Cysteine Conjugate β-Lyase-Dependent Biotransformation of the

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Chem. Res. Toxicol. 1997, 10, 811-819

811

Cysteine Conjugate β-Lyase-Dependent Biotransformation of the Cysteine S-Conjugates of the Sevoflurane Degradation Product 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A) Ramaswamy A. Iyer and M. W. Anders* Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, P.O. Box 711, Rochester, New York 14642 Received December 3, 1996X

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1, Compound A) is a fluoroalkene formed by the base-catalyzed degradation of sevoflurane that is nephrotoxic in rats. Fluoroalkene 1 is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate β-lyase-dependent bioactivation to reactive intermediates. The present experiments were designed to study the β-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]-L-cysteine (5) by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat kidney cytosol or a pyridoxal model system showed the formation of inorganic fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), the expected products of a β-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amount of acid 9 formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amount of pyruvate formed. Incubation of conjugate 4 with rat kidney cytosol and analysis by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (10); the formation of acid 10 was observed in the pyridoxal model only after prolonged incubation (>18 h). Lactic acid 10 was identified as a degradation product of acid 9. Cysteine S-conjugate 5 was not stable in pH 7.4 buffer and underwent a rapid cyclization reaction (t1/2 ≈ 5 min) to form 2-[1-(fluoromethoxy)-2,2,2trifluoroethyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid (14). These data show that fluoroalkene 1-derived cysteine S-conjugates are substrates for renal β-lyase and that acid 9 is formed as a terminal product. Acid 9 is, however, unstable and affords lactic acid 10 as a degradation product.

Introduction Sevoflurane (fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl)ethyl ether) is an inhalational anesthetic that is approved by the Food and Drug Administration for use in the United States and is now approved for use in 41 countries worldwide. Sevoflurane undergoes base-catalyzed elimination of hydrogen fluoride to give 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A, 1; Scheme 1) as the major degradation product, and the formation of fluoroalkene 1 in anesthesia circuits equipped with carbon dioxide scrubbers is well documented (1-3). Other halogenated anesthetics, i.e., halothane, also undergo base-catalyzed degradation (4). Fluoroalkene 1 is nephrotoxic in rats, and its nephrotoxicity is associated with damage to the corticomedullary stripe, elevated blood urea nitrogen concentrations, and the appearance of glucose, proteins, and ketone bodies in the urine (5-9). The mechanism by which fluoroalkene 1 causes nephrotoxicity has not been fully established but may be similar to the mechanism elucidated for a range of 1,1-difluoroalkenes (for reviews, see refs 10 and 11). Haloalkene-induced nephrotoxicity is associated with hepatic glutathione S-conjugate formation, enzymatic hydrolysis of the glutathione S-conjugates to cysteine S-conjugates, active uptake of the cysteine X

Abstract published in Advance ACS Abstracts, June 15, 1997.

S0893-228x(96)00196-8 CCC: $14.00

S-conjugates by the kidney, and bioactivation by renal cytosolic and mitochondrial cysteine conjugate β-lyase (10). β-Lyase-catalyzed β-elimination reactions of 1,1difluoroalkene-derived cysteine S-conjugates give unstable R-fluorothiolates that lose inorganic fluoride to afford thioacylating agents, whose reaction with cellular macromolecules is associated with cell damage and death (12-14). Fluoroalkene 1 undergoes glutathione-dependent metabolism in vivo: fluoroalkene 1-derived glutathione S-conjugates are eliminated in the bile of rats given fluoroalkene 1, and the corresponding mercapturic acids are excreted in the urine (9, 15). Also, 2-(fluoromethoxy)3,3,3-trifluoropropanoic acid (9), the expected terminal product of the β-lyase-catalyzed metabolism of the cysteine S-conjugates of fluoroalkene 1, was detected in the urine of rats given fluoroalkene 1 (16). Moreover, (aminooxy)acetic acid, which inhibits β-lyase (17), partly blocks the nephrotoxicity of fluoroalkene 1 (9). Thus, although considerable evidence indicates that the observed nephrotoxicity of fluoroalkene 1 is associated with glutathione conjugate formation and β-lyase-dependent bioactivation, some workers disclaim a role for the β-lyase pathway in the nephrotoxicity of fluoroalkene 1 (18). The objective of this work was to study the β-lyasedependent biotransformation of the fluoroalkene 1-derived cysteine S-conjugates S-[2-(fluoromethoxy)-1,1,3,3,3© 1997 American Chemical Society

812 Chem. Res. Toxicol., Vol. 10, No. 7, 1997 Scheme 1

pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5). The biotransformation of cysteine conjugate 4 by rat kidney cytosol or its chemical transformation by a pyridoxal model system was investigated by 19F NMR spectroscopy and GC/MS. Incubation of conjugate 4 with rat kidney cytosol or a pyridoxal model showed the formation of fluoride, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), and 3,3,3-trifluorolactic acid (10). Cysteine S-conjugate 5 was not stable in pH 7.4 buffer, and its degradation was followed by 19F NMR and UV spectroscopy. Also, the synthesis of acid 9 and its characterization by 1H and 19F NMR spectroscopy and GC/MS are described. Finally, the stoichiometry of pyruvate, fluoride, and acid 9 formation from cysteine S-conjugate 4 was measured, and the formation of trifluorolactic acid 10 as a degradation product of acid 9 was demonstrated.

Experimental Procedures Materials. 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (fluoroalkene 1) was provided by Abbott Laboratories (Abbott Park, IL). N-Dodecylpyridoxal bromide was synthesized by the method of Kondo et al. (19). Trifluoroacetaldehyde ethyl hemiacetal, mercury(II) trifluoroacetate, 15-crown-5, and silica gel (Merck, grade 60, 240-400 mesh, 60 Å) were purchased from

Iyer and Anders Aldrich Chemical Co. (Milwaukee, WI). Bakerbond octadecyl (C18, 40 µm, prep LC Packing) reverse-phase chromatographic column packing was obtained from J. T. Baker, Inc. (Phillipsburg, NJ). TLC plates (Whatman, silica gel, 250 µm, AL SIL G/UV) were purchased from VWR Scientific (Rochester, NY). Preparative TLC plates (Uniplate, silica gel GF, 2000 µm, 20 × 20 cm) were purchased from Analtech (Newark, DE). THF was dried over sodium metal and freshly distilled before use. Acetonitrile was dried overnight over activated 4 Å molecular sieves. N-Acetyl-L-cysteine was dried overnight on a Labconco 4.5 lyophilizer. All other reagents were obtained from commercial suppliers and used without further purification, except as noted. Analytical Methods. Melting points were determined with a Mel-Temp melting point apparatus and are uncorrected. 1H and 19F NMR spectra were recorded with a Bruker 270 MHz spectrometer operating at 270 MHz for 1H and 254 MHz for 19F. Chemical shifts, δ, are reported in parts per million (ppm). The internal standard (δ ) 0.0 ppm) for 1H NMR with CDCl3 as the solvent was tetramethylsilane. The HOD resonance at 4.7 ppm was used as the internal standard for 1H NMR spectra when D2O was the solvent. The solvent resonance peak at 2.1 ppm was used as the internal standard for 1H NMR spectra when acetone-d6 was the solvent. Trifluoroacetamide (δ ) 0.0 ppm) was used as the external standard for 19F NMR spectra. Electronic absorption spectra were recorded with a HewlettPackard 8453 diode-array spectrophotometer or a Beckman DU64 spectrophotometer. Pyruvate formation was measured after derivatization with 0.1 M o-phenylenediamine and analysis by HPLC (20). Samples were analyzed on a Hewlett-Packard 1090 liquid chromatograph fitted with a Radial-Pak cartridge packed with Resolve C-18 RCM column (8 mm i.d. × 100 mm; Millipore Corp., Milford, MA). The eluant was methanol/water/acetic acid (45:54:1) at a flow rate of 1 mL/min, and the fluorescent intensity of the eluate was measured with a Gilson model 121 fluorometer. Fluoride ion concentrations were measured with a fluoride-specific electrode (ATI Orion, Boston, MA) and a Corning pH meter (Corning Glass Works, Medfield, MA). Acid 9 and trifluorolactate 10 were converted to their pentafluorobenzyl esters and analyzed by GC/MS (21); dichloroacetic acid was used as the internal standard. The limit of detection of acid 9 and trifluorolactate 10 after conversion to their pentafluorobenzyl esters and analysis by GC/MS was about 15 µg. Mass spectra were recorded with a Hewlett-Packard 5880A gas chromatograph (25 m × 0.2 mm, 0.5-µm film thickness, HP-1 cross-linked methyl siloxane column; Hewlett-Packard, Wilmington, DE) coupled to a Hewlett-Packard 5970B mass selective detector; the injector and transfer-line temperatures were 240 and 285 °C, respectively. The methyl esters of acids 9 and 10 and tert-butyl sulfide 11 were analyzed with a temperature program of 30 °C for 1 min followed by a linear gradient of 10 °C/min to 200 °C. Methyl esters of acids were prepared for GC/ MS analysis by reaction with a solution of the acids in ether with diazomethane. For analysis of the benzyl ester of 9 and the methyl ester of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1propenyl]-N-acetyl-L-cysteine, the initial oven temperature was 50 °C for 1 min followed by a linear gradient of 10 °C/min to 200 °C. Caution: Diazomethane is toxic and mutagenic and should be used with care in an efficient fume hood. Elemental analyses were determined by Midwest Microlab (Indianapolis, IN). Silica gel or Bakerbond was used for column chromatography, and the columns were eluted by gravity flow. Cysteine S-conjugates on TLC plates were detected with a spray reagent of 0.3% ninhydrin in n-butanol/acetic acid (97:3). Syntheses. S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4; Scheme 2). Solid potassium hydroxide was added to a stirred suspension of L-cysteine (4.84 g, 40 mmol) and ethylenediaminetetraacetic acid disodium salt (144 mg, 0.4 mmol) in water (20 mL) to a pH of 9.6, when the suspension became a clear solution. A solution of butylated hydroxytoluene (84 mg, 0.4 mmol) in ethanol (20 mL) was added followed by 10 mL of water. The solution was cooled to 0 °C in an ice bath,

β-Lyase-Dependent Fluoroalkene Biotransformation and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1; 7.92 g, 44.0 mmol) in 10 mL of ethanol was added dropwise over 1 h to the stirred reaction mixture. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. Ethanol (80 mL) was added, and the solution was brought to pH 2 by addition of concentrated HCl. Evaporation of the solvent in vacuo gave a yellow oil that was loaded onto a C18 reverse-phase column. Elution of the column with acetonitrile/ water/acetic acid (15:84:1 and then 20:79:1) gave S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) as a pale yellow solid (5.8 g, 44%): mp 129-131 °C; TLC Rf ) 0.51 (nbutanol/water/acetic acid, 6:1:1); 1H NMR (D2O) δ 5.42 (d, 2H, J ) 54 Hz, -OCH2F), 5.05-5.15 (m, 1H, CF2CH(CF3)OCH2F), 3.90-3.98 (m, 1H, CH2CH(NH2)COOH), 3.20-3.32 and 3.423.52 (d of m, 2H, SCH2CH(NH2)COOH); 19F NMR (D2O) δ 2.973.01 (m, 3F, CF2CH(CF3)OCH2F), -7.90 to -2.90 (m, 2F, CF2CH(CF3)OCH2F), -75.70 (t, 1F, J ) 54 Hz, OCH2F). Anal. Calcd for C7H9NF6SO3: C, 27.91; H, 3.01; N, 4.65. Found: C, 28.18; H, 3.0; N, 4.43. S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-Lcysteine (5; Scheme 2). 1. S-[2-(Fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]-N-acetyl-L-cysteine. Triethylamine (810 mg, 8.1 mmol) was added to a stirred solution of N-acetylL-cysteine (653 mg, 4.0 mmol) in THF (20 mL) under a nitrogen atmosphere, and the solution was cooled to 0 °C. 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1; 900 mg, 5.0 mmol) in 5 mL of THF was added dropwise to the reaction mixture over 10 min, and the reaction mixture was stirred for 1.5 h at 0 °C. After addition of 30 mL of water, the mixture was extracted with ethyl acetate (2 × 40 mL). The organic layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a yellow viscous oil. The N-acetyl-Lcysteine S-conjugate thus obtained was hydrolyzed without purification. A sample of the N-acetyl-L-cysteine S-conjugate was purified by silica gel column chromatography for analysis. The column was eluted with methanol/ethyl acetate (1:9) containing 0.1% acetic acid. Evaporation of the solvent in vacuo gave S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-Nacetyl-L-cysteine as a yellow viscous oil: TLC Rf ) 0.2 (methanol/ ethyl acetate/acetic acid, 1:9:0.01); 1H NMR (CDCl3) δ 9.90 (s, 1H, COOH), 6.67 (d, 1H, CHNHCOCH3), 5.30-5.50 (d of d, 2H, J ) 54 Hz, OCH2F), 4.45-4.60 (m, 1H, CH2CH(NHCOCH3)COOH), 3.30-3.70 (m, 2H, SCH2CH(NHCOCH3)COOH), 2.00 (s, 3H, COCH3); 19F NMR (CDCl3) δ 10.60-10.90 (m, 3F, CFdC(CF3)OCH2F), -31.90 to -31.50 (m, 1F, CFdC(CF3)OCH2F), -74.90 (t, 1F, J ) 54 Hz, OCH2F); GC/MS (methyl ester) tR ) 20.1 min, m/z 337 (M+, 1), 278 (60), 236 (75), 192 (19), 144 (90), 88 (100). 2. S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]The N-acetyl-L-cysteine S-conjugate was dissolved in 1.2 N HCl/methanol (1:1, 30 mL) and heated in a water bath at 50 °C for 5 h. The solvent was removed in vacuo, and the white solid obtained was dissolved in water and loaded onto a C18 reverse-phase column. Elution with acetonitrile/water/acetic acid (10:89:1) gave S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1propenyl]-L-cysteine (5) as a white solid (340 mg, 25%): mp 9395 °C dec; TLC Rf ) 0.51 (n-butanol/water/acetic acid, 6:1:1); 1H NMR (D O) δ 5.32 (d of d, 2H, J ) 54 Hz, OCH F), 4.222 2 4.32 (m, 1H, CH2CH(NH2)COOH), 3.35-3.45 (m, 2H, SCH2CH(NH2)COOH); 19F NMR (D2O) δ 10.10-10.18 (m, 3F, CFdC(CF3)OCH2F), -31.90 to -31.30 (m, 1F, CFdC(CF3)OCH2F), -75.3 (t, 1F, J ) 54 Hz, OCH2F). L-cysteine.

2-(Fluoromethoxy)-3,3,3-trifluoropropanoic Acid (9; Scheme 3). 1. tert-Butyl 2-(Fluoromethoxy)-1,1,3,3,3pentafluoropropyl Sulfide (11). Potassium hydroxide (283 mg, 5.0 mmol) was dissolved in water/ethanol (1:4, 20 mL), and the solution was cooled to 0 °C in an ice bath. 2-Methyl-2propanethiol (451 mg, 5.0 mmol) was added, and the solution was stirred for 5 min. A solution of 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene (1; 918 mg, 5.1 mmol) in 5 mL of ethanol was added dropwise over 10 min to the reaction mixture. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. Water (30 mL) was added, and the

Chem. Res. Toxicol., Vol. 10, No. 7, 1997 813 reaction mixture was extracted with ether (3 × 40 mL). The ether layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a pale yellow liquid. The product was loaded onto a silica gel column, which was eluted with ether/hexane (5:95) to give tert-butyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide (11) as a colorless liquid (1.08 g, 80%): 1H NMR (CDCl3) δ 5.32-5.52 (d of d, 2H, J ) 54 Hz, OCH2F), 4.22-4.34 (m, 1H, CF2CH(CF3)OCH2F), 1.55 (s, 9H, SC(CH3)3); 19F NMR (CDCl3) δ 2.98-3.10 (m, 3F, CF2CH(CF3)OCH2F), -4.50 to -0.50 (m, 2F, CF2CH(CF3)OCH2F), -78.50 (t, 1F, J ) 54 Hz, OCH2F); GC/MS tR ) 10.8 min, m/z 270 (M+, 2), 181 (0.6), 57 (100). 2. Benzyl 2-(Fluoromethoxy)-3,3,3-trifluoropropanoate (12). Mercury(II) trifluoroacetate (2.7 g, 6.1 mmol) and anisole (0.4 mL) were added to a solution of tert-butyl 2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl sulfide (11; 1.35 g, 5.0 mmol) in water/glacial acetic acid (2:8, 30 mL). The solution was stirred at room temperature for 3 h, and 40 mL of water was added. The mercury was removed by precipitation as mercuric sulfide by bubbling H2S gas through the solution for 1 h. (Caution: Hydrogen sulfide is poisonous and should be handled with caution in a fume hood.) The suspension was filtered through a Celite pad. The filtrate was brought to pH 1.4 with concentrated HCl and extracted with ether (3 × 100 mL). The ether layers were combined, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield impure 2-(fluoromethoxy)-3,3,3trifluoropropanoic acid (9) as a pale yellow liquid (140 mg, about 16%). The formation of the acid 9 was confirmed by GC/MS analysis of the methyl ester and by 19F NMR spectroscopy (data not shown but similar to those reported below for the acid 9 obtained by debenzylation). To a solution of impure acid 9 (140 mg, 0.79 mmol) in acetonitrile (8 mL) was added anhydrous sodium carbonate (106 mg, 1.0 mmol), benzyl bromide (170 mg, 1.0 mmol), and 15-crown-5 (22 mg, 0.1 mmol). The suspension was heated in a water bath at 65 °C under a nitrogen atmosphere for 1 h. Water (30 mL) was added, and the aqueous solution was extracted with ether (2 × 75 mL). The organic layer was separated, dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield a yellow liquid that was loaded onto a silica gel column. Elution with ether/hexane (1:9) gave benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanoate (12) as a colorless liquid (35 mg, 1.3% from 11): TLC Rf ) 0.41 (ethyl acetate/ hexane, 1:9); 1H NMR (CDCl3) δ 7.35-7.40 (m, 5H, Ar), 5.40 (d of d, 2H, J ) 54 Hz, OCH2F), 5.31 (s, 2H, OCH2C6H5), 4.604.69 (m, 1H, CF3CH(OCH2F)COOCH2C6H5); 19F NMR (CDCl3) δ 2.21 (d, 3F, CF3CH), -77.34 (t, 1H, J ) 54 Hz, OCH2F); GC/ MS tR ) 13.6 min, m/z 266 (M+, 4), 108 (0.7), 91 (100). Anal. Calcd for C11H10F4O3: C, 49.64; H, 3.80. Found: C, 50.30; H, 4.04. 3. 2-(Fluoromethoxy)-3,3,3-trifluoropropanoic Acid (9). To a solution of benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanoate (12) (30 mg, 0.11 mmol) in 3 mL of ethyl acetate was added Pd/C (10%, 60 mg, 0.04 mmol), and the suspension was stirred under a hydrogen atmosphere (atmospheric pressure) at room temperature for 6 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo to give 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9) as a colorless oil (15 mg, 76%): 1H NMR (acetone-d6) δ 5.65 (d, 2H, J ) 54 Hz, OCH2F), 5.02-5.13 (m, 1H, CF3CH(OCH2F)COOH), 4.40 (bs, 1H, COOH); 19F NMR (acetone-d6) δ 2.30 (d, 3F, CF3CH(OCH2F)COOH), -77.5 (t, 1F, J ) 54 Hz, OCH2F); GC/MS (methyl ester) tR ) 5.5 min, m/z 190 (M+, 0.3), 142 (6), 131 (33), 59 (100). 3,3,3-Trifluorolactic Acid (10). 3,3,3-Trifluorolactic acid (10) was synthesized by the procedure of Burstein and Ringold (22) except that trifluoroacetaldehyde ethyl hemiacetal was used as the precursor for the preparation of the cyanohydrin instead of trifluoroacetaldehyde hydrate. 3,3,3-Trifluorolactic acid (10) was obtained as a white crystalline solid (500 mg, 35%): mp 67-69 °C (lit. mp 68-69 °C (22)); 1H NMR (acetone-d6) δ 4.92 (q, 1H, J ) 11 Hz, CF3CH(OH)COOH); 19F NMR (acetone-d6) δ 0.45 (d, 3F, J ) 11 Hz, CF3CH(OH)COOH); GC/MS (methyl

814 Chem. Res. Toxicol., Vol. 10, No. 7, 1997 ester) tR ) 4.2 min, m/z 159 (M+ + 1, 0.2), 99 (17), 59 (100). Methyl 2-[1-(Fluoromethoxy)-2,2,2-trifluoroethyl]-4,5dihydro-1,3-thiazole-4-carboxylate. A solution of conjugate 5 (65 mg, 0.23 mmol) in 0.1 M potassium phosphate buffer (pH 7.4, 20 mL) was stirred at room temperature for 24 h. The aqueous solution was lyophilized, and the resulting solid was treated with excess diazomethane in ether. The ether solution was filtered and loaded onto a preparative TLC plate. Development with ethyl acetate/hexane (2:9) gave a pale yellow oil (40 mg, 65%): TLC Rf ) 0.3 (ethyl acetate/hexane, 3:7); 1H NMR (CDCl3) δ 5.35-5.55 (m, 2H, OCH2F), 5.25-5.35 (m, 1H, CH2CHCOOH), 5.08-5.15 (m, 1H, NdCCH(CF3)OCH2F), 3.91 and 3.94 (2s, 3H, diastereomeric COOCH3), 3.65-3.82 (m, 2H, SCH2CHCOOH); 19F NMR (CDCl3) δ 1.09 and 1.14 (2s, 3F, diastereomeric CF3), -78.24 and -77.82 (2t, 1F, J ) 54 Hz, diastereomeric OCH2F); GC/MS tR ) 14.1 and 14.3 min (two diastereomeric peaks), m/z 275 (M + H, 0.2), 256 (2), 216 (94), 166 (100), 59 (94). Cyclization of S-[2-(Fluoromethoxy)-1,3,3,3-tetrafluoro1-propenyl]-L-cysteine (5). Preliminary studies showed that cysteine S-conjugate 5 underwent cyclization to give a 2-substituted thiazole-4-carboxylic acid (14) (see Results). Hence, the first-order rate constant (k) and t1/2 for the cyclization of cysteine S-conjugate 5 was determined. Cysteine S-conjugate 5 (100 µM) was incubated in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C in a quartz cuvette, and the decrease in absorbance at 247 nm was measured every 20 s for 3 min. The first-order rate constant was determined by fitting the data to the equation: A0 ) Ate-kt, where A0 is the absorbance at time t ) 0, At is the absorbance at time t, and k is the first-order rate constant. A plot of ln(A0 - At/At) vs t gave a straight line with a slope of -k. The t1/2 for the cyclization of conjugate 5 was calculated from t1/2 ) 0.693/k. The cyclization of cysteine S-conjugate 5 was also studied by 19F NMR spectroscopy. Conjugate 5 (2.0 mM) was dissolved in an NMR tube containing 0.1 M potassium phosphate buffer (pH 7.4) and 100 µL of D2O. 19F NMR spectra were recorded at 25 °C at several times. Biotransformation of S-[2-(Fluoromethoxy)-1,1,3,3,3pentafluoropropyl]-L-cysteine (4) by Rat Kidney Cytosol. Male Fischer 344 rats (180-220 g; Charles River Laboratories, Wilmington, MA) were anesthetized with ether and killed by cardiac puncture. The kidneys were removed and homogenized in 0.1 M phosphate buffer (pH 7.4), and cytosol was isolated by published procedures (23). The cytosol was dialyzed in Spectrapor dialysis membranes (MW cutoff of 3000; Spectrum Medical Laboratories, Houston, TX) for 36 h at 4 °C in 0.1 M phosphate buffer (pH 7.4). Protein concentrations were determined by the Bradford assay with bovine serum albumin as the standard (24). Heat-inactivated cytosol was prepared by heating the cytosolic fraction in a boiling water bath for 3-4 min. Cysteine S-conjugate 4 (4 mM) was incubated with rat kidney cytosol (5 mg of protein/mL) in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 2 h. The proteins were precipitated by addition of 0.6 N perchloric acid to pH 2.0. The precipitated proteins were removed by centrifugation. The supernatant fraction (450 µL) was analyzed by 19F NMR spectroscopy after mixing with 50 µL of D2O in an NMR tube. The supernatant fractions were also analyzed for pyruvate, fluoride, and acid 9 concentrations, as described under Analytical Methods above. Incubation of S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) with a Pyridoxal Model System. Cysteine S-conjugate 4 (4 mM) was incubated in 0.1 M phosphate buffer (pH 8.0) with N-dodecylpyridoxal bromide (0.25 mM), cetyltrimethylammonium chloride (3 mM), and EDTA (1 mM) at 37 °C for 2 h. The reaction mixture was analyzed by 19F NMR spectroscopy, and pyruvate, fluoride, and acid 9 concentrations were measured, as described above.

Results Syntheses. S-[2-(Fluoromethoxy)-1,1,3,3,3,-pentafluoropropyl]-L-cysteine (4) was obtained by the procedure

Iyer and Anders Scheme 2a

(a) L-Cysteine, KOH, EtOH-H2O; (b) N-acetyl-L-cysteine, Et3N, THF; (c) 1.2 N HCl, MeOH-H2O.

Scheme 3a

(a) (CH3)3CSH, KOH, EtOH-H2O; (b) 80% AcOH, Hg(OCOCF3)2; (c) H2S, H2O; (d) BnBr, Na2CO3, 15-crown-5, CH3CN; (e) H2, Pd/C, ethyl acetate.

used for other 1,1-difluoroalkenes (25) (Scheme 2). The attempted synthesis of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) in sodium and liquid ammonia, which has been used successfully for the synthesis of 1,1-dichloroalkene-derived vinylic cysteine S-conjugates (26), failed to give the expected product. Fluoroalkene 1 and its cysteine S-conjugates underwent degradation in the presence of sodium and liquid ammonia: 19F NMR spectroscopic analysis of the reaction mixture showed the loss of the fluoromethoxy group, indicating that this group was not stable under strongly basic conditions. Reaction of fluoroalkene 1 with Nacetyl-L-cysteine gave the mercapturate S-[2-(fluoromethoxy-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cysteine, which was hydrolyzed to give S-[2-(fluoromethoxy1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) (Scheme 2). 2-(Fluoromethoxy)-3,3,3-trifluoropropanoic acid (9) was obtained by the route shown in Scheme 3. (An alternative route to acid 9 has been reported (16).) Reaction of fluoroalkene 1 with 2-methyl-2-propanethiol in the presence of aqueous potassium hydroxide gave tert-butyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide (11). Removal of the tert-butyl group with mercury trifluoroacetate in 80% aqueous acetic acid followed by precipitation of the mercury as mercuric sulfide gave crude acid 9 (27). Attempts to purify acid 9 by distillation at 7080 °C under reduced pressure led to the degradation of the acid to give 3,3,3-trifluorolactic acid (10), whose formation was confirmed by comparison of the 19F NMR and mass spectra (methyl ester) with a synthetic standard. 3,3,3-Trifluorolactic acid (10) was prepared by the Strecker reaction (22). Attempts to purify acid 9 by column chromatography also led to its decomposition. Hence, acid 9 was converted to its benzyl ester 12, purified by column chromatography, and deblocked by hydrogenolysis to give acid 9 (Scheme 3). Incubation of

β-Lyase-Dependent Fluoroalkene Biotransformation

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Figure 1. 19F NMR spectra of reaction mixtures containing S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) (1 mM) incubated with rat kidney cytosol (5 mg of protein/mL) at 37 °C for 0 h (A) or 3 h (B) (see Experimental Procedures). The resonances at 3.80, -7.90 to -2.90, and -75.70 ppm were assigned to conjugate 4, the resonance at 2.20 ppm was assigned to acid 9, the resonance at 0.87 ppm was assigned to lactic acid 10, and the resonance at -43.2 ppm was assigned to inorganic fluoride. Chemical shifts were referenced to external trifluoroacetamide.

Figure 2. 19F NMR spectra of reaction mixtures containing S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) (4 mM) and incubated with a pyridoxal model system (see Experimental Procedures) at 37 °C for 0 h (A), or 2 h (B). The resonances at 3.80, -7.90 to -2.90, and -75.70 ppm were assigned to conjugate 4, the resonances at 2.20 and -76.50 ppm were assigned to acid 9, and the resonance at -43.2 ppm was assigned to inorganic fluoride. Chemical shifts were referenced to external trifluoroacetamide.

acid 9 in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C showed the release of fluoride and the formation of 3,3,3-trifluorolactic acid (10) by 19F NMR spectroscopy.1 Biotransformation Studies. Cysteine S-conjugate 4 was incubated with rat kidney cytosol at 37 °C. 19F NMR spectroscopy showed the formation of inorganic fluoride (Figure 1). The formation of fluoride is consistent with a β-lyase-catalyzed β-elimination reaction of cysteine S-conjugate 4 to give thiolate 6, which loses fluoride to form thioacyl fluoride 8. The hydrolysis of 8 is expected to give acid 9 as the terminal product. Inspection of the 19F NMR spectrum showed, however, 1

R. A. Iyer and M. W. Anders, unpublished results.

that little acid 9 was formed. Acids 9 and 10 could be detected by GC/MS analysis after pentafluorobenzyl ester formation, but the amounts were too small to be accurately quantified. Observed fragment ions of pentafluorobenzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanoate were (m/z, relative abundance): 356 (M + H, 0.04), 336 (M - HF, 1.6), and 181 (M - 174, 100). For pentafluorobenzyl 3,3,3-trifluorolactate, the observed fragment ions were (m/z, relative abundance): 324 (M + H, 2.5) and 181 (M - 143, 100). Another resonance upfield from the trifluoromethyl group of acid 9 was assigned to the trifluoromethyl group of lactic acid 10. Similar results were obtained with the pyridoxal model: little acid 9 was formed, but no resonances assignable to lactic acid 10

816 Chem. Res. Toxicol., Vol. 10, No. 7, 1997

Iyer and Anders

Figure 3. (A) UV spectra of S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) (100 µM) incubated in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 0 min (A), 5 min (B), or 10 min (C). (B) 19F NMR spectra of S-[2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]-L-cysteine (5) (2 mM) incubated in 0.1 M potassium phosphate buffer (pH 7.4) at 25 °C for 0 min (A), 10 min (B), 30 min (C), 60 min (D), or 90 min (E). The resonances at 10.7, -31.7, and -74.9 ppm were assigned to conjugate 5, the resonances at 0.82 and -76.7 ppm were assigned to thiazole 14, and the resonance at -43.2 ppm was assigned to inorganic fluoride. Chemical shifts were referenced to external trifluoroacetamide.

were seen after 2 h incubation (Figure 2); resonances for lactic acid 10 were, however, seen after longer incubation times (>18 h). When the reaction mixtures from rat kidney cytosol or the pyridoxal model incubated with conjugate 4 were analyzed for pyruvate and fluoride concentrations, pyruvate and fluoride formed from conjugate 4 amounted to 0.54 ( 0.04 and 1.24 ( 0.05 nmol min-1 (mg of protein)-1, respectively (n ) 3), and the ratio of fluoride to pyruvate formation was 2.3; for the pyridoxal model, pyruvate and fluoride formation from conjugate 4 were 8.9 ( 0.4 and 22.7 ( 1.9 nmol/min, respectively (n ) 3), and the ratio of fluoride to pyruvate formation was 2.5. The amounts of acid 9 and lactic acid 10 formed were too small to be accurately quantified in incubation mixtures of conjugate 4 with rat kidney cytosol. In the pyridoxal model, the rate of acid 9 (0.4 ( 0.01 nmol/min) formed was less than 5% of the rate of pyruvate formation. Cysteine S-conjugate 5 underwent cyclization in 0.1 M phosphate buffer (pH 7.4), which was accompanied by a change in its UV spectrum (Figure 3A). When conjugate 5 was incubated in 0.1 M phosphate buffer (pH 7.4)

at 25 °C and the 19F NMR spectrum was recorded at several times up to 90 min, the resonances assigned to conjugate 5 decreased in intensity and new resonances at 0.82 ppm (doublet, -CF3), -76.7 ppm (triplet, -OCH2F), and -43.2 ppm (singlet, F-) appeared (Figure 3B). Analysis of the reaction mixture by TLC showed the absence of ninhydrin-positive compounds, indicating the loss of conjugate 5. The reaction mixture was acidified, extracted with ether, and reacted with diazomethane. GC/MS analysis showed two peaks with identical molecular ions and similar fragmentation patterns (see Experimental Procedures). The information that the product formed retained its -CF3 and -OCH2F groups, lost fluoride, lacked an R-amino group, and gave two isomeric peaks by GC/MS analysis of the methyl esters indicated the possible formation of diastereomeric thiazole 14 (Scheme 4). The methyl ester of thiazole 14 was prepared (see Experimental Procedures) and characterized, thereby confirming its formation from conjugate 5. The firstorder rate constant for the cyclization of conjugate 5 to give thiazole 14 was 0.14 ( 0.00 min-1, and the t1/2 was 5.1 ( 0.15 min.

β-Lyase-Dependent Fluoroalkene Biotransformation Scheme 4

Discussion The β-lyase pathway has been established as an important mechanism for the bioactivation of a range of nephrotoxic haloalkenes (10, 11, 28). The first step in this multistep pathway is the glutathione S-transferasecatalyzed formation of glutathione S-conjugates, which undergo γ-glutamyltransferase- and dipeptidase-catalyzed hydrolysis to the corresponding cysteine S-conjugates. The cysteine S-conjugates may undergo detoxication by conversion to mercapturates or β-lyasedependent bioactivation to thioacylating intermediates that react with cellular nucleophiles. Several nephrotoxic 1,1-difluoroalkenes, including 2-bromo-2-chloro-1,1-difluoroethylene (29, 30), bromotrifluoroethylene (30), chlorotrifluoroethylene (31), 1,1-dichloro-2,2-difluoroethylene (30), hexafluoropropene (32), and tetrafluoroethylene (33), undergo β-lyase-dependent bioactivation. Fluoroalkene 1, which is nephrotoxic in rats (5-7, 9, 34), is structurally similar to other nephrotoxic fluoroalkenes and may, therefore, also undergo β-lyase-dependent bioactivation. Indeed, previous studies show that fluoroalkene 1 is metabolized to glutathione S-conjugates 2 and 3 and that the corresponding mercapturic acids are excreted in the urine of rats given fluoroalkene 1 (Scheme 1) (9, 15). Also, cysteine S-conjugates 4 and 5 are substrates for rat, human, and nonhuman primate renal β-lyase (35), and acid 9, the expected product of the β-lyase-catalyzed metabolism of conjugates 4 and 5, has been identified in the urine of rats given fluoroalkene 1 (16). Finally, glutathione S-conjugates 2 and 3 and cysteine S-conjugate 4 are nephrotoxic in rats (36). Although these findings implicate the β-lyase pathway in the bioactivation of fluoroalkene 1, evidence purporting to show that the β-lyase pathway is not involved in fluoroalkene 1-induced nephrotoxicity has been presented (18). The objective of the present experiments was to characterize the fluoroorganic products of the β-lyasecatalyzed biotransformation of fluoroalkene 1-derived cysteine S-conjugates 4 and 5. Hence, the fate of conjugates 4 and 5 was studied in incubation mixtures with rat renal cytosol or with a pyridoxal model system as catalysts, and the products were characterized by 19F NMR spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat renal cytosol and examination of the reaction mixture by 19F

Chem. Res. Toxicol., Vol. 10, No. 7, 1997 817

NMR spectroscopy showed the formation of inorganic fluoride and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9) (Figure 1). Although the formation of inorganic fluoride indicated that conjugate 4 had undergone a β-elimination reaction, little acid 9 was formed: only weak resonances assigned to the trifluoromethyl and fluoromethoxy groups of acid 9 were observed. Another resonance, upfield from the trifluoromethyl groups of conjugate 4 and acid 9, was observed and assigned to the trifluoromethyl group of 3,3,3-trifluorolactic acid (10). This assignment was confirmed by comparison with the 19F NMR spectrum of authentic acid 10. In studies with the pyridoxal model system, lactic acid 10 formation was not observed when conjugate 4 was incubated with the pyridoxal model system for 2 h (Figure 2) but was observed after prolonged incubation (>18 h). The formation of lactic acid 10 indicated that acid 9 was unstable. Incubation of synthetic acid 9 at pH 7.4 and 37 °C showed the formation of lactic acid 10 (data not shown). Although the mechanism of the conversion of acid 9 to lactic acid 10 has not been investigated, it is possible to speculate that intramolecular displacement of the fluoromethoxy group by the carboxylate of acid 9 may lead to the formation of 2-(trifluoromethyl)-R-lactone. Hydrolysis of the R-lactone would afford the observed 3,3,3-trifluorolactic acid (10). Alternatively, thioacyl fluoride 8 may be converted to the corresponding thiol acid, and intramolecular displacement of the fluoromethoxy group by the thiolate of the thiol acid would give 2-(trifluoromethyl)-R-thiolactone; hydrolysis of the R-thiolactone and the thiol acid would also give lactic acid 10. There is precedent for such intramolecular displacement reactions in chemical experience (37-39) and in the β-lyase-catalyzed bioactivation of bromine-containing cysteine S-conjugates (40). Acid 9 may also undergo an internal SN2 reaction in which the fluoride of the fluoromethoxy group is displaced by the carboxylate to give ether-lactone 4-oxo-5-(trifluoromethyl)-1,3-dioxolane. It is not known whether such substituted 1,3-dioxolanes are stable. Finally, a base-catalyzed R-elimination of the fluoromethoxy group of acid 9 to give a carbene followed by hydrolysis of the carbene to give lactic acid 10 was also considered. It is unlikely, however, that carbene formation would be favored under the conditions of the experiments. The lack of stability of acid 9 was also demonstrated in stoichiometry studies. Incubation of conjugate 4 with rat kidney cytosol or the pyridoxal model system showed that the pyruvate:fluoride ratio was about 1:2, as expected for the conversion of conjugate 4 to acid 9. In contrast, the pyruvate:acid 9 ratio was about 1:0.05, confirming the instability of acid 9. Cysteine S-conjugate 5 was not stable in buffer at pH 7.4 and 37 °C, and 19F NMR spectroscopic studies showed the release of fluoride (Figure 3B). The organic product was identified by 1H and 19F NMR spectroscopy and GC/ MS as 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazole-4-carboxylic acid (14). The proposed mechanism for the formation of thiazole 14 is shown in Scheme 4. Internal attack of the amino group of cysteine S-conjugate 5 on the vinylic side chain may give the intermediate thiazolidine 13, which may lose HF to give thiazole 14 (Scheme 4, pathway a) or thiazolidine 15 (Scheme 4, pathway b), which may tautomerize to thiazole 14. 19F NMR spectroscopic studies indicated that imine tautomer 14 predominates. The rapid cyclization of conjugate 5 (t1/2 ≈ 5 min) shows that cyclization is facile

818 Chem. Res. Toxicol., Vol. 10, No. 7, 1997

and may compete with β-lyase-catalyzed biotransformation in the kidney and may, thereby, reduce flux through the β-lyase pathway. Preliminary experiments indicate that thiazole 14 is present in the urine of rats given fluoroalkene 1.1 A thorough exploration of the fate of fluoroalkene 1 is important for understanding its potential nephrotoxicity. The available data on the metabolism and bioactivation of fluoroalkene 1, including the present results, are summarized in Scheme 1. Fluoroalkene 1 may undergo glutathione S-transferase-catalyzed glutathione S-conjugate formation to give diasteriomeric S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione (2) and (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1propenyl]glutathione (3). γ-Glutamyltransferase- and dipeptidase-catalyzed hydrolysis of glutathione S-conjugates 2 and 3 would give cysteine S-conjugates 4 and 5, respectively. Acetylation of conjugates 4 and 5 would give the corresponding mercapturic acids, which have been detected in the urine of rats given fluoroalkene 1 (9). The β-lyase-catalyzed transformation of conjugate 4 would be expected to afford 2-(fluoromethoxy)-1,1,3,3,3pentafluoropropanethiolate (6), which may lose fluoride to give 2-(fluoromethoxy)-3,3,3-trifluorothiopropanoyl fluoride (8). The expected product obtained from the β-lyasecatalyzed biotransformation of conjugate 5 is 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenethiolate (7), which may tautomerize to give thioacyl fluoride 8. Hydrolysis of thioacyl fluoride 8 would give acid 9. The present studies confirm that fluoroalkene 1-derived cysteine S-conjugate 4 undergoes β-lyase-catalyzed biotransformation and show that acid 9 is the product of the reaction. The data also show that acid 9 is unstable and gives lactic acid 10.2 The finding that acid 9 is not stable has implications for evaluating the β-lyase-dependent metabolism of fluoroalkene 1 in man: the formation and excretion of acid 9 may be used to quantify flux of fluoroalkene 1 through the β-lyase pathway in humans anesthetized with sevoflurane, but the instability of acid 9 may make such analysis quantitatively unreliable. Finally, the rapid cyclization of conjugate 5 may compete with its bioactivation by β-lyase. In previous studies, the β-lyase-catalyzed biotransformation of conjugate 5 was demonstrated (35); in these studies the conjugate was added last to the incubation mixture, which apparently allowed rapid enzyme-substrate complex formation and prevented cyclization. If the cyclization of conjugate 5 is a quantitatively significant reaction, conjugate 4 may prove to be of greater toxicological significance than conjugate 5. Hence, studies designed to quantify the several metabolites and rearranged products of cysteine S-conjugates of fluoroalkene 1 are warranted and may provide insight into the mechanism of nephrotoxicity of fluoroalkene 1.

Acknowledgment. The authors thank Ms. Sandra Morgan for her assistance in preparing the manuscript. This research was supported by National Institute of Environmental Health Sciences Grant ES03127 and by Abbott Laboratories. 2 Acid 10 has been detected in the urine of rats given fluoroalkene 1 (R. A. Iyer and M. W. Anders, unpublished results), and acids 9 and 10 have been detected in the urine of human subjects anesthetized with sevoflurane (R. A. Iyer, E. J. Frink, Jr., T. J. Ebert, and M. W. Anders, unpublished results).

Iyer and Anders

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