Synthesis of (25R)-26-hydroxycholesterol.

Article abstract of DOI:10.1016/S0039-128X(02)00060-0

We describe the synthesis of (25R)-cholest-5-en-3beta,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall, yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.

Full text of DOI:10.1016/S0039-128X(02)00060-0

Steroids 67 (2002) 1041–1044
Synthesis of (25R)-26-hydroxycholesterol
John R. Williams∗, Deping Chai, Dominic Wright
Department of Chemistry, Temple University, 13th & Norris Streets, Beury Hall (016-00), Philadelphia, PA 19122-2585, USA
Received 13 February 2002; received in revised form 9 April 2002; accepted 24 April 2002
Abstract
We describe the synthesis of (25R)-cholest-5-en-3␤,26-diol ((25R)-26-hydroxycholesterol) from diosgenin in four steps in 58% overall,
yield via a modified Clemmensen reduction followed by a Barton deoxygenation reaction.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Oxysteroids; 26-Hydroxycholesterol; Diosgenin; Modified Clemmensen reduction; Barton deoxygenation reaction; Synthesis
1. Introduction
were recorded in CDCl3 with chemical shift values (in ppm)
1
relative to the solvent peak (7.26 ppm for H and 77.0 ppm
for 13C). Coupling constants are in units of hertz (Hz). High
resolution mass spectra (HRMS) were obtained on a Fis-
sons ZAB HF double-focusing mass spectrometer at Drexel
University, Philadelphia, PA.
The role of oxysteroids including (25R)-cholest-5-en-
3␤,26-diol ((25R)-26-hydroxycholesterol) (1) in the regula-
tion of cholesterol homeostasis has been critically examined
[1]. (25R)-26-Hydroxycholesterol is an intermediate in the
metabolic pathway from cholesterol to the bile acids [2].
It is a potent inhibitor of cholesterol biosynthesis in vitro
as it is an effective inhibitor of HMG-CoA reductase [3],
but there is little direct evidence that it is important under
in vivo conditions. (25R)-26-Hydroxycholesterol has also
been shown to be an inhibitor of DNA synthesis [4].
(25R)-26-Hydroxycholesterol has been synthesized from
two readily available natural products, kryptogenin [5–8],
and diosgenin [9–11] and by addition of a side chain building
block to the steroid backbone [12].
Analytical thin layer chromatography (TLC) was con-
ducted using Analtech silica gel GF plates (250 ␮m) con-
taining a fluorescent indicator. Detection was performed by
observation under UV light or by iodine staining. Prepara-
tive TLC was carried out using 20 cm2 silica gel GF plates
(1000 ␮m). Flash column chromatography was performed
using Merck silica gel 50 (230–400 mesh). Elemental
analyses were performed by Galbraith Laboratories, Inc.
Knoxville, TN.
We now describe in Fig. 1, a convenient and higher yield-
ing method, using Barton’s dithiocarbonate deoxygenation
procedure [13], for the synthesis of (25R)-26-hydroxycho-
lesterol from diosgenin in four steps in 58% overall yield.
2.1. (25R)-Cholest-5-en-3β,16β,26-triol (2)
To a 100 ml three neck flask was added sequentially dios-
genin (0.22 g, 0.53 mmol), zinc dust (4.5 g, 68.8 mmol), and
50 ml absolute alcohol. After the mixture was stirred, heated
to reflux, and 40 ml of concentrated HCl was added drop-
wise during a 30-min period. The reaction was refluxed for
an additional 30 min, then filtered to remove zinc dust. The
solution was collected and distilled water was added until
a precipitate appeared. The solution was heated until trans-
parent, then slowly cooled. The precipitate, which formed,
was collected by suction filtration and washed three times
with cold water. The crystals were then dried to yield the
triol 2 as a white solid (0.18 g, 85%): mp = 172–174 ◦C, lit.
[9] mp = 176–178 ◦C. IR (KBr) (cm−1): 3377, 2934, 1464,
1378, 1043, 827. 1H NMR (CDCl3/CD3OD (2:1)), δ (ppm):
0.82 (s, 3H, Me-18), 0.83 (d, J = 7.0 Hz, 3H, Me-27),
2. Experimental
Melting points were determined on a Thomas–Hoover
capillary melting point apparatus and are uncorrected. IR
spectra were recorded on a Mattson 4040 FT-IR spectrom-
eter and the data reported in wavenumbers (cm−1). The 1H
NMR and 13C NMR spectra were recorded on a GE QE 300
(at 300 and 75.48 MHz, respectively) spectrometer. Spectra
∗
Corresponding author. Tel.: +1-215-204-7144; fax: +1-215-204-1532.
E-mail address: john.r.williams@temple.edu (J.R. Williams).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00060-0

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J.R. Williams et al. / Steroids 67 (2002) 1041–1044
Fig. 1. Synthesis of (25R)-26-hydroxycholesterol (1) from diosgenin.
0.91 (d, J = 6.5 Hz, 3H, Me-21), 0.95 (s, 3H, Me-19),
3.33–3.56 (m, 3H, H-3␣ and H-26), 4.28 (m, 1H, H-16␤),
5.28 (m, 1H, H-6). 13C NMR (CDCl3/CD3OD (2:1)), δ
(ppm): 13.40(C18), 17.01(C27), 18.63(C21), 19.98(C19),
21.25(C11), 23.97(C23), 30.29(C20), 31.67(C2), 32.04(C8),
32.04(C7), 32.35(C24), 35.89(C25), 36.44(C22), 37.06(C10),
37.12(C15), 37.75(C1), 40.42(C12), 42.74(C4), 42.38(C13),
50.66(C9), 54.97(C14), 61.99(C17), 68.23(C26), 71.77(C3),
72.50(C16), 121.83(C6), 141.55(C5).
24.47(C23), 30.41(C20), 26.64((CH3)3CSi), 26.69((CH3)3-
CSi), 32.17(C8), 32.54(C7), 32.75(C2), 34.27(C24), 36.46-
(C25), 36.96(C22), 37.23(C15), 37.33(C10), 38.01(C1),
40.60(C12), 43.50(C4), 42.86(C13), 50.86(C9), 55.26(C14),
62.13(C17), 69.11(C26), 72.96(C3), 73.22(C16), 121.61
(C6), 141.19(C5).
2.3. (25R)-3β,26-Bis[(tert-butyldimethylsilyl)oxy]-
cholest-5-ene (4)
2.2. (25R)-3β,26-Bis[(tert-butyldimethylsilyl)oxy]-
cholest-5-en-16β-ol (3)
To a solution of 3 (30 mg, 0.046 mmol) in 5 ml THF
were added 40 mg (1 mmol) NaH (65% suspension in min-
eral oil) and 4 mg imidazole. The mixture was refluxed
under argon for 30 min, then 0.4 ml (4.7 mmol) CS2 was
added. The mixture was refluxed for another hour, then
0.2 ml (3 mmol) of methyl iodide was added. The mixture
was refluxed for an additional 30 min. Then the mixture
was poured into 10 ml of water, and extracted with ether
(2 × 10 ml). The ether extracts were washed with brine,
dried (Na2SO4) and the solvent was removed in vacuo to
give a yellow oil. The oil was refluxed in toluene (10 ml)
containing a trace of AIBN, under argon. A solution of
tributylstannane (0.2 ml, 0.75 mmol) in toluene (3 ml) was
added over 30 min. The mixture was refluxed overnight. The
reaction was quenched with aqueous saturated NaHCO3,
and the aqueous layer was extracted with diethyl ether. The
combined organic phases were dried (Na2SO4), and the
solvent was evaporated in vacuo. The residue was purified
by flash chromatography (10:1 hexane:ethyl acetate) to
yield 4 as a white solid (22 mg, 75%): mp = 98–100 ◦C.
IR (CH2Cl2) (cm−1): 2933, 2857, 1464, 1254, 1097, 836,
To a solution of tert-butyldimethylsilyl chloride (0.9 g,
6 mmol) in 10 ml of dry DMF was added imidazole
(0.8 g, 12 mmol) and the mixture was stirred at room tem-
perature for 15 min under nitrogen 2 (0.5 g, 1.2 mmol)
was added and the mixture stirred at room temperature
for 16 h. A white precipitate was obtained by filtration,
which was purified by flash chromatography (20:1, hex-
ane:ethyl acetate) to afford 3 as a white solid (0.64 g,
93%): mp = 122–124 ◦C, lit. [10] mp = 123–124 ◦C.
IR (KBr) (cm−1): 3629, 2956, 1470, 1384, 1253, 1080,
1
837. H NMR (CDCl3), δ (ppm): 0.04 (s, 6H, (CH3)2–Si),
0.06 (s, 6H, (CH3)2–Si), 0.86 (s, 3H, Me-18), 0.891
(s, 9H, (CH3)3–Si), 0.896 (s, 9H, (CH3)3–Si), 0.98 (d,
J = 6.5 Hz, 3H, Me-21), 1.01 (s, 3H, Me-19), 3.33–3.56
(m, 3H, H-3␣ and H-26), 4.33 (m, 1H, H-16␤), 5.34 (m,
1H, H-6). 13C NMR (CDCl3), δ (ppm): −4.62(CH3Si),
−3.86(CH3Si), 13.71(C18), 17.45(C27), 18.87((CH3)3CSi),
18.92((CH3)3CSi), 19.02(C21), 20.12(C19), 21.38(C11),

J.R. Williams et al. / Steroids 67 (2002) 1041–1044
1043
1
774. H NMR (CDCl3), δ (ppm): 0.04 (s, 6H, (CH3)2–Si),
of a secondary alcohol to a hydrogen without the possi-
bility of a rearrangement that could occur if the reaction
proceeded via an ionic process of any type. We chose the
Barton deoxygenation reaction which proceeds via a radi-
cal mechanism [13]. The C-16 alcohol in 3 was converted
to the intermediate dithiocarbonate using sodium hydride,
carbon disulfide, and methyl iodide and then reduced with
n-Bu3SnH and AIBN to the 3␤,26-bis-silyl ether 4 in 75%
yield. Desilylation of 4 using 49% aqueous HF [14] afforded
26-hydroxycholesterol in 98% yield. Using this sequence
the 25R isomer of 26-hydroxycholesterol was synthesized
from diosgenin in 58% overall yield, an improvement over
the best previous yield of 45% [11].
0.06 (s, 6H, (CH3)2–Si), 0.67 (s, 3H, Me-18), 0.85 (d,
J = 3.5 Hz, 3H, Me-27), 0.889 (s, 9H, (CH3)3–Si), 0.895
(s, 9H, (CH3)3–Si), 0.90 (d, J = 6.5 Hz, 3H, Me-21), 1.00
(s, 3H, Me-19), 3.33–3.56 (m, 3H, H-3␣ and H-26), 5.34
(m, 1H, H-6). 13C NMR (CDCl3), δ (ppm): −4.91(CH3Si),
−4.16(CH3Si), 12.27(C18), 17.11(C27), 18.67((CH3)3CSi),
18.77((CH3)3CSi), 19.12(C21), 19.85(C19), 21.49(C11),
23.78(C23), 24.71(C15), 26.38((CH3)3CSi×2), 28.67(C16),
32.32(C2), 32.36(C8), 32.51(C7), 34.00(C24), 36.15(C20),
36.15(C25),36.61(C22),37.00(C10),37.81(C1),40.23(C12),
42.74(C4), 43.25(C13), 50.63(C9), 56.57(C17), 57.22(C14),
68.96(C26), 73.05(C3), 121.58(C6), 141.94(C5).
2.4. (25R)-Cholest-5-ene-3β,26-diol (1)
Acknowledgments
In a plastic tube was placed the disilyl ether 4 (0.064 g,
0.1 mmol), THF (2 ml), and 49% aqueous HF (1 ml). The so-
lution was allowed to stir for 20 h. Water was added and the
solution extracted with ethyl acetate (3×10 ml). The organic
layers were combined and washed to neutrality with a sat-
urated NaHCO3 solution, then with water and finally dried
(Na2SO4) and the solvent removed in vacuo. Recrystalliza-
tion from ethyl acetate yielded 26-hydroxycholesterol (4)
(40 mg, 98%), mp = 168–170 ◦C, lit. [6] mp = 172–173 ◦C.
Financial support for this research was provided by a
grant from the Temple University Research Incentive Fund,
a GlaxoSmithKline Fellowship (D.C.) and D.W. was sup-
ported in part by the Howard Hughes Medical Institute grant
to the Undergraduate Biological Sciences Education Pro-
gram at Temple University. We thank Dr. Stephen Wilson
of the Chemistry Department at New York University for
suggesting the modified Clemmensen reduction conditions.
IR (KBr) (cm−1): 3341, 2932, 1466, 1378, 1055. H NMR
1
(CDCl3), δ (ppm): 0.61 (s, 3H), 0.85 (d, J = 6.43 Hz, 3H),
0.90 (d, J = 6.5 Hz, 3H), 0.98 (s, 3H), 3.33–3.56 (m, 3H),
5.30 (m, 1H). 13C NMR (CDCl3 + CD3OD, two drops),
δ (ppm): 12.17(C18), 16.80(C27), 18.98(C21), 19.7(C19),
21.41(C11), 23.78(C23), 24.62(C15), 28.58(C16), 31.63(C2),
32.24(C7), 32.24(C8), 33.92(C24), 36.05(C20), 36.09(C25),
36.52(C22), 36.85(C10), 37.59(C1), 40.13(C12), 42.31(C4),
42.66(C13), 50.48(C9), 56.48(C17), 57.11(C14), 68.46(C26),
71.78(C3), 121.96(C6), 141.19(C5).
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3. Results and discussion
Diosgenin was chosen as the starting material for the
synthesis of (25R)-26-hydroxycholesterol (1) since it is
commercially available in high purity. Clemmensen reduc-
tion of diosgenin has been reported to yield (25R)-cho-
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[9], 45% [10] and 2 and recovered diosgenin ∼75% [11].
The yield for this reaction was improved to 85% by removing
the mercury and just using zinc dust in ethanol and adding
concentrated HCl acid dropwise. The product was then pure
enough to crystallize from aqueous ethanol instead of being
extracted with chloroform and chromatographed [10].
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zole as a catalyst to afford the 3␤,26-bis-silyl ether 3 in 93%
yield. Next, we needed a reaction for the deoxygenation
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