Dithiocarbazate Ligand-Based Cu(II), Ni(II), and Zn(II) Complexes: Synthesis, Structural Investigations, Cytotoxicity, DNA Binding, and Molecular Docking Studies

S-4-methylbenzyl-β-N-(2-methoxybenzylmethylene)dithiocarbazate ligand, 1, prepared from S-(4-methylbenzyl)dithiocarbazate, was used to produce a novel series of transition metal complexes of the type, [M (L)2] [M = Cu(II) (2), Ni(II) (3), and Zn(II) (4), L = 1]. The ligand and its complexes were investigated by elemental analysis, FTIR, 1H and 13C-NMR, MS spectrometry, and molar conductivity. In addition, single X-ray crystallography was also performed for ligand, 1, and complex 3. The Hirshfeld surface analyses were also performed to know about various bonding interactions in the ligand, 1, and complex 3. The investigated compounds were also tested to evaluate their cytotoxic behaviour. However, complex 2 showed promising results against MCF-7 and MDA-MB-213 cancer cell lines. Furthermore, the interaction of CT-DNA with ligand, 1, and complex 2 was also studied using the electronic absorption method, revealing that the compounds have potential DNA-binding ability via hydrogen bonding and hydrophobic and van der Waals interactions. A molecular docking study of complex 2 was also carried out, which revealed that free binding free energy value was −7.39 kcal mol−1.


Introduction
Coordination compounds have a long history of use as chemotherapeutic medicines and have advantages over organic compounds. e bioinorganic mechanism and standard pharmacokinetic parameters of uptake, distribution, and excretion are used to assess the potential of coordination compounds as effective anticancer agents (as drugs or modifiers of biological response) [1]. Furthermore, medicinal chemists can investigate the potential of metalcontaining compounds using a variety of strategies, such as coordination compounds with different coordination numbers, geometries, oxidation states, and ligand substitution thermokinetics [2,3]. Cisplatin (cis-diamminedichloroplatinum(II)), a platinum-based bifunctional reagent, is an extremely effective chemotherapeutic drug used to treat a variety of malignancies. However, despite their excellent efficacy, these drugs are only used in limited circumstances due to their toxicity, side effects, and acquired resistance in patients [4]. erefore, to improve therapeutic effectiveness and avoid drawbacks, there is a need for novel platinum and non-platinum-based compounds with potential chemotherapeutic properties [5].
Copper, nickel, and zinc are bio-essential elements and are required for performing various biological functions in the human body. In the field of non-platinum compounds with anticancer properties, complexes with copper, nickel, and zinc have provided a promising alternative to platinum in the development of anticancer drugs, exhibiting activity on cisplatin inactive tumours and the potential to be successful against platinum-resistant malignancies in patients. In addition, copper is also essential for the activity of cytochrome oxidase, superoxide dismutase, ascorbate oxidase, and tyrosinase, enzymes, and proteins involved in energy metabolism, respiration, and DNA synthesis [6]. Furthermore, these metal complexes are likely to have different modes of action, bio-distribution, and toxicity than platinum-based medicines. In addition, the design of ligands associated with metal ions has a considerable impact on drug transport to the target position [7,8]. Schiff bases, also known as "privileged ligands," have a characteristic imine linkage and are frequently used as pharmacophores for drug design because of their easy synthesis and high solubility in organic solvents. Furthermore, Schiff bases exhibit significant biological activity owing to the interaction of azomethine nitrogen with protein amino acid residues or DNA nucleobases [9][10][11].
Over the past several years, dithiocarbazate Schiff bases have gained considerable interest because of their promising bioactivities against various cancer cells [12][13][14][15]. However, the biological applications of these compounds become changed by the addition of different organic substituents, resulting in even minor structural modifications [12,[16][17][18]. Furthermore, dithiocarbazate Schiff bases coordinate with the metal ions to form a wide range of metal complexes with numerous biological applications [12,13,[15][16][17][18]. erefore, considering the diverse significance of dithiocarbazate Schiff bases and their role in a variety of biological applications, herein we are interested to design a novel dithiocarbazate Schiff base ligand ( Figure 1) and its complexes with Cu(II), Ni(II), and Zn(II) ions. e ligand and its complexes are characterized by various spectroscopic methods and singlecrystal X-ray crystallography in the case of ligand and complex 2. All the synthesized compounds have been tested to evaluate anticancer activity against MCF-7 and MDA-MB-213 cancer cell lines. Single-crystal X-ray diffraction and Hirshfeld surface studies were used to better define the geometric structure of 1 and 3. e experimental data were investigated using density functional theory (DFT), and the shape of complex 2 was optimized for molecular docking simulations using DFT. An electrothermal digital melting point equipment was used to measure the melting points, whereas the magnetic susceptibilities were determined at 25°C using a Sherwood Scientific MSB-AUTO magnetic susceptibility balance. A JENWAY 4310 conductivity meter was used to determine the molar conductivities of the complexes in DMSO at 10 −3 M at 27°C. PerkinElmer Spectrum 100 with universal ATR polarization was used to measure FTIR spectra at 4000-280 cm −1 . A LECO CHNS-932 analyzer was used to perform analyses of C, H, and N. A Perkin-Elmer Plasma 1000 Emission Spectrometer was used to do metal measurements. Shimadzu UV-1650PC UV-Visible Spectrophotometer was used to measure the electronic spectra at 1000-200 nm. An NMR JNM ECA400 spectrometer was used to record the 1 H and 13 C NMR spectra of 1, while those of 3 and 4 were determined by a Bruker Ascend ™ 700 MHz spectrometer. A Shimadzu GC-MS QP2010 plus mass spectrometer was used to collect the mass spectra.

Synthesis of S-4-Methylbenzyl
e dithiocarbazate derivative, S-(4-methylbenzyl)dithiocarbazate (S4MBDTC), was synthesized using a reported procedure [12,18,19]. S4MBDTC (2.12 g, 0.01 mol) was dissolved (100 mL) in the mixture of hot ethanol and acetonitrile (Scheme 1). An equimolar amount of 2-methoxybenzaldehyde was added to the hot dithiocarbazate solution and refluxed at 70°C for 5 h and then stirred for another 5 h until a precipitate is formed at room temperature, which was then filtered and dried over silica gel, yielding yellow crystals on recrystallization in acetonitrile.
Yield   2 Zn.2H 2 O] in 1:1 molar ratio (Scheme 1). e reaction mixture was stirred and refluxed at 70°C for 3 hours, which was subsequently allowed to cool at room temperature, resulting in the formation of a precipitate. e precipitate was separated and purified by drying it over silica gel, followed by recrystallization in methanol. We were successful in obtaining single crystals for 3. However, despite our best efforts, we failed to grow single crystals suitable for single-crystal X-ray diffraction for 2 and 4.

Single-Crystal X-Ray Structure Determination.
e intensity data for the single crystal of the ligand, 1, were obtained on a CrysAlisPro Oxford diffractometer fitted with graphite monochromated Cu-K radiation (λ �1.54Å), at 296 K, whereas the measurements for complex 3 were processed on a CCD area detector APEXII Duo diffractometer operating at 50 kV and 30 mA with Mo-Kα radiation (λ � 0.71073Å) at 296 K.
SAINT and APEXII software packages were used to integrate the data, and the SADABS tool was used to perform the empirical absorption correction [20]. Direct methods using SHELXS-2014 were applied to find the solution and subsequently refined by applying the full-matrix leastsquares method to F2 using SHELXL-2014 [21]. X-Seed was used as a graphic interface for SHELXL [22]. Non-hydrogen atoms were refined anisotropically, while the refinement for hydrogen atoms was isotropic. Table 1 shows the entire crystal data and refinement details. Bioinorganic Chemistry and Applications 3

Density Functional eory (DFT) Calculation.
Gaussian09 [23] and GaussView5 [24] software packages were used to conduct DFT calculations. e DFT approach was used to fully optimize the structure of complexes 2 and 4 utilizing the B3LYP [25,26] hybrid exchange correlation functional with LanL2DZ pseudopotential on Cu and Zn [27][28][29] and the 6-311G (d, p) Pople basis set for all other atoms. e initial structures and geometries of 1 and 3 were obtained from single-crystal X-ray diffraction analysis. e DFT calculation for 1 and 3 was analysed with the same functional and basis sets. A scaling factor of 0.9682 was used to scale vibrational frequencies [30]. e HOMO-LUMO energies of the optimized geometries were calculated with time-dependent DFT (TD-DFT) measurements on the same basis set [31,32], and the solvation effects (DMSO) were added using the polarizable continuum method (PCM) [33][34][35].

Hirshfeld Surface Analysis.
e Hirshfeld surfaces and 2D fingerprint plots were generated using CrystalExplorer 17.5 [36]. e structural input files for 1 and 3 were obtained in the CIF format. A normalized contact, denoted by d norm , is a ratio that takes into account the distances between any surface point and the closest internal (d i ) and exterior (d e ) atoms, as well as the van der Waals radii of the atoms.

Cell Culture and Viability
Assay. ATTC, Virginia, USA, provided human breast cancer cells with both estrogenpositive (MCF-7) and triple-negative estrogen (MDA-MB-231) receptors. Cells were grown at 37°C in RPMI-1640 (high-glucose) media with 10% fetal bovine serum comprising 1% penicillin in a moistened environment of 5% CO 2 in air. e 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the inhibitory effect of the ligand, 1, and its complexes 2-4 on the proliferation of breast cancer cells [12,18,37]. It was observed that ligand, 1, and its complexes 2-4 were nontoxic to cancer cells when they were dispersed in DMSO and diluted in culture medium to a final DMSO concentration of less than 0.05% (v/v). Cells were sown in 96-well plates at a density of 6000 cells per well for 24 hours before being exposed to various doses of chemicals for 72 hours. e wells were rinsed with 200 μL of phosphate buffer saline after the medium was removed. Each well was filled with aliquots of 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and incubated at 37°C for 4 hours. Following that, each well that had 2 μL of the medium was replaced with 200 μL of DMSO. An ELISA plate reader was used to determine optical density at 570 nm. In all of the experiments, there were control wells (100% viability) filled with medium and DMSO. e average of triplicate assays was used to generate all of the data points. GI 50 was used to measure cytotoxicity, which was defined as the concentration that reduced the absorbance of treated cells by 50% when compared to the control (untreated cells) [38].

DNA-Binding Studies via UV-Visible Absorption
Spectroscopy. UV-Vis absorption studies were carried out to ensure the CT-DNA interaction of the ligand, 1, and complex 2 at room temperature. roughout the absorption titrations, the concentration of the bioactive complex 2 solutions was kept constant at 50 μM, while the concentration of CT-DNA was gradually increased (4.21 × 10 −5 M). At room temperature, the complex was dispersed in DMSO and diluted in Tris-HCI buffer [39], with wavelength measured at 230 to 600 nm.
e absorbance values were taken ten minutes after the DNA solution was injected. e following equation was used to calculate the binding constant, K b : where [DNA] denotes the base-pair concentration, ε a is the apparent molar extinction coefficient A abs / [M], and ε f is the extinction coefficient for free complex 2 [M], while ε b is the extinction coefficient for the completely bound complex 2. e DNA-binding absorption studies followed the same procedure as those described in our earlier publications [19].

Molecular Docking.
e Protein Data Bank [40] provided the coordinates for dodecamer d (CGCGAATTCGCG) 2 of B-DNA (PDB ID : 1BNA). e coordinates of 2 were determined after utilizing the DFT approach to minimize energy. e AutoDock Tools version 1.5.6 and 4.2.5.1 programs were used for molecular docking investigations [41]. Water molecules were removed from the receptor and replaced with polar hydrogen atoms and Kollman charges (DNA sequences). e AutoDock parameter file was updated to include van der Waals interactions and other 2-specific parameters retrieved from the AutoDock website [42,43]. e conformation with the lowest binding energy from the highest cluster was chosen as the best ligand-receptor binding conformation [19,44].

Results and Discussion
e synthesis of dithiocarbazate Schiff base, 1, and its complexes with Cu(II), Ni(II), and Zn(II) ions is shown in Scheme 1. e isolated complexes were obtained in good yield (70-83%) and were soluble in DMSO, DMF, acetone, and chloroform at room temperature. e molar conductance values for the isolated complexes at 0.30-2.04 Ω −1 cm 2 mol −1 suggested the complexes to be nonelectrolytic [45].
e IR spectra of the ligand, 1, exhibit the characteristic bands due to v (NH) at 3086 and 3105 cm −1 [46,47]. However, the stretching bands due to v (NH) disappeared when ligand, 1, is complexed with metal ions, suggesting its deprotonation [46,47]. Furthermore, the strong band appearing at 1600-1595 cm −1 in the spectra of the ligand is assigned to v (C�N) vibration [46][47][48][49][50][51][52], which, however, shifted to lower wavenumber, suggesting the complexation of 1 to metal(II) ions via azomethine nitrogen atoms [46][47][48][49][50][51][52]. However, the same pattern was observed for the hydrazine v (N−N) band in the spectra of metal(II) complexes, suggesting the coordination of the ligand, 1, via the nitrogen lone pair, where the electron delocalization occurs through the 5-membered chelate ring [46,47]. e disappearance of vibrations due to v (C�S) and the appearance of the splitting v (CSS) bands in the spectra of complexes provide strong evidence that the ligand has been coordinated to metal ion through the thiolate sulphur [49,53,54]. e 1 H NMR spectrum of 1 did not show any signal at ca. δ 4.00 ppm, attributed to S-H proton, confirming that 1 exists as thione tautomer even in solution [55]. e occurrence of a proton connected to the nitrogen atom is shown by a singlet signal at 13.29 ppm, which, however, was absent in the spectra of complexes 3 and 4, indicating the coordination of the Schiff bases to the metal ion. e 13 C NMR spectrum of 1 showed a downfield chemical shift at δ ∼ 196 ppm, indicating the existence of the C � S thione tautomer in solution. In the spectra of complexes 3 and 4, there was an upfield shift of this signal, suggesting a drop in electron density at the carbon atom as a result of the sulphur atom complexing with metal ion, confirming the structures suggested for the complexes [46][47][48][49][50][51][52] (Supplementary Information Figures S5-S10).
e MS spectral data revealed that the molecular ion peak identified in the spectra matches the proposed structure of the ligand, 1. e mass spectra revealed the presence of a molecular ion peak [C 17 Figure S11). e UV-Vis spectra of 1 in DMSO at 10 −3 M showed a high-intensity peak at 356 nm, which was assigned to n ⟶ π * and π ⟶ π * transitions and agrees well with the TD-DFT electron excitation at 348 nm [56]. is excitation of electrons occurs at lone pair of nitrogen atoms and aromatic phenyl ring (Figure 2). For 2-4, the HOMO is mainly located on the 3-methoxybenzyl backbone of each dithiocarbazate Schiff bases and the metal centre, whereas the LUMO mainly resides on 3-methoxybenzyl and backbone of dithiocarbazate Schiff bases. Table S2 contains the UV/Vis data for all compounds.

Hirshfeld Surface Analysis.
e intermolecular interaction of crystal structures of 1 and 3 was quantified through the Hirshfeld surface analysis and 2D fingerprint plots using CrystalExplorer 17.5 [36]. e Hirshfeld surface mapped over d norm 1 and 3 is shown in Figures 6(a) and 6(b), respectively. ere are bright-red spot regions in 1, demonstrating the occurrence of a strong hydrogen bond connecting the neighbouring molecules via N2-H2· · ·S1.

DNA-Binding Studies
3.5.1. Absorption Spectroscopic Studies. UV-Vis absorption spectral titrations were performed to explore the interactions between cytoactive complex 2 and CT-DNA. Figure 7 shows the absorption spectra of 2 in the absence and presence of CT-DNA as a function of concentration. Complex 2 exhibited a strong absorption peak at λ max 342, corresponding to π ⟶ π * intra-ligand transition of the aromatic chromophore.
e intensity of the prominent absorption was reduced as the concentration of CT-DNA was increased, and a bathochromic shift (1 to 5 nm) was observed in 2. e changes reveal that the aromatic chromophore of 2 exhibits promising interactions with the aromatic base pairs of the DNA, suggesting a good binding with a 25.51% hypochromic shift. e intrinsic binding constant (K b ) of 2 was 2.55 × 10 4 M −1 , which is consistent with previous reports showing intermediate binding interactions between complexes and CT-DNA [63]. e obtained K b values were lower than those of the classical intercalator (ethidium-DNA, 3 × 10 6 M −1 ) [64]. is indicates that 2 has a less binding affinity to the CT-DNA than ethidium bromide [63][64][65].

Molecular Docking
Simulation. Molecular docking simulation plays a significant role in the development of new chemotherapeutic drugs. It is used to understand the interactions between synthesized drugs and DNA, supporting the experimental investigation of DNA binding. e conformation of docked complex 2 was analysed based on binding energy and non-covalent (hydrogen bonding, major or minor groove, and electrostatic) interactions between complex 2 and DNA. e labelled DNA duplex of sequenced (CGCGAATTCGCG) 2 dodecamer used is given in Figure 8(a). e molecular docking of 2 with DNA duplex yielded the most suitable docked poses (Figure 8(b)). As illustrated in Figure 8(c), we can express that the 2 fit closely into the G-C region of DNA via hydrogen bonding and hydrophobic and van der Waals interactions with binding energy −7.39 kcal/mol. is demonstrates the higher binding affinity in the G-C region compared with the adeninethymine (AT)-rich region with a binding energy of −4.38 kcal/mol. e G-C region is crucial in DNA stability   because three hydrogen bonds stabilize guanine and cytosine, and it has been suggested that targeting these regions could have a key role in anticancer activity [66,67]. Complex 2 forms two carbon-hydrogen bonds between the C atom of complex 2 with phosphate groups of adenine-18 and cytosine-11 with the bond distance of 3.32 and 3.44Å, respectively. e other non-covalent bonds between 2 and DNA bases are shown in Figure 8(c). Significant cytotoxicity was observed, although DNA-binding studies revealed that complex 2 did not strongly bind to DNA. e mechanism of action of complex 2 remains unknown and is a subject for future research.

Conclusions
A number of physicochemical studies and single-crystal X-ray diffraction analysis were performed to investigate the dithiocarbazate Schiff base ligand 1 and its complexes 2, 3, and 4. e in vitro cytotoxic activity of the synthesized compounds against MCF-7 and MDA-MB-231 cells was tested. However, only complex 2 demonstrated significant cytotoxic potency against both cancer cells. e absorbance and molecular docking simulations were used to further investigate the DNA-binding studies of 2, which reveal that 2 binds to DNA rather effectively via hydrogen bonding and hydrophobic, and van der Waals interactions. e obtained data correlate well with the cytotoxicity of the complex, indicating that 2 has the potential to be a promising anticancer candidate.

Data Availability
CCDC 2106915 (1) and 2106916 (3) contain all crystallographic data and can be obtained free of charge via the Cambridge Crystallographic Data Centre.