New metabolites from fungal biotransformation of an oral contraceptive agent: Methyloestrenolone

Article abstract of DOI:10.1016/j.steroids.2013.01.002

Fungal cell cultures were used for the first time for the biotransformation of methyloestrenolone (1), an oral contraceptive. Fermentation of 1 with Macrophomina phaseolina, Aspergillus niger, Gibberella fujikuroi, and Cunninghamella echinulata produced eleven metabolites 2-12, six of which 2-5, 11 and 12 were found to be new. These metabolites were resulted from the hydroxylation at C-1, C-2, C-6, C-10, C-11, and C-17α-CH₃, as well as aromatization of ring A of the steroidal skeleton of substrate 1. The transformed products were identified as 17α-methyl-6β,17β- dihydroxyestr-4-en-3-one (2), 17α-(hydroxymethyl)-11β,17β- dihydroxyestr-4-en-3-one (3), 17α-methyl-2α,11β,17β- trihydroxyestr-4-en-3-one (4), 17α-methyl-1β,17β-dihydroxyestr- 4-en-3-one (5), 17α-methyl-11α,17β-dihydroxyestr-4-en-3-one (6), 17α-methyl-11β,17β-dihydroxyestr-4-en-3-one (7), 17α-methyl-10β,17β-dihydroxyestr-4-en-3-one (8), 17α-(hydroxymethyl)-17β-hydroxyestr-4-en-3-one (9), 17α-methylestr-1,3,5(10)-trien-3,17β-diol (10), 17α-methyl-3, 17β-dihydroxyestr-1,3,5(10)-trien-6-one (11), and 17α-methyl-6β, 10β,17β-trihydroxyestr-4-en-3-one (12).

Full text of DOI:10.1016/j.steroids.2013.01.002

Steroids 78 (2013) 418–425
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New metabolites from fungal biotransformation of an oral contraceptive agent:
Methyloestrenolone
Salman Zafar a,b, Marium Bibi a, Sammer Yousuf a, M. Iqbal Choudhary a,c,
⇑
a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b Department of Chemistry, Abdul Wali Khan University, Mardan 23200, Pakistan
c Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 August 2012
Received in revised form 15 December 2012
Accepted 15 January 2013
Available online 26 January 2013
Fungal cell cultures were used for the first time for the biotransformation of methyloestrenolone (1), an
oral contraceptive. Fermentation of 1 with Macrophomina phaseolina, Aspergillus niger, Gibberella fujikuroi,
and Cunninghamella echinulata produced eleven metabolites 2–12, six of which 2–5, 11 and 12 were
found to be new. These metabolites were resulted from the hydroxylation at C-1, C-2, C-6, C-10, C-11,
and C-17
formed products were identified as 17
methyl)-11b,17b-dihydroxyestr-4-en-3-one (3), 17
a
-CH3, as well as aromatization of ring A of the steroidal skeleton of substrate 1. The trans-
-methyl-6b,17b-dihydroxyestr-4-en-3-one (2), 17 -(hydroxy-
-methyl-2 ,11b,17b-trihydroxyestr-4-en-3-one (4),
-methyl-1b,17b-dihydroxyestr-4-en-3-one (5), 17 -methyl-11 ,17b-dihydroxyestr-4-en-3-one (6),
-methyl-11b,17b-dihydroxyestr-4-en-3-one (7), 17 -methyl-10b,17b-dihydroxyestr-4-en-3-one (8),
-(hydroxymethyl)-17b-hydroxyestr-4-en-3-one (9), 17 -methylestr-1,3,5(10)-trien-3,17b-diol (10),
-methyl-3,17b-dihydroxyestr-1,3,5(10)-trien-6-one (11), and 17 -methyl-6b,10b,17b-trihydroxy-
a
a
Keywords:
a
a
Methyloestrenolone
Oral contraceptive
Biotransformation
Macrophomina phaseolina
Aspergillus niger
17
17
17
17
a
a
a
a
a
a
a
a
a
Gibberella fujikuroi
estr-4-en-3-one (12).
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
viz. acetylcholinesterase, butyrylcholinesterase, urease, phospho-
diesterase, and
a-chymotrypsin enzymes inhibition, cytotoxicity
Microbial transformation has been extensively employed for
the synthesis of fine chemicals for years [1]. Microbial biotransfor-
mation of steroids is a well established method for the large scale
manufacturing of steroidal drugs [2].
Methyloestrenolone (1) belongs to the estrogen agonist phar-
macological group. On the basis of mechanism of action, it is also
classified as sex hormone. Compound 1 is one of the most effective
inhibitors of progesterone formation during pregnancy [3]. No re-
port concerning the microbial transformation of 1 has been pub-
lished to date.
(PC-3 cell lines), immunomodulatory, anti-glycation and anti-bac-
terial activities, but no significant activity was observed. The
metabolites 2–12 were also evaluated for their acetylcholinester-
ase, butyrylcholinesterase and urease inhibitory potential but none
of them showed any significant activity in available assays.
2. Experimental
2.1. General
In the last decade, we have extensively studied the structural
modifications of several steroids especially the steroidal drugs
[4–23]. Here we report the biotransformation of another steroidal
drug, methyloestrenolone (1) for the first time. Small scale exper-
iments showed that Macrophomina phaseolina, Aspergillus niger,
Gibberella fujikuroi, and Cunninghamella echinulata were able to
efficiently transform 1 into a diverse array of metabolites. The sub-
strate (1) was subjected to a variety of bioactivity screening tests
Methyloestrenolone (1) was purchased from local market in the
form of tablets (Gyanecosid, Efroze Chemical Industries, Pakistan).
The substrate (1) was extracted from the tablets with dichloro-
methane and purified on silica gel column by using pet. ether/
EtOAc (7:3) isocratic solvent system. Pure methyloestrenolone
(1) was then used for experiments. Precoated TLC (silica gel,
PF254; 20 ꢀ 20, 0.25 mm) were purchased from Merck, Germany.
Column chromatography was carried out by using silica gel (70–
230 mesh, Merck). A Buchi-535 melting point apparatus was used
to measure the melting points of compounds. A JASCO P-2000
polarimeter was employed to measure optical rotations in metha-
nol. UV Absorption data (in nm) were obtained in methanol with a
⇑
Corresponding author at: H. E. J. Research Institute of Chemistry, International
Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270,
Pakistan. Tel.: +92 21 34824924; fax: +92 21 34819018/19.
E-mail address: iqbal.choudhary@iccs.edu (M.I. Choudhary).
Hitachi U-3200 spectrophotometer. Infrared (IR) spectra (in cmꢁ1
)
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.01.002

S. Zafar et al. / Steroids 78 (2013) 418–425
419
were recorded with an FT-IR-8900 spectrophotometer. 1H and 13C
NMR spectra were recorded in CD3OD and deuterated DMSO on a
Bruker Avance NMR spectrophotometer with solvent signal as
the internal standard. Chemical shifts (d values) were reported as
ppm (parts per million), relative to TMS (0 ppm). The coupling con-
stants (J values) were reported in hertz. JEOL JMS-600H mass spec-
trometer was used to record electron impact mass spectra (EI-MS)
and high-resolution mass spectra (HREI-MS) in m/z (rel.%). Single-
crystal X-ray diffraction study was performed on a Bruker Smart
APEX II, CCD 4-K area detector diffractometer [24]. Data reduction
was performed by using SAINT program. The structure was solved
by direct method [25] and refined by full-matrix least squares on
F2 by using the SHELXTL-PC package [26]. The figures were plotted
with the aid of ORTEP program [27].
(Rint = 0.0128). The R values were: R1 = 0.0325, wR2 = 0.0888 for
I > 2r(I), and R1 = 0.0329, wR2 = 0.0896 for all data; max/min resid-
ual electron density: 0.193/ꢁ0.241 e Åꢁ3. Crystallographic data for
compound 2 has been deposited in the Cambridge Crystallographic
Data Center. The crystallographic information can directly be ob-
tained free of charge from CCDC data center (CCDC 836387 refer-
ence code).
2.4.2. 17a-(Hydroxymethyl)-11b,17b-dihydroxyestr-4-en-3-one (3)
Colorless crystalline solid (8.0 mg, 0.008%). Mp: 114–116 °C. UV
(MeOH) kmax nm (log): 243 (4.3). ½a2D5ꢃ: +88.0 (c = 0.1, MeOH). IR
(CHCl3) mmax cmꢁ1: 3410, 1657. 1H NMR (CD3OD, 500 MHz): See
Table 1. 13C NMR (CD3OD, 125 MHz): See Table 2. HREI-MS m/z
(mol. formula, calcd value): 320.1992 (C19H28O4, 320.1988). EI-
MS m/z (rel. int.,%): 320 [M+] (21), 302 (18), 289 (13), 271 (32),
228 (29), 215 (34), 159 (13), 105 (13), 85 (64), 83 (95), 44 (100).
2.2. Microorganisms and culture medium
The fungi were obtained either from Karachi University Culture
Collection (KUCC) or American Type Culture Collection (ATCC).
M. phaseolina (KUCC 730) and C. echinulata (ATCC 9244) were
grown in a medium, composed of the following ingredients dis-
solved in distilled H2O (4.0 L): glucose (40.0 g), glycerol
(40.0 mL), peptone (20.0 g), yeast extract (20.0 g), KH2PO4
(20.0 g), and NaCl (20.0 g).
2.4.3. 17a-Methyl-2a,11b,17b-trihydroxyestr-4-en-3-one (4)
Colorless amorphous solid (6.0 mg, 0.006%). UV (MeOH) kmax
nm (log): 242 (4.0). ½aD25ꢃ: +131.0 (c = 0.1, MeOH). IR (CHCl3) mmax
cmꢁ1: 3416, 1674. 1H NMR (CD3OD, 500 MHz): See Table 1. 13C
NMR (CD3OD, 125 MHz): See Table 2. HREI-MS m/z (mol. formula,
calcd value): 320.1986 (C19H28O4, 320.1988). EI-MS m/z (rel.
int.,%): 320 [M+] (28), 302 (100), 284 (45), 276 (91), 244 (96), 232
(48), 226 (61), 200 (73), 185 (42), 159 (31), 121 (60), 105 (37),
91 (42), 43 (50).
The medium (4 L) for A. niger (ATCC 10549) was the same as
above, except 12 g yeast extract was used.
Four liters of fermentation medium for G. fujikuroi (ATCC 10704)
was composed of glucose (80.0 g), NH4NO3 (4.0 g), KH2PO4 (20.0 g),
MgSO4ꢂ7H2O (4.0 g) and trace element solution (8.0 mL). The Gib-
berella trace element solution was prepared by dissolving Co(NO3)3
(0.01 g), FeSO4ꢂ7H2O (0.1 g), CuSO4ꢂ5H2O (0.1 g), ZnSO4ꢂ7H2O
(0.16 g), MnSO4ꢂ7H2O (0.01 g), Mo(NH4)3 (0.01 g) in 100 mL of dis-
tilled water.
2.4.4. 17
a-Methyl-1b,17b-dihydroxyestr-4-en-3-one (5)
Colorless crystalline material (45.0 mg, 0.045%). Mp: 203–
205 °C. UV (MeOH) kmax nm (log): 239 (4.2).
½
a2D5ꢃ: +18.3°
(c = 0.12, MeOH). IR (CHCl3) mmax cmꢁ1: 3410, 1663. 1H NMR (CD3-
OD, 500 MHz): See Table 1. 13C NMR (CD3OD, 125 MHz): See Ta-
ble 2. HREI-MS m/z (mol. formula, calcd value): 304.2043
(C19H28O3, 304.2038). EI-MS m/z (rel. int.,%): 304 [M+] (10), 286
(100), 247 (30), 213 (72), 160 (53), 121 (10), 91 (10), 55 (7), 43 (16).
2.3. General fermentation and extraction conditions
Two stage fermentation protocol was followed for all biotrans-
formation experiments, which involves the preparation of seed
flasks, followed by inoculation of all the remaining flasks. Extrac-
tion conditions were the same as that used previously in our labo-
ratory [4,5,15].
2.4.5. 17a-Methyl-11a,17b-dihydroxyestr-4-en-3-one (6)
Amorphous material (4.5 mg, 0.0045%). UV (MeOH) kmax nm
(log): 241 (4.2). ½aD25ꢃ: +81.4 (c = 0.09, MeOH). IR (CHCl3) mmax
cmꢁ1: 3410, 1655. 1H NMR (CD3OD, 500 MHz): See Table 1. 13C
NMR (CD3OD, 125 MHz): See Table 2. HREI-MS m/z (mol. formula,
calcd value): 304.2043 (C19H28O3, 304.2038). EI-MS m/z (rel.
int.,%): 304 [M+] (55), 286 (56), 247 (100), 228 (60), 213 (39), 159
(29), 123 (39), 110 (63), 91 (33), 71 (36), 55 (16), 43 (38).
2.4. Fermentation of methyloestrenolone (1) with M. phaseolina
1.0 g of methyloestrenolone (1) was used for fermentation with
M. phaseolina. The fermentation was continued for 8 days. The
crude extract, obtained after filtration and extraction, was fraction-
ated on a silica gel column with pet. ether/ethyl acetate (10% gra-
dient) solvent system to obtain five main fractions. The fractions
were subjected to isocratic RP-HPLC (1:1 H2O/MeOH) to obtain
six pure compounds 2–7 (Fig. 1).
2.4.6. 17a-Methyl-11b,17b-dihydroxyestr-4-en-3-one (7)
Colorless crystalline material (14.0 mg, 0.014%). Mp: 224–
225 °C (Reported 219–224 °C [28]). UV (MeOH) kmax nm (log):
239 (4.1). ½aD25ꢃ: +61.4 (c = 0.14, MeOH). IR (CHCl3) mmax cmꢁ1
:
3398, 1657. 1H NMR (CD3OD, 500 MHz): See Table 1. 13C NMR
(CD3OD, 125 MHz): See Table 2. HREI-MS m/z (mol. formula, calcd
value): 304.2043 (C19H28O3, 304.2038). EI-MS m/z (rel. int.,%): 304
[M+] (12), 286 (71), 268 (17), 228 (100), 215 (45), 159 (18), 121
(12), 91 (42), 55 (7).
2.4.1. 17a-Methyl-6b,17b-dihydroxyestr-4-en-3-one (2)
Colorless crystalline solid (18 mg, 0.018%). Mp: 197–199 °C. UV
(MeOH) kmax nm (log): 237 (4.0). ½a2D5ꢃ: – 76.2 (c = 0.08, MeOH). IR
(CHCl3) mmax cmꢁ1: 3406, 1658. 1H NMR (CD3OD, 500 MHz): See
Table 1. 13C NMR (CD3OD, 125 MHz): See Table 2. HREI-MS m/z
(mol. formula, calcd value): 304.2032 (C19H28O3, 304.2038). EI-
MS m/z (rel. int.,%): 304 [M+] (22), 286 (66), 253 (19), 228 (100),
215 (98), 159 (23), 122 (12), 91 (12), 55 (7), 43 (20). Crystal data:
2.5. Fermentation of methyloestrenolone (1) with A. niger
Incubation of 1 g (in 20 mL acetone/40 flasks) of compound 1
with a fully grown culture of A. niger was carried out for 14 days.
2.5 g of a brown gum was obtained after extraction. The extract
was subjected to fractionation on silica gel column with pet.
ether–ethyl acetate (10% gradient) solvent system to obtain three
main fractions which on repeated isocratic RP-HPLC (1:1 H2O/
MeOH) yielded three pure compounds 2, 8 and 9 (Fig. 2). Metabo-
lite 2 had also been obtained by fermentation with M. phaseolina,
as discussed above (Fig. 1).
C
19H88O3,
a = 9.2433(6) Å, b = 9.4562(7) Å, c = 9.2871(6) Å, b = 90.8090(10),
V = 1447.41(16) Å3, Z = 2, calc = 1.246 mg/m3, F(000) = 332,
(Mo K = 0.71073 Å, max/min transmission 0.9601/0.9539, crystal
Mr = 304.41,
Monoclinic,
space
group
P21,
q
l
a
dimensions 0.58 ꢀ 0.50 ꢀ 0.50, 1.96° < h < 25.5°, 4792 reflections
were collected, of which 1608 reflections were observed

420
S. Zafar et al. / Steroids 78 (2013) 418–425
OH
18
12
17
15
13
11
9
16
H
H
14
1
10
8
2
3
H
H
5
7
4
6
O
1
OH
OH
OH
OH
OH
OH
HO
OH
H
H
H
H
H
H
H
H
H
H
H
H
O
O
2
3
OH
HO
H
OH
H
H
HO
O
H
H
H
O
4
5
HO
H
HO
H
H
H
H
H
O
O
7
6
Fig. 1. Biotransformation of methyloestrenolone (1) with Macrophomina phaseolina.
2.5.1. 17
a
-Methyl-10b,17b-dihydroxyestr-4-en-3-one (8)
ether/EtOAc), and 11 (7:3 Pet. ether/EtOAc) (Fig. 3) which were
Colorless crystalline material (8.0 mg, 0.008%). Mp: 190–192 °C
purified by column chromatography (silica gel).
(Reported 182–190 °C [29]). UV (MeOH) kmax nm (log): 230 (4.1).
½
a2D5ꢃ: +51.2 (c = 0.08, MeOH) (Reported = ½a2D5ꢃ = + 44.6 (c 0.269,
dioxane) [29]). IR (CHCl3) mmax cmꢁ1
:
3433, 1662. 1H NMR
2.6.1. 17a-Methylestr-1,3,5(10)-trien-3,17b-diol (10)
(DMSO-d6, 500 MHz): See Table 1. 13C NMR (DMSO-d6,
125 MHz): See Table 2. HREI-MS m/z (mol. formula, calcd value):
304.2043 (C19H28O3, 304.2038). EI-MS m/z (rel. int.,%): 304 [M+]
(87), 286 (67), 228 (83), 216 (100), 215 (87), 160 (37), 124 (43),
91 (26), 55 (24), 43 (43).
Colorless crystalline solid (14.0 mg, 0.014%). Mp: 190–191 °C
(Reported 184–186 °C [32]). ½a2D5ꢃ: +37.2 (c = 0.11, MeOH). IR
(CHCl3) mmax cmꢁ1: 3344, 3300, 1610, 1498. 1H NMR (CD3OD,
500 MHz): See Table 1. 13C NMR (CD3OD, 125 MHz): See Table 2.
HREI-MS m/z (mol. formula, calcd value): 286.1937 (C19H26O2,
286.1933). EI-MS m/z (rel. int.,%): 286 [M+] (35), 272 (100), 244
(6), 213 (51), 186 (18), 172 (33), 160 (40), 146 (28), 133 (20),
107 (98), 91 (6).
2.5.2. 17a-(Hydroxymethyl)-17b-hydroxyestr-4-en-3-one (9)
Colorless crystalline material (12.0 mg, 0.012%). Mp: 136–
142 °C. (Reported Mp: 140 °C) ½a2D5ꢃ +26.5 (CHCl3) [30]) UV (MeOH)
kmax nm (log): 239 (3.9). IR (CHCl3) m
max cmꢁ1: 3406, 1658. 1H NMR
(CD3OD, 500 MHz): See Table 1. 13C NMR (CD3OD, 125 MHz): See
Table 2. HREI-MS m/z (mol. formula, calcd value): 304.2043
(C19H28O3, 304.2038). EI-MS m/z (rel. int.,%): 304 [M+] (87), 286
(44), 273 (100), 255 (73), 231 (25), 215 (85), 159 (24), 91 (35),
55 (15), 41 (17). Single-crystal X-ray diffraction analysis [31].
2.6.2. 217a-Methyl-3,17b-dihydroxyestr-1,3,5(10)-trien-6-one (11)
Colorless crystalline solid (10.0 mg, 0.01%). Mp; 219–221 °C. UV
(MeOH) kmax nm (log): 245 (4.2). ½a2D5ꢃ: ꢁ28.0 (c = 0.1, MeOH). IR
(CHCl3) mmax cmꢁ1
:
3410, 1664, 1606. 1H NMR (CD3OD,
500 MHz): See Table 1. 13C NMR (CD3OD, 125 MHz): See Table 2.
HREI-MS m/z (mol. formula, calcd value): 300.1782 (C19H24O3,
300.1773). EI-MS m/z (rel. int.,%): 300 [M+] (33), 272 (100), 244
(7), 213 (51), 186 (18), 172 (33), 160 (40), 146 (28), 133 (20),
107 (98), 91 (6).
2.6. Fermentation of methyloestrenolone (1) with G. fujikuroi
Incubation of 1 (1 g/20 mL acetone) with G. fujikuroi culture in
40 flasks for 11 days produced three metabolites 7, 10 (4:6 Pet.

Table 1
Selected 1H NMR chemical shifts of compounds 1–12 (d in ppm; J in Hz; W1/2 in Hz).
COMPOUNDS
C
1a
2a
3a
4a
5a
6a
7a
8b
9a
10a
11a
12a
1
2
2.26, m; 2.37, m 1.36c, m; 2.29c, m 1.57, m; 2.32c, m
1.73, m; 2.21, m 2.31, m; 2.36, m 2.28, m; 2.37, m
1.42c, m; 2.52c, m 4.41, q (3.0)
2.28, m; 2.43, m
2.23, m; 2.41, m
2.29, m; 2.35, m
1.75, qd (14.0, 3.5);
2.24, m
1.77, m; 2.04, m 1.36, m; 1.96, m
2.12, m; 2.49, m 2.28, m; 2.37, m
7.06, d (8.0)
7.33, d (8.5)
1.88, m; 2.18, m
4.13, m
2.57, d (3.0)
6.52, dd (8.5, 2.5) 7.00, dd (8.5, 2.5) 2.28, dt (8.2, 2.2),
2.64, ddd (8.7,
6.2, 2.5)
3
4
5
6
–
–
–
–
–
–
–
–
–
–
–
–
5.80, m
–
2.30, m; 2.45, m 4.28, t (2.5)
5.85, d (2.0)
–
5.82, s
–
5.80, t (1.5)
–
5.83, t (2.0)
–
5.78, s
–
2.38, m; 2.47,
dq (13.5, 2.0)
5.82, s
–
2.31, m; 2.48, dt
(6.0, 3.0)
1.05, qd (13.0, 4.0);
1.92, m
5.61, s
–
5.78, s
–
6.46, d (2.5)
–
2.73, m; 2.79, m
7.36, d (3.0)
–
–
5.81, s
–
4.38, t (1.5)
2.31c, m; 2.47,
dt (14.0, 3.0)
2.28c, td (14.0, 5.0); 2.35c, m; 2.53,
2.47c, dt (14.5, 3.0) dt (15.0, 3.5)
2.18c, m; 2.56c, m 2.27, m; 2.38, m
7
1.01, m; 1.82, m 1.25c, m; 1.96c, m 1.05, qd (13.5, 4.0); 1.05, qd (13.0, 4.0); 1.05, m; 1.80, m 1.07, m; 1.93, m
0.89c, m; 1.73c, m 1.61, m; 1.96, m
1.26, m; 1.85, m 2.24, dd
1.25, m; 2.02, dt
1.88, m
1.91, m
(16.5, 13.0); 2.63, (10.5, 7.0)
dd (17.0, 3.5)
1.91, m
2.45, m
–
8
9
10
11
12
1.75, m
0.82, m
2.10, m
1.92, m
0.85, m
2.55, m
1.75, qd (11.0, 3.5) 1.75, qd (11.5, 3.5) 1.46, m
0.98, td (11.0, 3.5) 0.96, td (11.0, 3.5) 1.34c, m
1.52, qd (11.0, 3.0) 2.34, m
1.72, m
0.93, m
4.93,s (OH)
2.19, m
0.85, qd (10.5, 5.0) 2.10, m
2.50, dt (14.5, 3.0)
1.39, m
2.14, m
1.05, td (5.7, 2.2)
–
1.03, m
2.42, m
0.96, td (11.0, 3.0)
2.62, m
2.63, br. t (10.5)
2.82c, m
4.15, m
2.33c, m
–
1.62, m; 1.87, m 1.62c, m; 1.87c, m 4.16, q (3.0)
1.37, m; 1.54, m 1.37, m; 1.54, m 1.42, m; 1.55, m
1.38c, m; 1.96, m 3.83, td (10.5, 5.5) 4.18, m (W1/2 = 10 Hz) 1.53, m; 1.55, m 1.05, m; 1.55, m
1.45, m; 2.30, m 1.58, m; 2.40, m 1.60, m; 1.70, m
1.47, m; 1.63, m 1.57, m; 1.70, m 1.35, m; 1.57, m
1.48, dd (14.0, 3.5); 1.39c, m; 1.55, m 1.30, m; 1.85, m
1.82, dd (14.0, 2.5)
1.48, dd (14.0, 3.5);
1.82, dd (14.0, 2.5)
1.18, m; 1.43, m 1.36, m; 1.64, m
13
14
15
16
17
18
17
–
–
–
–
–
–
–
–
–
–
–
–
0.82, m
1.27, m
1.35, m
1.26, m
1.33c, m
1.35, m
1.28, m
1.15, m
1.37, m
1.40, m
1.64, m
1.32, m
1.28, m; 1.55, m 1.28, m; 1.57, m 1.39, m; 1.58, m
1.37, m; 1.62, m
1.34c, m; 1.62, m 1.32, m 1.62, m
1.40, m; 1.62, m
1.65, m; 1.87, m
–
1.12, s
1.16, s
1.15, m; 1.47, m 1.37, m; 1.62, m
1.48, m; 1.74, m 1.84, m; 1.94, m
4.03, s (OH)
0.77, s
1.34, m; 1.65, m 1.34, m; 1.65, m 1.32, m; 1.62, m
1.70, m; 1.89, m 1.72, m; 1.89, m 1.66, m; 1.86, m
–
0.88, s
1.23, s
1.64, m; 1.85, m 1.67c, m; 1.85c, m 1.59, m; 1.92, m
1.65c, m; 1.88c, m 1.66, m; 1.88, m 1.69, m; 1.88, m
–
–
–
–
–
–
–
–
–
0.90, s
0.93, s
1.18, s
1.14, s
3.38, d (11.0)
3.57, d (11.5)
1.12, s
1.16, s
0.93, s
1.19, s
0.91, s
1.20, s
0.95, s
3.42, d (11.0);
3.60, d (11.0)
0.88, s
1.26, s
0.93, s
1.18, s
a
-CH3 1.20, s
1.05, s
a
b
c
500 MHz, CD3OD.
500 MHz, DMSO-D6.
Exchangeable assignments.

422
S. Zafar et al. / Steroids 78 (2013) 418–425
Table 2
13C NMR Chemical shifts of compounds 1–12.
COMPOUNDS
C
1a
26.9
2a
27.1
3a
27.0
4a
35.7
5a
66.3
6a
28.5
7a
27.1
8b
33.0
9a
27.0
10a
11a
12a
34.0
1
2
3
4
5
6
7
8
127.2
113.8
156.0
116.0
139.0
30.5
28.6
41.2
45.1
132.5
27.2
32.8
47.0
51.0
24.0
39.0
82.0
14.8
26.0
127.8
122.5
157.0
113.2
134.5
200.8
45.0
42.2
43.8
140.0
26.8
32.4
46.8
50.6
23.7
39.1
82.0
14.2
26.0
37.3
199.6
124.5
166.6
36.4
32.4
37.1
55.1
42.5
27.2
32.6
45.6
49.5
23.9
39.0
81.3
14.8
26.2
37.0
203.9
125.6
169.1
72.6
39.3
35.7
50.8
39.6
27.2
32.6
47.0
50.7
23.9
39.2
82.3
14.7
26.1
37.3
203.0
124.8
172.1
36.4
32.4
37.1
55.1
39.0
67.4
40.1
46.4
52.2
24.3
33.5
84.5
17.5
67.8
73.0
202.1
122.7
171.0
35.5
31.9
36.9
55.8
39.2c
67.1
40.0
46.4
51.8
23.9
39.1c
82.5
17.1
26.4
46.9
201.7
124.7
166.1
36.2
30.7
41.9
43.9
48.4
26.5
32.5
46.8
50.8
24.0
39.1
82.3
14.6
26.0
36.5
204.1
124.5
172.1
37.8
33.5
42.2
55.8
45.3
73.0
44.0
47.2
50.0
24.1
39.1
81.5
15.5
25.6
37.1
203.1
124.5
172.1
36.2
32.2
37.2
55.1
39.1
68.0
40.0
46.0
51.9
24.2
39.0
82.0
17.0
26.2
33.5
198.5
123.1
165.0
31.5c
31.2c
35.5
52.7
69.1
19.5
30.7
45.0
49.0
23.5
38.1
79.5
14.0
26.5
37.0
202.5
125.1
171.0
36.5
33.8
43.9
50.9
35.5
27.3
32.5
46.5
51.2
24.4
32.2
84.0
15.0
67.8
34.8
202.8
126.0
162.5
73.8
39.5
31.2
54.2
72.0
21.0
32.4
46.8
50.8
24.2
39.2
82.0
14.5
25.8
9
10
11
12
13
14
15
16
17
18
17
a-CH3
a
b
c
125 MHz, CD3OD.
125 MHz, DMSO-D6.
Exchangeable assignments.
OH
18
12
17
15
13
11
16
H
H
14
9
1
10
8
2
3
H
H
5
7
4
6
O
1
OH
OH
OH
H
H
OH
H
H
H
H
H
O
O
O
2
8
OH
H
OH
H
H
H
9
Fig. 2. Biotransformation of methyloestrenolone (1) with Aspergillus niger.
2.7. Fermentation of methyloestrenolone (1) with C. echinulata
2.7.1. 17
a-Methyl-6b,10b,17b-trihydroxyestr-4-en-3-one (12)
Colorless amorphous solid (5.0 mg, 0.006%).
½
a2D5ꢃ: +15
900 mg of compound 1 (20 mL acetone) was fed equally to 40
cultured flasks of C. echinulata, yielding metabolites 6 (6:4 Pet.
ether/EtOAc) and 12 (1:1 Pet. ether/EtOAc) after 12 days of fermen-
tation (Fig. 4). Metabolite 12 was found to be a new compound.
(c = 0.0018, MeOH). UV (MeOH) kmax nm (log): 239 (4.2). IR (CHCl3)
mmax cmꢁ1: 3399, 1658. 1H NMR (CD3OD, 500 MHz): See Table 1.
13C NMR (CD3OD, 125 MHz): See Table 2. HREI-MS m/z (mol. for-
mula, calcd value): 320.2033 (C19H28O4, 320.2046). EI-MS m/z

S. Zafar et al. / Steroids 78 (2013) 418–425
423
18
OH
12
17
15
13
11
9
16
H
H
14
1
4
10
8
2
3
H
6
H
5
7
O
1
OH
OH
OH
HO
H
H
H
H
H
O
H
H
H
H
H
HO
O
HO
11
10
7
Fig. 3. Biotransformation of methyloestrenolone (1) with Gibberella fujikuroi.
OH
HO
OH
18
H
H
H
12
17
15
13
11
9
H
H
16
H
H
14
1
4
OH
10
8
O
O
2
3
H
H
5
7
6
6
O
OH
1
H
H
12
OH
Fig. 4. Biotransformation of methyloestrenolone (1) with Cunninghamella echinulata.
(rel. int.,%): 320 [M+] (35), 286 (71), 268 (17), 228 (100), 215 (45),
159 (18), 121 (12), 91 (42), 55 (7).
The 1H NMR spectrum (Table 1) of 2 showed a downfield
methine signal at d 4.28 as a triplet (J6e,7a,e = 2.5 Hz), with corre-
sponding carbon at d 72.6 in the 13C NMR spectrum (Table 2).
The C-4 olefinic proton resonated as a close doublet at d 5.85
(J4,6e = 2.0 Hz), due to its weak allylic couplings with C-6 allylic
proton (d 4.28), which is geminal to the hydroxyl group. The proton
at d 4.28 also showed HMBC correlations with C-4 (d 125.6), and C-
10 (d 39.6). It showed vicinal coupling with H2-7 (d 1.25, d 1.96) in
COSY spectrum. The carbon at d 72.6 was HMBC correlated with H-
4 (d 5.85) and H-8 (d 1.92). From the above observations, the hy-
droxyl-bearing methine carbon was identified as C-6. H-6 did not
show any NOESY interaction with H-10 (d 2.55) and H-8 (d 1.92),
3. Results and discussion
Microbial transformation of the oral contraceptive, methyloest-
renolone (1), C19H28O4, was investigated for the first time. Fermen-
tation of 1 with M. phaseolina, A. niger, G. fujikuroi and C. echinulata
yielded eleven metabolites 2–12 (Figs. 1–4). Metabolites 2–5, 11
and 12 were found to be new which are discussed below in detail.
The composition of metabolite 2 was deduced as C19H28O3 (M+
m/z 304.2032, calcd 304.2038), suggesting the incorporation of an
oxygen to the substrate 1. The UV analysis indicated the retention
of chromophore (kmax = 241 nm), while hydroxyl (3410 cmꢁ1) and
conjugated ketone (1660 cmꢁ1) functionalities were deduced from
the IR spectrum.
therefore it was assigned an
a-orientation. Finally the structure
of 2 was identified as a new compound, 17
a
-methyl-6b,17b-
dihydroxyestr-4-en-3-one (2). The assigned structure was
unambiguously deduced by single-crystal X-ray diffraction studies

424
S. Zafar et al. / Steroids 78 (2013) 418–425
(Fig. 5). The ORTEP diagram of compound 2 (Fig. 5) showed that
molecule consists of four fused rings A (C1–C5/C10), B (C5–C10),
C (C8–C9/C11–C14) and D (C13–C17). Ring A exists in a half chair
conformation, whereas trans fused rings B and C are in chair con-
formations. Ring D is folded like an envelope. The hydroxy substit-
uents at C-6 and C-17 adopt axial and pseudo equatorial
orientations, respectively. In the crystal structure, the molecules
are linked by O2–H2Aꢂ ꢂ ꢂO3 and O3–H2Aꢂ ꢂ ꢂO2 interactions to form
infinite chains running parallel to a-axis (Fig. 6). The bond dimen-
sions (bond lengths and angles) were in normal range and similar
to those found in structurally related steroidal compounds [31,33].
The HREI-MS analysis of metabolite 3 supported the molecular
composition C19H28O4 [M+ = m/z 320.1992 (calcd 320.1988)], with
32 amu increment from substrate 1, suggesting the incorporation
of two oxygen atoms. An enone (1657 cmꢁ1, kmax 238 nm), and hy-
droxyl functionality (3410 cmꢁ1) were deduced from the UV and IR
spectral analyses, respectively.
Fig. 6. The crystal packing of compound 2.
The 1H NMR spectrum of 3 displayed a hydroxyl-bearing
methine proton signal at d 4.16 (q, J9a,12a,e = 3.0 Hz) and downfield
methylene protons at d 3.38 (d, J20a,20b = 11.0 Hz) and 3.57 (d,
J20b,20a = 11.5 Hz), while the 13C NMR spectrum (Table 2) displayed
corresponding peaks at d 67.4 (CH) and 67.8 (CH2), respectively.
The spectrum showed only a single methyl carbon signal at d
17.5. This suggested the hydroxylation at the other methyl group,
67.1, respectively, in the 13C NMR spectrum (Table 2). The chemical
shift and multiplicity of the methine proton signal at d 4.15 was
similar to the H-11 of the metabolite 3. Thus after careful examina-
tion of the HMBC and COSY spectra, the –OH was placed at C-11
with a
a-oriented geminal methine proton. The other hydroxyl-
most probably C-17
ported by the HMBC correlations of the methylene protons (d
3.38 and 3.57) with C-16 (d 33.5).
a
-CH3. This initial inference was further sup-
bearing methine C-2 (d 73.0) was HMBC correlated with H2-1 (d
1.42, d 2.52) and H-4 (d 5.80). On the other hand, H-2 (d 4.13)
showed HMBC cross peaks with C-3 (d 202.1) and C-10 (d 39.2).
Based on these observations, this -OH was placed at C-2. The COSY
cross peaks of H-2 (d 4.13) with H2-1 (d 1.42, 2.52) further sup-
ported this inference. The H-2 (d 4.13) showed NOE interaction
with H-10 (d 2.82), which in turn showed NOE with H-8 (d 1.75).
Thus H-2 was deduced to be b-oriented. The structure of this
The other hydroxyl-bearing methine proton (d 4.16) showed
HMBC cross peaks with C-9 (d 55.1) and C-13 (d 46.4). It was there-
fore assigned to C-11 proton. H-11 (d 4.16) also showed vicinal
couplings in COSY spectrum with H-9 (d 0.98) and H-12 (d 1.42,
d 1.55), further confirming the assignment. The stereochemistry
at C-11 was deduced by 2D-NOE techniques. H-10 (d 2.63) showed
NOE interaction with H-8 (d 1.75), which in turn showed NOE with
H-18 (d 1.14). All the above protons are axially oriented, and none
showed any NOE correlation with H-11 (d 4.16). This suggested
new metabolite 4 was elucidated as 17a-methyl-2a,11b,17b-tri-
hydroxyestr-4-en-3-one.
Metabolite 5 with M+ at m/z 304.2043 (calcd 304.2038) was in
agreement with the formula C19H28O3. This suggested the incorpo-
ration of an oxygen atom into substrate 1. The presence of an en-
that H-11 is probably equatorially oriented (
a). The new com-
pound was thus identified as 17a-(hydroxymethyl)-11b,17b-
one moiety in ring A was inferred from the UV spectrum
dihydroxyestr-4-en-3-one (3).
(kmax = 239 nm), while the IR spectrum exhibited absorption bands
Molecular composition of 4 was deduced as C19H28O4 (M+ m/z
320.1986, calcd 320.1988), which also suggested the incorporation
of two oxygen atoms in substrate 1. The presence of hydroxyl func-
tional group (3416 cmꢁ1) and an enone system (kmax = 242 nm,
1674 cmꢁ1) was inferred from the IR and UV spectral analyses.
The 1H NMR spectra of metabolite 4 (Table 1) showed two hy-
droxyl-bearing methine proton signals at d 4.13 (m) and 4.15
(m). Their corresponding carbons were resonated at d 73.0 and
at 3410 (OH) and 1663 cmꢁ1 (C@C–C@O).
The 1H NMR spectrum of 5 (Table 1) showed a methine proton
signal at d 4.41 (q, J1a,2a,e,10a = 3.0 Hz), with corresponding carbon at
d 66.3 in the 13C NMR spectrum (Table 2). From the HMQC, COSY
and HMBC spectral analyses, the methine signals (d 4.41/d 66.3)
were assigned to H/C-1. The H-1 (d 4.41) showed HMBC correla-
tions with C-3 (d 201.7), C-5 (d 166.1) and C-10 (d 48.4). Moreover,
the position of this methine proton (d 4.41) was deduced by COSY
cross peaks of H-1 (d 4.41) with H2-2 (d 2.57) and H-10 (d 2.33).
The stereochemistry at C-1 was determined by NOESY spectral
analyses. The H-1 showed strong NOESY interaction with H-9 (d
1.34), which indicated that H-1 is axial and
a-oriented. This new
metabolite 5 was thus identified as 17a-methyl-1b,17b-dihydroxy-
estr-4-en-3-one.
The HREI-MS supported the molecular composition of 11 as
C
19H24O3. The M+ appeared at m/z 300.1782 (calcd 300.1773).
The compound showed a UV absorption (kmax) at 245 nm. The IR
spectrum showed major absorptions at 3410 (OH), 1664 (C@O)
and 1606 (C@C) cmꢁ1
.
The 1H NMR spectrum of metabolite 11 (Table 1) was distinctly
similar to metabolite 10. The spectrum showed protons in the aro-
matic region at d 7.00 (dd, J1,2 = 8.5 Hz, J2,4 = 2.5 Hz), 7.33 (d,
J2,1 = 8.5 Hz) and 7.36 (d, J4,2 = 3.0 Hz). The 13C NMR spectrum (Ta-
ble 2) showed the corresponding aromatic carbon signals at d
122.5, 127.8, and 113.2, respectively. An additional carbon signal
at d 200.8 was for ketonic carbonyl group. Comparison with the
NMR data of metabolite 10 indicated the aromatization of ring A.
Fig. 5. The molecular structure of the compound 2 with displacement ellipsoids
drawn at 30% probability level. Hydrogen atoms are omitted for clarity.

S. Zafar et al. / Steroids 78 (2013) 418–425
425
Macrophomina phaseolina and b-glucuronidase inhibitory activity of
transformed products.. J Enz Inhib Med Chem 2012;27:348–55.
[5] Choudhary MI, Erum S, Atif M, Malik R, Khan NT, Atta-ur-Rahman.
Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four
filamentous fungi. Steroids 2011;76:1288–96.
These aromatic carbons were identified as C-1 (d 127.8), C-2 (d
122.5), and C-4 (d 113.2), based on COSY and HMBC interactions.
H-1 (d 7.33) and H-8 (d 1.91) showed HMBC cross peaks with the
ketonic carbonyl carbon (d 200.8), therefore it was assigned to
the C-6. The rest of the structural features were distinctly similar
with metabolite 10. Thus the new metabolite 11 was characterized
[6] Choudhary MI, Shah SAA, Atta-ur-Rahman, Khan SN, Khan MNT. a-Glucosidase
and tyrosinase inhibitors from fungal hydroxylation of tibolone and
hydroxytibolones. Steroids 2010;75:956–66.
[7] Choudhary MI, Atif M, Atta-ur-Rahman. Oxidation of lynestrenol by the fungus
Cunninghamella elegans. Nat Prod Res 2010;24:1–6.
[8] Choudhary MI, Mohammad YM, Musharraf SG, Parvez M, Al-Aboudi A, Atta-ur-
Rahman. New oxandrolone derivatives by biotransformation using Rhizopus
stolonifer. Steroids 2009;74:1040–4.
[9] Al-Aboudi A, Mohammad MY, Haddad S, Al-Far R, Choudhary MI, Atta-ur-
Rahman. Biotransformation of methyl cholate by Aspergillus niger. Steroids
2009;74:483–6.
[10] Choudhary MI, Siddiqui ZA, Nawaz SA, Atta-ur-Rahman. Microbial
transformation of 18b-glycyrrhetinic acid by Cunninghamella elegans and
Fusarium lini, and lipoxygenase inhibitory activity of transformed products.
Nat Prod Res 2009;23:507–13.
as 17a-methyl-3,17b-dihydroxyestr-1,3,5(10)-trien-6-one.
The molecular composition of 12 was found to be C19H28O4
[M+ = m/z 320.2033, calcd 320.2046], suggesting dihydroxylation,
as in cases of metabolites 3 and 4. The UV spectrum showed
absorption maximum at 239 nm. The IR spectrum displayed
absorptions at 3399 (OH) and 1658 (C@O) cmꢁ1
.
The 1H NMR spectrum of compound 12 (Table 1) showed a
deshielded triplet at d 4.38 (J6e,7a,e = 1.5 Hz) with corresponding
carbon appearing at d 73.8 in the 13C NMR spectrum (Table 2). This
observation was very similar to metabolite 2. The hydroxylation
[11] Azizuddin,
Saifullah,
Khan
S,
Choudhary
MI,
Atta-ur-Rahman.
Biotransformation of dydrogesterone by cell suspension cultures of
Azadirachta indica. Turk J Chem 2008;32:141–6.
[12] Al-Aboudi A, Mohammad MY, Musharraf SG, Choudhary MI, Atta-ur-Rahman.
Microbial transformation of testosterone by Rhizopus stolonifer and Fusarium
lini. Nat Prod Res 2008;22:1498–509.
was thus proposed to have occurred at C-6 (a-oriented). Another
downfield quaternary carbon also appeared at d 72.0. Rest of the
spectrum did not show any major difference with the substrate
1. The downfield methine carbon (d 73.8) and the quaternary car-
bon (d 72.0) were HMBC correlated with the C-4 olefinic proton (d
5.81). The HMBC of H-6 (d 4.38) with the downfield quaternary
carbon (d 72.0) suggested that the other hydroxyl group was
substituted at C-10 of the steroidal skeleton. C-10 (d 72.0) was also
HMBC correlated with H2-2 (d 2.28, dt, J2a,2e = 8.2 Hz, J2a,1a/
1e = 2.2 Hz, d 2.64, ddd, J2e,2a = 8.7 Hz, J2e,1a = 6.2 Hz, J2e,1e = 2.5 Hz)
which also supported the assignment. The structure of a new
[13] Choudhary MI, Shah SAA, Atta-ur-Rahman. Microbial oxidation of anabolic
steroids. Nat Prod Res 2008;22:1289–96.
[14] Choudhary MI, Azizuddin, Jalil S, Musharraf SG, Atta-ur-Rahman. Fungal
transformation of dydrogesterone and inhibitory effect of its metabolites on
the respiratory burst in human neutrophils. Chem Biodiv 2008;5:324–31.
[15] Choudhary MI, Khan NT, Musharraf SG, Anjum S, Atta-ur-Rahman.
Biotransformation of adrenosterone by filamentous fungus Cunninghamella
elegans. Steroids 2007;72:923–9.
[16] Choudhary MI, Nasir M, Khan SN, Atif M, Ali RA, Khalil SM, Atta-ur-Rahman.
Microbial hydroxylation of hydroxyprogesterone and a-glucosidase inhibition
activity of its metabolites. Z Naturforsch 2006;62b:593–9.
[17] Choudhary MI, Yousuf S, Samreen, Shah SAA, Ahmed S, Atta-ur-Rahman.
metabolite 12 was finally deduced as 17a-methyl-6b,10b,17b-tri-
hydroxyestr-4-en-3-one.
Biotransformation of physalin
H and antileishmanial activity of its
Metabolites 6–10 were characterized as known compounds
based on detailed spectral analyses. Compound 6 has been earlier
obtained via the oxygenation of steroids by Mucorales fungi, re-
ported in a patent obtained by the Upjohn Co. in 1955 [34]. Metab-
olite 7 was prepared by Magerlein et. al., in 1961 through several
step synthetic manipulation of 3-methoxy-1,3,5(10)-estratrien-
17-one [28]. Sasaki synthesized metabolite 8 from 3,3-ethylenedi-
oxy-5(10)-estren-17b-ol through several synthetic steps [29]. 3,17-
and 20 steroid ketones produced metabolite 9 upon synthetic func-
tional group modifications, which was patented in 1964 [30].
Metabolite 10 was a synthetic product of estrone, reported by
Haack et al. [32].
In conclusion, we report here an efficient method for the trans-
formation of an oral contraceptive steroid, methyloestrenolone (1),
by using fungal cultures. This strategy can be used effectively for
the synthesis of libraries of new oral contraceptive steroids by
the in vitro metabolism.
transformed products. Chem Pharm Bull 2006;54:927–30.
[18] Choudhary MI, Batool I, Shah SAA, Nawaz SA, Atta-ur-Rahman. Microbial
hydroxylation of pregnenolone derivatives. Chem Pharm Bull 2005;53:
1455–9.
[19] Choudhary MI, Sultan S, Khan MNT, Atta-ur-Rahman. Microbial
transformation of 17a-ethynyl-and 17a-ethylsteroids, and tyrosinase
inhibitory activity of transformed products. Steroids 2005;70:798–802.
[20] Choudhary MI, Sultan S, Jalil S, Anjum S, Rahman AA, Fun H-K, Atta-ur-
Rahman. Microbial transformation of mesterolone. Chem Biodiv 2005;2:
392–400.
[21] Choudhary MI, Sultan S, Khan MTH, Yasin A, Shaheen F, Atta-ur-Rahman.
Biotransformation of (+)-androst-4-ene-3,17-dione. Nat Prod Res 2004;
18:529–35.
[22] Musharraf SG, Atta-ur-Rahman, Choudhary MI, Sultan S. Microbial
transformation of (+)-adrenosterone. Nat Prod Lett 2002;16:345–9.
[23] Atta-ur-Rahman, Choudhary MI, Asif F, Farooq A, Yaqoob M. Fungal
transformations of steroids by Cephalosporium aphidicola and Trichothecium
roseum. Nat Prod Lett 2000;14:217–24.
[24] Siemens. SMART and SAINT. Madison, WI, USA: Siemens Analytical X-ray
Instruments Inc; 1996.
[25] Altomare A, Cascarano G, Giacovazzo C, Guagliardi A. Completion and
refinement of crystal structures with SIR92. J Appl Cryst 1993;26:343–50.
[26] Sheldrick GM. SHELXTL-PC (Version 5.1). Madison, WI, USA: Siemens
Analytical Instruments Inc; 1997.
Acknowledgements
[27] Jhonson CK. ‘ORTEPII’ Report ORNL-5138. TN, USA: Oak Ridge National
Laboratory; 1976.
[28] Magerlein BJ, Hogg JA. 11-b-Hydroxy-17a-alkyl estrogens, vol. 2. Upjohn Co.;
1961. US 2991295 19610704.
[29] Sasaki K. 10Hydroxy-4-estren-3-one derivatives, vol. 7. Jpn. Tokyo
One of the authors, Salman Zafar, acknowledges the Higher
Education Commission, Pakistan, for providing financial support
through, ‘‘Indigenous Ph. D. Scholarship’’.
Koho: Shionogi and Co., Ltd.; 1967. CODEN: JAXXAD JP 42016299 19670904
Showa.
References
[30] Muller G, Poittevin A. Steroid Derivatives. Roussel-UCLAF 1964;18:FR
1359646.
[1] Straathof AJJ, Panke S, Schmid A. The production of fine chemicals by
biotransformations. Curr Opin Biotech 2002;13:548–56.
[31] Yousuf S, Zafar S, Choudhary MI, Ng SW. 17b-Hydroxy-17a-
(hydroxymethyl)estr-4-en-3-one. Acta Cryst 2010;E66:o2894.
[2] Fernandes P, Cruz A, Angelova B, Pinheiro HM, Cabral JMS. Microbial
conversion of steroid compounds: recent developments. Enzyme Microb
Technol 2003;32:688–705.
[3] Saure A, Teravainen T, Karjalainen O. The effect of synthetic gestagens on
progesterone formation in vitro in human placenta of early pregnancy. Reprod
Fert 1977;51:369–73.
[32] Haack E, Stoeck G, Voigt H. 17-Methylestradiol, an orally very active estrogen.
Naturwissenschaften 1954;41:429.
[33] Allen FH, Kennard O, Watson DG, Brammer L, Orpen AG, Taylor R. Tables of
bond lengths determined by X-ray and neutron diffraction. Part 1. Bond
lengths in organic compounds. J Chem Soc Perkin Trans 1987;2(12):S1–S19.
[34] . Oxygenation of Steroids by Mucorales Fungi. Upjohn Co.; 1955. GB 724149
19550216.
[4] Choudhary MI, Zafar S, Khan NT, Ahmad S, Noreen S, Marisini BP, Al-Khedairy
AA, Atta-ur-Rahman. Biotransformation of dehydroepiandrosterone with