Synthesis and Antimicrobial Activity of 4-S-Methyl-1,3,4-Oxadiazole Derivatives of Some Natural Amino Acids Bearing Secondary Quaternary Ammonium Salt Moieties

Secondary quaternary ammonium salts (QAS) derived from three natural amino acids (L-Leucine, LPhenylalanine and L-Methionine) bearing 1,3,4-oxadiazole and acetic acid moieties have been synthesized and characterized by IR, 1H and 13C NMR. All the synthesized compounds were evaluated for their preliminary in vitro antibacterial and antifungal activity against different bacterial and fungal strains. All the synthesized compounds showed varying degrees of inhibition against the tested microorganisms.

Amino acids play a crucial role in diverse biological functions [13], they are the building blocks for proteins in all living organisms [14].
Some Amino acids and their derivatives are used therapeutically [15] and those bearing 1,3,4oxadiazole ring display a broad spectrum of biological activities for new drug development [16]. Such as antibacterial [17], antimycobacterial [18], antifungal [19], antiviral [20], anticancer [21], anti-inflammatory [22], antituberculosis [23] and diverse pharmacological activities [24]. This paper is focused on the synthesis and evaluating antimicrobial activities of secondary quaternary ammonium salts (QAS) derived from three natural amino acids (L-leucine, Lphenylalanine and L-methionine) bearing 1,3,4oxadiazole and acetic acid moieties. This work is a part of big project to cover all natural and unnatural effective amino acids.

Results and Discussion
The target compounds 6(a-c) were synthesized by a multiple-step procedure as shown in Scheme 1. Amino acid methyl ester 2(a-c) were synthesized using starting amino acid in methanol and concentrated sulfuric acid in good yields (67-87%), see schema1. Esters then converted to acid hydrazides using hydrazine in ethanol. The hydrazides were converted to substituted 1,3,4-oxadiazole-2-thione 4(a-c) using carbon disulfide and ethanolic potassium hydroxide, the compounds were alkylated with methyl iodide in ethanolic sodium hydroxide to give the S-methyl derivative 5(a-c). Compound 5(a-c) were quaternized with bromoacetic acid to form the final products 6(a-c).

In vitro antimicrobial activities
In vitro antimicrobials activities of the tested compounds 6(a-c) were screened for antibacterial activity against both reference strains and clinical isolates. Were taken from collection of pure cultures of Laboratory of Biotoxicology, Pharmacognosy and Biological recovery of plants, University of Dr Moulay Taher, Saida.

Antibacterial screening
The Antimicrobial activities of the tested compounds 6(a-c) were screened for in vitro antimicrobial activity by measuring the Minimum Inhibitory concentrations (MIC) and the minimum bactericidal concentration (MBC) which gives the lowest concentrations of compound inhibiting visible growth, according to the broth macrodilution method of the National Committee for Clinical Laboratory Standards (NCCLS) recommendations [26]. Three Gram-positive bacteria and three Gram-negative bacteria, stock solutions of different test compounds (100 μg per mL) were made in DMSO. A series of dilutions of each tested compound in the range concentration of 10, 5, 2.5, 1.5, 0.625, 0.3125, 0.156, 0.078, and 0.039 mg/mL were prepared. Fresh cultures of bacteria obtained by inoculating bacteria in Muller-Hinton broth. The Petri-dishes containing bacterial species were incubated at 37 °C for 24 h, and observed for antibacterial activity. Test was repeated twice and the average values of MIC and MBC results are presented in Table 1.
As indicated in Table1 all synthesized compounds are more active against Grampositive microorganism as compared to gram negative. The results of antimicrobial evaluation suggest that the compounds 6a have very good potential to act as antibacterial agents against Gram-positive bacteria. Compounds 6c exhibited good inhibitory effects against Bacillus subtilis and Campylobactr fetus. Compound 6b showed growth inhibition against Bacillus subtilis and Enterobacter cloacae. Samples with CMI˃10 mg /mL were considered not determined nd

Anti-yeast activities
Compounds 6(a-c) assayed for anti-yeast activities against two registered yeast species, Candida albicans ATCC 10231 and Candida albicans IPP444. They are maintained by subculturing on Sabouraud agar and stored at 4 °C, and the plates were incubated at 25 °C for 2-5 days. A Reference method for broth dilution antifungal susceptibility testing of yeasts was used to determine minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Test was performed twice and average values of MIC and MFC are shown in Table 2. Regarding the Anti-yeast activity the tested compounds exhibited significant Anti-yeast activities, compounds 6b has maximum activity against Candida albicans ATCC 10231 and compound 6a displayed good activity against Candida albicans IP 444.

Antifungal Activities
In order to investigate the antifungal activity of the final compounds 6(a-c), four fresh cultures of fungal strains were used such as Aspergillus niger, Alternaria alternata, Rhizopus stolinifer and Aspergillus flavus. Dimethylsulphoxide (DMSO) was used as a solvent for all the compounds and as negative control. Compounds were tested with the following concentrations: 2.5; 1.25; 0.5 and 0.25 μL/mL in 20 mL of tepid PDA medium in the test tube. The test plates were incubated at 27 ˚C. When the mycelium of fungi reached the edge of the control plate, the antifungal index was calculated as follows: Where Da is the diameter of the growth zone in the experimental dish (cm) and Db is the diameter of the growth zone in the control dish (cm). The average results of the antifungal activities of the tested compounds 6(a-c) for two times are presented in Table 3. Table 3. Antifungal indices (%) of tested compounds 6(a-c). The results listed in Table 3 indicated that the compounds 6a, 6b and 6c exhibited more than 80% inhibitory activity against A. niger, compounds 6b showed prominent activity showed prominent activity against A. flavus, the tested compounds exhibited slightly lower activity against R. stolinifer

General
All solvents and reagents used in this study were obtained from Sigma Aldrich and BIOCHEM. The purity of the compounds was routinely checked by thin layer chromatography (TLC) using silica gel F254 supplied by MERCK, while spots were observed by using iodine as visualizing agent. All Melting points were determined in open capillary tubes on a BÜCHI 540 melting point apparatus and are uncorrected. The Infrared spectra as potassium bromide discs recorded on a Shimadzu FTIR-8300 Fourier Transform infrared spectrophotometer and expressed in cm -1 . The 1 H-and 13 C-NMR Spectra were measured in protic deuterated solvents such as methanol and water on Bruker AM 300 MHZ Spectrometer (University of Oran, Essenia), relative to the internal standard tetramethylsilane (TMS), and chemical shift values are expressed in parts per million (δ, ppm).

General procedure for the preparation of amino acid methyl esters 2(a-c):
Amino acid methyl esters was prepared according to literature [25], into a solution of the corresponding amino acid 1(a-c) (0.018 mole) in absolute methanol and concentrated sulfuric acid (2 ml) was heated at 80°C in an oil bath. Reaction followed with TLC to obtain the desired compound. The excess of acid neutralized with sodium bicarbonate, washed with methylene chloride. Solvent evaporated and the product collected.

General procedure for the preparation of amino acidhydrazide 3(a-c):
Amino acid methyl esters 2(a-c) (0.011 mole) were refluxed with hydrazine hydrate (0.1 mole, 3 ml) in absolute ethanol for 4-5 hours. Reactions were monitored by TLC. Mixtures were cooled down, solvent evaporated and the products collected.

General procedure for synthesis of 5-Subtituted -1, 3, 4-oxadiazoles-2-methylthio N-carboxymethyl ammonium bromides 6(a-c):
Secondary quaternary ammonium salts were prepared by direct quaternisation of primary amine with molar equivalent quantity of compound 5(a-c) (0.0042 mole) and bromoacetic acid (0.58g, 0.0042 mole) dissolved in dry acetone (50 mL), these mixture were gently refluxed for 8 hours. After that, solvent was evaporated the residue were washed several times with cold diethyl ether and allowed to dry.

Conclusions
In this paper, new QAS compounds containing active moiety 1,3,4-oxadiazole Lamino acids, L-Leucine, L-Phenylalanine and L-Methionine attached to secondary ammonium bromide group were successfully synthesized and characterized. Physical and antimicrobial (antibacterial, antiyeast and antifungal) properties were studied. The synthesized compounds showed promising antimicrobial potential against some of the tested microorganisms.
The newly synthesized compounds were prepared with an objective of developing better antimicrobial activity and for future investigations.