Journal of Medicinal Chemistry
Article
Fraction eluted over a RP18 column with MeOH gave a mixture
that was further purified by HPLC on a Nucleodur 100−5 C18 (5 μm;
4.6 mm i.d. × 250 mm) with MeOH/H2O (83:17) as eluent (flow rate
1 mL/min), to give 26 mg of compound 12 (41%, tR = 5.4 min);
s), 0.65 (3H, s). 13C NMR (100 MHz CDCl3): δ 63.7, 56.7, 56.3, 43.9,
42.8, 40.6 (2C), 40.3, 37.8, 36.0, 35.6, 31.9, 29.5, 28.3, 27.6, 27.3 (2C),
27.0, 26.8, 24.4, 21.4, 20.9, 18.7, 12.2. HRESIMS m/z 347.3321 [M +
H]+, C24H43O requires 347.3314.
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[α]D = +21.4 (c 1.46, CH3OH). Selected 1H NMR (400 MHz
5β-Cholan-24-yl-24-sodium sulfate (15). At a solution of 14 (5
mg, 14.5 × 10−3 mmol) in DMF dry (3 mL) was added
triethylamine−sulfur trioxide complex (12 mg, 72.5 × 10−3 mmol)
under an argon atmosphere, and the mixture was stirred at 80 °C for
24 h. Most of the solvent was evaporated, and the residue was poured
over a RP18 column to remove excess SO3·NEt3. Fraction eluted with
MeOH gave 1.5 mg of compound 15 (23%). An analytic sample was
obtained by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. ×
250 mm) with MeOH/H2O (98:2) as eluent (flow rate 1 mL/min, tR
CD3OD): δ 4.28 (1H, m), 3.96 (2H, m), 0.99 (3H, s), 0.98 (3H, d,
ovl), 0.72 (3H, s). 13C NMR (100 MHz CD3OD): δ 81.0, 70.9, 56.6
(2C), 45.4, 44.8, 42.6, 41.3, 40.5, 38.3, 36.8, 35.6, 35.2, 33.1, 29.5, 28.8,
28.2, 27.0, 26.9, 24.6, 22.1, 21.8, 19.1, 12.5. HRESIMS m/z 441.2681
[M − Na]−, C24H41O5S requires 441.2675.
Methyl 3α,7β-Ditosyloxy-5β-cholan-24-oate (36). To a solution of
UDCA methyl ester (300 mg, 0.73 mmol) in dry pyridine (20 mL),
tosyl chloride (1.39 g, 7.3 mmol) was added and the mixture was
stirred at room temperature for 6 h. It was poured into cold water (30
mL) and extracted with CH2Cl2 (3 × 30 mL). Purification by silica gel
eluting with ethyl acetate−hexane (8:2) gave 36 (521 mg, quantitative
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1
= 9.9 min); [α]D = +9.0 (c 0.88, CH3OH). Selected H NMR (400
MHz CD3OD): δ 3.64 (2H, m), 0.96 (3H, d, ovl), 0.95 (3H, s), 0.69
(3H, s). 13C NMR (100 MHz CD3OD): δ 69.7, 57.6, 47.9, 45.2, 43.8,
41.9, 41.6, 38.7, 37.3, 36.8, 36.5, 33.2, 29.3, 28.6, 28.4, 28.2, 27.7, 27.1,
25.3, 24.8, 22.4, 22.0, 19.2, 12.5. HRESIMS m/z 425.2732 [M − H]−,
C24H41O4S requires 425.2726.
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1
yield) as a white solid; [α]D = −11 (c 0.48, CH3OH). Selected H
NMR (400 MHz CDCl3): δ 7.79 (2H, d, J = 8.3 Hz), 7.75 (2H, d, J =
8.3 Hz), 7.36 (2H, d, J = 8.3 Hz), 7.32 (2H, d, J = 8.3 Hz), 4.58 (1H,
m), 4.38 (1H, m), 3.67 (3H, s), 2.47 (3H, s), 2.45 (3H, s), 2.33 (1H,
m), 2.22 (1H, m), 0.91 (3H, d, J = 6.5 Hz), 0.88 (3H, s), 0.61 (3H, s).
HRESIMS m/z 715.3333 [M + H]+, C39H55O8S2 requires 715.3338.
Methyl 5β-Cholan-24-oate (37) and 8α Epimer 38. Lithium
bromide (45 mg, 0.52 mmol) and lithium carbonate (38 mg, 0.52
mmol) were added to a solution of 36 (190 mg, 0.26 mmol) in dry
DMF (20 mL), and the mixture was refluxed for 3 h. After cooling to
room temperature, the mixture was slowly poured into 10% HCl
solution (50 mL) and extracted with CH2Cl2 (3 × 30 mL). The
combined organic layer was washed successively with water, saturated
NaHCO3 solution, and water and then dried over anhydrous MgSO4
and evaporated to dryness to give 124 mg of oily residue (quantitative
yield), which was subjected to next step without any purification.
An oven-dried 25 mL flask was charged with 10% palladium on
carbon (10 mg) and the crude product (124 mg, 0.23 mmol), and the
flask was evacuated and flushed with argon. Absolute methanol (5 mL)
and dry THF (5 mL) were added, and the flask was flushed with
hydrogen. The reaction was stirred at room temperature under H2 (1
atm) overnight. The mixture was filtered through Celite, and the
recovered filtrate was concentrated to give 95 mg of crude product,
which was further purified by HPLC on a Nucleodur 100−5 C18 (5
μm; 10 mm i.d. × 250 mm) with MeOH:H2O (995:5) as eluent (flow
rate 3 mL/min), to give 25 mg of compound 38 (26%, tR = 25.5 min)
and 32 mg of compound 37 (33%; tR= 33 min).
Compound 16. Compound 38 (12 mg, 32.0 × 10−3 mmol) was
hydrolyzed with a methanol solution of sodium hydroxide (5%, 5 mL)
in H2O (1 mL) overnight under reflux. The resulting solution was then
concentrated under vacuum, diluted with water, acidified with HCl 6
N, and extracted with ethyl acetate (3 × 50 mL). The collected organic
phases were washed with brine, dried over Na2SO4 anhydrous, and
evaporated under reduced pressure to give 9 mg of carboxyl acid 16
(78%). An analytic sample was obtained by HPLC on a Nucleodur
100−5 C18 (5 μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (99:1)
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as eluent (flow rate 1 mL/min, tR = 21 min); [α]D = +12.7 (c 0.28,
1
CHCl3). Selected H NMR (400 MHz CDCl3): δ 2.42 (1H, m), 2.28
(1H, m), 0.97 (3H, d, J = 6.8 Hz), 0.85 (3H, s), 0.81 (3H, s).
HRESIMS m/z 359.2958 [M − H]−, C24H39O2 requires 359.2950.
Compound 17. Compound 17 (18 mg, 97%) was synthesized,
starting from compound 38 (20 mg, 53.5 × 10−3 mmol), by analogous
procedures to those detailed above for compound 14. An analytic
sample was obtained by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6
mm i.d. × 250 mm) with MeOH/H2O (999.5:0.5) as eluent (flow rate
1 mL/min, tR = 15.8 min); [α]D25 = +2.9 (c 0.18, CHCl3). Selected 1H
NMR (400 MHz CDCl3): δ 3.64 (2H, m), 0.97 (3H, d, J = 6.3 Hz),
0.85 (3H, s), 0.81 (3H, s). 13C NMR (100 MHz CDCl3): δ 63.6, 56.9,
44.1 (2C), 42.6, 37.4 (2C), 36.7, 36.1, 35.3, 34.2, 31.7, 29.3, 27.7, 27.2,
27.1, 26.6, 25.8, 24.9, 24.6, 21.7, 19.5, 19.1, 18.1. HR ESIMS m/z
347.3318 [M + H]+, C24H43O requires 347.3314.
Compound 18. Compound 18 (8.8 mg, 43%) was synthesized,
starting from compound 17 (16 mg, 46.2 × 10−3 mmol), by an
analogous procedure to that detailed above for compound 15. An
analytic sample was obtained by HPLC on a Nucleodur 100−5 C18 (5
μm; 4.6 mm i.d. × 250 mm) with MeOH/H2O (98:2) as eluent (flow
rate 1 mL/min, tR = 9 min); [α]D25 = +9.7 (c 0.15, CH3OH). Selected
1H NMR (400 MHz CD3OD): δ 3.96 (2H, m), 0.98 (3H, d, J = 6.3
Hz), 0.87 (3H, s), 0.81 (3H, s). HRESIMS m/z 425.2733 [M − Na]−,
C24H41O4S requires 425.2726.
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Methyl 5β-Cholan-24-oate (37). [α]D = −3.0 (c 0.03, CH3OH).
1
Selected H NMR (400 MHz CDCl3): δ 3.66 (3H, s), 2.33 (1H, m),
2.20 (1H, m), 0.91 (6H, ovl), 0.65 (3H, s). 13C NMR (100 MHz
CDCl3): δ 171.9, 56.6, 56.0, 51.5, 43.8, 40.5 (2C), 40.3, 37.7, 35.9,
35.4 (2C), 31.0 (2C), 28.2, 27.5, 27.3, 27.1, 26.5, 24.2 (2C), 21.3, 20.8,
18.2, 12.0. HRESIMS m/z 375.3267 [M + H]+, C25H43O2 requires
375.3263.
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Compound 38. [α]D = +19.7 (c 0.02, CH3OH). Selected 1H
NMR (400 MHz CDCl3): δ 3.68 (3H, s), 2.46 (1H, m), 2.35 (1H, dd,
J = 10.1, 5.3 Hz), 0.95 (3H, d, J = 6.5 Hz), 0.84 (3H, s), 0.81 (3H, s).
13C NMR (100 MHz CDCl3): δ 172.2, 56.6, 51.5, 43.9 (2C), 37.3,
37.1, 36.7, 35.9, 35.3 (2C), 34.1, 31.0, 30.8, 27.7, 27.1 (2C), 26.6, 25.7,
24.9, 24.6, 21.7, 19.4, 18.7, 18.3. HRESIMS m/z 375.3265 [M + H]+,
C25H43O2 requires 375.3263.
Methyl 3α-Tosyloxy-7α-hydroxy-5β-cholan-24-oate (39). To a
solution of CDCA methyl ester (400 mg, 1.0 mmol) in dry pyridine
(10 mL), tosyl chloride (382 mg, 2.0 mmol) was added and the
mixture was stirred at room temperature for 4 h. It was poured into
cold water (20 mL) and extracted with CH2Cl2 (3 × 50 mL). The
combined organic layer was washed with saturated NaHCO3 solution
(30 mL) and water (30 mL) and then dried over anhydrous MgSO4
and evaporated in vacuo to give 555 mg of 39 (quantitative yield) in
5β-Cholan-24-ol (14). Dry methanol (8 μL, 0.19 mmol) and LiBH4
(95 μL, 2 M in THF, 0.19 mmol) were added to a solution of 37 (10
mg, 26.7 × 10−3 mmol) in dry THF (5 mL) at 0 °C under argon, and
the resulting mixture was stirred for 3 h at 0 °C. The mixture was
quenched by addition of NaOH (1 M, 55 μL) and then allowed to
warm to room temperature. Ethyl acetate was added, and the separated
aqueous phase was extracted with ethyl acetate (3 × 15 mL). The
combined organic phases were washed with water, dried (Na2SO4),
and concentrated to give 7.8 mg of 14 (84%). An analytic sample was
obtained by HPLC on a Nucleodur 100−5 C18 (5 μm; 4.6 mm i.d. ×
250 mm) with MeOH/H2O (999.5:0.5) as eluent (flow rate 1 mL/
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the form of colorless oil; [α]D = +22.6 (c 0.93, CH3OH). Selected
1H NMR (500 MHz CDCl3): δ 7.80 (2H, d, J = 8.5 Hz), 7.34 (2H, d, J
= 8.5 Hz), 4.38 (1H, m), 3.83 (1H, s), 3.62 (3H, s), 2.40 (3H, s) 2.35
(1H, m), 2.23 (1H, m), 0.93 (3H, d, J = 6.4 Hz), 0.89 (3H, s), 0.65
(3H, s). HRESIMS m/z 561.3288 [M + H]+, C32H49O6S requires
561.3283.
Methyl 3β,7α-Dihydroxy-5β-cholan-24-oate (40). A solution of 39
(555 mg, 0.99 mmol) and CH3COOK (97 mg, 0.99 mmol) dissolved
in water (2 mL) and N,N′-dimethylformamide (DMF, 10 mL) was
refluxed for 2 h. The solution was cooled at room temperature, and
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min, tR = 18 min); [α]D = +20.5 (c 0.20, CH3OH). Selected H
NMR (400 MHz CDCl3): δ 3.62 (2H, m), 0.94 (3H, d, ovl), 0.92 (3H,
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dx.doi.org/10.1021/jm501273r | J. Med. Chem. 2014, 57, 8477−8495