A ferrocene-tagged benzimidazolium salt and a method of preparation thereof

Abstract

The present invention discloses a ferrocene-tagged benzimidazolium salt, N-ferrocenylmethyl-N′-(2-pyridylmethyl)benzimidazolium iodide, and a method of preparation thereof. The compound accumulates in mitochondria, promotes ROS and singlet-oxygen generation, and disrupts mitochondrial membrane potential triggering cancer cell death more effectively than ferrocene and standard drug doxorubicin.

Description

FIELD OF THE INVENTIONThe present invention relates to the field of organometallic chemistry and cancer biology. Inparticular, the present invention relates to a ferrocene-tagged benzimidazolium salt and its useas mitochondria-targeting, redox-active anticancer agent that induces reactive oxygen species(ROS) and mitochondrial membrane disruption. The present invention also relates to a methodof preparation of ferrocene-tagged benzimidazolium salt.BACKGROUND OF THE INVENTIONCancer is a complex group of diseases characterized by the uncontrolled growth, proliferation,and spread of abnormal cells within the body. These malignant cells evade normal regulatorymechanisms, acquire the ability to invade surrounding tissues, and may metastasize to distantorgans, ultimately impairing essential physiological functions. Despite decades of advancementin diagnostics and therapeutics, cancer remains one of the leading causes of morbidity andmortality worldwide. Its heterogeneity across tissue types, genetic mutations, and tumourmicroenvironments continues to pose significant challenges for effective, targeted, andaffordable treatment.Beyond its biological complexity, cancer imposes a substantial and multidimensional burdenon individuals, families, healthcare systems, and national economies. The financial impactarises from direct medical expenses (including diagnostics, surgeries, chemotherapy, radiationtherapy, immunotherapy, and long-term follow-up care), indirect costs such as loss of incomeand productivity, and intangible costs associated with reduced quality of life. For manyhouseholds, the economic strain begins at the point of diagnosis and intensifies throughouttreatment due to recurring hospital visits, medication expenses, and supportive carerequirements.In low- and middle-income countries, including India, cancer treatment costs frequently exceedannual household incomes, resulting in catastrophic health expenditure for a significant portionof the population. Even individuals with health insurance often face high out-of-pocketspending due to limitations in coverage, non-standardized treatment pricing, and the risingadoption of advanced but costly therapies such as targeted biologics and precision medicines.At a systemic level, cancer leads to substantial productivity losses as patients and caregiverswithdraw from the workforce during treatment and recovery. This cumulative economic burdencontributes to widening health inequities, delayed diagnosis, and reduced access to timely andeffective care.Given the rising incidence of cancer and the escalating cost of conventional treatmentmodalities, there is a pressing need for innovative, cost-effective, and accessible solutions thatcan enhance early detection, improve therapeutic precision, reduce treatment complexity, orlower the financial toxicity experienced by patients.Anticancer drugs are crucial because they kill or slow the growth of cancer cells, which isessential for treating cancer and improving patient outcomes. These medications can shrinktumors, prevent the spread of cancer, and help relieve cancer-related symptoms. They are alsoused to destroy any remaining cancer cells after surgery or radiation therapy and can enhancethe effectiveness of other treatments. Organometallic compounds, especially those based onferrocene, have garnered a lot of interest as new prospects for anticancer medicationdevelopment. With its distinct redox potential, aromatic stability, and advantageouslipophilicity, ferrocene, an iron-containing sandwich compound, is a promising scaffold for thedevelopment of bioactive drugs. Its capacity to engage in redox cycling can result in theproduction of reactive oxygen species (ROS), which can cause oxidative stress to surpass thecellular threshold and trigger apoptosis when they are increased in cancer cells.US 20180354909 discloses substituted benzimidazolium compounds as chemotherapeutics, itssynthesis, product form, and biological evaluation, including cytotoxicity and mechanism ofaction. US 20160331727 discloses methods for treating cancer with imidazolium andimidazolinium compounds, their cellular localization, cytotoxicity, and mechanism of action.CN 118946358 A discloses specific tetradentate copper chelators as anticancer agents. US12134626 discloses a ferrocene derivative, its preparation method, and use. US 20240207414discloses a disulfide-based prodrug compound. US 20220340601 discloses functionalized goldcarbene naphthaquinone complexes for cancer treatment.Thus, there is a strong need in the art for developing next-generation organometallic anticanceragents that possess adequate solubility, stability, and ease of synthesis.OBJECTIVES OF THE INVENTIONIn view of the foregoing disadvantages inherent in the existing arts, the primary objective ofthe present invention is to provide a ferrocene-tagged benzimidazolium salt.Another objective of the present invention is to provide a ferrocene-tagged benzimidazoliumsalt which shows mitochondria-targeting and induces mitochondrial membrane disruption.Yet another objective of the present invention is to provide a ferrocene-taggedbenzimidazolium salt which shows selective anticancer efficacy by inducing oxidative stressthrough reactive oxygen species.Still another objective of the present invention is to provide a method for the synthesis offerrocene-tagged benzimidazolium salt.These and other objects and advantages of the present subject matter will be apparent to aperson skilled in the art after consideration of the following detailed description, taking intoconsideration accompanying drawings in which preferred embodiments of the present subjectmatter are illustrated.SUMMARY OF THE INVENTIONAn aspect of the present invention provides a ferrocene-tagged benzimidazolium salt havingthe structure formula IFormula I.Another aspect of the present invention provides a ferrocene-tagged benzimidazolium salt, Nferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide.Yet another aspect of the present invention provides a method for preparation of a ferrocenetagged benzimidazolium salt having the structure formula IFormula Icomprising the steps:a. mixing 1-(2-pyridinylmethyl)-1H-benzimidazole and (ferrocenylmethyl)trimethylammonium iodide and dissolving in a solvent to obtain a reaction mixture;b. refluxing the reaction mixture at a temperature in the range of 80-90°C for 18-20hours;c. allowing the reaction mixture to cool to room temperature and adding ice-cold waterto the reaction mixture;d. adding chloroform to the reaction mixture and extracting an organic phase; ande. drying the organic phase over anhydrous sodium sulfate to obtain the ferrocenetagged benzimidazolium salt.These and other aspects of the disclosed subject matter, as well as additional novel features,will be apparent from the description provided herein. The intent of this summary is not to bea comprehensive description of the claimed subject matter, but rather to provide a shortoverview of some of the subject matter's functionality. Other systems, methods, features andadvantages here provided will become apparent to one with skill in the art upon examinationof the following figures and detailed description. It is intended that all such additional systems,methods, features and advantages that are included within this description be within the scopeof any claims.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:It is to be noted, however, that the appended drawings illustrate only typical embodiments ofthe present subject matter and are therefore not to be considered for limiting its scope, for theinvention may admit to other equally effective embodiments. The detailed description isdescribed concerning the accompanying figures. Some embodiments of system or methods inaccordance with embodiments of the present subject matter are now described, by way ofexample, and with reference to the accompanying figures, in which:Figure 1 illustrates (i) Solid-state structure of FBP, (ii) Space-filling model of FBP, (iii) 1HNMR spectrum of FBP in DMSO-d6 at RT, (iv) 13C NMR spectrum of FBP in DMSO-d6 at RT.Figure 2 shows Biocompatibility Studies of FBP on NIH3 / T3 cells (a, b) and L929 cells (c, d).Figure 3 shows Cytotoxicity Studies of FBP on A549 cells (a, b); Cytotoxicity Studies of FBPon HeLa cells (c, d); Cytotoxicity Studies of FBP on B16 cells (e, f); Cytotoxicity Studies ofFBP on 4T1 (g, h).Figure 4 (a, b) shows Uptake Studies of FBP, Ferrocene and Doxorubicin on HeLa cervicalcancer cells (Scale bar: 50 μm).Figure 4 (c, d, and e) shows Live-dead assay in HeLa cells after treatment with FBP, Ferroceneand Doxorubicin (Scale bar: 100 μm).Figure 5 (a, c) shows ROS generation evaluation of Ferrocene, Doxorubicin and FBP on HeLacervical cancer cells by a DCFDA assay (Scale bar: 100 μm).Figure 5(b, d) shows Singlet oxygen species generation evaluation in HeLa cells after treatmentwith Ferrocene, Doxorubicin, and FBP by a SOS G assay (Scale bar: 100 μm).Figure 5(e, f, and g) shows Mitochondrial damage evaluation by JC-1 staining in HeLa cellsafter treatment with Ferrocene, Doxorubicin, and FBP (Scale bar: 100 μm).DETAILED DESCRIPTION OF THE INVENTIONA detailed description of various exemplary embodiments of the disclosure is described herein.It should be noted that the embodiments are described herein in such detail as to communicatethe disclosure. However, the amount of details provided herein is not intended to limit theanticipated variations of embodiments; on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spirit and scope of the presentdisclosure.It is also to be understood that various substitutions / arrangements / permutations orcombinations may be devised that, although not explicitly described or shown herein, embodythe principles of the present disclosure. Moreover, all statements herein reciting principles,aspects, and embodiments of the present disclosure, as well as specific examples, are intendedto encompass equivalents thereof.The terminology used herein is to describe particular embodiments only and is not intended tobe limiting to example embodiments. As used herein, the singular forms "a", "an" and "the"are intended to include the plural forms as well, unless the context indicates otherwise. It willbe further understood that the terms "comprise", "comprising", "includes" and / or "including"when used herein, specify the presence of stated features, steps, operations, elements and / orcomponents, but do not preclude the presence or addition of one or more other features, steps,operations, elements, components and / or groups thereof.The term 'further' is used in the embodiments and claims of the present application. The saidterm is a well-accepted term to narrow down any principal feature. Therefore, the person skilledin the art would clearly understand the scope of the said term in the context of the presentdisclosure.As used herein, the term 'FBP' refers to the N-ferrocenylmethyl-N'-(2-pyridylmethyl)benzimidazolium iodide salt.As used herein, the term 'ferrocene-tagged benzimidazolium salt' refers to anybenzimidazolium salt in which one nitrogen atom is substituted with a ferrocenyl-containingmoiety, providing redox-active organometallic functionality.As used herein, the term 'reactive oxygen species (ROS)' refers to chemically reactive oxygencontaining molecules generated intracellularly, including but not limited to superoxide,hydrogen peroxide, and hydroxyl radicals, which contribute to oxidative stress.As used herein, the term 'IC₅₀' refers to the concentration of the compound required to inhibit50% of cellular metabolic activity in vitro, as determined using assays such as MTT.As used herein, the term 'pharmaceutical composition' refers to a formulation containing thecompound of the invention and one or more pharmaceutically acceptable carriers, diluents,stabilizers, or excipients.As used herein, the term 'anticancer agent' refers to a compound capable of inhibitingproliferation or inducing death of cancer cells through mechanisms including but not limited toROS induction and mitochondrial disruption.The present invention is directed towards a ferrocene-tagged benzimidazolium salt having thestructure formula IFormula I.In an embodiment of the present invention there is provided a ferrocene-taggedbenzimidazolium salt, wherein the salt is N-ferrocenylmethyl-N'-(2-pyridylmethyl)benzimidazolium iodide.Another embodiment of the present invention provides a method for the preparation of aferrocene-tagged benzimidazolium salt having the structure formula IFormula Icomprising the steps:a. mixing 1-(2-pyridinylmethyl)-1H-benzimidazole and (ferrocenylmethyl)trimethylammonium iodide and dissolving in a solvent to obtain a reaction mixture;b. refluxing the reaction mixture at a temperature in the range of 80-90°C for 18-20hours;c. allowing the reaction mixture to cool to room temperature and adding ice-cold waterto the reaction mixture;d. adding chloroform to the reaction mixture and extracting an organic phase; ande. drying the organic phase over anhydrous sodium sulfate to obtain the ferrocenetagged benzimidazolium salt.In yet another embodiment of the present invention there is provided a method for thepreparation of a ferrocene-tagged benzimidazolium salt, wherein the solvent is acetonitrile.In still another embodiment of the present invention there is provided a method for preparationof a ferrocene-tagged benzimidazolium salt, wherein 1-(2-pyridinylmethyl)-1H-benzimidazoleand (ferrocenylmethyl) trimethylammonium iodide are mixed in a ratio of 1 : 1.2 equivalents.Another embodiment of the present invention provides a method for the preparation of aferrocene-tagged benzimidazolium salt having the structure formula IFormula Icomprising the steps:a. mixing 1-(2-pyridinylmethyl)-1H-benzimidazole and (ferrocenylmethyl)trimethylammonium iodide and dissolving in acetonitrile to obtain a reactionmixture;b. refluxing the reaction mixture at a temperature of 85°C for 18 hours;c. allowing the reaction mixture to cool to room temperature and adding ice-cold waterto the reaction mixture;d. adding chloroform to the reaction mixture and extracting an organic phase; ande. drying the organic phase over anhydrous sodium sulfate to obtain the ferrocenetagged benzimidazolium salt.The present invention discloses a ferrocene-tagged benzimidazolium salt, N-ferrocenylmethylN'-(2-pyridylmethyl) benzimidazolium iodide having the structure formula IFormula I.The salt N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide is engineered toenhance selective anticancer activity through mitochondrial targeting and oxidative stressinduction. The salt is synthesized by mixing 1-(2-Pyridinylmethyl)-1H-benzimidazole and(ferrocenylmethyl) trimethylammonium iodide, dissolving in acetonitrile to obtain a reactionmixture and refluxing the reaction mixture for 18 hrs at 85°C. After the completion of thereaction, the reaction mixture is allowed to attain room temperature, and ice-cold water is addedto the reaction mixture. After that, chloroform (3 times volume) is added and an organic phaseis extracted. This organic phase is dried over anhydrous Na2SO4, and chloroform is removedunder reduced pressure to obtain N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazoliumiodide. Scheme 1 below provides a synthesis route.Scheme 1The salt is soluble in both water and various organic solvents, including chloroform, DCM,acetonitrile, and DMSO. The UV-Vis absorption spectrum exhibits a strong absorption peak atλabs = 210 nm and a relatively weak absorption peak at λabs = 254 nm, accompanied by ashoulder peak at λabs = 280 nm. The salt is found to be non-emissive in solution (methanol) aswell as in the crystalline state.The salt is engineered to enhance selective anticancer activity through mitochondrial targetingand oxidative stress induction. The salt is first evaluated for biocompatibility using normalmurine fibroblast cell lines (NIH3 / T3 and L929), revealing tolerability at therapeuticconcentrations. Anticancer efficacy is assessed in four cancer cell lines -HeLa (cervical), 4T1(breast), B16 (melanoma), and A549 (lung), where HeLa cells demonstrate the highestsensitivity, with a favourable IC₅₀ (180 μg / mL) value and selectivity index (1.61).To investigate cellular behaviour, fluorescence microscopy is employed using Hoechst 33342and MitoTracker Green, confirming efficient cellular uptake and mitochondrial localization.To further probe the mechanism of action, a series of functional assays is performed. DCFDAstaining and Singlet Oxygen Sensor Green (SOSG) assays reveal substantial ROS and singletoxygen generation. Additionally, JC-1 dye-based assays indicate significant mitochondrialmembrane potential disruption, a key marker of apoptosis initiation. The salt consistentlyoutperforms both doxorubicin, a clinically used chemotherapeutic agent, and native Ferrocene,indicating that structural modification confers enhanced bioactivity and selectivity.ADVANTAGES OF THE PRESENT INVENTION- The N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide salt of thepresent invention shows better biological activity than other chemotherapy drugs andFerrocene.- The N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide salt of thepresent invention shows good biocompatibility in the normal cell lines (L929 andNIH3 / T3).- The N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide salt of thepresent invention shows a potent cytotoxic effect on cancer cell lines, and moreselectivity towards cervical cancer cells.- The N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide salt of thepresent invention causes cancer cell death by ROS generation and mitochondrialmembrane disruption.EXAMPLESThe following examples are given by way of illustration, therefore, should not be construed tolimit the scope of the invention.EXAMPLE 1Synthesis of N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide (FBP)1-(2-Pyridinylmethyl)-1H-benzimidazole (500 mg, 2.39 mmol, 1 equivalent) and(ferrocenylmethyl) trimethylammonium iodide (1.10 g, 2.87 mmol, 1.2 equivalent) were mixedand dissolved in 15 mL acetonitrile to obtain a reaction mixture. The reaction mixture wasrefluxed for 18 hrs at 85°C. After the completion of the reaction, the reaction mixture wasallowed to attain room temperature, and ice-cold water was added to the reaction mixture.Chloroform (15 mL x 3 times) was added to the reaction mixture, to extract an organic phasewhich was dried over anhydrous Na2SO4. The chloroform was removed under reduced pressureto obtain yellowish powdered N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazoliumiodide (FBP).N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodideEXAMPLE 2CHARACTERIZATIONThe formation of the salt was confirmed by SCXRD, 1H NMR, and 13C NMR spectroscopytechniques.2.1 SCXRDThe Bruker D8 Venture Single Crystal X-ray diffractometer was used to determine the crystalstructure of the FBP molecule, utilising a Mo-Kα (0.71073 Å) source at 298 K. Figure 1 shows(i) Solid-state structure of FBP, and (ii) the Space-filling model of FBP.2.2 1H NMRThe 1H NMR data were collected using a Bruker Ultra Shield 400 MHz spectrometer at RT (25°C) IN CDCl3.Figure 1 shows (iii) 1H NMR spectrum of FBP in DMSO-d6 at RT. The presence of the N-CHN peak at 9.92 ppm in the ¹H NMR spectrum confirms the formation of the product in thesolution state, supporting the solid-state structure of FBP.2.3 13C NMRFigure 1 (iv) shows 13C NMR spectrum of FBP in DMSO-d6 at RT.2.4 UV-VisUV-Vis absorption data are collected using a Lab India 2000U UV-Vis spectrometer at RT (25°C) in Methanol. The UV-Vis absorption spectrum exhibits a strong absorption peak at λabs =210 nm and a relatively weak absorption peak at λabs = 254 nm, accompanied by a shoulderpeak at λabs = 280 nm. FBP is found to be non-emissive in solution (methanol) as well as in thecrystalline state.EXAMPLE 3BIOCOMPATIBILITYL929 fibroblasts (procured from National Centre for Cell Science (NCCS), Pune, Maharashtra,India) and NIH3 / T3 murine fibroblasts (procured from National Centre for Cell Science(NCCS), Pune, Maharashtra, India) were used to establish the cytocompatibility of FBP.Cells were exposed to a concentration gradient of 1-500 μg / mL (50, 100, 200, 300, 400, and500 μg / mL) of FBP for 24 hours under standard culture conditions.Figure 2 shows Biocompatibility Studies of FBP on NIH3 / T3 cells (a, b) and L929 cells (c, d).Dose-response analyses revealed negligible cytotoxicity (cell viability ≥ 80 %) atconcentrations ≤ 200 μg / mL across all both types, with IC50 values exceeding 220-290 μg / mL,indicating favourable biocompatibility within therapeutically relevant dosage ranges. Thispreliminary profiling in diverse normal cell models supports the suitability of the salt forsubsequent anticancer screening while highlighting its potential selectivity toward malignantversus non-transformed tissues.EXAMPLE 4CYTOTOXICITYThe cytotoxic profiles of FBP were evaluated in 4T1 (murine mammary carcinoma) cell line,A549 (human lung adenocarcinoma) cell line, B16 cell line, and HeLa (human cervicalcarcinoma) cell line via MTT assay. All above mentioned cell lines were procured fromNational Centre for Cell Science (NCCS), Pune, Maharashtra, India.Cells were seeded in 96-well plates at a density of 1 x 10 cells / well and incubated at 37°C with5% CO2 for 24 hours to promote cell adhesion. Subsequently, cells were treated with varyingconcentrations (50, 100, 200, 300, 400, and 500 μg / mL) of the FBP. Untreated cells served asthe negative control. After 24 hours of FBP exposure, the medium was replaced with freshDMEM or RPMI containing MTT reagent (5 mg / mL), and cells were incubated for anadditional 3 hours to allow for formazan crystal formation. The resulting crystals weredissolved in DMSO, and absorbance was measured at 570 nm and 630 nm using a multimodeplate reader to assess cell viability.Figure 3 shoes Cytotoxicity Studies of FBP on A549 cells (a, b); Cytotoxicity Studies of FBPon HeLa cells (c, d); Cytotoxicity Studies of FBP on B16 cells (e, f); Cytotoxicity Studies ofFBP on 4T1 (g, h). Dose-dependent cytotoxicity analyses revealed notable differences insensitivity: FBP exhibited the highest IC50 potency in HeLa cells (180 μg / mL) (Table 1).Untreated cells served as negative controls, with viability normalised to 100%.Selectivity indices (SI) were calculated to assess therapeutic specificity using the formula: SI= (IC50 value in normal cells) / (IC50 value in cancer cells) Thresholds define SI as follows: SI< 1 (normal cell toxicity), SI = 1 (equivalent toxicity), SI > 1 (cancer cell selectivity), and SI>> 1 (high specificity).At a threshold of SI ≥ 1, FBP showed an SI of 1.61 in HeLa cells relative to NIH3 / T3 murinefibroblasts, as shown in Table 2. These values indicate preferential cytotoxicity towardmalignant cells, underscoring their potential as selective chemotherapeutic agents with anexpanded therapeutic window.Table 1. IC50 of FBP in different cell lines.Table 2. Selectivity index of FBP in different cell lines.EXAMPLE 5INTRACELLULAR UPTAKE STUDIESThe intracellular uptake and localisation of FBP, Ferrocene and Doxorubicin were evaluated inHeLa cells. Hoechst was used to stain the nucleus. Mitogreen was specifically used to stainmitochondria to understand the localisation of these compounds.HeLa cervical cancer cells were used to assess intracellular uptake of Ferrocene, Doxorubicin(Dox), and FBP through imaging studies. Approximately 1 x 10⁵ cells were seeded ontocoverslips placed in a 12-well plate. After allowing the cells to adhere for 12 hours, they weretreated with 300 μg / mL of Ferrocene, the IC50 concentration of Dox, and 180 μg / mL of FBPfor 5 hours. Following treatment, the cells were stained with Hoechst & Mitogreen dyes andincubated in the dark for 10 minutes. Imaging was performed using the Zoe Fluorescent CellImager.Figure 4 (a, b) shows Uptake Studies of FBP, Ferrocene and Doxorubicin on HeLa cervicalcancer cells (Scale bar: 50 μm). The blue and green fluorescence in Figure 4a shows theintracellular uptake of FBP after 3h of incubation in HeLa cells. Quantitative analysis of theuptake study was done using Image J (Figure 4a, 4b).EXAMPLE 6LIVE / DEAD ASSAYThe anticancer efficacy of Ferrocene, Doxorubicin and FBP was qualitatively assessed viaFDA / PI dual staining, a fluorescence-based live / dead assay.Following 24-hour exposure to Ferrocene, FBP and Doxorubicin (300 μg / mL of Ferrocene,180 μg / mL of FBP and 2μM of Doxorubicin), HeLa cells were incubated with FDA (5 μg / mL)and PI (2 μg / mL) for 15 minutes. FDA, hydrolysed by viable cells to emit green fluorescence(λex = 488 nm, λem = 530 nm), indicated metabolic activity, while PI selectively stained nucleiof membrane-compromised cells with red fluorescence (λex = 535 nm, λem = 617 nm).Figure 4 (c, d, and e) shows Live-dead assay in HeLa cells after treatment with FBP, Ferroceneand Doxorubicin (Scale bar: 100 μm). Fluorescence microscopy revealed a pronouncedincrease in PI-positive cells (bright red fluorescence) in FBP-treated HeLa cultures comparedto untreated controls and other treatment groups. The untreated groups exhibited predominantlygreen fluorescence, consistent with >90% viability, whereas doxorubicin and Ferro-cenepositive controls showed intermediate PI staining. These observations correlate with priorcytotoxicity data, confirming the selective induction of cell death by FBP in a cell linedependent manner.EXAMPLE 7DCFDA ANALYSISThe intracellular ROS generation mediated by FBP in HeLa cells was quantified via the 2',7'-dichlorofluorescein diacetate (DCFDA) assay, a widely employed fluorometric method fordetecting oxidative stress.Following 24-hour exposure to the Ferrocene, Doxorubicin and FBP (300 μg / mL of Ferrocene,180 μg / mL of FBP and 2μM of Doxorubicin), cells were incubated with 10 μM DCFDA inserum-free medium, allowing the cell-permeable probe to undergo de-esterification byintracellular esterases to non-fluorescent DCFH, which is subsequently oxidized by ROS tofluorescent dichloro fluorescein (DCF).Figure 5 (a, c) shows ROS generation evaluation of Ferrocene, Doxorubicin and FBP on HeLacervical cancer cells by a DCFDA assay (Scale bar: 100 μm). Fluorescence microscopyrevealed a marked elevation in DCF-derived green fluorescence intensity (λex = 488 nm, λem =530 nm) in treated cells compared to untreated controls and other treatment groups. HeLa cellstreated with FBP exhibited a prominent increase in fluorescence signal relative to controls,indicating ROS accumulation in a cell line- and complex-dependent manner. Untreated cellsdemonstrated minimal fluorescence, consistent with basal ROS levels maintained byendogenous antioxidant systems. These findings validate the pro-oxidant efficacy of FBP indestabilising redox homeostasis, correlating with their observed cytotoxic effects.The efficiency of singlet oxygen (1O2) generation by Ferrocene, Doxorubicin (Dox), and FBPwas assessed in real time using the Singlet Oxygen Sensor Green (SOSG) probe. HeLa cervicalcancer cells were treated with each of the test (300 μg / mL of Ferrocene, 180 μg / mL of FBPand 2μM of Doxorubicin) and control groups.Figure 5(b, d) shows Singlet oxygen species generation evaluation in HeLa cells after treatmentwith Ferrocene, Doxorubicin, and FBP by a SOS G assay (Scale bar: 100 μm). Notable greenfluorescence was observed in the FBP-treated samples under a fluorescence microscope. Thisfluorescence indicated the activation of SOSG, confirming the production of singlet oxygen(1O2).EXAMPLE 8MITOCHONDRIAL MEMBRANE POTENTIAL ANALYSISMultiple cellular processes, including ATP synthesis, redox equilibrium, cell cyclemanagement, and cellular apoptosis, are regulated by the cell's powerhouse, "mitochondria."The membrane potential in mitochondria is a marker for the functioning of mitochondria.Decreased membrane potential might trigger a cascade of events correlating with programmedcell death.The JC1 staining method was used to analyze the FBP activity on the membrane potential ofthe mitochondria. The JC1 dye fluoresces green as a monomer when it encounters an unhealthyor dying mitochondrial mem brane, but it aggregates and fluoresces red when it meets a healthymitochondrial membrane.HeLa cells, upon treatment with Ferrocene, Dox, and FBP (300 μg / mL of Ferrocene, 180μg / mL of FBP and 2μM of Doxorubicin), were incubated for 24 hours. Post incubation, JC1stain was added to the plate and left undisturbed for 30 min in an incubator. J monomer (λex =485 nm, λem = 535 nm) and J aggregate (λex = 535 nm, λem = 595 nm) readings were recordedby a microplate reader. To assess mitochondrial membrane potential, green fluorescenceindicates unhealthy mitochondria, and red fluorescence indicates healthy mitochondria. Theresultant red-green fluorescence was then analyzed to indicate mitochondrial damage.Figure 5(e, f, and g) shows Mitochondrial damage evaluation by JC-1 staining in HeLa cellsafter treatment with Ferrocene, Doxorubicin, and FBP (Scale bar: 100 μm). Figure 5g indicatesthat cells subjected to FBP had significantly damaged mitochondria due to increased ROSproduction compared to other control groups and test groups. The figure shows that the red-to-green ratio was found to be lower in the cells exposed to the FBP than in the other groups(Figure 5e, 5f). The quantitative analysis of JC1 staining is represented in 5e, respectively,revealing a statistical significance of p < 0.001. Thus, the FBP can be used as a potential anti-cancer agent.Although embodiments for the present subject matter have been described in languagespecific to features, it is to be understood that the present subject matter is not necessarilylimited to the specific features described. Rather, the specific features and methods aredisclosed as embodiments for the present subject matter. Numerous modifications andadaptations of the system / device of the present invention will be apparent to those skilled inthe art, and thus it is intended by the appended claims to cover all such modifications andadaptations which fall within the scope of the present subject matter.It will be further appreciated that functions or structures of a plurality of components or stepsmay be combined into a single component or step, or the functions or structures of one-step orcomponent may be split among plural steps or components. The present invention contemplatesall of these combinations. Unless stated otherwise, dimensions and geometries of the variousstructures depicted herein are not intended to be restrictive of the invention, and otherdimensions or geometries are possible. In addition, while a feature of the present invention mayhave been described in the context of only one of the illustrated embodiments, such feature maybe combined with one or more other features of other embodiments, for any given application.It will also be appreciated from the above that the fabrication of the unique structures hereinand the operation thereof also constitute methods in accordance with the present invention. Thepresent invention also encompasses intermediate and end products resulting from the practiceof the methods herein.

Claims

1. A ferrocene-tagged benzimidazolium salt having the structure formula I Formula I.

2. The ferrocene-tagged benzimidazolium salt as claimed in claim 1, wherein the salt is N-ferrocenylmethyl-N'-(2-pyridylmethyl) benzimidazolium iodide.

3. A method for preparation of a ferrocene-tagged benzimidazolium salt having the structure formula I Formula I comprising the steps: a. mixing 1-(2-pyridinylmethyl)-1H-benzimidazole and (ferrocenylmethyl) trimethylammonium iodide and dissolving in a solvent to obtain a reaction mixture; b. refluxing the reaction mixture at a temperature in the range of 80-90°C for 18-20 hours; c. allowing the reaction mixture to cool to room temperature and adding ice cold water to the reaction mixture; d. adding chloroform to the reaction mixture and extracting an organic phase; and e. drying the organic phase over anhydrous sodium sulfate to obtain the ferrocenetagged benzimidazolium salt.

4. The method as claimed in claim 3 wherein the solvent is acetonitrile.

5. The method as claimed in claim 3, wherein 1-(2-pyridinylmethyl)-1H-benzimidazole and (ferrocenylmethyl) trimethylammonium iodide are mixed in a ratio of 1: 1.2 equivalent.