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-alpha-
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.