Design, Synthesis, and Characterization of 7-Methoxy-4-(aminomethyl)coumarin as a Novel and Selective Cytochrome P450 2D6 Substrate Suitable for High-Throughput Screening

In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M. J. De Groot et al. [(1997J Chem. Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized. The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes. Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly. HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence. It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 muM with only P450 IA2 contributing significantly to the formation of HAMC. P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2EI, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 muM. Km and Vmax values of MAMC for P450 2D6 were found to be 26.2 ± 2.8 muM and 2.9 ± 0.07 min-1 respectively. For P450 IA2, MAMC was found to have a Km value of 29.7 ± 6.2 muM and a Vmax of 0.57 ± 0.07 min -1 Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 muM selective for P450 2D6, and furafylline, at aconcentration of 30 muM selective for P450 1A2. Inconclusion, O-demethylation of 7-methoxy4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.