704
steroids 7 3 ( 2 0 0 8 ) 702–707
and to 3-acetoxystigmast-5-ene-7-one (6) (1.21 g, 2.57 mmol,
yield: 47% (72%/3-acetoxystigmast-5-ene present in the start-
ing reaction mixture).
brine (30 mL) and dried over magnesium sulfate. After fil-
tration and removal of the solvent (25 ◦C, 15 mmHg), the
crude mixture was chromatographed on a silica gel column
(30 g SiO2, EtOAc/petroleum ether: 5/95) leading to 3-
acetoxy-5,6-epoxystigmastane (10) (0.642 g, 1.30 mmol, yield:
89%).
Spectral data: see Ref. [1].
2.6.
3ˇ-Hydroxystigmast-5-ene-7-one (7)
Spectral data: see Ref. [1].
To a solution of 3-acetoxystigmast-5-ene-7-one (6) (1.21 g,
2.55 mmol) in MeOH (40 mL) was added at room temperature
Na2CO3 (0.40 g, 3.85 mmol) followed by the addition of H2O
(4 mL). The reaction mixture was stirred 20 h at room temper-
ature, hydrolyzed with water (20 mL), extracted with CH2Cl2
(3 × 20 mL). The organic layers were washed with a saturated
aqueous NaCl solution (10 mL) and dried over Na2SO4, fil-
tered and the solvent was removed under reduced pressure
(25 ◦C, 15 mmHg). The crude product was purified on a silica
gel column (30 g SiO2, EtOAc/petroleum ether: 20/80) leading to
3-hydroxystigmast-5-ene-7-one (7) (1.11 g, 2.44 mmol, yield:
95%; purity determined by GC: ≥95%).
2.9.
3ˇ-Hydroxy-5,6ˇ-epoxystigmastane (11)
To
a
solution of 3-acetoxy-5,6-epoxystigmastane (10)
(0.642 g, 1.30 mmol) in methanol (70 mL) was added sodium
carbonate (0.245 g, 2.30 mmol). The reaction mixture was
stirred 4 h at room temperature then the solvent was removed
under reduced pressure (25 ◦C, 15 mmHg) and water (20 mL)
was added. After extraction with CH2Cl2 (3 × 20 mL), the
organic phase was washed with brine (30 mL) and dried over
magnesium sulfate. After filtration and removal of the solvent
(25 ◦C, 15 mmHg), the crude mixture was chromatographed on
a silica gel column (30 g SiO2, EtOAc/petroleum ether: 20/80)
leading to 3-hydroxy-5,6-epoxystigmastane (11) (0.542 g,
1.20 mmol, yield: 93%).
Spectral data: see Ref. [1].
2.7.
3ˇ-Acetoxy-5˛-bromo-6ˇ-hydroxystigmastane (8)
a mixture of 3-acetoxystigmast-5-ene (4) and 3-
Spectral data: see Ref. [1].
To
acetoxystigmastanol (5) (1.47 g, ∼.20 mmol) in dioxan (40 mL),
water (2 mL) and three drops of perchloric acid (70–74%) was
added at 0 ◦C freshly prepared N-bromoacetamide (0.532 g,
3.8 mmol) [9]. The reaction was maintained 1 h at 0 ◦C then
2 h at 20 ◦C under light exclusion. The solution was then
treated with a solution of sodium thiosulfate until decol-
oration and extracted with Et2O (3 × 30 mL). The organic phase
was washed with brine (30 mL) and dried over magnesium
sulfate. After filtration and removal of the solvents (25 ◦C,
15 mmHg), the crude mixture was chromatographed on a
silica gel column (100 g SiO2, EtOAc/petroleum ether: 10/90)
leading to 3-acetoxystigmastane (5) (0.400 g, 0.89 mmol,
yield: 24%; 72%/starting material) along with an unsepara-
ble mixture of 3-acetoxy-5␣-hydroxy-6-bromostigmastane
(9), 3-acetoxy-5,6␣-epoxystigmastane (10a), 3-acetoxy-5,6-
epoxystigmastane (10) (0.310 g; ratio (9)/(10 + 10a): 1.3/1
determined by NMR) and finally to 3-acetoxy-5␣-bromo-
6-hydroxystigmastane (8) as a pure compound. (0.864 g,
1.60 mmol, yield: 49%; 72%/starting material).
3.
Results and discussion
As mentioned above, we needed to have in our hands
preparative amounts of highly pure oxyphytosterol derivatives
especially 7keto-,7␣-hydroxysitosterol, 7-hydroxysitosterol
and 5,6-␣ (and )-epoxysitosterol. For that purpose, we started
to study the regioselective hydrogenation of the 22–23 dou-
ble bond of stigmasterol (1) which was already described
by Kircher and Rosenstein in the early seventies [6]. These
authors obtained several interesting results: when the hydro-
genation of stigmasterol and derivatives was carried out in
the presence of 5% Pd/C (entries 1–3), the starting material
was still present (2–6%). More recently, Lichtfouse and Albrecht
described the regioselective hydrogenation of the 22–23 dou-
ble bond of 3b-acetoxystigmast-5-ene (obtained from 95% pure
stigmasterol) over platinum dioxide: these authors claimed
that sitosterol acetate was isolated in 86% yield with 91%
purity as determined by GC [7].
3-Acetoxy-5␣-bromo-6-hydroxystigmastane (8): 1H NMR
(CDCl3): ı 5.60–5.40 (m, 1H); 4.18 (broad s, 1H); 2.60–2.40 (m, 1H);
2.35–2.10 (m, 2H); 2.10–1.50 (m, 10H); 1.50–0.70 (m, 35H); 0.61 (s,
3H). 13C NMR (CDCl3): ı 170.16, 86.44, 75.49, 71.85, 55.71, 55.43,
47.13, 45.54, 42.41, 40.06, 39.37, 38.14, 35.85, 34.82, 34.31, 33.60,
30.29, 28.84, 27.94, 26.07, 25.75, 23.79, 22.78, 21.08, 21.01, 19.53,
18.74, 18.45, 17.74, 11.90, 11.69. Analysis for C31H53BrO3: calcd
C, 67.25; H, 9.65. Found C, 67.56; H 9.71.
According to these literature results, we first decided to
check if we could reproduce them. For that purpose, commer-
cially available stigmasterol was first purified on a silica gel
column leading to the following mixture of products as deter-
mined by GC: 1.0% brassicasterol; 0.5% campesterol; 95.0%
stigmasterol; 3.5% sitosterol. Thus, we assumed that stigmas-
terol has a purity ≥95%.
In the presence of Pd/C (entries 1–3) the hydrogenation
of the starting material always delivered a mixture of three
the other hand, in the presence of PtO2, we were not able to
reproduce the Lichtfouse and Albrecht results [7], the start-
ing material being the main product recovered (entries 4 [7]–4)
(Table 1). This hydrogenation process was also utilized by
Heupel and Nes but these authors worked only with analyti-
cal amounts of products and did not separate sitosterol from
campesterol [8].
2.8.
3ˇ-Acetoxy-5,6ˇ-epoxystigmastane (10)
The mixture of 3-acetoxy-5␣-bromo-6-hydroxystigmastane
(8) (0.844 g, 1.50 mmol) and sodium acetate (1.55 g, 1.9 mmol)
in ethanol (30 mL) was refluxed under argon during 3 h.
The solvent was removed under reduced pressure (25 ◦C,
15 mmHg) and after water addition (20 mL), extraction with
CH2Cl2 (3 × 20 mL), the organic phase was washed with