Synthesis , Antimicrobial Activities of New Sulfonamidobenzoxazoles and Molecular Docking Studies on Escherichia coli TEM-1 β-Lactamase

β-Lactam antibiotics are frequently used for treatment of multi-drug resistant microbial infections and the most common mechanism of resistance against these antibiotics is bacterial β-lactamase production. Herein, we reported the design, synthesis and in vitro antimicrobial activities of some new 2-substituted-5-(2,4-dinitrophenylsulfonamido)benzoxazole derivatives. Compounds TN1, TN2, and TN3 were found to be significantly active against E. coli isolate which contains extended spectrum β-lactamase enzyme at the MIC value of 8 μg mL–1 and that is 4-fold higher than the reference drug ampicillin. We performed molecular docking studies into active site of Escherichia coli TEM-1 β-lactamase enzyme in order to predict the protein-ligand interactions. According to the docking results, compounds TN1, TN2, and TN3 showed strong interactions between the important active site residues which are responsible for the catalytic mechanism of TEM-1 β-lactamase enzyme and a good correlation is found with the experimental data.


INTRODUCTION
HE need for new effective antimicrobial agents to prevent the diseases caused by the fastest-growing prevalence of multi-drug resistant microbial infections, still maintains its importance.For treatment of these infections β-lactam antibiotics are frequently used and the most common mechanism of resistance against these antibiotics is bacterial β-Lactamase production.The Extended Spectrum β-lactamase (ESBL) are enzymes that produced by Enterobacteriaceae family including Escherichia coli [1][2][3] and the enzymes are derived from broad-spectrum beta lactamase TEM-1, TEM-2 or SHV-1 by a limited number of mutations. [4]ESBL enzymes hydrolyze the amide bond of βlactam ring of penicillins, cephalosporins and related antibiotics, thereby inactivating them and often cause diseases in clinics and hospitals worldwide. [5]ulbactam, tazobactam, and clavulanate are known as efficient β-lactamase inhibitors however, their efficiency is restricted to class A β-lactamases. [4,6,7]Escherichia coli TEM-1 β-lactamase enzyme belongs to class A β-lactamases and have a broad substrate specificity. [8]In recent years, numerous compounds have been reported as β-lactamase inhibitors.Eidam et al. synthesized sulfonamide boronic acid derivatives and reported their higher β-lactamase inhibitory activities. [9]dditionally, a series of phenylethenesulfonamide derivatives found to be the potent inhibitors of TEM-1 β-lactamase enzyme. [10]13][14][15] Herein, we designed and synthesized some new 2substituted-5- (2,4-dinitrophenylsulfonamido)benzoxazole derivatives, which consist of both benzoxazole and sulfonamide moieties, and tested their in vitro antimicrobial activities.Furthermore we performed molecular docking studies into active site of Escherichia coli TEM-1 β-lactamase enzyme in order to predict their protein-ligand interactions.

Chemistry
All of the solvents and chemicals were purchased from commercial vendors and were used without purification.The melting points were uncorrected and measured on Buchi B540.FTIR spectra were obtained on a Agilent Technologies Cary 630 FTIR spectrometer. 1 H NMR and 13 C NMR spectra were obtained on a VARIAN Mercury 400 MHz FT spectrometer, chemical shifts were expressed as ppm, and coupling constants (J) were expressed as hertz.Mass spectra were obtained on a Waters Micromass ZQ using the ESI method.Analytical data were obtained on elemental analyzer system Leco CHNS-932 CHNS-O analyzer and the results (C, H, N, S) were found within ± 0.4 % of the calculated amounts.

Molecular Docking
The crystal structure of the TEM-1 β-lactamase enzyme of E. coli was selected for molecular docking studies (PDB ID: 1ERQ). [20]Protein and ligands were prepared by using Accelrys Discovery Studio 3.5 software. [21]Target protein, TEM-1 β-lactamase enzyme was taken, hydrogens were added the ligand was extracted, and optimized using the all atom CHARMm forcefield and the Adopted Basis set Newton Raphson method until the root mean deviation (RMS) gradient was < 0.05 kcal / mol / Å 2 .By using the binding site module, minimized protein was defined as the receptor.The binding site was defined from the cavity finding method and modified that contain all of the important active site residues of the β-lactamase enzyme.Binding sphere (40.78, 36.48,31.78,8.27) was selected from the active site.The most active compounds against E. coli isolate (TN1, TN2, TN3) and the boronate inhibitor that is the ligand of 1ERQ.pdbcrystal structure were sketched, all atom CHARMm forcefield parameterization was assigned and then minimized using the ABNR method as described above.Conformational searches of the ligands were performed using a simulated annealing molecular dynamics approach.The ligands were heated to a temperature of 700 K and then annealed to 200 K. CDOCKER method was performed by using Discovery Studio 3.5. [22]TEM-1 β-lactamase enzyme was held rigid but the ligands were allowed to be flexible during refinement.At first the methodology was validated by docking of boronate inhibitor.The docked position of boronate inhibitor overlaps well with an RMSD of 1.3 Å with the crystal structure position.Afterwards molecular docking studies were performed on the compounds TN1, TN2, TN3.All docked poses were scored by applying Analyze Ligand Poses subprotocol and binding energies were calculated by using in situ ligand minimization step (ABNR method) and using implicit solvent model (GBMV) in Discovery Studio 3.5 software.The lowest binding energy was taken as the best-docked conformation of the compounds for the macromolecule.The pictures were taken by using Discovery Studio 4.1 visualizer.

Chemistry
In this study, 2-(4-substitutedphenyl/benzyl)-5- (2,4dinitrophenylsulfonamido)benzoxazole derivatives (TN1-TN14) were synthesized for the first time.16] Then 2,4-dinitrobenzenesulfonyl chloride and 5-amino-2substitutedbenzoxazole derivatives were treated in pyridine and dichloromethane to obtain 5-(2,4dinitrophenyl-sulfonamido)benzoxazole derivatives (TN1-TN14) (Scheme 1). [16,17]Synthesized structures were characterized by 1 H NMR, 13 C NMR, Mass Spectra and Elemental Analysis and the results are in agreement with the proposed structures.According to the 1 H NMR spectra of the compounds the signals of NH (SO2NH) proton of the compounds was observed at 11.05-11.17ppm.Aromatic CH3 and benzylic CH2 protons were appeared at 2.24-2.40ppm and 2.71-4.30ppm, respectively.All the aromatic protons were observed at 7.07-8.39ppm. 13C NMR spectra were appropriate to formulas of the synthesized compounds and Mass spectra showed M + + H peaks in accordance with their formulas.Additionally, elemental analyses results of C, H, N, S were found within ± 0.4 % of the calculated amounts.Table 1.The structures of the tested benzoxazoles and their in vitro antimicrobial activities as MIC values (μg mL -1 ).

CONCLUSION
Herein, we designed and synthesized some new 2substituted-5-(2,4-dinitrophenylsulfonamido)benzoxazoles and tested their in vitro antimicrobial activities.All of the tested compounds showed significant activity against the tested microorganisms especially extended spectrum β-lactamase containing E. coli isolate.Compounds TN1, TN2, and TN3 were found to be significantly active with the MIC value of 8 µg mL -1 against the extended spectrum β-lactamase containing E. coli isolate, which was resistant to all β-lactam antibiotics.In order to predict the protein-ligand interactions we performed molecular docking studies into active site of E. coli TEM-1 β-lactamase enzyme.According to the docking results, compounds TN1, TN2, and TN3 showed strong interactions between the important active site residues which are responsible for the catalytic mechanism of TEM-1 β-lactamase enzyme, such as Ser70, Ser130, and Asn132.A good correlation was noticed between the docking scores and the microbiological data.It can be concluded that these compounds could show their activity by inhibiting the β-lactamase enzyme.The compounds obtained from this study can be useful in designing of new potent β-lactamase inhibitors by using them as lead compounds.