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

Abstract

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 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 concentrationdependent incorporation of radioactivity in calf thymus DNA after incubations with [ 14 C]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.Advances in molecular biology and pharmacology have led to the identification of 14 5-hydroxytryptamine (serotonin, 5-HT) receptor subtypes (Hoyer et al., 2002). These subtypes are classified into seven receptor families (5-HT 1 to 5-HT 7 ) according to their structure, function, and signal transduction properties and are distributed widely in the central and peripheral nervous systems. The 5-HT 2 receptor subfamily comprises three subtypes, namely 5-HT 2A , 5-HT 2B , and 5-HT 2C . These receptors exhibit 46 to 50% sequence homology and belong to the large family of seven-transmembrane domain G proteincoupled receptors. Each of these receptor subtypes has been implicated in the control of food intake. In particular, the 5-HT 2C receptor has been the focus of many studies investigating feeding behavior.Evidence from transgenic mice with a targeted deletion of the 5-HT 2C receptor (Tecott et al., 1995), and pharmacological studies using specific 5-HT 2C receptor ligands (Bickerdike, 2003) supports a potential therapeutic utility of 5-HT 2C receptor agonists as antiobesity agents. For instance, 5-HT 2C receptor-deficient mice are obese and hyperphagic and exhibit impaired satiety. They also display elevated insulin and leptin levels and impaired gl...

We Need 2C but Not 2B: Developing Serotonin 2C (5‐HT2C) Receptor Agonists for the Treatment of CNS Disorders

“…[13] However, CP-809101 is relatively potent at 5-HT 2B receptors (EC 50 =65.3 n m , E max =57%), and its development has been discontinued due to the observation of genotoxicity in preclinical studies. [14] …”

Deleterious Effects of Reactive Metabolites

“…In certain cases the metabolites are so reactive that they do not escape the enzyme that formed them and covalently bind to the enzyme leading to irreversible inhibition 39,40. Because metabolism precludes enzyme inactivation, these compounds fall into the category of mechanism-based inactivators.…”

“…Because a good correlation has been established between in vitro metabolism dependent mutagenic response and the outcome of rodent carcinogenicity evaluations, drug candidates intended for non-life-threatening indications are generally discontinued from development, when they exhibit a positive response in the in vitro assays in the presence of S-9/NADPH. An example of this phenomenon was highlighted with a study on the anti-obesity agent and 5-hydroxytryptamine (5-HT) 2C agonist 2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine ( 1; Figure 6 ) 40. The attractive in vitro/in vivo pharmacology and pharmacokinetic attributes of 1 were offset by its S-9/NADPH-dependent genotoxic effects in the bacterial Salmonella Ames assay, which led to its discontinuation from clinical development.…”

“…It is noteworthy to point out that not all reactive metabolites can be trapped with GSH. Hard electrophiles including DNA-reactive metabolites (e.g., electrophilic carbonyl compounds) will preferentially react with hard nucleophiles such as amines (e.g., semicarbazide and methoxylamine), amino acids (e.g., lysine), and DNA bases (e.g., guanine and cytosine) affording the corresponding Schiff bases 40. Likewise, the cyanide anion, N-acetyllysine and methoxylamine are ‘hard’ nucleophiles that can be used to trap hard electrophiles such as electrophilic iminium species that are generated via metabolism of tertiary amines 99,100…”

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure–activity relationship, in vivo evaluation, mechanistic and bioactivation studies

“…20 The trapping reagents used include glutathione (for soft electrophiles such as the quinone imine) as well as methoxylamine and potassium cyanide (for hard electrophiles such as the iminium ion). [21][22][23] Electrochemical oxidation online with electrospray ionization mass spectrometry (EC-ESI/MS) is a relatively new technique that avoids the complexity of working with biological matrices. Electrochemical oxidation has been found to successfully mimic CYP450 benzylic hydroxylation, hydroxylation of aromatic rings containing electron-donating groups, Ndealkylation, S-oxidation, dehydrogenation and, less efficiently, N-oxidation and Odealkylation.…”