62
V. Sepe et al. / Steroids 105 (2016) 59–67
MeOH/H2O (83:17) as eluent (flow rate 1 mL/min), to give 3.5 mg of
38.7, 37.3, 36.5, 35.0, 29.3, 28.6, 28.4, 28.2, 27.7, 25.3, 24.8, 22.4,
21.9, 20.0, 12.5; HR ESIMS m/z 347.2947 [M+H]+, C23H39O2 requires
347.2950.
D
compound 4 (tR = 11 min); [a]25 = +22.5 (c 0.05,CH3OH); selected
1H NMR (400 MHz,CD3OD): d 3.58 (2H,t,J = 7.0 Hz), 2.95 (2H,t,
J = 7.0 Hz), 2.24 (1H,m), 2.08 (1H,m), 0.95 (3H,d,ovl), 0.94 (3H,s),
0.68 (3H,s). HR ESIMS m/z 480.3150 [MꢂNa]ꢂ, C27H46NO4S requires
480.3153.
2.2.9. Methyl cholan-3,5-dien-24-oate (21)
At a solution of the ditosylate 20 (500 mg, 0.7 mmol) in water
(3 ml) and N,N0-dimethylformamide (DMF; 27 ml) was added
CH3COOK (206 mg, 2.1 mmol) and the mixture was refluxed for
36 h. The solution was cooled at room temperature, then water
and ethyl acetate were added and the separated aqueous phase
was extracted with ethyl acetate (3 ꢁ 20 mL). The combined
organic phases were washed with water, dried (Na2SO4) and con-
centrated to give a mixture. Purification by silica gel eluting with
hexane–ethyl acetate (99:1) gave the diene 21 (200 mg, 78%).
selected 1H NMR (400 MHz CD3OD): d 5.92 (1H, d,J = 9.7 Hz),
5.58 (1H,d,J = 11.3 Hz), 5.38 (1H,m), 3.66 (3H,s), 2.34 (1H,m),
2.22 (1H,m), 0.94 (3H,d,J = 5.8 Hz), 0.92 (3H,s), 0.70 (3H,s); 13C
NMR (100 MHz CD3OD): d 174.8, 141.5, 129.1, 125.2, 123.2, 57.0,
55.8, 51.5, 48.4, 43.4, 39.8, 35.4, 33.8, 31.9, 31.8, 31.7, 31.0, 30.9,
28.1, 24.2, 23.9, 23.0, 20.9, 18.8, 12.0. HR ESIMS m/z 371.2947
[M+H]+, C25H39O2 requires 371.2950.
2.2.5. 3
A solution of lithocholic acid 11 (500 mg, 1.3 mmol) in 10 mL of
90% formic acid containing 25 L of 70% perchloric acid was stirred
a-formyloxy-5b-cholan-24-oic acid (14)
l
at 47–50 °C for 12 h. The temperature of the heating bath was low-
ered to 40 °C, then 5 mL of acetic anhydride was added and the
mixture was stirred for 15 min. The solution was cooled to room
temperature, poured into 50 mL of water and extracted with
diethyl ether. The organic layers were washed with saturated
NaHCO3 solution (50 mL) and water to neutrality, dried over
Na2SO4, and evaporated to give 540 mg of 14 (quantitative yield).
An analytic sample was obtained by silica gel chromatography
eluting with CH2Cl2:MeOH 9:1. Selected 1H NMR (400 MHz CD3OD):
d 8.04 (1H,s), 4.85 (1H,m), 2.39 (1H,m), 2.25 (1H,m), 0.93 (3H,s),
0.92 (3H,d,J = 6.7 Hz), 0.65 (3H,s); 13C NMR (100 MHz CD3OD): d
178.1, 160.8, 74.4, 56.5, 55.9, 41.9, 40.5, 40.2, 40.1, 35.8 (3C),
35.4 (2C), 32.3, 30.8, 30.7, 28.2, 27.0, 26.3, 24.2, 23.4, 20.8, 18.2,
12.1; HRMS-ESI m/z 405.2997 [M+H]+, C25H41O4 requires 405.2999.
2.2.10. Methyl 5a-cholan-24-oate (6)
A
solution of methyl chol-3,5-dien-24-oate 21 (150 mg,
0.40 mmol) in absolute methanol (5 mL) and dry THF (5 mL) was
added in an oven-dried 50 mL flask, that was charged with 10% pal-
ladium on carbon (10 mg). The flask was evacuated and flushed
first with argon and then with hydrogen. The reaction was stirred
at room temperature under H2 (1 atm) for 1 h. The mixture was
2.2.6. 3a-formyloxy-24-nor-5b-cholan-23-nitrile (15)
Crude 14 (500 mg, 1.2 mmol), 3.8 mL of cold trifluoroacetic acid,
and 1 mL (7.2 mmol) of trifluoroacetic anhydride were stirred at
0 °C until dissolution. Sodium nitrite (248 mg, 3.6 mmol) was
added at the solution. The reaction mixture was stirred first at
0–5 °C for 1 h, then at 45–50 °C for 3 h. When the reaction was
completed, it was neutralized with NaOH 2 N, then the product
was extracted with 50 mL of diethyl ether (3 ꢁ 50 mL), followed
by washing with brine and dried over anhydrous Na2SO4. The ether
was removed under reduced pressure to afford 380 mg of 15 (85%),
that was subjected to next step without any purification.
filtered through celite, and the recovered filtrate was concentrated
D
to give 147 mg of compound 6 (quantitative yield). [
a
]25 = ꢂ2.9 (c
2.54,CH3OH); selected 1H NMR (400 MHz CDCl3): d 3.66 (3H,s),
2.34 (1H,m), 2.21 (1H,m), 0.91 (3H,d,J = 6.4 Hz), 0.77 (3H,s), 0.64
(3H,s); 13C NMR (100 MHz CDCl3): d 176.0, 56.9, 56.2, 55.0, 51.6,
47.3, 40.4, 39.0 (2C), 35.7 (3C), 32.5, 31.4, 31.3, 29.4, 29.3, 28.4,
27.2, 24.5, 22.6, 21.2, 18.6, 12.5, 12.4; HR ESIMS m/z 375.3269
[M+H]+, C25H43O2 requires 375.3263.
2.2.7. 24-nor-lithodeoxycholic acid (16)
Compound 15 (350 mg, 0.94 mmol) was refluxed in ca. 50 mL of
methanol–water 1:1 with 30% KOH. After 2 h, the basic aqueous
solution was neutralized with HCl 6 N. Then methanol was evapo-
rated and the residue was extracted with ethyl acetate (3 ꢁ 50 mL)
and then with CH2Cl2 (3 ꢁ 50 mL). The organic layers were washed
with brine, dried and evaporated to dryness to give white solid
residue, that was purified by silica gel chromatography, eluting
with CH2Cl2:MeOH 9:1 (340 mg, quantitative yield). An analytic
2.2.11. 5a-cholan-24-oic acid (7)
Compound 6 (50 mg, 0.13 mmol) was hydrolyzed with a metha-
nol solution of sodium hydroxide (5%, 5 mL) in H2O (1 mL) over-
night under reflux. The resulting solution was then concentrated
under vacuum, diluted with water, acidified with HCl 6 N and
extracted with ethyl acetate (3 ꢁ 30 mL). The collected organic
phases were washed with brine, dried over Na2SO4 anhydrous
and evaporated under reduced pressure to give compound 7
(40 mg, 87%). An analytic sample was obtained by HPLC on a
sample was purified by HPLC on a Nucleodur 100–5 C18 (5
lm;
4.6 mm i.d. ꢁ 250 mm) with MeOH/H2O (95:5) as eluent (flow rate
Nucleodur 100–5 C18 (5
l
m; 4.6 mm i.d. ꢁ 250 mm) with MeOH/
D
D
1 mL/min), to give compound 16 (tR = 10.5 min). [
a]
25 = +21.9
H2O (92:8) as eluent (flow rate 1 mL/min, tR = 12 min); [
a
]
=
25
(c 0.58,CH3OH); selected 1H NMR (400 MHz CD3OD): d 3.54 (1H,m),
2.41 (2H,m), 1.00 (3H,d, J = 7.0 Hz), 0.94 (3H,s), 0.71 (3H,s). 13C
NMR (100 MHz CD3OD): d 178.0, 72.5, 57.9, 57.5, 43.6, 42.5, 41.9,
41.4, 37.2, 37.1, 36.5, 35.7, 34.9, 31.1, 29.3, 28.3, 27.6, 25.2, 23.9,
21.9, 19.9, 12.5; HRMS-ESI m/z 363.2895 [M+H]+, C23H39O3
requires 363.2899.
+17.7 (c 0.14,CH3OH); selected 1H NMR (400 MHz,CD3OD): d
2.31 (1H,m), 2.20 (1H,m), 0.94 (3H,d,J = 6.2 Hz), 0.81 (3H,s), 0.69
(3H,s). HR ESIMS m/z 359.2953 [MꢂH]ꢂ, C24H39O2 requires
359.2950.
2.2.12. 5a-cholan-24-oyl-taurine sodium salt (8)
Compound 7 (10 mg, 27.8 ꢁ 10ꢂ3 mmol) in DMF dry (3 mL) was
2.2.8. 24-nor-5b-cholanoic acid (5)
treated with DMT-MM (16 mg, 58.2 ꢁ 10ꢂ3 mmol) and triethy-
Compound 5 (210 mg, 75% over five steps) was synthesized,
starting from compound 16 (300 mg, 0.82 mmol) as described in
Scheme 2, by an analogous procedure to that detailed above for
compound 3. An analytic sample was obtained by HPLC on a Nucle-
lamine (70 lL, 0.49 mmol) and the mixture was stirred at room
temperature for 10 min. Then to the mixture was added taurine
(15 mg, 0.16 mmol). After 24 h, the reaction mixture was concen-
trated under vacuo and dissolved in water (5 mL). The mixture
odur 100–5 C18 (5
l
m; 10 mm i.d. ꢁ 250 mm) with MeOH/H2O
was purified by HPLC on a Nucleodur 100–5 C18 (5 lm; 4.6 mm
25
(999.5:0.5) as eluent (flow rate 3 mL/min, tR = 20 min); [
a]
=
i.d. ꢁ 250 mm) with MeOH/H2O (83:17) as eluent (flow rate
D
+24.6 (c 0.03,CH3OH); selected 1H NMR (400 MHz CD3OD): d 2.41
(1H,m), 1.00 (3H,d,J = 6.0 Hz), 0.95 (3H,s), 0.72 (3H,s); 13C NMR
(100 MHz CD3OD): d 178.0, 58.0, 57.6, 45.2, 44.0, 42.8, 41.9, 41.5,
1 mL/min), to give 3.5 mg (27%) of compound 8 (tR = 10.4 min);
[a]
25 = +37. (c 0.03,CH3OH); selected 1H NMR (400 MHz,CD3OD):
D
d 3.58 (2H,t,J = 7.0 Hz), 2.96 (2H,t,J = 7.0 Hz), 2.24 (1H,m), 2.09