Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones

Article abstract of DOI:10.1016/j.steroids.2010.05.017

Sixteen new and one known metabolites 4-20 were obtained by incubation of tibolone (1) and hydroxytibolones (2 and 3) with various fungi. Their structures were elucidated by means of a homo and heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by 2D NOESY experiment. Metabolites of tibolone (1) exhibited significant inhibitory activities against α-glucosidase and tyrosinase enzymes. Hydroxylations at C-6, C-10, C-11, C-15 positions and α,β-unsaturation at C-1/C-2, C-4/C-5 showed potent inhibitory activities against these enzymes.

Full text of DOI:10.1016/j.steroids.2010.05.017

Steroids 75 (2010) 956–966
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of
tibolone and hydroxytibolones
M. Iqbal Choudharya, S. Adnan Ali Shaha,b,∗, Atta-ur-Rahmana, Shamsun-Nahar Khana,
Mahmud Tareq Hassan Khana
a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b Institut Kajian Ubat Semulajadi (iKUS), Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Puncak Alam, Selangor Darul Ehsan, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen new and one known metabolites 4–20 were obtained by incubation of tibolone (1) and hydrox-
ytibolones (2 and 3) with various fungi. Their structures were elucidated by means of a homo and
heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by
2D NOESY experiment. Metabolites of tibolone (1) exhibited significant inhibitory activities against
␣-glucosidase and tyrosinase enzymes. Hydroxylations at C-6, C-10, C-11, C-15 positions and ␣,␤-
unsaturation at C-1/C-2, C-4/C-5 showed potent inhibitory activities against these enzymes.
© 2010 Elsevier Inc. All rights reserved.
Received 19 March 2010
Received in revised form 21 May 2010
Accepted 24 May 2010
Available online 1 June 2010
Keywords:
Microbial transformation
Tibolone
␣-Glucosidase inhibitors
Tyrosinase inhibitors
Enzyme inhibition assay
Hydroxytibolone
1. Introduction
In the current study, tibolone (1) was subjected to microbial
transformation, and difference between microbial transformation
Microbial transformation is an effective tool to synthesize many
steroidal drugs with potential biological activities [1–9]. Such
studies are primarily useful in the generation of hydoxylated
metabolites for drug toxicity studies [11,12]. Fungi, bacteria and
yeast have been utilized successfully as in vitro models to mimic and
predicts the metabolic fate of drugs and other xenobiotics in mam-
malian systems [10,12–14]. Previously, many biotransform studies
on various 17␣-ethynyl steroids had been carried out with vari-
ous fungal and bacterial strains, which afforded hydroxylation at
various positions [9,15–17].
Tibolone (1) is a synthetic steroid that combines oestrogenic and
progestogenic properties with androgenic property, which mimic
the action of a male sex hormone [18]. The in vivo metabolism of
tibolone (1) in human had been studied and three metabolites, 3␤-
hydroxytibolone (2), 3␣-hydroxytibolone (3), and ꢀ4-tibolone (6)
were identified [19–24].
and human metabolism was studied. The fungal transformation
of 1 led to novel hydroxylation at various positions (metabolites
4–20), in which only metabolite 6 was reported as a metabolite in
human metabolism [19–24]. Our research group has been focus-
ing on the structural modifications of bioactive natural products
by using microorganisms, in order to obtain potent inhibitors of
various enzymes [1–9,25–37]. Tibolone (1) is used effectively for
the treatment of menopausal symptoms and in the prevention of
osteoporosis, as a hormone replacement therapy (HRT). The hor-
mone replacement therapy (HRT) effects on glucose metabolism
in non-diabetic obese postmenopausal women [38–43]. Com-
pound 1 when incubated with Rhizopus stolonifer, Fusarium lini,
Cunninghamella elegans and Gibberella fujikuroi, resulted in the
formation of a library of hydroxyl derivatives. These hydrox-
ytibolones were then evaluated for inhibition of ␣-glucosidase
and tyrosinase enzymes.␣-Glucosidase (EC 3.2.1.20) is a typical
exo-type glycosidase that catalyze the releases of ␣-glucosides
from the no reducing end side of the substrates [44]. Diabetes
mellitus is a chronic metabolic disorder characterized by high
blood glucose levels. Tyrosinase (EC 1.14.18.1) is a multifunctional,
copper-containing enzyme widely distributed in plants and ani-
mals. Tyrosinase inhibitors are clinically useful for the treatment
of some dermatological disorders, associated with melanin hyper
pigmentation [45].
∗
Corresponding author at: Institut Kajian Ubat Semulajadi (iKUS), Faculty of Phar-
macy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Puncak
Alam, Selangor Darul Ehsan, Malaysia. Tel.: +60 3 3258 4616; fax: +60 3 3258 4602.
E-mail addresses: syedadnan@salam.uitm.edu.my, benzene301@yahoo.com
(S.A.A. Shah).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.05.017

M.I. Choudhary et al. / Steroids 75 (2010) 956–966
957
We are reporting here a new class of tyrosinase and ␣-
2.4. Fermentation of tibolone (1) with R. stolonifer (TSY-0471)
glucosidase inhibitors, i.e. 17␣-ethynyl steroids, as transformed
products of compound 1. These compounds exhibited various level
of activity against the enzymes, tyrosinase and ␣-glucosidase in
comparison to the standard inhibitors.
Compound 1 (500 mg), dissolved in 15 mL acetone and dis-
tributed among 40 flasks, and kept for fermentation. All the media
were filtered after 3 days and extracted with dichloromethane
and evaporated under reduced pressure to finally obtain a brown
thick extract (0.90 g), and the transformed metabolites were iso-
lated by using column chromatography. Metabolite 4 (20 mg) was
eluted with pet. ether and EtOAc (60:40), compound 5 (17 mg)
with pet. ether–EtOAc (58:42) and compound 6 (40 mg) with
pet. ether–EtOAc (55:45). There were more transformed prod-
ucts in crude mixture but due to the insufficient quantities,
we did not able to determine the structure of these metabo-
lites.
2. Experimental
2.1. General
Melting points were determined on a Yanaco MP-S3 apparatus.
UV spectra were measured on a Shimadzu UV 240 spectrophotome-
ter. IR spectra were recorded on a JASCO A-302 spectrophotometer
in CHCl3. 1H- and 13C NMR spectra were recorded on a Bruker
Avance AM-400 spectrometer with tetramethylsilane (TMS) as an
internal standard. 2D NMR spectra were recorded on a Bruker
Avance AMX 500 NMR spectrometer. Optical rotations were mea-
sured on JASCO DIP-360 digital polarimeter by using 10 cm cell
tube. Mass spectra (EI and HREI-MS) were measured in an elec-
tron impact mode on Varian MAT 12 or MAT 312 spectrometers
and ions are given in m/z (%). TLC was performed on a pre-coated
silica gel card (E. Merck), spots were viewed with ultraviolet light at
254 nm for florescence quenching spots and at 366 nm for fluores-
cent spot and stained by spraying with a solution of ceric sulphate
in 10% H2SO4. For column chromatography, silica gel (E. Merck,
230–400 mesh). Tibolone (1) was extracted from Livial-Organon
using dichloromethane.
2.4.1. 6ˇ-Hydroxytibolone (4)
White amorphous solid (20 mg); mp 186–188 ◦C; [␣]25 −17 (c
D
0.35, CHCl3); UV (MeOH) ꢁmax (log ε) 204 (3.7) nm; IR (CHCl3) ꢂmax
3381, 2150, 1705, 1668, 1043 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 1 and 3; EI MS m/z (rel. int. %) 328 (M+, 6), 309 (5), 241 (16),
226 (9), 169 (14), 149 (23), 138 (28), 121 (28), 109 (23), 107 (100),
97 (20), 93 (21), 81 (26), 71 (22), 69 (41), 55 (64); HREI MS m/z
328.2171 (calcd for C21H28O3, 328.2143).
2.4.2. 15ˇ-Hydroxytibolone (5)
White solid (17 mg); mp 202–205 ◦C; [␣]25D +16 (c 0.31, CHCl3);
UV (MeOH) ꢁmax (log ε) 203.4 (3.4) nm; IR (CHCl3) ꢂmax 3383,
2162, 1708, 1663, 1050 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 1 and 3; EI MS m/z (rel. int. %) 328 (M+, 3), 312 (100), 245
(27), 229 (36), 203 (17), 189 (14), 187 (17), 174 (24), 161 (28), 149
(26), 135 (24), 121 (25), 96 (38), 81 (23), 69 (21), 67 (24), 55 (59);
HREI MS m/z 328.2070 (calcd for C21H28O3, 328.2038).
2.2. Fungi and culture conditions
Microbial cultures of the F. lini (NRRL 68751), R. stolonifer
(TSY 0471), C. elegans (TSY 0865) and G. fujikuroi were grown on
Sabouraud-4% glucose-agar (Merck) at 25 ◦C and stored at 4 ◦C.
R. stolonifer (TSY 0471) medium was prepared by adding glucose
(100 g), peptone (25 g), KH2PO4 (25 g) and yeast extract (15 g) into
distilled water (4 L) and pH was maintained at 5.6. F. lini (NRRL
68751) and C. elegans (TSY 0865) media were prepared by mixing
the following ingredients into distilled H2O (3.0 L) in each case:
glucose (30.0 g), glycerol (30.0 g), peptone (15.0 g), yeast extract
(15.0 g), KH2PO4 (15.0 g), and NaCl (15.0 g). G. fujikuroi medium
was prepared by adding the following ingredients into distilled
H2O (3.0 L): glucose (80.0 g), KH2PO4 (5.0 g), MgSO4·2H2O (1.0 g),
NH4NO3 (0.5 g) and Gibberella trace element solution (2 mL).
The Gibberella trace element solution was prepared by mixing
Co(NO3)2·6H2O (0.01 g), FeSO4·7H2O (0.1 g), CuSO4·5H2O (0.1 g),
ZnSO4·7H2O (0.161 g), MnSO4·4H2O (0.01 g) and NH4 molybdate
(0.01 g) into distilled water (100 mL).
2.4.3. ꢀ4-Tibolone (6)
Crystalline solid (40 mg); mp 206–208 ◦C; [␣]25 −145 (c 0.21,
D
CHCl3); UV (MeOH) ꢁmax (log ε) 235 (3.2) nm; IR (CHCl3) ꢂmax 3402,
2150, 1687, 1667, 1017 cm−1 1H NMR data in CDCl3, Table 1; 13
; C
NMR (100 MHz, CDCl3) ı 25.2 (C-1), 38.3 (C-2), 198.1 (C-3), 126.4
(C-4), 161.4 (C-5), 47.2 (C-8), 45.4 (C-9), 38.2 (C-10), 26.2 (C-11),
79.1 (C-17), 87.5 (C-20), 74.3 (C-21); EI MS m/z (rel. int. %) 312 (M+,
34,), 245 (53), 229 (33), 187 (17), 173 (18), 161 (20), 147 (28), 135
(56), 121 (23), 109 (32), 107 (43), 105 (39), 95 (24), 91 (62), 81
(34), 79 (59), 67 (58), 55 (100); HREI MS m/z 312.2023 (calcd for
C21H28O2, 312.2089).
2.5. Fermentation of tibolone (1) by F. lini (NRRL 68751) and C.
elegans (TSY 0865)
Compound 1 (600 mg), dissolved in 18 mL acetone and dis-
tributed among 50 flasks, was kept for fermentation, for 6 days
and then filtrates were extracted with dichloromethane and evap-
orated under reduced pressure to afford a brown thick extract
(1.02 g). Column chromatography was used for the separation of
metabolites 7–10 from C. elegans crude, while F. lini yielded one
major metabolite 6. Compound 7 (6.2 mg) was eluted with pet.
ether–EtOAc (42:58), compound 8 (15.5 mg) with pet. ether–EtOAc
(38:62), 9 (5.2 mg) with pet. ether–EtOAc (40:60), whereas com-
pound 10 (10.2 mg) with pet. ether–EtOAc (30:70).
2.3. General fermentation and extraction conditions
The fungal media were transferred into 250 mL conical flasks
(100 mL each) and autoclaved at 121◦ C. Seed flasks were prepared
from 3-day-old slant and fermentation was allowed for 2 days on
a shaker at 25◦ C. The remaining flasks were inoculated from seed
flasks. After 2 days, tibolone (1) was dissolved in acetone and trans-
ferred in each flask (15 mg/0.5 mL) and the flasks were placed on a
rotatory shaker (128 rpm) at 22◦ C for fermentation period. The time
course study was carried out after 2 days and the transformation
was analyzed on TLC. The culture media were filtered and extracted
with CH2Cl2. The extract was dried over anhydrous Na2SO4, evap-
orated under reduced pressure and the brown gummy crude was
analyzed by thin layer chromatography.
2.5.1. ꢀ1,4-Tibolone (7)
Amorphous solid (6.2 mg); mp 196–200 ◦C; [␣]25D −172 (c 0.32,
CHCl3); UV (MeOH) ꢁmax (log ε) 241.5 (4.1) nm; IR (CHCl3) ꢂmax
3303, 2154, 1691, 1666, 1044 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 1 and 3; EI MS m/z (rel. int. %) 310 (M+, 42), 241 (100), 230
(11), 199 (17), 187 (27), 161 (22), 149 (34), 145 (17), 119 (18), 109

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M.I. Choudhary et al. / Steroids 75 (2010) 956–966
(14), 91 (62), 67 (62), 55 (100); HREI MS m/z 310.2004 (calcd for
21H26O2, 310.2011).
C
2.5.2. 10ˇ-Hydroxy-ꢀ4-tibolone (8)
White powdered solid (15.5 mg); mp 198–201 ◦C; [␣]25 +12 (c
D
0.25, CHCl3); UV (MeOH) ꢁmax (log ε) 239 (2.9) nm; IR (CHCl3) ꢂmax
3345, 2149, 1698, 1649, 1018 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 1 and 3; EI MS m/z (rel. int. %) 328 (M+, 13), 310 (14), 229
(20), 187 (25), 171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109
(44), 107 (43), 91 (55), 83 (28), 67 (47), 57 (50), 55 (100); HREI MS
m/z 328.2090 (calcd for C21H28O3, 328.2123).
2.5.3. 11˛,15ˇ-Dihydroxytibolone (9)
White powdered solid (5.2 mg); mp 208–210 ◦C; [␣]25 −81 (c
D
0.24, CHCl3); UV (MeOH) ꢁmax (log ε) 203 (3.3) nm; IR (CHCl3) ꢂmax
3342, 2142, 1718, 1636, 1028 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 1 and 3; EI MS m/z (rel. int. %) 344 (M+, 13), 310 (14), 229
(20), 187 (25), 171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109
(44), 107 (43), 91 (55), 83 (28), 67 (47), 57 (50), 55 (100); HREI MS
m/z 344.2212 (calcd for C21H28O4, 344.2234).
2.5.4. 11˛,15ˇ-Dihydroxy–ꢀ5–tibolone (10)
White powdered solid (10.2 mg); mp 207–211 ◦C; [␣]25 +27 (c
D
0.28, CHCl3); UV (MeOH) ꢁmax (log ε) 202.4 (3.4) nm; IR (CHCl3)
ꢂmax 3312, 2102, 1722, 1652, 1057 cm−1 1H and 13C NMR data in
;
CDCl3, Tables 1 and 3; EI MS m/z (rel. int. %) 344 (M+, 8), 310 (14),
229 (20), 187 (25), 171 (26), 161 (54), 149 (87), 136 (32), 124 (55),
109 (44), 107 (43), 91 (55), 83 (28), 67 (74), 57 (50), 55 (100); HREI
MS m/z 344.2341 (calcd for C21H28O4, 344.2316).
2.6. Fermentation of tibolone (1) by G. fujikuroi (ATCC 10704)
Compound 1 (850 mg) was dissolved in 20 mL acetone and
distributed among 30 flasks for fermentation for 12 days. After
fermentation, media was extracted with dichloromethane and
evaporated to get a crude extract (1.22 g). Column chromatogra-
phy was used for the separation of metabolites 11–16 from crude
extract. There were more transformed products in crude mixture
but due to the insufficient quantities, we did not able to deter-
mine the structure of these metabolites. Metabolite 11 (5.6 mg)
was eluted with pet. ether–EtOAc (54:46), compound 12 (10.2 mg)
with pet. ether–EtOAc (50:50), compound 13 (11.2 mg) with pet.
ether–EtOAc (45:55), compound 14 (8.3 mg) with pet. ether–EtOAc
(38:58), compound 15 (9.5 mg) with pet. ether–EtOAc (30:70) and
compound 16 (10.3 mg) with pet. ether–EtOAc (35:75).
2.6.1. ꢀ5-Tibolone (11)
White powdered solid (5.6 mg); mp 201–205 ◦C; [␣]25 − 17 (c
D
0.20, CHCl3); UV (MeOH) ꢁmax (log ε) 203.6 (3.9) nm; IR (CHCl3)
ꢂmax 3341, 2157, 1728, 1642, 1088 cm−1 1H and 13C NMR data in
;
CDCl3, Tables 1 and 3; EI MS m/z (rel. int. %) 312 (M+, 42), 242 (100),
227 (63), 187 (27), 161 (22), 149 (34), 124 (26), 91 (21), 67 (23), 55
(100); HREI MS m/z 312.1456 (calcd for C21H28O2, 312.1513).
2.6.2. 6ˇ-Hydroxy-ꢀ4-tibolone (12)
White powdered solid (10.2 mg); mp 189–93 ◦C; [␣]25 −101
D
(c 0.41, CHCl3); UV (MeOH) ꢁmax (log ε) 239.5 (2.1) nm; IR (CHCl3)
ꢂmax 3332, 2128, 1698, 1651, 1063 cm−1 1H and 13C NMR data in
;
CDCl3, Tables 1 and 3; EI MS m/z (rel. int. %) 328 (M+, 13), 312 (13),
245 (17), 229 (34), 189 (17), 187 (17), 161 (28), 149 (49), 121 (45),
91 (38), 69 (35), 67 (36), 55 (100); HREI MS m/z 328.2176 (calcd for
C21H28O3, 328.2116).
2.6.3. 6˛-Hydroxy-ꢀ4-tibolone (13)
White powdered solid (11.2 mg); mp 187–190 ◦C; [␣]25 + 66.2
D
(c 0.35, CHCl3); UV (MeOH) ꢁmax (log ε) 240.1 (3.7) nm; IR (CHCl3)

M.I. Choudhary et al. / Steroids 75 (2010) 956–966
959
ꢂmax 3342, 2106, 1683, 1659, 1061 cm−1 1H and 13C NMR data in
;
CDCl3, Tables 2 and 3; EI MS m/z (rel. int. %) 328 (M+, 56), 312 (11),
245 (45), 229 (6), 201 (17), 187 (14), 171 (20), 161 (34), 149 (57),
135 (24), 121 (46), 91 (34), 81 (34), 67 (4), 55 (59); HREI MS m/z
328.2132 (calcd for C21H28O3, 328.2205).
2.6.4. 15˛-Hydroxy-ꢀ4-tibolone (14)
White powdered solid (8.3 mg); mp 203–205 ◦C; [␣]25 −21.3
D
(c 0.34, CHCl3); UV (MeOH) ꢁmax (log ε) 237.6 (2.6) nm; IR (CHCl3)
ꢂmax 3401, 2176, 1691, 1662, 1060 cm−1 1H and 13C NMR data
;
in CDCl3, Tables 2 and 4; EI MS m/z (rel. int. %) 328 (M+, 3),
312 (100), 245 (27), 229 (36), 203 (17), 189 (10), 187 (17), 174
(14), 161 (56), 149 (26), 135 (5), 121 (67), 96 (38), 81 (23), 69
(15), 67 (34), 55 (51); HREI MS m/z 328.2212 (calcd for C21H28O3,
328.2283).
2.6.5. 6˛-Hydroxy-ꢀ1,4-tibolone (15)
White powdered solid (9.5 mg); mp 193–196 ◦C; [␣]25 +44 (c
D
0.25, CHCl3); UV (MeOH) ꢁmax (log ε) 244 (3.7) nm; IR (CHCl3)
ꢂmax 3441, 2123, 1680, 1637, 1045 cm−1 1H and 13C NMR data
;
in CDCl3, Tables 2 and 4; EI MS m/z (rel. int. %) 326 (M+, 13),
312 (34), 245 (27), 229 (67), 203 (5), 189 (14), 187 (14), 174
(24), 161 (56), 149 (26), 135 (24), 121 (100), 96 (45), 81 (23), 69
(12), 67 (24), 55 (34); HREI MS m/z 326.2231 (calcd for C21H28O3,
326.2292).
2.6.6. 6ˇ-Methoxy-ꢀ4-tibolone (16)
White powdered solid (10.3 mg); mp 206–209 ◦C; [␣]25D −18 (c
0.42, CHCl3,); UV (MeOH) ꢁmax (log ε) 242.1 (3.1) nm; IR (CHCl3)
ꢂmax 3323, 2153, 1691, 1661, 1101 cm−1 1H and 13C NMR data
;
in CDCl3, Tables 2 and 4; EI MS m/z (rel. int. %) 343 (M+, 6),
312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187 (17), 174
(25), 161 (28), 149 (26), 135 (24), 121 (25), 91 (100), 81 (23), 69
(21), 67 (23), 55 (45); HREI MS m/z 343.2356 (calcd for C22H30O3,
343.2338).
2.7. Reduction of tibolone (1) with sodium borohydride
Compound
1 (1 g) was dissolved in dry dichloromethane
(100 mL) and cooled in an ice bath to 0 ◦C. Sodium borohydride
(500 mg) was added to the solution in portions whilst stirring. The
mixture was stirred at room temperature for 5 h. Acetic acid (50 mL)
was by destroyed the excess sodium borohydride (NaBH4) and the
dichloromethane was removed in vacuo. Water (100 mL) was added
to the resulting oily product, and the suspension was extracted
with ethyl acetate (1 L). The ethyl acetate extract was washed
with sodium hydrogen carbonate solution (300 mL), water and
brine. The removal of the solvent on a rotary evaporator afforded
crude extract (1.2 g). Column chromatography was used for the
separation of these hydroxytibolones 2 and 3 [47]. Compound 2
(300 mg) was eluted with pet. ether–EtOAc (50:50) and compound
3 (400 mg) was eluted with pet. ether–EtOAc (48:52).
2.8. Fermentation of 3ˇ-hydroxytibolone (2) by C. elegans (TSY
0865)
Compound 2 (300 mg), dissolved in 15 mL acetone and dis-
tributed among 40 flasks, was kept for fermentation. Fermentation
was continued for 12 days and then filtrates were extracted with
CH2Cl2 and evaporated under reduced pressure to afford brown
thick crude (0.81 g). Column chromatography was used for the
separation of one hydroxyl metabolites 17 from crude extract. Com-
pound 17 (4.2 mg) was eluted with pet. ether–EtOAc (40:60).

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M.I. Choudhary et al. / Steroids 75 (2010) 956–966
Table 3
13C NMR data of compounds 4–13 (100 and 125 MHz; CDCl3).
No. of C
4
5
7
8
9
10
24.5 (t)
11
24.9 (t)
12
26.7 (t)
13
25.2 (t)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
25.5 (t)
38.9 (t)
25.2 (t)
38.9 (t)
153.4 (d)
127.0 (d)
199.4 (d)
125.2 (d)
162.1 (s)
30.7 (t)
32.9 (d)
43.5 (d)
46.4 (d)
43.0 (d)
29.7 (t)
32.4 (t)
46.0 (s)
47.2 (d)
22.6 (t)
39.0 (t)
79.9 (s)
13.2 (q)
12.7 (q)
87.6 (s)
74.0 (d)
30.6 (t)
33.8 (t)
24.7 (t)
38.1 (t)
38.1 (t)
198.7 (s)
41.2 (t)
162.2 (s)
124.1 (d)
33.6 (d)
39.7 (d)
58.1 (d)
38.8 (d)
66.3 (d)
45.1 (t)
40.1 (s)
49.2 (d)
67.0 (d)
44.9 (t)
79.5 (s)
12.4 (q)
12.2 (q)
87.2 (s)
74.1 (d)
37.6 (t)
199.1 (s)
40.9 (t)
162.1 (s)
123.4 (d)
34.6 (d)
44.2 (d)
45.9 (d)
43.4 (d)
31.9 (t)
33.1 (t)
46.2 (s)
48.7 (d)
22.4 (t)
39.8 (t)
79.1 (s)
13.0 (q)
12.5 (q)
87.5 (s)
73.9 (d)
36.6 (t)
199.8 (s)
126.5 (d)
164.8 (s)
65.9 (d)
42.0 (d)
39.1 (d)
46.0 (d)
38.7 (d)
29.8 (t)
32.3 (t)
46.3 (s)
46.9 (d)
22.2 (t)
39.5 (t)
79.7 (s)
12.8 (q)
12.6 (q)
87.3 (s)
74.2 (d)
38.8 (t)
198.5 (s)
126.7 (d)
163.9 (s)
70.0 (d)
40.0 (d)
38.1 (d)
46.2 (d)
39.7 (d)
29.7 (t)
33.6 (t)
46.5 (s)
45.9 (d)
22.4 (t)
39.9 (t)
79.2 (s)
12.4 (q)
12.2 (q)
87.2 (s)
74.1 (d)
199.5 (s)
33.1 (t)
122.5 (s)
65.9 (d)
41.6 (d)
39.4 (d)
46.2 (d)
128.4 (s)
29.8 (t)
32.9 (t)
47.4 (s)
48.6 (d)
222.2 (t)
38.9 (t)
79.9 (s)
13.1 (q)
12.5 (q)
87.6 (s)
73.8 (d)
200.1 (s)
43.6 (t)
123.0 (s)
31.5 (t)
32.1 (d)
40.4 (d)
46.3 (d)
128.2 (s)
29.7 (t)
32.6 (t)
45.6 (s)
51.2 (d)
65.5 (d)
48.6 (t)
79.1 (s)
13.1 (q)
13.0 (q)
87.4 (s)
74.1 (d)
198.7 (s)
126.7 (d)
162.2 (s)
31.9 (t)
33.5 (d)
38.1 (d)
53.7 (d)
70.3 (s)
22.9 (t)
33.0 (t)
45.9 (s)
49.7 (d)
22.4 (t)
39.2 (t)
79.6 (s)
12.4 (q)
12.2 (q)
87.2 (s)
74.1 (d)
198.7 (s)
42.1 (t)
122.5 (s)
31.8 (t)
33.5 (d)
40.0 (d)
58.8 (d)
126.2 (s)
66.1 (d)
47.5 (t)
40.9 (s)
49.7 (d)
65.4 (d)
45.1 (t)
79.6 (s)
12.4 (q)
12.2 (q)
87.6 (s)
74.6 (d)
Multiplicities were assigned from DEPT experiments in parentheses: s: quaternary, d: CH, t: CH2, and q: Me C-atoms.
2.8.1. 3ˇ,6ˇ-Dihydroxytibolone (17)
Compound 18 (4.6 mg) was eluted with pet. ether–EtOAc (40:60).
Metabolite 19 (4.2 mg) was eluted with pet. ether–EtOAc (54:46)
and compound 20 (5.1 mg) with pet. ether–EtOAc (50:50).
White powdered solid (4.2 mg); mp 206–209 ◦C; [␣]25 +105
D
(c 0.31, CHCl3); UV (MeOH) ꢁmax (log ε) 201 (3.1) nm; IR (CHCl3)
ꢂmax 3353, 2164, 1663, 1078 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 2 and 4; EI MS m/z (rel. int. %) 330 (M+, 7), 312 (17), 245 (27),
229 (32), 203 (17), 189 (14), 187 (17), 174 (25), 161 (28), 149 (26),
135 (24), 121 (25), 91 (100), 81 (23), 69 (21), 67 (23), 55 (45); HREI
MS m/z 330.2346 (calcd for C21H32O3, 328.2374).
2.9.1. 3˛-Hydroxy-ꢀ5-tibolone (18)
White powdered solid (4.6 mg); mp 206–207 ◦C; [␣]25 +52.2
D
(c 0.23, CHCl3); UV (MeOH) ꢁmax (log ε) 204.2 (4.0) nm; IR (CHCl3)
ꢂmax 3313, 2154, 1646, 1038 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 2 and 4; EI MS m/z (rel. int. %) 314 (M+, 8), 312 (17), 245 (27),
229 (32), 203 (17), 189 (14), 187 (17), 174 (25), 161 (28), 149 (21),
135 (24), 121 (23), 91 (11), 81 (23), 69 (21), 67 (21), 55 (45); HREI
MS: m/z 314.2541 (calcd for C21H32O2, 314.2597).
2.9. Fermentation of 3˛-hydroxytibolone (3) by C. elegans (TSY
0865)
Compound 3 (400 mg), dissolved in 18 mL acetone and dis-
tributed among 50 flasks, was kept for fermentation. Fermentation
was continued for 12 days and then filtrates were extracted with
dichloromethane and evaporated under reduced pressure to afford
a brown thick crude (0.89 g). Column chromatography was used for
the separation of hydroxy metabolites 18–20 from crude extract.
2.9.2. 3˛,6ˇ-Dihydroxy-ꢀ4-tibolone (19)
White powdered solid (4.2 mg); mp 201–205 ◦C; [␣]25 −18 (c
D
0.31, CHCl3); UV (MeOH) ꢁmax (log ε) 201.2 (2.9) nm; IR (CHCl3)
ꢂmax 3323, 2156, 1665, 1118 cm−1 1H and 13C NMR data in CDCl3,
;
Tables 2 and 4; EI MS m/z (rel. int. %) 330 (M+, 6), 312 (17), 245 (27),
Table 4
13C NMR data of compounds 14–20 (100 and 125 MHz; CDCl3).
No. of C
14
25.3 (t)
15
16
17
18
25.1 (t)
19
25.1 (t)
20
25.2 (t)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
154.2 (d)
126.5 (d)
198.9 (s)
125.7 (d)
161.9 (s)
69.8 (d)
39.0 (d)
38.1 (d)
45.8 (d)
39.3 (d)
29.2 (t)
31.9 (t)
46.3 (s)
46.5 (d)
22.0 (t)
38.9 (t)
79.3 (s)
12.9 (q)
12.2 (q)
87.8 (s)
73.7 (d)
25.0 (t)
38.7 (t)
25.1 (t)
38.8 (t)
67.0 (d)
40.2 (t)
122 (s)
74.2 (d)
43.3 (d)
38.3 (d)
46.2 (d)
127 (d)
29.7 (t)
33.6 (t)
46.5 (s)
45.9 (d)
22.4 (t)
39.9 (t)
79.2 (s)
15.1 (q)
14.2 (q)
87.2 (s)
74.0 (d)
38.8 (t)
199.5 (s)
126.5 (d)
164.9 (s)
30.7 (t)
33.1 (d)
39.7 (d)
46.0 (d)
43.5 (d)
29.9 (t)
32.3 (t)
45.2 (s)
52.3 (d)
65.9 (d)
46.9 (t)
79.9 (s)
13.0 (q)
12.8 (q)
87.7 (s)
73.8 (d)
38.4 (t)
78.0 (d)
40.4 (d)
147.0 (s)
132.0 (d)
39.1 (d)
43.2 (d)
46.2 (d)
43.0 (d)
29.0 (t)
33.6 (t)
46.5 (s)
45.9 (d)
22.4 (t)
39.9 (t)
79.1 (s)
15.1 (q)
14.2 (q)
87.3 (s)
74.2 (d)
38.2 (t)
78.0 (d)
124.0 (t)
147.0 (s)
74.0 (d)
43.2 (d)
39.1 (d)
46.2 (d)
44.2 (s)
29.7 (d)
33.6 (t)
46.1 (s)
45.9 (d)
22.5 (d)
39.9 (t)
79.1 (s)
15.1 (q)
14.1 (q)
87.0 (s)
74.2 (d)
38.8 (t)
78.1 (d)
126.0 (t)
147.0 (s)
31.9 (t)
33.2 (d)
41.2 (d)
51.0 (d)
44.2 (d)
66.3 (d)
36.6 (t)
46.5 (s)
45.9 (d)
22.4 (d)
39.9 (t)
79.1 (s)
15.2 (q)
13.9 (q)
87.4 (s)
74.1 (d)
198.7 (s)
126.7 (d)
162.2 (s)
70.2 (d), 57.6 (OMe)
38.7 (d)
39.1 (d)
45.9 (d)
44.7 (d)
29.6 (t)
32.9 (t)
46.6 (s)
46.2 (d)
22.4 (t)
39.6 (t)
79.6 (s)
12.4 (q)
12.2 (q)
87.2 (s)
74.2 (d)
Multiplicities were assigned from DEPT experiments in parentheses: s: quaternary, d: CH, t: CH2, and q: Me C-atoms.

M.I. Choudhary et al. / Steroids 75 (2010) 956–966
961
229 (32), 203 (17), 189 (14), 187 (14), 174 (25), 161 (21), 149 (26),
3. Results and discussion
135 (22), 121 (25), 91 (100), 81 (23), 69 (21), 67 (23), 55 (45); HREI
MS m/z 330.2356 (calcd for C21H32O3, 330.2338).
Fermentation of tibolone (1) with R. stolonifer (TSY 0471) yielded
two new mono-hydroxylated metabolites, 4 and 5, and a known
metabolite 6 (Fig. 1). The HREI MS of metabolite 4 exhibited the
molecular ion (M+) at m/z 328.2171, corresponding to the for-
mula C21H28O3, which indicated that a new oxygen functionality
was introduced during fermentation period. The IR absorptions
2.9.3. 3˛,11˛-Dihydroxy-ꢀ4-tibolone (20)
White powdered solid (5.1 mg); mp 199–201 ◦C; [␣]25 +61 (c
D
0.45, CHCl3); UV (MeOH) ꢁmax (log ε) 202.6 (3.5) nm; IR (CHCl3)
ꢂmax 3323, 2143, 1668, 1105 cm−1 1H and 13C NMR data in CDCl3,
;
were attributed to hydroxyl (3381 cm−1) and carbonyl (1705 cm−1
)
Tables 2 and 4; EI MS m/z (rel. int. %) 330 (M+, 4), 312 (14), 245 (27),
229 (42), 203 (17), 189 (34), 187 (17), 174 (19), 161 (28), 149 (26),
135 (35), 121 (25), 91 (98), 81 (23), 69 (67), 67 (23), 55 (51); HREI
MS m/z 330.2256 (calcd for C21H32O3, 330.2238).
functionalities. The 1H NMR spectrum, when compared with the
substrate (Table 1), showed a new signal of OH-bearing methine
proton at ı 4.04, resonating as a doublet (J6e,7e = 4.0 Hz) with its
corresponding carbon at ı 65.9 in 13C NMR spectrum, which was
assigned to C-6 on the basis of HMBC correlations of H-6 (ı 4.04)
with C-5 (ı 122.5) and C-10 (ı 128.4) (Table 3). In the COSY 45◦
spectrum, the aforementioned methine proton showed correlation
with the C-7 methine proton (ıH 2.0). The stereochemistry of C-6
hydroxyl group was determined to be axial by the NOESY correla-
tions between H-6 (ı 4.04) and H-19 (ı 0.76). The above spectral
data indicated that new metabolite 4 has an –OH group at C-6
position, as compared to compound 1.
2.10. Material and methods for tyrosinase inhibition assay
Tyrosinase inhibition assays were performed in 96-well
microplate format using the SpectraMax 340 microplate reader
(Molecular Devices, CA, USA) according to the method developed by
Hearing (1987) [48]. Briefly, first the compounds were screened for
the o-diphenolase inhibitory activity of tyrosinase using L-DOPA as
the substrate. All the active inhibitors from the preliminary screen-
ing were subjected to IC50 studies. Compounds were dissolved in
methanol to a concentration of 2.5%. 30 units of mushroom tyrosi-
nase (28 nM from Sigma Chemical Co., USA), first preincubated with
the compounds in 50 nM Na-phosphate buffer (pH 6.8) for 10 min at
25 ◦C. Then, the L-DOPA (0.5 mM) was added to the reaction mix-
ture and the enzyme reaction was monitored by measuring the
change in absorbance at 475 nm (at 37 ◦C) due to the formation of
the DOPA chrome for 10 min. The percent inhibition of the enzyme
was calculated as follows, by using MS Excel®TM 2000 (Microsoft
Corp., USA) based program developed for this purpose:
The HREI MS of metabolite 5 showed the M+ at m/z 328.2070
(C21H28O3), indicating an increment of 16 mass units as compared
to compound 1. The 1H and 13C NMR data of 5 revealed the presence
of a new OH-bearing methine group, that resonated at ıH 4.06 (m,
W1/2 ∼ 10.8 Hz) and ıC 65.5 and deduced to be C-15 on the basis
of HMBC of C-16 protons (ıH 2.24, 1.7) and C-14 methine proton
(ıH 1.85) with C-15 (ıC 65.5) (Tables 1 and 3). The stereochemistry
of C-15 hydroxyl group was deduced as ␤ on the basis of NOESY
correlations between H-15 (ıH 4.06) and H-14 (ıH 1.85). From these
spectral data, the new compound 5 was identified as 7␣-methyl-
17␣-ethynl-15␤,17␤-dihydroxy-19-norandrost-5(10)-en-3-one.
The incubation of compound 1 with F. lini (NRRL 68751) for
6 days also led to the isolation of a UV active metabolite 6 with
exhibiting the M+ at m/z 312.2023 in HREI MS spectrum (C21H28O2).
The 1H NMR spectrum showed a singlet for an olefinic proton at
ı 5.82. Its broad-band decoupled 13C NMR spectrum showed, in
comparison with that of the substrate 1, the disappearance of one
quaternary carbon signal resonating at ı 128.2 for C-10 and appear-
ance of an olefinic methine carbon at ı 126.4 which was assigned
to the C-4 on the basis of HMQC spectrum, indicating the migra-
tion of the C-5/C-10 double bond to C-4/C-5 (Tables 1 and 3). This
indicated ␣,␤-unsaturated ketone moiety in metabolite 6. The axial
orientation of C-10 methine proton was assigned on the basis of
NOESY coupling between H-10 (ı 2.31) and H-8 (ı 1.64) (Fig. 2).
The above spectral data supported the structure of metabolite
6 as 7␣-methyl-17␣-ethynl-17␤-hydroxy-19-norandrost-4-en-3-
one, previously obtained from human metabolism of tibolone
[19].
ꢀ
ꢁ
B − S
Percent inhibition =
× 100
S
where B and S are absorbances for the blank and samples,
respectively. After screening, the compounds median inhibitory
concentration (IC50) was also calculated. All the studies have been
carried out at least in triplicates and the results represent the
mean S.E.M. (standard error of the mean). Kojic acid and L-
mimosine were used as standard inhibitors for the tyrosinase, and
both of them were purchased from Sigma Chem. Co., USA [48].
2.11. ˛-Glucosidase enzyme inhibition assay
␣-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay has been
performed according to the slightly modified method of Mat-
sui et al. ␣-Glucosidase (E.C.3.2.1.20) from Saccharomyces sp. was
purchased from Wako Pure Chemical Industries Ltd. (Wako 076-
02841). The inhibition was measured spectrophotometrically at
pH 6.9 and at 37 ◦C using 0.7 mM p-nitrophenyl ␣-D glucopy-
ranoside (PNP-G) as a substrate and 250 m units/mL enzyme
in 50 mM sodium phosphate buffer containing 100 mM NaCl. 1-
Deoxynojirimycin (0.425 mM) and acarbose (0.78 mM) were used
as positive controls. The increment in absorption at 400 nm due to
the hydrolysis of PNP-G by ␣-glucosidase was monitored contin-
uously with the spectrophotometer (Molecular Devices, CA, USA)
[49].
The incubation of 1 (600 mg) with C. elegans (TSY 0865) for
6 days yielded metabolites 7–10 (Fig. 1). The HREI MS of com-
pound 7 showed the M+ at m/z 310.2004, in accordance with the
formula C21H26O2. Comparison of the 1H and 13C NMR data of com-
pounds 6 and 7, metabolite 7 showed the presence of two additional
olefinic proton signals at ı 7.12 (dd, J1,2 = 8.4 Hz, J1,10 = 4.7 Hz, H-1)
and 6.62 (dd, J2,1 = 8.4 Hz, J2,4 = 2.6 Hz, H-2) with the correspond-
ing carbons resonated at ı 153.4 (C-1) and 127.0 (C-2), respectively
(Tables 1 and 3). The presence of a double bond between C-1 and
C-2 was deduced from COSY 45◦ correlations between H-1 (ı 7.12),
H-2 (ı 6.62) and H-10 (ı 2.42). While HMBC interactions of H-1
with C-2 (ı 127.0) and C-10 (ı 43.0) were observed. The structure
of metabolite 7 was finally deduced to be 7␣-methyl-17␣-ethynl-
17␤-hydroxy-19-norandrost-1,4-dien-3-one.
2.11.1. Estimation of IC50 values
The concentrations of the test compounds, which inhibited the
hydrolysis of PNP-G by ␣-glucosidase by 50% (IC50), were deter-
mined by monitoring the effect of increasing the concentration of
these compounds on the inhibition values. The IC50 values were
then calculated by using EZ-Fit enzyme kinetics program (Perrella
Scientific Inc., Amherst, MA, USA).
The HREI MS of metabolite 8 showed the M+ at m/z 328.2090,
in agreement with the formula C21H28O3 indicating an introduc-
tion of a new oxygen in the molecule, probably as OH. The 13C

962
M.I. Choudhary et al. / Steroids 75 (2010) 956–966
Fig. 1. Structures of substrates and their metabolites 1–20.
NMR spectrum showed a downfield oxygen-bearing quaternary
carbon resonated at ı 70.3, which was assigned to C-10 through
its HMBC interactions with H-1 (ı 2.36, 2.29) and H-4 (ı 5.77)
(Tables 1 and 3). The 10␤-hydroxylation was also deduced by the
␤-SCS (substituents chemical shift) of −4.5, −9.1 and −5.3 ppm
for C-2, C-8 and C-11, respectively, and by the downfield shifts of
C-1 and C-9 (+5.4 and +8.3, respectively) with respect to the 13C
NMR chemical shifts in compounds 6 and 8 [16]. The spectral data
supported the structure of a new metabolite 8 as 7␣-methyl-17␣-
ethynl-10␤,17␤-dihydroxy-19-norandrost-4-en-3-one.

M.I. Choudhary et al. / Steroids 75 (2010) 956–966
963
Fig. 2. Key correlations of compound 6 in NOESY spectrum.
Fig. 4. Key correlations of compound 10 in NOESY spectrum.
The HREI MS of metabolite 9 showed the M+ at m/z 344.2212,
supporting the formula C21H28O4, indicated that two oxygen had
been incorporated into the molecule, as compared to 1. The 1H and
13C NMR displayed two OH-bearing methine groups at ıH 3.43 (ddd,
J11e,9a = 15.1 Hz, J11a,12a = 11.1 Hz, J11a,12e = 5.0 Hz); and 4.10 (m,
W1/2 ∼ 8.82 Hz) and ıC 66.1 and 65.4, respectively (Tables 1 and 3).
The COSY 45◦ spectrum showed correlations of H-11 (ı 3.43) with
H-9 (ı 1.62) and H2-12 (ı 2.05, 1.51), and of H-15 (ı 4.10) with
H-14 (ı 1.80) and H2-16 (ı 2.01, 1.55). Hydroxylations at C-11
and C-15 was further supported by HMBC correlations of H2-12
(ı 2.05, 1.51) and Me-18 (ı 0.90) with C-11 (ı 66.1), and corre-
lation of H-14 (ı 1.80) with ı 65.4 (C-15). The axial orientation
of C-11 proton was deduced on the basis of NOESY correlation
of H-11 (ı 3.43) with Me-18 (ı 0.90) and multiplicity of H-11
(ıH 3.43, ddd, J11e,9a = 15.1 Hz, J11a,12a = 11.1 Hz, J11a,12e = 5.0 Hz) [6],
while ␤-stereochemistry of the newly introduced OH group at C-
15 was deduced by the NOESY correlations between H-14 (ı 1.80)
and H-15 (ı 4.10) (Fig. 3). According to this spectral data, the
structure was deduced to be 7␣-methyl-17␣-ethynl-11␣,15␤,17␤-
trihydroxy-19-norandrost-5(10)-en-3-one.
H-11 (ı 3.40) and H-18 (ı 0.94) and larger coupling constants
(J11e,9a = 15.3 Hz) of H-11 signal [6]. The ␤-orientation of the OH
group at C-15 was deduced on the basis of NOESY between H-
14 (ı 1.85) and H-15 (ı 3.91) (Fig. 4). The ␤-orientation (axial) of
C-10 proton was deduced through NOESY cross peaks between
H-10 (ı 2.46) and H-8 (ı 1.59) (Fig. 4). Based on the above
mentioned spectral data, the structure was deduced as 7␣-methyl-
17␣-ethynl-11␣,15␤,17␤-trihydroxy-19-norandrost-5-en-3-one.
Tibolone (1) was fermented with G. fujikuroi (ATCC 10704) for
12 days yielding six new metabolites 11–16 (Fig. 1). The HREI
MS of metabolite 11 showed the M+ at m/z 312.1456, with cor-
responding formula C21H28O2. The 1H NMR spectrum showed an
upfield doublet of olefinic proton at ı 5.33 (J6,7e = 4.7 Hz), which
was assigned to H-6 through its COSY 45◦ correlation with H-7 (ı
1.85). The 13C NMR spectrum showed downfield methine carbon
at ı 123.4 (Tables 1 and 3). A double bond between C-5 and C-6
was deduced through HMBC between H-6 (ı 5.33) and C-7 (ı 34.6).
The structure was thus deduced as 7␣-methyl-17␣-ethynl-17␤-
hydroxy-19-norandrost-5-en-3-one.
Metabolites 12 and 13 were found to be epimers and differ-
entiated on the basis of 1H NMR and NOESY experiments. The
1H NMR spectrum of metabolite 12 displayed a doublet at ı 4.05
(J6e,7e = 4.0 Hz), while 13 exhibited a doublet at ı 3.60 (J6a,7e = 6.4 Hz)
(Tables 1 and 2). The 13C NMR spectra of both the isomers 12 and
13 showed OH-bearing methine carbons resonating at ı 65.9 and
70.0, respectively (Table 3). The position of the newly introduced
hydroxyl at C-6 in both isomers was inferred from the HMBC cou-
pling. The relative configuration of the new hydroxyl group at C-6
in compound 12 was inferred on the basis of coupling pattern and
NOESY correlations between H-6 (ı 4.05) and C-19 methyl protons
(ı 0.75), while NOESY spectrum of compound 13 displayed corre-
lations between H-6 (ı 3.60) and C-7 methine proton (ı 1.98). The
above spectral data concluded that metabolites 12 and 13 have an
–OH group at C-6 position with different orientations.
The HREI MS of metabolite 10 showed the M+ at m/z 344.2341,
supporting the formula C21H28O4, with an increment of 32 a.m.u.
The UV spectrum showed a weak absorption at 202 nm, while IR
showed absorptions at 3312 (OH), 1722 (C O) and 1652 (C C)
cm−1. The 1H NMR spectrum showed an upfield doublet for an
olefinic methine proton at ı 5.42 (J6,7e = 4.2 Hz, H-6), which showed
COSY 45◦ correlations with H-7 (ı 1.83). Two additional OH-bearing
methine protons at ı 3.40 (ddd, J11e,9a = 15.3 Hz, J11a,12a = 11.0 Hz,
J11a,12e = 4.75 Hz) and 3.91 (m, W1/2 ∼ 9.9 Hz) were unambiguously
assigned to H-11 and H-15, respecting through 2D NMR and 13C
NMR spectra (Tables 1 and 3). The stereochemistry of C-11 OH
was deduced to be ␣ (equatorial) on the basis NOESY between
The molecular formula of metabolite 14 was deduced as
C
21H28O3 by HREI MS (m/z 328.2132). The 1H and 13C NMR spectra
of compound 14 exhibited a OH-bearing methine group, res-
onated at ıH 4.02 (m, W1/2 ∼ 14.9 Hz); ıC 65.9 was unambiguously
assigned to C-15 on the basis of two-dimensional NMR experiments
(Tables 2 and 4). In the COSY 45◦ spectrum, the aforementioned
methine proton showed correlation with the C-14 methine proton
(ıH 1.72). This was further supported by the HMBC of C-14 pro-
ton (ıH 1.72) with C-15 (ıC 65.9). The ␣-relative configuration of
OH group at C-15 was deduced on the basis of NOESY correlations
between H-15 (ı 4.02) and C-18 methyl protons (ı 0.84) (Fig. 5).
This spectral data led to the structure 14 as 7␣-methyl-17␣-ethynl-
15␣,17␤-dihydroxy-19-norandrost-4-en-3-one.
Fig. 3. Key correlations of compound 9 in NOESY spectrum.

964
M.I. Choudhary et al. / Steroids 75 (2010) 956–966
Fig. 5. Key correlations of compound 14 in NOESY spectrum.
Fig. 6. Key correlations of compound 17 in NOESY spectrum.
The HREI MS of metabolite 15 showed the M+ at m/z 326.2231
(C21H28O3). Two additional doublets in the 1H NMR spectrum
for two olefinic protons appeared at ı 7.08 (dd, J1,2 = 8.2 Hz,
J1,10 = 4.2 Hz) and 6.76 (d, J2,1 = 8.2 Hz) and one new hydroxyme-
thine proton (ı 3.81, d, J6a,7e = 6.4 Hz). The DEPT spectrum of
compound 15 showed two olefinic methine carbon signals at ı
154.2 and 126.5, corresponding to a double bond between C-1/C-
2, as deduced on the basis of HMBC of H-1 (ı 7.08) with C-2 (ı
126.5) and of H-2 (ı 6.76) with C-1 (ı 154.2) and C-4 (ı 125.7)
(Tables 2 and 4). One OH-bearing methine carbon resonated at ı
69.8 was identified as C-6 based on 3J correlations between H-6
(ı 3.81) and C-4 (ı 125.7). The ␣-orientation of newly hydroxyl
group at C-6 was deduced to be equatorial on the basis of NOESY
correlations between H-6 (ı 3.81) and H-7 (ı 1.95). According to
these spectral studies, the structure of metabolite 15 was deduced
as 7␣−methyl-17␣-ethynl-6␣,17␤-dihydroxy-19-norandrost-1,4-
dien-3-one.
tion of the new hydroxyl group at C-6 was deduced through HMBC
interactions. The ␣ (equatorial) orientation of C-6 proton, gemi-
nal to the hydroxyl group, was deduced on the basis of NOESY
between H-6 (ı 3.90) and C-19 methyl protons (ı 0.73) (Fig. 6).
The structure of metabolite 17 was identified as 7␣-methyl-17␣-
ethynl-3␤,6␤,17␤-trihydroxy-19-norandrost-5(10)-en-3-one.
Fermentation of 3␣-hydroxytibolone (3) (400 mg) with C. ele-
gans for 12 days yielded three polar metabolites 18–20 (Fig. 1).
Metabolite 18 showed the M+ at m/z 314.2541 (C21H30O2) in HREI
MS, while the 1H and 13C NMR spectra showed an olefinic proton
at ı 5.38 (d, J6,7e = 4.5 Hz) with corresponding carbon at ı 132.0, as
compared to substrate 3, and was assigned to C-6 methine car-
bon (Tables 2 and 4). The ␤ (axial) orientation of C-10 proton
was deduced on the basis of NOESY correlations. From these data,
the new compound 18 was identified as 7␣-methyl-17␣-ethynl-
3␣,17␤-dihydroxy-19-norandrost-5-en-3-one.
The HREI MS of metabolite 16 showed the M+ at m/z 343.2356
corresponding to the formula C22H30O3. The 1H NMR spectrum
showed the presence of a methoxy singlet at ı 3.47, while geminal
methoxy protons were resonated at ı 3.93 (d, J6e,7e = 3.9 Hz). The
13C NMR spectrum showed a methoxy carbon signal at ı 57.6 and
a methoxy-bearing carbon at ı 70.2 (Tables 2 and 4). The position
of the methoxy group at C-6 was deduced through HMBC of H-6 (ı
3.93) with C-4 (ı 126.7). The ␤-orientation of the newly introduced
OCH3 group at C-6 was deduced on the basis of NOESY correla-
tions between H-6 (ı 3.93) and C-19 methyl protons (ı 0.77). The
metabolite 16 was thus identified as 7␣-methyl-17␣-ethynl-6␤-
methoxy-17␤-hydroxy-19-norandrost-4-en-3-one.
In order to check the effect on hydroxylation at various positions
in presence of C-3 hydroxyl group (␣ and ␤ isomers) tibolone (1)
(1 g) was reduced with NaBH4 in dichloromethane yielded reduced
products, i.e. 3␤-hydroxytibolone (2) and 3␣-hydroxytibolone (3)
[47]. The hydroxyl group at C-3 promoted the hydroxylation at C-6
as in case of metabolites 17 and 19 and better yield of these metabo-
lites was produced as compared to tibolone (1). The 1H NMR spectra
of both the isomers 2 and 3 showed OH-bearing geminal methine
protons at ı 3.80 (m, W1/2 ∼ 21.5 Hz) and 4.04 (m, W1/2 ∼ 10.7 Hz),
which indicated the reduction of the C-3 ketonic group. The ori-
entation of C-3 methine proton in the both isomers 2 and 3 was
deduced from the multiplicity of C-3 proton signals resonated at ı
3.80 (m, W1/2 ∼ 21.5 Hz, H-3␣) and 4.04 (m, W1/2 ∼ 10.7 Hz, H-3␤),
respectively [46].
The HREI MS of metabolite 19 showed the M+ at m/z 330.2356
corresponding to the formula C21H30O3. The 1H NMR spectrum
of 19 showed an additional OH-bearing methine proton at ı
4.13 (br. s), with corresponding methine carbon at ı 74.0 (C-6)
(Tables 2 and 4). The COSY 45◦ spectrum also showed an allylic
coupling between H-4 and H-6 (ı 4.13). The ˛ (equatorial) orien-
tation of C-6 proton, geminal to OH, was deduced on the basis
NOESY correlations between H-6 and Me-19 protons (ı 0.87).
The structure of metabolite 19 was identified as 7␣-methyl-17␣-
ethynl-3␣,6␤,17␤-trihydroxy-19-norandrost-4-en-3-one.
Metabolite 20 showed the M+ at m/z 330.2256 in HREI MS
in agreement with the formula C21H30O3. The 1H and 13C NMR
spectra of 20 showed an OH-bearing methine proton at ıH 3.80
(ddd, J11e,9a = 13.5 Hz, J11a,12a = 9.5 Hz, J11a,12e = 3.3 Hz) and ıC 66.3
and assigned C-11 through COSY between H-11 (ı 3.80), H-18
(ı 1.04) and H2-12 (ı 2.1, 1.64). The HMBC correlations revealed
the connectivities between H-11, H-18 (ı 1.04) and C-11 (ı 66.3)
(Tables 2 and 4). The ˇ (axial) orientation of C-11 proton, geminal
to an OH, was deduced on the basis of larger coupling con-
stant of H-11 signal (ıH 3.82, ddd, J11e,9a = 13.5 Hz, J11a,12a = 9.5 Hz,
J11a,12e = 3.3 Hz) and NOESY correlations between H-11 and Me-
18 protons (ı 0.88) (Fig. 7) [6]. The structure of metabolite 20
was proposed as 7␣-methyl-17␣-ethynl-3␣,11␣,17␤-trihydroxy-
19-norandrost-4-en-3-one.
Incubation of 3␤-hydroxytibolone (2) (300 mg) with C. elegans
for 12 days yielded a hydroxyl-bearing metabolite 17 (Fig. 1).
Metabolite 17 showed the M+ at m/z 330.2346 corresponding to the
formula C21H30O3 by HREI MS. The 1H NMR spectrum showed a res-
onance for OH-bearing methine proton at ı 3.90 (d, J6e,7e = 3.5 Hz),
with corresponding carbon at ı 74.2 (Tables 2 and 4). The posi-
Some hydroxy metabolites of tibolone (1) showed significant
inhibitory activity against enzyme tyrosinase, the OH at C-6, C-
11 and C-15 as in metabolites 4, 9 and 10, respectively showed
pronounced inhibitory activity the against enzyme, while ␣,␤-
unsaturated system (C-4/C-5) in metabolite 6 also showed a good
activity (Table 6).

M.I. Choudhary et al. / Steroids 75 (2010) 956–966
965
potent inhibitors of ␣-glucosidase enzyme, while metabolites 10
and 11 containing unsaturation in ring B and hydroxylations at C-
11 and C-15 in metabolites 9 and 10 showed no inhibitory activities
against ␣-glucosidase enzyme. This concluded that both unsatura-
tion in ring A and hydroxylations in ring A and hydroxylations in
ring B enhanced the inhibition of ␣-glucosidase enzyme.
4. Conclusions
In conclusion, the transformation of tibolone (1) by fungus
yielded thirteen metabolites 4–16. While incubation of hydroxyti-
bolones (2 and 3) with C. elegans yielded four polar metabolites
17–20. The main hydroxylations occurred in rings B and D, espe-
cially at C-6 and C-15 positions. The metabolites 4, 5, 9, 10, 12,
13, 14, 15, and 16 were identified as the main metabolites of the
fermentations. Hydroxylations at C-6, C-10 and C-15 positions in
metabolites 4, 8, 12, 13, 14, and 15 showed pronounced inhibitory
activity against ␣-glucosidase enzyme, while ␣,␤-unsaturated sys-
tem (C-4/C-5) in metabolite 6 and C-1/C-2, C-4/C-5 in metabolite
7 also showed a good inhibitory activity (Table 5). The analogs of
tibolone (1) also exhibited a mild to potent inhibition of the enzyme
tyrosinase, except compounds 7, 11, 13, and 14. Compound 9 was
the most potent inhibitor of the tyrosinase (Table 6).
Fig. 7. Key correlations of compound 20 in NOESY spectrum.
Table 5
␣-Glucosidase inhibitory activities of tibolone (1) and its analogs 4–16, as compared
to the reference inhibitors.
Compounds
IC50 S.E.M.a (in ␮M)
1
4
5
NAb
225.0 0.00
NAb
Acknowledgment
6
7
8
9
10
11
343.0 0.00
227.0 0.02
877.0 0.03
NAb
We acknowledge the help of Syed Khawaja Karim-Ullah for pro-
viding product Livial-Organon of Zafa Pharma, Karachi, Pakistan.
NAb
NAb
12
13
653.0 0.02
<70.0
References
14
15
16
<70.0
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Table 6
Tyrosinase inhibitory activities of tibolone (1) and its analogs 4–16, as compared to
the reference inhibitors.
Compounds
IC50 S.E.M.a (in ␮M)
1
4
5
6
7
8
9
10
11
12
13
14
NAb
7.45 0.21
50.86 0.36
8.19 0.54
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119.44 0.20
5.12 0.67
7.10 0.13
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36.14 0.21
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16
25.15 0.24
16.67 0.51
3.68 0.022
Kojic acid (KA)c
L-Mimosine (LM)c
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a
S.E.M. is the standard error of the mean.
The inactive compounds.
The standard inhibitors of the tyrosinase.
b
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