title: Metabolic activation of a pyrazinone-containing thrombin inhibitor. Evidence for novel biotransformation involving pyrazinone ring oxidation, rearrangement, and covalent binding to proteins.
abstract: Compound I, (2-[3-[(2,2-difluoro-2(2-pyridyl)ethyl)amino]-6-methyl-2-oxohydropyrazinyl]-N-[(3-fluoro(2-pyridyl))methyl]acetamide, is a potent competitive inhibitor of thrombin that reacts stoichiometrically with the protease. Compounds of this class possess therapeutic potential as anticoagulation agents. During the metabolic characterization of compound I, evidence was obtained for extensive metabolic activation of the pyrazinone ring system. Following administration of (14)C-labeled I to rats, significant levels of irreversibly bound radioactivity to proteins were detected in rat plasma and liver. LC/MS/MS analysis of metabolites formed in rat and human liver microsomes fortified with glutathione (GSH) revealed the presence of two structurally distinct GSH adducts. It is proposed that the first of these GSH conjugates derives from a two electron oxidation of the 6-methyl-2-oxo-3-aminopyrazinone moiety to afford an electrophilic imine-methide intermediate, while the second is formed by addition of GSH to an epoxide formed by P-450-mediated oxidation of the double bond at the 5-6 position of the pyrazinone ring. The addition of GSH to the proposed epoxide facilitates opening of the pyrazinone ring and a rearrangement to afford a stable, rearranged imidazole-containing metabolite. Elucidation of the metabolic activation pathways of I provides structural guidance for the design of thrombin inhibitors with decreased potential for the generation of chemically reactive intermediates.
authors.affiliation: Department of Drug Metabolism, Merck Research Labs, West Point, Pennsylvania, USA. romi_singh@merck.com
authors.forename: Rominder
authors.initials: R
authors.lastname: Singh
authors.forename: Maria V
authors.initials: MV
authors.lastname: Silva Elipe
authors.forename: Paul G
authors.initials: PG
authors.lastname: Pearson
authors.forename: Byron H
authors.initials: BH
authors.lastname: Arison
authors.forename: Bradley K
authors.initials: BK
authors.lastname: Wong
authors.forename: Rebecca
authors.initials: R
authors.lastname: White
authors.forename: Xiao
authors.initials: X
authors.lastname: Yu
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Burgey
authors.forename: Jiunn H
authors.initials: JH
authors.lastname: Lin
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Baillie
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001667:Binding, Competitive; D001711:Biotransformation; D002250:Carbon Radioisotopes; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011480:Protease Inhibitors / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D011485:Protein Binding; D011719:Pyrazines / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D002250:Carbon Radioisotopes; D004852:Epoxy Compounds; D007097:Imines; D011480:Protease Inhibitors; D011719:Pyrazines; D009249:NADP; D013917:Thrombin; D005978:Glutathione