SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF S-SUBSTITUTED DERIVATIVES OF 1,2,4-TRIAZOL-3-THIOL

The aim of the work. 1,2,4-triazole derivatives possess a wide range of pharmacological activity, so they are used for the development of drugs and active pharmaceutical ingredients. Due to the reactivity of 1,2,4-triazoles there are many options for their further structural modification on different reaction centers. Therefore, the aim of the work was to obtain new S-substituted derivatives of 1,2,4-triazole-3-thiols, study physicochemical parameters of the substances synthesized, evaluate the antimicrobial activity of new S-derivatives of the 4-R-5-((3-(pyridin-4-yl)-1H-1,2,4-triazol-5yl)thio)methyl)-4H-1,2,4-triazole-3-thiol series, and study some regularities of the “structure – biological activity” relationship for the synthesized compounds as well. Materials and methods. The subject of the study was new S-substituted 1,2,4-triazoles containing 2-oxopropan-1-yl and 2-aryl-2-oxoethan-1-yl substituents. The antimicrobial activity was studied by double serial dilutions on test cultures of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 885-653). The results of the biological screening showed that at a concentration of 125 g/mL, all synthesized substances showed activity (MIC – in the range of 31.25 – 62.5 μg/mL, MBCK – in the range of 62.5–125 μg/mL) against strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans. Variation of substituents on the sulfur atom did not lead to a significant change in antimicrobial and antifungal activities among derivatives of 4-R-5-((3(pyridin-4-yl)-1H-1,2,4-triazole-5-yl) thio)methyl)-4H-1,2,4-triazole-3-thiols. Conclusions. Biological screening data indicate the prospects for the search for new antimicrobial substances among the abovementioned derivatives of 1,2,4-triazoles. The most active compounds were 1-((4-ethyl-5-((3-(pyridin-4-yl)-1H-1,2,4triazol-5-yl)thio)methyl)-4H-1,2,4-triazol-3-yl)thio)propan-2-one and 1-(4-methoxyphenyl)-2-(4-ethyl-5-(((3-(pyridin-4-yl)1H)-1,2,4-triazol-5-yl)thio)methyl)-4H-1,2,4-triazol-3-yl)thio)ethanone, which showed the most pronounced antimicrobial activity against the Pseudomonas aeruginosa strain (MIC – 31.25 μg/mL, MBcK – 62.5 μg/mL)


Introduction
Numerous new strains of microorganisms resistant to antimicrobial drugs have recently been identified all over the globe, creating a serious public health threat [1]. Improvement of the existing and development of new effective antimicrobial and antifungal synthetic medicines is one of the priorities of modern pharmaceutic industry.
1,2,4-triazoles are of a great interest due to being an inexhaustible source for search and development of chemical compounds with a variety of biological activities [1,2]. Derivatives of 1,2,4-triazole possess a relatively low toxicity, have a wide spectrum of biological activity and, at the same time, are rather reactive [3,4]. A high probability of finding a pharmacologically valuable substances among 1,2,4-triazoles and the broad possibilities for their chemical modifications are a solid foundation for the search for new synthetic drugs among them [5,6]. Compounds containing 1,2,4-triazole moiety have been demonstrated to have antimicrobial, antitumor, analgesic, hypolipidemic, neuroleptic and other kinds of activities [7][8][9][10][11][12]. Hence, the synthesis of new highly effective antimicrobial medicines containing this heterocyclic fragment is a promising branch in pharmaceutical chemistry investigations.

Planning (methodology) of the research
To achieve the aim the design of the experiment was done (Fig. 1).

Materials and methods
The identity of S-substituted derivatives of 1,2,4triazole 3a-3f, 4a-4f has been confirmed by gas chromatography via Agilent 7890B GC system connected with Agilent 5977B mass spectrometry detector (USA). The column used for separation was DB-5ms with the following dimensions: 30 m × 250 μm × 0.25 μm. Carrier gas (helium) flow rate was 2.0 mL/min. Injection volume: 0.5 μL. Flow split was 1:20. Temperature of the injection system was programmed as follows: 300 °C → →10 °C/s → 310 °C. Oven temperature: programmable, with the initial temperature of 130°C (1 minute delay) → →20 °C/min → 250 °C (5 min delay). Total time of chromatographic run was 12 min. The temperature of the GC-MS interface was maintained at 280°C; ion source temperature was 230 °C; temperature of quadrupole mass analyzer was 150 °C. Type of ionization: electron impact (EI) with electron energy of 70 eV. Range of scanned mass ratios: 50-500 m/z.
Melting points were determined according to open capillary method using OptiMelt MPA100 apparatus (USA) equipped with platinum RTD sensor and temperature measurement possibility of up to 400°C and 0.1°C resolution.
Elemental analysis of the synthesized compounds was afforded using Elementar Vario L cube multipurpose elemental analyzer (CHNS) produced by Analysen systeme GmbH (Germany) using sulfanilamide as the standard. 1 H NMR spectra were recorded on Varian MR-400 spectrometer operating at 400 MHz frequency using DMSO-d 6 as a solvent and tetramethylsilane (TMS) as an internal standard. Spectra were processed via ADVASP™ Analyzer software (Umatek International Inc.). Chemical shifts are reported in ppm (δ scale) downfield with the residual protons of the solvent (DMSO-d 6 ) observed at δ=2.49 ppm.
Starting compounds 1, 2 (Scheme 1) are crystalline substances of white colour, insoluble in water and soluble in N,N-dimethylformamide, dimethyl sulfoxide. For analytical purposes, the compounds were recrystallized from water-methanol mixture. The synthesis of the starting thiols 1, 2 have been previously described by other authors [13].
The compounds 1, 2, 3a-f, 4a-f were synthesized on the Department of Natural Sciences for Foreign Students and Toxicological Chemistry of Zaporizhzhia State Medical University, according to a known procedure [12]; the compounds were further used in the investigation of their antimicrobial activity [14,15].
In the literature tautomeric equilibrium of 1,2,4triazole derivatives was reported [16,17]. Thione form is usually dominant in neutral and acidic media, whereas in an alkaline solution the equilibrium is shifted towards the formation of thiol form which can easily react with alkyl halides.

Experimental biological part
Antibacterial and antifungal activities were studied on the Department of Microbiology, Virology and Immunology of ZSMU. ATCC 25923 strain of Staphylococcus aureus, АТСС 25922 strain of Escherichia coli, ATCC 27853 strain of Pseudomonas aeruginosa, and ATCC 885-653 strain of Candida albicans were used as the test cultures. The activities were investigated according to the methodological recommendations [9] using a double serial dilutions method in a liquid nutrient medium considered optimal for the test cultures' growth at the concentration of 10 6 cells/mL. The evaluated compounds were dissolved in dimethyl sulfoxide (1 mg/mL) prior to the experiment. A minimum inhibitory concentration (MIC) was determined by the absence of a visible growth in a test tube at the minimum concentration of the studied substance sample.
Minimum bactericidal or fungicidal concentrations were determined by seeding the contents of the wells of the plate with dilutions on appropriate dense nutrient media (for bacteria -meat-peptone agar, for yeast-like fungi -dense Saburo medium), and then incubated in the thermostat at the optimal temperature for fungi (37 °C) and bacteria (28 °C) growth. Additionally, control tests of growth media and the solvent were performed in accordance with the known procedures [18].

Discussion
The investigation of antimicrobial activity of the obtained compounds revealed the fact that at the concentration of 125 µg/mL all of the synthesized compounds were active against the strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans.
Study limitations. The serial dilution method is a useful technique in laboratories, but it faces some limitations. Because serial dilution is performed in a stepwise manner, it requires a more extended period. Prepared environments must be deployed immediately, with no storage capability. It is limiting the efficiency of the method.
4. Certain relationships between the chemical structure and antimicrobial and antifungal activities of the synthesized compounds have been determined. It indicates that the search for compounds expressing bactericidal activity in the studied range of derivatives is relevant for research.