Synthesis, Molecular Docking and Antimicrobial Evaluation of Some Benzothiazoles

Abstract The new series of 2-(substituted amino)-N-(6- substituted-1,3-benzothiazol-2yl) acetamide BTC(a-t) has been synthesized by appropriate synthetic route from substituted 2-amino benzothiazole. The synthesized compounds were screened experimentally for its antimicrobial property against gram positive, gram negative bacteria and fungi. Zone of inhibition and minimum inhibitory concentration of compounds was determined against selected bacterial and fungal strains. Compound BTC-j N-(6-methoxy-1,3-benzothiazol-2-yl)-2-(pyridine-3-yl amino) acetamide and compound BTC-r N-(6-nitro-1,3-benzothiazol-2-yl)-2-(pyridine-3-yl amino) acetamide found to have good antimicrobial potential. The compound BTC-j has shown good antibacterial activity against S. aureus at MIC of 12.5 µg/ml, B. subtilis at MIC of 6.25µg/ml, E. coli at MIC of 3.125µg/ml and P. aeruginosa at MIC of 6.25µg/ml. No statistical difference in antimicrobial activity of standard and test compounds was found indicating test compounds have comparable activity. Further docking study was carried out to check the probable interactions with the selected protein using V-life MDS 3.5 software. (DNA gyrase, PDB: 3G75). The dock score of compounds and antimicrobial activity found to be consistent.


Introduction
The emergence and spread of resistance are one of the important problems which is needed to be consider as early as possible. This resistance development has limited the selection of antimicrobials to treat the disease. As the incidences of multidrug resistance increases and number of newer drugs that reaches to market are very less. This situation shows the urgent necessity of newer drugs in market.
DNA gyrase is classified as topoisomerase II, enzyme that play crucial role in the transcription and replication process of DNA molecule. It plays a vital role in all type of bacteria except higher eukaryotes and which makes DNA gyrase as attractive targets for designing new antimicrobial drugs [22].

Chemistry
All the chemicals and reagents used for synthesis were of laboratory grade. The completion of the reaction and purity of all the synthesized compounds was supervised by using chromatography technique. Shimadzu FTIR 8400S was used to record IR spectra by using KBr and the NMR spectra were recorded in NMR Varian-Mercury 300 MHz spectrometer in CDCl3 and values are expressed in ppm.

General synthetic procedures
The general reaction sequence for different title compounds is outlined in Scheme 1.
2.2.1. General synthetic procedure for 6-substituted-2-aminobenzothiazoles (BT [1][2][3][4][5] Method A: BT 1 To 25 ml (0.02mol) Aniline, 25 ml conc. hydrochloric acid was added in a 250 ml beaker and the solution was heated till it becomes warmed. A saturated solution of ammonium thiocyanate in water was prepared and added slowly to the above and boiled until the solution become turbid. The turbid solution was poured into cold water with continuous stirring. Phenylthiourea, separated as precipitate was ltered and recrystallized from aqueous ethanol (80%). To 250 ml round bottom ask containing 75 ml of chloroform, phenylthiourea (0.098mol) was added and stirred it. Bromine in chloroform (5%) was added dropwise to above suspension till an orange-yellow colour appeared, the temperature was maintained at 0-5℃. The reaction mixture was stirred overnight (20 h). The precipitate obtained was ltered and washed with chloroform until the colour disappeared. This precipitate, the hydrobromide salt, was dissolved in acidi ed water (5% HCl) and basi ed with concentrated ammonia solution. The precipitate obtained was ltered, dried and recrystallized using ethanol and water mixture [23].

Biological Activity Evaluation
All target compounds, BTC(a-t) were screened for their antibacterial, antifungal activity. The synthesized compounds were evaluated for their antimicrobial activity against gram positive, gram negative bacteria and fungi by determining-

Minimum Inhibitory Concentration (MIC)
The synthesized compounds were evaluated for their antibacterial activity against two-gram positive (Staphylococcus aureus, Bacillus subtillis) and two-gram negative bacteria (E. coli, Pseudomonas aeruginosa) by using cipro oxacin as reference antibacterial agent. Antifungal activity was evaluated against Candida albicans and Aspergillus niger. uconazole was used as reference drug. The microbial strains were collected from microbiology laboratory of Waghire college, saswad.

Zone of inhibition using cup plate method
The compounds were evaluated for the antibacterial activity by cup plate method. The results were recorded for each tested compound as average diameter of inhibition zone of bacterial and fungal growth around the cup in mm. The nutrient agar medium containing peptone (1%), beef extract (0.5%), sodium chloride (0.8%) and agar (2.5%) in distilled water. The solution was sterilized for 20 min in an autoclave at 15 psi pressure at 121 o C. The basal medium 15-20 ml was poured in the sterile petri-dishes. After solidi cation of medium, the microorganisms were sub-cultured and then holes of 6mm diameter were bored. To these cups, concentration of 50, 100, 150 μg/ml of the test compounds were added by micropipette. Petri dishes were kept in refrigerator to facilitate the diffusion for about 2 h. These plates were then incubated at 37°C for 48 h. The extent of inhibition was determined by measuring the diameter of the inhibition zone in mm. For antibacterial and antifungal study, the cipro oxacin 150 μg/ml and 150 μg/ml of uconazole were used as reference drugs respectively.

Minimal inhibitory concentration using two folds dilution method (MIC)
The broth dilution was performed by using microtiter plates. The nutrient broth was prepared using peptone (1%), beef extract (0.5%), sodium chloride (0.8%). It was added in microtiter plates except rst well. First well of microtiter plate was used to check the sterility of the medium as negative control in which inoculum was not added. Stock solution of test compounds was prepared in DMSO (200μg/ml) followed by twofold dilution at concentrations of (100, 50, 25….3.125 μg/ml). The inoculums were added to the other all wells containing test compounds ranging from 100, 50, 25….3.125 μg/ml. The micro titer plates were then incubated at 37°C for 24 h for bacteria and 48 h for fungi and minimal inhibitory concentration were measured for the growth in the form of turbidity. The cipro oxacin (150μg/ml) was used as reference drug for antibacterial study while uconazole (150μg/ml) was used for antifungal study [26].

Molecular Docking
To check the type of interactions exists between the enzyme and the ligand the docking study is a promising tool. The prediction of predominant binding modes of a ligand with a protein of known threedimensional structure is one of the important goals of ligand-protein docking. In present study, V Life MDS 3.5 software was used for docking purpose [27][28][29][30][31]. The structure of DNA gyrase protein (PDB: 3G75) was obtained from Protein Data Bank (http://www.rcsb.org).

Results And Discussion
The compounds BT (1-5) were prepared as per reported scheme 1. 4-substituted anilines (BT 1-5) were reacted with ammonium thiocyanate and bromine in acetic acid by stirring. The reaction mainly involves formation of intermediate phenylthiourea and cyclisation of phenylthiourea gives 6-substituted 2-amino benzothiazole. The target compounds BTC(a-t) were synthesized by the scheme indicated in scheme 1. The compounds were prepared by re uxing solution of BTC (1-5) and amino derivatives. The synthesized compounds structures were con rmed by spectral analysis using 1 H NMR, IR and Mass were found consistent with the spectral data. IR spectra of BTC(a-t) showed characteristic aromatic CH stretch between 3140-3000 cm -1 . The C=N is seen at 3394-3201 cm-1. The carbonyl (C=O) peak is observed between at 1778-1625 cm-1. The absence of C-Cl stretch in the IR spectrum also con rms the formation of 2-(substituted amino)-N-(6-substituted-1,3-benzothiazo-2yl) acetamide BTC (a-t) 1 H NMR of synthesized compounds are taken in DMSO. Which shows sharp solvent peak at 2.5 and 3.38 ppm. The compounds display aromatic protons between 6.6-8.5 ppm. The compound revealed a characteristic N-H proton of amide around 10.28-12.59 ppm and presence of singlet for N-H proton of substituted amines between 4.5-8.6ppm con rmed the formation of target compounds.

Antimicrobial activity
The minimum inhibitory concentration of BTC (a-t) compounds was determined using a double dilution method. Cup plate method was used to determine the zone of inhibition. Cipro oxacin and uconazole were used as standard drugs for antibacterial and antifungal activity respectively.
The synthesized compounds were evaluated for antibacterial activity by using gram positive bacteria: B. subtilis, S. aureus, gram negative bacteria: E. coli, P. aeruginosa and antifungal activity by using A. niger and C. albicans.
The compound BTC-j has shown good antibacterial activity against S. aureus at MIC of 12.5 µg/ml, B. subtilis at MIC of 6.25 µg/ml, E. coli at MIC of 3.125µg/ml and P. aeruginosa at MIC of 6.25 µg/ml. The compound BTC-j also shown good antifungal activity against A. niger and C. albicans at MIC of 3.125 and 6.25 µg/ml respectively. For BTC (a-t) zone of inhibition against gram positive bacteria was observed using cup plate method. Cipro oxacin was used as reference drug and the zone of inhibition observed for gram positive (S. aureus and B. subtilis) and gram-negative (E. coli and P. aeruginosa) bacteria are shown in Table 2 and 3 respectively. Zone of inhibition in mm, Conc.in µg/ml.
The compounds BTC-j and BTC-r have shown signi cant zone of inhibition amongst all 20 synthesized compounds. Compounds BTC-j was found to have comparable activity with cipro oxacin at 150µg/ml against both of gram-positive bacteria. The compound BTC-p was found to be minimum acting among synthesized compounds. Zone of inhibition in mm, Conc.in µg/ml.
Zone of inhibition study for gram negative bacteria shows that compounds BTC-j and BTC-r possess better zone of inhibition in comparison with reference drug cipro oxacin among the twenty synthesized compounds. Compound BTC-j showed signi cant zone of inhibition and compound BTC-a possess least activity compared to reference drug.
Zone of inhibition of synthesized compounds BTC(a-t) was measured against two species of fungi.
Fluconazole was used as reference drug. The observations are reported in Table 4  Zone of inhibition in mm, Conc.in µg/ml.
Compound BTC-j was found to be more active as antifungal in all synthesized compounds when compared with uconazole as reference drug. Compound BTC-a was shown least activity as compared to other synthesised compounds.
Among the screened compounds, compound BTC-j showed good antifungal activity as compared to standard. BTC-f and BTC-r showed a moderate antifungal activity.
It was observed that compounds possessing methoxy substitution were found to hold better antibacterial activity against gram-positive and gram-negative bacteria among the synthesized compounds, whereas most of the compounds did not show good antifungal activity against C. albicans species compared to uconazole.

Molecular docking
The docking study was carried out to check the interactions of target compounds. All the synthesized compounds BTC(a-t) were docked in DNA gyrase (pdb 3G75) protein. The dock score and binding interactions were recorded Table 5. The docking study revealed that the compound BTC-j possess -5.54 Kcal/mol as highest dock score. The compound BTC-j forms hydrogen bond between N-H of amide and N-H of amine with amino group of GLU58A and ASP57A. The compound was held in active site with hydrophobic interactions with ASP57A, GLU58A and ALA 61A amino acid residue. The antibacterial activity was found to be in correlation with dock score.
The compound BTC-j has shown good antibacterial activity against S. aureus at MIC of 12.5 µg/ml, B. subtilis at MIC of 6.25 µg/ml, E. coli at MIC of 3.125µg/ml and P. aeruginosa at MIC of 6.25 µg/ml.
The compound BTC-j also shown good antifungal activity against A. niger and C. albicans at MIC of 3.125 and 6.25 µg/ml respectively.
The docking study was carried out to check the interactions of target compounds with selected protein.
All the synthesized compounds BTC(a-t) were docked in DNA gyrase (pdb 3G75) protein. The dock score and binding interactions were recorded in Table 5. The docking study revealed that the compound BTC-j possess -5.54 Kcal/mol as highest dock score. The compound BTC-j forms hydrogen bond between N-H of amide and N-H of amine with amino group of GLU58A and ASP57A. The compound was held in active site with hydrophobic interactions with ASP57A, GLU58A and ALA61A amino acid residue. The antibacterial activity was found to be in correlation with dock score.
The results of antimicrobial activity and docking study indicates that methoxy substituted benzothiazole with pyridine substitution have shown good antimicrobial activity compared to other synthesized compounds, nitro substituted benzothiazole with pyridine substitution shown moderate activity when compared with other synthesized compounds.
Thus, we conclude that the synthesized compounds have potential for further development as novel antimicrobial agents.