Journal of Medicinal Chemistry
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The combined organic phases were washed with water, dried
(Na2SO4), and concentrated. Purification by silica gel (CH2Cl2/
MeOH 9:1) gave triol derivative as a white solid (27 mg, quantitative
5β-Cholan-3α,7α,24-tryl-3,7,24-sodium Trisulfate (20). The
triethylamine−sulfur trioxide complex (91 mg, 0.5 mmol) was added
to a solution of triol 16 (40 mg, 0.1 mmol) in dry DMF (3 mL) under
an argon atmosphere, and the mixture was stirred at 95 °C for 3 h.
Most of the solvent was evaporated, and the residue was poured over a
RP18 column to remove excess SO3·NEt3. The fraction eluted with
H2O/MeOH, 9:1, 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 (35:65) as eluent (flow rate 1 mL/min) to give 20 (44
1
yield). [α]D25 +1.97 (c 0.5, CH3OH). Selected H NMR (400 MHz,
CDCl3): δ 3.67 (1H, br s), 3.60 (2H, m), 3.43 (1H, m), 0.93 (3H, d, J
= 6.6 Hz), 0.88 (3H, s), 0.87 (3H, t, J = 7.1 Hz), 0.67 (3H, s). 13C
NMR (100 MHz, CDCl3): δ 73.2, 71.2, 60.8, 57.9, 51.7, 51.6, 46.9,
43.9, 43.1, 41.5, 41.0, 39.8, 36.7, 36.6, 34.5, 34.4, 34.2, 31.2, 29.3, 24.5,
23.7, 23.5, 21.9, 19.5, 19.3, 12.2, 12.0. HRMS-ESI m/z 393.3367 [M +
H+], C25H45O3 requires 393.3369.
mg, tR = 8 min, 65%). [α]2D5 +4.78 (c 0.12, CH3OH). H NMR (400
1
Triethylamine−sulfur trioxide complex (127 mg, 0.7 mmol) was
added to triol (27 mg, 0.07 mmol) in DMF dry (10 mL) under an
argon atmosphere, and the mixture was stirred at 95 °C for 48 h. The
reaction mixture was quenched with water (1.6 mL), and the solution
was poured over a C18 silica gel column to remove excess Et3N·SO3.
The fraction eluted with H2O/MeOH (9:1) gave a mixture, which was
further purified by HPLC on a Nucleodur 100-5 C18 (5 μm, 10 mm
i.d. × 250 mm) with MeOH/H2O (35:65) as eluent (flow rate 3 mL/
min) to give compound 13 as a white solid (47 mg, 72%). [α]D25 −2.3
MHz, CD3OD): δ 4.45 (1H, br s), 4.15 (1H, m), 3.95 (2H, t, J = 6.5
Hz), 2.35 (1H, d, J = 12.5 Hz), 2.3 (1H, d, J = 14.0 Hz), 0.97 (3H, d
ovl), 0.97 (3H, s), 0.70 (3H, s). HR ESIMS m/z 661.1395 [M − Na],
C24H39Na2O12S3 requires 661.1399.
The same sequence of reactions and purification procedure was
carried out on 6α-ethylchenodeoxycholic acid (15) to give a mixture of
compounds 19 and 21. Purification by HPLC on a Nucleodur 100-5
C18 (5 μm, 4.6 mm i.d. × 250 mm) with MeOH/H2O (65:35) as
eluent (flow rate 1 mL/min) gave compound 19 (tR = 22.0 min) as a
white solid (15 mg). Purification by HPLC on a Nucleodur 100-5 C18
(5 μm, 4.6 mm i.d. × 250 mm) with MeOH/H2O (35:65) as eluent
(flow rate 1 mL/min) gave compound 21 (tR = 24.0 min) as a white
solid (27 mg).
1
(c 0.24, CH3OH). Selected H NMR (400 MHz, CD3OD): δ 4.62
(1H, br s), 4.11 (1H, m), 4.03 (2H, m), 3.19 (18H, q, J = 7.2 Hz), 1.30
(27H, t, J = 7.2 Hz), 0.99 (3H, d, J = 6.5 Hz), 0.96 (3H, s), 0.93 (3H,
t, J = 7.4 Hz), 0.71 (3H, s). HR ESIMS m/z 833.4322 [M − Et3NH],
C37H73N2O12S3 requires 833.4326.
6α-Ethyl-3α,7α-dihydroxy-5β-cholan-24-yl-24-sodium Sul-
fate (19). [α]D25 +3.18 (c 0.51, CH3OH). Selected H NMR (400
1
Chenodeoxycholan Sulfate Derivative Synthesis. Methyl
3α,7α-Dihydroxy-5β-cholan-24-oate (14). A mixture of CDCA
(3, 100 mg, 0.25 mmol) and p-toluenesulfonic acid (237 mg, 1.25
mmol) in dry methanol (10 mL) was left to stand at room
temperature for 1 h. The mixture was quenched by addition of
NaHCO3 solution until neutrality. Most of the solvent was evaporated,
and the residue was extracted with EtOAc. The combined extract was
washed with brine, dried with Na2SO4, and evaporated to give the
desired methyl ester as colorless amorphous solids (102 mg,
quantitative yield). An analytic sample was obtained by silica gel
chromatography, eluting with CH2Cl2/MeOH 95:5. [α]D25 +7.81 (c
MHz, CD3OD): δ 3.96 (2H, t, J = 6.3 Hz), 3.65 (1H, br s), 3.30 (1H,
m ovl), 0.97 (3H, d, J = 6.5 Hz), 0.92 (3H, s), 0.90 (3H, t, J = 7.0 Hz),
0.70 (3H, s). 13C NMR (100 MHz, CD3OD): δ 72.2, 70.9, 69.6, 56.4,
50.5, 45.9, 42.5, 41.1, 39.9, 39.6, 35.7, 35.6 (2C), 35.4, 33.4, 32.0, 31.9,
29.3, 28.2, 23.6 (2C), 23.1, 20.5, 18.9, 12.2, 11.9. HR ESIMS m/z
485.2935 [M − Na], C26H45O6S requires 485.2937.
6α-Ethyl-5β-cholan-3α,7α,24-tryl-3,7, 24-sodium Trisulfate
(21). [α]D25 −1.9 (c 0.23, CH3OH). Selected H NMR (400 MHz,
1
CD3OD): δ 4.55 (1H, br s), 4.09 (1H, m), 3.96 (2H, t, J = 6.0 Hz),
0.97 (3H, d, J = 6.4 Hz), 0.92 (3H, s), 0.90 (3H, t, J = 7.0 Hz), 0.70
(3H, s). HR ESIMS m/z 689.1715 [M − Na], C26H43Na2O12S3
requires 689.1712.
1
2.33, CHCl3). Selected H NMR (400 MHz, CDCl3): δ 3.73 (1H, br
s), 3.57 (3H, s), 3.30 (1H, m ovl), 2.26 (1H, m), 2.13 (1H, m), 0.84
(3H, d, J = 6.0 Hz), 0.82 (3H, s), 0.60 (3H, s). 13C NMR (100 MHz,
CDCl3): δ 174.4, 71.2, 67.6, 55.3, 51.0, 49.8, 42.0, 41.1, 39.2, 38.8
(2C), 34.9, 34.8, 34.5, 34.2, 32.3, 30.5, 30.4, 29.9, 27.7, 23.0, 22.3, 20.1,
17.7, 11.2. HRMS-ESI m/z 407.3165 [M + H+], C25H43O4 requires
407.3161.
Bis-homochenodeoxycholan Sulfate Derivatives. 3α,7α-Di-
(tert-butyldimethylsilyloxy)-5β-cholan-24-ol (22). 2,6-Lutidine
(290 μL, 2.5 mmol) and tert-butyl dimethylsilyltrifluoromethane-
sulfonate (171 μL, 0.75 mmol) were added at 0 °C to a solution of
methyl ester 14 (100 mg, 0.25 mmol) in 10 mL of CH2Cl2. After the
mixture was stirred for 2 h at 0 °C, the reaction was quenched by
addition of aqueous NaHSO4 (1 M, 50 mL). The layers were
separated, and the aqueous phase was extracted with CH2Cl2 (3 × 50
mL). The combined organic layers were washed with NaHSO4, water,
saturated aqueous NaHCO3, and brine. Purification by flash
chromatography on silica gel using hexane/ethyl acetate, 99:1, and
0.5% of triethylamine as eluent gave the corresponding methyl ester
(131 mg, 83%) as a clear, colorless oil. To a solution of methyl ester
(100 mg, 0.16 mmol) in dry THF (5 mL) at 0 °C were added dry
methanol (45 μL, 1.12 mmol) and LiBH4 (560 μL, 2 M in THF, 1.12
mmol). The resulting mixture was stirred for 3 h at 0 °C. The mixture
was quenched by addition of 1 M NaOH (320 μL) and then ethyl
acetate. The organic phase was washed with water, dried (Na2SO4),
and concentrated. Purification by silica gel (hexane/ethyl acetate 8:2)
gave compound 22 as a white solid (97 mg, quantitative yield). [α]2D5
+7.47 (c 0.12, CHCl3). Selected 1H NMR (400 MHz, CDCl3): δ 3.82
(1H, br s), 3.47 (2H, t, J = 6.5 Hz), 3.41 (1H, m), 0.93 (3H, d, J = 6.0
Hz), 0.88 (9H, s), 0.88 (3H, s ovl), 0.86 (9H, s), 0.65 (3H, s), 0.08
(6H, s), 0.04 (6H, s). 13C NMR (100 MHz, CDCl3): δ 72.8, 69.7,
63.4, 56.3, 50.1, 42.5, 42.0, 40.7, 40.6, 39.8, 36.0, 35.8, 35.2, 34.8, 32.4,
32.2, 31.2, 29.7 (3C), 28.3, 26.3 (3C), 26.0 (3C), 24.1, 23.0, 20.7, 18.8,
12.0, −2.2, −4.4, −4.5, −5.4. HRMS-ESI m/z 607.4946 [M + H+],
C36H71O3Si2 requires 607.4942.
3α,7α,24-5β-Cholantriol (16). Methyl ester 14 (100 mg, 0.25
mmol) was dissolved in dry THF (5 mL) at 0 °C in the presence of
dry methanol (70 μL, 0.84 mmol) and LiBH4 (875 μL, 2 M in THF,
1.75 mmol). After 8 h, a solution of 1 M NaOH (500 μL) was added
and then allowed to warm to room temperature. Ethyl acetate was
added, and the separated aqueous phase was extracted with ethyl
acetate (3 × 30 mL). The combined organic phases were washed with
water, dried (Na2SO4), and concentrated. Purification by silica gel
(CH2Cl2/MeOH 95:5) gave triol derivative 16 as a colorless oil (90
mg, 96%). [α]D25 +0.82 (c 2.07, CH3OH). Selected H NMR (400
1
MHz, CDCl3): δ 3.80 (1H, br s), 3.56 (2H, m), 3.40 (1H, m), 0.91
(3H, d, J = 6.0 Hz), 0.87 (3H, s), 0.63 (3H, s). 13C NMR (100 MHz,
CDCl3): δ 71.8, 68.4, 63.2, 56.0, 50.3, 42.5, 41.5, 39.6, 39.3 (2C), 35.5,
35.3, 34.9, 34.5, 32.8, 30.5 (2C), 29.3, 28.2, 23.6, 22.7, 20.5, 18.6, 11.7.
HRMS-ESI m/z379.3216 [M + H+], C24H43O3 requires 379.3212.
3α,7α-Dihydroxy-5β-cholan-24-yl-24-sodium Sulfate (18). At
a solution of triol 16 (40 mg, 0.1 mmol) in DMF dry (3 mL) was
added triethylamine−sulfur trioxide complex (36 mg, 0.2 mmol) under
an argon atmosphere, and the mixture was stirred at 95 °C for 1 h.
Most of the solvent was evaporated, and the residue was poured over a
RP18 column to remove excess SO3·NEt3. The fraction eluted with
H2O/MeOH 1:1 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 (65:35) as eluent (flow rate 1 mL/min) to give 35 mg (73%) of
compound 18 (tR = 11.2 min). [α]D25 +4.93 (c 0.05, CH3OH). Selected
1H NMR (400 MHz, CD3OD): δ 3.95 (2H, t, J = 6.4 Hz), 3.78 (1H,
br s), 3.30 (1H, m ovl), 0.96 (3H, d ovl), 0.95 (3H, s), 0.70 (3H, s).
HR ESIMS m/z 457.2627 [M − Na], C24H41O6S requires 457.2624.
Ethyl 3α, 7α-Di(tert-butyldimethylsilyloxy)-25,26-bis-homo-
5β-chol-24-en-26-oate (23). DMSO (4.18 mL, 0.75 mmol) was
added dropwise for 5 min to a solution of oxalyl chloride (14.7 mL,
0.37 mmol) in dry dichloromethane (5 mL) at −78 °C under argon
atmosphere. After 30 min, a solution of 22 (90 mg, 0.15 mmol) in dry
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dx.doi.org/10.1021/jm401873d | J. Med. Chem. 2014, 57, 937−954