Design and synthesis of androgen receptor antagonists with bulky side chains for overcoming antiandrogen resistance

Article abstract of DOI:10.1021/jm801218k

Incorporation of curcumin and β-ionone into one chemical entity led to identification of a novel antiandrogen with two bulky side chains, 6, which is a pure antagonist of the wild-type and the T877A, W741C, and H874Y mutated androgen receptors (AR), showi

Full text of DOI:10.1021/jm801218k

5546 J. Med. Chem. 2009, 52, 5546–5550
DOI: 10.1021/jm801218k
Design and Synthesis of Androgen Receptor Antagonists with Bulky Side Chains
for Overcoming Antiandrogen Resistance
Jinming Zhou,†,‡ Guoyan Geng,†,‡ Qingwen Shi,‡,§ Francoise Sauriol, and Jian Hui Wu*,†,‡,^
‡
†Montreal Centre for Experimental Therapeutics in Cancer, Segal Cancer Center and Lady Davis Institute for Medical Research,
Sir Mortimer B. Davis-Jewish General Hospital, McGill University, 3755 Cote-Ste-Catherine, Road, Montreal, Quebec H3T 1E2, Canada,
§Hebei Medical University, Hebei, China, Queen’s University, Kingston, Ontario K7L 3N6, Canada, and ^Department of Oncology,
McGill University, 546 Pine Avenue W, Montreal, Quebec H2W 1S6, Canada
Received December 1, 2008
Incorporation of curcumin and β-ionone into one chemical entity led to identification of a novel
antiandrogen with two bulky side chains, 6, which is a pure antagonist of the wild-type and the T877A,
W741C, and H874Y mutated androgen receptors (AR), showing no cross-reactivity with progesterone
receptor and low micromolar cytotoxicity in LNCaP, PCa-2b, 22Rv1, and C4-2B prostate cancer cells.
Molecular modeling indicates 6 adopts a “Y”-shape conformation and forms multiple hydrogen bonds
with AR backbone.
Introduction
utilized for the design of estrogen receptor antagonists.6
However, several DHT-derived molecules bearing one bulky
chain surprisingly turned out to be potent agonists of the AR.7
In the present work, we report the design, synthesis, and
biological characterization of a novel class of antiandro-
gens with two bulky side chains. Our computational study
revealed that chemical compounds with two bulky side
chains could be obtained by incorporating two dietary
agents, β-ionone and curcumin, into one chemical entity.
The β-ionone is a phytochemical present in many fruits,
vegetables, and grains. It is found to exert in vitro antic-
arcinogenic and antitumor activities.8 Curcumin, the major
pigment in the dietary spice turmeric, is found to possess
diverse pharmacological effects including anti-inflamma-
tory, antioxidant, antiangiogenic, and anticancer activ-
ities.9 Several curcumin analogues possess antiandrogenic
activity.10 We hypothesize that the hybrid molecule of
β-ionone and curcumin could furnish a novel class of
antiandrogens (Scheme 1). Eleven compounds (1-11) were
synthesized. Among them, compound 6 shows low micro-
molar cytotoxicity in a panel of five prostate cancer cell lines
and potently suppress DHT-induced transactivation of the
WT and the T877A, W741C, and H874Y mutated ARs,
showing no cross-reactivity with human progesterone re-
ceptor (PR). AR fluorescence polarization assays indicate
compound 6 binds to the AR-LBD at the hormone-binding
pocket. Molecular modeling indicated that compound 6
forms multiple hydrogen bonds with the backbone of AR
and adopts a “Y” shape conformation.
The androgen receptor (ARa) is a critical mediator of
prostate cancer, even at the castration-resistant status.1 Once
prostate cancer becomes castration-resistant, hormonal ther-
apy with current antiandrogens is not effective. Mutation in
AR is an important mechanism that accounts for the devel-
opment of resistance to the current clinically used antiandro-
gens such as flutamide and bicalutamide.2 One particular
mutation is the T877A in the ligand-binding domain (LBD)
of AR, which actually results in paradoxical activation by
hydroxyflutamide, an active metabolite of antiandrogen flut-
amide.3 The W741C AR mutant is activated by antiandrogen
bicalutamide.4 Yoshida et al. have demonstrated the agonistic
effect of bicalutamide to a xenograft with the clinically
induced W741C mutated AR.5 Thus, in laboratory setting,
AR mutations, such as T877A and W741C, have turned the
growth-inhibitory effect of the current clinically used anti-
androgens into a growth-promoting effect. To develop novel
pan-antiandrogens effective against the wild type (WT) and
multiple clinically relevant mutant ARs represents an attrac-
tive strategy for circumventing antiandrogen resistance.
The helix-12 (H12) at the AR ligand-binding domain (AR-
LBD) plays a critical role in AR transactivation. On binding
of androgen, such as dihydrotestosterone (DHT), H12 is
repositioned to cover the hormone-binding pocket, forming
the activation function-2 surface. One way to antagonize AR
function is to design compounds bearing an extending bulky
arm to displace H12. Such a strategy has been successfully
Results
*To whom correspondence should be addressed. Phone: (514) 340-
8222. Fax: (514) 340-8717. E-mail: jian.h.wu@mcgill.ca. Address: Lady
Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish
General Hospital, 3755 Cote-Ste-Catherine, Road, Montreal, Quebec H3T
1E2, Canada.
Chemistry. Eleven β-ionone derivatives 1-11 (Tables 1
and 2) were synthesized as outlined in Scheme 2. Condensa-
tion of (E)-6-(2,6,6-trimethylcyclohex-1-enyl)hex-5-ene-2,4-
dione with aromatic aldehyde furnished compounds 1-5
(Table 1).11 Compounds 6-11 (Table 2) were synthesized via
a two-step procedure. Condensation of the second aromatic
a Abbreviations: AR, androgen receptor; Bic, bicalutamide; CS-FBS,
charcoal-stripped fetal bovine serum; DHT, dihydrotestosterone; FP,
fluorescence polarization; H12, helix-12; LBD, ligand-binding domain;
OHF, hydroxyflutamide; PR, progesterone receptor; WT, wild type.
r
pubs.acs.org/jmc
Published on Web 08/12/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5547
aldehyde with the middle methylene (-CO-CH2-CO-) was
accomplished by Knoevenagel condensation in methanol
with catalytic amount of piperidine (Scheme 2). Purifications
of the crude products were achieved by silica gel CC (elutant:
n-hexane and ethyl acetate). Some of the products were
further purified by preparative TLC.
We have tried to synthesize 6-9 by a one-pot reaction
starting from (E)-6-(2,6,6-trimethylcyclohex-1-enyl)hex-5-
ene-2,4-dione, with molar ratio of the dione and aromatic
aldehyde as 1:2.3, using a method similar to that for curcu-
min derivatives but B2O3 was taken out.11 This one-pot
reaction afforded small amount of the 1:2 condensation
product. Eventually, we have employed a two-step proce-
dure for the syntheses of 6-11, which involves condensation
with the first aromatic aldehyde to obtain the corresponding
diketone and condensation of the diketone with the second
aromatic aldehyde (Scheme 2). This two-step procedure
allows us to synthesize compounds bearing two different
R groups, such as 10 and 11.
The NMR analysis revealed 6 is a mixture of the cis and
trans isomers that interconvert at room temperature
(Scheme 3). For the cis isomer, the proton H20 has
NOE with H12, and for the trans isomer, the H20 has
NOE with H8. The ratio of the two isomers is about 3:2,
with the cis isomer more favorable. The substituted
C7dC8 and C12dC13 double bonds are both trans in the
two isomers (Tables S1 and S2, Supporting Information
(SI)). There is only one peak in the HPLC analysis of 6
(Table S3, SI).
Scheme 1. Design of the Hybrid of β-Ionone and Curcumin
Scheme 2. Syntheses of Compounds 1-5 and 6-11a
Table 1. Cytotoxicity of Compounds 1-5 in LNCaP and PC-3 Cells
a Reagent and conditions: (a) CH3ONa, ethyl acetate; (b) B2O3,
(nBuO)3B, nBuNH2; (c) HCl; (d) piperidine, CH3OH, room temp.
Scheme 3. Compound 6 Exists in Both the Cis and Trans
Isomers
a IC50 is the concentration of compounds which causes a 50%
inhibition as compared to the vehicle control (0.5% DMSO); b N.D.,
not determined.
Table 2. Cytotoxicity of Compounds 6-11 in Prostate Cancer Cell Lines
substituents
cellular IC50 (μM)a
Compound
R2
R3
R4
R5
R6
LNCaP
PCa 2b
22Rv1
C4-2B
PC-3
4.2
6
H
H
H
H
OCH3
OC2H5
H
OH
H
H
H
H
H
1.3
14.9
4.5
8.7
2.3
8.5
8.5
151
2.5
6.8
2.2
9.3
1.6
4.5
4.0
8.5
2.3
5.3
6.1
ND
7
OC2H5
OH
OH
H
14.1
6.7
8
H
OCH3
4.7
4.7
9
10
OCH3
15.3
8.1
ND b
29.5
ND
17.6
2.4
12.5
7.0
11
curcuminc
5.1
15.2
16.8
inactived
18.3
ND
β-ionone
a IC50 is the concentration of compounds which causes a 50% inhibition as compared to the vehicle control (0.5% DMSO). b ND, not determined.
cCurcumin was synthesized according to the literature.11 d Maximum concentration tested is 150 μM.

5548 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Zhou et al.
Figure 1. Growth inhibitory effects of compound 6, hydroxyflut-
amide (OHF), and bicalutamide (Bic) in the PCa 2b and 22Rv1 cells.
Figure 3. Competitive binding of compound 6 to the AR-LBD
evaluated by AR fluorescence polarization (FP) assay, using Polar-
Screen AR competitor assay kit (P3018, Invitrogen). Fluorescence
polarization was used as a read-out and normalized to that of DMSO
vehicle. Results are reported as average ( sd of experiments per-
formed in triplicate.
DHT Rescue of the Growth Suppression of LNCaP Cells by
Compound 6. LNCaP cells in phenol red-free RPMI 1640
medium supplemented with 10% charcoal-stripped FBS
(CS-FBS) were treated with DMSO vehicle, DHT, as well
as compound 6 at designated concentrations in the presence
of 0.1 or 1 nM DHT (Figure S1, SI). The 0.1 nM DHT-
stimulated growth of LNCaP cells was suppressed by com-
pound 6 in a dose-dependent manner (gray bars, Figure S1,
SI). Importantly, the growth suppression of LNCaP cells by
compound 6 at 0.1, 1, and 5 μM, but not 10 μM, was at least
partially reversed by increasing the DHT concentration to
1 nM (Figure S1, SI). In contrast, this DHT rescue of growth
suppression by compound 6 was not observed in the AR-
negative PC-3 cells (Figure S1, SI). These results indicate that
the cytotoxicity of compound 6 in LNCaP cells is at least in
part attributable to its antiandrogenic activity.
Competitive Binding Assay and Cross-Reactivity with
Other Steroid Receptors. The possible competitive binding
of compound 6 to the hormone-binding pocket of the rat
AR-LBD was evaluated by PolarScreen AR competitor
assay kit (P3018, Invitrogen). If the test compound binds
to AR-LBD, it will prevent the formation of the AR/tracer
complex, and the tracer will be free in solution. When the
tracer is free in solution, its rotational mobility is greater
than when bound to the receptor, resulting in a low fluores-
cence polarization value. We have controlled the assay for
minimal competition (DMSO vehicle), which has a max-
imum value of fluorescence polarization and for no receptor
(tracer only), which represents the minimum value of the
fluorescence polarization that can possibly be reached by a
competitor. The DHT was included as a positive control
(Figure 3). Compound 6 has reduced the fluorescence polar-
ization value in a dose-dependent manner, indicating it binds
to AR-LBD by competing with the tracer over the hormone-
binding pocket (Figure 3).
Figure 2. Effect of compound 6 at 0.1 μM (gray bar) and 1 μM
(black bar), OHF at 1 μM, and Bic at 1 μM on the transactivation of
WT and the T877A, W741C, and H874Y mutant ARs in the
presence (black and gray bars) and absence (white bars) of 0.1 nM
DHT. Plasmids expressing human ARs are transiently transfected
in PC-3 cells. The results are reported as mean ( sd. Relative
luciferase activity is standardized to the Renilla luciferase control
and normalized to the 0.1 nM DHT RLU, relative luciferase units.
Antiproliferative Activity of 1-11. The in vitro cytotoxicity
of 1-11 were evaluated by MTT assays in a panel of prostate
cancer cell lines, including LNCaP, MDA PCa 2b, 22Rv1,
C4-2B, and PC-3. The IC50 values were computed from cell
survival curves (Tables 1 and 2). The effect of hydroxyfluta-
mide, bicalutamide, and 6 in MDA PCa 2b and 22Rv1 cells
are shown in Figure 1. Among 1-11, compound 6 shows the
most potent cytotoxicity in LNCaP cells and is substantially
more potent than curcumin and β-ionone (Table 2). In
LNCaP cells, the para-hydroxyl substituent appears to be critical
for the cytotoxicity of 6 and removal of the meta-methoxy group
reduces the activity (compare 6,7,and8). Adding a second meta-
methoxy to 6 does not improve the activity (compare 9 and 6).
Cytotoxic activity of 10 in LNCaP, 22Rv1, and C4-2B is
comparable to that of 6, indicating R group at chain B could
be varied significantly (Table 2, Scheme 1).
Antiandrogenic Activity of Compound 6. To investigate
antiandrogenic activity of compound 6, AR-dependent re-
porter assays were performed in PC-3 cells using ARE-
driven MMTV-luc reporter and AR-expressing plasmids
(Figure 2). Antiandrogen hydroxyflutamide and bicaluta-
mide were included as the control. We have tested the effect
of compound 6 against the WT and the T877A, W741C, and
H874Y mutated ARs. In accordance with previous stu-
dies,3,4 our reporter assays revealed that in the absence of
DHT, the T877A and W741C mutated ARs were activated
by hydroxyflutamide and bicalutamide, respectively (white
bars, Figure 2b,c). In contrast, compound 6 is a nonagonist
for the WT and the W741C, T877A, and H874Y mutated
ARs (white bars, Figure 2). Further, compound 6 at 1 μM
demonstrates potent antiandrogenic activity in suppressing
DHT-induced transactions of the WT and the T877A and
W741C AR mutants, as well as modest antiandrogenic
activity against the H874Y mutant (black bars, Figure 2).
To evaluate the cross-reactivity of 6 with PR, we have
performed PR-dependent luciferase assays in PC-3 (Figure
S2, SI). Compound 6 at 0.1 and 1 μM is inactive in suppres-
sing 10 nM R5020-induced transactivation of the B form of
human PR, indicating 6 is not the antagonist of PR. In the
absence of R5020, compound 6 at 0.1 and 1 μM is incapable
of activating PR, indicating 6 is a nonagonist of PR. The
basal control revealed there is no detectable endogenous PR
in PC-3 cells (Figure S2, SI).
Molecular Modeling. Despite the fact that the crystal
structures of the T877A mutant in complex with hydroxy-
flutamide and the W741L mutant in complex with bicaluta-
mide are available, the H12 of both of the two complexes are

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5549
has been found in patients who were treated with flutamide
and eventually became refractory to the treatment.2 The
functional significance of the W741C mutation was demon-
strated by the bicalutamide-stimulated tumor growth of a
novel prostate xenograft model derived from a bicalutamide-
treated patient.5 It appears that aberrant AR activation due to
AR mutations is an important mechanism that accounts for
development of the resistance to the current clinically used
antiandrogens. Thepan-antagonistic property of compound6
against multiple AR mutants (Figure 2) is of clinically
significance as it would be harder for prostate tumor to
acquire resistance to an antiandrogen effective against
multiple mutated ARs. To date, reports of antiandrogens
capable of antagonizing multiple mutant ARs are limited.
McGinley et al. reported identification of bicalutamide
derivatives that show potent antiandrogenic activity against
the WT and the W741L and T877A mutated ARs.13 We have
recently identified two novel chalcones as potent pan-anti-
androgens.14,15
Figure 4. (A) Predicted binding mode of compound 6 in the
structural model of AR-LBD at the antagonistic form; and (B)
H12 at the agonistic form (orange ribbon), taken from the AR-
LBD/DHT complex (PDB entry: 1t65), was merged into the struc-
tural model, showing steric clash of chains A and B with the
agonistic H12. Compound 6 and side chains are colored according
to the atomic-coloring scheme (O in red, N in blue, C in cyan for 6
but in green for side chains). The vdW surfaces of V889, L884, and
L880 are in orange grid. Hydrogen bond is indicated by dash lines.
Both LNCaP and MDA PCa 2b cell lines are androgen-
dependent and express functional mutated ARs, with the
T877A mutated AR in LNCaP cells and the T877A and
L701H double mutated AR in PCa 2b cells. Both C4-2B
and PC-3 cells are androgen-independent, but C4-2B
cells express T877A mutated AR and PC-3 cells lack endo-
genous AR. Cellular growth of 22Rv1 cells, which express
H874Y mutated AR, are stimulated by DHT and EGF.
Therefore, these five prostate cancer cell lines constitute a
panel of diverse cellular models for prostate cancer. Signifi-
cantly, compound 6 shows low micromolar cytotoxicity in
LNCaP, PCa 2b, 22Rv1, C4-2B, and PC-3 (Table 2). The
following line of evidence suggests that the activity of com-
pound 6 in AR-positive prostate cell lines is at least in part
mediated via AR inhibition: the growth suppression of
LNCaP cells by compound 6 at 0.1, 1, and 5 μM was at least
partially reversed by increasing the DHT concentration to
1 nM, but at a higher dose of compound 6 (10 μM), the 1 nM
DHT can not rescue the growth suppression (Figure S1, SI).
In contrast, this DHT rescue of growth suppression by
compound 6 was not observed in the AR-negative PC-3 cells
(Figure S1, SI).
Molecular modeling suggested possible molecular basis for
the pan-antagonistic property of compound 6 (Figure 4, SI
Figures S3, S4). Analyses of the crystal structures of a series of
mutant AR-LBDs (PDB entries: 1z95, 2ax6, 2q7k, 1gs4, etc.)
indicates the backbone conformation of the mutated AR-
LBD remains essentially the same with the WT receptor.
Consequently, single point mutation can not easily break
the hydrogen bonds between 6 and the backbone of the
receptor (Figure 4, SI Figure S4) and compound 6 thus
remains as an antagonist in the AR mutants (Figure 2).
Indeed, the “backbone targeting strategy” has been success-
fully utilized in the development of inhibitors against HIV
protease mutants.16
at the agonist form (PDB entries: 2ax6 and 1z95). To date,
crystal structure of AR-LBD at the antagonist form has not
been obtained. To investigate molecular basis for the finding
that compound 6 remains as a pure antiandrogen in the WT
and multiple mutated ARs, we have built a structural model
of the WT AR-LBD with H12 at the antagonist form, using
crystal structures of the AR-LBD/DHT and ER/antagonist
complexes (PDB entries: 1t65 and 3ert) as templates (SI).
Figure 4A demonstrates the predicted binding mode of
compound 6 in the antagonistic model of WT AR-LBD.
To investigate possible steric clash between the bulky anti-
androgen 6 with the H12 at the agonist form, H12 from the
crystal structure of the agonistic AR-LBD/DHT complex
(PDB entry: 1t65) was merged with the antagonistic AR-
LBD model and shown in Figure 4B.
Inspection of the predicted binding mode indicated that
6 adopts a “Y” shape conformation, with the β-ionone core
anchoring inside the hormone-binding pocket and chains A
and B protruding toward the agonist form of H12 (orange
ribbon, Figure 4B). The para-hydroxyl in chain A of com-
pound 6 forms multiple hydrogen bonds with the backbones,
but not the side chains, of residues Q738 (at carbonyl
oxygen), W741 (at amide nitrogen), and M742 (at amide
nitrogen), whereas chain B forms a hydrogen bond with
E709 and interacts with a hydrophobic pocket formed by
L880, L881, and V889 (Figure 4). This is in consistent with
the finding that the para-hydroxyl in chain A is critical for the
cytotoxic activity of 6 in LNCaP cells (Table 2). Compounds
6 and 10, which demonstrate similar cytotoxicity in LNCaP
cells are predicted to have similar binding mode in chain A
(Figure S3, SI). In contrast, the para substituents of chain A
in 7, 9, and 11 do not form multiple hydrogen bonds with the
backbone of AR-LBD (Figure S3, SI). In addition, com-
pound 6 is predicted to have similar binding modes in the WT
and the T877A, W741C, and H874Y mutated ARs, forming
hydrogen bonds with the backbones of Q738 and M742, as
well as side chain of E709 (Figure 4 and SI Figure S4).
Conclusion
Compound 6 is a novel antiandrogen with two bulky side
chains, which is a pure antagonist of the WT and the clinically
relevant T877A, W741C, and H874Y mutated ARs and has
no cross-reactivity with PR. Molecular modeling indicates the
backbone-targeting characteristic and the “Y” shape confor-
mation, bearing two bulky side chains, are important for the
pure antagonistic activity of 6 in the multiple AR mutants.
Discussion
A series of AR mutations, such as T877A, H874Y, and
W741C, were identified from tissue specimens of patients with
castration-resistant prostate cancer. The incidence of AR
mutation in advanced prostate cancer is estimated to be in
the range of 10-40%.2,3,12 In particular, the T877A mutation

5550 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Zhou et al.
Further, 6 shows low micromolar cytotoxicity in AR-positive
LNCaP, PCa 2b, 22Rv1, and C4-2B cells. Its cytotoxicity in
AR-negative PC-3 cells suggests 6 is a multitarget agent. The
possible additional target(s) of 6 is not clear at this stage, and
this is the subject of our further work. Taken together, 6 is a
lead compound to be further optimized as a novel antiandro-
gen for advanced prostate cancer.
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Experimental Section
General. All reagents for chemical syntheses were purchased
from Sigma-Aldrich (Oakville, ON, Canada). Hydroxyflutamide,
bicalutamide and DHT were purchased from Toronto Research
Chemicals (North York, ON, Canada). R5020 was purchased
from PerkinElmer Inc. (Woodbridge, ON, Canada). All the 1H
NMR spectra of compounds 1-5 and 7-11 were recorded on an
Avance Bruker NMR spectrometer operating at 500 MHz on
proton. The NMR spectra of compound 6 were recorded on an
Avance Bruker NMR spectrometer operating at 600.17 MHz on
proton and 150.93 MHz on carbon-13. Mass measurements
were performed on a LC-MSD-TOF instrument from Agilent
Technologies in positive electrospray mode. Purity was deter-
mined by HPLC (Waters Alliance 2695-2996) and purity of
compound 1-11 was g95%.
General Procedure for the Synthesis of Compounds 6-11.
Diketone 1 (0.25 mmol, 92 mg) and 4-hydroxy-3-methoxyben-
zaldehyde (0.3 mmol, 47 mg) were dissolved in 6 mL of
methanol. After adding piperidine (25 μL), the mixture was
stirred for 48 h at room temperature. The solvent was distilled
off, and the crude product was purified by silica gel column
chromatography (elutant: EtOAc/hexane, 1:2) to afford com-
pound 6. Yellow solid (26 mg, 21%). ESI-TOF MS m/z 503.24
[M þ H]þ. The 1H and 13C NMR analyses for both cis and trans
isomers of compound 6 were summarized in Tables S1 and S2
(SI). Compounds 7-11 were synthesized by the same procedure
for compound 6.
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Biology. Details of antiproliferative assays in five prostate
cancer cell lines, DHT rescue of the growth suppression of
LNCaP cells by compound 6, luciferase reporter assays, and
AR fluorescence polarization assay are described in the Sup-
porting Information.
Acknowledgment. We thank Dr. Gerald Batist for his
insightful comments of the manuscript. We thank Dr.
Liang-Nian Song (Columbia University), Dr. S Srivastava
(Uniformed Services University), and Dr. Osmu Ogawa
(Kyoto University, Japan) for providing AR expressing
plasmids. This work was supported by the Canadian Institute
of Health Research via an operating grant to J.H.W. (grant
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Supporting Information Available: Syntheses of (E)-6-(2,6,6-
trimethylcyclohex-1-enyl)-hex-5-ene-2,4-dione and diketones
1-5, the predicted binding modes of compounds 6-11, NMR
and MS analyses, biological assays details, and molecular
modeling methods. This material is available free of charge via
the Internet at http://pubs.acs.org.