Two water-soluble copper(II) complexes: Synthesis, characterization, DNA cleavage, Protein Binding Activities and in vitro anticancer activity studies Jing Lu, Qian Sun, Jun-Ling Li, Lin Jiang, Wen Gu, Xin Liu, Jin-Lei Tian, Shi-Ping Yan PII: DOI: Reference:

S0162-0134(14)00092-0 doi: 10.1016/j.jinorgbio.2014.03.015 JIB 9497

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

23 September 2013 25 March 2014 27 March 2014

Please cite this article as: Jing Lu, Qian Sun, Jun-Ling Li, Lin Jiang, Wen Gu, Xin Liu, Jin-Lei Tian, Shi-Ping Yan, Two water-soluble copper(II) complexes: Synthesis, characterization, DNA cleavage, Protein Binding Activities and in vitro anticancer activity studies, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.03.015

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ACCEPTED MANUSCRIPT Two water-soluble copper(II) complexes: Synthesis, characterization, DNA cleavage, Protein Binding Activities and in vitro anticancer

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activity studies

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Jing Lua,b,c,d, Qian Suna,b,c,d, Jun-Ling Lia,b,c,d, Lin Jianga,b,c,d,Wen Gua,b,c,d, Xin Liua,b,c,d, Jin-Lei Tiana,b,c,d and Shi-Ping Yana,b,c,d a

Tianjin Key Laboratory of Metal and Molecule Based Material Chemistry, Tianjin 300071,

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Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China

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People’s Republic of China Key Laboratory of Advanced Energy Materials Chemistry (MOE), Tianjin 300071, People’s

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Republic of China

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Collaborative Innovation Center of Chemical Science and Engineering (Tianjin)

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Correspondence to: X. Liu, J.-L. Tian, Department of Chemistry, Nankai University, Tianjin 300071, P.R. China. E-mail: [email protected], [email protected] -1-

ACCEPTED MANUSCRIPT Abstract Two water-soluble ternary copper(II) complexes of [Cu(L)Cl](ClO4) (1) and [Cu(L)Br2] (2) (L=(2-((quinolin-8-ylimino)methyl)pyridine) were prepared and characterized by various

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physico-chemical techniques. Both 1 and 2 were structurally characterized by X-ray crystallography. The crystal structures show the presence of a distorted square-pyramidal CuN3Cl2

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(1) or CuN3Br2 (2) geometry in which Schiff-base L act as a neutral tridentate ligand. Both complexes present intermolecular π-π stacking interactions between quinoline and pyridine rings. The interaction of two complexes with CT-DNA (calf thymus-DNA) and BSA (bovine serum

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albumin) was studied by means of various spectroscopy methods, which revealed that 1 and 2 could interact with CT-DNA through intercalation mode, and could quench the intrinsic

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fluorescence of BSA in a static quenching process. Furthermore, the competition experiment using Hoechst 33258 indicated that two complexes may bind to CT-DNA by minor groove. DNA

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cleavage experiments indicate that the complexes exhibit efficient DNA cleavage activities

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without any external agents, and hydroxyl radical (HO.) and singlet oxygen (1O2) may serve as the major cleavage active species. Notably, the in vitro cytotoxicity of the complexes on three human

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tumor cells lines (HeLa, MCF-7, and A549) demonstrate that two compounds have broad-spectrum antitumor activity with a quite low IC50 ranges of 0.43-1.85 μM. Based on the cell cycle experiments, 1 and 2 could delay or inhibit cell cycle progression through the S phase.

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Keywords: water-soluble, cytotoxicity, chemical nuclease, quinoline ring, copper complex 1. Introduction

Metal-based pharmaceuticals are of considerable interest due to their application in DNA molecule probes and chemotherapeutic reagents for the confrontation against cancer [1-3]. Cisplatin, one of the world’s most important metal-based drugs, acts mainly through induction of DNA unwinding and DNA-protein binding which is widely used in clinical introduced in the 1970s [4-6]. But the serious side effects such as asneuro-, hepato- and nephrotoxicity and the insurmountable frequent development of resistance had limited its effectiveness [7-10]. Therefore, numerous attempts have been devoted to develop alternative strategies, based on different metals, with improved pharmacological properties and aimed at different targets. Recently, copper complexes with physiologically endogenous transition metal element centers showed various geometries and coordination numbers, various oxidation states, better -2-

ACCEPTED MANUSCRIPT solubility, higher affinity for the nucleobases [11-14]. Investigation of the literature revealed that copper complexes showed encouraging perspectives and displayed a significantly higher level of

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show general lower host toxicity than platinum compounds [15-18].

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anticancer, antibacterial, antiproliferative and antimitotic activities for solid tumor metastases, and

As we all known, the nucleic acid is the basic genetic material of life which is the main target

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of anti-cancer drugs into the human body, therefore, study of the interaction model between drugs and DNA is important to make a breakthrough in the mechanism of anti-cancer drugs cure. In recent years, understanding the mechanism of DNA cleavage, and exploring the application in

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antineoplastic medication were drawing more attention in molecular biology and bioengineering areas. There is enough evidence that the antitumor activity is due to these compounds intercalation

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with the base pair of DNA and interference with normal functioning of the enzyme topoisomerase II, which involved in the breaking and releasing of DNA strands [19].

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On the other hand, serum proteins play an essential role in the transport and metabolism of

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drug. Therefore, the interactions of metallo-drug with serum albumins have caught more and more attention in the scientific community by studying antitumoral metallo-pharmaceutical

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pharmacokinetics and structure−activity relationships [20]. Due to its structural homology with human serum albumin (HSA), BSA is the most extensively studied serum albumin. This fact further supports the value of studying the interaction behavior of the metal complexes with BSA

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protein, while evaluating their anticancer properties. In our study, we have synthesized a new Schiff base ligand characterized with quinoline ring systems and the planar aromatic heterocyclic chromophore which positions itself between two consecutive base pairs of the double helix though non covalent and reversible, involving van der Waal’s forces with the base pairs. It has been well studied that quinoline ring system and its synthetic derivatives are widely existed in alkaloids, such as camptothecin with potential biological effects including antibacterial, antihyperglycemic, antiinflammatory, antimalarial, antiasthmatic activities [21-25]. They have been used PDGF-RTK (platelet derived growth factor receptor tyrosine kinase) inhibiting agents [26]. Two copper complexes were synthesized and characterized. Also, we have investigated their interesting pharmacological properties such as DNA binding, DNA cleavage, protein binding, and cytotoxicity. 2. Experimental section -3-

ACCEPTED MANUSCRIPT 2.1 Materials and instrumentation Caution! Perchlorate salts of metal complexes are potentially explosive and therefore should be prepared in small quantities.

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Chemicals and solvents were purchased from commercial sources and used as received. Ligand L was synthesized according to a previously reported procedure [27，28]. Plasmid pBR322

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DNA, agarose, ethidium bromide (EB), bovine serum albumin, calf thymus-DNA, 3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), crystal violet, hoechst 33342, propidium iodide (PI) and rhodamine 123 (Rh123) were obtained from Sigma. HeLa,

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MCF-7, and A549 cells cells were obtained from the American. Stock solutions of copper(II) complexes (5.0 × 10−4 M in H2O for electronic spectra, the circular dichroism spectra; 1.0× 10−3 M

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in H2O for fluorescence spectra, and viscosity measurements ) were stored at 4 °C and prepared to required concentrations for all experiments. Tris-HCl and phosphate buffer solution were prepared

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using ultrapure water. Fetal bovine serum (FBS) was obtained from Hyclone.

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CT-DNA stock solution was prepared by diluting DNA to Tris-HCl/NaCl buffer (pH=7.2, 5 mM Tris–HCl, 50 mM NaCl), and kept at 4 °C for no longer than a week. The UV absorbance at

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260 and 280 nm of CT-DNA solution in Tris buffer give a ratio of 1.8-1.9, indicating that the DNA was sufficiently free of protein [29]. The concentration of CT-DNA was determined from its absorption intensity at 260 nm with a molar extinction coefficient of 6600 M−1 cm−1 [30].

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Elemental analyses for C, H and N were obtained on a Perkin-Elmer analyzer model 240. Infrared spectroscopy on KBr pellets were performed on a Bruker Vector 22 FT-IR spectrophotometer in the 4000–400 cm−1 regions. Electronic spectra were measured on a JASCO V-570 spectrophotometer. Fluorescence spectral data were obtained on a MPF-4 fluorescence spectrophotometer at room temperature. The circular dichroism (CD) spectra were taken on a JASCO-J715 spectropolarimeter. Viscosity measurements were carried out on an Ubbelodhe viscometer maintained at a constant temperature (37.0±0.1 °C) in a thermostatic water-bath. The Gel Imaging and documentation DigiDoc-It System were assessed using Labworks Imaging and Analysis Software (UVI, UK). Electrospray ionization mass spectrometry (ESI-MS) was obtained on Agilent 6520 Q-TOF LC/MS. The MTT assay was determined by measuring the absorbance of each well at 570 nm and 630 nm on a Biotek Microplate Reader (Bio-Rad, USA). The cell cycle

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ACCEPTED MANUSCRIPT distribution was analyzed by a flow cytometer (BD FACS Calibur). The data were acquired and analyzed by a ModFit 1.2 software.

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2.2. Preparation of complexes

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2.2.1. Synthesis of L

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Ligand L was obtained according to a previously reported procedure [27, 28]. Hot ethanolic solution of 8-aminoquinoline (1.44g, 0.01 mol) and ethanolic solution of 2-Pyridinecar boxaldehyde (0.95 mL, 0.01 mol) were mixed. This mixture was refluxed at 60-70 ℃ for 6h under

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constant stirring. On cooling the reaction mixture, yellow-colored product were precipitated out. They were filtered, washed with cold EtOH. (yield 73%)

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2.2.2. Synthesis of [Cu(L)Cl] (ClO4 )(1)

An aqueous solution (10 mL) of CuCl2·2H2O (340 mg, 0.2 mmol) was added an ethanol solution (10 mL) of L (0.2 mmol). The resulting mixture was stirred for 5h at room temperature.

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After filtered, the solid was dissolved in methanol, then 0.2 mmol NaClO4 solid was added to the

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solution. Green block crystals suitable for X-ray diffraction were obtained by slow evaporation of the filtrate after two weeks, washed with diethyl ether and dried in air ( yield: 41%). Elemental

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analysis (%): calc. for C15H10Cl2CuN3O4: C, 41.84; H, 2.34; N, 9.76. Found: C, 41.76; H, 2.38; N, 9.69. FT-IR (KBr, ν, cm− 1): 3506 s, 1637 vs, 1507m, 1397m, 1120 m, 618 s. The stability of complex 1 in aqueous solution was investigated by ESI-MS. The major peak at m/z 330.99 in the

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ESI-MS spectrum could be assigned to the molecular ion peak [CuLCl]+. 2.2.3. Synthesis of [Cu(L)Br2] (2) Complex 2 was prepared by a procedure similar to that given in the case of 1, but adding CuBr2 instead of CuCl2·2H2O to the reaction mixture. Green flaky crystals suitable for X-ray diffraction were obtained by slow evaporation of the filtrate after ten days, washed with diethyl ether and dried in air ( yield: 47%). Elemental analysis (%): calc. for C15H10Br2CuN3: C, 39.54; H, 2.21; N, 9.22. Found: C, 39.62; H, 2.18; N, 9.16. FT-IR (KBr, ν,cm− 1): 3497 s, 1636 vs, 1118 m, 615 s. The state of complex 2 in aqueous solution was investigated by ESI-MS. The major peak at m/z 341.05 in the ESI-MS spectrum could be assigned to the molecular ion peak [CuL(CH3CH2O)]+. 2.3. X-ray crystallography -5-

ACCEPTED MANUSCRIPT Single-crystal X-ray diffraction data of copper complexes were collected at 293(2) K, on a Bruker Smart 1000 CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å) with the ω scan technique. An empirical absorption correction was applied to the raw intensities. The structures

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were solved by direct methods (SHELX- S-97) [31] and refined with full-matrix least-squares

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technique on F2 using the SHELXL-97 [32]. The hydrogen atoms were added theoretically, and

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riding on the concerned atoms and refined with fixed thermal factors. The details of crystallographic data and structure refinement parameters are summarized in Table 1.

2.4. DNA-binding experiments 2.4.1. Absorption spectrophotometric studies

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(Insert Table 1)

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Absorption spectra titrations were performed at room temperature in Tris-HCl/NaCl buffer (50 mM Tris-HCl/1 mM NaCl buffer, pH 7.5) to investigate the binding affinity between CT-DNA

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and complexes. 2 mL solutions of the blank Tris–HCl/NaCl buffer and the Cu(II) complexes

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samples ([complex] = 5 × 10−4 M) were placed into two 1 cm path cuvettes, respectively. Then one aliquot (10 μL) of buffered CT-DNA solution (0.01 M) was added to each cuvette in order to

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eliminate the absorbance of DNA itself. Before the absorption spectra were recorded, the Cu(II)-DNA solutions were incubated at room temperature for 5 min in order to full reaction. 2.4.2. Competitive Binding Experiments

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The relative binding of complexes to CT-DNA were determined with an EB-bound CT-DNA solution in Tris–HCl/NaCl buffer (pH=7.2, 5 mM Tris–HCl, 50 mM NaCl). The experiments were carried out by adding a certain amount of a solution of complexes ([complex] = 1.0 × 10−3 M) step by step into the EB–DNA solution (2.4 μM EB and 48 μM CT-DNA). The influence of the addition of complexes to the EB-DNA complex have been obtained by recording the variation of fluorescence emission spectra with excitation at 510 nm and emission at 602 nm. Before the emission spectra were recorded, the Cu(II)-DNA solutions were incubated at room temperature for 5 min in order to full reaction. The procedure was the same for the Hoechst 33258 reactions using the following conditions: working solutions were 20 μ M DNA and 2 μ M Hoechst 33258; λex = 338 nm and λem = 350−650 nm (with λmax∼ 600 nm). 2.4.3. CD spectrophotometric studies -6-

ACCEPTED MANUSCRIPT The CD spectra of CT-DNA in the presence or absence of complexes were collected in Tris –HCl buffer (pH = 7.2) containing 50 mM NaCl at room temperature. All CD experiments were performed on a JASCO-J715 spectropolarimeter at room temperature from 350 to 230 nm.

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2.4.4 Viscosity experiments

Viscosity measurements were carried out on CT-DNA by varying the concentration of the

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complexes in 5mM Tris–HCl/50mM NaCl buffer (pH = 7.2), which using an Ubbelodhe viscometer maintained at a constant temperature (37.0±0.1 °C) in a thermostatic water-bath. Flow

average flow time was calculated. 2.5. DNA cleavage and mechanism studies

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time was measured with a digital stopwatch, and each sample was measured three times, and an

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The DNA cleavage experiments were done by agarose gel electrophoresis, which were performed by incubation at 37 °C as follows: pBR322 DNA (0.1 μg/ μL) in 50 mM Tris–HCl/18

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mM NaCl buffer (pH = 7.2) was treated with two complexes. The samples were incubated for 3 h,

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and loading buffer was added. Then the samples were electrophoresed for 2 h at 80 V on 0.9% agarose gel using Tris–boric acid–EDTA buffer. After electrophoresis, bands were visualized by

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UV light and photographed. The extent of cleavage of the SC DNA was determined by measuring the intensities of the bands using the Gel Documentation System. Cleavage mechanistic investigation of pBR322 DNA was carried out in the presence of

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standard radical scavengers and reaction inhibitors. The reactions were carried out by adding standard radical scavengers of DMSO, KI, NaN3, L-Histidine, SOD, EDTA, methyl green and SYBR Green to pBR322 DNA prior to the addition of complexes. Cleavage was initiated by the addition of complexes and quenched with 2 μL of loading buffer. Further analysis was carried out by the above standard method. 2.6. Protein binding studies The protein binding study was performed by fluorescence quenching experiments using bovine serum albumin stock solution (BSA, 1.5 mM) in 10 mM phosphate buffer ( pH = 7.0). A concentrated stock solution of the compounds were prepared as used for the DNA binding experiments, except that the phosphate buffer was used instead of a Tris-HCl buffer for all of the experiments. The fluorescence spectra was recorded at room temperature with excitation wavelength of BSA at 280 nm and the emission at 342 nm by keeping the concentration of BSA -7-

ACCEPTED MANUSCRIPT constant (30 μM) while varying the complexes concentration from 0 to 35 μ M. In addition, Absorption titration experiments were carried out by keeping the concentration of complexes constant (10 μ M) while varying the BSA concentration from 0 to 15 μ M.

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2.7. Cytotoxicity assays

The cytotoxicity assays of 1 and 2 were performed as follows: All cells were grown in a

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humidified incubator at 37 °C under 5% CO2. HeLa, MCF-7, and A549 were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/mL penicillin/streptomycin.

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2.7.1. MTT assay Cytotoxicity measurements:

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A density of 2 x 103 cells per well cells in 100 μL medium was seeded in 96-well microassay culture plates (Costar) and then incubated for 48 h. The cells were subsequently treated with

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different concentrations of freshly prepared complexes for 72 h. Upon completion of incubation,

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the medium was removed and 200 μL serum-free medium containing 1 mg/mL MTT was added into each well. After 4 h incubation at 37 °C, the medium was aspirated. A volume of 200 μL

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DMSO was added to dissolve the formed formazan crystals. The absorbance was read at 570 nm and 630 nm on a Biotek Microplate Reader. Experiments were carried out in triplicate, and IC50

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values were calculated from plots of cell viability against dose of compound added. 2.7.2. Cell cycle assay: HeLa cells were planted in 6-well plates (Nunc) at a density of 5 × 105 cells per well. After 24 h, cells were treated with test complexes at 40, 80, and 160 μM. After 24 h incubation, cells were harvested by trypsinization, centrifuged, resuspended in PBS (phosphate buffered saline), and fixed with 70% EtOH at 4 C overnight. The cells were subsequently centrifuged, washed with 5 mL PBS, and resuspended in 1 mL PI staining solution (0.1% Triton X-100, 200 μg/mL RNase A, and 20 μg/mL PI in PBS). The cells were incubated in the dark at 37 °C for 30 min and kept at 4 ˚C until measured. 3. Results and discussion 3.1. X-ray structure characterization -8-

ACCEPTED MANUSCRIPT The crystallographically independent monomeric unit is shown in Figure 1 (a) with the atom-numbering scheme. A view of a segment of the uniform spaced chain growing through a screw axis parallel to c is depicted in Figure 1 (b). It crystallizes in the monoclinic system, with

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space group Cc. The X-ray crystal structures shows that complex 1 forms a zigzag chain of

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polymeric [Cu(L)Cl]+ units linked by equatorial-apical chloride bridges. The coordination

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polyhedron around the copper(II) ion could be best described as square pyramidal, which is reflected by the τ [33]( τ = ( β – α ) / 60 and α and β being the two largest coordination angles) value (0.04) defined by Addison et al. (τ = 0 for an ideal square pyramid, and 1 for an ideal

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trigonal-bipyramid). The basal plane is built by three nitrogen atoms from the tridentate Schiff base ligand and one chloride. The chain is propagated through the axial chloride, which is part of

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the base of the adjacent square pyramid. The five coordinating atoms N1, N2, N3 and Cl1 are nearly coplanar, with the largest deviation from the mean plane is 0.672Å for Cl1. The basal sites

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are occupied by the N of the quinoline ring, the nitrogen of pyridine groups and chloride atoms,

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while the chloride atom of the next repeating unit occupies the apical position. As is commonly observed in square pyramidal complexes, the copper(II) ion lies at 0.249Å, above the least-squares

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plane of N1, N2, N3, and Cl1. The Cu···Cl(apical) and inter-monomer metal···metal distances are 2.594(3) and 3.7878(15) Å, respectively. The axial Cu(1)–Cl distance of 2.594(3)Å is shorter than that in a number of other

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mono-chlorido-bridged copper(II) complexes [34, 35]. In complex 1, the axial Cu–Cl bond distance, 2.594(3) Å, is longer than the equatorial Cu–Cl distance due to the square pyramidal geometry [36]. All the three N of the tridentate ligand are at almost equidistance from the central copper(II) ion. The Cu(1) - Cl(1) bond, which is 2.244 Å long, however is slightly longer than the three Cu - N bonds. The Jahn-Teller distortion in this case is observed in the Cu(1) - Cl(#) bond and as a result Cu(1) - Cl(#) bond length is 2.594 Å.

(Insert Figure 1(a), (b), and Table 2) The structure of complex 2 is depicted in Figure 1 (c) and the selected bond lengths and angles are listed in table 3. It crystallizes in the triclinic system, with space group P1¯ . Copper ion is surrounded by three nitrogen and two bromine atoms, resulting in a distorted square-pyramidal geometry, rather than a distorted trigonal-bipyramidal geometry. This is also suggested by the -9-

ACCEPTED MANUSCRIPT stuctural index τ which has been introduced by Addison to show the relative amount of trigonality in five-coordinate compounds [37]. For [CuLBr2] the value of τ equals 0.04. So in the case of 2, the Cu(II) square-pyramid is defined by the three nitrogen donors of the main ligand and two

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bromide ligands, with Br1 in the apical position and the three nitrogens of the ligand and the other bromide (Br2) in the equatorial plane. The copper(II) ion lies at 0.373(1) A, above the

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least-squares plane of N1, N2, N3, and Br2.

The tridentate ligand L utilizes all its potential N-donors for coordination. The copper-nitrogen distances are normal for this kind of compound (see Table 3). The apical bromide has a bond

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distance of 2.5667(12) Å to copper, in the usual range for square-pyramidal geometries. The equatorial bromide, with a distance of 2.3718(11) Å to copper, deforms the ideally square basal

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plane into a brolly-like conformation [38], and the angles of Br(l)-Cu(1)-Br(2) and N(2)-Cu(1)-Br(2) to respectively 105.31(4) and 155.19(12). In complex 2 all the three N of the

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tridentate ligand are at almost equidistance from the central copper(II) ion. The Cu(1) - Br(2) bond,

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which is 2.3718 Å long, however is slightly longer than the three Cu - N bonds. The Jahne-Teller distortion in this case is observed in the Cu(1) - Br(1) bond and as a result Cu(1) - Br(1) bond

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length is 2.5667 Å.

(Insert Figure 1(c) and Table 3)

3.2. Electronic absorption titration

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Electronic absorption spectroscopy is one of the most useful techniques in examining the binding mode of DNA with the metal complexes [39]. Firstly, the potential binding ability of the complexes 1 and 2 to CT-DNA was studied by UV spectroscopy. The typical titration curves for 1 and 2 in the absence and presence of CT-DNA at different concentrations are given in Figure 2. The absorption peaks at 232 nm and 369 nm for complex 1, at 230 nm and 368 nm for complex 2, are attributed to intraligand π –π * transition. With the concentration of CT-DNA being increased, hypochromismand and a small amount of red shifts are observed, which indicate partial intercalation between the complexes and DNA, because intercalation would lead to hypochromism and bathochromism in UV absorption spectra owning to a strong stacking interaction between an aromatic chromophore and the base pairs of DNA [40, 41]. These observations can be rationalized by the following reasons. When the copper(II) complexes intercalate the base pairs of CT-DNA， the π* -orbital of the intercalated ligand in the complexes can couple with the π-orbital of the base - 10 -

ACCEPTED MANUSCRIPT pairs of CT-DNA, thus decreasing the π –π * transition energy and resulting in bathochromism. Furthermore, the coupling π-orbital is partially filled by electrons, thus decreasing the transition probabilities and concomitantly resulting in hypochromism. Usually, intercalation between the metal

non-intercalative/electrostatic interaction causes hyperchromism [42].

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complexes and DNA results in hypochromism with or without red/blue shift; on the other hand,

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In order to determine the binding strength of the complexes with CT-DNA, intrinsic binding constants (Kb) and binding site (s) were calculated according to the equation [43, 44]: a

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  f /  b   f   b  b 2  2 K b Ct DNA/ s 2

b  1  K bCt  K b DNA/ 2s

1b 

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1/ 2

/ 2K C b

t

1a 

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

The Kb and s values (Kb = 3.57 × 105 M -1 , s = 0.66 for complex 1; Kb = 3.45 × 106 M -1, s =

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1.28 for complex 2) compare lower than those observed for classical intercalators ( EB: Kb = 1.4 × 106 M-1 in 25 mM Tris-HCl / 40 m M NaCl buffer) [45] but higher than those of mononuclear

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complexes [46, 47]. The low value of s ( 1, indicating that complex 2 intercalates more strongly than complex 1. A larger aromatic ring surface area of quinoline rings leads to the intercalative interaction of the complexes with DNA. For complex 2, two terminal ligands Br- are all dissociated from copper center which

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lead to the smaller steric hindrance. Compared with 1 of [CuLCl]+, the actual effects of steric hindrance of 2 are smaller than 1 in aqueous solution, which lead to 2 has higher DNA binding affinity.

(Insert Figure 2) 3.3. Competitive Binding Experiments There is no luminescence observed for complex 1 and 2 at room temperature in aqueous solution. In order to further clarifying the binding of these complexes with DNA, the competitive binding experiments were carried out on EB-CT-DNA by varying the concentration of the complexes. As a sensitive fluorescence probes, EB is a planar cationic dye emits intense fluorescence at about 600 nm in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs [49]. The enhanced fluorescence can be quenched upon the addition of the second molecule which could replace the bound EB or break the secondary structure of the - 11 -

ACCEPTED MANUSCRIPT DNA [13]. The interaction of complex 1 and 2 with CT-DNA were studied with an EB-bound CT-DNA solution in 5 mM Tris–HCl/NaCl buffer (pH=7.2). Fluorescence intensities at 602 nm (510 nm excitation) were measured at different complex concentrations which suggested that

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complexes displace DNA-bound EB and bind to DNA in the binding sites available for EB (Figure

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3). According to the Stern-Volmer equation [50], I 0 / I  1  K Q  the relative binding propensity of the complex to CT-DNA was determined from the slope of straight line obtained from the plot of the fluorescence intensity versus the complex concentration. The fluorescence quenching curve

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of EB-bound CT-DNA by complexes showed that the quenching plot illustrates that the quenching of EB bound to CT-DNA by complex is in good agreement with the classical Stern–Volmer equation. From the equation KEB [EB] = Kapp [complex] where the complex concentration was the

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value at a 50% reduction of the fluorescence intensity of EB and KEB=1.0 × 107 M−1, ([EB] =2.4 μM). The calculated apparent binding constant values at room temperature (Kapp) for complexes -1

for complex 1; 3.46 × 105 M-1 complex 2，the binding

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were calculated to be 3.16 ×105 M

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constants of the complexes with DNA are more or less equal to the binding strength of the previous reported mononuclear copper complexes [51-53], but are two orders of magnitude less

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than that of classical intercalator ethidium bromide (EB) [54], so the binding mode between the complexes and DNA were non-classical intercalative. (Insert Figure 3)

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Furthermore, we carried out a similar competition experiment using Hoechst 33258(a minor groove binder). This dye binds to DNA in two concentration dependent ways [55]. The first type of binding occurs in the minor groove at low dye-to DNA ratios [56, 57]. The fluorescence yield of Hoechst 33258 increases significantly in presence of DNA. The displacement of bound Hoechst 33258 from its binding site on CT-DNA is implicated from a decrease in its fluorescence intensity on addition of the complexes.When the complexes are added to Hoechst-DNA solution we observed a decrease (∼ 46% for 1 and ∼ 59% for 2, respectively) in the fluorescence (Figure 4). This suggests that especially complex 1 and 2 are capable of binding in the minor groove of DNA. (Insert Figure 4) 3.4. Circular dichronism studies CD spectroscopy is sensitive in monitoring the conformation changes of the helix in solution

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ACCEPTED MANUSCRIPT and provides detailed information about the binding of the small molecules with DNA. The CD spectra of CT-DNA exhibits a positive band at 275 nm due to base stacking and a negative band at 245 nm due to the helicity of B-type DNA [58]. When treated with complex 1 and 2, the decrease

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of intensity in both the positive and negative bands of DNA was observed, which showed a clear indication of the interactions between the complexes and DNA (Figure 5). The decreased intensity

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in the negative band suggests the complex can unwind the DNA helix and lead to loss of helicity [59].

(Insert Figure 5)

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3.5. Viscosity activities

Viscosity measurements were carried out to further verify the interaction of the complexes

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with CT-DNA. In classical intercalation, the complexes result in a lengthening and stiffening of the double helix of DNA, leading to an increase in the viscosity of DNA[60]. In contrast, a partial

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and/or non-intercalation of the ligand could result in less pronounced effect on the viscosity[61].

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The plots of relative specific viscosity (η/η0)1/3 versus [complex]/[DNA] ratio for 1 and 2 are given in Figure 6. It shows that the relative viscosity of CT-DNA was increase with increasing the

intercalation.

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concentration of the complexes. The result suggests that the complexes could bind to CT-DNA by

(Insert Figure 6)

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3.6. The cleavage of pBR322 DNA The DNA cleavage activities of complex 1 and 2 have been studied using supercoiled pBR322 plasmid DNA as a substrate in a medium of 50 mM Tris–HCl/NaCl buffer (pH = 7.2) in the absence of external agents under physiological conditions for 3h. When the original supercoiled form (Form I) of plasmid DNA is nicked, an open circular relaxed form (Form II) will exist in the system and the linear form (Form III) can be found upon further cleavage [62]. When conducted by electrophoresis, the compact Form I migrates relatively faster while the nicked Form II migrates slowly, and the linearized form (Form III) migrates between Forms I and II. Firstly, the concentration-dependent DNA cleavage by complexes 1 and 2 without any external additives in natural light were performed. The results of gel electrophoretic separations of plasmid pBR322 DNA induced by increasing concentration was shown in Figure 7. With increase of concentrations of the complexes, Form I plasmid DNA is gradually converted into Form II, - 13 -

ACCEPTED MANUSCRIPT complex 2 shows more obvious nuclease activity than complex 1. At 200 μM, complex 2 cleaves about 87% of the plasmid to yield the nicked circular form, whereas complex 1 cause less than 60% of the plasmid to yield the nicked circular form(shown in Figure 8 and Figure 9). This result

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indicates that the copper complexes can efficiently cleave plasmid DNA. (insert Figure 7, Figure 8 and Figure 9)

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Furthermore, the time-dependent cleavage of DNA by complexes 1 and 2 was also studied under similar conditions. With increased reaction time, the amounts of forms II increased and form I gradually decreased. The results show that cleavage of DNA by the two complexes is dependent

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on reaction time (shown in Figure 10).

(insert Figure 10)

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3.7. DNA cleavage mechanism

In order to obtain a good knowledge of the active chemical species that were responsible for

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the DNA damage activities by complexes, we further investigated the influence of different

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potentially inhibiting agents including hydroxyl radical scavenger (DMSO), singlet oxygen quenchers (NaN3 , L -histidine), superoxide scavenger (SOD, superoxide dismutase), hydrogen

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peroxide scavenger (KI) and chelating agent (EDTA) under our experimental conditions. No obvious inhibitions are observed for complexes in the presence of SOD (lane 4) in Figure 11. These results rule out the possibility of DNA cleavage by superoxide. The addition of NaN3 (Lane

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2), L –histidine (Lane 3), DMSO (Lane 5), KI (Lane 6) diminishes significantly the nuclease activity of the compounds which is indicative of the involvement of the singlet oxygen a (singlet oxygen-like) or hydroxyl radical in the cleavage process. The chelating agent EDTA can efficiently inhibit DNA cleavage (lane 7), indicating Cu II complex play the key role in the DNA breakage. In order to assure the sites of interaction between the complexes and DNA, we added the SYBR Green and methyl green, which are known to interact to DNA at minor and major grooves. The addition of SYBR Green could inhibit DNA cleavage while methyl green does not, the result suggests that the complexes preferred to bind to DNA minor groove. (insert Figure 11) 3.8. Protein binding studies Qualitative analysis of the binding of chemical compounds to BSA is usually detected by inspecting the fluorescence spectra. Generally, the fluorescence of BSA is caused by two intrinsic - 14 -

ACCEPTED MANUSCRIPT characteristics of the protein, namely tryptophan and tyrosine. Changes in the emission spectra of tryptophan are common in response to protein conformational transitions, subunit associations, substrate binding, or denaturation [63]. Therefore, the intrinsic fluorescence of BSA can provide

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considerable information on their structure and dynamics and is often utilized in the study of protein folding and association reactions. The interaction of BSA with our compounds was studied

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by fluorescence measurementat room temperature. A solution of BSA (30 μM) was titrated with various concentrations of the compound (0 −35μM). Fluorescence spectra were recorded in the range of 290−450 nm upon excitation at 280 nm. The effects of the compound on the the

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fluorescence emission spectrum of BSA are shown in Figure 12.

The fluorescence quenching is described by the Stern–Volmer equation and the quenching

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rate constant Kq was calculated with the plot of I0 / I versus [Q]. Quenching can occur by different mechanisms, which are usually classified as dynamic quenching and static quenching; dynamic

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quenching refers to a process in which the fluorophore and the quencher come into contact during

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the transient existence of the excited state. Static quenching refers to fluorophore-quencher complex formation in the ground state. A simple method to explore the type of quenching is

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UV−visible absorption spectroscopy. UV-visible (UV-Vis) spectra of BSA in the absence and presence of the compounds (Figure 12) show that the absorption intensity of BSA was enhanced as the compounds were added, and there was a little blue shift. It revealed that there exists a static

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interaction between BSA and the added compounds due to the formation of the ground state complex of the type of BSA-compound reported earlier [64]. (insert Figure 12, Figure 13 )

If it is assumed that the binding of compounds with BSA occurs at equilibrium, the equilibrium binding constant can be analyzed according to the Scatchard equation[63]:

logI 0  I  / I   log K bin  n logQ  ，where Kbin is the binding constant of the compound with DNA and n is the number of binding sites (shown Figure 14 and table 4). The calculated value of n is around 1 for all of the compounds, indicating the existence of just a single binding site in BSA for all of the compounds [63]. (insert Figure 14) 3.9. Antitumor activity

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ACCEPTED MANUSCRIPT 3.9.1. MTT assay The in-vitro cytotoxicity of the complexes has been evaluated against a 3-cell line panel consisting of HeLa (human cervical carcinoma), MCF -7 (human breast adenocarcinoma), and

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A549 (lung adenocarcinoma carcinoma), respectively. The cells have been exposed to different concentrations of complexes 1 and 2 for 72 h and the growth inhibitions of the cells have been

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determined. The IC50 values also indicate that complex 1 and 2 has the highest cytotoxicity in all three cell lines (Table 5). Comparing to the previous reported the IC50 values for Copper(II) complexes, 1 and 2 are less one order of magnitude as reported by [Cu(tpy)(Cl)2], et al. (37.5 ±

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3.0 μM, 40.4±2.0 μM and 41.2±3.0 μM for A549) [63]. For the MCF-7 cell lines, with IC50 value of 0.46 μ M are much less than cisplatin (IC50=3.92 μ M) and carboplatin (IC50=36.65 μ M)

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[66]. It is well known that the quinoline ring system and analogues are important structural units widely existing in alkaloids which are very potent cytotoxin with IC50 in the micromole range

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[67-69]. All of these suggest that our complexes have enormous potential to act as anticancer

3.10. Cell cycle analysis

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agents.

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We further investigated the effects of complexes 1 and 2 on cell cycle distribution. HeLa cells were treated with various concentrations of these complexes for 24 h prior to the cells being stained with propidium iodide and analyzed for their DNA content by flow cytometry. Compounds

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induced cell cycle arrest in a concentration dependent manner as shown in Figure 15, Table 6 and 7. The results indicated that both complex 1 and 2 induced S phase arrest. A moderate increase of S phase and decrease of G0-G1 phase were observed at a concentration of 1.2 µM complex 1 and 2. There are also an increased numbers of cells at G2-M phase after treatment of complexes. Cell cycle arrest of quinoline complex treatment have been reported previously, which showed the ability to inhibit the cell cycle in G2/M phase [70]. Complexes 1 and 2 induced cell cycle arrest at the same manner may indicate the same mechanism of behavior of those complexes in vitro. (insert Figure 15) 4. Conclusions In conclusion, two copper(II) complexes have been synthesized and structurally characterized. Complex 1 shows a zigzag chain structure, while complex 2 exists as a monoclear species showing a slightly distorted square-pyramidal geometry around Cu. The DNA binding properties - 16 -

ACCEPTED MANUSCRIPT have been investigated by electronic absorption titration, ethidium bromide-DNA displacement experiments, Hoechst 33258 competiting experiments and circular dichronism studies. The results suggest complex 1 and 2 bind to DNA through intercalation modes. They cleave plasmid pBR322

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DNA without addition of any external additives by an oxidative mechanism. They bind to bovine sermalbumin and are responsible for quenching of BSA fluorescence by dynamic quenching

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mechanism. In addition, the two complexes have been found to exhibit in vitro cytotoxicity towards HeLa, MCF-7, and A549 cells. The IC50 values of the complexes have been determined by MTT assay. As a result of Cell cycle analysis experiments, we find that the two complexes may

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intercalate DNA, induces cell cycle arrest at S phase against HeLa cell lines, which is probably same to that of cisplatin.

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5. Abbreviations: EB

Ethidium bromide Calf thymus DNA

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CT-DNA

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TBE SOD

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PBS ESI-MS

HSA FBS

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BSA

Tris-boracic-EDTA Superoxide dismutase Phosphate buffered saline Electrospray ionization massspectrometry bovine serum albumin human serum albumin Fetal bovine serum

Acknowledgements This work was supported by the National Natural Science Foundation of China (21171101, 21001066 and 20771062) and Tianjin Science Foundation (No. 12JCYBJC13600).

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Wilson, W. A. Denny, J. Med. Chem. 52 (2009) 6822-6834. [68] C.-H. Tseng, Y.-L. Chen, K.-Y. Chung, C.-M. Cheng, C.-H. Wang, C.-C. Tzeng, Bioorg. Med. Chem. 17 (2009) 7465-7476. [69] C.-M. Lu, Y.-L. Chen, H.-L. Chen, C.-A. Chen, P.-J. Lu, C.-N. Yang, C.-C. Tzeng, Bioorg. Med. Chem. 18 (2010) 1948-1957. [70] C.-H. Tseng, Y.-L. Chen, C.-L. Yang, C.-M. Cheng, C.-H. Han, C.-C. Tzeng, Bioorg. Med. Chem. 20 (2012) 4397-4404.

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Table 1 Crystallographic data for complexes 1 and 2 Complex 1

Complex 2

Chemical formula Formula Mass Crystal system a/Å b/Å c/Å α/° β/° γ/° Unit cell volume/Å3 Temperature/K Space group No. of formula units per unit cell, Z No. of reflections measured No. of independent reflections Rint Final R1 values (I > 2σ(I)) Final wR(F2) values (I > 2σ(I)) Final R1 values (all data) Final wR(F2) values (all data)

C15H10Cl2CuN3O4 430.70 Monoclinic 10.495(2) 24.121(5) 7.4342(15) 90.00 123.29(3) 90.00 1573.2(5) 293(2) Cc 4 4449 2197 0.0630 0.0668 0.1608 0.0801 0.1716

C15H10Br2CuN3 455.62 Triclinic 7.1548(14) 8.3552(17) 12.514(3) 97.64(3) 98.94(3) 92.87(3) 730.5(3) 293(2) P1¯ 2 6102 2563 0.0434 0.0456 0.0726 0.0695 0.0775

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Table 2 Selected bond lengths (Å) and angles (°) for complex 1 Bond distances (Å) Cu(1)-N(1) Cu(1)-Cl(1)

82.0(3) 161.5(4) 97.4(3) 100.4(2) 95.4(3)

N(2)-Cu(1)-N(3) N(2)-Cu(1)-Cl(1) N(3)-Cu(1)-Cl(1) N(1)-Cu(1)-Cl(1)#1 Cl(1)-Cu(1)-Cl(1)#1

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Bond angles (deg) N(2)-Cu(1)-N(1) N(1)-Cu(1)-N(3) N(1)-Cu(1)-Cl(1) N(2)-Cu(1)-Cl(1)#1 N(3)-Cu(1)-Cl(1)#1

1.989(8) 2.244(3)

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1.959(9) 2.016(8) 2.594(3)

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Cu(1)-N(2) Cu(1)-N(3) Cu(1)-Cl(1)#1

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Symmetry transformations used to generate equivalent atoms: # x, -y, 0.5+z

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79.8(4) 159.0(2) 98.5(3) 90.9(2) 100.60(11)

ACCEPTED MANUSCRIPT Table 3 Selected bond lengths (Å) and angles (°) for complex 2 Bond distances (Å) Cu(1)-N(3) Cu(1)-Br(2)

78.90(17) 157.51(17) 97.70(13) 99.51(12) 95.03(13)

N(2)-Cu(1)-N(1) N(2)-Cu(1)-Br(2) N(1)-Cu(1)-Br(2) N(3)-Cu(1)-Br(1) Br(2)-Cu(1)-Br(1)

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Bond angles (deg) N(2)-Cu(1)-N(3) N(3)-Cu(1)-N(1) N(3)-Cu(1)-Br(2) N(2)-Cu(1)-Br(1) N(1)-Cu(1)-Br(1)

2.023(4) 2.3718(11)

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1.974(4) 2.023(4) 2.5667(12)

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Cu(1)-N(2) Cu(1)-N(1) Cu(1)-Br(1)

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80.34(17) 155.19(12) 97.51(13) 96.81(13) 105.31(4)

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Table 4 The quenching constant, binding constant and number of binding sites for the interactions of complex 1 and 2 with BSA

4

4

4.2*10 3.9*104

3.4*10 8.1*104

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Kbin (M-1)

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Kq (M-1)

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n 0.98 1.07

ACCEPTED MANUSCRIPT Table 5 IC50values (μM) of complexes 1 and 2 with different cell lines HeLa

MCF -7

A549

1 2 Cisplatin carboplatin

1.85±0.14 1.72±0.14 3.8 ---

0.46±0.03 0.46±0.04 3.92±0.56 36.65±6.56

0.90±0.04 0.43±0.06 4.13±0.35 ---

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ACCEPTED MANUSCRIPT Table 6 Cell cycle distribution of cells after treatment for 24 h with various doses of complex 1 Distribution (% HeLa cells) G0-G1

S

G2-M

0 (control) 0.4 0.8 1.2

70.2 74.84 57.99 42.63

19.6 17.51 30.31 39.26

10.13 7.64 11.7 18.11

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Treatment ( μ M)

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ACCEPTED MANUSCRIPT Table 7 Cell cycle distribution of cells after treatment for 24 h with various doses of complex 2 Distribution (% HeLa cells) G0-G1

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G2-M

0 (control) 0.4 0.8 1.2

70.2 75.35 65.4 45.03

19.6 16.72 24.29 38.58

10.13 7.93 10.3 16.4

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Treatment ( μ M)

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Fig. 1 Perspective view of the complex 1 (a) (b) and 2 (c). Hydrogen atoms and dissociative small molecules are omitted for clarity.

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Fig. 2 Absorption spectra of complexes (25 μM) 1 (A) and 2 (B) in the absence (dashed line) and presence (solid line) of increasing amounts of CT-DNA at room temperature in 5mM

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Tris–HCl/50mM NaCl buffer (pH = 7.2). The arrow shows the absorbance changes on increasing

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CT-DNA concentration (a). The inset shows the plot of (εa−εf) /(εb−εf) vs [DNA], obtained from

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absorption spectral titration of the complexes (b). Fig. 3 Effect of addition complexes 1 (A) and 2 (B), on the emission intensity of ethidium

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bromide bound to DNA(2.4μM) at different concentrations in a 5mM Tris–HCl/50mM NaCl buffer (pH = 7.2), inset: Plot of I0/I vs. [complex]. Fig. 4 Fluorescence spectra of the Hoechst-bound ct-DNA in aqueous buffer in the absence and presence of increasing amounts of complex 1 (A) and 2 (B). λex = 338 nm, [Hoechst] = 2 μM, [DNA] = 20 μ M, [complexes] (μM): 0− 30 in 12.5 μ M increments. T = 298 K. Fig. 5 CD spectra of CT-DNA in the buffer solution (Tris-HCl) at 0.6 mM in the absence (Black dashed line) and presence of 0.3 mM complex 1 and 0.3 mM complex 2 Fig. 6 Effect of increasing amounts of the complexes 1 and 2 on the relative viscosity of CT- DNA at 37 (± 0.1) °C in 5mM Tris–HCl/50mM NaCl buffer (pH = 7.2, [DNA] = 0.1 mM).

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ACCEPTED MANUSCRIPT Fig. 7 Agarose (1%) gel electrophoresis showing concentration-dependent cleavage of pBR322 DNA (200ng) by complexes 1 and 2 in different condition for an incubation time of 3.0 h at 37℃. a)

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Lane 0: DNA control; lanes 1–5: DNA+ 0.025, 0.1, 0.2, 0.25, 0.4 mM of 1 .b) Lane 0: DNA only;

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lanes 1–5: DNA+ 0.025, 0.10, 0.2, 0.25, 0.4 mM of 2. Fig. 8 The histogram gives relative amounts for complex 1

Fig. 9 The histogram gives relative amounts for complex 2.

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Fig. 10 Agarose (1%) gel electrophoresis showing time-dependent cleavage of pBR322 DNA

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(200ng) by complexes 1 and 2 in different condition at 37℃. a) Lane 0: DNA control; lanes 1–5: DNA+ 0.25 mM of 1 for 0, 15, 45, 75, 135, 180 min. b) Lane 0: DNA only; lanes 1–5: DNA+ 0.20

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Fig. 11 Agarose (1%) gel electrophoresis showing cleavage of pBR322 DNA (200ng) by

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complexes 1 and 2 in different condition for an incubation time of 3.0 h at 37℃. a) lane 0, DNA control; lane 1, DNA + Complex 1 (0.25 mM); lane 2, DNA + Complex 1 (0.25 mM) + NaN3 (20

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mM); lane 3, DNA + Complex 1 (0.25 mM) + L-histidine (20 mM); lane 4, DNA + Complex 1 (0.25 mM) + SOD (20 U/ml); lane 5, DNA + Complex 1 (0.25 mM) + DMSO (1 mM); lane 6, DNA + Complex 1 (0.25 mM) + KI (1 mM); lane 7, DNA + Complex 1 (0.25 mM) + EDTA (10 mM); lane 8, DNA + Complex 1 (0.25 mM) +Methyl Green; lane 9, DNA + Complex 1 (0.25 mM) + SYBR; b) lane 0, DNA control; lane 1, DNA + Complex 2 (0.2 mM); lane 2, DNA + Complex 2 (0.2 mM) + NaN3 (20 mM); lane 3, DNA + Complex 2 (0.2 mM) + L-histidine (20 mM); lane 4, DNA + Complex 2 (0.2 mM) + SOD (20 U/ml); lane 5, DNA + Complex 2 (0.2 mM) + DMSO (1 mM); lane 6, DNA + Complex 2 (0.2 mM) + KI (1 mM); lane 7, DNA + Complex 2 (0.2 mM) + EDTA (10 mM); lane 8, DNA + Complex 2 (0.2 mM) +Methyl Green; lane 9, DNA + Complex 2

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ACCEPTED MANUSCRIPT (0.2 mM) + SYBR; Fig. 12 The emission spectrum of BSA (30 μM; λexi = 280 nm; λemi = 345 nm) in the presence of

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increasing amounts of compounds 1 (A) and 2 (B). The dash line shows the intensity in the

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absence of complexes. The arrow shows the fluorescence quenching upon increasing the concentrations of the compound (a). The inset shows the Stern−Volmer plots (b) and Scatchard plots (c) of the complex with BSA.

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Fig. 13 Absorption spectra of BSA (15 μM) in the buffer solution (Tris-HCl) in the absence and

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Fig. 14 Plot of log (F0− F)/ F vs.log[ Q ] for BSA in the presence of complex 1 and 2

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Fig. 15 cell cycle phase distribution of untreated cells and cells treated with complex 1 (a) and 2

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Graphical abstract

The complexes exhibit efficient DNA cleavage activity without any

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external agents. They could quench the intrinsic fluorescence of BSA in a

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ACCEPTED MANUSCRIPT Highlights Two water-soluble copper(II) complexes were successfully synthesized. Complexes 1 and 2 bind to CT-DNA with an intercalative mode.

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Complexes 1 and 2 could quench the fluorescence of BSA in a static quenching process. Complexes 1 and 2 are more potential anticancer drug candidate with low IC50 values.

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Complexes 1 and 2 could delay or inhibit cell cycle progression through the S phase.

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