European Journal of Medicinal Chemistry 80 (2014) 316e324

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

Original article

Ruthenium(II) complexes as apoptosis inducers by stabilizing c-myc G-quadruplex DNA Zhao Zhang a,1, Qiong Wu a,1, Xiao-Hui Wu a, Fen-Yong Sun b, *, Lan-Mei Chen c, Jin-Chan Chen c, Shu-Ling Yang a, Wen-Jie Mei a, * a b c

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, PR China Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai 200072, PR China School of Pharmacy, Guangdong Medical College, Zhanjiang 524023, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 August 2013 Received in revised form 23 April 2014 Accepted 23 April 2014 Available online 25 April 2014

Two ruthenium(II) complexes, [Ru(L)2(p-tFMPIP)](ClO4)2 (L ¼ bpy, 1; phen, 2; p-tFMPIP ¼ 2-(4-(trifluoromethyphenyl)-1H-imidazo[4,5f][1,10] phenanthroline)), were prepared by microwave-assisted synthesis technology. The inhibitory activity evaluated by MTT assay shown that 2 can inhibit the growth of MDA-MB-231 cells with inhibitory activity (IC50) of 16.3 mM, which was related to the induction of apoptosis. Besides, 2 exhibit low toxicity against normal HAcat cells. The inhibitory growth activity of both complexes related to the induction of apoptosis was also confirmed. Furthermore, the studies on the interaction of both complexes with c-myc G4 DNA shown that 1 and 2 can stabilize the conformation of c-myc G4 DNA in groove binding mode, which has been rational explained by using DFT theoretical calculation methods. In a word, this type of ruthenium(II) complexes can act as potential apoptosis inducers with low toxicity in clinic by stabilizing c-myc G4 DNA. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Ruthenium(II) complexes Microwave-assisted synthesis Apoptosis induction c-myc G4 DNA

1. Introduction Ruthenium(II) complexes have attracted extensively attentions for their potential utility in chemotherapy [1,2]. For example, NAMI-A [3] and KP1019 [4,5], have been successfully entered into clinical trials. It’s found that ruthenium(II) complexes have been usually exhibit high selectivity to tumor cells [5,6], but low cytotoxicity toward human normal cells [7]. More and more attentions were focused on ruthenium (II) complexes with large aromatic intercalating ligand [8e10], which exhibit promise inhibitory activity against various tumors. In last decades, numbers of evidence indicated that this type of ruthenium(II) complexes can bind to DNA molecules with high affinity [9e11]. For example, Gasser et al. reported that [Ru(dppz)2(CppH)]2þ(dppz ¼ dipyrido[3,2-a:20 ,30 -c] phenazine; CppH ¼ 2-(20 -pyridyl)pyrimidine-4-carboxylic acid) exerted its toxicity through a mitochondria related pathway [12]. Xu et al. also confirmed that ruthenium (II) complexes coordinated by b-carboline alkaloids exhibit effective cell growth inhibition by triggering G0/G1 phase arrest and inducing apoptosis through a ROS-mediated mitochondrial dysfunction pathway [13]. In recent, * Corresponding authors. E-mail addresses: [email protected] (F.-Y. Sun), [email protected] (W.-J. Mei). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.ejmech.2014.04.070 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

studies by our laboratory demonstrated that chiral ruthenium(II) complexes coordinated by tFMPIP (2-(trifluoromethyphenyl)-1Himidazo[4,5f][1,10] phenanthroline) can regulate the expression of Bcl-2 family protein to induce apoptosis of tumor cells through the caspase signal pathway [14]. More recently, Chen [15] and Yu [16] et al. indicated that ruthenium (II) complexes can act as stabilizer of G-quadruplex DNA. In general, G-quadruplex DNA, which has been potential target for small molecule drugs [17], will constructed for those G-rich DNA sequences via Hoogsteen hydrogen bonds in the presence of monovalent cations [18,19]. It’s found that the promoter of oncogene c-myc, which over-expressed in up to 80% of solid tumors [19,20] can also form a G-quadruplex conformation in the presence of potassium or sodium ion, and play a key role in the proliferation and apoptosis of tumor cells [19]. Those complexes, which can stabilize the conformation of c-myc G4 DNA usually exhibit high inhibitory activity against tumor cells [21,22]. For example, CX3543, a fluoroquinolone-based antitumor agent by inducing apoptosis of tumor cells, can selected interact with c-myc G4 DNA [23]. Evidences also indicated that transition complexes, such as platinum(II) complexes coordinate by schiff base, can also repress the expression of c-MYC by stabilizing the G-quadruplexes of c-myc [24]. Besides, Thomas et al. indicated that dinuclear ruthenium(II) complexes could bind to G-quadruplex DNA with higher affinities

Z. Zhang et al. / European Journal of Medicinal Chemistry 80 (2014) 316e324

than that of duplex DNA molecules [25], and which has been also confirmed by the DNA competition FRET-melting assay [15]. In this paper, two ruthenium(II) complexes [Ru(bpy)2(ptFMPIP)](ClO4)2 and [Ru(phen)2(p-tFMPIP)](ClO4)2 (1 and 2, Fig. 1) have been synthesized by microwave-assisted technology. The studies by MTT assay shown that 2 exhibit excellent inhibitory activity against MDA-MB-231 cells, and low toxicity against HAcat cells. It’s also demonstrated that 2 can induce apoptosis of human breast cancer cells. The further studies by using spectroscopic, ITC, NMR spectra and FRET melting point assay, as well as the DFT calculations and the frontier molecular orbital theory analysis, shown that the interaction of these complexes with c-myc G4 DNA may act as a key role in the mechanism of this type of complexes, resulting the expression levels of relevant protein were markedly suppressed. 2. Results and discussion 2.1. Synthesis and characterization The target compounds 1 and 2 have been prepared by microwave-assisted synthesis technology [7]. It’s observed that the temperature of reaction system instantly reach 140  C in 30 s under microwave irradiation, after then, it kept almost unchanged during the whole process (Fig. S1). The yield for 1 and 2 under the irradiation of microwave is about 83 and 80%, respectively. The chemical shift of 1 and 2 in 1H NMR at 7.87 (d, 2H) and 8.55(d, 2H) can be attributed to trifluoromethyl-benzene (Fig. S2). For complex 1, the chemical shift at 8.85, 8.12, 7.33 and 7.40 ppm can be attributed to H1, H2, H3 and H4 in bpy ligands, respectively. The chemical shift attribute to the phenanthroline ring appeared at (7.87e9.09) of 8.03 (d, 2H), 7.87 (t, 2H), and 9.09 (d, 2H). For complex 2, the chemical shift at 8.41 ppm can be attributes to H5 and H6 in co-ligand phenanthroline [26]. 2.2. Biological activity The in vitro inhibitory activities of 1 and 2 against human breast cells (MDA-MB-231, MCF-7), human esophageal carcinoma cells

Table 1 The inhibitory activity (IC50/mM) of the Ru(II) complexes and cisplatin against selected cell lines. Complex

MDA-MB-231

MCF-7

EC-1

HAcat

1 2 Cisplatin

88.8  3.3 16.3  2.6 36.1  1.9

>100 74.5  2.3 /

>100 >100 >100

>100 >100 7.48  2.9

(EC-1), normal cuticle cells (HaCat) were evaluated by MTT assay after a 48 h treatment, and the results were listed in Table 1. Besides, the alterations chart of morphological and viability of MDAMB-231 cells in the absence and presence of 2 also shown in Fig. 2. As shown in Fig. 2, in the phase-contrast observation, the cells treated with 2 for 72 h displayed reduction in cell number, cell shrinkage and loss of cell-to-cell contact. The reduction of cell viability and the change in cell morphology both proved the growth inhibitory effect of 2 on MDA-MB-231 cells. As shown in Table 1, both 1 and 2 exhibit acceptable inhibitory activity breast cancer cells. The IC50 values determined for 2 (16.3 mM), which was cytotoxicity toward MDA-MB-231 cells in this study, is close or even lower than for cisplatin at the same conditions (IC50 ¼ 36.1 mM). Moreover, 2 was found to be less cytotoxic than cisplatin on the healthy cell line (HaCat) studied in this work is suggestive of a better therapeutic profile than cisplatin (IC50 ¼ 7.5 mM). These data indicate that this type of ruthenium(II) complexes have a potential utility in clinic with acceptable inhibitory activity against tumor cells but low toxicity [14]. 2.3. Apoptosis induction Harmless removal of cancer cells, apoptosis induced by compounds, is one of the considerations in drug development [14]. Inhibition of cancer cell growth by cytotoxic drugs could be the result of apoptosis inducing, cell cycle arrest or a combination of these two modes. The apoptotic cells usually shown apoptotic features such as nuclear shrinkage and chromatin condensation, DNA fragmentation and formation of apoptotic bodies [27e29]. To

CF3

CF3

26

20

25

19

24

18 23

17

20

14 13 HN

21 HN

N15

12 10

317

N19

22

16

18 17

11

16 15

9

4

5

8

N

N

6

3

N

2

N

N

7

Ru

N

13

Ru

9 8

N

7 11

N

6

N

N

1 2

1

10

12

N

1 N

14

2

Fig. 1. The target compounds molecular structure of 1 and 2.

4 3

5

318

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Fig. 2. The morphological change of human breast cancer cells in the absence and presence of 2.

clarify whether 1 and 2 can induce apoptosis of tumor cells, and determine the effect of both complexes on cell growth, a TUNEL analysis, a quantitative method which detects early stages of apoptosis, was firstly performed. As shown in Fig. 3A, to the control (with 1% DMSO), exposure of MDA-MB-231 cells induced apoptosis in 4.2% of the cells after 12 h. However, exposure of MDA-MB-231 cells to 1 and 2 induced

apoptosis in 21.3 and 65.4% of the cells, respectively, suggesting that the cytotoxic activity of this type of ruthenium(II) complexes in MDA-MB-231 cells may occurs via apoptosis. Besides, the effect of both 1 and 2 on the expression of bcl-2 and caspase-3, which can account for arrested cell growth and apoptosis were also evaluated by quantitative PCR (RT-qPCR). As seen in Fig. 3B, the results of RT-qPCR demonstrate a 6.5 and 13.8-

Fig. 3. Apoptosis of MDA-MB-231 cells induced by 1 and 2. A) Flow-cytometric analysis of MDA-MB-231 cells treated with 1 and 2 (20 mM). Generation of free 30 -OH DNA fragments was determined using TUNEL analysis and quantitated by flow cytometry. B) The regulation of bcl-2 and caspase-3 after dealing with different concentration 1 and 2 in cell level tested by quantitative “real-time” PCR (RT-qPCR). The amplification plot and dissociation curve of RT-qPCR of bcl-2 and caspase-3 transcript expression profiles of complexes 1 and 2 at various confluences as shown in Figs. S3 and S4.

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319

Fig. 4. Western blot analysis of c-Myc expression in the transduced cells with 1 and 2.

fold increase in the level of expression of bcl-2 and caspase-3 activation in MDA-MB-231 cells after treatment with complexes 2, a essential in apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner, with both complexes treatment compared to non-treatment. Those data of above suggested clearly that the cytotoxic activity of 1 and 2 in MDA-MB231 cells occurs via apoptosis [30,31].

Upon the addition of c-myc G4 DNA, obviously hypochromism and red shift were observed for both complexes. For 1, the hypochromism at MLCT and IL absorption is about 15.4 (Dl ¼ 1 nm) and 20.1% (Dl ¼ 2.5 nm), respectively. As for 2, the hypochromism at MLCT and IL transition is about 6.5 (Dl ¼ 1 nm) and 28.0% (Dl ¼ 4.5 nm), respectively. Compared with [Ru(bpy)2(bppp)]2þ, these data indicated that both 1 and 2 could bind to c-myc G4 DNA with high affinity, and 2 was more stronger.

2.4. Induced apoptosis through c-myc mediated mechanisms Having established that both complexes, especially 2, exert inhibitory by inducing apoptosis of tumor cells, then we examined the effect of 1 and 2 on the c-myc protein levels in MDA-MB231 cells, which is widely known as a crucial regulator in proliferation and apoptosis of tumor cells [17e19,21]. Thus, western blot analysis was further tested to determine whether c-myc activation occurs after treatment with 1 and 2. As shown in Fig. 4, the expression levels of c-myc in the MDAMB-231 cells treatment were markedly suppressed by both 1 and 2, by western blotting analysis after 24 h. These results indicate that this type of ruthenium(II) complexes can induce apoptosis through a c-myc mediated mechanism [32].

2.5.2. Isothermal titration calorimetry (ITC) methods To further certify the binding behavior of both complexes with cmyc G4 DNA at high salt concentrations, and dissect the observed binding free energy and entropic components, we have conducted ITC experiments at 293 K (Fig. S5) [25]. And the standard molar enthalpy change for the binding (DH0m ), the binding constant (Kb), and the binding stoichiometry (n) thus obtained were listed in Table 2. DbH0m values for 1 and 2 interacting with c-myc G4 DNA was 2.49 and 3.48 kcal M1, respectively. The Gibbs free energy change (DbG0m ) for binding reaction were calculated by the equations of thermodynamics as equations: DbG0m ¼ DH0m  TDbS0m (Isothermal). The Kb for 1 and 2 obtained from ITC was 1.78  0.30 and 3.22  0.74  104 mol/L, respectively.

2.5. The binding behavior of both ruthenium(II) complexes with cmyc G4 DNA It’s found that small drugs stabilizing the conformation of c-myc G4 DNA can interface the expression of c-myc oncogene [20,24]. Thus the binding behavior of both complexes with c-myc G4 DNA have been further investigated to clarify this.

1.00

1.00

0.75

0.75 ABS

ABS

2.5.1. Electronic spectra The interaction of both 1 and 2 with c-myc G4 DNA has been firstly investigated by the electronic spectra, as shown in Fig. 5. As shown in Fig. 5, the characterized MLCT transition was observed for 1 at 457 nm, and a strong characterized IL transition was observed at 284 nm. As for complex 2, the characterized MLCT and IL transition was appeared at 455 and 263 nm, respectively.

2.5.3. Circular dichroism spectra CD spectra can offer some useful information on the interaction of small molecules with biology macro-molecules. The CD spectra of c-myc G4 DNA in the absence and in the presence of 1 and 2 were illustrated in Fig. 6. As shown in Fig. 6, a positive CD signal for c-myc G4 DNA was observed in rang of 240e280 nm with the maximum at 264 nm. Upon the addition of 1 and 2, the positive CD signal strengths of cmyc G4 DNA decreased 27.4 and 66.7%, respectively. Besides, a positive induced CD signal was appeared in rang of 290e300 nm, with the maximum at 293 nm following the concentration increase for complex 1. Those data suggested that those complexes may bind to c-myc G4 DNA in groove binding mode [33].

0.50 0.25 0.00

0.50 0.25

300

400 500 Wavelength/ nm

1

600

0.00

300

400 500 Wavelength/ nm

600

2

Fig. 5. The electronic spectras of 1 and 2 in the absence and in the presence of c-myc G4 DNA. [Ru] ¼ 5  106 M.

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Table 2 Thermodynamic parameters for the binding of 1 and 2 to c-myc G4 DNA measured by ITC at 298.15 K. Complex 1 2

Kb  104 (M1)

DbH0m

DbG0m

DbS0m

1.78  0.30 3.22  0.74

2.49 3.48

21.5 25.1

63.9 96.0

(kcal mol1)

(kcal mol1)

(cal mol1 K1)

2.5.4. NMR spectroscopy Nuclear magnetic resonance is a powerful tool for the analysis of interactions between small molecules and biology macromolecules [34]. The 1H NMR spectroscopy experiments was carried out to clarify the binding mode of both complexes with c-myc G4 DNA, and the results were shown in Fig. 7. As shown in Fig. 7, 1H NMR spectra of c-myc G4 DNA was characterized by the chemical shift at 8.56 (s, 1H) and 8.46 (s, 1H) ppm which attributed to the protons for aromatic region of A15. The similar situation, chemical shift at 7.74, 7.10 and 7.52, 7.41 ppm, was also occurred on the A24 and A25. The separate signals, for the G16, G17, G21, G22, attributed to the protons of aromatic region appeared at 8.10, 7.86, 7.89 and 7.65 ppm, respectively [34]. Upon the addition of 1 and 2 to a solution of c-myc G4 DNA(0.5 mM) at a [c-myc G4 DNA]/[Complex] ratio of 1:0.5, the change of aromatic region proton signals was observed and clearly assigned. The key changes are focused on T14wG17 and G21wA25, the signal of which almost all were disappear or wider, while the G7wG9 signal did not show any variation. The results suggested that both complexes bind closely to the 50 -terminal face of the Gquadruplex DNA by the groove constitute T14wG17 and G21wA25 in c-myc G4-DNA molecules. Thus, the 1H NMR spectroscopy experiments provided additional evidence (besides the CD spectroscopy) that both 1 and 2 can stabilize of c-myc G4 DNA by groove binding mode [35].

6 4 2 0 -2 -4 240

2.6. FRET melting point curves and DNA competition measurements The FRET melting point assay was carried out to investigate the thermodynamic stability of c-myc G4 DNA in the presence of 1 and 2, and the results were shown in Fig. 9. As shown in Fig. 9, the melting point of c-myc G4 DNA was about 45.1  C, while the Tm (the melting point of c-myc G4 DNA) in the presence of 3.0 mM of 1 and 2 increased to 48.5 (DTm ¼ 3.4) and 52.7 (DTm ¼ 7.6)  C, respectively. At the concentration of [Ru] ¼ 6.0 mM, the Tm in the presence of 1 and 2 increased to 51.9 (DTm ¼ 6.8) and 56.0 (DTm ¼ 10.9)  C, respectively. These data indicated that both complexes can stabilize the G-formation of c-myc oligomer [15,16], and 2 was more stronger. Besides, the DNA competition FRET-melting assay was also carried out to show the DTm change for 3.0 mM of the complexes with 0.2 mM c-myc by adding 10 mM the double-stranded DNA. As shown in Table 4, the values of DTm do not change even though the double-stranded DNA was present at a concentration 50 times that of c-myc G4 DNA. This results indicated that both complexes have extremely high selectivity for the c-myc G4 DNA over the duplex DNA [15]. 3. Conclusions In summary, two ruthenium(II) complexes have been prepared in high yield under microwave irradiation, and characterized by ESI-MS, 1H NMR and 13C NMR. The investigation by using MTT

Circular Dichroism/mdeg

Circular Dichroism/mdeg

2.5.5. Theoretical The molecular configuration of optimized for both complexes was illustrated in Fig. S6, and the mean coordination bond length and bond angle were listed in Table S1. It’s observed that the calculated selective geometric data of both complexes has not a substantial difference. We can speculate that the different binding affinity for both complexes can attribute to the difference of hydrophobic of co-ligand bpy and phen, which can be reasonably explained by the DFT computations and the frontier molecular orbital theory. Based on the computed results, some frontier molecular orbital energies and total energies were listed in Table 3, the molecular orbital stereographs of 2 were illustrated in Fig. S7, and the schematic diagram of the energies and related 1MLCT transitions was shown in Fig. 8.

For a reaction controlled by orbital interactions between reactant molecules, a higher HOMO energy of one reactant molecule and a lower LUMO energy of the other are more advantageous to the reaction between the two molecules, because electrons more easily transfer from the HOMO of one reactant to the LUMO of the other in the orbital interaction [9]. In fact, we can see that the related frontier MO contour plots of the two complexes are very alike, in particular, no substantial difference in the LUMO population appears. However, from Table 3 and Fig. 8, we can clearly see that the energies of LUMO and L þ x (L ¼ LUMO; x ¼ 1, 2, etc.) of 2 are all higher than those of 1, i.e. DεLeH(2) > DεLeH(1), DεLeNH(2) > DεLeNH(1), Lþ1(2) > Lþ1(1), etc. Since lower energies of LUMO and L þ x must be advantageous to accepting the electrons of HOMO and H  x (H ¼ HOMO; x ¼ 1, 2, etc.) of c-myc G4 DNA based on the frontier MO theory, the interaction between 2 and c-myc G4 DNA must be stronger than that between 1 dose [9]. The experimental results that in vitro anticancer activities and the DNA-binding constant (Kb) of c-myc oligomer of 2 were greater, can be reasonably explained.

270 300 Wavelength/ nm

1

330

6 4 2 0 -2 -4 240

270 300 Wavelength/ nm

2

Fig. 6. The CD spectras of c-myc G4 DNA with the addition of 1 and 2. [c-myc G4 DNA] ¼ 2  106 M.

330

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321

Fig. 7. 1H NMR spectras of c-myc G4 DNA in 1 and 2 titration experiments.

Table 3 Some frontier molecular orbital energies (εi/au) and total energies (Etotal/au) of 1 and 2. Comp.

H-3

H-2

H-1

HOMOa

LUMOb

Lþ1

Lþ2

DεLeHc

DεLeNH

1 2

0.4083 0.4043

0.4080 0.4025

0.4018 0.3983

0.3922 0.3899

0.2780 0.2723

0.2747 0.2684

0.2712 0.2676

0.1142 0.1176

0.1238 0.126

a b c

HOMO (or H): the highest occupied molecular orbital; NHOMO (or NH): the next HOMO (or H-1). LUMO (or L): the lowest unoccupied molecular orbital; NLUMO (or NL): the next LUMO (or L þ 1). DεLeH: energy difference between LUMO and HOMO; DεLeNH: energy difference between LUMO and NHOMO, etc.

assay shown that both complexes, especially 2, exhibit excellent inhibitory activity against MDA-MB-231 cells by inducing apoptosis of tumor cells. The further studies on the interaction of these complexes with c-myc G4 DNA shown that both 1 and 2 can stabilize the G-quadruplex conformation of c-myc oncogene promoter via groove binding mode, resulting the expressing of c-myc to down-regulated. In a word, this type of ruthenium(II) complexes can be used as potential inducers apoptosis with low toxicity by

Fig. 8. Schematic diagram of the energies and related energy transitions of some frontier molecular orbits of 1 and 2.

stabilizing the c-myc G4 DNA, and the detail mechanism was under further investigation. 4. Experimental 4.1. Materials and physical measurements All reagents and solvents were purchased commercially and used without further purification unless specially noted. Distilled water was used in all experiments. c-myc oligomers strand (50 T4G5A6G7G8G9T10G11G12G13T14A15G16G17G18T19 G20G21G22T23A24A25-30 ) was purchased from Shenggong Biotech(Shanghai) Co., Ltd. and formed G-quadruplex conformation as literature by renaturation for 24 h at 4  C, after 90  C, denaturation for 5 min. All aqueous solutions were prepared with doubly distilled water. The compounds cis-Ru(bpy)2Cl2$2H2O, cis-Ru(phen)2Cl2$2H2O and ptFMPIP were prepared according to the literature [7,9] methods. The c-myc G4 DNA-binding experiments were performed at room temperature. TriseHCleKCl (Buffer solution) consisting of Tris and KCl, and the pH value was adjusted to 7.2 by HCl solution, which was applied to UV titration, CD spectra and ITC experiments. These complexes were synthesized by using Anton Paar monowave 300 microwave reactor (an Initiator single mode microwave cavity at 2450 MHz (Biotage)). The 1H NMR, 13C NMR spectra were recorded in d6-DMSO solution on a Bruker DRX 2500 spectrometer, and ESI-MS spectra were obtained in methanol on Agilent 1100 ESIMS system operating at room temperature. Microanalyses (C, H, and N) were carried out on a PerkineElmer 240Q elemental analyzer. UVevis absorption spectra were recorded on a Shimadzu UV-2550 spectrophotometer using 1 cm path length quartz cuvettes (3 mL) at room temperature. Circular dichroism (CD) spectra were measured on a Jasco J-810 spectrophotometer. ITC (isothermal titration calorimetry) experiments were carried out on an isothermal titration calorimeter (VP-ITC; MicroCal, Northampton, USA) at 298.15 K with stirring at 394 rpm. Fluorescence melting curves were measured by a Roche Lightcycler 2.0 real-time PCR detection system. RT-qPCR assay was measured by PCR (biored IQ5).

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Fig. 9. FRET melting profiles of c-myc G4 DNA in the absence and in presence of 1(A) and 2(B) and the competition FRET-melting assay with ds26 in the presence of 3 mM 1(C) and 2(D). [c-myc G4 DNA] ¼ 2  107 M.

Table 4 The Tm of 1 and 2 with c-myc G4 DNA system in the presence of ds26 (Competition FRET-melting assay). R ([ds26]/[c-myc)]

0

5

10

50

[Ru] [ 3 mM 1 2

48.5 52.7

47.1 52.8

46.1 51.0

51.8 49.1

4.2. Cell culture Human cancer cell lines including MDA-MB-231, MCF-7 and EC1 were purchased from American Type Culture Collection (ATCC, Manassas, VA). The normal human cuticle HACat cells were also obtained from ATCC. All cell lines were maintained in either RPMI1640 or DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/mL) and streptomycin (50 units/mL) at 37  C in CO2 incubator (95% relative humidity, 5% CO2). 4.3. Synthesis and characterization 4.3.1. Synthesis of [Ru(bpy)2(p-tFMPIP)](ClO4)2 (1) A mixture of [Ru(bpy)2(Cl)2] (105 mg, 0.2 mmol), p-tFPIP (79.2 mg, 0.3 mmol) and in ethylene glycol was irradiated by microwaves for 30 min at 140  C. The cooled reaction mixture was diluted with water. Added sodium perchlorate to the filter liquor, lots of orange suspended solid will obtain. The orange suspended solid was collected and washed with small amounts of water and diethyl ether, and purified by Al2O3 column chromatography. The solvent was removed under reduced pressure and red microcrystals were obtained; yield: 83.6%. ESI-MS (in CH3CN, m/z): 777.3 ([M2ClO4eH]þ, cal: 777.7). UVevis [l (nm), ε (M1 cm1)) (in 5% DMSO/ Buffer solution]: 457.0(84500), 284(80583). 1H NMR (500 MHz, in d6-DMSO, d/ppm) 9.09 (dd, J ¼ 8.2 Hz, 2H), 8.85 (d, J ¼ 8.2 Hz, 4H), 8.12 (t, J ¼ 8.1, 1.3 Hz, 4H), 8.01 (s, 2H), 7.87 (d, J ¼ 5.3 Hz, 2H), 7.40e 7.33 (m, 8H). 13C NMR (126 MHz, in d6-DMSO, d/ppm) 157.26, 157.06, 151.87, 145.48, 138.41, 138.25, 130.86, 128.36, 128.22, 127.50,

126.67, 124.92, 124.85, 56.50 (Found: C 50.16%, H 2.82%, N 11.70%. Calcd. for C40H27F2N8Cl2O8Ru: C 49.27%, H 3.13%, N 11.70%). 4.3.2. Synthesis of [Ru(phen)2(p-tFMPIP)](ClO4)2 (2) The complex [Ru(phen)2(p-tFMPIP)](ClO4)2 (2) was obtained in the same method as above, but use [Ru(phen)2(Cl)2] (113 mg, 0.2 mmol) instead, yield: 80.9%. ESI-MS (in CH3CN, m/z): 825.2 ([M2ClO4eH]þ, cal: 825.8). UVevis [l (nm), ε (M1 cm1)) (in 5% DMSO/Buffer solution]: 455 (16667), 263.5(82417), 358.5(9750). 1H NMR (500 MHz, in d6-DMSO, d/ppm) 9.09 (d, J ¼ 2.6 Hz, 2H), 8.79 (d, J ¼ 0.8 Hz, 2H), 8.78 (t, J ¼ 2.1 Hz, 2H), 8.55 (d, J ¼ 8.2 Hz, 2H), 8.41 (s, 2H), 8.15 (dd, J ¼ 5.3 Hz, 2H), 8.10 (dd, J ¼ 5.2 Hz, 2H), 8.03 (d, J ¼ 4.1 Hz, 2H), 7.83 (d, J ¼ 2.2 Hz, 2H), 7.82 (d, J ¼ 2.9 Hz, 2H), 7.80 (d, J ¼ 2.7 Hz, 2H), 7.78 (d, J ¼ 2.9 Hz, 2H). 13C NMR (126 MHz, in d6DMSO, d/ppm) 153.28, 147.72, 147.63, 146.11, 137.29, 134.41e134.01, 130.92, 130.20e130.09, 128.54, 127.59, 127.03e126.57, 127.10e 126.52, 125.69, 123.52, 56.50 (Found: C 55.60%, H 1.23%, N 14.38%. Calcd. for C44H27F2N8Cl2 O8Ru: C 52.54%, H 2.69%, N 11.14%). 4.4. MTT assay The tested compounds were dissolved in DMSO with stock solution at 1 mM. Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye. Cells were seeded in 96-well tissue culture plates (5  103 cells/well) for 24 h. The cells were then incubated with the tested compounds at different concentrations for 72 h. After incubation, 20 mL/well of MTT solution (5 mg/mL phosphate buffered saline) was added and incubated for 5 h. The medium was aspirated and replaced with 150 mL/well DMSO to dissolve the formazan salt. The absorbance intensity, which reflects the cell growth condition, was measured at 570 nm using a microplate spectrophotometer (Versamax). 4.5. Flow cytometry analysis of apoptotic cells (TUNEL assay) MDA-MB-231 cells were firstly washed with PBS and fixed in 4% paraformaldehyde prepared freshly in PBS after treating with/ without both complexes (20 mM). The cells were permeabilised

Z. Zhang et al. / European Journal of Medicinal Chemistry 80 (2014) 316e324

using 0.1% triton X-100 in 0.1% sodium citrate for 2 min on ice. After washing, the cells were incubated in the TdT incubation buffer (fluorescein/dNTP mix, TdT and labeling buffer) for 1 h at 37  C in the dark and then washed and re-suspended in PBS. In humid chamber terminal deoxynucleotidyl transferase (TdT) catalyzes polymerization of fluorescein-labeled deoxynucleotides to free 30 OH DNA ends in a template-independent manner. Free 30 -OH DNA in apoptotic cells was detected and quantified based on red fluorescence by flow cytometer.

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4.8.3. Isothermal titration calorimetry (ITC) measurements About 1.43 mL of c-myc G4 DNA solution was titrated with the complexes solution. A typical titration experiment consisted of 30 consecutive injections of 10 mL volume and 20 s duration each, with a 3 min interval between injections. Heats of dilution of the complex were determined by injecting the complex solution into the buffer alone and the total observed heats of binding were corrected for the heat of dilution. Micro-Cal Origin software was used to determine the site-binding model that gave a good fit to resulting data.

4.6. Quantitative reverse transcription-PCR assay Total RNA was isolated using Trizol (Invitrogen) according to the manufacture’s recommendation. 2 mg of total RNA from each samples were reverse transcribed using oligo (dT) primers at 37  C for 90 min. The relative mRNA levels were evaluated by quantitative PCR using SYBR green PCR kit (Takara). The signals were normalized to 18 S as internal control. The quantity of bcl-2 in each BC relative to the average expression in 40 NATs was calculated using the equation RQ ¼ 2DDCT, where DDCT ¼ (CT c-myc  CT U6 RNA)S  (CT c-myc  CT U6 RNA)MeanC. All of the conditions of the quantitative reverse transcriptionPCR assay for casepase-3 expression assay reaction system were similar to the bcl-2, except casepase-3 were replaced. 4.7. Western blot analysis MDA-MB-231 cells were seeded and incubated with different agents at 10 or 40 mM in the presence of 10% FBS for 24 h. Total cellular proteins were extracted by incubating cells in lysis buffer obtained from Cell Signaling Technology and protein concentrations were determined by BCA assay. SDS-PAGE was done in 10% tricine gels loading equal amount of proteins per lane. After electrophoresis, separated proteins were transferred to nitro-cellulose membrane and blocked with 5% non-fat milk in TBST buffer for 1 h. Then the membranes were incubated with primary antibodies at 1:5000 dilutions in 5% non-fat milk overnight at 4  C, and then secondary antibodies conjugated with horseradish peroxidase at 1:2000 dilution for 1 h at room temperature. The blots were visualized with the Amersham ECL Plus western blotting detection reagents according to the manufacturer’s instructions. 4.8. The binding behavior of ruthenium(II) complexes with c-myc G4 DNA 4.8.1. UV titration The electronic absorption spectra were carried out at room temperature to determine the binding affinity between c-myc G4 DNA and the target complexes. Initially, 3.0 mL solutions of the ruthenium(II) complexes sample was placed in the reference and then first spectrum was recorded in the range of 200e800 nm. The titration processes were repeated with 2 mL of c-myc G4 DNA solutions(100 mM) until there was no change in the spectra for four titrations at least, indicating binding saturation had been achieved. 4.8.2. CD spectrum Circular dichroism spectra were measured on a Jasco J-810 spectropolarimeter. Initially, 1.0 mL solutions of the c-myc G4 DNA sample was placed in the reference, and then the titration processes were repeated with 2 mL of ruthenium(II) complexes solutions until there was no change in the spectra. For each sample, at least three spectrum scans were accumulated over the wavelength range of 200e500 nm at the room temperature in a 1.0 cm path length cell at a scanning rate of 50 nm/min.

4.8.4. NMR experiments NMR experiments were performed on a Bruker DRX-2500 spectrometer. All titration experiments were carried out at 30  C in a 90% H2O/10% D2O solution containing 150 mM KCl, 25 mM KH2PO4, and 1 mM EDTA (pH 7.0). A standard 1-1 echo pulse sequence with a maximum excitation centered at 12.0 ppm was used for water suppression. Thirty-two scans were acquired for each spectrum with a relaxation delay of 2 s. 4.8.5. FRET melting point curves The fluorescent-labeled oligonucleotide c-myc was prepared as a 100 mM stock solution in buffer and annealed with heat at 90  C for 5 min. After heating, the annealed sample was cooled to room temperature slowly. Fluorescence melting curves were measured by a Roche Lightcycler 2.0 real-time PCR detection system using a total reaction volume of 25 mL, 200 nM of labeled oligonucleotide and different concentrations of complexes. Fluorescence readings with excitation at 470 nm and detection at 530 nm were taken at intervals of 1  C from 30 to 95  C. The temperature was maintained for 30 s before each reading to ensure that the sample had reached equilibrium. All of the conditions of the DNA competition FRET-melting assay reaction system were similar to the FRET melting point assay, except different concentrations of duplex DNA ds26 were added. 4.8.6. Theoretical section Each of the octahedral complexes forms from ruthenium(II) and one main co-ligand (bpy and phen) or intercalated ligand and one co-ligands (p-tFMPIP). It is symmetry in these complexes. The full geometry optimization computations were performed for these complexes applying the DFT-B3LYP method and LanL2DZ basis set. All computations were performed with the G98 quantum chemistry program-package. In order to vividly depict the detail of the frontier molecular orbital interactions, the stereographs of some related frontier MO of the complexes were drawn with the Moldenv3.6 program based on the obtained computational results. Acknowledgment This work was supported by the Science and Technology Item Foundation of Guangzhou (2013J4100072), excellent discipline leader training plan of Shanghai health system and Young Teachers Foundation of Guangdong Pharmaceutical University. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.04.070. References [1] V. Brabec, O. Nováková, DNA binding mode of ruthenium complexes and relationship to tumor cell toxicity, Drug Resistance Updates 9 (2006) 111e 122.

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Ruthenium(II) complexes as apoptosis inducers by stabilizing c-myc G-quadruplex DNA.

Two ruthenium(II) complexes, [Ru(L)2(p-tFMPIP)](ClO4)2 (L = bpy, 1; phen, 2; p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazo[4,5f][1,10] phenanthrol...
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