Assessment of the Metabolism and Intrinsic Reactivity of a Novel

Chem. Res. Toxicol. 2008, 21, 1125–1133

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Assessment of the Metabolism and Intrinsic Reactivity of a Novel Catechol Metabolite J. Matthew Hutzler,*,† Roger J. Melton,† Jeanne M. Rumsey,† David C. Thompson,‡ Dan A. Rock,†,§ and Larry C. Wienkers†,§ Pharmacokinetics, Dynamics, and Metabolism (PDM), and Drug Safety Research and DeVelopment (DSRD), Pfizer Global Research and DeVelopment, 700 Chesterfield Parkway West T3A, Chesterfield, Missouri 63017 ReceiVed December 6, 2007

PH-302 (1) demonstrates potent inhibitory activity against the inducible form of nitric oxide synthase (iNOS). The primary metabolite of PH-302 is a catechol (2) resulting from oxidative demethylenation of the methylenedioxyphenyl moiety by cytochrome P450 3A4. Concerns regarding subsequent two-electron oxidation of 2 to an electrophilic quinone species and the potential for resulting toxicity prompted additional studies to examine the reactivity and metabolic fate of this metabolite. Contrary to literature reports of catechol reactivity, 2 appeared to be resistant to quinone formation in human liver microsomal incubations, as determined by the lack of detectable glutathione (GSH) adducts and no covalent binding to microsomal proteins. In addition, 2 showed no evidence of depletion of intracellular glutathione or cytotoxicity at concentrations up to 1 mM in primary human and rat hepatocytes. In the presence of tyrosinase, spectral evidence indicated that 2 was oxidized to the ortho-quinone, and upon incubation in the presence of GSH, two conjugates were detected and characterized by LC/MS/MS. Lastly, the metabolic pathways of 2 were investigated in rat and human hepatocytes and found to be similar, proceeding via glucuronidation, sulfation, and methylation of the catechol. Collectively, these studies demonstrate that 2 appears to be resistant to further oxidation to quinone in liver microsomes, as well as spontaneous redox cycling, while the formation of phase II metabolites in hepatocytes suggests that multiple detoxication pathways may provide added protection against toxicity in the liver. Introduction PH-302 (1) is a pyrimidineimidazole that was investigated in preclinical development for potent and selective inhibition of the inducible form of nitric oxide synthase (iNOS),1 an enzyme believed to play a role in the pathogenesis of pain and inflammation associated with osteoarthritis (1). The iNOS hemecontaining enzyme is active as a dimer, and PH-302 acts to prevent dimerization of the respective iNOS monomers via direct interaction of the imidazole moiety with the heme prosthetic (2): a novel mechanism for inhibiting this enzyme. We have recently demonstrated that, parallel to this pharmacological mechanism, PH-302 also inhibits cytochrome P450 enzymes, most notably P450 3A4. Not surprisingly, inhibition of P450 3A4 occurred primarily through coordination of the imidazole moiety to the P450 heme iron (i.e., type II inhibition) (3). In addition to the aforementioned mechanism, PH-302 is also a mechanism-based inactivator of P450 3A4 through the formation of a metabolite-inhibitory (MI) complex. This latter mechanism * To whom correspondence should be addressed: Pharmacokinetics, Dynamics, and Metabolism (PDM), Pfizer Global Research and Development, 700 Chesterfield Parkway West, T3A, Lab 319E, Chesterfield, MO 63017. Telephone: 314-274-0261. Fax: 314-274-4426. E-mail: j.matt.hutzler@ pfizer.com. † Pharmacokinetics, Dynamics, and Metabolism (PDM). ‡ Drug Safety Research and Development (DSRD). § Present address: Amgen, Inc., Pharmacokinetics and Drug Metabolism, AW2-D/2381, 1201 Amgen Court West, Seattle, WA 98119-3105. 1 Abbreviations: iNOS, inducible nitric oxide synthase; P450 3A4, cytochrome P450 3A4; GSH, glutathione; MI, metabolite-inhibitory complex; DPM, disintegrations per minute; MDP, methylenedioxyphenyl; UGT, uridine glucuronosyltransferase; SULT, sulfotransferase; COMT, catecholo-methyl transferase; LC/MS/MS, liquid chromatography/tandem mass spectrometry; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid).

requires metabolism at the methylene carbon of the methylenedioxyphenyl moiety, resulting in the formation of a carbene intermediate that noncovalently complexes with the heme iron of P450, effectively inactivating the enzyme in a time-dependent fashion (3). With this mechanism of enzyme inactivation, a thermodynamic branch-point exists, where the carbene intermediate ultimately forms a catechol metabolite that is released from the active site (3). Additional studies investigating the metabolic profile of PH-302 in liver microsomes indicated that the catechol (2) was indeed the primary metabolite. As a result, the reactivity of 2 and the potential formation of quinone(s) became the main focus of concern. It has been well-established that catechol-containing drugs, metabolites, and endogenous molecules may be susceptible to oxidation by a number of key oxidative enzymes to form electrophilic quinones, including cytochrome P450, tyrosinase, and peroxidase enzymes (4). Exposure to these oxidative products may lead to various forms of cytotoxicity and cellular damage because of alkylation of nucleophilic proteins and/or DNA (5–7). The formation of quinone-type intermediates from a number of exogenous chemicals has been demonstrated, including catechol estrogens (8, 9) the nonsteroidal antiestrogen tamoxifen (7), the flavonoid catechin (10), and the recreational drug of abuse 3,4-methylenedioxymethamphetamine (ecstasy) (11). Interestingly, the endogenous neurotransmitter dopamine has also been implicated in forming quinone-type intermediates that cause neurological damage, proposed to play a role in the development of Parkinson’s disease (12). In addition, o-quinones are known to be redox-active molecules that can form significant amounts of reactive oxygen species (ROS), leading to severe

10.1021/tx700429v CCC: $40.75  2008 American Chemical Society Published on Web 04/12/2008

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oxidative stress and the formation of oxidized cellular components, such as lipids and DNA (5). In the present study, the metabolic pathways and intrinsic reactivity of the previously uncharacterized catechol metabolite of PH-302 were assessed, in an effort to understand the safety liability of exposure to this metabolite in humans. Multiple in Vitro approaches were used, including glutathione trapping, spectroscopic techniques, liver microsomal covalent binding, and assessment of cytotoxicity and metabolism in isolated hepatocytes.

Experimental Procedures Materials. Potassium phosphate buffer, Krebs-Henseleit buffer modified, NADPH, magnesium chloride, tolbutamide, eugenol, glutathione, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and tyrosinase (mushroom) were purchased from Sigma-Aldrich (St. Louis, MO). [14C]-Diclofenac was purchased from Amersham (specific activity of 54 µCi/µmol). Pooled human liver microsomes (0.33 nmol/mg of protein) were purchased from BD Biosciences (San Jose, CA), and purified P450 3A4 was purchased from Invitrogen (Carlsbad, CA). All other chemicals were obtained from commercial sources and were of the highest purity available. Synthesis of PH-302 Catechol (2) and [3H]-PH-302. PH-302 was synthesized according to methods reported in McMillan et al. (13) (U.S. Patent 6,432,947) and provided to us by David Davey at Berlex Biosciences (Richmond, CA). The catechol of PH-302 [N-(3-((3,4-dihydroxybenzyl)(methyl)amino)propyl)-N-(2-(1H-imidazol-1-yl)-6 methylpyrimidin-4-yl)glycinamide hydrochloride] was synthesized internally at Pfizer (St. Louis, MO). Briefly, PH-302 (437 mg, 1.0 mmol) was dissolved in CH2Cl2 (40 mL) and cooled to -70 °C. A solution of BCl3 (1.0 M in xylenes, 4.0 mL, 4.0 mmol) was slowly added. The mixture was allowed to slowly warm to room temperature over 8 h and was then stirred an additional 14 h. The reaction was quenched with water (20 mL) and methanol (20 mL). The reaction mixture was concentrated, and the residue was suspended in 1 M aqueous hydrochloric acid (40 mL). The mixture was heated to 50 °C, allowed to cool to room temperature, and filtered, and the resulting filtrate was concentrated to afford 485 mg (91%) of the product as a white solid (1H NMR characterization in the Supporting Information). [3H]-PH-302 was synthesized with a uniform tritium-radiolabel by an outside vendor, received as a total of 300 mCi in 0.5 mL of MeOH, and then purified internally at Pfizer (St. Louis, MO) to 99.7% radiochemical purity (specific activity of 34.3 µCi/nmol). As previously reported (3), [3H]-PH-302 was separated using a YMC ODS-AQ S-3 120 A 5 µm, 4.6 × 150 mm column and a gradient of 90% mobile phase A (0.1% TFA)/10% B (acetonitrile) initially flowing at 1.5 mL/min, followed by a slow gradient to 100% B over 15 min, and holding until 19 min, then returning to initial conditions, and detected by UV (254 nm, retention time of 3.06 min) and radioactivity monitoring (retention time of 3.40 min). Microsomal Incubations. Reactive intermediate trapping studies were performed using human liver microsomes (1 mg/mL) with 100 µM PH-302 or 25 µM of 2 (synthetic standard) and 2 mM glutathione (GSH) in 100 mM potassium phosphate buffer at pH 7.4 containing 3 mM MgCl2. Raloxifene (100 µM) was incubated as a positive control for GSH conjugate formation. Incubations were performed in microcentrifuge tubes (200 µL final volume), preincubated for 3 min at 37 °C, followed by the addition of 1 mM NADPH, and allowed to proceed for 30 min, where 200 µL of ice-cold acetonitrile was added to quench the reaction. Samples were then centrifuged at 10 000 rpm for 10 min, and the supernatant was analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Tyrosinase Incubations. Incubations containing 2 (100 µM) and tyrosinase (10–25 µg/mL) were carried out at room temperature in a stirred cuvette containing 1 mL of 100 mM potassium phosphate buffer (pH 7.4) and 3 mM MgCl2. Absorbance changes were detected on a Hewlett-Packard 8453 diode array spectrophotometer.

Hutzler et al. Subsequent experiments used similar conditions and measured the effect of glutathione on the formation of quinone. Reactions were initiated by the addition of tyrosinase, and absorbance at 386 nm was monitored for up to 5 min. Where indicated, glutathione (1 mM) was present at the beginning of the experiment or was added 3 min following the start of the incubation. In subsequent tyrosinase experiments to characterize GSH conjugate(s), incubations were carried out in glass borosilicate tubes with similar conditions as described above, and samples were quenched with 2 mL of acetonitrile after 10 min of incubation at room temperature. Samples were then centrifuged at 2000 rpm for 10 min, and the top layer of acetonitrile was transferred to a separate glass tube and evaporated to dryness under a stream of nitrogen. The residue was then resuspended in 200 µL of mobile phase and analyzed by LC/MS/ MS for characterization of potential GSH adducts. Glutathione Depletion. The stability of the catechol at physiological pH was assessed by measuring its effect on the rate of glutathione depletion over time. Facile oxidation of 2 to a quinone under these conditions is expected to deplete glutathione via either the formation of quinone conjugates or oxidized glutathione. Reactions contained 1 mM glutathione and 100 or 200 µM 2 (in DMSO). Incubations were carried out at 37 °C in 0.1 M sodium phosphate buffer (pH 7.4) for up to 4 h. At the indicated time points, glutathione was measured using a calorimetric assay and Ellman’s reagent (DTNB), with absorbance at 412 nm monitored using a Hewlett-Packard 8453 diode array spectrophotometer (Palo Alto, CA). In Vitro Covalent Binding to Human Liver Microsomes. [3H]PH-302 (3 µCi) and [14C]-diclofenac (0.11 µCi) were incubated at 10 µM in 100 mM potassium phosphate buffer at pH 7.4 with 1 mg/mL human liver microsomes and 3 mM MgCl2, and 1 mM NADPH was added to initiate incubation for 15 and 30 min at 37 °C, with control incubations being conducted without NADPH. Protein was precipitated by adding 100 µL of cold acetonitrile, followed by 700 µL of cold acetone. After centrifugation, the supernatant was removed and the pellet was washed with 100 µL of 1 M NaOH, followed by sonication for 10 min. The suspension was then repelleted with 700 µL of 4% acetic acid in acetonitrile, washed with 700 µL of 80% methanol, and sonicated for 10 min. The aforementioned steps were repeated until no radioactivity above the background could be detected in the supernatant, usually 5 times. Once background levels were achieved, the pelleted protein was solubilized with 150 µL of 3 M NaOH at 50 °C and 120 µL was placed into a scintillation vial. Samples were then neutralized with acidified acetonitrile, followed by the addition of 5 mL of scintillation cocktail and analysis on a Packard 2700TR scintillation counter (Meriden, CT). Also, 30 µL of the protein was analyzed for the protein concentration via the Bradford assay. Data were normalized for the amount of protein recovered and are reported as picomoles of drug bound per milligram of protein, as calculated using the equation

[

(dpm) × substrate concentration (nmol/mL) ( radioactivityradioactivity ) of substrate (dpm) microsomal protein concentration (mg/mL)

]

× 1000 (1)

Spectrophotometric Analysis. Spectral binding titration studies were carried out using purified P450 3A4 (1 µM) in a 100 µM potassium phosphate buffer at pH 7.4. Incubation mixtures were evenly divided between 1.5 mL reference and sample black-walled cuvettes, and experiments were performed at room temperature by titrating in 1 µL aliquots of 2 (DMSO solution) into sample cuvette (0–12 µM), with an aliquot of DMSO added to the reference cuvette (DMSO < 1%, v/v). After each titration, cuvettes were mixed and allowed to equilibrate for 1 min, and difference spectra were recorded between 350 and 500 nm using a Hitachi (Danbury, CT) U3300 dual-beam spectrophotometer. Data were then analyzed using UV solutions 1.2 software. Metabolite Profiling and Cytotoxicity in Hepatocytes. Fresh human and rat hepatocytes were obtained from Cellz Direct (Pittsboro, NC) in suspension in media without phenol red. For

Metabolism and ReactiVity of a NoVel Catechol

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Scheme 1. Metabolism of PH-302 (1) by P450 3A4 to Its Catechol Metabolite (2), with further Oxidation to the Reactive o-Quinone in the Presence of Tyrosinase

metabolite profiling, hepatocyte incubations were conducted in a 24-well polystyrene plate (1 × 106 viable hepatocytes/mL). For each test compound, 950 µL of prewarmed William’s Medium E (37 °C) was placed into two wells of a 24-well plate. Control incubations contained an additional 450 µL of William’s Medium E, whereas 450 µL of fresh rat hepatocytes were placed into experimental incubations. After a 5 min prewarming incubation at 37 °C, 100 µL of a 15× solution of 2 (and midazolam as a positive control) was added to both the control well and the experimental well containing hepatocytes (1500 µL of total volume). The incubations were allowed to proceed for 120 min in a Thermo Mixer at 37 °C at 400 rotations per minute. At the end of the incubation, 700 µL was taken from each well, and the reaction was quenched by adding 700 µL of ice-cold acetonitrile. The reaction was then centrifuged at 14 000 rpm (Eppendorf 5417R), and the supernatant was transferred to a fresh tube and then analyzed by LC/MS/MS as described below. For cytotoxicity studies, the viability of fresh hepatocytes was improved using a hepatocyte isolation kit (product K2000) obtained from Xenotech (Lenexa, KS) according to the instructions of the manufacturer. Hepatocytes were resuspended (106 cells/mL) in Krebs-Henseleit buffer; medium was supplemented with HEPES (12.5 mM); and pH was adjusted to 7.4. Hepatocytes were placed in a 24-well flat-bottom plate in a 37 °C incubator and mixed continually at 300 rpm for 30 min. Aliquots from test compound stock solutions prepared in DMSO were added to the appropriate wells and incubated under the aforementioned conditions. After incubation for 4 h, hepatocyte viability was assessed by counting the number of cells that excluded trypan blue (0.1%, w/v). To ensure metabolic activity of the hepatocytes, midazolam was also incubated as a control in this study. LC/MS/MS Analysis. Analysis of PH-302, catechol (2), and corresponding phase II conjugates was performed using a Finnigan LCQ Deca XPPLUS ion-trap mass spectrometer (Thermoelectron Corporation, San Jose, CA) connected in line with a ThermoFinnigan Surveyor HPLC system. An Agilent Zorbax Eclipse XDB 5 mm C18 (2.1–150 mm) was used with mobile phase A (0.1% heptafluorobutyric acid in water) and mobile phase B (0.1% heptafluorobutyric acid in acetonitrile) delivered at 0.3 mL/min using gradient elution starting at 0% B for 3 min, ramped to 50% B over 17 min, and then held for 5 min. The system was returned to initial conditions over 0.1 min and allowed to equilibrate for 5 min. Mass spectral analysis was performed using MS and MS/MS for structural elucidation. An electrospray source was set in positive ionization mode with a spray voltage of 4.5 kV, a capillary temperature set to 250 °C, and tube lens set to 10 V. A sheath gas of 45 (arbitrary units) and auxiliary gas of 10 (arbitrary units) were used. For MS/MS characterization, normalized collision energy was 30%. Disappearance of midazolam was measured on a Sciex 3000 triple-quadrupole instrument using multiple reaction monitoring (MRM), with MRM transition m/z 326/291 for midazolam and m/z 271/155 for the internal standard (tolbutamide). The mass spectrometer was equipped with an electrospray ionization (ESI) interface in positive-ion mode and was connected to a Shimadzu

LC20AD HPLC pump and a Leap Technologies CTC PAL (Carrboro, NC) autosampler, with the source temperature set at 400 °C. Analytes were separated using a Zorbax 3.5 µm Eclipse Plus C18 (2.1 × 50 mm) column with a gradient elution profile. The mobile phase was flowing at 0.4 mL/min, and the gradient was initiated and held for the first 0.2 min at 95% A/5% B (A, 0.1% formic acid in H2O; B, 0.1% formic acid in acetonitrile) and was then ramped linearly to 5% A/95% B over the next 0.8 min and held for 0.5 min. The profile was then immediately returned to initial conditions and allowed to re-equilibrate for 1.5 min. All data were analyzed using PE Sciex Analyst 1.4.1 software. Calculation of LogD. LogD values were predicted using the ACD/LogD Suite within the Pallas Combi Suite software package offered by CompuDrug International (Sedona, AZ).

Results Microsomal Incubations. As previously reported (3), when PH-302 (1) was incubated with human liver microsomes, the primary site of metabolism was at the methylenedioxyphenyl moiety, resulting in ring opening and the formation of a catechol metabolite (2) (Scheme 1). This was confirmed by a similar retention time as the authentic catechol standard (Figure 1). Upon incubation with human liver microsomes, there was no apparent disappearance of 2, as indicated by a similar peak area from extracted ion m/z 426 after a 30 min incubation (data not shown). In addition, upon supplementing incubations with 2 mM GSH, there was no evidence of GSH conjugate formation, indicating that 2 was stable in the oxidative environment of human liver microsomes. Raloxifene was employed as a positive control, and a GSH conjugate (M + H+ at m/z 779) was observed and characterized by LC/MS (data not shown). This identified conjugate is consistent with data reported by Chen et al. (14), suggesting that raloxifene is bioactivated by P450 3A4 to either an epoxide or a quinone intermediate and subsequently trapped by GSH conjugation. Tyrosinase Incubations. To further characterize the inherent susceptibility to oxidation, solutions of 2 were incubated with tyrosinase, an enzyme known to catalyze the oxidation of catechols to quinones. Initial experiments demonstrated the timedependent formation of a metabolite that absorbed at 386 nm (trace A in Figure 2), which suggests the formation of a quinone species, likely the o-quinone, although it is known that oquinones may spontaneously isomerize to quinone-methide intermediates (15, 16), and thus, we cannot rule out the formation of quinone-methide based on absorbance properties alone. Interestingly, when GSH was added 3 min after the reaction was initiated, quenching of the absorbance at 386 nm was observed (trace B in Figure 2), while GSH addition prior to catechol resulted in no change in absorbance at 386 nm (trace

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Figure 1. Extracted ion chromatogram (XIC) showing the metabolism of PH-302 (1) to catechol (2) in human liver microsomes (HLMs), matching the retention time of the authentic catechol standard (retention time of 11 min).

Figure 2. Incubation of 2 in the presence of tyrosinase showing the increase in absorbance at 386 nm, indicating the time-dependent formation of o-quinone (trace A), with subsequent quenching by the addition of glutathione (GSH) either 3 min after initiation of incubation (trace B) or at the beginning of incubation (trace C).

C in Figure 2). When the mixture from a subsequent incubation was analyzed by LC/MS/MS, two peaks of similar intensities consistent with GSH conjugate formation (M + H+ at m/z 731) were observed (Figure 3A), which suggests trapping of a quinone intermediate. Mass fragmentation of both ions yielded a spectrum that included characteristic fragment ions at m/z 602 [M + H+ - 129]+ (loss of glutamic acid, Glu), as well as m/z 656 [M + H+ - 75]+ (loss of glycine, Gly) (Figure 3B), confirming the formation of GSH conjugates arising from tyrosinase oxidation of 2. No GSH diadduct (calculated M + H+ at m/z 1036) was formed with tyrosinase incubations. GSH Depletion Assay. To assess the potential for 2 to undergo redox cycling at a physiological pH and form reactive oxygen species (ROS), the rate of glutathione depletion was measured. Data from reactions consisting of 1 mM GSH and 100 or 200 µM 2 indicated that this metabolite does not result in measurable depletion of GSH, as indicated by a decrease in absorbance at 412 nm with time that resembled that observed by the control incubation (Figure S1 in the Supporting Information). The slight loss in absorbance in control incubations was attributed to oxidation of GSH by itself under the reaction conditions. Thus, it does not appear that 2 is redox-active. Microsomal Covalent Binding. [3H]-PH-302 (3 µCi, 10 µM) was incubated in human liver microsomes to investigate whether metabolic activation of 1 resulted in the covalent modification of microsomal protein. Consistent with our stability assessment of 2, we found no evidence of measurable covalent binding to microsomes (0.05 and 0.07 pmol of drug/mg of protein after 15 and 30 min incubations, respectively). In contrast, diclofenac, which has been proposed to be bioactivated to benzoquinone

imine intermediates (17), was found to covalently bind to microsomal protein at a level of 63 pmol of drug/mg of protein, consistent with literature values (57 pmol of drug/mg of protein) (18). An aliquot of this incubation mixture was analyzed by LC-radiomatic detection, according to methods reported in previous studies (3), to confirm enzymatic activity and the formation of catechol in this study (Figure S2 in the Supporting Information). Spectrophotometric Analysis. Upon titration of 2 (0–12 µM) with purified P450 3A4, it was observed that 2 induces a type II spectral shift (Figure 4), indicative of imidazole coordination with the heme iron of P450. This data confirms access and binding by 2 in the P450 3A4 active site. Mass Spectrometry Analysis of Hepatocyte Incubations. The metabolic fate of 2 was assessed in fresh human and rat hepatocytes. Overall, similar metabolic profiles were observed, depicted in the representative chromatogram in Figure 5 and Scheme 2. As shown in Figure 6A, the fragmentation spectra of 2 yielded a diagnostic daughter fragment ion at m/z 304, which is present in the MS/MS spectra of each characterized metabolite and suggests that all biotransformation pathways occur on the catechol moiety. This fragment ion was also observed in the MS scan of all identified metabolites, which was also useful for characterization. As might be expected, glucuronidation of 2 was observed (m/z 602) and characterized in Figure 6B. Upon MS/MS fragmentation, the facile loss of the glucuronide moiety (176 Da) was observed, yielding the parent mass of 2 (m/z 426), consistent with fragmentation properties of glucuronide conjugates. Similarly, a sulfate metabolite was also observed (m/z 506) and shown in Figure 6C, where MS/MS fragmentation yielded the mass of 2 (m/z 426) and the diagnostic ion at m/z 304. In addition, a methylated glucuronide conjugate was observed (m/z 616), which upon MS/ MS fragmentation yielded a daughter ion m/z 440, proposed to be the methylated 2 (Figure 6D). Interestingly, no methylated catechol was observed in our hepatocyte incubations. As a result, we are assuming that any methylated catechol was rapidly glucuronidated, although the order of these metabolic events was not clearly defined. In addition, it was not determined which phenolic position that these various conjugates were attached. Lastly, there were no GSH conjugates (m/z 731) observed from hepatocyte incubations, corroborating the findings in human liver microsomes. Hepatocyte Cytotoxicity. Studies using freshly isolated rat and human hepatocytes demonstrated no evidence of cytotoxicity when cells were exposed to elevated concentrations of 2 (0.3 and 1 mM) for up to 4 h, as measured by trypan blue exclusion (Table 1). Incubation with eugenol, a known cytotoxic



Figure 3. (A) Extracted ion chromatogram showing two mono-GSH conjugates (m/z 731), measurable after oxidation of the catechol to o-quinone by tyrosinase in the presence of GSH. (B) Spectra of product ions obtained by mass fragmentation of the parent GSH conjugates (m/z 731). The m/z 602 product ion is characteristic of a GSH conjugate (neutral loss of 129 mass units from the glutamate moiety). No di-GSH conjugate was observed.

Figure 4. Type II spectrum resulting from the titration of 2 with purified P450 3A4, confirming that 2 does access the active site of P450 3A4, with the preferred binding orientation being imidazole positioned to complex with the heme iron.

agent shown to form a quinone methide that leads to three isomeric GSH conjugates, led to an expected concentrationdependent level of cytotoxicity, with up to 60% loss in viability following 4 h of incubation (Table 1).

Discussion PH-302 is a potent and selective inhibitor of the inducible form of nitric oxide synthase (iNOS) (2), an enzyme involved

in the excess production of nitric oxide (NO) and believed to play a key role in the pathogenesis of inflammation and osteoarthritis (1). PH-302 possesses within its chemical structure an imidazole and a methylenedioxyphenyl (MDP) moiety, demonstrated previously by our laboratory to simultaneously contribute to the inhibition of cytochrome P450 3A4 via distinct mechanisms: type II heme coordination and time-dependent metabolite-inhibitory (MI) complex formation, respectively (3). The latter mechanism is consistent with PH-302 being a mechanism-based inactivator of P450 3A4, in which a carbene intermediate coordinates with the heme of P450 (i.e., MI complex). In addition to the risk of time-dependent inhibition of P450 3A4, competition with this catalytic branch point resulted in the formation of a diffusible catechol metabolite (2). The identification of 2 as the only metabolite represents an additional risk for subsequent oxidation to an electrophilic quinone species. Thus, in Vitro investigations were performed to characterize the reactivity and metabolic fate of this catechol metabolite. It has been well-established that the methylenedioxyphenyl (MDP) functional group is susceptible to bioactivation by P450 enzymes, leading to ring opening and catechol formation (19–21). In turn, catechols have been linked to numerous adverse events, including hepatotoxicity and carcinogenesis, believed in part to be associated with the in situ formation of o-quinones

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Figure 5. Representative total ion chromatograms (TICs) from metabolite profiling samples of 2 in human and rat hepatocyte incubations, showing peaks for 2 (m/z 426), sulfate conjugate (m/z 506), glucuronide conjugate (m/z 602), and a methylated glucuronide (m/z 616). BP ) base peak.

and quinone-methides (5, 6, 22), reactive species capable of alkylating key cellular proteins and/or DNA. Moreover, quinones are redox-active molecules that can undergo futile redox-cycling, producing reactive oxygen species (ROS), such as superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (HO•), causing oxidative stress and damage to macromolecules (5). Specifically, investigations around understanding risk factors associated with estrogen-replacement therapy have shown that the equine estrogens equilin, equilenin, and 8,9-dehydroestrone are metabolized to catechols, which then undergo further oxidation to cytotoxic quinones that cause oxidative stress and alkylation of DNA, ultimately linked to breast and endometrial cancer in women who have undergone long-term estrogenreplacement therapy (8, 23). Additional work has shown that tamoxifen, a nonsteroidal antiestrogen used in the treatment of breast cancer, is metabolized to the catechol 3,4-dihydroxytamoxifen, which reacts with GSH to form multiple conjugates in the presence of microsomal P450 and in human breast cancer cells (24), presumably via its o-quinone. Furthermore, metabolism of the recreational drug of abuse MDMA (ecstasy) to its

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catechol metabolite (N-Me-R-MeDA) has been proposed to lead to numerous adverse events, including neurotoxicity, hepatotoxicity, and cardiotoxicity, attributed to the loss of GSH homeostasis because of conjugation with GSH, as well as the formation of aminochromes (11, 25–27). The most recent demonstration of catechol susceptibility to oxidation is the methylenedioxyhenyl-containing selective serotonin re-uptake inhibitor paroxetine, shown by Kalgutkar et al. to oxidize from catechol to an electrophilic quinone and covalently bind to human liver microsomes and S-9 fractions (28). Given the precedence for the reactivity of catechols, it was particularly surprising to find that 2 did not appear to undergo oxidation to quinone, either spontaneously or enzymatically. First, no glutathione (GSH) conjugates could be characterized from either human liver microsome (Figure S3 in the Supporting Information) or hepatocyte incubations (data not shown) with 1 or 2. Corroborating the lack of GSH conjugate formation was the finding of negligible covalent binding to human liver microsomal protein following incubation of [3H]-PH-302, whereas [14C]-diclofenac, proposed to be bioactivated to a benzoquinone imine intermediate (17, 29), bound covalently to microsomal protein at levels consistent with literature values (18). This data was particularly informative because, even in the most sensitive in Vitro system for evaluating covalent binding (i.e., the absence of competing phase II elimination and other clearance pathways), no covalent binding was observed, which suggests that 2 was stable in a matrix with high P450 enzymatic activity. Lastly, in both rat and human hepatocyte incubations with 2, no cytotoxicity was observed at concentrations up to 1 mM, with incubations lasting up to 4 h, indicating the lack of a species difference, whereas incubation with 1 mM eugenol, reported to form a quinone methide, lead to approximately 30–60% cytotoxicity (Table 1), consistent with literature data (30). While low flux into the hepatocyte resulting from a low logD may normally be a viable explanation for the lack of oxidation, it does not seem plausible in this case, because a number of phase II metabolites were observed, indicating the presence of sufficient concentrations of 2 in the hepatocyte. In addition to a glucuronide and sulfate conjugate, a methylated glucuronide was also characterized, although concentrations were relatively low based on the level of noise observed in the representative chromatogram (Figure 5). This data is consistent with other catechols that have been shown to be substrates of glucuronosyltransferase (UGT), sulfotransferase (SULT), and catechol-O-methyl (COMT) phase II conjugating enzymes (20, 31, 32). It was noted that no methylated catechol was observed in our incubations; therefore, we propose that this species was likely rapidly glucuronidated, and while the order

Scheme 2. Metabolism of 2 in Human and Rat Hepatocytes (Regiochemistry and Order of Methylation/Glucuronidation Undefined)



Figure 6. (A) Spectra of product ions obtained by CID of 2 (m/z 426), showing m/z 304 being the predominant characteristic daughter ion. (B) Spectra of product ions obtained by CID of the glucuronide conjugate of 2 (m/z 602), showing daughter ion m/z 426 upon cleavage of the glucuronide moiety. (C) Spectra of product ions obtained by CID of the sulfate conjugate of 2 (m/z 506), showing daughter ion m/z 426 upon cleavage of the sulfate moiety. (D) Spectra of product ions obtained by CID of the parent methylated glucuronide conjugate of 2 (m/z 616), showing diagnostic daughter ion m/z 440 upon cleavage of the glucuronide moiety. Exact regiochemistry of glucuronide, sulfate, and methylation addition to the catechol was not defined.

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Table 1. Cytotoxicity of 2 in Freshly Isolated Rat and Human Hepatocytes after 4 h of Incubationa treatment

percent cytotoxicity of rat hepatocytes

percent cytotoxicity of human hepatocytes

2 (300 µM) 2 (1 mM) eugenol (300 µM) eugenol (1 mM)

1.8 (1.9) 2.6 (1.3) 13 (2.3) 60 (1.0)

3.3 (1.4) 0.84 (1.1) 19 (2.6) 31 (1.4)

a Cell cytotoxicity was assessed by Trypan blue exclusion as described in the Experimental Procedures. Values are presented as the mean (standard deviation) from three determinations, and viability was g70% after 4 h for all control cells.

of methylation/glucuronidation was not defined, it is assumed that methylation occurs prior to glucuronidation based on literature examples (28, 33). Ultimately, in addition to 2 not appearing to be susceptible to P450-mediated quinone formation or redox cycling, it is likely that the relative rates of the operative phase II metabolic pathways of elimination may serve as added protection against cytotoxicity in the hepatocyte. This argument was nicely demonstrated recently by showing an increase in the cytotoxicity induced by polychlorinated biphenyl catechols in UGT- and COMT-inhibited hepatocytes (33). It is not clear based on the current data why 2 does not undergo P450-mediated oxidation to quinone, because the majority of catechols identified in the literature appear to oxidize readily to quinone species. From a physicochemical properties standpoint, a particularly interesting study by Moridani et al. showed that, in general, lipophilicity (logD7.4) and pKa were two key properties of catechols that were likely to impact their reactivity and cytotoxicity toward rat hepatocytes (34). In this work, catechols with higher logD7.4 and pKa values tended to be more cytotoxic toward hepatocytes when compared to other less lipophilic catechols, although the exact mechanism of toxicity (covalent binding after metabolic activation versus production of ROS) was not defined. One simple hypothesis may be that catechols with higher lipophilicity relative to 2 (logD7.4 ) -1.3) are better substrates for P450 and are thus more rapidly oxidized. For example, other higher logD catecholic compounds, such as the flavenoid catechin (logD7.4 ) 2.8) and tamoxifen (logD7.4 ) 6.6), are readily oxidized to their quinone intermediates by P450 (10, 35). Arguing against this hypothesis, however, is the fact that dopamine and ecstasy (MDMA), following demethylenation to form catechol, are both reported to readily oxidize to quinone intermediates (6, 11, 26), despite having low logD values (logD7.4 ∼ –2.0). In addition, 2 is apparently able to access the P450 3A4 active site, demonstrated by the type II spectra that is induced when titrated with purified P450 3A4 in a cuvette (Figure 4). This type II spectra is similar to the one previously demonstrated by the parent PH-302 (3). However, it was found that, despite the type II binding orientation (i.e., imidazole complexing to heme iron) being preferred, metabolism at the methylenedioxyphenyl carbon still occurred, albeit at a slow rate, as indicated by a slow rate of P450 3A4 inactivation (3). This observation is consistent with work demonstrating that compounds exhibiting type II binding with P450 3A4 tend to be more metabolically stable compared to compounds that bind in a type I fashion (36), although exceptions likely exist. In this case, oxidation of 2 does not appear to be operable to any substantial degree, despite access to the P450 3A4 active site, which may be related to 2 being bound primarily in a type II orientation. It would be interesting to investigate whether an analogue of 1 or 2 that did not have preferred type II binding (i.e., imidazole replacement) would be more susceptible to oxidation to quinone, a potential result of the P450 3A4 enzyme not being predominately in a “low-

spin” state. We did not have a sufficient number of suitable analogues available to answer this question or the importance of logD and pKa. As such, this will be the focus of future investigations. In addition to cytochrome P450 enzymes, tyrosinase is a copper-containing human enzyme that has been shown to oxidize catechols to their reactive quinone species, specifically o-quinones (6, 10, 37). Thus, we were interested in whether 2 may be oxidized to quinone by this enzyme. When incubating 2 in buffer in the presence of tyrosinase, an increase in spectral absorbance at 386 nm was observed, indicating time-dependent formation of quinone (Figure 2), and two mono-GSH adducts (m/z 731) were readily identified and characterized (Figure 3). Interestingly, only mono-adducts were observed, and neither di-adduct nor a rearranged di-adduct ortho-benzoquinone imine, which has been characterized previously by Samuel et al. with o-quinones (38), was observed. This observation of GSH adducts with tyrosinase was extremely valuable, because the stability of potential GSH conjugates of 2 is unknown, making it difficult to definitively conclude that no GSH conjugates formed in liver microsomes and hepatocytes based solely on the inability to detect conjugates. With this data in hand, we were confident that, if GSH conjugates of 2 formed in microsomal or hepatocyte studies, our LC/MS/MS methods would have been sufficient for characterization. The question still remains as to why 2 appears resistant to oxidation to quinone by P450, even with evidence showing its presence in the active site, but is readily oxidized to o-quinone by tyrosinase. Tyrosinase acts similarly to P450, in that the oxidation of catechol to o-quinone proceeds by a reduction of molecular oxygen, with the production of H2O (39). However, the tyrosinase active site is defined by a dicopper peroxo oxidizing species (oxy-tyrosinase), which likely possesses a different oxidation potential than the P450 heme iron-oxo species, possibly explaining the catalytic differences observed in our studies and maybe arguing for a more complex electronic explanation. In summary, a previously uncharacterized catechol metabolite has been found to be stable to further oxidation to electrophilic quinone species in both liver microsomes and hepatocytes, as indicated by several in Vitro investigations including the lack of cytotoxicity in isolated hepatocytes, which challenges the general dogma of catechol reactivity. While it is not entirely clear why 2 is not susceptible to further oxidation to quinone(s) or redox cycling in microsomes/hepatocytes, it is reasonable to suggest that, with preferred type II binding to P450 and multiple phase II elimination pathways, exposure to this metabolite may not lead to severe adverse events, such as hepatotoxicity. Acknowledgment. The authors would like to thank Mark Schnute for synthesizing the catechol metabolite of PH-302, Evan Smith (Pfizer, La Jolla) for assisting with GSH-trapping studies in liver microsomes, and Jeffrey C. Stevens and J. Scott Daniels for thoughtful review of our manuscript. U.S. Patent 6,432,947. Supporting Information Available: Spectral data demonstrating that 2 did not appear to deplete GSH relative to the control and thus does not appear to be redox-active (Figure S1), radiochromatogram of the sample taken from microsomal covalent binding studies ensuring catechol (2) formation (Figure S2), extracted ion chromatogram from human liver microsome incubation with PH-302 (1) (Figure S3), and 1H NMR of PH302 catechol. This material is available free of charge via the Internet at http://pubs.acs.org.



References (1) Salerno, L., Sorrenti, V., Di Giacomo, C., Romeo, G., and Siracusa, M. A. (2002) Progress in the development of selective nitric oxide synthase (NOS) inhibitors. Curr. Pharm. Des. 8, 177–200. (2) Blasko, E., Glaser, C. B., Devlin, J. J., Xia, W., Feldman, R. I., Polokoff, M. A., Phillips, G. B., Whitlow, M., Auld, D. S., McMillan, K., Ghosh, S., Stuehr, D. J., and Parkinson, J. F. (2002) Mechanistic studies with potent and selective inducible nitric oxide synthase dimerization inhibitors. J. Biol. Chem. 277, 295–302. (3) Hutzler, J. M., Melton, R. J., Rumsey, J. M., Schnute, M. E., Locuson, C. W., and Wienkers, L. C. (2006) Inhibition of cytochrome P450 3A4 by a pyrimidineimidazole: Evidence for complex heme interactions. Chem. Res. Toxicol. 19, 1650–1659. (4) O’Brien, P. J. (1991) Molecular mechanisms of quinone cytotoxicity. Chem.-Biol. Interact. 80, 1–41. (5) Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000) Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135–160. (6) Cavalieri, E. L., Li, K. M., Balu, N., Saeed, M., Devanesan, P., Higginbotham, S., Zhao, J., Gross, M. L., and Rogan, E. G. (2002) Catechol ortho-quinones: The electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases. Carcinogenesis 23, 1071–1077. (7) Liu, X., Pisha, E., Tonetti, D. A., Yao, D., Li, Y., Yao, J., Burdette, J. E., and Bolton, J. L. (2003) Antiestrogenic and DNA damaging effects induced by tamoxifen and toremifene metabolites. Chem. Res. Toxicol. 16, 832–837. (8) Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Role of quinoids in estrogen carcinogenesis. Chem. Res. Toxicol. 11, 1113–1127. (9) Bolton, J. L., and Chang, M. (2001) Quinoids as reactive intermediates in estrogen carcinogenesis. AdV. Exp. Med. Biol. 500, 497–507. (10) Moridani, M. Y., Scobie, H., Salehi, P., and O’Brien, P. J. (2001) Catechin metabolism: Glutathione conjugate formation catalyzed by tyrosinase, peroxidase, and cytochrome P450. Chem. Res. Toxicol. 14, 841–848. (11) Carvalho, M., Remiao, F., Milhazes, N., Borges, F., Fernandes, E., Monteiro Mdo, C., Goncalves, M. J., Seabra, V., Amado, F., Carvalho, F., and Bastos, M. L. (2004) Metabolism is required for the expression of ecstasy-induced cardiotoxicity in vitro. Chem. Res. Toxicol. 17, 623– 632. (12) Hastings, T. G., and Zigmond, M. J. (1994) Identification of catecholprotein conjugates in neostriatal slices incubated with [3H]dopamine: Impact of ascorbic acid and glutathione. J. Neurochem. 63, 1126– 1132. (13) McMillan, K., Adler, M., Auld, D. S., Baldwin, J. J., Blasko, E., Browne, L. J., Chelsky, D., Davey, D., Dolle, R. E., Eagen, K. A., Erickson, S., Feldman, R. I., Glaser, C. B., Mallari, C., Morrissey, M. M., Ohlmeyer, M. H., Pan, G., Parkinson, J. F., Phillips, G. B., Polokoff, M. A., Sigal, N. H., Vergona, R., Whitlow, M., Young, T. A., and Devlin, J. J. (2000) Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry. Proc. Natl. Acad. Sci. U.S.A. 97, 1506–1511. (14) Chen, Q., Ngui, J. S., Doss, G. A., Wang, R. W., Cai, X., DiNinno, F. P., Blizzard, T. A., Hammond, M. L., Stearns, R. A., Evans, D. C., Baillie, T. A., and Tang, W. (2002) Cytochrome P450 3A4-mediated bioactivation of raloxifene: Irreversible enzyme inhibition and thiol adduct formation. Chem. Res. Toxicol. 15, 907–914. (15) Bolton, J. L., Acay, N. M., and Vukomanovic, V. (1994) Evidence that 4-allyl-o-quinones spontaneously rearrange to their more electrophilic quinone methides: Potential bioactivation mechanism for the hepatocarcinogen safrole. Chem. Res. Toxicol. 7, 443–450. (16) Sugumaran, M., and Bolton, J. (1995) Direct evidence for quinonequinone methide tautomerism during tyrosinase catalyzed oxidation of 4allylcatechol. Biochem. Biophys. Res. Commun. 213, 469–474. (17) Tang, W., Stearns, R. A., Wang, R. W., Chiu, S. H., and Baillie, T. A. (1999) Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac. Chem. Res. Toxicol. 12, 192– 199. (18) Evans, D. C., Watt, A. P., Nicoll-Griffith, D. A., and Baillie, T. A. (2004) Drug-protein adducts: An industry perspective on minimizing the potential for drug bioactivation in drug discovery and development. Chem. Res. Toxicol. 17, 3–16. (19) Murray, M. (2000) Mechanisms of inhibitory and regulatory effects of methylenedioxyphenyl compounds on cytochrome P450-dependent drug oxidation. Curr. Drug Metab. 1, 67–84. (20) Beaumont, K. C., Causey, A. G., Coates, P. E., and Smith, D. A. (1996) Pharmacokinetics and metabolism of zamifenacin in mouse, rat, dog and man. Xenobiotica 26, 459–471. (21) Kumagai, Y., Wickham, K. A., Schmitz, D. A., and Cho, A. K. (1991) Metabolism of methylenedioxyphenyl compounds by rabbit liver

(22)

(23)

(24)

(25) (26)

(27)

(28)

(29)

(30) (31)

(32)

(33)

(34) (35)

(36)

(37)

(38)

(39)

preparations. Participation of different cytochrome P450 isozymes in the demethylenation reaction. Biochem. Pharmacol. 42, 1061–1067. Zhang, F., Chen, Y., Pisha, E., Shen, L., Xiong, Y., van Breemen, R. B., and Bolton, J. L. (1999) The major metabolite of equilin, 4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyequilenin-o-quinone. Chem. Res. Toxicol. 12, 204–213. Zhang, F., Yao, D., Hua, Y., van Breemen, R. B., and Bolton, J. L. (2001) Synthesis and reactivity of the catechol metabolites from the equine estrogen, 8,9-dehydroestrone. Chem. Res. Toxicol. 14, 754– 763. Zhang, F., Fan, P. W., Liu, X., Shen, L., van Breeman, R. B., and Bolton, J. L. (2000) Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-Dihydroxytamoxifen-o-quinone. Chem. Res. Toxicol. 13, 53–62. Monks, T. J., Bai, F., Miller, R. T., and Lau, S. S. (2001) Serotonergic neurotoxicity of methylenedioxyamphetamine and methylenedioxymetamphetamine. AdV. Exp. Med. Biol. 500, 397–406. Carvalho, M., Milhazes, N., Remiao, F., Borges, F., Fernandes, E., Amado, F., Monks, T. J., Carvalho, F., and Bastos, M. L. (2004) Hepatotoxicity of 3,4-methylenedioxyamphetamine and R-methyldopamine in isolated rat hepatocytes: Formation of glutathione conjugates. Arch. Toxicol. 78, 16–24. Carvalho, M., Hawksworth, G., Milhazes, N., Borges, F., Monks, T. J., Fernandes, E., Carvalho, F., and Bastos, M. L. (2002) Role of metabolites in MDMA (ecstasy)-induced nephrotoxicity: An in vitro study using rat and human renal proximal tubular cells. Arch. Toxicol. 76, 581–588. Zhao, S. X., Dalvie, D. K., Kelly, J. M., Soglia, J. R., Frederick, K. S., Smith, E. B., Obach, R. S., and Kalgutkar, A. S. (2007) NADPHdependent covalent binding of [3H]paroxetine to human liver microsomes and S-9 fractions: Identification of an electrophilic quinone metabolite of paroxetine. Chem. Res. Toxicol. 20, 1649–1657. Tang, W., Stearns, R. A., Bandiera, S. M., Zhang, Y., Raab, C., Braun, M. P., Dean, D. C., Pang, J., Leung, K. H., Doss, G. A., Strauss, J. R., Kwei, G. Y., Rushmore, T. H., Chiu, S. H., and Baillie, T. A. (1999) Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: Identification of glutathione conjugated metabolites. Drug Metab. Dispos. 27, 365–372. Thompson, D. C., Constantin-Teodosiu, D., and Moldeus, P. (1991) Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes. Chem.-Biol. Interact. 77, 137–147. Antonio, L., Xu, J., Little, J. M., Burchell, B., Magdalou, J., and Radominska-Pandya, A. (2003) Glucuronidation of catechols by human hepatic, gastric, and intestinal microsomal UDP-glucuronosyltransferases (UGT) and recombinant UGT1A6, UGT1A9, and UGT2B7. Arch. Biochem. Biophys. 411, 251–261. Loureiro, A. I., Bonifacio, M. J., Fernandes-Lopes, C., Almeida, L., Wright, L. C., and Soares-Da-Silva, P. (2006) Human metabolism of nebicapone (BIA 3-202), a novel catechol-o-methyltransferase inhibitor: Characterization of in vitro glucuronidation. Drug Metab. Dispos. 34, 1856–1862. Sadeghi-Aliabadi, H., Chan, K., Lehmler, H. J., Robertson, L. W., and O’Brien, P. J. (2007) Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes. Chem.-Biol. Interact. 167, 184–192. Moridani, M. Y., Siraki, A., Chevaldina, T., Scobie, H., and O’Brien, P. J. (2004) Quantitative structure toxicity relationships for catechols in isolated rat hepatocytes. Chem.-Biol. Interact. 147, 297–307. Yao, D., Zhang, F., Yu, L., Yang, Y., van Breemen, R. B., and Bolton, J. L. (2001) Synthesis and reactivity of potential toxic metabolites of tamoxifen analogues: Droloxifene and toremifene o-quinones. Chem. Res. Toxicol. 14, 1643–1653. Chiba, M., Jin, L., Neway, W., Vacca, J. P., Tata, J. R., Chapman, K., and Lin, J. H. (2001) P450 interaction with HIV protease inhibitors: Relationship between metabolic stability, inhibitory potency, and P450 binding spectra. Drug Metab. Dispos. 29, 1–3. Naish, S., Holden, J. L., Cooksey, C. J., and Riley, P. A. (1988) Major primary cytotoxic product of 4-hydroxyanisole oxidation by mushroom tyrosinase is 4-methoxy ortho-benzoquinone. Pigm. Cell Res. 1, 382– 385. Samuel, K., Yin, W., Stearns, R. A., Tang, Y. S., Chaudhary, A. G., Jewell, J. P., Lanza, T., Jr., Lin, L. S., Hagmann, W. K., Evans, D. C., and Kumar, S. (2003) Addressing the metabolic activation potential of new leads in drug discovery: A case study using ion trap mass spectrometry and tritium labeling techniques. J. Mass Spectrom. 38, 211–221. Sanchez-Ferrer, A., Rodriguez-Lopez, J. N., Garcia-Canovas, F., and Garcia-Carmona, F. (1995) Tyrosinase: A comprehensive review of its mechanism. Biochim. Biophys. Acta 1247, 1–11.

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