Effect of structure and composition of nickel(II) complexes with salicylidene Schiff base ligands on their DNA/protein interaction and cytotoxicity Peng li, MeiJu Niu, Min Hong, Shuang Cheng, JianMin Dou PII: DOI: Reference:

S0162-0134(14)00110-X doi: 10.1016/j.jinorgbio.2014.04.005 JIB 9505

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

14 January 2014 2 April 2014 2 April 2014

Please cite this article as: Peng li, MeiJu Niu, Min Hong, Shuang Cheng, JianMin Dou, Effect of structure and composition of nickel(II) complexes with salicylidene Schiff base ligands on their DNA/protein interaction and cytotoxicity, Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.04.005

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ACCEPTED MANUSCRIPT Effect of structure and composition of nickel(II) complexes

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with salicylidene Schiff base ligands on their DNA/protein

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interaction and cytotoxicity

ABSTRACT

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Peng li,a MeiJu Niu,  a Min Hong,a Shuang Chengb and JianMin Doua

Three new salicylidene Schiff base nickel(II) complexes [Ni(L1)(CH3COOH)2]2 (1),

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[Ni2(L1)2(CH3OH)] (2), [Ni(L2)2]·3H2O (3) {H2L1 =N,N’-bis(salicylidene)-3,6-dioxa-1,8-diaminooctane, HL2=2ethyl-2- (2-hydroxybenzylideneamino)propane-1,3-diol} were synthesized and characterized fully by structural, analytical, and spectral methods. The single-crystal X-ray structures of complexes 1 and 2 exhibit the symmetrical

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ligands coordinated to the nickel(II) ion in a tetradentate fashion via ONNO donor atoms, while the unsymmetrical ligand L2 presented a ONO tridentate coordination mode in complex 3. The nickel(II) ions lie in the six-

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coordinated octahedral environment for the mononuclear complexes 1 and 3, along with dinuclear complex 2. The interaction of the complexes with calf thymus DNA (CT-DNA) has been explored by absorption and emission

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titration methods, which revealed that complexes 1-3 could interact with CT-DNA through intercalation. The interactions of the complexes with bovine serum albumin (BSA) were also investigated using UV-Vis, fluorescence and synchronous fluorescence spectroscopic methods. The results indicated that all of the complexes could quench the intrinsic fluorescence of BSA in a static quenching process. Further, the in vitro cytotoxic effect of the complexes examined on cancerous cell lines such as human lung carcinoma cell line (A549), human colon carcinoma cell lines (HCT-116), human promyelocytic leukemia cells (HL-60) and colonic cancer cell line Caco-2 showed that all three complexes exhibited substantial cytotoxic activity.

Keywords: Schiff base, vitro cytotoxic, crystal structure, DNA-binding, BSA-binding

1. Introduction

 Corresponding author Tel: +866358230615; Fax: +866358239121. E-mail address: [email protected] (Prof. Niu) a

Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell Technology, School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, 252059, P. R. China. b School of Agriculture, Liaocheng University, Liaocheng, 252059, P. R. China.

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ACCEPTED MANUSCRIPT Nickel(II) complexes of a Schiff base ligand containing mixed donors have attracted much attention because of their fascinating structural diversities shown in the metal-organic frameworks and their wide applications in catalysis [1], functional materials [2], biological activities [3]etc. Also nickel is an essential element of life by

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being present in a series of enzymes [4]. On the other hand, nickel(II) complexes are regarded as one of the most promising alternatives to the traditional cisplatin as anticancer drugs; an idea supported by a considerable number

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of research articles describing the synthesis, DNA binding, and cytotoxic activities of numerous nickel(II)

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complexes [5-7]. However the exact mechanism of antitumor activities has not been fully elucidated. In general, anticancer agents that are approved for clinical use are molecules which damage DNA, block DNA synthesis indirectly through inhibition of nucleic acid precursor biosynthesis, or disrupt hormonal stimulation of cell growth [8,9]. Therefore, considerable attempts are being made to research the interaction of nickel(II) complexes with

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DNA and their cytotoxic activities [10-13].

On the other hand, drug interactions at the protein binding level significantly affect the apparent distribution volume and their elimination rate. Therefore, the interactions of metal complexes with serum albumins have

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received much attention in the scientific community by studying antitumoral metallopharmaceutical pharmacokinetics and structure−activity relationships [14]. Although studies have been carried out to understand

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the interaction of metal complexes, metal ions and Schiff base ligands with albumins, corresponding investigations on nickel(II) Schiff base complexes seems to be limited [10,15-17]. BSA is the most extensively studied serum

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albumin, due to its structural homology with human serum albumin (HSA). This fact further supports the value of studying the interaction behavior of the metal complexes with BSA, while evaluating their anticancer properties. Based on the above facts and considering the role and activity of nickel and its complexes in biological systems, along with the significance of Schiff base in medicine, we present in this work a systematic study on the synthesis and molecular structure of nickel(II) complexes containing salicylidene Schiff base ligands and their interaction with DNA and proteins, along with cytotoxicity. Further, the effect of the substituent group present on the ligand as well as the structure of the nickel(II) complexes on the above said properties were also studied in detail. The synthetic routes of the ligands and the Ni(II) complexes are shown in Scheme 1.

2. Experimental 2.1 Materials and physical measurements 3,6-Dioxa-1,8-diaminooctane,2-amino-2-ethyl-1,3-propanediol and salicylaldehyde were purchased from aladdin-reagent. The ligands H2L1 and HL2 were prepared according to the references [18,19].

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ACCEPTED MANUSCRIPT [Ni(CH3COO)2]·4H2O, NiCl2·6H2O, calf thymus DNA, BSA, and ethidium bromide (EB) were obtained from Sigma-Aldrich and used as received. All starting precursors were of analytical grade, and double-distilled water was used throughout the experiments. Elemental analyses (C, H, N) were performed on Perkin-Elmer 2400 II

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analyzer. Electrospray ionization mass spectroscopic (ESI-MS) analyses are performed with a Bruker microTOFQ mass spectrometer (Bruker Daltonics Inc., Billerica, MA), and the mass spectra are obtained in the positive

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mode. IR spectra (4000−400 cm−1) were recorded on a Nicolet Avatar Model FT-IR spectrophotometer. Melting

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points (M.p.) of the complexes were determined with a Lab India instrument. Electronic absorption spectra were recorded using a HP-8453A diode array spectrophotometer. Emission spectra were measured with LS55 spectrofluorometer. Circular dichroism (CD) spectra measurements were measured on a Jasco J-810 spectropolarimeter.

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[Insert Scheme 1] 2.2 X-Ray crystallography

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Diffraction data for the title complexes were obtained on a Bruker Smart 1000 CCD diffractometer (graphite monochromized Mo Kα radiation, λ = 0.71073 Å) and collected by the

-2θ scan technique at 298(2) K. A

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semiempirical absorption correction was applied to the data. The structure was solved by direct methods using SHELXS-97 and refined against F2 by full-matrix least squares using SHELXL-97. Hydrogen atoms were placed

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in calculated positions. Crystal data and experimental details of the structure determinations are listed in Table S1. 2.3 Synthesis

2.3.1 Synthesis of [Ni(L1)(CH3COOH)2]2 (1) The methanol solution (15 ml) of [Ni(CH3COO)2]·4H2O (0.1245 g, 0.5 mmol) was dropwise added to a stirring methylene chloride solution (15 mL) containing H2L1 (0.1782 g, 0.5 mmol). The mixture was stirred for 5 h at room temperature and then filtered. Slow evaporation of complex 1 in the filtration afforded single crystals suitable for X-ray diffraction studies. Yield: 74%. M.p.: 132 ℃. Anal. Calc. (%) for C24H30N2NiO8 (Mr = 533.21): C 54.06; H 5.67; N 5.25. Found: C 53.88; H 5.49; N 5.41. ESI-MS, m/z: 413.42 [L1 + Ni + H]+, 435.50 [L1 + Ni + Na]+, 825.25 [2L1 + 2Ni + H]+, 847.33 [2L1 + 2Ni + Na]+. Selected IR (KBr pellet, cm-1): 3438 (s, O-H), 1614 (m, C=N), 567 (m, Ni-N), 447 (m, Ni-O). UV-visible (UV-Vis) (CH3OH), λmax/nm: 268, 356. 2.3.2 Synthesis of [Ni2(L1)2(CH3OH)] (2) Complex 2 was prepared by a procedure similar to that described above using NiCl2·6H2O (0.1689 g, 0.5 mmol) and ligand H2L1 (0.1782 g, 0.5 mmol). The slow evaporation of complex 2 in the filtration afforded green crystals suitable for X-ray diffraction studies. Yield: 68%. M.p.: 122 ℃.

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ACCEPTED MANUSCRIPT Anal. Calc. (%) for C41H48N4Ni2O9 (Mr = 858.25): C, 57.37; H, 5.64; N, 6.53. Found: C, 57.34; H, 5.59; N, 6.52. ESI-MS, m/z: 413.33 [L1 + Ni + H]+, 435.33 [L1 + Ni + Na]+, 825.17 [2L1 + 2Ni + H]+, 847.08 [2L1 + 2Ni + Na]+. Selected IR (KBr pellet, cm-1): 3471 (s, O-H), 1612 (m, C=N), 617 (m, Ni-N), 431 (m, Ni-O). UV-Vis (CH3OH),

2.3.3 Synthesis of [Ni(L2)2]·3CH3OH (3)

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λmax/nm: 266, 358.

Complex 3 was prepared by stirring equimolar quantities of

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[Ni(CH3COO)2]·4H2O (0.1241 g, 0.5 mmol) and the ligand HL2 (0.1116 g, 0.5 mmol) in 30 mL of

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CH3OH/CH2Cl2 solution. After a few minutes of mixing of the above reactants, 0.5 mL tetraethyl ammonium hydroxide (TEAOH) aqueous solution (20%) was added to the reaction mixture and continuously stirred for 5 h at room temperature, and then the resulting product was filtered off. The green crystals for X-ray diffraction studies were obtained at room temperature by the slow evaporation of the filtration. Yield: 86%. M.p.: 181 ℃. Anal. Calc.

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(%) for C27H44N2NiO9 (Mr = 599.35): C 54.11; H 7.40; N 4.67. Found: C 54.09; H 7.35; N 4.69. ESI-MS, m/z: 503.17 [2L2 + Ni + H]+. Selected IR (KBr pellet, cm-1): 3394 (s, O-H), 1651 (m, C=N), 533 (m, Ni-N), 437 (m, NiUV-Vis (CH3OH), λmax/nm: 266, 360.

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O).

2.4 DNA binding studies

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The DNA-binding experiment was carried out in the buffer solution of 10 mM Tris–HCl, 10 mM NaCl, pH = 7.2. For the fluorescence quenching experiments, the EB solution was added to the prepared buffer solution of CT-

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DNA for 2h, and then added to the methanol solution of nickel(II) complex from 0 to 120 μM. The sample was excited at 258 nm and emission spectra were recorded at 500-700 nm. The spectrum was recorded at the scan speed of 150 nm/min with excitation/emission slit width 10.0/10.0 nm. UV-Vis absorbance was performed by keeping the concentration of the nickel(II) complex (10 μM) constant while varying the CT-DNA concentrations from 0 to 10 μM. The sample solution was scanned in the range of 200–500 nm. For the CD spectra of CT-DNA was carried out in the absence and presence of the nickel(II) complex at the room temperature with a quartz cell of 1 cm path length. Each sample solution was scanned in the range of 220–320 nm with a scan speed of 100 nm/min and 1 s response time. Each spectrum was the average of three accumulations from which the buffer background had been subtracted. 2.5 Protein binding studies The BSA-binding experiments with nickel(II) complexes were studied from the fluorescence spectra in 10mM Tris–HCl, 10mM NaCl, pH 7.2 buffer solution recorded with an excitation at 280 nm and corresponding emission at 346 nm. Fluorescence spectra were measured at a scan speed of 150 nm/min and slit width of 5 nm both the

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ACCEPTED MANUSCRIPT excitation and emission monochromators. For synchronous fluorescence spectra also, the same concentration of BSA and the complexes were used, and the spectra were measured at two different Δλ values (difference between the excitation and emission wavelengths of BSA), such as 15 and 60 nm. In the measurement of UV spectra, the

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concentration of BSA was kept at 5×10-7 M and the complex was kept at 1.0×10-6 M.

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2.6 Cytotoxicity

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In vitro cytotoxicities of the complexes were studied using standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium Bromide) assay bioassay in different cancer cells at 24 h of drug administration. The test complexes were prepared to the experiment by dissolving in 0.1% DMSO and diluted with medium. Cell lines of human lung carcinoma cell line (A549), human colon carcinoma cell lines (HCT-116), human promyelocytic leukemia cells (HL-60) and colonic cancer cell line (Caco-2) were cultured in 96-well culture plate in RPMI-1640

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medium containing 10% FBS and 1% antibiotics, maintain culture at 37 ℃, 5% CO2 and 95% air in the CO2 incubator for 24 h. Various concentrations of prepared complexes were added to the cells and incubation were

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continued for 24 h. Then the media was removed, MTT was dissolved in medium and added to each well, and incubated for another 4 h. The purple formazan crystals were solubilized by the addition of 100 L DMSO. A

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reading was taken on a plate reader, and the absorbance was measured at 570 nm by the ELISA reader after the plate was shaken for 5 min. The values are the averages from at least three independent experiments, which were

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measured as the percentage ratio of the absorbance of the treated cells to the untreated controls. The IC50 values were determined by non-linear regression analysis.

3. Results and discussion

3.1 Syntheses of nickel complexes In this work, we have prepared two different nickel(II) complexes with ligand H2L1 by the reaction of the free ligand and starting nickel(II) salt, [Ni(CH3COO)2]·4H2O or NiCl2·6H2O. X-ray crystal structure determination reveals that complex 1 contains a [Ni(L1)] part and two acetic acid molecules which were formed by the protonation of CH3COO-, while 2 presents a binuclear complex composed of a bridged CH3OH and a [Ni2(L1)2] part. Although complex 3 was prepared by stirring reaction mixtures containing a 1 : 1 ratio of [Ni(CH3COO)2]· 4H2O and ligand HL2 in methanol-dichloromethane solution, for the best coordination mode (six-coordinated nickel center) complex 3 constructed with a L2-Ni-L2 chelation fashion. The positive ion ESI-MS of the mononuclear nickel(II) complex [Ni(L1)(CH3COOH)2]2 (1) showed four distinct peaks because of the formation of different ions, m/z = 413.42 [L1 + Ni + H]+, 435.50 [L1 + Ni + Na]+,

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ACCEPTED MANUSCRIPT 825.25 [2L1 + 2Ni + H]+, 847.33 [2L1 + 2Ni + Na]+. Obviously, the coordinated solvent molecules CH3COOH are detached, and meanwhile apart from the monomeric ion there exist the dimeric ions expectedly in the solution. Similarly, in the ESI-MS of the symmetrical binuclear nickel(II) complex [Ni2(L1)2(CH3OH)] (2) shows the same

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four peaks with the identical assignments compared to complex 1. For complex 3, its structure is not disturbed obviously in solution shown in the ESI-MS analysis only the departure of the non-coordinated solvent methanol

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molecules in their crystal structure.

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3.2 Single crystal X-ray studies [Insert Fig. 1]

Complex 1. Complex 1 exhibits as an mononuclear [Ni(L1)(CH3COOH)2] molecules which consists of one Ni(II) ion and a deprotonated Schiff base ligand (Fig. 1). In the asymmetric unit, there are two independent

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molecules. The center of unit include a Ni(II) ion and a [N2O4] slightly distorted octahedral environment. The [N2O4] part is formed by a Schiff base ligand which provide two imine nitrogen atoms [N(1) and N(2)] and two

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deprotonated phenolic oxygen atoms O(3) and O(4); The other two oxygen atoms O(5)and O(7) are from two acetic acid molecules. The bond distance around Ni(1) ion range from 2.0049(19) Å to 2.159(2) Å. The N-Ni-O

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and O-Ni-O angles are from 84.75(9)° to 94.90(9)° in the equatorial plane and the axial angle N(2)-Ni(1)-O(7) is 172.76(9)°. Selected angles and bond lengths are listed in Table S2.

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Complex 2. Complex 2 contains a disorder CH3OH and a [Ni2(L1)2] part (Fig. 2). Charge-balance within the part of [Ni2(L1)2], unlike complex 1 Cl- cannot participate in coordination, so that the CH3OH molecule was introduced to complex 2. Half of the molecule forms the asymmetric unit. The two halves of the molecules are related through center of inversion. The nickel(II) atom(s) have distorted octahedral geometry with approximate square planar base [N(2), O(3), O(3)#1, O4] (symmetric operation codes: -x, y, -z + 1/2) and fifth and sixth coordination sites are occupied by one imine nitrogen atom and one methanol oxygen atom. The bond distance around Ni(II) ion range from 1.986(2) Å to 2.240(3) Å. The equatorial bond lengths are in the range 1.986(2) Å to 2.041(3) Å, while the axial bond lengths are 2.179(2) Å and 2.240(3) Å. The N-Ni-O and O-Ni-O angles are from 79.02(11)° to 96.73(11)° in the equatorial plane and the axial angle N(1)-Ni(1)-O(5) is 166.44(11)°. The distance between Ni···Ni centers was found to be 2.976 Å. A similar structure has been reported for series of macrocyclic dinuclear nickel(II) complexes [20], however, the Ni···Ni distance in the analogous nickel(II) complexes (3.039 and 3.059 Å) [20] is longer than that in complex 2. Selected angles and bond lengths are listed in Table S2. [Insert Fig. 2] Complex 3. Complex 3 contains three free methanol molecules and a [Ni(L2)2] part per asymmetric unit (Fig.

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ACCEPTED MANUSCRIPT 3). And the six-coordinated Ni(II) atom is the center of slightly distorted octahedron. The slightly distorted octahedral environment of [N2O4] is formed by two Schiff base ligands which provide two imine nitrogen atom N(1) and N(2), two deprotonated phenolic oxygen atoms O(2) and O(4), two hydroxyl oxygen O(3) and O(5). The

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O(4), O(5), N(1) and N(2) atoms occupy the equatorial plane and O(2), O(3) atoms lie in the axial positions. The N-Ni-O and O-Ni-O angles are from 79.65(17)° to 96.71(19)° in the equatorial plane and the axial angle O(2)-

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Ni(1)-O(3) is 170.52(18)°. Selected angles and bond lengths are listed in Table S2.

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[Insert Fig. 3] [Insert Fig. 4]

The 2D supramolecular sheet structure is held together through weak H-bonding O3-H3···O8 (Symmetry code: -x + 1, y + 1/2, -z), O8-H8···O6, O6-H6···O9, O9-H9···O2, O5-H5···O1 (Symmetry code: -x + 2, y - 1/2, -z),

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O1-H1···O7 (Symmetry code: x + 1, y, z) and O7-H7···O4 (Symmetry code: x - 1, y, z) interactions. Methanol molecules act as pillars to connect and stabilize the 2D supramolecular structure as shown in Fig. 4. H-bonding is shown in dashed line and the parameters are presented in Table S3.

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3.3 DNA binding studies

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3.3.1 Competitive binding between EB and complexes for CT-DNA Fluorescence spectrum analysis is found to be an effective tool to examine the binding mode of metal

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complexes with EB-DNA system [21]. EB is a weak fluorescent reagent and belongs to aromatic fluorescent compounds, when in the presence of DNA its emission intensity can be enormously enhanced because of its strong intercalation between the adjacent DNA base pairs. The interactions of complexes with DNA were evaluated by the EB–DNA compound system, which can be used to distinguish intercalating and nonintercalating complexes [22]. If EB which has bound to CT-DNA was replaced by the metal complexes, fluorescence emission intensity of the EB–DNA system will be observably quenched [9]. The fluorescence spectra of EB–DNA system quenched by complex 1 and the plots of I0/I vs. r (C[complex]/C[DNA]) are shown in Fig. 5 (respective spectra of complexes 2 and 3 are presented as Fig. S1 in the ESI†). With increasing the concentrations of complexes 1-3, the intensity of the fluorescence spectra emission band at 590 nm of the EB-DNA system obviously decreased. It can be inferred that the title complexes 1-3 may bind to CT-DNA with an intercalative mode [21,22]. The observed linearity in the plot supported the fact that the quenching of EB bound to DNA by the test complex is in good agreement with the linear Stern-Volmer equation [23]:

I/I0 = 1 + Kspr

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Where, I0 and I represent fluorescence intensities in the absence and presence of the samples, respectively; r corresponds to the concentration ratio of the sample to DNA. Ksq, the linear Stern-Volmer quenching constant, can

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be obtained from the slope of I/I0 versus r linear plot. The calculated values of Ksq for complexes 1-3 are 3.74, 4.67

values are higher than those for some other complexes [24,25].

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[Insert Fig. 5]

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and 2.38, respectively, and indicating stronger interaction between complex 2 and DNA than 1 and 3. These Ksq

3.3.2 UV-Vis absorption studies

Electronic absorption spectroscopy is usually employed to determine the binding ability of metal complexes with DNA helix. Complexes that bound to DNA through intercalation are characterized by a change in absorbance

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(hypochromism) and bathochromic shift in wavelength, due to a strong stacking interaction between the aromatic chromophore of the test complexes and DNA base pairs. The extent of hypochromism is commonly consistent

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with the strength of intercalative interaction [3]. The absorption spectra of complexes 1-3 displayed two wellresolved bands ~266 nm and ~358 nm, which are assigned to intraligand charge transfer (ILCT) transitions and

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ligand-to-metal charge transfer (LMCT) transitions, respectively. The absorption spectra of complexes 1-3 in the absence and presence of CT-DNA are shown in Figs. 6 and S2,

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respectively. From the electronic absorption spectral data, it was clear that increasing the concentration of DNA added to the nickel(II) complexes 1-3, all of the above mentioned absorption bands showed hypochromism accompanied with bathochromic shifts. These observations are similar to those reported earlier for various metallointercalators [10]. To compare the binding strength of the complexes with CT-DNA, the intrinsic binding constant Kb were calculated according to the equation [3]:

[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εb − εf)

The Kb values were calculated using the above equation and were found to be 5.04 × 103 M-1, 9.46 × 103 M-1 and 3.62 × 103 M-1 corresponding to the complexes 1-3. On the whole, the binding constant Kb indicates medium binding strength of the complex with CT-DNA. The dinuclear complex 2 shows a higher Kb value in comparison to 1 and 3. Once the test complex intercalates to the base pairs of DNA, the π* orbital of the intercalators may couple with the π orbital of the base pairs, thus decreasing the π→π* transition probabilities and hence a hypochromism is observed in the above cases. These results are similar to those reported earlier for hydrazone

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ACCEPTED MANUSCRIPT metallointercalators [3]. [Insert Fig. 6]

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3.3.3 Circular dichroism The CD spectrum is one of the most common means to monitor the conformation of DNA in solution. The CD

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spectrum of DNA exhibits a positive peak at 278 nm because of base stacking and a negative peak at 246 nm

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because of the helicity of B-type DNA [26,27]. The observed CD spectra of CT-DNA in the presence of complex are shown in Fig. 7. In the presence of complexes 1–3, both the positive (ca. 278 nm) and negative (ca. 246 nm) peaks decreased in intensity, which is a clear information of non-classical intercalation between the complexes 1–3 and CT-DNA. The CD-spectra also indicated that the binding of the complexes 1–3 to CT-DNA lead to a

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significant change in the base stacking and can not unwind the DNA helix conformation. [Insert Fig. 7]

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3.4 Protein binding studies

3.4.1 Fluorescence quenching of BSA by metal complexes 1-3

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Fluorescence spectroscopy is an effective method to qualitative analysis of the binding of complexes to BSA. Generally, the native fluorescence of BSA is caused by three protein residues, namely tryptophan, tyrosine, and

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phenylalanine. Fluorescence quenching refers to that the decrease of the fluorophore induced by the environmental alteration around the fluorophore, which can reveal the nature of BSA binding reaction [28,29]. Fig. 8 shows the effect of that increasing the concentration of the complex 1 on the fluorescence emission of BSA (for complexes 2 and 3, see Fig. S3, ESI†). Addition of respective nickel(II) Schiff base complexes to BSA resulted in the quenching of fluorescence emission intensity, revealing quenching due to the complex-BSA complex system formed between the nickel(II) Schiff base complexes and BSA. The quenching process of the complexes on BSA fluorescence can be analyzed by the Stern–Volmer equation [30]: [Insert Fig. 8] I0 /I = 1 + Ksv[Q]

where the I0 and I are the fluorescence intensities of fluorophore at 346 nm in the absence and presence of the nickel(II) complexes, respectively. [Q] is the concentration of quencher and Ksv is the Stern–Volmer quenching constant. As shown in Fig. 8, the insets plot of I0/I versus [Q] exhibits a good linear relationship and the linear

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ACCEPTED MANUSCRIPT correlation coefficient R1 = 0.991, R2 = 0.993, R3 = 0.985. Also the Ksv value obtained from the slope of the linear are 1.57 × 105 M-1, 1.02 × 105 M-1 and 1.04 × 105 M-1, respectively. [Insert Fig. 9]

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General fluorescence quenching usually occur by two different mechanisms which are classified as dynamic quenching and static quenching. Dynamic quenching refers to that the fluorophore and the quencher come into

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contact during the transient existence of the excited state and the static quenching refers to fluorophore–quencher

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form a ground state. In order to confirm the above types of quenching mechanism of BSA by these nickel(II) complexes, UV–Vis absorption spectra were recorded (Fig. 10). The weak absorption peak at about 278 nm in the absence of metal complexes showed an increase in the intensity, which revealed that fluorescence quenching of BSA by these complexes are mainly a static quenching procedure by forming complex-BSA ground state

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complexes [3,10]. [Insert Fig. 10]

For static quenching interaction, the fluorescence intensity data can also be used to analyze the apparent

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binding constant (Kb) and the number of binding sites (n) for the complex and BSA system by the following

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equation [31,32]:

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log((I0 - I)/I) = logKb + nlog[Q]

where, Kb is the equilibrium constant and n is the number of binding sites per albumin that is calculated from the intercept and slope in Fig. 9 log((I0 - I)/I) versus log[Q]. The values of Kb were obtained to be 1.54 × 106 M-1, 1.17 × 105 M-1 and 2.05 × 106 M-1, and n were found to be 1.21, 1.02 and 1.27, respectively. These values of n are approximately equal to 1, suggest that there is only one binding site for these complexes on the BSA molecule. [Insert Table 1] ESI-MS studies reveal that in solution the coordinated solvent molecules have detached from the crystal structures. It is interesting to note that the order of the binding affinities determined here is dependent of their ligand structure. For three nickel(II) complexes, each metal cation lies in the intersection of two mutually perpendicular salicylidene-nickel planars. This structure belongs to the classical metallo-intercalators, and the flexibility of the oxygen-containing alphatic chain linker of the ligand H2L1 contributes to the formation of the stable mutually perpendicular planars in complexes 1 and 2. For complex 3, the coordinated -OH group also induces the stability of two mutually perpendicular salicylidene-nickel planars. It is generally known that the planarity and the number of the hetero atom in the structure of the complexes have also influenced their affinity

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ACCEPTED MANUSCRIPT towards DNA and BSA binding. From the DNA and BSA binding results (Table 1), it is clear that complexes 1 and 2 showed higher binding constant (Kb) with DNA when compared to complex 3 which may be due to the existence of more phenyl ring, and the dinuclear complex 2 show stronger DNA-binding interaction than

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mononuclear complex 1. But in general, due to the bad planarity of two ligands, the DNA-binding constants with the magnitude of 103 for the titled three complexes 1-3 (determined by the UV-Vis absorption studies) are lower

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BSA which may be due to the two hydroxyl oxygen atoms.

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than those reported elsewhere (~104) [20,33,34]. Furthermore, complex 3 showed strongest binding constant with

3.4.2 Characteristic of synchronous fluorescence spectra

It is well known that the difference synchronous fluorescence spectroscopy between excitation and emission wavelength (Δλ = λem − λex) reflects to a different nature of chromophores. If the Δλ value is 15 nm the

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synchronous fluorescence of BSA is characteristic of a tyrosine residue and large Δλ values such as 60 nm is characteristic of tryptophan [3]. In order to study the structural changes of BSA in present of nickel(II) complexes,

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we measured synchronous fluorescence spectra with the addition of complexes 1-3. The synchronous fluorescence spectra of BSA with nickel(II) complexes 1-3 were recorded at both Δλ = 15 nm and 60 nm. When increasing the

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concentration of nickel(II) complexes, the fluorescence intensity of emission corresponding to tryptophan was found to decrease with a bathochromic shift of emission wavelength. And the tyrosine fluorescence emission also

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showed a decrease in the intensity but with a hypsochromic shift of emission wavelength. These results show that the complexes 1-3 affected both the micro environments of tryptophan and tyrosine residues. The spectrum of complex 1 is given in Fig. 11 (for complexes 2 and 3, see Figs. S4 and S5, ESI†). These results indicate that the metal complexes increase the polarity around the tryptophan residues and also the hydrophobicity around the tyrosine residues is strengthened. This increase observed in synchronous fluorescence spectroscopy confirmed the effective binding of the complexes with the BSA [10]. [Insert Fig. 11] 3.5 Cytotoxicity The functions of complexes of Schiff base to suppress cell growth and promote apoptosis were well documented [35-37]. In vitro cell culture studies are valuable tools for screening of chemotherapy agents and provide preliminary data for further relative studies. The cytotoxicities of the complexes to cells were evaluated through the loss of cell viability using MTT assay [38]. The inhibition effects of complexes 1, 2 and 3 against the four cells lines at a concentration of 20.0 μg/mL are listed in figure 12. The IC50 values against four cell lines A549, HCT-116, HL-60 and Caco-2 are shown in Table 2.

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ACCEPTED MANUSCRIPT Dinuclear complex 2 is remarkable in displaying the most prominent cytotoxicity against the tested cell lines. While complexes 1 and 3 show different trend for two cell lins, thus the complexes are selective toward different cancerous cells. In special, three compounds show completely inactive for colonic cancer cell line Caco-2 and the

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ligand H2L1 present obvious cytotoxicity against HL-60 cell line particularly. Also as shown in Table 2 the tested three complexes were found to be more potent than cisplatin. All the above observations clearly reveal that the

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cytotoxicity of dinuclear complex 2 is higher than mononuclear nickel complexes 1 and 3, which is consistent with

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the high ability of 2 to bind to DNA in an intercalation and causes a conformational change on DNA. In addition to these studies we also verified the importance of the introduction of the nickel ions on inhibiting the cancer cell proliferation. The cytotoxic activity of all starting materials (free ligands: H2L1 and HL2; nickel salts: [Ni(CH3COO)2]·4H2O, NiCl2·6H2O ) against four cancer cell lines were determined as described previously,

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and their corresponding IC50 values obtained exhibit inactive (as shown in Table 2) except for the free ligand H2L1 against HL-60 cell line, clearly pointing out the necessity of the coordination action on the biological properties on the respective complex.

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[Insert Table 2]

4. Conclusions

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[Insert Fig. 12]

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It appears from the study that the changes in the substituent group present on the Schiff base ligand as well as the structure of the nickel(II) complexes have significant effects on their interaction with CT-DNA, BSA and cancer cell lines. Studies reveal that the cytotoxicity of dinuclear complex 2 is higher than mononuclear nickel complexes 1 and 3, which is consistent with the high ability of 2 to bind to DNA in an intercalation and causes a conformational change on DNA. Also, it emerges from our present studies that the nickel salicylidene Schiff base complexes, which can effect the structural changes on DNA and bind to BSA, can act as the potential anticancer drugs.

5. Abbreviations BSA

Bovine serum albumin

CT-DNA Calf thymus DNA CD

Circular dichroism

EB

Ethidium bromide

ESI-MS

Electrospray ionization mass spectroscopic

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FBS

Fetal bovine serum

H2L1

N,N’-bis(salicylidene)-3,6-dioxa-1,8-diaminooctane

HL2

2-ethyl-2- (2-hydroxybenzylideneamino)propane-1,3-diol

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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Bromide

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tetraethyl ammonium hydroxide

Acknowledgment

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We acknowledge the financial support of the Natural Science Foundation of Shandong Province (Nos. ZR2013BM017), the Natural Science Foundation of China (No. 20971063 and 21105042).

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Appendix A. Supplementary material

CCDC No. 763659, 922024 and 922029 for complexes 1, 2 and 3. These data can be obtained free of charge from

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the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Electronic Supplementary Information (ESI) available: [Crystal and structure refinement data (Table S1), selected

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bond lengths (Å) and angles () (Table S2), hydrogen bonding geometries for complex 3 (Table S3), effects of

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complexes 2 and 3 on the fluorescent spectra of EB-DNA system (Fig. S1), UV–Vis absorption spectrum of complexes 1 and 2 in the absence and presence of CT-DNA (Fig. S2), fluorescence emission spectra of BSA in the absence and presence of the complexes 2 and 3 (Fig. S3), synchronous spectra of BSA as a function of concentration of the complexes 2 and 3 (Fig. S4 and S5)]. See DOI: 10.1039/b000000x/

References

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ACCEPTED MANUSCRIPT [8] W.O. Foye, Cancer Chemotherapeutic Agents; American Chemical Society: Washington, DC. 1995. [9] H.D. Yin, H. Liu, M. Hong, J Organomet Chem. 713 (2012) 11-19. [10] P. Sathyadevi, P. Krishnamoorthy, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh, N. Dharmaraj, Dalton Trans. 40 (2011)

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[12] F. Bisceglie, M. Baldini, M. Belicchi-Ferrari, E. Buluggiu, M. Careri, G. Pelosi, S Pinelli, P. Tarasconi, Eur. J. Med. Chem. 42 (2007) 627–634.

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[13] G. Barone, N. Cambino, A. Ruggirello, A. Silvestri, A. Terenzi, V.T. Liveri, J. Inorg. Biochem. 103 (2009) 731–737. [14] B.P. Esposito, R. Najjar, Coord. Chem. Rev. 232 (2002) 137−149.

[15] J.S. Stamler,D.J. Singel, J. Loscalzo, Science 258 (1992) 1898–1902.

[16] H.Y. Shrivastava, M. Kanthimathi, B.U. Niar, Biochem. Biophys. Res. Commun. 265 (1999) 311–314. [17] D.S. Raja, N.S.P Bhuvanesh, K. Natarajan, Inorg. Chem. 50 (2011) 12852−12866.

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[18] D.B. Etemadi, A. Taeb, H. Sharghi, A. Tajarodi, H. Naeimi, IRAN J SCI TECHNOL A 28 (2004) A1. [19] H.H. Li, M.J. Niu, D.W. Sun, S.N. Wang, S. Cheng, Inorg. Chem. Commun. 27 (2013) 97100.

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[20] S. Anbu, M. Kandaswamy, B. Varghese, Dalton Trans. 39 (2010) 3823–3832. [21] J.F. Dong, L.Z. Li, D.Q. Wang, Chin J Chem. 29 (2011) 259-266. [22] J.H. Wen, C.Y. Li, Z.R. Geng, X.Y. Ma, Z.L. Wang, Chem. Commun. 47 (2011) 11330–11332.

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[23] J.R. Lakowicz, G. Weber, Biochemistry. 12 (1973) 4161–4170. [24] M. Baldini, M. Belicchi-Ferrari, F. Bisceglie, G. Pelosi, S. Pinelli, P. Tarasconi, Inorg. Chem. 43 (2004) 7170-7179.

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[25] F. Wang, H.D. Yin, C.H. Yue, S. Cheng, M. Hong, J Organomet Chem. 738 (2013) 35-40. [26] H.Y. Liu, L.Z. Li, Q. Guo, J.F. Dong, J.H. Li, Transition Met Chem. 38 (2013) 441–448. [27] A. Rajendran, B.U. Nair, Biochim. Biophys. Acta 1760 (2006) 1794–1801. [28] W.S. VanScyoc, B.R. Sorensen, E. Rusinova, W.R. Laws, J.B.A. Ross, Biophys J 83 (2002) 2767–2780. [29] L.Z. Li, Q. Guo, J.F Dong, T Xu, J. H Li, J Pphotoch Photobio B 12 (2013) 556–62. [30] C.X Wang, F.F. Yan, Y.X. Zhang, L Ye, J Photochem Photobiol A Chem. 192 (2007) 23-28. [31] B. Ahmad, S. Parveen, R.H. Khan, Biomacromolecules 7 (2006) 1350-1356. [32] Y.J. Hu, Y. Liu, J.B. Wang, X.H. Xiao, S.S. Qu, J. Pharm. Biomed. Anal 36 (2004) 915–919. [33] S. Ramakrishnan, E. Suresh, A. Riyasdeen, M.A. Akbarshad, M. Palaniandavar, Dalton Trans. 40 (2011) 3245-3256. [34] P.P. Silva, W. Guerra, G.C. Santos, N. Fernandes, J.N. Silveira, M.C. Ferreira, T. Bortolotto, H. Terenzi, A.J. Bortoluzzi, A. Neves, E.C. Pereira-Maia, J. Inorg. Biochem. 132 (2014) 67-76. [35] R. Katwal, H. Kaur, B.K. Kapur, Sci. Revs. Chem. Commun. 3 (2013) 1-15. [36] B.S. Jayashree, M. Kaur, A. Pai, Elixir Org. Chem. 52 (2012) 11317-11322. [37] A. Noureen1, S. Saleem, T. Fatima, H.M. Siddiqi, B.M. Pak, J. Pharm. Sci. 26 (2013) 113-124. [38] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.

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ACCEPTED MANUSCRIPT Table 1. Comparison of interaction study results between complexes 1-3 on CT-DNA and protein. DNA binding

Protein binding

Complex Ksq

Kb (M-1)

Ksv (M-1) 3

Kb (M-1)

5

n 6

1.21 1.02

3.74

5.04 × 10 1.57 × 10 1.54 × 10

2

4.67

9.46 × 103 1.02 × 105 1.17 × 105

3

2.38

3.62 × 103 1.04 × 105 2.05 × 106

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ACCEPTED MANUSCRIPT Table 2. IC50 (μM) of all compounds against human lung carcinoma cell line (A549), human colon carcinoma cell lines (HCT-116), human promyelocytic leukemia cells (HL-60) and colonic cancer cell line (Caco-2) for 24 h treatment. A549

HCT-116

HL-60

Caco-2

1

65.38 ± 2.4

34.85 ± 0.6

24.859±1.4

>100

2

20.2 ± 0.5

22.64 ± 1.5

12.469±1.1

>100

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31.98 ± 1.6

59.84 ± 3.3

>100

1

H2L

>100

>100

25.46±1.4

HL2

>100

>100

>100

>100

>100

>100

>100

NiCl2·6H2O

>100

>100

>100

>100

Cisplatin

>100

>100

>100

>100

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[Ni(CH3COO)2]

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·4H2O

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ACCEPTED MANUSCRIPT Figure Caption Scheme 1. Preparation routes of the ligands (H2L1, HL2) and nickel(II) complexes (1, 2 and 3) Fig.1 Molecular structure of complex 1, hydrogen atoms are omitted for clarity. Fig.2 Molecular structure of complex 2, hydrogen atoms are omitted for clarity.

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Fig.3 Molecular structure of complex 3, hydrogen atoms and methanol molecules are omitted for clarity. Fig.4 2D supramolecular net structure of complex 3, in which hydrogen bonds are shown in dashed line.

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Fig.5 Effects of complex 1 on the fluorescent spectra of EB-DNA system (λex = 258 nm); CDNA = 30μM; CEB = 3μM; from 1 to 8 CVOL =0, 3, 9, 15, 30, 60,

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90, 120μM, respectively. Arrow shows changes in the emission intensity upon addition of increasing concentration of the complex. Inset: plot of I0/I vs r ( r = CVOL/ CDNA) for complexes 1-3.

Fig.6 UV–vis absorption spectrum of complex 3 (1.0 × 10-5 M) in the absence and presence of CT-DNA, from 1 to 6, [DNA] ＝0, 2.0 × 10-5, 4.0 × 10-5, 6.0 × 10-5, 8 × 10-5 and 10 × 10-5 M, respectively. Arrows show the changes in absorbance with respect to an increase in the DNA concentration (Inset: plot between [DNA] and [DNA]/[εa−εf]).

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Fig.7 CD-spectra of CT-DNA in the absence and presence of the complex, [DNA] =1.0 × 10-4 M, [complex] = 0 and 4.0×10–5 M. Fig.8 Fluorescence emission spectra of BSA in the absence and presence of complex 1. [BSA] = 1.0 × 10-6 M, [Complex] = 0, 2.0 × 10-6 M , 4.0 × 10-6 M,

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6.0 × 10-6 M, 8.0 × 10-6 M, 10.0 × 10-6 M, 12.0 × 10-6 M, 14.0 × 10-6 M, respectively; λex = 280 nm, both excitation and emission slits were 5 nm. (Inset: Plot of [Q] vs. I0/I). Fig.9 Plot of log[(I0-I)/I] vs. log [Q].

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Fig.10 UV–vis absorption spectra of BSA in the absence and presence of the complex. [BSA] = 1×10–6 M, [complex] = 0 and 1×10–6 M. Fig.11 Synchronous spectra of BSA as a function of concentration of the complex 1 with wavelength difference of Δλ = 15 nm and Δλ = 60 nm.

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Fig.12 Inhibition [%] of complexes 1, 2, and 3 [dose level of 20.0 μg/mL] against human tumor cells.

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ACCEPTED MANUSCRIPT O

O

N

N

Ni(CH 3COO)2·4H 2O

(1)

Ni O

O O

H2N

NH 2

N

HO

O

N

OH

HO NiCl2 ·6H 2O

O

O

O

N

O

Ni

CH

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OH OH HO

NH 2 OH

N

O

HO

Ni(CH3 COO)2 ·4H 2O TEAOH

C HO

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OH

N

N

Ni

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C

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O

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O O

O

OH

N

O

Ni

N

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(3)

O O

Preparation routes of the ligands (H2L1, HL2) and nickel(II) complexes (1, 2 and 3)

Scheme 1

(2)

OH

OH HO N

O O

Scheme 1.

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Figure 1

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Figure 11

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Figure 12

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Graphical abstract

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ACCEPTED MANUSCRIPT Synopsis for the Graphical abstract Three new salicylidene Schiff base nickel(II) complexes were synthesized and characterized and the effect of structure and composition of these complexes on DNA/protein interaction and cytotoxicity

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experiments.

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ACCEPTED MANUSCRIPT Highlights

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1. Three new salicylidene Schiff base nickel(II) complexes were synthesized and characterized. 2. The interaction between these complexes with DNA has been studied. 3. The interaction of the Ni(Ⅱ) complex with BSA was also investigated. 4. The cytotoxicities of free ligands; nickel salts and three complexes are investigated.

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