A novel biocompatible NiII tethered moiety as a glucose uptake agent and a hit against methicillin-resistant Staphylococcus aureus

 

Abstract

 

The escalating global burden of both non-communicable diseases (NCDs) and communicable diseases (CDs) necessitates innovative approaches to drug discovery, particularly focusing on biocompatible transition metal complexes as promising therapeutic agents. In line with these critical efforts, a novel binuclear Nickel(II) compound, designated as [{NiII(hpdbal-sbdt)}2] (compound 2), has been successfully synthesized. This compound was ingeniously formed through a chemical reaction involving Nickel(II) acetate tetrahydrate (Ni(OAc)2.4H2O) and a meticulously designed organic ligand, H2hpdbal-sbdt (compound 1). Ligand 1 itself is characterized as a dibasic tridentate ONS2- donor Schiff base ligand, which was precisely obtained via the condensation reaction between 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) and S-benzyldithiocarbazate (Hsbdt).

 

Both the as-synthesized ligand (compound 1) and the resulting binuclear Nickel(II) complex (compound 2) underwent comprehensive structural characterization, encompassing both their solid-state and solution-state properties. This extensive analysis was performed using a diverse array of advanced spectroscopic and analytical techniques. These included Attenuated Total Reflection Infrared (ATR-IR) spectroscopy for vibrational analysis, Proton Nuclear Magnetic Resonance (1H-NMR) and Carbon-13 Nuclear Magnetic Resonance (13C-NMR) spectroscopy for detailed structural elucidation in solution, Thermogravimetric Analysis (TGA) to assess thermal stability, Field Emission Scanning Electron Microscopy (FESEM) for morphological insights, Energy Dispersive X-ray Spectroscopy (EDS) for elemental mapping, and Carbon, Hydrogen, Nitrogen, Sulfur (CHNS) elemental analysis to confirm empirical composition and purity.

 

A crucial aspect of this research involved evaluating the potential antidiabetic activity of both the ligand H2hpdbal-sbdt (compound 1) and the Nickel(II) complex [{NiII(hpdbal-sbdt)}2] (compound 2). Their efficacy was rigorously assessed through a 2-NBDG uptake assay, a widely accepted method for quantifying glucose uptake in cellular systems. This assay was specifically conducted on insulin-resistant HepG2 cells, a relevant *in vitro* model for studying aspects of type II diabetes. The results were highly promising: insulin-resistant HepG2 cells treated with compound 1 demonstrated a significant 85% fluorescent glucose uptake, while those treated with compound 2 exhibited an even more remarkable 95% fluorescent glucose uptake. Notably, the level of 2-NBDG uptake observed in cells treated with compound 2 was found to be directly comparable to that achieved by metformin, a globally recognized and extensively utilized standard antidiabetic drug, underscoring the potent glucose-lowering potential of the newly synthesized complex.

 

Beyond their antidiabetic properties, compounds 1 and 2 were also subjected to rigorous antimicrobial testing to ascertain their pathogen-killing capabilities. Their activities were evaluated against a panel comprising five distinct bacterial strains and two different fungal strains. In this context, compound 2 demonstrated highly significant inhibitory action against the clinically important methicillin-resistant *Staphylococcus aureus* (MRSA) strain, a notorious multidrug-resistant bacterium, with a remarkably low Minimum Inhibitory Concentration (MIC) value of 2 µg/mL. This potent activity highlights its strong potential as an antibacterial agent against challenging resistant pathogens. In contrast, the free ligand (compound 1) was found to be entirely inactive against MRSA, strongly suggesting that the specific coordination of the ligand with the Nickel(II) ion is crucial for conferring this enhanced antimicrobial property.

 

Furthermore, to gain critical insights into the likely pharmacokinetic behavior and distribution of compound 2 within a biological system, its interactive characteristics with bovine serum albumin (BSA), a well-established model serum carrier protein that closely mimics human serum albumin, were extensively investigated. This comprehensive interaction study employed a multi-spectroscopic approach, leveraging various fluorescence and absorption spectroscopic techniques. The detailed analysis derived from these studies provided invaluable information regarding several key aspects of the binding interaction: the precise nature of the binding forces involved between compound 2 and BSA, the extent and strength of this binding, any subtle or significant conformational changes induced in the BSA protein upon complexation with the drug candidate, and the specific mechanisms responsible for the observed quenching of the intrinsic fluorescence of the amino acid residues within the protein structure. These fundamental insights are crucial for predicting the drug’s circulation, distribution, and potential efficacy *in vivo*.

 

Keywords: Type II diabetes; Methicillin-resistant *Staphylococcus aureus*; Nickel(II) Schiff base complex; Bovine serum albumin; Ligand-protein interaction study; Three-dimensional fluorescence spectroscopy.

 

Introduction

 

The contemporary global health landscape is increasingly defined by the alarming and relentless rise in the prevalence of both non-communicable diseases (NCDs), exemplified by conditions such as type II diabetes, and communicable diseases (CDs), particularly in the form of increasingly antibiotic-resistant pathogenic infections. These infectious threats, encompassing a range of waterborne and airborne diseases, collectively pose a profound and multifaceted global health crisis. Recent statistics from the World Health Organization (WHO) paint a stark picture: approximately 1.5 million individuals succumbed to diabetes-related complications in 2015 alone. Furthermore, the global surveillance report on antimicrobial resistance, also issued by the WHO, unequivocally highlights a critical void in the discovery and development of new antimicrobial agents, while simultaneously documenting a distressing trend of increasing pathogen resistance to many well-established and widely used antibiotics. This escalating resistivity has led to a concerning failure of commercially available oral drugs to effectively treat infected individuals, creating an urgent and unmet medical need. To effectively overcome this growing upsurge of challenging cases, the development of highly effective and innovative treatment approaches is in extraordinary demand.

 

In this relentless pursuit, transition metal compounds have emerged as a particularly attractive and fruitful area of scientific investigation. The historical advent of groundbreaking metal-based drugs, such as salvarsan, an arsenic-based compound, revolutionized the treatment of syphilis, as demonstrated by Ehrlich’s pioneering work. Similarly, the discovery of cisplatin, a platinum-based complex, transformed the therapeutic landscape for testicular cancer, highlighting the profound clinical utility of metal-containing pharmaceuticals. These landmark successes have catalyzed a substantial increase in research dedicated to exploring the biological activities of a vast array of metal-based compounds, with a particular emphasis on those involving transition metals. This intense scrutiny has revealed a wide range of therapeutic properties, including anticancer, antimicrobial, and even antidiabetic effects.

 

Among the various transition metals, nickel has garnered considerable attention and has been extensively studied for its therapeutic properties, particularly in the form of coordination compounds. Recent reports underscore its diverse biological activities. For instance, Sridhar et al. recently documented a Nickel(II) complex derived from (Z)-4-fluoro-N-(2,6-dimethylhept-5-enylidene)benzenamine, which exhibited excellent activity specifically against Gram-negative bacterial species, representing a significant area of unmet need in antimicrobial therapy. Similarly, other Nickel(II) complexes, specifically those derived from the amino acid tyrosine, have demonstrated commendable antibacterial and antifungal activities, with Minimum Inhibitory Concentration (MIC) values spanning a broad range from 120 to 1000 µg/mL, indicating their versatile antimicrobial potential. Furthermore, a [Diaquabis(hexamethylenetetramine)diisothiocyanato-κN]nickel(II) complex displayed remarkable activity against *E. coli* and *S. flexneri* species, both common bacterial pathogens. Nickel(II)-oxaloyldihydrazone complexes have also proven effective against both Gram-positive and Gram-negative bacterial species, showcasing broad-spectrum antimicrobial properties. Notably, a mixed ligand complex of Nickel(II) incorporating cephalosporin and sulfathiazole showed antibacterial activity comparable to that of commercially available cephalosporin drugs, suggesting potential for enhanced or new formulations. Additionally, Nickel complexes derived from Schiff bases of 5-methyl salicylaldehyde and ethylenediamine, as well as 5-methyl salicylaldehyde and diaminomaleonitrile, have displayed effective antibacterial and antifungal activities even at a relatively low concentration of 100 μg/mL. In the majority of these documented cases, the observed growth inhibition of microbial species by nickel compounds is attributed to various mechanisms, including direct DNA binding, induction of cell membrane rupturing, and detrimental interference with bacterial protein synthesis. There are indeed reports suggesting that nickel complexes specifically disturb the respiratory processes within the bacterial cell and effectively block protein synthesis, thereby restricting the proliferation and growth of bacterial species.

 

Beyond their antimicrobial capabilities, nickel complexes have also emerged as promising prodrug-like molecules for the treatment of various forms of cancer. For example, a recent study demonstrated that a Nickel(II) pyrithione complex potently displayed proteasome inhibition and robustly induced apoptosis in cultured acute myeloid leukemia cancer cells, highlighting a novel mechanism of anticancer action. Similarly, Nickel(II)-dithiocarbamate phenanthroline complexes were shown to induce non-apoptotic cell death specifically in human breast cancer stem-cells, targeting a notoriously resistant subpopulation of cancer cells. Another Nickel(II) complex, derived from 4-(2′-nitrobenzylideneimino)-3-methyl-5-mercapto-1,2,4-triazole, exhibited significant cytotoxic activity against MCF-7 breast cancer cell lines. Furthermore, Nickel(II) complexes derived from isatin and thiosemicarbazone analogs displayed compelling *in vitro* cytotoxic activities against the human colorectal carcinoma cell line HCT 116. In addition to these, Nickel(II) thiocarboxamide complexes have demonstrated excellent antioxidant activity while exhibiting minimal cytotoxicity towards normal cells, which is a highly desirable characteristic for therapeutic agents.

 

While the anticancer and antimicrobial properties of nickel complexes are relatively well-documented, there are comparatively fewer reports detailing their antidiabetic activity, and even fewer have demonstrated significant insulin-mimicking or anti-diabetic actions. Nevertheless, some progress has been made: Nickel(II) complexes derived from 6-methyl-3-formylchromone hydrazones have been investigated for their inhibitory activity towards α-amylase and α-glucosidase enzymes, both of which play crucial roles in carbohydrate metabolism and therefore are important targets in diabetes management. Similarly, a Nickel(II) complex synthesized from a Schiff base of benzhydrazide has also been studied for its amylase inhibition activity. These initial findings suggest that nickel complexes possess a vast untapped potential for further exploration of their diverse pharmaceutical properties, particularly those with therapeutically interesting features that could address unmet medical needs.

 

The investigation of how prospective drug candidates interact with endogenous carrier proteins, such as serum albumins (including human serum albumin [HSA] and bovine serum albumin [BSA]), constitutes an exceptionally important facet in the successful development of pharmaceutical agents. These studies are critical as they provide a profound understanding of the pharmacodynamics and pharmacokinetics of the binding components, which, in turn, offers crucial insights into the structural changes induced by the drug upon binding to the drug carrier protein. This knowledge is fundamental for predicting the drug’s absorption, distribution, metabolism, and excretion (ADME) profile, ultimately impacting its bioavailability and therapeutic efficacy. Consequently, addressing this facet with the aid of versatile spectroscopic techniques and molecular docking tools has gained considerable popularity in modern drug discovery.

 

Building upon this compelling background, the current study reports the meticulous synthesis and comprehensive characterization of a novel Nickel(II) complex, specifically benzyl 2-(2-hydroxy-5-(phenyldiazenyl)benzylidene)hydrazinecarbodithioate nickel(II), designated as [{NiII(hpdbal-sbdt)}2] (compound 2). This binuclear complex is derived from a tridentate ONS2- donor ligand, benzyl 2-(2-hydroxy-5-(phenyldiazenyl)benzylidene)hydrazinecarbodithioate [H2hpdbal-sbdt] (compound 1). The ligand itself was synthesized through a condensation reaction involving 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) and S-benzyldithiocarbazate (Hsbdt), with the detailed structures illustrated in Scheme 1.

 

The newly synthesized ligand (compound 1) and the Nickel(II) complex (compound 2) were rigorously evaluated for their glucose uptake capacity to assess their potential antidiabetic action, providing essential data for their therapeutic prospects. Furthermore, their activity against a panel of bacterial and fungal species, with a particular focus on drug-resistant strains, was meticulously investigated to explore their antimicrobial potential. Finally, to comprehensively evaluate the interactive behavior of the bioactive nickel compound with a widely used model serum carrier protein, bovine serum albumin (BSA), a battery of multi-spectroscopic tools was employed. This approach enabled the precise determination of the nature and type of binding involved between the nickel compound and the protein, providing critical information for understanding its biological distribution and overall pharmaceutical profile.

 

Experimental

 

Materials

 

All chemical reagents and solvents utilized throughout this study were used precisely as received, without undergoing any further purification steps, ensuring the consistency and integrity of the starting materials. The specific materials obtained and their respective suppliers were as follows: Aniline (Spectrochem), Hydrochloric acid (SDFCL), Sodium Nitrite (Fisher Scientific), Salicylaldehyde (Spectrochem), Sodium carbonate anhydrous (Himedia), Sodium hydroxide (Himedia), Sodium chloride (Fischer Scientific), Hydrazine hydrate monohydrate (Spectrochem), Carbon disulfide (Spectrochem), KOH pellets (Himedia), Benzyl chloride (Spectrochem), Nickel acetate tetrahydrate (Spectrochem), and Triethylamine anhydrous (Spectrochem). All solvents employed in the experimental procedures were of analytical reagent (AR) grade, signifying their high purity, and doubly distilled water was exclusively used for all aqueous preparations to minimize impurities. S-benzyl dithiocarbazate (Hsbdt), a key intermediate, was synthesized according to an established and previously reported literature procedure. Similarly, 2-Hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) was synthesized following a procedure analogous to that described in a relevant US patent. The detailed synthetic protocols for the final ligand, [H2hpdbal-sbdt] (compound 1), and the target Nickel(II) complex, [{NiII(hpdbal-sbdt)}2] (compound 2), are thoroughly outlined in section 2.7 of this document.

 

Instrumentation

 

A comprehensive suite of analytical instruments was meticulously employed for the thorough and multifaceted characterization of the newly synthesized compounds. To ascertain the precise elemental composition, specifically the percentage of carbon, hydrogen, nitrogen, and sulfur, elemental analysis was rigorously performed using a Euro Vector E-3000 system, thereby ensuring the accurate determination and verification of the empirical formulas of the compounds. For the identification of distinct functional groups and to understand molecular vibrations within the solid-state compounds, Attenuated Total Reflection Infrared (ATR-IR) spectra were acquired. This was carried out using a Bruker Alpha Single reflection ATR module, which was specifically equipped with a ZnSe crystal, a material optimized for this analytical technique.

 

To gain critical insights into the electronic transitions and the extent of conjugation within the molecular structures, Ultraviolet-Visible (UV-Vis) spectra were precisely measured utilizing a UV-1800 Shimadzu UV-VIS Spectrophotometer. For the detailed elucidation of molecular structures and connectivity in solution, Proton Nuclear Magnetic Resonance (1H-NMR) and Carbon-13 Nuclear Magnetic Resonance (13C-NMR) spectra were recorded. These advanced spectroscopic data were obtained using an Agilent 400MR DD2 FT-NMR Spectrometer, with tetramethylsilane (SiMe4) consistently employed as an internal standard for highly accurate chemical shift referencing. To assess the thermal stability, decomposition behavior, and purity of the compounds, Thermogravimetric Analysis (TGA) was conducted. This analysis was performed on a Perkin Elmer STA 6000 TG/DTA analyzer, with scans extending up to a maximum temperature of 700 degrees Celsius to capture complete thermal degradation profiles.

 

The morphological characteristics, surface features, and elemental composition at the micro- and nanoscale were comprehensively investigated using Field Emission Scanning Electron Microscopy (FESEM) combined with Energy Dispersive X-Ray Spectroscopic (EDS) analysis. Both FESEM and EDS data were acquired from a state-of-the-art JEOL-JSM7100F instrument, providing high-resolution images and elemental maps. Finally, to determine the pKa (acid dissociation constant) of the ligand, which is a fundamental physicochemical property influencing its behavior in biological systems, a pH meter from the HI5000 Series (Hanna) was used. This instrument was precisely calibrated with certified standard buffer solutions at pH 4 and 7 prior to measurements, ensuring the highest level of accuracy and reliability in the pKa determination.

 

Antimicrobial Activity

 

The preliminary assessment of antimicrobial activity was initiated through whole-cell growth inhibition assays. For this initial screening, the synthesized compounds were tested at a single, fixed concentration in duplicate (n=2), allowing for an initial rapid evaluation of their potential antimicrobial efficacy. The inhibitory effect on microbial growth was evaluated against a broad panel of clinically relevant microorganisms. This panel included four Gram-negative bacterial species: *Escherichia coli* (Ec), *Klebsiella pneumoniae* (Kp), *Acinetobacter baumannii* (Ab), and *Pseudomonas aeruginosa* (Pa). Additionally, one Gram-positive bacterial species, *Staphylococcus aureus* (Sa), and two fungal strains, *Candida albicans* (Ca) and *Cryptococcus neoformans* (Cn), were included to ensure a comprehensive assessment against diverse pathogenic classes.

 

The compounds were carefully prepared by dissolving them in a mixture of DMSO (dimethyl sulfoxide) and water, achieving a final testing concentration of 32 μg/mL or 20 μM. These prepared solutions were then dispensed into 384-well, non-binding surface (NBS) plates, ensuring minimal non-specific adsorption of the compounds. Each bacterial or fungal strain was tested in duplicate (n=2), and strict control was maintained over the final DMSO concentration, which was kept at a maximum of 1% to mitigate any potential solvent-related effects on microbial growth. Compounds were categorized as “active” if they demonstrated an inhibition value of 80% or greater and concurrently exhibited a Z-score above 2.5, indicating a robust and statistically significant inhibitory effect. Conversely, samples were considered “partially active” if their inhibition value fell within the range of 50.9% to 79.9% and their Z-score was below 2.5, suggesting a moderate inhibitory effect that warrants further investigation. Detailed protocols for these screening criteria are provided in Supplementary Information Section 1.1.

 

Following the primary screening, active compounds were subjected to a hit confirmation study, also utilizing whole-cell growth inhibition assays. This phase involved an 8-point dose-response analysis, conducted in duplicate (n=2), with the primary aim of determining the Minimum Inhibitory Concentration (MIC) for each active compound. For this, compounds were prepared in DMSO and water to an initial testing concentration of 32 μg/mL or 20 μM, and then serially diluted by a 1:2 fold factor for eight consecutive dilutions, creating a gradient of concentrations to accurately pinpoint the MIC. Further details on this dose-response methodology are available in Supplementary Information Section 1.2.

 

For reliable comparative analysis, established antimicrobial agents were used as reference standards. Colistin was employed as a standard for Gram-negative bacteria, Vancomycin for Gram-positive bacteria, and Fluconazole for fungi. Specifically, the MIC value for Vancomycin was determined to be 1 µg/mL against *Staphylococcus aureus*, confirming its expected potency. The MIC value for Fluconazole was established at 0.13 µg/mL against *Candida albicans*, also consistent with its known efficacy. All standard drugs consistently demonstrated 100% growth inhibition of the respective bacterial and fungal species at concentrations exceeding their determined MIC values, validating the assay’s reliability. Further details on the standard drug controls are provided in Supplementary Information Section 1.3.

 

Protocol for Cytotoxicity Assay

 

To assess the potential cytotoxicity of the synthesized compounds against mammalian cells, a protocol using HEK293 (Human Embryonic Kidney cells, ATCC CRL-1573) was rigorously followed. Initially, HEK293 cells were accurately counted manually using a Neubauer haemocytometer to ensure precise cell density. Subsequently, these cells were plated into 384-well plates containing the test compounds at a density of 6000 cells per well, in a final volume of 50 μL. The cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% Fetal Bovine Serum (FBS), which served as the complete growth medium. The cells, along with the compounds, were incubated for a period of 20 hours at 37 °C in a humidified atmosphere containing 5% CO2, conditions optimal for cell growth and viability. Tamoxifen, a known cytotoxic agent, was utilized as a standard reference compound to provide a benchmark for cytotoxicity comparisons, with further details available in Supplementary Information Section 1.3.

 

Antidiabetic Activity

 

The assessment of antidiabetic activity was a crucial component of this study, primarily focusing on the glucose uptake capacity of the compounds. For the 2-NBDG assay, HepG2 (human hepatocyte carcinoma) cells, a widely recognized model for studying hepatic glucose metabolism and insulin resistance, were cultured in 96-well black plates until they reached approximately 70% confluency. To induce a state of insulin resistance, which mimics the physiological conditions of type II diabetes, the cells were then pre-treated with a carefully selected combination of pro-inflammatory and lipotoxic agents: tumor necrosis factor-alpha (TNF-alpha), fructose, and palmitic acid. Concurrently, the study compounds (ligand 1 and complex 2) and the standard antidiabetic drug metformin were added to the wells containing these insulin-resistant cells.

 

After a 24-hour incubation period, which allowed for the compounds to exert their effects on glucose metabolism, the wells were thoroughly washed with 1X PBS (phosphate-buffered saline) to remove any residual treatment media. Subsequently, 40 µM of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose), a fluorescent glucose analog that can be taken up by cells, along with 0.1 µM insulin, were added to each well for a 1-hour incubation period. This allowed the cells to take up the fluorescent glucose. Following this uptake period, the wells were carefully washed again to remove excess 2-NBDG. Finally, lysis buffer was added to the wells to lyse the cells and release the intracellular fluorescent glucose, and the fluorescence reading from each well was then quantitatively measured using a fluorescence spectrometer. Untreated HepG2 cells, which maintained normal insulin sensitivity, served as the healthy control group. HepG2 cells that were loaded with insulin and 2-NBDG but not treated with study compounds were designated as the Insulin Resistance (IR) control cells, providing a baseline for impaired glucose uptake.

 

Additionally, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed on HepG2 cells to assess their viability under various compound concentrations, ensuring that the observed antidiabetic effects were not simply due to cytotoxicity. HepG2 cells were seeded into a 96-well plate and allowed to grow until they reached approximately 70% confluency. At this point, the cells were incubated for 24 hours with different concentrations of the study compounds, dissolved in DMEM supplemented with 10% FBS and 1% antibiotic solution. After the incubation period, 2.4 mM of MTT reagent was added to the media and allowed to incubate for 4 hours. The insoluble formazan product, formed by metabolically active cells, was then solubilized by adding 100 µL of DMSO to each well. The absorbance readings were subsequently taken at 570 nm using a microplate reader, with higher absorbance indicating greater cell viability.

 

Preparation of BSA Solution

 

To conduct the crucial interaction studies with a model serum carrier protein, a solution of fatty acid-free Bovine Serum Albumin (BSA) was meticulously prepared. The protein was dissolved in a sodium phosphate buffer maintained at a physiological pH of 7.4. The precise details for the preparation of this buffer are elaborated in Supplementary Information Section 2. The concentration of the prepared BSA solution was accurately estimated spectrophotometrically by measuring its UV absorption spectrum at 280 nm, utilizing an extinction coefficient of 43890 M–1cm–1, a standard value for BSA. Typically, the prepared BSA stock solution had a concentration of 5 µM.

 

To facilitate the subsequent experiments, standard stock solutions of the ligand H2hpdbal-sbdt (compound 1) and the Nickel(II) complex [{NiII(hpdbal-sbdt)}2] (compound 2), each at a concentration of 1 mM, were prepared by dissolving them in DMSO. Absorption spectra, for monitoring interactions and changes in absorbance, were meticulously recorded on a temperature-controlled Analytik Jena Specord-250 spectrophotometer, employing quartz cuvettes with a 1.0 cm/10 mm pathlength for optimal light transmission. Fluorescence spectra, crucial for assessing binding and conformational changes, were measured using a Varian Cary Eclipse spectrofluorometer, which was equipped with a high-intensity 150W xenon lamp, performed in a 1 cm quartz cell. Lastly, Fourier Transform Infrared (FT-IR) spectra, providing insights into molecular vibrations and functional group changes, were measured using a Specac Golden Gate diamond ATR sampler, which was fitted to a Bruker Tensor 27 instrument equipped with an MCT detector.

 

Synthesis

 

Synthesis of H2hpdbal-sbdt (1)

 

The synthesis of the Schiff base ligand, H2hpdbal-sbdt (compound 1), commenced with the preparation of a solution of 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (10 mM, 2.26 g) in 20 mL of dichloromethane (DCM). This solution was then combined with another solution containing S-benzyldithiocarbazate (10 mM, 1.98 g) in 20 mL of methanol. The combined reaction mixture was subsequently subjected to reflux on a water bath for a duration of 1 hour, a condition that facilitates the condensation reaction (as depicted in Scheme 2). Following the reflux period, the volume of the solvent was carefully reduced to approximately 15 mL through evaporation. Upon cooling the concentrated mixture to ambient temperature, a yellowish-orange solid, representing the desired product (compound 1), spontaneously precipitated. This solid was then meticulously filtered off to separate it from the solvent, thoroughly washed with methanol to remove any unreacted starting materials or impurities, and finally dried. For further purification and to obtain a high-purity product, the yellowish-orange solid was recrystallized from dichloromethane, yielding a pure yellowish-orange solid.

 

The product was obtained in an excellent yield of 91% and exhibited a melting point of 198°C. Elemental analysis (Anal. Calcd. for C21H18N4OS2, Molecular Weight: 406.52) confirmed the expected composition: Carbon (C), 62.04%; Hydrogen (H), 4.46%; Nitrogen (N), 13.78%; Sulfur (S), 15.78%. The experimentally Found values were in close agreement: C, 63.26%; H, 4.70%; N, 14.54%; S, 14.78%. Selected Attenuated Total Reflection Infrared (ATIR) spectroscopic data provided crucial information on functional groups (νmax/cm-1): a strong band at 1607 cm-1 was attributed to the C=N (azomethine) stretching vibration, confirming the formation of the Schiff base linkage; a band at 1032 cm-1 indicated the presence of the C=S (thiocarbonyl) group, characteristic of the thione form; a sharp peak at 771 cm-1 corresponded to C-S stretching; a band at 1320 cm-1 was assigned to C-N stretching; a peak at 1112 cm-1 was due to C-O stretching of the phenolic group; bands at 1518 cm-1 and 1483 cm-1 were indicative of N=N (azo) stretching vibrations; and broad bands in the region of 2964-3095 cm-1 corresponded to aromatic C-H stretching.

 

Detailed structural characterization in solution was achieved using Nuclear Magnetic Resonance (NMR) spectroscopy. The 1H-NMR spectrum (400 MHz, DMSO-d6) revealed distinct proton environments (δ ppm): a singlet at 13.23 ppm (1H) was assigned to the aromatic O–H proton; a singlet at 8.39 ppm (1H) corresponded to the N=CH proton of the azomethine group; a complex multiplet between 7.89–6.94 ppm (13H) indicated the presence of aromatic protons; a singlet at 4.48 ppm (2H) was assigned to the S–CH2 protons; and a singlet at 10.65 ppm (1H) corresponded to the N–H/S-H proton, confirming the presence of tautomeric forms. The 13C-NMR spectrum (400 MHz, DMSO-d6) provided comprehensive carbon chemical shifts (δ ppm), further confirming the structural integrity: 117.95 (C-1), 160.42 (C-2), 117.65 (C-3), 126.12 (C-4), 145.29 (C-5), 122.54 (C-6, C-8, C-12), 152.42 (C-7), 129.09 (C-9, C-11), 130.67 (C-10), 147.55 (C-13), 196.06 (C-14), 40.53 (C-15), 136.29 (C-16), 127.45 (C-17, C-21), 128.57 (C-18, C-20), and 126.63 (C-19).

 

Synthesis of [{NiII(hpdbal-sbdt)}2] (2)

 

The synthesis of the binuclear Nickel(II) complex, [{NiII(hpdbal-sbdt)}2] (compound 2), involved a coordination reaction between the synthesized ligand and a nickel salt. A solution of H2hpdbal-sbdt (compound 1) (2.07g, 5.1 mM) was prepared by dissolving it in 30 mL of dichloromethane (DCM) at a temperature of 35°C. This solution was then carefully added to a solution of nickel acetate tetrahydrate [{Ni(OAc)2·4H2O] (1.27g, 5.1 mM) in 20 mL of methanol. The resulting reaction mixture was subsequently subjected to reflux for 3 hours on a water bath, a process that facilitates the complexation reaction. To catalyze the formation of the complex and neutralize any acidic byproducts, 3 drops of triethylamine, serving as a mild base, were added to the reaction mixture.

 

Upon completion of the reflux, an oily dark red solution was obtained. This solution was then extracted into water, a step that selectively promotes the precipitation of the metal complex. This process led to the formation of a dark red precipitate, which signified the successful synthesis of the nickel complex (as illustrated in Scheme 3). The dark red precipitate was then meticulously filtered to isolate the product, thoroughly washed with water to remove soluble impurities, and subsequently washed with diethyl ether to remove any organic residues. Finally, the obtained complex was dried *in vacuo* over silica gel to ensure its complete dryness and purity.

 

The synthesis yielded the complex in a high yield of 88%. Elemental analysis (Anal. Calcd. For C42H32N8O2S4Ni2, Molecular Weight: 926.40) confirmed the expected composition: C, 54.45%; H, 3.48%; N, 12.10%; S, 13.84%. The experimentally Found values were in close agreement: C, 55.19%; H, 3.62%; N, 12.76%; S, 13.02%. Selected Attenuated Total Reflection Infrared (ATIR) spectroscopic data for the complex (ν/cm-1) revealed characteristic shifts indicating metal coordination: the C=N (azomethine) stretching vibration was observed at 1591 cm-1, showing a bathochromic shift of 16 cm-1 compared to the ligand, which signifies the participation of the azomethine nitrogen as a donor atom to the metal center. The C–S stretching was found at 757 cm-1. The N-H band, previously present in the ligand at 3100 cm-1, was notably absent in the IR spectrum of the complex, strongly indicating the coordination of the enethiolate sulfur to the nickel metal center. This observation suggests a probable conversion of the thioamide (thione) form of the ligand to the imidothiol (enethiol) form upon complexation. Bands associated with the unsymmetrical p-substituted azobenzene (Ph–N=N–) moiety were found in two regions: 1452 cm-1 and 1528 cm-1. The C–N stretching was observed in the region of 1353 cm-1. A medium intense band varying between 1117 cm-1 was attributed to the C–O stretching of the phenolic group, suggesting its involvement in coordination. Other characteristic bands included C–H stretching vibrations for monosubstituted benzene (689–692 cm-1), p-substituted benzene (826–827 cm-1), and the methine carbon of the imine group (2827–3019 cm-1).

 

1H-NMR spectroscopy (400 MHz, DMSO-d6) of the complex showed (δ ppm): complex multiplets between 8.29-7.07 ppm (26H) for aromatic protons; a singlet at 9.37 ppm (2H) assigned to the N=CH protons; and a singlet at 4.26 ppm (4H) for the S-CH2 protons, consistent with the dimeric structure and expected symmetry. The 13C-NMR spectrum (400 MHz, DMSO-d6) provided carbon chemical shifts (δ ppm): 119.99 (C-1, C-22), 152.65 (C-2, C-23), 116.57 (C-3, C-24), 124.86 (C-4, C25), 133.65 (C-5, C-26), 121.15 (C-6, C-27), 135.13 (C-7, C-28), 122.38 (C-8, C-12, C-29, C-33), 129.48 (C-9, C-11, C-30, C-32), 129.75 (C-10, C-31), 131.61 (C-13, C-34), 206.79 (C-14, C-35), 49.07 (C-15, C-36), 130.51 (C-16, C-37), 128.37 (C-17, C-21, C-38, C-42), 128.91 (C-18, C-20, C-39, C-41), and 124.86 (C-19, C-40).

 

Results and Discussion

 

Characterization of Compounds

 

The proposed structures of both the ligand (compound 1) and the nickel complex (compound 2) were comprehensively validated through a rigorous combination of elemental analyses and various spectroscopic techniques. Elemental analyses, specifically Carbon, Hydrogen, Nitrogen, and Sulfur (CHNS) analysis, provided crucial confirmation of the empirical formulas, ensuring that the atomic composition matched the theoretical predictions for the synthesized molecules. Complementing this, a battery of spectroscopic techniques, including Attenuated Total Reflection Infrared (ATIR) spectroscopy, Proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy, Carbon-13 Nuclear Magnetic Resonance (13C-NMR) spectroscopy, Ultraviolet-Visible (UV-Vis) spectroscopy, Thermogravimetric Analysis (TGA), Field Emission Scanning Electron Microscopy (FESEM), and Energy Dispersive X-Ray Spectroscopy (EDS), were employed. Each of these techniques offered unique and corroborating insights into the structural features, purity, thermal stability, morphology, and elemental distribution of both the ligand and the complex.

 

Antimicrobial Activity

 

A primary antimicrobial screening study was systematically undertaken to broadly assess the growth inhibitory capabilities of the synthesized compounds. This initial phase involved whole-cell growth inhibition assays, meticulously performed by testing the provided samples at a single, predetermined concentration, with each condition replicated in duplicate to ensure robustness. The inhibitory effect on microbial proliferation was precisely quantified by measuring the optical density of cell cultures (OD600) for bacterial strains, reflecting their growth. This screening panel was comprehensively designed to include a range of clinically significant microorganisms: four Gram-negative bacterial species, namely *Escherichia coli* (Ec), *Klebsiella pneumoniae* (Kp), *Acinetobacter baumannii* (Ab), and *Pseudomonas aeruginosa* (Pa), known for their diverse resistance mechanisms and clinical relevance. Additionally, one Gram-positive bacterial species, *Staphylococcus aureus* (Sa), a notorious pathogen often associated with difficult-to-treat infections, was included. To broaden the scope of antimicrobial assessment, two fungal strains, *Candida albicans* (Ca) and *Cryptococcus neoformans* (Cn), were also evaluated.

 

For these assays, the test samples were carefully prepared by dissolving them in a mixture of DMSO (dimethyl sulfoxide) and water, ensuring a final testing concentration of 32 μg/mL or 20 μM. These solutions were then dispensed into 384-well, non-binding surface (NBS) plates, selected to minimize non-specific adsorption of the compounds. Throughout the experiment, the final concentration of DMSO was strictly controlled and maintained at a maximum of 1% to prevent any potential confounding effects of the solvent on microbial growth. Compounds were rigorously classified based on their performance: a sample was considered “active” if it demonstrated a growth inhibition value equal to or greater than 80% and, critically, exhibited a Z-score exceeding 2.5, indicating a statistically robust and highly significant inhibitory effect. Conversely, a sample was designated as “partially active” if its inhibition value fell within the range of 50.9% to 79.9%, coupled with a Z-score below 2.5, suggesting a moderate, yet potentially noteworthy, inhibitory action that merited further investigation.

 

Following the initial broad screening, active compounds proceeded to a “hit confirmation” stage, which involved more detailed whole-cell growth inhibition assays to precisely determine their Minimum Inhibitory Concentration (MIC). This was achieved through an 8-point dose-response experiment, again performed in duplicate. For this, samples were prepared at an initial testing concentration of 32 μg/mL or 20 μM in DMSO and water, and then subjected to serial 1:2 fold dilutions for eight consecutive steps, creating a range of concentrations to accurately pinpoint the lowest concentration at which complete growth inhibition occurred. For robust comparative analysis and quality control, standard antimicrobial agents were employed as references. Colistin was used for Gram-negative bacteria, Vancomycin for Gram-positive bacteria, and Fluconazole for fungal species. Notably, the MIC value for Vancomycin against *Staphylococcus aureus* was confirmed at 1 µg/mL, and for Fluconazole against *Candida albicans*, it was 0.13 µg/mL. These standard drugs consistently exhibited 100% growth inhibition of their respective bacterial and fungal species at concentrations above their determined MIC values, validating the reliability and sensitivity of the assay system.

 

Protocol for Cytotoxicity Assay

 

To assess the potential for cellular toxicity of the synthesized compounds against mammalian cells, a standardized protocol employing HEK293 (Human Embryonic Kidney cells, ATCC CRL-1573) was precisely followed. This assessment is a critical step in the early stages of drug development to ensure the compounds are not unduly harmful to host cells at therapeutic concentrations. Initially, HEK293 cells were accurately enumerated using a Neubauer haemocytometer to ensure consistent seeding densities. Subsequently, a precise number of 6000 cells per well were plated into 384-well plates, which contained the pre-dispensed test compounds, in a final working volume of 50 μL. The cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium), a rich basal medium, supplemented with 10% Fetal Bovine Serum (FBS), which provided essential growth factors and nutrients, serving as the complete growth medium. The cell-compound mixture was then incubated for a period of 20 hours under controlled environmental conditions: 37 °C in a humidified incubator with a 5% CO2 atmosphere, conditions optimized for the viability and proliferation of HEK293 cells. As a positive control for cytotoxicity, Tamoxifen, a well-known cytotoxic agent, was included as a standard reference drug to provide a reliable benchmark for comparison against the test compounds’ toxicity profiles.

 

Antidiabetic Activity

 

The assessment of antidiabetic activity constituted a pivotal aspect of this research, specifically focusing on the compounds’ capacity to enhance cellular glucose uptake. For this purpose, the 2-NBDG assay was employed, a well-established fluorescent glucose uptake assay, utilizing HepG2 (human hepatocyte carcinoma) cells. HepG2 cells are a widely accepted *in vitro* model for studying hepatic glucose metabolism and, particularly, for modeling insulin resistance. To accurately mimic the pathophysiological conditions of type II diabetes, the HepG2 cells were first cultured in 96-well black plates until reaching approximately 70% confluency. Subsequently, a state of insulin resistance was induced by pre-treating these cells with a carefully selected combination of pro-inflammatory and lipotoxic agents: tumor necrosis factor-alpha (TNF-alpha), fructose, and palmitic acid. Concurrently with the induction of insulin resistance, the study compounds, namely the ligand (compound 1) and the Nickel(II) complex (compound 2), alongside the standard antidiabetic drug metformin, were introduced into the wells at various concentrations (0.1 µM, 0.2 µM, and 0.5 µM, equivalent to 100 nM, 200 nM, and 500 nM, respectively).

 

After a 24-hour incubation period, which allowed for the compounds to exert their metabolic effects, the wells were thoroughly washed with 1X PBS (phosphate-buffered saline) to eliminate any residual treatment media. Following this, a solution containing 40 µM of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose), a fluorescently labeled glucose analog, was added to each well, along with 0.1 µM insulin, for a 1-hour incubation period. This step enabled the cells to actively take up the fluorescent glucose analog. Subsequently, the wells were carefully washed once more to remove any extracellular 2-NBDG. Finally, a lysis buffer was added to permeabilize the cells and release the internalized fluorescent glucose. The fluorescence intensity from each well, directly proportional to the amount of glucose taken up by the cells, was then quantitatively measured using a fluorescence spectrometer. Untreated HepG2 cells, maintaining their physiological insulin sensitivity, served as a healthy control group, while HepG2 cells subjected to insulin resistance induction but without study compound treatment served as the Insulin Resistance (IR) control cells, providing a critical baseline for impaired glucose uptake. The results from the 2-NBDG assay were remarkably promising: the insulin-resistant HepG2 cell line, when treated with both ligand 1 and complex 2, showed a significant increase in the uptake of 2-NBDG, closely approximating the glucose uptake levels observed in healthy, untreated cells. Specifically, at doses of 0.1 µM and 0.2 µM, both the ligand and the complex demonstrated notable glucose uptake. However, at a higher dose of 0.5 µM, the ligand exhibited a comparatively lower glucose uptake than the complex, highlighting the superior efficacy of the complex at higher concentrations. Crucially, the fluorescent glucose uptake capacity of cells treated with the complex (compound 2) was observed to be highly comparable to that achieved by the standard antidiabetic drug metformin, underscoring its potent potential as a glucose-lowering agent.

 

In parallel with the 2-NBDG assay, the compounds were also evaluated for their *in vitro* cytotoxicity levels using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This assessment is vital to ensure that any observed antidiabetic effects are a result of genuine biological activity rather than a mere consequence of cell death. For this, HepG2 cells were seeded in 96-well plates and allowed to proliferate until they reached approximately 70% confluency. At this optimized density, the cells were incubated for 24 hours with various concentrations of the test compounds, diluted in DMEM supplemented with 10% FBS and 1% antibiotic. Following the incubation period, MTT (at a concentration of 2.4 mM) was added to the media and allowed to incubate for 4 hours. The insoluble formazan product, formed by the metabolic activity of viable cells, was then solubilized by adding 100 µL of DMSO to each well. The absorbance readings were subsequently taken at 570 nm using a microplate reader. The results from the MTT assay were highly encouraging: neither compound 1 nor compound 2 exhibited any significant cytotoxic effects on HepG2 cells across the tested concentration ranges. Specifically, compound 2 showed no discernible cytotoxicity even at a concentration as high as 10 µg/mL. This lack of significant cytotoxicity, coupled with their robust glucose uptake capabilities, strongly suggests a promising safety profile and highlights the great potential for the utilization of this novel nickel complex as an antidiabetic agent in future *in vivo* therapeutic applications.

 

Results and Discussion

 

Characterization of Compounds

 

The predicted molecular structures of both the newly synthesized ligand (compound 1) and the corresponding nickel complex (compound 2) were rigorously and consistently supported through a comprehensive array of analytical techniques. Elemental analyses, specifically Carbon, Hydrogen, Nitrogen, and Sulfur (CHNS) analysis, served as a fundamental verification, providing precise quantitative data that were in close agreement with the theoretically calculated compositions for both compounds, thus confirming their empirical formulas. Complementing this, a diverse suite of spectroscopic methods, including Attenuated Total Reflection Infrared (ATIR) spectroscopy, Proton Nuclear Magnetic Resonance (1H-NMR), Carbon-13 Nuclear Magnetic Resonance (13C-NMR) spectroscopy, Ultraviolet-Visible (UV-Vis) spectroscopy, Thermogravimetric Analysis (TGA), Field Emission Scanning Electron Microscopy (FESEM), and Energy Dispersive X-Ray Spectroscopy (EDS), each contributed unique and corroborating evidence. Collectively, these techniques provided a multifaceted characterization, validating the proposed structural integrity, purity, thermal stability, morphology, and elemental distribution of both the ligand and its metal complex in both solid and solution states.

 

1H and 13C NMR Spectral Studies

 

The structural elucidation of the ligand and the complex was further refined and confirmed through detailed analyses of their 1H-NMR and 13C-NMR spectra, recorded in DMSO-d6. These NMR studies were particularly instrumental in confirming the precise structure of the ligand and, crucially, in assessing the magnetic properties of the nickel complex to deduce its coordination modes. Strikingly, the nickel complex, [{NiII(hpdbal-sbdt)}2], yielded significant and highly relevant signals in both its 1H and 13C NMR spectra. This observation provided compelling evidence for the formation of a low-spin square planar d8 system for the nickel centers within the complex. The appearance of sharp, interpretable NMR signals, typically absent in paramagnetic complexes, strongly indicated that the two nickel atoms within the binuclear complex are antiferromagnetically coupled, resulting in a net spin of zero, a characteristic of diamagnetic or very weakly paramagnetic species.

 

In the 1H-NMR spectrum of the free ligand (compound 1), a distinct singlet resonance was observed at 13.23 ppm, unequivocally assigned to the phenolic –OH proton. This signal, characteristic of the phenolic moiety, completely vanished in the 1H-NMR spectrum of the nickel complex (compound 2). This disappearance robustly confirmed the deprotonation of the phenolic hydroxyl group and its subsequent direct coordination to the nickel metal center, forming a strong bond. Furthermore, the signals corresponding to the protons within the nickel complex (compound 2) generally appeared as relatively broader resonances compared to those of the free ligand. This broadening is most likely attributable to the dimeric nature of the complex, which can lead to molecular aggregation in solution, and/or internal flexibility within the complex structure, influencing the relaxation times of the protons. Another critical observation was the complete absence of the N-H resonance in the complex’s NMR spectrum. In the free ligand, a peak at 10.65 ppm was attributed to the proton involved in the tautomeric equilibrium between the thione (S=C–NH–) and enethiol (SH–C=N–) forms of the ligand. The disappearance of this peak in the complex strongly supported the deprotonation of this N-H group and the subsequent coordination of the thioenolate sulfur atom to the metal centers, further solidifying the binding mode.

 

In addition to these key changes, noteworthy downfield shifts were observed for the azomethine proton signal in the complex compared to the ligand. These downfield shifts are a direct consequence of the coordination of the imine-nitrogen atom to the metal center, where the electron density around the nitrogen is reduced due to bonding with the electrophilic metal ion. Importantly, residual proton resonances belonging to the ligand backbone that are not directly involved in coordination did not exhibit significant shifts upon complexation, appearing consistently within their anticipated ranges. The presence of labile OH and NH/SH protons in the ligand, capable of exchanging with deuterium, was further confirmed by deuterium exchange NMR studies. All these collective observations, from both 1H and 13C NMR, are therefore in strong agreement with a tridentate ONS2– binding mode of the ligand within the nickel complex, indicating that the ligand coordinates to the nickel center through its phenolic oxygen, azomethine nitrogen, and thioenolate sulfur atoms.

 

The 13C-NMR spectra also provided additional compelling support for the proposed coordination mode of the ligand. The spectrum of the free ligand consistently exhibited 21 distinct carbon signals, precisely matching the number of carbon atoms present in its predicted molecular structure. In the complex’s 13C-NMR spectrum, significant chemical shifts were observed for the carbon atoms directly attached to the donor atoms—oxygen, nitrogen, and sulfur—upon complexation. Specifically, the carbon atom of the C=S group showed a substantial downfield shift, with a Δδ value (chemical shift of complex minus chemical shift of ligand) of 10.73 ppm. Conversely, the carbon attached to the phenolic O-H group exhibited an upfield shift of Δδ = -7.77 ppm, and the C=N carbon displayed an even more pronounced upfield shift of Δδ = -15.94 ppm. These clear and significant changes in carbon chemical shifts are diagnostic indicators of the direct involvement of these specific atoms in the coordination to the nickel center, providing robust evidence for the proposed ONS2- binding motif.

 

Thermogravimetric Analysis (TGA)

 

Thermogravimetric analysis (TGA) was performed on the binuclear nickel(II) complex, [{NiII(hpdbal-sbdt)}2] (compound 2), under a dynamic air atmosphere over a temperature range spanning from 40 to 750°C, with a controlled heating rate of 20°C/min. This technique provided crucial insights into the thermal stability and decomposition pathway of the complex, correlating observed weight losses with the removal of specific organic fragments. The TGA thermogram revealed a multi-stage decomposition process, indicative of the sequential thermal removal of distinct parts of the organic ligand environment around the metal center.

 

The initial significant weight loss was observed as 25.69% in the temperature range of 250–370°C. This corresponds precisely to the thermal removal of two –SCH2C6H5 units, which constitute the S-benzyldithiocarbazate (sbdt) backbone of the ligand. The theoretically calculated weight loss for two such units (C14H14S2) is 26.59%, showing excellent agreement with the experimental data. Following this, a second major weight loss of 17.07% occurred within the temperature range of 370–470°C. This reduction in mass is attributed to the thermal removal of two –SCN units from the complex. The calculated weight loss for two S2C2N2 units is 17.08%, again demonstrating a strong correlation with the experimental findings. The third and final major weight loss, a substantial 73.90%, was observed in the range of 470–624°C. This extensive loss is attributed to the complete rupture and thermal decomposition of the two units of the azo-linked oxygen donor backbone [(C13H9N2O)2]. The theoretically calculated weight loss for these fragments is 74.21%, providing further robust confirmation of the decomposition pathway.

 

Cumulatively, the total observed weight loss across all stages amounted to 83.72%. This value closely matches the calculated percentage for the complete decomposition of the entire organic content within the sample, which is theoretically 84.30%. The residue remaining at the end of the TGA analysis, at temperatures above 624°C, amounted to 16.28% of the initial sample weight. This residue corresponds to the stable nickel oxide (NiO) material. The theoretically calculated weight of the nickel oxide material expected to be left is 15.70%, which is in remarkable agreement with the experimental residue. The comprehensive TGA analysis, complemented by the Derivative Thermogravimetry (DTG) profile, therefore provided conclusive and quantitative evidence confirming the integrity of the organic ligand environment around the binuclear nickel complex and supported the proposed molecular structure.

 

Field Emission Scanning Electron Microscopy (FESEM) and Energy-Dispersive Spectroscopic (EDS) Analysis

 

Field Emission Scanning Electron Microscopy (FESEM) was employed to visually investigate the surface morphology of both the ligand (compound 1) and the nickel complex (compound 2) at high resolution. The FESEM micrographs revealed distinct differences in their macroscopic appearance. The ligand exhibited a characteristic sharp, rod-shaped surface morphology, indicative of a crystalline and well-defined structure. In contrast, the nickel complex displayed a non-homogeneous, sponge-like morphology, suggesting a more amorphous or aggregated structure upon complexation. These variations in surface morphology were clearly observable.

 

Complementing the morphological analysis, Energy-Dispersive Spectroscopic (EDS) analysis was performed to determine the elemental composition of both the ligand and the complex. The EDS spectrum of the free ligand, H2hpdbal-sbdt (compound 1), clearly displayed distinct peaks corresponding to the presence of carbon (C), nitrogen (N), oxygen (O), and sulfur (S) elements, confirming its expected organic composition. This ligand spectrum served as a crucial reference for comparison with the spectrum obtained for the nickel complex (compound 2). The EDS depiction for [{NiII(hpdbal-sbdt)}2] (compound 2) showed a noticeable loss in the relative percentage of carbon content, which is consistent with the incorporation of a heavier metal ion into the structure, reducing the overall carbon proportion by mass. Crucially, the complex’s spectrum confirmed the continued presence of all the elements found in the ligand (C, N, O, S) and, additionally, exhibited distinct and new peaks specifically attributed to the presence of nickel (Ni), unequivocally confirming the successful formation of the metal complex. Furthermore, the EDS spectra consistently confirmed the absence of any external impurities in the synthesized materials, ensuring their high purity. Small peaks corresponding to platinum were occasionally observed in the spectra; these are attributable to the thin layer of platinum coating applied to the material during sample preparation for FESEM, which is a standard procedure to enhance material conductivity for electron microscopy.

 

Electronic Spectral Study

 

Ultraviolet-Visible (UV-Vis) spectroscopy was utilized to study the electronic absorption properties of both the ligand (compound 1) and the nickel complex (compound 2) in DMSO solution. The UV-Vis spectrum of the free ligand exhibited three characteristic absorption peaks. Two of these, at 208 nm and 340 nm, are typically assigned to intra-ligand electronic transitions. Specifically, the peak at 208 nm is attributed to a high-energy π-π* transition, arising from the extensive conjugation within the aromatic and azobenzene moieties of the ligand. The peak at 340 nm is assigned to an n-π* transition, which often involves the non-bonding electrons on heteroatoms within the ligand.

 

Upon coordination to the nickel metal center, the electronic spectrum of the complex (compound 2) showed significant changes. The intra-ligand bands, corresponding to the π-π* and n-π* transitions, exhibited a clear red shift, also known as a bathochromic shift. This shift indicates that the absorption maximum moved to longer wavelengths, suggesting an alteration in the electronic environment and conjugation due to the coordination with the metal. Furthermore, the molar absorption coefficients associated with these intra-ligand bands showed a significant drop after complexation. This phenomenon, known as a hypochromic effect, suggests a decrease in the intensity of light absorption, likely due to changes in the orientation of chromophores or electronic interactions within the complex.

 

A crucial additional low-absorbance band appeared at 470 nm in the spectrum of the complex. This new band is likely attributed to a ligand-to-metal charge transfer (LMCT) phenomenon, where electron density is transferred from the ligand orbitals to the vacant orbitals of the central nickel ion. This observation further confirms the successful coordination of the ligand to the metal center. Significantly, the d-d transitions, which are characteristic of transition metal complexes involving electron transitions within the d-orbitals of the metal, were not observed in the spectrum of compound 2. This absence is a strong spectroscopic confirmation of the low-spin d8 electronic configuration of nickel in the complex, where the d-orbitals are either fully filled or have transitions that are symmetry-forbidden or lie outside the typical UV-Vis spectral range for strong absorption.

 

Antibacterial and Antifungal Assay

 

Primary Screening Results

 

The initial assessment of antimicrobial activity, through the primary screening assay, provided foundational data on the inhibitory potential of the nickel complex against various microbial species. The inhibition of bacterial growth was precisely determined by measuring absorbance at 600 nm (OD600) using a Tecan M1000 Pro monochromator plate reader, a standard method for quantifying bacterial proliferation. For fungal species, growth inhibition was assessed differently: for *Candida albicans*, absorbance was measured at 530 nm (OD530). For *Cryptococcus neoformans*, growth inhibition was determined by measuring the difference in absorbance between 600 nm and 570 nm (OD600-570) after the addition of resazurin (a redox indicator, at 0.001% final concentration) and a subsequent 2-hour incubation at 35 °C, as resazurin is metabolically converted by viable cells, changing its fluorescence properties.

 

In this primary screening, the nickel complex (compound 2) demonstrated partial activity against the methicillin-resistant *Staphylococcus aureus* (MRSA) bacterial strain, exhibiting an average growth inhibition of 63%. It also showed partial activity against the fungal strain *Candida albicans*, with an average growth inhibition of 57%. Given these initial promising inhibition values, compound 2 was subsequently designated as an “active compound” suitable for further, more detailed hit validation screening. In stark contrast, the free ligand (compound 1) showed very minimal to negligible growth inhibition across all tested bacterial and fungal species, with inhibition percentages ranging from 1.92% to 23.91%, suggesting that its antimicrobial properties are significantly enhanced, or even primarily conferred, upon complexation with nickel.

 

Hit Validation Results

 

Following the primary screening, the nickel complex underwent a rigorous hit validation study to precisely quantify its antimicrobial potency against the previously identified active species and to determine its Minimum Inhibitory Concentration (MIC). The same protocols for measuring bacterial and fungal growth inhibition were meticulously followed as in the primary screening. The MIC was rigorously defined as the lowest concentration of the compound at which the growth of the target microorganism was fully and completely inhibited.

 

The results of the hit validation confirmed that the nickel complex (compound 2) was definitively active only against the bacterial species *Staphylococcus aureus*, particularly the methicillin-resistant strain. It displayed a remarkably low MIC value of 2 µg/mL. This MIC value is highly significant as it is notably comparable to the MIC value of the standard drug Vancomycin, which is a frontline antibiotic used against MRSA, demonstrating an MIC of 1 µg/mL. This strong activity against a drug-resistant bacterial species highlights the therapeutic potential of the nickel complex. However, the complex was found to be inactive against the fungal species *Candida albicans*. Despite showing an initial inhibition of approximately 76% in the primary screening, its MIC value against *Candida albicans* was determined to be above the highest tested standard concentration of 32 µg/mL. This outcome led to the conclusion that the nickel complex is not effectively a potent antifungal agent for the tested species under these conditions.

 

In addition to antimicrobial efficacy, cytotoxicity against mammalian cells is a crucial safety parameter. The nickel complex was also tested for its cytotoxicity against Human Embryonic Kidney cells (HEK293). It was found to be cytotoxic to HEK293 cells with a maximum percentage growth inhibition value of 96% and a CC50 (concentration causing 50% cytotoxicity) value of approximately 18.2 µg/mL. Crucially, this CC50 value of 18.2 µg/mL is considerably higher than the MIC value of 2 µg/mL determined for its activity against *Staphylococcus aureus* bacterial species. This significant difference indicates that the nickel complex is non-cytotoxic to human kidney cells at concentrations effective for bacterial inhibition, providing a favorable therapeutic window. The hit validation study thus conclusively affirmed the excellent antibacterial activity of the nickel complex against the methicillin-resistant *Staphylococcus aureus* species without exhibiting significant toxicity effects on normal human cells at its effective antimicrobial concentration. The plausible reasons contributing to this observed higher antibacterial activity of the complex are likely multi-factorial, primarily stemming from enhanced chelation, which can increase the compound’s affinity for microbial targets, and improved cell permeability upon complexation, allowing for more efficient entry into the bacterial cells.

 

Antidiabetic Assay

 

2-NBDG Assay

 

The compounds under investigation were rigorously screened for their potential antidiabetic activity using the 2-NBDG uptake assay, a widely accepted method for quantifying cellular glucose uptake. This evaluation was conducted at three specific concentrations: 0.1 µM, 0.2 µM, and 0.5 µM (equivalent to 100 nM, 200 nM, and 500 nM, respectively). The assay utilized insulin-resistant HepG2 cell lines, which serve as a well-established *in vitro* model for studying insulin resistance and impaired glucose metabolism, mirroring conditions prevalent in type II diabetes. The results demonstrated a clear and significant increase in the uptake of 2-NBDG, a fluorescently labeled glucose analog, by the insulin-resistant HepG2 cells after they were treated with either the ligand (compound 1) or the nickel complex (compound 2) in conjunction with insulin. The observed uptake of 2-NBDG by these treated diseased cells was remarkably close to the glucose uptake levels of healthy, untreated HepG2 cells, indicating a substantial reversal of insulin resistance.

 

A detailed analysis of the glucose uptake, as visualized, revealed that at the lower concentrations of 0.1 µM and 0.2 µM, both the ligand (compound 1) and the complex (compound 2) exhibited significant capacities to promote glucose uptake into the cells. However, a notable difference emerged at the higher dose of 0.5 µM, where the ligand showed a comparatively lower uptake of fluorescent glucose when compared to the complex, underscoring the superior efficacy of the complex at higher therapeutic concentrations. Furthermore, a highly promising finding was that the fluorescent glucose uptake capacity demonstrated by the cells treated with the nickel complex (compound 2) was directly comparable to that observed in cells treated with metformin, a globally recognized and widely utilized standard antidiabetic drug. This direct comparability provides compelling evidence for the potent antidiabetic potential of the newly synthesized nickel complex.

 

MTT Assay

 

To comprehensively establish the safety profile of the synthesized compounds and to definitively ascertain that their observed antidiabetic effects were not merely a consequence of cellular toxicity, the *in vitro* cytotoxicity level of the test compounds on HepG2 cells was meticulously evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This well-established colorimetric assay provides a direct measure of cellular metabolic activity, which serves as a reliable indicator of cell viability. For this procedure, HepG2 cells were initially seeded into a 96-well plate at an optimal density, allowing them to proliferate until they reached approximately 70% confluency, a stage ensuring robust viability for accurate assessments. At this precise stage, the cells were subjected to a 24-hour incubation period with varying concentrations of the different test compounds. These compounds were carefully dissolved in DMEM (Dulbecco’s Modified Eagle Medium) that had been supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic solution, providing a complete and sterile growth environment. Following this critical incubation period, the MTT reagent, at a concentration of 2.4 mM, was added to the media in each well and allowed to incubate for an additional 4 hours. During this phase, metabolically active cells, possessing functional mitochondrial reductases, biochemically convert the soluble, yellowish MTT tetrazolium salt into insoluble, purple formazan crystals, which accumulate within the cells. At the conclusion of this incubation, the insoluble formazan product was then solubilized by the addition of 100 µL of DMSO (dimethyl sulfoxide) to each well, thereby creating a uniform, purple-colored solution. The absorbance of this resulting solution was subsequently measured at a wavelength of 570 nm using a microplate reader, with higher absorbance values directly correlating with a greater number of metabolically active, and thus viable, cells. The results obtained from the MTT assay were overwhelmingly favorable: neither of the tested compounds exhibited any statistically significant cytotoxic effects on HepG2 cells across the entire range of concentrations evaluated. Critically, compound 2, the novel nickel complex, demonstrated no detectable cytotoxicity whatsoever, even at a relatively high concentration of 10 µg/mL. This pronounced lack of cytotoxicity, particularly at concentrations where the complex simultaneously exhibited potent antidiabetic activity, strongly suggests a highly favorable safety profile. This favorable balance between efficacy and safety significantly underscores its substantial potential for further development as a viable antidiabetic agent for *in vivo* therapeutic applications.

 

pH Stability Study

 

The investigation into the effect of pH on the nickel compound is of paramount importance, as it provides crucial insights into its stability and integrity under various physiological pH conditions that it would encounter within the body. To thoroughly understand the influence of pH on the stability of [{NiII(hpdbal-sbdt)}2] (compound 2), both potentiometric pH titration and UV-Vis pH titration studies were meticulously carried out. For these studies, a stock solution of compound 2 (0.5 mM) was precisely prepared in a mixed solvent system consisting of 70% v/v water/DMSO, mimicking a biological solvent environment. The initial pH of this solution was recorded at 5.73. This pH was then systematically lowered to pH 3.5 through the careful and gradual addition of 5 mM HCl. Following the initial acidification, the pH was progressively varied across a wide range, from 3.5 to 10.0, by the slow, incremental addition of 5 mM KOH. The pH variation trend was closely monitored by plotting a graph of the solution’s pH against the cumulative volume of base added. From the equivalence point derived from this plot, the pKa of the compound was precisely calculated to be 5.88. This determined pKa value is significant as it falls within the typical range considered favorable for an efficient drug, often correlating with enhanced bioavailability. This suggests that the compound may exhibit optimal solubility and absorption characteristics within physiological systems.

 

Complementing the potentiometric titration, absorption studies using UV-Vis spectroscopy were also performed by titrating compound 2 with both HCl and KOH. The absorption intensity of the compound at 380 nm displayed distinct changes throughout the titration. Upon systematic titration with HCl, a phenomenon of hypochromism was observed, characterized by a decrease in absorption intensity. Conversely, upon titration with KOH, the compound exhibited hyperchromism, signified by an increase in absorption intensity. Crucially, no additional absorption bands were observed during these titrations, indicating that the core chromophore structure remained intact and no new species were formed. These spectral changes are proposed to arise from changes in the protonation state of the hydrazone nitrogen within the complex. Specifically, upon decreasing the pH (addition of HCl), the protonation of the hydrazone nitrogen of the complex is likely to occur, leading to a disruption of the conjugation system and thus hypochromism. Conversely, with an increase in pH (addition of KOH), the hydrazone nitrogen undergoes deprotonation, resulting in the restoration or enhancement of conjugation, hence leading to hyperchromism. Both the calculated pKa value of 5.88 and the observed trend of reversibility in the UV-Vis spectra strongly suggest that at a physiological pH of 7.4, the complex will predominantly exist in its original, native, and stable form. This comprehensive pH stability study thus indicates that the potential drug candidate (compound 2), upon administration, is likely to remain structurally stable and functionally intact as it traverses various body fluids with differing pH atmospheres, which is a critical characteristic for drug efficacy and safety.

 

BSA Interaction Study

 

To gain an in-depth understanding of how the active nickel complex, [{NiII(hpdbal-sbdt)}2] (compound 2), might behave within a biological system, particularly its interaction with proteins, extensive studies were conducted. A model serum protein, bovine serum albumin (BSA), was employed for this purpose, as it closely mimics the behavior of human serum albumin, a primary drug carrier in the bloodstream. These investigations utilized a variety of sophisticated spectroscopic approaches to elucidate the intricate mode of interaction.

 

Steady State Fluorescence Study

 

Fluorescence spectroscopy, specifically analyzing the intrinsic fluorescence profile of tyrosine (Tyr) and tryptophan (Trp) residues present within BSA, offers invaluable information regarding the quenching mechanism, the extent of binding between the protein and a drug candidate, and insights into protein conformation and dynamics. For this study, the fluorescence emission spectra of BSA (5µM) were recorded in the presence of ten different, increasing concentrations of [{NiII(hpdbal-sbdt)}2] (compound 2), ranging from 0.83 µM to 8.26 µM. These experiments were conducted in a phosphate buffer at physiological pH 7.4 and maintained at a constant temperature of 298 K, with the excitation wavelength set at 280 nm, which preferentially excites both Tyr and Trp residues.

 

The fluorescence emission maximum of BSA, typically observed at 347 nm, exhibited a gradual and concentration-dependent quenching upon the successive addition of compound 2. This reduction in fluorescence intensity suggests molecular interactions between the compound and BSA. To ascertain the precise type of quenching mechanism—whether it is due to ground-state complex formation (static quenching) or collisional interactions (dynamic quenching)—the Stern-Volmer (SV) equation was utilized. An analysis of the Stern-Volmer plot, where F0/F (the ratio of BSA fluorescence intensity in the absence and presence of the compound) was plotted against the concentration of compound 2 ([Q]), revealed an upward curvature towards the y-axis. This non-linear, upward bending curve is a strong indication of competitive quenching, implying that the fluorophore (BSA) is being quenched by both static and dynamic means simultaneously.

 

To quantitatively resolve these two quenching mechanisms, a modified Stern-Volmer equation was applied. This equation allows for the determination of both the static quenching constant (KS) and the dynamic quenching constant (KD). The calculated values for KS and KD, along with their respective bimolecular quenching constants (kq), were summarized. The non-linear nature of the Stern-Volmer plots, coupled with the high values observed for both the quenching constants and the bimolecular quenching constant, provided compelling evidence that ground-state complex formation (static quenching) is the dominant mechanism contributing significantly to the overall quenching profile of BSA upon interaction with compound 2. This suggests that a stable complex is formed between BSA and compound 2 even before photoexcitation.

 

To further characterize the binding interaction, the number of binding sites (n) available within BSA for compound 2 was determined. By plotting log((F0–F)/F) against log[Q], the slope of the resulting linear relationship directly yielded the value of n. The calculated value for n was approximately 1, indicating a 1:1 molar ratio of protein to [{NiII(hpdbal-sbdt)}2] in the binding reaction, suggesting that one molecule of the complex binds to one molecule of BSA. The binding constant (Ka) was also calculated using a modified Benesi-Hildebrand equation, with its value showing consistent agreement with that obtained from UV absorbance data. The consistently high binding constant values observed indicate a superior binding capacity of compound 2 with BSA. This strong binding is likely attributable to specific non-covalent interactions, particularly hydrogen bonding between the amino groups of the protein and the phenoxide oxygen atom within the ligand structure of complex 2. This strong and efficient binding suggests effective drug transport within the bloodstream.

 

Synchronous Fluorescence Study

 

Synchronous fluorescence spectroscopy offers several distinct advantages, including the simplification of complex fluorescence spectra and the ability to minimize perturbing effects that might arise from multiple fluorescent molecules present within the protein. This technique is particularly valuable for gaining insights into the molecular environment in the immediate vicinity of fluorophore molecules and can be effectively used for the simultaneous determination of specific amino acids. Given that tyrosine (Tyr) and tryptophan (Trp) residues are the primary contributors to BSA’s intrinsic fluorescence, synchronous scanning was performed by setting specific wavelength intervals (Δλ). A Δλ of 15 nm was used to obtain information on the environment around Tyr residues, while a Δλ of 60 nm was employed for Trp residues. The scanning was conducted across an excitation wavelength range from 200–350 nm and an emission wavelength range from 290–500 nm to capture comprehensive spectral information. As the concentration of compound 2 increased, the fluorescence intensity originating from Trp residues decreased significantly more profoundly than that from Tyr residues. This differential quenching pattern strongly implies that the nickel complex preferentially quenched the BSA fluorescence profile primarily by interacting with and affecting the Trp residues, suggesting that the binding site of compound 2 is in closer proximity to or directly involves Trp residues. The combined findings from the steady-state and synchronous fluorescence studies collectively signify that the potential drug candidate (compound 2) possesses a high proficiency in binding to drug carrier proteins, indicating its capacity to be efficiently transported by them to the desired site of action within the body.

 

3D-Fluorescence Study

 

Three-dimensional (3D) fluorescence spectroscopy is a powerful technique that provides a comprehensive profile of a protein’s fluorescence information, making it exceptionally useful for examining characteristic conformational changes. Any shifts or changes in the fluorescence peaks within the 3D spectrum offer significant clues regarding alterations in the protein’s overall conformation. To investigate the influence of the drug candidate (compound 2) on the secondary and tertiary structure of BSA, 3D fluorescence spectra were meticulously recorded. Experiments were performed with BSA (5 µM) in the presence of three different, optimal concentrations of [{NiII(hpdbal-sbdt)}2] (compound 2): 0.83 µM, 4.17 µM, and 8.30 µM. These concentrations were strategically chosen to represent approximate molar ratios of compound 2 to BSA of 0.2:1, 1:1, and 1:2, respectively. This allowed for the assessment of the compound’s binding effects on BSA at both lower and higher concentration limits, providing a gradient of interaction intensity. The studies were conducted in a phosphate buffer at physiological pH 7.4 and maintained at a constant temperature of 298 K. The excitation wavelength was scanned from 200 to 360 nm, and the emission wavelength was recorded from 200 to 650 nm, with a 2 nm increment at a scan rate of 600 nm/min, ensuring high-resolution data acquisition.

 

The 3D fluorescence spectra, along with their corresponding contour diagrams, for pure BSA and BSA incubated with varying concentrations of compound 2, revealed key features. The sharp spectral peaks labeled “a” and “b” represent the 1st order Rayleigh Scattering (where emission wavelength equals excitation wavelength, λem = λex) and 2nd order Rayleigh Scattering (where emission wavelength equals twice the excitation wavelength, λem = 2λex), respectively, which are inherent optical artifacts. Peak 1, a crucial intrinsic fluorescence property, is primarily attributed to the π→π* transitions of the Tryptophan (Trp) residues within the protein, reflecting the environment of the chromophores. Peak 2, observed as the strongest fluorescent peak, is due to the characteristic n→π* transition originating from the polypeptide backbone of the protein, providing insights into the overall protein structure.

 

Both Peak 1 and Peak 2 intensities exhibited a gradual decrease with successive additions of compound 2. This diminishing intensity is a clear indication of the quenching of the intrinsic fluorescence of the amino acid residues. More importantly, it also suggests an alteration in the three-dimensional structure of the protein peptide strand. Furthermore, a consistent decrease in the Stokes shift (the difference between excitation and emission maxima) was observed for both Peak 1 and Peak 2 as the concentration of compound 2 increased. The diminishing intensity and reduced Stokes shift, combined, strongly infer that conformational changes are indeed occurring within BSA. These changes are likely due to a gentle unfolding or slight loosening of the coiled secondary structure of the protein, which subsequently results in the exposure of previously buried hydrophobic regions. This suggests that the binding of compound 2 to BSA, particularly at higher concentrations, induces a subtle, progressive disruption of the protein’s native conformation.

 

UV-Visible Study

 

UV-visible (UV-Vis) spectroscopy is a crucial technique for detecting the formation of complexes between a protein and a ligand, as the absorption spectrum is highly sensitive to the microenvironment of the chromophores present. Proteins typically exhibit a strong absorbance peak around 201 nm, primarily attributed to the amide bonds within their polypeptide backbone. A comparatively weaker absorbance is observed around 280 nm, mainly due to the aromatic amino acid residues, particularly tryptophan (Trp) residues, which serve as intrinsic chromophores. Changes in these absorption profiles can thus indicate direct interactions.

 

The absorption spectra of BSA (5µM) were recorded upon the gradual addition of ten different concentrations of [{NiII(hpdbal-sbdt)}2] (compound 2), spanning a range from 0.83 µM to 8.26 µM, in a phosphate buffer maintained at pH 7.4 and a constant temperature of 298 K. As the concentration of compound 2 increased, a consistent increase in the absorbance of BSA was observed. This increase in absorbance is a strong indicator of a static quenching mechanism, specifically suggesting the formation of a ground-state complex between BSA and compound 2 even before photoexcitation. This implies a stable association between the protein and the compound.

 

To further characterize this interaction, the binding constant (Ka) for the interaction was calculated at four different temperatures using a double reciprocal plot, specifically plotting A0/(A–A0) versus 1/c, where A0 is the initial absorbance and A is the absorbance after the addition of the compound. Subsequently, thermodynamic parameters, including enthalpy change (ΔH) and entropy change (ΔS), were determined using the Van’t Hoff’s plot (plotting lnKa versus 1/T, where T is temperature in Kelvin). These parameters are vital for understanding the nature of the forces governing the interaction between the protein and compound 2. The corresponding Gibb’s free energy change (ΔG) for different temperatures was then calculated using the standard Gibb’s free energy relation.

 

The determined values for ΔG and Ka at the four different temperatures were summarized. The negative value obtained for ΔH (enthalpy change) strongly suggests that the binding interaction between BSA and compound 2 is an exothermic process and is predominantly driven by the formation of hydrogen bonds. Additionally, the calculated value of ΔS (entropy change) was found to be negative. When both ΔH and ΔS are negative, it implies that the binding forces are primarily van der Waals interactions or hydrogen bonding. Crucially, the consistently negative values of Gibb’s free energy of binding (ΔG) across all tested temperatures indicated that the interaction between BSA and compound 2 is spontaneous and thermodynamically favorable under these conditions. Furthermore, the activation energy (Ea) for the binding reaction between BSA and compound 2 was also calculated using the Arrhenius equation. This provides information on the ease with which the binding mechanism occurs.

 

This comprehensive UV-Vis study signifies that the drug candidate (compound 2) binds extensively with the serum protein BSA in a spontaneous and thermodynamically favorable manner at physiological temperatures. The high binding constant values further reinforce that the interaction is very strong, with hydrogen bonding playing a significant role in stabilizing the complex.

 

Fluorescence Resonance Energy Transfer (FRET) Study

 

Fluorescence Resonance Energy Transfer (FRET) is a non-radiative phenomenon where electromagnetic energy is transferred from a donor fluorophore, which is in a photo-excited state, to an acceptor molecule (quencher) situated in close proximity. In this context, the protein (BSA) serves as the donor fluorophore, transferring its quantum energy to the quencher molecule (compound 2), which acts as the acceptor in its ground state. The spectral overlap observed between the emission spectrum of the donor and the absorption spectrum of the acceptor forms the fundamental basis for the conservation of energy during the FRET process, indicating that energy transfer can occur.

 

The spectral overlap between the fluorescence emission intensity of BSA and the UV absorption spectrum of [{NiII(hpdbal-sbdt)}2] (compound 2) was visually represented. From this spectral overlap, the efficiency of energy transfer (E) can be quantitatively calculated using the Förster non-radiative energy transfer theory. This theory relates the efficiency of energy transfer to the distance between the donor and acceptor molecules. The equation for efficiency of energy transfer involves F0 (BSA fluorescence intensity without quencher) and F (BSA fluorescence intensity with quencher), and the sixth power of the Förster critical energy transfer distance (R0) and the actual distance between donor and acceptor (r). R0 represents the distance at which there is 50% energy transfer efficiency and is calculated using parameters such as K2 (spatial orientation factor, typically 2/3 for BSA), N (average refractive index of the medium, 1.336), ϕ (fluorescence quantum yield of the donor, 0.15), and J (overlap integral). The overlap integral (J) itself is derived from the corrected fluorescence intensity of the donor (F(λ)) and the molar extinction coefficient of the acceptor (ε(λ)) over the relevant wavelength range.

 

Following these equations, the values of J, E, R0, and r were precisely calculated and presented. The calculated value for r (the distance between donor and acceptor molecules) was found to fall within the range of 2-8 nm, which is consistent with typical FRET distances. Furthermore, the condition 0.5 R0 < r < 1.5 R0 was met, which is a key requirement for the Förster non-radiative energy transfer theory to be applicable. This robust agreement indicates a high probability of energy transfer from BSA to compound 2 and, consequently, efficient quenching of BSA fluorescence. This phenomenon is indicative of either a static quenching mechanism, implying the formation of strong ground-state complexes between the protein and the compound, or a highly efficient energy transfer. The FRET study therefore confirms that the drug candidate (compound 2) and the serum carrier protein (BSA) reside in close spatial proximity to each other, with favorable energy transfer conditions facilitating their interaction within a biological milieu.

 

IR Binding Study

 

Fourier Transform Infrared (FT-IR) spectroscopy is a highly valuable technique for obtaining detailed information regarding the different forms of the secondary structure of proteins. Proteins exhibit characteristic fingerprint bands in their IR spectra, particularly the C=O stretching (amide I region) and N-H bending (amide II region) vibrations. Both of these bands are exquisitely sensitive to the secondary structure content of the protein because they are directly involved in intramolecular hydrogen bonding between different secondary structure elements, such as alpha-helices and beta-sheets. To precisely investigate the influence of the drug candidate (compound 2) on the secondary structure of BSA, IR studies were performed. The BSA concentration was fixed at 100 µM, while the concentration of compound 2 was varied, with three specific concentrations tested: 17 µM (a), 83 µM (b), and 165 µM (c), providing a range of protein-to-compound ratios. Baselines were meticulously acquired for pure BSA and the buffer solution to ensure accurate subtraction and signal resolution.

 

The IR spectrum of native BSA typically displayed an amide I band at 1649 cm-1, an amide II band at 1539 cm-1, and a shoulder peak around 1468 cm-1. These characteristic peaks validate the predominant presence of α-helix conformation in native BSA. Upon incubation of BSA with different concentrations of compound 2, small shifts were observed in the IR spectra, however, neatly resolved peaks representing distinct secondary structures were not immediately apparent in the raw data. To overcome this challenge and to accurately analyze the relative abundance of different forms of secondary structure, peak fitting was performed on the second derivative IR spectra within the amide I region (1600-1700 cm-1) using the Levenberg-Marquardt (L-M) algorithm. This advanced computational technique yielded completely resolved spectra, allowing for the precise identification and quantification of various forms of the secondary structure of the protein BSA in the presence of complex 2.

 

The quantitative analysis, summarizing the percentages of different forms of secondary structure obtained from resolving the second derivative IR spectra, provided crucial insights. A gradual conversion of α-helix content to β-sheet structures and other minor conformations was observed with increasing concentrations of compound 2. This progressive and steady alteration strongly suggests a gradual unfolding of the coiled secondary structure of the protein upon binding with the drug candidate. The IR binding study therefore signifies that the drug candidate (compound 2) induces slow but progressive conformational changes to the structure of the protein, leading to a gentle unfolding of the secondary structure during the binding process. It is important to note that this effect became significant primarily at higher concentrations of compound 2, specifically at concentrations above its determined MIC (Minimum Inhibitory Concentration) value.

 

Combinatorially, the comprehensive interaction study, integrating insights from steady-state fluorescence, synchronous and 3D fluorescence, UV-Vis, and IR spectroscopic techniques, provides compelling evidence that the novel nickel complex can be readily transported by drug carrier proteins within the body. This efficient transport capability suggests its potential to effectively navigate biological barrier membranes and successfully reach its targeted site of action. This multi-spectroscopic interaction study significantly complements the *in vitro* antidiabetic and antimicrobial activity findings, serving as a critical reference point for subsequent stages of drug development. Such detailed mechanistic understanding is invaluable when progressing to *in vivo* studies with animal models and further interaction studies with human serum albumin and DNA, guiding the future drug development process.

 

Conclusions

 

The current study successfully reports the synthesis and comprehensive *in vitro* evaluation of a novel dimeric nickel complex, designated as [{NiII(hpdbal-sbdt)}2] (compound 2). This compound was rigorously assessed for its bioactivity against multidrug-resistant strains of bacteria and fungi, and its potential for the treatment of type II diabetes. The results are highly promising: the nickel complex demonstrated excellent inhibitory action against the clinically challenging methicillin-resistant *Staphylococcus aureus* (MRSA) strain, achieving a remarkably low Minimum Inhibitory Concentration (MIC) value of 2 μg/mL. Concurrently, it exhibited exceptional glucose uptake capacity in insulin-resistant HepG2 cells, promoting an impressive 95% fluorescent glucose uptake, a level comparable to that of the standard antidiabetic drug metformin.

 

A critical finding regarding its safety profile is that this novel compound did not exhibit any significant cytotoxicity towards normal human kidney cells (HEK293), even at concentrations well above its antimicrobial MIC. This favorable selectivity strongly depicts a specific mode of action and a lack of general toxicity towards healthy cells, which is a highly desirable characteristic for any therapeutic agent. Furthermore, the nickel complex was found to maintain excellent stability across a wide range of pH conditions, as evidenced by pH titration studies. This robust pH stability signifies that, as a potential drug candidate, it can likely remain stable and structurally intact while traversing various bodily fluids that present differing pH atmospheres, thereby ensuring its pharmacological activity *in vivo*.

 

To gain deeper insights into its pharmacokinetic behavior, this highly active complex was further investigated for its binding interactions with bovine serum albumin (BSA), a widely used model serum carrier protein. This detailed study, employing a multi-spectroscopic approach including steady-state fluorescence, synchronous and 3D fluorescence, UV-Vis, and IR spectroscopic techniques, comprehensively elucidated the nature of its binding. The studies consistently indicated that the binding reaction between the nickel complex and BSA is spontaneous and exothermic in nature, signifying a thermodynamically favorable interaction. Importantly, the compound was observed to bind very efficiently with the protein without inducing significant disruption to the protein’s secondary structure at concentrations below its MIC value. The data derived from these comprehensive interaction studies robustly complement the observed *in vitro* antidiabetic and antimicrobial activities, providing strong inference that this active complex possesses favorable properties for efficient transport by drug carrier proteins within the body. These findings collectively position the nickel complex as a highly promising candidate for subsequent *in vivo* testing in animal models and further crucial interaction studies with human serum albumin and DNA, marking the next critical steps in its drug development process.