Formation of Unusual Glutamate Conjugates of 1-[3- (aminomethyl)phenyl]-n-[3-fluoro-2 -(methylsulfonyl)-[1,1 -biphenyl]- 4-yl]-3-(trifluoromethyl)-1h-pyrazole-5-carboxamide (dpc 423) and Its Analogs: the Role of -glutamyltranspeptidase in the Biotransformation of Benzylamines

The role of -glutamyltranspeptidase (GGT) in transferring glutamate from endogenous glutathione (GSH) to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3fluoro-2 -(methylsulfonyl)-[1,1 -biphenyl]-4-yl]-3-(trifluoromethyl)1H-pyrazole-5-carboxamide (DPC 423), is described. Studies were performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotopelabeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate transfer to the benzylamines in the presence of GGT. To further demonstrate that the protons on the benzylamines and glutamate (as part of glutathione) were unaffected during the transpeptidation, these protons were replaced with deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, was used to abolish the formation of the glutamate conjugates of DPC 423 in vitro and in vivo. This provided further evidence of the role of GGT in forming the glutamate conjugates of benzylamines. This study demonstrated conclusively that GGT was responsible for mediating the transfer of glutamic acid from GSH to the benzylamine moiety of a series of structurally related compounds. The ability to characterize minor and unusual metabolites has been greatly accelerated with the introduction of versatile analytical techniques, such as liquid chromatography/mass spectrometry (LC/MS) and LC/NMR. Recently, we described the isolation and characterization of unique acetaminophen peptide conjugates using these techniques (Mutlib et al., 2000b). The coupling of these acetaminophen peptide conjugates with glutamic acid was described. The elucidating of these unusual metabolites prompted us to investigate the nature of the enzyme(s) involved in such metabolic reactions. The involvement of -glutamyltranspeptidase (GGT) was proposed but not confirmed. The postulated role of GGT in forming some of these unusual metabolites of acetaminophen led us to investigate whether this enzyme plays an even greater role in disposing xenobiotics than we had previously envisioned. GGT was first identified in kidney tissue and later shown to be present in serum and in all cells except muscle cells (Hanigan and Pitot, 1985). Evidence to date has demonstrated that the GGT is involved in the catabolism of GSH-conjugates of xenobiotics (Curthoys and Hughey, 1979). GGT cleaves GSH and GSH-conjugates extracellularly, leading to catabolites that can be reabsorbed into cells. GGT plays an important role in maintaining high-intracellular GSH concentrations through its role in the -glutamyl cycle (Griffith et al., 1978). The -glutamyl cycle, originally proposed by Meister (1973), is involved in the biosynthesis and degradation of glutathione. GGT has also been postulated to be involved in transporting amino acids into cells via this glutamyl cycle (Meister, 1973; Tate and Meister, 1981; Meister and Anderson, 1983; Smith et al., 1991; Coomes, 1997; Griffith and Mulcahy, 1999). This translocation mechanism is mediated through the concerted action of several enzymes, one of them being GGT, which is located on the external part of the cell membrane. Here it forms -glutamyl amino acids from extracellular amino acids and intracellular GSH. The -glutamyl amino acids are translocated into the cell, where the intracellular enzyme -glu1 Abbreviations used are: LC, liquid chromatography; MS, mass spectroscopy; MS/MS, tandem mass spectroscopy; GGT, -glutamyltranspeptidase; GSH; glutathione; Fmoc, N-(fluorenyl)methoxycarbonyl; TFA, trifluoroacetic acid; DMF, dimethylformamide; HPLC, high-pressure liquid chromatography; HBTU, 2(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; ESIMS, electrospray ionization-mass spectroscopy; amu, atomic mass unit; HMBC, heteronuclear multiple bond correlation. Address correspondence to: Dr. A. E. Mutlib, Drug Metabolism and Pharmacokinetics Section, DuPont Pharmaceuticals Company, P.O. Box 30, 1094 Elkton Road, Newark, DE 19714. E-mail address: [email protected] 0090-9556/01/2910-1296–1306$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 10 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 413/929774 DMD 29:1296–1306, 2001 Printed in U.S.A. 1296 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from tamyl cyclotransferase catalyzes the conversion of -glutamyl amino acids to 5-oxoproline and the corresponding free amino acid. The active transport of amino acids by GGT was clearly demonstrated in vitro using Caco-2 monolayers (Smith et al., 1991). In this study, we describe the involvement of GGT in the disposition of structurally related benzylamines; specifically by the transfer of glutamate from endogenous GSH to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2 (methylsulfonyl)-[1,1 -biphenyl]-4-yl]-3-(trifluoromethyl)-1Hpyrazole-5-carboxamide (DPC 423). Studies were also performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine moiety. The structures of three compounds, which produced the glutamate conjugates, are shown in Fig. 1. The transfer of glutamate from GSH in the presence of GGT was demonstrated unequivocally by using deuterium-labeled glutathione. Likewise, to demonstrate that the protons on benzylamines and glutamate (as part of glutathione) were unaffected during transpeptidation, these protons were replaced with deuterium in one of the analogs (Fig. 1). Materials and Methods Chemicals and Supplies. DPC 423 and its analogs were synthesized and characterized by the DuPont Pharmaceuticals Company. Resin Fmoc-Gly was obtained from Advanced Chemtech (Louisville, KY). Fmoc-d3 Glu-O-tbutyl ester was obtained from Anaspec, Inc. (San Jose, CA). All other amino acids and synthesizer reagents were obtained from Applied Biosystems (Foster City, CA). Trifluoroacetic acid (TFA), phenol, ethanedithiol, N-acetylglutamate, and thioanisole were obtained from Aldrich (Milwaukee, WI). Glutathione was purchased from Sigma (St. Louis, MO). Bond Elut C18 cartridges (10 g/60 ml) were obtained from Varian Sample Preparation Products (Harbor City, CA). All general solvents and reagents were the highest grade available commercially. Synthesis of N-Acetylglutamate Conjugate of DPC 423. Dicyclohexylcarbodiimide (8 mg, 0.04 mmol) was added to a solution of N-acetylglutamate (8 mg, 0.04 mmol) in DMF (1 ml). The solution was stirred at room temperature for 30 min. To another vial, DPC 423 (22 mg. 0.04 mmol) and dimethylaminopyridine (6 mg, 0.05 mmol) were added in 1 ml of DMF and stirred at room temperature for 30 min. The entire solution in the first vial was added to the second vial, and the mixture was subsequently stirred for 30 min. At the end of the reaction, the organic solvents were removed under a stream of nitrogen, and the residue was reconstituted in 1 ml of 1:4 mixture of acetonitrile and 0.1% acetic acid. Aliquots of 100 l were injected onto a semipreparative HPLC column (Beckman C18; 250 10 mm) (Beckman Instruments, Inc., Fullerton, CA). The separation of products was achieved using an isocratic mobile phase consisting of 1:1 mixture of acetonitrile and 0.1% acetic acid delivered at 3.5 ml/min. The products were monitored using a variable wavelength detector set at 254 nm. Two products, showing retention times at 7.3 and 8.6 min, were collected and submitted for LC/MS and NMR analyses. Furthermore, to confirm the identity of the metabolite present in rat bile, LC/MS/MS of the pseudomolecular ion at m/z 704 was done for both the standards and for the metabolite present in the bile. The retention times and mass spectral fragmentation patterns were then compared. The two isomers were spiked in bile samples containing the N-acetylglutamate conjugate of DPC 423 and the retention times compared. Synthesis of Deuterated Glutathione, D3-GSH The deuterated glutathione was made using custom synthesized Fmoc-d3 Glu-O-t-butyl ester obtained from Anaspec, Inc. The peptide was synthesized on solid phase using Fmoc protection and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) activation on an ABI 433A peptide synthesizer from Applied Biosystems. During the first step, the Fmoc group was removed from the Fmoc-Gly-resin (0.13 g, 0.1 mmol) by treatment with 20% piperidine/ DMF; the resin was then washed with DMF and dichloromethane. The deprotection was followed by the conductance of the solution. After deprotection was completed, the next amino acid in the sequence (Fmoc-Cys, 1 mmol) was preactivated using a solution of HBTU (1 mmol) in DMF/N-hydroxybenzotriazole and diisopropylethylamine. The activated amino acid solution was added to the resin. When the coupling was completed, the resin was washed, and the instrument was started on another cycle. The last coupling was carried out manually using Fmoc-d3 Glu-O-t-butyl ester (0.171 g, 0.4 mmol), HBTU (0.152 g, 0.4 mmol) and diisopropylethylamine (14 l, 0.8 mmol). The reaction was shaken for 2 h. Completion of the reaction was determined by a negative ninhydrin test. The resin was deprotected using 20% piperidine/DMF and was washed. TFA-catalyzed cleavage of the peptide from the resin was done using reagent K (King et al., 1990) [TFA (4 ml), H2O (0.2 ml), thioanisole (0.2 ml), ethanedithiol (0.1 ml), and melted phenol (0.28 ml)]. This was followed by precipitation from ethyl ether and freeze drying, which afforded the crude peptide that was purified by reverse phase HPLC (see below) to yield 3.8 mg of D3-GSH. Electrospray ionization-mass spectroscopy (ESI-MS) showed MH at m/z 311.1, as expected. HPLC purification was performed on a C18 semipreparative Vydac column (250 22 mm, 10 m; Vydac, Hesperia, CA). The solvent system consisted of two components (solvent A, 0.1% TFA in water; solvent B, 90% aqueous acetonitrile containing 0.1% TFA). HPLC was performed using isocratic elution for 10 min with solvent A followed by a gradient from 0 to 30% B in 20 min. The solvent flow rate was 18 ml/min, and the components were detected by a UV detector with the wavelength set at 220 nm. The peak was collected from several injections and dried under vacuum before analyses by mass spectrometry. Synthesis of [CD2]Benzylamine, B The synthesis of compound B , labeled with C and deuterium on aminomethyl group, is depicted in Fig. 2. Cyclization of 2-bromophenylhydrazine (1) with 4,4,4-trifluoro-1-(2-furyl)1,3-butanedione (2) provided 3. Refluxing 3 with potassium cyanide (Clabeled) and catalytic amount of cuprous iodide in N-methyl pyrrolidone (Carr et al., 1994) yielded 4. Lithium aluminum deuteride reduction of 4 followed by treatment with trifluoroacetic anhydride afforded 5. Oxidation of 5 with sodium chlorite led to carboxylic acid, the acid chloride of which was coupled with biphenyl aniline to provide 8. Careful alkaline hydrolysis of 8 followed by acidification yielded the desired hydrochloride 9 (Galemmo et al., 1999). H NMR (500 MHz, dimethyl sulfoxide-d6) showed the absence of proton signals from the aminomethyl side chain as expected, with the rest of the H NMR spectrum being identical to benzylamine B. The ESI-MS mass spectrum showed an increment of 3 amu (two deuterium molecules and one C) giving MH at m/z 537.0. Liquid Chromatography/Mass Spectrometry. The metabolites were separated on a Waters Symmetry C18 column (2.1 150 mm; Waters, Milford, MA) 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 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 reequilibrating with the initial mobile phase. 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 FIG. 1. Structures of DPC 423 (A) and its analogs. , C-labeled. 1297 DISPOSITION OF BENZYLAMINES BY -GLUTAMYLTRANSPEPTIDASE at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from