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 u sing a small molecule model developed by M. J. De Groot et al. [(1997) Chem . Res. Toxicol. 10, 41-48]. The substrate, 7-methoxy-4-(aminomethyl)coumari n (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminometh yl)coumarin (HAMC) were synthesized, and their respective fluorescence prop erties were characterized. The selectivity of MAMC for P450 2D6 was charact erized using microsomes containing single human P450 isoenzymes and human l iver microsomes. Formation of the metabolic product HAMC was easily assesse d in real time with fluorescence spectroscopy, since MAMC and HAMC excitati on 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 mic rosomes from yeast or lymphoblastoid cells selectively expressing P450 isoe nzymes, MAMC was selective for P450 2D6 at a concentration of 25 mu M with only P450 1A2 contributing significantly to the formation of HAMC. P450s 2A 6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 mu M. K-m and v(max) values of MAMC for P450 2D6 were found to be 26.2 +/- 2.8 mu M and 2.9 +/- 0.07 min(-1), respectively. For P450 1A2, MAMC was found to have a K-m value of 29.7 +/- 6.2 mu M and a v(max) of 0.57 +/- 0.07 min(-1). Formation of HAMC in human liver microsom es could be completely inhibited by quinidine, at a concentration of 0.5 mu M selective for P450 2D6, and furafylline, at a concentration of 30 mu M s elective for P450 1A2. In conclusion, O-demethylation of 7-methoxy-4-(amino methyl)coumarin is a rapid and easily determined parameter for P450 2D6 act ivity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes.