2
F. Balssa et al. / Steroids 86 (2014) 1–4
Whatman silica gel coated plates. Melting points were measured
with a Reichert apparatus.
CH3), complex multiplets between 1.0 and 3.0 ppm. 13C NMR
ppm: 166.13 (C@O), 157.50 (3-C), 137.95, 132.76, 132.58, 130.91,
129.54, 128.37, 126.39, 113.85, 111.53, 82.65 (17-C), 55.21 (–
OCH3), 49.55, 45.43, 43.71, 39.16, 32.18, 30.29, 29.95, 28.12,
26.23, 24.49, 16.82 (18-C).
2.1. 17b-Estradiol-3-methyl ether 1
2.1.1. Starting from 17b-estradiol
A mixture of 17b-estradiol (2.86 g, 10.5 mmol), sodium carbon-
ate (11.1 g, 105 mmol) and iodomethane (17 mL, 38.8 g, 273 mmol)
in methanol (50 mL) was heated under reflux with stirring. After
16 h, iodomethane (1 mL, 2.28 g, 16 mmol) was added and the mix-
ture was heated for 48 h. This suspension was then filtered and the
inorganic salts were washed with methanol (150 mL). The metha-
nolic solution was distilled under vacuum to give a white solid
which was redissolved in boiling methanol (50 mL). Water
(250 mL) was slowly added to the boiling solution of methanol with
vigorous stirring, leading to the precipitation of a white solid. After
2 h stirring at room temperature, the precipitate was recovered by
filtration, washed with water (250 mL) and dried under vacuum at
90 °C to give 2.92 g (yield: 97%) of 17b-estradiol-3-methyl ether 1.
2.3. 5(10)-Estren-17
a-ol,3-one 3
A solution of 3-methoxy-estra-1,3,5(10)-trien-17
a-ol-17-ben-
zoate 2 (1.42 g, 3.63 mmol) in 1,4-dioxane/diethyl ether (1:1,
100 mL) was added to a solution of lithium (1.42 g, 204.6 mmol)
in ammonia (250 mL). Ethanol (30 mL) was added dropwise over
a period of 20 min (until decoloration), ammonia was allowed to
evaporate and water (200 mL) was added. This mixture was
extracted with diethyl ether (2 Â 300 mL). The organic fractions
were combined, washed with water (2 Â 300 mL) dried with brine
(300 mL), sodium sulfate and evaporated under vacuum to give a
white solid.
This solid was dissolved in methanol (120 mL) and a solution of
oxalic acid dihydrate (1.4 g, 11.1 mmol) in water (20 mL) was
added. After 2 h stirring at room temperature, the volume was
reduced under vacuum at 40 °C to 50 mL. Diethyl ether (200 mL)
was added and washed with sodium hydroxide solution (1 N,
200 mL). The aqueous layer was back extracted with diethyl ether
(200 mL) and the combined organic fractions were washed with
sodium hydroxide solution (1 N, 3 Â 200 mL) and water (200 mL).
The organic layer was dried with brine (200 mL), sodium sulfate
and evaporated to give a white solid. Recrystallization from a hex-
ane/ethyl acetate mixture gave 443.6 mg of 3 (white crystals, yield:
44.5%).
2.1.2. Starting from estrone
A mixture of estrone (2.0 g, 7.4 mmol), sodium carbonate (9.0 g,
84.9 mmol) and iodomethane (10 mL, 22.8 g, 160.6 mmol) in
methanol (50 mL) was heated under reflux with stirring. After
16 h, this mixture was cooled to room temperature and water
(100 mL) was slowly added with vigorous stirring, leading to the
precipitation of a white solid. After 1 h stirring, the precipitate
was recovered by filtration, washed with water (300 mL) and dried
under vacuum to give 2.049 g of estrone-3-methyl ether (yield:
97.6%). A solution sodium borohydride (0.5 g, 13.2 mmol) in water
(10 mL) was added to a suspension of estrone-3-methyl ether
(2.049 g, 72 mmol) in ethanol (350 mL) and stirred at room tem-
perature. After 3 days, hydrochloric acid (1 N, 20 mL) was slowly
added, the volume was reduced under vacuum to 50 mL (a crystal-
line precipitate appeared) and water (250 mL) was slowly added
under vigorous stirring. The precipitate was collected by filtration,
washed with water (150 mL) and dried under vacuum to give b-
estradiol-3-methyl ether 1 as a white solid (1.982 g, 96%).
These 17b-estradiol-3-methyl ether preparations were identical
to a reference standard, as evidenced by GC/MS.
MS (EI), bisTMS derivative, m/z (%): 418 (100), 403 (9), 327 (3),
285 (4), 259 (7), 246 (6), 233 (4), 220 (7), 195 (8), 182 (38). HRMS
(APCI, MH+) calc.: 275.20056, found: 275.20076. 1H NMR ppm:
3.77 (d, J = 6 Hz, 1H 17b-H), 0.68 (s, 3H 18-CH3), complex multi-
plets between 1.0 and 3.0 ppm. 13C NMR ppm: 211.43 (3-C),
131.12 (10-C), 126.45 (5-C), 79.91 (17-C), 47.32, 45.87, 44.68,
39.35, 39.13, 32.55, 31.81, 30.83, 27.56, 27.30, 25.04, 24.05, 17.31
(18-C). Melting point: 149–150 °C (Litt.: 144–150 °C [5]).
2.4. 5(10)-Estrene-3b,17a-diol 6
2.2. 3-Methoxy-estra-1,3,5(10)-trien-17
a
-ol-17-benzoate 2
A solution of potassium hydroxide (19.5 mg, 0.35 mmol) in
isopropanol (1.95 mL) was added to solution of
5(10)-estren-17 -ol,3-one (392 mg, 1.43 mmol) and chlor-
a
A solution of diisopropyl-azodicarboxylate (1.6 mL, 7.86 mmol)
in toluene (10 mL) was added dropwise to solution of
a
3
a
o{[(1S,2S)-(+)-2-amino-1,2-diphenylethyl](4-toluenesulfo-
nyl)amido}(p-cymene)ruthenium(II) (204 mg, 0.32 mmol) in
isopropanol (40 mL). After 20 h stirring at room temperature, the
solution was diluted with ethyl acetate (300 mL) and washed with
hydrochloric acid (1 N, 200 mL). The aqueous phase was back
extracted with ethyl acetate (200 mL). The organic extracts were
combined and washed with hydrochloric acid (1 N, 3 Â 200 mL),
saturated sodium hydrogen carbonate solution (3 Â 200 mL), dried
with brine (250 mL) and sodium sulfate.
Evaporation of the solvent under vacuum gave a black solid.
This residue was dissolved in ethyl acetate (50 mL), adsorbed on
SiO2, loaded onto a silica gel column (3 Â 25 cm) and eluted by
hexane/ethyl acetate mixture (80/20, v/v). Three fractions were
17b-estradiol-3-methyl ether 1 (1.50 g, 5.24 mmol), benzoic acid
(1.0 g, 8.18 mmol) and diphenyl-(2-pyridyl)-phosphine (2.0 g,
7.59 mmol) in toluene (50 mL) in an ice bath. After 24 h heating
at 80 °C, the mixture was cooled at room temperature and washed
with hydrochloric acid (1 N, 4 Â 100 mL), saturated aqueous
sodium hydrogencarbonate solution (3 Â 100 mL), dried with brine
(100 mL) and anhydrous sodium sulfate.
After distillation of the solvent under vacuum, the residual
brown gum was purified by chromatography on a silica gel column
(5 Â 50 cm, elution: hexane/ethyl acetate, 95:15, v/v) to give a light
yellow solid which was recrystallized from boiling ethanol (10 mL)
to give white crystals (1.168 g, yield: 57.1%).
TLC: Rf = 0.5 (hexane/ethyl acetate, 8:1, revelation: UV 254 nm).
Melting point: 115 °C (Litt.: 114–115 °C [6]). MS (EI), m/z (%): 390
(100), 268 (20), 253 (6), 241 (12), 239 (12), 225 (12), 211 (7), 186
(14), 174 (44), 160 (27), 147 (23), 105 (64). HRMS (APCI, MH+)
calc.: 391.22677, found: 391.22696. 1H NMR ppm: 8.23
(d, J = 8 Hz, 2H benzoate o-H), 7.57 (t, J = 6 Hz, 1H benzoate p-H),
7.49 (t, J = 8.5 Hz, 2H benzoate m-H), 7.24 (d, J = 8 Hz 1H 1-H),
6.74 (dd, J = 2.75 Hz, J = 8.5 Hz, 1H 2-H), 6.68 (d, J = 3 Hz, 1H 4-H),
5.15 (d, J = 6 Hz, 1H 17b-H), 3.81 (s, 3H, –OCH3), 0.89 (s, 3H 18-
collected: the first one, containing pure 5(10)-estrene-3b,17a-diol
6 (166 mg), the second one, containing target compound 6 contam-
inated with 2% of undesired epimer (147.1 mg) and the third one,
containing a small amount of a 1/1 mixture of the two epimers
(this fraction was discarded). Combined: 313.1 mg, yield: 79.7%.
Chemo-enzymatic purification: 100 mg of the second fraction
(containing 2% of the 3
a epimer) was dissolved in a solution of
vinyl acetate (1 mL, 10.8 mmol) in anhydrous toluene (35 mL).
Novozym 435Ò (3 g) was added and this mixture was stirred at