The oxidation of Δ2, Δ2,4 and Δ4,6 steroids with RuO4

Article abstract of DOI:10.1016/j.steroids.2003.11.003

In order to find new ways for the functionalization of the A and B rings of the steroid nucleus, the reaction of 5α-androst-2-en-17β-ol 17-acetate (1), cholesta-2,4-diene (4) and cholesta-4,6-dien-3β-ol 3-acetate (7) was examined using stoichiometric amounts of ruthenium tetraoxide to yield 1,2-cis diols and/or α-hydroxy ketones. The reaction of 5α-cholest-2-en-3-ol 3-acetate (9) with ruthenium tetraoxide was also carried out and afforded, apart from an α-hydroxy ketone, also a diketone and a seco-dicarboxylic acid. The structures of all new steroids, including stereochemical details, were deduced by analysis of spectral data.

Full text of DOI:10.1016/j.steroids.2003.11.003

Steroids 69 (2004) 173–179
The oxidation of ꢀ2, ꢀ2,4 and ꢀ4,6 steroids with RuO4
ଝ
Domenica Musumeci, Giovanni N. Roviello, Donato Sica∗
Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli “Federico II”,
Complesso Universitario Monte Sant’Angelo, Via Cinthia, I-80126 Napoli, Italy
Received 24 April 2003; received in revised form 6 November 2003; accepted 14 November 2003
Abstract
In order to find new ways for the functionalization of the A and B rings of the steroid nucleus, the reaction of 5␣-androst-2-en-17␤-ol
17-acetate (1), cholesta-2,4-diene (4) and cholesta-4,6-dien-3␤-ol 3-acetate (7) was examined using stoichiometric amounts of ruthenium
tetraoxide to yield 1,2-cis diols and/or ␣-hydroxy ketones. The reaction of 5␣-cholest-2-en-3-ol 3-acetate (9) with ruthenium tetraoxide
was also carried out and afforded, apart from an ␣-hydroxy ketone, also a diketone and a seco-dicarboxylic acid. The structures of all new
steroids, including stereochemical details, were deduced by analysis of spectral data.
© 2004 Elsevier Inc. All rights reserved.
Keywords: Ruthenium tetraoxide; Oxidation; 1,2-Diols; ␣-Ketols
1. Introduction
tetrahydropyran [19] moiety, respectively, by intramolecular
cyclization.
As a continuation of our study on the reactivity of
unsaturated organic substrates with metal transition ox-
ides in high oxidation states and at the same time on the
functionalization of particular positions on the steroid nu-
cleus through oxidative methods [9–11,13–16,18,20–23],
in the present paper we report the reactions of ruthenium
tetraoxide with 5␣-androst-2-en-17␤-ol 17-acetate (1),
cholesta-2,4-diene (4), cholesta-4,6-dien-3␤-ol 3-acetate (7)
and 5␣-cholest-2-en-3-ol 3-acetate (9). These oxidations
conducted to the formation of the polyoxygenated steroids
2, 3, 5, 6, 8, 10, 11 and 12, reported in Schemes 1–4.
Transition metal oxides have been successfully used for
oxidation of unsaturated organic compounds [1–8]. Ruthe-
nium tetraoxide is a powerful oxidizing agent and has
been employed under very mild reaction conditions to ox-
idize a great variety of substrates [1–4]. The reaction with
alkenes gives carbonyl compounds via oxidative cleavage of
carbon–carbon double bonds [1–4]. In short reaction times,
alkenes give almost exclusively 1,2-diols and/or ␣-hydroxy
ketones, as we have revealed on oxidation with RuO4 of a
number of monoene steroids [9], conjugated diene steroids
[10] and alkenes [11]. The same results can be achieved
in a biphasic solvent system if RuO4 is present in catalytic
amounts and stoichiometric amounts of sodium periodate
were used [12]. Interestingly, some steroidal 1,3-dienes
furnished mainly epoxydiols and epoxyketols products by
treatment with RuO4 when the double bonds were located
in a hindered position [10,13]. We have also demonstrated
that the reactions of alkenes with RuO4 proceed through
bis-cyclic oxoruthenium(VI) diesters, the initially formed
intermediate in the oxidation process [14–16]. Oxidation
of 1,5- and 1,6-dienes with catalytic amount of RuO4 in a
biphasic solvent system at room temperature gives in few
minutes products containing tetrahydrofuran [17,18] and
2. Experimental
2.1. General methods
Steroidal starting materials of high purity were obtained
from Steraloids Inc. Cholesta-4,6-dien-3␤-ol 3-acetate (7)
was prepared from cholesta-4,6-dien-3␤-ol by following
standard acetylation conditions.
Melting points were determined using a Zeiss Axioskop
microscope apparatus equipped with a Mettler FP82HT mi-
crofurnace. Fourier transform IR (FT-IR) spectra were ob-
tained with a Perkin-Elmer 1760-X FT-IR spectrophotome-
ଝ
Part 10 in the series “Reaction of RuO4 with carbon–carbon double
1
ter. H NMR and 13C NMR spectra of all the samples in
bonds”. For part 9 see Ref. [18].
CDCl3 and [2H5]pyridine solutions were recorded on a Var-
ian 200 MHz spectrometer. Proton chemical shifts were ref-
∗
Corresponding author. Tel.: +39-081-674001; fax: +39-081-674393.
E-mail address: sica@unina.it (D. Sica).
0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2003.11.003

174
D. Musumeci et al. / Steroids 69 (2004) 173–179
OAc
H
1
RuO4
Acetone/H2O
10 min
OAc
OAc
HO
O
HO
HO
H
H
2
3
Scheme 1.
H17C8
Scheme 4.
impact mass spectra (HREIMS) were obtained by electron
impact at 70 eV on a Kratos AEI-MS 902 mass spectrome-
ter. High-performance liquid chromatography (HPLC) was
performed on a Varian 2510 apparatus equipped with a
Waters R403 dual cell refractometer, using semipreparative
(250 mm × 10 mm) and analytical (250 mm × 4 mm) Hibar
Lichrosorb Si-60 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 consumed.
4
RuO4
Acetone/H2O
15 min
H17C8
H17C8
HO
O
OH
O
2.2. RuO4 oxidation of 5α-androst-2-en-17β-ol
17-acetate (1)
5
6
Scheme 2.
To a stirred solution of 5␣-androst-2-en-17␤-ol 17-acetate
(1, 28 mg, 0.0886 mmol) in acetone (10 ml) at −15 ◦C
was added a solution of ruthenium tetraoxide in 5:1
(v/v) acetone–water (12 ml). The oxidant was prepared
by stirring at room temperature RuO2·2H2O (45 mg,
0.266 mmol, 3 equiv.) with a suspension of NaIO4 (340 mg,
1.590 mmol, 18 equiv.) in 5:1 (v/v) acetone–water (8 ml)
until the reaction mixture became yellow (1 h) followed
by centrifugation. The mixture was stirred for 10 min,
then quenched with few drops of 2-propanol. The black
precipitate formed was centrifuged and washed with chlo-
roform/methanol. Then the solvents were removed by
evaporation and the crude reaction mixture was fraction-
ated by HPLC on a semipreparative Si-60 column eluted
with hexane/AcOEt (8:2, v/v, F0 = 2.5 ml/min) to give
the new sterols 2␣,17␤-dihydroxy-5␣-androstan-3-one
erenced to residual CHCl3 (δ = 7.26) and C5HD4N (δ =
8.71, 7.55, 7.19) signals. 13C NMR chemical shifts were
referenced to the solvent (CDCl3: δ = 77.0; C5D5N: δ =
149.9, 135.5, 123.5). The multiplicity of 13C NMR reso-
nances was determined by DEPT experiments. Electron im-
pact mass spectra (EIMS) were recorded on a Trio 2000
mass spectrometer. FAB mass spectra were obtained on a
Kratos MS 50 mass spectrometer. High-resolution electron
H17C8
H17C8
RuO4
Acetone/H2O
5 min
OH
OAc
OAc
OH
8
17-acetate (2, 13.9 mg, 45%, tR
=
17.8 min) and
7
5␣-androstan-2␣,3␣,17␤-triol 17-acetate (3, 9.3 mg, 30%,
tR = 21.4 min) (Scheme 1).
Scheme 3.

D. Musumeci et al. / Steroids 69 (2004) 173–179
175
Table 1
1H NMR (200 MHz) spectral data of compounds 2, 3 and 8
2
3
8
(C)
δH(CDCl3)a
δH(CDCl3)a
δH(C5D5N)a
δH(CDCl3)a
δH(C5D5N)a
1eq
2ax
2.47, dd (12.2, 6.7)
4.22, dd (11.7, 6.7)
3.75, bd (12.1)
4.03, ddd (10.2, 5.5, 3.0)
3eq
3ax
3.96, bs (W1/2 = 7.7)
4.32, bs (W1/2 = 7.7)
5.23, ddd (9.0, 6.4, 1.7)
5.47, ddd (9.9, 6.6, 2.2)
4eq
4ax
2.27, dd (14.0, 3.7)
2.41, dd (14.0, 14.0)
5.57, bs (W1/2 = 4.3)
5.67, bs (W1/2 = 4.3)
6eq
4.09, d (3.4)
4.45, d (3.8)
7ax
3.29, dd (9.8, 3.4)
3.52, dd (9.9, 3.8)
16
2.16, m
17␣
18
19
4.58, dd (8.5, 8.5)
0.80, s
1.10, s
4.59, dd (8.8, 8.8)
0.77
0.80
4.59, dd (8.8, 8.8)
0.78
0.81
0.71
1.25
0.73
1.48
21
0.92, d (6.8)
0.86, d (6.8)
2.06
0.99, d (6.6)
0.87, d (6.6)
2.04
26 and 27
CH3COO–
OH-2
OH-3
2.04, s
3.50, bd (2.4)
2.03
2.01
5.75, bd (5.5)
5.54, bs
a δ values are in ppm from the residual solvent signal (CHCl3: δ 7.26; C5D4HN: δ 8.71, 7.55, 7.19); coupling constants (in Hz) are given in
parentheses; 1H assignments aided by selective H–H decoupling experiments.
2.2.1. 2α,17β-Dihydroxy-5α-androstan-3-one 17-acetate
(2)
White needles; mp 134–136 ◦C (MeOH). FT-IR (CHCl3)
Table 2
13C NMR (50.1 MHz) spectral data of compounds 2, 3 and 8
(cm−1): 1715 (C O), 1740, 3440 (OH). H NMR (CDCl3,
1
=
200 MHz): see Table 1. 13C NMR (CDCl3, 50.1 MHz): see
Table 2. MS (FAB) m/z: (assignment, relative intensity) 349
(MH+, 45), 331 (MH+–H2O, 100), 289 (MH+–AcOH, 20),
271 (MH+–AcOH–H2O, 15). HRMS (EI) m/z: 348.2320
(M+, calcd. for C21H32O4, 348.2301).
2
3
8
(C)
δC(CDCl3)a
δC(C5D5N)a
δC(CDCl3)a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
CH3COO
CH3COO
48.5b
72.7
210.9
42.4b
48.3
28.4
31.2
34.5
53.7
37.1
21.08
36.7
42.6
50.4
23.5
27.5
82.6
12.1
12.8
41.9
69.1
69.8
37.24
38.7
28.1
31.8
34.8
54.6
37.20
20.8
35.6
42.9
50.8
23.7
27.9
82.9
12.3
12.7
29.2
37.0
71.0
125.1
149.2
77.44
77.37
38.3
51.9
36.7
21.1
40.2
43.7
56.2b
25.7
27.9
55.8b
12.4
19.2
36.1
21.3
36.6
24.3
39.8
28.2
22.7b
22.9b
21.2
170.5
2.2.2. Synthesis of 2α,17β-dihydroxy-5α-androstan-3-one
17-acetate (2)
Commercial 2␣-bromo-5␣-androstan-17␤-ol-3-one 17-
acetate (10 mg) in 83% aqueous acetone (3 ml) was
treated with K2CO3 (1 equiv.) at reflux, following the
procedure described by Numazawa and Nagaoka for
2␣,17␤-dihydroxy-5␣-androstan-3-one [24]. This reaction
afforded after 30 min a product which had mp and 1H NMR
spectrum identical to compound 2.
2.2.3. 5α-Androstane-2α,3α,17β-triol 17-acetate (3)
Amorphous powder; FT-IR (CHCl3) (cm−1): 1750,
1
1
3400 (OH). H NMR (CDCl3, 200 MHz): see Table 1. H
NMR (C5D5N, 200 MHz): see Table 1. 13C NMR (C5D5N,
50.1 MHz): see Table 2. MS (FAB) m/z: (assignment, rel-
ative intensity) 351 (MH+, 100), 333 (MH+–H2O, 30),
291 (MH+–AcOH, 9), 273 (MH+–AcOH–H2O, 15), 255
(MH+–AcOH–2H2O, 20). HRMS (EI) m/z: 350.2449 (M+,
calcd. for C21H34O4, 350.2457).
21.13
171.1
21.0
170.7
2.3. RuO4 oxidation of cholesta-2,4-diene (4)
a δ values are in ppm from CDCl3 (δ 77.0) and C5D5N (δ 149.9, 135.5,
123.5) signals; 13C assignments were assisted by DEPT experiments.
b Assignments may be interchanged within a column.
A stirred solution of cholesta-2,4-diene 4 (17.7 mg,
0.048 mmol, 1 equiv.) in 10 ml acetone, cooled at −15 ◦C,

176
D. Musumeci et al. / Steroids 69 (2004) 173–179
was treated with a solution of ruthenium tetraoxide pre-
pared, as described above, from RuO2·2H2O (8.1 mg,
0.048 mmol, 1 equiv.) and NaIO4 (56.25 mg, 0.263 mmol,
5.5 equiv.), in 5:1 (v/v) acetone–water (12 ml). After
15 min, the reaction was quenched by adding 2-propanol
and worked up as described above. The crude reac-
tion mixture was fractionated by HPLC on an analytical
Si-60 column, eluting with hexane/AcOEt (87:13, v/v,
F0 = 1 ml/min) to give pure samples of the known sterols
5-hydroxy-5␣-cholest-2-en-4-one 5 (6.5 mg, 34% yield,
tR = 7.0 min) [25] and 2␣-hydroxycholest-4-en-3-one 6
(4.7 mg, 15%, tR = 8.3 min) [26] (Scheme 2).
(M+–AcOH–2H2O, 37). HRMS (EI) m/z: 460.3510 (M+,
calcd. for C29H48O4, 460.3553).
2.5. RuO4 oxidation of 5α-cholest-2-en-3-ol 3-acetate (9)
Enol acetate 9 (40 mg, 0.093 mmol, 1 equiv.) in 10 ml
acetone, cooled at −15 ◦C, was mixed with a solution
(12 ml acetone–water, 5:1) of ruthenium tetraoxide pre-
pared from RuO2·2H2O (17.4 mg, 0.103 mmol, 1.1 equiv.)
and NaIO4 (133.3 mg, 0.623 mmol, 6.7 equiv.). The reac-
tion mixture was stirred for 10 min and then quenched
by adding 2-propanol and worked up as described above.
After removal of the solvent under reduced pressure the
crude reaction mixture was fractionated by HPLC on a
semipreparative Si-60 column, eluting with CHCl3/CH3OH
(9:1, v/v, F0 = 2.5 ml/min) to give two principal frac-
tions. The last eluted fraction contained the known
2,3-seco-5a-cholestane-2,3-dicarboxylic acid 12 (7.7 mg,
19% yield, tR = 10.1 min) [27]. The first eluted fraction
(16.0 mg, tR = 4.0 min) was found to be a mixture of two
compounds which were separated by HPLC on an analyt-
ical Si-60 column, eluting with hexane/AcOEt (95:5, v/v,
F0 = 1 ml/min) to give pure solid samples of the known
sterols 5␣-cholestane-2,3-dione 10 (8.9 mg, 24% yield,
tR = 6.5 min) [28] and 2␣-hydroxy-5␣-cholestan-3-one 11
(4.5 mg, 12%, tR = 17.0 min) [24,29] (Scheme 4).
2.3.1. 5-Hydroxy-5α-cholest-2-en-4-one (5)
Amorphous powder; FT-IR (CHCl3) (cm−1): 1680
1
=
(C O), 3422 (OH). H NMR (CDCl3, 200 MHz): δ = 0.66
(3H, s, CH3-18), 0.86 (6H, d, J = 6.7 Hz, CH3-26 and -27),
0.91 (3H, d, J = 6.7 Hz, CH3-21), 0.92 (3H, s, CH3-19),
2.12 (1H, bdd, J = 19.5, 6.1 Hz, H␤-1), 2.43 (1H, bd,
J = 19.5 Hz, H␣-1), 5.98 (1H, dd, J = 10.4, 3.0 Hz, H-3),
6.83 (1H, ddd, J = 10.4, 6.1, 2.4 Hz, H-2).
2.3.2. 2α-Hydroxycholest-4-en-3-one (6)
Crystallization from methanol gave the pure product as
colorless needles; mp 146–148 ◦C (lit. [26] 147–148 ◦C).
FT-IR (CHCl3) (cm−1): 1610 (C C), 1674 (C O), 3350
(OH). 1H NMR (CDCl3, 200 MHz): δ = 0.71 (3H, s,
CH3-18), 0.86 (6H, d, J = 6.7 Hz, CH3-26 and -27), 0.91
(3H, d, J = 6.7 Hz, CH3-21), 1.17 (3H, s, CH3-19), 2.48
(1H, dd, J = 13.4, 5.5 Hz, H␤-1), 4.18 (1H, bdd, J = 14.0,
5.5 Hz, H␤-2), 5.80 (1H, s, H-4).
=
=
2.5.1. 5α-Cholestane-2,3-dione (10)
1
Amorphous powder; H NMR (CDCl3, 200 MHz): δ =
0.68 (3H, s, CH3-18, diketone form), 0.77 (3H, s, CH3-18,
3-enol form), 1.00–0.90 (overlapping methyl signals), 1.10
(3H, s, CH3-19, 1-enol form), 2.85–2.30 (8H, overlapping
AB systems relative to CH2-1 and CH2-4 of diketone form,
CH2-1 of 3-enol form and CH2-4 of 1-enol form), 5.70 (1H,
bd, J = 3.0 Hz, H-4, 3-enol form), 6.36 (1H, bs, H-1, 1-enol
form), 6.45 (1H, bs, OH-2, 1-enol form), 6.73 (1H, bs, OH-3,
3-enol form).
2.4. RuO4 oxidation of cholesta-4,6-dien-3β-ol 3-acetate
(7)
To a stirred solution of the diene steroid 7 (40 mg,
0.094 mmol, 1 equiv.) in 10 ml acetone at −15 ◦C was
added a solution of ruthenium tetraoxide prepared, as de-
scribed above, from the hydrate form of RuO2 (17.6 mg,
0.104 mmol, 1.1 equiv.) and NaIO4 (135 mg, 0.631 mmol,
6.7 equiv.), in 12 ml acetone–water (5:1, v/v). After 5 min,
the reaction was quenched by adding 2-propanol and the
usual work-up was performed. The crude reaction mixture
was purified by HPLC separation of the reaction mixture
on a semipreparative Si-60 column, eluting with hex-
ane/AcOEt (75:25, v/v, F0 = 2.5 ml/min) to give the new
sterol cholest-4-ene-3␤,6␤,7␤-triol 3-acetate 8 (17.4 mg,
40%, tR = 18.3 min) (Scheme 3).
2.5.2. 2α-Hydroxy-5α-cholestan-3-one (11)
Colorless needles; mp 125–127 ◦C (acetone) (lit. [29]
126.5–128.5 ◦C). FT-IR (CHCl3) (cm−1): 1720 (C O),
=
1
3445 (OH). H NMR (CDCl3, 200 MHz): δ = 0.67 (3H, s,
CH3-18), 0.86 (6H, d, J = 6.8 Hz, CH3-26 and -27), 0.90
(3H, d, J = 6.8 Hz, CH3-21), 1.09 (3H, s, CH3-19), 2.26
(1H, dd, J = 13.7, 3.9 Hz, Heq-4), 2.40 (1H, dd, J = 13.7,
13.7 Hz, Hax-4), 2.47 (1H, dd, J = 12.7, 6.6 Hz, Heq-1),
3.50 (1H, bs, OH-2), 4.22 (1H, dd, J = 11.7, 6.8 Hz, Hax-2).
2.4.1. Cholest-4-ene-3β,6β,7β-triol 3-acetate (8)
2.5.3. Synthesis of 2α-hydroxy-5α-cholestan-3-one (11)
Commercial 2␣-bromo-5␣-cholestan-3-one was sub-
jected to alkaline hydrolysis to obtain the 2␣-hydroxy-3-one
derivative following the procedure described by Numazawa
and Nagaoka [24]. The reaction afforded after 40 min a
Amorphous powder; FT-IR (CHCl3) (cm−1): 1740,
3455 (OH). 1H NMR (CDCl3, 200 MHz): see Table 1.
1H NMR (C5D5N, 200 MHz): see Table 1. 13C NMR
(C5D5N, 50.1 MHz): see Table 2. MS (EI) m/z: (assign-
ment, relative intensity) 460 (M+, 5), 442 (M+–H2O, 35),
400 (M+–AcOH, 100), 382 (M+–AcOH–H2O, 97), 364
1
product which had mp and H NMR spectrum identical to
compound 11.

D. Musumeci et al. / Steroids 69 (2004) 173–179
177
2.5.4. 2,3-Seco-5a-cholestane-2,3-dicarboxylic acid (12)
lest-4-en-3-one 6 (15%) [26] that were identified on compar-
ison of their spectroscopic and chemical physical properties
with those reported in literature (see Section 2).
Colorless needles; mp 190–193 ◦C (acetone) (lit. [27]
1
192–195 ◦C). H NMR (C5D5N, 200 MHz): δ = 0.67 (3H,
s, CH3-18), 0.89 (6H, d, J = 6.8 Hz, CH3-26 and -27),
0.90 (3H, s, CH3-19), 0.94 (3H, d, J = 6.3 Hz, CH3-21),
2.36 (1H, dd, J = 15.1, 11.2 Hz, Hax-4), 2.65 (1H, d,
J = 14.2 Hz, B part of an AB system centered at 2.68,
CH2-1), 2.71 (1H, d, J = 14.2 Hz, A part of an AB system
centered at 2.68, CH2-1), 2.85 (1H, btt, J = 11.2, 3.0,
3.0 Hz, H-5), 3.23 (1H, bdd, J = 15.1, 3.0 Hz, Heq-4).
Cholesta-4,6-dien-3␤-ol 3-acetate 7 (Scheme 3), on oxi-
dation with RuO4, furnished the new sterol cholest-4-ene-
3␤,6␤,7␤-triol 3-acetate 8 in 40% yield. Interpretation of
13C and DEPT spectra of 8 allows us to obtain its com-
plete 13C NMR assignments (see Table 2). In particular,
the combination of 13C NMR and DEPT experiments, indi-
cated the presence in the molecule of a trisubstituted dou-
ble bond [13C NMR: δ 125.1 (CH-4) and 149.2 (C-5)] and
of two secondary alcoholic functions [13C NMR: δ 77.37
1
3. Results
(CH-7) and 77.44 (CH-6)]. The H NMR spectrum of 8
(see Table 1) contained, in addition to the signal of the ace-
toxymethine proton H␣-3 at δ = 5.23 (1H, ddd, J = 9.0,
6.4 and 1.7 Hz), an olefinic resonance at δ = 5.57 (1H, bs,
W1/2 = 4.3 Hz) and two mutually coupled oxymethine sig-
nals at δ = 3.29 (1H, dd, J = 9.8 and 3.4 Hz) and 4.09 (1H,
d, J = 3.4 Hz), assigned to H-7 and H-6, respectively, on
the basis of selective homonuclear irradiation experiments.
The 6␤,7␤-stereochemistry for the two hydroxyl groups was
established, besides of coupling constant and chemical shift
values of the H-6 and H-7 proton signals, on the basis of
RuO4 oxidation of 5␣-androst-2-en-17␤-ol 17-acetate
(1, Scheme 1) in acetone–water, as solvent, gave in 10 min
the new sterols 2␣,17␤-dihydroxy-5␣-androstan-3-one
17-acetate (2, 45% yield) and 5␣-androstane-2␣,3␣,17␤-triol
17-acetate (3, 30%), identified on the basis of their spec-
troscopic data. Specifically, the HREIMS spectrum of 2
showed an M+ ion peak at m/z 348.2320 indicating the
molecular formula C21H32O4. The strong IR absorption at
1715 cm−1 and the carbon signal at δ = 210.9 in the 13C
NMR spectrum provided evidence for the presence of a ke-
1
the H NMR spectrum of 8 recorded in [2H5]pyridine (see
tone group whereas the broad IR absorption at 3440 cm−1
,
Table 1) which showed the expected pyridine-induced down-
field shifts for the H-6 (ꢀδ = −0.36), H-7 (ꢀδ = −0.23)
and CH3-19 (ꢀδ = −0.23) with respect to the spectrum
recorded in CDCl3 [30]. Furthermore, the stereochemistry
at C-6 and C-7 stereogenic centers was also corroborated
by Dreiding molecular modeling analysis and by comparing
the spectral data of 8 with those reported in literature for the
analogous 7-epimer, cholest-4-ene-3␤,6␤,7␣-triol [20]. In
particular, in compound 8, the small coupling constant value
of 3.4 Hz between H-6 and H-7 oxymethine signals jus-
tify an axial/equatorial relationship for the hydroxyl groups
OH-6 and OH-7, while the large 1H NMR coupling constant
value for H-6 and H-7 of the 7␣-epimer proved an equato-
rial/equatorial relationship between OH-6 and OH-7.
Finally, oxidation of enol acetate 9 with RuO4 (Scheme
4) afforded the known sterols 5␣-cholestane-2,3-dione 10
[28] and 2␣-hydroxy-5␣-cholestan-3-one 11 [24,29] and the
known secosterol 2,3-seco-5␣-cholestane-2,3-dicarboxylic
acid 12 [27] which were identified by comparison of
their spectroscopic data with those reported in litera-
ture. Furthermore, the structure of 11 was confirmed by
comparison of its spectral data with those of an authen-
tic sample synthesized by stereoselective hydrolysis of
2␣-bromo-5␣-cholestan-3-one [24].
the 13C NMR signal at δ = 72.7 and the peak at m/z
331 (MH+–H2O) in the FABMS spectrum revealed the
1
presence of an hydroxyl group in the molecule. H NMR
spectrum of 2 in CDCl3 included a signal of an oxyme-
thine proton at δ = 4.22 (1H, dd, J = 11.7 and 6.7 Hz),
assigned to the H-2 proton with ␤ stereochemistry accord-
ingly with its coupling constant values. The structure of
2 was proven by its independent synthesis starting from
2␣-bromo-5␣-androstan-17␤-ol-3-one 17-acetate following
the procedure described by Numazawa and Nagaoka for
2␣,17␤-dihydroxy-5␣-androstan-3-one [24] (see Section 2).
The spectroscopic data of the oxidation product 3 were in
good agreement with the proposed structure. The molecular
formula of 3 was determined as C21H34O4 on the basis of
HREIMS, FABMS and 13C NMR spectra, implying two
OH functionalities [HREIMS m/z: 350.2449; FABMS: m/z
255 (MH+–AcOH–2H2O); 13C NMR: δ 69.8 and 69.1]
and the pre-existed acetoxy group [13C NMR: δ 170.7
(CH3COO)]. The positions and the stereochemistry of the
CH(OH) groups was deduced on the basis of the multi-
plicities, chemical shifts and coupling constants values of
1
the H-2 and H-3 proton signals and by the study of the H
NMR spectrum of 3 recorded in [2H5]pyridine, compared
to that recorded in CDCl3 [30]. Furthermore, structure of
compound 3 was confirmed by comparison of its 13C NMR
chemical shift values with those reported in literature of the
analogous (25R)-5␣-spirostane-2␣,3␣-diol: A and B rings
δC values of the compounds are superimposable [31].
4. Discussion
The oxygenation of double bonds can be accom-
plished using transition metal oxidants in high oxida-
tion states such as osmium tetraoxide [5,6], alkaline
permanganate [8], ruthenium tetraoxide [1–4,9–19] and
The reaction of cholesta-2,4-diene
4 with RuO4
(Scheme 2) afforded the known sterols 5-hydroxy-5␣-
cholest-2-en-4-one 5 (34% yield) [25] and 2␣-hydroxycho-

178
D. Musumeci et al. / Steroids 69 (2004) 173–179
H
C
C
H
C
C
H
C
O
H
C
C
O
O
O
O
O
O
H2O
O
O
O
C
C
oxidative
decomposition
Ru
Ru
R
R
R
OH
R
O
C
R
OH
H
13
16
14
H2O
H
R
R
RuO4
C
O
+
C
O
C
C
H
HO
OH
aldehydes, ketones, carboxylic acids
15
Scheme 5.
methyltrioxorhenium–hydrogen peroxide system [7,20–23].
In this paper RuO4 oxidation was applied to monoene
steroids 1 and 9, and to the diene steroids 4 and 7. The
reason of such functionalization reactions on steroids as
substrates relies in the objective of synthesizing polyoxy-
genated steroids which is an important class of compounds
since they have been isolated from marine organisms [32]
and are intermediates in the synthesis of secosterols [33]
and biologically active compounds [34].
The formation of all RuO4 oxidation products can be
explained, accordingly to our previous results concerning
the oxidation of monoene steroids [9], linear and cyclic
alkenes [11] and conjugated diene steroids [10], assuming
that the initially formed bis-cyclic oxoruthenium(VI) diester
13 [14–16] of the Scheme 5 may undergo hydrolysis to the
cyclic diester derivative 14. This intermediate may undergo
further hydrolysis to the 1,2-diol 15 or may undergo oxida-
tive decomposition to give ␣-hydroxycarbonyl compound
16. This last could also derive by oxidation of 15 with a
second molecule of RuO4. Finally, fragmentation of the in-
termediate 13 affords scission products, namely aldehydes,
ketones or carboxylic acids [1–4,17]. This mechanicistic hy-
pothesis was supported by the formation, during the above
reported reactions, of highly unstable brownish precipitates,
very likely the oxoruthenium(VI) ester intermediate [14–16],
detectable by TLC analysis, that however, do not survive to
the work-up stage.
di Spettrometria di Massa del CNR e dell’Università di
Napoli; the assistance of the staff is gratefully acknowl-
edged. High-resolution mass spectra were kindly provided
by Prof. F. Zollo.
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