Bioactive Cr(III), Co(II), and Mn(II) Complexes with N′((3-Hydroxynaphthalen-2-yl)methylene)picolinohydrazide: Structural, Computational, and Biological Studies

Abstract

The present study focuses on the synthesis and characterization of novel bioactive complexes of Cr­(III), Co­(II), and Mn­(II) derived from N′-((3-hydroxynaphthalen-2-yl)­methylene)­picolinohydrazide (H₂L), a Schiff-base ligand featuring multifunctional donor sites. The primary objective was to investigate the coordination behavior, stability, and biological efficacy of these metal chelates. The complexes were synthesized via direct metal–ligand reactions and characterized using elemental analysis, FT-IR spectroscopy, UV–Vis, PXRD, ¹H NMR spectroscopy, MS, magnetic susceptibility measurements, and thermogravimetric analysis. Spectroscopic evidence supported tetrahedral geometries for the Co­(II) and Mn­(II) complexes, while the Cr­(III) complex exhibited an octahedral arrangement. Thermogravimetric and kinetic studies yielded positive activation free energy (ΔG*) values, indicating nonspontaneous decomposition pathways and high thermal stability. Quantum Theory of Atoms in Molecules (QTAIM) and reduced density gradient (RDG) analyses were performed to elucidate the nature of ligand–receptor interactions. Biological assessments revealed promising results, as DNA degradation assays demonstrated notable nuclease-like activity, particularly for the Co­(II) complex. Antibacterial potency was evaluated via minimum inhibitory concentration (MIC), where the Mn­(II) complex exhibited the strongest activity (0.313 mg/mL), followed by Co­(II) and Cr­(III) (0.625 mg/mL each), and the free ligand (1.250 mg/mL). Cytotoxicity testing against HeLa, HCT-116, and MCF-7 cancer cell lines showed high anticancer efficacy for the [CoL]·2H₂O complex with IC₅₀ values of 7.76  ±  0.4, 10.23  ±  0.8, and 6.88  ±  0.4 μM, respectively. Molecular docking studies using an induced fit protocol highlighted the strong noncovalent interactions of the complexes with DNA targets relevant to each cell line.

1. Introduction

Exploring coordination chemistry is an important goal in the search for innovative materials with distinct properties for a wide range of applications. Hydrazones are a distinct type of the Schiff-base ligand defined by the presence of the reactive (HN–NCH) functional group within their molecular formulas. Hydrazones are a specialized subclass of the Schiff base family, with a diverse range of biological and physiological activities. These actions include antitumor, antitubercular, antifungal, antioxidant, antibacterial, anti-inflammatory, and antimalarial effects. − Picolinate hydrazones are characterized by the existence of additional donor atoms, including the oxygen atom of the (CO) group and nitrogen atoms of both the (CN)_Py and (CN)_az groups. Previous studies have demonstrated that picolinic acid, a metabolite of L-tryptophan, exhibits significant biological potential, functioning as a neuroprotective, immunomodulatory, antimicrobial, and antiproliferative agent within the body. ,− Chromium picolinate has been identified as a dietary supplement that facilitates fat reduction and muscle enhancement in humans while also demonstrating efficacy in lowering blood glucose levels in diabetic patients. − Our research introduces a structurally distinct picolinate-based hydrazone ligand (H₂L) featuring pyridine-N-oxide functionality. This design not only enriches the coordination chemistry landscape with multiple donor sites (CO, CN_py, and CN_az) but also enhances the ligand’s versatility in forming stable tridentate, dibasic complexes with transition metals. Compared to earlier reports by Deepa et al., Toyama et al., and Parvarinezhad et al., which involved simpler ligand systems, our synthesized ligand facilitates more robust coordination environments, as confirmed by extensive spectral, thermal, and magnetic analyses (IR, UV–visible, XRD, ¹H NMR, MS, and TGA).

Furthermore, the prepared compounds transcend the isolated biological evaluations seen in previous works ,, by demonstrating a comprehensive profile of bioactivities, including anticancer, antibacterial, and antioxidant effects, DNA degradation, and BSA binding properties for the Co­(II), Cr­(III), and Mn­(II) chelates. Molecular docking analysis provides additional insights into protein–ligand interactions, reinforcing the biomedical relevance of our complexes. Taken together, this work presents a distinctive contribution through the integration of an innovative ligand architecture, diverse coordination behavior, and broad-spectrum biological efficacy, clearly distinguishing it from prior studies and advancing the field of bioinorganic chemistry.

2. Experimental Section

2.1. Instruments

All of the chemical compounds involved in this investigation were of analytical grade and utilized directly without undergoing any further purification. The chemicals, reagents, and instruments used in the characterization are described in the Supporting Information. The DFT calculations of the ligand were performed using Gaussian 09 software with B3LYP/6-311G (d) basis set, while the isolated metal chelates were achieved with B3LYP/6-311G (d) for the C, H, N, and O atoms, and LANL2DZ basis set for the metal atom.

2.2. Preparation of the H₂L Ligand and Its Complexes

The preparation of the H₂L ligand involved the condensation of 3-hydroxy-2-naphthaldehyde and picolinohydrazide in a 1:1 molar ratio (Scheme ) at 90 °C for 4 h. An orange precipitate of the H₂L ligand was obtained. The obtained precipitate was separated through filtration, subsequently recrystallized from ethyl alcohol, and dried under vacuum over anhydrous calcium chloride (CaCl₂). The metal chelates were synthesized through refluxing a 1:1 molar ratio of the ligand and the corresponding metal salts, including CoCl₂, CrCl₃·6H₂O, and MnCl₂·4H₂O, at 150 °C for 6 h (Scheme ). The solid precipitate was separated by filtration, washed, and subsequently dried over anhydrous CaCl₂.

1. Preparation of H₂L and Its Complexes.

2.3. Biological Applications

2.3.1. Antioxidant Activity Using the DPPH Assay

The antiradical activity of the examined compounds was evaluated using the DPPH^• colorimetric assay with ascorbic acid as the standard. Serial dilutions were prepared by mixing equal volumes of the sample solution and methanol. A 0.135 mM solution of DPPH^• was introduced to each dilution, followed by incubation of the resulting mixtures in a dark environment at 37 °C for a duration of 30 min. − Absorbance was measured at 517 nm. The residual DPPH^• percentage was determined by using the stratified application of the following equation:

$$\%\mathrm{inhibition}=\frac{A_{\mathrm{control}}-A_{\mathrm{test}}}{A_{\mathrm{control}}}\times 100$$ 1

The % DPPH^• remaining was plotted against the sample concentration (mg/mL) using an exponential curve to determine the IC₅₀ value, representing the antioxidant quantity required to decrease the primary DPPH^• concentration by 50%. Lower IC₅₀ values correspond to higher antioxidant capacity. The reported total antioxidant activity values represent the mean of three independent experimental replicates.

2.3.2. Antibacterial Activities

The antibacterial potency of the investigated compounds was assessed utilizing the well diffusion assay technique outlined in previously published literature. , The antibacterial potency of agents against various bacterial strains was estimated by an agar well diffusion assay technique. The agar plate surface is prepared by evenly spreading a specific volume of the bacterial inoculum. A 9²³ mm diameter hole is then aseptically created using a sterile cork borer or tip. A 100 μL sample at the desired concentration is introduced into this well. The agar plates are then incubated under optimal conditions tailored to the specific test microorganism. During incubation, the antibacterial agent diffuses through the agar medium, effectively inhibiting the growth of the tested microbial strains.

2.3.3. Determination of the Minimum Inhibitory Concentration (MIC)

Serial dilutions of the sample at concentrations ranging from 5 to 0.078 mg/mL were used to establish the minimum concentration of the sample that would still exhibit antibacterial potency against Escherichia coli. The control consisted solely of inoculated broth, which was incubated for 1 day at 37 °C. The MIC endpoint is defined as the lowest sample concentration at which no visible microbial growth is observed in the tubes. The visual turbidity of the tubes was observed both prior to and following incubation to validate the MIC value, and the optical density (O.D.) was recorded at 600 nm to confirm the findings.

2.3.4. DNA Degradation Activity

The DNA degradation property of the examined compounds was estimated using Escherichia coli (E. coli) DNA as a substrate. Electrophoretic analysis was performed in a 1% agarose gel, prestained with ethidium bromide, under the conditions of 80 V for 2 h in Tris-(hydroxymethyl)­methane buffer. Visualization and documentation of the gel were conducted using a UV transilluminator for precise observation of degradation patterns. ,

2.3.5. Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Electrophoresis was performed using 10 mg of bovine serum albumin (BSA) treated with H₂L ligands or metal complexes at a concentration of 5 mM. The prepared mixtures were incubated at 37 °C for 2 h, after which a six times sample buffer was added to the incubated mixtures. The stacking gel was made using a 4.0% (w/v) polyacrylamide solution in Tris buffer (pH 6.8), whereas the separating gel was made with a 15% (w/v) polyacrylamide solution in Tris buffer (pH 8.8) and 0.1% (w/v) SDS. Protein sample migration was conducted at 45 V for 15 min following the loading of samples in the stacking gel. Electrophoretic separation was then induced at 90 V in the running gel for 3 h. The protein bands were visualized by staining the SDS-PAGE gel with bromophenol blue, followed by destaining with a solution composed of methanol, glacial acetic acid, and distilled water.

2.3.6. Antitumor Activities

The antitumor potency of the tested samples was meticulously investigated by an MTT assay. The evaluation targeted specific cancer cell lines, including HeLa (epithelioid carcinoma of the cervix), MCF-7 (mammary gland breast adenocarcinoma), and HCT-116 (human colorectal adenocarcinoma). The inhibitory properties of the tested samples on cell proliferation were assessed by the MTT assay. Cells were cultured in RPMI-1640 medium with supplements and placed at a density of 1.0 × 10⁴ cells/well. After treatment with varying compound concentrations, MTT solution was added, and formazan crystals were dissolved in DMSO solution. The absorbance was measured at 570 nm, and the cell viability (%) was calculated. ,

2.4. Molecular Docking with Receptors 5IAE, 2W3L, and 3W2S

Molecular docking simulations, a key component of computational drug design, offer mechanistic insights into drug–target interactions. By providing detailed information on binding affinity and orientation, these simulations support the rational development of new pharmaceutical entities.

2.4.1. Ligand and Protein Preparation

For molecular docking studies, the synthesized compounds underwent optimization using Ligprep (Schrödinger suite) with default settings. The three-dimensional structures of HeLa (PDB ID: 5IAE) https://www.rcsb.org/structure/5IAE, HCT-116 (PDB ID: 1YWN) https://www.rcsb.org/structure/1YWN, and MCF-7 (PDB ID: 3W2S) https://www.rcsb.org/structure/3W2S were retrieved from the protein database. − Protein structure preparation was performed using the Protein Preparation Wizard (Maestro v. 14.1, Schrödinger suite), encompassing error correction, side-chain reconstruction, and structural repair. The protonation states were determined using PROPKA at pH 7.0, followed by energy minimization with the OPLS4 force field until the heavy atom RMSD converged to 0.30 Å. The bond orders were determined, and hydrogen atoms were incorporated into the structure at a controlled pH of 7.0 ± 2.0. Final restrained minimization, employing the OPLS 2005 force field, was performed with heavy atom RMSD convergence set at 0.3 Å.

2.4.2. Induced Fit Docking

Molecular docking simulations were conducted utilizing the Schrödinger induced fit docking (IFD) protocol. A 10 Å grid box, centered on the native ligand coordinates of 5IAE, 1YWN, and 3W2S, was generated. Ligand flexibility was addressed with Glide (v. 10.4) using a 2.5 kcal/mol ring conformation sampling window, while receptor flexibility was accounted for using Prime. The IFD protocol employed modified van der Waals and Coulombic parameters and temporary side-chain removal. The docking of the minimized compounds was performed with standard sampling, a potential scaling factor of 0.5, and a maximum of five retained poses. IFD scores, reflecting protein–ligand interaction and total system energy, were used for pose ranking.

2.4.3. Docking Validation

The reliability of the docking procedure was assessed through redocking experiments. The cocrystallized ligands were removed and redocked into their respective binding sites using the Schrödinger suite software, without altering any function parameters. This validation step was performed to ensure the accurate reproduction of ligand binding within the active site and to minimize deviations from the experimentally determined cocrystallized structures.

3. Results and Discussion

The metal complexes (10^–4 M in DMF) exhibited molar conductance values in the range of 6.32–9.44 Ω^–1 cm² mol^–1, indicative of their nonelectrolytic nature. Table S1 provides an overview of the various physical properties and analytical data of the isolated compounds. Elemental analysis data, combined with spectral studies, were utilized to infer the structural formulas and propose the geometries of the complexes. Moreover, all complexes are stable in air and insoluble in most organic solvents, except DMSO and DMF. Additionally, the M–Cl bond in our system functions as a terminal coordinating halide, contributing to the stabilization of the metal center through σ-donation.

3.1. Infrared Spectra

The IR spectra of H₂L and the synthesized complexes were assigned from 4000 to 400 cm^–1 (Table S2) and are represented graphically in Figure S1. The spectrum of H₂L displays two strong vibrational peaks at about 1662 and 1528 cm^–1, which are attributed to the ν­(CO) and ν­(CN)_py groups, respectively. The band at 1619 cm^–1 is attributed to the azomethine group. The broad bands at 3172 and 3464 cm^–1 are assigned to the ν­(NH) and ν­(OH) groups, respectively. On the other hand, the strong peak at 1033 cm^–1 is assigned to the ν­(N–N) vibration. The infrared spectra of the studied complexes were discussed by comparing their spectra with those of the free ligands. Consequently, the IR spectrum of the [CoL]·2H₂O complex shows that the H₂L ligand acts as a binegative tetradentate Schiff base (ONNO) by deprotonating both OH groups of the enolic and aromatic rings and (CN)_az, (CN)_Py groups. This bonding mode is obtained through the following:

• (i)The absence of vibration bands of the (CO) and (NH) groups, alongside the emergence of new bands associated with ν­(CN) and ν­(C–O) vibrations, indicates the enolization of the carbonyl group.
• (ii)The absence of OH vibrations serves as evidence of deprotonation.
• (iii)The observed shift of the ν­(CN)_az and ν­(N–N) vibrations to a new position signifies their sharing in the coordination.
• (iv)The movement of the ν­(CN)_Py vibration suggests its involvement in complex formation.

Although in the [Cr­(L)­(H₂O)₂Cl]·H₂O complex, the H₂L ligand acts as a bibasic tridentate Schiff base (ONO) via the nitrogen of the azomethine group and deprotonated of both enolic and phenolic OH groups, this coordination mode is proposed by the following:

• (i)The disappearance of the ν­(NH) and ν­(CO) bands, accompanied by the emergence of a new band attributed to ν­(–CN), confirms the enolization form.
• (ii)The absence of an (OH) group signal suggests that deprotonation occurred.
• (iii)The observed shift of the ν­(CN)_az and ν­(N–N) vibrations to a new position signify their participation in the coordination.
• (iv)There were no changes in the positions of the ν­(CN)_py and δ­(CN)_py bands, indicating that this functional group was not involved in the chelation process.

In the [Mn­(HL)] complex, the H₂L ligand acts as a mononegative tetradentate Schiff base (ONNO) via the (CO) and (CN)_az groups, and a deprotonated OH group of the aromatic rings. This bonding mode is explained as follows:

• (i)The presence of the stretching vibrations of amine and carbonyl groups validates the keto structure, while the observed shift of the ν­(CO) vibrational band to another position provides evidence of its involvement in the chelation process.
• (ii)The disappearance of the OH vibrational band provides compelling evidence for the occurrence of deprotonation.
• (iii)The observed shift of the ν­(CN)_az and ν­(N–N) vibrations to a new position signifies their participation in the coordination.
• (iv)The movement of the position of the ν­(CN)_Py group reveals that this group is employed in complexation.

3.2. Nucleic Magnetic Resonance (NMR) Analysis

The NMR spectrum of the synthesized H₂L was obtained in DMSO-d ₆ solvent. The proton NMR spectrum of the H₂L ligand (Figure S2) exhibited a singlet signal at 12.214 ppm, which was attributed to the phenolic proton. This upper chemical shift value may be attributed to the influence of hydrogen bonding. The singlet peak at 9.83 ppm is assigned to the amino proton. These signals disappeared upon deuteration (Figure S3). The characteristic signal appears at 8.77 ppm, which is related to the azomethine proton. The multiple peaks observed within the chemical shift range of 7.24–8.22 ppm correspond to the aromatic protons. The H₂L ligand was analyzed by recording its ¹³C NMR spectrum, as presented in Figure S4. Multiple bands were seen in the downfield region, within the range of 119.42–138.61 ppm, relating to the aromatic nature of the carbons present in the ligand structure. The triplet signals at 149.03, 149.12, and 149.42 ppm are attributed to the carbon of the (CN)_py, (CN)_az, and (OC–NH) groups, respectively. The signals at 160.52 and 158.83 ppm are assigned to the carbon of the (CO) and C–OH groups, respectively.

3.3. Mass Spectra

The EI-MS spectra were analyzed to validate the proposed molecular structures of the H₂L ligand and its complexes. The mass spectrum of H₂L (Figure S5) revealed a molecular ion peak [M]⁺ at m/z = 291.31, which corresponds to its molecular weight. The proposed fragmentation pathway of H₂L is outlined and visually presented in Scheme . The EI-MS of the the [CoL]·2H₂O complex (Figure S6) offered strong evidence supporting its suggested molecular formula and a molecular weight of 384.26. The molecular ion peak at m/z = 384.30 (69.70%) corresponds to (C₁₇H₁₅CoN₃O₄). The EI-MS spectrum of this complex shows distinct fragments that produce peaks of varying intensities at different m/z values, such as at 128.17 (100%) (C₁₀H₈), 221.07 (17.40%) (C₇H₅CoN₃O₂), 142.04 (7.31%) (C₁₀H₆O₂), 205.98 (18.45%) (C₇H₅CoN₃O), 120.03 (1027%) (C₆H₄N₂O), and 227.99 (13.39%) (C₁₁H₇CoNO). The mass spectrum of the [Cr­(L)­(H₂O)₂Cl]·H₂O complex (Figure S7) confirmed the proposed molecular formula. The molecular ion peaks at m/z = 430.79 (52.66%) correspond to (C₁₇H₁₇ClCrN₃O₅). Finally, EI-MS of the [Mn­(HL)] complex (Figure S8) confirmed its molecular formula with a molecular ion peak at m/z = 345.54 (65.21) corresponding to (C₁₇H₁₂MnN₃O₂). Other fragments appear at different m/z values, such as at 57.01 (100%) (CHN₂O), 263.37 (26.11%) (C₁₄H₁₀MnNO), 113.95 (27.32%) (C₉H₆), and 306.08 (31.57%) (C₁₄H₉MnN₃O₂).

2. Mass Fragmentation Path of the H₂L Ligand.

3.4. Electronic Spectrum and Magnetic Susceptibility Studies

The electronic spectra of the H₂L ligand and its metal complexes were recorded in DMF solvent at room temperature with a concentration of 10^–4 M. The magnetic moments and spectral absorption bands are shown graphically in Figure S9 and are reported in Table S3. The electronic spectrum of H₂L exhibits three absorption bands: the first one, located at 270 nm, is attributed to the π→π* transition of the aromatic rings. The two bands at 329 and 361 nm are attributed to the n→π* transition of carbonyl and (CN) of both the pyridyl and azomethine groups. The other bands at higher wavelengths within the range of 384–430 nm are due to charge transfer. These bands show a shift toward higher wavenumbers in the metal chelates due to chelation. , In the cobalt complex, the bands at 275 and 327 nm are assigned to the π→π* and n→π* transitions, respectively. The two bands at 358 and 382 nm are due to charge transfer, while the absorption band at 450 nm is attributable to the ⁴A₂→⁴T₁ (P) transition. These results indicate the existence of a tetrahedral geometry for the [CoL]·2H₂O complex, proven by its magnetic moment of 4.36 (B.M.). This value lies within the expected range for the tetrahedral arrangement of divalent cobalt complexes, typically between 4.2 and 4.8 μ_B. The UV-visible spectrum of the [Cr­(L)­(H₂O)₂Cl]·H₂O complex exhibits absorption bands in the wavelength range of 275–497 nm. The two bands at 275 and 336 nm correspond to the ligand field transitions. The bands at 464 and 498 nm are assigned to the ⁴A_2g(F) →⁴T_1g(F) and ⁴A_2g(F) →⁴T_2g(F) transitions, respectively. , As expected, the υ3 band at about 300 nm, which corresponds to the ⁴A_2g→ ⁴T_1g(P) transition, is obscured by the charge transfer bands. , Furthermore, the μ_eff value of 3.56 μ_B supports the octahedral geometry proposed for this complex. In the [Mn­(HL)] complex, the bands at 362 and 382 nm correspond to the LMCT transition. The absorption at 465 nm is related to ⁶A₁→⁴T₂, corresponding to the tetrahedral Mn­(II) environment. , The measured magnetic moment μ_eff was calculated to be 5.58 μ_B, corroborating the characterization of a high-spin tetrahedral Mn­(II) complex.

3.5. Powder XRD Studies

Due to the inability to separate single crystals of the synthesized compounds for X-ray crystallographic analysis, powder X-ray diffraction data were obtained for structural description. X-ray diffraction analysis was conducted to provide comprehensive insights into the crystalline nature of H₂L and its chelates. The diffractograms of the studied compounds (Figure ) display several sharp peaks, indicating their polycrystalline nature. The computer software BIOVIA Material Studio, the reflex powder indexing module, and the TREOR9 indexing algorithm were employed to calculate the theoretical values of 2θ^o, crystal system, space group, and lattice parameters. Table S4 presents the crystallite-particulate parameters, including the 2θ^o values, interplanar spacing (d-values, Å), relative intensity, dislocation density (δ), and crystal strain (ε) of the investigated complex, all calculated using standard equations. − The diffraction data was employed to determine the average crystallite size (D) using the Scherrer equation

$$D=\frac{0.9\lambda }{\beta \cos \theta }$$ 2

where λ= 1.5406 Å, θ represents the Bragg diffraction angle, and β denotes the full width at half-maximum (fwhm) of the peak. , Based on the highest intensity peaks, the grain sizes of the studied compounds were found to be 57.65, 18.42, 81.75, and 81.21 nm for the H₂L ligand, [CoL]·2H₂O, [Cr­(L)­(H₂O)₂Cl]·H₂O, and [Mn­(HL)] complexes, respectively. According to the δ values, the polycrystalline phase of the ligand and its complexes increased in the following order: [Cr­(L)­(H₂O)₂Cl]·H₂O (1.79 × 10^–3) > [Mn­(HL)] (2.05 × 10^–3) > H₂L­(2.84 × 10^–3) > [CoL]·2H₂O (7.63 × 10^–3 nm^–2).

1.

Experimental and simulated diffractograms of the H₂L ligand (a), [CoL]·2H₂O (b), [Cr­(L)­(H₂O)₂Cl]·H₂O (c), and [Mn­(HL)] (d) complexes.

3.6. Thermal Studies

3.6.1. Thermogravimetric Analysis (TG)

A detailed thermal investigation of the Cr­(III), Co­(II), and Mn­(II) chelates was performed using thermogravimetric analysis (TGA) in the temperature range of 25–800 °C (Table S5). The goal of this analysis was to establish the type of water molecules found inside these complexes, namely, whether they exist in crystalline form or are coordinated to the metal centers. The structural characteristics and thermal stability of the complexes are clarified in this work. The thermogram of the [CoL]·2H₂O complex displayed five decomposition steps (Figure ). The first stage of fragmentation appeared in the temperature range of 28 to 137 °C, which corresponded to the loss of two hydrated water molecules (weight loss: found 8.93%; calcd 9.36%). The second thermal step, occurring in the temperature range of 218–326 °C, is accompanied by a measured weight loss of 11.25% after the elimination of CO + 0.5N₂ molecules from the complex structure. The third step within the temperature range of 327–450 °C corresponds to the loss of the C₆H₄CNOH fragment (weight loss: found 31.40%; calcd 31.02%). The fourth stage of the thermal curve occurred in the temperature range of 451–481 °C, with a weight loss percentage of 20.98%, corresponding to the removal of the C₅H₅N composite. The final residue upon decomposition, attributed to cobalt, , was detected at temperatures greater than 670 °C, and the residual weight was 15.75% (calcd: 15.34%). The thermodecomposition behavior of the [Cr­(L)­(H₂O)₂Cl]·H₂O complex is marked by four steps of decomposition, as shown in Figure S10. The initial stage of decomposition, observed in the range of 24–100 °C, is due to the loss of a hydrated water molecule, and a mass loss value of 4.56% (calcd: 4.18%) was recorded. The second thermal degradation, occurring within the 126–360 °C temperature range, is associated with the evolution of two coordinated water and HCl molecules from the complex, and consequently an observed mass loss of 16.16% (calcd: 16.82%). The third thermolysis step in the temperature range of 361–515 °C is attributed to the elimination of the C₆H₄CNOH fragment with the release of N₂ gas, and the weight loss observed is 34.40% (calcd: 34.14%). The final degradation process, in the temperature range of 516–733 °C, is attributed to the loss of the C₅H₅ moiety, with a mass loss observed of 14.88%. The remaining part at temperatures greater than 734 °C accounted for 30.00% of the original mass and is due to the presence of residual organic carbon species and chromium monoxide (CrO). The thermogravimetric trace shows no noticeable mass loss below 200 °C, which indicates that there are no coordinated or lattice water molecules in the structure of the Mn­(II) complex (Figure S11). The first step of the thermal decomposition occurs within the temperature range of 204–333 °C and involves the release of N₂ and H₂ gases, with a weight loss of 17.03%. The second thermal decomposition stage, occurring between 334 and 413 °C, corresponds to the elimination of the C₅H₄N fragment from the complex structure, accompanied by an observed mass loss of 21.74%. The final thermodecomposition stage, between 414 and 664 °C, corresponds to the loss of the C₆H₄ moiety and the concomitant release of C₂H₂ gas from the complex structure with a mass loss of 30.03%. At temperatures exceeding 665 °C, the remaining residue consists predominantly of manganese oxide (MnO) and residual organic carbon, accounting for 31.02% of the initial sample mass.

2.

Thermogram of the [CoL]·2H₂O complex.

3.6.2. Thermodynamics and Kinetics Studies

Based on the thermogram, the kinetic and thermodynamic parameters of the complexes were determined by using the Coats–Redfern and Horowitz–Metzger equations. The data supporting these calculations are documented in Tables and S6 and are visually represented in Figures S12–S17. The Eyring equations were mathematically applied to investigate and analyze the kinetic and thermodynamic parameters of the complexes. The negative values of ΔS* observed for the investigated complexes suggest that they are more ordered than the original ligand, indicating a slow thermal decomposition process. Conversely, the positive values of activation entropy (ΔS*) may imply that the disorder of the disintegrated fragments increases notably quicker than that of the undecomposed species. , The endothermic nature of all fragmentation processes is evidenced by the positive values of activation enthalpy. The complexes under study display thermal stability, and the positive signs of ΔG* demonstrate that all decomposition steps are nonspontaneous. ,

1. Kinetic Parameters Calculated by the Coats–Redfern Method for the Co­(II) and Cr­(III) Complexes.

complex	peak	mid temp (K)	E a (kJ/mol)	A (S–1)	ΔH* (kJ/mol)	ΔS* (kJ/mol·K)	ΔG* (kJ/mol)
[CoL]·2H2O	1st	317	113.23	6.63 × 1016	110.60	0.0766	86.31
	2nd	509	416.68	4.78 × 1040	412.44	0.5294	142.97
	3rd	647	1057.1	7.44 × 1083	1052.1	1.354	175.94
	4th	738	1843.1	9.62 × 10128	1836.9	2.216	201.0
	5th	899	784.68	4.45 × 1043	777.21	0.5815	254.42
[Cr(L)(H2O)2Cl]·H2O	1st	311	101.41	1.29 × 1015	98.83	0.0440	85.13
	2nd	521	371.75	3.47 × 1034	367.41	0.4117	152.92
	3rd	704	231.19	7.46 × 1014	225.34	0.0326	202.33
	4th	817	320.17	3.14 × 1018	313.38	0.1008	231.00
[Mn(HL)]	1st	572	210.07	1.08 × 1017	205.32	0.0757	162.00
	2nd	637	329.21	7.16 × 1024	323.92	0.2246	180.84
	3rd	748	270.64	4.25 × 1016	264.42	0.0658	215.21

3.7. DFT Studies

3.7.1. Geometry Optimization

Figure S18 displays the optimized structures of the H₂L ligand and [CoL]·2H₂O, [Cr­(L)­(H₂O)₂Cl]·H₂O, and [Mn­(HL)] complexes. Tables S7 and S14 comprehensively detail the bond lengths and angles of the synthesized compounds. The bond angles and lengths vary depending on the metal center and the coordinating environment. The bond angles in the [CoL]·2H₂O and [Mn­(HL)] complexes show tetrahedral structures with sp³ hybridization, similar to those of tetrahedral Co­(II) and Mn­(II) complexes. The bond angles in the [Cr­(L)­(H₂O)₂Cl]·H₂O chelate are typical of the octahedral environment observed in Cr­(III) chelates with d²sp³ hybridization. Consistency in bond length elongation for active groups such as CO, CN, and OH was observed upon coordination with metal ions. When the metal–nitrogen (M–N) bond forms, the carbon–nitrogen (C–N) bond weakens due to coordination with the imine group’s nitrogen atom.When the metal–nitrogen (M-N) bond is formed, the carbon–nitrogen (C–N) bond becomes weaker because of coordination with the imine group’s nitrogen atom (CN). This weakening is caused by the nitrogen lone pair’s participation in the formation of the coordination bond with the metal ion, which previously contributed to the double-bond nature of the CN bond. As a result, the electron density of the CN bond is lowered, resulting in decreased strength. The formation of the (M–O) bond induces weakening, thereby facilitating chelation of the carbonyl group (CO).

3.7.2. Frontier Molecular Analysis

The HOMO and LUMO energies define a molecule’s capacity to donate or accept electrons. The chemical reactivity is determined by the energy gap between these two orbital levels. The small energy band gap suggests that the compound demonstrates strong chemical reactivity, considerable biological potency, and strong polarizability. The obtained HOMO and LUMO of the studied compounds are shown in Figures and S19. The computed energies of the HOMO and LUMO are used to calculate various chemical parameters, including electronegativity (χ), chemical potential (μ), hardness (η), softness (σ), and electrophilicity index (ω) (Table S15). A higher ΔE value suggests hardness, whereas a lower ΔE value indicates softness. A soft molecule exhibits greater reactivity compared to a hard molecule due to its ability to donate electrons readily to electron acceptors. The ionization potential is relative to the energy level of the HOMO, whereas the electron affinity is relative to the energy level of the LUMO, both of which are positive. An increase in the ionization potential and electron affinity indicates that the element has greater electronegativity. The metal complexes display smaller band gap energies compared to those of the H₂L ligand. This demonstrates their enhanced electron transfer capacity, decreased kinetic stability, increased chemical reactivity, and significant biological activity. Because of their improved electron transport capabilities, these complexes are expected to interact more effectively with macromolecular structures, allowing for easier binding to proteins or DNA. All investigated compounds demonstrate a negative chemical potential (μ), revealing that the inclusion process occurs spontaneously and that the compounds are thermodynamically stable. The chemical potential quantitatively represents the inclination of electrons to diverge from an equilibrium configuration, which decreased as follows: [Mn­(HL)] complex (−3.54) > H₂L ligand (−4.05) > [CoL]·2H₂O complex (−4.17) > [Cr­(L)­(H₂O)₂Cl]·H₂O (−4.41) eV.

3.

Frontier molecular orbitals (HOMO and LUMO) of the H₂L ligand.

3.7.3. Molecular Electrostatic Potential (MEP)

MEP diagrams are a valuable index for visualizing the distribution of electric charges on a molecule’s surface. This understanding helps us understand the molecule’s chemical and physical features. The establishment of many intermolecular interactions, including hydrogen bonding, drug–receptor binding, and enzyme–substrate interactions, is mostly dictated by the electrostatic potential distribution of the molecule. MEP is a well-known method for providing a comprehensive understanding of the reactive sites of compounds, especially identifying areas that react to electrophilic and nucleophilic interactions. The MEP mapping of the H₂L Schiff base and its chelates may be classified into three distinct regions based on their color representation, as seen in Figure . The red regions correspond to areas of high electronic density, which aid electrophilic attacks. In contrast, the green regions represent zones of neutral electrostatic potential, while the blue region signifies regions of low electronic density, indicating nucleophilic attack behavior. The regions of negative electrostatic potential are concentrated around electronegative atoms with red color, including the oxygen atoms in carbonyl and hydroxyl groups, nitrogen of the pyridine ring, and nitrogen in the imine group. The hydrogen atom bonded to nitrogen in the (NH) group is positioned within the blue region, which displays a distinctly positive electrostatic potential, rendering it highly vulnerable to nucleophilic attack.

4.

ESP maps of the studied compounds.

3.8. Topology Analyses

3.8.1. Quantum Theory of Atoms in Molecules (QTAIM)

QTAIM analysis was conducted to characterize ligand–receptor interactions, focusing on bond critical points (BCPs). Topological parameters (Table ) and electron density maps (Figure ) were generated. Contour maps showed a decrease in electron density (ρ) from the nuclear centers, and relief maps identified BCPs as the electron density minimum along the bond paths. Negative values of the electronic energy density H(r) and Laplacian of electron density ∇²ρ_r confirmed the covalent bond characteristics, while positive values indicated noncovalent interactions. The covalent bond character was supported by the calculated negative H(r) and ∇²ρ_r values (Table ). The G(r)/V(r) ratio, used to differentiate the interaction types, revealed covalent interactions G(r)/V­(r) < 0.5 for most bonds, except for BCP 6 (N3–H27) in ligand H₂L, which exhibited a noncovalent (van der Waals) interaction G(r)/V(r) > 1.

2. Calculated Topological Parameters (in a.u) of Ligand H₂L.

BCP no.	atoms	ρr	∇2ρr	V r	G r	H r	G r/V r
1	C2–N3	0.339	–0.933	–0.747	0.257	–0.490	0.344
2	C1–H23	0.282	–0.975	–0.319	0.037	–0.281	0.118
3	C1–C2	0.311	–0.876	–0.419	0.100	–0.319	0.239
4	N8–H27	0.333	–1.653	–0.513	0.050	–0.463	0.097
5	N3–C4	0.338	–0.964	–0.719	0.239	–0.480	0.332
6	N3–H27	0.020	0.082	–0.014	0.017	0.003	1.228
7	C7–N8	0.313	–0.882	–0.630	0.205	–0.425	0.325
8	C1–C6	0.310	–0.874	–0.419	0.100	–0.318	0.239
9	N8–N9	0.359	–0.691	–0.520	0.173	–0.346	0.334
10	C4–C7	0.258	–0.633	–0.272	0.057	–0.215	0.209
11	C5–C6	0.311	–0.879	–0.422	0.101	–0.321	0.240
12	C4–C5	0.311	–0.874	–0.419	0.100	–0.319	0.240
13	C6–H26	0.283	–0.984	–0.318	0.036	–0.282	0.113
14	N9–C21	0.370	–0.692	–0.991	0.409	–0.582	0.413
15	C7–O10	0.410	–0.253	–1.318	0.627	–0.690	0.476
16	C5–H25	0.285	–0.999	–0.319	0.035	–0.284	0.108
17	C11–C21	0.273	–0.699	–0.313	0.069	–0.244	0.221
18	C11–C12	0.294	–0.796	–0.369	0.085	–0.284	0.231
19	C12–C13	0.321	–0.913	–0.459	0.115	–0.344	0.251
20	C13–H28	0.276	–0.928	–0.316	0.042	–0.274	0.132
21	C13–C14	0.295	–0.792	–0.376	0.089	–0.287	0.237
22	C11–C16	0.314	–0.880	–0.433	0.107	–0.327	0.246
23	C14–C15	0.291	–0.774	–0.363	0.085	–0.278	0.233
24	C17–H30	0.280	–0.957	–0.318	0.039	–0.279	0.124
25	C17–C18	0.319	–0.909	–0.447	0.110	–0.337	0.246
26	C15–C16	0.300	–0.820	–0.387	0.091	–0.296	0.235
27	C14–C17	0.296	–0.801	–0.377	0.088	–0.289	0.234
28	C18–C19	0.297	–0.807	–0.380	0.089	–0.291	0.235
29	C18–H31	0.282	–0.969	–0.319	0.038	–0.281	0.120
30	C15–C20	0.295	–0.797	–0.373	0.087	–0.286	0.233
31	C19–C20	0.320	–0.915	–0.450	0.111	–0.339	0.246
32	C16–H29	0.284	–0.988	–0.318	0.036	–0.283	0.112
33	C19–H32	0.282	–0.967	–0.319	0.039	–0.281	0.121
34	C12–O22	0.279	–0.358	–0.695	0.303	–0.392	0.436
35	C2–H24	0.286	–0.999	–0.319	0.034	–0.284	0.108
36	C20–H33	0.281	–0.967	–0.318	0.038	–0.280	0.120
37	C21–H34	0.280	–0.955	–0.311	0.036	–0.275	0.116
38	O22–H35	0.366	–2.535	–0.776	0.071	–0.705	0.092

5.

Relief and contour maps of ligand H₂L.

3.8.2. Reduced Density Gradient

RDG analysis was employed to characterize the noncovalent interactions. RDG, a dimensionless quantity, serves as a metric for evaluating weak interactions in real space, according to electron density, and is defined by the equation

$$\mathrm{R}\mathrm{D}\mathrm{G}=(r)=\frac{1}{1{\left(3\pi r^2\right)}^{1/3}}\frac{\left|{\mathrm{\Delta }}^2{\rho }_{\mathrm{r}}\right|}{{\rho }_{\mathrm{r}}^{4/3}}$$ 3

This method enables the discovery of areas with less electron density, indicating weak interactions, while high-density gradient values correspond to strong interactions. Scatter plots and color-filled contour maps of the RDG for the investigated compounds were generated using the Multiwfn program (Figure ). The second eigenvalue of the Hessian matrix (λ₂) was employed as a criterion to differentiate between bonded interactions, characterized by λ₂ < 0, and nonbonded interactions, marked by λ₂ > 0. In this investigation, the ρ function associated with the λ₂ sign exhibited a range spanning from −0.05 to 0.05 atomic units (a.u.). The RDG spectra were color-coded as red, green, and blue. The red peaks in the λ₂ > 0 region indicate steric repulsion within the ring. The peaks observed in the λ₂ ≈ 0 region represent London dispersion or dipole–dipole forces. The blue peaks in the ρ > 0 and λ₂ < 0 regions signify electrostatic interactions, such as hydrogen and halogen bonds.

6.

NCI and RDG graphs of ligand H₂L.

3.9. Biological Applications

3.9.1. Antioxidant Potency

The antiradical potency of the prepared compounds was assessed by measuring the IC₅₀ values, with lower IC₅₀ values indicating greater antioxidant activity. We evaluated the antiradical activity of ascorbic acid as a reference antioxidant, and the findings are shown in Figure . H₂L exhibited moderate antioxidant properties with an IC₅₀ of 0.156 ± 0.0099 mg/mL. The [Cr­(L)­(H₂O)₂Cl]·H₂O, [Mn­(HL)], and [CoL]·2H₂O complexes are more active than the ligand alone, with IC₅₀ values of 0.0258 ± 0.0058, 0.0341 ± 0.0082, and 0.0839 ± 0.0057 mg/mL, respectively. This suggests that the complex formation-induced structural changes redistribute the electrostatic charge, thereby increasing the activity. The interaction between the ligand and the positively charged metal induces a transfer of electron density away from the ligand, thereby enhancing its polarization. Overall, the findings suggest that complexation can greatly enhance the bioactivity of organic ligands, with the [Cr­(L)­(H₂O)₂Cl]·H₂O complex identified as the most potent antioxidant among the compounds tested, suggesting their potential for further investigation and development as therapeutic agents targeting pathological conditions associated with oxidative stress. ,

7.

DPPH scavenging activities (IC₅₀ values) of the studied compounds.

This enhancement can be attributed to metal–ligand complexation that leads to structural and electronic modifications that redistribute electron density, thereby augmenting the free radical scavenging ability of the ligand. Structural modifications of the ligand induced by coordination with 3d-transition metals result in distinct variations in the biological activity of the corresponding complexes. Existing literature corroborates that metal complexation significantly influences the microbiological and cytotoxic profiles of ligands. The reaction mechanism is likely associated with electron transfer and radical stabilization initiated via coordination of the ligand to the positively charged metal centers. Transition metal complexation increases the electron-withdrawing nature of the ligand, enhancing its polarization and enabling hydrogen atom transfer or single-electron transfer mechanisms. For example, the presence of solvent molecules in [Cr­(L)­(H₂O)₂Cl]·H₂O and the tendency of Cr­(III) to strongly accept an electron raise the redox potential of the complex, making it the most effective antioxidant compound among those investigated. The interaction with the positively charged metal results in an electron density shift away from the ligand, thereby polarizing it further. The findings concur with previous observations that biologically active metal complexes of ligands have greater antioxidant activity compared to free ligands due to synergistic metal–ligand interactions.

Overall, the findings suggest that complexation can greatly enhance the bioactivity of organic ligands, with the [Cr­(L)­(H₂O)₂Cl]·H₂O complex identified as the most potent antioxidant among the compounds tested, suggesting their potential for further investigation and development as therapeutic agents targeting pathological conditions associated with oxidative stress. ,

3.9.2. Antibacterial Activity

The synthesized ligand (H₂L), along with its [Cr­(L)­(H₂O)₂Cl]·H₂O, [CoL]·2H₂O, and [Mn­(HL)] chelates, were evaluated for their in vitro antibacterial potency against various microbial strains comparable to Ciprofloxacin antibiotic as the reference documented in Table .

• For the Escherichia coli strain, the findings demonstrate that the [Mn­(HL)] chelate exhibits greater antibacterial potential than the ligand and other complexes. The antibacterial activity for this study decreased as Mn­(II) > Cr­(III) = Co­(II)> H₂L.

• For the Salmonella typhimurium and Enterobacter cloacae strains, all the tested compounds did not exhibit antibacterial activities.

• For the Klebsiella pneumonia strain, only the Mn­(II) complex exhibited antibacterial activity.

• For the Bacillus subtilis strain, Cr­(III) exhibits higher activity than H₂L, while the [Mn­(HL)] and [CoL]·2H₂O complexes are inactive against Bacillus subtilis.

• For the Bacillus cereus strain, the order of activity was Cr­(III) > Mn­(II) > H₂L, while the cobalt complex did not exhibit antibacterial activity.

• For Staphylococcus aureus and Staphylococcus epidermidis strains, the antibacterial activities decreased in the order Cr­(III) > Co­(II) > Mn­(II) > H₂L.

3. Antibacterial Activities of the Studied Complexes .

	inhibition zones in mm
microorganisms	H2L	Mn(II)	Cr(III)	Co(II)	control (DMSO)	antibiotic
Gram-negative Bacteria
Escherichia coli	14	20	18	16		25
Salmonella typhimurium						14
Klebsiella pneumonia		14				20
Enterobacter cloacae
Gram-positive Bacteria
Bacillus subtilis	14		19			25
Bacillus cereus	11	16	17			15
Staphylococcus aureus	12	15	17	16		23
Staphylococcus epidermidis	12	16	17	16		23

^aNo inhibition zone means <9 mm.

The antibacterial application of metal complexes varies due to several factors, including the metal’s biological compatibility, its interactions with enzymatic systems, the generation of oxidative stress, the complex’s stability, and the inherent toxicity of the metal. The results demonstrated that most of the studied metal complexes exhibited greater antibacterial activity than H₂L. This enhanced activity can be attributed to the improved lipophilicity of the complexes resulting from chelation.

3.9.3. Determination of Minimum Inhibitory Concentration (MIC)

All of the complexes showed strong antibacterial activity against the Escherichia coli strain. Given their strong antibacterial properties, these complexes were subjected to minimum inhibitory concentration (MIC) testing against Escherichia coli (Table ). After 24 h of incubation at 37 °C, turbidity was observed starting from a concentration of 0.625 mg/mL H₂L, indicating an MIC of 1.250 mg/mL. In the Mn­(II) complex, turbidity was observed starting from a concentration of 0.156 mg/mL, indicating an MIC of 0.313 mg/mL. In the Cr­(III) complex, turbidity was observed starting from a concentration of 0.313 mg/mL, indicating an MIC of 0.625 mg/mL. In Co­(II), turbidity was observed starting from a concentration of 0.313 mg/mL, indicating an MIC of 0.625 mg/mL. Based on the MIC values, the activity of the tested samples decreased in the following order: Mn­(II) (0.313 mg/mL) > Co­(II) (0.625 mg/mL) = Cr­(III) (0.625 mg/mL) > L (1.250 mg/mL). Overall, the findings indicate that Mn­(II) has strong efficacy against bacterial pathogens, indicating that it could be a viable candidate for antimicrobial drug development.

4. Measured Optical Densities at a Wavelength of 600 nm for E. coli for the Investigated Compounds.

	OD600
concentration (mM)	Cr(III)	Mn(II)	Co(II)
5.000	0.02	0.091	0.066
2.500	0.08	0.120	0.067
1.250	0.04	0.074	0.112
0.625	0.014	0.053	0.042
0.313	1.074	0.037	0.940
0.156	0.981	0.022	0.985
0.078	1.040	0.817	1.242

3.9.4. DNA Degradation Activity

DNA degradation activity was assessed by using agarose gel electrophoresis. In the experiment, DNA alone and DNA with DMSO solvent were utilized as controls, and no damage to DNA was observed in lanes 1 and 2 (Figure S20). DNA cleavage activity gradually increased with increasing concentrations of the investigated substances. Figure S20 shows the results using ligand concentrations of 1, 3, and 5 mM and their complexes. The findings reveal a concentration-dependent influence on DNA cleavage. At a higher concentration of 5 mM, all the compounds exhibited complete DNA degradation. At a concentration of 3 mM, significant DNA cleavage activity was observed, with the highest effect for the Co­(II) complex. Finally, at a low concentration of 1 mM, the examined compounds exhibited moderate DNA degradation activity. DNA intercalation refers to the insertion of planar molecules between the stacked base pairs of double-stranded genomic DNA, disrupting its native structure. This process leads to a notable decrease in the helical twist of the DNA structure. The observed differences in cleavage efficiency among the compounds might be attributed to variations in their DNA-binding affinities. Therefore, based on the demonstrated DNA-binding properties of the investigated samples, it was deemed pertinent to evaluate their potential anticancer properties.

3.9.5. Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The effect of the examined samples on the BSA protein was assessed, and the results are shown in Figure S21. The disappearance of the band in the [Cr­(L)­(H₂O)₂Cl]·H₂O complex suggests strong BSA binding and potential aggregation that prevents the protein from entering the gel, as observed in lane six. A significant effect is observed in lane four, which is assigned to the [CoL]·2H₂O complex. A weak effect was observed for the [Mn­(HL)] complex, while the H₂L ligand displayed no effect compared to the control. The varied effects of metal complexes on BSA may be influenced by multiple factors, including the chemical structure of the complexes, the type of metal ions, and protein–metal interactions.

3.9.6. Anticancer Activity

The anticancer properties of the H₂L Schiff base and its corresponding metal chelates were assessed utilizing the MTT assay, targeting HeLa, MCF-7, and HCT-116 cell lines. The isolated compounds were prepared at different concentrations (1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM) in DMSO solvent. The effects of the compounds on the three cell lines were evaluated based on the IC₅₀ values, representing the drug concentration required to reduce cell viability by 50%. The percentages of relative cell viability at various concentrations are shown in Figure , and the IC₅₀ values are tabulated in Table . The results were compared with those of doxorubicin, which served as the standard reference.

• The [CoL]·2H₂O complex exhibited very strong potency against the HeLa, HCT-116, and MCF-7 cell lines with IC₅₀ values of 7.76 ± 0.4, 10.23 ± 0.8, and 6.88 ± 0.4 μM, respectively.

• The [Mn­(HL)] complex displayed very strong anticancer potency against the MCF-7 cell line with an IC₅₀ of 9.46 ± 0.7 μM. Moreover, it demonstrated strong activity against HeLa and HCT-116 cell lines with IC₅₀ values of 13.47 ± 1.2 and 15.56 ± 1.3 μM, respectively.

• The [Cr­(L)­(H₂O)₂Cl]·H₂O complex exhibited strong antitumor properties against MCF-7 cells, with an IC₅₀ value of 17.79 ± 1.3 μM and moderate activities against HeLa and HCT-116 cell lines with IC₅₀ values of 26.10 ± 1.8 and 22.49 ± 1.5 μM, respectively.

• The H₂L ligand exhibited moderate anticancer properties against HeLa and MCF-7 cells with IC₅₀ values of 42.65 ± 2.5 and 29.74 ± 1.9 μM, respectively, while it exhibited weak activity against cancer HCT-116. According to the results, the anticancer potency of the tested compounds follows the order [CoL]·2H₂O> [Mn­(HL)]> [Cr­(L)­(H₂O)₂Cl]·H₂O > H₂L ligand.

8.

Cell viability of the studied compounds against the human cancer (A) HeLa, (B) HCT-116, and (C) MCF-7 cell lines.

5. Anticancer Activities of the Studied Compounds against Human Cell Lines.

	in vitro cytotoxicity, IC50 (μM)
compound	HeLa	HCT-116	MCF-7
doxorubicin	5.57 ± 0.4	5.23 ± 0.3	4.17 ± 0.2
H2L ligand	42.65 ± 2.5	67.15 ± 3.6	29.74 ± 1.9
[CoL]·2H2O	7.76 ± 0.4	10.23 ± 0.8	6.88 ± 0.4
[Cr(L)(H2O)2Cl]·H2O	26.10 ± 1.8	22.49 ± 1.5	17.79 ± 1.3
[Mn(HL)]	13.47 ± 1.2	15.56 ± 1.3	9.46 ± 0.7

The results indicate that the metal complexes exhibit greater efficacy in inhibiting cancer cell proliferation than their corresponding free ligands. These findings suggest that the chelation of metal ions to H₂L Schiff bases markedly improves their anticancer efficacy and alters their biological activity profile. Enhanced activity may be attributed to the influence of the metal ions, which increase the acidity of the ligand, thereby promoting the formation of strong hydrogen bonds. The high activity of the Co­(II) and Mn­(II) complexes in influencing cancer cell processes can be attributed to their adequate lipid solubility, which facilitates metal transport across cellular membranes and significant thermodynamic stability to ensure that they reach the target site intact. The reduced activity of the Cr­(III) complex could be attributed to its low lipid solubility, which hinders the transport of the metal atom to the precise target site. As a result, the cancer cell wall remains unaffected, and no interference occurs with cellular processes, allowing normal cell activity to proceed uninterrupted.

3.10. Molecular Docking

3.10.1. Induced Fit Docking Simulation

Molecular docking simulations were performed to estimate the binding affinities of the synthesized compounds toward the target DNA helices of the selected receptors, providing detailed insights into their interaction profiles (Figures and S22–S25). The induced fit docking (IFD) protocol was employed, which accounts for both ligand and receptor flexibility. This iterative procedure involves initial Glide docking with softened potentials, followed by prime side-chain prediction and minimization, and subsequent redocking of the ligands into the refined receptor structures. The IFD methodology, which utilizes the Refinement module in Prime v 7.7, was selected to accurately predict the binding modes of the studied compounds within the active sites of 5IAE, 1YWN, and 3W2S (Tables and ). For HeLa and HCT-116 inhibition, the [Cr­(L)­(H₂O)₂Cl]·H₂O complex exhibited significant negative G _emodel values (−38.119 kcal/mol for HeLa and −53.954 kcal/mol for HCT-116), indicative of stable binding. These values were attributed to substantial electrostatic (G _eCoul = −29.795 kcal/mol for HeLa and −21.777 kcal/mol for HCT-116) and van der Waals (G _evdW = −18.418 kcal/mol for HeLa and −17.213 kcal/mol for HCT-116) energy contributions, as determined by the OPLS4 force field. Furthermore, favorable G _score (−7.765 and −6.415 kcal/mol), RMSD (1.662 and 2.486 Å), hydrophobic (G _lipo = −1.256 and −1.092 kcal/mol), and IFD_score (−559.43 and −618.63 kcal/mol) values confirmed the high binding affinity of [Cr­(L)­(H₂O)₂Cl]·H₂O with the 5IAE and 1YWN receptors. Interaction analysis revealed a single aromatic ring interaction with TYR204 (5.09 Å) in HeLa and a hydrogen bond between CN and ARG840 (2.49 Å) in HCT-116 (Figure S22).

9.

3D poses of free ligand H₂L with the (a) HeLa, (b) HCT-116, and (c) MCF-7 targets.

6. Molecular Docking IFD Scores of the Investigated Compounds against Selected Targets.

compound	IFD score	RMSD (Å)	interactions	type	distance (Å)
HeLa (PDB ID: 5IAE)
original ligand	–576.22	0.920	PHE250→(CO)	H-bond	1.88
			PHE250←(H2O)→(CO)	H-bond	1.90. 1.73
			TRP214→(C–O–)	H-bond	2.39
			SER65←(H2O)→(CO)	H-bond	1.97, 1,93
			SER65→(C–O–)	H-bond	2.34
			ARG64---(C–O–)	salt bridge	2.96
			GLY122→(CO)	H-bond	2.31
			GLY122←(H2O)→(CO)	H-bond	1.74, 1.70
			GLN161→(CO)	H-bond	1.89
			CYS163→(CO)	H-bond	2.79
			NH→SER205	H-bond	2.38
			ARG207---(C–O–)	salt bridge	2.98
			ARG207---(C–O–)	salt bridge	3.94
			ARG207→(CO)	H-bond	1.80
			NH→ARG207	H-bond	1.60
			ARG207←(H2O)→(C–O–)	H-bond	1.98, 2.25
H2L ligand	–555.87	1.157	SER209→CN	H-bond	2.34
			ARG207←(H2O)→(C–O–)	H-bond	1.91, 1.97
			ARG207←(H2O)→(CO)	H-bond	1.91, 1.84
			ARG207---(C–O–)	salt bridge	2.84
[Cr(L)(H2O)2Cl]·H2O	–559.43	1.662	TYR204---Ar ring	π–π stacking	5.09
[CoL]·2H2O	–547.37	1.464	TRP206---Ar ring	π–π stacking	5.23
			ARG207---(C–N–)	salt bridge	4.62
[Mn(HL)]	–549.25	0.905	ARG207→(C–O–)	H-bond	2.40
			TRP206---Ar ring	π–π stacking	3.81
			TRP206---Ar ring	π–π stacking	4.11
			TRP206---Ar ring	π–cation	5.66
doxorubicin	–553	1.942	TYR214→(H2O)→(OH)	H-bond	1.81, 1.55
			TYR204←(H2O)→(CO)	H-bond	1.81, 1.87
			OH→SER205	H-bond	2.07
			ARG207→(OH)	H-bond	2.10
HCT-116 (PDB ID: 1YWN)
original ligand	–623.21	0.590	NH→GLU883	H-bond	2.15
			NH→GLU883	H-bond	2.05
			ASP1044→(CO)	H-bond	1.86
			NH2→GLU915	H-bond	1.80
H2L ligand	–611.89	2.352	ASP1044←H2O→(CN)	H-bond	1.99, 1.87
			ASP1044→(CO)	H-bond	2.57
			LYS866→H2O→(CN)	H-bond	2.11, 1.87
			LYS866---(C–O–)	salt bridge	2.85
[Cr(L)(H2O)2Cl]·H2O	–618.63	2.486	ARG840→(CN)	H-bond	2.49
[CoL]·2H2O	–606.82	2.845	LYS866---(C–O–)	salt bridge	3.63
[Mn(HL)]	–608.94	2.647	solvent exposure
doxorubicin	–607.98	1.913	ASP1044→NH2	H-bond	1.98
			ASP1044←H2O→(C–O–)	H-bond	1.92, 1.77
			OH→GLU915	H-bond	1.82
MCF-7 (PDB ID: 3W2S)
original ligand	–673.03	0.823	MET893→(CN)	H-bond	1.94
			CYS775→(H2O)→(CN)	H-bond	2.48, 1.94
			THR854→(H2O)→(CN)	H-bond	1.86, 2.39
			THR854←(H2O)→(CN)	H-bond	2.62, 2.39
			NH→PHE856	H-bond	2.07
			PHE856---Ar ring	π–π stacking	5.31
			NH→GLY857	H-bond	2.37
			LYS745→(CO)	H-bond	1.67
			Cl→LEU788	halogen bond	3.09
ligand	–658.63	1.812	PHE856---Ar ring	π–π stacking	4.86
			LYS745---Ar ring	π–cation	3.64
[Cr(L)(H2O)2Cl]·H2O	–658.17	1.308	LYS745---(C–N–)	salt bridge	2.82
			PHE723---Py-ring	π–π stacking	5.37
[CoL]·2H2O	–649.25	1.368	solvent exposure
[Mn(HL)]	–663.18	1.278	LYS745→ (C–O–)	H-bond	3.09
			NH→GLY857	H-bond	1.97
doxorubicin	–658.35	1.079	LYS745→(C–O–)	H-bond	1.84
			OH→GLY857	H-bond	1.74
			OH→GLU749	H-bond	1.87
			(NH3)→ASP855	H-bond	2.07
			(NH3)---ASP855	salt bridge	2.91
			(NH3)→ASN842	H-bond	2.73
			OH→ASN842	H-bond	1.85

7. Induced Fit Docking Scores of the Investigated Compounds against Selected Targets (kcal/mol).

compound	G score	G evdW	G eCoul	G energy	G emodel	G hbond	G lipo	IFDscore
HeLa (PDB ID: 5IAE)
original ligand	–17.346	–37.314	–79.701	–117.014	–244.430	–4.547	–1.354	–576.220
H2L ligand	–6.484	–25.547	–20.673	–46.220	–69.196	–0.416	–1.259	–555.870
[Cr(L)(H2O)2Cl]·H2O	–7.765	–18.418	–29.795	–48.213	–38.119	–0.780	–1.256	–559.430
[CoL]·2H2O	–4.599	–32.146	–0.454	–32.601	–42.487	0.000	–0.958	–547.370
[Mn(HL)]	–4.974	–27.744	–10.759	–38.503	–44.176	–0.223	–1.735	–549.250
doxorubicin	–11.095	–21.689	–34.161	–55.850	–99.426	–0.231	–1.497	–553.000
HCT-116 (PDB ID: 1YWN)
original ligand	–14.678	–59.698	–18.167	–77.865	–140.736	–1.126	–3.953	–623.210
H2L ligand	–7.615	–38.024	–6.092	–44.116	–63.179	–0.456	–1.985	–611.890
[Cr(L)(H2O)2Cl]·H2O	–6.415	–17.213	–21.777	–38.990	–53.954	–0.563	–1.092	–618.630
[CoL]·2H2O	–7.716	–31.411	–6.259	–37.670	–51.285	0.000	–1.650	–606.820
[Mn(HL)]	–6.681	–37.771	–7.835	–45.606	–61.006	0.000	–2.744	–608.940
doxorubicin	–8.996	–43.242	–13.954	–57.196	–59.170	–0.045	–1.926	–607.980
MCF-7 (PDB ID: 3W2S)
original ligand	–14.377	–73.002	–22.734	–95.737	–154.482	–1.119	–5.353	–673.030
H2L ligand	–5.561	–37.043	–0.627	–37.670	–49.019	–0.133	–1.646	–658.630
[Cr(L)(H2O)2Cl]·H2O	–6.901	–38.553	–20.461	–59.014	–32.654	–0.096	–2.085	–658.170
[CoL]·2H2O	–4.081	–42.893	–1.996	–44.889	33.088	0.000	–2.332	–649.250
[Mn(HL)]	–12.402	–40.857	–10.445	–51.302	–75.511	–0.520	–2.114	–663.180
doxorubicin	–11.259	–51.537	–21.390	–72.927	–107.495	–0.232	–2.222	–658.350

^aGlide score.

^bGlide van der Waals energy.

^cGlide Coulomb energy.

^dGlide energy.

^eGlide model energy.

^fGlide hydrogen bonding.

^gGlide lipophilic contact plus phobic attractive term in the glide score.

^hInduced fit docking score.

For MCF-7 inhibition, the free ligand H₂L demonstrated a highly negative G _emodel (−49.019 kcal/mol), driven by significant electrostatic (G _eCoul = −0.627 kcal/mol) and van der Waals (G _evdW = −37.043 kcal/mol) energy contributions, indicating stable binding. Additionally, favorable G _score (−5.591 kcal/mol), RMSD (1.812 Å), hydrophobic (G _lipo = −1.646 kcal/mol), and IFD_score (−658.63 kcal/mol) values indicated strong binding affinity with the 3W2S receptor. Interaction analysis showed π–π stacking with PHE856 (4.86 Å) and π–cation stacking with LYS745 (3.64 Å) (Figure c).

Although molecular docking is acknowledged as a valuable tool, it has inherent limitations due to its dependence on algorithms, scoring functions, and active site sensitivity. Moreover, it does not fully account for experimental factors, microbial characteristics, or drug transport mechanisms.

3.10.2. Docking Validation

Redocking experiments were performed to validate the docking protocol and assess its efficiency using identical docking parameters. The native peptide inhibitor in the 5IAE receptor exhibited precise binding within the active site pocket, characterized by three salt-bridge interactions with ARG64 and ARG207, and 13 hydrogen bonds involving residues PHE250, TRP214, SER65, ARG64, GLY122, GLN161, CYS163, SER205, and ARG207. The redocked native ligand in the 1YWN receptor formed six hydrogen bonds with distances ranging from 1.80 to 2.15 Å. The native inhibitor in the 3W2S receptor interacted with the active site pocket through seven hydrogen bonds, including three water-mediated interactions, a π–π stacking interaction between an aromatic ring and PHE856 (5.31 Å), and a halogen bond between Cl and LEU788 (3.9 Å). An overlay analysis of the redocked ligands with the native cocrystallized structures was conducted (Figure ). The redocked inhibitors bound to the 5IAE, 1YWN, and 3W2S receptors exhibited IFD scores of −576.22, −623.21, and −673.03 kcal mol^–1, respectively, and RMSD values of 0.920, 0.590, and 0.823 Å, respectively. The low RMSD values of 0.920, 0.590, and 0.823 Å upon redocking confirm the reliability and accuracy of the chosen IFD protocol for the SIAE, 1YWN, and 3W2S receptors, respectively.

10.

Superimposition of the redocked original ligand (represented in gray) and the native cocrystallized ligand (depicted in green) within the binding pocket of the (a) HeLa, (b) HCT-116, and (c) MCF-7 receptors.

4. Conclusion

In summary, the effective synthesis and full characterization of H₂L and its transition metal complexes were carried out through an extensive series of physicochemical, analytical, and spectroscopic investigations. Spectroscopic evidence confirmed the tetrahedral geometries of the Co­(II) and Mn­(II) complexes and octahedral coordination spheres of the Cr­(III) complexes. Quantum chemical calculations with QTAIM and RDG provided a wealth of information on bonding and noncovalent interactions, which proved to be decisive for the stabilization of ligand–receptor complexes and rational drug design. Thermogravimetric analysis, supported by activation energy calculations, revealed variations in the thermal stabilities of the complexes. The optical band gap values also indicate that these materials are well-suited for solar energy collection as well as the assembly of photovoltaic devices. Owing to its pronounced DNA-binding affinity and superior anticancer activity, the Co­(II) complex emerges as a promising candidate for in vivo application as an antitumor agent. Its potential mechanism involves the inhibition of DNA replication within malignant cells, thereby contributing to the suppression of tumor development. The complexes exhibited structure-dependent and selective interactions with bovine serum albumin (BSA). The rise in anticancer activity upon metal coordination arises from the increased ligand acidity involving strong hydrogen bonding interactions. Remarkably, computational docking demonstrated that the [Cr­(L)­(H₂O)₂Cl]·H₂O complex exhibited high binding affinities with HeLa and HCT-116 DNA helices and a selective affinity of the free ligand with the MCF-7 cell line. These multifaceted findings affirm the promise of these compounds as multifunctional candidates for photovoltaic and therapeutic applications.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05921.

• Chemicals, instruments, ¹H NMR, ¹³C NMR, FT-IR, UV–vis, and mass spectra of the ligand and its complexes; figures and tables; and details on the XRD, thermal curves, kinetic data, and theoretical studies. (PDF)

Yasmeen G. Abou El-Reash: Data curation, project administration, and review and editing; Saja Abdulrahman Althobaiti: data curation; Sahar Abdalla: data curation; Gaber M. Abu El-Reash: conceptualization and formal analysis; and Mahdi A. Mohammed: methodology, writing original draft, software, visualization, and review and editing.

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501).

The authors declare no competing financial interest.