Influence of N-substitution of 7-methoxy-4-(aminomethyl)-coumarin on cytochrome P450 metabolism and selectivity

A series of six structural analogs of 7-methoxy-4-(aminomethyl)-coumarin (M AMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metab olism by cytochrome P450s, as well as on P450 selectivity. The analogs were obtained by introducing alkyl substituents at the amino group of MAMC and by replacing this moiety with a pyridine group. Competition experiments usi ng heterologously expressed CYP2D6 demonstrated that the introduction and e longation of alkyl substituents strongly decreased the IC50 values toward d extromethorphan O-demethylation. Metabolism studies showed that the regiose lectivity of metabolism was unaffected by the varying N substituents, as on ly O-dealkylation of the analogs and no N-dealkylation was observed. In exc ellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affini ty of the compounds toward CYP2D6, as indicated by an up to 100-fold decrea se in K-m values. The V-max values displayed a much less pronounced decreas e with an increasing N-alkyl chain, resulting in as much as a 30-fold incre ase in the V-max /K-m value. Interestingly, due to the higher fluorescent y ield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dea lkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fol d higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 a nd CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both h eterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K-m value s of O-dealkylation by CYP1A2, but it did result in higher V-max values. Fi nally, CYP1A2 also N-dealkylated the analogs.