Design, Synthesis, Molecular Docking and Antibacterial Screening of Some Quinolone Compounds

Some 6,8-dichloro-quinolone compounds were designed and synthesized; by comparing with 6-fluoro-8-chloro-quinolone compounds, the influence of the nature of the halogen atom from six position of the quinolone ring on the molecular properties and on the antimicrobial activity was studied. The DFT/B3LYP/6-311G* level of basis set was used for the computation of molecular structure of optimized compounds. The calculations of characteristics and molecular properties were performed using Spartan’14 Software from Wavefunction, Inc. Irvine, CA. The HOMO-LUMO energies and orbitals, global reactivity descriptors, various thermodynamic parameters, and dipole moment (μ) were calculated to determine the molecular properties of quinolone compounds. Molecular docking studies were realized to identify and visualize the most likely interaction ligand (quinolone/fluoroquinolone compounds) with the protein receptor. The score and hydrogen bonds formed with the amino acids from group interaction atoms are used to predict the binding modes, the binding affinities, and the orientation of the docked quinolone/fluoroquinolone derivatives in the active site of the protein receptor. The protein-ligand complex was realized based on the X-ray structure of Bacillus cereus (PDB ID: 1VEN) using CLC Drug Discovery Workbench 2.4 software. The quinolone compounds were characterized by physical-chemical methods (elemental analysis, IR spectral analysis, 1H-NMR, 13C-NMR spectra, UVVis, thin layer chromatography) and by antimicrobial activity against some Gram-positive and Gramnegative microorganisms: Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Micrococcus luteus, Escherichia coli and Pseudomonas aeruginosa.

Fluoroquinolones are also, antibacterial agents used in the treatment of some bacterial infections, including hospital acquired infections and the infections caused by bacterial pathogens multi drug resistant [23][24][25][26][27]. Infections caused by Gram-positive rods are occasional in comparison with the infections caused by Gram-positive cocci or Gram-negative rods. It was observed that Gram-positive spore-forming rods (Bacillus spp.) are common contaminants of cultures but they may cause grave infections such as septicemia, endocarditis, endophthalmitis and wound infections. The most common species, Bacillus cereus, is resistant to β-lactam antibiotics, but it has been treated effectively with clindamycin, ciprofloxacin, vancomycin, and imipenem. Food poisoning caused by the Bacillus cereus does not need antibiotic treatment [28,29].
In this work we have realized design studies of some bicyclic quinolone compounds [30][31][32]. The most common substituents attached to the quinolone ring are shown in Figure 1.
Many quinolone compounds contain fluorine atom in 6 position, because C-6 fluorine is quite crucial for both high DNA gyrase complex binding activity and great bacterial cell penetration of the quinolone derivatives [33]. We have extended our research to design and synthesis new 6,8-dichloro-quinolone compounds for evaluated the influence of the nature of the halogen atom from six position of the quinolone ring on the molecular properties and on the antimicrobial activity. For the new quinolones was chosen an ethyl group for N-1 position and a piperidinyl moiety for 7 position. The introduction of the chlorine atom at 8 position was motivated by the fact that the presence of this halogen atom in this position leads to a decrease in toxicity [34].

Molecular modeling
Molecular, topological, conformational characteristics on 3D optimized structure have been calculated using Spartan 14 Software [35]. The DFT/B3LYP/6-311G* level of basis set has been used for the computation of molecular structure, vibrational frequencies, and energies of optimized structures.

Docking studies
Molecular docking studies was realized using CLC Drug Discovery Workbench Software. In the docking simulation the quinolone compounds (ligands) was fitted into predictable binding site on the surface of the protein target. The score docking and hydrogen bonds formed with the amino acid residues from the active site of the receptor are used to predict the binding modes, the binding affinities and the orientation of the ligands in the active site of the protein-receptor. The protein-ligand complex has been realized based on the X-ray structure of Bacillus cereus, who was taken from the Protein Data Bank (PDB ID: 1VEN) [36].

Minimum inhibitory concentration (MIC) determination
All the quinolone compounds: 6ClPQ30, 6ClPQ33, FPQ30 and FPQ33 have been screened for their in vitro antibacterial activity against a variety of bacteria: Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus Subtilis ATCC 6633, Bacillus Cereus IP 64 and Micrococcus luteus ATCC 9341 by using a standard twofold serial dilution method with agar media [38].

Ligand preparation
The ligands (quinolone derivatives) have been prepared using SPARTAN'14 software package [35]. The DFT/B3LYP/6-311 G* level of basis set has been used for the computation of molecular structure, vibrational frequencies and energies of optimized structures (Figure 2). To realized structure-activity relationship (SAR) studies, some electronic properties, such as HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energy values, HOMO and LUMO orbital coefficients distribution, molecular dipole moment, polar surface area (PSA) (a descriptor who allows the prediction of transport properties of the drugs because has been shown to correlate well with passive molecular transport through membranes), the ovality, polarizability (descriptor useful to predict the interactions between non-polar atoms or groups and other electrically charged species), the octanol water partition coefficient (logP) ( Table 1) habe been calculated. Table 1 display also chemical potential (μ) and electronegativity (χ) as well as global (softness (S), hardness (η) and electrophilicity index (ψ) [39][40][41]. The chemical softness (S) parameter for fluoroquinolones (FPQ30, FPQ33) is less than chloroquinolones (6ClPQ30, 6ClPQ33), therefore the stability of fluoroquinolones is grate than chloroquinolones. The positive value of electrophilicity (ψ) shows the tendency of the system to accept electron from the environment. The electrophilicity increases in the order FPQ33<6ClPQ33<FPQ30< 6ClPQ30. The FPQ33 is more stable because this fluoroquinolone possesses the smallest value of the electrophilicity from all the compounds.  Frontier molecular orbital analysis Frontier molecular orbital's (FMOs) show a decisive role in the chemical stability of a compound and in the interactions between atoms and are considered to be operative in determining characteristics of the compounds such as optical properties and biological activities. The most important FMOs are the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). The HOMO represents the ability of a molecule to donate an electron, while the LUMO is an electron acceptor. The graphic has "blue and red" regions that are corelated to positive and negative values of the orbitals. Figure 3 displays distribution and energy levels of the HOMO and LUMO orbitals. For the HOMO of 6ClPQ33, electron density is localized on piperidine heterocyclic (N1, C-11, C-12), on C-6, C-5, C-4 and C-2 atoms from aromatic ring, on O-1, Cl-1 and on Cl-2, on the same as with fluoroquinolones FPQ 30 and FPQ33. In the case of quinolone 6ClPQ30, electron density is localized on piperidine heterocyclic (N, C-11, C-12), on C-6, C-5 and C-8 atoms from aromatic ring, and on chlorine atom, Cl-2. For the LUMO of 7-substituted-8-chlorquinolones 6ClPQ30 and 6ClPQ33, electron density is localized on 4-piridinona ring, on aromatic ring and on chlorine atom (Cl-2), on the same as with fluoroquinolones FPQ 30 and FPQ 33. The frontier orbital gap (E) give information about the chemical reactivity of the molecule. The higher value of HOMO-LUMO gap (E), for the all compounds, shows the all quinolones have a chemically stable molecule.
Molecular Electrostatic Potential (MEP) is important for the determination of the reactive sites of nucleophilic or electrophilic attack in hydrogen-bonding interactions and for the figure out of the process of biological recognition. An electrostatic potential map displays hydrophilic regions in red (negative potential) and blue (positive potential) and hydrophobic regions in green. The electrostatic potential increases in the order red<orange<yellow<green<blue. Figure 4 showed the molecular electrostatic potential maps of quinolone compounds 6ClPQ30 and 6ClPQ33 and fluoroquinolones FPQ30 and FPQ33 for B3LYP/ 6-311G* levels. For all compounds, red regions are localized on the O atoms and the blue regions are localized on N atoms.

Docking studies
In the docking simulation, the quinolone compounds (ligands) was fitted into predictable binding site on the surface of the protein target. The score docking and hydrogen bonds formed with the amino acid residues from the active site of the receptor are used to predict the binding modes, the binding affinities, and the orientation of the ligands in the active site of the receptor. The protein-ligand complex has been realized based on the X-ray structure of Bacillus cereus, who was imported from the Protein Data Bank (PDB ID: 1VEN). All investigated compounds have been virtually docked on Bacillus Cereus according to the protocol described in previous works [42,43]. The docking studies revealed that all the compounds presented good docking score ( Table 2). The better score docking has been obtained from quinolone 6ClPQ 33 (score: -62.80; RMSD 0.04 Å).  Table 2 also shows the amino acids residues from group of interaction of all compounds docked in the binding site of 1VEN. The synthesis of the quinolone derivatives is shown in Scheme1. The quinolone compounds were synthesized by the reaction of the compound (1) (QA: R6= F, 6ClQA: R6=Cl) [37] with piperidine or 4-methyl-piperidine. The 8-unsubstituted quinoline-3-carboxylic acid (FPQ32 [43], FPQ24 [37], 6ClPQ32, 6ClPQ24 [37] was chlorinated with sulfuryl chloride. The final quinolone compounds, 6ClPQ33, 6ClPQ30, FPQ33 [43] and FPQ30 [38] were analyzed by physic-chemical techniques (elemental analysis, 1H-NMR, 13C-NMR, FT IR, UV-Vis).

Scheme 1. Synthesis of quinolone compounds
Quinolone derivatives were evaluated for "in vitro" activity" by determining minimum inhibitory concentration against some Gram-positive: Staphylococcus aureus ATCC 29213, Bacillus cereus IP 64, Bacillus subtilis ATCC 6633, Micrococcus luteus ATCC 9341 and Gram-negative microorganisms: Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 ( Table 3). The introduction of a chlorine atom into the sixth position of the quinolone nucleus leads to the decrease of biological activity against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, but does not affect the biological activity against Bacillus cereus, Bacillus subtilis and Micrococcus luteus. In the case of compound FPQ 30, a compound that has a good activity against all the studied microorganisms, the replacement of the fluorine atom with chlorine atom, leads to the improvement of the activity towards the Bacillus cereus.

Conclusions
Some 6,8-dichloro-quinolone compounds were designed and synthesized, and we studied the influence of the change of the fluorine atom from six position on the quinolone ring with a chlorine atom, on the molecular properties and on the antimicrobial activity. The quinolone compounds were characterized by physical-chemical methods (elemental analysis, IR spectral analysis, 1H-NMR, 13C-NMR spectra, UV-Vis, thin layer chromatography) and by antimicrobial activity against some Grampositive and Gram-negative microorganisms: Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Micrococcus luteus, Escherichia coli and Pseudomonas aeruginosa. As a result of docking simulations, the score and hydrogen bonds formed with the amino acids residues were used to predict the binding modes, the binding affinities, and the orientation of the docked quinolone compounds. The docking studies reveals that the all compounds presented good docking score. The better score docking was obtained from quinolone 6ClPQ 33 score: -62.80 (RMSD 0.04 Å). A correlation of the predicted data with the experimental data obtained from the evaluation of the antimicrobial activity against Bacillus cereus of quinolone compounds were observed. In conclusion, structural modifications of this class of antimicrobial agents have afforded compounds with better activity against Bacillus cereus, Bacillus subtilis and Micrococcus luteus.