Cytochrome
P450 (CYP) enzymes constitute an essential xenobiotic metabolizing
system that regulates the elimination of lipophilic compounds from the
body. Convenient and affordable assays for CYP enzymes are important for
assessing these metabolic pathways.

In
this study, 10 novel profluorescent coumarin derivatives with various
substitutions at carbons 3, 6 and 7 were developed. Molecular modeling
indicated that 3-phenylcoumarin offers an excellent scaffold for the
development of selective substrate compounds for various human CYP
forms, as they could be metabolized to fluorescent 7-hydroxycoumarin
derivatives. Oxidation of profluorescent coumarin derivatives to
fluorescent metabolites by 13 important human liver
xenobiotic-metabolizing CYP forms was determined by enzyme kinetic
assays.

Four of the coumarin derivatives
were converted to fluorescent metabolites by CYP1 family enzymes, with
6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively
by CYP1A2 in human liver microsomes. Another set of four compounds were
metabolized by CYP2A6 and CYP1 enzymes.
7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by
CYP2C19 and CYP2D6 in a non-selective fashion.

The
advantages of the novel substrates were (1) an excellent
signal-to-background ratio, (2) selectivity for CYP1 forms, and (3)
convenient multiwell plate measurement, allowing for precise
determination of potential inhibitors of important human hepatic forms
CYP1A2, CYP2C19 and CYP2D6.