Abstract The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogues (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritiumlabeled compounds to rat liver microsomal protein was time-and NADPH-dependent, and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant cytochrome (CYP) 3A1 and 3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) enhanced the extent of bioactivation, which in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5-and 9-fold increase, respectively). Herein, we describe studies which demonstrate that the fluoropyrrolidine ring is prone to metabolic activation, and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation
