Preference for O -demethylation reactions in the oxidation of 2´-, 3´-, and 4´-methoxyflavones by human cytochrome P450 enzymes.

2 '-, 3 '-, and 4 '-Methoxyflavones (MeFs) were incubated with nine forms of recombinant human cytochrome P450 (P450 or CYP) enzymes in the presence of an NADPH-generating system and the products formed were analyzed with LC-MS/MS methods. CYP1B1.1 and 1B1.3 were highly active in demethylating 4 ' MeF to form 4 '-hydroxyflavone (rate of 5.0 nmol/min/nmol P450) and further to 3 ',4 '-dihydroxyflavone (rates of 2.1 and 0.66 nmol/min/nmol P450, respectively). 3 ' MeF was found to be oxidized by P450s to m/z 239 (M-14) products (presumably 3 '-hydroxyflavone) and then to 3 ',4 '-dihydroxyflavone. P450s also catalyzed oxidation of 2 ' MeF to m/z 239 (M-14) and m/z 255 (M-14, M-14 + 16) products, presumably mono- and di-hydroxylated products, respectively. At least two types of ring oxidation products having m/z 269 fragments were formed, although at slower rates than the formation of mono- and di-hydroxylated products, on incubation of these MeFs with P450s; one type was products oxidized at the C-ring, having m/z 121 fragments, and the other one was the products oxidized at the A-ring (having m/z 137 fragments). Molecular docking analysis indicated the preference of interaction of O-methoxy moiety of methoxyflavones in the active site of CYP1A2. These results suggest that 2 '-, 3 '-, and 4 '-methoxyflavones are principally demethylated by human P450s to form mono- and di-hydroxyflavones and that direct oxidation occurs in these MeFs to form mono-hydroxylated products, oxidized at the A- or B-ring of MeF.