SELECTIVELY DEUTERATED WARFARIN - SUBSTRATE PROBE FOR THE MECHANISM OF AROMATIC HYDROXYLATION CATALYZED BY CYTOCHROME-P450

The effect of branch pathways on the observed intramolecular isotope e ffect and deuterium retention associated with 6- and 7-hydroxylation o f selectively monodeuterated (R)- and (S)-warfarin with cytochrome P45 0 (CYP) 2C9 and CYP1A2 were studied, cDNA-expressed CYP2C9 was incubat ed with enantiomerically pure (S)-7d(1)- and (S)-6d(1)-warfarin, and e xpressed CYP1A2 was incubated with enantiomerically pure (R)-7d(1) and (R)-6d(1)-warfarin. A high degree of deuterium retention was observed in all metabolites, independent of the stereochemistry of the substra te or CYP isoform. No deuterium kinetic isotope effect was observed fo r the formation of 6-hydroxy- or 7-hydroxywarfarin in the case of the (S)-6d(1)-warfarin metabolism by CYP2C9, or for the formation of 6-hyd roxy-, 7-hydroxy-, and 8-hydroxywarfarin in the case of the (R)-6d(1)- warfarin metabolism by CYP1A2. Deuterium isotope effects of 1.17 and 1 .23 accompanied formation of 7-hydroxywarfarin from (S)-7d(1)-warfarin by CYP2C9 and from (R)-7d(1)-warfarin by CYP1A2, respectively. These observations are consistent with the addition-rearrangement pathway fo r aromatic hydroxylation, in which a triplet-like active oxygen specie s initially adds to the pi system, resulting in a tetrahedral intermed iate, The intermediate subsequently rearranges to generate the phenol, the final product of the reaction.