Heterocyclizations based on N-(R-hydrazine-1-carbonothioyl)cycloalkancarboxamides: functionalized azoles and their antimicrobial activity

Synthesis and structural modification of azoles remains an important area of medical chemistry and allows to obtain new compounds with a wide range of biological activity. Among the significant number of azoles, 1,3,4-thiadiazoles and 1,2,4-triazoles attract special attention, among which are known drugs, larvicides, insecticides, growth regulators, etc. Even though heterocyclizations of functionally substituted hydrazines for their synthesis are well studied, N-(R-hydrazine-1-carbonothioyl)cycloalkanecarboxamides, and nowadays, remain reagents with undiscovered potential. Moreover, the introduction of lipophilic “pharmacophore” fragments (cycloalkanes) in the structure of 1,3,4-thiadiazoles and 1,2,4-triazoles is a promising direction for their modification. That should provide additional intermolecular interactions with enzymes and may lead to enhancement or alteration of the biological activity vector. Thus, the synthesis of new derivatives of this class of compounds and the study of their antibacterial properties remains an urgent problem of medical and organic chemistry.

Diverse and high biologically active azoles have given them the status of "privileged" and pharmacologically attractive compounds. These heterocycles are characterized by anti-inflammatory, analgesic, antiepileptic, diuretic, antimicrobial, antiviral, antitumor, antituberculous, and many other types of activity [5][6][7][8][9][10][11][12][13][14][15]. However, at this stage, the synthesis and modification of azoles remains an important area of medical chemistry and allows to obtain of new compounds with a wide range of pharmacological activity. Moreover, the original N-(R-hydrazine-1-carbonothioyl)cycloalkanecarboxamides still remain reagents with undiscovered potential for the synthesis of functionalized azoles with lipophilic "pharmacophore" fragments (cycloalkanes), and their introduction will undoubtedly provide additional interactions with enzymes and may increase or alter the vector of biological activity.

Aim
Therefore, the aim of this work is to study the heterocyclization reactions of N-(R-hydrazine-1-carbonothioyl)cycloalkanecarboxamides and to establish the structure and study the antimicrobial activity of the synthesized compounds.

Materials and methods
The melting point of the compounds was determined by the capillary method on the device "Mettler Toledo MR 50". Determination of the elemental composition of the compounds was performed on an elemental analyzer "ELE-MENTAR Vario EL cube". The components on the thermal conductivity detector (TCD) were quantified. The error rate was ±0.3 %. IR spectra were recorded on a Bruker Alpha spectrophotometer in the range of 7500-400 cm -1 , using an ATR prefix (direct injection). 1 H and 13 C NMR spectra was on a nuclear magnetic resonance spectrophotometer "Mercury 500", solvent DMSO-d 6 , internal standard -TMS. Chromato-mass spectra were obtained on a high-performance liquid chromatography "Agilent 1100 Series", equipped with diode-matrix and mass-selective detector "Agilent LC/MSD SL". Ionization method -chemical ionization at atmospheric pressure (APCI). Ionization mode -simultaneous scanning of positive and negative ions in the mass range of 80-1000 m/z.
Synthetic studies were conducted according to general approaches to the search for potential biologically active substances, using reagents from Merck (Darmstadt, Germany), Sigma-Aldrich (Missouri, USA) and Enamine (Kyiv, Ukraine). Substituted N-(acylhydrazine-1-carbonotioyl) cycloalkanecarbocamides (1.1-1.14) for the synthetic part of the work were obtained by known methods with constants that correspond to the literature [16][17][18].
Method B. 0.01 Mol of the corresponding cycloalkanecarboxylic acid (3.1-3.6) and 0.91 g (0.01 mol) of thiosemicarbazide were placed in a flat-bottomed flask, then 5 ml of conc. sulfuric or phosphate acids were added, mixed, and kept at a temperature of 80 °C for 8 hours. Then the mixture was cooled, poured into water, neutralized. The formed precipitates were filtered off. Crystallized from DMF.  Antimicrobial test. The sensitivity of the microorganisms to the synthesized compounds was evaluated according to the described methods [19]. The assay was conducted on Mueller-Hinton agar by two-fold serial dilution of the compound in 1 ml. After which, 0.1 ml of microbial seeding (106 cells/ml) was added. Minimal inhibition concentration of the compound was determined by the absence of visual growth in the test tube with a minimal concentration of the substance. Minimal bactericide/fungicide concentration was determined by the absence of growth on agar medium after inoculation of the microorganism from the transparent test tubes. DMSO was used as a solvent, initial solution concentration was 1 mg/ml. For preliminary screening of the abovementioned standard test, cultures were used: S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and C. albicans ATCC 885-653.

Discussion
It was shown 5-substituted 2-amino-1,3,4-thiadiazoles (2.1-2.5, scheme) by the result of heterocyclization of compounds 1.1-1.8 in concentrated sulfuric or phosphate acids, according to chromato-mass spectra. This heterocyclization was realized by a known mechanism, namely through the nucleophilic attack of the electron pair of the sulfur atom on the carbonyl group of the semicarbazide fragment, followed by dehydration of the resulted intermediate and intramolecular proton migration to form an aromatic system [20]. Unfortunately, the cycloalkane-urea fragment of the molecule was additionally hydrolyzed to form an amino group in the process of cyclization, regardless of its size. In addition, this method is not preparative due to the formation of a significant number of side products, and crystallization leads to final products with low yields (2-30 %). It was carried out their counter-synthesis by cyclization of carboxylic acids (3.1-3.6) with thiosemicarbazide under these conditions to prove the structure of compounds 2. The reaction was carried out by the above mentioned mechanism through the stage of formation of intermediate, 2-(cycloalkanecarbonyl) hydrazine-1-carbothioamides, followed by the formation of 5-R-2-amino-1,3,4-thiadiazoles (2.1-2.7) with a yield of 41-70 % (scheme) [20]. It was also found that diacyl hydrazides with phenoxy-(1.4, 1.6), phenylthio-(1.10, 1.11) acetylhydrazide or semicarbazide (1.8) fragments in the molecule undergo spontaneous heterocyclization refluxing prolonged heating in butanol (scheme). However, for compounds with benzoyl-(1.12, 1.13) and heteroyl-(1.7, 1.14) hydrazide fragments, this transformation was not characteristic [11]. It can probably be explained either by their lower solubility or by the effect on the redistribution of electron density in the molecule (hydrazide-hydrazone tautomerism) and, therefore, a decrease in the electrophilicity of the carbonyl group. It was important that the yield of final products in this reaction is not significant (35-58 %).
There is no doubt that the heterocyclization of diacylthiosemicarbazides (1.7, 1.1-1.14) in the presence of alkalis was rate through intermediate 4-cycloalkanecarbonyl-triazole-3-thiones, which under these conditions are hydrolyzed to the target compounds 5 (scheme).
The structure and individuality of synthesized compounds were confirmed by elemental analysis, chromato-mass, and 1 H NMR spectrometric. A quasimolecular ion [M+1] was registered in the chromato-mass spectra of azoles, which confirms their structure and individuality. 1 H NMR spectra also was indicated their unambiguous formation. Thus, in the 1 H NMR spectra of 5-R-1,3,4-thiadiazol-2-amines (2) the two-proton singlet of the amino group was characteristic, which was registered at 7.12-6.83 ppm. Whereas, in the 1 H NMR spectra of compounds 4, single-proton singlet of protons at the 1 st position of the heterocycle were observed, which resonate at 12.75-12.43 ppm. In compound 4.5, this proton undergoes a significant diamagnetic shift (11.93 ppm) due to the presence of an amino group at the 3 rd position (positive mesomeric effect). The 1 H NMR spectra of compounds 5 were also characterized by weak-field singlet protons of the 2 nd and 4 th positions of the heterocycle at 13.24-12.28 and 10.19-9.14 ppm, respectively. Significant weak field shifts of these protons in compounds 4 and 5, clearly indicate their existence in the form of thions. Additionally, signals of axial and equatorial protons of methylene and methine groups of the cycloalkyl