CORD-19:0a04d377a83b28dbc0d97c90acd829255796f69c JSONTXT 9 Projects

Journal Pre-proof Anticancer activity studies of novel metal complexes of ligands derived from polycyclic aromatic compound via greener route Abstract Remedy for cancer is certainly one of the big medical challenges to the scientists of 21 st century and is one of the current research hotspots. The escalating research to fight against cancer is getting us closer to a future, where cancer becomes a curable disease. As a researcher, we dream of curing cancer as easy as the deadly diseases viz., chickenpox, smallpox, polio, measles, sars (severe acute respiratory syndrome), yellow fever, malaria etc., [1] . Although existing drugs show efficacy, their lack of selectivity for tumor cells over normal cells can lead to severe side effects [2] . Cancer is caused by mutations that transform healthy cells into tumor cells. Recent studies have demonstrated that there will not be a match between tumors of two patients. It implies that the scientists should work on medicines that are tailored to cure a specific patient. Platinum based drugs are actively used in treating various types of cancers; however, drug resistance, adverse side effects and low specificity are common in platinum chemotherapy [3, 4] . To overcome these abnormalities to great extent, in recent years other metal based drugs have been developed and tested for their anticancer efficacy [5] . Important ones are the coordination compounds of Ni(II), Cu(II), Zn(II) and Ru(II) which can overcome the limited activity of cisplatin and its analogous [6, 7] . Our current research puts in effort to crack a new anticancer drug candidate which could prove to be a better medicine. In our present work, the hydrazone Schiff base ligands derived from 9-oxo-9H-fluorene-1-carboxylic acid and their respective Co(II), Cu(II) and Ni (II) complexes are synthesized impeccably and analyzed. Hydrazones play an important role in inorganic medicinal chemistry. They can easily form stable complexes with transition metal ions and can exhibit interesting photophysical and biological properties [8] . They exhibit biological activities such as antibacterial [9] , antioxidant [10] , antifungal [11] , antitubercular [12] , antiinflammatory [13] and anticancer [14] . Fluorenone is no doubt a polycyclic aromatic compound, one could doubt about its therapeutic property. But the literature reveals that derivatives of fluorenone have exhibited interesting biological activities. Arylsulfonylspiro[fluorene-9,5΄-imidazolidine]-2΄,4΄-diones are found to be effective as aldose reductase inhibitors [15] . Azabenzo[a]fluorene-5,6-diones have comparable cytotoxicities against cancer cell lines in vitro, with that of standard drug doxorubicin [16] . Our previous publication [17] have successfully established the anticancer efficacy of copper complex of fluorenone based ligand; which exhibited better anticancer activity against human breast carcinoma (MCF-7) cell line when compared to that of standard drug paclitaxel. These results prompted us to synthesize new ligands which are embedded with fluorenone moiety, carboxylic acid, carboxamide and hydrazone groups. The metal complexes are synthesized from these ligands via eco-friendly routes employing green solvents. The ligands and respective metal complexes were tested for their anticancer activities with MTT assay against breast cancer (MCF-7) cell line, DNA cleavage study was performed using pBR322 plasmid DNA. Antioxidant activity study was carried out through DPPH free radical-scavenging ability assay. Molecular docking studies were also carried out to examine the bonding mode of synthesized compounds. All the raw materials including reagents, catalysts, solvents and drying agents were of fine chemical grade and utilized as received by the vendor. The MTT [2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazol-3-ium bromide] dye was purchased from Sigma, St. Louis, MO, USA. Infrared (IR) spectra of the synthesized materials were recorded in the range of 4000-550 cm -1 with PerkinElmer Spectrum Two FT-IR spectrometer. By using TMS as internal reference compound, 1 H and 13 C NMR spectra were recorded in DMSO-d 6 solvent on JEOL 400 MHz and Bruker 400 MHz spectrometer at room temperature. Using ThermoQuest Elemental Analyzer, elemental analyses of the compounds was carried out. Electronic spectra of ligands and their metal complexes were recorded on a PerkinElmer LAMBDA 365 UV-Vis spectrophotometer in the range of 1100-200 nm. Thermal analysis of the metal complexes was carried out with an SDT Q600 analyzer, by increasing the temperature from RT to 1000 °C at the rate of 10 °C min -1 . The molar conductance measurements were made on ELICO CM 180 conductivity meter with a cell constant of 1.0 after calibration with standard KCl solution at 25 °C. LC-ESI-MS spectra were recorded on LCMS 2010A, SHIMADZU instrument. 9-Oxo-9H-fluorene-1-carboxylic acid is prepared from fluoranthene using potassium dichromate as an oxidizing agent as per the protocol reported in our previous publication [18] An equimolar mixture of 9-oxo-9H-fluorene-1-carboxylic acid (2.5 g, 11.15 mmol) and benzohydrazides (4-methoxy and 4-tert-butyl derivatives) (11.15 mmol) is refluxed in 50 mL methanol at 70 °C for 8 h (Scheme 1) [19] . The contents are partially soluble at RT. About 30 min of heating at 70 °C, the reaction mixture turns orange to yellow and yellow solids separate, which indicates the formation of desired product. The reaction is monitored by TLC in 9: 1 chloroform: methanol. The solids are filtered and washed with cold methanol and dried under vacuo at 40 °C. L 1 H: Yield: 3.9 g; 94%. Color: Canary yellow. L 2 H: Yield: 4.1 g; 92%. Color: Canary yellow. Copper (II) complex: The copper(II) complexes are synthesized in water at room temperature. The effect of ligands and their metal complexes on the viability of breast cancer cells was determined using the standard colorimetric MTT [2-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-2Htetrazol-3-ium bromide] assay [20] . The monolayer cell culture was trypsinized and the cell count was adjusted to 1.0 X 10 5 cellsmL -1 using DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS and seeded to 96-well microtiter plates (Falcon, Becton-Dickinson, Franklin Lakes, NJ, USA). After 24 h; of plating, cells were serum starved for 24 h. Respective concentrations (10, 20, 30, 40 and 50 µgmL -1 ) of ligands and their metal complexes were added to serum free medium and the assay was terminated after 48 h. Medium was removed and 200 µL of DMSO was added and the amount of formazan formed was measured at 595 nm on a 6 Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The percentage growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit cell growth by 50% (IC 50 ) is generated from the dose-response curves for each cell line. This assay is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product [21] . Inhibition Percentage = OD of Test sample ÷ OD of control × 100 All experiments were performed in triplicates (n = 3) and the numerical data are presented as mean ± standard deviation (SD). The DNA cleavage activity of the newly synthesized ligands and their metal complexes was monitored by Agarose gel electrophoresis on pBR322 DNA (200 ng) in TAE buffer (40 mM Tris-Acetate, 1 mM EDTA) to yield a total volume of 10 µL and then incubated in dark for 30 min at 37 °C. Different concentrations of compounds were tested; 10, 30, 50 and 100 µgmL -1 . The reaction was incubated at 37 °C for 2 h and 3 µL of loading buffer (bromophenol blue in H 2 O) was added to each tube and the mixed samples were loaded on 1% Agarose gel. The electrophoresis was carried out for 2 h at 50 V in Tris-acetate-EDTA buffer (pH 8.3). After electrophoresis, the gel was stained with Ethidium bromide (EB) for 30 min prior to being photographed under UV light [22, 23] . The cleavage properties were determined based on the ability of compounds to convert the supercoiled form (Form I) into nicked form (Form II). The results were compared with standard DNA marker and control DNA. In the present anticancer docking study B-cell lymphoma-extra large (BCl-XL) protein (PDB ID: 3ZK6) was used as drug target in molecular docking studies to computationally prove the anticancer potentialities of newly synthesized metal complexes and ligands. The crystal structure of the BCL-XL protein bound to its respective inhibitor was collected from Protein Data Bank (PDB) database. The site to which the inhibitor molecule was bound was identified using PDBSum server. Optimization of target protein by removal ligands and hetero atoms was carried out using Discovery Studio tool. 3D coordinates files of newly synthesized compounds in ".pdb" format were generated using marvin sketch tool. Further these files were converted from ".pdb" to ".pdbqt" file format using Autodock 4 tool. Automated molecular docking was carried out to determine the best orientation of the newly synthesized compounds possessing anti-cancer property was hypothesized as inhibitors bound in the active site of BCL-XL protein. A Lamarckian genetic algorithm implemented in the Auto Dock 4.0 program was employed [24] . In the present study, radical scavenging activity of ligands and their respective metal complexes was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical as a reagent [26] . Required volume of a DPPH radical solution in ethanol (60 µM) was mixed with different concentrations of testing samples. The mixture was incubated for 30 min in dark at room temperature. After the incubation, the absorbance of the reaction mixture was measured at 517 nm using a UV-Vis Spectrophotometer. For the positive control ascorbic acid was used as a reference standard. The DPPH scavenging activity of each sample was calculated using the following equation: Where, A c is the absorbance of the control reaction (100 µL of ethanol with 100 µL of the DPPH solution) and A t is the absorbance of the test sample. The experiment was done in triplicate. The IC 50 value was calculated for all the samples. Lower absorbance of the reaction mixture indicates higher free radical activity. To identify the bonding mode as well as the complexation behavior, the IR spectra were recorded for the prepared hydrazone Schiff base ligands and their respective metal complexes. The IR Table 1 . coordination to the central metal ion [27] . The absence of broad band in the region 2400-3400 cm -1 infers the coordination of carboxylic acid oxygen via deprotonation. Further, the strong band due to ν(C=O) of carboxylic acid group has disappeared upon complex formation, since in carboxylate ion the negative charge is equally distributed on two oxygen due to resonance, thereby decreasing the bond order. This implies that the carboxylate ion is involved in bond formation [28] . Simultaneously, two new bands have appeared in the range of 1520-1580 cm -1 and 1370-1380 cm -1 which are assigned to the asymmetric and symmetric stretching frequencies of coordinated carboxylate ion respectively. In Cu(L 1 ) 2 The 1 H NMR spectrum of ligand 1 (L 1 H, spectrum 5 of supplementary material) shows a broad singlet centered at 12.15 ppm due to amido proton (-NH-). Proton attached to carboxylic acid oxygen sometimes due to exchange with moisture in the solvent will not appear. The characteristic singlet at 3.87 ppm is due to the resonance of methoxy protons. Remaining protons are aromatic and appeared as multiplets between 7.0-9.0 ppm [31] . 1 H NMR (400MHz, DMSO- Electronic spectra of ligands 1 (L 1 H) and 2 ( respectively, of supplementary material) shows one broad peak due to spin-allowed d-d transition centered at 707 and 717 nm assignable to 2 T 2g 2 E g as is the consequence of distorted octahedral environment around copper (II) ion [36, 37] . Thermal behavior of all the metal complexes was studied over a temperature range of 25 Molar conductance of all the metal complexes was measured at 25 °C by preparing mmol solutions in DMF using the conductivity bridge (G* = 1). The values obtained for the complexes not exceeded 20 Ω -1 cm 2 mol -1 which confirms the non-electrolytic nature of metal complexes [39] . The molar conductance values have been reproduced in Table 3 along with the elemental analyses data which are in well agreement with the calculated percentages of each element. The detailed experimental results of molar conductance study have been tabulated in Table 4 . In the present investigation, in vitro MTT cell viability model was taken to study the cytotoxic In case of all tested drugs, dose dependant activity was observed with salient features. The IC 50 value of L 1 H, Cu(L 1 ) 2 , Cu(L 2 ) 2 , Ni(L 1 ) 2 and Ni(L 2 ) 2 compounds is found to be less than 10µgmL -1 and other tested drugs also demonstrated very promising activity (Table 7) . Compared with control group, the cells treated with ligands and metal complexes showed significant detach in culture medium. There was a decrease in number of cells as well as observable abnormalities in the treated group of cells such as turgidity, shrunken in shape, cell breakage, cell shrinkage and apoptotic bodies are observed compared to untreated control cells. Microscopic examination revealed that morphological changes and shrinkage of cells occurred, leading to cell apoptosis induced by tested compounds [40, 41] . IC 50 values of ligands and their respective metal complexes in µgmL -1 for the MCF-7 cell line are compiled in Table 5 . cell line v/s concentration in µgmL -1 . Designing small molecules help in targeting specific sites on a DNA strand and can lead to novel therapeutic agents. Photocleavage study of DNA also helps in various applications like photodynamic therapy of cancers, DNA foot printing agents and in genomic research [42, 43] . Interaction of metal complexes with pBR322 DNA was studied using Agarose gel breaks and smear appearance [44, 45] . Overall findings have revealed that the tested compounds can successfully cleave the DNA as it is evident from the images reproduced in Fig. 9 . In Table 8 . Free radicals lead to the severe causes and implications in the occurrence of various diseases such as liver cirrhosis, atherosclerosis, cancer, diabetes and aging [47] . Antioxidants play an important role in the control of free radicals by delaying or inhibiting its activity. Table 9 Experimental results of L 1 H and its metal complexes on DPPH radical scavenging model. In the current study, two new methoxy and t-butyl substituted hydrazone Schiff base ligands and their metal complexes were synthesized. The key precursors, ligands and their transition metal complexes were characterized by spectral ( 1 H and 13