DESIGN, SYNTHESIS, MOLECULAR DOCKING, ADMET STUDIES AND BIOLOGICAL EVALUATION OF PYRAZOLINE INCORPORATED 1, 2, 3-TRIAZOLE BENZENE SULPHONAMIDES

Objective: The main objective of this work was to design, synthesize and evaluate the novel pyrazoline incorporated 1,2,3-triazole benzene sulphonamides for cytotoxic and anti-gout activities also to perform Insilco molecular docking studies. Methods: Designed compounds were synthesized by condensation of different substituted chalcones (3a-i) with hydrazine hydrate and substituted phenylhydrazines. All the synthesized compounds were characterized on the basis of physical and spectral data. To predict the affinity and activity of the ligand molecule Libdock program was employed to generate different bioactive binding poses of designing molecules at the active site of protein Phosphatidylinositol 3-kinase (PI3Kα). Title compounds were evaluated for cytotoxic activity by using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and anti-gout activity by potassium oxonate induced assay. Results: All the synthesized compounds showed characteristic peaks in FTIR, 1 H, 13 C NMR and MASS spectral analysis. In molecular docking studies, compound 3i has shown good binding affinity to the active site of PI3Kα with a docking score of 145.031 and 4 hydrogen bonding interactions with least hepatotoxicity and good bioavailability when compared with that of reference ligand KKR exhibited a Libdock score of 88.35. Remaining compounds also have a good binding affinity with a minimum of 2 bonding interactions and having better absorption, distribution, metabolism, elimination and toxicity (ADMET) profile. The same compound (3i) exhibited the highest cytotoxic activity with an IC 50 value of 4.54µg/ml. Compound 4d was evaluated for anti-inflammatory activity and it has significantly ameliorated against potassium oxonate induced gout in mice when compared with that of standard drug allopurinol due to its anti-inflammatory property. Conclusion: We designed and synthesized a novel series of title compounds in quantitative yields and performed docking studies. New derivatives have a good binding affinity towards PI3Kα enzyme, good bioavailability, least hepatotoxicity and significant cytotoxic activity. The level of xanthine oxidase was determined by. Uric acid level was determined in serum by using peroxidase endpoint assay. Blood urea nitrogen level was determined by using berthelot end point assay. Present study indicated that the group of mice given standard reference compound allopurinol at 5 mg/kg body significantly (p<0.05) reduced serum creatinine, uric acid, blood urea nitrogen, and xanthine oxidase enzyme level in hyperuricemic mice.


INTRODUCTION
Cancer is a multifactorial disease, arising from the uncontrolled proliferation of a cell with the potential to invade to other organs of the body [1]. PI3Kα is one of the proto-oncogenes, which has a vital role in the regulation of many important cell signaling pathways including cellular replication, cell proliferation leading to growth and apoptosis [2,3]. The enzyme PI3Kα appeared to involve in 40% of all types of human cancers including breast, gastric, cervical, urinary tract, colon, non-small-cell lung and squamous cell lung carcinomas [4]. Considerable evidence suggest that PI3K (P110α) subunit protein is expected to mutate in these cancers that bring about constitutive downstream pathways leading to defective control of cellular proliferation and malignant transformation, thus PI3Kα is drawing attention in cancer biology [5,6]. Hence targeting PI3Kα may be considered as a promising approach in the design of new anticancer drugs.
Pyrazoles are a class of heterocyclic compounds characterized by 5membered aromatic ring structure composed of three carbon atoms and two nitrogen atoms in adjacent positions. Pyrazole derivatives have a long history of applications in agriculture as herbicides and insecticides as well as in the pharmaceutical industry as antipyretic and anti-inflammatory agents [7][8][9][10][11][12]. Pyrazole derivatives have been reported to show a broad spectrum of biological activity including antibacterial [13], antifungal [14], analgesic [15], anti-inflammatory [16][17][18], neuroprotective [19], estrogen receptor binding [20], antineoplastic [21], activities. Due to their wide range of biological activities, pyrazoles received considerable interest in the field of drug discovery and therefore pyrazole ring constitutes a relevant synthetic target in the pharmaceutical industry. On the other hand, 1,2,3-triazole is an important heterocycle which has gained a lot of interest for researchers in view of its high potency, low toxicity with broad spectrum of activities. Triazole derivatives have been reported to have anticancer [22], anti-inflammatory [23], antibacterial [24], antiviral [25], anti-human immuno virus (HIV) [26], fungicidal [27] and insecticidal [28][29][30] activities. The objective of the current work was to synthesize novel pyrazoline incorporating 1,2,3-triazole benzene sulphonamide derivatives as potent antiproliferative and anti-inflammatory agents. We were well known about the synthesis of simple pyrazolines from chalcones but we designed pyrazoline incorporating 1,2,3-triazolyl benzene sulphonamide derivatives. Inspired by the biological properties of both pyrazole and triazole heterocycles in the present study, we thought of synchronizing both these moieties into a single molecule in order to obtain new hybrid molecules with improved biological activity and low toxicity.

MATERIALS AND METHODS
All the chemicals and solvents used were of synthetic grade from SD fine chemicals Ltd., (Mumbai, India), and avra chemicals pvt ltd Hyderabad. Completion of the reaction was monitored by analytical thin layer chromatography (TLC) using E-Merck 0.25 mm silica gel plates. Visualization was accomplished with ultraviolet (UV) light (256 nm) and iodine chamber. Synthesized compounds were purified by the re-crystallization process. The purity of the compounds was checked by a single spot in TLC and solvent system for TLC was determined on trial and error basis. Melting points were determined in open capillary tubes using ANALAB melting point apparatus and were uncorrected. All the 1 H NMR spectra were recorded on varian 400 MHz spectrometer using DMSO-d6 as solvent and tetramethylsilane (TMS) as an internal standard. Chemical shift values are listed in δ scale. The FT-IR spectra were recorded on schimadzu FT-IR spectrophotometer by using 1% potassium bromide discs. Mass spectra of the compounds were recorded on electronic spin ionization mass spectra (ESI-MS) on aglient 1100 series.
Synthesis of 4-(4-acetyl-5-methyl-1H-1,2,3-triazol-1-yl) benzene sulfonamide (2) Sodium metal was dissolved in 10 ml of absolute ethanol in a dry flask to which azido benzene sulfonamide and 2 ml of acetylacetone were added and stirred for 1 h on magnetic stirrer. Transferred to the round-bottomed flask (RBF) and refluxed at 140 °C for 12-16 h. The completion of the reaction was monitored by TLC. The reaction mixture was poured into crushed ice to obtain the solid product. Then the precipitate was filtered under suction, washed thoroughly with water and recrystallized from aq. methanol.

Molecular docking and ADMET studies
The molecular docking study of synthesized compounds was done by employing the Libdock protocol in order to identify binding interactions with the target protein PI3Kα (PDB ID: 3ZIM). ADMET studies were carried out by using discovery studio software.

Cytotoxic activity
The title compounds were evaluated for In vitro antiproliferative activity via MTT ([3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]) based cytotoxic assay [31] against MCF-7 breast cancer cell line with taxol as standard reference. Cell lines were purchased from the national institute of nutrition (NIN) Hyderabad. Cells were harvested from the logarithmic phase of cultures and re-suspended in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS). The cell counts were adjusted and equal number of cells were plated into each well of 96-well culture plates and allowed to grow overnight at 37 °C, in presence of 5% CO2. The cells were treated with test substances at various concentrations as indicated for 72h. In vehicle control culture wells, a maximum of 0.5% DMSO was added. Culture medium was renewed at every 24h with fresh culture medium supplemented with test substances. Thereafter, 0.5 mg/ml of MTT reagent was added to each well and the microplate was incubated further for 4h at 37 °C in presence of 5% CO2. Finally, the cells were solubilized by adding solubilizing solution and allowed to incubate at 37 °C overnight. After complete solubilization of the formazan crystals the absorbance was read at 540 nm in a microplate reader (Bio-Rad, USA). The results (mean OD±SD) obtained from quadruplicate wells were used in the calculation to determine the cytotoxicity (50% of inhibitory concentration, IC50) of the test compounds.

Evaluation of anti-inflammatory effect of compound 4d against potassium oxonate induced gout in mice
Anti-inflammatory activity of compound 4d in potassium oxonate induced gout in mice was investigated in the present study. Intraperitoneal injections of potassium oxonate 250 mg/kg daily for 28 d has lead to increased levels of serum concentration of creatinine, uric acid, blood urea nitrogen, and xanthine oxidase enzyme level as an indicator of impaired kidney function and increased plasma concentration of the blood parameters such as total leukocyte count, differential leukocyte count and erythrocyte sedimentation rate (ESR) indicates inflammation. Treatment with compound 4d in doses 50 mg/kg and 100 mg/kg for 28 d exhibited a significant improvement in gout disease in mice as evidenced by the decrease in biochemical parameters and inflammation in joints. The total leukocyte count, differential leukocyte count and erythrocyte sedimentation rate were estimated as per reported methods. Creatinine level was determined in serum by using modified jaffe's reaction. The level of xanthine oxidase was determined by. Uric acid level was determined in serum by using peroxidase endpoint assay. Blood urea nitrogen level was determined by using berthelot end point assay. Present study indicated that the group of mice given standard reference compound allopurinol at 5 mg/kg body weight significantly (p<0.05) reduced serum creatinine, uric acid, blood urea nitrogen, and xanthine oxidase enzyme level in hyperuricemic mice.
Male albino mice were obtained from Sainath Agencies Hyderabad. They were acclimatized to laboratory conditions (22±2 ˚C of temperature, 12-hr light/dark cycle), food and water were given ad libitum. After an acclimatization period of 2 w, they were randomly divided into 5 experimental groups.
To evaluate Anti-inflammatory effect of Quinoline-3-carbonitrile derivative against potassium oxonate induced gout in mice. 30 Male albino mice weighing 25-30 gm were randomly divided into five groups of six mice in each group such as:   All the groups except normal control have received potassium oxonate 250 mg/kg intraperitoneally for 28 d and 1 hour later, standard group and treatment groups have received allopurinol (5 mg/kg p. o) and quinoline-3-carbonitrile (low dose-50 mg/kg and high dose-100 mg/kg p. o) respectively. Differential leukocyte count and total leukocyte count were estimated on 7 th , 14 th and 29 th day by collecting blood through retro-orbital plexus. On 29 th day blood samples were collected for estimating biochemical parameters and later on all animals were sacrificed for histopathological studies.
The 1 H NMR spectrum of final compounds contains a singlet around 2.61 ppm for CH3 protons, protons of CH2 and CH of pyrazoline ring appeared as doublet of doublets, triplet of doublets in the range of 3-5 ppm, aromatic protons appeared in the range of 6.5-8.2 ppm and two protons of-NH2 appeared as a singlet at 7.6 ppm whereas one proton of pyrazoline NH appeared as singlet at 7.7 ppm.        -(1,5-Diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl) Table 1   The ADMET screening results obtained for synthesized compounds are summarized in table 2 and compared with standard levels. As per discovery studio (DS) parameters, standard comparable values of human intestinal absorption as level 0, solubility as level 3 and 4, blood-brain barrier (BBB) penetration as level 3, non-inhibitory property with cytochrome-P(CYP450 2D6) as level 0, and non-toxicity as level 0. An ADMET plot was generated for the blood-brain barrier penetration and the intestinal absorption using descriptors AlogP98 and 2D polar surphase area (PSA) that comprise confidence ellipses of 95% and 99%. These ellipses elucidate zones where welloccupied compounds are expected to be settled. The compounds are found to be in the range of 95 and 99 % confidence ellipse for both the intestinal absorption and BBB as shown in fig. 4.

Fig. 3: The hydrogen bond interactions of the compound 3i with the protein PI3Kα
From the analyzed results, all the synthesized compounds showed a BBB level of 4 except for 4k which is 3. The BBB level 4 showing undefined penetration and level 3 indicating a little penetration across the central nervous system (CNS) hence it reduces the side effects linked to CNS. The absorption level was found to be 0 and 1 for all the compounds revealing good and moderate intestinal absorption. For all the compounds, the calculated hepatotoxic level was 1 implying the compounds as toxic. The solubility level 3 indicates very good solubility, level 2 indicates low solubility and level 1 indicates very low solubility or no solubility. All the compounds are found to be having the solubility level 2 except 3l, which was 3, compound 5b was 1. Similarly, compounds having level 0 were found to be satisfactory with respect to CYP 450 2D6 liver enzyme, suggesting that the compounds are non-inhibitors of the metabolic enzyme and those having level 1 suggests that all the compounds are inhibitors of the metabolic enzyme. Finally, the PPB value found to be 2 for most of the compounds indicates that the compounds have binding ≥ 90 % and the compounds 3i, 4l and 5k are found to have 0 which denotes that the compounds have binding ≤90 % clearly reveal that the compounds have good bioavailability and are not likely to be highly bound to carrier proteins in the blood.     All the data are expressed as mean±SEM (n=6). All the data are expressed as mean±SEM (n=6). All the data are expressed as mean±SEM (n=6). All the data are expressed as mean±SEM (n=6). All the data are expressed as mean±SEM (n=6). All the data are expressed as mean±SEM (n=6). Data analysed by one way ANOVA followed by tukeys test. α P<0.05, when compared to normal control. β P<0.05 when compared to disease control. γ P<0.05 when compared to low dose.

CONCLUSION
In this study, we have designed and synthesized a novel series of pyrazoline incorporated 1, 2, 3-triazole benzene sulphonamides and performed docking simulations in order to identify their binding affinity towards the selected target protein PI3Kα and tested for their ADMET profiles. Among all the compounds, compound 3i displayed preferred binding orientations along with strong affinities towards the active site of PI3Kα with better ADMET profile. The synthesized compounds were examined for cytotoxic activity and compound 3i exhibited higher activity with an IC50 value of 4.54 µg/ml. In Anti-inflammatory activity compound, 4d has significantly shown an anti-inflammatory effect on potassium oxonate induced gout and this was mediated by suppressing the inflammatory responses.