Epoxidation and reduction of cholesterol, 1,4,6-cholestatrien-3-one and 4,6-cholestadien-3β-ol

Article abstract of DOI:10.1016/j.steroids.2004.11.003

Many naturally occurring polyhydroxylated sterols and oxysterols exhibit potent biologic activities. This paper describes reagent and position selectivity of epoxidation and reduction of cholesterol derivatives. Cholesterol was reacted with m-chloroperoxybenzoic acid (m-CPBA) to form 5α,6α-epoxycholestan-3β-ol, but in reaction with 30% H ₂O₂, it did not reacted. 1,4,6-Cholestatrien-3-one was obtained from cholesterol and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. 1,4,6-Cholestatrien-3-one was reacted with 30% H₂O ₂ and 5% NaOH in methanol to give 1α,2α-epoxy-4,6- cholestadien-3-one, which was stereoselectively reduced with NaBH₄ to form 1α,2α-epoxy-4,6-cholestadien-3β-ol and reduced with Li metal in absolute ethanol to give 2-ethoxy-1,4,6-cholestatrien-3-one. And 1,4,6-cholestatrien-3-one was epoxidized with m-CPBA in dichloromethane to afford 6α,7α-epoxy-1,4-cholestadien-3-one, which was reacted with NaBH₄ to synthesize 6α-hydroxy-4-cholesten-3-one and reduced Li metal in absolute ethanol to form 2-ethoxy-1,4,6-cholestatrien-3-one, respectively. 1,4,6-Cholestatrien-3-one was reduced with NaBH₄ in absolute ethanol to form 4,6-cholestadien-3β-ol, which was reacted with 30% H₂O₂ to leave original compound, but was reacted with m-CPBA to give 4β,5β-epoxy-6-cholesten-3β-ol as the major product and 4β,5β-epoxy-6α,7α-epoxycholestan-3β-ol as the minor product.

Full text of DOI:10.1016/j.steroids.2004.11.003

Steroids 70 (2005) 245–250
Epoxidation and reduction of cholesterol, 1,4,6-cholestatrien-3-one
and 4,6-cholestadien-3␤-ol
∗
Eunsook Ma , Haksoon Kim, Eunjeong Kim
College of Pharmacy, Catholic University of Daegu, 330 Geumrak 1-ri, Hayang-eup, Gyongsan-si Gyongbook 712-702, Korea
Received 25 May 2004; received in revised form 11 November 2004; accepted 21 November 2004
Abstract
Manynaturallyoccurringpolyhydroxylatedsterolsandoxysterolsexhibitpotentbiologicactivities. Thispaperdescribesreagentandposition
selectivity of epoxidation and reduction of cholesterol derivatives. Cholesterol was reacted with m-chloroperoxybenzoic acid (m-CPBA) to
form 5␣,6␣-epoxycholestan-3␤-ol, but in reaction with 30% H
and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in dioxane. 1,4,6-Cholestatrien-3-one was reacted with 30% H
to give 1␣,2␣-epoxy-4,6-cholestadien-3-one, which was stereoselectively reduced with NaBH to form 1␣,2␣-epoxy-4,6-cholestadien-3␤-ol
and reduced with Li metal in absolute ethanol to give 2-ethoxy-1,4,6-cholestatrien-3-one. And 1,4,6-cholestatrien-3-one was epoxidized with
m-CPBA in dichloromethane to afford 6␣,7␣-epoxy-1,4-cholestadien-3-one, which was reacted with NaBH to synthesize 6␣-hydroxy-4-
cholesten-3-one and reduced Li metal in absolute ethanol to form 2-ethoxy-1,4,6-cholestatrien-3-one, respectively. 1,4,6-Cholestatrien-3-one
was reduced with NaBH in absolute ethanol to form 4,6-cholestadien-3␤-ol, which was reacted with 30% H to leave original compound,
2
O
2
, it did not reacted. 1,4,6-Cholestatrien-3-one was obtained from cholesterol
2
O
2
and 5% NaOH in methanol
4
4
4
2 2
O
but was reacted with m-CPBA to give 4␤,5␤-epoxy-6-cholesten-3␤-ol as the major product and 4␤,5␤-epoxy-6␣,7␣-epoxycholestan-3␤-ol
as the minor product.
©
2005 Published by Elsevier Inc.
Keywords: Stereoselective epoxidation; m-CPBA; H2O2; Cholesterol; 1,4,6-Cholestatrien-3-one; 4,6-Cholestadien-3␤-ol
1
. Introduction
OH-cholesterol and 7␣-OH-cholesterol, 7-ketocholesterol,
lowered the levels of 3-hydroxy-3-methylglutaryl coenzyme
A (HMG-CoA) reductase activity in mouse L cells and in
primary cultures of fecal mouse liver cells [9]. 25-OH-,
26-OH-, and 24,25(S)-epoxycholesterols are representa-
tive oxysterols in HMG-CoA reductase regression assays
[10–14].
Many naturally occurring polyhydroxylated sterols and
oxysterols exhibit potent biologic activities [1–3]. Some
polyhydroxylated sterols showed potent cytotoxicity to can-
cer cells [4–7]. 7␤-Hydroxycholesterol has been shown to be
more toxic towards tumorous than non-tumorous cells [8].
2
4-Methylenecholest-4-en-3␤,6␤-diol was obtainedfrom the
Epoxides are among the most frequently occurring func-
tional groups found in natural products and their synthetic
analogues. Not only are these compounds easily prepared
from a variety of starting materials, but the inherent polarity
and strain of their three-membered ring makes them suscepti-
ble to reaction with a large number of reagents-electrophiles,
nucleophiles, acid, bases, reducing agents, and oxidizing
agents [15,16].
soft coral Alcyonium patagonicum and showed potent cy-
totoxicity to murine leukemia cells with an IC50 value of
1
ug/mL [5].
Furthermore, oxysterols are very important factors in
atherogenesis [1]. In 1973, Kandutsch and Chen reported
the three 7-oxygenated derivatives of cholesterol, i.e., 7␤-
∗
In order to study the reagent selectivity and position
selectivity of the epoxidizing agents for the synthesis of
Corresponding author. Tel.: +82 53 8503621; fax: +82 53 8503602.
E-mail address: masook@cu.ac.kr (E. Ma).
0
039-128X/$ – see front matter © 2005 Published by Elsevier Inc.
doi:10.1016/j.steroids.2004.11.003

2
46
E. Ma et al. / Steroids 70 (2005) 245–250
◦
−1
:
epoxycholesterol derivatives, 30% H2O2 and m-chloro-
peroxybenzoic acid were used as simple epoxidizing agents
without the presence of catalyst. NaBH4 and Li metal in
absolute ethanol were taken as reducing agents, respectively.
Yield: 3.2 g (65%), mp: 89.5–91.5 C, IR (KBr) cm
1
2
923, 2850, 1646, 1458, 1373 and 1281, H NMR (CDCl3)
δ: 0.81 (3H, s, H-18), 0.87 (3H, s, H-26), 0.90 (3H, s, H-27),
.93 (3H, d, J = 6.8 Hz, H-21), 1.21 (1H, s, H-19), 6.06 (2H,
d, J = 14.0 Hz, H-6, H-7), 6.19–6.30 (2H, m, H-2, H-4), 7.08
1H, d, J = 10.0 Hz, H-1), 13C NMR (CDCl3) δ: 186.4, 162.8,
153.1, 138.8, 128.1, 127.6, 123.7, 56.1, 53.7, 48.5, 43.1, 41.4,
9.6, 38.4, 36.2, 35.9, 28.3, 28.2, 24.0, 23.9, 23.0, 22.9, 22.0,
0
(
2
. Experimental
3
+
2
.1. General methods
16.8, 12.2, FABMS (M + H) : 381.2.
All non-aqueous reactions were performed under a dry
2.4. 1α,2α-Epoxy-4,6-choestadien-3-one (3)
atmosphere of nitrogen. The commercial reagents were pur-
chased from Aldrich, Fluka, or Sigma. Solvents were puri-
fied and dried prior to use. Melting points were measured on
Thomas–Hoover melting point apparatus in open capillary
According to a procedure by Liu et al. [19], to a solution of
the trienone 2 (500 mg, 1.32 mmol) in methanol 60 mL was
added a mixture of 5% NaOH–MeOH 0.6 mL and 30% H2O2
6 mL. The reaction mixture was stirred at room temperature
for 22 h, at which time TLC indicated reaction completion,
and then extracted with dichloromethane. The organic layers
were combined and were washed with brine, and H2O, dried
over anhydrous MgSO4, filtered, and concentrated to leave
crude solid. Column chromatography on silica gel, eluting
ethyl acetate/n-hexane (1:5), gave the pure white crystals of
epoxide.
1
13
tubes and were not corrected. H and C NMR spectra were
obtained on Gemini 200 or 50 MHz spectrometer in CDCl3,
chemical shifts δ in ppm are relative to tetramethylsilan, cou-
pling constants J in Hz. NOESY spectra were recorded using
a Bruker AM-500 MHz spectrometer. IR spectra were deter-
mined on a Jasco FT-IR 300E spectrometer as KBr pellet.
Low FAB mass spectra were obtained on a Tandem mass
spectrometer. An analytical TLC was performed on Merck
precoated silica gel 60 F254 plates. Solvent systems for TLC
were ethyl acetate/n-hexane mixtures. Column chromatog-
raphy was carried out on Merck silica gel 9385 (230–400
mesh).
◦
Yield: 444 mg (85%), mp: 109.3–110.9 C, IR (KBr)
cm 1: 2942, 2864, 1670, 1617, 1462, 1377, 1284, H NMR
−
1
(
CDCl3) δ: 0.87 (3H, s, H-18), 0.87 (3H, s, H-26), 0.90 (3H,
s, H-27), 0.95 (3H, d, J = 6.4 Hz, H-21), 1.12 (3H, s, H-19),
.42-3.50 (1H, m, H-1), 3.60 (1H, d, J = 4.0 Hz, H-2), 5.66
3
1
3
(1H, d, J = 1.2 Hz, H-4), 6.10 (2H, s, H-6, H-7), C NMR
CDCl3) δ: 194.8, 159.0, 140.8, 127.8, 119.5, 59.6, 56.1, 54.8,
2
.2. 5α,6α-Epoxy-cholestan-3β-ol (1)
(
5
2
3
3.4, 46.1, 43.1, 39.6, 39.4, 39.0, 37.7, 36.2, 29.8, 28.2, 28.1,
4.0, 23.9, 23.0, 22.7, 21.3, 18.8, 18.7, FABMS (M + H) :
97.1.
To a solution of cholest-5-en-3␤-ol (cholesterol, 1 g,
.59 mmol) in CHCl3100 mL, m-chloroperoxybenzoic acid
483 mg, 2.80 mmol) was added at room temperature. The
+
2
(
mixture was stirred at room temperature for 18 h under ni-
trogen gas. The solution was diluted with CHCl3 and treated
with aqueous sodium sulfite, and then the aqueous layer was
extracted with several portions of CHCl3 and the organic ex-
tracts were combined. The organic extracts were washed with
water and dried with anhydrous MgSO4, filtered, and evap-
orated to leave a crude solid which was recrystallized from
ethyl acetate and n-hexane (1:5) to afford white crystals.
2
.5. 6α,7α-Epoxy-1,4-cholestadien-3-one (4)
m-Chloroperoxybenzoic acid (490 mg, 2.84 mmol) was
added to a solution of trienone 2 (1 g, 2.63 mmol) in CHCl3
00 mL at room temperature. The reaction mixture was
1
stirredatthesametemperatureuntilnostartingmaterialcould
be detected by TLC. The mixture was stirred with H2O, and
then extracted with dichloromethane. The extract was dried
over anhydrous MgSO4, filtered, and evaporated to obtain
pale yellow precipitate. Column chromatography on silica
gel, eluting with ethyl acetate/n-hexane (1:5) gave the pure
white crystals of epoxide.
◦
◦
Yield: 947 mg (91%), mp: 145–146 C. (mp: 142–144 C,
Ref. [17]).
2
.3. 1,4,6-Cholestatrien-3-one (2)
◦
−1
Yield: 376 mg (72%), mp: 127–129 C, IR (KBr) cm
:
1
2
0
925, 1665, 1455, H NMR (CDCl3) δ: 0.78 (3H, s, H-18),
.87 (3H, s, H-26), 0.90 (3H, s, H-27), 0.93 (1H, d, J = 6.6 Hz,
A solution of the cholesterol (5 g, 12.95 mmol) and 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (10 g, 88 mmol) in
dioxane 125 mL was refluxed for 24 h under nitrogen accord-
ingtotheproceduresreportedbyFurstetal. [18]. Thereaction
mixture was cooled down, and the resulting precipitate was
filtered off and washed with dichloromethane. The filterate
was rotary evaporated to dryness and the dark brown residue
was purified by column chromatography on silica gel (ethyl
acetate/n-hexane, 1:7) to afford white crystals.
H-21), 1.22 (3H, s, H-19), 3.65–3.80 (1H, m, H-7), 3.64 (1H,
d, J = 3.6 Hz, H-6), 6.23 (1H, dd, J = 1.8 Hz, 10.0 Hz, H-4),
6
.48 (1H, d, J = 1.6 Hz, H-2), 7.03 (1H, d, J = 10.4 Hz, H-1),
C NMR (CDCl3) δ: 185.4, 160.2, 153.6, 131.1, 127.8, 55.9,
4.1, 51.8, 43.1, 41.3, 39.6, 39.4, 38.3, 36.2, 35.9, 35.4, 28.4,
13
5
2
8.2, 24.0, 23.7, 23.0, 22.7, 22.1, 20.8, 18.8, 12.1, FABMS
+
(
M + H) : 397.1.

E. Ma et al. / Steroids 70 (2005) 245–250
247
2
.6. 4,6-Cholestadien-3β-ol (5)
trated by rotary evaporation to afford white solid. The resid-
ual precipitate was stirred with water and d-HCl solution
for 30 min, and then extracted with dichloromethane from
aqueous layer. The organic layer was washed with water and
dried with MgSO4, filtered, and concentrated to give white
solid, whichwas purifiedwithcolumnchromatography(ethyl
acetate/n-hexane, 1:5) on silica gel.
To a solution of compound 2 (1 g, 2.63 mmol) in absolute
ethanol 100 mL, NaBH4 (370 mg, 9.8 mmol)was addeddrop-
wise at room temperature. The resulting mixture was stirred
overnight at same temperature, and then was concentrated by
rotary evaporation to afford white solid. It was stirred with
water for 30 min, and then extracted with dichloromethane
from aqueous layer. The organic layer was washed with aque-
ous sodium hydrogen carbonate, water and dried with anhy-
drous MgSO4, filtered, and concentrated to give a residue,
which was recrystallized from ethyl acetate and n-hexane
◦
Yield: 286 mg (57%), mp: 103.5–106.5 C, IR (KBr)
cm 1: 3410, 2924, 2874, 1461, H NMR (CDCl3) δ: 0.74
(3H, s, H-18), 0.85 (3H, s, H-26), 0.88 (3H, s, H-27), 0.92
(3H, d, J = 6.0 Hz, H-21), 1.14 (3H, s, H-19), 3.22 (1H, d,
J = 6.6 Hz, H-1), 3.34 (1H, t, H-2), 4.58 (1H, d, J = 5.2 Hz,
H-3), 5.39 (1H, d, J = 5.8 Hz, H-4), 5.70 (1H, d, J = 2.4 Hz,
−
1
(1:3) to afford white crystals.
◦
13
Yield: 933 mg (92%), mp: 119.8–121.5 C, IR (KBr)
H-6), 5.91 (1H, d, J = 9.8 Hz, H-7), C NMR (CDCl3) δ:
cm 1: 3400, 2935, 2865, 1464, 1378, H NMR (CDCl3) δ:
0
0
−
1
142.4, 133.2, 128.0, 118.7, 64.2, 57.0, 56.1, 54.1. 53.8, 45.8,
43.3, 39.7, 36.9, 36.3, 36.0, 28.4, 28.2, 24.1, 23.0, 22.8, 22.5,
.73 (3H, s, H-18), 0.85 (3H, s, H-26), 0.89 (3H, s, H-27),
.92 (3H, d, J = 6.0 Hz, H-21), 0.99 (3H, s, H-19), 4.10–4.15
+
21.0, 19.0, 18.9, 18.6, 12.1, FABMS (M + H) : 381.1.
(
1H, m, H-3), 5.27 (1H, s, H-4), 5.60 (1H, d, J = 8.0 Hz, H-6),
5
1
3
2
3
.95 (1H, dd, J = 2.4 Hz, 8.2 Hz, H-7), 13C NMR (CDCl3) δ:
44.8, 131.5, 128.1, 126.2, 68.0, 56.3, 54.2, 51.5, 43.6, 40.0,
9.7, 37.5, 36.3, 36.0, 35.2, 33.8, 29.1, 28.4, 28.2, 24.1, 24.0,
2.9. 2-Ethoxy-1,4,6-cholestatrien-3-one (8)
To a solution of 3 (500 mg, 1.26 mmol) in absolute ethanol
50 mL, lithium metal 300 mg was added slowly and vigor-
ously stirred at the room temperature for 18 h. The reaction
+
3.0, 22.7, 20.9, 18.8, 18.2, 12.2, FABMS (M + H H2O) :
67.2.
◦
mixture was cooled down to 0 C and slowly added with
2
4
.7. 4β,5β-Epoxy-6-cholesten-3β-ol (6) and
β,5β-epoxy-6α,7α-epoxycholestan-3β-ol (6a)
water and stirred to clear solution. The mixture was evap-
orated to remove ethanol, and then the aqueous layer was
extracted with ethyl acetate for several times. The combined
organic layer was washed with H2O and dried with anhy-
drous MgSO4, filtered and evaporated to crude oils, which
was purified with column chromatography on silica gel (ethyl
acetate/n-hexane, 1:5).
m-Chloroperoxybenzoic acid (260 mg, 1.51 mmol) was
added to a solution of dienol 5 (500 mg, 1.30 mmol) in
CHCl3100 mL at room temperature and then treated in a man-
ner similar to synthesis of compound 1. The pure compound
Yield: 331 mg (62%), IR (KBr) cm 1: 2918. 2851, 1646,
−
6
as a major product and 6a as a minor product were obtained
1
white crystals by column chromatography (ethyl acetate/n-
1454, H NMR (CDCl3) δ: 0.79 (3H, s, H-18), 0.85 (3H, s,
hexane, 1:3) on silica gel, respectively.
H-26), 0.88 (3H, s, H-27), 0.92 (3H, d, J = 6.4 Hz, H-21),
1.20 (3H, s, H-19), 1.43(3H, t, OCH2CH3), 3.82–3.86 (2H,
m, OCH2CH3), 5.94 (1H, s, H-4), 6.06 (2H, d, J = 5.2 Hz,
H-6, H-7), 6.12 (1H, dd, J = 2.8 Hz, 6.8 Hz, H-1), 13C NMR
(CDCl3) δ: 181.5, 163.1, 150.1, 139.3, 127.0, 123.4, 121.1,
63.3, 56.0, 53.8, 49.5. 43.1, 41.4, 39.7, 36.9, 36.3, 36.2, 35.9,
28.3, 28.2, 24.0, 23.8, 23.0, 22.7, 22.5, 22.1, 18.8, 15.5, 12.2,
◦
6
; Yield: 312 mg (48 %), mp: 136.0–138.1 C, IR (KBr)
cm : 3439, 2927, 2857, 1642, 1457, 1249, 1H NMR
−
1
(
CDCl3) δ: 0.76 (3H, s, H-18), 0.85 (3H, s, H-26), 0.86 (3H,
s, H-27), 0.94 (3H, d, J = 6.0 Hz, H-21), 1.34 (3H, s, H-19),
.58–3.64 (1H, m, H-3), 4.16 (1H, d, J = 2.6 Hz, H-4), 5.30
H, d, J = 8.6 Hz, H-6), 5.64 (1H, s, H-7), 13C NMR (CDCl3)
δ: 127.4, 125.5, 80.3, 71.5, 68.8, 68.1, 50.1, 42.7, 39.8, 39.4,
3
(
+
FABMS (M + H) : 425.2
3
2
7.1, 36.8, 35.8, 35.0, 34.6, 29.5, 28.9, 28.5, 25.1, 24.5, 22.8,
+
2.5, 20.1, 18.0, 17.8, 13.1, FABMS (M + H H2O) : 383.2.
2.10. 6α-Hydroxy-cholest-4-en-3-one (9)
◦
−1
6
a; Yield: 72 mg (12%), mp: 109–110 C, IR (KBr) cm
:
1
3
0
425, 2953, 1699, 1418, 1305, 1263, H NMR (CDCl3) δ:
To a solution of 4 (500 mg, 1.26 mmol) in absolute ethanol
100 mL, NaBH4 (177 mg, 4.70 mmol) was added and stirred
at room temperature until the starting material was disap-
peared in TLC test. The reaction mixture was evaporated
to dry solid and the residue was added to H2O and stirred.
The aqueous layer was extracted with ethyl acetate, and the
organic layer was dried with anhydrous MgSO4, filtered,
concentrated to crude precipitate. The purified product was
obtained from column chromatography on silica gel (ethyl
acetate/n-hexane, 1:3).
.72 (3H, s, H-18), 0.85 (6H, s, H-26, H-27), 0.88 (3H, s,
H-21), 0.94 (3H, s, H-19), 2.65 (1H, d, J = 4.0 Hz, H-6), 3.20
1H, d, J = 4.0 Hz, H-7), 3.42 (1H, s, H-4), 3.92–4.08 (1H, m,
(
+
H-3), FABMS (M + H) : 399.2.
2
.8. 1α,2α-Epoxy-4,6-cholestadien-3β-ol (7)
To a solution of compound 3 (500 mg, 1.26 mmol) in abso-
lute ethanol 100 mL, NaBH4 (177 mg, 4.70 mmol) was added
dropwise at room temperature. The resulting mixture was
stirred overnight at same temperature, and then was concen-
◦
−1
Yield: 258 mg (52 %), mp: 84–87 C, IR (KBr) cm
:
1
3422, 2916, 2845, 1657, 1447, H NMR (CDCl3) δ: 0.75

2
48
E. Ma et al. / Steroids 70 (2005) 245–250
(
(
(
5
3
1
3H, s, H-18), 0.91 (3H, s, H-26), 0.93 (3H, s, H-27), 1.05
3H, s, H-21), 1.29 (3H, s, H-19), 4.00 (1H, m, H-6), 5.84
1H, s, H-4), 13C NMR (CDCl3) δ: 183.5, 155.3, 126.9, 68.6,
solution at room temperature, the only 1,2-double bond of
2 was epoxidized to form 1␣,2␣-epoxy-4,6-cholestadien-3-
one (3), position selectively. The stereochemistry of 3 was
identified with NOESY spectrum. The cross peak of H-
1 (3.42–3.50 ppm) and H-19 (1.12 ppm) was observed in
NOESY spectrum, it means that the relationship of C-1 hy-
drogen (H-1) and C-10 methyl hydrogen (H-19) is cis config-
uration. Therefore, H-1 was identified as ␤ configuration and
6.1, 50.6, 45.4, 42.6, 41.0, 39.9, 39.6, 39.3, 38.6, 36.2, 35.9,
5.6, 34.1, 29.8, 28.2, 23.9, 23.8, 23.0, 22.7, 21.0, 18.8, 17.2,
+
2.0, FABMS (M + H) : 401.2
2
.11. 7β-Ethoxy-6α-hydroxy-1,4-cholesadien-3-one (10)
1,2-epoxide was identified as ␣ configuration. 2 was epox-
To a solution of 4 (500 mg, 1.26 mmol) in absolute ethanol
0 mL, lithium metal 300 mg was added slowly and vigor-
idized with m-chloroperoxybenzoic acid in chloroform at
room temperature, the 6,7-double bond was epoxidized to
form 6␣,7␣-epoxy-1,4-spirostadien-3-one (4), stereoselec-
tively. Initial attack by a peroxy acid should to be directed
to a pseudo-axial site on the face opposite to the C-10 methyl
group to form ␣-epoxide. Allylic interaction between the ad-
jacent double bond and the intermediate in the epoxidation
might favour a particular site of attack by stabilizing an ad-
jacent carbocationic intermediate [22].
5
ously stirred at the room temperature, until the starting mate-
rial was disappeared in TLC test. The following procedures
were treated with the same synthetic methods of compound 8.
The purified product was obtained as white crystals from col-
umn chromatography on silica gel (ethyl acetate/n-hexane,
1
:3).
◦
−1
:
Yield: 357 mg (64%), mp: 160–161 C, IR (KBr) cm
3
0
2
383, 2954, 1663, 1617, 1460, 1H NMR (CDCl3) δ:
.78 (3H, s, H-18), 0.85 (3H, s, H-26), 0.88 (3H, s, H-
7), 0.89 (3H, s, H-21), 1.17 (3H, s, H-19), 1.19 (3H,
Compound 2 was reduced with NaBH4 in absolute ethanol
to form 4,6-cholestadien-3␤-ol (5) as 1,2- and 1,4 addi-
tion product, stereoselectively [23]. The attack of hydride
anion to the axial direction of the carbonyl group pro-
duced the 3␤-hydroxy configuration. IR spectrum showed
t, OCH2CH3), 3.38–3.52 (2H, m, H-6, H-7), 3.86–3.92
(
2H, q, OCH2CH3), 6.21 (1H, m, H-1), 6.25 (1H, d,
13
−1
1
J = 1.4 Hz, H-4), 7.09 (1H, dd, J = 1.6 Hz, 11.2 Hz, H-2),
C
band at 3400 cm corresponding to OH group. H NMR
spectrum of 5 showed peaks at 4.10–4.15 ppm (H-3), and
5.27 ppm (H-4), 5.60 ppm (H-6) and 5.95 ppm (H-7) as dou-
ble bond hydrogens, respectively. 13C NMR spectrum was
assigned four double bond carbons at 144.8, 131.5, 128.1
and 126.2 ppm. The FABMS spectrum indicated a proto-
nated ion [M + H H2O] at m/z 367.2 consistent with the loss
of H2O. The stereochemistry of compound 5 was identified
with NOESY spectrum. The cross peak between hydrogens
of C-10 methyl group (H-19) on the ␤-face and H-3 was not
observed in NOESY spectrum. H-3 and C-10 angular methyl
group is positioned trans form, therefore OH group on C-3
is directed ␤-position. Compound 5 was reacted with 30%
H2O2 and 5% NaOH in methanol, but it also did not reacted.
And 5 was reacted with m-chloroperoxybenzoic acid to give
4␤,5␤-epoxy-cholest-6-en-3␤-ol(6)asthemajorproductand
4␤,5␤-epoxy-6␣,7␣-epoxycholestan-3␤-ol (6a) as the minor
NMR (CDCl3) δ: 186.0, 162.3, 157.8, 130.4, 126.7, 85.3,
7
3
1
2.6, 64.5, 56.1, 49.9, 43.5, 42.8, 39.6, 39.3, 36.3, 36.0,
5.5, 28.3, 28.2, 23.9, 23.0, 22.8, 22.5, 19.2, 18.8, 15.2,
+
+
2.1, FABMS (M + H) : 443.2, (M + H C2H OH) : 397.2,
5
+
(
M + H C2H OH H2O) : 379.2.
5
3
. Results and discussion
In the synthesis of cholesterol derivatives, the commer-
cially available cholesterol was used as a starting material.
The planned synthetic route from cholesterol was shown in
Scheme 1. First, cholesterol was reacted with 30% H2O2
and 5% NaOH in MeOH solution at room temperature, but
it did not react. 5␣,6␣-Epoxycholestan-3␤-ol (1) was al-
®
ready synthesized by the method of oxone -mediated epox-
−
1
idation [20]. In order to investigate the simple epoxidzing
method, cholesterol was treated with m-chloroperoxybenzoic
acid to give compound 1 in good yield, which was formed
by ␣-face attack of peroxy acid to the 5,6-double bond.
product. We identified OH band at 3439 cm in IR spec-
1
trum of 6. H NMR data for 6 showed evidence that a dou-
blet of epoxy hydrogen at 4.16 ppm had appeared instead
of singlet at 5.27 ppm (H-4) of 5 and double bond hydro-
1
3
[
21].
gens at 5.30 (H-6) and 5.64 ppm (H-7) were identified.
C
In order to synthesize various epoxycholesteol and hy-
droxycholesterol derivatives, 1,4,6-cholestatrien-3-one (2)
was synthesized from cholesterol and 2,3-dichloro-5,6-
NMR spectrum showed two double bond carbon at 127.4 and
125.5 ppm, respectively. FABMS spectrum showed a proto-
nated molecular ion [M + H H2O] at m/z 383.2 consistent
with the loss of H2O. In first epoxidation, the presence of the
3␤-hydroxy group of the allylic alcohol directed the epox-
idation exclusively to the ␤-face of the double bond of the
molecule [24,25]. Once the initial epoxide has been formed,
the stereochemistry of the second epoxidation may be deter-
mined by a repulsive interaction between the non-bonding
electrons of the epoxide oxygen and the ␲-electrons of the
alkene. This would lead to a higher electron density on the
−
1
dicyano-1,4-benzoquinone. IR spectrum band at 1646 cm
1
indicates the presence of a carbonyl group. In the H NMR
spectrum, signals at 6.06, 6.19–6.30 ppm, and 7.08 ppm ap-
pearsixhydrogensofdoublebond. Inthe13CNMRspectrum,
signal at 186.4 ppm is corresponded to carbonyl carbon, and
signals at 162.8, 153.1, 138.8, 128.1, 127.6 and 123.7 ppm
were showed the six double bond carbons. Compound 2
was epoxidized with 30% H2O2 and 5% NaOH in MeOH

E. Ma et al. / Steroids 70 (2005) 245–250
249
Scheme 1. Synthesis of cholesterol derivatives. (a) H2O2, 5%NaOH/MeOH, MeOH, rt; (b) m-CPBA, CHCl3, rt; (c) DDQ, dioxane, reflux; (d) NaBH4, absolute
ethanol, rt and (e) Li, absolute ethanol, rt.
face of the alkene, that is trans to the first epoxide. Hence at-
(H-4), 6.06 ppm (H-6, H-7) and 6.12 ppm (H-1), and methyl
and methylene hydrogens of ethoxy group were observed at
1.43 and 3.82–3.86 ppm, respectively. The six double bond
carbons were identified at 163.1, 150.1, 139.3, 127.0, 123.4
and 121.1 ppm in 13C NMR spectrum. Compound 4 was re-
acted with NaBH4 to afford 6␣-hydroxy-4-cholesten-3-one
(9). This compound was obtained by 1,4-addition of hydride
to ␣,␤-unsaturated carbonyl group and by ␤-face attack of
hydride to 7 position of 6␣,7␣-epoxide ring. In IR spec-
trum, bands of OH group and carbonyl group were observed
tack on the second double bond by the peroxy acid might be
1
directed to take place trans to the first epoxide [22]. H NMR
spectrum of 6a showed two doublet at 2.65 and 3.20 ppm as
H-6, H-7 epoxy hydrogens and singlet at 3.42 ppm as H-4, re-
spectively. FABMS spectrum showed a protonated molecular
ion [M + H] at m/z 399.2.
Compound3and4werereducedwithNaBH4 andLimetal
in absolute ethanol, respectively. 3 was reacted with NaBH4
to form 1␣,2␣-epoxy-4,6-cholestadien-3␤-ol (7). NaBH4 se-
lectively attack the carbonyl group of the compound 3 to
−
1
−1
1
at 3422 cm and 1657 cm , respectively. H NMR spec-
trum showed one double bond hydrogen at 5.84 ppm and
−
1
give 3␤-ol. We observed broadband at 3410 cm as OH
1
13
group in IR spectrum, and H NMR spectrum showed dou-
C NMR spectrum showed two double bond carbons at
blet at 4.58 ppm (H-3). 13C NMR spectrum showed four dou-
ble bond carbons at 142.4, 133.2, 128.0 and 118.7 ppm, but
the carbonyl carbon at 194.8 ppm was not observed. And 3
was also reacted with Li metal in absolute ethanol to form
155.3 and 126.9 ppm, respectively. 7␤-Ethoxy-6␣-hydroxy-
1,4-cholestadien-3-one (10) was obtained from the reaction
of 4 with Li metal in absolute ethanol at room temperature.
The ␤-face attack of ethoxide ion to the 7-position of epoxide
1
2
-ethoxy-1,4,6-cholestatriene-3-one (8). In this case, ethox-
might be directedto take place cleavage of epoxide ring. In H
ide anion, formed from Li metal and absolute ethanol, at-
tacked the 2-position of 1␣,2␣-epoxide to form 2␤-ethoxy-
NMR spectrum of 10, three double bond hydrogens were ob-
served at 6.21 ppm (H-1), 6.25 ppm (H-4) and 7.09 ppm (H-2)
and in 13C NMR spectrum, four double bond carbons were
identified at 162.3, 157.8, 130.4 and 126.7 ppm. FABMS
spectrum showed a protonated molecular ion [M + H] at m/z
1
␣-hydroxy-4,6-cholestadien-3-one at first, and then H2O
1
was eliminated to give compound 8. In H NMR spectrum of
, four hydrogens of double bond were observed at 5.94 ppm
8

2
50
E. Ma et al. / Steroids 70 (2005) 245–250
4
43.2 and [M + H C2H OH] at m/z 397.2 consistent with the
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