Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites

Article abstract of DOI:10.1016/j.steroids.2014.04.004

Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2-6, while M. phaseolina transformed compound 1 into polar metabolites 7-11. These metabolites were identified as 6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12-14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1-14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC₅₀ values between 49.0 and 84.9 μM.

Full text of DOI:10.1016/j.steroids.2014.04.004

Accepted Manuscript
Biotransformation of dianabol with the filamentous fungi and β-Glucuronidase
inhibitory activity of resulting metabolites
Naik T. Khan, Salman Zafar, Shagufta Noreen, Abdullah M. Al Majid, Zeid A.
Al Othman, Saud Ibrahim Al-Resayes, Atta-ur-Rahman, M. Iqbal Choudhary
PII:
S0039-128X(14)00075-0
http://dx.doi.org/10.1016/j.steroids.2014.04.004
STE 7547
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 July 2013
15 March 2014
10 April 2014
Please cite this article as: Khan, N.T., Zafar, S., Noreen, S., Al Majid, A.M., Al Othman, Z.A., Al-Resayes, S.I.,
Atta-ur-Rahman, Iqbal Choudhary, M., Biotransformation of dianabol with the filamentous fungi and β-
Glucuronidase inhibitory activity of resulting metabolites, Steroids (2014), doi: http://dx.doi.org/10.1016/j.steroids.
2014.04.004
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Biotransformation of dianabol with the filamentous fungi and β-glucuronidase
inhibitory activity of resulting metabolites
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Naik T. Khan1, Salman Zafar1, Shagufta Noreen1, Abdullah M. Al Majid2, Zeid A. Al
Othman2, Saud Ibrahim Al-Resayes2, Atta-ur-Rahman*1, M. Iqbal Choudhary*1,2
1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
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2Department of Chemistry, College of Science, King Saud University, Riyadh-11451, Saudi
Arabia
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* Corresponding authors. Tel.: +92-21-34824924, 34824925; fax: +92-21-34819018; 34819019
E-mail: hej@cyber.net.pk
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Abstract
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Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the
filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied.
Incubation of 1 with C. elegans yielded five hydoxylated metabolites 2−6, while M. phaseolina
transformed compound 1 into polar metabolites 7−11. These metabolites were identified as
6β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17β-dihydroxy-17α-methylandrost-
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1,4-dien-3-one (3), 11α,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6β,12β,17β-
trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6β,15α,17β-trihydroxy-17α-methylandrost-1,4-
dien-3-one (6), 17β-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7β,17β,-dihydroxy-17α-
methylandrost-1,4-dien-3-one (8), 15β,17β-dihydroxy-17α-methylandrost-1,4-dien-3-one (9),
17β-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11β,17β-dihydroxy-17α-
methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12
and oximes 13, and 14. Compounds 6 and 12−14 were identified as new derivatives of dianabol (1).
The structures of all transformed products were deduced on the basis of spectral analyses.
Compounds 1−14 were evaluated for β-glucuronidase enzyme inhibitory activity. Compounds 7,
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13, and 14 showed a strong inhibition of β-glucuronidase enzyme, with IC50 values between 49.0
and 84.9 µM.
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Keywords: Dianabol, Anabolic Steroid, Biotransformation, Cunninghamella elegans,
Macrophomina phaseolina, Hydroxylation, β-Glucuronidase inhibition.
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1. Introduction
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Structural modifications of steroids by using microbial whole cells are now well established [1].
Microbial strains are capable of performing specific chemical transformations in steroids, such as
reduction, hydrolysis, oxidation, rearrangement, epimerization, isomerization, reverse aldol and
Michael addition reaction, with high regio- and stereo-selectivity [2].
In continuation of our studies on bioactive steroids [3−7], we report here the biotransformation
of an anabolic-androgenic steroid, dianabol (17β-hydroxy-17α-methylandrost-1,4-dien-3-one,
C20H28O2) (1), with filamentous fungi. Small scale screening indicated that Cunninghamella
elegans and Macrophomina phaseolina efficiently transformed 1 into many polar metabolites.
Subsequent large scale fermentation of 1 with these two fungi produced nine known metabolites
2−5 and 7−11, and a new compound 6. Metabolite 3 was obtained in sufficient amount and was
chemically transformed into diketone 12 and oximes 13 and 14 for SAR studies.
Previously we have reported several steroids having strong inhibitory activity against the enzyme
β-glucuronidase [6, 8]. β-Glucuronidase is found in both prokaryotic and eukaryotic organisms.
It is responsible of the hydrolysis of β-glucuronide compounds in vivo [9]. Increase in the levels
of β-glucuronidase enzyme has been found to be associated with some diseases, including
hepatic disorders, cancer, AIDS and inflammatory joint disease. The enzyme also has a role in
the development of cholelithiasis in humans [10]. Compounds 1−14 were also tested for their β-
glucuronidase inhibitory potential.
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2. Experimental
2.1. General
Dianabol (1) and pyridinium chlorochromate (PCC) were purchased from Fluka (USA), and
hydroxylammonium chloride from Wako (Japan). Column chromatography was carried out with
Silica gel (Merck, 70-230 mesh). Precoated silica gel plates (Merck, PF254) were used to perform
thin layer chromatography (TLC). Melting points were measured on Buchi-535 instrument.
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Infrared (IR) spectra (in cm-1) were recorded with an FT-IR-8900 spectrophotometer. UV Spectra
(in nm) was recorded on U-3200 spectrophotometer (Hitachi). The optical rotations were
measured in chloroform or methanol in a P-2000 polarimeter (JASCO). Jeol JMS-600H mass
spectrometer, in m/z (rel. %), was used for electron impact mass spectra (EI-MS) and high-
resolution electron impact mass spectra (HREI-MS). NMR experiments were measured in CDCl3
or CD3OD on a Bruker Avance spectrometer with standard Bruker pulse sequences. The
chemical shifts (δ) and coupling constants (J) were reported in ppm and Hertz, respectively. The
transformed products were purified on recycling HPLC (JAI, Japan) equipped with YMC L-80
(4-5 µm, 20 × 250 mm i.d.), with H2O-MeOH as mobile phase and UV detector at 254 nm.
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2.2. Microorganisms and culture medium. Macrophomina phaseolina (KUCC 730) was
provided by Karachi University Culture Collection (KUCC) and Cunninghamella elegans
(NRRL 1392) was purchased from Northern Regional Research Laboratories (NRRL). The
mentioned fungi were grown on sorbitol dextrose agar (SDA) at 25 °C, and stored at 4 °C. The
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media for both fungi C. elegans and M. phaseolina was made in distilled H2O (4.0 L) by adding
20.0 g of each yeast extract, potassium phosphate mono basic, sodium chloride, and peptone,
40.0 g of glucose and 40.0 mL of glycerol.
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2.3. General Fermentation and Extraction Conditions. Fermentation experiments were
conducted as previously described [3−8]. Briefly, substrate was incubated with fungi in sterilized
liquid medium. When fermentation was complete the biotransformed products were separated
from broth by liquid-liquid extraction (CH2Cl2), followed by evaporation of organic extract
followed by chromatographic separation of pure compounds.
2.4. Biotransformation of Dianabol (1) with Cunninghamella elegans. Substrate 1 (1 g in 20
mL acetone) was uniformly incubated with C. elegans in 40 flasks for 3 days. The finally
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obtained gummy material (3.7 g) was purified by silica gel column chromatography with
increased polarity of petroleum ether and acetone. The fractions obtained were pooled together
into five fractions (DH-1-5) on the basis on TLC analysis. Fractions DH-1 and -2 were subjected
to RPHPLC (L-80, MeOH-H2O = 2:1, 3 mL/min.) to afford pure compounds 2 (Rt: 32 min., p: 65
barr) and 3 (Rt: 32 min., p: 60 barr), while fraction DH-5 yielded compound 6 (L-80, MeOH:H2O
= 1:1, 3 mL/min., Rt: 38 min., p: 61 barr). Compound 4 was recrystallized from fraction DH-3 in
MeOH:H2O. Fraction DH-4 was further subjected to silica gel column chromatography (CC)
(pet. ether: acetone = 5.5:4.5) to obtain pure compound 5.
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2.4.1. 6β,17β-Dihydroxy-17α-methylandrost-1,4-dien-3-one (2)
Colorless crystalline solid (37 mg). m.p.: 227-229 °C. [Literature 230-231 °C] [11]. [α]25 : −42.6
D
(c = 0.1, CHCl3) [Literature −44, c = 1.0, CHCl3]. UV (MeOH): λmax nm (log ε) 245 (4.2). IR
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(KBr); νmax: 3386, 1658, 1609 cm-1. H-NMR (CDCl3, 400 MHz): Table-1. C-NMR (CDCl3, 75
MHz): Table-2. EI-MS: m/z 316 (29, M+), 298 (87), 283 (73), 258 (63), 243 (41), 240 (58), 225
(86), 197 (43), 186 (55), 172 (64), 159 (99), 157 (100), 147 (86), 108 (48), 79 (35). HREI-MS:
m/z 316.2026 (M+, [C20H28O3]+; calcd 316.2038).
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2.4.2. 15α,17β-Dihydroxy-17α-methylandrost-1,4-dien-3-one (3)
Colorless crystalline solid (186 mg). m.p.: 110−111 °C. [α]25D : +54.4 (c = 0.2, CHCl3). UV
(MeOH): λmax nm (log ε) 245 (4.4). IR (KBr); νmax: 3404, 1658, 1607 cm-1. 1H-NMR (CDCl3, 400
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MHz): Table-1. C-NMR (CDCl3, 75 MHz): Table-2. EI-MS: m/z 316 (5, M+), 298 (6), 280 (7),
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159 (37), 122 (100), 121 (98), 119 (51), 107 (30), 83 (84). HREI-MS: m/z 316.2033 (M+,
[C20H28O3]+; calcd 316.2038).
2.4.3. 11α,17β-Dihydroxy-17α-methylandrost-1,4-dien-3-one (4)
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Colorless crystalline solid (130 mg) . m.p.: 249-251 °C. [Literature 250−253 °C] [12]. [α]25
:
D
−35.3 (c = 0.25, CHCl3) [Literature -33, c = 1.0, CHCl3]. UV (MeOH): λmax nm (log ε) 248 (4.5).
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IR (KBr); νmax: 3405, 1657 cm-1. H-NMR (CDCl3, 400 MHz): Table-1. C-NMR (CDCl3, 75
MHz): Table-2. EI-MS: m/z 316 (36, M+), 298 (22), 240 (22), 227 (29), 195 (94), 181 (26), 163
(36), 147 (71), 134 (41), 122 (100), 91 (19), 77 (23), 55 (4). HREI-MS: m/z 316.1998 (M+,
[C20H28O3]+; calcd 316.2038).
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2.4.4. 6β,12β,17β-Trihydroxy-17α-methylandrost-1,4-dien-3-one (5)
Amorphous material (40 mg). [α]25 : +37.2 (c = 0.06, CHCl3). UV (MeOH): λmax nm (log ε) : 245
D
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13
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(4.2). IR (KBr): νmax 3406, 1658, 1602, 1054 cm-1. H-NMR (CDCl3, 400 MHz): Table-1. C-
NMR (CDCl3, 75 MHz): Table-2. EI-MS: m/z 332 (24, M+), 314 (25), 298 (9), 280 (22), 238
(41), 225 (33), 174 (100), 159 (68), 134 (72), 121 (77), 105 (48). HREI-MS: m/z 332.2002 (M+,
[C20H28O4]+; calcd 332.1987).
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2.4.5. 6β,15α,17β-Trihydroxy-17α-methylandrost-1,4-dien-3-one (6)
Colorless crystalline solid (32 mg). m.p.: 168−169 °C. [α]25 : +44.0 (c = 0.1, CHCl3). UV
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(MeOH): λmax nm (log ε) 247 (4.4). IR (KBr): νmax 3406, 1657, 1610. H-NMR (CD3OD, 500
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MHz): Table-1. 13C-NMR (CD3OD, 125 MHz): Table-2. EI-MS: m/z 332 (18, M+), 314 (33), 298
(12), 243 (34), 160 (28), 147 (44), 137 (70), 121 (57), 109 (100), 87 (32). HREI-MS: m/z
332.1964 (M+, [C20H28O4]+; calcd 332.1987).
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2.5. Fermentation of Dianabol (1) with Macrophomina phaseolina. M. phaseolina was grown
for 48 h and distributed equally into 40 flasks. The compound 1 (1 g) was dissolved in acetone
(20 mL) and divided equally among 40 flasks and incubated at 25 °C for 8 days. The obtained
gum (3.2 g) was purified on silica gel column chromatography by increasing the polarity of pet.
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ether and acetone. The two main fractions (DH-6, and 7) were pooled. Fraction DH-6 was further
purified by applying CC (silica gel), isocratic system of pet. ether- acetone (3:1), to afford
metabolites 7-9. Faction DH-7 was subjected to RPHPLC (L-80, MeOH:H2O = 1:1, 4 mL/min.,
p: 64 barr) to afford pure compounds 10 (Rt: 25 min.) and 11 (Rt: 22 min.).
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2.5.1. 17β-Hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7)
Colorlees crystalline solid (12 mg). m.p.: 159−160 °C. [Lit. 161−161.5 oC] [11]. [α]25 : −177.1 (c
D
= 0.3, CHCl3). [Literature −181, c = 1.0, CHCl3]. UV (MeOH) λmax nm (log ε) : 254 (2.8). IR
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(KBr): νmax 3447, 1660, 1612 cm-1. 1H-NMR (CDCl3, 400 MHz): Table-1. 13C-NMR (CDCl3, 100
MHz): Table-2. EI-MS: m/z 314 (91, M+), 296 (15), 238 (94), 225 (61), 202 (29), 160 (49), 147
(58), 137 (37), 121 (100), 82 (47). HREI-MS: m/z 314.1888 (M+, [C20H26O3]+; calcd 314.1882).
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2.5.2. 7β,17β,-Dihydroxy-17α-methylandrost-1,4-dien-3-one (8)
Colorless crystalline solid (110 mg). m.p.: 117−118 °C. [α]25D : +66.1 (c = 0.05, CHCl3). UV
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(MeOH) λmax nm (log ε): 243 (3.5). IR (KBr): νmax 3385, 1657, 1606 cm-1. H-NMR (CDCl3, 400
MHz): Table-1. C-NMR (CDCl3, 150 MHz): Table-2. EI-MS: m/z 316 (8, M+), 298 (13), 280
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(9), 240 (24), 177 (27), 159 (38), 122 (98), 82 (100), 43 (38). HREI-MS: m/z 316.2040 (M+,
[C20H28O3]+; calcd 316.2038).
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2.5.3. 15β,17β-Dihydroxy-17α-methylandrost-1,4-dien-3-one (9)
Colorlees crystalline solid (30 mg). m.p.: 161−162 °C. [α]25 : +46.5 (c = 0.05, CHCl3). UV
D
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(MeOH) λmax nm (log ε) : 245 (3.6). IR (KBr): νmax 3342, 1656, 1616 cm-1. 1H-NMR (CDCl3, 400
MHz): Table-1. C-NMR (CDCl3, 150 MHz): Table-2. EI-MS: m/z 316 (5, M+), 240 (8), 215
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(21), 147 (21), 122 (45), 121 (48), 91 (14), 44 (100). HREI-MS: m/z 316.2032 (M+, [C20H28O3]+;
calcd 316.2038).
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2.5.4. 17β-Hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10)
Colorless crystalline solid (17 mg). m.p.: 226−227 °C. [α]25 : +38.3 (c = 0.07, CHCl3). UV
D
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(MeOH): λmax nm (log ε) 242 (3.9). IR (KBr): νmax 3454, 1706, 1661 cm-1. H-NMR (CDCl3, 400
MHz): Table-1. 13C-NMR (CDCl3, 100 MHz): Table-2. EI-MS: m/z 314 (100, M+), 296 (14), 278
(10), 225 (47), 160 (53), 147 (61), 135 (31), 121 (83). HREI-MS: m/z 314.1871 (M+, [C20H26O3]+,
calcd 314.1882).
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2.5.5. 11β,17β-Dihydroxy-17α-methylandrost-1,4-dien-3-one (11)
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Colorlees crystalline solid (35 mg). m.p.: 262−263 °C. [Literature 262−265 °C] [13]. [α]25
:
D
+37.7 (c = 0.07, CHCl3) [Literature + 40, c = 1.0, CHCl3]. UV (MeOH) λmax nm (log ε) : 243
(3.6).
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2.6. Chemical Transformation of metabolite 3.
2.6.1. 17β-Hydroxy-17α-methylandrost-1,4-dien-3,15-dione (12)
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Compound 3 (16 mg, 0.05 mmol) was dissolved in distilled CH2Cl2 (10 mL), and pyridinium
chlorochromate (PCC) (30 mg, 0.14 mmol) was added to this solution. The reaction was stirred
at room temperature for 30 min. The solvent was removed by evaporation, and the crude was
subjected to silica gel CC with acetone- pet. ether (2:8) to afford pure compound 12 as a
crystalline solid (13.3 mg, 85%). m.p.: 209−210 °C. [α]25 : +63.8 (c = 0.06, CHCl3). UV
D
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(MeOH): λmax nm (log ε) 243 (4.3). IR (KBr): νmax 3428, 1731, 1659 cm-1. H-NMR (CDCl3, 500
MHz): Table-1. C-NMR (CDCl3, 75 MHz): Table-2. EI-MS: m/z 314 (98, M+), 296 (23), 229
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(25), 175 (33), 147 (100), 134 (32), 122 (64). HREI-MS: m/z 314.1873 (M+, [C20H26O3]+; calcd
314.1882).
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2.6.2. (3Z)-Hydroxyimino-17α-methylandrost-1,4-dien-15α,17β-diol (13)
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Compound 3 (50 mg, 0.16 mmol) was dissolved in pyridine (10 mL) and hydroxylammonium
chloride (20 mg, 0.3 mmol) was added to it in portions. The solution was stirred at reflux
temperature for 24 hour. The solvent was evaporated in vacuo, and the crude was
chromatographed on silica gel with 50% acetone in pet. ether to obtain a mixture of E / Z oximes
(43 mg, 76%). This was further purified by repeated RPHPLC (L-80, MeOH: H2O = 2:1, Flow
rate = 3 mL / min., p: 58 barr) to obtain pure compounds 13 (10 mg, Rt: 77 min.) and 14 (29 mg,
Rt: 82 min.). m.p.: 225−226 °C. [α]25 : +66.0 (c = 0.06, MeOH). UV (MeOH): λmax nm (log ε)
D
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13
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259 (4.1). IR (KBr): 3353, 1643, 1604, 933 cm-1. H-NMR (CD3OD, 500 MHz): Table-1. C-
NMR (CD3OD, 125 MHz): Table-2. EI-MS: m/z 331 (100, M+), 296 (47), 278 (38), 159 (34), 136
(45), 135 (74), 121 (89), 120 (93). HREI-MS: m/z 331.2153 (M+, [C20H29NO3]+, calcd 331.2147).
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2.6.3.
(3E)-Hydroxyimino-17α-methylandrost-1,4-dien-15α,17β-diol (14)
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m.p.: 202−204 °C. [α]25 : +45.2 (c = 0.08, MeOH). UV (MeOH): λmax nm (log ε) 259 (3.6). IR
D
1
13
(KBr): 3382, 1649, 1602, 949 cm-1. H-NMR (CD3OD, 600 MHz): Table-1. C-NMR (CD3OD,
150 MHz): Table-2. EI-MS: m/z 331 (57, M+), 296 (15), 159 (26), 146 (26), 135 (32), 121 (100),
120 (51). HREI-MS: m/z 331.2148 (M+, [C20H29NO3]+, calcd 331.2147).
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2.7. β-Glucuronidase inhibition Assay.
The spectroscopic method was used to determine the inhibition of β-glucuronidase. The
absorbance of p-nitophenol formed from the substrate was measured at 405 nm [6, 8]. The
reaction mixture (250 µL) containing 0.1 M acetate buffer (pH 7.0, 185 µL), test compound (5
µL, different concentration) and enzyme solution (10 µL) was incubated at 37 °C for 30 min.
After incubation, 50 µL of p-nitophenyl-β-D-glucuronide was added in the reaction mixture and
absorbance was measure at 405 nm by using SpectraMax plus 384 (Molecular Devices, CA,
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USA). All the experiments were performed in triplicate. The IC50 values were then calculated by
using the EZ-Fit Enzyme Kinetic program (Perrella Scientific Inc., Amherst, MA, USA).
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3. Results
Incubation of compound 1 with C. elegans and M. phaseolina produced ten metabolites 2−11.
Metabolites 2−4, 8, 9 and 11 were previously reported as biotransformed products [12−24],
while 5 was identified earlier as in-vivo metabolite of 1 through GC-MS [25]. Compounds 7 and
10 were synthesized chemo-enzymatically previously [11, 26].
The HREI-MS analysis of metabolite 5 showed an M+ at m/z 332.2002 for formula C20H28O4
(calcd 332.1987), 32 a.m.u. higher than the substrate 1. Absorption bands in IR spectrum at
3406 and 1658 cm-1 indicated the presence of OH and conjugated ketone functionalities,
respectively. The UV spectrum showed λmax at 245 nm for a conjugated ketone. The 1H NMR
spectrum (Table-1) of 5 showed a downfield methine triplet at δ 4.52 (J6α αβ = 2.6 Hz), and a
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,7
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methine double doublet at δ 3.74 (J β = 10.9 Hz, J α = 4.8 Hz), indicating hydroxylations at
12α,11
12α,11
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two sites. The C-NMR spectrum (Table-2) also showed two additional hydroxyl-bearing
methine carbons at δ 73.7 and 73.0, as compared to substrate 1. The methine signal at δ 4.52
showed HMBC correlations with C-4 (δ 126.2), C-10 (δ 43.3) and C-8 (δ 30.0), while H-4 (δ
6.14) showed HMBC correlations with δ 73.7. Thus these signals were assigned to OH-bearing
H/C-6. The methine signals at δ 3.74/δ 73.0 were assigned to OH-bearing H/C-12, as H2-11 (δ
1.89, 1.76), H-14 (δ 1.15), H-9 (δ 1.12) and Me-18 (δ 1.02) exhibited HMBC correlations with
C-12 (δ 73.0) (Fig. 3). Moreover the upfield shift of C-18 methyl carbon signal by about 5.4 ppm
further indicated a hydroxylation at C-12. The relative stereochemistry at C-12 was deduced
from NOESY correlations of H-12 (δ 3.74) with 17α-Me (δ 1.32), H-14 (δ 1.15) and H-9 (δ 1.12)
(Fig. 3). On the basis of the above observations, metabolite 5 was identified as 6β,12β,17β-
227
228
trihydroxy-17α-methylandrost-1,4-dien-3-one. No spectral data for 5 were reported earlier [25].
10

229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
The molecular formula (C20H28O4) for new metabolite 6 was deduced from HREI-MS ([M+ =
m/z 332.1964], calcd 334.1987), 32 a.m.u. higher than the substrate 1. The UV spectrum
showed λmax at 247 nm, suggesting the presence of an enone system. The IR spectrum showed
absorptions at 3406 and 1657 cm-1. The 1H NMR spectrum (Table-1) exhibited two downfield
methine signals at δ 4.46 (t, J α α β = 2.4 Hz) and 4.02 (td, J
α = 9.5 Hz, J
= 3.5 Hz),
15β,14α/16
15β,16β
6
,7 ,
with their respective methine carbons resonated at δ 74.4 and 72.7 (Table-2) in HMQC spectra.
This indicated the introduction of two OH- substituents at two methylenes of steroidal
skeleton. The proton signals at δ 4.46 and 4.02 were assigned to H-6 and H-15, respectively, on
the basis of COSY correlations with H-4 (δ 6.13)/ H2-7 (δ 2.44, 1.34) and H2-16 (δ 2.40, 1.57)/
H-14 (δ 1.24), respectively. Moreover H-6 (δ 4.46) showed correlations with C-4 (δ 125.6), C-
10 (δ 45.4) and C-8 (δ 31.9) in the HMBC spectrum. Similarly H-15 (δ 4.02) showed HMBC
interactions with C-17 (δ 79.6), C-14 (δ 58.1) and C-8 (δ 31.9) (Fig. 4). The β (axial)
stereochemistry of C-6 OH was deduced from the splitting pattern of geminal H-6 signal (δ
4.46, t, J6α αβ = 2.4 Hz). The small J-value indicated an equatorial (α) disposition of H-6 and
,7
hence an axial (β) orientation of OH. The α-stereochemistry of C-15 OH was deduced through
NOESY correlations of H-15 (δ 4.02) with H-8 (δ 2.33) and Me-18 (δ 0.96) (Fig. 4). On the
basis of the above data, compound 6 was characterized as a new metabolite, 6β,15α,17β-
245
246
247
248
249
250
trihydroxy-17α-methylandrost-1,4-dien-3-one.
The most abundant metabolite 3 was smoothly oxidized to the diketone 12 (Fig. 5), as evident
from the EI-MS [M+ = m/z 314]. The hydroxyl-bearing methine proton signal of H-15 in the
substrate 3 (Table-1) was also absent in the 1H-NMR spectrum of diketone 12, while 13C-NMR
(Table-2) showed a quaternary ketonic carbonyl signal at δ 216.1.
251
252
253
Metabolite 3 was transformed to E- and Z-oximes (13 and 14) (Fig. 5) (C20H29NO3), EI-MS M+
1 13
= m/z 331. The H- and C-NMR (Tables-1 and -2) spectra of both stereoisomers 13 and 14
were very similar in rings BCD of the steroidal skeleton; however there was a marked
11

1
13
254
255
256
257
258
259
260
difference in the H- and C-NMR chemical shifts in ring A (Tables-1 and -2). These
differences in chemical shifts were used to deduce the E- and Z-configuration of oximes 13 and
14, as reported elsewhere [27]. In Z-isomer (13), H-2 (δ 6.09) was shifted upfield as compared
to H-2 (δ 6.84) of E-isomer (14), while H-4 (δ 6.62) in 13 was shifted downfield than H-4 (δ
5.87) of 14. On the contrary, C-2 (δ 121.9) in 13 was shifted downfield by about 7.5 ppm, as
compared to 14, while C-4 (δ 110.0) was shifted upfield by about 6.6 ppm in 13 in comparison
to 14.
261
262
The results of β-glucuronidase inhibitory activity assay on transformed products 2−14 and
substrate 1 are summarized in Table-3.
263
264
265
266
4. Discussion
In the present study, the microbial transformation of an important anabolic steroid, dianabol (1)
with Cunninghamella elegans and Macrophomina phaseolina was investigated. We obtained
metabolites 2−11 from the transformation of 1 by C. elegans and M. phaseolina.
267
268
269
270
271
272
Interestingly both the fungal strains showed completely different metabolic profiles. In general,
M. phaseolina furnished hydroxyl from the β-face of steroidal skeleton, while C. elegans was
less selective, however, the major metabolites 3 and 4, were obtained as a result of hydroxylation
from the α-face by C. elegans. Moreover, both the fungi produced C-11 and C-15 hydroxylated
metabolites as epimers, unique to each of the two fungi. Furthermore, M. phaseolina also
produced C-6 and C-11 keto products 7, and 10 which were not observed for C. elegans.
273
274
275
276
Transformation of 1 with various microorganisms had been previously reported [12-24].
Penicillium notatum transformed 1 into metabolites 2 and 3 [14], while Trichoderma hamatum
yielded 4, along with 12β-hydroxydianabol and 11α-hydroxyandrost-1,4-dien-3,17-dione [15].
Similarly Beauveria bassiana [16], Aspergillus ochraceus [17], Colletotrichum lagenarium [18]
12

277
278
279
280
281
282
283
284
and Sporotrichum sulfurescens [12, 19, 20] produced metabolite 4 from 1. Absidia glauca
metabolized 1 into the metabolites 2, 8, and 9 [21], on the contrary, biotransformation of 1 with
A. coerulea yielded metabolite 11, along with 7α-OH and 14α,15α-epoxy products [22], while
Botrytis cinerea yielded 7α-hydroxydianabol as a sole product [23]. Moreover a 3,5-hexahydro
product was reported from 1 with Clostridium papraputrificum and metabolite 3 with Aspergillus
flavus [24]. None of the above microorganisms were found to produce trihydroxy metabolites 5
and 6. Physical and / or spectral data for the known compounds 3, 5, and 7−10 was not reported
previously and we are reporting here the essential data of these compounds for the first time.
285
286
287
288
289
290
291
292
293
294
295
296
297
Compounds 1−14 were screened for their ability to inhibit the β-glucuronidase activity in vitro
(Table-3). β-Glucuronidase is involved in a number of important biological events [10]. Some
steroids were reported to inhibit this enzyme [6, 8, 28]. The substrate 1 and hydroxy metabolites
2-6, 8, 9, and 11 were found to be inactive, whereas the diketone metabolite 7 exhibited a strong
inhibition of β-glucuronidase activity (IC50 = 60.7 µM) as compared to the standard (IC50 = 48.4
µM) used. The oximes 13 and 14 were also found to be strong inhibitors of the enzyme, where
activity of Z-oxime 13 (IC50 = 84.9 µM) was less than E-oxime 14 (IC50 = 49.0 µM) and standard.
The activity of E-oxime 14 was comparable to the standard (IC50= 48.4 µM).
In conclusion, biotransformation of dianabol (1) with C. elegans provides an efficient route for
the synthesis of 11α- and 15α- hydroxy steroidal derivatives, which can be exploited for the
synthesis of steroidal drugs. Compounds 6, 13 and 14 showed a strong inhibitory potential
against β-glucuronidase enzyme which can be further investigated for possible therapeutic
applications.
13

298
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300
Acknowledgements: Two of us (Naik T. Khan and Salman Zafar) acknowledge the enabling
role of the Higher Education Commission, Islamabad, Pakistan, by a financial support through,
“Indigenous Ph. D Fellowship Program (5000 Scholarships)”.
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305
306
307
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310
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313
314
315
316
317
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degradation of the side chain. German patent DE 1048579; 15 January 1959.
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Sporotrichum. US Patent US 2877162; 10 March 1959.
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Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in
Absidia coerulea culture. J Steroid Biochem Mol Biol 1996; 57: 357−362.
[23] Huszcza E, Dmochowska-Gładysz J. Transformation of testosterone and related steroids
by Botrytis cinerea. Phytochemistry 2003; 62: 155−158.
[24] Schubert K, Schlegel J, Groh H, Rose G, Hoerhold C. Metabolism of steroid drugs. VIII.
Structure-metabolism relations in the microbial hydrogenation of various substituted
testosterone derivatives. Endokrinologie 1972; 59: 99−114.
[25] Schäenzer W, Geyer H, Donike M. Metabolism of metandienone in man: Identification
and synthesis of conjugated excreted urinary metabolites, determination of excretion rates
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androstatrienes. US patent US 2899447; 11 August 1959.
[27] Sohar P, Bodor A, Schwartz R. Study on steroidal oximinoethers: Synthesis and
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17

Fig. 1: Biotransformation of dianabol (1) with Cunninghamella elegans.
1

Fig. 2: Biotransformation of dianabol (1) with Macrophomina phaseolina.
2

Fig. 3: Key HMBC (a) and NOESY (b) correlations in metabolite 5.
Fig. 4: Key HMBC (a) and NOESY (b) correlations in metabolite 6.
3

Fig. 5: Chemical transformation of metabolite 3.
4

Table-1: Selected 1H-NMR Chemical Shifts of Compounds 1-14 (δ in ppm; J in Hz).
Compound
H-1
H-2
H-4
Me-17α
Me-18
Me-19
CHOH
7.03, d
(J = 10.1)
7.03, d
(J = 10.1)
7.03, d
(J = 10.1)
7.72, d
(J = 10.3)
6.20, dd
(J = 10.1, J = 1.5)
6.18, dd
(J = 10.1, J = 1.8)
6.21, dd
(J = 10.1, J = 1.3)
6.13, dd
(J = 10.3, J = 1.8)
1a
2a
3a
4b
6.05, br. s
1.17, s
1.18, s
1.31, s
1.19, s
0.91, s
0.95, s
0.94, s
0.93, s
1.23, s
1.44, s
1.24, s
1.31, s
-
6.13, d
(J = 1.8)
4.52, t (J = 2.5)
4.08, td
(J = 9.3, J = 3.4)
4.08, td
(J = 10.3, J = 4.9)
4.52, t (J = 2.6)
3.74, dd (J = 10.9,
J = 4.8)
4.46, t (J = 2.4)
4.02, td (J = 9.5,
J = 3.5)
6.05, br. s
6.07, br. s
7.01, d
(J = 10.1)
6.20, dd
(J = 10.1, J = 1.7)
6.14, d
(J = 1.7)
5a
1.32, s
1.28, s
1.02, s
0.96, s
1.45, s
1.47, s
7.26, d
(J = 10.1)
6.19, dd
(J = 10.1, J = 1.6)
6.13, d
(J = 1.5)
6c
7a
7.07, d
(J = 10.2)
7.02, d
(J = 10.4)
7.04, d
(J = 10.0)
7.66, d
(J = 10.2)
7.27, d
(J = 10.2)
7.66, d
(J = 10.2)
6.27, d
(J = 10.2)
6.45, d
(J = 10.3)
6.28, dd
(J = 10.2, J = 1.8)
6.20, br. d
(J = 10.0)
6.21, dd
(J = 10.4, J = 1.2 )
6.18, dd
(J = 10.2, J = 1.8)
6.24, dd
(J =10.2, J = 1.5)
6.18, dd
(J = 10.2, J = 1.8)
6.09, dd
(J = 10.2, J = 1.5)
6.84, dd
(J = 10.3, J = 1.5)
6.35, d
(J = 1.8)
1.22, s
1.18, s
1.14, s
1.25, s
1.14, s
1.25, s
1.27, s
1.27, s
0.93, s
0.94, s
1.17, s
0.87, s
1.16, s
0.87, s
0.97, s
0.90, s
1.20, s
1.26, s
1.27, s
1.44, s
1.45, s
1.44, s
1.20, s
1.20, s
-
3.44, m
(W1/2 = 19.2)
4.17, m
a
6.07, br. s
6.06, br. s
6.06, br. s
5.99, br. s
6.06, br. s
6.62, br. s
5.87, br. s
8
a
9
(W1/2 =19.0 )
10d
-
4.43, br. t
(J =3.0 )
b
11
12a
13c
14e
-
3.97, td (J = 9.5,
J = 3.6)
3.97, td (J = 9.5,
J = 3.5)
a400 MHz, CDCl3; b300 MHz, CDCl3; c500 MHz, CD3OD; d500 MHz, CDCl3; e600 MHz, CD3OD.
1

Table-2: 13C-NMR Chemical Shifts of Compounds 1-14.
d
d
1a
2a
3b
4a
5b
156.4
126.9
186.4
126.2
165.0
73.7
39.0
30.0
50.2
43.3
30.9
73.0
49.6
48.1
23.2
38.2
81.9
8.5
6c
7a
10b
154.4
127.8
186.2
124.2
168.2
33.1
30.8
33.6
45.3
42.8
22.8
31.6
46.0
56.1
216.1
50.7
76.4
15.2
18.7
21.4
11e
155.4
127.5
186.2
122.2
168.3
32.1
33.6
32.5
55.8
43.8
70.2
40.7
44.6
51.0
23.5
38.8
81.6
16.3
20.9
25.8
12a
155.5
127.6
186.2
124.6
166.4
32.4a
33.2a
37.5
13c
143.0
121.9
151.0
110.0
159.6
34.0a
35.2a
37.7
54.7
44.2
22.7
32.9
47.8
58.5
72.8
50.9
79.6
16.1
20.5
26.3
14f
147.1
114.4
151.1
116.6
155.0
33.4
35.1
37.7
54.3
44.1
22.7
32.9
47.8
58.4
72.8
50.9
79.6
16.1
20.3
26.3
C
8
9
1
2
155.1
127.6
186.4
123.9
168.9
32.8
33.4
36.3
52.4
43.5
22.4
31.3
45.6
49.8
23.3
38.8
81.1
13.9
18.7
25.8
157.1
126.7
186.6
125.9
165.7
73.9
39.7
30.9
51.9
43.6
22.3
31.3
45.6
49.8
23.3
38.8
81.5
14.1
20.4
25.8
155.1
127.6
186.3
123.8
168.8
32.8
33.8
35.9
52.5
43.5
22.4
31.4
46.8
57.6
72.1
50.5
79.0
15.5
18.7
26.2
158.7
125.1
186.7
124.6
167.8
33.3
33.1
35.5
60.6
44.0
68.4
43.4
46.0
49.2
23.3
38.4
80.8
15.0
18.7
25.9
160.5
126.9
189.0
125.6
170.2
74.4
41.5
31.9
53.9
45.4
23.4
32.7
47.9
58.1
72.7
50.9
79.6
16.1
20.8
26.2
154.4
128.1
186.2
125.2
162.0
201.8
46.2
35.8
49.9
44.8
22.7
31.3
45.4
50.8
23.2
38.7
81.3
14.2
18.8
25.8
156.2
127.5
186.7
124.5
165.8
42.5
75.9
43.2
49.4
49.0
22.9
31.1
46.3
48.8
25.8
38.7
80.5
14.1
18.8
25.7
155.8
127.6
186.4
123.9
169.0
32.6
32.7
32.2
52.7
43.7
22.5
32.8
45.0
54.2
69.2
51.8
81.2
16.7
18.7
25.3
3
4
5
6
7
8
9
60.2
10
11
12
13
14
15
16
17
18
19
17α-Me
42.3
209.3
50.5
49.8
49.3
23.0
38.8
80.4
14.7
20.4
25.7
18.6
25.9
a100 MHz, CDCl3; b75 MHz, CDCl3; c125 MHz, CD3OD; d150 MHz, CDCl3; e125 MHz, CD3OD; f150 MHz, CD3OD.
gexchangeable assighnments in a column
2

Table-3: β-Glucuronidase Inhibitory Activities of Compounds 1-14.
Compound
IC50 (μM)
NAa
NAa
NAa
NAa
NAa
NAa
60.7
NAa
NAa
NAa
NAa
NAa
84.9
49.0
48.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
D-Saccharic acid-1,4-lactoneb
anot active
bstandard used
3

Highlights:
.
.
Biotransformation of the anabolic steroid dianabol (1) by fungi was studied.
Incubation of 1 with C. elegans yielded one new 6 and four known 25
metabolites.
.
Transformation of 1 with M. phaseolina resulted into five known 711
metabolites.
.
.
Metabolite 3 was transformed chemically into diketone 12 and oximes 13, and 14.
Compounds 7, 13, and 14 showed a strong inhibition of β-glucuronidase enzyme.