Biotransformation of mestanolone and 17-methyl-1-testosterone by Rhizopus stolonifer
MOHAMMAD YASIN MOHAMMAD 1 , SYED GHULAM MUSHARRAF 1 , ABDULLAH M. AL-MAJID 2, ATTA- UR-RAHMAN 1 & MUHAMMAD IQBAL CHOUDHARY 1,2
1 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan and 2 Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia
Abstract Microbial transformation of mestanolone ( 1 ) using the plant pathogenic fungus, Rhizopus stolonifer , resulted in the produc-tion of two known metabolites, identifi ed as 11 α -hydroxymestanolone ( 3 ) and 6 α -hydroxymestanolone ( 4 ). Transformation of 17-methyl-1-testosterone ( 2 ) by R. stolonifer yielded two known metabolites, methandrostenolone ( 5 ) and 11 α ,17 β -dihydroxy-androsta-1,4-diene-3-one ( 6 ). These transformations included α -hydroxylations at C-11 and C-6, dehydro-genation at C-4, androsta and a demethylation at C-17 positions. Structures of transformed products were determined using spectroscopic techniques.
Correspondence: Muhammad Iqbal Choudhary, H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. Tel: � 92-21-4824924, 4824925. Fax: � 92-21-4819018. E-mail: [email protected]
(Received 11 October 2012 ; revised 29 December 2012 ; accepted 27 April 2013 )
Introduction
Microorganisms have been used extensively for hydroxylation of steroids as their enzymes can cata-lyze reactions with high regio- and stereospecifi ty. Their ability to oxidize steroidal compounds has immense synthetic and commercial importance. The hydroxylation of a large number of substances, including steroids, has been studied by employing a variety of microorganisms (Choudhary et al. 2005). However no report concerning the transformation of mestanolone ( 1 ) by Rhizopus stolonifer , and 17-methyl-1-testosterone ( 2 ) using microorganisms or plant cell culture has been published.
Mestanolone (17 β -hydroxy-17 α -methylandrostan-3-one, C 20 H 32 O 2 ) ( 1 ) is an anabolic synthetic derivative of testosterone that acts on androgen receptors (Pappo and Jung 1962). It has been previously synthesized by the oxidation of 17 β -meth-ylandrostan-3 β ,17 β -diol (Counsell et al. 1962). 17-methyl-1-testosterone (17 β -hydroxy-17 α -methyl-1-androsten-3-one, C 20 H 30 O 2 ) ( 2 ) is an anabolic synthetic derivative of mestanolone ( 1 ), obtained by bromination of mestanolone ( 1 ), followed by
dehydrobromination using 2-iodoxybenzoic acid (Ginotra et al. 2004). Mestanolone ( 1 ) and 17-methyl-1-testosterone ( 2 ) are the main intermediates in the synthesis of an anabolic synthetic steroid, oxandrolone (Ginotra et al. 2004).
In continuation of our biotransformation studies on bioactive compounds (Choudhary et al. 2003; Choudhary et al. 2005a – f; Choudhary et al. 2006; Devkota et al. 2007; Choudhary et al. 2009; Al-Aboudi et al. 2009), we describe here the syn thesis of hydroxylated derivatives of mestanolone ( 1 ) and 17-methyl-1-testosterone ( 2 ) by a plant pathogenic fungus Rhizopus stolonifer . Oxidation of 1 by R. stolonifer resulted in the formation of two hydroxylated metabolites which were identi-fi ed as 11 α -hydroxymestanolone ( 3 ) and 6 α -hydroxymestanolone ( 4 ), while oxidation of 2 by R. stolonifer resulted in the formation of two metabo-lites which were identifi ed as methandrostenolone ( 5 ) and 11 α ,17 β -dihydroxy-androsta-1,4-diene-3-one ( 6 ). Metabolites 3-6 have already reported as biotransformed products from various steroidal compounds (Schaenzer et al. 1991; Stewart et al.
Biocatalysis and Biotransformation, 2013; 31(4): 153–159
2009; Bartmanska et al. 2007; Ullah et al. 2012; Faramarzi et al. 2007; Al-Aboudi et al. 2008).
Experimental
General
Mestanolone ( 1 ) and 17-methyl-1-testosterone ( 2 ) were obtained from the Hikma Pharmaceuticals Limited, 11118 Amman, Jordan. Silica gel precoated plates (Merck, PF 254 ; 20 � 20, 0.25 mm) were used for TLC. Silica gel (70 – 230 mesh, Merck) was used for column chromatography. UV Spectra were recorded in methanol with a Hitachi U-3200 spec-trophotometer. Infrared (IR) spectra were recorded in KBr discs with an FT-IR-8900 spectrophotome-ter. 1 H- and 13 C-NMR spectra were recorded in CDCl 3 on a Bruker Avance-300 NMR spectrometer at 300 and 75 MHz, respectively, with tetramethyl-silane (TMS) as the internal standard. Standard pulse sequences were used for distortionless enhan-cement by polarization transfer (DEPT) and 2D-NMR experiments. The chemical shifts ( d values) are reported in parts per million, relative to TMS at 0 ppm. The coupling constants ( J values) are reported in Hertz. Electron impact mass spectra (EI-MS) and high-resolution electron impact mass spectra (HREI-MS) were recorded on a Jeol JMS-600H mass spectrometer; in m/z (rel. %).
Microorganisms and culture medium
Rhizopus stolonifer (NRRL 1392) was purchased from the Northern Regional Research Laboratories (NRRL), Peoria, Illinois, U.S.A., and grown on
Sabouraud-4% potato dextrose-agar (Merck) at 28 ° C and stored at 4 ° C. The medium for Rhizopus stolonifer was prepared by mixing the following ingredients into distilled H 2 O (4.0 L): glucose (80.0 g), peptone (20.0 g), yeast extract (20.0 g), KH 2 PO 4 (20.0 g), and NaCl (20.0 g).
Fermentation and extraction conditions for Compound 1
The fungal medium was transferred into 250 mL conical fl asks (100 mL each) and autoclaved at 121 ° C. Mycelia of R. stolonifer were transferred to all the fl asks and incubated at 28 ° C for 3 days with rotary shaking (128 rpm). After 3 days, compound 1 (1.0 g, 3.29 mmol) was dissolved in 40 mL acetone and added to each fl ask (25 mg/1.0 mL acetone) and the fl asks were placed on a rotary shaker (128 rpm) at 28 ° C for fermentation. Parallel control experi-ments were conducted which included incubation of the fungus without sample 1 and another incubation of 1 in a medium without fungus. Time course stud-ies were carried out by sampling after every 24 h and the degree of transformation was analyzed using TLC. After 7 days, the culture medium was fi ltered and extracted with ethyl acetate (12 L) in three por-tions. The extract was dried over anhydrous Na 2 SO 4 , evaporated under reduced pressure, and the brown gummy crude residue (1.5 g) was analyzed using thin-layer chromatography.
Fermentation and extraction conditions for compound 2
The fungal medium was transferred into 250 mL conical fl asks (100 mL each) and autoclaved at 121 ° C.
Table I. 1 H-NMR data of Compound 1 and its Metabolites 3 and 4 (300 MHz; CDCl 3 ).
No. 1 d H
3 d H
4 d H
1 1.56 a (1H, m), 1.67 b (1H, m) 1.56 (1H, m), 1.76 a (1H, m) 1.23 a (1H, m), 1.60 b (1H, m)2 2.08 c (1H, m),
2.73 (1H, ddd, J � 14.0, 6.5, 2.0 Hz)2.10 c (1H, m), 2.53 (1H, m)
4 2.02 c (1H, m), 2.25 (1H, m) 2.07 (1H, dt, J � 15.0, 15.0, 3.0 Hz), 2.26 (1H, m)
1.96 (1H, m), 2.08 c (1H, m)
5 1.53 (1H, m) 1.54 (1H, m) 1.28 (1H, m)6 1.35 d (1H, m), 1.50 a (1H, m) 1.30 b (1H, m), 1.50 (1H, m) 3.30 (1H, m)7 1.23 e (1H, m), 1.32 d (1H, m) 1.14 (1H, m), 1.32 b (1H, m) 0.75 (1H, m), 1.88 (1H, m)8 1.44 (1H, m) 1.45 (1H, m) 1.43 (1H, m)9 0.70 (1H, td, J � 10.8, 10.8, 3.2 Hz) 0.81 (1H, t, J � 10.5 Hz) 0.83 (1H, m)
11 1.27 e (1H, m), 1.30 d (1H, m) 4.01 (1H, td, J � 10.0, 10.0, 5.0 Hz) 1.22 a (1H, m), 1.26 (1H, m)12 1.19 e (1H, m), 1.60 (1H, m) 1.25 b (1H, m), 1.83 a (1H, m) 1.20 a (1H, m), 1.38 d (1H, m)14 1.21 (1H, m) 1.26 (1H, m) 1.13 (1H, m)15 1.22 e (1H, m), 1.59 a (1H, m) 1.27 b (1H, m), 1.57 (1H, m) 1.11 (1H, m), 1.37 d (1H, m)16 1.69 b (1H, m), 1.79 (1H, m) 1.71 (1H, m), 1.80 a (1H, m) 1.59 b (1H, m), 1.63 b (1H, m)18 0.85 (3H, s) 0.87 (3H, s) 0.80 (3H, s)19 1.01 (3H, s) 1.21 (3H, s) 1.16 (3H, s)20 1.19 (3H, s) 1.13 (3H, s) 1.07 (3H, s)
a, b, c, d, e: These values are interchangeable.
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Fungal transformation of steroids 155
Mycelia of Rhizopus astolonifer were transferred to all the fl asks and incubated at 28 ° C for 3 days with rotary shaking (128 rpm). After 3 days, compound 2 (1.0 g, 3.31 mmol) was dissolved in 40 mL acetone and added to each fl ask (25 mg/1.0 mL acetone) and the fl asks placed on a rotary shaker (128 rpm) at 28 ° C for fermentation. Parallel control experiments and time course experiments were conducted as described above. After 7 days, the culture medium was treated as described above, yielding a brown gummy residue (2.0 g) which was analyzed by thin-layer chromatography.
Isolation of transformed products
The crude extract was dissolved in chloroform, meth-anol (8:2 v/v), absorbed on silica (2.0 g) and subjected to column chromatography. The eluant consisted of gradient mixtures of chloroform and methanol. Com-pounds 1 (50 mg), 2 (80 mg), and 4 (150 mg) were eluted with CHCl 3 /MeOH (9.8:0.2 v/v), while com-pounds 3 (120 mg), 5 (180 mg), and 6 (80 mg) were eluted with CHCl 3 /MeOH (9.5:0.5 v/v).
2.5.1. 11 a -Hydroxymestanolone (11 α ,17 β -dihydroxy-17 α -methyl-5 α -androstan-3-one) ( 3 ), white
Table II. 1 H-NMR data of Compound 2 and its Metabolites 5 and 6 (300 MHz; CDCl 3 ).
No. 2 d H
5 d H
6 d H
1 7.13 (1H, d) 7.30 (1H, d) 7.74 (1H, d, J � 10.3 Hz)2 5.84 (1H, d) 5.80 (1H, d) 6.12 (1H, dd, J � 10.3, 1.4 Hz)4 2.21 (1H, m), 2.35 (1H, m) 5.71 (1H, s) 6.06 (1H, s)5 1.90 (1H, m) – – 6 1.40 (1H, m), 1.44 a (1H, m) 2.23 (1H, m), 2.37 (1H, m) 2.32 (1H, m), 2.44 (1H, m)7 1.26 (1H, m), 1.72 b (1H, m) 1.26 a (1H, m), 1.54 b (1H, m) 1.03 a (1H, m), 1.95 (1H, m)8 1.50 (1H, m) 1.58 (1H, m) 1.68 (1H, m)9 0.97 (1H, m) 0.90 (1H, m) 1.05 (1H, m)
11 1.45 a (1H, m), 1.80 (1H, m) 1.42 (1H, m), 1.50 b (1H, m) 4.04 (1H, td, J � 10.4, 10.4, 5.0 Hz)12 1.32 (1H, m), 1.55 c (1H, m) 1.16 (1H, m), 2.00 (1H, m) 1.07 a (1H, m), 2.20 (1H, m)14 1.22 (1H, m) 1.17 (1H, m) 1.00 (1H, m)15 1.24 (1H, m), 1.59 c (1H, m) 1.30 a (1H, m), 1.59 (1H, m) 1.28 (1H, m), 1.57 (1H, m)16 1.68 (1H, m), 1.78 b (1H, m) 1.69 (1H, m), 1.82 (1H, m) 1.46 (1H, m), 2.07 (1H, m)18 0.87 (3H, s) 0.87 (3H, s) 0.83 (3H, s)19 1.10 (3H, s) 1.19 (3H, s) 1.30 (3H, s)20 1.20 (3H, s) 1.20 (3H, s) –
a, b, c: These values are interchangeable.
Table III. 13 C-NMR data of Compounds 1 – 6 (300 MHz; CDCl 3 ).
No. 1 d c
2 d c
3 d c
4 d c
5 d c
6 d c
1 38.6 a ( t ) 158.4 ( d ) 38.9 a ( t ) 38.2 a ( t ) 155.0 ( d ) 159.1 ( d )2 38.1 a ( t ) 127.4 ( d ) 38.4 a ( t ) 39.3 a ( t ) 128.6 ( d ) 125.0 ( d )3 212.0 ( s ) 200.2 ( s ) 211.0 ( s ) 213.3 ( s ) 211.5 ( s ) 186.9 ( s )4 44.7 ( t ) 41.0 ( t ) 45.2 ( t ) 44.5 ( t ) 123.9 ( d ) 124.4 ( d )5 46.8 ( d ) 44.4 ( d ) 47.5 ( d ) 52.9 b ( d ) 171.2 ( s ) 168.3 ( s )6 31.6 b ( t ) 27.6 ( t ) 31.3 ( t ) 69.1 ( d ) 32.8 ( t ) 33.1 ( t )7 31.4 b ( t ) 31.0 a ( t ) 29.7 ( t ) 40.6 ( t ) 31.4 ( t ) 34.7 ( t )8 36.3 ( d ) 36.5 ( d ) 35.8 ( d ) 34.9 ( d ) 36.5 ( d ) 37.0 ( d )9 53.8 ( d ) 50.0 b ( d ) 60.0 ( d ) 53.1 b ( d ) 53.8 ( d ) 60.8 ( d )
10 35.8 ( s ) 35.9 ( s ) 37.5 ( s ) 36.3 ( s ) 36.1 ( s ) 30.5 ( s )11 21.1 ( t ) 20.9 ( t ) 69.7 ( d ) 20.8 ( t ) 20.7 ( t ) 67.9 ( d )12 28.8 ( t ) 31.6 a ( t ) 44.0 ( t ) 31.3 ( t ) 35.8 ( t ) 45.0 ( t )13 45.5 ( s ) 45.7 ( s ) 45.8 ( s ) 45.4 ( s ) 45.2 ( s ) 49.5 ( s )14 50.5 ( d ) 50.6 b ( d ) 49.7 ( d ) 50.1 ( d ) 50.2 ( d ) 47.8 ( d )15 23.3 ( t ) 23.2 ( t ) 23.6 ( t ) 23.0 ( t ) 23.2 ( t ) 23.4 ( t )16 38.9 a ( t ) 39.0 ( t ) 40.1 ( t ) 38.3 a ( t ) 38.9 ( t ) 37.1 ( t )17 81.6 ( s ) 81.6 ( s ) 81.2 ( s ) 81.2 ( s ) 81.5 ( s ) 80.7 ( d )18 14.0 ( q ) 14.1 ( q ) 15.0 ( q ) 13.7 ( q ) 17.4 ( q ) 18.7 ( q )19 11.5 ( q ) 13.1 ( q ) 11.9 ( q ) 12.4 ( q ) 13.9 ( q ) 12.2 ( q )20 25.8 ( q ) 25.9 ( q ) 25.9 ( q ) 25.3 ( q ) 25.8 ( q ) –
Multiplicities were determined by DEPT experiments. a, b: These values are interchangeable.
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156 M. Y. Mohammad et al.
solid. UV (MeOH): λ max (log ε ) 246 nm (3.1). IR (MeOH): 3409, 2932, 1706, and 754 cm � 1 . 1 H- and 13 C- NMR: Tables I and III. EI-MS: m/z 320 (4, M � ), 302 (13), 284 (6), 244 (53), 229 (11), 187 (9), 161 (12), 135 (18), 107 (41), 79 (45), and 55 (100). HREI-MS: m/z 320.2360 (M � , [C 20 H 32 O 3 ]
� ; calc. 320.2351).
6 a -Hydroxymestanolone (6 α ,17 β -dihydroxy-17 α -methyl-5 α -androstan-3-one) ( 4 ), white solid. UV (MeOH): λ max (log ε ) 247 nm (3.1). IR (MeOH): 3408, 2930, 1706, and 753 cm � 1 . 1 H- and 13 C-NMR: Tables I and III. EI-MS: m/z 320 (9, M � ), 302 (11), 287 (5), 244 (50), 229 (8), 234 (27), 176 (6), 153 (33), 125 (88), 91 (58), 70 (100), and 55
O
O
HO
1
3
O
4OH
12
34
56
7
8
9
10
1112
13
14 1516
17
18
19
20HO
HO HO
H
H
H
H
H
H H
H
H
H H
H
Figure 1. Transformation of mestanolone ( 1 ) by Rhizopus stolonifer .
O O
2 5
6O
1
2
34
56
7
8
9
10
1112
13
14 15
1617
18
19
20HO HO
OH
HO
H
H H
H H
H H
H H
H
Figure 2. Transformation of 17-methyl-1-testosterone ( 2 ) by Rhizopus stolonifer .
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Fungal transformation of steroids 157
(88). HREI-MS: m/z 320.2349 (M � , [C 20 H 32 O 3 ] � ;
( 6 ), white solid. IR (MeOH): 3411, 2918, 1725, and 707 cm � 1 . 1 H- and 13 C-NMR: Tables II and III. EI-MS: m/z 302 (24, M � ), 284 (12), 266 (4), 251 (4), 225 (6), 197 (3), 181 (33), 161 (11), 147 (15), 134 (25), 122 (100), 107 (15), 91 (22), 77 (11), 67 (6), and 55 (7).
Results and discussions
Screening-scale experiment showed that Rhizopus stolonifer (NRRL 1392) had the capacity to trans-form compound 1 into its hydroxylated derivatives, compound 2 into 5 , and compound 5 into 6 , thus a large-scale experiment was performed. Incubation of mestanolone ( 1 ) and 17-methyl-1-testosterone ( 2 ) with Rhizopus stolonifer yielded four metabolites 3 – 6 (Figures 1 and 2). Metabolites 3 and 6 were obtained
as major products with 11.4% and 18.0% yields, respectively. A time course analysis of the transforma-tion of 1 revealed that metabolite 3 was formed after 48 h of incubation, while metabolite 4 was detected only after 72 h. A time course analysis of the trans-formation of 2 revealed that metabolite 5 was formed after 24 h of incubation, while metabolite 6 was favoured only after 72 h. Structures of the metabolites were elucidated spectroscopically.
The HREI-MS of metabolite 3 exhibited an M � at m/z 320.2360, corresponding to the formula C 20 H 32 O 3 (calc. 320.2351), 16 a.m.u. higher than 1 , indicating incorporation of an oxygen atom. The 1 H-NMR spectrum showed an additional OH-bearing methine proton signal at δ 4.01 (td, J β H11, α H9 � 10.0 Hz, J β H11, α H12 � 10.0 Hz, and J β H11, β H12 � 5.0 Hz), while the 13 C-NMR spectrum showed an OH-bearing methine carbon at δ 69.7, along with disap-pearance of the C-11 methylene signal at δ 21.1, in comparison to compound 1 . Moreover the downfi eld shift of C-9 ( δ C 60.0) and C-12 ( δ C 44.0) suggested that the hydroxylation occurred at C-11. The HMBC spectrum of metabolite 3 showed correlations of H-8 ( δ H 1.45), H-9 ( δ H 0.81) and H 2 -12 ( δ H 1.25 and 1.83) with C-11 ( δ C 69.7), while H-11 ( δ H 4.01) showed correlations with C-10 ( δ C 37.5), and C-12 ( δ C 44.0) which further supported evidence for hydroxylation at C-11 . The COSY 45 o spectrum showed correlations of H-11 with H 2 -12 and H-9. The α -stereochemistry of the C-11 OH was deduced
C-6 ( δ C 69.1). The COSY 45 o spectrum showed correlations of H-5, and H-7 with H-6, which further supported the hydroxylation at C-6. NOESY spectrum showed correlations between β Me-19 ( δ 1.16) with H-6 ( δ 3.30), supporting the presence of an OH substituent at the α (equatorial)-position of C-6 (Figure 4). These data indicated the structure of compound 4 as 6 α - hydroxymestanolone.
The HREI-MS of metabolite 5 exhibited an M � at m/z 300.2075, corresponding to the formula C 20 H 28 O 2 (calc. 300.2089), 2 a.m.u. less than 2 , indi-cating the introduction of a double bond. The 1 H-NMR spectrum showed an additional signal at δ H 5.71, while the 13 C-NMR spectrum showed a methine carbon at δ 123.9 and quaternary carbon at δ 171.2, along with the disappearance of C-4 methylene ( δ 41.0) and methine signals ( δ 44.4), in comparison to compound 2 . HMBC Spectrum of metabolite 5 showed the correlations of H-4 ( δ 5.71) with C-2 ( δ 128.6), C-3 ( δ 211.5), and C-5 ( δ 171.2) which fur-ther supported the dehydrogenation at C-4 (Figure 5). The structure of compound 5 was thus deduced as 17 β -hydroxy-17 α -methyl-androst-1,4-diene-3-one.
The EI-MS of metabolite 6 exhibited an M � at m/z 302, corresponding to the formula C 19 H 26 O 3 . The 1 H-NMR spectrum showed an additional signal at δ H 4.04 (td, J β H11, α H9 � 10.4 Hz, J β H11, α H12 � 10.4 Hz, and J β H11, β H12 � 5.0 Hz), while the 13 C-NMR spectrum showed two methine carbons at δ 67.9 and 80.7, and disappearance of C-11 methylene ( δ C 20.9) and quaternary C-17 ( δ C 81.6) signals, in
H
H
OH
HO
H
H
2
3 56 7
89
10
1112 13
14 15 16
17
18 CH3
19 CH3
1
4O
Figure 7. Key NOESY correlations of compound 6 .
O
OH
HH
H
O
OOH
HH
H OH
O
OH
HH
H
O
OH
HH
H
HO
[O]
[O]
5 5c
5d6
Dec
arbo
xyla
tion
_ C
O2
Hydroxylation
Scheme 1. Proposed pathway to the oxidized metabolite 6 .
from NOESY correlations of β H-8 ( δ 1.45), β Me-18 ( δ 0.87), and β Me-19 ( δ 1.21) with H-11 ( δ H 4.01), suggesting hydroxylation at the α -position (equa-torial) at C-11 (Figure 3). The structure of com-pound 3 was deduced as 11 α -hydroxymestanolone.
The HREI-MS of metabolite 4 showed an M � at m/z 320.2349, corresponding to the formula C 20 H 32 O 3 (calc. 320.2351) . The 1 H-NMR spectrum showed an additional OH-bearing methine proton at δ H 3.30 (m). The 13 C-NMR spectrum showed 20 carbon signals, with an additional methine carbon resonating at δ 69.1, and the disappearance of C-6 methylene signal, in comparison to compound 1 , which indicated hydroxylation of C-6. The HMBC spectrum showed correlations of the C-6 methine proton ( δ H 3.30) with C-4 ( δ C 44.5), C-5 ( δ C 52.9), and C-7 ( δ C 40.6), while H-4 ( δ H 2.08), H-5 ( δ H 1.28), and H-7 ( δ H 1.88) showed correlations with
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Fungal transformation of steroids 159
comparison to compound 2 , which indicated the hydroxylation and demethylation. HMBC Spectrum of metabolite 6 showed the correlations of H-8 ( δ H 1.68), H-9 ( δ H 1.05), and H 2 -12 ( δ H 1.07 and 2.20) with C-11 ( δ C 67.9), while H-11 ( δ H 4.04) showed correlations with C-10 ( δ C 30.5) and C-12 ( δ C 45.0) which further supported an OH at C-11 (Figure 6) . The COSY 45 o spectrum showed correlations of H-11 with H 2 -12 and H-9. The stereochemistry of the C-11 OH was deduced by NOESY correlations of β H-8 ( δ H 1.68), β Me-18 ( δ H 0.83), and β Me-19 ( δ H 1.30) with H-11 ( δ H 4.04) suggesting an α -OH (equatorial) (Figure 7). The structure of compound 6 was deduced as 11 α , 17 β -dihydroxy-androst-1,4-diene-3-one. The proposed biotransformation to the oxidized metabolite 6 is shown in Scheme 1.
Biotransformation by Rhizopus stolonifer proved to be effective for dehydrogenation, demethylation, and α -hydroxylation of C-11 and C-6. Metabolites 3 and 5 were obtained as major products, which may be used to synthesize new compounds with interest-ing biological activities.
Acknowledgement
We acknowledge the Hikma Pharmaceuticals Limi-ted, 11118 Amman, Jordan, for gift of substrates 1 and 2 .
One of us (Mohammad Yasin Mohammad) is grateful to the University of Karachi for fi nancial support during his Ph.D. studies.
Declaration of interest: The authors report no dec-larations of interest. The authors alone are responsi-ble for the content and writing of the paper.
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