Eco-Friendly Synthesis of a New Class of Pyridinium-Based Ionic Liquids with Attractive Antimicrobial Activity

The present study reports a green synthesis of a new family of ionic liquids (ILs) based on functionalized 4-dimethylaminopyridinium derivatives. The structures of 23 newly synthesized ILs (2–24) were confirmed by FT-IR, 1H-, 13C-, 11B-, 19F-, and 31P-NMR spectroscopy and mass spectrometry. The antimicrobial activity of all novel ILs was tested against a panel of bacteria and fungi. The results prove that all tested ILs are effective antibacterial and antifungal agents, especially 4-(dimethylamino)-1-(4-phenoxybutyl)pyridinium derivatives 5 and 19.


Introduction
Ionic liquids (ILs) have received increased attention in recent years due to their outstanding and unique properties, such as negligible vapor pressure, non-volatility, non-flammability, excellent thermal stability, and high electrical conductivity [1][2][3][4][5][6][7].Generally, ILs are defined as organic salts with a melting point below 100 °C that contain an organic cation combined with various anions, such as halides or fluorinated anions [8].An extensive range of applications of ILs has been reported based on the above-cited characteristics.For example, as an alternative solvent of volatile organic compounds [9,10], as media for the electrodeposition of metals [11], catalysts and biocatalysts [12][13][14], potential corrosion inhibitors [15,16], and in food chemical science [17].Additionally, the antimicrobial activity of various families of ILs against both environmental and clinically important microorganisms has been studied by different research groups [18,19].

OPEN ACCESS
In our previous research, we investigated green procedures, including microwave and ultrasound irradiation, to provide a clean synthesis of ILs compared with their conventional preparation.The reduction in reaction times and the increase in the product yields were the most important advantages from using these eco-friendly technologies [20,21].
Continuing our interest in the design and synthesis of potential antimicrobial agents based on ionic liquids [22,23], we herein present an interesting preparation of a new series of ILs based on 4-(dimethylamino)pyridinium derivatives.All newly-synthesized ILs were screened for their antibacterial and antifungal activity against eight pathogenic strains.

Chemistry
ILs 2-24 were synthesized under ultrasound irradiation, as shown in Schemes 1 and 2.  To the best of our knowledge, all are novel ILs except 4-(dimethylamino)-1-(2-hydroxyethyl) pyridinium bromide 1 [24].Initially, the nucleophilic alkylation of 4-dimethylaminopyridine (DMAP) with various functionalized alkyl halides in toluene was carried out under ultrasound irradiation for 5 h at 80 °C, and afforded the desired ILs 1-6 in 79%-85% yield as solids (Table 1).In the second step, three fluorine-containing anions were introduced to obtain low melting point ILs.This metathesis reaction consisted of a halide anion exchange using sodium tetrafluoroborate, potassium hexafluorophosphate or sodium trifluoroacetate under ultrasonic irradiation (Scheme 2).
The desired ionic liquids 7-24 were synthesized by reacting the mixture of 4-dimethylaminopyridinium ILs 1-6 and different metal salts in a closed vessel exposed to ultrasound irradiation for 45 min at 70 °C.The excellent yields for this step are summarized in Table 1.
The structures of ILs 1-6 were confirmed by 1 H-NMR, 13 C-NMR, FT-IR, and LCMS.The 1 H-NMR spectrum contained a singlet around δH 3.20 ppm corresponding to the six protons for N(CH3)2.The protons of the different methylene groups (CH2) of all the ILs were observed at their usual chemical shifts.In addition, the signals of the pyridinium protons appeared as two doublets around δH 7 and 8 ppm.For IL 5, more aromatic protons for the phenyl group were observed as a multiplet at δH 6.89-6.93ppm.It is also important to note the disappearance of the singlet around δH 5.1 ppm for the OH proton in the spectra of ILs 1 and 2, as the NMR solvent was D2O.
All of the 13 C-NMR spectra of ILs 1-6 showed the CH2 and CH3 signals at their usual chemical shifts.For example, the signals for the N(CH3)2, (OCH2), and (NCH2) carbons of IL 2 appeared at δC 39.6, 54.6, and 57.9 ppm, respectively.Furthermore, the aromatic carbons and the C=N gave signals between δC 107-158 ppm.
The IR spectra of ILs 1 and 2 showed a major absorption band at 3213 cm −1 , indicating the presence of hydroxyl group (OH).In addition, the FT-IR spectra of ILs 1-5 contained peaks around 1160 cm −1 , which is consistent with the presence of a C-O bond belonging to an ether or hydroxyl group.To support the NMR evidence, the band at 2247 cm −1 (characteristic of a cyano group), clearly confirms the structure of IL 6.
The structures of ILs 7-24 were also fully characterized.The 1 H-and 13 C-NMR spectra were essentially the same as those recorded for the parent ILs 1-6, and the 11 B-, 19 F-, and 31 P-NMR were also recorded to confirm the success of the metathesis reactions for these compounds.All peaks related to B or F in BF4 appeared around δB −1 ppm and δF −148 ppm.The 31 P-NMR and 19 F-NMR spectra contained a septuplet at δP −131 to −157 ppm related to PF6, and a doublet around δF −69 to −71 ppm related to PF6.Finally, the presence of CF3COO was also confirmed by the 19 F NMR, and gave a peak around δF −73 ppm.

Antimicrobial Activity
As mentioned, one of the aims of the current work was to test the antibacterial and antifungal activities of all newly-synthesized ILs.ILs 1-24 were screened in vitro for their antibacterial activity against a panel of bacteria and fungi.These were two Gram-positive bacteria (Streptococcus pneumonia and Bacillus subtilis) and two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) using an agar diffusion method with Mueller-Hinton agar medium for the bacteria [25].The ILs 1-24 were also screened against four fungal strains (Aspergillus fumigates, Syncephalastrum racemosum, Geotrichum candidum, and Candida albicans) using an agar diffusion method with Sabouraud's agar medium for the fungi [26].
The mean values for inhibition zone diameter summarized in Table 2 show that, except IL 4, 7-10 and 22-24, which did not show any antimicrobial activity against all the tested bacterial and fungal strains, all ILs displayed good to excellent antibacterial activities against the growth of all selected bacteria compared with the standards Amphotericin B, ampicillin and Gentamicin.

Minimum Inhibitory Concentration (MIC)
Based on the excellent results obtained in the inhibition zone test, it seemed appropriate to evaluate the Minimum Inhibitory Concentration (MIC), which is the highest dilution of the compound that shows a clear fluid with no development of turbidity.For this, eight ILs were selected based on their activity, and the results are summarized in Table 3.
From the MIC values obtained, all compounds exhibited antibacterial activity of varying degrees as well as spectrum.In general and as expected, all tested ILs (2, 5, 12, 13, 14, 16, 19, and 20) possessed similar antibacterial activities.
IL 5 (4-(dimethylamino)-1-(4-phenoxybutyl)pyridinium bromide) and IL 19 (4-(dimethylamino)-1-(4-phenoxybutyl)pyridinium tetrafluoroborate) exhibited particularly impressive antimicrobial activities in the series against all tested bacteria and fungi, with MIC values significantly lower than those of the standard controls.The excellent antibacterial activity of ILs 5 and 19 confirm our recently published results and allows us to unambiguously attribute this to the presence of the butylphenoxy group [20].
However, in this case, exchanging the halides (Br or Cl) with fluorinated anions (BF4, PF6 or CF3CO2) did not cause any obvious trends in the activity, and different activities were observed depending on the bacteria or fungi and the ionic liquid tested.

Apparatus
All new compounds were characterized by 1 H-NMR, 13 C-NMR and IR spectroscopy, and LCMS. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were measured in DMSO and D2O at room temperature.Chemical shifts (δ) were reported in ppm, with tetramethylsilane (TMS) as an internal standard (Bruker, Faellanden, Switzerland).The LCMS spectra were measured with a Micromass LCT mass spectrometer (Agilent Technologies, Waldbronn Germany).IR spectra were recorded on a KBr disc with a Shimadzu 8201 PC FT-IR spectrophotometer (νmax in cm −1 ) (Shimadzu Scientific Instruments INC, Canby, OR, USA).The elemental analyses were given by using the 2400 Series II CHNS/O Elemental Analyzer (Perkin Elmer, Waltham, MA, USA).Ultrasound-assisted reactions were performed with a high-intensity ultrasonic processor SUB Aqua 5 Plus-Grant with a temperature controller (750 W), microprocessor controlled-2004.The ultrasonic frequency of the cleaning bath used is 25 KHz (Grant Scientific, Cambridgeshire, UK).

Synthesis
General procedures for the synthesis of imidazolium halides (1-6).To a solution of 4-dimethylaminopyridine (2 g, 0.0163 mol) in 20 mL of toluene, was added the appropriate alkyl halide (1.1 eq) at room temperature.The mixture was placed in a closed vessel and exposed to ultrasound irradiation for 5 h at 80 °C using a sonication bath.The completion of the reaction was marked by the separation of oil or a solid from the initially obtained clear and homogenous mixture of 4-dimethylaminopyridine and alkyl halide in toluene.The product was isolated by extraction or filtration to remove the unreacted starting materials and solvent.Subsequently, the pyridinium IL was washed with (3 × 20 mL) of ethyl acetate followed by drying under reduced pressure.

Table 2 .
Antimicrobial activities (inhibition zone; diameter in mm) of ILs 1-24 against four fungi and four bacteria.