Synthesis of quaternary ammonium salts based on quinuclidin-3-ol and pyridine-4-aldoxime with alkyl chains

Methods: The commercially available quinuclidine-3-ol and pyridine-4-aldoxime were used for the synthesis of new derivatives with the appropriate alkyl chains. The purity of the synthesized salts was tested by thin-layer chromatography on an aluminum plate where aluminum oxide was used as a stationary phase. Melting points were determined in open capillaries using an instrument for melting point determination and the obtained values were left uncorrected. The FTIR spectra were recorded with a spectrometer and the data were analyzed in cm-1.


Introduction
Quaternary ammonium salts have a wide range of applications across a variety of industries; due to their pronounced antimicrobial activity, they can be used as ingredients in surfactants, such as cationic surfactants, or incorporated into drugs, such as antibiotics [1]. The structure of these heterocyclic compounds features a nitrogen atom, most commonly as part of a cyclic structure, and may occasionally contain other heteroatoms. These compounds also play a major biological role as participants in various metabolic processes and as building blocks of some of the most important molecules in nature, such as DNA and RNA [2]. Heterocyclic compounds are present in the structure of naturally occurring bioactive compounds, but they are also commonly found in synthesized compounds that can be used to research biological properties. Pyridine and quinuclidine are among the various heterocyclic compounds whose derivatives, for example pyridinium oximes or 3-substituted quinuclidine derivatives, have a widespread application in the pharmacological industry [3].
Commercially available quaternary ammonium salts, such as benzalkonium chloride (BAC), cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC) and dodecyldimethylammonium chloride (DDAC), are weakly reactive, resulting in their accumulation in the environment. Consequently, due to heavy exposure to these compounds, bacteria may develop resistance to commercially available antimicrobial agents [4,5]. With technological advancement, attempts have been made to produce more effective alternatives to said agents so as to overcome bacterial resistance. Previously synthesized quinuclidin-3-ol-based quaternary ammonium salts have exhibited good antimicrobial activity against Gram-positive and Gram-negative bacteria in combination with various aryl reagents [6]. The aim of this paper is to present a synthesis of new quaternary ammonium salts, which had not been described in the literature. For this purpose, we used quaternization reactions of quinuclidin-3-ol and pyridine-4-aldoxime with alkyl chains containing three to eight carbon atoms and one or two terminal bromine atoms.

Quaternization reactions of quinuclidin-3-ol and pyridine-4-aldoxime
The quaternization reactions of quinuclidin-3-ol and pyridine-4-aldoxime with appropriate alkyl reagents took place in anhydrous acetone. Quinuclidin-3-ol or pyridine-4-aldoxime, respectively, were dissolved in a minimum volume of acetone. An appropriate alkyl reagent was then added directly into the solution in small portions. Alkyl bromide was added in equimolar ratio (1:1) and alkyl dibromide in double excess with respect to the heterocyclic compound. The reaction mixture was left for two or three days in a dark place on a magnetic stirrer in an air-tight environment.

Purity testing of newly synthesized quaternary salts
After synthesis, the purity of the produced quaternary ammonium salts was tested by thin-layer chromatography, where aluminum oxide on an aluminum plate was used as a stationary phase (Merck, Darmstadt, Germany) and the mobile phase consisted of the solvent mixtures of chloroform and methanol in the 9:1 and/or 5:1 ratio. Detection was performed using a UV lamp for all synthesized compounds and pyridine-4-oxime. Treatment with iodine vapor was applied to quinuclidine-3-ol as the structure of this compound lacks conjugated double bonds. The melting point was determined for each newly synthesized solid using the Melting Point SMP1 (Stuart Scientific) device. IR spectra were recorded using the Shimadzu FTIR-8400S spectrophotometer, with wave numbers n expressed in cm -1 .

Synthesis of quinuclidin-3-ol-based quaternary salts
30.5 mg of quinuclidin-3-ol was weighed using an analytical balance (M = 127.18 g mol -1 ; n = 0.2398 mmol) in a penicillin vial and dissolved in 900 μL of acetone. An equimolar amount of an appropriate alkyl reagent (1-bromopropane, 1-bromobutane, 1-bromohexane, 1-bromooctane) was added directly into the prepared solution, whereas 1,3-dibromopropane was added in double excess. The reaction mixture was left in the dark for 48-72 hours at room temperature until the formation of white crystals, which were washed several times with ether (Figure 1). Thin-layer chromatography determined a R f value corresponding to the formation of a quaternary ammonium salt which, due to the high polarity, did not deviate from the starting value, unlike the reactants.
The quaternization reaction of quinuclidin-3-ol with an appropriate alkyl dibromide was performed using the same procedure, with the exception being that the amount of the appropriate alkyl reagent was doubled and pre-dissolved in a minimum volume of acetone (Figure 2). The product was a white solid. Table 1 shows data on the newly synthesized quinuclidin-3-ol-based quaternary ammonium salts.

Synthesis of pyridine-4-aldoxime-based quaternary salts
Fifty mg of pyridine-4-aldoxime was weighed using an analytical balance (M = 122.12 gmol -1 ; n = 0.4094 mmol) in a penicillin vial and dissolved in 400 μL of acetone. Double that amount of an appropriate alkyl reagent was dissolved in a minimum volume of acetone.
The penicillin vial containing the dissolved pyridine-4-aldoxime was placed on a magnetic   Figure 3). Using thin layer chromatography, R f value corresponding to the formation of a quaternary ammonium salt was determined in this case as well, which, due to the high polarity, did not deviate from the starting value, unlike the reactants. Table 2 shows data on the newly synthesized pyridine-4-aldoxime-based quaternary ammonium salts.

Discussion
The purpose of synthesizing quaternary ammonium salts as described in this paper was to test their biological properties -primarily, their antimicrobial activity -for the pur-  The purity of synthesized compounds was determined by TLC chromatography and all compounds were obtained in very good yields. The quaternary bromides were identified by melting point and IR spectroscopy which shows characteristic vibration of functional groups heterocyclic compounds but also vibration characteristic for C-Br bond. The weakness of our study is the lack of a more detailed structure analysis of newly synthetized compounds using NMR or MS spectroscopies. FTIR spectroscopy and determination of melting point do not provide proof of the chemical structure and prior of biological testing the compounds will be sent for further structure analysis.
All the synthesized salts described in this paper featured a positively charged nitrogen atom as part of their polar "head" and a long hydrocarbon chain as the non-polar "tail".
Due to their surface-tension decreasing property and electrostatic attraction to a negatively charged bacterial cell wall, these compounds penetrate the bacterial membrane bilayer.
The consequence of this interaction between the quaternary salt and the bacterial membrane is the disruption of the membrane and leakage of the cytoplasmic content, or the "lysis" of the bacterial cell ( Figure 5). Therefore, these compounds are frequently used as ingredients of surfactants with membrane activity [8]. Moreover, we have shown that quaternization with long alkyl chains results in compounds with good antimicrobial activity, more specifically against ESKAPE human pathogens [6] for which reason, newly synthesized compounds could have industrial and medical application.