Synthesis and pharmacological evaluation of naftopidil-based arylpiperazine derivatives containing the bromophenol moiety

Article abstract of DOI:10.1007/s43440-019-00041-w

Background: Prostate cancer (PCa) is the most common malignancy in men and in the absence of any effective treatments available. Methods: For the development of potential anticancer agents, 24 kinds of naftopidil-based arylpiperazine derivatives containin

Full text of DOI:10.1007/s43440-019-00041-w

Pharmacological Reports
https://doi.org/10.1007/s43440-019-00041-w
ARTICLE
Synthesis and pharmacological evaluation of naftopidil‑based
arylpiperazine derivatives containing the bromophenol moiety
Hong Chen1,2 · Yuna Qian3,4 · Huixia Jia2 · Yuzhong Yu1 · Haibo Zhang1 · Shanchao Zhao1 · Jianliang Shen3,4
Received: 7 July 2019 / Revised: 19 July 2019 / Accepted: 25 September 2019
© Maj Institute of Pharmacology Polish Academy of Sciences 2020
Abstract
Background Prostate cancer (PCa) is the most common malignancy in men and in the absence of any efective treatments
available.
Methods For the development of potential anticancer agents, 24 kinds of naftopidil-based arylpiperazine derivatives contain-
ing the bromophenol moiety were synthesized and characterized by using spectroscopic methods. Their pharmacological
activities were evaluated against human PCa cell lines (PC-3 and LNCaP) and a1-adrenergic receptors (a1-ARs; α1a, α1b,
and α1d-ARs). The structure–activity relationship of these designed arylpiperazine derivatives was rationally explored and
discussed.
Results Among these derivatives, 3c, 3d, 3h, 3k, 3o, and 3s exhibited the most potent activity against the tested cancer cells,
and some derivatives with potent anticancer activities exhibited better a1-AR subtype selectivity than others did (selectivity
ratio >10).
Conclusion This work provided a potential lead compound for the further development of anticancer agents for PCa therapy.
Graphic abstract
Keywords Prostate cancer · Synthesis · Arylpiperazine derivatives · Anticancer activity · Antagonistic activity
Hong Chen and Yuna Qian have contributed equally to this work.
Introduction
Electronic supplementary material The online version of this
Prostate cancer (PCa) is the most common malignancy and
the second leading cause of cancer mortality in men [1].
In Europe, around 416,700 new PCa cases are diagnosed
annually [2]. In the U.S. alone, 161,300 new cases of PCa
and 26,730 deaths due to this disease were reported in 2017.
Approximately one of six males in the U.S. may be aficted
with this type of cancer, and the risk is increased remarkably
article (https://doi.org/10.1007/s43440-019-00041-w) contains
supplementary material, which is available to authorized users.
* Shanchao Zhao
zhaoshanchao@263.net
* Jianliang Shen
shenjl@wibe.ac.cn
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

H. Chen et al.
for older males. Genetics, age, race, diet, family history, and
even lifestyle may contribute to the risk of PCa [3]. Cur-
rent therapies (radical prostatectomy, chemotherapy, local
radiotherapy, or hormonotherapy) are successful in treat-
ing localized diseases (androgen-dependent PCa) [4]. How-
ever, for nonorgan-confned diseases, especially metastatic
PCa (androgen-independent PCa), no signifcantly efective
therapies exist [5–8], and androgen ablation therapy has
been the major therapeutic modality for advanced PCa [9].
Consequently, novel anticancer drugs are needed to stop the
progression of PCa at later stages.
and pharmaceutical agents because of their potent activities
and unique structures. Other studies have also shown that
various bromophenols isolated from marine organisms, as
well as synthesized derivatives from natural bromophenol,
demonstrate an excellent anticancer activity against a panel
of cancer cell lines [40–46].
Based on previous results, our current hypothesis is that
the introduction of the bromophenol moiety into the pip-
erazine skeleton might favor the pharmacological activity
of piperazine derivatives. Herein, we designed and syn-
thesized a series of novel naftopidil-based arylpiperazine
derivatives containing the bromophenol moiety (Scheme 1).
The design strategy of naftopidil-based arylpiperazine
derivatives containing the bromophenol moiety is shown in
Fig. 2. All of the derivatives were bioassayed against the
PCa cell lines PC-3 and LNCaP and the normal prostate
epithelial cell line WPMY-1. The antagonistic activities of
the representative compounds with potent anticancer activi-
ties toward a1-adrenergic receptors (a1-ARs) were further
evaluated through dual-luciferase reporter assays. A simple
structure–activity relationship (SAR) study was also con-
ducted to facilitate the further development of arylpiperazine
derivatives. As expected, some arylpiperazine derivatives
exhibited signifcant cytotoxic activities against the PC-3
and LNCaP cells. Some of these derivatives also showed a
better a1-AR subtype selectivity than others did.
Piperazines and substituted piperazines are key pharma-
cophores that are crucial in many marketed drugs, such as
the Merck HIV protease inhibitor Crixivan and other drugs
under development [10]. Piperazine derivatives also exhibit
receptor-blocking properties [11–15] and antiproliferative
properties [16–23]. Naftopidil (Fig. 1), an arylpiperazine
derivative, is an α1-adrenoceptor blocker used for treat-
ing lower urinary tract symptoms associated with benign
prostatic hyperplasia (BPH) [24], which is a widely used
α1-adrenergic receptor antagonist for the treatment of BPH
in Japan. Other studies have demonstrated that naftopidil
inhibits cell proliferation and causes cell cycle arrest in PC-3
and LNCaP cells [25]. It also induces apoptosis in malignant
mesothelioma cell lines independent of α1-adrenoceptor
blocking [26]. These fndings indicate that naftopidil may
be useful as an anticancer drug. Recently, we investigated
compounds with a piperazine moiety as anticancer drugs for
the site-directed chemotherapy of PCa. These new hybrids
show a moderate to strong cytotoxic activity in PCa cell
lines [27–31]. In vitro and in vivo evidence has shown that
piperazine derivatives may be promising anticancer com-
pounds. Hence, we further designed and synthesized a new
naftopidil-based class of piperazine derivatives as potential
anticancer agents.
Materials and methods
Apparatus and analysis
All chemicals and reagents used in the current study were
of analytical grade. Melting points (uncorrected) were
measured on a SGW X-4 micro melting point apparatus.
NMR spectra were obtained on a Bruker AVANCE-400
spectrometer in CDCl3, with TMS as an internal standard,
and chemical shift values were reported in δ (ppm) and cou-
pling constants in Hertz. HRMS spectra were recorded on
the AB Sciex X500R QTOF mass spectrometer (Foster, CA,
USA). The completion of all reactions was monitored by
TLC on precoated silica-gel 60 F254 TLC plates (VWR).
The chromatograms were viewed under UV light at 254 and/
or 365 nm.
Bromophenols, which are natural marine products iso-
lated from marine organisms, such as algae, ascidian,
sponges, jellyfsh, and mollusks, possess various potent
activities, including antioxidation; α-glucosidase inhibi-
tion; protein tyrosine kinase inhibition; protein tyrosine
phosphatase 1B inhibition; antithrombosis; antimicrobial,
anti-infammatory, antibacterial, antifungal, and antiviral
properties; free radical scavenging; aldose reductase inhibi-
tion; and anticancer activities [32–39]. Such products have
been widely investigated in the felds of functional foods
Synthesisof2‑bromo‑1‑(3‑bromo‑4‑methoxyphenyl)
ethan‑1‑one (1) [47]
OH
O
To a stirred solution of acetophenones 3-bromo-4-methoxy-
acetophenone (5 g, 21.9 mmol) in THF (80 mL) was added
trimethylphenylammonium tribromide (9.07 g, 24.1 mmol)
at 0 °C. The reaction mixture was stirred at room tempera-
ture for 12 h. The solid was fltered, and to the fltrate was
N
N
O
Fig. 1 Structures of naftopidil
1 3

Synthesis and pharmacological evaluation of naftopidil-based arylpiperazine derivatives…
O
O
O
(i)
(ii)
O
O
O
Br
N
N Ar
1
2
Br
Ar
Br
Br
OH
O
(iii)
N
N
Br
3
N
3d
, Ar
=
3b
3g
3e
, Ar
, Ar
=
=
3c
3a
, Ar
, Ar
, Ar
=
=
=
F
F
Cl
O
O
3i
, Ar
=
3j
, Ar
, Ar
3f
, Ar
=
=
=
3h
F
O
F
3n
3s
3x
, Ar
F
3k
3p
=
=
=
, Ar
3o
3t
F
3l
, Ar =
O
, Ar
=
=
=
3m
, Ar
=
F
Cl
Cl
Cl
Cl
3q
, Ar
, Ar
, Ar
=
, Ar
, Ar
Cl
CN
=
=
3r
, Ar
=
F
3C
3u
, Ar
3v
CF3
, Ar
=
Br 3w
, Ar
=
Cl
Scheme 1 Reagents and conditions are as follows: (i) 1.1 times of PhN+Me3·Br3−, THF, rt. (ii) 1.2 times of arylpiperazines, 4.0 times of K2CO3,
CH3CN, refux; (iii) 2.0 times of NaBH4, EtOH, rt
acetate=10:1, v/v) to obtain the intermediates 1. White solid
OH
O
OH
(ethanol); Yield: 82%; Mp 112–113 °C; 1H NMR (400 MHz,
CDCl3) δ 8.19 (d, J=2.2 Hz, 1H), 7.94 (dd, J=8.6, 2.2 Hz,
1H), 6.95 (d, J = 8.6 Hz, 1H), 4.37 (s, 2H), 3.98 (s, 3H).
HRMS (ESI) m/z [M+1]+: Calcd for C9H9Br2O2, 306.8964,
found, 306.8962.
R1
R
N
N
N
N
O
Naftopidil
R2
HO
Br
Br
OH
General synthetic procedure of intermediates (2)
O
N
N
Ar
Arylpiperazines (1.2 equiv) and potassium carbonate (4.0
equiv) were added to a solution of 1 (1 equiv) in acetonitrile
(CH3CN, 20 mL). The reaction mixture was heated to 85 °C
and stirred for 12 h. Afterward the mixture was cooled to
room temperature. The reaction mixture was fltered, and the
fltrate was concentrated in vacuo. The residue was extracted
with ethyl acetate (60 mL) and water (20 mL). After drying
the organic layer with anhydrous Na2SO4 and evaporating
the solvent under reduced pressure, a solid was appeared.
The solid was recyrstallized from ethanol to obtain inter-
mediates 2.
3a-3x
Fig. 2 Design strategy for naftopidil-based arylpiperazine derivatives
containing the bromophenol moiety 3a–3x
added EtOAc (150 mL). The organic layer was washed
successively with H2O (50 mL) and brine (50 mL). The
organic layer was then dried over Na2SO4, fltered and con-
centrated in vacuo. The crude residue was then purifed by
chromatography on silica-gel column (petroleum ether: ethyl
1 3

H. Chen et al.
1H), 3.87 (s, 3H), 2.75 (br s, 2H), 2.57–2.32 (m, 8H); 13
C
General synthetic procedure of target compounds
NMR (101 MHz, CDCl3) δ 163.07, 160.63, 155.25, 138.14,
135.75, 130.91, 129.27, 129.20, 126.06, 115.57, 115.36,
111.75, 111.65, 74.48, 67.75, 65.96, 56.31, 51.82, 29.72;
HRMS (ESI) m/z [M + 1]+: Calcd for C26H28BrF2N2O2,
517.1297, found, 517.1295.
3a–3x
NaBH4 (2 equiv) was added to a stirred solution of the
intermediates 2 (1 equiv) in ethanol (20 mL). The reaction
mixture was left overnight at room temperature. The sol-
vent was removed in vacuo. Then the residue was purifed
by chromatography on silica-gel column (petroleum ether:
ethyl acetate=1:1, v/v) to obtain the corresponding products
3a–3x, and the solid was further recyrstallized from ethanol.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(pyridin‑2‑yl)pipera-
zin‑1‑yl)ethan‑1‑ol (3e) White solid (ethanol); Yield: 68%;
Mp 101–102 °C; 1H NMR (400 MHz, CDCl3) δ 8.20 (dd,
J = 4.8, 1.2 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.49 (ddd,
J = 8.8, 7.2, 2.0 Hz, 1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H),
6.88 (d, J = 8.4 Hz, 1H), 6.76–6.54 (m, 2H), 4.73 (dd,
J = 9.9, 3.6 Hz, 1H), 3.89 (s, 3H), 3.68–3.51 (m, 4H),
2.97–2.78 (m, 2H), 2.62–2.54 (m, 2H), 2.55–2.49 (m, 2H);
13C NMR (101 MHz, CDCl3) δ 159.22, 155.11, 147.80,
137.36, 135.37, 130.74, 125.88, 113.38, 111.58, 111.48,
106.96, 67.65, 65.99, 56.12, 52.66, 45.14; HRMS (ESI)
m/z [M + 1]+: Calcd for C18H23BrN3O2, 392.0968, found,
392.0970.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑phenylpiperazin‑1‑yl)
ethan‑1‑ol (3a) White solid (ethanol); Yield: 85%; Mp 147–
148 °C; 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J=2.0 Hz,
1H), 7.36–7.27 (m, 3H), 6.95 (d, J=7.9 Hz, 2H), 6.88 (dd,
J = 7.9, 5.0 Hz, 2H), 4.71 (dd, J = 10.0, 3.9 Hz, 1H), 3.90
(s, 3H), 3.35–3.11 (m, 4H), 3.06–2.83 (m, 2H), 2.70–2.59
(m, 2H), 2.57–2.45 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 155.12, 150.97, 135.41, 130.75, 128.99, 125.88, 119.80,
116.01, 111.59, 111.50, 67.64, 65.91, 56.14, 52.85, 49.16;
HRMS (ESI) m/z [M + 1]+: Calcd for C19H24BrN2O2,
391.1016, found, 391.1020.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(o‑tolyl)piperazin‑1‑yl)
ethan‑1‑ol (3f) White solid (ethanol); Yield: 80%; Mp 116–
117 °C; 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J=2.0 Hz,
1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H), 7.18 (t, J = 6.8 Hz,
2H), 7.07–6.96 (m, 2H), 6.89 (d, J=8.4 Hz, 1H), 4.71 (dd,
J=10.4, 3.5 Hz, 1H), 3.89 (s, 3H), 3.13–2.79 (m, 6H), 2.67–
2.48 (m, 4H), 2.31 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ 155.28, 151.29, 135.76, 132.61, 131.12, 130.95, 126.62,
126.08, 123.31, 119.01, 111.79, 111.68, 67.79, 66.17, 56.33,
51.84, 29.72, 17.89; HRMS (ESI) m/z [M+1]+: Calcd for
C20H26BrN2O2, 405.1172, found, 405.1178.
2‑(4‑Benzylpiperazin‑1‑yl)‑1‑(3‑Bromo‑4‑methoxyphenyl)
ethan‑1‑ol (3b) White solid (ethanol); Yield: 82%; Mp 107–
108 °C; 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J=2.0 Hz,
1H), 7.32 (d, J=4.4 Hz, 4H), 7.29–7.25 (m, 2H), 6.86 (d,
J = 8.5 Hz, 1H), 4.64 (dd, J = 10.4, 3.7 Hz, 1H), 3.88 (s,
3H), 3.53 (d, J = 2.5 Hz, 2H), 2.77 (br s, 2H), 2.59–2.40
(m, 8H); 13C NMR (101 MHz, CDCl3) δ 155.23, 138.00,
135.79, 130.92, 129.21, 128.27, 127.13, 126.06, 111.75,
111.64, 67.73, 66.02, 63.01, 56.31, 53.13, 29.72; HRMS
(ESI) m/z [M + 1]+: Calcd for C20H26BrN2O2, 405.1172,
found, 405.1147.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(m‑tolyl)piperazin‑1‑yl)
ethan‑1‑ol (3g) Colorless oil liquid; Yield: 72%; 1H NMR
(400 MHz, CDCl3) δ 7.59 (d, J = 2.0 Hz, 1H), 7.29 (dd,
J = 8.4, 2.0 Hz, 1H), 7.17 (t, J = 7.7 Hz, 1H), 6.88 (d,
J=8.5 Hz, 1H), 6.80–6.66 (m, 3H), 4.74 (dd, J=9.4, 3.8 Hz,
1H), 3.89 (s, 3H), 3.36–3.13 (m, 4H), 3.03–2.81 (m, 2H),
2.74–2.60 (m, 2H), 2.61–2.48 (m, 2H), 2.33 (s, 3H); 13C
NMR (101 MHz, CDCl3) δ 155.33, 151.14, 138.90, 130.93,
129.03, 126.07, 120.99, 117.12, 113.39, 111.79, 67.81,
66.04, 56.33, 53.09, 49.33, 29.72, 21.79; HRMS (ESI)
m/z [M + 1]+: Calcd for C20H26BrN2O2, 405.1172, found,
405.1176.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑((4‑chlorophenyl)(phe-
nyl)methyl)piperazin‑1‑yl)ethan‑1‑ol (3c) Colorless oil liq-
1
uid; Yield: 76%; H NMR (400 MHz, CDCl3) δ 7.55 (s,
1H), 7.39–7.33 (m, 4H), 7.32–7.26 (m, 3H), 7.25–7.17 (m,
3H), 6.86 (d, J = 8.5 Hz, 1H), 4.62 (dt, J = 10.0, 3.0 Hz,
1H), 4.22 (s, 1H), 3.88 (s, 3H), 2.75 (br s, 2H), 2.59–2.33
(m, 8H); 13C NMR (101 MHz, CDCl3) δ 155.24, 142.05,
141.28, 135.76, 132.64, 130.92, 129.18, 128.73, 128.66,
127.82, 127.25, 126.06, 111.75, 111.64, 75.44, 67.73, 65.97,
56.31, 51.89, 29.72; HRMS (ESI) m/z [M+1]+: Calcd for
C26H29BrClN2O2, 515.1095, found, 515.1097.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(p‑tolyl)piperazin‑1‑yl)
ethan‑1‑ol (3h) White solid (ethanol); Yield: 82%; Mp
2 ‑ ( 4 ‑ ( B i s ( 4 ‑ f l u o r o p h e n y l ) m e t h y l ) p i p e r -
azin‑1‑yl)‑1‑(3‑bromo‑4‑methoxyphenyl)ethan‑1‑ol
(3d) Colorless oil liquid; Yield: 78%; 1H NMR (400 MHz,
CDCl3) δ 7.55 (d, J = 2.0 Hz, 1H), 7.37–7.33 (m, 4H),
7.25 (dd, J=7.9, 2.0 Hz, 1H), 7.00–6.95 (m, 4H), 6.86 (d,
J = 8.5 Hz, 1H), 4.62 (dd, J = 10.1, 3.7 Hz, 1H), 4.23 (s,
1
146–147 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
J = 2.0 Hz, 1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H), 7.09 (d,
J=8.4 Hz, 2H), 6.87 (t, J=8.6 Hz, 3H), 4.71 (dd, J=10.1,
3.8 Hz, 1H), 3.89 (s, 3H), 3.31–3.09 (m, 4H), 3.02–2.82
(m, 2H), 2.78–2.58 (m, 2H), 2.58–2.44 (m, 2H), 2.28 (s,
1 3

Synthesis and pharmacological evaluation of naftopidil-based arylpiperazine derivatives…
3H); 13C NMR (101 MHz, CDCl3) δ 155.30, 149.07, 135.65,
130.94, 129.70, 129.53, 126.07, 116.54, 111.78, 111.68,
67.83, 66.10, 56.33, 53.07, 49.91, 20.46; HRMS (ESI)
m/z [M + 1]+: Calcd for C20H26BrN2O2, 405.1172, found,
405.1175.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2‑fuorophenyl)pipera-
zin‑1‑yl)ethan‑1‑ol (3m) White solid (ethanol); Yield: 78%;
1
Mp 105–106 °C; H NMR (400 MHz, CDCl3) δ 7.60 (d,
J = 2.0 Hz, 1H), 7.29 (dd, J = 8.5, 2.0 Hz, 1H), 7.13–6.92
(m, 4H), 6.88 (d, J=8.5 Hz, 1H), 4.71 (dd, J=10.4, 3.5 Hz,
1H), 3.89 (s, 3H), 3.23–3.09 (m, 4H), 3.03–2.88 (m, 2H),
2.70–2.61 (m, 2H), 2.61–2.47 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 156.97, 155.31, 139.98, 139.89, 135.62, 130.94,
126.08, 124.53, 124.49, 122.70, 122.62, 118.99, 118.96,
116.28, 116.07, 111.78, 111.70, 67.80, 66.10, 56.33,
53.10, 50.67, 50.64; HRMS (ESI) m/z [M+1]+: Calcd for
C19H23BrFN2O2, 409.0921, found, 409.0925.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2‑methoxyphenyl)piper-
azin‑1‑yl)ethan‑1‑ol (3i) Colorless oil liquid; Yield: 74%; 1H
NMR (400 MHz, CDCl3) δ 7.60 (d, J = 1.9 Hz, 1H), 7.29
(dd, J=8.4, 1.9 Hz, 1H), 7.06–6.99 (m, 1H), 6.99–6.92 (m,
2H), 6.93–6.85 (m, 2H), 4.71 (dd, J=10.2, 3.5 Hz, 1H), 3.89
(s, 3H), 3.87 (s, 3H), 3.14 (br s, 4H), 3.02–2.89 (m, 2H),
2.71–2.63 (m, 2H), 2.62–2.44 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 155.40, 152.39, 141.21, 135.87, 131.07, 126.22,
123.27, 121.16, 118.38, 111.91, 111.80, 111.34, 67.89,
66.27, 56.45, 55.52, 50.88; HRMS (ESI) m/z [M + 1]+:
Calcd for C20H26BrN2O3, 421.1121, found, 421.1118
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(4‑fuorophenyl)pipera-
zin‑1‑yl)ethan‑1‑ol (3n) White solid (ethanol); Yield: 86%;
1
Mp 126–127 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
J=2.0 Hz, 1H), 7.29 (dd, J=8.4, 2.0 Hz, 1H), 7.04–6.93 (m,
2H), 6.91–6.85 (m, 3H), 4.71 (dd, J=9.9, 3.6 Hz, 1H), 3.89
(s, 3H), 3.25–3.03 (m, 4H), 3.02–2.85 (m, 2H), 2.74–2.59
(m, 2H), 2.59–2.44 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 156.56, 155.76, 148.23, 136.00, 131.36, 126.50, 118.45,
118.38, 116.14, 115.92, 112.23, 112.13, 68.28, 66.47,
56.76, 53.47, 50.77; HRMS (ESI) m/z [M+1]+: Calcd for
C19H23BrFN2O2, 409.0921, found, 409.0917.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(3‑methoxyphenyl)
piperazin‑1‑yl)ethan‑1‑ol (3j) White solid (ethanol); Yield:
82%; Mp 133–134 °C; 1H NMR (400 MHz, CDCl3) δ 7.59
(d, J=2.0 Hz, 1H), 7.29 (dd, J =8.5, 2.0 Hz, 1H), 7.19 (t,
J=8.2 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 6.55 (dd, J=8.2,
2.0 Hz, 1H), 6.48 (t, J = 2.2 Hz, 1H), 6.44 (dd, J = 8.1,
2.1 Hz, 1H), 4.71 (dd, J=9.7, 3.6 Hz, 1H), 3.89 (s, 3H), 3.80
(s, 3H), 3.39–3.10 (m, 4H), 3.03–2.85 (m, 2H), 2.66–2.58
(m, 2H), 2.58–2.45 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 160.62, 155.31, 152.53, 135.59, 130.93, 129.86, 126.07,
111.79, 111.69, 108.97, 104.67, 102.67, 67.84, 66.08, 56.33,
55.22, 52.99, 49.22; HRMS (ESI) m/z [M+1]+: Calcd for
C20H26BrN2O3, 421.1121, found, 421.1120
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2,4‑difuorophenyl)piper-
azin‑1‑yl)ethan‑1‑ol (3o) White solid (ethanol); Yield: 75%;
1
Mp 103–104 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
J=2.1 Hz, 1H), 7.28 (dd, J=8.5, 2.0 Hz, 1H), 7.02–6.86 (m,
2H), 6.87–6.76 (m, 2H), 4.70 (dd, J=10.4, 3.6 Hz, 1H), 3.89
(s, 3H), 3.34–3.00 (m, 4H), 3.06–2.84 (m, 2H), 2.70–2.60
(m, 2H), 2.59–2.45 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 155.32, 135.57, 130.93, 126.07, 119.44, 111.78, 111.70,
110.87, 110.62, 105.02, 104.76, 104.52, 67.81, 66.06, 56.33,
53.09, 51.02, 50.99; HRMS(ESI)m/z[M + 1]+: Calcd for
C19H22BrF2N2O2, 427.0827, found, 427.0825.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(4‑methoxyphenyl)piper-
azin‑1‑yl)ethan‑1‑ol (3k) White solid (ethanol); Yield: 76%;
1
Mp 148–149 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
J=2.0 Hz, 1H), 7.29 (dd, J=8.5, 2.0 Hz, 1H), 6.96–6.85 (m,
5H), 4.70 (dd, J=10.0, 3.6 Hz, 2H), 3.90 (s, 3H), 3.78 (s,
3H), 3.17–3.12 (m, 4H), 2.95–2.85 (m, 2H), 2.69–2.59 (m,
2H), 2.59–2.49 (m, 2H); HRMS (ESI) m/z [M+1]+: Calcd
for C20H26BrN2O3, 421.1121, found, 421.1116.
4‑(4‑(2‑(3‑Bromo‑4‑methoxyphenyl)‑2‑hydroxyethyl)piper-
azin‑1‑yl)‑3‑fuorobenzonitrile (3p) White solid (ethanol);
Yield: 67%; Mp 132–133 °C; 1H NMR (400 MHz, CDCl3)
δ 7.59 (d, J = 1.9 Hz, 1H), 7.38 (dd, J= 8.4, 1.2 Hz, 1H),
7.30 (dd, J = 3.9, 1.9 Hz, 1H), 7.27 (d, J = 2.0 Hz, 1H),
6.98–6.84 (m, 2H), 4.71 (dd, J = 10.1, 3.7 Hz, 1H), 3.90
(s, 3H), 3.38–3.18 (m, 4H), 3.06–2.87 (m, 2H), 2.77–2.61
(m, 2H), 2.61–2.43 (m, 2H); 13C NMR (101 MHz, CDCl3)
δ 155.38, 155.26, 152.79, 143.90, 135.37, 130.92, 129.47,
126.06, 119.88, 119.64, 118.78, 118.74, 118.37, 118.35,
111.80, 111.73, 103.97, 67.87, 66.02, 56.33, 52.77,
49.75, 49.70, 29.71; HRMS (ESI) m/z [M+1]+: Calcd for
C20H22BrFN3O2, 434.0874, found, 434.0879.
4‑(4‑(2,4‑Difluorophenyl)piperazin‑1‑yl)‑2H‑benzo[h]
1
chromen‑2‑one (3l) Colorless oil liquid; Yield: 75%; H
NMR (400 MHz, CDCl3) δ 7.60 (d, J = 2.0 Hz, 1H), 7.29
(dd, J=8.4, 2.0 Hz, 1H), 7.02–6.96 (m, 1H), 6.95–6.84 (m,
4H), 4.72 (dd, J=10.4, 3.5 Hz, 1H), 4.08 (q, J=7.0 Hz, 2H),
3.89 (s, 3H), 3.16 (br s, 4H), 3.07–2.89 (m, 2H), 2.82–2.63
(m, 2H), 2.60–2.42 (m, 2H), 1.46 (t, J = 7.0 Hz, 3H); 13C
NMR (101 MHz, CDCl3) δ 155.27, 151.57, 141.11, 135.77,
130.94, 126.07, 122.95, 121.02, 118.21, 112.47, 111.78,
111.67, 67.77, 66.18, 63.58, 56.32, 50.64, 29.72, 14.96;
HRMS (ESI) m/z [M + 1] + : Calcd for C21H28BrN2O3,
435.1278, found, 435.1276.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2‑chlorophenyl)pip-
erazin‑1‑yl)ethan‑1‑ol (3q) White solid (ethanol); Yield:
1 3

H. Chen et al.
80%; Mp 105–106 °C; 1H NMR (400 MHz, CDCl3) δ 7.60
(d, J = 2.0 Hz, 1H), 7.37 (dd, J = 7.9, 1.5 Hz, 1H), 7.29
(dd, J = 8.5, 2.0 Hz, 1H), 7.26–7.19 (m, 1H), 7.06 (dd,
J=8.0, 1.4 Hz, 1H), 6.99 (td, J=7.8, 1.5 Hz, 1H), 6.88 (d,
J = 8.5 Hz, 1H), 4.71 (dd, J = 10.5, 3.5 Hz, 1H), 3.89 (s,
3H), 3.12 (br s, 4H), 3.01–2.87 (m, 2H), 2.75–2.62 (m, 2H),
2.60–2.45 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 155.41,
149.18, 135.79, 131.05, 130.81, 128.91, 127.76, 126.20,
123.98, 120.52, 111.90, 111.81, 67.90, 66.19, 56.44, 51.42;
HRMS (ESI) m/z [M + 1]+: Calcd for C19H23BrClN2O2,
425.0626, found, 425.0628.
1H), 6.95–6.85 (m, 2H), 6.81 (d, J=8.5 Hz, 1H), 4.63 (dd,
J=10.4, 3.6 Hz, 1H), 3.82 (s, 3H), 3.03–2.70 (m, 6H), 2.65–
2.33 (m, 4H), 2.18 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ 155.30, 152.35, 135.66, 132.00, 131.78, 130.94, 130.82,
126.08, 123.14, 119.54, 111.79, 111.69, 67.80, 66.11, 56.33,
51.66, 29.72, 17.50. HRMS (ESI) m/z [M +1]+: Calcd for
C20H25BrClN2O2, 439.0782, found, 439.0776.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(4‑bromophenyl)pipera-
zin‑1‑yl)ethan‑1‑ol (3v) White solid (ethanol); Yield: 73%;
1
Mp 131–132 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
J=2.0 Hz, 1H), 7.44–7.32 (m, 2H), 7.28 (dd, J=8.5, 2.0 Hz,
1H), 6.88 (d, J=8.5 Hz, 1H), 6.85–6.76 (m, 2H), 4.71 (dd,
J=9.5, 3.8 Hz, 1H), 3.89 (s, 3H), 3.33–3.11 (m, 4H), 3.01–
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(3‑chlorophenyl)pipera-
zin‑1‑yl)ethan‑1‑ol (3r) White solid (ethanol); Yield: 73%;
1
Mp 107–108 °C; H NMR (400 MHz, CDCl3) δ 7.59 (d,
2.82 (m, 2H), 2.72–2.59 (m, 2H), 2.57–2.45 (m, 2H); 13
C
J = 2.0 Hz, 1H), 7.28 (dd, J = 8.4, 2.0 Hz, 1H), 7.17 (t,
J=8.1 Hz, 1H), 6.88 (dd, J=5.4, 3.0 Hz, 2H), 6.84–6.78 (m,
2H), 4.71 (dd, J=9.6, 3.7 Hz, 1H), 3.89 (s, 3H), 3.33–3.12
(m, 4H), 2.99–2.80 (m, 2H), 2.70–2.57 (m, 2H), 2.57–2.46
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 155.34, 152.17,
135.49, 135.01, 130.93, 130.09, 126.07, 119.57, 115.93,
114.01, 111.79, 111.71, 67.87, 66.04, 56.33, 52.85, 48.86;
HRMS (ESI) m/z [M + 1]+: Calcd for C19H23BrClN2O2,
425.0626, found, 425.0616.
NMR (101 MHz, CDCl3) δ 155.33, 150.15, 135.50, 131.93,
130.92, 126.06, 117.76, 112.12, 111.79, 111.70, 67.87,
66.03, 56.33, 52.86, 49.14; HRMS (ESI) m/z [M + 1]+:
Calcd for C19H23Br2N2O2, 469.0121, found, 469.0115.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2‑(trifluoromethyl)
phenyl)piperazin‑1‑yl)ethan‑1‑ol (3w) Colorless oil liq-
1
uid; Yield: 75%; H NMR (400 MHz, CDCl3) δ 7.63 (d,
J=7.9 Hz, 1H), 7.60 (d, J=2.0 Hz, 1H), 7.53 (t, J=7.4 Hz,
1H), 7.39 (d, J=8.0 Hz, 1H), 7.28 (dd, J=8.4, 2.0 Hz, 1H),
7.23 (t, J=7.7 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 4.70 (dd,
J=10.5, 3.6 Hz, 1H), 3.89 (s, 3H), 3.07–2.93 (m, 4H), 2.90
(d, J=5.1 Hz, 2H), 2.60 (d, J=3.5 Hz, 2H), 2.59–2.41 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 155.28, 152.33, 135.74,
132.79, 130.93, 127.28, 127.23, 126.08, 124.94, 123.99,
111.78, 111.70, 67.78, 66.08, 56.32, 53.52; HRMS (ESI)
m/z [M+1]+: Calcd for C20H23BrF3N2O2, 459.0890, found,
459.0888.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(4‑chlorophenyl)pipera-
zin‑1‑yl)ethan‑1‑ol (3s) White solid (ethanol); Yield: 78%; Mp
126–127 °C; 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J=2.0 Hz,
1H), 7.29 (dd, J=8.5, 2.0 Hz, 1H), 7.24–7.18 (m, 2H), 6.95–6.80
(m, 3H), 4.71 (dd, J=9.8, 3.8 Hz, 1H), 3.89 (s, 3H), 3.30–3.10
(m, 4H), 2.96–2.84 (m, 2H), 2.68–2.58 (m, 2H), 2.59–2.46 (m,
2H); 13C NMR (101 MHz, CDCl3) δ 155.34, 149.75, 135.49,
130.92, 129.02, 126.06, 124.84, 117.38, 111.79, 111.71, 67.85,
66.03, 56.33, 52.90, 49.33; HRMS (ESI) m/z [M+1]+: Calcd
for C19H23BrClN2O2, 425.0626, found, 425.0608.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(4‑(trifuoromethyl)phe-
nyl)piperazin‑1‑yl)ethan‑1‑ol (3x) White solid (ethanol);
Yield: 83%; Mp 125–126 °C; 1H NMR (400 MHz, CDCl3)
δ 7.59 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.29
(dd, J=8.5, 2.0 Hz, 1H), 6.94 (d, J=8.5 Hz, 2H), 6.88 (d,
J=8.5 Hz, 1H), 4.72 (dd, J=9.3, 3.9 Hz, 1H), 3.89 (s, 3H),
3.41–3.24 (m, 4H), 2.95–2.84 (m, 2H), 2.67–2.59 (m, 2H),
2.58–2.47 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 155.36,
153.14, 135.44, 130.93, 126.47, 126.43, 126.07, 114.69,
111.80, 111.72, 67.90, 66.05, 56.33, 52.76, 48.16; HRMS
(ESI) m/z [M+1]+: Calcd for C20H23BrF3N2O2, 459.0890,
found, 459.0892.
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(2,3‑dichlorophenyl)piper-
azin‑1‑yl)ethan‑1‑ol (3t) White solid (ethanol); Yield: 68%;
1
Mp 128–129 °C; H NMR (400 MHz, CDCl3) δ 7.60 (d,
J=2.0 Hz, 1H), 7.28 (dd, J=8.5, 2.0 Hz, 1H), 7.22–7.13 (m,
2H), 6.97 (dd, J=6.6, 3.0 Hz, 1H), 6.89 (d, J=8.5 Hz, 1H),
4.71 (dd, J=10.5, 3.5 Hz, 1H), 3.89 (s, 3H), 3.11 (br s, 4H),
2.93 (d, J=4.3 Hz, 2H), 2.66 (d, J=5.9 Hz, 1H), 2.61–2.46
(m, 2H); 13C NMR (101 MHz, CDCl3) δ 155.32, 151.05,
135.61, 134.12, 130.94, 127.57, 127.51, 126.08, 124.79,
118.64, 111.79, 111.71, 67.81, 66.05, 56.33, 51.43; HRMS
(ESI) m/z [M+1]+: Calcd for C19H22BrCl2N2O2, 459.0236,
found, 459.0232.
Biological assays
1‑(3‑Bromo‑4‑methoxyphenyl)‑2‑(4‑(5‑chloro‑2‑methylphenyl)
piperazin‑1‑yl)ethan‑1‑ol (3u) Colorless oil liquid; Yield:
In vitro cytotoxic assay
1
72%; H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 2.0 Hz,
Cell culture PC-3 and WPMY-1 cells were cultured in Dul-
becco’s modifcation Eagle’s medium (DMEM, Invitrogen,
1H), 7.22 (dd, J = 8.4, 2.0 Hz, 1H), 7.02 (d, J = 8.0 Hz,
1 3

Synthesis and pharmacological evaluation of naftopidil-based arylpiperazine derivatives…
Carlsbad, CA, USA) supplemented with 10% fetal bovine
serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL peni-
cillin and 0.1 mg/mL streptomycin (Invitrogen). LNCaP
cells were cultured in F12 media supplemented with 10%
fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin
and 0.1 mg/mL streptomycin (Invitrogen). The cells were
incubated at 37 °C in a humidifed atmosphere with 5% CO2.
between 3-bromo-4-methoxyacetophenone and trimethyl-
phenylammonium tribromide was carried out by using THF
as the solvent to obtain intermediate 1. Second, intermedi-
ate 2 was obtained by applying the nucleophilic substitution
reaction between intermediate 1 and arylpiperazines, adding
4 times of K2CO3 in acetonitrile. Lastly, the reduction of
intermediate 2 with NaBH4 in ethanol led to the formation
of the fnal arylpiperazine derivatives containing the bromo-
phenol moiety. All of the synthesized arylpiperazine deriva-
tives were confrmed via 1H-NMR, 13C-NMR, and HRMS.
Assessment of antitumor activity by CCK‑8 assay Cell pro-
liferation was measured with the Cell Counting Kit-8 (CCK-
8) assay kit (Dojindo Corp., Kumamoto, Japan). Cells were
harvested during logarithmic growth phase and seeded in
96-well plates at a density of 1×105 cells/mL, and cultured
at 37 °C in a humidifed incubator (5% CO2) for 24 h, fol-
lowed by exposure to various concentrations of compounds
tested for 24 h. Subsequently 10 μL of CCK-8 (Dojindo)
was added to each well, the cells were then incubated for an
additional 1 h at 37 °C to convert WST-8 into formazan. Cell
growth inhibition was determined by measuring the absorb-
ance (Abs) at λ=450 nm using amicroplate reader. Three
independent experiments were performed. Cell growth inhi-
bition was calculated according to the following equation:
Biological evaluation
Antitumor activity
All of the target compounds were screened in terms of their
in vitro cytotoxicity against PC-3 and LNCaP, and compared
with their efects on WPMY-1 via the CCK-8 assay [28,
29]. Naftopidil and fnasteride [49] were used as reference
compounds, and the results were reported in terms of IC50.
The results are summarized in Table 1.
As shown in Table 1, the cytotoxic activities of all the
derivatives (IC50 < 10 μM) except 3p, 3t, and 3u against
PC-3 and LNCaP cells were strong, and their activities
were also higher than those of fnasteride. Among these
compounds, 3c, 3d, 3h, 3k, 3o and 3s exhibited the most
potent activity against PC-3 cells with IC50 of 0.67, 0.25,
0.16, 0.08, 0.79, and 0.55 μM, which were 26-, 71-, 111-,
222-, 22-, and 356-fold more active than fnasteride, respec-
tively. They also exhibited a weak cytotoxic efect on nor-
mal human prostate epithelial cell (WPMY-1) with IC50 of
>50 μM.
Growth inhibition = (1 − OD of treated cells/OD
of control cells) × 100%.
The half maximal inhibitory concentrations (IC50) were
obtained from liner regression analysis of the concentra-
tion–response curves plotted for each tested compound.
Antagonistic activity in a1‑ARs by dual‑luciferase reporter
gene assay [48] Firefy and Renilla luciferase activi-
ties, which are indicated as RLUs, were determined using
Dual-Glo luciferase assay kits (Promega) according to the
manufacturer’s instructions. RLUs were measured using a
luminometer (GloMaxTM 96-Microplate Luminometer, Pro-
mega) and are reported as the mean SEM of three individ-
Antagonistic activity in a1‑ARs (α1a, α1b, and α1d
)
ual experiments. For agonists, fold of induction=LUinduced
/
PCa and BPH are common diseases in elderly males, and
studies have shown that androgen receptor-mediated andro-
gen afects the incidence of BPH and PCa, and derivatives
with piperazine moiety may act as potential α1a-AR- and/
or α1a-AR + α1d-AR-selective ligands for the treatment of
BPH [48, 50–52]. Therefore, to evaluate antagonistic action
of arylpiperazine derivatives with bromophenol moiety on
a1-ARs (α1a, α1b, and α1d), the derivatives with potent anti-
cancer activities were selected for further investigating their
antagonistic activities using dual-luciferase reporter assays
[48] to identify a1-AR subselective antagonist candidates to
treat BPH from arylpiperazine derivatives. The results are
shown in Table 2.
RLUuninduced. For antagonists, % of control=100×RLU
(agonist+antagonist)/RLU (agonist alone). All RLUs were
normalized against frefy RLUs/Renilla RLUs. Data are
expressed as EC50/IC50 values in μM, and the IC50 of phe-
nylephrine (μM) was calculated by plotting the data using
nonlinear regression analysis in Graph-Pad Prism 5 software.
Results
Chemistry
The synthetic route of compounds 3a–3x followed the
general pathway outlined in Scheme 1. These compounds
were prepared in three steps. First, α-bromination reaction
1 3

H. Chen et al.
Table 1 In vitro cytotoxicity of arylpiperazine derivatives 3a–3x
Table 2 Antagonistic activities (IC50) on α1-ARs (α1a, α1b, and α1d) of
arylpiperazine derivatives
Compd.
IC50 (μM)a
PC-3b
Compd.
IC50 (nM)a
Selectivity ratio
LNCaPb
WPMY-1b
α1a
α1b
α1d
α1b/α1a
α1b/α1d
3a
3.05 0.03
1.23 0.16
0.67 0.12
0.25 1.27
4.71 0.16
1.07 0.32
0.89 0.21
0.16 0.02
2.73 0.14
1.45 0.19
0.08 0.02
7.14 0.13
6.38 1.09
2.13 0.15
0.79 0.09
11.03 0.78
4.03 0.17
1.32 0.42
0.05 0.04
15.31 0.67
10.37 0.54
4.42 0.15
7.38 1.06
2.25 0.15
42.10 0.79
17.83
2.05 0.12
1.09 0.15
0.56 0.27
0.17 0.54
3.62 1.04
5.73 0.17
3.46 0.26
1.04 0.14
4.08 1.10
2.02 0.53
0.72 0.22
5.47 0.78
5.42 0.26
3.42 0.18
1.27 0.34
9.27 0.84
5.93 0.32
1.85 0.18
0.18 0.12
8.29 1.04
12.57 1.24
5.77 0.14
8.78 0.65
3.25 0.62
22.36 0.61
14.53
49.2 1.12
>50
>50
>50
45.7 0.46
>50
3b
412.12
660.36
393.85
62.18
467.63
77.84
573.92
483.29
92.64
473.62
555
926.31
982.46
764.43
892.67
1132.35
972.05
1045.63
683.45
963.72
1053.35
634
765.26
826.57
472.98
347.12
92.54
873.27
87.36
393.25
673.56
87.47
2.2
1.5
1.9
14.3
2.4
12.5
1.8
1.4
10.4
2.2
1.2
1.1
1.6
2.6
12.2
1.1
11.9
1.7
1.4
12.0
11.48
3b
3c
3c
3d
3d
3g
3e
3h
3f
3j
3g
>50
>50
3k
3h
3o
3i
32.2 0.66
42.8 1.25
>50
38.4 1.07
47.4 0.65
>50
>50
>50
37.7 0.26
>50
>50
>50
47.9 1.05
>50
>50
>50
>50
–
3r
3j
3s
3k
Naftopidil
55.2
1.1
3l
3m
aIC50 values are taken as means from three experiments
3n
3o
to the antitumor activity. (2) After the phenyl group was
replaced with the pyridinyl group, the resultant compound
3e displayed a comparable activity with that of 3a against
the tested cancer cells, and these compounds also elicited a
weak cytotoxic efect on WPMY-1 with IC50 of > 50 μM.
(3) The position of the substituent on the phenyl group also
afected the cytotoxic activities. Among the compounds
containing a methyl substituent, the order of the cytotoxic
activities of compounds 3f (2-CH3), 3g (3-CH3), and 3h
(4-CH3) against PC-3 and LNCaP cells could be placed as
follows: 3h>3g>3f. A similar order of antitumor activity
was observed in 3i (2-OCH3), 3j (3-OCH3), 3k (4-OCH3),
3q (2-Cl), 3r (3-Cl), and 3s (4-Cl). Namely, the activity of
the p-substituted phenyl group derivatives against LNCaP
and PC-3 cells was better than that of the substituted groups
in other positions. In addition, the same order of antitumor
activity against PC-3 and LNCaP cells was observed in 3m
(2-F) versus 3n (4-F) and in 3w (2-CF3) versus 3x (4-CF3).
(4) The efectiveness of the compounds with difuorosub-
stituents on the phenyl group was higher than that of the
compounds with monofuorosubstituents. For example, the
cytotoxic activity of 3o (2,4-F2, IC50 =0.79 and 1.27 μM)
against PC-3 and LNCaP cells was more efective than those
of 3m and 3n. Moreover, the cytotoxic activity of 3o against
normal WPMY-1 was weak with IC50 of >50 μM. However,
the inhibitory activity of other disubstituted compounds 3p,
3t, and 3u against cancer cells was relatively weak. (5) The
cytotoxic activity of 3v (4-Br) against PC-3 and LNCaP
cells was relatively lower than those of 3n (4-F) and 3s
(4-Cl). The activity profles indicated that the introduction
of a bromo group at the p-position in the phenyl group was
inauspicious for anticancer activity. (6) The activity of 3k
(4-OCH3) with electron-donating groups against LNCaP
and PC-3 cells was relatively better than that of 3x (4-CF3)
3p
3q
3r
3s
3t
3u
3v
3w
3x
Naftopidil
Finasteride
aIC50 values are taken as mean standard deviation from three exper-
iments
bPC-3, androgen-insensitive human prostate cancer cell line; LNCaP,
androgen-sensitive human prostate cancer cell line; WPMY-1, normal
non-cancer human prostate epithelial cell line
Discussion
The SAR of these designed arylpiperazine derivatives
was thoroughly discussed. With 3a as the lead, the SAR
investigation mainly focused on the variation in the phenyl
group at the 4-position of the piperazine ring with other
aryl groups and the substitute’s type and position on the
phenyl group as a required group for antitumor activities.
(1) First, the resultant compound 3b displayed an improved
cytotoxic activity against PC-3 and LNCaP cells with IC50
values of 1.23 and 1.09 μM, respectively, after the phenyl
group at the 4-position of the piperazine ring was replaced
with the benzyl group. Especially, compounds 3c and 3d
demonstrated potent activities against PC-3 and LNCaP
cells. These results suggested that a larger group substi-
tuted at the 4-position of the piperazine ring was benefcial
1 3

Synthesis and pharmacological evaluation of naftopidil-based arylpiperazine derivatives…
3. Nelson WG, De Marzo AM, Isaacs WB. Mechanisms of disease.
with electron-withdrawing groups in the phenyl group. The
activity profles indicated that the introduction of electron-
donating groups at the p-position in the phenyl group con-
tributed to anticancer activities.
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SAR studies revealed that a larger group substituted at
the 4-position of the piperazine ring and the p-substituted
phenyl group in the arylpiperazine derivatives displayed a
relatively improved activity against the tested cancer cells.
As shown in Table 2, although arylpiperazine derivatives
(3b, 3c, and 3d) with a larger group substituted at the 4-posi-
tion of the piperazine ring exhibited strong cytotoxic activi-
ties against PC-3 and LNCaP cells, they demonstrated no
a1-ARs subtype selectivity. The o-substituted phenyl group
arylpiperazine derivatives (3g, 3j, and 3r) showed potent
cytotoxic activities against the tested cancer cells. Their a1a
subtype selectivity was better than a1b subtype selectivity
(a1b/a1a ratio>10). By contrast, the a1d subtype selectivity
of p-substituted phenyl group arylpiperazine derivatives (3h,
3k, and 3s) with potent anticancer activities was better that
the a1b subtype (a1b/a1d ratio=12.2, 11.9, and 12.0).
In summary, the majority of the derivatives exhib-
ited strong cytotoxic activities against PC-3 and LNCaP
cells, and possessed higher activities than fnasteride, and
derivatives with potent anticancer activities exhibited bet-
ter a1-ARs subtype selectivity (selectivity ratio> 10) than
others did. Overall, the results of this study suggested that
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Acknowledgements This work was financially supported by the
National Natural Science Foundation of China (No. 31800833),
Zhejiang Provincial Natural Science of Foundation of China (Nos.
LZ19H180001 and LQ19C100001), the Wenzhou Medical Uni-
versity and Wenzhou Institute of Biomaterials and Engineering
(WIBEZD2017001-03), the Science and Technology Planning Pro-
jects of Guangdong Province (No. 2016A020215175), the Natural Sci-
ence Foundation of Guangdong Province (No. 2016A030313583), the
Medical Scientifc Research Foundation of Guangdong Province (No.
A2016555), the Outstanding Youths Development Scheme of Nanfang
Hospital, Southern Medical University (No. 2015J005) and the Science
and Technology Planning Project of Guangzhou (No. 201704020070).
15. Ananthan S, Saini SK, Zhou G, Hobrath JV, Padmalayam I,
Zhai L, Bostwick JR, Antonio T, Reith ME, McDowell S,
Cho E, McAleer L, Taylor M, Luedtke RR. Design, synthe-
sis, and structure-activity relationship studies of a series of
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1-(2-pyridyl)piperazines as Delta(8)-Delta(7) sterol isomerase
(emopamil binding protein) selective ligands with antiprolifera-
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Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
17. Abate C, Niso M, Contino M, Colabufo NA, Ferorelli S, Per-
rone R, Berardi F. 1-Cyclohexyl-4-(4-arylcyclohexyl) pipera-
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with antiproliferative and P-glycoprotein inhibitory activity.
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Publisher’s Note Springer Nature remains neutral with regard to
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Afliations
Hong Chen1,2 · Yuna Qian3,4 · Huixia Jia2 · Yuzhong Yu1 · Haibo Zhang1 · Shanchao Zhao1 · Jianliang Shen3,4
1
Department of Urology, Nanfang Hospital, Southern
Medical University, Guangzhou 510515, China
and Optometry, School of Biomedical Engineering, Wenzhou
Medical University, Wenzhou 325035, China
2
3
4
Luoyang Key Laboratory of Organic Functional Molecules,
College of Food and Drug, Luoyang Normal University,
Luoyang 471934, China
Wenzhou Institute of Biomaterials and Engineering, Chinese
Academy of Science, Wenzhou 325001, China
State Key Laboratory of Ophthalmology, Optometry
and Vision Science, School of Ophthalmology
1 3