Biotransformation of tissue-specific hormone tibolone with fungal culture Trichothecium roseum

Article abstract of DOI:10.1016/j.molstruc.2013.03.046

Whole cells based biotransformation is an important tool for bioconversion of steroids. It can be used to synthesize biologically potent compounds with diverse structures. Biotransformation of tissue-specific hormone tibolone (1) with Trichothecium roseum

Full text of DOI:10.1016/j.molstruc.2013.03.046

Journal of Molecular Structure 1042 (2013) 118–122
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Biotransformation of tissue-specific hormone tibolone with fungal culture
Trichothecium roseum
Syed Adnan Ali Shah a,b, , Sadia Sultan a,b, M. Zaimi bin Mohd Noor b
⇑
a Department of Pharmacology and Chemistry, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan,
Malaysia
b Atta-ur-Rahman Institute for Natural Products Discovery (RiND), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan,
Malaysia
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Biotransformation of tibolone (1) with Trichothecium roseum has being carried out for the first time.
Two new and three known metabolites 2–6 were isolated from fermentation of tibolone (1).
Effect of fungal cultures on compound 1 modification has been studied.
Functionalization could be difficult by conventional chemical methods.
Structures of new metabolites 5 and 6 were characterized by 2D NMR spectroscopy and mass spectrometry.
a r t i c l e i n f o
a b s t r a c t
Article history:
Whole cells based biotransformation is an important tool for bioconversion of steroids. It can be used to
synthesize biologically potent compounds with diverse structures. Biotransformation of tissue-specific
hormone tibolone (1) with Trichothecium roseum (ATCC 13411) has being carried out for the first time.
Two new and three known metabolites 2–6 were isolated from fermentation of tibolone (1) with Trich-
othecium roseum and their structures were characterized by 2D NMR spectroscopy and mass spectrom-
etry. The relative stereochemistry of new metabolites 5 and 6 was deduced by 2D NOESY experiments.
The effect of cultures on tibolone structural modifications and time-course studies has also been
conducted.
Received 25 February 2013
Received in revised form 25 March 2013
Accepted 25 March 2013
Available online 2 April 2013
Keywords:
Biotransformation
Tibolone
Trichothecium roseum
Hydroxytibolone
2D NMR spectroscopy
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
Our research group has been focused on the structural modifi-
cations of 17a-ethynyl steroids by using fungi, in order to investi-
Fungi have found wide applications in biotransformation of ste-
roids in order to obtain high regio- and stereoselective products.
They can transform fine chemicals through environmental friendly
gate the effect of culture on substrate [4–7]. As a result of these
studies, we have been reported the effect of four fungal cultures
on tibolone (1) and hydroxytibolones previously [5]. In the present
study, we have chosen to investigate the effect of Trichothecium
roseum on tibolone and it has being carried out for the first time.
Tibolone (1), a synthetic steroid that is used for hormone
replacement in menopausal women, is metabolized to three pri-
and cost effective way. Fungal transformation of 17a-ethynyl ste-
roids has been extensively investigated and hydroxylation at vari-
ous position have been produced on large scale. The
transformation mostly carried out by fungi included hydroxylation,
oxidation and reduction of alcohols, ketones and C@C bond [1–3].
mary active agents: 3
a-hydroxytibolone, 3b-hydroxytibolone and
D
4-tibolone (2). It can exhibit estrogenic, progestogenic or andro-
genic activity, depending primarily on the target tissue involved;
for this reason, tibolone is said to have tissue-specific activity
and structurally related to 19-nortestosterone derivatives [8,9].
Tibolone (1) and its metabolites also showed tissue specific inhibi-
tion of sulfatase enzyme which could be protective against devel-
opment of mammary carcinomas, whereas it retains favorable
estrogenic effects on bone [10].
⇑
Corresponding author at: Department of Pharmacology and Chemistry, Faculty
of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300
Bandar 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. Sha.
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.03.046

S.A.A. Shah et al. / Journal of Molecular Structure 1042 (2013) 118–122
119
In continuation of our biotransformational studies on bioactive
compounds [4–7,11–27], structural transformation of tibolone (1)
with T. roseum was studied which afforded five polar metabolites
2–6. Compounds 2–4 were previously reported as metabolites of
compound 1 [5].
from three-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 3 days, tibolone (1) was dissolved in acetone
and transferred in each flask (10 mg/500 L) and the flasks were
placed on a rotatory shaker (128 rpm) at 25 °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 ethyl acetate. The extract was dried
over anhydrous Na2SO4, evaporated under reduced pressure and
the brown gummy crude was analyzed by thin layer
chromatography.
2. Experimental
2.1. General
Tibolone (1) was purchased from Sigma–Aldrich (USA). Melting
points were determined on a Yanaco MP-S3 apparatus. UV spectra
were measured on a Shimadzu UV 240 spectrophotometer. JASCO
DIP-360 Digital polarimeter was used to measure the optical rota-
tions in chloroform by using 10 cm cell tube. FTIR-8900 Spectro-
photometer was used to record IR spectra in CHCl3. The 1H NMR
and 2D NMR spectra were recorded on a Bruker Avance III 600 As-
cend spectrometer using BBO probe and Protasis CapNMR ICG
probe with CTC/LEAP liquid handler, while 13C NMR spectra were
recorded on Bruker Avance III 600 Ascend spectrometer operating
at 150 MHz using CDCl3 as solvent. Chemical shifts were reported
in d (ppm), relative to SiMe4 as internal standard, and coupling
constants (J) were measured in Hz. The EI-MS and HREIMS were
measured on Jeol HX 110 mass spectrometer. TLC was performed
on Si gel precoated plates (PF254, 20 ꢁ 20, 0.25 mm, Merck, Ger-
many). Ceric sulfate in 10% H2SO4 spraying reagent was used for
the staining of compounds on TLC. All reagents used were of ana-
lytical grades.
2.4. Incubation of tibolone (1) with T. roseum (ATCC 13411)
Compound 1 (200 mg), dissolved in 10 mL acetone and distrib-
uted among 20 flasks, and kept for fermentation. Five of the flasks
were extracted after days in order to carry out time course study.
Rest of the media flasks were filtered after 12 days of fermentation
and extracted with ethyl acetate and evaporated under reduced
pressure to finally obtain a brown thick extract (0.40 g), and the
transformed metabolites were then isolated by using column chro-
matography. Metabolite 2 (20 mg) was eluted with pet. Ether-
EtOAc (70:30), compound
3 (7 mg) with pet. Ether–EtOAc
(65:35), compound 4 (6.4 mg) with pet. Ether-EtOAc (55:45), com-
pound 5 (3 mg) with pet. Ether-EtOAc (50:50) and compound 6
(3.2 mg) with pet. Ether-EtOAc (45:55).
2.4.1. 12b-hydroxy-D
4-tibolone (5)
White powdered solid (3 mg); mp 202–205 °C; ½a D25
ꢃ 13 (c
ꢂ
0.42, CHCl3); UV (MeOH) kmax (loge) 240 (2.3) nm; IR (CHCl3) mmax
3432, 2152, 1690 and 1670 cmꢃ1 1H and 13C NMR data in CDCl3,
;
2.2. Fungi and culture conditions
Tables 1 and 2; EI MS m/z (rel. int.%) 328 (4, M+), 312 (15), 244
(22), 229 (26), 202 (16), 189 (14), 187 (14), 176 (24), 161 (21),
149 (26), 135 (22), 121 (25), 91(91), 81 (23), 69 (21), 67 (23), 55
(40); HREI-MS [M+ m/z 328.2116 (calcd 328.2104)].
Microbial cultures of the T. roseum (ATCC 13411) was grown on
Potato dextrose agar (PDA) at 25° C and stored at 4 °C. T. roseum
(ATCC 13411) medium was prepared by adding glucose (20 g),
peptone (10 g), KH2PO4 (10 g), yeast extract (10 g), NaCl (10 g)
and glycerol (10 mL) into distilled water (2 L).
2.4.2. 3a,6a-dihydroxy-D
4-tibolone (6)
White powdered solid (3.2 mg); mp 199–201 °C; ½a D25
ꢃ 36 (c
ꢂ
2.3. General fermentation and extraction conditions
0.22, CHCl3); UV (MeOH) kmax (loge) 203 (1.7) nm; IR (CHCl3) mmax
3403, 2151, and 1665 cmꢃ1; 1H and 13C NMR data in CDCl3, Tables
1 and 2; EI MS m/z (rel. int.%) 330 (2, M+), 312 (5, [M–H2O]+), 241
(21), 228 (22), 202 (16), 189 (14), 187 (13), 176 (24), 161 (21), 149
The fungal media were transferred into 250 mL conical flasks
(100 mL each) and autoclaved at 120 °C. Seed flasks were prepared
Table 1
1H NMR data of metabolites 2–6 at 600 MHz in CDCl3; d in ppm, J and W1/2 in Hz.
No. of C
2
3
4
5
6
1
2
3
4
1.84, 2.01 m
2.22, 2.28 m
–
5.78 s
–
7.11, dd (8.3, 4.7)
6.61 dd (8.3, 2.5)
–
6.55 d (2.6)
–
2.16, 2.26 m
2.25, 2.25 m
–
5.80 s
–
2.10, 2.25 m
2.30, 2.31 m
–
5.80 s
–
1.95, 2.14 m
2.08, 2.25 m
4.08 m (W1/2 = 10.4)
5.60 br s
–
5
6
7
8
9
2.15, 2.20 m
1.66 m
1.68 m
1.51 m
2.32 m
1.83, 2.27 m
1.59, 1.75 m
–
1.67 m
1.40, 1.61 m
2.31, 1.87 m
–
2.15, 2.25
1.62 m
1.64 m
1.58 m
2.32 m
1.82, 2.26 m
1.55, 1.72 m
–
1.97, 2.14 m
1.96 m
1.76 m
1.68 m
2.46 m
1.87, 2.27 m
1.64, 1.66 m
–
2.16, 2.15 m
1.60 m
1.62 m
1.31 m
2.11
1.88, 2.34 m
4.14 d (4.65, 10.83)
–
4.28 br s
1.90 m
1.68 m
1.62 m
2.12
1.89, 2.24 m
1.62, 1.67 m
–
10
11
12
13
14
15
16
17
18
19
20
21
1.65 m
1.72 m
1.66 m
1.70 m
1.36, 1.68 m
2.23, 1.80 m
–
0.89 s
0.82 d (7.01)
–
4.06 m (W1/2 ꢄ 15.2)
1.72, 2.24 m
–
0.90 s
0.81 d (7.02)
–
1.44, 1.53 m
2.21, 2.24 m
–
0.89 s
0.78 d (7.04)
–
1.47, 1.51 m
1.72, 2.20 m
–
0.91 s
0.80 d (7.02)
–
0.91 s
0.81 d (7.2)
–
2.57 s
2.58 s
2.55 s
2.56 s
2.57 s

120
S.A.A. Shah et al. / Journal of Molecular Structure 1042 (2013) 118–122
Table 2
(26), 135 (22), 121 (25), 91(87), 81 (23), 69 (11), 67 (23), 57 (41);
13C NMR data of metabolites 2–6 at 150 MHz in CDCl3; d in ppm.
HREI-MS [M+ m/z 330.2165, (calcd 330.2132)].
No. of C
2
3
4
5
6
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)
199.3 (s)
33.2 (t)
123.0 (s)
30.4 (d)
33.6 (d)
39.4 (d)
46.2 (d)
128.4 (s)
29.8 (t)
32.9 (t)
47.4 (s)
48.6 (d)
22.2 (t)
38.9 (t)
79.8 (s)
13.1 (q)
12.5 (q)
87.5 (s)
73.5 (d)
152.9 (d)
127.2 (d)
199.6 (d)
125.3 (d)
162.4 (s)
30.7 (t)
32.9 (d)
43.5 (d)
46.4 (d)
43.0 (d)
29.7 (t)
32.4 (t)
46.2 (s)
47.2 (d)
22.6 (t)
39.0 (t)
79.9 (s)
13.2 (q)
12.7 (q)
87.6 (s)
74.1 (d)
25.3 (t)
38.8 (t)
198.5 (s)
126.2 (d)
164.5 (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)
66.1 (d)
47.1 (t)
79.9 (s)
13.1 (q)
12.7 (q)
87.7 (s)
73.5 (d)
26.1 (t)
37.9 (t)
201.5 (s)
125.2 (d)
163.2 (s)
31.9 (t)
42.6 (d)
38.4 (d)
47.1 (d)
44.0 (s)
42.1 (t)
74.2 (d)
44.2 (s)
48.2 (d)
24.2 (t)
39.1 (t)
79.2 (s)
13.1 (q)
12.2 (q)
88.1 (s)
73.3 (d)
25.2 (t)
38.9 (t)
78.1 (d)
128.0 (t)
148.0 (s)
69.1 (d)
42.6 (d)
40.1 (d)
46.2 (d)
44.2 (s)
29.4 (t)
33.1 (t)
45.6 (s)
50.2 (d)
23.1 (t)
40.1 (t)
79.1 (s)
14.2 (q)
12.5 (q)
87.4 (s)
73.7 (d)
2.5. 1D hydrogen–deuterium exchange NMR experiment
1D hydrogen–deuterium exchange experiments were carried
out by adding a drop of D2O to a CDCl3 solution in the NMR tube,
mixing thoroughly and run the NMR experiments on a Bruker
Avance III 600 Ascend spectrometer using Protasis CapNMR ICG
probe with CTC/LEAP liquid handler.
3. Results and discussion
Tibolone (1) C21H28O2, when subjected to microbial transforma-
tion with T. roseum (ATCC 13411) for 12 days, affording five polar
products (2–6). Two hydroxytibones 5 (12b-hydroxy-
D
4-tibolone)
and (3 ,6 -dihydroxy-
6
a
a
D
4-tibolone) were deduced as new
metabolites (Fig. 1). Present study also showed that the effect of
cultures on tibolone (1) resulted different regio- and stereoselec-
tive hyroxyl products.
Fig. 1. Biotransformation of tibolone (1) by a fungal culture of Trichothecium Roseum.

S.A.A. Shah et al. / Journal of Molecular Structure 1042 (2013) 118–122
121
The isolated metabolites (2–4) were unambiguously identified
by 2D NMR spectroscopy and mass spectrometry as:
4-Tibolone
(2),
1,4-tibolone (3) and 15 -hydroxy-D4 tibolone (4). The pro-
D
D
a
tons of hydroxyl group of isolated metabolites (2-4) were reso-
nated ranging from d 2.09-2.2, which was selectively removed
from 1H NMR spectrum by 1D hydrogen–deuterium exchange
experiment in order to confirm the exchange between OH proton
and heavy atom deuterium. Metabolites 2–4 have been previously
reported as metabolites of compound 1, when incubated with Rhi-
zopus stolonifer, Fusarium lini, Cunninghamella elegans and Gibber-
ella fujikuroi but fungal transformation with T. roseum has being
carried out for the first time. All spectroscopic data were showed
excellent agreement with the previously published results [5]
(Tables 1 and 2). These metabolites also exhibited various level
Fig. 3. Key NOESY ( ) and HMBC ( ) interactions in compound 6.
The molecular composition C21H30O3 for metabolite 6 was ob-
tained from the HREI-MS [M+ m/z 330.2165, (calcd 330.2132)],
18 mass units higher than substrate 1 (Fig. 1). The 18 a.m.u. incre-
ment in the M+ of the metabolite 6, as compared to substrate 1,
could be attributed to the addition of a water molecule. The UV
spectrum showed a weak absorption at 203 nm, suggesting lack
of any conjugated system, while IR showed absorptions at 3403
(OH), 2151 (C„C), and 1665 (C@C) cmꢃ1 with absence of carbonyl
absorption. This also confirmed that the C-3 ketonic group has
been reduced during fermentation of compound 1. This was con-
firmed with the help of 1H and 13C NMR spectra (Tables 1 and 2).
The 1H and 13C NMR of metabolite 6 (Tables 1 and 2), when com-
pared with the substrate 1, displayed two OH-bearing methine
groups at dH 4.08 (m, W1/2 ꢄ 10.4 Hz); 4.28 (br. s) and dc 78.1
and 69.1, whereas protons of hydroxyl group were resonated at d
2.11, respectively [5]. The selective removal of resonance of
protons of hydroxyl group was carried through 1D hydrogen–
deuterium exchange experiment, which resulted absence of
hydroxyl proton resonances. The 1H NMR also showed a broad
singlet resonated at d 5.60, which indicating a migration of the
double bond from C-5/C-10 to C-4/C-5 position (Table 1). The
DQF-COSY spectrum showed an weak allylic coupling between
H-3 (d 4.08), H-4 (d 5.60) and H-6 (d 4.28). Hydroxylations at
C-6 position was further supported by HMBC correlations of H-6
(d 4.28) with C-4 (d 128.0), C-5 (148) and C-7 (d 43.2) (Fig. 3).
H-6 (d 4.28) showed NOESY interactions with H-10 (d 2.12) and
H-7 (d 1.90) indicating that the C-6 proton was b-oriented, thus
of activity against the enzymes, tyrosinase and a-glucosidase [5].
The fermentation of compound 1 with T. roseum (ATCC 13411)
for 12 days led to the isolation of a UV active metabolite 5 showed
a strong absorption at 240 nm, characteristic of an a,b-unsaturated
carbonyl compound [5], while the IR spectrum showed the absorp-
tions at 3432 (OH), 2152 (C„C), 1690 (conjugated C@O) and 1670
(C@C) cmꢃ1. The molecular formula for metabolite 5 (C21H28O3)
was obtained from the HREI-MS [M+ m/z 328.2116 (calcd
328.2104)], which was 16 a.m.u. higher than the substrate 1. The
16 a.m.u. increment in the M+ of the metabolite 5, as compared
to substrate 1, could be attributed to the addition of an oxygen
atom. The 1H NMR of 5 (Table 1), when compared with the sub-
strate 1, showed an olefinic proton signal resonated at d 5.80 indi-
cating a migration of the double bond from C-5/C-10 to C-4/C-5
position and a new signal of OH-bearing methine proton at d
4.14, resonating as
a
double doublet (J12a,11e = 4.65 Hz,
J12a,11a = 10.83 Hz), whereas protons of hydroxyl group were reso-
nated at d 2.20, with its corresponding carbon at d 74.2 in 13C
NMR spectrum (Table 2), which was assigned to C-12 on the basis
of HMBC correlations of H-12 (d 4.14) with C-11 (d 42.1) and C-13
(d 44.2) (Tables 1 and 2) and HSQC assignments, respectively
(Fig. 2) [18]. The selective removal of resonance of protons of hy-
droxyl group was carried through 1D hydrogen–deuterium ex-
change experiment, which resulted absence of hydroxyl group
proton resonances. The H-12 showed COSY interaction with the
C-11 methine protons (d 1.88, 2.34) in DQF-COSY experiment. This
confirmed that the hydroxylation had occurred at C-12 of the tibo-
lone skeleton. The b-orientation (equatorial) of C-12 hydroxyl
group was determined by the 2D NOESY correlations between
Ha-12 (d 4.14), Ha-9 (d 1.31) and Ha-14 (d 1.66) (Fig. 2). The axial
orientation of C-10 methine proton was also assigned on the basis
of NOESY coupling between H-10 (d 2.11) and H-8 (d 1.62) [5].
Metabolite 5 was characterized as a new compound (12b-hydro-
the geminal hydroxyl group was
these spectral data, the new compound 6 was identified as
,6 -dihydroxy-
4-tibolone (Fig. 1).
Effect of cultures on compound 1 modification with C. elegans,
G. fujikuroi and T. roseum have also been studied. In case of C. ele-
gans, tibolone (1) underwent hydroxylation at C-11 and C-15b
and C-10b with ,b-unsaturated ketone at C-3. Compound 1, when
incubated with G. fujikuroi resulted the production of hydroxylated
products at C-6 and C-15 with ,b-unsaturated ketone at C-3 as
a-oriented (Fig. 3). From
3a
a
D
a
D
4-tibolone) Fig. 1.
a
xy-
a
a
a
previously reported [5]. Whereas compound 1 when incubated
with T. roseum resulted the regio- and stereoselective hydroxyl-
ation at C-3a, C-6a and C-12b positions, respectively.
4. Conclusions
In conclusion, the biotransformation of tibolone (1) by fungal
culture T. roseum yielded five metabolites 2–6. The hydroxylations
were mainly underwent in rings A, B and C, especially at C-3, C-6
and C-12 positions. The metabolites 5 and 6 were identified as
the new metabolites from present fermentation experiment. Effect
of cultures on compound 1 modification with C. elegans, G. fujikuroi
and T. roseum have also been studied. As a result of these studies,
the regio- and stereoselective hydroxylations at C-3
a, C-6a and
Fig. 2. Key NOESY ( ) and HMBC ( ) interactions in compound 5.
C-12b positions were found. These transformed products provide

122
S.A.A. Shah et al. / Journal of Molecular Structure 1042 (2013) 118–122
[12] M.I. Choudhary, S. Erum, M. Atif, R. Malik, N. Khan, Steroids 76 (2011) 1288–
1296.
[13] S.A.A. Shah, S. Sultan, H.S. Adnan, Int. J. Pharm. Pharm. Sci. 3 (Suppl. 1) (2011)
1–6.
accesses to regions of the steroidal skeletons, which are difficult to
be functionalized by conventional chemical methods.
[14] M.I. Choudhary, M.Y. Mohammad, G.S. Musharraf, M. Parvez, A. Al-Aboudic,
Steroids 74 (2009) 1040–1044.
Acknowledgment
[15] M.I. Choudhary, S.A.A. Shah, Nat. Prod. Res. 22 (2008) 1289–1296.
[16] M.I. Choudhary, I. Batool, S.A.A. Shah, S.N. Khan, Nat. Prod. Res. 22 (2008) 489–
494.
[17] M.I. Choudhary, S. Yousuf, Chem. Pharm. Bull. 54 (2006) 927–930.
[18] M.I. Choudhary, S.A.A. Shah, A. Sami, A. Ajaz, F. Shaheen, Chem. Biodivers. 2
(2005) 516–524.
This work was funded by the Fundamental Research Grant
Scheme (FRGS) (600-RMI/FRGS 5/3, 12/2012), KPT, Malaysia.
References
[19] M.I. Choudhary, I. Batool, S.A.A. Shah, S.A. Nawaz, Chem. Pharm. Bull. 53 (2005)
1455–1459.
[20] M.I. Choudhary, S.A.A. Shah, S.G. Musharraf, F. Shaheen, Nat. Prod. Res. 17
(2003) 215–220.
[1] H.N. Bhatti, R.A. Khera, Steroids 77 (2012) 1267–1290.
[2] A. Bartma´nsk, J. Dmochowska-Gładysz, Enzyme Microb. Technol. 40 (2007)
1615–1621.
[3] M.V. Donova, O.V. Egorova, Appl. Microbiol. Biotechnol. 94 (2012) 1423–1447.
[4] S. Zafar, Yousuf, H.A. Kayani, S. Saifullah, S. Khan, A.M. Al-Majid, M.I.
Choudhary, Chem. Central J. 6 (2012) 109–116.
[5] M.I. Choudhary, S.A.A. Shah, Steroids 75 (2010) 956–966.
[6] Atta-ur-Rahman, M.I. Choudhary, F.Z. Basha, G. Abbas, S.N. Khan, S.A.A. Shah,
Pure Appl. Chem. 79 (2007) 2263–2267.
[7] M.I. Choudhary, M. Atif, S.A. Nawaz, M.Q. Fatmi, Nat. Prod. Res. 20 (2006)
1074–1081.
[8] A.J. Ederveen, H.J. Kloosterboer, J. Bone Miner. Res. 1 (1999) 1963–1970.
[9] S. Palacios, Eur. Heart J. Suppl. 3 (Supplement M) (2001) M12–M16.
[10] M.E. de Gooyer, G.T. Kleyn, K.C. Smits, A.G. Ederveen, H.A. Verheul, H.J.
Kloosterboer, Mol. Cell. Endocrinol. 183 (2001) 55–62.
[21] M.I. Choudhary, Chem. Biodivers. 5 (2008) 324–331.
[22] M.I. Choudhary, M. Nasir, S.N. Khan, M. Atif, R.A. Ali, S.M. Khalil, Atta-ur-
Rahman, Zeitschrift für Naturforschung B 62b (2007) 593–599.
[23] M.I. Choudhary, S. Sultan, S. Jalil, S. Anjum, A.A. Rahman, H.K. Fun, Chem.
Biodivers. 2 (2005) 392–400.
[24] M.I. Choudhary, S. Sultan, M.T.H. Khan, Steroids 70 (2005) 798–802.
[25] M.I. Choudhary, Z.A. Siddiqui, S.A. Nawaz, Nat. Prod. Res. 23 (2009) 507–513.
[26] Y. Chenru, F. Huafang, Y. Yuan, G. Jinming, Chin. J. Chem. 31 (1) (2013) 127–
131.
[27] S. Sultan, M.I. Choudhary, S.N. Khan, U. Fatima, M. Atif, R.A. Ali, Eur. J. Med.
Chem. 62 (2013) 764–770.
[11] S.S. Azam, Med. Chem. Res. 21 (2012) 1677–1683.