Synthesis and properties of S-derivatives of 4-amino-5-(5-methylpyrazol-3-yl)-1,2,4-triazole-3-thiol

Materials and methods. Experimental methods of organic chemistry: synthesis using microwave activation, physical and chemical methods for the analysis of organic compounds (determination of the melting point, elemental analysis, 1H NMR, IR spectroscopy and chromatography-mass spectrometry). Methods for in silico pre-screening studies to establish the biological potential in several synthesized compounds (molecular docking).

Актуальные вопросы фармацевтической и медицинской науки и практики. 2021. Т. 14, № 3(37). С. 268-274 Today, many research teams are studying the synthetic and biological properties of new compounds based on the nitrogen-containing system 1,2,4-triazole [1][2][3][4][5][6]. It is known that the introduction of various substituents in the structure of the nucleus of 1,2,4-triazole has a positive effect not only on the increase of existing and the emergence of new pharmacological activity but also allows offering more options for chemical transformations [4][5][6][7][8]. It is important to note the fact that 1,2,4-triazole derivatives, in addition to high biological activity, are mostly low-toxic or virtually non-toxic compounds. Analysis of the available literature data revealed that the combination of triazole and pyrazole fragments within one molecule has a certain level of practical significance and is interesting.

Aim
The aim of this work was to develop a preparative method for the synthesis of S-derivatives of 4-amino-5-(5-methylpyrazol-3-yl)-1,2,4-triazole-3-thiol using microwave irradiation, followed by study of physical and chemical properties and establishing the biological potential of the obtained compounds.
S-alkylation of the starting compound was performed under microwave irradiation using a microwave synthesis system Milestone FlexiWave (time -30 min, temperature -160 °C, pressure -11.2 bar, power -400 W) in propan-2-ol medium without the addition of equivalent amount of alkali, which allowed to obtain pure alkylthio derivatives with high yield (Fig. 1).
Synthesized haloalkanes were white crystalline substances, soluble in alcohols, insoluble in water. The structure of the obtained compounds was confirmed by a package of modern physical and chemical methods of analysis ( 1 H NMR spectroscopy, IR spectroscopy) and their individuality by chromato-mass spectrometry.
Docking studies were performed on all compounds 2.0-2.10 using the AutoDock Vina ® software package. Enzyme structures for in silico studies were obtained from Protein Data Bank (PDB). Preliminary optimization of 2.0-2.10 molecules was performed using the HyperChem 7.5 program by the MM + molecular mechanics' method until the RMS gradient was less than 0.1 kcal/(mol•Å). The final minimization of the energies of the studied structures was carried out by the semi-empirical quantum chemical method PM3 until the RMS gradient was less than 0.01 kcal/(mol•Å).

Results
Analysis of 1 H NMR spectra showed that the protons of S-alkyl fragments resonate in a strong part of the field in the form of signals with different intensities in the range 3.14-0.82 ppm. For example, singlet signals of methyl protons of the thiomethyl fragment were present in the range of 3.14-3.08 ppm. Multiple proton signals of methylene fragments were recorded in a stronger field (1.97-1.20 ppm). A gradual increase in the length of the S-alkyl chain leads to a slight shift in the signals of the protons of the methyl group in the stronger part of the field.
Proton signals of methylene moieties of S-alkyl substituents were conducted in a similar way but were difficult to differentiate because they form mostly multiproton multiplets. Exceptions were only signals of protons of the methylene group directly with the sulfur atom. In this case, there was a signal in the form of a triplet. The formation of a positive inductive effect contributed to these changes. Thus, the signal of protons of the methyl group gradually shifts to 0.83 ppm.
The IR spectrum of the synthesized thiol (2.0) was characterized by the presence of clear bands of deformation and valence oscillations of strong and medium intensity of the main fragments of the molecule: planar deformation oscillations CH in the region 1229-950 cm -1 (bands of low intensity at 1229-1182 cm -1 , 1045-1029 cm -1 , 1013-998 cm -1 , 975-960 cm -1 ), out-of-plane deformation oscillations CH in the region 998-663 cm -1 (bands of strong intensity at 781-765 cm -1 , 687-672 cm -1 ). There was the presence of a band of valence vibrations of the SH group in the range of 3298-3280 cm -1 . The oscillation bands of the C = N fragment in the region of 1548-1530 cm -1 were also recorded.
In the spectra of the synthesized alkyl derivatives (2.0-2.10) deformation oscillations of alkyl groups in the range from 645 cm -1 to 1390 cm -1 and the H-C-H fragment in the narrow frequency range 1485-1360 cm -1 were observed. In the spectra of the synthesized alkyl derivatives (2.1-2.10) deformation oscillations of alkyl groups in the range from 645 cm -1 to 1390 cm -1 and the H-C-H fragment in the narrow frequency range 1485-1360 cm -1 were observed.   Individual peaks of molecular ions [M+1] were recorded in the chromato-mass spectra, which had a high intensity, which confirms the structure and individuality of the compounds (Fig 2, 3). Molecular docking. It is noteworthy that a significant number of antifungal drugs contain a fragment of 1,2,4-triazole (fluconazole, itraconazole, voriconazole, posaconazole). Triazole-containing anastrozole and letrozole were also quite effective anticancer drugs. On the other hand, the presence of pyrazole enzyme indicates the feasibility of testing for anti-inflammatory activity.

4-Amino-5-(5-methylpyrazol-3-yl)-1,2,4-triazole-3-thiol (2.0).
Molecular docking was performed to obtain structural information on the interaction of the synthesized compounds and the corresponding biological structure. For this purpose, the Х-ray crystal structures of the corresponding biological targets from the protein database (PDB-ID) in complex with the standard ligand were previously downloaded: cyclo oxygenase-1 with diclofenac (3N8Y), lanosterol 14-α-demethylase with ketoconazole (3LD6), kinases of anaplastic lymphoma in the complex of crizotinib (2XP2) [11][12][13]. The use of cyclooxygenase-1 as a model enzyme is dictated by the need to determine the possible impact on the complex of processes, and not only on the inflammatory response. At the same time, a few highly effective antifungal agents have a 1,2,4-triazole fragment in their structure, so the choice in favor of 14-α-demethylase as a model enzyme is obvious. The choice of aplastic lymphoma kinase to determine the affinity of the synthesized substances to its active center is due to the use of known anticancer drugs created based on nitrogen-containing heterocycles.
The ligands (diclofenac, ketoconazole, crizotinib) were previously removed from the primary structures. It was carried out the joining of different ligands to the protein using AUTODOCK. The conformations of the ligand were analyzed in terms of energy, hydrogen bonding and hydrophobic interaction between the ligand and the receptor protein. A detailed analysis of the ligand-receptor interactions was performed, and the final coordinates of the ligand and receptor were saved as pdb files. The free binding energy (FEB) of all compounds was calculated [14][15][16][17].
In order to investigate the probability of detection of molecules with molecules with anti-inflammatory activity, the interaction parameters with the active center of cyclo oxygenase-1 (COX-1) were studied ( Table 1) (Fig. 4). The estimated free energy of binding of the synthesized substances of their lowest energy positions with lanosterol-14α-demethylase was calculated ( Table 2). Synthesized substances 2.6-2.10 showed a good range of binding energies from -8.1 to -9.4 kcal/mol.
The obtained substances were stabilized in the active center of anaplastic lymphoma kinase due to intermole cular hydrogen chemical bond with MET A: 1199, alkyl hydrophobic   5). Moreover, attention is drawn to the presence of a certain amount of π-alkyl and π-anion hydrophobic interactions with the active site of the enzyme (with ALA A: 1200, LEU A: 1122 and GLU A: 1122), which had an immediate effect on the stability of a particular biologically active substance in the active site (Fig. 5).
Calculations of the free binding energy showed that an increase in the length of the S-alkyl fragment of the synthesized substances can have a positive effect on the affinity with the active site of the enzyme ( Table 3). The most significant level of interaction with the active center of the enzyme were demonstrated by substances 2.8 and 2.10 with values of the free energy of interaction -8.0 kcal/mol and -8.4 kcal/mol accordingly ( Table 3).

Discussion
The performed docking studies suggest that the synthesized S-alkyl derivatives exhibit the ability to bind to the active sites of COX-1, lanosterol 14-α-demethylase, and anaplastic lymphoma kinase.
It is also necessary to emphasize the participation of all fragments of molecules of new substances in interactions with the active site of enzymes.

Conventional Hydrogen Bond
Alkyl Pi-Alkyl

Interactions
Pi-Sulfur  Comparison of the calculated E min values in the series of synthesized substances made it possible to establish the effect of the length of the S-alkyl fragment on the affinity with the active site of the enzymes under consideration. Moreover, the transition from a methyl substituent to a decyl substituent was accompanied by an increase in this affinity. Alkyl hydrophobic interactions of the synthesized substances with amino acid residues of the corresponding enzymes had a significant influence on the formation of this dependence.
2. The performed docking studied suggest that an increase in the length of the S-alkyl fragment increases the likelihood of anti-inflammatory, antifungal, and anticancer activity. Moreover, in molecules with an even number of carbon atoms in the alkyl substituent, this probability will only increase.
Prospects for further research. According to the research results it is planned to expand the line of this class of compounds to identify promising biologically active compounds among them.