Metabolism-Dependent Mutagenicity of a Compound Containing a

a possible link between metabolism and mutagenicity, a structural analogue 1b ... to 1a, 1b did not undergo the metabolic cleavage (loss of indazole r...
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Chem. Res. Toxicol. 2006, 19, 1341-1350

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Metabolism-Dependent Mutagenicity of a Compound Containing a Piperazinyl Indazole Motif: Role of a Novel P450-Mediated Metabolic Reaction Involving a Putative Oxaziridine Intermediate† Hao Chen,*,‡ Joel Murray,§ Brian Kornberg,| Lloyd Dethloff,§ David Rock,⊥ Sham Nikam,| and Abdul E. Mutlib‡ Departments of Pharmacokinetics, Dynamics and Metabolism, World Wide Safety Sciences, Medicinal Chemistry, and Pharmacology, Pfizer Global Research & DeVelopment, 2800 Plymouth Road, Ann Arbor, Michigan 48105 ReceiVed December 20, 2005

Compound 1a (6-chloro-5-{3-[4-(1H-indazol-3-yl)-piperazin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydroindol-2-one) was mutagenic to Salmonella typhimurium TA98 in the presence of rat liver S9 subcellular fraction. The metabolism of 1a in rat liver S9 or microsomes demonstrated that it underwent a P450mediated N-deindazolation (loss of indazole ring) as a predominant metabolic pathway. To investigate a possible link between metabolism and mutagenicity, a structural analogue 1b (6-chloro-5-{3-[4-(1Hindazol-3-yl)-piperidin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydro-indol-2-one), the cleaved product 2a (6chloro-3,3-dimethyl-5-(3-piperazin-1-yl-propyl)-1,3-dihydro-indol-2-one), and the core motif 3a (3piperazinyl indazole) were evaluated in the Ames assay. It was found that 1b was not mutagenic to Salmonella typhimurium TA98 in the absence or presence of a metabolic activating system. In contrast to 1a, 1b did not undergo the metabolic cleavage (loss of indazole ring). Marginal mutagenicity of 2a to TA98 was observed with rat liver S9, whereas 3a was shown to be a promutagen. It was further demonstrated that 1a inactivated P450 3A, the principle enzyme catalyzing the N-deindazolation reaction, in an NADPH-, time-, and concentration-dependent manner. The kinetics of inactivation was characterized by a KI of 8.1 µM and kinact of 0.114 min-1. The differences in mutagenicity between 1a and 1b suggest that a chemical bond extending from the 3-position of the indazole to a heteroatom (as part of another cyclic ring) is a prerequisite for the toxicity. The metabolic process leading to the elimination of the indazole from the rest of the molecule apparently plays a key role in causing mutagenicity. It is postulated that the N-deindazolation of 1a proceeds Via an oxaziridine intermediate, the formation of which is indirectly inferred from the presence of benzoic acid in microsomal incubations. Benzoic acid is thought to be derived from the hydrolysis of 3-indazolone, an unstable product generated from the oxaziridine. Evidence suggests that the electrophilic oxaziridine intermediate may be responsible for the mutagenicity and inactivation of P450 3A. Introduction The Salmonella reverse mutation test (1, 2) has been used for several decades as a useful screening tool to detect potentially mutagenic chemicals, including new chemical entities (NCE) discovered in early stages of drug discovery and development. The mutagenic potential of an NCE is generally evaluated in genetically different strains of the Salmonella typhimurium, such as TA98, TA100, TA1535, and TA1537. These test strains all carry some type of defective (mutant) gene that prevents them from synthesizing the amino acid histidine in a minimal bacterial culture medium. In the presence of mutagenic chemicals, the defective gene may be mutated back to the functional state that allows the bacterium to grow in the medium. It has been reported that 1, 2-indazoles are nonmutagenic in the absence or presence of metabolic activation (3). However, † An abstract was published in Drug Metabolism ReView 2004, 36, 197. Part of this work was presented at the seventh International Meeting of the International Society for the Study of Xenobiotics (ISSX), Vancouver, Canada, 2004. * Corresponding author. Tel: (734) 622-4517. Fax: (734) 622-5115. E-mail: [email protected]. ‡ Department of Pharmacokinetics, Dynamics and Metabolism. § Department of World Wide Safety Sciences. | Department of Medicinal Chemistry. ⊥ Department of Pharmacology.

the mutagenic potential of 1,2-indazoles that are linked to other chemical functional groups such as piperazine remains unknown. A number of NCEs that possesses 3-piperazinyl indazole as the core structure were screened for potential mutagenicity to Salmonella typhimurium tester strain TA98 and TA100 in the absence or presence of metabolic activation. The results demonstrated that these compounds were mutagenic to Salmonella typhimurium TA98 but not to TA100 when the assay was carried out in the presence of rat liver S9 homogenate. Mutagenicity to TA98 and TA100 was not observed in the absence of an activating system. Interestingly, the metabolism of these compounds in rat liver S9 or microsomes exhibited a previously undescribed metabolic pathway (4), involving the cleavage of the indazole moiety from the rest of the molecule to produce an N-deindazolated metabolite (Scheme 1). The metabolism of compound 1a (Figure 1), which was chosen as a model compound to investigate the relationship between metabolism and mutagenicity, showed that the metabolic cleavage was a predominant pathway to produce the N-deindazolated metabolite 2a (Figure 1) in rat liver S9 or microsomes. The metabolic pathway leading to the cleaved product 2a was shown to be NADPH- and liver S9/microsomesdependent, indicating the possible involvement of cytochrome P450 enzymes. However, the structurally related heterocyclic

10.1021/tx050354+ CCC: $33.50 © 2006 American Chemical Society Published on Web 09/20/2006

1342 Chem. Res. Toxicol., Vol. 19, No. 10, 2006 Scheme 1. N-deindazolation of Compounds Containing 3-Piperazinyl Indazole

analogues possessing piperazinyl isothiazole (1c, Figure 1) and piperazinyl isoxazole (1d, Figure 1) were not mutagenic to TA98 and TA100 in the absence or presence of metabolic activation. It was subsequently shown that the major route of in vitro metabolism for 1c and 1d was S-oxidation and hydroxylation,1 respectively. Furthermore, the metabolic cleavage of 1c or 1d, resulting in metabolite analogues of 2a, was not demonstrated in the microsomal incubations. These apparent differences in the metabolism of these heterocyclic analogues (indazole, isothiazole, and isoxazole) suggested a possible link between the metabolism and mutagenicity of compounds carrying 3-piperazinyl indazole group. Once it was established that 1a was mutagenic in the presence of metabolizing enzymes, further studies were designed and conducted to establish a possible link between the unique N-deindazolation metabolic pathway and toxicity. It was hypothesized that during the P450-mediated cleavage reaction (loss of indazole) of 1a, a reactive intermediate or mutagenic metabolite was formed that contributed to the mutagenicity. It was postulated that an oxaziridine intermediate, which is an oxidant and a hard electrophile capable of oxidizing or covalently binding to DNA molecules, was formed prior to metabolic cleavage leading to the loss of indazole. The replacement of piperazine with piperidine would not result in this apparent displacement of indazole as a result of potential oxaziridine formation. Even though an oxaziridine could be formed, the overall reaction would not proceed favorably because piperidine, in contrast to piperazine, is not a good leaving group. Consequently, 1b (Figure 1) was considered as a good probe to test the possible link between metabolism and mutagenicity. The objectives of the current study were to (i) evaluate 1b, 2a, and 3a for mutagenicity to Salmonella typhimurium TA98 and TA100 in the absence or presence of metabolic activation; (ii) identify specific P450 isoforms responsible for the N-deindazolation of 1a; (iii) conduct enzyme kinetics study of the N-deindazolation of 1a using male rat liver microsomes or specific cDNA-expressed rat P450 enzyme(s); and (iv) investigate the potential of 1a to act as a mechanismbased inhibitor of the P450 involved in the N-deindazolation reaction.

Materials and Methods Chemicals and Reagents. All test compounds (Figure 1) were synthesized by the Pfizer Medicinal Chemistry department (Ann Arbor, MI) and fully spectroscopically characterized. The purity of each compound was >95% on the basis of HPLC analysis. The chemicals, including NADPH, N-benzylimidazole, 1-aminobenzotrizole (ABT), furafylline, cimetidine, troleandomycin (TAO), 6βhydroxytestosterone (6β-OHT), testosterone (TST), cortexolone (CTXL), 3-indazolinone, and benzoic acid (BA), were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Symmetry C18 columns (2.1 × 150 mm and 2.1 × 50 mm, 5 µm) were obtained from Waters Corporation (Milford, MA). The Luna C18 column (3.0 × 150 mm, 5 µm) and the Luna CN column (4.6 × 150 mm, 5 µm) were purchased from Phenomenex (Torrence, CA). All solvents and reagents were of the highest grade commercially available. Salmonella typhimurium test strains, TA98 and TA100, 1

Unpublished results.

Chen et al. were obtained from Dr. Bruce Ames (Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA) and processed for mutagenicity assay by the Pfizer World Wide Safety Sciences department (Ann Arbor, MI). Male SpragueDawley rat liver S9 (pool of 118 animals) and microsomes (pool of 162 animals), and cDNA-expressed rat P450 microsomes were obtained from BD Sciences (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, 2C13, 2D1, 3A1, and 3A2. Preimmune IgG and poly clonal antibody (PAb) against the respective rat P450 1A1/2, 2B1/2, and 3A2 were also obtained from BD Sciences. Mutagenicity Assay. Compounds were tested for mutagenicity using Salmonella typhimurium tester strain TA98 and TA100 in the absence or presence of an Aroclor-induced male SpragueDawley rat liver S9 fraction. The assay was run in six-well plates. The test compound was dissolved in Me2SO at 50 mg/mL, and each test compound was tested at concentrations of 60, 120, 241, 481, and 962 µg/plate. Two replicates (per chemical) were tested with both tester strains at each respective concentration. Duplicate solvent (Me2SO) was included for each Salmonella typhimurium tester strain as the vehicle control. In the absence of metabolic activation, 2-nitrofluorene (0.4 µg/well) for TA98 and sodium azide (1.9 µg/well) for TA100 were used as the positive controls. In the presence of metabolic activation, 2-aminoanthracene (0.8 µg/well) for TA98 and TA100 was used as the positive control. Assays were carried out in the absence or presence of rat liver S9 subcellular fraction. Mutagenicity was assessed by comparing the number of experimentally induced revertants to the number of spontaneous revertants in the vehicle control. For each tester strain, the mean number of revertants for the two replicates was calculated at each concentration in the absence or presence of the exogenous S9 metabolism system. The main criteria used for classifying a chemical as positive (i.e., mutagenic) was that the mean number of revertants in any strain at any exposure concentration had to be at least two times greater than the mean of the number of revertants in the concurrent negative control (vehicle). An additional criteria was the presence of a concentration-related increase in the mean revertants per plate in that same strain. The use of these classification criteria was based on generally accepted methods (5-8). Metabolism in Vitro in Rat Liver S9 Fractions and Microsomes. Incubations with rat liver S9 or microsomal subcellular fractions were carried out using the following protocol: rat liver S9 protein (2 mg) or microsomes (1 mg), ( NADPH (2 mM), ( GSH (5 mM), MgCl2 (3 mM), and test compound (5 and 50 µM) with the final volume adjusted to 0.5 mL with 0.1 M phosphate buffer (pH 7.4). The reaction was performed for 20 min at 37 °C and terminated by the addition of 1 mL of ice-cold acetonitrile. The samples were vortexed and centrifuged at 1600g for 5 min. The supernatant was separated, transferred to clean glass tubes, and dried under nitrogen. The dried extracts were reconstituted in 200 µL of the HPLC mobile phase and aliquots analyzed by LC/MS operated in a full scan and tandem mass spectrometry (MS/MS) modes. The identity of metabolites was confirmed by comparing the LC/MS retention times and MS/MS data with the synthetic standards. Characterization of the P450 Responsible for the Formation of 2a from 1a. The formation of 2a was studied by conducting incubations with commercially available cDNA-expressed rat P450 enzymes, P450-selective chemical inhibitors, and poly clonal antibodies against the rat P450 enzymes (see below). The enzymatic reactions described below were terminated by the addition of 50 µL of ice-cold acetonitrile containing 2 µg/mL of the internal standard (2b, Figure 1). The samples were vortexed and centrifuged at 1600g for 5 min. The supernatant was separated and aliquots analyzed by LC/MS operated in the multiple reaction monitoring (LC/MS-MRM) mode to quantitate 2a. 1. Incubations with cDNA-Expressed Rat P450 Enzymes. To screen for the rat P450 enzyme(s) responsible for the formation of 2a, incubations (in triplicate) were carried out with 10 commercially available cDNA-expressed rat P450 microsomes. The incubation

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Figure 1. Chemical structures of compounds.

mixtures consisted of the microsomes containing the P450 (10 pmol). cytochrome b5 (10 pmol) and P450 reductase, NADPH (0.5 mM), 1a (5 µM), 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 10 min at 37 °C. 2. Studies with Chemical Inhibitiors. The incubations, done in triplicate, consisted of male rat liver microsomal protein (0.2 mg), NADPH (0.5 mM), MgCl2 (3 mM), and various chemical inhibitors. The stock solutions of chemical inhibitors were prepared in Me2SO. The organic concentration in the incubation mixture was below 1%. The following inhibitors were examined: N-benzylimidazole (100 µM), ABT (100 µM), furafylline (20 and 50 µM), cimetidine (50 and 100 µM), and TAO (50 and 100 µM). Each chemical inhibitor, except for N-benzylimidazole, was preincubated for 15 min with the microsomes and NADPH before adding 1a (final concentration of 5 µM). 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 10 min at 37 °C. 3. Studies with Inhibitory Antibodies. Incubations were performed in triplicate consisting of male rat liver microsomal protein (0.2 mg), PAb to rat P450 (20 µL, 0.2 mg protein), MgCl2 (3 mM), NADPH (1 mM), and 1a (5 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 30 min at room temperature, followed by the addition of other components. The incubations were carried out for 10 min at 37 °C. Enzymes Kinetics of 2a Formation. The kinetics of 2a formation from compound 1a in male rat liver microsomes and cDNA-expressed rat P450 3A2 were determined separately. The consumption of substrate at all concentrations was kept at less than 10%. It was determined that the production of 2a was linear up to 10 min for both liver microsomes and cDNA-expressed rat P450 3A2. The formation of 2a was linear with microsomal protein concentration up to 2.5 mg/mL and with less than 100 pmol/mL of cDNA-expressed rat P450 3A2. The formation of 2a in liver microsomes was determined using substrate concentrations ranging from 0.10 to 500 µM. Incubations (in triplicate) were performed using the following protocol: 0.4 mg of male rat liver microsomal protein, 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 reaction was carried out for 10 min at 37 °C and terminated by the addition of 50 µL ice-cold acetonitrile containing 2b (2 µg/mL). The formation of 2a in cDNA-expressed rat P450 3A2 was determined at substrate concentrations ranging from 0.05 to 500 µM. Incubations (in triplicate) were performed using the following protocol: P450 3A2 (10 pmol), 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 reaction was carried out for 7 min at 37 °C and terminated by the addition of 50 µL of ice-cold acetonitrile containing 2b (2 µg/mL). After terminating the reaction, the incubation mixtures were vortexed and centrifuged at 1600g for 5 min. The supernatant was removed and aliquots analyzed by LC/ MS-MRM. The concentration of 2a produced at each substrate level was calculated from the calibration curve of 2a prepared in the range of 0.01-0.80 µg/mL. Kinetic Data Analysis. The data (rate of 2a formation vs 1a concentrations) obtained from either rat liver microsomes or P450 3A2 were initially fitted to a typical Michaelis-Menten equation

V ) (Vmax‚S)/(Km + S) (eq 1), where V and Vmax are the observed and maximal rates of 2a formation, respectively, S is the concentration of substrate, and Km is the concentration of 1a at half Vmax. Subsequently, non-Michaelis-Menten kinetics such as substrate inhibition was tested to fit data using the following equation V ) Vmax/(1 + Km/S + S/Ki) (eq 2) (9, 10), where Ki represents the dissociation constant for the substrate molecule binding to the inhibitory site of the enzyme that results in a reduction of the rate. All data fitting for nonlinear regression analyses was performed using the Enzyme Kinetics Module of SigmaPlot 7.0 (SPSS, Inc., Chicago, IL). Inactivation of P450 3A by 1a. The ability of 1a to act as an inactivator of P450 3A was assessed in cDNA-expressed rat P450 3A2. A primary incubation, consisting of the test compounds 1a, 1b, 1c, 1d, and 3a (20 µM), NADPH (1 mM), and the P450 3A2 (0.25 µM), was conducted at 37 °C for 10 min in a total volume of 20 µL. TAO (20 µM) was used as a positive control to inactivate P450 3A. Control incubations were also performed by omitting NADPH. Subsequently, the primary incubation mixture was diluted 25-fold with 0.1 M phosphate buffer (pH 7.4) containing TST and NADPH, to give final concentrations of 50 µM and 0.5 mM, respectively. Further incubation of this diluted mixture was conducted for 10 min at 37 °C and stopped by the addition of icecold acetonitrile containing the internal standard (CTXL, 0.25 µg/ mL). In addition, the inclusion of GSH (5 mM) or TST (200 µM) in the incubation mixtures was performed to show any inhibition of inactivation of P450 3A. The formation of 6β-OHT, as a marker of P450 3A activity, was determined and quantified using LC/MSMRM assay. To study the kinetics of P450 3A inactivation, 1a was tested at a range of concentrations (0-20 µM) in the primary incubations with cNDA-expressed rat P450 3A2 (250 nM) as described above. At intervals of 0, 2, 5, 8, 12, and 18 min, the primary incubation was then diluted and assayed for P450 3A activity as described above. LC/MS for Metabolite Identification and Quantitation. Identification of metabolites was achieved by LC/MS analyses of samples using a LCQ Deca XP+ quadrupole ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) that was coupled to an Agilent HP1100 HPLC system (Agilent Technologies, Palo Alto, CA). The HPLC effluent was introduced into the source using a positive ion electrospray interface (ESI). The mass spectrometer was operated in the positive ion mode to achieve optimum sensitivity for metabolites. The output signal from the mass spectrometer was interfaced to the computer operating Xcalibur v.1.3 (ThermoFinnigan, San Jose, CA) for data collection, peak area integration, and analysis. The metabolites were separated on a Waters Symmetry C18 column (2.1 × 150 mm, 5 µm) by a gradient solvent system consisting of acetonitrile and 10 mM ammonium acetate (pH 5). The percentage of acetonitrile was increased from 10 to 60% over 14 min with the solvent flow rate set at 0.4 mL/min. After 14 min, the percentage of acetonitrile was increased to 80% within 2 min before re-equilibrating with the initial mobile phase. Aliquots (10-40 µL) of the reconstituted samples were directly injected onto the column. To quantitate 2a present in the incubation mixtures, the mass spectrometer was operated in the MRM mode with a dwell time of 200 ms. The mass transitions monitored during the LC/MS-MRM

1344 Chem. Res. Toxicol., Vol. 19, No. 10, 2006 analysis included m/z 322 f 208 for 2a and m/z 336 f 250 for 2b. For quantitation purposes, 2a and 2b were eluted on a Waters Symmetry C18 column (2.1 × 50 mm, 5 µm) by a gradient solvent system consisting of a mixture of acetonitrile and 10 mM ammonium acetate (pH 5). The percentage of organic was increased linearly from 10 to 50% over 6 min. The column was brought back to the initial conditions within the next minute and equilibrated for 4 min before the next injection. The solvent flow rate was set at 0.4 mL/min. Quantitation was carried out using a standard calibration curve of 2a over a concentration range of 0.01 to 0.80 µg/mL. A weighted (1/x) linear least-squares regression of 2a concentrations and measured peak area ratios of 2a to 2b was used to construct the calibration curve. LC/MS for Detection of BA and Quantitation of 6β-OHT. The LC/MS system consisted of a triple-stage quadrupole mass spectrometer API 4000 Q-Trap (PE-Sciex, Toronto, Ontario) and a LC-10 AD HPLC system (Shimadzu, Columbia, MD). The mass spectrometer was interfaced to a computer operating Analyst 1.4 software (PE-Sciex, Toronto, Ontario) for data collection, peak integration, and analysis. To detect BA, the atmospheric pressure chemical ionization (APCI) interface was utilized, and the mass spectrometer was operated in the negative ion mode (11). The mass spectrometer was operated in the MRM mode to achieve specificity and maximum sensitivity for BA. The mass transition m/z 121 f 77 was used to detect BA. The chromatographic analysis was achieved on a Phenomenex Luna C18 column (3.0 × 150 mm, 5 µm). The mobile phase consisted of acetonitrile and 10 mM ammonium acetate (pH 5), which was delivered using a gradient program. The percentage of acetonitrile was increased from 10 to 35% (v/v) over 9 min. After 9 min, the percentage of acetonitrile was changed back to 10% over 1 min. The column was reequilibrated for 4 min before the next injection. The flow rate was set at 0.4 mL/min. To quantitate 6β-OHT, a turbo ionspray interface was used to introduce the HPLC effluent to the mass spectrometer being operated in the positive ion mode. The method of quantitation was developed on the basis of the reported LC/MS-MRM procedures (12) with minor modifications. The mass transitions m/z 305 f 269 and m/z 347 f 109 were used for 6β-OHT and CTXL, respectively. The chromatographic analysis was achieved on a Phenomenex Luna CN column (4.6 × 150 mm, 5 µm). The mobile phase consisted of methanol and 0.5 mM ammonium acetate (pH ∼5), which were delivered using a gradient program. The percentage of methanol was increased from 35 to 90% (v/v) over 8 min. After 8 min, the percentage of methanol was kept at 90 for 2 min before being reduced back to 35% for re-equilibration before the next injection. The flow rate was set at 0.8 mL/min. The flow from the column was split 1:1, with 0.4 mL/min directed to the mass spectrometer. Quantitation was carried out using a standard calibration curve of 6β-OHT over a concentration range of 0.01 to 0.80 µg/mL. A weighted (1/x) linear least-squares regression of 6β-OHT concentrations versus measured peak area ratios of 6βOHT to CTXL was used to construct the calibration curve.

Results Mutagenicity Assay. In the absence of S9 metabolism, none of the compounds including 1a-d, 2a, and 3a were mutagenic to Salmonella typhimurium test strains TA98 and TA100 (data not shown). However, in the presence of S9 metabolism, 1a and 3a were shown to be mutagenic to TA98, whereas none of the compounds tested were mutagenic to TA100. As shown in Figure 2, 1a exhibited the highest mutagenic potency to TA98 at 60 and 120 µg/plate. The revertant frequency of 1a increased 133- and 58-fold per µmol over the vehicle control at these concentrations, respectively. Cytotoxicity by 1a was observed at concentrations higher than 120 µg/plate. At these concentrations, cell precipitation and absence of bacterial growth were observed. In contrast, structural analogues including 1b, 1c, and 1d were neither mutagenic nor cytotoxic to TA98 at the concentrations that led to mutagenicity in 1a.

Chen et al.

Figure 2. Mutagenicity of 1a, 1b, 1c, 1d, 2a, and 3a in Salmonella typhimurium TA98 in the presence of rat liver S9 subcellular fraction. Assay conditions were described in Materials and Methods. The symbols shown (9-1a; 2-1b; (-1c; •-1d; ∆-2a; 0-3a) indicate the mean number of two replicates tested at each concentration.

It was found that 1a underwent a novel metabolic reaction N-deindazolation (loss of indazole) to give 2a in the presence of rat liver S9 or microsomes. To investigate if the mutagenicity of 1a was metabolism-mediated and to determine the nature of the potential toxicophore, 2a was chemically synthesized and evaluated for mutagenicity. It was found that 2a was only marginally mutagenic to TA98 in the presence of metabolic activation. At the lowest mutagenic concentration of 2a (120 µg/plate), the revertant frequency increased 7.2-fold per µmol over the vehicle control, which accounted for approximately 12% mutagenic potency of 1a (Figure 2). As a result, the mutagenicity of 1a could not be attributed to 2a. Interestingly, 3-piperazinyl indazole (3a), which was the core chemical motif of 1a, was mutagenic to TA98 in the entire concentration range examined. Nevertheless, the potency appeared to be lower than that of 1a on the basis of micromolar equivalence (Figure 2). In contrast to 1a, the cytotoxicity of 3a was not observed at any concentration. Metabolite Identification. Incubation of 1a with male rat liver microsomes resulted in the formation of a metabolite (2a) eluting at 5.2 min (Figure 3). The formation of 2a was shown to be microsomal protein- and NADPH-dependent. On the basis of LC/MS total ion current (TIC, Figure 3A) and the LC/UV chromatogram (Figure 3B), 2a, the most abundant component, had a pseudomolecular ion [M + H]+at m/z 322, which exhibited one-chlorine M + 2 isotope cluster in the mass spectra. The mass difference (116 Da) between 2a (m/z 322) and 1a (m/z 438) suggested that 2a was produced by a loss of the indazole ring in 1a. MS/MS analyses of 2a showed major fragment ions at m/z 279, 236, and 208 (Figure 4). The origin of these fragment ions is postulated (Figure 4). The structure of 2a was subsequently confirmed by comparing the LC/MS retention time and MS/MS spectral data with the synthetic standard. Similarly, the presence of 2a in S9 subcellular fraction incubation was demonstrated to be protein- and NADPHdependent. LC/MS analyses of incubation extracts of 1a in Aroclor-induced rat liver S9 fraction, which was used in the standard mutagenic assay, demonstrated a similar metabolite profile, in which 2a was present as the predominant metabolite (data not shown). Incubations of 1b with male rat liver microsomes or S9 fraction were performed under the same experimental conditions. LC/MS analysis of incubation extracts showed no evidence of a deindazolated metabolite, as was seen with 1a. Incubation of 1a, 1b, or 3a in the presence of GSH

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Figure 3. LC/MS/UV trace of extracts from rat liver microsomal incubation of 1a: (A) LC/MS total ion current (TIC); (B) UV trace at 240 nm.

Figure 4. MS/MS spectra of 2a produced from 1a in rat liver microsomes.

was also carried out. LC/MS analyses of incubation extracts did not show the presence of any compound-related GSH conjugates. Detection of BA. The microsomal incubation extracts were analyzed for the presence of BA, an expected product resulting from the hydrolysis of 3-indazolone. It is postulated that the metabolic cleavage (loss of indazole ring) of 1a Via an oxaziridine intermediate leads to the 3-indazolone (Scheme 2). As shown in Figure 5, the presence of BA with tR at 7.1 min (Figure 5A) was demonstrated by conducting LC/MS-MRM

analyses of the microsomal extracts of 1a and comparing the HPLC retention time and MRM transition with the standard BA (Figure 5B). Similarly, it was found that BA was present in the incubation extracts of 3a (Figure 5C). However, BA was found not to be produced by 1b in the presence of rat liver microsomes (Figure 5D) using the LC/MS-MRM analysis (limit of detection ∼0.005 µg/mL). Characterization of P450 Enzymes Responsible for 2a Formation. A sensitive and specific LC/MS-MRM assay was developed to quantitate 2a present in various biological matrices

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Chen et al.

Scheme 2. Postulated Mechanism for the N-deindazolation of 1a Wia a Putative Reactive Oxaziridine Intermediate

(e.g., rat liver microsomes). No matrix-associated ion suppression as well as matrix-induced interference was observed. The limit of detection (LOD, ratio of signal-to-noise ∼3) was 0.005 µg/mL, and the limit of quantitation (LOQ) was determined to be 0.010 µg/mL. The calibration curve was demonstrated to be linear over the range of 0.01-0.80 µg/mL of 2a with r2 > 0.99. Preliminary experiments showed that incubation of 1a with rat liver microsomes in the presence of either N-benzylimidazole (100 µM), a nonspecific competitive inhibitor of P450, or ABT (100 µM), a nonspecific mechanism-based inhibitor of P450, resulted in 57% and 29% inhibition on the formation of 2a, respectively. These results indicated the participation of P450 enzymes in this particular metabolic reaction. Studies conducted using ten cDNA-expressed rat P450 enzymes demonstrated a marked difference in the formation of 2a by various enzymes. It was found that P450 3A1 and P450 3A2 were the major enzymes contributing toward the formation of 2a, using the same P450 concentration of 0.05 µmol/mL per enzyme (Figure 6). Preincubation with TAO (20 and 50 µM), a mechanism-based inhibitor of P450 3A, exhibited 75% and 82% inhibition (Table 1) on the formation of metabolite 2a, respectively. In contrast, furafylline (20 and 50 µM), a mechanism-based inhibitor of P450 1A, and cimetidine (50 and 100 M), a mechanism-based inhibitor of P450 2C11 (13), did not show any significant inhibition. These results supported the role of the rat P450 3A family, particularly P450 3A2, one of the major constitutive P450 enzymes present in untreated male rat liver microsomes (14-16) in forming 2a. In addition, studies conducted with specific anti-rat P450 antibodies provided further evidence for the role of the P450 3A enzyme in forming 2a. The production of 2a was reduced by approximately 80% (as compared to preimmune IgG control). Microsomes treated with the antibody against rat P450 1A enzymes produced 40% less 2a than the control (Table 1). In contrast, the microsomes treated with the antibody against rat P450 2B1/2 showed little or no effect on the formation of 2a (Table 1). Overall, these results clearly indicated that rat CYP3A2 was the principle P450 enzyme in male rat liver microsomes mediating N-deindazolation of 1a to 2a. Kinetics of 2a Formation in Male Rat Liver Microsomes and in cDNA-Expressed Rat CYP3A2. The kinetics of 2a formation Via N-deindazolation of 1a was studied in male rat liver microsmes. It was observed that the substrate-velocity curve for 2a formation was non-hyperbolic, although a hyperbolictype curve was apparent at low substrate concentrations. There was clearly no defined plateau at high substrate concentrations

Figure 5. Detection of BA in rat liver microsomal incubations of 1a, 1b, and 3a by LC/MS-MRM analyses: (A) incubation mixture of 1a (the peak eluted with tR at 5.4 min remains unknown); (B) standard BA 0.1 µg/mL spiked in the incubation medium; (C) incubation mixture of 3a; (D) incubation mixture of 1b.

and it was observed that rates decreased at higher substrate concentrations. Figure 7 shows a convex-type rate plot for 2a formation in rat liver microsomes. Furthermore, the kinetics of 2a formation was studied in cDNA-expressed rat CYP3A2, which was demonstrated to be the principle P450 enzyme mediating the metabolism of 1a to 2a. Consistent with the results

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Figure 6. Formation of 2a from 1a in the presence of various cDNAexpressed rat P450 enzymes.

Figure 7. Substrate-velocity curve of 2a produced from 1a in male rat liver microsomes. Table 1. Inhibition of 2a Formation in Male Rat Liver Microsomes in the Presence of Chemical Inhibitors or Antibodies Against Rat P450 Enzymes 2a formationa

inhibitor furafylline cimetidine troleandomycin PAb to CYP1A1/2 PAb to CYP2B1/2 PAb to CYP3A2

20 µM 50 µM 50 µM 100 µM 20 µM 50 µM

100 98 95 92 25 18 60 100 18

a The values are the average of triplicate and are expressed as percentages relative to the control without chemical inhibitors or the antibody.

obtained with liver microsomes, a substrate concentrationdependent inhibition profile was demonstrated for 2a formation in rat P4503A2 (data not shown). The data (rate of 2a formation vs 1a concentrations) from liver microsomes and the pure enzyme could not be fitted to the Michaelis-Menten equation. It appeared that the data was better described by non-MichaelisMenten kinetics corresponding to the substrate inhibition profile (eq 2, in Materials and Methods), suggesting the possibility of inhibition by substrate or product formed during the Ndeindazolation of 1a. Inactivation of P450 3A by 1a. The potential of 1a and its analogues including 1b, 1c, 1d, and 3a as an inactivator of P450 3A was evaluated. The activity of P450 3A was estimated on the basis of the rate of 6β-OHT formation under defined conditions. The experiment was performed such that cDNAexpressed rat P450 3A2 was preincubated with the test compound in the presence of NADPH followed by a 25-fold dilution of the incubation prior to assessment of the P450 3A activity. As shown in Figure 8, the preincubation of 1a in the

microsomes resulted in significant inhibition of the activity of testosterone 6β-hydroxylation (approximately 30% of control activity). The inclusion of GSH in the primary incubation of 1a did not protect P450 3A2 to any appreciable extent. However, the preincubation of other compounds, such as 1b, 1c, 1d, or 3a, did not result in the inhibition of P450 3A2 activity. The requirement for NADPH indicated that 1a had to be metabolized to a reactive species that was responsible for the inactivation. A similar result was obtained when preincubation was conducted in rat liver microsomes, where only 1a caused a significant inhibition of the activity of P450 3A (data not shown). It was found that P450 3A2 was inactivated by 1a in a timeand concentration-dependent manner (Figure 9). The inactivation followed pseudo-first-order kinetics with respect to time between 2 and 20 µM of 1a. There was no lag time associated with the inactivation as can be seen in the time-dependent loss of testosterone 6β-hydroxylase activity with increasing 1a concentrations. Linear regression analysis of the data in Figure 9 was used to determine the rate constants of inactivation. The plot of the reciprocals of the initial rate constants as a function of the reciprocals of the inactivator concentrations (Figure 9, insert) was used to determine the kinetic constants. The concentration of 1a required for the half-maximal rate of inactivation (KI) was 8.1 µM, and the time required for onehalf of the enzyme molecules to become inactivated (t1/2) was 6.1 min. The maximal rate of inactivation at saturation (kinact) was 0.114 min-1. The partition ratio (ratio of non-inactivating activity to inactivating activity) was also obtained (17). The percent of P450 3A2 activity remaining (as determined by 6βOHT formation) following preincubation at the last time-point tested (t ) 18 min) was plotted against the ratio of the concentration of 1a to P450 3A2. A partition ratio of ∼38 was calculated from the intercept of the two lines, representing the lower and higher concentrations of 1a.

Discussion Compound 1a (Figure 1), a potential therapeutic agent, was evaluated by the Ames test as part of routine safety assessment studies. Unexpectedly, this molecule was shown to be mutagenic to one test strain of Salmonella typhimurium. As a result, further development of this compound was terminated. In the Ames test, 1a was shown to be positive to Salmonella typhimurium TA98 only in the presence of rat liver S9 subcellular fraction, indicating that the mutagenicity was metabolism-mediated. Subsequently, it was found that 1a underwent a unique metabolic N-deindazolation reaction, in which the indazole ring was cleaved to produce 2a. This metabolic reaction was demonstrated to be S9/microsomal protein- and NADPHdependent, and 2a appeared to be the only metabolite produced by these liver subcellular fractions. Compound 1b was produced by replacing piperazine (in 1a) with piperidine, essentially replacing the heteroatom nitrogen with carbon at the position where the indazole moiety was linked to the rest of the molecule (Figure 1). This minimal structural alteration led to the desired inhibition of the deindazolation reaction as well as eliminating the mutagenic liability of the piperazine analogue. Although this approach exemplified success in eliminating potential toxicological consequences associated with 1a, the desired pharmacological properties of 1b were compromised. Despite this, the finding that 1b was devoid of the mutagenic liability associated with 1a provided some insight into the mechanism involved in the N-deindazolation reaction and its potential contribution to the toxicity.

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Chen et al.

Figure 8. Inhibition of testosterone oxidation to 6β-hydroxyl testosterone by TAO, 1a, 1b, 1c, 1d, and 3a.

Figure 9. Time- and concentration-dependent inactivation of P450 3A2 by 1a following incubation with NADPH. Incubation conditions were described in Materials and Methods. The concentrations of 1a were (() 0, (9) 2, (2) 5, (x) 8, (•) 10, and (∆) 20 µM. Values shown are the means of three determinations, and in all cases, the standard deviation was less than 18%. The insert is a Kitz-Wilson double reciprocal linearization plot from which KI and kinact were determined.

To investigate metabolism-mediated toxicity, the cleaved product 2a, the most abundant metabolite of 1a produced through the N-deindazolation, was evaluated in the Ames test and demonstrated to be only marginally mutagenic to TA98. Consequently, the mutagenic and toxic properties of 1a to TA98 could not be attributed to the presence of 2a. Hence, it was speculated that during the metabolism of 1a to 2a, a reactive intermediate with a finite half-life and capable of interacting with macromolecules was formed. It was postulated that this reactive intermediate was either formed preceding the ring cleavage or was one of the products such as 3-indazolone (Scheme 2) not detected by the LC/MS analyses. Further studies were conducted to investigate this scenario. Experiments using GSH as a trapping agent were conducted and no compoundrelated GSH adduct was found. However, the kinetics of 2a formation in rat liver microsomes, which was investigated to understand the process of N-deindazolation of 1a, was shown to be nonhyperbolic in nature. It was shown that the velocity

of 2a formation decreased once a particular substrate concentration was surpassed. A similar kinetic profile of 2a formation was also observed with the recombinant rat P450 3A2, the principle P450 enzyme catalyzing the N-deindazolation of 1a. In both cases, the data (velocity of 2a formation vs 1a concentrations) could not be fitted to the standard MichaelisMenton kinetics profile, whereas good fits were apparently obtained by assuming a second binding of substrate. However, to date, there is no independent evidence that suggests the existence of a second binding site within rat P450 3A2. One possible explanation for the kinetic patterns observed with the N-deindazolation of 1a is that an inhibitory intermediate was being generated during the process of N-deindazolation. This metabolic intermediate may be competitive or could inactivate the P450 enzyme catalyzing the reaction. Further studies were conducted to gain a better understanding of the inactivation of P450 3A by 1a. It was observed that the preincubation of 1a with the recombinant rat P450 3A2 led to a decrease in P450 3A activity (Figure 8). This enzyme inhibition was characterized by a dependence upon NADPH, the cofactor required for P450-catalyzed reactions. The degree of inhibition was found to be time- and substrate concentrationdependent (Figure 9). The loss of P450 3A activity could not be prevented by co-incubation with an exogenous nucleophile GSH, although an alternative substrate such as testosterone afforded partial protection. These data collectively suggested that 1a was a potential mechanism-based inhibitor of P450 3A, which was responsible for the formation of reactive metabolites. Such metabolite(s) could either alkylate the heme moiety of P450 3A, form a metabolic-intermediate (MI) complex, or alternatively, bind covalently to the apoprotein, thereby impairing enzyme function. However, the nature of inactivation of P450 3A by 1a was not demonstrated by the studies presented herein. In contrast to 1a, compounds including 1b, 1c, and 1d, which do not undergo deindazolation, did not inactivate P450 3A (Figure 8). Consequently, it was convincing that a reactive metabolite formed during the N-deindazolation of 1a was responsible for the inactivation of P450 3A and perhaps contributed toward the toxicity observed with 1a in the Ames test. N-dearylation as a metabolic reaction has been observed with benzoisoxazoles and benzisothiazoles (18, 19). P450-mediated N-deindazolation described herein appears to be a previously

Mutagenicity of Piperazinyl Indazole

undescribed pathway. A plausible mechanism is proposed for the N-deindazolation of 1a, which would involve an initial P450mediated formation of an oxaziridine intermediate followed by the ring opening and subsequent elimination to give the N-deindazolated metabolite 2a (Scheme 2). To demonstrate the occurrence of an oxaziridine intermediate in the present work, studies were conducted to trap such an intermediate with a nucleophile such as GSH. As described before, no GSH conjugate was detected from incubations of 1a with rat liver microsomes or recombinant rat P450 3A2. However, this is not totally surprising because oxaziridine being a hard electrophile will not react easily with a soft nucleophile such as GSH. Theoretically, 3-indazolone would be generated from the process of oxaziridine ring opening and elimination (Scheme 2). As reported in the literature, 3-indazolone is not stable, and it reacts rapidly with nucleophiles such as water to produce benzoic acid and nitrogen gas (20). Therefore, the production of benzoic acid in microsomal incubations of 1a would indirectly indicate the existence of an oxaziridine intermediate. As expected, the presence of benzoic acid in microsomal incubations of 1a was clearly demonstrated, and the formation of benzoic acid was shown to be concentration- and incubation time-dependent (data not shown). Hence, the mechanism involving an oxaziridine intermediate appears to be a logical explanation for the observed P450-mediated N-deindazolation of 1a. Oxaziridines, a class of heterocyclics containing oxygen, nitrogen, and carbon atoms in a three-membered ring, were first reported five decades ago. Extensive investigations of oxaziridines have revealed their unusual reactivity, which is very useful synthetically (21). Oxaziridines are known to be electrophiles and potent oxidizing agents (21) and are capable of reacting with DNA molecules. As mentioned above, the other product of N-deindazolation of 1a, 3-indazolone, has been reported to be unstable and shown to react with nucleophiles (20). However, the Ames test of 3-indazolinone (3b, Figure 1), a possible metabolic precursor of 3-indazolone, demonstrated that this compound was not mutagenic to TA98 in the absence or presence of metabolic activation (data not shown), although it apparently underwent P450-mediated biotransformation to 3-indazolone, as demonstrated by the formation of benzoic acid (data not shown). In addition, 3-indazolinone was shown not to be an inactivatior of rat P450 3A (data not shown). These data indicate that 3-indazolone rapidly undergoes a detoxification process such as by reacting with water present in the active site of P450 enzyme after its formation. Thus, 3-indazolone, as a reactive species produced during N-deindazolation of 1a, would not likely contribute to toxicity and inactivation. As a result, the reactive intermediate, oxaziridine, produced during the N-deindazolation is most likely the ultimate species responsible for the toxicity and inactivation of P450 3A. As determined from the inactivation studies, the partition ratio of ∼38 suggests that the oxaziridine intermediate of 1a is a relatively weak inactivator of P450 3A compared to some potent P450 3A inactivators reported (22, 23). The relatively low reactivity of this intermediate as a hard electrophile provides an opportunity for it to escape out of the active site of P450 3A. Subsequently, because of its finite half-life, it is capable of reacting with hard nucleophiles such as DNA, resulting in mutagenicity. Although there are only a few reports of biologically generated oxaziridines and their significance to macromolecules, recent studies of photochemical DNA cleavage by antitumor agent 3-amino1,2,4-benzotriazine-1,4-dioxide demonstrated that the reactive intermediate responsible for DNA cleavage was reasonably expected to be an oxaziridine intermediate that was trapped by

Chem. Res. Toxicol., Vol. 19, No. 10, 2006 1349

p-anisidine to give 3-(4-methoxyanilino)-1,2,4-benzotriazine1,4-dioxide (24). Studies are currently underway to demonstrate the covalent binding of 1a to DNA molecules and to determine the chemical nature of binding. Compound 3a, a substructure in 1a, was found to be mutagenic to TA98 in the presence of rat liver S9, but the potency appeared to be lower (approximately 60% potency of 1a), on the basis of the increase of revertant frequency per micromole at the active concentrations. Although the presence of N-deindazolated product, piperizine, was not demonstrated because of analytical difficulties, the presence of benzoic acid in the microsomal incubation of 3a was clearly shown, again indicating the existence of an oxaziridine intermediate. As a result, the mutagenicity of 3a could be attributed to the reactive oxaziridine as described for 1a. Reasons for the discrepancy of mutagenicity between these two chemicals 1a and 3a are unclear at this time. However, it should be appreciated that many factors can influence mutagenic potency. Differences in the rate of transport of the chemicals across the cell wall of Salmonella strains, the binding of chemicals to the activating enzymes, and kinetics of metabolism to reactive intermediate(s) and of metabolic detoxification are but several considerations that must be made in assessing differences in mutagenic potencies. In summary, P450-mediated N-deindazolation reaction of 1a that possesses a 3-piperazinyl indazole is postulated to be responsible for the mutagenicity observed with Salmonella typhimurium TA98. It is highly unlikely that the toxicity is due to the N-deindazolated metabolite 2a, but it may result from a putative oxaziridine intermediate that precedes the formation of 2a. The oxaziridine, an electrophilic and highly reactive intermediate, could be the ultimate species responsible for the toxicity exhibited by 1a. In addition, the finding that 3-piperazinyl indazole is a promutagen as opposed to a nonmutagenic indazole itself is interesting, and perhaps it is a general feature for indazoles having a good leaving group at the 3-position, which certainly deserves further investigation.

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