Synthesis, Spectroscopic, X-ray Diffraction and Theoretical Studies of some Novel Nickel(II) Chelates of a Heterocyclic Based Tridentate NNO donor Aroylhydrazone: In vitro DNA Binding and Docking studies

Five new nickel(II) complexes have been synthesised with an NNO donor tridentate aroylhydrazone (HFPB) employing the chloride, nitrate, acetate and perchlorate salts, and all the complexes were physiochemically characterized. Elemental analyses suggested a deprotonated iminolate form of the ligand in four of the complexes, however in one case ( 2 ), two aroylhydrazone moieties are binding to the metal centre in the neutral and anionic forms. The structure of the bisligated complex 5, characterized using X ray diffraction studies affirmed that the metal has a distorted octahedral N 4 O 2 coordination environment, with each of the two ligands coordinating through the pyridine nitrogen, imino-hydrazone nitrogen and the deprotonated oxygen of the hydrazone moiety. In order to compare and study the elctronic interactions and stabilities of the metal complexes various quantum chemical parameters were calculated. Moreover, Hirshfeld surface analaysis was carried out for complex 5 in order to determine the intermolecular interactions. The biophysical attributes of the ligand and complex 5 have been investigated with CT DNA and experimental outcomes show that the Ni(II) complex exhibited higher binding propensity towards DNA as compared to ligand . Furthermore, in order to specifically understand the type of interactions of the meatal complexes with DNA molecular docking studies were carried out. In addition, the electronic and related reactivity behaviors of the ligand and five Ni(II) complexes were effectuated using B3LYP/6-31++g**/LANL2DZ level. As expected, the obtained results from NBO computations displayed that the resonance interactions (n→π * and π→π *) play a determinant role in evaluating the chemical attributes of the reported compounds.


INTRODUCTION
The coordination chemistry of nickel proves to be an outgrowing field which provoked extensive research because of the anomalous behavior of Ni(II) containing metal complexes [1]. The tetra-coordinated Ni(II) complexes proved to be promising candidates, with numerous examples possessing several exploitable physical and chemical properties including redox, optical, magnetic, catalytic and electronic activity [2][3][4][5]. To explain this, diverse models have been introduced which include solute-solvent interactions, magnetically non-equivalent sites in the unit cell, configurational equilibrium and so on [2]. Numerous nickel dependent enzymes are found throughout the natural world and are catalysts in many crucial biosynthetic routes [6]. The reaction mechanism of all the enzymatic reactions proved that the catalytic property of nickel atom present in the active site of an enzyme can be attributed to its redox activity [7]. Moreover, in the hardness to softness scale, Ni(II) is a borderline element which accounts for the formation of covalent bonds with the nitrogenous bases and phosphate groups of DNA [8]. It is found that nickel atoms covalently bind to the N7 atom in guanine and adenine which proved the strong interactions of nickel-based metal complexes with DNA [9,10]. This property of nickel-based complexes is well exploited in the development of pharmacophores which possess antineoplastic properties, which in turn are largely used in cancer therapy.
Aroylhydrazones are polydentate ligands with donor atoms integrated into an aromatic chromophore. They cover a wide range of functions and complexity, and they have been proven to have substantial biological activity, making them significant in a variety of metabolic processes. The hydrazone functional group is undoubtedly inevitable since it has uses in a multitude of areas, from medicinal chemistry to supramolecular chemistry, and has been utilized as a catalyst in a variety of organic syntheses [11]. These compounds are derived from the condensation reaction between hydrazides and carbonyl compounds which includes aldehydes and ketones [12]. Their popularity among the scientific community can be attributed to its simple synthetic procedure, stability towards hydrolysis and modularity [13]. The physical and chemical properties of hydrazones are determined by the azomethine group characterized by the triatomic structure (-C=N-N-) [14]. The structural characteristics that make the hydrazone group a unique functional moiety is the presence of an imine carbon that is both nucleophilic and electrophilic, a nucleophilic imine and amino type nitrogen, and acidic hydrogen attached to the amino nitrogen [15]. In comparison with simple hydrazones, aroylhydrazones containing heterocyclic rings possess additional donor sites which present a more extensive scope of properties for these molecules [16]. Pyridine-based hydrazones have drawn a lot of interest among heterocyclic aroylhydrazones because they are frequently employed in asymmetric catalysis and serve as a good ligand with a high degree of flexibility in coordination [17].
In the present study, a detailed characterization of the newly synthesised metal complexes has been performed by combining experimental and high-level theoretical calculations in order to understand the structure and properties to an electronic level [18]. The theoretical studies carried out provide all the indispensable information about the structure, bonding, electron distribution and reactivity of the metal complexes. Moreover, the study of the interactions between transition metal complexes and DNA offers the opportunity to explore systematically how factors like molecular shape and hydrogen bonding stabilize the test molecules on DNA.
These minuscule molecules bind to DNA through a series of weak interactions, including pistacking interactions associated with intercalation of aromatic chromophore between base pairs, hydrogen bonding and van der waals interactions of functionalities bound along the DNA helical groove. The physicochemical properties of ligand and one of the complexes (5) were studied using CT DNA, and the results showed that the complex had a higher affinity for DNA binding than the ligand. The findings are expected to spur the development of a newer metal complex with DNA as its ultimate target.
In this work, five novel Ni(II) complexes derived from HFPB were synthesised and characterised using several physicochemical methods. In particular, DFT calculations were performed to validate the experimental results and to explore the structural properties in detail.
Further, hirshfeld surface analysis was also carried out, as this provides an additional insight into weak intermolecular interactions influencing the packing of molecules in crystals. Notably in this work we could also develop a halogen bonding directed molecular assembly in one of the complexes as evident from its crystal structure.

General Remarks
All starting materials were commercially available and were used without further purification. Elemental analyses of the aroylhydrazone and the complexes were performed

Synthesis of the aroylhydrazone and its Ni(II) chelates
The aroylhydrazone of our interest N′- benzohydrazide monohydrate (HFPB·H2O) was synthesized by adapting a previously reported procedure [19] via condensation between 3-flouropicolinaldehyde and the corresponding aroylhydrazide (Scheme 1). The synthesis and crystal structure of HFPB·H2O have been published earlier [20]. To validate the utility of this methodology, the reaction was also performed with different stoichiometric ratios of the reactants.

[Ni(FPB)(NO3)]·2H2O (1)
This complex has been prepared by mixing hot methanolic solution of HFPB·H2O (0.2612 g, 1 mmol) with methanolic solution of nickel(II) nitrate hexahydrate (0.2908, 1 mmol) (Scheme 2). The resulting mixture was then refluxed for 5 hours. The solution was then cooled at room temperature and green color product obtained was filtered, washed with methanol followed by ether and dried over P4O10 in vacuo.

[Ni(HFPB)(FPB)]Cl (2)
A solution of HFPB·H2O (0.2612 g, 1 mmol) in methanol was treated with a methanolic solution of nickel(II) chloride hexahydrate (0.2377 g, 1 mmol). The resulting green color solution was then refluxed for 4 hours. The solution was cooled at room temperature and after slow evaporation, brown colored product separated out was filtered, washed with methanol followed by ether and dried over P4O10 in vacuo. (2)

[Ni(FPB)(OAc)(DMF)] (3)
A solution of HFPB·H2O (0.2612 g, 1 mmol) in methanol was treated with a methanolic solution of Ni(OAc)2·4H2O (0.2480 g, 1 mmol). The resulting solution was then refluxed for about 4 hours. The precipitated complex was then recrystallized from DMF, brown colored product separated out was filtered, finally washed with ether and dried over P4O10 in vacuo.

[Ni(FPB)(ClO4)]·DMF (4)
To a methanolic solution of HFPB·H2O (0.2612 g, 1 mmol), nickel(II) perchlorate hexahydrate (0.3650 g, 1 mmol) dissolved in methanol was added. The resulting solution was stirred and refluxed for about 5 hours. On slow evaporation at room temperature, green color needle shaped crystals were separated. The precipitated complex was then recrystallized from DMF, filtered, washed with methanol followed by ether and dried over P4O10 in vacuo.

[Ni(FPB)2] (5)
A methanolic solution of Ni(OAc)2·4H2O (0.1240 g, 0.5 mmol) was treated with a solution of HFPB·H2O (0.2612 g, 1 mmol) in methanol. The resulting solution was then refluxed for 4 hours. The brown product formed immediately was filtered. The solution was cooled at room temperature and after slow evaporation, brown colored product separated out was filtered. It was then recrystallized from DMF solution, dark brown shining crystals separated were filtered, finally washed with ether and dried over P4O10 in vacuo.

X-ray crystallography
Single crystals of compound 5 of X-ray diffraction quality were grown from its DMF solution by slow evaporation at room temperature in air. A single crystal of suitable dimensions of the complex 5 was selected and mounted on a Bruker SMART APEXII CCD diffractometer with graphite-monochromated MoKα (λ = 0.71073 Å) radiation at the Sophisticated Analytical Instruments Facility (SAIF), Cochin University of Science and Technology, Kochi-22, Kerala, India. The crystallographic data and structure refinement parameters are given in Table 4.3. Bruker SMART software was used for data acquisition and Bruker SAINT software for data integration [21]. Absorption corrections were carried out using SADABS based on Laue symmetry using equivalent reflections . The cell refinement was done using APEX2 and SAINT. The data was reduced using SAINT and XPREP. The structure was solved by direct methods using SHELXS97 and refined by full-matrix least-squares refinement on F 2 using SHELXL2018/1 [22]. The molecular and crystal structures were plotted using DIAMOND version 3.2g [23].

Hirshfeld surface analysis
It is possible to get a clear picture of intermolecular interactions present in the molecule from Hirshfeld surface and 2D fingerprint analysis [24]. Crystal Explorer v 3.1 software package is used for the analysis. The electronic distribution results from the sum of isotopic atom electron densities are the concept behind HSs. Three functions, dnorm, shape index and curvedness are used for the mapping of HSs. dnorm is the normalized contact distance which indicates the ratio covering the distance of any surface point to the nearest interior (di) and exterior (de) atom and the van der Waals radii (r vdw ) of the atoms. It is given by the expression

Computational aspects
All DFT/B3LYP [25,26] computations of HFPB precursor and five Ni(II) complexes were performed by using the G09W package [27] at LANL2DZ [28] for heavy atoms and 6-31++g** [29,30] for the remaining atoms. In theoretical chemistry, the Koopmans' Theorem [31] is the first important step in the prediction of the chemical reactivity behavior through using the FMO energies to calculate the ionization energy "I" and electron affinities "A" as follow

DNA binding studies
The interaction of DNA with Ni(II) complex of octahedral geometry (5) is studied as it is found to be a potential candidate due to its geometry and steady state emission. The binding efficiency of complex was monitored using absorption and fluorometric studies.

Electronic absorption titration
The exposing CT DNA is sufficiently free from protein contamination.

Fluorescent spectroscopic studies
To examine whether the complex can displace ethidium bromide (EB) from its CT-DNA-EB complex, the fluorescent titration was carried out. The EB solution was made from Tris HCl/NaCl buffer, maintaining the pH at 7.

Docking Studies
Docking Server system was used in carrying out all the docking calculations [40]. Geometry optimization of the complexes was carried out using the MMFF94 method. The Gasteiger charge calculation method was applied to the complexes. A pH which is equal to 7.0 is preferred in all calculations. Grid maps 90 × 90 × 90Â (x, y and z) and Lamarckian genetic algorithm (LGA) methods were used in the placement calculations [41]. At the time of insertion, the population size was set to 150. A translation step of 0.2 Å and a 5 Å quaternion and torsion steps were applied during the search for the appropriate region of the target protein of the molecules studied.

Synthesis, molar conductivity, and magnetic susceptibility measurements
The complexes were obtained from direct reaction between the metal salts and the ligand using methanol as solvent. Elemental analyses suggested a deprotonated iminolate form of the ligand is present in most of the complexes, however in few cases ligand is binding to the metal center in the neutral amido form, with the anions of the metal salt satisfying the Ni(II) valency. The complexes prepared were green or dark brown in color and are found to be readily soluble in solvents like DMF and DMSO but less soluble in solvents like ethanol, methanol and chloroform. All the isolated complexes were quite stable and could be stored for several weeks without any appreciable change. X-ray quality single crystals of one of the complexes, [Ni(FPB)2] (5) were obtained by the recrystallization of the compound from its DMF solution.
Molar conductivity of the complexes was measured in the 10 -3 M DMF solution and were found to be non-electrolytic in nature except complex 2 which is a 1:1 electrolyte [42]. The molar conductivity value of this complex is found to be in the range 87 ohm -1 cm 2 mol -1 .
The magnetic properties of Ni complexes provide valuable information for distinguishing their geometry. All the nickel complexes were found to be paramagnetic which excludes the possibility of a square planar configuration. At room temperature the effective magnetic moments (μeff) of complexes 2, 3 and 5 are found to be in the range 2.6-2.8 B.M., consistent with octahedral geometry for these complexes [43]. In regular octahedral complexes of Ni(II), consideration of spin-orbit coupling and contribution from the 3 A2g and the next higher 3 T2g states give a higher magnetic moment than the spin-only moment of 2.83 B.M. For complexes 1 and 4, magnetic moments were found to be in the range 2.8-3.2 B.M. which are slightly higher than their spin only values, suggesting some orbital contributions in this case and are similar to that of other reported tetrahedral Ni(II) complexes [44].

Infrared spectroscopy
The IR spectrum of the hydrazone and their Ni(II) complexes were recorded in the solid state as KBr discs. The characteristic IR bands for the free ligand when compared with metal complexes provide meaningful information regarding the bonding sites of the ligands. The selected IR bands of the hydrazones and complexes are represented in Table 1. The IR spectrum of the aroylhydrazone exhibit two bands in the range 3058 and 1682 cm -1 due to the ν(NH) and ν(C=O), respectively, and these bands are absent in the case of complexes.
This indicates that the ligand coordinates to the metal in an iminolate form. The aroylhydrazone has a band at 1597 cm -1 due to azomethine group. This suffers a shift to lower wave number in the complexes. This indicates the coordination of azomethine nitrogen to Ni(II) in the complexes [14].
According to Stefov et al., the broad band above 3200 cm -1 in complex 1 indicates the presence of lattice water and a new medium intensity band in the region 1353 cm -1 may be assigned to ν(C-O) stretching vibration [45]. The IR spectrum of [Ni(FPB)(NO3)]·2H2O (1) (Fig. S1) exhibits two bands at 1448 and 1325 cm -1 corresponding to N=O stretching vibrations of the nitrate ion.
The separation of these bands by 120 cm -1 indicates the unidentate nature of the nitrate ion in the complex [43]. respectively. The frequency difference Δν of acetate group is found at ~150 cm -1 which is consistent with the bidentate nature of the acetate group [46]. In complex 4 (Fig. S4), the band at 1597 cm -1 assignable to ν(C=N) in the free ligand was shifted to lower values indicating its participation in the chelation with π-electron delocalization. The perchlorate assignments in and ν4(ClO4) respectively [47]. Both show considerable splitting due to a reduced symmetry of the perchlorate species suggesting some interaction of perchlorate anion ClO4with the metal. In complex 5 (Fig. S5), the ligand coordinates the metal center with nitrogen atoms from the pyridine and imino moieties and one oxygen atom coming from its iminolic counterpart.

Electronic spectroscopy
The electronic spectral assignments of the complexes in DMF solutions are summarized in Table 2 and shown in Fig. 1. The absorption spectrum exhibits a charge transfer transition (CT) at 22000-29500 cm -1 region which may be assigned to the ligand-to-metal charge transfer from the coordinated unsaturated ligand to the metal ion. The absorption bands due to n→π* and π→π* transitions of free hydrazone suffered considerable shift upon complexation and the bands at ~ 36100 cm -1 can be assigned to shifted intraligand transitions. This is due to the weakening of the C=O bond and the extension of conjugation upon complexation [48]. T1(P)← 3 T1(F); however we could not locate any of these bands, probably hidden under highintensity charge transfer bands [43]. The electronic spectra of the complexes in DMF solution are not well resolved owing to very intense charge transfer bands extending into visible portion of spectra.

Crystal structure description
Dark brown crystals of complex [Ni(FPB)2] (5) suitable for X-ray analysis were grown by recrystallization of the compound from dimethylformamide in a week time. Crystal data and structural refinement for the complex 5 are tabulated in the Table 3. A perspective view on the molecular structure of compound 5 along with atom numbering scheme is presented in Fig. 3 and selected bond lengths and bond angles are summarized in Table 4. The complex 5 crystallized in the monoclinic system with the space group P21/c.    The packing of the molecules is also stabilized by some other weak intermolecular contacts C-H···π, C-F···π and π···π interactions (Fig. 5) and are presented in Table S10. The π-stacking interactions found between the aromatic rings of adjacent molecules connect them in a head to tail fashion with a Cg•••Cg contact distance of 3.7048(17) Å. In addition to the weak π-stacking interactions, significant C-F···π and C-H···π interactions further reinforce the unit cell packing (Fig. 6).

Hirshfeld surface analysis
CIF from the SCXRD studies is used to map Hirshfeld surfaces with different functions. The surfaces are kept transparent so that we can easily observe the complexes. We get globularity and asphericity of the surfaces from the HSs. Globularity describes the deviation from spherical surface. Anisotropy of objects is obtained by term asphericity [52].

NBO Analysis
The optimized geometry of HFPB and the corresponding nickel complexes (1)(2)(3)(4)(5) with the original atom numbering scheme are given in Fig. 10. The results of the second orderperturbative energy analyses of the reported compounds are provided in Table S11.
Although the influence of electron delocalization in lowering the stabilization energy was determined to be smaller than the resonance interactions, as expected, the anomeric interactions contributed significantly towards the stabilization energy.
It is clear from the Table S11 that

Chemical reactivity and Frontier molecular orbitals
In contemporary research, the electronic-structure-related quantum chemical parameters have been implemented successfully to evaluate the reactivity phenomenon of many kinds of chemical species which include a wide spectrum of molecules from basic systems [55,56] to complex molecular structures [57,58]. All the calculated physical and QCPs of precursor HFPB and five Ni(II) complexes are given in Table 5.  The electrophilic and nucleophilic attack regions of precursor HFPB and five Ni(II) complexes are given in Fig. 11  there is an increase in electron density over the phenyl rings when compared to the ligand.

Electronic absorption titration
Fixed amount (63 μL, 10 -5 M) of metal complex (5)  LMCT transition bands respectively (Fig. 12a). These spectral characteristics reveal an intercalative mode of binding of the complex to DNA helix through a strong stacking between aromatic hydrazone ligands and DNA base pairs [59].
In the present study, the intrinsic binding constant (Kb) for the complex (5) is calculated following the hypochromism of LMCT band observed at 372 nm. The Kb value of complex 5 is found to be 0.8418x 10 4 M -1 (Fig. 12b), however, the observed Kb value of the complex is much less than that of the classical intercalators like EB (Kb=7.7 x 10 7 M -1 ), confirming a nonclassical mode of intercalation, similar to other reported compounds in the literature (Table 6) [60]. The octahedral complex binds to DNA in three dimensions, the extended aromatic surface of the Ni complex due to the presence of two hydrazone moieties intercalate into DNA base pairs and the π* orbital of the intercalated complex would be expected to couple with π orbitals of base pairs resulting in hypochromism. .

Fluorescence spectroscopic studies
Ethidium bromide (EB) demonstrates a dramatic enhancement of its fluorescence when complexed to DNA and in the present study EB replacement studies by the addition of test molecule (complex 5) as a quencher have been carried out to support the binding of complex to DNA.
Results of fluorescence measurements obtained for the present system (5) are consistent with a mechanism for quenching that does not involve appreciable displacement of ethidium from the nucleic acid. Instead, ethidium ion and the complex simultaneously bind to DNA forming an adduct and the enhanced hydrophobicity due to the flanking aromatic rings in the DNA bound complex would be expected to perturb DNA helix thereby displacing EB [61]. Fig. 13a shows the emission spectra of the DNA-EB system with incremental addition of the metal complex. The addition of the test molecule to the DNA-bound EB solution caused an appreciable reduction in emission intensity and the fluorescence data were analysed using the Stern-Volmer equation (Eq. 3, Fig. 13b).
The binding strength of the test molecule was also evaluated in terms of equilibrium DNA binding constant, Kb, employing Scatchard equation, which includes number of binding sites, by regression analysis (Fig. 13c).
The KSV, Kq, Kb and n values for the interactions of 5 with DNA are shown in Table 6. The experimental results show that Ni(II) complex displays strong binding propensity with DNA which also validates the electronic absorption spectral results.  maintains genomic stability and prevents tissue reduction in multiple organs. In some cancer types, it provides anti-cancer drug resistance caused by cross-links between DNA strands [62].
The binding energies of the investigated compounds 1-5 according to their preferred location on human DNA (PDB ID: 4REA) and the interaction types with the nucleotides at the binding sites resulting from their interaction with DNA are given in Table 7. The binding modes of compounds 1-5 interacting with DNA are shown in Fig. 14. hydrophobic, π-π interactions and halogen bonds with DNA and got effectively intercalated between the DNA base pairs. Hydrogen bonding contributes significantly towards binding energy. It is clear from Table 4 that complex 4 forms two hydrogen bonds with DNA base pairs which results in high binding energy. Moreover, hydrophobic interactions of the test compounds with DNA are quite prominent with the amino acid residues of the target proteins.
The test compounds also exhibit stacking interactions with DNA due to the extended aromatic surfaces that can effectively form π-π interactions with DNA base pairs [63].
When the interaction poses of the investigated complexes and target proteins representing DNA binding were examined, it was observed that the hydrazone ligands forming the skeletal structure were in approximately the same region of the DNA groove. The nitrogen and oxygen atoms of hydrazone ligands are involved in strong hydrogen bonding interactions with the nucleotides of DNA. Phenyl groups in all compounds favor π-π stacking interactions as well as hydrophobic and polar interactions with amino acid residues. Moreover, the fluorine atom present in the ligand is found to form halogen bonds with the DNA base pairs. Altogether, hydrogen bonding, hydrophobic interactions, π-π stacking and halogen bonding contribute significantly towards the binding of the respective metal complexes with DNA.

Conclusion
The synthesis, characterization, and theoretical studies of some novel Ni (II) chelates  and DNA. Additionally, molecular docking studies reveals the existence of various interactions like hydrogen bonding, hydrophobic, π-π stacking and halogen bonding between the metal complex and DNA base pairs.