Structurally modified dipyrazinylpyridine-based homoleptic Cu(II) complexes: comparative cytotoxic evaluation in breast cancer cell lines

Abstract

The DNA interaction ability of transition metal complexes is closely related to their ligand structure, which plays a crucial role in therapeutic applications. In this context, two mononuclear homoleptic Cu(II) complexes, [Cu(L1)₂](ClO₄)₂, 1, and [Cu(L2)₂](ClO₄)₂, 2 (where L1 = 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-dimethylaniline and L2 = 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-diphenylaniline), were synthesised and characterised using various spectroscopic and analytical techniques. SCXRD data indicate that in both complexes, the central Cu(II) unit is oriented in a distorted octahedral manner. The redox performance of the complexes was investigated using various voltammetric techniques. The DNA-binding ability of the complex was investigated using absorbance spectroscopy and ethidium bromide (EB) fluorescence quenching studies with double-stranded salmon sperm DNA (ss-DNA). The binding constant (K_b) and Stern–Volmer constant (K_sv) were obtained in the range of 10⁴ M⁻¹. Molecular docking studies were performed to understand the interactions between B-DNA and the complexes. Furthermore, DNA nicking activity was monitored by in vitro electrophoresis analysis of the supercoiled plasmid DNA. The effects of cell viability on MCF-7 and MDA-MB-231 cell lines were investigated. Cellular production of reactive oxygen species (ROS) was determined using a fluorescent probe (Calcein-AM). Notably, co-administration of 1 with Camptothecin (CPT) enhanced the overall cytotoxic effect, suggesting a synergistic therapeutic response. Interestingly, these results could lead to the development of cost-effective non-platinum transition metal-based chemotherapeutic agents to overcome the lacuna of the present conventional drugs.

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Introduction

DNA serves as a key element for cancer therapies as it is essential for various cellular functions involved in cell replication and differentiation.¹ Therefore, studies related to DNA binding properties by small molecules are gaining much attention in recent years. cis-Platin is the most commonly utilised clinical drug.² Along with cis-platin, other conventional medications like oxaliplatin and carboplatin can inhibit the process of cell replication by forming intrastrand crosslinks with the base pairs of DNA.³ However, issues like low water solubility, intrinsic resistance by certain types of tumors, and adverse effects like bone marrow restraint, nausea, and nephrotoxicity restrict the applications of such drugs in the current scenario.⁴ Hence, there is an urgent need for some better alternatives that can overcome the lacuna of the present conventional drugs. As the malfunctioning of mitochondria leads to apoptosis and necrosis, researchers have adopted synthetic strategies to develop systems that can selectively target the particular organelle for therapeutic applications.⁵ In this regard, several transition metal complexes with appropriate biocompatible ligands have shown improved activity towards real applications.^6,7 The DNA binding efficacy of such complexes majorly depends on different factors such as coordinating metal ions, the variety of donor-atoms in the ligand framework, steric hindrance and ligand planarity.⁸

Copper is a crucial trace element for living organisms, and its complexes enable wide biological applications owing to its less toxic nature and redox-active characteristics.^9,10 These complexes are very much biocompatible and are found to exert promising antitumor activity for a number of diverse cancer cells via discrete pathways. In many instances, these Cu(II) complexes can readily generate ROS, leading to cell damage, oxidative stress, and, ultimately, apoptotic cancer cell death by shuttling the redox states, as needed.^4,11 Based on the properties of the ligand architecture, these complexes show DNA binding properties through different modes, mainly groove binding, electrostatic binding, and DNA intercalation.^12,13 The electrostatic interaction in general is relatively weak, and hence, the focus in recent times is more inclined towards the synthesis of such complexes capable of binding with DNA either through groove interaction or via intercalation. Most of the Cu(II) complexes also exhibit comparable effectiveness towards DNA nicking applications via oxidative, photolytic and hydrolytic approaches.^3,14 The ability of Cu(II) complexes to control drug circulation and metabolism by modifying their overall structural architecture results in improved efficacy with minimal side effects.¹⁵ Under these circumstances, polypyridyl ligands such as 2,2′-bipyridine (bpy),¹⁶ 1,10-phenanthroline (phen),¹⁷ dipyridophenazine (dppz),¹⁸ and 2,2′:6′,2″-terpyridine (tpy)³ show promising activity in DNA binding, nicking and therapeutic anticancer applications. The mixed ligand-based Cu(II) tridentate–bidentate systems have been extensively studied in the past decade using bpy, phen, and dppz ligands as potential bidentate ligands.^19–21 In the recent past, our group has also explored the same heteroleptic Cu(II) polypyridyl complexes for DNA interaction and anticancer activities.^10,22 However, the reports of Cu(II) complexes with only tridentate systems are limited and need to be explored for better replacements for the stated purposes.²³ The use of tridentate ligands is more advantageous than bidentate systems; for example, in bis-tridentate complexes, the coordination sites are fully occupied, facilitating ease of synthesis and simplifying the issues related to base binding.²⁴

Terpyridine is a well-known tridentate meridional coordinating ligand, and its different derivatives are a vital type of chelating ligand that can readily form transition metal complexes with high structural stability.¹⁵ The planar nature of the terpyridine molecule helps in better DNA intercalation by tuning the ligand framework via different substitutions at the various positions of the aromatic pyridine ring and hence can easily regulate different biological activities.²⁵ The substituted terpyridine complexes are particularly important due to their ability to stabilize the DNA G–quadruplex structures.²⁶ Other than pharmacological applications, metal complexes derived from terpyridine are also useful in various fields, such as molecular-level sensing,²⁷ dye-sensitized solar cells,²⁸ and catalysis in recent years, especially in the area of solar fuel research, such as water splitting, H₂ generation, and CO₂ reduction chemistry,²⁹ as well as organic light-emitting (OLED) devices.³⁰ Recently, Nair et al. reported imidazole-substituted bis-terpyridine Cu(II) complexes and studied their DNA binding properties.²⁴ Liu and co-workers synthesised a Cu(II) complex using 4′-phenyl-terpyridine as a tridentate ligand and investigated its anticancer properties against two different cell lines (A549 and MCF-7), showing impressive activity in both cases.³¹ Rahiman et al. explored how substituted terpyridine-based Cu(II) complexes interact with DNA, demonstrating effective binding and notable cytotoxic performance.³² Analogous to terpyridine, dipyrazinyl pyridines (dppy) and their derivatives have promising biological uses because of their comparable pincer-like structure and the extra nitrogen atoms located on the pyrazine ring.^33,34 Most recently, Ghosh and co-workers synthesised bis-tridentate Cu(II) complexes using 3,4-diethoxyphenyl-2,6-di(pyrazine-2-yl)pyridine and investigated its DNA/BSA interaction and cytotoxicity towards Dalton's lymphoma cancer cell line.³⁵ Despite its structural properties analogous to terpyridine, reports related to biological properties using metal complexes with dppy-type ligands are still limited.³⁶

Herein, we report two mononuclear Cu(II) complexes of type [Cu(L1)₂](ClO₄)₂; 1 and [Cu(L2)₂](ClO₄)₂; 2, where L1 = 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-dimethylaniline and L2 = 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-diphenylaniline. Both complexes were well-characterised with diverse analytical and spectroscopic tools along with single-crystal X-ray diffraction (SCXRD). The primary objective of the current investigation is to demonstrate the consequence of extended conjugation by donor–acceptor chromophoric moieties like N,N-dimethylaniline (for L1) and N,N-diphenylaniline (for L2) on complexes 1 and 2, respectively, towards their DNA interaction. The DNA interaction study with both complexes has been explored using various spectroscopic techniques and molecular docking studies. The biological functions of the Cu(II) complexes were evaluated using cancer cell lines. Camptothecin (CPT), a well-known anticancer drug, exerts its effects by targeting DNA topoisomerase I and stabilizing the cleavage complex, leading to DNA breakage. Therefore, CPT was used as a positive control to examine whether Cu(II) complexes could enhance CPT-induced cytotoxicity. In addition, their biological activity was assessed based on their ability to reduce cancer cell viability and induce ROS production.

Results and discussion

Synthesis

Ligands L1 and L2 are synthesised utilizing a previously reported method.³⁷ Initially, the 2-acetylpyrazine and the respective aldehyde derivatives (4-(dimethylamino)benzaldehyde for L1 and 4-(diphenylamino)benzaldehyde for L2) were dissolved in ethanol. Afterward, KOH and NH₄OH solutions were added, followed by stirring for 24 h at room temperature. The solid product formed was filtered and washed with distilled water and finally with diethyl ether. Finally, recrystallization in dichloromethane (DCM) leads to the formation of a pure product (Scheme 1). Both L1 and L2 were characterised using UV-vis, FT-IR, ¹H-NMR, and mass spectrometric analysis. Metal complexes (1 and 2) were synthesised by dissolving Cu(ClO₄)₂·6H₂O and the respective ligands (L1 and L2) in methanol solvent in a 1 : 2 molar ratio (Scheme 2). Subsequently, 1 and 2 were recrystallised by dissolving them in an acetonitrile–methanol 2 : 1 solvent blend, resulting in dark orange (for 1) and brown (for 2) crystals.

Scheme 1 Synthesis of L1 and L2.

Scheme 2 Synthesis of 1 and 2.

Additionally, we synthesise [Cu(en)₂]SO₄ (en = ethylenediamine) complex as a negative control in viscosity measurement studies, as it lacks sufficient planarity to interact with DNA. The synthetic route adopted for the synthesis of [Cu(en)₂]SO₄ is given in the SI. To confirm the successful complex formation, mass spectral analysis was carried out in H₂O solvent, which gives the molecular ion peak at m/z = 318.0587 [M + K]⁺ (calculated: 317.9825) (Fig. S1).

Spectral aspects

Both ligands were well characterised by ¹H-NMR spectroscopy. For ligand L1, the six-proton singlet for the two equivalent methyl protons of –NMe₂ moiety was observed at 3.06 ppm, and a sharp singlet for the most deshielded two equivalent meta protons of the central pyridine unit was found at 9.86 ppm. Other signals for the rest of the aromatic protons were observed in the range of 6.80–9.00 ppm. Likewise, for L2, a sharp singlet for two equivalent meta-protons of the central pyridine unit was observed at 9.87 ppm. All the other aromatic protons were found in the range of 7.00–9.00 ppm (Fig. S2). Further, the ¹³C NMR spectra of both L1 and L2 were recorded in CDCl₃ solvent. For L1, the aliphatic carbon consists of an –NMe₂ unit found at 40.27 ppm, and the remaining signals of other distinct aromatic carbons of both L1 and L2 are observed in their desired range of 100–160 ppm (Fig. S3). Solid-state FT-IR spectra of both ligands show a peak of ν(C–H) at 3034 cm⁻¹ and 3004 cm⁻¹, respectively. The aromatic ν(C=C) and ν(C=N) sharp stretching were noticed at 1594 and 1468 cm⁻¹ for L1 and at 1582 and 1481 cm⁻¹ for L2, respectively.¹ For complexes 1 and 2, the presence of the perchlorate (ClO₄⁻) counterion was verified by the peaks at ∼1080 cm⁻¹ and ∼625 cm⁻¹, respectively.³⁸ Other representative bands like ν(C–H), ν(C=C) and ν(C=N) were somewhat altered in comparison with the free ligands (Fig. S4). The absorption profiles of ligands and their respective metal complexes have been measured in Tris-HCl buffer medium (Fig. 1). Ligands L1 and L2 show distinct absorption bands within the 200–400 nm wavelength range. The bands observed near 271 nm, 293 nm (for L1) and 288 nm (for L2) correspond to the π–π* transition, while the bands near 330 nm and 377 nm correspond to the n–π* transition of ligands L1 and L2, respectively.³⁹ The absorption spectra of 1 and 2 show a close resemblance to L1 and L2, respectively. In both cases, the electronic transition bands are slightly shifted compared to the ligand-based bands. For complex 1, the π–π* transition bands were found at 270 nm and 294 nm, and the n–π* transition band was found at 326 nm. Similarly, for 2, π–π* and n–π*, electronic bands appeared at 270 nm and 351 nm, respectively. Another low-intensity band at a higher wavelength for 2 was also observed at 462 nm, which might correspond to the metal-induced intra-ligand charge transfer transition.⁴⁰ The alteration in electronic bands upon complexation compared to their respective free ligands (L1 and L2) signifies successful complexation. All the mass spectrometric data were taken in acetonitrile solvent (Fig. S5). The molecular ion peak of L1 was obtained at m/z = 355.1659 [L1 + H]⁺ (calculated: 354.1593), that of L2 was found at 479.1950 [L2 + H]⁺ (calculated: 478.1960), that of 1 was found at m/z = 386.1758 [1]²⁺ (calculated: 386.1900), and that of 2 was found at m/z = 509.1305 [2]²⁺ (calculated: 509.6554). Stability studies of 1 and 2 were also checked by absorption spectroscopy in buffer solution, which reveals that both 1 and 2 were stable for at least 72 h (Fig. S6).

Fig. 1 Absorption spectra of L1, L2, 1 and 2 (25 μM) in 5 mM Tris-HCl buffer (pH 7.4).

EPR studies

The paramagnetic characteristics of the complexes were validated using room temperature EPR investigation in the solid state under the following conditions: microwave frequency = 9.450 GHz, center field = 326 mT, and width± = 250 mT. The EPR spectra of 1 and 2 are axial with g_∥ > g_⊥, which signifies the existence of an unpaired electron in the d_x²−y² orbital of the central Cu(II) unit.⁴¹ Four-line, well-resolved hyperfine signal was obtained for both complexes, indicating the intermolecular interaction between the electron spin (S = 1/2) and the nuclear spin of the copper(II) center (I = 3/2) (Fig. 2).

Fig. 2 EPR spectra of (a) 1 and (b) 2 at room temperature in the solid state.

The obtained g_∥ and g_⊥ values are found to be 2.228 and 2.061 (for 1) and 2.242 and 2.091 (for 2), respectively. For both 1 and 2, the trend of g_∥ > g_⊥ > g_e (g_e = 2.0023) is validated, suggesting anisotropic spectra in both cases. For both 1 and 2, the g_∥ and g_⊥ values are near 2 (g_∥ > g_⊥), which implies a significant deviation from octahedral geometry.⁴ The observed g_∥ and g_⊥ values are comparable with the reports from previous studies.^24,35

Crystallographic description

The structures of the complexes were verified by SCXRD analysis. The ORTEP diagram of both 1 and 2 is depicted in Fig. 3. Complex 1 was crystallised as a triclinic structure under the space group of P1̄, while 2 was crystallised in a monoclinic arrangement with a space group of P2₁/c. Significant crystallographic parameters, the key bond lengths (Å) and bond angles (°) are presented in Tables S1 and S2, respectively. In both complexes, the central Cu(II) unit is six-coordinated, where the N atoms N1, N1, and N3 of ligands L1 (for 1) and L2 (for 2) occupied the first three coordination and the remaining three coordinations are fulfilled by the N4, N5, and N6 atoms from another distinct L1 and L2 ligands for 1 and 2, respectively. Although a majority of Cu(II) complexes with a six-coordination structure are found to have an elongation along the axial direction, herein, complexes 1 and 2 behave differently.³ In both complexes, the central Cu(II) unit is oriented in a distorted octahedral structure. It was observed that the N atoms of the pyrazine rings of L1 and L2 (N2, N3, N4 and N6) sit approximately in the basal plane, while the N atoms of the central pyridine unit (N1 and N5) are oriented towards the axial direction of the octahedron. A clear deviation was noticed in the trans (N1–Cu–N5) angles from the linearity (180°) (N1–Cu1–N5 = 176.56(11)° for 1 and N1–Cu1–N5 = 176.98(14)° for 2), which shows the deviation from the perfect octahedral structure in both cases. As a result of the rigidity in both structures of 1 and 2, the average axial Cu–N bond lengths (1.967 Å for 1 and 1.971 Å for 2) are noticeably smaller in comparison to the mean Cu–N bond distance in the equatorial positions (2.192 Å for 1 and 2.194 Å for 2), indicating that both complexes have rhombic distortion with a compressed octahedral geometry.³ Various terpyridine-based Cu(II) complexes with similar structural geometries like 1 and 2 have been documented in the previous studies.^24,42

Fig. 3 ORTEP diagram of (a) 1 and (b) 2. All the H atoms, counteranions and solvent molecules are omitted for simplicity. Thermal ellipsoids are drawn at a 30% probability.

Two separate displaced π–π stacking interactions between the terminal pyrazine ring and the extended conjugative phenyl ring were positioned above the central pyridine ring in the packing diagram of 1. The stacking interaction distances are found to be 3.811 Å and 3.674 Å, respectively. Conversely, no similar stacking interactions are observed for complex 2. The stacking distances for 2 are calculated to be much higher, 4.555 Å and 4.922 Å, beyond the permissible distance (∼4.0 Å) of such interactions (Fig. S7).⁴³

Several H-bonding interactions have been found for both 1 and 2. For 1, the O1 atom of the first ClO₄⁻ counter anion participates in forming H-bonding with the H15 atom (H15–O1 = 2.699 Å) of the terminal pyrazine ring of L1. Similarly, the O2 atom of the perchlorate also takes part in the H-bonding interaction with the H14 and H15 atoms of the pyrazine ring (H14–O2 = 2706 Å; H15–O2 = 2.477 Å), while the O3 atom forms an interaction with H41 of another terminal pyrazine moiety coordinated to the central Cu(II) unit. The fourth oxygen (O4) atom also participates in several interactions with the phenyl ring H9 hydrogen and H16, H42 atoms of the pyrazine unit (H9–O4 = 2.645 Å, H16–O4 = 2.621 Å and H42–O4 = 2.493 Å). Similar to the O1 to O4 atoms of the first perchlorate (ClO₄⁻) anion, the O atoms of the second ClO₄⁻ group also engage in multiple H-bonding interactions with the appropriate hydrogen atoms of L1. The O5 atom forms H-bonds with the H12 of the pyrazine unit (H12–O5 = 2.538 Å) and the H43C atom of the methyl (–NMe₂) proton of L1 (H43C–O5 = 2.598 Å). The O7 atom is involved in the H-bonding with the pyrazine H40 atom, with a distance of H40–O7 = 2.578 Å. Similar to the O4 atom, the O8 atom is also involved in various H-bonding interactions with the H26 atom of the phenyl proton (H26–O8 = 2.636 Å) and the H13 and H40 atoms of the terminal pyrazine units (H13–O8 = 2.621 Å and H40–O8 = 2.646 Å). For 2, the pyrazine H-atoms, H10, H14, and H16, are involved in forming H-bonding interactions with the O atoms (O2, O5 and O6) of the ClO₄⁻ moiety (H10–O2 = 2.660 Å, H14–05 = 2.435 Å, and H16–O6 = 2.601 Å). The O8 atom engaged in H-bonding with the H9 atom of the phenyl ring was positioned above the central pyridine moiety, which was directly coordinated to the Cu(II) center (H9–O8 = 2.418 Å). Additionally, another H-bonding between the H19 of the central pyridine unit and the O8 atom of the ClO₄⁻ anion (H19–O8 = 2.665 Å) was observed in the structure of 2. The H-bonding pictorial of both complexes is illustrated in Fig. S8.

Electrochemical investigation

The electrochemical characteristics of both complexes were studied using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) analyses. The experiments were performed under N₂-saturated atmosphere using 1 mM of 1 and 2 in dry CH₃CN solvent, where the supporting electrolyte is 0.1 M Bu₄N(ClO₄) [TBAP] and the glassy carbon served as the working electrode. All the redox potential values were referred to as SCE. For both 1 and 2, reversible one-electron Cu(III)/Cu(II) reduction was observed at 1.01 V and 0.89 V, respectively (Fig. 4).²² For complex 1, another one-electron reduction was observed at a less positive redox value of 0.06 V, demonstrating the Cu(II)/Cu(I) reduction. An irreversible Cu(I)/Cu(0) reduction was observed at a higher negative potential of −1.20 V.^44,45 The other cathodic peaks at −1.55 V and −1.81 V are due to the reduction of L1. In case 2, successive one-electron reduction associated with the Cu(II)/Cu(I) and Cu(I)/Cu(0) events is observed correspondingly at 0.08 V and −1.07 V, respectively. Both complexes exhibit a peak at −0.28 V and −0.39 V, respectively, signifying the deposition of Cu at the electrode surface.⁴⁶ Additionally, peaks at higher cathodic regions of −1.16 V and −1.80 V are due to ligand (L2)-based reductions (Fig. S9). All the redox potential values of 1 and 2 are presented in Table 1.

Fig. 4 CV (black line) and DPV (red line) of (a) 1 and (b) 2 in dry CH₃CN solvent using 0.1 M TBAP as a supporting electrolyte under a nitrogen atmosphere for the Cu(III)/Cu(II) couple.

Table 1 Redox potential values

Complex	Cu(III)/Cu(II)^a	Cu(II)/Cu(I)^a	Cu(I)/Cu(0)^a	Ligand-reduction
a Potentials vs. SCE in CH3CN/0.1 M TBAP.
1	1.01 V	0.06 V	−1.20 V	−1.55 V, −1.81 V
2	0.89 V	0.08 V	−1.07 V	−1.16 V, −1.80 V

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DNA binding studies

Absorption spectroscopy is a widely recognized method used to examine the DNA binding properties, which was utilised to study the interaction of ss-DNA with our synthesised complexes. For this purpose, a 5 mM tris-HCl buffer (pH = 7.4) solution was utilised. Metal complexes are mainly found to engage with DNA through non-covalent interactions, such as electrostatic binding, groove binding and intercalation.^47,48 In the electronic spectrum, when there is an interaction between a metal complex and DNA, generally a red shift or blue shift of absorption maxima is typically observed, accompanied by an increase or decrease in absorbance. As a result of intercalative binding, generally a hypochromic, and/or bathochromic change was noticed in the UV-vis spectrum due to the interactions involving the planar ligand framework and the DNA base pairs.²²

The representative diagrams of the binding studies of 1 and 2 are displayed in Fig. 5. The sequential rise in the strength of ss-DNA and hyperchromism was noticed in the intra-ligand (π–π*) transition (∼273 nm and 225 nm) bands with a minor (∼1–2 nm) hypsochromic change in the spectrum of 1. This suggests that complex 1 might follow the groove (either major or minor groove) binding mode to interact with DNA. However, for 2, the spectral changes are completely reversed from 1 under similar experimental conditions. Hypochromism was observed in the UV-vis spectral transitions (π–π* at 293 nm and n–π* at 378 nm) with a small bathochromic change of ∼2 nm, which signifies an intercalative binding in this case.

Fig. 5 Absorption spectra of (a) 1 and (b) 2 (both 50 μM) in the absence (black) and presence (colour) of ss-DNA with different concentrations (10, 20, 30, 40, 50, 60 and 70 μM). Inset: Plot of A₀/A − A₀vs. 1/[DNA] for the determination of the binding constant of complexes 1 and 2.

The Donor–Acceptor (D–A) nature of the ligand framework greatly influences the DNA interaction and cell cytotoxicity of different complexes.⁴⁹ In this present scenario, the pyrazine–pyridine part of both L1 and L2 is electron-withdrawing in nature, which acts as a strong π-acceptor.⁵⁰ The N,N-dimethylaniline group in L1 (for 1) acts as a stronger electron donor compared to diphenylaniline (for 2) owing to the delocalization of nitrogen's lone pair in the phenyl rings of L2, making both the complexes have a donor–acceptor nature.⁵¹

The N,N-diphenylaniline moiety in 2 has a less donating nature and can stabilise the ligand (L2) centered charge transfer (LCCT) states. This factor enhances stacking interactions with base pairs of DNA, promoting an intercalative mode of binding in 2.⁵² The extended conjugative N,N-diphenylaniline moiety can be easily incorporated between the DNA-base pairs unlike the N,N-dimethylaniline {–PhN(Me)₂} moiety in 1. Conversely, the greater electron-donating N,N-dimethylaniline moiety in L1 favours the metal-to-ligand charge transfer (MLCT) process. It drives down the π-conjugation efficiency in L1, resulting in the weakening of DNA intercalation while enabling the groove binding interactions.⁵³

The binding constant (K_b) was calculated to compare the binding ability of 1 and 2 with DNA. Eqn (1) is utilised to determine the binding constant (K_b) using a spectrophotometric method.

where A₀ and A denote the absorbance of metal complexes in the absence and presence of the DNA, respectively. The ε_G and ε_H−G denote the respective molar extinction coefficients. The graph of A₀/(A − A₀) vs. 1/[DNA] gives a linear fit, which validates the 1 : 1 adduct formation with DNA. Employing eqn (1), the intercept and slope ratio provide K_b values of 0.93 × 10⁴ M⁻¹ and 1.34 × 10⁴ M⁻¹, for 1 and 2, respectively. These values suggest that both complexes are moderately interacting with DNA compared to the conventional DNA intercalator EB, which has a K_b value in the range of 10⁶ M⁻¹.⁵⁴ A higher K_b value suggests a better interaction with DNA for 2. This might be due to the presence of an extended conjugative N,N-diphenylaniline {–PhN(Ph)₂} unit in the ligand framework of L2 in 2, which leads to better DNA interaction compared to 1. There are a few reports from different research groups on Cu(II) homoleptic complexes, where substitutions at the 4′-position of terpyridine (tpy) with different planar moieties have occurred, and their electronic and structural effects on DNA interaction have been studied. The K_b values of our synthesized complexes were found to be in good agreement with the earlier reported analogous Cu(II) complexes from the literature (Chart 1, Table 2).^24,32,35

Chart 1 Ligands addressed along with documented Cu(II) complexes.

Table 2 Copper complexes and their comparison of binding constant values

Complexes studied	Binding constant (Kb)/M^−1	Ref.
[Cu(La)2]Cl2	1.10 × 10^6	35
[Cu(Lb)2](ClO4)2	4.26 × 10^3	24
[Cu(Lc)2]Cl2	1.53 × 10^5	32
[Cu(Ld)2]Cl2	1.62 × 10^5	32
[Cu(Le)2]Cl2	1.02 × 10^5	32
[Cu(Lf)2]Cl2	1.09 × 10^5	32
[Cu(L1)2](ClO4)2	0.93 × 10^4	This work
[Cu(L2)2](ClO4)2	1.34 × 10^4	This work

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Viscosity measurement studies

To obtain further knowledge about the binding properties of both complexes, viscosity measurement studies were conducted using ss-DNA with increasing concentrations of added complexes. The experiment is based on determining the flow time measurement using a viscometer capillary tube. Generally, such molecules that interact with DNA via the intercalation mode lead to an increase in the viscosity of the DNA solution due to the expansion of the length of the DNA helix. However, the surface interactions (groove binding) induce minor changes in the viscosity. Eqn (2) was employed for the measurement of the relative viscosity coefficient (η):⁵⁵

η = t − t₀/t₀, (2)

where t denotes the time taken for the DNA-bound complex solution to pass through the viscometer capillary and t₀ denotes the time required for the buffer only. Fig. 6 shows the plot of (η/η₀)^1/3vs. [complex]/[DNA], which reveals that the viscosity of the DNA solution gradually enhances with the enhancement of the strengths of metal complexes. This implies that both complexes interact with ss-DNA in the solution. For 2, the extent of the increase in the relative viscosity is much higher than that of 1, which again suggests that 2 binds with DNA via intercalation.²⁴ The minor alteration in the viscosity in the presence of 1 signifies that it undergoes a DNA binding interaction via groove binding interaction. As a positive control of viscosity, we utilised EB, as this is considered the classical DNA intercalator, so its viscosity increases gradually with the concentration of EB. Similarly, a proper negative control is also useful to better understand the influence of the synthesised metal complexes on viscosity change. For this purpose, we synthesised [Cu(en)₂]SO₄ (en = ethylenediamine) complex.⁵⁶ As complex [Cu(en)₂]SO₄ lacks the planarity to interact with DNA, the only possible interaction mode is electrostatic interaction, and hence, no significant increase in DNA solution viscosity will occur; thus, it is well consistent with the role as a negative control.^57,58 Additionally, the obtained trend of EB > 2 > 1 > baseline ≥ [Cu(en)₂]SO₄ from the viscosity experiment aligns closely with the findings of the DNA binding experiment by applying the absorption spectrophotometric technique.

Fig. 6 Alteration in the viscosity of the ss-DNA solution under the influence of metal complexes. For (η/η₀)^1/3vs. [complex]/[DNA], the concentration of DNA was 200 μM and complex concentration was varied from 10 to 80 μM.

Ethidium bromide (EB) emission quenching studies

This investigation provides valuable information about the competitive interaction involving the EB and the complexes with DNA. Ethidium bromide (EB) is commonly non-luminescent in buffer medium due to the solvent-induced quenching phenomenon.²² Although in the presence of a DNA solution, EB exhibits a strong enhancement of fluorescence intensity due to its rapid intercalative binding with the adjacent DNA base pairs, and for this reason, EB is generally termed a classical DNA intercalator. Both 1 and 2 were tested for their ability to displace EB from EB-bound DNA (EB-DNA) solution. Upon successive addition of the complexes, a gradual decrease in fluorescence intensity was observed. This signifies that our synthesised complexes can readily substitute the EB from the EB-DNA solution. For both 1 and 2 initially, a rapid decrease in fluorescence maxima was observed, but afterward, the complexes came to a saturation point at different added concentrations of the respective complexes. The EB-displacement diagrams of both complexes are shown in Fig. 7, where complex 2 was found to displace the EB from the EB-DNA species at a faster rate, as evident from the rapid decrease in the emission maxima of EB-DNA species at a lower complex concentration (50 μM) compared to complex 1 (80 μM). To calculate the extent of quenching ability of both 1 and 2, the Stern–Volmer quenching constant (K_SV) was determined by employing eqn (3):⁵⁹

Fig. 7 Fluorescence quenching plot of EB-bound DNA with incremental concentrations of (a) 1 and (b) 2. [DNA] = 100 μM, [EB] = 10 μM. [Quencher] (μM) for 1 (1–80 μM), and 2 (1–50 μM). Inset: Plot of I₀/I vs. [quencher].

The findings of this study again conclude that 2 has better DNA intercalation ability than 1. Here, I and I₀ denote the emission intensity of EB-DNA solution in the presence and absence of the complex, respectively, and [Q] represents the strength of the metal complex. The graph between I₀/I vs. [Q] was linear, and the K_SV values were deduced from the ratio between the slope and intercept values. The higher K_SV value of 2 (3.7 × 10⁴ M⁻¹) than 1 (2.0 × 10⁴ M⁻¹) indicates that it has better DNA intercalation ability than 1.

Circular dichroism (CD) spectral analysis

The investigation related to the alteration of DNA morphology by the impacts of 1 and 2 was carried out using CD spectral analysis. The ss-DNA in its right-handed B form shows one positive and one negative band at ∼275 nm and ∼246 nm in its CD spectrum due to base stacking and DNA helicity, respectively.⁶⁰ Generally, complexes that prefer to engage with DNA interaction via groove binding or electrostatic interactions lead to less prominent changes in both the CD spectral bands. In contrast, an intercalative mode of interaction leads to major alterations in the intensities of the above-mentioned bands.³² The CD spectral diagram of ss-DNA in the presence and absence of 1 and 2 is shown in Fig. 8. Upon incorporation of 1 in the DNA solution, it leads to an almost negligible change in the base stacking band and a minor change (increase in intensity) in the helicity band, suggesting that it undergoes DNA interactions via the groove binding pathway. Moreover, in the case of 2, visible changes were evident in both the positive (decrease in intensity) and negative (increase in intensity) bands in the CD spectrum of ss-DNA, suggesting its intercalative mode of binding.⁶¹ The spectral alterations indicate that the interaction of 2 with DNA is significant enough to cause conformational perturbation in the secondary structure of ss-DNA. All these results for the CD spectral studies are found to be in good agreement with their respective modes of engagement, as suggested by the documented literature.¹⁵

Fig. 8 CD spectra of free ss-DNA (100 μM) alone (black line) in the presence of complexes (red and blue line) 1 and 2 (50 μM).

Lipophilicity studies

The membrane permeability and cellular uptake capabilities of complexes 1 and 2 were further examined based on their lipophilicity partition coefficient values (log P_oct/buff). Using the shaking flask method, the partition coefficient values of complexes 1 and 2 in n-octanol and water (tris HCl buffer) (log P_oct/buff, where P = [oct]/[buff]) were determined. The log P values are calculated to be −0.103 and −0.307 for 1 and 2, respectively. The negative values in both cases signify that both complexes are slightly hydrophilic; thus, the rate of lipophilicity follows the order of 1 > 2.⁶² The experimental plots of lipophilicity experiments are illustrated in Fig. S10 and S11, respectively.

Molecular docking (MD) studies

To understand the possible interaction sites and orientation with respect to DNA and metal complexes, we explored in silico binding studies (Fig. 9). The resultant docked figures highlight the portion of DNA involved in π–anion, π–alkyl, C–H, π–π stacked bond and van der Waals interactions. Complexes 1 and 2 were observed to partially accommodate within the major groove, engaging with the pyrazine, pyridine, and phenyl groups along the inner edge while maintaining the integrity of the DNA double helix. The binding free energies were calculated to be −5.60 and −6.65 kcal mol⁻¹ for 1 and 2, respectively. Overall, this docking study supports our spectroscopic findings and provides additional confirmation regarding duplex DNA interaction. In general, this molecular docking investigation supports our spectroscopic findings and offers additional proof of the duplex DNA interaction.

Fig. 9 MD of (a) 1 and (b) 2 with the B-DNA (PDB ID:1BDW). Binding of nucleotides dA3, dG4, dA13, and dC12 with our metal complexes depicted where the interactions are denoted by dotted lines (C–H bond: light grey, π–anion: orange, π–σ: light pink, π–π: pink, conventional N–H hydrogen bond: green). The backbone of DNA is depicted with light grey lines, while the ligand backbone of the complexes is shown in dark grey. The adenine, cytosine, thymine, and guanine nucleotides are denoted in red, pink, cyan and green, respectively.

DNA nicking analysis

Agarose gel electrophoresis was used to examine the DNA nicking capability of 1 and 2. Nicking was determined by utilizing circular plasmid DNA. It was observed that 1 was able to convert supercoiled circular (SC) DNA to its corresponding nicked circular (NC) DNA conformation at the concentration tested (50 and 100 μM) (Fig. 10a, lane 3). Notably, complex 2 also exhibited a property similar to that of DNA cleavage (Fig. 10d, lane 3). Since the degree of DNA nicks relies on the Cu(II)-catalysed ROS production, the external supply of H₂O₂ to the reaction might enhance the ability of 1 and 2 to nick DNA. To examine this, the reaction was performed in the presence of H₂O₂. It was observed that the DNA nicking ability of 1 and 2 was not further improved in the presence of H₂O₂ (Fig. 10b and e). This is not surprising because Cu(II) complexes themselves can efficiently generate reactive oxygen species through redox cycling, and the addition of external H₂O₂ does not significantly contribute to further DNA cleavage under experimental conditions.

Fig. 10 DNA nicking activity of 1 and 2. (a) Supercoiled circular (SC) DNA was incubated with 1 (50 and 100 μM), and the appearance of nicked circular (NC) DNA was monitored by agarose gel electrophoresis. (b) Effects of H₂O₂ (1 mM) and EDTA (2 mM) on DNA cleavage by 1. (c) Effects of H₂O₂ (1 mM) and azide (5 mM) on DNA cleavage by 1. (d) Analysis of DNA nicking by 2. (e) Effects of H₂O₂ (1 mM) and EDTA (2 mM) on DNA cleavage by 2. (f) Effect of H₂O₂ (1 mM) and azide (5 mM) on DNA cleavage by 1. The DNA was analysed by agarose gel electrophoresis, followed by EtBr staining.

To confirm the essential role of Cu(II) in the catalysis of DNA cleavage, the chelating agent EDTA was added to the reaction. As EDTA forms stable complexes and effectively sequesters Cu(II) from 1, DNA cleavage was significantly abolished by EDTA (Fig. 10b, lanes 4 and 6). EDTA prevented DNA cleavage by 2 (Fig. 10e, lanes 4 and 6), indicating the involvement of hydroxyl radicals (˙OH) as ROS during DNA cleavage. To examine whether azide (N₃⁻) could compete with the Cu(II) complexes by ligand exchange or coordination competition and prevent DNA cleavage, supercoiled circular DNA was incubated with H₂O₂ and sodium azide (NaN₃). It was observed that the DNA nicking activity of 1 could not be abated by the azide (Fig. 10c, lanes 4 and 6). Similar results were also found with 2 (Fig. 10f, lanes 4 and 6). These results suggest a strong interaction of Cu(II) with 1 and 2, preventing displacement by azide.

Cytotoxicity analysis and evaluation of intracellular ROS production

The in vitro cytotoxicity of complexes 1 and 2 was evaluated on two different human breast cancer (MDA-MB-231 and MCF-7) cell lines using the MTT assay. The graph of cell viability vs. concentrations of 1 and 2 for the two cell lines revealed that both cell lines were slightly more sensitive to 1 than to 2 (Fig. 11a). IC₅₀ values of 1 and 2 were found to be 5.43 μM and 20.25 μM, respectively, in the MDA-MB-231 cell line and 7.44 μM and 25.58 μM, respectively, in the MCF7 cell line. IC₅₀ values of 1 and 2 were found to be around 5.08 μM and 1.8 μM, respectively, in the non-cancer HEK293 cell line, which is comparable with the IC₅₀ value of cis-platin as well.⁶³ Further confirmation of cytotoxicity was obtained by Calcein-AM probe analysis (Fig. 11b). This assay is based on the activity of intracellular esterase in live cells that converts Calcein into Calcein-AM, which subsequently produces green fluorescence as long as the plasma membrane remains intact. Although complex 2 exhibits a higher DNA binding affinity compared to complex 1, the scenario is completely reversed when we compare their cytotoxicity performance. This might be due to the presence of bulky diphenyl groups, and their special arrangement creates steric hindrance, which leads to reduced membrane permeability and cellular uptake and hence shows lower cytotoxicity.^64,65 However, complex 1 has less steric bulk in the N,N-dimethylaniline moiety, assisting faster membrane permeability and higher intracellular accumulation, which accounts for its higher cytotoxicity.^66,67

Fig. 11 Cellular response of cells treated with 1 and 2. (a) Survival of breast cancer cell lines MCF7 and MDA-MB-231 and non-cancer cell line HEK293. Cells were treated with increasing concentrations of 1 and 2, and cell survival was determined by MTT assay. (b) Cell viability analysis: MCF7 and MDA-MB-231 cells were incubated with 1 and 2 for 48 h, and viability was measured by observing the cells under a fluorescent microscope using DAPI and Calcein-AM dye. (c) Evaluation of intracellular ROS production in MDA-MB-231 cells through DCFDA staining upon treatment with 1 and 2. (d) Quantification of cellular fluorescence of DCFDA-treated cells in (c). The scale bar represents 400 µm.

The induction of intracellular ROS production by the complexes was evaluated using DCFDA staining in the MDA-MB-231 cell line. The non-fluorescent dichloro-dihydro-fluorescein diacetate (DCFDA) is oxidised by intracellular ROS and converted to highly fluorescent DCF. The intensity of fluorescence is directly related to the quantity of ROS. Here, the minimal fluorescence intensity was displayed by the control cells, which was attributed to their negligible ROS content. However, upon treatment with 1 and 2, the intensity increased significantly, among which 1 showed slightly higher activity (Fig. 11c and d). Together, the results suggest that compounds 1 and 2 have a strong ability to generate DNA nicks and ROS, with compound 1 showing comparatively little improvement in the induction of cytotoxicity.

Camptothecin (CPT) is a tryptophan-derived quinoline that is used as a cytotoxic anti-cancer drug. CPT exerts its effect by targeting the DNA topoisomerase I-DNA complex, which subsequently generates DNA breakage. We were interested in knowing if 1 could enhance the cytotoxic effect of CPT. We examined the cytotoxicity of CPT alone and in combination with 1. Interestingly, increased cytotoxicity was observed when the cells were treated with CPT in the presence of a sub-toxic concentration of 1 (1 μM). The IC₅₀ of CPT changed from 38.23 μM to 0.27 μM in the presence of 1 (Fig. 12).

Fig. 12 Cytotoxicity analysis of cancer cells treated with camptothecin (CPT) alone and in combination with 1. Breast cancer cell line MDA-MB-231 was treated with an increasing concentration of CPT in the presence of a sub-toxic concentration of 1 (1 μM), and cell viability was determined by XTT assay.

Conclusions

The present study outlines the synthesis of two homoleptic Cu(II) complexes of type [Cu(L1)₂](ClO₄)₂, 1, and [Cu(L2)₂](ClO₄)₂, 2. The ligand frameworks of L1 and L2 were appended to the chromophoric units of N,N-dimethylaniline and N,N-diphenylaniline, respectively. Characterization of both complexes was carried out using several spectroscopic and analytical methods. The molecular architectures of 1 and 2 were validated by utilizing the single SCXRD technique, confirming that the central Cu(II) is oriented in a distorted octahedral geometry in both cases, which has been further confirmed via EPR spectroscopy. The redox properties of both complexes were examined. The DNA binding properties were investigated by applying the absorption spectroscopic method. Competitive EB replacement studies were conducted using fluorescence spectroscopy for both 1 and 2. Significant binding parameters, such as K_b and K_sv, were calculated for both complexes, and the findings show higher K_b and K_sv values for 2 compared to 1. To further verify the binding properties of both complexes, viscosity and CD spectral analyses were performed. The superior DNA interaction ability of 2 is due to the presence of a more planar extended conjugative N,N-diphenylaniline chromophoric moiety in the ligand framework of L2, compared to the N,N-dimethylaniline {–PhN(Me)₂} unit in 1. The involvement of major groove-mediated interactions in both complexes was validated through molecular docking studies. DNA nicking activity was examined through in vitro electrophoresis analysis of supercoiled plasmid DNA, and both 1 and 2 showed significant DNA nicking ability. Analysis of cell viability using cancer cell lines revealed that both complexes were cytotoxic to cancer cells, but 1 is slightly more cytotoxic than 2 probably because 1 produced more ROS compared to 2. Furthermore, 1 could increase the cytotoxicity of the cancer drug, Camptothecin (CPT). Thus, these results could be further explored as the design and development of potentially novel and economical non-platinum transition metal-based chemotherapeutic agents towards anticancer drugs that can overcome the deficiencies of current conventional drugs.

Experimental section

Materials

Cu(ClO₄)₂·6H₂O, ethidium bromide, salmon sperm DNA (ss-DNA), 2-acetylpyrazine, 4-(dimethylamino)benzaldehyde, and 4-(diphenylamino)benzaldehyde were bought from Sigma Aldrich India. All the solvents utilised for synthesis and experimental purposes are of reagent-grade. The acetonitrile utilised for the redox investigations was dried using CaH₂ as a drying agent. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Dulbecco's phosphate-buffered saline (DPBS), trypsin–EDTA solution, antibiotic solution, calcein AM, and NucBlue™ Reagent (Hoechst 33342) were bought from Thermo Fisher Scientific. XTT cell proliferation kit and 2′,7′-Dichlorofluorescein Diacetate (DCFDA) were procured from Sigma Aldrich India. Dimethyl sulfoxide (DMSO) was purchased from SRL Laboratories. MCF-7 (human breast cancer) and MDA-MB-231 (human triple-negative breast adenocarcinoma) cell lines were obtained from the National Centre for Cell Science, India.

Physical measurements

¹H NMR spectra of ligands L1 and L2 were recorded using a BRUKER AVANCE III-400 MHz spectrometer in CDCl₃ solvent. All the coupling constants (Hz) along with their corresponding chemical shift values (δ, ppm) are assigned in an accustomed manner with tetramethylsilane (TMS) (δ(H) 0.00 ppm) as a reference standard. All the UV-vis electronic absorption spectra of L1, L2, 1 and 2 were recorded using a JASCO V-730 spectrophotometer at room temperature. A Bruker Alpha-P spectrometer was used for all the FT-IR spectral measurements in the solid state at room temperature. An Agilent Technologies mass spectrometer (model: HRMS Q-TOF 6538) was used to obtain the high-resolution mass spectra in the electrospray ionisation (ESI) mode. A Horiba Flouromax-4 fluorescence spectrophotometer was used to obtain the emission spectral data at room temperature. Elemental analysis was recorded using a BRUKER EURO EA. A JOEL EPR spectrometer was used for all the EPR measurements at room temperature. The redox behaviour was analysed using a CHI-660 potentiostat device. A one-pot three-electrode setup was used in which a glassy carbon electrode served as the working electrode. The counter and reference electrodes are a platinum wire and a saturated calomel electrode (SCE), respectively. All the electrochemical studies are conducted under an inert atmosphere. The 0.1 M TBAP (tetrabutylammonium perchlorate) was used as a supporting electrolyte during the entire electrochemical analysis. Circular dichroism spectra were measured using a JASCO J-815 CD spectrophotometer ranging from 190 to 500 nm.

Synthesis of 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-dimethylaniline (L1)

A mixture of 2-acetylpyrazine (2.5 g, 20.47 mmol) and 4-(dimethylamino)benzaldehyde (1.5 g, 10.05 mmol) reacted together in the presence of 25 mL of ethanol, followed by the addition of KOH (2.0 g, 35.65 mmol) and 10 mL of NH₄OH solution. Then, the resulting mixture was stirred for 24 h at room temperature. Afterward, the solid formed was filtered and washed thoroughly with distilled water and diethyl ether. Afterwards, the crude product was purified using recrystallization in a dichloromethane (DCM) solvent, resulting in an off-white crystalline product of ligand L1. Yield: 2.42 g, (68%). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) = 9.86 (s, 2H), 8.71–8.63 (m, 6H), 7.84 (d, J = 8.9 Hz, 2H), 6.82 (d, J = 8.9 Hz, 2H), 3.06 (s, 6H, –(CH₃)₂N). ¹³C NMR (151 MHz, CDCl₃) δ 154.08, 151.35, 151.19, 150.40, 144.52, 143.68, 143.48, 128.01, 124.56, 118.32, 112.29, 40.27. Anal. calcd for C₂₁H₁₈N₆: C, 71.17; H, 5.12; N, 23.71; found: C, 70.74; H, 5.21; N, 23.41. Melting point ∼211 °C. ESI-MS (CH₃CN): m/z = 355.1659 [L1 + H⁺] (calc.: 354.1593). FT-IR (solid, ν, cm⁻¹): 754 ν(aromatic =C–H), 1468 ν(C=C), 1594 ν(C=N), 3034 ν(sp² C–H). UV-vis (Tris-HCl buffer) λ_max (nm) [ε (L mol⁻¹ cm⁻¹)]: 271 [39 704], 293 [17 970], 330[10 350].

Synthesis of 4-(2,6-di(pyrazin-2-yl)pyridin-4-yl)-N,N-diphenylaniline (L2)

Ligand L2 was synthesised by employing a synthetic route similar to L1, but in this case, 4-(diphenylamino)benzaldehyde (2.73 g, 10.00 mmol) was used in place of 4-(dimethylamino)benzaldehyde. The purification of the crude product via further recrystallisation in DCM solvent led to the formation of an off-white colour crystalline product of ligand L2. Yield: 3.11 g, (65%). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) = 9.87 (s, 2H), 8.70–8.64 (m, 6H), 7.75 (d, J = 8.7 Hz, 2H), 7.37–7.27 (m, 4H), 7.17 (dd, J = 8.1, 5.5 Hz, 6H), 7.09 (t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 154.30, 150.92, 150.12, 149.27, 147.21, 144.70, 143.64, 143.55, 130.66, 129.47, 127.99, 125.04, 123.70, 122.80, 119.10. Anal. calcd for C₃₁H₂₂N₆: C, 77.80; H, 4.63; N, 17.56; found: C, 77.42; H, 4.71; N, 17.40. Melting point ∼233 °C. ESI-MS (CH₃CN): m/z = 479.1950 [L2 + H⁺] (calc.: 478.1906). FT-IR (solid, ν, cm⁻¹): 752 ν(=C–H), 1481 ν(C=C), 1582 ν(C=N), 3004 ν(sp² C–H). UV-vis (Tris-HCl buffer) λ_max (nm) [ε (L mol⁻¹ cm⁻¹)]: 288[30 768], 377[21 415].

General procedure for the synthesis of copper(II) complexes 1 and 2

In a methanolic solution of Cu(ClO₄)₂·6H₂O (0.25 mmol), another methanolic solution of corresponding ligands (0.5 mmol) (L1 for 1 and L2 for 2) was added slowly with constant stirring. The subsequent mixture was then stirred vigorously for 4 h at room temperature, resulting in the formation of a precipitate. Then, the formed precipitate was filtered and washed carefully with a minimum amount of cold methanol, and finally with diethyl ether. Finally, complexes 1 and 2 were recrystallised using a solvent blend of (2 : 1 v/v) acetonitrile and methanol.

[Cu(L1)₂](ClO₄)₂ (1). Dark orange crystals. Yield: 148 mg (61%). Anal. calcd for C₄₂H₃₆Cl₂CuN₁₂O₈: C, 51.94; H, 3.74; N, 17.31; found: C, 51.98; H, 3.76; N, 17.41. Melting point >250 °C. ESI-MS (CH₃CN): m/z = 386.1900 [1]²⁺ (calc.: 386.1758). FT-IR (solid, ν, cm⁻¹): 1079, 621 (Cl–O), 1589 (C=N stretching), 1488 (C=C stretching). UV-vis (Tris-HCl buffer) λ_max (nm) [ε (L mol⁻¹ cm⁻¹)]: 270 [37 548], 294 [13 034], 326 [2820].

[Cu(L2)₂](ClO₄)₂ (2). Brown crystals. Yield: 192 mg (63%). Anal. calcd for C₆₂H₄₄Cl₂CuN₁₂O₈: C, 61.06; H, 3.64; N, 13.78; found: C, 61.13; H, 3.69; N, 13.73. Melting point >250 °C. ESI-MS (CH₃OH): m/z = 509.1305 [2]²⁺ (calc.: 509.6554). FT-IR (solid, ν, cm⁻¹): 1081, 624 (Cl–O), 1590 (C=N stretching), 1467 (C=C stretching). UV-vis (Tris-HCl buffer) λ_max (nm) [ε (L mol⁻¹ cm⁻¹)]: 270 [28 674], 351 [28 674], 462 [4643].

Caution! Perchlorate salts are generally explosive and should be handled with proper precautions. Additionally, it is recommended to use in small quantities.

Crystallography

Crystals of both complexes were obtained from a solvent mixture of CH₃CN and CH₃OH (2 : 1 v/v). The Bruker D8 Venture instrument using a Photon III mixed mode detector with graphite-monochromatic Mo-Kα (λ = 0.71073 Å) radiation was utilised for data collection purposes. To mount single crystals, a cryoloop (Hampton Research Corp.) in a minimal quantity of parabar oil was utilised. SHELXT-97 is used for crystal solving, and SHELXL was employed for the refinement of all the non-H atoms on F² by utilizing full-matrix least-squares methods.^68,69 H atoms were positioned at geometrically appropriate sites, and all the non-H atoms were refined anisotropically. The calculations were performed by the Olex2 software, and for the ORTEP representations, we utilised the Mercury 2024.1.0 program.^70,71 The CCDC numbers of 1 and 2 in this study are 2450766 and 2450767, respectively.

Experimental methods

Details of the experimental methods, such as DNA binding studies, viscosity measurement, ethidium bromide (EB) emission quenching studies, CD spectra analysis, lipophilicity studies, molecular docking studies, DNA nicking analysis, and cell-based studies, are provided in the SI.

Author contributions

I. Roy: synthesis, investigation, data curation, formal analysis, and writing original draft. K. L.: visualization and formal analysis. S. Deb: spectroscopic data analysis, writing – review and editing. S. S. V.: cytotoxicity studies. J. Jangra: DNA nicking analysis. S. Patra: molecular docking. A. Roy and S. Maji: funding acquisition, conceptualization, methodology, validation, supervision, writing – review and editing the final manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. Details of the experimental methods such as, DNA binding studies, viscosity measurement, ethidium bromide (EB) emission quenching studies, CD spectra analysis, lipophilicity studies, molecular docking studies, DNA nicking analysis, and Cell-based studies, ¹H-NMR spectra of L1 and L2, all FT-IR spectra, UV-vis stability of 1 and 2, all mass spectrometric data, crystal packing diagrams of 1 and 2, H-bonding interactions in 1 and 2 and cyclic voltammograms of 1 and 2, lipophilicity plots of 1 and 2 are given in the supporting information. See DOI: https://doi.org/10.1039/d5dt01877c.

CCDC 2450766 and 2450767 contain the supplementary crystallographic data for this paper.^72a,b

Acknowledgements

Financial support received from Science and Engineering Research Board (SERB), Anusandhan National Research Foundation (ANRF), Government of India: project no. CRG/2023/003259, India: project no. CRG/2023/003259 and for (fellowship to K. L.); Ministry of Education (MoE) (fellowship to I.R.), institute post-doctoral fellowship (IPDF) IIT Hyderabad (fellowship to S. D.), Funding from Indian Council of Medical Research (ICMR), Grant EMDR/SG/13/2023-0897, Govt of India (fellowship to R. A.) and Indian Institute of Technology Hyderabad are gratefully acknowledged.

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