Characterization of Novel Glutathione Adducts of a Non-Nucleoside

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Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne Abdul Mutlib,*,† Hao Chen,† John Shockcor,† Robert Espina,† Sy Chen,† Kevin Cao,† Alicia Du,† Greg Nemeth,‡ Shimoga Prakash,† and Liang-Shang Gan† Drug Metabolism and Pharmacokinetics Section and Department of Chemical and Physical Sciences, DuPont Pharmaceuticals Company, Stine-Haskell Research Center, P.O. Box 30, 1094 Elkton Road, Newark, Delaware 19714 Received February 14, 2000

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with 13C stable isotope labels. Rats were orally administered [13C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the 13C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of 14C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNAexpressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the R,β-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Introduction The effective treatment of HIV infection and AIDS is still difficult despite tremendous advances in our understanding of the pathogenesis of the disease and the arrival of potent drugs aimed at different, critical targets in the life cycle of the virus (1). Efavirenz (Sustiva) is a potent non-nucleoside reverse transcriptase inhibitor (NNRTI)1 that was approved recently for the treatment of AIDS. Clinical trials have demonstrated a durable, long-lasting reduction in the levels of HIV RNA after * To whom correspondence should be addressed: Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Co., P.O. Box 30, 1094 Elkton Rd., Newark, DE 19714. Telephone: (302) 451-4830. Fax: (302) 366-5769. E-mail: [email protected]. † Drug Metabolism and Pharmacokinetics Section. ‡ Department of Chemical and Physical Sciences.

once-a-day treatment in combination with other drugs (2). In the search for other compounds that exhibit better antiviral activity, a couple of new NNRTIs, including (S)6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), have been introduced into clinical trials. DPC 961 is a potent and specific inhibitor of HIV-1 reverse transcriptase and is currently in the early stages of clinical trials. DPC 961 is a structural analogue of efavirenz differing only by one atom (see Figure 1); the former is a cyclourea, while the later is a cyclocarbamate. 1 Abbreviations: NNRTI, non-nucleoside reverse transcriptase inhibitor; ESI-LC/MS, electrospray ionization-liquid chromatography/ mass spectrometry; MS/MS, mass spectrometry/mass spectrometry; SPE, solid-phase extraction; TOCSY, total correlated spectroscopy; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum coherence.

10.1021/tx000029g CCC: $19.00 © 2000 American Chemical Society Published on Web 08/01/2000

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Scheme 1. Proposed Mechanism for the Formation of Glutathione Conjugates from the Postulated Oxirene Metabolite of DPC 961

Clear differences in the metabolic disposition of efavirenz by a number of species have been previously reported (3). Efavirenz was metabolized extensively by rats, cynomolgus monkeys, and humans with the major metabolite being the 8-OH efavirenz glucuronide. LC/MS and NMR analyses of bile and urine samples from rats showed the presence of glutathione-derived adducts formed by enzymatic addition of glutathione across the triple bond of the acetylene moiety (4). The formation of such conjugate has also been linked to the species-specific nephrotoxicity observed in rats (5, 6). These observations with efavirenz led us to search for and characterize any glutathione adducts produced from DPC 961 in rats and other species. DPC 961 was found to be metabolized extensively by rats, cynomolgus monkeys, and humans with the major urinary metabolite being the glucuronide conjugate of the aromatic ring hydroxylated metabolite. There were significant differences in the nature of metabolites that were excreted in the urine of all three species (7). In addition to the glucuronides and sulfates of hydroxylated metabolites, a number of unusual glutathione-derived adducts were found in the bile and urine of rats treated with DPC 961. The structures of these glutathione conjugates were determined from NMR analyses of isolated metabolites. Interestingly, the structures of these adducts pointed to a totally different mechanism for the formation of the glutathione adducts with DPC 961 as compared to that with efavirenz. The glutathione conjugate of efavirenz was formed by enzymatic addition of glutathione across the triple bond after the initial hydroxylation on the methine carbon of the cyclopropyl ring (4). It was postulated that the glutathione adducts of DPC 961 found in rat bile were produced as a result of oxidation of the triple bond leading to an unstable oxirene intermediate which subsequently rearranged to an R,β-unsaturated cyclobutenyl compound. Such an expansion of the cyclopropyl ring via metabolic activation of an adjacent triple bond has not been previously reported in the literature. This observation led us to further investigate the nature of the rat

liver P450s capable of carrying out such a reaction. The formation of epoxides from P450-catalyzed oxidations of olefins and aromatic rings is well-documented. However, the oxidation of alkynes leading to oxirenes has not received much attention in the past due to both the difficulties in oxidizing such bonds and the high reactivity of the end products. A limited number of reports have appeared in the literature describing the metabolic oxidation of terminal acetylenes leading to product(s) capable of inhibiting P450 enzymes (8). The formation of oxirenes from these terminal acetylenes was invoked, but not demonstrated conclusively by any spectroscopic means. With the application of LC/MS, high-field NMR, and improved isolation techniques, it has become feasible to isolate and characterize such unusual metabolites such as those derived from oxirenes.

Materials and Methods Chemicals and Supplies. DPC 961 was synthesized as described by Corbett et al. (9). Bond-Elute C18 cartridges (10 g/60 cm3) were obtained from Varian Sample Preparation Products (Harbor City, CA). Dexamethasone- and phenobarbital-induced rat liver microsomes, polyclonal antibodies against rat P450s 1A1, 2B1/2B2, and 3A1/3A2, and preimmune IgG were obtained from Xenotech (Kansas City, KS). Polyclonal antibody against rat P450 1A2 was obtained from Daiichi Pure Chemicals Co. (Tokyo, Japan). Rat P450 1A1, P450 1A2, P450 3A1, and P450 3A2 supersomes were purchased from Gentest Corp. (Woburn, MA). The human P450 chemical inhibitors tranylcypromine (2A6), sulfaphenazole (2C9), quinidine (2D6), troleandomycin (3A4), and diethyldithiocarbamate (2E1) were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI), while furafylline (1A2) was obtained from Research Biochemicals International (Natick, MA). Other reagents were either HPLC or analytical grade and were used without further purification. Liquid Chromatography/Mass Spectrometry. The metabolites were separated on a Waters Symmetry C18 column (2.1 mm × 150 mm) by a gradient solvent system consisting of acetonitrile and 10 mM ammonium formate (pH 3.5). The percentage of acetonitrile was increased from 15 to 80% over

Glutathione Adducts of a Disubstituted Alkyne the course of 20 min with the solvent flow rate set at 0.4 mL/ min. After 20 min, the column was washed with 90% acetonitrile for 5 min before re-equilibration with the initial mobile phase was carried out. Aliquots of bile and urine samples were injected directly onto the HPLC column, and the eluent was introduced into the source of the mass spectrometer. To detect the metabolites in the fractions from C18 cartridges and from the semipreparative HPLC column, aliquots (20-50 µL) were introduced into the mass spectrometer using a flow injection analyses method. The mobile phase consisted of a mixture of acetonitrile and 10 mM ammonium formate (pH 3.5) (1:1 v/v) delivered at a rate of 0.35 mL/min. LC/MS was carried out by coupling a Hewlett-Packard HPLC system (HP1100) to either a Finnigan LCQ ion trap mass spectrometer or a PE Sciex API300 quadrupole mass spectrometer. Electrospray ionization-liquid chromatography/mass spectrometry (ESI-LC/MS) was performed on both mass spectrometers operated in the positive ion mode. The glutathione adducts were detected by operating the mass spectrometer either in the full scan mode or by selected ion monitoring of the pseudomolecular ions of the conjugates (MH+ at m/z 638). MSn spectra of fragment ions on the LCQ mass spectrometer were obtained with a relative collision energy of 20-25%. High-Field NMR. All the spectra were obtained on a Bruker Avance 500 MHz NMR spectrometer equipped with either a 2.5 mm 1H/13C inverse LC/NMR flow probe (cell volume of 120 µL) or a 2.5 mm 1H/13C inverse conventional NMR probe. Suppression of the residual water and acetonitrile signals was carried out using the WET solvent suppression method in all the LC/ NMR experiments. Chemical shifts were referenced to DMSO at 2.49 ppm and to acetonitrile at 2.0 ppm. An HP1100 LC system was used with a Bruker diode array detector set at 254 nm. A 3.9 mm × 150 mm Waters Symmetry C18 column was used. A gradient from 75% D2O and 25% acetonitrile-d3 to 50% D2O and 50% acetonitrile-d3 over the course of 20 min at a flow rate of 0.8 mL/min was employed for separation. Both solvents contained 0.05% TFA. The structures of DPC 961 metabolites were determined from proton and carbon one-dimensional NMR as well as proton-proton total correlated spectroscopy (TOCSY), proton-carbon heteronuclear single-quantum coherence (HSQC), and long-range proton-carbon heteronuclear multiple-bond correlation (HMBC) two-dimensional NMR. Synthesis of [13C]DPC 961. DPC 961 labeled with 13C at the acetylenic carbons was prepared utilizing the procedure outlined in Scheme 2 (9). Monoalklyation of [13C2]acetylene (1) with 1-chloro-3-bromopropane (2) provided 5-chloro[1,2-13C]pentyne (3) (10). Lithium cyclopropyl acetylide (4) was prepared by treatment of lableled 3 with n-butyllithium in THF. The

Scheme 2. Synthesis of

13C-Labeled

DPC 961

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Figure 1. Structures of efavirenz and DPC 961. imine 6, obtained by dehydration of 5 with benzenesulfonic acid, was reacted with labeled lithium cyclopropylacetylide (4) to provide racemic 7. Chiral chromatographic separation of 7 on a Chiral Pak As column afforded pure 7a (greater than 99% pure as determined by HPLC; see below for HPLC conditions) which was further diluted with DPC 961 to about 10% 13C enrichment. 1H NMR (500 MHz, CDCl ): δ 0.81 (2H, m, cyclopropyl-CH ), 3 2 0.95 (2H, m, cyclopropyl-CH2), 1.43 (1H, m, cyclopropyl-CH), 5.55 (1H, s, broad, NH), 6.72 (1H, d, Ar-H), 7.34 (1H, dd, Ar-H), 7.55 (1H, s, Ar-H), 7.67 (1H, s, broad, NH). 13C NMR (125 MHz, CDCl3): δ 68 (d, 187.5 Hz), 92.5 (d, 187.5 Hz). ESI-MS: MH+ at m/z 317.5. Synthesis of [14C]DPC 961. The route used to prepare [14C]DPC 961 was analogous to that of [13C]DPC 961, except intermediate 5 (see Scheme 2) was prepared from labeled potassium cyanate. The 14C label on the final product is shown in Figure 1. The purity of [14C]DPC 961 was confirmed by TLC and HPLC. TLC was carried out on a silica gel (5 cm × 20 cm, Baker) using a mixture of hexane and ethyl acetate (1:1 v/v) as the solvent. The product (Rf ) 0.24) was detected using a TLC linear analyzer (Bethold, model Tracemaster 20). The radiochemical purity was found to be >99.8%. An HPLC experiment was performed using a C18 column (Zorbax SB-C18, 0.46 cm × 25 cm). The eluting solvent consisted of phase A (45% methanol in water) and phase B (85% methanol in water). A gradient elution from 100% A to 50% B over the course of 30 min was employed using a flow rate of 1.5 mL/min. The eluent from the column was monitored using on-flow radioactivity (Berthold LB 507) and variable UV (λ ) 254 nm) detectors. The radiochemical purity of the sample was found to be >99.9%. The specific activity (gravimetric) of the purified material was determined to be 54.5 mCi/mmol (171.8 µCi/mg). 1H NMR (500 MHz, DMSOd6): δ 0.73 (2H, m, cyclopropyl-CH2), 0.87 (2H, m, cyclopropylCH2), 1.5 (1H, m, cyclopropyl-CH), 6.88 (1H, d, Ar-H), 7.38 (1H, s, Ar-H), 7.43 (1H, d, Ar-H), 8.51 (1H, s, NH), 9.87 (1H, s, NH). Animal Studies. In separate experiments, bile duct-cannulated Sprague-Dawley rats (300-350 g) housed in metabolic cages were given an oral DPC 961 suspension twice daily at a dose of 30 mg/kg for 3 days before treatment with either the unlabeled DPC 961 (50-500 mg/kg), [14C]DPC 961 (350 mg/kg), or a 13C-enriched compound (300 mg/kg). Both male and female rats were treated with DPC 961 to demonstrate sex differences in the disposition of the compound. Urine and bile were collected over ice on a daily basis and stored frozen at -20 °C until they were analyzed. Bile and urine were collected at 24 h intervals for 3 consecutive days after administration of 14C-labeled DPC 961. Feces were not collected during this study. Isolation of Glutathione and Cysteinylglycine Conjugates of DPC 961 from Rat Bile. Bile pooled from several rats was pooled, diluted 1:1 with distilled water, and loaded onto a C18 cartridge (10 g/60 cm3). The sample was allowed to elute under gravity at a rate of less than 1 mL/min. After the sample had been loaded, the column bed was washed with 30 mL of deionized water followed by elution with different proportions of methanol in water. The fractions containing the metabolites (5-25% methanol) were dried and re-extracted on C18 cartridges using different proportions of methanol in 0.2% acetic acid as the eluent. The second C18-extracted samples containing the glutathione adducts (60-70% methanol fraction) were dried and re-extracted on a C18 cartridge (2 g/10 cm3) using 5-80%

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methanol in 2% acetic acid as the eluent. Aliquots of 4 mL were collected, and the presence of metabolites was confirmed by LC/ MS analyses. The fractions (50-60% methanol) containing the glutathione and cysteinylglycine adducts of DPC 961 were purified twice on a semipreparative HPLC column (Beckman C18, 250 mm × 10 mm) using a mixture of either acetonitrile and 0.1% acetic acid or acetonitrile and 10 mM ammonium formate (pH 3.4) (30:70 v/v) as the mobile phases delivered at a rate of 3.5 mL/min. After the fractions containing the metabolites had been pooled, the samples were dried under vacuum and submitted for NMR analyses. Approximately 1-2 mg of each metabolite was isolated for NMR analyses. The isolation of 13C-labeled glutathione conjugates of DPC 961 was carried out in the same manner as described above. Isolation of N-Acetylcysteine Conjugates of DPC 961 from Rat Urine. The corresponding N-acetylcysteine conjugates of the two glutathione adducts found in rat bile were isolated from urine of female rats given DPC 961 at a dose of 50-500 mg/kg. Urine from 20 female Sprague-Dawley rats was pooled and extracted on Megabond C18 (10 g/60 cm3) cartridges as described for the bile samples. The 40% methanol fraction which contained one of the N-acetylcysteine conjugates was dried and re-injected onto the semipreparative column (Beckman C18, 250 mm × 10 mm) using a mixture of acetonitrile and 0.1% acetic acid as the mobile phase. The percentage of acetonitrile was increased from 35 to 60 over the course of 20 min with the solvent delivered at a rate of 4.7 mL/min. The peak at 5.4 min (tR) was collected, dried, and repurified on the same semipreparative column using the isocratic HPLC system. The mobile phase consisted of 38% acetonitrile in 0.1% acetic acid delivered at a rate of 4.7 mL/min. The N-acetylcysteine conjugate was isolated as a pure metabolite (1 mg after drying the solvents) at 4.7 min (tR). Similarly, the isomeric form of this conjugate, which appeared in the 60-70% fraction from the initial C18 extraction, was re-injected onto the same semipreparative column as described above. This metabolite had a retention time tR of 7.8 min on the HPLC column. The fractions corresponding to this metabolites were pooled and re-injected on an analytical column (Beckman C18, 250 mm × 4.6 mm) using a gradient from 45 to 60% acetonitrile over the course of 10 min at a flow rate at 1.0 mL/min. The N-acetylcysteine conjugate was obtained as a white powder (0.7 mg) after drying the solvents in a Savant Centrifuge (model SC110) overnight. Radiochemical Profiles of Rat Bile and Urine following Oral Treatment with [14C]DPC 961. Analysis of rat bile and urine was conducted by HPLC using a 500TR series Radiomatic Flow Scintillation Analyzer (model D515F04, Packard Instruments). To obtain the metabolite profiles, aliquots of bile and urine (50 and 80 µL, respectively) were injected onto a Zorbax C18 column (250 mm × 4.6 mm) and components resolved using a gradient HPLC system. The mobile phase consisted of two solvents, A (acetonitrile) and B [10 mM ammonium formate (pH 4.0)]. The proportion of A was increased from 20 to 80% over the course of 40 min with a solvent flow rate of 1.0 mL/min. The scintillant cocktail was introduced at a flow rate of 3.0 mL/ min to a mixer where it was mixed with the HPLC effluent and subsequently introduced into the 500 µL flow cell of the on-line radiochemical chemical detector. The recovery of the injected radioactivity was checked by comparing the disintegration per minute (dpm) values obtained from counting the same aliquots of the samples on a liquid scintillation counter (LSC). To obtain the percentage of the excreted dose, aliquots of bile and urine (50 and 100 µL, respectively) were added in duplicates to 5 mL of scintillant (Ultima GOLD, Packard) and the amount of radioactivity was determined by LSC (model Tri-Carb 1900CA, Packard Instruments, Meriden, CT). The amount of radioactivity (dpm) was calculated using a quench curve after the subtraction of the background. Demonstrating the Reversibility of the GS-1 and GS-2 Adducts. The glutathione adduct (GS-2) was incubated either in phosphate buffer (0.1 M, pH 7.4) or in basic aqueous solution (adjusted to pH 9.0 with ammonium hydroxide) in the presence

Mutlib et al. of N-acetylcysteine. Control experiments were set in which the N-acetylcysteine was omitted from the incubations. After 2 h, the reaction was terminated by carrying out solid-phase extraction (SPE) of the mixture on a C18 cartridge. The eluent from the cartridge was dried, reconstituted in the HPLC mobile phase, and subsequently analyzed by LC/MS using the full scan mode. In Vitro Metabolism Studies. Unless otherwise stated, all in vitro incubations were carried out using the following protocol: dexamethasone- or phenobarbital-induced rat liver microsomes (0.2 mg) or cDNA-expressed supersomes (50 pmol of P450), with or without NADPH (2 mM), with or without glutathione (4 mM), MgCl2 (3 mM), DPC 961 (50 µM), with the volume adjusted to 0.5 mL with 0.1 M phosphate buffer (pH 7.4). Chemical inhibitors were added in volumes of less than 6 µL of water or DMSO. Unless stated otherwise, incubations were carried out for 45 min and reactions terminated by the addition of 2 mL of acetonitrile. The samples were vortexed and centrifuged, and the supernatant was separated. The dried extracts were reconstituted in 200 µL of HPLC mobile phase and aliquots analyzed by LC/MS. Characterization of the P450 Responsible for the Formation of GS-1 and GS-2 Adducts. The postulated oxirene metabolic intermediate in rat microsomal incubations was detected by trapping the reactive cyclobutenyl ketone intermediate with glutathione. The adducts produced in vitro were confirmed by LC/MS/MS to be the same as those isolated from in vivo studies. The formation of these GSH adducts was demonstrated by carrying out incubations with cDNA-expressed rat P450 enzymes. The in vitro formation of these GSH adducts was inhibited with commercially available polyclonal antibodies (5 × the microsomal protein concentration) and with chemical inhibitors. (1) Chemical Inhibitors. While specific chemical inhibitors for human P450s have been well characterized (11), few studies have been carried out regarding the specificity of the same inhibitors on P450s from other species. We attempted to use these chemical inhibitors so that we could gain preliminary information about the rat P450 that might be involved in the production of the postulated oxirene intermediate from DPC 961. The incubation mixtures consisted of phenobarbital-induced rat liver microsomes (0.2 mg/mL), NADPH (2 mM), MgCl2 (3 mM), glutathione (2 mM), and various chemical inhibitors used at two concentrations [tranylcypromine (2.5 and 25 µM), sulfaphenazole (2.5 and 25 µM), quinidine (2.5 and 25 µM), troleandomycin (5 and 50 µM), diethyldithiocarbamate (5 and 50 µM), and furafylline (1 and 5 µM)]. Troleandomycin, diethyldithiocarbamate, and furafylline were preincubated for 10 min with the microsomes and NADPH before adding DPC 961 (48 µM). The volume of each incubation was adjusted to 1 mL with 0.1 M sodium phosphate buffer (pH 7.4). Incubations were carried out in duplicate at 37 °C for 60 min. At the end of the incubations, 2 mL of acetonitrile was added to each incubation mixture and the samples were centrifuged. The supernatants were transferred and dried in 12 mm culture tubes at 26 °C under a stream of nitrogen. The residues were reconstituted in acetonitrile and 10 mM ammonium formate (pH 4.0) (1:4 v/v) and analyzed via LC/MS. Once it was demonstrated that troleandomycin (P450 3A4) inhibited the formation of glutathione conjugates, experiments were carried out with ketoconazole (10 and 25 µM) to confirm the involvement of the rat P450 3A enzyme in forming the postulated oxirene metabolite. (2) Inhibition of Glutathione Conjugate Formation with Polyclonal Antibodies. Preliminary studies with either monoclonal or polyclonal antibodies directed against rat P450 1A1, P450 2B1, and P450 3A1 showed no inhibition in the formation of glutathione conjugates, GS-1 and GS-2. From the studies conducted with rat supersomes, it was found that at least two enzymes, P450 3A1 and P450 1A2, were involved in the formation of the postulated oxirene metabolite (see below). Hence, studies were carried out in which the rat microsomes were preincubated with either individual antibodies or a mixture

Glutathione Adducts of a Disubstituted Alkyne


Scheme 3. Proposed Metabolic Pathways for DPC 961 in Rats

of P450 1A2 and P450 3A1 antibodies. Incubations consisted of dexamethasone-induced microsomes (0.15 mg/mL), MgCl2 (3 mM), NADPH (2 mM), glutathione (4 mM), DPC 961 (25 µM), antibodies (0.5 mg/mL), and 0.1 M phosphate buffer in a final volume of 0.25 mL. Preimmune IgGs were used in control experiments. The antibodies were added to the microsomes and preincubated for 15 min prior to adding the rest of the incubation mixture. Incubations were carried out for 35 min, after which the reaction was terminated by addition of 1 mL of acetonitrile. The samples were analyzed by LC/MS after drying and reconstituting the extracts as described above. (3) Glutathione Conjugate Formation in the Presence of Rat Supersomes. To confirm what enzyme was responsible for the formation of the postulated oxirene metabolite and subsequently the glutathione conjugates, experiments were carried out with commercially available rat supersomes P450s 3A1, 3A2, 1A1, and 1A2. Each of these supersomes consisted of the cytochrome P450, the P450 reductase, and cytochrome b5. Incubation mixtures consisted of the supersomes (50 pmol of P450), NADPH (2 mM), glutathione (4 mM), MgCl2 (3 mM), DPC 961 (48 µM), and 0.1 M phosphate buffer adjusted to a final volume of 1 mL. The mixtures were incubated for 35 min, after which they were extracted as described above.

was identified as the cysteinylglycine conjugates derived from M2 via the mercapturic acid pathway. The analyses of urine samples showed M1 as the principal metabolite in both sexes. However, while the male rats showed the urinary excretion of M1 only, the female rats showed significant quantities of the 8-OH sulfate conjugate (M7) as well as detectable levels of the N-acetylcysteine (M5 as two isomers, NAC-1 and NAC-2) conjugates and free phenol, M6. The HPLC/radiochemical profile of bile (1224 h) from a male rat treated with 350 mg/kg of DPC 961 is shown in Figure 2.

Results Characterization of GSH and Cysteinylglycine Adducts in Rat Bile. The LC/MS and radiochemical profiles of bile and urine were similar for male and female rats given equivalent doses of DPC 961. The principal metabolite excreted in the bile of male and female rats was the 8-OH glucuronide conjugate (M1). Metabolite M1 accounted for greater than 90% of the excreted dose in the bile of both male and female rats. Much smaller quantities of the glutathione-related adducts (M2 and M3) were found in the bile of both sexes (see Scheme 3 for the structures). Metabolite M2 appeared as two separate peaks which were later confirmed as stereoisomers (GS-1 and GS-2) of the glutathione conjugate. Metabolite M3 also appeared as two distinct peaks and

Figure 2. HPLC/radiochemical/UV profiles of DPC 961 metabolites present in the 12-24 h bile sample of a male rat treated with 350 mg/kg of DPC 961. The following metabolites were identified (tR) at 8.0 min (M2, isomer GS-1), 8.4 min (M3), 10.4 min (M2, isomer GS-2), 11.1 min (M1), 15.1 min (M7), and 21.0 min (M6). The HPLC/radiochemical/UV profiles of urine samples from rats treated with 350 mg/kg of DPC 961 showed that >90 and 95% of the radioactivity eluted as M1 (tR ) 11.1 min) in female and male rats, respectively. See Scheme 3 for the structures of metabolites.

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Figure 5. Total correlated spectroscopy (TOCSY) of the glutathione adduct (GS-1). Protons d-g of the cyclobutane ring are observed as part of one spin system.

Figure 3. MS/MS (top) and MS/MS/MS (bottom) spectra of the glutathione adduct (GS-1) isolated from bile of rats treated with DPC 961.

Figure 6. HSQC spectrum of GS-1 showing the correlations between the carbons and the attached protons.

Figure 4. 1H NMR spectrum of one of the glutathione adduct isomers (GS-1) isolated from rat bile.

The structures of two major glutathione adducts (M2, identified as GS-1 and GS-2) isolated from rat bile are shown in Scheme 1. The LC/MS analysis of these adducts exhibited MH+ at m/z 638 with characteristic ion fragments at m/z 563, 509, 492, 406, and 331 (Figure 3). The ions at m/z 563 and 509 are characteristic fragments produced on losses of glycine and glutamate moieties from glutathione conjugates (12). Further mass spectrometry/mass spectrometry (MS/MS) studies showed that the aromatic ring of DPC 961 was intact, indicating that the glutathione conjugation was on the cyclopropyl acetylene side chain. The structures of these two glutathione conjugates were elucidated after a series of NMR experiments. The NMR data of one of the adducts (GS1) are shown in Figures 4-7 (1H NMR, TOCSY, HSQC, and HMBC, respectively). The 1H NMR spectrum clearly showed the characteristic signals h, i, j, k, and l for the

Figure 7. HMBC spectrum of GS-1 depicting the correlations between the carbons of carbonyls 2-6 with adjacent protons (2J and 3J coupling).

glutathione protons (Figure 4). The protons of the cyclobutane ring were demonstrated to be part of the same spin system by TOCSY (Figure 5). The correlations between the carbons and protons of the methyl, methylene, and methine are shown in Figure 6. Quaternary carbons 2-6 were assigned by correlations to the neighboring protons by HMBC experiments (Figure 7). As a comparison, the 1H NMR spectrum of the other glutathione adduct (GS-2) is shown in Figure 8; note the differences in the chemical shifts and the split patterns of the cyclobutyl ring protons of the two isomers (compare protons e, f, and g in Figures 4 and 8).


Figure 8. 1H NMR spectrum of the glutathione adduct (GS-2) isolated from rat bile.

Figure 9. 13C spectra of the labeled DPC 961 (top) and the isolated glutathione adduct (bottom).

The structure of one of the cysteinylglycine conjugates (M3) of DPC 961 was confirmed by mass spectral and NMR analyses. The pseudomolecular ion (MH+) was at m/z 509 with a mass spectral fragmentation pattern similar to those of the glutathione adducts (M2). The 1H NMR spectrum was similar to that of GS-1 (M2) except for the distinct absence of glutamate protons at δ 1.90 (2H, glu β - l), 2.30 (2H, glu γ - k), and 3.25 (1H, glu R - m) (see Figure 4 for the 1H NMR spectrum of the corresponding GS-1 adduct). Characterization of Cysteine- and N-Acetylcysteine-Derived Adducts in Rat Urine. The cysteine and N-acetylcysteine adducts of DPC 961 produced MH+ at m/z 452 (data not shown) and 494 (see Figure 10 for the mass spectrum), respectively. The cysteine conjugates present in rat urine were not characterized further. The N-acetylcysteine adducts (M4 identified as NAC-1 and NAC-2) were isolated from rat urine and characterized by LC/NMR. The 1H NMR spectra of the N-acetylcysteine adducts were similar to those obtained for the corresponding GSH adducts isolated from bile, except the signals for the glutamate and glycine moieties were absent in the former as expected. The signals for the protons (designated as a-i) of N-acetylcysteine adducts matched those of the corresponding GSH adducts (see Figure 4 for the assignment of signals). The following chemical shifts were observed for the protons of one of the isolated N-acetylcysteine adducts (NAC-1): δ 1.75 (1H, m, cyclobutyl - g), 1.98 (2H, m, cyclobutyl - f), 2.08


Figure 10. LC/MS profile of the incubation mixture in which GS-2 was exchanged with N-acetylcysteine at physiological pH 7.4 (top). The mass spectrum of one of the N-acetylcysteine adducts (bottom) shows MH+ at m/z 494.

(1H, m, cyclobutyl - g), 2.47 (1H, m, cysteine β - h), 2.70 (1H, m, cysteine β′ - h), 3.37 (1H, q, cyclobutyl - e), 3.48 (1H, q, cyclobutyl - d), 3.90 (1H, m, cysteine R - i), 6.80 (1H, s, Ar - a), 6.98 (1H, d, Ar - c), 7.40 (1H, dd, Ar b). The singlet for the CH3 group of the N-acetyl appeared at 1.80 ppm. The 1H NMR spectrum of the other isomer of the N-acetylcysteine adduct (NAC-2) exhibited chemical shifts at δ 1.55 (1H, m, cyclobutyl - g), 1.72 (2H, m, cyclobutyl - f and g), 2.08 (1H, m, cyclobutyl - f), 2.70 (1H, m, cysteine β - h), 2.92 (1H, m, cysteine β′ - h), 3.60 (1H, m, cyclobutyl - e), 3.75 (1H, q, cyclobutyl - d), 4.18 (1H, m, cysteine R - i), 6.88 (1H, s, Ar - a), 6.95 (1H, d, Ar - c), and 7.45 (1H, dd, Ar - b). The singlet for the CH3 group of the N-acetyl appeared at 1.83 ppm. Characterization of the 13C-Enriched Glutathione Adduct. Figure 9 shows the one-dimensional spectrum of the 13C-labeled DPC 961 (top) and the spectrum of the 13 C-enriched glutathione adduct, GS-1 (bottom). The spectrum of 13C-labeled DPC 961 shows acetylene 13C chemical shifts at 68 and 92.5 ppm. The spectrum of the labeled GS-1 adduct shows a ketone 13C chemical shift (200 ppm) for carbonyl 2 and a methine 13C chemical shift (49.5 ppm) for carbon d. The spectrum of both compounds exhibits the 13C-13C coupling as expected. Radiochemical Profiles of Rat Bile and Urine. Most of the radioactivity was eliminated within 48 h in the bile of rats. Approximately 90% of the recovered radioactivity was found in the bile of both male and female rats with the rest being eliminated in the urine. Total recovery of the radioactivity could not be obtained since feces were not collected. At the end of 3 days, approximately 46 and 75% of the administered doses were recovered in the bile and urine of male and female rats, respectively. It appears that due to the high dose administered to the animals, a significant portion of the radioactivity was eliminated as unabsorbed drug in the feces. Further mass balance studies have shown full recovery of the radioactivity in bile, urine, and feces after administration of [14C]DPC 961 to rats (data not shown). The radiochemical profiles of bile samples (0-12 and 1224 h samples for 3 days) from rats treated with [14C]DPC 961 showed that the major metabolite excreted by both males and females was the 8-OH glucuronide conjugate (M1). There were a number of minor components in the bile, consisting mostly of the glutathione-derived adducts,

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M2 (GS-1 and GS-2) and M3 (see Figure 2). The GSHderived adducts accounted for less than 20% of the eliminated dose in each sample. The HPLC/radiochemical profile of urine samples showed that the females excreted M1 (approximately 90% of the recovered radioactivity) as well as small quantities (less than 10% of the radioactivity) of M7. The males, on the other hand, eliminated M1 as the only major metabolite which accounted for greater than 95% of the excreted dose in every 0-12 and 12-24 h sample from each rat. Reversibility of the Glutathione Adducts. The GS-2 adduct was found to regenerate the cyclobutenyl ketone intermediate as demonstrated by the exchange of GSH with N-acetylcysteine. The LC/MS profile of the reaction mixture showed the presence of N-acetylcysteine adducts with MH+ at m/z 494 (Figure 10). The retention time and mass spectral pattern of one of the N-acetylcysteine adducts were identical to those of the isolated metabolite, M5. Similarly, it was found that when the other isomer, GS-1, was incubated in the presence of N-acetylcysteine the same two mercapturic acid conjugates (Figure 10) were formed. Epimerization of GS-2 to GS-1 under the same experimental conditions was also demonstrated if N-acetylcysteine was omitted from the incubation mixture. Characterization of Enzymes Responsible for the Formation of Glutathione Adducts, GS-1 and GS2. Initial studies with chemical inhibitors specific for human P450 enzymes [furafylline (1A2), sulfaphenazole (2C9), tranylcypromine (2C19), quinidine (2D6), diethydithiocarbamate (2E1), and ketoconazole/troleandomycin (3A4)] indicated the possibility that rat P450 3A1/2 was involved in the formation of the postulated oxirene intermediate. When DPC 961 (48 µM) was incubated with dexamethasone- or phenobarbital-induced rat liver microsomes in the presence of troleandomycin (10 and 50 µM) or ketoconazole (10 and 20 µM), the extent of formation of GS-1 and GS-2 was reduced. The glutathione adducts were formed in the presence of commercially available rat P450 3A1 and P450 1A2 enzymes, while little or no GSH adducts were produced by P450 3A2 and P450 1A1. Studies carried out with the antibody against P450 2B1/2 did not prevent the formation of the glutathione adducts. Polyclonal antibodies to P450 3A1/2 and P450 1A2, when added together to the incubations, were shown to inhibit the formation of the glutathione adducts. Incubations carried out with polyclonal antibodies added separately did not inhibit the formation of the postulated oxirene intermediate (Figure 11). This was probably due to the contribution made by the other isozyme capable of catalyzing the same metabolic reaction. These studies clearly showed that at least two isozymes present in livers of rats were responsible for the formation of the postulated oxirene metabolite. Due to the short half-life of the oxirene metabolite, it was trapped by addition of glutathione to the incubation mixtures.

Discussion DPC 961 was metabolized extensively by rats to a number of metabolites, including the glucuronide and sulfate conjugates of the 8-hydroxylated metabolite (7). In addition to these metabolites, several glutathionederived adducts were found in bile and urine of rats treated with DPC 961. In our previous studies with the

Mutlib et al.

Figure 11. Selected ion monitoring of the glutathione adducts formed in the presence of P450 3A1 and P450 1A2 antibodies.

structural analogue of DPC 961, efavirenz (Figure 1), we had elucidated the metabolic pathways in different species (3, 4). As with DPC 961, a glutathione conjugate was detected in the bile of rats treated with efavirenz. This glutathione conjugate (or its breakdown products) has been implicated as the agent responsible for selective nephrotoxicity observed in rats given high doses of efavirenz (5, 6). Due to a slight structural modification of DPC 961 as compared to efavirenz, we were interested to see if a similar glutathione conjugate would be produced by DPC 961. On characterization of the DPC 961 glutathione conjugates present in bile of rats, it was found that the structure of the adduct was totally different from that produced by efavirenz. In the case of efavirenz, the compound was hydroxylated first on the cyclopropyl ring (on the methine carbon) before an enzyme-catalyzed addition of glutathione could take place. This glutathione transferase-catalyzed addition of glutathione to the triple bond of efavirenz occurred in a specific manner only in rats and did not take place in cynomolgus monkeys and humans (3). For DPC 961, it appears that oxidation took place preferentially on the triple bond, probably giving rise to an unstable oxirene metabolic intermediate which underwent rapid conversion to an R,β-unsaturated carbonyl compound. The possibility of formation of an oxirene intermediate from DPC 961 which has a disubstituted alkyne functional group was demonstrated by isolating and characterizing novel glutathione adducts. A postulated mechanism leading to the formation of the glutathione adducts is shown in Scheme 1. Rat P450 3A1 and P450 1A2 catalyzed the oxidation of the triple bond to the postulated oxirene intermediate which rearranges to form a reactive cyclobutenyl ketone. The


rearrangement to the cyclobutenyl ketone could take place via the ketocarbene a or through the intermediate b as shown in Scheme 1. GSH adds to this metabolic intermediate via 1,4-Michael addition, producing the two isomeric glutathione adducts GS-1 and GS-2 (collectively called M2). The existence of oxirenes as an oxidized product of alkynes has been previously reported (13-16). The chemistry of oxirenes has been reviewed in detail by Lewars (14). It has been suggested that oxirenes do exist as intermediates on oxidation of carbon-carbon triple bonds, and are not present as mere transition states. On chemical oxidation of alkynes with peroxy acids, the formation of oxo carbenes, R,β-unsaturated ketones and ketenes have been described (14). Biochemically, a number of studies in the past with terminal alkynes suggested the formation of oxirenes as metabolic intermediates (17, 18). The formation of highly reactive oxirene species from acetylene, leading to the inactivation of hepatic P450, has been proposed previously (17). Also, the substrateinduced inactivation of aromatase by the formation of oxirene intermediates from acetylinic steroids had been proposed previously (19). The oxidation of 4-ethynylbiphenyl to the carboxylic acid was postulated to involve an oxirene, since the labeled alkyne gave an acid product with complete retention of deuterium (8, 18, 20). The mechanism for the retention of deuterium of terminal acetylene was believed to be due to the formation of a ketene with the migration of deuterium. Similarly, biotransformation studies carried out with 17R-ethynylestradiol in the rats led its authors to conclude that an oxirene intermediate was probably responsible for irreversible bindings (21). Recently, in a study designed to describe the tissue distribution and metabolism of an ethynesulfonamide in rats, the formation of oxirene from a nonterminal alkyne was postulated (22). However, in all these studies, the authors did not substantiate the findings by providing any further spectroscopic evidence for the direct attachment of oxygen onto one of the carbons of the triple bond. The addition of oxygen to one of the carbons of the triple bond in DPC 961 was demonstrated by characterizng the glutathione adduct isolated from rats treated with 13C-labeled DPC 961. The NMR studies confirmed that oxygen was attached to one of the labeled carbons. This was demonstrated by comparing the 13C resonances of the alkyne carbons of 13C-labeled DPC 961 with those of the isolated glutathione adduct. The 13C resonances of the labeled carbons had apparently changed due to the change in the spin states from sp-hydridized carbons to sp2 and sp3. Figure 9 shows the one-dimensional 13C NMR spectra of the labeled DPC 961 (top) and the labeled metabolite (bottom). The spectrum of DPC 961 shows acetylene 13C chemical shifts (68 and 92.5 ppm). The spectrum of the glutathione conjugate shows a ketone 13C chemical shift (200 ppm) for carbonyl 2 and a methine 13C chemical shift (49.5 ppm) for carbon d. The spectrum of both compounds shows the 13C-13C coupling. The possibility of formation of an oxirene intermediate from DPC 961 was demonstrated by trapping the reactive intermediate with added glutathione in the microsomal incubation. The addition of GSH to the cyclobutenyl ketone intermediate was found to be nonenzymatic since the GS-1 and GS-2 conjugates were formed in the presence of cDNA-expressed isozymes which did not have any glutathione transferase activities. It was found that


the naı¨ve microsomes (saline-treated animals) produced only minute quantities of the glutathione adducts. However, in the presence of microsomes obtained from phenobarbital- or dexamethasone-treated rats, significant quantities of the glutathione conjugates (M2) were obtained. These results were not surprising since it is known that mature male or female rats do not have P450 3A1 (also previously classified as cytochrome P450p; 23). However, on treatment of rats with dexamethasone and phenobarbital, the levels of this enzyme increase dramatically (23). Similarly, from in vivo studies, it was observed that induction of the hepatic enzymes was needed before significant levels of the M2 were observed. Addition of polyclonal antibodies specific to rat P450 3A1/2 and P450 1A2 together in the incubation mixture led to 70-80% inhibition in the formation of GSH adducts. The involvement of these isozymes in the formation of the postulated oxirene intermediate was demonstrated by using commercially available rat P450 3A1 and P450 1A2 supersomes. The reversibility of the glutathione adducts was demonstrated by an exchange experiment with N-acetylcysteine under physiological and slightly basic conditions. This study demonstrated that the glutathione conjugate of DPC 961 may act as a transporter molecule of the R,βcyclobutenyl ketone intermediate by dissociation at sites distant from the site of formation. Examples of GSH conjugates of electrophiles that act as transporters of electrophiles include isothiocyanates (24), isocyanates (25), R,β-unsaturated aldehydes (26), and aldehydes (27). The reversibility of glutathione conjugates derived from an isocyanate metabolic intermediate of a formamide has been described previously (28). Overall, the metabolism of DPC 961 was similar to that of efavirenz (4) in that it is principally converted to the glucuronide conjugate of the ring-hydroxylated metabolite. However, changes in the fused cyclic ring of DPC 961, whereby oxygen was replaced with a nitrogen (Figure 1), led to significant differences in the metabolism of the cyclopropyl acetylene side chain. The most notable difference was the nature of glutathione adducts produced by the two compounds. In rats, an initial hydroxylation on the cyclopropyl ring of efavirenz was found to be a prerequisite for enzymatic addition of glutathione across the triple bond (4). However, in the case of DPC 961, rats were able to directly oxidize the triple bond, resulting in an intermediate that was capable of 1,4Michael addition with glutathione. The oxidation of the triple bond in DPC 961 was found to be specifically catalyzed by rat P450s 1A2 and 3A1.

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Mutlib et al. (17) Ortiz de Montellano, P. R., and Kunze, K. L. (1980) Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates. J. Biol. Chem. 255, 5578-5585. (18) Ortiz de Montellano, P. R. (1985) Alkenes and alkynes. In Bioactivation of Foreign Compounds (Anders, M., Ed.) pp 121155, Academic Press, New York. (19) Metcalf, B. W., Wright, C. L., Burkhan, J. P., and Johnston, J. O. (1981) Substrate-induced inactivation of aromatase by allenic and acetylenic steroids. J. Am. Chem. Soc. 103, 32213222. (20) McMahon, R. E., Turner, J. C., Whitaker, G. W., and Sullivan, H. R. (1981) Deuterium isotope effect in the biotransformation of 4-ethynylbiphenyls to 4-biphenylacetic acids by rat hepatic microsomes. Biochem. Biophys. Res. Commun. 99, 662-667. (21) Maggs, J. L., Grabowski, P. S., Rose, M. E., and Park, K. (1982) The biotransformation of 17R-ethynyl [H] estradiool in the rat: irreversible binding and biliary metabolites. Xenobiotica 12, 657668. (22) Radembino, N., Dessalles, M.-C., Trouvin, J.-H., Loiseau, P. M., Gayral, P., Mahuzier, G., Rapp, M., Labarre, P., Godeneche, D., Madelmont, J.-C., Maurizis, J.-C., Veyre, A., and Chabard, J.-L. (1997) Tissue distribution and metabolism in rat of an ethynesulphonamide with filaricidal activity. Xenobiotica 27, 73-85. (23) Cooper, K. O., Reik, L. M., Jayyosi, Z., Bandiera, S., Kelley, M., Ryan, D. E., Daniel, R., McCluskey, S. A., Levin, W., and Thomas, P. E. (1993) Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats. Arch. Biochem. Biophys. 301, 345-354. (24) Bruggeman, I. M., Temmink, J. H. M., and Van Bladeren, J. P. (1986) Glutathione and cysteine mediated cytotoxicity of allyl and benzyl isothiocyanate. Toxicol. Appl. Pharmacol. 83, 349359. (25) Slatter, J. G., Rashed, M. S., Pearson, P. G., Han, D.-G., and Baillie, T. A. (1991) Biotransformation of methylisocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities. Chem. Res. Toxicol. 4, 157-161. (26) Witz, G. (1989) Biological interaction of R,β-unsaturated aldehydes. Free Radical Biol. Med. 7, 333-349. (27) Fennell, T. R., Deal, F. H., and Swenberg, J. A. (1988) Crosslinked adducts on reaction of formaldehyde with amino acids or glutathione, and deoxyribonucleosides or DNA. Proc. Am. Assoc. Cancer Res. 29, 88. (28) Mutlib, A. E., Talaat, R. E., Slatter, J. H., and Abbott, F. S. (1990) Formation and reversibility of S-linked conjugates of N-(1-methyl3,3-diphenylpropyl)isocyanate, an in vivo metabolite of N-(1methyl-3,3-diphenylpropyl)formamide. Drug Metab. Dispos. 18, 1038-1045.

TX000029G

Characterization of Novel Glutathione Adducts of a Non-Nucleoside

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