The investigation of antimicrobial activity of some s-substituted bis-1,2,4-triazole-3-thiones

New S-substituted 4-alkyl-5-((3-(pyridin-4-yl)-1H-1,2,4-triazole-5-yl)thio)methyl)-4H-1,2,4-triazole-3-thiol derivatives have been designed, synthesized and studied their antimicrobial activity on 11 standard Gram-positive and Gram-negative microorganism strains. Their spectral and physicochemical parameters were established using modern comprehensive methods of analysis, including 1H NMR spectroscopy, GC-MS and elemental analysis.It has been found that compound 2a exhibits strong suppression of 5 test strains (MBC = 15.6 μg/mL). Compound 4a showed moderate inhibition of Salmonella pullorum, Escherichia coli O2, Salmonella enteritidis strains (MBC = 31.25 μg/mL) Compound 6a was sensitive toward ten tested bacteria at 31.25 μg/mL concentration.


Introduction
The currently rising numbers of drug-resistant bacterial pathogen strains disrupts the effectiveness of the existing treatments and amplifies the incidence of new bacterial infections. This circumstance urges researchers around the world to search for new effective and at the same time low-toxic medicines distinguished by innovative mechanisms of action, which is of the most significance for modern pharmaceutical science.
Organic compounds containing nitrogen atoms represent a large group of chemical structures, of both natural and synthetic origin, which may possess a broad range of biological activities. In this respect, 1,2,4-triazole system, a five-membered nitrogen-containing heterocycle, has arisen strong scientific interest (Shcherbyna et al. 2016;Jethwa 2016). Therefore, compounds that contain a 1,2,4-triazole cycle in their structure are of the most promising subjects of pharmacological research.
The derivatives of 1,2,4-triazole are widely used in pharmaceutical and medicinal chemistry as well as in organic synthesis (Gotsulya et al. 2015;Alrawashdeh 2018). For instance, such drugs as fluconazole, bevacizumab (Avestim), trifuzol-neo, itraconazole, thiotriazoline and many other substances containing 1,2,4-triazole ring have found applications in clinical and veterinary practices.
In our opinion, the investigation of the structures constructed using two bridged 1,2,4-triazole cycles belonging to various chemical classes may lead to the discovery of quite promising compounds that exhibit versatile antibacterial effects. The increase in the number of the system's reaction sites enables sufficient variations of chemical modification of bis-1,2,4-triazole, which multiples the probability of finding active derivatives. During the recent years, there have been numerous publications on the syntheses of 1,2,4-triazole derivatives containing various pharmacophore frameworks that exhibit antimicrobial activity (Düğdü et al. 2014;Saadeh et al. 2010).

Purpose
The purpose of this work was to synthesize new compounds based on 4-alkyl-5-(((3-(pyridin-4-yl)-1Н-1,2,4triazole-5-yl)thio)methyl)-4Н-1,2,4-triazole-3-thiol, in which isopropyl 2-chloroacetate, 1-bromopentane and 2-chloroacetamid were used as alkylation agents, study the ammonolysis of the previously obtained isopropyl ester with methylamine, ethylamine, dimethylamine, diethylamine, morpholine and 2-methylpiperidine, study their physicochemical properties and confirm the structure of these derivatives using modern multidimensional methods of identification. Along with that, we attempted to investigate the antimicrobial activities of the compounds on standard Gram-positive and Gram-negative microorganism strains to evaluate the pharmacological potential of the substances. The ultimate purpose was to conduct the comparative analysis of the relationships between the antimicrobial activity and functional groups of these molecules.

Planning (methodology) of research
To achieve this aim and find the answers to the queries, the experiment was designed in the way illustrated in Fig. 1.

Generalities
Melting points were determined on 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 was performed on a Elementar Vario L cube multipurpose elemental analyzer (CHNS) produced by Analysen systeme GmbH (Germany) using sulfanilamide as the standard. The mass-tocharge ratio of S-derivatives of bis-1,2,4-triazole was been determined by gas chromatography via Agilent 7890B GC system equipped with Agilent 5977B mass spectrometry detector (USA). The column used for separation was DB-5ms. Type of ionization: electron impact (EI) with electron energy of 70 eV. 1 H NMR spectra were recorded at 400 MHz and 100 MHz using Varian MR-400 spectrometer with DMSO-d 6 as the solvent. Spectra were processed via ADVASP Analyzer software (Umatek International Inc.). Chemical shifts are reported in ppm (δ scale) down field with residual protons of the solvent (DMSO-d6) present at δ=2.49 ppm and using a common internal standard. Molecular docking was performed using Autodock 4.2.6. The screening was performed on the crystallographic structure of the enzyme "beta-lactamase cTEM-19m" (4R4S) (Gobeil et al. 2019). We applied automatic docking: the 4R4S structure was utilized in the subsequent docking experiments with other parameters established by default in the software.
Serial dilutions method was used to assess the sensitivity of the isolated microorganism strains to the experimental samples. The activity was studied according to the methodological recommendations (Volyansky et al. 2004), using standard diffusion method in Mueller-Hinton agar in media optimized for growing the test cultures at the concentration of 10 6 cells/mL. The compounds of interest were dissolved in dimethyl sulfoxide (1 mg/mL) prior to the experiment. A minimum inhibitory concentration (MIC) was assessed by observing the growth in a test tube, where the absence of growth represented the minimum concentration of the studied substance required to suppress the culture. In addition, control tests of the growth media and solvent were conducted by following common procedures.
Based on the previous research, it was approved empirically that the introduction of functional groups at Sulfur atom of bis-1,2,4-triazole-3-thione leads to the increase of solubility in more polar substances due to the charge redistribution in the molecule and formation of intermolecular hydrogen bonds. The derivatives containing one 1,2,4-triazole cycle are practically insoluble in water and poorly soluble in highly polar solvents. However, a number of bis-1,2,4-triazoles are soluble in water, acetic acid, DMF and alcohols. This system is of great interest with regard to its biological activity since its reactivity is increased meaning a broader range of possible chemical modifications in various directions.

Results
The results of antimicrobial and antifungal activity of the alkylated bis-derivatives of 1,2,4-triazole with regard to the test cultures are presented in Table 1. A solution without the addition of the compounds was used as a control substance in the trials of antimicrobial activity.

Molecular docking
For molecular docking, the cTEM-19m beta-lactamase enzyme (PDB ID: 4R4S, 1.1 Å) was chosen, which is synthesized by the bacteria E. coli О 2 and K. pneumoniae, due to which the strains acquire resistance to many antibiotics. Compounds 2a and 5b were selected based on analysis of data of antimicrobial activity as potential inhibitors of beta-lactamase. The interaction of the ligand with the active center of the enzyme is complex and is provided mainly by alkyl bonds, ionic and conventional hydrogen bonds with water molecules and amino acid residues of the enzyme. An important point for binding to the enzyme is the presence of a sulfur atom in the molecules of the test compounds (Fig. 3).
Visualization of the 3D structure of the "ligand-enzyme" complex is shown in (Fig. 4.) In Fig. 4 shows the surface drawn around the active center, indicating the region of the donor H bond, and the acceptor region of the hydrogen bond. As can be seen in Figure 4, the main structural element of the inhibitor that is complementary to the ligand is the 1,2,4-triazole ring and the side ether and amide radical.

Study limitations
The main disadvantages that may be pointed out from the serial dilution include the time restriction for conducting the research and expansion of the error during the consecutive dilutions. In the first case, time limitation is specific to the serial dilution method, due to which the prepared media must be used immediately, without the possibility of storage. As for the second case, the most considerable error is assigned to the most concentrated solution. In order to compensate for the error, longer mixing times are required, which makes the serial dilution more time-consuming.

Prospects for further research
The results of the preliminary research of antimicrobial activity of the synthesized S-substituted bis-1,2,4-triazoles demonstrate the promise of further studies of their biological properties, while also the targeted synthesis of new 1,2,4-triazoles possessing bactericidal activity represents great scientific interest.