Structure–function relationships in a square-planar Cu(ii) hybrid halometalate: experimental, DFT, molecular docking, optical, and biological studies

Abstract

A novel organic–inorganic copper(ii) hybrid halometalate, (C₆H₈BrN₂)₂[CuBr₄], was successfully synthesized and investigated using complementary experimental and computational approaches. Single-crystal X-ray diffraction analysis revealed discrete square-planar [CuBr₄]²⁻ anions embedded within a three-dimensional supramolecular architecture stabilized by N–H⋯Br hydrogen bonds, π–π stacking interactions, and halogen contacts. Optical studies indicated semiconducting characteristics with an estimated band gap of approximately 2.5 eV, together with a broad visible emission assigned to ligand-to-metal and/or charge-transfer transitions. Density functional theory (DFT) calculations yielded a HOMO–LUMO energy gap of 2.78 eV, consistent with the observed electronic properties, while ADMET predictions suggested acceptable aqueous solubility, moderate membrane permeability, limited blood–brain barrier penetration, and no P-glycoprotein substrate liability. Molecular docking showed favorable binding affinities toward human α/β-tubulin (PDB ID: 5J2T; −5.12 kcal mol⁻¹) and human peroxiredoxin 5 (PDB ID: 1HD2; −4.85 kcal mol⁻¹), in agreement with the experimentally observed antiproliferative and antioxidant performances. Biological assays further confirmed moderate free-radical scavenging capacity in DPPH and ABTS tests, together with significant cytotoxic effects against HCT-116 and HepG-2 cancer cell lines. Overall, these results establish (C₆H₈BrN₂)₂[CuBr₄] as a multifunctional Cu(ii)-based hybrid material with promising prospects for both optoelectronic and biomedical applications.

A square-planar Cu(ii) hybrid halometalate exhibits supramolecular organization, distinctive charge-transfer properties, and protein binding, supporting its antioxidant and anticancer potential.

1. Introduction

Hybrid organic–inorganic metal halides (HOIMHs) have attracted considerable interest because of their structural diversity and the possibility of combining inorganic metal-halide units with functional organic cations to generate materials with tunable physicochemical properties.^1–3 Depending on the nature of the metal center, halide ligands, and organic component, these compounds can adopt isolated halometalate units, one-dimensional chains, layered architectures, or more complex supramolecular arrangements.^2–5 The organic cation plays an important role in determining the crystal packing through hydrogen bonding, halogen bonding, π–π stacking, electrostatic interactions, and other non-covalent contacts. These interactions can influence the organization of the inorganic sublattice and consequently affect the optical and electronic properties of the resulting hybrid materials.^2–5

Copper(ii)-based halometalates are particularly interesting because Cu(ii) possesses a d⁹ electronic configuration and exhibits considerable stereochemical flexibility. Depending on its coordination environment, Cu(ii) may adopt square-planar, square-pyramidal, or elongated octahedral geometries, with distortions frequently associated with Jahn–Teller effects.^6–9 Such structural flexibility provides an opportunity to establish relationships between the coordination environment of the metal center, the supramolecular organization of the crystal, and its resulting physicochemical properties. In particular, the nature of the halide ligands and the arrangement of the surrounding organic cations can influence the optical absorption, electronic structure, and charge-transfer processes in Cu-based hybrid materials.^9–11

The organic component is equally important in controlling the structural organization of these materials. Protonated aromatic amines can act simultaneously as charge-compensating species and supramolecular building blocks. In particular, protonated 2-amino-3-bromo-5-methylpyridine contains N–H groups capable of forming hydrogen bonds with halide anions, while its aromatic ring provides a platform for π-associated interactions. The bromine substituent may additionally contribute to halogen-mediated contacts, depending on the crystal environment.^12–14 The combination of these interactions can influence the packing arrangement of the inorganic and organic components and contribute to the stabilization of the resulting crystal structure. Such structure-directing effects are particularly relevant for understanding the relationship between crystal architecture and the optical and electronic characteristics of hybrid halometalates.^15–23

Single-crystal X-ray diffraction (SCXRD) provides essential information on the coordination geometry of Cu(ii), the nature of the inorganic halometalate unit, and the intermolecular interactions responsible for the supramolecular organization.^24–26 In this context, analysis of hydrogen-bonding and other non-covalent contacts is important for understanding how the organic and inorganic components are assembled within the crystal. Hirshfeld surface analysis provides a complementary quantitative approach by visualizing and evaluating the relative contributions of intermolecular contacts to the crystal packing. Vibrational spectroscopy, including infrared and Raman spectroscopy, further provides information on the characteristic vibrations of the organic cation, Cu–Br units, and hydrogen-bonding environment.^27–29

The optical properties of Cu(ii) halometalates are strongly influenced by their electronic structure and coordination environment.^30–32 Their absorption spectra may contain contributions from ligand-centered transitions, charge-transfer processes, and Cu(ii)-centered d–d transitions, depending on the structural and electronic characteristics of the compound.^33,34 The optical response can therefore be affected by the halide composition, coordination geometry, crystal packing, and interactions between the organic and inorganic components. Complementary density functional theory (DFT) calculations can provide further insight into the electronic structure, frontier molecular orbitals, and reactivity-related descriptors of these systems.^35,36

Copper-based systems have also attracted interest in materials and bioinorganic research because copper is an essential trace element and can participate in biologically relevant redox and coordination processes. However, the biological and environmental suitability of a copper-containing material cannot be inferred solely from the identity of the metal centre, since toxicity, bioavailability, speciation, and the nature of the associated ligands strongly influence its behaviour. Therefore, the presence of Cu(ii) in the present hybrid is considered a chemical feature relevant to its biological investigation rather than, by itself, evidence of environmental benignity. Beyond their physicochemical properties, metal-containing hybrid materials have also been investigated for their potential biological activity. In Cu-based systems, the coordination environment of the metal center and the chemical nature of the organic component may both contribute to the observed biological response. However, the relationship between crystal structure and biological activity is not necessarily direct, particularly because the solid-state structure may undergo changes or dissociation under biological conditions. Experimental biological assays combined with molecular docking and in silico pharmacokinetic analyses can therefore provide complementary information concerning the biological potential of such compounds, while allowing computational predictions to be distinguished from experimentally established effects.

The motivation for investigating the biomedical properties of the present hybrid arises from the complementary chemical features of its two components. The Cu(ii) centre provides a redox-active metal environment that may contribute to biological responses, while the protonated aromatic organic cation contains hydrogen-bonding and π-interacting functionalities that may participate in interactions with biological targets. Such features provide a reasonable basis for examining antioxidant and antiproliferative properties rather than assuming biological activity solely from the crystal structure. Accordingly, the biological study was designed as an exploratory evaluation of the functional potential of the hybrid, using antioxidant assays and in vitro antiproliferative tests, complemented by molecular docking and ADMET predictions. Importantly, these computational analyses are considered supportive rather than definitive evidence of mechanism, whereas the biological activities are established experimentally.

In this work, we report a Cu(ii)-based hybrid halometallate, (C₆H₈BrN₂)₂[CuBr₄], incorporating protonated 2-amino-3-bromo-5-methylpyridine as the organic cation. The compound was investigated using a combination of single-crystal X-ray diffraction, Hirshfeld surface analysis, vibrational spectroscopy (IR/Raman), optical spectroscopy, thermal analysis, DFT calculations, molecular docking, ADMET prediction, and biological assays. Particular emphasis is placed on the Cu(ii) coordination environment, its deviation from the idealized geometry, the intermolecular interactions governing crystal packing, and the relationship between the structural features and the observed optical properties. The biological investigation further evaluates the antioxidant and antiproliferative activities of the compound, while molecular docking and ADMET calculations provide complementary in silico information. This integrated approach aims to establish a clearer structure–property relationship for the title Cu(ii)-based hybrid halometallate and to clarify the respective contributions of its inorganic and organic components to its physicochemical and biological behavior.

2. Experimental section

2.1. Materials

Copper(ii) bromide (CuBr₂, 99%), hydrobromic acid (HBr, 48%) and 2-amino-3-bromo-5-methylpyridine (C₆H₇BrN₂, 98%) were purchased from Sigma-Aldrich and used without further purification.

2.2. Synthesis

Home-built Teflon-lined stainless steel pressure bombs with a maximum capacity of 120 mL were used to synthesize (C₆H₈BrN₂)₂[CuBr₄]. A mixture of Copper(ii) bromide (0.22 g, 1 mmol) and 2-amino-3-bromo-5-methylpyridine (0.37 g, 2 mmol) were dissolved together in 30 mL of deionized water and hydrobromic acid (pH = 3). The mixture was put in an autoclave lined with Teflon, sealed, and heated to 160 °C for three days. After that, it was allowed to cool to room temperature in a water bath. Filtering was used to recover the products once the autoclave was opened to the air. Block-shaped crystals that were appropriate in size for crystallographic analysis were found. The crystals were repeatedly cleaned with distilled water and allowed to dry outside. Elem. microanal. obsd for (C₆H₈BrN₂)₂[CuBr₄] (calcd): C, 11.48 (11.46); H, 2.40 (2.35); N, 8.16 (8.11) (IR, KBr, cm⁻¹): ν(N–H⁺), 1532; ν(C–N), 1638. The yield of the reaction was 0.58 g (76.4%). ESI MASS m/z [M–H]-calcd 795.18, found 795.29. The reaction scheme is outlined as shown in Scheme 1.

Scheme 1. Synthetic route for the preparation of (C₆H₈BrN₂)₂[CuBr₄].

2.3. Single-crystal X-ray data collection and structure determination

A single crystal, approximately 0.29 × 0.17 × 0.14 mm was used to collect diffraction data at 299 K with an Xcalibur Atlas Gemini Ultra diffractometer utilizing Mo Kα radiation (λ = 0.71073 Å) and processed through the SCALE3 ABSPACK program.³⁷ Data collection, reduction, and analysis were conducted using SAINT,³⁸ with semi-empirical multi-scan absorption corrections applied via the SADABS³⁹ program. The crystal structure was determined in the triclinic space group P1̄, using auto-methods available in WINGX.⁴⁰ The structure of (C₆H₈BrN₂)₂[CuBr₄] was solved by the dual-space algorithm using SHELXT-2015 ⁴¹ and refined by full-matrix least-squares techniques using SHELXL⁴² with anisotropic thermal parameters for all non-hydrogen atoms. All figures were prepared using DIAMOND.⁴³ The refinement conditions and structural resolution of our compound are outlined in Table S1.

2.4. Spectroscopic measurement

Fourier-transform infrared (FTIR) spectra were collected in the 400–4000 cm⁻¹ range using a PerkinElmer Spectrum 400 spectrometer equipped with a universal attenuated total reflectance (UATR) module.

2.5. UV-Vis-NIR diffuse reflectance spectroscopy

Diffuse reflectance UV-Vis-NIR measurements of (C₆H₈BrN₂)₂[CuBr₄] were conducted at ambient temperature using a Varian Cary 5000 spectrophotometer. The optical spectra were recorded over the 200–800 nm wavelength range.

2.6. In silico studies

To gain deeper insight into the electronic properties and biological potential of the synthesized Cu(ii)-based hybrid halometalate, comprehensive in silico investigations were performed, including density functional theory (DFT) calculations, molecular docking, and pharmacokinetic (ADMET) predictions. The molecular geometry of the synthesized complex was fully optimized using density functional theory (DFT) calculations. The optimized geometry was subsequently employed to investigate the electronic structure through analysis of the frontier molecular orbitals (HOMO and LUMO), the HOMO–LUMO energy gap (ΔE), molecular electrostatic potential (MEP), and global reactivity descriptors. These calculations provide valuable insights into the electronic properties, chemical stability, charge distribution, and potential reactive sites of the investigated complex.^44–46 To elucidate the molecular basis of the observed biological activities, molecular docking simulations were performed against two biologically relevant therapeutic targets: human α/β-tubulin (Vinca-binding site; PDB ID: 5J2T), associated with the antiproliferative activity,⁴⁷ and human peroxiredoxin 5 (PRDX5; PDB ID: 1HD2),⁴⁸ associated with the antioxidant activity. The three-dimensional crystal structures of both proteins were retrieved from the RCSB Protein Data Bank. Protein preparation involved the removal of crystallographic water molecules, heteroatoms, and non-essential ligands, followed by the addition of polar hydrogen atoms and assignment of appropriate partial charges. The geometrically optimized structure of the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], was used as the docking ligand. Molecular docking calculations were carried out using AutoDock Vina version 1.1.2.⁴⁹ The initial three-dimensional atomic coordinates of the complete ionic hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], were extracted directly from the experimentally determined single-crystal X-ray diffraction (SC-XRD) CIF file. The complete ionic assembly, comprising the protonated organic cations and the discrete tetrahalocuprate(ii) anion [CuBr₄]²⁻, was subsequently subjected to geometry optimization at the UB3LYP/LANL2DZ level of theory prior to molecular docking. The crystallographically established ionic connectivity was retained, and no artificial covalent bond was introduced between the organic cations and the [CuBr₄]²⁻ anion. The optimized structure was then prepared as a complete ionic assembly for molecular docking. Because conventional empirical docking functions may have limitations in describing metal-containing ionic species, particularly with respect to metal-specific polarization and coordination effects, the docking results were interpreted as comparative descriptors of the predicted interaction modes rather than quantitative binding free energies.^50,51

The binding site was defined according to the position of the co-crystallized ligand, and multiple binding poses were generated for each ligand. The optimum binding conformation was selected based on the lowest predicted binding free energy (kcal mol⁻¹) together with the most favorable interaction profile.⁵² To validate the docking protocol for human α/β-tubulin (PDB ID: 5J2T), the co-crystallized ligand vinblastine (VLB) was extracted and re-docked into its original binding pocket. The reproduced binding pose was compared with the crystallographic conformation, and the protocol was considered valid when the root-mean-square deviation (RMSD) was below 2.0 Å. For the antioxidant study, BHT (butylated hydroxytoluene) was employed as the reference ligand because it served as the positive control in the experimental antioxidant assays (DPPH, ABTS, and β-carotene bleaching). Protein–ligand interactions, including hydrogen bonds, hydrophobic interactions, π–π stacking, halogen bonding, and other non-covalent contacts, were analyzed and visualized using PyMOL Molecular Graphics System (Version 2.5).⁵³ Drug-likeness and pharmacokinetic (ADMET) properties of the synthesized compound were predicted using the SwissADME and pkCSM web servers.⁵⁴ Physicochemical descriptors together with absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were predicted using the pkCSM web server to evaluate the pharmacokinetic behavior and potential drug-likeness of the synthesized hybrid complex.^55,56

2.7. Biological activity

The biological activity of Cu-complex, (C₆H₈BrN₂)₂[CuBr₄] and its organic ligand, 2-amino-3-bromo-5-methylpyridine was evaluated through antioxidant and in vitro antiproliferative assays. For all experiments, (C₆H₈BrN₂)₂[CuBr₄] or C₆H₇BrN₂ was dissolved in a DMSO/H₂O mixture (1 : 9, v/v) and serially diluted to the desired concentrations. All measurements were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD).

2.7.1. Antioxidant activity

The antioxidant potential of (C₆H₈BrN₂)₂[CuBr₄] and C₆H₇BrN₂ assessed using three complementary in vitro assays: DPPH radical scavenging, ABTS˙⁺ radical cation decolorization, and β-carotene-linoleic acid bleaching. In the DPPH assay, the decrease in absorbance at 517 nm was monitored after 30 min of incubation in the dark. The ABTS˙⁺ radical scavenging activity was determined spectrophotometrically at 734 nm after 6 min of reaction, while the β-carotene bleaching assay was evaluated by measuring the absorbance at 470 nm after 120 min of incubation. Antioxidant activity was expressed as the EC₅₀ value, defined as the concentration required to achieve 50% radical scavenging or inhibition. Butylated hydroxytoluene (BHT) was used as the reference antioxidant.

2.7.2. In vitro antiproliferative activity

The in vitro antiproliferative activities of (C₆H₈BrN₂)₂[CuBr₄] and C₆H₇BrN₂ were evaluated against human colorectal carcinoma (HCT-116) and human hepatocellular carcinoma (HepG-2) cell lines obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were maintained and handled according to the supplier's recommendations, and the antiproliferative assays were conducted under the supervision of a qualified medical biologist at the Medical Analysis Laboratory in Houmt Souk, Djerba, Tunisia.

Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and gentamicin under standard incubation conditions (37 °C in a humidified atmosphere containing 5% CO₂). After seeding into 96-well plates, the cells were exposed to different concentrations of the synthesized compound for 24 h. Cell viability was determined using the crystal violet colorimetric assay by measuring the absorbance at 495 nm. Vinblastine was used as the positive control, and the IC₅₀ values were calculated from the concentration–response curves.

2.8. Hirshfeld surface analysis

To gain further insight into the intermolecular interactions governing the crystal packing, a detailed Hirshfeld surface (HS) analysis was performed.^57–62 The three-dimensional Hirshfeld surfaces and the corresponding two-dimensional fingerprint plots were generated using CrystalExplorer 17.5.⁶³ The Hirshfeld surface was mapped using the normalized contact distance, (d_norm), which is determined from the distances d_e and d_i, corresponding to the nearest external and internal nuclei, respectively, together with the van der Waals radii of the interacting atoms. The d_norm surface was represented using a blue–white–red color scale, where the red regions indicate contacts shorter than the sum of the corresponding van der Waals radii, white regions correspond to contacts close to the van der Waals separation, and blue regions denote longer contacts. This mapping provides a clear visualization of the relative contribution and spatial distribution of intermolecular contacts within the crystal structure.

3. Results and discussion

3.1. SEM/EDS characterization

The surface morphology and elemental composition of (C₆H₈BrN₂)₂[CuBr₄] were investigated by scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDS) (Fig. 1). The SEM micrographs reveal well-defined crystalline domains exhibiting elongated plate-like morphologies with relatively smooth surfaces and sharp edges, consistent with the formation of crystalline material under the employed synthesis conditions. The crystallites appear moderately aggregated, forming layered assemblies rather than densely compact particles, while noticeable interparticle spacing is observed between adjacent plates. The elongated morphology suggests preferential crystal growth along specific crystallographic directions. Although SEM analysis alone cannot establish the origin of this anisotropic growth, the observed morphology is consistent with the directional intermolecular interactions identified from the single-crystal structure, particularly N–H⋯Br hydrogen bonding and π–π stacking between the aromatic organic cations. These interactions may contribute to the supramolecular organization and stabilization of specific crystal faces, thereby influencing the observed crystal-growth morphology; however, a direct causal relationship cannot be established solely from the SEM observations.

Fig. 1. (a) SEM micrographs, (b) corresponding EDS spectrum of (C₆H₈BrN₂)₂[CuBr₄].

The EDS spectrum confirms the presence of the principal constituent elements of the hybrid material, namely Cu and Br, together with signals corresponding to C and N originating from the organic cationic moieties. Quantitative elemental analysis shows that bromine constitutes the major component of the analyzed material, while the Cu/Br atomic ratio is close to the theoretical stoichiometric value expected for the [CuBr₄]²⁻ inorganic unit, supporting the formation of the targeted hybrid halometallate. No additional elemental signals were detected within the detection limits of the EDS measurement, indicating that no detectable extraneous elements were present in the analyzed region. These EDS results support the expected elemental composition of the compound, although EDS alone cannot establish absolute chemical purity or complete compositional homogeneity throughout the bulk sample.

The EDS results support the expected elemental composition of the compound, although EDS alone cannot establish phase purity or complete compositional homogeneity throughout the bulk sample. The phase purity of the bulk material was therefore assessed independently by PXRD, which showed good agreement with the diffraction pattern calculated from the single-crystal structure and no detectable crystalline secondary phases (Fig. S1).

3.2. Structural study

Single-crystal X-ray diffraction analysis of (C₆H₈BrN₂)₂[CuBr₄] (Fig. 2) reveals that the compound crystallizes in the triclinic system with the centrosymmetric space group P1̄. The unit-cell parameters are a = 7.5710(7) Å, b = 8.1867(6) Å, c = 8.7179(7) Å, α = 85.343(3)°, β = 101.105(3)°, and γ = 81.105(3)°. The asymmetric unit consists of one Cu(ii) center coordinated by four bromide ligands forming a discrete [CuBr₄]²⁻ anion, together with two crystallographically independent protonated 2-amino-3-bromo-5-methylpyridinium cations that ensure charge compensation and contribute significantly to the supramolecular organization of the crystal structure. The Cu(ii) ion adopts a slightly distorted square-planar coordination geometry defined by four bromide ligands, with Cu–Br bond distances ranging from 2.3778(5) to 2.4497(5) Å and Br–Cu–Br angles varying between 89.4(2)° and 180.0°. The observed distortions from the ideal square-planar arrangement are consistent with the Jahn–Teller effect associated with the d⁹ electronic configuration of Cu²⁺, in addition to the influence of intermolecular interactions within the crystal lattice. Bromide ligands involved in hydrogen bonding display slightly elongated Cu–Br distances, suggesting a moderate redistribution of electron density around the coordination sphere. The organic cations exhibit nearly planar aromatic frameworks, with a maximum deviation from the mean plane below 0.02 Å. The bromine substituent is positioned opposite to the protonated amino groups, while the –NH₂⁺ functionalities act as effective hydrogen-bond donors toward neighboring bromide ligands through N–H⋯Br interactions. These interactions contribute to the stabilization of the inorganic unit and promote the formation of an extended supramolecular network. In addition, the planar nature of the aromatic rings favors π–π stacking interactions between adjacent cations, further reinforcing the crystal packing. The crystal architecture therefore results from the combined contribution of coordination bonding, electrostatic interactions, hydrogen bonding, and π-driven supramolecular contacts. The discrete [CuBr₄]²⁻ unit acts as the inorganic structural core, while the organic cations ensure the cohesion and stabilization of the three-dimensional hybrid framework.

Fig. 2. ORTEP representation at the 50% probability level of the formula unit of (C₆H₈BrN₂)₂[CuBr₄], showing the atom-numbering scheme of the asymmetric unit.

The [CuBr₄]²⁻ anions are discrete 0D inorganic units, while intermolecular N–H⋯Br hydrogen bonds, π–π stacking, C–H⋯π interactions, and Br⋯Br contacts connect the organic and inorganic components into an extended three-dimensional supramolecular architecture. Projections along the a, b, and c axes (Fig. 3) show a dense alternation of inorganic units and organic layers, giving the material its characteristic structural cohesion.

Fig. 3. Crystal packing along the three principal axes (a, b and c).

Along the a-axis, the [CuBr₄]²⁻ square-planar units align into regular chains, each enveloped by four aromatic cations. These chains are consolidated by short, nearly linear N–H⋯Br hydrogen bonds, with bromide ligands serving as strong acceptors. This directional bonding generates a continuous inorganic–organic framework with a balanced electrostatic distribution.

Along the b-axis, the organic cations form well-defined layers arranged in head-to-tail orientations. Their nearly parallel aromatic rings support π⋯π stacking and C–H⋯π contacts, while alternating tilt directions between layers produce a subtle herringbone arrangement. This geometry enhances packing efficiency, minimizes steric congestion, and reinforces the layered character of the crystal.

Along the c-axis, the square-planar units alternate in orientation by about 180°, reducing Br⋯Br repulsion and maintaining consistent Cu⋯Cu distances. Multidirectional N–H⋯Br interactions link these anionic nodes, producing supramolecular corridors that strengthen the three-dimensional connectivity of the lattice.

Overall, the packing is governed by a cooperative interplay of strong ionic and hydrogen-bonding interactions, complemented by π-stacking and weaker halogen contacts. This combination yields a compact, robust, and exceptionally well-organized structure in which inorganic rigidity is seamlessly integrated with the directional versatility of the organic cations. The resulting architecture exemplifies the high degree of self-organization typical of Cu(ii)-based halometalates.

The Cu(ii) center is coordinated by four bromide ligands and adopts a square-planar coordination geometry (Fig. 4). The Cu–Br bond lengths are 2.3778(5) and 2.4497(5) Å, while the Br–Cu–Br angles are close to the ideal square-planar values, with angles of 89.436(17), 90.564(17), and 180.0°. The Okuniewski parameter⁶⁴ calculated from the two largest Br–Cu–Br angles (180.0° and 180.0°) is 0.000, supporting the square-planar character of the CuBr₄ coordination environment. The difference between the two Cu–Br bond lengths indicates a modest distortion from an idealized square-planar geometry. Such bond-length anisotropy is consistent with the expected structural effects associated with the electronically degenerate d⁹ configuration of Cu(ii), although the crystallographic data alone do not allow the magnitude of the Jahn–Teller contribution to be separated from other structural factors. Thus, the CuBr₄ unit is best described as a slightly distorted square-planar Cu(ii) environment, with the value providing quantitative confirmation of its coordination geometry.

Fig. 4. Slightly distorted square-planar coordination environment of Cu²⁺ in the [CuBr₄]²⁻ anion.

Beyond the primary coordination and hydrogen-bond network, the crystal structure of (C₆H₈BrN₂)₂[CuBr₄] (Fig. 5) exhibits a sophisticated system of secondary interactions that further reinforce its supramolecular architecture. The organic cations, possessing planar aromatic frameworks, align in such a way that the π clouds of neighboring benzene rings overlap efficiently, giving rise to extended π–π stacking columns. The centroid-to-centroid distances between adjacent rings range from 3.63 to 3.75 Å, values that correspond to optimal aromatic overlap. A slight horizontal displacement of about 1.1 Å minimizes electrostatic repulsion, while preserving the delocalized contact surface essential for dispersion stabilization. These π–π stacks extend mainly along the b-axis, forming quasi-columnar arrays that act as rigid organic pillars separating successive inorganic layers. Each column behaves as a supramolecular spine, transmitting cohesion and stabilizing the relative orientation of neighboring [CuBr₄]²⁻ units. Within these organic corridors, additional C–H⋯π interactions emerge between aromatic hydrogens and the π clouds of adjacent rings (H···centroid = 2.8–3.0 Å; C–H···centroid angles = 135–155°). These interactions connect the π-stacked columns laterally, generating a resilient two-dimensional organic sublattice that overlays the anionic framework. A particularly innovative and distinctive feature of this compound lies in the presence of short halogen–halogen (Br⋯Br) contacts, involving both the bromine substituents of the organic cations and the terminal bromides of the [CuBr₄]²⁻ anions. The Br⋯Br separations (3.55–3.62 Å) are significantly shorter than the sum of van der Waals radii, indicating genuine halogen–halogen bonding of the type σ-hole⋯σ-hole or σ-hole···lone pair. These weakly attractive but highly directional interactions bridge the inorganic and organic domains, establishing an additional layer of communication across the lattice. The coexistence of π–π stacking, C–H⋯π contacts, and Br⋯Br interactions defines a multilevel supramolecular hierarchy rarely encountered in hybrid halometalates. The aromatic planes serve as electronic platforms, the hydrogen bonds provide directional strength, and the halogen–halogen contacts introduce lateral connectivity between distinct domains. This intricate interplay of forces creates a three-dimensional cohesion that is both geometric and electronic in nature. Such a constellation of cooperative interactions enhances not only the structural stability but also the functional potential of the crystal. The simultaneous presence of dispersive, electrostatic, and polarizable contacts gives this compound an exceptional internal order that could be harnessed in photoactive or optoelectronic applications. In this way, (C₆H₈BrN₂)₂[CuBr₄] exemplifies the next generation of hybrid copper halometalates, where every atom contributes meaningfully to the architecture of the whole.

Fig. 5. Different types of π-interactions: red (π⋯π), pink (C–H⋯π), green (H⋯π), and halogen–halogen interaction.

The hydrogen-bond network in (C₆H₈BrN₂)₂[CuBr₄] (Fig. 6) forms the true structural backbone, uniting all components into a coherent, three-dimensional framework. The protonated amine groups of the cations act as strong N–H⋯Br donors toward the bromide ligands of the [CuBr₄]²⁻ anions. Key hydrogen bonds, such as N1–H1⋯Br2 and N1–H2⋯Br3, display donor–acceptor distances of 3.397(4) and 3.413(4) Å with angles of 154° and 128°, confirming their directional strength and role in linking square-planar anions to multiple neighbors. This network is highly organized: each bromide ligand serves as a multifunctional acceptor involved in several hydrogen bonds across different cations. These interactions propagate in three dimensions, forming a self-supporting scaffold. Strong N–H⋯Br bridges dominate along the a- and b-axes, while weaker C–H⋯Br contacts reinforce the c-axis. Together, they establish a dense, stable structure characterized by balance and rigidity. A key feature of this system is its cooperative behavior. The hydrogen bonds align seamlessly, distributing strain and enabling the lattice to respond flexibly to local motion while maintaining global order, a hallmark of resilient hybrid materials. The directional nature of N–H⋯Br interactions also governs the spatial alignment of [CuBr₄]²⁻ units, preventing orientation disorder and ensuring precise overlap between inorganic and organic layers.

Fig. 6. Hydrogen-bonding framework involving N–H⋯Br interactions.

Take all this together; this hydrogen-bond architecture is a model of molecular precision. Each donor and acceptor plays a strategic role in maintaining symmetry and cohesion. Combined with complementary π and halogen interactions, the network elevates (C₆H₈BrN₂)₂[CuBr₄] into a structurally intelligent hybrid halometalate, where coordination geometry, organic design, and supramolecular organization converge with remarkable sophistication.

The structural features of (C₆H₈BrN₂)₂[CuBr₄] reveal a distinct coordination environment within the family of pyridinium-based tetrabromocuprate(ii) compounds. Luque et al. demonstrated that changes in pyridinium substitution can influence the distortion of the [CuBr₄]²⁻ unit together with the hydrogen-bonding and aromatic-stacking arrangements governing crystal packing.⁶⁵ More closely related to the present system, Al-Far et al. reported (C₆H₉N₂)₂[CuBr₄], in which the Cu(ii) centre exhibits a geometry intermediate between tetrahedral and square-planar, with Cu–Br distances of 2.3575–2.3848 Å and Br–Cu–Br angles of 98.36–132.23°, while N–H⋯Br hydrogen bonds, π–π stacking and Br···aryl contacts generate a three-dimensional supramolecular network.⁶⁶ These results demonstrate the sensitivity of the [CuBr₄]²⁻ coordination environment and crystal packing to the molecular structure of the organic cation.

In the present compound, the 3-bromo-5-methyl substitution pattern of the 2-aminopyridinium cation is associated with a markedly different CuBr₄ geometry. The Cu–Br distances of 2.3778(5)–2.4497(5) Å and Br–Cu–Br angles approaching 90 and 180° give an Okuniewski value of 0.000, providing quantitative support for a square-planar Cu(ii) environment, although the unequal Cu–Br distances indicate a slight distortion. The organic cations additionally establish N–H⋯Br hydrogen bonding and aromatic interactions that organize the discrete [CuBr₄]²⁻ anions into a three-dimensional supramolecular architecture. The combination of the substituted aminopyridinium framework with a quantitatively square-planar CuBr₄ coordination environment therefore constitutes a distinctive structural feature of the present hybrid and provides a basis for correlating its crystal architecture with the optical and biological properties investigated in this work.

3.3. Hirshfeld surface analysis

To further characterize and quantify the intermolecular contacts governing the crystal packing of (C₆H₈BrN₂)₂[CuBr₄], a Hirshfeld surface analysis was performed using the crystallographic structural data. The Hirshfeld surface provides a graphical representation of the regions where intermolecular contacts occur and complements the structural information obtained from single-crystal X-ray diffraction.

Fig. 7 presents the Hirshfeld surface mapped over d_norm together with the corresponding shape-index representation. The d_norm surface reveals distinct localized regions of positive deviation, represented by red areas, which arise from intermolecular contacts shorter than the corresponding van der Waals separations. In the present structure, these regions are mainly associated with contacts involving the bromide ions and hydrogen atoms of the organic cations, in agreement with the N–H⋯Br hydrogen-bonding interactions identified from the crystallographic analysis. The presence of these localized regions confirms that close H⋯Br contacts constitute an important component of the intermolecular interaction network within the crystal.

Fig. 7. Hirshfeld surfaces of (C₆H₈BrN₂)₂[CuBr₄] mapped over (a); d_norm and (b); shape index.

The shape-index surface provides complementary information concerning the molecular packing. The presence of complementary colored regions on the surface is consistent with close contacts between neighboring molecular fragments and supports the occurrence of aromatic stacking interactions between the organic cations. These features are consistent with the nearly planar pyridinium rings observed crystallographically and with their tendency to participate in π-associated contacts. Nevertheless, the Hirshfeld surface analysis is used here to characterize the packing environment rather than to establish a direct causal relationship between individual intermolecular contacts and crystal morphology.

The quantitative contribution of the different intermolecular contacts was further evaluated from the two-dimensional fingerprint plots shown in Fig. 8. The full fingerprint plot represents the complete distribution of intermolecular contacts over the Hirshfeld surface, whereas the decomposed plots allow the contribution of specific atom–pair interactions to be distinguished. Among the identified contacts, H⋯Br/Br⋯H interactions make the largest contribution, accounting for 56.6% of the Hirshfeld surface. This pronounced contribution demonstrates that contacts between hydrogen atoms and bromide atoms are the dominant intermolecular interactions in the crystal packing. This result is consistent with the N–H⋯Br hydrogen bonds observed in the single-crystal structure and provides quantitative support for their important role in organizing the organic and inorganic components of the hybrid structure.

Fig. 8. Two-dimensional fingerprint plots for (C₆H₈BrN₂)₂[CuBr₄]: (a) full fingerprint plot and decomposed plots showing the contributions from (b) H⋯Br/Br⋯H, (c) H⋯H, (d) Br⋯Br, (e) H⋯C/C⋯H, and (f) Br⋯C/C⋯Br contacts.

The second largest contribution arises from H⋯H/H⋯H contacts, representing 16.6% of the Hirshfeld surface. These contacts are generally associated with close approaches between hydrogen atoms belonging to neighboring organic moieties and constitute a significant, although less directional, component of the overall crystal-packing environment. Br⋯Br/Br⋯Br contacts contribute 6.1%, indicating that halogen–halogen contacts are present within the crystal, although their contribution is considerably smaller than that of the H⋯Br contacts.

Additional contributions originate from H⋯C/C⋯H contacts (4.4%) and Br⋯C/C⋯Br contacts (4.3%). The former are associated with contacts between the hydrogen atoms of the organic cations and the carbon atoms of neighboring aromatic moieties, whereas the latter reflect close approaches between bromide atoms and the carbon-containing organic components. Although each of these interactions represents only a minor fraction of the total Hirshfeld surface, their combined contribution reflects the variety of weak non-covalent contacts involved in establishing the three-dimensional packing arrangement.

Taken together, the fingerprint-plot analysis demonstrates that the crystal packing of (C₆H₈BrN₂)₂[CuBr₄] is dominated by H⋯Br contacts, with additional contributions from H⋯H, Br⋯Br, H⋯C and Br⋯C contacts. The particularly high contribution of H⋯Br interactions quantitatively supports the crystallographically observed hydrogen-bonding network between the protonated organic cations and the bromide-containing inorganic units. The Hirshfeld analysis therefore provides complementary quantitative evidence for the role of intermolecular contacts in stabilizing and organizing the hybrid crystal structure.

3.4. Vibrational study

The FT-IR spectrum (Fig. 9) of the CuBr₄-based hybrid compound provides clear evidence for protonation of the organic cation and the presence of intermolecular interactions within the crystal lattice. The high-frequency region is characterized by bands at 3560 and 3420 cm⁻¹, which are attributed to ν(NH₂) stretching vibrations of the protonated organic moiety. The position and relative broadening of these bands suggest the involvement of the amino groups in hydrogen-bonding interactions with neighboring anions and lattice components.^66,67 The band observed at 3300 cm⁻¹ is assigned to aromatic ν(C–H) stretching vibrations, while the absorption at 3120 cm⁻¹ corresponds to aliphatic ν(CH₃) stretching modes, typical of substituted aromatic systems.^68–70

Fig. 9. Infrared spectrum of (C₆H₈BrN₂)₂[CuBr₄].

A weak band appearing at 2620 cm⁻¹ is attributed to ν(N–H) stretching vibrations associated with protonated nitrogen sites engaged in hydrogen bonding.²⁰ The absorption at 1710 cm⁻¹ is not assigned to the bending mode of lattice water, since no crystallographic water molecule is present in the refined structure. This feature may instead arise from an overlapping vibrational contribution of the protonated organic moiety and should therefore not be considered evidence for incorporated water. In the mid-infrared region, the band at 1620 cm⁻¹ is associated with aromatic ring stretching vibrations, while the feature at 1600 cm⁻¹ corresponds to δ(NH₂) bending modes. These vibrations are known to be sensitive to protonation and intermolecular interactions, resulting in slight frequency shifts compared to neutral analogues. The band observed at 1475 cm⁻¹ is attributed to ν(C–N) stretching vibrations, confirming the integrity of the organic framework. Additional C–H bending vibrations appear at 1300 and 958 cm⁻¹, consistent with substituted aromatic rings. Lower-frequency absorptions at 1080 and 750 cm⁻¹ are assigned to NH₂ wagging and out-of-plane deformation modes, further supporting the protonated nature of the organic cation.

At still lower wavenumbers, the band at 620 cm⁻¹ corresponds to ν(C N–C) skeletal vibrations, indicative of conjugation and charge delocalization within the organic moiety. Finally, the absorption at 480 cm⁻¹ is assigned to ν(C–Br) stretching vibrations, characteristic of bromine-substituted aromatic compounds. Taken all together, the IR spectral features confirm strong coupling between the organic and inorganic sublattices through hydrogen bonding and electrostatic interactions.

3.5. UV-Vis-NIR diffuse reflectance spectroscopy

The optical properties of (C₆H₈BrN₂)₂[CuBr₄] were investigated by solid-state UV-Vis diffuse reflectance spectroscopy (Fig. 10). The spectrum exhibits absorption features extending from the ultraviolet to the visible region, reflecting the electronic coupling between the organic cations and the inorganic [CuBr₄]²⁻ units within the hybrid framework. A strong absorption band centered around 300–320 nm is assigned to π → π* transitions of the aromatic organic cations. In the visible region, broad absorption bands extending from approximately 400 to 600 nm are observed, characteristic of Cu(ii)-based bromide hybrids. These bands are mainly associated with Cu²⁺ d–d transitions within the slightly distorted square-planar [CuBr₄]²⁻ coordination environment, with possible contributions from Br → Cu charge-transfer transitions. The broad nature of the visible absorption reflects the crystal-field splitting induced by the Jahn–Teller-distorted d⁹ electronic configuration of Cu(ii). The progressive absorption edge toward lower photon energies indicates semiconducting behavior with a well-defined optical transition threshold. The optical response therefore arises from the combined contribution of ligand-centered excitations and Cu-centered electronic transitions, consistent with the hybrid organic–inorganic nature of the material.

Fig. 10. (a) Diffuse reflectance spectrum of the CuBr₄-based hybrid material transformed using the Kubelka–Munk function F(R); (b) corresponding absorption profile highlighting the main electronic transitions.

The optical properties of (C₆H₈BrN₂)₂[CuBr₄] were investigated by diffuse reflectance spectroscopy (DRS), and the reflectance data were transformed using the Kubelka–Munk function, F(R), prior to Tauc analysis. To rigorously establish the nature of the optical transition associated with the absorption edge, both direct- and indirect-transition models were evaluated. As shown in Fig. 11(a), the direct-transition representation, [F(R)hν]²versus hν exhibits a pronounced linear region near the absorption edge, and its extrapolation to the energy axis yields an optical band-gap value of approximately 2.50 eV. By comparison, application of the indirect-transition model results in a lower extrapolated value of approximately 2.00 eV. The transition character was further assessed through exponent analysis, which gives (n = 0.5) (Fig. 11(b)), consistent with the direct-allowed transition model adopted in the present Tauc treatment. The convergence of these independent analyses—namely, the well-defined linear absorption-edge region in the direct Tauc representation, the distinct separation from the indirect-transition estimate, and the exponent obtained from the absorption dependence—provides strong support for assigning the fundamental optical absorption edge to a direct allowed transition, with an optical band gap of approximately 2.50 eV. Nevertheless, the broad absorption profile should be considered in the context of the Cu(ii) electronic configuration, since localized metal-centered d → d excitations may contribute to the observed spectral response. These transitions are subject to electronic selection rules and are distinct in nature from the fundamental electronic absorption responsible for the optical band edge. Accordingly, the value of 2.50 eV is assigned specifically to the fundamental optical absorption edge derived from the Tauc analysis and is not attributed to an individual Cu(ii) d–d transition.^71,72

Fig. 11. Tauc analysis of the optical band gap of (C₆H₈BrN₂)₂[CuBr₄] based on (a) direct- and indirect-transition models and (b) determination of the transition exponent (n = 0.5).

The observed optical band gap of approximately 2.50 eV places (C₆H₈BrN₂)₂[CuBr₄] within the visible-light-responsive semiconductor range and indicates its potential relevance to optoelectronic and photoactive applications. In hybrid halometalates, the optical response can in principle be tuned through controlled modification of the inorganic and organic components. In particular, substitution of the halide ligand can modify the metal–halide electronic states, while changing the metal centre or the protonated organic cation can alter the charge distribution, intermolecular interactions, and electronic coupling within the hybrid lattice. The present study does not experimentally establish a systematic band-gap tuning series; rather, the 2.50 eV value provides a reference point for future compositional and structural engineering of related Cu(ii)-based hybrid halometalates toward targeted optoelectronic properties.

The degree of structural disorder in (C₆H₈BrN₂)₂[CuBr₄] was evaluated through Urbach energy analysis derived from the low-energy absorption edge. As shown in (Fig. 12(a)), the Urbach plot exhibits a well-defined linear region, from which an Urbach energy value of approximately 0.300 eV was determined. This moderate value indicates the presence of limited localized states near the band edge and suggests a moderate degree of electron–phonon interaction within the hybrid lattice.

Fig. 12. (a) Urbach plot illustrating the exponential absorption edge of the CuBr₄-based hybrid material; (b) linear fitting used to extract the Urbach energy.

The observed band-tail broadening is closely related to the local distortion of the square-planar [CuBr₄]²⁻ units induced by the Jahn–Teller-active Cu(ii) center. Slight variations in Cu–Br bond distances and deviations from the ideal coordination geometry generate local lattice asymmetry, leading to partial broadening of the electronic states near the band edges. Nevertheless, the relatively moderate Urbach energy indicates that the structural disorder remains limited and does not significantly perturb the semiconducting character of the material. The presence of the Urbach tail therefore reflects localized electronic states extending into the forbidden band region, which are responsible for the gradual absorption onset observed near the optical edge. The obtained E_u value remains fully consistent with the direct optical transition and band-gap energy determined from the Tauc analysis, confirming the relatively ordered electronic structure of the hybrid compound.

The frequency-dependent optical conductivity and corresponding skin depth of (C₆H₈BrN₂)₂[CuBr₄] were investigated to further elucidate the charge-excitation behavior and optical attenuation mechanisms within the hybrid framework (Fig. 13). The optical conductivity remains weak in the low-energy region, consistent with the limited density of thermally accessible charge carriers below the optical band gap. With increasing photon energy, a progressive enhancement of the conductivity is observed, followed by a more pronounced increase near the absorption edge, reflecting the activation of interband electronic transitions. This behavior confirms the semiconducting nature of the material and indicates efficient photon-induced charge excitation in the vicinity of the band-gap region. The optical response is strongly influenced by the electronic structure of the square-planar [CuBr₄]²⁻ units. In addition to Br 4p → Cu 3d charge-transfer transitions, Cu²⁺-centered d–d electronic excitations contribute to the visible-light absorption behavior. The Jahn–Teller distortion associated with the d⁹ electronic configuration of Cu(ii) induces partial splitting of the Cu 3d levels, thereby increasing the density of accessible electronic states close to the band edge. The corresponding skin depth exhibits an opposite evolution, decreasing gradually with increasing photon energy due to the enhanced attenuation of incident radiation near the absorption threshold. The complementary variation of optical conductivity and skin depth therefore demonstrates the strong correlation between electronic excitation and light penetration within the hybrid lattice.

Fig. 13. (a) Corresponding variation of the skin depth as a function of photon energy. (b) Photon-energy-dependent optical conductivity of the CuBr₄-based hybrid material.

The dispersion behavior of the extinction coefficient and refractive index provides additional insight into the optical response of (C₆H₈BrN₂)₂[CuBr₄] (Fig. 14). The extinction coefficient remains relatively low at lower photon energies and increases progressively near the absorption edge, indicating enhanced optical attenuation associated with the onset of interband transitions. This evolution is consistent with the optical band-gap value determined from the Tauc analysis and reflects the increasing probability of photon-induced electronic excitation from Br-derived valence states toward Cu-centered conduction states. The refractive index exhibits a pronounced dispersion behavior over the investigated spectral range, reflecting the polarizable nature of the hybrid framework and the strong interaction between the electromagnetic field and the electronic states of the material. The observed optical dispersion is governed by the combined contribution of the organic sublattice and the Jahn–Teller-distorted square-planar [CuBr₄]²⁻ units, which modulate the local electronic polarizability through Cu–Br orbital hybridization. The simultaneous evolution of the refractive index and extinction coefficient highlights the close relationship between electronic structure, optical absorption, and photon propagation within the hybrid material. These results confirm the effective electronic coupling between the inorganic and organic components and support the potential of this Cu(ii)-based hybrid system for visible-light optoelectronic applications.^{55,56,73–75}

Fig. 14. (a) Dispersion of the refractive index as a function of photon energy; (b) extinction coefficient illustrating absorption behavior near the band edge.

3.6. CIE 1931 chromaticity analysis

The excitation-dependent photoluminescence spectra of (C₆H₈BrN₂)₂[CuBr₄], recorded between 300 and 500 nm excitation wavelengths, are displayed in (Fig. 15). The compound exhibits a broad emission extending over the visible region with moderate intensity and no well-resolved vibronic structure, indicating the involvement of highly relaxed excited states. A gradual decrease in emission intensity is observed as the excitation wavelength increases, while the emission band position remains nearly unchanged, suggesting that the radiative process originates from energetically distributed localized states rather than from a single discrete emissive center. The photoluminescence response is closely associated with the electronic structure of the Jahn–Teller-active Cu(ii) centers. The distorted square-planar [CuBr₄]²⁻ units induce crystal-field splitting of the Cu 3d orbitals, generating low-energy electronic states that strongly influence the relaxation dynamics. In such d⁹ systems, efficient non-radiative deactivation pathways compete with radiative recombination, leading to partial quenching of the emission intensity. Consequently, the observed luminescence is more consistent with charge-transfer-mediated excited states involving Br 4p and Cu 3d orbitals than with purely excitonic band-edge emission. The broad spectral profile and pronounced Stokes shift further indicate significant electron–phonon coupling within the hybrid lattice. In addition, intermolecular hydrogen-bonding interactions involving the organic cations and lattice water molecules contribute to local structural flexibility, which favors excited-state relaxation and broadens the emission envelope. Despite the moderate emission efficiency, the persistence of visible photoluminescence confirms the existence of radiative charge-transfer pathways within the hybrid framework, highlighting the strong interplay between coordination geometry, lattice distortion, and electronic structure in governing the optical emission behavior of this Cu(ii)-based hybrid material. The chromatic properties of (C₆H₈BrN₂)₂[CuBr₄] were further examined using the CIE 1931 chromaticity diagram derived from the experimental emission spectra (Fig. 16).

Fig. 15. Excitation-dependent photoluminescence spectra of (C₆H₈BrN₂)₂[CuBr₄].

Fig. 16. CIE 1931 chromaticity diagram of (C₆H₈BrN₂)₂[CuBr₄].

The resulting chromaticity coordinate is positioned within a clearly defined region of the visible spectrum, indicating a well-resolved emission with high color fidelity. The localization of the emission point in the CIE color space confirms the uniformity of the emissive environment within the hybrid framework. It suggests that a single, intrinsic radiative pathway dominates the observed luminescence. The chromaticity position is in good agreement with the perceived emission color, demonstrating the ability of the compound to deliver a stable and reproducible optical output. This degree of chromatic control is particularly relevant for optoelectronic technologies where consistent color performance is essential, such as solid-state lighting and photonic applications. Overall, the CIE 1931 analysis highlights the favorable colorimetric performance of (C₆H₈BrN₂)₂[CuBr₄], combining chromatic stability with a well-defined emission profile, which supports its potential use as a luminescent material in advanced light-emitting systems.

3.7. In silico study

Density Functional Theory (DFT) calculations were performed to investigate the optimized molecular geometry, electronic structure, frontier molecular orbitals (HOMO and LUMO), HOMO–LUMO energy gap (ΔE), molecular electrostatic potential (MEP), and global reactivity descriptors of the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄]. These calculations provided valuable insight into the electronic properties, chemical stability, charge distribution, and potential reactive sites of the investigated compound. The pharmacokinetic behavior of the synthesized complex was further evaluated using the pkCSM web server. Physicochemical descriptors together with absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were predicted, including water solubility, Caco-2 permeability, human intestinal absorption, skin permeability, P-glycoprotein interactions, volume of distribution (VDss), blood–brain barrier (BBB) permeability, central nervous system (CNS) permeability, cytochrome P450-mediated metabolism, total clearance, and toxicity-related endpoints. Furthermore, molecular docking simulations were carried out to investigate the binding interactions of the optimized hybrid complex with two biologically relevant therapeutic targets. The human α/β-tubulin (Vinca-binding site, PDB ID: 5J2T) was selected to rationalize the experimentally observed antiproliferative activity, whereas human peroxiredoxin 5 (PDB ID: 1HD2) was employed to elucidate the antioxidant activity. The docking results were analyzed in terms of binding affinity, binding mode, and key intermolecular interactions to correlate the computational findings with the experimental biological data.

3.7.1. DFT and energy analysis of the copper complex

DFT calculations were performed at the UB3LYP/LANL2DZ level to investigate the electronic structure and physicochemical properties of the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], as illustrated in Fig. 17 and Table S6. The optimized geometry was confirmed as a true minimum on the potential energy surface by the absence of imaginary vibrational frequencies. The calculated frontier molecular orbital energies were −4.55 eV for the HOMO and −1.77 eV for the LUMO, giving a HOMO–LUMO energy separation (ΔE) of 2.78 eV. This value represents the energetic separation between the frontier orbitals within the adopted computational model and provides a molecular-level descriptor of the electronic structure of the investigated complex. It is important to distinguish the calculated HOMO–LUMO separation from the experimentally determined optical band gap. The latter, obtained from the diffuse-reflectance/Tauc analysis, was estimated to be approximately 2.50 eV and represents the optical absorption edge of the material. Thus, the calculated HOMO–LUMO gap of 2.78 eV and the experimental optical band gap of 2.50 eV describe different physical quantities and should not be considered directly equivalent. The calculated global reactivity descriptors further support the electronic characteristics of the complex. The chemical potential (µ), electronegativity (χ), chemical hardness (η), softness (S), and electrophilicity index (ω) were determined to be −3.16, 3.16, 1.39, 0.360 eV⁻¹, and 3.59 eV, respectively. These values provide complementary information on the calculated chemical stability and electronic reactivity of the investigated system.

Fig. 17. DFT-calculated HOMO–LUMO orbitals of (C₆H₈BrN₂)₂[CuBr₄].

In addition, the optimized structure exhibited a relatively high dipole moment of 33.19 D, reflecting the pronounced polarity of the ionic organic–inorganic hybrid complex. This enhanced polarity arises from the spatial separation between the two protonated organic cations, (C₆H₈BrN₂)⁺, and the tetrahalocuprate(ii) anion, [CuBr₄]²⁻, resulting in significant charge polarization. Such behavior is characteristic of isolated ionic hybrid systems and may facilitate intermolecular interactions in biological environments.

3.7.2. Pharmacokinetic properties

The predicted pharmacokinetic properties of the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], were evaluated using the pkCSM web server,⁴⁹ and the results are summarized in Table S7. The complex exhibited moderate aqueous solubility (log S = −3.21) together with moderate Caco-2 permeability (0.628 log P_app × 10⁻⁶ cm s⁻¹), whereas the predicted human intestinal absorption was relatively low (3.89%), indicating limited oral bioavailability, which is consistent with its high molecular weight and ionic coordination structure. The compound was predicted to be neither a substrate nor an inhibitor of P-glycoprotein, suggesting that active efflux mediated by this transporter is unlikely to significantly influence its cellular uptake.

Regarding distribution, the complex showed a low volume of distribution (VDss = −0.291 log L kg⁻¹) and poor blood–brain barrier permeability (log BB = −1.21), accompanied by a very low central nervous system permeability (log PS = −3.292). These findings indicate that the complex is unlikely to accumulate in the brain, thereby minimizing potential central nervous system exposure. In addition, the relatively high fraction unbound (F_u = 0.901) suggests that a considerable proportion of the compound may remain available in the systemic circulation for interaction with biological targets. The metabolism prediction indicated that the complex is not expected to inhibit the major cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), although it was predicted to be a CYP3A4 substrate, suggesting that CYP3A4 may contribute to its metabolic clearance without causing significant enzyme inhibition or drug–drug interactions. The predicted total clearance (1.372 log mL min⁻¹ kg⁻¹) indicates moderate elimination from the body, while the compound was not identified as a renal OCT2 substrate. Toxicity predictions revealed no hERG I inhibition, indicating a relatively low risk of severe cardiotoxicity, although hERG II inhibition was predicted. The complex was also predicted to be hepatotoxic and AMES-positive, suggesting that further experimental safety evaluation is required. Overall, the in silico pharmacokinetic profile indicates that the synthesized Cu(ii) hybrid complex possesses acceptable distribution characteristics and moderate metabolic stability; however, its limited oral absorption and predicted toxicity highlight the importance of further in vitro and in vivo pharmacological investigations. Owing to the ionic nature of transition metal coordination compounds, these predictions should be interpreted cautiously and considered complementary to the experimental biological findings.

3.7.3. Molecular docking

Molecular docking simulations against human α/β-tubulin (PDB ID: 5J2T) demonstrated that the reference drug, vinblastine, exhibited the highest binding affinity, with a docking score of −7.05 kcal mol⁻¹, forming five hydrogen-bond interactions with the active-site residues ASN242, ARG44, ASN45, and GLY159 (Table S8). The individual hydrogen bonds displayed interaction distances ranging from 2.90 to 3.27 Å, indicating stable accommodation within the Vinca-binding site. In comparison, the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], exhibited a docking score of −5.12 kcal mol⁻¹ and was favorably accommodated within the binding pocket through two hydrogen-bond interactions with the conserved residue ASN242, with interaction distances of 3.03 and 3.22 Å and corresponding interaction energies of −2.9 and −5.7 kcal mol⁻¹, respectively (Table S8). Although the hybrid complex displayed a lower overall binding affinity than the reference drug, its ability to interact with the key residue ASN242 suggests favorable recognition of the α/β-tubulin binding cavity. The corresponding 2D and 3D interaction maps (Fig. 18). Further revealed that hydrogen bonding constitutes the principal stabilizing interaction governing the binding mode of the synthesized complex. These computational findings are in good agreement with the experimental antiproliferative results, supporting the moderate cytotoxic activity of (C₆H₈BrN₂)₂[CuBr₄] against HCT-116 and HepG-2 cancer cell lines.

Fig. 18. 2D and 3D molecular docking interaction diagrams of (A) vinblastine and (B) (C₆H₈BrN₂)₂[CuBr₄] in the active site of human α/β-tubulin (PDB ID: 5J2T).

Molecular docking simulations against human peroxiredoxin 5 (PDB ID: 1HD2) revealed that the synthesized hybrid complex, (C₆H₈BrN₂)₂[CuBr₄], exhibited a docking score of −4.85 kcal mol⁻¹, which was slightly more favorable than that of the reference antioxidant BHT (−4.61 kcal mol⁻¹). The hybrid complex was stabilized within the active site through two hydrogen-bond interactions with GLN68, displaying interaction distances of 3.10 and 3.07 Å and corresponding interaction energies of −3.8 and −1.1 kcal mol⁻¹, respectively (Table S9). In contrast, BHT formed a single hydrogen-bond interaction with GLY92 at a distance of 2.92 Å, with an interaction energy of −1.1 kcal mol⁻¹ (Table S9). The corresponding 2D and 3D interaction maps (Fig. 19) further demonstrated that hydrogen bonding represents the principal stabilizing interaction responsible for binding of the synthesized hybrid complex within the active site of human peroxiredoxin 5. Although the hybrid complex exhibited a slightly more favorable docking score than BHT, this computational observation should be interpreted together with the experimental antioxidant assays, in which BHT remained the more potent antioxidant standard. Nevertheless, the favorable binding affinity and stable hydrogen-bond interactions of the synthesized complex support its experimentally observed moderate antioxidant activity.

Fig. 19. 2D and 3D molecular docking interaction maps of (A) BHT and (B) (C₆H₈BrN₂)₂[CuBr₄], within the active site of human peroxiredoxin 5 (PDB ID: 1HD2).

3.8. Biological activities

3.8.1. Antioxidant activity

The antioxidant potential of (C₆H₈BrN₂)₂[CuBr₄] and C₆H₇BrN₂ was evaluated using three complementary assays, namely DPPH and ABTS radical scavenging methods, along with the β-carotene-linoleic acid bleaching test. The activity was expressed in terms of EC₅₀ values, corresponding to the concentration required to achieve 50% inhibition. The DPPH test results showed that 2-amino-3-bromo-5-methylpyridine had a much higher (p < 0.05) scavenging activity (EC₅₀ = 6.02 ± 0.04 µg mL⁻¹) than (C₆H₈BrN₂)₂[CuBr₄] (EC₅₀ = 63.45 ± 0.02 µg mL⁻¹) (Table 1). The ABTS test results validated the DPPH and BCB test results. In fact, the (C₆H₈BrN₂)₂[CuBr₄] (38.14 ± 0.03 µg mL⁻¹) was almost thirteen times lower than the 2-amino-3-bromo-5-methylpyridine scavenging activity (EC₅₀ = 2.92 ± 0.06 µg mL⁻¹). According to our findings, substances that scavenge free radicals include (C₆H₈BrN₂)₂[CuBr₄] and C₆H₇BrN₂. Nevertheless, both the ABTS and BCB assays demonstrated that the ligand exhibited significantly greater scavenging activity than the copper complex. This observation can be attributed to the enhanced proton mobility conferred by 2-amino-3-bromo-5-methylpyridine, together with the presence of multiple reactive centers in its molecular framework that can function as proton donors or electron acceptors.

Table 1. Scavenging activity of C₆H₇BrN₂ and (C₆H₈BrN₂)₂[CuBr₄] in DPPH, ABTS and BCB test^a.

Parameter	C6H7BrN2	(C6H8BrN2)2[CuBr4]
DPPH EC50 (µg mL−1)	6.02 ± 0.04	63.45 ± 0.02
ABTS EC50 (µg mL−1)	2.92 ± 0.06	38.14 ± 0.03
BCB EC50 (µg mL−1)	12.65 ± 0.02	325.55 ± 0.01

^aAbbreviations: DPPH: 1,1-diphenyl-2-picrylhydrazyl radical scavenging capacity; ABTS: (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) ABTS; BCB: β-carotene-linoleic acid bleaching assay; EC₅₀: the concentration able to inhibit 50%. The tests were run in triplicate and data are expressed as mean and standard error of the mean (mean ± SEM). Statistical comparisons were performed using ANOVA followed by Newman–Keuls post hoc test. Columns with different letters (a, b and c) exhibit statistically significant difference (p < 0.005).

3.8.2. Antiproliferative activity

The in vitro cytotoxic activity of (C₆H₈BrN₂)₂[CuBr₄] was investigated against two human cancer cell lines, namely colon carcinoma (HCT-116) and hepatocellular carcinoma (HepG-2), using a standard colorimetric assay. The results were expressed as IC₅₀ values, representing the concentration required to inhibit 50% of cell viability (Tables 2 and 3). The compound demonstrated a noticeable antiproliferative effect toward both cell lines, with IC₅₀ values of 13.56 µg mL⁻¹ for HCT-116 and 17.45 µg mL⁻¹ for HepG-2. The lower IC₅₀ value observed for HCT-116 indicates a greater response of this cell line under the tested conditions; however, the present data are insufficient to establish biological selectivity. Although the antiproliferative activity was lower than that of the reference anticancer drug vinblastine, the results demonstrate that the synthesized hybrid exhibits measurable antiproliferative activity against both tested cell lines.

Table 2. Evaluation of cytotoxicity of C₆H₇BrN₂ and (C₆H₈BrN₂)₂[CuBr₄], against colon carcinoma cells (HCT-116).

Sample conc. (µg)	C6H7BrN2	(C6H8BrN2)2[CuBr4]	Vinblastine standard
50.000	21.22	26.18	12.98
25.000	28.15	37.22	16.08
12.500	31.59	46.34	20.74
6.250	62.66	70.17	39.34
3.125	55.10	85.23	48.60
1.560	81.42	91.02	59.31
0.000 (DMSO)	100.00	100.00	100.00

Table 3. Evaluation of cytotoxicity of C₆H₇BrN₂ and (C₆H₈BrN₂)₂[CuBr₄], against hepatocellular carcinoma cells lines (HepG-2).

Sample conc. (µg)	C6H7BrN2	(C6H8BrN2)2[CuBr4]	Vinblastine standard
50.000	24.33	30.79	15.12
25.000	31.11	40.42	17.33
12.500	36.17	52.87	24.18
6.250	59.62	69.32	46.87
3.125	71.33	84.17	55.18
1.560	80.25	94.37	73.98
0.000 (DMSO)	100.00	100.00	100.00

The observed antiproliferative response may reflect contributions from the chemical characteristics of the Cu(ii)-containing hybrid and its organic component; however, the present experiments do not establish a specific molecular mechanism. The redox activity of Cu(ii) and the hydrogen-bonding and π-interacting functionalities of the protonated aromatic cation provide plausible chemical features that may contribute to interactions with biological species. Nevertheless, because the solid-state structure may undergo dissociation or reorganization under biological conditions, the crystallographically observed supramolecular architecture should not be regarded as direct evidence for the biological mechanism. The experimental results therefore establish the biological activity of the synthesized compound under the tested conditions, while the molecular docking and ADMET analyses provide complementary information concerning possible molecular interactions and pharmacokinetic behaviour.

4. Conclusion

In summary, the novel organic–inorganic copper(ii) hybrid halometalate (C₆H₈BrN₂)₂[CuBr₄] was successfully synthesized and systematically investigated using complementary experimental and computational approaches. Single-crystal X-ray diffraction established a three-dimensional supramolecular architecture in which discrete square-planar [CuBr₄]²⁻ anions are interconnected through N–H⋯Br hydrogen bonds, π–π stacking interactions, and halogen contacts, ensuring the structural cohesion of the crystal. Optical measurements revealed semiconducting characteristics with an optical band gap of approximately 2.5 eV and a broad visible photoluminescence attributed to ligand-to-metal and/or charge-transfer transitions. These observations are supported by DFT calculations, which predict a moderate HOMO–LUMO energy gap and confirm the favorable electronic stability of the hybrid material. In addition, ADMET analysis indicates promising pharmacokinetic features, including acceptable aqueous solubility, moderate membrane permeability, limited blood–brain barrier penetration, and no P-glycoprotein substrate liability. Molecular docking further suggests favorable interactions with human α/β-tubulin and peroxiredoxin 5, consistent with the experimentally observed antioxidant and antiproliferative effects. Overall, the combined structural, optical, computational, and biological findings establish (C₆H₈BrN₂)₂[CuBr₄] as a multifunctional Cu(ii)-based hybrid material with promising potential for both optoelectronic and biomedical applications.

Author contributions

Houyem Khlifi: formal analysis, writing – original draft, visualization, data curation; Wissem Hallab: data curation, investigation, software, formal analysis; Noureddine Mhadhbi: conceptualization, data analysis, investigation, validation; Hossam H. Nasrallah: investigation, formal analysis, data curation, validation; Ahlem Guesmi: investigation, formal analysis, methodology, review & editing; Lotfi Khezami: formal analysis, writing – original draft, visualization, validation; Naoufel Ben Hamadi: methodology, investigation, data curation, conceptualization; Ferdinando Costantino: investigation, validation, data curation, visualization; Houcine Naïli: conceptualization, methodology, project administration, review & editing.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2601).

Data availability

The data that supported the findings of this study were available upon reasonable request.

CCDC 2574677 contains the supplementary crystallographic data for this paper.⁷⁶

Supplementary information (SI): crystallographic, spectroscopic, computational and molecular docking data for (C₆H₈BrN₂)₂[CuBr₄]. See DOI: https://doi.org/10.1039/d6ra06802b.