Synthesis, molecular modelling and antibacterial activity of 4-aryl-thiosemicarbazides


 N-Substituted phenyl/cyclohexyl-2-(pyridine-4-carbonyl) hydrazine-1-carbothioamides (2a–r) were synthesized, characterized by spectral and analytical data. The compounds were evaluated for antibacterial activity by the disc diffusion method. Most of the compounds showed activity against Gram-positive bacteria. Compound 2h with 4-Sulfapyrimidine phenyl substitution was found to be the most promising candidate, active against Gram-positive and methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory concentration (MIC) of (2–7 μg/mL). From the docking study, we predicted that compounds (2r, 2g, 2h, 2o, 2p and 2e) possess better antibacterial activity by having a good binding affinity with target protein and they could be used as potential drugs as antimicrobials. Amongst all the docked compounds, the compound 2h presented near binding affinity & interaction docking score with DNA gyrase enzymes with reference to ciprofloxacin.


INTRODUCTION
Antibiotic resistance of bacterial pathogens has been increased for many years, both in Gram-positive and Gram-negative bacteria 1 . Therefore, many research groups have been working to improve the potency and spectrum of available drugs. There is always an urgent need of fi nding new chemical entities to curb drug resistance. In antimicrobial research, bacterial topoisomerases have found importance since the discovery of Escherichia coli topoisomerase II (DNA gyrase) 2 . DNA gyrase and topoisomerase IV are attractive targets in the search for new antibiotics 3 . During bacterial DNA replication and chromosome segregation, these two homologous enzymes play an important role. DNA gyrase and topoisomerase IV are structurally related to each other. Both the enzymes are heterotetramers. GyrA comprises the DNA binding, cleavage and relegation activity, while GyrB comprises the ATPase activity. Quinolones are highly effective antimicrobial drugs 4 . Because of the widespread use of quinolones, there is an emergence of drug-resistant bacteria due to changes in the DNA gyrase or topoisomerase IV.
To discover potent antibacterial compounds, a new set of thiosemicarbazides (2a-r) were synthesized, characterized and in vitro antibacterial activity was studied.
Pyridyl ring, an important scaffold present in many bioactive molecules, has played an important role in the development of different medicinal agents 25−27 . The thiosemicarbazide pharmacophore was preserved and indole moiety was replaced by pyridine moiety of the isoniazid (Fig. 1).

Chemistry
All the solvents were procured from Merck. The purity of the compounds was determined by thin-layer chromatography (TLC) performed on Silica gel G coated plates (Merck). The FT IR spectra were determined in KBr pellets on a (Spectrum BX) Perkin Elmer FT-IR spectrophotometer. Gallenkamp melting point apparatus was used for the determination of the melting point of compounds and thermometer was uncorrected. NMR Spectra were scanned in DMSO-d 6 on a Bruker NMR spectrophotometer operating at 500 MHz for 1 H and 125.76 MHz for 13 C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. High--resolution mass of the compounds was determined by HR MS.

General method for the synthesis of 4-arylthiosemicarbazides
To a solution of appropriate aniline (0.01 mol) in absolute ethanol (25 mL), was added KOH (0.01 mol) and carbon disulfi de CS 2 (0.75 mL). The mixture was stirred at 0-5 o C for one hour to form the potassium salt of substituted phenyl dithiocarbamate. To the stirred mixture, was added isoniazid (INH) (0.01 mol) and stirring was continued at 80 o C for one hour. Crushed ice was added to obtain 4-arylthiosemicarbazides.

Antibacterial assay
The antibacterial activities for the compounds were tested against representative pathogenic Gram-positive strains by using the paper disc diffusion method 28 . In this method, nutrient agar for bacteria was prepared and sterilized by autoclaving at 120 o C and 1.5 atmospheric pressure for 20 minutes 29 . The agar plates were poured, left to cool down and after solidifi cation they were inoculated with the bacterial strains by streaking. The synthesized compounds were dissolved in methanol at a fi nal concentration of 10 mg/mL and 5 μL of each compound was loaded on a sterile fi lter paper disc (5 mm diameter; 50 μg per disc). The fi lter paper disc was then transferred aseptically into the inoculated agar plates along with commercially available ciprofl oxacin disc as positive controls and methanol as the negative control for comparison. After incubation, diameters of the inhibition zones around the paper discs were measured in mm as an indication of the antibacterial activities of the compounds.

Molecular docking
Docking for the synthesized compounds (2a-r) and references compound (ciprofl oxacin) CIP was conducted via their 3D structures built and energy minimized using MMFF94x of Molecular Operating Environment energy minimization module (MOE, Version 2015, Chemical Computing Group Inc., Montreal, Quebec, Canada) (MOE Chemical Computing Group). (PDB ID: 4ZVI) that represents 3D crystal structure of DNA gyrase was selected from Protein Data Bank database (http://www. rcsb.org). The missing residues and side chains of the crystal structure were repaired, the 3D protonation of the target was achieved for molecular docking and the energy was minimized using MMFF94x 24. The docking procedure was followed using the standard protocol implemented in MOE 2015 and the geometry of resulting complexes was studied using the MOE's Pose Viewer utility. The enzyme was prepared for docking as follows: The Co-crystallized ligand and water molecules were removed. The enzyme was 3D protonated, where hydrogen atoms were added at their standard geometry, the partial charges were computed and the system was optimized. Flexible ligand-rigid receptor docking of the most stable conformers was done with MOE-DOCK using triangle matcher as placement method and London dG as a scoring function. The obtained poses were subjected to force fi eld refi nement using the GBVI/ WSA dG rescoring function. Thirty of the most stable 8H, Ar-H), 8

RESULTS AND DISCUSSIO N
The synthesis of isoniazid derivatives was carried out in one pot as shown in (Scheme 1). Substituted anilines were reacted with carbon disulfi de in presence of alcoholic KOH in equimolar quantities. Potassium salt of substituted phenyl dithiocarbamate was obtained by stirring at 0-5 o C for one hour. Equimolar quantity of isoniazid was added with stirring and refl uxed for one hour at 80 o C. 4-Aryl-thiosemicarbazides were obtained by adding crushed ice to the mixture in good yield (60-80%). The purity of the compounds was confi rmed by the elemental analysis and thin-layer chromatography (TLC). The compounds were identifi ed by spectral data e.g., FTIR, 1 H NMR, 13  which were used in this study were: Bacillus subtilis ATCC 6633; Bacillus pumilus ATCC 14884; Bacillus cereus ATCC 10876; Staphylococcus aureus ATCC 6538 and methicillin--resistant Staphylococcus aureus ATCC 33591 (MRSA). Initially, antibacterial activity of 4-aryl-thiosemicarbazides was screened based on growth inhibition zones by the disc diffusion method. Minimum inhibitory concentrations (MIC) were determined using the agar dilution method. Ciprofl oxacin (25 μg) was used as standard drug for comparison of zone of inhibition of synthesized compounds. All compounds of this series were showing activity against Gram-positive strains. Compound 2h with sulfapyrimidine substitution was found to be effective against Gram-positive an d MRSA strains. Compound 2r with 2-nitro substitution was found to be most active against MRSA ATCC 33591. Compound 2h was selected for minimal inhibitory concentration (MIC) calculation against all strains. Compound 2h was found to be the most promising candidate active against Gram-positive and MRSA strains with minimum inhibitory concentration MIC (2-7 μg/mL).

Docking studies
Considering the well obtained in vitro results, it was thought worthy to perform molecular docking studies, hence screening the compounds, inculcating both in silico and in vitro results. Considering DNA gyrase as the target receptor, comparative and automated docking studies with newly synthesized compounds were performed to determine the best in silico conformation.  (Fig. 2) shows the docked images of selected ligands

Anti-bacterial activity
All the compounds of this series were screened for antibacterial activity (Table 1). The synthesized compounds were evaluated against the strains of Gram-positive and methicillin-resistant Staphylococcus aureus. The microorganisms   (Fig. 3). From the docking study, we predicted that compounds (2r, 2g, 2h, 2o, 2p and 2e) possess better antibacterial activity by having good binding affi nity with target protein and it could be used as potential drugs as antimicrobial. Amongst all the docked compounds, the compound (2r) shows near binding affi nity & interaction docking score of -6.61570501 with DNA gyrase enzymes with reference to ciprofl oxacin.

CONCLUSION
In conclusion, in the present study, eighteen 4-aryl--thiosemicarbazides (2a-r) were synthesized by one-pot method in good yield from substituted anilines and were fully characterized by spectral data. The compounds were evaluated for their antibacterial activity against Gram--positive and MRSA strains. Amongst all the docked compounds, the compound 2h showed near binding affi nity & interaction docking score of -6.28270531 with DNA gyrase enzymes with reference to ciprofl oxacin. The present study revealed new lead compound 2h with signifi cant activity and the same results were refl ected in docking studies also. Compound 2h was found to be the most promising candidate active against Gram-positive and MRSA strains. Finally, the compound 2h represents a good lead for the development of pyridine-based aryl--thiosemicarbazides as antibacterial agent.

Data Availability
Samples of the compounds (2a-r) in pure form are available from the authors.

Confl ict of interest
The authors declare that there is no confl ict of interest.