Antiproliferative studies of transition metal chelates of a pyrazolone based hydrazone derivative

Abstract Pyrazolone derivatives play a significant role in the treatment of cancer. The synergic effect which emerges from the combination of pyrazolone moiety with hydrazone functionality was investigated. The objective of this study was to explore the antiproliferative potential of copper(II), cobalt(II), nickel(II) and zinc(II) metal chelates synthesized from pyrazolone based hydrazone derivative. The ligand and the metal chelates were characterized by various spectroscopic and analytical studies. The ligand was characterized by single crystal X-ray diffraction analysis.The results were in line with the spectroscopic methods. The geometry optimization of ligand and metal chelates were performed using density functional theory (DFT). The invitro cytotoxicity of ligand and metal chelates against different cancer cell lines was investigated by MTT assay. The cell-viability experiments showed that copper(II) complex is an efficient cytotoxic agent against HeLa cell line. Moreover, possible inhibition mechanism of synthesized compounds was evaluated in silico against HPV16-E6 receptor. Communicated by Ramaswamy H. Sarma


Introduction
Ti C h i Â 100% G ¼ g k À2:0023 À Á = g ? À 2:0023 ð Þ The World Health Organization defines cancer as an abnormal growth of cells beyond their usual boundaries and it can invade adjoining parts of the body and spread to other organs. Cancer is a disease which displays traits such as uncontrolled cell growth, invasion, and metastasis. The healthy cells in our bodies divide continuously throughout our lives while cancer multiplies in the cell in an uncontrolled manner. These abnormal cells constitute a mass called the tumor. The cancer is caused due to several reasons such as the abnormalities in the genetic material, exposure to external agents such as radiation, some chemicals and so on. It has become the second fatal disease after the cardiovascular disease. The statistical reports suggested that in 2012, there were about 8.2 million cancer deaths and every year about 4 lakh deaths take place. This might be due to the poor accessibility, availability of prevention, diagnosis and cure of the disease. These reports explain the fact that both in developing and developed countries cancer had become a significant life-threatening disease. The decisive role of metal complexes dates back with the development of cis-platin as an effective anticancer drug. The survey of the National Cancer Registry Programme indicated that the primary sites of cancer are oral cavity, lungs, oesophagus, stomach, uterine cervix and breast. There are about 200 different kinds of cancer, each classified based on organ or type of cell from which it originates.
Hydrazones are one of the well-known functional organic compounds because of various binding modes and lone pair of electrons on the imine nitrogen (Corey & Enders, 1976). These hydrazones possess a wide range of biological properties such as antimicrobial, anti-inflammatory, analgesic, antifungal, anti-tubercular, antiviral, anticancer, antiplatelet, antimalarial, anticonvulsant, cardioprotective, antihelmintic, antiprotozoal activities (Rollas & Kucukguzel, 2007;Şenkardeş et al., 2021). The structural motifs such as nucleophilic imine and amino-type nitrogens, acidic N-H proton give the hydrazone group its characteristic physical and chemical properties. They are ready to coordinate with various metal ions, and it has attracted increasing interest by the structural, novel functional and natural aesthetics.
These hydrazone-based ligands have drawn much attention due to their versatility in chelation with transition metals. A large number of hydrazones can be prepared by condensation of different kinds of hydrazides and carbonyl compounds. The hydrazones derived from pyrazolone derivatives have acquired only sporadic attention. There are several reports of hydrazone derivatives with promising anticancer potential (Dandawate et al., 2012;Hamada et al., 2021;Mohareb & Al-Omran, 2012).The vital role of metal complexes in cancer treatment dates back with the development of cisplatin as an effective anticancer drug (Jung & Lippard, 2007). The high systemic toxicity and acquired resistance are the two major drawbacks associated with platinum-based drugs .This clearly indicate the need for the development of new metal-based anticancer drugs .In the present work; we have made an attempt to study the anticancer potential of a hydrazone derivative and its metal complexes. The availability of a broad spectrum of coordination numbers, geometries, accessible redox states, thermodynamic and kinetic properties make the metal complexes suitable candidates of drug action. The excellent photophysical and photochemical properties make these metal complexes as ideal scaffold in the treatment of cancer. In the present communication, we have taken a modest attempt to study the anticancer potential of transition metal complexes of a hydrazone derivative derived from isonicotinic acid hydrazide and antipyrine-4carboxaldehyde.

Materials and methods
All chemicals used in this work were of analytical reagent grade, purchased from Aldrich, Fischer, SISCO (India), etc. Standard methods purified solvents used for physicochemical measurements. Elemental analysis for C, H, and N were carried out using a Elemental Vario EL-II-CHN Analyzer. Molar conductance measurements were carried out using 10 À3 M solutions of the complexes in suitable solvent at room temperatures using a Systronic direct reading conductivity meter Type 305. Infrared spectra were recorded using KBr discs on a Shimadzu FTIR 8000 spectrophotometer, and electronic spectra were recorded on a Hitachi 320 UV-Vis spectrophotometer. Magnetic susceptibilities were measured at 25 C using the Gouy method with mercuric tetrathiocyanatocobaltate(II) as the magnetic susceptibility standard (Figgis et al., 1958). Melting points were determined with a Stuart melting point apparatus. NMR spectra were recorded using a JEOL GSX 400 NB 400 MHz spectrometer in DMSO-d 6 . The EPR spectrum of the copper(II) complex was recorded using a JES -FA200 ESR spectrometer with an X band employing manganese as reference material. The fluorescence property was measured using JASCO 8300 spectrofluorometer.

Synthesis of ligand (ISAP)
The ligand was prepared by microwave irradiation. About 0.01 mol of antipyrine-4-carboxaldehyde was mixed with 0.01 mol of isonicotinic acid hydrazide and ground in a mortar. The reaction mixture was then dissolved in 2 ml of ethanol and irradiated for 6 min. A pale yellow product was formed, which is then recrystallized with ethanol, dried under vacuum at room temperature. The purity of the reaction was studied using TLC (Scheme 1).

Synthesis of metal complexes
The hot methanolic solution of the ligand (0.01 mmol) was mixed with the corresponding methanolic solution of cobalt chloride (0.01 M)/nickel chloride (0.01 M)/copper chloride (0.01 M)/zinc chloride (0.01 M)solution. The resulting solution was magnetically stirred for 1 hr followed by refluxing for 6-8 hr at 80 C. The resulting solution was concentrated and allowed to cool. The metal complex formed was filtered, washed successively with methanol and diethyl ether and dried over.

Determination of cytotoxicity
The cytotoxicity was determined by using an SRB assay (Orellana & Kasinski, 2016). The procured cell lines were grown in RPMI 1640 medium containing 15% fetal bovine serum and 2 mM L-glutamine. Then the cells were inoculated into 96 well microtiter plates in 100 mL. After inoculation, the microtiter plates were incubated at 37 C, 5% CO2, and 100% relative humidity for 24 h before the addition of synthesized compounds. The compounds were initially dissolved in dimethyl sulfoxide at 100 mg/ml and diluted to 1 mg/ml using water and stored frozen prior to use. Aliquots of 10 ml of the different dilutions were added to the appropriate microtiter wells already containing 90 ml of the medium, resulting in the required final drug concentrations i.e.10 lg/ ml, 20 lg/ml, 40 lg/ml, 80 lg/ml. The plates were incubated at standard conditions for 48 hours and the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 ml of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4 C. The supernatant was discarded; the plates were washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (50 ml) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered, and the residual dye was removed by washing five times with 1% acetic acid. The plates were air-dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was measured at a wavelength of 540 nm with a 690 nm reference wavelength. The growth percentage was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells multiplied by 100. The absorbance measurements were used for calculating percentage growth was calculated at each of the drug concentration levels.
Percentage growth inhibition was calculated as: Apoptosis assay by acridine orange (AO) and ethidium bromide (EB) staining The HeLa cells were seeded in well plates and allowed to reach 70% confluence (Liu et al., 2015). The cells were then treated with IC 50 concentrations of the complex for 24 hours. The cells were trypsinized and pelleted, and then suspended in PBS. A drop of suspension of the cell was placed on a glass slide and stained with Acridine Orange (AO) and Ethidium Bromide (EtBr), and a cover slip was laid over to reduce light diffraction. The cells after staining were washed twice with PBS and observed by a fluorescence microscope with a blue filter of a fluorescent microscope (Olympus CKX41 with Optika Pro5 camera).

Apoptosis assay by flow cytometry
The HeLa cells were cultured as per standard procedures described earlier and treated with manganese(II) complex (50 mg/mL) and incubated for 24 hours (Liu et al., 2015).The cells were trypsinized after incubation, and 100 lL of cells in suspension were transferred to separate tubes. To the tubes, added 100 lL of the Muse TM Annexin V and dead cell reagent. The tubes were mixed thoroughly by pipetting up and down or vortexing at medium speed for 3 to 5 seconds, followed by incubation for 20 minutes at room temperature in the dark. The cells were analyzed in a flow cytometer and analyzed using muse flow cytometry software. Cells were gated against untreated control cells and analyzed for apoptosis using Muse FCS 3.0 software.

Agarose gel electrophoresis
The gel electrophoresis method is used for the separation and visualization of DNA fragments (Meyers et al., 1976).The fragments are separated by charge and size and move through an agarose gel matrix when subjected to an electric field. The electric field generation occurs by applying a potential across an electrolyte solution .The agar dissolved when boiled in an aqueous buffer and solidifies to gel upon cooling. 1.5% agarose gel was prepared in 1x TE buffer and melted in a hot water bath at 90 C. The melted agarose was cooled down to 45 C. About 6 mL of 10 mg/mL of ethidium bromide was added and poured into gel casting apparatus with the gel comb. After setting, the comb was removed from the gel. The electrophoresis buffer was poured in the gel tank and the platform with the gel was placed in it so as to immerse the gel. The gel was loaded with the samples and run at 50 V for 30 minutes. The stained gel was visualized using a gel documentation system.

Invitro ROS measurement using DCFDA
The cells were washed with PBS and added with 50 mL of DCFDA and incubated for 30 minutes. After incubation, the excess dye was washed with PBS, and fluorescence was imaged in a fluorescent microscope (Olympus CKX41 with optika pro5 CCD camera), and fluorescence was measured

Lipid peroxidation (LPO)
Lipid peroxidation was performed using the thiobarbituric acid-reactive substance TBARS method (Buege & Aust, 1978).The cells were collected by centrifugation and were sonicated in ice-cold KCl solution (1.15%) and were then centrifuged for 10 min at 3000xg. The resulting supernatant (1 mL) was added to 2 ml of thiobarbituric acid (TBA) reagent and was heated at 100 C for 15 min in a boiling bath. The samples were placed in the cold and were centrifuged at 1000 Â g for 10 min. At 550 nm, the absorbance of the supernatant was measured.

Glutathione level
After treatment, treated cells were trypsinized with Trypsin-EDTA Solution (HiMedia), collected, and transferred into eppendorf tubes. The tubes were centrifuged at 5000 pm for five minutes.The supernatant was discarded, and the pellet was suspended in 200 mL of lysis buffer (0.1 M tris, 0.2M EDTA, 2 M NaCl, 0.5% Triton). The samples were incubated at 4 C for 20 minutes. The cell lysate thus obtained was used for the study. About 0.1 mL of cell lysate 0.5 mL of phosphate buffer (0.2 M pH 8), 1.3 mL distilled water, and 0.2 mL of DTNB (0.6 mM) were added. The contents were mixed well and read on a spectrophotometer at 420 nm (Moron et al., 1979).

Computational details
The geometry optimization of the structure of ligand and metal complexes was performed using the B97d hybrid density functional method with 6-311þþG(d,p) basis set (Qi et al., 2016).The Natural Bond Orbital analysis was also done at B97d/6-311þþG(d,p) level. All the calculations were performed using Gaussian 09 program package with the help of the Gauss View visualization program. During optimization no symmetry constraints were applied. No imaginary frequency was obtained after frequency calculations.

Molecular docking
The X-ray crystal structures of HPV16-E6 with PDB ID 4GIZ at 2.55 Å resolution was retrieved from the RCSB protein data bank (Huang et al., 2018).The 3 D-structures of ligand and metal complexes were drawn using Chem Sketch 2017.2.1 version. The in silico docking studies were carried out by using the Schrodinger suite 2018-2 (Initially there was a pretreatment process for both the protein and the ligands. After the preparation of the PDB, the grids were generated around the better binding pocket analyzed by site map analysis. Molecular docking of the ligand with the receptors was carried out using Glide programme of Schrodinger suite. After the preparation of the PDB, the grids were generated around the better binding pocket analyzed by site map analysis. Molecular docking of the ligand with the receptors was carried out using Glide programme of Schrodinger suite. . The optimized geometries of the ligands were used for docking after fixing zero bond order to bonds with metal. After the preparation of the PDB, the grids were generated around the better binding pocket analyzed by site map analysis. Molecular docking of the ligand with the receptors were carried out using Glide programme of Schrodinger suite.

Result and discussion
The analytical data of ISAP and metal complexes were determined (Table 1). ISAP was found to be soluble in organic solvents like methanol, ethanol, benzene, toluene, etc. The complexes were stable and soluble in DMSO and DMF. The molar conductance values of metal complexes were evaluated and were in the range of 10.4 to 12.7 ohm À1 cm 2 mol À1 in the DMSO solution which indicated the non-electrolytic nature of complexes (Geary, 1971).

IR spectra
The IR spectrum provides the information regarding the coordination modes observed in the ligand and its metal complexes (Figures 1 and 2). A strong absorption band was observed at 3207 cm À1 which corresponds to the m(N-H) vibration .The m(N-H) band was slightly shifted in the complexes indicating the existence of ligand in the hydrazo form. Two sharp bands observed at 1672 cm À1 and 1628 cm À1 can be assigned to pyrazolone and amide m(C ¼ O) vibrations, respectively. It was found that amide C ¼ O vibrations remain unchanged in complexes while the pyrazolone m(C ¼ O) vibrations were considerably shifted to lower wavenumber in the range of 1620-1626 cm À1 .A strong band at 1156 cm À1 correspond to m (N-N) vibration mode. The absence of m (C-O) confirms the existence of ligand in the hydrazo form rather than azo form. The m(C ¼ N) band which is the characteristic of azomethine linkage, occurred at 1581 cm À1 in the ligand was shifted in the range of 1573-1564 cm À1 in complexes. The complexes showed non-ligand bands in the  (Ferraro, 1971).The IR spectral data indicated that ISAP behaves as a bidentate ligand and coordinate to metal ion through hydrazo nitrogen and the pyrazolone oxygen.

NMR spectra
The proton NMR spectra of ISAP and Zn(II) complex was recorded and are given in Figures 3 and 4. The ligand showed a singlet at 14.261 ppm due internally hydrogenbonded hydrazone proton which remained unchanged in the complex. This downfield shift is due to the presence of adjacent electron rich oxygen atom of carbonyl group which is involved in hydrogen bonding. The signal due to azomethine proton which appeared at 8.76 ppm was shifted to 8.71 ppm in the complex .Two singlets were observed at 2.46, and 3.35 ppm were due to methyl protons of C-CH 3 and N-CH 3 groups, respectively (Philip et al., 2018). The NMR signal in the region 7.26 À 7.62 ppm corresponds to aromatic protons, which were slightly shifted in the range of 7.21-7.59 ppm in the case of metal complexes.

Electronic spectra
The hydrazones can be differentiated from azo compounds using the electronic spectrum. Usually, hydrazone compounds give a weak band at around 295 nm and a strong absorption band at a wavelength higher than 320 nm while azo compounds show a strong band at $ 280 nm. In this case, the ligand (ISAP) showed two bands at 267 nm and 326 nm corresponding to G -P Ã and n-G Ã transitions, respectively. The broadband observed at 326 nm arise due to the hydrazo linkage in the ligand. The metal complexes showed only a marginal shift in the electronic spectrum in the range of 350-357nm which clearly indicated that the metal complexes are in the same structural form as that of the ligand. The copper(II) complex exhibited a band at 730 nm and has a magnetic moment value of 1.86 BM, which corresponds to the square planar geometry for the complex. The cobalt(II) complex exhibited bands at 605 nm and 518 nm, which corresponds to 4 A 2 ! 4 T 1 (F) (t2) and 4 A 2 ! 4 T 1 (P) (t3), supporting a tetrahedral geometry for the cobalt(II) complex . It possesses a magnetic moment value of 4.30 BM, which is in agreement with a tetrahedral environment. The Ni(II) complex showed bands at 560 nm and 725 nm which give rise to 1 A 1g ! 1 B 1g and 1 A 1g ! 1 A 2g transitions, respectively indicating a square planar geometry ( Table 2). The magnetic moment measurements showed the diamagnetic nature of the nickel(II) complex. The zinc(II) complex preferred a tetrahedral geometry due to its fully filled configuration.

ESR studies
The ESR spectrum of the copper(II) complex was recorded at liquid nitrogen temperature in DMSO ( Figure 5). It showed axial symmetry with g | and g ? values obtained from the spectrum were 2.27 and 2.05 respectively. The g jj value was found to be greater than g ? which indicated the presence of unpaired electron probably in the d X 2 -y 2 orbital. The g jj values provided an idea about the covalent nature of the metal-ligand bond. The g av value is 2.12 which further supported the covalency (Table 3).
The geometric parameter G was calculated using the equation,   According to Hathaway, G value gives an idea of the exchange interaction between metal centers (Hathaway & Tomlinson, 1970).The copper(II) complex showed a G value of 5.61 which suggests that the exchange interaction between metal centers is negligible .The bonding parameter, a 2 value, was found to be which indicated the covalent nature of the M-L bond. The index of tetragonal distortion (f) showed value in the range of 135 cm À1 , which is expected for tetragonally distorted complexes.

Single-crystal XRD studies
The single-crystal XRD studies of the ligand ISAP were carried out, and it confirms its structure. The details are deposited in Cambridge Crystallographic System (CCDC) with CCDC 1868932. Single-crystal XRD showed that the compound crystallises in orthorhombic space group P212121 with unit cell dimensions, a ¼ 5.4563(3) Å, b ¼ 17.0047(10) Å, c ¼ 17.0725(9) Å, Z ¼ 4. The direct methods were used to evaluate the crystal structure and refined by full-matrix least-squares procedures to a final R-value of 0.0573 with 25127 observed reflections. The crystal has a volume of 1584.03(15) Å 3 . The ORTEP diagram of ISAP is shown in Figure 6. The details of structural data and hydrogen bond interactions of ISAP are given in Tables 4 and 5, respectively. The single-crystal data of the metal complexes were not obtained .We have done the powder XRD studies of metal complexes and were found to be amorphous in nature.

Thermogravimetric analysis
The thermal studies of copper(II) complex was carried out and the thermogram was recorded in Figure 7. The complex was stable upto 160 C and there are three decomposition stages. The first decomposition occurred in the temperature range 170 C and 220 C with a peak temperature of 199.33 C.The estimated mass loss was found to be 32% which might be due to the loss of moiety of the ligand. The second decomposition stage was in the range of 230 C and 340 C with a peak temperature of 286.73 C. The mass loss was found to be 52.46% which might be due to the loss of remaining ligand moiety. The third decomposition stage was in the range of 360 C to 495 C corresponding to the loss of coordinated chlorine atoms and oxidation of Cu to CuO. The result obtained was used to characterize the metal complex. From the various spectral studies the following structures have been proposed for the ligand and its metal complexes (Figures 8 and 9).

Computational studies
The DFT studies are widely used to predict various properties of ligand and the metal complexes. In the present study, the geometry of ligand and its metal complexes were optimized using B97D density functional method with 6-311þþG(d, p) basis sets as incorporated in the Gaussian 09 programme in gas phase. The optimized structures of the ligand and the metal complexes were given in Figure 10. The spectral studies revealed that the ligand coordinated to the metal ion through the O20 atom of the amide group and N16 atom of the hydrazo moiety which is further confirmed by the considerable shift in their bond lengths observed in the theoretical studies ( Table 6). The M-Cl bond lengths were also found to be shifted to a considerable extent confirming their participation in the complex formation. The frontier molecular contour surfaces of ISAP and metal complexes are given in Figure 11. The LUMO of ISAP is spread over the whole ligand moiety while the HOMO is mainly spread over the hydrazo unit and pyrazolone moiety.
It was observed that in the metal(II) complexes, HOMO encompasses the whole metal moiety along with the chloride groups and also the hydrazone unit in the case of copper(II) complex while in cobalt(II), nickel(II) and zinc(II) complex were solely concentrated on the metal region. The LUMO of the metal complexes encompasses mainly the pyridine moiety and the hydrazone group. The extensive overlapping of these orbitals might be attributed to the high reactivity of the metal complexes which lead to its  effective interaction with the biological molecules. The NBO analysis was carried out to understand the atomic charge distribution in the synthesized compounds and is given in Table 7. The calculated atomic charge was found to be lower than the formal charge on metals which was a clear evidence for charge transfer from ligand to metal centers. Thus the NBO analysis also confirmed the bidentate coordination of ligand through the hydrazo nitrogen and the   amide carbonyl group. The atoms with negative charge act as donor atoms and those with positive charge act as acceptor atoms. The details regarding the global reactivity parameters such as global hardness (g), global softness (S), absolute softness (r) and global electrophilicity index (x) were obtained from the extent of HOMO and LUMO energy gap. The lower energy gap indicated the soft nature of the metal complexes with higher reactivity (Table 8). The chemical hardness of a compound provide information regarding its biological efficiency . The copper(II) complex showed lowest hardness which might be reason for its enhanced biological activity. The mathematical expression of g, º, r can be written as (Arab & Habibzadeh, 2015) g The global electrophilicity index (x) (Pearson, 1986) x ¼ l 2 2g The higher electrophilicity index is an indication of promising biological activity and the metal complexes were found has a higher value compared to ISAP.

Invitro cytotoxicity assay
The antiproliferative actions of ISAP and its metal complexes were examined in MDA -MB-231, K-562, HeLa, HT-29, and   Hep -G2 cell lines and are evaluated by SRB assay (Jung & Lippard, 2007).The effect of the newly synthesized compounds at different concentrations ie. 10, 20, 40, 80 mg/mL on cell viability of these cell lines were studied. The results clearly indicated that the cytotoxicity effect increased in a dose dependent manner (Fouad & Omima, 2021). The cobalt(II) complex showed IC 50 value of 45.5 lg/mL towards K-562 cell line. The metal complexes showed significant activity towards HeLa cell line (Table 9). The copper(II) complex showed promising activity with a IC 50 value of 13 mg/ mL. The other metal complexes showed better activity than the ligand with a cell viability of 85-90% for the concentrations up to 10 lg/mL. The cell viability of the copper(II) complex showed an effective decrement when compared to the control cells and the ISAP. The morphological images showed that the cells became rounded and undergo cell shrinkage along with the loss of interaction with the nearby cells ( Figure 12). It is also interesting to note that all the compounds showed a weak cytotoxic effect on normal cell line MCF-10A which clearly indicate the selective action of compounds towards the cancer cells.

DNA fragmentation analysis
The metal complexes are reported to be stronger DNA binding agents and were found to induce several changes in the conformation of DNA (Kulkarni et al., 2011). Moreover the metal complexes are known for its ability to cleave DNA which in turn throws light on the anticancer potential of the compound. In the present work, the interaction mode between the synthesized compounds and plasmid DNA were studied using agarose gel electrophoresis experiments. The circular plasmid DNA on electrophoresis exhibits relatively fast migration resulting in an intact supercoiled form (form I).
When scission occurs the supercoil will relax to form a slower moving open circular form (form II). When both the strands are cleaved, a linear form i.e. form III migrate between form I and II will be generated (Li et al., 2011;Nejedl y et al., 1998). Thus the Figure 13 showed that the cleavage activity of plasmid pBR322 DNA after incubation with metal complexes at 37 C for 1 h at dark condition. The form (II) cannot be    The metal complexes exhibited form I and form II cleavage which is more significant the control in which no cleavage of plasmid DNA occurred. The agarose gel electrophoresis assay of copper(II) complex showed that there is a obvious mobility shift on supercoiled DNA which implies its strong intercalation interaction to copper(II) complex .These observations clearly depict the DNA cleavage ability of copper(II) complex which might be attributed to its significant cytotoxicity. The higher nucleobase affinity of copper(II) ion might also contribute to its cleavage efficiency (Pankaj et al., 2012).

Apoptosis studies by acridine orange and ethidium bromide (AO & EB) staining
The apoptosis can be qualitatively studied using AO & EB staining method based on the perspective of fluorescent emission (Smith et al., 2012).The IC 50 value of copper(II) complex was injected into the HeLa cell line and incubated for 24 hours followed by staining with the acridine orange/ethidium bromide. The morphological features of cells clearly indicated the presence of orange fluorescing nuclei with condensed or fragmented chromatin which is an indication of late apoptosis (Figure 14).

Apoptosis studies by flow cytometry
The percentage of apoptosis can be determined quantitatively using flow cytometric technique (Wlodkowic et al., 2009). The morphological studies showed copper(II) complex can induce apoptosis of HeLa cells. To determine the percentages of apoptotic cells, HeLa cells without sample was taken as the control. In the control, the proportions of living cells and apoptotic cells were 94.12% and 2.98%, respectively. The IC 50 concentration of copper(II) complex was added to the HeLa cells and incubated for 24 hours .The proportions of living cells and apoptotic cells were 63.92% and 27.40%, respectively ( Figure 15). It was observed that on comparing with the control, the proportion of living cells was decreased in the copper(II) complex which in turn increased the proportion of apoptotic cells. The cell death induced by the complex was found to follows a pathway from the viable cells to the early apoptotic cells and to the late apoptotic cells .This is a clear evidence for induced cell death which occurs mainly through apoptosis. The ROS generation can lead to oxidative damage to the cell which adversely affect signalling and the biological functions of cell (Liu et al., 2014).The ROS targeting is considered as a suitable strategy for cancer treatment. The primary source of production of ROS is mitochondrion and its excess production is an indication of mitochondrial damage, genetic instability, and ultimately  apoptotic cell death (Wang et al., 2017).The ROS production upon 24 h treatment with Cu(II) complex was determined using DCFDA assay. Cisplatin was taken as the positive control. It was observed that Cu(II) complex showed intense green fluorescence indicating high level of ROS generation while the control showed an insignificant effect. There are reports that the level of induction of cytotoxicity depends on the level of ROS generation (Rodriguez-Fanjul et al., 2018).The results clearly suggested the possibility of ROSinduced apoptosis on exposure to the copper complex which might be the reason for its antiproliferative action.

Intracellular glutathione depletion
The glutathione (GSH) level have a vital role in various biological processes such as transcription activation of specific genes, regulation of redox-related signal transduction pathways, and control of cell proliferation and apoptosis (Rahman & MacNee, 2000). There are reports that GSH has a significant effect in anticancer therapy as cytotoxicity depends directly on intracellular GSH level. The GSH level of cells that had been exposed to copper(II) complex, control and ISAP were determined ( Figure 16).The studies showed that the copper(II) complex is capable of reducing GSH levels to a significant extent when compared to ISAP .The GSH depletion can facilitate ROS accumulation and thereby enhance the potential of an antitumour drug (Cuadrado et al., 2003).

Lipid peroxidation
Lipid peroxidation (LPO) is a natural process in the cellular system which involves oxidative degradation of lipids which accelerate ROS generation in cell resulting in cell damage.   The lipid peroxidation can also contribute to cell proliferation, differentiation, maturation and apoptosis (Cejas et al., 2004).The lipid peroxidation is determined by TBARS method. It was observed that copper(II) complex showed increased lipid peroxidation level which proves its role in the enhancement of reactive oxygen species in the HeLa cell and is given in Figure 17. The potential activity of copper(II) complex might be attributed to its high redox potential which stimulate the lipid peroxidation.

Molecular docking studies
The molecular docking studies of ISAP and metal complexes were carried out using PDB ID-4GIZ (Zanier et al., 2013).The docked pose of each ligand was analyzed for interactions with HPV16-E6 receptor. Among the complexes copper(II) complex has a binding affinity which is in good agreement with the experimental results. The docking studies indicated that ISAP showed hydrophobic interactions with ALA 64, TRP 63, TRP 64, TRP 341, PRO 155, TYR 156, PHE 157, TYR 211 and TRP 231 and a pi-cation interaction and has a binding energy of À 4.67 KCalmol À1 (Figure 18). The copper(II) complex exhibited hydrophobic interactions with PHE 230, PHE 263, CYS 283, VAL 284, TYR 275, LEU 38, MET 87, ASN 86. It also showed polar interactions with ASN 86, SER 262, SER 282, GLN 8 and positive charged interactions with ARG 285 (Konakanchi et al., 2021). These interactions are responsible for the promising binding affinity of À8.7 Kcalmol À1 for the copper(II) complex (Table 10).This suggests that copper(II) complex might interact effectively with HPV16-E6 receptor and this may contribute to their anticancer activity. The weak intermolecular hydrophobic interactions are responsible for stabilizing the compound on interaction with protein structure and also have a vital role in determining the binding affinity between proteins and ligands.

Conclusion
The present investigation highlights the synthesis of new copper(II), cobalt(II),nickel (II) and zinc(II) metal chelates bearing bidentate N,O pyrazolone based hydrazone ligand. The characterization of ligand and metal complexes was accomplished using analytical and spectroscopic methods such as IR, 1 H, 13 C NMR, ESR and MS. The cytotoxicity studies showed that copper(II) metal chelate mediated cancer cell death is highly dependent on the ROS generation which was confirmed by invitro studies.The observed cytotoxicity is correlated with the nature of the metal ion. The results indicated that copper(II) complex is the most valuable cytotoxic compound from the four metal complexes with lowest IC 50 values and high selectivity index against the cervical cancer cell line, with no cytotoxic effect on normal human mammary epithelial cell line (MCF-10A).Molecular docking studies was utilized to screen the binding affinity of the synthesized compounds towards the HPV16-E6 receptor. The computational prediction was in agreement with the findings achieved from the experimental studies. Hence, the synthesized copper(II) metal chelate can be suggested as a potential anticancer candidate worthy of further investigation.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.