutic agents. In the present study, the intrinsic reactivity and associated structure–property relationships of cathepsin K inhibitors featuring substituted pyridazines [6-phenylpyridazine-3-carbonitrile, 6-(4-fluorophenyl)­pyridazine-3-carbonitrile, 6-(4-methoxyphenyl)­pyridazine-3-carbonitrile, 6-<i>p</i>-tolylpyridazine-3-carbonitrile], pyrimidines [5-<i>p</i>-tolylpyrimidine-2-carbonitrile, 5-(4-fluorophenyl)­pyrimidine-2-carbonitrile], and pyridines [5-<i>p</i>-tolylpicolinonitrile and 5-(4-fluorophenyl)­picolinonitrile] were evaluated using a combination of computational and analytical approaches to establish correlations between electrophilicity and levels of metabolites that were formed in glutathione- and <i>N</i>-acetylcysteine-supplemented human liver microsomes. Metabolites that were characterized in this study featured substituted thiazolines that were formed following rearrangements of transient glutathione and <i>N</i>-acetylcysteine conjugates. Peptidases including γ-glutamyltranspeptidase were shown to catalyze the formation of these products, which were formed to lesser extents in the presence of the selective γ-glutamyltranspeptidase inhibitor acivicin and the nonspecific peptidase inhibitors phenylmethylsulfonyl fluoride and aprotinin. Of the chemical series mentioned above, the pyrimidine series was the most susceptible to metabolism to thiazoline-containing products, followed, in order, by the pyridazine and pyridine series. This trend was in keeping with the diminishing electrophilicity across these series, as demonstrated by <i>in silico</i> modeling. Hence, mechanistic insights gained from this study could be used to assist a medicinal chemistry campaign to design cysteine protease inhibitors that were less prone to the formation of covalent adducts