Microbial transformation of epiandrosterone by Aspergillus sydowii

Article abstract of DOI:10.3184/174751916X14786062524888

Incubation of epiandrosterone with Aspergillus sydowii MRC 200653 afforded ten metabolites. The fungal dehydrogenation of epiandrosterone is reported for the first time. The formation of the major metabolite, 6?-hydroxyandrost-4-ene-3,17-dione, involved first dehydrogenation to give a 4-ene and then hydroxylation at C-6?. Small amounts of the substrate were hydroxylated at C-1α, C-7α, C-7β and C-11α.

Full text of DOI:10.3184/174751916X14786062524888

718 RESEARCH PAPER
VOL. 40 DECEMBER, 718–721
JOURNAL OF CHEMICAL RESEARCH 2016
Microbial transformation of epiandrosterone by Aspergillus sydowii
Kudret Yildirim* and Ali Kuru
Chemistry Department, Sakarya University, 54187 Sakarya, Turkey
Incubation of epiandrosterone with Aspergillus sydowii MRC 200653 afforded ten metabolites. The fungal dehydrogenation of
epiandrosterone is reported for the first time. The formation of the major metabolite, 6β-hydroxyandrost-4-ene-3,17-dione, involved first
dehydrogenation to give a 4-ene and then hydroxylation at C-6β. Small amounts of the substrate were hydroxylated at C-1α, C-7α, C-7β
and C-11α.
Keywords: epiandrosterone, Aspergillus sydowii, dehydrogenation, biotransformation
The remarkable regio- and stereoselectivities of microbial
steroid biotransformations have been used for a long time
in order to synthesise more valuable steroidal drugs and
hormones. There continue to be many attempts to perform more
effective microbial steroid biotransformations and to detect
new, useful microorganisms and reactions.1 Aspergillus species
are well known for their mycotoxin formation. They have been
used in studies of fundamental eukaryotic genetics and in
biotechnological exploration.2 These species are widely found
in soil, water and decaying materials. A few Aspergillus species
are regarded as pathogenic organisms for humans and animals.3
Aspergillus sydowii is considered to be a mesophilic soil
and marine saprophyte, a food contaminant and a potential
opportunistic pathogen for humans.4 There has been only
one steroid biotransformation by A. sydowii reported in
the literature.5 In the present work, epiandrosterone 1 was
incubated with A. sydowii MRC 200653 in order to investigate
its metabolism by the fungus.
The second metabolite was identified as androsta-1,4-dien-
3,17-dione 3. The 1H NMR spectrum of 3 showed a significant
downfield shift (∆ 0.41 ppm) for the 19-methyl group and had
characteristic resonances7 at δH 6.10 ppm (1H, s), δH 6.24 ppm
(1H, d, J = 10.0 Hz) and δH 7.10 ppm (1H,d, J = 10.0 Hz), which
were assigned to the 4-H, 2-H and 1-H protons, respectively.
These resonances were in accordance with the presence of 1,2-
and 4,5-double bonds. The 13C NMR spectrum of 3 lacked the
C-3 resonance of 1 at δC 71.06 ppm and showed a new resonance
at δC 184.85 ppm, confirming the presence of a new carbonyl
group conjugated with two double bonds. The C-17 resonance
1
of 1 was maintained at δC 220.07 ppm. The H and 13C NMR
data for 3 were consistent with the literature.7
The third metabolite was identified as 17β-hydroxyandrost-
1
4-en-3-one 4. The H NMR spectrum of 4 lacked the 3α-H
resonance (1H, tt, J = 5.0 and 11.0 Hz) of 1 at δH 3.59 ppm,
demonstrated a significant downfield shift (∆ 0.35 ppm) for
Incubation of epiandrosterone 1 with A. sydowii MRC 200653
for five days yielded 10 metabolites (Table 1). The first metabolite
was identified as 5α-androst-1-ene-3,17-dione 2 (Fig. 1). The 1H
NMR spectrum of 2 had a downfield shift (∆ 0.21 ppm) for the
19-methyl group and demonstrated characteristic 1,2-double
bond resonances6 at δH 5.86 ppm (1H, d, J = 10.0 Hz) and
δ 7.15 ppm (1H, d, J = 10.0 Hz). The 13C NMR spectrum of
2H showed resonances at δ 157.82 and δC 127.63 (Table 2),
confirming the presence ofCa 1,2-double bond. In addition to
this, the 13C NMR spectrum of 2 lacked the C-3 resonance of 1
at δ 71.06 ppm and showed a new resonance at δC 200.00 ppm,
indiCcating the presence of a new carbonyl group conjugated with
a 1,2-double bond. The C-17 resonance of 1 was retained at δC
220.57 ppm. Both the melting point and the 1H NMR data for 2
were comparable with the values reported in the literature.6
Table 1 Metabolite yields following chromatography
Substrate
Metabolite
Yield/%
Epiandrosterone 1
2
5
2
5α-Androst-1-ene-3,17-dione 2
Androsta-1,4-diene-3,17-dione 3
17β-Hydroxyandrost-4-en-3-one 4
3
17β-Hydroxyandrosta-1,4-dien-3-one 5
6β-Hydroxyandrost-4-en-3,17-dione 6
6β,17β-Dihydroxyandrost-4-ene-3-one 7
3β,11α-Dihydroxy-5α-androstan-17-one 8
1α,3β-Dihydroxy-5α-androstan-17-one 9
3β,7β-Dihydroxy-5α-androstan-17-one 10
3β,7α-Dihydroxy-5α-androstan-17-one 11
32
2
2
5
3
2
R2
O
O
R1
R3
R1
H
H
H
H
O
HO
R2
O
O
H
H
R1
1 R1 = R3 = H, R2 = H2
2
3 R1 = O
4 R1 = H2, R2 = βOH,αH
6 R1 = βOH,αH, R2 = O
7 R1 = R2 = βOH,αH
8 R1 = H, R2 = H2, R3 = OH
9 R1 = OH, R2 = H2, R3 = H
5 R1 = βOH,αH
10 R1 = H, R2 = βOH,αH, R3 = H
11 R1 = H, R2 = βH,αOH, R3 = H
Fig. 1 Chemical structures of compounds 1–11.
* Correspondent. E-mail: kudrety@sakarya.edu.tr

JOURNAL OF CHEMICAL RESEARCH 2016 719
Table 2 13C NMR data of compounds 1–11 determined in CDCl3
Carbon atom
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
10
36.77
31.35
70.91
37.56
42.03
38.76
74.76
42.88
52.49
35.10
20.70
31.50
48.27
51.01
24.92
36.01
221.00
14.04
12.43
11
36.63
31.31
71.00
37.53
37.06
35.70
66.78
39.06
46.07
36.00
20.28
31.12
47.57
45.81
21.33
36.63
221.10
13.50
11.20
36.87
31.35
71.06
37.98
44.76
28.32
30.83
34.97
54.35
35.57
20.43
31.47
47.76
51.34
21.72
35.80
221.50
13.76
12.25
157.82
127.63
200.00
40.85
44.28
27.32
30.08
35.17
51.28
38.97
20.46
31.44
47.77
50.08
21.70
35.79
220.57
13.88
13.03
155.54
127.54
184.85
123.97
168.32
32.21
31.06
34.98
52.15
43.41
22.01
32.47
47.62
50.28
21.83
35.56
220.07
13.72
18.61
35.61
33.85
199.72
123.73
171.51
33.07
31.44
35.55
53.81
38.59
20.55
36.32
42.72
50.37
23.25
30.20
81.31
10.99
17.32
156.05
127.33
186.44
123.73
169.40
32.73
32.73
35.47
52.42
43.59
22.43
36.23
43.02
50.00
23.45
30.28
81.47
11.10
36.88
34.04
200.58
126.20
168.31
72.40
37.09
29.27
53.48
37.95
20.13
31.11
47.55
50.72
21.59
35.69
220.85
13.66
19.40
36.28
34.08
200.80
126.01
168.90
72.57
36.90
29.65
53.58
37.96
20.46
37.96
42.77
50.32
23.16
30.17
81.46
11.07
19.37
38.41
31.63
70.72
38.41
44.94
28.80
30.68
34.13
60.34
37.07
68.56
42.82
47.98
50.18
21.65
35.84
220.16
14.49
12.70
72.74
38.36
66.36
37.80
37.25
28.15
30.44
34.92
46.86
39.56
19.78
31.28
47.77
51.24
21.75
35.78
221.63
13.72
12.87
8
9
10
11
12
13
14
15
16
17
18
19
18.63
the 19-methyl group, and had a characteristic resonance8 at
δH 5.72 ppm (1H, s), suggesting the presence of a 4-en-3-keto
moiety in ring A. The 13C NMR spectrum of 4 showed new
resonances at δ 199.72 ppm (C-3), δC 171.51 ppm (C-5) and δ
123.73 ppm (C-C4), further suggesting the presence of a 4-en-3C-
keto moiety. The 13C NMR spectrum of 4 also lacked the C-17
resonance of 1 at δC 221.50 ppm and showed a new resonance at
δ 81.31 ppm, indicating the presence of a 17β-hydroxyl group.
CThe fourth metabolite was identified as 17β-hydroxyandrosta-
presence of a 4-en-3-keto moiety in ring A. The NMR spectra
of 7 also contained new resonances at δH 4.35 ppm (1H, bs)
and δC 72.57 ppm, which are typical of a 6β-hydroxyl group.
The 13C NMR spectrum of 7 showed a downfield shift for C-7
(∆ 6.07 ppm), whereas it showed a γ-gauche upfield shift for C-8
(∆ 5.32 ppm), suggesting the presence of a 6β-hydroxyl group.
The 13C NMR spectrum of 7 also lacked the C-17 resonance
of 1 at δC 221.50 ppm and had a new resonance at δC 81.46,
confirming the presence of a 17β-hydroxyl group.
1
1,4-dien-3-one 5. The H NMR spectrum of the metabolite
The seventh metabolite was identified as 3β,11α-dihydroxy-
5α-androstan-17-one 8. The NMR spectra of 8 had new
resonances at δ 3.96 ppm (1H, dt, J = 5.0 and 10.0 Hz) and
δC 68.56 ppm, Hsuggesting the presence of an 11α-hydroxyl
group.10 The 13C NMR spectrum of 8 showed a downfield shift
for C-9 (∆ 5.99 ppm), whereas it showed a γ-gauche upfield
shift for C-8 (∆ 0.84). These results further suggested that an
11α-hydroxylation had taken place. The 3α-H resonance (1H,
tt, J = 5.0 and 11.0 Hz) of 1 remained at δ 3.57 ppm.
showed a significant downfield shift (∆ 0.41 ppm) for the
19-methyl group and had characteristic resonances7 at δ
6.08 ppm (1H, s), δH 6.21 ppm (1H, dd, J = 1.8 and 10.0 HzH)
and δH 7.05 ppm (1H, d, J = 10.0 Hz), suggesting the presence
of 1,2- and 4,5-double bonds in ring A. The 13C NMR spectrum
of 5 lacked the C-3 resonance of 1 at δ 71.06 ppm and showed
a new resonance at δC 186.44 ppm, indCicating the presence of a
new carbonyl group conjugated with two double bonds. The 13C
NMR spectrum of 5 also lacked the C-17 resonance of 1 at δC
221.50 ppm and had a new resonance at δC 81.47, confirming
The eighth metabolite was identified Has 1α,3β-dihydroxy-
5α-androstan-17-one 9. The 1H NMR spectrum of 9 had
characteristic resonances11 at δH 3.83 ppm (1H, bs) and
1
the presence of a 17β-hydroxyl group. The H and 13C NMR
data for 5 were consistent with the literature.7
δ 4.03 ppm (1H, tt, J = 5.0 and 11.0 Hz), indicating the
The fifth metabolite was identified as 6β-hydroxyandrost-
pHresence of a 1α-hydroxyl group and a 3β-hydroxyl group,
respectively. The 13C NMR spectrum of 9 showed downfield
shifts for C-2 (∆ 7.01 ppm) and C-10 (∆ 3.99 ppm), whereas it
showed γ-gauche upfield shifts for C-5 (∆ 7.51 ppm) and C-9
(∆ 7.49 ppm), confirming the presence of a 1α-hydroxyl group.
1
4-ene-3,17-dione 6. The H NMR spectrum of 6 showed a
significant downfield shift (∆ 0.55 ppm) for the 19-methyl group
and had a new resonance at δ 5.82 ppm (1H, bs). The 13C NMR
spectrum of 6 lacked the C-H3 resonance of 1 at δC 71.06 ppm
and showed a new resonance at δC 200.58 ppm. These results
indicated the presence of a 4-en-3-keto moiety. NMR spectra
of 6 had characteristic resonances8,9 at δH 4.36 ppm (1H, bs) and
δC 72.40 ppm, suggesting the presence of a 6β-hydroxyl group.
The 13C NMR spectrum of 6 showed a downfield shift for C-7
(∆ 6.26 ppm), whereas it showed a γ-gauche upfield shift for
C-8 (∆ 5.70 ppm), further suggesting that a 6β-hydroxylation
had taken place. The C-17 resonance of 1 remained at δC
220.85 ppm.
The sixth metabolite was identified as 6β,17β-
dihydroxyandrost-4-en-3-one 7. The NMR spectra of 7
lacked the C-3 resonance of 1 at δ 71.06 ppm and revealed a
downfield shift (∆ 0.54 ppm) for thCe 19-methyl group, together
with a new resonance at δH 5.81 ppm (1H, bs), indicating the
1
The H NMR spectrum of 9 showed a significant downfield
shift (∆ 0.44 ppm) for the 3α-H resonance (1H, tt, J = 5.0 and
11.0 Hz) of 1, suggesting the presence of a 1α-hydroxyl group in
close proximity. Both the melting point12 and the 1H NMR data13
for 9 were comparable with the values reported in the literature.
The ninth metabolite was identified as 3β,7β-dihydroxy-
5α-androstan-17-one 10. NMR spectra of 10 showed new
resonances at δH 3.45 ppm (1H, m) and δC 74.76 ppm, which
are typical of a 7β-hydroxyl group.14 The 13C NMR spectrum
of 10 showed downfield shifts for C-6 (∆ 10.44 ppm) and C-8
(∆ 7.91 ppm), whereas it showed γ-gauche upfield shifts for C-5
(∆ 2.73 ppm) and C-9 (∆ 1.86 ppm). These shifts are consistent
1
with the presence of a 7β-hydroxyl group. The H NMR
spectrum of 10 had a resonance (1H, tt, J = 5.0 and 11.0 Hz)

720 JOURNAL OF CHEMICAL RESEARCH 2016
A. sydowii MRC 200653 was obtained from the TÜBİTAK Marmara
Research Center, Food Science and Technology Research Institute,
Culture Collection Unit, Kocaeli, Turkey. Stock cultures were
maintained at 4 °C on PDA slopes. The biotransformation experiment
was performed in duplicate and run with control flasks containing
non-inoculated sterile medium and the substrate. After 5 days of
incubation, all controls were harvested and analysed by TLC. No
metabolites were detected in the controls.
at δ 3.60 ppm, indicating that the 3β-hydroxyl group was
retaiHned.
The tenth metabolite was identified as 3β,7α-dihydroxy-5α-
androstan-17-one 11. NMR spectra of 11 had characteristic
resonances10 at δ 3.95 ppm (1H, bs) and δC 66.78 ppm, indicating
the presence of Ha 7α-hydroxyl group. The 13C NMR spectrum
of 11 showed downfield shifts for C-6 (∆ 7.38 ppm) and C-8
(∆ 4.09 ppm), whereas it showed γ-gauche upfield shifts for
C-5 (∆ 7.70 ppm) and C-9 (∆ 8.28 ppm), further indicating the
presence of a 7α-hydroxyl group. The 3α-H resonance (1H, tt,
J = 5.0 and 11.0 Hz) of 1 was maintained at δH 3.58 ppm.
As can be seen from Table 1, most of epiandrosterone 1 was
dehydrogenated and then hydroxylated mainly at C-6β by A.
sydowii MRC 200653, whereas some of the remaining substrate
was hydroxylated at C-1α, C-7α, C-7β and C-11α.
According to the literature, the dehydrogenation of steroids by
some fungal species has been observed.5,7,15–22 Dehydrogenation
in ring A of steroids is widely used in the production of
corticosteroids.23 Synthetic steroidal drugs with a 1,2-double
bond have a greater affinity for the glucocorticoid receptors,
and the presence of a 1,2-double bond together with a 3-keto-4-
ene moiety gives these compounds higher therapeutic potency
by reducing their rate of metabolic degradation.7,23 Introduction
of a 1,2-double bond into the steroids is very rare in fungi
although it is very common in bacteria.24 Only a few fungi,
such as Phycomyces blakesleeanus,15 Nectria haematococca,16
Trichoderma hamatum,17 Cephalosporium aphidicola,18
Fusarium lini,7,18 Fusarium oxysporum,19 Acremonium
strictum20 and Aspergillus niger,21 were known to perform this
type of dehydrogenation. In this work, A. sydowii carried out
the introduction of both a 1,2-double bond and a 4,5-double
bond into the substrate simultaneously as well as separately.
Furthermore, this work is the first epiandrosterone incubation
affording dehydrogenated metabolites as far as fungal steroid
biotransformations are concerned.
Biotransformation of epiandrosterone 1
The liquid medium for A. sydowii MRC 200653 was prepared by
mixing malt extract (30 g) and peptone (3 g) in 1 L of artificial
seawater containing CaCl2.2H2O (1.36 g), MgCl2.6H2O (9.68 g), KCl
(0.61 g), NaCl (30 g), Na2HPO4 (0.014 mg), Na2SO4 (3.47 g), NaHCO3
(0.17 g), KBr (0.1 g), SrCl2.6H2O (0.04 g) and H3BO3 (0.03 g).26 The
final pH of the medium was adjusted to 8.0 by the addition of 3M
KOH. The medium was evenly distributed among ten culture flasks
of 250 mL capacity (100 mL in each) and autoclaved for 20 minutes
at 121 °C. Spores freshly obtained from a PDA slope were transferred
aseptically to each flask containing sterile medium in a biological
safety cabinet. After cultivation at 32 °C for three days on a rotary
shaker (150 rpm), epiandrosterone 1 (1 g) dissolved in 10 mL of
dimethylformamide (DMF) was evenly distributed aseptically among
the flasks and the biotransformation was carried out in ten flasks
for five days under the same conditions. The fungal mycelium was
separated from the broth by filtration under vacuum, and the mycelium
was rinsed with ethyl acetate (500 mL). The broth was extracted three
times each with 1 L of ethyl acetate. The organic extract was dried over
anhydrous sodium sulfate, and the solvent evaporated in vacuo to give
a brown gum (2847 mg) which was then chromatographed on silica
gel. Elution with 20% ethyl acetate in n-hexane yielded 5α-androst-
1-ene-3,17-dione 2 (20 mg, 2%), which crystallised from ethyl acetate
as needles, m.p. 141–142 °C (lit.6 139.5–140.5 °C), vmax/cm−1 1740 and
1671; δH 0.93 (3H, s, 18-H), 1.04 (3H, s, 19-H), 5.86 (1H, d, J = 10.0 Hz,
2-H), 7.15 (1H, d, J = 10.0 Hz, 1-H).
Elution with 30% ethyl acetate in n-hexane yielded the unreacted
starting material (155 mg), which was identified by comparison of its
1H and 13C NMR spectra with those of an authentic sample.
Further elution with 30% ethyl acetate in n-hexane yielded androsta-
1,4-dien-3,17-dione 3 (49 mg, 5%), which crystallised from acetone–n-
hexane as needles, m.p. 143–144 °C (lit.27 141–142 °C), vmax/cm−1 2920,
2860, 1738 and 1660; δH 0.94 (3H, s, 18-H), 1.24 (3H, s, 19-H), 6.10
(1H, s, 4-H), 6.24 (1H, d, J = 10.0 Hz, 2-H), 7.10 (1H, d, J = 10.0 Hz,
1-H).
Elution with 40% ethyl acetate in n-hexane yielded
17β-hydroxyandrost-4-en-3-one 4 (20 mg, 2%), which crystallised
from acetone as prisms, m.p. 157–158 °C (lit.28 154–155 °C), vmax/cm−1
3200, 1655 and 1615; δH 0.80 (3H, s, 18-H), 1.18 (3H, s, 19-H), 3.65
(1H, t, J = 8.5 Hz, 17α-H), 5.72 (1H, s, 4-H).
Most of the dehydrogenated material was hydroxylated
only at C-6β, whilst a minor hydroxylation of the remaining
epiandrosterone
1 occurred at C-1α, C-7α, C-7β and
C-11α. In the previous work, A. sydowii dehydrogenated
dehydroepiandrosterone predominantly and then hydroxylated
it at C-6β, whilst the remaining dehydroepiandrosterone
was hydroxylated only at C-7α or C-7β.5 In this work, the
1α-hydroxylation of epiandrosterone 1 by A. sydowii could
have been due to the hydration of the 1,2-double bond, as in the
testosterone catabolism by Steroidobacter denitrificans.25
Experimental
Further elution with 40% ethyl acetate in n-hexane yielded
17β-hydroxyandrosta-1,4-dien-3-one 5 (31 mg, 3%), which crystallised
from acetone as needles, m.p. 172–73 °C (lit.28 169–170 °C), vmax/cm−1
3520, 1660, 1620 and 1600; δH 0.82 (3H, s, 18-H), 1.24 (3H, s, 19-H),
3.64 (1H, t, J = 8.5 Hz 17α-H), 6.08 (1H, s, 4-H), 6.21 (1H, dd, J = 1.8
and 10.0 Hz, 2-H), 7.05 (1H, d, J = 10.0 Hz, 1-H).
Epiandrosterone 1 was purchased from Sigma-Aldrich (Istanbul,
Turkey). Solvents were of analytical grade and were purchased from
Merck (Istanbul). Potato dextrose agar (PDA) and agar for PDA slopes
and ingredients for liquid media were also purchased from Merck. The
steroids were separated by column chromatography on silica gel 60
(Merck 107734), eluting with increasing concentrations of ethyl acetate
in n-hexane. Thin layer chromatography (TLC) was carried out with
0.2 mm thick Merck Kieselgel 60 F254 TLC plates using ethyl acetate/n-
hexane (1:1) as eluent. TLC plates were dipped into an anisaldehyde/
H2SO4 reagent and heated to 120 °C for 3 minutes in order to visualise the
spots. Infrared spectra were recorded using a PerkinElmer Spectrum Two
spectrometer. 1H NMR spectra were recorded in deuteriochloroform with
tetramethylsilane as internal reference at 300 MHz with a Varian Mercury
300 spectrometer unless otherwise specified. 13C NMR spectra were
recorded in deuteriochloroform at 75 MHz with a Varian Mercury 300
spectrometer unless otherwise specified. Chemical shifts are given in ppm
(δ scale) and coupling constants (J) are given in Hz. Melting points were
determined by an Electrothermal IA 9200 melting point apparatus and are
uncorrected.
Elution with 50% ethyl acetate in n-hexane yielded
6β-hydroxyandrost-4-ene-3,17-dione
6 (333 mg, 32%), which
crystallised from acetone as prisms, m.p. 193–194 °C (lit.29
190–193 °C), vmax/cm−1 3420, 1735 and 1670; δH 0.93 (3H, s, 18-H), 1.38
(3H, s, 19-H), 4.36 (1H, bs, 6α-H), 5.82 (1H, bs, 4-H).
Elution with 60% ethyl acetate in n-hexane yielded 6β,17β-
dihydroxyandrost-4-en-3-one 7 (21 mg, 2%), which crystallised from
ethyl acetate as prisms, m.p. 213–214 °C (lit. 29 215–220 °C), vmax/cm−1
3495, 1660 and 1630; δH 0.83 (3H, s, 18-H), 1.37 (3H, s, 19-H), 3.64
(1H, t, J = 8.5 Hz, 17α-H), 4.35 (1H, bs, 6α-H), 5.81 (1H, bs, 4-H).
Further elution with 60% ethyl acetate in n-hexane yielded 3β,11α-
dihydroxy-5α-androstan-17-one 8 (22 mg, 2%), which crystallised
from acetone as plates, m.p. 105–106 °C (lit.10 103–106 °C), vmax/cm−1

JOURNAL OF CHEMICAL RESEARCH 2016 721
7
8
9
A. Al-Aboudi, M.Y. Mohammed, S.G. Musharraf, M.I. Choudhary and
A.U. Rahman, Nat. Prod. Res., 2008, 22, 1498.
D.N. Kirk, H.C. Toms, C. Douglas, K.A. White, K.E. Smith, S. Latif and
R.W.P. Hubbard, J. Chem. Soc. Perkin Trans. 2, 1990, 1567.
J.W. Blunt and J.B. Stothers, Org. Magn. Reson., 1977, 9, 439.
3473 and 1740; δH 0.92 (3H, s, 18-H), 1.01 (3H, s, 19-H), 3.57 (1H, tt,
J = 5.0 and 11.0 Hz, 3α-H), 3.96 (1H, dt, J = 5.0 and 10.0 Hz, 11β-H).
Elution with 70% ethyl acetate in n-hexane yielded 1α,3β-dihydroxy-
5α-androstan-17-one 9 (53 mg, 5%), which crystallised from ethyl
acetate as prisms, m.p. 204–205 °C (lit.12 200–201 °C), vmax/cm−1 3495,
3430 and 1730; δH 0.86 (3H, s, 18-H), 0.83 (3H, s, 19-H), 3.83 (1H, bs,
1β-H), 4.03 (1H, tt, J = 5.0 and 11.0 Hz, 3α-H).
Elution with 80% ethyl acetate in n-hexane yielded 3β,7β-dihydroxy-
5α-androstan-17-one 10 (32 mg, 3%), which crystallised from acetone
as cubes, m.p. 238–239 °C (lit.14 240–244 °C), vmax/cm−1 3425 and
1740; δH 0.86 (3H, s, 18-H), 0.89 (3H, s, 19-H), 3.45 (1H, m, 7α-H),
3.60 (1H, tt, J = 5.0 and 11.0 Hz, 3α-H).
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Further elution with 80% ethyl acetate in n-hexane yielded 3β,7α-
dihydroxy-5α-androstan-17-one 11 (21 mg, 2%), which crystallised
from acetone as prisms, m.p. 197–198 °C (lit.10 194 °C), vmax/cm−1 3600,
3420 and 1740; δH 0.84 (3H, s, 18-H), 0.87 (3H, s, 19-H), 3.58 (1H, tt,
J = 5.0 and 11.0 Hz, 3α-H), 3.95 (1H, bs, 7β-H).
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Acknowledgements
This work was financially supported by the Sakarya
University Scientific Research Foundation, Turkey (Project
No. 20160204016). We are grateful to Rasit Yilmaz and Yavuz
Derin for running the 1H and 13C NMR spectra.
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Received 31 August 2016; accepted 4 October 2016
Paper 1604294 doi: 10.3184/174751916X14786062524888
Published online: 14 November 2016
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