Design, synthesis, biomedical investigation, DFT calculation and molecular docking of novel Ru(II)-mixed ligand complexes

Abstract A novel series of bioactive water-soluble mononuclear Ru(II)-mixed ligand complexes of 2,2′-bipyridyl and V-shaped Schiff base ligands were synthesized and structurally characterized. Biomedical activities of Ru(II) complexes have been tested in view of antioxidant activities, interaction with calf thymus DNA (CT-DNA), and anticancer performance. The optimized structure of these complexes has been further supported by density functional theory (DFT) calculations. Further, validation of the interaction studies of some complexes was accomplished by carrying out molecular docking studies with DNA using molecular operating environment (MOE) software are reported. Communicated by Ramaswamy H. Sarma


Introduction
Today, cancer is one of the most serious medical problems, leading cause of death worldwide, and is a primary target of therapeutic chemistry (Bray et al., 2018). The development of novel anticancer, antibiotic, and antiviral therapeutics is a major research goal in several worldwide laboratories. Many existing compounds that are clinically useful against diseases such as cancer are thought to exert their primary biological effects by interacting with DNA to inhibit template function and hence replication, or by interfering with transcriptional machinery (Propst & Perun, 1992). Therefore, the metal-based cancer drugs are a focus of research in bioinorganic chemistry. Since cisplatin and its platinum complexes were first used as anticancer agents successfully, more active transition metal complexes with better anticancer activity have been studied (Zhang et al., 2019).
Platinating agents, including cisplatin, carboplatin, and oxaliplatin, have been used clinically for many years as part of anticancer drug to treat many types of cancers (Muggia et al., 2015). However, treatment with these agents is characterized by severe side effects systemic toxicities like nephrotoxicity, neurotoxicity, ototoxicity inflicts serious disorders or injuries and acquired resistance caused by prolonged treatment have resulted in the search for alternatives to circumvent drug resistance (Rabik & Dolan, 2007).
Development of more efficient anticancer drugs with better selectivity but less toxic side effects is currently an area of hot research topic in bioinorganic chemistry and with this goal, as promising alternatives to platinum compounds. Ru(II) complexes have attracted extensive attention as new anticancer drugs because of its variable oxidation states, selectivity for cancer cells, low toxicity, and ability to mimic iron when binding to biomolecules (Xu et al., 2014). A number of ruthenium complexes display unique antitumor properties, and the treatment is not accompanied by major drug-related side effects (Levina et al., 2009;Li et al., 2015). Due to the unique spectroscopic and electrochemical properties of Ru(II) bearing diamine 2,2 0 -bipyridyl (bpy) derivatives complexes, their interaction with DNA has been widely studied (Song et al., 2012), which is the basis for the design of new antitumor chemotherapeutic drugs (Shi et al., 2006). Additionally, in general, ternary complexes, i.e., mixed ligand metal ion complexes, can be considered as models (Dutta & Bhattacharya, 2000) for enzymemetal ion-substrate complexes (Soliman et al., 2019). Furthermore, such ternary complexes probably occur in biological fluids that contain several ligands as well as different metal ions.

Synthesis of V-shaped Schiff bases
The Schiff bases (L 1-4 ) are prepared according to, previously reported procedure . The synthetic route to L 1-4 is shown in Scheme 1.
2.4. General procedure for the synthesis of mixed ligand complexes (1-4) Synthesis of complexes (1-4) consist of two steps. The first step is the preparation of a [Ru(bpy) 2 Cl 2 ] precursor. The compound was prepared according to the literature (Sullivan et al., 1978). 1.0 mmol of cis-[Ru(bpy) 2 Cl 2 ] and appropriate Schiff base ligand (1.0 mmol) in ethanol (20 mL) was refluxed with constant stirring for 13 h. The reaction mixture was then allowed to cool, and the solvent was removed by slow evaporation. The isolated crude product was then washed several times by hot petroleum ether, recrystallized from hot water then dried under vacuum. The synthetic route for Ru(II) complexes (1-4) is depicted in Scheme 2.

Pharmacological activity
The biological activity analysis was carried out in central laboratory, Faculty of Science, Al-Azhar University, Egypt.

Antioxidant activity evaluation
A standard method is employed using using a,a-diphenylb-picrylhydrazyl (DPPH) free radical (Jagadeesh et al., 2015) to evaluate the radical scavenging activity of complexes, which is based on the conversion of DPPH into 1,1-diphenyl-2-picrylhydrazine. First, a qualitative approach was done to check whether the complexes are active or not by applying $ 50 mg/mL of the complex as a spot-on TLC plate and after the development of chromatogram using MeOH mobile phase, 0.2% DPPH (w/v) solution is sprayed on the plate. A yellow spot on the purple background indicates the antioxidant activity. For the quantitative estimation of scavenging activity, in different test tubes, 1.0 mL of each complex solution (5-25 mM), 3.0 mL of DPPH solution (0.1 mM) was added, and the mixture was shaken vigorously for $ 5 min. After 20 min of incubation in dark room, the absorbance of test solutions was recorded at 517 nm (purple color with e ¼ 8.32 Â 10 3 L mol À1 cm -1 ) at room temperature. The control experiment was carried out as above without the test samples. Ascorbic acid was used as standard, whereas DPPH was used as positive control and Tris-HCl buffer (pH 7.2) was used as negative control. The reduction of DPPH was calculated relative to the measured absorbance of control. The DPPH radical scavenging activity of the compounds was calculated using the Equation (1): where A 0 is the control absorbance (blank), and A c is the sample absorbance. All the analyses were made in triplicate for each and the results were compared with control. 1.7-1.9, indicating that CT-DNA was sufficiently free of protein (Chalkidou et al., 2012). The DNA concentration per nucleotide (NP) was determined by absorption spectroscopy using the reported molar extinction coefficient of DNA, 6600 L mol À1 cm -1 at 260 nm (Purtaş et al., 2016).

Preparation of tris-hydrochloride buffer solution.
Tris-HCl (197 mg, 5 mM) and NaCl (730 mg, 50 mM) were weighed accurately and made up to 250 mL in a Standard Measuring Flask using double distilled water. The pH of this solution was adjusted to 7.2 using 1 mM NaOH solution with the help of pH meter (EUTECH INSTRUMENT, pH 510) before making up. This buffer pH 7.2 was used for all DNA studies.

UV-visible absorption titration experiments.
In electronic absorption titration experiments, a fixed amount of each Ru(II) complex (4.0 mM) was titrated by increasing the concentrations of CT-DNA (0.0-12 mM). In order to measure the absorption spectra, an equal amount of DNA was added to both the test solution and the reference solution to omit the absorbance of DNA itself. Before recording the absorption spectra, the solutions were allowed to incubate for 10 min. Quantitative DNA-binding affinities of the complexes are estimated by calculating their intrinsic binding constants (K b ) with CT-DNA using the using modified Wolfe-Shimer Equation (2) (Jahani et al., 2016): 2.5.2.3. Competing binding fluorescence studies. The fluorescence titration experiment has been widely used to understand the mode of DNA interaction with the complexes (Mansouri-Torshizi et al., 2017). In this experiment, in a fluorescence cell containing 2.0 mL Tris-HCl buffer, 50 mL of CT-DNA (5 mM) and 50 mL aqueous EtBr (5 mM) were added and allowed to equilibrate for 15 min at room temperature. After equilibration time, the EtBr-DNA solution was excited at 471 nm at which maximum quantum yield for EtBr was achieved. Then, the effects of each Ru(II) complex on the emission intensity of interacted EtBr-DNA adduct were studied by adding increasing concentrations of complex (0-15 mM). Using the data pertaining to each complex and Equation (3), the Stern-Volmer quenching constant, K SV (a measure of the effectiveness of the metal complex as quencher) was determined (Yousefi et al., 2015) using Equation (3): where I o and I are the emission intensity in the absence and presence of Ru (II) complexes. The relative binding affinity of the complexes with DNA was determined by measuring the quenching constant (K SV ) from the slopes of the lines from the plot of fluorescence ratio I o /I versus [Ru (II) complex]. To further elucidate the binding extent, apparent DNA binding values (Kapp) were analyzed by Equation (4) (Patra et al., 2007): where K EB (1.0 Â 10 7 M -1 ) is the DNA binding constant of EtBr, the concentration of EtBr is 10 mM, and [complex] is the concentration of the compound used to obtain at 50% decrease in the fluorescence intensity of EtBr.
2.5.2.4. Viscosity measurements (hydrodynamic measurements). For further identification of the binding mode, viscosity measurements were carried out using Ubbelohde viscometer using an Ubbelohde viscometer at a temperature of 30.0 ± 0.1 C in a thermostatic water bath (Patra et al., 2021). Viscosities of CT-DNA at different complex concentrations dissolved in Tris-HCl buffer (pH 7.2) and to minimize complexities arising from CT-DNA flexibility, CT-DNA samples with an approximate average length of 200 base pairs were prepared by sonication (Ali et al., 2020). Titrations were performed by adding Ru(II) complexes to CT-DNA (10 lM) and the concentration of Ru(II) complexes gradually increased from 0 to 20 lM. The relative viscosity of DNA in the presence and absence of the metal complex was calculated from Equation (5): where t 0 and t are the flow time observed in the absence (Tris-HCl buffer alone) and presence of Ru(II) complexes. For each sample, average flow time was obtained in triplicates, using a digital stopwatch. The data are presented graphically as (g/g 0 ) 1/3 versus the binding ratio of [CT-DNA]/[Ru(II) complex, where g is the viscosity of the CT-DNA in the presence of the complex and g o is the viscosity of the DNA alone.

In vitro anticancer activity
Antitumar activity of all complexes' toward carcinoma cell lines (MCF-7, HCT-116, and Hep-G2) was tested by the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Ferrari et al., 1990). The cell lines were obtained from National Cancer Institute, Cairo University. Cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 mg/mL gentamycin at 37 C in a humidified atmosphere of 5% CO 2 and were sub-cultured two times a week. Standard 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay procedures were used to estimate the capacity of tested Ru(II) complexes to interfere with the growth of HCT-116, MCF-7, and Hep-G2 cell lines. Cell lines with a 5 Â 10 4 -10 5 cell/well were precultured into 96-well microtiter plates for 24 h at 37 C. Cis-platin, Ru(II) complexes dissolved in the culture medium with 1% DMSO were added in microwells containing the cell culture at final concentrations of 0, 1.56, 3.125, 6.25, 12.5, 25, and 50 lM. Then, each well was loaded with 10 lL MTT solution (5 mg mL À1 in phosphate buffer saline, pH ¼ 7.4) for 4 h at 37 C. Control wells contained supplemented media with 1% DMSO. The insoluble formazan was dissolved in 100 lL DMSO, and the cell viability was determined by recording the optical density (OD) of each well that was measured at 570 nm a Bio-Rad 680 microplate reader (Bio-Rad, USA) with an ELIZA microplate reader (Meter Tech. R 960, USA). . All experiments were performed in triplicate, and the percentage of cell viability was calculated according to Equation (6): The IC 50 value is the concentration required to produce 50% inhibition of cell growth. It was calculated using the linear fit equation of the linear part of the cytotoxicity graphs (cell viability versus concentration) for each compound. The values of IC 50 were used for the comparison of the cytotoxicity and chemotherapeutic characteristics among the synthesized compound.

Computational studies
All computations were carried out using Gaussian 09 W software (Frisch et al., 2009). All the calculations were performed using the hybrid density functional theory (DFT-B3LYP) method. Double zeta plus polarization basis set 6-31 G (d,p) for organic ligands atoms and LANL2DZ basis set for ruthenium complexes were used to calculate the geometries of the molecules. Energy gap, electronegativity, chemical hardness, chemical softness, chemical potential, electrophilicity index, ionization potential, and electron affinity were calculated by using the HOMO and LUMO energy values.

Molecular docking of compounds
The docking studies were performed using Molecular Operating Environment (MOE) package version 2016.08. Xray crystal structure of a B-DNA dodecamer d(CGCGAATTCGCG) 2 running 30-50 direction (PDB ID: 1BNA) at 1.9 Å resolution (Drew et al., 1981). The DNA structure was opened in MOE, hydrogen atoms were inserted, and subsequently energy optimization was carried out. The resulting model afforded to systematic conformational research using default parameters in the Site Finder tool implicated in MOE software.

Results and discussion
Four Ru(II) mixed complexes of general formula [Ru(bpy) 2 (L 1-4 )Cl]Cl are synthesized via thermal reaction between the precursor [cis-Ru(bpy) 2 Cl 2 ] and four V-shaped Schiff bases ligands (L 1-4 ). All of the complexes are air-stable for extended periods. The complexes are remarkably soluble in water and common polar organic solvents.The structures of the complexes were characterized by physicochemical techniques including, elemental analysis, FT-IR, 1 H NMR, and ES-Ms. The elemental analyses are shown in Table 1, which are in good agreement with the proposed formula (calculated). The molar conductivities in DMSO solution indicate that the complexes are in the range expected for 1:1 electrolyte (Ali et al., 2013).

Infrared spectra
The FT-IR spectra of the ruthenium complexes (1-4) were recorded in the region 4000-400 cm -1 . The FT-IR spectra of the complexes are dominated by vibrational bands associated with bpy and Schiff base ligands coordinated to Ru(II) center. At first, the IR spectra of the free Schiff base ligands L 1-4 showed a strong absorption band at around 1669-1681 cm -1 , which are assigned to the stretching vibration of the m (C¼O) acetyl group are nearly unchanged upon complexes formation due to non-participating in coordination but became characteristic with weak intensity due to change in conjugation system in complex relative to that in the parent free ligands. The coordination of the Schiff bases in the titled complexes is indicated by the appropriate downward shift of strong stretching vibrations around the region 1602-1627 cm -1 , which are assigned to the stretch vibration of the azomethine group m (HC¼N) were shifted to 1595-1596 cm -1 upon complexation, reflecting coordination of the nitrogen atom of the Schiff base to ruthenium (Booysen et al., 2014). Furthermore, the characteristic stretching vibration due to m (C¼N) ring stretching in bpy was shifted to lower wave numbers (1548-1558 cm -1 ) after the formation of the titled mixed complexes (Sun et al., 2015). This clearly indicates that the nitrogen atoms of bpy ligand participate in the coordination to Ru(II) ion (Ye et al., 2010). Furthermore, the appearance of new weak non-ligands around 422-423 cm -1 and 441-487 cm -1 was assigned to vibration bands of (Ru-N) is supportive of the involvement of N donor atoms of HC ¼ N and C ¼ N, in both the Schiff base and bpy, respectively (Mishra et al., 2008). The vibrational frequencies and their assignments for the bpy and Schiff base ligands as well as its ruthenium complexes are given in Table 2. The FT-IR spectra of the ligands and their Ru(II) complexes are delineated in supplementary materials as Supplementary Figure S1-S8.

1 H NMR spectra
The 1 H NMR spectra of Ru(II) compounds clearly show the diamagnetic nature of these Ru(II)-low-spin complexes, confirming the reduction of the Ru(II) ion upon the reaction of [cis-Ru(bpy) 2 Cl 2 ] with the co-ligands L 1 -L 4 . The 1 H NMR signal assignment for Ru(II) complexes (1, 3; 2 & 4) are shown in Figures 1-4, respectively, and the data are given in Table 3. The signal numbered as 8 (orange) at d ¼ 8.94, 8.97, 8.88, and 8.83 ppm was assigned to imine (-CH ¼ N) proton upon complexation from the Schiff base ligands. Additionally, the NMR region above 6.5 ppm exhibits a series of superimposed signals. The signals assigned by the number 3, 3 // 0 , 4, 4 // , 5, 5 // , 6, and 6 // (blue) are attributed to hydrogens present on bpy ligand. The signals numbered by 10, 11, 13, and 14 (blue) correspond to aromatic ring bearing dimethyl amine or dimethoxy groups. Finally, the signals numbered by 16, 18, 19, and 20 in complexes 1 and 2; 16, 17, 18, and 20 (red), correspond to aromatic ring bearing acetyl group. The signals numbered by 3 close to 2.0 ppm correspond to aliphatic hydrogens.

Electron spray ionization mass (ESI-MS) spectra
ESI-MS spectroscopy has proven to be very helpful for identifying metal complexes with high molecular masses. The empirical formula of the complexes was established by ESI-MS spectrometry. The complexes 1 and 2 are considered as representative examples (Figures 5 and 6). The mass spectra of both complexes exhibit well-defined molecular ion peak due to molecular ions [M] þ which is in good agreement with the stoichiometric composition of these complexes.

Electronic absorption spectra
The UV-Vis spectra relevant to synthesized complexes (1.0 mM) were recorded at room temperature in Tris-HCl  buffer (pH 7.2) solution in region 200-800 nm. The UV-vis spectra of the complexes (Figure 7) displayed distinctive bands at about 300 nm and 378-384 nm corresponding to p!p* and n!p* transitions of ligands, respectively. Additionally, all Ru(II) complexes display a broad absorption band in the region 558-562 nm, which is ascribed to  ruthenium(II) d!p* MLCT in all respective complexes (Zhang et al., 2018). The similarity between the spectra established similar geometry and electronic environment in all complexes.
Based on the analytical, spectroscopic data (FT-IR, UV-Vis, 1 H NMR, and ESI-mass), the following tentative mixed mononuclear octahedral structure has been proposed for all of the new complexes ( Figure 8).

DPPH radical scavenging ability
Oxidative reactions of biological molecules induce a variety of pathological events such as cellular injury and the aging process, and these damaging events are caused by free radicals (Shobana et al., 2015). The antioxidant activity of an inhibitor mainly depends on the way it participates in neutralizing the radical centers that are generated in the biological systems by donating an electron or hydrogen. The  structure and properties of the inhibitor play a prominent role in showing the activity. Therefore, to prevent free radical damage in the body, it is important to administer drugs that may be rich in antioxidants. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay was used to evaluate the antioxidant activities of the synthesized complexes according to the established method. The results of the DPPH radical scavenging activity for the compounds on the basis of percent inhibition are presented in Figure 9. The results have shown that a considerable increase in the percent of scavenging activity is found with increase in the concentration of the complexes and generally, displayed better DPPH radical scavenging activities compared to the ascorbic acid (AA). A critical examination of the values indicates that Ru(II) complex (3) is found more efficient in decolorizing the pink color of the DPPH solution than other complexes. Moreover, based on IC 50 (Figure10) which lie in the range of 5-25 mg/mL, the complexes (1-4) can be ranked as follows: 3 > 4 > 1 > 2. Those values allow the possibility of using the new complexes for further studies in the design of drugs for the treatment of pathological diseases arising from oxidative stress.

DNA binding studies
To design effective chemotherapeutic agents and effective anticancer drugs, DNA interaction with ruthenium ternary complexes is currently being investigated (Thota et al., 2018).

UV-Vis. absorption titration. The interaction of the
Ru(II) complexes (1-4) with CT-DNA helix in the absence and presence of different concentrations of the Ru(II) complexes was monitored by UV spectroscopy since the changes observed in the UV spectra upon titration may provide evidence of the existing interaction mode . The observed decrease in absorption intensity, with change in the peak positions (bathochromic), revealed the intercalative binding mode between complexes and CT-DNA (Karami  et al., 2015). As can be seen in Figure 11, after increasing amounts of CT-DNA to complexes solutions (1-4), the MLCT bands exhibited a gradual reduction in the maximum absorbance (hypochromism) with a red shift by about 5, 3, 9, and 7 nm for the complexes (1), (2), (3), and (4), respectively. Such features are characteristic to DNA intercalation binding due to the involving a strong p-p Ã stacking interaction between an aromatic intercalative ligand and the base pairs of CT-DNA . To determine quantitatively the CT-DNA binding strengths of the complexes, the intrinsic binding constants (K b ) were calculated by Equation (1) (Supplementary Figures S9-S12). The calculated K b values in the interaction of the complexes (1-4) with CT-DNA are found to be 7.71 Â 10 5 M -1 , 4.70 Â 10 5 M -1 , M -1 , 9.12 Â 10 5 M -1 , and 7.88 Â 10 5 M -1 , respectively. The K b values obtained here for DNA are higher than those reported for classical intercalator (EB-DNA, 3.3 Â 10 5 M -1 in 20 mM Tris-HCl, 20 mM NaCl, pH 7.2), which is indicative of a  stronger binding of DNA with the reported Ru(II) complexes than those of the classical intercalators (Mallepally et al., 2016). The intrinsic binding constant values (K b , M -1 ) of all Ru(II)complexes (1-4) with CT-DNA, the hyperchromatism extent (H%) and red shift (Dk, nm) have been summarized in Table 4.

Fluorescence quenching measurements. Next, to
further support the mode of interaction of Ru(II) complexes (1-4) to CT-DNA, competitive-binding versus ethidium bromide (EtBr) were monitored. EtBr is a sensitive fluorescence probe for CT-DNA structure detection, and its fluorescence intensity is increased in the presence of CT-DNA due to strong intercalation between the DNA base pairs (Protogeraki et al., 2014). Subsequently, the changes observed in the fluorescence emission spectra of a solution DNA-induced EtBr upon the addition of the complexes may be used to investigate the ability of the compounds to displace EtBr from the EtBr-DNA adduct. Emission spectra of EtBr bound to CT-DNA during titration with the increasing concentration were monitored in the range of 525 to 725 nm, where the emission intensities of complexes were found to be very small and negligible (Figure 12). The spectra show that, upon successive addition of metal complex to EB-DNA adduct, the fluorescence intensity is reduced decreases with increase in the concentration of complexes, reflecting the competition of complexes with EtBr for DNAbinding. Moreover, the quenching of fluorescence intensity is an indication of binding of complexes to the hydrophobic pockets of DNA and chelates can be protected efficiently by the hydrophobic environment inside the DNA helix. Since EtBr intercalates DNA through interactions with the minor groove, the displacement of EtBr (confirmed by the decrease in fluorescence) can be suggestive of an intercalative or minor groove binding (Deepika et al., 2013). Quantitatively, the fluorescence quenching constants (K SV ) of the complexes have been calculated using the Stern-Volmer equation, and the values are 1.87 Â 10 5 M -1 , 2.24 Â 10 5 M -1 , 3.45 Â 10 5 Â 10 5 M -1 , and 2.52 Â 10 5 M -1 for (1), (2), (3), and (4) complexes, respectively. The Stern-Volmer plot between I o /I and [CT-DNA] for the quenching of the fluorescence is shown in inset graph in Supplementary Figure S13-S16. This implies that (3) exhibits stronger DNA-binding properties than other complexes, consistently with UV-Vis's titration data. To further elucidate the binding extent, the apparent binding constant Kapp was calculated. The trend in Kapp values of 1-4 is in line with the trend in the K b values obtained from the UV-vis absorption spectral studies (Table 4). 3.5.2.3. Viscosity measurements. Although optical photophysical methods are employed to offer essential information about the binding monitor the binding mode of Ru(II) complex with the DNA, they are lacking evidence to support an intercalative binding model (Pandya et al., 2019). Hydrodynamic measurement which is sensitive to the change in length (i.e., viscosity) is considered as one of the least uncertain and most critical tests of the binding model in a solution (Sun et al., 2008). The effects of complexes on the viscosity of CT-DNA were also studied. The plot of (g/g 0 ) 1/3 versus [DNA]/[complex] gives a measure of the viscosity change. As illustrated in Figure 13, a significant increase in the relative viscosity of CT-DNA solution was observed upon addition of all complexes, suggesting intercalation binding nature of the complexes (Hmoud Alotaibi & Abdalla Momen, 2020). Such an intension in viscosity is consistent with that, the complexes classically intercalate between the doublestranded DNA, that causes the unwinding and lengthening of the said DNA to accommodate the bound complex, leading to the increase of DNA viscosity (Gao et al., 2006). The viscosity results are consistent with the above spectroscopic results, where the increased degree of relative viscosity caused by complex (1) is stronger than other complexes.

In vitro antiproliferative activity
Cell viability study is the primary procedure to understand the cytotoxicity profile of any new chemical entity (Bollu et al, 2019). The positive results obtained from the DNA binding for complexes (1-4) encouraged us to test their anticancer  properties. The chemotherapeutic potential of Ru(II) complexes (1-4) was evaluated by measuring their abilities to kill HCT-116 MCF-7, and Hep-G2 cells (Figure 14) using cis-platin as a reference drug control. With regard to selectivity, the prepared Schiff base complexes have a positive impact against both cancer cell lines, cancer cell lines, and in both cases the impact was higher than that of their subsequent Schiff base ligands. The results revealed that all the Ru(II) complexes attenuated the three types of cancer cells proliferation in different selectivity, and the inhibition effects were enhanced in a concentration dependent fashion. As a measure of therapeutic potential, the inhibitory concentration of the tested compounds and standard drug (cis-platin) required for 50% cell inhibition (IC 50 ). The corresponding IC 50 values were calculated for complexes (1-4) using the cell viability against the concentration of the complexes and are given in Table 5. The analysis data indicated that these Ru (II) complexes have selectivity for different cancer cell lines. It has been found that complex (1) has the most powerful anticancer effect against colon cancer (HCT-6 cell lines) and breast cancer (MCF-7 cell lines), with an IC 50 value of 21.61 mM. With respect to breast cancer (MCF-7 cell lines), Ru(II) complex (3) exhibits the best activity with IC 50 ¼ 27.24 mM. In addition, with respect to Hep G2, the antitumor activity order can be arranged as follows: (1) < (2) < (4) < (3), with IC 50 values of 25.42 lM, 25.31 lM, 23.44, and 21.31 lM, respectively. These observations reflect the effect of the studied compounds on changing the morphology of the cancer cell lines, and this behavior may be associated to the higher DNA binding affinity. Consequently, the prepared complexes can be used as effective potential anticancer drugs.
3.6. Stereochemistry and chemical reactivity prediction 3.6.1. Molecular orbital calculations The stereochemistry and molecular optimization of the most stable structures of L 1 , L 2 , [Ru(bpy) 2 L 1 Cl] þ , and [Ru(bpy) 2 L 2 Cl] þ derivatives were investigated using the   density function theory (DFT). It was focused first on the structure of the Schiff base molecules; more specifically, the orientation of its functional groups with respect to each other and with respect to the central azomethine (HC ¼ N) moiety. The optimized structure of the Schiff bases showed that they adopted a characteristic V-shaped conformation at the azomethine nitrogen ( Figure 15). The C2-N1-C4 angle of the azomethine group was 128.70 and 128.74 for L 1 and L 2 , respectively. Also, the C2 ¼ N1 bond length was found to be 1.29 Å for both the two ligands. These bond displacements are shorter than the normal single C-N bond and indicated the presence of double bond character (Tsai et al., 2013). The X-ray structural of the Schiff base (E)-1-(4-((4-bromobenzylidene)amino)phenyl)ethanone (similar to the reported ligands) showed that the C ¼ N bond length is 1.27 Å, which is comparable to the reported ligands . Also, the bond length of C ¼ N in the imine group of the Schiff base N'-(2-hydroxybenzylidene)-2phenylacetohydrazid was found to be 1.289 Å (Ramadan et al., 2021). Furthermore, the C15-O17, C21-O18, and C20-O19 bond lengths in L 2 ligand ( Figure 15) were found to be 1.25 Å, 1.47 Å, and 1.47 Å, which confirmed the presence of a double and a single bond characteristic for the two groups. From Figure 15, it can be observed that the structure is not planar, where the two phenyl moieties were bent in a reverse direction to reduce the steric repulsion with a dihedral angle between the two planes found to be approximately 76.8 . This orientation suggested a monodentate characteristic of the ligands to be able to coordinate ruthenium through the azomethine nitrogen. All the other bond   lengths and angles were found to be in the normal ranges reported before (Sankaraperumal et al., 2013). The optimized structures of the two ruthenium complexes [Ru(bpy) 2 L 1 Cl] þ and [Ru(bpy) 2 L 2 Cl] þ are presented in Figure  16. In these complexes, the Ru(II) species adopted a distorted octahedral structure and coordinated to two bipyridine and a chloride moiety along with one Schiff base ligand. The distortion of the octahedral arrangement was confirmed from the computed M-N bond lengths (1.94 Å for the Ru-Bpy and 1.96 Å for Ru-L). The experimental bond distances for Ru-N, which were found to be common in different ruthenium complexes, ranged from 1.94 Å to 2.19 Å (El-Samanody, 2018). On the other hand, the calculated Ru-N distances for some ruthenium complexes were found to be in the range of 1.8-1.9 Å (Adeniyi & Ajibade, 2016;Al-Noaimi & AlDamen, 2012). The bond angles between the metal species and the binding sites in the coordination sphere vary between 71.66 and 177.18 . The lowest bond angles in the coordination sphere were those contained the bipyridine chelates (71.66-78.63 ). The bond length between Ru and Cl in the reported complexes was found to be 2.25 Å. The common experimental bond separation for Ru-Cl in different ruthenium complexes ranged from 2.30 Å to 2.37 Å (Mohamed et al., 2018), while the calculated Ru-Cl distances for some ruthenium complexes were found to be in the range of 2.17-2.21 Å (Cebri an-Losantos et al., 2008).

Quantum global reactivity descriptors
The global properties of the reported compounds including HOMO and LUMO, energy gap (Eg), chemical hardness (g), electronegativity (v), chemical potential (V), electron affinity (A), ionization potential (I), and chemical softness (S) were computed and presented in Table 6 (Raja et al., 2012). The Eg separation between the HOMO and LUMO of the compounds characterizes the molecular chemical reactivity. The smaller energy gap reflects the ease of the charge transfer and polarization within the compound. The donating properties, E HOMO , of the reported two derivatives follow the order L 1 > L 2 > [Ru(bpy) 2 L 1 Cl] þ % [Ru(bpy) 2 L 2 Cl] þ . Also, the accepting properties, E LUMO , follow almost the same order: L 1 > L 2 > [Ru(bpy) 2 L 1 Cl] þ > [Ru(bpy) 2 L 2 Cl] þ and, thus, it is the order of increasing chemical reactivity. From the HOMO and LUMO energies, ionization potential, and electron affinity are expressed as I$ -E HOMO and A $ -E LUMO . The variations in electronegativity (v) values are sustained by the electrostatic potential. The results showed that the order of decreasing electronegativity, i.e., increasing charge transfer within the

molecules, is [Co
Smaller g values for the two Schiff base derivatives imitated the ability of charge transfer inside the molecules. The order of increasing charge transfer within them is [Ru(bpy) 2 L 2 Cl] þ > [Ru(bpy) 2 L 1 Cl] þ > L 1 > L 2 .

Molecular docking of some ligands and complexes
Molecular docking is an excellent tool to understand the interaction between molecularly designed bioactive molecules (promised drugs) and biological macro target. Analysis of the docking data is useful in predicting the conformational changes associated with the amino acid residues at the binding position to accommodate the docked hydrophobic inhibitors. The two ligands L 1 and L 2 and the two complex ions, [Ru(bpy) 2 L 1 Cl] þ , [Ru(bpy) 2 L 2 Cl] þ , as examples, were subjected to molecular docking studies using the MOE version 2016.08 to understand the compound-DNA interactions and to explore the potential binding mode and energy. The docked ligands conformations were rated according to the binding energy, hydrogen bonding, and hydrophobic interactions between the ligands and the complexes, and the B-DNA (PDB ID:1BNA). The docking studies determine the way by which the docked compounds fundamentally fit in the DNA minor groove and comprise of hydrophobic, ionic, and hydrogen bonding interactions with the DNA bases. It was found that the optimal docking results were mostly in the guanine-cytosine (GC) region, and with less extent in adenine region. These theoretical studies support the experimental findings of the fluorescence quenching and viscosity measurements, which indicated the intercalative mode for DNA interaction. Figure 17 indicated that the free ligands L 1 and L 2 showed very good binding scores with high negative e-values, which represented high binding affinity between the receptor and ligand molecules and consequently indicated the high efficiency of these ligands as bioactive compounds (-5.70 and -5.32 kcal/mol for L 1 and L 2 , respectively). In the case of L 1 , the binding interaction comes from hydrophobic interaction between the amino acid residues such as DG A12, DG B16, DC B15, DC A11, and DA B18 with the aromatic and methyl moieties of the ligand. In addition, a hydrogen bond was formed between the ketonic oxygen of the ligand and DG A10 moiety. On the other hand, the binding interaction of L 2 came from the hydrogen bond formed between DG B16 and the oxygen of ketone group, interaction of DA B17 with the phenyl moiety and the hydrophobic interaction between the amino acid residues of the DNA such as DG A12, DG A10, DC A9, and DA B18 with the aromatic and methyl moieties of ligand L 2 . In case of the two ruthenium complexes, they also exhibited good binding scores (-5.57 and -4.60 kcal/mol for [Ru(bpy) 2 L 1 Cl] þ and [Ru(bpy) 2 L 2 Cl] þ , respectively). From Figure18, it can be noticed that the two complexes interacted with the DNA from almost the same regions like the free ligands. Both the two complexes interacted hydrophobically with the DG, DC, and DA regions. Also, the ketonic oxygen of L 1 and L 2 parts in the two complexes underwent hydrogen bonding with DG B22 region of the DNA. Therefore, it can be concluded that these bioactive ligands and complexes were able to interact with the available binding sites of the target proteins effectively. The different biological studies as well as molecular docking were correlated to each other and supported the fact that the complexes can bind to DNA via intercalative mode and showed various DNA binding potency. The compounds may, therefore, be considered as promising potential drugs for therapeutic intervention in various diseases.

Conclusions
In summary, four new mixed ligands Ru(II) complexes have been synthesized and characterized via physicochemical, spectral analysis as well as DFT and molecular docking studies of some Ru(II) were performed. The scavenging activity of the compounds toward DPPH radical was noteworthy. The interaction of compounds with CT DNA indicated that investigated complexes exhibited good greater DNA binding. Moreover, in vitro cytotoxicity of the complexes toward HCT-116, MCF-7, and Hep G2 cell lines showed significant cytotoxicity against selected cell lines. The order of cytotoxicity inhibition differs from cell line to each other, reflecting the selectivity of the as synthesized complexes. Briefly, it was concluded that the reported complexes may be used as promising anticancer agent in future.