772
R.B.R. Porter et al. / Steroids 64 (1999) 770–779
extracts were combined, dried with sodium sulfate, and the
solvent was removed in vacuo. The mycelial residue was
dried at 90°C in an oven for 12 h.
2.4. 17-Hydroxyandrost-4-en-3-one (5)
The biotransformation of 5, 1 g, yielded 49.4 g of dried
mycelium and 1.91 g of organic extract on harvesting.
Column chromatography on the organic extract using in-
creasing concentrations of ethyl acetate in petrol gave 228
mg of fed and 713 mg of transformed steroid. The fraction
eluting in 20% ethyl acetate was 3,17-dihydroxyandrost-
2.2. 3-Hydroxyandrost-5-en-17-one (1)
The fermentation of 1, 1 g, yielded 1.05 g of organic
extract and 55.31 g of dry mycelium on harvesting. The
extract was subjected to column chromatography using
increasing concentrations of ethyl acetate in petrol. The
fraction eluting in 10% ethyl acetate was untransformed
steroid (55 mg). Further elution using 20% ethyl acetate
in petrol afforded 3,17-dihydroxyandrost-5-ene (3),
115 mg, which crystallized from chloroform as cubes, mp
169–171°, [␣]D Ϫ 47° (c ϭ 0.11, EtOH); lit. [16] mp
4-ene (6), 15 mg, which resisted crystallization; IR
max
3418, 2954, 1457 cmϪ1; HRMS (EI) m⁄
z
(%) 290.2238 (100)
[C19H30O2], 272 (13), 231 (50), 107 (27); 1H NMR (CDCl3)
␦ 0.82 (3H, s, H-18), 0.90 (3H, s, H-19), 3.98 (1H, m, w⁄
18 Hz, H-17␣), 5.20 (1H, m, w⁄
ϭ 12 Hz, H-3␣), 6.25 (1H,
2
ϭ
2
d, J ϭ 6 Hz, H-4). Further elution with 20% ethyl acetate in
petrol enabled the purification of 17-hydroxy-5␣-andro-
stan-3-one (7), 4 mg, which crystallized from methanol as
cubes, mp 184–186°, [␣]D ϩ 37° (c ϭ 0.02, EtOH); lit. [19]
177–179°, [␣]D Ϫ 55° (c ϭ 0.4, ipa); IR max 3381, 3308,
2972, 1467 cmϪ1; HRMS (EI)
⁄ (%) 290.2248 (100)
z
m
mp 181°, [␣]D ϩ 32° (EtOH); IR
3434, 2925, 1738,
max
1467 cmϪ1; HRMS (EI) m⁄
(%) 290.2244 (83) [C19H30O2],
z
[M]ϩ [C19H30O2], 275 (18), 272 (62), 257 (41), 205 (58),
272 (24), 231 (27); 1H NMR (CDCl3) ␦ 0.75 (3H, s, H-18),
1.00 (3H, s, H-19), 3.65 (1H, t, J ϭ 6 Hz, H-17␣). Increas-
ing the polarity of the solvent system to 40% then to 50%
ethyl acetate in petrol gave 3␣-hydroxy-5-androstan-17-
one (9), 104 mg. Crystallization from chloroform afforded
cubes, mp 171–173°, [␣]D ϭ ϩ84° (c ϭ 0.3, EtOH); lit. [20]
145 (54), 107 (80); 1H NMR (CDCl3) ␦ 0.75 (3H, s,
w
H-18), 1.05 (3H, s, H-19), 3.50 (1H, m,
⁄2 ϭ 14 Hz,
H-3), 3.63 (1H, t, J ϭ 8.5 Hz, H-17), 5.30 (1H, d, J ϭ
5.6 Hz, H-6). Further elution of the transformed products
with 50% ethyl acetate in petrol gave 3,6-dihydroxy-
5-androstan-17-one [17] (2), 93 mg, which crystallized
from chloroform as cubes, mp 226–230°, [␣]D ϩ 104°
mp 175–176°, [␣]D ϩ 89° (MeOH); IR
3404, 2932,
max
1738, 1465 cmϪ1; HRMS (EI)
[C19H30O2], 272 (56), 257 (50), 244 (50); 1H NMR (CDCl3)
␦ 0.80 (3H, s, H-18), 0.94 (3H, s, H-19), 3.62 (1H, m, w⁄
⁄ (%) 290.2247 (100)
z
m
(c ϭ 0.25, EtOH); IR
resolution mass spectrometry (HRMS) (EI)
3479, 3397, 1735 cmϪ1; high
max
m
⁄
z
(%)
2
ϭ
306.2182 (5) [C19H30O3], 288 (12), 270 (2), 233 (100),
138 (2), 94 (9); Acetylation of 2, 80 mg, followed by
column chromatography in 20% ethyl acetate in petrol
enabled the purification of 3,6-diacetoxy-5-andro-
16 Hz, H-3). Increasing the polarity to 80% ethyl acetate
in petrol gave 3␣,17-dihydroxy-5-androstane (8), 268
mg, which crystallized from chloroform as plates, mp 234–
236°, [␣]D ϭ ϩ18° (c ϭ 0.25, EtOH); lit. [21] mp 236°,
stan-17-one (71.3 mg), which resisted crystallization; IR
[␣]D ϩ 25° (c ϭ 0.07, EtOH); IR
3632, 3346, 2937,
max
1451 cmϪ1; HRMS (EI) m⁄
(%) 292.2390 (16) [C19H32O2],
2944, 1738, 1366, 1236 cmϪ1; MS (CI)
⁄z (%) 390
m
max
z
(0.1) [C23H34O5], 331 (0.1), 272 (2.5), 270 (100); 1H
NMR (CDCl3) ␦ 0.80 (3H, s, H-18), 1.01 (3H, s, H-19),
2.00 (3H, s, CH3CO2), 2.04 (3H, s, CH3CO2), 4.65 (1H,
274 (100), 259 (25), 256 (68), 241 (33), 215 (96). Com-
pound 8, 100 mg, was acetylated in the usual manner.
Column chromatography on the acetylated mass, 114 mg,
using 10% ethyl acetate in petrol gave pure 3␣,17-diace-
toxy-5-androstane, 99 mg, which resisted crystallization;
w
w
m, ⁄ ϭ 18 Hz, H-3␣); 4.75 (1H, m, ⁄ ϭ 9 Hz, H-6␣).
2
2
IR max 2934, 1736, 1378, 1027 cmϪ1; MS (CI) m⁄
(%) 316
z
2.3. 3-Hydroxypregn-5-en-20-one (4)
(20), 256 (33); 1H NMR (CDCl3) ␦ 0.75 (3H, s, H-18), 0.91
(3H, s, H-19), 2.0 (6H, s, 2 ϫ CH3CO2), 4.55 (1H, t, J ϭ 8
The fermentation of 4, 1 g, yielded an organic extract of
1.01 g and a dry mycelial weight of 43.6 g. The organic
extract was subjected to acetylation. This was then purified
by column chromatography using increasing concentrations
of ethyl acetate in petrol. The fraction eluting in 15% ethyl
acetate in petrol contained 3,17-diacetoxyandrost-5-ene,
142 mg, which crystallized from methanol as plates, mp
150–153°, [␣]D Ϫ 48° (c ϭ 0.11, EtOH); lit. [18] mp
w
Hz, H-17␣), 4.70 (1H, m, ⁄
2
ϭ 16 Hz, H-3).
2.5. 17␣,21-Dihydroxypregn-4-ene-3,11,20-trione (10)
Fermentation of 10, 1 g, yielded 21.2 g of dry mycelium
and 2.19 g of organic extract at harvest. Column chroma-
tography on the extract with 30% ethyl acetate in petrol
gave 3␣-hydroxy-5-androstane-11,17-dione (12), 47 mg,
which crystallized from methanol as cubes, mp 189–192°,
[␣]D ϩ 141.3° (c ϭ 0.23, EtOH); lit. [22] mp 188–189°,
[␣]D ϩ 144° (EtOH); IR max 3428, 2926, 1742, 1705, 1452,
158–159°, [␣]D Ϫ 56°; IR
2939, 1734, 1364, 1246
max
cmϪ1; MS (CI)
⁄ (%) 374 (0.6), 314 (9), 270 (100), 254
z
m
1
(2.7); H NMR (CDCl3) ␦ 0.80 (3H, s, H-18), 1.05 (3H, s,
1076 cmϪ1; HRMS (EI) m⁄
286 (62), 271 (51), 232 (100), 199 (76); H NMR (CDCl3)
z
(%) 304.2041 (64) [C19H28O3],
H-19), 2.05 (6H, s, 2 ϫ CH3CO2), 4.60 (2H, m, w⁄
H-3␣, 17␣), 5.38 (1H, d, J ϭ 8 Hz, H-6).
2
ϭ 16 Hz,
1