Design, synthesis, and biological evaluation of 2-(5-methyl-1H-pyrazol-1-yl) acetamide derivatives as androgen receptor antagonists

Article abstract of DOI:10.1007/s00044-019-02291-y

Androgen receptor (AR) signaling is often activated in prostate cancer (PCa) cells, and blockage of this signaling by AR antagonists is an important strategy in PCa therapy. In this study, we designed and synthesized a series of 2-(5-methyl-1H-pyrazol-1-y

Full text of DOI:10.1007/s00044-019-02291-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02291-y
ORIGINAL RESEARCH
Design, synthesis, and biological evaluation of 2-(5-methyl-1H-
pyrazol-1-yl) acetamide derivatives as androgen receptor
antagonists
Junze Dong1 Jingya Zhang1 Zilu Li1 Solomon Asnake2 Daoguang Zhang1 Per-Erik Olsson2 Guisen Zhao1
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Received: 25 October 2018 / Accepted: 11 January 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Androgen receptor (AR) signaling is often activated in prostate cancer (PCa) cells, and blockage of this signaling by AR
antagonists is an important strategy in PCa therapy. In this study, we designed and synthesized a series of 2-(5-methyl-1H-
pyrazol-1-yl) acetamide derivatives, and evaluated their biological activities. AR luciferase reporter assay revealed
compound 6f (59.7%) as a potent AR antagonist. Some compounds in this series showed higher anti-proliferative activity
against LNCaP cells than Bicalutamide (IC50 = 35.0 μM), especially 6g with IC50 value of 13.6 μM.
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Keywords Androgen receptor Prostate cancer Antagonists Pyrazole derivatives
Introduction
AR, and intratumoral de novo steroidogenesis (Locke et al.
2008; Montgomery 2008; Mostaghel 2007). This indicates that
AR signaling is a critical driver for the development of CRPC
(Taplin 2007). Research of drugs targeting CRPC is mainly
divided into two directions, one is blockage of endogenous
synthesis of androgen by the CYP17A inhibitor Abiraterone
(De Bono et al. 2011; Reid 2008) and the other is to combat
drug resistance that emerges after prolonged usage of first-
generation AR antagonists (e.g., Bicalutamide, Fig. 1).
The latter has led to the development of the second-generation
AR antagonists such as Enzalutamide (MDV3100, Fig. 1)
(Tran et al. 2009; Oudard 2013).
Early prostate cancer (PCa) can be effectively remitted by
androgen deprivation therapy (ADT), which is a combination
of surgical or medical castration with administration of
androgen receptor (AR) antagonist (Mercader 2007; Harris
et al. 2009). However, the disease often relapses after a period
of ADT treatment and progresses to a new stage termed
castration-resistant prostate cancer (CRPC) (Katzenwadel and
Wolf 2015). Multiple mechanisms for the relapse have been
proposed, most of them suggesting reactivation of the AR
signaling pathway, including over-expression or mutations of
Our previous work focused on the pharmacological char-
acterization of molecules based on a pyrazole scaffold as
potential anti-PCa agents. Among the pyrazole derivatives,
compound 10e showed potent anti-proliferative activity in
LNCaP cells with an IC50 value of 17.8 μM (Guo et al. 2016).
However, in a later study, 10e was found to be rapidly
metabolized by human liver microsomes (HLMs) with a half-
life (T1/2) of ~30 min. The methylene amino-linker bridging
the pyrazole core and terminal phenyl ring was a possible
metabolism site, as the amino group could undergo de-
alkylation or oxidation via the catalysis of cytochrome P450
that generally existed in HLMs. In this work, structural
modification focused on this methylene amino-linker motif,
which was replaced by an acetamido structure (Fig. 2).
Therefore a novel series of 2-(5-methyl-1H-pyrazol-1-yl)
acetamide derivatives was designed and synthesized.
These authors contributed equally to this work: Junze Dong and Jingya
Zhang
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02291-y) contains supplementary
material, which is available to authorized users.
* Guisen Zhao
guisenzhao@sdu.edu.cn
1
Department of Medicinal Chemistry, Key Laboratory of Chemical
Biology (Ministry of Education), School of Pharmaceutical
Sciences, Shandong University, Jinan 250012 Shandong, PR
China
2
Biology, The Life Science Center, School of Science and
Technology, Örebro University, Örebro, SE-70182
Örebro, Sweden

Medicinal Chemistry Research
Fig. 1 Representative AR
antagonists in clinical
application
Results and discussion
Anti-proliferative activity in PCa cell lines
Chemistry
The anti-proliferative property of the new compounds was
tested by using MTT assay in two PCa cell lines, LNCaP
(with a T877A mutation in AR) and PC-3 (with wild-type
AR) (Table 1). Most compounds showed good responses
in LNCaP cells, and exhibited more potent anti-
proliferative activity in LNCaP cells than the positive
control, bicalutamide (IC50 = 35.0 μM). It was worth
noticing that compounds (6h–6n) with a p-cyano sub-
stitution at the R1-position showed high selection for
LNCaP cells. Analysis of structure–activity relationship
showed that a p-fluoro group on the 3-phenyl ring of the
pyrazole core favored improved growth inhibition in PC-3
Synthesis of novel compounds was performed following the
route depicted in Scheme 1. As the starting material, a p-
substituted benzaldehyde (1) was condensed with chlor-
oacetyl chloride to obtain the substituted (E)-4-phenylbut-3-
en-2-one as intermediate 2 via an aldol condensation.
Cyclization of intermediate 2 with p-toluenesulfonyl
hydrozide under alkaline conditions generated the sub-
stituted 5-methyl-3-phenyl-1H-pyrazole as intermediate 3.
A substituted aniline (4) was utilized as an additional
starting material, which when condensed with chloroacetyl
chloride resulted in substituted 2-chloro-N-phenylacetamide
as intermediate 5. Finally, a substitution of intermediates 3
and 5 under alkaline condition afforded novel compounds 6.
cells. In this series, the most potent compound, 6g (IC50
=
13.6 μM), showed a more than twofold higher anti-
proliferative activity in LNCaP cells than bicalutamide
(IC50 = 35.0 μM), as well as a more than threefold higher
activity (IC50 = 20.2 μM) compared with bicalutamide
(IC50 = 63.1 μM) in PC-3 cells. The most potent AR
antagonist, 6f, also showed superior anti-proliferative
activity in both LNCaP and PC-3 cells with IC50 values of
28.2 and 28.6 μM, respectively.
Biological activity
AR antagonistic activity
AR antagonistic activity of the new compounds was
quantified via an AR luciferase reporter assay in MDA-Kb2
cells (Fig. 3). The MDA-Kb2 cell line stably expresses an
androgen-responsive reporter, and exposure to compounds
with AR antagonistic activity will result in a reduction of
the luciferase reporter levels. All the compounds in this
series were tested at 1 µM concentration. Among them, five
compounds, 6a, 6f, 6h, 6l, and 6n, exhibited AR antag-
onistic activities with inhibitory rates in a range of 34.7–
59.7%, which was higher than that of bicalutamide (32.0%
inhibition).
Substituents on both terminal phenyl rings affected the
AR antagonistic activity. Generally, most of compounds
with a p-fluoro group on the phenyl ring at 3-position of the
pyrazole core showed higher AR antagonistic activity than a
p-cyano substitution at the same position (6a vs. 6h, 6c vs.
6j). However, if there was a p-substitution at the R2-posi-
tion, opposite effects were observed (6d vs. 6k, 6e vs. 6l,
and 6g vs. 6n). In this series, compound 6f showed the
highest AR antagonistic activity (59.7%), which was nearly
twofold higher than bicalutamide (32.0%).
Materials and methods
All materials and reagents were commercially available
and used without further purification. Thin-layer chromato-
graphy (TLC) performed on silica gel HSGF254 plates was
used to monitor the reaction. Column chromatography was
performed using silica gel (60 Å, 200–300 mesh). Melting
points were determined in a capillary melting point appa-
ratus and are uncorrected. All 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance DRX-400
Spectrometer (Bruker, Rheinstetten, Germany) with
tetramethyl-silane (TMS) as the internal standard, and
compounds were dissolved in deuterated dimethylsulfoxide
(DMSO-d6). The high-resolution mass spectra (HRMS) were
measured with X500R QTOF (AB SCIEX, USA). All tested
compounds were more than 95% pure on the HPLC analy-
sis. The method for HPLC analysis and a table of the data
for all tested compounds are in Supporting Information.

Medicinal Chemistry Research
Fig. 2 Design of novel 2-(5-methyl-1H-pyrazol-1-yl) acetamide derivatives
General synthetic procedure of intermediate 2
addition, the reaction mixture was stirred for one more hour
at room temperature, then EA (30 mL) and H2O (30 mL)
were added for an extraction. The organic contract was
washed with water (15 mL × 3) and saturated brine (15 mL),
dried over Na2SO4 and evaporated under reduced pressure
to afford intermediate 5.
A mixture of substituted benzaldehyde (20.0 mmol) and
morpholine trifluoroacetate (0.8 g, 4 mmol) in acetone (50
mL) was sealed in a tube and refluxed at 80–90 °C for 72 h.
It was then cooled down, and excess acetone was evapo-
rated in vacuo. The residue was dissolved in ethyl acetate
(EA, 50 mL), washed with H2O (20 mL), brine (20 mL),
dried over Na2SO4, filtered, and concentrated to afford
intermediate 2, which was directly used for the next step
without further purification.
2-chloro-N-(3-cyanophenyl)acetamide (5a)
1
White solid; yield: 73%; mp: 153–155 °C. H NMR (400
MHz, DMSO-d6) δ 10.77 (s, 1H, NH), 8.08 (s, 1H, Ar–H),
7.82–7.79 (m, 1H, Ar–H), 7.57–7.56 (m, 2H, Ar–H), 4.09
(s, 2H, CH2).
(E)-4-(3-oxobut-1-en-1-yl)benzonitrile (2a)
Light yellow solid; yield: 80%; mp: 93–96 °C .1H NMR
(400 MHz, DMSO-d6) δ 7.93–7.89 (m, 4H, Ar–H), 7.68 (d,
J = 16.4 Hz, 1H, C1–H), 6.97 (d, J = 16.4 Hz, 1H, C2–H),
2.37 (s, 3H, CH3).
2-chloro-N-(4-cyano-3-(trifluoromethyl)phenyl)acetamide
(5b)
1
White solid; yield: 76%; mp: 127–130 °C. H NMR (400
MHz, DMSO-d6) δ 11.18 (s, 1H, NH), 8.25 (d, J = 1.5 Hz,
1H, Ar–H), 8.14 (d, J = 8.5 Hz, 1H, Ar–H), 7.99 (dd, J =
8.5, 1.6 Hz, 1H, Ar–H), 4.13 (s, 2H, CH2).
General synthetic procedure of intermediate 3
A
mixture of intermediate 2 (20.0 mmol) and 4-
toluenesulfonyl hydrazide (4.1 g, 22.0 mmol) in acetoni-
trile (50 mL) was stirred at 40 °C for 3 h. NaOH (1.2 g, 30.0
mmol) was added, and the reaction mixture was refluxed for
12 more hours. It was then cooled down, and EA (50 mL)
was added to the solution, washed with water (20 mL × 3),
and dried over Na2SO4. The solvent was removed in vacuo
to afford intermediate 3.
General synthetic procedure of target compounds
NaH (60%, 120 mg, 3.0 mmol) was added slowly into a
solution of intermediate 3 (1.0 mmol) in N, N-dimethylfor-
mamide (DMF, 2.5 mL) and stirred for 30 min at room
temperature. Then intermediate 5 (1.0 mmol) was added and
the reaction mixture was stirred for another 12 h. It was then
poured into ice-cold water (35 mL) to form precipitate that
was collected by filtration, washed with water, and dried.
The crude product was dissolved with DCM and purified
using column chromatography (petroleum ether: EA = 3:1)
on silica gel to obtain target compounds.
4-(5-methyl-1H-pyrazol-3-yl)benzonitrile (3a)
1
Light yellow solid; yield: 89%; mp: 130–133 °C. H NMR
(400 MHz, DMSO-d6) δ 12.84 (s, 1H, NH), 7.95 (d, J = 8.2
Hz, 2H, Ar–H), 7.83 (d, J = 8.2 Hz, 2H, Ar–H), 6.60 (s, 1H,
C4–H), 2.28 (s, 3H, CH3).
N-(3-fluorophenyl)-2-(3-(4-fluorophenyl)-5-methyl-1H-
pyrazol-1-yl)acetamide (6a)
General synthetic procedure of intermediate 5
1
White solid; yield: 30%; mp: 167–169 °C. H NMR (400
A substituted aniline (5.0 mmol) and triethylamine (1.87 g,
18.5 mmol) were dissolved by dichloromethane (10 mL),
and stirred under an ice–water bath, to which chloroacetyl
chloride (1.29 g, 11.5 mmol) was added dropwise. After
MHz, DMSO-d6) δ 10.58 (s, 1H, CONH), 7.84 – 7.72 (m,
2H, Ar–H), 7.58 (dt, J = 11.5, 2.0 Hz, 1H, Ar–H), 7.35 (dt,
J = 17.8, 8.3 Hz, 2H, Ar–H), 7.20 (t, J = 8.9 Hz, 2H, Ar–
H), 6.91 (td, J = 8.5, 2.0 Hz, 1H, Ar–H), 6.52 (s, 1H, C4–

Medicinal Chemistry Research
Scheme 1 Reagents and conditions: (i) morpholine trifluoroacetate, acetone, 85 °C; (ii) p-toluenesulfonyl hydrazide, NaOH, acetonitrile, 50 °C;
(iii) chloroacetyl chloride, triethylamine, CH2Cl2, r.t.; (iv) NaH, DMF, r.t
H), 5.01 (s, 2H, CH2), 2.29 (s, 3H, CH3). 13C NMR (100
5.04 (s, 2H, CH2), 2.32 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6) δ 166.71 (CO), 162.07 (d, JCF = 242.0 Hz),
1
1
MHz, DMSO-d6) δ 166.40 (CO), 162.61 (d, JCF = 240.0
1
Hz), 162.06 (d, JCF = 242.0 Hz), 148.79 (C-3), 141.98 (C-
148.85 (C-3), 142.01 (Ar–C), 139.82 (C-5), 13.87 (Ar–C),
130.46 (d, 4JCF = 3.0 Hz), 127.70 (CN), 127.33 (d, 2C, 3JCF
= 8.0 Hz), 124.30 (Ar–C), 122.41 (Ar–C), 119.05 (Ar–C),
3
3
5), 140.76 (d, JCF = 11.0 Hz), 131.03 (d, JCF = 10.0 Hz),
4
3
130.48 (d, JCF = 3.0 Hz), 127.32 (d, 2C, JCF = 8.0 Hz),
115.88 (d, 2C, JCF = 21.0 Hz), 115.43 (d, JCF = 2.0 Hz),
110.59 (d, JCF = 21.0 Hz), 106.50 (d, JCF = 27.0 Hz),
102.96 (C-4), 52.68 (CH2), 11.32 (CH3). HRMS (ESI+) m/
z C18H15ON3F2 (M+H+) calcd 328.1256, obsd 328.1250.
2
4
2
115.88 (d, 2C, JCF = 22.0 Hz), 112.19 (Ar–C), 103.00 (C-
2
2
4), 52.67 (CH2), 11.31 (CH3). HRMS (ESI+) m/z
C19H15ON4F (M+H+) calcd 335.1303, obsd 335.1288.
N-(4-fluorophenyl)-2-(3-(4-fluorophenyl)-5-methyl-1H-
2-(3-(4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(3-
(trifluoromethyl)phenyl) acetamide (6b)
pyrazol-1-yl)acetamide (6d)
1
White solid; yield: 27%; mp: 181–183 °C. H NMR (400
1
White solid; yield: 26%; mp: 145–147 °C. H NMR (400
MHz, DMSO-d6) δ 10.42 (s, 1H, NH), 7.81 – 7.73 (m, 2H,
Ar–H), 7.62 (ddd, J = 7.1, 5.3, 2.8 Hz, 2H, Ar–H), 7.19 (dt,
J = 12.5, 9.0 Hz, 4H, Ar–H), 6.52 (s, 1H, C4–H), 4.99 (s,
2H, CH2), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-
MHz, DMSO-d6) δ 10.73 (s, 1H, NH), 8.11 (s, 1H, Ar–H),
7.88 – 7.69 (m, 3H, Ar–H), 7.59 (t, J = 8.0 Hz, 1H, Ar–H),
7.44 (d, J = 7.8 Hz, 1H, Ar–H), 7.20 (t, J = 8.9 Hz, 2H, Ar–
H), 6.53 (s, 1H, C4–H), 5.04 (s, 2H, CH2), 2.30 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6) δ 166.67 (CO),
1
d6) δ 165.91 (CO), 162.05 (d, JCF = 242.0 Hz), 158.65 (d,
1JCF = 239.0 Hz), 148.73 (C-3), 141.93 (C-5), 135.46 (d,
1
4
162.08 (d, JCF = 242.0 Hz), 148.86 (C-3), 142.00 (Ar–C),
4JCF = 3.0 Hz), 130.51 (d, JCF = 3.0 Hz), 127.31 (d, 2C,
4
3
139.82 (C-5), 130.60 (Ar–C), 130.49 (d, JCF = 3.0 Hz),
3JCF = 8.0 Hz), 121.47 (d, 2C, JCF = 8.0 Hz), 115.92 (d,
2
3
130.06 (q, JCF = 31.7 Hz), 127.32 (d, 2C, JCF = 8.1 Hz),
2C, 2JCF = 22.0 Hz), 115.87 (d, 2C, 2JCF = 21.0 Hz), 102.94
(C-4), 52.63 (CH2), 11.21 (CH3). HRMS (ESI+) m/z
C18H15ON3F2 (M+H+) calcd 328.1256, obsd 328.1253.
1
124.51 (q, JCF = 271.0 Hz), 123.25 (Ar–C), 120.41 (q,
3JCF = 4.0 Hz), 115.86 (d, 2C, JCF = 22.0 Hz), 115.77 (q,
2
3JCF = 4.0 Hz), 102.96 (C-4), 52.69 (CH2), 11.30 (CH3).
HRMS (ESI+) m/z C19H15ON3F4 (M+H+) calcd 378.1224,
obsd 378.1208.
2-(3-(4-fluorophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(4-
(trifluoromethyl)phenyl) acetamide (6e)
1
N-(3-cyanophenyl)-2-(3-(4-fluorophenyl)-5-methyl-1H-
White solid; yield: 37%; mp: 150–152 °C. H NMR (400
pyrazol-1-yl)acetamide (6c)
MHz, DMSO-d6) δ 10.73 (s, 1H, NH), 7.86 – 7.74 (m, 4H,
Ar–H), 7.70 (d, J = 8.7 Hz, 2H, Ar–H), 7.20 (t, J = 8.9 Hz,
2H, Ar–H), 6.53 (s, 1H, C4–H), 5.05 (s, 2H, CH2), 2.30 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 166.69 (CO),
1
White solid; yield: 26%; mp: 180–182 °C. H NMR (400
MHz, DMSO-d6) δ 10.72 (s, 1H, NH), 8.08 (d, J = 1.1 Hz,
1H, Ar–H), 7.87 – 7.69 (m, 3H, Ar–H), 7.62 – 7.50 (m, 2H,
Ar–H), 7.20 (t, J = 8.9 Hz, 2H, Ar–H), 6.53 (s, 1H, C4–H),
1
162.08 (d, JCF = 242.0 Hz), 148.85 (C-3), 142.62 (Ar–C),
4
141.99 (C-5), 130.47 (d, JCF = 3.0 Hz), 127.32 (d, 2C,

Medicinal Chemistry Research
AR antagonistic activity
120
100
80
60
40
20
0
DHT
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
Bic-1
Fig. 3 AR antagonistic activity of compounds at the concentration of 1 μM (DHT: DHT at 10 nM, Bic-1 bicalutamide at 1 μM)
Table 1 Anti-proliferative activity in LNCaP and PC-3 cell lines
H), 6.21 (s, 1H, C4–H), 4.95 (s, 2H, CH2), 2.19 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6) δ 167.20 (CO),
1
1
162.65 (d, JCF = 245.0 Hz), 162.07 (d, JCF = 243.0 Hz),
3
147.63 (C-3), 141.97 (C-5), 131.07 (d, 2C, JCF = 8.0 Hz),
130.45 (d, 4JCF = 2.0 Hz), 127.32 (d, 3JCF = 8.0 Hz), 126.32
4
3
Code
Structure
R1
Anti-proliferative activity (IC50
μM)
,
(d, JCF = 2.0 Hz), 125.55 (d, JCF = 4.0 Hz), 120.36 (d,
2JCF = 16.0 Hz), 116.23 (d, 2C, JCF = 21.0 Hz), 115.90 (d,
2
2JCF = 22.0 Hz), 106.29(C-4), 52.41 (CH2), 13.72 (CH3).
HRMS (ESI+) m/z C18H14ON3F2Cl (M+H+) calcd
362.0866, obsd 362.0855.
R2
LNCaP
PC-3
>80
6a
6b
6c
6d
6e
6f
F
3-F
38.66 0.50
25.45 19.33
51.10 5.92
41.20 17.68
21.52 11.16
28.20 13.30
13.58 3.18
40.85 18.07
21.09 21.17
28.18 12.01
13.86 3.33
32.55 8.02
24.84 7.32
52.35 17.93
34.96 4.45
F
3-CF3
3-CN
19.03 0.33
>80
F
F
4-F
>80
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-(3-(4-
fluorophenyl)-5-methyl-1H-pyrazol-1-yl) acetamide (6g)
F
4-CF3
2-F-3-Cl
3-CF3-4-CN
3-F
34.92 1.45
28.58 4.39
20.21 1.06
>80
F
6g
6h
6i
F
1
White solid; yield: 44%; mp: 150–152 °C. H NMR (400
CN
CN
CN
CN
CN
CN
CN
MHz, DMSO-d6) δ 11.17 (s, 1H, NH), 8.28 (s, 1H, Ar–H),
8.13 (d, J = 8.6 Hz, 1H, Ar–H), 7.97 (dd, J = 8.5, 1.8 Hz,
1H, Ar–H), 7.77 (dd, J = 8.8, 5.6 Hz, 2H, Ar–H), 7.20 (t, J
= 8.9 Hz, 2H, Ar–H), 6.54 (s, 1H, C4–H), 5.09 (s, 2H,
CH2), 2.25 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ
3-CF3
3-CN
>80
6j
>80
6k
6l
4-F
>80
4-CF3
2-F-3-Cl
3-CF3-4-CN
>80
6m
6n
>80
>80
1
167.49 (CO), 162.10 (d, JCF = 243.0 Hz), 149.00 (C-3),
Bicalutamide
63.13 2.49
143.57 (Ar–C), 142.05 (Ar–C), 137.05 (C-5), 132.30 (q,
4
2JCF = 31.3 Hz), 130.39 (d, JCF = 3.0 Hz), 127.32 (d, 2C,
3JCF = 8.0 Hz), 124.20 (CN), 122.84 (q, JCF = 272.0 Hz),
1
3
3
3JCF = 8.0 Hz), 126.64(q, 2C, JCF3 = 3.3 Hz), 124.78 ((q,
117.02 (q, JCF = 5.0 Hz), 116.13 (Ar–C), 115.85 (d, 2C,
1JCF = 269.7 Hz), 124.16(q, JCF = 31.7 Hz), 119.64 (2C,
2JCF = 21.0 Hz), 103.04 (Ar–C), 102.54 (C-4), 52.79 (CH2),
11.24 (CH3). HRMS (ESI+) m/z C20H14ON4F4 (M+H+)
calcd 403.1176, obsd 403.1188.
2
Ar–C), 115.86 (d, 2C, 2JCF = 21.0 Hz), 102.97 (C-4), 52.75
(CH2), 11.29 (CH3). HRMS (ESI+) m/z C19H15ON3F4 (M
+H+) calcd 378.1224, obsd 378.1212.
2-(3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(3-
N-(3-chloro-2-fluorophenyl)-2-(3-(4-fluorophenyl)-5-methyl-
fluorophenyl)acetamide (6h)
1H-pyrazol-1-yl) acetamide (6f)
1
White solid; yield: 11%; mp: 218–219 °C. H NMR (400
1
White solid; yield: 30%; mp: 157–159 °C. H NMR (400
MHz, DMSO-d6) δ 10.26 (s, 1H, NH), 7.58–7.49 (m, 2H,
Ar–H), 7.34–7.27 (m, 2H, Ar–H), 7.25–7.16 (m, 3H, Ar–
MHz, DMSO-d6) δ 10.61 (s, 1H, NH), 7.93 (d, J = 8.5 Hz,
2H, Ar–H), 7.83 (d, J = 8.5 Hz, 2H, Ar–H), 7.62 – 7.53 (m,
1H, Ar–H), 7.35 (dt, J = 19.8, 8.4 Hz, 2H, Ar–H), 6.92 (td,

Medicinal Chemistry Research
3
J = 8.5, 2.8 Hz, 1H, Ar–H), 6.70 (s, 1H, C4-H), 5.07 (s, 2H,
CH2), 2.31 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ
(2C, Ar–C), 121.50 (d, 2C, JCF = 8.0 Hz), 119.47 (CN),
2
115.94 (d, 2C, JCF = 23.0 Hz), 110.00 (Ar–C), 104.06 (C-
1
166.16 (CO), 162.61 (d, JCF = 240.0 Hz), 148.02 (C-3),
4), 52.85 (CH2), 11.36 (CH3). HRMS (ESI+) m/z
3
142.53 (Ar–C), 140.71 (d, JCF = 11.0 Hz), 138.28 (C-5),
C19H15ON4F (M+H+) calcd 335.1303, obsd 335.1299.
133.16 (2C, Ar–C), 131.04 (d, 3JCF = 10.0 Hz), 125.97 (2C,
4
Ar–C), 119.47 (CN), 115.46 (d, JCF = 2.0 Hz), 110.64 (d,
2-(3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(4-
(trifluoromethyl)phenyl) acetamide (6l)
2JCF = 21.0 Hz), 110.02 (Ar–C), 106.54 (d, 2JCF = 26.0 Hz),
104.08 (C-4), 52.91 (CH2), 11.34 (CH3). HRMS (ESI+) m/
z C19H15ON4F (M+H+) calcd 335.1303, obsd 335.1303.
1
White solid; yield: 30%; mp: 230–231 °C. H NMR (400
MHz, DMSO-d6) δ 10.76 (s, 1H, NH), 7.93 (d, J = 8.4 Hz,
2H, Ar–H), 7.86 – 7.77 (m, 4H, Ar–H), 7.71 (d, J = 8.7 Hz,
2H, Ar–H), 6.71 (s, 1H, C4–H), 5.10 (s, 2H, CH2), 2.32 (s,
3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 166.47 (CO),
148.05 (C-3), 142.54 (Ar–C), 138.26 (C-5), 133.15 (2C, Ar–
C), 126.69 (q, 2C, 3JCF = 3.7 Hz), 125.96 (2C, Ar–C), 124.77
(q, 1JCF = 269.7 Hz), 124.16 (q, 2JCF = 32.0 Hz), 123.42 (Ar–
C), 119.65 (2C, Ar–C), 119.47 (CN), 110.03 (Ar–C), 104.09
(C-4), 52.95 (CH2), 11.33 (CH3). HRMS (ESI+) m/z
C20H15ON4F3 (M+H+) calcd 385.1271, obsd 385.1259.
2-(3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(3-
(trifluoromethyl)phenyl) acetamide (6i)
1
White solid; yield: 29%; mp: 199–201 °C. H NMR (400
MHz, DMSO-d6) δ 10.75 (s, 1H, NH), 8.10 (s, 1H, Ar–H),
7.94 (d, J = 8.5 Hz, 2H, Ar–H), 7.84 (d, J = 8.5 Hz, 2H, Ar–
H), 7.77 (d, J = 7.9 Hz, 1H, Ar–H), 7.59 (t, J = 8.0 Hz, 1H,
Ar–H), 7.44 (d, J = 7.8 Hz, 1H, Ar–H), 6.71 (s, 1H, C4–H),
5.09 (s, 2H, CH2), 2.32 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6) δ 166.43 (CO), 148.08 (C-3), 142.55 (Ar–C),
139.76 (C-5), 138.28 (Ar–C), 133.15 (2C, Ar–C), 130.64 (Ar–
C), 130.05 (q, 2JCF = 31.3 Hz), 125.97 (2C, Ar–C), 124.50 (q,
N-(3-chloro-2-fluorophenyl)-2-(3-(4-cyanophenyl)-5-methyl-
1H-pyrazol-1-yl) acetamide (6m)
3
1JCF = 271.0 Hz), 123.28 (Ar–C), 120.47 (q, JCF = 4.0 Hz),
3
1
119.45 (CN), 115.78 (q, JCF = 4.0 Hz), 110.04 (Ar–C),
White solid; yield: 9%; mp: 165–167 °C. H NMR (400
104.08 (C-4), 52.90 (CH2), 11.33 (CH3). HRMS (ESI+) m/z
MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 7.94 (d, J = 8.2 Hz,
2H, Ar–H), 7.89 – 7.77 (m, 1H, Ar–H), 7.71 (d, J = 8.4 Hz,
2H, Ar–H), 7.36 (t, J = 7.5 Hz, 1H, Ar–H), 7.19 (t, J = 8.2
Hz, 1H, Ar–H), 6.37 (s, 1H, C4–H), 5.04 (s, 2H, CH2), 2.20
(s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 167.03
C20H15ON4F3 (M+H+) calcd 385.1271, obsd 385.1269.
N-(3-cyanophenyl)-2-(3-(4-cyanophenyl)-5-methyl-1H-
pyrazol-1-yl)acetamide (6j)
1
(CO), 149.73 (d, JCF = 246.0 Hz), 148.01 (C-3), 143.71
1
White solid; yield: 31%; mp: 219–220 °C. H NMR (400
(Ar–C), 135.18 (C-5), 133.20 (2C, Ar–C), 129.48 (2C, Ar–
3
MHz, DMSO-d6) δ 10.75 (s, 1H, NH), 8.08 (s, 1H, Ar–H),
7.93 (d, J = 8.5 Hz, 2H, Ar–H), 7.87 – 7.80 (m, 3H, Ar–H),
7.56 (s, 2H, Ar–H), 6.71 (s, 1H, C4–H), 5.09 (s, 2H, CH2),
2.32 (s, 3H, CH3).13C NMR (100 MHz, DMSO-d6) δ
166.48 (CO), 148.08 (C-3), 142.56 (Ar–C), 139.77 (C-5),
138.26 (Ar–C), 133.15 (2C, Ar–C), 130.87 (Ar–C), 127.74
(Ar–C), 125.97 (2C, Ar–C), 124.31 (Ar–C), 122.44 (Ar–C),
119.46 (CN), 119.04 (Ar–C), 112.20 (Ar–C), 110.04 (Ar–
C), 104.10 (C-4), 52.89 (CH2), 11.33 (CH3). HRMS (ESI+)
m/z C20H15ON5 (M+H+) calcd 342.1349, obsd 342.1343.
C), 127.54 (d, JCF = 11.0 Hz), 126.36 (Ar–C), 125.51 (d,
2
4JCF = 4.0 Hz), 123.15 (Ar–C), 120.38 (d, JCF = 16.0 Hz),
119.04 (CN), 111.45 (Ar–C), 107.18 (C-4), 52.81(CH2),
13.65 (CH3). HRMS (ESI+) m/z C19H14ON4FCl (M+H+)
calcd 369.0913, obsd 369.0906.
N-(4-cyano-3-(trifluoromethyl)phenyl)-2-(3-(4-
cyanophenyl)-5-methyl-1H-pyrazol-1-yl) acetamide (6n)
1
White solid; yield: 29%; mp: 244–247 °C. H NMR (400
MHz, DMSO-d6) δ 11.19 (s, 1H, NH), 8.28 (d, J = 1.9 Hz,
1H, Ar–H), 8.13 (d, J = 8.6 Hz, 1H, Ar–H), 7.99 – 7.90 (m,
3H, Ar–H), 7.84 (d, J = 8.5 Hz, 2H, Ar–H), 6.72 (s, 1H,
C4–H), 5.14 (s, 2H, CH2), 2.31 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6) δ 167.29 (CO), 148.20 (C-3), 143.54
(Ar–C), 142.61 (Ar–C), 138.19 (C-5), 137.13 (Ar–C),
133.16 (2C, Ar–C), 132.30 (q, 2JCF = 31.7 Hz), 125.98 (2C,
2-(3-(4-cyanophenyl)-5-methyl-1H-pyrazol-1-yl)-N-(4-
fluorophenyl)acetamide (6k)
1
White solid; yield: 17%; mp: 247–250 °C. H NMR (400
MHz, DMSO-d6) δ 10.43 (s, 1H, NH), 7.93 (d, J = 8.5 Hz,
2H, Ar–H), 7.83 (d, J = 8.5 Hz, 2H, Ar–H), 7.61 (dd, J =
9.1, 5.0 Hz, 2H, Ar–H), 7.17 (t, J = 8.9 Hz, 2H, Ar–H),
6.70 (s, 1H, C4–H), 5.04 (s, 2H, CH2), 2.31 (s, 3H, CH3).
13C NMR (100 MHz, DMSO-d6) δ 165.67 (CO), 158.67 (d,
1JCF = 239.0 Hz), 147.96 (C-3), 142.49 (Ar–C), 138.32 (C-
1
Ar–C), 122.86 (q, JCF = 271.7 Hz), 122.68 (CN), 119.44
3
(CN), 117.10 (q, JCF = 5.0 Hz), 116.14 (Ar–C), 110.08
(Ar–C), 104.16 (Ar–C), 102.59 (C-4), 53.00 (CH2), 11.29
(CH3). HRMS (ESI+) m/z C21H14ON5F3 (M+H+) calcd
410.1223, obsd 410.1235.
4
5), 135.41 (d, JCF = 2.0 Hz), 133.16 (2C, Ar–C), 125.96

Medicinal Chemistry Research
Biological analysis
revealed that compound 6f was a potent AR antagonist.
Some compounds in this series also showed higher anti-
proliferative activity against LNCaP cells than Bicaluta-
mide. These compounds can be used to discover novel
potent AR antagonists after further optimization.
Cell culture
MDA-Kb2, LNCaP, and PC-3 cells were obtained from the
American Type Culture Collection (Manassas, VA, USA).
MDA-Kb2 cells were cultured in L15 media containing L-
glutamine (Hyclone), Na pyruvate (Hyclone), penstrep
(Hyclone), and 10% FBS (Hyclone). LNCaP cells were
cultured in RPMI-1640 media (Gibco, with addition of
NaHCO3 1.5 g/L, glucose 2.5 g/L, and sodium pyruvate
0.11 g/L) with 10% FBS (Gibco). PC-3 cells were cultured
in F12 media (Hyclone) with 10% FBS (Hyclone). All cells
were propagated in an incubator at 37 °C with 5% CO2.
Acknowledgements This work was financially supported by the
National Natural Science Foundation of China (No. 21272140), Key
Research and Development Project of Shandong Province (No.
2017CXGC1401), and the Knowledge Foundation (No. 201550084)
of Sweden.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Luciferase reporter assay
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
MDA-Kb2 cells were seeded in 96-well plates (Nunc) at a
density of 3 × 104 cells/well and incubated for 24 h. The
cells were treated with the test compounds at 1 µM in
combination with 10 nM DHT. Cells only exposed to 10
nM DHT were used as a control group. After 24 h of
incubation, cells were lysed using reporter lysis buffer
(Promega) and the luciferase levels were determined with
luciferase assay kit (Promega) in a TD 20/20 luminometer
(Turner Designs, USA). The antagonism rates of the syn-
thesized compounds were calculated using the luciferase
activities.
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Conclusion
In summary, a series of 2-(5-methyl-1H-pyrazol-1-yl)
acetamide derivatives were developed via structural mod-
ification of a pyrazole based lead compound 10e. We
determined the AR antagonistic activity of the compounds
and evaluated their growth inhibitory effects using the
LNCaP and PCa cell lines. The AR luciferase reporter assay