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W. Lu et al. / Steroids 69 (2004) 803–808
pared according to the literature [4] and further dried in vacuo
(3 mm Hg) for 2 h at 100 ◦C. The key intermediate 24-keto-
cholesterylacetate4waspreparedaccordingtothepreviously
reported method [5] in five steps with an overall 64% yield.
(d, 3H, J = 7.0 Hz, 26-CH3 or 27-CH3), 3.54 (tt, 1H, J = 11.5
and 5.0 Hz, 3-CH), 4.66 (d, 1H, J = 1.5 Hz, 28-CHa), 4.71 (d,
1H, J = 1.5 Hz, 28-CHb), 5.35 (dt, 1H, J = 3.0 and 2.5 Hz, 6-
CH); 13C NMR (CDC13, 125 MHz) δ: 156.9 (C), 140.7 (C),
121.7 (CH), 105.9 (CH2), 71.9 (CH), 56.8 (CH), 56.0 (CH),
50.1 (CH), 42.3 (CH2), 42.3 (C), 39.8 (CH2), 37.2 (CH2),
36.5 (C), 35.7 (CH), 34.7 (CH2), 33.8 (CH), 31.9 (CH2),
31.9 (CH), 31.6 (CH2), 30.9 (CH2), 28.2 (CH2), 24.3 (CH2),
22.0 (CH3), 21.8 (CH3), 21.1 (CH2), 19.4 (CH3), 18.7 (CH3),
11.9 (CH3). Analysis calculated for C28H46O: C, 84.42; H,
11.56. Found: C, 84.42; H, 11.57.
2.2. 24-Keto-cholesterol 5
24-Keto-cholesteryl acetate 4 (220 mg, 0.5 mmol) was re-
fluxed with 15 ml of 3% KOH in MeOH for 15 min. The
solvent was evaporated to a small volume and then diluted
with 20 ml ethyl acetate. The organic layer was washed with
water and brine. After drying over anhydrous Na2SO4, the
solvent was removed under reduced pressure, and the result-
ing crude product was purified by flash chromatography on
silica gel using petroleum ether (bp 60–90 ◦C)/EtOAc (4:1)
as eluent and recrystallized from acetone to afford com-
pound 5 (172 mg, 86%). m.p. 128–130 ◦C. FABMS m/z:
401 (M + H)+. IR (KBr) ν: 3416, 2866, 1707, 1640, 1581,
2.4. 24-Methylene-7-keto-cholesteryl acetate 7 and
7,24-Diketo-cholesteryl acetate 8
24-Methylene-cholesterol 6 (50 mg, 0.13 mmol) was dis-
solved in 2 ml pyridine, and l ml acetic anhydride was added.
The mixture was incubated at room temperature for 24 h.
Water (10 ml) was added to the mixture to quench the ex-
cess acetic anhydride, and the mixture was extracted with
ethyl acetate (10 ml × 4). The combined organic layer was
washedsuccessivelywithwater, 1 MHC1solution, andbrine,
and then dried over anhydrous Na2SO4. The solvent was re-
moved and the dried residue was dissolved in 15 ml ben-
zene, containing 50 mg molecular sieves (3A). PCC/Al2O3
(2.0 g) was added, and the mixture was stirred with in re-
flux for 24 h under nitrogen. After cooling to room temper-
ature, the mixture was thoroughly extracted with CH2Cl2.
After evaporation to dryness under reduced pressure, the
residue was subjected to flash chromatography on silica gel
using petroleum ether (bp 60–90 ◦C)/EtOAc (7:1) as eluent,
and the acetate of substrate 6 (Rf = 0.75) 11 mg was recov-
ered. The followed portion (Rf = 0.30) was collected and re-
crystallized from ether/methanol to afford 7 (29 mg, 51%).
m.p. 154–155 ◦C. FABMS m/z: 455 (M + H)+. IR (KBr) ν:
3078, 2959, 2872, 1731, 1673, 1639, 1465, 1376, 1263,
1
1461, 1377, 1243, 1103, 1053, 1025, 955 cm−1. H NMR
(CDC13, 500 MHz) δ: 0.68 (s, 3H, 18-CH3), 0.92 (d, 3H,
J = 7.0 Hz, 21-CH3), 1.01 (s, 3H, 19-CH3), 1.09 (d, 6H, two
overlapping, J = 7.0 Hz, 26-CH3 and 27-CH3), 2.61 (m, 1H,
J = 7.0 Hz, 25-CH), 3.53 (tt, 1H, J = 12.0 and 5.5 Hz, 3-CH).
5.35 (dt, 1H, J = 3.0 and 2.5 Hz, 6-CH). Analysis calcu-
lated for C27H44O2: C, 81.00; H, 11.00. Found: C, 80.95;
H, 11.01.
2.3. 24-Methylene-cholesterol 6
NaH-paraffin (60%; 84 mg, 2.10 mmol of sodium hydride)
was placed in a two-neck flask. After the paraffin was washed
away with anhydrous petroleum ether (30–60 ◦C), 4 ml of an-
hydrous dimethyl sulfoxide were added to the dried NaH un-
der an argon atmosphere. The mixture was stirred at 80 ◦C for
40 min, and a dark green solution was produced. After cool-
ing to room temperature, a solution of methyltriphenylphos-
phonium bromide (500 mg, 1.25 mmol) in 4 ml anhydrous
dimethyl sulfoxide was added, and the solution immediately
turned yellowish in color. After 10 min, a solution of 24-
keto-cholesterol 5 (45 mg, 0.12 mmol) in l ml of anhydrous
dioxane was added, and the resulting mixture was stirred
at 80 ◦C for 2 h. The reaction mixture was cooled, diluted
with 15 ml water, and extracted with ether (10 ml × 4). The
combined organic extracts were washed with water and brine
and then, dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure, and the resulting crude prod-
uct was purified by flash chromatography on silica gel using
petroleum ether (bp 60–90 ◦C)/EtOAc (5:1) as eluent and
recrystallized from methanol to afford colorless crystal nee-
dles of 6 (39 mg, 85%). m.p. 140–142 ◦C (literature value:
143 ◦C [6]); [␣]D = −36◦ (c, 0.15, CHCl3) (literature value:
−34.8◦ (c, 0.26, CHCl3) [6]). FABMS m/z: 399 (M + H)+.
1R (KBr) ν: 3416, 3078, 2942, 1644, 1461, 1377, 1053, 955,
885, 842, 800 cm−1. 1H NMR (CDCl3, 500 MHz) δ: 0.68 (s,
3H, 18-CH3), 0.95 (d, 3H, J = 6.5Hz, 21-CH3), 1.01 (s, 3H,
19-CH3), 1.02(d, 3H, J = 7.0 Hz, 26-CH3 or 27-CH3), 1.03
1239, 1185, 1044, 887 cm−1. H NMR (CDCl3, 500 MHz)
1
δ: 0.69 (s, 3H, 18-CH3), 0.96 (d, 3H, J = 6.5 Hz, 21-CH3),
1.02 (d, 3H, J = 7.0 Hz, 26-CH3 or 27-CH3), 1.03 (d, 3H,
J = 7.0 Hz, 26-CH3 or 27-CH3), 1.21 (s 3H, 19-CH3), 2.06
(s, 3H, 3-CH3COO), 4.66 (d, 1H, J = 1.5 Hz, 28-CHa), 4.72
(d, 1H, J = 1.5 Hz, 28-CHb), 4.72 (m, 1H, 3-CH), 5.71 (d,
1H, J = 1.5 Hz, 6-CH). Analysis calculated for C30H46O3:
C, 79.30; H, 10.13. Found: C, 79.23; H, 10.18. The por-
tion (Rf = 0.12) was collected using petroleum ether (bp
60–90 ◦C)/EtOAc (4:1) as eluent and recrystallized from
ether/methanol to afford 8 (9 mg, 16%). m.p. 141–143 ◦C.
FABMS m/z: 457 (M + H)+. IR (KBr) ν: 2949, 2872, 1733,
1
1711, 1671, 1631, 1465, 1379, 1243, 1183, 1038 cm−1. H
NMR (CDCl3, 500 MHz) δ: 0.68 (s, 3H, 18-CH3), 0.92
(d, 3H, J = 6.5 Hz, 21-CH3), 1.09 (d, 6H, two overlapping,
J = 7.0 Hz, 26-CH3 and 27-CH3), 1.21 (s, 3H, 19-CH3), 2.06
(s, 3H, 3-CH3COO), 2.61 (m, 1H, J = 7.0 Hz, 25-CH), 4.72
(m, 1H, 3-CH), 5.71 (d, 1H, J = 2.0 Hz, 6-CH). Analysis cal-
culated for C29H44O4: C, 76.32; H, 9.65. Found: C, 76.31;
H, 9.70.