Discovery of novel 5-methyl-1H-pyrazole derivatives as potential antiprostate cancer agents: Design, synthesis, molecular modeling, and biological evaluation

Article abstract of DOI:10.1111/cbdd.13173

Androgen receptor (AR) signaling functions as a core driving force for the progression of prostate cancer (PCa), and AR has been proved to be an effective therapeutic target even for castration-resistant prostate cancer (CRPC). Herein, structural modification via a fragments splicing strategy was performed based on two lead compounds T3 and 10e, leading to the discovery of a series of 5-methyl-1H-pyrazole derivatives. AR reporter gene assay revealed compounds A13 and A14 as potent AR antagonists. Some of the compounds in this series inhibited growth of PCa LNCaP cells more efficiently than enzalutamide. A13 and A14 also showed improved metabolic stability compared with 10e in human liver microsomes.

Full text of DOI:10.1111/cbdd.13173

DR DAOGUANG ZHANG (Orcid ID : 0000-0002-4627-5400)
Article type
: Research Article
Discovery of novel 5-methyl-1H-pyrazole derivatives as potential
anti-prostate cancer agents: design, synthesis, molecular modeling and
biological evaluation
Daoguang Zhanga, , Solomon Asnakeb, , Jingya Zhanga, Per-Erik Olssonb,*, Guisen Zhaoa,*
aKey Laboratory of Chemical Biology (Ministry of Education), Department of Medicinal
Chemistry, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong, PR
China
bBiology, The Life Science Center, School of Science and Technology, Örebro University,
Örebro, Sweden
These authors contributed equally to this work.
*Corresponding Author
aDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong
University, Jinan, Shandong, PR China. E-mail: guisenzhao@sdu.edu.cn.
bBiology, The Life Science Center, School of Science and Technology, Örebro University,
Örebro, Sweden. E-mail: Per-Erik.Olsson@oru.se
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13173
This article is protected by copyright. All rights reserved.

ABSTRACT
Androgen receptor (AR) signaling functions as a core driving force for the progression
of prostate cancer (PCa), and AR has been proved to be an effective therapeutic target even
for castration resistant prostate cancer (CRPC). Herein, structural modification via a
fragments splicing strategy was performed based on two lead compounds T3 and 10e, leading
to the discovery of a series of 5-methyl-1H-pyrazole derivatives. AR reporter gene assay
revealed compounds A13 and A14 as potent AR antagonists. Some of the compounds in this
series inhibited growth of PCa LNCaP cells more efficiently than enzalutamide. A13 and A14
also showed improved metabolic stability comparing with 10e in human liver microsomes.
Key words: prostate cancer, androgen receptor, AR antagonist, pyrazole derivative,
luciferase reporter gene assay.
1. INTRODUCTION
Androgen receptor (AR) signaling functions as a core driving force for the progression
of prostate cancer (PCa). AR, belonging to the nuclear receptor superfamily, is a crucial
ligand-induced transcription factor in this signaling1. Generally, androgen deprivation therapy
(ADT) by surgical or chemical castration is the standard treatment for PCa2. However,
patients develop resistance to this androgen blockade strategy and tumor progresses into a
state termed castration resistant PCa (CRPC)3. Massive efforts have been dedicated exploring
the resistance mechanism of CRPC. Several mechanisms have been illuminated, including
ligand independent AR activation4, AR gene amplification5, conversion of adrenal androgen
to dihydrotestosterone (DHT)6, and intratumoral de-novo androgen synthesis7. It has been
confirmed that even in the castration resistant state, prostate cancer remains hormone driven.
Targeting AR by antiandrogens remains an effective therapeutic option against CRPC.
Development of non-steroidal AR antagonists targeting the AR ligand binding domain (LBD)
has experienced two generations. The first generation, represented by flutamide and
This article is protected by copyright. All rights reserved.

bicalutamide (Fig. 1), achieved significant clinical success. However, drug resistance seems
inevitable after long time usage. A T877A point mutation of AR LBD was identified to be
responsible for the resistance to hydroxyflutamide, while a W741L mutation was responsible
for an antagonist to agonist switch of bicalutamide8. Seeking the next generation of AR
antagonists prompted the discovery of enzalutamide (Enz, Fig. 1) which was optimized from
a screening for nonsteroidal antiandrogens showing activity under condition of increased AR
expression. Enzalutamide is a more potent AR-LBD ligand with an 8-fold higher affinity than
bicalutamide. Enzalutamide can also prevent AR nuclear translocation and impair both DNA
binding to androgen response elements (AREs) and recruitment of coactivators9. More
candidates have entered into clinical stages, like ARN-509 and ODM-201 (Fig. 1) both in
phase 3 clinical trials.
Our laboratory once identified a pyrazole derived molecule T3 (Fig. 2) as a potential
AR-LBD ligand via a pharmacophore-based virtual screening10. In vitro evaluation revealed
that T3 could effectively inhibit the proliferation of PCa LNCaP cells and downregulate the
level of prostate specific antigen (PSA, a classic biomarker of PCa). A structural modification
of T3 was subsequently carried out, leading to the discovery of a
3-(4-fluorophenyl)-1H-pyrazole derivative 10e (Fig. 2) which maintained high anti-prostate
cancer potency against both PCa cell proliferation and PSA expression11. Compounds T3 and
10e both possessed a 4-phenylpyrazole moiety which was also shared by ODM-201, a highly
affinitive AR antagonist in phase 3 clinical stage. Combining with the benzamide motif of
Enzalutamide/ARN-509, a series of 5-methyl-1H-pyrazole derivatives were designed using a
fragments splicing strategy (Fig. 2). 22 compounds were thus synthesized and evaluated for
their AR antagonistic activity and antiproliferative activity against two PCa cell lines (LNCaP
and PC-3). In vitro AR activation study was conducted to determine their ability as either AR
agonists or antagonists, as well as an in silico docking study to analyze for their ability to
bind with the AR-LBD.
This article is protected by copyright. All rights reserved.

2. Materials and methods
2.1. Chemistry
All the chemical reagents and solvents were purchased from commercial sources and
used without further purification. Reactions were monitored using thin-layer chromatography
(TLC) performed on SGF254 plates. Chromatographic separations were performed using
column chromatography on silica gel (60 Å, 200–300 mesh). Melting points were determined
on a Büchi capillary melting point apparatus (Buchi Labortechnik AG, Switzerland) without
correction. The 1H and 13C NMR spectra were recorded at 400 MHz for 1H and 100 MHz for
13C on a Bruker Avance DRX-400 Spectrometer (Bruker, Germany). Deuterated
dimethylsulfoxide (DMSO-d6) was used as the NMR solvent. The chemical shifts (δ) were
reported in parts per million (ppm) using tetramethylsilane as internal standard. Peak
multiplicities are expressed as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), broad singlet (br s), doublet of doublets (dd), doublet of triplet (dt), and quartet of
doublets (qd). All compounds tested in biological assays were > 95% pure.
2.1.1. General procedure for the synthesis of intermediate 2
Proper substituted benzaldehyde (1, 5.0 mmol) and morpholine trifluoroacetate (201 mg,
1.0 mmol) were dissolved in excess acetone (30 ml), this mixture was sealed in a vial, heated
to 75 , and stirred for 12 hrs. Then the reaction mixture was cooled to ambient temperature
and excess acetone was evaporated under vacuum. Saturated aqueous sodium hydrogen
carbonate solution (NaHCO3, 30 ml) and ethyl acetate (EA, 30 ml) were added. After phase
separation the aqueous phase was extracted three times by EA (3 × 15 ml), the organic layers
were joint together, washed by saturated brines (20 ml), dried over sodium sulfate (Na2SO4)
and concentrated under vacuum to give intermediate 2 which was directly used for the next
step without further purification.
This article is protected by copyright. All rights reserved.

(E)-4-(4-bromophenyl)but-3-en-2-one (A3-2)
Light yellow solid, yield 89%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.68 (d, J = 8.4 Hz,
2H), 7.65 – 7.59 (m, 3H), 6.84 (d, J = 16.4 Hz, 1H), 2.34 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 198.56, 142.27, 134.24, 132.42, 130.78, 128.44, 124.25, 27.89.
(E)-4-(3-oxobut-1-en-1-yl)benzonitrile (A-2)
White solid, yield 93%. 1H NMR (400 MHz, DMSO) δ 7.93 – 7.89 (m, 4H), 7.68 (d, J = 16.4
Hz, 1H), 6.97 (d, J = 16.4 Hz, 1H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO) δ 197.55,
140.37, 138.53, 132.18, 129.53, 128.41, 117.99, 111.65, 26.99.
2.1.2. General procedure for the synthesis of intermediate 3
Intermediate 2 (3.0 mmol) and p-toluenesulfonhydrazide (615 mg, 3.3 mmol) were
dissolved in acetonitrile (10 ml) and stirred at room temperature for 3 hrs. Then sodium
hydroxide (NaOH, 132 mg, 3.3 mmol) were added and the reaction mixture was heated to
reflux for an additional 12 hrs. The reaction mixture was then cooled to ambient temperature,
EA (30 ml) and water (30 ml) were added for an extraction. The organic layer was then
washed with brine (20 ml), dried over sodium sulfate, filtrated and concentrated under
vacuum. The crude product was purified by column chromatography (petroleum ether/EA =
5/1).
3-(3,4-difluorophenyl)-5-methyl-1H-pyrazole (A9-3)
Yellow solid, yield 55%. 1H NMR (400MHz, DMSO-d6) δ (ppm):7.76 (ddd, J1 = 12.0 Hz, J2
= 8.0 Hz, J3 = 2.0 Hz, 1H), 7.61 – 7.58 (m, 1H), 7.44 (dt, J1 = 10.4 Hz, J2 = 8.8 Hz, 1H), 6.50
(s, 1H), 2.25 (s, 3H). 13C NMR (101 MHz, DMSO) δ 151.43, 151.30, 150.40, 150.27, 149.06,
149.04, 149.00, 148.87, 147.96, 147.84, 140.36, 132.37, 122.02, 121.99, 118.19, 118.02,
114.17, 114.00, 101.75, 10.89.
This article is protected by copyright. All rights reserved.

2.1.3. General procedure for the synthesis of 5
4-bromomethylbenzoic acid (4, 1.08 g, 5.0 mmol) was suspended in dichloromethane
(DCM, 30 ml), excess oxalyl chloride (2.54 g, 20.0 mmol) was added following by two drops
of N,N-dimethylformamide (DMF). This mixture was stirred at room temperature until
insoluble substance completely dissolved. Then the solvent was evaporated under vacuum,
and the residue was dissolved in tetrohydrofuran (THF, 10 ml). This acyl chloride solution
was then added drop wise to a THF (20 ml) solution of appropriate amine (5.0 mmol) and
triethylamine (TEA, 1.01 g, 10.0 mmol) cooled in an ice-water bath. The reaction mixture
was stirred for an additional hour, then diluted with EA (50 ml), and washed with water (30
ml) and brine (30 ml). The organic layer was dried over Na2SO4. After filtration, removing
solvent under vacuum afforded intermediate 5 which can be used for the next step without
further purification.
4-(bromomethyl)-N-methylbenzamide (A13-5)
White solid, yield 87%. 1H NMR (400 MHz, DMSO) δ 8.25 (d, J = 7.6 Hz, 1H), 7.82 (d, J =
8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 4.74 (s, 2H), 4.14 – 4.05 (m, 1H), 1.16 (d, J = 6.6 Hz,
6H). 13C NMR (101 MHz, DMSO) δ 166.60, 141.36, 134.79, 129.65, 127.90, 34.04, 26.72.
4-(bromomethyl)-N-(tert-butyl)benzamide (A-5)
White solid, yield 91%. 1H NMR (400 MHz, DMSO) δ 7.78 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H),
7.49 (d, J = 8.2 Hz, 2H), 4.74 (s, 2H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.33,
141.07, 136.19, 129.40, 128.20, 51.28, 34.14, 29.03.
This article is protected by copyright. All rights reserved.

2.1.4. General procedure for the synthesis of target compound A
Pyrazole intermediate 3 (1.0 mmol) and 4-bromomethylbenzamide intermediate 5 (1.0
mmol) were dissolved in acetonitrile (10 ml), and NaOH (60 mg, 1.5 mmol) was added
subsequently. This mixture was stirred at room temperature for 2 hrs. Then EA (30 ml) and
water (30 ml) were added, and the organic layer was washed with brine (20 ml), dried over
anhydrous Na2SO4, filtered, and concentrated under vacuum. Purification by chromatography
on silica gel (petroleum ether/EA = 5/1 ~ 1/1) afforded the desired compound A.
N-(tert-butyl)-4-((3-(4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide (A1)
White solid, yield 62%. Mp: 155 – 158 . 1H NMR (400 MHz, DMSO) δ 7.82 - 7.77 (m,
2H), 7.74 (d, J = 8.3 Hz, 2H), 7.69 (s, 1H), 7.24 - 7.18 (m, 4H), 6.54 (s, 1H), 5.39 (s, 2H),
2.23 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.52, 163.27, 160.85, 148.74,
140.64, 140.54, 135.60, 130.55, 130.52, 128.16, 127.36, 127.28, 126.96, 115.99, 115.78,
103.29, 52.32, 51.20, 29.03, 11.26.
N-(tert-butyl)-4-((3-(4-chlorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide (A2)
White solid, yield 50%. Mp: 161 – 163 . 1H NMR (400 MHz, DMSO) δ 7.78 (d, J = 8.5 Hz,
2H), 7.75 (d, J = 8.2 Hz, 2H), 7.70 (s, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H),
6.58 (s, 1H), 5.40 (s, 2H), 2.24 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.52,
148.49, 140.77, 140.44, 135.63, 132.84, 132.22, 129.08, 128.17, 127.09, 126.99, 103.53,
52.39, 51.21, 29.03, 11.27.
This article is protected by copyright. All rights reserved.

4-((3-(4-bromophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-(tert-butyl)benzamide (A3)
Off white solid, yield 52%. Mp: 132 – 134 . 1H NMR (400 MHz, DMSO) δ 7.74 (d, J = 8.3
Hz, 2H), 7.72 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 6.58 (s,
1H), 5.39 (s, 2H), 2.24 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.51, 148.51,
140.79, 140.42, 135.62, 133.18, 131.99, 128.17, 127.42, 126.99, 120.75, 103.53, 52.40,
51.21, 29.03, 11.27.
N-(tert-butyl)-4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide (A4)
Off white solid, yield 57%. Mp: 172 – 174 . 1H NMR (400 MHz, DMSO) δ 7.95 (d, J = 8.5
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.3 Hz, 2H), 7.70 (s, 1H), 7.21 (d, J = 8.3 Hz,
2H), 6.73 (s, 1H), 5.43 (s, 2H), 2.26 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ
166.50, 147.96, 141.20, 140.19, 138.32, 135.69, 133.17, 128.20, 127.04, 125.96, 119.49,
109.99, 104.45, 52.58, 51.21, 29.03, 11.29.
N-(tert-butyl)-4-((5-methyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)methyl)benza
mide (A5)
White solid, yield 58%. Mp: 152 – 155 . 1H NMR (400 MHz, DMSO) δ 7.98 (d, J = 8.1 Hz,
2H), 7.75 (d, J = 3.5 Hz, 2H), 7.73 (d, J = 3.4 Hz, 2H), 7.70 (s, 1H), 7.20 (d, J = 8.3 Hz, 2H),
6.69 (s, 1H), 5.43 (s, 2H), 2.26 (s, 3H), 1.35 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.50,
148.17, 141.05, 140.30, 137.83, 135.66, 128.19, 127.02, 126.07, 126.03, 125.89, 123.51,
104.15, 52.51, 51.21, 29.03, 11.29.
N-(tert-butyl)-4-((3-(3-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide (A6)
White solid, yield 54%. Mp: 100 – 102 . 1H NMR (400 MHz, DMSO) δ 7.75 (d, J = 8.3 Hz,
2H), 7.70 (s, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.54 (ddd, J = 10.6, 2.5, 1.5 Hz, 1H), 7.42 (td, J =
This article is protected by copyright. All rights reserved.

8.0, 6.3 Hz, 1H), 7.20 (d, J = 8.3 Hz, 2H), 7.13 - 7.08 (m, 1H), 6.63 (s, 1H), 5.40 (s, 2H), 2.24
(s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.51, 164.26, 161.85, 148.50, 148.48,
140.79, 140.41, 136.47, 136.38, 135.63, 131.13, 131.04, 128.17, 126.98, 121.45, 121.43,
114.52, 114.31, 111.95, 111.73, 103.82, 52.42, 51.21, 29.03, 11.28.
N-(tert-butyl)-4-((3-(3-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide (A7)
White solid, yield 66%. Mp: 110 – 112 . 1H NMR (400 MHz, DMSO) δ 8.17 (s, 1H), 8.12 -
8.09 (m, 1H), 7.76 - 7.73 (m, 3H), 7.70 (s, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 8.2 Hz,
2H), 6.71 (s, 1H), 5.42 (s, 2H), 2.25 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ
166.50, 147.71, 141.05, 140.27, 135.67, 135.14, 131.30, 130.44, 129.86, 128.65, 128.19,
127.03, 119.24, 112.32, 103.97, 52.50, 51.21, 29.03, 11.31.
N-(tert-butyl)-4-((5-methyl-3-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)methyl)benza
mide (A8)
White solid, yield 75%. Mp: 117 – 120 . 1H NMR (400 MHz, DMSO) δ 8.07 - 8.05 (m,
2H), 7.75 (d, J = 8.3 Hz, 2H), 7.70 (s, 1H), 7.64 - 7.63 (m, 2H), 7.20 (d, J = 8.3 Hz, 2H), 6.73
(s, 1H), 5.44 (s, 2H), 2.26 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.49,
148.13, 141.03, 140.35, 135.63, 134.94, 130.29, 130.15, 129.83, 129.27, 129.26, 128.18,
126.94, 126.07, 124.25, 124.21, 123.36, 121.44, 121.40, 103.88, 52.47, 51.20, 29.02, 11.29.
N-(tert-butyl)-4-((3-(3,4-difluorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide
(A9)
White solid, yield 49%. Mp: 165 – 167 . 1H NMR (400 MHz, DMSO) δ 7.78 - 7.73 (m,
3H), 7.70 (s, 1H), 7.63 - 7.60 (m, 1H), 7.44 (dt, J = 10.6, 8.6 Hz, 1H), 7.20 (d, J = 8.3 Hz,
2H), 6.62 (s, 1H), 5.40 (s, 2H), 2.24 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ
166.51, 151.46, 151.33, 150.53, 150.41, 149.03, 148.90, 148.09, 147.97, 147.78, 140.92,
This article is protected by copyright. All rights reserved.

140.36, 135.64, 131.77, 131.73, 131.70, 131.67, 128.18, 126.98, 122.10, 122.07, 122.04,
122.00, 118.30, 118.13, 114.22, 114.04, 103.71, 52.40, 51.20, 29.02, 11.26.
N-(tert-butyl)-4-((3-(3-chloro-4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzam
ide (A10)
Off white solid, yield 53%. Mp: 160 – 162 . 1H NMR (400 MHz, DMSO) δ 7.92 (dd, J =
7.3, 2.1 Hz, 1H), 7.78 - 7.74 (m, 3H), 7.70 (s, 1H), 7.43 (t, J = 9.0 Hz, 1H), 7.20 (d, J = 8.3
Hz, 2H), 6.63 (s, 1H), 5.40 (s, 2H), 2.24 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ
166.51, 158.21, 155.76, 147.51, 140.95, 140.35, 135.64, 131.92, 131.88, 128.18, 127.05,
127.00, 125.91, 125.84, 120.39, 120.21, 117.75, 117.54, 103.70, 52.41, 51.21, 29.03, 11.28.
4-((3-(3-bromo-4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-(tert-butyl)benza
mide (A11)
Light yellow solid, yield 53%. Mp: 162 – 165 .1H NMR (400 MHz, DMSO) δ 8.05 (dd, J =
6.8, 2.1 Hz, 1H), 7.80 (ddd, J = 8.5, 4.8, 2.1 Hz, 1H), 7.75 (d, J = 8.3 Hz, 3H), 7.40 (t, J = 8.7
Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H), 6.64 (s, 1H), 5.40 (s, 2H), 2.24 (s, 3H), 1.36 (s, 9H). 13C
NMR (101 MHz, DMSO) δ 166.51, 159.24, 156.81, 147.39, 140.93, 140.37, 135.60, 132.20,
132.17, 129.83, 128.20, 126.99, 126.60, 126.52, 117.59, 117.36, 108.94, 108.73, 103.70,
52.38, 51.20, 29.01, 11.29.
N-(tert-butyl)-4-((3-(3,4-dimethoxyphenyl)-5-methyl-1H-pyrazol-1-yl)methyl)benzamide
(A12)
Light yellow solid, yield 68%. Mp: 75 – 77 . 1H NMR (400 MHz, DMSO) δ 7.74 (d, J = 8.2
Hz, 2H), 7.70 (s, 1H), 7.33 (d, J = 1.8 Hz, 1H), 7.28 (dd, J = 8.3, 1.9 Hz, 1H), 7.17 (d, J = 8.2
Hz, 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.51 (s, 1H), 5.38 (s, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 2.21
(s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO) δ 166.52, 149.69, 149.32, 148.83, 140.78,
This article is protected by copyright. All rights reserved.

140.34, 135.52, 128.14, 126.96, 126.79, 117.93, 112.38, 109.04, 103.08, 55.99, 55.94, 52.21,
51.20, 29.03, 11.25.
4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-methylbenzamide (A13)
White solid, yield 63%. Mp: 201 – 204 .1H NMR (400 MHz, DMSO) δ 8.43 (d, J = 4.5 Hz,
1H), 7.96 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 8.2 Hz, 2H), 7.22 (s, 2H),
6.74 (s, 1H), 5.45 (s, 2H), 2.77 (d, J = 4.5 Hz, 3H), 2.27 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 166.73, 147.98, 141.26, 140.55, 138.28, 134.24, 133.19, 127.92, 127.23, 125.96,
119.51, 109.97, 104.46, 52.49, 26.69, 11.30.
4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-ethylbenzamide (A14)
White solid, yield 71%. Mp: 196 – 197 . 1H NMR (400 MHz, DMSO) δ 8.43 (t, J = 5.4 Hz,
1H), 7.95 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.22 (d, J =
8.3 Hz, 2H), 6.74 (s, 1H), 5.44 (s, 2H), 3.30 - 3.23 (m, 2H), 2.26 (s, 3H), 1.10 (t, J = 7.2 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 166.03, 147.97, 141.24, 140.52, 138.28, 134.42, 133.19,
127.98, 127.21, 125.96, 119.50, 109.97, 104.46, 52.52, 34.47, 15.27, 11.30.
4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-isopropylbenzamide (A15)
Off white solid, yield 82%. Mp: 195 – 197 . 1H NMR (400 MHz, DMSO) δ 8.17 (d, J = 7.7
Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.3 Hz, 2H), 7.23 (d,
J = 8.3 Hz, 2H), 6.73 (s, 1H), 5.44 (s, 2H), 4.08 (dq, J = 13.3, 6.6 Hz, 1H), 2.26 (s, 3H), 1.15
(d, J = 6.6 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 165.52, 147.98, 141.22, 140.42, 138.32,
134.65, 133.17, 128.10, 127.16, 125.97, 119.48, 109.99, 104.45, 52.57, 41.43, 22.78, 11.29.
This article is protected by copyright. All rights reserved.

4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N,N-dimethylbenzamide (A16)
Off white solid, yield 69%. Mp: 152 – 155 . 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.5
Hz, 2H), 7.85 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.75 (s,
1H), 5.44 (s, 2H), 2.96 (s, 3H), 2.87 (s, 3H), 2.28 (s, 3H). 13C NMR (101 MHz, DMSO) δ
170.22, 147.98, 141.27, 138.82, 138.28, 136.10, 133.20, 127.84, 127.15, 125.96, 119.51,
109.96, 104.47, 52.46, 35.17, 11.32.
4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N,N-diisopropylbenzamide
(A17)
Off white solid, yield 74%. Mp: 192 – 193 . 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.5
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 6.75 (s,
1H), 5.42 (s, 2H), 3.59 (br s, 2H), 2.29 (s, 3H), 1.40 (br s, 6H), 1.07 (br s, 6H). 13C NMR
(101 MHz, DMSO) δ 169.88, 147.96, 141.25, 138.54, 138.29, 137.93, 133.19, 127.42,
126.21, 125.95, 119.51, 109.96, 104.44, 52.45, 20.79, 11.35.
4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-(2-hydroxyethyl)benzamide
(A18)
White solid, yield 66%. Mp: 167 – 170 . 1H NMR (400 MHz, DMSO) δ 8.40 (t, J = 5.6 Hz,
1H), 7.95 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 6.4 Hz, 2H), 7.82 (d, J = 6.1 Hz, 2H), 7.23 (d, J =
8.3 Hz, 2H), 6.73 (s, 1H), 5.44 (s, 2H), 4.71 (t, J = 5.6 Hz, 1H), 3.50 (q, J = 6.1 Hz, 2H), 3.32
(q, overlapped with water, J = 6.0 Hz, 2H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSO) δ
166.44, 147.99, 141.25, 140.58, 138.31, 134.32, 133.18, 128.07, 127.20, 125.98, 119.49,
110.00, 104.47, 60.22, 52.55, 42.62, 11.30.
This article is protected by copyright. All rights reserved.

4-(5-methyl-1-(4-(pyrrolidine-1-carbonyl)benzyl)-1H-pyrazol-3-yl)benzonitrile (A19)
Light yellow solid, yield 63%. Mp: 150 – 152 .1H NMR (400 MHz, DMSO) δ 7.96 (d, J =
8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.75
(s, 1H), 5.44 (s, 2H), 3.44 (t, J = 6.8 Hz, 2H), 3.34 (t, J = 6.5 Hz, 2H), 2.28 (s, 3H), 1.88 -
1.74 (m, 4H). 13C NMR (101 MHz, DMSO) δ 168.28, 147.98, 141.26, 139.18, 138.28,
136.83, 133.19, 127.95, 127.07, 125.95, 119.50, 109.97, 104.46, 52.48, 49.35, 46.37, 26.41,
24.36, 11.31.
4-(5-methyl-1-(4-(piperidine-1-carbonyl)benzyl)-1H-pyrazol-3-yl)benzonitrile (A20)
Off white solid, yield 61%. Mp: 170 – 172 . 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.5
Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.1 Hz, 2H), 6.74 (s,
1H), 5.43 (s, 2H), 3.55 (br s, 2H), 3.23 (br s, 2H), 2.28 (s, 3H), 1.59 – 1.43 (m, 6H). 13C
NMR (101 MHz, DMSO) δ 168.98, 147.99, 141.25, 138.72, 138.29, 136.15, 133.16, 127.51,
127.28, 125.96, 119.48, 109.97, 104.44, 52.47, 24.48, 11.32.
4-(5-methyl-1-(4-(morpholine-4-carbonyl)benzyl)-1H-pyrazol-3-yl)benzonitrile (A21)
Off white solid, yield 72%. Mp: 121 – 123 . 1H NMR (400 MHz, DMSO) δ 7.96 (d, J = 8.4
Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 6.75 (s,
1H), 5.44 (s, 2H), 3.60 - 3.31 (m, overlapped with water, 8H), 2.28 (s, 3H). 13C NMR (101
MHz, DMSO) δ 169.19, 148.01, 141.29, 139.14, 138.27, 135.17, 133.20, 127.97, 127.32,
125.96, 119.51, 109.98, 104.46, 66.52, 52.43, 11.33.
This article is protected by copyright. All rights reserved.

4-((3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)methyl)-N-(2-morpholinoethyl)benzam
ide (A22)
Light yellow solid, yield 57%. Mp: 150 – 152 . 1H NMR (400 MHz, DMSO) δ 8.37 (t, J =
5.6 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.23
(d, J = 8.2 Hz, 2H), 6.73 (s, 1H), 5.44 (s, 2H), 3.57 - 3.54 (m, 4H), 3.39 - 3.34 (m, 2H), 2.46 -
2.40 (m, 6H), 2.27 (s, 3H). 13C NMR (101 MHz, DMSO) δ 166.27, 148.01, 141.25, 140.62,
138.30, 134.33, 133.17, 128.00, 127.26, 125.98, 119.48, 110.00, 104.46, 66.66, 57.80, 53.75,
52.53, 37.01, 11.30.
2.2. Luciferase reporter assay
The human breast cancer cell line, MDA-Kb2, which stably expresses an
androgen-responsive reporter were purchased from ATCC (USA)12. Cells were grown at 37
in L-15 media (Hyclone) supplemented with 10% FBS (Hyclone), L-glutamine (Hyclone),
Na pyruvate (Hyclone) and penstrep (Hyclone) without additional CO2. 3 × 104 cells/well
were seeded on 96 well plates (Nunc). At 24 hrs, the medium was replaced with a medium
containing the test compounds alone at 10 nM, 100 nM and 1 µM for the agonist study. For
the antagonist study, cells were exposed to medium containing 10 nM DHT alone or in
combination with 10 nM, 100 nM and 1 µM of test compounds. Following 24 hrs exposure
cells were lysed by using reporter lysis buffer (Promega) and luciferase levels were measured
using the luciferase assay kit (Promega) in a TD 20/20 luminometer (Turner Designs, USA).
For the agonist study, the compounds were compared to control which was set to 100%
whereas the antagonist study were compared to DHT luciferase levels set to 100%.
2.3. Antiproliferative activity
In vitro antiproliferative activity was evaluated via a standard MTT assay. PCa cell
lines, LNCaP and PC-3, were purchased from American type culture collection (ATCC,
USA) and cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum (FBS,
This article is protected by copyright. All rights reserved.

Gibco), 100 IU/ml penicillin, and 100 μg/ml streptomycin at 37 in a 5% CO2 humidified
incubator. The cells were seeded in 96-well plates at density of 4000 cells/well. After
attachment of the cells to the bottom, compounds to be tested were dissolved to
concentrations of 5, 10, 20, 40, and 80 μM by DMSO and then added to the plates with a no
more than 0.5% final percentage of DMSO. The treatment lasted for four days, and MTT (20
μl, 5 mg/ml) was added, following by another four hrs of incubation. Then the supernatant
was removed and DMSO (200 μl) was added to dissolve the generated formazan. The
absorbance at 570 and 630 nm was measured using an ELX800 Microplate Reader (BioTek,
USA) for three times of each plate.
2.4. Stability in human liver microsomes
In vitro metabolic stability in human liver microsomes was assessed using testosterone,
diclofenac and propafenone as control. A DMSO solution of the tested compound (10 mM,
10 μl/well) and solution of microsome (80 μl/well) were added to a 96 well plate, this mixture
was first incubated at 37 for 10 min. Then potassium phosphate buffer (100 mM, 10
μl/well) was added and No Co-Factor (NCF) remaining was evaluated after a further
incubation of 60 min. After this pre-warming process, NADPH regenerating system (10
μl/well) was added and remaining of each compound was tested at 5 time points (5 min, 10
min, 20 min, 30 min and 60 min). At each time point, stop solution (Including 100 ng/ml
Tolbutamide and 100 ng/ml Labetalol, cold in 4 , 300 μl/well) was added to terminate the
reaction. The sampling plates were shaked for approximate 10 min, and then samples were
centrifuged at 4000 rpm for 20 min under 4 to afford the supernatant (100 μl) for LC/MS
test. Intrinsic clearance (CLint) and half-life (T1/2) values were then calculated. CLint(mic)
=
0.693/half-life/mg microsome protein per ml. CLint(liver) = CLint(mic) × (45 mg microsomal
protein/g liver weight) × (20 g liver weight/kg body weight).
This article is protected by copyright. All rights reserved.

2.5. 4D docking
In silico molecular docking study was performed by using the internal coordinate
mechanics (ICM, Version 3.8.4) software. All compounds were constructed using the ligand
editor application in ICM. In the 4D docking, first all ligands from the receptors were
removed, the receptors were then superimposed and prepared for docking by choosing the
ligand binding pocket. After this, a 4D grid was set up and finally the compounds were
docked and ICM scores were calculated. The ECEPP/3 force field was used for optimizing
the receptors and ligands and for the calculation of scoring energy. The ICM score is a sum of
the hydrogen bond energy, hydrophobic energy, van der Waals interaction energy, internal
conformational energy of the ligand, solvation electrostatics energy change upon binding and
desolvation energy of exposed hydrogen bond donors and acceptors.
3. Results and discussion
3.1. Chemistry
The synthesis route of this class of compounds is illustrated as in scheme 1. Using a
commercial available benzaldehyde with proper substitution (1) as starting material, a Claisen
condensation with acetone afforded the α,β-unsaturated ketone intermediate 2. Then
cyclization with p-toluenesulfonhydrazide under basic condition generated the pyrazole
intermediate 3. Intermediate 5 was obtained from another starting material
4-bromomethylbenzoic acid (4) by chlorization and following coupling with proper aliphatic
amine. Finally an SN1 substitution reaction between 3 and 5 generated target compound A.
3.2. AR mediated transcriptional activity
Inhibition of AR mediated transcriptional activity was investigated via an AR luciferase
reporter assay in MDA-Kb2 cells (Tab. 1, Fig. S1). Compounds were tested for their AR
antagonistic potency at concentrations of 0.01, 0.1 and 1 μM. Some of the synthesized
This article is protected by copyright. All rights reserved.

compounds exhibited potent AR antagonistic activity, and compounds A6 and A14 showed
the highest AR inhibition rates of 54.8% and 53.4% at 1 μM. Various isosteres of fluorine on
ring A (eg. chlorine, bromine, cyano group and trifluoromethyl group) were tested. Among
them, compound A6 with a meta-fluorine exhibited highest AR inhibition with antagonistic
rates in a range of 38% ~ 55% at all three concentrations. Substitution position also
influenced AR antagonistic potency on ring A, as compound A1 with para-fluorine only
showed slight AR antagonistic activity with antagonistic rate of 13.8% at 1 μM, comparing
with its high AR antagonistic meta-fluorine analogue A6 (54.8% at 1 μM). Introducing an
additional halogen atom adjacent to the para-fluorine also made a difference. Compound A10
with a meta-chlorine exhibited partial agonistic activity (-21.4%) at 0.1 μM, which
dramatically switched to antagonism (44.8) as its concentration increasing to 1.0 μM.
Meanwhile, compound A9 with meta fluorine exhibited the highest antagonism (39.5%) at
0.1 μM which dropped rapidly to 4.3% at 1 μM. For compounds with different amide side
chains, it was discovered that smaller sized groups were more functional for antagonistic
performance. Compounds A13 – A15 exhibited high AR inhibition of more than 41% at 1.0
μM. On the other hand, compounds A17 – A20 and A22 enhanced the activation of AR
signaling by upregulation of luciferase level in a range of 15% – 82%.
All compounds were further evaluated for their AR agonistic activity by testing the
luciferase levels without DHT induction (Tab. 1, Fig. S2). Compounds A4 – A8 exhibited
significant AR agonistic activity in this test, especially for A4 with AR agonistic rate of
143.5% at 1 μM, and A5 with AR agonistic rate of 122.4% and 139.2% at 0.01 μM and 0.1
μM, respectively. The potent AR antagonist A6 also showed a degree of AR agonistic
activity with a 75% of AR activation at 1 μM in this test. Compounds A13 and A14 showed
partial AR agonistic activity with agonism rate of 30.5% and 32.9% at 1 μM. Interestingly,
compounds A17 – A20 and A22 exhibited no AR agonistic activity in this test. Since these
compounds could enhance the activation of AR signaling as shown in the AR antagonism
assay, these data would suggest that these compounds did not directly interact with AR itself,
and their functional mechanism remained to be discovered.
This article is protected by copyright. All rights reserved.

3.3. Antiproliferative activity in PCa cells
In vitro inhibition of PCa cell growth was evaluated in both AR positive LNCaP cells
and AR negative PC-3 cells (Tab. 2). All the compounds were tested via a standard MTT
colorimetric assay. Part of the compounds exhibited enhanced antiproliferative activity
comparing to enzalutamide. Compound A4 exhibited the highest cell growth inhibition in
LNCaP cells with IC50 value of 21.9 μM. Two mono halogen substituted compounds, A2
with a para-chlorine and A3 with a para-bromine substitution, showed high potency with IC50
values of 23.9 μM and 27.6 μM, respectively. Para-halogen substitution behaved better than
meta substitution on ring A, as compounds A7 and A8 with meta substitutions lost their
potency in LNCaP cells, compared with their para substituted analogues A4 and A5 with IC50
values of 21.9 μM and 56.1 μM, respectively. Compound A1 with poor AR antagonistic
activity also showed higher growth inhibition than its meta-fluoro analogue A6 which was a
potent AR antagonist. Compounds A13 – A14 showing high AR antagonistic activity
maintained potent antiproliferative activity with IC50 values of 34.2 and 30.6 μM,
respectively. It is worth mentioning that compounds A18 – A20 and A22 also exhibited
potent growth inhibition in LNCaP cells with A20 showing the highest antiproliferative
activity (IC50 = 20.4 μM). Since these compounds could enhance the activation of the AR
signaling (as shown in AR antagonism assay) without direct function on AR itself (as shown
in AR agonism assay), their functional mechanism was worthy to be further investigated.
3.4. Metabolic stability in human liver microsomes
Compounds A13 and A14, exhibiting potent AR antagonistic activity and superior
antiproliferative activity than enzalutamide in PCa cell lines, were selected for a further
evaluation of stability in human liver microsomes (Tab. 3). Improved metabolic stability was
detected after a 60 min incubation by high total remaining of 82.2% and 75.1% for A13 and
A14, respectively, comparing with our lead compound 10e of which only 27.1% was left.
Intrinsic clearance (CLint) and half-life (T1/2) values were calculated. Both A13 and A14
showed much lower clearance (< 9.6 μL/min/mg for A13 and 10.8 μL/min/mg for A14) and
This article is protected by copyright. All rights reserved.

much longer half-life (> 145 min for A13 and 128.8 min for A14), while compound 10e
showing high clearance and short half-life with values of 42.3 μL/min/mg and 32.7 min,
respectively. The high in vitro metabolism stability of A13 and A14 in human liver
microsomes forms a foundation for further pharmacokinetics studies.
3.5. In silico docking study
In silico molecular docking studies were performed by using the internal coordinate
mechanics (ICM, Version 3.8.4) software. A 4D docking process was implemented where a
number of crystallized structures of AR LBDs (wild type, T877A and W741L mutated)
complexed with either DHT or non-steroidal agonists/antagonists were used (Tab. 4). This
was done because the compounds synthesized were much bigger for the pocket of a crystal
structure complexed with DHT and it also gives freedom to the compounds as they can
choose their own receptor with best fit.
Preliminary 4D docking studies of the chosen receptors, docked against their own
ligands, showed that DHT, S-1, R-3, HN-1, hydroxyflutamide and bicalutamide recognized
their own receptors (Tab. 5). The other representative non-steroidal AR antagonists like
enzalutamide, ARN-509 and ODM-201 preferred the T877A mutated AR. The two
previously identified lead compounds T3 and 10e preferred the W741L and T877A mutated
AR, respectively. ICM scores also showed that ODM-201 had the highest score followed by
DHT, bicalutamide and HN-1.
Among the newly synthesized compounds, fourteen compounds preferred the T877A
mutated receptor complex with the non-steroidal agonist receptor S1 (PDB: 2AX7) and eight
compounds preferred the WT AR (Tab. 1). Compound A18, preferring the WT AR (PDB:
1T63), had the highest score of -44.6 Kcal/mol, followed by A13 – A15, A17 and A20 – A22
with scores ranging from -36.1 to -39.9 Kcal/mol. A1 – A12 and A16 had lower ICM scores
of between -23.2 and -27.5 Kcal/mol. Comparison of the in silico study and the in vitro
analysis showed that compounds A13 – A15 with substantial antagonistic activity also had
This article is protected by copyright. All rights reserved.

high ICM scores between -37 to -39.3 Kcal/mol. Compounds with agonistic activity (A4 –
A8) showed lower ICM scores between -23.4 and -27 Kcal/mol. There was also a general
trend where compounds with a terminal cyano group showed high ICM scores, above 30
Kcal/mol, with the exception of A4 and A16. The cyano group aided in hydrogen bond
formation with AR, resulting in higher ICM scores.
To better understand the binding mode of this series of compounds, molecular docking
was performed using the Surflex-Dock program of the Sybyl X software package (version
2.0). Compound A13 with high AR antagonistic activity and high ICM score was selected for
this study. Using a T877A mutational AR-LBD (PDB code: 2AX7), the binding orientation
and interactions of compound A13 with AR ligand binding pocket were predicted as shown
in figure 3. The terminal cyano group acted as a hydrogen bond acceptor, interacting with
both arginine 752 and glutamine 711. Another hydrogen bond interaction occurred between
the amide group on the other end and methionine 742. The co-crystal ligand S-1 adopted a
bent conformation based on its chain linker structure between the two terminal rings.
Superimposed structures of A13 and S-1 exhibited high consistency especially on the two
terminal rings. The necessary flexibility of A13 was mainly provided by the rotatable
methylene linking the pyrazole and carbamyl phenyl rings.
4. Conclusion
In summary, this study outlines the design, synthesis and biological evaluation of a
novel class of 5-methyl-1H-pyrazole derivatives as potential anti-prostate cancer agents. This
series of compounds was designed via a fragments splicing strategy based on a structural
modification of two lead compounds T3 and 10e in our previous work. AR reporter gene
assay revealed compounds A13 and A14 as potent AR antagonists. Some of the compounds
in this series showed higher growth inhibition in AR positive LNCaP cells as compared to
enzalutamide. Selected compounds A13 and A14 also exhibited satisfied metabolic stability
in human liver microsomes. In silico docking study showed that compounds with a terminal
cyano group possessed higher binding affinity to AR. This type of compounds has the
This article is protected by copyright. All rights reserved.

potential to contribute to the discovery of novel potent AR antagonists after further
optimization.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (no.
21272140).
Figure legends
Figure 1. Representative non-steroidal AR antagonists.
Figure 2. Design of novel 5-methyl-1H-pyrazole derivatives via a fragments splicing
strategy.
Figure 3. Predicted binding mode of A13 (ball & stick model), superimposing mode of A13
and S-1 (red stick model).
Scheme 1. Route for the synthesis of designed compounds. Reagents and conditions: (a)
acetone, morpholine trifluoroacetate, 70 ~ 90 ; (b) p-Toluenesulfonhydrazide, sodium
hydroxide, 60 ~ 70 ; (c) Oxalyl chloride, N,N-dimethylformamide, dichloromethane, 20 ~
30 ; appropriate amine, triethylamine, tetrahydrofuran, 0 ~ 5 ; (d) sodium hydroxide,
acetonitrile, 20 ~ 30 .
Conflict of interest
The authors declare no conflicts of interest in this work.
This article is protected by copyright. All rights reserved.

References
1. (a) Culig, Z.; Klocker, H.; Bartsch, G.; Hobisch, A., Androgen receptors in prostate cancer. Endocr Relat
Cancer 2002, 9 (3), 155-70; (b) Wang, D.; Tindall, D. J., Androgen action during prostate carcinogenesis.
Methods Mol Biol 2011, 776, 25-44.
2. Huggins, C.; Hodges, C. V., Studies on prostatic cancer. I. The effect of castration, of estrogen and
androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CA Cancer J Clin 1972, 22
(4), 232-40.
3. Harris, W. P.; Mostaghel, E. A.; Nelson, P. S.; Montgomery, B., Androgen deprivation therapy: progress in
understanding mechanisms of resistance and optimizing androgen depletion. Nat Clin Pract Urol 2009, 6 (2),
76-85.
4. Ceraline, J.; Cruchant, M. D.; Erdmann, E.; Erbs, P.; Kurtz, J. E.; Duclos, B.; Jacqmin, D.; Chopin, D.;
Bergerat, J. P., Constitutive activation of the androgen receptor by a point mutation in the hinge region: a new
mechanism for androgen-independent growth in prostate cancer. Int J Cancer 2004, 108 (1), 152-7.
5. (a) Edwards, J.; Krishna, N. S.; Grigor, K. M.; Bartlett, J. M., Androgen receptor gene amplification and
protein expression in hormone refractory prostate cancer. Br J Cancer 2003, 89 (3), 552-6; (b) Koivisto, P.;
Kononen, J.; Palmberg, C.; Tammela, T.; Hyytinen, E.; Isola, J.; Trapman, J.; Cleutjens, K.; Noordzij, A.; Visakorpi,
T.; Kallioniemi, O. P., Androgen receptor gene amplification: a possible molecular mechanism for androgen
deprivation therapy failure in prostate cancer. Cancer Res 1997, 57 (2), 314-9.
6. Stanbrough, M., Increased expression of genes converting adrenal androgens to testosterone in
androgen-independent prostate cancer. Cancer Res 2006, 66, 2815-2825.
7. (a) Culig, Z.; Hobisch, A.; Cronauer, M. V.; Cato, A. C.; Hittmair, A.; Radmayr, C.; Eberle, J.; Bartsch, G.;
Klocker, H., Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by
adrenal androgens and progesterone. Mol Endocrinol 1993, 7 (12), 1541-50; (b) Culig, Z.; Stober, J.; Gast, A.;
Peterziel, H.; Hobisch, A.; Radmayr, C.; Hittmair, A.; Bartsch, G.; Cato, A. C.; Klocker, H., Activation of two
mutant androgen receptors from human prostatic carcinoma by adrenal androgens and metabolic derivatives
of testosterone. Cancer Detect Prev 1996, 20 (1), 68-75.
8. Bohl, C. E.; Gao, W.; Miller, D. D.; Bell, C. E.; Dalton, J. T., Structural basis for antagonism and resistance of
bicalutamide in prostate cancer. Proc Natl Acad Sci U S A 2005, 102 (17), 6201-6.
9. Tran, C.; Ouk, S.; Clegg, N. J.; Chen, Y.; Watson, P. A.; Arora, V.; Wongvipat, J.; Smith-Jones, P. M.; Yoo, D.;
Kwon, A.; Wasielewska, T.; Welsbie, D.; Chen, C. D.; Higano, C. S.; Beer, T. M.; Hung, D. T.; Scher, H. I.; Jung, M.
E.; Sawyers, C. L., Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate
Cancer. Science 2009, 324 (5928), 787-790.
This article is protected by copyright. All rights reserved.

10. Liu, J.; Liu, B.; Guo, G.; Jing, Y.; Zhao, G., Discovery of novel androgen receptor antagonists: a hybrid
approach of pharmacophore-based and docking-based virtual screening. Anti-Cancer Drugs 2015, 26 (7),
747-753.
11. Guo, G.; Liu, J.; Wang, G.; Zhang, D.; Lu, J.; Zhao, G., Synthesis and biological evaluation of
3-(4-fluorophenyl)-1H-pyrazole derivatives as androgen receptor antagonists. Anti-Cancer Drugs 2016, 27 (4),
278-285.
12. Wilson, V. S.; Bobseine, K.; Lambright, C. R.; Gray, L. E., Jr., A novel cell line, MDA-kb2, that stably
expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor
agonists and antagonists. Toxicol Sci 2002, 66 (1), 69-81.
This article is protected by copyright. All rights reserved.

Table 1. AR antagonism/agonism study and in silico docking study for compounds in series
A.
AR antagonism rate (%) AR agonism rate (%)
Docking study by ICM
Preferred ICM Score
receptor (Kcal/mol)
Code
0.01 μM 0.1 μM 1.0 μM 0.01 μM 0.1 μM 1.0 μM
A1
A2
A3
A4
A5
A6
A7
A8
A9
-4.6
-7.1
-5.3
31.0
29.8
49.3
29.6
29.9
16.6
7.3
13.8
2.4
-9.0
-1.8
-5.3
60.3
122.4
43.8
101.3
94.6
-8.8
2.0
-3.6
-4.5
10.4
55.0
143.5
69.9
75.0
68.0
98.9
8.9
2AX7
3V49
2AX7
3V49
3V49
2AX9
2AX7
2AX7
2AX7
2AX7
2AX7
2AX7
2AX7
2AX7
2AX7
3V49
2AX7
1T63
-26.6
-23.9
-23.2
-27.0
-23.6
-25.8
-23.4
-25.6
-27.3
-25.3
-27.1
-25.9
-39.3
-37.0
-38.3
-27.5
-38.9
-44.6
-30.7
-37.3
-36.1
-39.9
-33.6
10.5
35.9
24.3
27.7
38.7
22.6
16.2
39.5
-21.4
11.7
-27.5
35.1
26.8
21.5
13.0
-26.9
-23.6
-57.6
-40.1
-22.1
-22.5
40.9
4.5
25.3
37.6
35.9
54.8
29.7
30.8
4.3
-15.6
49.8
139.2
53.4
119.1
101.7
-6.3
A10 -9.2
A11 8.0
44.8
18.1
21.8
46.2
53.4
41.0
28.2
-82.0
-55.6
-38.2
-33.8
-7.5
14.3
5.1
-5.2
14.5
18.9
30.5
32.9
5.2
21.3
39.2
17.2
20.4
7.3
A12 -6.7
A13 1.7
15.5
18.3
20.5
24.7
-6.6
A14 1.7
A15 -7.8
A16 -13.5
A17 -52.8
A18 -69.2
A19 -67.0
A20 -42.8
A21 1.8
3.1
-19.2
-16.2
-21.2
-18.8
-5.8
-10.4
-10.8
-
-23.8
-2.7
-21.7
4.4
-11.2
5.1
-29.1
-13.0
-14.5
-16.6
-
3V49
2AX7
2AX7
3V49
2AX7
-1.0
-5.2
-
A22 -15.2
Enz 24.0
-34.7
80.3
This article is protected by copyright. All rights reserved.

Table 2. Antiproliferative activity of the new compounds in LNCaP and PC-3 cells.
Cell growth inhibition (IC50/μM)
Code -R1
-NR2R3
LNCaP
PC-3
>80
A1
A2
A3
A4
A5
A6
A7
A8
A9
4-F
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
-NHtBu
32.54 4.39
23.90 1.35
27.63 2.28
21.96 3.54
56.08 3.83
40.01 2.74
>80
4-Cl
>80
4-Br
>80
4-CN
4-CF3
3-F
>80
>80
>80
3-CN
3-CF3
3-F-4-F
>80
>80
>80
47.21 5.28
28.84 2.56
49.90 0.68
>80
>80
A10 3-Cl-4-F
A11 3-Br-4-F
>80
>80
A12 3-MeO-4-MeO -NHtBu
>80
A13 4-CN
A14 4-CN
A15 4-CN
A16 4-CN
-NHMe
-NHEt
34.18 1.78
30.62 0.64
38.58 5.88
51.13 5.59
>80
25.04 1.27
>80
-NHiPr
-N(Me)2
>80
This article is protected by copyright. All rights reserved.

A17 4-CN
A18 4-CN
-N(iPr)2
42.44 5.93
24.76 0.36
>80
>80
-NHC2H4OH
A19 4-CN
A20 4-CN
A21 4-CN
30.15 0.49
20.38 0.52
36.37 1.97
>80
>80
>80
N
A22 4-CN
29.51 2.60
45.25 4.85
>80
Enzalutamide
64.95 0.95
Table 3. Metabolic stability of A13, A14 and 10e in human liver microsomes. R2 is the
correlation coefficient of the linear regression for the determination of kinetic constant,
CLint(mic) is the intrinsic clearance.
HLM 0.5
Sample name
CLint(mic)
CLint(liver)
Remaining
(T=60min)
Remaining
R2
T1/2 (min)
(μL/min/mg)
(mL/min/kg)
(*NCF=60min)
A13
0.8099
0.8437
0.9967
0.9990
0.9986
0.9792
>145
128.8
32.7
15.9
10.1
6.5
<9.6
10.8
<8.6
9.7
82.2%
75.1%
27.1%
6.9%
98.1%
98.6%
93.4%
82.8%
84.4%
92.9%
A14
10e
42.3
38.1
78.6
124.0
193.3
Testosterone
Diclofenac
Propafenone
87.4
137.8
214.7
1.6%
0.2%
*NCF: the abbreviation of no co-factor. No NADPH regenerating system is added into NCF sample
(replaced by buffer) during the 60 min-incubation.
This article is protected by copyright. All rights reserved.

Table 4. Ensemble of AR LBD crystal structures used for 4D docking.
PDB Code Type of mutation Ligand
Structure
1T63
WT
WT
DHT
3L3X
2AXA
2AX7
2AX8
WT
T877A
W741L
S-1
R-3
2AX9
3V49
WT
WT
HN-1
2AX6
1Z95
T877A
W741L
Hydroxyflutamide Structure shown in Fig. 1
Bicalutamide Structure shown in Fig. 1
This article is protected by copyright. All rights reserved.

Table 5. ICM scores of the docked natural compound DHT and representative non-steroidal
AR antagonists.
Code
DHT
S-1
Original receptor
Preferred receptor ICM score (Kcal/mol)
3L3X
1T63/3L3X
-47.1
-37.6
-34.7
-43.0
-34.4
-43.8
-33.6
-29.6
-50.6
-32.8
-25.4
2AXA/2AX7/2AX8 2AXA
R-3
2AX9
3V49
2AX9
3V49
2AX6
1Z95
HN-1
Hydroxyflutamide 2AX6
Bicalutamide
Enzalutamide
ARN-509
ODM-201
T3
1Z95
-
-
-
-
-
2AX7
2AX7
2AX7
1Z95
10e
2AX7
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.