Design, Synthesis And Invitro Biological Evaluation Of Benzothiazole, Indole And Imidazole Derivatives As Anti Tubercular Agents Targeting Glutamine Synthetase-1

Due to the emergence of multidrug-resistant TB (MDR-TB) and extensively drug resistant (XDR) TB, the disease becomes a major health security threat globally 2 . In order to eliminate DRTB there is an urgent need of novel molecules which are very effective with lesser side effects. Hence this research work focused to work on synthesis and biological evaluation of benzothiazole, indole and imidazole derivatives as anti tubercular agents. Benzothiazole, indole and imidazole moieties were designed and docked against glutamine synthetase (PDB ID-3ZXR) using Autodock ® tools software. Compounds with minimum binding energy were selected and screened for in silico toxicity prediction using osiris ® software and in silico drug likeness prediction by using molinspiration ® software. The compounds were synthesized and characterized by IR, 1 H NMR and LC-MS. Anti-tubercular activity was evaluated by Microplate Alamar Blue Assay (MABA) method. Among the synthesized compounds, indole derivatives and imidazole derivatives was found to be active at micro gram level. point and Thin layer chromatography. The Melting point of the synthesized compounds was determined in open capillary tube and values are reported uncorrected. Thin layer chromatography was done to assess the course of reaction and the purity of the intermediates and the nal compounds. Visualization of the compounds on chromatographic plates was done by exposure to iodine vapors. The IR absorption spectra were recorded by ABB MB 3000-PH FT-IR Spectrometer using KBr disk. 1 H-NMR spectra were recorded on Bruker Advance 500 (300MHz) Spectrometer in CDCl3/DMSO-d6 as a solvent, the chemical shifts are expressed in ppm using TMS as internal standard. Mass spectra were measured using a high-resolution GC-MS.


Introduction
Tuberculosis is an infectious disease that usually attacks the lungs and also spread to other parts of the body, like brain and spine. Today TB has become a major health threat due to the emergence of Multi-drug resistance tuberculosis (MDR-TB), Extensively drug resistance tuberculosis(XDR-TB) and Totally Drug Resistant (TDR) Benzothiazole is a privileged bicyclic ring system consists of phenyl ring fused with thiazole ring. Derivatives of benzothiazole possess important pharmacological activities like anti-diabetice, anticonvulsant, analgesic and antiin ammatory activities [3]. Indole nucleus continuously drawing interest for development of newer drug moiety due to its wide range of pharmacological activities. Indole derivatives were reported to have antioxidant, antibacterial and antitubercular activities [4,5]. A new series of 1H-indole-2, 3-dione derivatives were reported for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv [10]. As per literature surveys, Imidazole derivatives are also well known for its pharmacological activities including antimycobacterial, antitubercular [6,7].
Hence we decided to design and synthesis benzothiazole, Indole and Imidazole derivatives as anti tubercular agents against target enzyme.
There are various biosynthetic enzymes that are essential for the survival of the Mycobacterium and are considered as potential drug targets. As per reported, a comprehensive in silico target identi cation pipeline for Mycobacterium tuberculosis, there are a total of 451 high-con dence targets [8]. Glutamine synthetase-1 is one of the key enzymes, which is critical for the survival and growth of MTB [9]. Glutamine synthetase, takes part in nitrogen metabolism, catalyses the formation of glutamine from glutamate and ammonia in the presence of adenosine triphosphate (ATP). MtGS plays an important role in cell wall biosynthesis, speci cally via the production of a poly-L-glutamateglutamine component found exclusively in pathogenic mycobacteria. Hence forth, the inhibition of Glutamine Synthetase secreted by M. tuberculosis is su cient to inhibit the growth of the bacterium, suggesting that MtGS might be a valid target for anti-tubercular agents [10]. Thus Glutamine synthetase selected as target of interest. The enzyme was downloaded from the Protein Data Bank (ID 3ZXR) in the pdb format and used for in silico molecular docking study.     Standard values for the Anti-Tb test which was performed.
Pyrazinamide-3.125µg/ml Cipro oxacin-3.125µg/ml Streptomycin-6.25µg/ml Experimental Molecular Docking Studies: Drug discovery is the process by which new agents are designed or discovered.
Computer Aided Drug Design uses Computational tools and software, helps in the identi cation of new compounds.
The designed molecules were docked against the target protein Glutamine synthetase-1 using AutoDock ® tools 1.5.6 software. It is an automated procedure for predicting the interaction of ligands with biomacromolecular targets. The current version of AutoDock ® tool using the Lamarckian Genetic Algorithm and empirical free energy scoring function, typically will provide reproducible docking results for ligands with approximately 10 exible bonds. The quality of any docking results depends on the starting structure of both the protein and the potential ligand [11,12]. The IR absorption spectra were recorded by ABB MB 3000-PH FT-IR Spectrometer using KBr disk. 1 H-NMR spectra were recorded on Bruker Advance 500 (300MHz) Spectrometer in CDCl3/DMSO-d6 as a solvent, the chemical shifts δ are expressed in ppm using TMS as internal standard. Mass spectra were measured using a high-resolution GC-MS.

Biological Evaluation
The designed and synthesized molecules need to be screened for their activity to inhibit the growth of the Mycobacterium tuberculosisusing microplateAlamar Blue assay (MABA).Alamar blue dye is used as an indicator for the determination of viable cells.The oxidized form, Resazurin is non-toxic, non-uorescent and blue in colour which becomes pink and uorescent upon reduction to resoru n by viable cells [13,14].

Conclusion
Page 8/10 The research work concludes that the synthesized anti-tubercular compounds might effectively inhibit the chosen target Glutamine Synthetase 1 which is essential for the Mycobacterial Tuberculosis.
To conclude, a series of benzothiazole Schiff bases were designed, docked, synthesized and evaluated against Glutamine synthetase-1 enzyme which is critical for the survival and growth of MTB. The results shown Minimum Inhibitory Concentration in the range between 100-6.25µg/ml. Compound "d" was found to be as sensitive as streptomycin (6.25 µg/ml) and other compounds found to have less activity when compared to the standard drugs.
A series of Schiff bases of isatin derivatives as Schiff bases were designed, docked, synthesized and evaluated for anti tb activity. The Minimum Inhibitory Concentration of the synthesized compounds ranges from 50-1.6μg/ml. The compounds "g" and "i" showed activity at 1.6 µg/ml concentrations which is compared to the activity of the standard drugs. A series of imidazole derivatives synthesized and tested against mtb activity. The Minimum Inhibitory Concentration was found in the range between 3.125-50µg/ml. Compound "o" showed better antitubercular activity at 3.125 µg/ml concentrations which are comparable to the activity of the standard drug pyrazinamide. The standard drugs Pyrazinamide, Cipro oxacin and Streptomycin shows anti-mycobacterial activity at 3.125µg/ml, 3.125µg/ml and 6.25µg/ml concentration respectively. Further in vivo and clinical studies required to con rm the activity of the synthesized compounds. Figure 1 Molecule a Vs (pdb id-3ZXR) Figure 2 Molecule e Vs pdb id-3ZXR)

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. meenaSupporting les.docx Schemes.pdf