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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

(Received November 1, 2006; accepted March 2, 2007)

	Abstract

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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.

In the course of our efforts to identify novel 5-HT_2C receptor agonists, we discovered a new class of selective 5-HT_2C agonists as exemplified with 2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) (Fig. 1). 3 is a potent 5HT_2C receptor agonist, which displays functional selectivity for the 5HT_2C receptor over the other 5HT₂ receptor subtypes. Whereas 3 binds to human 5HT_2C, 5HT_2A, and 5HT_2B receptors subtypes with K_i values of 1.0, 8.4, and 34.0 nM, respectively, functional agonist activity (EC₅₀ of 54 nM and 90% of maximal 5-HT activation) is observed only at the 5HT_2C receptor. In acute in vivo studies, oral administration of 3 to Wistar rats resulted in the dose-dependent inhibition of both spontaneous, nocturnal food intake and fasting-induced refeeding. Furthermore, oral administration of 3 at 30 mg/kg daily to Wistar rats for 4 days resulted in 8% inhibition of cumulative food intake and 24% reduction in body weight over the 4-day period. Unfortunately, 3 displayed a serious genetic safety liability that manifested as a positive finding in the Salmonella reverse mutation assay in the presence of metabolic activation. The Salmonella assay has become an integral part of the safety evaluation of drug candidates and is required by regulatory agencies for drug approvals worldwide. Because positive findings in the Salmonella reverse mutation assay have a good correlation with the outcome of rodent carcinogenicity testing, a positive result leads to the discontinuation of development, particularly for drugs intended for non-life-threatening indications (Zeiger, 1998; Kim and Margolin, 1999). To design follow-on 5-HT_2C agonists devoid of this issue, we set out to investigate the mutagenic mechanisms that led to a positive response in the Salmonella assay with 3. The results of these studies are described herein.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Synthetic scheme for the title compounds used in the study.

	Experimental Procedures

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Chemicals. All chemicals and solvents used in chemical synthesis were purchased from the Aldrich Chemical Co. (Milwaukee, WI). The purity of products was judged to be at least 99% on the basis of their chromatographic homogeneity. ¹H NMR spectra in CDCl₃ or CD₃OD were recorded on a Varian Unity M-400 MHz spectrometer (Varian, Inc., Palo Alto, CA); chemical shifts are expressed in parts per million () calibrated to the deuterium lock signal for CDCl₃ and CD₃OD. Spin multiplicities are given as s (singlet), d (doublet), and m (multiplet). NADPH, methoxylamine, glutathione, and potassium cyanide were purchased from Sigma-Aldrich (St. Louis, MO). Aroclor 1254-induced Sprague-Dawley rat liver S9 mixture was purchased from Molecular Toxicology (Boone, NC). The synthesis of 2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3) and 6-(3-chlorobenzyloxy)-N-(2-aminoethyl)pyrazin-2-amine (5), the major metabolite of 3 in rat S9 fractions is shown in Fig. 1.

Preparation of 2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine (3). Step I. tert-Butyl-4-(6-chloropyrazin-2-yl)piperazine-1-carboxylate (1). A mixture of 2,6-dichloropyrazine (2.98 g, 20 mmol), piperazine-1-carboxylic acid-tert-butyl ester (3.72 g, 20 mmol), and sodium carbonate (2.12 g, 20 mmol) in tert-butanol (50 ml) was heated under nitrogen for 65 h. The solution was concentrated in vacuo. The residue was partitioned between ethyl acetate (50 ml) and water (50 ml). The aqueous phase was extracted with ethyl acetate (3 x 30 ml). The combined organic extracts were washed with brine (50 ml), dried over magnesium sulfate, filtered, and concentrated in vacuo to give 1 as a white solid. ¹H NMR (CDCl₃) 7.96 (s, 1H), 7.82 (s, 1H), 3.52-3.60 (m, 8H), 1.46 (s, 9H). MS (ES⁺) Calculated: 298.1; found: 299.1 (M + 1). Step II. tert-Butyl-4-(6-(3-chlorobenzyloxy)pyrazin-2-yl)piperazine-1-carboxylate (2). A mixture of 1 (300 mg, 1.0 mmol), 3-chlorobenzyl alcohol (0.142 ml, 1.2 mmol), potassium hydroxide (191 mg, 3.4 mmol), and 18-crown-6 (10.6 mg, 0.04 mmol) in toluene (6 ml) was stirred under reflux for 5.5 h and concentrated in vacuo. The residue was partitioned between water (30 ml) and dichloromethane (30 ml). The aqueous phase was separated and extracted with dichloromethane (2 x 30 ml). The combined organic extracts were dried over magnesium sulfate, filtered, and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography (5% methanol in dichloromethane) to afford 380 mg of 2. ¹H NMR (CDCl₃) 7.63 (s, 1H), 7.59 (s, 1H), 7.40 (s, 1H), 7.27 (s, 3H), 5.26 (s, 2H), 3.51 (s, 8H), 1.46 (s, 9H). MS (ES⁺) Calculated: 404.1; found: 405.0 (M + 1). Step III. Fumarate salt of 3. To a solution of 2 (380 mg, 0.94 mmol) in dichloromethane (6 ml) was added trifluoroacetic acid (1.4 ml). After stirring at room temperature for 2 h, the solution was concentrated in vacuo. The residue was partitioned between 1 M HCl (20 ml) and ethyl acetate (30 ml). The aqueous phase was washed with ethyl acetate (2 x 30 ml), basified with 3 N NaOH, and extracted with ethyl acetate (3 x 30 ml). The combined organic extracts were concentrated in vacuo to afford the free base of 3 (252 mg) as an oil. ¹H NMR (CDCl₃) 7.62 (s, 1H), 7.56 (s, 1H), 7.40 (s, 1H), 7.28-7.25 (m, 3H), 5.26 (s, 2H), 3.49 (t, 4H), 2.95 (t, 4H), 1.96 (s, 1H). To a solution of the free base form of 3 (252 mg, 0.83 mmol) in a mixture of isopropyl ether (8 ml) and methanol (1 ml) was added 0.5 M fumaric acid in methanol (1.86 ml, 0.93 mmol) in a single portion. The resulting mixture was stirred at room temperature for 1 h. The white solid was collected by filtration and washed with isopropyl ether followed by hexane and dried in vacuo to afford the fumarate salt of 3. ¹H NMR (CD₃OD) 7.77 (s, 1H), 7.59 (s, 1H), 7.44 (d, 1H), 7.34-7.28 (m, 3H), 6.67 (s, 2H), 5.35 (s, 2H), 3.82-3.79 (m, 4H), 3.29-3.24 (m, 4H). MS (ES⁺) Calculated: 304.1; found: 305.4 (M + 1).

Synthesis of 6-(3-Chlorobenzyloxy)-N-(2-aminoethyl)pyrazin-2-amine (5), the Major Metabolite of 3 in Aroclor 1254-Induced Rat Liver S9 Protein. Step I. 2-(3-Chlorobenzyloxy)-6-chloropyrazine (4). To a solution of 3-chlorobenzyl alcohol (0.16 µl, 1.4 mmol) in tetrahydrofuran (5 ml) was added sodium hydride (60% in mineral oil; 67.2 mg, 1.68 mmol). After stirring at room temperature for 30 min, 2,6-dichloropyrazine (200 mg, 1.34 mmol) was added in one portion. The resulting mixture was refluxed for 1.5 h, cooled to room temperature, and poured into water (50 ml). The aqueous solution was extracted with ethyl acetate (2 x 30 ml). The combined organic extracts were washed with brine, dried under magnesium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (30% ethyl acetate in hexanes) to give 4 (328 mg).¹H NMR (CDCl₃) 8.18 (2s, 2H), 7.44 (s, 1H), 7.31 (2s, 3H), 5.34 (s, 2H). MS (ES⁺) Calculated: 254.0, Found: 255.2 (M + 1). Step II. A mixture of 4 (290 mg, 1.1 mmol) and ethylene diamine (1.14 ml, 17.1 mmol) in dioxane (4 ml) was refluxed for 15 h. The solution was cooled to room temperature, poured into ice water (30 ml), and extracted with ethyl acetate (3 x 20 ml). The combined organic extracts were extracted with 1 N HCl, and the aqueous solution was basified with 1 N NaOH and extracted with ethyl acetate (3 x 30 ml). The combined organic extracts were washed with brine, dried under magnesium sulfate, filtered, and concentrated in vacuo to give 5 (258 mg). ¹H NMR (CDCl₃) 7.48 (s, 1H), 7.44 (s, 1H), 7.39 (s, 1H), 7.25 (m, 3H), 5.24 (s, 2H), 5.12 (m, 1H), 3.32-3.37 (m, 2H), 2.89 (m, 2H), 1.81 (broad s, 2H). MS (ES⁺) Calculated: 278.1; found: 279.3 (M + 1).

Synthesis of [¹⁴C]-2-(3-Chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine ([¹⁴C]-3). Step I. Preparation of [¹⁴C]-2-(3-chlorobenzyloxy)-6-chloropyrazine ([¹⁴C]-4). Sodium tert-butoxide (42 mg, 0.44 mmol) was added to a solution of 3-chlorobenzyl alcohol (52 µl, 0.44 mmol) in 3 ml of tetrahydrofuran. A 1.5-ml portion of this solution was added dropwise to 2,6-dichloro-[2,3-¹⁴C]pyrazine (7.75 mCi, 55 mCi/mmol, 0.14 mmol) (ChemSyn Laboratories, Lenexa, KS) over 2.5 h, and the resulting solution was stirred at room temperature overnight. The reaction was then concentrated in vacuo, and the resulting residue was purified by silica gel flash column chromatography, eluting with 5% acetone in hexane, to afford 4.56 mCi of [¹⁴C]4. Step II. Piperazine (48 mg, 0.56 mmol) was added to a solution of [¹⁴C]4 (4.56 mCi, 55 mCi/mmol, 0.08 mmol) in 1 ml of water. The resulting suspension was heated at 90°C for 32 h. The reaction was then cooled to room temperature, diluted with 5 ml of water, and extracted twice with 5-ml portions of ethyl acetate. The combined ethyl acetate extracts were concentrated in vacuo, and the resulting residue was purified by silica gel flash column chromatography, eluting with 10% methanol in dichloromethane, to afford 1.94 mCi of [¹⁴C]3.

Genetic Toxicity Studies. The Salmonella reverse mutation assay was performed using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 (Maron and Ames, 1984). Briefly, the Salmonella cells were treated in soft agar overlays with 3 dissolved in dimethyl sulfoxide (DMSO) at concentrations ranging from 0.015 to 5 mg/plate or an appropriate amount of DMSO in the presence or absence of an Aroclor 1254-induced rat liver S9 mixture and a NADPH-regenerating system. Incubations comprising tester strain TA100, 3 (0.5 mg/plate), methoxylamine (1 mM), or glutathione (1 mM) in the presence or absence of Aroclor 1254-induced rat liver S9 mixture and NADPH were also conducted to assess the effect of exogenously added nucleophiles on the mutagenic response of 3. The number of visible revertant colonies present after 72 h of incubation at 37°C was recorded, and the fold of change over DMSO-treated control plates was calculated. At least three independent experiments with each Salmonella tester strain were performed. The analysis of the data included calculation of the average and S.E.M. The statistical significance was determined using the t test. A 2-fold statistically significant (p 0.05) increase of number of revertant colonies over the DMSO-treated controls was considered as a positive response in the Salmonella assay.

In Vitro Covalent Binding of [¹⁴C]3 to DNA. To emulate conditions of the Salmonella assay, calf thymus DNA (Sigma-Aldrich) was incubated with appropriate concentrations of [¹⁴C]3 (specific activity 55 mCi/mmol) in the bacterial growth medium (Oxiod Nutrient Broth 2, BD Biosciences, Franklin Lakes, NJ) in the presence or absence of metabolic activation for 3 h at 37°C. In the first set of experiments, metabolic activation was performed using NADPH as cofactor. Incubations (volume = 1 ml) consisted of calf thymus DNA (1.0 mg/ml), 3 (0.5 µM), and 20 µl of Aroclor 1254-induced rat liver S9 protein that was prepared separately by combining 0.5 ml of S9 (protein concentration 38.4 mg/ml), 1.25 ml of phosphate buffer, and 36.5 mg of NADPH. In the second set of experiments, a NADPH self-regenerating system was used instead of NADPH. Incubations (volume = 1 ml) comprised calf thymus DNA (1.0 mg/ml), 3 (0.5 and 5 µM), and 139 µl of S9 protein. The mixture was prepared by combining 100 µl of S9 protein (protein concentration = 38.4 mg/ml), NADP monosodium (4 mM), glucose 6-phosphate (5 mM), potassium chloride (33 mM), and magnesium chloride (8 mM) in 900 µl of 100 mM sodium phosphate buffer (pH 7.4). All incubations in both test conditions were conducted in duplicate or triplicate and experiments included positive ([¹⁴C]benzo[a]pyrene, 100 µM) and vehicle (0.5% DMSO) controls. In addition, incubations were also conducted in the presence or absence of S9 protein. Incubations lacking NADPH cofactor were also included to test cytochrome P450-independent mechanisms of DNA binding by [¹⁴C]3. After incubation, unbound [¹⁴C]3 was removed via extraction of the calf thymus DNA with phenol-chloroform-isoamyl alcohol (25:24:1) followed by two additional extractions with chloroform-isoamyl alcohol (24:1). The DNA was precipitated by the addition of 1/10 volume of 2 M NaCl and 2x volume of ethanol. After centrifugation (2000g for 10 min), the resulting DNA pellet was washed twice with ethanol, dissolved in 1x Tris-EDTA, and quantified by ultraviolet spectroscopy ( = 260 nm). Remaining DNA-bound radioactivity was measured on a Beckman LS6000IC liquid scintillation counter (Beckman Coulter, Fullerton, CA). The results are expressed as mean counts (disintegrations per minute) per 20 µg of calf DNA.

Metabolism Studies. Stock solutions of the test compounds were prepared in methanol. The final concentration of methanol in the incubation media was 0.2% (v/v). Incubations were carried out at 37°C for 60 min in a shaking water bath. The incubation volume was 1 ml and consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4) containing MgCl₂ (10 mM), Aroclor 1254-induced rat liver S9 fraction (final protein concentration = 1 mg/ml), NADPH (1 mM), substrate (20 µM), and methoxylamine (1 mM) or potassium cyanide (1 mM) or glutathione (1 mM). Incubations that lacked either NADPH or trapping agents served as negative controls, and reactions were terminated by the addition of ice-cold acetonitrile (1 ml). The solutions were centrifuged (3000g, 15 min), and the supernatants were dried under a steady nitrogen stream. The residue was reconstituted with mobile phase and analyzed for metabolite formation by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Bioanalytical Methodology for Metabolite Identification. The separation of metabolites was achieved at ambient temperature on a Kromasil C4 100A column (3.5 µm, 150 x 2.0 mm; Phenomenex, Torrance, CA) by reverse-phase chromatography. The mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile (solvent B) and was delivered at 0.200 ml/min. A gradient was used to separate 3 and its metabolites as well as conjugates derived from trapping of reactive intermediates with exogenous nucleophiles. The initial composition of solvent B was maintained at 1% for 10 min and then increased in a linear manner as follows: 30% at 28 min; 50% at 30 min, and 90% at 35 min. It was then maintained at 90% for up to 37 min and then decreased to 1% in the next 3 min. The column was allowed to equilibrate at 1% solvent B for 5 min before the next injection. The high-performance liquid chromatography effluent going to the mass spectrometer was directed to waste through a divert valve for the initial 5 min after sample injection. Mass spectrometric analyses were performed on a ThermoFinnigan Deca XP ion trap mass spectrometer, which was interfaced to an HP-1100 high-performance liquid chromatography system (Agilent Technologies, Palo Alto, CA) and equipped with an electrospray ionization source. The values for electrospray ionization were as follows: capillary temperature, 270°C; spray voltage, 4.0 kV; capillary voltage, 4.0 V; sheath gas flow rate, 90; and auxiliary gas flow rate, 30. The mass spectrometer was operated in a positive ion mode with data-dependent scanning. The ions were monitored over a full mass range of m/z 100 to 1000. For a full scan, the automatic gain control was set at 5.0 x 10⁸, maximum ion time was 100 ms, and the number of microscans was set at 3. For MSⁿ scanning, the automatic gain control was 1.0 x 10⁸, maximum ion time was 400 ms, and the number of microscans was set at 2. For data-dependent scanning, the default charge state was 1, default isolation width was 3.0, and normalized collision energy was 45.0. Metabolites were identified by comparing t = 0 samples to t = 60 min samples (with or without NADPH cofactor), and structural information was generated from collision-induced dissociation (CID) spectra of the corresponding protonated molecular ions and/or comparison with synthetic standards. Apart from full scan analysis, methoxylamine conjugate formation for 3 was also assessed by multiple reaction monitoring (MRM) of the anticipated mass transition corresponding to the loss of the 3-chlorobenzyl moiety (loss of 125 mass units) from the parent molecular ion of the respective amine conjugates.

View larger version (8K): [in this window] [in a new window]   FIG. 2. Evaluation of piperazine 3 in the Salmonella reverse mutation assay. The mutagenic potential of 3 was tested using Salmonella tester strains TA100 (), TA98 (), TA 1535 (), and TA 1537 (). The experiments were performed in the presence (A) and absence (B) of metabolic activation (Aroclor 1254-induced rat liver S9 mixture and a NADPH-regenerating system). The data represent the average of three independent experiments ± S.E.M. Statistical significance was evaluated using a t test: *, p 0.05.

	Results

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To assess whether the increased incidence of reverse mutations in the Salmonella assay is a consequence of covalent modification of DNA by a reactive metabolite of 3, [¹⁴C]3 was synthesized (Fig. 1) and reacted with calf thymus DNA in the presence or absence of a metabolic activation system. The well-established reaction of [¹⁴C]benzo[a]pyrene with calf thymus DNA in the presence of metabolic activation (Szeliga and Dipple, 1998) was also included in this analysis as a positive control. Treatment of calf thymus DNA with [¹⁴C]benzo[a]pyrene led to a 6- to 7-fold increase of DNA-bound radioactivity over the vehicle control or samples without Aroclor 1254-induced rat liver S9 (Table 1). Treatment of calf thymus DNA with [¹⁴C]3 in the absence of S9 or presence of S9 fraction without NADPH cofactor resulted in an 2-fold increase of radioactivity over the background levels. In contrast, inclusion of a complete S9/NADPH activation system resulted in dose-dependent increase of the covalently bound radioactivity over the DNA samples treated with [¹⁴C]3 in the absence of S9 or the presence of S9 fraction without cofactor (Table 1).

Reactive Metabolite Trapping Studies. LC-MS/MS analysis of S9 incubations containing 3, an NADPH-regenerating system, and methoxylamine led to the detection of a single conjugate 6 (MH⁺ = 350) (Fig. 3B) that eluted after the parent compound (Fig. 3C). The methoxylamine conjugate 6 was detected in the full scan mode and in the MRM mode monitoring for the mass transition 350 125, which represents the loss of the 3-chlorobenzyl group from the anticipated mass of 6. The observed molecular ion (MH⁺ = 350 Da) of 6 was consistent with the addition of one molecule of methoxylamine to 3. The formation of 6 was abolished when the NADPH-regenerating system (Fig. 3A) and/or methoxylamine were omitted from the incubations.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Extracted ion chromatogram (MRM mode 350 125) of the methoxylamine conjugate 6 (Rt = 14.40 min) of piperazine derivative 3 (B) after incubation of 3 with NADPH-supplemented Aroclor 1254-induced rat liver S9 protein in the presence of excess methoxylamine. A, lack of conjugate formation in the absence of NADPH. C, extracted ion chromatogram (MRM mode 305 125) of the parent piperazine 3 (Rt = 13.82 min) in these incubations.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Product ion spectra obtained by CID of the MH+ ion (m/z 350) of the methoxylamine conjugate 6 (Rt = 14.40 min) (A). B, product ion spectra obtained by CID of the MH+ ion (m/z 305) of the parent piperazine 3 (Rt = 13.82 min). The origins of the diagnostic ions are as indicated.

Full scan LC-MS/MS analysis of S9 incubations containing 3, an NADPH-regenerating system, and potassium cyanide also led to the detection of a single conjugate 7 [MH⁺ = 346, retention time (R_t) = 28 min] (Fig. 5A). The observed molecular ion (MH⁺ = 346 Da) of 7 was consistent with the addition of one molecule of cyanide to a monohydroxylated metabolite of 3. The formation of 7 was abolished when the NADPH-regenerating system and/or potassium cyanide were omitted from the incubations (data not shown). The product ion spectrum obtained by CID of the MH⁺ at m/z 346 for the cyano conjugate 7 is shown in Fig. 5B. MS³ fragmentation of m/z 319 (arising from elimination of cyanide) (Fig. 5C) afforded additional diagnostic fragment ions at m/z 248, 194, and 178, respectively. The fragment ions at m/z 194 and 178 suggested that the 3-chlorobenzyl portion in the conjugate was intact and that hydroxylation and subsequent cyanide addition had occurred on the pyrazinyl-piperazine motif. Furthermore, the fragment ion at m/z 248 ruled out the internal tertiary piperazine nitrogen and the pyrazine ring as the sites of modification. A proposed structure for 7 that is consistent with the observed mass spectrum is shown in Fig. 5.

View larger version (19K): [in this window] [in a new window]   FIG. 5. Extracted ion chromatogram of the cyano conjugate 7 (Rt = 28.00 min) of piperazine derivative 3 after incubation of 3 with NADPH-supplemented Aroclor 1254-induced rat liver S9 protein in the presence of excess potassium cyanide (A). B, product ion spectrum obtained by CID of the MH+ ion (m/z 346) of 7. C, MS3 spectrum of fragment ion m/z 319 observed in the product ion spectrum of 7.

View larger version (17K): [in this window] [in a new window]   FIG. 6. Extracted ion chromatogram of the glutathione conjugate 8 (Rt = 16.3 min) of piperazine derivative 3 after incubation of 3 with NADPH-supplemented Aroclor 1254-induced rat liver S9 protein in the presence of excess glutathione (A). B, product ion spectrum obtained by CID of the MH+ ion (m/z 448) of 8. The origins of the characteristic ions are as indicated.

General Metabolic Profile of Piperazine (3) in NADPH-Supplemented Aroclor 1254-Induced Rat Liver S9 Protein. Figure 7A depicts a full scan analysis of the various metabolites of piperazine 3 observed in Aroclor 1254-induced rat liver S9 protein in a NADPH-dependent fashion. A total of eight metabolites eluting before parent were observed. The major metabolite M7 eluted at R_t of 24.5 min and displayed a molecular ion (MH⁺) at 279, which was 26 Da lower than the molecular mass of the parent compound 3. The mass spectral characteristics of M7 (Fig. 7B) are consistent with P450-mediated piperazine ring scission in 3 to yield the diamine metabolite M7. The retention time and mass spectral properties of M7 were identical to those of the synthetic standard of the diamine metabolite 5. The product ion spectra obtained by the CID of the MH⁺ ions for the remainder of the metabolites provided some insight into their respective structures. For instance, the metabolite M4 exhibited a molecular ion (MH⁺) at 295, which indicated that M4 was a monohydroxylated metabolite of M7. The mass spectrum of M4 suggested that the site of monohydroxylation was on the 3-chlorophenyl ring (Fig. 7C). Metabolites M1, M2, and M5 were derived from monohydroxylation of parent 3 since their molecular mass (MH⁺ = 321) represented the addition of 16 Da to the molecular ion of 3 (MH⁺ = 305). The CID spectra of M1 and M5 indicated the presence of base fragment ions at m/z 193 and m/z 141, suggesting that the pyrazinylpiperazine motif was intact and that the site of monohydroxylation was the 3-chlorophenyl ring in 3 (Fig. 8). The CID spectra of M2 revealed the presence of a base fragment ion at m/z 196, which is the addition of 16 Da to the ion fragment at m/z 179 in the mass spectrum of 3. This observation suggested that the pyrazinylpiperazine group in M2 had un-dergone monohydroxylation. The additional diagnostic ion fragments at m/z 293 and 278 lead us to suspect the pyrazine ring as the site of oxidation. The molecular ion (MH⁺ = 337) of metabolite M3 was consistent with dihydroxylation of 3. The CID spectra of M3 indicated the presence of a base fragment ion at m/z 209, suggesting that the one site of oxidation was the pyrazinylpiperazine group. The lack of additional diagnostic mass fragments leads us to speculate that the second site of oxidation is the 3-chlorophenyl ring in 3.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Total ion chromatogram (A) of the various metabolites of 3 after incubation in NADPH-supplemented Aroclor 1254-induced rat liver S9 protein. B, product ion spectrum obtained by CID of the MH+ ion (m/z 279) of the major metabolite of 3, i.e., M7. C, product ion spectrum obtained by CID of the MH+ ion (m/z 295) of the M4, the monohydroxylated metabolite of M7.

View larger version (16K): [in this window] [in a new window]   FIG. 8. Product ion spectra obtained by the CID of the MH+ ions of the M1--M3 and M5 metabolites of 3 in NADPH-supplemented Aroclor 1254-induced rat liver S9 protein.

Metabolite M6 (R_t = 22.1 min) exhibited a molecular ion (MH⁺) at m/z 307, which was 2 mass units greater than the molecular weight of 3. The mass spectrum of M6 at m/z 307 revealed a fragment ion at m/z 289 in the MS² spectrum, suggesting the loss of a water molecule (Fig. 9). The fragment ion at m/z 289 was subjected to further fragmentation in the data-dependant scanning mode. This spectrum gave major fragment ions at m/z 262, 246, 219, 164, and 125 as shown in Fig. 9. The fragmentation pattern and the molecular ion led us to speculate that M6 is derived from a unique piperazine ring-opening pathway leading to the ring-contracted imidazolidinol isomers shown in Fig. 9.

View larger version (13K): [in this window] [in a new window]   FIG. 9. Product ion spectra obtained by the CID of the MH+ ion of the M6 metabolite of 3 in NADPH-supplemented Aroclor 1254-induced rat liver S9 protein.

View larger version (10K): [in this window] [in a new window]   FIG. 10. Product ion spectra obtained by the CID of the MH+ ion of the M8 metabolite of 3 in NADPH-supplemented Aroclor 1254-induced rat liver S9 protein.

	Discussion

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Reactive metabolite trapping studies in NADPH-supplemented Aroclor 1254-induced rat S9 incubations of 3 in the presence of amine, cyanide, and glutathione nucleophiles led to the detection of conjugates of 3 with each of these trapping agents. This observation suggested that 3 was bioactivated to both hard and soft electrophilic intermediates capable of forming stable adducts with corresponding hard (methoxylamine and cyanide) and soft (glutathione) nucleophiles. The observed molecular ions of the methoxylamine and the cyanide conjugates of 3, i.e., 6 and 7, were consistent with the addition of one molecule of methoxylamine to 3 and addition of one molecule of cyanide to monohydroxylated 3, respectively. Furthermore, the mass spectrum of 6 and 7 suggested that the site of adduction of both the amine and the cyanide nucleophile occurred on the piperazine ring system. Overall, the in vitro reactive metabolite trapping studies using methoxylamine, the data on the covalent adduction of [¹⁴C]3 to calf thymus DNA, and the observation that inclusion of methoxylamine in the Ames assay results in a statistically significant attenuation in the mutagenic response strengthen the link between piperazine ring bioactivation and the mutagenic response with 3. A bioactivation sequence is proposed in which the initial P450-mediated hydroxylation on the carbon to the secondary piperazine nitrogen in 3 would afford the unstable carbinolamine intermediate, which would spontaneously open to the electrophilic aldehyde intermediate (Fig. 11, pathway a). Condensation of this carbonyl intermediate with methoxylamine would give rise to the Schiff base conjugate 6, capable of existing in the ring-opened or ring-closed forms. Alternately, a direct two-electron oxidation of the -carbon-nitrogen bond in 3 will generate the electrophilic iminium intermediate (Fig. 11, pathway b), which upon reaction with methoxylamine would also afford 6. The proposed mechanism for the formation of the cyano conjugate 7 is shown in Fig. 11, pathway c. Initial oxidation of the secondary piperazine nitrogen in 3 would furnish the hydroxylamine intermediate, which, upon a two-electron oxidation on the -carbon-nitrogen bond would lead to the nitrone derivative capable of reacting with a cyanide ion and generating conjugate 7 in a manner similar to that observed for related acyclic analogs (Clark and Cawkill, 1975; Kim et al., 1999). Considering that exogenously added amine-trapping agents such as methoxylamine are well established model nucleophiles for assessing the reactivity of hard electrophiles with DNA bases in in vitro microsomal incubations (Ames et al., 1977; Chen et al., 1995; Chen et al., 1997; Schnetz-Boutaud et al., 2000), we further propose that the electrophilic aldehyde or iminium intermediate undergoes similar chemistry with an appropriate base in DNA, leading to a mutagenic response. Besides these bioactivation mechanisms, there also exists the possibility of an additional pathway that would lead to an aldehyde intermediate capable of reacting with exogenously added nucleophiles including DNA. Thus, the P450-catalyzed piperazine ring scission in 3 to yield the diamine metabolite 5 would in theory result in the concomitant liberation of the electrophilic glyoxal (Fig. 11, pathway d). The contribution of this pathway to the mutagenic response of 3 cannot be ruled out despite the lack of detection of the corresponding methoxylamine conjugate(s) of glyoxal in NADPH-supplemented rat S9 (data not shown) and the observed incorporation of radioactivity into calf thymus DNA (¹⁴C radiolabel was on the pyrazine motif). Finally, the observation that methoxylamine significantly decreases the mutagenic response of 3 suggests that this hard nucleophile effectively competes with DNA bases and scavenges electrophilic carbonyl intermediates derived from piperazine ring scission.

View larger version (10K): [in this window] [in a new window]   FIG. 11. Proposed P450-catalyzed bioactivation pathways involving the piperazine ring in 3.

View larger version (7K): [in this window] [in a new window]   FIG. 12. Proposed P450-catalyzed bioactivation pathways involving the 3-chlorobenzyl ring in 3.

From a structure-mutagenicity relationship point of view, the P450-catalyzed formation of the diamine metabolite 5, the methoxylamine conjugate 6, the cyano conjugate 7, and the metabolite M8 suggests that electrophilic carbonyl/iminium intermediates (derived from the piperazine ring metabolism) as well as the quinone-methide (derived from the bioactivation of the 3-chlorobenzyl group) possess the ability to react with DNA. Although evidence linking the involvement of the aldehyde intermediate derived from the piperazine ring scission in 3 toward reaction with DNA is strengthened on the basis of the DNA covalent binding studies and the decrease in mutagenic response in the Salmonella Ames assay after coincubation with methoxylamine, additional studies will be needed to decipher the contribution of other reactive species, especially the quinone-methide, in the mutagenic response of 3. Structure-toxicity relationship studies are in progress and will involve an assessment of the mutagenic potential of analogs of 3 specifically designed to avoid quinone-methide formation while maintaining the rest of the chemical architecture (pyrazinyl-piperazine) constant. Likewise, a parallel effort involving the replacement of the secondary piperazine nitrogen with suitable bioisosteres and/or introduction of -carbon alkyl substituents on the piperazine ring in 3 while maintaining the 3-chlorobenzyl motif constant is also in the works. These studies will be reported in due course.

	Footnotes

 
doi:10.1124/dmd.106.013649.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine, serotonin; 3, 2-(3-chlorobenzyloxy)-6-(piperazin-1-yl)pyrazine; NMR, nuclear magnetic resonance; 5, 6-(3-chlorobenzyloxy)-N-(2-aminoethyl)pyrazin-2-amine; 1, tert-butyl-4-(6-chloropyrazin-2-yl)piperazine-1-carboxylate; MS, mass spectrometry; 2, tert-butyl-4-(6-(3-chlorobenzyloxy)pyrazin-2-yl)piperazine-1-carboxylate; 4, 2-(3-chlorobenzyloxy)-6-chloropyrazine; DMSO, dimethyl sulfoxide; LC-MS/MS, liquid chromatography tandem mass spectrometry; CID, collision-induced dissociation; MRM, multiple reaction monitoring; R_t, retention time; P450, cytochrome P450.

Address correspondence to: Dr. Amit S. Kalgutkar, Pharmacokinetics, Dynamics, and Metabolism Department, Pfizer Global Research and Development, Groton, CT 06340. E-mail: amit.kalgutkar{at}pfizer.com

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