Pharmacological exploitation of the peroxisome proliferator-activated receptor γ agonist ciglitazone to develop a novel class of androgen receptor-ablative agents

Article abstract of DOI:10.1021/jm701212m

On the basis of our finding that the peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone at high doses was able to mediate PPARγ-independent transcriptional repression of androgen receptor (AR) in a tumor cell-specific manner, we used

Full text of DOI:10.1021/jm701212m

2100
J. Med. Chem. 2008, 51, 2100–2107
Pharmacological Exploitation of the Peroxisome Proliferator-Activated Receptor γ Agonist
Ciglitazone To Develop a Novel Class of Androgen Receptor-Ablative Agents
Jian Yang, Shuo Wei, Da-Sheng Wang, Yu-Chieh Wang, Samuel K. Kulp, and Ching-Shih Chen*
DiVision of Medicinal Chemistry, College of Pharmacy, 336 Parks Hall, The Ohio State UniVersity, 500 West 12th AVenue,
Columbus, Ohio 43210
ReceiVed September 25, 2007
On the basis of our finding that the peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone
at high doses was able to mediate PPARγ-independent transcriptional repression of androgen receptor (AR)
in a tumor cell-specific manner, we used ∆2CG, a PPARγ-inactive analogue of ciglitazone, to conduct lead
optimization to develop a novel class of AR-ablative agents. Structure–activity analysis indicates a high
degree of flexibility in realigning ∆2CG’s structural moieties without compromising potency in AR repression,
as evidenced by the higher AR-ablative activity of the permuted isomer 9 [(Z)-5-(4-hydroxybenzylidene)-
3-(1-methylcyclohexylmethyl)thiazolidine-2,4-dione]. Further modificiations of 9 gave rise to 12 [(Z)-5-(4-
hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexylmethyl)thiazolidine-2,4-dione], which com-
pletely inhibited AR expression in LNCaP cells at low micromolar concentrations. This AR down-regulation
led to growth inhibition in LNCaP cells through apoptosis induction. Moreover, the role of AR repression
in the antiproliferative effect of compound 12 was validated by the differential inhibition of cell viability
between androgen-responsive and androgen-nonresponsive cells.
Introduction
mediated PPARγ-independent transcriptional repression of AR
in a tumor cell-specific manner.17 This PPARγ-independent
suppression of AR expression might, in part, underlie the
antiproliferative activity of troglitazone in prostate cancer cells
and is of translational value to the development of troglitazone
and ciglitazone into potent AR-ablative agents. Our early
optimization of troglitazone has generated STG28 (Figure 1A)
with moderate potency in suppressing AR expression in LNCaP
prostate cancer cells.17 In this study, the lead optimization of
ciglitazone gave rise to compound 12, which exhibited the ability
to mediate AR ablation at low micromolar concentrations.
Mounting evidence indicates that dysregulation of androgen
receptor (ARa) through gene amplification or mutations plays
a key role in the development of androgen-refractory prostate
cancer,1–8 a hallmark of incurable and lethal prostate cancer
progression. These molecular changes enhance AR sensitivity
or permit AR activation by antiestrogen, thus allowing prostate
cancer cells to become resistant to androgen ablation-induced
apoptosis.9,10 From a clinical perspective, targeting AR expres-
sion represents an important strategy to improve the treatment
of androgen-independent prostate cancer and ultimately to
increase the survival of prostate cancer patients. A recent study
indicates that knocking down the AR protein level by a small
interfering RNA (siRNA) resulted in significant apoptotic cell
death in LNCaP androgen-responsive prostate cancer cells but
not in the AR-null PC-3 cells.11 Moreover, in a LNCaP tumor
xenograft model, short hairpin RNA (shRNA) mediated AR
knockdown was effective in blocking tumor growth and delaying
tumor progression,12 which provides a proof-of-principle of this
AR-targeted therapy.
Although a number of natural-product-based, small-molecule
agents exhibit the ability to suppress AR expression, including
resveratrol,13 vitamin E succinate,14 genistein,15 and curcumin,6
their therapeutic use in humans is limited by unattainable
therapeutic concentrations as a result of low potency. Thus, it
is of urgency to develop potent AR-ablative agents in the pursuit
of new strategies for prostate cancer treatment. During the course
of our investigation of the effect of the thiazolidinedione
family of peroxisome proliferator-activated receptor γ (PPARγ)
agonists on repressing prostate specific antigen (PSA),16 we
demonstrated that troglitazone and ciglitazone at high doses
Chemistry
The lead optimization of ciglitazone to develop compound
12 consisted of three stages (Figure 1B). Stage 1 was to abrogate
ciglitazone’s PPARγ agonist activity by introducing a double
bond adjoining the terminal thiazolidinedione ring, leading to
the PPARγ inactive analogue ∆2CG.16 Stage 2 was to structur-
ally modify ∆2CG via three distinct strategies: (a) regioisomer-
ization of the (1-methylcyclohexyl)methyl moiety to yield
compound 1, (b) phenyl ring substitutions to give compounds
2–8, and (c) permutational rearrangement of the terminal
cyclohexyl moiety to generate compound 9. In stage 3,
compound 9 underwent modifications at the terminal phenyl
ring, generating two series of compounds, i.e., 10 and 11, and
12-19. These ∆2CG derivatives were synthesized according
to general procedures described in Figure 1C, and their ability
to suppress AR expression in LNCaP cells was assessed by the
AR promoter-luciferase reporter gene assay followed by Western
blot analysis.
Results
* To whom correspondence should be addressed. Phone: (614) 688-4008.
Fax: (614) 688-8556. E-mail: chen.844@osu.edu.
Dissociation of the PPARγ activity does not affect the
ability of ∆2CG to inhibit AR expression at both mRNA
and protein levels in LNCaP cells. Dose- and/or time-
dependent effects of ciglitazone and ∆2CG on suppressing AR
expression were assessed in LNCaP cells in 10% fetal bovine
a Abbreviations: PPARγ, peroxisome proliferator-activated receptor γ;
AR, androgen receptor; shRNA, short hairpin RNA; PSA, prostate specific
antigen; FBS, fetal bovine serum; RT-PCR; reverse transcription-polymerase
chain reaction; PPRE, peroxisome proliferator-activated receptor response
element; TBST, Tris-buffered saline containing 0.1% Tween-20.
10.1021/jm701212m CCC: $40.75
2008 American Chemical Society
Published on Web 03/13/2008

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2101
Figure 1. (A) Structures of troglitazone (TG) and STG28. (B) Schematic representation of the course of structural optimization of ciglitazone to
develop AR-ablative agents. (C) General synthetic procedure for ciglitazone derivatives. Reaction conditions: a, LiAlH4, THF; b, (CF3SO2)2O,
pyridine, CH2Cl2; c, K2CO3, DMF; d, AcOH, piperidine, ethanol/reflux; e, AcOH, piperidine, pyridine/reflux; f, K2CO3, DMF.
serum (FBS) by Western blotting and reverse transcription-
polymerase chain reaction (RT-PCR). These analyses indicate
that ∆2CG, albeit lacking PPARγ agonist activity, exhibited
modestly higher potency than ciglitazone in mediating tran-
scriptional repression of AR. For example, the concentrations
required for complete suppression of AR protein expression were
approximately 30 and 60 µM for ∆2CG and ciglitazone,
respectively (Figure 2A).
Furthermore, RT-PCR analysis indicates that the down-
regulation of AR expression occurred at the transcriptional level
(Figure 2B). Together, these findings confirmed the ability of
ciglitazone to ablate AR independently of PPARγ activation,
which provided a molecular rationale to use ∆2CG as a starting
point for lead optimization to generate potent AR-ablative
agents. To expedite the screening of AR-ablative agents, we
used a luciferase reporter assay17 to analyze the effect of
individual derivatives on suppressing AR transcription by using
LNCaP cells transiently transfected with the AR promoter-linked
luciferase reporter plasmid.
Lead Optimization of ∆2CG. As aforementioned, ∆2CG
underwent three types of structural modifications, leading to
compounds 1–9. Individual derivatives at 10 µM, compared to
ciglitazone and ∆2CG, each at 20 µM, were evaluated in the
luciferase reporter assay in the transiently transfected LNCaP
cells. Relative to ∆2CG, these derivatives showed improved
potency in suppressing the activity of the AR promoter,
suggesting that a high degree of flexibility existed in the
structure–activity relationship. This premise was borne out by
the regioisomer 1 and the permuted isomer 9, both of which
showed enhanced AR-ablating activity despite substantial con-
figuration changes (Figure 3A, left panel). Moreover, examina-
tion of the IC50 values of individual derivatives in suppressing
the viability of LNCaP cells after 48 h of treatment indicates a
positive correlation between the ability to suppress AR mRNA
transcription and that of inhibiting cell viability (Figure 3A).
Of these derivatives, we chose compounds 1, 6, and 9 as
representatives to conduct Western blot analysis. As shown in
Figure 3B, these three derivatives showed a dose-dependent

2102 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Yang et al.
Figure 2. Effect of ciglitazone (CG) and ∆2CG on AR ablation in
LNCaP cells. (A) Dose- and time-dependent effects of CG and ∆2CG
on suppressing AR protein expression levels. Cells were exposed to
CG or ∆2CG under the indicated conditions in 10% FBS-supplemented
medium, and the lysates were subjected to Western blot analysis. (B)
Time-dependent effect of CG (60 µM) and ∆2CG (30 µM) on
suppressing the mRNA levels of AR. Cells were treated with either
agent in 10% FBS-supplemented medium for the indicated times. Total
RNA was isolated and subjected to RT-PCR analysis as described in
the Experimental Section.
Figure 4. Differential effect of compounds 10–19 on suppressing AR
expression in LNCaP cells. (A, left) Analysis of the effects of DMSO
vehicle (D) or individual compounds on the transcriptional repression
of the AR gene by the AR promoter-luciferase reporter assay. LNCaP
cells were transiently transfected with an AR promoter-linked luciferase
reporter plasmid and exposed to compounds 10–19 (10 µM) in 10%
FBS-supplemented RPMI 1640 medium for 48 h. Analysis of luciferase
activity was carried out as described in the Experimental Section:
(columns) mean (n ) 3); (bars) SD. (A, right) IC50 values of individual
agents in inhibiting the cell viability of LNCaP cells. Cells were exposed
to individual agents at various concentrations in 5% FBS-supplemented
RPMI 1640 medium for 48 h, and cell viability was assessed by MTT
assays. (B) Dose-dependent effect of ciglitazone (CG), ∆2CG, and
compounds 12 and 16, relative to that of 10 µM troglitazone (TG), on
PPARγ activation in PC-3 cells. PC-3 cells were transiently transfected
with PPRE-x3-TK-Luc reporter vector and then exposed to individual
agents or DMSO vehicle (D) in 10% FBS-supplemented RPMI 1640
medium for 48 h. Analysis of luciferase activity was carried out as
described in the Experimental Section: (columns) mean (n ) 6); (bars)
SD. (C) Western blot analysis of the dose-dependent effect of
compounds 12 and 16 on reducing AR protein levels. Cells were
exposed to individual agents at the indicated concentrations in 10%
FBS-supplemented medium for 72 h, and the lysates were subjected
to Western blot analysis. (D) Immunocytochemical analysis of the effect
of 5 µM compound 12 on suppressing AR expression after 24 h of
exposure. The nuclear counterstaining was achieved using a 4′,6-
diamino-2-phenylindole (DAPI)-containing mounting medium.
Figure 3. Differential effects of ciglitazone, ∆2CG, and compounds
1–9 on suppressing AR expression in LNCaP cells. (A, left) Analysis
of the effects of individual compounds on the transcriptional repression
of the AR gene by the AR promoter-luciferase reporter assay. LNCaP
cells were transiently transfected with an AR promoter-linked luciferase
reporter plasmid and exposed to DMSO vehicle (D), ciglitazone (CG,
20 µM), ∆2CG (∆2, 20 µM), or compounds 1–9 (10 µM) in 10% FBS-
supplemented RPMI 1640 medium for 48 h. Analysis of luciferase
activity was carried out as described in the Experimental Section:
(columns) mean (n ) 3); (bars) standard deviation (SD). (A, right)
IC50 values of individual agents in inhibiting the cell viability of LNCaP
cells. Cells were exposed to individual agents at various concentrations
in 5% FBS-supplemented RPMI 1640 medium for 48 h, and cell
viability was assessed by MTT assays. (B) Western blot analysis of
the dose-dependent effect of compounds 1, 6, and 9 on reducing AR
protein levels. Cells were exposed to individual agents at the indicated
concentrations in 10% FBS-supplemented medium for 72 h, and the
lysates were subjected to Western blot analysis.
analysis and cell viability assay indicated a subtle structure–
activity relationship among these derivatives (Figure 4A).
For example, moving the terminal para OH function to the
ortho or meta position (compounds 10 and 11) abolished the
ability to suppress AR promoter-luciferase activity and cell
viability, indicating its important role in interacting with the
target protein. Moreover, substitutions of the phenyl ring with
CF3 or Br led to substantially higher potency in AR repression,
while those with NO2 or electron-donating groups attenuated
the activity (Figure 4A). Of these derivatives, compounds 12
effect on suppressing AR protein expression (Figure 3B). In
light of the unique structural feature of compound 9, we used
this permuted derivative to carry out further structural optimiza-
tion, generating compounds 10-19. The luciferase reporter

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Figure 5. Antitumor effects of compound 12 in LNCaP cells. (A) Differential dose-dependent effects of compound 12 on the inhibition of cell
viability of LNCaP versus PC-3 cells at 48 h (inset) and 72 h of treatment. Cells were exposed to the indicated concentrations of compound 12 in
5% FBS-containing RPMI 1640 medium for 48 and 72 h, and cell viability was determined by MTT assays: (points) mean (n ) 6); (bars) SD. (B)
Dose- and time-dependent antiproliferative effects of compound 12 in LNCaP (left) and PC-3 (right) cells. Cells were seeded into six-well plates
(250 000 cells/well), incubated for 24 h, and exposed to compound 12 at the indicated concentrations in 5% FBS-supplemented medium for different
time intervals. Cells were harvested and counted using a Coulter counter. (C) Flow cytometric analysis of LNCaP cells after treatment with DMSO
or the indicated concentrations of compound 12 for 72 h. Percentages of cell cycle distribution represent the mean of two independent determinations.
(D) Western blot analysis of the dose-dependent effects of compounds 12 and 16 on PARP cleavage, caspase 3 activation, and caspase 7 activation
in LNCaP cells after 72 h of treatment.
and 16 represented the optimal agents in inhibiting AR mRNA
transcription and LNCaP cell viability.
µM compound 12 for 48 h led to a substantial decrease in AR
levels in the nucleus.
To demonstrate that this drug-induced transcriptional repres-
sion of AR was independent of PPARγ, we examined the ability
of compounds 12 and 16 versus troglitazone, ciglitazone, and
∆2CG to transactivate PPARγ by using the PPAR response
element (PPRE) luciferase reporter assay. In PC-3 cells trans-
fected with a reporter construct (PPRE-x3-TK-Luc), troglitazone
and ciglitazone at 10 µM exhibited a significant effect on
increasing luciferase activity, ranging from 2.5-fold to 4-fold
(P < 0.05). In contrast, compounds 12 and 16, like their parent
compounds ∆2CG, lacked appreciable activity in PPARγ
activation.
Western blot analysis indicates that the IC50 values for
suppressing AR expression by compounds 12 and 16 after 72 h
of exposure were approximately 2 and 4 µM, respectively
(Figure 4C). The ability of compound 12 to suppress AR
expression was further demonstrated by immunocytochemical
analysis (Figure 4D). As shown, exposure of LNCaP cells to 5
Antitumor Effects of Compound 12 in Prostate Cancer
Cells. We assessed the antitumor effects of compound 12 in
both LNCaP androgen-responsive and PC-3 androgen-nonre-
sponsive prostate cancer cells via three different methods,
including the MTT assay for cell viability, cell counting for
cell proliferation, and flow cytometric analysis for cell cycle
distribution. Because of the lack of AR expression, PC-3 cells
exhibited substantially lower sensitivity to the antiproliferative
activities of compound 12 compared to LNCaP cells. The IC50
values for suppressing cell viability were 8 and 3 µM at 48 and
72 h of drug treatment, respectively, in LNCaP cells and were
15 and 12 µM, respectively, in PC-3 cells (Figure 5A).
This differential susceptibility was also manifest in the cell
counting assay, in which compound 12 exhibited at least 2-fold
higher potency in inhibiting the proliferation of LNCaP cells
compared to PC-3 cells (Figure 5B). Moreover, cell cycle
analysis was carried out after exposing LNCaP cells to different

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Yang et al.
of the AR expression suggests a reversible nature of this
ligand-protein interaction.
Discussion
In light of the pivotal role of PPARγ in prostate cell proliferation
and differentiation, the chemopreventive activity of thiazolidinedi-
ones in prostate cancer has been attributed to their ability to activate
PPARγ signaling, leading to the terminal differentiation and growth
arrest of tumor cells.19–21 However, mounting evidence suggests
that the antiproliferative ability of these agents is independent of
their PPARγ agonist activity.16,17,22–31 In our laboratory, we have
identified several “off-target” mechanisms that might underlie the
antitumor effects of thiazolidinediones, including Bcl-2/Bcl-xL
inhibition, proteasomal degradation of cyclin D1,25 ꢀ-catenin,32 and
Sp1,17 and transcriptional repression of PSA16 and AR.17 Separation
of these pharmacological effects from PPARγ activation provides
a mechanistic rationale for using thiazolidinediones as a scaffold
to develop potent molecularly targeted agents. Considering the
importance of AR in prostate tumorigenesis and tumor progression,
we carried out lead optimization of ciglitazone and its PPARγ-
inactive derivative ∆2CG to develop potent AR-ablative agents.
There existed a high degree of tolerance for the substructural
rearrangement of ∆2CG without compromising the AR-ablative
activity, as evidenced by the improved potency of compounds 1
and 9. In contrast, modifications of the phenyl ring exhibited a
subtle effect on the AR-ablative potency. For example, changing
the orientation of the terminal hydroxyl function of compound 9
completely abrogated the ability of the resulting compounds 10
and 11 to suppress AR expression, while the CF3 substitution or
di-Br substitution led to enhanced potency. Together, these findings
suggest that the benzylidene-thiazolidinedione substructure played
a crucial role in interacting with the target protein.
Figure 6. Evidence that the ability of compound 12 to inhibit AR
expression in LNCaP cells is not mediated through an irreversible
mechanism. (A) Structure and the dose-dependent effect of compound
20 on suppressing AR expression in LNCaP cells. Cells were exposed
to compound 20 at the indicated concentrations in 10% FBS-
supplemented medium for 72 h, and the lysates were subjected to
Western blot analysis. (B) Dose-dependent effects of compound 12
versus compound 20 in suppressing the viability of LNCaP cells. Cells
were exposed to individual agents at various concentrations in 5% FBS-
supplemented RPMI 1640 medium for 48 h, and cell viability was
assessed by MTT assays. (C) Restoration of AR expression in LNCaP
cells after compound 12 was washed out. The effect of 5 µM compound
12 on AR repression in LNCaP cells was examined at different intervals
throughout a 72 h period in two different manners. For continuous
exposure experiments, cells in T-25 flasks were incubated in drug-
containing 10% FBS-supplemented medium for 72 h. For washout at
48 h of treatment, cells in T-25 flasks were exposed to the agent for
44 h, followed by incubation in drug-free medium for an additional
24 h. AR levels in cell lysates were analyzed by Western blot analysis.
Among all derivatives examined, compound 12 represented a
structurally optimized derivative with an order-of-magnitude higher
potency than ciglitazone in suppressing AR expression. This AR
down-regulation led to growth inhibition in LNCaP cells through
apoptosis induction, as evidenced by flow cytometry, PARP
cleavage, and caspase activation. The role of AR repression in the
antiproliferative effect of compound 12 was supported by the
differential inhibition of cell viability between LNCaP androgen-
responsive and PC-3 androgen-nonresponsive cells. Because thia-
zolidinediones mediate AR repression through down-regulation of
Sp1,17 compound 12 also suppresses the transcription of many Sp1-
targeted genes (data not shown), which accounts for the ability of
compound 12 to inhibit PC-3 cell viability.
Relative to many natural-product-based agents that suppress
AR expression/function, such as resveratrol,13 vitamin E suc-
cinate,14 genistein,15 and curcumin,6 compound 12 is substan-
tially more effective in down-regulating AR expression. Thus,
this AR-ablative agent has translational potential to foster new
therapeutic strategies for prostate cancer treatment as a single
agent or in combination with other molecularly targeted agents.
doses of compound 12 for 72 h (Figure 5C). As shown,
compound 12 caused a dose-dependent increase in the sub-G1
population, accompanied by decreases in the G2/M phase (Figure
5C). Furthermore, the ability of compounds 12 and 16 to induce
apoptotic death in LNCaP cells was demonstrated by their dose-
dependent effects on modulating various apoptosis-related
biomarkers, including PARP cleavage, and the proteolytic
activation of caspase 3 and caspase 7 (Figure 5D).
Our earlier study indicates that thiazolidinediones mediated
the transcriptional repression of AR by facilitating the degrada-
tion of the transcription factor Sp1.17 However, it was reported
that compounds containing a 5-arylidene-3-aryl-2,4-thiazo-
lidinedione substructure underwent conjugation addition with
p-thiocresol in the presence of piperidine upon heating.18 This
report raised the possibility that compound 12 and other
derivatives might act as “Michael acceptors” by covalently
modifying the target enzyme/protein upon binding. Here, we
obtained two lines of evidence to refute this possibility. First,
compound 20, a saturated counterpart of compound 12, retained
the ability to suppress AR expression and cell viability, though
with slightly lower potency, in LNCaP cells (Figure 6A,B).
Second, the expression level of AR in drug-treated LNCaP cells
would be rapidly restored once compound 12 was removed (or
washed out) from the medium (Figure 6C). This rapid restoration
Conclusion
The in vivo efficacy of targeting AR expression to block
tumor growth and delaying tumor progression has recently been
demonstrated in a LNCaP tumor xenograft model by using
shRNA-mediated AR knockdown.12 This finding provides a
proof-of-principle that inhibition of AR expression represents
a therapeutically relevant strategy for prostate cancer treatment.
Relative to shRNAs, small-molecule agents prove to be
advantageous in many aspects of cancer therapy. Consequently,

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Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2105
Hz, 1H), 7.63 (d, J ) 8.42 Hz, 1H), 7.71 (s, 1H), 7.80 (s, 1H),
8.09–8.12 (br, 1H); HRMS exact mass of (M + Na)+, 422.1014
amu; observed mass of (M + Na)+, 422.1019 amu.
in vivo testing of compound 12 to suppress prostate tumor
growth is currently underway.
Experimental Section
(Z)-5-[3-Methoxy-4-(1-methylcyclohexylmethoxy)benzylidene]-
1
thiazolidine-2,4-dione (5). H NMR (300 MHz, CDCl3) δ 1.09
Chemical reagents and organic solvents were purchased from
Sigma-Aldrich unless otherwise mentioned. Nuclear magnetic
resonance spectra (1H NMR) were measured on a Bruker DPX 300
model spectrometer. Chemical shifts (δ) were reported in parts per
million (ppm) relative to the TMS peak. Electrospray ionization
mass spectrometry analyses were performed with a Micromass
Q-Tof II high-resolution electrospray mass spectrometer. Elemental
analyses were performed by the Atlantic Microlab, Inc. (Norcross,
GA) and were reported to be within 0.4% of calculated values.
Flash column chromatography was performed with silica gel
(230–400 mesh). ∆2CG and the two series of compounds, 1–8 and
9–19, were synthesized according to the general methods described
in Figure 1B, which are illustrated by the synthesis of compounds
1 and 9 as examples.
(s, 3H), 1.40–1.58 (m, 10H), 3.75 (s, 2H,), 3.96 (s, 3H), 6.96 (d, J
) 8.40 Hz, 1H), 7.00 (s, 1H), 7.11 (d, J ) 8.42 Hz, 1H), 7.79 (s,
1H), 8.55 (s, 1H); HRMS exact mass of (M + Na)+, 384.1245
amu; observed mass of (M + Na)+, 384.1239 amu.
(Z)-5-[3-Ethoxy-4-(1-methylcyclohexylmethoxy)benzylidene]-
1
thiazolidine-2,4-dione (6). H NMR (300 MHz, CDCl3) δ 1.08
(s, 3H), 1.40–1.58 (m, 13H), 3.74 (s, 2H), 4.10 (q, J ) 6.9 Hz,
2H), 6.95 (d, J ) 8.40 Hz, 1H), 7.01 (d, J ) 2.10 Hz, 1H), 7.11
(dd, J ) 8.40, 2.10 Hz, 1H), 7.79 (s, 1H), 8.42 (s, 1H); HRMS
exact mass of (M + Na)+, 389.1402 amu; observed mass of (M +
Na)+, 389.1402 amu.
(Z)-5-[3,5-Dimethyl-4-(1-methylcyclohexylmethoxy)benzylidene]-
1
thiazolidine-2,4-dione (7). H NMR (300 MHz, CDCl3) δ 1.13
(s, 3H), 1.32–1.59 (m, 10H), 2.42 (s, 6H), 3.48 (s, 2H), 7.17 (s,
2H), 7.76 (s, 1H), 8.26 (s, 1H); HRMS exact mass of (M + Na)+,
382.1453 amu; observed mass of (M + Na)+, 382.1448 amu.
(Z)-5-[4-(1-Methylcyclohexylmethoxy)naphthalen-1-ylmeth-
(Z)-5-[3-(1-Methylcyclohexylmethoxy)benzylidene]thiazolidine-
2,4-dione (1). Step a. To a stirring solution of LiAlH4 (20 mmol)
in anhydrous THF (10 mL) at 4 °C was added 1-methylcyclohex-
anecarboxylic acid (i, 7.0 mmol) in 50 mL of THF dropwise over
a period of 1 h. The solution was stirred at refluxing temperature
under N2 for 6 h. The solution was cooled to 4 °C by ice bath, and
1 mL of 1 N NaOH (1 mL) followed by H2O (2 mL) was slowly
added to the solution to quench the reaction. The solution was stirred
at 23 °C for 1 h and then filtered to remove solid material. The
solution was concentrated. Purification by flash silica gel chroma-
tography (ethyl acetate/hexanes, 1:2) gave the product, (1-methyl-
cyclohexyl)methanol (ii), in 82% yield.
Step b. A solution of compound ii (1 mmol) in dry CH2Cl2 (5
mL) was cooled to 4 °C, to which was added pyridine (1.1 mmol)
and triflate anhydride (1.1 mmol). After being stirred at 4 °C for
2 h, the solution was concentrated, and the residue was purified by
flash silica gel column chromatography (ethyl acetate/hexanes, 1:10)
to afford trifluoromethanesulfonic acid 1-methyl-cyclohexylmethyl
ester (iii) in 35% yield.
1
ylene]thiazolidine-2,4-dione (8). H NMR (300 MHz, CDCl3) δ
1.18 (s, 3H), 1.51–1.59 (m, 10H), 3.91 (s, 2H), 6.915 (d, J ) 8.70
Hz, 1H), 7.55–7.69 (m, 3H), 8.12 (d, J ) 8.70, 1H), 8.39 (d, J )
8.40, 1H), 8.59 (s, 1H); HRMS exact mass of (M + Na)+, 404.1296
amu; observed mass of (M + Na)+, 404.1299 amu.
(Z)-5-(4-Hydroxybenzylidene)-3-(1-methylcyclohexylmeth-
yl)thiazolidine-2,4-dione (9). Step e. A mixture of p-hydroxyben-
zaldehyde (vi, 0.5 mmol), 2,4-thiazolidinedione (0.6 mmol), and
catalytic amounts of piperidine and AcOH was refluxed in toluene
(5 mL) for 24 h. The precipitated product was filtered, washed with
toluene (3 × 10 mL), and dried in vacuo at 60 °C overnight,
yielding 5-(4-hydroxybenzylidene)thiazolidine-2,4-dione (vii) in a
85% yield.
Step f. A solution of compound vii (0.5 mmol), compound iii
(0.6 mmol), and K2CO3 (0.65 mmol) was stirred in DMF (3 mL)
at 80 °C for 4 h, poured into water, extracted with ethyl acetate (3
× 10 mL), dried, and concentrated. The residue was purified by
Step c. A mixture of compound iii (0.5 mmol), 3-hydroxyben-
zaldehyde (iv, 0.6 mmol), and K2CO3 (0.7 mmol) was dissolved in
DMF (3 mL). The solution was heated to 80 °C for 4 h. The solution
was poured into water, extracted with ethyl acetate (10 mL) three
times, and concentrated. The residue was purified by chromatog-
raphy and resulted in 0.22 mmol of 3-(1-methylcyclohexylmethoxy)-
benzaldehyde (v) with a 44% yield.
1
chromatography, affording compound 9 in 42% yield. H NMR
(300 MHz, CDCl3) δ 0.94 (s, 3H), 1.14–1.86 (m, 10H), 3.63 (s,
2H), 5.69 (s, 1H), 6.94(d, J ) 8.40 Hz, 2H), 7.43(d, J ) 8.40 Hz,
2H), 7.83 (s, 1H); HRMS exact mass of (M + Na)+, 354.1140
amu; observed mass of (M + Na)+, 354.1141 amu.
(Z)-5-(3-Hydroxybenzylidene)-3-(1-methylcyclohexylmethyl)-
Step d. A mixture consisting of compound v (0.5 mmol), 2,4-
thiazolidinedione (0.6 mmol), and catalytic amounts of piperidine
was refluxed in EtOH (5 mL) for 24 h and then concentrated. The
oily product was dissolved in ethyl acetate, poured into water, and
acidified with AcOH. The solution was extracted with ethyl acetate,
dried, and concentrated. The residue was purified by silica gel
1
thiazolidine-2,4-dione (10). H NMR (300 MHz, CDCl3) δ 0.96
(s, 3H), 1.24–1.67 (m, 10H), 3.65 (s, 2H), 5.24 (s, 1H), 6.70 (d, J
) 1.5 Hz, 1H), 6.93(dd, J ) 8.10, 1.5 Hz, 1H), 7.10 (d, J ) 7.80
Hz, 1H), 7.36 (dd, J ) 7.80, 7.50 Hz, 1H), 7.84 (s, 1H); HRMS
exact mass of (M + Na)+, 354.1140 amu; observed mass of (M +
Na)+, 354.1143 amu.
1
chromatography, providing compound 1 in 67% yield. H NMR
(Z)-5-(2-Hydroxybenzylidene)-3-(1-methylcyclohexylmeth-
yl)thiazolidine-2,4-dione (11). 1H NMR (300 MHz, CDCl3) δ 0.95
(s, 3H), 1.22–1.65 (m, 10H), 3.66 (s, 2H), 6.44 (d, J ) 0.9 Hz,
1H), 6.91 (dd, J ) 8.10, 0.9 Hz, 1H), 7.04 (td, J ) 7.2, 0.6 Hz,
1H), 7.32 (tdd, J ) 7.5, 1.5, 0.6 Hz, 1H), 7.46 (dd, J ) 7.80, 1.5
Hz, 1H), 8.42 (s, 1H); HRMS exact mass of (M + Na)+, 354.1140
amu; observed mass of (M + Na)+, 354.1145 amu.
(Z)-5-(4-Hydroxy-3-trifluoromethylbenzylidene)-3-(1-methyl-
cyclohexylmethyl)thiazolidine-2,4-dione (12). 1H NMR (300
MHz, CDCl3) δ 0.95 (s, 3H), 1.46–1.56 (m, 10H), 3.64 (s, 2H),
6.08–6.38 (br, 1H), 7.09 (d, J ) 8.40 Hz, 1H), 7.59 (d, J ) 8.40
Hz, 1H), 7.69 (s, 1H), 7.83 (s, 1H); HRMS exact mass of (M +
Na)+, 422.1014 amu; observed mass of (M + Na)+, 422.1012 amu.
Anal. (C19H20F3NO3S) C, H, N, S, O, F.
(Z)-5-(4-Hydroxy-3-nitrobenzylidene)-3-(1-methylcyclohexyl-
methyl)thiazolidine-2,4-dione (13). 1H NMR (300 MHz, CDCl3)
δ 0.96 (s, 3H), 1.23–1.57 (m, 10H), 3.68 (s, 2H), 7.31(d, J ) 8.40
Hz, 1H), 7.74 (dd, J ) 8.40, 2.1 Hz, 1H), 7.81 (s, 1H), 8.29 (d, J
) 2.1 Hz, 1H); HRMS exact mass of (M + Na)+, 399.0991 amu;
observed mass of (M + Na)+, 399.0991 amu.
(300 MHz, CDCl3) δ 1.04 (s, 3H), 1.46–1.56 (m, 10H), 3.69 (s,
2H), 6.78–7.28 (m, 2H), 7.08 (d, J ) 8.40 Hz, 1H), 7.39 (dt, J )
2.10, 8.40 Hz, 1H), 7.84 (s, 1H), 8.21–8.78(br, 1H); HRMS exact
mass of (M + Na)+, 354.1140 amu; observed mass of (M + Na)+,
354.113 amu.
(Z)-5-[3-Bromo-4-(1-methylcyclohexylmethoxy)benzylidene]-
1
thiazolidine-2,4-dione (2). H NMR (300 MHz, CDCl3) δ 1.11
(s, 3H), 1.40–1.61 (m, 10H), 3.77 (s, 2H), 6.95 (d, J ) 8.42 Hz,
1H), 7.41 (dd, J ) 2.10, 8.42 Hz, 1H), 7.69 (d, 1H, J ) 2.10),
7.74 (s, 1H), 8.38 (s, 1H); HRMS exact mass of (M + Na)+,
432.0245 amu; observed mass of (M + Na)+, 432.0247 amu.
(Z)-5-[4-(1-Methylcyclohexylmethoxy)-3-nitrobenzylidene]thi-
1
azolidine-2,4-dione (3). H NMR (300 MHz, CDCl3) δ 1.08 (s,
3H), 1.42–1.59 (m, 10H), 3.79 (s, 2H,), 7.23 (d, J ) 8.40 Hz, 1H),
7.85 (d, J ) 8.42 Hz, 1H), 7.92 (s, 1H), 8.10 (s, 1H), 8.33 (s, 1H);
HRMS exact mass of (M + Na)+, 399.0991 amu; observed mass
of (M + Na)+, 399.0995 amu.
(Z)-5-[4-(1-Methylcyclohexylmethoxy)-3-trifluoromethylben-
zylidene]thiazolidine-2,4-dione (4). 1H NMR (300 MHz, CDCl3)
δ 1.08 (s, 3H), 1.42–1.59 (m, 10H), 3.79 (s, 2H), 7.10 (d, J ) 8.40

2106 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Yang et al.
(Z)-5-(3-Bromo-4-hydroxybenzylidene)-3-(1-methylcyclohexyl-
methyl)thiazolidine-2,4-dione (14). 1H NMR (300 MHz, CDCl3)
δ 0.79 (s, 3H), 1.17–1.46 (m, 10H), 3.36 (s, 2H), 7.01 (d, J ) 8.40
Hz, 1H), 7.37 (d, J ) 8.40 Hz, 1H), 7.73 (s, 2H); HRMS exact
mass of (M + Na)+, 432.0245 amu; observed mass of (M + Na)+,
432.0245 amu. Anal. (C18H20BrNO3S) C, H, N, O.
(Z)-5-(4-Hydroxy-3-methoxybenzylidene)-3-(1-methylcyclo-
hexylmethyl)thiazolidine-2,4-dione (15). 1H NMR (300 MHz,
CDCl3) δ 0.92 (s, 3H), 1.21–1.58 (m, 10H), 3.62 (s, 2H), 3.97 (s,
3H), 5.95 (br, 1H), 6.90–7.03 (m, 2H), 7.10 (d, J ) 7.80 Hz, 1H),
7.82 (s, 1H), HRMS exact mass of (M + Na)+, 384.1245 amu;
observed mass of (M + Na)+, 384.1245 amu. Anal. (C19H23NO4S)
C, H, N, O.
(Z)-5-(3,5-Dibromo-4-hydroxybenzylidene)-3-(1-methylcyclo-
hexylmethyl)thiazolidine-2,4-dione (16). 1H NMR (300 MHz,
CDCl3) δ 0.94 (s, 3H), 1.32–1.56 (m, 10H), 3.63 (s, 2H), 6.22(s,
1H), 7.62 (s, 2H), 7.68(s, 1H); HRMS exact mass of (M + Na)+,
511.9330 amu; observed mass of (M + Na)+, 511.9329 amu. Anal.
(C18H19Br2NO3S) C, H, N, S, O, Br.
(Z)-5-(4-Hydroxy-3-iodo-5-methoxybenzylidene)-3-(1-methyl-
cyclohexylmethyl)thiazolidine-2,4-dione (17). 1H NMR (300
MHz, CDCl3) δ 0.94 (s, 3H), 1.22–1.62 (m, 10H), 3.63 (s, 2H),
3.96 (s, 3H), 6.44 (s, 1H), 6.97 (s, 1H), 7.50 (s, 1H), 7.73 (s, 1H),
HRMS exact mass of (M + Na)+, 510.0212 amu; observed mass
of (M + Na)+, 510.0213 amu.
(Z)-5-(4-Hydroxy-3,5-dimethylbenzylidene)-3-(1-methylcyclo-
hexylmethyl)thiazolidine-2,4-dione (18). 1H NMR (300 MHz,
CDCl3) δ 0.94 (s, 3H), 1.22–1.66 (m, 10H), 2.30 (s, 6H), 3.62 (s,
2H), 5.06 (s, 1H), 7.17 (s, 2H), 7.78 (s, 1H); HRMS exact mass of
(M + Na)+, 382.1453 amu; observed mass of (M + Na)+, 382.1454
amu.
(Z)-5-(4-Hydroxynaphthalen-1-ylmethylene)-3-(1-methylcy-
clohexylmethyl)thiazolidine-2,4-dione (19). 1H NMR (300 MHz,
CDCl3) δ 0.98 (s, 3H), 1.20–1.66 (m, 10H), 3.67 (s, 2H), 5.91 (s,
1H), 6.91 (d, J ) 7.80 Hz, 1H), 7.56–7.67 (m, 3H), 8.15 (d, J )
8.40 Hz, 1H), 8.29 (d, 1H, J ) 7.20 Hz), 8.60 (s, 1H); HRMS
exact mass of (M + Na)+, 404.1296 amu; observed mass of (M +
Na)+, 404.1296 amu.
solubilized in DMSO (200 µL/well). Absorbance was determined
at 570 nm by a 96-well plate reader.
Transfection and Luciferase Assay. The 3.6-kilobase AR
promoter-linked reporter plasmid p-3600ARCAT was kindly pro-
vided by Dr. Chawnshang Chang (University of Rochester Medical
Center, Rochester, NY). The AR promoter gene (-3600 to +550)
encompassing the transcription start site was isolated by using PCR
to generate hAR-luc with the following primers: 5′- TACAGG-
TACCGGTATCTCGACCTGCAGGTC-3′ and 5′-TGTTAGATCT-
TGCTGAAGCCGCTCCCCAGT-3′. The fragment was subcloned
into the pGL3 luciferase reporter vector (Promega, Madison, WI)
at KpnI and BglII in the multiple cloning site. The PPRE-x3-TK-
Luc reporter vector contains three copies of the PPAR-response
element (PPRE) upstream of the thymidine kinase promoter-
luciferase fusion gene and was kindly provided by Dr. Bruce
Spiegelman (Harvard University, Cambridge, MA). The pCMVSp1
plasmid was purchased from Origene Technologies, Inc. (Rockville,
MD). LNCaP or PC3 cells were transfected with 5 µg of individual
plasmids in an Amaxa Nucleofector using a cell-line-specific
Nucleofector kit according to the manufacturer’s protocol (Amaxa
Biosystems, Cologne, Germany) and were then seeded in six-well
plates at 5 × 105 cells per well for 48 h. The transfection efficiency
was determined to be 70–80% by transfecting cells with 2 µg of
pmaxGFP plasmid, followed by fluorescence microscopy to measure
GFP expression. For each transfection, herpes simplex virus
thymidine kinase promoter-driven Renilla reniformis luciferase was
used as an internal control for normalization.
For the reporter gene assay, after transfection, cells were cultured
in 24-well plates in 10% FBS-supplemented RPMI 1640 medium
for 48 h, subject to different treatments for the indicated times,
collected, and lysed with passive lysis buffer (Promega). Then 50
µL aliquots of the lysates were added to 96-well plates, and
luciferase activity was monitored after adding 100 µL of luciferase
substrate (Promega) to each well by using a MicroLumatPlus
LB96V luminometer (Berthold Technologies, Oak Ridge, TN) with
the WinGlow software package. All transfection experiments were
carried out in six replicates.
Cell Cycle Analysis. LNCaP cells were seeded in six-well plates
(2.5 × 106 cells/well) and treated with different concentrations of
compound 12 for 72 h. After being extensively washed with PBS,
cells were trypsinized followed by fixation in ice-cold 80% ethanol
at 4 °C overnight. Cells were then centrifuged for 5 min at 1500g
at room temperature and stained with propidium iodide (50 µg/
mL) and RNase A (100 units/mL) in PBS. Cell cycle phase
distributions were determined on a FACScort flow cytometer and
analyzed by the ModFitLT V3.0 program.
5-(4-Hydroxy-3-trifluoromethylbenzyl)-3-(1-methylcyclohexyl-
methyl)thiazolidine-2,4-dione (20). A mixture of compound 12
(20 mg) and Pd-C (40 mg) in methanol (5 mL) was stirred under
hydrogen (50 psi) overnight, filtered, and concentrated to dryness
under vacuum. The residue was purified by silica gel flash
chromatography and recrystallized with ethyl acetate–hexane (1:
1
8), giving compound 20 (14 mg). H NMR (250 MHz, CDCl3) δ
0.81 (s, 3H), 1.16–1.59 (m, 10H), 3.13 (dd, 1H, J ) 9.3 Hz, 8.7
Hz), 3.45 (s, 2H), 3.51 (dd, 1H, J ) 9.3 Hz, 3.6 Hz), 4.44 (dd, 1H,
J ) 3.6 Hz, 8.7 Hz), 5.53 (s, 1H), 6.92 (d, 1H, J ) 8.40 Hz), 7.33
(d, 1H, J ) 8.40 Hz), 7.39 (s, 1H), HRMS exact mass of (M +
Na)+, 422.1170 amu; observed mass of (M + Na)+, 422.1173 amu.
Cell Culture. LNCaP androgen-responsive (p53+/+) and PC-3
androgen-nonresponsive (p53-/-) prostate cancer cells were obtained
from the American Type Culture Collection (Manassas, VA) and were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum
at 37 °C in a humidified incubator containing 5% carbon dioxide.
Cell Counting and Cell Viability Assay. LNCaP or PC-3 cells
were placed in six-well plates (2.5 × 105 cells/well) in 10% FBS-
supplemented RPMI 1640 for 24 h and treated with various
concentrations of compound 12 for additional 24, 48, and 72 h.
Cells were then trypsinized and counted by using a Coulter counter
(model Z1 D/T, Beckman Coulter, Fullerton, CA). Cell viability
was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) assay in six replicates in 96-well
plates. LNCaP or PC-3 cells were seeded at 6000 cells per well in
10% FBS-supplemented RPMI 1640 for 24 h, followed by
treatments with various compounds in 5% FBS-supplemented RPMI
1640 at the indicated concentrations. Controls received DMSO at
a concentration equal to that in drug-treated cells. After the end of
incubation, MTT (0.5 mg/mL) in 10% FBS-supplemented RPMI
1640 was added to each well, and cells were incubated at 37 °C
for 2 h. Medium was removed, and the reduced MTT dye was
RT-PCR and Immunoblotting. LNCaP cells were cultured in
T25 flasks at an initial density of 1 × 106 cells/flask. After exposure
to various compounds at the indicated conditions, cells were subject
to total RNA isolation by using an RNeasy mini-kit (QIAGEN,
Valencia, CA). RNA concentrations were determined by measuring
absorption at 260 nm in a spectrophotometer. Aliquots of 6 µg of
total RNA from each sample were reverse-transcribed to cDNA
using an Omniscript RT kit (QIAGEN) according to the manufac-
turer’s instructions. The PCR primers used were as follows.
AR: 5′-ACACATTGAAGGCTATGAATGTC-3′ (forward)
5′-TCACTGGGTGTGGAAATAGATGGG-3′ (reverse)
ꢀ-actin:5′-TCTACAATGAGCTGCGTGTG-3′ (forward)
5′-GGTCAGGATCTTCATGAGGT-3′ (reverse)
PCR reaction products were separated electrophoretically in 1.5%
agarose gels. For immunoblotting, protein extracts were prepared
by M-PER mammalian protein extraction reagent (Pierce, Rockford,
IL) with freshly added 1% phosphatase and protease inhibitor
cocktails (Calbiochem) followed by centrifugation at 13000g for
10 min. Supernatant was collected, and protein concentration was
determined by protein assay reagent (Bio-Rad, CA). Protein extracts
were then suspended in 2× SDS sample buffer and subject to 10%
SDS-polyacrylamide gels. After electrophoresis, proteins were
transferred to nitrocellulose membranes using a semidry transfer
cell. The transblotted membrane was washed twice with Tris-
buffered saline containing 0.1% Tween-20 (TBST). After blocking

NoVel AR-AblatiVe Agents
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with TBST containing 5% nonfat milk for 1 h, the membrane was
incubated with mouse monoclonal anti-AR (Santa Cruz, CA) or
anti-ꢀ-actin (MP Biomedicals) antibodies (diluted 1:1000) in 1%
TBST nonfat milk at 4 °C overnight. After incubation with the
primary antibody, the membrane was washed three times with TBST
for a total of 30 min, followed by incubation with horseradish
peroxidase conjugated goat antimouse IgG (diluted 1:2500) for 1 h
at room temperature. After three thorough washes with TBST for
a total of 30 min, the immunoblots were visualized by enhanced
chemiluminescence.
Immunocytochemical Analysis. Cells were seeded onto cov-
erslips in six-well plates (2.5 × 105 cells/well) for 24 h followed
by exposure to 5 µM compound 12 for an additional 48 h. After
extensive washing with PBS, cells were fixed and permeabilized
with PBS containing 0.1% Triton X-100 for 1 h and then incubated
with anti-AR (1:100 dilution) in PBS containing 0.1% Triton X-100,
0.2% bovine serum albumin, 0.5 mM PMSF, and 1 mM DTT at
room temperature for 12 h followed by Alexa Fluor 488-conjugated
goat antimouse IgG (1:100, Molecular Probes) for 2 h. Nuclear
counterstaining was performed by mounting with 4,6-diamidino-
2-phenylindole (DAPI)-containing medium. Images of immunocy-
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microscope (Eclipse TE300).
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Acknowledgment. This work is supported by National
Institutes of Health Grant CA112250, Department of Defense
Prostate Cancer Research Program Grant W81XWH-05-1-0089,
and grants from William R. Hearst Foundation and Prostate
Cancer Foundation, and the Lucius A. Wing Endowed Chair
Fund at The Ohio State University.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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