Sequential Metabolism and Bioactivation of the Hepatotoxin

Chem. Res. Toxicol. 2007, 20, 1833–1842

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Sequential Metabolism and Bioactivation of the Hepatotoxin Benzbromarone: Formation of Glutathione Adducts From a Catechol Intermediate Matthew G. McDonald and Allan E. Rettie* Department of Medicinal Chemistry, School of Pharmacy, UniVersity of Washington, Seattle, Washington 98195 ReceiVed April 18, 2007

Benzbromarone (BBR) is a uricosuric agent that has been used as a treatment for chronic gout. Although never approved in the United States, BBR was recently withdrawn from European markets due to several clinical cases linking the drug to an idiosyncratic hepatotoxicity that is sometimes fatal. We report here a possible mechanism of toxicity that involves the bioactivation of BBR through sequential hydroxylation of the benzofuran ring to a catechol, which can then be further oxidized to a reactive quinone intermediate capable of adducting protein. NADPH-supplemented human liver microsomes generated a single metabolite that was identified as 6-OH BBR by comparison with the synthesized chemical standard. CYP2C9 was the major recombinant enzyme capable of catalyzing the formation of 6-OH BBR, although CYP2C19 also showed a lower degree of activity. Further oxidation of either 6-OH BBR or 5-OH BBR by human liver microsomes resulted in the formation of a dihydroxy metabolite with identical chromatographic and mass spectral properties. This product of sequential metabolism of BBR was identified as the catechol, 5,6-dihydroxybenzbromarone. Incubation of the catechol with liver microsomes, in the presence of glutathione, resulted in the formation of two glutathione adducts that could derive from a single orthoquinone intermediate. Isoform profiling with recombinant human P450s suggested that CYP2C9 is primarily responsible for the formation of this reactive quinone intermediate. Introduction Benzbromarone (BBR, Figure 1)1 is a uricosuric agent that has been used therapeutically for the treatment of chronic gout. Gout is characterized by a painful buildup of uric acid crystals in the joints or soft tissues that can result from hyperuricemia, an often asymptomatic condition of elevated serum urate levels, which has also been hypothesized to play a role in cardiovascular disease and hypertension (1). Currently, allopurinol (a xanthine oxidase inhibitor) is the preferred oral treatment for patients suffering from chronic gout, but there are few effective alternatives for those patients allergic to this drug or for whom allopurinol is contraindicated (such as solid organ transplant recipients with cyclosporin-induced gout) (2–4). Until recently, BBR had been a quite useful drug in Japan and Europe for the treatment of allopurinol-intolerant patients suffering from this disorder. In fact, several studies have shown that the efficacy of BBR in lowering uric acid levels is superior to allopurinol in patients with both normal and impaired renal function––an effect that might be further improved by dispensing the drugs in combination (3–8). BBR is not approved for use in the United States and has recently been withdrawn from European markets (9) due to several reported cases linking the drug to liver damage, including at least three instances of fatal fulminant hepatic failure (10–13). However, the suggestion has been made * To whom correspondence should be addressed. Tel: 206-685-0615. Fax: 206-685-3252. Email: [email protected]. 1 Abbreviations: P450, cytochrome P450; BBR, benzbromarone; OHBBR, hydroxybenzbromarone; CAT, 5,6-dihydroxybenzbromarone; GSH, glutathione; CAT-SG, glutathione adduct of 5,6-dihydroxybenzbromarone; DCM, dichloromethane; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; LC/ESI-MS, LC-MS, liquid chromatography/electrospray ionization mass spectrometry; GC-MS, gas chromatography–mass spectrometry detection.

Figure 1. BBR.

that BBR may have been removed from the market too soon and that an effective drug has been lost due to overreaction to a few cases of idiosyncratic hepatotoxicity (14). Liver toxicity is often associated with P450-mediated bioactivation (15), and so, it may be instructive to consider what is known about the metabolism of BBR. Early metabolic studies identified two major hydroxylated metabolites, M1 and M2, in urine, bile, and plasma (16–18). M1 was shown to be 1′hydroxybenzbromarone (1′-OH-BBR) based on comparison to a synthetic standard (17). M2 was determined to result from oxidation on the benzofuran ring of BBR (i.e., at the C4-, C5-, C6-, or C7-positions), although the specific site of oxidation was not discernible by mass spectral fragmentation analysis (19). Subsequently, M2 was identified as 6-OH-BBR, based on proton NMR data obtained on a derivatized urinary metabolite (20). This same study also reported the finding of two other minor primary metabolites of BBR, which apparently resulted from hydroxylation at other positions on the benzofuran ring. Later reports identified a number of other plasma and urinary sequential metabolites of BBR, essentially all of which appear to derive from multiple oxidation reactions at the 1′ and the C4–7 ring positions, although structures could not be assigned

10.1021/tx7001228 CCC: $37.00  2007 American Chemical Society Published on Web 11/20/2007

1834 Chem. Res. Toxicol., Vol. 20, No. 12, 2007

unambiguously (21, 22). In vitro metabolic studies involving BBR are restricted to one report that describe a single metabolite formed by CYP2C9 and CYP2C19-dependent hydroxylation of the benzofuran moiety tentatively identified as 6-OH BBR (23). Therefore, aromatic hydroxylation appears to be an important early step in the metabolism of BBR and, as such, would represent an attractive starting point for the further generation of reactive intermediates. The goal of the present study was to elucidate pathways and enzymes involved in microsomal metabolism and reactive intermediate formation from BBR. To this end, we report the first synthesis of the putative major BBR metabolite, 6-OH BBR, NMR-based characterization of a catechol derived from sequential metabolism of 6-OH-BBR, evidence for formation of a reactive ortho-quinone that may be relevant to the hepatotoxicity exhibited by the drug, and a prominent role for CYP2C9 in sequential metabolism of this drug.

Experimental Procedures Materials. All chemicals used in this study were purchased either from Aldrich Chemical Co. (Milwaukee, WI) or from Sigma (St. Louis, MO), unless otherwise noted. Deuterated solvents used for NMR analysis were procured from Cambridge Isotope Laboratories, Inc. (Andover, MA), while nonlabeled solvents were obtained from J. T. Baker, Inc. (Phillipsburg, NJ) or Fischer Scientific (Springfield, NJ). All recombinant human P450 Supersomes were purchased from Gentest BD Biosciences (Woburn, MA). Instrumentation. HPLC was carried out on a Shimadzu system equipped with two LC10ADvp pumps, an SPD-M10Avp UV–vis detector, an SCL-10Avp controller, and an SIL-10ADvp autosampler (Shimadzu Scientific Instruments, Inc., Columbia, MD) using either a Thermo Hypersil Kromasil 5 µm, 2.0 mm × 250 mm C18 column (Keystone Scientific Operations, Bellefonte, PA) with a flow rate of 0.3 mL/min for analytical separation or an Ultrasphere 5 µm, 10 mm × 250 mm ODS column (Beckman-Coulter, Inc., Fullerton, CA), with a flow rate of 2.5 mL/min, for preparative HPLC. Data acquisition and analysis were performed on EZSTART chromatography software (v 7.2, Shimadzu). Three gradient methods were used for metabolite analysis and purification. In each method, solvent A consisted of a 70:30 solution of aqueous 1% acetic acid:acetonitrile, while solvent B was 100% acetonitrile. Method A (analytical) was programmed for a linear increase in solvent B from 15 to 55% over 17 min, followed immediately by another increase in B from 55 to 80% from 17 to 20 min. Solvent B was run at 80% for 2 min and then decreased to 30% from 22 to 23.5 min. Method B (analytical) consisted of an isocratic flow for 8 min at 5% solvent B, followed by a linear increase to 60% from 8 to 17 min. From 17 to 20 min, solvent B was maintained at 60% and then decreased back to 5% over an additional 3 min. Method C (preparative) involved a linear increase from 20 to 65% solvent B over 21 min, followed by a decrease to 20% B over the next 3 min. Peaks were monitored by UV at 285 nm. LC/ESI-MS and LC/ESI-MS/MS analyses were conducted on a Micromass Quattro II Tandem Quadrupole Mass Spectrometer (Micromass, Ltd., Manchester, United Kingdom) connected to a Shimadzu HPLC system (similar to the system described above). The mass spectrometer was run in electrospray positive ionization mode (ESI+) at a cone voltage of between 20 and 45 V, a source block temperature of 150 °C, and a desolvation temperature of 350 °C. In tandem MS mode, the collision energy was set to 45 V. Quantitative analysis was performed in selected ion monitoring (SIM) mode, and monitoring channels were set at either m/z 283 (internal standard) and 441 for 6-OH BBR quantification or at m/z 478 (internal standard) and 762 for the quantification of 5,6dihydroxybenzbromarone-glutathione (CAT-SG) adducts. Data analyses were carried out on Windows NT-based MicromassMassLynxNT, v 3.2, software. 1H NMR data were acquired on a Brucker AV300 300 MHz or Brucker DPX200 200 MHz spec-

McDonald and Rettie trometer at 25 °C. Spectra were referenced by chemical shift to residual proton peaks within the following deuterated solvents: CDCl3 δ 7.24 (s), CD3COCD3 δ 2.04 (quintet), CD3SOCD3 δ 2.49 (quintet), and CD3OD δ 3.30 (quintet). All chemical shift values, δ, are given in ppm. GC-MS experiments were conducted using a Shimadzu GC-17A gas chromatograph, incorporating an XTI-5 30 m, 0.25 µm i.d. capillary column (Restek, Co., Bellefonte, PA) and equipped with an AOC-20i autoinjector, coupled to a Shimadzu QP5050A mass spectrometer. Microsomal Incubations. Incubations were performed with a pool of human liver microsomes (HLM) consisting of equal quantities of total microsomal protein from 13 different liver samples, prepared as described previously (24, 25). Typical incubation mixtures contained 1 mg/mL microsomal protein from the HLM pool (or 20–50 pmol of recombinant P450), 1 mM NADPH, and 100 µM substrate [5 µL from a 10 mM stock in dimethyl sulfoxide (DMSO)], made up to 500 µL total volume with 100 mM potassium phosphate buffer, pH 7.4. Metabolic reactions were carried out at 37 °C and 70 rpm, in a water bath shaker, for 30 min, and were then quenched with either an equal volume of 10% HCl or 50 µL of 15% w/v ZnSO4. The quenched solutions were extracted twice with ethyl acetate, the combined organics were evaporated, and the residues were taken up in 100 µL of acetonitrile for HPLC analysis (method A). Experiments for the determination of BBR binding and turnover kinetics in the HLM pool were performed using 75 pmol of total P450 per incubation [determined by CO difference spectroscopy (26)] in 100 mM KPi buffer, pH 7.4, at the following concentrations of BBR: 2, 5, 10, 20, 50, 100, 200, 300, 400, and 500 µM. Reactions were initiated by the addition of NADPH to a 1 mM final concentration. Incubation reactions for each substrate concentration were performed in triplicate. The steady-state kinetics of 6-OH-BBR formation from BBR catalyzed by recombinant CYP2C9 and CYP2C19 were determined in a similar manner, except that only 20 pmol of P450 was used per incubation. Data (as either HPLC or LC-MS peak areas, method A) were quantified by fitting product/internal standard peak area ratios to a standard curve with 5-hydroxybenzarone being utilized as an internal standard. Synthesis of 5-OH-BBR and 6-OH-BBR (Scheme 1), 2-Acetyl-5-methoxybenzofuran (2a). 5-Methoxysalicylaldehyde, 1a (4.1 mL, 3.29 mmol), and chloroacetone (3.01 mL, 3.78 mmol) were successively added to a mixture of 3.75 g of K2CO3 in 12 mL of anhydrous acetone with stirring under a nitrogen atmosphere. The reaction was heated to reflux for 90 min. After it was cooled to room temperature, the dark red product mixture was diluted in 250 mL of deionized water and acidified to pH 1–2 with 10% HCl. The solution was then extracted several times with ether, and the combined organic extracts were washed with 1 N NaOH, to remove starting material, and brine. The solvent was removed in vacuo to yield relatively pure 2a as a bright yellow solid (4.5g, 70%). GCMS m/z 178 [M]+. 2-Ethyl-5-methoxybenzofuran (3a). A solution containing KOH (2.5 g, 45 mmol), hydrazine monohydrate (85%, 1.8 mL), and 2-acetyl-5-methoxybenzofuran, 2a (2.5 g, 13.2 mmol), dissolved/ suspended in diethylene glycol (40 mL), was heated to reflux for 90 min. At this time, the reflux condenser was removed from the reaction allowing the water, formed as a byproduct of the modified Wolff–Kishner reaction (27), to boil off. The condenser was replaced when the reaction temperature had eclipsed 195 °C. After the reaction was refluxed for an additional 90 min, another 1.8 mL of hydrazine monohydrate and 2.5 g of KOH were added and the reaction was refluxed for 2 more hours before cooling to room temperature. The solution was next diluted with 300 mL of water and extracted several times with ethyl acetate. Both phases from the extractions were saved. The combined organic phase was washed several times with water to remove any diethylene glycol and then with brine before drying over MgSO4. The solvent was removed via rotavap to give 3a as a brown oil (1.6 g, 69%). The basic aqueous phase from the original extraction was acidified with 6N HCl and then re-extracted (3×) with ethyl acetate. The combined organic phase from these extractions was also washed

BioactiVation of Benzbromarone

Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1835 Scheme 1. Scheme for the Synthesis of 5-OH BBR and 6-OH BBR

several times with water and then with brine prior to concentration in vacuo, which provided 4a as a brown solid (320 mg, 15%). 2-Ethyl-5-hydroxybenzofuran (4a). A solution of 2-ethyl-5methoxybenzofuran, 3a (1.26 g, 7.16 mmol), in 10 mL of anhydrous dichloromethane (DCM), stirring under an inert nitrogen atmosphere, was cooled to -78 °C in a dry ice/acetone bath. One equivalent of boron tribromide (7.16 mL of a 1 M solution in DCM) was added slowly to the cooled solution. After complete addition of the Lewis acid, the cold bath was removed and the reaction was allowed to warm to room temperature where it was stirred for an additional 35 min. The resultant solution was poured onto an ice–water slush, diluted with an equal volume of brine (to break up emulsions), and extracted several times into DCM. The product phenol was then extracted from the combined organic phase into 1 N NaOH (3×), and the basic extract was acidified with 10% HCl to pH 1–2. The phenol was finally re-extracted into DCM, which was washed with brine, and dried over magnesium sulfate, and the solvent was removed to afford a brown solid, 4a (990 mg, 86%). 2-Ethylbenzofuran-5-acetate (5a). 2-Ethyl-5-hydroxybenzofuran, 4a (990 mg, 6.1 mmol), was dissolved in 5 mL of 3 M NaOH and cooled with the addition of 15 g of crushed ice. The addition of acetic anhydride (1.5 mL, 14.7 mmol), with vigorous stirring, caused the immediate formation of a brown precipitate. The mixture was diluted with water (50 mL) and acidified with 10% HCl prior to ether extraction. The ether was dried over MgSO4 and concentrated in vacuo to give 1.0 g (80%) of the ester, 5a, as a brown solid. 2-Ethyl-3-(4-methoxybenzoyl)benzofuran-5-acetate (6a). Compound 5a, 2-ethylbenzofuran-5-acetate (910 mg, 4.5 mmol), was dissolved in anhydrous DCM (20 mL) and cooled to -78 °C in a dry ice/acetone bath stirred under nitrogen. AlCl3 (890 mg, 6.7 mmol) was carefully added into the mixture, followed by the dropwise addition of anisoyl chloride (1 mL, about 6.7 mmol). Over 1 h, the reaction was allowed to warm to approximately -20 °C. A second aliquot of AlCl3 (590 mg, 4.4 mmol) was added, and the solution continued to warm to room temperature over another hour. The reaction was then stirred an additional 2 h at room temperature. At this point, the resulting Friedl–Craft solution was cooled back down to -78 °C and carefully quenched with water. After it was warmed back to room temperature, the mixture was diluted with DCM, transferred to a separatory funnel, and washed successively with water, 5% NaOH, and saturated NaCl. Finally, the organic fraction was dried over MgSO4, and the solvent was removed in vacuo to yield a dark brown oil. Flash chromatography of the

product residue (SiO2, 100% hexane to 30% EtOAc:hexane step gradient) resulted in the isolation of 6a as a brown oil (1.45 g, 95%). 2-Ethyl-3-(4-methoxybenzoyl)-5-hydroxybenzofuran (7a). Lithium hydroxide monohydrate (160 mg, 3.75 mmol) was added to a suspension of compound 6a (500 mg, 1.5 mmol) in a 50:50 mixture of methanol/water (5 mL). The reaction was heated to reflux until the oil starting material had completely dissolved into solution (approximately 3 h). It was then taken off the heat and allowed to stir overnight at room temperature. Work up consisted of first diluting the reaction solution with 0.1 N NaOH and washing with ether. The basic aqueous phase was acidified to pH 1–2 with 10% HCl and then extracted several times with DCM. The combined DCM extracts were washed with 50% saturated sodium bicarbonate and brine before drying over magnesium sulfate. The solvent was removed under reduced pressure to afford 7a (370 mg, 85%) as a white solid. 2-Ethyl-3-(4-methoxybenzoyl)-6-hydroxybenzofuran (7b) was produced from 1b in a manner analogous to the stepwise synthesis of 7a. 2-Ethyl-3-(4-hydroxybenzoyl)-5-hydroxybenzofuran (8). Aluminum chloride (675 mg, 5.06 mmol) was added to a stirred solution of compound 7a (500 mg, 1.69 mmol) dissolved in anhydrous chlorobenzene (10 mL) under a nitrogen atmosphere. The reaction was heated to 70 °C for 4 h. After the solution was cooled to 0 °C, excess aluminum chloride was quenched carefully by the slow addition of water to the reaction flask. The resultant mixture was diluted with DCM, washed with water, and extracted with 1 N NaOH. After it was acidified with 10% HCl, the aqueous fraction was extracted with DCM, which was dried over anhydrous MgSO4, and then concentrated under reduced pressure to give 386 mg (81%) of 8 as a white solid. 5-OH-BBR (9). To a vigorously stirred solution of compound 8 (20 mg, 71 µmol) in 5 mL of methanol, cooled in an ice–water bath, was added 16.2 µL (1.0 equiv) of a 25% solution of sodium methoxide/methanol. After 5 min, 35.5 µL (0.5 equiv) of a 1 M solution of methanolic bromine was added; the orange bromine color was immediately lost upon mixing. The solution was stirred for another 20 min prior to the addition of 8.1 µL (0.5 equiv) of sodium methoxide solution, followed by the addition of another 35.5 µL (0.5 equiv) of 1 M bromine. This was repeated twice more until a total of 2.5 equiv of base and 2 equiv of bromine had been added. The reaction was then stirred at room temperature for another hour prior to workup. A 1 N concentration of HCl was added to quench, and the solution was diluted with water and extracted with

1836 Chem. Res. Toxicol., Vol. 20, No. 12, 2007 DCM. The organic solution was then extracted twice with a 10% saturated solution of sodium bicarbonate. Acidification of the bicarbonate extracts to pH 2, followed by another extraction with DCM, yielded 87 mg of a white solid residue upon solvent removal. This solid was seen by LC-MS to contain starting material as well as at least eight different mono- to tetrabrominated analogues of 5-hydroxybenzarone (8). Compound 9 (4.5 mg, 13.7%) was purified from this mixture by semipreparative HPLC (method C). ESI+ m/z 439 (50%), 441 (100%), and 443 (50%) [M + H]+ cluster. 2-Ethyl-3-(4-methoxybenzoyl)benzofuran-6-tert-butyl Ether (10). Compound 7b (296 mg, 1.0 mmol) was dissolved in 20 mL of anhydrous DCM, and the solution was cooled to -78 °C in a dry ice/acetone bath stirring under a nitrogen environment. Concurrently, 2-methylpropene gas was added to a separate, evacuated, 250 mL round-bottom flask, also cooled to -78 °C, to induce liquification. The nitrogen line was taken off the organic solution and replaced with an empty balloon. Five milliliters of liquid 2-methylpropene was then added, followed by 92 µL (10% molar equiv) of CF3SO3H as a catalyst. After 2 h, the reaction was warmed to -20 °C and stirred for 4 more hours. The reaction was then allowed to warm to room temperature. Triethylamine (170 µL) was added to quench the acid catalyst, and the resultant product mixture was diluted with DCM and then washed successively with 1 N HCl, 1 N NaOH, and brine. After it was dried over MgSO4, the solvent was evaporated to give 10 in good purity and almost quantitative yield (342 mg, 97%). 2-Ethyl-3-(4-hydroxybenzoyl)benzofuran-6-tert-butyl Ether (11). Sodium ethanethiolate (119 mg, 1.42 mmol, 2.5 equiv) was added to a stirred solution of compound 10 (200 mg, 0.57 mmol) in 10 mL of anhydrous N,N-dimethylformamide (DMF) under nitrogen. The reaction was refluxed for 3 h and then cooled to room temperature. Following dilution with ether, the solution was extracted several times with 1 N NaOH. The combined aqueous layers were acidified and extracted with DCM, and the resultant organics were washed twice with water and then with brine prior to drying over MgSO4. The solvent was evaporated in vacuo, and the resultant product was recrystallized from water/methanol to give 11 (135 mg, 70%). BBR-6-tert-butyl Ether (12). Bromine (15.9 µL, 0.31 mmol) was added to a solution of 11 (50 mg, 0.15 mmol) and triethylamine (43.3 µL, 0.31 mmol) in 5 mL of methanol, stirring at -5 °C (CaCl2/ice bath). The reaction was stirred for 2 h at this temperature and then quenched by the addition of 1 N HCl. The quenched solution was diluted with 100 mL of DCM and washed with 1 N HCl, and the organic phase was then extracted with 1 N NaOH. The resultant basic solution was next carefully acidified with 10% HCl causing a white solid to precipitate. This solid was collected by filtration and was recrystallized (ether/petroleum ether) to give 12 (62 mg, 85%). 6-OH-BBR (13). Compound 12 (118 mg, 0.24 mmol) was dissolved in 2 mL of anhydrous TFA and was stirred under nitrogen overnight at room temperature. The reaction was diluted with water and extracted into ethyl acetate. The organic layer was then extracted with 1 N NaOH, which was then acidified, and the precipitate was collected. Recrystallization with methanol/water afforded 13 (59 mg, 57%) as a white solid. ESI-MS m/z 439 (50%), 441 (100%), and 443 (50%) [M + H]+ cluster. Large Scale Conversion of 5-OH-BBR to Catechol in Rat Liver Microsomes. Five 250 mL triple-baffled Erlenmeyer flasks, each containing a 50 mL solution of 100 µM 5-OH-BBR, rat liver microsomes (at 800 pmol total P450/mL), and a NADPH regenerating system (consisting of 1 mM NADPH, 1 mM glucose-6phosphate, 5 mM MgCl2, and 26.6 units/flask of glucose-6phosphate dehydrogenase) in 100 mM KPi buffer, pH 7.4, were incubated at 37 °C and 150 rpm for 75 min. Reaction mixtures were acidified with HCl, to pH 2, and extracted twice with ethyl acetate. The ethyl acetate was evaporated to a residue, which was purified by HPLC (method C) for NMR analysis. 1H NMR (Figure 6) was carried out on a Brucker AF 300 MHz NMR instrument using CD3OD as solvent. ESI-MS m/z 455 (50%), 457 (100%), and 459 (50%) [M + H]+ cluster.

McDonald and Rettie GSH Adduction of an Oxidative Intermediate of CAT. GSH adduction experiments contained 500 µM GSH, 50 µM CAT (1.25 µL from 5 mM DMSO stock), and either pooled HLM (1 mg/mL microsomal protein) or 25 pmol of P450 Supersomes (1A2, 2C9, 2C19, 2D6, or 3A4) in 100 mM KPi buffer (pH 7.4, 125 µL final volume). Reactions were preincubated at 37 °C and 70 rpm for 3 min and were then initiated by the addition of NADPH, to a final concentration of 1 mM. Negative control reactions, lacking NADPH, were run in parallel. After 30 min of incubation at 37 °C, the reactions were quenched with 12.5 µL of 10% HCl and then centrifuged to remove the protein precipitate prior to LC-MS analysis (HPLC analytical method B). The ratio of the GSH adduct peaks produced in the P450 assays was determined by taking the peak area ratio for the adducts relative to that of 3-glutathionyl menadione (thiodione), prepared from menadione by a literature procedure (28), which was added to the quenched incubation mixtures as an internal standard.

Results Synthesis of Hydroxy-BBR Metabolites. We prepared the 1′-OH-BBR according to a literature procedure (17); however, neither 5-OH-BBR nor 6-OH-BBR had been previously synthesized. BBR analogues have been made with varying structure at the 2-alkyl and 3-benzoyl positions (29, 30), but no modifications have so far been attempted on the larger ring in the benzofuran moiety. Using methodology similar to that employed in the synthesis of BBR itself (31), we began with 4-methoxy, 1a, or 5-methoxy salicylaldehyde, 1b, for the synthesis of 5-OH-BBR and 6-OH-BBR, respectively (Scheme 1). The starting material, 1, was first condensed with chloroacetone to form a 2-acetylbenzofuran, 2, which was reduced with hydrazine in refluxing alkaline ethylene glycol to generate compound 3. Some phenolic demethylation also occurred in this step, generating a small amount of compound 4. The phenolic ether, 3, as the major product from the hydrazine reduction, was then deprotected with boron tribromide, and the combined product 4 was further converted to the acyl ester, 5, so that the subsequent Friedl–Craft acylation, with p-methoxyanisoyl chloride, would be directed to the 3-position of the benzofuran ring, 6. Following removal of the ester, 7, and ether phenolic protecting groups, 5-OH-BBR, 9, was obtained from 5-hydroxybenzarone, 8, in low yield and purity, through bromination in the presence of a base promoter. The presence of an additional benzofuran hydroxy group on the benzarone starting material provides for multiple sites of bromination leading to a highly impure product, although a small amount of the 5-OH-BBR product could be purified by preparative HPLC. This problem of multiple bromination sites was overcome in the synthesis of 6-OH-BBR by protecting the benzofuran hydroxyl moiety of 7b with a bulky tert-butyl group, 10. Demethylation was then carried out with sodium ethanethiolate, 11, and bromination was subsequently achieved selectively to give the desired dibromo derivative, 12. The t-butyl protecting group was then cleaved with trifluoroacetic acid to generate 6-OH-BBR, 13. BBR Metabolism in HLMs and Recombinant P450s. Metabolic reactions were conducted with BBR and either HLMs or commercially available recombinant human P450 Supersomes. Recombinant enzymes tested for metabolic activity included CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, 3A4, 4A11, 4F2, 4F3B, and 4F12. HLMs formed a single NADPH-dependent metabolite that exhibited chromatographic (Figure 2B) UV (λmax ) 241, 285 nm) and ESI-MS (m/z 277, 279, 281; 439, 441, 443) properties identical to synthetic 6-OHBBR. Incubations of BBR with recombinant P450s identified CYP2C9 and CYP2C19 as major and minor BBR 6-hydroxy-


Figure 2. BBR metabolism by pooled HLMs and recombinant P450 Supersomes. The figure shows HPLC analyses (at λ ) 285 nm) of incubations of BBR (100 µM) with (A) HLM – NADPH, (B) HLM + NADPH, (C) CYP2C19 + NADPH, (D) CYP2C9 + NADPH, and (E) synthetic standards of 1′-OH BBR, 5-OH BBR, and 6-OH BBR. Other recombinant enzymes that were tested and failed to produce observable metabolites of BBR include the following P450s: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6*1, CYP2E1, CYP2J2, CYP3A4, CYP4A11, CYP4F2, CYP4F3B, and CYP4F12. The unmarked peak at 17.6 min is due to a minor impurity in the BBR substrate.

Figure 3. Benzbromarone metabolism by human liver microsomes (Km ) 30 µM), recombinant CYP2C9 (Km ) 6.8 µM) and CYP2C19 (Km ) 35 µM) all exhibit strong substrate inhibition kinetics. Data points represent the average results from triplicate incubations.

lases, respectively (Figure 2D, 2C). BBR metabolism by HLMs (as well as by CYP2C9 and CYP2C19) exhibited nonlinear kinetics consistent with a model for substrate inhibition (Figure 3). Km, Ki, and Vmax values for 6-OH-BBR formation in HLMs were calculated, using a two-substrate binding site model (32), to be 30 µM, 360 µM, and approximately 2.9 pmol/min/pmol microsomal P450, respectively.2 Metabolism of 6-OH-BBR and 5-OH-BBR by HLMs. Incubation of 6-OH-BBR with HLMs resulted in the NADPHdependent generation of two metabolites, 1 and 2 (Figure 4A). Metabolites 1 and 2 both exhibited [M + H]+ peaks at m/z 455, 457, and 459, indicative of further hydroxylation of 6-OH-BBR. In contrast, 5-OH-BBR formed a single dihydroxylated microsomal metabolite, which coeluted with metabolite 2 (Figure 4B). Metabolites 1 and 2 from 6-OH-BBR (as well as the sole metabolite formed from 5-OH BBR) possessed essentially identical mass spectral properties, as seen by both ESI-MS and tandem mass spectrometry data generated from the daughter 2 The following molar solubility values for BBR were taken from the registry database listed in SciFinder Scholar: 0.27 mM at 25 °C and pH 7.0; 1.2 mM at 25 °C and pH 8.0. Our incubations were carried out at 37 °C and pH 7.4; therefore, we believe that the observed inhibition kinetics at >0.05 mM BBR are not the result of substrate insolubility.

Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1837

Figure 4. Oxidation products from the metabolism of 5-OH BBR and 6-OH BBR by HLMs. The figure shows LCMS spectra, monitored at m/z 457, for incubations of HLM with (A) 6-OH BBR + NADPH, (B) 5-OH BBR + NADPH, and (C) 6-OH BBR – NADPH. Metabolite 2 from 6-OH BBR oxidation coelutes exactly with the product of 5-OH BBR hydroxylation. All incubations were carried out at substrate concentrations of 100 µM.

ions of the m/z 457 peaks (Figure 5). Incubation of 6-OH-BBR with an array of recombinant P450 enzymes showed that only CYP2C9 was capable of metabolizing this substrate and resulted in the production of both dihydroxybenzbromarone metabolites 1 and 2 (data not shown). The peak at 17.1 min in Figure 4A could not be positively identified, although it has an [M + H]+ cluster at m/z 453, 455, and 457. This compound only periodically appears in certain incubations of 6-OH-BBR with HLM and might be an artifact of the workup procedure. Scale-up Formation and NMR Analysis of a Dihydroxybenzbromarone Metabolite. Large-scale microsomal biosynthesis of 6-OH-BBR-derived metabolites 1 and 2 for structural assignment was complicated by a number of factors. First, 6-OH-BBR metabolized by human (and rat) liver microsomes at rates only 1/10 that of 5-OH-BBR. Second, substrate inhibition was again observed at the higher (>100 µM) concentrations for both 6-OH- and 5-OH-BBR, resulting in drastically reduced metabolite formation. However, the incubation of 5-OH-BBR with either rat or HLMs generated essentially the same metabolic profile. Therefore, for convenience, a large scale (250 mL) microsomal incubation of 100 µM 5-OH-BBR was conducted with rat liver microsomes at an incubation concentration of 800 pmol/mL total P450. The product metabolite, deductively identified as metabolite 2, was isolated from the acidified reaction mixture by organic extraction and then purified by preparative HPLC, yielding approximately 200–300 µg of the dihydroxybenzbromarone. Analysis of the product by 1 H NMR spectroscopy showed three singlet peaks in the aromatic region of the spectrum at chemical shift 6.76, 6.96, and 7.93 ppm with integration values of one, one, and two protons, respectively (Figure 6). The peak at δ 7.93 corresponds to the two equivalent protons on the dibromophenol ring, while the signals at δ 6.76 and 6.96 are due to hydrogen atoms on the unsubstituted positions of the benzofuran ring. The fact that these latter two signals are sharp singlets suggests that the two hydrogens occupy positions para to each other; if they were in an ortho or meta arrangement, peak splitting due to coupling would occur. Therefore, the structure of metabolite 2 was assigned as CAT.


McDonald and Rettie

Figure 5. ESI-MS (top) and ESI-MS/MS (bottom, daughter ions of m/z 457) of (A) metabolite 1 and (B) metabolite 2 isolated from the incubation of 6-OH BBR with HLM.

Figure 6. 1H NMR spectrum of CAT isolated from the large scale incubation of 5-OH BBR with rat liver microsomes.

Similar attempts to characterize metabolite 1, from scaled up incubations of 6-OH-BBR with either rat or HLMs, failed to produce enough compound for NMR analysis/structure determination. However, on the basis of the foregoing structural analysis of metabolite 2 and the similarities between the daughter ion spectra shown in Figures 5A and 5B, metabolite 1 must be either 6,7- or 4,6-dihydroxy-BBR. Further attempts to discriminate between these two possibilities involved GC-MS analysis of cyclic boronate esters (33), whose formation would be indicative of a catechol metabolite. Although 3,4-dihydroxycinnamic acid yielded the expected cyclic boronate, neither metabolite 1 nor 2 was successfully derivatized, suggesting that their higher molecular weights may have hindered volatilization of the compounds.

Formation of a GSH Adduct from Further Bioactivation of CAT. CAT was incubated with HLMs in the presence of GSH as trapping agent. LC/ESI-MS analysis of the reaction showed that two adducted oxidative metabolites, termed CAT-SG1 and CAT-SG2, were produced in an NADPHdependent manner (Figure 7). Mass spectral analysis of both metabolites showed clustered (dibromo-containing) peaks centered at m/z 762 and m/z 800, corresponding, respectively, to [M + H]+ and [M + K]+ ions of a catechol-GSH adduct. Additional clustered peaks could be discerned in the spectrum of the CAT-SG2, indicating dibromo-containing fragment ions, which occurred at m/z 685, 687, 689 and 631, 633, 635 and are due to the loss of glycine or pyroglutamate from [M + H]+, respectively (Figure 8). Loss of both of these fragments from


Figure 7. LC-MS analysis, monitored at m/z 762, of incubations of CAT with HLM and GSH, in the presence (top) or absence (bottom) of NADPH cofactor.

Figure 8. ESI-MS spectrum and hypothetical structure of CAT-SG2.

the molecular ion, in combination with the additional loss of carbon monoxide, would generate an ion cluster centered at m/z 530, while cleavage adjacent to the sulfur ion on the side of the GSH chain would result in an ion cluster at m/z 485, 487, 489 (Figure 8). Incubations of BBR and 6-OH-BBR with HLMs in the presence of GSH failed to produce any observable GSH adducts. P450 Isoform Selectivity for CAT-SG Formation. CATSG1 and CAT-SG2 were generated following bioactivation of CAT by several recombinant human P450s in the presence of GSH, although CYP2C9 again appeared to be the dominant enzyme involved (Figure 9). While we are unable to quantify the concentrations of the GSH conjugates produced in these incubations, it is significant that the peak area ratio of CATSG2 to CAT-SG1 remains essentially invariant (at ∼10:1) across all of the P450s examined.

Discussion The present study was initiated with the primary intent of characterizing metabolic pathways of BBR metabolism in HLMs that might lead to bioactivation of the drug and, thereby, contribute to its hepatotoxicity. A secondary goal was to gain some insights into the identity of P450 enzymes that metabolize BBR. From the enzymatic perspective, it is well-recognized that BBR enhances the anticoagulant effect of warfarin by lowering the clearance of (S)-warfarin while having no effect upon metabolism of the (R)-enantiomer (34, 35). This is because BBR acts as a potent competitive inhibitor of CYP2C9-dependent (S)-warfarin 7-hydroxylation, exhibiting a Ki value of ap-


Figure 9. Incubations of CAT with GSH in the presence and absence of recombinant P450, and NADPH cofactor, results in the isozyme selective production of two GSH adducts. The relative CAT-SG adduct ratios were determined by comparison of the adduct ESI-MS [M + H]+ peak areas at m/z 762 relative to the [M + H]+ peak area of an internal standard (thiodione, m/z 478). In all incubations, the amount of CAT-SG1 formed relative to CAT-SG2 remained essentially invariable at a ratio of ∼1 to 10. The graph represents the results from triplicate incubations (standard deviations are indicated by error bar).

proximately 20 nM (29, 30). BBR is also known to inhibit CYP2C19 activity, exhibiting a Ki of 3.7 µM for the inhibition of 3-O-methylfluorescein demethylation (23). Because BBR must be binding to these enzymes to elicit these inhibitory effects, it seemed reasonable to expect that one, or both, of these enzymes might play a role in human liver microsomal metabolism of BBR. HLMs formed a single NADPH-dependent product that was clearly identified as 6-OH-BBR based on comparison with the synthesized chemical standard. Isoform profiling with recombinant human P450s demonstrated further that CYP2C9, and to a lesser extent CYP2C19, contributed to microsomal 6-hydroxylation, confirming an earlier preliminary report that these two enzymes generated a hydroxylated metabolite on the benzofuran nucleus that was not 1′-OH-BBR (23). CYP2C9 was a more active BBR 6-hydroxylase than CYP2C19 and was generally present at higher concentrations in human liver. Consequently, CYP2C9 was likely to be the dominant P450 enzyme catalyzing the 6-hydroxylation pathway in vivo. We should note, however, that the kinetics of BBR metabolism in HLMs (and with CYP2C9 and CYP2C19) were nonlinear, exhibiting significant inhibition at high susbstrate concentrations. While atypical kinetics are well-recognized to complicate in vitro–in vivo predictions of drug clearance (36), early in vivo metabolic studies, conducted in a European population, are consistent with a prominent role for CYP2C9 in BBR clearance. A large population study of 153 individuals found that BBR was metabolized rapidly in 148 subjects, while four showed an intermediate rate of metabolism and a single subject metabolized the drug very slowly (37). A related family study by the same laboratory reported that, despite the fact that only one subject of the 153 examined had been clearly defined as a poor metabolizer of BBR, two brothers, from a family of seven, were found to be slow metabolizers of the drug. These data prompted the conclusion that deficient BBR elimination was a genetically controlled disorder (38). In retrospect, a “poor metabolizer” frequency of ∼1:150 is quite consistent with CYP2C9 being the dominating metabolic enzyme, given that the major inactivating mutation, CYP2C9*3, has an allele frequency of ∼8% in European Americans (39).


McDonald and Rettie

Figure 10. Proposed mechanism(s) of bioactivation for BBR in HLMs. (A) A route by which BBR could be sequentially converted to CAT (metabolite 2) and then on to the experimentally observed GSH adducts, CAT-SG1 and CAT-SG2. (B) An additional hypothetical mechanism of bioactivation, which could occur from metabolite 1 if the identity of this metabolite is the catechol, 6,7-dihydroxybenzbromarone. Dashed arrows represent multiple positions on putative reactive intermediates that could be susceptible to nucleophilic attack by a thiol reagent.

Further metabolism of 6-OH-BBR in HLMs consistently generated two oxidized metabolites, as seen upon analysis by LC/ESI-MS, both of which were again formed predominantly by CYP2C9. The major metabolite 1 and minor metabolite 2 both exhibited [M + H]+ peaks at m/z 455, 457, 459. Comparison of MS data for substrate and products showed that hydroxylation could not have occurred on the dibromohydroxybenzoyl ring, as both metabolites 1 and 2 still gave fragment ions at m/z 277, 279, 281. Moreover, if 6-OH-BBR had undergone hydroxylation on either carbon (1′ or 2′) of the ethyl moiety, we would have expected to see significant fragmentation peaks at [M + H]+ – 18 in the ESI-MS of the product metabolites, due to the loss of water. The fact that neither metabolite 1 nor metabolite 2 exhibited such fragmentation, in either the ESI-MS or the tandem MS of their respective m/z 457 daughter ions, indicates that both compounds were formed by hydroxylation on the aryl ring of the benzofuran moiety of 6-OH-BBR. Because the further hydroxylation of both 5-OHBBR and 6-OH-BBR by HLM generated the same product, that is, metabolite 2, this metabolite was expected to be CAT, a hypothesis that was confirmed by 1H NMR spectroscopy. By extrapolation, metabolite 1 must be either 4,6-dihydroxybenzbromarone or a second catechol, 6,7-dihydroxybenzbromarone. Unfortunately, attempts to distinguish between these two possibilities were unsuccessful due to the low yield of metabolite 1 in bioreactor systems.

On the basis of the foregoing findings, a reasonable scenario for BBR bioactivation could involve successive CYP2C9mediated aryl oxidations at adjacent carbons of the benzofuran ring, generating a catechol that could then be further oxidized to a reactive ortho-quinone intermediate. The ortho-quinone might then adduct cellular protein at cysteine residues in a manner similar to that proposed to occur in the bioactivation of numerous other compounds, including phenytoin (40–43). This proposal is supported by the experimental data, which shows that CAT can undergo further microsomal oxidation to a reactive intermediate, which can be trapped with GSH. In HLMs, this process was strictly NADPH-dependent and resulted in the formation of the two GSH adducts, CAT-SG1 and CATSG2. Incubation of the catechol with the major drug-metabolizing recombinant human P450 enzymes showed that adduct formation was isozyme selective with CYP2C9, increasing the yield of CAT-SG 3–5-fold with respect to the other enzymes tested. In all recombinant P450 reactions, regardless of the isozyme tested, the ratio of CAT-SG2 to CAT-SG1 produced remained essentially invariant. This is strong evidence that the two adducts are formed from a single high-energy intermediate in a spontaneous chemical reaction. A recently proposed mechanism of bioactivation for the selective estrogen receptor modulator, raloxifene, is quite similar to that outlined above for BBR. Raloxifene is reported to undergo CYP3A4-mediated oxidation to a reactive diquinone


methide intermediate (44). The diquinone methide is capable of then undergoing Michael addition reactions at a number of different locations on the molecule, leading to the formation of three distinct monothiol adducts from incubations with either human or rat liver microsomes in the presence of GSH as a trapping agent (44, 45). The catechol, CAT, which bears a striking structural resemblance to raloxifene, is oxidatively converted to an ortho-quinone, through catalysis by CYP2C9. The quinone could then react with thiol via Michael addition at either the 4- or the 7-position of the benzofuran ring to form the two observed GSH adducts (Figure 10A). A second hypothetical pathway for bioactivation of BBR is shown in Figure 10B. Metabolite 1 has been identified as either 4,6dihydroxybenzbromarone or 6,7-dihydroxybenzbromarone. If it is, in fact, the latter of these two compounds, then it also can undergo P450-mediated oxidation to a reactive quinone metabolite capable of adducting thiol reagents/cysteine residues. Because it appears that much more of metabolite 1 is formed from 6-OH-BBR in HLM as compared to metabolite 2, this hypothetical pathway could potentially be more significant for bioactivation of BBR. Alternative bioactivation routes should also be considered. For instance, bioactivation might occur through direct, nonoxidative, thiol adduction of the highly conjugated quinone-enol tautomer of a putative 1′-oxo-6-hydroxybenzbromarone metabolite. However, 1′-oxo-aryl-OH BBR was reported to be only a very minor metabolite in vivo (20, 22), and we did not observe P450-dependent oxidation at the C-1 position in the present studies. The origin of 1′-hydroxylated metabolites of BBR remains to be determined. Another possibility may be through biotransformation and adduction of the halogenated ring of BBR (or any of its metabolites), which bears some resemblance to the known hepatotoxicant, bromobenzene. Bromobenzene undergoes a series of P450-mediated oxidations resulting in the formation of a number of reactive intermediates (46, 47), which have been found to covalently modify over 40 distinct microsomal (48) and cytosolic (49) liver proteins in rat in vivo studies. However, as there are no known in vitro or in vivo metabolites of BBR resulting from oxidative elaboration of the halogenated ring and because we failed to see any GSH adduction arising from BBR itself or 6-OH-BBR products upon coincubation with HLMs, it seems doubtful that toxicity results from biotransformation of the halogenated ring of BBR or its analogues. In summary, BBR is metabolized via 6-OH-BBR to two aryldihydroxy metabolites, one of which, metabolite 2, has been identified as the catechol, CAT. This catechol is further oxidized to a reactive intermediate that can be trapped with GSH to generate two adducts, CAT-SG1 and CAT-SG2. These conjugates likely result from thiol attack at either the 4- or the 7-position of an ortho-quinone intermediate. The identity of the other microsomal aryldihydroxy metabolite of 6-OH-BBR, metabolite 1, is either 4,6-dihydroxy or 6,7-dihydroxybenzbromarone. If metabolite 1 is, in fact, the 6,7-dihydroxy derivative of BBR, it represents a potential additional pathway of bioactivation for the drug. Finally, experiments conducted with various recombinant P450 isozymes indicate that CYP2C9 plays a key role in the sequential metabolism and bioactivation of BBR in human liver. Acknowledgment. This work was funded in part by NIH Grant GM32165. Supporting Information Available: 1H NMR data for all synthesized compounds. This material is available free of charge via the Internet at http://pubs.acs.org.


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TX7001228

Sequential Metabolism and Bioactivation of the Hepatotoxin

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