Water-soluble nickel (II) Schiff base complexes: Synthesis, structural characterization, DNA binding affinity, DNA cleavage, cytotoxicity, and computational studies

Abstract Two water-soluble nickel (II) Schiff base complexes were prepared and their interaction with fish sperm DNA (FS-DNA) was investigated by various methods including UV–vis spectroscopy, fluorescence spectroscopy, cyclic voltammetry, and viscometric measurements. Complex 1: [N,N′-bis{5-[(triphenyl phosphonium chloride)-methyl] salicylidine}-3,4-diaminobenzophenone]nickel(II) perchloride dihydrate: [Ni(5-CH2PPh3-3,4-salophen)] (ClO4)2.2 H2O was synthesized as a new complex and characterized by elemental analysis, IR, 1H NMR, thermal gravimetric analysis (TGA) and UV–vis spectroscopy. Complex 2: sodium [(N,N′-bis(5-sulfosalicyliden)-3, 4-diaminobenzophenone)aqua] nickel(II) hydrate: Na2[Ni (5-SO3-3,4-salbenz)(H2O)]. H2O was already synthesized by our research team, but in this study, its function as a DNA-binding compound was tested, and compared with the results of complex 1-DNA binding. The calculation of different constants using absorption and emission data, all confirmed the stronger binding ability of complex 1 than complex 2 with DNA. Different thermodynamic parameters showed the interactions between DNA and complexes were the type of hydrophobic interaction for complex 1 and electrostatic interaction for complex 2. Also, the negative values of free energy changes proved a spontaneous DNA binding process. Based on cell toxicity assay against two different cell lines including Jurkat and MCF-7, the effect of complex 1 was comparable to cisplatin, and the toxicity mechanism was further justified by bright field microscopy, flow cytometry, and cleavage of DNA in the presence of H2O2. Besides, the docking calculations suggested intercalation after measuring the lowest-energy between the complexes and DNA. For both complexes, all analytical, spectroscopic, and molecular modeling methods supported partial intercalation as the main binding mode between the complexes and DNA.


Introduction
Schiff bases are an important class of ligands that have been attracted the interest of chemists as well as biologists. This fact can be due to the wide range of applications of Schiff bases in various fields such as organic chemistry, inorganic chemistry, and also biochemistry. Structurally, Schiff bases as polydentate ligands result from the condensation of a ketone or aldehyde with a primary amine which forms the active imine or azomethine group. [1] Over the years, many articles on the subject of Schiff bases and their derivatives have reported a wide range of corresponding biological activities including anti-inflammatory, [2] antioxidant, [3] antifungal, [4] antibacterial, [5] antimalarial, [6] anticonvulsant, [7] antiviral, [8] antimicrobial, [9] antitumor [10] and anticancer [11] activities.
The presence of the azomethine groups and different donor atoms such as oxygen and sulfur give Schiff bases capable to connect to metal centers and lead to synthesize the complexes with stable structures.
Researchers have been trying for years to find the best drugs to treat cancer. In fact, the discovery of metal complexes with these types of wonderful and varied chelating ligands was the main step to realize the important role of inorganic chemistry in improving anticancer drugs.
One of the best metal complexes is cisplatin, [12] which is still used as a covalent binder anti-cancer drug, but its serious side effects, high general toxicity, and inherent or acquired resistance [13] lead to an effort to develop new drugs with other transition metals. Among the different metal derivatives, nickel Schiff base complexes have always been considered and applied as an antioxidant [14,15] antimicrobial, [16,17] antibacterial, [18][19][20] and antitumor [21,22] agents. The anticancer properties of these versatile complexes have received a lot of attention and many articles have been published accordingly, in recent years. [23][24][25] Unlike cisplatin, nickel Schiff base complexes bind to DNA as a non-covalent binder through three modes of binding which are classified as intercalation, [26][27][28][29] electrostatic, and minor/major groove bindings. [30][31][32][33] Intercalation binding occurs as a result of strong π-π stacking interactions between aromatic rings of complexes and DNA base pairs. Interaction of complexes can also involve DNA grooves via hydrogen bonding or van der Waals interactions. Electrostatic binding refers to external interaction between the phosphate groups of nucleic acid and small molecules with positive charge. [34,35] One of the greatest advantages of non-covalent binders is their little side effects compared to covalent binders. [36] In this work, the main focus is on the synthesis and characterization of two water-soluble Schiff base complexes of nickel as well as the study of their interaction with FS-DNA. These complexes were characterized by using 1 H NMR, IR, elemental analysis (CHN), UV-vis, and TGA. Complex 1: Ni(5-CH 2 PPh 3 -3,4-salophen)](ClO 4 ) 2 .2H 2 O is new but our research group previously synthesized complex 2: Na 2 [Ni (5-SO 3 -3,4-salbenz)(H 2 O)]. H 2 O and investigated in detail its interaction with human serum albumin (HSA) which is one of the important target proteins for designing more effective drugs. [37] In order to complete the biological studies on this complex and also to compare it with the newly synthesized complex 1, its interaction with DNA was also studied. The types of possible binding modes were investigated by UV-vis and fluorescence spectroscopy, electrochemical measurements, viscometric method, and also molecular docking process. The effectiveness of these complexes was evaluated more accurately through anticancer and DNA cleavage test. The purposeful design of these complexes was associated with the presence of various groups with the positive or negative charges on ligands in order to increase their solubility in water and their proper function in biological environments. These different groups (positive triphenylphosphonium and negative sulfonate groups) also provided the conditions for comparing the tendency of complexes to interact with DNA.

Preparation of the compounds
The new complex (complex 1) was synthesized as follows:

Synthesis of (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride
In order to the preparation of this aldehyde, 0.08 mol of salicylaldehyde was mixed with 0.05 mol paraformaldehyde in 50 ml of conc. HCl, and was stirred at room temperature. After 48 h, the precipitate of 5-(chloromethyl)salicylaldehyde with dark red color was obtained and washed with NaHCO 3 (0.5%) and water. Heating of mixture of this red powder (0.06 mol) and triphenylphosphine (0.06 mol) under reflux in 200 ml acetonitrile for 4 h resulted in the production of the white precipitate of phosphonium

Solubility and stability
The solubility of the complexes in water and ethanol were significant. During the research, the complexes in the solid phase were stable at room temperature and in light and did not require any special storage conditions (Scheme 2). [37]

DNA binding experiments
The proportion of DNA absorbance at 260 to absorbance at 280 nm, A 260 / A 280 , was 1.98, this ratio clearly showed that DNA existed without any protein. The studies were conducted at different temperatures (298 and 304 K) and each absorption spectrum was recorded after 4 minutes. In all UV-vis absorbance experiments, the concentration of complexes was fixed, whereas DNA amount was constantly changing through several injections of 20 μl for complex 1 and 10 μl for complex 2. The proper concentration of DNA was reported 1 × 10 −2 M which was calculated by Beer's law (ε = 6600 M −1 cm −1 and λ max = 260 nm). The solution of DNA is stored at 4 °C for a short time for reusing. The final solutions of complexes were prepared in the buffer (1 mM Tris-HCl, 5 mM NaCl at pH 7.2), and each of them equaled to 1 × 10 −4 M. In fluorescence spectroscopy, two synthesized complexes including complex 1 and complex 2 with concentrations equal to 5 × 10 −6 M and 1 × 10 −4 M, respectively, were prepared in1mM Tris buffer, 5 mM NaCl, at pH 7.2. Emission spectra of complex 1 was recorded in the presence of 0-110 µl DNA (1 × 10 −3 M), while a competitive experiment to displace EtBr was performed by injecting 10 µl of complex 2 into the solution containing EtBr and DNA with the concentration ratio of [EtBr]/[DNA] = 1. There was the 4-minute interval between injections.
Because of the inner filter effect, the observed fluorescence intensities (F obs ) were corrected (F corr ) according to Equation (1): [38] F F corr obs The absorbance of the complexes at the excitation wavelength (A ex ) and emission wavelength (A em) have been used in this equation. The slit width was selected 10.0 nm for excitation and emission, and the scanning speed was considered 500 nm per min.
The results from the viscosity of DNA in the absence (η o ) and presence (η) of complexes were provided by an Oswald-type viscometer which made it possible to measure (η/η o ) 1 The electrochemical cell consist of a three-electrode system that could provide more information to elucidate the electrochemical behaviors of synthesized complexes after binding to DNA. Various types of electrodes including platinum working electrode, Ag/Ag + reference, and platinum counter electrodes were utilized. All solutions including complex 1 and complex 2 (1 × 10 −3 M) were prepared in H 2 O/DMSO. Voltammetric studies of DNA-complex binding were done in the range of −1 V to 1 V at a potential scanning rate of 0.10 Vs −1 , and in the presence of 10 µl of DNA (1 × 10 −2 M).

Theoretical studies
Docking calculations were performed by using the crystal structure of DNA (PDB id:1z3f). This structure was provided from the Protein Data Bank (PDB) (http://www.pdb.org) and the protein molecules were removed from it to be selected as a receptor. In order to obtain the molecular geometry of the complexes with the lowest energy, their structure was optimized by B3LYP [39][40][41] method using the LANL2DZ and 6-31G basis sets for the metal centers and other atoms, respectively. The stable structures were selected for the docking study. All computations were performed by GAUSSIAN 03. [42] Molegro Virtual Docker (MVD) [43][44][45] was used as an integrated platform to identify the types of complex-DNA interactions. This software can give the optimized configurations from DNA and complexes according to Moldock and Rerank scores. The Moldock score [GRID] scoring method was used and the GRID resolution was 0.3 Å. The selected molecular docking algorithm was Moldock with 10 runs, and 10 different poses were considered for each docking calculation. Other parameters were defined based on the default values.

Analysis of anticancer properties of the complexes by MTT assay
The MTT assay as a colorimetric assay was used to assess the cytotoxicity of the synthesized complexes. [46] Briefly, this process was performed in several steps:

2.5 × 10 4 of Jurkat (Human Leukemia) cell line and 1 × 10 4 MCF-7
cell line (breast cancer cells) were seeded into each well of 96-well plates for 24 h. 2. The cells treated with varying ranges of the nickel complexes concentrations (5-100 μg ml −1 ) in fresh culture medium (90 μl). 3. After 24h and 48h of treatment, and adding the solution of MTT in PBS buffer (10 μl, 4.50 mg ml −1 ) to wells and covering the plates with aluminum foil, the final mixture, was incubated for 4 h at 37 °C. 4. In this step, as a result, MTT is reduced to formazan. The solubilization solution with pH = 4.7 (100 μl, DMF 40% (v/v), and SDS 16% (w/v)) was added to dissolve this insoluble purple product and provide the colored solution.
Finally, the absorbance was monitored via plate reader (Biotek Instruments, Inc., USA (at 570 nm, and the reference wavelength is taken at 620 nm. Data were collected for three replicates each and were used to calculate the mean value. The percentage of inhibition was calculated from these data using Equation (2):

Flow cytometry analysis
The total population of 2 × 10 5 of Jurkat and MCF-7 cell lines are preseeded in 24 well plates for 16 h. The cells were then exposed of complex 1 (32.5 and 65.0 µM), and cisplatin (21.2 and 42.1 µM) for 48 hours. The AnnexinV/PI staining was then performed using eBioscience TM Annexin V apoptosis detection kit (Invitrogen). After harvesting the cells with 0.25% of trypsin, cells were washed twice with phosphate-buffered saline (PBS), and once with 500 μL binding buffer. In the next step, 5 μL of Annexin V-fluorescein isothiocyanate is added to 100 μL of binding buffer for at least 15 minutes. Then, cells were washed again with 500 μL of binding buffer and incubated for an additional 5 min in 200 μL of the binding buffer containing 5 μL Propodium Iodide (PI) solution. The apoptosis rates were then monitored using BD FACS Calibur™ flow cytometry (BD Biosciences, San Jose, CA, USA). The apoptosis rates were finally estimated as the sum of early apoptosis and late apoptosis.

Gel electrophoresis study
In order to carry out the gel electrophoresis experiments, supercoiled pBlu2KSM DNA (500 ng, in 10 mM Tris-HCl buffer with pH 7.2) in 1% DMSO and the synthesized complexes (50 µM of complex 1 and 70 µM of complex 2) were prepared. The complexes were incubated with DNA for 1 h at 310 K in the presence of H 2 O 2 (50 µM). In the ensuing step, after loading onto 1% agarose gel in running buffer, Tris-acetate-EDTA (TAE), containing 1.0 µg ml −1 EtBr at 60 V for 1 h, the gel was photographed under UV light. The experimental results were used to investigate the effect of the complexes on DNA.

Spectral properties of the complexes
The synthesized complex, complex 1, was identified by elemental analysis, 1 H NMR, FT-IR, UV-vis spectroscopy and TGA and complex 2 was characterized according to the reference. [37] Proton NMR spectra of compounds indicated the structures which were expected. In order to determine the structure of (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride, the regions of aldehydic and aromatic protons were investigated. As shown in Fig. S1, the signals related to aromatic protons were seen at 6.95-7.90 ppm. The peaks attributed to HC = O and OH groups were revealed at 10.13 ppm and 11.18 ppm, respectively. Also, the signals of CH 2 -P protons appeared at 5.09-5.15 ppm.
According to the 1 H NMR spectra of complex 1, the protons of unsymmetrical azomethine groups were assigned to 8.46 and 8.58 chemical shifts (ppm). The protons attributed to aromatic rings appeared at a range of 6.45-7.95 ppm. Also, protons of CH 2 -P groups in the structure of the complex were exhibited by signals at 4.89-5.1 ppm (Fig. S2).
IR spectrum of (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride shows a medium band at 1674.1 cm −1 which describes the carbonyl group of aldehyde. Also, O-H available in the structure was proved by a broad and moderate-intensity band at 3741.6 cm −1 (Fig. S3). The presence of several main functional groups confirms the structure of complex 1, as explained below.
Analysis of electronic spectral data is one of the helpful ways for the primary characterization of compounds. Based on Fig. S5, the peaks attributed to (π→π*) transitions cover a wavelength range of 250-450 nm. The corresponding absorption bands for carbonyl and azomethine chromophores were appeared in longer wavelengths than (C = C) in aromatic rings. The broad absorption bands with weak intensity in 450-600 nm range can evaluate n-π* transitions which are related to the carbonyl group or oxygen atoms of Schiff base attached to the phenyl rings. [49] The TG/DTA curve in Fig. S6 shows two main steps of thermal decomposition of complex 1 which are as follows: The first mass loss at 250-380 °C is due to the loss of perchlorate groups and C 13 H 8 ON 2 (bridging diamine) (Calc. 33.18%, found 33%), and the second step was related to the mass loss of remaining part of the complex (Calc. 66.81%, found 67%). the resulted data from complex 1 has been listed in Table S2.

Electronic structure determination
The optimized molecular structures of complex 1 and complex 2 are shown in Fig. S7. Also, geometrical parameters including bond lengths, bond angles, and energies of frontier orbitals, metal charge, and other parameters of the complex based on B3LYP/6-31G calculations [50] are listed in Tables S3 and S4. All of the bond lengths and bond angles of the atoms were in the normal range. The optimized structure suggested a distorted square planar geometry around the central atom with two N atoms from diamine moiety and two O atoms of phenolic groups.

HOMO-LUMO energy gap calculation
Both HOMO and LUMO are the main orbitals that take part in the chemical stability. The frontier molecular orbitals play an important role in the reactivity of the compounds and in many electric and optic properties. A Large HOMO-LUMO gap automatically means high excitation energies for many excited states, good stability, and high chemical hardness for the complex. Therefore, measurement of the difference between these molecular orbitals can serve as a measure of the excitability of the molecule, the smaller the energy, the more easily it will be excited.
The 3D plots of the HOMOs and the LUMOs of the complexes are shown in Fig. S8.

Absorption spectroscopic studies
The investigation of spectral changes can supply the information to specify the mode of DNA binding. Two absorbance peaks of DNA at 260 and 230 nm are attributed to the conjugated double bond and the rings of the purines and pyrimidines bases. [51] Therefore, the assessment of the spectral changes in these wavelengths is meaningless.
If the spectral changes lead to decreasing in the absorbance (hypochromism), it can be a sign of an intercalation binding mode in which the strong noncovalent interactions occur between aromatic rings of DNA base pairs and intercalated complexes (π-π stacking interactions) . [52,53] In addition, increasing absorbance (hyperchromism) can demonstrate various binding modes. The absorption of single strand DNA is higher than the absorbance of double strand DNA, so the interactions which unwind double-stranded DNA or damage to the structure of DNA result in hyperchromism. Also, groove binding and electrostatic binding make hyperchromic effect. [34,54] Figs. 1 and 2 show the decrease in the absorbance of two complexes at the maximum wavelengths of 390 nm and 373 nm continuously after adding DNA which suggests an intercalation mode of binding. The isosbestic point which has been displayed at 304 nm demonstrates an equilibrium between the free and bound complex 1 in the ground state. It is worth noting that these results are not conclusive, and should be consistent with other investigations. Absorption titrations were also done for the complexes at 304 K. The corresponding curves are shown in Figs. S9 S10 and S10.
In order to find out the differences between complexes in terms of the strength of binding to DNA, Wolfe-Shimmer equation [55] (Eq. (3)) was used to calculate the intrinsic-binding constant (K b ).
where [DNA] shows the concentration of DNA, ε is the extinction coefficient of the free form of the complex in the absence of DNA (ε f ), bound   Table 1 shows the values of constants. The intrinsic binding constants of two nickel complexes are in the range of values of DNA-binding constant for intercalators or groove binder complexes with various metals and show weak intercalation compared to EtBr as a classical intercalator. [56][57][58][59][60][61] The binding affinity of complex complex 1 is approximately six times larger than complex 2 at 298 K. It can be attributed to the presence of more aromatic rings as well as the presence of -CH 2 -PPH 3 + groups with positive charges which lead to more effective binding of this complex to DNA.
Thermodynamic parameters including enthalpy change (ΔH) and entropy change (ΔS) can be helpful to identify the mode of biomolecule-drug binding. These non-covalent interactions are classified according to the signs of ΔH and ΔS. When ΔH < 0 and ΔS < 0, van der Waals force or hydrogen bond play an important role. Additionally, ΔH > 0 and ΔS > 0 and ΔH ∼ 0 and ΔS > 0 suggest hydrophobic and electrostatic interactions, respectively. [62] The negative values of free energy changes (ΔG) indicated that the interaction between complexes and DNA proceed spontaneously. The relationship between temperature and thermodynamic parameters was defined by Van't Hoff equation (Eq. (4)): [63] ln K T S b ' ' H R R (4) And free energy changes (ΔG) at different temperatures can be calculated from Equation (5): [63] ' ' ' G H T S (5)  where T is the corresponding temperature in the Kelvin scale, K is the binding constant at T, R shows the gas constant which is equal to 8.314 J mol −1 K −1 . All calculations have been listed in Table 1.

Fluorescence binding studies
Fluorescence spectroscopy is a practical and useful technique that can provide a more in-depth look at the interactions between DNA and other compounds. There are important distinctions between fluorescence spectra of complexes before and after adding DNA that lead to the identification of how to interact with DNA. The compounds that can fit themselves in between base pairs of DNA are limited to the hydrophobic internal environment of DNA, in consequence, they are out of reach of solvent molecules. In the absence of solvent molecules, decreasing the vibrational modes of relaxation results in an increase in the intensity of fluorescence emission. Fig. 4 shows the increase in the intensity of complex 1 at 758 nm Table 1. Apparent binding constant, relative thermodynamic parameters, and the model of interaction between complexes and Fs-dNA at two temperatures.
complexes  after adding DNA. Hence this complex can be considered as an intercalator for the reasons stated above. Some complexes do not be able to show the fluorescence emission band in the absence and presence of DNA. In these cases, EtBr as a probe can be applied to provide the needed information for finding the mode of binding. [64] EtBr is an aromatic molecule with weak fluorescence that can intercalate into DNA base pairs. [65] For the reasons mentioned above, which described the characteristic of intercalation binding mode, the intensity of Ethidium Bromide's fluorescence emission is greatly increased after adding DNA because it acts as a strong DNA intercalator. [66,67] In fact, a compound-DNA binding competition is formed between the complex and EtBr. If EtBr molecules are replaced by the complex, decreasing in the fluorescence intensity is observed. As can be seen from Fig. 5, decreasing in the emission of EtBr-DNA at 603 nm proved complex 2 -DNA intercalation binding mechanism.
In general, there are two mechanisms. The Static process occurs based on the formation of a non-fluorescent complex. This complex is the result of strong contacts between a fluorophore and a quencher before electron excitation (a ground-state complex), while in a dynamic process, the fluorescence intensity of a fluorophore is quenched non-radiatively after colliding to a quencher in the excited state. [68] Stern-Volmer equation describes the mechanism of quenching [69] (Eq. (6)). The measured emission intensity of a fluorophore (F o ) is changed to (F) in the presence of several injections of a quencher which its total concentration is equal to [Q]. Based on equation 5, the slope of the linear plot of F o /F versus [Q] gives easily Stern-Volmer constant (K SV ). Also, the quenching rate constant (k q ) can be calculated by K SV = k q τ 0 which τ 0 refers to the lifetime of a biomolecule in the absence of quencher (τ 0 =10 −8 s).
The calculated values of K SV and k q. (K SV =5.79 × 10 4 M −1 and k q = 5.79 × 10 12 M −1 s −1 ) show clearly that the complex has a much larger value of K q than the limiting diffusion rate constant of biomacromolecule (2.0 × 10 10 M −1 s −1 ). Therefore, complex 2 prefers a static mechanism (Fig.  6a). [70,71] The high K SV value of complex 2 shows the ability of this complex to displace EthBr and bind to DNA. [72][73][74] Like the quenching process, the enhancement constant (K E ) for complex 1 can be calculated by following equation: [75] Based on the inset of Fig. 6-b, the plot of F o /F versus 10 −4 [E] indicates K E =2.00 × 10 6 M −1 .

Viscosity measurements
The dependence of DNA viscosity on changes in its length provides helpful conditions for examining the type of binding between DNA and other compounds. When a complex can be placed between the base pairs of DNA, makes DNA longer resulting in an increase in its viscosity. This is similar to the behavior of the classical intercalators such as EtBr after binding to DNA. In contrast, the compounds which lead to the kink of DNA through binding decrease the relative viscosity. Additionally, some kinds of binding modes such as groove binding show almost no effect on the viscosity of DNA. [76,77] As shown in Fig. 7, the pair of nickel complexes behave like DNA -intercalators and show a partial intercalative binding mode. According to the slope of viscosity changes in Figure 5, the relative viscosity of DNA is more influenced by complex 1 than complex 2. [78]

Electrochemical studies
Changes in the electrochemical behaviors of complexes after binding to DNA, such as current changes and shifts of anodic and cathodic peaks to negative or positive potentials, can help to determine the type of binding mode. Basically, the decrease in the current intensity upon the addition of DNA confirms the formation of complex-DNA binding. As Bard and coworkers reported, [79] if the peak potential shifts to higher values, indicates the presence of a complex between the base pairs of DNA. In other words, such a complex prefers intercalation as a DNA binding mode. According to Fig. 8, the cyclic voltammogram of complex 1 shows the decrease in the current with no shift in potential after adding DNA, which confirms the existence of DNA-complex interaction, but complex 2 also shows weak intercalation because the anodic peak shifts to positive potential slightly (from 0.48 to 0.51 V). [80]

Molecular docking
In the field of molecular modeling, docking plays an important role in designing the structure of drugs. [81] Docking as one of the most common methods can indicate the best binding-conformation and preferred orientation of small molecules to form the stable complex with the appropriate binding site of biomolecules and predict the strength of association or binding affinity between two molecules involved in the molecular binding. [82][83][84] For this purpose, the calculated binding scores are present in Table S5. The lowest MolDock score conformation for complexes 1 and 2 are −281.693 and −219.503, respectively. Comparing these calculated energies with the energy of EtBr (−170.657) can propose intercalation as one of the possible binding modes (Fig. 9). [85]

Anticancer activity studies
The studies on the anticancer activity of the complexes against Jurkat and MCF-7 cancer cell lines have demonstrated satisfactory results. All the data listed in Table 2 are recorded in different concentrations of the compounds from 5 to 100 μM and also at two different times, 24 and 48 h of incubation. According to the IC 50 values, complex 1 has been very effective against two cell lines, so that its molar concentration required to inhibit 50% of cancerous cells, is almost equal to half the amount of cisplatin against the same cell lines. While, 100 μM of complex 2 was not effective even after 48 h. The remarkable anticancer activity of this complex, in comparison with complex 2 shows the important effect of the structure of the coordinated ligand on the complex-DNA binding strength, the inhibition ability of cell proliferation, and its further medicinal properties.

Bright field microscopy
The morphological alternations of Jurkat and MCF-7 cancerous cells after being exposed to 30 µM of both complex 1 and cisplatin was also presented in Fig. 10. Accordingly, the rounded shape and cellular shrinkage showed the induction of programmed cell death in both complex 1 and cisplatin.

Flow cytometry analysis
The comparative apoptosis/necrosis analysis of complex 1 and cisplatin was also performed. As illustrated in Fig. 11, the complex 1 showed comparable apoptosis induction with Cislatin with maximum apoptotic population percentage of 70.77 and 71.59, respectively. Additionally, a negligible necrosis is seen after in both complex 1 and cisplatin with 5.25 and 2.81%, respectively. Hence, complex 1 is capable to induced programmed cell death equal with known anticancer agent, cisplatin.

DNA cleavage study
The gel electrophoresis process provides the conditions for comparing the band intensity of supercoiled form DNA (Form I) with the initial amount. The complexes that are able to cleave DNA, can induce the single-strand or double-strand breaks of DNA. In fact, their ability to convert supercoiled plasmid DNA into other forms can be a measure of their efficiency of the cleavage activity. When samples are loaded into wells, the supercoiled form DNA has the fastest migration. According to lane 2 in Fig.  12, circular DNA exposed to complex 1 is trapped in the well and acted similar to genomic DNA. These data significantly show the ability of complex 1 to alter DNA structure and it may be responsible for its therapeutic efficacy and cytotoxic effects on cancerous cells. Moreover, no structural change was observed in circular DNA when treated with

Conclusion
Taken together, synthesis, characterization, and DNA-binding studies of two nickel Schiff base complexes were considered. One of them, complex 2: Na 2 [Ni (5-SO 3 -3,4-salbenz)(H 2 O)]. H 2 O was prepared by our research team in 2014 and its interaction with HSA was examined carefully. This complex was synthesized and identified in the same way as the similar procedure of the literature in order to further study with other biomolecules such as DNA, and also compare its binding mode and binding affinity with the newly designed complex from the family of nickel complexes. In this work, the new water-soluble complex which is complex 1: Ni(5-CH 2 PPh 3 -3,4-salophen)](ClO 4 ) 2 ·2H 2 O, was also synthesized and characterized by different techniques including elemental analysis, 1 H NMR, IR, UV-vis spectroscopy, and thermogravimetric analysis.
The results of absorption and emission titration methods, viscosity measurement, and cyclic voltammetry were in good conformity and showed both complexes can bind to DNA by intercalative mode. The higher binding constant (K b ) of complex 1 calculated from absorption data in comparison with complex 2, the large value of enhancement constant (K E ), and the extreme effect on the viscosity of DNA displayed the important role of the ligand in determining the type and strength of DNA binding. The obtained results from Van't Hoff equation showed spontaneous hydrophobic interaction for complex 1 and electrostatic interaction for complex 2. The large value of quenching rate constant which is equal to 5.59 × 10 12 M −1 s −1 showed that complex 1-DNA complex was formed through a static mechanism.
The cytotoxicity of the complexes against Jurkat and MCF-7 cell lines, and also their DNA cleavage activity was evaluated by gel electrophoresis. The results displayed anticancer activity and the ability of complex 1 to induce changes in DNA structure under oxidative condition (in the presence of H 2 O 2 ), while complex 2 did not reveal any cytotoxicity and had no effect on DNA cleavage. Additionally, Complex 1 is capable to induce apoptotic programmed cell death similar to known anticancer complex, Cisplatin. Both experimental and theoretical results were in agreement on the type of binding mode. Molecular docking models showed the complexes in a position that can bind to DNA by intercalation mode.
Finally, complex 1 can be suggested as the appropriate alternative candidate for anticancer drugs. The presence of -CH 2 -PPH 3 + groups with several aromatic rings and positive charge played a pivotal role in this success and helped this complex to interact with DNA base pairs strongly by π-π stacking. So the binding affinity of this complex is much larger than the other, about six folds.