A bufadienolide derived androgen receptor antagonist with inhibitory activities against prostate cancer cells

Article abstract of DOI:10.1016/j.cbi.2013.10.020

Molecular docking studies have shown that Δ^8,14- anhydrobufalin (1) exhibited more potent binding affinity on androgen receptor (AR) than Δ^14,15-anhydrobufalin (2) and bufalin (3). To validate the docking results, compounds 1 and 2 were synthesized. The AR competitive binding assay indicated that the IC₅₀ values of 1-3 were 1.9, >50 and >50 μM (relative binding affinity), respectively, which confirmed that our theoretical binding mode was reliable and predictable. Furthermore, compound 1 was found to show more potent inhibitory activity against the androgen dependent LNCaP cancer cells than the androgen independent PC3 cancer cells, but exhibited less inhibition on the Na⁺/K ⁺ ATPase as compared with the parent compound 3. To the best of our knowledge, compound 1 represented the first AR antagonist derived from bufadienolide discovered through a series of combined approaches of molecular docking and actual experimental validation.

Full text of DOI:10.1016/j.cbi.2013.10.020

Chemico-Biological Interactions 207 (2014) 16–22
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
A bufadienolide derived androgen receptor antagonist with inhibitory
activities against prostate cancer cells
a,
Hai-Yan Tian a,1, Xiao-Feng Yuan a,1, Lu Jin a,1, Juan Li a, Cheng Luo b, Wen-Cai Ye a, , Ren-Wang Jiang
⇑
⇑
a Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Molecular docking studies have shown that
D
8,14-anhydrobufalin (1) exhibited more potent binding
affinity on androgen receptor (AR) than
results, compounds 1 and 2 were synthesized. The AR competitive binding assay indicated that the IC50
values of 1–3 were 1.9, >50 and >50 M (relative binding affinity), respectively, which confirmed that our
D
14,15-anhydrobufalin (2) and bufalin (3). To validate the docking
Received 8 September 2012
Received in revised form 4 October 2013
Accepted 24 October 2013
Available online 5 November 2013
l
theoretical binding mode was reliable and predictable. Furthermore, compound 1 was found to show
more potent inhibitory activity against the androgen dependent LNCaP cancer cells than the androgen
independent PC3 cancer cells, but exhibited less inhibition on the Na+/K+ ATPase as compared with the
parent compound 3. To the best of our knowledge, compound 1 represented the first AR antagonist
derived from bufadienolide discovered through a series of combined approaches of molecular docking
and actual experimental validation.
Keywords:
Androgen receptor
Antagonist
Bufadienolide
Molecular docking
Na+/K+ ATPase
Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
on Na+/K+-ATPase [8], which greatly hindered the clinical applica-
tion [9].
Androgen receptor (AR) belongs to the steroid nuclear receptor
super-family [1]. It is activated by endogenous androgens as tes-
tosterone and 5a-dihydrotestosterone (DHT) or exogenous com-
pounds, and regulates genes for male differentiation and
development [2]. However, high levels of AR expression may lead
to severe diseases like prostate cancer (PCa). Recently, AR was
found to play a critical role in PCa since approximately 80–90%
of PCa are androgen dependent at initial diagnosis [3,4]. Thus it
has become an attractive target for the treatment of PCa. Although
several pure AR antagonists, such as bicalutamide and flutamide,
were developed for PCa therapy, they could not completely blocks
binding of DHT to AR and the small amount of free DHT still stim-
ulate prostate cancer growth [5]. Furthermore, bicalutamide can
exhibit some agonist activity in cells containing mutant AR [6].
Thus, a sustained effort for the development of new and more
effective AR antagonist has been undertaken.
Close examination of the structure of 3 revealed the similarity
to those of steroidal AR antagonists, such as VN/85-1 [10]. Both
of them possessed a steroidal skeleton with an unsaturated substi-
tution at C-17. However, the steroid moiety of bufalin is saturated
in contrast to the presence of at least one double bond in the ste-
roidal AR antagonists. We hypothesized that introduction of a dou-
ble bond in bufalin would increase the interactions with androgen
receptor.
In order to test the hypothesis, firstly, we virtually introduced a
double bond around the hydroxyl group at C-14 of 3 considering
that it was in the middle of the molecule and important for the
conformation of the whole molecule. Then, the molecular docking
method was used to compare the interactions of 3 and the two
derivatives, i.e.,
D D
8,14-anhydrobufalin (1) and 14,15-anhydrobufa-
lin (2, Scheme 1) with androgen receptor. Finally, the actual deriv-
atives were synthesized and their activities toward the two
molecular targets were tested.
Bufalin (3, Scheme 1), a typical bufadienolide, has been reported
to show potent antineoplastic activity against human prostate can-
cer cells LNCaP and DU145 [7]. However, it was reported to be five
times more lethal than ouabain due to its much stronger inhibition
2. Materials and methods
2.1. Molecular docking study of compounds 1–3 to AR
2.1.1. Homology modeling
⇑
Corresponding authors. Tel./fax: +86 20 85221559.
E-mail addresses: chyewc@gmail.com (W.-C. Ye), trwjiang@jnu.edu.cn,
The 3D model of the androgen receptor (AR) in its inactive form
was constructed based on the known antagonist form of human
glucocorticoid receptor (PDB ID: 3H52) [11], a homologous protein
rwjiang2008@126.com (R.-W. Jiang).
1
These authors contribute equally to this work.
0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbi.2013.10.020

H.-Y. Tian et al. / Chemico-Biological Interactions 207 (2014) 16–22
17
Scheme 1. Structure formulae of compounds 1–3.
of AR (they share >50% sequence similarity) and an agonist form of
2.1.3. Molecular docking
AR (PDB ID: 2PIT) [12]. Sequence alignment and homology model-
ing were performed using Modeller V9.10 [13]. The final sequence
alignment was visualized using ESpript [14] as shown in Fig. 1.
2.1.3.1. Protein preparation and grid generation. Protein structure
was prepared with Protein Preparation Wizard [18] in Maestro
9.0, energy was minimized using OPLS force-field and default set-
ting. The A chain of antagonist human glucocorticoid receptor was
overlapped into above mentioned structure, and the position of co-
crystallized ligand was used as a reference for next step. Grid was
generated by Glide 5.5 using default setting.
2.1.2. Molecular dynamic (MD) simulation
In order to obtain a more stable conformation of AR in solution,
MD simulation was performed. Simulations were conducted with
the OPLS all-atom force-field implemented in GROMACS 4.5.3
[15]. Topology files were generated using pdb2gmx module in Gro-
macs, then this system was solvated by water suing TIP4P water
model in a cubic box extending 10 Å around the receptor. In addi-
tion, the system was neutralized using sodium chloride and the
concentration was adjusted to 0.17 mM by genion (in Gromacs).
Long-range electrostatic interactions were treated using the parti-
cle-mesh Ewald method [16]. Periodic boundary conditions were
applied to avoid edge effects in all calculations. The temperature
was kept constant at 300 K by separately coupling the water, ions,
and protein in a thermal bath using the Berendsen thermostat
method [17] with a coupling time of 2 ps. Berendsen pressure cou-
pling was used for the equilibration of the systems. The solvated
system was underwent two energy minimizations with protein po-
sition constrained and none restrain at all. Following, energy min-
imized solvated system was equilibrated by 100 ps protein
position restrained NVT and NPT process at 300 K. Finally, a
10 ns NPT equilibration was conducted without restriction. After
10 ns equilibration, the final conformation was extracted and used
for docking.
2.1.3.2. Ligand preparation. Ligand was drawn using Maestro 9.0
[19] and prepared using LigPrep [20] application. MMFFs force-
field was chosen. Other parameters use default values.
2.1.3.3. Docking procedure. Molecular docking was performed using
Induced Fit Docking (IFD) [21] and Rigid Docking [22] modules in
Maestro 9.0 (SP mode). The ligand flexibility was considered in
both approaches. Grid box was centered on R752, R711, L704 and
W741, which was based on crystal structure of AR bound to DTH
(PDB ID: 2PIT) [12]. Since the binding site of AR is a very large bind-
ing cavity, we extend the outer box length to 24.6 Å (from 18.7 Å).
Flexible residues were defined using residues within 5.0 Å distance
from the reference ligand for the Induced Fit Docking method,
while for the Rigid Docking method, all residues were kept rigid.
Best pose of each molecule was extracted, and final result was
visualized using PyMol 1.3 [23] and LigPlot [24].
Fig. 1. Sequence alignment was performed in Modeller 9.10 and drawn with ESPript. The sequence of androgen receptor (PDB code: 2PIT) was shown on the upper side and
the sequence of glucocorticoid receptor (PDB code: 3H52) was shown downside.

18
H.-Y. Tian et al. / Chemico-Biological Interactions 207 (2014) 16–22
2.2. Chemistry
l(Cu-Ka
) = 0.601 mmꢁ1, 4.176 h 662.57, unique reflections = 3230,
R = 0.0580, S = 1.038, CCDC No. 898321.
2.2.1. General methods
ESI-MS spectra were carried out on a Finnigan LCQ Advantage
Max ion trap mass spectrometer. HR-ESI-MS data were obtained
on an Agilent 6210 ESI/TOF mass spectrometer. NMR spectra were
measured on Bruker AV-400 spectrometer. The solvents used in
column chromatography and HPLC were of analytical grade
(Shanghai Chemical Plant, Shanghai, P.R. China) and chromato-
graphic grade (Fisher Scientific, New Jersey, U.S.A.), respectively.
2.3. AR competitive binding assay
The fluorescence polarization (FP) technique was used to deter-
mine the binding affinity of compounds 1–3 using PolarScreenTM
Androgen Receptor Competitor Assays kit (Catalog # P3018) pur-
chased from Invitrogen [25]. Essentially, the protocol involved
titration of different concentration of competitive ligand against
the pre-formed complex of Fluormone AL green (2 nM) and the
AR-LBD (50 nM). The assay mixture was allowed to equilibrate at
20–25 °C in 384-well plates for 4 h, after which the polarization
values are measured at room temperature using the Perkin Elmer
EnVision Multilabel Reader. The excitation and emission wave-
length values for the Fluormone were 480 and 535 nM, respec-
tively. The data analysis for the ligand binding assays was done
using GraphPad Prism 5 software. The IC50 values were calculated
by the equation:
2.2.2. Synthesis of bufalin derivatives 1 and 2
To a solution of bufalin (50 mg) dissolved in 10 ml of dioxane,
35% hydrochloric acid solution (500 ll) was added, which was
sealed and then heated at 120 °C in an oil bath with a magnetic
stirrer for 4 h. The solution was neutralized with 27% ammonia
water, and evaporated under reduced pressure to give a residue,
which was subsequently partitioned between water and CHCl3.
The CHCl3 fraction was separated using preparative HPLC using
acetonitrile–water (60:40) as the mobile phase to give
1
Y ¼ mP100% þ ðmP0% ꢁ mP100%Þ=1 þ 10ððLogIC50 ꢁ XÞ ꢂ HillslopeÞ;
(20.8 mg, 48.4%) and 2 (3.0 mg, 6.9%).
D
8,14-Anhydrobufalin (1): Colorless crystals, 1H NMR (CDCl3,
where: Y = mP, X = Log [inhibitor], mP100% = 100% inhibition, and
mP0% = 0% inhibition [25].
400 MHz) d = 1.47 (2H, m, H-1), 1.57 (2H, m, H-2), 4.02 (1H, br s,
H-3), 1.39 (1H, m, H-4 ), 2.08 (1H, m, H-4b), 1.82 (1H, m, H-5),
1.23 (1H, m, H-6 ), 1.87 (1H, m, H-6b), 2.36 (1H, m, H-7 ), 1.88
(1H, m, H-7b), 2.40 (1H, m, H-9), 1.54 (1H, m, H-11 ), 1.45 (1H,
m, H-11b), 1.15 (1H, m, H-12 ), 1.61 (1H, m, H-12b), 1.88 (1H, m,
H-15 ), 2.36 (1H, m, H-15b), 1.88 (1H, m, H-16 ), 2.18 (1H, m, H-
a
a
a
2.4. Inhibition of Na+/K+-ATPase activity
a
a
The Na+/K+ ATPase inhibitory activities of these derivatives
were determined according to the reported method [26].
a
a
16b), 2.24 (1H, dd, J = 12.6, 6.9 Hz, H-17), 0.71 (3H, s, H-18), 0.83
(3H, s, H-19), 7.27 (1H, br s, H-21), 7.30 (1H, dd, J = 9.5, 2.6 Hz, H-
22), 6.29 (1H, br d, J = 9.5 Hz, H-23) ppm; 13C NMR (CDCl3,
100 MHz) d = 29.4 (C-1), 28.3 (C-2), 67.0 (C-3), 33.2 (C-4), 36.6 (C-
5), 26.8 (C-6), 25.3 (C-7), 129.3 (C-8), 35.7 (C-9), 37.0 (C-10), 19.5
(C-11), 43.4 (C-12), 49.5 (C-13), 138.6 (C-14), 25.3 (C-15), 24.7
(C-16), 51.8 (C-17), 18.7 (C-18), 24.1 (C-19), 118.4 (C-20), 148.8
(C-21), 145.3 (C-22), 115.4 (C-23), 162.1 (C-24) ppm.
2.5. Cytotoxicity of 1 against prostate cancer cells
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide] assay was done as described previously [27] with
taxol served as the positive control. Briefly, 3 ꢂ 103 cells per well
for PC3 cell line and 5 ꢂ 103 cells per well for LNCaP cell line were
plated into the 96-well plates, respectively and placed in the incu-
bator overnight using the standard culture conditions as reported
[28]. A series diluted compound 1 was added to each well. After
48 h of exposure, the cells were stained with MTT. Cell growth (via-
bility) was determined by measuring optical density at 570 nM
with a microplate reader (TECAN Spectra II Plate Reader, Research
Triangle Park, N.C.). The relative cell growth (%) was expressed as a
percentage relative to the untreated control cells. The experiments
were repeated twice, each in triplicate.
D
14,15-Anhydrobufalin (2): Colorless crystals, 1H NMR (CD3OD,
400 MHz) d = 4.02 (1H, br s, H-3), 5.28 (1H, m, H-15), 2.82 (1H,
dd, J = 10.6, 8.2 Hz, H-17), 0.75 (3H, s, H-18), 0.96 (3H, s, H-19),
7.54 (1H, br s, H-21), 7.58 (1H, dd, J = 9.6, 2.6 Hz, H-22), 6.32 (1H,
br d, J = 9.6 Hz, H-23); 13C NMR (CD3OD, 100 MHz) d = 30.8 (C-1),
28.6 (C-2), 67.7 (C-3), 34.2 (C-4), 36.4 (C-5), 25.1 (C-6), 27.5 (C-
7), 36.7 (C-8), 41.2 (C-9), 37.8 (C-10), 22.8 (C-11), 42.2 (C-12),
49.5 (C-13), 155.5 (C-14), 117.9 (C-15), 34.5 (C-16), 54.1 (C-17),
19.3 (C-18), 24.1 (C-19), 120.9 (C-20), 150.6 (C-21), 147.9 (C-22),
115.8 (C-23), 164.6 (C-24).
3. Results and discussion
2.2.3. X-ray crystallographic analyses of compound 1 and 2
Colorless crystals of both compounds 1 and 2 were obtained via
slow evaporation of a methanol solution. Data collections were
performed on an Agilent Gemini S Ultra CCD diffractometer. The
crystal structures were solved by direct methods using SHELXS-
97 and refined by full-matrix least-squares method on F2 using
SHELXTL v.510. Non-hydrogen atoms were subjected to aniso-
tropic refinement. Hydrogen atoms bonded to carbons were placed
at their idealized positions with assigned isotropic thermal param-
eters and included in the calculation of structure factors.
Androgens and anti-androgens bind to the Ligand binding do-
main (LBD) of AR in different manners. The former (e.g. DHT or
R1881) serves as agonists, and the binding is often accompanied
by activation of associated gene expression and many other related
biological responses [29,30]. In contrast, the latter serves as antag-
onists, and the binding often induces a conformational change of
AR LBD and leads to impairment or loss of functions [31].
D
8,14-Anhydrobufalin (1) and 14,15-anhydrobufalin (2) were
D
virtually modified from 3 through dehydration around the hydro-
xyl group at C-14. Because of the similar structures of 1–2 with
those of AR antagonists, e.g. VN/85-1 [10] and abiraterone [32],
we inferred that 1–2 might target the AR as antagonists. Thus a
three-dimensional structure describing the inactive conformation
of AR LBD was required. However, currently, the crystal structure
of steroidal antagonist-AR LBD was not available. So a homology
model of antagonist complex AR was constructed based on the
known agonist complex of androgen receptor and antagonist
Compound 1, C24H32O3ꢀH2O, M = 386.51, Orthorhombic,
space group P2(1)2(1)2(1), a = 7.50080(10), b = 10.4630(2),
c = 26.5100(4)Å, V = 2080.53(6)Å3, Z = 4, Dc = 1.279 Mg/m3,
F(000) = 840,
l(Cu-Ka , 3.336 h 6 62.67, unique
) = 0.653 mmꢁ1
reflections = 2867, R = 0.0439, S = 1.055, CCDC No. 898322.
Compound 2, C24H32O3, M = 368.50, Orthorhombic, space group
P2(1)2(1)2(1), a = 8.46030(10), b = 12.7309(2), c = 19.1062(3)Å,
V = 2057.88(5)Å3,
Z = 4,
Dc = 1.189 Mg/m3,
F(000) = 800,

H.-Y. Tian et al. / Chemico-Biological Interactions 207 (2014) 16–22
19
complex of human glucocorticoid receptor which was a close re-
lated hormone receptor.
Bufalin (3) and its unsaturated derivatives,
lin (1) and D
D
8,14-anhydrobufa-
14,15-anhydrobufalin (2) were subsequently docked
In order to refine our initial model and obtain a more stable
structure in the solvated environment, a 10 ns molecular dynamics
simulation was performed [33,34]. The relative structural drift was
measured by recording the RMSD of all heavy atoms versus the
simulation time. It was found that the protein structure drifted
rapidly from the initial structure within the first 1 ns, and still fluc-
tuated until 4.2 ns (Fig. S1). Then the RMSD value stabilized around
1.5 Å, indicating that the structure was relatively stable and 10 ns
simulation was sufficient for stabilizing a fully relaxed model.
Superimposing the refined structure with initial structure, we
found that the H12 Helix was most flexible which was consistent
with the literature [35] and other characteristic substructures,
such as H3, H7 and H10 helix were relatively stable during the sim-
ulation (Fig. S2). The average RMSD value between two structures
reads 1.43 Å. Considering the improved stability and similarity to
the initial structure, the structure acquired from MD simulation
was used for docking experiments.
into the LBD of the modeled AR (Fig. 2) using an Induced Fit Dock-
ing methods. Compounds 1–3 adopted obvious different poses
when penetrated into the binding pocket. In the complex of com-
pound 1 with AR, the lactone ring was close to Arg 752 (R 752)
and Gln 711 (Q 711), generating a bifurcated H-bond from 24-car-
bonyl group to both of the two amino acid residues. Besides, the
hydroxyl group at C-3 served as a hydrogen donor to form the third
hydrogen bond with Thr 877 (T877), which was believed to play a
role in both ligand binding and receptor activation [36]. Compound
2 was in the same direction as 1 when inserted into the pocket, but
the hydrogen bond with Arg 752 was lost. In contrast, compound 3
took an absolutely opposite gesture in the AR pocket. The carbonyl
group at C-24 was near to Thr 877 (T 877), but no hydrogen bond
was formed between the hydroxyl group at C-3 of the ligand and
the receptor. Furthermore, close examination of the ligand confor-
mations in the bound conformations obtained by docking revealed
that compound 1 was more bended to form an arched shape than
the other two compounds, which is favorable for the AR LBD. It was
observed that 1 was more tightly wrapped up by a hydrophobic
pocket composed of I899, M780, F764, L704, M745, M749, V746,
L873, W741 and F876, thereby increasing the hydrophobic con-
tacts with the LBD (Fig. 3). In addition, we also used the rigid dock-
ing method to compare the binding patterns of 1–3 with AR, which
showed the same tendency for those compounds as the Induced Fit
Docking method (Table S1, Supporting information). The binding
poses of 1 obtained from rigid and flexible docking also share sim-
ilar binding pattern (Fig. S3, Supporting information). However, it
is noteworthy that the flexible docking protocol did improve the
prediction theoretical binding affinity (for example, the 3-OH in
the ligand formed hydrogen bond with T877 of AR). These above
results suggested 1 could be a potential inhibitor of androgen
receptor.
In order to validate the results of molecular docking, we initi-
ated an experiment to synthesize the derivatives 1 and 2. A diox-
ane solution of bufalin was heated under an acid condition (HCl)
to yield two major peaks, which were subsequently separated by
preparative HPLC (Fig. 4). The structures of the two peaks were
elucidated to be
D D
8,14-anhydrobufalin (1) and 14,15-anhydrobuf-
alin (2) based on their HR-ESI-MS and NMR data. The structures
of these two compounds were reported before [37]; however,
only 1H NMR data was available and the stereochemistry was
unknown. In the present study, the full assignments of the
NMR data of 1 and 2 were achieved by extensive 1D and 2D
NMR analysis. Furthermore, the complete structure and stereo-
chemistry of 1 and 2 were confirmed by single-crystal X-ray
analysis (Fig. 5).
The relative affinity of compounds 1, 2 and 3 on AR were eval-
uated by a Polar Screen Androgen Receptor Competitor Assay Kit
[25]. Compound 1 showed the highest relative affinity with the
IC50 value of 1.9
lM, which was comparable to the positive control
(progesterone IC50 value of 2.40
lM), indicating that it competed
with the high affinity AR ligand (Fluormone™ AL Green). In con-
trast, compounds 2 and 3 were nearly inactive on AR with IC50 va-
lue larger than 50 lM. The dose-dependent curves for 1–3 were
shown in Fig. 6. The relative binding affinities of 1–3 were consis-
tent with the geometrical complementarities and stabilizing inter-
actions in the binding pose as predicted by molecular docking
analysis. It is noteworthy that though experimental IC50 values
indicated a much higher relative binding affinity for compound 1
than for 2 and 3, the docking scores (Table S1, supporting informa-
tion) did not differentiate the relative affinity of the three com-
pounds very much, which confirmed that the scoring functions of
the docking programs might not make a definite prediction of li-
gand’s relative binding affinity [38].
Fig. 2. Binding mode of compounds 1 (A), 2 (B) and 3 (C) in AR: dashed lines
represent hydrogen bonds.

20
H.-Y. Tian et al. / Chemico-Biological Interactions 207 (2014) 16–22
Fig. 3. Hydrophobic interactions of compound 1 with AR.
Fig. 4. HPLC chromatogram of bufalin derivatives 1 and 2.
The active 1 was further tested for the cytotoxic activities on the
value of 5.6 lM than bufalin (3, IC50 value of 0.022 lM), indicating
androgen dependent prostate cancer cells LNCaP and androgen
independent cells PC3 with taxol served as the positive control. It
was found that 1 showed more potent inhibitory activity against
two hundred fold decrease of inhibition on Na+/K+ ATPase.
In summary, the unsaturated derivatives of bufalin were virtu-
ally constructed and docked into the ligand binding domain of AR
using an flexible method (both the ligand and receptor were trea-
ted flexible), which suggested that the derivative 1 with a double
bond between C8 and C14 showed strong hydrogen bonding and
hydrophobic interactions with AR. Subsequently, the active
derivative was synthesized and the relative AR binding activity
LNCaP cells with an IC50 value of 6.8
lM than PC3 cells with an
IC50 value of 16.4 M. The dose-dependent curves of compound 1
l
on cell growth was shown in Fig. 7.
The inhibitory activity of 1 on Na+/K+ ATPase was also explored.
It was found that 1 showed much weaker inhibition with an IC50

H.-Y. Tian et al. / Chemico-Biological Interactions 207 (2014) 16–22
21
Fig. 7. The dose response curve of compound 1 by plotting the cell growth vs series
concentrations. PC3 cancer cells (3 ꢂ 103 cells per well) and LNCaP cancer cells
(5 ꢂ 103 cells per well) were plated into the 96-well plates, respectively and placed
in the incubator overnight. A series diluted compound 1 was added to each well.
After 48 h of exposure, the cells were stained with MTT. The relative cell growth (%)
was determined by measuring optical density at 570 nM with a microplate reader,
and was expressed as a percentage relative to the untreated control cells.
a cardiac steroid-like antagonist through combined approaches of
molecular docking and validation by chemical synthesis and bioas-
says. Further work is warranted to investigate compound 1’s ef-
fects on AR-mediated functional gene expression.
Conflict of interest
None declared.
Fig. 5. Crystal structures of compounds 1 and 2.
Acknowledgements
This work is dedicated to Professor James Trotter in celebration
of his 80th birthday, and was financially supported by the Guang-
dong High Level Talent Scheme to R.W.J., the Fundamental Re-
search Funds for the Central Universities (11612603), National
Natural Science Foundation of China (81102518), and Postdoctoral
Granted Financial Support (20110490915).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cbi.2013.10.020.
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[39,40], to the best of our knowledge, it is the first time to discover

22
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