Synthesis of polyhydroxysterols (I): Synthesis of 24-methylenecholest-4-en-3β,6β-diol, a cytotoxic natural hydroxylated sterol

Article abstract of DOI:10.1016/S0039-128X(00)00132-X

Starting from stigmasterol (2), 24-methylenecholest-4-en-3β,6β-diol (1), a cytotoxic natural dihydroxylated sterol, was synthesized via 10 steps in 20% overall yield. The introduction of a side-chain of sterol was achieved by solid-liquid phase-transfer Wittig reaction using (3-methyl-2-oxo)butyltriphenylarsonium bromide (12) and K₂CO₃. Construction of the steroidal nucleus was finished by the addition of 3β-acetoxycholest-5,6-en-24-one (7) with NBA in dioxane under ambient temperature and by the elimination of 3β, 6β-diacetoxy-5a-bromocholestane-24-one (9). The spectral data of the synthetic product (1) are completely consistent with those of the natural compound (1). Copyright (C) 2001 Elsevier Science Inc.

Full text of DOI:10.1016/S0039-128X(00)00132-X

Steroids 66 (2001) 33–38
Synthesis of polyhydroxysterols (I): synthesis of 24-methylenecholest-
4-en-3␤,6␤-diol, a cytotoxic natural hydroxylated sterol૾
JianGuo Cuia,b,*, LongMei Zenga, JingYu Sub, WeiGang Lua
aDepartment of Chemistry, Zhongshan University, Guangzhou 510275, P.R. of China
bDepartment of Chemistry, Guangxi Teachers College, Nanning 530001, P.R. of China
Received 29 November 1999; received in revised form 22 May 2000; accepted 26 May 2000
Abstract
Starting from stigmasterol (2), 24-methylenecholest-4-en-3␤,6␤-diol (1), a cytotoxic natural dihydroxylated sterol, was synthesized via
10 steps in 20% overall yield. The introduction of a side-chain of sterol was achieved by solid-liquid phase-transfer Wittig reaction using
(3-methyl-2-oxo)butyltriphenylarsonium bromide (12) and K2CO3. Construction of the steroidal nucleus was finished by the addition of
3␤-acetoxycholest-5,6-en-24-one (7) with NBA in dioxane under ambient temperature and by the elimination of 3␤, 6␤-diacetoxy-5a-
bromocholestane-24-one (9). The spectral data of the synthetic product (1) are completely consistent with those of the natural compound
(1). © 2001 Elsevier Science Inc. All rights reserved.
Keywords: Polyhydroxylated sterol; 24-Methylenecholest-4-en-3␤,6␤-diol; Synthesis
1. Introduction
2. Experimental
Stigmasterol was purchased from the Merck Co. (West
Point, PA, USA). All chemicals and solvents were analyti-
cal grade and solvents were purified by general methods
before use. Melting points were determined on a X4 appa-
ratus and were uncorrected. Infrared spectra were measured
with Nicolet 205 FT-IR and Nicolet FT-360 Spectropho-
tometers. 1H NMR spectra were recorded on either a JEOL
FX-90Q (90 MHz) or a Varian UnityInova 500 NB (500
MHz) spectrometer in CDCl3 using tetramethylsilane
(TMS) as the internal standard. Mass spectra were measured
with a VG-ZAB-HS mass spectrometer using a 70 eV elec-
tron impact ion source.
Many naturally occurring polyhydroxylated sterols and
oxysterols exhibit potent biologic activities. In our previous
study on the soft coral and the sponge, some new polyhy-
droxylated sterols were isolated, and they showed potent
cytotoxicity to cancer cells [1–3]. 24-Methylenecholest-4-
en-3␤,6␤-diol (1) was obtained from the soft coral Alcyo-
nium patagonicum and showed potent cytotoxicity to mu-
rine leukemia cells with an IC50 value of 1 ␮g/ml [1]. This
spurred our interest in studying the synthesis of compound
(1). This paper reports the synthesis of 24-methylenecho-
lest-4-en-3␤,6␤-diol (1) using stigmasterol as the starting
material (Scheme 1).
2.1. Stigmasteryl acetate (3)
Acetic anhydride (1.86g) was added to a solution of
stigmasterol (2) (2.44g, 5.9 mmol) in 10 ml of pyridine, and
the mixture was incubated at room temperature for 30 h. To
this mixture, 30 ml of water were added to quench the
excess acetic anhydride, and the mixture was subsequently
extracted with ethyl acetate (20 ml ϫ 4). The combined
organic layer was washed successively with water, 1 M HCl
solution, and brine, and then dried over anhydrous Na2SO4
and evaporated in vacuo. The residue was recrystallized
૾ The authors thank the Natural Science Foundation of China (Project:
29932030) and the Natural Science Foundation of Guangdong Province
(Project: 970154) for their support.
* Corresponding author. Tel.: ϩ86-771-5333317 (home), ϩ86-771-
3132288, ext. 8065 (office).
E-mail address: cuijg@public.nn.gx.cn (JG. Cui).
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00132-X

34
JG. Cui et al. / Steroids 66 (2001) 33–38
Scheme 1.
from EtOAc to give 3 as colorless needle crystals (2.75 g,
96%), m.p. 148–149°C. IR (KBr)␯: 1728, 1651, 1461,
tored by TLC. When the 5␣,6␤-dibromostigmastan-3␤-yl
acetate disappeared (ca. 24 min), the ozonization reaction
was stopped. The reaction mixture was immediately poured
into a mixture of glacial AcOH (3.8 ml) and Zn dust (2.47
g) and stirred at room temperature for 4 h. The solution was
filtered, washed successively with water, 10% sodium car-
bonate, 5% sodium hydroxide, and brine, and then dried
over anhydrous Na2SO4. Evaporation of the solvent gave a
residue, which was dissolved in a small amount of MeOH
and then combined with 15 ml of saturated NaHSO3 solu-
tion. The mixture was stirred at room temperature for 6 h.
Ether (15 ml) was added and the mixture was stirred for an
additional 10 min. The suspension was filtered, and the solid
was washed with water and dried under vacuum to give
0.60 g of the bisulfite addition product.
The bisulfite addition product was suspended in water (2
ml) and ether (40 ml), and then 10% Na2CO3 solution (20
ml) was added. The mixture was stirred until two clear
layers formed. The organic layer was separated, and the
aqueous layer was extracted with ether. The ether solution
was washed with brine and dried over anhydrous Na2SO4.
The solvent was removed under reduced pressure and dried
to give 0.46 g (82%) of 5, m.p. 107–108°C. IR (KBr) ␯:
2945, 2846, 1728, 1461, 1370, 1257, 1039 cm Ϫ1. 1H NMR
(CDCl3, 90 MHz): ␦ ϭ 0.73 (s, 3H, 18-CH3), 1.03 (s, 3H,
19-CH3), 1.13 (␦, 3H, J ϭ 7.0 Hz, 21-CH3), 2.03 (s, 3H,
3-OCOCH3), 4.59 (m, 1H, 3a-CH), 5.38 (brd, 1H, 6-CH),
9.57(d, 1H, J ϭ 3.2 Hz, 22-CH).
1370, 1039, 969, 800 cmϪ1
.
2.2. 5a,6␤-Dibromostigmastan-3␤-yl acetate (4)
Bromine (0.54 g, 3.4 mmol) was added to a solution of
iodobenzene (0.66 g, 3.2 mmol) in dry n-hexane (10 ml),
and the solution was cooled to Ϫ5°C.
A solution of 3 (1.35 g, 3.0 mmol) in dry n-hexane (70
ml) was also cooled to Ϫ5°C, stirred vigorously, and the
reagent prepared above was added dropwise over 2.5 h
under an argon atmosphere to maintain the solution a pale
yellow color. The resulting solution was stirred for 2 h
more, and then filtered. The solution was concentrated
under vacuum until most of the bromosterol was precip-
itated. The mixture was heated, and the clear solution was
allowed to stand overnight in the refrigerator. The result-
ing 5a,6␤-dibromostigmastan-3␤-yl acetate was filtered
off and washed with MeOH. 1.78 g (97%) of 4 was
obtained as colorless crystals, m.p. 128–130°C. IR (KBr)
1
␯: 1735, 1461, 1370, 1236, 1032, 969, 561 cm Ϫ1. H
NMR (CDCl3, 90 MHz): ␦ ϭ 0.72 (s, 3H, 18-CH3), 0.78
and 0.85 (d, 6H, J ϭ 4.4 Hz, 26-CH3 and 27-CH3), 1.01
(d, 3H, J ϭ 6.8 Hz, 21-CH3), 1.46 (s, 3H, 19-CH3), 2.05
(s, 3H, 3-OCOCH3), 4.84 (brd, 1H, 6␣-CH), 5.04 to 5.16
(m, 2H, 22- and 23-CH), 5.32 to 5.56 (m, 1H, 3-CH).
2.3. Cholest-5-en-3␤-ol-22-al (5)
2.4. 3␤-Acetoxycholest-5, 22-diene-24-one (6)
A solution of 5␣,6␤-dibromostigmastan-3␤-yl acetate
(4) (0.93 g; 1.5 mmol) in dried CH2Cl2 (30 ml) and pyridine
(0.35 ml) was cooled in a liquid nitrogen/ethyl acetate bath.
A stream of ozone-rich oxygen was passed into the solution
through a sintered-glass sprayer. The reaction was moni-
(3-Methyl-2-oxo)butyltriphenyl-arsonium
bromide
(12) (0.31 g, 0.66 mmol), K2CO3 (90 mg, 0.66 mmol),
and 0.02 ml of H2O were added to a solution of 5 (0.18
g, 0.48 mmol) in 4 ml of MeCN, and the mixture was

JG. Cui et al. / Steroids 66 (2001) 33–38
35
stirred at room temperature for 28 h under argon atmo-
sphere. Water (15 ml) was added to the suspension, and
the solution was extracted with ether. The ether solution
was washed with water and brine and then dried over
Na2SO4. The solvent was removed under vacuum, and
the residue was purified by flash chromatography on
silica gel using petroleum ether (b.p. 60–90°C)/EtOAc
(19:1) as eluent to give 0.18 g (85%) of colorless needle
crystals 6, m.p. 143–144°C. IR (KBr)␯: 2938, 1735,
1.318 (s, 3H, 19-CH3), 2.039 (s, 3H, C3-CH3CO), 2.608 (m,
1H, 25-CH), 4.181 (brs, 1H, 6-CH), 5.471 (m, 1H, 3-CH).
2.7. 3␤, 6␤-Diacetoxy-5␣-bromocholestane-24-one (9)
Acetic anhydride (0.90 g) was added to a solution of 8
(0.12 g, 0.22 mmol) in 1.5 ml of pyridine, and the mixture
was stirred at room temperature for 40 h. 10 ml of water
were then added and the mixture was extracted with ethyl
acetate (8 ml ϫ 4). The combined organic layer was
washed successively with water, 1 M HCl solution, wa-
ter, and brine and then dried over anhydrous Na2SO4.
After the solvent was evaporated in vacuo, the solid
residue was dissolved again using petroleum ether (b.p.
60–90°C)/EtOAc (1:1). The solution was put in the re-
frigerator overnight. The resulting colorless crystals were
filtered and washed with petroleum ether and dried under
vacuum to yield 0.11 g (85%) of 9, m.p. 162–163°C. IR
(KBr)␯: 2947, 1750, 1734, 1707, 1561, 1456, 1361, 1241,
1
1693, 1623, 1243, 1039, 990 cmϪ1. H NMR (CDCl3,
90Hz): ␦ ϭ 0.72 (s, 3H, 18-CH3), 1.02 (s, 3H, 19-CH3),
1.10 (d, 9H, J ϭ 6.8 Hz, 21-CH3, 26-CH3, 27-CH3), 2.03
(s, 3H, CH3CO), 2.84 (m, 1H, 25-CH), 4.60 (m, 1H,
3-CH), 5.38 (m, 1H, 6-CH), 6.06 (d, 1H, J ϭ 16.1Hz,
23-CH), 6.73 (dd, 1H, J ϭ 16.1Hz and 9.0Hz, 22-CH).
2.5. 3␤-Acetoxycholest-5-en-24-one (7)
PtO2 (16 mg) was added to a solution of 6 (0.22 g; 0.5
mmol) in 30 ml of EtOAc. Hydrogen was passed into the
stirred mixture at room temperature. After 0.5 mmol H2
was absorbed, the reaction was stopped, and the mixture
was filtered. The solvent was evaporated to dryness,
leaving a white solid, which was recrystallized with
CH3OH to give 216 mg (98%) of colorless needle crys-
tals 7, m.p. 126–127°C. IR (KBr)␯: 2938, 1728, 1707,
1209, 1156, 1031, 668 cmϪ1 1H NMR (CDCl3, 500
.
MHz): ␦ ϭ 0.680 (s, 3H, 18-CH3), 0.906 (d, 3H, J ϭ 6.5
Hz, 21-CH3), 1.091 (d, 6H, J ϭ 6.5 Hz, 26-CH3
and 27-CH3), 1.284 (s, 3H, 19-CH3), 2.031 (s, 3H,
C3-CH3CO), 2.092 (s, 3H, C6-CH3CO), 2.608 (m,
1H, 25-CH), 4.324 (brs, 1H, 6-CH), 5.454 (m, 1H,
3-CH).
1644, 1468, 1370, 1243, 1032, 962 cmϪ1 1H NMR
.
2.8. 3␤, 6␤-Diacetoxycholest-4-en-24-one (10)
(CDCl3, 500 MHz): ␦ ϭ 0.674 (s, 3H, 18-CH3), 0.915 (d,
3H, J ϭ 6.5 Hz, 21-CH3), 1.016 (s, 3H, 19-CH3) 1.091 (d,
6H, J ϭ 7.5 Hz, 26-CH3, 27-CH3), 2.033 (s, 3H,
CH3CO), 2.610 (m, 1H, 25-CH), 4.607 (m, 1H, 3-CH),
5.374 (d, 1H, J ϭ 4.5 Hz, 6-CH).
The solution of bromo-ketone (9) (80 mg) in 1.5 ml of
dimethylacetamide (DMA) was added to a stirred suspen-
sion of calcium carbonate (140 mg) in refluxing DMA (3
ml). Refluxing and stirring were continued for 30 min. The
cooled solution was filtered, and the calcium carbonate was
washed with ether. To the filtrate, 8 ml of water were added,
and the resulting solution was extracted with ether (10 ml ϫ
3). The combined organic layer was washed with water and
brine, and then dried over anhydrous Na2SO4. After the
solvent was evaporated in vacuo, the residue was chromato-
graphed on silica gel using petroleum ether (60–90°C)/
acetone (7:1) as eluent to give 65 mg (95%) of 10 as
colorless needle crystals, m.p. 143–145°C. IR (KBr)␯:
2938, 1735, 1700, 1468, 1370, 1243, 1166, 1096, 1032, 969,
934, 892 cmϪ1. 1H NMR (CDCl3, 500 MHz): ␦ ϭ 0.719 (s,
3H, 18-CH3), 0.912 (d, 3H, J ϭ 6.5 Hz, 21-CH3), 1.092 (d,
6H, J ϭ 6.5 Hz, 26-CH3 and 27-CH3), 1.163 (s, 3H, 19-
CH3), 2.030 (s, 3H, C3-CH3CO), 2.061 (s, 3H, C6-CH3CO),
2.609 (m, 1H, 25-CH), 5.239 (ddd, 1H, J ϭ 10.0, 7.0 and 2.0
Hz, 3-CH), 5.284 (dd, 1H, J ϭ 3.0 and 1.5 Hz, 6-CH), 5.606
(s, 1H, 4-CH).
2.6. 3␤-Acetoxy-5a-bromo-6␤-hydroxycholestane-24-one
(8)
In the dark and at room temperature, four portions of
N-bromoacetamide (NBA) (0.22g, 1.6 mmol) were added
stepwise at 10-min interrals to a solution of 3␤-acetoxycho-
lest-5-en-24-one (7) (0.24 g, 0.54 mmol) in 2 ml of dioxane
containing 70% perchloric acid (0.01 ml) and water (0.10
ml). The mixture was stirred for 30 min, and then 4 ml of
freshly prepared 5% sodium sulfite were added into the
reaction mixture. The mixture was extracted with methylene
chloride (7 ml ϫ 3), and the combined organic layer was
successively washed with H2O, 5% KHCO3, and brine, and
then dried over anhydrous Na2SO4. The solvent was re-
moved under reduced pressure, and the residue was chro-
matographed on silica gel using petroleum ether (60–90°C)/
acetone (7:1) as eluent to give 0.20 g (76%) of 8 as white
solid (m.p. 145–147°C), and 25 mg of 7 were recovered. IR
(KBr)␯: 3409, 2945, 1735, 1707, 1574, 1461, 1370, 1271,
2.9. Cholest-4-en-3␤,6␤-diol -24-one (11)
1
1243, 1159, 1039, 969, 610 cmϪ1. H NMR (CDCl3, 500
A total of 1.6 ml of 10% potassium carbonate was
added to a solution of 3␤, 6␤-diacetoxycholest- 4-en-24-
one (10) (55 mg, 0.11 mg) in 6 ml of MeOH, and the
MHz): ␦ ϭ 0.675 (s, 3H, 18-CH3), 0.907 (d, 3H, J ϭ 6.5 Hz,
21-CH3), 1.091 (d, 6H, J ϭ 7.0 Hz, 26-CH3 and 27-CH3),

36
JG. Cui et al. / Steroids 66 (2001) 33–38
mixture was refluxed for 2.5 h. The MeOH was evapo-
rated under vacuo, and the residue was dissolved in 20 ml
of CH2Cl2. The methylene chloride layer was washed
with water and brine, dried over anhydrous sodium sul-
fate, and evaporated in vacuo to give a white solid. The
crude product was recrystallized with acetone/CH3OH
(1:1) to give 35 mg (77%) of colorless needle crystals 11,
m.p. 211–212°C. IR (KBr)␯: 3291, 2931, 1716, 1635,
(decomposition). IR (KBr) ␯: 2797, 1693, 1440, 1032, 744,
688, 477 cmϪ1. 1H NMR (CDCl3, 90 MHz): ␦ ϭ 1.16 (d, 6H,
J ϭ 7.1 Hz, C(CH3)2), 2.96 to 3.44 (m, 1H, CH), 5.97 (s, 2H,
CH2), 7.33 to 7.96 (m, 15H, Ph-H).
2.12. Methyltriphenylphosphonium iodide (13)
Triphenylphosphine (5.24 g, 20 mmol) was dissolved in
20 ml dry benzene. Iodomethyl (2.67 g, 21 mmol) was
added, and the mixture was stirred at room temperature for
16 h. The resulting white solid was isolated with suction and
washed with benzene. The crude product was recrystallized
from CHCl3/EtOAc (1:1) to afford 7.53 g (97%) of colorless
crystals 13, m.p. 193–194°C. IR (KBr) ␯: 1586, 1484, 1439,
1
1466, 1382, 1093, 1036, 1019, cmϪ1. H NMR (CDCl3,
500 MHz): ␦ ϭ 0.707 (s, 3H, 18-CH3), 0.907 (d, 3H, J ϭ
7.0 Hz, 21-CH3), 1.090 (d, 6H, J ϭ 6.5 Hz, 26-CH3 and
27-CH3), 1.255 (s, 3H, 19-CH3), 2.33–2.50 (m, 2H, 23-
CH2), 2.607 (m, 1H, 25-CH), 4.180 (ddd, 1H, J ϭ 9.0, 6.5
and 1.0 Hz, 3-CH), 4.227 (dd, 1H, J ϭ 3.0 and 2.5 Hz,
6-CH), 5.543 (s, 1H, 4-CH).
1
1116, 915, 749 cmϪ1. H NMR (CDCl3, 500 MHz): ␦ ϭ
3.211 (d, 3H, J ϭ 13.0 Hz, CH3), 7.699 to 7.840 (m, 15H,
arom-H).
2.10. 24-Methylenecholest-4-en-3␤,6␤-diol (1)
Of 80% NaH-paraffin (2.33 mmol of sodium hydride), 56
mg was placed in a two-neck flask. After the paraffin was
washed away with petroleum ether (30–60°C), 4 ml of anhy-
drous dimethyl sulfoxide were added to the dried NaH under
an argon atmosphere. The mixture was stirred at 80°C for 40
min, and a dark green solution was produced. After cooling to
room temperature, a solution of 350 mg (0.88 mmol) of meth-
yltriphenylphosphonium iodide (13) in 2 ml of anhydrous
dimethyl sulfoxide was added, and the solution turned yellow-
ish in color immediately. After 10 min, a solution of 28 mg
(0.067 mmol) of cholest-4-en-3␤,6␤-diol -24-one (11) in 2 ml
of anhydrous dimethyl sulfoxide was added, and the resulting
mixture was stirred at 80°C for 4 h. The reaction mixture was
cooled, diluted with 15 ml of cold water, and extracted with
ether. The combined organic extracts were washed with water
and then brine. After drying over anhydrous sodium sulfate,
the solvent was removed under reduced pressure, and the
resulting crude product was purified by flash chromatography
on silica gel using petroleum ether (b.p. 60–90°C)/acetone
(3:1) as eluent to give 18 mg (65%) of colorless needle crystals
1, m.p. 224–225°C. IR (KBr)␯: 3301, 1639, 1463, 1382, 1274,
3. Results and discussion
In the synthesis of steroids, the commercially available
stigmasterol is often used as a starting material because the
double bond in the side chain of stigmasterol can be easily
oxidized to an aldehyde by ozonization. The advantages of this
process are that various types of side chains can be constructed
via this aldehyde, and the configuration of C-17 and C-20 of
the natural steroids will be maintained [4–5]. Therefore, stig-
masterol was chosen as a starting material for the synthesis of
24-methylenecholest-4,5-ene-3␤,6␤-diol (1).
The planned synthetic route from stigmasterol (2) to the
target compound 24-methylenecholest-4-en-3␤,6␤-diol (1) is
shown in Scheme 1. The 3␤-hydroxy group of 2 was protected
by acetylation, then bromination of 3␤-acetoxystigmasterol (3)
was performed by using iodobenzene dibromide to give the
5,6-dibromide (4), which is pure enough for ozonolysis [6].
When the ozonide was decomposed with zinc and acetic acid
at room temperature, debromination of 4 was simultaneously
carried out to give compound (5). For the conversion of alde-
hyde 5 to ketone 6, a solid-liquid phase-transfer Wittig reaction
was used [7]. The reaction was performed at room temperature
using (3-methyl-2-oxo)butyltriphenylarsonium bromide (12)
as reagent and K2CO3 as base in CH3CN. The addition of a
trace of H2O or HCONH2 to the reaction mixture greatly
1
1158, 1089, 1029, 879, 836 cmϪ1; H NMR (CDCl3, 500
MHz): ␦ ϭ 0.715 (s, 3H, 18-CH3), 0.941 (d, 3H, J ϭ 6.5 Hz,
21-CH3), 1.017 (d, 3H, J ϭ 2.5 Hz, 26-CH3 or 27-CH3), 1.031
(d, 3H, J ϭ 2.5 Hz, 26-CH3 or 27-CH3), 1.259 (s, 3H, 19-CH3),
2.227 (m, 1H, 25-CH), 4.181 (ddd, 1H, J ϭ 9.0, 6.5 and 1.0
Hz, 3␣-CH), 4.231 (brt, 1H, J ϭ 2.5 Hz, 6␣-CH), 5.545 (brs,
1H, 4-CH).
accelerates this reaction. The coupling constant (3JH-H
ϭ
15.5Hz) at the 22, 23-double bond revealed that this reaction
was stereoselective and produced only the trans-isomer. Cata-
lytic hydrogenation of 6 gave nearly quantitative amounts of
compound (7).
2.11. (3-Methyl-2-oxo)butyltriphenylarsonium bromide
(12)
A solution of triphenylarsine (8.80 g, 29 mmol) and 1-bro-
mo-3-methyl-2-butanone (4.50 g, 27 mmol) in 3 ml of benzene
was stirred at about 65°C under an argon atmosphere for 10 h.
The white solid formed was filtered and washed with benzene.
The crude product was recrystallized from CHCl3 /EtOAc (2:
1) to give 7.61 g (60%) of colorless crystals 12, m.p. 182
The 3␤-acetoxycholest-5-en-24-one (7) was converted to
bromohydrin (8) with NBA containing a catalytic amount of
70% HClO4 in dioxane/water under dark conditions. Rode-
wald et al. studied the reaction of compound (14) with NBA
and showed that in addition to the major product bromohy-
drin (15), the reaction mixture contained the minor, more

JG. Cui et al. / Steroids 66 (2001) 33–38
37
Scheme 2.
mobile products (16), (17), and (18) (Scheme 2). The mech-
anism of this reaction was also discussed [8].
pose that in compound (20), the C6-OH trans to C5-Br might
accelerate the formation of 5,6-␤-epoxide (21). Therefore, the
C6-OH of 20 was protected by acetylation to give 23. In
compound (23), HBr can be eliminated by boiling in dimethy-
lacetamide (DMA) containing calcium carbonate to give 24 in
87% overall yield (Scheme 5). Referring to the reaction from
20 to 24, compound 10 was prepared from the bromohydrin 8
in 81% yield. The preparation of 1 directly from compound 10
was unsuccessful. Therefore, 10 was hydrolyzed to compound
11, methylenation of 11 was completed by Wittig reaction with
methylenetriphenylphosphonium iodide 13, and the target
compound 1 was obtained. The IR and 1H NMR spectra data
of 1 are completely consistent with those of the natural com-
pound 1. This synthesis unambiguously confirmed the struc-
The model compound (19) was used to study this reaction,
and 61% of bromohydrin (20) was obtained when the ratio of
19 (0.50 mmol): 70% HClO4 (0.01 ml): H2O (0.05 ml) was
used (Scheme 3). This showed that the amount of HClO4 had
a key effect on the reaction. If the acidity was either too strong
or too weak, it was unfavorable for the formation of 20. A
better reaction condition was obtained through the model re-
action. Using the same reaction condition as for compound
(19), bromohydrin (8) was obtained in 76% yield from 7.
The elimination of HBr from 20 to 22 using a method from
the literature [9–10] yield compound (21) as the major prod-
uct, but not the desired compound (22) (Scheme 4). We sup-
Scheme 3.
Scheme 4. a. Heated at 105°C for 30 min in glacial acetic acid containing the catalytic amount of HCl. b. Heated under reflux for 9 h in dry pyridine.

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JG. Cui et al. / Steroids 66 (2001) 33–38
Scheme 5.
[5] David MP, Jerzy W. Various approaches to the construction of
aliphatic side chains of steroids and related compounds. Chem Rev
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ture of the natural product 1 as 24-methylenecholest-4-en-
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