Synthesis and characterization of 1-carbalkoxymethyl-4-hydroxy-1-methylpiperidinium chlorides

The synthesis of new 1-carbalkoxymethyl-4-hydroxy-1-methylpiperidinium chlorides derived from 4-hydroxy-1-methylpiperidine and alkyl chloroacetates was described. Salts were characterized by MS, FTIR, 1 H and 13 C NMR spectroscopies. Compounds investigated were tested against Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger.


Introduction
The antibacterial properties of quaternary ammonium compounds were first observed at the end of the 19-th century.The effects of substituents, structures and anions of quaternary salts on the antimicrobial activity have been extensively studied. 1 The germicidal activity is the greatest in the compounds containing a long alkyl chain from 12 to 16 carbon atoms. 1 To increase solubility of the quaternary salts an ester group is introduced to compounds of a series of N-methylmorpholinium, N,N'-dimethyl-piperazinium, N-methyl-piperidinium salts and they were tested against same bacteria.A series of piperidinium salts has been shown to have maximum activity. 2n recent years a permanent increase in the number of bacterial strains resistant to disinfectants has been observed.Cationic surfactants are of interest because of their membranedisruptive properties, rapid antimicrobial activities, and activity against a broad range of bacteria and other cell. 3This is the reason why the compounds belonging to this group are still synthesized.
Recently we have synthesized N-carbalkoxylmethyl-N-alkyl-piperidinium chlorides and tested them against nine bacteria species and two Candida-type yeasts. 4The most active are Ncarbdodecyloxymethyl-N-methylpiperidinium chloride and N-carbethoxymethyl-Ndodecylpiperidinium chloride, whose activity is greater than that of dodecyl-trimethylammonium chloride, usually used as a reference substance in the microbiological tests.The promising pharmacological properties of this type of salts have prompted a synthesis of a series of piperidinium salts in which the hydroxyl group is at the piperidinium ring.[7][8][9][10][11][12] The aim of this work is the synthesis of new 1-carbalkoxymethyl-4-hydroxy-1methylpiperidinium chlorides (Scheme 1) and their spectral characterization by FTIR, MS, 1 H and 13 C NMR.The hydroxyl group at the piperidinium ring and a long alkyl chain in the ester group are expected to increase the solubility of salts and their antimicrobial properties.
In the previous work, we have studied the structures of two stereoisomers of 1carboxymethyl-4-hydroxy-1-methylpiperidinium chloride. 13According to the X-ray analysis these stereoisomers differ in the conformation of the hydroxyl group at the C(4) atom, which can be in the axial or equatorial position, while the methyl group attached to the nitrogen atom is in the axial position and the carboxymethyl substituent in the equatorial one.

Results and Discussion
Starting from 4-hydroxy-1-methylpiperidine and the corresponding alkyl chloroacetates a series of eighth 1-carbalkoxymethyl-4-hydroxy-1-methylpiperidinium chlorides (1-8) was synthesized (Scheme 1).A mixture of stereoisomers, with the OH group in the axial (a) and the equatorial (e) positions was obtained.Only esters 1e and 2a were isolated as the pure stereoisomer by a fractional crystallization. 14Esters 3-8 are very soluble in water and in organic solvents, hence a separation of stereoisomers (a) and (e) by a multiple recrystallization from acetonitrile was not successful.The melting points of 3-8 were constant and could not be raised by further treatment.The 1 H NMR spectra show that the stereoisomer with the equatorial OH group is predominant (60 %) in the reaction mixture.It has been shown earlier that in the quaternization of piperidine derivatives the ratio of equatorial to axial isomers depends on solvent, alkylation agent and substrate structure.

Scheme 1
The proton chemical shifts of the compounds investigated are listed in Table 1.The numbering of atoms is shown in Scheme 1.The significant difference in the 1 H NMR spectra of isomers (e) and (a) appears in the region of the resonance signals assigned to the protons at the C(2,6) carbon atoms.The spectrum of the stereoisomer with the axial OH group reveals two multiplets, while that with the equatorial OH group only one broad multiplet. 14he X-ray diffraction study of 1 confirmed the chair conformation of the piperidinium ring with the OH and CH 2 COOCH 3 groups in the equatorial positions and the CH 3 group in the axial one. 16The 1 H NMR spectrum of 1(e) is characterized by a broad multiplet at 3.71 ppm, attributed to the equatorial and axial protons attached to C(2,6) atoms (Figure1a).* The piperidinium ring with the hydroxyl group in the axial position Two separate multiplets corresponding to the equatorial and axial protons at 3.86 and 3.56 ppm are observed in the spectrum of 2 (Figure1b).Such behavior of the equatorial and axial protons at C(2,6) is similar to that observed in the spectrum of α-4-hydroxy-1-methylpiperidine betaine hydrochloride, in which the orientation of the hydroxyl group was estimated by X-ray analysis. 13,14However, three multiplets are observed in the region of the resonance of protons attached to C(2,6) atoms in the spectra of the 3 -8 esters (Figure 1c).Two of these at ca. 3.85 and 3.61 ppm are due to the isomers with the axial OH group, 3(a) -8(a), while the more intensive multiplet at ca. 3.73 ppm is attributed to the stereoisomers with the equatorial OH group, 3(e) -8(e).This behavior of the resonance signals suggests that the compounds 3 -8 exist as a mixture of stereoisomers (e) and (a) (Scheme 1).
As follows from the 1 H NMR spectra, the steroisomers with the equatorial OH group, 3(e) -8(e) are predominant in the reaction mixture and their population has been determined as ca.60 %, from the intensities of the resonance signal of the N + CH 3 protons.This signal appears in the lower magnetic field in the spectra of stereoisomers (e), with the equatorial OH group (compare Figures 1a and 1b).Table 2. 13 C chemical shifts (ppm) of 1-carbalkoxymethyl-4-hydroxy-1-methylpiperidinium chlorides ( * The piperidinium ring with the hydroxyl group in the axial position The carbon chemical shifts of the compounds investigated in D 2 O are listed in Table 2 and the 13 C NMR spectrum of 6 is shown in Fig. 2. The assignments are based on the 1 H- 13 C two dimensional correlation. In the carbon-13 spectra the double signals attributed to the ring carbon atoms C(2,6), C(3,5) and to the carbon atoms of N + CH 3 , N + CH 2 and COO groups confirm the existence of two stereoisomers in the reaction between 4-hydroxy-1-methylpiperidine and alkyl chloroacetate (Figure 2) (Scheme 1).The resonance signals of the carbon atoms of the N + CH 3 and N + CH 2 groups are less intense than the signals of the ring carbon atoms and the alkyl chain.To study the antimicrobial activity of the compounds investigated two strains of bacteria: Escherichia coli and Staphylococcus aureus, one yeast-type: Candida albicans and one funge Aspergillus niger have been used.The minimum inhibitory concentration (MIC) of the title compounds vary from 0.1% [2.6 mM] to above 5% [119 mM] (Table 3).The lowest values of MIC are observed for the compounds with the decyl, undecyl and dodecyl alkyl chain (Figure 5).The data indicate that the compound 6 shows the highest antibacterial and antifungal activity.The biocidal concentration of 6 is 0.1 % and 0.2 % for Staphylococcus aureus and Escherichia coli, respectively.Fungicidal concentrations are slightly higher, and both for Candida albicans and Apergillus niger are 0.5%.The mechanism of the biocidal action of quaternary ammonium compounds is based on the adsorption of positively charged alkylammonium salt moiety on the negatively charged cell wall and its penetration by alkyl chain which leads to a leak of low molecular components of cell.In a consequence the microorganism cell dies.It has been previously shown that the most effective are the ammonium derivatives with hydrocarbon chain between C 12 -C 14 . 18-21A similar correlations are observed for N-methyl-N-carbalkoxymethylpiperidinium chlorides. 4perimental Section General Procedures.Elemental analyses were carried out with a Vario EL III instrument and the values found were within ±0.2 % of theoretical values.Mass spectra were run on a Waters and Micromass Spectrometer 2Q, using direct inlet system under positive ion electrospray ionization source in methanol. 1H and 13 C NMR spectra were recorded on a Varian-Gemini 300VT spectrometer operating at 300.07 ( 1 H) and 75.4614 ( 13 C) MHz, respectively.The spectra were measured in D 2 O relative to an internal standard of 3-(trimethylsilyl)propionic-d 4 acid sodium salt and in CDCl 3 relative to TMS.The 2D spectra were obtained with standard Varian software.FTIR spectra were measured on a Bruker IFS 113v instrument, evacuated to avoid water and CO 2 absorption.Solid state spectra were recorded in Nujol and Fluorolube suspensions using KBr plates.Each spectrum consists of 128 scans.General procedure A Alkyl chloroacetates were prepared by refluxing chloroacetic acid (0.5 mol) with corresponding alkyl alcohol (0.5) in the presence of sulfuric acid (5 dcm 3 ) and benzene (100 dm 3 ) as solvent for 7 h.The mixture was extracted with two 50 portions of 25% sodium carbonate.The benzene layer was dried over sodium sulfate and the solvent distilled off.The esters of chloroacetic acid were purified by vacuum distillation and obtained in yields from 65 to 95%. 2,22The esters as colorless liquids were further characterized by comparison of their boiling point with reported data from the literature and their NMR spectra.Octyl chloroacetate, b.p. 110-111 o C/2 mm Hg, 23 ; 91-93 o C/5 mm Hg; 24  General procedure B 1-Carbalkoxymethyl-4-hydroxy-1-methylpiperidinium chlorides were prepared by treating 4hydroxy-1-methylpiperidine (1 M) with the corresponding alkyl chloroacetates (1 M) in diethyl ether solution.The mixture was stirred at room temperature till formation of precipitate and finally left to stand for 24 h.The crude product was filtered off and washed with diethyl ether.

Figure 5 .
Figure 5.The plot of the minimum inhibitory concentration (MIC) of salts (in mM) on (a) Escherichia coli (○) and Staphylococcus aureus (∆) and (b) Candida albicans (□) and Aspergillus niger ( ), versus the number of the methylene groups in the hydrophobic chain.
Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538, Candida albicans ATCC 10231 and Aspergillus niger ATCC 16484 were taken from Collection of Pure Cultures (ŁOCK 105) of Institute of Fermentation Technology and Microbiology of Technical University of Łódź, Poland.

Table 3 .
Activity of the compounds studied with microorganisms in mM (% w/v) isolated as stereoisomer with the OH group in the equatorial position at the piperidine ring; recrystallized from methanol, m.p. 184-185 o C. Anal.