Biomacromolecular Interactions, Cytotoxicity, and Theoretical Studies of Aldimine-Based Vanadyl Complexes

Abstract

Four vanadyl complexes (1–4) based on methyl, nitro, chloro, and fluoro-substituted aldimine ligands, respectively, have been designed, and the structures and geometries are optimized through DFT studies. The interactions of the complexes with DNA, as per UV absorption titration, indicate that complexes 1–3 show a hyperchromic shift and complex 4 shows a hypochromic shift, implying groove and intercalation modes of binding, respectively. The highest binding constant (K_b) value, 9.84 × 10⁵ M^–1, is obtained for the methyl-substituted complex 1. The competitive binding of the complexes (1–4), through a fluorescence assay, shows the highest Stern–Volmer constant (K_SV) value of 7.81 × 10³ M^–1 for methyl-substituted complex 1. The interaction of the complexes with BSA, as determined by the tryptophan emission quenching method, indicates a dose-dependent quenching of the intrinsic fluorescence of BSA with increasing complex concentration. All complexes exhibit moderate radical scavenging activity compared to the standard, as per antioxidant studies. Furthermore, the predictions of molecular docking studies with both DNA and BSA support the experimental observations. The complexes cleave pBR322 DNA based on gel electrophoretic studies. The cytotoxicity study, against MCF-7 cancer cells, implies that nitro-substituted complex 2 has a higher cytotoxicity than other complexes.

1. Introduction

Schiff’s bases have long been regarded as one of the key ligand systems in coordination chemistry, and the associated metal complexes have been investigated for a variety of uses. − Salicylaldehyde derivative-based Schiff bases strongly coordinate to metals and have been thoroughly investigated for various biological functions. , The conjugated aromatic–azomethine scaffolds of the ligands chosen for this investigation are structurally linked to show antibacterial, antioxidant, anticancer, and enzyme–inhibitory properties. When related to transition metals, such as metal chelation, the azomethine (−C = N−) functionality is enhanced for biological activity. Their intriguing coordination chemistry is enhanced by the electronic effects brought about by the presence of electron-donating or electron-withdrawing groups, which support their diverse biological roles. Salophen, salen, and salan are examples of N₂O₂ ligand-based compounds that are considered potent chemotherapeutic agents. Halogen-substituted medications are considered advantageous and effective, increasing solubility across cell membranes. Vanadyl-Schiff base complexes have been extensively explored as insulin mimetics and for enzyme inhibitory action. Additionally, the compounds possess antibacterial, anti-inflammatory, and anticancer potential. − Numerous research groups have established a correlation between DNA interaction and anticancer effect against distinct cancer cell types. Further, DNA-cleaving ability has been observed using gel electrophoresis, suggesting that the complex and DNA have binding interactions. − Bovine serum albumin, called BSA, is a hydrophilic protein that is widely utilized in biochemical research. The interaction of metal complexes with BSA is one of the crucial factors to be assessed for drug-like compounds. Through their interaction with the protein, the drugs are efficiently distributed and made accessible at the site of action to treat the disease. − Antioxidants protect the biological systems against the oxidative stress caused due to the imbalance that occurs between production and the removal of reactive oxygen species (ROS). There are numerous techniques for evaluating antioxidant activity, including DPPH and H₂O₂ tests. The MCF-7 cell line, one of the popular models for breast cancer, has been used to evaluate the cytotoxic efficacy of the test compounds, acting as chemotherapeutic agents to destroy cancer cells. , It is understood that the electronic, steric, and lipophilic characteristics of compounds are influenced by the electronic effects of the substituents. This would rather impact the binding affinity with nucleic acid and protein, cytotoxic potential, causing apoptosis. It has been reported that the complexes with electron-donating groups and large aromatic moieties may direct the compounds to bind to DNA either through intercalation or grooves. However, the complexes with electron-donating groups may promote ROS-mediated apoptosis. , The structural elucidation and biological characteristics, including antibacterial and antioxidant properties, as well as interaction studies with CT-DNA, BSA, and HSA, of Schiff base vanadyl complexes have been investigated. A study on the synthesis of chiral Schiff base complexes V­(V) (S)-[VO­(OMe)­L] and (R)-[VO­(OMe)­L] (L = 2-((E) ((10S,20R)-20-hydroxy-10,20-diphenylethylimino)­methyl)­phenol), exhibiting groove binding with DNA, and cytotoxicity against HepG2 cells have been reported. Similar studies have been reported with mononuclear oxovanadium compounds with tridentate O^N^O Schiff base ligands and dibenzofuran. The DNA/BSA binding studies have been assessed through various techniques. Several such reports based on multiple Schiff base vanadium compounds and their anticancer applications have been investigated. ,−

The above investigations have significance in various domains, including molecular biology, supramolecular chemistry, drug transportation, and pharmaceutical design. , In the current work, we are reporting hitherto unreported, aldimine-based vanadyl complexes toward investigating the cytotoxic potential. Experimental observations are supported by theoretical investigations, wherein the optimized geometry of the complexes is given by density functional theory. The interactions with DNA and BSA are carried out both experimentally and using molecular docking studies.

2. Materials and Methods

Each chemical was purchased from a commercial supplier and used without further purification. Sigma-Aldrich provided 3,5-dichlorosalicylaldehyde, 4-chloro-o-phenylenediamine, 4-fluoro-o-phenylenediamine, 4-nitro-o-phenylenediamine, 4-methyl ortho phenylenediamine, and [VO (acac)₂]. The AR-grade methanol, acetonitrile, and ethanol solvents were supplied by SD Fine Chemicals. We purchased ethidium bromide (EtBr), bovine serum albumin (BSA), calf thymus DNA (CT-DNA), pBR322, Tris­(hydroxymethyl)­methylamine agarose, and TBE buffer from Merck Millipore.

2.1. Instruments

Using the JASCO UV–vis-NIR V-670 spectrophotometer, the UV–visible absorption spectra were recorded. FT-IR spectra were captured using a Shimadzu IR Affinity-1CE model with a IV resolution in the 4000–400 cm- range. The elemental tests were conducted using a PerkinElmer CHNS (2400 II, U.S.A.). Electron paramagnetic resonance (EPR) spectra were recorded using a JEOL Model JES FA200 EPR spectrometer operating in the X-band (8.5–9.5 GHz) with a magnetic field as high as 2 T. A Hitachi F-7000 FL spectrophotometer was used to record fluorescence spectra. Gel electrophoresis and gel recording using a 24-megapixel digital camera (CMOS). The mass spectra were recorded with an ESQUIRE3000_00037 spectrometer.

2.2. Preparation of Ligands and Vanadyl Complexes

2.2.1. Ligands

The preparation of the four ligands, HL1-HL4, has been reported earlier by our group elsewhere. , The ligands possess varying substituents, such as HL1 – methyl substituted, HL2 – nitro substituted, HL3 – chloro substituted, and HL4 – fluoro substituted, as shown in Scheme .

1. Preparation of Complexes.

Complex 1: 20 mL of methanol was used to dissolve the methyl-substituted ligand, HL1 (1.0 mmol, 0.1 g). Fifteen mL of methanol was used to dissolve the [VO (acac)₂] (1.0 mmol, 0.124 g), which was added dropwise to the methanolic solution of the ligand. The resultant mixture was refluxed for 8 h at 70 °C. Subsequently, it was filtered, washed with methanol, and the obtained red precipitate was vacuum-dried. (Scheme ) Anal. Calc. for C₂₁H₁₂Cl₄N₂O₃V: Mol.wt. 533.0795, Yield (82%), UV–visible (DMSO, 1 × 10^–3 M): λ_max (nm) 275 (ε = 11460 M^–1 cm^–1), 320 (ε = 83700 M^–1 cm^–1), 432 (ε = 61900 M^–1 cm^–1). Mass: 530.9045 (m/z), FT-IR (ATR mode); 1641 cm^–1 (ν_C=N); 1583 cm^–1, 1507 cm^–1 (ν_C=C); 1202 cm^–1 (ν_C–O); 962 cm^–1 (ν_V=O); 504 cm^–1 (ν_V–N). Elemental analysis, found (calculated): C, 47.04 (47.32); H, 2.09 (2.27); N, 5.01 (5.26)

Complex 2: 20 mL of methanol was used to dissolve the nitro-substituted ligand, HL2 (1.0 mmol, 0.1 g). Fifteen mL of methanol was used to dissolve the [VO (acac)₂] (1.0 mmol, 0.082 g), which was added dropwise to the methanolic solution of the ligand (1:1). The resultant mixture was refluxed for 8 h at 70 °C. Subsequently, it was filtered, washed with methanol, and the obtained pale brown precipitate was vacuum-dried. Anal. Calc. for C₂₀H₉Cl₄N₃O₅V: Mol.wt. 533.0495, Yield (64%), UV–visible (DMSO, 1 × 10^–3 M): λ_max (nm) 271 (ε = 14170 M^–1 cm^–1), 328 (ε=12980 M^–1 cm^–1), 420 (ε=11080 M^–1 cm^–1), Mass: 561.8742 (m/z), FT-IR (ATR mode); 1636 cm^–1 (ν_C=N); 1588 cm^–1, 1501 cm^–1 (ν_C=C); 1261 cm^–1 (ν_C–O); 979 cm^–1 (ν_V=O); 492 cm^–1 (ν_V–N). Elemental analysis, found (calculated): C, 41.98 (42.59); H, 1.53 (1.61); N, 6.90 (7.45)

Complex 3: 20 mL of methanol was used to dissolve the chloro-substituted ligand, HL3 (1.0 mmol, 0.1 g). Fifteen mL of methanol was used to dissolve the 1.0 mmol (0.146 g) of [VO (acac)₂], which was added dropwise to it. The resulting mixture was refluxed for 8 h at 70 °C. The pale brown precipitate obtained was filtered, washed with methanol, and vacuum-dried. Anal. Calc. for C₂₀H₉Cl₅N₂O₃V: Mol. wt. 553.4945, Yield (71%), UV–visible (DMSO, 1 × 10^–3 M): λ_max (nm) 264 (ε = 25870 M^–1 cm^–1), 315 (ε = 22760 M^–1 cm^–1), 421 (ε = 17670 M^–1 cm^–1), Mass: 550.8494 (m/z), FT-IR (ATR mode); 1739 (ν C = N); 1460, 1369 (ν C = C); 1244 (ν C–O); 936 (ν V = O); 566 (ν V–N). Elemental analysis, found (calculated): C, 42.51 (43.40); H, 1.30 (1.64); N, 4.86 (5.06)

Complex 4: 20 mL of methanol was used to dissolve the fluoro-substituted ligand, HL4 (1.0 mmol, 0.1 g). Fifteen mL of methanol was used to dissolve the 1.0 mmol (0.096 g) of [VO (acac)₂], which was added dropwise. The resulting mixture was refluxed for 8 h at 70 °C 8 h at 70 °C. The pale brown precipitate obtained was filtered, washed with methanol, and vacuum-dried. Anal. Calc. for C₂₀H₉Cl₄FN₂O₃V: Mol. wt. 537.0429, Yield (83%), UV–visible (DMSO,1 × 10^–3 M): λ_max (nm) 262 (ε = 55000 M^–1 cm^–1), 313 (ε = 44380 M^–1 cm^–1), 418 (ε = 36120 M^–1 cm^–1), Mass: 534.8795 (m/z), FT-IR (ATR mode); 1750 (ν C = N); 1449, 1369 (ν C = C); 1244 (ν C–O); 942 (ν V = O); 572 (ν V–N). Elemental analysis, found (calculated): C, 43.89 (44.73); H, 1.48 (1.69); N, 5.16 (5.22)

2.3. DFT Study

The Gaussian 09 computational codes, which used density functional theory (DFT) to apply Becke’s three-parameter hybrid exchange and the Lee–Yang–Parr nonlocal correlation functional (B3LYP), were used for all calculations. All calculations use the conventional basis set 6–311G (d, p) for lighter elements (C, H, N, O, F, and Cl) and the LanL2DZ effective core potential for metal­(V). For vanadium­(IV) oxo (V = O) Schiff base and coordination complexes, the B3LYP/6–311G (d, p)/LANL2DZ level of theory was chosen because of its extensive and effective application, which yields dependable predictions for structural parameters, vibrational frequencies, and electrical characteristics. The vanadium center is effectively and accurately treated by the LANL2DZ effective core potential, while the ligand environment is well described by the 6–311G (d, p) basis set. − The d¹ configuration of VO²⁺ was taken into consideration by analyzing the system in the +2 charge and doublet spin state. All optimized geometries were verified to be true minima by harmonic vibrational frequency analysis (no imaginary frequencies), representing only the local minima associated with positive eigenvalues. Vertical electronic excitations based on B3LYP will be generated using the ground-state optimized geometry and time-dependent density functional (TD-DFT) theory. The DFT and TD-DFT were coupled with the conductor-like polarizable continuum model (CPCM) in a DMSO medium to account for the solvent impact. The fractional molecular orbital contributions are computed using the Gauss Sum.

2.4. DNA Interaction Studies: Absorption Titration

The binding properties of the complexes (1–4) to calf thymus DNA (CT-DNA) have been examined using electronic absorption spectroscopy, where the concentration is taken as 1 mg/1 mL of complexes in acetonitrile. The CT-DNA stock solution was prepared in 10 mM Tris–HCl/50 mM NaCl buffer (pH = 7.2), and the ratio of UV absorbance at 260 and 280 nm (A260/A280) and the UV absorbance at 260 nm (ε = 6600 M 1 cm 1), which is used to determine the concentration. Electronic absorption spectra were obtained by fixing the complex concentration (1.00 × 10 mol L ¹) and gradually increasing the CT-DNA concentration (0–10 μL) using 1 cm quartz cuvettes at 25 °C. The absorbance of CT-DNA itself was eliminated by adding an equivalent quantity of CT-DNA to the chemical solutions and the reference buffer solution. The absorbance of CT-DNA itself was eliminated by adding an equivalent quantity of CT-DNA to the chemical solutions and the reference buffer solution. When the CT-DNA is put in both the front and back cuvettes in an absorption titration, a correction for the absorbance of the CT-DNA itself is typically used. When the absorbance difference between the two cuvettes is determined, the absorbance of the DNA is effectively canceled out by ensuring that the concentration of CT-DNA in each cuvette is the same. ,

2.5. Competitive Displacement AssayFluorescence Technique

The binding affinities of complexes were examined using the fluorescence quenching method. Ethidium bromide (EtBr, 3,8-diamino-5-ethylphenanthridinium bromide), an organic cationic dye, is a well-known DNA intercalator that also serves as a fluorescent marker. The displacement test employed an EtBr-bound CT-DNA solution (EtBr = 10 μM and DNA = 100 μM) as a probe. The interactions of complexes with CT-DNA were investigated using an EB-bound CT-DNA solution in a 5 mM Tris-HCl/NaCl buffer (pH = 7.2). The fluorescence experiment employed a reference solution of EtBr-bound DNA with concentrations ranging from 0 to 100 μM. The emission happened at 600 nm when excited at 530 nm. With a binding constant of 1.0 × 10⁷ M^{– 1}, EtBr exhibits strong fluorescence when it intercalates between the closest DNA base pairs. Because of its tendency to intercalate into CT-DNA base pairs, it exhibits strong fluorescence when CT-DNA is added.

2.6. BSA Interaction Studies

Tryptophan fluorescence quenching was used to assess the interaction of complexes with bovine serum albumin (BSA). The complex concentration (0–20 μM) was raised at room temperature while the BSA (2 μM) concentration in PBS (pH = 7.4) buffer was constant. Additionally, (λ_ex = 290 nm) and (λ_em = 340 nm) were found to be quenched.

2.7. Antioxidant Studies

2.7.1. DPPH Assay

The DPPH method was used to evaluate the ability of complexes to scavenge free radicals at varying concentrations (50, 100, 150, 200, and 250 μg mL^–1). A solution of 1.0 mL of 0.1 mM DPPH in methanol was vortexed with additional amounts of the test chemicals, and it was then incubated in the dark for 30 min at room temperature. The decrease in absorbance of DPPH at 517 nm was assessed in comparison to a blank. The scavenging activity was measured using (eq )­

$$\mathrm{D}\mathrm{P}\mathrm{P}\mathrm{H}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=[(A_{control}-A_{sample})/A_{control}]\times 100$$ 1

Where Ac and As stand for the absorbances of the test and control samples, respectively. The results are displayed in the plot as mean ± SD, and the experiment was conducted in triplicate.

2.7.2. Hydrogen Peroxide Assay

The ability of the complex to scavenge hydrogen peroxide was investigated by combining hydrogen peroxide, FAS, and 1,10-phenanthroline at different concentrations (50,100, 150, 200, and 250 μg mL^–1). Control solutions were made by dissolving 1 mg of 1,10-phenanthroline (180.21 g/mol) in 10 mL of solvent and 1 mg of FAS (392.14 g/mol) in 10 mL of solvent. The standard used was ascorbic acid. Using (eq ), the scavenging activity was evaluated.

$$\mathrm{H}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=\frac{A_{blank}-A_{test}}{A_{blank}}\times 100$$ 2

where A_test represents the absorbance of the test substance in the presence of hydrogen peroxide, and A_blank indicates the absorbance of the blank. The experiment was conducted in triplicate.

2.8. Molecular Docking Studies

If it can predict noncovalent interactions by identifying preferential binding sites for metal complexes, molecular docking is a viable technique for creating medicinal compounds. The Autodock 1.5.7 computational tool and the Lamarckian genetic algorithm (LGA) were used to perform docking studies on complexes , with BSA and DNA. Using Autodock 1.5.7, the crystal structures of DNA (PDB ID: 1BNA) and BSA (PDB ID: 4F5S) were obtained and improved using the Protein Data Bank. Gaussian 12 (G 12) was used to design complex structures, which were then converted into PDB format and thoroughly examined. Considering the inherent limitations associated with metal parametrization and rigid-receptor assumptions, used in the standard docking protocols, the docking results for VO²⁺ complexes are qualitative in nature. Program Discovery Studio was used to make the graphic.

2.9. DNA Cleavage Study

A popular method for assessing the cleavage activity of DNA complexes is gel electrophoresis. To identify which reactive oxygen species (ROS) were involved in the cleavage reaction, the radical scavenger DMSO was introduced. The interaction and effectiveness of the vanadyl complexes on CT-DNA were investigated using the agarose gel electrophoresis method. The reaction mixtures, which each contained 20 mL of CT-DNA and 20 mL of vanadium complexes, were separately incubated in 1X TBE buffer (pH 8) solution for 1 h at 310 K. The tracking gel loading blue dye (1 mL) and all of the prepared solutions (5 mL) were then added to the wells of a 1% agarose gel that had been prepared well in advance and included 1 μM ethidium bromide. The reaction mixture of complexes and CT-DNA was then separately loaded into the wells using 0.25% gel loading blue dye. The electrophoresis mobility experiment was then carried out for 120 min at a constant 50 V electrical voltage in a Tris Borate EDTA buffer (1X TBE) until the dye had covered 50% of the gel distribution area. The degree of DNA binding capacity of the vanadyl complexes was then evaluated by taking pictures of the resultant bands under UV light using a Gel Documentation System. − The reactions were used to examine oxidative cleavage activity carried out at 37 °C for 3 h. The reaction mixture, which contained 200 ng of pBR322 in a 50 mM Tris-HCl (pH 7) buffer solution with auxiliary reagent, was supplemented with H₂O₂.

2.10. Cytotoxicity StudiesMTT Assay

The 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT) assay was used to examine the complexes’ cytotoxicity against MCF7 breast cancer cell lines in vitro. A mitochondrial enzyme known as succinate dehydrogenase transforms MTT, a yellow tetrazolium salt, into an insoluble purple formazan. The number of viable cells is directly correlated with the quantity of formazan generated. After the complexes’ known weight was dissolved in DMSO and added to the culture media, they were cultured for a full day. In MCF7 cells, the complexes decreased cell growth, and death occurred at doses between 6.5 and 100 μg/mL in a dose-dependent manner. After being incubated for 48 h, each well was filled with 15 μL of MTT (5 mg/mL) in PBS and incubated at 37 °C for 4 h. After removing the MTT medium, 100 μL of DMSO was used to dissolve the formazan crystals. A SpectraMaxL microplate reader was used to detect the absorbance at 570 nm. , The experiment was conducted in triplicate.

3. Results and Discussion

3.1. Spectral Characterization of Vanadyl Complexes

The electronic absorption spectra of the complexes (Figures S1–S4) were measured in DMSO. The electronic nature of the substituents on the Schiff base ligands causes visible alterations in the complexes’ absorption maxima. The π→π* bands in complexes with electron-donating groups (EDGs), −CH₃, show a bathochromic (red) shift, whereas those with electron-withdrawing groups (EWGs), −NO₂, −Cl, and −F, show a hypochromic (blue) shift. The strong σ-donation ability of phenolate oxygen is responsible for the O → V charge transfer, which occurs when the nonbonding 2p electrons of the coordinated phenolic oxygen are excited into the vacant d_xy or d_x² – y² orbital of the V­(IV) center. The N → V contribution involves the transfer of electron density from the imine nitrogen lone pair to the vanadium d-orbitals. The complexes exhibited three absorption bands. Complex 1 – 275 (ε = 11460 M^–1 cm^–1) & 320 (ε = 83700 M^–1 cm^–1) nm; Complex 2 – 271 (ε = 14170 M^–1 cm^–1) & 328 (ε = 12980 M^–1 cm^–1) nm; Complex 3 – 264 (ε = 25870 M^–1 cm^–1) & 315 (ε = 22760 M^–1 cm^–1) nm; Complex 4 – 262 (ε = 55000 M^–1 cm^–1) & 313 (ε = 44380 M^–1 cm^–1) nm. These bands correspond to π–π∗ intraligand transitions of the aromatic rings and ligand to metal charge transfer (LMCT) transitions of complexes (1–4). The absorption bands at 432 (ε = 61900 M^–1 cm^–1), 420 (ε = 11080 M^–1 cm^–1), 421 (ε = 17670 M^–1 cm^–1) & 418 (ε = 36120 M^–1 cm^–1) nm correspond to the d¹ system, the d-d transition of complexes (1–4).

IR spectra for (1–4) complexes, the peak that represents the O–H stretching frequency disappears. Furthermore, the complex (1) displayed the > CN, > CC<, and > C–O–, V–N stretching frequencies at 1641, 1583, 1202, 504 cm^–1. The stretching vibrations of oxovanadium (V = O) are represented by the peaks at 962 cm^–1. The > CN, > CC<, and > C–O-, V–N stretching frequencies were observed at 1636, 1588, 1261,557 cm^–1, respectively, in complex (2). Oxovanadium (V = O) stretching vibrations are exhibited by the peaks at 979 cm^–1. In complex (3), the > CN-, > CC, and > C–O-, V–N stretching frequencies were observed at 1739, 1460, and 1244, 566 cm^–1, respectively. The peaks at 936 cm^–1 show stretching vibrations of oxovanadium (V = O). The stretching frequencies of > CN-, > CC<, and > C–O-, V–N were found in complex (4) at 1750, 1449, 1244, and 572 cm^–1. The peaks at 942 cm^–1 correspond to oxovanadium (V = O) stretching vibrations , (Table and Figures S5–S8).

1. FT-IR Imine Ν­(CN) Shifts of Vanadyl Complexes and Their Aldimine Ligands.

Compounds	Complexes ν(CN) (cm–1)	Ligands ν(CN) (cm–1)	Δν (cm–1)
Complex 1	1641	1594	+47
Complex 2	1636	1602	+34
Complex 3	1739	1608	+131
Complex 4	1750	1659	+91

Vanadyl complexes show distinctive isotopic signatures (⁵¹V). The mass spectra of complexes (1–4) exhibit molecular ion peaks (M+) at 530.9045 m/z, 561.8742 m/z, 550.8494 m/z, and 534.8795 m/z, respectively, corresponding to the molecular formula [C₂₁H₁₂Cl₄N₂O₃V] for complex 1, [C₂₀H₉Cl₄N₃O₅V] for complex 2, [C₂₀H₉Cl₅N₂O₃V] for complex 3, and [C₂₀H₉Cl₄FN₂O₃V] for complex 4 (Figures S9–S12).

3.1.1. Thermal Gravimetric Analyses

Thermogravimetric analysis (TGA) was performed for all four vanadium complexes to assess their thermal stability and to verify the absence of coordinated or lattice solvent molecules. All complexes display a similar behavior at low temperatures, characterized by a small initial mass loss between 0.2–3.5% occurring around 60 °C. This feature is consistently attributed to physisorbed moisture or trace solvent residues, reflecting the mildly hygroscopic nature of the samples. Importantly, none of the complexes exhibits a defined mass-loss event that would indicate the presence of coordinated solvent molecules, thereby, supporting the proposed formulas are C₂₁H₁₂Cl₄N₂O₃V, C₂₀H₉Cl₄N₃O₅V, C₂₀H₉Cl₅N₂O₃V, and C₂₀H₉C_l4FN₂O₃V. It has seen two stages of decomposition for complex (1–4), as depicted in Figures S13 and S14. Following the initial desorption event, the complexes exhibit their main decomposition stages in the 90–200 °C temperature range, although the extent of mass loss differs depending on the ligand environment. Complexes 1 and 3 show minimal total mass losses (3–5%), in the temperature range recorded, indicating relatively rigid frameworks and higher thermal robustness. In contrast, Complex 2 undergoes decomposition, resulting in a higher overall mass loss (29%), which may probably due to the more flexible ligand structure. Complex 4 shows a moderate weight loss of 13%. Thus, the TGA data provide supportive indication that the analytical deviations originate from surface moisture uptake rather than errors in empirical formula assignment, reaffirming the compositional correctness of the complexes.

The X-band ESR spectra of complexes were obtained at room temperature in the solid state, with a single peak, and at liquid nitrogen temperature (LNT) in DMSO. The room-temperature spectrum displays a single peak, whereas at LNT it exhibits eight lines of hyperfine splitting (Figure ). due to the connection of the d --electron to the ⁵¹V (I = 7/2) nucleus. The nature of the bonding and the core metal ion environment can be determined using the metal complexes’ EPR spectra. This suggests that the exchange contacts in the RT solid state are broken during dilution. Solid-state spectra exhibited signals with S = 1/2. The obtained g value complexes 1–4, 1.978,1.974,1.963, 1.961, and the oxidation state of the metal is +4, found to be comparable with that of reported vanadyl complexes. , The complexes g∥, g⊥, and A∥ values are shown in Table . The parameters g⊥ = [2.08, 2.07, 2.14, and 2.15], g∥ = [2.00, 2.06, 2.04, and 2.03], and A∥ = [123, 244, 239, and 236] × 10^–4 cm^–1 indicate a square-pyramidal VO²⁺ environment. For vanadyl complexes with strong equatorial donor atoms, the amplitude of A∥ is typically >160 × 10^–4 cm^–1, indicating a short V = O bond and a mainly $d_{x^2-y^2}$ ground state. Together, the measured g and A characteristics support the axially suggested square-pyramidal coordination around vanadium­(IV). The EPR spectra of complexes 2,3, and 4 are shown in Figures S15 and S16, which exhibited a similar splitting pattern. These results support the hypothesized V­(IV) coordination environment and confirm a d electronic configuration, falling within the usual range reported for square-pyramidal VO²⁺ complexes. Thus, the EPR spectra of complexes (1–4) in DMSO at LNT display eight lines, demonstrating the paramagnetic character of the vanadyl ion.

1.

ESR spectra of complex 1 at RT and LNT.

2. Spin Hamiltonian Units of Vanadyl Complexes (1–4) in DMSO Medium at LNT.

	g-tensor
Compounds	g⊥	g∥	A×10–4 (cm–1) A∥
Complex 1	2.08	2.00	123
Complex 2	2.07	2.06	244
Complex 3	2.14	2.04	239
Complex 4	2.15	2.03	236

3.1.2. Density Functional Theory

Computational studies of the four vanadyl complexes based on Schiff base derivatives were performed utilizing the B3LYP and Gaussian 09 computational programs. Various quantum-chemical characteristics were computed using B3LYP/6–31G**/LanL2DZ ECP methods, such as geometry optimization, molecular energy, ESP charges, the energy of border molecular orbitals, and bandgap. Vanadyl complexes exhibit a distorted pyramidal shape, as illustrated in Figure . Table presents the relevant bond lengths. The electrostatic potential was projected onto a surface with constant electron density for the optimized van der Waals surface shape. The catalytic activity of vanadyl complex in the epoxidation of alkenes. Polyhedron, 31(1), pp.265–271. It is also useful in molecular structure studies due to its photophysical property relationship with hydrogen bonding interactions in free ligands and vanadyl complexes. The largest negative region, which is the favored site for electrophilic assault, is marked in red. The entire positive area, the favored place for nucleophilic attack, is blue (Figure ). The difference between the energy of the complex and the sum of the energies of its constituent parts (the ligand and the metal ion) was estimated to represent the binding energy of the ligand with the vanadium­(IV) ion.

2.

Optimized molecular geometries of complexes using the DFT/B3LYP method.

3. Optimized Bond Lengths of Tetrahedral Complexes (Å).

	NL···V	NR···V	OL···V	OR···V	VO
Complex 1	1.9472	2.0478	1.8436	1.8227	1.5958
Complex 2	1.9467	1.9228	1.8482	1.8607	1.7489
Complex 3	2.0411	1.9608	1.8449	1.8179	1.5933
Complex 4	2.0952	1.9731	1.8492	1.8116	1.6119

3.

Electrostatic potential mapped on the surface of optimized molecular geometries of vanadyl complexes by the DFT/B3LYP 6–31 G­(d,p) method.

The frontier molecular orbitals (FMOs), which compare the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), as well as the energy gap between HOMO and LUMO of all complexes, were calculated and are shown in Figure and Table . The energy gap (ΔE) is crucial for understanding a molecule’s chemical reactivity and kinetic stability. The HOMO–LUMO energy gap should normally be between 1.06 and 3.18 eV to support charge transfer transitions because such tiny energy separations enable effective intramolecular charge migration and electronic excitation. Table shows the energies of frontier molecular orbitals (E_HOMO and E_LUMO), energy band gap (ΔE), electronegativity (χ), chemical potential (μ), global hardness (η), global softness (S), and global electrophilicity index (ω). , These values explain the charge transfer interaction within the molecule. The purpose of these metrics is to determine the stability and reactivity of molecules. The electrophilicity index is one of the most important quantum chemical metrics for defining the toxicity of different contaminants based on their reactivity and site selectivity. Furthermore, the electrophilicity property measures the biological activity of drug-receptor interactions.

4.

Frontier molecular orbitals of ligands and complexes using the DFT/B3LYP method.

4. Calculated Molecular Electronic Parameters .

S.No	Code	Energy	DM	HOMO (eV)	LUMO (eV)	Gap	χ	μ	η	S	ω
1	Complex 1	–1924831.92	13.34	–4.73	–3.67	1.06	4.20	–4.20	–0.53	–0.27	–4.70
2	Complex 2	–2028892.13	9.43	–5.68	–3.90	3.18	4.79	–4.79	–0.89	–0.44	–10.19
3	Complex 3	–2189613.25	10.55	–4.94	–3.69	1.25	4.32	–4.32	–0.62	–0.31	–5.82
4	Complex 4	–1962584.76	11.11	–4.91	–3.67	1.24	4.29	–4.29	–0.62	–0.31	–5.69

^aDipole moment (DM), Energy of HOMO (EH), energy of LUMO (EL), energy band gap (ΔE), electronegativity (χ), global hardness (η), chemical potential (μ), global electrophilicity index (ω), and global softness (S).

The ground-state optimized geometries were used for TD-DFT computations, and the related electronic transitions with their associated orbital contributions were calculated. In comparison, the experimental and projected absorbance bands are matched, and Table shows an update to their characteristic electronic transition. The oscillator strength (f) values are 0.0998, 0.1556, 0.1559, 0.1885, 0.1071, 0.139, 0.1019, and 0.0921 for the primary electronic transitions. The comparatively high f values (≥0.15) are indicative of strong ligand-centered (π→π*) transitions involving HOMO and LUMO orbitals that are situated on the aromatic Schiff base moiety. Due to electron excitation from ligand π orbitals to unoccupied V­(3d)/V = O antibonding orbitals, the transitions with intermediate oscillator strengths (0.09–0.14) are ascribed to ligand-to-metal charge transfer (LMCT) processes. Since there were no discernible low-intensity (f < 0.05) transitions, the examined complexes showed minimal contributions from d–d transitions, which are often weak and spin-forbidden. The orbital composition from TD-DFT analysis and the observed experimental absorption bands, where the π→π* transitions occur in the 262–275 nm region and the LMCT transitions in the 313–328 nm range, are both in agreement with these designations. In addition, Figure S17 depicts the predicted UV–vis spectra of its metal complexes, which are closely related to the measured absorbance band.

5. Comparison of Experimental and Theoretical Excitation Spectral Details .

	Experimental		Theoretical prediction
Code	Abs (nm)	Mol. abs (L.m–1.cm–1)	Abs. max (nm)	Oscillator strength (f)	State
Complex 1	275,320	432	465.21,398.53	0.0998,0.1556	S0→S11, S0→S15
Complex 2	271,328	420	426.6, 365.41	0.1559,0.1885	S0→S9, S0→S14
Complex 3	264,315	421	422.07,340.45	0.1071,0.139	S0→S7, S0→S12
Complex 4	262,313	418	419.17,360.73	0.1019,0.0921	S0→S9, S0→S12

^aOS- oscillator strength.

3.2. DNA Interaction Studies: Absorption Titration

The binding efficacy of vanadyl complexes was investigated using the UV absorption technique with calf thymus DNA (CT-DNA). We have prepared the test solutions in Tris-HCl buffer at pH 7.2 and analyzed the absorption spectra of the complexes without and with CT-DNA. The UV spectra of ligands show intraligand transition bands. Hyperchromic or hypochromic effects occur when the DNA double helix structure changes due to the binding of test compounds. The concentration of the complex was maintained at 40 μM throughout the experiment, while the concentration of DNA increased with subsequent additions. With the gradual addition of DNA (0–10 μL), the phenolic chromophore of the ligands interacts with the base pairs of DNA, thereby increasing the absorbance. At 40 μM, the two bands in the UV region exhibit hyperchromic shifts, occurring at 210 and 259 nm for Complex 1, 208, and 253 nm for Complex 2, and 209 and 257 nm for Complex 3. However, for complex 4, there is a decrease in absorbance observed for both absorption bands at 247 and 314 nm, thus exhibiting a hypochromic shift. This study discusses two primary processes of DNA binding: intercalation, in which the complex inserts between adjacent base pairs of the double helix, and groove binding, in which the complex accommodates within the major or minor grooves of DNA. A red shift and hyperchromic changes (increased absorbance intensity) support the possibility of groove binding. On the other hand, hypochromic shift (decreased absorbance intensity), either with or without a wavelength shift, suggests intercalation mode of binding. The intrinsic binding constant values (K_b) are calculated to evaluate the relative binding efficiencies of the complexes with CT-DNA using the given eq . ,

$$\frac{[DNA]}{({\epsilon }_a-{\epsilon }_f)}=\frac{[DNA]}{({\epsilon }_b-{\epsilon }_f)}+\frac{1}{K_b({\epsilon }_a-{\epsilon }_f)}$$ 3

where [DNA] represents the CT-DNA concentration in base pairs, ε_a represents the complex extinction coefficient at a specific DNA concentration, ε_f represents the complex extinction coefficient in free solution, and ε_b represents the complex extinction coefficient when fully bound to DNA. , The trend in the absorbance changes and the binding constants (K_b) values is given in Table . The complex 1 at 40 μM is shown in Figure . Complexes (2–4) are shown (Figures S18–S20). Based on the above observations, Complexes 1 and 3 show a higher binding affinity than Complexes 2 and 4. The complexes mean ± SD is 8.27 ± 1.42.

6. Binding Constant Values of the Complexes with CT-DNA.

Compounds	Concentration (μM)	λmax (nm)	Kb (M–1)
Complex 1	40	210	9.18 × 105
		259	9.84 × 105
Complex 2	40	208	3.30 × 105
		253	7.57 × 105
Complex 3	40	209	7.62 × 105
		257	9.02 × 105
Complex 4	40	247	4.09 × 105
		314	6.67 × 105

5.

(a) UV–visible spectrum at 40 μM with DNA (0–10 μL), which was recorded at room temperature. (b and c) wolfe-shimmer plots for complex 1.

3.2.1. Stability Study

To conduct stability tests on the newly synthesized vanadyl complexes over 24 h, acetonitrile: Tris-HCl buffer (7.2 pH) medium (1:9, v/v) was used. The stability study of complexes (1–4) was performed by measuring the absorbance at room temperature at different intervals of time, say, 0, 24, 48, and 72 h. The complex mainly stays intact as there is no substantial change observed in absorbance values, indicating that the complex remains stable without any structural changes (Figure S21).

3.2.2. Viscosity Study

The viscosity experiment is a helpful method for determining the bonding between complexes and DNA. The intercalative binding mode changes the structure of DNA by interacting with small molecules, extending it, and increasing its relative viscosity. However, the groove and electrostatic binding modes would prefer that there be little to no change in the viscosity of DNA. , The viscosity of DNA treated with buffer and complex (4) significantly increases, suggesting an intercalative binding mechanism. In contrast, complexes 1–3 exhibit relatively small changes in viscosity as concentration rises, support the possibility of groove binding of complexes with DNA (Figure S22).

3.3. Competitive Displacement AssayFluorescence Technique

The concentration of EtBr-bound DNA remained constant while the fluorescence spectra were captured as the complex concentration increased. When excited at 530 nm, the emission wavelengths for complexes (1–4) were measured in the range of 600 nm. A decrease in fluorescence intensity has shown the ability of the complexes to bind with DNA as the complex concentration increased gradually (0–100 μM). This is caused by the complexes displacing EtBr from DNA. The spectra show the relative binding ability of the compounds with CT-DNA and are used to determine the Stern–Volmer constant, K_SV, as per eq .

$$\frac{F_0}{F}=1+K_{SV}[Q]$$ 4

where F₀ and F represent the fluorescence in the absence and the presence of the complexes, respectively (Figure ). The slope of the plot F₀/F vs Q is used to determine the Stern–Volmer constant (K_SV) values, which are found to be 7.81 × 10³ M^–1 for Complex 1; 3.08 × 10³ M^–1 for Complex 2, 1.57 × 10³ M^–1 for Complex 3, and 2.83 × 10³ M^–1 for Complex 4. As observed, Complex 1, with a methyl substituent, exhibits higher Stern–Volmer constant values than other complexes (Figures S23–S26).

6.

(a) Fluorescence spectrum of complex 1 (0–100 μM), which was recorded at room temperature. (b) Linear plot of complex 1.

3.4. BSA Binding Study

3.4.1. Fluorescence Quenching Studies

Anticancer medications also target proteins apart from nucleic acids as molecular targets. Fluorescence spectroscopy is the most effective method for assessing the efficacy of the interaction of BSA with metal complexes. BSA can be used as the substrate in tryptophan fluorescence quenching experiments with PBS (phosphate-buffered saline, pH 7.4) to examine the binding capacity of the complex. Three aromatic amino acid residues, including tryptophan, tyrosine, and phenylalanine, are found in proteins; however, two tryptophan residues, Trp-134 and Trp-212, are primarily responsible for the fluorescence of BSA. Trp-134 is found on the surface of subdomain IB, while Trp-212 is found inside a hydrophobic binding pocket in subdomain IIA. The wavelength maxima for both fluorescence emission and absorption are found at 295 and 340 nm, respectively. The BSA fluorescence spectra were captured both with and without increasing complex concentrations. As the complex concentration increases, the protein fluorescence intensity, which is measured at about 340 nm, decreases without any change in λ_max (0–20 μM). This shows that the local dielectric environment of BSA is unchanged, which would prevent shifts in emission maxima. The subunit interactions, protein structural changes, denaturation, or substrate binding are the probable factors responsible for the quenching effect.

The quenching constant (K_BSA) is determined using the Stern–Volmer equation. Applying corrected fluorescence data that accounts for dilution, Stern–Volmer graphs of I₀/I vs (complexes) are produced (Figure ) . eq can be used to fit the data to create a linear fit.

$$\frac{I_0}{I}=1+K_{BSA}[Q]=1+K_q{\tau }_0[Q]$$ 5

7.

(a) Fluorescence spectra of complex 1 (0–20 μM), which were recorded at room temperature. (b) Linear plot – Stern–Volmer, (c) linear plot – Scatchard.

The quenching constant (K_BSA) was determined using Origin Pro 8.5 software, where I and I₀ represent the BSA emission intensities in the presence and absence of a quencher at a concentration (Q), respectively. kq is the quenching rate constant, and τ0 is the average lifetime of tryptophan in BSA without a quencher, which is reported to be 1 × 10^–8 s. Fluorescence quenching mechanisms are divided into several types, including mixed static, dynamic, and static quenching. The process of static quenching includes the creation of a fluorophore-quencher complex. During the brief life of the excited state, the quencher and fluorophore come into contact through a process known as dynamic quenching. The Scatchard eq shows the quencher’s potential to bind with the corresponding serum proteins. ,

$$\log \frac{I_0-I}{I}=\log K+n\log [Q]$$ 6

The values of n and K are obtained from the slope and the intercept by “fitting” the log (I₀–I)/I vs log (Q) plot linearly. The complex 4, fluoro substituents, have higher binding constant values than complexes 1,2 and 3 (Table ) (Figures S27–29).

7. Binding Parameters of Complexes from Fluorescence Studies of BSA.

Compounds	(KSV) (M–1)	(Kq) (M–1 S1–)	(K) × 106 M–1	Number of binding sites (n)
Complex 1	0.0121 × 104	1.21 × 1012	0.07	0.5
Complex 2	0.0142 × 104	1.42 × 1012	0.05	0.6
Complex 3	0.0160 × 104	1.60 × 1012	0.07	0.5
Complex 4	0.0324 × 104	3.24 × 1012	0.05	0.9

3.5. Antioxidant Activities

3.5.1. DPPH Assay

The antioxidant activity was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) technique by measuring the change in absorbance using the UV–vis spectrophotometric technique. The ability of the complexes to scavenge the DPPH radicals was studied at various concentrations using this method. − The absorbance of the mixture of complexes with DPPH was measured at 517 nm. The test compounds typically transfer one electron by acting as a reducing agent. The study is conducted in the presence of a standard antioxidant, Ascorbic acid, and is performed in triplicate (Figure ). The results of the study indicate the IC₅₀ values, such as the standard Ascorbic acid is 173.61 μg mL^–1; Complex 1, with methyl substituent, 216.06 μg mL^–1; Complex 2, with nitro substituent, 190.67 μg mL^–1; Complex 3, with chloro substituent, 224.79 μg mL^–1; Complex 4, with fluoro substituent, 182.91 μg mL^–1. Compounds with high antioxidant activity exhibit lower IC₅₀ values. It is observed that complex 4 shows relatively better antioxidant activity than the other complexes.

8.

DPPH scavenging activities of vanadyl complexes.

3.5.2. Hydrogen Peroxide Scavenging Activity

H₂O₂ is a significant ROS molecule because, despite not being harmful by itself, it can transform into a more hazardous radical, such as •OH. The volume of chromogen generated in the reaction between H₂O₂, 1,10-phenanthroline, and ferrous ammonium sulfate (FAS) decreased in a dose-dependent manner because the compounds have the antioxidant capacity to scavenge H₂O₂ radicals. The ability of the complexes to scavenge radicals is thus determined. The results show IC₅₀ values: Ascorbic acid is 167.68 μg mL^–1, Complex 1 – 190.29 μg mL^–1; Complex 2 – 207.30 μg mL^–1; Complex 3 −174.46 μg mL^–1; Complex 4 – 182.73 μg mL^–1. From the results, it is observed that Complex 3, with a chloro substituent, demonstrates relatively better antioxidant activity than other complexes (Figure ).

9.

Hydrogen peroxide scavenging activities of vanadyl complexes.

3.6. Molecular Docking Studies with DNA

The docking technique reveals the preferred binding mode of metal complexes with DNA, better understanding of their connection with biological macromolecules. The study was carried out with the proposed structures used for the complexes. The docking simulations included major and minor grooves as potential binding sites for metal complexes. To conduct blind docking investigations on complexes with DNA (PDB ID: 1BNA), the grid dimensions were adjusted to x = 14.780, y = 20.976, and z = 9.218. The complexes possess comparable binding energies. However, the nitro-substituted complex 2 has a higher affinity for the DNA macromolecule compared to Complexes 1, 3 and 4, which is consistent with experimental results. Complexes with extended aromatic rings display many features, including H bonding and interpolated charges, as given in (Figures , S30–S32). The complexes 1–3 have major groove binding, and complex 4 has minor intercalation binding. The dock scores of the complexes are −7.7 to −8.5 kcal/mol for Complex 1; −8.2 to −9.4 kcal/mol for Complex 2; −7.8 to −8.8 kcal/mol for Complex 3; −7.8 to −9.0 kcal/mol for Complex 4.

10.

Molecular docking of complex 1 with DNA.

3.7. Molecular Docking Studies with BSA

The complexes were docked into the 3D structure of BSA (PDB ID: 4F5S) using Autodock 1.5.7 to determine their preferred binding locations. Trp213 and Trp134 are the major binding sites in the BSA molecule. Trp213 is located in a hydrophobic pocket, whereas Trp134 is on the hydrophilic surface. The grid dimensions of x = 34.885, y = 23.976, and z = 98.792 were used for blind docking investigations on the complexes with BSA. The BSA consists of 583 amino acid residues with a molecular mass of 66 200 Da in a single polypeptide chain. The complexes possess comparable binding free energies. According to their dock score (−8.4 to −10.1 kcal/mol for Complex 1; −8.2 to −9.8 kcal/mol for Complex 2; −7.8 to −9.3 kcal/mol for Complex 3; −7.9 to −9.2 kcal/mol for Complex 4), the methyl-substituted Complex 1 has a higher affinity for the BSA protein than complexes 2, 3, and 4. This is consistent with the computational results and supports the binding of BSA in site IA. Complexes with extended aromatic rings display many characteristics, including aromatic qualities, H bonding, and hydrophilicity, as demonstrated in Figures , S33–S35.

11.

Molecular docking of complex 1 with BSA: a) aromatic, b) H-bond, c) hydrophobicity.

3.8. DNA Cleavage (Gel Electrophoresis Method)

3.8.1. Hydrolytic DNA Cleavage

The agarose gel electrophoresis method was used to assess the ability of the vanadyl complexes to cleave pBR322 plasmid DNA. In the gel, the molecules move according to their mass, charge, and shape. DNA cleavage reveals that supercoiled circular (form I) DNA has transformed into linear (form III) and nicked circular (form II). − Form I showed the fastest migration, Form II the slowest, and Form III showed a migration speed halfway between Form I and Form II. Lane 1 contains untreated pBR322 DNA that is still intact, while Lanes 2–10 are treated with metal compounds, ligands, and complexes to cause cleavage from the supercoiled form (I). The proportion of supercoiled DNA (Form I, SC) that is cleaved by the ligands and complexes is assessed. Lane 1 (pBR322 DNA)-65.5%; Lane 2 (methyl substituted) – 68%; Lane 3 (nitro substituted) 67.2%; Lane 4 (chloro substituted) 46.5%; Lane 5 (fluoro substituted) 47%; the corresponding vanadium complexes, Lane 6 (methyl substituted) 62%; Lane 7 (nitro-substituted) 59.1%; Lane 8 (chloro substituted) 52%; Lane 9 (fluoro substituted) 51%; Lane 10 (vanadyl acetylacetonate) 49.3%;

Similarly, the linear DNA (Form III) also displays the percentage cleavage for pBR322 DNA, methyl, nitro, chloro, fluoro, and substituted ligands as lane 1 (88.4%), lane 2 (89%), lane 3 (87%), lane 4 (82%), and lane 5 (49%). Additionally, lane 6 (72%) displays the corresponding vanadium complexes, while lanes 7 (66.3%), 8 (70.5%), 9 (61%), and 10 display the cleavage percentage of 59.4% for Vanadyl acetylacetonate. The nicked form II in Lanes 2 and 4 has a cleavage percentage of 45% and 38%, respectively. The results support that, in comparison to other ligands and complexes, the methyl-substituted ligand exhibits the highest cleaving ability. Complexes, however, only exhibit a limited capacity for cleavage. The gel image is shown in Figure .

12.

Hydrolytic cleavage pattern of pBR322 DNA by complexes (1–4) Lane 1: DNA pBR322, Lane 2: DNA + HL1; Lane 3: DNA + HL2; Lane 4: DNA + HL3; Lane 5: DNA + HL4; Lane 6: DNA + complex 1; Lane 7: DNA + complex 2; Lane 8: DNA + complex 3; Lane 9: DNA + complex 4; Lane 10: DNA + [VO (acac)₂].

3.8.2. Oxidative DNA Cleavage

In the presence of hydrogen peroxide (30 mM), complex 1’s ability for oxidative cleavage was assessed using pBR322 in Tris–HCl buffer (pH 7.2). On incubating for 2 h at 37 °C, Complex 1 exhibits a cleavage of pBR322 into supercoiled (Form I), nicked circular (Form II), and linear (Form III). The plasmid was subjected to control trials both with and without hydrogen peroxide for comparison. The concentration of complex 1 had a significant impact on the plasmid’s oxidative cleavage. Complex 1 (20 and 40 μM) demonstrated cleavage of the supercoiled form to the nicked form to 57 and 42%, respectively, as shown in Figure (lanes 2 and 3). The supercoiled form was shown to be cleaved to the nicked form by Complex 1 (60, 80, and 100 μM) to 34, 47, and 29%, respectively (lanes 2 and 3). The presence of methyl groups in Complex 1 enables improved stacking between base pairs, resulting in a linear shape with enhanced cleaving ability.

13.

Concentration-dependent oxidative cleavage of pBR322 in the presence of complex 1.

3.9. MTT Assay

The cytotoxicity of the complexes was assessed using the MTT assay technique against MCF7 cell lines, which have been employed for cytotoxic activity against diverse compounds in vitro for several decades. , (Figures , S36–S38) illustrates the microscopic images of MCF7 cells treated with different concentrations of complexes along with cisplatin (Figure S39). Figure shows the % cell inhibition of the complexes, and that for cisplatin is shown in Figure S40. The observed IC₅₀ values are as follows: for Complex 1, 50.75 μg/mL; for Complex 2, 35.84 μg/mL; for Complex 3, 47.53 μg/mL; and for Complex 4, 40.01 μg/mL, for cisplatin, 14.91 μg/mL, respectively. From the results, it has been observed that the nitro-substituted complex 2 exhibits relatively higher cytotoxicity than the other complexes, but is found to be lower than cisplatin. An African green monkey’s kidneys, normal Vero cells were used to test complex 2. The findings suggest that the complexes have selective cytotoxicity only against cancer cells, but are not harmful to the normal Vero cell lines, exhibiting 90% cell viability as shown in Figure S41.

14.

Microscopic images of MCF7 cells treated with different concentrations of complex 1.

15.

MTT assay – % cell inhibition a) complex 1, b) complex 2, c) complex 3, d) complex 4.

Previously reported vanadyl-Schiff base complexes have been included for comparison as shown in Table .

8. Comparing Our K_b/K_SV and Cytotoxicity with the Literature.

S. No	Complexes	DNA binding Kb values	BSA quenching KSV values	Cytotoxicity IC50 values	References
1	Vanadyl Schiff base complex	5.40 × 104 M–1	1.7 × 104 M–1	MDA-MB231 (4.5 μM)	Sedighipoor et al. (2019)
2	Vanadyl Schiff base complex	0.98 × 104 M–1	-	-	Salomanravi et al. (2023)
3	Vanadyl Schiff base complex	7.53 × 104 M–1	7.65 × 104 M–1	-	Kalantari et al. (2020)
4	Vanadyl Schiff base complex	2.3 × 104 M–1	7.5 × 105 M–1	MCF-7 (7.8 μM)	Mohamadi et al. (2015)
5	Vanadyl Schiff base complex	9.84 × 105 M–1	0.0324 × 104 M–1	MCF-7 (67.24 μM)	Our work

4. Conclusion

In the current study, we have designed vanadyl complexes and optimized their structures using DFT studies. The ability of the complexes to bind to DNA is assessed by UV absorption titration and fluorescence techniques. The results indicate that complexes efficiently bind to CT-DNA, with Complexes 1–3 exhibiting hyperchromism, showing preferential groove binding, whereas Complex 4 exhibits hypochromism, showing an intercalation mode of binding. The obtained binding constant values imply that the electron-releasing methyl-substituted complex 1 shows the highest value, followed by the chloro-substituted complex 3, which also shows electron-donating ability due to the presence of a lone pair of electrons. However, both nitro-substituted complex 2 and fluoro-substituted complex 4 have shown relatively lower binding constant values due to their electron-withdrawing abilities. According to fluorescence spectra, fluorescence intensity decreased with increasing complex concentration. Competitive Displacement Assay, the electron-withdrawing nitro-substituted complex 2, the chloro-substituted complex 3, and the fluoro-substituted complex 4 had lower quenching constant values than the highly electron-releasing methyl-substituted complex 1. As per the BAS binding studies, the fluoro-substituted compound 4 has higher quenching constant values than the other complexes. The complexes have also demonstrated good scavenging abilities, both by the DPPH and H₂O₂ methods. The docking analysis indicates that the complexes have good binding propensity with both DNA and BSA, supporting spectral evidence. Gel electrophoresis also suggests good cleaving ability of the complexes. Further, the MTT assay indicates that nitro-substituted complex 2 exhibits higher cytotoxicity against breast cancer cell lines than other complexes.

The data that support the findings of this study are available throughout the manuscript and supporting files.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c08913.

• Spectral characterizations of vanadyl complexes (UV, IR, HRMS, TGA, ESR), DNA/BSA binding studies (UV, FL, and viscosity measurements), theoretical evaluation (DFT and molecular docking ), and microscopic images of vanadyl complexes treated with MCF-7 cell lines (PDF)

T.S.: Methodology, validation, data curation, original draft preparation. A. S.: Conceptualization, resources, visualization, investigation, supervision, writing-reviewing and editing.

The authors declare no competing financial interest.