In Vitro Metabolism of Chloroprene: Species Differences, Epoxide

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 1,3-dimethyl-3,4,5 .... EI-MS m/z 106/104 (3.3/10, M+), 55 (100, M+ − CH2Cl), 27 (31, M+ − CH2Cl − CO)...
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Chem. Res. Toxicol. 2001, 14, 1552-1562

In Vitro Metabolism of Chloroprene: Species Differences, Epoxide Stereochemistry and a De-chlorination Pathway Lisa Cottrell,† Bernard T. Golding,*,‡ Tony Munter,‡ and William P. Watson*,† Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, SK10 4TJ, U.K., and Department of Chemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, U.K. Received July 10, 2001

Chloroprene (1) was metabolized by liver microsomes from Sprague-Dawley rats, Fischer 344 rats, B6C3F1 mice, and humans to the monoepoxides, (1-chloro-ethenyl)oxirane (5a/5b), and 2-chloro-2-ethenyloxirane (4a/4b). The formation of 4a/4b was inferred from the identification of their degradation products. With male Sprague-Dawley and Fischer 344 rat liver microsomes, there was a ca. 3:2 preference for the formation of (R)-(1-chloroethenyl)oxirane (5a) compared to the (S)-enantiomer (5b). A smaller but distinct enantioselectivity in the formation of (S)-(1-chloro-ethenyl)oxirane occurred with liver microsomes from male mouse (R:S, 0.90:1) or male human (R:S, 0.86:1). 2-Chloro-2-ethenyloxirane was very unstable in the presence of the microsomal mixture and was rapidly converted to 1-hydroxybut-3-en-2-one (11) and 1-chlorobut-3-en-2-one (12). An additional rearrangement pathway of 2-chloro-2ethenyloxirane gave rise to 2-chlorobut-3-en-1-al (14) and 2-chlorobut-2-en-1-al (15). Further reductive metabolism of these metabolites occurred to form 1-hydroxybutan-2-one (17) and 1-chlorobutan-2-one (18). In the absence of an epoxide hydrolase inhibitor, the microsomal incubations converted (1-chloroethenyl)oxirane to 3-chlorobut-3-ene-1,2-diol (21a/21b). When microsomal incubations were supplemented with glutathione, 1-hydroxybut-3-en-2-one was not detected because of its rapid conjugation with this thiol scavenger.

Introduction Chloroprene (1) (Chart 1) is a large-scale petrochemical with a worldwide production of 4.0 × 106 kg/annum, which compares with butadiene (2) and isoprene (3) at ca. 5.4 × 109 and 1.3 × 109 kg/annum, respectively. The primary use of these dienes is in the manufacture of synthetic rubbers. Chloroprene is used almost exclusively in the production of polychloroprene, a solvent resistant elastomer for automotive rubber goods and personal protection (1). The principal sources of exposure to chloroprene are therefore of an occupational nature (2). There is concern over the possible human health effects from exposure to chloroprene, particularly its potential carcinogenicity. A 2-year rodent cancer study using F344/N rats and B6C3F1 mice exposed to chloroprene by inhalation indicated that it was carcinogenic in both species at all concentrations studied (12-80 ppm) (3). There were, however, differences in organ specificity between species. In the rat, chloroprene was carcinogenic to oral cavity, thyroid gland, lung, kidney, and mammary gland, whereas in mice the susceptible organs were lung, circulatory system, Harderian gland, kidney, forestomach, liver, mammary gland, skin, mesentery, and Zymbal’s gland. On the basis of the data for the induction of lung neoplasms in female mice, the study concluded that chloroprene has a similar carcinogenic potency to butadiene, which is substantially greater than isoprene (4). * To whom correspondence should be addressed: E-mail: (B.T.G.) [email protected]; (W.P.W.) [email protected]. † Syngenta Central Toxicology Laboratory. ‡ Department of Chemistry.

There is conflicting data on the mutagenicity of chloroprene: both positive (5) and negative (6) results have been reported. However, it seems likely that impurities may have contributed to the positive findings in the absence of mammalian enzymes (7). In in vivo studies with B6C3F1 mice, it was reported that chloroprene did not increase chromosomal aberrations or sister chromosome exchanges in bone marrow cells or raise the frequency of micronucleated erythrocytes in peripheral blood (8). A recent review by the International Agency for Research on Cancer concluded that there is sufficient evidence in experimental animals for the carcinogenicity of chloroprene but inadequate evidence in humans. The overall evaluation was that chloroprene is possibly carcinogenic to humans and it was therefore classified as a Group 2B carcinogen (9). The toxicology of chloroprene has recently been reviewed in detail (10). It may be expected that the rodent carcinogenicity of chloroprene is derived from one or more of its metabolites. However, there are no comprehensive studies on the metabolism of chloroprene. It was suggested that 2-chloro2-ethenyloxirane (4a/4b) and/or (1-chloroethenyl)oxirane (5a/5b) could be intermediates in the biotransformation of chloroprene (11). This conclusion was based on the finding that 4-(4-nitrobenzyl)pyridine trapped a volatile metabolite produced by the action of mouse liver microsomes on chloroprene. In a preliminary report, Himmelstein et al. (12) claimed to have identified (1-chloroethenyl)oxirane (5a/5b) from the liver microsomal metabolism of chloroprene, but did not specify the relative amounts of the enantiomers or identify other metabolites.

10.1021/tx0155404 CCC: $20.00 © 2001 American Chemical Society Published on Web 10/13/2001

Metabolism of Chloroprene Chart 1

Rac-(1-chloroethenyl)oxirane has recently been shown to be mutagenic in a range of strains of Salmonella typhimurium, but to be nonclastogenic in cultured Chinese Hamster V79 cells (13). In this paper, we define the structures, and where appropriate, stereochemistry of chloroprene metabolites from rodent species and humans. The principal metabolites have been identified by comparison with synthetic reference standards. The standards include the monochiral (S)-(1-chloroethenyl)oxirane (5b), for which a concise synthetic route has been devised. The data are compared with results from studies of butadiene and isoprene.

Materials and Methods Caution. All work involving chloroprene, its epoxides, and derived chloroaldehydes and ketones, should be performed with protective clothing and in a well-ventilated fume hood. Chemicals. 3-Chloroperoxybenzoic acid (m-CPBA)1 and butadiene monoxide (ethenyloxirane) were purchased from Aldrich Chemical Co. (Poole, Dorset, U.K.). The commercial m-CPBA was purified by washing with phosphate buffer (pH 7.5), 1 Abbreviations: DBU,1,8-diazabicyclo[5.4.0]undec-7-ene; m-CPBA, 3-chloroperoxybenzoic acid; DMPU, 1,3-dimethyl-3,4,5,6-tetrahydro2(1H)-pyrimidinone; PEG, poly(ethylene glycol); PNP, p-nitrophenol; SIM, selected ion monitoring.

Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1553 filtering, and drying the residue in a vacuum desiccator (14). Iodometric assay indicated 99+% purity. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), Dess-Martin periodinane, 2-acetoxyisobutyryl chloride, mesitylene, Dowex 50WX8-200 resin, 1-hydroxybutan-2-one, and (E/Z)-3-chlorobut-2-en-1-ol were purchased from Lancaster Synthesis (Eastgate, Lancashire, U.K.). 3,4-Dichloro-1-butene was purchased from Acros Organics (Fischer Scientific, Loughborough, Leicestershire, U.K.). 2-Chlorobut-3-en-1-ol was prepared from butadiene monoxide (ethenyloxirane) according to the method of Kadesch (15). 1-Chlorobutan-2-one was prepared as described (16). Petrol refers to the solvent with boiling range 40-60 °C. Chromatographic Methods. GC-analyses were performed with a Pye Unicam GCD gas chromatograph containing a 1.5 m × 4 mm i.d. 10% PEG-terephthalate column and a flame ionization detector. Preparative GC was performed with a Varian series 2700 gas chromatograph containing a 2.5 m × 4 mm i.d. 10% poly(ethylene glycol) (PEG) 20 M column and a thermal conductivity detector. Nitrogen was used as carrier gas for both instruments. Spectrometric Methods. The 1H and 13C NMR spectra were recorded with either a 500 MHz JEOL-JNM-LA 500 spectrometer, a 200 MHz Bruker AC 200F or a 400 MHz Bruker Avance DPX spectrometer operating at frequencies given with the spectral data for each compound. The compounds were dissolved in CDCl3 or D2O. Residual proton signals from the deuterated solvents were used as references. The electron impact mass spectra of reference compounds were recorded with a Micromass Auto Spec-M mass spectrometer at an ionizing voltage of 55 eV. Elemental analyses were performed on a Carlo Erba Instrumentazione model 1106 analyzer. Optical rotations were recorded with an Optical Activity PolAAr 2001 automatic polarimeter. GC-MS Analysis of Reference Compounds and Metabolites. Reference standards were analyzed by a GC-MS system comprising of a Hewlett-Packard 5973 mass selective detector and Hewlett-Packard 6890 GC equipped with a split/splitless injector. Compounds were separated using a Chiraldex G-PN (gamma cyclodextrin propionyl) capillary column (30 m × 0.25 mm i.d.) with a helium flow of 1.1 mL/min and an injector temperature of 200 °C with a split ratio of 100:1. The oven temperature was held at 40 °C for 10 min and then increased at 10 °C/min for the following program: 60 °C (4 min), 100 °C (10 min), and 150 °C (1 min). The MS was operated in scan mode in order to determine the ions used for selected ion monitoring (SIM) of components in the microsomal incubations. Biochemical Reagents. Biochemicals were obtained from Sigma (Poole, Dorset, U.K.). Coomassie blue protein reagent was obtained from Bio-rad Laboratories Ltd., Hemel Hempstead, Hertfordshire, U.K. Animals. Male and female Sprague-Dawley rats (8-9 weeks old) and B6C3F1 mice (5-7 weeks old) were supplied by Charles River (Manston, Kent, U.K.) and were acclimatized for at least 4 days before use. Human Microsomes. Pooled microsomes from male and female human liver were obtained from In Vitro Technologies (Baltimore, MD). The pools were prepared from 15 donors. Preparation of 2-Chloro-1,3-butadiene (Chloroprene) (1). DBU (11.47 g, 75 mmol) was added dropwise with stirring to a solution of 3,4-dichloro-1-butene (6) (7.25 g, 58 mmol) in acetonitrile (50 mL) cooled in an ice-water bath. The reaction mixture was stirred at room temperature for 15 h. Mesitylene was added (10 mL) and the resulting solution was washed with saturated NaCl solution (7 × 50 mL), dried (Na2SO4), and filtered. To the filtrate was added 0.2 g of hydroquinone. Distillation through a Vigreux column (130 mm × 13 mm) and collection of the distillate (bp 55-58 °C) in a receiver flask cooled in dry ice afforded 2.0 g (39%) of chloroprene as a colorless liquid. The purity of the chloroprene was >99% according to GCanalysis. The distilled chloroprene was cooled in dry ice, sealed into glass ampules in vacuo (15 mm Hg) and stored at -80 °C. The 1H NMR spectral data were in agreement with literature

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data (17). 13C NMR (125.65 MHz, CDCl3): δ 139.0 (C-2), 133.8 (C-3), 118.6 (C-1 or C-4), 116.1 (C-4 or C-1). Preparation of rac-(1-Chloroethenyl)oxirane (5a/5b). Racemic 3,4-dichloro-1-butene (5.8 g, 46 mmol) was added dropwise to a solution of m-CPBA (10.7 g, 62 mmol) in dichloromethane (100 mL). The mixture was heated at reflux for 80 h. The progress of the epoxidation was followed by GC [column at 80-180 °C, 8 °C/min; 3,4-dichloro-1-butene, retention time 4.7 min; 2-(1,2-dichloro-ethyl)-oxirane (7) as two diastereoisomers, retention times 10.8 and 11.4 min, respectively]. The mixture was cooled in an ice-water bath and the precipitated 3-chlorobenzoic acid was filtered off. The filtrate was washed with 10% Na2SO3 (30 mL), saturated NaHCO3 solution (3 × 50 mL), water (2 × 50 mL), and saturated NaCl solution (2 × 50 mL). The organic phase was dried (Na2SO4) and filtered. Careful removal of dichloromethane by rotary evaporation under reduced pressure (200 mbar) without heating afforded 5.1 g (79%) of 2-(1,2-dichloro-ethyl)-oxirane (7) as a pale yellow liquid. DBU (6.7 g, 44 mmol) was added dropwise to a solution of the 2-(1,2dichloro-ethyl)-oxirane (7) in DMPU (15 mL). The mixture was stirred at room temperature for 3 h and the precipitate was filtered off. Distillation of the filtrate under reduced pressure (15 mm Hg) and collecting the distillate in a receiver flask cooled in dry ice afforded 816 mg (22%) of (1-chloroethenyl)oxirane (5a/ 5b) as a colorless liquid. The purity of the distilled epoxide was 94% according to GC-analysis. 1H NMR (500 MHz, CDCl3): δ 5.54 (d, 1 H, H-2′a, J ) 1.8 Hz) 5.38 (d, 1 H, H-2′b, J ) 1.5 Hz), 3.46 (m, 1 H, H-2), 2.86 (dd, 1 H, H-3a, J ) 5.8, 3.9 Hz), 2.82 (dd, 1 H, H-3b, J ) 5.5, 2.4 Hz). 13C NMR (125.65 MHz, CDCl3): δ 138.2 (C-1′), 115.3 (C-2′), 52.9 (C-2), 47.8 (C-3). Preparation of rac-3-Chlorobut-3-ene-1,2-diol (21a/21b). This compound was essentially prepared according to a published method (18). A solution of (1-chloroethenyl)oxirane (68 mg, 0.65 mmol) in 97% formic acid (0.5 mL) was stirred at 60 °C for 1.5 h. Water (0.9 mL) was added and the solution was heated at 105-110 °C for 2 h. After cooling, the mixture was extracted with chloroform (3 × 0.7 mL). The aqueous phase was concentrated in vacuo to afford 61 mg of a crude diol. This was purified by chromatography on silica gel eluting with diethyl ether-petrol (4:1, vol/vol) to give 37 mg (46%) of 3-chlorobut-3ene-1,2-diol (21) as a colorless oil. 1H NMR (500 MHz, CDCl3): δ 5.53 (m, 1 H, H-4a), 5.37 (m, 1 H, H-4b), 4.24 (m, 1 H, H-2), 3.76 (dd, 1 H, H-1a, J ) 11.6, 3.7 Hz), 3.68 (dd, 1 H, H-1b, J ) 11.5, 6.3 Hz), 2.95 (br, 1 H, OH), 2.27 (br, 1 H, OH). 13C NMR (125.65 MHz, CDCl3): δ 140.3 (C-3), 113.9 (C-4), 74.8 (C-2), 64.3 (C-1). EI-MS m/z 106/104 (17/55, M+ - H2O), 93/91 (33/100, M+ - CH2OH), 56 (36, M+ - Cl - CH2OH). Small Scale Reaction of Chloroprene with m-CPBA. Chloroprene (15 mg, 0.17 mmol) was added to a solution of m-CPBA (29 mg, 0.17 mmol) in CDCl3 (0.55 mL). The mixture was held at room temperature for 70 h and was monitored by 1H NMR and GC. The NMR spectra showed the formation of two epoxides, (1-chloroethenyl)oxirane (5a/5b) and 2-chloro-2ethenyloxirane (4a/4b) in a ratio of 1:1.8. 1H NMR (200 MHz, CDCl3) for 2-chloro-2-ethenyloxirane: δ 5.81 (dd, 1 H, H-1′, J ) 16.8, 10.0 Hz), 5.60 (dd, 1 H, H-2′Z, J ) 16.9, 1.0 Hz), 5.33 (dd, 1 H, H-2′E, J ) 9.9, 1.0 Hz), 3.20 (d, 1 H, H-3a, J ) 5.4 Hz), 2.90 (d, 1 H, H-3b, J ) 5.4 Hz). The 1H NMR signals corresponding to (1-chloroethenyl)oxirane were identical with those for a standard sample of the compound. GC-analysis (column at 80-180 °C, 8 °C/min) of the mixture showed two major product peaks in a ratio of 1:1.8 and with retention times of 3.8 and 6.8 min, respectively. The compound at 3.8 min had the same retention time as a standard sample of (1-chloroethenyl)oxirane. After 70 h, D2O (50 µL) was added and the mixture was agitated on a roller mixer at room temperature for 70 h. D2O (550 µL) was added and the mixture was shaken. The D2Ophase was separated and analyzed by 1H NMR, which showed the presence of mainly 1-hydroxy-but-3-en-2-one (11) (comparison of the 1H NMR with data from an authentic synthetic sample of 11).

Cottrell et al. Large Scale Reaction of Chloroprene with m-CPBA. Chloroprene (1.1 g, 12.5 mmol) was added dropwise to a stirred solution of m-CPBA (2.15 g, 12.5 mmol) in dichloromethane (25 mL). The mixture was stirred at room temperature for 48 h. GC-analysis showed the presence of two major product peaks with the same retention times as the compounds observed in the small scale reaction. The solution was cooled in an ice-water bath and the precipitate was filtered off. The filtrate was concentrated to about 2.5 mL by careful removal of dichloromethane. The compound with the retention time of 6.8 min (analytical GC) was isolated from the resulting mixture by preparative GC (injection volume was 50 µL, column temperature 130 °C, retention time 8 min). Collecting the compound from 12 successive separations afforded 61 mg of 1-chlorobut3-en-2-one (12) as a pale yellow liquid. 1H NMR (500 MHz, CDCl3): δ 6.52 (dd, 1 H, H-3, J ) 17.4, 10.7 Hz), 6.32 (d, 1 H, H-4Z, J ) 17.4 Hz), 5.90 (d, 1 H, H-4E, J ) 10.7 Hz), 4.21 (s, 2 H, 2 × H-1). 13C NMR (125.65 MHz, CDCl3): δ 191.4 (C-2), 132.5 (C-3), 130.8 (C-4), 46.7 (C-1). EI-MS m/z 106/104 (3.3/10, M+), 55 (100, M+ - CH2Cl), 27 (31, M+ - CH2Cl - CO). Highresolution mass spectrometry gave the molecular formula as C4H5ClO (M+ 104.0031, calcd 104.0029). Epoxidation of (1-Chloroethenyl)oxirane with m-CPBA. (1-Chloro-ethenyl)oxirane (15 mg, 0.14 mmol) was added to a solution of m-CPBA (27 mg, 0.16 mmol) in CDCl3 (0.55 mL). The mixture was held at room temperature for 140 h and was monitored by 1H NMR (200 MHz). The spectra showed the formation of new signals as multiplets centered at δ 3.38, 3.00, 2.90, and 2.73 ppm in addition to the signals of (1-chloroethenyl)oxirane. The reaction proceeded slowly and it was estimated that after 48 h about 25% and after 140 h about 40% of (1-chloroethenyl)oxirane had reacted. Preparation of (E/Z)-3-Chlorobut-2-en-1-al (13a/13b). A solution of (E/Z)-3-chlorobut-2-en-1-ol (50 mg, 0.47 mmol) in dry dichloromethane (3 mL) was added to a stirred solution of Dess-Martin periodinane (19) (239 mg, 0.56 mmol) in dry dichloromethane (3 mL) under nitrogen. The reaction mixture was stirred for 1 h at room temperature in the dark. Purification by chromatography (silica, elution with dichloromethane) and evaporation of the solvent yielded (Z)- and (E)-3-chlorobut-2en-1-al (13a/13b) (40 mg, 81%) in a ratio of 8:1. Major isomer (Z), 13a, 1H NMR (500 MHz, CDCl3): δ 9.93 (d, 1 H, CHO, J ) 7 Hz), 6.04 (m, 1 H, H-2), 2.29 (m, 3 H, CH3). Minor isomer (E), 13b, 1H NMR (500 MHz, CDCl3): δ 9.77 (d, 1 H, CHO, J ) 7.3 Hz), 6.21 (m, 1 H, H-2), 2.52 (m, 3H, CH3). EI-MS: m/z 106/104 (29/97, M+), 105/103 (11/47, M+ - H), 77/75 (7/12, M+ - CHO), 69 (26, M+ - Cl), 39 (100, M+ - HCl - CHO). High-resolution mass spectrometry gave the molecular formula as C4H5ClO (M+ 104.0024, calcd 104.0029) Preparation of rac-2-Chlorobut-3-en-1-al (14) and (Z)2-Chlorobut-2-en-1-al (15). A solution of rac-2-chlorobut-3-en1-ol (139 mg, 1.3 mmol) in dry dichloromethane (3 mL) was added to a stirred solution of Dess-Martin periodinane (19) (668 mg, 1.58 mmol) in dry dichloromethane (6 mL) under nitrogen. The mixture was stirred for 2 h at room temperature in the dark. The mixture was purified by chromatography (silica 5.2 g, column 140 mm × 13 mm, elution with dichloromethane). Fractions of about 8 mL were collected. The solvent was rotary evaporated and the fractions were analyzed by 1H NMR. Fraction 3 (21 mg) was a mixture of 2-chlorobut-3-en-1-al (14) and (Z)-2-chlorobut-2-en-1-al (15) in a ratio of 1:7 according to 1H NMR. Fraction 4 (103 mg) was a mixture of 14 and 15 in a ratio of 7:3. An attempt to separate the aldehydes from fraction 4 by column chromatography (silica 18.5 g, column 100 mm × 28 mm, elution dichloromethane) yielded only aldehyde 15 (54 mg, 40%) from isomerization of 14. The experiment was repeated with attempts to isolate pure 2-chlorobut-3-en-1-al (14) by the workup procedure described by Dess and Martin (19) or by distillation of the reaction mixture under reduced pressure. These attempts were unsuccessful and a mixture of 14 and 15 was again obtained. The isomers gave only a single peak on analytical GC. 2-Chlorobut-3-en-1-al (14) 1H NMR (500 MHz,

Metabolism of Chloroprene CDCl3): δ 9.36 (d, 1 H, CHO, J ) 2.4 Hz), 5.85 (m, 1 H, H-3), 5.48 (d, 1 H, H-4Z, J ) 17.1 Hz), 5.42 (d, 1 H, H-4E, J ) 10.1 Hz), 4.63 (m, 1 H, H-2). 13C NMR (50.3 MHz, CDCl3): δ 192.3 (CHO), 129.9 (C-3), 122.2 (C-4), 64.3 (C-2). (Z)- 2-Chlorobut-2en-1-al (15) 1H NMR (500 MHz, CDCl3): δ 9.31 (s, 1 H, CHO), 6.91 (q, 1 H, H-3, J ) 6.7 Hz), 2.07 (d, 3 H, CH3, J ) 6.7 Hz). 13C NMR (50.3 MHz, CDCl ): δ 185.7 (CHO), 147.2 (C-3), 136.9 3 (C-2), 15.4 (CH3). EI-MS m/z 106/104 (28/93, M+), 105/103 (10/ 16, M+ - H), 77/75 (7/22, M+ - CHO), 69 (13, M+ - Cl), 39 (100, M+ - HCl - CHO). High-resolution mass spectrometry gave the molecular formula as C4H5ClO (M+ 104.0027, calcd 104.0029). Preparation of (4R,5R)-trans-4,5-Bis(bromomethyl)-2,2dimethyl-(1,3)dioxolane (8). This compound was synthesized according to the procedure of Townsend et al. (20) except that dimethyl (2R,3R)-L-tartrate was used as starting material instead of diethyl (2R,3R)-L-tartrate and the reduction of dimethyl (2R,3R)-2,3-O-isopropylidenetartrate was performed with LiAlH4 in ether. Preparation of (2R,3R)-1,4-Dibromobutane-2,3-diol (9). Dowex 50WX8-200 resin (20 g) was added to a solution of (4R,5R)-trans-4,5-bis(bromomethyl)-2,2-dimethyl-(1,3)dioxolane (4.8 g, 16.7 mmol) in methanol (50 mL). The mixture was stirred at room temperature for 23 h and the resin was filtered off. Evaporation of the solvent and purification of the residual crude product by chromatography [silica, elution with ethyl acetate/petrol (1/1 vol/vol)] yielded a white solid, which was recrystallized from ethyl acetate/petrol to afford (2R,3R)-1,4dibromobutane-2,3-diol (9) (3.33 g, 80%), mp 76-77 °C. 1H NMR (200 MHz, CDCl3): δ 3.94 (m, 2 H, H-2, H-3), 3.46 (m, 4 H, 2 × H-1, 2 × H-4), 2.59 (d, 2 H, 2 × OH, J ) 6.0 Hz). 13C NMR (50.3 MHz, CDCl3): δ 71.7 (C-2, C-3), 35.0 (C-1, C-4). EI-MS m/z 249/ 247 (9/5, M+), 233/231/229 (24/87/35, M+ - H2O), 168/166 (93/ 85, M+ - Br), 155/153 (32/30, M+ - CH2Br), 137/135 (18/20, M+ - CH2Br - OH), 125/123 (97/100, M+ - CH2Br - CHOH). Anal. calcd for C4H8Br2O2: C, 19.38; H, 3.25. Found: C, 19.50; H, 2.94. [R]20D +14.4° (c 5.1 in methanol). Preparation of (2S,3R)-3-Acetoxy-1,4-dibromo-2-chlorobutane (10). 2-Acetoxyisobutyryl chloride (2.47 g, 15 mmol) in dichloromethane (10 mL) was added to a solution of (2R,3R)1,4-dibromobutane-2,3-diol (3.1 g, 12.5 mmol) in dichloromethane (75 mL). The reaction mixture was stirred at room temperature for 5 h. The resulting solution was washed with 5% NaHCO3 solution (3 × 60 mL) and with H2O (2 × 80 mL), dried (MgSO4), and filtered. The solvent was removed to yield a pale yellow oil which crystallized upon cooling to -15 °C. Recrystallization from methanol afforded of (2S,3R)-3-acetoxy-1,4-dibromo-2-chlorobutane (10) as white crystals (2.9 g, 75%), mp 40-42 °C. 1H NMR (200 MHz, CDCl3): δ 5.10 (m, 1 H, H-3), 4.35 (m, 1 H, H-2), 3.70 (m, 4 H, 2 × H-1, 2 × H-4), 2.09 (s, 3 H, CH3). 13C NMR (50.3 MHz, CDCl3): δ 169.4 (CO), 72.6 (C-3), 58.7 (C-2), 33.9 (C-1 or C-4), 31.7 (C-1 or C-4), 20.9 (CH3). EI-MS: m/z 252/250/ 248/246 (6/40/55/20, M+ - CH3CO2), 231/229/227 (16/62/44, M+ - Br), 215/213/211 (5/15/11, M+ - CH2Br), 167/165 (15/16, M+ - C2H3BrCl), 43 (100, M+ - C4H6Br2ClO). Anal. calcd for C6H9Br2ClO2: C, 23.37; H, 2.94. Found: C, 23.63; H, 2.69. [R]20D +21.5° (c 5.1 in methanol). Preparation of (S)-(1-Chloroethenyl)oxirane (5b). To a solution of sodium ethane-1,2-diolate [from sodium (330 mg, 14 mmol)] in dry ethane-1,2-diol (8 mL) under high vacuum (0.07 mm Hg) was added (2S,3R)-3-acetoxy-1,4-dibromo-2-chlorobutane (1.0 g, 3.25 mmol). The reaction mixture was stirred at 30 °C for 2 h at 0.07 mm Hg, and the epoxide (287 mg, 85%) was collected in a receiver cooled in liquid nitrogen. Purification by preparative GC [column at 100 °C for 5 min, 100-230 °C, 16 °C/min] afforded (S)-(1-chloroethenyl)oxirane (5b) (125 mg, 37%) as a colorless liquid. The purity was >99% according to GC and NMR analysis. The enantiopurity of 5b was determined as 99.6% ee by GC analysis (Chiraldex G-PN chiral column). The 1H and 13C NMR data were the same as those reported above for racemic (1-chloroethenyl)oxirane. EI-MS: m/z 106/104 (5/15, M+), 105/103 (3/14, M+ - H), 77/75 (5/13, M+ - CHO), 69

Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1555 (23, M+ - Cl), 63/61 (2/7, M+ - C2H3O), 39 (100, M+ - HCl CHO). High-resolution mass spectrometry gave the molecular formula as C4H5ClO (M+ 104.0033, calcd 104.0029). [R]20D -25.2° (c 5.1 in methanol). Preparation of 1-Hydroxybut-3-en-2-one (11). This compound was prepared essentially according to a published method (21). To a solution of 1,4-dihydroxybut-2-yne (30 g, 0.349 mol) in water (260 mL) was added mercury(II) sulfate (1.56 g, 5.26 mmol) and concentrated sulfuric acid (3.5 g, 35 mmol). The mixture was stirred for 2 h at 50 °C. After cooling, the acid was neutralized by careful addition of barium carbonate. Filtration through Celite and concentration of the filtrate at 40 °C in vacuo gave an aqueous solution of 1-hydroxybut-3-en-2-one (11). 1H NMR (400 MHz, D2O), 4.62 (s, 2 H, 2 × H-1), 6.04 (dd, 1 H, H-4 E to CdO, JVic ) 10.5 Hz, JGem ) 1.0 Hz), 6.37 (dd, 1 H, H-4 Z to CdO JVic ) 17.6 Hz, JGem ) 1.0 Hz), 6.48 (dd, 1 H, H-3, JE ) 17.6 Hz, JZ ) 10.5 Hz); 13C NMR: δ 202.7 (CO), 132.6 (C-4 or C-3), 131.3 (C-3 or C-4), 66.2 (C-1). Preparation of Rat and Mouse Liver Microsomes. Animals were sacrificed in a slow rising concentration of CO2 followed by cardiac puncture. Blood was taken and the liver was quickly removed, blotted, weighed, and placed in ice-cold 1.15% KCl solution. Tissue was minced with scissors and washed with 1.15% KCl solution to remove blood. After draining the 1.15% KCl solution, potassium phosphate (0.1 M)/5 mM EDTA (pH 7.4) was added and the liver homogenized with a Potter tissue press. The homogenate was centrifuged at 16900g for 15 min at 4 °C and the pellet was discarded. The supernatant was centrifuged at 105000g for 70 min at 4 °C. The supernatant (cytosol) was decanted and stored at -80 °C. The pellet was resuspended in 0.1 M potassium phosphate and the homogenate was centrifuged at 105000g for 70 min at 4 °C. The pellet was resuspended in 0.1 M potassium phosphate buffer (pH 7.4, 1 mL/2 g of original tissue). The prepared microsomes were stored at -80 °C. This procedure was used for the preparation of microsomes from male and female rats, e.g. Sprague-Dawley (male, n ) 3, 220-260 g body weight and female, n ) 6, 160200 g body weight) and male and female B6C3F1 mice (n ) 6 for male and female, 16-20 g body weight). The microsomal protein concentration was determined by Coomassie blue protein reagent. Assay for Epoxide Hydrolase Activity. The enzymatic microsomal epoxide hydrolase activity in mouse and rat liver microsomes was measured with styrene oxide as the substrate, essentially as described (22). The reaction was performed in a total volume of 400 µL and contained 1.2 mg of microsomal protein in 0.1 M potassium phosphate buffer (pH 7.4). After initiation of the reaction by the addition of 40 µL of styrene oxide (15 mM), the mixture was incubated at 37 °C for 5 min. The reaction was terminated by the addition of ethyl acetate (1 mL). The resulting mixture was centrifuged. The organic layer was removed and concentrated by blowing with a stream of nitrogen. The residue was reconstituted in methanol/water (50:50). Samples were analyzed by HPLC using a C18 Luna 250 mm × 4.6 mm (Phenomenex) column eluted with a linear gradient of aqueous methanol 50 to 80% (v/v) over 10 min with detection by UV at 254 nm. The analyses were carried out in duplicate, and the levels of phenylethane-1,2-diol formed were determined by reference to a standard curve. Controls were prepared by the addition of styrene oxide to boiled microsomes. Assay for Cytochrome P450 2E1. Levels of cytochrome P450 2E1 for rat and mouse liver microsomes were determined as described (23). Microsomal Incubation of Chloroprene for Species Comparison. The microsomal incubations were carried out in 2 mL vials with a total incubation volume of 1 mL. Each incubation contained 4.2 mg of NADPH, 0.1 M potassium phosphate buffer (pH 7.4), 14 mM cyclohexene oxide, 2.3 mg of microsomal protein, and a substrate concentration ranging from 1 to 40 mM. The reaction mixture was incubated on a roller mixer for 30 min at 37 °C. The reaction was terminated by the addition of a saturating amount of NaCl followed by addition

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of cycloheptanone (2 µL, 17 mM) as an internal standard. The samples were extracted with ethyl acetate (2 × 1 mL) and the combined extracts were concentrated to a volume of ca. 200 µL. Control samples were run in the absence of substrate. Headspace gas sampling analysis was carried out on the test samples, but extraction of the samples with ethyl acetate gave better sensitivity for detection of metabolites. Samples were analyzed by GC-MS in SIM mode using the GC conditions described above in section entitled “GC-MS Analysis of Reference Compounds and Metabolites”. The ions in the mass spectra selected for the comparison of metabolism between species were m/z 55, 57, and 104. For full identification of metabolites, a number of incubations using microsomes from male SD rats were carried out and the samples pooled prior to analysis by GC-MS in scan mode. Incubation of (1-Chloroethenyl)oxirane with Epoxide Hydrolases. For identification of the enzymatic hydrolysis products of monoepoxide metabolites in microsomal incubations, crude rat liver epoxide hydrolase, 50 µL (1.6 mg, specific activity 2.29 pmol/min/mg) (gift from Dr C. Morisseau and Professor B. Hammock, University of California, Davis), was incubated with (1-chloroethenyl)oxirane (10 mM) in a total volume of 300 µL potassium phosphate buffer (0.1 M, pH 7.4). The samples were prepared in duplicate and incubated at 37 °C for 15 min. The reaction was stopped by adding a saturating amount of NaCl followed by extraction with ethyl acetate (3 × 1 mL). The samples were concentrated under a stream of nitrogen before analysis by GC-MS. The incubation was repeated with 10 µL of human soluble liver epoxide hydrolase (0.12 mg, specific activity 31 pmol/min/mg), following the same procedure as for the rat. The samples were analyzed by GC-MS using the conditions described above in section “GC-MS Analysis of Reference Compounds and Metabolites”. The MS was operated in scan mode to determine the ions used for SIM of the microsomal incubations. Microsomal Incubations without Epoxide Hydrolase Inhibitor. Liver microsomes from male SD rats were incubated with 30 mM chloroprene using the same conditions as described above for incubations for species comparison, except that epoxide hydrolase inhibitor was omitted. The samples were prepared in duplicate with controls performed in the absence of substrate. Samples were incubated at 37 °C for 15, 30, 60, and 120 min. Samples were analyzed by GC-MS using the same conditions as described for incubations with epoxide hydrolase with the MS in SIM mode and ions selected of m/z 91, 93, 75, 55, and 57. A sample and control at 120 min were also analyzed in scan mode. Microsomal Incubations in the Presence of Glutathione. Male SD rat liver microsomes (2.33 mg of protein/mL) were incubated for 25 min at 37 °C in 0.1 M potassium phosphate buffer pH 7.4 (1 mL) containing 4.2 mg NADPH, cyclohexene oxide (14 mM) and chloroprene (30 mM). An aqueous solution of glutathione (100 µL of 1 mg/mL), was added and the reaction mixture was incubated for a further 5 min. The samples were analyzed using the procedure described in the section “Microsomal Incubation of Chloroprene for Species Comparison”. Control samples were run in the absence of glutathione.

Results and Discussion Synthesis of Chloroprene. Chloroprene is no longer commercially available, and it was therefore necessary to develop an efficient small-scale preparative method for this compound. Commercially available racemic 3,4dichloro-1-butene (6) was dehydrochlorinated using DBU. The main problem was to isolate the chloroprene free of organic solvent. This was accomplished by performing the reaction with DBU in acetonitrile and then adding mesitylene (bp 163-165 °C). An aqueous wash removed the acetonitrile and careful fractional distillation gave chloroprene that was over 99% pure by GC analysis. Synthesis of Racemic 5a/5b and (S)-(1-Chloroethenyl)oxirane (5b). Epoxidation of racemic 3,4-dichloro-

Cottrell et al. Scheme 1a

a Reagents: (i) Dowex 50WX8-200, MeOH; (ii) 2-acetoxyisobutyryl chloride, CH2Cl2; (iii) NaOCH2CH2OH, HOCH2CH2OH.

1-butene with 3-chloroperoxybenzoic acid gave 2-(1,2dichloro-ethyl)-oxirane as a diastereoisomeric mixture, which was dehydrochlorinated with DBU to give racemic (1-chloroethenyl)oxirane (5a/5b). The route to the monochiral (S)-(1-chloroethenyl)oxirane is shown in Scheme 1. Acid-catalyzed hydrolysis of (4R,5R)-trans-4,5-bis(bromomethyl)-2,2-dimethyl-(1,3)dioxolane (8) gave (2R,3R)-1,4dibromobutane-2,3-diol (9), which was converted into (2S,3R)-3-acetoxy-1,4-dibromo-2-chlorobutane (10) using 2-acetoxyisobutyryl chloride (24). Treatment of 10 with sodium ethane-1,2-diolate in ethane-1,2-diol brought about ring closure to an intermediate epoxide followed by dehydrobromination to give (S)-(1-chloroethenyl)oxirane (5b). The conversion of 10 into 5b is modeled on the reported treatment of 3-acetoxy-1,2,4-trichlorobutane with base to give 2-(1,2-dichloro-ethyl)-oxirane, 2,3-bis(chloromethyl)-oxirane and 3-chloro-2-(chloromethyl)-oxetane (25). The use of bromo in place of chloro as leaving group enhances the cyclization to an epoxide compared to an oxetane. The assignment of the absolute configuration of 5b is based on that of the starting material 8, which was derived from (R,R)-tartaric acid, and the known stereochemistry of the formation of vicinal chloroacetates from 1,2-diols using 2-acetoxyisobutyryl chloride (24). The final ring closure of 10 to 5b is therefore presumed to proceed with retention of configuration at the oxygen-substituted chiral center. The enantiomeric excess of 5b was determined as 99.6% by GC analysis with the Chiraldex G-PN column. Syntheses of Chloroaldehydes 13a/13b, 14, and 15. The chloroaldehydes 13a/13b, 14, and 15 were prepared by oxidation of the corresponding alcohols 2-chlorobut3-en-1-ol and (E/Z)-3-chlorobut-2-en-1-ol with DessMartin periodinane (19). The assignment of the configuration of aldehydes 13a as (Z)-3-chlorobut-2-en-1-al (major isomer) and 13b as (E)-3-chlorobut-2-en-1-al (minor isomer) was based on the 1H NMR chemical shifts of the methyl groups. In the E-isomer, the methyl group is cis to the carbonyl group and as expected, the methyl group was observed 0.23 ppm downfield from that of the Z-isomer (26). The oxidation of 2-chlorobut-3-en-1-ol gave a mixture of 2-chlorobut-3-en-1-al (14) and the conjugated aldehyde (Z)-2-chlorobut-2-en-1-al (15). The Z-configuration of 15 was assigned by comparison of the 1H NMR shifts with those reported (27). Several attempts to isolate pure 14 by silica gel chromatography, distillation of the reaction mixture under reduced pressure, or by diluting the reaction mixture with ether and extraction with aqueous sodium bicarbonate and sodium thiosulfate (19) were unsuccessful and mixtures of 14 and 15 were always obtained. The EI mass spectra of 13a/13b, 14, and 15 displayed the same fragmentation pattern. The molecular

Metabolism of Chloroprene

ion was observed at m/z 106/104 and cleavage of the formyl group, chlorine, and of HCl plus the formyl group from M+ produced the fragment ions at m/z 77/75, 69, and 39, respectively. Biomimetic Chemical Oxidation of Chloroprene: Formation and Rearrangement of 2-Chloro2-ethenyloxirane (4a/4b). Epoxidation of chloroprene with m-CPBA was monitored by 1H NMR, which clearly showed the formation of both 2-chloro-2-ethenyloxirane (4a/4b) and (1-chloroethenyl)oxirane (5a/5b) over a period of 70 h at room temperature in a ratio of ca. 1.8:1 (4/5). The 2-chloro-2-ethenyloxiranes were relatively stable in CDCl3, but addition of water and agitation at room temperature for a further 70 h caused hydrolysis to 1-hydroxybut-3-en-2-one (11). A preparative scale epoxidation of chloroprene was carried out in dichloromethane. Attempted isolation of 4a/4b by preparative GC (column temperature 130 °C) brought about rearrangement of 4a/4b to 1-chlorobut-3-en-2-one (12). Such a thermal rearrangement is well-known for chlorooxiranes (28). Formation of Chloro-2,2′-bioxiranes (16). The epoxidation of rac-(1-chloroethenyl)oxirane (5a/5b) with m-CPBA at room temperature was followed by 1H NMR. The spectrum after 23 h clearly showed new signals as complex multiplets centered at δ 3.38, 3.00, 2.90, and 2.73 ppm in addition to the signals from (5a/5b). The reaction proceeded slowly and it was estimated that after 48 h about 25% and after 140 h about 40% of (1-chloroethenyl)oxirane had reacted with m-CPBA to form chloro-2,2′bioxiranes (16) as a mixture of diastereoisomers. Analyses of Chloroprene Metabolites. Gas chromatographic separation of chloroprene metabolites was achieved with a Chiraldex-G-PN column. Using synthetic reference standards of 2-chloro-2-ethenyloxirane (4a/4b) and (1-chloroethenyl)oxirane (5a/5b), conditions were developed for complete separation of the regioisomeric epoxides 4 and 5 and complete resolution of epoxide enantiomers 5a and 5b. Enantiomers 4a and 4b were not resolved. Using this column for coupled GC-MS, we employed selective ion monitoring and scanning modes for MS detection and identification of mono-epoxides and other metabolites (see section “Identification of Metabolites in Microsomal Incubations of Chloroprene”). These conditions were also used to compare the stereochemistry of (1-chloroethenyl)oxirane (5a/5b) formed by liver microsomes from male and female Sprague-Dawley and Fischer 344 rats, B6C3F1 mice, and humans. Identification of Metabolites in Microsomal Incubations of Chloroprene. Rat liver microsomes from Sprague-Dawley rats were principally used for the identification of metabolites formed from chloroprene. All the metabolites identified using rat liver microsomes were also detected in incubations with mouse and human microsomes. The profiles of SIM of metabolites formed from chloroprene by liver microsomes from SpragueDawley rats are shown in Figure 1. Ions monitored were at m/z 104, 55, and 57. Metabolites were not detected in any of the control experiments: liver microsomes in the absence of NADPH, inactivated (boiled) microsomes, the absence of microsomes, and microsomes in absence of substrate. The proposed in vitro metabolism is shown in Scheme 2. Epoxides. To determine the enantioselectivity of microsomal epoxidations, inhibition of epoxide hydrolases was necessary, because catalytic hydrolysis may be a

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Figure 1. Profiles of selected ion monitoring m/z 104 (a), 55 (b), 57 (c) mass spectrometry of metabolites formed from chloroprene by liver microsomes from Sprague-Dawley rats.

competing enantioselective process (29, 30). Metabolites were analyzed by headspace analysis (31) and by extraction of the incubation mixtures with ethyl acetate. By monitoring for ions at m/z 75 and 104, (1-chloroethenyl)oxirane (5a/5b) was detected as the major mono-epoxide metabolite designated as (Ia/b). The R-enantiomer was formed preferentially compared to the S-enantiomer (R:S ratio 3:2) and was identified by gas chromatographic and mass spectrometric comparison with authentic standards of the R- and S -enantiomers (Figure 2). This epoxide was found to be stable toward hydrolysis in the absence of epoxide hydrolase. It was not possible to detect 2-chloro2-ethenyloxirane in the microsomal incubations because this epoxide readily underwent hydrolysis to 1-hydroxybut-3-en-2-one (11) (lifetime of the epoxide in water was