Synthesis , Antimicrobial , Antioxidant and Molecular Docking Study of Some Novel Bis-1 , 2 , 4-Triazolo [ 3 , 4-b ]-1 , 3 , 4 -

A novel series of 1-aryl-3,4-bis-(3-alkyl/phenyl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazol)-1H-pyrazole (5a-i) are synthesized by the cyclocondensation of 1-(aryl)-1H-pyrazol-3,4-dicarboxylic acids with 3-alkyl/aryl-4-amino-5-mercapto-1,2,4-triazoles. Pyrazole dicarboxylic acids were prepared by the 1, 3-dipolar cyclo addition of 3-aryl sydnones with dimethylacetylenedicarboxylate (DMAD). The newly synthesized compounds were studied for their antibacterial, antifungal and antioxidant activities. Particularly compounds 5a and 5g showed considerable antibacterial activity against the standard drug, while all the tested compounds displayed poor inhibitory effect against fungi. Compound 5d exhibited good antioxidant activity. The docking study was performed with Acinetobacter baumannii penicillinbinding protein target using AutoDock 4.2, which proved H-bond interaction and strong binding affinity.


Materials and Methods
All the reagents and solvents were purchased from Sigma-Aldrich or Hi-Media and used after distillation/ recrystallization. 1 H NMR spectra were recorded on Bruker Avance II NMR spectrometer operating at 400 MHz and all the chemical shift values were reported in parts per million (ppm) relative to tetramethylsilane (TMS).Mass spectra were acquired on a SHIMADZU LCMS-8030 mass spectrometer.Melting points of the synthesized compounds were determined in open capillary tubes in Innovative DTC-967A digital melting point apparatus.SHIMADZU FT-IR 157 spectrophotometer was used for recording IR spectra.C H N analysis was performed with Vario-EI Elementar-III model analyzer.In-silico study was done using Auto Dock 4.2.

Introduction
The present scenario in synthetic chemistry has been focused on designing new molecules by the lead hybridization-based synthesis of different pharmacophore fragments in a single molecule with improved biological efficacy.

Assay of in vitro antibacterial activity
Bacterial and fungal strains were purchased from National collection of industrial microorganisms, Pune, India.Antibacterial activity was tested against Gram-positive bacteria Staphylococcus aureus (NCIM -5021), Bacillus subtilis (NCIM 2197) and Gram-negative bacteria Escherichia coli (NCIM-2931), Pseudomonas aeruginosa (NCIM-2036) using Ciprofloxacin as the reference drug.Antifungal activity of the newly synthesized compounds was tested against two fungi namely Candida albicans (NCIM 3471) and Aspergillus niger (NCIM 3452) using Fluconazole as the reference drug.
The sterilized nutrient agar medium was distributed 100 mL each in two 250 mL conical flasks and allowed to cool to room temperature.To these media, 18-24 h grown bacterial/fungal sub-cultures were added and shaken thoroughly to ensure uniform distribution of organisms throughout the medium.Then, agar medium was distributed in equal portions, in sterilized Petri dishes, ensuring that each Petri dish contains about 45-50 mL of the medium.The medium was then allowed for solidification.The cups were made with the help of a sterile cork borer (6 mm diameter) punching into the set of agar media.The solutions of required concentrations (100 µg/mL) of test compounds were prepared by dissolving the compounds in DMSO were filled into the cups with 1 mL of respective solution.Then, the Petri dishes were kept for incubation in an inverted position for 24-48 h at 37 °C in an incubator.When growth inhibition zones were developed surrounding each cup, their diameter in mm was measured and compared with that of the standard drugs [17,18].Each experiment was made in triplicate using DMSO as a control.

Molecular Docking studies
The binding interaction between macromolecule and ligands was done using AutoDock 4.2.Lamarkian genetic algorithm was used to study the docking calculation generated few poses for ligand molecules with the protein target [19].Polar hydrogen bond network was optimized and the systematic Kollaman charges were added by means of a cluster-based approach.The grid map which was centered was predicted from the ligplot.In all the cases, we have used grid maps with a grid box size of 60×60×60 Ǻ 3 points with a grid-point spacing of 0.375 Ǻ.During docking, centre grid parameters were specified for x, y and z axis as -55.15, -35.444 and 42.741, respectively.The Lamarckian genetic algorithm, the pseudo-Solis and Wets methods were applied for minimization using default parameters.Binding energy, torsional energy, intermolecular energy, number of H-bonds and RMS value were recorded in each ligand bound.

Assay of in vitro antioxidant activity
The free radical scavenging activity of test sample was measured by DPPH scavenging assay.Free radical scavenging activity of the test compounds was carried based on the scavenging activity of stable DPPH. 100 µg/mL of each test sample and standard BHA was taken in different test tubes and the volume was adjusted to 1mL using DMSO.Freshly prepared 1mL of 0.1 mM DPPH solution was mixed and vortexed thoroughly and left in dark for 30 min.The absorbance of stable DPPH radical was measured at 517 nm.The DPPH control was prepared using the same procedure.Radical scavenging activity was expressed as the inhibition percentage and was calculated using the equation [20] .

DPPH radical scavenging activiy (%) = (A
Where A Control is the absorbance of DPPH radical+methanol; A Sample is the absorbance of DPPH radical+test sample/standard BHA.

Results and Discussion
Synthesis 3-Aryl substituted sydnones 1 were obtained by the reaction of appropriately substituted aniline with ethyl chloroacetate followed by hydrolysis, nitrosation and cyclization with acetic anhydride [21] (Scheme 1).These sydnones 1 when treated with DMAD underwent 1, 3-dipolar cycloaddition reaction to give 1-aryl-1H-pyrazole-3, 4-dimethylcarboxylate 2. Hydrolysis of The structure of newly synthesized compounds was confirmed by 1 H-NMR, IR, LCMS and C, H, N analysis.The absence of S-H and NH 2 absorption bands confirmed the formation of product.The IR spectra of compound 5a-5i showed absorption peak at 1506-1516 cm -1 which is attributed to the stretching vibration of C=N.The characteristic absorption band due to C-S stretching was observed at 1070-1082 cm -1 , whereas C-H stretching bands at 3059-3076 cm -1 associated with the aromatic rings were observed in all the molecules. 1H-NMR spectrum of compound 5e showed multiplet of methyl protons at δ, 1.36-1.39ppm integrating for six protons.A multiplet due to four methylene protons was observed at δ, 3.05-3.17ppm.
The peaks due to aromatic protons were seen at δ, 7.62-7.66,8.04 and 7.5-7.54ppm pertain to ortho, meta and para protons of phenyl ring of pyrazole.While the proton of pyrazole ring displayed as a singlet at δ, 9.58 ppm.Further evidence for the formation of triazolo-thiadiazoles (5a-i) was obtained by recording mass spectra, where molecular ion peaks obtained were in consistence with their molecular formula.

Antimicrobial Studies
All the newly synthesized triazolo-thiadiazoles (5a-i) were investigated for antibacterial and antifungal activity.Compound 5a showed good activity against Bacillus subtilis and compound 5g showed good activity against Pseudomonas aeruginosa.None of the compounds showed any considerable antifungal activity (Table 1).
The ligand molecules, 5a, 5b and 5g revealed binding energy of -8.32, -8.13 and -7.83 kJ/mol, with ligand efficiency of -0.27, -0.25 and -0.26, respectively.The completely wrapping of the molecules by amino acid residues at the active site pocket region as displayed in Figure 1.In 5a, the oxygen atom of methoxy group displayed H-bonding interaction with the hydrogen atom of Ser434 at a distance of (2.174) Å, while the sulphur atom present in thiadiazolotriazole ring of the compound 5d, 5e and 5g was involved in the H-bonding with the active site of amino acid residue Tyr485 at a distance of (2.667), (2.872) and (2.816) Å, respectively as depicted in Figure 2. The docking study results showed that the molecules 5a-5h has good inhibition constant, vdW + H-bond + desolv energy with best RMSD value.The details of docked score results of the molecules are given in Table 2.