Mechanism of cytochrome P4503A4- and 2D6-catalyzed dehydrogenation of ezlopitant as probed with isotope effects using five deuterated analogs.

Ezlopitant is metabolized by cytochrome P450 primarily to two metabolites: a benzyl alcohol and a corresponding alkene. The alkene arises as a direct product of metabolism of ezlopitant rather than through dehydration of the benzyl alcohol. The mechanism of this cytochrome P450 (P450)-catalyzed dehydrogenation reaction was probed with five different deuterium-labeled analogs of ezlopitant. At saturating ezlopitant concentrations, deuterium substitution resulted in small differences in reaction velocity. When deuterium was incorporated into the benzylic position ([d(1)]ezlopitant and [d(7)]ezlopitant), low isotope effects on the formation of both the benzyl alcohol and alkene were observed (1.25-1.55 for CYP3A4 and 1.48-2.61 for CYP2D6), suggesting that abstraction of the benzylic hydrogen is obligatory in the formation of both metabolites. A small amount of metabolic switching occurred because isotope effects were slightly higher for alkene and alcohol formation than for ezlopitant consumption. Intramolecular deuterium isotope effects of the dehydrogenation reaction for tri- and tetradeuterated analogs were very low (1.13-1.15) for both CYP3A4 and CYP2D6, whereas intramolecular isotope effects for the chemical dehydration of correspondingly deuterated ezlopitant benzyl alcohol (CJ-12,764) were 3.8 to 5.9. Thus, dehydrogenation does not appear to occur via enzyme-mediated general acid catalysis of the benzyl alcohol. A mechanism for the dehydrogenation of ezlopitant is proposed in consideration of the data presented.