Benzophenone-nucleoside derivatives as telomerase inhibitors: Design, synthesis and anticancer evaluation in vitro and in vivo

European Journal of Medicinal Chemistry 124 (2016) 729e739

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Benzophenone-nucleoside derivatives as telomerase inhibitors: Design, synthesis and anticancer evaluation in vitro and in vivo Jing Bo Shi a, Liu Zeng Chen a, Yang Wang a, Cheng Xiou a, Wen Jian Tang a, Hai Pin Zhou c, Xin Hua Liu a, c, *, Qi Zheng Yao b, ** a b c

School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China School of Pharmacy, China Pharmaceutical University, NanJing, 210009, PR China School of Material Science Chemical Engineering, ChuZhou University, ChuZhou, 239000, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 July 2016 Received in revised form 22 August 2016 Accepted 3 September 2016 Available online 5 September 2016

Based on telomerase, thirteen novel phenstatin moiety linked stavudine derivatives (8a~8e and 11a~11f) were synthesized. The structures were determined by NMR and TOF-HRMS. The screening results showed that some compounds had better anti-cancer activity in vivo and in vitro. Among them, Compound 8d showed high inhibitory activity against telomerase and showed good antiproliferative activity against SGC-7901 cell with IC50 value 0.77 mM by inducing cell cycle arrest at G2 phase. It also could improve SGC-7901 cell apoptosis, mitochondrial membrane potential assay indicated that the dissipation of MMP might participate in apoptosis induced by title compound. In vivo studies showed that compound 8d displayed potent anticancer activity with inhibition tumor growth. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Benzophenone-nucleoside derivatives Anticancer activity Telomerase Inhibitor

1. Introduction Telomerase plays important role in early stage of lifemaintaining telomere and chromosomal integrity of frequently dividing cells [1]. About 90% of cancer cells from a large range of different cancer types have detectable telomerase activity, telomerase is hence believed as a potential anticancer target [2e5]. As the component of telomerase, expression of telomerase reverse transcriptase (TERT) is the rate-limiting factor for telomerase activity, and most human somatic cells do not show detectable telomerase activity due to lack of TERT, TERT is hence regarded as the key role for the drug discovery [6,7]. Target telomerase, a lot of works have been carried out in recent years [8e10]. Unfortunately, highly efficient and selective activity molecule has not been designed so far. Combretastatin A-4 (Fig. 1A) is a natural product, its cis-stilbene shows potent cytotoxicity against a broad spectrum of human cancer cell lines [11]. Based on CA-4, large analogues [12e14] and

its structure-activity relationships (SARs) studies of this compound have been widely carried out [15e17]. Among them, phenstatin (Fig. 1B) strongly inhibited cancer cellular growth and tubulin polymerization and caused significant arrest of mitosis. BIBR1532, a selective TERT inhibitor shows the mechanism of action similar to inhibitors of HIV-1 reverse transcriptase. Known to all, stavudine was demonstrated potent anti-HIV-1 activity. Motivated by the afore-mentioned findings, taking into account the similarity of their mechanistic and structural similarities between TERT and HIV-1 reverse transcriptase, we anticipate that the presence of stavudine in the phenstatin moiety should play an important role in the telomerase activity. Herein, in continuation to extend our research for searching effective telomerase inhibitors [18e20], some novel compounds of phenstatin moiety linked stavudine (Fig. 1C) will been designed and optimized in this study. 2. Results and discussion 2.1. Chemistry

* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China. ** Corresponding author. E-mail addresses: [email protected] (X.H. Liu), [email protected] (Q.Z. Yao). http://dx.doi.org/10.1016/j.ejmech.2016.09.011 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

According to known procedure [21], benzophenones, analogues of phenstatin (5a~5e) were prepared (Scheme 1) from guaiacol 1. 13 novel benzophenone-nucleoside hybrid derivatives were designed

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Fig. 1. The workflow of the general design strategy in this study.

Scheme 1. Synthesis of compounds 5a~5e Reagents and conditions: (A) ClCH2COCl, 120e130
C, 5e8 h; (B) MeSO3H/P2O5, 40
C, 8e10 h, (C) NaOAc, MeOH, reflux, 30 min.

and synthesized (Schemes 2e3). In order to optimize the reaction conditions, we applied a modified methodology in the second and third stage. Thus, Eaton's reagent (MeSO3H/P2O5) at 40e60
C was used, afforded the precursors 4a~4e with high yields. Deprotection of chloroacetyl group was achieved with sodium acetate and obtained benzophenone analogues (5a~5e). Title compounds 8a~8e were obtained with steps: First, succinic anhydride was introduced at the 50 -position of stavudine in dry dichloromethane (DCM) in the presence of triethylamine (TEA) to obtain compound 7. The compounds were treated with corresponding benzophenones (5a~5e) in dry DCM, in the presence of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to afford compounds 8a~8e, as show in Scheme 2. The target compounds 11a~11h were synthesized according to Scheme 3. Treatment of the key benzophenones 5a~5e with ethyl bromoacetate in dry acetone in the presence of K2CO3 produced the corresponding benzophenone derivatives 9a~9h. They were converted to corresponding carboxylic acid derivatives 10a~10h in base. Finally, stavudine 6 was treated with corresponding 10a~10h in dry DCM, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) car-bodiimide (EDC$HCl), N-hydroxybenzotriazole monohydrate (HoBt) and triethylamine (TEA) to afford target compounds

11a~11h. All compounds were characterized by HR-MS, 1H NMR and 13C NMR spectral analysis. 2.2. Anticancer activity At first, all title compounds were evaluated for their antiproliferative activities against cervical cancer cell (Hela), human hepatocellular carcinoma cell (SMMC-7721), human gastric cancer cell (SGC-7901) and human hepatocellular carcinoma (HepG2) cell lines. Also included was the activity of reference AMD. The cells were allowed to proliferate in presence of tested material for 48 h, and the results were reported in terms of IC50 values (Table 1). From Table 1, it is obvious to see that compounds 8a and 8d exhibited high activity against four cell lines. Among them, compound 8d possessed potent activity against Hela, SMMC-7721 and SGC-7901 cell lines with IC50 ¼ 1.58 ± 0.20, 0.82 ± 0.11 and 0.77 ± 0.33 mM, respectively, which were even better than that of the commercial AMD. But, compounds 11a~11f exhibited poor inhibitory activity against all the tested cells. The preliminary results show that the length of the linker between phenstatin and stavudine had a great influence on the activities.

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Scheme 2. Synthesis of title compounds 8a~8e Reagents and conditions: (A) Succinic anhydride, Et3N, CH2Cl2, r.t., 12 h; (B) DCC, DMAP, CH2Cl2, r.t., 12 h.

Scheme 3. Synthesis of title compounds 11a~11h Reagents and conditions: (A) Ethyl bromoacetate, K2CO3, Acetone, reflux, 4e5 h; (B) 2 N NaOH, MeOH, r.t., 2e3 h; (C) EDC$HCl, HoBt, DCM, Et3N, r.t., 12 h.

2.3. Annexin V/propidium iodide assay To determine whether the cells death induced by the compound 8d was due to apoptosis or necrosis, the compound was further investigated to SGC-7901 cells using an Annexin V-FITC/propidium iodide (PI) assay. As phosphatidylserine (PS) exposure usually precedes loss of plasma membrane integrity in apoptosis, the presence of AnnexinVþ/PI- cells can be considered as an indicator of apoptosis. The apoptosis ratios induced by compound 8d were quantitatively determined by flow cytometry. And SGC-7901 cells not treated with compound 8d were used as control. Four quadrant images were observed by flow cytometric analysis: the Q1 area represented damaged cells appearing in the process of cell collection, the Q2 area represented necrotic cells and later period apoptotic cells, Q3 area represented early apoptotic cells, and the normal cells were located in the Q4 area. The results were given in Fig. 2. After 48 h, apoptosis ratios (including the early and late apoptosis ratios) were obtained by treatment at the concentration of 1 mM, 2 mM and 4 mM. The apoptosis of SGC-7901 cells treated with compound 8d increased gradually in a concentration manner.

The apoptosis ratios of compound 8d measured at different concentration points were found to 13.3% (1 mM), 38% (2 mM) and 59.6% (4 mM), respectively, while that of control was 3.23%. The results revealed that compound 8d could induce SGC-7901 cells apoptosis. 2.4. Mitochondrial membrane potential (MMP) assay The loss of mitochondrial membrane potential (Dj) is regarded as a limiting factor in the apoptotic pathway, which has been considered as an antitumor target and is of high importance to the control of the cell apoptosis. To investigate the role of mitochondria in the apoptosis induced by compound 8d, the effect of compound on MMP was measured by flow cytometry after SGC-7901 cells were stained with JC-1, which is capable of selectively entering mitochondria to form monomers that emit green fluorescence at low MMP, and form JC-1 aggregates that emit red fluorescence at high MMP. After treated for 48 h with compound 8d, compared with the control group, the number of treated cells emitting red fluorescence significantly decreased while the number of treated cells emitting green fluorescence obviously increased which

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Table 1 In vitro anticancer activity of compounds 8 and 11 against Hela, SMMC-7721, HepG2 and SGC-7901 cell linesb. Comp

8a 8b 8c 8d 8e 11a 11b 11c 11d 11e 11f 11g 11h AMDc a

IC50/mm Hela

SMMC-7721

HepG2

SGC-7901

2.65 ± 0.39 17.80 ± 2.35 21.11 ± 2.11 1.58 ± 0.20 6.51 ± 0.88 12.80 ± 1.03 28.60 ± 1.87 36.31 ± 2.01 da 50.21 ± 2.89 e 30.21 ± 2.33 45.80 ± 1.65 3.99 ± 0.35

2.86 ± 0.39 22.59 ± 1.93 30.51 ± 1.80 0.82 ± 0.11 3.21 ± 0.98 21.15 ± 2.02 20.11 ± 2.20 40.29 ± 1.28 e e e 32.33 ± 2.95 40.60 ± 2.51 1.95 ± 0.17

6.85 ± 1.22 20.30 ± 4.00 24.02 ± 1.55 3.15 ± 0.78 11.09 ± 1.40 14.96 ± 1.06 28.57 ± 2.41 37.99 ± 1.87 e e e e e 2.67 ± 0.31

2.27 ± 1.48 8.25 ± 1.81 10.20 ± 1.67 0.77 ± 0.33 4.19 ± 1.62 23.39 ± 1.60 31.42 ± 2.97 29.10 ± 1.95 e 48.14 ± 2.51 e e e 3.41 ± 0.20

Not observed in the tested concentration range. The standard deviation (SD) of three time independent tests. The IC50 value was defined as the concentration at which 50% survival of cells was observed. Inactive at 50 mM (highest concentration tested). c AMD used as a positive control. b

suggested the disruption of MMP. The results indicated that dissipation of MMP might participate in apoptosis induced by compound 8d (Figs. 3e4).

2.5. Cell cycle analysis To understand whether the reduced cell proliferation was due to cell cycle, the effect of compound 8d on the cells cycle was investigated by flow cytometry. Cytometric profiles of the PI-stained DNA showed cell cycle arrest of SGC-7901 cells treated with compound 8d for 48 h at different concentrations. As shown in Fig. 5, the treatment of SGC-7901 cells with compound 8d enhanced cell cycle arrest at the G2 phase at different concentrations, resulting in concomitant population increase in the G2 phase (30%, 42.6% and 60.9%) compared with the control cells (24.6%). The population of the G1 phase decrease at a certain extent (32.9%, 28.5% and 15.8%) compared with the control cells (38.1%). For S phase, the cell percentages were decreased obviously from 37.4% to 37.1%, 28.9% and 23.2%. These data indicated that compound 8d significantly induced cells cycle arrest at G2 phase.

Fig. 2. Apoptosis ratio detected by Annexin V/PI assay on the SGC-7901 cells treated with compound 8d of 1 mM, 2 mM and 4 mM for 48 h, respectively. Analyses were performed at least 3 times, and a representative experiment was presented.

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Fig. 3. MMP assay on the SGC-7901 cells treated with compound 8d of 1 mM, 2 mM and 4 mM for 48 h, respectively. Analyses were performed at least 3 times, and a representative experiment was presented.

2.6. Anticancer activity evaluation in vivo Mice sarcoma cells S180 and hepatoma cells HepG2 models are recognized as the reliable research models for study of antitumor pharmacodynamics. To further verify the inhibitory against the growth of tumor cells in vivo, sarcoma cells S180 and hepatoma cells HepG2 models were selected to evaluate its antitumor effects. The inhibitory effects of compound 8d against the growth of the transplanted S180 or HepG2 carcinoma were presented in Table 2. The results revealed that compound 8d significantly decreased the tumor weights of S180 and HepG2 tumor-bearing mice. The potent activity was showed with inhibitory rates 40.6% and 48.5% for S180 and HepG2 at the dosage of 60 mg/kg/day, respectively. 2.7. Telomerase activity To confirm if the synthesized compounds performed anticancer activity via telomerase inhibition as designed, some title compounds were evaluated by a modified TRAP assay using an extraction from SGC-7901 cells. Modified TRAP is a powerful technique to determine small molecules inhibiting telomere elongation qualitatively and quantitatively [22]. The results were summarized in Table 3. Among them, compounds 8a and 8d showed potent inhibitory activities against telomerase with IC50 values at 6.43 and 7.01 mM, better than positive control staurosporine.

Therefore, the length of the linker between phenstatin and stavudine played an important role for the telomerase activity. This preliminary SAR could offer the useful information for further structural optimization. 3. Conclusions In this study, two series of phenstatin-stavudine derivatives were designed, synthesized and biologically evaluated as potential telomerase inhibitors. Among the 13 compounds, compounds 8d exhibited potent activities against Hela, SMMC-7721, HepG2 and SGC-7901 cells. In vivo studies showed that compound 8d displayed potent anticancer activity with inhibition tumor growth of S180 and HepG2 tumor-bearing mice. Moreover, compound 8d showed high inhibitory activity against telomerase. Annexin V/propidium iodide assay results revealed that compound 8d could induce apoptosis in SGC-7901 cells. Further mitochondrial membrane potential assay indicated that the dissipation of MMP might participate in apoptosis induced by compound 8d. 4. Experimental section 4.1. Chemistry The reactions were monitored by thin layer chromatography

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Fig. 4. Collapse of mitochondrial membrane potential of SGC-7901 cells treated by compound 8d of 1 mM, 2 mM and 4 mM for 24 h, respectively, and stained by JC-1. Selected fields illustrated the corresponding live cells (orange-red), apoptotic cells (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(TLC) on pre-coated silica GF254 plates. Melting points were determined on a XT4MP apparatus (Take Corp., Beijing, China), and are uncorrected. 1H and 13C NMR spectra were recorded on a Brucker AM-300 (300 MHz) spectrometer with CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard. Chemical shifts are reported in d (parts per million) values. Coupling constants J were reported in Hz. Mass spectra were performed on an Agilent 12606221 TOF mass spectrometer. Stavudine was purchased from WuXi AppTec, the stereo is known: 1-((2R,5S)-5-(hydroxymethyl)-2,5dihydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione. All reagents were purchased from standard commercial suppliers and treated with standard methods before use. Solvents were dried in a routine way and redistilled.

4.2. Synthesis of benzophenone analogues intermediates 5 Guaiacol (10.2 mmol) and chloroacetyl chloride (14.6 mmol) were placed into a flask under N2 atmosphere. The mixture was heated to 120e130
C for 5e8 h with stirring. The mixture was cooled to r.t. and carefully added to cold water, then was extracted with CH2Cl2 (3  30 mL). The combined organic phase was washed sequentially with water, saturated NaHCO3, brine, dried with anhydrous Na2SO4. The solvent was evaporated in vacuo and yielded yellow oil, which was recrystallized with ethanol to afford white needle-like crystals compound 2. To an Eaton's reagent (50 mL) [23] ice bath solution was added benzoic acid derivatives 3 (67.4 mmol) and compound 2 (45 mmol). The reaction mixture was allowed to stand at 25e50
C for 8e10 h, then cooled and poured into ice water (150 mL). The mixture was extracted with CH2Cl2 (3  30 mL). The combined organic phase was washed sequentially with water, saturated NaHCO3, brine, dried with anhydrous Na2SO4. The solvent was evaporated in vacuo and the obtained residue was refluxed in ethanol, then cooled to r.t. and filtered through a Büchner funnel to afford off-white powders 4a~4e.

To a solution of compounds 4a~4e (8 mmol) in MeOH (25 mL) was added NaOAc (36 mmol). The reaction mixture was refluxed for 30e60 min, then the solvent was evaporated. The obtained residue was added H2O (10 mL) with stirring for 2e5 h, filtered, and dried to afford compounds 5a~5e as white or off-white powders. 4.3. General procedure for preparation of compounds 8 To a dry DCM (10 mL) solution of stavudine (4.46 mmol) and succinic anhydride (6.69 mmol) in an ice bath was added TEA (10.8 mmol). The mixture was stirred for 8e12 h at room temperature, then distilled to remove DCM and TEA. The residue was purified by crystallization from ethanol to give stavudine succinic acid monoester 7. To a DCM (10 mL) solution of 7 (0.5 mmol) was added DCC (0.7 mmol), DMAP (0.5 mmol), the mixture was stirred 10 min at 0
C, then corresponding benzophenone analogues 5 (1.0 eq.) was added. The reaction solution was stirred overnight at room temperature. The mixture was filtered and filtrate was washed twice with water, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate, 10:1 / 1:1) to give title crude compounds, the product was recrystallized from suitable solvent to give title compounds 8a~8e (Scheme 2) as solids. 4.3.1. 8a: 2-methoxy-5-(2,4,5-trimethoxybenzoyl)phenyl((2S,5R)5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5dihydrofuran-2-yl)methyl succinate Light yellow powder solid, yield, 85%; mp 177e178
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.30 (s, 1H, NH), 7.71 (dd, J ¼ 8.6, 2.1 Hz, 1H, ArH), 7.52 (d, J ¼ 2.1 Hz, 1H, ArH), 7.24 (s, 1H, pyrimidine C6eH), 6.97 (m, 3H, ArH and 10 -H), 6.56 (s, 1H, ArH), 6.27 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.88 (d, J ¼ 6.0 Hz, 1H, 20 -H), 5.04 (m, 1H, 40 -H), 4.47 (dd, J ¼ 12.3, 4.0 Hz, 1H, 50 -H), 4.25 (dd, J ¼ 12.3, 3.1 Hz, 1H, 500 -H), 3.96

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Fig. 5. Inhibition of cell cycle progress in SGC-7901 cells treated with compound 8d for 48 h. Cells were fixed with ethanol and stained with PI. Cell cycle distribution was analyzed by flow cytometry. Analyses were performed at least 3 times, and a representative experiment was presented.

Table 2 Antitumor effects of compound 8d against tumor growth on the S180 and HepG2 xenograft mice.a Models

Groups

Animal number (End, n)

Body weight (g)

S180

Control 8d Control 8d

10 10 10 10

19.38 19.99 19.15 19.87

Beginning

HepG2

± ± ± ±

1.07 1.26 1.01 0.51

Tumor weight (g)

Inhibition rate (%)

End 24.00 24.25 25.17 26.34

± ± ± ±

1.78 1.55 1.60 1.67

2.02 1.20 2.01 1.04

± ± ± ±

0.19 0.17* 0.09 0.28**

40.6 48.5

a

Mice were inoculated with S180 or HepG2 subcutaneously into the right front armpit and randomly divided into four test groups. The mice were daily treated by compound 8d (60 mg/kg/day), or normal saline (NS, 10 mL/kg) by oral gavage for ten consecutive days. Data were analyzed using SPSS11.0. Significant difference between each treatment and the control are shown as P < 0.05 (*) and P < 0.01 (**).

(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.04e2.89 (m, 2H, CH2), 2.74 (m, 2H, CH2), 1.92 (s, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C31H32N2NaO12 [MþNa]þ, 647.1849; found 647.1849. 4.3.2. 8b: 5-(4-fluorobenzoyl)-2-methoxyphenyl((2S,5R)-5-(5methyl-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-2,5dihydrofuran-2-yl)methyl succinate Off-white powder solid, yield, 90%; mp 192e193
C; 1H NMR

(300 MHz, CDCl3) d (ppm): 8.36 (s, 1H, NH), 7.80 (dd, J ¼ 8.6, 5.5 Hz, 2H, ArH), 7.71 (dd, J ¼ 8.5, 2.1 Hz, 1H, ArH), 7.57 (d, J ¼ 2.1 Hz, 1H, ArH), 7.24 (s, 1H, pyrimidine C6eH), 7.16 (t, J ¼ 8.6 Hz, 2H), 7.03 (d, J ¼ 8.5 Hz, 1H, ArH), 6.98 (m, 1H, 10 -H), 6.28 (d, J ¼ 5.9 Hz, 1H, 30 -H), 5.89 (d, J ¼ 5.9 Hz, 1H, 20 -H), 5.04 (m, 1H, 40 -H), 4.47 (dd, J ¼ 12.3, 4.0 Hz, 1H, 50 -H), 4.26 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.91 (s, 3H, OCH3), 3.04e2.87 (m, 2H, CH2), 2.75 (m, 2H, CH2), 1.92 (s, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C28H25FN2NaO9 [MþNa]þ, 575.1427; found 575.1427.

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of

some

compounds

against

Compound

IC50 (mM)

8a 8c 8d 11f Staurosporineb

6.43 ± 1.19 28.91 ± 1.95 7.01 ± 1.14 noc 8.50 ± 0.37

a

Telomerase supercoiling activity. Staurosporine is reported as a control. c The standard deviation (SD) of three time independent tests. No, not observed in the tested concentration range 0e60 mm. b

4.3.3. 8c: 2-methoxy-5-(4-nitrobenzoyl)phenyl((2S,5R)-5-(5methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5dihydrofuran-2-yl)methyl succinate Off-white powder solid, yield, 92%; mp 180e181
C;1H NMR (300 MHz, CDCl3) d (ppm): 8.39 (s, 1H, NH), 8.34 (d, J ¼ 8.7 Hz, 2H, ArH), 7.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.72 (dd, J ¼ 8.6, 2.1 Hz, 1H, ArH), 7.59 (d, J ¼ 2.1 Hz, 1H, ArH), 7.23 (s, 1H, pyrimidine C6eH), 7.05 (d, J ¼ 8.6 Hz, 1H, ArH), 6.98 (m, 1H, 10 -H), 6.28 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.89 (d, J ¼ 5.9 Hz, 1H, 20 -H), 5.05 (s, 1H, 40 -H), 4.44 (dd, J ¼ 12.3, 4.1 Hz, 1H, 50 -H), 4.28 (dd, J ¼ 12.3, 3.1 Hz, 1H, 500 -H), 3.93 (s, 3H, O CH3), 3.04e2.91 (m, 2H, CH2), 2.75 (m, 2H, CH2), 1.92 (s, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C28H25N3NaO11 [MþNa]þ, 602.1383; found 602.1383. 4.3.4. 8d: 2-methoxy-5-(2-methoxybenzoyl)phenyl((2S,5R)-5-(5methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5dihydrofuran-2-yl)methyl succinate Off-white powder solid, yield, 90%; mp 187e188
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.38 (s, 1H, NH), 7.70 (dd, J ¼ 8.6, 2.1 Hz, 1H, ArH), 7.56 (d, J ¼ 2.1 Hz, 1H, ArH), 7.45 (dd, J ¼ 11.2, 4.5 Hz, 1H, ArH), 7.33 (dd, J ¼ 7.5, 1.6 Hz, 1H, ArH), 7.25 (s, 1H, pyrimidine C6eH), 7.08e6.93 (m, 4H, ArH and 10 -H), 6.27 (d, J ¼ 6.0 Hz, 1H, 30 H), 5.88 (d, J ¼ 5.7 Hz, 1H, 20 -H), 5.04 (s, 1H, 40 -H), 4.48 (dd, J ¼ 12.3, 4.0 Hz, 1H, 50 -H), 4.25 (dd, J ¼ 12.3, 3.2 Hz, 1H, 500 -H), 3.88 (s, 3H, O CH3), 3.74 (s, 3H, O CH3), 3.05e2.85 (m, 2H, CH2), 2.73 (dd, J ¼ 9.1, 4.9 Hz, 2H, CH2), 1.92 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 194.21 (C]O, diphenyl ketone), 171.65 (OeC]O), 170.00 (OeC]O), 163.64 (NHeC]O,dihydropyrimidine), 157.10, 155.06 (NeC]O,dihydropyrimidine), 150.74, 139.21, 135.56 (CH2dihydropyrimidine), 133.34, 131.74, 130.86, 130.07, 129.36, 128.63, 127.20, 124.36, 120.51, 111.45, 111.36, 111.09 (CH-dihydropyrimidine), 89.81 (CH, 2-furan), 84.16 (CH, 5-furan), 64.97 (OCH2), 56.12 (OCH3), 55.62 (OCH3), 28.92 (CH2), 28.67 (CH2), 12.53 (Medihydropyrimidine). TOF-HRMS: calcd for C29H28N2NaO10 [MþNa]þ, 587.1636; found 587.1632. 4.3.5. 8e: 5-(2-chlorobenzoyl)-2-methoxyphenyl((2S,5R)-5-(5methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2,5dihydrofuran-2-yl)methyl succinate Off-white powder solid, yield, 85%; mp 175e176
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.34 (s, 1H, NH), 7.66 (dd, J ¼ 8.6, 2.1 Hz, 1H, ArH), 7.58 (d, J ¼ 2.0 Hz, 1H, ArH), 7.47e7.40 (m, 2H, ArH), 7.37e7.28 (m, 2H, ArH), 7.25 (s, 1H, pyrimidine C6eH), 6.97e6.99 (m, 2H, ArH and 10 -H), 6.27 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.89 (d, J ¼ 5.9 Hz, 1H, 20 -H), 5.04 (s, 1H, 40 -H), 4.46 (dd, J ¼ 12.3, 4.0 Hz, 1H, 50 -H), 4.25 (dd, J ¼ 12.3, 3.2 Hz, 1H, 500 -H), 3.89 (s, 3H, O CH3), 2.99e2.88 (m, 2H, CH2), 2.79e2.67 (m, 2H, CH2), 1.91 (s, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C28H25ClN2NaO9 [MþNa]þ, 591.1141; found 561.1143.

4.4. General procedure for the synthesis of title compounds 11a~11h To an acetone (15 mL) solution of corresponding benzophenone analogues (0.314 mmol) in an ice bath was added anhydrous K2CO3 (0.377 mmol) and ethyl bromoacetate (0.377 mmol). The mixture was heated to 60e65
C with stirring. The reaction was monitored by TLC. When the starting material had completely disappeared, the mixture was distilled to remove acetone. The residue was added H2O (20 mL) and ethyl acetate (20 mL), then stirred over 10e15 min. The product was extracted into ethyl acetate (2  20 mL), the organic extract washed with water, and the resulting organic liquor evaporated to dryness on a rotavaporator and obtained crude compounds 9. The crude compounds 9 (1 mmol) were dissolved in methanol (10 mL), 2 N NaOH (2 mL) solution and stirred at r.t. for 2e3 h. After completion of the reaction, the mixture was acidified with HCl (1 N), then the solvent was concentrated to a low volume. The precipitated product was granulated at 10
C for a further hour. The title compounds 10 was collected by filtration, washed with water, and dried to give as solid. To a DCM (15 mL) solution of compounds 10 (0.266 mmol) was added EDC$HCl (0.319 mmol), HOBt (0.319 mmol), and Stavudine (0.266 mmol), the mixture was stirred 30 min at 0
C. After TEA (0.319 mmol) was added, the reaction solution was stirred overnight at r.t. The mixture was washed twice with saturated NH4Cl, NaHCO3 and water, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was recrystallized from suitable solvent to give title compounds 11a~11h (Scheme 3) as solids. 4.4.1. 11a: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(3,4,5trimethoxybenzoyl)phenoxy)acetate Off-white powder solid, yield, 82%; mp 170e171
C; 1HNMR (300 MHz, CDCl3) d (ppm): d 8.57 (s, 1H, NH), 7.49 (dd, J ¼ 8.6, 1.9 Hz, 1H, ArH), 7.35 (d, J ¼ 1.9 Hz, 1H, ArH), 7.22 (d, J ¼ 1.2 Hz, 1H, pyrimidine C6eH), 7.00 (s, 2H, ArH), 7.00e6.97 (m, 1H, 10 -H), 6.95 (d, J ¼ 8.6 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.91 (d, J ¼ 5.4 Hz, 1H, 20 -H), 5.05 (m, 1H, 40 -H), 4.78 (s, 2H, CH2), 4.51 (dd, J ¼ 12.2, 4.3 Hz, 1H, 50 -H), 4.35 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.96 (s, 3H, O CH3), 3.94 (s, 3H, O CH3), 3.89 (s, 6H, 2OCH3), 1.91 (d, J ¼ 1.0 Hz, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C29H30N2NaO11 [MþNa]þ, 605.1744; found 605.1744. 4.4.2. 11b:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(2,4,5trimethoxybenzoyl)phenoxy)acetate Off-white powder solid, yield, 82%; mp 172e174
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.30 (s, 1H, NH), 7.39 (m, 2H, ArH), 7.25 (s, 1H, pyrimidine C6eH), 6.99 (m, 1H, 10 -H), 6.93 (s, 1H, Ar), 6.88 (d, J ¼ 9.0 Hz, 1H, ArH), 6.57 (s, 1H, ArH), 6.25 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.91 (d, J ¼ 6.0 Hz, 1H, 20 -H), 5.05 (s, 1H, 40 -H), 4.76 (s, 2H, CH2), 4.53 (dd, J ¼ 12.2, 4.1 Hz, 1H, 50 -H), 4.35 (dd, J ¼ 12.3, 3.0 Hz, 1H, 500 -H), 3.97 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 1.90 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 193.87 (C]O, diphenyl ketone), 168.35 (OeC]O), 163.64 (NHeC]O,dihydropyrimidine), 153.50, 152.72, 152.13, 150.74 (NeC]O,dihydropyrimidine), 146.73, 143.02, 135.47 (CH2-dihydropyrimidine), 132.98, 131.32, 127.50, 126.86, 119.88, 114.14, 113.19, 111.30, 110.53 (CH-dihydropyrimidine), 97.58, 89.84 (CH, 2-furan), 83.86 (CH, 5-furan), 66.04 (OCH2), 65.44 (OCH2), 56.74 (OCH3), 56.51 (OCH3), 56.17 (OCH3), 56.01 (OCH3), 12.42 (Me-dihydropyrimidine). TOF-HRMS: calcd for C29H30N2NaO11 [MþNa]þ, 605.1744; found 605.1744.

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739

4.4.3. 11c:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(5-(4-fluorobenzoyl)-2methoxyphenoxy)acetate Off-white powder solid, yield, 80%; mp 180e181
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.44 (s, 1H, NH), 7.78 (dd, J ¼ 8.7, 5.4 Hz, 2H, ArH), 7.43 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.37 (d, J ¼ 1.9 Hz, 1H, ArH), 7.22 (d, J ¼ 1.2 Hz, 1H, pyrimidine C6eH), 7.16 (t, J ¼ 8.6 Hz, 2H, ArH), 7.00e6.96 (m, 1H, 10 -H), 6.94 (d, J ¼ 8.4 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.91 (d, J ¼ 6.0 Hz, 1H, 20 -H), 5.06 (s, 1H, 40 -H), 4.78 (s, 2H, CH2), 4.52 (dd, J ¼ 12.2, 4.4 Hz, 1H, 50 -H), 4.36 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.95 (s, 3H, OCH3), 1.90 (d, J ¼ 0.9 Hz, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 193.59 (C] O, diphenyl ketone), 168.27 (OeC]O), 163.68 (NHeC]O,dihydropyrimidine), 153.59 (NeC]O,dihydropyrimidine), 150.75, 147.02, 135.38 (CH2-dihydropyrimidine), 134.09, 134.05-132.83132.36, 132.24, 129.90, 127.60, 126.78, 115.57, 115.28, 115.02, 111.29 (CH-dihydropyrimidine), 110.65, 89.92 (CH, 2-furan), 83.85 (CH, 5furan), 66.05 (CH2), 65.50 (CH2), 56.11 (OCH3), 12.45 (Me-dihydropyrimidine). TOF-HRMS: calcd for C26H23FN2NaO8 [MþNa]þ, 533.1331; found 533.1333. 4.4.4. 11d:((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(5-(4-bromobenzoyl)-2methoxyphenoxy)acetate Off-white powder solid, yield, 80%; mp 177e179
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.45 (s, 1H, NH), 7.62 (s, 4H, ArH), 7.42 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.37 (d, J ¼ 1.9 Hz, 1H, ArH), 7.22 (d, J ¼ 1.1 Hz, 1H, pyrimidine C6eH), 7.01e6.96 (m, 1H, 10 -H), 6.94 (d, J ¼ 8.4 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.91 (d, J ¼ 5.6 Hz, 1H, 20 -H), 5.06 (s, 1H, 40 -H), 4.78 (s, 2H, CH2), 4.52 (dd, J ¼ 12.2, 4.4 Hz, 1H, 50 -H), 4.36 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.95 (s, 3H, OCH3), 1.90 (d, J ¼ 0.8 Hz, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 192.87 (C]O, diphenyl ketone), 167.22 (OeC]O), 162.71 (NHeC]O,dihydropyrimidine), 152.73 (NeC] O,dihydropyrimidine), 149.76, 146.03, 135.62 (CH2-dihydropyrimidine), 134.35, 131.79, 130.54, 130.25, 128.57, 126.61, 126.02, 125.86, 113.89, 110.26, 109.66 (CH-dihydropyrimidine), 88.89 (CH, 2-furan), 82.82 (CH, 5-furan), 65.00 (OCH2), 64.49 (OCH2), 55.11 (OCH3), 11.45 (Me-dihydropyrimidine). TOF-HRMS: calcd for C26H23BrN2NaO8 [MþNa]þ, 593.0537; found 593.0537. 4.4.5. 11e: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(4nitrobenzoyl)phenoxy)acetate Yellow powder solid, yield, 78%; mp 170e171
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.40 (s, 1H, NH), 8.31 (d, J ¼ 8.6 Hz, 2H, ArH), 7.87 (d, J ¼ 8.6 Hz, 2H, ArH), 7.40 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.35(d, J ¼ 1.9 Hz, 1H, ArH), 7.20 (s, 1H, pyrimidine C6eH), 7.02e6.93 (m, 2H, 10 -H and ArH), 6.22 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.89 (d, J ¼ 5.6 Hz, 1H, 20 -H), 5.05 (s, 1H, 40 -H), 4.75 (s, 2H, CH2), 4.51 (dd, J ¼ 12.2, 4.3 Hz, 1H, 50 -H), 4.34 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.93 (s, 3H, OeCH3), 1.88 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 193.12 (C]O, diphenyl ketone), 168.26 (OeC]O), 163.79 (NHeC]O,dihydropyrimidine), 154.49 (NeC]O,dihydropyrimidine), 150.84, 149.69, 147.43, 143.50, 135.46 (CH2-dihydropyrimidine), 132.85, 130.46, 128.91, 127.79, 127.43, 123.60, 114.80, 111.32 (CH-dihydropyrimidine), 110.88, 90.09 (CH, 2-furan), 83.96 (CH, 5-furan), 66.11 (CH2), 65.65 (CH2), 56.32 (OCH3), 12.59 (Me-dihydropyrimidine). TOF-HRMS: calcd for C26H23N3NaO10 [MþNa]þ, 560.1276; found 560.1276. 4.4.6. 11f: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)-methyl 2-(5-(5-bromo-2chlorobenzoyl)-2-methoxyphenoxy)acetate Off-white powder solid, yield, 80%; mp 168e169
C; 1H NMR

737

(300 MHz, CDCl3) d (ppm): 8.36 (s, 1H, NH), 7.55 (dd, J ¼ 8.5, 2.2 Hz, 1H, ArH), 7.47 (d, J ¼ 2.3 Hz, 1H, ArH), 7.43 (d, J ¼ 1.7 Hz, 1H, ArH), 7.27e7.35 (m, 2H, ArH), 7.24 (s, 1H, pyrimidine C6eH), 6.99 (brs, 1H, 10 -H), 6.90 (d, J ¼ 8.5 Hz, 1H, ArH), 6.25 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.93 (d, J ¼ 5.9 Hz, 1H, 20 -H), 5.07 (s, 1H, 40 -H), 4.78 (s, 2H, CH2), 4.54 (dd, J ¼ 12.2, 4.2 Hz, 1H, 50 -H), 4.36 (dd, J ¼ 12.2, 3.0 Hz, 1H, 500 -H), 3.94 (s, 3H, OCH3), 1.91 (s, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 191.77 (C]O, diphenyl ketone), 168.11 (OeC]O), 163.66 (NHeC]O,dihydropyrimidine), 154.76 (NeC]O,dihydropyrimidine), 150.75, 147.35, 140.18, 135.40 (CH2-dihydropyrimidine), 133.92, 132.89, 131.53, 131.50, 130.07, 128.74, 127.73, 127.59, 120.54, 113.42, 111.32 (CH-dihydropyrimidine), 110.94, 89.89 (CH, 2-furan), 83.84 (CH, 5-furan), 65.93 (CH2), 65.54 (CH2), 56.18 (OCH3), 12.47 (Me-dihydropyrimidine). TOF-HRMS: calcd for C26H22BrClN2NaO8 [MþNa]þ, 627.0145; found 627.0145. 4.4.7. 11g: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)methyl 2-(2-methoxy-5-(2methoxybenzoyl)phenoxy)acetate Off-white solid, purified by flash chromatography (PE/EtOAc, 1:1); yield, 77%; mp 170e171
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.54 (s, 1H, NH), 7.54e7.41 (m, 2H, ArH), 7.35 (dd, J ¼ 8.4, 1.9 Hz, 1H, ArH), 7.31e7.27 (m, 1H, ArH), 7.26 (d, J ¼ 1.2 Hz, 1H, pyrimidine C6eH), 7.09e6.98 (m, 3H, ArH and 10 -H),6.86 (d, J ¼ 8.5 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.91 (d, J ¼ 5.8 Hz, 1H, 20 -H), 5.06 (s, 1H, 40 -H), 4.77 (s, 2H, CH2), 4.53 (dt, J ¼ 13.4, 4.9 Hz, 1H, 50 -H), 4.34 (dd, J ¼ 12.3, 3.0 Hz, 1H, 500 -H), 3.91 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 1.90 (d, J ¼ 0.8 Hz, 3H, pyrimidine C5eCH3). TOF-HRMS: calcd for C27H26N2NaO9 [MþNa]þ, 545.1533; found 545.1533. 4.4.8. 11h: ((2S,5R)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin1(2H)-yl)-2,5-dihydrofuran-2-yl)-methyl 2-(5-(2-chlorobenzoyl)-2methoxyphenoxy)acetate Off-white powder solid, yield, 78%; mp 180e181
C; 1H NMR (300 MHz, CDCl3) d (ppm): 8.53 (s, 1H, NH), 7.48e7.38 (m, 3H, ArH), 7.29e7.37 (m, 3H, ArH), 7.25 (d, J ¼ 1.0 Hz, 1H, pyrimidine C6eH), 7.01e6.96 (m, 1H, 10 -H), 6.89 (d, J ¼ 8.5 Hz, 1H, ArH), 6.24 (d, J ¼ 6.0 Hz, 1H, 30 -H), 5.92 (d, J ¼ 5.7 Hz, 1H, 20 -H), 5.06 (s, 1H, 40 -H), 4.78 (s, 2H, CH2), 4.54 (dd, J ¼ 12.2, 4.2 Hz, 1H, 50 -H), 4.35 (dd, J ¼ 12.2, 3.1 Hz, 1H, 500 -H), 3.93 (s, 3H, OCH3), 1.91 (d, J ¼ 0.8 Hz, 3H, pyrimidine C5eCH3). 13C NMR (75 MHz, CDCl3) d (ppm): 193.53 (C] O, diphenyl ketone), 168.18 (OeC]O), 163.63 (NHeC]O,dihydropyrimidine), 154.44 (NeC]O,dihydropyrimidine), 150.72, 147.22, 138.55, 135.44 (CH2-dihydropyrimidine), 132.93, 131.05, 131.00, 130.02, 129.36, 128.85, 127.62, 127.54, 126.71, 113.52, 111.31 (CH-dihydropyrimidine), 110.85, 89.86 (CH, 2-furan), 83.84 (CH, 5furan), 65.95 (CH2), 65.50 (CH2), 56.12 (OCH3), 12.44 (Me-dihydropyrimidine). TOF-HRMS: calcd for C26H23ClN2NaO8 [MþNa]þ, 549.1035; found 549.1035. 4.5. Cell proliferation assays The antiproliferative activity evaluation was conducted by using a modified procedure as described in the literature [24]. Briefly, target tumor cells were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 3  104 cells mL1 with the complete medium, 100 mL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was performed at 37
C, 5% CO2 atmosphere for 24 h before subjecting to antiproliferation assessment. Tested samples at pre-set concentrations were added to 6 wells with Doxorubicin (AMD) co-assayed as a positive reference. After 48 h exposure period, 25 mL of PBS containing 2.5 mg mL1 of MTT was added to each well. After 4 h, the medium was replaced by

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150 mL DMSO to dissolve the purple formazan crystals produced. The absorbance at 570 nm of each well was measured on an ELISA plate reader. The data represented the mean of three experiments in triplicate and were expressed as means ± SD using Student's test. The IC50 value was defined as the concentration at which 50% of the cells could survive. 4.6. Apoptosis analysis Apoptosis was detected by flow cytometric analysis of annexin V staining. Annexin V-FITC/PI assay was performed as previously described. Briefly, adherent SGC-7901 cells were harvested and suspended in the annexin-binding buffer (1  106 cells/mL). Then, cells were incubated with V-FITC buffer and incubated with annexin V-FITC for 15 min and then stained by PI for 5 min at 4
C in the dark and immediately analyzed by flow cytometry. The data are presented as biparametric dot plots showing PI red fluorescence vs annexin V-FITC green fluorescence. 4.7. MMP assays Exponentially growing gastric cancer cells were cultured in sixwell plates (2  105/well) and treated with 1, 2, 4 mM compound 8d for 48 h. After incubation, adherent cells were detached with 0.5% trypsin. The detached and suspended cells were harvested in the DMEM medium. Then, cells were stained with JC-1 in a humidified incubator with 5% CO2, 37
C for 0.5 h in the dark. At last cells were washed in JC-1 dyeing buffer twice and analyzed by FACSVERSE (BD) Flow Cytometer.

Sciences, and the cells were cultured in RPMI-1640 medium, which was supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin and cultured in an atmosphere of 5% CO2 at 37
C. Cells were collected for the experiments in the logarithmic growth phase. To establish the tumorbearing mouse model, the cell lines were harvested and inoculated subcutaneously into the right armpit region of the mice. On the 7th day, the tumor ascrites were obtained and washed with sterile PBS. Under sterile condition, the tumor ascrites were diluted with sterile normal saline to 1  1010/L cell suspension. Tumor ascites were maintained in vivo in mice by transplantation of 0.2 mL of ascites (2  106 cells) from the infected mice to the non-infected mice. 4.9.2. Tumor xenograft model in vivo Each Kunming mouse (male or female, weight 20 ± 2 g) were inoculated with seven-day-old ascrite (0.2 mL, 2  106 cells) subcutaneously into the right front armpit. 24 h after implantation of tumor cells, the mice were randomly divided into four test groups with 10 mice per group. Each mouse was weighed immediately after inoculation. The mice were treated by oral gavage with test samples (60 mg/kg/day) or normal saline (NS, 10 mL/kg) for ten days once daily. On day 11, the mice were sacrificed via cervical dislocation, and the mouse and tumor were excised and weighed for evaluating the tumor growth inhibition. The tumor inhibitory rate was calculated by the following formula:

 Tumor inhibitory rate rate ð%Þ ¼

4.9. Evaluation of the antitumor activities in vivo 4.9.1. Animals and cell lines Kunming mice (SPF, male or female, 20 ± 2 g) were purchased from the experimental animal center of China Pharmaceutical University. Animals were housed in a temperature (22 ± 2
C) and relatively humidity (50%)-controlled room on a 12 h light/dark cycle, given free access to food and water, and acclimatized for at least one week prior to use. All the animal experiments were performed in accordance with the Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of China. Efforts were made to minimize the number of animals used and their suffering. Animals were maintained in accordance with the Guides of Center for Developmental Biology, Anhui Medical University for the Care and Use of Laboratory Animals and all experiments used protocols approved by the institutions' subcommittees on animal care. Cell lines used for evaluation of the in vivo antitumor activity in this study included two tumor cell lines, namely S180 (sarcoma tumor cell line), HepG2 (liver carcinoma cell line). All of cell lines were purchased by the Shanghai Institutes for Biological Sciences, Chinese Academy of

  100% (1)

4.8. Cell cycle analysis The distribution of cells at specific cell cycle stages was evaluated by Flow Cytometry. 2  105 cells/well were cultured in six-well plates and treated with 1, 2, 4 mM compound 8d in a humidified incubator with 5% CO2, 37
C for 48 h, and cells incubated in medium without compound 8d served as controls. After incubation, adherent cells were detached with 0.5% trypsin. The detached and suspended cells were harvested in complete medium and centrifuged at 300 g for 5 min. Pellets were washed with PBS twice and fixed with ice cold 70% ethanol for 1 h at 20
C, treated with RNAse and subsequently stained with propidium iodide. The cells were analyzed by FC 500 (BECKMAN) Flow Cytometer analyzer.

Wcontrol  Wtreated Wcontrol

Wcontrol and Wtreated were the average tumor weights of the control and treated mice, respectively. 4.10. Telomerase activity assays Compounds 8a, 8c, 8d and 11f were tested in a search for small molecule inhibitors of telomerase activity by using the TRAP-PCRELISA assay. In detail, the SGC-7901 cells were firstly maintained in RPMI 1640 buffer (Hyclone, Miami, FL, USA), supplemented with 10% fetal bovineserum (GIBCO, New York, USA), streptomycin (0.1 mg/mL) and penicillin (100 IU/mL) at 37
C in a humidified atmosphere containing 5% CO2. After trypsinization, 5  104 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New York, USA) and cultured to allow to adherence. The cells were then incubated with Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with a series of concentration as 60, 20, 6.67, 2.22, 0.75, 0.25 and 0.082 l g/mL, respectively. After 24 h treatment, the cells were harvested by cell scraper orderly following by washed once with PBS. The cells were lysed in 150 mL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g for 20 min at 4
C and stored at 80
C. The TRAP-PCR-ELISA assay was performed using a telomerase detection kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. In brief, 2 mL of cell extracts were mixed with 48 mL TRAP reaction mixtures. TRAP primers and Taq polymerase and incubated at 25
C for 30 min. PCR was then initiated at 94
C, 120 s for predenaturation and performed using 35 cycles each consisting of 94
C for 30 s, 50
C for 30 s, 72
C for 90 s. Then 20 mL of PCR products were hybridized to a digoxigenin (DIG)-labeled telomeric repeat specific detection probe. And the PCR products were immobilized via the biotin-labeled primer to a streptavidincoated microtiter plate subsequently. The immobilized DNA

J.B. Shi et al. / European Journal of Medicinal Chemistry 124 (2016) 729e739

fragments were detected with a peroxidase-conjugated anti-DIG antibody and visualized following addition of the stop regent. The microtitre plate was assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland) at a wavelength of 490 nm, and the final value were presented as mean ± SD. Acknowledgments The authors wish to thank the National Natural Science Foundation of China (No. 21272008, 21572003), Science and Technological Fund of Anhui Province for Outstanding Youth (1408085J04). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.09.011. References [1] [2] [3] [4] [5] [6] [7]

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