Sulfaphenazole derivatives as tools for comparing cytochrome P450 2C5 and human cytochromes P450 2Cs: identification of a new high affinity substrate common to those enzymes.

The inhibitory effects of a series of sulfaphenazole (SPA) derivatives were studied on two modified forms of rabbit liver cytochrome P450 2C5 (CYP2C5), CYP2C5dH, and structurally characterized CYP2C5/3LVdH and compared to the previously described effects of these compounds on human CYP2C8, 2C9, 2C18, and 2C19. SPA and other negatively charged compounds that potently inhibit CYP2C9 had very little effect on CYP2C5dH, whereas neutral, N-alkylated derivatives exhibited IC50 values between 8 and 22 microM. One of the studied compounds, 4, that derives from SPA by replacement of its NH(2) substituent with a methyl group and by N-methylation of its sulfonamide moiety, acted as a good substrate for all CYP2Cs used in this study. Hydroxylation of the benzylic methyl of 4 is the major reaction catalyzed by all of these CYP2C proteins, whereas hydroxylation of the N-phenyl group of 4 was observed as a minor reaction. CYP2C5dH, 2C5/3LVdH, 2C9, 2C18, and 2C19 are efficient catalysts for the benzylic hydroxylation of 4, with K(m) values between 5 and 13 microM and k(cat) values between 16 and 90 min(-1). The regioselectivity observed for oxidation of 4 by CYP2C5/3LVdH was easily interpreted on the basis of the existence of two different binding modes of 4 characterized in the experimentally determined structure of the complexes of CYP2C5/3LVdH with 4 described in the following paper [Wester, M. R. et al. (2003) Biochemistry 42, 6370-6379].