B. Ruan et al. / Steroids 64 (1999) 385–395
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2.4. (20R,22R)-Cholest-5-ene-3,20,22-triol 3-acetate (3a)
and (20S,22S)-cholest-5-ene-3,20,22-triol 3-acetate (3b)
1), 501 (20), 359 (69, M-TMSOCH(CH2)2CH(CH3)2), 341
(12, 359-H2O), 299 (74, 359-CH3COOH), 281 (25, 359-
CH3COOH-H2O), 255 (17, M-SC-CH3COOH), 173 (100,
TMSOCH(CH2)2CHCH3)2), 83 (54), 73 (73, TMS); 1H and
13C NMR, Tables 1 and 2.
To AD-mix- (7 g) in a mixture of tert-butanol (20 ml)
and water (20 ml) was added methanesulfonamide (30 mg)
and the resulting mixture was cooled to 0°C. The ⌬5,20(22) E
diene 2a (100 mg; 0.23 mmol) in tert-butanol (3 ml) was
added. Then, tert-butanol (2 ml) and water (5 ml) were
added and the mixture was kept at 4°C for 24 h. After the
disappearance of 2a (as judged by TLC), sodium sulfite (7
g) was added, and, after stirring for 1 h at room temperature,
the mixture was extracted with chloroform (4 ϫ 50 ml). The
combined extracts were washed once with brine (20 ml),
dried over anhydrous sodium sulfate, and evaporated to
dryness. The residue was subjected to silica gel MPLC
(50 ϫ 1 cm; solvent, 10% ethyl acetate in hexane). Frac-
tions 10–22 were combined and evaporated to dryness to
give, as judged by normal phase HPLC, Agϩ-HPLC, and 1H
NMR, an ϳ1:1 mixture (72 mg; 67%) of 3a and 3b.† Sterols
3a and 3b were incompletely separated on normal phase
HPLC (solvent, 3% acetone in hexane; tR 52.3 min and 54.8
min, respectively) but baseline resolved on analytical Agϩ-
HPLC (300 ϫ 3.2 mm; solvent, 10% acetone in hexane; tR
16.8 min for 3a and 17.9 min for 3b). Accordingly, 3a and
3b were separated by semipreparative Agϩ-HPLC (250 ϫ
10 mm; solvent, 7.5% acetone in hexane; tR 47.6 min for 3a
and 52.6 min 3b).‡ Fifteen injections [5–7 mg each, dis-
solved in the mobile phase (500 l)] furnished 3a (34 mg)
and 3b (32 mg).
Characterization of the 20R,22R isomer 3a: mp, 196–
197°C (lit. [9] 192–194°C); single component on analytical
Agϩ-HPLC (250 ϫ 4.6 mm; solvent, 9.1% acetone in hex-
ane; tR 16.6 min); MS, m/z 424 (2, M-2H2O), 400 (1,
M-CH3COOH), 382 (61, M-H2O-CH3COOH), 367 (6,
M-H2O-CH3COOH-CH3), 364 (15, M-2H2O-CH3COOH),
359 (52, M-HOCH(CH2)2CH(CH3)2), 349 (6), 341 (13,
359-H2O), 299 (100, 359-CH3COOH), 281 (27, 359-
CH3COOH-H2O), 255 (19, M-SC-CH3COOH), 159 (38);
single major component (99.8%; tR 82.6 min)§ by GC-MS
of the TMS derivative, m/z 517 (6, M-CH3), 516 (9, M-CH3-
Characterization of the 20S,22S isomer 3b: glassy solid;
single component on analytical Agϩ-HPLC (250 ϫ 4.6 mm;
solvent, 9.1% acetone in hexane; tR 17.6 min); MS, 424
(2, M-2H2O), 382 (14, M-CH3COOH-H2O), 364 (9, M-
CH3COOH-2H2O), 359 (31, M-HOCH(CH2)2CH(CH3)2),
341 (8, 359-H2O), 299 (100, 359-CH3COOH), 281 (25, 359-
CH3COOH-H2O), 255 (15, M-SC-CH3COOH); single major
component (99.5%; tR 77.1 min)§ by GC-MS of the TMS
derivative, m/z 517 (4, M-CH3), 516 (6, M-CH3-1), 501 (13),
359 (62, M-TMSOCH(CH2)2CH(CH3)2), 341 (12, 359-H2O),
299 (84, 359-CH3COOH), 281 (22, 359-CH3COOH-H2O),
255 (15, M-SC-CH3COOH), 173 (100, TMSOCH(CH2)2
CH(CH3)2), 83 (54), 73 (81, TMS); 1H and 13C NMR, Tables
1 and 2.
2.5. (20R,22R)-Cholest-5-ene-3,20,22-triol (4a) and
(20S,22S)-cholest-5-ene-3,20,22-triol (4b)
The 20R,22R isomer 3a (13 mg) was heated with 15%
KOH in 95% ethanol (2 ml) for 2 h at 70°C. After the
addition of water (2 ml), the mixture was extracted with
methylene chloride (3 ϫ 8 ml), and the combined extracts
were washed once with brine (4 ml), dried over anhydrous
sodium sulfate, and evaporated to dryness. The residue was
applied to a silica gel column (5 ϫ 0.5 cm), and, after
washing the column with methylene chloride (20 ml) to
remove a nonpolar yellowish impurity, 2-propanol was used
to elute the triol. Recrystallization from acetone-hexane
gave 4a as a white solid (10 mg; 84%): mp, 176–178°C (lit.
[8] 178–180°C; lit. [12] 179–180°C, lit. [22] 175–178°C);
single component on TLC (solvent, 50% acetone in hexane;
Rf 0.9); single component on normal phase HPLC (solvent,
9.1% acetone in hexane; tR 22.3 min); MS, m/z 400 (6,
M-H2O), 382 (9, M-2H2O), 367 (4, M-2H2O-CH3), 349 (3,
M-3H2O-CH3), 317 (100, M-HOCH(CH2)2CH(CH3)2), 299
(56, 317-H2O), 281 (12, 317-2H2O), 271 (7), 255 (16,
M-SC-H2O), 213 (10), 159 (32), 145 (37); single major
component (98.5%; tR 67.2 min)§ by GC-MS of the TMS
derivative, m/z 546 (15, M-CH3-1), 531 (20), 473 (5), 389
(96, M-TMSOCH(CH2)2CH(CH3)2), 371 (12), 370 (11),
299 (58, 389-TMSOH), 281 (18), 255 (14, M-SC-TMSOH),
173 (100, TMSOCH(CH2)2CH(CH3)2), 129 (41), 83 (43),
† When AD-mix-␣ [17] was used with the same reaction conditions and
workup, analyses of the product by normal phase HPLC, Agϩ-HPLC and
1H NMR showed a 1:99 mixture of 3a and 3b.
‡ Compound 3a can also be isolated by repeated crystallization of the
mixture of 3a and 3b. An ϳ1:1 mixture (41 mg) of 3a and 3b was
recrystallized from 5% acetone in hexane to give 3a, 95% purity. A second
recrystallization gave 3a (15 mg), 99% purity (as judged by Agϩ-HPLC
and 1H NMR).
1
73 (TMS); H and 13C NMR, Tables 1 and 2.
§ Minor components with mass spectra compatible with the acetate or
TMS ether of pregnenolone were observed in GC-MS analyses of 3a
[0.2%; tR 19.4 min; m/z 298 (100, M-CH3COOH)], 3b [0.5%; tR 19.5 min;
m/z 298 (100, M-CH3COOH)], 4a [1.5%; tR of 15.8 min; 388 (34, Mϩ),
373 (9, M-CH3), 332 (18), 298 (33, M-TMSOH), 283 (16, M-TMSOH-
CH3), 259 (25), 241 (9), 159 (11), 145 (15), 129 (100), 75 (42), 73 (60,
TMS)], and 4b [two minor components of 0.5% each; tR 14.2 min and 15.7
min; m/z 388 (Mϩ), 298 (M-TMSOH), 283 (M-TMSOH-CH3), 259, 129,
73]. The minor components in 4b may represent a pair of C-17 epimers. 1H
Saponification of 3b (20 mg) and workup (as described
above for 3a) gave 4b (15 mg; 82%): mp, 168–170°C (lit.
[8] 169–171°C); single component on TLC (solvent, 50%
acetone in hexane; Rf 0.9) and on normal phase HPLC
NMR analysis of 4a and 4b indicated no signals for pregnenolone at a
detection limit of 0.1%.