Microwave-assisted Quaternization of Various Pyridine Derivatives and their Antibacterial Activity

In this study, reactions of quaternization under microwave heating of pyridine, α-picoline, pyridine-4-aldoxime, pyridine-2aldoxime, nicotinamide, isonicotinamide and pyridoxal oxime with different electrophiles: 2-bromo-4'-nitroacetophenone, 2-amino-4chloro-methylthiazole hydrochloride, methyl iodide, 1,3-diiodopropane and 1,3-dibromopropane are reported. The synthesis yield by microwave dielectric heating is improved and reaction time shortened compared to conventional heating. The structure of obtained molecules were analyzed and determined by 1D and 2D NMR spectroscopy methods, IR spectroscopy and mass spectrometry. The highest antibacterial activity against two Gram-positive and two Gram-negative bacteria strains has been found for 1-[2-(4-nitrophenyl)-2oxoethyl]pyridinium bromide (2).


INTRODUCTION
N recent decades, a large number of reports on synthesis of heterocycles compounds containing N, O and S have been published due to their wide range of biological activity.−3] Pyridinium salts belong to the category of cationic surfactants consisting of a hydrophilic part, such as a quaternary nitrogen moiety which is able to interact in polar chemical milieu, and a hydrophobic part which can penetrate into non-polar molecular agglomerates.These are unsaturated heterocyclic compounds with different functional groups present either on pyridine ring or on nitrogen atom.A great deal of pyridinium derivatives have been investigated concerning their biological and pharmacological activities.Their importance lies in their effective antimicrobial, [4][5][6][7][8][9] antiviral, [10][11][12] antihypertensive and immunostimulating activities. [13]16][17] The advantages of microwave heathing compared to conventional synthesis are: a shorter reaction time (from hours or days to minutes), better utilization and decrease in by-product production. [18] simple and efficient method of preparation of five 1-(4-bromophenacyl)azoles under the influence of MW irradiation in solvent-free conditions was described.Quaternization reactions were performed by using pyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, indazole and the benzotriazole with 4-bromophenacyl bromide.In all examples, only N-1 alkylated products were obtained as a consequence of the solvent absence whereas equimolar parts of phenacylbromide and azoles were used. [19]22] Since the conventional methods of quaternization reactions were performed with a large excess of solvent, and as they are time consuming to obtain acceptable yields, we tried to perform the reactions in a different way.Therefore, new methods were developed for the synthesis of different pyridine derivatives (Scheme 1).The reaction time was shortened, a better yield of the product was obtained and the whole reaction was more energy efficient.
Since pyridinium derivatives are most frequently used as antimicrobial or antibacterial agents, some of the synthesized compounds were tested in order to determine their potential antibacterial activity against two Grampositive and two Gram-negative bacteria.

MW Synthesis and Analysis of Pyridinium Salts
All reactions were performed in a controllable single-mode microwave reactor, Microwave Synthesis Labstation Start S (Milestone, Shelton, Connecticut, USA).The reactor is equipped with a magnetic stirrer as well as with temperature and power controls (220 V/50-60 Hz, 2.4 kW).Solvents and reagents were purchased from Fluka and Aldrich and used without further purification.IR spectra were measured on FTIR -8400S, SHIMADZU spectrophotometer in KBr pellets (Shimadzu Corporation, Japan).
The one-and two-dimensional homo-and heteronuclear 1 H, 13 C NMR spectra were recorded with a Bruker AV 300 and AV 600 spectrometers, operating at 300.132 MHz and 600.135MHz for the 1 H nucleus and 75.475MHz and 150.917MHz for the 13 C nucleus (Bruker, Rheinstetten, Germany).Samples were measured from DMSO-d6 solutions at 25 °C (298 K) in 5 mm NMR tubes.Chemical shifts, in ppm, are referred to TMS.FID resolutions in 1 H and 13 C NMR spectra were 0.29 and 0.54 Hz per point, respectively.The following measurement techniques were used: standard 1 H, 13 C APT, COSY, HMQC and HMBC.The 2D NMR spectra were measured in pulsed field gradient mode (z-gradient).The enumeration of carbon atoms used for the assignment is displayed in Figure 1.The API 2000 LC-MS/MS (Applied Biosystems, USA) is used to provide information about the molecular ions of synthesized compounds in Q1 MS scan mode.Melting points were determined with Stuart melting point apparatus SMP 3 (Mettler Toledo, Croatia).

4-hydroxyiminomethyl-1-methylpyridinium iodide (4).
Pyridine-4-aldoxime (0.871 g, 4 mmol) and methyl iodide (0.765 mL) were dissolved in 5 mL of acetone.The reaction mixture was subjected to MW irradiation (2 minutes at 250 W) until the product was not visible on TLC.As a result, the reaction mixture changed from yellow to orange and during cooling yellow crystals immediately precipitated.

Antibacterial Activity of Selected Compounds
Regeneration of microbial cultures was performed on Tryptic Glucose Yeast extract agar (TGK agar; Biolife, Italy) composition: peptone bios D 5.0 g, yeast extract 2.5 g, 1.0 g of glucose, agar BiosLL 15 g; 23.5 g agar (dissolved in demineralized water, heated to boiling, and sterilized by autoclaving at 121 °C for 15 minutes at pH: 7.0±0.2).Prior to analysis, all cultures were regenerated by a successive subculturing on TGK agar for three days.Determination of minimum bactericidal (MBC) and minimum inhibitory concentrations (MIC) was performed in the Brain heart infusion broth (BHI broth, Biolife, Italy).Composition of BHI broth extract: 200 g of brain tissue, heart tissue extract 250 g, peptocomplex 10 g, D-(+) glucose 2.0 g, NaCl 5.0 g, Na2HPO4 2.5 g.BHI broth was prepared by dissolving of 37 g of culture medium in 1000 ml of demineralized water, heated to boiling, and sterilized by autoclaving (121 °C/15 minutes).The pH of the medium was 7.4±0.2.Selected compounds (1, 2, 3, 6) were dissolved (0.04 g) in 4 mL of DMSO and 10 mL of 6 % Tween 80 before testing.Solubility was amended by ultrasound bath.After dissolving, the solutions were sterilized by filtration through a filter with 0.22 µm pore size.The solution of the selected compounds was serially diluted in BHI broth to achieve following concentrations: 5000 mg L −1 , 2500 mg L −1 , 1250 mg L −1 and 650 mg L −1 .To each tube, a suspension of bacterial cells (0.1 mL; 1×10 6 CFU mL −1 ) in saline was transferred.After incubation for 24 h ours at 37 °C (25 °C for P. syringae), 0.1 mL from the tubes with no visible growth was transferred to a sterile BHI broth.After an additional 24 h incubation at 37 °C (25 °C for P. fluorescens), in case of no growth, the concentration of the compound was MBC (minimum bactericidal concentration).If growth was visible, the concentration was MIC (minimum inhibitory concentration).At MIC concentration the cells survive in the presence of the tested compound, but they do not proliferate.Gentamicin sulfate (Sigma-Aldrich ® , Saint Louis, USA) was used as positive control.

RESULTS AND DISCUSSION
In this paper, MW assisted quaternization of pyridine, αpicoline, pyridine-4-aldoxime, pyridine-2-aldoxime, nicotinamide, isonicotinamide and pyridoxal oxime with various electrophiles: 2-bromo-4'-nitroacetophenone, 2amino-4-chloromethylthiazole hydrochloride, methyl iodide, 1,3-diiodopropane and 1,3-dibromopropane has been investigated.The obtained results are shown in Table 1, which includes yields, and optimal conditions in reactions of quaternization influenced by MW irradiation.On the basis of our previous research [24] the synthesis was carried out under microwave powers of 250 W and 440 W, and different irradiation time intervals (from 1 minute to 10 minutes).These reactions were performed under the influence of MW irradiation in a variety of solvents (absolute ethanol, acetonitrile and acetone), depending on the solubility of the starting materials and the obtained products.For the synthesis of compound (1) solvent was not necessary because the thiazole reactant was soluble in α-picoline.
Ten compounds were prepared and identified by FTIR, NMR and MS spectra.The structures of the salts were confirmed by one-and two-dimensional homo-and heteronuclear 1 H and 13 C NMR spectroscopy analyses.The assignment of spectra was performed on basis of chemical and substituent shifts, coupling constants and connectivity in two-dimensional homo-and heteronuclear spectra.The correlation of 1 H chemical shifts of pyridine moiety in COSY spectra and C-H correlations in HMBC spectra over three bonds between H-2/6 and C-7 and H-10/14 and C-8 unambiguously proved the structures of obtained compounds.
Quaternization of α-picoline with 2-amino-4chloromethylthiazole was successful and gave compound (1).The product was synthesized in significantly shorter reaction time (4 min) with higher yield (23 %) compared to the conventional synthesis (72 hours, 10 %), (Scheme 2). [23]n our previous work we synthesized nine quaternary salts of pyridoxal oxime using microwave heating in solvent (acetone) and under solvent-free conditions. [24]An attempt was made to perform quaternization reaction of pyridoxal oxime with 2-amino-4-chloromethylthiazole under MW irradiation (Scheme 3), but the synthesis was not successful.It could be assumed that the reaction did not occur for steric reasons and short linker between two heterocyclic rings.
Quaternization reaction was also performed on isonicotinamide using two electrophiles: dibromopropane and diiodopropane.In very short time products (7) and (8)  in moderate yields were obtained (Scheme 7).
Similar synthesis of isonicotinamide in a conventional way was examined.It was performed quaternization reaction of isonicotinamide with 1, 4-dibromobutane in acetonitrile and after 18 hours at 65 to 70 °C the product in 96 % yield was obtained. [27]yridine-2-aldoxime methyl iodide (2-PAM) is the most common nerve agent.According to Poziomiek et al., 2-PAM was synthesized for the first time in ethanol.The yield given by Poziomek et al., was 34.8 % after 160 hours and ethyl instead of the methyl group was added to the pyridinium nitrogen.Even though it is not directly comparable, one would expect that addition of methyl to  the pyridinium nitrogen would result in similar yields as compared to the yield for ethyl addition in the same location.This deviation could be caused by steric hindrance.An ethyl group is a larger than methyl group, thus it is possible that smaller groups like methyl may be added to the pyridine nitrogen, with higher yields than larger groups like ethyl. [28]The synthesis of 2-PAM iodide was also performed in acetone where a pressure bottle was used and the temperature kept at 95 °C for 6 hours obtaining moderate yield (44 %). [26]2-PAM was synthesized by the conventional method; reflux in acetone for about 4 hours at 60 °C gave 69 % yield. [29]In our study a simple method for the efficient synthesis of the same compound, 2-hydroxyiminomethyl-1-methyl-pyridinium iodide ( 9), was used.The synthesis was performed under MW irradiation at 250 W with a shorter reaction time (4 minutes) and in a higher yield of 94 % (Scheme 8).
Quaternization reaction of pyridoxal oxime with methyl iodide was successfully performed resulting in methyl-3-hydroxy-4-hydroxyiminomethyl-5-hydroxy methyl-1,2-dimethylpyridinium iodide formation (10) (Scheme 9).Compared to the conventional way, it was obtained in higher yield and shorter reaction time. [30]ew methods of synthesis under the influence of MW irradiation resulted in higher yields as well as considerable reduction of the reaction time.

Antibacterial Activity
Antibacterial properties of four synthesized quaternary pyridinium salts were examined.The minimum inhibitory (MICs) and minimum bactericidal concentration (MBCs) for two Gram-positive and two Gram-negative bacteria are shown in Table 2.The synthesized compounds were the most active against Pseudomonas syringae (Gramnegative) bacteria.As a plant pathogen, it can infect a wide range of species, as it comprises more than 50 different pathovars.A variety of symptoms are associated with woody plants infected by Pseudomonas syringae. [31]−34] Compound (1) with thiazole moiety showed a weak antibacterial activity against all tested bacteria.The results are consistent with the literature data in which the results revealed that most of the heterocycles containing benzothiazole moiety displayed weak effects on the growth of the tested Gram-positive and Gram-negative bacterial strains.The tested compounds acted similarly to fungal strains. [35]A similar study of antimicrobial activity of some thiazole derivatives was conducted where most of the compounds showed a moderate degree of potent antimicrobial activity against Gram-positive, Gramnegative bacteria and fungi. [36]n our study, the lowest minimum bactericidal concentration (MBC) has been observed in compounds ( 2) and (6) (625 mg L −1 ), while the lowest minimum inhibitory concentration (MIC) has been determined for compounds (2) and ( 6), (625 mg L −1 ).The results indicate that quaternary pyridinium salts exhibit weak antibacterial activity and, as such, cannot be used to control bacterial Scheme 8. Preparation of 2-hydroxyiminomethyl-1-methylpyridinium iodide (9).Scheme 9. Preparation of 3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-1,2 dimethylpyridinium iodide (10).

Figure 2 .
Figure 2. Proposed mode of antimicrobial action for pyridinium compounds.

Table 1 .
Optimal conditions in quaternization reaction under MW irradiation BUŠIĆ et al.: Microwave-assisted Quaternization of Pyridine Derivatives … V.

Table 2 .
Minimum inhibitory and minimum bactericidal concentration of the substance on the selected Gram-positive and Gram-negative bacteria of different analogues of pyridine (MIC and MBC are expressed in mgL −1 ) (a)EF -Enterococcus faecalis; SA -Staphylococcus aureus; SE -Salmonella enteritidis; PS -Pseudomonas syringae.