and 1725 (C=O), 1100 (C-O); 1H NMR (600 MHz, CDCl3) δ 5.18
(dt, J = 18.6, 6.6Hz, 1H, 6β-H), 4.22 (dd, J = 13.0, 6.3 Hz, 1H,
2-H), 3.66 (s, 3H, -OCH3), 3.48 (s, 1H, 2-OH), 2.04 (s, 3H,
CH3CO), 1.08 (s, 3H, 19-H), 0.92 (d, J = 6.5 Hz, 3H, 21-H), 0.68
(s, 3H, 18-H); 13C NMR (151 MHz, CDCl3) δ 211.3 (C-3), 174.6
(C-24), 170.1, 71.1 (C-6), 70.2 (C-2), 56.1, 55.9, 51.5, 48.7, 46.3,
42.9, 41.5, 39.7, 38.5, 35.3, 35.1, 34.5, 31.1, 31.0, 30.9, 28.1, 24.0,
22.5, 21.2, 21.1, 18.3, 12.0; HRMS (ESI, m/z) Calcd for C27H42O6
462.2981; found: 485.2878 [M + Na]+ (Calcd 485.2879).
(O-H), 1783 and 1740 (C=O), 1172 (C-O); HRMS (ESI, m/z)
Calcd for C27H44O6 464.3138; found: 487.3024 [M + Na]+ (Calcd
487.3036). Compound H8 was then purified by column
chromatography through silica gel (eluted with 8: 1 acetone:
hexane) to afford compound H8 (3ɑ, 50% yield) as a white solid
1
and compound H8 (3β, 50% yield) as a white solid; H8 (3ɑ) H
NMR (600 MHz, CDCl3) δ 5.19 (dt, J = 12.1, 4.8 Hz, 1H, 6-H),
4.07-3.99 (m, 1H, 2-H), 3.72-3.67 (m, 1H, 3-H), 3.65 (s, 3H,
-OCH3), 2.00 (s, 3H, CH3CO), 1.01 (s, 3H, 19-H), 0.90 (d, J = 6.5
Hz, 3H, 21-H), 0.63 (s, 3H, 18-H); 13C NMR (151 MHz, CDCl3) δ
174.8 (C-24), 170.5, 70.9 (C-3), 68.9 (C-6), 67.4 (C-2), 56.2, 55.9,
51.5, 42.9, 40.6, 39.9, 39.4, 38.5, 38.3, 35.3, 34.6, 31.2, 31.1,
30.9, 28.1, 26.3, 24.0, 23.6, 21.4, 21.1, 18.3, 12.0. H8 (3β) 1H
NMR (600 MHz, CDCl3) δ 5.12 (dt, J = 12.3, 4.8 Hz, 1H, 6-H),
3.65 (s, 3H, -OCH3), 3.53 (ddd, J = 12.6, 8.9, 4.1 Hz, 1H, 2-H),
3.39 (ddd, J = 11.4, 8.9, 5.0 Hz, 1H, 3-H), 2.02 (s, 3H, CH3CO),
1.00 (s, 3H, 19-H), 0.90 (d, J = 6.5 Hz, 3H, 21-H), 0.63 (s, 3H,
18-H); 13C NMR (151 MHz, CDCl3) δ 174.8 (C-24), 170.5, 76.3
(C-3), 71.2 (C-6), 70.9 (C-2), 56.0, 55.9, 51.5, 45.4, 43.2, 42.8,
41.4, 39.8, 38.8, 35.3, 34.7, 31.3, 31.1, 30.9, 28.1, 27.9, 24.1,
23.2, 21.4, 20.9, 18.3, 12.0.
2.2.6. Methyl 6α-acetoxyl-2-hydroxyl-5β-cholanoate (H6)
PTSH (0.0411g, 0.2 mmol) was added by ten portions to a
stirred solution of compound H5 (0.1011 g, 0.2 mmol) in MeOH
(15 mL) over 15 min. The mixture was stirred at rt for 15 min, and
heated to reflux temp to maintain for 45 min, and then allowed to
stir at rt for 15 min. The reaction process was monitored by TLC.
After compound H5 was depleted, a mixture of NaBH3CN
(0.0213 g, 0.3 mmol) and ZnCl2 (0.0212 g, 0.2 mmol) in MeOH (2
mL) was added to the reaction solution. After the mixture was
allowed to stand for 3 h at reflux, the reaction was quenched with
0.1 M NaOH aq (10 mL) and evaporated under reduced pressure.
After extracting with Et2O (20 mL) by three portions, the
combined organic layers were washed with H2O (10 mL×2), and
brine (10 mL×3), and dried by anhyd MgSO4. After filtered, the
filtrate was concentrated under reduced pressure to give the crude
product, which was purified by column chromatography through
silica gel (eluted with 5: 1 hexane: ethyl acetate) to afford
compound H6 (0.0663 g, 68% yield) as colorless oily liquid;
2.2.9. 2, 3, 6α-Trihydroxyl-5β-cholanoate (H9/H10)
To a stirred solution of compound H8 (3)(0.1011 g, 0.2 mmol)
in MeOH (4 mL) and H2O (1 mL) was added NaOH (0.0441 g, 1.1
mmol) at room temperature. The mixture was heated at reflux for
1 h, and then adjusted to pH 1-2 with 1 M HCl aq to afford
compound 2, 3, 6α - trihydroxyl - 5β - cholanoate H9 (0.0794 g,
[]2D0
= -12.9 (c = 0.5, CH2Cl2); FTIR (KBr, cm–1) 3462 (O-H),
[]2D0
90% yield) as white solid; mp 52-55 °C;
= -9.2 (c = 0.5,
1740 (C=O), 1170 (C-O); 1H NMR (600 MHz, CDCl3) δ 5.17 (dt,
J = 18.0, 6.0 Hz, 1H, 6β-H), 3.72-3.66 (m, 1H, 2-H), 3.65 (s, 3H,
-OCH3), 2.04 (s, 3H, CH3CO), 1.1 (s, 3H, 19-H), 0.90 (d, J = 6.6
Hz, 3H, 21-H), 0.70 (s, 3H, 18-H); 13C NMR (151 MHz, CDCl3) δ
174.7 (C-24), 170.6, 71.8 (C-6), 66.1 (C-2), 56.1, 55.9, 51.5, 46.3,
46.2, 42.9, 41.4, 39.9, 38.9, 35.4, 35.3, 34.7, 31.3, 31.0, 30.9, 28.1,
24.1, 23.8, 21.4, 20.9, 19.8, 18.3, 12.0; HRMS (ESI, m/z) Calcd
for C27H44O5 448.3189; found: 471.3065 [M + Na]+ (Calcd
471.3086).
CH2Cl2); FTIR (KBr, cm–1) 3443 (O-H), 1704 (C=O), 1025 and
1023 (C-O); HRMS (ESI, m/z) Calcd for C24H40O5 408.2876;
found: 431.2751 [M + Na]+ (Calcd 431.2773). H9 H NMR
1
(600 MHz, CD3OD) δ 4.08 (dt, J = 11.1, 4.2 Hz, 1H, 6-H),
4.02-3.96 (m, 1H, 2-H), 3.68-3.61 (m, 1H, 3-H), 1.00 (s, 3H,
19-H), 0.98 (d, J = 6.5 Hz, 3H, 21-H), 0.72 (s, 3H, 18-H); 13C
NMR (151 MHz, CD3OD) δ 176.7 (C-24), 68.9 (C-3), 67.1 (C-6),
67.0 (C-2), 56.1, 55.9, 42.6, 42.1, 40.4, 39.9, 37.8, 37.7, 35.3,
34.8, 33.9, 30.9, 30.6, 27.8, 25.5, 23.8, 22.9, 20.9, 17.4, 11.0.
2.2.7. 2, 6α-Dihydroxyl-5β-cholanoate (H7)
2, 3, 6α-Trihydroxyl-5β-cholanoate H10 was prepared as a
white solid according to the similar synthetic method of
compound H9. 1H NMR (600 MHz, CD3OD) δ 4.02 (dt, J = 11.4,
4.4 Hz, 1H, 6-H), 3.55-3.42 (m, 1H, 2-H), 3.33-3.27 (m, 1H, 3-H),
0.98 (d, J = 7.1 Hz, 6H,19,21-H), 0.72 (s, 3H, 18-H); 13C NMR
(151 MHz, CD3OD) δ 176.8 (C-24), 75.9 (C-3), 70.4 (C-6), 67.0
(C-2), 56.1, 55.9, 48.4, 43.7, 42.6, 41.3, 39.9, 37.9, 35.3, 34.8,
34.1, 30.9, 30.6, 27.8, 27.4, 23.8, 22.5, 20.7, 17.3, 11.0.
To a stirred solution of compound H6 (0.0399 g, 0.1 mmol) in
MeOH (3 mL) and H2O (1 mL) was added NaOH (0.0115 g, 0.3
mmol) at rt. The mixture was heated at reflux for 1 h, and then
adjusted to pH 1-2 with 1 M HCl aq to afford compound H7 as
[]2D0
white solid (0.0301 g, 86% yield). mp 169-172 °C;
= -12
(c = 0.5, CH2Cl2); FTIR (KBr, cm–1): 3512 and 3405 (O-H), 1714
1
(C=O), 1025 (C-O); H NMR (600 MHz, DMSO-d6) δ 11.96 (s,
1H), 4.28 (s, 1H), 4.25 (s, 1H), 3.91-3.83 (m, 1H, 6-H),
3.50-3.39(m, 1H, 2-H), 0.88 (t, J = 3.1 Hz, 6H, 19, 21-H), 0.61 (s,
3H, 18-H); 13C NMR (151 MHz, DMSO-d6) δ 175.3 (C-24), 66.6
(C-6), 64.8 (C-2), 56.1, 55.9, 49.8, 47.3, 42.8, 41.3, 39.9, 38.4,
36.1, 35.3, 35.3, 34.8, 31.2, 31.1, 28.1, 24.6, 24.3, 21.1, 19.1, 18.6,
12.3; HRMS (ESI, m/z) Calcd for C24H40O4 392.2927; found:
415.2815 [M + Na]+ (Calcd 415.2824).
2.2.10. Methyl 3α-hydroxyl-6-oxo-5β-cholanoate (H2-1)
IBX (0.5201 g, 1.9 mmol) was added to a stirred solution of
compound H1 (0.5003 g, 1.2 mmol) in tert-BuOH (25 mL) at rt.
The mixture was heated at reflux for 1 h, and then quenched with
10% Na2SO3 aq (25 mL), and the mixture was evaporated under
reduced pressure. EtOAc (40 mL) was added to the residue and
filtered. The filtrate was washed with 10% Na2SO3 aq (25 mL×2),
saturated NaHCO3 aq (25 mL×2), and brine (20 mL×3). The
organic layer was dried by anhyd MgSO4, filtered, and
concentrated under reduced pressure to give the crude product,
which was purified by column chromatography through silica gel
(eluted with 4: 1 hexane: ethyl acetate) to afford compound H2-1
2.2.8. Methyl 6α-acetoxyl-2, 3-dihydroxyl-5β-cholanoate (H8)
NaBH4 (0.0412 g, 1.1 mmol) was added to a solution of H5
(0.1021 g, 0.2 mmol) containing MeOH (10 mL) at 0 °C. Then the
mixture was allowed to warm to rt and quenched after 1 h with
H2O (30 mL). After extracting with EtOAc (20 mL) by three
portions, the organic layer was washed with saturated NaHCO3 aq
(15 mL×3), and brine (15 mL×3), and dried by anhyd MgSO4.
After filtered, the solvent was evaporated under reduced pressure
to afford compound H8 (0.0953 g, 95% yield) as a white oily
[]2D0
(0.3582 g, 72% yield) as a white solid; mp 134-137 °C;
=
-47.2 (c = 0.5, CH2Cl2); FTIR (KBr, cm-1) 3507 (O-H), 1730 and
1679 (C=O), 1100 (C-O); 1H NMR (600 MHz, CDCl3) δ 3.68 (s,
3H, -OCH3), 3.66-3.59 (m, 1H, 3β-H), 0.94 (d, J = 5.9 Hz, 3H,
21-H), 0.85 (s, 3H, 19-H), 0.66 (s, 3H, 18-H). 13C NMR (151
[]2D0
liquid;
= -9.2 (c = 0.5, CH2Cl2); FTIR (KBr, cm–1) 3452