Synthesis and in vitro characterization of ionone-based compounds as dual inhibitors of the androgen receptor and NF-κB

Article abstract of DOI:10.1007/s10637-013-0040-y

Current therapeutic strategy for advanced prostate cancer is to suppress the androgen receptor (AR) signaling. However, lethal castration-resistant prostate cancer (CRPC) arises due to AR reactivation via multiple mechanisms, including mutations in the AR and cross-talk with other pathways such as NF-κB. We have previously identified two ionone-based antiandrogens (SC97 and SC245), which are full antagonists of the wild type and the clinically-relevant T877A, W741C and H874Y mutated ARs. Here, we discovered SC97 and SC245 also inhibit NF-κB. By synthesizing a series of derivatives of these two compounds, we have discovered a novel compound 3b that potently inhibits both AR and NF-κB signalling, including the AR F876L mutant. Compound 3b showed low micromolar antiproliferative activites in C4-2B and 22Rv1 cells, which express mutated ARs and are androgen-independent, as well as DU-145 and PC-3 cells, which exhibit constitutively activated NF-κB signalling. Our studies indicate 3b is effective against the CRPC cells.

Full text of DOI:10.1007/s10637-013-0040-y

Invest New Drugs (2014) 32:227–234
DOI 10.1007/s10637-013-0040-y
PRECLINICAL STUDIES
Synthesis and in vitro characterization of ionone-based
compounds as dual inhibitors of the androgen
receptor and NF-κB
Weiguo Liu & Jinming Zhou & Guoyan Geng &
Rongtuan Lin & Jian Hui Wu
Received: 4 September 2013 /Accepted: 8 October 2013 /Published online: 23 October 2013
#
Springer Science+Business Media New York 2013
Summary Current therapeutic strategy for advanced prostate
cancer is to suppress the androgen receptor (AR) signaling.
However, lethal castration-resistant prostate cancer (CRPC)
arises due to AR reactivation via multiple mechanisms,
including mutations in the AR and cross-talk with other
pathways such as NF-κB. We have previously identified two
ionone-based antiandrogens (SC97 and SC245), which are full
antagonists of the wild type and the clinically-relevant T877A,
W741C and H874Y mutated ARs. Here, we discovered SC97
and SC245 also inhibit NF-κB. By synthesizing a series of
derivatives of these two compounds, we have discovered a
novel compound 3b that potently inhibits both AR and
NF-κB signalling, including the AR F876L mutant. Compound
3b showed low micromolar antiproliferative activites in C4-2B
and 22Rv1 cells, which express mutated ARs and are
androgen-independent, as well as DU-145 and PC-3 cells,
which exhibit constitutively activated NF-κB signalling. Our
studies indicate 3b is effective against the CRPC cells.
Abbreviations
AR
Bic
Androgen receptor
Bicalutamide
CRPC Castration-resistant prostate cancer
DHT
WT
Di-hydrotestosterone
Wild type
Introduction
Proliferation and survival of prostate cancer cells are critically
dependent on the androgen receptor (AR) signaling. This
provides the rationale for androgen ablation therapy, which
aims to suppress the AR signaling by reducing level of
androgens (via castration) and antagonizing the AR by
antiandrogens, such as flutamide, nilutamide and bicalutamide
[1]. However, lethal castration-resistant prostate cancer
(CRPC) arises due to AR reactivation. Current treatment
modalities for patients with established CRPC are limited to
docetaxel-based chemotherapy, with the median survival time
<2 years [2, 3]. Enzalutamide (MDV3100) and abiraterone
acetate were recently approved by FDA for treating CRPC
patients. Enzalutamide is a second-generation antiandrogen
that acts as a full antagonist even under elevated level of the
AR. Abiraterone acetate is an oral 17α-hydroxylase inhibitor
that blocks steroid biosynthesis in the adrenal gland and
possibly within the tumor [4]. Clinical data from phase I/II
and III clinical trials of both Abiraterone and enzalutamide
have reported a high level of antitumor activity in CRPC
patients, revealing that AR signaling is a driver of the CRPC
cells [5–7]. It has been established that, in CRPC cells, AR is
activated by multiple mechanisms that can no longer be
effectively suppressed by castration and conventional
antiandrogen agents, such as bicalutamide [6, 8]. Mutations
.
.
Keywords Prostate cancer Androgen receptor
.
.
Antiandrogen NF-κB IKKβ
Electronic supplementary material The online version of this article
(doi:10.1007/s10637-013-0040-y) contains supplementary material,
which is available to authorized users.
:
:
:
:
W. Liu J. Zhou G. Geng R. Lin J. H. Wu
Segal Cancer Center, Lady Davis Institute for Medical Research,
Sir Mortimer B. Davis-Jewish General Hospital, 3755
Cote-Ste-Catherine, Rd., Montreal, QC H3T 1E2, Canada
:
R. Lin J. H. Wu (*)
Departments of Oncology and Medicine,
McGill University, 546 Pine Ave. W, Montreal,
QC H2W 1S6, Canada
e-mail: jian.h.wu@mcgill.ca

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Invest New Drugs (2014) 32:227–234
in the AR ligand-binding domain and crosstalk of the AR with
other deregulated pathways (such as NF-κB) are two
important mechanisms that may account for persistent AR
activation in the CRPC cells [9–11].
whereas chains A and B are protruding toward the helix-12
of the AR ligand-binding domain (Scheme 1) [27]. We have
synthesized a series of ionone-based compounds with
chemical modifications at side chains [27–29]. In the present
work, we discovered SC97 and SC245 also inhibit NF-κB
activation. To optimize SC97 and SC245, we have
synthesized a series of their derivatives with modifications at
ionone cores. Our work has led to discovery of a novel
compound 3b that potently inhibits both AR and NF-κB
signaling.
The incidence of AR mutation in advanced prostate cancer
is estimated to be in the range of 10−40% [9, 12]. The T877A
and H874Y mutations are two most commonly identified
variants in tissue specimens of patients with advanced prostate
cancer. The T877A and H874Y mutated ARs were reported to
be activated by antiandrogen hydroxyflutamide, androgens
and a series of other sex steroids [11]. The T877A mutant is
activated by nilutamide, another clinically used antiandrogen
[13]. The W741C mutation was derived from liver metastatic
tissue of a patient treated with bicalutamide and died of
CRPC. Yoshida et al. have demonstrated that bicalutamide is
paradoxically an agonist of the W741C mutant [10]. Thus, it
appears the AR mutations could contribute to aberrant AR
activation in the CRPC cells. Importantly, the AR F876L
mutant was recently found to confer resistance to
enzalutamide [14, 15].
The NF-κB plays a key role in inflammation, immune
response, cell proliferation and protection against apoptosis
[16]. Constitutive NF-κB activity is common in solid tumors
[17], including metastatic prostate cancer [18]. Androgen-
independent prostate cancer cell lines PC-3 and DU145
exhibit a high constitutive activation of IκB kinase (IKKβ)
and NF-κB signaling [19]. IKKβ is critical for the NF-κB
activation and a series of IKKβ inhibitors are currently in
preclinical development for various cancers [20].
Accumulating evidence indicate there are important crosstalk
between AR signaling and NF-κB pathway: i) NF-κB/p65
induces AR expression. Forced over-expression of NK-κB
increased sensitivity of AR-positive prostate cancer cells to
low concentrations of androgen, enhancing their growth and
survival [21]; ii) The NF-κB-regulated IL-6 stimulates growth
of prostate cancer in vitro and in vivo through activation of the
AR [22]. Further, IL-6 regulates androgen synthesis in
prostate cancer cells [23], and increases prostate cancer cells’
resistance to bicalutamide [24]. Increased serum levels of IL-6
have been reported in patients with CRPC [25]. It was
demonstrated that the NF-κB pathway controls the
progression of prostate cancer to androgen-independent
growth in vitro and in vivo [26]. Therefore, IKKβ/NF-κB/
IL-6 axis is likely contributing to aberrant activation of the AR
in CRPC cells.
Materials and methods
General All reagents for chemical syntheses were purchased
from Sigma-Aldrich (Oakville, ON, Canada). Bicalutamide
and enzalutamide were purchased from Toronto Research
1
Chemicals (North York, ON, Canada). All of the H NMR
spectra were recorded on a Bruker Avance NMR spectrometer
operating at 500 MHz on proton. Mass measurements
were performed on a LC-MSD-TOF instrument from
Agilent technologies in positive electrospray mode. Purity
was determined by HPLC (Waters Alliance 2695–2996)
and the purities of compounds 3a-3d and 4a-4f were
≥ 95 %.
Luciferase assays for AR activation Point mutation of human
AR (F876L) was introduced in pCMV-AR plasmid using
Quickchange mutagenesis according to the manufacturer’s
instructions (Stratagene) (referred to as pCMV-AR-F876L).
Plasmid pCMV-AR-W741C was kindly provided by Dr
Osmu Ogawa (Kyoto University, Kyoto, Japan). PC-3 cells
was seeded at a density of 1×105 cells per well in 24-well
microtiter plates and 24 h later, the cells were cotransfected
with PSA-luc and pCMV-AR-W741C or pCMV-AR-F876L
expressing plasmid and Renilla null luciferase using
LipofectamineTM 2000 reagent (Invitrogen) following
manufacturer’s protocol. Five hours after transfection, the
medium was changed to phenol red-free RPMI 1640
supplemented with 10 % charcoal-stripped FBS and 16 h later,
the cells were exposed to DMSO vehicle, 10 nM DHT or
compounds in the presence 10 nM DHT. After further 24 h,
the reporter gene activities were measured by dual-luciferase
reporter assay, according to the manufacturer’s instructions
(Promega). All experiments were performed in triplicate and
repeated at least twice.
Our previous work has identified two novel ionone-based
antiandrogens (referred to as SC97 and SC245), which are full
antagonists of the wild type and the T877A, W741C and
H874Y mutated ARs (Scheme 1) [27, 28]. Using PolarScreen
AR competitor assay kit (P3018, Invitrogen), we showed that
SC97 binds with the AR ligand-binding domain. Our
molecular modeling studies indicated that the ionone portion
of SC97 is anchoring inside the hormone binding pocket,
Luciferase assays for NF-κB activation Transfections for
luciferase assay were carried out in HEK293 cells.
Subconfluent HEK293 cells seeded on 24-well plates were
transiently co-transfected with 30 ng of pRLTK reporter
(Renilla luciferase for internal control), 100 ng of NF-κB-
Luc reporter (firefly luciferase, experimental reporter) and

Invest New Drugs (2014) 32:227–234
229
General procedure for the synthesis of 3a−3d After the
n
mixture of di-ketone (2a−2d) (6.4 mmol) and Bu3BO3
(3.08 mL, 11.4 mmol) in 50 mL of anhydrous ethanol was
stirred at 40°C for 0.5 h, 4-hydroxy-3-methoxycyclo-
hexanecarbaldehyde (2.4 g, 15.4 mmol) was added into the
n
mixture and stirred for 10 min. Next, BuNH2 (278 μL,
2.82 μmol) was added into the mixture. The mixture reacted
at room temperature for 4 days. The reaction was monitored
by TLC. The solvent was distilled off under reduced pressure
when the reaction completed. Ethyl acetate (30 mL) and 10 %
HCl (aq) (8 mL) were added and stirred for 0.5 h at 60 °C.
Saturated brine (30 ml) was added to the above mixture. The
aqueous phase was extracted with ethyl acetate (20 mL×3).
The combined organic phase was dried over Na2SO4. The
organic solvent was distilled off under reduced pressure after
filtering out the solid. The residue was purified through
chromatography on silica gel to give 3a−3d (elutant: n-
hexane and ethyl acetate).
Scheme 1 Chemical structures of ionone-based antiandrogens SC97 and
SC245 and the diagram showing the two bulky sidechains of SC97
100 ng of plasmid encoding IKKβ by calcium phosphate co-
precipitation method. The total amounts of DNA were kept
constant by supplementation with an empty vector (pcDNA3.1).
At 24 h after transfection, the reporter gene activities were
measured by dual-luciferase reporter assay, according to the
manufacturer’s instructions (Promega).
3a (Yellow solid, 19 % in yield), 1H NMR (500 MHz,
CD3COCD3): δ 8.00 (1H, s), 7.79 (1H, d, J =15.5 Hz),
7.66 (1H, d, J =16.1 Hz), 7.54 (1H, m), 7.53 (1H, m),
7.46 (1H, d, J =2.0 Hz), 7.42 (1H, d, J =1.5 Hz), 7.05
(1H, d, J =9.3 Hz), 6.84 (1H, d, J =8.3 Hz), 6.75 (1H, td,
J =10.0, 15.5 Hz), 6.41 (1H, d, J =16.0 Hz), 5.61 (1H, s),
4.09 (3H, s), 3.99 (3H, s), 2.57 (1H, d, J =10.0Hz), 1.43–
1.85 (4H, m), 1.14 (3H, s), 1.06 (3H, s), 1.01 (3H, s). ESI-
TOF-MS, m/z: 503.24 [M + H]+.
3b (Yellow solid, 17 %), 1H NMR (500 MHz,
CD3COCD3): δ 7.91 (1H, s), 7.69 (1H, d, J =15.5 Hz),
7.41 (1H, d, J =16.0 Hz), 7.38 (1H, m), 7.26 (1H, m), 7.22
(1H, d, J =2.0 Hz), 7.14 (1H, d, J =1.5 Hz), 7.06 (1H, d, J =
9.0 Hz), 6.94 (1H, d, J =8.5 Hz), 6.55 (1H, d, J =15.5 Hz),
6.23 (1H, d, J =16.0 Hz),3.99 (1H, s), 3.96 (3H, s), 3.87
(3H, s), 1.44–1.85 (4H, m), 1.75 (3H, s), 1.07 (3H, s), 0.96
(3H, s). ESI-TOF-MS, m/z: 519.24 [M + H]+.
3c (Yellow solid, 23 % in yield), 1H NMR (500 MHz,
CD3COCD3): δ 7.85 (1H, s), 7.82 (1H, d, J =15.5 Hz),
7.71 (1H, d, J =16.0 Hz), 7.61 (1H, m), 7.59 (1H, m), 7.52
(1H, d, J =16.0 Hz), 7.44 (1H, d, J =1.5 Hz), 7.35 (1H, d,
J =9.5 Hz), 7.21 (1H, d, J =2.0 Hz), 6.89 (1H, d, J =
8.5 Hz), 6.75 (1H, d, J =15.5 Hz), 5.21 (1H, s), 3.99 (3H,
s), 3.91 (3H, s), 1.61 (3H, s), 1.43–1.84 (4H, m), 1.11 (3H,
s), 0.98 (3H, s). ESI-TOF-MS, m/z: 519.24 [M + H]+.
3d (Yellow solid, 18.5 % in yield), 1H NMR (500 MHz,
CD3COCD3): δ 7.96 (1H, s), 7.67 (1H, d, J =15.5 Hz),
7.56 (1H, d, J =16.1 Hz), 7.41 (1H, d, J =2.0 Hz), 7.32
(1H, d, J =1.5 Hz), 7.23 (1H, m), 7.16 (1H, m), 7.06 (1H,
d, J =8.5 Hz), 6.85 (1H, d, J =8.5 Hz), 6.75 (1H, d, J =
15.5 Hz), 6.38 (1H, d, J =16.0 Hz), 3.96 (3H, s), 3.87
(3H, s), 1.43–1.85 (6H, m), 1.16 (3H, s), 1.02 (3H, s),
0.97 (3H, s). ESI-TOF-MS, m/z: 519.24 [M + H]+.
Quantification of IL-6 protein concentration DU145 cells
were exposed to DMSO vehicle and compounds at designated
doses for 24 h. The IL-6 level of cell culture supernates was
determined using an enzyme-linked immunosorbent assay
(ELISA) kit according to the manufacture’s protocol (R&D
Systems, Inc., Minneapolis, MN, USA). Each sample was
tested in duplicate. After development of the colorimetric
reaction, the absorbance at 450 nm was quantitated by means
of a microplate reader. The absorbance readings were then
converted to picograms per milliliter (pg/mL) based on
standard curves obtained with recombinant cytokines. The
lower limit of sensitivity of this kit was 3.1 pg/mL.
Western blot analysis HEK293 cells were pretreated with
compounds for 12 h, then 10 ng/mLTNFα for 30 min. LNCaP
cells in RPMI 1640 medium supplemented with 10 % FBS
were exposed to DMSO vehicle or test compounds at the
designated concentrations for 24 h. Additional details were
provided in Supporting Materials.
MTT assays The growth inhibition assay was performed as
described [27]. Briefly, LNCaP, C4-2B, 22Rv1, PC-3 and
Du145 are maintained in RPMI 1640 supplemented with
10 % FBS. Cells were seeded at a density of 6–7×103 cells
per well in 96-well plates. After overnight incubation, cells in
fresh RPMI 1640 supplemented with 10 % FBS were exposed
to DMSO vehicle control (0.5 %) and test compounds at
designated concentrations for 72 h. Viable cells were
evaluated by MTT assays. Experiments were performed in
triplicate and repeated at least twice.

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Starting material α-ionone (1a) and β-ionone (1c) are
commercially available (Sigma-Aldrich). Procedures for
preparation of the intermediates 1b, 1d and 2a−2d as well
as preparation of compounds 4a−4f were reported in the
Supplementary Materials.
Results
1) SC97 and SC245 inhibit NF-κB activation and suppress
IL-6 secretion
To evaluate effect of SC97 and SC245 on the NF-κB
signaling, HEk293 cells were transiently transfected with
NF-κB-luc and IKKβ-expressing plasmid or pcDNA3.1
empty vector. Transfected cells were exposed to SC97
and SC245 at designated doses for 24 h. The study
indicated that SC97 and SC245 inhibit NF-κB activation
in a dose-dependent manner (Fig. 1a). By Western blot
analysis in HEK293 cells, we further showed that SC97,
like PS-1145 (a known IKKβ inhibitor), suppresses
TNFα-induced degradation of the IκBα protein, a key
substrate for IKKβ (Fig. 1b), indicating SC97 is an
inhibitor of IKKβ. In particular, SC97 at 5 μM has
substantially reduced IKKβ protein expression
(Fig. 1b). By ELISA assays, we showed that SC97 and
SC2454 dose-dependently suppress IL-6 secretion of
DU145 prostate cancer cells (Fig. 1c).
2) Synthesis of novel derivatives of SC97 and SC245
To explore ionone core, we have synthesized four
SC97 derivatives 3a−3d and six SC245 derivatives
4a−4f (Schemes 2, 3 and 4). Compounds 3a−3d were
synthesized as outlined in Scheme 2. Condensation of
(E)-6-(2,6,6-trimethylcyclohex-2-enyl)hex-5-ene-2,4-
dione (2a) with 4-hydroxy-3-methoxybenz aldehyde
furnished compounds 3a. Compounds 4a−4c were
synthesized according to Scheme 3, and 4d−4f were
synthesized according to Scheme 4.
Fig. 1 a Compounds SC97 and SC245 dose-dependently suppress NF-ĸB
activation. HEK293 cells were transiently transfected with NF-ĸB-luc,
IKKβ-expressing plasmids (black block) or pcDNA3.1 vector (white
block) and pRL-TK. Fold of induction related to the cells that were
transfected with pcDNA3.1 vector and exposed to DMSO vehicle was
shown; b Western blot analysis indicates that SC97 and PS1145 suppress
TNFα-induced degradation of the IκBα protein in HEK293 cells. HEK293
cells were pretreated with compounds for 12 h, then 10 ng/mL TNFα for
30 min; c SC97 and SC245 suppress IL-6 secretion of DU-145 prostate
cancer cells. The IL-6 level of cell culture supernates was determined using
ELISA kit (R&D Systems, Inc., MN, USA)
3) Compound 3b potently inhibits NF-kB and AR signaling
To evaluate effect of compounds 3a−3d and 4a−4f on
the NF-κB signaling, HEK293 cells were transiently
transfected with NF-κB-luc and IKKβ-expressing
plasmid. Transfected cells were exposed to the
compounds at designated concentrations for 24 h. As
shown in Fig. 2, Compounds 3c and 3d at 5 μM are
more potent than SC97 in suppressing NF-κB activation,
while 3b is comparable to that of SC97. Among SC245
analogues 4a−4f, compound 4e is more potent than
SC245 (Fig. 2).
bicalutamide (Bic) was included as a positive control
(Fig. 3). As indicated by the fold of suppression,
compound 3b at 2.5 μM is much more potent than SC97
in suppressing DHT-induced AR activation, whereas
compounds 4d and 4e are more potent than SC245
(Fig. 3). These studies indicated that introduction of a
hydroxyl group to position 4 of the ionone ring of SC97
(Scheme 1) has substantially increased its anti-androgenic
activity, while such modification only has minor impact on
the inhibitory activity against the NF-κB signaling
(compare 3b and SC97 in Figs. 2 and 3). By AR-
dependent reporter assay in PC3 cells, we showed that
3b significantly suppresses DHT-induced activation of
the W741C and F876L mutated ARs (Fig. 4).
To study effect of compounds 3a−3d and 4a−4f on the
AR signaling, LNCaP cells were transiently transfected
with PSA-luc reporter and were exposed to DMSO vehicle
control, 1 nM DHT or compounds at designated
concentrations in the presence of 1 nM DHT. Antiandrogen

Invest New Drugs (2014) 32:227–234
231
Scheme 2 Synthesis of
compounds 3a–3d and their
intermediates: i) CH3ONa, ethyl
acetate, 380C; ii) n-Bu3BO3, n-
BuNH2, room temperature (R.T.),
4 days; and iii) MCPBA, CH2Cl2,
R.T.
4) SC97 and Compound 3b suppressed PSA expression and
induced apoptosis in LNCaP cells
LNCaP and C4-2B prostate cancer cells express
endogenous T877A mutated AR. LNCaP cells are
androgen-dependent and C4-2B cells are androgen-
independent. The 22Rv1 cells are AR-positive and
androgen-independent, whereas PC-3 and DU-145 cells
are AR-negative and androgen-independent. The
cytotoxicity effect of compounds 3a−3d and 4a−4f in a
panel of prostate cancer cell lines, including LNCaP, C4-
2B, 22Rv1, PC-3 and DU-145, were evaluated by MTT
assay (Table 1). Among the SC97 analogues 3a−3d,
compound 3b showed potent cytotoxicity in LNCaP,
C4-2B and 22Rv1 cells, with IC50 values in the range of
1.2–3.8 μM (Table 1). Among the SC245 analogues 4a
−4f, compound 4e showed low micromolar activities in
LNCaP, C4-2B and 22Rv1 cells (Table 1).
To determine whether SC97 and compound 3b
suppress PSA expression and induce apoptosis in LNCaP
cells, LNCaP cells in complete medium were exposed to
DMSO vehicle control, SC97 and compound 3b at 2.5,
and 5 μM for 24 h. Bic at 2.5 μM was included as a
positive control. Western blot analysis indicated SC97
and 3b dose-dependently suppressed PSA protein
expression and induced caspase-3 activation and specific
proteolytic cleavage of poly(ADP-ribose) polymerase
(PARP) in LNCaP cells (Fig. 5). The study indicated that
3b is more potent than SC97 in suppressing PSA
expression and inducing apoptosis in LNCaP cells.
5) Antiproliferative activities in a panel of prostate cancer
cell lines
Discussion
Current therapeutic strategy for advanced prostate cancer is to
suppress the AR signalling. Despite of initial response to
castration and conventional antiandrogens, such as bica-
lutamide, lethal CRPC arises and remains addicted to the
AR signalling [6, 30]. In particular, abiraterone and
enzalutamide were recently approved by FDA for the
treatment of CRPC. However, reports to date suggest that
resistance to these agents invariably develops within 1−3
years in CRPC patients and is characterized with a rising
Scheme 3 Synthesis of compounds 4a–4c. CTAB, cetyltrimethyl
ammonium bromide

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Invest New Drugs (2014) 32:227–234
Scheme 4 Synthesis of
compounds 4d–4f
serum prostate-specific antigen (PSA), suggesting aberrant re-
activation of the AR [8, 30, 31]. The key challenge in the field
is how to suppress the continued AR signalling in CRPC cells
[32]. To date, intensive research in CRPC has revealed that the
AR in CRPC cells could be activated by multiple
mechanisms, including mutations in AR, rendering the AR
promiscuous so that it can be activated by a broad range of
non-androgen ligands, even antiandrogens [33] and androgen-
independent activation of the AR via cross-talk with other
factors [34, 35]. In particular, it was demonstrated that IL-6
(a NF-κB regulated cytokine) stimulates prostate cancer
growth in vitro and in vivo through activation of the AR and
increase prostate cancer cell resistance to bicalutamide [22,
24]. Further, Yemelyanov et al. found that IKKβ kinase, a
master regulator of the NF-κB activation, is constitutively
active in tissue specimens from advanced prostate cancer
[36]. Thus, co-inhibition of the AR and NF-κB appears to
be an attractive strategy for battling advanced prostate cancer.
Our previous work has identified two novel ionone-based
antiandrogens SC97 and SC245, which are full antagonists of
Fig. 3 Effect of compounds SC97, SC245, 3a–3d and 4a–4f on the
DHT-induced AR activation in LNCaP cells. LNCaP cells were
transiently transfected with PSA-luc (reporter) and pRL-TK and were
exposed to DMSO vehicle, 1 nM DHT or compounds at the designated
concentrations (μM) in the presence of 1 nM DHT for 24 h. Fold of
induction related to the DMSO vehicle control was shown. Fold of
suppression related to the cells that were exposed to 1 nM DHT alone
was also indicated. Bars, standard deviations. Bic (bicalutamide) was
included as a positive control
Fig. 2 Effect of compounds SC97, SC245, 3a–3d and 4a–4f on the
NF-ĸB-dependent luciferase activity induced by expression of IKKβ.
HEK293 cells were transiently cotransfected with NF-ĸB-Luc, IKKβ-
expressing plasmids and pRL-TK (internal control) and were exposed to
DMSO vehicle or compounds at designated doses (μM) for 24 h. NT,
HEK293 cells were cotransfected with NF-ĸB-Luc, pcDNA3.1 empty
vector and pRL-TK. Fold of induction related to the NTwas shown. Fold
of suppression related to the DMSO vehicle control was also indicated

Invest New Drugs (2014) 32:227–234
233
Ta b l e 1 The cytotoxicity of compounds 3a–3d and 4a–4f in five prostate
cancer cell lines (IC50, μM)
LNCaP
C4-2B
22Rv1
PC-3
DU145
SC97
SC245
3a
1.3
0.6
4
1.6
0.7
5.7
2.8
3.3
4.1
>20
9.0
5.6
13.1
4.1
8.7
2.2
1.1
9.2
3.8
5.1
8.1
15.3
6.8
4.2
15.6
4.1
8.2
4.2
2.9
2.0
N.D.
8.5
4.5
3b
1.2
1.7
2.1
8.1
2.9
1
3.1
5.6
3c
3.9
8.3
3d
4.2
7.8
4a
>20
N.D.
N.D.
N.D.
N.D.
6.6
>20
6.9
4b
4c
3.7
4d
9.2
1.8
5.8
17.2
6.3
4e
4f
12.6
Fig. 4 Compound 3b dose-dependently suppressed DHT-induced
activation of the AR W741C (a) and F876L (b) mutants. ENZ,
enzalutamide. For NT, only the PSA-luc, pCMV empty vector and
pRL-TK were co-transfected into PC3 cells. For all other wells, the
PSA-luc, AR mutant W741C or F876L expressing plasmid and pRL-
TK were co-transfected into PC3 cells. The cells were exposed to DMSO
vehicle, 10 nM DHT or compounds in the presence of 10 nM DHT for
24 h. Fold of induction were calculated relative to the DMSO wells. ***p
<0.0001 when compared with DHT alone
N.D. not determined. Values for SC97 and SC245 were taken from
references [27, 28]
inactive against the W741C mutant, whereas the second-
generation antiandrogen enzalutamide is inactive against the
F876L mutant (Fig. 4). This could have clinically significant
as the W741C mutant was characterized from CRPC patients
who were treated with bicluamide [10]. The F876L mutant
was recently found in CRPC patients who progress on
treatment of an enzalutamide analogue [14, 15]. Overall, our
studies indicated that 3b is a potent inhibitor of both AR and
NF-κB signaling. The C4-2B and 22Rv1 cells are AR-
positive and are androgen-independent. C4-2B cells express
T877A mutated AR, whereas 22Rv1 cells express H874Y
mutated ARs. Androgen-independent PC-3 and DU145 cells
are AR-negative and exhibit a high constitutive activation of
IKKβ and NF-κB signaling [19]. Compound 3b showed low
micromolar antiproliferative activities in C4-2B, 22Rv1, PC-3
and DU-145 cells (Table 1), indicating compound 3b is an
effective agent against the CRPC cells.
the wild type and the T877A, W741C and H874Y mutated
ARs [27, 28]. Using PolarScreen AR competitor assay kit
(P3018, Invitrogen), we showed that SC97 binds with the
AR ligand-binding domain [27]. In this work, we discovered
that SC97 and SC245 also inhibits NF-κB and dose-
dependently suppress IL-6 secretion in DU-145 prostate
cancer cells. Our study indicated that SC97 suppresses IKKβ
expression (Fig. 1b). Further work is needed to characterize
molecular mechanism that account for this activity.
In an effort to optimize SC97 and SC245, we have focused
on chemical modifications at the ionone ring and discovered
compound 3b that is more potent than SC97 in suppressing
AR signalling in LNCaP cells (Figs. 3 and 5) and as potent as
SC97 in inhibiting NF-κB activation (Fig. 2). Importantly, 3b
dose-dependently suppressed DHT-inducted activation of the
AR W741C and F876L mutants. In contrast, bicalutamide is
Conclusion
In present study, we discovered that ionone-based antian-
drogen SC97 and SC245 also inhibit NF-κB activation and
dose-dependently suppress IL-6 secretion in DU-145 prostate
cancer cells. By introducing a hydroxyl group into position-4
of the ionone ring at SC97, we have obtained compound 3b
that is substantially more potent than SC97 in suppressing
DHT-induced AR activation and as potent as SC97 in
inhibiting NF-κB activation. Importantly, compound 3b
significantly inhibits F876L mutant which confers resistance
to enzalutamide. Further, compound 3b showed potent
cytotoxicity in both AR-positive and AR-negative androgen-
independent prostate cancer cells.
Fig. 5 SC97 and compound 3b dose-dependently suppressed PSA
expression and induced apoptosis in LNCaP cells. LNCaP cells were
exposed to DMSO vehicle or compounds for 24 h. Bic was included as a
positive control

234
Invest New Drugs (2014) 32:227–234
Acknowledgments This work was supported by The Cancer Research
Society (J.W.). Postdoctoral fellowship from the CIHR/MCETC Strategic
Training Program is grateful acknowledged (J.Z.).
17. Shen HM, Tergaonkar V (2009) NF + ¦B signaling in carcinogenesis
and as a potential molecular target for cancer therapy. Apoptosis 14:
348–363
18. Setlur SR, Royce TE, Sboner A et al (2007) Integrative microarray
analysis of pathways dysregulated in metastatic prostate cancer.
Cancer Res 67:10296–10303
Conflict of interest The authors declare that they have no conflict of
interest.
19. Gasparian AV, Yao YJ, Kowalczyk D et al (2002) The role of IKK in
constitutive activation of NF- + ¦B transcription factor in prostate
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related kinases for cancer therapy. Clin Cancer Res 14:5656–5662
21. Zhang L, Altuwaijri S, Deng F et al (2009) NF- + ¦B regulates
androgen receptor expression and prostate cancer growth. Am J
Pathol 175:489–499
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