Novel Synthetic Routes to Quaternary Pyridinium Salts and their Antifungal Activity

: Eleven pyridine derivatives were prepared by quaternization reactions by different synthetic routes: conventional, microwave, and ultrasound. Since acetone an d other solvents used in conventional quaternization reactions are harmful, attempts were made to replace the organic solvents with more environmentally friendly alternative - deep eutectic solvents. The reactions were carried out using pyridine -3- aldoxime, pyridine-4 -aldoxime, isonicotinamide and nicotinamide as nucleophiles and three different dihaloalkanes as electrophiles: diiodopro-pane, dibromopropane and diiodohexane. The results showed that the microwave method using acetone as solvent was significa ntly faster and gave higher yields than the conventional method. In contrast, synthesis in the eutectic solvents choline chloride : urea gave significantly lower yields. The structures of the synthesized compounds were confirmed by 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis. The antifungal activity of all compounds was tested at two different concentrations (10 and 100 µg mL –1 ) against Botrytis cinerea, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiorum in vitro . All tested compounds showed excellent inhibitory activity against the studied phytopathogenic fungal species at a concentration of 100 µg mL –1 .


INTRODUCTION
YRIDINIUM salts are the main topic of scientific research due to their physicochemical properties and various biological activities. Their antimicrobial, [1][2][3][4] antimalarial, [5,6] and antileishmanial [7][8][9] activities have been reported in the literature. Pyridinium-based oxime compounds are used worldwide as antidotes following exposure to anticholinesterase agents. [10,11] Bioassay results have shown that some of them have excellent antifungal, [12,13] insecticidal and herbicidal activities. [14] They are involved in a wide variety of synthetically useful reactions in organic chemistry and serve as important intermediates for the preparation of pharmacologically active heterocycles. [15][16][17] Numerous reviews on quaternary pyridinium salts have described the synthetic pathways, reactivity, and importance of pyridinium compounds as antimicrobial, antimalarial, anticancer, and anticholinergic inhibitors. [18][19][20][21][22][23][24] Since the compounds are of huge importance to industry, various routes of their synthesis need to be considered. In today's world, where the chemical industry produces a lot of waste, it has never been more important that chemical processes be performed in a more environmentally friendly manner. Therefore, methods for the production of pyridinium salts must be in line with green chemistry methods. The classical synthetic route for the preparation of quaternary pyridinium salts involves the quaternization reaction of pyridine with organic halides. The aim of our work was to carry out the quaternization reaction with different pyridine-aldoximes, nicotinamide and isonicotinamide as nucleophiles, and three different dihaloalkanes as electrophiles: diiodopropane, dibromopropane and diiodohexane. For this purpose, several methods for the preparation of quaternary pyridinium salts were investigated: conventional method, microwave method, ultrasound method. Acetonitrile, [25] anhydrous benzene, [26] acetone and anhydrous dimethylformamide [27] are used as organic solvents for the conventional quaternization reactions of pyridinium salts. Inspired by our previous research [28] in which we successfully substituted classical organic solvents with eutectic solvents in the quaternization reaction, this study presents different synthetic routes to quaternary pyridinium salts and an attempt of quaternization in a deep eutectic solvent, choline-chloride (ChCl) : urea. Since the quaternary pyridinium salts have shown antifungal activity in our previous study, [13] in this paper we investigated whether the obtained products with the quaternary nitrogen atom and the hydrophobic tail are effective fungicides compared to commercial agricultural ones.

Synthesis and Analysis of Quaternary Salts
Microwave-assisted synthesis (MW) was performed in The general scheme of the quaternary pyridinium salts synthesis is shown in Scheme 1, and the products studied are shown in Scheme 2.

CONVENTIONAL METHOD
The quaternization reactions of pyridine derivatives (pyridine-4-aldoxime, pyridine-3-aldoxime, nicotinamide, isonicotinamide) with an appropriate dihaloalkane reagents took place in acetone. In pyridine derivative solution (0.04 g, 4 mmol) dissolved in 10 mL of acetone, the corresponding dihaloalkane (20 mmol) was added in small portions. The dihaloalkanes were added in a molar ratio of 5:1 to the heterocyclic compound. The reaction mixture was heated under reflux for 3 h and then cooled to room temperature. The progress of the reaction was monitored by thin layer chromatography (TLC) in the mobile phase consisting of the solvent mixtures chloroform and methanol in the ratio 6 : 2. Slow cooling of the reaction mixture promoted the formation of crystals. The crude product was washed with diethyl ether to remove the residual dihaloalkane and with acetone to remove the starting pyridine derivatives, and recrystallized from the mixture (ethyl acetate : ethanol = 1 : 1) to obtain the pure product. Scheme 1. General scheme of the quaternary pyridinium salts synthesis.
Scheme 2. Series of studied quaternary pyridinium salts.

US SYNTHESIS
To a pyridine derivative solution (0.12 g, 1 mmol) dissolved in 5 mL of acetone, dihaloalkane (5 mmol) was added. The reaction mixture was subjected to US irradiation, heated at reflux for 3 h and then cooled to room temperature. Slow cooling of the reaction mixture promoted the formation of crystals. The crude product was purified by column chromatography using the solvent system chloroform : methanol = 6 : 1.

MW SYNTHESIS IN ACETONE
Pyridinium starting compound (0.12 g, 1 mmol) was dissolved in 5 mL of acetone. Dihaloalkane (5 mmol) was added in solution. The reaction mixture was subjected to MW irradiation (20 minutes at 250 W). Slow cooling of the reaction mixture promoted the formation of crystals. The crude product was purified by column chromatography (chloroform : methanol = 6 :1).

SYNTHESIS IN DES Preparation of DES ChCl : urea
DES was prepared by mixing choline chloride (5 g), previously dried at 65 °C for 24 h, with urea in a 1:2 molar ratio at 80 °C on a magnetic stirrer for 3 h. Stable, homogeneous solutions were cooled and used without further purification.
The mixture of pyridine derivatives (1 mmol) and dihaloalkanes (5 mmol) was dissolved in DES ChCl : urea (reactant : choline chloride = 1:10). The reaction mixture was heated to 80 °C and stirred for 3 h on a magnetic stirrer. Absolute ethanol was then added and the product was precipitated over the next 24 h. The crude product was filtered off and washed with diethyl ether to remove the residual dihaloalkane. The crude product was purified by column chromatography carried out with the solvent system chloroform : methanol = 6 : 1.
The optimization of the quaternization reaction in DES ChCl: urea was carried out on the model reaction of nicotinamide and diiodopropane by the conventional method at 40, 60 and 80 °C for 1 h in the solvent ChCl: urea (Table 2). Then, optimization was performed at a temperature of 80 °C for 30 min, 1 h, 2 h, 3 h and 4 h to determine the reaction time which gave the highest yield ( Table 3).
The experimental parameters for compounds 1, 3 and 7 were already published in our paper Bušić et al. [3] In this work, they were synthesized from acetone and deep eutectic solvents, and their antifungal activity was also studied.

ANTIFUNGAL TEST
The antifungal test was performed on four cultures of phytopathogenic fungi (Macrophomina phaseolina, Sclerotinia sclerotiorum, Fusarium culmorum, and Botrytis cinerea) from the culture collections of the Department of Phytopathology, Faculty of Agrobiotechnical Sciences Osijek, University of Osijek. The fungicidal activity of eleven synthetic compounds at concentrations of 10 µg mL -1 and 100 µg mL -1 was tested. For the antifungal tests, the method of Bušić et al. [28] was used. Each treatment was performed in four replicates for each fungal species. An untreated PDA was used as a control. The commercial fungicide was used as a positive control. For B. cinerea, the fungicide from the hydroxynalide group with the active ingredient fenhexaminet was used. For M. phaseolina, S. sclerotiorum and F. culmorum, the fungicide from the tebuconazole group with the active ingredient difenconazole was used. The fungicides were mixed with PDA at the recommended concentration. The first diameter of mycelial growth of each culture was measured 48 h after inoculation. Measurements were taken for up to 168 h (depending on the fungal species) in two directions to determine the average colony growth in millimeters. Based on the measured mycelial growth, the inhibition rate was calculated. The inhibition rate of the synthetic compounds on the four fungal species was calculated based on the inhibition index (I, %): Statistical analysis of the experimental results was performed using factorial analysis of variance ANOVA, in which the data were grouped according to the inhibition rate and the applied concentration. To estimate the statistical significance of the differences between the synthetic compounds at different concentrations, the Fisher LSD test was applied using the SAS 9.2 statistical package. Means were considered significantly different when p ≤ 0.05.
The structures of the synthesized compounds were confirmed by 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis. The chemical shifts of the NMR signals in the spectra (δ = 9.30 -8.  4-6, 8-11). The atom labels of the synthesized compounds used for assignment in the NMR spectra, as well as the 1 H and 13 C spectra of 11 with labeled signals are shown in Figures 1. The results show a different reaction yield depending on the method and the reaction performed (Table 5). In acetone as solvent, the lowest yields were obtained by the conventional method.
The highest yield obtained by conventional method was achieved for product 1 (85 %) formed by the quaternization reaction of pyridinium-4-aldoxime and 1,3-diiodopropane. The lowest yield was obtained for product 7 obtained by the quaternization reaction of isonicotinamide and 1,3-dibromopropane. Iodides are generally found to be better nucleophiles than bromides.
Ultrasound method showed significantly higher yields than the conventional method, but not as high as with the microwave method. The highest yield by ultrasound method was also achieved for product 1 (91 %).
The optimization of the quaternization reaction under the influence of microwave irradiation was carried out on the model reaction of nicotinamide and diiodopropane for 2.5, 5, 10, 20, and 40 min and 250 and 500 W, respectively. The optimization showed that the most favorable reaction conditions where a time of 20 min and a power of 250 W ( Table 1). The highest yields were obtained with this synthetic method. The quaternization reaction of pyridinium-4-aldoxime and diiodopropane gave compound 1 in almost quantitative yield (98 %).
To determine the reaction parameters for performing the quaternization reaction in the deep eutectic solvent ChCl : urea (1 : 2), the model reaction of nicotinamide and diiodopropane was performed at different temperatures and times. The optimization showed that the most suitable temperature for the quaternization reaction is 80 °C ( Table  2) and a reaction time of 3 h for the conventional method (Table 3). To carry out microwave synthesis in DES, an optimization method based on time and power was obtained. From this study, the most suitable time in the MW method is 30 min at a power of 250 W (Table 4). Quaternary salts were formed in the eutectic solvent ChCl : urea. However,    the reaction yields were significantly lower than in acetone as solvent. It was also surprising that lower yields were obtained by the microwave method in the eutectic solvent than as opposed to the same in acetone. The highest yields for conventional and microwave synthesis in CHCl : urea were obtained for compound 1 (75 % and 80 %, respectively).

Antifungal Activity
The inhibitory activity of all eleven compounds at a concentration of 10 µg mL -1 (Table 6)     index of 44.48 %, while compounds 7 and 9 showed the weakest inhibitory effect with 19.57 %. However, compared to the control variant, the inhibitory effect of these compounds was statistically significantly higher. Compound 4 with an inhibitory effect of 54.46 % and compound 1 with 50.21% had the strongest effect on the growth of the pathogen M. phaseolina at a concentration of 10 µg mL -1 . Compound 9 had the weakest inhibitory effect with 22.63 %.
Considering the highest and lowest inhibition index of each pathogen, it can be concluded that the effect of all compounds at a concentration of 10 µg mL -1 had the weakest inhibition effect on the pathogens M. phaseolina (22.63 -54.46 %) and F. culmorum (19.57 % -44.48 %).
All eleven compounds at a concentration of 100 µg mL -1 (Table 7) again showed very strong growth inhibition for the pathogen S. sclerotiorum. The growth inhibition for this pathogen ranged from 79.62 to 90.76 %.

CONCLUSIONS
The studies showed that the quaternization reaction could be successfully carried out in acetone as solvent. The highest yields were obtained with the microwave method, slightly lower with the ultrasound method, while low to medium yields were obtained with the conventional method. When syntheses were carried out in eutectic solvents as an environmentally friendly alternative to conventional organic solvents, products were formed but the yields were lower. Therefore, the quaternization of pyridine compounds with dihaloalkanes in other choline chloride based deep eutectic solvents remains to be explored. From the obtained results, it can be concluded that three compounds (4, 6, 8) have excellent inhibitory activity on the studied phytopathogenic fungal species at a concentration of 100 µg mL -1 . However, it should be noted  that compound 5 has a very good inhibitory effect on S. sclerotiorum at both concentrations, while the same compound has the weakest effect on the pathogens F. culmorum and M. phaseolina at a concentration of 100 µg mL -1 . Thus, it can be said that the effect of a single compound depends on the pathogen species, but it can also have a stimulatory effect on them.