BIOACTIVATION OF THE 3-AMINO-6-CHLOROPYRAZINONE RING IN A THROMBIN INHIBITOR LEADS TO NOVEL DIHYDRO-IMIDAZOLE AND IMIDAZOLIDINE DERIVATIVES: STRUCTURES AND MECHANISM USING ¹³C-LABELS, MASS SPECTROMETRY, AND NMR

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Thrombin is a serine protease that plays a key role in the blood coagulation cascade. Compound I [2-[6-chloro-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide] is a potent, selective, and orally bioavailable thrombin inhibitor that is being studied as a possible anticoagulant. Biotransformation studies in rats revealed that 84% of an i.v. dose of I was excreted in the form of two metabolites. Both metabolites were formed by metabolic activation of the pyrazinone ring in I and subsequent rearrangement leading to two novel dihydro-imidazole and imidazolidine derivatives. The structures of these metabolites and their mechanism of formation were elucidated by additional use of two 13 C single labels in the pyrazinone ring of I in combination with mass spectrometry and NMR techniques. The metabolite structures described here illustrate the rich metabolic chemistry of the amino-pyrazinone heterocycle.Thrombin is a serine protease that plays multifarious roles in the complex blood coagulation cascade (Goldsack et al., 1998;Mann et al., 2003). Existing anticoagulation therapy (Majerus and Tollefsen, 2001;Weitz and Hirsh, 2001) primarily consists of low-molecularweight heparin and warfarin. Both therapeutic agents suffer from significant limitations: low-molecular-weight heparin requires either continuous intravenous infusion or subcutaneous injection, and warfarin has a delayed onset of action, narrow therapeutic index, and a high potential for drug-drug interactions. Hence, a small-molecule thrombin inhibitor represents an alternative target in the search for orally administered anticoagulants that could offer a safer alternative to existing therapies for the treatment and prevention of thromboembolic disorders (Vacca, 2000;Burgey et al., 2003b).is a potent, selective and orally bioavailable thrombin inhibitor from the amino-pyrazinone acetamide series (Burgey et al., 2003a,b). In this study, we provide details of the in vivo biotransformation of I in rats. The structures and mechanisms leading to novel dihydro-imidazole and imidazolidine derivatives have been delineated by MS 1 and NMR analysis and the additional use of two singly labeled 13 C analogs of I. Materials and Methods Chemicals.Compound I-A and its 14 C analog, shown in Fig. 1, were synthesized at Merck Research Laboratories (West Point, PA) (Burgey et al., 2003). The 14 C label was incorporated on the carbon 17 (see Fig. 1 for nomenclature) having a specific activity of 47.7 Ci/mg and a radiochemical purity of Ͼ98% as determined by HPLC. Compounds I-B and I-C are structurally identical to compound I-A except for a mono 13 C label at carbon labeled 13 or 12 (Fig. 1), respectively, of the pyrazinone ring. Both I-B and I-C were prepared at Merck Research Laboratories (Labeled Compound Synthesis group, Rahway, NJ) and were determined to have a purity of Ͼ99% by NMR. (Burgey et al., 2003b) of 5 mg/kg (10 mg/ml in DMSO) to bile...

Comparative Biotransformation of Pyrazinone-Containing Corticotropin-Releasing Factor Receptor-1 Antagonists: Minimizing the Reactive Metabolite Formation

“…Pyrazinones have been incorporated into other drug candidate molecules such as inhibitors of human immunodeficiency virus-1 reverse transcriptase (Heeres et al, 2005), caspase-3 (Han et al, 2005), mast cell tryptase (Hopkins et al, 2004), and thrombin (Sanderson et al, 1998). Although metabolism of the majority of the pyrazinone-containing compounds remains unknown, metabolic activation of a series of thrombin inhibitors containing the 6-position substituted pyrazinone was published Singh et al, 2003;Subramanian et al, 2003;Deng et al, 2005;Lin et al, 2005). Of these thrombin inhibitors, a 3-amino-6-chloro-pyrazinone analog underwent extensive bioactivation in BDC rats after intravenous administration, leading to formation of a number of pyrazinone ring oxidation metabolites, such as the ring-opened metabolite and rearranged products, presumably via a pyrazinone epoxide intermediate (Subramanian et al, 2003).…”

“…Although metabolism of the majority of the pyrazinone-containing compounds remains unknown, metabolic activation of a series of thrombin inhibitors containing the 6-position substituted pyrazinone was published Singh et al, 2003;Subramanian et al, 2003;Deng et al, 2005;Lin et al, 2005). Of these thrombin inhibitors, a 3-amino-6-chloro-pyrazinone analog underwent extensive bioactivation in BDC rats after intravenous administration, leading to formation of a number of pyrazinone ring oxidation metabolites, such as the ring-opened metabolite and rearranged products, presumably via a pyrazinone epoxide intermediate (Subramanian et al, 2003). In addition, GSH adducts of a 3-amino-6-methyl-pyrazinone analog were detected in human liver microsomal incubations supplemented with NADPH and GSH as a result of pyrazinone oxidation followed by ring scission and subsequent rearrangements .…”

Lipid Peroxide–Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples

“…We also note that an oxygen anion is naturally formed for the proposed ring closure (2, Scheme 1). Previously reported mechanisms for P450-mediated oxidation of a pyrazinone ring are related in that they involve either hydrolysis or GSH addition to an epoxide Subramanian et al, 2003;Zhuo et al, 2010), presumably catalyzed by epoxide hydrolases or glutathione S-transferases present in liver (Parkinson, 2001). These led to the pyrazinone ring opening by the breakage of one or both C-N bonds, resulting in multiple products.…”

“…The ROP formation is distinct in terms of both the cause of oxidation and subsequent rearrangements from two previous reports of rearranged oxidative metabolites of pyrazinone-containing thrombin inhibitors developed at Merck Subramanian et al, 2003). Oxidative rearrangements in the Merck cases occurred via cytochrome P450 (P450)-mediated epoxidation at the C=C bond in a pyrazinone ring (of 3-amino-6-methyl-pyrazinone and 3-amino-6-chloro-pyrazinone derivatives) Subramanian et al, 2003).…”

“…Oxidative rearrangements in the Merck cases occurred via cytochrome P450 (P450)-mediated epoxidation at the C=C bond in a pyrazinone ring (of 3-amino-6-methyl-pyrazinone and 3-amino-6-chloro-pyrazinone derivatives) Subramanian et al, 2003). This is no surprise because P450 enzymes are known to be capable of epoxidation dx.doi.org/10.1124/dmd.115.065136. s This article has supplemental material available at dmd.aspetjournals.org.…”