Synthesis and biological evaluation of estrone 3-O-ether derivatives containing the piperazine moiety

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Synthesis and biological evaluation of estrone 3-O-ether derivatives containing the piperazine moiety Hong Chena,1, Xue Liangb,1, Tao Suna, Xiaoguang Qiaoa, Zhan Zhoua, Ziyong Lia, Chaojun Hea, ⁎ ⁎ Huiyuan Yaa, , Mu Yuanc, a

College of Food and Drug, Luoyang Normal University, 6# Jiqing Road, Luoyang 471934, Henan Province, China The Fifth Affiliated Hospital of Guangzhou Medical University, 621# Gangwan Road, Guangzhou 510700, Guangdong Province, China c Pharmaceutical Research Center, Guangzhou Medical University, 195# Dongfengxi Road, Guangzhou 511436, Guangdong Province, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Synthesis Estrone derivatives Prostate cancer Piperazines Structure-activity relationship

A series of new estrone derivatives were designed and synthesized, and their structures were confirmed by spectroscopic methods. All new estrone derivatives were investigated for their in vitro cytotoxic efficacies against a panel of three human prostate cancer cell lines (PC-3, LNCaP, and DU145). The derivatives 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 showed important cytotoxic actions against individual carcinoma cell line collections. Moreover, antagonistic activities of compounds (7, 15, 16 and 21) towards a1-ARs (α1A, α1B, and α1D) were further evaluated using dual-luciferase reporter assays, and the compounds 16 and 21 exhibited better a1-ARs subtype selectivity. The structure–activity relationship (SAR) suggested that the substitute’s type and position on the phenyl group leads to the interesting variations within pharmacological effects of resultant molecular systems.

1. Introduction Prostate cancer is the most common non-skin cancer in men and the second-leading cause of cancer-related deaths in the US [1]. Although current therapies (radical prostatectomy, chemotherapy, local radiotherapy, or hormonotherapy) are effective against prostate cancer at their early stages, upon the onset of prostate cancer metastasis no significantly effective therapies exist [2–5], and androgen ablation therapy has been the major therapeutic modality for advanced prostate cancer [6]. Therefore, developing novel anti-prostate cancer drugs that are effective against the progression of prostate cancer at later stages is now urgently required. Steroids are a biologically important class of natural compounds with a large range of pharmacological activities, such as inotropic, antihypercholesterolemic, anti-inflammatory and antioxidant properties [7–11], and the anticancer capacity is one of the most intensively investigated characters of natural steroid products and synthetic analogs. Moreover, steroids as well as their derivatives have been found to have the potential application as drugs for treating a large number of diseases, including cardiovascular [12], auto immune diseases [13], brain tumors, breast cancer, prostate cancer, osteoarthritis, etc. [14]. The promise of using steroids for development of lead molecules relies

⁎

1

on their regulation of a variety of biological processes and being a fundamental class of signaling molecules [15]. Estrone (Fig. 1), one of the three naturally occurring estrogens, is of interest for the treatment and prevention of breast cancer [16–18], and various types of substituted estrone analogs or homoestrones have been reported as antiprostate cancer agents [19–23], and cytotoxic or cytostatic (antiproliferative) anticancer agents to treat cancer [24–27]. Thus, the modification of estrone structure is very important for developing new anticancer agents. 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 [28]. Moreover, piperazine derivatives hold a spectrum of biological importance, such as antiarrhythmic [29], diuretic [30], antiallergic [31], antidepressant [32], anxiolytic [33], antipsychotic [34], antimalarial [35], antiplasmodial [36], receptor-blocking properties [37–41] and anti-proliferative properties [42–49]. In our previous studies, the compounds with piperazine moiety have been reported as anticancer drugs for the site-directed chemotherapy of prostate cancer [50,51]. Above results inspired our current hypothesis that the introduction of piperazine moiety into the estrone skeleton may favour the biological activity of estrone derivatives. Herein, we designed and

Corresponding authors. E-mail addresses: [email protected] (H. Ya), [email protected] (M. Yuan). These authors contributed equally to this work.

https://doi.org/10.1016/j.steroids.2018.02.002 Received 6 December 2017; Received in revised form 25 January 2018; Accepted 9 February 2018 0039-128X/ © 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Chen, H., Steroids (2018), https://doi.org/10.1016/j.steroids.2018.02.002

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recorded on SGW X-4 apparatus and are uncorrected. NMR spectra were obtained by using a Bruker AVANCE-500 apparatus in CDCl3 using TMS as internal standard, and chemical shifts (δ) are expressed in parts per million (ppm), the coupling constants (J) are expressed in hertz (Hz). ESI mass spectra were recorded on an Agilent 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA), and HRMS spectra were recorded on the AB Sciex 5600 Triple TOF mass spectrometer (Foster, CA, USA). Flash column chromatography was performed with silica gel (Qing Dao Ocean Chemical Factory, 300–400 mesh) eluted with petroleum ether–ethyl acetate.

Fig. 1. Structures of estrone.

synthesized a series of new estrone 3-O-ether derivatives containing the piperazine moiety. All the title compounds were bioassayed against three prostate cancer cell lines (PC-3, LNCaP and DU145) and one normal prostate epithelial cell line (WPMY-1). Furthermore, antagonistic activities of representative compounds towards a1-adrenergic receptors (a1-ARs) were further evaluated using dual-luciferase reporter assays. A simple SAR study was also explored to facilitate the further development of the estrone derivatives. As we expected, some compounds exhibited strong anti-cancer activities against the tested cancer cells and superior potency than finasteride.

2.1.1. Synthesis of 3-O-(4-(2-hydroxyethyl)benzyl) estrone (2) Compound 2 was synthesized using methods reported previously in the literature [50]. White solid. Yield: 85%; Mp 114–115 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.38 (d, J = 7.6 Hz, 2H), 7.25 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 5.01 (s, 2H), 3.86 (d, J = 5.3 Hz, 2H), 2.88 (t, J = 6.3 Hz, 4H), 2.61 – 2.33 (m, 2H), 2.30 – 1.86 (m, 5H), 1.70 – 1.34 (m, 7H), 0.91 (s, 3H); MS (ESI, m/z): 405.1 [M + 1]+.

2. Experimental section 2.1.2. Synthesis of 3-O-(4-methyl)phenethyl 4-methylbenzenesulfonate estrone (3) Compound 3 was synthesized using methods reported previously in the literature [50]. White solid. Yield: 90%; Mp 85–86 °C; 1H NMR (500 MHz, CDCl3) δ in ppm 7.68 (d, J = 8.0 Hz, 2H), 7.31 (d,

2.1. Chemistry Reagents and solvents were commercially available. The organic solvents were distilled before used. The melting points (Mp) were

Scheme 1. Synthesis of estrone 3-O-ether derivatives. Reagents and conditions: (a) Estrone, K2CO3, CH3CN, 85 °C, 16 h; (b) TsCl, Et3N and 4-dimethylaminopyridine, Cl2CH2, 0 °C, 16 h; (c) Arylpiperazines, K2CO3, CH3CN, 85 °C, 16 h; (d) Piperidines, K2CO3, CH3CN, reflux, 16 h.

2

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in ppm: 7.38 – 7.29 (m, 7H), 7.21 (d, J = 8.6 Hz, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.78 (dd, J = 8.6, 2.6 Hz, 1H), 6.72 (d, J = 2.6 Hz, 1H), 4.99 (s, 2H), 3.53 (s, 2H), 3.03 – 2.71 (m, 4H), 2.72 – 2.33 (m, 12H), 2.33 – 1.91 (m, 5H), 1.76 – 1.32 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.32, 157.30, 140.48, 138.44, 138.17, 135.34, 132.66, 129.62, 129.29, 128.61, 128.07, 127.45, 126.73, 115.26, 112.76, 70.21, 63.46, 60.84, 53.57, 53.43, 50.81, 48.40, 44.39, 38.75, 36.27, 33.70, 31.98, 30.10, 30.05, 26.94, 26.31, 21.98, 14.25; MS (ESI, m/z): 563.0 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O2, 563.3632, found, 563.3627.

Table 1 In vitro cytotoxicity of compounds 4–26. Compd.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Finasteride

IC50 (μM)a PC-3b

LNCaPb

DU145b

WPMY-1b

> 50 18.15 ± 1.12 > 50 15.13 ± 0.32 > 50 3.41 ± 0.21 12.32 ± 0.59 32.62 ± 0.12 > 50 5.46 ± 0.13 48.49 ± 0.12 31.58 ± 0.07 14.14 ± 0.80 > 50 12.19 ± 0.23 > 50 12.32 ± 1.87 > 50 35.18 ± 1.27 23.24 ± 1.15 > 50 31.58 ± 0.07 24.17 ± 1.23 17.83

8.15 ± 0.24 > 50 1.42 ± 0.15 0.83 ± 0.13 15.83 ± 1.83 > 50 1.28 ± 0.41 7.28 ± 1.21 10.21 ± 1.22 > 50 9.22 ± 1.42 1.08 ± 0.72 > 50 > 50 > 50 9.28 ± 1.27 1.28 ± 0.13 0.78 ± 0.34 17.84 ± 0.52 > 50 > 50 10.11 ± 0.93 1.42 ± 1.02 14.53

7.57 ± 0.14 > 50 2.12 ± 0.17 0.55 ± 0.25 > 50 > 50 2.31 ± 0.61 41.12 ± 1.34 > 50 > 50 10.38 ± 0.14 0.94 ± 0.10 1.09 ± 0.16 > 50 10.05 ± 2.36 41.12 ± 1.31 2.05 ± 0.46 > 50 1.13 ± 0.27 > 50 2.12 ± 0.63 > 50 2.53 ± 1.09 13.53

> 50 NDc > 50 > 50 > 50 NDc > 50 NDc NDc 42.35 ± 0.34 32.24 ± 1.54 > 50 > 50 NDc NDc NDc > 50 > 50 > 50 NDc > 50 > 50 > 50 –

2.1.3.3. 3-O-(4-(2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzyl) estrone (6). White solid; Yield: 72%; Mp 135–136 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 8.27 – 8.14 (m, 1H), 7.48 (ddd, J = 8.6, 7.0, 2.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 6.66 (d, J = 8.6 Hz, 1H), 6.63 (dd, J = 7.0, 5.0 Hz, 1H), 5.00 (s, 2H), 3.59 (t, J = 4.8 Hz, 4H), 3.00 – 2.80 (m, 4H), 2.68 – 2.65 (m, 6H), 2.60 – 2.35 (m, 2H), 2.33 – 1.87 (m, 5H), 1.78 – 1.34 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.63, 159.25, 156.63, 147.71, 139.65, 137.51, 137.18, 134.76, 132.01, 128.65, 127.44, 126.07, 114.59, 113.08, 112.09, 106.80, 69.54, 60.18, 52.75, 50.14, 47.73, 44.92, 43.72, 38.08, 35.60, 32.99, 31.31, 29.43, 29.39, 26.27, 25.64, 21.31, 13.58; MS (ESI, m/z): 550.0 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C36H44N3O2, 550.3428, found, 550.3424. 2.1.3.4. 3-O-(4-(2-(4-(o-tolyl)piperazin-1-yl)ethyl)benzyl) estrone (7). White solid; Yield: 82%; Mp 140–141 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 1H NMR (500 MHz, CDCl3) δ 7.36 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.98 – 6.87 (m, 4H), 6.78 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.16 (t, J = 4.8 Hz, 4H), 2.91 – 2.84 (m, 4H), 2.79 – 2.61 (m, 6H), 2.58 – 2.33 (m, 2H), 2.22 – 1.87 (m, 5H), 1.67 – 1.39 (m, 6H), 1.25 (s, 3H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.04, 158.28, 157.04, 156.38, 148.10, 140.06, 137.94, 135.18, 132.44, 129.05, 127.85, 126.49, 118.00, 117.94, 115.74, 115.56, 115.00, 112.50, 69.95, 60.47, 53.35, 50.56, 50.30, 48.14, 44.14, 38.50, 36.01, 33.43, 31.73, 29.84, 29.80, 26.68, 26.06, 21.73, 13.99; MS (ESI, m/z): 563.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O2, 563.3632, found, 563.3627.

a

IC50 values are taken as means ± standard deviation from three experiments. PC-3, androgen-insensitive human prostate cancer cell line; LNCaP, androgen-sensitive human prostate cancer cell line; DU145, androgen-insensitive human prostate cancer cell line; WPMY-1, normal non-cancer human prostate epithelial cell line. c ND = not determined. b

J = 7.7 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 7.7 Hz, 2H), 6.78 (d, J = 8.3 Hz, 1H), 6.72 (s, 1H), 4.99 (s, 2H), 4.21 (t, J = 6.9 Hz, 2H), 3.02 – 2.83 (m, 4H), 2.50 – 2.46 (m, 2H), 2.43 (s, 3H), 2.31 – 1.89 (m, 5H), 1.75 – 1.33 (m, 6H), 0.91 (s, 3H); MS (ESI, m/z): 559.0 [M + 1]+. 2.1.3. General procedure for the preparation of estrone 3-O-ether derivatives 4–26 A mixture of 3 (100 mg, 0.28 mmol), arylpiperazines or piperidines, K2CO3, (1.2 equiv), potassium carbonate (6.0 equiv) and CH3CN (25 mL) were placed in 50-mL round-bottomed flask equipped with magnetic stirrer for 14 h at 85 °C (monitored by TLC). After completion of reaction, 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 = 5:1, v/v) to obtain the corresponding products (4–26), and all compounds were recrystallized from dichloromethane and n-hexane.

2.1.3.5. 3-O-(4-(2-(4-(p-tolyl)piperazin-1-yl)ethyl)benzyl) estrone (8). White solid; Yield: 80%; Mp 118–119 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 7.6 Hz, 2H), 7.25 (d, J = 7.36 Hz, 2H), 7.20 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H), 5.00 (s, 2H), 3.19 (br s, 4H), 2.98 – 2.77 (m, 4H), 2.67 (dd, J = 15.7, 6.0 Hz, 6H), 2.58 – 2.34 (m, 2H), 2.28 (s, 3H), 2.20 – 1.90 (m, 5H), 1.77 – 1.36 (m, 6H), 0.91 (s, 3H); MS (ESI, m/z): 563.2 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O2, 563.3632, found, 563.3633.

2.1.3.1. 3-O-(4-(2-(4-phenylpiperazin-1-yl)ethyl)benzyl) estrone (4). White solid. Yield: 75%; Mp 152–153 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.25 – 7.22 (m, 4H), 7.20 (d, J = 8.6 Hz, 1H), 6.94 (d, J = 8.0 Hz, 2H), 6.86 (t, J = 7.4 Hz, 1H), 6.78 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.24 (t, J = 4.8 Hz, 4H), 2.97 – 2.79 (m, 4H), 2.76 – 2.55 (m, 6H), 2.55 – 2.34 (m, 2H), 2.30 – 1.88 (m, 5H), 1.70 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.06, 157.04, 151.43, 140.12, 137.93, 135.16, 132.43, 129.25, 129.06, 127.85, 126.48, 119.88, 116.21, 115.01, 112.50, 69.95, 60.55, 53.37, 50.55, 49.29, 48.15, 44.14, 38.50, 36.01, 33.45, 31.72, 29.85, 29.80, 26.69, 26.05, 21.73, 13.99; MS (ESI, m/z): 549.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H45N2O2, 549.3476, found, 549.3473.

2.1.3.6. 3-O-(4-(2-(4-(2,5-dimethylphenyl)piperazin-1-yl)ethyl)benzyl) estrone (9). White solid; Yield: 75%; Mp 117–118 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.7 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 6.85 (s, 1H), 6.80 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.74 (s, 1H), 5.01 (s, 2H), 2.97 (br s, 4H), 2.94 – 2.81 (m, 4H), 2.68 (dd, J = 9.5, 6.1 Hz, 6H), 2.54 – 2.38 (m, 2H), 2.31 (s, 3H), 2.27 (s, 3H), 2.21 – 1.90 (m, 5H), 1.72 – 1.36 (m, 6H), 0.91 (s, 3H); MS (ESI, m/z): 577.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C39H49N2O2, 577.3789, found, 577.3788. 2.1.3.7. 3-O-(4-(2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl)benzyl) estrone (10). White solid; Yield: 75%; Mp 152–153 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 7.16 (t, J = 7.7 Hz, 1H), 6.83 – 6.68 (m, 5H), 5.01 (s, 2H), 3.28 (br s, 4H), 2.96 – 2.86 (m, 4H),

2.1.3.2. 3-O-(4-(2-(4-benzylpiperazin-1-yl)ethyl)benzyl) estrone (5). White solid; Yield: 60%; Mp 147–148 °C (HCl salt); 1H NMR (500 MHz, CDCl3) δ 3

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Fig. 2. Estrone derivatives 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 inhibited cell viability (percent relative to control) in prostate cell lines. The tested cells were exposed to escalating concentrations of estrone derivatives for 24 h, respectively, and the cell viability was detected by CCK-8 assay.

2.76 (br s, 6H), 2.56 – 2.36 (m, 2H), 2.33 (s, 3H), 2.20 – 1.90 (m, 5H), 1.74 – 1.38 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.05, 157.02, 138.99, 137.94, 132.45, 129.13, 129.07, 127.90, 126.49, 117.21, 115.01, 113.50, 112.50, 69.92, 60.36, 53.27, 50.56,

49.12, 48.15, 44.14, 38.50, 36.01, 31.73, 29.85, 29.80, 26.68, 26.05, 21.91, 21.73, 13.99, 0.14; MS (ESI, m/z): 579.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O3, 579.3568, found, 579.3572.

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2.1.3.12. 3-O-(4-(2-(4-(4-fluorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (15). White solid; Yield: 69%; Mp 110–111 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.37 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.22 – 7.15 (m, 3H), 7.05 (d, J = 7.6 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 2.99 (t, J = 4.8 Hz, 4H), 2.97 – 2.82 (m, 4H), 2.70 – 2.67 (m, 6H), 2.53 – 2.37 (m, 2H), 2.30 – 1.88 (m, 5H), 1.79 – 1.35 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.05, 157.05, 151.59, 140.22, 137.93, 135.14, 132.74, 132.43, 131.19, 129.07, 127.86, 126.72, 126.49, 123.30, 119.15, 115.12, 115.01, 112.51, 69.97, 60.69, 53.87, 51.82, 50.56, 48.15, 44.14, 38.51, 36.01, 33.50, 31.73, 29.85, 29.81, 26.69, 26.06, 21.73, 18.02, 14.00; MS (ESI, m/z): 567.0 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H44FN2O2, 567.3381, found, 567.3377.

Table 2 Agonistic activities (IC50) on α1-ARs (α1A, α1B, and α1D) of compounds 7, 15, 16 and 21. Compd.

7 15 16 21 naftopidil a

IC50 (nM)a

Selectivity ratio

α1a

α1b

α1d

α1b/α1a

α1b/α1d

412.62 1210.62 873.85 65.30 555

577.24 987.29 1086.54 958.20 634

925.38 298.37 89.63 875.08 55.2

1.4 0.8 1.2 14.7 1.1

0.6 3.3 12.1 1.1 11.48

IC50 values are taken as means ± standard deviation from three experiments.

2.1.3.8. 3-O-(4-(2-(4-(2-ethoxyphenyl)piperazin-1-yl)ethyl)benzyl) estrone (11). White solid; Yield: 80%; Mp 111–112 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.37 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.02 – 6.88 (m, 3H), 6.86 (d, J = 7.9 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 4.08 (q, J = 7.0 Hz, 2H), 3.25 (br s, 4H), 3.05 – 2.67 (m, 10H), 2.56 – 2.32 (m, 2H), 2.32 – 1.88 (m, 5H), 1.67 – 1.49 (m, 6H), 1.46 (t, J = 7.0 Hz, 3H), 0.91 (s, 3H); 13 C NMR (126 MHz, CDCl3) δ in ppm: 220.93, 156.88, 151.52, 137.81, 132.32, 128.95, 127.79, 126.36, 121.04, 118.32, 114.88, 112.42, 112.37, 69.78, 63.56, 53.27, 50.43, 48.02, 44.01, 38.36, 35.88, 31.59, 29.71, 29.67, 26.55, 25.92, 21.59, 14.96, 13.86; MS (ESI, m/z): 593.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C39H49N2O3, 593.3738, found, 593.3735.

2.1.3.13. 3-O-(4-(2-(4-(2,4-difluorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (16). White solid; Yield: 55%; Mp 103–104 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.96 – 6.80 (m, 3H), 6.78 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.16 (t, J = 4.8 Hz, 4H), 2.91 – 2.83 (m, 4H), 2.79 – 2.61 (m, 6H), 2.57 – 2.33 (m, 2H), 2.28 – 1.86 (m, 5H), 1.78 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.92, 170.56, 156.92, 139.98, 137.81, 135.05, 132.31, 128.93, 127.73, 126.36, 119.45, 114.88, 112.38, 110.79, 110.59, 104.90, 104.69, 104.50, 69.83, 60.39, 53.26, 50.94, 50.44, 48.02, 44.02, 38.38, 35.89, 33.30, 31.60, 29.72, 29.68, 26.56, 25.93, 21.60, 13.87; MS (ESI, m/z): 585.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H43F2N2O2, 585.3287, found, 585.3287.

2.1.3.9. 3-O-(4-(2-(4-(3-methoxyphenyl)piperazin-1-yl)ethyl)benzyl) estrone (12). White solid; Yield: 76%; Mp 145–146 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 7.7 Hz, 2H), 7.26 – 7.11 (m, 4H), 6.79 (d, J = 8.0 Hz, 1H), 6.74 (s, 1H), 6.56 (d, J = 7.0 Hz, 1H), 6.48 (s, 1H), 6.43 (d, J = 8.0 Hz, 1H), 5.01 (s, 2H), 3.80 (s, 3H), 3.24 (br s, 4H), 2.96 – 2.75 (m, 4H), 2.68 – 2.61 (m, 6H), 2.59 – 2.32 (m, 2H), 2.20 – 1.88 (m, 5H), 1.76 – 1.34 (m, 6H), 0.91 (s, 3H); MS (ESI, m/ z): 579.2 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O3, 579.3581, found, 579.3577.

2.1.3.14. 3-O-3-fluoro-4-(4-(4-methyl)phenethyl)piperazin-1-yl)benzonitrile estrone (17). White solid; Yield: 47%; Mp 119–120 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.1 Hz, 3H), 7.29 (d, J = 8.1, 1H), 7.24 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.93 (t, J = 8.1 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.27 (t, J = 4.8 Hz, 4H), 2.96 – 2.75 (m, 4H), 2.69 (m, 6H), 2.61 – 2.33 (m, 2H), 2.30 – 1.81 (m, 5H), 1.72 – 1.32 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.90, 156.91, 137.83, 135.14, 132.35, 129.45, 128.91, 127.76, 126.37, 119.80, 119.60, 118.71, 114.87, 112.36, 69.81, 60.23, 52.90, 50.43, 49.61, 48.02, 44.02, 38.38, 35.88, 31.60, 29.72, 29.68, 26.56, 25.93, 21.60, 13.87; MS (ESI, m/z): 592.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H43FN3O2, 592.3334, found, 592.3330.

2.1.3.10. 3-O-(4-(2-(4-(4-methoxyphenyl)piperazin-1-yl)ethyl)benzyl) estrone (13). White solid; Yield: 69%; Mp 155–156 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.37 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 9.1 Hz, 2H), 6.85 (d, J = 9.1 Hz, 2H), 6.78 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.77 (s, 3H), 3.25 (s, 4H), 2.99 – 2.84 (m, 10H), 2.62 – 2.32 (m, 2H), 2.31 – 1.84 (m, 5H), 1.72 – 1.35 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.92, 156.87, 137.83, 132.36, 128.95, 127.86, 126.38, 118.70, 114.89, 114.52, 112.38, 69.75, 55.58, 53.05, 50.43, 48.02, 44.02, 38.37, 35.89, 31.61, 29.68, 26.56, 25.93, 21.61, 13.88; MS (ESI, m/z): 579.0 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H47N2O3, 579.3581, found, 579.3575.

2.1.3.15. 3-O-(4-(2-(4-(2-chlorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (18). White solid; Yield: 57%; Mp 103–104 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.25 – 2.19 (m, 4H), 7.07 (d, J = 7.5 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 6.74 (br s, 1H), 5.01 (s, 2H), 3.12 (br s, 4H), 2.87 – 2.2.83 (m, 4H), 2.81 – 2.60 (m, 6H), 2.58 – 2.34 (m, 2H), 2.33 – 1.88 (m, 5H), 1.77 – 1.35 (m, 6H), 0.91 (s, 3H); MS (ESI, m/z): 583.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H44ClN2O2, 583.3086, found, 583.3083.

2.1.3.11. 3-O-(4-(2-(4-(2-fluorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (14). White solid; Yield: 65%; Mp 104–105 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.12 – 6.90 (m, 4H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 3.16 (br s, 4H), 3.00 – 2.83 (m, 4H), 2.81 – 2.64 (m, 6H), 2.60 – 2.34 (m, 2H), 2.32 – 1.83 (m, 5H), 1.79 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.05, 157.05, 156.86, 154.91, 137.93, 135.18, 132.43, 129.07, 127.86, 126.49, 124.62, 124.59, 122.59, 119.09, 119.06, 116.33, 116.17, 115.12, 115.02, 112.51, 69.96, 60.54, 53.40, 50.63, 50.57, 48.15, 44.15, 38.51, 36.02, 33.38, 31.73, 29.85, 29.81, 26.69, 26.06, 21.73, 14.00; MS (ESI, m/z): 567.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H44FN2O2, 567.3381, found, 567.3378.

2.1.3.16. 3-O-(4-(2-(4-(3-chlorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (19). White solid; Yield: 62%; Mp 151–152 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 6.89 (t, J = 2.1 Hz, 1H), 6.82 – 6.77 (m, 3H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.24 ((t, J = 4.8 Hz, 4H)), 2.96 – 2.78 (m, 4H), 2.78 – 2.59 (m, 6H), 2.58 – 2.35 (m, 2H), 2.31 – 1.91 (m, 5H), 1.71 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.04, 157.05, 152.45, 137.94, 135.20, 135.11, 132.45, 130.17, 129.06, 127.86, 126.50, 119.43, 115.89, 115.01, 114.00, 112.51, 69.96, 60.45, 53.15, 50.56, 48.80, 48.15, 44.15, 38.51, 36.02, 33.43, 31.73, 29.85, 29.81, 26.69, 26.06, 21.73, 14.00; MS (ESI, m/z): 583.2 [M + 1]+; HRMS 5

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(ESI) m/z [M + 1]+: Calcd for C37H44ClN2O2, 583.3086, found, 583.3082.

J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 3.32 (t, J = 4.9 Hz, 4H), 2.91 – 1.86 (m, 4H), 2.77 – 2.62 (m, 6H), 2.59 – 2.36 (m, 2H), 2.30 – 1.86 (m, 5H), 1.72 – 1.39 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.92, 156.91, 153.28, 139.86, 137.82, 135.10, 132.34, 128.92, 127.75, 126.42, 126.37, 114.88, 114.52, 112.37, 69.82, 60.30, 52.93, 50.43, 48.02, 48.00, 44.02, 38.38, 35.88, 33.30, 31.60, 29.72, 29.68, 26.56, 25.93, 21.60, 13.87; MS (ESI, m/z): 617.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H44F3N2O2, 617.3349, found, 617.3345.

2.1.3.17. 3-O-(4-(2-(4-(4-chlorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (20). White solid; Yield: 47%; Mp 165–166 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 9.0 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 1H), 3.20 (t, J = 4.8 Hz, 4H), 2.91 – 2.83 (m, 4H), 2.77 – 2.62 (m, 6H), 2.58 – 2.33 (m, 2H), 2.33 – 1.89 (m, 5H), 1.78 – 1.34 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.92, 156.92, 149.92, 139.93, 137.82, 135.07, 132.33, 128.97, 128.93, 127.74, 126.37, 124.56, 117.24, 114.88, 112.38, 69.83, 60.34, 53.08, 50.44, 49.18, 48.03, 44.02, 38.38, 35.89, 33.32, 31.61, 29.73, 29.68, 26.57, 25.94, 21.61, 13.88; MS (ESI, m/z): 583.0 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H44ClN2O2, 583.3086, found, 583.3083.

2.1.3.22. 3-O-(4-(2-(4-methylpiperidin-1-yl)ethyl)benzyl) estrone (25). White solid; Yield: 78%; Mp 127–128 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.34 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.78 (dd, J = 8.6, 2.7 Hz, 1H), 6.72 (d, J = 2.7 Hz, 1H), 4.99 (s, 2H), 2.98 (d, J = 11.0 Hz, 2H), 2.94 – 2.82 (m, 4H), 2.64 – 2.35 (m, 4H), 2.32 – 1.91 (m, 8H), 1.72 – 1.32 (m, 10H), 0.94 (d, J = 6.3 Hz, 3H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.06, 157.07, 137.92, 135.00, 132.41, 129.06, 127.81, 126.48, 115.01, 112.51, 69.98, 61.11, 54.13, 50.57, 48.16, 44.15, 38.51, 36.02, 34.46, 33.64, 31.74, 30.93, 29.85, 29.81, 26.70, 26.06, 22.05, 21.74, 14.00; MS (ESI, m/z): 486.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C33H44NO2, 486.3367, found, 486.3363.

2.1.3.18. 3-O-(4-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethyl)benzyl) estrone (21). White solid; Yield: 45%; Mp 146–147 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.6 Hz, 1H), 7.19 – 7.11 (m, 2H), 6.98 (dd, J = 6.8, 2.7 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 3.11 (br s, 4H), 2.91 – 2.84 (m, 4H), 2.79 – 2.65 (m, 6H), 2.59 – 2.36 (m, 2H), 2.32 – 1.89 (m, 5H), 1.76 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.30, 157.30, 151.64, 140.38, 138.18, 135.41, 134.43, 132.69, 129.31, 128.11, 127.92, 127.85, 126.74, 124.99, 119.00, 115.26, 112.76, 70.21, 60.77, 53.67, 51.71, 50.81, 48.40, 44.39, 38.75, 36.26, 33.72, 31.98, 30.06, 26.94, 26.31, 21.98, 14.25; MS (ESI, m/z): 617.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H43Cl2N2O2, 617.2696, found, 617.2710.

2.1.3.23. 3-O-(4-(2-(4-phenylpiperidin-1-yl)ethyl)benzyl) estrone (26). White solid; Yield: 82%; Mp 125–126 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.36 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.26 – 7.15 (m, 6H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.73 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 3.16 (d, J = 11.0 Hz, 2H), 2.92 – 2.86 (m, 4H), 2.67 – 2.64 (m, 2H), 2.59 – 2.45 (m, 2H), 2.43 – 2.35 (m, 1H), 2.31 – 1.73 (m, 11H), 1.71 – 1.40 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 220.96, 156.95, 146.27, 140.21, 137.83, 135.00, 132.32, 129.24, 128.97, 128.74, 128.48, 128.07, 127.75, 126.91, 126.38, 126.22, 114.91, 112.41, 69.87, 60.91, 54.37, 50.46, 48.05, 44.04, 42.71, 38.40, 35.91, 33.44, 31.63, 29.75, 29.70, 26.59, 25.95, 21.63, 13.90; MS (ESI, m/z): 548.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H46NO2, 548.3523, found, 548.3524.

2.1.3.19. 3-O-(4-(2-(4-(4-bromophenyl)piperazin-1-yl)ethyl)benzyl) estrone (22). White solid; Yield: 55%; Mp 165–166 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.38 – 7.32 (m, 4H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.83 – 6.77 (m, 3H), 6.73 (d, J = 2.7 Hz, 1H), 5.00 (s, 2H), 3.32 (t, J = 4.8 Hz, 4H), 2.91 – 2.83 (m, 4H), 2.77 – 2.57 (m, 6H), 2.58 – 2.35 (m, 2H), 2.32 – 1.88 (m, 5H), 1.73 – 1.39 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.05, 157.04, 150.44, 140.04, 137.94, 135.20, 132.45, 132.00, 129.05, 127.86, 126.49, 117.76, 115.01, 112.50, 111.96, 69.95, 60.45, 53.17, 50.56, 49.13, 48.15, 44.14, 38.50, 36.01, 33.44, 31.73, 29.85, 29.81, 26.69, 26.06, 21.73, 14.00; MS (ESI, m/z): 627.1 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C37H44BrN2O2, 627.2581, found, 627.2576.

2.2. In vitro cytotoxic assay 2.2.1. Cell culture PC-3 and WPMY-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 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.

2.1.3.20. 3-O-(4-(2-(4-(2-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) benzyl) estrone (23). White solid; Yield: 65%; Mp 137–138 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.63 (d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 7.6 Hz, 1H), 7.21 (d, J = 8.6 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 5.01 (s, 2H), 3.01 (t, J = 4.8 Hz, 4H), 2.97 – 2.82 (m, 4H), 2.70 – 2.67 (m, 6H), 2.61 – 2.35 (m, 2H), 2.33 – 1.88 (m, 5H), 1.78 – 1.36 (m, 6H), 0.91 (s, 3H); 13C NMR (126 MHz, CDCl3) δ in ppm: 221.31, 157.31, 152.95, 140.43, 138.19, 135.39, 133.13, 132.68, 129.32, 128.12, 127.61, 127.56, 126.74, 125.18, 124.42, 115.27, 112.77, 70.22, 60.88, 53.94, 53.81, 50.82, 48.41, 44.40, 38.76, 36.27, 33.74, 31.99, 30.10, 30.06, 26.95, 26.31, 21.98, 14.25; MS (ESI, m/z): 617.2 [M + 1]+; HRMS (ESI) m/z [M + 1]+: Calcd for C38H44F3N2O2, 617.3349, found, 617.3347.

2.2.2. Assessment of antitumor activity by CCK-8 assay Cell proliferation was measured with the Cell Counting Kit-8 (CCK8) 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 a microplate 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%

2.1.3.21. 3-O-(4-(2-(4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)ethyl) benzyl) estrone (24). White solid; Yield: 72%; Mp 148–149 °C; 1H NMR (500 MHz, CDCl3) δ in ppm: 7.49 (d, J = 8.7 Hz, 2H), 7.36 (d, 6

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(2-OCH3) and 13 (4-OCH3) against LNCaP and DU145 cells. Namely, the o-substituted phenyl group derivatives showed better activity for LNCaP and DU145 cells than did the p-substituted group for LNCaP and DU145 cells. However, the compounds with electron-withdrawing groups on the phenyl group showed another rule, for instance, the cytotoxic activities of compounds 14 (2-F) and 15 (4-F) against LNCaP and DU145 cells could be placed as following: 14 (IC50 = 9.22 μM and 10.38 μM, respectively) < 15 (IC50 = 1.08 μM and 0.94 μM, respectively). The similar results were also found in compounds 18 (2-Cl) vs. 20 (4-Cl). In addition, compounds 22 (IC50 = 1.13 μM) and 24 (IC50 = 2.12 μM) with a substituent at the 4-position on the phenyl group also exhibited potent anticancer activity against DU145 cells. Interestingly, dimethyl-substituted phenyl compound 9 displayed potent activity against PC-3 cells, whereas, dichloro-substituted phenyl compound 21 showed strong cytotoxic activities against LNCaP cells. Compounds with difluoro-substituents on the phenyl showed excellent cytotoxic activities. For example, compound 16 (2,4-F2) exhibited excellent cytotoxic activities against DU145 cells. Besides, compound 16 also exhibited excellent selective activity for DU145 cells over other tested cancer cells, and displayed low cytotoxic character toward normal human prostate epithelial cell (WPMY-1) with > 50 μM of IC50. Piperidine derivatives 25 and 26 (Scheme 1) were synthesized to compare the anti-cancer activity of estrone derivatives containing the piperazine moiety. As shown in Table 1, compounds 26 exhibited strong cytotoxic activities against LNCaP and DU145 cells. These results suggest that the introduction of phenyl piperidine ring was beneficial for anti-cancer activity. Taken together, the o-substituted phenyl group derivatives and p-substituted phenyl group derivatives displayed improved cytotoxic activity against LNCaP and/or DU145 cells. The studies of SAR can lead to a tool which can facilitate further rational in drug designing.

The half maximal inhibitory concentrations (IC50) were obtained from liner regression analysis of the concentration-response curves plotted for each tested compound. 2.3. 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. 3. Results and discussion 3.1. Chemistry The synthetic pathways to this series of target compounds are shown in Scheme 1. Nucleophilic substitution of 1 with estrone in the presence of potassium carbonate (K2CO3) in acetonitrile (CH3CN) gave 2 (85% yield). Subsequently, compound 2 was treated with 4-toluenesulfonyl chloride in the presence of triethylamine and a catalytic amount of 4-dimethylaminopyridine at 0 °C for 16 h to obtain compound 3 (90% yield). Finally, the desired compounds 4–26 were produced by reacting 4 with various arylpiperazines or piperidines in the presence of K2CO3 in CH3CN at 85 °C with 45–82% yield. All the target structures were confirmed by 1H NMR, 13C NMR, MS and HRMS.

3.2.2. Antagonistic activity in a1-ARs (α1A, α1B, and α1D) Many studies have shown that arylpiperazine derivatives may act as potential α1A-AR and/or α1A-AR + α1D-AR selective ligands for the treatment of benign prostatic hyperplasia (BPH) [53–57]. So, in this work, we further evaluated antagonistic activities of estrone derivatives with the arylpiperazine moiety (7, 15, 16 and 21) towards a1-ARs (Table 2) using dual-luciferase reporter assays [53,58] to identify a1-AR subselective antagonist candidates to treat BPH from estrone derivatives. As shown in Table 2, compound 16 (2,4-F2) exhibited excellent cytotoxic activities against DU145 cells (IC50 = 1.09 μM), and demonstrated better a1D subtype selectivity over a1B (a1B/a1D ratio = 12.1). Whereas, dichloro-substituted phenyl compound 21 (IC50 = 0.78 μM) showed strong cytotoxic activities against LNCaP cells, and exhibited better a1A subtype selectivity over a1B (a1B/a1A ratio = 14.7).

3.2. Biological evaluation 3.2.1. Antitumor activity and structure–activity relationship (SAR) analysis All the target compounds were screened for in vitro cytotoxicity against a panel of three human prostate cancer cell lines including PC-3, LNCaP, and DU145 in comparison to their effects in normal non-cancer human prostate epithelial WPMY-1 cell line using the CCK-8 assay [50]. Finasteride [52] were taken as reference compounds and the results are reported in terms of IC50 values. The results are summarized in Table 1. As shown in Table 1, compounds 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 exhibited strong cytotoxic activities against LNCaP and/or DU145 cells (Fig. 2), and possessed higher activities than finasteride (IC50 < 3 μM). Among these compounds, compound 7 exhibited the potent activity against LNCaP and DU145 cells with IC50 values of 0.83 and 0.55 μM, which were 17- and 25-fold more active than finasteride, respectively. For DU145 cells, compound 15 (IC50 = 0.94 μM) is 14 folds more active than finasteride, and compound 21 displayed 18-fold more active than finasteride against LNCaP cells. Moreover, these compounds exhibited low cytotoxic character toward normal human prostate epithelial cell (WPMY-1) with > 50 μM of IC50. Taking compound 4 as a lead, the SAR investigation was mainly focused on the variation of phenyl group at the 4-position of piperazine ring with other aryl group and the substitute’s type and position on the phenyl group as a required group for antitumor activity. Firstly, in replacing the phenyl group at the 4-position of piperazine ring with pyridinyl, the resultant compound 6 displayed improved cytotoxic activity against LNCaP and DU145 cells with the IC50 value of 1.42 and 2.21 μM, respectively. The position of the substituent on the phenyl also affected the anticancer activities. For instance, the compounds with a methyl substituent at the 2-position on the phenyl group (7) exhibited higher anticancer activity than compounds with a methyl substituent at the 4-position on the phenyl group (8) against LNCaP and DU145 cells. Also, the same order of antitumor activity was observed upon screening the compounds 10

4. Conclusion This paper has reported the synthesis of a novel class of estrone derivatives containing the piperazine moiety and their antitumor activities against several classical prostate cancer cell lines including PC3, LNCaP, and DU145, as well as normal non-cancer human prostate epithelial cell line (WPMY-1). The results showed that compounds 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 exhibited strong cytotoxic activities against LNCaP and/or DU145 cells, and possessed higher activities than finasteride (IC50 < 3 μM). Moreover, compounds 16 (a1B/a1D ratio = 12.1) and 21 (a1B/a1A ratio = 14.7) exhibited better a1-ARs subtype selectivity. The SAR results suggested that the o-substituted phenyl group derivatives and p-substituted phenyl group derivatives could improve cytotoxic activity against LNCaP and/or DU145 cells. This results provides a new way for the further research of other novel class of estrone derivatives.

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Acknowledgments

[25] M.P. Leese, B. Leblond, S.P. Newman, A. Purohit, M.J. Reed, B.V.L. Potter, Anticancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates, J. Steroid Biochem. Mol. Biol. 94 (2005) 239–251. [26] G.E. Agoston, J.H. Shah, T.M. LaVallee, X. Zhan, V.S. Pribluda, A.M. Treston, Synthesis and structure-activity relationships of 16-modified analogs of 2-methoxyestradiol, Bioorg. Med. Chem. 15 (2007) 7524–7537. [27] J.H. Shah, G.E. Agoston, L. Suwandi, K. Hunsucker, V. Pribluda, X.H. Zhan, G.M. Swartz, T.M. LaVallee, A.M. Treston, Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure-activity relationships compared to 2-methoxyestradiol, Bioorg. Med. Chem. 17 (2009) 7344–7352. [28] P. Chaudhary, R. Kumar, A.K. Verma, D. Singh, V. Yadav, A.K. Chhillar, G.L. Sharma, R. Chandra, Synthesis and antimicrobial activity of N-alkyl and N-aryl piperazine derivatives, Bioorg. Med. Chem. 14 (2006) 1819–1826. [29] N. Szkaradek, A. Rapacz, K. Pytka, B. Filipek, A. Siwek, M. Cegła, H. Marona, Synthesis and preliminary evaluation of pharmacological properties of some piperazine derivatives of xanthone, Bioorg. Med. Chem. 21 (2013) 514–522. [30] V. Cecchetti, A. Fravolini, F. Schiaffella, O. Tabarrini, G. Bruni, G. Segret, oChlorobenzenesulfonamidic derivatives of (aryloxy)propanolamines as betablocking/diuretic agents, J. Med. Chem. 36 (1993) 157–161. [31] D.A. Walsh, Y.H. Chen, J.B. Green, J.C. Nolan, J.M. Yannit, The synthesis and antiallergy activity of 1-(aryloxy)-4-(4-arylpiperazinyl)-2-butanol derivatives, J. Med. Chem. 33 (1990) 1823–1827. [32] H.J. Seo, E.J. Park, M.J. Kim, S.Y. Kang, S.H. Lee, H.J. Kim, K.N. Lee, M.E. Jung, M. Lee, M.S. Kim, E.J. Son, W.K. Park, J. Kim, J. Lee, Design and synthesis of novel arylpiperazine derivatives containing the imidazole core targeting 5-HT(2A) receptor and 5-HT transporter, J. Med. Chem. 54 (2011) 6305–6318. [33] R. Kikumoto, A. Tobe, H. Fukami, M. Egawa, Synthesis and antianxiety activity of (omega-piperazinylalkoxy)indan derivatives, J. Med. Chem. 26 (1983) 246–250. [34] J.C. Jaen, L.D. Wise, T.G. Heffner, T.A. Pugsley, L.T. Meltzed, Dopamine autoreceptor agonists as potential antipsychotics. 1. (Aminoalkoxy) anilines, J. Med. Chem. 31 (1988) 1621–1625. [35] R.M. Cross, N.K. Namelikonda, T.S. Mutka, L. Luong, D.E. Kyle, R. Manetsch, Synthesis, antimalarial activity, and structure-activity relationship of 7-(2-phenoxyethoxy)-4(1H)-quinolones, J. Med. Chem. 54 (2011) 8321–8327. [36] C. Clarkson, C.C. Musonda, K. Chibale, W.E. Campbella, P. Smitha, Synthesis of totarol amino alcohol derivatives and their antiplasmodial activity and cytotoxicity, Bioorg. Med. Chem. 11 (2003) 4417–4422. [37] M. Leopoldo, E. Lacivita, E. Passafiume, M. Contino, N.A. Colabufo, F. Berardi, R. Perrone, 4-[omega-[4-arylpiperazin-1-yl]alkoxy]phenyl)imidazo[1,2-a]pyridine derivatives: fluorescent high-affinity dopamine D3 receptor ligands as potential probes for receptor visualization, J. Med. Chem. 50 (2007) 5043–5047. [38] X. Chen, M.F. Sassano, L.Y. Zheng, V. Setola, M. Chen, X. Bai, S.V. Frye, W.C. Wetsel, B.L. Roth, J. Jin, Structure-functional selectivity relationship studies of β-arrestin-biased dopamine D₂ receptor agonists, J. Med. Chem. 55 (2012) 7141–7153. [39] L.A. Romeiro, M. Da Silva Ferreira, L.L. Da Silva, H.C. Castro, A.L. Miranda, C.L. Silva, F. Noël, J.B. Nascimento, C.V. Araújo, E. Tibiriçá, E.J. Barreiro, C.A. Fraga, Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative with potent alpha 1A/D-adrenergic receptor blocking properties, Eur. J. Med. Chem. 46 (2011) 3000–3012. [40] M. Baran, E. Kepczynska, M. Zylewski, A. Siwek, M. Bednarski, M.T. Cegla, Studies on novel pyridine and 2-pyridone derivatives of N-arylpiperazine as α-adrenoceptor ligands, Med. Chem. 10 (2014) 144–153. [41] S. Ananthan, S.K. Saini, G. Zhou, J.V. Hobrath, I. Padmalayam, L. Zhai, J.R. Bostwick, T. Antonio, M.E. Reith, S. McDowell, E. Cho, L. McAleer, M. Taylor, R.R. Luedtke, Design, synthesis, and structure-activity relationship studies of a series of [4-(4-carboxamidobutyl)]-1-arylpiperazines: insights into structural features contributing to dopamine D3 versus D2 receptor subtype selectivity, J. Med. Chem. 57 (2014) 7042–7060. [42] F. Berardi, C. Abate, S. Ferorelli, A.F. De Robertis, M. Leopoldo, N.A. Colabufo, M. Niso, R. Perrone, Novel 4-(4-aryl)cyclohexyl-1-(2-pyridyl)piperazines as Delta (8)-Delta(7) sterol isomerase (emopamil binding protein) selective ligands with antiproliferative activity, J. Med. Chem. 51 (2008) 7523–7531. [43] C. Abate, M. Niso, M. Contino, N.A. Colabufo, S. Ferorelli, R. Perrone, F. Berardi, 1Cyclohexyl-4-(4-arylcyclohexyl) piperazines: mixed σ and human Δ(8)-Δ(7) sterol isomerase ligands with antiproliferative and P-glycoprotein inhibitory activity, ChemMedChem 6 (2011) 73–80. [44] W.H. Liu, J.X. Chang, Y. Liu, J.W. Luo, J.W. Zhang, Design, synthesis and activities of novel benzothiazole derivatives containing arylpiperazine, Acta Pharmaceut. Sin. 48 (2013) 1259–1265. [45] H.H. Lin, W.Y. Wu, S.L. Cao, J. Liao, L. Ma, M. Gao, Z.F. Li, X. Xu, Synthesis and antiproliferative evaluation of piperazine-1-carbothiohydrazide derivatives of indolin-2-one, Bioorg. Med. Chem. Lett. 23 (2013) 3304–3307. [46] S.L. Cao, Y. Han, C.Z. Yuan, Y. Wang, Z.K. Xiahou, J. Liao, R.T. Gao, B.B. Mao, B.L. Zhao, Z.F. Li, X. Xu, Synthesis and antiproliferative activity of 4-substitutedpiperazine-1-carbodithioate derivatives of 2,4-diaminoquinazoline, Eur. J. Med. Chem. 64 (2013) 401–409. [47] C.K. Arnatt, J.L. Adams, Z. Zhang, K.M. Haney, G. Li, Y. Zhang, Design, syntheses, and characterization of piperazine based chemokine receptor CCR5 antagonists as anti-prostate cancer agents, Bioorg. Med. Chem. Lett. 24 (2014) 2319–2323. [48] Y.B. Lee, Y.D. Gong, H. Yoon, C.H. Ahn, M.K. Jeon, J.Y. Kong, Synthesis and anticancer activity of new 1-[(5 or 6-substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives, Bioorg. Med. Chem. 18 (2010) 7966–7974. [49] F.J. Guo, J. Sun, L.L. Gao, X.Y. Wang, Y. Zhang, S.S. Qian, H.L. Zhu, Discovery of phenylpiperazine derivatives as IGF-1R inhibitor with potent antiproliferative

The work was supported by the Natural Science Foundation of China (No. 21705072), Henan Province Science and Technology Attack Plan Foundation (Nos. 162102310477 and 172102310478), the Key Scientific Research Project of Higher Education of Henan Province (Nos. 16A350008, 17A150039 and 17B350001) and National Scientific Research Foundation of Luoyang Normal University (No. 2015-PYJJ005). References [1] R.T. Greenlee, T. Murray, M.B. Hill-Harmon, M.J. Thun, Cancer statistics, CACancer J. Clin. 51 (2001) (2001) 15–36. [2] T.B. Dorff, L.M. Glode, Current role of neoadjuvant and adjuvant systemic therapy for high-risk localized prostate cancer, Curr. Opin. Urol. 23 (2013) 366–371. [3] D.A. Loblaw, C. Walker-Dilks, E. Winquist, S.J. Hotte, 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.) 25 (2013) 406–430. [4] J.Y. Han, F.Q. Zhu, X. Xu, H. Huang, W.Q. Huang, W.H. Cui, H. Dai, J.X. Jiang, S.L. Wang, Tetramethylpyrazine hydrochloride inhibits proliferation and apoptosis in human prostate cancer PC3 cells through Akt signaling pathway, J. Third Mil. Med. Univ. 35 (2013) 105–108. [5] C. Zou, X. Li, R. Jiang, The progress of molecular mechanism studies for Chinese traditional medicine on prostate cancer therapy, Chin. J. Androl. 26 (2012) 66–68. [6] B. Akduman, E.D. Crawford, The management of high risk the prostate cancer, J. Urol. 169 (2003) 1993–1998. [7] C.N. Ahlem, T.M. Page, D.L. Auci, M.R. Kennedy, K. Mangano, F. Nicoletti, Y. Ge, Y.G. Huang, S.K. White, S. Villegas, D. Conrad, A. Wang, C.L. Reading, J.M. Frincke, Novel components of the human metabolome: the identification, characterization and anti-inflammatory activity of two 5-androstene tetrols, Steroids 76 (2011) 145–155. [8] A. Aperia, New roles for an old enzyme: Na, K-ATPase emerges as an interesting drug target, J. Intern. Med. 261 (2007) 44–52. [9] R. Minorics, T. Szekeres, G. Krupitza, P. Saiko, B. Giessrigl, J. Wölfling, É. Frank, I. Zupkó, Antiproliferative effects of some novel synthetic solanidine analogs on HL60 human leukemia cells in vitro, Steroids 76 (2011) 156–162. [10] K. Prokai-Tatrai, P. Perjesi, N.M. Rivera-Portalatin, J.W. Simpkins, L. Prokai, Mechanistic investigations on the antioxidant action of a neuroprotective estrogen derivative, Steroids 73 (2008) 280–288. [11] M. Rocha, C. Banuls, L. Bellod, A. Jover, V.M. Victor, A. Hernandez-Mijares, A review on the role of phytosterols: new insights into cardiovascular risk, Curr. Pharm. Des. 17 (2011) 4061–4075. [12] R.K. Dubey, S. Oparil, B. Imthurn, E.K. Jackson, Sex hormones and hypertension, Cardiovasc. Res. 53 (2002) 688–708. [13] K.A. Latham, A. Zamora, H. Drought, S. Subramanian, A. Matejuk, H. Offner, E.F. Rosloniec, Estradiol treatment redirects the isotype of the autoantibody response and prevents the development of autoimmune arthritis, J. Immunol. 171 (2003) 5820–5827. [14] (a) P.J. Sheridan, K. Blum, M. Trachtenberg, Steroid Receptors and Disease, Marcel Dekker, New York, 1988, pp. P289–564; (b) V.K. Moudgil, Steroid Receptors in Health and Disease, Plenum Press, New York/London, 1987. [15] (a) B.W. O’Malley, Hormones and Signaling, Academic Press, San Diego, 1998; (b) M.G. Parker, Steroid Hormone Action, IRL Press, Oxford, 1993. [16] A. Picazo, R. Salcedo, Carcinogenic activity in estrone and its derivatives: a theoretical study, J. Mol. Struct (Theochem) 624 (2003) 29–36. [17] L. McCarthy-Morrogh, P.A. Townsend, A. Purohit, H.A.M. Hejaz, B.V.L. Potte, M.J. Reed, G. Packham, Differential effects of estrone and estrone-3-O-sulfamate derivatives on mitotic arrest, apoptosis, and microtubule assembly in human breast cancer cells, Cancer Res. 60 (2000) 5441–5450. [18] S. Ahmed, K. James, C.P. Owen, The design, synthesis, and biochemical evaluation of derivatives of biphenyl sulfamate-based compounds as novel inhibitors of estrone sulfatase, Biochem. Biophys. Res. Commun. 294 (2002) 180–183. [19] K.M. Kasiotis, P. Magiatis, H. Pratsinis, A. Skaltsounis, V. Abadji, A. Charalambous, P. Moutsatsou, S.A. Haroutounian, Synthesis and biological evaluation of novel daunorubicin-estrogen conjugates, Steroids 66 (2001) 785–791. [20] D. Milic, T. Kop, J. Csanadi, Z. Juranic, Z. Zizak, M.J. Gasic, B. Solaja, Estrone derived steroidal diepoxide: biologically active compound and precursor of a stable steroidal A,B-spiro system, Steroids 74 (2009) 890–895. [21] I.V. Rassokhina, Y.A. Volkova, A.S. Kozlov, A.M. Scherbakov, O.E. Andreeva, V.Z. Shirinian, I.V. Zavarzin, Synthesis and antiproliferative activity evaluation of steroidal imidazo[1,2-a]pyridines, Steroids 113 (2016) 29–37. [22] X. Shi, Z. Wang, F. Xu, X. Lu, H. Yao, D. Wu, S. Sun, R. Nie, S. Gao, P. Li, L. Xia, Z. Zhang, C. Wang, Design, synthesis and antiproliferative effect of 17β-amide derivatives of 2-methoxyestradiol and their studies on pharmacokinetics, Steroids 128 (2017) 6–14. [23] A. Kaise, K. Ohta, Y. Endo, Novel p-carborane-containing multitarget anticancer agents inspired by the metabolism of 17β-estradiol, Bioorg. Med. Chem. 25 (2017) 6371–6378. [24] A. Stander, F. Joubert, A. Joubert, Docking, synthesis and in vivo evaluation of antimitotic estrone analogs, Chem. Biol. Drug Res. 77 (2011) 173–181.

8

Steroids xxx (xxxx) xxx–xxx

H. Chen et al.

[55] L.A. Romeiro, M. da Silva Ferreira, L.L. da Silva, H.C. Castro, A.L. Miranda, C.L. Silva, F. Noël, J.B. Nascimento, C.V. Araújo, E. Tibiriçá, E.J. Barreiro, C.A. Fraga, Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative with potent alpha 1A/D-adrenergic receptor blocking properties, Eur. J. Med. Chem. 46 (2011) 3000–3012. [56] F.M. Awadallah, W. el-Eraky, D.O. Saleh, Synthesis, vasorelaxant activity, and molecular modeling study of some new phthalazine derivatives, Eur. J. Med. Chem. 52 (2012) 14–21. [57] A. Prandi, S. Franchini, L.I. Manasieva, P. Fossa, E. Cichero, G. Marucci, M. Buccioni, A. Cilia, L. Pirona, L. Brasili, Synthesis, biological evaluation, and docking studies of tetrahydrofuran- cyclopentanone- and cyclopentanol-based ligands acting at adrenergic α1- and serotonine 5-HT1A receptors, J. Med. Chem. 55 (2012) 23–36. [58] F. Xu, H. Chen, X.L. He, J.Y. Xu, B.B. Xu, B.Y. Huang, X. Liang, M. Yuan, Identification of two novel α1-AR agonists using a high-throughput screening model, Molecules 19 (2014) 12699–12709.

properties in vitro, Bioorg. Med. Chem. Lett. 25 (2015) 1067–1071. [50] H. Chen, X. Liang, F. Xu, B.B. Xu, X.L. He, B.Y. Huang, M. Yuan, Synthesis and cytotoxic activity evaluation of novel arylpiperazine derivatives on human prostate cancer cell lines, Molecules 19 (2014) 12048–12064. [51] H. Chen, F. Xu, X. Liang, B.B. Xu, Z.L. Yang, X.L. He, B.Y. Huang, M. Yuan, Design, synthesis and biological evaluation of novel arylpiperazine derivatives on human prostate cancer cell lines, Bioorg. Med. Chem. Lett. 25 (2015) 285–287. [52] A.H. Banday, A.K. Giri, R. Parveen, N. Bashir, Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents against LNCaP, PC-3 and DU-145 cells, Steroids 87 (2014) 93–98. [53] F. Xu, H. Chen, J.Y. Xu, X. Liang, X.L. He, B.H. Shao, X.Q. Sun, B. Li, X.L. Deng, Mu. Yuan, Synthesis, structure-activity relationship and biological evaluation of novel arylpiperzines as α1A/1D-AR subselective antagonists for BPH, Bioorg. Med. Chem. 23 (2015) 7735–7742. [54] G. Romeo, L. Materia, M.N. Modica, V. Pittalà, L. Salerno, M.A. Siracusa, F. Manetti, M. Botta, K.P. Minneman, Novel 4-phenylpiperidine-2,6-dione derivatives. Ligands for α1-adrenoceptor subtypes, Eur. J. Med. Chem. 46 (2011) 2676–2690.

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