Design, Synthesis, Molecular Docking, and Antibacterial Evaluation of Some Novel Flouroquinolone Derivatives as Potent Antibacterial Agent

Objective. Quinolone moiety is an important class of nitrogen containing heterocycles widely used as key building blocks for medicinal agents. It exhibits a wide spectrum of pharmacophores and has bactericidal, antiviral, antimalarial, and anticancer activities. In view of the reported antimicrobial activity of various fluoroquinolones, the importance of the C-7 substituents is that they exhibit potent antimicrobial activities. Our objective was to synthesize newer quinolone analogues with increasing bulk at C-7 position of the main 6-fluoroquinolone scaffold to produce the target compounds which have potent antimicrobial activity. Methods. A novel series of 1-ethyl-6-fluoro-4-oxo-7-{4-[2-(4-substituted phenyl)-2-(substituted)-ethyl]-1-piperazinyl}-1,4-dihydroquinoline-3-carboxylic acid derivatives were synthesized. To understand the interaction of binding sites with bacterial protein receptor, the docking study was performed using topoisomerase II DNA gyrase enzymes (PDB ID: 2XCT) by Schrodinger's Maestro program. In vitro antibacterial activity of the synthesized compounds was studied and the MIC value was calculated by the broth dilution method. Results. Among all the synthesized compounds, some compounds showed potent antimicrobial activity. The compound 8g exhibited good antibacterial activity. Conclusion. This investigation identified the potent antibacterial agents against certain infections.


Introduction
In recent years microbial infections are associated with high rates of attributable morbidity and mortality. Infections caused by microbial species are common in immune compromised patients and have significant treatment costs and mortality. The increasing rate of bacterial resistance to clinical antimicrobial agents and its impact on treatment of infectious diseases have begun to present a unique problem throughout the world. Drug resistant, multiple drug resistant (MDR), and extensively drug resistant (XDR) infectious bacterial pathogens put a greater risk on the population at large due to the risk of pandemic illness. Increasing complication is the fact that many antibacterial agents can induce mutations and resistance, often by different mechanisms. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) are the most important infections caused by bacteria which have been found worldwide in hospitals [1,2]. The emerging resistance of some microbial species to some synthetic antimicrobial agents makes it necessary to continue the search for new antimicrobial agents. Quinolone moiety is an important class of nitrogen containing heterocycles widely used as key building blocks for medicinal agents. It exhibits a wide spectrum of pharmacophores and has bactericidal [3], antiviral [4], antimalarial [5], and anticancer [6] activities. Among these, flouroquinolone derivatives play an important role in the field of medicine. Fluoroquinolones are the most widely used antibacterial agents in modern therapy due to their broad spectrum and excellent oral bioavailability. In 2 The Scientific World Journal this work our interest is to study the antibacterial activity of novel 1-ethyl-6-fluoro-4-oxo-7-{4-[2-(4-substituted-phenyl)-2-(substituted)-ethyl]-1-piperazinyl}-1,4-dihydroquinoline-3-carboxylic acid derivatives. The present investigation deals with the clinically isolated different Gram-positive and Gram-negative bacteria against synthesized compounds and most of the tested compounds act as potent antibacterial agents. To understand the interactions of tested compounds at active sites of protein receptors, topoisomerase II DNA gyrase enzymes (PDB ID: 2XCT) molecular docking studies were also performed and reported in this paper.

Materials and Method
Melting points ( ∘ C, uncorrected) of all the synthesized compounds were checked in capillary tubes by using a digital melting point apparatus (Veego melting point apparatus). All the reactions were monitored by thin layer chromatography (TLC silica gel 0.25 mm, 60 G F254; eluting solvents were chloroform : methanol : hexane 9 : 0.5 : 0.5). All the compounds were characterized by FT-IR spectrometer (IR Prestige-21, Shimadzu, Japan) using KBr pellets; 1 HNMR spectra were obtained in CDCl 3 on Bruker Avance-II 400 MHz instrument and chemical shifts were measured as parts per million downfield from tetramethylsilane (TMS) as internal standard. Mass spectra were recorded on LCMS 2010 EV S Shimadzu mass spectrometer.

Preparation of Protein.
The structure of topoisomerase II DNA gyrase enzyme (PDB ID: 2XCT) was downloaded from the Protein Data Bank and imported and prepared by a multistep process through the protein preparation wizard of Maestro (version 9.7). The protocol was especially used to obtain the optimised and minimised energy conformation of the protein. Firstly, the bond order in the protein was assigned; hydrogen atoms were added and the water molecules which did not participate in interactions were removed. Following the above steps of preparation, the protein was subjected to energy minimisation using Schrodinger implementation of OPLS-2005 force field with implicit solvation.

Preparation of Ligands.
The ligands were prepared using the LigPrep 3.0 [12] module of the Schrodinger suite [13] using Merck molecular force field (MMFF). MMFF was used for 2D to 3D conversion of ligand molecules while the optimized potential for liquid simulation (OPLS) force field was used for conformation analysis through ConfGen [14].

Active Site Prediction.
Active site is a pocket pouch present on the protein structure that has the tendency to accept the ligand molecules within it. The Sitemap applies theoretical methods and predicts the most accurate binding site. The OPLS-AA force field generates site points, possible for ligand interaction within the protein. Sitemap gives an idea about positions favorable for a donor, acceptor, and hydrophobic group to be present in the receptor. The maps were very useful for analysing the interactions of ligands with the receptor. In order to find the active site volume, surface area, and character of binding sites of the enzymes (2XCT), Sitemap was performed by choosing the top ranked potential receptor binding sites and the other parameters were kept as default. OPLS-2005 was used as the force field. Five sites with different site scores were obtained as output and the site with the highest site score was selected.
Docking studies on LigPrep treated ligands were carried out to predict the binding pocket of 2XCT using the docking program, Glide. Glide used a series of hierarchical filters to search for possible locations for the ligand in the active site region of the receptor. For the grid-based ligand docking, the receptor grid generation file was used. For protein structure, a grid box of 30 × 30 × 30Å with a default inner box (10 × 10 × 10Å) was centered on the corresponding ligand. The receptor grid was defined as an enclosing box at the centroid of the cocrystallized ligand (i.e., 2XCT) to include the cofactor and substrate binding sites. In the initial Glide docking stage, a softened potential docking with the van der Waals radii scaling of 0.7 for the proteins was performed to retain the maximum number of 20 poses per ligand. Residues within 5.0Å of ligand poses were kept free to move in the Prime refinement step, and the side chains were further minimized. Then single low energy 3D structure of ligands with correct chiralities was docked with the binding site using the "Extra Precision" Glide algorithm in Schrodinger.

In Vitro Antibacterial Activity.
All the synthesized compounds were studied for their antibacterial activity against two clinically isolated Gram-positive strains (S. aureus and S. pneumoniae) and two Gram-negative strains (P. aeruginosa and Escherichia coli) using conventional broth dilution method. The minimum inhibitory concentration (MIC) values were calculated by comparison to norfloxacin as the reference bacterial drug and they are shown in Table 2.
All the cultures were prepared by Mueller-Hinton agar and the turbidity of all the bacterial cultures was adjusted to 0.5 McFarland standard by preparing bacterial suspension of 3-5 well-isolated colonies of the same morphological type selected from an agar plate culture. The cultures were further diluted 1000-fold to get an inoculum size of 1 × 10 6 CFU. The synthesized compounds and standard bacterial drugs (50 mg) were dissolved in dimethyl sulphoxide (DMSO) (0.5 mL) and the solution was diluted with water (4.5 mL) to get a stock solution of 10,000 mg/L of each compound. Further progressive double dilution with melted Mueller-Hinton agar was performed to obtain the required concentrations of 100, 50, 25, 12.5, 6.25, and 1 g/mL [15,16]. To ensure that the solvent had no effect on the bacterial growth, a control test was performed with a test medium supplemented with DMSO at the same dilution as that used in the experiment. Incubation of the cultures was done at 37 ∘ C for 24 hrs and comparison with blank was done in terms of turbidity which is due to microbial growth.    Against Gram-Negative Strain. Compounds 8i, 8n, 8q, and 8s have shown potent antibacterial activity with MIC value in the range of 6.25-50 g/mL against Gram-negative E. coli and P. aeruginosa. The remaining compounds have shown antibacterial activity with MIC value in the range of 12.5-50 g/mL against Gram-negative E. coli and ≥100 g/mL against Gram-negative P. aeruginosa. It was observed that 8i, 8l, 8n, 8q, and 8s have shown potent antibacterial activity against Gram-positive S. aureus and S. pneumonia and against Gram-negative E. coli strains and P. aeruginosa.

Results and Discussion
In terms of structure activity relationship, results suggest that compounds having 4-nitro and 4-methoxy on phenyl ring at C-7 piperazinyl side chain exhibited the most potent antibacterial activity. It was also observed that compounds with imino nitrogen substituted with a semicarbazide or amine ( 2 = -NHCONH 2 and NH 2 ) exhibited potent antibacterial activity.

Molecular Docking Studies of Derivatives.
To understand the interaction of all the synthesized molecules (8a-8t) with topoisomerase II DNA gyrase enzymes, the crystal structure of topoisomerase II was downloaded from Protein Data Bank (PDB ID: 2XCT) and the molecular docking studies were performed using the Glide program. The protein ligand interaction plays a significant role in structural based drug designing. In this approach, H-bonding, Glide energy score, Emodel, and -score are kept as a support for the present work. The preferred ligands 8g, 8i, 8e, 8c, 8s, 8q, 8h, 8n, and 8a had a -score value of −8.51 to −7.43. The -score values of the above mentioned standard compound (norfloxacin) were comparatively lesser than their analogues with the scoring of −7.29.
The minimum Glide energy required for the formation of complex between ligand and the receptor indicates excellent binding affinity. Very low energy indicates that the ligand is buried in the cavity of the receptor [17]. The Glide binding energy of the fluroquinolone analogues as ligands was found to be −54.38 to −74.83. Standard compounds such as norfloxacin possessed Glide energy −54.23.
In the present investigation, the nine fluoroquinolone analogues ligands had high quality binding affinity with the enzyme. Hydrogen bond interaction can act either as antagonist or as agonist for a ligand with receptor. On analyzing the docking results, all the seven compounds had H-bond interactions with the receptor indicating that the ligand binding affinity increased together with the number of the H-bond interactions. All nine ligands had a significant number of H-bond interactions with the enzyme.
Emodel had a more significant weighting of the force field components (electrostatic and van der Waals energies), which made it well suited for comparing conformers. Therefore, Glide uses Emodel to pick the "best" pose of a ligand (pose selection) and then ranks these best poses against one another with Glide score. A low Emodel value indicates a good binding affinity between protein and ligand. Glide Emodel values of −119.918 to −84.85, for the nine best fluroquinolone analogues, were calculated (Table 3) indicating that these compounds engage in energetically more favourable interactions than norfloxacin (Emodel score −83.0725, Table 3) within the active site.
From the docking study we predicted that fluoroquinolone analogues (8g, 8i, 8e, 8c, 8s, 8q, 8h, 8n, and 8a) possess better antibacterial activity than the standard drugs by having good binding affinity with target protein and it could be used as potential drug as antimicrobial. Amongst all the docked compounds, compound 8g shows good binding affinity and interaction with topoisomerase II DNA gyrase enzymes (2XCT) with reference to norfloxacin shown in Figures 1 and 2.

Conclusion
In summary, a new series of novel 1-ethyl-6-fluoro-4oxo-7-{4-[2-(4-substituted phenyl)-2-(substituted)-ethyl]-1-piperazinyl}-1,4-dihydroquinoline-3-carboxylic acid derivatives were synthesized and characterized by FT-IR, 1 HNMR, and high resolution mass (HRMS-EI) spectral analyses. All the molecules were studied for their interactions with topoisomerase II DNA gyrase enzymes by molecular docking protocol. Among the tested molecules, compound 8g exhibited a good Glide score value of -8.51 and Glide energy −54.38 with Emodel value of −105.2. In vitro antibacterial activity of the tested compounds shows improved activity against all the microorganisms used. In particular, compound 8g exhibits marked activity against two microorganisms. The results of antibacterial activity are supported by docking analysis. The