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Genotoxicity of 2-(3-Chlorobenzyloxy)-6-(piperazinyl)pyrazine, a Novel 5-Hydroxytryptamine_2c Receptor Agonist for the Treatment of Obesity: Role of Metabolic Activation

Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut (A.S.K., C.V., M.E.L., T.S.M., V.F.S.) and La Jolla, California (D.K.D., E.B.S.); and Safety Sciences (J.A., S.L.C., J.R.C.), Cardiovascular and Metabolic Diseases Chemistry (P.C., K.F.M.), and Chemical Research and Development (K.S., R.V.C.), Pfizer Global Research and Development, Groton, Connecticut

2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) is a potent and selective 5-HT_2C agonist that exhibits dose-dependent inhibition of food intake and reduction in body weight in rats, making it an attractive candidate for treatment of obesity. However, examination of the genotoxicity potential of 3 in the Salmonella Ames assay using tester strains TA98, TA100, TA1535, and TA1537 revealed a metabolism (rat S9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon coincubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA after incubations with [¹⁴C]3 in the presence of rat S9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S9/NADPH in the presence of methoxylamine and cyanide led to the detection of amine and cyano conjugates of 3. The mass spectrum of the amine conjugate was consistent with condensation of amine with an aldehyde metabolite derived from hydroxylation of the secondary piperazine nitrogen--carbon bond. The mass spectrum of the cyano conjugate suggested a bioactivation pathway involving N-hydroxylation of the secondary piperazine nitrogen followed by two-electron oxidation to generate an electrophilic nitrone, which reacted with cyanide. The 3-chlorobenzyl motif in 3 was also bioactivated via initial aromatic ring hydroxylation followed by elimination to a quinone-methide species that reacted with glutathione or with the secondary piperazine ring nitrogen in 3 and its monohydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.