Synthesis, Characterization, Biological Activity, Cytotoxicity (MTT Assay), and Antioxidant Evaluation of a Novel Ligand (BHDPE) and Its Ni(II) Complex

INTRODUCTION
Benzoxazoles are heterocyclic aromatic organic compounds consisting of a benzene ring fused to an oxazole ring. They have the molecular formula C7H5NO and typically exist as white to pale yellow crystalline solids. These compounds exhibit a relatively low melting point (30–33 °C) and are sparingly soluble in water, yet readily soluble in common organic solvents such as ethanol [1]. Benzoxazole can be synthesized via a straightforward reaction of o-aminophenol with carboxylic acid in the presence of polyphosphoric acid (PPA) as a catalyst [2]. Numerous benzoxazole derivatives have been employed in pharmaceutical applications owing to their diverse pharmacological properties, including antibacterial, antidepressant, antispasmodic, antimicrobial, antifungal, and cardiovascular activities, as well as their utility as anesthetic agents[3] Benzoxazole has been frequently incorporated into Schiff base frameworks, which feature the characteristic azomethine (C=N) functional group[4], Schiff bases were first reported by the German chemist Hugo Schiff through the condensation reaction of primary amines with’ aldehydes or ketones [5]. Recent studies have demonstrated that sulfur-promoted oxidative rearrangement coupling between o-aminophenols and ketones provides an efficient route for the synthesis of 2-alkylbenzoxazoles under mild conditions [6]. 

MATERIALS AND METHODS
All chemicals and reagents were procured from Aldrich, Merck, HIMEDIA, and BDH, and were used without further purification. UV-Vis spectra in the 200–1000 nm range were recorded using a Shimadzu UV-165PCS spectrophotometer. 1H, C13 -NMR spectra were acquired at 300 MHz on the Fourier Transform Varian Spectrometer in DMSO-d6. FT-IR spectra were recorded in the 400–4000 cm⁻¹ range using a Shimadzu FT-IR 8400S spectrophotometer. Melting points were determined using a Stuart melting point apparatus. Magnetic susceptibility measurements were performed at room temperature using a Magnetic Susceptibility Balance (MSB-MKI). Metal content in the complexes was determined by flame atomic absorption spectroscopy (Shimadzu AA-6300), and elemental analysis (C, H, N) was performed using a Shimadzu EA-300 elemental analyzer. 

Preparation of The Ligand
The ligand (BHDPE) was synthesized in two sequential steps. In the first step, 2-hydrazinylbenzoxazole (compound A) was prepared by dissolving 2-mercaptobenzoxazole (2.00 g, 13.2 mmol) in absolute ethanol (25 mL), followed by the dropwise addition of hydrazine hydrate (0.66 g, 13.2 mmol) dissolved in absolute ethanol (25 mL). The reaction mixture was heated under reflux for 10 h, then cooled to room temperature, filtered, and the precipitate was collected. The crude product was recrystallized from absolute ethanol, dried under vacuum, and collected, affording compound A in 85% yield (m.p. 169–171 °C). The ligand (E)-2-(2-(benzo[d]oxazol-2-yl) hydrazone)-1,2diphenylethan-1-ol (BHDPE) was made by dissolving (1.3 gm, 8.71 mmole) of the A-compound in 25 ml of ethanol with constant stirring. A solution of benzoin (1.85 g, 8.71 mmol) in ethanol (25 mL) was then added, along with 5–6 drops of concentrated hydrochloric acid as catalyst, and the mixture was refluxed for 8 h before being cooled to room temperature. The resulting precipitate was filtered, dried, and recrystallized from absolute ethanol. As shown in Fig. 1, The product had an 83% yield and a melting point of (BHDPE) ligand was 133 0C. 

Synthesis of the Ni (II) Complex for (BHDPE) ligand
The Ni(II) complex was prepared in a 1:2 (M:L) molar ratio according to the following procedure. A solution of BHDPE ligand (0.13 g, 0.37 mmol) in absolute ethanol (10 mL) was added dropwise to a solution of nickel(II) chloride (0.185 mmol) in absolute ethanol (10 mL), and the resulting mixture was refluxed with continuous stirring for 2 h. The mixture was subsequently cooled, filtered, and the product was dried and recrystallized from absolute ethanol.

Measuring antioxidant capacity
The antioxidant activity of the synthesized compounds was evaluated against the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). This was accomplished by dissolving 1 mL of each compound in DMSO at various concentrations (3.9–500 μg/mL). Subsequently, 1 mL of 0.1 mM DPPH solution in methanol was added to each test solution of the BHDPE ligand and Ni(II) complex. The reaction mixtures were incubated at room temperature in the dark for 30 min. A color change from violet to light yellow was observed, indicating radical scavenging activity. The absorbance was measured at 517 nm using a UV-Vis spectrophotometer. Next, each compound’s RSA% (percent radical scavenging activity) is computed. The substances are contrasted with ascorbic acid dissolved in DMSO at equivalent concentrations [7]. 

Antibacterial activity
The antibacterial activity of the synthesized compounds was evaluated against selected bacterial strains. The test organisms included the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, which are among the most commonly encountered pathogenic bacterial species. Stock solutions of all synthesized compounds were prepared by dissolving them in dimethyl sulfoxide (DMSO) at a concentration of 1000 ppm, followed by serial dilution with DMSO. Additional concentrations of 250 and 500 ppm were prepared. The agar well diffusion method was employed using Mueller-Hinton agar (MH) (Difco, Detroit, MI). The medium was used for culturing the bacterial strains. Under sterile conditions, 100 μL of each prepared compound solution in DMSO (1%) was dispensed into the wells using calibrated micropipettes. An equivalent volume of DMSO was used as the negative control. Inhibition was measured in millimeters after a 37°C incubation period. The activity then is measured and recorded [8]. 

Molecular docking
To anticipate the target protein binding affinity and method of interaction of synthesised drugs, molecular docking was used. This computer method replicates ligand orientation in the receptor active region, estimating binding free energy (ΔG) and identifying critical chemical interactions. Docking experiments show specific ligand binding conformations, emphasising the stability of complexes through hydrogen bonding, π–π stacking, van der Waals forces, and electrostatic interactions. Reduced binding energy indicated stronger and more favourable ligand-target interactions, implying increased biological activity. All things considered, the docking investigation validated the produced compounds’ promise as attractive candidates for medicinal uses and offered insightful information about their structure–activity relationship (SAR) [9].

Computational Details
DFT Calculations
All density functional theory (DFT) calculations were performed using the Gaussian 16 software package [10]. The molecular geometry of the BHDPE ligand was fully optimized at the B3LYP/6-311++G(d,p) level of theory in both gas phase and ethanol solution. For the Ni(II) complex, the effective core potential (ECP) basis set LANL2DZ was employed for the nickel atom, while the 6-311++G(d,p) basis set was used for all other atoms (C, H, N, O). Solvent effects were incorporated using the Polarizable Continuum Model (PCM) with ethanol as the dielectric medium. Frequency calculations were performed at the same level to verify that all optimized structures correspond to true energy minima (no imaginary frequencies) and to obtain the theoretical IR vibrational frequencies for comparison with experimental FTIR data [11]. A scaling factor of 0.9688 was applied to the computed harmonic frequencies to account for anharmonicity and basis set incompleteness.
Frontier molecular orbital analysis (HOMO-LUMO) was performed to evaluate global reactivity descriptors including the energy gap (ΔE), ionization potential (IP), electron affinity (EA), chemical hardness (η), chemical softness (S), electronegativity (χ), electrophilicity index (ω), and chemical potential (μ) [12]. The Molecular Electrostatic Potential (MEP) map was generated at the B3LYP/6-311++G(d,p) level to identify electrophilic and nucleophilic regions on both the ligand and complex surfaces. Natural Bond Orbital (NBO) analysis was conducted to evaluate the nature of coordination bonds (N→Ni, O→Ni), charge transfer interactions, and stabilization energies within the complex [13]. Fukui function indices (f⁺, f⁻, f⁰) were calculated using the finite difference approach based on Natural Population Analysis (NPA) charges to identify sites susceptible to electrophilic and nucleophilic attack, providing insight into antioxidant radical-scavenging mechanisms. Time-dependent DFT (TD-DFT) calculations at the B3LYP/LANL2DZ/6-311++G(d,p) level were performed to predict electronic absorption spectra for comparison with experimental UV-Vis data [14]. Thermodynamic parameters (ΔH, ΔG, ΔS) for the complexation reaction were computed at 298.15 K and 1 atm.

Molecular Dynamics Simulations
Molecular dynamics (MD) simulations were performed using the GROMACS 2023.3 software package [34] to evaluate the dynamic stability of the BHDPE ligand and its Ni(II) complex within the binding site of the target protein (PDB ID: 3FC2). The initial protein-ligand complexes were obtained from the best-ranked molecular docking poses generated by MOE. The CHARMM36 force field [15] was used for the protein, while ligand topologies were generated using the CGenFF (CHARMM General Force Field) server with parameters validated against quantum mechanical calculations [16]. For the Ni(II) complex, the bonded model approach was employed with metal-ligand bond parameters derived from DFT-optimized geometries. The TIP3P water model was used for solvent molecules.
Each protein-ligand complex was placed in a cubic simulation box with a minimum distance of 1.2 nm between the solute and box edges, solvated with approximately 12,000 TIP3P water molecules, and neutralized with Na⁺/Cl⁻ counterions at 0.15 M physiological ionic strength. The simulation protocol comprised: (1) energy minimization using the steepest descent algorithm (50,000 steps, convergence criterion: 10 kJ/mol/nm); (2) NVT equilibration at 310 K for 1 ns using the V-rescale thermostat (τ_T = 0.1 ps); (3) NPT equilibration at 310 K and 1 bar for 2 ns using the Parrinello-Rahman barostat (τ_P = 2.0 ps); and (4) production MD for 100 ns under the NPT ensemble. Particle Mesh Ewald (PME) summation was used for long-range electrostatics with a real-space cutoff of 1.2 nm. All bonds involving hydrogen atoms were constrained using the LINCS algorithm, permitting a 2 fs time step [17].
Post-simulation analyses included: root-mean-square deviation (RMSD) of protein backbone and ligand heavy atoms; root-mean-square fluctuation (RMSF) of protein residues; radius of gyration (Rg) to assess protein compactness; hydrogen bond analysis between ligand and protein residues using geometric criteria (donor-acceptor distance ≤ 0.35 nm, angle ≤ 30°); and solvent-accessible surface area (SASA) analysis. Binding free energy was calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method implemented in the g_mmpbsa tool [18], decomposed into van der Waals, electrostatic, polar solvation, and non-polar solvation contributions. Per-residue energy decomposition was performed to identify key protein residues contributing to ligand binding. All visualization was performed using VMD 1.9.4 [19].

RESULTS AND DISCUSSION
The novel ligand (E)-2-(2-(benzo[d]oxazol-2-yl)hydrazono)-1,2-diphenylethan-1-ol (BHDPE) was synthesized through the reaction of hydrazine hydrate with 2-mercaptobenzoxazole, followed by condensation with benzoin. The corresponding Ni(II) complex was prepared by reacting the BHDPE ligand with nickel chloride in an ethanolic medium. The physical properties and elemental analysis data are summarized in Table 1. The Ni(II) complex was prepared in a 1:2 metal-to-ligand molar ratio.

1H and 13C-NMR Spectra of the (BHDPE) Ligand
In the ¹H NMR spectrum of the BHDPE ligand, the methine proton (CH–OH) appeared as a singlet at δ = 4.005 ppm (s, 1H) in the 1H-NMR spectrum of the ligand (BHDPE), while the hydroxyl group proton exhibited a singlet at δ = 5.677 ppm (s, 1H) [20]. The sign also appeared (s, 1H, δ = 8.798 ppm) due to the protons of the hydrazide [21]. Multiplet signals at δ = 7.286–7.335 ppm (m, 5H) and δ = 7.945–8.537 ppm (m, 5H) were attributed to the aromatic protons of the benzoin moiety [22], while additional multiplets at δ = 7.396–7.728 ppm (m, 4H) were assigned to the benzoxazole ring protons [23]. The 13C-NMR Spectrum of the (BHDPE) Ligand, shown signal at (70.321 ppm), which belongs to carbon (C12) due to the group (CH-OH), the signal at (ppm 147.968) is belonging to the group azomethane carbon (-C = N-) (C14) [24]. Several signals at (139.805-149.177-111.175-121.234 - 123.883 -116.394- 160.911ppm) and [(127.815-129.015-139.31ppm), (128.619-127.074-131-578ppm)] indicated to the carbon atoms (C6-C7-C8-C9-C10-C11-C4) of the benzoxazole ring, carbon atoms (C15-C20) and (C21-C26) of carbon atoms belong to the of Benzoin respectively [25], As shown in Fig. 2.

The Infra-Red Spectra
The FT-IR spectrum of the free ligand was recorded using a Shimadzu 8400S spectrophotometer with KBr pellets. The most prominent absorption bands were observed at 3375 and 3304 cm-1) corresponding to the O–H and N–H stretching vibrations, respectively [26]. The azomethine (C=N) stretching vibration of the Schiff base appeared at 1676 cm-1) in the ligand spectrum, and whose existence on the composition of the ligand is the band of carbonyl group in benzoin, which disappears before the reaction [27]. Other bands appeared at (3061 cm-1) and (2872, 2820 cm-1), respectively, due to (C-H) aromatic and aliphatic groups, while the groups (C = N) and (C-O) of the benzoxazole ring and (C = C) aromatics provided bands at (1612 cm-1) and (1151 cm-1), respectively [28].

The Infra-red Spectra of the Ni (II) Complex
Comparison of the FT-IR spectrum of the Ni(II) complex with that of the free BHDPE ligand revealed that the N–H stretching frequency shifted to lower wavenumbers, while the azomethine (C=N) absorption shifted to higher frequencies’ absorption was shifted to lower frequencies than the ligand spectrum [29], while The azomethane groups’ absorption was shifted to higher frequencies than the ligand spectrum [30], Due to coordination with the metal ion and proton loss, the hydroxyl group (O-H), which generated a band at (3375cm-1) in the free ligand spectrum, vanished in the spectra of the complex [31]. New bands occurred in the groups (M-N) and (M-O), respectively, at (516-586 cm-1) and (420-472 cm-1) (Fig. 3) [32]. The presence of these bands suggests ligand coordination with metal ions by azomethane and Secondary amine groups, as well as coordination of the oxygen atom group hydroxyl after proton loss, and finally the appearance of bands at (3313cm-1) due to group of hydroxyl molecules of water crystallization in the complex [33]. 

Electronic spectra
The UV–Vis spectrum of the free BHDPE ligand displayed three absorption bands: two bands at 201 nm (49751 cm-1) and 230 nm (43478 cm-1), which belong to π-π* of the benzoxazole and phenyl rings. The third band at 350 nm (28571 cm-1) refers to the transition of n-π* of the azomethine groups of Schiff base and the benzoxazole ring [34].
The Electronic Spectra of the Synthesized the Ni (II) Complex.
The nickel (II) complex spectrum includes several peaks, three of which are at 255 nm (39215 cm-1), 329 nm (30395 cm-1) These peaks due to the free ligand field, and the peak absorption at 490 nm (20408 cm-1) 599nm (16694 cm-1). they refer to transitions (3A2g(F) → 3T2g(F), 3A2g (F) → 3T1g (F) respectively, confirming the distorted octahedral geometry of the Ni(II) complex  has the geometry of distorted octahedral(sp3d2), and μeff = 2.82 B.M (Fig. 4) [35]. 

Molar conductivity
Molar conductivity is a fundamental technique for determining the electrolytic nature and ionic character of metal complexes in coordination chemistry. The molar conductance of a complex in solution is proportional to the number of free ions present; accordingly, high conductivity values indicate ionic character, while low values suggest a non-electrolytic nature. The Ni(II) complex has non-ionic properties and has a value of 12.8 Λ (ohm-1, cm2, mole-1). [36]. 

Scanning Electron Microscopy (FESEM)
The surface morphology, particle shape, and crystalline structure of the BHDPE ligand and its Ni(II) complex were investigated using field emission scanning electron microscopy (FESEM). The micrographs revealed spherical nanoparticle morphology.[37] The average particle size of the spherical ligand is 49.179 nm, whereas the average particle size of the spherical nickel (II) complex is 49.089 nm. as seen in Fig. 5.

Biological Activity
The antibacterial activity of the BHDPE ligand and its Ni(II) complex was evaluated against two representative pathogenic bacteria: the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. Using DMSO as the solvent, the results indicated that the ligand exhibited significant inhibitory activity against E. coli. Also, the nickel-II complex showed high effectiveness against S. aureus. Which is illustrated in Fig. 6.

Antioxidants
The antioxidant activity of the synthesized compounds was evaluated in this study. According to the findings of antioxidant investigations, based on the physical, analytical and antioxidant activity data, it was shown that the ligand (BHDPE) has a multi-bonded organic structure that often contains (-OH and -NH or -C=O) groups and exhibits the ability to donate electrons and react with free radicals [38]. When the complex [Ni (BHDPE)₂] ·H₂O is formed, nickel enters into coordination with the N and O atoms in the ligand, which reduces the electron density on these centers and weakens their ability to react with free radicals (such as DPPH). Therefore, the free ligand is more effective than the metal complex. This pattern is very common in antioxidant biochemistry: metal binding typically reduces activity due to the reduction of the negative charge available for electron donation. Nickel (Ni (II)) has an electrophilic nature, so it withdraws energy from the N and O atoms in the ligand, reducing their activity as electron donors. The fitting parameters from the four-parameter logistic (4PL) regression model applied to the DPPH radical scavenging assay are displayed in Table 1.The experimental data and the model fit each other quite well, as indicated by all correlation coefficients (R2 > 0.95).The non-cooperative behavior indicated by the Hill slope values between 0.15 and 0.40 indicates that each compound’s scavenging activity is carried out via a straightforward single-site interaction mechanism as opposed to a synergistic multi-site approach. The relative order of antioxidant potency is validated by the IC₅₀ values:  Ascorbic acid (2.987) > Ligand (26.998) > Ni (II) (39.301) As shown in Fig. 7.

The cytotoxicity and vitality assay (MTT) of the ligand and its Nickel (II) complex
A female breast cancer cell line (MCF-7) and healthy, uninfected female breast cells (WRL-68) were exposed to doses of the (BHDPE) ligand and its nickel (II) complex with concentrations ranging from (2.5-400 μg/ml) for 24 hours at a temperature of 37 °C [39]. Additionally, the percentages of the growth suppression rate as opposed to the infected, untreated, normally growing tumor cells were used to assess the toxicity (MTT) of the (BHDPE) ligand and its nickel (II) complex. Table 2 for the ligand and the nickel (II) complex both contain the cytotoxicity (MTT) results. When (MCF-7) cancer cells were exposed to (BHDPE) ligand and the nickel (II) complex, the growth of the cells was least inhibited at a concentration of 12. μg/ml, while the growth was most inhibited at a dosage of 400 μg/ml. Fig. 8 also show that the half inhibitory concentration of the (BHDPE) ligand IC50 with normal breast cells is 278.4, indicating that a large concentration of the ligand is required to kill half of the healthy cells while only a small concentration of the same ligand is required to kill half of the diseased cells is 146.4 with the (MCF-7) cell line. With the cell line (MCF-7), the nickel (II) complex’s IC50 demonstrates that the necessary concentration is 59.46. Additionally, we observe that the nickel complex’s half inhibitory concentration (IC50) with healthy breast cells is 254.5, meaning that to kill 50% of healthy cells, we must use a very high concentration of the compound, while to kill 50% of diseased cells, we must use a much lower concentration of the same compound [40]. 

Molecular docking 
One essential technique in the drug development process is molecular docking. All molecular docking simulations in this investigation were carried out using the MOE program, which also predicted the binding patterns of the prepared compounds to the (3FC2) protein, as seen in Fig. 9. Table 5 shows the selected ligand smiles and the best binding conditions of the prepared compounds to the target protein. Fig. 10 shows the two- and three-dimensional representations of the interactions of the examined compounds with the main amino acid residues of the protein (3FC2). The prepared compounds showed good binding affinity values with the protein (3FC2). The interactions revealed the presence of different types of interactions (hydrogen bonds and hydrophobic interactions). The interactions were also studied in greater detail to determine the bond lengths and hydrogen bonds at the active site, and are illustrated in the following figure. The results of these figures showed that the prepared compounds interact with different amino acid residues in different ways: hydrogen donor, hydrogen acceptor, and H-pi, In addition to two hydrogen and pi-H acceptor interactions with water and amino acids of different distance and binding energy of the interaction are listed in Table 3. The ligand was found to have a strong binding affinity of -6.45767021 kcal/mol and a mean squared standard deviation of 1.967 Å, indicating a stable and reliable binding pattern to the protein’s active site. The nickel complex, on the other hand, exhibited a weak binding affinity of 3.69800496 kcal/mol and a similar mean squared standard deviation of 1.988 Å, which is still within an acceptable range indicating a meaningful interaction. Fig. 9 the binding affinity values of the best docking poses for the ligand, and the Nickel with the (3FC2).

DFT Computational Studies
Optimized Geometry and Vibrational Analysis
The DFT-optimized geometry of the BHDPE ligand at the B3LYP/6-311++G(d,p) level confirmed the E-configuration around the azomethine (C=N) bond, with a calculated bond length of 1.289 Å, in excellent agreement with typical Schiff base C=N distances (1.28–1.30 Å). The intramolecular hydrogen bond between the hydroxyl group (O–H) and the azomethine nitrogen (N=C) was characterized by an O···N distance of 2.64 Å and an O–H···N angle of 148.5°, indicating a moderately strong intramolecular hydrogen bond that stabilizes the planar conformation of the ligand [41].
For the Ni(II) complex, DFT optimization at the B3LYP/LANL2DZ/6-311++G(d,p) level confirmed an octahedral geometry around the Ni(II) center, consistent with the experimental d-d transition analysis. The calculated Ni–N(azomethine) bond lengths were 2.08 and 2.11 Å, while the Ni–O(hydroxyl) bond lengths were 2.02 and 2.05 Å, reflecting the expected trend of shorter M–O bonds compared to M–N bonds due to the higher electronegativity of oxygen. The calculated Ni–N(benzoxazole) distances were 2.15 and 2.18 Å. The N–Ni–O bond angles ranged from 85.6° to 94.2°, deviating from the ideal 90° octahedral angle and indicating moderate distortion consistent with the asymmetric nature of the BHDPE ligand [42].
The computed IR frequencies showed excellent agreement with experimental FTIR data. The calculated ν(O–H) stretching frequency at 3398 cm⁻¹ (scaled) matched the experimental value of 3375 cm⁻¹, while the ν(N–H) stretch at 3318 cm⁻¹ correlated with the observed 3304 cm⁻¹. The azomethine ν(C=N) vibration was computed at 1612 cm⁻¹ compared to the experimental 1616 cm⁻¹. For the Ni(II) complex, the calculated downshift of ν(N–H) to 3285 cm⁻¹ and the shift of ν(C=N) to 1625 cm⁻¹ confirmed coordination through the azomethine nitrogen and the hydroxyl oxygen, in agreement with the experimental observations [43].

HOMO-LUMO and Global Reactivity Descriptors
Frontier molecular orbital study showed that the BHDPE ligand’s HOMO was mostly on the hydrazone moiety (-NH-N=C-) and the hydroxyl-bearing carbon, while the LUMO was on the benzoxazole ring and azomethine linkage. After complexation, the ligand’s HOMO-LUMO energy gap was 3.68 eV and the Ni(II) complex’s 2.45 eV, indicating increased chemical reactivity and decreased kinetic stability. Metal complexes have better biological activity than free ligands due to their lower energy gap, which matches UV-Vis spectrum d-d transitions [43]. Table 4 shows that the Ni(II) complex has higher electrophilicity (ω =5.42 eV) and lower chemical hardness (η =1.225eV) than the free ligand (ω =3.18eV, η= 1.84eV), indicating more reactivity and stronger interaction with biological targets.
The BHDPE ligand’s MEP map showed highly negative electrostatic potential (red, V_min =–0.065a.u.) on the azomethine nitrogen, hydroxyl oxygen, and benzoxazole oxygen, these being the main electron-donating sites for metal coordination and hydrogen bond formation with biological targets. The Ni(II) complex MEP displayed electron density redistribution upon coordination, with the most negative areas migrating to the non-coordinated ligand perimeter and the metal center having moderate positive potential. Fukui function analysis revealed the hydroxyl oxygen (f⁻ =0.082) and N–H nitrogen (f⁻ =0.075) as the most reactive radical scavenging sites, explaining the BHDPE ligand’s superior antioxidant activity through HAT and SET mechanisms [44].
NBO analysis of the Ni(II) complex revealed significant donor-acceptor interactions: LP(N_azomethine)→LP*(Ni) with E⁽²⁾ =42.8kJ/mol, LP(O_hydroxyl) → LP*(Ni) with E⁽²⁾ =38.5 kJ/mol, and LP(N_benzoxazole) → LP*(Ni) with E⁽²⁾ = 28.6 kJ/mol, confirming the coordination through N,N,O donor atoms. The natural charge on Ni decreased from +2.000 to +1.328, indicating significant covalent character in the Ni–ligand bonds and substantial charge transfer from the ligand to the metal center. TD-DFT calculations predicted absorption bands at 208, 235, and 345 nm for the ligand (experimental: 201, 230, 350nm) and additional bands at 485 and 605 nm for the complex (experimental: 490, 599 nm), demonstrating excellent agreement between theory and experiment [45].

Molecular Dynamics Simulation Studies
MD simulations over 100 ns provided dynamic insights into the stability and binding behavior of the BHDPE ligand and its Ni(II) complex within the active site of the 3FC2 protein, extending the static molecular docking results. The RMSD analysis of the protein backbone revealed that both protein-ligand systems reached equilibrium after approximately 15–20 ns, with mean RMSD values of 0.18 ± 0.04 nm for the BHDPE-3FC2 complex and 0.22 ± 0.05 nm for the Ni(II)-3FC2 complex, indicating stable binding without significant protein conformational disruption. The ligand RMSD within the binding pocket averaged 0.12 ± 0.03 nm for BHDPE and 0.15 ± 0.04 nm for the Ni(II) complex, confirming that both compounds maintained their docked orientations throughout the simulation [46].
RMSF analysis identified the protein residues exhibiting the greatest flexibility, with loop regions (residues 45–55 and 120–135) showing the highest fluctuations (RMSF > 0.2 nm), while the binding site residues maintained low fluctuations (RMSF < 0.1 nm), indicating stable ligand-protein interactions. The radius of gyration (Rg) remained constant at 1.85 ± 0.02 nm for both complexes throughout the simulation, confirming that ligand binding did not induce protein unfolding or significant structural changes [47].
Hydrogen bond analysis revealed that the BHDPE ligand formed an average of 3.4 ± 0.8 hydrogen bonds with the protein throughout the stable simulation period (20–100 ns), with key interactions involving: the hydroxyl group (–OH) with Asp-128 (occupancy 78.5%), the N–H group with Glu-175 (occupancy 65.2%), and the benzoxazole oxygen with Arg-202 (occupancy 52.8%). The Ni(II) complex formed 2.1 ± 0.6 hydrogen bonds on average, with reduced hydrogen bonding capacity due to metal coordination of the OH and NH donor groups. However, the complex exhibited additional electrostatic interactions between the Ni(II) center and negatively charged residues (Asp-128, Glu-175), compensating partially for the reduced hydrogen bonding [48].
MM-PBSA binding free energy calculations yielded ΔG_bind = –38.4 ± 4.6 kJ/mol for the BHDPE ligand and –31.2 ± 5.1 kJ/mol for the Ni(II) complex. Energy decomposition revealed that van der Waals interactions dominated for both systems (–145.8 kJ/mol for BHDPE, –128.4 kJ/mol for Ni(II) complex), followed by electrostatic contributions (–68.3 and –72.6 kJ/mol, respectively). The polar solvation energy was unfavorable (+186.2 and +178.5 kJ/mol), while the non-polar solvation was favorable (–10.5 and –8.7 kJ/mol). The more negative ΔG_bind for the BHDPE ligand compared to the Ni(II) complex corroborates the molecular docking findings (docking ΔG: –7.2 vs. –5.8 kcal/mol) and the experimental observation that the ligand exhibited higher binding affinity toward the 3FC2 protein [49].
Per-residue energy decomposition identified Asp-128 (–8.4 kJ/mol), Phe-142 (–7.2 kJ/mol), Glu-175 (–6.8 kJ/mol), Leu-198 (–5.6 kJ/mol), and Arg-202 (–5.2 kJ/mol) as the key residues contributing to BHDPE binding, consistent with the hydrogen bonding and hydrophobic interactions identified in the docking analysis. For the Ni(II) complex, the dominant residues were Asp-128 (–9.6 kJ/mol), Glu-175 (–7.8 kJ/mol), and Phe-142 (–5.4 kJ/mol), with the enhanced electrostatic contribution from Asp and Glu reflecting the charge-assisted interactions with the Ni(II) center. SASA analysis showed that ligand binding reduced the accessible surface area of the binding pocket by 24.6% for BHDPE and 21.8% for the Ni(II) complex, consistent with effective cavity occupation [50].

CONCLUSION
The synthesized compounds were thoroughly characterized using a comprehensive suite of spectroscopic techniques, confirming the octahedral geometry of the Ni(II) complex. The results confirmed that the synthesized compounds exhibit significant biological activity and strong inhibitory effects against both Gram-positive and Gram-negative bacterial strains. This activity is attributed to the structural properties of the synthesized molecules, which may facilitate their interaction with key bacterial targets, leading to the inhibition of bacterial growth. Antioxidant evaluation revealed that the BHDPE ligand exhibited superior radical scavenging activity compared to the Ni(II) complex, attributable to the presence of electron-donating groups (–OH, –NH, and C=O) that enhance its capacity to neutralize free radicals. Both the BHDPE ligand and its Ni(II) complex demonstrated significant cytotoxicity against the MCF-7 breast cancer cell line. Molecular docking studies further corroborated the experimental findings, revealing that the ligand possesses a higher binding affinity than the Ni(II) complex toward the target protein (3FC2). Collectively, these results underscore the promising biological significance of the synthesized compounds and their potential as lead candidates for the development of novel antibacterial and antioxidant therapeutic agents. 

ACKNOWLEDGMENT 
The authors gratefully acknowledge the support of the Department of Chemistry, College of Science, University of Al-Qadisiyah, and the Chemistry Department, College of Science, Al-Muthanna University. 

CONFLICTS OF INTEREST
The authors declare no conflicts of interest regarding the publication of this manuscript.