Synthesis and biological evaluation of arylpiperazine derivatives as potential anti-prostate cancer agents

Article abstract of DOI:10.1016/j.bmc.2018.11.029

A novel scaffold of arylpiperazine derivatives was discovered as potent androgen receptor (AR) antagonist through rational drug designation based on our pre-work, leading to the discovery of a series of new antiproliferative compounds. Compounds 10, 16, 2

Full text of DOI:10.1016/j.bmc.2018.11.029

Accepted Manuscript
Synthesis and Biological Evaluation of Arylpiperazine Derivatives as Potential
Anti-prostate Cancer Agents
Hong Chen, Yu-Zhong Yu, Xiu-Mei Tian, Cai-Lu Wang, Yu-Na Qian, Zai-An
Deng, Jing-Xiao Zhang, Dao-Jun Lv, Hai-Bo Zhang, Jian-Liang Shen, Mu
Yuan, Shan-Chao Zhao
PII:
S0968-0896(18)31614-6
https://doi.org/10.1016/j.bmc.2018.11.029
BMC 14633
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Revised Date:
Accepted Date:
14 September 2018
28 October 2018
20 November 2018
Please cite this article as: Chen, H., Yu, Y-Z., Tian, X-M., Wang, C-L., Qian, Y-N., Deng, Z-A., Zhang, J-X., Lv,
D-J., Zhang, H-B., Shen, J-L., Yuan, M., Zhao, S-C., Synthesis and Biological Evaluation of Arylpiperazine
Derivatives as Potential Anti-prostate Cancer Agents, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.11.029
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Synthesis and Biological Evaluation of Arylpiperazine
Derivatives as Potential Anti-prostate Cancer Agents
Hong Chena,b,†, Yu-Zhong Yua†, Xiu-Mei, Tiane, †, Cai-Lu Wangb, Yu-Na Qiand,
Zai-An Dengc, Jing-Xiao Zhangb, Dao-Jun Lva, Hai-Bo Zhanga, Jian-Liang
Shenc,d,, Mu Yuane, Shan-Chao Zhaoa,
a Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, PR
China
b College of Food and Drug, Luoyang Normal University, Luoyang, Henan, 471934, PR China
c School of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou Medical
University, Wenzhou, 325035, PR China
d Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Science,
Wenzhou,325001, PR China
e School of Basic Medical Sciences; Pharmaceutical Research Center, Guangzhou Medical
University, Guangzhou 511436, PR China
ABSTRACT
A novel scaffold of arylpiperazine derivatives was discovered as potent androgen
receptor (AR) antagonist through rational drug designation based on our pre-work,
leading to the discovery of a series of new antiproliferative compounds. Compounds
10, 16, 27, 29 and 31 exhibited relatively strong antagonistic potency against AR and
exhibited potent AR binding affinities, while compounds 5, 6, 10, 14, 16, 19, 21, 27
and 31 exhibited strong cytotoxic activities against LNCaP cells (AR-rich) as well as
also displayed the higher activities than finasteride toward PC-3 (AR-deficient) and
DU145 (AR-deficient). Docking study suggested that the most potent antagonist 16
mainly bind to AR ligand binding pocket (LBP) site through hydrogen bonding
interactions. The structure-activity relationship (SAR) of these designed
arylpiperazine derivatives was rationally explored and discussed. These results
indicated that the novel scaffold compounds demonstrated a step towards the
development of novel and improved AR antagonists, and promising candidates for
future development were identified.
Keywords Prostate cancer; Synthesis; Arylpiperazine derivatives; Antagonistic
activity; Binding affinities; Docking study
1. Introduction
Prostate cancer (PCa) is the most common malignancy and the second leading
 Corresponding author. Tel./fax: +86 0577 88017536 (J.-L. Shen)
E-mail address: zhaoshanchao@263.net (S.-C. Zhao); shenjl@wibe.ac.cn (J.-L. Shen)
†These authors contributed equally to this work.
1

cause of cancer mortality in men.1,2 Androgen deprivation therapy (ADT) has been
proved to be effective initially, but the tumor will eventually progress and develop
into the lethal castration resistant prostate cancer (CRPC),3 upon the onset of prostate
cancer metastasis no significantly effective therapies exist.4–7 Although various
chemotherapeutic agents8 are used solely or in combination with radiotherapy to treat
advanced diseases, none of the conventional approaches to cancer therapy have been
proven to be highly successful for prostate cancer.
The androgen receptor (AR) is a ligand-dependent transcription factor belonging
to the nuclear receptor superfamily and plays a critical role in the progression of
normal prostate cells. The development and progression of prostate cancer is directly
related to the nuclear steroidal AR,9–12 and overexpression of AR was found in most
CRPC, which is essential for CRPC to adapt to the low levels of androgens. As AR
contributes significantly to the resistance to castration, which has been recognized as
an attractive target for the treatment of CRPC.13–17 AR mediates gene expression and
regulates the binding of androgens, such as testosterone (T) and its active metabolite
dihydrotestosterone (DHT). Testosterone is the principal androgen in the blood, while
DHT is the most potent androgen in the cells.18 In order to induce their biological
effects, androgens have to bind to the AR: the hormone-receptor complex binds DNA
and modulates gene expression.19 Upon androgen stimulation, the proliferation of
prostate cells is increased and a malignant tumor can develop.19 The drugs that are
designed and expected to have high affinity to the novel targets, and they not only
inhibit the proliferation but also the differentiation of tumor cells and speed up their
death.20 However, AR antagonists in clinical use, such as flutamide and bicalutamide
(Fig. 1), encounter resistance after several years of hormone therapy, predominantly
due to mutations of AR. Thus, although some new-generation AR antagonists have
been developed, novel types of AR antagonists are still required to treat drug-resistant
prostate cancer. Therefore, as the second-most common cancer worldwide for males,
prostate cancer is a challenge for researchers because of the absence of no effective
therapies.
Fig. 1. Structures of flutamide and bicalutamide
Piperazines and substituted piperazines are the key pharmacophores, and play an
important role in many marketed drugs as the Merck HIV protease inhibitor Crixivan,
and other drugs under development.21 Moreover, piperazine derivatives hold a
spectrum of biological importance, such as antiarrhythmic,22 diuretic,23 antiallergic,24
2

antidepressant,25 anxiolytic,26 antipsychotic,27 antimalarial,28 antiplasmodial,29
receptor-blocking properties30–34 and anti-proliferative properties.35–42 Recently, we
have reported a series of arylpiperazine derivatives as anticancer drugs for
site-directed chemotherapy of prostate cancer,43,44 and some of these compounds were
found to be able to inhibit growth of human cancer cells potently. Previous designed
strategy of arylpiperazine derivatives was fastening aromatic phenol ring (A) at one
end, and introducing different aryl piperazine tail (C) at other end. Our group have
long been devoted to the synthesis of novel arylpiperazine derivatives and evaluation
of their biological actions. To obtain more effective full antagonism toward AR, in
this work, we have designed and synthesized a series of new arylpiperazine
derivatives through exchanging aromatic phenol ring (A) and aryl piperazine tail (C).
Namely, aryl piperazine tail (C) fastened was at one end, and introducing different
aromatic phenol ring (A) based on pre-work (Fig. 2, Scheme 1). A simple SAR study
was also explored to facilitate the further development of the novel arylpiperazine
derivatives. Thus simple strategy for prostate cancer therapeutics would open a new
avenue for research academic institute and pharmaceutical industries.
Fig. 2. Design strategy for the target compounds 5–31
2. Results and Discussion
2.1 Chemistry
3

Scheme 1 illustrated the synthesis of arylpiperazine derivatives 5–31 via a
four-step reaction starting from 2-(4-(bromomethyl)phenyl)acetic acid 1. The first step
involved a reduction reaction between 1 and borane–methyl sulfide complex (2 M in
tetrahydrofuran) to synthesize 2, and the obtained crudes were directly used in the next
step without further purification. After the nucleophilic substitution reaction was
carried out between compound 2 and 1-phenylpiperazine using CH3CN as solvent in
the presence of potassium carbonate at 85 oC for 16 h gave 3 (70% yield from 1).
Subsequently, compound 4 (80% yield) was obtained by reacting 3 with
4-toluene-sulfonyl chloride using CH2Cl2 as solvent in the presence of trimethylamine
and a catalytic amount of 4-dimethylaminopyridine at 0 °C for 16 h. Finally, compound
4 was treated with various phenol (1.5 equiv) in the presence of K2CO3 (6 eq) to obtain
derivatives 5–31 in moderate yields. (53–75%). All synthesized products (HCl salts)
were confirmed based on their expected m/z of [M+1]+, 1H-NMR, 13C-NMR spectra
and elemental analyses (C, H, and N).
Scheme 1. Reagents and conditions are as follows: (i) BH3.S(CH3)2,anhydrous THF, 11 h;
(ii)1-phenylpiperazine, K2CO3, CH3CN, 85 oC, 16 h; (iii) TsCl, Et3N and
4

4-dimethylaminopyridine (catalytic amount), Cl2CH2, 0 oC, 16 h; (iv) Phenol, K2CO3, CH3CN,
85 oC, 16 h; (v) HCl, AcOEt, rt, 0.5 h
2.2 SAR analysis for Antiproliferative and androgen receptor antagonist assay
Antiproliferative activity of the newly synthesized compounds was initially
evaluated in LNCaP-hr cell line using the CCK-8 assay.44 Additionally, AR-negative
DU145 cell line and AR-negative PC-3 cell line were also used to examine whether
these compounds depend AR to exert inhibitory activity as well as potent cytotoxicity
using naftopidil and finasteride45 were taken as reference compounds. Moreover,
normal non-cancer human prostate epithelial RWPE-1 cell line was used to compare
their effects. Then, to clarify whether the antiproliferative activity is related to any
interference with the AR function, the AR antagonist effect was evaluated using the
luciferase reporter gene assay. 46–49 The AR luciferase assays were examined in the
presence of 1 nM AR agonist R1881 co-treatment and the antagonistic activity was
measured as inhibition of R1881 induced luciferase expression.
Table 1. Antiproliferative and AR antagonist activity of compounds 5–31
PC-3a
IC50 (μM)b
(μM)b
DU145a
IC50 (μM)b
LNCaPa
IC50
RWPE-1a
IC50 (μM)b
AR antagonistic activity
Compound
% (10 μM)c
8.2 ± 0.14
4.72 ± 0.21
17.71 ± 0.15
>50
9.82 ± 0.82
15.95 ± 0.23
12.15 ± 0.13
40.39 ± 0.23
29.17 ± 0.21
10.11 ± 0.16
6.4 ± 0.23
6.67 ± 0.06
8.92 ± 0.05
19.28 ± 0.12
>50
>50
40.6 ± 1.2
32.5 ± 3.2
N.D
5
12.15 ± 0.07
27.84 ± 0.13
>50
6
7
N.D
8
15.1 ± 0.16
1.47 ± 0.07
>50
34.87 ± 0.18
5.62 ± 0.03
>50
>50
N.D
9
>50
54.7 ± 0.9
40.2 ± 2.4
N.D
10
11
12
13
14
15
16
17
18
19
20
21
22
23
>50
4.86 ± 0.17
6.72 ± 0.17
2.99 ± 0.14
>50
27.97 ± 0.21
15.61 ± 0.23
3.44 ± 0.13
>50
17.92 ± 0.14
17.59 ± 0.14
7.43 ± 0.14
>50
21.09 ± 0.13
12.26 ± 0.14
4.86 ± 0.12
>50
N.D
16.7 ± 2.6
N.D
8.95 ± 0.13
>50
9.79 ± 0.46
14.23 ± 0.15
7.21 ± 0.20
6.86 ± 0.14
38.65 ± 0.23
9.2 ± 0.12
7.46 ± 0.02
>50
>50
67.1 ± 1.4
N.D
>50
48.83 ± 0.13
5.7 ± 0.05
>50
17.82 ± 0.16
7.83 ± 0.13
>50
19.67 ± 0.14
4.62 ± 0.08
>50
N.D
35.1 ± 0.8
N.Dd
3.59 ± 0.16
22.96 ± 0.07
10.1 ± 0.08
8.64 ± 0.13
19.84 ± 0.14
19.45 ± 0.15
4.76 ± 0.11
>50
32.4 ± 1.6
41.1 ± 2.2
N.D
5.36 ± 0.13
13.15 ± 0.25
47.21 ± 0.17
5

>50
11.58 ± 0.17
10.07 ± 0.23
9.43 ± 0.15
4.81 ± 0.14
2.51 ± 0.25
46.24 ± 0.24
0.77 ± 0.11
8.74 ± 0.57
34.58 ± 0.31
14.53
41.87 ± 0.32
13.47 ± 0.09
32.16 ± 0.18
8.76 ± 0.13
>50
>50
N.D
24
6.69 ± 0.14
>50
15.65 ± 0.13
>50
N.D
25
N.D
26
4.96 ± 0.14
>50
13.15 ± 0.12
>50
48.2 ± 0.8
32.1 ± 1.7
47.2 ± 1.8
24.5 ± 2.1
50.1 ± 1.3
N.D
27
28
1.05 ± 0.04
>50
43.58 ± 0.13
>50
>50
29
>50
30
9.23 ± 0.05
42.10 ± 0.79
17.80
8.51 ± 0.15
22.36 ± 0.61
13.53
3.87 ± 0.14
>50
31
Naftopidil
Finasteride
R1881
Enzalutamide
N.D
N.D
N.D
N.D
N.D
N.D
N.E
N.D
N.D
N.D
N.D
84.7 ± 1.4
N.D = not determined.
N.E = no antagonistic effect.
a PC-3 and DU145, androgen-insensitive human prostate cancer cell line; LNCaP,
androgen-sensitive human prostate cancer cell line; RWPE-1, normal non-cancer
human prostate epithelial cell line.
b IC50 values are taken as means ± standard deviation from three experiments.
c Inhibition rate was shown as a ratio to the R1881 control.
As shown in Table 1, compounds 5, 6, 10, 14, 16, 19, 21, 27 and 31 exhibited
strong cytotoxic activities against LNCaP-hr cells as well as against AR-negative
PC-3 and AR-negative DU145 cells (Fig. 3), and possessed higher activities than
finasteride. However, to our delight, compounds 10, 16, 27 and 31 exhibited relatively
strong antagonistic potency against AR (inhibition >45%). Among these compounds,
compounds 5, 10 and 16 exhibited low cytotoxic character toward normal human
prostate epithelial cells (RWPE-1) with >50 μM of IC50. However, compounds 6, 14,
19, 21, 27 and 31 also exerted marked cytotoxic effect on RWPE-1 cells. Moreover,
compounds 12, 13, 25 and 29 displayed strong cytotoxic activities against
AR-negative PC-3 cells, and exhibited excellent selective activity for PC-3 cells over
other tested cancer cells. But, only compound 29 exhibited low cytotoxic character
toward normal human prostate epithelial cells (RWPE-1) and exhibited potent
antagonistic potency against AR (inhibition = 47.2%). However, compounds 11, 18,
22, 26, 28 and 30 displayed strong cytotoxic activities against DU145 cells, and
exhibited excellent selective activity for DU145 cells over other tested cancer cells.
Interestingly, these compounds exhibited low cytotoxic character toward RWPE-1
cells except 18.
The SAR of these designed arylpiperazine derivatives was fully and thoroughly
explored and discussed. Taking compound 5 as a lead, the SAR investigation was
mainly focused on the variation of the substitute’s type and position on the phenyl
group as a required group for antitumor activity. Firstly, in replacing the phenyl group
with naphthyl group, the resultant compound 6 displayed decreased cytotoxic activity
6

against DU145 and LNCaP cells with the IC50 value of 15.95 and 8.92 μM,
respectively and exhibited cytotoxic activities against RWPE-1 cells. Compounds 7
and 8 exhibited moderate to weak cytotoxic activities against the tested cancer cell
lines compared with compound 6. The activity profiles indicated that hydrogenation
on the benzene ring was inauspicious for anti-cancer activity. The position of the
substituent on the phenyl also affected the cytotoxic activities. Compared to
compound 5, the p-substituted phenyl group derivatives displayed relatively strong
effect. For example, compounds 10, 16, 27 and 31 exhibited strong antagonistic
potency, which was consistent with the LNCaP cell antiproliferation activity. However,
compounds with halogen group (19 and 21) at the 4-position of benzene ring
demonstrated weak antagonistic potency. In addition, amongst the compounds
containing a methyl substituent, the order of the cytotoxic activities of compounds 9
(2-CH3) and 10 (4-CH3) against PC-3, DU145, and LNCaP cells could be placed as
following: 10>9. The similar results were also found in compounds 15 (2-OCH3)
vs.16 (4-OCH3), 18 (2-F) vs.19 (4-F), 20 (2-Cl) vs.21 (4-Cl), 24 (2-Br) vs.25 (4-Br), as
well as 26 (2-CF3) vs.27 (4-CF3) for PC-3, DU145, and LNCaP cells. To our delight,
the antagonistic activity of compound 16 with electron-donating group demonstrated
the highest antagonistic potency (67.1% inhibition). Compound 11 displayed potent
activity against DU145 cells, and compound 12 showed strong cytotoxic activities
against PC-3 cells. These results suggest that the introduction of a larger group at the
4-position of benzene ring was beneficial for improving selectivity for the tested
cancer cells. Compared to compound 13 (3,4-CH3),3,5-Dimethyl-substituted phenyl
compound 14 displayed potent activity against the tested cancer cells. However,
3,5-dichloro-substituted phenyl compound 23 showed moderate cytotoxic activities
against the tested cancer cells, and compound 14 exhibited highly cytotoxic activities
against RWPE-1 cells. Compound 28 (2-CN) displayed improved cytotoxic activity
against DU145 cells. But, compound 29 (4-CN) showed strong cytotoxic activities
against PC-3 cells. In addition, compounds 28 and 29 exhibited excellent selective
activity for the tested cancer cells, and low cytotoxic character toward RWPE-1 cells.
Compound 30 (2-F, 4-CN, IC50 = 0.77 μM) exhibited higher cytotoxic activity than
compounds 18 (2-F) and 29 (4-CN) against DU145 cells, and exhibited excellent
selective activity for DU145 cells over PC-3 and LNCaP cells. Although compound
31 (4-NO2) with electron-withdrawing groups on the phenyl group showed strong
cytotoxic activities against the tested cancer cells and also exhibited potent
antagonistic potency against AR (50.1% inhibition), this compound exhibited highly
cytotoxic activities against RWPE-1 cells. Taken together, the studies of SAR
indicated that the p-substituted phenyl group derivatives displayed improved
cytotoxic activity against the tested cancer cells and displayed relatively strong
antagonistic effect, and above results can lead to a tool which can further design
arylpiperazine derivatives as AR antagonists for in vitro and in viro studies.
The derivatives 10, 16, 27 and 31 exhibited relatively strong antagonistic potency,
7

which was consistent with the LNCaP-hr cell antiproliferation activity. But, those
compounds also exhibited toxicity against AR-negative PC-3 and AR-negative
DU145 cells. The studies reported that the toxicity of the drugs toward AR-negative
cells may be due to promoted uptake by GPCR6A, a cell surface AR-like receptor
overexpressed in cells, and which has been implicated in the cytotoxicity of
antiandrogen-tagged nanoparticles.50,51 In addition, the androgen independent
antiproliferative activity was also confirmed using CRPC model LNCaP subline
LNCaP-SF,52 demonstrating that the drugs could overcome CRPC by inhibiting cell
growth AR-independently.53 The above studies indicated that the synthesized
derivatives may showed the androgen independent antiproliferative activity against
PC-3 and DU145 cells.
8

Fig. 3. Arylpiperazine derivatives 5, 6, 10, 14, 16, 19, 21, 27 and 31 inhibited cell viability
(percent relative to control) in prostate cell lines PC-3, DU145 and LNCaP. The all cells were
exposed to escalating concentrations of arylpiperazine derivatives respectively for 24 h, and the
cell viability was detected by CCK-8 assay.
2.3 Binding affinity assay of compounds 10, 16, 27, 29 and 31
To further investigate the binding affinity of the representative compounds
against the AR. Here, we examined the AR binding affinity of the compounds 10, 16,
27, 29 and 31 with fluorescence polarization (FP) based on the fluorescent tracer and
nonfluorescent antagonist competing for binding to AR by binding assay, The results
were showed in Table 2. All the tested compounds exhibited potent binding affinities
against the AR (IC50 <5 μM), and compound 16 (IC50 = 1.15 μM) displayed
comparable binding affinity to enzalutamide (IC50 = 1.32 μM) for the AR. Among all
the tested compounds, we also found a correlation between the effect on antagonistic
activity and binding affinity of these the tested compounds. For example, compound
16 with highest affinity (IC50 = 1.15 μM) for receptor also showed the highest
antagonistic activity (67.1% inhibition). In addition, the order of the binding affinity
of compounds 10, 27, 29 and 31 (binding affinity: 10 > 31 > 27 >29) against the AR is
consistent with the results of antagonistic activity of these compounds. The above
results may be attributed to the fundamental role binding affinity plays in promoting
the antagonistic activity. The results indicated that some arylpiperazine derivatives
9

may be efficient AR antagonist, and the most potent compound 16 was selected for
further investigating the binding site of compound and the AR.
Table 2. Binding affinity of 10, 16, 27, 29 and 31 to mutant AR
Compound
IC50/µMa
3.66 ± 0.78
1.15 ± 0.34
4.03 ± 0.85
4.22 ± 1.01
3.82 ± 1.09
1.32 ± 0.78
10
16
27
29
31
Enzalutamide
a The data represent the mean of at least three independent determinations.
2.4 Docking study
In order to better understand the binding site of these compound targets, as well
as to explore the detail information about their dominant interactions with AR,54
docking simulation is performed. The most potent antagonist, compound 16 was taken
as the template molecule in this process, and three binding sites of AR, including
ligand binding pocket (LBP), activation function-2 (AF2) and binding function 3
(BF3),55,56 were all used to explore the binding affinities of this compound. The
lowest docked energy values were summarized in Table 3.
Table 3. The binding affinities (kcal/mol) of compound 16 in three binding sites of AR
Binding site
Compound 16
LBP (PDB ID: 2OZ7)
AF2 (PDB ID: 2YHD)
BF3 (PDB ID: 2YLO)
-8.2
-6.0
-6.4
As displayed in Table 3, LBP site had the highest binding affinity of -8.2 kcal/mol,
which proved that AR LBP was the major binding site for compound 16. To decipher
these binding interactions, the number of hydrogen bonds of this antagonist was
calculated, and the result was summarized in Fig. 4. As shown in Fig. 4, compound 16
could fit into the AR LBP site by the formation of three hydrogen bonding
interactions with Arg752 and Asn705, which are the key residues as reported by
Eileen, et al (2015).57 The results suggested that the compound 16 mainly bind to AR
LBP site through hydrogen bonding interactions.
10

Fig. 4. The stereo view of compound 16-AR interaction (A), and compound 16-AR
interaction plots (B) generated by LigPlot+.
3. Conclusion
In this paper, we report the synthesis and biological in vitro evaluation of a novel
class of arylpiperazine derivatives as potent androgen receptor antagonists, leading to
the discovery of a series of new antiproliferative compounds. Compounds 10, 16, 27,
29 and 31 exhibited relatively strong antagonistic potency against AR and exhibited
potent AR binding affinities, and on the same time compounds 10, 16, 27 and 31
exhibited strong cytotoxic activities against LNCaP cells (AR-rich) as well as also
displayed the higher activities than finasteride toward PC-3 (AR-deficient) and
DU145 (AR-deficient). In addition, other compounds also displayed strong cytotoxic
activities against the tested cancer cells. Docking study indicated that the compound
16 mainly bind to AR LBP site through hydrogen bonding interactions. The studies of
SAR indicated that the p-substituted phenyl group derivatives displayed improved
biological activity. Those results can lead to a tool which can further design
arylpiperazine derivatives for in vitro and in vtro studies.
4.1 General chemistry
Tetrahydrofuran was distilled from sodium, and other reagents were obtained from
commercial sources and were used as received. Melting points were measured on an
uncorrected SGW X-4 micro melting point apparatus. NMR spectra were obtained on
a Bruker AVANCE-500 spectrometer in CDCl3 or DMSO-d6, with TMS as an internal
standard, and chemical shift values are reported in δ (ppm) and coupling constants in
11

Hertz. ESI mass spectra were recorded on the AB Sciex 5600 Triple TOF mass
spectrometer (Foster, CA, USA). Elemental analyses (EA) data were recorded on an
Elementar Vario EL elemental analyzer. 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.
4.1.1 Synthesis of 2-(4-(Bromomethyl)phenyl)ethanol (2)
Compound 2 was synthesized using methods reported previously in the literature [44].
4.1.2 2-(4-((4-phenylpiperazin-1-yl)methyl)phenyl)ethan-1-ol (3)
Compound 3 was synthesized using methods reported previously in the literature
[44], and sesamol was substituted with 1-phenylpiperazine. White solid
Yield: 70% from compound 1; Mp 64.2–64.6 oC; 1H NMR (500 MHz, CDCl3) δ in
ppm: 7.33 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 6.95
(d, J = 7.9 Hz, 2H), 6.88 (t, J = 7.3 Hz, 1H), 3.89 (t, J = 6.6 Hz, 2H), 3.58 (s, 2H),
3.23 (t, J = 5.0 Hz, 4H), 2.90 (t, J = 6.6 Hz, 2H), 2.64 (t, J = 5.0 Hz, 4H); MS (ESI,
m/z): 297.2 [M+1]+.
.
4.1.3 4-((4-phenylpiperazin-1-yl)methyl)phenethyl 4-methylbenzenesulfonate (4)
Compound 4 was synthesized using methods reported previously in the
literature [44]. White solid
. Yield: 80%. Mp 72.3–72.9 oC; 1H NMR
(500 MHz, CDCl3) δ in ppm: 7.71 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.28 –
7.21 (m, 4H), 7.08 (d, J = 7.9 Hz, 2H), 6.91 (d, J = 7.9 Hz, 2H), 6.84 (t, J = 7.3 Hz,
1H), 4.20 (t, J = 7.1 Hz, 2H), 3.53 (s, 2H), 3.19 (t, J = 5.0 Hz, 4H), 2.94 (t, J = 7.1 Hz,
2H), 2.59 (t, J = 5.0 Hz, 4H), 2.43 (s, 3H); MS (ESI, m/z): 451.2 [M+1]+.
4.1.4 General Procedure for the Preparation of Arylpiperazine Derivative
Hydrochloride Salts 5–31
Phenol (1.5 equiv) and potassium carbonate (6.0 equiv) were added to a solution
of 4 (100 mg, 0.22 mmol) in acetonitrile (CH3CN, 15 mL). The reaction mixture was
heated to 85 oC and stirred for 16 h. Afterward the mixture was cooled to room
temperature. The reaction mixture was filtered, and the filtrate was concentrated in
vacuo. Then the residue was purified by chromatography on silica-gel column
(petroleum ether: ethyl acetate = 20:1, v/v) to obtain the corresponding products
(5–31), and then to a solution of above corresponding products in ethyl acetate was
added dropwise 4 M HCl solution in ethyl acetate (50 mL), keeping stirring for 0.5 h.
Then the resulting solid was collected by filtration to give corresponding
hydrochloride salts as a white solid.
4.1.4.1 1-(4-(2-phenoxyethyl)benzyl)-4-phenylpiperazine dihydrochloride (5)
12

White solid
; Yield: 56%; Mp 85.5–85.9 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.64 (s, 1H, N+H), 7.62 (d, J = 7.7 Hz, 2H), 7.41 (d,
J = 7.7 Hz, 2H), 7.26 (q, J = 7.8 Hz, 4H), 6.97 (d, J = 8.1 Hz, 2H), 6.95 – 6.81 (m,
4H), 4.33 (d, J = 4.8 Hz, 2H), 4.20 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.5 Hz, 2H), 3.33
(d, J = 11.4 Hz, 2H), 3.27 – 3.11 (m, 4H), 3.07 (t, J = 6.6 Hz, 2H); 13C NMR (126
MHz, DMSO-d6) δ in ppm: 158.00, 149.07, 139.59, 131.19, 129.15, 128.96, 128.79,
127.17, 120.22, 119.72, 115.67, 114.13, 67.34, 57.76, 49.67, 44.87, 34.31; MS (ESI,
m/z): 373.2 [M+1]+; Anal. Calcd for C25H28N2O·2HCl: C, 67.41; H, 6.79; N, 6.29.
Found: C, 67.27; H, 6.76; N, 5.94.
4.1.4.2 1-(4-(2-(naphthalen-1-yloxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(6)
White solid
; Yield: 60%; Mp 95.5–95.7 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.52 (s, 1H, N+H), 8.09 (dd, J = 6.0, 2.8 Hz, 1H),
7.85 (dd, J = 6.0, 2.8 Hz, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.49
– 7.36 (m, 4H), 7.24 (t, J = 7.8 Hz, 2H), 6.97 (dd, J = 11.9, 8.0 Hz, 3H), 6.85 (t, J =
7.5 Hz, 1H), 4.39 (t, J = 6.3 Hz, 2H), 4.33 (d, J = 4.8 Hz, 2H), 3.76 (d, J = 12.5 Hz,
2H), 3.32 (d, J = 11.2 Hz, 2H), 3.23 (t, J = 6.3 Hz, 2H), 3.20 – 3.03 (m, 4H); 13C
NMR (126 MHz, DMSO-d6) δ in ppm: 154.34, 149.96, 140.76, 134.48, 132.01,
129.94, 129.60, 128.00, 127.93, 126.88, 126.71, 125.75, 125.37, 121.91, 120.44,
116.45, 105.77, 68.79, 58.63, 50.53, 45.66, 35.26; MS (ESI, m/z): 423.2 [M+1]+;
Anal. Calcd for C29H30N2O·2HCl: C, 70.30; H, 6.51; N, 5.65. Found: C, 70.43; H,
6.69; N, 5.51.
4.1.4.3
1-phenyl-4-(4-(2-((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)ethyl)benzyl)piperazine
dihydrochloride (7)
White solid
; Yield: 64%; Mp 89.5–90.8 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.45 (s, 1H, N+H), 7.60 (d, J = 8.0 Hz, 2H), 7.41 (d,
J = 8.0 Hz, 2H), 7.25 (dd, J = 8.2, 7.6 Hz, 2H), 7.00 (t, J = 8.0 Hz, 1H), 6.97 (d, J =
8.0 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.62 (d, J = 7.6 Hz,
1H), 4.34 (d, J = 4.8 Hz, 2H), 4.15 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.8 Hz, 2H), 3.33
(d, J = 11.4 Hz, 2H), 3.26 – 3.10 (m, 4H), 3.07 (t, J = 6.6 Hz, 2H), 2.65 (t, J = 6.0 Hz,
2H), 2.47 (t, J = 6.0 Hz, 2H), 1.76 – 1.56 (m, 4H); 13C NMR (126 MHz, DMSO-d6) δ
in ppm: 156.45, 149.94, 140.80, 138.23, 131.90, 129.90, 129.61, 127.89, 126.25,
125.44, 121.62, 120.49, 116.46, 108.56, 68.31, 58.62, 50.53, 45.69, 35.35, 29.46,
23.17, 22.84, 22.81; MS (ESI, m/z): 427.3 [M+1]+; Anal. Calcd for C29H34N2O·2HCl:
C, 69.73; H, 7.26; N, 5.61. Found: C, 69.57; H, 7.32; N, 5.25.
13

4.1.4.4
1-phenyl-4-(4-(2-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)ethyl)benzyl)piperazine
dihydrochloride (8)
White solid
; Yield: 66%; Mp 80.7–81.5 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.46 (s, 1H, N+H), 7.60 (d, J = 7.5 Hz, 2H), 7.40 (d,
J = 7.5 Hz, 2H), 7.25 (t, J = 7.4 Hz, 2H), 6.97 (d, J = 7.9 Hz, 2H), 6.92 (d, J = 8.0 Hz,
1H), 6.85 (t, J = 7.2 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H), 6.61 (s, 1H), 4.33 (d, J = 4.8
Hz, 2H), 4.14 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.3 Hz, 2H), 3.33 (d, J = 10.7 Hz, 2H),
3.23 – 3.11 (m, 4H), 3.03 (t, J = 6.6 Hz, 2H), 2.65 (br s, 2H), 2.61 (br s, 2H),1.68 (s,
4H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 156.09, 149.40, 140.02, 137.57,
131.47, 129.67, 129.25, 129.10, 128.55, 127.43, 120.01, 115.97, 114.13, 112.31,
67.69, 58.09, 50.01, 45.19, 34.69, 28.98, 27.91, 22.94, 22.65; MS (ESI, m/z): 427.3
[M+1]+; Anal. Calcd for C29H34N2O·2HCl: C, 69.73; H, 7.26; N, 5.61. Found: C,
69.52; H, 7.22; N, 5.46.
4.1.4.5 1-phenyl-4-(4-(2-(o-tolyloxy)ethyl)benzyl)piperazine dihydrochloride (9)
White solid
; Yield: 54%; Mp 96.5–97.5 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.60 (s, 1H, N+H), 7.62 (d, J = 7.9 Hz, 2H), 7.42 (d,
J = 7.9 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.12 (dd, J = 9.7, 8.0 Hz, 2H), 6.97 (d, J =
8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 1H), 6.81 (t, J = 7.4 Hz, 1H), 6.81 (t, J = 7.4 Hz, 1H),
4.34 (d, J = 4.8 Hz, 2H), 4.19 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.7 Hz, 2H), 3.33 (d, J
= 11.2 Hz, 2H), 3.25 – 3.06 (m, 6H), 2.08 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ
in ppm: 156.40, 149.31, 140.17, 131.46, 130.35, 129.41, 129.15, 127.47, 126.94,
125.67, 120.20, 116.08, 111.26, 67.91, 58.08, 49.95, 45.26, 34.87, 15.95; MS (ESI,
m/z): 387.2 [M+1]+; Anal. Calcd for C26H30N2O·2HCl: C, 67.97; H, 7.02; N, 6.10.
Found: C, 67.62; H, 7.15; N, 5.87.
4.1.4.6 1-phenyl-4-(4-(2-(p-tolyloxy)ethyl)benzyl)piperazine dihydrochloride (10)
White solid
; Yield: 65%; Mp 89.7–90.2 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.56 (s, 1H, N+H), 7.61 (d, J = 7.6 Hz, 2H), 7.40 (d,
J = 7.6 Hz, 2H), 7.25 (t, J = 7.6 Hz, 2H), 7.06 (d, J = 8.1 Hz, 2H), 6.97 (d, J = 8.1 Hz,
2H), 6.86 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 8.2 Hz, 2H),, 4.33 (br s, 2H), 4.16 (t, J = 6.6
Hz, 2H), 3.78 (d, J = 12.4 Hz, 2H), 3.33 (d, J = 11.1 Hz, 2H), 3.27 – 3.08 (m, 4H),
3.04 (t, J = 6.6 Hz, 2H), 2.21 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ in ppm:
155.92, 148.85, 139.64, 131.22, 129.50, 128.95, 128.85, 127.16, 120.05, 115.85,
114.02, 67.49, 57.74, 49.60, 45.03, 34.37, 19.74; MS (ESI, m/z): 387.2 [M+1]+; Anal.
Calcd for C26H30N2O·2HCl: C, 67.97; H, 7.02; N, 6.10. Found: C, 67.77; H, 7.05; N,
5.88.
14

4.1.4.7 1-(4-(2-(4-isopropylphenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(11)
White solid
; Yield: 58%; Mp 90.0–90.7 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.55 (s, 1H, N+H), 7.61 (d, J = 8.0 Hz, 2H), 7.41 (d,
J = 8.0 Hz, 2H), 7.25 (dd, J = 8.5, 7.6 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.96 (d, J =
8.1 Hz, 2H), 6.84 (d, J = 8.5 Hz, 3H), 4.33 (d, J = 4.8 Hz, 2H), 4.17 (t, J = 6.7 Hz,
2H), 3.78 (d, J = 12.9 Hz, 2H), 3.33 (d, J = 11.5 Hz, 2H), 3.25 – 3.10 (m, 4H), 3.05 (t,
J = 6.7 Hz, 2H), 2.80 (dt, J = 13.8, 6.9 Hz, 1H), 1.15 (d, J = 6.9 Hz, 6H); 13C NMR
(126 MHz, DMSO-d6) δ in ppm: 156.45, 149.44, 140.45, 140.01, 131.51, 129.28,
129.12, 127.48, 127.12, 120.01, 115.98, 114.28, 67.78, 58.11, 50.03, 45.20, 34.69,
32.54, 24.10; MS (ESI, m/z): 415.3 [M+1]+; Anal. Calcd for C28H34N2O·2HCl: C,
68.98; H, 7.44; N, 5.75. Found: C, 68.74; H, 7.48; N, 5.62.
4.1.4.8 1-(4-(2-(4-(tert-butyl)phenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (12)
White solid
; Yield: 68%; Mp 81.1–82.0 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.47 (s, 1H, N+H), 7.61 (d, J = 7.7 Hz, 2H), 7.41 (d,
J = 7.7 Hz, 2H), 7.28 – 7.23 (m, 4H), 6.96 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.5 Hz,
3H), 4.34 (d, J = 4.8 Hz, 2H), 4.18 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.2 Hz, 2H), 3.33
(d, J = 11.0 Hz, 2H), 3.27 – 3.09 (m, 4H), 3.05 (t, J = 6.6 Hz, 2H), 1.23 (s, 9H); 13C
NMR (126 MHz, DMSO-d6) δ in ppm: 156.08, 149.37, 142.71, 139.97, 131.51,
129.26, 129.12, 127.48, 126.04, 120.07, 116.01, 113.93, 67.74, 58.08, 49.99, 45.21,
34.67, 33.71, 31.32; MS (ESI, m/z): 429.3 [M+1]+; Anal. Calcd for C29H36N2O·2HCl:
C, 69.45; H, 7.64; N, 5.59. Found: C, 69.27; H, 7.88; N, 5.37.
4.1.4.9 1-(4-(2-(3,4-dimethylphenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (13)
White solid
; Yield: 64%; Mp 97.9–98.6 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.45 (s, 1H, N+H), 7.60 (d, J = 7.9 Hz, 2H), 7.41 (d,
J = 7.9 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 7.00 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 8.0 Hz,
2H), 6.86 (d, J = 6.6 Hz, 1H), 6.73 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 8.0, 2.4 Hz, 1H),
4.33 (d, J = 4.8 Hz, 2H), 4.15 (t, J = 6.7 Hz, 2H), 3.78 (d, J = 12.2 Hz, 2H), 3.33 (d, J
= 11.2 Hz, 2H), 3.26 – 3.09 (m, 4H), 3.04 (t, J = 6.6 Hz, 2H), 2.16 (s, 3H), 2.12 (s,
3H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 156.44, 149.47, 140.07, 137.25,
131.49, 130.15, 129.29, 129.12, 127.97, 127.45, 119.98, 115.97, 111.41, 67.69, 58.15,
50.07, 45.20, 34.71, 19.57, 18.39; MS (ESI, m/z): 401.3 [M+1]+; Anal. Calcd for
C27H32N2O·2HCl: C, 68.49; H, 7.24; N, 5.92. Found: C, 68.76; H, 7.45; N, 5.78.
15

4.1.4.10 1-(4-(2-(3,5-dimethylphenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (14)
White solid
; Yield: 60%; Mp 88.2–89.2 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.56 (s, 1H, N+H), 7.61 (d, J = 7.9 Hz, 2H), 7.40 (d,
J = 7.9 Hz, 2H), 7.25 (t, J = 8.0 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 6.85 (t, J = 7.2 Hz,
1H), 6.54 (s, 2H), 6.37 (s, 1H), 4.34 (d, J = 4.8 Hz, 2H), 4.16 (t, J = 6.6 Hz, 2H), 3.78
(d, J = 12.7 Hz, 2H), 3.33 (d, J = 11.3 Hz, 2H), 3.27 – 3.08 (m, 4H), 3.04 (t, J = 6.6
Hz, 2H), 2.21 (s, 3H), 2.15 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 158.22,
157.07, 149.27, 138.42, 138.10, 131.33, 129.12, 128.96, 122.00, 120.27, 119.86,
115.82, 112.83, 111.99, 67.39, 57.95, 49.86, 45.04, 34.55, 20.85, 20.82; MS (ESI,
m/z): 401.3 [M+1]+; Anal. Calcd for C27H32N2O·2HCl: C, 68.49; H, 7.24; N, 5.92.
Found: C, 68.68; H, 7.39; N, 5.64.
4.1.4.11 1-(4-(2-(2-methoxyphenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (15)
White solid
; Yield: 73%; Mp 94.1–95.1 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.58 (s, 1H, N+H), 7.61 (d, J = 8.0 Hz, 2H), 7.42 (d,
J = 8.0 Hz, 2H), 7.25 (t, J = 7.9 Hz, 2H), 6.97 – 6.94 (m, 4H), 6.91 – 6.84 (m, 3H),
4.33 (d, J = 4.8 Hz, 2H), 4.17 (t, J = 6.8 Hz, 2H), 3.78 (d, J = 12.9 Hz, 2H), 3.73 (s,
3H), 3.32 (d, J = 11.5 Hz, 2H), 3.24 – 3.12 (m, 4H), 3.06 (t, J = 6.8 Hz, 2H); 13C
NMR (126 MHz, DMSO-d6) δ in ppm: 150.09, 149.76, 148.61, 140.59, 132.14,
130.01, 129.79, 128.14, 121.71, 121.44, 120.70, 116.66, 114.24, 113.12, 69.32, 58.77,
56.26, 50.68, 45.86, 35.45; MS (ESI, m/z): 403.2 [M+1]+; Anal. Calcd for
C26H30N2O2·2HCl: C, 65.68; H, 6.78; N, 5.89. Found:C, 65.73; H, 7.07; N, 5.91.
4.1.4.12 1-(4-(2-(4-methoxyphenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (16)
White solid
; Yield: 62%; Mp 94.7–95.1 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.47 (s, 1H, N+H), 7.60 (d, J = 8.0 Hz, 2H), 7.40 (d,
J = 8.0 Hz, 2H), 7.25 (t, J = 7.9 Hz, 2H), 6.97 (d, J = 7.9 Hz, 2H), 6.87 – 6.82 (m, 5H),
4.33 (d, J = 4.8 Hz, 2H), 4.14 (t, J = 6.7 Hz, 2H), 3.78 (d, J = 12.7 Hz, 2H), 3.68 (s,
3H), 3.33 (d, J = 11.1 Hz, 2H), 3.24 – 3.10 (m, 4H), 3.03 (t, J = 6.6 Hz, 2H); 13C
NMR (126 MHz, DMSO-d6) δ 153.03, 152.02, 149.10, 139.71, 131.17, 128.96,
128.80, 127.13, 119.70, 115.66, 115.07, 114.27, 67.97, 57.80, 55.01, 49.71, 44.88,
34.41; MS(ESI, m/z): 403.2 [M+1]+; Anal. Calcd for C26H30N2O2·2HCl: C, 65.68; H,
6.78; N, 5.89. Found: C, 65.70; H, 6.85; N, 5.61.
4.1.4.13 1-(4-(2-(4-ethoxyphenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(17)
16

White solid
; Yield: 54%; Mp 92.3–93.3 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.49 (s, 1H, N+H), 7.60 (d, J = 8.0 Hz, 2H), 7.40 (d,
J = 8.0 Hz, 2H), 7.25 (dd, J = 8.4, 7.6 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 6.90 – 6.76
(m, 5H), 4.33 (d, J = 4.8 Hz, 2H), 4.13 (t, J = 6.7 Hz, 2H), 3.92 (q, J = 7.0 Hz, 2H),
3.78 (d, J = 12.8 Hz, 2H), 3.33 (d, J = 11.4 Hz, 2H), 3.24 – 3.06 (m, 4H), 3.03 (t, J =
6.7 Hz, 2H), 1.27 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ in ppm:
152.59, 152.28, 149.46, 140.05, 131.49, 129.28, 129.12, 127.45, 120.00, 115.97,
115.38, 115.22, 68.28, 63.29, 58.13, 50.04, 45.20, 34.75, 14.73; MS (ESI, m/z): 417.3
[M+1]+; Anal. Calcd for C27H32N2O2·2HCl: C, 66.25; H, 7.00;N, 5.72. Found: C,
65.92; H, 6.97; N, 5.37.
4.1.4.14 1-(4-(2-(2-fluorophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(18)
White solid
; Yield: 76%; Mp 89.9–90.6 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.60 (s, 1H, N+H), 7.62 (d, J = 8.0 Hz, 2H), 7.42 (d,
J = 8.0 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 7.22 – 7.14 (m, 2H), 7.10 (t, J = 7.8 Hz, 1H),
6.97 (d, J = 8.0 Hz, 2H), 6.95 – 6.88 (m, 1H), 6.85 (t, J = 7.3 Hz, 1H), 4.34 (d, J = 4.8
Hz, 2H), 4.28 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.8 Hz, 2H), 3.33 (d, J = 11.4 Hz, 2H),
3.28 – 3.11 (m, 4H), 3.09 (t, J = 6.6 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ in
ppm: 152.68, 150.75, 149.13, 146.33, 146.25, 139.62, 131.58, 129.34, 129.20, 127.62,
124.85, 124.82, 121.10, 121.04, 120.46, 116.24, 116.04, 115.90, 115.11, 68.91, 58.04,
49.91, 45.40, 34.62; MS (ESI, m/z): 391.2 [M+1]+; Anal. Calcd for
C25H27FN2O·2HCl: C, 64.79; H, 6.31; N, 6.04. Found: C, 64.69; H, 6.37; N, 5.87.
4.1.4.15 1-(4-(2-(4-fluorophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(19)
White solid
; Yield: 65%; Mp 88.1–89.2 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.55 (s, 1H, N+H), 7.62 (d, J = 7.8 Hz, 2H), 7.42 (d,
J = 7.8 Hz, 2H), 7.26 (t, J = 7.8 Hz, 2H), 7.10 (t, J = 8.7 Hz, 2H), 7.04 – 6.90 (m, 4H),
6.86 (t, J = 7.2 Hz, 1H), 4.34 (d, J = 4.8 Hz, 2H), 4.19 (t, J = 6.6 Hz, 2H), 3.79 (d, J =
12.5 Hz, 2H), 3.34 (d, J = 11.2 Hz, 2H), 3.24 – 3.10 (m, 4H), 3.06 (t, J = 6.6 Hz, 2H);
13C NMR (126 MHz, DMSO-d6) δ in ppm: 157.88, 156.00, 155.16, 149.47, 140.29,
132.04, 129.75, 129.68, 127.99, 121.06, 116.76, 116.37, 116.27, 116.20, 68.88, 58.52,
50.35, 45.94, 35.11; MS(ESI, m/z): 391.2 [M+1]+; Anal. Calcd for C25H27FN2O·2HCl:
C, 64.79; H, 6.31; N, 6.04. Found: C, 64.72; H, 6.34; N, 5.83.
4.1.4.16 1-(4-(2-(2-chlorophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(20)
White solid
; Yield:57%; Mp 95.8–96.9 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.51 (s, 1H, N+H), 7.61 (d, J = 8.0 Hz, 2H), 7.45 (d,
17

J = 8.0 Hz, 2H), 7.41 (dd, J = 8.0, 1.2 Hz, 1H), 7.30 – 7.24 (m, 3H), 7.16 (dd, J = 8.0,
1.2 Hz, 1H), 6.97 (d, J = 8.0 Hz, 2H), 6.94 (td, J = 7.8, 1.2 Hz, 1H), 6.86 (t, J = 7.3
Hz, 1H), 4.34 (d, J = 4.8 Hz, 2H), 4.28 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.9 Hz, 2H),
3.33 (d, J = 11.5 Hz, 2H), 3.26 – 3.11 (m, 4H), 3.11 (t, J = 6.6 Hz, 2H); 13C NMR
(126 MHz, DMSO-d6) δ in ppm: 153.35, 149.10, 139.41, 131.12, 129.56, 129.15,
128.79, 127.99, 127.22, 121.15, 120.94, 119.70, 115.66, 113.51, 68.51, 57.78, 49.70,
44.87, 34.25; MS (ESI, m/z): 407.2 [M+1]+; Anal. Calcd for C25H27ClN2O·2HCl: C,
62.57; H, 6.09; N, 5.84. Found: C, 62.56; H, 6.07; N, 5.56.
4.1.4.17 1-(4-(2-(4-chlorophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(21)
White solid
; Yield: 65%; Mp 95.0–95.7 oC (HCl salt); 1H NMR
(400 MHz, DMSO-d6) δ in ppm: 11.59 (s, 1H, N+H), 7.61 (d, J = 7.9 Hz, 2H), 7.41 (d,
J = 7.9 Hz, 2H), 7.30 (d, J = 8.9 Hz, 2H), 7.25 (t, J = 7.8 Hz, 2H), 6.98 (d, J = 3.0 Hz,
2H), 6.95 (d, J = 3.0 Hz, 2H), 6.85 (t, J = 7.2 Hz, 1H), 4.33 (d, J = 4.8 Hz, 2H), 4.21
(t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.7 Hz, 2H), 3.33 (d, J = 11.3 Hz, 2H), 3.26 – 3.11
(m, 4H), 3.06 (t, J = 6.6 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 157.24,
149.10, 139.74, 131.58, 129.25, 129.06, 127.58, 124.26, 120.51, 116.99, 116.31,
116.26, 68.21, 58.06, 49.91, 45.43, 34.54; MS (ESI, m/z): 407.2 [M+1]+; Anal. Calcd
for C25H27ClN2O·2HCl: C, 62.57; H, 6.09; N, 5.84. Found: C, 62.29; H, 6.03; N, 5.49.
4.1.4.18 1-(4-(2-(3,4-dichlorophenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (22)
White solid
; Yield: 53%; Mp 94.1–95.1 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.41 (s, 1H, N+H), 7.60 (d, J = 7.9 Hz, 2H), 7.50 (d,
J = 8.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 2H), 7.25 (t, J = 7.8 Hz, 3H), 6.96 (d, J = 8.0 Hz,
3H), 6.85 (t, J = 7.2 Hz, 1H), 4.34 (d, J = 4.8 Hz, 2H), 4.26 (t, J = 6.7 Hz, 2H), 3.78
(d, J = 12.1 Hz, 2H), 3.33 (d, J = 11.1 Hz, 2H), 3.24 – 3.06 (m, 4H), 3.07 (t, J = 6.7
Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 158.37, 149.99, 140.09, 132.09,
132.00, 131.43, 129.81, 129.60, 128.07, 122.89, 120.43, 116.89, 116.43, 116.05,
69.06, 58.62, 50.57, 45.66, 34.86; MS (ESI, m/z): 441.1 [M+1]+; Anal. Calcd for
C25H26Cl2N2O·2HCl: C, 58.38; H, 5.49; N, 5.45. Found: C, 58.49; H, 5.75; N, 5.17.
4.1.4.19 1-(4-(2-(3,5-dichlorophenoxy)ethyl)benzyl)-4-phenylpiperazine
dihydrochloride (23)
White solid
; Yield: 64%; Mp 79.4–80.4 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.47 (s, 1H, N+H), 7.61 (d, J = 8.0 Hz, 2H), 7.41 (d,
J = 8.0 Hz, 2H), 7.25 (t, J = 7.9 Hz, 2H), 7.14 (d, J = 1.6 Hz, 1H), 7.05 (d, J = 1.6 Hz,
2H), 6.97 (d, J = 8.2 Hz, 2H), 6.89 – 6.80 (m, 1H), 4.34 (d, J = 4.8 Hz, 2H), 4.29 (t, J
18

= 6.6 Hz, 2H), 3.78 (d, J = 12.6 Hz, 2H), 3.33 (d, J = 11.3 Hz, 2H), 3.16 (m, 4H), 3.06
(t, J = 6.6 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 160.34, 149.89, 139.99,
135.08, 132.01, 129.80, 129.62, 128.08, 120.82, 120.55, 116.50, 114.95, 114.42,
69.14, 58.58, 50.50, 45.70, 34.80; MS (ESI, m/z): 441.1 [M+1]+; Anal. Calcd for
C25H26Cl2N2O·2HCl: C, 58.38; H, 5.49; N, 5.45. Found: C, 58.06; H, 5.56; N, 5.18.
4.1.4.20 1-(4-(2-(2-bromophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(24)
White solid
; Yield: 75%; Mp 94.5–95.4 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.49 (s, 1H, N+H), 7.60 (d, J = 8.0 Hz, 2H), 7.55
(dd, J = 7.9, 1.2 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.36 – 7.28 (m, 1H), 7.24 (dd, J =
8.2, 7.4 Hz, 2H), 7.12 (dd, J = 8.2, 1.2 Hz, 1H), 6.97 (d, J = 8.0 Hz, 2H), 6.92 – 6.80
(m, 2H), 4.33 (d, J = 4.8 Hz, 2H), 4.26 (t, J = 6.6 Hz, 2H), 3.78 (d, J = 12.9 Hz, 2H),
3.33 (d, J = 11.4 Hz, 2H), 3.24 – 3.11 (m, 4H), 3.10 (t, J = 6.6 Hz, 2H); 13C NMR
(126 MHz, DMSO-d6) δ in ppm: 154.04, 148.95, 139.29, 132.43, 130.93, 129.05,
128.62, 128.49, 127.04, 121.51, 119.51, 115.49, 113.21, 110.45, 68.46, 57.62, 49.54,
44.69, 34.10; MS (ESI, m/z): 452.1 [M+2]+; Anal. Calcd for C25H27BrN2O·2HCl: C,
57.27; H, 5.57;N, 5.34. Found: C, 57.29; H, 5.56; N, 5.05.
4.1.4.21 1-(4-(2-(4-bromophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(25)
White solid
; Yield: 61%; Mp 101.4–101.7 oC (HCl salt); 1H
NMR (500 MHz, DMSO-d6) δ in ppm: 11.51 (s, 1H, N+H), 7.61 (d, J = 8.0 Hz, 2H),
7.42 (t, J = 8.4 Hz, 4H), 7.25 (t, J = 7.7 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 6.91 (d, J =
8.8 Hz, 2H), 6.85 (t, J = 7.2 Hz, 1H), 4.33 (d, J = 4.8 Hz, 2H), 4.21 (t, J = 6.6 Hz, 2H),
3.78 (d, J = 12.5 Hz, 2H), 3.33 (d, J = 11.3 Hz, 2H), 3.27 – 3.09 (m, 4H), 3.06 (t, J =
6.6 Hz, 2H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 158.15, 149.93, 140.24,
132.61, 132.01, 129.79, 129.61, 128.03, 120.50, 117.31, 116.47, 112.43, 68.60, 58.61,
50.53, 45.69, 34.97; MS (ESI, m/z): 452.0 [M+2]+; Anal. Calcd for
C25H27BrN2O ·2HCl: C, 57.27; H, 5.57; N, 5.34. Found: C, 57.00; H, 5.60; N, 5.16.
4.1.4.22 1-phenyl-4-(4-(2-(2-(trifluoromethyl)phenoxy)ethyl)benzyl)piperazine
dihydrochloride (26)
White solid
; Yield: 59%; Mp 96.0–96.8 oC (HCl salt); 1H NMR
(500 MHz, DMSO-d6) δ in ppm: 11.54 (s, 1H, N+H), 7.60 (t, J = 7.6 Hz, 4H), 7.42 (d,
J = 7.9 Hz, 2H), 7.25 (dd, J = 14.7, 7.6 Hz, 3H), 7.07 (t, J = 7.6 Hz, 1H), 6.97 (d, J =
8.2 Hz, 2H), 6.85 (t, J = 7.2 Hz, 1H), 4.33 (t, J = 6.5 Hz, 4H), 3.78 (d, J = 12.8 Hz,
2H), 3.32 (d, J = 11.4 Hz, 2H), 3.27 – 3.08 (m, 6H); 13C NMR (126 MHz, DMSO-d6)
δ in ppm: 156.34, 149.28, 139.74, 134.40, 131.62, 129.58, 129.35, 127.75, 126.87,
19

126.83, 120.62, 120.41, 116.39, 113.71, 69.00, 58.18, 50.04, 45.55, 34.72; MS (ESI,
m/z): 441.2 [M+1]+; Anal. Calcd for C26H27F3N2O·2HCl: C, 60.82; H, 5.69; N, 5.46.
Found: C, 61.18; H, 5.71; N, 5.20.
4.1.4.23 1-phenyl-4-(4-(2-(4-(trifluoromethyl)phenoxy)ethyl)benzyl)piperazine
dihydrochloride (27)
White solid
; Yield: 62%; Mp 95.0–96.1 oC; 1H NMR (500 MHz,
DMSO-d6) δ in ppm: 11.54 (s, 1H, N+H), 7.63 (dd, J = 8.0, 5.3 Hz, 4H), 7.43 (d, J =
7.9 Hz, 2H), 7.25 (t, J = 7.9 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.2 Hz,
2H), 6.85 (t, J = 7.3 Hz, 1H), 4.33 (d, J = 4.8 Hz, 2H), 4.30 (t, J = 6.7 Hz, 2H), 3.36
(m, 4H), 3.25 – 3.00 (m, 6H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 161.42,
149.60, 139.76, 131.80, 129.49, 129.33, 127.84, 127.14, 127.11, 120.27, 116.21,
115.21, 68.43, 58.23, 52.28, 50.19, 45.39, 34.60; MS (ESI, m/z): 441.2 [M+1]+; Anal.
Calcd for C26H27F3N2O·2HCl: C, 60.82; H, 5.69; N, 5.46. Found: C, 55.03; H, 6.83; N,
4.70.
4.1.4.24 2-(4-((4-phenylpiperazin-1-yl)methyl)phenethoxy)benzonitrile
dihydrochloride (28)
White solid
; Yield: 74%; Mp 80.1–81.3 oC; 1H NMR (500 MHz,
DMSO-d6) δ in ppm: 11.52 (s, 1H, N+H), 7.71 (dd, J = 7.7, 1.5 Hz, 1H), 7.66 –7.57
(m, 3H), 7.46 (d, J = 8.0 Hz, 2H), 7.32 – 7.20 (m, 3H), 7.08 (t, J = 7.8 Hz, 1H), 6.97
(d, J = 8.0 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 4.37 (t, J = 6.6 Hz, 2H), 4.33 (d, J = 4.8
Hz, 2H), 3.78 (d, J = 12.9 Hz, 2H), 3.34 (d, J = 11.5 Hz, 2H), 3.21 (t, J = 12.4 Hz,
2H), 3.12 (t, J = 6.6 Hz, 4H); 13C NMR (126 MHz, DMSO-d6) δ in ppm: 160.90,
160.62, 150.11, 140.12, 135.77, 135.28, 134.32, 133.82, 132.17, 130.19, 129.79,
128.32, 121.77, 120.70, 120.11, 117.01, 116.88, 116.67, 113.73, 101.20, 69.63, 58.76,
50.70, 45.88, 35.05; MS (ESI, m/z): 398.2 [M+1]+; Anal. Calcd for C26H27N3O·2HCl:
C, 66.38; H, 6.21; N, 8.93. Found: C, 65.99; H, 6.08; N, 8.92.
4.1.4.25 4-(4-((4-phenylpiperazin-1-yl)methyl)phenethoxy)benzonitrile
dihydrochloride (29)
White solid
; Yield: 67%; Mp 93.2–93.7 oC; 1H NMR (500 MHz,
DMSO-d6) δ in ppm: 11.37 (s, 1H, N+H), 7.75 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.0 Hz,
2H), 7.42 (d, J = 8.0 Hz, 2H), 7.25 (t, J = 8.0 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 6.96
(d, J = 8.0 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1H), 4.33 (t, J = 6.5 Hz, 4H), 3.78 (d, J = 11.8
Hz, 2H), 3.33 (d, J = 10.8 Hz, 2H), 3.26 – 2.94 (m, 6H); 13C NMR (126 MHz,
DMSO-d6) δ in ppm: 162.33, 150.03, 139.94, 134.68, 132.07, 129.78, 129.59, 128.15,
120.37, 119.61, 116.40, 116.10, 103.30, 68.81, 58.55, 52.54, 50.53, 45.59, 34.82; MS
20

(ESI, m/z): 398.2 [M+1]+; Anal. Calcd for C26H27N3O·2HCl: C, 66.38; H, 6.21; N,
8.93. Found: C, 66.28; H, 6.37; N, 8.71.
4.1.4.26 3-fluoro-4-(4-((4-phenylpiperazin-1-yl)methyl)phenethoxy)benzonitrile
dihydrochloride (30)
White solid
; Yield: 59%; Mp 96.9–98.0 oC; 1H NMR(500 MHz,
DMSO-d6) δ in ppm: 11.54 (s, 1H, N+H), 7.81 (dd, J = 11.3, 2.0 Hz, 1H), 7.64 (dd, J
= 8.6, 1.6 Hz, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 8.2
Hz, 1H), 7.23 (dd, J = 8.6, 7.6 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 6.83 (t, J = 7.3 Hz,
1H), 4.39 (t, J = 6.7 Hz, 2H), 4.31 (d, J = 4.8 Hz, 2H), 3.76 (d, J = 12.9 Hz, 2H), 3.30
(d, J = 11.5 Hz, 2H), 3.19 (t, J = 12.5 Hz, 2H), 3.10 (t, J = 6.7 Hz, 4H); 13C NMR
(126 MHz, DMSO-d6) δ in ppm: 152.39, 151.17, 151.09, 150.43, 149.15, 139.74,
132.19, 131.00, 130.98, 129.90, 129.83, 128.26, 121.66, 120.31, 120.14, 118.64,
117.11, 116.09, 103.35, 103.28, 69.86, 58.55, 50.34, 46.28, 34.83; MS (ESI, m/z):
416.2 [M+1]+; Anal. Calcd for C26H26FN3O·2HCl: C, 63.94; H, 5.78; N, 8.60. Found:
C, 63.76; H, 5.81; N, 8.29.
4.1.4.27 1-(4-(2-(4-nitrophenoxy)ethyl)benzyl)-4-phenylpiperazine dihydrochloride
(31)
White solid
; Yield: 66%; Mp 90.1–91.1 oC; 1H NMR (500 MHz,
DMSO-d6) δ in ppm: 11.45 (s, 1H, N+H), 8.13 (d, J = 9.2 Hz, 2H), 7.56 (d, J = 8.0 Hz,
2H), 7.37 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.9 Hz, 2H), 7.10 (d, J = 9.2 Hz, 2H), 6.91
(d, J = 8.0 Hz, 2H), 6.79 (t, J = 7.3 Hz, 1H), 4.33 (t, J = 6.6 Hz, 2H), 4.28 (d, J = 4.8
Hz, 2H), 3.72 (d, J = 12.5 Hz, 2H), 3.27 (d, J = 11.1 Hz, 2H), 3.17-3.17 (m, 4H), 3.06
13
(t, J = 6.6 Hz, 2H)； C NMR (126 MHz, DMSO-d6) δ 164.17, 149.55, 141.28, 139.81,
132.10, 129.79, 129.66, 128.17, 126.35, 120.95, 116.70, 115.56, 69.34, 58.48, 50.37,
45.86, 34.79; MS (ESI, m/z): 418.2 [M+1]+; Anal. Calcd for C25H27N3O3·2HCl: C,
61.23; H, 5.96; N, 8.57. Found: C, 60.90; H, 5.96; N, 8.25.
4.2 Biological evaluation
4.2.1 In Vitro Cytotoxic Assay
4.2.1.1 Cell Culture
PC-3 and RWPE-1 cells were cultured in Dulbecco’s modification Eagle’s
medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 0.1 mg/mL
streptomycin (Invitrogen). DU145 cells were cultured in RPMI1640 media
supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin and
0.1 mg/mL streptomycin (Invitrogen). LNCaP cells were cultured in F12 media
21

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
humidified atmosphere with 5% CO2.
4.2.1.2 Assessment of Antitumor Activity by CCK-8 Assay
Cell proliferation 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 humidified incubator (5% CO2) for 24 h, followed 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 absorbance (Abs) at λ = 450 nm using amicroplate reader. Three independent
experiments were performed. Cell growth inhibition was calculated according to the
following equation:
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 concentration-response curves plotted for each tested compound.
4.2.2 Antagonistic Activity in a1-ARs by Dual-luciferase Reporter Gene Assay
Firefly and Renilla luciferase activities, 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, Promega) and are reported as the mean ± SEM of three
individual experiments. For agonists, fold of induction = LUinduced/RLUuninduced. For
antagonists, % of control = 100 × RLU (agonist + antagonist)/RLU (agonist alone).
All RLUs were normalized against firefly RLUs/Renilla RLUs. Data are expressed as
EC50/IC50 values in μM, and the IC50 of phenylephrine (μM) was calculated by
plotting the data using nonlinear regression analysis in Graph-Pad Prism 5 software.
4.2.3 Fluorescence polarization (FP)
The fluorescence polarization techniquewas used to analyze the binding of 10, 16,
27, 29, 31 and enzalutamide to the AR using the PolarScreen™ AR Competitor Assay,
Green (lifetechnologies, A15880) according to the manufacturer's instructions. Briefly,
the assay entails titration of the test compound against a preformed complex of
Fluormone™AL Green and the AR-LBD (GST). The assay mixture was allowed to
equilibrate at room temperature in 384-well black plates for 4 h, after which the
fluorescence polarization values were measured in a SpectraMax®Paradigm®
Multi-Mode Detection Platform (Molecular Devices) using an excitation wavelength
of 485 nm and an emission wavelength of 535 nm. Data analysis for the ligand
binding assays was performed using Prism software (GraphPad Software, Inc.).
22

4.3 Molecular docking simulation
Until now, three binding sites of androgen receptor have been reported, including
LBP, AF2 and BF3. In order to explore the mechanism of androgen receptor
antagonism by the target compound 16, a docking simulation was carried out using
AutoDock Vina software.58 The crystal structure of androgen receptor downloaded
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do)59 was
taken as the template protein to engage in docking simulation. In prepare, the
exogenous ligand was removed and the hydrogen atoms were added to the system. To
ensure the reliability of docking simulation, a redock process for the exogenous ligand
was performed before docking analysis. Finally, one compound target (i.e. compound
16) with high AR antagonistic activity was docked into three potential binding sites
(including LBP, AF2 and BF3) with 10 configurations.
Acknowledgments
The work was supported by the Natural Science Foundation of China (No.
81401462), the Wenzhou Medical University and Wenzhou Institute of Biomaterials
& Engineering (WIBEZD2017001-03), Henan Province Science and Technology
Attack Plan Foundation (No. 162102310477), the Science and Technology planning
Projects of Guangdong Province (No. 2016A020215175), the Natural Science
Foundation of Guangdong Province (No. 2016A030313583) and the Science and
Technology planning Project of Guangzhou (No. 201704020070).
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