CH3ReO3-catalyzed oxidation of cholesta-5,7-dien-3β-yl acetate with the urea-hydrogen peroxide adduct under various conditions. Synthesis of the natural epoxy sterol 9α,11α-epoxy-5α-cholest-7-en-3β,5,6β-triol

Article abstract of DOI:10.1016/S0039-128X(02)00017-X

This article describes the oxidation of cholesta-5,7-dien-3β-yl acetate (4) with the urea-hydrogen peroxide adduct (UHP) using methyltrioxorhenium (MTO) as catalyst, under various conditions. Specifically, the effects of using different solvents (CHCl₃ and ethers) and additives (EtOH and pyridine) on the course of the MTO-catalyzed oxidation of 4 were investigated. Some new steroids (6, 9, 10 and 11), obtained from this oxidation, were isolated and characterized on the basis of chemical evidence and interpretation of spectroscopic data including H-H COSY and HMBC experiments. The optimal solvent for the oxidation of 4 with MTO/UHP oxidizing system was diethyl ether. In this solvent the reaction is clean and gave as the main product 5,6β-dihydroxy-5α-cholest-7-en-3β-yl acetate (8, 65% yield), obtained with a more simple procedure and with a higher yield than that reported in literature. Sterol 8 is a key intermediate compound in the synthesis of many steroids of marine origin, biologically active, oxygenated at the B/C rings. In fact, starting from diol 8, we performed the synthesis of the natural cytotoxic epoxy sterol 9α,11α-epoxy-5α-cholest-7-en-3β,5,6β-triol (15, 21% yield) with an improvement in yield and number of steps over a synthesis of the same natural product previously reported. When the oxidation of 4 with the MTO/UHP system in diethyl ether was performed in the presence of pyridine as ligand, the unsaturated epoxide 5,6α-epoxy-5α-cholest-7-en-3β-yl acetate (10, 90% yield) was obtained after only 5 min in good yield. In fact, pyridine, besides having beneficial effect on the reaction rate, shuts down the ring opening reactions, as reported in literature.

Full text of DOI:10.1016/S0039-128X(02)00017-X

Steroids 67 (2002) 661–668
CH3ReO3-catalyzed oxidation of cholesta-5,7-dien-3␤-yl acetate with
the urea–hydrogen peroxide adduct under various conditions.
Synthesis of the natural epoxy sterol 9␣,11␣-epoxy-5␣-cholest-7-en-
3␤,5,6␤-triol
Domenica Musumeci, Donato Sica*
Dipartimento di Chimica Organica e Biochimica, Universita` degli Studi di Napoli “Federico II,” Complesso Universitario Monte Sant’Angelo,
Via Cintia, I-80126 Napoli, Italy
Received 15 October 2001; received in revised form 7 January 2002; accepted 17 January 2002
Abstract
This article describes the oxidation of cholesta-5,7-dien-3␤-yl acetate (4) with the urea–hydrogen peroxide adduct (UHP) using
methyltrioxorhenium (MTO) as catalyst, under various conditions. Specifically, the effects of using different solvents (CHCl3 and ethers)
and additives (EtOH and pyridine) on the course of the MTO-catalyzed oxidation of 4 were investigated. Some new steroids (6, 9, 10 and
11), obtained from this oxidation, were isolated and characterized on the basis of chemical evidence and interpretation of spectroscopic data
including H–H COSY and HMBC experiments. The optimal solvent for the oxidation of 4 with MTO/UHP oxidizing system was diethyl
ether. In this solvent the reaction is clean and gave as the main product 5,6␤-dihydroxy-5␣-cholest-7-en-3␤-yl acetate (8, 65% yield),
obtained with a more simple procedure and with a higher yield than that reported in literature. Sterol 8 is a key intermediate compound in
the synthesis of many steroids of marine origin, biologically active, oxygenated at the B/C rings. In fact, starting from diol 8, we performed
the synthesis of the natural cytotoxic epoxy sterol 9␣,11␣-epoxy-5␣-cholest-7-en-3␤,5,6␤-triol (15, 21% yield) with an improvement in
yield and number of steps over a synthesis of the same natural product previously reported. When the oxidation of 4 with the MTO/UHP
system in diethyl ether was performed in the presence of pyridine as ligand, the unsaturated epoxide 5,6␣-epoxy-5␣-cholest-7-en-3␤-yl
acetate (10, 90% yield) was obtained after only 5 min in good yield. In fact, pyridine, besides having beneficial effect on the reaction rate,
shuts down the ring opening reactions, as reported in literature. © 2002 Elsevier Science Inc. All rights reserved.
Keywords: Methyltrioxorhenium; Catalysis; Oxidation; Unsaturated diols; Epoxides; Cytotoxic sterol
1. Introduction
catalyzed by MTO to be described [4]. This reaction was
subsequently explored by other research groups as in ep-
oxidation of styrene [5], [60]fullerene [6] and trans-cy-
clooctene [7], in regio- and diastereoselective oxidation of
geraniol [8] and in diastereoselective epoxidation of cyclic
allylic alcohols [9]. The active species involved in the ox-
ygen transfer to the olefinic double bonds are probably the
two Re(VII) peroxo complexes 2 and 3, obtained by the
addition of one or two H2O2 molecules to MTO, respectively
(Scheme 1) [10]. However, depending on the constitution of
the epoxide, a ring opening catalyzed by the electropositive
Re(VII) center can also occur to give 1,2-diols [4]. This prob-
lem can to some extent be overcome by replacing hydrogen
peroxide with the urea–hydrogen peroxide adduct (UHP) as
primary oxidant in non aqueous media [11,12] or by adding
pyridine to the oxidative system [13].
Recently, the oxidation using transition metal complexes
as catalysts attracted much attention, on the one hand, due to
the ability of these complexes to activate oxidants such as
molecular oxygen or hydrogen peroxide and, on the other
hand, for the possibility to control the regio- and stereose-
lectivity of these reactions [1,2]. Specifically, methyltrioxo-
rhenium (CH3ReO3, MTO, 1, Scheme 1) is a highly effi-
cient and selective catalyst for the oxidation of a large
number of organic compounds [3]. The epoxidation of ole-
fins with hydrogen peroxide was the first type of reaction
* Corresponding author. Tel.: ϩ39-081-674001; fax: ϩ39-081-
674393.
E-mail address: sica@unina.it (D. Sica).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00017-X

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D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
refractometer, using semipreparative Hibar Lichrosorb
Si-60 (250 ϫ 10 mm) and ␮Bondapak C18 (7.8 ϫ 300 mm)
columns. TLC analyses were performed on precoated F254
silica gel plates (0.25 mm, Merck). The reactions were
monitored by TLC until all starting material had been con-
sumed. Tetrahydrofuran and diethyl ether were distilled
over sodium/benzophenone prior to use. Chloroform was
purified by passage over aluminum oxide, activated, basic
Brockmann I.
Scheme 1.
In continuing our studies concerning the oxidation of
conjugated diene steroids with the UHP in the presence of
catalytic amounts of methyltrioxorhenium [14,15], in this
paper we describe the effects of using different solvents
(CHCl3 and ethers) and additives (EtOH and pyridine) in
the course of the MTO-catalyzed oxidation of cholesta-5,7-
dien-3␤-yl acetate (4). This study is part of our research
lines regarding either the functionalization of particular po-
sitions on the steroid nucleus through oxidative methods
[16,17] and the oxidation of unsaturated organic compounds
with transition metal oxides [16–23].
The new steroids 6, 9, 10 and 11, obtained from the
oxidation of 4 with MTO/H2O2-urea under various condi-
tions, were isolated and characterized. Moreover, the oxi-
dation product 8, obtained in 65% yield from the reaction of
4 with MTO/UHP in diethyl ether, was used as an interme-
diate in the synthesis of 9␣,11␣-epoxy-5␣-cholest-7-en-
3␤,5,6␤-triol (15), a cytotoxic epoxy sterol isolated from
the marine mollusk Planaxis sulcatus [24]. This synthesis
represents an improvement in yield and number of steps
over a synthesis of the same natural product previously
reported [25].
2.2. Oxidation of cholesta-5,7-dien-3␤-yl acetate 4 with
the MTO/UHP system in CHCl3
To a suspension of the urea–hydrogen peroxide adduct
(UHP, 79.4 mg, 0.845 mmol, 4 equiv.) in CHCl3 (2 ml)
methyltrioxorhenium (MTO, 5.3 mg, 0.021 mmol, 0.1
equiv.) was added at 25°C. After stirring for 10 min, a
solution of cholesta-5,7-dien-3␤-yl acetate (4, 90 mg, 0.211
mmol, 1 equiv.) in CHCl3 (2 ml) was added to the yellow
mixture and the stirring was continued for 1 h, after which
10 ml water was added. The reaction mixture was extracted
with CHCl3 (3 ϫ 15 ml), the combined organic layers were
dried over MgSO4 and the solvent was evaporated under
reduced pressure to give 110 mg of crude products. Sepa-
ration of the reaction mixture by HPLC on a Si-60 column,
eluting with hexane/AcOEt (8:2 v/v, Fo ϭ 3.5 ml/min),
afforded pure samples of the known sterols 5-hydroxy-6-
oxo-5␣-cholest-7-en-3␤-yl acetate (5, 6.8 mg, 7%, tR ϭ 8.8
min) [26], 5,6␣-dihydroxy-5␣-cholest-7-en-3␤-yl acetate
(7, 14.0 mg, 14%, tR ϭ 15.8 min) [21] and 5,6␤-dihydroxy-
5␣-cholest-7-en-3␤-yl acetate (8, 8.7 mg, 9%, tR ϭ 23.5
min) [26], and the new sterol 9␣-hydroxy-6-oxo-cholesta-
4,7-dien-3␤-yl acetate (6, 12.3 mg, 13%, tR ϭ 10.2 min),
(Fig. 1 and Table 1), as white solids. The structures of
compounds 5, 7 and 8 were established by direct compari-
son with authentic materials.
2. Experimental
2.1. General methods
When the oxidation of 4 with MTO/UHP in CHCl3 was
performed at 0°C, the same results as the reaction per-
formed at 25°C were obtained, apart from a small increase
in the reaction time. However, the reaction of 4 in CHCl3
produced a complex mixture of products, as is evident from
the HPLC profile of the reaction mixture, with a total yield
in main products of 43%.
Fourier transform IR (FTIR) spectra were obtained with
a Perkin-Elmer 1760-X FT-IR spectrophotometer. Ultravi-
olet (UV) spectra were recorded on a Perkin-Elmer Model
1
550S spectrophotometer. H NMR and 13C NMR spectra
were recorded in CDCl3 and [2H5]pyridine solutions on a
Bruker AMX-500 and Varian 200 spectrometers. Proton
chemical shifts were referenced to residual CHCl3 (␦ ϭ
7.26) and C5HD4N (␦ ϭ 8.71, 7.55, 7.19) signals. 13C NMR
chemical shifts were referenced to the solvent (CDCl3: ␦ ϭ
2.2.1. 9␣-Hydroxy-6-oxo-cholesta-4,7-dien-3␤-yl acetate
(6)
1
77.0; C5D5N: ␦ ϭ 149.9, 135.5, 123.5). H and 13C NMR
FTIR (CHCl3): 3420, 1733, 1668, 1631 cmϪ1. UV
1
assignments were based on DEPT, COSY and HMBC ex-
periments. The multiplicity of 13C NMR resonances was
determined by DEPT experiments. Electron impact mass
spectra (EIMS) were recorded on a Trio 2000 mass spec-
trometer. High-resolution electron impact mass spectra
(HREIMS) were obtained by electron impact at 70 eV on a
Kratos AEI-MS 902 mass spectrometer. High-performance
liquid chromatography (HPLC) was performed on a Varian
2510 apparatus equipped with a Waters R403 dual cell
(CHCl3): ␭
261 nm (⑀ ϭ 8748). H NMR (CDCl3, 500
max
MHz): see Table 2. 1H NMR (C5D5N, 500 MHz): see Table
2. 13C NMR (CDCl3, 125 MHz): ␦ ϭ 16.6 (C-18), 18.9,
20.4, 20.5, 21.1 (CH3COO), 22.5 (C-25 or C-26), 22.8
(C-25 or C-26), 24.0 (C-23), 24.3, 24.6, 26.5, 28.0 (C-25),
29.7, 34.2, 35.5, 36.1, 38.1 (C-10), 39.4 (C-24), 43.8 (C-14),
47.8 (C-13), 50.9 (C-17), 69.4 (C-3), 96.5 (C-9), 127.4 (C-4
or C-7), 130.4 (C-4 or C-7), 144.3 (C-5), 161.2 (C-8), 170.8
(CH3COO), 188.0 (C-6). MS (EI) m/z: (assignment, relative

D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
663
2.3. MTO/UHP oxidation of 4 in diethyl ether and
tetrahydrofuran
A solution of the diene steroid 4 (100 mg, 0.235 mmol,
1 equiv.) in 2 ml diethyl ether was added dropwise to a
mixture, prepared as described before, of MTO (5.8 mg,
0.023 mmol, 0.1 equiv.) and UHP (88.3 mg, 0.939 mmol, 4
equiv.) in 4 ml diethyl ether, and the suspension was stirred
at 25°C. After 20 min, the reaction was complete and the
mixture was diluted with H2O and extracted with diethyl
ether. The organic layer was dried over MgSO4 and the
solvent was evaporated to dryness under reduced pressure.
After separation of the reaction mixture by HPLC on a Si-60
column, eluting with hexane/AcOEt (8:2 v/v, Fo ϭ 3.5
ml/min), pure solid samples of the new sterol 5-hydroxy-
5␣-cholesta-6,8(14)-dien-3␤-yl acetate (9, 15.2 mg, 15%, tR
ϭ 5.8), and the known sterols 5␣,6␣-diol 7 (4.2 mg, 4%
yield, tR ϭ 15.8 min) and 5␣,6␤-diol 8 (70.0 mg, 65%, tR ϭ
23.5 min) (Fig. 1 and Table 1) were obtained with an
increase in the overall yield (84%) with respect to the
reaction in CHCl3. Performing the reaction at 0°C, the same
results as the reaction at 25°C were obtained, but a longer
time was required.
The behavior of the MTO/UHP oxidative system toward
4 in tetrahydrofuran as solvent was practically the same as
in diethyl ether. The products, 7, 8 and 9 obtained also in
diethyl ether, were formed in 1:16:10 ratios, but total yield
in these products was only of 60% for solubility problems.
2.3.1. 5-Hydroxy-5␣-cholesta-6,8(14)-dien-3␤-yl acetate
(9)
UV (CHCl3): ␭
254 nm (⑀ ϭ 15 000). 1H NMR
max
(CDCl3, 200 MHz): see Table 2. 13C NMR (CDCl3, 50.1
MHz): see Table 2. MS (EI) m/z: 442 (Mϩ, 3), 424 (Mϩ–
H2O, 4), 409 (Mϩ–H2O–CH3, 2), 364 (Mϩ–H2O–AcOH,
100), 349 (Mϩ–H2O–AcOH–CH3, 64), 251 [Mϩ–H2O–
AcOH–C8H17(side chain), 82]. HRMS (EI) m/z: 442.3419
(Mϩ, calcd for C29H46O3, 442.3447).
Fig. 1.
intensity) 456 (Mϩ, 30), 438 (Mϩ–H2O, 4), 428 (Mϩ–CO,
4), 414 (Mϩ–CH2CO, 100), 396 (Mϩ–AcOH, 19), 381
(Mϩ–AcOH–CH3, 19), 325 [Mϩ–C8H17(side chain)–H2O,
10], 283 (Mϩ–C8H17–AcOH, 5). HRMS (EI) m/z: 456.3262
(Mϩ, calcd for C29H44O4, 456.3240).
2.4. MTO/UHP oxidation of 4 in diethyl ether in the
presence of pyridine
To a suspension of MTO (5.8 mg, 0.023 mmol, 0.1
equiv.), UHP (88.3 mg, 0.939 mmol, 4 equiv.) and pyridine
Table 1
Yields of compounds 5–11 by oxidation of 4 (Fig. 1) with MTO/UHP under various reaction conditions
Reaction conditionsa
Yields (%)b
Solvent
Time (min)
Additives
5
6
7
8
9
10
11
CHCl3
Et2O
Et2O
Et2O
60
20
5
7
13
14
4
9
65
4
15
2
22
Pyr
EtOH
90
40
75
a Reactions were performed at 25°C, using 0.1 equiv. of MTO and 4 equiv. of UHP.
b Yields are for isolated (HPLC) products.

664
D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
(190 ␮l, 2.350 mmol, 10 equiv.) in 4 ml diethyl ether a
solution of the diene steroid 4 (100 mg, 0.235 mmol, 1
equiv.) in 2 ml diethyl ether was added whilst stirring at
25°C. After only 5 min, the reaction was complete and the
usual extractive work-up was performed. HPLC separation
of the reaction mixture on Si-60 column, eluting with hex-
ane/AcOEt (8:2, v/v, Fo ϭ 2.2 ml/min), resulted in the
isolation of the unsaturated epoxide 5,6␣-epoxy-5␣-cholest-
7-en-3␤-yl acetate (10, 93.5 mg, 90% yield, tR ϭ 4.1 min),
as white solid, as well as compounds 9 (2.1 mg, 2%, tR ϭ
6.2 min) and 8 (4.3 mg, 4%, tR ϭ 24.5 min), with an overall
yield of 96% (Fig. 1 and Table 1).
Hz, CH3-26 and -27), 0.94 (3H, d, J ϭ 6.2 Hz, CH3-21),
1.12 (3H, t, J ϭ 6.8 Hz, CH3CH2OC-6), 1.24 (3H, s,
CH3-19), 2.03 (3H, s, CH3COO), 2.21 (1H, bdd, J ϭ 12.6
and 4.5 Hz, Heq-4), 2.60 (1H, t, J ϭ 12.6 Hz, Hax-4), 3.57
(2H, CH3CH2OC-6), 3.68 (1H, bd, J ϭ 5.0 Hz, H-6), 5.63
(1H, m, H-7), 5.76 (1H, m, H-3). 13C NMR (CDCl3, 50.1
MHz): ␦ ϭ 12.1 (C-18), 15.7 (CH3CH2OC-6), 18.2 (C-19),
18.8 (C-21), 21.5 (CH3COO), 22.1 (C-11), 22.5 (C-26 or
C-27), 22.8 (C-26 or C-27), 22.9 (C-15), 23.0 (C-4), 23.9
(C-23), 26.9 (C-2), 27.8 (C-16), 28.0 (C-25), 32.4 (C-1),
35.8 (C-20), 36.0 (C-22), 37.2 (C-10), 39.5 (C-24 and
C-12), 43.7 (C-9), 43.9 (C-13), 54.8 (C-14 or C-17), 56.2
(C-14 or C-17), 65.3 (CH3CH2OC-6), 71.4 (C-3), 76.0 (C-
5), 80.3 (C-6), 115.6 (C-7), 143.1 (C-8), 170.6 (CH3COO).
MS (EI) m/z: 471 (Mϩ–OH, 22), 455 (Mϩ–H2O–CH3, 3),
442 (Mϩ–EtOH, 2), 428 (Mϩ–AcOH, 4), 411 (Mϩ–OH–
AcOH, 100), 395 (Mϩ–H2O–CH3–AcOH, 64), 382 (Mϩ–
AcOH–EtOH, 7), 380 (Mϩ–AcOH–H2O–2CH3, 8), 365
(Mϩ–AcOH–EtOH–OH, 38), 364 (Mϩ–AcOH–H2O–
EtOH, 19), 349 (Mϩ–AcOH–H2O–EtOH–CH3, 11), 297
[Mϩ–C8H17(side chain)–H2O–AcOH, 4], 269 (Mϩ–C8H17–
AcOH–EtOH, 6), 254 (Mϩ–C8H17–AcOH–EtOH–CH3, 6),
251 (Mϩ–C8H17–AcOH–EtOH–H2O, 16), 209 [Mϩ–
C8H17–AcOH–EtOH–H2O–C3H6(D ring), 15].
2.4.1. 5,6␣-Epoxy-5␣-cholest-7-en-3␤-yl acetate (10)
1H NMR (CDCl3, 200 MHz): ␦ ϭ 0.54 (3H, s, CH3-18),
0.84 (6H, d, J ϭ 6.6 Hz, CH3-26 and -27), 0.91 (3H, d, J ϭ
6.2 Hz, CH3-21), 1.03 (3H, s, CH3-19), 2.02 (3H, s,
CH3COO), 2.35 (1H, t, J ϭ 12.4 Hz, Hax-4), 3.00 (1H, d,
J ϭ 4.0 Hz, H-6), 4.98 (1H, m, H-3), 5.42 (1H, m, H-7). 13
C
NMR (CDCl3, 50 MHz): ␦ ϭ 11.8 (C-18), 16.3 (C-19), 18.8
(C-21), 20.9, 21.2, 22.5 (C-26 or C-27), 22.7 (C-26 or
C-27), 23.1, 23.8, 27.4, 27.7, 27.9, 33.1 (C-1), 34.9 (C-10),
36.1 (C-3), 38.7 (C-12), 39.4 (C-24), 41.3 (C-9), 42.2 (C-
13), 54.5 (C-14), 54.6 (C-6), 55.9 (C-17), 66.3 (C-5), 71.0
(C-3), 114.7 (C-7), 147.9 (C-8), 170.1 (CH3COO). MS (EI)
m/z: 442 (Mϩ, 2), 424 (Mϩ–H2O, 6), 382 (Mϩ–AcOH, 6),
364 (Mϩ–AcOH–H2O, 100), 349 (Mϩ–AcOH–H2O–CH3,
33), 251 [Mϩ–AcOH–H2O–C8H17(side chain), 42], 209
[Mϩ–AcOH–H2O–C8H17–C3H6(D ring), 32]. HRMS (EI)
m/z: 442.3428 (calcd for C29H46O3, 442.3447).
2.6. Synthesis of the natural epoxy sterol 15 (Scheme 2)
2.6.1. 5-Hydroxy-5␣-cholesta-7,9(11)-diene-3␤,6␤-diol
3,6-diacetate (13)
MTO (29 mg, 0.117 mmol), UHP (441 mg, 4.69 mmol)
and cholesta-5,7-dien-3␤-yl acetate (4, 500 mg, 1.174
mmol) in 9 ml diethyl ether were maintained at 25°C with
stirring for 20 min, as described before. After the usual
extractive work-up, the reaction mixture was separated by
HPLC on a semipreparative Si-60 column, eluting with
hexane/AcOEt (7:3, v/v, Fo ϭ 2.5 ml/min), to give com-
pound 8 (350 mg, 65% yield, tR ϭ 12.2 min). This last (8,
350 mg, 0.761 mmol) was acetylated by treatment with 5 ml
2.5. MTO/UHP oxidation of 4 in diethyl ether/ethanol
To a suspension, prepared as described before, of MTO
(2 mg, 0.008 mmol, 0.1 equiv.) and UHP (31 mg, 0.329
mmol, 4 equiv.) in 2 ml Et2O/EtOH (1:1 v/v) the diene
steroid 4 (35 mg, 0.082 mmol, 1 equiv.), dissolved in 2 ml
diethyl ether, was added and the mixture was stirred at 25°C
for 40 min. After the usual extractive work-up, HPLC sep-
aration on a Si-60 column, eluting with hexane/AcOEt
(85:15 v/v, Fo ϭ 2.5 ml/min), furnished compounds 9 (8
mg, 22%, tR ϭ 8.0 min) and 6␤-ethoxy-5-hydroxy-5␣-
cholest-7-en-3␤-yl acetate (11, 30 mg, 75%, tR ϭ 9.2 min)
(Fig. 1 and Table 1), as white solids.
2.5.1. 6␤-Ethoxy-5-hydroxy-5␣-cholest-7-en-3␤-yl acetate
(11)
1H NMR (CDCl3, 200 MHz): ␦ ϭ 0.59 (3H, s, CH3-18),
0.87 (6H, d, J ϭ 6.6 Hz, CH3-26 and -27), 0.93 (3H, d, J ϭ
6.2 Hz, CH3-21), 1.02 (3H, s, CH3-19), 1.15 (3H, t, J ϭ 7.0
Hz, CH3CH2OC-6), 1.75 (1H, bdd, J ϭ 12.4 and 4.5 Hz,
Heq-4), 2.04 (3H, s, CH3COO), 2.25 (1H, t, J ϭ 12.4 Hz,
Hax-4), 3.25 (1H, bd, J ϭ 5.0 Hz, H␣-6), 3.53 (2H, two 8
line multiplets, AB part of an ABX3 system, CH3CH2OC-
6), 5.13 (1H, m, H-3), 5.36 (1H, m, H-7). 1H NMR (C5D5N,
200 MHz): ␦ ϭ 0.61 (3H, s, CH3-18), 0.86 (6H, d, J ϭ 6.6
Scheme 2.

D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
665
pyridine/(Ac)2O (2:1, v/v) at 25°C, overnight. After work-
up, the crude residue was purified by HPLC to give 5-hy-
droxy-5␣-cholest-7-en-3␤,6␤-diol 3,6-diacetate in almost
was stirred at 25°C for 6 h, after which CH3OH was re-
moved under reduced pressure and the reaction mixture was
diluted with water and extracted with CHCl3. 107 mg (21%
overall yield) of a compound having 1H and 13C NMR data
exactly superimposable to those of natural epoxy sterol 15
[24] were obtained.
1
quantitative yield (378 mg): H NMR (CDCl3, 200 MHz):
␦ ϭ 0.58 (3H, s, CH3-18), 0.87 (6H, d, J ϭ 6.6 Hz, CH3-26
and -27), 0.93 (3H, d, J ϭ 6.0 Hz, CH3-21), 1.07 (3H, s,
CH3-19), 2.04 (3H, s, CH3COO), 2.06 (3H, s, CH3COO),
4.82 (1H, bd, J ϭ 4.9 Hz, H-6), 5.14 (1H, m, H-3), 5.27
(1H, m, H-7). Subsequently, to a solution of 5-hydroxy-5␣-
cholest-7-en-3␤,6␤-diol 3,6-diacetate (378 mg, 0.753
mmol) in 6 ml CHCl3, Hg(OAc)2 (479 mg, 1.507 mmol) in
CH3COOH (4.2 ml) was added, and the suspension was
maintained at 25°C whilst stirring for about 24 h. The
reaction mixture was then filtered, concentrated at 70–80°C
in vacuo and filtered once again. The filtered was washed
with aqueous solution of NaHCO3 and extracted with
CHCl3. The organic phase was finally washed with H2O,
dried over MgSO4 and concentrated in vacuo. HPLC sepa-
ration of the crude products on Lichrosorb Si-60 column,
eluting with hexane/AcOEt (6:4, v/v, Fo ϭ 2.5 ml/min),
afforded a peak at tR ϭ 4.2 min that was subjected to further
purification on ␮Bondapak C18 column, eluting with
CH3OH/H2O (85:15 v/v, Fo ϭ 3.5 ml/min), to give 169 mg
5-hydroxy-5␣-cholesta-7,9(11)-diene-3␤,6␤-diol 3,6-diac-
etate (13, 45% yield, tR ϭ 28.3 min) and 113 mg of the
3. Results
3.1. MTO-catalyzed oxidation of cholesta-5,7-dien-3␤-yl
acetate with UHP under various conditions
3.1.1. Structural elucidation of new sterols
The spectroscopic data of the oxidation product 6 (Fig.
1), obtained from the reaction between 4 and the MTO/UHP
system in CHCl3, were in good agreement with the pro-
posed structure. Specifically, the HREIMS spectrum
showed an Mϩ ion peak at m/z 456.3262 (calcd 456.3240)
indicating the molecular formula C29H44O4. The UV max-
imum at 261 nm (⑀CHCl ϭ 8748), the strong IR absorption
3
at 1668 cmϪ1 and the carbon signal at ␦ ϭ 188.0 in the 13
C
NMR spectrum provided evidence for the presence of an
␣,␤-unsaturated ketone group whereas the broad IR absorp-
tion at 3420 cmϪ1 and the peak at m/z 438 (Mϩ–H2O) in the
EIMS spectrum revealed the presence of an hydroxyl group
in the molecule. 1H NMR spectrum of 6 in CDCl3 included
signals of an acetoxymethine proton at ␦ ϭ 5.39 (ddd, J ϭ
9.2, 5.1 and 1.6 Hz), assigned to the H␣-3 proton, and of two
olefinic protons at ␦ ϭ 6.37 (t, J ϭ 1.6 Hz) and ␦ ϭ 6.01 (d,
J ϭ 2.4 Hz), attributed to H-4 and H-7, respectively, on the
1
starting monoene product (30%, tR ϭ 37.3 min). 13: H
NMR (CDCl3, 200 MHz): ␦ ϭ 0.57 (3H, s, CH3-18), 0.86
(6H, d, J ϭ 6.5 Hz, CH3-26 and -27), 0.91 (3H, d, J ϭ 6.0
Hz, CH3-21), 1.25 (3H, s, CH3-19), 2.03 (3H, s, CH3COO),
2.05 (3H, s, CH3COO), 2.15 (1H, bd, J ϭ 17.8 Hz, Hax-12),
2.29 (1H, m, H-14), 2.39 (1H, dd, J ϭ 17.8 and 6.2 Hz,
Heq-12), 4.94 (1H, dd, J ϭ 5.7 and 1.7 Hz, H-6), 5.16 (1H,
m, H-3), 5.38 (1H, bd, J ϭ 5.7 Hz, H-7), 5.75 (1H, bd, J ϭ
6.2 Hz, H-11).
1
basis of H–1H COSY experiment (see Table 2). The pres-
ence of two double bonds was confirmed by the analysis of
the 13C NMR data that showed four olefinic carbon signals
at ␦ ϭ 161.2 (quaternary C), 144.3 (quaternary C), 130.4
(CH) and 127.4 (CH). The signal of an exchangeable proton
2.6.2. 9␣,11␣-Epoxy-5␣-cholest-7-en-3␤,5,6␤-triol (15)
To a solution of the diene 13 (169 mg, 0.339 mmol) in 3
ml CHCl3 a solution of m-chloroperbenzoic acid (75 mg,
0.441 mmol) in 2 ml CHCl3 was added with stirring at 0°C.
After 6 h the reaction mixture was diluted with CHCl3,
washed with aqueous solution of Na2SO3 and NaHCO3
successively, and dried over MgSO4. The solvent was evap-
orated and the crude residue was separated by HPLC on a
semipreparative Si-60 column, eluting with hexane/AcOEt
(6:4, v/v, Fo ϭ 2.5 ml/min), to give, as main product,
9␣,11␣-epoxy-5-hydroxy-5␣-cholest-7-en-3␤,6␤-diol 3,6-
1
at ␦ ϭ 7.85 (bs, variable in the range 7.37–.85) in the H
NMR spectrum and the resonance at ␦ ϭ 96.5 in the 13C
NMR spectrum, both recorded in CDCl3, denoted the pres-
ence in the molecule of a tertiary hydroxyl group in allylic
position. Furthermore, the pyridine-induced shifts for sev-
1
eral protons in the H NMR spectrum of 6 recorded in
[2H5]pyridine (see Table 2), were in good agreement with
the presence in the molecule of a 9␣-OH group [27].
The structure of compound 9 (Fig. 1) was determined by
deacetylation of 9 with K2CO3 in H2O/MeOH at 40°C to
1
1
diacetate (131 mg, 75% yield, tR ϭ 5.7 min): H NMR
afford 5␣-cholesta-6,8(14)-diene-3␤,5-diol whose H NMR
(CDCl3, 200 MHz): ␦ ϭ 0.61 (3H, s, CH3-18), 0.85 (6H, d,
J ϭ 6.6 Hz, CH3-26 and -27), 0.90 (3H, d, J ϭ 6.4 Hz,
CH3-21), 1.28 (3H, s, CH3-19), 2.02 (3H, s, CH3COO), 2.08
(3H, s, CH3COO), 2.18 (1H, dd, J ϭ 14.9 and 5.6, Heq-12),
2.33 (1H, m, H-14), 3.16 (1H, d, J ϭ 5.6 Hz, H-11), 5.06
(1H, dd, J ϭ 5.2 and 2.3 Hz, H-6), 5.11 (1H, m, H-3), 5.66
(1H, dd, J ϭ 5.4 and 2.7 Hz, H-7). To this epoxy steroid
(131 mg, 0.254 mmol), dissolved in 4 ml CH3OH, an
aqueous solution of K2CO3 (3 ml) was added. The reaction
data were indistinguishable from those reported in literature
[28]. However, interpretation of 1H, 13C and HMBC spectra of
1
9 allows us to obtain its complete H and 13C NMR assign-
1
ments, not reported before (see Table 2). Specifically, the H
NMR spectrum of 9 contained, in addition to the signal of the
acethoxymethine proton H␣-3 at ␦ ϭ 5.18 (seven line multip-
let), two mutually coupled olefinic doublets at ␦ ϭ 6.22 and
6.46 (J ϭ 9.6 Hz). The 13C NMR data indicated the presence
in the molecule of two double bonds, a disubstituted one with

666
D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
Table 2
NMR spectral data of compounds 6 and 9 [1H (500 MHz), 13C (125 MHz)]
C
6
9
c
␦H(CDCl3)a,b
␦H(C5D5N)a
␦H(CDCl3)a,b
␦
HMBC: H/C
C
1ax
2.25, ddd
(11.6, 9.2, 1.9)
2.056
2.065, 1.68
5.39, ddd
(9.2, 5.1, 1.6)
6.37, t (1.6)
—
2.61, bdd
(11.6, 9.2)
28.8
1eq
2
3
26.8
70.8
5.26, m
5.18, m
4, CH3COO
4
5
6.54, bs
—
1.89
37.9
73.5
eq
—
—
6
7
8
9
10
11
12eq
—
—
6.22, d (9.6)
5.46, d (9.6)
—
2.41
—
129.6
128.0
124.3
39.5
38.9
19.1
4, 5, 8, 10
5, 9, 8, 14
—
8, 14
—
6.01, d (2.4)
—
—
6.40, d (2.4)
—
—
—
—
2.33
14
36.7
12ax
13
14
2.05
—
—
9, 11, 13, 14
—
—
—
—
44.1
151.7
2.65, ddd
(10.2, 7.8, 2.4)
1.75, 1.66
2.80, bdd
(10.2, 7.8)
15
16
17
18
2.39
25.0
27.3
55.7
15.6
8, 13, 14, 16
0.77, s
0.77, s
1.10, s
0.90, s
12, 13, 14,
17
1, 5, 9, 10
13, 16, 21,
22
19
20
1.17, s
1.43
0.78, s
1.20
19.6
34.8
21
22
23
24
25
26
0.89, d (6.8)
1.34, 1.00
1.37, 1.13
1.15, 1.13
1.53
0.95, d (6.0)
0.94, d (6.5)
1.35, 0.99
1.34, 1.12
1.18, 1.13
1.52
18.9
35.8
23.7
39.5
28.0
22.6*
17, 20, 22
0.86*, d (6.4)
0.86*,
d (6.4)
0.87*,
d (6.4)
2.01, s
—
0.86, d (6.5)
24, 25, 26,
27
24, 25, 26,
27
27
0.87*, d (6.4)
0.86, d (6.5)
22.8*
CH3CO
CH3CO
OH
2.07, s
—
7.85, bs
2.02, s
—
21.5
170.5
—
8.53, bs
a ␦ values are in ppm from the residual solvent signal; coupling constants (in Hertz) are given in parentheses. b 1H assignments aided by 1H–1H COSY
experiment. c ␦ values are in ppm from CDCl3 signal (77.0); 13C assignments were assisted by DEPT and HMBC experiments. * means the assignments may
be interchanged within a column.
1
resonances at ␦ ϭ 129.6 and 128.0, attributed, on the basis of
HMBC experiment, to C-6 and C-7, respectively, and a tetra-
substituted double bond with signals at ␦ ϭ 151.7 (C-14) and
124.3 (C-8). Furthermore, the 13C NMR spectrum showed a
signal at ␦ ϭ 73.5, attributed to C-5 according to HMBC
correlations (see Table 2), relative to a carbon bearing a tertiary
hydroxyl group, whose presence was also confirmed by EIMS
peaks derived from loss of H2O.
The spectroscopic data of the epoxy steroid 10 (Fig. 1),
obtained from the oxidation of 4 with MTO/UHP in diethyl
ether in the presence of pyridine, were in good agreement
with the proposed structure. Molecular peak at m/z
442.3428 in HREIMS spectrum, unsaturation count, and
13C and H NMR analysis [13C epoxide signals: ␦ ϭ 66.3
(quaternary C) and 54.6 (CH); 1H epoxide signal: ␦ ϭ 3.00
(1H, d, J ϭ 4.0 Hz, H-6)] confirmed the presence of an
oxirane ring in the molecule. Furthermore, the H-6 proton
signal at ␦ ϭ 3.00 showed correlation with the olefinic
signal at ␦ ϭ 5.42 (1H, m, H-7), as revealed by selective
H–H decoupling experiments, according to the unsaturated
epoxide structure. The 5␣,6␣-stereochemistry of the epox-
ide ring was established on the basis (a) of coupling con-
stant values of the H-6 and H-7 proton signals, (b) of the
H-3 and CH3-19 proton chemical shifts and (c) by compar-
ing the spectral data of 10 with those reported in literature
for the analogous deacetylated compound [28].

D. Musumeci, D. Sica / Steroids 67 (2002) 661–668
667
Compound 11 (Fig. 1) was easily identified to be the
6-ethyl derivative of compound 8. Comparison of H and
1 and Table 1), a key intermediate compound in the syn-
thesis of many biologically active natural steroids oxygen-
ated at the B/C rings. In fact, starting from sterol 8 we
performed an improvement in the synthesis of the cytotoxic
sterol 15 (21% yield, Scheme 2) over a synthesis of the
same natural product previously reported [25].
When the oxidation of 4 in diethyl ether was performed
in the presence of pyridine as ligand the unsaturated epoxide
5,6␣-epoxy-5␣-cholest-7-en-3␤-yl acetate (10, 90% yield,
Fig. 1 and Table 1) was obtained after only 5 min. In fact,
pyridine, besides having a strong accelerating effect on the
epoxidation rate, prevents decomposition of the epoxide
products, as previously noted [13]. The addition of a nu-
cleophile, such as ethanol, in oxidation using diethyl ether
as solvent, afforded the expected product of trans-diaxial
epoxide ring opening in high yield (11, 75%, Fig. 1 and
Table 1).
1
13C NMR spectra showed the two compounds to be identi-
cal except for the additional ethyl group in 11 and the
corresponding highfield shift of the H␣-6 signal from ␦ ϭ
3.60 in 8 to 3.25 in 11. Further proof for the presence of the
ethoxide group in the molecule came from analysis of the
EIMS spectrum of 11 which included peaks deriving from
loss of ethanol (m/z 442) or ethanol with other groups such
as H2O, AcOH, CH3 and side chain. The ␣ stereochemistry
for the C-5 hydroxyl group was confirmed by the 1H NMR
spectrum recorded in [2H5]pyridine which showed the ex-
pected pyridine-induced downfield shifts for the H-3, H-6,
H2-4 and CH3-19 with respect to the spectrum recorded in
CDCl3 [27].
3.2. Synthesis of the natural epoxy sterol 15 (Scheme 2)
Taking these data into consideration, we have reached
the conclusion that the MTO-catalyzed hydrogen peroxide
oxidation of the conjugated diene sterol 4 stereoselectively
gave epoxide 10, which is a precursor of the steroids 7, 8, 9
and 11. Compounds 5-hydroxy-6-oxo-5␣-cholest-7-en-
3␤-yl acetate (5) and 9␣-hydroxy-6-oxo-cholesta-4,7-dien-
3␤-yl acetate (6) (Fig. 1) probably derive through a more
complex mechanism.
The new compound 6, obtained from oxidation of 4 in
CHCl3 with a 13% yield, has the C-4/C-8 segment identical
to that of pinnasterol, an ecdysone-like metabolite extracted
from the red alga Laurencia pinnata Yamada, which shows
biologic activity as moulting hormones by a Sarchophaga
method [29]. Therefore, bioassay is in progress to evaluate
the biologic relevance of 6.
The synthetic strategy that we have employed to obtain
the natural epoxy sterol 15 (Scheme 2) was based on the
preparation of the 5␣,6␤-diol 8. This compound was ob-
tained with a 65% yield following oxidation of 4 with the
MTO/UHP system in diethyl ether at 25°C. This procedure
was simpler and provided a higher yield of diol 8 than the
two-step-procedure reported in literature [28]. The 6␤-hy-
droxyl group of 8 was acetylated to give compound 12 that
was subjected to dehydrogenation using Hg(OAc)2 (2
equiv.) in glacial acetic acid and CHCl3 at 25°C for 24 h
affording conjugated diene 13 with a 45% yield. Subse-
quently, epoxidation of 13 with m-chloroperbenzoic acid in
CHCl3 at 0°C yielded 14 (75% yield). The epoxide 14 was
finally deacetylated with K2CO3 in MeOH/H2O to give 15,
whose spectral data were identical to those reported in
literature for natural compound 9␣,11␣-epoxy-5␣-cholest-
7-ene-3␤,5,6␤-triol, active against the L1210 cell line at a
0.6 mg/ml level [24]. Therefore, Scheme 2 represent a more
convenient procedure to prepare natural epoxy sterol 15,
obtained with an overall yield of 21%, than that yet reported
in literature employing eight synthetic steps and a final yield
in 15 of 6% [25].
In addition, this article furnishes the spectroscopic
data, not reported before, of the new steroids 6, 9, 10
and 11, obtained from the oxidation of 4 under different
conditions.
Acknowledgments
This work has been supported by MURST. The facil-
ities of the Centro di Metodologie Chimico-Fisiche
dell’Universita` degli Studi di Napoli were used for the
NMR work. Mass spectral data were obtained from the
4. Discussion
Methyltrioxorhenium-catalyzed oxidation of cholesta-
5,7-dien-3␤-yl acetate (4) with the UHP using different
temperatures (25°C and 0°C), solvents (CHCl3 and ethers)
and additives (EtOH and pyridine) was investigated. Tem-
perature does not significantly affect the course of the oxi-
dation process: the same products with the same yields
obtained at 25°C were also obtained at 0°C with a small
increase in the reaction time.
Servizio di Spettrometria di Massa del CNR
e
dell’Universita` di Napoli; the assistance of the staff is
gratefully acknowledged. High-resolution mass spectra
were kindly provided by Prof. F. Zollo.
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