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A. M€antyl€a et al. / Bioorg. Med. Chem. 12 (2004) 3497–3502
4.2. Chemistry
dropwise. The mixture was refluxed for 4 h and evapo-
rated to dryness under high vacuum. The crude product
was purified by column chromatography (petroleum
ether/ethyl acetate 10:1) affording 4 (Rf ¼ 0:42) as a
yellow oil (2.04 g, 6.0 mmol 98.7%): 1H NMR (CDCl3) d
0.81 (9H, s, –C(CH3)3), 0.82–1.10 (5H, m, CHC(CH3)3,
Hax), 1.49 (1H, br m, CH–CH2), 1.74 (4H, m, Heq), 2.50
(2H, d, J ¼ 7:3 Hz, CH2–CH), 4.11 (3H, s, OCH3), 7.69
(2H, m, H-6, H-7), 8.05 (2H, m, H-5, H-8).
4.2.1. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-hydroxy-
[1,4]naphthoquinone-1-oxime (2). To a solution of bu-
parvaquone (1) (1.00 g, 3.06 mmol) and hydroxylamine
hydrochloride (0.53 g, 5.66 mmol) in ethanol (100 mL)
was added sodium acetate (0.76 g, 9.19 mmol) in water
(2 mL) and the mixture was refluxed for 3 days. The
solvent was removed under reduced pressure, and a
crude product was purified by silica gel column chro-
matography (petroleum ether/ethyl acetate, 3:1) afford-
ing 2 (5:1 mixture of E/Z isomers) (Rf ¼ 0:35) as a
yellow solid (0.92 g, 2.69 mmol, 88.1%): mp (decom-
4.2.4. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-methoxy-
[1,4]naphthoquinone-1-oxime (5). Compound 5 was pre-
pared as a product of compound 2 from 4 (2.18 g,
6.4 mmol) to give a brown oil, which was purified by
column chromatography (petroleum ether/ethyl acetate
20:1) yielding 5 (Rf ¼ 0:25) as a viscose oil (10:1 mixture
of E/Z isomers) (0.3 g, 0.84 mmol, 13.1%). 1H NMR (d6-
DMSO) E-isomer d 0.79 (9H, s, –C(CH3)3), 0.82–1.00
(5H, m, CHC(CH3)3, Hax), 1.41 (1H, br m, CH–CH2),
1.69 (4H, m, Heq), 2.40 (2H, d, J ¼ 7:2 Hz, CH2–CH),
3.92 (3H, s, OCH3), 7.62 (1H, td, J ¼ 7:5, 1.2 Hz, H-6),
7.72 (1H, td, J ¼ 7:4, 1.5 Hz, H-7), 8.08 (1H, dd,
J ¼ 7:8, 1.5 Hz, H-5), 8.93 (1H, d, J ¼ 7:9 Hz, H-8),
13.47 (1H, br s, OH); Z-isomer d 3.76 (3H, s, OCH3),
7.55 (1H, td, J ¼ 7:3, 1.2 Hz, H-6), 7.65 (1H, td, J ¼ 7:3,
1.6 Hz, H-7), 7.95 (1H, dd, J ¼ 7:9, 1.4 Hz, H-5), 8.04
(1H, d, J ¼ 7:9 Hz, H-8) (other signals overlap with the
E-isomer). 13C NMR (d6-DMSO) E-isomer d 27.3, 27.6,
31.2, 32.4, 34.0, 37.6, 48.1, 62.0, 126.9, 127.0, 129.2,
130.1, 130.7, 131.2, 132.6, 141.6, 161.0, 185.7; Z-isomer
d 37.7, 48.1, 61.1, 123.4, 126.3, 129.4, 130.2, 131.3,
132.3, 133.1, 142.7, 156.4, 185.6 (other signals overlap
with the E-isomer). ESI-MS: 356.4 (M+1). Anal. Calcd
(C22H29NO3Æ0.5EtOAc) C: 75.16, H: 9.08, N: 3.51;
found C: 75.14, H: 7.37, N: 3.73.
1
posed). H NMR (d6-DMSO) E-isomer d 0.80 (9H, s,
–C(CH3)3), 0.82–1.01 (5H, m, CHC(CH3)3, Hax), 1.46
(1H, br m, CH–CH2), 1.69 (4H, m, Heq), 2.39 (2H,
d, J ¼ 7:2 Hz, CH2–CH), 7.61 (1H, td, J ¼ 7:5; 1:3 Hz,
H-6), 7.67 (1H, td, J ¼ 7:7, 1.6 Hz, H-7), 8.09 (1H, dd,
J ¼ 7:6, 1.6 Hz, H-5), 8.97 (1H, d, J ¼ 8:1 Hz, H-8), 9.61
(1H, br s, OH), 13.57 (1H, br s, OH); Z-isomer d 7.56
(1H, td, J ¼ 7:5, 1.4 Hz, H-6), 7.60 (1H, td, J ¼ 7:6,
1.6 Hz, H-7), 7.98 (1H, dd, J ¼ 7:6, 1.6 Hz, H-5), 8.08
(1H, d, J ¼ 7:8 Hz, H-8), (other signals overlap with the
E-isomer). 13C NMR (d6-DMSO) E-isomer d 26.8, 27.3,
29.9, 32.0, 33.3, 37.1, 47.5, 116.1, 125.8, 125.9, 129.2,
130.3, 130.4, 132.0, 139.7, 158.7, 183.3; Z-isomer d 29.3,
32.0, 33.3, 36.9, 47.5, 122.1, 125.2, 129.3, 130.7, 131.4,
(other signals overlap with the E-isomer). ESI-MS:
340.7 (M)1). Anal. Calcd (C21H27NO3Æ0.1H2O) C:
73.31, H: 7.97, N: 4.10; found C: 73.48, H: 7.99, N: 4.08.
4.2.2. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-hydroxy-
[1,4]naphthoquinone-1-(O-methyloxime) (3). To a solu-
tion of 1 (3.01 g, 9.22 mmol) and O-methyl-hydroxyl-
amine hydrochloride (1.95 g, 23.35 mmol) in ethanol
(200 mL) was added sodium acetate (2.28 g, 27.80 mmol)
in a mixture of water (5 mL) and refluxed for 5 days. The
solvent was removed under vacuum. 50 mL of water was
added, and the product was extracted with DCM
(3 · 75 mL). The combined organic layers were dried
with sodium sulfate and evaporated to give a yellow
solid as a 20:1 mixture of E/Z isomers (3.2 g, 9.00 mmol,
4.2.5. Preparation of hepatic microsomes. Wistar rats
(150–200 g) were treated ip for 4 days with either
phenobarbital (80 mg/kg/day in 0.9% NaCl solution), 3-
methylcholanthrene (20 mg/kg/day in olive oil), dexa-
methasone (50 mg/kg/day in olive oil) or 0.5 mL olive oil
(control rats).16 Microsomes were prepared as previ-
ously reported30 and stored at )80 ꢁC until use. Protein
concentrations were determined by using the Biorad
Protein Assay (BioRad, Hercules, USA), and cyto-
chrome P450 contents were determined as reported
earlier.31
1
97.6%): mp 150 ꢁC. H NMR (CDCl3) E-isomer d 0.80
(9H, s, –C(CH3)3), 0.82–1.10 (5H, m, CHC(CH3)3, Hax),
1.55 (1H, br m, CH–CH2), 1.75 (4H, m, Heq), 2.50 (2H,
d, J ¼ 7:3 Hz, CH2–CH), 4.28 (3H, s, OCH3), 7.57 (2H,
m, H-6, H-7), 7.66 (1H, s, OH), 8.26 (1H, m, H-5), 8.74
(1H, m, H-8); E-isomer: d 7.52 (2H, m, H-6, H-7), 8.12
(2H, m, H-5, H-8), 9.55 (1H, s, OH) (other signals
overlap with the E-isomer). 13C NMR (CDCl3, E-iso-
mer) d 27.3, 27.6, 30.5, 32.4, 33.8, 37.6, 48.1, 65.0, 118.3,
125.8, 127.1, 129.6, 131.2, 131.5, 132.0, 139.8, 155.9,
184.5. ESI-MS: 354.5 (M)1). Anal. Calcd (C22H29NO3)
C: 74.33, H: 8.22, N: 3.94; found C: 74.10, H: 8.26, N:
3.90.
4.2.6. Enzymatic incubations in microsomes. A stock
solution of buparvaquone-oxime (2) was prepared in
ethanol. 20 lL of the stock solution (final substrate
concentration of 0.95 mM), MgCl2 solution (5 mM),
liver microsomes (protein concentration of 4.4–6.3 mg/
mL) and 50 mM phosphate buffer pH 7.4 (total volume
1 mL) were incubated for 5 min at 37 ꢁC before adding
NADPH solution (final concentration of 4 mM). For the
blank sample, liver microsomes were replaced by the
same volume of water. Reactions were incubated in a
water bath at 37 ꢁC for 24 h, and then stopped either by
mixing 1 mL of ice-cold acetonitrile with the sample for
4.2.3. 3-(trans-4-tert-Butyl-cyclohexylmethyl)-2-methoxy-
[1,4]naphthoquinone (4). Compound
1
(2.00 g,
6.10 mmol) and anhydrous potassium carbonate (3.50 g,
24.4 mmol) were suspended in 100 mL of dry acetone,
and dimethyl sulfate (2.3 mL, 24.4 mmol) was added