Novel 2H-chromen derivatives: design, synthesis and anticancer activity

Abstract

A series of novel dihydropyrazole derivatives linked with 2H-chromen were designed and synthesized. All of the compounds have been screened for their antiproliferative activity against MGC-803, Bcap-37, SGC-7901 and HepG₂ cell lines in vitro. The results revealed that compounds 4a and 10a exhibited strong inhibitory activity against HepG₂ cell and manifested obvious un-toxic effect on GES-1 and L-02 cell lines. Some title compounds were tested against telomerase, compound 10a showed the most potent inhibitory activity with IC₅₀ value at 0.98 ± 0.11 μM, it could fit well into the active site of TERT. The further molecular mechanism of antiproliferation was explored, the data suggested that compound 10a could inhibit hTERT expression and Wnt/β-catenin signaling.

---

Introduction

Many 4,5-dihydropyrazole-based derivatives have shown several biological activities as seen for cannabinoid receptor 1 antagonist, monoamine oxidase inhibitors, and tumor necrosis inhibitors,^1–7 several dihydropyrazole derivatives used as anticancer agents were described in recent publications.^8–12 In our recent work,¹³ several dihydropyrazole derivatives were designed, which had potent anticancer activity as potential telomerase inhibitors (Fig. 1A). Recently, 2H-chromen and its derivatives were extensively studied to treat various types of cancer, their derivatives with antitumor effect and could cause significant change in the regulation of immune responses, cell growth and differentiation (Fig. 1B). Motivated by the afore-mentioned findings, when we introduced the chromen moiety into the dihydropyrazole skeleton, the compound of dihydropyrazole linked with 2H-chromen showed better anticancer activity (Fig. 1C).^14–16

Fig. 1 Moieties with high activity against cancer cells.

In this study, it was of our interest to utilize rational chemical approaches to generate and identify novel derivatives as potential hTERT inhibitors for cancer therapy. Our group recently disclosed the first X-ray crystal structure of a small molecule inhibitor bound to hTERT motor domain construct (compound dihydropyrazole-chromen targeted with TERT, Fig. 2A). Since structural information of the binding site was available to us, we initiated an effort to leverage molecular modeling in combination with available data to provide guidance for future target selection. To this end, computer-generated molecular models of dipropylamino-acetyl-trifluoromethylphenyl dihydropyrazole-chromen moiety docked into the allosteric site of hTERT were analyzed. Is that what we expected, when trifluoromethylphenyl was introduced (Fig. 2B), which would form a halogen bond, the dihydropyrazole ring projected into a more stable hydrophobic region, should enhance the anticancer activity.

Fig. 2 (A) Molecular docking modeling of compound with telomerase (B) molecular modeling for target selection.

Since dominant-negative mutants of hTERT was demonstrated to have selective anticancer effects in tumor cells. So, the mechanism of title compound inhibiting hTERT was investigated.

Results and discussion

Chemistry

Started from substituted-salicylaldehyde and acetoacetate, piperazine used as catalyst at 25–30 °C (Scheme 1), compound 1 was synthesized. Claisen–Schmidt condensation 3-acetyl-2H-chromen-2-one and substituted-benzaldehyde using mild catalyst piperidine, proved to be an efficient method for the synthesis of α, β unsaturated ketone 2. Using 2-bromoacetic acid and 4-nitrobenzenesulfonyl chloride, proved to be an efficient alternative method for synthesis of title compound 3. In addition, in order to facilitate the synthesis of title compounds 4a–l, KI of catalytic amount was added. Compounds 1–3 were prepared according to a previously published report.⁶ The synthesis of title compounds 10a–e started from phenol. The general synthetic method for the key intermediate compound 7 was shown in Scheme 2. Firstly, Compound 5 was prepared according to the procedure reported by Blaquiere, N. and Evans, D. A. et al. with some modifications. Secondly, compound 5 reacted with Eaton's reagent to produce compound 6. Thirdly, compound 7 was synthesized by Vilsmeier reaction.

Scheme 1 Synthesis of title compounds 4a–l. Reagent and conditions: (A) piperazine, 25–30 °C, 20 min. (B) 2-(Trifluoromethyl) benzaldehyde, piperidine, butanol, reflux, 14 h. (C) NH₂–NH₂·H₂O, C₂H₅OH, BrCH₂COOH, 4-nitrobenzenesulfonyl chloride 40–60 °C, 2 h. (D) Dipropylamine, DMAP, KI, 40–50 °C, 4 h. R¹ = H, R² = 2-CF₃ (4a); R¹ = 6-NO₂, R² = 4-Br (4f); R¹ = H, R² = 3-CF₃ (4k); R¹ = H, R² = 4-Br (4b);R¹ = 6-NO₂, R² = 4-Me (4g); R¹ = 6-Me, R² = 4-CF₃ (4l). R¹ = H, R² = 4-OMe (4c); R¹ = 6-Me, R² = 2-CF₃ (4h); R¹ = H, R² = 4-F (4d);R¹ = 6-Me, R² = 4-F (4i); R¹ = 6-NO₂, R² = 2-CF₃ (4e); R¹ = 6-Me, R² = 4-Br (4j).

Scheme 2 Synthesis of title compounds 10a–e. Reagents and conditions: (E) Meldrum's acid, 90–100 °C; (F) Eaton's reagent, 60–70 °C; (G) POCl₃, DMF, 50–60 °C; (H) substituted-acetophenone, piperidine, butanol, reflux, 8 h. (I) NH₂–NH₂·H₂O, C₂H₅OH; BrCH₂COOH, 4-nitrobenzenesulfonyl chloride, reflux, 4 h. (J) Dipropylamine, DMAP, KI, 40–50 °C, 5 h. R³ = 4-CF₃ (10a); R³ = 4-Me (10b); R³ = 2-Br (10c); (10d); R³ = 4-NO₂ (10b); R³ = H (10e).

Crystal structure analysis

The structure of compound 4a was determined by X-ray crystallography. Crystal data of 4a: colorless crystals, yield, 76%; mp 191–193 °C; C₂₇H₂₈F₃N₃O₃, monoclinic, space group P2₁/c; a = 7.673(5), b = 18.385(11), c = 17.392(11) (Å); α = 90, β = 92.942(7), γ = 90 (°), V = 2450(3) nm³, T = 293(2) K, Z = 4, D_c = 1.343 g cm⁻³, F(000) = 1032, reflections collected/unique = 11282/4441, data/restraints/parameters = 4441/1/313, goodness of fit on F² = 1.039, fine, R₁ = 0.1055, wR(F²) = 0.2152. The molecular structure of compound 4a was shown in Fig. 3.

Fig. 3 ORTEP drawing of compound 4a.

In vitro anticancer activity

Our preliminary results indicated that compounds containing dihydropyrazole moiety displayed certain activity against human gastric cancer cell, so gastric cancer cells MGC-803 (human gastric cancer cell line), SGC-7901 (gastric cancer cell line) were chosen. In this screening assay study, all compounds were evaluated for their cytotoxic activity against SGC-7901, MGC-803, Bcap-37 (human breast cancer cell line) and human hepatoma (HepG₂) cell lines. The results were reported in terms of IC₅₀ values (Table 1). In the initial phase of our SAR study, seventeen compounds were divided into two types, one was the 3-coumarin-dihydropyrazole skeleton (compounds 4a–l), another was the 5-coumarin-dihydropyrazole skeleton (compounds 10a–e). In general, 5-coumarin-dihydropyrazole moiety leded to increase in inhibitory activity, among them, compound 10a was the most potent activity against HepG₂ cell with IC₅₀ value of 1.12 ± 0.06 μM, surpassing that of the positive control 5-fluorouracil. Furthermore, it is obvious that compounds 4a, 4e and 10a, 10c exhibited the strong inhibitory activity against the MGC-803 cell (with IC₅₀ values of 3.11 ± 0.37, 3.38 ± 0.41, 2.81 ± 0.30, 3.88 ± 0.71 μM respectively) and the values could compare with that of the 5-fluorouracil. All compounds showed poor activity against Bcap-37 cell comparable to that of the positive control.

Table 1 Cytotoxic activity of the synthesized compounds against SGC-7901, MGC-803, Bcap-37 and HepG₂ cell lines^a

Compound	IC50^b (μM)
	SGC-7901	MGC-803	Bcap-37	HepG2
a The data represented the mean of three experiments 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.c Used as a positive control, negative control 0.1%DMSO, no activity.
4a	7.01 ± 0.42	3.11 ± 0.37	32.75 ± 2.85	9.42 ± 0.58
4b	10.27 ± 1.22	11.41 ± 1.40	26.30 ± 2.50	31.08 ± 1.07
4c	14.90 ± 1.28	15.91 ± 1.33	30.71 ± 1.48	12.30 ± 0.35
4d	14.61 ± 0.97	23.50 ± 1.19	36.81 ± 1.82	26.31 ± 0.67
4e	9.34 ± 1.01	3.38 ± 0.41	40.52 ± 2.68	6.20 ± 0.81
4f	13.20 ± 0.70	12.68 ± 0.75	28.25 ± 2.21	37.0 ± 1.36
4g	30.31 ± 2.31	28.97 ± 2.08	33.55 ± 2.72	35.27 ± 1.02
4h	5.66 ± 0.58	4.70 ± 0.49	17.30 ± 1.21	5.68 ± 0.20
4i	18.73 ± 1.09	20.00 ± 1.22	36.50 ± 1.41	22.33 ± 0.90
4j	9.30 ± 0.69	7.00 ± 0.25	18.71 ± 0.94	4.52 ± 0.21
4k	38.55 ± 1.26	40.09 ± 1.37	36.84 ± 2.30	42.17 ± 1.98
4l	39.87 ± 2.08	26.51 ± 0.98	31.31 ± 1.58	33.75 ± 1.89
10a	4.31 ± 0.28	2.81 ± 0.30	9.04 ± 0.39	1.12 ± 0.06
10b	9.14 ± 0.60	6.03 ± 0.51	10.75 ± 0.90	12.08 ± 0.65
10c	5.42 ± 0.46	3.88 ± 0.71	11.05 ± 0.82	9.87 ± 0.44
10d	8.22 ± 0.54	5.90 ± 0.60	14.13 ± 0.55	21.31 ± 0.75
10e	9.12 ± 0.44	5.07 ± 0.29	10.58 ± 0.74	15.10 ± 0.35
5-Fluorouracil^c	6.89 ± 0.33	3.55 ± 0.30	5.04 ± 0.39	2.85 ± 0.17

---

The rationale behind selecting a large number of compounds bearing different functionalities was to establish a definite structure activity relationship pattern and emphasize the role of fluorine in imparting bioactivity. Scanning Table 1, we found that there was clear SAR against MGC-803 cell. Inspection of the chemical structure of the final compounds (Scheme 1) suggested that the introduction of trifluoromethyl group in the title compounds could significantly influence the antitumor activity. For example, CF₃ group was introduced into the 2-position of the phenyl ring in the series of 4, the compounds exhibited higher activity against MGC-803 cell (4a, 4e, 4h) while it was on the 3 or 4-position of the phenyl ring, the compounds showed poor activity against above cancer cells (4k and 4l); However, the presence of 4-CF₃ of the phenyl ring group in the series of 10, which played an important role in the antitumor activity (10a).

In order to explore that title compounds whether with high selectivity against tumor cells versus human somatic cells. We subsequently conducted a proliferative inhibition assay with human normal gastric mucosa cell (GES-1) and human normal liver cell (L-02). As given in Table 2, all compounds manifested obvious un-toxic effect on GES-1 and L-02 cells with IC₅₀ between the 1.6 to 2.5 mM. The data indicated that our compounds with high selectivity against tumor cells versus human somatic cells.

Table 2 IC₅₀ values of selected compounds against human normal cells L-02 and HK-2 proliferation^a

Compound	GES-1 (IC50, mM)	L-02 (IC50, mM)
a MTT assays were used for evaluation, and values were expressed as mean IC50 of the triplicate experiment.
4a	1.6	2.0
4e	1.6	2.0
10a	2.5	2.2
10c	2.2	1.8

---

Telomerase activity

Some compounds were assayed for telomerase inhibition, using HepG₂ cells extract, also included the activity of reference compounds Ethidium bromide and BIBR1532. The results (Table 3) suggested that compounds 4a and 10a showed strong telomerase inhibitory activity with IC₅₀ values of 1.09 ± 0.17, 0.98 ± 0.11 μM respectively, which surpassing that of the positive control ethidium bromide, comparable to that of positive control BIBR1532, furthermore, there was a good correlation between antiproliferative activity and telomerase activity of compounds 4a and 10a (Tables 1 and 3). Among them, trifluoromethylphenyl-5-coumarin-4,5-dihydropyrazole derivative 10a showed higher inhibitory activity than the others and with high selectivity against tumor cells versus human somatic cells. This also pointed out the direction for us to further optimize the structure of dihydropyrazole with anticancer activity as potential telomerase inhibitor.

Table 3 Inhibitory effects of selected compounds against telomerase

Compound	IC50 (μM) telomerase^a
a Telomerase supercoiling activity.b Ethidium bromide and BIBR1532 are reported as a control. The inhibition constant of ethidium toward telomerase has been reported previously.
4a	1.09 ± 0.17
4e	9.85 ± 0.67
4h	16.11 ± 1.55
10a	0.98 ± 0.11
10c	2.60 ± 0.32
Ethidium bromide^b	2.25 ± 0.18
BIBR1532^b	0.25 ± 0.12

---

Molecular docking

In an effort to elucidate the mechanism by which the title compound can inhibit telomerase, molecular docking of the potent inhibitor 10a into binding site of telomerase was performed to simulate a binding model derived from telomerase TERT (3DU6. pdb). The binding model of compound 10a with TERT was depicted in Fig. 4. In general, compound 10a could fit well into the catalytic subunit of telomerase (TERT) and inhibit telomerase activity as a substitute of substrate nucleotides. Six obvious interactions were shown. First, we found hydrogen bonds between LYS406 and two F atoms of CF₃; second, hydrogen bond (LYS372 with the F atom of CF₃) was found; third, Pi-cation interaction was formed (LYS372 with benzene ring, respectively); another two hydrogen bonds were formed between LYS189 and oxygen atom of compound 10a.

Fig. 4 The binding mode between the active conformation of compound 10a and the target protein TERT (PDB code: 3DU6). We employed 3D interaction map to display the interaction.

Inhibited hTERT expression and Wnt/β-catenin signalling

Because compound 10a showed the most potent activity against HepG₂ cells and strong telomerase inhibitory activity, molecular docking also showed that compound 10a could fit well into the TERT of telomerase, since the expression of hTERT is a rate-limiting step for telomerase activity, so we determined the effect of compound 10a on hTERT and further explored whether compound 10a inhibited the proliferation of HepG₂ cells by inhibiting hTERT through the canonical Wnt/β-catenin pathway, we studied the expression of the critical gene β-catenin and downstream effector genes cyclin D1 and c-myc in HepG₂ cells treated with compound 10a.

Analysis of hTERT by RT-PCR and western blotting showed inhibition of hTERT expression in response to compound 10a treatment for 48 h in HepG₂ cells. The β-actin unaffected by compound 10a was used as an internal control (Fig. 5). Meanwhile, results of RT-PCR and Western blotting showed that the expression of β-catenin, cyclin D1 and c-myc at both protein and mRNA levels in HepG₂ cells treated with compound 10a was obviously lowed compared with the control group. Based on these findings, it was reasonable to conclude that compound 10a might inhibit the proliferation of HepG₂ cells by inhibiting hTERT through Wnt/β-catenin signal pathway.

Fig. 5 Compound 10a inhibited hTERT expression and Wnt/β-catenin signalling, and the effect of AZT (telomerase inhibitor, 3.0 mM) and iCT (β-catenin inhibitor, 40 μM) on Wnt/β-catenin pathway in HepG₂ cells. (A) The mRNA expression of hTERT, β-catenin, cyclin D1 and c-myc was analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). (B) The protein expression of hTERT, β-catenin and downstream moleculars was analyzed by Western blotting. The results are expressed as relative expression against control expression. n = 3. Results are the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 vs. control.

Cell cycle analysis

It has been reported that the inhibition of hTERT expression significantly abrogated the cell viability in the short term, which may impact the cell cycle of cancer cells.¹⁷ To understand whether the reduced cell proliferation was due to cell cycle, we used flow cytometric analyses to measure the effect of compound 10a on induction of cell cycle. As shown in Fig. 6, the cells in G0/G1 phase in the HepG₂ control group accounted for about 46.2%, while after cells treated with compound 10a for 48 h, the ratio was approximately 54.8%, confirming that after down-regulating of hTERT, cells in G0/G1 phase increased and cell viability decreased by compound 10a. So the results indicated that compound 10a suppressed cell proliferation through inducing cell cycle arrest in G0/G1 phase. Furthermore, it has been reported that hTERT of telomerase regulated the expression of cyclin D1.¹⁸ Therefore, compound 10a may down-regulate cyclin D1 expression by hTERT, resulting in cell cycle arrest in G0/G1 phase.

Fig. 6 Cells cycle analysis by flow cytometry in HepG₂ cells. Cells were incubated with PI and examined by flow cytometry. The cells in G0/G1 phase in the HepG2 cells control group accounted for about 46.2%, while after cells treated with 10a for 48 h, the ratio was approximately 54.8%, suggesting that compound 10a increased the number of cells in G0/G1 phase and decreased cell viability. Control; DMSO (1‰), negative control; 5-Fu (2.8 μM), positive control; compound 10a (1.1 μM). n = 3. Results are the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01 vs. control.

Conclusions

In brief, based on reasonable molecular design, we designed some novel 3 or 5-coumarin-4,5-dihydropyrazole derivatives, followed by chemical synthesis and biological evaluated for them. The results revealed that compound 10a exhibited strong inhibitory activity against HepG₂ cells, and showed the most potent telomerase inhibitory activity with IC₅₀ value at 0.98 ± 0.11 μM. The docking simulation was performed to get the probable binding models and poses. The results indicated that compound 10a, which acted as potential telomerase inhibitor, could bind well into the active site of TERT. The study also indicated that our compounds with high selectivity against tumor cells versus human somatic cells. Compared with our previous studies, the advantage of this study was that our experiments demonstrated that title compound 10a could inhibit the proliferation of HepG₂ cells by modulating TERT through Wnt/β-catenin signal pathway. These results are of help in the rational design of higher selectivity telomerase TERT inhibitors in further.

Experimental section

Chemistry

The reactions were monitored by thin layer chromatography (TLC) on Merck pre-coated silica GF254 plates. Melting points (uncorrected) were determined on a XT4MP apparatus (Taike Corp., Beijing, China). ¹H NMR, ¹³C NMR spectrum were measured on a Mercury Plus-500 MHz spectrometer in CDCl₃ solution with TMS as the internal standard. Elemental analyses were performed on a CHN–O-Rapid instrument, and were within ±0.4% of the theoretical values. Compound 1 was prepared according to literature method as described.⁶

General synthetic procedure process for compound 4a–l

To a chloroform (20 mL) solution of 3-(1-(2-bromoacetyl)-5-(substituted)-4,5-dihydro-1H-pyrazol-3-yl)-6-substituted-2H-chromen-2-one 3 (10 mmol) was added dipropylamine (12 mmol), DMAP (10 mmol) and catalytic KI, the reaction mixture was allowed to stand at 40–50 °C for 4 h. The mixture was cooled, washed with water. The product was collected by filtration and the crude residue was purified by chromatography on SiO₂ (dichloromethane–methanol, v:v= 60 : 1) to give title compounds 4a–l (Scheme 1) as colorless solids.

4a. 3-(1-(2-(Dipropylamino)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 76%; mp 191–193 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.88 (t, 6H, J = 7.5 Hz), 1.51 (t, 4H, J = 7.5 Hz), 2.62 (q, 4H, J = 6.3 Hz), 3.27 (dd, 1H, J₁ = 19.2 Hz, J₂ = 5.7 Hz, pyrazole 4-H_a), 3.76 (d, 1H, J = 16 Hz), 3.93 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.2 Hz, pyrazole 4-H_b), 4.00 (d, 1H, J = 16.1 Hz), 5.93 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 7.14–7.67 (m, 8H, ArH and chromen H), 8.42 (s, 1H, chromen C₄–H). ¹³C NMR (CDCl₃, 125 MHz): δ 11.94, 21.09, 44.38, 54.79, 56.47, 57.22, 116.78, 118.84, 119.62, 125.01, 125.14, 126.45, 127.66, 128.87, 132.71, 133.05, 140.40, 141.15, 150.28, 154.22, 159.10, 169.32; ESI-MS: 499.89 (C₂₇H₂₈F₃N₃O₃, [M + H]⁺); anal. calcd for C₂₇H₂₈F₃N₃O₃: C, 64.92; H, 5.65; N, 8.41%. Found: C, 65.11; H, 6.00; N, 8.15%.

4b. 3-(5-(4-Bromophenyl)-1-(2-(dipropylamino)acetyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 70%; mp 183–184 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.92 (t, 6H, J = 7.5 Hz), 1.47 (t, 4H, J = 7.5 Hz), 2.55 (q, 4H, J = 6.2 Hz), 3.11 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.5 Hz, pyrazole 4-H_a), 3.54 (d, 1H, J = 16 Hz), 4.02 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.11 (d, 1H, J = 16.0 Hz), 5.85 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.5 Hz, pyrazole 5-H), 6.94–7.42 (m, 8H, ArH and chromen H), 8.31 (s, 1H, chromen C₄–H). ESI-MS: 509.91 (C₂₆H₂₈BrN₃O₃, [M + H]⁺); anal. calcd for C₂₆H₂₈BrN₃O₃: C, 61.18; H, 5.53; N, 8.23%. Found: C, 60.87; H, 5.75; N, 8.54%.

4c. 3-(1-(2-(Dipropylamino)acetyl)-5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 72%; mp 177–178 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.94 (t, 6H, J = 7.5 Hz), 1.49 (t, 4H, J = 7.5 Hz), 2.63 (q, 4H, J = 6.2 Hz), 3.06 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.2 Hz, pyrazole 4-H_a), 3.62 (d, 1H, J = 16 Hz), 3.77 (s, 3H, –OMe), 3.99 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.08 (d, 1H, J = 16.0 Hz), 5.77 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 6.79–7.18 (m, 8H, ArH and chromen H), 8.44 (s, 1H, chromen C₄–H). ESI-MS: 460.84 (C₂₇H₃₁N₃O₄, [M + H]⁺); anal. calcd for C₂₇H₃₁N₃O₄: C, 70.26; H, 6.77; N, 9.10%. Found: C, 69.92; H, 6.44; N, 9.19%.

4d. 3-(1-(2-(Dipropylamino)acetyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 64%; mp 196–198 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.40 (t, 4H, J = 7.5 Hz), 2.69 (q, 4H, J = 6.2 Hz), 3.17 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.78 (d, 1H, J = 16 Hz), 3.92 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.01 (d, 1H, J = 16.0 Hz), 5.65 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 6.87–7.40 (m, 8H, ArH and chromen H), 8.34 (s, 1H, chromen C₄–H). ESI-MS: 450.33 (C₂₆H₂₈FN₃O₃, [M + H]⁺); anal. calcd for C₂₆H₂₈FN₃O₃: C, 69.47; H, 6.28; N, 9.35%. Found: C, 69.19; H, 6.01; N, 9.00%.

4e. 3-(1-(2-(Dipropylamino)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-nitro-2H-chromen-2-one, colorless crystals, yield, 64%; mp 196–198 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.40 (t, 4H, J = 7.5 Hz), 2.69 (q, 4H, J = 6.2 Hz), 3.17 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.78 (d, 1H, J = 16 Hz), 3.92 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.01 (d, 1H, J = 16.0 Hz), 5.65 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 7.00–7.46 (m, 5H, ArH and chromen H), 8.11 (d, 1H, chromen 7-H), 8.29 (s, 1H, chromen 5-H), 8.34 (s, 1H, chromen C₄–H). ESI-MS: 545.08 (C₂₇H₂₇F₃N₄O₅, [M + H]⁺); anal. calcd for C₂₇H₂₇F₃N₄O₅: C, 59.55; H, 5.00; N, 10.29%. Found: C, 59.87; H, 4.74; N, 10.03%.

4f. 3-(5-(4-Bromophenyl)-1-(2-(dipropylamino)acetyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-nitro-2H-chromen-2-one, colorless crystals, yield, 73%; mp 190–191 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.98 (t, 6H, J = 7.5 Hz), 1.44 (t, 4H, J = 7.5 Hz), 2.58 (q, 4H, J = 6.2 Hz), 3.11 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.2 Hz, pyrazole 4-H_a), 3.65 (d, 1H, J = 16 Hz), 3.97 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.08 (d, 1H, J = 16.0 Hz), 5.73 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 6.98–7.34 (m, 5H, ArH and chromen H), 8.08 (d, 1H, chromen 7-H), 8.27 (s, 1H, chromen 5-H), 8.44 (s, 1H, chromen C₄–H). ESI-MS: 555.37 (C₂₆H₂₇BrN₄O₅, [M + H]⁺); anal. calcd for C₂₆H₂₇BrN₄O₅: C, 56.22; H, 4.90; N, 10.09%. Found: C, 56.54; H, 5.28; N, 10.01%.

4g. 3-(1-(2-(Dipropylamino)acetyl)-5-p-tolyl-4,5-dihydro-1H-pyrazol-3-yl)-6-nitro-2H-chromen-2-one, colorless crystals, yield, 62%; mp 181–182 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.92 (t, 6H, J = 7.5 Hz), 1.51 (t, 4H, J = 7.5 Hz), 2.27 (s, 3H, 6-Me), 2.55 (q, 4H, J = 6.2 Hz), 3.15 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.69 (d, 1H, J = 16 Hz), 3.92 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.01 (d, 1H, J = 16.0 Hz), 5.77 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 7.01–7.26 (m, 5H, ArH and chromen H), 8.04 (d, 1H, chromen 7-H), 8.21 (s, 1H, chromen 5-H), 8.41 (s, 1H, chromen C₄–H). ESI-MS: 489.78 (C₂₇H₃₀N₄O₅, [M + H]⁺); anal. calcd for C₂₇H₃₀N₄O₅: C, 66.11; H, 6.16; N, 11.42%. Found: C, 65.85; H, 6.00; N, 11.73%.

4h. 3-(1-(2-(Dipropylamino)acetyl)-5-(2-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-methyl-2H-chromen-2-one, colorless crystals, yield, 62%; mp 181–182 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.98 (t, 6H, J = 7.5 Hz), 1.55 (t, 4H, J = 7.5 Hz), 2.42 (s, 3H, 6-Me), 2.63 (q, 4H, J = 6.2 Hz), 3.11 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.2 Hz, pyrazole 4-H_a), 3.73 (d, 1H, J = 16 Hz), 3.99 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.12 (d, 1H, J = 16.0 Hz), 5.66 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 6.87–7.49 (m, 7H, ArH and chromen H), 8.45 (s, 1H, chromen C₄–H). ESI-MS: 513.21 (C₂₈H₃₀F₃N₃O₃, [M + H]⁺); anal. calcd for C₂₈H₃₀F₃N₃O₃: C, 65.49; H, 5.89; N, 8.18%. Found: C, 65.25; H, 6.14; N, 8.03%.

4i. 3-(1-(2-(Dipropylamino)acetyl)-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-methyl-2H-chromen-2-one, colorless crystals, yield, 71%; mp 185–186 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.48 (t, 4H, J = 7.5 Hz), 2.48 (s, 3H, 6-Me), 2.74 (q, 4H, J = 6.2 Hz), 3.15 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.77 (d, 1H, J = 16 Hz), 3.91 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.16 (d, 1H, J = 16.0 Hz), 5.78 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 6.95–7.18 (m, 7H, ArH and chromen H), 8.43 (s, 1H, chromen C₄–H). ESI-MS: 463.01 (C₂₇H₃₀FN₃O₃, [M + H]⁺); anal. calcd for C₂₇H₃₀FN₃O₃: C, 69.96; H, 6.52; N, 9.06%. Found: C, 70.13; H, 6.65; N, 8.89%.

4j. 3-(5-(4-Bromophenyl)-1-(2-(dipropylamino)acetyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-methyl-2H-chromen-2-one, colorless crystals, yield, 62%; mp 175–177 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.98 (t, 6H, J = 7.5 Hz), 1.44 (t, 4H, J = 7.5 Hz), 2.39 (s, 3H, 6-Me), 2.77 (q, 4H, J = 6.2 Hz), 3.11 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.2 Hz, pyrazole 4-H_a), 3.68 (d, 1H, J = 16 Hz), 3.94 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.11 (d, 1H, J = 16.0 Hz), 5.65 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 6.88–7.51 (m, 7H, ArH and chromen H), 8.43 (s, 1H, chromen C₄–H). ESI-MS: 525.04 (C₂₇H₃₀BrN₃O₃, [M + H]⁺); anal. calcd for C₂₇H₃₀BrN₃O₃: C, 61.83; H, 5.77; N, 8.01%. Found: C, 62.05; H, 6.02; N, 7.72%.

4k. 3-(1-(2-(Dipropylamino)acetyl)-5-(3-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-2H-chromen-2-one, colorless crystals, yield, 70%; mp 186–188 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.47 (t, 4H, J = 7.5 Hz), 2.70 (q, 4H, J = 6.3 Hz), 3.25 (dd, 1H, J₁ = 19.2 Hz, J₂ = 5.5 Hz, pyrazole 4-H_a), 3.79 (d, 1H, J = 16 Hz), 3.87 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.2 Hz, pyrazole 4-H_b), 4.12 (d, 1H, J = 16.0 Hz), 5.81 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 7.07–7.35 (m, 8H, ArH and chromen H), 8.47 (s, 1H, chromen C₄–H). ESI-MS: 500.04 (C₂₇H₂₈F₃N₃O₃, [M + H]⁺); anal. calcd for C₂₇H₂₈F₃N₃O₃: C, 64.92; H, 5.65; N, 8.41%. Found: C, 64.63; H,5.46; N, 8.75%.

4l. 3-(1-(2-(Dipropylamino)acetyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-3-yl)-6-methyl-2H-chromen-2-one, colorless crystals, yield, 60%; mp 178–179 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.62 (t, 4H, J = 7.5 Hz), 2.45 (s, 3H, 6-Me), 2.67 (q, 4H, J = 6.2 Hz), 3.07 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.2 Hz, pyrazole 4-H_a), 3.65 (d, 1H, J = 16 Hz), 4.04 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 4.17 (d, 1H, J = 16.0 Hz), 5.65 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.2 Hz, pyrazole 5-H), 6.92–7.49 (m, 7H, ArH and chromen H), 8.61 (s, 1H, chromen C₄–H). ESI-MS: 513.84 (C₂₈H₃₀F₃N₃O₃, [M + H]⁺); anal. calcd for C₂₈H₃₀F₃N₃O₃: C, 65.49; H, 5.89; N, 8.18%. Found: C, 65.66; H, 6.00; N, 8.45%.

General synthetic procedure process for compound 10a–e

To a chloroform (20 mL) solution of 3-(1-(2-bromoacetyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-4-chloro-2H-chromen-2-one 9 (10 mmol) was added dipropylamine (12 mmol), DMAP (12 mmol) and catalytic KI, the reaction mixture was allowed to stand at 40–50 °C for 5 h. The mixture was cooled, washed with water. The product was collected by filtration and the crude residue was purified by chromatography on SiO₂ (dichloromethane–methanol, v:v= 68 : 1) to give title compounds 10a–e (Scheme 2) as colorless solids.

10a. 4-Chloro-3-(1-(2-(dipropylamino)acetyl)-3-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one, colorless crystals, yield, 65%; mp 195–197 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.46 (t, 4H, J = 7.5 Hz), 2.65 (q, 4H, J = 6.3 Hz), 3.14 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.56 (d, 1H, J = 16 Hz), 3.71 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 3.87 (d, 1H, J = 16.0 Hz), 5.70 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 6.97–7.72 (m, 8H, ArH and chromen H). Anal. calcd for C₂₇H₂₇ClF₃N₃O₃: C, 60.73; H, 5.10; N, 7.87%. Found: C, 61.00; H, 4.83; N, 8.14%.

10b. 4-Chloro-3-(1-(2-(dipropylamino)acetyl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one, colorless crystals, yield, 61%; mp 190–191 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.89 (t, 6H, J = 7.5 Hz), 1.55 (t, 4H, J = 7.5 Hz), 2.29 (s, 3H, –CH₃), 2.69 (q, 4H, J = 6.3 Hz), 3.11 (dd, 1H, J₁ = 18.5 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.51 (d, 1H, J = 16 Hz), 3.67 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 3.97 (d, 1H, J = 16.0 Hz), 5.64 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 7.06–7.41 (m, 8H, ArH and chromen H). Anal. calcd for C₂₇H₃₀ClN₃O₃: C, 67.56; H, 6.30; N, 8.75%. Found: C, 67.85; H, 5.95; N, 9.01%.

10c. 3-(3-(2-Bromophenyl)-1-(2-(dipropylamino)acetyl)-4,5-dihydro-1H-pyrazol-5-yl)-4-chloro-2H-chromen-2-one, colorless crystals, yield, 57%; mp 182–183 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.85 (t, 6H, J = 7.5 Hz), 1.49 (t, 4H, J = 7.5 Hz), 2.72 (q, 4H, J = 6.3 Hz), 3.17 (dd, 1H, J₁ = 19.0 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.43 (d, 1H, J = 16 Hz), 3.72 (dd, 1H, J₁ = 12.0 Hz, J₂ = 19.0 Hz, pyrazole 4-H_b), 3.84 (d, 1H, J = 16.0 Hz), 5.71 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 7.00–7.48 (m, 8H, ArH and chromen H). Anal. calcd for C₂₆H₂₇BrClN₃O₃: C, 57.31; H, 4.99; N, 7.71%. Found: C, 57.59; H, 5.27; N, 8.06%.

10d. 4-Chloro-3-(1-(2-(dipropylamino)acetyl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one, colorless crystals, yield, 75%; mp 194–195 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.88 (t, 6H, J = 7.5 Hz), 1.52 (t, 4H, J = 7.5 Hz), 2.64 (q, 4H, J = 6.3 Hz), 3.19 (dd, 1H, J₁ = 18.5 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.52 (d, 1H, J = 16 Hz), 3.65 (dd, 1H, J₁ = 12.0 Hz, J₂ = 18.5 Hz, pyrazole 4-H_b), 3.77 (d, 1H, J = 16.0 Hz), 5.60 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 7.09–7.79 (m, 8H, ArH and chromen H). Anal. calcd for C₂₆H₂₇ClN₄O₅: C, 61.11; H, 5.33; N, 10.96%. Found: C, 61.46; H, 5.00; N, 11.29%.

10e. 4-Chloro-3-(1-(2-(dipropylamino)acetyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-2H-chromen-2-one, colorless crystals, yield, 70%; mp 190–191 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm): 0.91 (t, 6H, J = 7.5 Hz), 1.46 (t, 4H, J = 7.5 Hz), 2.67 (q, 4H, J = 6.3 Hz), 3.15 (dd, 1H, J₁ = 18.5 Hz, J₂ = 5.0 Hz, pyrazole 4-H_a), 3.45 (d, 1H, J = 16 Hz), 3.62 (dd, 1H, J₁ = 12.0 Hz, J₂ = 18.5 Hz, pyrazole 4-H_b), 3.79 (d, 1H, J = 16.0 Hz), 5.71 (dd, 1H, J₁ = 12.0 Hz, J₂ = 5.0 Hz, pyrazole 5-H), 6.97–7.58 (m, 9H, ArH and chromen H). Anal. calcd for C₂₆H₂₈ClN₃O₃: C, 67.02; H, 6.06; N, 9.02%. Found: C, 66.80; H, 5.77; N, 9.00%.

Crystallographic studies

A colorless single crystal of title compound 4a with dimensions of 0.21 mm × 0.16 mm × 0.15 mm was chosen for X-ray diffraction analysis performed on a BRUCKER SMART APEX-CCD diffractometer equipped with a graphite monochromatic MoKa radiation (λ = 0.71073 A) radiation at 296(2) K. A total of 11282 reflections were collected in the range of 2.35 < θ < 25.30° by using a ψ–ω scan mode with 4441 independent ones (R_int = 0.1034), of which 1698 with I > 2σ(I) were observed and used in the succeeding refinements. The data set was corrected by SADABS program; the structure was solved by direct methods with SHELXS-97 and refined by full-matrix least-squares method on F² with SHELXL-97.¹⁹ The non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were added according to theoretical models. The structure was refined by full-matrix least-squares method on F² with SHELXT-97. The final refinement gave R = 0.1055, wR = 0.1499 (w = 1/[σ²(F₀²) + (0.1008P)² + 0.8341P], where P = (F₀²+ 2F_c²)/3), S = 1.039, (Δρ)_max = 0.265 and (Δρ)_min = –0.326 e Å⁻³.

Anticancer assay

The cytotoxicity evaluation was conducted by using a modified procedure as described in the literature. 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 mL⁻¹ with the complete medium, 100 μL 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 5-fluorouracil co-assayed as a positive reference. After 48 h exposure period, 25 μL of PBS containing 2.5 mg mL⁻¹ of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. After 4 h, the medium was replaced by 150 μL 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 t test. The IC₅₀ value was defined as the concentration at which 50% of the cells could survive.

Telomerase activity assay

Compounds 4 and 10 were tested in a search for small molecule inhibitors of telomerase activity by using the TRAP-PCR-ELISA assay. In detail, the MGC-803 cells were firstly maintained in DMEM medium (GIBCO, New York, USA) supplemented with 10% fetal bovine serum (GIBCO, New York, USA), streptomycin (0.1 mg mL⁻¹) and penicillin (100 IU mL⁻¹) 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, 0.74, 0.25 and 0.0821 g mL⁻¹, 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 μL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12000 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 μL of cell extracts were mixed with 48 μL 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 μL 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.

General procedure for molecular docking

Discovery Studio 3.1 (DS 3.1, Accelrys Software Inc., San Diego, California, USA). Crystal structure of telomerase (PDB entry 3DU6) was used as template. Hydrogen atoms were added to protein model. The added hydrogen atoms were minimized to have stable energy conformation and to also relax the conformation from close contacts. The active site was defined and sphere of 5 Å was generated around the active site pocket, with the active site pocket of BSAI model using C-DOCKER, a molecular dynamics (MD) simulated-annealing based algorithm module from DS 3.1. Random substrate conformations are generated using high-temperature MD. Candidate poses are then created using random rigid-body rotations followed by simulated annealing. The structure of protein, substrate were subjected to energy minimization using CHARMm force field as implemented in DS 3.1. A full potential final minimization was then used to refine the substrate poses. Based on C-DOCKER, energy docked conformation of the substrate was retrieved for post docking analysis.

Western blotting

Mouse anti-TERT monoclonal antibody and rabbit anti-cyclin D1 monoclonal antibody were purchased from Abcam (Cambridge, UK) and vector (Switzerland), respectively. Secondary antibodies for goat anti-rabbit immunoglobulin (Ig) G horse radish peroxidase (HRP), goat anti-mouse IgG HRP was purchased from Santa Cruz Biotechnology (California, USA). β-Actin antibody was obtained from Santa Cruz Biotechnology (California, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma Inc. (St. Louis, MO, USA). AZT as telomerase inhibitor was obtained from Sigma-Aldrich (Poole, UK). And iCRT, a non-special β-catenin inhibitor, was produced by Merck Millipore Company (Darmstadt, Germany).

Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNAs were extracted from MGC-803 cells with a TRIzol reagent (Invitrogen, California, USA). The first-strand cDNA was synthesized from total RNA using Thermoscript RT-PCR synthesis kit (Fermentas, Canada) according to the manufacturer's instructions. RT-PCR was carried out under standard protocol. PCR was performed at 94 °C for 2 min, followed by 30–33 cycles of amplification at 94 °C for 30 s, 56 °C for 40 s and 72 °C for 1 min by using ABI9700. The band intensities were measured by adensitometer and the results were normalized with β-actin. The results were repeated by at least three times independently from three different pools of templates, while each pool of template was extracted from at least eight ventricles. PCR primers were as follows in Table 4.

Table 4 Primers used for RT-PCR^a

Gene	Accession no.	Forward	Reverse
a Nucleotide sequence of primers used for RT-PCR. Position is defined as the 5-nucleotide of the respective primer related to the source sequence.
hTERT	NM_198253.2	5′-GGAGCAAGTTGCAAAGCATTG-3′	5′- TCCCACGACGTAGTCCATGTT-3′
β-Catenin	NM_001904.3	5′-A̲G̲G̲A̲A̲G̲G̲G̲A̲T̲G̲G̲A̲A̲G̲G̲T̲C̲T̲C̲-3′	5′-C̲G̲C̲T̲G̲G̲G̲T̲A̲T̲C̲C̲T̲G̲A̲T̲G̲T̲G̲C̲-3′
Cyclin D1	NM_053056.2	5′-A̲C̲G̲A̲A̲G̲G̲T̲C̲T̲G̲C̲G̲C̲G̲T̲G̲T̲T̲-3′	5′-C̲C̲G̲C̲T̲G̲G̲C̲C̲A̲T̲G̲A̲A̲C̲T̲A̲C̲C̲T̲-3′
c-myc	NM_002467.4	5′-G̲T̲C̲T̲C̲C̲A̲C̲A̲C̲A̲T̲C̲A̲G̲C̲A̲C̲A̲A̲C̲T̲-3′	5′-T̲G̲T̲T̲T̲C̲A̲A̲C̲T̲G̲T̲T̲C̲T̲C̲G̲T̲C̲G̲T̲T̲-3′
β-Actin	NM_001101.3	5′-C̲C̲C̲A̲C̲A̲C̲T̲G̲T̲G̲C̲C̲C̲A̲T̲C̲T̲A̲C̲G̲-3′	5′-G̲C̲C̲A̲T̲C̲T̲C̲T̲T̲G̲C̲T̲C̲G̲A̲A̲G̲T̲C̲C̲-3′

---

Cell cycle analysis

For cell cycle analysis, we performed Cell Cycle Kit (Beyotime, China). HepG₂ cells 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⁻¹ RNase A, 50 μg mL⁻¹ PI, at 37 °C for 30 min in the dark. Analyses were performed on a BD LSR flowcytometer (BD Biosciences) and data were processed with CellQuest Pro (BD Biosciences). All experiments were repeated at least three times.

Acknowledgements

The authors wish to thank the National Natural Science Foundation of China (no. 21272008), Science and Technological Fund of Anhui Province for Outstanding Youth (1408085J04), Anhui Provincial Natural Science Foundation, (1308085MH137), China Postdoctoral Science Foundation funded project (2012M511948), The Governor Fund Of Guizhou Province (20120037) and the Research Foundation of Doctor, Anhui Medical University (XJ201120, XJ201121).

References and notes

1. A. B. Need, R. J. Davis, J. T. Alexander-Chacko, B. Eastwood, E. Chernet, L. A. Phebus, D. K. Sindelar and G. G. Nomikos, Psychopharmacology, 2006, 184, 26–35.
2. H. Dmytro, Z. Borys, V. Olexandr, Z. Lucjusz, G. Andrzej and L. Roman, Eur. J. Med. Chem., 2009, 44, 1396–1404.
3. F. Manna, F. Chimenti, R. Fioravanti, A. Bolasco, D. Secci, P. Chimenti, C. Ferlinib and G. Scambia, Bioorg. Med. Chem. Lett., 2005, 15, 4632–4635.
4. B. Insuasty, A. Tigreros, F. Orozco, J. Quiroga, R. Abonía, M. Nogueras, A. Sanchez and J. Cobo, Bioorg. Med. Chem., 2010, 18, 4965–4974.
5. K. S. Girisha, B. Kalluraya and N. Vijaya, Eur. J. Med. Chem., 2010, 45, 4640–4644.
6. X. H. Liu, J. Zhu, A. N. Zhou, B. A. Song, H. L. Zhu, L. S. Bai, P. S. Bhadury and C. X. Pan, Bioorg. Med. Chem., 2009, 17, 1207–1213.
7. M. Shaharyar, M. M. Abdullah, M. A. Bakht and J. Majeed, Eur. J. Med. Chem., 2010, 45, 114–119.
8. P. K. Ashish, D. H. Girish, H. T. Rajesh, H. R. Atish and M. K. Vandana, Bioorg. Med. Chem. Lett., 2012, 22, 6611–6615.
9. C. Xiao, X. Y. Luo, D. J. Li, H. Lu, Z. Q. Liu, Z. G. Song and Y. H. Jin, Eur. J. Med. Chem., 2012, 53, 159–167.
10. E. Maccioni, S. Alcaro, F. Orallo, M. C. Cardia, S. Distinto, G. Costa, M. Yanez, M. L. Sanna, S. Vigo, R. Meleddu and D. Secci, Eur. J. Med. Chem., 2010, 45, 4490–4495.
11. J. H. M. Lange, A. Attali, H. C. Wals, A. Mulder, H. Zilaout, A. Duursma, H. H. M. Aken and B. J. Vliet, Bioorg. Med. Chem. Lett., 2010, 20, 4992–4998.
12. X. H. Liu, B. F. Ruan, J. Li, B. A. Song, H. L. Zhu, P. S. Bhadury and J. Zhao, Mini-Rev. Med. Chem., 2011, 11, 771–821.
13. X. H. Liu, H. F. Liu, J. Chen, Y. Yang, B. A. Song, L. S. Bai, J. X. Liu, H. L. Zhu and X. B. Qi, Bioorg. Med. Chem. Lett., 2010, 20, 5705–5708.
14. T. Raj, R. K. Bhatia, A. Kapur, M. Sharma, A. K. Saxena and M. P. S. Ishar, Eur. J. Med. Chem., 2010, 45, 790–794.
15. Y. Z. Dong, K. Nakagawa-Goto, C. Y. Lai, L. S. Morris-Natschke, K. F. Bastow and K. H. Lee, Bioorg. Med. Chem. Lett., 2011, 21, 546–549.
16. C. Xiao, X. Y. Luo, D. J. Li, H. Lu, Z. Q. Liu, Z. G. Song and Y. H. Jin, Eur. J. Med. Chem., 2013, 62, 545–555.
17. J. N. Zheng, D. S. Pei, F. H. Sun, B. F. Zhang, X. Y. Liu, J. F. Gu, Y. H. Liu, X. L. Hu, L. J. Mao, R. M. Wen, J. J. Liu and W. Li, Cancer Biol. Ther., 2009, 8, 84–91.
18. J. Li, X. Huang, X. Xie, J. Wang and M. Duan, Acta Oto-Laryngol., 2011, 131, 546–551.
19. G. M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and Refinement, University of Göttingen, Göttingen, Germany, 1997.

---

Footnotes

† CCDC 861076. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c3ra47252c

‡ These authors contributed equally to this paper.

---