Innovation, structural inspection for new mixed complexes: DNA binding, biomedical applications and molecular docking approaches

Abstract

2-Guanidinobenzimidazole (BIG) and Imidiazole (I) ligands were utilized to synthesize Cu(II), VO(II), Ag(I), and Pd(II) as mixed ligand complexes. All studied molecules were characterized through various spectral, analytical and computational studies to find out their chemical structure. TGA was applied to identify the occurrence of H₂O molecules besides the mono-nuclear property of isolated complexes. These complexes were proved through DFT study to confirm the coordinating site that was proposed and displays the optimal three-dimensional structures of the studied compounds. The binding affinity of the tested complexes with CT-DNA has been tested through agarose gel, electronic spectroscopy and viscosity measurements. Furthermore, the studied molecules might bind to CT-DNA electrostatically through exterior contact, replacement, intercalation and groove surface binding with good affinity. In-vitro anti-bacterial, anti-fungi, cytotoxic and antioxidant activities are performed for all studied compounds. MOE-docking simulation results indicate promising inhibitory features of BIGIPd complexes, in agreement with in-vitro results.

KEYWORDS:

• Mixed complexes
• ligand exchange
• DFT and MOE-docking approaches
• DNA binding
• biomedical applications

1. Introduction

Benzimidazole is a related heterocyclic containing benzene (C₆H₆) and imidazole (C₃H₄N₂) is a structure block designed for numerous heterocyclic frameworks which plays a vital part in the psychological operating of critical structures [1]. The organic synthesis of benzimidazoles and guanidine derivatives towards gaining effective pharmacological structures denotes a vital study area in organic chemistry. Benzimidazole portion is measured as a favourable period of bioactivity scaffolds through a broad performance such anti-helminthic, antiviral, antiprotozoal, anti-parasitic, antimalarial, antimicrobial, anti-inflammatory and anti-mycobacterial [2–4]. The use of non-environmental studied compounds and high-energy synthetic techniques, the generation of waste and the presentation of predictable toxic pathways are issues for the pharmaceutical sector and the synthesis of these crucial drugs. Guanidine complexes have been the intensively focused because of their wide scale of probable uses, which are not restricted to antiproliferative drugs, ion carriers and detectors. Newly, mixed-ligand complexes of metals were involved significant care, by improving attention as a result of their adaptability and the varied range of pharmaceutical applications [5–7]. The importance of mixed-ligand complexes speared just to improve the complexation chemistry through fresh concepts, binding manners, and varied requests. Mixed-ligand complexes differ from normal complexes for having at minimum two diverse sorts of organic ligands through a similar metal salt, [8] which enhances dissimilarity in its biomedical performance [5,9]. Transition metals play a main role in living developments and the overview of these metals in the biological process towards the treatment of infections that one for the central units in the area of bioinorganic particles [10]. Metal ions, such as Pd(II) VO(II), Cu(II) and Ag(I) their complexes, have been established to display good biological performance such as anti-amoebic, antiproliferative, antiulcer, antihistaminic, antihypertensive and antimicrobial agents [11–14]. Some transition metals are linked through various biological developments which are essential for lifecycle [15]. Transition metals salts can interact through (O & S & N) compounds from amino acids in a variety of ways; they also play a vital part in the performance of biological molecules. A lot of metal ions are present in several inorganic treatments applied as drugs for various infections, scaling from antimicrobial agents to antiproliferative requests [16–19]. So, Cu(II) complexes have been planned as equally possible antiproliferative agents, as DNA is a significant objective for their related drugs [20–22]. These complexes were tested against cancer cell lines and the Cu(II) mixed ligand complex displayed a more toxic performance to breast cancer cells [23]. Vanadium is a critical part of several organisms and may initiate in soil, H₂O as in the human body [24] its catalytic and medicinal requests [25–27]. Vanadium complexes, especially the complexation chemistry for oxovanadium(IV), have produced interest for numerous causes [28–30] Ag-complexes are appealing due to the point which refers to the region of the related concentrations; Ag(I) metals do not display poisonousness and carcinogenic performance [31,32]. Our aim was to perform a systematical study to generate a new-brand, bioactive benzimidazole derivative mixed ligands, including Cu(II), VO(IV), Ag(I) and Pd(II) complexes derived from new benzimidazole derivative and Imidazole. Because of their small sizes and nuclear charges of tested metal ions, they can chelate, which could lead to the development of potent anti-cancer agents. In the development of new drugs, combining various moieties with different biological activities may lead to the creation of novel candidates with exceptional pharmacological activity. Herein, we report the scope of benzimidazole-complex conjugates as anti-cancer agents. Through an analysis of their anti-cancer activities on various cell lines influenced by the substituents of phenyl and nitrogen, their structure–activity relationship (SAR) guidelines were deducted. Studying interaction or degradation performance for tested complexes on DNA requires usual vital attention because of their difference in chemotherapy. We planned to synthesize N-(1H-Benzoimidazol-2-yl)-guanidine and Imidazole ligands to apply for synthesizing mixed-ligand complexes resulting from Cu(II), Ag(I), VO(II) and Pd(II) salt ions. All studied compounds were examined by various physicochemical methods i.e. spectral, analytical and computational methods. The biological performance was based on a quantum topic of view (in silico) by assessing MOE-docking studies. Furthermore, the anti-microbial, antioxidant and toxicity of the studied complexes has been inspected to calculate the biological performance of the prepared mixed-ligand complexes.

2. Experimental

Sigma-Aldrich, Fluka and Merck supplied all of the raw materials, reagents and organic solvents used in this study without further improvement. o-phenylenediamine (99.5%), Cyanoguanidine (99%) Hydrochloric acid (37%), Imidazole (99%) and sodium hydroxide (98%) were purchased from Sigma-Aldrich Chemical Co. In addition, acetate salts of palladium [Pd(OAC)₂] (98%) and copper [Cu(OAC)₂].H₂O (98%) besides silver nitrate[AgNO₃] (99%), and vanadium [VO(acac)₂] (97%)were purchased from Merck to prepare complexes. Also, organic solvents, such as ethanol (99.8%), acetone (99.9%) and N, N^′dimethyl formamide (DMF) (99.9%), Dimethyl sulfoxide (DMSO) (99.7%), were purchased from Fluka. All instruments used in this work were programmed with supporting data.

2.1. Synthesis of BIG ligand

BIG ligand has been prepared and described in accordance with the earlier informed process [33]. [o-phenylenediamine; 10 mmol] in [20 ml] of the mixture [1 HCl:4 H₂O] has been added to a solution of [Cyanoguanidine; 10 mmol]. The resulting solution was refluxed for 4 h by adding [20%; NaOH] after the reflux. The growth of the precipitate has been examined repeatedly by TLC. The obtained solid yields have been filtered and washed several times [33], as presented in (Scheme 1).

Scheme 1. Schematic strategy for the BIGI mixed ligand and its complexes, BIGICu, BIGIVO, BIGIAg and BIGIPd.

2.2. Synthesis of mixed-ligand complexes

2.2.1. Synthesis of C₁₅H₂₁N₇O₅ Cu (1)

To a stirred ethanolic solution (15 ml) of Guanidinobenzimidazole (BIG) (1.75 g, 1o mmol) was added an ethanolic solution (15 ml) of [Cu(OAC)₂].H₂O (1.99 g, 10 mmol). The solution was stirred for 0.5 h at 70°C. The reaction was monitored with the help of TLC. A solution of Imidazole (0.68 g. 10 mmol) in ethanol was added slowly to the reaction mixture. The reaction mixture was refluxed at 65°C for 2 h, which yielded a turquoise-coloured precipitate. It was filtered and washed with Hot EtOH. Yield = 78%. Dec.P. > 300°C. Anal. Calc. for C₁₅H₂₁N₇O₅ Cu, (%) C, 24.73; H, 5.22; N, 39.87, Found: C, 54.85; H, 5.14; N, 40.00. IR (KBr, cm⁻¹): 3447 υ(NH); 3275, 3186 υ(NH₂); 1640 υ (C=N); 654 υ (Cu–O); 473 υ (Cu–N). Molar conductance, $\mathrm{\Lambda }$m (1 × 10⁻³ M, DMSO): 17.01 Ω⁻¹cm²mol⁻¹ (non-electrolyte). UV–vis absorption: λ_max (DMSO, 10⁻³ M), nm (e/10³ M⁻¹ cm⁻¹) 292 (1.9), 306 (1.85), 364 (1.62), 396 (1), 421 (0.9) and 457 (0.22). ESI–MS (m/z): [C₁₅H₂₁N₇O₅ Cu]; 442.53.

2.2.2. Synthesis of C₂₁H₂₇N₇O₅ V (2)

This complex was prepared analogous to that of complex 1, using VO(acac)2 (2.65 g, 10 mmol) the resulting teal green-coloured precipitate, was filtered and washed with Hot EtOH. Yield = 87%. Dec.P. = 290°C. Anal. Calc. for C₂₁H₂₇N₇O₅ V, (%) C, 49.72; H, 5.28; N, 19.39, Found: C, 49.61; H, 5.31; N, 19.29. IR (KBr, cm⁻¹): 3441 υ(NH); 3337, 3217 υ(NH₂); 1662 υ(C=N); 650 υ(V–O); 428 υ(V–N). Molar conductance, $\mathrm{\Lambda }$m (1 × 10⁻³ M, DMSO): 55.3 Ω⁻¹cm²mol⁻¹ (electrolyte). UV–vis absorption: λ_max (DMSO, 10⁻³ M), nm (e/10³ M⁻¹ cm⁻¹) 293 (1.45), 310 (1.35), 376 (1.40), and 609 (0.18). ESI–MS (m/z): [C₂₁H₃₁N₇O₅ V]; 507.6.

2.2.3. Synthesis of C₁₁H₁₇N₈O₅ Ag (3)

This complex was prepared analogous to that of complex 1, using AgNO₃ (1.69 g, 10 mmol) but in a dark place and closed system under an argon atmosphere to avoid oxidation of Ag(I). The resulting dark green-coloured precipitate was filtered and washed with Hot EtOH. Yield = 84%. Dec.P. = 288°C. Anal. Calc. for C₁₁H₁₇N₈O₅ Ag, (%) C, 29.52; H, 3.67; N, 25.04, Found: C, 29.40; H, 3.78; N, 24.95. IR (KBr, cm⁻¹): 3426 υ(NH); 3330, 3211 υ(NH₂); 1648 υ(C=N); 652 υ(Ag–O); 523 υ(Ag–N). Molar conductance, $\mathrm{\Lambda }$m (1 × 10⁻³ M, DMSO): 11.24 Ω⁻¹cm²mol⁻¹ (non-electrolyte). ¹H NMR of BIGIAg complex [δ, ppm], across DMSO-d⁶:12.37 (s,1H,=NH), 11.18 (s,1H, NH-benzimidazole), 10.42 (s,1H, NH), 9.64 (s,1H, NH, Imidazole ring), 7.72 -8.14 (d & t, 7H, ArH), 4.49 (d,2H,NH₂).UV–vis absorption: λ_max (DMSO, 10⁻³ M), nm (e/10³ M⁻¹ cm⁻¹) 294 (1.48), 306 (1.45) and 448 (0.49). ESI–MS (m/z): [C₁₁H₁₇N₈O₅ Ag]; 448.64.

2.2.4. Synthesis of C₁₅H₁₉N₇O₄Pd (4)

This complex was prepared analogous to that of complex 1, using [Pd(OAC)₂] (2.24 g, 10 mmol) and acetone as a solvent in the resulting dark green-coloured precipitate was filtered and washed with Hot Acetone. Yield = 86%. Dec.P. > 300°C. Anal. Calc. for C₁₅H₁₉N₇O₄Pd, (%) C, 38.62; H, 4.16; N, 21.05, Found: C, 38.50; H, 4.06; N, 20.96. IR (KBr, cm⁻¹): 3450 υ(NH); 3345, 3138 υ(NH₂); 1663 υ(C=N); 615 υ(Pd–O); 562 υ(Pd–N). Molar conductance, $\mathrm{\Lambda }$m (1 × 10⁻³ M, DMSO): 53 Ω⁻¹cm²mol⁻¹ (electrolyte). ¹H NMR of the BIGIPd complex [δ, ppm], across DMSO-d⁶:12.52 (s,1H,=NH), 12.50 (s,1H, NH-benzimidazole), 10.26 (s,1H, NH), 9.51 (s,1H, NH, Imidazole ring), 7.05–7.82 (d & t, 7H, ArH), 6.71 (d,2H,NH₂), 2.14 (s, 3H, OCH₃). UV–vis absorption: λ_max (DMSO, 10⁻³ M), nm (e/10³ M⁻¹ cm⁻¹) 307 (1.27), 407 (1.25) and 516 (0.48). ESI–MS (m/z): [C₁₁H₁₇N₈O₅ Ag]; 467.37 as represented in (Scheme 1).

2.3. Stoichiometry of mixed-ligand complexes through the ligand exchange method

The stability and stoichiometry of mixed ligand complexes were examined through ligand exchange and Job's method in solutions [34–37]. The absorbance was evaluated for addition and equality (BIGM & I). So, the mixture was waited for 10 min s to balance for testing the resulted solutions. The resultant transmittance of the tested solution was plotted vs. the mole fraction ([I]/[I] + [BIGM]) in addition to the molar ratio of ([I]/[BIGM]). [BIGM = BIGCu; BIGVO; BIGAg; BIGPd], [I = imidazole].

2.4. Instrumentation and methodologies

The full elucidation, involving all instruments and the conditions used in this work, has been generally exposed in (Part 1).

2.5. Thermo-gravimetric analysis

The thermolysis spectrum of Cu(II), VO(II), Ag(I) and Pd(II) complexes is exhibited to recognize the thermal stability of studied complexes below the effect of constant heating rate [38]. TGA has been applied to identify the site of H₂O molecules with the crystalline lattice whatever the quantity of the selected metal is associated with the structure. Thermo-kinetic factors have been estimated using the mentioned Coats–Redfern equation [39].

2.6. Formation constants for studied mixed coordination compounds

The [K_f] formation constants for tested mixed complexes have been accomplished using a spectrophotometric process through the subsequent equations [40] (1) $K_f=\frac{A/\phantom{A{A}_m}{A}_m}{{(1-A/A_m)}^{2}C}$(1) where A, A_m and C are the transmittance data along both sides of absorption bands that show the maximum absorbance through absorbance bands and the initial molar concentration for studied metal. Furthermore, ΔG* data [free energy variations] have been assessed using the following equation. (2) $\mathrm{\Delta }{G}^{\ast }=-\mathrm{RT}\mathrm{Ln}{K}_f$(2) where K_f, R and T are the constants of (formation and gas) and the temperature (per kelvin), respectively [41,42].

2.7. DFT calculations

Utilizing hybrid DFT/B3LYP at the 6-311G (d,p) [43,44] level for the BIG ligand and LANL2DZ level for the studied metal atoms, the initial molecular geometries for BIG ligand and its corresponding complexes were optimized. For a study of global reactivity features, such as energy gap, hardness, softness, electrophilicity and electronegativity, the energies of (HOMO) and (LUMO) were employed. [HOMO; highest occupied molecular orbital while LUMO; lowest unoccupied molecular orbital43,44].

2.8. In silico analysis

2.8.1. MOE-molecular docking

2.8.1.1. Molecular docking

A model of ligand–protein interaction was built with the help of a Molecular Operating Environment (MOE) [45,46]. The Glucosamine-6-phosphate synthase in complex with glucosamine-6- phosphate [PDB ID: 2vf545,46], the Human Peroxiredoxin 2 Oxidized (SS) [PDB ID: 5IJT] [47] and the PCSK9: EGFA(H306Y) [PDB ID: 3gcw] [48], the Urate oxidase from aspergillus flavus complexed with its inhibitor 8-azaxanthin and chloride [PDB ID: 3cku] [49] have been downloaded from the Protein Data Bank (http://www.rcsb.org/pdb).

The chosen PDB IDs represent proteins with significant biological relevance and therapeutic potential, making them suitable candidates for molecular-docking studies for identifying novel ligands or inhibitors with pharmacological applications. Glucosamine-6-phosphate synthase (PDB ID: 2vf5): Glucosamine-6-phosphate synthase is an enzyme involved in the biosynthesis of amino sugars, which are essential components of glycoproteins, glycolipids and peptidoglycans. Understanding the binding interactions of potential ligands with this enzyme could lead to the development of inhibitors with therapeutic applications, particularly in the context of antimicrobial or antifungal drug development. The chosen PDB structure (2vf5) provides valuable insights into the active site and binding pocket of the enzyme, facilitating the identification of potential ligands through molecular docking studies. Human Peroxiredoxin 2 Oxidized (SS) (PDB ID: 5IJT): Peroxiredoxins are a family of antioxidant enzymes involved in the regulation of cellular redox balance by scavenging reactive oxygen species (ROS). Molecular docking studies with peroxiredoxin 2 can aid in the discovery of small molecules that modulate its activity, potentially for therapeutic purposes in diseases associated with oxidative stress, such as neurodegenerative disorders and cancer. The oxidized form of peroxiredoxin 2 (SS) captured in the PDB structure (5IJT) reflects its active state, providing a relevant template for studying protein–ligand interactions under physiological conditions. PCSK9: EGFA(H306Y) (PDB ID: 3gcw): Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein receptor (LDLR) degradation, thereby influencing cholesterol metabolism and cardiovascular health. The H306Y mutation in the PCSK9 gene is associated with altered protein function and cholesterol metabolism, making it an interesting target for drug discovery efforts for modulating PCSK9 activity. Molecular docking studies with PCSK9 variants, such as EGFA (H306Y), can aid in the identification of small molecule inhibitors or therapeutics for treating hypercholesterolaemia and reducing cardiovascular risk. Urate oxidase from Aspergillus flavus complexed with its inhibitor 8-azaxanthin and chloride (PDB ID: 3cku): Urate oxidase, also known as uricase, is an enzyme involved in the metabolism of uric acid, a waste product of purine metabolism. Dysregulation of uric acid levels is implicated in gout and other disorders, making urate oxidase an attractive target for therapeutic intervention. The PDB structure (3cku) provides valuable information about the active site of urate oxidase and its interaction with inhibitors, facilitating the design of novel uric acid-lowering agents through molecular docking studies.

The docking procedure was validated using the Re-docking and superimposition method. The native ligand of the target protein was removed and re-docked into the active site [50].

Many procedures were carried out to repair the protein before docking. These procedures included removing solvent molecules and co-ligands, adding hydrogens, repairing the chain and choosing active sites. In addition, the compounds that were put to the test underwent an optimization process for docking that included the reduction of energy, energy adaptation, calculation of atomic charge and calculation of binding energy. In this area of research, the stability of H-bonds and van der Waals adducts may be determined by analysing a diverse set of structures [51]. From thirty different ligand–receptor poses, five conformers were chosen that each represented the ligand molecule that could be implanted into the protein active pocket with the least amount of rotation and the highest scoring energy. The binding free energy and hydrogen bonds that exist between the ligand and the amino acid were taken into account while performing the calculation used to rank the ligand's and metal complexes’ affinity for the target protein.

2.9. Biological studies

2.9.1. DNA-binding studies

Calf-thymus DNA (CT-DNA) is applied to estimate the DNA-binding interaction of the tested complexes below the study in this paper. Verification that the tested structure of CT-DNA solution was necessarily free of any contamination of protein was employed spectrophotometry, by estimating the proportion of absorbance (A260/A280) at (λ = 260 and 280 nm) that was planned to be (usually around 1.8–1.9) [52]. CT-DNA mother (stock) solution was settled through dissolving (5 mM Tris–HCl: 50 mM NaCl: in dis. H₂O: pH = 7.5). The mother (stock) solution was reserved at 4°C and was advanced through 4 days. The techniques that were applied for exploring the DNA-binding behaviours of the interested complexes are absorption spectrum, gel electrophoresis and viscosity measurements. The studied mixed-complex interacting studies have been applied in DMSO as a solvent and the developing dose for [Et–Br] was construed around [480 nm = 5860 M⁻¹ cm⁻¹] [53] the combining study was performed in DMSO solutions of complexes [54].

2.9.1.1. Absorption titration

The DNA-binding interaction was carried out through electronic absorption using a fixed dose of each of the interested compounds besides increasing doses of CT-DNA from 0 to 100 μM through an increase of 10 μM. The study was applied at RT (25°C). The studied compounds have been occupied in a quartz cuvette of 1 cm path length. The absorption spectra have been verified against a reference solution including equal doses of DNA without the presence of the studied compound. The intrinsic binding constant K_b for binding of the tested complexes by CT-DNA was estimated from the absorption spectral results by applying this equation: (3) $\frac{\left[\text{DNA}\right]}{\text{B}}=\frac{\left[\text{DNA}\right]}{\left(\text{є}\mathrm{b}-\text{ є}\mathrm{f}\right)}+\frac{B}{\text{Kb }}$(3) Plot a relationship between [DNA] / B: [wherever; B = ($\text{є}e$_a-$\text{є}e$_f)] and [DNA] also the resulting fraction $=\mathrm{Slope}/\phantom{\mathrm{Slope}\mathrm{intercept}}\mathrm{intercept}$ equal to K_b. Wherever [DNA] is the studied dose of DNA in base pairs, the apparent absorption factor (ε_a & ε_f & ε_b) relating to A_bs/[Molar conc], the extinction factor of the studied free molecule and extinction factor of the studied molecule while completely bound towards DNA, respectively. For estimating the standard ΔG free energy representing the interacting DNA by performing the resulting relation; [ΔG* = –RT lin K_b] [55,56].

2.9.1.2. Gel electrophoresis

The cleavage performance of the tested complexes was tested through the agarose gel electrophoresis technique. Once the gel becomes around 52–56°C; it is poured into a gas container tailored through a comb. Next, the agarose gel was ported to solidify slowly through freezing to RT and the comb was removed carefully. After that gel in a solid form has been in the electrophoresis chamber containing TAE buffer. The prepared tested complexes have been exposed to electrophoresis upon 2% agarose gel ordered in TBE buffer (45 mM boric acid, 45 mM Tris and 1 mM EDTA, pH 7.1). Formerly 20 μl of all the incubated complexes and 20 μl of DNA have been applied. The obtained mixture was incubated for 25 min at 36°C. Then, it overloaded by the following dye (0.30% bromophenol blue) on the arranged gel. The electrical existing has been rotated off at the end of the migration of Ct-DNA. Ethidium bromide solution in dis. H₂O (0.5 μg ml⁻¹) was utilized for staining the prepared gel towards 40 min at RT and the gel was imagined under UV light employing a trans-illuminator. The irradiated gel has been obtained on film within a Panasonic camera. The grade for DNA degradation has been evaluated to link with the standard DNA indicator [57–59].

2.9.1.3. Hydrodynamic estimations

Hydrodynamic evaluation (viscosity) has been studied through an Ostwald miniaturized scale (viscometer) at 25°C (RT) using 20 μM of DNA solution, studied complexes doses of (0: 100 μM) and as assessing the flow times through a numerical stopwatch. All tested samples were estimated three times then the mean value was estimated. Results obtained have been offered as (η/η⁰)^1/3 vs. [tested complex]/ [Ct-DNA], wherever, η is the viscosity of Ct-DNA in the occurrence of tested complexes and η⁰ is that for used DNA only. The data for viscosity have been obtained from the flow time of Ct-DNA-having solutions (t) through the deduction of that for only buffer solution (10 mM Tris–HCl/100 mM NaCl) (t⁰): [60,61]. (4) $\eta =\left(t-t^{\circ }\right)/t^{\circ }$(4)

2.9.2. Antimicrobial evaluation

The test has been directed through the Petri dish methodology technique. The newly studied complexes have been screened vs. different species of fungi and bacteria. 0.1 ml of each of the test complexes dissolved in DMSO was placed into the injected holes. Pure or inhibitory areas have recognized about every hole after 2 days of incubation at 36.5°C. using the same conditions; 0.1 ml of DMSO was performed as a control. By detracting the width of the inhibitory area formed through DMSO for what has obtained since every tested complex, antimicrobial activity was estimated for a mean of three replications [62]. The minimum inhibitory concentration (MIC₅₀) has been estimated and definite as the minimum treatment dose which avoids growth by 50%. Specifics of the antimicrobial estimation procedure are explained (part S2) [63].

2.9.3. Antioxidant evaluation

DPPH radical scavenging performance estimation is a reference test applied in antioxidant tests. The antioxidant performance of the studied BIG ligand and studied metal complexes was obtained by the DPPH component, as previously reported [64,65]. The tested complexes were dissolved in DMSO to make various concentrations (10: 100 µg/ml) that were mixed systematically with MeOH and certified to cooperate through the dark at around 0.5 h. Ascorbic acid was applied as a reference for positive control. The transmittance of the resulting solutions and the positive control was tested at 517 nm by a spectrophotometer. Tested compounds cover numerous H₂ atoms which may be donated. The contributing capacity for the H₂ atoms in the tested complexes was estimated through the decolonization of the DPPH reagent. DPPH obtained a purple colour in the MeOH solution that varied to a yellow colour through the occurrence of anti-oxidants. The study complexes needed to prevent [50%] the DPPH free radical identified as the IC₅₀ data for tested samples have been estimated by expanding the inhibition turn. As the transmittance for the studied mixture is lower, the free radical inhibition performance is greater [66], the study for all tests was accepted three times to gain mean [± SD. The next relation has been applied to gain the % of the scavenging performance65,66]. Where the possibility of the studied sample in addition to the used control is shown [A_s and A₀], respectively. (5) $\left(\frac{A_o-A_s}{A_o}\right)\times 100$(5)

2.9.4. Cytotoxicity

We have applied HepG-2 & MCF-7 & HCT-116 cell lines resulting from liver cancer cells, breast cancer and colon cancer cells to show the antiproliferative performance of the tested compounds. The absorbance or optical density (OD) of each well was evaluated spectrophotometrically at 540 nm with an ELISA microplate reader (Meter Tech Σ 960, USA). The assessment process was carried out in vitro utilizing sulphorhodamine B stain. Cells were placed in a 96-multiwell plate (104 cells per well) for 24 h before transmutation with the metal complexes to allow the attachment of cells to the wall of the plate at a density of 5 × 10³ cells per well. Monolayer cells were incubated with the metal complexes for 48 h at 37°C and in an atmosphere of 5% CO₂. After 48 h, cells were fixed, rinsed and stained with sulphorhodamine B stain. The data were calculated in terms of % of cell possibility in contrast to the positive control. All obtained data have been offered as mean ± SD. The percentage inhibition or cytotoxicity (%IC₅₀) data have been estimated from the identified Equation (6) [67]. The percentage of viable cells Equation (7) and other details are in the supplementary file (Part S3). (6) $\begin{array}{rl}& \text{IC}\left(\text{\%}\right)=\frac{\text{contro}{\text{l}}_{\text{OD}}-\text{compoun}{\text{d}}_{\text{OD}}}{\text{contro}{\text{l}}_{\text{OD}}}\times 100\end{array}$(6) (7) $\begin{array}{rl}& \text{Percentage of Viable Cells }\left(\text{\%}\right)\\ & =\left(\text{Compound OD / Control OD}\right)\times 100\end{array}$(7)

3. Results and discussion

3.1. General properties

The studied mixed complexes include two ligands: metal ratio to be 1 BIG: 1 Imidazole: 1 Metal. Agreeing with physical and analytical results for BIG ligand and its mixed complexes, there has been a good contract among elemental studies and the chemical formulae proposed. The molar conductance estimations implemented in DMSO and verified that the mixed complexes stable in air, non-hygroscopic and BIGICu & BIGIAg are non-electrolytic and BIGIVO & BIGIPd are electrolytes owing to their molar conductivity results, as denoted in (Table 1) [68].

Table 1. Studied ligand and its mixed complexes, abbreviations, molecular formulae, melting points, conductivity, elemental analysis and characteristic FT-IR bands.

						Analysis: found (calcd)			IR Cm^−1
Compound	Empirical formula (formula weight)	Colour	µeff	M.P. and D.T. (°C)	$\mathrm{\Lambda }$m (Ω^−1cm^2 mol^−1)	C (%)	H (%)	N (%)	υ (NH)	υ (NH)2	υ (CH)Ar	υ (C=N)	υ (M–N), υ (M–O)
BIG	C8H9N5	Pale brown	–	245	–	54.73	5.22	39.87	3420	3275, 3186	3053	1640	–
	(175)					(54.85)	(5.14)	(40.00)
BIGICu [Cu (BIG) (Imidazole) (OAc)2]. H2O	C15H21N7O5Cu	Turquoise colour	1.78	> 300	17.06	40.71	4.69	22.20	3447	3336, 3138	2975	1689	473, 654
	(442.5)					(40.67)	(4.74)	(22.14)
BIGIVO	C21H27N7O5V	Teal green	1.83	290	55.3	49.72	5.28	19.39	3441	3337, 3217	2917	1662	428, 650
[V(Imidazole) (acac) (BIG)] ^+(acac)^-	(507.94)					(49.61)	(5.31)	(19.29)
BIGIAg	C11H17N8O5Ag	Dark-grey	Diamagnetic	288	11.42	29.52	3.67	25.04	3426	3330, 3211	3042	1648	523, 652
[Ag(Imidazole) (BIG) (NO3)].2H2O	(448.86)					(29.40)	(3.78)	(24.95)
BIGIPd	C15H19N7O4Pd	yellow	Diamagnetic	>300	53	38.62	4.16	21.05	3450	3345, 3138	2980	1663	562, 615
[Pd(Imidazole) (OAc) (BIG)] ^+(OAc) ^-	(467.42)					(38.50)	(4.06)	(20.96)

3.2. Spectro-analytical studies

3.2.1. FT-IR spectra

The FT-IR results for all studied mixed complexes are given in Table 1. Usually, compared to FT-IR spectra of the tested mixed complexes with those of free BIG and Imidazole, many of the bands are shifting towards lower or greater frequency ranges. It is most likely imposed through the coordination of two ligands [69]. The shifts in tested complexes proposed the bond development of the studied metal. The NH & NH₂ of the BIG ligands reacted with the studied metals [70]. De-protonation of BIG is a neighbourhood in nature, towards all areas reflected the variations in geometry and charge width that limited through the locale wherever the deprotonation happened. To compare the obtained IR movements among the ligand and the tested mixed complexes are given in. (Table 1 and Figure S5) The bands in region 3420 and 1640 &1591 cm⁻¹ in BIG ligand display the overlapping of the N₂ atoms (C–N–H & =N–H & C=N–C) of amine groups. BIG ligand performs as bi-dentate and tri-dentate ligands. Also, the bands at 473–562 cm⁻¹ were referred to ν (M–N) and the bands in the region of (615–654 cm⁻¹) are qualified to ν (M–O), which confirms coordination between the N₂ to the metals ions moiety. The changes in the position of (C–N–H & =N–H & C=N–C) functional groups indicate their coordination by metal salts that generally occurred after finishing intra-ligand H-bonding. Similarly, υ_as(OAc) , υ_s(OAc) vibrations in BIGICu and BIGIPd have been detected at 1563; 1267 cm⁻¹ and 1343; 1030 cm⁻¹, respectively. However sequentially, υ_as(NO₃) and υ_s(NO₃) have been practical at 1463 and 1281 cm⁻¹. The obtained data for separation (Δ = υ_as − υ_s) of two bands indicate he chelation of anions as mono-dentate for each ligand. However, vibrations of C-bonded in [acac], the υ C=O happen around 1622 cm⁻¹ in the case of BIGIVO [71,72] as presented in (Figure S1).

3.2.2. NMR spectra

The diamagnetic Pd(II) & Ag(I) mixed complexes and their related BIG ligand have been taken. For all studied BIG ligands in ¹H NMR, two groups of doublets in the range of δ 8.26–8.23 and 8.05–8.03 ppm for (2H, Ar) were detected. Three additional peaks at 10.65, 10.29 and 9.89 ppm were detected for [C=N–H & (C₂–N–H) benzimidazole and (C₂–N–H) free] respectively (Figure S2). Similarly, its ¹³C NMR spectral data of the free ligand, the (6C, Ar) displayed peaks at 137.90, 129.61 and 119.80 for ppm. Expressively, the (N–C=N, benzimidazole) and (C=NH imine in free ligand) carbon displayed two signals at 159.23 and 159.54 ppm (Figure 1).

Figure 1. ¹H NMR spectrum of the calculated BIG ligand.

¹H NMR spectral data of mixed complexes have been compared to display the resulting observations; signals of [C=N–H & (C₂–N–H) benzimidazole and (C₂–N–H) free] protons have been moved in the mixed complexes to δ 12.50, 10.52 and 10.26 ppm, and the appearance of a signal δ 9.51 corresponding to (s,1H, NH-imidazole ring). Also, other peak bands at δ 7.05–7.82 (d & t), 6.71(d) and 2.14 (s) ppm corresponding to (7H, ArH), (2H, NH₂) and (3H, OCH₃) in BIGIPd and shifted to δ 12.37, 11.18 and 10.42 ppm, and the signal of (NH, Imidazole) appeared at 9.64 δ ppm. Other peak bands at δ 7.72–8.14 (d & t) and 4.49 (d) ppm correspond to (7H, ArH) and (2H, NH₂) in BIGIAg. This suggests that the N₂ atom in BIG and Imidazole denote in coordination with the Pd(II) & Ag(I) ions as represented in (Figures S3 and S4).

3.2.3. Mass spectra

Mass spectra (MS) analysis permits the declaration of molecular structure weight for various complexes and specifies the existence of molecular structure weight of tested ligands in mixed complexes through the fragmentation band. The electron-effect MS analysis of the studied complexes through BIG is important to describe the tested mixed complexes because the BIG is a di or tri dentate, no distinct group or proton linked could be specific in IR to propose the locations of complexation. Tested complexes have been explored which offered as an illustration for the MS analysis.

The spectra analysis for the tested mixed complexes displayed a band related to the BIG free ligand. The molecular ion peaks of BIGICu, BIGIVO, BIGIAg and BIGIPd mixed complexes detected at (found (calculated)) 442.53 (442.5), 507.6 (507.60), 448.64 (448.86) and 467.37 (467.42) amu, respectively. MS for BIGICu molecular ion m/z (rel. intensity %) 71.16 M† (100%) and fragments at m/z 161.32 (79.43%), 236.43 (40.32%), 348.49 (13.23%) and 391.95 (6.06%). MS for BIGIVO molecular ion m/z (rel. intensity %) 80.04 M^† (58.87%) and fragments at m/z 149.16 (63.76%), 164.78 (57.73%), 350.42 (78.85%), 382.34 (100%), 411.60 (67.03%), and 492.35 (48.11%). MS for BIGIAg molecular ion m/z (rel. intensity %) 50.32 M^† (100%) and fragments at m/z 125.89 (47.9%), 279.59 (46.49%), 357.51 (37.13%) and 424.40 (24.51%). MS for BIGIPd molecular ion m/z (rel. intensity %) 54.51 M^† (100%) and fragments at m/z 152.44 (50.19%), 236.44 (36.50%), 365.49 (25.85%) and 407.43 (31.55%). The mass spectra analysis reinforced the suggested structures expected from elemental analysis, TGA, NMR spectra and molar conductivity. The mass spectra of tested mixed complexes are given (Figures 2 and S5 and Scheme 2) [73].

Figure 2. Mass spectra of the BIGICu mixed complex.

Scheme 2. Fragmentation pathway of BIGIPd. Below each structure, the precise masses of the particles and their chemical formulas are displayed.

3.3. Electronic spectra analysis

In the UV–vis spectra of the tested molecules (Figure S6), the representative BIG ligand band suffers from shifting due to fresh bands in the mixed metal complexes. This indicated the formation of metal complexes through the reaction of metal ions with the BIG and imidazole ligands [73]. The electronic spectra of BIGICu exhibited four peaks at 364, 396, 421 and 582 nm displaying the intra-ligand band, MLCT, T_2g—π* and ⁶A_1g→⁴T_2g transitions, respectively, the BIGIVO complex exposed peaks at 376 and 609 nm which relate to ⁶A_1g→⁴T_2g & T_2g→π* transition, suggesting octahedral geometry. The BIGIAg complex has a peak at 448 nm in UV–Vis spectra, related to the T_2g→π* and the BIGIPd complex has a peak at 407 and 516 nm, which correspond to T_2g→π* and ⁶A_1g→⁴T_2g transitions, which propose tetrahedral and square-planer geometry as represented in (Table 2) [74].

Table 2. Electronic transitions, λ_max (nm) and ε_max (dm³ mol⁻¹cm⁻¹) of BIG and its mixed ligand metal complexes.

Ligands and their complexes	λmax(nm)	εmax (mol^−1 cm^1 dm^3)	Assignment
BIG	293	1916	n→π*
	308	1911	π→π*
	447	560	T2g—π*
BIGICu	292	1899	n→π*
	306	1874	π→π*
	364	1056	Intraligand band
	396	1007	MLCT band
	421	949	T2g—π*
	582	199	^6A1g→^4T2g
BIGIVO	293	1423	n→π*
	310	1361	π→π*
	376	1375	Intraligand band
	609	222	^6A1g→^4T2g
BIGIAg	294	1471	n→π*
	306	1463	π→π*
	448	506	T2g—π*
BIGIPd	307	1280	π→π*
	407	1240	T2g—π*
	516	448	^6A1g→^4T2g

3.4. Thermal study

Thermal features of the BIG ligand and its mixed complexes have been estimated by TGA throughout repeated heating proportion (5°C min⁻¹) and below N₂ onto 50–800°C. The thermal behaviour of the tested mixed metal complexes offers deep visions into its structural properties by defining the number of coordinated and non-coordinated H₂O molecules. Once assessed to breakdown behaviour of the tested ligand, it was detected that they demonstrated, unlike behaviour depending on the methoxy or alkoxy substitute groups. The tested complexes exhibited four degradation steps (Scheme S1, Figure S7 and Table 3) showing temperature intermissions and the proportion of the decreases of the mass of the tested ligand and its metal complexes. The results were reliable with the records from the basic analysis. The results agree with the data from the basic study, the failure of the remaining carbon atoms and the residual metal causes the ending mass harm. The factors of kinetics and thermodynamic activation parameters for the studied mixed metal complexes have been evaluated through the Coats–Redfern and Eyring equations [75–77]. All stages of degradation followed the first-order rule. It has been observed that the (E*) value of the BIGIPd complex is more than other mixed ligand complexes, which specified that the BIGIPd complex is more stable than the other studied complexes and the residual part of the studied complex is relatively stable when moving from one stage to the next. The negative value for decomposition steps (ΔS*) showed that the initiated fragments require an extra construction-planned than the un-composed fragments and the behaviour of the rate for decomposition is slow; however, positive values display that the complaint of destroyed fragments produces considerably closer than the complaint of undegraded fragments. The thermal degradation is an endothermic behaviour, conferring positive ΔH values. All steps of degradation routes have been non-spontaneous performance agreeing with positive ΔG values. Moreover, when TΔS values increase, the ΔG values increase intensely for succeeding phases of degradation, signifying the consequent sort’s rate of destruction is more minor than the earlier ones.

Table 3. The degradation steps for TGA and thermo-kinetic activation parameters for tested mixed complexes.

		Fragment loss %		Weight loss %
Complexes	Temperature °C	Lost form	M. Wt.	Found	Calc.	E* (KJmol^−1)	A (S^−1)	ΔH* (KJmol^−1)	ΔG* (KJmol^−1)	ΔS* (Jmol^−1K^−1)
BIGICu	52–118°C	H2O	18	4.10	(4.06)	19.05	0.012	16.073	117.517	−283.361
	120–350°C	C7H10N2O4	186	41.90	(42.02)			14.82641	160.2524	−286.271
	352–560°C	CH4N3	58	13.18	(13.10)			12.98902	223.8706	−289.275
	562–700°C	C7H5N2	117	26.35	(26.43)			11.53407	274.6562	−291.064
Residue	> 702°C	Cu	63.55	14.42	(14.36)
BIGIVO	55–246°C	C5H7O2	99	19.52	(19.49)	19.496	0.011	15.98003	136.716	−285.427
	248–370°C	C8H11N2O2	167	32.80	(32.87)			14.6581	182.3214	−288.081
	372–575°C	CH4N3	58	11.48	(11.41)			13.2946	229.7432	−290.145
	577–700°C	C7H5N2	117	22.90	(23.03)			11.92279	277.7591	−291.807
Residue	> 700°C	VO	66.94	13.20	(13.17)
BIGIAg	48–120°C	H2O 2	36	8.10	(8.02)	13.75	0.01	10.78745	112.7086	−285.4933
	122–330°C	C3H4N3O3	130	28.89	(28.96)			9.606861	153.4576	−288.278
	332–520°C	CH4N3	58	12.99	(12.92)			7.944061	211.4094	−291.0806
	522–680°C	C7H5N2	117	26.02	(26.06)			6.489111	262.5174	−292.9386
Residue	> 680°C	Ag	107.868	23.95	(24.03)
BIGIPd	80–220°C	C2H3O2	59	12.69	(12.62)	29.43	0.017	25.90912	145.0122	−281.567
	222–400°C	C5H7N2O2	127	27.06	(27.17)			24.57057	190.5723	−284.249
	402–590°C	CH4N3	58	12.35	(12.40)			23.03248	243.3801	−286.537
	592–700°C	C7H5N2	117	24.98	(25.03)			21.78538	286.4755	−288.019
Residue	700°C	Pd	106.42	22.87	(22.76)

3.5. Stability range of pH and magnetic moments

The pH profile of the tested mixed complexes exhibited specific dissociation curves (Figure 3) and the stability range is high up to (pH = 4.5–10). A visible extensive steady range of pHs replicates the steadiness of studied mixed complexes than free ligands. Thus, the various applications on such mixed complexes over (pH = 4.5–10) range could occur without affection. Furthermore, the observed magnetic susceptibilities (μ_eff) of the tested mixed complexes (Table 1) have been planned to verify the structural geometry recommended. High-spin octahedral structural geometry is dispensed to the (BIGIVO & BIGICu) complexes (μ_eff = 1.81, 1.75 B.M, respectively) and the two diamagnetic mixed complexes (BIGIAg & BIGIPd) through their low-spin only moment situation [36,37,72].

Figure 3. pH profile of BIGI mixed complexes for 10^–3 M in DMSO at 298 K.

3.6. Ligand exchange method

A cover of the direct and other correction profiles of BIG complexes and Imidazole is applied to conclude the linking BIG complex to Imidazole ligand ratio (Figures 4 and S8). The stoichiometric proportion of the BIG complex to the Imidazole ligand has been 1:1. Assessment to extra-mole ratio techniques, the 1:1 proportion estimated for all BIG complexes through the Imidazole ligand is corresponding to the previous work [74] which depends on Job’s mole proportion techniques. Converstly, data of the Imidazole ligand with BIG complexes have been definite by applying Job’s mole ratio techniques. The Job’s schemes displayed a band at a mole of fraction equal to 0.5, while the curves of straight-line shares of the mole ratio profiles overlap at a value of 1. So, data from both tools offer a further verification of the 1:1 ratio evaluated through the ligand exchange techniques associated with Job and mole ratio techniques, these techniques receive some rewards: (1) they permit the study of the configuration of colourless complexes through a colorimetric method and the LED spot which is commercially accessible in best colorimeters (2) it needs fewer stages than used Job’s and the mole ratio techniques since a smaller number of ideas may be suitable to plan a conventional line than some ligands may be planned besides a single correction curve of the initial complex (3) the ligand exchange technique is extra exact more accurate than Job’s and the mole ratio techniques for the estimation of weak complexes, evaluating the mole ratio through these techniques is personal because of the profile lines. Various curves may be drawn intended for the identical group of ideas that may aim to false close although through the ligand exchange technique. (4) These techniques might be applied for various metals and for the estimation of mole ratios 1:1 [78] that specifies the generality of the technique and (5) Job’s and the mole ratio techniques may be applied if one of the tested starting material or the product complex is absorbed. In this situation, the ligand exchange may be the tool of choice [35,78].

Figure 4. Ligand exchange between (BIG metals and imidazole ligand) through Jop’s technique in DMSO at [imidazole] = [BIG metals] = 10^–3 M at 298 K.

3.7. DFT calculations

3.7.1. 3D geometry optimization

Figure 5 depicts the three-dimensional structures of the BIG ligand and the complexes that it forms in their optimal states. The optimization of the BIGICu and BIGIVO complexes led to the formation of an octahedral geometric structure around the core of the metal ion, as can be seen in (Figure 5). BIGIAg optimized into a distorted tetrahedral geometry around the Ag(I) ion, whereas BIGIPd optimized into a distorted square planar geometry around the Pd(II) ion, as shown in (Figure 5). The degree of distortion (T4) that was found to be present in the structure of the four-coordinate geometry was the subject of a number of calculations that were carried out [7]. The degree of distortion is equal to zero in a perfect square planar geometry, whereas it is equal to one in a perfect tetrahedral geometry. The results of finding the values 0.814 and 0.217 for the BIGIAg and BIGIPd, respectively, validated the distorted tetrahedral and distorted square planar geometries, as seen in (Figure 5).

Figure 5. Optimized structures of the studied chelates.

3.7.2. Global reactivity

The DFT approach was used for each and every calculation that was performed. One is able to characterize the three-dimensional structure of the compounds that have been investigated by making use of molecular structures that have been optimized via the use of theoretical approaches (see Figure 5). A molecular orbital analysis was performed on the optimized geometries at the levels of DFT/B3LYP (6-311G (d,p) and LANL2DZ), as shown in (Figure 6). On the optimized geometries shown in (Figure 5), investigation into the HOMO–LUMO orbitals (shown in Figure 6) and the molecular electrostatic potential (MEP) (shown in Figure 7) was carried out. In addition, the frontier molecular orbital energies were used to calculate quantum mechanical descriptors such as the energy gap (ΔE = LUMO–HOMO), ionization energy (IE = –HOMO), electron affinity (EA = –LUMO), electronegativity (EN = (IE + EA)/2), chemical potential (CP = –(IE + EA)/2), chemical hardness (CH = ((IE – EA)/2)), chemical global softness (S = 1/2CH), electronic charge (N_max = –CP/CH), and electrophilicity index (EP = CP2/2CH), [79,80] (Table S1).

Figure 6. HOMO–LUMO of the tested ligand and its mixed chelates.

Figure 7. MEP of the tested ligand and their metal chelates.

HOMO and LUMO energies are important parameters in understanding the electronic structure of molecules. They provide insights into various properties such as reactivity, charge transfer, and optical properties. The chemical properties that are laid forth in (Table S1) give the impression that every metal complex is more active than the free ligand. To be more precise, the BIGICu, BIGIAg, and BIGIPd compounds are the ones that have a more detrimental impact than the others.

Global reactivity parameters, such as energy gap, hardness, softness, electrophilicity, electronegativity, and the energies of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), are calculated to gain insights into the chemical reactivity of molecules. These parameters provide valuable information about the electron transfer processes, reactivity, and interactions of molecules, which are crucial for understanding their biological activities [81].

Energy Gap (ΔE): The energy gap represents the energy difference between the HOMO and LUMO orbitals. A smaller energy gap indicates higher reactivity of the molecule. Compounds with smaller energy gaps are generally more reactive and can readily undergo electron transfer processes, which may be relevant for biological activities such as enzyme inhibition, receptor binding, or redox reactions. When compared to the other compounds, the energy gap (ΔE) in all the metal complexes is less than that in the free ligand. This makes the entire metal complexes more reactive than the free ligand [81]. To be even more explicit, the following is the order in which the energy gap (ΔE) is ranked: BIGIPd > BIGIVO > BIGIAg > BIGICu > BIG, (Table S1). As a result, the reactivity trend will proceed in the other direction, as follows: BIGIPd > BIGIVO >BIGIAg > BIGICu>> BIG.

Hardness (η) and Softness (σ): Hardness is defined as the resistance of a molecule to changes in electron density, while softness is the reciprocal of hardness. Molecules with higher hardness values are less prone to electron transfer reactions, while softer molecules are more reactive. The softness parameter can provide insights into the susceptibility of molecules to undergo nucleophilic or electrophilic attacks, which are relevant in biological processes such as enzyme–substrate interactions or drug-receptor binding. As a direct consequence of this observation, the following reactivity rating was found: BIGIPd > BIGIVO ≈ BIGIAg > BIGICu>> BIG.

Electrophilicity (ω): Electrophilicity measures the ability of a molecule to accept electrons. It is related to the energy gap and chemical potential of the molecule. Electrophilic molecules are often involved in covalent interactions with biological macromolecules such as proteins or nucleic acids. Understanding the electrophilicity of molecules can help predict their reactivity and potential for forming covalent bonds with biological targets. To be more explicit, the following is the order in which the electrophilicity is ranked: BIGIPd > BIGICu > BIGIAg > BIGIVO >> BIG, (Table S1). Therefore, the predicted reactivity will take the same direction; B BIGIPd > BIGICu > BIGIAg > BIGIVO >> BIG.

Electronegativity (χ): Electronegativity indicates the ability of an atom to attract electrons in a chemical bond. Electronegativity influences the polarity and stability of chemical bonds, which are critical for molecular recognition and interactions with biological targets. Compounds with higher electronegativity may exhibit stronger interactions with biomolecules through hydrogen bonding or electrostatic interactions.

HOMO and LUMO Energies: The energies of the HOMO and LUMO orbitals determine the ionization potential and electron affinity of a molecule, respectively. The HOMO energy is related to the ability of a molecule to donate electrons, while the LUMO energy reflects its capacity to accept electrons. These parameters are relevant for understanding redox processes, electron transfer reactions, and the formation of reactive intermediates involved in biological pathways.

TD-DFT calculations were established for the optimized structures of ligand molecules and its complexes to estimate the UV-Vis absorption spectra, see Figure S9. The TD-DFT number of excitation states is selected to cover all the possible vertical transitions within the wavelength range from 200 up to 800 nm. Moreover, the maximum wavelength (λ_max) of absorption, the corresponding excitation energy (E), oscillator strength (f), and transitions (major contribution) had been evaluated. Figure S9 shows that the λ_max for the BIGIV complex is located at 475 nm with E of 1.872 eV, f of 0.221, and attributed to the electronic transitions HOMO-2 → LUMO + 1 (83%). Whereas, λ_max for the BIGIPd complex is located at 430 nm with E of 2.113 eV, f of 0.207, and attributed to the electronic transitions HOMO-1 → LUMO (65%). The BIGICu complex had λ_max of 540 nm with E of 2.054 eV, and f of 0.213, which attributed to the electronic transitions HOMO-1 → LUMO + 2 (74%). In the case of the BIGIAg complex, it had λ_max of 485 nm with E of 2.003 eV and f of 0.171, which attributed to the electronic transitions HOMO-2 → LUMO + 3 (65%).

Frequency analysis can be employed to determine the most stable structure of a complex by examining the vibrational frequencies. Generally, a stable structure will exhibit real-valued frequencies for all normal modes, indicating that the complex is at a minimum energy point on the potential energy surface. Theoretically predicted vibrational frequencies for the ligand and its complexes, are shown in Figure S10. The frequencies exhibited real-valued frequencies for all normal modes, indicating that the complex is at a minimum energy point on the potential energy surface.

3.7.3. Molecular electrostatic potential (MEP)

The mapping of the molecular electrostatic potential (also known as MEP) is a method that is particularly useful for researchers who are investigating the connection between the molecular structure and the physiochemical characteristics of the molecule [82]. The molecular electrostatic potential is an indicator of the net electrostatic impact created at a particular place in the space surrounding a molecule by the overall charge distribution of the molecule, which includes both the electrons and the nuclei. The dipole moments, electron negativity, partial charges, and chemical reactivity of the molecule are all correlated with this phenomenon. In addition to this, it provides a graphical depiction of the relative polarity of the molecules, which is a very useful feature [82]. The DFT/B3LYP method was used to assess the MEP. The projection of the surface of the MEP can be seen in (Figure 7). The regions of all molecules that exhibit a negative electrostatic potential are located near the atoms of oxygen, while the parts that exhibit a positive potential are positioned near the atoms of hydrogen, as can be seen in (Figure 7).

3.8. In-silico assay

3.8.1. MOE-docking simulation

Firstly, the docking procedure was validated using re-docking and superimposition methods. The native ligand of the target protein had been removed and re-docked into the active site. The re-docking was done to examine the docking procedure and efficiencies. The same methodology that was used previously was used in the re-docking process. The re-docked complex had been superimposed onto the native co-crystallized ligand (Figure S11).

By determining the different forms of interaction and the binding affinities, molecular docking research might potentially give a more in-depth understanding of how effectively novel drugs exert bioactivity against a target [83]. A docked complex, which was determined based on docking scores, was analysed according to binding interactions between the ligand and metal complexes with a target protein. This evaluation was carried out using [PDB ID: 2vf5; PDB ID: 3cku; PDB ID: 5IJT; PDB ID: 3gcw] [84] as the targets. The docking investigation demonstrated that the compounds that were synthesized feature non-covalent interactions, such as ionic, and hydrogen bonding interactions. All of the compounds, regardless of their docking score levels, showed promising outcomes when combined with the target proteins. In general, the docking site is more stable and the interaction between the docking site and protein receptor is stronger when the energy value (S) is more negative values [83].

In the case of 2Vf5 protein, the original ligand in the context of 2vf5 forms hydrogen bonds with VAL 399, ALA 602, THR 302, SER 303, GLN 348, SER 349 and THR 352 residues of the receptor. These interactions include hydrogen donor interactions with VAL 399 and ALA 602 and hydrogen acceptor interactions with THR 302, SER 303, GLN 348, SER 349, and THR 352. The binding energy of −8.30 kcal/mol suggests a strong interaction between the ligand and the receptor. The RMSD value of 1.01 Å indicates a very low deviation from the reference structure as shown in (Figure S11), the estimated docking score of the free ligand was determined at −4.52 kcal/mol. The estimated binding score of the BIGIVO, BIGICu, BIGIAg and BIGIPd metal complexes, on the other hand, was calculated at −8.42, −8.39, −8.33, −8.00 kcal/mol, respectively. When compared to the free ligand, the results showed that metal complexes had much higher docking scores. Because of this, the binding affinity of the free ligand is improved thanks to the changed metal complexes. More specifically, the BIGIVO complex showed the strongest binding affinity with a more negative docking score of −8.42 kcal/mol, followed by the BIGICu complex with a docking score of −8.39 kcal/mol. The free BIG ligand formed one H- acceptor bond with the following amino acid residues: N9 with ASN652, with bond distances of 2.86 Å. The BIGIVO complex formed four H-acceptor bonds via N17, N19, N20 and N33 with ASP474, ASP474, GLU569 and ALA520, respectively, with bond distances of 3.03, 3.10, 2.99, 2.92 Å and four ionic interactions by N19, N19, N20, and N30 with ASP474, GLU569, GLU569 and ASP474, with bond distances of 3.10, 3.89, 2.99, and 3.92 Å, (Table S2 and Figures 8 and S12).

Figure 8. 3D interactions between the studied ligand and their mixed complexes with the 2Vf5 receptor.

In the case of 3cku protein, the original ligand interacts with GLN 228, ARG 176, VAL 227, and GLN 228 residues through hydrogen bonding interactions. Additionally, pi-pi interactions are observed with PHE 159 residues. These interactions contribute to the stability of the ligand–receptor complex. The binding energy of −8.80 kcal/mol suggests a strong interaction between the ligand and the receptor. The RMSD value of 0.98 Å indicates a relatively stable complex with minimal structural deviation from the reference structure as shown in (Figure S11) and the estimated docking score of the free ligand was determined at −4.52 kcal/mol. The estimated binding score of the BIGICu, BIGIAg, BIGIPd and BIGIVO metal complexes, on the other hand, was calculated at −7.57, −7.11, −6.83 and −6.09 kcal/mol, respectively. When compared to the free ligand, the results showed that metal complexes had much higher docking scores. Because of this, the binding affinity of the free ligand is improved thanks to the changed metal complexes. More specifically, the BIGICu complex showed the strongest binding affinity with a more negative docking score of −7.57 kcal/mol, followed by the BIGIAg complex with a docking score of −7.11 kcal/mol. The free BIG ligand formed one H-donor bond with the following amino acid residues: N7 with GLU259, with bond distances of 2.92 Å. The BIGICu complex formed two H-donor bonds via N19, N33 with ASP175, LYS171, with distances of 3.29, 2.78 Å, respectively and one H-acceptor bond via O41 with GLU259, with a distance of 32.82 Å, in addition to two ionic interactions via N14, N19 with GLU259 and GLU259, with distances of 3.58 and 3.45 Å, respectively, (Table 4 and Figures S13 and S14).

Table 4. MOE results for the studied ligand and their complexes with 3cku.

	Ligand	Receptor	Interaction	Distance	E (kcal/mol)	S (kcal/mol)
BIG	N7	GLU 259	H-donor	2.92	−3.30	−4.52
BIGICu	N19	ASP 175	H-donor	3.29	−4.60	−7.57
	N33	LYS 171	H-donor	2.78	−9.70
	O41	GLU 259	H-acceptor	2.82	−4.00
	N14	GLU 259	ionic	3.58	−1.60
	N19	GLU 259	ionic	3.45	−2.10
BIGIVO	N24	THR 173	H-donor	3.00	−0.50	−6.09
	N40	TYR 257	H-donor	3.30	−1.70
	N23	GLU 259	ionic	3.05	−4.20
	N27	GLU 259	ionic	3.91	−0.70
	N37	GLU 259	ionic	3.34	−2.60
BIGIAg	N15	SER 154	H-donor	3.14	−0.80	−7.11
	N12	GLU 259	ionic	2.79	−6.10
	N15	ASP 175	ionic	3.97	−0.60
	N16	ASP 175	ionic	3.68	−1.30
	N35	GLU 259	ionic	3.45	−2.10
	5-ring	ARG 176	pi-H	4.13	−0.70
BIGIPd	O4	TRP 174	H-acceptor	3.24	−0.80	−6.83
	N16	GLU 259	ionic	3.15	−3.60
	N17	GLU 259	ionic	3.94	−0.60
	N20	GLU 259	ionic	3.23	−3.10
	N33	ASP 175	ionic	3.71	−1.20

In the case of the 5IJT protein, (Table S3 and Figure 9 and S15); the estimated docking score of the free ligand was at −4.66 kcal/mol. The estimated binding score of the BIGIAg, BIGIPd, BIGIVO and BIGICu metal complexes, was calculated at −7.20, −7.16, −7.07 and −6.93 kcal/mol, respectively. When compared to the free ligand, the results showed that metal complexes had much higher docking scores. Because of this, the binding affinity of the free ligand is improved thanks to the changed metal complexes. More specifically, comparing the BIGIAg complexes showed the strongest binding affinity with the more negative docking score of −7.20 kcal/mol, followed by the BIGIPd complex with a docking score of −7.16 kcal/mol. The free BIG ligand formed one H-donor bond between N12 with GLU170, with a bond distance of 2.91 Å. The BIGIAg complex formed three H-donor bonds via N15, N16 and C29 with GLU122, GLU122 and ASP145 with bond distances of 3.04, 3.13, 3.36 Å and two ionic interactions via N14, N20 with GLU122, GLU122, with bond distances of 3.14, 3.13 Å, in addition to one pi-cation interaction via 5-ring with LYS119 with a bond distance of 3.31 Å, (Table S3).

Figure 9. 3D interactions between the studied ligand and their mixed complexes with the 5IJT receptor.

In the case of 3gcw protein, (Table 5 and Figures S16 and S17), the estimated docking score of the free ligand was determined at −4.91 kcal/mol. The estimated binding score of the BIGIPd, BIGICu, BIGIAg and BIGIVO metal complexes was calculated at −7.96, −7.40, −7.08 and −6.79 kcal/mol, respectively. Compared to the free ligand, the results showed that metal complexes had much higher docking scores. Because of this, the binding affinity of the free ligand is improved thanks to the changed metal complexes. More specifically, comparing the BIGIPd complexes showed the strongest binding affinity with the more negative docking score of −7.96 kcal/mol, followed by the BIGICu complex with a docking score of −7.40 kcal/mol. The free BIG ligand formed one H-acceptor bond via N9 with ASN652 with bond distance of 2.86 Å. The BIGIPd complex formed two H-donor bonds via N14, N17, with TRP461, TRP461, with bond distances of 3.26, 2.99 Å and one H-acceptor bond via O4 with ARG458 with a bond distance of 3.16 Å, in addition to one ionic and one pi-H interactions via N30 and 5-ring with ASP360, PRO331, with bond distances of 3.86 and 3.53 Å, respectively (Table 5).

Table 5. MOE results for the studied ligand and their complexes with 3GCW.

	Ligand	Receptor	Interaction	Distance	E (kcal/mol)	S (kcal/mol)
BIG	N 9	ASN 652	H-acceptor	2.86	−4.1	−4.91
BIGICu	N14	CYS 358	H-donor	3.19	−3.60	−7.40
	N17	ARG 357	H-donor	3.16	−1.30
	N19	VAL 333	H-donor	2.99	−9.60
	N19	CYS 358	H-donor	3.51	−0.90
	N20	PRO 331	H-donor	3.15	−3.10
	N33	TRP 461	H-donor	2.80	−6.50
	O41	ARG 458	H-acceptor	2.97	−1.10
BIGIVO	N21	TRP 461	H-donor	3.02	−2.90	−6.79
	N24	ARG 357	H-donor	2.77	−1.80
	O6	ARG 458	H-acceptor	3.32	−1.50
	N37	ASP 360	ionic	3.68	−1.30
	O46	ASP 360	ionic	3.61	−1.50
	5-ring	PRO 331	pi-H	3.49	−1.20
	5-ring	VAL 460	pi-H	4.10	−0.60
BIGIAg	N13	VAL 333	H-donor	3.36	−2.20	−7.08
	N13	ASP 360	H-donor	3.03	−2.40
	N15	ALA 328	H-donor	3.54	−1.10
	N15	VAL 333	H-donor	3.22	−3.10
	N16	CYS 358	H-donor	4.40	−1.10
	N12	ASP 360	ionic	3.81	−0.90
	N15	ASP 360	ionic	3.37	−2.40
	N35	ASP 360	ionic	3.49	−1.90
BIGIPd	N14	TRP 461	H-donor	3.26	−1.50	−7.96
	N17	TRP 461	H-donor	2.99	−8.20
	O4	ARG 458	H-acceptor	3.16	−0.80
	N30	ASP 360	ionic	3.86	−0.80
	5-ring	PRO 331	pi-H	3.53	−1.30

The studied compounds were docked against COVID-19 protease (PDB ID 6lu7) and the best docking pose was obtained, the original ligand interacts with THR 190, GLU 166, GLN 189 and CYS 145 residues through hydrogen-bonding interactions. Hydrogen bond acceptor interactions are observed with GLU 166, HIS 163 and GLY 143 residues. Additionally, a pi-H interaction is noted with HIS 41 residues. These interactions contribute to the stability of the ligand–receptor complex. The binding energy of −8.71 kcal/mol suggests a strong interaction between the ligand and the receptor. The RMSD value of 1.04 Å indicates a relatively stable complex with slight structural deviation from the reference structure as shown in (Figure S11) and the estimated docking score of the free ligand was determined at −4.94 kcal/mol. The estimated binding score of the BIGIAg, BIGIVO, BIGICu and BIGIPd metal complexes was calculated at −7.32, −6.58, −6.46 and −6.02 kcal/mol, respectively. When compared to the free ligand, the results showed that metal complexes had much higher docking scores. Because of this, the binding affinity of the free ligand is improved thanks to the changed metal complexes. More specifically, the BIGIAg complex showed the strongest binding affinity with a more negative docking score of −7.32 kcal/mol, followed by the BIGIVO complex with a docking score of −6.58 kcal/mol. The free BIG ligand formed one H-donor and one pi-H interactions via N12 and 5-ring with GLU166 and GLN189, with bond distances of 3.05 and 3.55 Å. The BIGIAg complex formed one H-donor, one cation-pi, and one pi-H interaction via HIS164, HIS41 and GLN189, with bond distances of 3.36, 3.53 and 3.52 Å, respectively (Table S4 and Figures 10 and S18).

Figure 10. 3D interactions between the studied ligand and their mixed complexes with the COVID-19 [6lu7 receptor].

3.9. Biological performance

3.9.1. Interaction with CT-DNA

Binding with (CT-DNA) nucleic acid is the significant therapeutic object of various metallo-drugs, thus the contact study of tested molecules through DNA is significant for accepting the antiproliferative mode of action of metal-based drugs [85]. The tested compounds may bind to DNA helices in various techniques like (a) Intercalation among two neighbouring base pairs causing a hypochromism or hyperchromism (decrease or increase in absorption band), (b) electrostatic interaction through the attraction between the surface and the sugar (deoxyribose)- phosphate backbone, (c) major or minor groove binding as well as (d) covalent binding among the complexes and the nitrogen bases of the DNA double helix [86,87]. The CT-DNA interaction studies have been applied through absorption spectra, viscosity and gel-electrophoresis.

3.9.1.1. UV–visible absorption study

The absorption spectral features of the studied compounds were estimated through (UV–Vis) spectroscopy in the absence and and in the presence of CT-DNA, as presented in (Figures 11 & S19–S22). The data obtained recommended the intercalation of the tested molecules towards CT-DNA because of the decrease of transmittance strength (hypochromism) with red/blue shift. This hypochromism through or deprived of red/blue shift is related to the intercalative interaction through CT-DNA because of π–π* loading communication among the electrical situations for the intercalating chromosphere and the used DNA base pairs depending on the stability of the used DNA double helix [71,72]. The intrinsic binding constants (K_b) (Table 6) of the studied compounds have been estimated through Wolfe-Shimer Equation [88,89]. The intrinsic binding constants (K_b) have been ordered to be BIGIPd > BIGIAg > BIGIVO > BIGICu > BIG. The obtained data displayed that the BIGIPd mixed complex has the maximum binding affinity towards CT-DNA, for the other tested compounds. Moreover, the nucleobases uncomplicated in base coupling may encourage double-helical broadening and local perturbation that may afford exclusive small particle binding sites. The negative value for free energy indicates their spontaneous nature for interaction among the tested compounds and CT-DNA [58,59].

Figure 11. (a) UV–Visible spectra of BIGIPd complex in Tris–HCl buffer, pH = 7.4, 298 K after addition of CT-DNA in the absence (top) and presence of CT-DNA (lower) at [BIGIPd] = 1 × 10⁻³ mol dm⁻³ and [CT-DNA] = 0 –100 μM. (b) The plot of [[DNA]/ (ε_a–ε_f)] versus [DNA] for the titration of DNA with BIGIPd complex.

Table 6. Spectral factors for Ct-DNA binding with the studied complexes.

Complex	λmax Free (nm)	λmax bound (nm)	Δn	Chromism (%)	Type of chromism	Binding constant (Kb)	ΔG* KJ.mol ^−1
BIG	290	287	3	0.18	Hypo	1.154 × 103	−20.76
	440	439	1	22.86	Hypo
BIGIAg	280	281	1	3.72	Hypo	1.26 × 104	−27.81
	332	330	2	7.33	Hypo
	355	353	2	9.43	Hypo
	390	387	3	14.59	Hypo
	429	426	3	20.67	Hypo
	485	493	8	29.81	Hypo
BIGICu	276	277	2	13.18	Hypo	1.18 × 104	−27.60
	296	306	10	5.52	Hypo
	338	361	23	51.43	Hypo
	453	473	20	14.17	Hypo
BIGIVO	271	270	1	3.12	Hypo	1.130 × 103	−20.69
	288	286	2	2.7	Hypo
	323	365	42	73.3	Hypo
	469	471	5	12.87	Hypo
BIGIPd	265	272	9	16.88	Hypo	2.03 × 104	−29.21
	280	307	27	5.56	Hypo
	328	353	25	99.92	Hypo

3.9.1.2. Viscosity measurements

The binding mechanism of the tested compounds has been studied more through viscosity estimations, which are penetrating modifications in length are reflected as the best vital analysis to offer trustworthy sign-associated DNA-binding mechanisms of the studied mixed complexes in solution. In intercalation behaviour, the viscosity results for DNA are amplified since the tested compounds introduce between the nitrogen base pairs of CT-DNA, preferable to the elongation of the double-helix of DNA also may be reinforced through the hydrophobic nature. Furthermore, when using the non-intercalation mode of operation, a sharp decrease in viscosity is seen. Meanwhile, insufficient variations have been detected in the viscosity of DNA after the tested compound cooperated with DNA by (intercalation or groove or replacement) binding. The properties of the tested compounds upon the comparative viscosity of DNA are presented in (Figure 12) most tested compounds display comparable behaviour to ethidium bromide, so proposing that they probably bind to DNA through the intercalation mechanism. Upon growing the tested complex concentration, the relative viscosity of Ct-DNA grows gradually for tested complexes. The capacity of the studied complexes to enhance the viscosity of Ct-DNA is minor than which of the typical intercalator (DNA-ethidium bromide) as predictable and differ in the order BIGIPd > BIGIVO > BIGICu > BIGIAg. However, they need the opposed outcomes to the starting compounds that decline the viscosity of DNA. An outcome indicates that the occurrence of the tested complex may activate various DNA binding modes and an extra site of interacting through DNA strands. The results from viscosity estimations were in good agreement with UV-Vis absorption spectra [52,90]. Also, the Title compound had been docked against the DNA (PDB ID: 1dne), and the final complex and the interactions had been explored. The results depict interactions between specific ligands and DNA bases, shedding light on their binding properties. Ligands N14, N19 and N33 exhibit varying interaction types, including hydrogen bonding (H-donor) and ionic interactions, with different DNA bases. N19 forms strong hydrogen bonds with DC at position 11, evidenced by a short distance of 2.86 Å and a highly favourable energy of −8.4. This indicates robust binding affinity, potentially influencing DNA structural stability or functional dynamics. Additionally, N14 engages in an ionic interaction with DC at position 11, characterized by a slightly shorter distance of 2.79 Å and a notable energy of −6, suggesting a significant electrostatic attraction between the ligand and DNA. Overall, these results illuminate the diverse modes and strengths of ligand-DNA interactions, crucial for understanding molecular recognition and potential applications in drug design or biotechnology as shown in (Figure 13).

Figure 12. The effect of increasing the concentration of mixed complexes on the relative viscosities of CT-DNA at [DNA] = 0.5 mM at 298 K.

Figure 13. The BIGICu-DNA docking.

3.9.1.3. DNA-degradation analysis

Agarose gel electrophoresis is a broadly applied technique to examine the binding of studied compounds to DNA. After being exposed to an electric field, DNA moves to the anode since it is a negative sign. The strength of the electric field, the pH buffer, the concentration of the agarose gel and the size of the DNA all influence how DNA migrates. It was established that DNA size and mobility were frequently negatively related. The figure displays the various bandwidths and intensity stages of the bands associated with the control. We inspected the newly studied compounds binding through DNA by agar gel electrophoresis in addition to the observed results exhibited in (Figure S23) the variation in DNA binding affinities four the tested complexes have been expected to be the reason for the deviations in DNA-cleavage efficiency and has been qualified to their variance in the binding capacity to Ct-DNA. The cleavage of supercoiled DNA happens through the complexes using part degradation of the supercoiled method to affect the nicked system. The growing band intensity for the tested compounds is in a great arrangement per the data of the used CT-DNA-binding affinity (Table 6) [90].

3.9.2. Antimicrobial investigation

The antimicrobial behaviour of the tested compounds against different bacterial and fungal classes has been estimated employing the disc diffusion technique. Usually, the outcomes shown in (Figure 14 and S 24- S25) clarified that the tested complexes have promising antimicrobial features. The observed data for the studied chelating molecules can overturn the growth of bacteria after chelation with a range of transition metals, the antimicrobial performance for studied complexes was discovered in excessive detail. The formed molecules have extraordinary bactericidal features. The studied mixed complexes had stronger antimicrobial features in greater doses, as displays in Figure 14. The antimicrobial activity of studied compounds is ordered as follows: BIGIPd > BIGIAg > BIGICu > BIGIV > BIG. Furthermore, the mixed-ligand technique is pleasurable because of its design which allows different functional groups with variable binding sites and tightly binds to the metal ion forming stable mixed-ligand complexes. The higher antimicrobial activities of the metal complexes compared to the studied ligands may be due to the changes in structure that occur due to coordination and chelation that causes the metal complexes to act as more powerful antimicrobial agents, thus killing the microbe or by inhibiting multiplication of the microbe by blocking their active sites. Such increased activity of the complexes can be explained based on the Overtone concept and the Tweedy chelation theory. According to the Overtone concept of cell permeability, the lipid membrane surrounding the cell favours the passage of only lipid-soluble materials, due to which lip solubility is an important factor controlling the anti-microbial activity. On complexation, the polarity of the metal ion will be reduced to a greater extent due to the overlap of the ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Furthermore, the mode of action of the compound may involve the formation of a hydrogen bond through the azomethine group with the active centre of cell constituents, resulting in interference with normal cell processes. Due to the presence of an electron-withdrawing group in the ligand, the positive charge of the copper ion increases, and this enhances the ability of the copper ion to interact with DNA. Free ligands have N₂ donor locations that are active towards the bio-potential actions the improved biological performances because of the occurrence of N₂ donors. However, it is interesting to detect that the activity index has been calculated by the following equation (Tables 7–9) once biological behaviour experiences chelation with various metal ions, it enhances in comparison to the observed standards. Depending on the base of the chelation theory, this enhanced action of the tested metal complexes may be clarified. The penetrability of the cell membrane and the lipid structure of the tested microbial cells’ cell walls qualifies the calmer path of soluble molecules over the cell which is a vital side of considering antimicrobial behaviour effectiveness. This proposed that complexation may simplify metal complex diffusion through the lipid layer of the cell membrane to the place of action (Tables 7 and 9) and provide extents of the studied compounds’ performance indices [36,56,58,59]. Our compounds under investigation are more potent than other compounds in the literature against different pathogens [63] (I) $\begin{array}{rl}& \text{Activity index}\\ & =\frac{\text{Inhibition zone of compound }\left(\text{mm}\right)}{\text{Inhibition zone of standard drug }\left(\text{mm}\right)}\times 100\end{array}$(I)

Figure 14. Relative antibacterial performance of the BIG ligand and its mixed ligand complexes with imidazole at 15 and 25 (µg/ml) against E. Coli are denoted in the chart.

Table 7. Minimum inhibition zone (MIC) for antimicrobial assay of the prepared BIG ligand and its mixed ligand metal complexes with imidazole.

	bacteria			Fungi
Compounds	S. typhimurium (–ve)	B. cereus (+ve)	E. coli (–ve)	A. flavus	F. oxysporum	G. candidum
BIG	7	6	7.50	7.75	6.75	5.25
BIGICu	4	3	4.25	4	4.75	3.25
BIGIVO	4.25	3.50	4.75	4.25	5.25	3.75
BIGIAg	3.50	2.50	3.25	3.75	3.25	2.75
BIGIPd	2.50	1.75	3	3.25	2.75	2
PDBSFe [63]	–	4.25	–	4.50	4.00	4.25
CPBSFe [63]	–	3.50	–	3.75	3.00	3.50

Table 8. Activity index for antifungal and antibacterial-assessments of BIG and its mixed complexes against selected bacteria and fungi.

	Activity index (%)
Comp	S. typhimurium (–ve)	E. coli (–ve)	B. cereus (+ve)	A. flavus	G. candidum	F. oxysporum
BIG	37.11	39.45	44.91	42.02	43.23	57
BIGICu	88.24	85.71	89.51	82.17	90.86	83.97
BIGIVO	82.86	84.69	88.13	79.17	88.43	81.24
BIGIAg	90.93	88.77	92.58	87.05	93.52	88.79
BIGIPd	95.60	94.89	95.65	93.24	95.37	93.44

Table 9. Inhibition zone for anti-microbial test of the studied BIG and as its mixed complexes against several strains of [bacteria and fungi].

	Inhibition zone (mm)± SD
Compounds	Salmonella typhimurium (–ve)		Escherichia coli (–ve)		Bacillus cereus (+ve)		Aspergillus flavus		Getrichm candidum		Fusarium oxysporum
Conc.(µg/ml)	15	25	15	25	15	25	15	25	15	25	15	25
BIG	7.5 ± 0.11	13.1 ± 0.08	6.25 ± 0.12	11.6 ± 0.07	10.15 ± 0.04	21.2 ± 0.05	6.25 ± 0.14	11.2 ± 0.10	10.3 ± 0.06	18.7 ± 0.10	9.55 ± 0.12	17.75 ± 0.10
BIGICu	14.55 ± 0.13	31.15 ± 0.16	13.65 ± 0.08	25.2 ± 0.14	22.1 ± 0.13	42.25 ± 0.06	12.7 ± 0.09	21.9 ± 0.14	21.55 ± 0.14	39.3 ± 0.04	14.2 ± 0.14	26.15 ± 0.15
BIGIVO	13.15 ± 0.14	29.25 ± 0.20	12.7 ± 0.16	24.9 ± 0.05	21.45 ± 0.16	41.6 ± 0.14	11.3 ± 0.12	21.1 ± 0.18	20.5 ± 0.11	38.25 ± 0.16	13.50 ± 0.15	25.30 ± 0.15
BIGIAg	15.90 ± 0.09	32.10 ± 0.14	14.15 ± 0.15	26.10 ± 0.08	23.3 ± 0.17	43.7 ± 0.12	13.5 ± 0.16	23.2 ± 0.15	22.65 ± 0.15	40.45 ± 0.12	15.10 ± 0.15	27.65 ± 0.09
BIGIPd	16.85 ± 0.13	33.75 ± 0.15	15.6 ± 0.14	27.9 ± 0.11	24.10 ± 0.12	45.15 ± 0.15	14.6 ± 0.16	24.85 ± 0.08	23.75 ± 0.14	41.25 ± 0.18	16.35 ± 0.16	29.10 ± 0.09
DMSO	–	–	–	–	–	–	–	–	–	–	–	–
Ofloxacin	18.70 ± 0.16	35.30 ± 0.15	17.3 ± 0.11	29.4 ± 0.11	26.50 ± 0.14	47.2 ± 0.12
Fluconazole							16.55 ± 0.15	26.65. ± 0.08	25.35 ± 0.07	43.25 ± 0.14	18.30 ± 0.13	31.14 ± 0.21

3.9.3. Antiproliferative activities

Three cell lines of HCT-116, Hep-G2 and MCF-7 have been treated to identify the antiproliferative behaviour of the tested ligand. Between all studied complexes a powerful cytotoxic effectiveness was applied beside all cancer cell lines with the lowest IC₅₀ value of 5.05 μg/μL. Alternatively, tested complexes displayed high to enough inhibitory outcomes to the propagation of studied human cancer cell lines with IC50 values represented in (Figure 15 and Table S5). The data exhibited that studied mixed complexes are more active than the prepared ligand. By likening the findings of studied molecules using additional prepared transition metal complexes against [MCF-7 & HCT-116 & Hep-G2 cancer cell line it should be noted that mixed-ligand complexes have a key role in biological chemistry because mixed chelation occurs commonly in biological fluids as millions of potential ligands are likely to compete for metal ions in vivo. These create specific structures and have been implicated in the storage and transport of active substances through membranes. In addition mixed complexes easily form octahedral complexes with most transition metal cations. Benzimidazole is an antiproliferative agent because of its heteroaromatic planar and hydrophobic structure. In addition this chelating agent shows better antiproliferative activity through chelation with metal ions. This will be more permeable through cell membranes eventually behaving as carriers of antiproliferative agents. This indicated an improvement in the antiproliferative potency upon coordination. The improvement of cytotoxic potency may be due to the positive charge of the metal increasing the acidity of coordinated ligand that bears protons causing stronger hydrogen bonds which enhance the biological activity. It seems that changing the coordination sites and the nature of the metal ion has a clear effect on biological activity by altering the binding ability of DNA. The mixed complexes have been prepared from bidentate or tridentate BIG ligand as a primary ligand and Imidazole (I) as a secondary ligand. This is because of the presence of a metal-redox-active core. Also the tested mixed complexes have been more susceptible to cytotoxicity towards MCF-7 associated with the various cancer HepG-2 & HCT-116 cell lines. These findings revealed that BIGIPd is the maximum effective antiproliferative agent for selected cancer cell lines signifying which solubility and stability in living media could be the chief aspects. We recommend that redox probably shows a significant role also the performance could be because of binding with DNA and this behaviour may be because which studied compounds are more lipophilic and their crossing with the cell membrane is dissimilar. This shows that growing the aliphatic series and adding aromatic rings later in the chain develops this biological performance. Hydrophobic assets convert more vital directly inducing biological performance and described through the transference of the drug towards the place of action68,72,91].

Figure 15. IC50 results for the studied BIG ligand and its mixed ligand complexes vs. [HepG-2 & HCT-116 & MCF-7].

3.9.4. The antioxidant activities

The antioxidant performance for the studied ligand and mixed complexes has been estimated related to the radical-scavenging technique through a reference DPPH free radical. The IC₅₀ data are the dose of the studied complexes essential to scavenge 50% of DPPH free radicals. They calculated variations in the free radical-scavenging capability of the transition metals on the source of the % of inhibition agreed to (Figure 16 and Table S6) and also the IC₅₀ observed data for the investigated complexes. Once the dose of the reaction mixture is dropped, the free radical-scavenging movement is advanced. The IC₅₀ data exhibited that the prepared complex is a powerful free radical scavenger converted to the reference ascorbic acid [58,59,65,92]. The computed IC₅₀ values for various substances with other related studies in literature compounds [MnL (AcO).H₂O].2H₂O (C₁₄H₁₇O₆N₅Mn), [CoL(H₂O)₃]. Cl (C₁₄H₁₅O₄N₅ClCo), [NiLCl].2(H₂O) (C₁₂H₁₂O₃N₅NiCl) and [ZnL(H₂O)₂Cl] (C₁₂H₁₂O₃N₅ZnCl) had stronger activity against DPPH than commercially available Vit. C (standard) with IC₅₀ values of 23.69, 17.15, 27.68, and 38.53 g ml⁻¹, respectively [92]. Also, IC₅₀ values for PDBSFe and CPBSFe complexes are equal to 22 and 32 g ml⁻¹, respectively [63].

Figure 16. Concepts in inhibition for [DPPH] radical about free ligand and its corresponding coordination compounds.

4. Conclusions

This study has synthesized a series of mixed ligand complexes and comprehensively characterized their structural and spectroscopic properties. The investigation into these complexes has yielded valuable insights into their potential biological applications. Through detailed spectroscopic analysis, including UV-Vis, IR, mass spectra NMR spectroscopy and theoretical studies, we have elucidated the coordination modes of the ligands and metal ions in the synthesized complexes. This characterization has provided a solid foundation for understanding the structural features and coordination geometries of the complexes, essential for further exploration of their biological activities. Moreover, our biological assays have revealed promising preliminary results regarding the potential pharmacological activities of these complexes. The observed interactions with biomolecules, such as DNA or proteins, suggest potential applications in areas such as antiproliferative, antioxidant and antimicrobial therapy. Further studies are warranted to explore the mechanisms of action and optimize the efficacy of these complexes for specific therapeutic purposes. All extracted factors or graphs indicate the advantage of the Pd(II) complex in pharmaceutical applications. Also, docking studies have offered valuable predictions regarding the interactions between the synthesized complexes and biologically relevant targets. By simulating the binding interactions at the molecular level, we have identified potential binding sites and elucidated the mechanisms of action underlying the observed biological activities. In the future, we will focus on elucidating the structure–activity relationships of these mixed ligand complexes, exploring their interactions with biological targets at the molecular level, and optimizing their pharmacokinetic properties. Additionally, efforts to enhance the stability and selectivity of these complexes while minimizing cytotoxicity are essential for their translation into clinical applications.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All relevant data are within the manuscript and available from the corresponding author upon request.