Article
J. Agric. Food Chem., Vol. 58, No. 2, 2010 1167
chromatography on silica gel using ethyl acetate to give the pure triol 9
(93 mg, 32%) as a white solid: mp 252-254 °C (from ethyl acetate) [lit. (23)
256-257 °C]; [R]D20 -16.7 (c 0.096 in CHCl3) [lit. (23) -8]; found C 77.57,
H 11.08; C29H50O3 requires C 77.97, H 11.28%; νmax/cm-1 3432, 2941,
1654; δH (300 MHz; DMSO-d6) 0.66 (3H, s, 18-CH3), 0.71-2.12 [41H, m,
containing 0.77-0.82 (9H, m, 26-CH3, 27-CH3 and 29-CH3), 0.98 (3H, d,
21-CH3), 1.00 (3H, s, 19-CH3)], 3.65 (1H, br s, 6-H), 3.73-3.91 (1H, m,
3R-H), 4.20 (1H, d, J 5.6, OH), 4.41 (1H, d, J 4.0, OH), 5.02 (1H, dd, J 15.1,
8.5, one of 22-H or 23-H), 5.16 (1H, dd, J 15.1, 8.5 one of 22-H or 23-H);
11.90 (CH3), 12.61 (CH3), 16.25 (CH3), 17.22 (CH3), 19.75 (CH3), 20.33
(CH3), 22.09 (CH2), 22.14 (CH2), 24.60 (CH2), 27.19 (CH2), 29.94 (CH),
32.72 (CH2), 37.41 (CH2), 38.70 (CH), 42.33 (CH2), 51.44, 51.53 (2 ꢀ CH),
53.62 (CH), 55.97, 56.22 (2 ꢀ CH), 63.18 (quaternary C), 63.85 (CH),
69.48 (CH); m/z (ESIþ) 445.3690 (Mþ þ H, C29H49O3 requires 445.3682),
445 (Mþ þ H, 94%), 427 (100), 409 (9), 347 (36), 329 (8), 224 (5), 127 (16).
(22S,23S)-Stigmast-5-ene-3β,22,23-triol 12. (22S,23S)-Diol 19
(284 mg, 0.62 mmol) was dissolved in THF-water (9:1, 10 mL), and
5 M H2SO4 (0.20 mL) was added. The reaction mixture was stirred for 24 h
at room temperature. The solvent was removed, and the residue was
dissolved in ethyl acetate (20 mL), washed with saturated aqueous sodium
bicarbonate (7 mL) and brine (10 mL) and dried over magnesium sulfate.
Removal of the solvent under reduced pressure gave the crude product as a
white solid. Purification by column chromatography on silica gel, eluted
with 40% ethyl acetate-hexane, produced the (22S,23S)-triol 12 (240 mg,
87%) as a white solid: mp 139-141 °C; [R]D20 -22.7 (c 0.198 in MeOH);
δC (75.5 MHz; DMSO-d6) 12.46 (CH3), 12.49 (CH3), 16.63 (CH3), 19.20
(CH3), 21.08 (CH2), 21.33 (CH3), 21.47 (CH3), 24.28 (CH2), 25.27 (CH2),
29.01 (CH2), 30.35 (CH), 31.44 (CH2), 31.71 (CH), 32.37 (CH2), 34.83
(CH2), 38.12 (quaternary C), 40.02 (CH2), 40.49 (CH), 41.25 (CH2), 42.50
(quaternary C), 44.93 (CH), 50.98 (CH), 55.82 (CH), 56.28 (CH), 66.10
(CH, CHOH), 74.46 (CH, CHOH), 74.65 (quaternary C, C-5), 129.05
(CH), 138.56 (CH).
ν
max/cm-1 3391, 2954, 1630; δH (300 MHz; DMSO-d6) 0.63-2.24 [45H, m,
3,6,-di-TMS Ether of 5r-Stigmasterol-3β,5r,6β-triol 23. 5R-
Stigmasterol-3β,5R,6β-triol 9 (40 mg, 0.09 mmol) and pyridine (1 mL)
were added under nitrogen, followed by hexamethyldisilizane (0.30 mL)
and chlorotrimethylsilane (0.15 mL). The reaction mixture was stirred
for 18 h at room temperature. Water (15 mL) was then added, and the
sample was extracted into hexane (2 ꢀ 30 mL). The organic layer was
washed with water (2 ꢀ 15 mL) and brine (2 ꢀ 15 mL) and dried over
magnesium sulfate. The solvent was then removed under reduced pressure
to yield the TMS ether 23 (32 mg, 55%) as a brown solid: mp 110-113 °C;
containing 0.63 (3H, s, 18-CH3), 0.73 (3H, d, 21-CH3), 0.77-0.80 (9H, m,
26-CH3, 27-CH3 and 29-CH3), 0.80 (3H, s, 19-CH3)], 3.11-3.42 (1H, m,
3R-H), 3.76 (1H, d, J 7.9), 4.21 (1H, d, J 6.2), 4.63 (1H, d, J 4.1), 5.27 (1H,
br s, 6-H); δC (75.5 MHz; DMSO-d6) 11.93 (CH3), 14.65 (CH3), 14.73
(CH3), 17.85 (CH3), 18.60 (CH2), 19.48 (CH3), 20.99 (CH2), 22.22 (CH3),
24.53 (CH2), 26.53 (CH), 27.89 (CH2), 31.69 (CH2), 31.76 (CH2), 31.84
(CH), 36.42 (quaternary C), 37.28 (CH2), 39.57 (CH2), 42.31 (CH), 42.66
(CH2), 49.06 (CH), 49.96 (CH), 52.53 (CH), 56.22 (CH), 69.84 (CH) 70.35
(CH), 71.42 (CH), 120.76 (CH, C-6), 141.57 (quaternary C, C-5), one of
the quaternary carbon obscured by DMSO signals; m/z (ESIþ) 429.3741
(MþþH - H2O, C29H49O2 requires 429.3733), 470 (MþþNa, 5%), 429
(Mþþ H - H2O, 4), 325 (4), 115 (9), 105 (78), 64 (100), 56 (14).
ν
max/cm-1 3435, 2956, 1637, 1251, 1080; δH (300 MHz; CDCl3) 0.07 [9H, s,
Si(CH3)3], 0.12 [9H, s, Si(CH3)3], 0.57 (3H, s, 18-CH3), 0.69-1.81 [38H, m,
containing 0.62-0.67 (9H, m, 26-CH3, 27-CH3 and 29-CH3), 0.94 (3H, d,
21-CH3), 0.98 (3H, s, 19-CH3)], 1.88-2.17 (3H, m), 3.44 (1H, br s), 4.04
(1H, septet, J 5.4, 3R-H), 5.01 (1H, dd, J 15.1, 8.5, one of 22-H or 23-H),
5.15 (1H, dd, J 15.1, 8.5, one of 22-H or 23-H); δC (75.5 MHz; CDCl3) 0.44
(3 ꢀ CH3), 0.53 (3 ꢀ CH3), 12.48 (CH3), 12.53 (CH3), 17.10 (CH3), 19.19
(CH3), 21.32 (CH3), 21.37 (CH2), 21.39 (CH3), 24.44 (CH2), 25.64 (CH2),
29.12 (CH2), 30.37 (CH), 31.28 (CH2), 32.11 (CH), 32.78 (CH2), 34.92
(CH2), 38.71 (quaternary C), 40.11 (CH2), 40.73 (CH), 41.67 (CH2), 42.83
(quaternary C), 46.46 (CH), 51.45 (CH), 56.14 (CH), 56.24 (CH), 68.58
(CH), 76.21 (CH), 77.44 (quaternary C), 129.46 (CH), 138.55 (CH); m/z
(ESIþ) 501.4106 (Mþ - TMSOH, C32H57O2Si requires 501.4128), 501
(Mþ - TMSOH, 38%), 429 (54), 411 (100).
(22R,23R)-Stigmast-5-ene-3β,22,23-triol 13. (22R,23R)-Diol 20
(210 mg, 0.46 mmol) was dissolved in THF-water (9:1, 20 mL), and
5 M H2SO4 (0.65 mL) was added. The reaction mixture was stirred for 42 h
at room temperature. The volatiles were removed under reduced pressure,
and the residue was taken up in ethyl acetate (50 mL), washed with
saturated aqueous sodium bicarbonate (40 mL) and brine (40 mL) and
dried over magnesium sulfate. Removal of the solvent gave the crude
product as a yellow solid. Purification by column chromatography on
silica gel, eluted with 40% ethyl acetate-hexane, produced the (22R,23R)-
triol 13 (137 mg, 67%) as a white solid: mp 210-212 °C; νmax/cm-1 3401,
2936, 1640, 1470; δH (300 MHz, MeOD) 0.74 (3H, s, 18-CH3), 0.82-2.31
[42H, m, containing 0.88-0.93 (9H, m, 26-CH3, 27-CH3 and 29-CH3),
0.95 (3H, d, 21-CH3), 1.06 (3H, s, 19-CH3)], 3.23-3.48 (2H, m, containing
3R-H), 3.56 (1H, d, J 8.4), 3.67 (1H, dd, J 8.7, 1.2), 5.34 (1H, d, J 4.8, 6-H);
δC (75.5 MHz, MeOD) 12.37 (CH3), 12.80 (CH3), 14.39 (CH3), 20.02
(CH3), 20.31 (CH3 and CH2), 21.80 (CH3), 22.38 (CH2), 25.36 (CH2),
28.99 (CH2), 30.56 (CH), 32.45 (CH2), 33.15 (CH2), 33.49 (CH), 37.82
(quaternary C), 38.70 (CH2), 38.83 (CH), 41.38 (CH2), 43.17 (CH2), 43.46
(quaternary C), 48.40 (CH), 51.87 (CH), 54.07 (CH), 58.33 (CH), 72.58
(CH), 73.83 (CH), 75.81 (CH), 122.54 (CH), 142.40 (quaternary C);
m/z (ESIþ) 429.3741 (Mþþ H - H2O, C29H49O2 requires 429.3733), 430
(Mþþ H - H2O, 13%), 412 (10), 326 (6), 116 (4), 63.8, 115 (84),74 (100).
Stigmasterol Acetate 14. Acetic anhydride (22.9 mL, 242.3 mmol)
and pyridine (19.5 mL, 242.3 mmol) were combined, and stigmasterol 1
(2.00 g, 4.85 mmol) was then added portionwise. The reaction mixture was
stirred under nitrogen for 5 days at room temperature. The reaction
mixture was then poured onto 10% aqueous HCl (200 mL) and was
extracted with ethyl acetate (4 ꢀ 50 mL). The organic layer was then
washed with water (100 mL), saturated aqueous sodium bicarbonate
(100 mL), and brine (100 mL) and dried over magnesium sulfate. The
solvent was removed under reduced pressure to give stigmasterol acetate
14 as a white solid, which was used without further purification (1.95 g,
89%): mp 141-143 °C (from ethyl acetate-hexane) [lit. (24) 141 °C];
found C 82.24, H 10.95, C31H50O2 requires C 81.88, H 11.08%; νmax/cm-1
2961, 1730, 1459; δH (300 MHz; CDCl3) 0.70 (3H, s, 18-CH3), 0.74-0.89
(9H, m, 26-CH3, 27-CH3 and 29-CH3), 0.89-2.14 [32H, m, including 1.01
(3H, d, 21-CH3), 1.03 (3H, s, 19-CH3) 2.03 (3H, s, CO2CH3)], 2.31 (2H, d,
J 7.9), 4.50-4.68 (1H, m, 3R-H), 5.01 (1H, dd, J 15.2, 8.5, one of 22-H or
23-H), 5.16 (1H, dd, J 15.2, 8.5, one of 22-H or 23-H), 5.37 (1H, d, J 4.3, 6-
H); δC (75.5 MHz; CDCl3) 12.26 (CH3), 12.47 (CH3), 19.20 (CH3), 19.52
(CH3), 21.22 (CH2), 21.31 (CH3), 21.44 (CH3), 21.65 (CH3), 24.57 (CH2),
25.63 (CH2), 27.98 (CH2), 29.13 (CH2), 32.07 (CH), 32.10 (CH2), 32.10
5,6,22,23-Diepoxystigmastane 10. Stigmasterol 1 (2.00 g, 4.85
mmol) was dissolved in dichloromethane (35 mL), and the solution was
cooled to 0 °C. m-CPBA (77.1%, 4.38 g, 17.83 mmol) in dichloromethane
(50 mL) was added dropwise, and the reaction mixture was stirred
for 48 h at room temperature. The reaction mixture was worked up
by washing with 5% sodium thiosulfate (3 ꢀ 60 mL), saturated aqueous
sodium bicarbonate solution (2 ꢀ 60 mL), and brine (60 mL), dried over
magnesium sulfate and concentrated under reduced pressure to give the
crude product. This was purified by chromatography on silica gel using
60% ethyl acetate-hexane to yield the bisepoxide as a mixture of
diastereomers, 10a, 10b, 10c, and 10d, in a ratio of 4.5:4.5:1:1 (1.06 g,
54%) as a white solid: mp 135-148 °C (as a mixture of diastereomers);
ν
max/cm-1 3435, 2960, 2871, 1466, 1379; δH (300 MHz; CDCl3) 5R,6R
isomers 10a, 10b, 0.53-2.16 [44H, m, containing 0.61 (3H, m, 18-CH3),
0.80-0.85 (9H, m, 26-CH3, 27-CH3 and 29-CH3), 0.95 (3H, m, 21-CH3),
1.05 (3H, s, 19-CH3), 2.08 (1H, dd, J 12.7, 11.4, one of 4-H)], 2.40-2.55
(3H, m, 22R-H, 22β-H, one of 23-H), 2.73 (1H, dd, J 7.1, 2.4, one of 23-H),
2.91 (1H, d, J 4.3, 6-H), 3.81-3.99 (1H, m, 3R-H), 5β,6β isomers 10c, 10d
distinguishable signals, 2.40-2.55 (3H, m, 22R-H, 22β-H, one of 23-H),
2.73 (1H, dd, J 7.1, 2.4, one of 23-H), 3.06 (1H, d, J 2.1, 6-H), 3.61-3.78
(1H, m, 3R-H); δC (75.5 MHz; CDCl3) signals for both diastereomers of
the 5R,6R-bisepoxide 10a, 10b, 12.01, 12.15 (2 ꢀ CH3), 12.53, 12.66 (2 ꢀ
CH3), 16.08 (2 ꢀ CH3), 16.36, 16.45 (2 ꢀ CH3), 19.51, 19.59 (2 ꢀ CH3),
19.68, 20.38 (2 ꢀ CH3), 20.75, 20.80 (2 ꢀ CH2), 20.98, 21.07 (2 ꢀ CH2),
24.42, 24.27 (2 ꢀ CH2), 27.04, 27.94 (2 ꢀ CH2), 28.91 (2 ꢀ CH2), 29.29,
29.46 (2 ꢀ CH), 30.03 (2 ꢀ CH), 31.17 (2 ꢀ CH2), 32.56 (2 ꢀ CH2), 35.01
(2 ꢀ quaternary C), 38.89, 39.01 (2 ꢀ CH), 39.34, 39.35 (2 ꢀ CH2), 39.96
(2 ꢀ CH2), 42.70, 42.81 (2 ꢀ CH), 42.81, 42.83 (2 ꢀ quaternary C), 48.43,
48.91 (2 ꢀ CH), 53.26, 55.91 (2 ꢀ CH), 56.58, 56.61 (2 ꢀ CH), 58.74 (CH,
one of C-23), 59.41, 59.48 (2 ꢀ CH, C-6), 62.26 (CH, one of C-23), 62.42,
63.28 (2 ꢀ CH, C-22), 65.96 (2 ꢀ quaternary C, C-5), 68.74 (2 ꢀ CH, C-3),
distinguishable signals for both diastereomers of the 5β,6β isomer 10c, 10d,