Mechanistic investigation of cytochrome P450 1A2 catalyzed metabolism of 8-alkylxanthines

Abstract

The mechanism of CYP1A2 (cytochrome P450 1A2) catalyzed metabolism of 8-alkylxanthines is explored using deuterium isotope effect studies and isotope tracing experiments with an 8-isopropyl analog (isopropylcyclohexylline, IPC) of the CYP1A2 mechanism-based inactivator furafylline. Desaturation of furafylline to an iminium reactive intermediate has been proposed to account for formation of both 1:1 CYP1A2-protein adduct and 8'-carbinol with the majority (80%) of the oxygen sourced to the medium. Reactive intermediate formation with 8-ethyl and 8-isopropyl analogs of furafylline is evident due to H2O incorporation into their 8'-carbinol metabolites (60% and 43%, respectively), though enzyme inactivation is virtually eliminated. Additionally, 35% and 85%, respectively, of 8-ethyl and 8-isopropyl analog metabolism results in side chain desaturation. Competitive intermolecular deuterium isotope effect studies with IPC in the presence of H2 18O suggest that both dual hydrogen atom abstraction and base-catalyzed rearrangement of the reactive intermediate are contributing to olefin metabolite formation, in contrast to the results from similar experiments with the 8-ethyl compound in which rearrangement of the reactive intermediate appears to be the only mechanism of side-chain desaturation. Intramolecular isotope effect analysis of IPC side chain desaturation in the presence of H2 18O did not clearly differentiate between the two proposed desaturation pathways. Several mechanistic possibilities to explain the experimental results are discussed. Lastly, the effect of reducing equivalent transfer rate on the sequential metabolism of IPC by two different CYP1A2 enzyme sources was investigated using isotope-tracing experiments.