Metabolism of Chloroprene
Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1555
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-2-
(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).
1
en-1-al (15) H NMR (500 MHz, CDCl3): δ 9.31 (s, 1 H, CHO),
P r ep a r a tion of 1-Hyd r oxybu t-3-en -2-on e (11). This com-
pound 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, J Vic ) 10.5 Hz, J Gem ) 1.0 Hz), 6.37 (dd, 1 H, H-4 Z
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, CDCl3): δ 185.7 (CHO), 147.2 (C-3), 136.9
(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).
P r ep a r a tion of (4R,5R)-tr a n s-4,5-Bis(br om om eth yl)-2,2-
d im eth yl-(1,3)d ioxola n e (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.
to CdO J Vic ) 17.6 Hz, J Gem ) 1.0 Hz), 6.48 (dd, 1 H, H-3, J E
)
17.6 Hz, J Z ) 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).
P r ep a r a tion of Ra t a n d Mou se Liver Micr osom es.
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, 160-
200 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.
P r ep a r a tion of (2R,3R)-1,4-Dibr om obu ta n e-2,3-d iol (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)dioxo-
lane (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)-1,4-
dibromobutane-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]20 +14.4° (c 5.1 in methanol).
D
P r ep a r a tion of (2S,3R)-3-Acetoxy-1,4-d ibr om o-2-ch lo-
r obu ta n e (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-chlorobu-
tane (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 C6H9-
Assa y for Ep oxid e Hyd r ola se 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.
Br2ClO2: C, 23.37; H, 2.94. Found: C, 23.63; H, 2.69. [R]20
+21.5° (c 5.1 in methanol).
D
P r ep a r a tion of (S)-(1-Ch lor oeth en yl)oxir a n e (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-chlorobu-
tane (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
Assa y for Cytoch r om e P 450 2E1. Levels of cytochrome
P450 2E1 for rat and mouse liver microsomes were determined
as described (23).
Micr osom a l In cu b a t ion of Ch lor op r en e for Sp ecies
Com p a r ison . 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