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 (MAMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metabolism 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 using heterologously expressed CYP2D6 demonstrated that the introduction and elongation of alkyl substituents strongly decreased the IC(50) values toward dextromethorphan O-demethylation. Metabolism studies showed that the regioselectivity of metabolism was unaffected by the varying N substituents, as only O-dealkylation of the analogs and no N-dealkylation was observed. In excellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affinity of the compounds toward CYP2D6, as indicated by an up to 100-fold decrease in K(m) values. The V(max) values displayed a much less pronounced decrease with an increasing N-alkyl chain, resulting in as much as a 30-fold increase in the V(max)/K(m) value. Interestingly, due to the higher fluorescent yield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dealkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fold higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 and CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both heterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K(m) values of O-dealkylation by CYP1A2, but it did result in higher V(max) values. Finally, CYP1A2 also N-dealkylated the analogs.
Venhorst
J
J
Leiden/Amsterdam Center for Drug Research, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam.
Onderwater
R C
RC
Meerman
J H
JH
Commandeur
J N
JN
Vermeulen
N P
NP
D000478:Alkylation; D001667:Binding, Competitive / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003640:Dealkylation; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008954:Models, Biological; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism
C120605:7-methoxy-4-(aminomethyl)coumarin; D003374:Coumarins; D007527:Isoenzymes; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D019388:Cytochrome P-450 CYP1A2; D019389:Cytochrome P-450 CYP2D6