Abstract
The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-{[(4-trifluoromethoxyphenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-{[(2,4-difluorophenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled 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 CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16α-carbonitrile 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 that 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.
Footnotes
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↵1 Current address: DMPK, Amgen-Cambridge Research Center, Cambridge, MA 02139.
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↵2 Current address: RI-CEDD, GlaxoSmithKline, King of Prussia, PA 19406.
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doi:10.1124/dmd.104.001842.
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ABBREVIATIONS: DPP-IV, dipeptidyl peptidase-IV; MRL-A, (1S)-1-(trans-4-{[(4-trifluoromethoxyphenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride; MRL-B, (1S)-1-(trans-4-{[(2,4-difluorophenyl)sulfonyl]amino}cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride; MRL, Merck Research Laboratories; [3H]MRL-A, [benzenesulfonamide-2-3H]MRL-A; [3H]MRL-B, [2,4-difluorophenyl-5-3H]MRL-B; HPLC, high-performance liquid chromatography; NAC, N-acetylcysteine; GSH, glutathione; PCN, pregnenolone-16α-carbonitrile; 3-MC, 3-methylcholanthrene; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring.
- Received August 16, 2004.
- Accepted October 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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