Synthesis, characterization and biological evaluation of six highly cytotoxic ruthenium(ii) complexes with 4'-substituted-2,2':6',2''-terpyridine.

MedChemComm View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. RESEARCH ARTICLE Cite this: Med. Chem. Commun., 2018, 9, 525 View Journal | View Issue Synthesis, characterization and biological evaluation of six highly cytotoxic rutheniumĲII) complexes with 4′-substituted-2,2′:6′,2″terpyridine† Qi-Pin Qin, ‡ac Ting Meng,‡ac Ming-Xiong Tan, *ac Yan-Cheng Liu, Shu-Long Wang,*ac Bi-Qun Zou *b and Hong Liangc c Herein, six rutheniumĲII) terpyridine complexes, i.e. [RuCl2Ĳ4-EtN-Phtpy)ĲDMSO)] (Ru1), [RuCl2Ĳ4-MeOPhtpy)ĲDMSO)] (Ru2), [RuCl2Ĳ2-MeO-Phtpy)ĲDMSO)] (Ru3), [RuCl2Ĳ3-MeO-Phtpy)ĲDMSO)] (Ru4), [RuCl2Ĳ1Bip-Phtpy)ĲDMSO)] (Ru5), and [RuCl2Ĳ1-Pyr-Phtpy)ĲDMSO)] (Ru6) with 4′-(4-diethylaminophenyl)-2,2′:6′,2″terpyridine (4-EtN-Phtpy), 4′-(4-methoxyphenyl)-2,2′:6′,2″-terpyridine (4-MeO-Phtpy), 4′-(2-methoxyphenyl)-2,2′:6′,2″-terpyridine (2-MeO-Phtpy), 4′-(3-methoxyphenyl)-2,2′:6′,2″-terpyridine (3-MeO-Phtpy), 4′-(1-biphenylene)-2,2′:6′,2″-terpyridine (1-Bip-Phtpy), and 4′-(1-pyrene)-2,2′:6′,2″-terpyridine (1-PyrPhtpy), respectively, were synthesized and fully characterized. The MTT assay demonstrates that the in vitro Received 20th October 2017, Accepted 31st January 2018 DOI: 10.1039/c7md00532f anticancer activity of Ru1 is higher than that of Ru2–Ru6 and more selective for Hep-G2 cells than for normal HL-7702 cells. In addition, various biological assays show that Ru1 and Ru6, especially the Ru1 complex, are telomerase inhibitors targeting c-myc G4 DNA and also cause apoptosis of Hep-G2 cells. With the same Ru center, the in vitro antitumor activity and cellular uptake ability of the 4-EtN-Phtpy and 1-Bip- rsc.li/medchemcomm Phtpy ligands follow the order 4-EtN-Phtpy > 1-Bip-Phtpy. Introduction Recently, a variety of genes such as c-kit-2, POT1, bcl-2, c-myc, and c-kit-1 have been considered as appealing targets for drug intervention in the therapy of many diseases.1–8 These targets are often associated with human diseases such as HIV, cancer, and diabetes.1–8 In particular, it is widely accepted that the aberrant over-expression of c-myc is associated with a variety of malignant tumors.9 In addition, the nuclear hypersensitivity element III1, upstream of the P1 promoter of c-myc, controls 80–90% of the c-myc transcription level.10–12 In the past a Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, College of Chemistry and Food Science, Yulin Normal University, 1303 Jiaoyudong Road, Yulin 537000, PR China. E-mail: mxtan2018@126.com, shulonghs@163.com; Fax: +86 775 2623650; Tel: +86 775 2623650 b Department of Chemistry, Guilin Normal College, 21 Xinyi Road, Gulin 541001, PR China. E-mail: zoubiqun@163.com c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, PR China † Electronic supplementary information (ESI) available: Vendor codes for the tested compounds, 1H NMR, ESI-MS, IR, 13C NMR, UV-vis data and crystal data. CCDC no. 1563746 for Ru5 contains the supplementary crystallographic data for this study. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7md00532f ‡ These authors contributed equally to this work. This journal is © The Royal Society of Chemistry 2018 decade, a large number of planar aromatic molecules, such as the natural product telomestatin,13,14 cationic porphyrins (TMPyP4 or Se2SAP),15,16 quindolines,13,17,18 perylenes,19 and berberine,13,17,18 have been studied due to their binding and stabilization of the G-quadruplex DNA (G4 DNA) in the NHE III1 sequence to suppress c-myc transcription in tumor cell lines. Therefore, the inhibition of the transcription of c-myc via stabilization of G-quadruplex DNA formation is a promising strategy for the design and development of efficacious anticancer agents.1–19 An increasing number of metal complexes, such as NiĲII),20,21 PtĲII),22–30 PdĲII),31 RuĲII),32 CuĲII),33 CoĲII), and ZnĲII)34 complexes, have been reported recently as G-quadruplex DNA binders. In addition, it has been reported that a series of metal–terpyridine complexes with ZnĲII), RuĲII), CuĲII), PdĲII), iridiumĲIII), and PtĲII) ion centers can act as effective G4-DNA ligands.31,35–45 For example, Ang and coworkers designed four dinuclear platinumĲII)–terpyridine complexes and showed that they acted as efficient G4-DNA ligands with high selectivity (ΔTm up to 17.0 °C) over duplex DNA (ΔTm = 1.0 °C).43 A dinuclear copperĲII)–terpyridine complex has been reported to bind to G4 DNA with high affinity and stabilize the antiparallel topology, and its selectivity for G4 is 100-fold higher than that for duplex DNA.35,44,45 Bertrand and co-workers also designed a range of copper– Med. Chem. Commun., 2018, 9, 525–533 | 525 View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. Research Article terpyridine complexes, which displayed higher affinity and selectivity for G4 DNA than other complexes.35,36 However, RuĲII) complexes with 4′-substituted-2,2′:6′,2″-terpyridine have been rarely studied, and detailed studies on their molecular mechanisms of action are still lacking.46–52 In this study, we designed and synthesized six rutheniumĲII) terpyridine complexes: [RuCl2Ĳ4-EtN-Phtpy)ĲDMSO)] (Ru1), [RuCl2Ĳ4-MeO-Phtpy)ĲDMSO)] (Ru2), [RuCl2Ĳ2MeO-Phtpy)ĲDMSO)] (Ru3), [RuCl2Ĳ3-MeO-Phtpy)ĲDMSO)] (Ru4), [RuCl2Ĳ1-Bip-Phtpy)ĲDMSO)] (Ru5), and [RuCl2Ĳ1-PyrPhtpy)ĲDMSO)] (Ru6) with 4′-(4-diethylaminophenyl)-2,2′:6′,2″terpyridine (4-EtN-Phtpy), 4′-(4-methoxyphenyl)-2,2′:6′,2″terpyridine (4-MeO-Phtpy), 4′-(2-methoxyphenyl)-2,2′:6′,2″terpyridine (2-MeO-Phtpy), 4′-(3-methoxyphenyl)-2,2′:6′,2″terpyridine (3-MeO-Phtpy), 4′-(1-biphenylene)-2,2′:6′,2″terpyridine (1-Bip-Phtpy), and 4′-(1-pyrene)-2,2′:6′,2″-terpyridine (1-Pyr-Phtpy), respectively. These six RuĲII) complexes exhibit remarkable and different in vitro cytotoxicities, which also exert inhibitory effects on the telomerase activity by directly targeting c-myc promoter elements (c-myc G4 DNA). MedChemComm Fig. 1 ORTEP view of the molecular structure of Ru5. Thermal ellipsoids for non-hydrogen atoms are drawn at a 30% probability level. Results and discussion Synthesis The six ligands 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, and 1-Pyr-Phtpy were synthesized according to the procedures reported by Li et al.53 The title complexes Ru1, Ru2, Ru3, Ru4, Ru5, and Ru6 were prepared via the reaction of cis-RuĲDMSO)4Cl2 with 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, and 1-Pyr-Phtpy, respectively, in a 90% (v/v) methanol–water solution (Scheme 1). The complexes Ru1–Ru6 were characterized via elemental analysis, 1H and 13C NMR spectroscopy, 1H–1H COSY NMR spectra, IR spectroscopy, UV-vis spectroscopy, single-crystal X-ray diffraction analysis, and ESI-MS (Fig. S1–S40, Table S4†). Crystal structure characterization The single crystal X-ray analysis revealed that Ru5 crystallized as a monoclinic crystal system with the P21/c space group. As shown in Fig. 1, the central RuĲII) in Ru5 is six-coordinated by two chlorides, one bidentate chelating big planar ligand N′N′ N-1-Bip-Phtpy, and one DMSO, adopting a distorted octahedral geometry. In addition, the Ru–Cl, Ru–N, and Ru–S bond lengths are in the range of 2.4175Ĳ14)–2.4440Ĳ14), 1.949Ĳ4)– 2.081Ĳ5), and 2.2184Ĳ15)–2.2190Ĳ16) Å, respectively, which are within the normal range. The bite angles (N1–Ru1–Cl2 and N1–Ru1–Cl1) of the chelate ring are 87.03(13) and 102.66Ĳ12)°, respectively, which are smaller than the S1–Ru1– Cl2 bite angles (177.92Ĳ6)°). The details of the selected bond lengths for Ru5 are tabulated in Table S2,† and the structural refinement parameters and crystallographic data are summarized in Tables S1 and S3,† respectively. Stability of Ru1–Ru6 in TBS Ru1–Ru6 were tested for their stability in 10 mM TBS (TrisHCl buffer solution 10 mM, pH 7.35, containing 2.0% DMSO) via UV-vis spectroscopy. As shown in Fig. S41 and Table S5,† no obvious spectral changes were observed in the UV-vis spectra of Ru1–Ru6 for 48 h at 37 °C; this demonstrated that they were also stable in their coordinating mode in TBS. These results confirm that the six complexes Ru1–Ru6 (2.0 × 10−5 M) are stable under physiologically relevant conditions for 48 h, which ensure adequate cellular uptake of Ru1–Ru6 during the time course of the MTT assay. In vitro cytotoxicity Scheme 1 Synthetic routes for Ru1–Ru6 with 4′-substituted-2,2′:6′,2″terpyridine. Reagents and solvents are as follows: (a) ethanol, KOH, 25% NH3 (aq) and (b) cis-RuĲDMSO)4Cl2 (0.1 mmol, 0.0485 g), CH3OH : H2O (v : v = 9 : 1), 80 °C. 526 | Med. Chem. Commun., 2018, 9, 525–533 The in vitro cytotoxic activities of the six ligands 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, and 1-Bip-Phtpy,1Pyr-Phtpy, Ru1–Ru6, cisplatin, and cis-RuĲDMSO)4Cl2 against the BEL-7404, Hep-G2, HL-7702, NCI-H460, and MGC80-3 cell lines were assessed using the MTT assay. The concentrations of these compounds ranged from 1.25 μM to 20.0 μM. As shown in Tables 1 and S6,† the inhibitory rates of Ru1–Ru6 against the This journal is © The Royal Society of Chemistry 2018 View Article Online MedChemComm Research Article Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. Table 1 IC50a (μM) values of 4′-substituted-2,2′:6′,2″-terpyridine, Ru1–Ru6, and cisplatin towards the five selected cell lines after incubation for 48 h Compd. BEL-7404 Hep-G2 NCI-H460 MGC80-3 HL-7702c 4-EtN-Phtpy Ru1 4-MeO-Phtpy Ru2 2-MeO-Phtpy Ru3 3-MeO-Phtpy Ru4 1-Bip-Phtpy Ru5 1-Pyr-Phtpy Ru6 Cisplatinb 26.18 ± 0.95 2.89 ± 0.66 56.78 ± 0.65 19.26 ± 1.20 36.94 ± 0.80 17.54 ± 0.63 34.34 ± 1.02 10.17 ± 1.45 30.56 ± 0.53 8.52 ± 0.75 45.12 ± 1.74 5.09 ± 0.40 16.91 ± 1.01 19.12 ± 1.39 1.28 ± 0.44 28.43 ± 0.39 10.19 ± 1.01 26.40 ± 1.45 7.35 ± 0.59 19.67 ± 0.73 6.04 ± 0.94 19.98 ± 1.02 4.89 ± 1.49 19.47 ± 1.23 3.01 ± 0.91 18.68 ± 0.67 16.54 ± 1.61 3.56 ± 1.60 33.78 ± 1.76 16.08 ± 1.06 24.73 ± 1.10 9.34 ± 1.85 19.96 ± 0.35 8.89 ± 0.75 19.42 ± 1.56 6.72 ± 0.40 17.43 ± 1.04 5.89 ± 1.58 18.01 ± 1.23 13.62 ± 0.51 6.01 ± 1.68 35.71 ± 0.92 15.01 ± 1.44 23.02 ± 1.44 11.18 ± 0.62 20.96 ± 0.31 10.78 ± 1.47 18.45 ± 1.32 9.96 ± 0.59 16.18 ± 0.69 7.79 ± 1.43 13.99 ± 0.76 38.02 ± 1.95 (1.99) 31.18 ± 1.19 (24.94) 18.11 ± 1.07 (0.64) 21.17 ± 1.88 (2.08) 17.16 ± 1.03 (0.65) 16.89 ± 0.57 (2.30) 35.11 ± 0.81 (1.78) 40.16 ± 1.51 (6.65) 25.16 ± 1.34 (1.26) 35.21 ± 1.05 (7.20) 30.02 ± 1.19 (1.54) 27.56 ± 0.81 (9.16) 17.98 ± 1.94 (0.96) a IC50 values are presented as the mean ± SD (standard error of the mean) from six independent experiments. b Cisplatin was dissolved at a concentration of 1.0 mM in 0.154 M NaCl.57 c The selectivity index factor, defined as IC50 (HL-7702 normal cells)/IC50 (Hep-G2 tumor cells), is given in parentheses.54–56 tested cancer cells were higher than those of 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, 1-PyrPhtpy, and cis-RuĲDMSO)4Cl2. Importantly, Ru1 exhibited higher in vitro cytotoxicity against all the selected cancer cell lines than 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeOPhtpy, 1-Bip-Phtpy, 1-Pyr-Phtpy, and Ru2–Ru6, but relatively higher cytotoxicity than cisplatin, a widely used clinical antitumor drug. Overall, Ru1–Ru6 were more sensitive to Hep-G2 tumor cells and exhibited lower IC50 values on Hep-G2 cells with 1.28 ± 0.44, 10.19 ± 1.01, 7.35 ± 0.59, 6.04 ± 0.94, 4.89 ± 1.49, and 3.01 ± 0.91 μM values for Ru1, Ru2, Ru3, Ru4, Ru5, and Ru6, respectively. The different in vitro antitumor activities of Ru1–Ru6 against the tested cancer cells are related to 4′substituted-2,2′:6′,2″-terpyridine in the following order: 4-EtNPhtpy > 1-Pyr-Phtpy > 1-Bip-Phtpy > 3-MeO-Phtpy > 2-MeOPhtpy > 4-MeO-Phtpy. Compared with their toxicity towards cancer cells, Ru1–Ru6 were considerably less toxic towards the normal HL-7702 cells, with the IC50 values ranging from 21.17 ± 1.88 μM to more than 40.16 ± 1.51 μM, which were significantly higher than that of cisplatin (17.98 ± 1.94 μM). Remarkably, the cytotoxicities of Ru1–Ru6 against the Hep-G2 cells and HL-7702 cells (human normal cells) were characterized by remarkably high selectivity index factors of more than 3.0 (Table 1).54–56 Ru1 and Ru6 showed the best selectivity indices (highest selectivity index factor values) of 22.94 and 9.16, higher than those of 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, 1-Pyr-Phtpy, Ru2–Ru5, and cisplatin; this indicated the high selectivity of Ru1 and Ru6 for Hep-G2 cells. In addition, the MTT assay indicated that Hep-G2 cells exhibited higher selectivity for Ru1 and Ru6 than for other compounds; therefore, Ru1 and Ru6 were selected for the cellular uptake assay, apoptosis analysis, western blot, transfection assay, and RT-PCR in the Hep-G2 cells. metal, Hep-G2 cells have been incubated with Ru1 (1.3 μM) and Ru6 (3.0 μM). The uptakes and distributions of Ru1 (1.3 μM) and Ru6 (3.0 μM) in the Hep-G2 cells were studied in more detail using the method reported by Sadler, Chen, Liu, and Schreiber.58–61 At first, the total cellular accumulation of Ru from Ru1 (1.3 μM) and Ru6 (3.0 μM) was determined for Hep-G2 cells using the ICP-MS assay. Ru accumulation after 24 h of exposure under 5% CO2 was higher for Ru1 ((5.32 ± 0.15 ng Ru)/106 cells) than for Ru6 ((3.67 ± 0.24 ng Ru)/106 cells) and cisplatin (10.0 μM) in the Hep-G2 cells (Fig. 2A).58 In addition, Fig. 2B showed that the Ru accumulation for Ru1 (1.3 μM) and Ru6 (3.0 μM) reached a high level in the nuclear fraction and mitochondrial fraction, whereas cisplatin was only accumulated in the mitochondrial membrane fraction.58,61 Ruthenium accumulation and distribution in Hep-G2 cells Fig. 2 Accumulation of Ru in the Hep-G2 cells after incubation with Ru1 (1.3 μM) and Ru6 (3.0 μM) for 24 h at 37 °C. The total Ru content in the whole Hep-G2 cell (A) and in different fractions (B) was measured by ICP-MS. To investigate whether the nuclear fraction or/and mitochondria fraction plays a role in the cellular accumulation of Ru This journal is © The Royal Society of Chemistry 2018 Telomerase inhibition To examine the ability of Ru1 (1.3 μM) and Ru6 (3.0 μM) to inhibit the telomerase activity, the TRAP assay was performed.62–66 Fig. 3 clearly reveals that Ru1 (1.3 μM) exhibits greater telomerase inhibitory activity (inhibitory rate of 53.06%) than Ru6 (3.0 μM, inhibitory rate of 16.34%) and cisplatin (10.0 μM, inhibitory rate of 17.89%)58 in the Hep-G2 cells. Herein, one of the notable features was the variation Med. Chem. Commun., 2018, 9, 525–533 | 527 View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. Research Article MedChemComm Fig. 4 mRNA expression of the c-myc and hTERT genes after 24 h incubation of the Hep-G2 cells treated with Ru1 (1.3 μM) and Ru6 (3.0 μM), determined using the qRT-PCR assay. the results show different effects on the c-myc and hTERT promoter in the following order: Ru1 > Ru6, consistent with the in vitro cytotoxicity (MTT assay), telomerase inhibition, and cell uptake assay results. Transfection Fig. 3 Influence of Ru1 (1.3 μM) and Ru6 (3.0 μM) on the telomerase activity of Hep-G2 cells for 24 h. with different ligands (4-EtN-Phtpy > 1-Bip-Phtpy), which might contribute to their telomerase inhibitory activity and other antitumour actions. The binding ability of Ru1 to G-quadruplex DNAs and duplex DNA was studied using the FID assay and ESI-MS analysis.8,58,71 As shown in Fig. S42,† the FID assay results indicate that Ru1 exhibits better selectivity for Pu27 G-quadruplex DNAs (c-myc promoter) than for other DNAs, which are the most efficient TO displacers (Pu27DC50 as low as 0.88 μM with Ru1). As expected, the selectivity of Ru1 for G4-DNA over duplex DNA is moderate. Furthermore, to verify further whether Ru1 (1.3 μM) and Ru6 (3.0 μM) could directly interact with the c-myc promoter (Pu27 G4 DNA) and regulate the expression of hTRET in Hep-G2 cells, transfection of EGFP (enhanced green fluorescent protein) and c-myc gene vectors into these cells was carried out.8,58,71–74 After the successful transfection of the c-myc gene vectors in the HepG2 cells, Ru1 (1.3 μM) and Ru6 (3.0 μM) were added to the cells and incubated for 24 h and examined via fluorescence microcopy or the luciferase reporter gene assay.58,61 The transfection assay showed that the HepG2 cells emitted green Inhibition of hTERT and c-myc gene and protein expression by Ru1 and Ru2 Many groups have carried out research to identify or design c-myc G4-ligands, which specifically bind to the G4-DNA and inhibit cell proliferation.67–70 It is important to know whether c-myc and hTERT are involved in the inhibition of telomerase activity by Ru1 (1.3 μM) and Ru6 (3.0 μM) in the Hep-G2 cancer cells. Thus, we performed RT-qPCR analysis and Western blot assay using Hep-G2 tumor cells. As shown in Fig. 4 and 5, Ru1 (1.3 μM) was more active in inhibiting hTERT and c-myc gene and protein expression level than Ru6 (3.0 μM) and cisplatin (10.0 μM) in the Hep-G2 cells.58,61 In addition, 528 | Med. Chem. Commun., 2018, 9, 525–533 Fig. 5 (A) Protein expression of hTERT, β-actin, and c-myc proteins in the Hep-G2 tumor cells treated with Ru1 (1.3 μM) and Ru6 (3.0 μM), analyzed by Western blotting. (B) Densitometric analysis of hTERT or c-myc protein normalized with β-actin. The relative expression of each protein is represented by the ratio of the protein band density to the β-actin band. This journal is © The Royal Society of Chemistry 2018 View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. MedChemComm Fig. 6 (A) Successful transfection of EGFP plasmid vector in the HepG2 cells for 6 h. (B) The treatment of Hep-G2 cells with Ru1 (1.3 μM) and Ru6 (3.0 μM) for 24 h after 6 h of transfection from 2.0 μg c-myc plasmid by Lipofectamine 2000 (Invitrogen), which showed a significant reduction in green fluorescence emission when they were examined using a multimode plate reader with the luciferase reporter gene assay kit. fluorescence after EGFP plasmid transfection; this suggested that the transfection was successful (Fig. 6A). These cells were then transfected by c-myc plasmid and treated with Ru1 (1.3 μM) and Ru6 (3.0 μM). Consequently, a remarkable decrease in the emission of the bright green fluorescence was observed (Fig. 6B).58,61 As a result, the treatment of Ru1 (1.3 μM) reduced the fluorescence emission by 79.82%, whereas the treatment with Ru6 (3.0 μM) and cisplatin (10.0 μM) only reduced the fluorescence emission by 13.11% and 30.13%, respectively, under the same conditions.58,61 These results further demonstrate the higher activity of Ru1 (1.3 μM) to inhibit telomerase activity by directly down-regulating the c-myc promoter (c-myc/Pu27 G4 DNA) in the Hep-G2 cells. Apoptosis assay by flow cytometry To determine the percentage of Hep-G2 cells undergoing apoptosis after treatment with Ru1 (1.3 μM) and Ru6 (3.0 μM) Fig. 7 Cell apoptosis induction in HepG2 cells treated with Ru1 (1.3 μM) and Ru6 (3.0 μM) for 24 h. This journal is © The Royal Society of Chemistry 2018 Research Article for 24 h, cell apoptosis analyses using flow cytometry were performed after staining the Hep-G2 cells with Annexin-VFITC and propidium iodide (PI).58,61,75 After treatment with Ru1 (1.3 μM) and Ru6 (3.0 μM) for 24 h, the percentage of apoptotic cells (Q2 + Q3) increased from 2.22% to 38.60% and 29.00%, and the percentage of early apoptotic Hep-G2 cells was 14.90% and 12.70%, respectively (Fig. 7). Overall, these results indicate that Ru1, even at a low concentration of 1.3 μM, could also cause more cell apoptosis than Ru6 (3.0 μM) and cisplatin (10.0 μM, ca. 13.60%) in the HepG2 cells.32,35 Conclusions In this study, six rutheniumĲII) terpyridine complexes Ru1, Ru2, Ru3, Ru4, Ru5, and Ru6 with 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, and 1-Pyr-Phtpy, respectively, were synthesized and fully characterized. Ru1 showed higher in vitro cytotoxicity than Ru2–Ru6, 4-EtNPhtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, 1-Pyr-Phtpy, and cisplatin on Hep-G2 cells under the same conditions. Various biological assays revealed that Ru1 acted as a telomerase inhibitor targeting the c-myc G-quadruplex DNA and thus driving the Hep-G2 cells towards apoptosis. Importantly, this study demonstrated a novel class of potent c-myc G4 DNA signaling telomerase inhibitors. Experimental methods Synthesis Synthesis of ligands. Synthesis of 4-EtN-Phtpy, 4-MeOPhtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy, and 1-PyrPhtpy was performed as reported by Li et al.53 Typically, 2.61 mL of 2-acetylpyridine (2.813 g, 23.2 mmol, 2 equiv.) was added to a solution of 4′-substituted benzaldehyde (11.6 mmol, 1 equiv.) in 50 mL of ethanol. Then, KOH pellets (2.6 g, 85%, 46.5 mmol, 4 equiv.) were added to this solution. The reaction was stirred at RT for 10 min. Subsequently, 40 mL of 25% aq. NH3 was added to the flask dropwise. After 24 h of stirring at 34 °C, 5 mL of 25% aq. NH3 was added to the reaction mixture again. The flask was cooled to −20 °C, and then, the white precipitate was filtered and washed with cold ethanol. We further purified the ligands by subsequent recrystallization from ethanol–H2O. Each ligand was recovered by filtration, washed with cold ethanol and petroleum ether, and dried under high vacuum for 24 h (Scheme 1). Data for 4-EtN-Phtpy. The white product of 4-EtN-Phtpy was suitable for structural characterization. Yield (44.21%). ESI-MS m/z: 783.5 [2M + Na]+, m/z: 403.2 [M + Na]+. 1H NMR (500 MHz, DMSO-d6) δ 8.76 (d, J = 4.3 Hz, 2H), 8.65 (d, J = 6.2 Hz, 4H), 8.02 (t, J = 7.7 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.51 (s, 2H), 6.83 (d, J = 8.9 Hz, 2H), 3.43 (s, 4H), 1.14 (s, 6H). Elemental analysis calcd (%) for C25H24N4: C 78.92, H 6.36, N 14.73; found: C 78.89, H 6.38, N 14.70. Data for 4-MeO-Phtpy. The white product of 4-MeO-Phtpy was suitable for structural characterization. Yield (45.62%). ESI-MS m/z: 701.4 [2M + Na]+, m/z: 362.1 [M + Na]+. 1H NMR Med. Chem. Commun., 2018, 9, 525–533 | 529 View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. Research Article (500 MHz, DMSO-d6) δ 8.75 (d, J = 4.7 Hz, 2H), 8.65 (d, J = 9.1 Hz, 4H), 8.02 (t, J = 7.7 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 7.51 (s, 2H), 7.12 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H). Elemental analysis calcd (%) for C22H17N3O: C 77.86, H 5.05, N 12.38; found: C 77.89, H 5.07, N 12.33. Data for 2-MeO-Phtpy. The yellow product of 2-MeO-Phtpy was suitable for structural characterization. Yield (45.62%). ESI-MS m/z: 701.4 [2M + Na]+, m/z: 362.1 [M + Na]+. 1H NMR (500 MHz, DMSO-d6) δ 8.74 (d, J = 4.7 Hz, 2H), 8.67 (d, J = 7.9 Hz, 2H), 8.57 (s, 2H), 8.03 (t, J = 7.7 Hz, 2H), 7.51 (s, 4H), 7.23 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 3.84 (s, 3H). Elemental analysis calcd (%) for C22H17N3O: C 77.86, H 5.05, N 12.38; found: C 77.84, H 5.08, N 12.34. Data for 3-MeO-Phtpy. The white product of 3-MeO-Phtpy was suitable for structural characterization. Yield (45.62%). ESI-MS m/z: 701.4 [2M + Na]+, m/z: 362.1 [M + Na]+. 1H NMR (500 MHz, DMSO-d6) δ 8.76 (d, J = 4.6 Hz, 2H), 8.66 (d, J = 8.9 Hz, 4H), 8.03 (t, J = 7.7 Hz, 2H), 7.52 (s, 2H), 7.50 (d, J = 7.9 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.39 (s, 1H), 7.11 (d, J = 7.4 Hz, 1H), 3.88 (s, 3H). Elemental analysis calcd (%) for C22H17N3O: C 77.86, H 5.05, N 12.38; found: C 77.81, H 5.10, N 12.35. Data for 1-Bip-Phtpy. The yellow product of 1-Bip-Phtpy was suitable for structural characterization. Yield (40.11%). ESI-MS m/z: 741.4 [2M + Na]+, m/z: 382.1 [M + Na]+. 1H NMR (500 MHz, DMSO-d6) δ 8.74 (d, J = 7.9 Hz, 2H), 8.71 (d, J = 5.4 Hz, 2H), 8.55 (s, 2H), 8.09 (s, 2H), 8.05 (d, J = 7.8 Hz, 2H), 7.91 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 7.3 Hz, 2H), 7.63–7.55 (m, 2H), 7.54–7.50 (m, 2H). Elemental analysis calcd (%) for C25H17N3: C 83.54, H 4.77, N 11.69; found: C 83.50, H 4.80, N 11.66. Data for 1-Pyr-Phtpy. The yellow product of 1-Pyr-Phtpy was suitable for structural characterization. Yield (46.98%). ESI-MS m/z: 889.4 [2M + Na]+, m/z: 456.2 [M + Na]+. 1H NMR (500 MHz, DMSO-d6) δ 8.79 (d, J = 7.9 Hz, 2H), 8.74 (d, J = 4.6 Hz, 2H), 8.71 (s, 2H), 8.48 (d, J = 7.8 Hz, 1H), 8.40 (d, J = 7.7 Hz, 1H), 8.35 (d, J = 7.3 Hz, 1H), 8.31 (s, 2H), 8.27 (d, J = 9.3 Hz, 1H), 8.23 (d, J = 2.7 Hz, 1H), 8.15 (s, 2H), 8.09 (t, J = 6.9 Hz, 2H), 7.57–7.53 (m, 2H). Elemental analysis calcd (%) for C31H19N3: C 88.89, H 4.42, N 9.69; found: C 88.93, H 4.39, N 9.61. Synthesis of Ru1–Ru6. cis-RuĲDMSO)4Cl2 (0.0485 g, 0.1 mmol), 0.1 mmol 4-EtN-Phtpy, 4-MeO-Phtpy, 2-MeO-Phtpy, 3-MeO-Phtpy, 1-Bip-Phtpy or 1-Pyr-Phtpy, 0.10 mL H2O, and 0.90 mL CH3OH, were placed in a 25.0 cm long Pyrex tube that was then quenched in liquid N2 before being evacuated and sealed, which was then heated at 80 °C for three days. The resulting black products suitable for structural characterization were isolated, washed with ethanol and ether, and air-dried. Data for Ru1. The black product of Ru1 was suitable for structural characterization. Yield (0.0564 g, 89.50%). ESI-MS m/z: 862.4 [M − Cl + 3DMSO + CH3OH]+. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (d, J = 5.4 Hz, 2H), 8.87 (s, 2H), 8.79 (d, J = 8.0 Hz, 2H), 8.12 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 1.2 Hz, 2H), 7.51 (s, 2H), 6.88 (d, J = 9.0 Hz, 2H), 3.49 (d, J = 7.0 Hz, 4H), 530 | Med. Chem. Commun., 2018, 9, 525–533 MedChemComm 2.54 (s, 6H), 1.18 (t, J = 7.0 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 159.26, 156.76, 148.85, 147.97, 136.59, 128.78, 126.08, 123.16, 121.76, 116.89, 116.51, 111.48, 43.76, 40.43, 40.05, 39.93, 39.79, 39.65, 39.51, 39.38, 39.24, 39.10, 12.53. IR (KBr): 3573, 3405, 2973, 2920, 1592, 1532, 1469, 1419, 1355, 1254, 1220, 1158, 1077, 1044, 1010, 925, 888, 826, 756, 789, 726, 676, 517, 455, 430 cm−1. Elemental analysis calcd (%) for C27H30Cl2N4ORuS: C 51.43, H 4.80, N 8.88; found: C 51.40, H 4.85, N 8.84. In addition, the 1H–1H COSY cross-peaks (Fig. S34–S40†) of H-4/H-3/H-2 and H-2/H-1 indicated the presence of a carbon chain C-4–C-3–C-2–C-1 in Ru1. Similarity, the carbon chains C-4′–C-3′–C-2′–C-1′, C-11–C-12, C-14–C-15, C-17–C18, and C-19–C-20 were observed in Ru1. Data for Ru2. The black product of Ru2 was suitable for structural characterization. Yield (0.0501 g, 85.00%). ESI-MS m/z: 780.2 [M − Cl + 2DMSO + CH3CN + CH3OH]+. 1H NMR (600 MHz, DMSO-d6) δ 9.36 (d, J = 5.4 Hz, 2H), 8.96 (s, 2H), 8.81 (d, J = 7.9 Hz, 2H), 8.24 (d, J = 8.7 Hz, 2H), 8.01 (t, J = 7.7 Hz, 2H), 7.54 (t, J = 6.5 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H), 3.91 (s, 3H), 3.60 (s, 2H), 2.54 (s, 6H). 13C NMR (151 MHz, DMSOd6) δ 160.98, 159.01, 157.11, 155.69, 147.29, 136.67, 129.14, 128.44, 126.28, 123.36, 118.15, 114.64, 55.48, 44.61, 40.43, 40.05, 39.93, 39.79, 39.65, 39.52, 39.38, 39.24, 39.10. IR (KBr): 3452, 2999, 2919, 2835, 1601, 1524, 1468, 1435, 1411, 1300, 1271, 1247, 1195, 1078, 1053, 1026, 1005, 961, 884, 825, 787, 756, 728, 677, 594, 528, 427 cm−1. Elemental analysis calcd (%) for C24H23Cl2N3O2RuS: C 48.90, H 3.93, N 7.13; found: C 48.87, H 3.95, N 7.11. Data for Ru3. The black product of Ru3 was suitable for structural characterization. Yield (0.0471 g, 80.00%). ESI-MS m/z: 780.2 [M − Cl + 2DMSO + CH3CN + CH3OH]+. 1H NMR (600 MHz, DMSO-d6) δ 9.37 (d, J = 5.5 Hz, 2H), 8.78 (s, 2H), 8.65 (d, J = 7.9 Hz, 2H), 7.97 (dd, J = 12.1, 4.5 Hz, 2H), 7.72 (dd, J = 7.5, 1.5 Hz, 1H), 7.54 (dt, J = 13.5, 7.2 Hz, 3H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H), 3.93 (s, 3H), 3.61 (s, 2H), 2.52 (m, 6H). 13C NMR (151 MHz, DMSO-d6) δ 158.93, 156.58, 156.52, 155.69, 146.17, 136.81, 131.11, 130.89, 126.31, 123.16, 121.89, 121.01, 111.97, 55.83, 44.59, 40.43, 39.93, 39.79, 39.65, 39.52, 39.38, 39.24, 39.10. IR (KBr): 3854, 3433, 3048, 3014, 2927, 2836, 1608, 1496, 1469, 1416, 1294, 1250, 1066, 1047, 1016 888, 791, 759, 678, 633, 465, 429 cm−1. Elemental analysis calcd (%) for C24H23Cl2N3O2RuS: C 48.90, H 3.93, N 7.13; found: C 48.93, H 3.90, N 7.10. Data for Ru4. The black product of Ru4 was suitable for structural characterization. Yield (0.0530 g, 90.00%). ESI-MS m/z: 554.1 [M − Cl]+. IR (KBr): 3788, 3060, 1604, 1543, 1470, 1409, 1291, 1271, 1217, 1163, 1098, 1073, 1047, 1015, 910, 787, 757, 681, 428 cm−1. Elemental analysis calcd (%) for C24H23Cl2N3O2RuS: C 48.90, H 3.93, N 7.13; found: C 48.85, H 3.97, N 7.09. Data for Ru5. The black block crystals were suitable for structural characterization. Yield (0.0568 g, 93.20%). ESI-MS m/z: 652.2 [M − Cl + DMSO]+. 1H NMR (600 MHz, DMSO-d6) δ 9.44–9.38 (m, 2H), 8.89 (s, 2H), 8.70 (d, J = 8.0 Hz, 2H), 8.18– 8.13 (m, 2H), 7.97 (ddd, J = 7.7, 3.6, 1.4 Hz, 3H), 7.84 (dd, J = 7.0, 0.9 Hz, 1H), 7.77 (dd, J = 8.1, 7.2 Hz, 1H), 7.69–7.64 (m, This journal is © The Royal Society of Chemistry 2018 View Article Online Published on 02 February 2018. Downloaded by RSC Internal on 30/05/2018 13:59:09. MedChemComm 2H), 7.57–7.52 (m, 2H), 2.54 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 158.82, 157.01, 155.72, 147.96, 136.84, 136.08, 133.41, 130.41, 129.42, 128.65, 127.81, 127.42, 126.47, 125.65, 124.74, 123.49, 122.49, 40.42, 39.92, 39.78, 39.65, 39.51, 39.37, 39.23, 39.09. IR (KBr): 3854, 3444, 3054, 2361, 1630, 1605, 1541, 1480, 1416, 1388, 1240, 1160, 1065, 1046, 1015, 790, 677, 633, 539, 429 cm−1. Elemental analysis calcd (%) for C27H23Cl2N3ORuS: C 53.20, H 3.80, N 6.89; found: C 53.16, H 3.85, N 6.87. Data for Ru6. The black product of Ru6 was suitable for structural characterization. Yield (0.0542 g, 79.30%). ESI-MS m/z: 726.2 [M − Cl + DMSO]+. 1H NMR (600 MHz, DMSO-d6) δ 9.47–9.42 (m, 2H), 9.03 (s, 2H), 8.74 (d, J = 8.1 Hz, 2H), 8.55 (d, J = 7.8 Hz, 1H), 8.42 (d, J = 7.6 Hz, 1H), 8.38 (dd, J = 7.6, 4.1 Hz, 2H), 8.35–8.33 (m, 2H), 8.30 (d, J = 16.2 Hz, 2H), 8.17 (t, J = 7.6 Hz, 1H), 8.01–7.96 (m, 2H), 7.60–7.56 (m, 2H), 3.67 (s, 3H), 2.54 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 158.88, 157.08, 155.79, 148.25, 136.86, 133.32, 131.41, 130.92, 130.37, 128.91, 128.39, 127.95, 127.35, 126.77, 126.51, 126.01, 125.58, 125.08, 124.09, 123.88, 123.55, 122.92, 44.63, 40.43, 39.94, 39.80, 39.66, 39.52, 39.38, 39.24, 39.10. IR (KBr): 3751, 3445, 3046, 2361, 1603, 1584, 1565, 1468, 1400, 1316, 1048, 1016, 846, 791, 722, 681, 657, 563 cm−1. Elemental analysis calcd (%) for C33H25Cl2N3ORuS: C 57.98, H 3.69, N 6.15; found: C 57.96, H 3.75, N 6.12. Materials, instrumentation, and the experimental methods. Tris, RNase A, and propidium iodide (PI) were purchased from Sigma. The hTERT, β-actin, and c-myc antibodies were purchased from Abcam. The EGFP (enhanced green fluorescent protein) and c-myc gene vectors, total RNA isolation kit, and two-step RT-PCR kit were purchased from TIANGEN. All the human cell lines (BEL-7404, Hep-G2, NCIH460, MGC80-3, and HL-7702 cells) were obtained from the Shanghai Institute for Biological Science (China). ct-DNA and other DNA oligomers (highly polymerized stored at 4 °C and long-term storage at −20 °C) are listed in Table S7† and were obtained from Shanghai Sangon Biological Engineering Technology & Services (Shanghai, China). Stock solutions of all the compounds (2 mM) were made in DMSO, and further dilutions to working concentrations were made with the corresponding buffer. The X-ray crystallography structure of Ru5 was solved with direct methods and refined using the SHELX-97 program.76,77 The materials, instrumentation, RTPCR, cytotoxicity assay, cellular morphology, RNA extraction, uptake of Ru in Hep-G2 cells, cell apoptosis, western blot, and transfection assay of Ru1–Ru6 were performed as reported by Chao, Reed, Liang, and Chen.8,20,32,71,72,78,79 Conflicts of interest The authors declare no competing interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21261025, 21761033), the Key Foun- This journal is © The Royal Society of Chemistry 2018 Research Article dation Project of Colleges and Universities in Guangxi (No. ZD2014108), the Innovative Team & Outstanding Talent Program of Colleges and Universities in Guangxi (2014-49 and 2017-38), the PhD Research Startup Program of Yulin Normal University (No. G2017009), IRT_16R15, the project of Guibei characteristic medicine resources research center of Guangxi Province (KYA201703), the basic skills improvement project for the young and middle-aged teachers in Guangxi colleges and universities (KY2016YB595), the Yulin Normal University Research Grant (2018YJKY34 and 2015YJYB08), as well as the State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2017-B17 and CMEMR2014-B08). Notes and references 1 A. T. 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