Steroids 82 (2014) 1–6

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Synthesis and anticancer activity of novel C6-piperazine substituted purine steroid–nucleosides analogues Li-Hua Huang a,b,c, Hong-De Xu b,c, Zhuo-Ya Yang a, Yong-Fei Zheng b,c, Hong-Min Liu b,c,⇑ a

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China c New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, China b

a r t i c l e

i n f o

Article history: Received 22 August 2013 Received in revised form 4 December 2013 Accepted 13 December 2013 Available online 28 December 2013 Keywords: Nucleoside analogues Nucleosides Purine Piperazines Anticancer activity

a b s t r a c t Novel C6-piperazine substituted purine nucleoside analogues (2–9) bearing a modified pyranose-like D ring of the 4-azasteroid moiety were efficiently synthesized through nucleophilic substitution at C6 position of the steroid–nucleoside precursors (1) with versatile piperazines. All newly-synthesized compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 8b and 9b exhibited significant cytotoxicity on PC-3 cell lines. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Nucleoside analogues have played an important role in the development of antiviral and antitumour drugs [1–3]. Due to the pharmacological significance, nucleoside analogues have attracted extensive investigation for small molecule drug discovery through decades, and modifications have been made to both the heterocyclic base and the sugar moiety [1,4]. Purine nucleobases and nucleoside analogues with modifications by introduction of various substituents at the C6 or C2 positions of purine ring have received considerable attention [5–7]. Among these, C6-aminopurine derivatives possess wide range of biological properties and have been shown to have antitumour activity as inhibitors of various protein kinases [6,7]. In addition, piperazine derivatives displayed a broad spectrum of biological activities, such as anthelmintic [8], antimalarial [9], antidepressant [10], MC4R antagonists [11], MMP inhibitors [12] and anticancer activity [13]. Many currently used drugs contain a piperazine ring as part of their molecular structure and some C6-piperazine substituted purine derivatives show potent anticancer activity [3]. On the other hand, apart from naturally occurring substances, most of steroidal drugs in use today are semi-synthetic com-

⇑ Corresponding author at: New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, China. Tel./fax: +86 371 67781739. E-mail address: [email protected] (H.-M. Liu). 0039-128X/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2013.12.004

pounds via the modification of the steroid ring system and side chains [14–16]. Therefore, large numbers of steroids with unusual and interesting structures have been synthesized, some of which exhibited good antitumour activity [17–19]. In 1977, van Lier et al. reported a novel class of compounds prepared via coupling steroids with naturally occurring purines and pyrimidines by way of a C–N linkage [20]. These newly-synthesized steroid– nucleosides exhibited great anti-tumour activity and were less likely to have side effects. Subsequently, several other classes of steroidal nucleosides analogues were synthesized with nucleobases and nucleoside analogues linked through a spacer group or linked directly to the steroid skeleton [21–24]. However, this type of steroidal derivatives has not been explored extensively. Our group has been interested in the design and synthesis of novel modified steroids [25–28], and we have previously described a novel series of steroid–nucleoside analogues, in which the bases were directly attached to the D ring of the 4-azasteroid moiety [29]. The preliminary biological evaluation indicated that the purine nucleosides analogues 1a–c (Fig. 1) showed potent anticancer activity. In view of the therapeutic importance of C6-aminopurine and piperazine derivatives, we herein report the synthesis of novel C6-piperazine substituted steroid–nucleosides analogues by displacement of the C6 chloro on the purine ring of compounds 1a–c with versatile piperazines and their anticancer activity against PC-3, MCF-7 and Hela cell lines in vitro.

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L.-H. Huang et al. / Steroids 82 (2014) 1–6

N O

N N

Cl N R1

N H

O

1a: R1 = H; 1b: R1 = Cl; 1c: R1 = F

5.91 (s, 1H, 4N-H),4.26 (brs, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 3.04–2.97 (m, 4H, protons of piperazine), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 158.81 (d, J = 206.8 Hz), 154.74 (d, J = 19.8 Hz), 151.69 (d, J = 19.4 Hz), 136.33 (d, J = 2.7 Hz), 117.88 (d, J = 4.2 Hz), 77.75, 76.33, 60.33, 50.63, 48.90, 46.09, 39.16, 36.05, 35.78, 33.40, 33.17, 28.51, 28.24, 27.12, 21.87, 21.42, 16.47, 11.31. HRMS (ESI): m/z cacld for C27H39FN7O2 (M+H)+, 512.3149; found, 512.3146.

1 Fig. 1. Novel class of steroidal purine nucleosides analogues.

2. Experimental 2.1. General remarks All reagents and solvents used were of analytical grade purchased from commercial sources. Thin-layer chromatography (TLC) was carried out on glass plates coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized by UV light (254 nm). Melting points were determined on a Beijing Keyi XT4A apparatus and are uncorrected. All NMR spectra were recorded with a Bruker DPX 400 MHz spectrometer with TMS as internal standard in CDCl3. Chemical shifts are given as d ppm values relative to TMS. Mass spectra (MS) were recorded on Q-Tof (Waters) mass spectrometer by electrospray ionization (ESI). 2.2. General procedure for the synthesis of compounds 2–9 To a solution of the steroidal purine nucleoside analogues (1a–c, 0.2 mmol) in MeOH (2 mL) were added the appropriate piperazine (0.24 mmol) and Et3N (0.24 mmol). The reaction mixture was refluxed for 5–20 min and then concentrated in vacuo. The residue was dissolved in CH2Cl2 (10 mL), and then washed with water. The separated organic phase was dried over Na2SO4, and then evaporated to afford the corresponding product 2–9. 2.2.1. 17a-(60 -(Piperazine-1-yl)purin-90 -yl)-D-homo-17a-oxa-4-aza5a-androst-3-one (2a) White solid, yield 91%, mp 272–273 °C. 1H NMR (400 MHz, CDCl3): d 8.34 (s, 1H, 20 -H), 7.96 (s, 1H, 80 -H), 6.13 (m, 2H, 4N-H and 17b-H), 4.37 (s, 4H, protons of piperazine), 3.07 (dd, J = 12.2, 3.3 Hz, 1H, 5a-H), 3.03–2.91 (m, 4H, protons of piperazine), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 153.88, 152.33, 150.03, 136.08, 119.76, 77.64, 76.12, 60.31, 50.64, 48.93, 46.30, 39.18, 36.06, 35.76, 33.51, 33.19, 28.52, 28.25, 27.10, 21.87, 21.51, 16.46, 11.30. HRMS (ESI): m/z cacld for C27H40N7O2 (M+H)+, 494.3243; found, 494.3241. 2.2.2. 17a-(20 -Chloro-60 -(piperazine-1-yl)purin-90 -yl)-D-homo-17aoxa-4-aza-5a-androst-3- one (2b) White solid, yield 93%, mp 247–249 °C. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, 80 -H), 6.10–5.98 (m, 2H, 4N-H and 17b-H), 4.26 (brs, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 3.03–2.95 (m, 4H, protons of piperazine), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.14, 153.89, 153.81, 151.21, 136.38, 118.56, 77.74, 76.35, 60.31, 50.66, 48.96, 46.59, 46.26, 39.20, 36.06, 35.78, 33.67, 33.19, 28.50, 28.25, 27.11, 21.86, 21.37, 16.53, 11.29. HRMS (ESI): m/z cacld for C27H39ClN7O2 (M+H)+, 528.2854; found, 528.2853. 2.2.3. 17a-(20 -Fluoro-60 -(piperazine-1-yl)purin-90 -yl)-D-homo-17aoxa-4-aza-5a-androst-3-one (2c) White solid, yield 90%, mp 264–266 °C. 1H NMR (400 MHz, CDCl3): d 7.90 (s, 1H, 80 -H), 6.00 (dd, J = 11.3, 2.7 Hz, 1H, 17b-H),

2.2.4. 17a-(60 -(4-Ethylpiperazine-1-yl)purin-90 -yl)-D-homo-17a-oxa4-aza-5a-androst-3-one (3a) White solid, yield 94%, mp 171–173 °C. 1H NMR (400 MHz, CDCl3): d 8.34 (s, 1H, 20 -H), 7.96 (s, 1H, 80 -H), 6.11 (dd, J = 11.3, 2.7 Hz, 1H, 17b-H), 5.94 (s, 1H, 4N-H), 4.33 (s, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.7 Hz, 1H, 5a-H), 2.63–2.52 (m, 4H, protons of piperazine), 1.36 (s, 3H, 18-H), 1.13 (t, J = 7.2 Hz, 3H,-NCH2CH3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.28, 153.77, 152.32, 150.02, 136.13, 119.78, 77.62, 76.14, 60.28, 52.42, 50.66, 48.94, 45.04, 39.19, 36.07, 35.72, 33.50, 33.20, 28.51, 28.25, 27.04, 21.86, 21.50, 16.45, 11.90, 11.29. HRMS (ESI): m/z cacld for C29H44N7O2 (M+H)+, 522.3556; found, 522.3552.

2.2.5. 17a-(20 -Chloro-60 -(4-ethylpiperazine-1-yl)purin-90 -yl)-D-homo17a-oxa-4-aza-5a-androst-3-one (3b) White solid, yield 93%, mp 184–186 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.12 (s, 1H, 4N-H), 6.04 (dd, J = 11.1, 2.3 Hz, 1H, 17b-H), 4.31 (brs, 4H, protons of piperazine), 3.06 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 2.65–2.51 (m, 4H, protons of piperazine), 1.12 (t, J = 7.2 Hz, 3H, -NCH2CH3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.17, 153.79, 151.20, 136.43, 118.57, 77.73, 76.35, 60.30, 52.81, 52.33, 50.67, 48.96, 39.20, 36.06, 35.76, 33.66, 33.20, 28.50, 28.24, 27.08, 21.85, 21.36, 16.53, 11.88, 11.29. HRMS (ESI): m/z cacld for C29H43ClN7O2 (M+H)+, 556.3167; found, 556.3168.

2.2.6. 17a-(20 -Fluoro-60 -(4-ethylpiperazine-1-yl)purin-90 -yl)-D-homo17a-oxa-4-aza-5a-androst-3-one (3c) White solid, yield 93%, mp 176–177 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.13 (s, 1H, 4N-H), 6.00 (dd, J = 11.3, 2.6 Hz, 1H, 17b-H), 4.86–3.84 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 2.57 (s, 4H, protons of piperazine), 1.13 (t, J = 7.2 Hz, 3H, -NCH2CH3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 158.82 (d, J = 206.7 Hz), 154.66 (d, J = 19.8 Hz), 151.65 (d, J = 19.3 Hz), 136.31 (d, J = 2.6 Hz), 117.88 (d, J = 4.1 Hz), 77.73, 76.31, 60.30, 52.82, 52.35, 50.64, 48.89, 44.43, 39.16, 36.04, 35.75, 33.40, 33.19, 28.52, 28.24, 27.09, 21.86, 21.42, 16.47, 11.93, 11.31. HRMS (ESI): m/z cacld for C29H43FN7O2 (M+H)+, 540.3462; found, 540.3461.

2.2.7. 17a-(60 -(4-Isopropylpiperazine-1-yl)purin-90 -yl)-D-homo-17aoxa-4-aza-5a-androst-3-one (4a) White solid, yield 94%, mp 242–243 °C. 1H NMR (400 MHz, CDCl3): d 8.34 (s, 1H, 20 -H), 7.96 (s, 1H, 80 -H), 6.11 (m, 2H, 4N-H and 17b-H), 4.32 (s, 4H, protons of piperazine), 3.13–3.01 (m, 1H, 5a-H), 2.85–2.69 (m, 1H, -NCH(CH3)2), 2.64 (d, J = 4.3 Hz, 4H, protons of piperazine), 1.15–1.04 (m, 6H, -NCH(CH3)2), 0.89 (s, 3H, 19H). 13C NMR (100 MHz, CDCl3): d 172.10, 153.73, 152.36, 149.99, 136.06, 119.75, 77.61, 76.12, 60.31, 54.64, 50.65, 48.94, 48.83, 45.46, 39.19, 36.07, 35.79, 33.51, 33.20, 28.52, 28.25, 27.15, 21.86, 21.51, 18.43, 16.45, 11.29. HRMS (ESI): m/z cacld for C30H46N7O2 (M+H)+, 536.3713; found, 536.3705.

L.-H. Huang et al. / Steroids 82 (2014) 1–6

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2.2.8. 17a-(20 -Chloro-60 -(4-isopropylpiperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (4b) White solid, yield 95%, mp 254–255 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.17 (brs, 1H, 4N-H), 6.04 (dd, J = 11.2, 2.5 Hz, 1H, 17b-H), 4.20 (brs, 4H, protons of piperazine), 3.06 (dd, J = 12.1, 2.7 Hz, 1H, 5a-H), 2.84–2.67 (m, 1H, -NCH(CH3)2), 2.67–2.56 (m, 4H, protons of piperazine), 1.07 (d, J = 6.5 Hz, 6H, -NCH(CH3)2), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 153.82, 153.72, 151.18, 136.38, 118.54, 77.72, 76.34, 60.30, 54.60, 50.68, 48.97, 48.73, 39.20, 36.06, 35.76, 33.66, 33.20, 28.50, 28.24, 27.08, 21.85, 21.37, 18.38, 16.53, 11.29. HRMS (ESI): m/z cacld for C30H45ClN7O2 (M+H)+, 570.3323; found, 570.3322.

2.2.13. 17a-(60 -(4-Phenylpiperazine-1-yl)purin-90 -yl)-D-homo-17aoxa-4-aza-5a-androst-3-one (6a) White solid, yield 92%, mp 239–240 °C. 1H NMR (400 MHz, CDCl3): d 8.37 (s, 1H, 20 -H), 7.98 (s, 1H, 80 -H), 7.29 (t, J = 8.0 Hz, 2H, Ar-H), 6.98 (d, J = 8.0 Hz, 2H, Ar-H), 6.90 (t, J = 7.3 Hz, 1H, ArH), 6.12 (dd, J = 11.3, 2.6 Hz, 1H, 17b-H), 5.85 (s, 1H, 4N-H), 4.46 (s, 4H, protons of piperazine), 3.36–3.24 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.7 Hz, 1H, 5a-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.07, 153.76, 152.36, 151.24, 150.10, 136.33, 129.23, 120.29, 119.88, 116.52, 77.68, 76.19, 60.32, 50.64, 49.62, 48.94, 39.19, 36.08, 35.81, 33.53, 33.21, 28.54, 28.26, 27.18, 21.88, 21.52, 16.47, 11.31. HRMS (ESI): m/z cacld for C33H44N7O2 (M+H)+, 570.3556; found, 570.3553.

2.2.9. 17a-(20 -Fluoro-60 -(4-isopropylpiperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (4c) White solid, yield 93%, mp 194–196 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.08 (s, 1H, 4N-H), 6.00 (dd, J = 11.3, 2.7 Hz, 1H, 17b-H), 4.75–3.87 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.4, 3.7 Hz, 1H, 5a-H), 2.75 (m, 1H, -NCH(CH3)2), 2.64 (s, 4H, protons of piperazine), 1.07 (d, J = 6.5 Hz, 6H, -NCH(CH3)2), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.16, 158.85 (d, J = 206.8 Hz), 154.60 (d, J = 20.0 Hz), 151.63 (d, J = 19.3 Hz), 136.25 (d, J = 2.6 Hz), 117.85 (d, J = 4.2 Hz), 77.72, 76.30, 60.30, 54.59, 50.64, 48.90, 44.76, 39.17, 36.05, 35.76, 33.41, 33.20, 28.53, 28.24, 27.11, 21.86, 21.42, 18.40, 16.47, 11.30. HRMS (ESI): m/z cacld for C30H45FN7O2 (M+H)+, 554.3619; found, 554.3618.

2.2.14. 17a-(20 -Chloro-60 -(4-phenylpiperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (6b) White solid, yield 95%, mp 193–195 °C. 1H NMR (400 MHz, CDCl3): d 7.96 (s, 1H, 80 -H), 7.31 (t, J = 7.9 Hz, 2H, Ar-H), 6.98 (d, J = 8.3 Hz, 2H, Ar-H), 6.92 (t, J = 7.3 Hz, 1H, Ar-H), 6.15 (s, 1H, 4NH), 6.07 (dd, J = 11.2, 2.6 Hz, 1H, 17b-H), 4.45 (brs, 4H, protons of piperazine), 3.38–3.19 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.11, 153.84, 151.32, 151.09, 136.65, 129.24, 120.42, 118.70, 116.57, 77.77, 76.44, 60.32, 50.71, 49.60, 49.00, 39.23, 36.09, 35.80, 33.67, 33.24, 28.52, 28.27, 27.12, 21.87, 21.38, 16.54, 11.30. HRMS (ESI): m/z cacld for C33H43ClN7O2 (M+H)+, 604.3167; found, 604.3169.

2.2.10. 17a-(60 -(4-Hydroxyethylpiperazine-1-yl)purin-90 -yl)-D-homo17a-oxa-4-aza-5a-androst-3-one (5a) White solid, yield 90%, mp 266–268 °C. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, 20 -H), 7.95 (s, 1H, 80 -H), 6.27 (s, 1H, 4N-H), 6.09 (dd, J = 11.3, 2.4 Hz, 1H, 17b-H), 4.30 (s, 4H, protons of piperazine), 3.66 (t, J = 5.2 Hz, 2H, -CH2OH), 3.04 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 2.66–2.54 (m, 6H), 0.87 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.31, 153.73, 152.31, 150.02, 136.23, 119.76, 77.68, 76.13, 60.28, 59.49, 57.76, 53.05, 50.61, 48.90, 45.15, 39.16, 36.05, 35.71, 33.49, 33.16, 28.51, 28.24, 27.04, 21.86, 21.50, 16.46, 11.31. HRMS (ESI): m/z cacld for C29H44N7O3 (M+H)+, 538.3506; found, 538.3506. 2.2.11. 17a-(20 -Chloro-60 -(4-hydroxyethylpiperazine-1-yl)purin-90 yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (5b) White solid, yield 92%, mp 260–261 °C. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, 80 -H), 6.28 (s, 1H, 4N-H), 6.04 (d, J = 10.5 Hz, 1H, 17b-H), 4.30 (brs, 4H, protons of piperazine), 3.67 (d, J = 4.7 Hz, 2H, -CH2OH), 3.05 (d, J = 9.9 Hz, 1H, 5a-H), 2.60 (dd, J = 13.2, 4.7 Hz, 6H), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.23, 153.79, 151.25, 136.55, 118.59, 77.75, 76.39, 60.29, 59.52, 57.88, 53.01, 50.69, 48.97, 39.21, 36.06, 35.75, 33.63, 33.20, 28.49, 28.25, 27.04, 21.85, 21.36, 16.52, 11.28. HRMS (ESI): m/z cacld for C29H43ClN7O3 (M+H)+, 572.3116; found, 572.3115. 2.2.12. 17a-(20 -Fluoro-60 -(4-hydroxyethylpiperazine-1-yl)purin-90 yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (5c) White solid, yield 90%, mp 182–183 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.20 (s, 1H, 4N-H), 6.00 (d, J = 9.6 Hz, 1H, 17b-H), 4.20 (m, 4H, protons of piperazine), 3.78–3.63 (m, 2H, -CH2OH), 3.13–3.02 (m, 1H, 5a-H), 2.63 (d, J = 12.9 Hz, 6H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.24, 158.78 (d, J = 206.8 Hz), 154.75 (s), 151.68 (d, J = 23.4 Hz), 136.43 (d, J = 2.4 Hz), 117.89 (d, J = 4.2 Hz), 77.77, 76.33, 60.29, 59.44, 57.78, 52.95, 50.63, 48.89, 39.16, 36.04, 35.74, 33.39, 33.17, 28.52, 28.24, 27.07, 21.86, 21.41, 16.47, 11.31. HRMS (ESI): m/z cacld for C29H43FN7O3 (M+H)+, 556.3411; found, 556.3412.

2.2.15. 17a-(20 -Fluoro-60 -(4-phenylpiperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (6c) White solid, yield 94%, mp 177–178 °C. 1H NMR (400 MHz, CDCl3): d 7.93 (s, 1H, 80 -H), 7.30 (dd, J = 8.6, 7.4 Hz, 2H, Ar-H), 6.97 (d, J = 7.9 Hz, 2H, Ar-H), 6.91 (t, J = 7.3 Hz, 1H, Ar-H), 6.01 (dd, J = 11.3, 2.7 Hz, 1H, 17b-H), 5.95 (s, 1H, 4N-H), 4.38 (s, 4H, protons of piperazine), 3.39–3.21 (m, 4H, protons of piperazin), 3.07 (dd, J = 12.3, 3.7 Hz, 1H, 5a-H), 1.34 (s, 3H, 18-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.14, 158.82 (d, J = 207.1 Hz), 154.69 (d, J = 19.8 Hz), 151.75 (d, J = 19.3 Hz), 151.05, 136.53 (d, J = 2.5 Hz), 129.27, 120.48, 117.98 (d, J = 4.0 Hz), 116.60, 77.78, 76.39, 60.31, 50.64, 49.60, 48.91, 39.17, 36.06, 35.79, 33.42, 33.20, 28.53, 28.25, 27.14, 21.88, 21.43, 16.47, 11.31. HRMS (ESI): m/z cacld for C33H42FN7O2Na (M+Na)+, 610.3282; found, 610.3280. 2.2.16. 17a-(60 -(4-(4-Methoxyphenyl)piperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (7a) White solid, yield 92%, mp 216–219 °C. 1H NMR (400 MHz, CDCl3): d 8.36 (s, 1H, 20 -H), 7.98 (s, 1H, 80 -H), 6.94 (d, J = 9.0 Hz, 2H, Ar-H), 6.85 (d, J = 9.0 Hz, 2H, Ar-H), 6.12 (dd, J = 11.3, 2.5 Hz, 1H, 17b-H), 6.04 (s, 1H, 4N-H), 4.45 (s, 4H, protons of piperazine), 3.77 (s, 3H, -OCH3), 3.23–3.14 (m, 4H, protons of piperazine), 3.06 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.15, 154.20, 153.77, 152.35, 150.09, 145.59, 136.29, 119.86, 118.76, 114.50, 77.67, 76.17, 60.31, 55.57, 51.15, 50.64, 48.93, 45.23, 39.18, 36.07, 35.77, 33.52, 33.20, 28.53, 28.25, 27.12, 21.88, 21.52, 16.46, 11.31. HRMS (ESI): m/z cacld for C34H46N7O3 (M+H)+, 600.3662; found, 600.3661. 2.2.17. 17a-(20 -Chloro-60 -(4-(4-methoxyphenyl)piperazine-1-yl) purin-90 -yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (7b) White solid, yield 93%, mp 168–169 °C. 1H NMR (400 MHz, CDCl3): d 7.95 (s, 1H, 80 -H), 6.95 (d, J = 9.0 Hz, 2H, Ar-H), 6.87 (d, J = 8.9 Hz, 2H, Ar-H), 6.17 (m, 1H, 4N-H), 6.10–5.97 (m, 1H, 17bH), 4.45 (brs, 4H, protons of piperazine), 3.79 (s, 3H, -OCH3), 3.26–3.15 (m, 4H, protons of piperazine), 3.11–3.00 (m, 1H, 5aH), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.10,

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154.34, 153.83, 151.29, 145.43, 136.60, 118.83, 118.67, 114.55, 77.77, 76.41, 60.32, 55.57, 51.12, 50.69, 48.98, 39.22, 36.07, 35.79, 33.68, 33.22, 28.53, 28.26, 27.12, 21.87, 21.38, 16.55, 11.30. HRMS (ESI): m/z cacld for C34H45ClN7O3 (M+H)+, 634.3272; found, 634.3270. 2.2.18. 17a-(20 -Fluoro-60 -(4-(4-methoxyphenyl)piperazine-1-yl) purin-90 -yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (7c) White solid, yield 90%, mp 179–181 °C. 1H NMR (400 MHz, CDCl3): d 7.94 (s, 1H, 80 -H), 7.04–6.90 (m, 2H, Ar-H), 6.87 (d, J = 9.0 Hz, 2H, Ar-H), 6.35 (s, 1H, 4N-H), 6.02 (dd, J = 11.2, 2.4 Hz, 1H, 17b-H), 4.89–3.90 (m, 4H, protons of piperazine), 3.79 (s, 3H, -OCH3), 3.18 (d, J = 4.6 Hz, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.26, 158.82 (d, J = 207.0 Hz), 154.69 (d, J = 19.8 Hz), 154.32, 151.75 (d, J = 19.1 Hz), 145.37, 136.49 (d, J = 2.5 Hz), 118.84, 117.96 (d, J = 4.1 Hz), 114.53, 77.76, 76.37, 60.29, 55.56, 51.11, 50.66, 48.91, 44.63, 39.18, 36.05, 35.73, 33.40, 33.20, 28.53, 28.25, 27.04, 21.86, 21.42, 16.47, 11.31. HRMS (ESI): m/z cacld for C34H44FN7O3Na (M+Na)+, 640.3387; found, 640.3390. 2.2.19. 17a-(60 -(4-(2-Fluorophenyl)piperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (8a) White solid, yield 95%, mp 234–235 °C. 1H NMR (400 MHz, CDCl3): d 8.36 (s, 1H, 20 -H), 7.97 (s, 1H, 80 -H), 7.09–7.01 (m, 2H, Ar-H), 6.96 (d, J = 8.2 Hz, 2H, Ar-H), 6.17–6.05 (m, 2H, 4N-H and 17b-H), 4.47 (s, 4H, protons of piperazine), 3.27–3.15 (m, 4H, protons of piperazine), 3.06 (dd, J = 10.8, 4.7 Hz, 1H, 5a-H), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 155.83 (d, J = 246.0 Hz), 153.81, 152.34, 150.11, 139.93 (d, J = 8.5 Hz), 132.30, 124.54 (d, J = 3.5 Hz), 122.93 (d, J = 7.9 Hz), 119.86, 119.16 (d, J = 2.7 Hz), 116.24 (d, J = 20.7 Hz), 77.67, 76.18, 60.31, 50.86, 50.83, 50.65, 48.94, 45.27, 39.19, 36.07, 35.77, 33.51, 33.20, 28.53, 28.26, 27.12, 21.87, 21.52, 16.46, 11.31. HRMS (ESI): m/z cacld for C33H43FN7O2 (M+H)+, 588.3462; found, 588.3464. 2.2.20. 17a-(20 -Chloro-60 -(4-(2-fluorophenyl)piperazine-1-yl)purin90 -yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (8b) White solid, yield 93%, mp 186–187 °C. 1H NMR (400 MHz, CDCl3): d 7.95 (s, 1H, 80 -H), 7.21–6.88 (m, 4H, Ar-H), 6.38 (s, 1H, 4 N-H), 6.06 (d, J = 10.9 Hz, 1H, 17b-H), 4.46 (brs, 4H, protons of piperazine), 3.20 (s, 4H, protons of piperazine), 3.06 (d, J = 11.3 Hz, 1H, 5a-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.20, 155.84 (d, J = 246.0 Hz), 153.87, 153.82, 151.33, 139.75 (d, J = 8.5 Hz), 136.62, 124.54 (d, J = 3.7 Hz), 123.04 (d, J = 7.7 Hz), 119.22 (d, J = 2.5 Hz), 118.68, 116.26 (d, J = 20.7 Hz), 77.76, 76.43, 60.30, 50.75, 50.71, 48.99, 39.23, 36.08, 35.77, 33.66, 33.23, 28.52, 28.26, 27.07, 21.86, 21.38, 16.53, 11.29. HRMS (ESI): m/z cacld for C33H42ClFN7O2 (M+H)+, 622.3073; found, 622.3074. 2.2.21. 17a-(20 -Fluoro-60 -(4-(2-fluorophenyl)piperazine-1-yl)purin90 -yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (8c) White solid, yield 92%, mp 192–194 °C. 1H NMR (400 MHz, CDCl3): d 7.93 (s, 1H, 80 -H), 7.10–7.04.90 (m, 2H, Ar-H), 7.00–6.93 (m, 2H, Ar-H), 6.09 (s, 1H, 4N-H), 6.01 (dd, J = 11.3, 2.6 Hz, 1H, 17b-H), 4.99–4.06 (m, 4H, protons of piperazine), 3.28–3.13 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 1.35 (s, 3H, 18-H), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.23, 158.81 (d, J = 207.1 Hz), 155.82 (d, J = 244.0 Hz), 154.74 (d, J = 19.7 Hz), 151.76 (d, J = 19.1 Hz), 139.71 (d, J = 8.6 Hz), 136.50 (d, J = 2.4 Hz), 124.57 (d, J = 3.5 Hz), 123.12 (d, J = 8.0 Hz), 119.20 (d, J = 2.7 Hz), 117.96 (d, J = 4.2 Hz), 116.29 (d, J = 20.6 Hz), 77.78, 76.38, 60.31, 50.74, 50.64, 48.90, 39.17, 36.05, 35.76, 33.41, 33.18, 28.52, 28.25, 27.10, 21.87, 21.42, 16.47, 11.31. HRMS

(ESI): m/z cacld for C33H42F2N7O2 (M+H)+, 606.3368; found, 606.3366. 2.2.22. 17a-(60 -(4-(2-Pyrimidinyl)piperazine-1-yl)purin-90 -yl)-Dhomo-17a-oxa-4-aza-5a-androst-3-one (9a) White solid, yield 96%, mp 256–258 °C. 1H NMR (400 MHz, CDCl3): d 8.36 (s, 1H, 20 -H), 8.33 (d, J = 4.7 Hz, 2H, protons of pyrimidine), 7.98 (s, 1H, 80 -H), 6.52 (t, J = 4.7 Hz, 1H, proton of pyrimidine), 6.11 (dd, J = 11.3, 2.5 Hz, 1H, 17b-H), 6.00 (s, 1H, 4N-H), 4.37 (s, 4H, protons of piperazine), 4.08–3.83 (m, 4H, protons of piperazine), 3.06 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 0.88 (s, 3H, 19H). 13C NMR (100 MHz, CDCl3): d 172.11, 161.67, 157.77, 153.89, 152.31, 150.10, 136.34, 119.90, 110.22, 77.66, 76.17, 60.30, 50.63, 48.94, 43.79, 39.18, 36.07, 35.78, 33.51, 33.20, 28.52, 28.25, 27.14, 21.87, 21.51, 16.46, 11.30. HRMS (ESI): m/z cacld for C31H42N9O2 (M+H)+, 572.3461; found, 572.3463. 2.2.23. 17a-(20 -Chloro-60 -(4-(2-pyrimidinyl)piperazine-1-yl)purin-90 yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (9b) White solid, yield 95%, mp 256–257 °C. 1H NMR (400 MHz, CDCl3): d 8.35 (d, J = 4.6 Hz, 2H, protons of pyrimidine), 7.96 (s, 1H, 80 -H), 6.55 (t, J = 4.6 Hz, 1H, proton of pyrimidine), 6.30 (s, 1H, 4N-H), 6.05 (d, J = 11.0 Hz, 1H, 17b-H), 4.37 (brs, 4H, protons of piperazine), 3.97 (s, 4H, protons of piperazine), 3.07 (d, J = 10.0 Hz, 1H, 5a-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.24, 161.62, 157.76, 153.99, 153.80, 151.31, 136.67, 118.72, 110.35, 77.75, 76.42, 60.32, 50.70, 48.98, 43.73, 39.22, 36.07, 35.75, 33.65, 33.20, 28.52, 28.26, 27.04, 21.86, 21.37, 16.53, 11.29. HRMS (ESI): m/z cacld for C31H41ClN9O2 (M+H)+, 606.3072; found, 606.3073. 2.2.24. 17a-(20 -Fluoro-60 -(4-(2-pyrimidinyl)piperazine-1-yl)purin-90 yl)-D-homo-17a-oxa-4-aza-5a-androst-3-one (9c) White solid, yield 95%, mp 242–244 °C. 1H NMR (400 MHz, CDCl3): d 8.35 (d, J = 4.7 Hz, 2H, protons of pyrimidine), 7.94 (s, 1H, 80 -H), 6.56 (t, J = 4.7 Hz, 1H, proton of pyrimidine), 6.14 (s, 1H, 4 N-H), 6.02 (d, J = 9.2 Hz, 1H, 17b-H), 4.37 (brs, 4H, protons of piperazine), 3.97 (s, 4H, protons of piperazine), 3.07 (dd, J = 12.2 Hz, 3.1 Hz, 1H, 5a-H), 1.35 (s, 3H, 18-H), 0.90 (s, 3H, 19H). 13C NMR (100 MHz, CDCl3): d 172.24, 161.59, 158.79 (d, J = 207.0 Hz), 157.80, 154.86 (d, J = 19.7 Hz), 151.76 (d, J = 19.3 Hz), 136.56 (d, J = 2.3 Hz), 118.02 (d, J = 4.0 Hz), 110.41, 77.76, 76.39, 60.30, 50.65, 48.91, 43.71, 39.17, 36.06, 35.76, 33.38, 33.18, 28.50, 28.24, 27.08, 21.86, 21.42, 16.45, 11.30. HRMS (ESI): m/z cacld for C31H40FN9O2 (M+H)+, 590.3367; found, 590.3369. 2.3. Bioactivity All the synthesized new steroid–nucleoside analogues 2–9 were subjected to in vitro cytotoxic evaluation against PC-3, MCF-7 and Hela cell lines. The anticancer potency of test compounds was measured using the MTT assay. For the test procedure, cells were distributed to wells of 96-well plates (about 6000 cells per well). After 24 h of incubation at 37 °C and 5% CO2 to allow cell attachment, the cells were treated with various concentrations of test samples. Plates were returned to the incubator for 72 h under same conditions. Thereafter, 20 lL of the MTT (0.5 mg/mL) solution was added to each well, and the cells were incubated for 4 h. The medium was removed, 150 lL of DMSO per well was added to dissolve the purple formazan crystals formed and plates were gently shaken for 10 min on a mechanical shaker. The optical density (OD) of solubilized formazan was measured at 570 nm with an automatic microplate reader. All the data of the experiment were expressed as the IC50 (lM) values.

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L.-H. Huang et al. / Steroids 82 (2014) 1–6 Table 1 Synthesis of 4-azasteroidal purine nucleoside analogues 2–9a.

N O

N N

N

Cl N

HN

R1 O

N

N R2

O

O

N H

b

2-9

Comp

R1

R2

Yields (%)

Comp

R1

2a 2b 2c

H Cl F

H

91 93 90

6a 6b 6c

H Cl F

3a 3b 3c

H Cl F

-CH2CH3

94 93 93

7a 7b 7c

H Cl F

4a 4b 4c

H Cl F

-CH(CH3)2

94 95 93

8a 8b 8c

H Cl F

5a 5b 5c

H Cl F

-CH2CH2OH

90 92 90

9a 9b 9c

H Cl F

a

N R1

1

b

N

N N

Et3N, MeOH

N H

R2

Yieldsb (%)

R2

92 95 94

OCH3

F

N

N

92 93 90

95 93 92 96 95 95

Reaction conditions: piperazines, Et3N, MeOH, reflux, 5–20 min. Isolated yields.

3. Results and discussion 3.1. Chemistry In our previous work, we have described in detail the protocol for the synthesis of 17a-(60 -chloropurin-90 -yl)-D-homo-17a-oxa4-aza-5a-androst-3-one (1) starting from 4-aza-5a-androst-3,17dione [29]. The C6-piperazine substituted 4-azasteroidal purine nucleoside analogues 2–9 were synthesized as shown in Table 1, which involved the nucleophilic substitution at the C6 position of purine moiety of series 1 with appropriate piperazines. The reaction was carried out in the presence of Et3N as an auxiliary base in refluxing MeOH for 5–20 min to give the desired products 2–9 in excellent yields. The products resulting from substitution of the 2-chloro or 2-fluoro group (for 1b, 1c) were not observed. This substitution usually required higher temperature and extended reaction time [30]. All the newly-synthesized compounds were characterized by 1H, 13C NMR and HRMS spectra.

3.2. Biology The newly synthesized purine nucleoside analogues 2–9 were evaluated for their anticancer activity in vitro against PC-3 (human prostatic carcinoma), MCF-7 (human breast carcinoma) and Hela (human cervical carcinoma) cell lines. The inhibition of test compounds was determined using the MTT assay. The anticancer activity was indicated in terms of IC50 (lM) value and the results were presented in Table 2. As shown in Table 2, it was evident from the data that the substituents at C6-position of the purine ring and N4-position of piperazine moiety had a significant influence on the cytotoxicity. Replacement of the 6-chloro group of the purine ring with piperazine did not significantly enhance the inhibitory activity. On the contrary, for most compounds of series 2–9, introduction of a

Table 2 The in vitro anticancer activity of 4-azasteroidal purine nucleoside analogues 1–9. Compound

1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c 6a 6b 6c 7a 7b 7c 8a 8b 8c 9a 9b 9c

IC50 (lM) Hela

PC-3

MCF-7

17.67 7.57 13.32 59.24 18.96 43.94 83.02 28.00 56.20 >100 29.87 54.07 >100 >100 >100 23.14 22.84 16.22 16.82 >100 34.54 21.13 >100 16.64 >100 >100 >100

3.25 11.19 3.76 68.14 15.69 25.97 >100 75.12 61.31 >100 >100 36.08 >100 >100 >100 28.02 >100 >100 >100 >100 >100 27.99 5.13 23.08 >100 1.84 >100

20.92 34.21 16.72 >100 84.60 18.02 >100 32.75 >100 >100 63.94 >100 >100 >100 >100 26.98 25.72 >100 >100 19.95 >100 22.11 12.75 29.99 >100 >100 >100

Hela: human cervical carcinoma; PC-3: human prostatic carcinoma; MCF-7: human breast carcinoma.

piperazine group resulted in a marked decrease in potency compared to series 1. For example, replacement of the 6-chloro atom with 4-hydroxyethylpiperazine (compounds 5a–c) led to loss of potency against all tested cell lines.

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For N4-phenyl substituted piperazine purine nucleoside analogues 6–8, substituents on the phenyl ring did not show significant influence on the cytotoxic activity. Among them, compound 8b, which contained the 2-chloro group and N4-(2-fluorophenyl)piperazine group on the purine ring, displayed strong cell growth inhibitory activity against PC-3, with the IC50 values of 5.13 lM. It is interesting to note that compound 9b bearing the N4-(pyrimidin-2-yl) piperazine demonstrated the best anticancer activity against PC-3 cells, with the IC50 values of 1.84 lM. However, this compound exhibited no inhibitory activity on the cell lines of Hela and MCF-7, indicating the selective inhibition of PC3 cells. In addition, compounds 9a and 9c bearing the same substituent at C6-position exhibited no inhibitory activity against all tested cell lines, suggesting that substituent at the C2-position of purine moiety also affect the anticancer activity. 4. Conclusion In summary, novel C6-piperazine substituted 4-azasteroidal purine nucleoside analogues 2–9 were efficiently prepared and evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. For most compounds of 2–9, introduction of a piperazine substituent resulted in a loss of potency compared to series 1. Among them, compounds 8b and 9b exhibited significant and selective inhibition of PC-3 cells. The research on their possible mechanism of inhibiting proliferation of cancer cell lines is underway. Acknowledgements We are grateful to the National Natural Sciences Foundation of China (No. 81172937) for financial support. References [1] (a) Haines DR, Tseng CK, Marquez VE. Synthesis and biological activity of unsaturated carboacyclic purine nucleoside analogues. J Med Chem 1987;30:943–7; (b) Lin TS, Luo MZ, Liu MC, Clarke-Katzenburg RH, Cheng YC, Prusoff WH, et al. Synthesis and anticancer and antiviral activities of various 20 - and 30 methylidene-substituted nucleoside analogues and crystal structure of 20 deoxy-20 -methylidenecytidine hydrochloride. J Med Chem 1991;34:2607–15. [2] Huryn DM, Okabe M. AIDS-driven nucleoside chemistry. Chem Rev 1992;92:1745–68. [3] Tuncbilek M, Guven EB, Onder T, Cetin Atalay R. Synthesis of novel 6-(4substituted piperazine-1-yl)-9-(b-D-ribofuranosyl) purine derivatives, which lead to senescence-induced cell death in liver cancer cells. J Med Chem 2012;55:3058–65. [4] (a) Choi WJ, Lee HW, Kim HO, Chinn M, Gao ZG, Patel A, et al. Design and synthesis of N(6)-substituted-40 -thioadenosine-50 -uronamides as potent and selective human A(3) adenosine receptor agonists. Bioorg Med Chem 2009;17:8003–11; (b) Gupta PK, Daunert S, Nassiri MR, Wotring LL, Drach JC, Townsend LB. Synthesis, cytotoxicity, and antiviral activity of some acyclic analogues of the pyrrolo[2,3-d] pyrimidine nucleoside antibiotics tubercidin, toyocamycin, and sangivamycin. J Med Chem 1989;32:402–8. [5] (a) Huang H, Liu H, Chen K, Jiang H. Microwave-assisted rapid synthesis of 2,6,9-substituted purines. J Comb Chem 2007;9:197–9; (b) Qu GR, Zhao L, Wang DC, Wu J, Guo HM. Microwave-promoted efficient synthesis of C6-cyclo secondary amine substituted purine analogues in neat water. Green Chem 2008;10:287–9. [6] Norman TC, Gray NS, Koh JT, Schultz PG. A structure-based library approach to kinase inhibitors. J Am Chem Soc 1996;118:7430–1. [7] (a) Legraverend M, Grierson DS. The purines: potent and versatile small molecule inhibitors and modulators of key biological targets. Bioorg Med Chem 2006;14:3987–4006; (b) Bressi JC, Choe J, Hough MT, Buckner FS, VanVoorhis WC, Verlinde CLMJ, et al. Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: elucidation of a novel binding mode for a 2amino-N6-substituted adenosine. J Med Chem 2000;43:4135–50; (c) Wilson SC, Atrash B, Barlow C, Eccles S, Fischer PM, Hayes A, et al. Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors. Bioorg Med Chem 2011;19:6949–65. [8] Rips R, Boschi G, Trinh MC, Cavier R. Phenol-piperazine adducts showing anthelmintic properties. J Med Chem 1973;16:725–8.

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