P450-Mediated Metabolism of 1-[3-(Aminomethyl ... - ACS Publications

Chem. Res. Toxicol. 2002, 15, 63-75

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P450-Mediated Metabolism of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole5-carboxamide (DPC 423) and Its Analogues to Aldoximes. Characterization of Glutathione Conjugates of Postulated Intermediates Derived from Aldoximes Abdul E. Mutlib,* Shiang-Yuan Chen, Robert J. Espina, John Shockcor, Shimoga R. Prakash, and Liang-Shang Gan Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Company, Stine-Haskell Research Center, P.O. Box 30, 1094 Elkton Road, Newark, Delaware 19714 Received July 20, 2001

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond in forming this GSH adduct. The lack of isotope effect in forming the aldoxime from [13CD2]DPC 602 suggests its formation does not occur through the imine intermediate. Instead the data supports the postulated mechanism of hydroxylamine and nitroso intermediates as precursors to the aldoxime. However, the formation of the GSH adduct from [13CD2]DPC 602 did show a significant intramolecular kinetic isotope effect (kH/kD ) 2.3) since a carbon-deuterium bond had to be broken on the aldoxime prior to the formation of the adduct. A stable nitrile oxide derived from DPC 602 was postulated as the reactive intermediate responsible for forming this unique GSH adduct.

Introduction The metabolism of 1-[3-(aminomethyl)phenyl]-N-[3fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) (Figure * To whom correspondence should be addressed. Phone: (302) 451-4830. Fax: (302) 366-5769. E-mail: abdul.mutlib@dupontpharma. com.

1) and its analogues has been recently reported (1-3). The formation of unique metabolites, produced specifically by the metabolism of benzylamine moiety was described in detail. Structures of unusual glucuronide, sulfamate, glutamate, and glutathione conjugates were unequivocally assigned based on spectroscopic data as well as by comparisons with synthetic standards. The structure of one of the glutathione (GSH) conjugates

10.1021/tx0101189 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/07/2001

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Mutlib et al.

Figure 1. Structures of DPC 423, DPC 602, [13CD2]DPC 602, and SX 737. Figure 3. Structures of DPC 423 analogues that were used to elucidate the secondary metabolic pathways.

Figure 2. Structure of GSH conjugate of DPC 423. Similar GSH adducts were produced by DPC 602, [13CD2]DPC 602, and SX 737.

(Figure 2) produced by DPC 423 was postulated based only on mass spectral data and needed further confirmation. During our initial in vitro studies with DPC 423 (and its analogues), it was found that oximes were produced as significant metabolites by rat liver microsomes (2). Studies performed with microsomes fortified with GSH showed much lower levels of these oxime metabolites; however, an additional metabolite was observed during the LC/MS analyses of the microsomal extract. This metabolite corresponded to a GSH adduct as demonstrated by MS/MS1 experiments which showed typical losses of glutamate and glycine moieties from the protonated molecular ion. Apparently, a metabolite that was capable of reacting with a nucleophile such as GSH was produced by DPC 423 in the presence of rat liver microsomes. The in vitro GSH adduct produced identical LC/MS retention time and MS/MS fragmentation pattern as the metabolite isolated from bile of rats dosed with DPC 423. It was postulated that this GSH adduct was derived from an oxime metabolite of DPC 423. The possibility of a GSH adduct of an oxime was interesting since a metabolite of this type had not been reported before. Oximes have been previously described as one of the products formed during the metabolism of amines, amidines, and guanidines (4-8). However, very few studies have been performed to date describing the metabolic fates of oximes. To further elucidate the structure of this unique GSH adduct, metabolism studies were done in rats with closely related benzylamines, including SX 737, DPC 602, and [13CD2]DPC 602 (see Figure 1) and similar GSH adducts 1 Abbreviations: LC-ESI/MS, liquid chromatography-electrospray ionization mass spectrometry; 1H NMR, proton nuclear magnetic resonance; MS/MS, mass spectrometry/mass spectrometry; TOCSY, total correlated spectroscopy; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence.

isolated from bile. The long-range coupling experiments employing high-field NMR and the C13-labeled analogue were used to confirm the unique structures of these metabolites. The availability of deuterated DPC 602 not only aided the structural elucidation of the GSH adduct but also confirmed the intramolecular isotope effect in forming this conjugate. The GSH conjugate of a model compound, benzaldehyde oxime, was synthesized, and its characteristic mass spectral fragmentation pattern was compared to that produced by the in vivo oxime GSH adducts of DPC 423 (and its analogues). A comparison was also made between the long-range NMR spectra of the synthetic GSH adduct and the isolated metabolite. Due to the possibility of Lossen rearrangement reaction (9) that would have led to an isomeric isocyanate derived GSH adduct, a synthetic conjugate of benzyl isocyanate was also made and its mass spectral and NMR properties studied. The rat P450 enzymes responsible for converting the DPC 423 oxime to a GSH trappable reactive intermediate was also investigated. Results from our previous studies with cDNA-expressed rat P450 enzymes showed that P450 3A1/2 were capable of forming the oximes from DPC 423 (2). Hence we were interested in finding out if the same enzymes were also involved in further metabolism of the oximes. Metabolism studies were performed with DPC 423 and DPC 602 oximes to demonstrate that the oxime was a precursor to the GSH conjugate. Furthermore, to support our proposed mechanisms leading to this GSH adduct, in vitro and in vivo studies were also done with several possible metabolic intermediates of DPC 423, including a nitrile, an amide, and a hydroxamate (Figure 3). To demonstrate the occurrence of a similar GSH adduct with other aromatic aldoximes, microsomal incubations were performed with benzaldehyde oxime. The possible mechanism leading to these unique aldoxime GSH conjugates from the oxime are postulated and discussed in this report.

Materials and Methods Chemicals and Supplies. 1-[3-(Aminomethyl)phenyl]-N-[3fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)1H-pyrazole-5-carboxamide (DPC 423) and its analogues, DPC 602 and SX 737 were synthesized and characterized by the DuPont Pharmaceuticals Company. Glutathione, phenyl isocyanate and syn-benzaldehyde oxime, were purchased from Sigma

Glutathione Conjugates of Aldoximes Scheme 1. Synthesis of Metabolites M1 (aldehyde) and M13 (oxime)

Chemical Co. (St.Louis, MO). Dexamethasone induced rat liver microsomes were obtained from Xenotech (Kansas City, KS). The rat P450 supersomes were purchased from Gentest Corporation (Woburn, MA). Bond Elut C18 cartridges (10 g/60 cm3) were obtained from Varian Sample Preparation Products (Harbor City, CA). All general solvents and reagents were the highest grade available commercially. Synthesis of 1-[3-(Formyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423 aldehyde). The synthesis of DPC 423 aldehyde was carried out using the procedure described previously (10). To a solution of DPC 423 (5.7 g, 10.0 mmol), 4-pyridinecarboxaldehyde (4.0 g, 40.0 mmol), and triethylamine (1 mL) in DMF (100 mL) was added DBU (6 mL). The mixture was stirred for 16 h at room temperature. The mixture was diluted with water and filtered. The solid obtained was dissolved in CH2Cl2 and purified by flash chromatography on silica gel. Elution initially with CH2Cl2 followed by ethyl acetate provided the desired product (750 mg, 14%). 1H NMR (300 MHz, CDCl3): δ 10.6 (1H, s), 8.28 (1H, t), 8.2 (1H.d), 8.13 (1H, s), 8.0-8.08 (2H, m), 7.81 (1H, d), 7.54-7.76 (3H, m), 7.37.4 (2H, m), 7.21 (1H, s), 7.18 (1H, d). MS (APCI): [M + H]+, 533. Synthesis of 1-[3-(Hydroxyiminomethyl)phenyl]-N-[3fluoro-2′-(methylsulfonyl) [1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423 oxime). The synthesis of DPC 423 oxime from the aldehyde was carried out as shown in Scheme 1. To a solution of DPC 423 aldehyde (1.3 g, 2.5 mmol) in CH2Cl2 (25 mL) and methanol (25 mL), was added a solution of hydroxylamine hydrochloride (0.7 g) and sodium acetate (0.8 g) in water (10 mL). The mixture was stirred for 2 h and then evaporated to dryness. The residue was stirred with water (20 mL). Filtration followed by drying under vacuum afforded the oxime (0.9 g, 67%). 1H NMR (300 MHz, DMSO-d6): δ 11.44 (1H, s), 10.69 (1H, s), 8.23 (1H, s), 8.09 (1H, d, 7.6 Hz), 7.8-7.6 (5H, m), 7.56 (1H, s), 7.54 (1H, s), 7.42 (1H, d, 6.5 Hz), 7.37 (1H, d, 11.8 Hz), 7.23 (1H, d, 8.2 Hz), 2.92 (3H, s). MS (APCI): [M + H]+, 547. Synthesis of 1-[3-(N-hydroxycarboxamido)phenyl]-N-[3fluoro-2′-(methylsulfonyl) [1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423 hydroxamate). A mixture of DPC 423 acid (55.0 mg, 0.1 mmol),

Chem. Res. Toxicol., Vol. 15, No. 1, 2002 65 hydroxylamine hydrochloride (9.0 mg, 0.1 mmol), benzotriazol1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP, 53.0 mg, 0.1 mmol) and diisopropylethylamine (78.0 mg, 0.6 mmol) in dimethylformamide (2 mL) was stirred at room temperature for 16 h. The mixture was diluted with ethyl acetate (50 mL), acidified with acetic acid (1 mL), and washed with water (50 mL). The ethyl acetate layer was separated and evaporated to dryness. The residue was purified by HPLC (Zorbax Rx C8, gradient elution from 30% acetonitrile in 0.05% aqueous TFA to 80% acetonitrile in 0.05% aqueous TFA in 20 min, tR ) 12.31 min) to provide the title compound (40 mg, 71%). 1H NMR (300 MHz, DMSO-d6) δ: 8.09 (1H,d, 6.6 Hz), 7.95 (1 H, s), 7.83 (1H, d, 7.8 Hz), 7.8-7.6 (6H, m), 7.43 (1H, d, 6.6 Hz), 7.3 (1 H, d, 1.2 Hz), 7.2 (1H, d, 8 Hz), 2.9 (3H, s). MS (ESI): [M+Na]+, 585.1 Synthesis of DPC 423 Nitrile and Amide. The syntheses of these compounds have been described previously (11). Synthesis of [13CD2]DPC 602. This compound was synthesized as described before (2). Synthesis of DPC 602 Oxime. The synthesis of DPC 602 oxime was performed in the same manner as described for DPC 423 oxime. The structure of the isolated product was confirmed spectroscopically and shown to have similar 1H NMR as DPC 423 oxime except for the distinct absence of methyl group at δ 2.92. LC-ESI/MS showed MH+ at m/z 548. Synthesis of DPC 602 Nitrile Oxide. Nitrile oxides are normally prepared by dehydrohalogenation of hydroximoyl chlorides or bromides under basic conditions. The nitrile oxides being very reactive are always generated in situ. Attempts were made to prepare the proposed DPC 602 nitrile oxide intermediate. The DPC 602 oxime obtained from the aldehyde was reacted with N-bromosuccinimde and pyridine to provide the nitrile oxide. Attempts to isolate the nitrile oxide for further spectroscopic characterization was unsuccessful. LC/MS analysis of the reaction mixture indicated the presence of nitrile oxide, which showed MH+ at m/z 546. The MS/MS spectra of the standard and the nitrile oxide generated by rat liver microsomes were compared and shown to be same. Synthesis of GSH Conjugate of Benzaldehyde Oxime. To a solution of benzaldehyde oxime (0.5 g) and pyridine (0.2 mL) in methylene chloride (5 mL) at 0 °C was added Nchlorosuccinimide (0.5 g). The mixture was stirred for 1 h, and washed with water (3 mL). To this CH2Cl2 solution at 0 °C was added GSH (307.0 mg) in DMF (1 mL) and triethylamine (0.3 mL). This mixture was stirred for 1 h. The reaction mixture was dried under vacuum and redissolved in water for an extraction on a C18 cartridge (10 g/60 cm3, Bond Elut). The cartridge was eluted with 20 mL aliquots of solvent consisting of various proportions of methanol in water. The GSH adduct of interest eluted in the 20% fraction. The GSH adduct showed MH+ at m/z 427 which on MS/MS showed fragment ions at m/z 409 (-H2O), 352 (-75 amu, loss of glycine), 298 (-129 amu, loss of glutamate) and 265 (-33 amu, loss of NH2OH from the ion at m/z 298). This conjugate showed the same characteristic mass spectral pattern as the oxime-derived GSH adduct of DPC 423, SX 737 and DPC 602. The 20% fraction from C18 extraction was further purified on a semipreparative C18 column (Waters Symmetry, 7.8 × 300 mm) using a mixture of acetonitrile and 0.05% trifluoroacetic acid (TFA) (12:88 v/v) as the mobile phase. The flow rate was set at 3.5 mL/min. The GSH adduct eluted at 8.9 min. The peaks corresponding to this compound from several injections were pooled, dried and analyzed by NMR spectroscopy. Synthesis of GSH Adduct of Phenyl Isocyanate. To a round-bottom flask containing 4.3 g of GSH in 10 mL of phosphate buffer (pH 7.4) was added 0.25 mL of phenyl isocyanate (0.9 g). The mixture was stirred for 30 min after which the sample was dried under vacuum. The GSH adduct was purified on a semipreparative column (Beckman C18, 10 × 250 mm) using a mixture of acetonitrile and 0.05% TFA (20: 80 v/v) as the mobile phase. The solvent flow rate was 3.5 mL/ min. The GSH adduct showed MH+ at m/z 427. The peak

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Table 1. Intramolecular Kinetic Isotopic Effects Observed When the Aminomethyl Protons of DPC 602 Were Substituted with Deuteriuma [13CD2]DPC 602 metabolite M1′ M2′ M11′ M13′ M16′

DPC 602

(µM)

I

II

III

I

II

III

10 50 10 50 10 50 10 50 10 50

0.25 ( 0.02 1.31 ( 0.06

0.75 ( 0.00 1.97 ( 0.11 0.05 ( 0.00 0.03 ( 0.00

0.72 ( 0.02 1.49 ( 0.05 0.05 ( 0.01 0.03 ( 0.01 0.31 ( 0.01 0.32 ( 0.01 0.25 ( 0.00 0.38 ( 0.00 0.13 ( 0.00 0.11 ( 0.00

0.71 ( 0.03 3.19 ( 0.11

2.02 ( 0.18 4.89 ( 0.13 0.29 ( 0.02 0.19 ( 0.00

1.86 ( 0.08 4.41 ( 0.10 0.30 ( 0.01 0.20 ( 0.03 0.71 ( 0.03 0.55 ( 0.10 0.25 ( 0.00 0.46 ( 0.05 0.24 ( 0.00 0.24 ( 0.04

0.24 ( 0.01 0.41 ( 0.00 0.50 ( 0.02 0.49 ( 0.04

0.23 ( 0.02 0.43 ( 0.02 0.93 ( 0.02 0.94 ( 0.04

a The ratios of peak areas of ions corresponding to the MH+ of each metabolite to an internal standard (DPC 423) are given. The results are presented as mean ( SD of ratios of peak areas [metabolites to an internal standard (DPC 423)]. The incubations were done in duplicates at each concentration of the substrate. I, no NADPH added; II, complete system without GSH; III, complete system with added GSH. The structures of metabolites M1′ (aldehyde), M2′ (carboxylic acid), M13′ (oxime), and M16′ (nitrile oxide) were confirmed by comparison with synthetic standards; M11′ was isolated and characterized spectroscopically.

corresponding to the GSH adduct was collected, dried and analyzed by NMR. Liquid Chromatography/Mass Spectrometry. LC/MS of the metabolites were performed using the conditions described in the accompanying paper (2). High-Resolution Mass Measurement of the Metabolites. Accurate masses of the GSH conjugates present in the bile of rats dosed with 100 mg/kg of either DPC 423 or DPC 602 were obtained on the QSTAR hybrid (quadrupole/time-of-flight) LC/ MS/MS instrument (PE Sciex, Toronto, Canada) as described before (2). High-Field NMR. All the spectra were obtained on a Bruker Avance 500 MHz NMR spectrometer equipped with 2.5 mm 1H/ 13C inverse conventional NMR probe. Chemical shifts were referenced to DMSO at δ 2.5 ppm and to acetonitrile at δ 2.0 ppm. The structures of 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. Animal Studies. Male Sprague-Dawley rats (weighing between 250 and 350 g) with cannulated bile ducts were administered a single oral dose of either DPC 423, SX 737, DPC 602 or [13CD2] DPC 602 at 100 mg/kg. The dosing volume was 5 mL/kg. The rats were housed and fed as described previously (2). The bile and urine samples were collected at 0-8 and 8-24 h time intervals and stored at -20 °C until analyzed. In another study, 2 male bile duct-cannulated Sprague-Dawley rats were dosed with 300 mg/kg/day of DPC 423 oxime for 2 days. Bile and urine samples were collected over 0-8 and 8-24 h intervals during the course of the study. Microsomal Metabolism of DPC 423 and its Analogues. DPC 423 or its structural analogues SX 737, DPC 602 and [13CD2]DPC 602 were incubated with rat liver microsomes (phenobarbital or dexamethasone induced or naı¨ve, 1 mg/mL) using the following protocol: NADPH (2 mM), substrate (10 or 100 µM), ( GSH (3 mM), MgCl2 (3 mM) and 0.1 M phosphate buffer to a final volume of 1 mL. To delineate the metabolic pathways of DPC 423 leading to the GSH adduct (M11), microsomal incubations were conducted with a number of possible metabolic intermediates including the nitrile, amide, oxime and the hydroxamate. The oximes of DPC 423 and DPC 602 were also incubated with rat microsomes using the same conditions. The mixtures were incubated for 1 h at 37 °C after which 2 mL of cold acetonitrile was added and the proteins precipitated. After centrifuging the samples at 1,200 x g for 5 min, the supernatants were transferred to clean culture tubes and dried under a stream of nitrogen at 25 °C. The dried extracts were reconstituted in the HPLC mobile phase (15% acetonitrile: 85% ammonium formate, pH 4.0) and analyzed by LC/ MS as described earlier.

In Vitro Metabolism of Oximes by cDNA Expressed Rat P450 Enzymes. To a buffered solution (either sodium phosphate or TRIS, 100 mM, pH 7.4) of DPC 423 oxime or DPC 602 oxime (20 µM), MgCl2 (3 mM), and rat P450 enzyme (50 pmol) was added NADPH (2 mM) and incubated at 37 °C for 45 min. The reactions were terminated by the addition of 2 mL of acetonitrile. The supernatant was dried and analyzed by LC/ MS. The following is a list of rat cDNA expressed enzymes employed in the study:1A1, 1A2, 2A1, 2A2, 2B1, 2C6, 2C11, 2C12, 2D1, 2D2, 2E1, 3A1 and 3A2. Microsomal Metabolism of DPC 423 Oxime in the Presence of 18O2. DPC 423 oxime (20 µM) was added to an incubation mixture consisting of microsomal protein (1 mg), NADPH (2 mM), MgCl2 (3 mM), and 0.1 M phosphate buffer (1 mL) that was presaturated with labeled molecular oxygen (18O2). The reaction was carried out in an airtight screw-capped vial. The vessel was sealed and the mixture shaken gently in water bath for 40 min. The reaction was terminated by an addition of 2 mL of acetonitrile and the sample analyzed by LC/MS as described above. In Vitro Metabolism of Benzaldehyde Oxime. To demonstrate that the formation of GSH conjugate could occur with other simpler aromatic aldoximes, microsomal incubations with benzaldehyde oxime (20 µM) in the presence of GSH was done. The studies were done in a similar manner as described above. The in vitro formation of GSH conjugate from benzaldehyde oxime was confirmed by comparing the LC/MS retention time and mass spectral data of the metabolite to those of synthetic standard. Due to the highly polar nature of the metabolites, LC/MS was done using an initial mobile phase consisting of 5% acetonitrile instead of 15% as described for the rest of assays. Determination of the Intramolecular Deuterium Kinetic Isotope Effect on the Formation of Metabolites from DPC 602 and [13CD2]DPC 602. Due to 1 amu differences in molecular weights of the metabolites produced by DPC 602 and its isotopomer, microsomal incubations were done separately for the labeled and nonlabeled compounds using identical conditions. The incubations were done as described above using two different concentrations of the substrate (10 and 50 µM). Incubations were done in duplicates in the presence or absence of GSH. Control incubations were also done from which NADPH was excluded. At the end of incubation 2 mL of acetonitrile containing 500 ng of internal standard (DPC 423) was added. The samples were dried under a stream of nitrogen, reconstituted in 200 µL of acetonitrile:10 mM ammonium formate (pH 4.00) (10:90 v/v) and analyzed by LC/MS. The LC/MS conditions were same as described before, except the initial mobile phase consisted of lower percentage organic (10%). The full LC/MS profiles were obtained and the ratio of peak areas of each metabolite to the internal standard (DPC 423) obtained.

Glutathione Conjugates of Aldoximes Isolation of Metabolites from Rat Bile. Bile duct-cannulated male Sprague-Dawley rats were dosed with DPC 423, DPC 602, [13CD2]DPC 602, SX 737, and DPC 423 oxime and bile collected as described above. The bile samples were subjected to solid-phase extraction as described in the accompanying paper (2). Isolation of Oxime Derived GSH Conjugates of DPC 602, [13CD2]DPC 602, and SX 737. Rats were found to produce large quantities of this unusual GSH conjugate when dosed with either DPC 602 or SX 737 as compared to DPC 423. Hence the isolation of this conjugate with MH+ at m/z 852, 853, 854 was done only from bile of rats given either SX 737, DPC 602, or labeled [13CD2]DPC 602, respectively. However, it was shown that DPC 423 produced a similar metabolite (MH+ at m/z 852) that fragmented to similar ions during the MS/MS experiments. The samples from C18 extraction (70% methanol fraction) containing the metabolites were pooled, dried under nitrogen, and reconstituted in water for further purification on a 2 g C18 cartridge. The metabolites were eluted from the cartridge with 5 mL aliquots of solvents consisting of different percentages of acetonitrile in 0.05% TFA. Each fraction was analyzed by FIA LC/MS. It was shown that the GSH conjugate eluted in 40% fraction. The 40% fraction was dried and further purified on a semipreparative column (Beckman C18, 10 × 250 mm) using a mobile phase consisting of a mixture of acetonitrile and 0.05% TFA (2:3 v/v). The solvent flow rate was 3.5 mL/min. The GSH conjugates for these compounds appeared between 9 and 10 min. The peaks corresponding to the GSH conjugates were collected from several injections, pooled, dried, and the dry powder analyzed by NMR. The isolated metabolites were analyzed on LC/MS to ensure the purity of the samples. Isolation of Glucuronide (M19) and GSH-Derived Adduct (M21) from Bile of Rats Dosed with DPC 423 Oxime (M13). The 50-60% C18 methanol fractions from the purification of DPC 423 oxime-dosed rat bile were pooled and dried under a stream of nitrogen. The residues were subsequently chromatographed on a semipreparative column (Supleco LC18, 10 × 250 mm) using an isocratic solvent system consisting of acetontrile:0.05% TFA (1:1 v/v) delivered at 3.5 mL/min. The glucuronide conjugate (M19) appeared at 7.3 min. while M21 eluted at 15.6 min. The peaks were collected using a fraction collector (Waters Fraction Collector II), dried, and repurified on the same HPLC system. The peaks corresponding to the two metabolites were collected, the solvents removed under vacuum, and the dried powders were analyzed by NMR.

Results In Vivo Metabolism of DPC 423. The metabolites excreted by rats dosed with DPC 423 and its analogues have been described before (1-3). The pathways leading to the different metabolites of DPC 423 are discussed in the preceding paper in this issue (2). In Vivo Metabolism of DPC 423 Oxime (M13). The oxime of DPC 423, when administered to rats, was metabolized extensively to a number of metabolites. Analyses of bile samples showed that the GSH conjugate M11 was the major metabolite. The carboxylic acid (M2), glucuronide conjugates M6 and M19, and the cysteinylglycine conjugate M20, were also excreted in significant quantities (see Figure 4). The identities of M2 and M6 were confirmed by comparing the LC/MS retention times with previously isolated standards (2). The glucuronide conjugate M6 was present as a mixture of isomers as was observed from studies conducted with DPC 423 (2). The cysteinylglycine conjugate M20 was isolated and shown to have the same NMR pattern as the GSH adduct M11 except for the distinct absence of glutamate proton signals. An unusual metabolite (M21) with MH+ at m/z 690 was observed in the bile samples. The structure of

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Figure 4. LC/MS profile of 0-8 h bile sample obtained from a rat dosed with 100 mg/kg of DPC 423 oxime (M13). The top pane shows the presence of M2, M6, M11, M19, M20, and M21 in the total ion current (TIC). The bottom pane is a mass spectrum of the glucuronide conjugate, M19, of the DPC 423 oxime.

this metabolite was confirmed by LC/MS/MS and NMR experiments. The 1H NMR of this metabolite showed signals at δ 10.70 (1H, s, Ar-NH-CO), 8.23 (1H, t, CH2NHCO), 7.2-8.1 (12H, Ar-H), 5.30 (1H, t, Cys R), 3.85 (1H, dd, Cys β), 3.80 (2H, m, Gly R), and 3.60 (1H, dd, Cys β′). Because the cysteine moiety was incorporated in the cyclic structure, there was an apparent downfield shift of both Cys R and β protons. As expected, the NMR spectrum of the metabolite showed an apparent loss of the singlet corresponding to the imino proton (at δ 8.20 in the NMR spectrum of the DPC 423 oxime standard). The signals for the aromatic protons remained unchanged as compared to the parent compound. The glucuronide conjugate (M19), showed the same 1H NMR spectrum as that of parent oxime except for the additional signals from the glucuronic acid moiety at δ 5.00 (1H, d, 1′′), 3.78 (1H, d, 5′′), 3.0-3.5 (3H, m, 2′′, 3′′, 4′′). The signal for the imino proton singlet had moved downfield to δ 8.45 in the spectrum. Enzymatic hydrolysis of this metabolite with β-glucuronidase yielded an aglycone that matched in its retention time and MS/MS fragmentation pattern with the oxime (M13). In Vivo Metabolism of SX 737, DPC 602, and [13CD2]DPC 602 in Rats. The metabolism of SX 737, DPC 602 ,and [13CD2]DPC 602 in rats was found to be similar to DPC 423. The slight modifications made to the molecule of DPC 423 produced only quantitative differences in the levels of metabolites produced from these structural analogues. The carbamyl glucuronide and the oxime-derived GSH conjugates were the major metabolites in bile of rats dosed with these benzylamines. SX 737 and DPC 602 produced greater quantities of the oxime-derived GSH conjugates as compared to DPC 423. This large difference in the formation of GSH adducts from DPC 602 and SX 737 as compared to DPC 423 could be due to the stable nitrile oxide produced from these two compounds (see Discussion). The large quantities of the oxime-derived GSH conjugate from SX 737 and DPC 602 made it possible to isolate and characterize these unique adducts with relative ease. The introduction of deuterium in the molecule of DPC 602 reduced the apparent levels of this GSH conjugate excreted in bile of rats dosed with [13CD2]DPC 602.

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Figure 5. HPLC/UV (λ ) 254 nm) chromatograms of metabolites of DPC 423 oxime (M13) incubated with rat liver microsomes in the presence (A) and absence (B) of GSH.

In Vitro Metabolism of DPC 423. DPC 423 was metabolized to M1, M2, M3, M8, M13 (two isomers), and M14 in the presence of rat liver microsomes (2). It was shown that CYP3A1/2, 1A1, and 2D2 converted DPC 423 to metabolite M13 (2). In Vitro Metabolism of DPC 423 Oxime (M13) and DPC 602 Oxime. The oxime of DPC 423 (M13) was metabolized extensively by the rat liver microsomes to the acid (M2), benzyl alcohol (M3), amide (M14), hydroxamate (M17), and nitrile (M18). The carboxylic acid (M2) was the major metabolite produced in the presence of rat liver microsomes (Figure 5). In incubations fortified with GSH, the GSH adduct, M11 was detected as the major metabolite. Metabolism of DPC 423 oxime in the presence of various rat cDNA expressed enzymes showed that CYP3A1 and CYP3A2 were largely responsible for the conversion of the oxime to the reactive intermediate trapped as the GSH adduct. CYP2C11 was also capable of metabolizing the oxime to the GSH adduct, although not as extensively as CYP3A1 and 3A2. The metabolite profiles obtained with these enzymes were similar to those obtained using rat liver microsomes; i.e., metabolites M2, M3, M11 (with added GSH), M17, and M18. In the presence of GSH, DPC 602 oxime was metabolized by rat liver microsomes in much the same way as DPC

Mutlib et al.

423 oxime producing the acid (M2′), benzyl alcohol (M3′), GSH adduct (M11′), and hydroxamate (M17′) metabolites. However, in the absence of GSH, the postulated nitrile oxide was the major metabolite (MH+ at m/z 546 and M + NH4+ at m/z 563). The identity of this metabolite was established by comparing its retention time and MS/ MS data with a synthetic standard. This metabolic intermediate was not detected when incubations were performed with DPC 423. CYP3A1 and 3A2 were shown to be responsible for the conversion of DPC 602 oxime to the GSH adduct. In Vitro Metabolism of DPC 423 Hydroxamate (M17). The hydroxamate of DPC 423 was extensively converted to the carboxylic acid metabolite (M2) in the presence of rat liver microsomes. There was no GSH adduct (M11) formed when the incubation media was fortified with GSH. In Vitro Metabolism of DPC 423 Amide (M14) and Nitrile (M18). The in vitro incubations of the amide and nitrile analogues of DPC 423 with rat liver microsomes showed no metabolism of these compounds; with greater than 98% of substrates remaining after 1 h of incubation. In Vitro Metabolism of SX 737, DPC 602, and [13CD2]DPC 602 in Rat Liver Microsomes. The in vitro metabolism of SX 737 by rat liver microsomes produced similar metabolites as DPC 423. The formation of aldehyde, benzyl alcohol, carboxylic acid, and the oxime metabolites was postulated based on pseudomolecular ions (MH+ and [M + NH4]+) and the fragmentation patterns of these ions. The MS/MS data of these metabolites were found to be similar to those of DPC 423 metabolite standards. GSH adducts with MH+ at m/z 852, 853, and 854 were found in microsomal incubations (fortified with GSH) performed with SX 737, DPC 602, and [13CD2]DPC 602, respectively. A comparison of in vitro microsomal metabolism of DPC 602 and [13CD2]DPC 602 was made. DPC 602 was metabolized to the aldehyde, acid, alcohol, and the oxime metabolites as DPC 423 (2). However, in addition to these metabolites, a compound with MH+ at m/z 546 and 547 ([M + NH4]+ at m/z 563 and 564, respectively) was found during the LC/MS analysis of DPC 602 and [13CD2]DPC 602 microsomal extracts, respectively. This was demonstrated to be the stable nitrile oxide intermediate with which GSH reacted to form the GSH adduct. The identity of this nitrile oxide was established by comparing its retention time and mass spectral fragmentation pattern with synthetic standard. A comparison of the LC/MS profiles showed that the level of this metabolite was miniscule in extracts in which GSH was included. The ratios of metabolites to an internal standard, produced from these two compounds are shown in Table 1. The significant decrease in the levels of DPC 602 nitrile oxide in the presence of GSH is illustrated in Figure 6. The presence of deuterium had no effect on the formation of the oxime, while significant intramolecular deuterium isotope effects were observed for the formation of the aldehyde (kH/kD ) 2.7), acid (kH/kD ) 6.3), postulated nitrile oxide (kH/kD ) 1.9), and GSH adduct (kH/kD ) 2.30). As observed in Table 1, it appears that DPC 602 can undergo autooxidation to form the aldehyde. This autooxidation also shows similar kH/kD value of 2.6 as observed with the microsomal formation of the aldehyde. In Vitro Metabolism of Benzaldehyde Oxime in Rats. The in vitro metabolism of benzaldehyde oxime was performed to demonstrate that oximes other than those

Glutathione Conjugates of Aldoximes

Figure 6. HPLC/UV (λ ) 254 nm) chromatograms showing the appearance of DPC 602 GSH conjugate (M11′) in the rat microsomal incubations fortified with GSH (top). The bottom pane shows the LC/UV trace obtained when GSH was omitted from the incubation mixture. The peak at tR ) 16.4 min corresponds to the nitrile oxide (M16′) formed from the oxime (M13′).

from DPC 423 analogues were capable of producing GSH adducts. In the presence of GSH, a large quantity of GSH adduct (MH+ at m/z 427) was formed. This metabolite was found to have the same retention time and mass spectral fragmentation pattern as the synthetic standard that was used to elucidate the structures of GSH adducts produced by DPC 423 and DPC 602. Using the HPLC conditions described, the GSH adduct eluted at tR ) 5.8 min while the substrate appeared at tR ) 10.9 min. Characterization of GSH Conjugates of DPC 423 and Its Analogues. The characterization of the GSH conjugates of DPC 423, SX 737, DPC 602, and [13CD2]DPC 602, isolated from rat bile, is described below. Rats dosed with DPC 423, SX 737, and DPC 602 excreted significant quantities of unique GSH conjugates, which showed net addition of 319 amu to the parent molecular weights. The pseudomolecular ions (MH+) of these GSH conjugates from DPC 423, DPC 602, [13CD2]DPC 602, or SX 737 dosed rats were at m/z 852, 853, 854, and 852, respectively. DPC 423 and SX 737 being regioisomers produced GSH adducts with identical MH+ at m/z 852. The high-resolution mass spectrum of DPC 423 GSH adduct showed MH+ at m/z 852.1745 (852.1745 calculated). The MS/MS spectrum showed characteristic losses of pyroglutamate (-129) and glycine, giving ion fragments at m/z 723.1413 (calculated 723.1319) and 777.1505 (calculated 777.1424), respectively (see Scheme 2). The major fragment formed by the neutral loss of hydroxylamine from ion at m/z 723 produced ion at m/z 690.1138 (calculated 690.1104). Rats dosed with [13CD2]DPC 602 excreted GSH conjugate with MH+ at m/z 854. The MS/MS spectrum of the MH+ ions of the conjugates from animals dosed with labeled and nonlabeled DPC 602 is shown in Figure 7. The mass spectral data of the labeled DPC 602 conjugate suggested loss of the two deuteriums on the aminomethyl side chain, hence giving a net addition of only 1 amu to MH+. An addition of GSH to a reactive metabolic intermediate usually leads to a net gain of either 305, 307, 321, or 323 amu to the parent mass. The difference of 2 amu from the expected GSH


conjugate of a hydroxylated metabolite (319 vs 321 amu) suggested the possibility of an unsaturated double bond. The loss of two deuteriums combined with a net increase of 1 amu (corresponding to the mass increment as a result of retention of C13) to the molecular weight of the GSH adduct of [13CD2]DPC 602 suggested a structure such as shown in Figure 2. However, the structures of these GSH conjugates could only be confirmed by NMR experiments. The 1H NMR and the assignments of the proton signals of the GSH adduct of DPC 602 isolated from rat bile are shown in Figure 8. In the NMR studies, similar correlation of the cysteine methylene (β, β′) protons and the aromatic proton, a, with the quaternary imino carbon at 148 ppm was observed for the GSH conjugates of DPC 602 and [13CD2]DPC 602 (see Figures 9 and 10, respectively). The strong correlation observed in Figure 10 is due to the C13 label on the aminomethyl side chain. The presence of C13 label enabled the correlation data to be obtained in a relatively short period of time. The correlations clearly indicated that the benzyl carbon of the parent compound (DPC 602) was now a quaternary carbon with a chemical shift consistent with an imine. These correlations and the chemical shift of the quaternary carbon are consistent with the proposed structure. Similar NMR spectra were obtained for the GSH adducts produced by rats dosed with DPC 423 and SX 737 (data not shown). Characterization of GSH Conjugate of Benzaldehyde Oxime. The GSH adduct of benzaldehyde oxime was synthesized to serve as a further confirmation for the proposed structure of the GSH adducts. The GSH conjugate of benzaldehyde oxime was characterized and compared with the GSH adduct of DPC 423 and DPC 602. The LC-ESI/MS showed MH+ at m/z 427. MS/MS of MH+ produced a characteristic base peak at m/z 265. This loss of pyroglutamate moiety followed by the loss of hydroxylamine to produce a cyclic product with m/z 265 is similar to the fragmentation pattern observed for all the benzylamine GSH adducts isolated from in vivo. The HMBC spectrum of the synthetic GSH adduct showed a strong correlation of the cysteine methylene protons (β, β′) and the aromatic protons (a and b) with the quaternary imino carbon at 150 ppm (Figure 11). These correlations were nearly identical to those seen in the spectra of the metabolites. Characterization of GSH Conjugate of Phenyl Isocyanate. A synthetic GSH adduct of phenyl isocyanate was made, and its NMR spectra were obtained. The HMBC experiment, which was performed in much the same way as those for other GSH adducts, did not show the critical correlation seen in GSH adducts of benzaldehyde oxime, DPC 602, or [13CD2]DPC 602. This provided clear evidence that the GSH adducts isolated from bile of rats dosed with either DPC 602 or [13CD2]DPC 602 were not related to this structure and hence were not derived from isocyanate intermediates formed by Lossen rearrangement.

Discussion The metabolism of compounds possessing a benzylamine moiety has been previously described (1-3). The metabolism of two such compounds, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2′-(ami-

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Scheme 2. High Resolution MS/MS of the MH+ Ion of the GSH Conjugate, M11 (MH+ at m/z 852.1745)

Figure 7. LC/MS/MS of GSH conjugates present in bile of rats dosed with DPC 602 and [13CD2]DPC 602.

nosulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1Hpyrazole-5-carboxamide (DPC 602), in rodents was studied extensively. It was found that the aminomethyl of the benzylamine moiety was a metabolic soft spot, leading to a whole plethora of metabolites including aldehydes, carboxylic acids, benzyl alcohols, carbamyl glucuronides, sulfamates, hydroxylamines (2), and glutamates (3). The metabolism of these two compounds was found to be principally due to modification of the benzylamine moiety via a host of enzymes, including P450, semicarbazidesensitive amine oxidases (SSAO) and phase II transferases. Studies done with [13CD2]DPC 602 and SX 737, a regioisomer of DPC 423, showed identical pattern of metabolism as DPC 602 and DPC 423. One of the metabolites produced by these benzylamines was a GSH

adduct whose structure was previously proposed based on mass spectral data (2). These GSH conjugates showed loss of two hydrogens and addition of elements of glutathione and oxygen. The stucture of this conjugate was initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Stable isotope-labeled version of DPC 602 was prepared, and the NMR and MS data of this conjugate isolated from rats dosed with this labeled compound indicated an oxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon at 148 ppm. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. Earlier suspected structures and intermediacy of amide were ruled out by in vitro experiments with rat liver microsomes in the presence of GSH. No GSH adduct was observed. Studies with the oxime intermediates from DPC 423 and DPC 602, upon incubation with rat liver microsomes in the presence of GSH, led to adducts identical to the ones isolated from in vivo. This supported the intermediacy of an oxime as a precusor to the GSH adduct. The in vivo oxime GSH conjugates from DPC 423 and DPC 602 were predominantly one isomer, the stereochemistry of which could not be deduced. The postulated pathway for the formation of this conjugate from DPC 423 is shown in Scheme 3. The formation of hydroxylamine followed by its further oxidation to a nitroso intermediate has been previously postulated (2). Subsequent rearrangement of nitroso to the oxime is a well-known phenomenon (4). Once the oxime (M13) is formed, it can either be metabolized to the carboxylic acid (M2) or to the GSH adduct (M11). In vitro studies with the synthetic standard of oxime (M13) showed that, in the absence of GSH, the carboxylic acid was the major metabolite. However, if the incubation media was forti-


Figure 8.

1H


NMR of GSH adduct isolated from bile of rats dosed with 100 mg/kg of DPC 602.

Figure 9. Partial HMBC spectrum of oxime derived GSH adduct isolated from bile of rats dosed with 100 mg/kg of DPC 602. Note the correlation between the cysteine protons (position 18) and aromatic proton (position 8) to the imino carbon at 148 ppm [marked with an asterix (*)].

fied with GSH, the glutathione adduct (M11) was the major product identified in the microsomal extracts. The formation of oximes from primary amines, mediated principally by human FMO3 has been described before (4, 12, 13). In all of these studies, the formation of hydroxylamine was suggested as an intermediate. Further studies with 4-(hydroxyphenethyl) hydroxylamine, the postulated metabolic intermediate from tyramine, demonstrated the formation of the same oxime metabolite as was produced by tyramine (4). The proposed mechanisms for the formation of oxime metabolite from the hydroxylamine involved a dioxygenated intermediate,

which eliminated water to form the oxime metabolite. The studies done in our laboratory showed that FMO3 was not involved in the formation of M13 (2). Instead, it was shown that the formation of M13 was mediated principally by rat P450 3A1/2 and to a lesser extent by 2D2 and 1A1 enzymes. Furthermore, P450 3A1/2 were the major enzymes capable of converting the oxime (M13) to the reactive intermediate trappable by GSH to form M11. Further evidence from the deuterium-labeled DPC 602 and DPC 423 have been presented elsewhere (1, 2) that supports the proposed mechanism leading to the oxime metabolites. The results from the deuterium-

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Figure 10. Partial HMBC spectrum of oxime derived GSH adduct isolated from bile of rats dosed with 100 mg/kg of [13CD2]DPC 602. A strong correlation of the cysteine methylene protons (β, β′) and the aromatic proton (a) with the imino carbon (*) at 148 ppm was observed.

Figure 11. HMBC NMR spectrum of synthetic GSH adduct of benzaldehyde oxime showing strong correlation of the cysteine methylene protons (β, β′) and the aromatic protons (a and b) with the imino carbon (*) at 150 ppm.

labeled (DPC 602) studies suggested that oxime formation does not occur through the imine intermediate as postulated for some compounds (7), even though aldehyde and carboxylic acid metabolites were found as microsomal metabolites. An imine intermediate would have involved breaking a carbon-deuterium bond enzymatically. As evidenced, the formation of aldehyde (assumed to be from an imine intermediate) showed a significant deuterium isotope effect as expected (see Table 1). To assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond on the metabolism of DPC 602, the effect of replacement of C-H by

C-D was studied. Carbon-deuterium bonds have higher activation energy for cleavage than carbon-hydrogen bonds, due to the lower zero-point energy of the former (14). If a carbon-hydrogen bond is cleaved during the rate-determining step of a multistep process, then substitution of deuterium for hydrogen will have the effect of slowing the rate of formation of all metabolites downstream of this step. The formation of DPC 602 metabolites M1′, M2′, M11′, and M16′ was found to be subject to such an apparent intramolecular primary kinetic deuterium isotope effect in that they were slowed by a factor of 1.9-6 when the aminomethyl group was



Scheme 3. Oxime (M13) Was Demonstrated to Form from DPC 423 in Vitro through the Postulated Hydroxylamine and Nitroso Intermediatea

a The presence of the hydroxylamine intermediate was demonstrated by characterizing its glucuronide conjugate in bile of rats. In vitro metabolism with the oxime standard showed that the carboxylic acid was the major product. However, in the presence of GSH, the major metabolite was the GSH adduct (M11).

repaced by deuterium. The formation of aldehyde (M1′) which is produced via an imine intermediate (15) showed a significant isotope effect with an average kH/kD of 2.7. The formation of the carboxylic acid (M2′) was significantly reduced with [13CD2]DPC 602, showing the highest kH/kD ratio of 6.3. This was expected since the formation of the carboxylic acid requires two consecutive steps in breaking the carbon-deuterium bond, once during the formation of the aldehyde and the other during the oxidation of aldehyde. The formation of aldoxime, M13′, showed no deuterium isotope effect at both concentrations of substrates. This is consistent with the postulated mechanism of an initial hydroxylamine formation with subsequent oxidation to a nitroso intermediate (see Scheme 3). The tautomerism of the nitroso intermediate to the aldoxime is not expected to be affected by the presence of deuterium on the molecule as it is a nonenzymatic process. There is precedence for such tautomerism of nitroso functional group with an adjacent R protons (9). However, it is also possible that further N-oxygenation of the hydroxylamine could lead to an intermediate that would dehydrate nonenzymatically leading to the same product (4). Oximes have been identified as common intermediates during the oxidative deamination of R-branched primary amines and were reported to be hydrolyzed rapidly to the corresponding ketone, which in turn is partially reduced to the alcohol (16, 17). Oximes of DPC 423 and its analogues appear to form via a different route (i.e., hydroxylamine and nitroso intermediates) as evidenced by the lack of isotope effect. The formation of GSH adduct, M11′, did show a significant kinetic isotope effect (kH/kD of 2.3) since a carbondeuterium bond has to be broken on the aldoxime prior to the formation of the adduct (see Scheme 4). The observation and mechanistic significance of primary

kinetic deuterium isotope effects occurring in biological systems have been reviewed by several workers (18-21). The results confirm that the GSH adduct formation lies downstream of the rate-limiting step in this metabolic pathway. Interestingly enough, the postulated nitrile oxide intermediate from DPC 602, M16′, shows a parallel isotope effect (kH/kD of 1.9) to that shown by M11′. Furthermore, as shown in Table 1, the levels of M16′ appear to be reduced 3-4-fold in incubations done in the presence of GSH. The significant reduction in the levels of this nitrile oxide intermediate in the presence of GSH is illustrated in Figure 6. The dramatic reduction in the M16′ level is accompanied by a significant increase in the level of the GSH adduct, M11′. The ratios of other metabolites to the internal standard remained fairly unchanged in both sets of incubations. This is consistent with the hypothesis that M16′ being the reactive intermediate from the DPC 602 aldoxime, reacts with GSH and forms M11′. An attempt to isolate M16′ failed due to its instability in solution. The formation of the GSH adduct from the oxime was interesting since a reaction of this type has not been reported previously in the literature. The oxime can potentially be oxidized to the free radical, as shown in Scheme 4. This radical can potentially react with a GSH radical and form an imino thioether linkage immediately. Alternatively, the oxime radical can be further oxidized by loss of second electron (and hydrogen) to form a highly reactive nitrile oxide (M16) capable of nucleophilic addition. This nitrile oxide can also be hydrolyzed to hydroxamate (M17) and subsequently to the carboxylic acid, M2. In vitro studies with the DPC 423 oxime showed that the hydroxamate (M17) was a significant metabolite in incubations done with rat liver microsomes and cDNA-expressed CYP3A1/2. In addition, the oxime

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Scheme 4. Postulated Mechanisms for the Formation of GSH Adduct (M11) from DPC 423 via One and Two Electron Abstractions Leading to a Free Radical and a Reactive Nitrile Oxide, Respectively

was found to produce the aldehyde (M1), carboxylic acid (M2), nitrile (M18), and GSH adduct, M11 (if the incubation media was fortified with GSH). The carboxylic acid (M2) was the major metabolite if GSH was omitted from the incubation mixtures. Surprisingly, the GSH adduct was the principal metabolite if GSH was added to the incubations (see Figure 4). Studies done in vivo with the oxime (M13) showed that the GSH adduct (M11) was the major metabolite followed by lesser quantities of the carboxylic acid M2, cysteinylglycine conjugate M20, and glucuronides M6 and M19. Interestingly, a cyclized product (M21) resulting from elimination of hydroxylamine from the cysteinylglycine conjugate was characterized as well. This metabolite is postulated to form via intramolecular cyclization of the cysteinylglycine conjugate (M20). A nucleophilic attack by the free amino group of the cysteine moiety at the benzylic carbon leads to a five-membered cyclic intermediate, which then eliminates hydroxylamine to form M21. Further in vitro metabolism studies with the hydroxamate (M17) showed that it was rapidly converted to the carboxylic acid, M2. The formation of carboxylic acid from the oxime was interesting and further studies with 18O2 were done to confirm the hydroxamate pathway leading to M2. In vitro microsomal incubations with M13 done in a vessel saturated with 18 O2 showed an incorporation of the label in the carboxylic acid metabolite. However, there was approximately 30% 18O -labeled carboxylic acid, while the rest of the mol2 ecules did not show any incorporation of the label. This could have been due to the direct hydrolysis of either the oxime or the nitrile oxide intermediate. The incorporation of 18O2 in the carboxylic acid metabolite formed from the oxime suggested P450-mediated hydroxylation of the imino carbon. Oxidation at this position would lead to a hydroxamate intermediate, which could exist as tautomers. Hydrolysis of this hydroxamate could then produce the carboxylic acid retaining the 18O label in the

molecule. The hydroxamate was shown as an in vitro microsomal metabolite of DPC 423 oxime. Further in vitro studies with the hydroxamate confirmed the formation of carboxylic acid as its major metabolite. However, the hydroxamate did not produce any GSH adduct when incubations were done in the presence of GSH. This clearly showed that the hydroxamate was not a precursor to the GSH adduct. Oxime derivatives are known to be good radical acceptors because of the extra stabilization of the aminyl radical by the lone pair on the adjacent oxygen atom. The formation of the GSH adduct could take place via P450catalyzed generation of a radical from the preformed oxime (see Scheme 4). Studies could be conducted using electron spin resonance to detect the existence of such a radical. On the basis of synthetic analogy, it is equally appealing to invoke the existence of a nitrile oxide resulting from successive two-electron oxidation of the oxime and loss of a proton. Indeed, studies with DPC 602, which coincidentally produces a lot more of the GSH adduct than DPC 423, showed the possible existence of nitrile oxide in the in vitro incubations. The identity of this nitrile oxide intermediate from DPC 602 was confirmed by comparing with a synthetic standard. Similar nitrile oxide from DPC 423 could not be chemically synthesized or produced in vitro using microsomal incubations. The greater stability of DPC 602 nitrile oxide as compared to the DPC 423 analogue could partially explain the much higher levels of GSH conjugate produced by DPC 602 (or any of the ortho-substituted benzylamines). The extra stability observed for DPC 602 nitrile oxide could be due to the close proximity of the nitrile oxide functional group to the five-membered ring. The metabolism of other oximes have been described previously (22-26). The P450-mediated dehydration of alkyl- and arylaldoximes leading to nitriles has been described (22). This is consistent with our observation


that the nitrile metabolite (M18) was produced from the DPC 423 oxime in vitro using rat liver microsomes or cDNA-expressed enzymes. The formation of nitrile and the glucuronide conjugate of oximes has also been reported (25). However, to date there are no reports on the formation of GSH adducts of oximes. In this study, we have demonstrated unequivocally the structures of unique GSH adducts of oximes formed by P450-mediated metabolism of benzylamines. Furthermore, we have shown that the formation of the oxime was most likely produced via N-oxygenation of the benzylamine with subsequent formation of a nitroso intermediate capable of tautomerizing to the oxime. The oxime metabolite is metabolized further by rat CYP3A1/2 to a putative nitrile oxide intermediate that can be trapped as a GSH adduct.

Acknowledgment. We are grateful to Dr. Takeo Sakuma for carrying out the high resolution mass spectral analysis of metabolites.

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