Novel metabolites of dehydroepiandrosterone and progesterone obtained in Didymosphearia igniaria KCH 6670 culture

Article abstract of DOI:10.1016/j.molcatb.2012.05.009

Dehydroepiandrosterone (DHEA) (10) and its five derivatives: testosterone (1), androstenedione (2), 17α-methyltestosterone (6), progesterone (13) and pregnenolone (14) were subjected to microbial transformation by the filamentous fungus Didymosphaeria igniaria KCH 6670. The predominant metabolism of the incubated 5-ene steroids (10 and 14) occurred through 3β-hydroxy-steroid dehydrogenase/5,4-en isomerase pathways resulting in the generation of a 4-en-3-oxo system on ring-A. The transformations of C ₁₉ steroids (1, 2, and 10) included a hydroxylation at 7α position, ketone-alcohol interconversion at C-17 and reduction of the double bond at C-4 and 3-keto group to the 3β-alcohol with 5α- stereochemistry at A/B ring. D. igniaria also carried out 6(7)-dehydrogenation and 6,7β-epoxidation during transformation of DHEA. Under these conditions transformation of DHEA (10) gave four products: 7α-hydroxyandrost-4-en-3, 17-dione (4), 17β-hydroxyandrost-4,6-dien-3-one (11), 17β- hydroxyandrost-6β-epoxy-4-en-3-one (12) and 3β,17β-dihydroxy- 5α-androstane (5). The compounds 11 and 12 are identified as DHEA metabolites for the first time. The transformation of C₂₁ steroids (13 and 14) led to the mixture of mono- (mainly 11α- and 15β-) and dihydroxy- (7α,15β-; 14α,15β-; 11α,15β-; 11α,14α-) products. 7α,15β-Dihydroxypregnan-4-en-3,20- dione (18) and 14α,15β-dihydroxypregnan-4-en-3,20-dione (19) were found to be new compounds. The main product of transformation of 17α-methyltestosterone (6) was 12β-hydroxy-17α- methyltestosterone (7). The results of these transformations demonstrate the dependence of hydroxylation position on the structure of steroid nucleus.

Full text of DOI:10.1016/j.molcatb.2012.05.009

Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Novel metabolites of dehydroepiandrosterone and progesterone obtained in
Didymosphearia igniaria KCH 6670 culture
Tomasz Janeczkoa,∗, Alina Swizdor , Jadwiga Dmochowska-Gładysza,b, Agata Białon´ skac,
´
a
Zbigniew Ciunikc, Edyta Kostrzewa-Susłowa
a Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
b Department of Cosmetology, Wrocław College of Physiotherapy, Ko´sciuszki 4, 50-038 Wrocław, Poland
c Faculty of Chemistry, University of Wrocław, F. Joliot-Curie 14, 50-383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Dehydroepiandrosterone (DHEA) (10) and its five derivatives: testosterone (1), androstenedione (2),
17␣-methyltestosterone (6), progesterone (13) and pregnenolone (14) were subjected to microbial trans-
formation by the filamentous fungus Didymosphaeria igniaria KCH 6670. The predominant metabolism
of the incubated 5-ene steroids (10 and 14) occurred through 3␤-hydroxy-steroid dehydrogenase/5,4-en
isomerase pathways resulting in the generation of a 4-en-3-oxo system on ring-A. The transfor-
mations of C19 steroids (1, 2, and 10) included a hydroxylation at 7␣ position, ketone–alcohol
interconversion at C-17 and reduction of the double bond at C-4 and 3-keto group to the 3␤-alcohol
with 5␣-stereochemistry at A/B ring. D. igniaria also carried out 6(7)-dehydrogenation and 6,7␤-
epoxidation during transformation of DHEA. Under these conditions transformation of DHEA (10) gave
four products: 7␣-hydroxyandrost-4-en-3,17-dione (4), 17␤-hydroxyandrost-4,6-dien-3-one (11), 17␤-
hydroxyandrost-6␤-epoxy-4-en-3-one (12) and 3␤,17␤-dihydroxy-5␣-androstane (5). The compounds
11 and 12 are identified as DHEA metabolites for the first time. The transformation of C21 steroids (13 and
14) led to the mixture of mono- (mainly 11␣- and 15␤-) and dihydroxy- (7␣,15␤-; 14␣,15␤-; 11␣,15␤-;
11␣,14␣-) products. 7␣,15␤-Dihydroxypregnan-4-en-3,20-dione (18) and 14␣,15␤-dihydroxypregnan-
4-en-3,20-dione (19) were found to be new compounds. The main product of transformation of
17␣-methyltestosterone (6) was 12␤-hydroxy-17␣-methyltestosterone (7). The results of these trans-
formations demonstrate the dependence of hydroxylation position on the structure of steroid nucleus.
Received 24 December 2011
Received in revised form 13 May 2012
Accepted 13 May 2012
Available online 22 May 2012
Keywords:
Biotransformation
DHEA
Didymosphaeria igniaria
Hydroxylation
Testosterone
Progesterone
Pregnenolone
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
about the metabolic pathway of a xenobiotic. The term “micro-
bial models of mammalian drug metabolism” was introduced by
Microbial transformation of steroids is an import method of
obtaining new steroid derivatives of potential pharmacological
activity, which fulfills “green chemistry” principles and meets
demands of “white biotechnology”. Microbial monooxygenases
reveal high similarity to mammal ones, therefore they may be used
to trace and preview metabolism of the tested compounds in higher
organisms [1–3].
The knowledge about the transformations that a certain com-
pound may undergo in living organisms is particularly important
in case of new biologically active compounds introduced into phar-
maceutical market. Microbial models of mammalian metabolism
may be an alternative, or at least may be complementary to tradi-
tional methods, because they provide a lot of valuable information
Smith and Rosazza in 1974 [1] and it means using microorgan-
ism in order to facilitate studies on metabolism of xenobiotics in
mammals [2–6]. Recent years have witnessed intensive ongoing
research on metabolism of 3␤-hydroxy-5-ene steroids – mainly
DHEA (dehydroepiandrosterone) (10), pregnenolone (14) and their
derivatives [7–11]
Our previous research on optically pure bicyclic enones [12]
showed that the strain of Didymosphaeria igniaria KCH 6670 con-
tained active monooxygenases. That was an inspiration for further
study on this microorganism as a biocatalyst in transformations
of steroid compounds. In this work we described the ability
of the strain D. igniaria to transformation of testosterone (1),
androstenedione (2), 17␣-methyltestosterone (6), DHEA (10), pro-
gesterone (13) and pregnenolone (14). Comparing the course of
transformation of the aforementioned substrates allowed us to
find how the position of hydroxylation depends on the compound
structure.
∗
Corresponding author. Tel.: +48 71 3205252; fax: +48 71 3284124.
E-mail address: janeczko13@interia.pl (T. Janeczko).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.009

T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
25
2. Materials and methods
CrysAlis CCD and CrysAlis Red programs [13]. Structure was solved
by direct methods (program SHELXS97) and refined by the full
matrix least-squares method on all F2 data using the SHELXL97
programs [14]. Non-hydrogen atoms were refined with anisotropic
displacement parameters; hydrogen atoms were placed in cal-
culated positions or found in ꢁꢂ maps. Before the last cycle of
refinement all H atoms were fixed and were allowed to ride on their
parent atoms. The Friedel pairs were merged before the final refine-
ment. The absolute structure was chosen on the basis of known
absolute configuration of the substrate. Crystallographic data for
crystal 18 in this paper have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication CCDC
869598. Copies of the data can be obtained, free of charge, on appli-
cation to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Tel.: +44
1223 762911 or e-mail: deposit@ccdc.cam.ac.uk).
2.1. Materials
The substrates: 17␤-hydroxyandrost-4-en-3-one (1), androst-
4-en-3,17-dione (2), 17␣-methyl-17␤-hydroxyandrost-4-en-3-
one (6), 3␤-hydroxyandrost-5-en-17-one (10), pregnan-4-en-
3,20-dione (13) and 3␤-hydroxypregnan-5-en-20-one (14) were
purchased from Sigma–Aldrich. The microorganism D. igniaria KCH
6670 used in this study was obtained from the collection of the
Department of Forest Phytopathology of the University of Agricul-
ture in Kraków, Poland. It was isolated from forest environment
(south Poland) from dead parts of leafy plants. The strain was
maintained on Sabouraud 4% dextrose-agar slopes and freshly sub-
cultured before use in the transformation experiments.
2.2. Screening procedure
2.5. Spectral data of isolated metabolites
Erlenmeyer flasks (300 ml), each containing 100 ml of the
medium consisting of 3 g glucose and 1 g aminobac dissolved in
water, were inoculated with a suspension of microorganisms and
then incubated for 3 days at 25 ◦C on a rotary shaker. After full
growth of the culture (about 12 g of cell dry weight/l) 20 mg of
a substrate dissolved in 1 ml of acetone was added. After 1, 3, 6
and 9 days of incubation under the above conditions, portions of
10 ml of the transformation mixture were taken out and extracted
with CHCl3 (3× 10 ml). The extracts were dried over MgSO4, con-
centrated in vacuo and analyzed by GC. All the experiments were
repeated three times.
The product structures were determined by means of elemental
analysis, 1H NMR, 13C NMR and correlation spectroscopy. 13C NMR
spectra of all the products obtained are summarized in Table 1. For
compound 18 was additionally performed crystallographic analysis
(Fig. 1).
2.5.1. 7˛-Hydroxytestosterone (3)
After 3 days of incubation of testosterone (1) (100 mg) and
androstenedione (2) (100 mg), 52 mg and 17 mg of compound 3
were isolated respectively. Anal. Calcd. for C19H28O3: C 74.96, H
9.27%; found: C 75.02, H 9.37%. Mass spectrum indicated a molec-
ular ion at m/z 305 of composition C19H
26O3; 1H NMR (CDCl3)
2.3. Preparative biotransformation
ı (ppm): 0.78 (s, 3H, 18-CH3); 1.19 (s, 3H, 19-CH3); 3.69 (t, 1H,
J = 8.47 Hz, H-17); 3.96 (m, 1H, Wh = 9.6 Hz, H-7␤); 5.78 (s, 1H, H-4).
The same transformations were performed on the preparative
scale in 2000 ml flasks, each containing 500 ml of the cultivation
medium. The cultures were incubated under the same conditions
and then 200 mg of substrate dissolved in 2 ml of acetone was added
to the grown cultures. After 3–9 days of incubation the mixtures
were extracted with CHCl3 (3× 300 ml), dried (MgSO4) and con-
centrated in vacuo. The transformation products were separated
by column chromatography and analyzed (TLC, GC, GC–MS).
2.5.2. 7˛-Hydroxyandrostenedione (4)
After
3 days of incubation of testosterone (1) (100 mg),
androstenedione (2) (100 mg) and DHEA (10) (100 mg), 16 mg,
39 mg and 21 mg of compound 4 were isolated respectively. Anal.
Calcd. for C19H26O3: C 75.46, H 8.67%; found: C 75.49, H 8.69%.
Mass spectrum indicated a molecular ion at m/z 303 of composition
C
19H26O3; 1H NMR (CDCl3) ı (ppm): 0.91 (s, 3H, 18-CH3); 1.21 (s,
2.4. Analytical methods
3H, 19-CH3); 4.09 (br s, Wh = 10.6 Hz, 1H, H-7␤); 5.82 (s, 1H, H-4).
The course of biotransformation was controlled by means of
TLC. Composition of product mixtures was established by GC. Prod-
ucts were separated by column chromatography using silica gel
(Kieselgel 60, 230–400 mesh, Merck) and hexane/acetone mix-
ture (2:1, v/v) as eluent. Analytical TLC was carried out on silica
gel G (Merck). Compounds were detected by spraying the plates
with H2SO4/CH3OH mixture (1:1, v/v). GC analysis was performed
using a Hewlett-Packard 5890A (Series II) GC instrument fitted with
a flame ionization detector (FID). The HP-5 (crosslinked phenyl
methyl siloxane) capillary column (30 m × 0.32 mm × 0.25 ␮m)
was used to determine the composition of product mix-
tures. The following temperature programme was used: 200 ◦C
(0 min)/10 ◦C/min/270 ◦C (0 min)/30 ◦C/min/300 ◦C (5 min). The
NMR spectra were recorded on DRX 500 MHz Bruker spectrometer
and measured in CDCl3. MS analyses were performed on a Varian
Chrompack GC CP-3800 Saturn 2000GC/MS/MS with an ionizing
energy of 70 eV. Elemental analysis was carried out on the Vario
EL III CHNS (Elementor). Crystallographic measurement was per-
formed at 100 K using an Oxford Cryosystem device on a Kuma
KM4CCD ␬-axis diffractometer with a graphite-monochromated
2.5.3. 3ˇ,17ˇ-Dihydroxy-5˛-androstane (5)
After
3 days of incubation of testosterone (1) (100 mg),
androstenedione (2) (100 mg) and DHEA (10) (100 mg), 6 mg, 5 mg
and 4 mg of compound 5 were isolated respectively. Anal. Calcd.
for C19H32O2: C 78.03, H 11.03%; found: C 77.89, H 11.19%. Mass
spectrum indicated a molecular ion at m/z 293 of composition
C19H32O2; 1H NMR (CDCl3) ı (ppm): 0.80 (s, 3H, 19-CH3); 0.83 (s,
3H, 18-CH3); 3.58–3.68 (m, 2H, H-3␣ and H-17␣).
2.5.4. 12ˇ-Hydroxy-17˛-methyltestosterone (7)
Six-day transformation of 17␣-methyltestosterone (6) (100 mg)
yielded 32 mg of compound 7. Anal. Calcd. for C20H30O3: C 75.43, H
9.50%; found: C 75.31, H 9.79%. Mass spectrum indicated a molec-
ular ion at m/z 319 of composition C20H30O3; 1H NMR (CDCl3) ı
(ppm): 0.95 (s, 3H, 18-CH3); 1.19 (s, 3H, 19-CH3); 1.34 (s, 3H, 17␣-
CH3); 3.74 (dd, 1H, J = 16.8, 7.2 Hz, H-12␣); 5.72 (s, 1H, H-4).
2.5.5. 7˛-Hydroxy-17˛-methyltestosterone (8)
Six-day transformation of 17␣-methyltestosterone (6) (100 mg)
yielded 25 mg of compound 8. Anal. Calcd. for C20H30O3: C 75.43, H
9.50%; found: C 75.59, H 9.28%. Mass spectrum indicated a molec-
ular ion at m/z 319 of composition C20H30O3; 1H NMR (CDCl3) ı
˚
Mo K␣ radiation (ꢀ = 0.71073 A). The data were corrected for
Lorentz and polarization effects. No absorption correction was
applied. Data reduction and analysis were carried out with the

26
T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
Table 1
13C NMR chemical shifts of products in CDCl3.
Atom number
Products
3
4
5
7
8
11
34.4
12
35.4
15
37.4
16
17
37.5
18a
35.5
19
35.9
20
36.3
21
37.4
22
35.7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
36.0
35.4
198.9
126.8
167.6
40.9
67.8
39,8
45.0
38.5
20.4
30.3
42.8
45.3
22.7
33.9
81.5
10.9
17.0
35.4
33.8
37.1
31.7
70.0
38.6
45.2
28.8
31.9
35.8
55.0
35.7
20.8
37.3
42.9
51.3
23.3
30.3
80.9
10.6
11.8
32.8
35.7
38.7
35.5
36.2
35.7
34.4
200.3
128.5
164.4
124.1
141.1
38.2
48.7
36.6
20.8
36.8
44.3
51.3
23.5
30.8
81.7
11.5
16.8
34.1
198.5
129.3
163.3
55.6
58.8
36.0
51.7
36.7
20.8
36.1
43.7
47.6
23.1
30.5
81.3
10.9
16.7
34.1
200.6
124.4
171.5
33.6
31.6
34.9
58.8
40.0
68.7
50.2
44.1
55.3
24.2
31.4
63.1
14.4
18.3
210.9
31.4
35.4
198.9
124.6
167.6
41.0
68.2
39.7
45.1
38.2
20.8
31.5
43.9
50.6
22.9
33.9
63.4
14.1
17.0
209.4
31.3
34.1
198.0
125.9
170.2
41.3
66.2
35.7
44.8
39.6
20.8
38.6
43.4
54.7
68.3
36.2
63.4
16.0
17.0
208.6
31.6
34.0
199.5
124.0
170.3
32.5
32.3
34.6
46.3
38.8
20.0
26.7
43.8
85.6
76.4
34.7
59.3
16.9
17.2
209.4
31.3
34.2
200.6
124.4
171.5
33.6
30.7
31.3
59.1
40.2
68.5
51.7
43.8
59.5
69.8
37.5
63.3
17.1
18.2
208.1
31.2
34.2
200.4
124.5
170.5
33.3
27.6
38.0
52.5
39.9
68.1
42.8
48.3
84.7
21.5
33.1
60.0
18.4
18.4
209.9
31.3
34.0
199.4
124.9
167.1
42.6
71.5
38.6
50.9
38.0
20.9
39.6
43.9
60.2
74.1
33.9
63.3
15.6
17.3
208.5
31.4
199.1
126.8
167.6
31.4
67.0
39.9
45.1
38.3
20.0
30.8
47.2
45.4
21.1
35.5
220.6
13.3
16.9
199.5
124.1
170.6
22.9
38.2
35.5
52.4
38.5
29.5
73.3
49.4
48.7
23.0
31.1
81.8
8.5
199.0
126.9
167.8
41.1
67.8
40.7
45.4
38.6
20.5
31.1
45.3
44.6
22.7
34.0
81.6
13.8
19.1
26.0
199.7
123.9
171.2
32.7
31.1
31.7
54.0
38.7
21.0
40.1
43.6
60,3
70.2
33.9
63.7
15.9
17.3
208.3
31.1
17.3
25.7
a
Compound 18 in DMSO.
(ppm): 0.89 (s, 3H, 18-CH3); 1.19 (s, 3H, 19-CH3); 1.22 (s, 3H, 17␣-
CH3); 3.95 (br s, 1H, Wh = 9.8 Hz, H-7␤); 5.78 (s, 1H, H-4).
C 75.14, H 8.82%. Mass spectrum indicated a molecular ion at m/z
303 of composition C19H26O3; 1H NMR (CDCl3) ı (ppm): 0.80 (s, 3H,
18-CH3); 1.17 (s, 3H, 19-CH3); 3.34 (d, 1H, J = 3.62 Hz, H-7); 3.45 (d,
1H, J = 3.62 Hz, H-6); 3.71 (t, 1H, J = 8.3 Hz, H-17); 6.10 (s, 1H, H-4).
2.5.6. 17˛-Methyl-3ˇ,17ˇ-dihydroxy-5˛-androstane (9)
Six-day transformation of 17␣-methyltestosterone (6) (100 mg)
yielded 5 mg of compound 9. Anal. Calcd. for C20H34O2: C 78.38, H
11.18; found: C 78.58, H 11.32%. Mass spectrum indicated a molec-
2.5.9. 11˛-Hydroxyprogesterone (15)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 128 mg
and 72 mg of compound 15 were isolated respectively. Anal. Calcd.
for C21H30O3: C 76.33, H 9.15; found: C 76.55, H 8.98%. Mass
spectrum indicated a molecular ion at m/z 331 of composition
ular ion at m/z 307 of composition C20H
34O2; 1H NMR (CDCl3) ı
(ppm): 0.83 (s, 3H, 19-CH3); 0.86 (s, 3H, 18-CH3); 1.24 (s, 3H, 17␣-
CH3); 3.63 (m, 1H, Wh = 31.0 Hz, H-3␣).
C
21H30O3; 1H NMR (CDCl3) ı (ppm): 0.69 (s, 3H, 18-CH3); 1.31
2.5.7. 6-Dehydrotestosterone (11)
Three-day transformation of DHEA (10) (100 mg) yielded 3 mg
of compound 11. Anal. Calcd. for C19H26O2: C 79.68, H 9.15; found:
(s, 3H, 19-CH3); 2.12 (s, 3H, 21-CH3); 4.01 (td, 1H, J = 9.6, 4.8 Hz,
H-11␤); 5.72 (s, 1H, H-4).
C 79.38, H 9.35%. Mass spectrum indicated a molecular ion at m/z
287 of composition C19H26O2; 1H NMR (CDCl3) ı (ppm): 0.82 (s, 3H,
18-CH3); 1.10 (s, 3H, 19-CH3); 3.67 (t, 1H, J = 8.47 Hz, H-17); 5.65 (s,
1H, H-6); 6.08 (s, 2H, H-4 and H-7).
2.5.10. 15ˇ-Hydroxyprogesterone (16)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 48 mg and
112 mg of compound 16 were isolated respectively. Anal. Calcd. for
C21H30O3: C 76.33, H 9.15; found: C 76.15, H 9.31%. Mass spectrum
indicated a molecular ion at m/z 331 of composition C21H30O3 1H
NMR (CDCl3) ı (ppm): 0.93 (s, 3H, 18-CH3); 1.21 (s, 3H, 19-CH3);
2.5.8. 6ˇ,7ˇ-Epoxytestosterone (12)
Three-day transformation of DHEA (10) (100 mg) yielded 8 mg
of compound 12. Anal. Calcd. for C19H26O3: C 75.46, H 8.67; found:
Fig. 1. Crystal structure of 7␣,15␤-dihydroxyprogesterone (18).

T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
27
OH
2.13 (s, 3H, 21-CH3); 4.31 (m, 1H, Wh = 15.4 Hz, H-15␣); 5.73 (s, 1H,
H-4).
2.5.11. 7˛-Hydroxyprogesterone (17)
OH
Three-day transformation of progesterone (13) (800 mg)
yielded 16 mg of compound 17. Anal. Calcd. for C21H30O3: C 76.33, H
9.15; found: C 76.35, H 9.11%. Mass spectrum indicated a molecular
ion at m/z 331 of composition C21H30O3 1H NMR (CDCl3) ı (ppm):
0.66 (s, 3H, 18-CH3); 1.19 (s, 3H, 19-CH3); 2.12 (s, 3H, 21-CH3); 3.98
(br s, Wh = 9.7 Hz, 1H, H-7␤); 5.79 (s, 1H, H-4).
O
HO
O
OH
3
OH
O
1
2.5.12. 7˛,15ˇ-Dihydroxyprogesterone (18)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 272 mg
and 16 mg of compound 18 were isolated respectively. Anal. Calcd.
for C21H30O4: C 72.80, H 8.73; found: C 72.75, H 8.70%. Mass
spectrum indicated a molecular ion at m/z 347 of composition
C21H30O4; 1H NMR (DMSO) ı (ppm): 0.79 (s, 3H, 18-CH3); 1.14 (s,
3H, 19-CH3); 2.06 (s, 3H, 21-CH3); 2.48 (t, 1H, J = 8.7 Hz, H-17␣); 4.08
(br s, Wh = 9.9 Hz, 1H, H-7␤); 4.21 (m, 1H, Wh = 15.4 Hz, H-15␣); 5.59
(s, 1H, H-4). Crystal data: C21H30O4, Mw = 346.45, colorless needles,
O
H
5
O
O
2
crystal size 0.20 mm × 0.05 mm × 0.05 mm, space group P212121,
3
OH
˚
˚
˚
˚
a = 11.525(2) A, b = 11.649(2) A, c = 13.719(2) A, V = 1841.8(5) A ,
Z = 4, Dc = 1.249 Mg m−3, T = 100(2) K, R = 0.046, wR = 0.102 (1787
reflections with I > 2ꢃ(I)), 226 parameters.
4
Scheme 1. Transformation of testosterone (1) and androstenedione (2) in the cul-
ture of D. igniaria. The composition of the products mixtures is presented in Table 2.
2.5.13. 14˛,15ˇ-Dihydroxyprogesterone (19)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 48 mg and
56 mg of compound 19 were isolated respectively. Anal. Calcd. for
3. Results and discussion
DHEA (10) and its five derivatives: testosterone (1), androstene-
dione (2), 17␣-methyltestosterone (6), progesterone (13) and preg-
nenolone (14) were subjected to microbial transformation by the
filamentous fungus D. igniaria KCH 6670. Transformation of testos-
terone (1) and androstenedione (2) led to a mixture of the same
products (Scheme 1): 7␣,17␤-dihydroxyandrost-4-en-3-one (3),
7␣-hydroxyandrost-4-en-3,17-dione (4) and 3␤,17␤-dihydroxy-
5␣-androstane (5), which varied only in quantitative composition
(Table 2). In order to investigate metabolic pathways of substrates
1 and 2 composition of mixtures sampled after 12, 24, 72 and 144 h
of transformation was studied. Gas chromatographic analysis of
mixture from 12 h transformation of androstenedione (2) indicates
that the reduction of the carbonyl group at C-17 took place and
then the resulting testosterone was hydroxylated at the 7␣ posi-
tion. Simultaneously, apart from the reduction, androstenedione
(2) was hydroxylated to 7␣-hydroxyandrost-4-en-3,17-dione (4).
Similarly, the oxidation of alcohol at C-17 was also observed
during transformation of testosterone (1). At the same time a com-
petitive process of testosterone hydroxylation occurred, leading
to 7␣,17␤-dihydroxyandrost-4-en-3-one (3). Thus, after transfor-
mation of androstenedione and testosterone in the culture of
D. igniaria, 7␣-hydroxyandrost-4-en-3,17-dione (4) was obtained
in the yields of 46% and 25%, respectively (Table 2). The enzy-
matic system of this strain was also capable of reduction of both
substrates to 3␤,17␤-dihydroxy-5␣-androstane (5), however that
compound was obtained in a low yield (below 10%). Our studies
indicated also that the compositions of reaction mixtures obtained
C
21H30O4: C 72.80, H 8.73; found: C 72.65, H 8.90%. Mass spectrum
indicated a molecular ion at m/z 347 of composition C21H30O4; 1
H
NMR (CDCl3) ı (ppm): 0.99 (s, 3H, 18-CH3); 1.24 (s, 3H, 19-CH3);
2.12 (s, 3H, 21-CH3); 3.15 (t, 1H, J = 8.7 Hz, H-17␣); 3.94 (dd, 1H,
J = 7.4, 1.7 Hz, H-15␣); 5.74 (s, 1H, H-4).
2.5.14. 11˛,15ˇ-Dihydroxyprogesterone (20)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 40 mg and
24 mg of compound 20 were isolated respectively. Anal. Calcd. for
C
21H30O4: C 72.80, H 8.73; found: C 72.89, H 8.71%. Mass spectrum
indicated a molecular ion at m/z 347 of composition C21H30O4; 1
H
NMR (CDCl3) ı (ppm): 0.94 (s, 3H, 18-CH3); 1.32 (s, 3H, 19-CH3);
2.13 (s, 3H, 21-CH3); 2.49 (t, 1H, J = 8.7 Hz, H-17␣); 4.02 (m, 1H,
Wh = 16.8 Hz, H-11␤); 4.29 (m, 1H, Wh = 15.3 Hz, H-15␣); 5.71 (s,
1H, H-4).
2.5.15. 11˛,14˛-Dihydroxyprogesterone (21)
After 3 days of incubation of progesterone (13) (800 mg) and
after 6 days of incubation of pregnenolone (14) (400 mg), 32 mg and
20 mg of compound 21 were isolated respectively. Anal. Calcd. for
C
21H30O4: C 72.80, H 8.73; found: C 72.74, H 8.74%. Mass spectrum
indicated a molecular ion at m/z 347 of composition C21H30O4; 1
H
NMR (CDCl3) ı (ppm): 0.79 (s, 3H, 18-CH3); 1.31 (s, 3H, 19-CH3);
2.13 (s, 3H, 21-CH3); 3.24 (t, 1H, J = 8.7 Hz, H-17␣); 4.04 (m, 1H,
Wh = 16.5 Hz, H-11␤); 5.74 (s, 1H, H-4).
2.5.16. 7ˇ,15ˇ-Dihydroxyprogesterone (22)
Table 2
Three-day transformation of progesterone (13) (800 mg)
yielded 7 mg of compound 22. Anal. Calcd. for C21H30O4: C 72.80, H
8.73; found: C 72.75, H 8.70%. Mass spectrum indicated a molecular
ion at m/z 347 of composition C21H30O4; 1H NMR (CDCl3) ı (ppm):
0.95 (s, 3H, 18-CH3); 1.24 (s, 3H, 19-CH3); 2.14 (s, 3H, 21-CH3); 3.62
(m, 1H, Wh = 17.4 Hz, H-7␣); 4.44 (m, 1H, Wh = 15.2 Hz, H-15␣); 5.76
(s, 1H, H-4).
Composition of crude mixture obtained in testosterone (1) and androstenedione (2)
transformations.
Substrate
Products (% by GC)
3
4
5
1
2
63
19
25
46
8
9

28
T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
OH
OH
OH
OH
OH
+
+
O
O
OH
8 (33%)
HO
O
H
9 (8%)
7 (49%)
6
Scheme 2. Transformation of 17␣-methyltestosterone (6) in the culture of D. igniaria.
after 1–9 days of transformations of testosterone and androsten-
dione did not differ in any significant way. The ability to perform
oxidation–reduction reactions by the strain D. igniaria has already
been described in our earlier papers, concerning enantioselective
reduction of low-molecular-weight ketones [15,16] and resolution
of racemic mixtures of bicyclic enones [17].
17␣-Methyltestosterone (6) was hydroxylated in a way that was
similar to androstenedione (2), i.e. at the 7␣ position (yield 33%),
but the main product was found to be 12␤-hydroxy derivative
7 (Scheme 2). The fact that we obtained that particular product,
unlike in the transformation of testosterone (1) leading mainly
to 7␣-hydroxyderivative, had probably arisen from the directing
effect of the methyl group at C-17 in the substrate 6. Additionally,
similarly to biotransformation of substrates 1 and 2, a product of
the reduction of both double bond at C-4 and carbonyl group at C-
3 – 3␤,17␤-dihydroxy-17␣-methyl-5␣-androstane (9) (yield 8%),
was obtained.
The predominant metabolism of both incubated 5-ene steroids:
dehydroepiandrosterone (DHEA) (10) and pregnenolone (14)
occurred through 3␤-hydroxy-steroid dehydrogenase/5,4-en iso-
merase pathways resulting in the generation of a 4-en-3-oxo
system on ring-A. DHEA (10) was transformed mainly to the deriva-
tives of androstenedione (7␣-hydroxyandrost-4-en-3,17-dione (4),
yield 38%) and testosterone (17␤-hydroxyandrost-4,6-dien-3-one
(11), yield 15%, and 6,7␤-epoxy-17␤-hydroxyandrost-4-en-3-
one (12), yield 14%). Similarly to the transformation of C19
4-en-3-oxo substrates, the reduction product (3␤,17␤-dihydroxy-
5␣-androstane (5), yield below 10%) was obtained as well
(Scheme 3). Products 11 and 12 were probably formed due to oxi-
dation/isomerization of the double bond and hydroxylation of C-7
atom in the substrate. They have not been known as biotransforma-
tion products of this substrate so far. All the products are especially
valuable due to a wide medical application of DHEA and because of
the similarity of microbial and human metabolisms.
The strain D. igniaria expressed capability of mono- and
dihydroxylation of both progesterone (13) (Scheme 4) and preg-
nenolone (14) (Scheme 5). The obtained results indicate that the
presence of an acetyl substituent at C-17 in the substrate has a
directing effect on hydroxylation mainly at 15␤- and 11␣-position;
the second hydroxyl group was introduced in 7␣- or 14␣-position.
Transformation of progesterone (13) gave also a product of 7␣-
hydroxylation, which was observed for transformations of C19
steroids by this strain. The lack of 7␣-hydroxylation in the case
of pregnenolone (14) suggests that hydroxylation at this position
may be attributed to 4-en-3-keto steroids and in this substrate an
isomerization of the double bond takes place before the hydrox-
ylation. Additionally, such a biotransformation route is confirmed
by the fact, that pregnenolone was transformed much slower than
dehydroepiandrosterone, which has the same A and B rings struc-
ture. The slower process of the double bond isomerization in 14
is probably the reason of the lack of the metabolites analogous to
those isolated after incubation of DHEA (10) and progesterone (13).
All the tested substrates play an important biochemical role in
higher organisms. Therefore this study is a valuable contribution to
the knowledge about their biosynthetic pathways.
3.1. Identification of products
3.1.1. 7˛,17ˇ-Dihydroxyandrost-4-en-3-one (3)
The signals of 4-H, 18-CH3 and 19-CH3 in the 1H NMR spec-
trum are shifted down-field compared to the substrate by 0.07,
0.01 and 0.01 ppm, respectively. These shifts and the presence of
a broad, singlet-like multiplet at ı = 3.96 ppm of an equatorial pro-
ton at the secondary carbon atom indicate the ␣ orientation of
the C-7 hydroxyl group. Additionally, there is a triplet at 3.69 ppm
(J = 8.47 Hz), which is the region typical for 17␤-steroid alcohols
[18] and therefore it unequivocally confirms the structure of the
isolated metabolite as 7␣-hydroxytestosterone (3) [19].
3.1.2. 7˛-Hydroxyandrost-4-en-3,17-dione (4)
In the 1H NMR of 4 we can also see a signal at ı = 4.09 ppm in
the shape typical for 7␣-hydroxyderivatives of 4-en steroids [20].
The presence of the C4 C5 double bond is additionally confirmed
by the signal at ı = 5.82 ppm, which is shifted toward lower field by
0.08 ppm compared to androstenedione (2) [21]. Chemical shifts of
O
O
O
OH
4 (38%)
OH
OH
O
11 (15%)
HO
10
O
O
12 (14%)
OH
HO
H
5 (9%)
Scheme 3. Transformation of DHEA (10) in the culture of D. igniaria.

T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
29
O
O
O
HO
+
+
+
+
+
OH
OH
OH
O
O
O
OH
17 (3%)
O
O
O
O
15 (20%)
16 (10%)
O
O
O
O
HO
OH
OH
OH
OH
O
O
19 (8%)
13
18 (41%)
20 (8%)
O
O
HO
OH
OH
22 (3%)
21 (7%)
Scheme 4. Hydroxylation of progesterone (13) in the culture of D. igniaria.
angular methyl groups are in accordance with the literature data
3.1.4. 17˛-Methyl-12ˇ,17ˇ-dihydroxyandrost-4-en-3-one (7)
for compound 4 [22,23].
The structure of product 7 was mainly confirmed by chemical
shifts of 4-H, 17-CH3, 18-CH3 and 19-CH3 which are in accordance
with the literature [18]. Additionally, the presence of a character-
istic signal of the axial proton at the secondary carbon atom at
ı = 3.74 ppm (dd, 1H, J = 16.8, 7.2 Hz) indicates that the hydroxyl
group is located at C-12 and is ␤-oriented. The spectral data are
consistent with the literature [25].
3.1.3. 3ˇ,17ˇ-Dihydroxy-5˛-androstane (5)
While comparing the 1H NMR spectrum of 5 to testosterone (1),
we may notice that the olefine signal of 4-H has disappeared, which
indicates that there is no double bond in compound 5. Additionally,
the presence of a multiplet of two protons at 3.58–3.68 ppm, which
is the region typical for protons attached to hydroxylated carbon
atom, shows that the reduction of the carbonyl group at C-3 took
place and that the second hydroxyl group at C-17 was left intact.
The signal of 18-CH3 is not shifted, whereas the signal of 19-CH3 is
shifted by 0.36 ppm, which unequivocally proves the structure of
3␤,17␤-dihydroxy-5␣-androstane [18,24].
3.1.5. 17˛-Methyl-7˛,17ˇ-dihydroxyandrost-4-en-3-one (8)
The presence of a hydroxyl group at 7␣ position is confirmed in
the 1H NMR spectrum by a characteristic, broad singlet-like multi-
plet at ı = 3.95 ppm of an equatorial proton attached to a secondary
carbon atom. Additional confirmation of the structure of 8 is the
O
O
O
HO
+
+
+
+
OH
OH
O
O
O
O
OH
18 (7%)
15 (22%)
16 (35%)
O
O
O
HO
HO
HO
14
OH
OH
OH
OH
O
O
O
19 (18%)
21 (9%)
20 (9%)
Scheme 5. Transformation of pregnenolone (14) to hydroxy derivatives of progesterone in the culture of D. igniaria.

30
T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
signals of 4-H, 17-CH3, 18-CH3 and 19-CH3, which, compared to the
substrate, are shifted down-field by 0.07, 0.03, 0.01 and 0.01 ppm,
respectively. The NMR data are in high accordance with the litera-
ture [18].
respectively. These shifts and the presence of a broad, singlet-like
multiplet at ı = 4.08 ppm of an equatorial proton at the secondary
carbon atom indicate the ␣ orientation of the C-7 hydroxyl group.
This, along with the chemical shift of 15␣ proton (4.21 ppm), indi-
cates hydroxylation in 15␤ position. The structure of 18 was clearly
confirmed by crystallographic analysis.
3.1.6. 17˛-Methyl-3ˇ,17ˇ-dihydroxy-5˛-androstane (9)
The lack of signals within the range of 4.50–6.00 ppm in the 1
H
NMR spectrum of 9 shows that there is no double bond in this prod-
uct. The presence of a multiplet at ı = 3.63 ppm, typical for H-3␣
indicates that the C-3 carbonyl group was reduced. The signal of
18-CH3 is not shifted, whereas the signal of 19-CH3 is shifted by
0.36 ppm toward upper field which unequivocally proves the struc-
ture of 17␣-methyl-3␤,17␤-dihydroxy-5␣-androstane. The NMR
data are consistent with the literature [18].
3.1.13. 14˛,15ˇ-Dihydroxypregnan-4-en-3,20-dione (19)
A considerable down-field shift (0.32 ppm) of 18-CH3 pro-
tons, compared to progesterone 13, indicates 15␤-hydroxylation,
whereas the shift of 17-CH signal by 0.59 ppm toward lower field
suggests introduction of 14␣-hydroxyl group. The change of the
chemical shift and the shape of H-15␣ signal, compared to 15␤-
hydroxyprogesterone (16), arise from the additional hydroxylation
in 14␣ position. Additionally, the dihydroxylation is confirmed by
13C NMR spectrum (Table 1) and by a much lower Rf in TLC, com-
pared to the monohydroxylation products.
3.1.7. 17ˇ-Hydroxyandrost-4,6-dien-3-one (11)
Chemical shifts and shapes of the signals of H-4 (s, ı = 6.08 ppm)
and H-17 (t, J = 8.3 Hz, ı = 3.67 ppm) unequivocally proved that the
substrate (DHEA) was transformed by the tested strain into a
testosterone derivative. New signals at ı = 5.65 ppm and 6.08 ppm
indicate that an additional double bond was introduced between
C-6 and C-7. Chemical shifts of the methyl groups of product are
consistent with the data reported earlier [26].
3.1.14. 11˛,15ˇ-Dihydroxypregnan-4-en-3,20-dione (20)
In the 1H NMR spectrum of 20 up-field shift of 4-H, 18-CH3,
19-CH3 and 21-CH3 signals (by 0.02, 0.29, 0.14, and 0.02 ppm,
respectively) and down-field shift of 17-H signal by 0.03 ppm
compared to progesterone were observed. These and the shapes
and positions of the multiplets of a protons attached to the
hydroxylated carbon atoms indicate the structure of 11␣,15␤-
dihydroxyprogesterone (20). The spectral data of this product are
in accordance with the literature [31,32].
3.1.8. 17ˇ-Hydroxyandrost-6ˇ-epoxy-4-en-3-one (12)
Chemical shifts and shapes of the signals of protons H-4 (s,
ı = 6.10 ppm) and H-17 (t, J = 8.3 Hz, ı = 3.71 ppm) in the 1H NMR
spectrum of 12 confirm the structure of A and D-rings being
the same as in compound 11. Lack of signals at ı = 5.65 ppm and
6.08 ppm, which were observed for 11, and new signals at ı = 3.34
and 3.45 ppm (d, J = 3.62 Hz and d, J = 3.62 Hz) in the spectrum of 12
indicate that there is an epoxide ring in this product. The chemi-
cal shifts of methyl groups are in good accordance with the earlier
published data [27].
3.1.15. 11˛,14˛-Dihydroxypregnan-4-en-3,20-dione (21)
The presence of a hydroxyl group at 11␣ position is confirmed in
the 1H NMR spectrum by a characteristic multiplet at ı = 4.04 ppm
of an axial CHOH. Additionally, the structure of 21 is confirmed by
the down-field shifted of 4-H, 17-H 18-CH3, 19-CH3 and 21-CH3 by
0.02, 0.71, 0.13, 0.12 and 0.01 ppm, respectively in comparison to
progesterone (13) [33].
3.1.9. 11˛-Hydroxypregnan-4-en-3,20-dione (15)
In the 1H NMR spectrum of 15 the following signals are shifted
up-field compared to progesterone: 4-H, 18-CH3, 19-CH3 and 21-
CH3, by 0.01, 0.03, 0.13 and 0.02 ppm, respectively. These and
the shape and position of the multiplet of a proton attached
to the hydroxylated carbon atom indicate the structure of 11␣-
hydroxyprogesterone (15) [28].
3.1.16. 7ˇ,15ˇ-Dihydroxypregnan-4-en-3,20-dione (22)
The structure of product 22 was mainly confirmed by chemical
shifts of 4-H, 18-CH3, 19-CH3 and 21-CH3, which are in accordance
with the literature [18]. Additionally, the presence of a charac-
teristic signal of the axial proton at the secondary carbon atom
at ı = 3.62 ppm (m, 1H, Wh = 17.4 Hz) indicates that the hydroxyl
group is located at C-7 and is ␤-oriented. Additionally, there is a
triplet at 4.44 ppm (m, 1H, Wh = 15.2 Hz), which is typical for 15␤-
steroid alcohols described in the literature [18] and therefore it
unequivocally confirms the structure of the isolated metabolite as
7␤,15␤-dihydroxyprogesterone (22) [34].
3.1.10. 15ˇ-Hydroxypregnan-4-en-3,20-dione (16)
In the 1H NMR of 16 a considerable down-field shift (by
0.26 ppm) of 18-CH3 signal was observed compared to proges-
terone 13. This, along with the chemical shift of 15␣ proton
(4.31 ppm) indicates hydroxylation in 15␤ position. Additional
confirmation of the structure of 16 is the shifts of 4-H, 17-CH3, 19-
CH3 and 21-CH3 signals compared to the substrate (progesterone),
which are identical with literature data [29].
Acknowledgement
This research was supported financially by the European Union
within the European Regional Development Fund (grant No.
POIG.01.03.01-00-158/09-05).
3.1.11. 7˛-Hydroxypregnan-4-en-3,20-dione (17)
In the 1H NMR of 17 we can also see a signal at ı = 3.98 ppm,
the shape of which is typical for 7␣-hydroxy derivatives of 4-ene
steroids [18]. The presence of the C4 C5 double bond is addition-
ally confirmed by the signal at ı = 5.79 ppm, which is shifted toward
lower field by 0.07 ppm compared to progesterone (13) [18]. Chem-
ical shifts of angular methyl groups are in accordance with the
literature data for compound 17 [30].
References
[1] R.V. Smith, J.P. Rosazza, Arch. Biochem. Biophys. 161 (1974) 551–559.
[2] A. Li, J.C. Bigelow, Steroids 75 (2010) 404–410.
[3] I. Lecroix, J. Biton, R. Azerad, Bioorg. Med. Chem. 7 (1999) 2329–2341.
[4] A.-C. Cotillon, J. Doostzadeh, R. Morfin, J. Steroid Biochem. Mol. Biol. 62 (1997)
467–475.
ˇ
[5] R. Hampl, M. Hill, I. Sterzl, L. Stárka, J. Steroid Biochem. Mol. Biol. 31 (2000)
3.1.12. 7˛,15ˇ-Dihydroxypregnan-4-en-3,20-dione (18)
211–218.
[6] N. Tagawa, Y. Hidaka, T. Takano, Y. Shimaoka, Y. Kobayasi, N. Amino, Clin. Chim.
Acta 340 (2004) 187–193.
NMR spectra of compound 18 were made in DMSO because of its
low solubility in CDCl3, TDF and CD3OD. The signals of 4-H, 18-CH3,
19-CH3 and 21-CH3 in the 1H NMR spectrum are shifted down-
field by 0.08, 0.28, 0.03 and 0.03 ppm compared to the substrate,
[7] M. Koshimura, T. Utsukihara, A. Hara, S. Mizobuchi, C.A. Horiuchi, M. Kuniyoshi,
J. Mol. Catal. B: Enzym. 67 (2010) 72–77.
´
[8] T. Kołek, A. Szpineter, A. Swizdor, Steroids 74 (2009) 859–862.

T. Janeczko et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 24–31
31
[9] A.C. Hunter, K.R. Watts, C. Dedi, H.T. Dodd, J. Steroid Biochem. Mol. Biol. 116
[22] L.-H. Huang, J. Li, G. Xu, X.-H. Zhang, Y.-G. Wang, Y.-L. Yin, H.-M. Liu, Steroids
75 (2010) 1039–1046.
(2009) 171–177.
[10] W. Ge, S. Wang, L. Shan, N. Li, H. Liu, J. Mol. Catal. B: Enzym. 55 (2008)
[23] M.A. Faramarzi, M. Aghelnejada, M.T. Yazdi, M. Amini, N. Hajarolasvadi, Steroids
73 (2008) 13–18.
37–42.
´
[11] T. Kołek, N. Milecka, A. Swizdor, A. Panek, A. Białon´ ska, Org. Biomol. Chem. 9
[24] M. Alam, J.R. Hanson, M. Liman, S. Nagaratnam, J. Chem. Res. (S) (1996) 56–57.
´
(2011) 5414–5422.
[25] A. Swizdor, T. Kołek, Steroids 70 (2005) 817–824.
[12] T. Janeczko, J. Dmochowska-Gładysz, A. Białon´ ska, Z. Ciunik, Biocatal. Biotrans-
[26] S. Al-Awadi, M. Afzal, S. Oommen, Steroids 70 (2005) 327–333.
[27] H.L. Holland, P.C. Chenchaiah, E.M. Thomas, B. Mader, M.J. Dennis, Can. J. Chem.
62 (1984) 2740–2747.
form. 24 (2006) 458–463.
[13] Oxford Diffraction, Poland Sp. z o.o., CrysAlis CCD and CrysAlis Red, V166, 2001.
[14] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112–122.
[15] T. Janeczko, J. Dmochowska-Gładysz, E. Kostrzewa-Susłow, Przem. Chem. 88
(2009) 458–460.
´
[28] A. Swizdor, T. Kołek, A. Panek, A. Białon´ ska, Biochim. Biophys. Acta 1811 (2011)
253–262.
[29] J.E. Bridgeman, P.C. Cherry, A.S. Clegg, J.M. Evans, E.R. Jones, A. Kasal, V. Kumar,
G.D. Meakins, Y. Morisawa, E.E. Richards, P.D. Woodgate, J. Chem. Soc. Perkin 1
(1970) 250–257.
´
[16] A. Swizdor, T. Janeczko, J. Dmochowska-Gładysz, J. Ind. Microbiol. Biotechnol.
37 (2010) 1121–1130.
[17] T. Janeczko, J. Dmochowska-Gładysz, E. Kostrzewa-Susłow, World J. Microbiol.
Biotechnol. 37 (2010) 1121–2051.
[18] D.N. Kirk, H.C. Toms, C. Douglas, K.A. White, J. Chem. Soc. Perkin Trans. 2 (1990)
1567–1594.
[30] D.W. Payne, C. Shackleton, H.H. Toms, I. Ben-Shlomo, S. Kol, M. deMoura, J.F.
Strauss, E.Y. Adashi, J. Biol. Chem. 270 (1995) 18888–18896.
[31] M.I. Choudhary, I. Batool, S.A.A. Shah, S.A. Nawaz, Atta-ur-Rahman, Chem.
Pharm. Bull. 53 (2005) 1455–1459.
[19] E. Huszcza, J. Dmochowska-Gładysz, Phytochemistry 62 (2003) 155–158.
[20] E.R.H. Jones, Pure Appl. Chem. 33 (1973) 39–52.
[21] T. Janeczko, J. Dmochowska-Gładysz, E. Kostrzewa-Susłow, A. Białon´ ska, Z. Ciu-
nik, Steroids 74 (2009) 657–661.
[32] T.C. McMorris, P.H. Le, M.W. Preus, S.R. Schow, G.R. Weihe, Steroids 53 (1989)
345–362.
[33] S.B. Mahato, S. Banerjee, N.P. Sahu, Steroids 5 (1984) 545–558.
[34] S. Garai, S. Banerjee, S.B. Mahato, J. Chem. Res. (S) 10 (1995) 408–409.