European Journal of Medicinal Chemistry 97 (2015) 155e163

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

Research paper

Novel myricetin derivatives: Design, synthesis and anticancer activity Wei Xue a, Bao-An Song a, **, Hong Ju Zhao a, Xing Bao Qi b, Yin Jiu Huang c, Xin Hua Liu a, b, * a

State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Guizhou University, Guiyang 55002, PR China School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China c Department of Bioscience, Bengbu Medical College, Bengbu, 233030, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 March 2015 Received in revised form 28 April 2015 Accepted 29 April 2015 Available online 1 May 2015

Telomere and telomerase were closely related to occurrence and development of some cancers. To enhance ability of myricetin moiety for inhibiting telomerase, we designed a series of novel myricetin derivatives based on reasonable analysis. The telomerase inhibition assay showed that compound 6d displayed the most potent inhibitory activity with IC50 value of 0.91 mM. The anticancer activity assay showed that 6d exhibited high activity against human breast cells MDA-MB-231. The docking simulation of compound 6d was performed to get the probable binding model, the results demonstrated that the furan ring inserted into the active site deeply and had hydrophobic interactions with residues of Phe 568, Pro 627, four methoxy groups had hydrophobic interactions with residues of Phe 568, Pro 627, Lys 902, Val 904 and Pro 929. Western blot results showed that expression of p65 and TERT protein was clearly down-regulated by compound 6d. These data support further studies for the rational design of more efficient p65 and TERT modulators. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Myricetin Anticancer activity Telomerase

1. Introduction Telomerase is expressed in 80e90% of tumors from all types of cancers [1]. The different telomerase expression levels, as well as the generally longer telomeres in healthy cells versus tumor cells, suggest a high degree of tumor specificity and a low risk of toxicity to normal tissues of compounds that selectively target telomerase [2e5]. Those facts resulted in significant efforts to validate telomerase as an anticancer drug target and to develop effective approaches toward its inhibition [6,7]. Based on searching for telomerase inhibitors, different approaches have been designed: drugs that inhibit telomerase enzymatic activity, active immunotherapy, gene therapy using telomerase promoter-driven expression of a suicide gene, agents that block telomerase biogenesis and G-quadruplex-stabilizing molecules. But, no drugs that inhibit telomerase activity and immunotherapy-based drugs have reached clinical trials so far [1,8]. Therefore, the pursuit of novel telomerase

* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China. ** Corresponding author. E-mail addresses: [email protected] (B.-A. Song), [email protected] (X.H. Liu). http://dx.doi.org/10.1016/j.ejmech.2015.04.063 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

inhibitors with better antitumor effects and more safety profile is still the main issue. Myricetin, flavonoid compounds (Fig. 1), are present in a wide variety of fruits. Interestingly, those myricetin derivatives are thought to show anticancer activity, which could decrease pancreatic cancer growth via induction of cell apoptosis [9,10]. There is also accumulating evidence to suggest that myricetin can also directly modulate activity of a large number of important signaling molecules, such as: protein kinase (ERK1/2) in lung cancer cells [11] and kinase B (Akt) in cervical and lung cancer cell lines [12e17]. These pathways, through their ability to affect growth and survival of cancer cells, lead to cell cycle progression and proliferation. It was of our interest to utilize rational chemical approaches to generate and identify novel compounds as potential hTERT (key protein of telomerase) inhibitors for cancer therapy [18]. Steczkiewicz et al. [19] have constructed a three-dimension human telomerase model by systematically utilizing computational methods for distant homology detection, comparative modeling and molecular docking. In this study, above three-dimension human telomerase model and an advanced docking method e IFD (Induced Fit €dinger were employed to explore the binding Docking) of Schro mode of BIBR1532, a potent hTERT inhibitor. Based on this,

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Fig. 1. Idea of design based on myricetin scaffold.

computer-generated molecular model of 5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen moiety docked into the model of hTERT was analyzed, we found four methoxy groups whereas had hydrophobic interactions with the key residues of Phe 568 and Pro 929, the chromen ring also projected into a more stable hydrophobic region. This is motivation provided in the design idea. So, in this design, methoxy (Fig. 1) was introduced. 2. Results and discussion 2.1. Chemistry Myricetrin was used as the raw material, and hydroxyl groups were protected with methyl iodide, 3-hydroxy-5,7-dimethoxy-2(3,4,5-trimethoxyphenyl)-4H-chromen-4-one 1 was prepared by removing of glycosides, and then reacted with alkyl halide containing different chains, in this way intermediate 2 was obtained, the next reaction was carried out in the presence of K2CO3, DMF was used as solvent at reflux condition (Scheme 1), finally, a series of title myricetin derivatives 3 containing nitrogen heterocycle moiety were synthesized. Myricetin hydrazone derivatives 6 were synthesized by hydrolysis, substitution and condensation reactions, Halogenated hydrocarbons, ethyl bromide, hydrazine hydrate and aromatic aldehyde were used as raw materials (Scheme 2).

1.86 mM, respectively, exhibited promising anticancer activity against Bcap-37 and MDA-MB-231 cell lines. The SAR indicated that all examined compounds showed good activity against human breast cancer cell lines (MDA-MB-231 and Bcap-37) but poor activity against human gastric cancer cells (SGC7901 and MGC-803 cell lines). Scanning from Table 1, it is obvious that all the examined compounds exhibited poor inhibitory activity against the SGC-7901 cells. Except compounds 6a and 6b, the activity of other compounds against MGC-803 cells was not high. In the further study, our examined compounds were divided into two series, one was 3-(3-(substituted) propoxy)-5,7dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one 3 (compounds 3a~3j), another was 2-(5,7-dimethoxy-4-oxo-2(3,4,5-trimethoxy-phenyl)-4H-chromen-3-yloxy)-N'-(2substituted) acetylhydrazine 6 (compounds 6a~6e). Although the majority compounds (3c, 3d, 3h, 3i and 3j) of series 3 exerted the moderate inhibitory activity, the most active agent against the tested cancer cell lines was compound 6d. Therefore, for this kind of structure moiety, this pointed out the direction for us to further optimize the structure of myricetin derivatives as potent anticancer agents. 2.3. Cells morphology effects MDA-MB-231 cell was chosen for examination of the cell morphology changes induced by compounds. Results of cells morphology analysis were shown in Figs. 2 and 3. Toxicity of doxorubicin on tumor cells was too serious, so that all cells were broke and cracked. But, damage of target cells caused by compound 6d was very small, so the inhibitory activity is mainly reflected in the inhibition of cells proliferation, the number of cells decreased obviously compared with the control group. From morphological observation we easily discovered that in reducing the number of cells at the same time, cells were distorted, but injury was not very obvious. 2.4. Cell cycle analysis To understand whether cell cycle arrest lead to decrease cell proliferation, we used flow cytometric analysis to measure the effect of compound 6d on induction of cell cycle. As shown in Fig. 4, the cells in G0/G1 phase in the MDA-MB-231 cells control group accounted for about 40.5%, while after MDA-MB-231cells treated with compound 6d for 48 h, the ratio was approximately 44.3%, this can be described as slight increase of the proportion of cells in G0/ G1 phase rather than as the arrest of cell cycle in this phase.

2.2. In vitro anticancer activity 2.5. Telomerase inhibition To test anticancer activity of the synthesized compounds, we evaluated activity of examined compounds 3 and 6 against Bcap-37 (human breast cancer cell), MDA-MB-231 (human breast adenoma cell), SGC-7901 (human gastric cancer cell) and MGC-803 (human gastric cancer cell) cell lines. The results were summarized in Table 1. From the results of MTT assay, it was found that some examined compounds showed remarkable anticancer effects mainly for MDA-MB-231 cells and Bcap-37 cells. The compounds 3c and 6d showed anticancer activities against Bcap-37 cells with IC50 values of 2.96, 3.11 mM, respectively, comparable to that of positive control ADM (Doxorubicin, 2.87 mM). Compounds 3c, 3d, 3h, 3j, 6a and 6f showed anticancer activities against MDA-MB-231 cells with IC50 values of 2.16, 2.90, 3.03, 3.87, 3.40, 3.75 mM, respectively, comparable to that of positive control ADM (2.01 mM). The compound 6a showed anticancer activity against MGC-803 cells with IC50 value 2.78 mM, comparable to that of positive control ADM (3.22 mM). Compounds 3h and 6d with the IC50 values of 2.62,

Based on the discovery of novel skeleton structure and in continuation to extend our research of telomerase inhibitors, some examined compounds were assayed for telomerase inhibition, using MDA-MB-231 cells extracts, ethidium bromide and BIBR1532 were used as the positive compounds. The results (Table 2) suggested that compounds 3h, 6a and 6d showed strong telomerase inhibitory activity with IC50 values of 2.00, 1.82, 0.91 mM, respectively, which surpassing that of the positive control ethidium bromide, and is comparable to that of positive control BIBR1532, Furthermore, there was a good correlation between anti-cancer activity and telomerase activity of compounds (Tables 1 and 2). 2.6. Down-regulated expression of p65 and TERT proteins hTERT and the protein p65 are essential catalytic core of telomerase. Interaction of the p65 C-terminal domain with TER is

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Scheme 1. Synthesis of title compounds 3. Reagent and conditions: (A) CH3I, K2CO3, DMF, reflux 2 d; (B) HCl, 95% ethanol, reflux, 2 h; (C) Br(CH2)nBr, acetone, K2CO3, reflux, 12 h; (D) R, K2CO3, DMF, reflux 24 h.

Scheme 2. Synthesis of title compounds 6. Reagent and conditions: (E) acetone, K2CO3, BrCH2COOC2H5, reflux, 10 h; (F) 80% NH2NH.2H2O, 95% ethanol, reflux, 2 h; (G) RCHO, 95% ethanol, acetic acid, reflux, 2 h.

necessary and sufficient for the hierarchical assembly of the TERTTER-p65 catalytic core [20,21]. In order to test whether compound 6d modulates the expression of p65 and TERT, we performed western blot. As shown in Fig. 5, compared with adjacent control

group, p65 and TERT proteins were expressed at lower level in MDA-MB-231 cells, which were treated with compound 6d. The results suggested that p65 and hTERT were clearly down-regulated within 48 h when the cells exposed to this title compound.

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Table 1 Cytotoxic activity of the examined compounds against MDA-MB-231, Bcap-37, SGC7901 and MGC-803 cellsa. Compound

IC50 (mM)b Bcap-37

3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 6a 6b 6c 6d 6e 6f ADMc

42.96 e 2.96 4.68 38.00 e 18.00 2.62 5.24 4.17 4.09 5.17 18.80 3.11 8.35 7.00 2.87

± 0.28 ± 0.28 ± 0.55 ± 1.21 ± ± ± ± ± ± ± ± ± ± ±

0.83 0.20 0.51 0.36 0.35 0.42 0.70 0.11 0.49 0.38 0.15

MDA-MB-231

SGC-7901

e e 2.16 2.90 30.09 12.97 10.40 3.03 4.17 3.87 3.40 4.57 9.89 1.86 4.47 3.75 2.01

56.85 e 16.51 11.20 e e 26.75 6.08 12.51 6.95 7.72 10.11 16.22 5.72 10.25 8.73 3.50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.14 0.25 1.33 0.77 0.39 0.25 0.40 0.21 0.25 0.29 0.81 0.09 0.20 0.33 0.17

± 1.69 ± 0.88 ± 1.32

± ± ± ± ± ± ± ± ± ± ±

1.41 0.61 1.75 1.00 1.05 1.00 1.02 0.29 0.52 0.58 0.26

MGC-803 40.21 32.09 10.01 21.04 e e 18.55 26.82 20.63 12.21 2.78 3.51 15.33 6.05 7.74 5.61 3.22

± ± ± ±

1.44 1.22 0.62 1.61

± ± ± ± ± ± ± ± ± ± ±

1.24 1.80 1.46 0.81 0.59 0.43 1.22 0.32 0.87 0.60 0.25

Negative control 0.1% DMSO, no activity. a The data represented the mean of three experiments done in triplicate and were expressed as means ± SD; Only descriptive statistics were done in the text. b The IC50 value was defined as the concentration at which 50% survival of cells was observed. Inactive at 60 mM (highest concentration tested). c ADM (doxorubicin) used as a positive control.

the receptor: one is between the carbonyl group and residue of Lys 710; another is between eNH of the hydrazide group with an oxygen atom of the DNA. The furan ring of 6d inserted into the active site deeply and had hydrophobic interactions with residues of Phe 568, Pro 627; the two benzene rings and four methoxy groups whereas had hydrophobic interactions with residues of Phe 568, Pro 627, Lys 902, Val 904, and Pro 929. 3. Conclusions In summary, based on reasonable analysis, some novel 3subsituted-5,7-dimethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one and 3,4,5-trimethoxy-phenyl-4H-chromen-Nʹ-acetylhydrazine derivatives were designed, followed by chemical synthesis and biological evaluation. The results revealed that some compounds exerted high activity against human breast cancer cells. Compound 6d exhibited strong inhibitory activity against MDAMB-231 cell, and showed the most potent telomerase inhibitory activity with IC50 value at 0.91 mM. Flow cytometric analysis indicated that the cells were arrested in G0/G1 phase by compound 6d. Changes of cell morphology caused by compound 6d indicated that inhibition of cell proliferation was the main mode of action. The docking simulation was performed to get the probable binding model. Our experiments preliminarily demonstrated that compound 6d could regulate the expression of protein p65 and hTERT. 4. Experimental section

2.7. Molecular docking 4.1. Chemistry To gain better understanding on the potency of the title compound and guide further SAR studies. The docked complex hTERTeBIBR1532 was then done a 10 ns MD simulation. Compound 6d was docked into the active site of the modeled hTERTeBIBR1532 complex to see its putative binding pose. As shown in Fig. 6, a typical docking pose demonstrated that the hydrazide group of compound 6d formed two hydrogen bonds with

The reactions were monitored by thin layer chromatography (TLC) on pre-coated silica GF254 plates. Melting points were determined on a XT4MP apparatus (Taike Corp., Beijing, China), and are uncorrected. 1H and 13C NMR spectra were recorded on a Brucker AM-500 (500 MHz) spectrometer with CDCl3 or DMSO-d6 as the solvent and TMS as the internal standard. Chemical shifts are

Fig. 2. Cell morphology effects of MDA-MB-231 treated with ADR and adryamicin at 1 mM and 10 mM for 24, 48 and 72 h. Myricetin (1 mM) was used for comparison.

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Fig. 3. Cell morphology effects of MDA-MB-231 treated with compound 6d at 1 mM and 10 mM for 24, 48 and 72 h.

Table 2 Inhibitory effects of selected compounds against telomerase. Compound

IC50 (mM) telomerasea

3a 3b 3c 3e 3f 3h 6a 6d Ethidium bromideb BIBR1532b

35.66 28.91 3.07 19.66 40.30 2.00 1.82 0.91 2.33 0.17

± ± ± ± ± ± ± ± ± ±

1.33 1.09 0.50 0.95 1.56 0.27 0.11 0.09 0.14 0.07

a

Telomerase supercoiling activity. Ethidium bromide and BIBR1532 are reported as a control [19]. The inhibition constant of ethidium toward telomerase has been reported previously. b

Fig. 4. Flow-cytometry analysis showed cell cycle of MDA-MB-231 cells after 48 h of incubation.10,000 cells were scored for the analysis (n ¼ 3) (1) Compound 6d (1 mM, IC50); (2) Control group.

reported in d (parts per million) values. Coupling constants J are reported in Hz. High-resolution electron impact mass spectra (HRMS) are recorded under electron impact (70 eV) condition using a MicroMass GCT CA 055 instrument. IR Spectra are recorded at Nicolet iS10 FT-IR instrument (ThermoScietific). All chemicals or 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. General procedure for the synthesis of compounds 3a e 3j To a solution of 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3(3,4,5-trihydroxy-6-methyl-tetra-hydro-2H-pyran-2-yloxy)-4H-

Fig. 5. Compound 6d inhibited p65 and TERT protein expression. Western blotting showed inhibition of p65 and TERT protein expression in response to compound 6d treatment for 48 h in MDA-MB-231 cells. AZT (3.0 mmol/L), positive control [22]; Compound 6d (1 mM IC50).

chromen-4-one (5 mmol) in DMF (20 mL) was added K2CO3 (16 mmol), After the mixture was stirred at 25
C for 10 min, methyl iodide (32 mmol) was slowly added, the resulting solution was allowed to 25
C and stirred for 48 h, filtrated, washed with ethyl ether, the filter liquor was then added into the water (100 mL), extracted with ethyl acetate three times, combined organic phase,

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54.1 (CH2), 26.4 (CH2), 23.4 (2CH2); MS (ESI, m/z): 500.3 [M þ H]þ.

€ dinger. Fig. 6. Binding mode of compound 6d to hTERT yielded by Glide 5.9 of Schro

concentrated, dissolved in ethanol (30 mL), refluxed, hydrochloric acid (8 mL) was added, continued to react for 2 h, cooled and filtered, compound 1 was obtain, 1 and 1,3-dibromopropane were refluxed for 12 h, K2CO3 used as acid-binding agent, intermediate 2a (4.7 mmol) was obtained. To a dimethylformamide (DMF, 25 mL) solution of 3-(3-bromopropoxy)-5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one 2a (0.5 mmol), anhydrous K2CO3 (1.0 mmol) and diethylamine (1.0 mmol) were added. The reaction mixture was allowed to stand at room temperature for 24 h. The mixture was added water (50 mL), and extracted three times with ethyl acetate (20 ml); combined organic phase, washed with HCl (20 mL, 1 mol/L), saturated NaHCO3 (20 ml) and saturated NaCl (20 ml), respectively. The combined organic layers were dried (Na2SO4) and concentrated under reduced pressure. The crude residue was purified by chromatography on SiO2 (chloroform: methanol ¼ 10:1, v/v) to give title compound 3a as a light yellow solid. Compounds 3b-3j were obtained by the same method (Scheme 1). 4.2.1. 3a: 3-(3-(diethylamino)propoxy)-5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 68.5%, m.p.195~197
C; IR (KBr, cm1): nmax 1622, 1600, 1558, 1506, 1417, 1346, 1244, 1213, 1122, 1039, 1001, 831; 1H NMR (500 MHz, CDCl3) d: 1.32 (t, J ¼ 14.3 Hz, 6H, CH3), 2.16e2.19 (m, 2H, CH2), 3.28 (q, J ¼ 21.75 Hz, 4H, CH2), 3.45 (t, J ¼ 12.6 Hz, 2H, CH2), 3.77e3.83 (m, 17H, 5  OCH3, OCH2), 6.24 (d, J ¼ 1.7 Hz, 1H, H-6), 6.48 (d, J ¼ 2.3 Hz, 1H, H-8), 7.16 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 174.9 (C-4), 165.1 (C7), 160.7 (C-9), 159.0 (C-2), 154.3 (C-5), 153.2 (C-30 , C-50 ), 140.6 (C40 ), 139.3 (C-3), 124.7 (C-10 ), 108.2 (C-10), 105.6 (C-60 , C-20 ), 96.5 (C6), 92.9 (C-8), 70.4 (OCH2), 61.0 (40 -OCH3), 56.7 (30 , 50 -2OCH3), 56.6 (7-OCH3), 56.4 (5-OCH3), 51.8 (CH2), 47.4 (2CH2), 22.4 (CH2), 9.0 (2CH3); MS (ESI, m/z): 502.3 [M þ H]þ. 4.2.2. 3b: 5,7-Dimethoxy-3-(3-(pyrrolidin-1-yl)propoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 55.6%, m.p. 207e209
C; IR (KBr, cm1): nmax 1624, 1598, 1558, 1506, 1411, 1348, 1211, 1122, 1014, 854, 829; 1 H NMR (500 MHz, CDCl3) d: 2.09 (m, 4H, CH2), 2.13e2.16 (m, 2H, CH2), 3.47 (m, 4H, CH2), 3.57 (t, J ¼ 12.6 Hz, 2H, CH2), 3.82e3.85 (m, 17H, 5  OCH3, OCH2), 6.29 (d, J ¼ 1.7 Hz, 1H, H-6), 6.50 (d, J ¼ 2.3 Hz, 1H, H-8), 7.20 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 175.1 (C-4), 165.0 (C-7), 160.7 (C-9), 159.0 (C-2), 154.2 (C-5), 153.3 (C-30 , C-50 ), 140.6 (C-40 ), 139.5 (C-3), 124.8 (C-10 ), 108.3 (C-10), 105.6 (C-60 , C-20 ), 96.6 (C-6), 92.9 (C-8), 70.6 (OCH2), 61.0 (40 -OCH3), 56.7 (30 , 5, 7- 3OCH3), 56.3 (5-OCH3), 54.3 (2CH2),

4.2.3. 3c: 5,7-Dimethoxy-3-(3-(piperidin-1-yl)propoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 67.4%, m.p. 166e168
C; IR (KBr, cm1): nmax 1625, 1597, 1558, 1506, 1417, 1354, 1209, 1126, 1016, 852, 815; 1 H NMR (500 MHz, CDCl3) d: 1.66 (brm, 2H, CH2), 1.89e1.91 (m, 4H, CH2), 2.21 (m, J ¼ 10.9 Hz, 2H, CH2), 3.38 (brm, 4H, CH2), 3.49 (t, J ¼ 12.6 Hz, 2H, CH2), 3.87e3.93 (m, 17H, 5  OCH3, OCH2), 6.34 (d, J ¼ 1.7 Hz, 1H, H-6), 6.55 (d, J ¼ 1.7 Hz, 1H, H-8), 7.26 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 175.0 (C-4), 165.2 (C7), 160.9 (C-9), 159.1 (C-2), 154.4 (C-5), 153.4 (C-30 , C-50 ), 140.8 (C40 ), 139.5 (C-3), 124.9 (C-10 ), 108.3 (C-10), 105.7 (C-60 , C-20 ), 96.6 (C6), 92.9 (C-8), 71.0 (OCH2), 61.1 (40 -OCH3), 56.7 (7-OCH3), 56.6 (30 , 50 -2OCH3), 56.4 (5-OCH3), 53.37 (2CH2), 53.6 (CH2), 24.6 (CH2), 23.4 (2CH2), 22.0 (CH2); MS (ESI, m/z): 514.3 [M þ H]þ. 4.2.4. 3d: 5,7-Dimethoxy-3-(3-morpholinopropoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 72.8%, m.p. 172e174
C; IR (KBr, cm1): nmax 1635, 1600, 1558, 1506, 1411, 1350, 1242, 1211, 1122, 1008, 858, 825; 1H NMR (500 MHz, CDCl3) d: 1.80e1.86 (m, 2H, CH2), 2.29 (brs, 4H, CH2), 2.38 (t, J ¼ 14.9 Hz, 2H, CH2), 3.59 (t, J ¼ 9.15 Hz, 4H, CH2), 3.84e3.88 (m, 15H, 5  OCH3), 4.01 (t, J ¼ 12.5 Hz, 2H, OCH2), 6.26 (d, J ¼ 2.3 Hz, 1H, H-6), 6.50 (d, J ¼ 1.9 Hz, 1H, H-8), 7.27 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 174.0 (C-4), 164.0 (C-7), 161.0 (C-9), 158.7 (C-2), 152.9 (C-5), 152.5 (C-30 , C-50 ), 140.6 (C-40 ), 139.8 (C-3), 126.1 (C-10 ), 109.4 (C-10), 105.9 (C-60 , C-20 ), 95.8 (C-6), 92.4 (C-8), 70.8 (OCH2), 66.9 (2CH2), 61.0 (40 -OCH3), 56.3 (CH2), 56.4 (30 , 50 -2OCH3), 55.8 (7-OCH3), 55.8 (5-OCH3), 53.7 (2CH2), 27.5 (CH2); MS (ESI, m/z): 516.3 [M þ H]þ. 3e: 5,7-dimethoxy-3-(3-(4-methylpiperazin-1-yl)propoxy)-2(3,4,5-trimethoxyphenyl)-4H-chromen-4-one. Light yellow solid, yield, 64.3%, m.p.150~152
C; IR (KBr, cm1): nmax 1627, 1600, 1558, 1506, 1417, 1348, 1213, 1126, 1012, 854, 817; 1 H NMR (500 MHz, CDCl3) d: 1.94e1.98 (m, 2H, CH2), 2.35 (s, 3H, CH3), 2.67e2.71 (m, 10H, CH2), 3.86e3.90 (m, 15H, 5  OCH3), 3.99 (t, J ¼ 12.05 Hz, 2H, OCH2), 6.30 (d, J ¼ 2.3 Hz, 1H, H-6), 6.50 (d, J ¼ 2.3 Hz, 1H, H-8), 7.27 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 174.0 (C-4), 164.1 (C-7), 161.0 (C-9), 158.8 (C-2), 153.0 (C-5), 152.9 (C-30 , C-50 ), 140.3 (C-40 ), 140.0 (C-3), 125.9 (C-10 ), 109.3 (C-10), 105.9 (C-60 , C-20 ), 95.9 (C-6), 92.5 (C-8), 70.4 (OCH2), 61.0 (40 -OCH3), 56.4 (30 , 50 ,7-3OCH3), 55.9 (5-OCH3), 55.1 (CH2), 53.8 (2CH2), 52.0 (CH2), 45.2 (CH3), 27.0 (CH2); MS (ESI, m/z): 529.3 [M þ H]þ. 4.2.5. 3f: 3-(4-(diethylamino)butoxy)-5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 74.8%, m.p.93~95
C; IR (KBr, cm1): nmax 1627, 1602, 1558, 1506, 1417, 1350, 1249, 1213, 1130, 1018, 856, 813; 1H NMR (500 MHz, CDCl3) d: 0.96 (t, J ¼ 14.3 Hz, 6H, CH3), 1.49e1.55 (m, 2H, CH2), 1.66e1.71 (m, 2H, CH2), 2.43 (t, J ¼ 15.45 Hz, 2H, CH2), 2.48 (q, J ¼ 21.8 Hz, 4H, CH2), 3.85e3.90 (m, 15H, 5  OCH3), 3.99 (t, J ¼ 13.15 Hz, 2H, OCH2), 6.28 (d, J ¼ 2.3 Hz, 1H, H6), 6.44 (d, J ¼ 2.3 Hz, 1H, H-8), 7.32 (overlapping s, 2H, H-20 , H-60 ); 13 C NMR (125 MHz, CDCl3) d: 174.1 (C-4), 164.0 (C-7), 161.0 (C-9), 158.8 (C-2), 152.9 (C-5), 152.5 (C-30 , C-50 ), 140.7 (C-40 ), 139.8 (C-3), 126.1 (C-10 ), 109.4 (C-10), 105.9 (C-60 , C-20 ), 95.8 (C-6), 92.4 (C-8), 72.4 (OCH2), 61.0 (40 -OCH3), 56.4 (30 , 50 -2OCH3), 56.3 (7-OCH3), 55.8 (5-OCH3), 52.4 (CH2), 46.7 (3CH2), 28.4 (CH2), 22.9 (CH2), 11.3 (2CH3); MS (ESI, m/z): 516.4 [M þ H]þ. 4.2.6. 3g: 5,7-Dimethoxy-3-(4-(pyrrolidin-1-yl)butoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 59.1%, m.p.111~113
C; IR (KBr, cm1):

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nmax 1635, 1602, 1558, 1506, 1417, 1350, 1246, 1211, 1126, 999, 852, 819; 1H NMR (500 MHz, CDCl3) d: 1.70e1.73 (m, 2H, CH2), 1.77 (m, 2H, CH2), 1.84 (m, 4H, CH2), 2.75 (m, 6H, CH2), 3.83e3.88 (m, 15H, 5  OCH3), 3.92 (t, J ¼ 11.05 Hz, 2H, OCH2), 6.29 (d, J ¼ 2.3 Hz, 1H, H6), 6.50 (d, J ¼ 2.3 Hz, 1H, H-8), 7.27 (overlapping s, 2H, H-20 , H-60 ); 13 C NMR (125 MHz, CDCl3) d: 174.1 (C-4), 164.1 (C-7), 160.9 (C-9), (158.8C-2), 153.0 (C-5), 152.7 (C-30 , C-50 ), 140.5 (C-40 ), 139.9 (C-3), 126.0 (C-10 ), 109.3 (C-10), 105.7 (C-60 , C-20 ), 95.9 (C-6), 92.5 (C-8), 71.6 (OCH2), 61.0 (40 -OCH3), 56.4 (7- OCH3), 56.3 (30 , 5-2OCH3), 55.9 (5-OCH3), 55.6 (CH2), 53.8 (2CH2), 27.9 (CH2), 24.2 (CH2), 23.3 (2CH2); MS (ESI, m/z): 514.3 [M þ H]þ. 4.2.7. 3h: 5,7-Dimethoxy-3-(4-(piperidin-1-yl)butoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 82.1%, m.p.71~73
C; IR (KBr, cm1): nmax 1625, 1602, 1558, 1506, 1417, 1350, 1244, 1213, 1128, 1016, 850, 813; 1H NMR (500 MHz, CDCl3) d: 1.33 (brm, 2H, CH2), 1.48e1.57 (m, 6H, CH2), 1.61e1.66 (m, 2H, CH2), 2.26e2.29 (m, 6H, CH2), 3.81e3.84 (m, 15H, 5  OCH3), 3.94 (t, J ¼ 12.6 Hz, 2H, OCH2), 6.23 (d, J ¼ 2.3 Hz, 1H, H-6), 6.39 (d, J ¼ 2.3 Hz, 1H, H-8), 7.27 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 174.0 (C-4), 163.9 (C7), 160.9 (C-9), 158.7 (C-2), 152.9 (C-5), 152.4 (C-30 , C-50 ), 140.6 (C40 ), 139.8 (C-3), 126.1 (C-10 ), 109.3 (C-10), 105.8 (C-60 , C-20 ), 95.8 (C6), 92.4 (C-8), 72.3 (OCH2), 61.0 (40 -OCH3), 58.9 (CH2), 56.3 (7OCH3), 56.3 (30 , 50 -2OCH3), 55.8 (5-OCH3), 54.3 (CH2), 28.5 (CH2), 25.5 (2CH2), 24.2 (CH2), 23.0 (CH2); MS (ESI, m/z): 528.4 [M þ H]þ. 4.2.8. 3i: 5,7-Dimethoxy-3-(4-morpholinobutoxy)-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 77.4%, m.p.109~111
C; IR (KBr, cm1): nmax 1635, 1600, 1558, 1506, 1417, 1348, 1244, 1213, 1134, 1114, 1016, 850, 812; 1H NMR (500 MHz, CDCl3) d: 1.50e1.56 (m, 2H, CH2), 1.66e1.71 (m, 2H, CH2), 2.26 (t, J ¼ 14.9 Hz, 2H, CH2), 2.3 (brs, 4H, CH2), 3.61 (t, J ¼ 9.15 Hz, 4H, CH2), 3.84e3.88 (m, 15H, 5  OCH3), 3.98 (t, J ¼ 13.15 Hz, 2H, OCH2), 6.26 (d, J ¼ 1.7 Hz, 1H, H-6), 6.50 (d, J ¼ 1.7 Hz, 1H, H-8), 7.30 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 174.0 (C-4), 164.0 (C-7), 161.0 (C-9), 158.7 (C-2), 152.9 (C-30 , C-50 ), 152.4 (C-5), 140.7 (C-40 ), 139.8 (C-3), 126.1 (C-10 ), 109.4 (C-10), 105.9 (C-60 , C-20 ), 95.8 (C-6), 92.4 (C-8), 72.3 (OCH2), 66.9 (2CH2), 61.0 (40 -OCH3), 58.6 (CH2), 56.4 (30 , 50 -2OCH3), 56.3 (7OCH3), 55.8 (5-OCH3), 53.6 (2CH2), 28.2 (CH2), 22.9 (CH2); MS (ESI, m/z): 530.3 [M þ H]þ. 4.2.9. 3j: 5,7-Dimethoxy-3-(4-(4-methylpiperazin-1-yl)butoxy)-2(3,4,5-trimethoxyphenyl)-4H-chromen-4-one Light yellow solid, yield, 76.7%, m.p.82~84
C; IR (KBr, cm1): nmax 1627, 1602, 1558, 1506, 1417, 1350, 1244, 1211, 1128, 1014, 852, 815; 1H NMR (500 MHz, CDCl3) d: 1.44e1.50 (m, 2H, CH2), 1.56e1.61 (m, 2H, CH2), 2.17 (s, 3H, CH3), 2.26 (t, J ¼ 14.9 Hz, 2H, CH2), 2.41 (brm, 6H, CH2), 3.74e3.78 (m, 15H, 5  OCH3), 3.87 (t, J ¼ 13.15 Hz, 2H, OCH2), 6.15 (d, J ¼ 2.3 Hz, 1H, H-6), 6.32 (d, J ¼ 2.3 Hz, 1H, H-8), 7.19 (overlapping s, 2H, H-20 , H-60 ); 13C NMR (125 MHz, CDCl3) d: 173.9 (C-4), 163.9 (C-7), 160.8 (C-9), 158.6 (C-2), 152.8 (C-30 , C-50 ), 152.3 (C-5), 140.5 (C-40 ), 139.7 (C-3), 126.0 (C-10 ), 109.2 (C-10), 105.7 (C-60 , C-20 ), 95.7 (C-6), 92.3 (C-8), 72.0 (OCH2), 60.9 (40 -OCH3), 57.7 (CH2), 56.2 (30 , 50 , 7-3OCH3), 55.8 (5-OCH3), 54.2 (2CH2), 52.2 (2CH2), 45.3 (CH3), 28.1 (CH2), 22.9 (CH2); MS (ESI, m/z): 543.4 [M þ H]þ.

161

for 10 h, compound 4 was obtained. To a solution of compound 4 (1.0 mmol) in ethanol (20 mL) was added hydrazine hydrate (2.0 mmol), the mixture was refluxed for 2 h, 2-(5,7-dimethoxy-4oxo-2-(3,4,5-trimethoxyphenyl)-4H-chromen-3-yloxy)aceto-hydrazide 5 was synthesized. To a solution of compound 5 (3.3 mmol) and 2-fluorobenzaldehyde (3.6 mmol) in ethanol (20 mL), catalytic amount of acetic acid was added, the mixture was refluxed for 2 h. After this time, the mixture was concentrated under vacuum and purified by recrystallization with ethanol to afford 6a. Compounds 6b~6f were synthesized with the same method (Scheme 2). 4.3.1. 6a: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'- (2-fluorinebenzyl)acetylhydrazine Light yellow solid, yield, 78.4%, m.p. 136e138
C; IR (KBr, cm1): nmax 1699, 1683, 1620, 1604, 1558, 1417, 1361, 1246, 1207, 1124, 1109, 1012, 819; 1H NMR (500 MHz, CDCl3) d: 3.92e3.99 (m, 15H, 5  OCH3), 4.35 (s, 2H, CH2), 6.40 (d, J ¼ 2.3 Hz, 1H, H-6), 6.54 (d, J ¼ 2.3 Hz, 1H, H-8), 7.07 (J ¼ 18.9 Hz, 1H), 7.16 (t, J ¼ 14.9 Hz, 1H), 7.22 (overlapping s, 2H, H-20 , H-60 ), 7.35 (q, J ¼ 20.6 Hz, 1H), 8.15 (t, J ¼ 13.7 Hz, 1H), 9.65 (s, 1H, NH), 12.35 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.7 (C-4), 165.6 (C]O), 164.7 (C-7), 161.1 (C9), 160.6 (C), 159.1 (C-2), 154.2 (C-5), 153.6 (C-30 , C-50 ), 142.6 (CH), 141.3 (C-40 ), 140.8 (C-3), 132.0 (CH), 127.6 (CH), 124.8 (C-10 ), 124.4 (CH), 121.8 (C), 115.7 (CH), 108.8 (C-10), 105.7 (C-60 , C-20 ), 96.5 (C-6), 92.7 (C-8), 73.2 (OCH2), 61.2 (40 -OCH3), 56.6 (7-OCH3), 56.5 (30 , 5’ eOCH3), 56.0 (8-OCH3); MS (ESI, m/z): 567.3 [M þ H]þ, 589.2 [M þ Na]þ. 4.3.2. 6b: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'- (4-methylbenzyl)acetylhydrazine White solid, yield, 82.3%, m.p. 152e154
C; IR (KBr, cm1): nmax 1699, 1683, 1620, 1604, 1558, 1506, 1417, 1354, 1247, 1219, 1124, 1109, 1011, 819; 1H NMR (500 MHz, CDCl3) d: 2.36 (s, 3H, CH3), 3.92e3.99 (m, 15H, 5  OCH3), 4.35 (s, 2H, CH2), 6.40 (d, J ¼ 2.3 Hz, 1H, H-6), 6.54 (d, J ¼ 2.3 Hz, 1H, H-8), 7.19 (d, J ¼ 8 Hz, 2H, 2CH), 7.21 (overlapping s, 2H, H-20 , H-60 ), 7.71 (d, J ¼ 8 Hz, 2H, CH), 8.35 (s, 1H, NH), 12.10 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.7 (C-4), 165.2 (C]O), 164.9 (C-7), 161.1 (C-9), 159.1 (C-2), 154.2 (C-5), 153.6 (C-30 , C-50 ), 149.5 (C),141.5 (C-40 ), 140.8 (C-3), 131.1 (C), 129.6 (CH), 129.4 (CH), 128.5 (CH), 127.9 (CH), 124.8 (C-10 ), 108.8 (C-10), 105.7 (C-60 , C-20 ), 96.5 (C-6), 92.8 (C-8), 73.3 (OCH2), 61.2 (40 -OCH3), 56.7 (7-OCH3), 56.5 (30 , 50 -OCH3), 56.0 (8-OCH3), 21.7 (CH3); MS (ESI, m/ z): 563.3 [M þ H]þ, 585.3 [M þ Na]þ. 4.3.3. 6c: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'-(2-pyridyl)acetylhydrazine Light yellow solid, yield, 55.6%, m.p. 176e178
C; IR (KBr, cm1): nmax 1699, 1683, 1602, 1588, 1506, 1417, 1361, 1244, 1211, 1126, 1012, 819; 1H NMR (500 MHz, CDCl3) d: 3.92e3.99 (m, 15H, 5  OCH3), 4.36 (s, 2H, CH2), 6.40 (d, J ¼ 2.3 Hz, 1H, H-6), 6.53 (d, J ¼ 2.3 Hz, 1H, H-8), 7.22 (overlapping s, 2H, H-20 , H-60 ), 7.27 (t, J ¼ 13.3 Hz, 1H), 7.71 (t, J ¼ 15.4 Hz, 1H), 8.20 (d, J ¼ 8 Hz, 1H), 8.53 (s, 1H, NH), 8.62 (d, J ¼ 4.6 Hz, 1H), 12.53 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.7 (C-4), 165.9 (C]O), 164.8 (C-7), 161.2 (C-9), 159.1 (C-2), 154.1 (C-5), 153.6 (C-30 , C-50 ), 153.3 (C), 149.6 (CH), 149.4 (CH),141.4 (C-40 ), 140.8 (C-3), 136.4 (CH), 127.6 (CH), 124.8 (C-10 ), 121.3 (CH), 108.8 (C-10), 105.7 (C-60 , C-20 ), 96.5 (C-6), 92.7 (C-8), 73.2 (OCH2), 61.2 (40 -OCH3), 56.6 (7-OCH3), 56.5 (30 , 50 -OCH3), 56.0 (8-OCH3); MS (ESI, m/z): 550.3 [M þ H]þ, 572.2 [M þ Na]þ.

4.3. General procedure for the synthesis of compounds 6a~6f To a solution of 3-hydroxy-5,7-dimethoxy-2-(3,4,5trimethoxyphenyl)-4H-chromen-4-one 1 (0.5 mmol) in acetone (20 mL) was added anhydrous K2CO3 (1.0 mmol) and ethyl 2bromoacetate (1.0 mmol), and the mixture was allowed to reflux

4.3.4. 6d: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'- (2-furan)acetylhydrazine White solid, yield, 73.2%, m.p. 172e174
C; IR (KBr, cm1): nmax 1699, 1683, 1608, 1558, 1506, 1417, 1359, 1246, 1217, 1126, 1066, 1014, 819; 1H NMR (500 MHz, CDCl3) d: 3.92e3.99 (m, 15H,

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5  OCH3), 4.32 (s, 2H, CH2), 6.41 (d, J ¼ 2.3 Hz, 1H, H-6), 6.48 (q, J ¼ 5.1 Hz, 1H, CH), 6.54 (d, J ¼ 2.3 Hz, 1H, H-8), 6.86 (d, J ¼ 3.4 Hz, 1H, CH), 7.20 (overlapping s, 2H, H-20 , H-60 ), 7.51 (d, J ¼ 1.15 Hz, 1H, CH), 8.30 (s, 1H, NH), 12.25 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.8 (C-4), 165.2 (C]O), 164.9 (C-7), 161.1 (C-9), 159.1 (C2), 154.2 (C-5), 153.6 (C-30 , C-50 ), 149.5 (C), 144.7 (CH), 141.5 (C-40 ), 140.8 (C-3), 138.8 (CH), 124.8 (C-10 ), 113.3 (CH), 111.9 (CH), 108.8 (C10), 105.7 (C-60 , C-20 ), 96.6 (C-6), 92.8 (C-8), 73.3 (OCH2), 61.2 (40 OCH3), 56.7 (7-OCH3), 56.5 (30 , 50 -OCH3), 56.0 (8-OCH3); MS (ESI, m/ z): 539.3 [M þ H]þ, 561.2 [M þ Na]þ. 4.3.5. 6e: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'-(3-methyl thiophene)acetylhydrazine White solid, yield, 65.9%, m.p. 132e134
C; IR (KBr, cm1): nmax 1699, 1681, 1620, 1606, 1558, 1506, 1417, 1354, 1246, 1217, 1126, 1107, 1064, 1010, 816; 1H NMR (500 MHz, CDCl3) d: 2.42 (s, 3H, CH3), 3.92e3.99 (m, 15H, 5  OCH3), 4.32 (s, 2H, CH2), 6.41 (d, J ¼ 2.3 Hz, 1H, H-6), 6.54 (d, J ¼ 2.3 Hz, 1H, H-8), 6.84 (d, J ¼ 5.15 Hz, 1H, CH), 7.21 (overlapping s, 2H, H-20 , H-60 ), 7.28 (d, J ¼ 5.15 Hz, 1H, CH), 8.63 (s, 1H, NH), 12.08 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.7 (C-4), 164.9 (C]O), 164.9 (C-7), 161.1 (C-9), 159.1 (C-2), 154.1 (C-5), 153.6 (C-30 , C-50 ), 144.1 (C),141.4 (C-40 ), 140.8 (C-3), 140.5 (C), 132.5 (CH), 130.5 (CH), 128.0 (CH), 124.8 (C-10 ), 108.8 (C-10), 105.7 (C-60 , C20 ), 96.5 (C-6), 92.8 (C-8), 73.3 (OCH2), 61.2 (40 -OCH3), 56.7 (7OCH3), 56.5 (30 , 50 -OCH3), 56.0 (8-OCH3), 14.2 (CH3); MS (ESI, m/ z): 569.2 [M þ H]þ, 591.2 [M þ Na]þ.

4.5. Cell morphology Cells were suspended and diluted to 3  104 cells mL1 with DMDM medium containing 10% FBS, and 100 mL cell suspension was added to each well of 96-well plates. The plates were incubated for 24 h at 37
C, 5% CO2 condition, then, 200 mL medium with different concentrations of compounds diluted in DMSO was added to each well. DMSO concentration in final culture medium should not exceed 0.1%. The plates were subsequently incubated for the certain period, then, cell morphological changes were observed under inverted microscope, and the pictures were taken in randomly selected fields. 4.6. Cell cycle analysis The cell cycle analysis, was performed by cell cycle kit (Beyotime, China). MDA-MB-231cells were incubated with compound 6d at 1 mM concentrations for 48 h. Untreated and treated cells were harvested, then, MDA-MB-231cells were washed three times by cold PBS, and then cells were fixed in 70% ethanol at 20
C for 12 h. After fixation, cells were washed with cold PBS and stained with 0.5 mL of propidium iodide (PI) staining buffer, which contain
200 mg/mL RNase A, 50 mg/mL PI, at 37 C for 30 min in the dark. Analyses were performed on FACScan flow cytometer. The experiments were repeated three times. 4.7. Telomerase activity assay

4.3.6. 6g: 2-(5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4Hchromen-3-yloxy) eN'-(2-pyrroly)acetylhydrazine White solid, yield, 72.4%, m.p. 135e137
C; IR (KBr, cm1): nmax 1699, 1681, 1620, 1602, 1558, 1506, 1417, 1354, 1244, 1213, 1126, 1107, 1039, 1006, 815; 1H NMR (500 MHz, CDCl3) d: 3.92e4.00 (m, 15H, 5  OCH3), 4.32 (s, 2H, CH2), 6.25 (d, J ¼ 2.3 Hz, 1H, H-6), 6.41 (d, J ¼ 2.3 Hz, 1H, H-8), 6.54 (m, 2H, 2CH), 6.91 (s, 1H, CH), 7.21 (overlapping s, 2H, H-20 , H-60 ), 8.23 (s, 1H, NH), 9.54 (s, 1H, NH), 12.11 (s, 1H, N]CH); 13C NMR (125 MHz, CDCl3) d: 174.8 (C-4), 165.2 (C]O), 164.9 (C-7), 161.0 (C-9), 159.1 (C-2), 154.1 (C-5), 153.6 (C-30 , C-50 ), 141.4 (C-40 ), 140.8 (C-3), 140.8 (CH), 127.2 (C), 124.8 (C-10 ), 124.3 (CH), 114.8 (CH), 109.8 (CH), 108.8 (C-10), 105.7 (C-60 , C-20 ), 96.5 (C-6), 92.8 (C-8), 73.2 (OCH2), 61.2 (40 -OCH3), 56.7 (7-OCH3), 56.5 (30 , 50 -OCH3), 56.0 (8-OCH3); MS (ESI, m/z): 538.3 [M þ H]þ, 560.2 [M þ Na]þ. 4.4. Anticancer assay Human tumor cell lines Bcap-37, MDA-MB-231, SGC-7901 and MGC-803 from laboratory of The First Affiliated Hospital of Anhui Medical University were used.The cytotoxicity evaluation was conducted by using a modified procedure described in the literature [22]. 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 cytotoxicity assessment. Tested samples at pre-set concentrations were added to 6 wells with ADM co-assayed as a positive reference. After 48 h exposure period, 25 mL of PBS containing 2.5 mg/mL of MTT (3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide) was added to each well. After 4 h, the medium was replaced by 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 done in triplicate and were expressed as means ± SD using Student t test. The IC50 value was defined as the concentration at which 50% of the cells could survive.

Compounds were tested in a search for small molecule inhibitors of telomerase activity by using the TRAP-PCR-ELISA assay [23]. In detail, the MDA-MB-231 cells were firstly maintained in DMEM medium (GIBCO, New York, USA) supplemented with 10% fetal bovineserum (GIBCO, NewYork, 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 examined compound with a series of concentration: as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.082 l mM, 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-PCRELISA 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. 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 streptavidin-coated microtiter plate subsequently. The immobilized DNA fragment 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. 4.8. Western blotting Mouse anti-TERT monoclonal antibody was purchased from Abcam (Cambridge, UK). Secondary antibodies for goat anti-rabbit

W. Xue et al. / European Journal of Medicinal Chemistry 97 (2015) 155e163

immunoglobulin (Ig) G horse radish peroxidase (HRP), goat antimouse IgG HRP was purchased from Santa Cruz Biotechnology (California, USA). b-actin antibody was obtained from Santa Cruz Biotechnology (California, USA). AZT as telomerase inhibitor was obtained from SigmaeAldrich (poole, UK). And iCRT, a non-special b-catenin inhibitor, was produced by Merck Millipore Company (Darmstadt, Germany). Human MDA-MB-231 cells were lysed with RIPA lysis buffer (Beyotime, China). Whole extracts were prepared, and protein concentration was detected using a BCA protein assay kit (Beyotime, China). Total protein (30 or 50 mg) from samples were separated by SDS-PAGE and blotted onto a PVDF membrane (Millipore Corp, Billerica, MA, USA). After blockade of nonspecific protein binding, nitrocellulose blots were incubated for 1 h with primary antibodies diluted in TBS/Tween20 (0.075%) containing 3% Marvel. Mouse monoclonal antibody recognizing TERT (Abcam, UK) was used 1:500 as was anti-b-actin (Santa Cruz, USA). Horseradish peroxidase conjugated anti-mouse and anti-rabbit antibodies were used as secondary antibodies correspondingly. After extensive washing in TBS/Tween-20, the blots were processed with distilled water for detection of antigen using the enhanced chemiluminescence system. Proteins were visualized with ECLchemiluminescent kit (ECL-plus, Thermo Scientific) [24]. 4.9. General procedure for molecular docking In this study, a three-dimension human telomerase model [19] €dinger's IFD (Induced Fit Docking) were used to and in silico Schro model the binding poses of our designed compounds with hTERT. Before doing the IFD, the structural errors of the model was first corrected manually and then the complex was treated by Protein €dinger. IFD is allowing incorporation of Prepared Wizard of Schro the protein and ligand flexibility in the docking protocol, which is consisted of the following steps: (i) constrained minimization of the protein with an RMSD cutoff of 0.18 Å; (ii) initial Glide docking of the ligand using a softened potential (Van der Waals radii scaling); (iii) one round of Prime side-chain prediction for each protein/ligand complex, on residues within defined distance of any ligand pose; (iv) prime minimization of the same set of residues and the ligand for each protein/ligand complex pose; (v) Glide redocking of each protein/ligand complex structure within a specified energy of the lowest energy structure; (vi) estimation of the binding energy (IFDScore) for each output pose. All docking calculations were run in the extra precision (XP) mode of Glide. The center of the grid box of the hTERT was defined by two residues in the active site: Lys 710 and Lys 902. The size of the grid box was set to 15 Å. Default values were used for all other parameters. A further 10 ns MD simulation was submitted using a docking pose which can account for the limited SAR of BIBR1532 and BIBR1591 by employing the program of Desmond, which was developed at D. E. Shaw Research to perform high-speed molecular dynamics simulations of biological systems on conventional commodity clusters. All suggested values of Desmond were employed to run this 10 ns MD simulation. The MD simulation include the following steps: (i) a solvated system for simulation was generated;

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