Synthesis and biological evaluation of 3-(4-fluorophenyl)-1H-pyrazole derivatives as androgen receptor antagonists

Article abstract of DOI:10.1097/CAD.0000000000000322

A novel series of 3-(4-fluorophenyl)-1H-pyrazole derivatives were synthesized and evaluated for their antiproliferative activity against two prostate cancer cell lines (LNCaP and PC-3) and androgen receptor target gene prostate-specific antigen (PSA) inhibitory activity in LNCaP cells. Several compounds showed potent antiproliferative activity against LNCaP cells and showed a promising PSA downregulation rate. Among these, compound 10e selectively inhibited LNCaP cell growth with an IC₅₀ value of 18 μmol/l and showed a PSA downregulation rate of 46%, which was better than the lead compound T3.

Full text of DOI:10.1097/CAD.0000000000000322

278 Preclinical report
Synthesis and biological evaluation of 3-(4-fluorophenyl)-
1H-pyrazole derivatives as androgen receptor antagonists
Guangzhu Guo*, Jianzhen Liu*, Guanjie Wang, Daoguang Zhang, Jinjie Lu and
Guisen Zhao
Anti-Cancer Drugs 2016, 27:278–285
A novel series of 3-(4-fluorophenyl)-1H-pyrazole derivatives
were synthesized and evaluated for their antiproliferative
activity against two prostate cancer cell lines (LNCaP and
PC-3) and androgen receptor target gene prostate-specific
antigen (PSA) inhibitory activity in LNCaP cells. Several
compounds showed potent antiproliferative activity against
LNCaP cells and showed a promising PSA downregulation
rate. Among these, compound 10e selectively inhibited
LNCaP cell growth with an IC50 value of 18 μmol/l and
showed a PSA downregulation rate of 46%, which was
better than the lead compound T3. Anti-Cancer Drugs
27:278–285 Copyright © 2016 Wolters Kluwer Health, Inc.
All rights reserved.
Keywords: androgen receptor, antagonist, prostate cancer,
pyrazole derivatives
Key Laboratory of Chemical Biology, Ministry of Education, Department of
Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University,
Jinan, People’s Republic of China
Correspondence to Guisen Zhao, PhD, Key Laboratory of Chemical Biology,
Ministry of Education, Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, China
Tel/fax: + 86 531 8838 2009; e-mail: guisenzhao@sdu.edu.cn
*Guangzhu Guo and Jianzhen Liu contributed equally to the writing of this article.
Received 29 September 2015 Revised form accepted 4 November 2015
Introduction
active site of AR ligand binding domain, was 58.1% at the
concentration of 10 μmol/l. In our efforts to improve the
selectivity to inhibit cell growth of LNCaP cell and the
ability to inhibit the expression of PSA, we performed
structure modification of T3. First, we replaced the
propyl chain of T3 to methylene to shorten the distance
between the pyrazole ring and the aromatic ring and
removed the methyl group of amino to increase the
flexibility of the heteroaromatic ring, generating com-
pound 10. Second, we changed the position of amino and
methylene of compound 10, obtaining compound 11.
Third, replacement of methylene of compound 11 to the
carbonyl group yielded compound 12 (Fig. 1).
Prostate cancer (PC) is one of the most frequently diag-
nosed cancers in men in the USA [1,2]. Androgen
receptor (AR) plays a vital role in PC, which regulates the
transcription and translation of downstream genes for the
survival of PC cells when activated by androgens such as
testosterone and dihydrotestosterone [3]. Blocking the
androgen signaling pathway by blocking the production
of testicular androgen through chemical or surgical cas-
tration [androgen deprivation therapy (ADT)] has been
the standard method for the treatment of PC for decades.
However, after several months of ADT, there may be a
relapse of PC and progression to a new stage called cas-
tration-resistant/recurrent prostate cancer (CRPC) [4,5].
Once CRPC occurs, ADT will no longer work, but the
survival of CRPC still relies on the androgen signaling
pathway even after castration [6–8]. Therefore, use of AR
antagonists to block the binding of androgens to AR
remains an attractive strategy to treat CRPC [1].
Materials and methods
Reagents and materials
All materials were commercially available and used
without further purification. Thin-layer chromatography
(TLC) was used to monitor reaction in SGF254 plates.
Column chromatography was performed on a silica gel
(60 Å, 200–300 mesh). Melting points were determined
on a Büchi capillary melting point apparatus (Buchi
Labortechnik AG, Flawill, Switzerland) without correc-
Our study began with compound T3 (Fig. 1), a
3-(4-fluorophenyl)-1H-pyrazole derivative, which was
reported in our previous work [5]. T3 was tested for
antiproliferative activity against the LNCaP cell line,
which expresses mutant AR, and the PC-3 cell line,
which is AR negative, and showed an IC50 of 17 and 57
μmol/l respectively, suggesting a certain degree of
selectivity against the LNCaP cell line. The inhibition of
prostate-specific antigen (PSA) expression was also
investigated, with an inhibition rate of 60.7% at the
concentration of 10 μmol/l. The binding affinity of T3,
evaluated by measuring the ability to displace a potent
fluorescently labeled androgen (fluorophore) from the
1
tion. The H NMR spectra were recorded on a Bruker
Avance DRX-600 Spectrometer (Bruker, Rheinstetten,
Germany) or a Bruker Avance DRX-400 Spectrometer.
The 13C NMR spectra were assessed on a Bruker Avance
DRX-400 spectrometer. The chemical shifts (δ) are
reported in parts per million (ppm) and the following
abbreviations are used: singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), doublet of doublets (dd), and
quartet of doublets (qd). The purity data were obtained
0959-4973 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.
DOI: 10.1097/CAD.0000000000000322
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3-(4-Fluorophenyl)-1H-pyrazole derivatives Guo et al. 279
Fig. 1
NC
H3C
CH3
N
CH3
N
CH3
O
N
N
N
N
NH
HN
N
N
N
NH
N
R
R
H
R
F
F
F
F
T3
10
11
12
Design strategy for target compounds 10, 11, and 12.
Scheme 1
NH
O
OH
N
O
O
O
i
iii
ii
Et
CH3
OEt
O
F
F
F
1
2
3
R1
R2
CH3
CH3
N
CH3
O
N
CH3
H2N
N
N
N
N
N
R3
F
HN
N
vii
viii
F
ix
OEt
R1
OH
C1
R3
F
R2
F
4
8
9
10
iv
R2
CH3
R2
CH3
CH3
Br
CH3
N
N
N
N
N
N
O
N
N
NH
NH2
v
x
vi
NH
OH
OEt
O
F
F
F
F
11
7
5
6
O
R
xi
Cl
CH3
O
N
N
R2
N
H
F
12
Synthesis of target compounds 10, 11, and 12. The reagents and conditions were as follows: (i) EtONa, diethyl oxalate, EtOH, reflux, 2 h; (ii) hydrazine
hydrate, acetic acid, EtOH, reflux, 3 h; (iii) dimethyl sulfate, NaH, DMF, 0°C; (iv) KOH, EtOH, reflux, 5 h; (v) DPPA, Et3N,1,4-dioxane, EtOH, reflux, 7 h;
(vi) NaOH, EtOH, reflux, 5 h; (vii) LiAlH4, THF, 0°C, 15 min; (viii) SOCl2, CH2Cl2, rt, 1 h; (ix) K2CO3, KI, acetone, rt, 8 h; (x) NaH, DMF, rt, 16 h; and (xi)
pyridine, THF, rt, 2 h. DMF, N,N-dimethylformamide; DPPA, diphenylphosphoryl azide; THF, tetrahydrofuran.
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

280 Anti-Cancer Drugs 2016, Vol 27 No 4
Table 1 Abilities of target compounds to inhibit prostate-specific antigen expression and growth of prostate cancer cell lines
CH3
R1
N
N
X
R2
R3
F
Cell growth inhibition (IC50/μmol/l)
Codes
X
R1
R2
R3
PSA expression inhibition rate (%) (5 μmol/l)
LNCaP
PC-3
Enzalutamide
T3
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
12a
12b
11a
–
–
–
–
–
–
–
67.7
36.0
33.8
11.9
46.8
39.8
46.3
41.3
36.3
63.6
30.3
43.2
24.5
37.5
27.8
33.0
54.7
6.1
32.1 6.5
26.9 7.9
26.2 8.7
20.4 3.8
57.1 2.7
27.9 7.0
17.8 0.1
20.1 5.8
27.9 1.8
61.8 2.9
26.6 2.9
30.7 9.9
25.0 1.8
31.6 9.4
41.1 9.5
25.4 2.1
37.2 3.5
43.6 9.0
30.8 7.6
30.0 5.1
> 80
–
4 8.4 8.0
38.7 2.1
21.6 3.3
> 80
> 80
> 80
51.6 1.6
60.3 7.3
> 80
38.7 2.8
26.0 9.9
36.6 5.3
68.4 5.8
48.3 3.0
37.4 6.4
64.9 5.2
> 80
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
CH2NH
NHCO
NHCO
NHCH2
H
H
CN
H
F
Br
CF3
CN
CN
H
Cl
H
H
H
H
CF3
F
Cl
Br
Cl
Br
CF3
Br
H
H
H
H
H
H
H
H
H
H
H
Br
F
H
Cl
CN
Br
CF3
Cl
H
H
H
F
H
CH3
H
H
H
H
H
H
2.6
1.4
> 80
26.9 3.3
PSA, prostate-specific antigen.
in a Shimadzu LC-20AT High-performance Liquid
Chromatograph (Shimadzu Corporation, Tokyo, Japan).
Ethyl 3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-carboxylate (4)
To a solution of 3 (4.00 g, 17.1 mmol) in N,N-dimethyl-
formamide (DMF) sodium hydride (1.03 g, 25.8 mmol)
was added in an ice bath. Dimethyl sulfate (3.25 g,
25.8 mmol) was added dropwise after the solution
became clear. The mixture was stirred for an additional
30 min then poured into 100 ml ice water. The aqueous
phase was extracted with ethyl acetate (30 ml × 3). The
organic layer was combined and washed with water
(30 ml × 3) and brine (30 ml × 1), and dried with anhy-
drous sodium sulfate. The solution was filtered. The
filtrate was purified by chromatography over a silica gel
(90% petroleum ether : ethyl acetate) to yield 4 as a white
solid (3.22 g, 76% yield). Mp: 54.0–56.0°C; MS (ESI) m/z
found: 249.3 [M + H]+, 1H NMR (600 MHz, DMSO-d6) δ
(ppm): 7.900(dd, J1 = 9.0 Hz, J2 = 5.4 Hz, 2H), 7.354(s,
1H), 7.247(t, J = 9.0 Hz, 2H), 4.333(q, J = 7.2 Hz, 2H),
4.132(s, 3H), 1.334(t, J = 7.2 Hz, 3H).
Synthesis
(Z)-Ethyl 4-(4-fluorophenyl)-2-hydroxy-4-oxobut-
2-enoate (2)
To
a solution of 4-fluoroacetophenone 1 (3.00 g,
21.74 mmol) and diethyl oxalate (6.35 g, 43.48 mmol) in
absolute anhydrous ethanol (10 ml) sodium ethoxide
(2.96 g, 43.48 mmol) was added dropwise in absolute
anhydrous ethanol (15 ml). The mixture was then heated
to 80°C and stirred for 5–7 h. The reaction mixture was
evaporated under reduced pressure and ice water was
added to the residue obtained. The pH of the mixture
was adjusted to 1 using 1 N HCl. The precipitation was
filtered and washed with water to yield compound 2 as a
yellowish white solid.
Ethyl 3-(4-fluorophenyl)-1H-pyrazole-5-carboxylate (3)
A solution of 2 (5.17 g, 21.72 mmol) in ethanol (15 ml)
was added to hydrazine hydrate (1.36 g, 21.72 mmol),
followed by the addition of glacial acetic acid (1 ml). The
mixture was then heated to 90°C and refluxed for 3 h.
The reaction mixture was concentrated under reduced
pressure to yield compound 3 as a faint yellow solid
(4.8 g, 93%). Mp: 161.0–163.0°C; MS (ESI) m/z found:
235.3 [M + H]+.
3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-carboxylic
acid (5)
To a solution of compound 4 (1.98 g, 8 mmol) in ethyl
alcohol (15 ml) a 10% potassium hydroxide aqueous
solution was added (20 ml). The reaction mixture was
refluxing for 5 h and monitored by TLC until no reactant
was left. The reaction was cooled to room temperature
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3-(4-Fluorophenyl)-1H-pyrazole derivatives Guo et al. 281
and then evaporated under reduced pressure to remove
ethyl alcohol. The residue was neutralized with saturated
citric acid solution until the pH was less than 2. The
suspension was filtered and the filter cake was washed
completely with water to yield compound 5 as a white
solid (1.74 g, 99%). Mp: 212.0–214.5°C; MS (ESI) m/z
found: 221.4 [M + H]+, 1H NMR (600 MHz, DMSO-d6) δ
(ppm): 13.473(s, 1H), 7.878(dd, J1 = 9.0 Hz, J2 = 5.4 Hz,
2H), 7.291(s, 1H), 7.243(t, J = 9.0 Hz, 2H), 4.119(s, 3H).
(50 ml × 1), dried with anhydrous sodium sulfate, filtered,
and concentrated to yield compound 8 as a white solid
(2.47 g, 100% yield). Mp: 112–114°C; 1H NMR
(600 MHz, DMSO-d6) δ (ppm): 7.783(dd, J1 = 8.4 Hz,
J2 = 5.4 Hz, 2H), 7.203(t, J = 9.0 Hz, 2H), 6.597(s, 1H),
5.323(t, J = 5.4 Hz, 1H), 4.518(d, J = 5.4 Hz, 2H), 3.824
(s, 3H).
5-(Chloromethyl)-3-(4-fluorophenyl)-1-methyl-
1H-pyrazole (9)
Ethyl (3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
carbamate (6)
A solution of compound 8 (165 mg, 0.8 mmol) in
dichloromethane was added to thionyl chloride (190 mg,
1.6 mmol) and stirred at room temperature for 1 h. The
solvent was removed under vacuum to yield compound 9
as a faint yellow solid (180 mg, 100% yield). Mp:
124–126°C; 1H NMR (400 MHz, DMSO-d6) δ (ppm):
7.799(t, J = 7.2 Hz, 2H), 7.227(t, J = 7.2 Hz, 2H), 6.800(s,
1H), 4.953(s, 2H), 3.891(s, 3H).
Compound 5 (1.00 g, 4.55 mmol) was dissolved in abso-
lute ethyl alcohol (15 ml) and anhydrous dioxane (15 ml).
Diphenylphosphoryl azide (DPPA, 1.53 g, 5.9 mmol) and
triethylamine (0.60 g, 5.9 mmol) were added and the
solution was heated to reflux for 7 h and monitored by
TLC. The reaction mixture was cooled to room tem-
perature and evaporated under reduced pressure. The
residue was then dissolved with ethyl acetate (30 ml) and
water (30 ml). The organic layer was washed with satu-
rated citric acid solution (30 ml × 3), aqueous sodium
hydrogen carbonate (30 ml × 3), and brine (30 ml × 1), and
dried with anhydrous sodium sulfate, filtered, and con-
centrated. The crude material was absorbed onto a silica
gel and purified using 75% of petroleum ether in ethyl
acetate to yield compound 6 as a white solid (0.9 g, 75%
General procedure for the preparation of 10a–10o
A mixture of substituted aniline (0.8 mmol), potassium
carbonate (553 mg, 4 mmol), and potassium iodide
(13 mg, 0.08 mmol) in acetone (12 ml) was sitrred at room
temperature. To the suspension, a solution of compound
9 (179 mg, 0.8 mmol) was added in acetone (6 ml) drop-
wise. The reaction was stirred for 8 h and monitored by
TLC. Solvent was removed under vaccum, followed by
the addition of ethyl acetate (40 ml) and water (30 ml).
The organic layer was washed with water (30 ml × 1) and
brine (30 ml × 1), dried with anhydrous sodium sulfate,
filtered, and concentrated. The crude material was
absorbed onto silica gel and purified using 90% of pet-
roleum ether in ethyl acetate to yield title compound 10.
1
yield). Mp: 124–126°C; H NMR (600 MHz, DMSO-d6)
δ (ppm): 9.678(s, 1H), 7.774(dd, J1 = 8.4 Hz, J2 = 6.0 Hz,
2H), 7.206(t, J = 9.0 Hz, 2H), 6.549(s, 1H), 4.154(q,
J = 7.2 Hz, 2H), 1.254(t, J = 7.2 Hz, 3H).
3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-amine (7)
Ten per cent sodium hydroxide aqueous solution (30 ml)
was added to a solution of compound 6 (1.29 g, 4.9 mmol)
in ethyl alcohol (30 ml). The reaction mixture was
refluxed for 5 h and monitored by TLC. After completion
of the reaction, ethyl alcohol was evaporated under
reduced pressure. Solid was precipated after ice water
was added. The the mixture was filtered to yield com-
pound 7 as a white solid (0.75 g, 80% yield). Mp:
129–131°C; 1H NMR (600 MHz, DMSO-d6) δ (ppm):
7.673(dd, J1 = 8.4 Hz, J2 = 6.0 Hz, 2H), 7.148(t,
J = 9.0 Hz, 2H), 5.563(s, 1H), 5.273(s, 2H), 3.553(s, 3H).
3-Bromo-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)aniline (10a)
White solid, 115 mg, yield 40%. Mp: 85–87°C, purity:
1
97.1%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.196(t, J = 8.8 Hz, 2H),
7.030(t, J = 8.0 Hz, 1H), 6.844(t, J = 1.6 Hz, 1H), 6.703
(dd, J1 = 7.6 Hz, J2 = 1.2 Hz, 1H), 6.669(dd, J1 = 8.4 Hz,
J2 = 2.0 Hz, 1H), 6.621(s, 1H), 6.447(t, J = 5.6 Hz, 1H),
4.324(d, J = 5.6 Hz, 2 H), 3.839(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 162.02(d, J = 242 Hz), 150.36,
147.99, 142.31, 131.21, 130.41(d, J = 3 Hz), 127.23(d,
J = 8 Hz), 122.77, 119.06, 115.93(d, J = 22 Hz), 114.99,
111.80, 103.00, 38.35, 36.95.
(3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methanol (8)
To a solution of compound 4 (2.98 g, 12 mmol) in tetra-
hydrofuran (THF) (40 ml), lithium aluminium hydride
(1.37 g, 36 mmol) was added under ice-cold conditions.
The mixture was stirred at room temperature for 15 min.
The reaction was quenched with ice water (50 ml) that
was added dropwise under ice-cold conditions. THF was
then removed under vacuum. The residue liquid was
neutralized with 1 N hydrochloric acid until the pH was
less than 1 and extracted with ethyl acetate (50 ml × 3).
Organic layers were combined and washed with brine
N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methyl)-3-(trifluoromethyl)aniline (10b)
White solid, 95 mg, yield 34%. Mp: 85–87°C, purity:
1
97.7%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.755
(dd, J1 = 8.4 Hz, J2 = 5.6 Hz, 2H), 7.299(t, J = 8.0 Hz, 1H),
7.193(t, J = 8.8 Hz, 2H), 6.959(s, 1H), 6.938(d, J = 8.8 Hz,
1H), 6.858(d, J = 7.6 Hz, 1H), 6.636(m, 2H), 4.385(s, 2H),
3.852(s, 3H). 13C NMR (100 MHz, DMSO) δ: 162.04(d,
J = 242 Hz), 149.16, 148.02, 142.15, 130.42(d, J = 3 Hz),
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

282 Anti-Cancer Drugs 2016, Vol 27 No 4
130.44(q, J = 31 Hz), 130.32, 127.22(d, J = 8 Hz), 124.98
(q, J = 271 Hz), 116.13, 115.92(d, J = 21 Hz), 112.65(q,
J = 4 Hz), 108.81(q, J = 4 Hz), 103.07, 38.33, 36.95.
4-Chloro-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)aniline (10g)
White solid, 119 mg, yield 47%. Mp: 106–108°C, purity:
1
96.4%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.758
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.197(t, J = 8.8 Hz, 2H),
7.114(d, J = 8.8 Hz, 2H), 6.684(d, J = 8.8 Hz, 2H), 6.618(s,
1H), 6.344(t, J = 5.6 Hz, 1H), 4.310(d, J = 6.0 Hz, 2H),
3.843(s, 3H). 13C NMR (100 MHz, DMSO) δ: 162.01(d,
J = 242 Hz), 147.95, 147.61, 142.46, 130.43(d, J = 3 Hz),
129.04, 127.22(d, J = 9 Hz), 120.06, 115.91(d, J = 22 Hz),
114.26, 103.02, 38.60, 36.93.
4-(((3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methyl)amino)phthalonitrile (10c)
White solid, 45 mg, yield 17%. Mp: 224–226°C, purity:
97.4%; 1H NMR (400 MHz, DMSO-d6) δ (ppm):
7.786 ∼ 7.712(m, 4H), 7.262(d, J = 2.4 Hz, 1H), 7.203(t,
J = 8.8 Hz, 2H), 7.042(dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H),
6.646(s, 1H), 4.500(d, J = 5.6 Hz, 2H), 3.843(s, 3H). 13C
NMR (100 MHz, DMSO) δ: 162.07(d, J = 242 Hz),
152.09, 148.15, 140.95, 135.24, 130.28(d, J = 3 Hz), 127.27
(d, J = 8 Hz), 117.86, 116.94, 115.94(d, J = 21 Hz), 103.13,
99.23, 37.82, 37.01.
4-(((3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methyl)amino)benzonitrile (10h)
White solid, 169 mg, yield 69%. Mp: 194–196°C, purity:
1
94.2%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.763
(dd, J1 = 9.2 Hz, J2 = 5.6 Hz, 2H), 7.491(d, J = 8.8 Hz,
2H), 7.199(t, J = 9.2 Hz, 2H), 6.763(d, J = 8.8 Hz, 2H),
6.636(s, 1H), 4.420(d, J = 6.0 Hz, 1H), 3.846(s, 3H). 13C
NMR (100 MHz, DMSO) δ: 162.04(d, J = 242 Hz, 152.14,
148.06, 141.70, 133.84, 130.34(d, J = 3 Hz), 127.25(d,
J = 8 Hz), 120.93, 115.93(d, J = 21 Hz), 112.75, 103.13,
97.00, 37.86, 36.98.
3-(((3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methyl)amino)benzonitrile (10d)
White solid, 196 mg, yield 80%. Mp: 129–131°C, purity:
1
97.6%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 8.4 Hz, J2 = 5.6 Hz, 2H), 7.275(d, J = 8.0 Hz,
1H), 7.193(t, J = 8.8 Hz, 2H), 7.016(s, 1H), 7.005(d,
J = 8.8 Hz, 1H), 6.964(d, J = 7.2 Hz, 1H), 6.662(t,
J = 7.6 Hz, 1H), 6.628(s, 1H), 4.374(d, J = 7.6 Hz, 2H),
3.845(s, 3H). 13C NMR (100 MHz, DMSO) δ: 162.03(d,
J = 242 Hz), 149.22, 148.01, 142.01, 130.52, 130.45(d,
J = 3 Hz), 127.22(d, J = 8 Hz), 119.94, 117.84, 115.92(d,
J = 22 Hz), 114.69, 112.17, 103.03, 38.23, 37.00.
4-Bromo-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)aniline (10i)
White solid, 182 mg, yield 63%. Mp: 117–119°C, purity:
1
97.7%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.751
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.208(t, J = 8.8 Hz, 2H),
7.190(d, J = 8.8 Hz, 2H), 6.454(d, J = 8.8 Hz, 2H), 6.607(s,
1H), 6.361(t, J = 5.6 Hz, 1H), 4.301(d, J = 6.0 Hz, 2H),
3.835(s, 3H). 13C NMR (100 MHz, DMSO) δ: 162.04(d,
J = 243 Hz), 148.06, 147.80, 142.59, 131.88, 130.19(d,
J = 3 Hz), 127.28(d, J = 8 Hz), 115.94(d, J = 22 Hz),
114.89, 107.56, 103.02, 38.39, 36.80.
4-Fluoro-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)aniline (10e)
White solid, 96 mg, yield 50%. Mp: 115–117°C, purity:
1
97.4%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.199(t, J = 8.8 Hz, 2H),
6.939(t, J = 8.8 Hz, 2H), 6.671(dd, J1 = 8.8 Hz,
J2 = 4.8 Hz, 2H), 6.626(s, 1H), 6.056(t, J = 6.0 Hz, 1H),
4.286(d, J = 6.0 Hz, 2H), 3.848(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 162.01(d, J = 242 Hz), 155.08(d,
J = 230 Hz), 147.95, 145.41(d, J = 1 Hz), 142.74, 130.46(d,
J = 3 Hz), 127.21(d, J = 8 Hz), 115.91(d, J = 21 Hz, 115.71
(d, J = 22 Hz), 113.74(d, J = 7 Hz), 103.01, 39.12, 36.91.
N-((3-(4-Fluorophenyl)-1-methyl-1H-pyrazole-5-yl)
methyl)-4-(trifluoromethyl)aniline (10j)
White solid, 81 mg, yield 29%. Mp: 109–111°C, purity:
1
91.4%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 9.2 Hz, J2 = 5.6 Hz, 2H), 7.399(d, J = 8.8 Hz,
2H), 7.191(t, J = 9.2 Hz, 2H), 6.893(s, 1H), 6.785(d,
J = 8.8 Hz, 2H), 6.634(s, 1H), 4.393(s, 2H), 3.849(s, 3H).
13C NMR (100 MHz, DMSO) δ: 162.03(d, J = 242 Hz),
151.67, 148.02, 142.03, 130.43(d, J = 3 Hz), 127.23(d,
J = 8 Hz), 126.68(q, J = 4 Hz), 125.77(q, J = 268 Hz),
116.41(q, J = 32 Hz), 115.90(d, J = 22 Hz), 112.23, 103.09,
38.12, 36.98.
3-Chloro-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)aniline (10f)
White solid, 159 mg, yield 63%. Mp: 77–79°C, purity:
1
97.8%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 8.4 Hz, J2 = 7.2 Hz, 2H), 7.201(t, J = 8.8 Hz, 2H),
7.095(t, J = 8.0 Hz, 1H), 6.703(t, J = 2.0 Hz, 1H), 6.648(d,
J = 1.6 Hz, 1H), 6.629(s, 1H), 6.578(dd, J1 = 8.0 Hz,
J2 = 1.2 Hz, 1H), 6.467(t, J = 5.6 Hz, 1H), 4.326(d,
J = 5.6 Hz, 2H), 3.846(s, 3H). 13C NMR (100 MHz,
DMSO) δ: 162.03(d, J = 242 Hz), 150.25, 147.99, 142.77,
134.11, 130.84, 130.47(d, J = 2 Hz), 127.22(d, J = 8 Hz),
116.15, 115.92(d, J = 21 Hz), 112.08, 111.53, 103.02,
38.42, 36.98.
4-Chloro-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)-3-(trifluoromethyl)aniline (10k)
White solid, 25 mg, yield 8%. Mp: 124–126°C, purity:
1
87.7%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.758
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.372(d, J = 8.8 Hz,
1H), 7.196(t, J = 8.8 Hz, 2H), 7.096(d, J = 2.8 Hz, 1H),
6.911(dd, J1 = 8.8 Hz, J2 = 2.8 Hz, 1H), 6.814(t,
J = 5.2 Hz, 1H), 6.629(s, 1H), 4.393(d, J = 5.2 Hz, 2H),
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

3-(4-Fluorophenyl)-1H-pyrazole derivatives Guo et al. 283
3.844(s, 3H), 13C NMR (100 MHz, DMSO) δ: 162.08(d,
J = 243 Hz), 148.19, 147.57, 142.03, 132.43, 130.02(d,
J = 3 Hz), 127.33(q, J = 30 Hz), 127.30(d, J = 8 Hz),
123.46(q, J = 271 Hz), 116.83, 116.55, 115.97(d,
J = 21 Hz), 111.63(q, J = 5 Hz), 103.08, 38.16, 36.77.
149.83(d, J = 133 Hz), 147.98, 146.31(d, J = 2 Hz), 142.22,
130.47(d, J = 3 Hz), 127.21(d, J = 8 Hz), 119.87(d,
J = 18 Hz), 117.37(d, J = 21 Hz), 115.92(d, J = 21 Hz),
113.16, 112.63(d, J = 6 Hz), 103.01, 38.84, 36.98.
N-(3-bromobenzyl)-3-(4-fluorophenyl)-1-methyl-
1H-pyrazole-5-amine (11a)
3-Bromo-4-fluoro-N-((3-(4-fluorophenyl)-1-methyl-
1H-pyrazole-5-yl)methyl)aniline (10l)
To a solution of compound 7 (132 mg, 0.69 mmol) in
DMF (15 ml) sodium hydride 60% (51 mg, 1.265 mmol)
was added. The mixture was stirred till no hydrogen was
generated. Then, a solution of 1-bromo-3-(bromomethyl)
benzene (190 mg, 0.76 mmol) in DMF (5 ml) was added
dropwise. The reaction was stirred for 16 h. The mixture
was poured into water and extracted with ethyl acetate
(30 ml × 3). The organic layer was combined and washed
with brine (30 ml × 1), dried with anhydrous sodium
sulfate, filtered, and concentrated. The crude material
was absorbed onto silica gel and purified using 75% of
petroleum ether in ethyl acetate to yield the title com-
pound as a faint yellow solid (67 mg, 27% yield). Mp:
White solid, 42 mg, yield 14%. Mp: 101–103°C, purity:
97.1%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.757
1
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.195(t, J = 8.8 Hz, 2H),
7.012(t, J = 8.8 Hz, 1H), 6.922(dd, J1 = 5.6 Hz,
J2 = 2.8 Hz, 1H), 6.932 ∼ 6.911(m, 1H), 6.616(s, 1H),
6.300(t, J = 5.6 Hz, 1H), 4.307(d, J = 6.0 Hz, 2H), 3.836(s,
3H). 13C NMR (100 MHz, DMSO) δ: 162.03(d,
J = 242 Hz), 150.91(d, J = 231 Hz), 148.00, 146.52(d,
J = 2 Hz), 142.27, 130.41(d, J = 2 Hz), 127.23(d, J = 6 Hz),
117.15(d, J = 23 Hz), 116.00(d, J = 7 Hz), 115.82, 113.24
(d, J = 7 Hz), 108.46(d. J = 21 Hz), 103.00, 38.82, 36.95.
1
3-Chloro-2-fluoro-N-((3-(4-fluorophenyl)-1-methyl-
1H-pyrazol-5-yl)methyl)aniline (10m)
117–119°C, purity: 97.8%; H NMR (600 MHz, DMSO-
d6) δ (ppm): 7.661(dd, J1 = 8.4 Hz, J2 = 5.4 Hz, 2H), 7.617
(s, 1H), 7.432(t, J = 8.4 Hz, 2H), 7.305(t, J = 7.8 Hz, 1H),
7.145(t, J = 8.4 Hz, 2H), 6.261(t, J = 6.0 Hz, 1H), 5.730(s,
1H), 4.261(d, J = 6.0 Hz, 2H), 3.621(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 161.83(d, J = 242 Hz), 149.93,
147.40, 143.27, 131.13(d, J = 3 Hz), 130.88, 130.46,
130.15, 126.94, 126.90(d, J = 8 Hz), 122.16, 115.54(d,
J = 2 Hz), 84.42, 48.26.
White solid, 91 mg, yield 34%. Mp: 87–89°C, purity:
1
98.0%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.749
(dd, J1 = 8.8 Hz, J2 = 5.6 Hz, 2H), 7.178(t, J = 8.8 Hz, 2H),
6.950(td, J1 = 8.0, J2 = 1.6 Hz, 1H), 7.758(td, J1 = 8.0 Hz,
J2 = 1.2 Hz, 1H), 6.688(td, J1 = 8.0 Hz, J2 = 1.2 Hz, 1H),
6.599(s, 1H), 6.453(td, J1 = 6.0 Hz, J2 = 1.2 Hz, 1H), 4.416
(d, J = 6.0 Hz, 2H), 3.858(s, 3H). 13C NMR (100 MHz,
DMSO) δ: 162.00(d, J = 242 Hz), 147.95, 146.72(d,
J = 239 Hz), 142.24, 138.00(d, J = 11 Hz), 130.43(d,
J = 3 Hz), 127.22(d, J = 8 Hz), 125.60(d, J = 4 Hz), 119.67
(d, J = 14 Hz), 116.79, 115.87(d, J = 22 Hz), 111.65(d,
J = 3 Hz), 102.94, 38.29, 36.99.
General procedure for the preparation of 12a–12b
To a solution of compound 7 (153 mg, 0.8 mmol) in THF
(15 ml) anhydrous pyridine (1 ml) was added, followed by
the addition of substituted benzoyl chloride (1 mmol) in
THF (20 ml) dropwise under an ice bath. The ice bath
was removed after addition was complete. The reaction
was stirred at room temperature for 2 h and monitored by
TLC. The sovent was removed under reduced pressure
and ethyl acetate was added. The organic layer was
washed completely with water (30 ml × 3) and brine
(30 ml × 1), dried with anhydrous sodium sulfate, filtered,
and concentrated. The crude material was absorbed onto
silica gel and purified using 85% of petroleum ether in
ethyl acetate to yield title compound 12.
3-Bromo-N-((3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)methyl)-2-methylaniline (10n)
White solid, 204 mg, yield 68%. Mp: 108–110°C, purity:
1
98.5%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.751
(dd, J1 = 8.4 Hz, J2 = 5.6 Hz, 2H), 7.179(t, J = 8.8 Hz, 2H),
6.903(t, J = 8.0 Hz, 1H), 6.825(d, J = 8.0 Hz, 1H), 6.606(d,
J = 8.0 Hz, 1H), 5.874(t, J = 4.8 Hz, 1H), 4.393(d,
J = 4.8 Hz, 1H), 3.867(s, 2H), 2.264(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 161.99(d, J = 242 Hz), 147.90,
147.75, 142.60, 130.52(d, J = 3 Hz), 128.21, 127.21(d,
J = 8 Hz), 125.32, 121.63, 120.48, 115.87(d, J = 21 Hz),
109.64, 102.96, 38.98, 37.03, 17.56.
3-Bromo-N-(3-(4-fluorophenyl)-1-methyl-1H-pyrazole-
5-yl)benzamide (12a)
3-Chloro-4-fluoro-N-((3-(4-fluorophenyl)-1-methyl-
1H-pyrazole-5-yl)methyl)aniline (10o)
White solid, 162 mg, yield 54%. Mp: 156–158°C, purity:
97.4%; H NMR (400 MHz, DMSO-d6) δ (ppm): 10.545
1
White solid, 150 mg, yield 56%. Mp: 121–123°C, purity:
99.6%; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.759
(dd, J1 = 8.4 Hz, J2 = 5.6 Hz, 2H), 7.198(t, J = 8.8 Hz, 2H),
7.134(t, J = 9.2 Hz, 1H), 6.791(dd, J1 = 6.0 Hz,
J2 = 2.4 Hz, 1H), 6.661 ∼ 6.623(m, 2H), 6.337(t,
J = 5.2 Hz, 1H), 4.309(d, J = 5.6 Hz, 2H), 3.837(s, 3H).
13C NMR (100 MHz, DMSO) δ: 162.01(d, J = 242 Hz),
(s, 1H), 8.185(t, J = 1.6 Hz, 1H), 7.994(d, J = 8.0 Hz, 1H),
7.966 ∼ 7.806(m, 3H), 7.539(t, J = 8.0 Hz, 1H), 7.230(t,
J = 8.8 Hz, 2H), 6.720(s, 1H), 3.758(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 164.90, 162.17(d, J = 242 Hz),
147.92, 137.81, 135.86, 135.40, 131.31, 130.97, 130.29(d,
J = 3 Hz), 127.50, 127.24(d, J = 8 Hz), 122.27, 115.97(d,
J = 21 Hz), 98.27, 36.34.
1
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

284 Anti-Cancer Drugs 2016, Vol 27 No 4
N-(3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-yl)-3-
(trifluoromethyl)benzamide (12b)
15 min by adding 50 μl of stopping solution to all wells.
The absorption was measured at 450 nm using the
ELX800 Microplate Reader.
White solid, 131 mg, yield 45%. Mp: 173–175°C, purity:
1
99.8%; H NMR (400 MHz, DMSO-d6) δ (ppm): 10.694
(s, 1H), 8.341(s, 1H), 8.305(d, J = 7.6 Hz, 1H), 8.027(d,
J = 7.6 Hz, 1H), 7.848 ∼ 7.214(m, J = 3H), 7.236(t,
J = 8.8 Hz, 2H), 6.7240(s, 1H), 3.768(s, 3H). 13C NMR
(100 MHz, DMSO) δ: 164.83, 162.17(d, J = 242 Hz),
147.89, 137.75, 134.70, 132.54, 130.39, 129.82(q,
J = 2 Hz), 129.19(q, J = 3 Hz), 127.22(d, J = 8 Hz), 125.01
(q, J = 4 Hz), 124.39(q, J = 271 Hz), 115.95(d, J = 21 Hz),
98.29, 36.44.
Results and discussion
Chemicals
Synthesis of target compounds started with commercially
available 4-fluorophenylacetophenone 1 as shown in
Scheme 1, which reacted with diethyl oxalate in the
presence of sodium ethylate to yield β-keto ester as
intermediate 2, whose condensation with hydrazine
hydrate catalyzed by acetic acid generated 3. Methylation
of 3 with dimethyl sulfate in DMF yielded intermediate
4, which was subsequently hydrolyzed with potassium
hydroxide to yield 5. Then, Curtius rearrangement was
performed in the presence of DPPA and triethylamine to
transform acid 5 into carbamate 6. Hydrolysis of 6 by
sodium hydroxide yielded key intermediate 7.
Intermediate 8 was produced by a complete reduction of
4 by LiAlH4 in THF, which was subsequently haloge-
nated to 9 using thionyl chloride at room temperature.
Then, a nucleophilic attack of substituted aniline to 9
yielded the target compound 10. Compound 11 was
obtained using 7 and substituted benzyl bromide in
DMF in the presence of sodium hydride. Target com-
pound 12 was obtained easily and completely by an
acylation reaction of 7 using a combination of substituted
benzoyl chloride and pyridine, which was used as an acid-
binding agent.
Antiproliferative activity
All target compounds were evaluated for their in-vitro
antiproliferative activity against two PC cell lines LNCaP
(androgen dependent) and PC-3 (androgen independent)
on the basis of the standard MTT assay methodology.
Cell lines were obtained from the American Type
Culture Collection (Manassas, Virginia, USA). Cells were
cultured in medium RPMI 1640 (Gibco, Carlsbad,
California, USA) containing 10% fetal bovine serum
(Gibco), 100 IU/ml penicillin, and 100 μg/ml streptomy-
cin in a humidified atmosphere with 5% CO2 at 37°C.
Cells were seeded in 96-well plates at a density of 4 × 103
cells/well. Compounds were dissolved in DMSO and
added to plates when cells were attached to the bottom.
The final concentrations of compounds were 5, 10, 20,
40, and 80 μmol/l and that of DMSO was less than 0.5%.
After 96 h of treatment with compounds, MTT (20 μl,
5 mg/ml) was added, followed by another 4 h of incuba-
tion; the absorbance at 570 and 630 nm was measured
using an ELX800 Microplate Reader (BioTek, Winooski,
Vermont, USA) three times for each plate.
Biological evaluation
All of the compounds were evaluated for their anti-
proliferative activity against two PC cell lines LNCaP
and PC-3, and the ability to inhibit AR target gene PSA
expression in LNCaP cells. The testing results are pre-
sented in Table 1. Most of the compounds from skeleton
10 showed increasing PSA expression inhibitory activity
at 5 μmol/l compared with the lead compound T3, such
as 10c, 10d, 10e, 10f, 10g, 10h, 10j, 10l, and 10o.
Among these, 10e showed promising antiproliferative
activity against the LNCaP cell line, with IC50 value of
18 μmol/l, and weak activity to the PC-3 cell line, which
is better than T3 in antiproliferative and PSA expression
inhibitory activity, worth of further study. Compound
10h has a PSA expression inhibitory rate of 63.6%, which
is higher than any other compounds in the three skele-
tons, although a little weaker than enzalutamide, worthy
of further structural modification.
Inhibition of prostate-specific antigen expression
measured by ELISA
The human PSA ELISA kit (catalog no. 1500) purchased
from Alpha Diagnostic International (San Antonio,
Texas, USA) was used to determine the PSA level in
medium according to the procedure of the manufacturer.
LNCaP cells (4 × 103/well), cultured in medium RPMI
1640 containing 10% fetal bovine serum, 100 IU/ml
penicillin, and 100 μg/ml streptomycin, were seeded in
96-well plates and incubated until cells attached to the
bottom. Then, the compounds were added at a final
concentration of 5 μmol/l. After 96 h treatment, 25 μl
supernatant, PSA, standards, and control were added to
appropriate wells of an ELISA plate. Then, 100 μl of
assay buffer was added to each well and incubated on a
plate shaker for 60 min at room temperature. The wells
were washed three times with 300 μl of 1 × wash buffer,
followed by the addition of 100 μl Ab–enzyme conjugate
buffer. After incubation and shaking for 30 min, the wells
were washed another three times with 1 × wash buffer.
100 μl 3,3′,5,5′-tetramethylbenzidine substrate was then
aspirated into each well. The reaction was stopped after
Compounds 10a, 10b, 10i, 10j, 10k, and 10n showed
good antiproliferative activity against both the cell lines,
some of which showed better activity than enzalutamide
and T3, such as 10b, 10i, 10k, and 10n. The good
antiproliferative activity, combined with their PSA inhi-
bitory activity, suggests that they may not only act on the
AR pathway but also on other targets that exist in both
PC-3 and LNCaP cells that can mediate apoptosis.
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.

3-(4-Fluorophenyl)-1H-pyrazole derivatives Guo et al. 285
Inhibition to PSA expression decrease markedly when
the position of amino and methylene was exchanged,
compared with compound 10a with 11a, both of which
were substituted by bromine on the meta-position of the
benzene ring. The inhibition increased slightly on
replacing methylene in the carbonyl group, and on
comparing compound 11a with 12a.
appreciate the cooperation given by the Institute of
Immunopharmacology and Immunotherapy at the School
of Pharmaceutical Sciences at Shandong University for
using the ELX800 Microplate Reader.
Conflicts of interest
There are no conflicts of interest.
Conclusion
References
In summary, a series of novel 3-(4-fluorophenyl)-
1H-pyrazole derivatives were synthesized and evaluated
for their antiproliferative activity against two PC cell lines
LNCaP and PC-3, and the ability to inhibit AR target
gene PSA expression in LNCaP cells. Some of the
compounds showed promising antiproliferative activity
against the androgen-dependent LNCaP cell line and a
higher PSA inhibition rate, better than the lead com-
pound T3. All compounds from skeleton 10 showed a
higher PSA downregulation level than compounds from
skeletons 11 and 12. Compounds 10a, 10b, 10i, 10j,
10k, and 10n may not only act on the AR pathway, but
on other targets that exist in both PC-3 and LNCaP cells.
Compound 10e selectively inhibited LNCaP cell growth
and showed promising PSA expression inhibitory activ-
ity, better than T3, and this is worth further study.
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Acknowledgements
This work was supported by the National Natural
Science Foundation of China (no. 21272140). We
8
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