4-chlorophenyl amide - ACS Publications - American Chemical Society

Chem. Res. Toxicol. 2008, 21, 1095–1106

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Bioactivation of a Dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide) Scaffold to a Putative p-Chlorophenyl Isocyanate in Rat Liver Microsomes and In ViWo in Rats Hao Chen,*,†,‡ Yanhua Zhang,†,§ Jeremy Edmunds,| Christopher Bigge,| and Abdul Mutlib†,‡ Departments of Pharmacokinetics, Dynamics, and Metabolism, and Chemistry, Pfizer Global Research and DeVelopment, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105 ReceiVed NoVember 8, 2007

Compound I (4,5-dihydropyrazole-1,5-dicarboxylic acid-1-[(4-chlorophenyl)-amide] 5-[(2-oxo-2H[1,3′]bipyridinyl-6′-yl)-amide] was found to undergo metabolic activation in rat liver microsomes in the presence of NADPH. A reactive intermediate, postulated to be p-chlorophenyl isocyanate (CPIC), was trapped by GSH in Vitro and characterized by liquid chromatography tandem mass spectrometry (LC/ MS/MS). Subsequently, the structure of the GSH conjugate was confirmed by a comparison with a synthetic standard. The GSH conjugate was also found in the bile of rats that received an oral dose (10 mg/kg) of compound I. Further analyses of rat bile and urine using online electrochemical derivatization coupled to LC/MS demonstrated the presence of p-chlorophenyl aniline (CPA), a hydrolytic product of the intermediate isocyanate. This provided further evidence for the potential existence of CPIC. Approximately 7% of the dose was accounted by the products of CPIC, which included the GSH conjugate and CPA excreted in bile and urine. Multiple rat cytochrome P450 enzymes, including P450 1A, P450 2C, and P450 3A, appeared to be responsible for the activation of compound I to CPIC. The activation kinetics of compound I to CPIC in male rat liver microsomes exhibited a biphasic profile, indicative of at least two contributing P450 enzymes. One enzyme showed a small value of Km at 42 µM and a low Vmax of 66 pmol min-1 mg-1, while the other exhibited a large value of Km at 148 µM and a high Vmax of 1200 pmol min-1 mg-1. The formation of a putative CPIC intermediate, a carbamoylating species known to be capable of covalent binding to macromolecules, suggests a potential liability associated with the compound, particularly the dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide) scaffold, which appears to be responsible for the generation of CPIC. The mechanism of bioactivation to the putative CPIC is postulated to involve an initial P450-mediated hydroxylation of the pyrazoline at the 3 position followed by subsequent decomposition to CPIC. This mechanistic insight into the bioactivation allowed for the development of a rational structural modification strategy to mitigate or minimize the reactive metabolite formation. One of the approaches included the introduction of a metabolically stable substituent with electron-donating character at the 3 position of pyrazoline to block CPIC formation. Introduction A significant issue faced by pharmaceutical companies today is the failure of new chemical entities in the later stages of development because of unexpected toxicities, such as hepatotoxicites. Toxicity often does not become apparent until the preclinical or clinical development stages when the testing is initiated in animal models or humans. It has been reported that approximately 30% of new chemical entities fail because of toxicity (1), resulting in increased costs and delays in successful drug development. Mechanisms of drug-induced toxicities are still poorly understood and remain a challenge for the pharmaceutical industry. Nevertheless, a large amount of circumstantial evidence suggests that chemically reactive metabolites of a drug, * To whom correspondence should be addressed: Wyeth Research, 500 Arcola Road, S3415, Collegeville, PA 19426. Telephone: (484) 865-2385. Fax: (484) 865-9404. E-mail: [email protected]. † Department of Pharmacokinetics, Dynamics, and Metabolism. ‡ Present address: Department of Drug Safety and Metabolism, Wyeth Research, 500 Arcola Rd., Collegeville, PA 19426. § Present address: Department of Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Groton, CT 06340. | Department of Chemistry.

rather than the parent drug, are responsible for toxicities (2–8). It has been recognized that reactive metabolites formed from the parent compound initiate damages to cellular macromolecules that, depending upon the nature of the damage (covalent binding, oxidative stress, etc.), cause changes in cellular signaling, regulatory, and other pathways that subsequently lead to cytotoxicity (9–14). Consequently, substantial efforts in several pharmaceutical companies have been made to minimize metabolic activation of new chemical entities to reactive metabolites at the early stages of discovery/lead optimization phase (15–19). In general, the formation of electrophilic reactive metabolites is often demonstrated through appropriate in Vitro trapping experiments using nucleophiles, such as GSH. The identity of reactive intermediate(s) is often inferred from the structural characterization of the trapped thioether conjugate(s). Such information is invaluable to chemists who are engaged in the process of structural optimization of lead chemical templates. Once a definitive structure of the reactive metabolite is established, the chemist can then seek ways to design out the potential metabolic liability in lead compounds. Hence, it is particularly important to establish the nature of the reactive metabolites as early as possible for chemists to mitigate the

10.1021/tx7004019 CCC: $40.75  2008 American Chemical Society Published on Web 04/05/2008

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Figure 1. Chemical structure of compound I.

reactive metabolite formation by either appropriate structural modification or redirecting a chemistry strategy to find compounds devoid of potential metabolic liability. Compound I (Figure 1) belongs to a series of dihydropyrazole dicarboxamide derivatives and displays desired pharmacological activities. The chemical scaffold in compound I, dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide), is a core substructure and appears to play an important role in the activity. As part of a preliminary screening strategy, compound I was tested for the formation of a GSH adduct(s). It was subsequently demonstrated that a GSH adduct was formed when the compound was incubated with rat liver microsomes fortified with GSH in the presence of NADPH. Furthermore, it was found that human liver microsomes fortified with GSH in the presence of NADPH also produced the same GSH adduct, the results of which are discussed in details elsewhere (20). This finding indicated the possible existence of a chemically reactive intermediate during the metabolism of compound I. Once the metabolic activation of compound I was established, further studies were designed and conducted to understand the metabolic activation through (1) elucidating the structure of the GSH conjugate of compound I produced in Vitro, (2) investigating the relevance of bioactivation of compound I in ViVo in rats, (3) quantitatively assessing the bioactivation of compound I by determining the concentrations of the GSH adduct and other related end products excreted in rat bile and urine, (4) assessing the role of P450 enzyme(s) in the activation of compound I and identifying specific P450 enzyme(s) responsible for the activation, and (5) investigating the enzyme kinetic behaviors in the activation of compound I employing male rat liver microsomes.

Materials and Methods Chemicals and Reagents. Compound I (Figure 1) were synthesized at Pfizer Global Research and Development (Ann Arbor, MI) and fully characterized spectroscopically. The purity of compound I was >95% based on high-performance liquid chromatography (HPLC) analysis. p-Chlorophenyl isocyanate (CPIC),1 phenyl isocyanate (PIC), p-chlorophenyl aniline (CPA), p-iodophenyl aniline (IPA), and GSH (reduced form) were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI). The chemicals including NADPH, 1-aminobenzotrizole (ABT), cimetidine (CMD), and troleandomycin (TAO) were also obtained from Sigma-Aldrich Chemical Co. Furafylline (FAL) was purchased from Research Biochemicals International (Natick, MA). Ketoconozole (KTO) was obtained from ICN Biomedicals, Inc. (Aurora, OH). Waters 1 Abbreviations: CPIC, p-chlorophenyl isocyanate; PIC, phenyl isocyanate; CPA, p-chlorophenyl aniline; IPA, p-iodophenyl aniline; SCPG, S-(N[p-chlorophenyl]carbamoyl)glutathione; SCPAC, S-(N-[p-chlorophenyl]carbamoyl)-N-acetyl-L-cysteine; SPG, S-(N-[phenyl]carbamoyl)glutathione; ABT, 1-aminobenzotrizole; CMD, cimetidine; TAO, troleandomycin; FAL, furafylline; KTO, ketoconozole; PAb, polyclonal antibody; ESI, electrospray ionization; MS/MS, tandem mass spectrometry; CID, collision-induced dissociation; LC/MS/MS, liquid chromatography tandem mass spectrometry; LC/MRM, liquid chromatography multiple reaction monitoring; LC/EC/ MRM, liquid chromatography electrochemistry multiple reaction monitoring.

Chen et al. Symmetry C18 columns (2.1 × 50 mm, 5 µm) were obtained from Waters Corporation (Milford, MA). A Synergi Max RP column (10 × 150 mm, 4 µm) was purchased from Phenomenex (Torrance, CA). Male rat liver microsomes (pooled from 162 animals), human liver microsomes (pooled from 10 individuals), cDNA-expressed rat P450 microsomes, and polyclonal antibody (PAb) against rat P450 1A1/2, 2B1/2, and 3A2 were purchased from BD Biosciences (Woburn, MA). Each cDNA-expressed P450 microsomes contained cDNA-expressed rat P450 reductase, human cytochrome b5, and a specific rat P450 enzyme, such as 1A1, 1A2, 2A2, 2B1, 2C6, 2C11, 2C12, 2C13, 2D1, 2D2, 3A1, and 3A2. All solvents and reagents were of the highest grade commercially available. Caution: CPIC, PIC, CPA, and IPA are highly toxic, and care should be taken in handling, analysis, and disposal of these substances. Synthesis of S-(N-[p-chlorophenyl]carbamoyl)glutathione (SCPG), S-(N-[p-chlorophenyl]carbamoyl)-N-acetyl-L-cysteine (SCPAC), S-(N-[phenyl]carbamoyl)glutathione (SPG). The synthesis and purification of these thioether conjugates were performed according to the procedures described in the literature (21). Briefly, a solution of CPIC (500 µmol) in acetone was added over 30 min to a stirred solution of either GSH or N-acetyl-L-cysteine (500 µmol) in acetonitrile/water (7:3, v/v) under nitrogen at ambient temperature. A sample of SPG was obtained by a similar synthetic procedure, using PIC in place of CPIC. After a period of 3 h, the solvent was removed under reduced pressure and the desired products were purified by HPLC using an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA) consisting of a quaternary pump coupled in sequence to an autosampler, a diode array detector, and an autofraction collector. The mobile phase consisted of two components: solvent A (1:1 acetonitrile/methanol, v/v) and solvent B (1 mM ammonium acetate containing 1% acetonitrile at pH 4). Purification of synthetic thioether conjugates was performed using a Phenomenex Synergi Max-RP C18 semipreparative column (15 cm × 10 mm, 4 µm) eluted with a specific isocratic solvent system for each thioether conjugate, with the flow rate set at 3.0 mL/min. The percentage of solvent A was set at 45, 27, and 30% for SCPG, SCPAC, and SPG, respectively. Under these conditions, the thioether conjugates were eluted with the retention times in the range of 2.5–5.0 min. The purified products exhibited nuclear magnetic resonance (NMR) and mass spectrometric characteristics fully consistent with the proposed structures. SCPG. NMR (Me2SO-d6) δ: 1.87 (m, 2H, Glu-β-CH2), 2.28 (t, J ) 7 Hz, 2H, Glu-γ-CH2), 2.94 (dd, J ) 10 and 14 Hz, 1H, Cysβ-CH), 3.46–3.52 (m, 2H, Glu-R-H and Cys-β-CH), 3.66 (d, J ) 6 Hz, 2H, Gly CH2), 4.32–4.48 (m, 1H, Cys-R-CH), 7.30 (d, J ) 9 Hz, 2H, phenyl), 7.47 (d, J ) 9 Hz, 2H, phenyl), 8.44 (d, J ) 6 Hz, 1H, Cys-NH), 8.60 (t, J ) 5 Hz, 1H, Gly NH), and 10.50 (s, 1H, CO-NH). MS: m/z 461/463 ([M + H]+, 35Cl/37Cl). Tandem mass spectrometry (MS/MS) analysis of [M + H]+ at m/z 461 employing collision-induced dissociation (CID) demonstrated the following fragment ions: m/z 443 ([M + H]+-H2O), m/z 386 ([M + H]+-Gly), m/z 332 (base peak, [M + H]+-γ-glutamyl moiety), m/z 205 ([M + H]+-ClC6H4NH), m/z 179 (cysteinylglycine + H+), m/z 162 (cysteinylglycine - NH3), m/z 128 (ClC6H4NH2 + H+). SCPAC. NMR (Me2SO-d6) δ: 1.91 (s, 3H, CH3), 3.26 (dd, J ) 9 and 14 Hz, 1H, Cys-β-CH), 3.54 (dd, J ) 9 and 14 Hz, 1H, Cys-β-CH), 4.65 (m, 1H, Cys-R-CH), 7.33 (d, J ) 9 Hz, 2H, phenyl), 7.60 (d, J ) 9 Hz, 2H, phenyl), 8.45 (d, J ) 6 Hz, 1H, Cys-NH), and 10.50 (s, 1H, CO-NH). MS: m/z 317/319 ([M + H]+, 35Cl/37Cl). MS/MS (CID of ([M + H]+ at m/z 317): m/z 275 ([M + H]+-acetyl group), m/z 164 (base peak, [M + H]+-N-acetyl cysteine), m/z 154 (ClC6H4NCO + H+), m/z 122 (Cys + H+). SPG. NMR (Me2SO-d6) δ: 1.89 (m, 2H, Glu-β-CH2), 2.28 (t, J ) 7 Hz, 2H, Glu-γ-CH2), 2.94 (dd, J ) 10 and 14 Hz, 1H, Cysβ-CH), 3.46–3.52 (m, 2H, Glu-R-H and Cys-β-CH), 3.67 (d, J ) 5 Hz, 2H, Gly CH2), 4.33–4.45 (m, 1H, Cys-R-H), 6.95 (t, J ) 9 Hz, 2H, phenyl), 7.30 (dd, J ) 11 Hz, 2H, phenyl), 7.48 (dd, J ) 9 Hz, 1H, phenyl), 8.45 (d, J ) 6 Hz, 1H, Cys-NH), 8.61 (t, J ) 6 Hz, 1H, Gly NH), and 10.50 (s, 1H, CO-NH). MS: m/z 427 ([M + H]+). MS/MS (CID of ([M + H]+ at m/z 427): m/z 352 ([M +

BioactiVation of Compound I H]+-Gly), m/z 298 ([M + H]+-γ-glutamyl moiety), m/z 179 (cysteinylglycine + H+). In Vitro Bioactivation in Rat or Human Liver Microsomes. Microsomal incubations were carried out using the following protocol: male rat or human liver microsomes ((0.5 mg), (NADPH (1 mM), GSH (0.5 mM), MgCl2 (3 mM), compound I, or its structural analogue (50 µM), with the final volume adjusted to 0.5 mL with 0.1 M phosphate buffer (pH 7.4). The reaction was run for 30 min at 37 °C and terminated by the addition of 1.0 mL of ice-cold acetonitrile containing acetic acid (0.1%, v/v). The samples were vortexed and centrifuged at 1600g for 5 min. The supernatant was separated, dried under N2, and reconstituted in 200 µL of HPLC mobile phase. Aliquots (10–20 µL) of the sample were analyzed by LC/MS and LC/MS/MS methods. Metabolic activation to reactive intermediate(s) was demonstrated by LC/MS detection of GSH conjugate(s) present in the extracts of incubation. To access the role of P450 enzymes in mediating the metabolic activation, preliminary inhibitory experiments using ABT and KTO were conducted. The stock solutions of ABT and KTO were prepared in Me2SO. The incubations, performed in triplicate, consisted of rat liver microsomes (0.5 mg), NADPH (1 mM), MgCl2 (3 mM), and GSH (0.5 mM). ABT (50 and 100 µM) was preincubated for 20 min with the microsomes, MgCl2, and NADPH before adding compound I (50 µM) and GSH. KTO (10 µM) was coincubated with the microsomes, compound I (50 µM), and NADPH in the presence of GSH. The volume of incubation was adjusted to 0.2 mL with 0.1 M phosphate buffer (pH 7.4). The incubations were carried out for 30 min at 37 °C. To terminate the reaction, 50 µL of ice-cold acetonitrile containing SPG (the internal standard, 1.0 µg/mL) and acetic acid (0.1%, v/v) was added to the incubation mixtures. The samples were extracted and centrifuged, and aliquots of supernatants were analyzed by liquid chromatography multiple reaction monitoring (LC/MRM) to quantitate SCPG as described below. In ViWo Bioactivation in Rats. Three adult bile duct-cannulated male Sprague–Dawley rats (250–300 g) were obtained from Charles River Laboratories (Wilmington, NC). Upon arrival, animals were acclimated following a 12 h light/dark cycle in a humidity- and temperature-controlled environment for 4 days in accordance with the National Institutes of Health (NIH) publication Guide for the Care and Use of Laboratory Animals. After acclimation, surgically implanted biliary catheters were connected for collection of bile under isoflurane anesthesia, and animals were kept unrestrained in metabolic cages. Control bile and urine samples were collected over ascorbic acid (a few milligrams) overnight. After overnight recovery, the rats were administrated compound I through gavages at a dose of 10 mg/kg. All of procedures in this study has been approved and conducted in compliance with the Animal Welfare Act Regulation (9 CFR, Parts 1, 2, and 3) as well as with all internal company policies and guidelines. Bile and urine samples were collected for 0–4 and 4–24 h in a tube containing ascorbic acid (a few milligrams) over ice. The samples were stored at -80 °C until analyzed. Prior to analyses, bile or urine samples were thawed at room temperature and centrifuged at 1600g for 5 min. Aliquots (10–20 µL) of supernatants were injected directly onto the column for LC/MS analysis. To quantitate SCPG present in bile, aliquots (50 µL) of supernatant were mixed with the solvent [acetonitrile/ 10 mM ammonium acetate at pH 5 (1:1, v/v)] containing SPG (the internal standard, 1.0 µg/mL) to a final volume of 500 µL. Aliquots (10 µL) of the samples were injected directly onto the column for LC/MRM analysis. To detect and quantitate CPA present in rat bile and urine, aliquots (50 µL) of supernatants were mixed with the solvent [acetonitrile/10 mM ammonium acetate at pH 8 (1:1, v/v)] containing IPA (the internal standard, 1.0 µg/mL) to a final volume of 200 µL. Aliquots (10 µL) of the samples were injected directly onto the column for liquid chromatography electrochemistry multiple reaction monitoring (LC/EC/MRM) analyses (see below). Characterization of the P450 Enzyme(s) Responsible for the Metabolic Activation. The P450 enzyme(s) responsible for the activation of compound I to CPIC was investigated by monitoring the formation of SCPG. The production of SCPG was

Chem. Res. Toxicol., Vol. 21, No. 5, 2008 1097 assessed quantitatively in each of following studies. The incubations were conducted in microcentrifuge tubes (1.5 mL) and terminated by the addition of 50 µL of ice-cold acetonitrile containing SPG (1.0 µg/mL) and acetic acid (0.1%, v/v). The samples were centrifuged at 1600g for 3 min. Aliquots (10 µL) of supernatants were analyzed by LC/MRM as described below. Incubations with cDNA-Expressed Rat P450 Enzymes. To screen rat P450 enzyme(s) responsible for the formation of SCPG, incubations (in triplicate) were carried out with 12 commercially available cDNA-expressed rat P450 microsomes. The incubation mixtures consisted of the microsomes containing the P450 (20 pmol), cytochrome b5 (20 pmol), P450 reductase, NADPH (1 mM), compound I (10 µM), GSH (0.5 mM), and MgCl2 (3 mM). The volume of incubation was adjusted to 0.2 mL with 0.1 M phosphate buffer (pH 7.4). The mixtures were incubated for 20 min at 37 °C. Experiments with Chemical Inhibitiors. The incubations, performed in triplicate, consisted of male rat liver microsomal protein (0.4 mg), NADPH (1 mM), GSH (0.5 mM), MgCl2 (3 mM), and various chemical inhibitors. The stock solutions of chemical inhibitors were prepared in Me2SO. The following inhibitors were examined: FAL (20 µM), CMD (50 µM), TAO (50 µM), and KTO (1 µM). Each chemical inhibitor, except KTO, was preincubated for 20 min with the microsomes and NADPH before adding compound I (final concentration of 10 µM). KTO was coincubated with the microsomes, compound I (10 µM), and NADPH in the presence of GSH. The volume of incubation was adjusted to 0.2 mL with 0.1 M phosphate buffer (pH 7.4). The organic concentration in the incubation mixture was below 1% (v/v). The incubations were carried out for 20 min at 37 °C. Experiments with Inhibitory Antibodies. Incubations were performed in triplicate consisting of male rat liver microsomal protein (0.2 mg), PAb against rat P450 (20 µL, 0.2 mg of protein), MgCl2 (3 mM), NADPH (1 mM), and compound I (10 µM) with the final volume adjusted to 0.2 mL using 0.1 M phosphate buffer (pH 7.4). Microsomes were preincubated with individual antibodies for 20 min at room temperature, followed by the addition of other components. The incubations were carried out for 20 min at 37 °C. Enzyme Kinetics Studies of Bioactivation. The enzyme kinetics of metabolic activation of compound I to CPIC that was trapped as a glutathione conjugate (SCPG) in male rat liver microsomes was studied. Incubations conducted in triplicate in microcentrifuge tubes (1.5 mL) consisted of male rat liver microsomal protein (0.2 mg), GSH (0.5 mM), MgCl2 (3 mM), and NADPH (1 mM) in a final volume adjusted to 0.2 mL with 0.1 M phosphate buffer (pH 7.4). The formation of SCPG was determined at substrate concentrations ranging from 1 to 2000 µM. The reaction was carried out for 10 min at 37 °C. The incubations were terminated by the addition of 50 µL of ice-cold acetonitrile containing SPG (1.0 µg/ mL) and acetic acid (0.1%, v/v). The respective microsomal samples were vortexed and centrifuged at 1600g for 3 min. Aliquots (10 µL) of supernatants were injected and analyzed by LC/MRM as described below. The concentrations of SCPG formed in the microsomal incubations were calculated from the standard curve prepared in the range of 10.0–800.0 ng/mL of SCPG in the matrix. Kinetic parameters (Km and Vmax) were obtained by fitting the data to the standard Michaelis–Menten equations using SigmaPlot version 7.0 (SPSS, Inc., Chicago, IL). NMR. All NMR spectra were recorded on a 600 MHz Bruker Avance NMR spectrometer (Bruker, Billerica, MA). Samples were dissolved in deuterated Me2SO, and chemical shifts are expressed as parts per million (δ) downfield from the reference tetramethylsilane. Signal multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiplet). LC/MS. To identify and characterize metabolites present in various biological matrices, a quadrupole-linear ion-trap mass spectrometer API 4000 Q-Trap (PE-Sciex, Toronto, Ontario, Canada) that was coupled to an Agilent 1100 HPLC system was used. The HPLC effluent was introduced into the ion source using a turbo ionspray interface. The mass spectrometer was operated in

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Figure 2. LC/MS detection of the GSH conjugate (tR ) 4.9 min) present in extracts of male rat liver microsomal incubation of compound I: (A) LC/MS total ion current (TIC), (B) extracted ion chromatography of [M + H]+ at m/z 461, and (C) mass spectrum of the GSH conjugate.

the positive-ion mode for LC/MS or LC/MS/MS analysis. The output signal from the mass spectrometer was interfaced to a computer operating Analyst 1.4 software (PE-Sciex) for data collection, peak integration, and analysis. The chromatography was conducted on a Waters Symmetry C18 column (2.1 × 50 mm, 5 µm) by a gradient solvent system consisting of acetonitrile and 10 mM ammonium acetate (pH 5). The percentage of acetonitrile was increased linearly from 10 to 28% over 4 min followed by a linear ramp to 65% in 1 min at a flow rate set at 0.20 mL/min. After 5 min, the column was re-equilibrated with the initial mobile phase for 2 min before the next injection. Quantitation of SCPG was achieved through LC/MRM analyses. The mass transitions monitored during the LC/MRM analysis included m/z 461 f 332 for SCPG and m/z 427 f 298 for SPG, respectively. The limit of detection (LOD, ratio of signal-to-noise ∼3) was 5.0 ng/mL, and the limit of quantitation (LOQ) was determined to be 10.0 ng/mL. Quantitation was conducted using a standard calibration curve of SCPG over a concentration range of 10.0-800.0 ng/mL. A weighted (1/x) linear least-squares regression of SCPG concentrations and measured peak area ratios of SCPG to SPG was used to construct the calibration curve (r2 > 0.99). LC/EC/MRM. To detect CPA in rat bile and urine, an electrochemical system (ESA Biosciences, Chelmsford, MA) coupled to LC/MS was employed. The application of electrochemistry coupled to LC/MS for detection and quantitation of CPA in biological samples has been previously described by us (22). Briefly, the electrochemical system consisted of a GuardStat potentiostat and a model 5021 conditioning cell. The cell contained a coulometric working electrode and a Pd reference electrode. For online coupling of LC/MS to electrochemical oxidation, the conditioning cell was placed between the column and the electrospray ionization (ESI) interface of the LC/MS system described above. The flow from the cell was introduced into the turbo ionspray interface. CPA was electrochemically converted to a dimer derivative, which produced a parent ion ([M + H]+) at m/z 217 with a characteristic one-chlorine M + 2 isotope cluster. The optimized voltage (1.0 V) of the cell was obtained through tuning the voltage versus the intensity of the dimer derivative acquired by LC/MRM analysis via flow inject analysis. Under the same conditions, IPA (the internal

standard) was electrochemically converted to a derivative that produced a parent ion ([M + H]+) at m/z 435. The chromatographic analysis was achieved on a Waters Symmetry C18 column (2.1 × 50 mm, 5 µm). The mobile phase consisted of acetonitrile and 10 mM ammonium acetate (pH 8.0), which was delivered through a gradient program. The percentage of acetonitrile was increased from 25 to 75% (v/v) over 4 min. After 4 min, the percentage of acetonitrile was ramped to 85% in 2 min and then back to 25% in 0.1 min to re-equilibrate for 4 min before the next injection. The flow rate was set at 0.25 mL/min. A quantitative assay of CPA in biological samples was developed through LC/MRM analyses of the electrochemical derivatives of CPA and IPA. The mass transitions monitored included m/z 217 f 182 for CPA and m/z 435 f 308 for IPA, respectively. LOD of CPA was 20.0 ng/mL (signal-to-noise ∼6), and LOQ was determined to be 50.0 ng/mL. Quantitation was carried out using a standard calibration curve of CPA over a concentration range of 50.0-800.0 ng/mL. A weighted (1/x) quadratic regression of CPA concentrations and measured peak area ratios of the CPA derivative to the IPA derivative was used to construct the calibration curve (r2 > 0.99).

Results Bioactivation to CPIC in Rat or Human Liver Microsomes. Incubation of compound I with male rat liver microsomes fortified with GSH in the presence of NADPH resulted in the formation of a GSH conjugate eluting at 4.9 min (Panels A and B of Figure 2). The formation of the GSH conjugate was shown to be microsomal protein- and NADPH-dependent. LC/MS analyses of the GSH conjugate showed a parent ion ([M + H]+) at m/z 461 and demonstrated a characteristic single chlorine M + 2 isotope cluster (Figure 2C). MS/MS analyses exhibited a prominent fragment ion at m/z 332 and a fragment ion at m/z 386 (Figure 3A), a loss of 129 and 75 Da from the parent ion at m/z 461, respectively. This corresponds to the loss of pyroglutamate and glycine, which is typical for GSH conjugates analyzed by ESI/MS (23).

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Figure 3. Tandem mass spectra of GSH conjugate (SCPG, [M + H]+ at m/z 461): (A) MS/MS of [M + H]+ at m/z 461 and (B) MS/MS of fragment ion at m/z 332. The tR and mass spectral characteristics of the GSH conjugate produced in rat liver microsomal incubation were identical to those of an authentic standard. The origins of the characteristic ions are proposed as indicated.

Further MS/MS analyses of the fragment ion at m/z 332 demonstrated a number of characteristic ions (Figure 3B) including those at m/z 314, which corresponds to the loss of a water molecule; m/z 257, which corresponds to the loss of glycine; m/z 205, which corresponds to the breakage of the amide bond; m/z 179, which corresponds to protonated cysteinylglycine because of the breakage of the carbonyl-sulfur bond; m/z 162, which corresponds to the loss of ammonia from the cysteinylglycine; and m/z 128, which corresponds to protonated p-chlorophenyl aniline. The origin of these fragment ions is postulated in Figure 3. The fragmentation patterns of the GSH conjugate were consistent to what was reported for S-(N-4-chlorophenylcarbamoyl)-glutathione [SCPG, (21)]. To further confirm the identity of the GSH conjugate, LC/MS/MS analyses were conducted with the synthetic standard. It was demonstrated that the LC/MS retention time and MS/MS spectral data were identical between the metabolite and synthetic standard (data not shown). The involvement of rat P450 enzymes in the activation of compound I was evaluated using chemical inhibitors of P450 enzymes, such as ABT and KTO. Preincubation of ABT (50 and 100 µM), a nonspecific mechanism-based inactivator of P450s, with rat liver microsomes led to 58 and 84% inhibition of SCPG formation, respectively, compared to the control (data

not shown), indicative of the participation of P450 enzymes. In addition, coincubation of KTO (10 µM), a competitive inhibitor of P450 enzymes, with compound I in rat liver microsomes resulted in 95% inhibition of SCPG formation (data not shown), providing further evidence of P450 enzymes mediating the bioactivation. The potential bioactivation of compound I to CPIC in humans was demonstrated by conducting studies with human liver microsomes. The presence of a GSH adduct, with [M + H]+ at m/z 461, was demonstrated by LC/MS analysis of human liver microsomal incubation extracts. MS/MS analyses of the GSH adduct demonstrated an identical fragmentation pattern that was produced by SCPG present in rat liver microsomes (data not shown). The identity of the GSH conjugate was further confirmed by comparing its HPLC retention time and mass spectral data with those of a synthetic standard (Figure 4). These results suggest the potential formation of CPIC as a reactive intermediate produced during the metabolism of compound I in human liver microsomes. Studies of the Bioactivation Mechanism to CPIC. To investigate the mechanism of activation, a structural analogue with a methyl group at the 3 position of pyrazoline was incubated with rat liver microsomes as performed with compound I. LC/MRM analyses of the incubation extracts showed

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Figure 4. LC/MRM analyses of SCPG produced in human liver microsomal incubation of compound I: (A) incubation extracts and (B) standard SCPG (10.0 ng/mL) spiked in the incubation medium.

Figure 5. Comparison of the SCPG formation in male rat liver microsomal incubations of compound I and its 3-CH3-substituted analogue.

minute levels of SCPG produced; approximately 95% reduction in the SCPG formation as compared to that produced from compound I (Figure 5). In contrast, the substitution of methyl group at the 4 and 5 positions of pyrazoline produced similar

levels of SCPG as compound I, showing that substitutions at these positions did not influence the formation of SCPG (data not shown). In addition, the structural analogue with a CF3 group at the 3 position of pyrazoline was found to be unstable in phosphate buffer (pH 7.4), releasing CPIC that was trapped with GSH to yield SCPG. However, after 30 min in the buffer at 37 °C, the yield of SCPG was still only 20% of the value obtained from rat liver microsomal incubation of compound I in the presence of NADPH at 37 °C for 30 min. The formation of SCPG from the 3-CF3 analogue was not significantly different between incubations conducted in phosphate buffer and in microsomes fortified with cofactors. This suggested the formation of CPIC, and subsequently, SCPG was not metabolism-dependent for the 3-CF3 analogue. However, the effect of an electron-withdrawing group (such as CF3) at the 3 position of pyrazoline reinforces our hypothesis that metabolic intervention at this position predisposes the compound toward forming the isocyanate intermediate. Bioactivation to CPIC in Rats. To verify the in ViVo relevance of bioactivation to CPIC, bile duct-cannulated rats were administered an oral dose (10 mg/kg) of compound I. Bile and urine samples were analyzed by LC/MS to demonstrate the presence of thioether conjugates. As shown in Figure 6, the



Figure 6. LC/MRM analyses of SCPG ([M + H]+ at m/z 461) present in the bile of rats administered compound I (10 mg/kg, po): (A) pre-dose bile, (B) post-dose bile at 0–4 h, and (C) pre-dose bile spiked with synthetic SCPG at 10.0 ng/mL.

upper chromatogram A represents the LC/MRM chromatogram of the predose rat bile. Chromatograms B and C represent bile collected after dosing with compound I and predose bile spiked with synthetic standard SCPG (10.0 ng/mL), respectively. These results clearly demonstrated the biliary excretion of SCPG by rats administered compound I. The corresponding N-acetyl cysteine conjugate (SCPAC) of CPIC was detected in urine of rats dosed with compound I as demonstrated by LC/MRM analyses. The LC/MRM chromatogram of predose rat urine is shown in Figure 7A. Chromatograms B and C (Figure 7) represent urine collected after dosing with compound I and predose urine spiked with synthetic standard SCPAC (10.0 ng/ mL), respectively. These results unequivocally demonstrate that the GSH and N-acetyl-cysteine conjugates of the proposed isocyanate, CPIC, were excreted into bile and urine, respectively, after administration of compound I to rats.

To detect CPA, a hydrolytic product of CPIC, a novel hyphenated LC/EC/MS system (22) was used to analyze rat bile and urine. Under electrochemical treatment, CPA undergoes oxidation to afford a dimer derivative (22) with an increased molecular weight and enhanced ionization potential, which can be easily detected by ESI/MS. As shown in Figure 8, the upper chromatogram A represents the LC/MRM chromatogram of predose rat bile. Chromatograms B and C represent bile collected after dosing with compound I and pre-dose bile spiked with standard CPA (50.0 ng/mL), respectively. These chromatograms reveal a component present in bile that corresponds to CPA, as indicated by the peak at a retention time of 3.4 min. The urine collected prior to and post-dosing was also analyzed by the same LC/EC/MRM technique, and the presence of CPA in urine after dosing was also clearly demonstrated (data not shown). The

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Figure 7. LC/MRM analyses of N-acetyl cysteine conjugate (SCPAC, [M + H]+ at m/z 317) present in the urine of rats administered compound I: (A) pre-dose urine, (B) post-dose urine at 0–4 h, and (C) pre-dose urine spiked with synthetic SCPAC at 10.0 ng/mL.

presence of CPA in rat bile and urine after administration of compound I provided further evidence for the existence of CPIC. Quantitation of SCPG and CPA. Quantitation of SCPG present in the bile of rats dosed with compound I was performed by LC/MRM analyses. By this approach, it was found that SCPG accounted for only a very small fraction of the dose (0.2%, Table 1) excreted in bile over 24 h. A validated assay of LC/EC/MRM (22) was used to quantitate CPA present in the bile and urine of rats dosed with compound I. It was found that a relatively high percentage of compound I was converted to CPA. As shown in Table 1, the fraction of the dose accounted for CPA in bile and urine over 24 h is 3.3 and 2.7%, respectively. Overall, the total percentage of the dose of compound I accounted for by the products of CPIC, including the GSH conjugate and CPA excreted in bile and urine, is approximately 7% (Table 1). Characterization of P450 Enzyme(s) Responsible for the Activation. Studies conducted using 12 cDNA-expressed rat P450 enzymes demonstrated a marked difference in the formation of SCPG by various enzymes (0.1 µmol of enzyme/

mL incubation). It was found that the highest yield of SCPG was obtained with P450 3A1 (Figure 9). Other enzymes, including P450 1A2, P450 2C11, and P450 3A2, also produced SCPG at much lower levels as compared to those of P450 3A1. Preincubation with FAL (20 µM, mechanism-based inactivator of P450 1A2), CMD [50 µM, mechanism-based inactivator of P450 2C11, (24)], and TAO (50 µM, mechanism-based inactivator of P450 3A) exhibited 29, 21, and 60% inhibition on the formation of SCPG, respectively (Table 2). Coincubation with KTO (1 µM) led to 45% inhibition of SCPG formation (Table 2). These results suggest that multiple rat P450 enzymes, including P450 1A2, P450 2C11, and P450 3A, were involved in the activation of compound I to CPIC. Furthermore, studies conducted with specific anti-rat P450 antibodies provided further evidence for the role of P450 enzyme(s) in forming SCPG (Table 2). The production of SCPG was reduced by approximately 90% (as compared to the preimmune IgG control) when the microsomes were treated with the antibody against rat P450 3A2. Microsomes treated with the antibody against rat P450 1A2 enzymes produced 30% less SCPG as compared



Figure 8. LC/EC/MRM analyses of CPA present in the bile of rats administered compound I (10 mg/kg, po): (A) pre-dose bile, (B) post-dose bile at 0–4 h, and (C) pre-dose bile spiked with standard CPA at 50.0 ng/mL.

Table 1. Percentage of Dose Estimated for SCPG and CPA Excreted in Bile and Urine of Three Rats Administered Compound I (10 mg/kg, po) products of CPIC (0–24 h) mean ((SD)

SCPG in bile

CPA in bile

CPA in urine

total percentage of dose

0.2 ((0.1)

3.3 ((2.2)

2.7 ((0.9)

6.7 ((1.2)

to the control. In contrast, the microsomes treated with the antibody against rat P450 2B1/2 showed little to no effect on the formation of SCPG. Collectively, these results indicate that several rat cytochrome P450 enzymes, including P450 1A2, P450 2C11, and P450 3A, are responsible for mediating the activation of compound I to CPIC. Particularly, rat P450 3A enzymes, constitutively expressed in male rat liver microsomes (25, 26), appeared to be the primary enzymes responsible for bioactivating compound I to CPIC. Enzyme Kinetics of SCPG Formation. The kinetics of SCPG formation from compound I was studied in male rat liver microsmes. As shown in Figure 10, the substrate-velocity curve of SCPG formation is hyperbolic when the data (rates of SCPG formation versus compound I concentrations) were fitted to a single-enzyme Michaelis–Menten equation. The Eadie-Hofstee

Figure 9. Incubations of compound I with cDNA-expressed rat P450 enzymes for SCPG formation (ctl ) control).

plot exhibits a biphasic profile (inset in Figure 10), indicating apparent multienzyme kinetics. As a result, the kinetic parameters of SCPG formation were obtained by fitting the data to a dual-enzyme Michaelis–Menten model (26). The apparent Km and Vmax of one enzyme were estimated to be 42 µM and 66 pmol min-1 mg-1, respectively. For the other enzyme, the apparent Km and Vmax were 148 µM and 1200 pmol min-1 mg-1, respectively. As a result, the turnover number (Vmax/Km) in male rat liver microsomes was 1.6 and 8.1 µL min-1 mg-1

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Table 2. Inhibition of SCPG Formation in Male Rat Liver Microsomes in the Presence of Chemical Inhibitors or Polyclonal Antibodies against Rat P450 Enzymes inhibitor

SCPG formationa

20 µM FAL 50 µM CMD 50 µM TAO 1 µM KTO PAb to P450 1A1/2 PAb to P450 2B1/2 PAb to P450 3A2

71 ((5) 79 ((9) 40 ((6) 55 ((9) 70 ((4) 107 ((9) 10 ((3)

a Values are the mean ((SD) of triplicates and are expressed as a percentage relative to the control without the chemical inhibitor or antibody.

Figure 10. Substrate-velocity curve of SCPG formation from compound I in male rat liver microsomes.

for these two corresponding enzymes, respectively. The activation efficiency of the enzyme with the larger Km is approximately 5-fold greater than that of the enzyme with a small Km.

Discussion Chemically reactive metabolites have been implicated in the toxicity for a number of drugs. Hence, the formation of reactive metabolic intermediates through biotransformation of new chemical entities may represent a potential liability. To evaluate potential activation to reactive metabolites, the current approach taken by the pharmaceutical companies (15–19) is to study lead compounds or drug candidates in animal and human liver microsomes fortified with GSH. Reactive metabolites capable of covalently binding to GSH can be trapped as GSH conjugate(s), whose structure is subsequently characterized to elucidate the nature of reactive metabolite(s). The establishment of identity of reactive metabolite(s) helps understand the chemical mechanism of bioactivation and provides guidance for structural modification to minimize the potential liability of new chemical entities. Compound I (Figure 1) was demonstrated to undergo the metabolic activation in Vitro in rat and human liver microsomes and in ViVo in rats. The GSH adduct produced from in Vitro and in ViVo studies was exhibited to be identical. Evidence obtained from these studies suggested the potential formation of CPIC as the reactive intermediate during the metabolism of compound I. The intermediate CPIC is known to be capable of covalently binding to macromolecules. Although protein covalently binding of compound I remains unknown, because of lack of a radiolabeled material, quantitative assessment of CPIC

Chen et al.

formation by measuring its GSH adduct was conducted. The result showed a fraction less than 0.2% of the dose given to the rats was excreted into bile over a 0–24 h period in the form of SCPG (Table 1). As described previously (21), the GSH conjugate of CPIC is not stable and CPIC can be released reversibly in a basic aqueous environment, such as the bile duct, a known chemical feature of carbamoylating species (28–30). Therefore, GSH conjugation of CPIC does not necessarily result in the detoxification of the reactive metabolite. In contrast, GSH may serve as a biological vehicle that transports the reactive metabolite from its primary site of formation to release at distant locations. In this regard, GSH conjugation may function to extend the effective biological lifetime of CPIC, as opposed to its more traditional role as a detoxification mechanism for electrophilic intermediates. Thus, the inherent instability or reversibility of CPIC S-linked conjugate indicates that GSH conjugation could have significant toxicological implications. It is known that aryl isocyanates undergo hydrolysis to produce aryl amines (31). Hence, the product of CPIC hydrolysis, CPA, which is an established rodent carcinogen (32), may serve as a useful marker of metabolic activation of new chemical entities leading to chlorophenyl isocyanate. Nevertheless, this small polar molecule is not detectable by the widely used LC/ MS systems equipped with electrospray ionization or atmospheric pressure chemical ionization capabilities, perhaps because of its unfavorable ionization potential. In a previous study (21), the formation of CPIC was indirectly demonstrated in ViVo in rats by LC/MS characterization of the thioether conjugates in bile and urine samples, but the presence of CPA was not reported. Consequently, efforts were made to develop an analytical method that allowed us to detect and quantitate CPA present in biological samples. As described before, a novel hyphenated analytical method based on the combination of electrochemical oxidation and ESI-MS/MS was developed for analysis of CPA (22). This took advantage of simplicity of online electrochemical derivatization of CPA with high sensitivity and specificity of MS/MS detection. With this sensitive, specific, and robust analytical technique, the presence of CPA in bile and urine of rats dosed with compound I was clearly demonstrated (Figure 8). It was found that a much higher fraction of dose (17-fold) was accounted for CPA excreted into bile in a period of 24 h compared to that of SCPG in bile (Table 1). The differences in quantities of products derived from CPIC were likely due to the instability or degradation of SCPG as well as the irreversible hydrolytic reaction to CPA. As demonstrated here, the chemical scaffold, dihydropyrazole1-carboxylic acid-(4-chlorophenyl)amide, is metabolically labile and apparently responsible for the generation of CPIC through P450-mediated biotransformation. As proposed in Scheme 1, the mechanism of bioactivation is postulated to involve an initial P450-mediated hydroxylation of the 3 position of pyrazoline, which leads to a 3-oxo derivative. The introduction of an electron-withdrawing amide-type group may destabilize the molecule in a manner that could involve deprotonation of urea linkage, which may undergo a general base-catalyzed process and result in decomposition to CPIC. The resultant CPIC reacts with GSH to form SCPG or with water to afford CPA. The prerequisite metabolism at the 3 position of compound I for the bioactivation to CPIC was demonstrated by conducting studies with substituted analogues. The replacement of 3-hydrogen with a methyl group led to a blockage of SCPG formation (Figure 5). The trace quantities of SCPG formed from the methyl-substituted analogue was likely due to the presence of residual CPIC, which was used as a synthetic agent. On the



Scheme 1. Proposed P450-Mediated Metabolic Activation of Compound I to CPIC (B ) General Base without a Known Identity)

other hand, substitution with a methyl group at the 4 and 5 position, respectively, did not affect the production of SCPG. The destabilizing effects associated with an electron-withdrawing group at the 3 position of pyrazoline were exemplified by a structural analogue that contained a CF3 group at the 3 position, from which fair quantities of SCPG were formed when the analogue was present in the buffer (pH 7.4). Similarly, as reported in the literature, hypoglycemic agents, such as chlorpropamide that contains arylsulfonyl ureas, were not stable to liberate the corresponding n-propyl isocyanate in aqueous solutions at physiological pH once the N1-sulfonamide nitrogen was acetylated (33). This observation appears to be in line with our results from the 3-CF3-substituted analogue that the introduction of an electron-withdrawing group at the 3 position of pyrazoline compounds may result in decomposition of the urea bond. Efforts were made to detect and identify the fate of the postulated leaving moiety, 3-oxo-pyrazolidine-5-carboxylic acid-(2-oxo-2H-[1,3′]-bipyridinyl-6′-yl)-amide, with no success. Additional studies employing kinetic isotope effects (replacement of 3-hydrogen with a deuterium in compound I) and methyl substitution of the urea bond of the 3-CF3-containing analogue are warranted to further elucidate the mechanism of bioactivation of the chemical scaffold, dihydropyrazole-1carboxylic acid-(4-chlorophenyl)amide, to CPIC. It is well-established that cytochrome P450 enzymes are involved in the biotransformation of both xenobiotics and endobiotic compounds, often leading to the bioactivation of certain promutagens and procarcinogens. Consequently, one of the goals of our study was to assess and characterize the P450 enzyme(s) responsible for the bioactivation of compound I. The

results from in Vitro studies revealed the involvement of multiple rat cytochrome P450 enzymes, including P450 1A2, P450 2C11, and P450 3A, in the activation of compound I (Figure 9 and Table 2). Particularly, P450 3A1 and P450 3A2, the constitutive P450 enzymes present in male rat liver microsomes, appeared to be principle enzymes responsible for catalyzing this activation. The finding that more than one P450 enzyme participated the activation of compound I was further demonstrated by kinetics studies of activation of compound I in male rat liver microsomes. A biphasic profile was exhibited by the Eadie-Hofstee plot (inset in Figure 10) of the data (rates of SCPG formation versus compound I concentrations), indicating two P450 components kinetically contributing to the reaction. At low substrate concentrations, the activation of compound I appeared to be mediated mainly by the enzyme with the small Km and low Vmax. The relative contribution of the other enzyme with the larger Km and higher Vmax in producing CPIC increased rapidly with an increasing substrate concentration. Both P450 enzymes exhibited Michaelis–Menten characteristics for the activation process, indicating the absence of a potential mechanism-based inactivation of enzyme(s), although a reactive intermediate CPIC was generated during the metabolism. The identities of these P450 enzymes exhibiting different catalytic efficiencies toward the activation of compound I remain to be established. In conclusion, the data presented here demonstrates the bioactivation of a novel chemical scaffold, dihydropyrazole-1carboxylic acid-(4-chlorophenyl amide), of compound I. This bioactivation pathway, which was demonstrated to be P450dependent in Vitro, may lead to a reactive CPIC species which,

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in turn, was trapped as a GSH adduct. In addition, the presence of CPA, a hydrolytic product of CPIC, was demonstrated in rat bile and urine. The formation of both SCPG and CPA has toxicological significance. The thioether conjugates of CPIC, including GSH, cysteine, and N-acetyl-cysteine derivatives, are susceptible to a thiol-exchange reaction, raising the possibility that these molecules may react with macromolecules, as demonstrated by other carbamate thioester derivatives (34, 35). The production of CPA, an established rodent carcinogen, is definitely an undesirable metabolic end product of new chemical entities. The work presented here represents an example of an ongoing effort between medicinal chemists and metabolism scientists in finding potentially safer and better therapeutic agents. Identification of bioactivating pathways and/or products often provides chemists with options to modify or select chemical templates to mitigate reactive metabolites. In this case, minimizing the bioactivation through a rational structural modification strategy to block the CPIC formation was attempted with success. The discovery team ultimately chose to steer away from the potentially problematic chemical scaffold because the pharmacological activity of these structurally modified analogues was compromised. Acknowledgment. The authors thank Mr. Greg Walker for providing NMR characterization of synthetic thioether conjugates of CPIC.

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