Depicting the DNA Binding and Cytotoxicity Studies against Human Colorectal Cancer of Aquabis (1-Formyl-2-Naphtholato- k 2 O,O (cid:48) ) Copper(II): A Biophysical and Molecular Docking Perspective

: In this study, we attempted to examine the biological activity of the copper(II)–based small molecule aquabis (1-formyl-2-naphtholato- k 2 O,O (cid:48) )copper(II) (1) against colon cancer. The characterization of complex 1 was established by analytical and spectral methods in accordance with the single-crystal X-ray results. A monomeric unit of complex 1 exists in an O 4 (H 2 O) coordination environment with slightly distorted square pyramidal geometry ( τ = ~0.1). The interaction of complex 1 with calf thymus DNA (ctDNA) was determined by employing various biophysical techniques, which revealed that complex 1 binds to ctDNA at the minor groove with a binding constant of 2.38 × 10 5 M –1 . The cytotoxicity of complex 1 towards human colorectal cell line (HCT116) was evaluated by the MTT assay, which showed an IC 50 value of 11.6 µ M after treatment with complex 1 for 24 h. Furthermore, the apoptotic effect induced by complex 1 was validated by DNA fragmentation pattern, which clariﬁed that apoptosis might be regulated through the mitochondrial-mediated production of reactive oxygen species (ROS) causing DNA damage pathway. Additionally, molecular docking was also carried out to conﬁrm the recognition of complex 1 at the minor groove.


Introduction
Regardless of the significant advancement in the treatment of colon cancer, its frequency is steadily rising, and it has become one of the leading causes of death worldwide [1][2][3]. It is often recognized that the development of cancer is linked with diverse genetic competencies attained throughout the growth of the tumour [4]. Apart from conventional chemotherapy based on organic and platinum-based drugs, other treatment regimes such as surgery, radiation, and immunotherapy therapy are also available to treat colon cancer [5][6][7][8]. Therefore, great efforts have been made to search for new metal-based agents to improve and optimize the action.
Recently, a remarkable study on small molecules binding with nucleic acids (DNA and RNA), which displayed significant impression owing to their vital roles in molecular biology, was carried out [9][10][11]. Several other cellular constituents have also been established as potential targets for metal-based drugs [12,13]. In particular, metal-based enzyme and protein inhibition plays a crucial role in metabolic pathways associated with cancer, which present common targets for the design of metal-based drugs [14,15]. Literature reports revealed that DNA remains a versatile target for metal-based therapeutic agents, and it has been examined by several factors in terms of the coordination environment and the nature of the organic frameworks [16,17]. Of all the transition metal complexes, the Cu(II) metal ion is the most widely studied and known to play a significant role in cancer research because some genetic pathways require a higher level of copper during cancer progression than can be found in normal tissues [18,19].
Hence, it is imperative to that we understand the DNA binding of the copper complex and its possible relationship to cytotoxicity in tumour cell lines [3]. Copper(II) complexes can identify DNA via non-covalent binding modes such as intercalation, groove, and external electrostatic contacts, leading to significant distortion in its structure and the proper biological function [20,21]. Intercalative binding, which is facilitated through welltailored aromatic structures able to stabilize the π-π interactions, is considered to be the most effective mode for DNA targeting drugs [22,23]. At the same time, in copper(II) complexes, the number of single ligands acts in a bidentate manner in combination with a fourth monodentate labile one, which enables the complex to covalently bind with the nucleobases or the phosphate backbone or other interactions with cellular targets, thereby exerting their cytotoxicity [24,25]. Previous results show that the copper(II) complexes contain a labile ligand substituted mainly by an N7 guanine nitrogen base of DNA [26][27][28].
In this scenario, copper(II) complexes are of specific concern, because of their involvement in respiration, energy metabolism, and capability of interacting with DNA [21,29]. It is evident that the cellular uptake of copper(II) complexes occurs largely through passive diffusion without any involvement with the human copper transporter1 protein (hCtr1), while free copper(II) ion requires hCtr1 [30][31][32]. This indicates that ligands play an important character in tempering its permeability by means of their lipophilicity, changing the redox properties of the metal ion and displaying intrinsic cytotoxicity [33,34]. Meanwhile, DNA damage induced by copper(II) complexes also depends on the ligand planarity, which promotes the diversification of the complex structures to enhance the binding propensity with DNA and influences the Cu 2+ -Cu + redox couples [35,36]. Thus, the combined effect of the metal ion and the ligand has a noticeable effect on the biological membranes as well as the DNA binding propensity.
Herein, we extend our discussion about analytical and spectroscopic characterization, topological analysis, and the Hirshfeld surface analysis of copper(II) complex (1) reported earlier [43]. Exploiting different biophysical methods, we have successfully shown that complex 1 avidly binds to a DNA minor groove. The binding propensity was further confirmed by molecular docking results. Furthermore, cytotoxicity towards the human colorectal cell line (HCT116) was evaluated by an MTT assay, and cellular uptake was monitored by fluorescence microscopy.

Synthesis of Aquabis
The copper(II) complex (1) was obtained by the slight alteration of the procedure mentioned earlier [43]. A solution of Cu(II) acetate monohydrate (0.199 g, 0.93 mmol) was mixed into a stirred methanolic solution (20 mL) of 2-hydroxy-1-naphthaldehyde (0.172 g, 1 mmol). The resulting mixture was refluxed at 80 • C for 2 h to obtain olive green colour solution. Slow evaporation of the clear solution led to block-shaped green colour crystals, which were collected after couple of days.

Crystallography
The details of single-crystal measurement have been given in supporting information (See the Supplementary Materials).

DNA Binding Studies
ctDNA was procured from Sigma-Aldrich (St. Louis, MO, USA) and kept at 4 • C. The purity ctDNA was assured by the absorbance ratio at A 260 /A 280 between~1.8 and 1.9. The concentration of ctDNA (ε 260 = 6600 cm −1 M −1 ) was determined from absorption spectroscopy [44].
Change in absorption pattern of a fixed amount of the complex 1 (60 µM) was monitored in presence of varying DNA concentrations. The binding constant (K b ) was calculated by employing the Wolf-Shimmer Equation (1) [45]; where [DNA] is the concentration of DNA, ε a , ε f , and ε b are the extinction coefficients of apparent, free, and bound metal complex, respectively. A plot of [DNA]/(ε a −ε f ) versus [DNA] provides K b is the slope to the intercept ratio. Ethidium bromide (EB) competitive ability of increasing amount of complex towards ctDNA was assessed for the solution of almost fully bound EB (20 µM) to DNA (100 µM) in aqueous buffer. The increasing concentration of 1 directly impacted the emission profile of EB-DNA system [16]. Stern-Volmer quenching constant (K SV ) can determined from the graph of F 0 /F verses [Q], where F 0 and F are the are the fluorescence intensities in the absence and the presence of the complex 1 as a quencher [Q], respectively [46].
Ostwald viscometer was used to monitor the change in specific viscosity of ctDNA (30 µM) at room temperature. The flow time inside the viscometer was recorded thrice, and the average values were used for obtaining the corresponding relative viscosity. The plot of (η/η 0 ) 1/3 versus r ([complex 1]/[ctDNA]; r = 0.0, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0) was obtained, where η and η 0 represent the viscosity of DNA in the presence and absence of complex 1, respectively [44]. Thermal denaturation was conducted on a Shimadzu Pharmaspec 1700 unit equipped with the Peltier controller (TMSPC-8) (Shimadzu, Kyoto, Japan). Absorbance versus temperature plot of DNA alone and complex 1-DNA system was detected with a rising temperature at a fixed wavelength (260 nm).

Cytotoxicity Studies
Studies related to cell line examinations (cell culture, MTT assay, quantification of apoptosis, and determination of mitochondrial ROS) are given in supporting information (See the Supplementary Materials).

Molecular Docking
Molecular docking of complex 1 with double-helical DNA was performed using AutoDock vina [47]. This docking program has been documented to carry out more accurate parameters than AutoDock within a shorter time [48,49]. Structure of DNA [PDB: 1BNA] was retrieved from RCSB Protein Data Bank. The water molecules from the structure were removed using AutoDock tools 1.5.6. Non-polar hydrogens were incorporated into the receptor molecules. The grid spacing was fixed at 1 Å, and details of grid are given in Supplementary Materials, Table S1. The PDB structure of complex 1 was converted from cif file using Mercury crystallographic software version 4.2. The post-docking analysis was performed using Discovery studio visualizer.

Synthesis and Characterization
The present investigation aims to ascertain the effect of Cu(II) complex bearing naphthalene rings on DNA binding events and in vitro anticancer activity towards colorectal cancer. The proposed geometry of complex 1 (Scheme 1) was proven by microanalytical, TGA, PXRD, FTIR, ToF-MS techniques, and electronic spectral studies in accordance with the single-crystal analysis.
Ostwald viscometer was used to monitor the change in specific viscosity of ctDNA (30 μM) at room temperature. The flow time inside the viscometer was recorded thrice, and the average values were used for obtaining the corresponding relative viscosity. The plot of (η/η0) 1/3 versus r ([complex 1]/[ctDNA]; r = 0.0, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0) was obtained, where η and η0 represent the viscosity of DNA in the presence and absence of complex 1, respectively [44].
Thermal denaturation was conducted on a Shimadzu Pharmaspec 1700 unit equipped with the Peltier controller (TMSPC-8) (Shimadzu, Kyoto, Japan). Absorbance versus temperature plot of DNA alone and complex 1-DNA system was detected with a rising temperature at a fixed wavelength (260 nm).

Cytotoxicity Studies
Studies related to cell line examinations (cell culture, MTT assay, quantification of apoptosis, and determination of mitochondrial ROS) are given in supporting information (See the Supplementary Materials).

Molecular Docking
Molecular docking of complex 1 with double-helical DNA was performed using Au-toDock vina [47]. This docking program has been documented to carry out more accurate parameters than AutoDock within a shorter time [48,49]. Structure of DNA [PDB: 1BNA] was retrieved from RCSB Protein Data Bank. The water molecules from the structure were removed using AutoDock tools 1.5.6. Non-polar hydrogens were incorporated into the receptor molecules. The grid spacing was fixed at 1 Å, and details of grid are given in Supplementary Materials, Table S1. The PDB structure of complex 1 was converted from cif file using Mercury crystallographic software version 4.2. The post-docking analysis was performed using Discovery studio visualizer.

Synthesis and Characterization
The present investigation aims to ascertain the effect of Cu(II) complex bearing naphthalene rings on DNA binding events and in vitro anticancer activity towards colorectal cancer. The proposed geometry of complex 1 (Scheme 1) was proven by microanalytical, TGA, PXRD, FTIR, ToF-MS techniques, and electronic spectral studies in accordance with the single-crystal analysis. Complex 1 displayed the characteristic ʋ (C-H) stretching vibration at 3100-3000 cm −1 due to presence of the aromatic ring. A medium intensity envelope at ⁓3400 cm −1 specified the coordinated water molecule, which was further confirmed by the presence of the rocking mode of water at 745 cm −1 [50]. The ligand exhibited stretching frequency due to carbaldehyde ʋ (HC=O) group and υ (C-O) at 1157 cm −1 and 1600 cm −1 moving to 1184 and 1620 cm −1 after complexation, indicating the coordination of the carbaldehyde and naphtholate oxygen at the metal centre [51,52]. The bands appearing at 980-838 cm −1 may correspond to the ring breathing mode, C-H, and C-C deformation. The band at 586 cm −1 Scheme 1. Synthesis of complex 1.
Complex 1 displayed the characteristic V (C-H) stretching vibration at 3100-3000 cm −1 due to presence of the aromatic ring. A medium intensity envelope at~3400 cm −1 specified the coordinated water molecule, which was further confirmed by the presence of the rocking mode of water at 745 cm −1 [50]. The ligand exhibited stretching frequency due to carbaldehyde V (HC=O) group and υ (C-O) at 1157 cm −1 and 1600 cm −1 moving to 1184 and 1620 cm −1 after complexation, indicating the coordination of the carbaldehyde and naphtholate oxygen at the metal centre [51,52]. The bands appearing at 980-838 cm −1 may correspond to the ring breathing mode, C-H, and C-C deformation. The band at 586 cm −1 because of the stretching vibration of V (Cu-O) further confirms the Cu 2+ ion coordination in a bidentate manner via naphtholate and carbonyl oxygen (Supplementary Materials, Figure S1).
The electronic spectrum of complex 1 in DMSO at room temperature has been provided. (Supplementary Materials, Figure S2). As can be seen, there are three obvious absorption bands in the spectrum. The bands at 230 and 314 nm can be assigned to the π−→ π * and n−→ π * transitions of the benzene rings, respectively. The broadening at 416 nm indicates ligand-to metal charge transfer (LMCT) transition, most probably from the naphtholate oxygen atoms [53]. However, the d-d band was observed at 595 nm, similar to other reported copper(II) ions (d 9 electronic configuration) in a distorted square pyramidal manner [54].
To examine the thermal stability of complex 1, thermogravimetric analysis (TGA) was performed (Supplementary Materials, Figure S4). The complex started to decompose after 260 • C. The thermogram of complex 1 showed single-step decomposition corresponding to the loss of two molecules of the ligand along with one coordinated water molecule at 310 • C (found: 85.06%; calc: 85.48%). This kind of single-stage pyrolysis of the complexes usually occurs because of electron delocalization as a result of regularity in the bond strength along a conjugated system. The final product of the mixture of CuO and C is produced at temperatures above 800 • C.
Powder-XRD analysis of complex 1 confirmed the bulk phase purity of the sample (Supplementary Materials, Figure S5). Nevertheless, some diffraction peaks of small intensity were detected due to the different orientation of the crystalline sample.

Structure Description
Crystal X-ray analysis revealed complex 1 contains a Cu(II) centre and crystalizes in a monoclinic P2 1 /c space group ( Table 1). The asymmetric unit of 1 involves two L1 ligands, one molecule of water, and a Cu(II) ion, as shown in     Table S3.  To obtain the involvement of even weak interactions existing in complex 1, we also performed topological analysis, which revealed the coordination formula as AB 01 2M 1 [55], where B 01 is C11H7O2 and M 1 is H2O. On the basis of a standard representation of valencebonded MOFs (Supplementary Materials, Figure S6 Figure S8). [010]. In the course of the simplification method based on H-and vdW-bonded molecular MOFs, one can find an account of the molecular packing. The calculation results expose that the underlying net is characterized by the 13T5 topological type (Supplementary Materials, Figure S8).
Various subnets can be acquired from the underlying net (contain edges of a weight no less than a specified value) by applying subroutine implemented in ToposPro. On the basis of a multilevel investigation [56], the following order of the subnets can describe the packing of the structure on different levels of the minimal solid angle (Ωi) (Supplementary Materials, Table S5). The formation of the 13-c net depends on the minimal solid angle value (Supplementary Materials, Figure S9).

Hirshfeld Surface Analysis
To quantitatively explore intermolecular interactions within the crystal structure, Hirshfeld surface (HF) analysis was performed [57]. HF surfaces can be utilized by generating three-dimensional images of d norm , curvedness, and shape index, while the associated two-dimensional fingerprint plot provides quantitative images of the involved close interactions [58][59][60]. The HF analysis of complex 1 was mapped with d norm , shape index, and curvedness in a range from −0.213 to 0.887, −1.0 to 1.0, and −4.0 to 0.40E, respectively (Supplementary Materials, Figure S10). The red, white, and blue colours in d norm showed the distance as shorter, equal to, and longer than the total of vdW separation, respectively. In Figure S10a

Absorption Titrations
Electronic absorption is an important tool for examining the drug-DNA interaction [61,62]. Figure 3 shows the absorption spectra of the complex 1 in the absence and presence of different amounts of ctDNA. Upon addition of ctDNA to the buffered solution of complex 1, the absorbance of these two bands at 314 and 416 nm decreased without any shift in wavelength maxima (λ max ). A small drop in absorbance and no alterations in λ max position were associated with groove binding [63]. The significant decrease in absorbance (hypochromism) of complex 1 in the presence of DNA without any shift suggests that 1 binds avidly with ctDNA via groove-binding mode. The presence of hypochromism (22%) may indicate the DNA complex stabilization due to π-π* stacking occurs between the aromatic naphthalene rings, which approaches partial intercalation of the base moiety through non-covalent interaction [64].
The binding strength of complex 1 was quantitatively determined by calculating the intrinsic binding constant K b , and it was found to be 2.38 × 10 5 M -1 . The K b value of the complex was smaller than that reported by classical intercalators [65] and comparable with the other Cu(II) complexes [61,66,67].
shift in wavelength maxima (λmax). A small drop in absorbance and no alterations in λmax position were associated with groove binding [63]. The significant decrease in absorbance (hypochromism) of complex 1 in the presence of DNA without any shift suggests that 1 binds avidly with ctDNA via groove-binding mode. The presence of hypochromism (22%) may indicate the DNA complex stabilization due to π-π* stacking occurs between the aromatic naphthalene rings, which approaches partial intercalation of the base moiety through non-covalent interaction [64]. The binding strength of complex 1 was quantitatively determined by calculating the intrinsic binding constant Kb, and it was found to be 2.38 × 10 5 M -1 . The Kb value of the complex was smaller than that reported by classical intercalators [65] and comparable with the other Cu(II) complexes [61,66,67].

Ethidium Bromide Displacement
Fluorescence quenching experiments were carried out to assess the relative binding affinity of complex 1 to ctDNA with respect to ethidium bromide (EB) as a probe. Upon excitation at 510 nm, the conjugated planar EB had weak fluorescence in the aqueous buffer due to the solvent quenching effect. However, the emission intensity of EB increased at a higher DNA concentration (100 μM), where the alteration of fluorescence intensity was minimal. This indicates that EB completely stacked onto double-stranded DNA. The addition of another DNA-binding molecule quenched the EB emission by replacing DNA-bound EB [68]. The emission spectrum of EB bound to DNA in the absence and presence of complex 1 is shown in Figure 4. It is evident from Figure 4

Ethidium Bromide Displacement
Fluorescence quenching experiments were carried out to assess the relative binding affinity of complex 1 to ctDNA with respect to ethidium bromide (EB) as a probe. Upon excitation at 510 nm, the conjugated planar EB had weak fluorescence in the aqueous buffer due to the solvent quenching effect. However, the emission intensity of EB increased at a higher DNA concentration (100 µM), where the alteration of fluorescence intensity was minimal. This indicates that EB completely stacked onto double-stranded DNA. The addition of another DNA-binding molecule quenched the EB emission by replacing DNA-bound EB [68]. The emission spectrum of EB bound to DNA in the absence and presence of complex 1 is shown in Figure 4. It is evident from Figure 4 that increasing concentrations of complex 1 to the EB-DNA system caused obvious quenching (ca. 20%) of the emission maxima. These findings indicate that the DNA-bound EB is partially substituted by complex 1, leading to partial intercalation into the DNA minor groove.   The K sv determined using Equation (2) was found to be 4.10 × 10 5 M −1 , which further confirms the outcomes obtained from absorption titrations. The apparent DNA-binding constant (K app ) was obtained from the following equation: (3)

UV Optical Melting
A melting experiment was performed to predict the mode and relative binding strength of the metal complex to DNA [69]. Upon increasing the temperature of the DNA solution, hydrogen bonding of the DNA double helix was affected, resulting in the disruption of the helical structure. The damage which caused the transformation of ds-DNA into ss-DNA can be distinguished as the "hyperchromic effect" [70]. The range of ∆Tm is the midpoint between the values triggered by different binding modes. Only a subtle change (1-3 • C) is observed in the case of groove binding or electrostatic mode of binding, while a high value is suggestive of an intercalative mode of binding (5-6 • C). With this consideration, the melting curve of ct-DNA (70 µM) in the absence and presence of complex 1 (10 µM) are presented in Figure 5. The melting temperature of free ctDNA was estimated to be 71.3 ± 0.3 • C. The addition of complex 1 to the DNA solution led to 74.6 ± 0.3 • C ( Figure 5).
The ∆Tm values of DNA in the presence of complex 1 (3.3 • C) suggest that the complex binds to the DNA groove.

Viscosity Measurements
The viscosity measurement of DNA is the most reliable tool to explain the DNAbinding mode in absence of a crystal structure. Regarding intercalation, the enhancement of DNA viscosity is associated with the DNA length, owing to the stacking of the planar ring of the molecule between DNA base pairs [20]. On the contrary, the electrostatic and groove-binding mode leads to a twisting or bending in the DNA helix, with a slight decrease in the viscosity [71,72]. A plot between relative specific viscosity (η/ηo) 1/3 versus the concentration of the complex 1/DNA ratio (r = 0.0, 0.05, 0.1, 0.15, 0.2, 0.25) is shown in Figure 6. A nominal rise in the DNA solution viscosity was observed in the presence of complex 1, suggesting the intercalation of their planar parts into the base pairs of doublehelical DNA. However, the relative viscosity increments are much smaller than expected for a classical intercalator, indicating groove binding.

Viscosity Measurements
The viscosity measurement of DNA is the most reliable tool to explain the DNAbinding mode in absence of a crystal structure. Regarding intercalation, the enhancement of DNA viscosity is associated with the DNA length, owing to the stacking of the planar ring of the molecule between DNA base pairs [20]. On the contrary, the electrostatic and groove-binding mode leads to a twisting or bending in the DNA helix, with a slight decrease in the viscosity [71,72]. A plot between relative specific viscosity (η/ηo) 1/3 versus the concentration of the complex 1/DNA ratio (r = 0.0, 0.05, 0.1, 0.15, 0.2, 0.25) is shown in Figure 6. A nominal rise in the DNA solution viscosity was observed in the presence of complex 1, suggesting the intercalation of their planar parts into the base pairs of doublehelical DNA. However, the relative viscosity increments are much smaller than expected for a classical intercalator, indicating groove binding. groove-binding mode leads to a twisting or bending in the DNA helix, with a slight decrease in the viscosity [71,72]. A plot between relative specific viscosity (η/ηo) 1/3 versus the concentration of the complex 1/DNA ratio (r = 0.0, 0.05, 0.1, 0.15, 0.2, 0.25) is shown in Figure 6. A nominal rise in the DNA solution viscosity was observed in the presence of complex 1, suggesting the intercalation of their planar parts into the base pairs of doublehelical DNA. However, the relative viscosity increments are much smaller than expected for a classical intercalator, indicating groove binding.

In Vitro Cytotoxicity
The cytotoxicity of complex 1 was performed at various concentrations (0-20 μM) against human colon cancer (HCT116) and human embryonic kidney 293 cells (HEK 293) using an MTT assay (Figure 7). In the case of the HCT116 cell line, more than 70% of cell

In Vitro Cytotoxicity
The cytotoxicity of complex 1 was performed at various concentrations (0-20 µM) against human colon cancer (HCT116) and human embryonic kidney 293 cells (HEK 293) using an MTT assay (Figure 7). In the case of the HCT116 cell line, more than 70% of cell deaths were observed at the concentration of 20 µM after 24 h. The data reveal that at 30%, 50%, and 70% HCT 116 cell deaths were observed at 7.6 µM, 11.6 µM, and 15.7 µM, respectively, of the complex 1 treatment after 24 h, whereas in the case of HEK 293, IC 30 , IC 50 , and IC 70 were detected at 11.5 µM, 22.6 µM, and 30.02 µM, which is almost double compared to HCT 116, which exposes that complex 1 exhibits selective cytotoxicity towards HCT 116 cancer cells, leaving behind normal cells that are scarcely affected. Therefore, we preferred the IC 50 value (11.6 µM) for further evaluation against HCT 116.

Quantification of Apoptosis and Necrosis using Flow Cytometry
To determine the necessary mechanism of the anticancer effectiveness of complex 1 on HCT 116 cells, the flow cytometric technique was used to analyse using Annexin V-FITC/PI (Figure 8). The percentage of the viable cell population in the control was 88% following mild early apoptosis of 4.8%, late apoptosis of 2.9%, and necrosis of 4.2%. After 24 h of 11.6 μM of complex 1 treatment, the viable cell population was altered to 62.8%, resulting in mild early apoptosis of 17%, late apoptosis of 17.1% and necrosis of 1.7%. These data confirm that the cause of cytotoxicity of complex 1 was due to apoptosis.

Quantification of Apoptosis and Necrosis Using Flow Cytometry
To determine the necessary mechanism of the anticancer effectiveness of complex 1 on HCT 116 cells, the flow cytometric technique was used to analyse using Annexin V-FITC/PI (Figure 8). The percentage of the viable cell population in the control was 88% following mild early apoptosis of 4.8%, late apoptosis of 2.9%, and necrosis of 4.2%. After 24 h of 11.6 µM of complex 1 treatment, the viable cell population was altered to 62.8%, resulting in mild early apoptosis of 17%, late apoptosis of 17.1% and necrosis of 1.7%. These data confirm that the cause of cytotoxicity of complex 1 was due to apoptosis.

Quantification of Apoptosis and Necrosis using Flow Cytometry
To determine the necessary mechanism of the anticancer effectiveness of complex 1 on HCT 116 cells, the flow cytometric technique was used to analyse using Annexin V-FITC/PI (Figure 8). The percentage of the viable cell population in the control was 88% following mild early apoptosis of 4.8%, late apoptosis of 2.9%, and necrosis of 4.2%. After 24 h of 11.6 μM of complex 1 treatment, the viable cell population was altered to 62.8%, resulting in mild early apoptosis of 17%, late apoptosis of 17.1% and necrosis of 1.7%. These data confirm that the cause of cytotoxicity of complex 1 was due to apoptosis.

Quantification of Mitochondrial Reactive Oxygen Species (ROS)
We assessed the mitochondrial ROS by DCF-DA in the presence of Mitotracker Red (Figure 9) to elucidate the anticancer potential of complex 1. The microscopic data show that after 24 h of complex 1 (11.6 µM) treatment, mitochondrial ROS generation increased compared to the untreated one. This conformation exposes that the generation of ROS is the prime cause of apoptosis in HCT 116 cell line induced by complex 1.

Quantification of Mitochondrial Reactive oxygen Species (ROS)
We assessed the mitochondrial ROS by DCF-DA in the presence of Mitotracker Red (Figure 9) to elucidate the anticancer potential of complex 1. The microscopic data show that after 24 h of complex 1 (11.6 μM) treatment, mitochondrial ROS generation increased compared to the untreated one. This conformation exposes that the generation of ROS is the prime cause of apoptosis in HCT 116 cell line induced by complex 1.

DNA Fragmentation Assay
Elevated levels of reactive oxygen species (ROS) prompt oxidative damage to the DNA and other cell's components and ultimately leads to cell death. Therefore, a DNA fragmentation assay was undertaken, in which the cells treated with complex 1 at different concentrations (0-20 μg/mL) were analysed for 24 h. Then, the resultant fragmented DNA was analysed using commercially accessible kits at 405 nm according to the earlier protocols [73,74]. The colorimetric data display that DNA fragmentation increased with the increasing concentration of 1 ( Figure 10). Our experimental investigations revealed that cell death provoked by complex 1 is associated with typical apoptotic changes such as the

DNA Fragmentation Assay
Elevated levels of reactive oxygen species (ROS) prompt oxidative damage to the DNA and other cell's components and ultimately leads to cell death. Therefore, a DNA fragmentation assay was undertaken, in which the cells treated with complex 1 at different concentrations (0-20 µg/mL) were analysed for 24 h. Then, the resultant fragmented DNA was analysed using commercially accessible kits at 405 nm according to the earlier protocols [73,74]. The colorimetric data display that DNA fragmentation increased with the increasing concentration of 1 ( Figure 10). Our experimental investigations revealed that cell death provoked by complex 1 is associated with typical apoptotic changes such as the externalization of phosphatidylserine and DNA fragmentation. In brief, the results clarify that complex 1's facilitated apoptosis might be controlled through a mitochondrial ROS-driven DNA damage route, which has significant potential as an anticancer agent.

Molecular Docking
Molecular docking is an imposing computational tool that can be used visualize the molecules-DNA interaction to validate the experimental findings. The interaction of complex 1 with DNA was explored using AutoDock vina, which offered nine various conformations. Among these, the lowest binding energy conformation is presented in Figure  11a. The outcomes revealed that complex 1 bound to a minor groove of DNA with the binding energy of −10.0 kcal mol −1 . Additionally, complex 1 formed two hydrogen bonds with dC11 and dD16 of the DNA (Figure 11b). Other nucleotides such as dC9, dG10, dG12, dG14, dC15, A17, etc. were also involved in the complexation.

Molecular Docking
Molecular docking is an imposing computational tool that can be used visualize the molecules-DNA interaction to validate the experimental findings. The interaction of complex 1 with DNA was explored using AutoDock vina, which offered nine various conformations. Among these, the lowest binding energy conformation is presented in Figure 11a. The outcomes revealed that complex 1 bound to a minor groove of DNA with the binding energy of −10.0 kcal mol −1 . Additionally, complex 1 formed two hydrogen bonds with dC11 and dD16 of the DNA (Figure 11b). Other nucleotides such as dC9, dG10, dG12, dG14, dC15, A17, etc. were also involved in the complexation.

Molecular Docking
Molecular docking is an imposing computational tool that can be used visualize the molecules-DNA interaction to validate the experimental findings. The interaction of complex 1 with DNA was explored using AutoDock vina, which offered nine various conformations. Among these, the lowest binding energy conformation is presented in Figure  11a. The outcomes revealed that complex 1 bound to a minor groove of DNA with the binding energy of −10.0 kcal mol −1 . Additionally, complex 1 formed two hydrogen bonds with dC11 and dD16 of the DNA (Figure 11b). Other nucleotides such as dC9, dG10, dG12, dG14, dC15, A17, etc. were also involved in the complexation.

Conclusions
Herein, we have discussed the structural elucidation and topological analysis of a Cu(II) complex (1), derived from 2-hydroxy-1-naphthaldehyde under basic conditions. To

Conclusions
Herein, we have discussed the structural elucidation and topological analysis of a Cu(II) complex (1), derived from 2-hydroxy-1-naphthaldehyde under basic conditions. To observe the outcome of complex 1 on DNA-binding propensity, several biophysical techniques were employed. The experimental results reveal the partial intercalation between the DNA nucleotides. Additionally, a molecular docking study was also performed, which shows that the complex binds to the minor groove of DNA via hydrogen bonding in the C-G region. Complex 1 showed potential inhibition activity against human colon cancer cell line (HCT116) in a dose-dependent mode. Moreover, complex 1 showed higher intracellular ROS levels and induced apoptosis via mitochondrial-mediated pathways. Therefore, it is established that a synergistic combination of ligands and Cu(II) metal ions is important for the realization Cu(II)-based anticancer drug.