Structural-Dependent N,O-Donor Imine-Appended Cu(II)/Zn(II) Complexes: Synthesis, Spectral, and in Vitro Pharmacological Assessment

Abstract

Four mononuclear bioefficient imine-based coordination complexes, [(L¹)₂Cu], [(L¹)₂Zn], [(L²)Cu(H₂O)], and [(L²)Zn(H₂O)], were synthesized using ligands [L¹ = 2-(((3-hydroxynaphthalen-2-yl)methylene)amino)-2-methylpropane-1,3-diol and L² = 4-(1-((1,3-dihydroxy-2-methylpropan-2-yl)imino)ethyl)benzene-1,3-diol]. The formation of the complexes was ascertained by elemental analysis, Fourier transform infrared, ¹H NMR, ¹³C NMR, electrospray ionization–mass spectroscopy, electron paramagnetic resonance, and thermogravimetric analysis. The comparative binding propensity profiles of the above-synthesized complexes with the DNA/human serum albumin (HSA) were investigated via UV absorption, fluorescence, and Förster resonance energy-transfer studies. On the basis of extended conjugation and planarity, L¹ complexes exhibited superior bioactivity with greater calculated DNA binding constant values, (K_b) 2.9444 × 10³[(L¹)₂Cu] and 2.2693 × 10³[(L¹)₂Zn], as compared to L² complexes, 1.793 × 10³[(L²)Cu(H₂O)] and 9.801 × 10²[(L²)Zn(H₂O)]. The competitive displacement assay of complexes was performed by means of fluorogenic dyes (EtBr and Hoechst), which corroborates the occurrence of minor groove binding because of the enhanced displacement activity with Hoechst 33258. The minor groove binding of the [(L¹)₂Cu] complex is further confirmed by the molecular docking study. Moreover, the HSA study demonstrated effective static quenching of complexes with substantial K_sv values. The [(L¹)₂Cu] complex was found to have pronounced cleavage efficiency as evaluated from sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis. Furthermore, in vitro antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals further proclaimed the remarkable bioefficiency of compounds, which make them promising as active chemotherapeutic agents.

1. Introduction

Coordination complexes of imine scaffolds are an indispensable class of chemistry because of their structural divergence and binding protocols. Imines are rationalized as novel ligands, which are eminently used because of their flexible entanglement, striking versatility, and facile solubility in common solvents.^1,2 They are broadly employed for their greater yield and are generally synthesized by the condensation of the ketone or aldehyde with an amine under variable conditions of temperature, pH, and solvents. The presence of azomethine linkage (−C=N) in the imines has gained interest because of their stability, chelating property, and diverse biological applications.³⁻⁵

Structural–activity approach has been anticipated as an efficacious platform for the discovery of potent drugs in the area of therapeutic science.⁶ Nowadays, various protocols are under extensive research for the refinement of such prototypes in which a molecular hybridization process has emerged as a powerful and advanced approach for rational designing of ligand scaffolds, which depend on the pharmacophoric entities present in the ligand framework. Thus, it leads to the formation of a new framework, which bears intrinsic properties of the initial pattern and manifests an innovative formulation with enhanced affinity and efficacy in comparison to the parent drug.^7,8 The prudent selection of the two precursors controls the resulting ligand’s denticity, donor atom’s nature, and the number of chelating moieties.⁹ An enormous number of imines has been designed and widely studied as they have some emblematic properties such as striking biological properties, flexible synthesis, thermal stability, novel structural and medicinal efficacies, etc. Imine-based metal complexes are progressively studied because of facile synthesis, crystallographic features, biological activity, and redox and catalytic properties. Earlier research has shown that metal complexes have better bioactivity than the free inorganic ligands (imines) as insertion of active metal centers enhances the stability of the metal complexes, thereby making them extremely valuable probes for the biological system.^10,11 Imine-based N,O donor metal complexes are extensively utilized as snipping agents for DNA binding studies focused on antitumor medications. In a recent study, Reedjik et al. described DNA binding and the cleaving capacity of a complex comprising Cu(II) Schiff base, which exhibited huge potential in cytotoxic consequences for HL 60 (leukemia) disease cells. Lou and co-workers have also synthesized a new cytotoxic copper(II) complex, which displayed promising antitumor activity over the breast tumor cell line (MCF-7cells).^12,13 The investigation of DNA interaction with the Schiff base complex is a preliminary step to develop and symphonize a new pharmaceutical molecule. The small-molecule moieties incorporating drugs can associate with DNA via three binding models cooperation with sections of DNA by hydrogen bonding, electrostatic binding amidst negatively charged DNA phosphate residues and cation species, or intercalative and van der Waals interactions.¹⁴⁻¹⁷ Imine-based complexes are also considered as promising candidates against malignancy drugs and have high proclivity for human serum albumin (HSA) binding. Remarkably, HSA is the amplest plasma protein, which is related to binding and drug transportation in the blood. The literature demonstrated that albumins from blood plasma could interact with most imine-based complexes.¹⁸ The interaction of drug with protein makes a stabilized drug–protein complex, which consequently makes an effect on drug distribution and its metabolism in the blood. The distribution of drugs is generally taken up by HSA as mainly drugs move in the plasma and achieve the targeted tissues through binding to albumin, hence functionalize as a most preferential substitute for the purpose of drug delivery.¹⁸ Among all the transition metals reported for their biological relevance, copper was found to have a vital role in chemotherapy.¹⁹ Copper exists as a central metal ion in many metalloproteins and affects their functioning by adjusting the geometrical arrangement of ligands around it.¹ It exists in two biologically active forms, +1 and +2, out of which +2 is the most stable form and was found to have more potential to alter various biological processes. Direct interaction of copper with DNA causes site-specific cleavage, thereby generating reactive oxygen species (ROS) under required optimum conditions. Copper-based synthetic compounds have the tendency to facilitate the cleavage of nucleic acid (DNA); therefore, they are gaining a lot of interest for both in vitro and in vivo advanced oncogenic studies.²⁰⁻²³ The copper complexes were also reported as the potential alternatives to anticancer agents(cisplatin)^24,25 and play an indispensable role in cell physiology as potent-free radical scavengers. Zinc is also an integral cofactor in many biological processes and is the second most abundant 3d metal in human body. Zinc complexes have various rich coordination affinities with a variety of macromolecules which mechanistically have dynamic influence on transcription and DNA replication.²⁶⁻²⁸

Nowadays, imine-based ligands having mixed donor atoms (N and O) are preferred as their derivatives are versatile and have the ability to form high nuclearity compounds. Polydentate pharmacophore such as 2-amino-2-methyl-1,3 propanediol has been intrinsically explored because of their diverse structural and pharmacological applications.^29,30 Imine bases derived from hydroxy naphthaldehyde have been broadly examined because of their substantial uses in therapeutic field. They outline stable adducts with metal particles owing to the occurrence of ortho phenolic hydroxyl group, which reconciliate to the metal particle by means of deprotonation. Hydroxy naphthaldehyde exhibits strong steric hindrance and high conjugating property because of the naphthyl ring, thereby forming imine-based metal complexes with more coordination sites and higher stability.^1,31

In the light of the above given details and in the expansion of our current research on imine-based complexes using hybrid pharmacophore approach,³² here we design and synthesize N,O donor imine ligands and their Cu(II) and Zn(II) complexes derived from bioactive scaffolds, 2-amino-2-methyl-1,3 propane-diol and 3-hydroxy-2-naphthaldehyde or 2,4-dihydroxyacetophenone. The formulated compounds were structurally and spectroscopically characterized by elemental analysis, Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR), electrospray ionization–mass spectrometry (ESI-MS), and thermogravimetric analysis (TGA). The pharmacological assessment of all synthesized compounds based on their structural features was illustrated by biophysical techniques (DNA/HSA binding), Förster resonance energy transfer (FRET), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, docking, and antioxidant studies.

2. Results and Discussion

The mononuclear complexes {[(L¹)₂Cu], [(L¹)₂Zn], [(L²)Cu(H₂O)], and [(L²)Zn(H₂O)]} were derived from the imine-based ligands L¹ and L², respectively. These ligands were synthesized via condensing 3-hydroxy-2-naphthaldehyde and 2,4-dihydroxyacetophenone with 2-amino-2-methyl-1,3-propanediol taking methanol as a solvent. All the synthesized complexes exhibited good stability at room temperature and were soluble in dimethyl sulfoxide (DMSO). The physical and analytical data of compounds are well demonstrated in Table 1. The nonelectrolytic nature of complexes (DMSO/1 × 10^–4 M) was confirmed from the outcomes of the molar conductance values (11–16 Ω^–1 cm² mol^–1).³³ The spectrochemical features as well as geometrical aspects of synthesized compounds were ascertained by spectral studies such as FTIR, NMR, ESI-MS, UV, TGA, and elemental analysis. The geometry surrounding Cu(II) ions in the complexes was elucidated from the electron paramagnetic resonance (EPR), while the absorption bands of Cu(II) complexes were detected by the electronic spectra. Moreover, binding studies of DNA and HSA were carried out to determine various biophysical parameters. Additionally, other biological studies such as gel electrophoresis, molecular docking, and antioxidant activity were also performed to further validate the biocompatible behavior of the complexes.

Table 1. Physical and Analytical Data (Elemental Analyses, m/z Values, Color, Yield, Molar Conductance, and Melting Point) of Imine-Based Ligands and Their Cu and Zn Complexes.

					calcd (found) (%)
compounds	F.W. (m/z+)	color	yield (%)	mp (°C)	C	H	N	M	molar conductivitym(mol–1 cm2 Ω–1)
ligand, L1 [C15H17NO3]	259.30 (260.43)	pale yellow	65	>250	69.41 (69.34)	6.60 (6.53)	5.39 (5.35)
ligand, L2 [C12H17NO4]	239.11 (240.13)	brown	75	>250	60.21 (60.15)	7.15 (7.10)	5.85 (5.75)
[(L1)2Cu] [C31H30CuN2O6]	577.1.4 (578.12)	dark green	70	>300	62.33 (62.10)	5.23 (5.00)	4.58 (4.40)	10.99	15.33
[(L1)2Zn] [C30H30ZnN2O6]	579.21 (580.95)	black	78	>300	62.13 (62.01)	5.21 (5.05)	4.83 (4.40)	11.27	12.25
[(L2)Cu(H2O)] [C12H16CuN2O5]	318.65 (319.61)	black	70	>300	45.21 (45.16)	5.37 (5.31)	4.39 (4.29)	19..93	13.10
[(L2)Zn(H2O)] [C12H16ZnNO5]	320.11 (321.49)	reddish brown	78	>300	44.95 (44.84)	5.34 (5.26)	4.38 (4.25)	20.39	11.23

2.1. IR Spectra

The infrared spectra of the newly synthesized compounds (400–4000 cm^–1) were performed to assign the functional groups existing in compounds. The IR spectra of synthesized ligands (L¹ and L²) exhibited a broad band (3416–3287 cm^–1) because of stretching vibration of aliphatic −OH. The medium intensity band (1352–1361 cm^–1) signifies the presence of ν(C–O) phenolic vibration, whereas the peak (3287–3176 cm^–1) suggests the occurrence of aromatic −OH. A new strong intensity band (1616–1625 cm^–1) attributes the presence of azomethine group (−CH=N−), which consequently confirms the formation of proposed imine ligands.⁷ The absence of broad band of phenolic −OH (3287–3176 cm^–1) in [(L¹)₂Cu], [(L¹)₂Zn], [(L²)Cu(H₂O)], and [(L²)Zn(H₂O)] complexes signifies the coordination between metal center and phenolato oxygen atom via deprotonation.¹ On subsequent metalation, the azomethine peak (1616–1625 cm^–1) was shifted to a lesser frequency from its original position, signifying the lone pair donation to metal ions by nitrogen in all complexes. This negative shift affirms the impeccable coordination of azomethine nitrogen toward the metal ions, which is further affirmed by the presence of vibration peaks in the region 464–490 cm^–1 ν(M–O) and 520–585 cm^–1 ν(M–N), respectively.^7,31 Furthermore, the peak detected (3300–3400 cm^–1) in [(L²)Cu(H₂O)] and [(L²)Zn(H₂O)] complexes corresponds to the occurrence of coordinated water molecules,⁷ while bands (1435–1490, 1012–1095, and 738–769 cm^–1) are related to aromatic ring vibrations.³⁴

2.2. ¹H NMR and ¹³C NMR

The ¹H NMR spectra of free ligands (L¹ and L²) and their corresponding complexes, [(L¹)₂Zn] and [(L²)Zn(H₂O)], were documented using DMSO-d₆ as a solvent The ¹H NMR spectra of free ligands (L¹ and L²) showed a singlet at 8.58 and 8.49 ppm, respectively (Figure 1a,b), affirming the presence of −CH=N– linkage, which eventually confirms the formation of Schiff base ligands.³⁵ The singlet hydroxyl proton appeared at 14.32 ppm for L¹ and 10.19 ppm for L².^7,36 The peak appeared at 5.20 and 5.17 ppm displayed the presence of aliphatic −OH group and the peak at 3.46, 1.37, and 3.33, 1.35 ppm displayed the aliphatic −CH₂ and −CH₃ groups of both ligand moieties. The multiplets in the range 6.67–8.16 and 6.15–7.57 ppm signified the presence of naphthalene and acetophenone ring protons, respectively.⁷ Unlike free ligands, in the ¹H NMR spectra of [(L¹)₂Zn] and [(L²)Zn(H₂O)], the hydroxyl peaks at 14.32 and 10.19 ppm are completely disappeared, which indicates the ligation between metal center and phenolato oxygen atom via deprotonation.^27,31 The characteristic azomethine peak at 8.58 and 8.49 ppm undergoes a negative shift in [(L¹)₂Zn] and [(L²)Zn(H₂O)], demonstrating the coordination of zinc(II) toward amine nitrogen.³⁷ Furthermore, multiplet peaks for aromatic protons undergoes downfield shift, which further confirmed the coordination of ligands with metal center. A new peak detected at 3.49 ppm is assigned to the coordinated water molecules in [(L²)Zn(H₂O)] (Figure S1a,b).³⁸

Figure 1.

¹H NMR spectra of ligands L¹ (a) and L² (b).

The ¹³C NMR results of the free ligands (L¹ and L²) displayed a sharp characteristic peak due to azomethine carbon, which resonates at 161.68 and 160.96 ppm, respectively (Figure 2a,b). The signals observed at 117.97, 121.72, 124.87, 126.23, 126.46, 127.64, 128.69, 134.52, 137.01, and 154.85 ppm may be assigned for naphthalene carbon moieties in the ligand framework while signals at 102.46, 109.39, 111.25, 132.98, 157.65, and 158.28 ppm may correspond to acetophenone carbons, respectively. The signals displayed at 61.42, 64.71, 64.82, 54.47, 65.93, and 66.17 ppm may reasonably be assigned to the aliphatic carbons, and the signals at 18.56 and 20.76 ppm belong to methyl carbon in both ligand frameworks. The characteristic azomethine peaks at 161.68 and 160.96 ppm for L¹ and L², respectively, undergo a downfield shift in both complexes, invoking the coordination via azomethine nitrogen and phenolic oxygen of ligand frameworks with zinc(II).⁷ The other signals mentioned above were also found to be downfield-shifted in both these complexes corresponding to further metalation (Figure S2a,b).

Figure 2.

¹³C NMR spectra of ligands L¹ (a) and L² (b).

2.3. Mass-Spectroscopy

The proposed molecular formulae of the synthesized ligands L¹ and L² and their [(L¹)₂Zn] and [(L²)Zn(H₂O)] complexes are confirmed by the presence of molecular ion peaks at m/z⁺ 260.30, 240.13, 580.95, and 321.49, respectively. The fragmentation of L¹ confirms a medium intensity peak at m/z 170.05 and 89.06, which are due to disintegration of [C₁₁H₈NO] and [C₄H₉O₂] moieties, respectively, while L² peaks appeared at m/z 103.06 and 136.05 because of successive disintegration of [C₄H₉NO₂] and [C₈H₈O₂] moieties from the molecular ion peak (Figure 3a,b). The fragmentation pattern of [(L¹)₂Zn] complex displayed important peaks at m/z 306.09 and 112.11 because of disintegration of [C₂₂H₁₇N₂] and [C₈H₁₆O₂] moieties, whereas [(L²)Zn(H₂O)] complex exhibited peaks at m/z 119.04, 86.05, and 18.01 because of loss of [C₈H₇O], [C₄H₈NO], and [OH₂] moieties from the parent complex (Figure S3a,b). The proposed molecular formulae of [(L¹)₂Zn] and [(L²)Zn(H₂O)] complexes are in accordance with the 1:2 and 1:1 metal-to-ligand ratio, respectively.

Figure 3.

Mass spectra of ligands L¹ (a) and L² (b).

2.4. UV Spectra

The UV–vis spectroscopic studies of ligands (L¹ and L²) and their corresponding [(L¹)₂Cu] and [(L²)Cu(H₂O)] complexes were performed at room temperature in DMSO solution (250–700 nm). The bands observed in the range 305–445 nm and 220–260 nm may correspond to n−π* transition of the imine(azomethine) group and π–π* transition of the aromatic ring of ligand moieties (L¹ and L²), respectively.³⁹ The shifting of the bands to a higher wavelength in the case of copper complexes confirmed the linkage of ligand frameworks to metal ions. The intense bands at λ_max 635 and 615 nm for [(L¹)₂Cu] and [(L²)Cu(H₂O)] complexes may be attributed to ²E_g–T_2g and B_g¹–A_g transition, respectively, signifying an octahedral and square-planar geometry around the Cu(II) ion in the given complexes.^40,41 However, the geometries of both complexes were further confirmed by their observed magnetic moment values of 1.92 [(L¹)₂Cu] and 1.85 BM [(L²)Cu(H₂O)].³¹

2.5. Electron Paramagnetic Resonance

The EPR spectral studies of [(L¹)₂Cu] and [(L²)Cu(H₂O)] complexes were performed in DMSO solvent (Figure S4). The spin Hamiltonian parameter of copper complex assists to unravel the ground state of the metal. For axial octahedral geometry with the g tensor value g_⊥ > g_∥ > 2.0023, the unpaired electron was found in the d_z² orbital, and for g_∥ > g_⊥ > 2.0023, the position of unpaired electron lies in the d_x²–y² orbital in the ground state.³¹ The spectra of both the copper complexes demonstrated an isotropic signal along with the axial symmetrical line with g_∥ = 2.230 and 2.147, g_⊥ = 2.070 and 2.066, respectively. The g_av value was calculated from the expression g_av² = (g_∥2 + 2g_⊥2)/3 and was found to be 2.123 and 2.093, respectively. Here in the [Cu(L¹)₂] complex, the observed value of g_∥ > g_⊥ > 2.003 estimated to form an octahedral geometry around copper(II),⁴² while in the case of [Cu(L²)·H₂O] complex, the value of g_∥ > g_⊥ > 2.003 is suggested to form a square-planar geometry.⁴³ The exchange of interaction is measured by the geometric parameter (G), which can be evaluated through formula, G = g_∥ – 2/g_⊥ – 2 (Kneubuhl’s method).⁷ It is justified in the literature that a minimal exchange interaction if G > 4 and a significant exchange interaction if G < 4 occur between the copper centers.³¹ In our case, the value of G for the copper complexes is 3.285 [(L¹)₂Cu] and 2.227 [(L²)Cu(H₂O)], indicating the significant exchange behavior in the present complexes.

2.6. Thermogravimetric Analysis

The thermal curves of [(L¹)₂Cu] and [(L¹)₂Zn] complexes showed a two-step weight loss, while [(L²)Cu(H₂O)] and [(L²)Zn(H₂O)] complexes showed a three-step weight loss (Figure S5). The first decomposition of [(L¹)₂Cu] and [(L¹)₂Zn] complexes in the temperature range 220–480 °C displayed weight losses (obs = 52.64 and 52.72%, cal = 52.98 and 52.84%) assigned to the disintegration of the naphthalene part of the ligand framework at imine bond, while the second decomposition with weight losses (obs = 24.85, 24.76%, cal = 24.94 and 24.88%) in the temperature range 490–690 °C could be accredited to the loss of residual organic moieties. However, no visible change is observed in the graph of [(L¹)₂Cu] and [(L¹)₂Zn] complexes up to 200 °C, signifying the nonoccurrence of coordinated water moieties.⁴⁴ The initial thermal decomposition in [(L²)Cu(H₂O)] and [(L²)Zn(H₂O)] complexes revealed weight loss (obs = 5.52 and 5.49%, cal = 5.65 and 5.62%) in the temperature range 80–140 °C, corresponding to the coordinated water molecules, while the second disintegration step showed weight loss (obs = 27.23 and 26.70%, cal = 27.00 and 26.88%) in the temperature range 220–350 °C, which may be owing to the decomposition of propanediol moieties of L² ligand at the imine bond. The final disintegration step displayed weight loss (obs = 37.20 and 37.10%, cal = 37.35 and 37.18%) in the temperature range 380–650 °C, corresponding to the loss of the remaining part of organic moieties. Finally, horizontal lines in TGA curves above 700 °C indicates no further weight loss, suggesting the presence of metal oxide as a final residue in all the compounds.⁷ The thermal analysis results are in accordance with the outcomes of elemental analysis.

3. Biological Studies

3.1. DNA Binding

DNA binding analysis is considered to be one of the most crucial factors to examine the feasibility of a number of anticancer drugs as it is the vital transporter of genetic data related to the most cancers occurring via DNA damage.⁴⁵ The binding of metal to DNA could occur by two processes: either via covalent bonding in which the nitrogen base of DNA replaces the labile metal ion or by noncovalent interactions (electrostatic, intercalative, or groove binding of complexes to DNA helix).⁴³ Therefore, the interaction of drug–DNA is vital for the coherent designing and development of new DNA-targeted drugs.

3.1.1. Absorption Interaction Studies

Electronic absorption spectroscopy (EAS) provides a conducive platform to inspect the way by which metal complexes bind with CT-DNA. In general, bathochromic shift, hypsochromic shift, hyperchromic, and hypochromic effect have been detected in the UV–visible spectra after binding to DNA.²⁷ The absorption titration of all complexes (0–60 μM) were carried out (230–350 nm) at a fixed concentration of CT-DNA (60 μM) (Figure 4). After adding a variable concentration of complexes (0–60 μM), the absorption band at 260 nm exhibited enhancement in absorption intensity (hyperchromic) accompanied by a significant red shift (bathochromic shift) of 2–3 nm. Hyperchromism observed in absorption spectra generally occurs because of minor/major groove binding of complexes to DNA, which infers that the unstacking and unwinding of the DNA double helix along with the concomitant exposure of the bases provides numerous hydrogen bonding sites which are easily available for both major and minor groove interactions.⁴³ Thus, the spectral result suggests that the interaction amidst the complexes with CT-DNA either occurs electrostatically or through groove binding interaction.⁴⁶ In our case, the hydroxyl group of naphthyl and acetophenone ring interacts with the DNA base pair via hydrogen bonding. Therefore, the preferable binding mode of the complex to the DNA helix will be groove binding interaction.⁴³ The observed order of hyperchromic effect in complexes was found to be [(L¹)₂Cu] > [(L¹)₂Zn] > [(L²)Cu(H₂O)] > [(L²)Zn(H₂O)], which suggests that the complexes of L¹ framework have comparatively better prospects to become an imperative chemotherapeutic agent than L² complexes because of more aromatic nature, larger planarity, and greater biopotency.

Figure 4.

Absorption profiles of DNA (60 μM) with altering concentration of complexes (0–60 μM) [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d). Arrows show variation in intensity with increasing concentration of complexes.

3.1.2. Steady-State Quenching

A steady-state fluorescence quenching experiment was performed in the range 410–600 nm in order to ascertain the binding susceptibility of all complexes with CT-DNA. The process of energy transfer, excited state reaction, molecular rearrangement, as well as ground-state complex formation is responsible for the phenomena of fluorescence quenching in the fluorescent molecules.⁴⁰ The fluorescence quenching efficiency of complexes was evaluated from the Stern–Volmer equation

F and F₀—fluorescent intensity of molecule with and without quencher, respectively; Q—concentration of quencher; and K_sv—quenching constant.

The results showed that on elevating the CT-DNA concentration (0–60 μM), there is a substantial decline in the fluorescence intensity of each complex devoid of any remarkable shift in the emission wavelength. The quenching pattern observed in the complexes is basically owing to the formation of nonfluorescent adduct of complexes and DNA (Figure 5). To verify the results, the ratio of maximum fluorescent intensity with and without DNA (F₀/F) was calculated against the concentration of DNA, while the slope of this graph gives the value of K_sv, and Figure 6 shows the linear Stern–Volmer plot, which confirms the mode of binding either static or dynamic in nature.⁴⁷ The quenching mechanism was further established by calculating the values of bimolecular quenching rate constant (K_q).

τ₀—average fluorescent life time without a quencher (generally 10^–8 s).

Figure 5.

Intrinsic fluorescence quenching of complexes (60 μM) [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) with varying concentrations of DNA (0–60 μM). Arrow shows changes in intensity with the elevation of DNA concentration.

Figure 6.

Stern–Volmer plots for quenching of intrinsic fluorescence of DNA with [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) complexes.

The values of K_sv and K_q were calculated (Table 2) by using the above equation. Apparently, K_q of all complexes, 2.94 × 10¹¹[(L¹)₂Cu], 2.26 × 10¹¹[(L¹)₂Zn], 1.79 × 10¹¹[(L²)Cu(H₂O)], and 9.80 × 10¹⁰[(L²)Zn(H₂O)], showed greater values than the collision quenching constant of biomolecules (2.0 × 10¹⁰ L mol^–1 s^–1), which unambiguously suggested the static mode of quenching (Figure 7).⁴⁸

Table 2. Quenching Constant and Binding Constant (K_sv and K_b) Values of Complexes with the CT-DNA Interaction System.

complexes	Ksv	Kb	Kq	n
[(L1)2Cu]	2.61 × 103	2.94 × 103	2.94 × 1011	0.9945
[(L1)2Zn]	1.57 × 103	2.26 × 103	2.26 × 1011	0.9999
[(L2)Cu(H2O)]	1.50 × 103	1.79 × 103	1.79 × 1011	0.9914
[(L2)Zn(H2O)]	1.08 × 103	9.80 × 102	9.80 × 1010	0.9986

Figure 7.

Modified Stern–Volmer plots for quenching of intrinsic fluorescence of DNA with [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) complexes.

3.1.3. Fluorescence Interaction (EtBr and Hoechst)

In order to get further insight about the interaction mode of complexes with the CT-DNA, a fluorescence titration experiment was carried out (450–700 nm) taking ethidium bromide (EB) as a fluorescent probe. EB is a measure of intercalation, which can readily form soluble complexes with nucleic acid that emits enhanced fluorescence owing to the intercalation among adjacent base pairs on the double helix of DNA.^8,48 The increased fluorescence intensity of EB gets markedly quenched upon addition of another molecule either by the displacement of the EB or by acquiring the excited-state electron of EB through a photoelectron-transfer mechanism.⁴⁵ Therefore, an EB displacement technique provides a collateral evidence for the modes of DNA binding.¹⁶ In Figure 8, the increased concentration of complexes to DNA-bound EB did not exhibit any change on the fluorescence intensity. These results ruled out the probability of intercalative mode of binding, which further nullifies the feasibility of replacing EB from the DNA–EB adduct. Therefore, the possibility of groove binding mechanism was further checked by using Hoechst displacement studies.⁴⁸

Figure 8.

Emission spectra of DNA–EB adduct with varying concentrations of complexes (0–60 μM) [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d).

The Hoechst displacement experiment was carried out in the range 360–600 nm. Hoechst is a minor groove binder and was known to exhibit weak fluorescence because of quenching by the solvent molecule in the free form. This weak fluorescence intensity of Hoechst increases drastically upon binding with DNA.⁴⁸ The binding of the molecule to the minor groove of DNA leads to the replacement of Hoechst from the DNA–Hoechst system, which is marked by a gradual decrease in fluorescent intensity of the DNA–Hoechst complex.⁴⁷Figure 9 shows that on increasing the concentration of complex to the DNA–Hoechst system, a substantial decline in the fluorescence intensity was seen in all complexes. These results suggested that the mode of binding is minor groove binding, and the complex replaces the Hoechst from Hoechst DNA system in the order [(L¹)₂Cu] > [(L¹)₂Zn] > [(L²)Cu(H₂O)] > [(L²)Zn(H₂O)].

Figure 9.

Emission spectra of DNA–Hoechst system with varying concentrations of complexes (0–60 μM) [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d).

3.2. HSA Binding

3.2.1. UV Absorption

EAS offers a consistent approach to elucidate the structural modification of a protein and its HSA–drug binding capability. The absorption spectrum of HSA exhibited a peak at 220 nm due to which n−π* transition is associated with peptide bond (alpha helix), whereas weak signals at 278 nm correspond to the π–π* transition of aromatic acid (phenyl rings) residues.⁴³ The hypochromic effect in the HSA spectra (Figure 10) was observed upon incremental addition of complex concentration (0–10 μM) to a constant amount of HSA (10 μM). This indicates the exposure of aromatic amino acids residues of HSA in a hydrophobic void with an aqueous environment after binding to the complexes. Also, occurrence of hypochromism in the absorption spectra of HSA with increasing concentration of the complexes infers the role of π–π stacking interactions among the phenyl rings of aromatic acid residues and aromatic rings of the compound.⁴⁹ The observed trend of hypochromic effect is [(L¹)₂Cu] > [(L¹)₂Zn] > [(L²)Cu(H₂O)] > [(L²)Zn(H₂O)], suggesting the greater binding tendency and better conjugation of L¹-based complexes as compared to L² complexes.

Figure 10.

UV–visible absorption spectra of HSA (10 μM) with varying concentrations (0–10 μM) of complexes [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d).

3.2.2. Fluorescence Quenching

HSA occurs abundantly in the plasma and plays an imperative role by regulating the osmotic pressure. It exhibited an outstanding binding property, storage, as well as transportation of many endogenous and exogenous compounds. Therefore, interaction of drugs with HSA helps in better interpretation of protein–drug complex properties. It may also give significant information regarding transportation, absorption, distribution, as well as drug metabolism. Fluorescence spectroscopy imparts an eminent role in elucidating the interactions between the receptor and metal complexes. HSA displays fluorescence owing to the occurrence of these fluorophores (tryptophan, tyrosine, and phenyl alanine residues) among which the tryptophan residue exclusively contributes to the intrinsic fluorescence.⁵⁰ The fluorescence quenching happened to take place when the molecules excited (295 nm) and bind particularly to albumin in the region comprising Trp 214 residue.^51,52 Upon gradual increment in the concentration of complexes (0–10 μM) to a fixed amount of HSA (10 μM), there is a sequential decline in the intrinsic fluorescence intensity of HSA at 340 nm (Figure 11). From the results, it is quite evident that all the complexes significantly show quenching and follows the order-[(L¹)₂Cu] > [(L¹)₂Zn] > [(L²)Cu(H₂O)] > [(L²)Zn(H₂O)]. These quenching outcomes illustrate that the protein-binding affinity of each complex induces a conformational change in HSA as the intramolecular forces associated in maintaining the secondary structure can be modulated together with decreased hydrophobicity, indicating the more exposure of Trp residues to solvent.⁵¹⁻⁵³ Basically, quenching process can be widely categorized into dynamic and static type. In dynamic quenching mechanism, the diffusion includes transfer of energy and molecular collision, which is stimulated by a high range of temperature, while in the case of static quenching, the compound formed by protein and complexes becomes disrupted at higher temperatures. Therefore, both of these quenching phenomena can be differentiated easily by their different temperature dependences. Furthermore, the probable mechanism of HSA in the presence of complex can be elucidated using the Stern–Volmer (S–V) plot

F and F₀—fluorescence intensities of HSA with and without quencher, respectively; K_sv—a Stern–Volmer quenching constant; K_q—quenching rate constant of the biomolecules; τ—average life time of the molecule without a quencher (δ₀ = 10^–8 s); and Q—concentration of the quencher.

Figure 11.

Tryptophan quenching measurement assay of HSA (10 μM) with varying concentrations of complexes (0–10 μM) [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d).

Figure 12 shows the Stern–Volmer plots for quenching of HSA fluorescence, and the values of K_sv and K_b were calculated (Table 3). The value of K_q was found to be greater compared to the limiting diffusion constant of the biomolecules (K_diff = 2.0 × 10¹⁰ M^–1 s^–1), suggesting that the quenching happened owing to the interaction of HSA with a complex which corroborates the static quenching mechanism.⁵⁴ The binding constant (K_b) was calculated using the modified Stern–Volmer equation

F and F₀—fluorescence intensities of HSA with and without quencher, respectively; K_b—binding constant; and n—number of binding sites per molecule of HSA.

Figure 12.

Stern–Volmer plots for quenching of intrinsic fluorescence of HSA with [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) complexes.

Table 3. Quenching Constant and Binding Constant (K_sv, K_b) Values of Complexes with HSA Interaction System.

complexes	Ksv	Kb	Kq	n
[(L1)2Cu]	7.90 × 104	4.62 × 104	4.62 × 1012	0.9963
[(L1)2Zn]	6.26 × 104	1.20 × 104	1.20 × 1012	0.9889
[(L2)Cu(H2O)]	5.01 × 104	5.13 × 103	5.13 × 1011	0.9868
[(L2)Zn(H2O)]	3.74 × 104	1.58 × 103	1.58 × 1011	0.9791

The value of binding constant K_b was evaluated from the intercept of the graph (Figure 13) and was found to be 4.62 × 10⁴[(L¹)₂Cu], 1.20 × 10³[(L¹)₂Zn], 5.13 × 10³[(L²)Cu(H₂O)], and 1.58 × 10³[(L²)Zn(H₂O)] (Table 3). The K_b values of L¹-based complexes were found to be 4.26 × 10⁴ and 1.20 × 10⁴, which are physiologically favorable (10⁴ to 10⁶ M^–1) for any drug carrier in the blood.⁴⁰ These results showed that L¹-based complexes are potential avid binder and interacts with the HSA more firmly as compared to L² complexes, making them a potential bioactive entity for HSA. This striking bioactivity of L¹ complexes was owing to the occurrence of high planarity and extended conjugation of naphthaldehyde ring (π–π conjugation) as compared to acetophenone ring. Moreover, the presence of π–π interaction in L¹-based complexes provides better insight into the mechanism of drug activity⁷ and showed more quenching responses than other complexes. The outcome of the present study revealed that the Cu complex of ligand (L¹) has sound activity than other complexes as it depends on the nature of the metal present in the complex, chelate effect, and the nature of ligand, that is, the complex with the drug as ligand is expected to be more potent. Thus, the importance of metal ion is crucial in conjunction with bioactive ligand scaffold. In literature, a plethora of copper complexes with diverse ligands has been fully reviewed, which show significant biological activity.^51,55,56 Hence, biorelevant metal atoms such as Cu and simultaneous coexistence of potential synergetic effect of bioactive ligand scaffold, here L¹, presented pronounced activity with regard to its structural feature.

Figure 13.

Modified Stern–Volmer plots for quenching of intrinsic fluorescence of HSA with [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) complexes.

3.2.3. Förster Resonance Energy Transfer

FRET is the type of phenomenon based on interaction of excited molecule with its adjacent molecule. The transfer of energy between the complex and HSA can be explained by this theory. It involves transfer of absorbed energy from the donor molecule to the receptor molecule (Figure 14).⁵⁷ It is considered as a nonradiative distance-based process which involves flow of excitation energy from the donor moiety (protein) to the receptor moiety (complex) in the ground state. The emission spectra of donor molecules superimposes with the absorption profile of the acceptor molecule.⁵⁷ According to the FRET theory, the energy transfer pathway is regulated by the mentioned criterion; the distance (must lie under 7 nm) between the donor and acceptor, efficient spectral overlay between the donor (fluorescence emission) and the acceptor (absorption) and the high fluorescence quantum yield of the donor molecule. The energy-transfer (E) value can be evaluated by using the equation⁵⁷

F and F₀—fluorescence intensities (HSA) with and without complex, respectively; r—the distance in between the acceptor and donor molecule; and R₀—critical distance when the efficiency of energy transfer is 50%, which can be evaluated by using equation

K²—orientation factor associated with the dipoles; n—the average refractive index value of medium; ⌀—fluorescence quantum yield of the donor; J—extent of spectral overlap of donor (fluorescence) and acceptor (absorbance).

Figure 14.

FRET of HSA with [(L¹)₂Cu] (a), [(L¹)₂Zn] (b), [(L²)Cu(H₂O)] (c), and [(L²)Zn(H₂O)] (d) complexes.

The value of J can be obtained by using the equation.

F(λ)—donor fluorescence intensity at wavelength (λ), and ε(λ)—molar absorptivity coefficient of the acceptor at wavelength (λ).

Here, ⌀, n, and K² were considered to be 0.118, 1.336, and 2/3, respectively, and the values of E, J, R₀, and r are mentioned in Table 4. In general, the distance between the bound complex and tryptophan residue is less than 7 nm, which undoubtedly deciphers the occurrence of radiationless energy-transfer mechanism for quenching.⁵⁷ Moreover, all the complexes have r values <8 nm and in the range “0.5 R₀ < r < 1.5 R₀”, indicating the transfer of energy from HSA to complexes. Also, the values of r are higher than those of R₀, suggesting the occurrence of static quenching process upon binding.⁵⁸ As evaluated from this study, the magnitude of short distance amid the bound complex and tryptophan residue inferred the significant HSA–complex interaction.

Table 4. FRET Parameters (E, J, R₀, and r) of HSA.

complexes	E	J	R0 (nm)	r (nm)
[(L1)2Cu]	0.155	3.60697 × 10–15	2.074528008	2.752158685
[(L1)2Zn]	0.155	3.60709 × 10–15	2.074539232	2.752173575
[(L2)Cu(H2O)]	0.155	2.42459 × 10–15	1.941639585	2.575863149
[(L2)Zn(H2O)]	0.155	2.23664 × 10–15	1.915703673	2.541455446

3.2.4. HSA Cleavage Activity

To obtain information regarding the capability of [(L¹)₂Cu] complex acting as a photoactivated chemotherapeutic agent, HSA photocleavage assay was performed. The photoinduced cleavage activity was carried out using SDS-PAGE electrophoresis in 10% polyacrylamide gel. From Figure 15a, it is clearly observed that HSA in the absence of [(L¹)₂Cu] complex (lane 1) shows no significant cleavage. However, on substantial increase of [(L¹)₂Cu] complex concentration (100–300 μM), HSA displayed prominent cleavage activity along with remarkable splitting of band (lanes 2, 3, and 4), which further fades away (lanes 5 and 6), suggesting the complete photocleavage upon irradiation (Figure 15a). To further confirm the mechanistic approach of photocleavage, comparative HSA cleavage assay was done in the presence of various hydroxyl radical scavengers (OH^.). It is illustrated well that no significant change was observed in the photocleavage activity of [(L¹)₂Cu] complex (Figure 15b) in the presence of KI and DMSO (lanes 7 and 8), which infers the noninvolvement of singlet oxygen as a reactive species in HSA cleavage. On the other hand, introduction of standard superoxide scavenger [NaN₃, superoxide dismutase (SOD)] leads to the remarkable inhibition of the cleavage activity (lane 9) of [(L¹)₂Cu] complex. This result revealed that the HSA cleavage assay follows the photoinduced oxidative cleavage pathway.⁴³

Figure 15.

SDS-PAGE electrophoresis of [(L¹)₂Cu] complex in Tris–HCl buffer (pH 7.4) (a) at different concentrations; M; standard protein markers; lane 1, HSA control; lane 2, HSA + [Cu(L¹)₂] (100 mM); lane 3, HSA + [(L¹)₂Cu] (150 mM); lane 4, HSA + [(L¹)₂Cu] (200 mM); lane 5, HSA + [(L¹)₂Cu] (250 mM); lane 6, HSA + [(L¹)₂Cu] (300 mM); (b) with different hydroxyl radicals; lane 7, HSA + [(L¹)₂Cu] (300 mM) + KI (3 mM); lane 8, HSA + [(L¹)₂Cu] (300 mM) + DMSO (20 μL); lane 9, HSA + [(L¹)₂Cu] (300 mM) + NaN₃ (3 mM) + SOD (10 units).

4. Docking

To further validate the spectroscopic results and to envisage the desired orientation of the [(L¹)₂Cu] complex, molecular docking study was carried out using the DNA duplex of sequence d(CGCGAATTCGCG)₂ dodecamer (PDB 1D: 1BNA) (Figure 16). The computer-aided molecular modeling techniques give the chiral preference as well as energetically most stable conformation of the docked molecule [(L¹)₂Cu], which fits perfectly in the G–C region of the minor groove DNA target. This optimal minor groove binding facilitates the formation of the least sterically hindered conformation of complex 1 with the DNA.⁴⁵ The resulting docked pose gives the value of binding −239.06 kJ/mol, which further substantiates the potential binding propensity of [(L¹)₂Cu] complex with DNA. The more the negative value of binding energy, the better the binding tendency of the complex will be. Thus, these molecular docking results further compliment the spectroscopic observation. Similarly, the imine-based [(L¹)₂Cu] complex was subjected to docking with another protein sequence target-HSA (PDB ID: 1h9z) (Figure 17). The crystalline study of HSA revealed that it is composed of three homologous domains (I, II, and III): I (residues 1–195), II (196–383), and III (384–585). These three domains along with two subdomains (A and B) assemble together to form a heart-shaped structure.⁵⁹ In HSA, the primary region of binding is located inside the hydrophobic voids of subdomains IIA and IIIA, which correspond to site I and II and tryptophan residue (Trp 214) in subdomain IIA.⁶⁰ The molecular docked model of [(L¹)₂Cu] complex and HSA showed the location of complex in hydrophobic cavity of IIA domain of HSA and one-half is submerged in the adjacent hydrophobic residue Glu 292, Val 293, Cys 289, Glu 294, and Ala 291 of subdomain IIA of HSA, inferring the occurrence of hydrophobic interactions among them (Figure 17). Thus, the result provides an apt confirmation to substantiate the efficient fluorescence quenching of HSA emission by the [(L¹)₂Cu] complex. Additionally, there are electrostatic interaction and hydrogen bonding between various polar and ion groups in the vicinity of the molecule such as ARG 117, ARG 186, ILE 142, and TYR 161. These interactions further stabilize the molecule. Therefore, the results of molecular docking give insights into the mode of binding of the compound with DNA and HSA along with the conformational constraints for complex formation.

Figure 16.

Docked pose model of [(L¹)₂Cu] complex with the DNA dodecamer duplex of sequence d(CGCGAATTCGCG)₂ (PDB ID: 1BNA).

Figure 17.

Docked pose model of [(L¹)₂Cu] located in hydrophobic cavity in subdomain IIA of HSA (PDB ID: 1h9z).

5. Antioxidant Activity

Schiff base compounds are known to significant antioxidant activity.⁶¹ ROS produced various biochemical processes which impart deleterious effects on human health.⁴⁰ Specifically, imines having ortho hydroxy groups acted effectively in scavenging the free radicals and lead to the development of biopotential and effective drugs.⁷ The scavenging effect of compounds was explored to investigate the antioxidant behavior by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and SOD mimetics under standard reaction conditions using ascorbic acid as a control. The data of DPPH and SOD mimetics at varying concentrations (0–200 μM) are given in Table 5 (Figure 18).

Table 5. Antioxidant Activity by DPPH and SOD Mimetics.

	IC50 (μM)
compounds	DPPH assay	SOD mimetics
control	37.5	40.14
[(L1)2Cu]	69	82.25
[(L1)2Zn]	91.3	96.33
[(L2)Cu(H2O)]	107.4	108.43
[(L2)Zn(H2O)]	123.7	119.22
L1	142.8	127.13
L2	155.2	153.67

Figure 18.

Antioxidant activity of ligands and their metal complexes by (1) DPPH and (2) SOD: ascorbic acid (a), [(L¹)₂Cu] (b), [(L¹)₂Zn] (c), [(L²)Cu(H₂O)] (d), [(L²)Zn(H₂O)] (e), L¹ (f), and L² (g).

5.1. Scavenging Activity by DPPH

The DPPH radical scavenging activity was calculated in terms of IC₅₀ and occurs basically because of the hydrogen/electron donating ability of DPPH. The liberation of hydrogen leads to a color change from purple to yellow. The scavenging data revealed that L¹-based complexes have more pronounced antioxidant activity than L². The results also suggested that chelated metal complexes are more efficient scavengers than their respective free ligands. The redox attributes and the coordination environment of complexes are the two important factors responsible for the variation in the scavenging activities.¹⁶ It is given that redox properties depend on various criteria, namely, extent of unsaturation in the chelate ring, size of chelate ring, and axial ligation.⁶² The enhanced scavenging performance of complexes is due to the occurrence of azomethine group, which liberates H⁺ easily upon binding.¹⁶ A number of reports have appeared in literature where the metal complexes have been shown to act as a better antioxidant in comparison to ligands. The comparative antioxidant activity has been explained in terms of chelation effect of imines as well as the influence of metal ions.⁶³⁻⁶⁵

5.2. SOD Mimetics

The eradication of superoxide anion(O₂^–) and hydroxyl radical(OH^–) is extremely important as they give rise to the number of diseases.⁴⁰ It is earlier reported in literature that complexes possessing O and N donor bidentate ligands have better reactivity for dioxygen.⁶⁶ Therefore, scavenging effects on superoxide anion radical were carried out in terms of IC₅₀ (Table 5). The obtained data suggested that complexes exhibited superior scavenging effect in comparison to their respective imine moieties [(L¹)₂Cu] > [(L¹)₂Zn] > [(L²)Cu(H₂O)] > [(L²)Zn(H₂O)].

6. Conclusions

In the pursuit of designing biocompatible chemotherapeutic drug entities, we have synthesized four imine-based monometallic complexes {[(L¹)₂Cu], [(L¹)₂Zn], [(L²)Cu(H₂O)], and [(L²)Zn(H₂O)]} and systematically characterized by spectral and thermal techniques. The comparative binding affinities of all the synthesized complexes with CT-DNA and HSA were performed using biophysical studies (absorption and emission). The relative results of DNA/HSA binding studies suggested that L¹ acted as a superior avid binder as its complexes have demonstrated higher biopotency than L²-based complexes. The photocleavage assay also revealed that [(L¹)₂Cu] complex exhibited appreciable photocleavage activity, which makes it a potential photoactivated chemotherapeutic agent. Moreover, the software-assisted molecular docking study (DNA/HSA) further validates the effective binding of the [(L¹)₂Cu] complex to the G–C-rich minor groove of DNA helix and hydrophobic cavity of IIA domain of HSA. The targeted approach (hybrid pharmacophore) of our work involves successful intervention of biologically active metal ions into the domain of biorelevant pharmacophores (L¹ and L²), which can be explored for in vivo applications as chemotherapeutic in future.

7. Experiment

7.1. Materials and Measurement

2-Amino-2-methyl-1,3-propanediol, 2,4-dihydroxyacetophenone (Sigma-Aldrich), and 3-hydroxy-2-naphthaldehyde were synthesized according to the reported protocol.⁶⁷ EB, disodium salt of calf thymus DNA(CT-DNA) (stored as 4 °C. 6× loading dye), Hoechst 33258, HSA (fatty acid free, 99%), agarose gel, SOD, sodium azide (NaN₃), ascorbic acid, DMSO, and KI were acquired from Sigma-Aldrich. There was no further purification of the chemicals and solvents utilized for all the experiments and synthesis. A PerkinElmer 2400 elemental analyzer was utilized for the microanalyses of compounds, while a Systronic type 302 conductivity bridge equilibrated at 25 ± 0.01 °C was used to record the molar conductivity data of imine complexes (10^–4 M/DMSO) at room temperature. The infrared spectra (4000–400 cm^–1, KBr pellets) were analyzed using a PerkinElmer-2400 spectrometer.⁶⁸ A Bruker AVANCE 11 400 NMR spectrometer and a WATERS Q-TOF chief mass spectrometer were used to analyze ¹H NMR, ¹³C NMR, and mass spectra. EPR was recorded on a ES-DVT4 spectrometer (9.167 GHz, DPPH as a standard, g = 2.0036). Furthermore, metal ions were evaluated according to the given standard protocol.⁴⁰ A PerkinElmer lambda 40 UV–vis spectrophotometer (800–200 nm, a quartz cuvette of path length 1 cm) was used to record the electronic spectra of compounds at room temperature, whereas Faraday balance was used at room temperature to record the magnetic moment of complexes. TGA was performed on a Shimadzu Thermal Analyzer up to 800 °C with alumina as the reference at a heating rate of 20 °C min^–1.^69,70

7.2. Synthesis

7.2.1. Synthesis of L¹, 2-(((3-Hydroxynaphthalen-2-yl)methylene)amino)-2-methylpropane-1,3-diol

A methanolic solution (25 mL) of 2-amino-2-methyl-1,3-propanediol (2 mmol) was gradually added to the methanolic solution of 3-hydroxy-2-naphthaldehyde (25 mL, 2 mmol). The solution was stirred at 70–80 °C and then subjected to reflux with constant stirring for 5–6 h. After continuous stirring, a deep yellow-colored precipitate was obtained and then filtered. The obtained product was washed with methanol several times and dried in vacuo.

7.2.2. Synthesis of L², 4-(1-((1,3-Dihydroxy-2-methylpropane-2-yl)imino)ethyl)benzene-1,3-diol

A methanolic solution (25 mL) of 2-amino-2 methyl propanediol (2 mmol) was gradually added to another 25 mL methanolic solution of 2,4 dihydroxy acetophenone (2 mmol). The solution was stirred at 70–80 °C and then subjected to reflux with constant stirring for 5–6 h. After continuous stirring, a dark brown-colored precipitate was obtained and then filtered. The obtained product was washed with methanol and dried in vacuo.

7.2.3. Synthesis of [(L¹)₂Cu] and [(L¹)₂Zn] Complexes

A methanolic solution (20 mL) of L¹ was gradually added to another 20 mL methanolic solution of copper nitrate (1 mmol) and zinc nitrate (1 mmol). The solution was initially stirred at 70–80 °C and then subjected to reflux with constant stirring for 5–6 h. After continuous stirring, a dark brown and green-colored precipitate was obtained for [(L¹)₂Cu] and [(L¹)₂Zn], respectively, and then filtered. The obtained product was washed with methanol and dried in vacuo (Scheme 1).

Scheme 1. Schematic Illustration of the Proposed Imine-Based Ligand (L¹) and Its Complexes [(L¹)₂Cu] and [(L¹)₂Zn].

7.2.4. Synthesis of [(L²)Cu(H₂O)] and [Zn(L²)(H₂O)] Complexes

The synthetic procedure for [(L²)Cu(H₂O)] and [(L²)Zn(H₂O)] complexes was the same as mentioned above for [(L¹)₂Cu] and [(L¹)₂Zn] complexes only with the difference of L² (1 mmol) in the place of L¹. In this case, we obtained a black and brown-colored precipitate, which was then filtered. The obtained product was washed with methanol and finally dried in vacuo (Scheme 2).

Scheme 2. Schematic Illustration of the Proposed Imine-Based Ligand (L²) and Its Complexes [(L²)Cu(H₂O)] and [(L²)Zn(H₂O)].

7.3. DNA Binding

The investigation on binding interaction of CT-DNA with the synthesized complexes was conducted in Tris–HCl buffer (10 mM), pH 7.2, using UV absorbance at 260 and 280 nm, which provided a ratio of about 1:1.8, to get an idea of the presence of CT-DNA free from protein. Absorption spectroscopy at 260 nm was employed to unravel the CT-DNA concentration per nucleotide, taking molar extinction coefficient of 6600 M^–1 cm^–1 (after 1:100 dilution).⁷¹ The aliquots of CT-DNA were incubated at 4 °C for further use in experimental assays.

7.3.1. UV–Visible Interaction

The absorption titration was performed using a UV–1800 Shimadzu spectrophotometer in the wavelength range of 230–350 nm at a fixed amount of CT-DNA (60 μM) while altering the concentrations of the complexes in the range of 0–60 μM. In order to eliminate the noise obtained because of the absorbance of CT-DNA, an equal amount of CT-DNA was added to the control as well as complex solution during titration.

7.3.2. Fluorescence Quenching Interaction

The fluorescence quenching mechanism was performed by titration using a Shimadzu spectrophotometer-5301PC assembled with a constant temperature holder. This holder was connected to the Neslab RTE-110 water bath having a precision of ±0.1 °C. The excitation wavelength of the complexes was set at 390, 390, 320, and 322 nm for [(L¹)₂Cu], [(L¹)₂Zn], [(L²)Cu(H₂O)], and [(L²)Zn(H₂O)] complexes, respectively, while their emission spectra were obtained in the range 300–600 nm. The slit widths were set to 10 nm each. The fluorescence quenching experiment was performed by taking a constant concentration (60 μM) of complexes and variable concentrations of CT-DNA (0–60 μM). Each time, 20 μL was added to avoid any change in volume.

7.3.3. Displacement Assay

A Shimadzu model RF-5301 spectrofluorometer was employed to investigate the interactions of complexes to dye-bound CT-DNA in Tris-HCl buffer (10 Mm), pH 7.2. Two dyes were used for this purpose (EB, Hoechst). The emission and excitation wavelength for each dye was set distinctly while measuring the intrinsic fluorescence. The emission wavelength was recorded in the range of 520–700 nm while the excitation wavelength was set at 476 nm for the EB–DNA complex. In the case of Hoechst 33258-DNA complex, the excitation spectra were set at 343 nm, and the emission was obtained in the range of 375–600 nm. The concentration of complexes was altered from 0 to 60 μM, whereas DNA concentration (60 μM) was kept fixed during the displacement assays.

7.4. HSA Binding

The stock solution of HSA was made by dissolving 20 mg of HSA in 1 mL of 100 mM phosphate buffer at pH 7.4. The concentration (HSA) was determined at 278 nm by UV–vis spectroscopy, taking the molar extinction coefficient as 35,700 M^–1 cm^–1.⁴⁹ The complexes were also dissolved in 100 mM phosphate-buffered saline (pH 7.4).

7.4.1. UV–Visible Interaction

Spectrophotometric analysis of HSA was performed on a UV–1800 Shimadzu spectrophotometer in a cuvette of 1 cm path length. The absorption was measured taking different amounts of complexes (0–10 μM) and a constant concentration of HSA (10 μM) in 100 mM phosphate buffer (pH 7.4) within the range of 240–340 nm.

7.4.2. Fluorescence Quenching Interaction

Fluorescence experiments were performed on a Shimadzu spectroflurometer-5301 assembled with fixed temperature control via fluorometric titration. The excitation spectra were recorded at 295 nm, while the emission spectra were obtained (310–450 nm) on a dual-path length fluorescence. The slit widths were fixed to 10 nm for recording both spectra. Afterward, fluorescence titration studies were performed by taking a fixed content of HSA (10 μM) against variable concentrations (0–10 μM) of complexes. Each time, 10 μL was added to avoid any change in volume.

7.4.3. Förster Resonance Energy Transfer

Similarly, the absorption spectra of complexes and the HSA were determined as mentioned in the absorption and emission section (300–400 nm). The energy transfer depends upon the efficient overlap between the donor (HSA) and acceptor (complex) spectra and the Förster distance between them.

7.4.4. HSA Cleavage

The HSA photocleavage performance was assessed via SDS-PAGE electrophoresis (10% polyacrylamide gel) to assess the capability of the [(L¹)₂Cu] complex to function as a synthetic metalloprotease. The photoexposure to UV-A1 light (at 365 nm and for 25 min) by[(L¹)₂Cu] complex led to the photocleavage of HSA (15 μM) in Tris–HCl buffer, pH 7.4, at the various concentrations (0–300 μM).⁴³

7.5. Docking

The molecular docking was carried via HEX 6.1 software.⁵⁹ The structure of the synthesized [(L¹)₂Cu] complex was illustrated with the help of ChemDraw 12.0 software and adapted to pdb setup employing Mercury software (htttp://www.ccdc.cam.ac.uk/). The structure of the B-DNA dodecamer d(CGCGAATTCGCG)₂ (PDB ID: 1BNA) and HSA (PDB ID: 1h9z) was acquired from the protein data bank. The docked pose was visualized using Discovery Studio molecular graphic program and CHIMERA.

7.6. Antioxidant Activity

The synthetic procedure is described in the Supporting Information (7.6.).^72,73

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03762.

• Antioxidant activity (DPPH assay and SOD mimetic), ¹H NMR, ¹³C NMR, and mass spectra of Zn complexes, EPR spectra of Cu complexes, and TGA plots of complexes (PDF)

The authors declare no competing financial interest.