Synthesis and Characterization of Antibacterial Ionic Liquids Moieties under Multiple Routes and their Catalytic Responses

2-Methyl-4(5)-nitro(3-methylenebenzene)-imidazoliumbromide 1a: 4.60 g; 97%; Mp: 168-170 °C; 1H NMR (400 MHz, DMSO-d6): δ; 2.35 (s, 3H), 5.28 (s, 2H), 7.08-7.17 (m, 5H), 8.42 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ; 14.1, 50.1, 122.9, 127.6, 128.3, 129.3, 130.9, 136.0, 145.7; MS: m/z: 298.13; Elemental analysis: Molecular formula (C11H12BrN3O2) Calculated: C: 44.30; H: 4.02; N: 14.09; Found C: 44.26; H: 3.96; N: 14.04.

From the study, authors claimed that minimal inhibitory concentrations are varied depends on the substituents [23][24][25][26].Luczak and co-workers [27] investigated the cytotoxic experiments with hydrophobic cation with different counter anions.The properties of cytotoxicity are purely based on hydrophobicity of counter cation; whereas counter anion showed little importance.Eco-toxicological screening of several classes of ionic liquids demonstrated that, some alkyl substituted imidazolium/pyridinium salts showed cytotoxic behaviours against microorganism [28][29][30][31].Herein, we wish to report the synthesis of substituted imidazolium cation with various counter anions under conventional/solvent free silica supported Muffle furnace approach and studied its catalytic response for Pechmann reaction.Also we have studied the solvent effects for Pechmann reaction from less polar, high polar solvents.Alberto and co-workers prepared the toxic and poisonous behaviours of Selenium containing ionic liquids under multistep reaction in the presence of toxic organic chlorinated solvent.They have been studied the antimicrobial investigation of Selenium containing of ionic liquids against human pathogenic microorganisms.While compared their results, we could say our IS's are acted potential candidate and showed excellent antimicrobial activities under MBC and MIC methods.To the best of our knowledge, our IS's showed excellent antibacterial responses than the available reported work [32].

General procedure for solvent free Muffle furnace condition
The above procedure is repeated except solvent, we have used 5 g of (80-120 mesh) silica gel with fine grinding using mortar & pestle.The reaction mixture is kept in Muffle furnace at 100°C for required period.

General procedure for anion exchange reaction
N-alkylated product of imidazolium bromide 1a,1b (1.0 equi.) is treated with NaBF 4 /KPF 6 /LiCF 3 SO 3 (1.05equi.) in the presence of 10 mL of deionized water at room temperature with stirring for about 1 h afforded the anion exchanged product of imidazolium cation with different anion.After the anion exchanged reaction, we have used Soxhlet extraction to remove metal bromide from imidazolium salts using 100 mL of dry THF for about 1 h refluxtion to give respective imidazolium salts in quantitative yield.

General procedure for imidazolium salt assisted Pechmann reaction
Phenol/4-nitrophenol/4-chlorophenol (2.126 × 10 -3 mmol; 1.0 eq.), EAA (2.126 × 10 -3 mmol; 1.0 eq.) and solvent (5 mL) are mixed along with optimized concentration of imidazolium salt (2.021 × 10 -4 mmol) at room temperature with stirring.After disappearance of starting material monitored by TLC, the reaction mixture was poured into 5 mL and ice cold water and 100 mL of diethyl ether and stirred for 5 min.two layers are formed; the organic layer was then dried over anhydrous Na 2 SO 4 .The organic layer was evaporated to dryness under reduced pressure to obtain a pure compound.

Results and Discussion
2-Methyl-4(5)-nitroimidazole is treated with slight excess amount of benzyl/4-nitrobenzylbromide in the presence of dry MeCN under refluxing condition between 9-13 h afforded the N-alkylated IS's 1 (ab) in quantitative yield (Scheme 1).The above reaction is conducted under solvent free silica supported Muffle furnace.Equal molar ratios of substituted imidazole and benzyl bromide/4-nitrobenzyl bromide are grained with 5 g of silica gel (80-120 mesh) using mortar and pestle, and kept in Muffle furnace at 100°C; we have monitor the reaction by TLC.We found that solvent free silica supported Muffle furnace reactions were completed from 3-3.5 h.Hence solvent free silica supported reaction are more convenient than the conventional method because of its lesser reactions time, higher yield, solvent free condition and easy work up.N-Alkylated imidazolium bromides 1(a-b) are treated with various inorganic salts such as KPF 6 , NaBF 4 and LiCF 3 SO 3 in the presence of deionized water at room temperature for 1 h to afford the anion exchanged product of IS 2 (a-f) in quantitative yield.The separation of IS from metal bromide using organic solvent extraction is not easier due to its water-soluble nature.So we carried out the Soxhlet extraction with dry tetrahydrofuran (THF) for 2 h under refluxing condition to afford metal bromide-free IS with higher yield.After completion of Soxhlet extraction, we have confirmed with aqueous AgNO 3 solution.The synthesized compounds 2 (a-f) are thoroughly characterized by spectral and analytical data.

Catalytic activities
Calculated equivalent of Pechmann reactants under conventional method without catalyst takes 10 hours to give only 30% yield.The same reaction is tried with optimum concentration of our catalyst using various polar and non-polar solvents which is completed with lesser reaction time and higher yield.We have used varies solvents from less polar to more polar solvents for Pechmann reaction to find out the suitable solvent we observed that, reaction time and percentage of yields are varied from less to the more polar solvent (Tables 1-4).We have studied solvent effect with different solvents like dimethyl sulfoxide (DMSO), ethanol, methanol, acetone and tetrahydrofuran (THF).Among these solvents, DMSO showed less reaction time with higher yield than the other solvents.Polar solvent plays an important role to enhance the Pechmann reaction (Table 1).Reaction between phenol and ethylacetoacetate (EAA) with optimized concentration of our substituted IS under conventional approach afforded 4-methyl-2H-chromen-2-one (3), (Scheme 2; Table 1).
4-chloro phenol is much reactive than the 4-nitrophenol in Pechmann reaction due to its electron release towards the arene moiety and facilitate the reaction due to high electron (Table 3).Preparation of 6-chloro-4-methyl-2H-chromen-2-one (5) using solvent free Silica Supported Approach (SSA) under Muffle furnace at optimum temperature 100°C; gave the product within 11 minutes whereas under conventional approach took 30 min.So solvent free silica supported methodology is more suitable than conventional condition for Pechmann reaction (Table 3).
To avoid the organic solvents, we tried silica supported solvent free method to afford chromone derivatives under Muffle furnace within 15 min in 97% yield.Hence, Muffle furnace method is more convenient than the conventional method because of its solvent free non-toxic nature, high yield and easy workup.
We have used nearly eight different IS's to study the catalytic responses for the Pechmann reaction.Among these 1a and 2 (a-c) showed excellent catalytic behaviour than the others.Because of the Lewis character than the others.Imidazolium cation with bromide counter anion showed excellent catalytic activity than the other counter anions.The Pechmann reaction under solvent free condition is nearly fifty times faster than conventional approach.Open Access 4 that 2.012 × 10 -4 mmol concentration is the optimum catalyst concentration.While increasing the concentration from 2.012 × 10 -4 mmol to 2.683 × 10 -4 mmol, there is no appreciable changes.The results are described in table 6.
Our synthesized substituted IS are acted as efficient candidates to accelerate the Pechmann reaction as quicker with higher yield.
Synthesized substituted IS are recycled upon fourth cycles and used for preparation of Chromen-one derivatives under same reaction condition.Even after fourth recycle, the product obtained was same as we observed in the fresh use shown in the figure 1.  4).Under silica supported Muffle furnace at 100°C; formation of 1-methyl-3H-benzo[f]chromen-3-one (6) completed within 17 minutes but under conventional method required more reaction time.

Microbiological study
We have examined the Pechmann product formation from (schemes 2-6) without IS, there is no appreciable changes even for long periods (Table 5) To find out the optimum catalyst concentration for Pechmann reaction of our IS we have used 8.385 × 10 -5 , 1.341 × 10 -4 , 2.012 × 10 -4 and 2.683 × 10 -4 mmol concentrations of our IS.We have observed   Bacterial cultures: Five pathogenic bacteria's have been used for present study as test microorganisms such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris and Staphylococcus aureus.The bacterial cultures were obtained from Department of Microbiology, Presidency College, Chennai, India.The bacteria are subculture using Nutrient agar (Himedia, India) and stored at 4°C until required for the study.

Sterility test:
A loopful of IS were inoculated into Nutrient agar and Sabourauds dextrose agar plates and incubated at 37°C and 20°C for 24 h and 72 h respectively.The compound was found by steriled.

Antibacterial studies
Rajarajan et al. [33] reported that bacterial screening of their synthesized quinolium salts are purely based on length of side chain.While moving from carbon number 8 into 16 the antibacterial responses also enhanced.Based on this report we have screened the antibacterial screening against both Gram positive/negative pathogens, which revealed that our IS's are observed excellent inhibition than the reported literature [33].Antibacterial activities of IS's are studied by well/disc diffusion methods using Mueller Hinton Agar (MHA) [34].The stock solution of the IS's are prepared as 1 mg/mL and the dilution were prepared using dimethyl sulfoxide as 25 µg/well (5.091 × 10 -4 mmol/mL), 50 µg/well (1.02 × 0 -4 mmol/ mL), 75 µg/well (1.53 × 10 -4 mmol/mL) and 100 µg/well (2.04 × 10 -4 mmol/mL) concentrations.The bacterial inoculum was adjusted to 0.5 scale of McFarland standard.The dilutions of IS's were loaded into respective wells of MHA plate.The antibiotic Erythromycin (30 µg/well, Nalidixic acid (30 µg/well) and Amikacin (30 µg/ well) are used as standard drugs for studies.The MHA plates were incubated at 37°C for 18-24 h.The zone of inhibition was measured in mm using Vernier Caliper and compared with standard drug disc.
Recovery plate technique: 20 µl of each well was streaked onto the sterile nutrient agar plates.The plates were incubated at 37°C for 18-24 h.The presence of growth was confirmed by imidazolium salts which have no bactericidal activities and absence of growth is confirmed that that the IS's has bactericidal activities.

Well diffusion technique: Antimicrobial activities of IS's against Gram negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris) and Gram positive (Staphylococcus aureus) microorganisms under well diffusion technique. Antimicrobial screening is conducted by well diffusion
technique as zone of inhibition with diameters.IS's are screened for their microbial activities under well diffusion method (Figures 2-6).Eight IS's were spread on petri dish plate and screened well diffusion with five test pathogens (Table 7).From the bacterial screening, we found that imidazolium bromide [(MNMNBI) Br -] 1b showed effective zone of inhibition while compared with other synthesized drug molecules.

Microdilution technique:
We have screened the minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) activities of our IS against Gram negative and Gram positive human pathogens under micro dilution technique (Table 8).We have observed that 1b showed excellent anti-microbial activities against Escherichia coli than the Proteus vulgaris and other pathogens are observed from good to moderate activities.

Binding site prediction via docking studies:
Computer assisted molecular docking studies are carried out in order to find out the host-guest binding interaction between our synthesized unsubstituted/nitro substituted IS's with different proteins.Figures 7-11 showed clearly how the pathogens are effectively binds with our IS's via host-gust interactions (ligand bond, hydrogen bonding etc).Compound 2a showed effective hydrogen bonding with E.coli pathogens.There are two amino acids residue like Cys, 238 A°, Ser 70 A° interacts with 2a via hydrogen bonding (Figure 7).Similarly we have run the docking studies of other synthesized IS's to analysis host guest interaction with compound 1b against P. vulgaris and S. aureus observed hydrogen bonding with two amino acid residues (Figures 10 and 11).Compound 2f and 1b showed mild interaction with available amino acid residues (Figures 8 and 9).
HB plots of microorganism of E. coli (5DWK), K. pneumonia (5EEC), P. aeruginosa (4Y9P), P. vulgaris (1KOZ) and S. aureus (3VUA) for compound 2a, 2f and 1b respectively HB plots give additional evidence for effective binding between various human pathogenic microorganisms against our IS's (Figures 12-16).From the molecular docking analysis; showed hydrogen bonding, hydrophobicity as well as Vanderwall interactions with different a peptides are observed between E. coli with [(MNMBI) Br -] 1a (Figure 17).Molecular docking studies are extended for other pathogen like K. pnemoniae with [(MNMBI) PF 6 -] 2b showed effective hydrogen bonding with six different amino acids (Figure 18).Also we found that interesting host-guest interaction from docking analysis are shown effective binding against P. aeruginosa, P. vulgaris and S. aureus against compound 1b (Figures 19-21).From the bacterial as well as molecular docking studies; host-guest interaction like hydrogen bonding, Vanderwall bonding as well as hydrophobic interaction between test pathogens and IS are very effective.
From the docking studies, observed that effective binding between human pathogenic bacteria's and our synthesized compounds via hydrogen bonding under least energy levels.Nitro substituted imidazolium bromide showed effective hydrogen bonding (TYR 6 (-1.66),TYR arg (-1.224) under lowest intermolecular energy at -7.17 against P. vulgaris.Then we have extended the hydrogen bonding studies with our nitro substituted imidazolium bromide against various pathogens like E. coli, K. Pneumonia, P. aeruginosa, S. aureus.Among these five pathogens our IS's showed binding under least energy level as follows order P. vulgaris>P.aeruginosa>E.coli based on number of hydrogen bonding, intermolecular energy, other physical parameters are available in table 9. Rest of the other our synthesized IS's are showed host-guest between\ human pathogen from good to moderate value.

Conclusions
We have prepared variety of substituted IS's under conventional and silica supported Muffle furnace method.From the preparation, solvent free silica supported method is much appreciable than the conventional due to lesser reaction time, higher yield, non-toxic and easy work up.Catalytic activities of optimized concentration of our eight IS's for Pechmann reaction were studied.Among these, nitro substituted imidazolium cation with bromide counter ion showed effective catalytic response than the others.We have employed several phenols such as phenol, 2-naphthol, 4-chlorophenol, 4-nitrophenol for Pechmann reaction under conventional as well as solvent free approach in absence and presence of IS's.Among these 4-chlorophenol reacts faster than the others.Solvent free Muffle furnace in the presence of optimum concentration of IS's showed nearly 50 times faster than the conventional method.Antimicrobial activities of our synthesized IS's against human pathogenic organisms were studied.We found that our IS's gives much more effective response than the poisonous and toxic Selenium containing of ionic liquids which are reported in literature [35].Our nitro substituted imidazolium bromide 1b showed excellent antibacterial activity against tested organisms.Nitro substituted imidazolium cation with PF 6 -, BF 4 -and CF 3 SO 3 -counter anions showed good anti-bacterial responses.We found that unsubstituted IS showed moderate response under MIC studies.

Table 6 :
Different concentrations of IS

Table 7 :
Antibacterial screening of MNMBI and MNMNBI with different counter anions against human pathogens under disc diffusion method