Inhibitory effect of 4-aryl 2-substituted aniline-thiazole analogs on growth of human prostate cancer LNCap cells

Article abstract of DOI:10.5012/bkcs.2012.33.1.111

Androgen receptor (AR) is ligand-inducible nuclear hormone receptor which has been focused on key molecular target in growth and progression of prostate cancer. We synthesized a series of 4-aryl 2-substituted aniline-thiazole analogs and evaluated their anti-cancer activity in AR-dependent human prostate cancer LNCap cells. Among them, the compound 6 inhibited the tumor growth in LNCap-inoculated xenograft model.

Full text of DOI:10.5012/bkcs.2012.33.1.111

Inhibitory Effect of 4-Aryl 2-Substituted Aniline-thiazole Analogs
Bull. Korean Chem. Soc. 2012, Vol. 33, No. 1 111
http://dx.doi.org/10.5012/bkcs.2012.33.1.111
Inhibitory Effect of 4-Aryl 2-Substituted Aniline-thiazole Analogs on Growth
of Human Prostate Cancer LNCap Cells
†,a
Seung-hwa Baek, Nakjeong Kim, Seong Hwan Kim, Kwang Hwa Park,
‡,a
†
§,#
¶
Kyung-Chae Jeong, Bae Keun Park, and Nam Sook Kang
#,*
$,*
†
‡
Pharmacology Research Center, Metabolic Syndrome Therapeutics Research Center, Bio-Organic Science Division,
Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu, Daejeon 305-600, Korea
§
#
Department of Pathology, Institute of Lifestyle Medicine, Yonsei University Wonju College of Medicine,
*
Gangwon Province 220-701, Korea. E-mail: baekpark@yonsei.ac.kr
¶
Molecular Oncology Branch, National Cancer Center, Gyeonggi-do 411-769, Korea
$
Graduate School of New Drug Discovery and Development, Chungnam National University, Deajeon 305-764, Korea
*
E-mail: nskang@cnu.ac.kr
Received August 30, 2011, Accepted November 7, 2011
Androgen receptor (AR) is ligand-inducible nuclear hormone receptor which has been focused on key
molecular target in growth and progression of prostate cancer. We synthesized a series of 4-aryl 2-substituted
aniline-thiazole analogs and evaluated their anti-cancer activity in AR-dependent human prostate cancer
LNCap cells. Among them, the compound 6 inhibited the tumor growth in LNCap-inoculated xenograft model.
Key Words : Androgen receptor, Antagonist, LNCap cell, Prostate cancer
Introduction
going to identify new small molecule antagonists of the AR
that are more effective than the current AR antagonists. Our
primary screening efforts produced a series of thiazole-based
compound in AR dependent LNCap cell line, and then we
investigated in vivo activity.
Androgen receptor (AR) is androgen inducible nuclear
hormone receptor and a key molecular target in growth and
progression of prostate cancer, even at the castration-
1,2
resistant status. Prostate cancer (PCa) is the most common
3
cancer form related to death in men. Nonsteroidal AR
Experimental Section
antagonists, preventing androgens from binding AR in the
prostate, have been extensively developed during the past
The 4-aryl-2-anilino-thiazole derivative was identified as
putative androgen receptor (AR) antagonist for the treatment
of prostate cancer through high-throughput screening (HTS)
using the chemical library of Korea Chemical Bank (Figure
2). Furthermore, we synthesized its derivatives to address
moiety of which could be attributing to their biological
activity.
4,5
years. The results of research and development are
exemplified by several marketed drugs as androgen receptor
antagonist including Schering-Plough’ flutamide (1,
®
Eulexin ) launched in 1983, Sanofi-Aventis’ nilutamide (2,
®
Nilandron ) in 1987 and AstraZeneca’ bicalutamide (3,
®
Casodex ) in 1993, as shown in Figure 1. Recent trouble-
shooting in AR antagonist development is that reactivation
of the AR signaling pathway causes the development of
androgen independent prostate cancer. Therefore, we were
According to the Scheme 1, we synthesized 10 compounds,
as follows. All of the 10 compounds were synthesized
6
according to the similar methods described in literatures.
The synthetic route of 4-aryl 2-substituted aniline-thiazole
analogs is shown in Scheme 1. Commercially available
acetophenones 11 was brominated with Br2 in CH2Cl2 to
afford -bromo acetophenones 12. Reaction of -bromoke-
tones 12 with NaSCN in EtOH, followed by condensation
with appropriate substituted-anilines 13 under reflux condi-
tion gave the desired 2-anilino thiazoles 1-4 in moderate
yield. Alternatively, coupling reaction of benzoylisothio-
Figure 1. Marketed non-steroidal androgen receptor antagonists in
recent.
Figure 2. The early hit structure (89% inhibition at 10 uM) obtain-
a
These authors contributed equally to this work.
ed from the high-throughput screening using in-house library.

112 Bull. Korean Chem. Soc. 2012, Vol. 33, No. 1
Seung-hwa Baek et al.
Scheme 1. Synthesis of 1-10, reagents and conditions; (a) Br2, CH2Cl2, rt, 5 h, 85-95%; (b) i) NaSCN, EtOH, reflux, 1h; ii) appropriate
Anilines, EtOH, 16 h, 45-60%; (c) i) acetone, rt, 3 h; ii) 2N-NaOH, THF, rt, 4 h, 70-80%; (d) EtOH, reflux, 12 h, 80-95%.
Table 2. Physicochemical properties, metabolic stability, CYP
inhibition of thiazole derivatives
a
Physicochemical properties
CYP
MS
Compound
PAMPA
logP
permeability
3A4
2D6
(%)
rat,
Table 1. In vitro activities against LNcap cell
(%)
in vitro
IC50 (uM)
5
6
4.71 0.41 −7.0 0.05
4.22 0.40 −6.5 0.05
2.66 0.80 −5.4 0.02
13.55
0
65.20%
Compound
X
Y
LNCap Cell
25.83 17.25 > 99%
11
-
-
-
1
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
H
3-Cl, 4-CN
3-CF3, 4-CN
4-NO2
36.19 2.60
19.05 0.31
13.29 0.49
43.50 2.45
8.65 0.02
9.55 0.35
35.41 0.75
13.72 0.37
5.21 1.10
40.77 2.42
6.46 0.65
2
Inhibition at 10 uM CYP concentration (%)
3
4
3-CF3, 4-NO2
2,4-di-Cl
while compound 11 showed lower logP value though having
good activity. Here we decided the compound 6 and 7 with
proper logP value that well correlate the solubility and
permeability and similar activity to reference compound
come into the further study. Additionally, we tested meta-
bolic stability and CYP effect. Both compounds did not
show the strong CYP inhibition activity, but in evaluation
for metabolic stability in rat, the compound 6, with 99% of
the parent compound remaining after 1 h incubation, showed
to be more stable than the compound 5. Based on the
reported data, we chose the compound 6 for further in vivo
study.
5
6
2-Cl, 4-NO2
2,4-di-Cl
7
8
3-NHSO3Et
4-NO2
4-Cl
3-CN, 4-CF3
4-OH
9
10
4-COOH
Bicalutamide
cyanate with various anilines 13 was carried out in acetone,
and then hydrolyzed with 2N-NaOH to afford substituted
arylthioureas 14, which was reacted with 12 in refluxing
ethanol to give desired compounds 1-10, as shown in Table
7
1, in good yield.
Results and Discussion
The effect of compounds on the viability of human pro-
8
state AR-positive LNCap cells were evaluated. Cell viability
assay revealed that the compounds 5, 6, and 9 showed the
potent inhibitory activity in the viability of LNCap cells,
having stronger or similar activities to the reference compound,
bicalutamide. Before evaluating the therapeutic activity of 4-
aryl 2-substituted aniline-thiazole analogs in the AR-positive
prostate cancer, we further determined physicochemical
9,10
11
metabolic stability and CYP inhibition
properties,
12
activity of compound 5, 6, and 9 that strongly inhibited the
viability of LNCap cell. As shown in Table 2, two
compounds, 5 and 6 showed appropriate logP values and
medium permeability against artificial permeable membrane,
Figure 3. Effect of compound 6 on tumor growth in LNCap-
inoculated xenograft model.

Inhibitory Effect of 4-Aryl 2-Substituted Aniline-thiazole Analogs
Bull. Korean Chem. Soc. 2012, Vol. 33, No. 1 113
0014248).
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Figure 4. The calculated binding mode for compound 6 based on
the bicalutamide complex structure (pdb code; 1Z95). Green
element colored molecule, compound 6, gray element colored
molecule, bicalutamide. W represents water molecules. Hydrogen
bonding interactions are displayed in blue dashed lines.
6. (a) Bursavich, M. G.; Paker, D. P.; Willardsen, J. A.; Gao, Z.;
Davis, T.; Ostanin, K.; Robinson, R.; Peterson, A.; Cimbora, D.
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Aggarwal, R.; Prakash, O.; Singh, S. P. Synth. Commun. 2007, 37,
2501. (d) Korea Patent. 2008-0109206 (published No)
Next, the therapeutic activity of the compound 6 was
13
evaluated in LNCap-inoculated xenograft model. As shown
1
7. Analytical data for the compounds ( H NMR 300 MHz);
in Figure 3, the compound 6 attenuated the tumor growth as
observed in the bicalutamide-treated group, suggesting its in
vivo inhibitory effect on the growth of AR-positive prostate
cancer.
o
1
compound (1): yellow solid. mp 223-224 C. H NMR (DMSO-
d6) 10.94 (brs, 1H), 8.70 (s, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.18 (d,
J = 8.1 Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.85 (s, 1H), 7.81 (d, J =
+
8.7 Hz, 2H), 7.76 (dd, 1H): MS (m/e, 70 eV): 322 (M );
o
1
compound (2): yellow solid. mp 227-228 C. H NMR (DMSO-
d6) 11.43 (brs, 1H), 8.72 (m, 2H), 8.38 (d, J = 7.8 Hz, 1H), 8.27 (d,
J = 9.0 Hz, 1H), 8.20 (d, J = 7.2 Hz, 1H), 7.94 (s, 1H), 7.92 (d, J =
7.2 Hz, 1H), 7.75 (dd, J = 9.0, 7.8 Hz, 1H) ): MS (m/e, 70 eV):
Also, we carried out the docking study to confirm the AR
binding mode for compound 6. The AR complex structure as
reference was obtained from the x-ray crystal structure of the
mutant W741L AR bound to bicalutamide (pdb code:
+
o
1
357 (M ); compound (3): yellow solid. mp 235-236 C. H NMR
(DMSO-d6) 11.35 (brs, 1H), 8.70 (m, 2H), 8.35 (d, J = 7.8 Hz,
1H), 8.18 (d, J = 7.8, 1.8 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.91 (s,
1H), 7.84 (dd, J = 8.7, 1.5 Hz, 1H), 7.74 (dd, 1H): MS (m/e, 70
14
1Z95), replacing Leu741 in 1Z95 by the wild-type Trp,
with adjustment of the conformation of the nearby Met895.
15
Docking study was carried out using LigandFit module
+
o
1
eV): 390 (M ); compound (4): yellow solid. mp 216-217 C. H
NMR (DMSO-d6) 11.18 (brs, 1H), 8.72 (s, 1H), 8.44 (d, J = 7.8
Hz, 1H), 8.27 (d, J = 9.3 Hz, 2H), 8.20 (d, J = 8.1 Hz, 1H), 7.93
(d, J = 9.3 Hz, 2H), 7.91 (s, 1H), 7.76 (dd, J = 8.1, 7.8 Hz, 1H):
in DiscoveryStudio 3.1 with CHARMM force field. The
experimental binding conformation of bicalutamide was
well created showing score value of 65.28 with Dock_Score
in LigandFit. Compound 6 was well fitted in the binding site
with the Dock_Score of 50.91 as shown in Figure 4. The two
terminal nitro groups also have H-bonding interactions with
Arg752 and two water molecules such as bicalutamide. As
docking study, we can confirm compound 6 interacts with
AR binding site in vitro.
+
MS (m/e, 70 eV): 342 (M ); compound (5): yellow solid. mp 230-
o
1
232 C. H NMR (DMSO-d6) 11.15 (brs, 1H), 8.71 (s, 1H), 8.36
(d, J = 7.8 Hz, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.19 (dd, J = 8.1, 2.1
Hz, 1H), 7.90 (d, J = 8.7 Hz, 1H), 7.89 (s, 1H), 7.76 (dd, 1H), 7.64
+
(dd, J = 8.7, 1.8 Hz, 1H): MS (m/e, 70 eV): 410 (M ); compound
o
1
(6): yellow solid. mp 258-260 C. H NMR (DMSO-d6) 10.47
(brs. 1H), 8.91 (d, J = 9.3 Hz, 1H), 8.68 (m, 1H), 8.40 (d, J = 7.8
Hz, 1H), 8.35 (d, J = 2.7 Hz, 1H), 8.27 (dd, J = 9.3, 2.7 Hz, 1H),
8.17 (d, J = 7.8 Hz, 1H), 7.96 (s, 1H), 7.74 (dd, 1H) ): MS (m/e, 70
+
o
1
Conclusion
eV): 376 (M ); compound (7): yellow solid. mp 197-198 C. H
NMR (DMSO-d6) 9.95 (brs, 1H), 8.64 (s, 1H), 8.47 (d, J = 9.0 Hz,
1H), 8.33 (d, J = 7.8 Hz, 1H), 8.15 (dd, J = 7.8, 2.1 Hz, 1H), 7.76
(s, 1H), 7.71 (dd, 1H), 7.65 (d, J = 2.7 Hz, 1H), 7.46 (dd, J = 9.0,
In this paper, we synthesized a series of 4-aryl 2-sub-
stituted aniline-thiazole analogs showing the similar binding
mode to bicalutamide and evaluated their anti-cancer
activity in AR-dependent human prostate cancer LNCap
cells. Among them, the compound 6 inhibited the tumor
growth in LNCap-inoculated xenograft model. Furthermore,
we are going to deeply optimize for our compounds to
develop the drug for prostate cancer treatment.
+
2.7 Hz, 1H): MS (m/e, 70 eV): 366 (M ); compound (8): white
o
1
solid. mp 193-194 C. H NMR (DMSO-d6) 9.84 (brs, 1H), 8.57
(d, J = 8.6 Hz, 1H), 7.88 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 2.4 Hz,
+
1H), 7.41 (m, 4H), 7.30 (m, 1H): MS (m/e, 70 eV): 321 (M );
o
1
compound (9): yellow solid. mp 205-206 C. H NMR (DMSO-
d6) 9.95 (brs, 1H), 8.52 (d, J = 9.0 Hz, 1H), 8.25 (d, J = 8.7 Hz,
2H), 8.33 (d, J = 8.7 Hz, 1H), 7.79 (s, 1H), 7.63 (d, J = 2.4 Hz,
+
1H), 7.45 (dd, J = 9.0, 2.4 Hz, 1H) ): MS (m/e, 70 eV): 366 (M ).
8. Cell culture and viability assay. Human prostate cancer LNCap
cells were cultured in RPMI1640 supplemented with 10% fetal
bovine serum (FBS), 100 U/mL of penicillin and 100 µg/mL of
streptomycin in humidified atmosphere of 5% CO2 at 37 °C. The
culture medium was changed every 3 days. For viability assay,
Acknowledgments. This work was supported by Ministry
of Education, Science and Technology (MEST), Korea.
BKP was supported in part by the National Research
Foundation of Korea grant by MEST, Korea (No. 2010-

114 Bull. Korean Chem. Soc. 2012, Vol. 33, No. 1
Seung-hwa Baek et al.
3
cells were seeded in a 96-well plate at 4 × 10 cells/well, cultured
phosphate dehydrogenase in 10 mM KPO4, pH 8.0) was added to
each well of the microtiter plate followed by the vehicle
acetonitrile (control) and the test samples. The plate was covered
and then incubated at 37 °C for 20 min. Enzyme reaction was
initiated by the addition of enzyme/substrate (E/S) mixture (0.5
for 24 h and then incubated with a compound for 48 h. Then, cell
viability was measured in triplicates by the Cell Counting Kit-8
(Dojindo Molecular Technologies, ML) according to the manu-
facturer’s protocol. Absorbance was measured by using Wallac
EnVision microplate reader (PerkinElmer, Finland).
pmol recombinant human CYP3A4 enzyme and
5 mM
9. logP. LogP is measured by a pH-metric method, based on a two-
phase acid-base titration in a mixture of water and octanol using
GLpKa system by Sirius.
dibenzylfluorescein, DBF). The plate was further incubated for 20
min, followed by the addition of the stop solution to terminate the
enzyme activity. Background reading was measured in a similar
manner except for the E/S mixture which was added after the
enzyme reaction was terminated. The fluorescence of DBF
metabolite fluorescein was measured on a fluorescence plate
reader with an excitation wavelength of 485 nm and an emission
wavelength of 530 nm.
10. Parallel artificial membrane permeability (PAMPA) perme-
ability. Donor solution (500 µM) was prepared by diluting 1 mM
DMSO compound stock solution using the diluted system buffer
(pH adjusted to 7.4). Five microliters lipid solution was applied to
each filter membrane. Donor solution (150 µL) was added to each
well of the filter plate. The filter plate was then put onto a receiver
plate with preloaded acceptor sink buffer. The sandwich was
incubated at room temperature for 16 h. Samples were taken from
both receiver and donor sides at the end of the incubation and
analyzed using UV spectrometer. Donor solutions were also
analyzed for initial concentration.
13. Animal experiment was in accordance with the guidelines of the
Yonsei University Institutional Animal Care and Use Committee
at Wonju campus (IACUC approval number YWC-101015-1).
6
Human prostate cancer LNCap cells (1 × 10 ) were implanted
subcutaneously on the left and right flank region of each
immunodeficient male mouse (Orientbio Inc., Korea; six-week-
old, 17-19 g), respectively. Ten days after transplantation, five
mice per cohort were treated orally by gavage (p.o.) five times per
week for 4 weeks with the 200 µg or 1,000 µg of test compounds
including bicalutamide (AstraZeneca) for a model reference
control dissolved in 1% carboxylmethyl cellulose/0.1% Tween-
80/5% dimethyl sulfoxide (10 mg or 50 mg per kg body weight).
The volume of prostate tumor was quantified repeatedly every
three days from the day of treatment through oral route.
11. Metabolic stability. Using rat liver microsomes, the amount of
parent compound remaining after 1 h incubation. The concen-
tration of the used compound is 5 mM and protein concentration is
1 mg/mL.
12. CYP3A4 enzyme assay. The assay was carried out using fluoro-
metric enzyme assays with Vivid CYP3A4 assay kit (PanVera,
USA, CA) in a 96-well microtiter plate following the manu-
facturer’s instruction with some modification. The compounds
including ketoconazole known as CYP3A4 inhibitor were
prepared in acetonitrile to give final concentrations of 10 mM.
NADP generating solution (1.0 mM NADPþ, 3.3 mM glucose-6-
phosphate, 3.3 mM MgCl2·6H2O, and 0.4 U/mL glucose-6-
14. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T. Proc.
Natl. Acad Sci. U S A. 2005, 102, 6201.
15. Venkatachalam, C. M.; Jiang, X.; Oldfield, T.; Waldman, M.
Journal of Mol. Graphics and Modelling 2003, 21, 28.