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

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: DOI: Reference:

S0968-0896(18)31614-6 https://doi.org/10.1016/j.bmc.2018.11.029 BMC 14633

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: 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: [email protected] (S.-C. Zhao); [email protected] (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

Compound

PC-3a IC50 (μM)b (μM)b

DU145a IC50 (μM)b

LNCaPa IC50

5

8.2 ± 0.14

9.82 ± 0.82

6.67 ± 0.06

>50

40.6 ± 1.2

6

4.72 ± 0.21

15.95 ± 0.23

8.92 ± 0.05

12.15 ± 0.07

32.5 ± 3.2

7

17.71 ± 0.15

12.15 ± 0.13

19.28 ± 0.12

27.84 ± 0.13

N.D

8

>50

40.39 ± 0.23

>50

>50

N.D

9

15.1 ± 0.16

29.17 ± 0.21

34.87 ± 0.18

>50

N.D

10

1.47 ± 0.07

10.11 ± 0.16

5.62 ± 0.03

>50

54.7 ± 0.9

11

>50

6.4 ± 0.23

>50

>50

40.2 ± 2.4

12

4.86 ± 0.17

27.97 ± 0.21

17.92 ± 0.14

21.09 ± 0.13

N.D

13

6.72 ± 0.17

15.61 ± 0.23

17.59 ± 0.14

12.26 ± 0.14

N.D

14

2.99 ± 0.14

3.44 ± 0.13

7.43 ± 0.14

4.86 ± 0.12

16.7 ± 2.6

15

>50

>50

>50

>50

N.D

16

8.95 ± 0.13

9.79 ± 0.46

7.46 ± 0.02

>50

67.1 ± 1.4

17

>50

14.23 ± 0.15

>50

>50

N.D

18

48.83 ± 0.13

7.21 ± 0.20

17.82 ± 0.16

19.67 ± 0.14

N.D

19

5.7 ± 0.05

6.86 ± 0.14

7.83 ± 0.13

4.62 ± 0.08

35.1 ± 0.8

20

>50

38.65 ± 0.23

>50

>50

N.Dd

21

3.59 ± 0.16

9.2 ± 0.12

8.64 ± 0.13

4.76 ± 0.11

32.4 ± 1.6

22

22.96 ± 0.07

5.36 ± 0.13

19.84 ± 0.14

>50

41.1 ± 2.2

23

10.1 ± 0.08

13.15 ± 0.25

19.45 ± 0.15

47.21 ± 0.17

N.D

5

RWPE-1a IC50 (μM)b

AR antagonistic activity % (10 μM)c

24

>50

11.58 ± 0.17

41.87 ± 0.32

>50

N.D

25

6.69 ± 0.14

10.07 ± 0.23

13.47 ± 0.09

15.65 ± 0.13

N.D

26

>50

9.43 ± 0.15

32.16 ± 0.18

>50

N.D

27

4.96 ± 0.14

4.81 ± 0.14

8.76 ± 0.13

13.15 ± 0.12

48.2 ± 0.8

28

>50

2.51 ± 0.25

>50

>50

32.1 ± 1.7

29

1.05 ± 0.04

46.24 ± 0.24

43.58 ± 0.13

>50

47.2 ± 1.8

30

>50

0.77 ± 0.11

>50

>50

24.5 ± 2.1

31

9.23 ± 0.05

8.74 ± 0.57

8.51 ± 0.15

3.87 ± 0.14

50.1 ± 1.3

Naftopidil

42.10 ± 0.79

34.58 ± 0.31

22.36 ± 0.61

>50

N.D

Finasteride

17.80

14.53

13.53

N.D

N.D

R1881

N.D

N.D

N.D

N.D

N.E

Enzalutamide

N.D N.D N.D = not determined. N.E = no antagonistic effect.

N.D

N.D

84.7 ± 1.4

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 IC50/µMa

Compound

3.66 ± 0.78 10 1.15 ± 0.34 16 4.03 ± 0.85 27 4.22 ± 1.01 29 3.82 ± 1.09 31 Enzalutamide 1.32 ± 0.78 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)

-8.2

AF2 (PDB ID: 2YHD)

-6.0

BF3 (PDB ID: 2YLO)

-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 . o 1 Yield: 70% from compound 1; Mp 64.2–64.6 C; H 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); 13 C 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 (t, J = 6.6 Hz, 2H)；13C 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). References 1. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J, Rosso S, Coebergh JW, Comber H, Forman D, Bray F. Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer. 2013;49:1374–1403. 2. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29. 3. Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin. 2002;52:154–179. 4. Dorff TB, Glode LM. Current role of neoadjuvant and adjuvant systemic therapy for high-risk localized prostate cancer. Curr Opin Urol. 2013;23:366–371. 5. Loblaw DA, Walker-Dilks C, Winquist E, Hotte SJ, G.C.D.S.G. of C.C.O.P., in: E.-B. Care (Ed.). Systemic therapy in men with metastatic castration-resistant prostate cancer: a systematic review. Clin Oncol (R Coll Riodiol). 2013;25:406–430. 6. Han JY, Zhu FQ, Xu X, Huang H, Huang WQ, Cui WH, Dai H, Jiang JX, Wang SL. Tetramethylpyrazine hydrochloride inhibits proliferation and apoptosis in human prostate cancer PC3 cells through Akt signaling pathway. J Third Mil Med Univ. 2013;35:105–108. 23

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Highlights 1. A novel of arylpiperazine derivatives were synthesized. 2. Antagonistic potency of derivatives were investigated against AR. 3. The anti-prostate cancer activities of derivatives was also investigated. 4. Some derivatives exhibited strong activities against AR and cancer cells. 5. Molecular docking and SAR of derivatives were also studied.