Steroids 85 (2014) 13–17

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Synthesis and biological evaluation of novel C6-cyclo secondary amine substituted purine steroid-nucleosides analogues Li-Hua Huang a,b,c, Yang Li a, Hong-De Xu b,c, 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 23 January 2014 Received in revised form 22 March 2014 Accepted 29 March 2014 Available online 12 April 2014 Keywords: 4-Azasteroid Purine Steroid-nucleosides Cyclic secondary amines Anticancer activity

a b s t r a c t Novel C6-cyclo secondary amine substituted purine steroid-nucleoside analogues (2–9) were efficiently synthesized through displacement of the C6 chloro on the purine ring of series 1 with versatile cyclic secondary amines, including pyrrolidines, piperidine, morpholine, and piperazines. All the newly-synthesized compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 5c and 6b 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 antitumor 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 antitumor activity as inhibitors of various protein kinases [6,7]. On the other hand, apart from naturally occurring substances, most of steroidal drugs in use today are semi-synthetic compounds via the modification of the steroid ring system and side chains [8–10]. Therefore, large numbers of steroids with unusual and interesting structures have been synthesized, some of which exhibited good antitumor activity [11–13]. 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

⇑ 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). http://dx.doi.org/10.1016/j.steroids.2014.03.017 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

C–N linkage [14]. These newly-synthesized steroid-nucleosides exhibited great anti-tumor 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 [15–18]. Our group has been interested in the design and synthesis of novel modified steroids [19–22], and we have previously described a novel series of steroid-nucleoside analogues [23]. The preliminary biological evaluation indicated that the purine nucleosides analogues 1a-c (Fig. 1) showed great anticancer activity. In view of the therapeutic importance of C6-aminopurine derivatives, we herein report the synthesis of novel C6-cyclo secondary amine substituted steroid-nucleosides analogues by displacement of the C6 chloro on the purine ring of compounds 1a-c with versatile cyclic secondary amines and their anticancer activity against PC-3, MCF-7 and Hela cell lines in vitro.

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

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L.-H. Huang et al. / Steroids 85 (2014) 13–17

N O

Cl

N N N R1

O

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

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

(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. 2a: White solid, yield 92%, mp 251–253 °C. 1H NMR (400 MHz, CDCl3): d 8.33 (s, 1H, 20 -H), 7.93 (s, 1H, 80 -H), 6.22–5.94 (m, 2H, 4 N-H and 17b-H), 3.94 (m, 4H, protons of pyrrolidine), 3.05 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 0.87 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.16, 153.07, 152.79, 149.28, 136.46, 120.07, 77.59, 76.07, 60.31, 50.64, 48.93, 47.38, 39.19, 36.07, 35.76, 33.54, 33.20, 28.53, 28.25, 27.11, 21.87, 21.53, 16.46, 11.30. HRMS (ESI): m/z cacld for C27H39N6O2 (M+H)+, 479.3134; found, 479.3133. 3a: White solid, yield 93%, mp 266–267 °C. 1H NMR (400 MHz, CDCl3): d 8.31 (s, 1H, 20 -H), 7.93 (s, 1H, 80 -H), 6.21–5.99 (m, 2H, 4 N-H and 17b-H), 4.22 (s, 4H, protons of piperidine), 3.04 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 0.87 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.17, 153.82, 152.42, 149.90, 135.73, 119.65, 77.58, 76.06, 60.31, 50.65, 48.94, 46.39, 39.19, 36.07, 35.76, 33.51, 33.21, 28.53, 28.25, 27.11, 26.15, 24.84, 21.87, 21.53, 16.47, 11.30. HRMS (ESI): m/z cacld for C28H41N6O2 (M+H)+, 493.3291; found, 493.3289. 4a: White solid, yield 94%, mp 271–273 °C. 1H NMR (400 MHz, CDCl3): d 8.33 (s, 1H, 20 -H), 7.96 (s, 1H, 80 -H), 6.28 (s, 1H, 4 N-H), 6.10 (dd, J = 11.3, 2.5 Hz, 1H, 17b-H), 5.45 (brs, 2H, 60 C-N(CH)2-), 3.06 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 1.36 (s, 3H, 18-H), 0.99 (d, J = 5.7 Hz, 6H, 60 C-N(CH)2(CH3)2), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.24, 153.65, 152.39, 149.91, 135.76, 119.65, 77.55, 76.03, 60.30, 50.64, 48.95, 42.75, 39.19, 36.07, 35.73, 33.52, 33.20, 31.53, 28.53, 28.25, 27.07, 21.86, 21.53, 19.24, 19.22, 16.46, 11.30. HRMS (ESI): m/z cacld for C30H45N6O2 (M+H)+, 521.3604; found, 521.3607. 5a: White solid, yield 93%, mp 284–285 °C. 1H NMR (400 MHz, CDCl3): d 8.33 (s, 1H, 20 -H), 7.95 (s, 1H, 80 -H), 6.29–5.97 (m, 2H, 4 N-H and 17b-H), 4.28 (s, 4H, protons of morpholine), 3.90–3.70 (m, 4H, protons of morpholine), 3.05 (dd, J = 12.3, 3.5 Hz, 1H, 5aH), 0.87 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.17, 153.86, 152.29, 150.11, 136.36, 119.87, 77.68, 76.19, 67.06, 60.30, 50.65, 48.94, 45.63, 39.18, 36.07, 35.76, 33.51, 33.20, 28.52, 28.25, 27.09, 21.87, 21.50, 16.45, 11.30. HRMS (ESI): m/z cacld for C27H39N6O3 (M+H)+, 495.3084; found, 495.3052. 6a: White solid, yield 91%, mp 264–265 °C. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, 20 -H), 7.95 (s, 1H, 80 -H), 6.31 (s, 1H, 4 N-H),

6.10 (d, J = 9.9 Hz, 1H, 17b-H), 5.32 (d, J = 41.6 Hz, 2H, protons of piperazine), 3.13 (d, J = 11.3 Hz, 2H, protons of piperazine), 3.05 (d, J = 10.8 Hz, 1H, 5a-H), 2.91 (d, J = 10.4 Hz, 2H, protons of piperazine), 2.77 (s, 1H, protons of piperazine), 1.16 (d, J = 5.1 Hz, 3H, – NCHCH3), 0.87 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.28, 153.75, 152.30, 150.02, 136.09, 119.73, 77.63, 76.11, 60.29, 50.64, 48.92, 45.98, 39.17, 36.05, 35.71, 33.49, 33.17, 28.51, 28.24, 27.03, 21.85, 21.50, 19.53, 16.45, 11.30. HRMS (ESI): m/z cacld for C28H42N7O2 (M+H)+, 508.3400; found, 508.3401. 7a: White solid, yield 93%, mp 266–268 °C. 1H NMR (400 MHz, CDCl3): d 8.34 (s, 1H, 20 -H), 7.96 (s, 1H, 80 -H), 6.23 (s, 1H, 4 N-H), 6.11 (d, J = 10.8 Hz, 1H, 17b-H), 4.33 (s, 4H, protons of piperazine), 3.06 (d, J = 12.1 Hz, 1H, 5a-H), 2.53 (s, 4H, protons of piperazine), 2.34 (s, 3H, –NCH3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.19, 153.81, 152.31, 150.04, 136.15, 119.81, 77.63, 76.14, 60.29, 55.13, 50.65, 48.94, 46.19, 39.18, 36.07, 35.74, 33.49, 33.20, 28.51, 28.24, 27.08, 21.86, 21.50, 16.44, 11.29. HRMS (ESI): m/z cacld for C28H42N7O2 (M+H)+, 508.3400; found, 508.3402. 8a: White solid, yield 96%, mp 201–203 °C. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, 20 -H), 7.94 (s, 1H, 80 -H), 7.30 (m, 5H, Ar-H), 6.08 (m, 2H, 4 N-H and 17b-H), 4.30 (s, 4H, protons of piperazine), 3.54 (s, 2H, ArCH2N–), 3.05 (dd, J = 12.2, 3.3 Hz, 1H, 5a-H), 2.64– 2.48 (m, 4H, protons of piperazine), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.17, 153.77, 152.34, 149.99, 137.73, 136.07, 129.25, 128.31, 127.21, 119.76, 77.63, 76.11, 63.14, 60.31, 53.20, 50.64, 48.93, 45.10, 39.18, 36.06, 35.76, 33.51, 33.19, 28.53, 28.25, 27.12, 21.87, 21.51, 16.46, 11.31. HRMS (ESI): m/z cacld for C34H46N7O2 (M+H)+, 584.3713; found, 584.3711. 9a: White solid, yield 95%, mp 186–188 °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, 2.5 Hz, 1H, 17b-H), 5.98 (s, 1H, 4 N-H), 4.27 (s, 4H, protons of piperazine), 3.54 (s, 4H, protons of piperazine), 3.06 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 1.48 (s, 9H, –C(CH3)3), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.11, 154.79, 153.79, 152.30, 150.12, 136.37, 119.87, 80.10, 77.68, 76.18, 60.31, 50.64, 48.93, 39.18, 36.07, 35.78, 33.50, 33.20, 28.53, 28.42, 28.25, 27.14, 21.88, 21.51, 16.46, 11.31. HRMS (ESI): m/z cacld for C32H48N7O4 (M+H)+, 594.3768; found, 594.3766. 2b: White solid, yield 94%, mp 199–202 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.39 (s, 1H, 4 N-H), 6.04 (dd, J = 11.2, 2.7 Hz, 1H, 17b-H), 4.14 (s, 2H, protons of pyrrolidine), 3.74 (s, 2H, protons of pyrrolidine), 3.05 (dd, J = 12.4, 3.7 Hz, 1H, 5a-H), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.30, 154.03, 153.27, 150.36, 136.85, 118.96, 82.98, 77.71, 76.26, 60.28, 50.66, 48.93, 47.69, 39.20, 36.05, 35.71, 33.73, 33.19, 28.51, 28.24, 27.01, 21.86, 21.38, 16.56, 11.30. HRMS (ESI): m/z cacld for C27H37ClN6O2Na (M+Na)+, 535.2564; found, 535.2567. 3b: White solid, yield 94%, mp 248–249 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.29 (brs, 1H, 4 N-H), 6.04 (dd, J = 11.2, 2.1 Hz, 1H, 17b-H), 4.20 (brs, 4H, protons of piperidine), 3.06 (d, J = 10.2 Hz, 1H, 5a-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.29, 153.86, 153.81, 151.09, 136.06, 118.47, 77.68, 76.26, 60.30, 50.67, 48.96, 39.21, 36.05, 35.73, 33.66, 33.21, 28.54, 28.24, 27.03, 26.12, 24.63, 21.85, 21.38, 16.55, 11.30. HRMS (ESI): m/z cacld for C28H40ClN6O2 (M+H)+, 527.2901; found, 527.2908. 4b: White solid, yield 96%, mp 241–242 °C. 1H NMR (400 MHz, CDCl3): d 8.35 (s, 1H, 80 -H), 6.28 (s, 1H, 4 N-H), 6.10 (dd, J = 11.2, 2.4 Hz, 1H, 17b-H), 5.40 (brs, 2H, 60 C-N(CH)2–), 3.06 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 1.38 (s, 3H, 18-H), 0.99 (d, J = 5.7 Hz, 6H, 60 CN(CH)2(CH3)2), 0.91 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.58, 153.78, 152.45, 149.98, 135.83, 118.65, 77.56, 76.19, 60.30, 50.66, 48.97, 42.77, 39.21, 36.09, 35.75, 33.54, 33.22, 31.55, 28.55, 28.27, 27.09, 21.88, 21.55, 19.26, 19.24, 16.48, 11.32. HRMS (ESI): m/z cacld for C30H44ClN6O2 (M+H)+, 555.3214; found, 555.3212.

L.-H. Huang et al. / Steroids 85 (2014) 13–17

5b: White solid, yield 92%, mp 248–250 °C. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, 80 -H), 6.41 (d, J = 24.4 Hz, 1H, 4 N-H), 6.04 (d, J = 10.6 Hz, 1H, 17b-H), 4.28 (s, 4H, protons of morpholine), 3.81 (d, J = 3.1 Hz, 4H, protons of morpholine), 3.06 (d, J = 11.9 Hz, 1H, 5aH), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.20, 153.89, 153.79, 151.30, 136.69, 118.65, 77.78, 76.43, 66.95, 60.30, 50.69, 48.98, 39.21, 36.07, 35.77, 33.67, 33.22, 28.53, 28.25, 27.08, 21.86, 21.36, 16.53, 11.30. HRMS (ESI): m/z cacld for C27H37ClN6O3Na (M+Na)+, 551.2513; found, 551.2515. 6b: White solid, yield 93%, mp 220–221 °C. 1H NMR (400 MHz, CDCl3): d 7.93 (s, 1H, 80 -H), 6.31 (m, 1H, 4 N-H), 6.05 (dd, J = 11.2, 2.3 Hz, 1H, 17b-H), 3.99 (brs, 2H, protons of piperazine), 3.17 (d, J = 12.1 Hz, 2H, protons of piperazine), 3.07 (dd, J = 12.3, 3.2 Hz, 1H, 5a-H), 1.19 (d, J = 5.9 Hz, 3H, –NCHCH3), 0.90 (s, 3H, 19-H). 13 C NMR (100 MHz, CDCl3): d 172.40, 153.78, 151.24, 136.44, 118.55, 77.73, 76.35, 60.30, 50.88, 50.66, 48.96, 45.65, 39.20, 36.05, 35.76, 33.67, 33.15, 28.44, 28.24, 27.03, 21.85, 21.37, 19.24, 16.54, 11.30. HRMS (ESI): m/z cacld for C28H41ClN7O2 (M+H)+, 542.3010; found, 542.3008. 7b: White solid, yield 95%, mp 237–238 °C. 1H NMR (400 MHz, CDCl3): d 7.93 (s, 1H, 80 -H), 6.05 (dd, J = 11.2, 2.5 Hz, 1H, 17b-H), 5.84 (s, 1H, 4 N-H), 4.25 (brs, 4H, protons of piperazine), 3.08 (dd, J = 12.2, 3.5 Hz, 1H, 5a-H), 2.54 (s, 4H, protons of piperazine), 2.36 (s, 3H, –NCH3), 0.91 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.05, 153.84, 153.79, 151.22, 136.47, 118.60, 77.75, 76.36, 60.31, 55.04, 50.65, 48.95, 46.12, 39.19, 36.06, 35.80, 33.69, 33.21, 28.53, 28.25, 27.15, 21.87, 21.37, 16.55, 11.30. HRMS (ESI): m/z cacld for C28H41ClN7O2 (M+H)+, 542.3010; found, 542.3011. 8b: White solid, yield 95%, mp 183–187 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 7.35 (m, 5H, Ar-H), 6.05 (dd, J = 11.2, 2.4 Hz, 1H, 17b-H), 5.91 (m, 1H, 4 N-H), 4.21 (brs, 4H, protons of piperazine), 3.57 (s, 2H, ArCH2N–), 3.08 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 2.61–2.54 (m, 4H, protons of piperazine), 0.90 (s, 3H, 19H). 13C NMR (100 MHz, CDCl3): d 172.07, 153.80, 151.19, 137.61, 136.38, 129.21, 128.32, 128.24, 127.25, 118.58, 77.72, 76.35, 63.04, 60.32, 53.09, 50.68, 48.98, 39.21, 36.07, 35.81, 33.67, 33.22, 28.53, 28.26, 27.16, 21.87, 21.38, 16.54, 11.30. HRMS (ESI): m/z cacld for C34H45ClN7O2 (M+H)+, 618.3323; found, 618.3325. 9b: White solid, yield 94%, mp 192–194 °C. 1H NMR (400 MHz, CDCl3): d 7.93 (s, 1H, 80 -H), 6.29 (d, J = 8.7 Hz, 1H, 4 N-H), 6.04 (d, J = 10.6 Hz, 1H, 17b-H), 4.26 (brs, 4H, protons of piperazine), 3.55 (s, 4H, protons of piperazine), 3.06 (d, J = 10.2 Hz, 1H), 1.49 (s, 9H, –C(CH3)3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.18, 154.67, 153.88, 153.76, 151.32, 136.71, 118.67, 80.19, 77.77, 76.43, 65.80, 60.30, 50.70, 48.98, 45.04, 39.21, 36.07, 35.75, 33.63, 33.21, 28.50, 28.39, 28.25, 27.03, 21.85, 21.36, 16.52, 15.24, 11.29. HRMS (ESI): m/z cacld for C32H46ClN7O4Na (M+Na)+, 650.3198; found, 650.3196. 2c: White solid, yield 90%, mp 233–234 °C. 1H NMR (400 MHz, CDCl3): d 7.89 (s, 1H, 80 -H), 6.42 (s, 1H, 4 N-H), 5.99 (dd, J = 11.3, 2.4 Hz, 1H, 17b-H), 4.16 (t, J = 6.2 Hz, 2H, protons of pyrrolidine), 3.72 (t, J = 6.2 Hz, 2H, protons of pyrrolidine), 3.05 (dd, J = 12.3, 3.4 Hz, 1H, 5a-H), 0.88 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.30, 159.03 (d, J = 207.0 Hz), 154.08 (d, J = 20.4 Hz), 150.78 (d, J = 19.8 Hz), 136.75 (d, J = 2.3 Hz), 118.32 (d, J = 4.2 Hz), 77.69, 76.25, 60.28, 50.64, 48.96, 47.67, 39.17, 36.04, 35.70, 33.42, 33.18, 28.51, 28.23, 27.01, 26.21, 24.17, 21.85, 21.42, 16.47, 11.29. HRMS (ESI): m/z cacld for C27H38FN6O2 (M+H)+, 497.3040; found, 497.3041. 3c: White solid, yield 92%, mp 190–193 °C. 1H NMR (400 MHz, CDCl3): d 7.90 (s, 1H, 80 -H), 6.31 (d, J = 14.2 Hz, 1H, 4 N-H), 6.10– 5.94 (m, 1H, 17b-H), 3.86 (m, 4H, protons of piperidine), 3.06 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.14, 158.94 (d, J = 205.7 Hz), 154.66 (d,

15

J = 20.0 Hz), 151.54 (d, J = 19.5 Hz), 135.93 (d, J = 2.7 Hz), 117.79 (d, J = 4.2 Hz), 77.69, 76.24, 60.32, 50.64, 48.90, 45.53, 39.17, 36.05, 35.78, 33.40, 33.19, 28.53, 28.24, 27.14, 26.13, 24.63, 21.87, 21.44, 16.48, 11.30. HRMS (ESI): m/z cacld for C28H40FN6O2 (M+H)+, 511.3197; found, 511.3196. 4c: White solid, yield 91%, mp 271–273 °C. 1H NMR (400 MHz, CDCl3): d 7.90 (s, 1H, 80 -H), 6.08 (s, 1H, 4 N-H), 6.01 (dd, J = 11.3, 2.5 Hz, 1H, 17b-H), 4.97 (brs, 2H, 60 C-N(CH)2-), 3.07 (dd, J = 12.3, 3.6 Hz, 1H, 5a-H), 1.35(s, 3H, 18-H), 0.99 (d, 6.4,6H, 60 C-N(CH)2(CH3)2), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.16, 158.90 (d, J = 205.8 Hz), 154.50 (d, J = 19.8 Hz), 151.57 (d, J = 19.3 Hz), 135.95 (d, J = 2.6 Hz), 117.82 (d, J = 4.2 Hz), 77.64, 76.23, 60.31, 50.66, 48.93, 42.56, 39.19, 36.06, 35.77, 33.39, 33.21, 28.52, 28.25, 27.11, 21.86, 21.44, 19.13, 16.46, 11.30. HRMS (ESI): m/z cacld for C30H44FN6O2 (M+H)+, 539.3510; found, 539.3511. 5c: White solid, yield 90%, mp 188–190 °C. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, 80 -H), 6.03 (m, 2H, 4 N-H and 17b-H), 4.20 (s, 2H, protons of morpholine), 3.92–3.70 (m, 6H, protons of morpholine), 3.08 (dd, J = 12.4, 3.8 Hz, 1H, 5a-H), 0.89 (d, J = 12.3 Hz, 3H). 13C NMR (100 MHz, CDCl3): d 172.12, 158.77 (d, J = 207.3 Hz), 154.78 (d, J = 19.6 Hz), 151.76 (d, J = 19.1 Hz), 136.56 (d, J = 2.6 Hz), 117.95 (d, J = 4.2 Hz), 77.79, 76.39, 66.95, 60.31, 50.64, 48.90, 39.16, 36.05, 35.78, 33.41, 33.20, 28.52, 28.24, 27.13, 21.87, 21.41, 16.46, 11.31. HRMS (ESI): m/z cacld for C27H38FN6O3 (M+H)+, 513.2989; found, 513.3019. 6c: White solid, yield 91%, mp 230–232 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.10–5.93 (m, 2H, 4 N-H and 17b-H), 3.18 (d, J = 12.0 Hz, 2H, protons of piperazine), 3.08 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 2.96 (d, J = 8.8 Hz, 2H, protons of piperazine), 1.21 (d, 5.7 Hz, 3H, –NCHCH3), 0.90 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.13, 157.74, 154.53, 151.80, 136.32, 117.91, 77.74, 76.32, 60.33, 50.63, 48.90, 39.16, 36.04, 35.78, 33.40, 33.18, 28.53, 28.24, 27.12, 21.87, 21.42, 19.25, 16.47, 11.31. HRMS (ESI): m/z cacld for C28H41FN7O2 (M+H)+, 526.3306; found, 526.3308. 7c: White solid, yield 90%, mp 189–191 °C. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H, 80 -H), 6.17 (s, 1H, 4 N-H), 6.00 (dd, J = 11.3, 2.4 Hz, 1H, 17b-H), 4.82–3.82 (m, 4H, protons of piperazine), 3.07 (dd, J = 12.3, 3.5 Hz, 1H, 5a-H), 2.54 (s, 4H, protons of piperazine), 2.35 (s, 3H, –NCH3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.20, 157.77, 154.81, 151.78, 136.34, 117.93, 77.75, 76.32, 60.30, 55.01, 50.63, 48.89, 46.12, 39.16, 36.04, 35.75, 33.40, 33.19, 28.52, 28.24, 27.08, 21.86, 21.41, 16.47, 11.31. HRMS (ESI): m/z cacld for C28H41FN7O2 (M+H)+, 526.3306; found, 526.3304. 8c: White solid, yield 92%, mp 169–171 °C. 1H NMR (400 MHz, CDCl3): d 7.90 (s, 1H, 80 -H), 7.52–7.23 (m, 5H, Ar-H), 6.36 (s, 1H, 4 N-H), 6.00 (dd, J = 11.2, 2.2 Hz, 1H, 17b-H), 4.79–3.72 (m, 4H, protons of piperazine), 3.56 (s, 2H, ArCH2N), 3.07 (dd, J = 12.2, 3.1 Hz, 1H, 5a-H), 2.57 (s, 4H, protons of piperazine), 0.89 (s, 3H, 19-H). HRMS (ESI): m/z cacld for C34H45FN7O2 (M+H)+, 602.3619; found, 602.3618. 13C NMR (400 MHz, CDCl3) d 172.18, 158.78 (d, J = 206.8 Hz), 154.69 (d, J = 20.0 Hz), 151.72 (d, J = 19.3 Hz), 136.52 (d, J = 2.3 Hz), 117.94 (d, J = 4.4 Hz), 77.76, 76.35, 63.20, 60.31, 53.21, 50.63, 48.90, 44.50, 39.16, 36.05, 35.79, 33.40, 33.20, 28.51, 28.24, 27.08, 21.87, 21.41, 16.47, 11.31. HRMS (ESI): m/z cacld for C34H45FN7O2 (M+H)+, 602.3619; found, 602.3618. 9c: White solid, yield 93%, mp 188–190 °C. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, 80 -H), 6.44–6.19 (m, 1H, 4 N-H), 6.00 (dd, J = 11.3, 2.1 Hz, 1H, 17b-H), 4.78–3.74 (m, 4H, protons of piperazine), 3.54 (d, J = 15.7 Hz, 4H, protons of piperazine), 3.17–2.95 (m, 1H, 5a-H), 1.49 (s, 9H, -C(CH3)3), 0.89 (s, 3H, 19-H). 13C NMR (100 MHz, CDCl3): d 172.23, 159.76, 157.70, 154.85, 151.87, 136.57, 117.98, 80.26, 77.78, 76.37, 60.29, 50.64, 48.89, 44.46,

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L.-H. Huang et al. / Steroids 85 (2014) 13–17

39.16, 36.04, 35.73, 33.39, 33.19, 28.52, 28.40, 28.24, 27.03, 21.86, 21.41, 16.46, 11.31. HRMS (ESI): m/z cacld for C32H47FN7O4 (M+H)+, 612.3674; found, 612.3625. 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. 3. Results and discussion

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

R1

–NR2R3

1a 1b 1c 2a 2b 2c

H Cl F H Cl F



3a 3b 3c

H Cl F

4a 4b 4c

H Cl F

5a 5b 5c

H Cl F

O

6a 6b 6c

H Cl F

H N

IC50 (lM) Hela

PC-3

MCF-7

17.67 7.57 13.32 >100 >100 36.47

3.25 11.19 3.76 >100 >100 48.16

20.92 34.21 16.72 >100 >100 >100

>100 >100 32.80

>100 >100 32.18

>100 >100 >100

>100 16.30 63.65

>100 17.77 12.11

>100 34.92 10.78

>100 57.04 >100

>100 >100 1.89

>100 >100 >100

19.11 30.47 27.89

>100 2.90 74.94

>100 41.02 >100

>100 39.66 >100

>100 22.48 >100

>100 >100 68.57

Ph

47.04 20.75 45.28

28.67 15.64 23.64

29.52 20.93 31.83

O

24.91 21.51 14.58

31.32 23.72 34.24

15.48 12.75 14.97

N

N

N

N

N 3.1. Chemistry The protocol for the synthesis of C6-cyclo secondary amine substituted 4-azasteroidal purine nucleoside analogues 2–9 was outlined in Scheme 1, which involved the nucleophilic substitution at the C6 position of purine moiety with appropriate cyclic secondary amines including pyrrolidines, piperidine, morpholine, piperazines and N-substituted 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 purine nucleoside analogues 2–9 in excellent yields. The 2-halogen group on purine ring was less reactive and this substitution usually required higher temperature and extended reaction time [24]. Thus, the products resulting from substitution of the 2-chloro or 2-fluoro group (for 1b, 1c) were not observed. All the newly-synthesized compounds were characterized by 1H, 13C NMR and HRMS spectra.

7a 7b 7c

H Cl F

N N

8a 8b 8c

H Cl F

9a 9b 9c

H Cl F

N N

O N N

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 1. As shown in Table 1, it was evident from the data that change of substituents on the purine ring had a significant influence on the cytotoxicity. Replacement of the 6-chloro group of the purine ring with cyclic secondary amines did not

N O

N N

Cl N R1

O

N H

enhance the inhibitory activity. On the contrary, for most compounds of series 2–9, introduction of a cyclic amino group resulted in a marked decrease in potency compared to series 1, for example, compounds 2a-c. Hela: human cervical carcinoma; PC-3: human prostatic carcinoma, MCF-7: human breast carcinoma. Among the screened compounds 2–9, compound 5c, which contained the 2-fluoro group and 6-morpholine group on the purine ring, demonstrated the best anticancer activity against PC-3 cells, with the IC50 values of 1.89 lM. However, this compound exhibited no inhibitory activity on Hela and MCF-7 cell lines, indicating

N R3 HN R2

O

N

Et 3N, MeOH, ref lux, 5-20 min

N R1

O 1

N

R3 N R

N H 2-9

Scheme 1. Synthesis of steroidal C6-aminopurine nucleoside analogues 2–9.

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L.-H. Huang et al. / Steroids 85 (2014) 13–17

the selective inhibition of PC-3 cells. In addition, compounds 5a and 5b bearing the same substituent at C6-position exhibited no inhibitory activity on PC-3 cell lines, suggesting that substituent at the C2-position of purine moiety also affect the anticancer activity. Generally, the 2-chloro or 2-fluoro substituted analogues showed higher activity compared to C2-unsubstituted compounds (R1 = H). For C6-piperazine derivatives substituted purine nucleoside analogues 6–9, compound 6b bearing the 2-chloro-6-(3-methylpiperazine)purine ring displayed potent inhibitory activity against PC-3 cells with the IC50 values of 2.90 lM. For compounds 7–9, the results showed that N4-substituent at piperazine ring did not show significant influence on the cytotoxic activity. It was noteworthy that compounds 8 and 9 displayed moderate anticancer activity against all tested cell lines and comparable activity with series 1 on MCF-7 cell lines. 4. Conclusion In summary, novel C6-cyclo secondary amine substituted 4azasteroidal purine nucleoside analogues 2–9 were efficiently prepared in excellent yields 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 an amino substituent resulted in a loss of potency compared to series 1. Among the screened compounds 2–9, compounds 5c and 6b exhibited significant anticancer activity against PC-3 cell lines, indicating the selective inhibition of PC-3 cells. Acknowledgement 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.

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Synthesis and biological evaluation of novel C6-cyclo secondary amine substituted purine steroid-nucleosides analogues.

Novel C6-cyclo secondary amine substituted purine steroid-nucleoside analogues (2-9) were efficiently synthesized through displacement of the C6 chlor...
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