Received for publication November 1, 2006.
Revised February 21, 2007.
Accepted for publication March 2, 2007.

GENOTOXICITY OF 2-(3-CHLOROBENZYLOXY)-6-(PIPERAZINYL)PYRAZINE, A NOVEL 5-HT2C RECEPTOR AGONIST FOR THE TREATMENT OF OBESITY: ROLE OF METABOLIC ACTIVATION

Abstract

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 S-9/NADPH)- and dose-dependent increase of reverse mutations in strains TA100 and TA1537. The increase in reverse mutations was attenuated upon co-incubation with methoxylamine and glutathione. The irreversible and concentration-dependent incorporation of radioactivity in calf thymus DNA following incubations with [¹⁴C]-3 in the presence of rat S-9/NADPH suggested that 3 was bioactivated to a reactive intermediate that covalently bound DNA. In vitro metabolism studies on 3 with rat S-9/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 mono-hydroxylated metabolite(s). The metabolism studies described herein provide a mechanistic basis for the mutagenicity of 3.

Key words: bioactivation, covalent drug binding, cytochrome P450, DNA adducts, DNA mutagenesis, drug discovery, drug toxicity