Molecules 2016, 21, 1139
9 of 12
Methyl 2
α
,3
α
-dihydroxy-6-oxo-5
α
-cholan-24-oate (11). A solution of compound 10 (500 mg, 1.29 mmol) in
30 mL of acetone and 10 mL water was prepared. Later 10 mg of NMMNO, 2 mL 4.0% (0,314 mmol)
OsO4 solution and 2 mL of pyridine were added. The mixture was kept under constant stirring at
room temperature for 72 h. The end of reaction was verified by TLC, and then 25 mL of Na2S2O3
saturated solution were added and allowed to stir for 30 min. The reaction was concentrated to a
volume of approximately 15 mL under reduced pressure. Then AcOEt (40 mL) were added. The
organic layer was washed with water (2
solvent was evaporated under reduced pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and
chromatographed on silica gel with EtOAc/hexane mixtures of increasing polarity (0.2:9.8 8.2:1.8).
×
25 mL), dried over anhydrous Na2SO4, and filtered. The
→
Two fractions were obtained: Fraction I, 218 mg of unreacted compound 10 and Fraction II, 277 mg
(51.0% yield) of compound 11. Compound 11 was a colorless solid (m.p. = 178–179 ◦C, MeOH/Et2O).
IR (cm−1): 3392 (OH); 2944 (CH3-); 2867 (C-CH2-C); 1738 (C=O); 1709 (C=O); 1435 (CH2); 1376 (CH3);
1255, C-O); 1170 (C-O). 1H-NMR: 4.05 (b.s, 1H, H-3); 3.77 (b.d, J = 11.1 Hz, 1H, H-2); 3.66 (s, 3H, CH3O);
2.67 (dd, J = 3.0 and 12.6 Hz, 1H, H-5); 0.926 (d, J = 6.4 Hz, 3H, H-21); 0.752 (s, 3H, H-19); 0.659 (s, 3H,
1
H-18). 13C-NMR: See Table 2. H-NMR and 13C-NMR are shown in Supplementary Materials.
Methyl 3α-acetoxy-6-oxo-5α-cholan-24-oate (12). To a solution of compound 8 (2.00g, 4.94 mmol) in 20 mL
of DCM, 5 mg of DMAP, 1 mL of pyridine and 0.5 mL (5.3 mmol) of Ac2O were added. The reaction
mixture was kept under constant stirring and room temperature for 30 min. The end of reaction
was verified by TLC, the mixture was then concentrated to a volume approximately 5 mL under
reduced pressure. Then AcOEt (30 mL) were added. The organic layer was washed with water
(
2 × 15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and
the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with EtOAc/hexane
mixtures of increasing polarity (0.2:9.8 4.7:5.3). Compound 12 (1.9 g, 95% yield) was a colorless
→
solid (m.p. = 174–176 ◦C, MeOH/Et2O) IR (cm−1): 2943 (CH3); 2869 (CH2); 1737 (C=O); 1708 (C=O);
1435 (CH2); 1376 (CH3); 1262 (C-O); 1221 (C-O); 1172 (C-O). 1H-NMR: 5.12 (m, 1H, H-3); 3.66 (s, 3H,
CH3O); 2.56 (dd, J = 3.3 and 12.0 Hz, 1H, H-5); 2.38 (ddd, J = 5.4, 10.3 and 15.4 Hz, 1H, H-23); 2.04 (s,
3H, CH3CO); 0.94 (d, J = 6.4 Hz, 3H, H-21); 0.74 (s, 3H, H-19); 0.67 (s, 3H, H-18). 13C-NMR: See Table 1.
1H-NMR and 13C-NMR are shown in Supplementary Materials.
Acid 3α-hydroxy-6-oxo-5α-cholan-24-oic (13). A solution of compound 8 (5.0 g, 12.36 mmol) in 60 mL of
MeOH was prepared. Later 20 mL of 15% P/V K2CO3 solution were added, and the reaction mixture
was refluxed for 1 h. The end of reaction was verified by TLC, the mixture was then concentrated to
a volume approximately 15 mL under reduced pressure. This was acidified with 20 mL of 10% HCl
solution. Then AcOEt (40 mL) were added. The organic layer was washed with water (2
dried over Na2SO4, and filtered. The solvent was evaporated under reduced pressure and a colorless
solid (4.11 g, 82.2% yield) was obtained. Compound 13 (m.p. = 102–107 ◦C, MeOH/Et2O) IR (cm−1):
×
15 mL),
1
3403–2500 (O-H); 2945 (CH3); 2869 (CH2); 1712 (C=O); 1689 (C=O); 1442 (CH2); 1380 (CH3). H-NMR
(CD3OD): 4.04 (m, 1H, H-C3); 2.75 (t, J = 8.1 Hz, 1H, H-5); 2.31 (ddd, J = 5.3, 9.9 and 15.2 Hz, 1H, H-23);
1
0.96 (d, J = 6.5 Hz, 3H, H-21); 0.730 (s, 3H, H-19); 0.71 (s, 3H, H-18). 13C-NMR: See Table 1. H-NMR
and 13C-NMR are shown in Supplementary Materials.
Acid 3α-acetoxy-6-oxo-5α-cholan-24-oic (14). To a solution of compound 13 (5.0 g, 12.8 mmol) in 60 mL of
DCM, 10 mg of DMAP, 1 mL of pyridine and 1.2 mL (12.8 mmol) of Ac2O were added. The reaction
mixture was kept under constant stirring and room temperature for 1 h. The end of reaction was
verified by TLC, the mixture was then concentrated to a volume approximately 10 mL under reduced
pressure. Then AcOEt (40 mL) were added. The organic layer was washed with 5% HCl (1 × 10 mL),
water (2
×
15 mL), dried over Na2SO4, and filtered. The solvent was evaporated under reduced
pressure and the crude was re-dissolved in CH2Cl2 (5 mL) and chromatographed on silica gel with
EtOAc/hexane mixtures of increasing polarity (0.2:9.8
→
8.8:11.2). Compound 14 (3.64 g, 72.8% yield)
was a colorless solid (m.p. = 171–177 ◦C, MeOH/Et2O) IR (cm−1): 3373-2495 (O-H); 2947 (CH3); 2869
1
(CH2); 1736 (C=O); 1708 (C=O); 1444 (CH2); 1376 (CH3). H-NMR: 5.12 (m, 1H, H-3); 2.56 (dd, J = 3.3