Efficient Antibacterial Dimeric Nitro Imidazolium Type of Ionic Liquids from a Simple Synthetic Approach

Synthesis of dimeric nitro-substituted imidazolium salts under the conventional/solvent-free method is reported. The solvent-free method is more important than the conventional one because of its shorter reaction time, higher yield from easily available starting material, environmental safety, and so forth. Counter anion exchange is carried out using inorganic salt, which is dissolved in deionized water at room temperature. In antibacterial studies, dimeric nitro-substituted imidazolium cations with bromide counter anions showed excellent inhibition against E. coli and P. aeruginosa bacteria. These experimental results were further supported by molecular docking studies. All the compounds (3–6) (a–d) showed excellent antibacterial activity than the standard drugs (gentamycin, nalidixic acid, oflaxacin, ciproflaxacin, and amikacin). Molecular docking studies showed strong hydrogen bonding, polar and hydrophobic interactions between the dimeric imidazolium salts, and Escherichia coli/Pseudomonas aeruginosa/Proteus vulgaris/Staphylococcus aureus receptors.


INTRODUCTION
Ionic liquids are potential and interesting application-oriented molecules. Ionic liquids consist of hydrophobic units, which are pharmaceutically active and a hydrophilic unit, which is easily soluble in water; hence, ionic liquids are used in pharma industries. 1 Imidazolium/pyridinium salt formation from sp 2 nitrogen of heterocyclic aromatic moieties is more useful for making ionic liquids. 2 The sp 2 -hybridized nitrogen containing five-/six-membered heterocyclic components constitutes the larger segment in ionic liquid preparation. Imidazole/pyridine core-based organic cations are commonly used for the synthesis of ionic liquids. 3−5 Monomeric imidazolium/ pyridinium type of ionic liquids showed moderate to excellent anti-inflammatory, anticancer, and antifungal activities. 6,7 Imidazolium/pyridinium cations with various counter anion type of ionic liquids are used in various applications such as in catalysts, corrosion inhibitors, food chemistry, and pharma industries. 8−14 Alkyl propargyl-and silyl alkyl-substituted imidazolium, piperidinium, and pyrrolidinium types of ionic liquids are prepared and their antibacterial response against Gram-positive bacteria are tested. Flexible longer alkyl chainsubstituted ionic liquids showed higher cytotoxicity than the shorter alkyl-substituted imidazolium salt because of their lipophilicity behavior. 15 Pyrrolidinium and piperidinium salts showed lesser antibacterial response than the imidazolium salts. 16 Longer alkylsubstituted imidazolium salt, 1-decyl-3-cinnamylimidazolium chloride, is easily attracted to the phospholipid bilayer than the methyl-substituted −3-cinnamylimidazolium chloride because of its longer alkyl-substituted imidazolium salts, which are more hydrophobic. 17 Pernak and co-workers reported that substituted alkoxy imidazolium salts showed an antimicrobial response against fungi rods and cocci and mentioned that higher alkyl-substituted imidazolium salts showed a very active response against cocci. 18 Gram-negative and Gram-positive pathogens have negative charges in their surface, which is a phospholipid bilayer, so imidazolium/pyridinium types of ionic liquids are easily linked to the outer membrane. 19−21 Hadni and Elhallaoui reported that molecular modeling studies with substituted azaaurones derivatives showed an inhibitory response against cytochromes under two-dimensional (2D) and three-dimensional (3D)-quantitative structure-activity relationship (QSAR)analysis. 22 The application of molecular modeling calculation is a very useful and impressive finding in the drug discovery area. 23,24 Poly(lactic-co-glycolic acid)-based chitosan-functionalized nanoparticles are prepared using the imidazolium type of ionic liquids for the drug delivery process under the emulsion solvent diffusion process. 25 Structural alterations of adenosine deaminase were obtained using alkyl and allyl group fused imidazolium chloride with the assistance of the molecular dynamics and docking model. 26,27 Hydrophobic organic cations and hydrophilic inorganic anions of imidazolium/ pyridinium-based ionic liquids act as cationic surfactants in aqueous medium and also showed an antimicrobial response against Gram-positive and Gram-negative pathogens. Aljuhani et al. reported that 3/4 of methyl pyridinium iodide with amide linger units is prepared under conventional as well as microwave methods and its anticancer and molecular docking properties are studied. 28 Longer alkyl-substituted piperidinium salt showed excellent antimicrobial activity against human pathogenic microorganisms than the simple alkyl-substituted hydroxy piperidinium salts. 29,30 Based on the literature report, we wish to prepare dimericsubstituted imidazolium ionic liquids under the conventional and solvent-free solid-supported method and evaluate the hydrophilic and lipophilic segments containing dimeric-substituted imidazolium salts as antibacterial agents against human pathogenic microorganisms and docking analysis.

Synthesis of Flexible Substituted Dimeric Imidazolium Salts.
The N-alkylation reaction is carried out between 1,4-dibromo butane/m-xylene dibromide and 2methyl-5-nitro imidazole in the presence of one equivalence of NaOH in dry acetonitrile under refluxing conditions for 6−7 h and afforded N-alkylated dimeric imidazole 1 and 2 in 95% of yield after the purification process (Scheme 1). Compounds 1 and 2 are treated with benzyl/4-nitrobenzyl bromide in the presence of CH 3 CN under refluxing conditions for 10 h and afforded 84% of dimeric-substituted imidazolium bromide 3a− 6a (Scheme 1). The same reaction is tried without the solvent under solid-phase silica-supported muffle furnace conditions. Fortunately, the solvent-free method is a more advantageous method than the conventional method such as easy work up, absence of organic solvents, and lesser reaction period with higher yields (Scheme 1).
When we change various counter anions in the dimericsubstituted imidazolium bromide, the physical properties are also changed. Two equivalents of inorganic salts (KPF 6 , NaBF 4 , and LiCF 3 SO 3 ) and bromide counter anions containing dimeric-substituted imidazolium salt are dissolved in double-deionized water and stirred at room temperature for 1 h and it gives anion-exchanged product of compounds 3−6 (b−d) in good yield (Scheme 1). We have used Soxhlet extraction with dry THF to remove unwanted inorganic salts (LiBr, KBr, and NaBr) and then tested with an aqueous AgNO 3 solution. Fortunately, we did not get any pale-yellow precipitate. All the synthesized compounds are fully characterized by various spectral and analytical methods (Table 1).   31 Antibacterial efficacy is purely based on the length of the alkyl chain. While altering the alkyl chain length of quinolinium salts from the lower to higher alkyl group, the antibacterial activity is also changed. 32−35 We have studied the antibacterial screening of dimeric-substituted imidazolium cations with various counter anions against Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris) and Gram-positive (Staphylococcus aureus, Enterococcus faecalis) microorganisms and it showed excellent inhibition than that available in the literature. 36 Antibacterial screening of dimeric-substituted imidazolium salts has been examined by well/disc diffusion methods using Mueller Hinton Agar (MHA). 37 The stock solutions of samples are prepared using dimethyl sulfoxide (DMSO) as the solvent in 1 mg/mL. We have prepared various concentrations of stock solution from 25 μg/ well (7.692 × 10 −4 mmol/mL), 50 μg/well (1.54 × 10 −3 mmol/mL), 75 μg/well (2.31 × 10 −3 mmol/mL), and 100 μg/ well (3.08 × 10 −3 mmol/mL) in DMSO. The bacterial inoculum is adjusted to the 0.5 scale of McFarland standard. 38 The dilutions of dimeric-substituted imidazolium salts are loaded into respective wells of the MHA plate. Gentamicin (30 μg/well), nalidixic acid (30 μg/well), ofloxacin (30 μg/well), ciprofloxacin (30 μg/well), and amikacin (30 μg/well) are  42 Dimeric imidazolium salts of compound 3−6(a−d) are screened for their microbial activities under the well diffusion method (zone of inhibition in diameters). We have prepared 16 substituted dimeric imidazolium salts; among these, nitro-substituted dimeric imidazolium salts showed excellent inhibition than the simple dimeric imidazolium salts ( Table 2).

Microdilution
Technique. MIC and MBC of synthesized imidazolium salts against Gram-negative/Grampositive human pathogens are studied under the microdilution technique. 44 We have observed that compound 6a shows excellent antimicrobial activity against E. coli and P. vulgaris than that against S. aureus; other pathogens showed good to moderate activity (Table 2)
Molecular docking analysis suggested that significant binding affinity toward E. coli with compound 3b showed hydrogen bonding, hydrophobicity, and van der Waals bonding with different peptides (Tables 4, S5−S7; Figure 5a). Molecular docking studies are extended to P. aeruginosa with compound 3c which showed hydrogen bonding with nearly six different amino acids (Tables 3, S5−S7; Figure 5b). Interesting observations are made based on docking results which showed effective binding with P. vulgaris and S. aureus against compound 5a and 6a, respectively (Tables 2 and 3, S2−S7; Figure 5c,d). We conclude that the dimeric imidazolium salts showed effective antibacterial activity, hydrogen bonding, hydrophobicity, van der Waals bonding based on antibacterial and molecular docking studies.

CONCLUSIONS
Dimeric-substituted imidazolium salts are prepared using benzyl/4-nitro benzyl bromide under the conventional/ solvent-free method. We observed that the solvent-free method is more advantageous than the conventional one because of its shorter reaction time, higher yield, environmentally safe and easy work procedure, and so forth. The counter anion exchange is carried out using inorganic salt which is dissolved in deionized water at room temperature. Computer-assisted docking analysis was performed for unsubstituted/nitrosubstituted dimeric imidazolium salts. In docking studies, effective binding occurred between proteins and dimeric imidazolium salts 3−6 (a−d) via hydrogen bonding with least energy levels. Nitro-substituted imidazolium salt 4b shows effective hydrogen bonding between SER 49 and TRP 22 with the lowest intermolecular energy at −6.68 against E. coli. Hydrogen bonding studies with nitro-substituted imidazolium bromide against various pathogens like E. coli, P. aeruginosa, P. vulgaris, and S. aureus are carried out. Among these pathogens, the nitro-substituted dimeric imidazolium salts 4 (a−d) showed the highest intermolecular binding in the order E. coli > P. aeruginosa > P. vulgaris > S. aureus based on the number of hydrogen bonding, intermolecular energy, residues, and other physical parameters. Other dimeric imidazolium salts 3 (a−d) and 5 (a−d) showed good to moderate values.

General Procedures for Solid-Supported Solvent-Free Muffle Furnace
Conditions. Required equivalents, as mentioned in the conventional method, were added in the absence of the solvent with 5 g of (80−120 mesh) silica gel followed by fine grinding using a mortar and pestle. The reaction mixture was kept in a muffle furnace at 100°C.

General Procedure for the Anion Exchange
Reaction. The N-alkylated product of quaternary ammonium bromide (1.0 equiv) is treated with NaBF 4 , KPF 6 , and LiCF 3 SO 3 (2.05 equiv) in the presence of 10 mL of deionized water at room temperature under stirring for about 1 h to afford the anion-exchanged ionic liquids. After the anion exchange reaction, we have used Soxhlet extraction to remove metal bromide from ionic liquids using 100 mL of dry THF for about 1 h refluxtion to give ionic liquids 3−6 (b−d) in quantitative yield. (1)