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

Abstract

(S)-5-Chloro-1-(1-cyclopropylethyl)-3-(2,6-dichloro-4-(trifluoromethyl)-phenylamino)pyrazin-2(1H)-one (BMS-665053), a pyrazinone-containing compound, is a potent and selective antagonist of corticotropin-releasing factor receptor-1 (CRF-R1) that showed efficacy in the defensive withdrawal model for anxiety in rats, suggesting its use as a potential treatment for anxiety and depression. In vitro metabolism studies of BMS-665053 in rat and human liver microsomes revealed cytochrome P450-mediated oxidation of the pyrazinone moiety, followed by ring opening, as the primary metabolic pathway. Detection of a series of GSH adducts in trapping experiments suggested the formation of a reactive intermediate, probably as a result of epoxidation of the pyrazinone moiety. In addition, BMS-665053 (20 mg/kg i.v.) underwent extensive metabolism in bile duct-cannulated (BDC) rats. The major drug-related materials in rat plasma were the pyrazinone oxidation products. In rat bile and urine (0-7 h), only a trace amount of the parent drug was recovered, whereas significant levels of the pyrazinone epoxide-derived metabolites and GSH-related conjugates were detected. Further evidence suggested that GSH-related conjugates also formed at the dichloroarylamine moiety possibly via an epoxide or a quinone imine intermediate. Other major metabolites in BDC rat bile and urine included glucuronide conjugates. To reduce potential liability due to metabolic activation of BMS-665053, a number of pyrazinone analogs with different substituents were synthesized and investigated for reactive metabolite formation, leading to the discovery of a CRF-R1 antagonist with diminished in vitro metabolic activation.

Role of Metabolism in Toxicity of Drugs in Humans

“…The attractive in vivo pharmacology and disposition properties of the pyrazinone derivative 9 (Scheme 11.9) were offset by the finding that 9 was bioactivated by P450 to form reactive metabolites. In fact, a major component of the elimination mechanism of 9 in rats (∼40% of drug-related material recovered in rat bile) is composed of conjugation with GSH, which is consistent with reactive metabolite formation in vivo [76]. Two distinct bioactivation pathways were deciphered (Scheme 11.9): (i) oxidative metabolism on the chloropyrazinone ring to yield an electrophilic epoxide and (ii) O-dealkylation of the difluoromethylphenoxy moiety to yield a phenol metabolite 10, two electrons in which generated the reactive quinone-imine species in a manner similar to that discerned with the 2,6-dichloro-4-hydroxyaniline analogs and NSAIDs diclofenac and lumiracoxib [77,78].…”

Mitigating Heterocycle Metabolism in Drug Discovery

“…79 Additional metabolite ID studies on 142 revealed that the pyrazin-2(1H)-one was susceptible to oxidation and GSH conjugation. 78 The suggested intermediate that led to pyrazin-2(1H)-one metabolism was the chloroepoxide 146, which after hydrolysis or reaction with GSH led to the observed oxidized metabolites 149 and 150 and GSH adducts 147 and 148. Metabolism was also observed on the arylamine portion of 142 but to a lesser extent.…”

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…Instead, the most commonly utilized approach is to capture the reactive intermediate with a trapping agent, leading to formation of stable adducts of reactive metabolites that can be detected and characterized using LC-MS/MS techniques [4][5][6]. These methods have been extensively applied to efforts in the minimization of bioactivation for lead compounds at the drug discovery stage [7][8][9][10][11].…”