M.I. Choudhary et al. / Steroids 70 (2005) 798–802
799
and JMS-DA-500 mass spectrometers, m/z (rel. int.). PAAR
Hydrogenation apparatus 3916.EA was used for hydrogena-
tion.
on carbon) yielded a compound 2. Recovered yield was
95%.
2.6. Incubation of
2
.2. Microorganisms and chemicals
17α-Ethynyl-17β-hydroxyandrost-4-en-3-one (1)
Cunninghamella elegans (TSY-0865) and Cephalospo-
The substrate 1 (200 mg), distributed in 20 conical flasks,
was incubated as above for C. aphidicola. Chromatography
of the extract on Si gel yielded starting material (90 mg) and
compound 3 (11 mg).
rium aphidicola (IMI 68689) were maintained on Sabouraud-
◦
Glucose-Agar slants and stored in a refrigerator at 4 C. 17␣-
Ethynyl-17-hydroxyandrost-4-en-3-one (1) was purchased
from E. Merck, Germany.
2
.6.1. 17α-Ethynyl-17β-hydroxyandrosta-
1,4-dien-3-one (3)
White solid; mp 161–162 C, [α]D +14.2 (c 0.02, CHCl3);
UV (MeOH) λ
2
.3. Preparation of medium
◦
25
Medium (3 L) for C. elegans (TSY-0865) was prepared
(log ∈) 244 nm (3.4); lit value mp
max
26
◦
by mixing glucose (60 g), peptone (15 g), KH2PO4 (15 g)
163–164 C, [α]D 0 (c 1.150, CHCl ); λ 245 nm (15,600)
3
and yeast extract (9 g). The medium for C. aphidicola: (IMI
[10].
6
8689) was prepared by mixing these ingredients in dis-
Again the substrate 1, (300 mg) in 30 conical flasks was
incubated in the culture media of C. elegans as above. Chro-
matography of the extract on Si gel gave; starting material
(160 mg) and 17␣-ethynyl-11␣,17-dihydroxyandrost-4-en-
tilled H2O (2 L): glucose 100 g, KH2PO4 (2 g), KCl (2 g),
MgSO4·7H2O (4 g), glycine (4 g) and Gibberella trace ele-
ment solution (4 mL).
3-one (4) (5.5 mg).
2
.4. Culture and fermentation procedure
2.6.2.
The following procedures were used for each fungus:
-day-old spores were aseptically transferred into broth
17α-Ethynyl-11α,17β-dihydroxyandros-4-en-3-one (4)
Gummy [α]2 +4 (c 0.05, CHCl ); UV (MeOH) λ
5
2
D
3
max
medium flask (250 mL) containing 100 mL of freshly pre-
pared and autoclaved media. The seed flasks thus obtained
were incubated on a shake table at 30 C for 2 days. Two-day-
old broth cultures from 100 mL seed flask were inoculated
aseptically into 30 media flasks (250 mL) for C. elegans
and 20 media flasks (250 mL) for C. aphidicola containing
(log ∈) 240 nm (3.4); IR (CHCl ) ν
3407, 1710,
3
max
−
1
1
13
1659 cm ; H NMR (CD OD, 500 MHz), see Table 1;
3
C
◦
3
NMR (CD OD, 100 MHz), see Table 1; EIMS m/z (rel. int.)
+
+
328 [M ] (10), 310 (7) [M −18], 261 (17), 187 (15),145
(17), 121 (35), 119 (25), 109 (28), 91 (45), 79 (40), 55 (100);
HREIMS m/z 328.2045 (calcd for C H O , 328.2038).
21
28
3
1
00 mL of medium and fermentation was continued for fur-
ther 2 days.
Compounds 1 and 2 were dissolved in EtOH and solutions
2.7. Incubation of
17α-Ethyl-17β-hydroxyandrost-4-en-3-one (2)
were evenly distributed among 30 flasks (10 mg/0.25 mL in
each flask) for C. elegans, and 20 flasks (10 mg/0.25 mL in
each flasks) for C. aphidicola containing 48-h-old stage II
cultures. Fermentation was carried out for further 12 days on
The substrate 2 (200 mg) in 20 conical flasks was incu-
bated as described above for C. aphidicola. The extract was
chromatographed on Si gel affording; starting material 2
(120 mg) and compound 5 (4.4 mg).
◦
a rotatory shaker (200 rpm) at 29 C. In all experiments, one
control flask without biomass (for checking substrate stabil-
ity) and one flask without exogenous substrate (for checking
endogenous metabolite) were used. The culture media and
mycelium were separated by filtration. The mycelium was
washed with EtOAc (1 L) and the filtrate was extracted with
EtOAc (5 L). The combined organic extract was dried over
anhydrous Na2SO4, evaporated under reduced pressure, and
analyzed by thin layer chromatography. Control flasks were
also harvested and compared with test by TLC to confirm the
bio-transformed products.
2.7.1. 17α-Ethyl-17β-hydroxyandrosta-1,4-dien-3-one
(5)
◦
25
White solid; mp 168–170 C (uncorrected); [α]D +11.1
(c 0.07, CHCl3); UV (MeOH) λmax (log ∈) 242 nm (2.8).
The substrate 2 (300 mg) in 30 conical flasks was also
incubated as described above for C. elegans. The extract
was chromatographed on Si-gel affording starting material
(90 mg); compounds 6 (8.5 mg) and 7 (4.8 mg).
2.7.2. 17α-Ethyl-11α,17β-dihydroxyandrost-4-en-3-one
2
1
.5. Hydrogenation of
7α-Ethynyl-17β-hydroxyandrost-4-en-3-one (1)
(6)
Gummy; [α]25 +45 (c 0.06, CHCl3); UV (MeOH)
D
λmax (log ∈) 241 nm (3.5); IR (CHCl3) νmax 3401, 1708,
−
1
1
13
Sample (1.0 g) dissolved in 50 mL dry MeOH reduced
1658 cm ; H NMR (CD3OD, 400 MHz), see Table 1;
C
in a PAAR hydrogenator with a catalyst (5% palladium
NMR (CD3OD, 125 MHz), see Table 1; EIMS m/z (rel. int.)