Synthesis, Characterization, and Bioactivity of a Dioxime-Based Copper(II) Complex: SOD/Catalase Mimicry, DNA/HSA Binding, and In Silico Evaluation for Cuproptosis-Mediated Anticancer Activity

Abstract

Cisplatin’s chemotherapy is hindered by drug resistance and toxicity, making copper complexes a potential alternative. A novel copper(II) complex, [CuLBr], was synthesized from a tetradentate vicinal dioxime ligand (H₂L) and characterized. [CuLBr] features a distorted square pyramidal geometry with a CuN₄Br chromophore. DFT calculations showed a narrowed HOMO-LUMO gap and increased electrophilicity, enhancing its chemical reactivity. [CuLBr] exhibited potent biomimetic catalytic activity, functioning as an efficient superoxide dismutase mimic and catalase mimic. Biophysical studies (UV-Vis, fluorescence, and viscosity) demonstrated a strong, spontaneous affinity of [CuLBr] for calf thymus DNA and Human Serum Albumin, suggesting groove-binding and static quenching mechanisms. In vitro assays revealed superior anticancer activity against HepG-2, HCT-116, and MDA-MB-231 cell lines, with greater selectivity than the free ligand and doxorubicin. Molecular docking studies reveal a high binding affinity of [CuLBr] with key proteins, including ferredoxin-1 and VEGF. This may suggest potential dual mechanisms of action, involving the induction of cuproptosis and the inhibition of tumor angiogenesis. These findings position [CuLBr] as an effective multi-metal-based anticancer agent with advantageous selectivity.

1. Introduction

Recently, there has been considerable focus on metal-based anticancer agents in the quest for new chemotherapeutic medications [1]. This growing concern largely results from the limitations of cisplatin, the leading approved metal-based medication. Although cisplatin is the most frequently used anti-cancer drug today, it encounters limitations because of drug resistance as well as nephrotoxicity and cardiotoxicity [2,3]. Thus, there is a need to investigate more non-platinum metal-based anticancer agents that demonstrate lower drug resistance and higher efficacy. Metal complexes possess distinct characteristics that allow them to participate in redox reactions and exchange ligands, playing key roles in biological functions and their biomolecules’ interactions [4].

Copper is one of the transition metals that play a vital role in biological processes. It is essential for redox reactions, respiration, nucleic acid synthesis, and energy production. Copper serves as a cofactor for superoxide dismutase (SOD) and cytochrome oxidase [5]. Several copper complexes that act as anticancer agents combat drug resistance through unique mechanisms of action other than platinum-based drugs. These observations suggest that copper complexes may be preferred over platinum complexes in cancer chemotherapy [6,7,8,9].

Copper ions have excellent coordination properties and are less toxic than those of various other metals [10,11,12,13,14]. Copper is capable of forming bonds with a wide range of heterocyclic aromatic compounds that include nitrogen, sulfur, oxygen, and phosphorus, creating intricate structures and varied coordination modes of copper-based complexes [9,15,16]. Copper(II)-based complexes were proven to penetrate cells’ membranes and significantly induce cuproptosis in tumor cells [17] as well as inhibit cancer angiogenesis [18,19,20].

Cuproptosis is a newly detected type of regulated cell death which correlates with intracellular copper accumulation, marked by the lipoylated mitochondrial enzymes’ clustering and Fe–S proteins’ reduction [21]. Mitochondrion acts as a main focus for copper-induced cell death, leading to lipid peroxidation of the mitochondrial lipid bilayer membrane and impairment of enzymes in the tricarboxylic acid (TCA) cycle [22]. Ferredoxin-1 (FDX1) binds with copper as a copper reductase, converting Cu(II) into the more poisonous form, Cu(I), which is essential in the process of cuproptosis. FDX1 is crucial for the initiation of cuproptosis since this copper-triggered cell death pathway relies on FDX1 to cluster lipidated TCA cycle proteins and destabilize Fe-S cluster proteins when copper accumulates [23]. Furthermore, FDX1 expression is correlated with the tumor microenvironment, which influences proliferation, promotes angiogenesis, and prevents apoptosis [24].

VEGF is a vascular endothelial growth factor that plays a vital role in angiogenesis, coupled with increased vascular density and metastasis. VEGF produced by tumor cells initiates the growth and survival of endothelial cells, as well as inflammatory responses, thereby promoting angiogenesis [25,26]. In recent years, targeting VEGF has emerged as a prevalent approach in cancer chemotherapy, which inhibits angiogenesis and thus restricts the supply of blood and nutrients to cancer cells [27,28]. The interactions between FDX1 and VEGF that trigger cuproptosis in cancer therapy, along with their connection to cuproptosis, underscore a rapidly evolving area with significant therapeutic promise [28,29,30,31].

The selection of ligands, such as oximes, is significant because these nitrogen-based organic compounds, found in certain plants, possess antimicrobial and anticancer properties [32]. Additionally, oximes serve a crucial role in producing various drug derivatives, making them a significant category of chemotherapeutic compounds [33,34]. Oximes’ anticancer properties are associated with mechanisms that encourage programmed cell death, restrain cellular proliferation, and disturb DNA replication through groove binding or intercalation, production of reactive oxygen species (ROS), and enzyme inhibition [35,36,37]. Recently, a few oxime-based copper(II) complexes have been studied and shown promising anticancer properties, with lower toxicity to normal cells and decreased drug resistance [38,39]. Copper ionophores and complexes can exploit the unique metabolic conditions present in cancer cells, leading to cuproptosis [40,41].

The development of oxime copper complexes designed to selectively attack cancer cells via cuproptosis, while reducing toxicity to normal cells, represents a promising field of study. To our knowledge, there has been no research addressing the capability of oxime-based copper(II) complexes to trigger cuproptosis.

The incorporation of the bromide anion into metal complexes as an anionic ligand is an excellent strategic choice to take advantage of its unique physicochemical properties: (1) the heavy atom effect facilitates the determination of the molecular structure via X-ray crystallography by providing anomalous scattering, which is essential for definitively determining the structure of the metal complex; and (2) the electronic effect of the bromine atom is critical for tuning the redox potential, lipophilicity, and overall biological activity of the compound, allowing for a systematic study of structure-activity relationships.

The objectives of this study are to synthesize and characterize a new dioxime-based copper(II) complex, in addition to investigating its SOD/catalase mimetic activity, DNA/HSA interactions, and linking these properties to its ability to induce cuproptosis through a docking study with FDX1 and VEGF proteins.

2. Results and Discussion

2.1. H₂L and [CuLBr] Complex Synthesis and Characterization

The vicinal dioxime ligand (H₂L) was synthesized following a modified literature procedure [42], via a Schiff base condensation between 2,3-diaminotoluene and biacetyl monoxime. The crude product underwent purification through recrystallization using hot ethanol, yielding highly pure red-brown needles. The structural integrity and purity of the ligand H₂L were confirmed by analytical data and spectroscopic methods (Scheme 1).

A copper(II)-based complex was synthesized by reacting copper(II) bromide (CuBr₂) with the synthesized H₂L ligand in ethanol. Elemental analysis data (Experimental Section) are consistent with the proposed molecular formula, [CuC₁₅H₁₉N₄O₂Br]. This formulation indicates that the tetradentate dioxime ligand coordinates to the Cu(II) center through its four nitrogen atoms. Interestingly, analytical data suggest that despite the vicinal dioxime (H₂L) possessing two ionizable protons, the ligand binds to the metal in a monobasic fashion. This coordination mode allows for the binding of an anionic donor (Br⁻) in the axial position and facilitates the formation of an intramolecular hydrogen bond, as depicted in Scheme 2.

Further insight into the complex’s nature was gained from molar conductivity measurements. A solution of the complex in DMF at a concentration of 1 × 10⁻³ M exhibited a conductivity value of 18.89 Ω⁻¹ cm² mol⁻¹ at room temperature. This low value is characteristic of non-electrolytic behavior [43], confirming a covalent coordination bond between the bromo ligand and the copper(II) ion within the complex structure. This finding indicates that the present copper(II) complex is penta-coordinated and the coordination chromophore around the Cu(II) center is N₄Br.

2.2. Thermal Analysis

The thermal decomposition profile of the copper(II) complex (Figure S1), [CuLBr], was investigated using thermogravimetric analysis (TGA and DTG), with key data summarized in

Table 1. The thermal decomposition data of the [CuLBr] complex.

Metal Complex	Temperature °C	DTGmax °C	% Mass Loss Found (Calcd.)	Formed Formulas
[CuLBr]	230–320	295	18.50 (18.55)	[CuL]
	320–410	380	46.50 (46.70)	[CuL(0.25)]
	410–700	470	16.00 (15.56)	CuO

. The pyrolysis process occurs in three distinct and well-defined steps, indicating a sequential degradation mechanism.

The first decomposition step occurs within the temperature range of 230–320 °C, with a maximum rate of mass loss (DTG_max) at 295 °C. The observed mass loss of 18.50% aligns closely with the calculated value of 18.55%. This step is attributed to the elimination of the coordinated bromide anion (Br⁻), resulting in the formation of an intermediate species with the proposed formula [CuL]. The excellent agreement between the observed and calculated mass losses strongly supports this assignment.

Subsequently, the second and most significant mass loss step takes place between 320 and 410 °C (DTG_max = 380 °C). This step accounts for a mass loss of 46.50% (Calcd.: 46.70%), which corresponds to the thermal decomposition and volatilization of most of the organic ligand (L). The product at the end of this step is proposed to be a copper-containing species with a fractional ligand formula, [CuL_(0.25)], suggesting a partial, thermally stable residue of the original ligand framework before complete decomposition.

Finally, the third step, observed between 410 and 700 °C (DTG_max = 470 °C), involves a mass loss of 16.00% (Calcd.: 15.56%). This step is associated with the complete combustion of any remaining organic material, ultimately yielding copper oxide (CuO) as the final, stable residue. The close correlation between the experimental and theoretical mass losses for all three steps confirms the proposed decomposition pathway and the assigned formulas for the intermediate and final products.

In conclusion, the pyrolysis data demonstrates a clear, stepwise degradation mechanism for the [CuLBr] complex, culminating in the formation of CuO. The precise correlation between the experimental and calculated mass losses validates the proposed thermal decomposition sequence.

2.3. Bonding Pattern

FTIR spectra of the organic ligand (H₂L) and its Cu(II) chelates, [CuLBr] and [CuLBr BF₂] (Figures S2 and S3), along with the data presented in

Table 2. FTIR spectra (cm⁻¹) of dioxime ligand and its Cu(II) complex.

Compound	υ(OH)	υ(C=N)	υ(N-O)	υ(Cu-N)
H2L	3411	1655	1182	-
[CuLBr]	3451, υ(O–H…O)	1630	1182	570
[CuLBr BF2]	-	1630	1182	570

, offer crucial information regarding their molecular structures, coordination environments, and bonding interactions.

The FTIR spectrum of the free H₂L ligand confirms its successful formation via a Schiff base condensation reaction. This is evidenced by the absence of characteristic primary amine (υ(NH₂)) and carbonyl (υ(C=O)) stretching bands, which indicates the complete consumption of the precursor materials. Consequently, new absorption bands emerge at 1655 cm⁻¹ and 1182 cm⁻¹, which are assigned to the stretching vibrations of the azomethine υ(C=N) and oxime υ(N-O) groups, respectively [42,44,45]. A strong, sharp peak at 710 cm⁻¹ is attributed to the deformation vibration δ(C=N-O) of the oximato functional group [45].

The spectrum of the free ligand also exhibits characteristic bands associated with its hydroxyl and hydrocarbon moieties. A broad stretching band for the hydroxyl group υ(OH) is observed at 3411 cm⁻¹, with the corresponding in-plane bending δ(OH) and out-of-plane twisting τ(OH) modes appearing at 1317 cm⁻¹ and 661 cm⁻¹, respectively [45]. Vibrational modes for the aromatic carbon-hydrogen groups include the υ(CH) stretch at 3064 cm⁻¹ and the τ(CH) mode at 753 cm⁻¹. For the methyl (CH₃) groups, the antisymmetric stretch ν_as(CH₃) is identified at 2922 cm⁻¹, while the antisymmetric and symmetric deformation modes (δ_as(CH₃) and δ_s(CH₃) appear at 1451 cm⁻¹ and 1364 cm⁻¹, respectively [45].

Significant changes in the infrared spectra upon complexation with copper(II) provide clear evidence for metal coordination. In the spectrum of the [CuLBr] complex, the azomethine stretching vibration shifts from 1655 cm⁻¹ in the free ligand to 1630 cm⁻¹ and decreases in intensity. This indicates bonding of the imine nitrogen atoms to the copper(II) ion. This finding is further supported by the emergence of a new band at 570 cm⁻¹, which is assigned to the υ(Cu-N) stretching vibration [45]. The hydroxyl stretching band (υ(OH)) shifts to 3451 cm⁻¹, suggesting its potential involvement in hydrogen bonding. In contrast, the oxime stretching frequency υ(N-O) remains unchanged at 1182 cm⁻¹, indicating that the oxime oxygen atoms do not participate in coordination to the copper(II) ion.

The presence of an intramolecular hydrogen bond in the [CuLBr] complex was confirmed by its reaction with boron trifluoride etherate (BF₃·OEt₂). This reaction replaces the hydrogen-bonded proton with a BF₂ group, resulting in the formation of the macrocyclic [CuLBF₂Br] complex [42,46]. This transformation is evidenced in the FTIR spectrum by the disappearance of the characteristic broad band associated with the υ(O-H…O) vibration at approximately 2500 cm⁻¹ in the [CuLBF₂Br] derivative (Scheme 3). Furthermore, new vibrational modes appear at 1173 cm⁻¹ and 1026 cm⁻¹, which are assigned to boron-oxygen (B-O) and boron-fluorine (B-F) stretching vibrations, respectively [42,46]. The observed band at 298 cm⁻¹ in the spectra of both the [CuLBr] and [CuLBF₂Br] complexes is assigned to the υ(Cu-Br) stretching vibration [45].

Collectively, these FTIR spectroscopic data, supported by elemental analysis and conductivity measurements, provide strong evidence for the proposed structures of the copper(II) complexes, as illustrated in Scheme 2 and Scheme 3.

2.4. Geometry Determination

2.4.1. UV-Vis Spectra

The electronic absorption spectral data for the free oxime (H₂L) and its penta-coordinated Cu(II) complex, [CuLBr], provide significant insight into their electronic structures and the coordination geometry of the complex.

The spectrum of the free dioxime ligand (H₂L) exhibits two absorption bands at 340 nm and 390 nm (Figure S4a). These are assigned to intra-ligand transitions, specifically π → π* and n → π* transitions, respectively, which are characteristic of the conjugated oxime functional groups.

Upon complexation with Cu(II), the spectrum of [CuLBr] reveals significant changes. The ligand-centered bands are still present but are shifted, with the π → π* band at 350 nm and the n → π* band at 410 nm. This bathochromic shift in the n → π* transition is consistent with the coordination of the imine nitrogen atoms to the metal center, which reduces electron density on the ligand and lowers the energy of this transition.

More importantly, the complex spectrum displays three new, distinct low-energy bands in the visible region at 510, 585, and 685 nm (Figure S4b). These bands are not present in the free ligand and are characteristic of d-d transitions in five-coordinate Cu(II) complexes [47]. The assignments are as follows:

(i) The band at 705 nm is assigned to the transition dz² → dx² − y². (ii) The band at 605 nm is assigned to the transition dxy → dx² − y². (iii) The band at 525 nm is assigned to the transition dxz, yz → dx² − y².

The presence of these three well-resolved d-d transitions is a strong spectroscopic indication of a distorted square pyramidal geometry for the [CuLBr] complex [47]. In this geometry, the electronic degeneracy of the d-orbitals is lifted, resulting in multiple, observable transitions. The energy order of the transitions (dz² being lowest in energy) is consistent with a ligand field where the equatorial field is stronger than the axial one, which is typical for square pyramidal complexes [47].

In conclusion, the electronic absorption spectra confirm the coordination of the ligand to the copper(II) ion and strongly support a penta-coordinated, distorted square pyramidal structure for the [CuLBr] complex, similar to other square pyramidal copper(II) complexes [48,49].

2.4.2. Magnetic Moment and Electron Paramagnetic Resonance Analysis

The [CuLBr] complex’s geometric configuration was further elucidated by studying its magnetic behavior. At room temperature, the measured effective magnetic moment (µeff) was 1.95 Bohr magnetons (B.M.). This figure is somewhat higher than the spin-only value of 1.73 B.M. expected for an uncoupled copper(II) ion possessing a d⁹ electron configuration. The obtained moment is characteristic of a magnetically dilute material, implying negligible spin-spin coupling in the solid-state structure.

A more detailed examination of the complex’s geometry was conducted via X-band Electron Paramagnetic Resonance (EPR) spectroscopy. The room-temperature spectrum (Figure S5), recorded using a microcrystalline sample (frequency = 9.1 GHz, magnetic field = 3100 G), displayed a rhombic pattern with three resolved g-tensor components: g_x = 2.208, g_y = 2.158, and g_z = 1.986. The extent of geometric distortion was evaluated using the parameter R, defined as R = (g_x − g_y)/(g_y − g_z) [45]. The calculated R value of 0.290 falls between the theoretical extremes for a perfect trigonal bipyramidal (R = 1) and a perfect square-pyramidal (R = 0) geometry. This indicates that the copper(II) center resides in a coordination environment with a slight distortion from an ideal square-pyramidal structure.

The average g-value (g_av) was computed using the formula g_av = 1/3 (g_∥ + 2g_⊥), where g_⊥ = (g_y + g_z)/2, yielding a result of 2.117. This value is considerably larger than that of the free electron (g_e = 2.0023), suggesting a pronounced covalent nature in the bonding between the copper(II) ion and the donor atoms of the dioxime ligand [46]. Additionally, the exchange interaction parameter G, determined from the equation G = (g_∥ − 2)/(g_⊥ − 2) [47], was calculated as 4.736. A G value greater than 4 is indicative of weak magnetic exchange interactions between neighboring copper(II) centers in the solid state [50].

In summary, the data obtained from electronic absorption and Electron Paramagnetic Resonance spectroscopy are in strong agreement with the parameters reported for five-coordinate copper(II) complexes that possess a square-pyramidal geometry [49,51].

2.5. PXRD Structural Analysis

X-ray powder diffraction (PXRD), particularly when augmented by computational chemistry techniques, is increasingly recognized as a robust and practical methodology for determining the molecular structures of metal complexes, serving as a viable substitute for single-crystal X-ray diffraction [49,51,52,53]. In the present study, the powder X-ray diffraction data for the copper(II) complex were processed and analyzed utilizing the Expo 2014 software package. The structural model was subsequently refined using the Rietveld refinement method [54].

The structural solution was validated by the Cambridge Crystallographic Data Centre (CCDC), which issued the corresponding deposition number: 2493200. As exemplified by the PXRD profile of the [CuLBr] complex (see Supplementary Materials, Figures S6 and S7, a strong correlation was observed between the computationally derived model and the experimental data. The structure was successfully solved and refined by the Rietveld method, resulting in satisfactory reliability factors.

2.5.1. Crystallographic Data

X-ray powder diffraction analysis of the [CuLBr] complex confirmed the formation of a high-purity microcrystalline phase. The relevant crystallographic parameters are compiled in Table S1. The structure adopts a triclinic lattice within the centrosymmetric space group P-1 (No. 2). The unit cell parameters were determined as a = 11.92 Å, b = 11.34 Å, c = 8.51 Å, α = 92.62°, β = 102.67°, and γ = 102.63°. These values yield a unit cell volume of 1090 ų and a calculated crystal density of 1.312 g/cm³. The asymmetric unit comprises two formula units (Z = 2), which is consistent with the symmetric requirements of the space group. An estimated crystallite size of 10.27 nm, as shown in Table S1, confirms the nano-crystallite nature of the synthesized complex.

The final structural model, validated by Rietveld refinement, demonstrated satisfactory agreement factors (Rp, Rwp) and goodness of fit. The crystal structure has been archived with the Cambridge Structural Database under the deposition number CCDC 2493200, ensuring permanent access to the data for validation and further study. These crystallographic results unambiguously define the solid-state structure of the complex, providing a reliable basis for the subsequent structural analysis presented in this work.

2.5.2. The Molecular Packing

As shown in Figure 1, crystal packing is stabilized by a combination of non-covalent interactions, primarily hydrogen bonds and van der Waals forces. The space group’s centrosymmetric character results in the formation of inversion-related molecular dimers. This arrangement leads to an efficiently filled lattice, which is consistent with the relatively high calculated density and the low volume per atom (Table S1). The packing efficiency within the unit cell is therefore notably high.

Analysis of the packing likely reveals that the complex molecules arrange into layers or chains through specific non-covalent interactions. These may include weak hydrogen bonding, such as C–H…O or C–H…N interactions, and possibly π–π stacking interactions between the aromatic rings of adjacent ligand systems (C₁₅H₁₉N₄O₂). The specific dimensions of the unit cell, particularly the shorter c-axis (8.51 Å), may indicate a direction of preferential growth or a specific stacking motif along this axis. The relatively small crystallite size of 10.27 nm, as estimated from the diffraction data, may influence the overall morphology and surface properties of the crystalline material.

2.5.3. Structural Configuration Around the Metal Center

The local coordination environment of the Cu1 ion was examined through a combination of experimental X-ray diffraction (XRD) and theoretical Density Functional Theory (DFT) calculations. To quantitatively assess the geometry in this five-coordinate system, the τ₅ parameter was employed. This index, which ranges from 0 (perfect square pyramidal) to 1 (perfect trigonal bipyramidal), yielded a calculated value of 0.04 [53]. This result strongly indicates that the coordination sphere around Cu1 is essentially square pyramidal, as the negligible deviation from zero confirms a close alignment with the ideal geometric limit for this configuration.

The bond angles obtained from both experimental (XRD) and theoretical (DFT) methods are presented in

Table 3. Bond angle (°) around the Cu1 core of [CuLBr] complex.

Nearly Ideal Square Pyramidal Geometry Cu1 Center (τ5 = 0.04)
Type	XRD	DFT	Difference
N1–Cu1–N2	82.87	82.81912	0.05088
N1–Cu1–N3	80.51	80.50962	0.00038
N1–Cu1–N4	148.49	148.47169	0.01831
N1–Cu1–Br1	100.67	100.72734	0.05734
N2–Cu1–N3	146.23	146.18061	0.04939
N2–Cu1–N4	82.64	82.70510	0.0651
N2–Cu1–Br1	106.61	106.56457	0.04543
N3–Cu1–N4	96.68	96.63133	0.04867
N3–Cu1–Br1	105.24	105.33139	0.09139
N4–Cu1–Br1	110.25	110.20558	0.04442

. An excellent correlation is observed between the two sets of data. The differences between the XRD and DFT calculated angles are remarkably small, with all deviations being less than 0.1°. The largest discrepancy is observed for the N3–Cu1–Br1 angle (Δ = 0.09139°), while the smallest is for the N1–Cu1–N3 angle (Δ = 0.00038°). This minimal deviation underscores the high accuracy of the employed DFT method in replicating the experimental molecular structure. In the same regard, these slight deviations observed are within the expected range for such comparisons, attributable to the fact that the XRD structure represents a molecule in a crystalline lattice at a specific temperature, while the DFT calculation models an isolated molecule in its gas-phase equilibrium geometry.

The angular parameters further substantiate the square pyramidal assignment. The basal plane angles (e.g., N1–Cu1–N2, N2–Cu1–N4, N3–Cu1–N4) are all close to 90°, as expected for the base of a square pyramid. The angles involving the apical bromine ligand (Br1) and the basal nitrogen donors fall within the range of 100.67° to 110.25°, which is consistent with the distortion from 90° expected when an atom is positioned in the apical site.

In conclusion, the combined experimental and computational data robustly demonstrate that the Cu1 center in the complex possesses a slightly distorted square pyramidal geometry. The exceptional agreement between the XRD and DFT results validates the reliability of the computational model for this system.

Based on the provided data in Table 3 for the [CuLBr] complex, the geometry around the Cu1 center is confirmed to be nearly ideal square pyramidal, as evidenced by the τ₅ parameter of 0.04. For this square pyramidal complex, the equatorial plane typically consists of four donor atoms (N₄) with shorter bond distances, while the apical position features a longer bond due to reduced bonding interactions.

The bond distances from XRD measurements (

Table 4. The key bond lengths (Ǻ) of the [CuLBr] complex.

Type	Bond Length (Ǻ)
	XRD	DFT	Difference
Cu1–N1	2.001589	2.00385	0.002261
Cu1–N2	1.971297	1.97149	0.000193
Cu1–N3	1.985400	1.98660	0.0012
Cu1–N4	1.957957	1.95905	0.001093
Cu1–Br1	2.545548	2.54916	0.003612

) indicate that the four nitrogen atoms (N1, N2, N3, and N4) have Cu1–N distances ranging from 1.958 Å to 2.002 Å, which are characteristic of equatorial bonds. Specifically, the distances are: Cu1–N1 = 2.002 Å, Cu1–N2 = 1.971 Å, Cu1–N3 = 1.985 Å and Cu1–N4 = 1.958 Å. In contrast, the Cu1–Br1 bond distance is significantly longer at 2.546 Å, consistent with an apical position. This assignment is further supported by the close agreement between XRD and DFT calculations, with minimal differences in bond distances (≤0.004 Å), confirming the stability of the square pyramidal geometry. Thus, in the [CuLBr] complex, the equatorial plane is formed by N1, N2, N3, and N4, while Br1 occupies the apical site.

2.6. DFT Calculations

2.6.1. Geometrical Optimization

A density functional theory study was conducted to perform a geometrical optimization of the [CuLBr] complex, providing deeper insight into its structural characteristics. The principal bond angles and lengths resulting from the optimization are presented in Table 3 and Table 4, and a schematic of the structure is shown in Figure 2. A remarkable agreement is observed between the calculated parameters and the experimental data obtained from Rietveld-refined X-ray diffraction analysis. This consistency confirms the validity of the proposed structural model for [CuLBr] and aligns with findings in similar systems [49,51,52,53,55].

2.6.2. Global Reactivity Descriptors

The computed global reactivity descriptors with a schematic representation provided in Figures S8 and S9 and summarized in

Table 5. The computed global reactivity descriptors of free H₂L and its [CuLBr] complex.

	ELUMO	EHOMO	ΔE	χ	η	S	ω	ΔNmax	μ
H2L	0.991061	−3.43813	4.429192	1.2235	2.214596	0.225775	0.337993	0.552487	−1.22353
[CuLBr]	−2.98661	−5.15619	2.169583	4.0714	1.084792	0.460918	7.640314	3.753163	−4.0714

, provide profound insight into the electronic structure and chemical reactivity of the ligand and its resulting copper(II) complex. A primary observation is the significant reduction in the HOMO-LUMO energy gap (ΔE) from 4.43 eV for the free ligand to 2.17 eV for the complex. This narrowing of the band gap is a classical indicator of increased chemical reactivity and a decrease in kinetic stability, suggesting that the complex is more polarizable and generally more reactive than the parent ligand.

The analysis of frontier molecular orbitals reveals a substantial stabilization of both the HOMO and LUMO levels upon complexation. The LUMO energy decreases dramatically from 0.99 eV to −2.99 eV, indicating a greatly enhanced electrophilic character. Concurrently, the HOMO energy shifts to a more negative value (from −3.44 eV to −5.16 eV), which implies a reduced tendency for electron donation, albeit within a more stabilized overall electronic framework.

This electronic stabilization is further reflected in the increase in global electronegativity (χ) from 1.22 eV to 4.07 eV and a marked decrease in chemical hardness (η) from 2.21 eV to 1.08 eV. The lower hardness value for the complex confirms its softer nature according to Pearson’s Hard-Soft Acid-Base (HSAB) principle, characterizing it as more susceptible to polarizability and charge transfer interactions compared to the harder ligand. The corresponding increase in global softness (S) from 0.23 eV⁻¹ to 0.46 eV⁻¹ reinforces this conclusion.

The dramatic rise in the electrophilicity index (ω) from 0.34 eV to 7.64 eV is particularly noteworthy. This over twenty-fold increase classifies the complex as a strong electrophile, capable of participating in robust charge-transfer interactions. This is consistent with the significant increase in the maximum electron transfer capacity (ΔN_max), which jumps from 0.55 to 3.75, underscoring the complex’s superior ability to accept electron density from a nucleophile.

Finally, the negative value of the chemical potential (μ) for both systems confirms their thermodynamic stability relative to a free electron in a vacuum. The more negative value for the complex (−4.07 eV vs. −1.22 eV for the ligand) aligns with the overall stabilization of its electronic structure upon formation.

In conclusion, the coordination of the ligand to the metal center results in a profound electronic transformation. The resulting copper(II) complex is characterized by a softer, more electrophilic nature with a significantly lower HOMO-LUMO gap, predicting enhanced chemical reactivity, particularly in interactions where it acts as an electron acceptor.

2.6.3. The Relationship Between DNA-Binding Ability and Global Reactivity Descriptors

The interaction of small molecules with DNA is a critical aspect in the development of therapeutic agents, particularly in anticancer research. The synthesized oxime compounds, H₂L and [CuLBr], were evaluated for their DNA-binding affinity using spectral characteristics, and their chemical reactivity was assessed through global reactivity descriptors derived from conceptual density functional theory (CDFT). The results in Table 5 and

Table 6. Spectral characteristics for the synthesized Oxime complexes’ interactions with DNA.

Compound	λmax Free (nm)	λmax Bound (nm)	Δλ (nm)	Chromism Type	Chromism (%)	(Kb) mol−1 dm3	ΔG kJ mol−1
H2L	276	271	5	Hyper	96.0	0.68 × 105	−26.6
[CuLBr]	268	266	2	Hyper	67.7	1.4 × 105	−28.4

reveal a clear correlation between the DNA-binding strength and the computed reactivity parameters, which also provides insights into their potential behavior in biological systems, including normal cells.

The DNA-binding studies indicate that both compounds exhibit hyperchromism, suggesting interactions primarily through electrostatic or groove-binding modes rather than intercalation. The binding constant (K_b) and free energy change (ΔG) values demonstrate that [CuLBr] has a higher DNA-binding affinity (K_b = 1.39802 × 10⁵ M⁻¹, ΔG = −29.355 kJ/mol) compared to H₂L (K_b = 0.6815733 × 10⁵ M⁻¹, ΔG = −27.575 kJ/mol). This enhanced binding for [CuLBr] is consistent with its global reactivity descriptors. Specifically, [CuLBr] possesses a narrower HOMO-LUMO energy gap (ΔE = 2.169583 eV) than H₂L (ΔE = 4.429192 eV), indicating higher chemical reactivity and softer character. The softness (S) value for [CuLBr] (0.460918 eV⁻¹) is approximately double that of H₂L (0.225775 eV⁻¹), confirming its greater propensity for charge transfer interactions with DNA, a soft biological target.

Furthermore, the electrophilicity index (ω) of [CuLBr] (7.640314) is significantly higher than that of H₂L (0.337993), highlighting its strong electrophilic nature, which facilitates covalent or coordinate interactions with nucleophilic DNA bases. The higher electronegativity (χ) and chemical potential (μ) values for [CuLBr] also support its enhanced DNA-binding capability, as these parameters reflect a greater tendency to attract electrons and form stable complexes with DNA. Additionally, the maximum electron transfer capacity (ΔN_max) is substantially greater for [CuLBr] (3.753163) compared to H₂L (0.552487), further underscoring its superior ability to participate in electron-transfer processes during DNA binding.

In the context of normal cells, the heightened reactivity and DNA-binding affinity of [CuLBr] raise important considerations regarding selectivity and toxicity. While strong DNA binding is desirable for anticancer activity, it may also lead to off-target effects in normal cells, potentially causing DNA damage and cytotoxicity. The soft and electrophilic character of [CuLBr] could result in non-specific interactions with cellular macromolecules in normal cells, necessitating further evaluation of its therapeutic index. In contrast, H₂L, with its lower reactivity and DNA-binding affinity, may exhibit reduced cytotoxicity towards normal cells, but its efficacy as a therapeutic agent might be limited.

In conclusion, the global reactivity descriptors effectively rationalize the DNA-binding behavior of the compounds, with [CuLBr] demonstrating superior binding due to its soft, electrophilic, and electron-accepting properties. However, the potential impact on normal cells must be carefully assessed through cytotoxicity and selectivity studies to ensure safe application in drug design.

2.6.4. The Relationship Between Anticancer Activity and Global Reactivity Descriptors

The analysis of the biological and quantum-chemical data presented in Table 5 and Table 11 reveals a significant relationship between the global reactivity descriptors of the compounds and their observed anticancer activity and selectivity. The coordination of the free dioxime (H₂L) with Cu(II) ion to form [CuLBr] resulted in a dramatic enhancement of anticancer potency against the tested cancer cell lines, as evidenced by the lower mean IC₅₀ values of [CuLBr] compared to the free ligand. Crucially, this enhanced activity in cancer cells is coupled with a distinct effect on the normal WISH cell line. The free H₂L ligand exhibited considerable toxicity towards normal cells (IC₅₀ = 10.5 µM), which was even more pronounced than its effect on some cancer lines (e.g., HCT-116, IC₅₀ = 18.43 µM). In contrast, the [CuLBr] complex demonstrated significantly reduced toxicity against the normal WISH cell line (IC₅₀ = 27.49 µM), the highest IC₅₀ value reported in this study. This indicates that the copper complex possesses a more favorable selectivity profile, being more toxic to cancer cells while sparing normal cells to a greater extent than both the free ligand and the standard drug doxorubicin (DOX).

This marked improvement in activity and selectivity can be rationalized by examining the global reactivity descriptors. The key electronic parameter is the energy gap (ΔE = E_LUMO − E_HOMO). The [CuLBr] complex has a significantly narrower HOMO-LUMO gap (ΔE = 2.17 eV) compared to the free H₂L ligand (ΔE = 4.43 eV). A smaller ΔE generally implies higher chemical reactivity and lower kinetic stability, as it facilitates the charge transfer interactions crucial for interacting with biological targets like DNA or proteins in cancer cells.

Further supporting this, the [CuLBr] complex exhibits a higher electrophilicity index (ω = 7.64) compared to the ligand (ω = 0.34). A high ω value signifies a strong tendency of a molecule to accept electrons, making it a potent electrophile. This aligns with the enhanced cytotoxicity of [CuLBr], as it can more effectively engage in electron-accepting interactions with nucleophilic sites in cellular biomolecules. Additionally, the higher chemical potential (μ) and lower chemical hardness (η) of the complex confirm its softer, more reactive nature compared to the harder and less reactive free ligand.

In conclusion, the copper complexation engineered a more reactive molecular entity, as quantified by a lower HOMO-LUMO gap, higher electrophilicity, and lower hardness. This enhanced reactivity directly correlates with the superior anticancer efficacy of [CuLBr] across the panel of cancer cell lines. More importantly, this reactivity is leveraged selectively, as the complex shows the lowest toxicity against normal WISH cells, suggesting a potential therapeutic window. These findings underscore the utility of global reactivity descriptors as powerful computational tools for predicting and explaining the biological activity and selectivity of metal-based anticancer agents.

2.7. Electrochemical Characterization of the [CuLBr] Complex

The redox potential of copper complexes is a critical parameter for assessing their efficacy as functional mimics of copper enzymes, such as copper-zinc superoxide dismutase (SOD), which catalyze electron transfer reactions. The catalytic SOD-mimetic activity of the [CuLBr] complex is significantly influenced by the electrochemical properties of the Cu(II)/Cu(I) redox couple. To elucidate this relationship, the redox behavior of the complex was investigated using cyclic voltammetry.

Cyclic voltammetry was performed at room temperature on a 0.001 molar solution of the complex in methanol, utilizing a 0.01 molar tetra-n-butyl ammonium perchlorate as a supporting electrolyte.

The cyclic voltammogram (Figure S10) displayed a quasi-reversible redox process. A E_pc (cathodic peak potential) was observed at +110 millivolts, corresponding to the reduction in Cu(II) to Cu(I). The reverse scan showed a corresponding E_pa (anodic peak potential) at +340 millivolts, attributed to the oxidation of Cu(I) back to Cu(II). The peak potential separation was calculated to be 230 millivolts, which is indicative of a quasi-reversible electron transfer system with slow kinetics. The cathodic and anodic peak currents were 6.1 microamperes and 6.0 microamperes, respectively, yielding a ratio of anodic to cathodic peak current of approximately 0.98. This value, being close to unity, supports the assignment of a one-electron transfer process. The formal redox potential for the Cu(II)/Cu(I) couple, determined as the average of the anodic and cathodic peak potentials, was found to be +225 millivolts.

These electrochemical findings demonstrate that the dioxime ligand facilitates the formation of a stable copper complex capable of existing in both the +II and +I oxidation states. The observed quasi-reversible redox behavior confirms the potential of this vicinal dioxime-based copper(II) complex to act as a catalyst in chemical reactions involving electron transfer.

2.8. Antioxidant Mimetic Catalytic Activity

2.8.1. SOD Mimetic Activity

Copper(II) complexes with low molecular mass that replicate the catalytic function of the native copper-zinc superoxide dismutase enzyme present a safer therapeutic alternative to the protein itself, which can potentially induce immune responses [56]. Within this framework, the superoxide dismutase-like activity of the [CuLBr] complex was investigated. The employed assay utilizes phenazine methosulfate to photochemically generate superoxide radicals (O₂^•−), with nitroblue tetrazolium acting as a scavenger. The reduction in nitroblue tetrazolium to a blue formazan product is spectrophotometrically monitored [57]. The capacity of the [CuLBr] complex to inhibit this nitroblue tetrazolium reduction under physiologically relevant conditions provides a direct assessment of its superoxide dismutase-mimetic efficacy.

In the assay system, the superoxide radical reduces NBT to formazan as shown in Reaction (1). The inhibition of this reaction by the [CuLBr] signifies its catalytic role in the dismutation of O₂^•−.

NBT + O₂^•− → Formazan

(1)

The rate of formazan formation in the absence of the catalyst is given by

d[Formazane]₀/dt = k[NBT][O₂^•−].

(2)

Analogous to the native CuZn-SOD enzyme, which catalyzes the dismutation of O₂^•− (Reaction (3)), the [CuLBr] complex facilitates the same conversion and leading to the reduction of formazan.

CuZn-SOD + 2O₂^•− + 2H⁺ → CuZn-SOD + H₂O₂ + O₂

(3)

The catalytic activity of the complex can be described by

d[Formazan]c/dt = k_c [CuᴵᴵLBr-complex] [O₂^•−],

(4)

where k_c is the second-order catalytic rate constant. The relationship between the absorbance in the absence (A₀) and presence (A_c) of the complex is defined as

A₀/A_c = 1 + k_c [CuLBr]/k[NBT].

(5)

The half-maximal inhibitory concentration value, defined as the concentration of the [CuLBr] complex necessary to achieve 50% inhibition of nitroblue tetrazolium reduction, was determined graphically from a plot of the inhibition percentage against the complex concentration (Figure 3). The percentage inhibition of nitroblue tetrazolium reduction was computed based on Equation (6):

% NBT Inhibition = [(A₀ − A_c)/A₀] × 100

(6)

The catalytic rate constant (k_c) was subsequently calculated using the expression:

k_c = k[NBT]/IC₅₀.

Using the known value of k (5.94 × 10⁴ M⁻¹ s⁻¹) and [NBT] = 30 μM [58], the [CuLBr] complex exhibited an IC₅₀ of (4.180 ± 0.29) × 10⁻⁶ M and a k_c of 4.26 × 10⁵ M⁻¹ s⁻¹. These values place the [CuLBr] complex within the activity range typical of other high-performance copper(II) SOD mimetics, which generally display k_c values from 10⁵ to 10⁷ M⁻¹ s⁻¹ [59,60,61,62,63]. For comparison, the native CuZn-SOD enzyme, with an IC₅₀ of 4.00 × 10⁻⁹ M and a k_c of 2.52 × 10⁻⁹ M⁻¹ s⁻¹ [64], demonstrates significantly higher activity, underscoring the position of [CuLBr] as an effective, though less active, synthetic mimic.

A fundamental characteristic of functional copper-zinc superoxide dismutase mimics is the redox versatility of the copper center, which must cycle between the Cu⁺⁺ and Cu⁺ oxidation states during the catalytic cycle. Consequently, the redox potential of the Cu(II)/Cu(I) couple is a critical determinant, as it directly governs the catalytic efficiency. Previous studies have established that complexes possessing redox potential values close to that of the native enzyme demonstrate superior superoxide dismutase activity [64,65].

The mechanism of O₂^•− scavenging by both the native enzyme and the [CuLBr] complex involves a two-step redox cycle (Reactions (7) and (8)):

Cuᴵᴵ + O₂^•− → Cuᴵ + O₂,

(7)

Cuᴵ + O₂^•− + 2H⁺ → Cuᴵᴵ + H₂O₂.

(8)

For efficient catalysis, the E_1/2 of the complex must lie between the electrochemical potentials for the reduction and oxidation of O₂^•−, approximately −0.14 V and +0.89 V vs. NHE at pH 7 [65]. The [CuL] complex, with a measured E_1/2 of +0.24 V vs. NHE, fulfills this criterion, which accounts for its significant SOD-mimetic activity. Furthermore, the coordinatively unsaturated square-pyramidal geometry of [CuLBr] provides an accessible site for substrate binding at the copper center, facilitating the catalytic process.

2.8.2. Catalase-like Activity of [CuLBr]

Although catalase is an iron protein, numerous Cu(II) chelates have also been shown to exhibit catalase-mimetic activity [66]. Given that the catalytic function of superoxide dismutase (SOD) involves the conversion of superoxide (O₂^•−) to hydrogen peroxide (H₂O₂), the subsequent elimination of this reactive oxygen species is crucial for protecting living cells. This protective role is naturally performed by the enzyme catalase, which catalyzes the decomposition of H₂O₂ into water and oxygen. To evaluate the potential of the synthetic [CuLBr] complex to mimic this function, its catalase-like activity was investigated.

The catalytic interaction between the [CuLBr] complex and hydrogen peroxide (H₂O₂) was investigated using electronic absorption spectroscopy. In a neutral medium, the introduction of H₂O₂ to a solution of the complex led to the appearance of a new absorption band at 350 nm. The progression of this reaction was monitored over time at this specific wavelength (Figure S11).

To quantify the catalase-mimetic activity and elucidate its mechanism, a kinetic study was performed. The concentration of hydrogen peroxide varied between 0.05 and 0.8 M, while the concentration of the [CuLBr] complex was maintained constant at 0.001 M. The results, depicted in Figure 4, indicate that the initial reaction rate is dependent on the concentration of H₂O₂ at lower levels. However, at higher concentrations, the rate reaches a saturation point and becomes constant.

This kinetic profile indicates a transition from first-order dependence on H₂O₂ at low concentrations to a saturation-controlled regime at higher concentrations. Consequently, the data were analyzed using the Michaelis–Menten enzymatic kinetic model to determine the parameters V_max, k_cat, K_M, and the catalytic efficiency (k_cat/K_M). The calculated values are as follows: V_max = 12.204 M s⁻¹, k_cat = 12.20 × 10⁴ s⁻¹, and K_M = 0.02748 M. The resulting k_cat/K_M ratio, a key indicator of catalytic efficiency, is 4.44 × 10⁶ M⁻¹ s⁻¹.

To contextualize the performance of the [CuLBr] complex, its kinetic parameters were compared with those of other reported Cu(II)-based catalase functional models, [67,68,69,70,71,72,73] and the comparison reveals that the [CuLBr] complex exhibits competitive catalytic efficiency.

The [CuLBr] complex demonstrated significant antioxidant activities, functioning as both a superoxide dismutase and catalase mimic. This notable reactivity is likely attributed to the structural flexibility of the dioxime ligand upon its coordination to the copper(II) center [74].

2.9. Biological Studies

To verify that the observed biological effects were specific to the compounds being tested, control assays were conducted. These experiments mirrored the conditions of the bioactivity studies but omitted either the free ligand (H₂L) or the copper-based [CuLBr] complex. The control tests covering interactions with calf thymus DNA (ct-DNA) and human serum albumin (HSA), along with anticancer and antibacterial evaluations, produced no significant activity in any instance. These results indicate that the measured bioactivities originate from the compounds themselves and are not due to procedural artifacts or external variables.

2.9.1. Interaction Studies with Biomolecules (DNA and HSA)

For metallo-drugs, a correlation often exists between DNA-binding capability and anticancer potency, since metal coordination can influence cellular internalization, redox behavior, and selective targeting of biological macromolecules [74]. Accordingly, this study aims to explore the connection between the DNA-binding characteristics of both the free H₂L ligand and its [CuLBr] complex and their prospective anticancer effects.

Assessment of DNA-Binding Affinity Using UV-Vis Absorption Spectroscopy

The binding interactions of the synthesized oxime derivatives, H₂L and [CuLBr], with DNA were examined via UV-Vis absorption spectroscopy. The binding constants (K) for the DNA and HSA complexes were then determined by analyzing the changes in absorbance using the relevant equations and methodology, as detailed in the Supplementary Materials. Spectral alterations depicted in Figure 5 and Supplementary Figure S12 offer evidence regarding the mode and strength of binding between these compounds and DNA. Corresponding spectral data are compiled in Table 6.

Both compounds exhibited a hypsochromic shift (blue shift) in the absorption maximum (λ_max) upon binding to DNA, along with hyperchromism, an increase in absorption intensity. The free ligand H₂L showed a shift from 276 nm to 271 nm (Δλ = 5 nm), while the copper complex [CuLBr] displayed a smaller shift from 268 nm to 266 nm (Δλ = 2 nm). These spectral changes are characteristic of non-intercalative binding modes, such as electrostatic or groove binding, where the compound interacts with the DNA helix without significant base stacking.

The hyperchromic effect, quantified as chromism (%), was more pronounced for H₂L (96.01%) compared to [CuLBr] (67.7%), suggesting a stronger perturbation of the DNA structure by the free ligand. However, the DNA-binding constant (K_b) was higher for [CuLBr] (1.40 × 10⁵ M⁻¹) than for H₂L (0.68 × 10⁵ M⁻¹), indicating a greater binding affinity of the metal complex. This enhanced affinity may be attributed to the presence of the copper ion, which can facilitate additional coordination or electrostatic interactions with DNA phosphate groups.

The DNA-binding affinity of the [CuLBr] complex, quantified by a binding constant (K_b) of approximately 1.4 × 10⁵ M⁻¹, is consistent with values reported for groove-binding or surface-associated copper(II) complexes incorporating oxime or analogous Schiff base ligands. For example, copper(II) dioxime complexes exhibit K_b values on the order of 10⁴ M⁻¹, which is characteristic of groove binding interactions [75]. Likewise, numerous copper(II)-Schiff base complexes that engage with DNA through groove-binding modes display affinity constants spanning 10⁴ to 10⁶ M⁻¹ [76]. Additionally, copper(II) complexes bearing carboxylate ligands have demonstrated similar K_b magnitudes (e.g., ~9.8 × 10⁴ M⁻¹), also ascribed to groove-binding mechanisms [77]. In the same context, the DNA-binding constant (K_b) determined for the [CuLBr] complex positions it among the most potent synthetic metallo-DNA binders, demonstrating an affinity that rivals or exceeds that of known nickel(II) complexes. Specifically, its binding strength is comparable to [NiBPA] (K_b = 5.2 × 10⁴ M⁻¹) [75] and [NiNPA] (K_b = 5.8 × 10⁴ M⁻¹) [76] and is superior to a broader class of nickel(II) complexes with K_b values spanning 10³ to 10⁴ M⁻¹ [77,78,79,80]. The superior binding efficacy of [CuLBr] complex can be ascribed to the synergistic effect of its distinctive ligand architecture and the presence of the bromide co-ligand. Together, these structural elements are thought to facilitate a more intimate and stable association with the DNA double helix, potentially through enhanced electrostatic and hydrophobic contacts.

The binding processes for both compounds were spontaneous, as indicated by negative Gibbs free energy changes (ΔG). The ΔG value for [CuLBr] (−28.4 kJ mol⁻¹) was more negative than that for H₂L (−26.6 kJ mol⁻¹), correlating with its larger binding constant. This further supports the stronger DNA-binding affinity of the copper complex.

In conclusion, both compounds likely interact with DNA via a non-intercalative pathway. The copper(II) complex demonstrates enhanced binding affinity and greater spontaneity, which may be attributed to the central metal ion’s ability to stabilize the DNA–compound adduct.

DNA-Binding Affinity via Fluorescence Spectroscopy

The DNA-binding properties of the synthesized compounds, namely the ligand H₂L and its copper(II) complex [CuLBr], were examined through fluorescence spectroscopy. The observed spectral variations are presented in Figure 6 and Figure S13. Quantitative binding parameters, obtained from Stern–Volmer and fluorescence quenching analyses, are compiled in

Table 7. Quenching parameters of fluorescence in the binding interactions of free H₂L and [CuLBr] with DNA.

Compound	Ksv (M−1)	Kq (M−1 S−1)	R2	Kb mol−1 dm3	n	R2	ΔG kJ mol−1
H2L	2.75 × 104	2.76 × 1012	0.9620	1.449 × 105	1.203	0.991	−29.444
[CuLBr]	3.55 × 104	3.55 × 1012	0.9739	8.128 × 105	1.340	0.9844	−33.7173

and discussed in the following section.

Both compounds effectively quench the intrinsic fluorescence of DNA-bound, indicating a competitive binding interaction. The Stern–Volmer quenching constants (Ksv) were found to be on the order of 10⁴ M⁻¹ for both compounds. Specifically, the Ksv value for [CuLBr] (3.55 × 10⁴ M⁻¹) is higher than that of the free ligand H₂L (2.75 × 10⁴ M⁻¹), suggesting a more efficient quenching process and potentially stronger interaction with DNA for the metal complex.

The bimolecular quenching constant (K_q) for both compounds was determined to be approximately 10¹² M⁻¹ s⁻¹. This magnitude considerably exceeds the diffusion-controlled limit for collisional quenching (2.0 × 10¹⁰ M⁻¹ s⁻¹), indicating that the fluorescence quenching mechanism is static rather than dynamic. These results provide evidence for the formation of ground-state complexes between the compounds and DNA.

Analysis of the fluorescence quenching data yielded the binding constants (K_b) and binding site numbers (n). The K_b values for both compounds are relatively high, on the order of 10⁵ M⁻¹, reflecting a substantial affinity for DNA. Notably, the binding constant for the copper(II) complex [CuLBr] (8.128 × 10⁵ M⁻¹) is about 5.6-fold greater than that of the free ligand H₂L (1.449 × 10⁵ M⁻¹). This marked increase in DNA-binding affinity for the metal complex likely arises from the presence of the copper center, which can engage in additional interactions—such as coordination with DNA bases or the phosphate backbone beyond the groove-binding modes typically provided by the organic ligand.

The binding site number (n) was found to be approximately unity for both species, implying that one compound molecule binds per DNA-binding site (or per base pair). This observation aligns with classical intercalative or specific groove-binding behavior. Using the binding constants, the Gibbs free energy change (ΔG) for the binding process was calculated. The obtained negative ΔG values for H₂L (−29.444 kJ mol⁻¹) and [CuLBr] (−33.717 kJ mol⁻¹) confirm that the binding is spontaneous and thermodynamically favorable. The more negative ΔG for the copper complex further supports its stronger binding affinity relative to the free ligand, consistent with the K_b results.

In summary, fluorescence spectroscopic data demonstrate that both H₂L and [CuLBr] interact with DNA via a static quenching mechanism, leading to stable compound-DNA complexes. The copper(II) complex exhibits significantly enhanced DNA-binding affinity and a more spontaneous binding process compared to the free ligand. This improvement is attributed to the synergistic role of the metal ion, which provides supplementary binding interactions and stabilizes the complex. These results underscore the potential of metal-based compounds, particularly [CuLBr], as promising agents for applications requiring strong and specific DNA binding.

• Investigation of HSA Interactions via UV-Vis Absorption Spectroscopy

The binding affinity of the synthesized compounds—namely, the free oxime ligand H₂L and its copper(II) complex [CuLBr] toward Human Serum Albumin (HSA) was assessed using UV-Vis absorption spectroscopy. Key spectroscopic parameters, including absorption maxima for the unbound and bound states, wavelength shifts (Δλ), percent chromism, binding constant (K_b), and Gibbs free energy change (ΔG), are compiled in

Table 8. Spectral characteristics for the synthesized dioxime compounds’ interactions with HSA.

Compound	λmax Free (nm)	λmax Bound (nm)	Δλ (nm)	Type of Chromism	a Chromism (%)	(Kb) × 105 mol−1 dm3	ΔG# kJ mol−1
H2L	204	211	7	hyper	12.126	0.5396825	−26.997
[CuLBr]	278	278	0	hyper	35.11	1.717429	−29.865

^a Chromism (%) = [(Abs_bound − Abs_free)/Abs_free].

and elaborated below.

In its unbound state, HSA displays a prominent absorption peak at approximately 204 nm, corresponding to the π→π* electronic transition of the protein’s polypeptide backbone carbonyl groups (C=O). Titration with the free ligand H₂L resulted in a bathochromic shift of 7 nm, relocating the peak to 211 nm. This red shift was accompanied by a marked hyperchromic effect, with a 12.126% increase in absorption intensity (Figure S14).

The observed red shift is indicative of alterations in the local environment surrounding aromatic residues (tryptophan and tyrosine) or perturbations to the peptide backbone itself. Such shifts often arise from ligand incorporation into hydrophobic binding pockets or from conformational adjustments in protein secondary structure. The pronounced hyperchromic response further suggests a partial unfolding or structural loosening of HSA upon ligand binding, which likely enhances solvent exposure and accessibility of the intrinsic chromophore.

Together, these spectral modifications confirm a specific and substantive interaction between H₂L and HSA.

In contrast, the copper(II) complex [CuLBr] exhibits markedly different behavior. Its intrinsic absorption band at 278 nm, which is likely associated with intra-ligand or ligand-to-metal charge transfer (LMCT) transitions, shows no shift in wavelength (Δλ = 0 nm) upon binding to HSA (Figure 7). However, a hyperchromic effect of 35.11% is still observed. The absence of a spectral shift implies that the immediate electronic environment of the complex’s chromophore is not significantly perturbed upon binding to the protein. This could be indicative of a binding mode where the complex is situated in a location that does not strongly interact with the specific amino acid residues responsible for causing spectral shifts, or that the binding occurs through hydrophobic or van der Waals interactions without direct coordination to the metal center. The observed hyperchromism confirms that an interaction does occur, leading to a change in the extinction coefficient.

A critical difference in binding affinity, quantified by the binding constant (K_b), is observed between the two compounds. The binding constant for the copper complex, [CuLBr], is 171.7429 × 10³ M⁻¹, which is an order of magnitude larger than the value of 53.9682 × 10³ M⁻¹ for the free H₂L ligand. This result clearly demonstrates that the [CuLBr] complex possesses a significantly higher binding affinity for Human Serum Albumin compared to the unbound ligand. The higher affinity of the complex could be attributed to its larger size, specific three-dimensional structure, and the potential for additional hydrophobic or π-stacking interactions with the protein’s binding pockets.

The spontaneous nature of the binding events is demonstrated by the negative Gibbs free energy change (ΔG) calculated for both species. The ΔG value for the copper(II) complex [CuLBr] (–29.865 kJ mol⁻¹) is more negative than that of the free oxime ligand H₂L (–26.997 kJ mol⁻¹). This correlates with its larger binding constant and further supports a more spontaneous and thermodynamically favored interaction with Human Serum Albumin (HSA).

In conclusion, UV-Vis titration experiments verified the binding of both H₂L and [CuLBr] to HSA. The free ligand induces considerable protein conformational alterations, as indicated by a pronounced red shift and hyperchromic effect. In contrast, the copper complex exhibits a stronger binding affinity while maintaining its intrinsic electronic structure, implying a distinct binding mode. The enhanced binding constant and more favorable ΔG for [CuLBr] underscores its tighter association with serum albumin, a key determinant for assessing potential pharmacokinetic profiles.

2.

Fluorescence Quenching and Binding Interaction with HSA

The fluorescence quenching of Human Serum Albumin (HSA) by the compounds H₂L and its copper complex [CuLBr] was investigated to elucidate the binding mechanism and affinity. The results with a graphical representation are shown in Figure 8 and Figure S15 and summarized in

Table 9. Spectral quenching metrics derived from the binding studies of HSA with the free H₂L and [CuLBr] complex.

Compound	Ksv × 103 (M−1)	Kq × 1011 (M−1 S−1)	R2	Kb (M−1)	n	R2	ΔG kJ mol−1
H2L	7.566	7.566	0.9953	1.9275× 103	0.8099	0.9809	−18.7413
[CuLBr]	33.753	33.753	0.9802	3.614 × 106	1.480	0.9715	−37.414

, providing key thermodynamic and kinetic parameters derived from Stern–Volmer and binding constant analyses.

The Stern–Volmer quenching constant (Ksv) for [CuLBr] (33.75 × 10³ M⁻¹) is significantly higher than that for H₂L (7.56 × 10³ M⁻¹). This substantial difference indicates that the copper complex is a much more efficient quencher of HSA fluorescence. The bimolecular quenching rate constants (K_q) for both compounds were calculated to be in the range of 10¹¹ M⁻¹ s⁻¹. Since these values exceed the typical diffusion-controlled limit in aqueous solution (~10¹⁰ M⁻¹ s⁻¹), the quenching is attributed primarily to a static mechanism. This involves the formation of a non-fluorescent ground-state complex between HSA and the quencher, rather than dynamic collision-based quenching.

Further evidence for static quenching comes from the binding analysis. The association constant (K_b) for [CuLBr] (3.614 × 10⁶ M⁻¹) is approximately three orders of magnitude higher than that for H₂L (1.9275 × 10³ M⁻¹), indicating a much stronger interaction between the copper complex and HSA. The binding site number (n) was found to be close to unity for H₂L (n ≈ 0.8099), consistent with a single primary binding site. In contrast, the value for [CuLBr] n ≈ 1.48 suggests the possibility of more than one independent binding site on HSA for this complex.

The Gibbs free energy change (ΔG) for the binding process was negative for both compounds, confirming the spontaneity of the interactions. The more negative (ΔG) value for [CuLBr] (−37.414 kJ mol⁻¹) compared to H₂L (−18.7413 kJ mol⁻¹) aligns with its larger binding constant, reflecting a thermodynamically more favorable and stronger association with the protein.

In conclusion, both H₂L and [CuLBr] quench HSA fluorescence via a static mechanism. However, the copper complex demonstrates significantly enhanced binding affinity and a potentially distinct binding mode relative to the free ligand. This is supported by its higher (Ksv), substantially larger (K_b), and more negative (ΔG). The high correlation coefficients (R²) close to 1 for both analyses underscore the reliability of the fitted data and the proposed model.

Viscosity Measurements

Viscosity analysis provides a classical hydrodynamic approach for investigating the binding mechanisms of small molecules including metal chelates and organic ligands with DNA [78,79]. In this method, changes in the viscosity of a DNA solution are recorded upon the gradual introduction of the compound. A marked rise in relative viscosity is generally associated with intercalative binding, whereas slight increases often correspond to groove binding or electrostatic interactions [79]. Additionally, the viscosity of DNA solutions serves as a sensitive indicator of conformational transitions, such as elongation, condensation, or aggregation that may result from these molecular associations.

Figure S16 illustrates the changes in the cubed root of the relative viscosity (η/η₀)^1/3 of a DNA solution with successive additions of either the copper complex ([CuLBr]) or its corresponding free organic ligand (H₂L). A clear divergence in behavior is observed between the two compounds. The addition of ([CuLBr]) results in a more pronounced increase in viscosity compared to the free ligand. The values for ([CuLBr]) rise steadily over the concentration range, indicating a significant enhancement in DNA solution viscosity. In contrast, the free ligand (H₂L) induces only a modest increase, over the same range.

This marked difference suggests that the copper(II) complex interacts with DNA in a manner that leads to a greater increase in molecular volume or rigidity. Such behavior is often associated with (full or partial) intercalative binding or strong electrostatic interactions, which can elongate the DNA helix and reduce its flexibility, thereby increasing viscosity. The minimal effect of the free ligand implies a weaker or different mode of interaction, possibly groove-binding or external electrostatic association, which does not significantly alter the DNA’s hydrodynamic volume.

These results support the conclusion that the metal center in ([CuLBr]) plays a crucial role in facilitating a stronger and more structurally consequential interaction with DNA, potentially due to its planar geometry and positive charge, which favor insertion between base pairs or tight association with the phosphate backbone.

This viscosity study provides complementary evidence to other biophysical techniques, reinforcing the role of ([CuLBr]) as a potent DNA-binding agent with potential implications for its biological activity.

Kinetic and Thermodynamic Parameters for DNA Interaction

The kinetic data obtained from stopped-flow experiments, as shown in Figure 9, Figure 10 and Figure S17 and summarized in

Table 10. Comparative kinetic data and activation parameters for the binding interactions of DNA with both the free H₂L ligand and the synthesized [CuLBr] complex.

Reaction Phase	Kinetic Parameters	[CuLBr] Complex	Free H2L
1st phase	k1 (s−1)	449.6 ± 95.46	129.5 ± 8.658
	k−1 (s−1)	0.6367 ± 0.063	9.66 ± 0.0023
	ka1 (M−1)	706.141	1.34
	Kd1 (10−3 M)	1.4161	0.07459
	ΔG1 (kJ mol−1)	−16.253	−23.547
2nd phase	K2 (s−1)	73.19 ± 10.99	Irreversible step
	k−2 (10−3 s−1)	0.139 ± 0.007
	ka2 (104 M−1)	526,546.762
	Kd2 (10−3 M)	0.0018
	ΔG2 (kJ mol−1]	−32.641
Overall reaction	Kd [M]	1.418
	ka	1.0013
	ΔG# [kJ mol−1]	−48.894

, provides significant insight into the binding mechanism and affinity of both the free ligand (H₂L) and the [CuLBr] complex with DNA.

A notable two-phase reaction was observed for the [CuLBr] complex, indicating a multi-step binding process (Scheme 4). The first phase is characterized by a very high forward rate constant (k₁ = 449.6 s⁻¹), which is approximately 3.5 times faster than that of the free ligand (k₁ = 129.5 s⁻¹). This suggests that the complex associates with DNA more rapidly in the initial step. The reverse rate constant for the complex in this phase (k₋₁ = 0.64 s⁻¹) is an order of magnitude smaller than that of the free ligand (k₋₁ = 9.66 s⁻¹), implying that the initial adduct formed by the complex is significantly more stable. This is further corroborated by the first-phase dissociation constant (Kd₁), where the complex exhibits a value of 1.42 × 10⁻³ M, compared to 7.46 × 10⁻⁵ M for the free ligand. The more favorable (negative) Gibbs free energy (ΔG₁ = −23.55 kJ mol⁻¹ for H₂L vs. −16.25 kJ mol⁻¹ for [CuLBr]) for the free ligand in this phase suggests its initial binding is thermodynamically more spontaneous, likely due to simpler electrostatic or groove-binding interactions.

The most striking difference is observed in the second phase. While the interaction for the free ligand is reported as an irreversible step (Scheme 5), the [CuLBr] complex undergoes a well-defined, reversible second step (Scheme 4). This phase for the complex is defined by an exceptionally high association constant (ka₂ = 5.27 × 10⁸ M) and a remarkably low dissociation constant (Kd₂ = 1.8 × 10⁻⁶ M). The Kd₂ value, in the nano molar affinity range, indicates the formation of an extremely tight and specific final complex with DNA. The highly negative ΔG₂ value of −32.64 kJ mol⁻¹ for this step confirms it is a highly spontaneous and thermodynamically driven process.

The overall binding parameters for the [CuLBr] complex solidify its superior DNA-binding capability. The overall dissociation constant (K_d = 1.42 × 10⁻³ M) is dominated by the first, weaker step, but the overall free energy of binding (ΔG = −48.89 kJ mol⁻¹) is significantly more negative than the individual steps, indicating a strong cooperative binding mechanism. The large overall driving force underscores the stability of the final adduct.

In conclusion, the kinetic and thermodynamic profiles reveal that the [CuLBr] complex binds to DNA via a concerted, two-step mechanism. The initial association is fast, leading to an intermediate state, which then undergoes a slow, highly favorable conformational change or stronger interaction to form a final complex with very high affinity and stability. In contrast, the free ligand binding is either a simpler, single-phase process or proceeds to an irreversible product, with generally lower affinity and different kinetic properties. This enhanced and more complex binding behavior of the [CuLBr] complex is likely attributable to the coordinative versatility and specific geometry offered by the copper metal center, which may facilitate modes of interaction such as partial intercalation or groove binding that are not as accessible to the free ligand.

2.9.2. Antineoplastic Potential of [CuLBr]

The in vitro antineoplastic potential of the free oxime ligand (H₂L) and its copper(II) complex ([CuLBr]) was evaluated against a HepG-2 (liver carcinoma), HCT-116 (colon carcinoma), and MDA-MB-231 (breast adenocarcinoma), using the normal WISH (amniotic) cell line for comparison. For comparative purposes, the standard chemotherapeutic agent doxorubicin (DOX) was used as a positive control. The study findings reveal moderate to low cytotoxicity of the free H₂L ligand against the cancer cell lines, with the highest cytotoxicity observed against MDA-MB-231 cells (

Table 11. Mean IC₅₀ values in µM (mean ± SEM) of free H₂L and [CuLBr] against different human cell lines, as well as a normal cell line (WISH), compared with doxorubicin (DOX).

Compound	Free H2L	[CuLBr]	DOX	[CuL′]	[CuL″]
WISH	10.5 ± 0.409 *	27.49 ± 0.288 *	4.376 ± 0.347 *	-	-
HepG-2	10.48 ± 0.50 *	4.34 ± 0.338 *	5.425 ± 0.380 *	19.6 ± 0.433 *	12.3 ± 0.5 *
HCT-116	18.43 ± 0.506 *	6.24 ± 0.382 *	6.21 ± 0.393 *	16.6 ± 0.301 *	11.8 ± 0.11 *
MDA-231	6.937 ± 0.469 *	5.9 ± 0.377 *	5.66 ± 0.330 *	16.75 ± 0.450 *	11.7 ± 0.21 *

* Indicates that the p ≤ 0.05, statistically significant compared to the free oxime ligand, [CuLBr] complex and DOX.

). Notably, its toxicity towards the normal WISH cell line (IC₅₀ = 10.5 µM) was comparable to or even higher than its effect on some cancer cells, particularly HepG-2, suggesting a lack of selective cytotoxicity.

In contrast, the copper complexation significantly enhanced the antineoplastic potency. The [CuLBr] complex exhibited markedly lower IC₅₀ values against all cancer cell lines, indicating superior cytotoxicity. Its activity was especially strong against HCT-116 and MDA-MB-231, with the greatest cytotoxicity observed against MDA-MB-231 cells. This notable enhancement highlights the critical role of metal coordination in increasing the biological activity of the organic ligand. Although the exact mechanism is not yet fully understood, this phenomenon is commonly observed in similar Cu(II) complexes. It is often attributed to several factors, including improved cellular uptake [81,82], metal-mediated redox activity that leads to the generation of ROS [83], or direct interaction with DNA [84]. These mechanisms represent possible pathways through which coordination enhances the observed potency compared to the free ligand. Importantly, the complex showed a significantly higher IC₅₀ value compared to the normal WISH cell line, compared to both free H₂L and DOX. Also, the substantial difference in IC₅₀ values between the malignant and normal cells suggests that the [CuLBr] complex possesses a favorable selectivity profile, preferentially targeting cancer cells while exhibiting reduced toxicity towards normal cells.

Furthermore, [CuLBr] complex showed similar effectiveness against the HCT-116 and MDA-MB-231 cell lines compared to DOX. While DOX was the most toxic compound tested on the normal WISH cell line, this also indicates a high level of general toxicity, which is a well-known limitation of this drug.

The comparative antineoplastic activity of the copper complex [CuLBr] was evaluated against the reference complexes [CuL′] [85] and [CuL″] [86] across several human cancer cell lines, as summarized in Table 11. The complex [CuLBr] demonstrated superior anticancer potency compared to both [CuL′] and [CuL″], as evidenced by its significantly lower IC₅₀ values. Specifically, against the HepG-2 cell line, [CuLBr] showed substantially lower IC₅₀ values than those reported for [CuL′] and [CuL″]. A similar enhancement in potency for [CuLBr] was observed in the HCT-116 and MDA-MB-231 cell lines, with IC₅₀ values markedly lower than those of both [CuL′] and [CuL″]. This consistent pattern across multiple cancer cell lines suggests that the structural features of [CuLBr], likely due to the bromo ligand, contribute to its improved cytotoxic efficacy compared to the other copper complexes studied [85,86]. Furthermore, the higher IC₅₀ value of [CuLBr] in the normal WISH cell line indicates a more favorable selectivity profile towards cancer cells.

In conclusion, the complexation of the H₂L ligand with copper(II) significantly enhanced its cytotoxic efficacy and selectivity against the tested human cancer cell lines. The [CuLBr] complex emerges as a promising candidate with potent and selective anticancer activity, warranting further investigation into its mechanism of action.

The Therapeutic Coefficient (TC)

The evaluation of selective toxicity is paramount in anticancer drug development. The TC, defined as the percentage of the IC₅₀ in a normal cell line (WISH) to the IC₅₀ in a cancer cell line, provides a crucial metric for this purpose. A TC value greater than 1 indicates preferential toxicity against cancer over normal cells, with higher values denoting greater selectivity and a potentially wider therapeutic range.

Analysis of the TC values reveals significant differences in the selectivity profiles of the tested compounds. The copper complex, [CuLBr], demonstrated consistently high and favorable TC values of 6.334, 4.405, and 4.659 for HepG-2, HCT-116, and MDA-MB-231, respectively. These values, all substantially greater than 1, suggest that [CuLBr] possesses remarkable selective cytotoxicity, preferentially targeting cancer cells while exhibiting lower toxicity towards the normal WISH cells. This marks a significant improvement over the parent ligand.

In stark contrast, the free ligand H₂L showed minimal to no selective cytotoxicity. Its TC values were approximately 1 for HepG-2 (1.002) and MDA-MB-231 (1.513), and even below 1 for HCT-116 (0.569). A value below 1 implies that the compound is more toxic to normal cells than to the cancer cells, which is an undesirable trait for a chemotherapeutic agent. Notably, the standard chemotherapeutic drug DOX used for comparison exhibited TC values below 1 for all cancer cell lines tested (Table S2). This result is consistent with the well-documented severe side effects of DOX, which arise from its high toxicity to normal, rapidly dividing cells, thereby limiting its therapeutic window.

In conclusion, the data strongly indicate that the complexation of the H₂L ligand with copper(II) bromide drastically enhances its anticancer selectivity. The complex [CuLBr] demonstrates superior and consistent selective cytotoxicity against all evaluated cancer cell lines compared to both the free ligand and the standard drug DOX. Its high TC values suggest a potentially wider therapeutic window, warranting further investigation into its mechanism of action and in vivo efficacy.

2.10. The Correlation Between DNA Binding, Cytotoxicity, and Selectivity

The data in Table 6 and Table S2 reveal a clear and significant correlation between the cytotoxic behavior of the compounds and their DNA-binding affinity, particularly when selectivity between cancerous and normal cells is considered. The copper(II) complex [CuLBr], with its superior DNA-binding constant (K_b = 1.398 × 10⁵ M⁻¹) compared to the H₂L ligand (K_b = 0.682 × 10⁵ M⁻¹), consistently demonstrates enhanced cytotoxic potency against all tested cancer cell lines (HepG-2, HCT-116, MDA-MB-231). For instance, against HCT-116 cells, the IC₅₀ of [CuLBr] is approximately three times lower than that of H₂L. This strong positive correlation suggests that the primary mechanism of action for these compounds, especially the metal complex, likely involves binding to DNA and disrupting critical processes like replication or transcription, leading to cell death in malignant cells.

The most significant finding is the relationship between DNA binding and selective toxicity. Although stronger DNA binding typically correlates with higher cytotoxicity, the data show that this does not always result in increased toxicity across all cell types [87]. H₂L, despite its weaker binding to DNA, exhibits high and non-selective toxicity, showing potency similar to that against normal WISH cell lines as it does against certain cancer cells. In stark contrast, [CuLBr] demonstrates a remarkable distinction in its effects: it combines strong DNA-binding and high toxicity toward cancer cells while significantly reducing toxicity to normal WISH cells.

The enhanced DNA-binding affinity of the [CuLBr] complex is a necessary but not exhaustive explanation for its superior efficacy. Other factors influence selectivity beyond just binding strength. Potential explanations include: (i) Differential Cellular Uptake: Cancer cells often exhibit increased permeability and active transport mechanisms, which may lead to a preferential accumulation of the metalated complex [CuLBr] in malignant cells compared to normal cells. (ii) Intracellular Activation: Copper complexes can undergo redox cycling inside cells, which has the potential to disrupt intracellular redox homeostasis within the cell. Given their elevated metabolic stress and often compromised antioxidant defenses, cancer cells may be more susceptible to this oxidative damage than normal cells. (iii) Alternative Targets: The biological activity of [CuLBr] may not be limited to DNA. It might also interact with other crucial targets in cancer cells, such as specific proteins or enzymes, contributing to its selective toxicity [87].

In conclusion, our findings indicate that metalation to form the complex [CuLBr] significantly enhances DNA binding, which promotes cancer cell death. Importantly, this enhancement occurs alongside a remarkable improvement in selectivity. This suggests that the copper complex is not only more potent but also more strategic in its targeting, leveraging strong interactions with DNA while preferentially selecting cancer cells. As a result, it emerges as a much more promising candidate compared to the non-selective parent ligand.

Given these findings, we decided to analyze the effectiveness of the [CuLBr] complex in inducing cuproptosis, particularly since its accumulation raises copper levels within cancer cells. We studied the docking of the [CuLBr] complex with the key enzyme involved in cuproptosis, FDX1. Additionally, our data suggests that other proteins within cancer cells may also be susceptible to binding with the [CuLBr] complex. We also conducted a docking study of the [CuLBr] complex with VEGF to explore its potential role in inhibiting cancer proliferation.

2.11. Molecular Docking Analysis of the [CuLBr] Complex with FDX1 and VEGF: Implications for Cuproptosis and Angiogenesis Inhibition

The binding affinity and interactions of the synthesized [CuLBr] complex with human ferredoxin-1 (FDX1, PDB: 3P1M) and VEGF-binding IgG1-Fc (Fcab 448, PDB: 5K64) were evaluated through molecular docking simulations. The results (

Table 12. Values calculated for energy in ligand docking calculations with 3P1M receptor.

Compound	Free Energy of Binding (kcal/mol)	Intermolecular Energy (kcal/mol)	vdW + Hbond + Desolv Energy (kcal/mol)	Electrostatic Energy (kcal/mol)	Total Internal Energy (kcal/mol)	Torsional Free Energy (kcal/mol)	Unbound System’s Energy	Inhibition Constant, Ki (mM)
Citrate anion	−1.50	−3.29	−2.46	−0.83	+0.08	+1.79	+0.08	80.05
Fe2/S2 inorganic cluster	−2.11	−2.11	0.00	0.00	0.00	0.00	0.00	28.22
[CuLBr]	−4.06	−4.06	−3.92	−0.14	0.00	0.00	0.00	1.06

,

Table 13. Values calculated for energy in ligand docking calculations with 5K64 receptor.

Compound	Free Energy of Binding (kcal/mol)	Intermolecular Energy (kcal/mol)	vdW + Hbond + Desolv Energy (kcal/mol)	Electrostatic Energy (kcal/mol)	Total Internal Energy (kcal/mol)	Torsional Free Energy (kcal/mol)	Unbound System’s Energy	Inhibition Constant, Ki (mM)
2-(n-morpholino)-ethane sulfonic acid	−1.96	−2.85	−1.86	−0.99	−2.87	+0.89	−2.87	36.81
Glycerol	−1.86	−3.35	−3.05	−0.30	−3.29	+1.49	−3.29	43.23
[CuLBr]	−4.08	−4.08	−4.07	−0.01	0.00	0.00	0.00	1.03

, Tables S3 and S4) demonstrate strong and specific binding to both targets, suggesting a dual mechanism of action.

2.11.1. Interaction with FDX1 and Induction of Cuproptosis

Docking analysis revealed that [CuLBr] binds favorably to the active site of FDX1, forming two strong hydrogen bonds (Table S3) with key amino acid residues (Figure 11a–c). The calculated binding free energy was −4.06 kcal/mol, with a low inhibition constant (Ki) of 1.06 mM, indicating high affinity compared to native ligands such as the citrate anion (Ki = 80.05 mM) and the Fe2/S2 inorganic cluster (Ki = 28.22 mM) (Table 12). Although the citrate anion exhibited comparable intermolecular energy, it incurred a higher torsional free energy penalty (+1.79 kcal/mol) due to its greater number of rotatable bonds, thereby reducing its overall binding stability.

The strong binding of [CuLBr] to FDX1 is of particular interest given FDX1’s role as a mitochondrial reductase activated by intracellular copper accumulation, a process linked to Cuproptosis, a novel form of programmed cell death. The low Ki value suggests that [CuLBr] may act as a substrate for FDX1, potentially facilitating the reduction in Cu(II) to highly toxic Cu(I) within mitochondria. The resulting Cu(I) can induce reactive oxygen species generation and bind to lipoylated proteins in the TCA cycle, disrupting metabolic processes and cellular energy homeostasis, ultimately leading to cell death.

2.11.2. Interaction with VEGF and Angiogenesis Inhibition

Similarly, [CuLBr] exhibited strong binding to VEGF-binding IgG1-Fc (Fcab 448), with a binding free energy of −4.08 kcal/mol and an inhibition constant of 1.03 mM (Table 13). This affinity was superior to that of native co-crystallized ligands such as 2-(n-morpholino)-ethane sulfonic acid (Ki = 36.81 mM) and glycerol (Ki = 43.23 mM). The complex formed a strong hydrogen bond with Gln 118 at the active site (Figure 11d–f), along with multiple hydrophobic interactions (Table S4).

VEGF plays a critical role in promoting angiogenesis, vascular density, and metastasis in tumors. Inhibition of VEGF signaling is an established therapeutic strategy to restrict blood and nutrient supply to cancerous tissues. The high binding affinity of [CuLBr] to VEGF suggests its potential to disrupt VEGF-related pathways, thereby inhibiting angiogenesis. This interaction may be facilitated by the ability of copper(II) complexes to bind nitrogenous biomolecules, as supported by prior BSA binding studies.

The docking data collectively indicate that [CuLBr] can bind with high affinity to both FDX1 and VEGF, two key targets in cancer biology. Through its interaction with FDX1, [CuLBr] may induce cuproptosis by promoting toxic copper(II) accumulation in mitochondria. Concurrently, its binding to VEGF may inhibit tumor angiogenesis, cutting off the blood supply essential for tumor growth and metastasis. Thus, [CuLBr] emerges as a promising multi-target metallodrug capable of attacking cancer cells through complementary mechanisms, including the induction of copper-dependent cell death and suppression of tumor vascularization.

3. Experimental Section

3.1. Chemicals

All chemicals used in this work were of analytical grade and were employed without further purification. Copper(II) bromide salt (CuBr₂), 2,3-diaminotoluene, and diacetyl monoxime were procured from Fluka and Aldrich.

3.2. Synthesis of the Tetradentate Oxime Ligand (H₂L)

The tetradentate dioxime ligand (H₂L) was synthesized via a Schiff base condensation reaction according to a literature procedure [42]. The product was obtained as a pure solid in 87% yield with a melting point of 166 °C. Elemental analysis confirmed its composition: Found (%): C, 62.19; H, 6.65; N, 19.17. Calculated for C₁₅H₂₁N₄O₂ (%): C, 62.49; H, 6.94; N, 19.45.

3.3. Synthesis of [CuLBr] Complex

The copper(II) complex was synthesized by the dropwise addition of an ethanolic solution of copper(II) bromide (CuBr₂, 0.005 mol, 0.7177 g) to a hot ethanolic solution of an equimolar amount of the H₂L ligand (0.005 mol, 1.4402 g). The reaction mixture was stirred continuously at room temperature for one hour, yielding shiny green microcrystals. The product was isolated by filtration, washed thoroughly with hot ethanol and diethyl ether, and dried over CaCl₂. An 85% yield was obtained.

Elemental analysis confirmed the composition (CuC₁₅H₁₉N₄O₂Br): Found (%) C, 41.59; H, 4.44; N, 12.92 and Calcd: C, 41.80; H, 4.41; N, 13.01.

3.4. Physicochemical and Biological Investigations

Detailed procedures for the physicochemical and biological investigations conducted in this work are provided in Section S1.

3.5. Molecular Docking

Molecular Docking with human ferredoxin-1 (FDX1) and VEGF-binding IgG1-Fc (Fcab 448) are listed in Section S1.

4. Conclusions

In this study, a novel copper(II) complex, [CuLBr], synthesized from a tetradentate dioxime ligand (H₂L), was successfully prepared and comprehensively characterized. The structure was elucidated through a combination of analytical and spectroscopic methods, supported by density functional theory calculations and Rietveld-refined X-ray powder diffraction analysis. These techniques unequivocally established a five-coordinate, slightly distorted square pyramidal geometry around the copper(II) center, characterized by an N₄Br chromophore.

The complex exhibited significant biomimetic catalytic properties, serving as a proficient functional model for both SOD and catalase enzymes. Its notable superoxide dismutase activity, with a turnover rate k_cat = 4.26 × 10⁵ M⁻¹ s⁻¹, is facilitated by a favorable redox potential (E_1/2 = +0.24 V vs. NHE). Furthermore, the complex demonstrated potent catalase-like functionality in decomposing hydrogen peroxide, as evidenced by a high catalytic efficiency, k_cat/K_M = 4.44 × 10⁶ M⁻¹ s⁻¹.

Biophysical interaction studies revealed that [CuLBr] binds to calf thymus DNA (ct-DNA) and HSA more strongly than the free ligand. The DNA binding, characterized by high binding constants (e.g., K_b approximate 10⁵–10⁶ M⁻¹) and a two-step kinetic mechanism with nanomolar affinity in the final step, suggests a potent and stable interaction, potentially involving a groove-binding mode with some intercalative character, as supported by viscosity measurements. The strong HSA binding (K_b = 3.614 × 10⁶ M⁻¹) indicates good potential for transport in the bloodstream.

The in vitro anticancer evaluation revealed a dramatic enhancement in potency and selectivity upon complexation. The [CuLBr] complex exhibited superior cytotoxicity against HepG-2, HCT-116, and MDA-MB-231 cancer cell lines compared to the H₂L ligand. Crucially, it demonstrated an excellent selectivity profile, with significantly reduced toxicity towards normal WISH cells. This resulted in high TC values, substantially outperforming both the non-selective free ligand and the standard drug doxorubicin.

The enhanced biological profile of [CuLBr] is rationally explained by its electronic structure, as determined by DFT. The complex possesses a lower HOMO-LUMO gap, higher electrophilicity, and greater global softness than the ligand, correlating with its increased chemical reactivity and superior biomolecular binding and anticancer activity. Also, [CuLBr] complex may be promising as a targeted drug for inducing cuproptosis in cancer cells, as molecular docking suggests interaction with FDX1 that may promote mitochondrial dysfunction and trigger cuproptosis in cancer cells. In addition, the predicted binding of [CuLBr] to VEGF may interfere with angiogenesis, potentially limiting tumor growth and proliferation.

In summary, the [CuLBr] complex emerges as a multifaceted bioactive agent with potent antioxidant, DNA-binding, and selective anticancer properties. Its well-defined structure, robust biomimetic activity, and compelling in vitro efficacy and selectivity make it a highly promising candidate for further exploration in the development of metallodrugs. Furthermore, the extremely low inhibition constants (Ki) for both targets (FDX1 and VEGF) highlight the compound’s strong potential to be an effective multi-target metallodrug. This dual mechanism of action highlights the potential of [CuLBr] as a promising therapeutic agent for cancer treatment. By targeting both cuproptosis and angiogenesis, it offers a novel approach to disrupt cancer cell survival and tumor progression, paving the way for more effective and targeted therapies. However, further in vitro and in vivo studies are necessary to support [CuLBr] effectiveness and safety in preclinical and clinical settings.