Anticancer self-assembled metal-organic triangle complex and process for preparation thereof

Abstract

The present invention relates to a transition metal coordination complex, for therapeutic purposes, particularly in combating breast cancer. More particularly, the present invention relates to a trinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1), wherein M is Zn(II), L is di deprotonated form of a novel thiosemicarbazone ligand, N⁴-cyclohexyl-2-(2- hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) and DMF acts as an ancillary ligand stabilizing the coordination environment; and process for preparation thereof. Further, the present invention relates to an anticancer composition comprising a trinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1), against breast cancer cells. The complex 1 of the present invention is cytotoxic in nature and induce cell death of the cancer cells by apoptosis. The in vitro cytotoxicity of the complex against MCF-7 cell lines reveals an IC₅₀ value of 10.65 μM, which is found ~3.5 times better than that of the standard cisplatin (36.668 μM).

Description

FIELD OF INVENTIONThe present invention relates to a transition metal coordination complex, for therapeuticpurposes, particularly in combating breast cancer. More particularly, the present inventionrelates to a trinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1),wherein M is Zn(II), L is di deprotonated form of a novel thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) and DMFacts as an ancillary ligand stabilizing the coordination environment; and process for preparationthereof. Further, the present invention relates to an anticancer composition comprising atrinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1), havingactivity against breast cancer cells. The complex of formula 1 of the present invention iscytotoxic in nature and induce cell death of the cancer cells by apoptosis.BACKGROUND OF THE INVENTIONCancer remains a significant global health challenge and imposes a substantialeconomic burden worldwide. According to the World Health Organization (WHO), cancerrelated deaths are projected to reach twelve million annually. The incidence of breast cancer(BC), the most prevalent malignancy among women, has risen significantly over the pastdecade, remaining a leading cause of cancer in western countries while becoming increasinglycommon across Asia. High degree of heterogeneity, encompassing up to ten distinct molecularsubtypes, presents a major obstacle in the development of universally effective therapeuticstrategies. Conventional chemotherapy continues to be the mainstay of breast cancer treatment;however, the availability of targeted molecular therapies remains limited. Effective clinicalmanagement is further complicated by the disease's clinicopathological heterogeneity and thescarcity of therapeutic options. In breast cancer research, in vitro studies using human cell linesprovide crucial initial insights into tumor treatment and response. Among these, the MCF-7cell line derived from human breast adenocarcinoma is one of the most widely used estrogenreceptor-positive (ER⁺) models for evaluating the cytotoxic and pro-apoptotic effects of newtherapeutic agents. It closely mimics luminal-type breast cancer, making it a suitable model forexploring hormone-responsive breast cancer biology and treatment. Apoptosis is essential forthe growth and homeostasis of multicellular organisms, as it ensures the elimination ofdamaged or unwanted cells. When cells with DNA damage evade apoptosis, they can surviveand contribute to cancer progression. Apoptosis is triggered by both external and internalsignals through two primary pathways, the extrinsic (death receptor-mediated) and the intrinsic(mitochondria-mediated) mechanisms. These pathways ultimately lead to the activation ofcaspases, which cause irreversible damage to essential cellular components like proteins andDNA, resulting in programmed cell death. Due to its critical role in eliminating cancer cells,apoptosis is a key target in cancer therapy, and considerable research has been devoted todeveloping apoptosis-inducing anticancer drugs. Cancer cells undergoing apoptosis typicallydisplay distinct morphological features, including membrane blebbing, chromatincondensation, and the formation of apoptotic bodies. Hence, the discovery of compounds thatdisrupt cancer cell growth is crucial, as they not only serve as potential anticancer agents butalso aid in understanding the molecular mechanisms of tumor progression. Since DNA is aprimary target for many cytotoxic agents, metal complexes capable of binding and cleavingDNA under physiological conditions have emerged as promising candidates for anticancertherapy. Over time, this has driven the development of numerous DNA-targeting drugs.Notably, studies have shown that polynuclear transition metal complexes (PTMCs) oftenexhibit superior DNA binding, cleavage, and cytotoxic effects compared to their mononuclearcounterparts.The introduction of cisplatin marked a major breakthrough in cancer chemotherapy,establishing the therapeutic potential of metal-based drugs. Despite its widespread clinical useagainst various cancers, cisplatin therapy is often limited by serious side effects and theemergence of acquired resistance with prolonged treatment. These challenges have promptedthe development of alternative metal complexes with improved efficacy and reduced toxicity.To this end, a wide range of metal-ligand systems have been synthesized and evaluated fortheir anticancer activity through both in vitro and in vivo studies. In the search for newanticancer agents, thiosemicarbazones have gained considerable attention over the years due totheir flexible coordination behavior and strong chelating ability with transition metal ions.Their ability to coordinate with metal ions such as Zn(II), Cu(II), Fe(III), and Ni(II) allows forthe formation of stable complexes that can interfere with cellular processes crucial for cancercell survival. Among their various biological applications, the cytotoxic potential ofthiosemicarbazones has been widely investigated, with many studies demonstrating theirability to induce apoptosis, inhibit ribonucleotide reductase, generate reactive oxygen species(ROS), and disrupt mitochondrial function. Moreover, structure activity relationship (SAR)studies have revealed that modifying the thiosemicarbazone framework or introducing halogensubstituents can significantly enhance their antitumor efficacy. Schiff base ligands containingiodine are extensively studied for their roles in organic synthesis and biotechnology. Notably,iodine-coordinated thiosemicarbazone complexes have demonstrated significant cytotoxic andpro-apoptotic activity. The presence of iodine is known to enhance inhibitory effects, therebyimproving efficacy against drug-resistant cancer variants. Zinc(II) plays a critical role innumerous cellular processes, primarily through its involvement in the catalytic function of awide range of metalloenzymes. It also contributes to cellular regulation as a neuromodulatorand exhibits cytoprotective properties by modulating apoptotic pathways. These biologicalroles have prompted interest in zinc complexes as potential anticancer agents. However, despitethe broad therapeutic potential of thiosemicarbazone ligands, their zinc- based complexesremain largely underexplored in the context of anticancer activity.Metal-assisted self-assembly is among the most powerful and versatile syntheticstrategies for constructing metal-organic complexes (MOCs), well defined, discrete twodimensional (2D) or three dimensional (3D) molecular structures formed through thecoordination of metal centers with multidentate ligands. These processes enable the formationof a wide range of distinct molecular topologies with aesthetically appealing and high structuralprecision. MOCs are particularly attractive for biological applications due to their tunabledimensions, the ability to select metal ions with specific sizes and coordination geometries, andthe ease with which their overall architecture can be controlled. Among the various transitionmetals, zinc is considered a suitable candidate for the construction of multinuclear complexesdue to its flexible coordination behavior, which complements the adaptability of ligand systems.The Zn(II) ion possesses a highly flexible coordination environment, enabling it to formcomplexes with a variety of geometries from tetrahedral to octahedral often displaying notabledeviations from ideal polyhedral shapes. A common method for synthesizing PTMCs involvesthe use of polypodal ligands, which possess several binding sites capable of coordinatingsimultaneously with multiple metal ions. Thiosemicarbazones, with suitable functionalities,represent a key class of such ligands, featuring a thiourea unit that imparts strong chelatingability and significant pharmacological relevance. They typically coordinate through the sulfuratom and hydrazine nitrogen, forming stable, often vividly colored complexes. Their versatilecoordination behavior makes them ideal candidates for assembling multinuclear MOCs. Someof the existing references are given below:CN101260121A discloses complex compounds of hetero aromatic ringthiosemicarbazones and transition metal, and discloses an application of the same for preparingantineoplastic drugs. The compounds of the invention are externally tested by a plurality oftumor cell lines, showing that the compounds have quite good inhibitory effects on tumors.Moreover, the compounds of the invention can also be made into a plurality of forms of drugcombination, such as tablets, capsules, or injections, etc.Shivendra Kumar Pandey, et al, Inorganica Chimica Acta, 2025, report di μ-acetatobridged Zn(II) complexes {[Zn(μ-ac)CyHCT]2 and [Zn(μ-ac)CyBHCT]2} containingthiosemicarbazone alias hydrazine-1-carbothioamide ligands {N-Cyclohexyl-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-carbothioamide (HCyHCT) and N-Cyclohexyl-2-(phenyl(pyridin2-yl)methylene)hydrazine-1-carbothioamide (HCyBHCT)}. The crystal structure ofcomplexes shows that the Zn(II) centre is coordinated in a distorted trigonal bipyramidalconfiguration by three thiosemicarbazone atoms (two N and one S) and by an O atom fromeach of the two acetate groups. Further, di μ-acetato bridges in [Zn(μ-ac)CyBHCT]2 areshowing linkage isomerism. The ligand and complexes were further studied for their anticanceractivities against the HT-29 (human colon) and DL (Dalton's lymphoma cells) cancer cells. Theresults imply that these compounds have superior cytotoxic activity against HT-29 cells.Olga Garbuz, et al, Molecules. 2025; 30(9):2077, explore thiosemicarbazones andisothiosemicarbazones as innovative pharmacological agents against cancer, unlocking theirfull potential, significantly enhancing cancer treatment protocols and improving patientsurvival rates.Paterson, et al, Angew. Chem. Int. Ed. 2017, 56, 8370 -8374, discloses a Zn(II) complexof the dianionic tetradentate ligand formed by deprotonation of glyoxal-bis(4-phenyl-3-thiosemicarbazone) (H2gtsp), which is a [3+3] trinuclear triangular prism. Recrystallization ofthis complex in the presence of either CO2, CS2, or CH3CN leads to the formation of [4+4]open-ended charge-neutral tetranuclear coordination nanotubes, approximately 2 nm in lengthand with internal dimensions large enough to accommodate linear guest molecules, which serveto template their formation. Upon removal of the templating molecules the nanotubesdemonstrated reversible sorption of CO2 with an isosteric enthalpy of sorption of 28 kJ mol-1at low loading.Buşra Kaya, et al, Chemico-Biological Interactions, Volume 351, discloses synthesis ofnew thiosemicarbazone-based zinc(II) complexes to study their cytotoxicity on A375malignant melanoma cells. The complexes containing salicylidene (Zn1a), 3-methoxysalicylidene (Zn1b) or 4-methoxy-salicylidene (Zn1c) moiety were characterized by analyticaland spectroscopic methods. Anticancer potential of the complexes was determined by MTTtest and HUVEC endothelial cells line was used to comprehend the effect on normal cells. Zn1bwith an IC50 of 13 μM was found to be highly cytotoxic against A375 cancer cells, moreeffective than cisplatin (IC50: 37 μM). Zn1a and Zn1c did not have a negative effect on cellviability in the normal cells and gave the impression that they are more advantageous thancisplatin in this respect.Despite recent advancements in metal-based complexes, there remains a critical unmetneed for novel, structurally precise polynuclear transition metal complexes that offer superiorcytotoxic and pro-apoptotic activity against breast cancer cells and provide an alternativetherapeutic mechanism to overcome the limitations associated with the systemic toxicity andacquired resistance of standard platinum-based drugs like cisplatin.The present invention addresses this need by providing a novel Zn(II)-based trinuclearcoordination complex as an anticancer compound, demonstrating highly potent and selectiveactivity against breast cancer cells via induction of apoptosis.OBJECTIVES OF THE INVENTIONThe primary objective of the present invention is to provide a trinuclear transition metalcoordination complex.Another objective of the present invention is to provide a self-assembled trinucleartransition metal coordination complex of formula [M3(μ-L)3·3DMF] (1).Another objective of the present invention is to provide a trinuclear transition metalcoordination complex of formula [M3(μ-L)3·3DMF] (1), wherein M is Zn(II), L is dideprotonated form of a novel thiosemicarbazone ligand, N4-cyclohexyl-2-(2- hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) and DMF acts as an ancillary ligand.Another objective of the present invention is to provide a process for preparation of atrinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1).Another objective of the present invention is to provide an anticancer compositioncomprising a trinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF](1).Another objective of the present invention is to provide a process for preparation of ananticancer composition comprising a trinuclear transition metal coordination complex offormula [M3(μ-L)3·3DMF] (1).Another objective of the present invention is to provide a novel thiosemicarbazoneligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L).One another objective of the present invention is to provide process for synthesis of anovel thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L).One another objective of the present invention is to synthesize self-assembled noveltrinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1) using a novelthiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L).Yet another objective of the present invention is to synthesize self-assembled noveltrinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1) using a novelthiosemicarbazone ligand building block, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) and DMF.SUMMARY OF THE INVENTION The present invention discloses a complex of Formula 1:[M3(μ-L)3·3DMF] (1),wherein M is Zn(II);L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and(2)DMF is dimethylformamide. In a feature of the present invention, the L or H2L is a tetradentate-ON2S donor bridgingligand. In a feature of the present invention, the complex of Formula (1) possesses anticanceractivity against breast cancer cells; and the complex of Formula (1) is a self- assembledtransition metal organic triangle complex. The present invention discloses a process for preparing a complex of Formula 1:[M3(μ-L)3·3DMF] (1),wherein M is Zn(II);L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and(2)DMF is dimethylformamide;the process comprising the steps of: a) mixing an alcoholic solution of the ligand H2L and aDMF solution of ZnCl2 to obtain a reaction mixture; b) refluxing the reaction mixture for 7-9hours to obtain yellow wet precipitate of the complex; c) isolating the complex and washingwith alcohol and an organic solvent, followed by drying in air at room temperature and overP4O10 in vacuo to obtain the complex of Formula 1. In a feature of the present invention, alcohol for preparing the alcoholic solution isselected from methanol, ethanol, propanol and butanol; preferably the alcohol is methanol. In a feature of the present invention, the organic solvent is selected from diethyl ether,chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane (DCM); preferably theorganic solvent is diethyl ether. In a feature of the present invention, the complex of formula (1) is a self- assembledtransition metal organic triangle complex. The present invention also discloses a composition comprising the transition metalcoordination complex of Formula (1), and one or more pharmaceutically acceptable excipients. The present invention also discloses a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2): The present invention also discloses a process for preparation of a thiosemicarbazoneligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) ofFormula (2):the process comprising the steps of: i) refluxing 3, 5-diiodosalicylaldehyde and N (4)-cyclohexylthiosemicarbazide in a 1:1 molar ratio in alcohol for 3-5 hours to obtain light-yellow product in areaction mass; and ii) filtering the light-yellow product, followed by washing with alcohol and anorganic solvent, and drying over P4O10 in vacuo to obtain the ligand of Formula (2).In a feature of the present invention, the alcohol is selected from methanol, ethanol,propanol and butanol; preferably the alcohol is methanol; and the organic solvent is selectedfrom diethyl ether, chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane(DCM); preferably the organic solvent is diethyl ether.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS / FIGURESThe accompanying drawings, which are incorporated in and constitute a part of thespecification, illustrate the synthesis and structure / elements of the invention and, together withthe description, explain the principles / concepts of the present invention.Figure 1 illustrates 1H NMR spectrum of H2L.Figure 2 illustrates 13C NMR spectrum of H2L.Figure 3 illustrates mass spectrum of H2L.Figure 4 illustrates an ORTEP (Oak Ridge Thermal Ellipsoid Plot) diagram (left) of[Zn3(μ-L)3·3DMF] (1) with atom-numbering scheme and 30% probability ellipsoids. Hydrogenatoms are omitted for clarity. The coordination geometry around the Zn(II) centers of complex1 (right) showing triangular Zn(II) arrangement.Figure 5 illustrates packing of complex 1 showing molecules are packed along the aaxis and the formation of 3D motif through hydrogen bonding and other short contacts.Figure 6 illustrates experimental (left) and theoretical (right) IR spectra of the ligandH2L.Figure 7 illustrates experimental (left) and theoretical (right) IR spectra of the complex1.Figure 8 illustrates experimental (left) and theoretical (right) Far IR spectra of complex1.Figure 9 illustrates UV-vis spectra of ligand H2L and the complex 1.Figure 10 illustrates UV-vis diffuse reflectance spectrum of H2L (left) and complex 1(right). Inset shows the band gap calculations.Figure 11 illustrates optimized geometries of H2L (left) and complex 1 (right).Figure 12 illustrates (a) Frontier molecular orbitals, (b) MEP plots of H2L and complex1.Figure 13 illustrates UV spectral changes of (a) ligand H2L and (b) complex 1 with theincreasing concentration of CT-DNA {[complex] = 50 μM; [DNA] = 3.4-30 μM}. Plots ofDNA / (εa-εf) versus [DNA] for the titration of (c) ligand H2L and (d) complex 1 with DNA.Figure 14 illustrates emission spectra of EB bound to DNA at varying concentrations ofthe (a) ligand H2L, (b) complex 1. The red arrow indicates a decrease in fluorescence intensitywith increasing compound concentration. Plot of F0 / F versus [complex] depicting the titration of(c) ligand H2L and (d) complex 1 with DNA.Figure 15 illustrates effect of increasing concentration of (a) ligand H2L and (b) complex1 on the relative viscosity of CT-DNA.Figure 16 illustrates assessment of MCF-7 cell survival in the presence of (a) cisplatin,(b) ligand and (c) complex 1 was conducted by treating the cells with different concentrations ofthe compounds. (*) indicate p < 0.001 when compared to control group.Figure 17 illustrates Morphological changes induced by H2L and complex 1 againstMCF7 cell lines compared with cisplatin at 100 μg / mL.Figure 18 illustrates morphologies of MCF-7 cells treated with complex 1 at aconcentration of 21.278 μg / mL for 24 hours. The cells are visualized under a confocalmicroscope after staining with DAPI (blue color, nucleus). The scale bar corresponds to 50 μm.Figure 19 illustrates quadrant graphs of cell apoptosis analysis in MCF7 cells treatedwith control (left) and complex 1 (right).Figure 20 illustrates histograms of the four stage MCF7 cells distribution.Figure 21 illustrates generation of ROS induced by (a) control (b) complex 1 andcorresponding phase contrast images (c) control (d) complex 1 in MCF7 cancer cell.Figure 22 illustrates fluorescent intensity plot of DCFDA / DCF assay performed for thedetection of ROS generation in MCF7 cells by complex 1 compared with untreated control.Figure 23 illustrates (a) binding mode of [Zn3(μ-L)3·3DMF] (1) and its focused view ofinteractions with 1BNA and (b) 2D representation of [Zn3(μ-L)3·3DMF] (1) with the active siteresidues of 1BNA.Figure 24 illustrates (a) binding mode of [Zn3(μ-L)3·3DMF] (1) and its focused view ofinteractions with 3HB5 and (b) 2D representation of [Zn3(μ-L)3·3DMF] (1) with the active siteresidues of 3HB5.DETAILED DESCRIPTION OF THE INVENTIONThe present invention will now be described in detail in connection with certainpreferred and optional embodiments, so that various aspects thereof may be more fullyinterpreted and comprehended. However, any skilled person or artisan will appreciate the extentto which such embodiments could be generalized in practice.It is further to be understood that all terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to be limiting in any manner orscope. Unless defined otherwise, all technical and scientific expressions used herein have thesame meaning as commonly understood by one of ordinary skill in the art to whichembodiments of the invention pertain. In describing and claiming the embodiments of thepresent invention, the following terminology can be used in accordance with the definitions setout below which are known in the state of art.The present invention relates to the design, synthesis, characterization, structural studyand application of a Zn(II) based metal organic triangle complex, for therapeutic purposes,particularly in combating breast cancer. More particularly, the present invention relates to atrinuclear transition metal coordination complex of formula [M3(μ-L)3·3DMF] (1), wherein Mis Zn(II), L is di deprotonated form of a novel thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) and DMF acts as an ancillaryligand stabilizing the coordination environment; and process for preparation thereof. Theunique structure of the trinuclear zinc(II) complex, crystallized in a monoclinic crystal systemwith P21 / n space group, reveals distorted trigonal bipyramidal coordination geometry aroundeach Zn(II) ion. Further, the present invention relates to an anticancer composition, particularlyagainst MCF-7 breast cancer cell lines. The in vitro cytotoxicity of the complex evaluated usingthe MTT assay showed notable inhibition of MCF-7 cell proliferation, with an IC50 value of21.278 μg / mL (10.65 μM), which is found ~3.5 times better than that of 11.041 μg / mL (36.668μM) shown by the standard cisplatin. The complex 1 is cytotoxic in nature and induce cell deathin the cancer cell lines by apoptosis, which is further explored via a reactive oxygen species(ROS) generation assay. In an aspect of the present invention, the present invention discloses a complex ofFormula 1:[M3(μ-L)3·3DMF] (1),wherein M is Zn(II);L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and(2)DMF is dimethylformamide. In an embodiment of the present invention, the L or H2L is a tetradentate-ON2S donorbridging ligand. In an embodiment of the present invention, the complex of Formula (1) possessesanticancer activity against breast cancer cells; and the complex of Formula (1) is a selfassembled transition metal organic triangle complex. In another aspect of the present invention, the present invention discloses a process forpreparing a complex of Formula 1:[M3(μ-L)3·3DMF] (1),wherein M is Zn(II);L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and(2)DMF is dimethylformamide;the process comprising the steps of: a) mixing an alcoholic solution of the ligand H2L and aDMF solution of ZnCl2 to obtain a reaction mixture; b) refluxing the reaction mixture for 7-9hours to obtain yellow wet precipitate of the complex; c) isolating the complex and washingwith alcohol and an organic solvent, followed by drying in air at room temperature and overP4O10 in vacuo to obtain the complex of Formula 1. In an embodiment of the present invention, alcohol for preparing the alcoholic solutionis selected from methanol, ethanol, propanol and butanol; preferably the alcohol is methanol. In an embodiment of the present invention, the organic solvent is selected from diethylether, chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane (DCM);preferably the organic solvent is diethyl ether. In an embodiment of the present invention, the complex of formula (1) is a selfassembled transition metal organic triangle complex. In one another aspect of the present invention, the present invention discloses acomposition comprising the transition metal coordination complex of Formula (1), and one ormore pharmaceutically acceptable excipients. In yet another aspect of the present invention, the present invention discloses athiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2):(2) In yet another aspect of the present invention, the present invention discloses a processfor preparation of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2):;(2)the process comprising the steps of: i) refluxing 3, 5-diiodosalicylaldehyde and N (4)-cyclohexylthiosemicarbazide in a 1:1 molar ratio in alcohol for 3-5 hours to obtain light-yellow product in areaction mass; and ii) filtering the light-yellow product, followed by washing with alcohol and anorganic solvent, and drying over P4O10 in vacuo to obtain the ligand of Formula (2).In an embodiment of the present invention, the alcohol is selected from methanol, ethanol,propanol and butanol; preferably the alcohol is methanol; and the organic solvent is selected fromdiethyl ether, chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane (DCM);preferably the organic solvent is diethyl ether.EXAMPLES / EXPERIMENTS:Instruments and Reagents3,5-Diiodosalicylaldehyde (Merck), hydrazine hydrate (Sigma Aldrich),cyclohexylisothiocyanate (Thermo scientific), zinc chloride (Spectrochem), CT-DNA(Aldrich), tris-HCl (Aldrich), ethidium bromide (EB) (Sigma) methanol (Merck), N, Ndimethyl formamide (Spectrochem), glacial acetic acid (Spectrochem), are employed in thiswork. All reagents and solvents were used as received, without any further purification.Elemental analyses for carbon, hydrogen, and nitrogen were conducted using an ElementarVario EL III CHNS analyzer at Sophisticated Test and Instrumentation Centre (STIC), Kochi.FT-IR spectra (4000-400 cm-1) were recorded on a JASCO FT-IR 4100 spectrometer with KBrpellets at DAC, CUSAT, Kochi. Far-IR spectra (600-100 cm-1) were recorded on a ThermoNicolet iS50 FTIR spectrometer at STIC, CUSAT. MALDI mass spectra were recorded usinga Bruker Autoflex spectrometer at STIC, CUSAT. Solution UV-visible absorption spectra(200-900 nm in DMF) were taken on a Thermo Scientific Evolution 220 spectrometer, whileUV-visible diffuse reflectance spectra were recorded with an Ocean Optics DH-2000-BALinstrument at DAC, CUSAT. 1H and 13C NMR spectra were recorded on a JEOL 400 MHz FTNMR Spectrometer at Government College for Women, Thiruvananthapuram, Kerala, India.Synthesis of the Schiff base ligand and its complex:Synthesis of N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene hydrazine carbothioamide(H2L)This compound was synthesized through a two-step procedure as shown in Schemes1 and 2:Synthesis of N4-cyclohexylthiosemicarbazide.A methanolic solution (20 mL) of cyclohexylisothiocyanate (2.1616 g, 1 mmol) andhydrazine hydrate (4.5973 g, 1 mmol) were combined, and the resulting mixture was stirred atroom temperature for 3 hours. The reaction yielded a colorless product, which was isolated byfiltration, subsequently washed with methanol, and dried under vacuum. A white solid, N (4)-cyclohexyl thiosemicarbazide, precipitated out. The solid was collected, washed thoroughlywith methanol and diethyl ether, and subsequently dried over P4O10 in vacuo.Synthesis of N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene hydrazine carbothioamide(H2L)Schiff base H2L was synthesized by refluxing 3, 5-diiodosalicylaldehyde (0.373 g, 1mmol) and N (4)-cyclohexyl thiosemicarbazide (0.173 g, 1 mmol) in a 1:1 molar ratio inmethanol for 4 hours. The resulting light-yellow product was filtered, washed with methanoland diethyl ether, and dried over P4O10 in vacuo.Yield: 75%, M.P. 260 °C.Elemental Anal. Found (calc.): C, 31.18 (31.78); H, 2.58 (3.04); N, 7.50 (7.94); S, 5.91(6.05) %.1H NMR (500 MHz, DMSO-d6), ppm: 11.44 (1H, s, N-NH-C), 10.35 (1H, s, br, OH),8.26 (1H, d, J=8.0Hz), 8.18 (1H, d, J= 4.8 Hz), 7.91 (1H, d, J=2.1Hz), 7.89 (1H, s), 4.16 (s,1H), 1.86 (d, J=9.7Hz, 2H), 1.72 (d, J=12.7Hz, 2H), 1.60 (d, J=12.4Hz, 1H), 1.39(dd, 2H),1.26(dd, 2H), 1.13 (t, J=12.3Hz, 1H), (Figure 1).13C NMR (125MHz, DMSO-d6), ppm: δ= 25.46 (cyclo-C11, C13), 25.68 (cyclo-C12),32.22 (cyclo-C10, C14), 53.66 (cyclo-C9), 176.44 (C8=S), 155.42 (C7=N) azomethine,146.90, 141.56, 137.75, 123.00, 90.12, 84.10 (aromatic carbons C1-C6) (Figure 2).MALDI- MS m / z [Found (calc.)]: 553.594 (552.16) {[H2L+Na] +} (Figure 3).Scheme 1. Synthesis of cyclohexylthiosemicarbazide.Scheme 2. Synthetic pathway of the Schiff base ligand H2L.Synthesis of the Zn(II) coordination complex [Zn3(μ-L)3·3DMF](1)To a MeOH solution (15 mL) of ZnCl2 (1.0 mmol, 0.136 g), a DMF solution (10 mL)of the ligand (H2L) (1.0 mmol, 0.529 g) was added and refluxed for 8 hours (Scheme 3). Theresulting yellow product was filtered, washed with methanol and diethyl ether, and dried overP4O10 in vacuo. Yellow crystals suitable for X-ray data collection were formed through the slowevaporation of the resulting reaction mixture. Yield: 55%, M.P. 273°C. Elemental Anal. Found(calc.): C, 30.18 (30.64); H, 3.58 (3.43); N, 8.50 (8.41); S, 4.91 (4.81) %.Scheme 3. Synthetic route leading to the trinuclear zinc (II) complex [Zn3(μ-L)3·3DMF](1).Crystal structure determinationCrystallographic data for complex 1 were collected at 296(2) K using a Bruker AXS D8VENTURE diffractometer equipped with Mo Kα (λ = 0.71073) radiation and a PHOTON IIarea detector, at SAIF, IIT Madras, India. The APEX3-SAINT software (Bruker, 2016) wasemployed for frame integration, ensuring a four-fold redundancy per reflection to facilitateaccurate multi-scan absorption correction using SADABS (Bruker, 2016). The structure wassolved with SHELXT-2018 (Sheldrick, 2018) and subsequently refined using SHELXL-2018(Sheldrick, 2018), both integrated within the WinGX system (version 2018.3). Anisotropicrefinement was applied to all non-hydrogen atoms. The molecular structure was visualizedusing ORTEP, Mercury, and PLATON softwares.Computational MethodologyDensity Functional Theory (DFT) using the Gaussian 09 software was employed for thetheoretical study of the ligand and the complex. Geometry optimizations and frequencycalculations for the ligand and complex were carried out using the B3LYP hybrid functional,which includes Becke's three-parameter nonlocal exchange functional and the Lee-Yang-Parr correlation functional. The LanL2DZ basis set was used for the zinc and iodine atoms,while the 6-311G (d,p) basis set was applied for the other atoms.DNA interaction studies:Absorption spectral studyElectronic absorption titration experiments were performed using a constantconcentration of the compounds (50 μM in DMF) and by gradually increasing the concentrationof CT-DNA (10-100 μM) in a tris-HCl buffer at pH 7.4, following a previously establishedmethod. The purity of the CT-DNA was assessed using electronic absorption spectroscopy,based on the absorbance ratio at 260 nm and 280 nm, which was found to be 1.9, indicatingthat the DNA sample was largely free of protein contamination. The intrinsic binding constant(Kb) was calculated using absorption data and the corresponding equation (1).EQUATION (1)Here, εa, εb, and εf represent the extinction coefficients at a specific DNA concentration,for the fully bound compound, and for the free compound, respectively. By plotting [DNA] / (εa- εf) against [DNA], a straight line was obtained. This line had an intercept of 1 / ( kb (εb - εf))and a slope of 1 / (εb - εf). The binding constant (kb) was then determined from the ratio of theslope to the intercept.Fluorescence studyEthidium bromide (EB) displacement experiments were performed to investigate thebinding interaction between the compound and CT-DNA. Initially, DNA was pre-incubatedwith the standard intercalator EB, resulting in enhanced fluorescence. Upon incrementaladdition of the compound, a gradual decrease in fluorescence intensity was observed, indicatingdisplacement of EB and providing insight into the compound's binding mode with CT-DNA.The fluorescence quenching data was fitted using the Stern-Volmer equation (2).EQUATION (2)where F0 and F, are the fluorescence intensities in the absence and presence of quencher,respectively, [Q] is the quencher concentration (the ligand and zinc complex in the systems),kq is the Stern-Volmer quenching constant, obtained from the slope of the F0 / F vs. [Q] plot.Viscosity studyThe viscosity tests were conducted at 25°C using an Ostwald viscometer. Thecompound (0-120 μM) was introduced gradually while the CT-DNA concentration (50 μM)remained constant. Relative viscosity (η / η0)1 / 3 was plotted against [compound] / [DNA] afterflow durations were recorded twice. The specific viscosities of free CT-DNA and the CT-DNAcomplex adduct, denoted by η0 and η, were calculated as (t-t0) / t0 (t: observed flow time, t0:buffer flow time).In vitro cytotoxicityIn vitro cytotoxicity of the compounds was assessed against the MCF7 human breastcancer cell line using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay. MCF7 cells were cultured in a 25 cm2 tissue culture flask containing DMEMsupplemented with 10% FBS, L-glutamine, sodium bicarbonate, and an antibiotic mixture(penicillin 100 U / mL, streptomycin 100 μg / mL, and amphotericin B 2.5 μg / mL), and incubatedat 37 °C in a humidified 5% CO2 environment. A total of 5000 cells per well were seeded intoa 96-well plate and incubated at 37 °C in a humidified 5% CO2 incubator. After 24 hours, thegrowth medium was discarded, and each compound was freshly prepared (1 mg in 1 mL 0.1%DMSO) and serially diluted five times in DMEM by two-fold increments (100, 50, 25, 12.5,and 6.25 μg in 500 μL of DMEM). A 100 μL aliquot of each concentration was added intriplicates to the respective wells and incubated at 37 °C in a humidified 5% CO2 incubator.Non-treated control cells were also maintained. After 24 hours of incubation, the contents ofthe wells were removed, and 30 μL of reconstituted MTT solution (15 mg MTT in 3 mL PBS)was added to all wells. The plate was gently shaken and then incubated for an additional 4hours at 37 °C in a humidified 5% CO2 incubator. Afterward, the supernatant was discarded,and 100 μL of MTT solubilization solution (DMSO) was added to each well, which was thenmixed gently by pipetting to dissolve the formazan crystals. Absorbance values were measuredat a wavelength of 540 nm using a microplate reader. Any noticeable changes in cellmorphology, such as cell rounding, shrinking, granulation, or vacuolization in the cytoplasm,were considered signs of cytotoxicity. The percentage of cell viability was calculated using thefollowing equation 3.Nuclear Staining AssayNuclear morphological alterations and structural integrity during cell death wereexamined using DAPI staining. The MCF-7 cell line was cultured in 25 cm2 tissue culture flaskwith DMEM supplemented with 10% FBS, L-glutamine, sodium bicarbonate and antibioticsolution containing: Penicillin (100 U / mL), Streptomycin (100 μg / mL), and Amphotericin B(2.5 μg / mL). Cultured cell lines were kept at 37 ºC in a humidified 5% CO2 incubator. Afterattaining sufficient growth, sample of concentration (21.278 μg / mL) from a stock of 1 mg / mLwas added and incubated for 24 hours. Non treated control cells were also maintained. After24 hours, the culture medium was removed washed with Phosphate Buffered Saline (PBS) and100 μL fixing solution (4% formaldehyde) was added and kept for 10 minutes and again washedwith PBS. DAPI stain was added, and the cells were imaged under a Confocal Laser scanningMicroscope (Olympus Fluoview FV3000) at specific wavelengths to examine morphologicalchanges.Apoptosis AssayCell apoptosis is typically assessed using the Annexin V-FITC assay. MCF-7 cell linewas cultured as per standard procedures and treated with LD50 concentration of samples: IC50(21.278 μg / mL) of complex 1 and incubated for 24 hours. Non treated control were alsomaintained and incubated at 37 ºC in a humidified 5% CO2 incubator for 24 hours. The cellsample was transferred to a 12x75 mm polystyrene tube. The minimum recommended numberof cells for fixation in a tube is 1x106 cells. The samples were then centrifuged at 3000 rpm for5 minutes. The supernatant was removed without disturbing the pellet. After centrifugation, thecell pellet forms either a visible pellet or a white film on the bottom of the tube. To the tubesadded 100 μL of the Muse Annexin V and dead cell reagent to each tube. The tubes weremixed thoroughly by pipetting up and down or vortexing at a medium speed for 3 to 5 secondsfollowed by incubation for 20 minutes at room temperature in the dark. The cells were analyzedin a flow cytometer and analyzed using Muse flow cytometry software. Cells were gated againstuntreated control cells and analyzed for apoptosis using Muse FCS 3.0 software.ROS generation assayThe generation of intracellular reactive oxygen species was evaluated by staining withDCFH- DA. The cell culture conditions were consistent with those detailed in the MTT assayand after attaining sufficient growth, sample of concentration (21.278 μg / mL) from a stock of1 mg / mL was added and incubated for 24 hours. Non treated control cells were also maintained.The cells were washed with PBS and added with 50 μL of DCFDA and incubated for 30 minutes.After incubation excess dye was washed with PBS and fluorescence was imaged in afluorescent microscope (Olympus CKX41 with optika pro5 CCD camera) and fluorescence wasmeasured using a fluorimeter at 470 nm excitation and emission at 635 nm (Qubit 3.0, Lifetechnologies, USA) and expressed in arbitrary units.Molecular DockingMolecular docking studies predict and analyze the interactions between a protein and aligand within the protein's active site using AutoDock Vina 1.1. The 3D structures of DNA (PDBID: 1BNA, resolution: 1.90 Å), and the human estrogen receptor alpha ligand-binding domain(PDB ID: 3HB5), associated with breast cancer were made available via the protein data bank.The AutoDock Tools 1.5.6 software suite was used to prepare the AutoDock Vina input files.Protein structure was refined by adding polar hydrogen bonds and eliminating unbound watermolecules. After adding net Kollman charges and making further changes, a large grid size boxthat could encompass the complete macromolecule was created. The 3D structures of theprotein and ligand were subsequently transformed into pdbqt file formats for further use.AutoDock Vina's number of restrictions was left at default. The results of the compounds withthe highest affinity are visually examined by the Discovery Studio program.Results and discussionThe facile condensation of 3,5 diiodosalicylaldehyde and the substituted thiosemicarbazidein a 1:1 molar ratio resulted in the formation of the product, N4-cyclohexyl-2-(2-hydroxy-3,5-diiodobenzylidene hydrazine carbothioamide (H2L). This compound has been utilized as atetradentate chelating and bridging ligand in the present invention or study. The reaction ofH2L with ZnCl2, in the DMF solution resulted in the formation of a novel zinc(II) coordinationcomplex with the formula [Zn3(μ-L)3·3DMF] (1) where DMF acts as an ancillary ligandstabilizing the coordination environment, which was fully characterized via X-raycrystallography. Scheme 3 shows the synthetic route leading to the imine and amine nitrogenbridged trinuclear zinc(II) complex [Zn3(μ-L)3·3DMF] (1). The ligand H2L and the trinuclearzinc(II) complex are stable in solid state and were soluble in some organic solvents like DMF,DMSO, acetonitrile, etc. Structural determination was further supported by elemental analysis,along with 1H-NMR, 13C-NMR, FT-IR, and UV-vis spectroscopy techniques. Molarconductivity measurements for zinc complex were carried out in DMF at a concentration of 10-3 M at room temperature. The observed conductivity value is approximately 15 Ω-1cm2mol-1,indicating that this complex is non-electrolytic and remain undissociated in DMF.Single Crystal X-ray Diffraction of Complex 1The crystal structure of the homometallic trinuclear zinc complex 1 has been determinedthrough single-crystal X-ray diffraction analysis. Crystallographic data and parameters of thecomplex are summarized in Table 1. ORTEP view of the [Zn3(μ-L)3·3DMF] complex (1), alongwith the atom-labeling scheme are given in Figure 4. The structure consists of three zinc centers{Zn(1), Zn(2), and Zn(3)}, three units of the deprotonated reduced Schiff base ligand (L), andthree DMF solvent molecules. Each ligand unit coordinates to the metal center in itsdeprotonated thiolate form. The observed C-S bond lengths (≈1.74 Å) are appreciably longerthan a typical C=S double bond, whereas the adjacent C-N bonds (≈1.34 Å) arecorrespondingly shorter, confirming thione-thiol tautomerization and deprotonation uponcomplexation. Key bond lengths and bond angles are provided in Table 2. The deviations oflisted bond angles are presumably due to restrictions induced by chelation of ligand. Thesethree zinc centers are bridged by three imine and amine nitrogen atoms, {N(2), N(8),N(5) and N(7), N(6), N(1)}, of the ligands. The geometry of a penta-coordinated metal centerscan be evaluated using the trigonality index or Addison parameter (τ). A τ value of 0 correspondsto an ideal square pyramidal geometry, while a τ value of 1 indicates a perfect trigonalbipyramidal geometry. Trigonality indices for each zinc centre in the current complex [τ = 0.45for Zn(1), τ = 0.55 for Zn(2), and τ = 0.65 for Zn(3)] are significantly out of the optimal rangefor square pyramidal geometry, indicating that each penta-coordinated zinc's coordinationgeometry is significantly warped from square pyramid to trigonal bipyramid. The binding modeof the ligand is μ2 to form a Zn3N6 core. The Zn(1) center is equatoriallycoordinated by S(2), N(7) of a ligand and O(6) of DMF molecule. The phenolate oxygen atomO(2) and imine nitrogen atom N(2), of a second molecule of the ligand coordinates the Zn(1)center in an apical position to complete its distorted trigonal bipyramidal geometry. The Zn(2)center is coordinated by the nitrogen (N(6)), sulfur (S(3)), and oxygen (O(3)) atoms of atetradentate ligand, as well as by the imine nitrogen (N(8)) of a second ligand and the oxygen(O(5)) of a DMF molecule, completing its distorted trigonal bipyramidal geometry. Similarly,the Zn(3) center is also coordinated by the nitrogen (N(1)), sulfur (S(1)), and oxygen (O(1))atoms of a tetradentate ligand, along with the imine nitrogen (N(5)) from a second ligand andthe oxygen (O(5)) of a DMF molecule, completing its trigonal bipyramidal geometry. The Xray diffraction analysis confirms the presence of a novel Zn(II) trinuclear metal system. TheZn∙∙∙Zn distances between the nearest zinc atoms are 4.782, 4.850, and 4.868 Å. The anglesbetween three zinc(II) centers are ~60° {60.71°, 58.96° and 60.33° for Zn(2)∙∙∙Zn(1)∙∙∙Zn(3),Zn(1)∙∙∙Zn(2)∙∙∙Zn(3) and Zn(1)∙∙∙Zn(3)∙∙∙Zn(2) respectively} and indicate the trigonal planararrangement of zinc(II) centres. The stability of the crystal lattice is maintained through anetwork of intramolecular and intermolecular hydrogen bonds formed among the moleculeswhich significantly influence both the structural and spectral characteristics of thethiosemicarbazone derivative (Table 3). Significant C-H···π interactions are also observed.Figure 5 illustrates the packing along the a-axis, highlighting the formation of a threedimensional network through hydrogen bonding and other short contacts.Table 1. Crystal refinement parameters of the complex [Zn3(μ-L)3·3DMF] (1).Table 2. The bond lengths (Å) and bond angles (˚) of the complex [Zn3(μ-L)3·3DMF](1).Table 3. Interaction parameters of the complex [Zn3(μ-L)3·3DMF] (1).Spectral features of H2L and Complex of Formula 1The experimental FT-IR spectra of the ligand H2L and its complex [Zn3(μ-L)3·3DMF](1) are shown in Figures 6 and 7. H2L ligand showed vibrational frequency around 3138 (DFTcalculated 3493) cm-1 which was assigned for N-H stretching and at 1593 (calculated 1664)cm-1 for C=N stretching, and the characteristic thioamide band, ν(C=S), appear at 1220(calculated 1236) cm-1. A weak broad band in the region of 3344 (calculated 3841) cm-1 due toa hydrogen bonded OH group in the free Schiff base ligand H2L is not observed in the infraredspectra of complex 1. This indicates that the phenolic oxygen is deprotonated and coordinatedin zinc (II) complex. In the zinc (II) complex, the coordination also occurred via sulphur andimine nitrogen. The ν(C=N), 1525 (calculated 1604) cm-1 and ν(C-S) 1144 (calculated 1189)cm-1 vibrations of zinc complex are found shifted considerably towards lower frequenciescompared to that of the free Schiff base ligand H2L. Zn-N and Zn-S vibrations were observedat 519 (calculated 573) and 333 (calculated 393) cm-1 respectively for the zinc(II) complex inthe far IR region. A band observed at 411 cm-1 (calculated 454) for the complex can beattributed to the ν(Zn-O) vibration, providing additional evidence for coordination through thedeprotonated phenolic oxygen atoms (Figure 8).The electronic spectra of the H2L and its complex [Zn3(μ-L)3·3DMF] were recorded in10-5 M solution of DMF within the range 200-800 nm. The spectra (Figure 9) clearly displayeddistinct absorptions corresponding to the aromatic groups and the non-bonding electronspresent in the complex. The bands at 270 (62000 M-1 cm-1) and 324 (36450 M-1 cm-1) nm inthe thiosemicarbazone are due to π -> π* transitions, may be due to benzene ring, and imineand thiocarbonyl groups present in the compound. The band observed for the ligand at 408(23020 M-1 cm-1) nm is attributed to n -> π* transition. These bands have undergone marginalchanges during complexation, which is in agreement with Zn(II) coordination. In Zn complex,π -> π* transition bands are shifted to 275 (78350) and 344 nm (37110 M-1 cm-1) respectively,while n -> π* transition is shifted to 413 (18770 M-1 cm-1) nm. The solid-state UV-vis DRS ofligand and complex 1 are shown in Figure 10. The free ligand displays four absorption bands at238, 286, 352, and 381 nm. The bands at 238 and 286 nm are assigned to π -> π* transition,originating from the aromatic ring, azomethine (C=N), and thioamide groups. The lowerenergy absorptions at 352 and 381 nm are attributed to intra-ligand charge transfer (ILCT)transitions involving electron donation from donor atoms (N and S) to the di-iodinated aromaticsystem, while a weak band observed at 420 nm is attributed to and n -> π* transition. Uponcomplexation with Zn(II), the solid-state UV-DRS spectrum of the complex shows threeprominent bands at 240, 310, and 368 nm. The slight red shifts observed in the π -> π*transitions (to 240 and 310 nm, respectively) suggest a redistribution of electron density uponcoordination. The ILCT band also shifts from 381 nm (H2L) to 368 nm (complex 1) and ashoulder band at 400 nm may be attributed to shifted n -> π* transition in the ligand moiety.The direct band gaps (Eg) of the compounds were determined by plotting (hυF(R))2 againstphoton energy (hυ), using the Kubelka-Munk function defined as F(R) = (1 - R)2 / 2R. Thecalculated band gaps for the ligand and the Zn(II) complexes were 3.02 eV and 2.71 eV,respectively.Density Functional Theory (DFT) AnalysisDFT calculations were performed to investigate the electronic and structural propertiesof the ligand (H₂L) and its Zn(II) complex (1). The optimized geometries of these molecules(Figure 11) show that H₂L adopts a nearly planar conformation of the azomethine and salicylicmoieties, contributing to extended conjugation and molecular stability. Frontier MolecularOrbital (FMO) analysis (Figure 12) revealed that complexation with Zn(II) significantlymodifies the HOMO-LUMO distribution and reduces the energy gap, indicating enhancedcharge delocalization and chemical reactivity. The HOMO of H₂L is mainly localized over thephenolic ring, azomethine group, and thioamide moiety, while in complex 1, it extends overboth the ligand and metal center, suggesting strong electronic interaction upon coordination.The molecular electrostatic potential (MEP) map further shows that the azomethine nitrogenand phenolic oxygen atoms are the most electron-rich regions, serving as preferred coordinationsites for metal ions.For better understanding of the complex's reactivity, overall indices such as hardnessand softness were considered. Table 4 describes the global reactivity parameters of compounds.Ligand (H2L) has a hardness of 1.78 eV, which decreases to 0.49 eV upon complexation witha zinc ion. The Zn(II) complex itself has a hardness of 1.29 eV. Softness, which is inverselyrelated to hardness, increases from -0.56 eV for H2L to -0.775 eV for the Zn(II) complex. Thelower hardness and higher softness indicate greater reactivity. The electrophilicity index playsa key role in determining the reactivity and biological activity of molecules. For the ligand, theelectrophilicity index is 4.31 eV, which increases significantly to 6.70 eV upon coordination inthe zinc complex. This rise indicates enhanced reactivity, as a higher electrophilicity indexcorresponds to greater electrophilic nature. Additionally, the chemical potential a quantumchemical descriptor was also evaluated. Ligand has a chemical potential of -3.92 eV, whichslightly increases to -4.16 eV for the complex. A more negative chemical potential, however,suggests stability. These computed indices confirm that the Zn(II) complex can be morereactive than the free ligand. Overall, analysis of all quantum chemical descriptors reveals thatcoordination of the Schiff base ligand with a transition metal enhances chemical reactivity andbiological properties of the complex.Table 4. The Frontier molecular orbital energies and calculated chemical reactivity parametersDNA binding study:Absorption studiesDNA is a key cellular target for many metallodrugs used in treating diseases likecancer, highlighting the importance of metal binding to electron-rich nucleobases or phosphategroups in the DNA duplex. The most popular and useful technique for figuring out the nature ofthese interactions among the different approaches is electronic absorption titration. The studyinvolved progressively adding CT-DNA to a solution with a fixed compound concentration (50μM). Figure 13 shows that as the DNA concentration increased from 3.4 to 30 μM, noticeablechanges in the compound's absorption characteristics were detected. Specifically, theabsorption band of ligand at 274 nm displayed hyperchromism, while the bands at 326, 350and 412 nm exhibited hypochromism. For complex hyperchromism was exhibited by bands at277, 325 and 346 nm and band at 405 nm show hypochromism. The interesting finding was theemergence of an isosbestic point for ligand and complex at 290 and 374 nm respectively whichdenoted a significant change in the system and the occurrence of a unique chemical process.The idea that the DNA and the compounds have a strong relationship is further supported by theexistence of these isosbestic point. The calculated intrinsic equilibrium binding constants (Kb)of ligand and complex were found to be 3.1 x 105 and 4.9 x 105 M-1 respectively. The Kbvalues further demonstrate that the complex has a stronger binding affinity than the ligand.The compounds bind to DNA via a groove-binding mechanism based on the variations inabsorbance intensity without any discernible redshift. These results provide credence to the ideathat the substance has a substantial and meaningful interaction with DNA.Emission spectroscopyIn fluorescence spectroscopy, EB was used as an efficient fluorophore to examine thecompetitive intercalative binding of ligand and complex with CT-DNA. Because of its cationicand planar nature, EB only exhibits modest emission in buffer solution. However, when EBintercalates between DNA base pairs, it forms DNA-EB adducts that exhibit a pronouncedincrease in fluorescence intensity. Fluorescence gradually decreased when ligand and complexwere added incrementally to these DNA-EB complexes, which were indicative of thedisplacement of EB by ligand and complex from the double helix of DNA. Figure 14 illustratesa significant drop in EB-DNA fluorescence intensity as quencher concentration increases (0.3-5 μM) indicating a groove mode of binding between compounds and DNA. The quenchingconstant (Kq) for the compounds were found to be 1.2 x 105 and 1.5 x 105 M-1 respectively. These findings show that the complex binds to DNA more strongly than the free ligand, whichis consistent with the data obtained from electronic absorption studies. A higher Kq value canindicate a stronger interaction, possibly resulting from factors such as binding affinity or closeproximity between the interacting molecules.Viscosity ExperimentsHydrodynamic techniques that are sensitive to molecular length are thought to beamong the most accurate and instructive ways to evaluate binding modes in solution. When amolecule attaches to DNA by a traditional intercalation method, it generally results in aconsiderable rise in the viscosity of the DNA solution. This is because base pairs are forcedapart at the intercalation sites, causing the DNA helix to expand and so extend the DNA strand.On the other hand, substances that only bind inside the DNA grooves either by partial or nonclassical intercalation usually result in negligible viscosity changes, which could be slightlypositive, negative, or insignificant under similar circumstances. To further explore the bindingmode of ligand and complex, viscosity measurements were carried out on CT-DNA bound tovarying concentrations of compound. The results showed that it exerts essentially no effect onCT-DNA viscosity, which is consistent with DNA groove binding (Figure 15).Estimation of CytotoxicityThe cytotoxic effects of the ligand (H2L) and its complex (1) were assessed on the MCF7 cell line using the widely established MTT assay. The MCF-7 cells were treated with fivedifferent concentrations (6.25, 12.5, 25, 50, 100 μg / ml) of the ligand and the designedcomplex (1) for 24 hours. A dose-dependent inhibition of cell growth and induction of celldeath were observed in MCF-7 cells following treatment with the ligand and complex, withvarying degrees of effect (Figure 16). The IC50 values for H2L and complex 1 were found tobe 67.053 (126.75 μM) and 21.278 μg / mL (10.65 μM), respectively, indicating that the ligandexhibits lower cytotoxic potential compared to the complex. However, the significant potencyof Zn complex was found better than that of 11.041 μg / mL (36.668 μM) shown by cisplatin.At the highest evaluated concentration of 100 μg / mL, MCF-7 cells exhibited a cell survivalrate of approximately 42.78% for ligand and 19.01% for the complex, as shown in Table 5.Thus, the structural modulation of the ligand and Zn(II) triangle coordination frameworkformation leads to better cytotoxic effect on MCF-7 cancer cell lines. The photographs (Figure17) show the efficacy of H2L and complex against MCF7 cell lines compared with cisplatin.Table 5. The percentage of cell viability of MCF-7 cell with the concentrations of ligand H2L and complex 1.Apoptosis is often regarded as the most controlled form of cell death, as it preventsleakage of cellular contents, avoids inflammation, and allows efficient removal of dead cellsby the immune system. DAPI, a nuclear staining dye, is employed for detecting nuclearchanges, aiding in the investigation of cell death mechanisms induced by treated complex 1.Figure 18 presents the DAPI-stained images of complex 1 treated with MCF-7 cell line.Compared to the control cells, noticeable alterations in nuclear morphology were observedupon treatment with the complex. Prominent morphological features such as nuclear shrinkage,chromatin condensation, and membrane blebbing were observed. Several nuclei appearedintensely stained, fragmented, or condensed, indicating nuclear pyknosis and karyorrhexis. Thepresence of apoptotic bodies further confirmed apoptosis as the mode of cell death. In contrast,such features were absent in the control group. These findings were further supported byfluorescence microscopy, which demonstrated typical apoptotic nuclear morphology,consistent with internucleosomal DNA fragmentation.Apoptotic effect in MCF7 cancer cell lineApoptosis plays a vital role in regulating tumor development and influencing responsesto cancer treatments. Many therapeutic approaches such as radiation, chemotherapy, andsurgical interventions are designed to trigger apoptosis in cancer cells by activating specificapoptotic signaling pathways. Moreover, both apoptosis and cell cycle checkpoints are keyfactors in the molecular pathogenesis of cancer and can significantly impact how tumor cellsreact to different forms of therapy. After confirming that complex 1 effectively inhibits thegrowth of the MCF7 cancer cell line, the next phase of the study aimed to assess its potentialto induce apoptosis. DAPI staining revealed characteristic features of apoptotic cell death inMCF7 cells following treatment with complex 1. To further substantiate this observation andquantify the level of apoptosis, an annexin V-FITC / PI double staining assay was performed onMCF7 cells. For this assay, cells were treated with a concentration of complex 1 at their IC50value for 24 hours., where untreated cells served as the negative control for comparisonpurposes. In the early stages of apoptosis, a key biochemical change is the loss of plasmamembrane phospholipid asymmetry due to the translocation of phosphatidylserine (PS) fromthe inner to the outer membrane surface. This allows Annexin V (FITC-conjugated) to bind toexternalized PS. As cell death progresses, membrane integrity is lost, enabling propidiumiodide (PI) to enter and stain nuclear DNA. As illustrated in Figure 19, each histogram is dividedinto four quadrants. The upper left (UL) quadrant represents necrotic cells that lack AnnexinV-FITC staining. The upper right (UR) quadrant indicates late apoptotic cells, which are bothPI-permeable and Annexin V-FITC positive. The lower left (LL) quadrant corresponds toviable cells with intact membranes, showing no staining. The lower right (LR) quadrantrepresents early apoptotic cells, which are positive for Annexin V-FITC but impermeable toPI.After 24 hours incubation with complex 1, the total apoptosis rates were 50.7% which is2.9 fold higher than the cells treated with vehicle (17.15%). The proportion of late apoptosiscells rose by 43.45% for complex 1. From the Figure 20, it emerges that the sum of LR and URregions is increased in complex as compared to the control; these two regions stand for earlyand late apoptosis cell percentages, respectively. Complex 1 specifically induced necrosis(4.15%) in the treated MCF-7 cells, unlike the controls. These findings demonstratedunequivocally that the [Zn3(μ-L)3·3DMF] complex effectively induced apoptosis.ROS production in MCF7 cellsThe apoptosis-inducing potential of antitumor compounds is closely associated withtheir ability to generate reactive oxygen species (ROS), as numerous promising chemopreventive and anticancer agents have been shown to promote apoptosis through ROSproduction. A reactive oxygen species (ROS) detection kit utilizing 2',7'- dichlorofluoresceindiacetate (DCFH-DA) was employed to assess the ROS-generating capability of complex 1 inMCF7 cells by using flow cytometry. They are naturally produced as by-products of variousbiological processes, including both enzymatic and non-enzymatic reactions, and they play keyroles in regulating cellular signaling and maintaining homeostasis. DCFDA is a cell-permeable,non-fluorescent fluorogenic dye used to detect reactive oxygen species (ROS) within cells;once inside, it undergoes de-esterification and is oxidized to form 2',7'-dichlorofluorescein(DCF), a highly green-fluorescent compound. MCF7 cells were treated with complex 1, alongwith an untreated control containing only DCFDA. Fluorescence microscopy images of therespective cells reveal a pronounced generation of the intensely green-fluorescent DCF in cellstreated with complex 1, in stark contrast to the untreated control group (Figure 21). The markedincrease in fluorescence intensity confirms substantial ROS production, which subsequentlydrives MCF7 cells toward the apoptotic pathway. Figure 22 shows the fluorescent intensity plotperformed for the detection of ROS generation in MCF7 cells by complex 1 compared withuntreated control.Docking Perspective of Complex 1 with 1BNA and 3HB5 proteinMolecular docking is a powerful computational technique to predict the interactionbetween synthesized compounds and biological macromolecular targets at the molecular level.Molecular docking studies of Complex 1 were carried out to investigate its interaction with BDNA (PDB ID: 1BNA). This analysis aimed to predict the binding mode, orientation, andbinding energy of the complex within the DNA structure. The complex 1 bind effectively toDNA with a binding energy of -6.4 kcal / mol. Figure 23(a) provides graphical view of thedocked complex 1 within the active site of the DNA-binding protein along with focused viewshighlighting their interactions with nucleotide residues. The complex exhibits two stronghydrogen bonding interactions DA6(H7)∙∙∙S1 and DA6(H61)∙∙∙O4. Figure 23(b) shows 2Drepresentation of complex 1 with the active site residues of 1BNA.The inhibitory potential of zinc complex against breast cancer mutant oxidoreductase(3HB5) was also assessed through docking simulation. Higher binding affinity of the zinccomplex with 3HB5, having a binding energy of -6.9 kcal / mol, corroborate with theexperimental activity. The best-docked conformation of the complex [Zn3(μ-L)3·3DMF]against 3HB5 showed two H-bonding interactions ARG76∙∙∙ S1 and ARG-76∙∙∙ O6, along withother interactions involving VAL-79 and ARG-76 (Figure 24(a)). 2D representation ofcomplex 1 with the active site residues of 3HB5 is shown in Figure 24(b). The overallinteractions of complex 1 against 1BNA and 3HB5 with binding scores are given in Table 6.Table 6. Docking energy and various interactions of the [Zn3(μ-L)3·3DMF] (1) with duplex1BNA and 3HB5.ConclusionsChemotherapeutic research has increasingly focused on non-platinum-basedcompounds as anticancer agents to minimize systemic toxicity. A novel ON2S donorthiosemicarbazone ligand and its trinuclear zinc complex [Zn3(μ-L)3·3DMF] (1), aresynthesized and characterized. The molecular structure of complex 1 is confirmed by SCXRDanalysis. In vitro DNA binding studies revealed that the complex 1 interacts through groovebinding, exhibiting a binding constant (Kb) of 4.9 x 105 M-1 and a quenching rate constant (Kq) of 1.5 x 105M1 underscoring strong interaction with DNA, which was further supported by insilico study. The in vitro cytotoxicity studies against the MCF-7 breast cancer cell line revealedthat the Zn(II) complex exhibits promising anticancer activity, with an IC50 value of 10.6 μMoutperforming the standard cisplatin (36.66 μM). Further investigations using DAPI stainingand apoptosis assays confirmed nuclear condensation and fragmentation, indicating apoptoticcell death. ROS generation studies demonstrated that the complex induces oxidative stress,contributing to its cytotoxic mechanism. These results suggest that the novel Zn(II) trianglecomplex 1 holds potential as an effective alternative therapeutic agent against breast cancer.

Claims

1. A complex of Formula 1: [M3(μ-L)3·3DMF] (1), wherein M is Zn(II); L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and (2) DMF is dimethylformamide.

2. The complex as claimed in claim 1, wherein the L or H2L is a tetradentate-ON2S donor bridging ligand.

3. The complex as claimed in claim 1, wherein the complex of Formula (1) possesses anticancer activity against breast cancer cells; and the complex of Formula (1) is a selfassembled transition metal organic triangle complex.

4. A process for preparing a complex of Formula 1: [M3(μ-L)3·3DMF] (1), wherein M is Zn(II); L is di deprotonated form of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2); and (2) DMF is dimethylformamide; the process comprising the steps of: a) mixing an alcoholic solution of the ligand H2L and a DMF solution of ZnCl2 to obtain a reaction mixture; b) refluxing the reaction mixture for 7-9 hours to obtain yellow wet precipitate of the complex; and c) isolating the precipitate of the complex and washing with alcohol and an organic solvent, followed by drying in air at room temperature and over P4O10 in vacuo to obtain the complex of Formula 1.

5. The process as claimed in claim 4, wherein alcohol for preparing the alcoholic solution is selected from methanol, ethanol, propanol and butanol; preferably the alcohol is methanol; and the organic solvent is selected from diethyl ether, chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane (DCM); preferably the organic solvent is diethyl ether.

6. The process as claimed in claim 4, wherein the complex of formula (1) is a selfassembled transition metal organic triangle complex.

7. A composition comprising the complex as claimed in claim 1 or 4, and one or more pharmaceutically acceptable excipients.

8. A thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy-3,5- diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2):

9. A process for preparation of a thiosemicarbazone ligand, N4-cyclohexyl-2-(2-hydroxy3,5-diiodobenzylidene)hydrazinecarbothioamide (H2L) of Formula (2): 36 ; (2) the process comprising the steps of: i) refluxing 3, 5-diiodosalicylaldehyde and N (4)-cyclohexyl thiosemicarbazide in a 1:1 molar ratio in alcohol for 3-5 hours to obtain light-yellow product in a reaction mass; and ii) filtering the light-yellow product, followed by washing with alcohol and an organic solvent, and drying over P4O10 in vacuo to obtain the ligand of Formula (2).

10. The process as claimed in claim 9, wherein the alcohol is selected from methanol, ethanol, propanol and butanol; preferably the alcohol is methanol; and the organic solvent is selected from diethyl ether, chloroform, ethyl acetate, tetrahydrofuran (THF) and dichloromethane (DCM); preferably the organic solvent is diethyl ether.