SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF 3-ARYLAMINOMETHYL-1-(2- OXO-2-ARYLETHYL)-6,7,8,9-TETRAHYDRO-5H-[1,2,4]TRIAZOLO[4,3-a] AZEPIN- 1-IUM BROMIDES AND ARYL-(4-R1-PHENYL-5,6,7,8-TETRAHYDRO-2,2a,8a- TRIAZACYCLOPENTA[cd]AZULEN-1-YLMETHYL)-AMINES

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Introduction
Unreasonable usage of antimicrobial drugs leads to appearance and spread of microorganism strains which are resistant to antimicrobials [1,2]. In addition to antimicrobial resistance spreading, existing drugs also have some drawbacks. Some of them display narrow spectrum of antimicrobial activity, unsatisfactory pharmacokinetic, high rate of side effects etc [3,4]. These facts are the reasons that medicine is in urgent need of novel antimicrobial substances [5,6] The core of 5H- [1,2,4]triazolo [4,3-a]azepine is a part of many compounds possessing analgesic, anxiolyt-ic, anti-inflammatory and antitumor activities Additionally, structurally close to them 3-biphenyl-3H-imidazo[1,2-a]azepin-1-ium bromides display antibacterial and antifungal activity [7].

Planning (methodology) of the research
For planning research, the following algorithm of actions was developed: І stage. Synthesis of selected series of compounds and establishment of its chemical characteristics.
Antimicrobial activity screening of the compounds synthesized was performed by measuring their minimum inhibitory concentration (MIC) values.

Materials and methods
Chemical experiments. 1 Н NMR spectra were recorded on a Bruker 400 (Germany) spectrometer in DMSO-d 6 solutions. 13 C NMR spectra on a Varian Mercury-400 100 MHz in DMSO-d 6 using tetramethylsilane as an internal standard. Chemical shifts were reported in ppm units using δ scale. The mass spectra were recorded on an Agilent 1200 LC/MSD SL instrument (Santa Clara, CA, USA). Elemental analysis was performed on a Eu-roVector EA-3000 instrument. Melting points were determined on a Kofler bench. All solvents and reagents were commercially available.
[ 4 -( 4 -C h l o r o p h e n y l ) -5 , 6 , 7, 8 -t e t r a h ydro-2,2a,8a-triazacyclopenta[cd]azulen-1-ylmethyl] [ Antimicrobial activity screening All compounds were dissolved in DMSO to give stock solutions with concentration of 10 mg/mL, and aliquots were diluted in water and 5 µL dispensed into empty 384-well plates in duplicates for each strain. Once cells were added to the plates, this gave a final compound concentration of 32 µg/mL, or in case of a serial dilution assay compound concentrations from 50 to 0.78 µg/mL, in both cases with a maximum DMSO concentration of 0.3 %, which does not show antimicrobial activity and does not affect the results of microbiological screening.
The inhibition level of the bacterial test-cultures growth treated with a specific concentration of the compounds tested was determined by measuring the optical density at wavelength of 600 nm (OD 600 ) using a multifunctional monochromator-based microplate reader Tecan M1000 Pro. The percentage of inhibition was calculated for each well using values obtained for negative control wells (containing only growth medium) and for positive control wells (containing bacterial/fungi inoculum suspension without the compounds (reference drugs) tested).
The inhibition level of C. albicans strain growth treated with the compounds tested was determined by measuring the optical density at wavelength of 530 nm (OD 530 ). The inhibition level of C. neoformans strain growth was determined by measuring the difference in optical density at wavelengths of 600 and 570 nm (OD 600-570 ) after adding resazurin solution (to its final concentration of 0.001 %) and incubation of the solutions at 35 °C for 2 h. The optical density of the mixtures containing fungi cultures was measured using multi-mode microplate reader Biotek Synergy HTX. The percentage of fungi growth inhibition was calculated in the same way as for the bacterial cultures [13,14].
Negative growth inhibition values (Table 2) indicate lower growth rate (lower OD 600 values) as compared to the negative control wells (inhibition level is set to 0 %). The growth rate of all bacterial and fungal strains was in the range of +/-10 % which is within normal growth rate distribution stated for bacteria/ fungi [15,16].
The tests were initially carried out using compounds 3, 6 in concentration of 32 µg/mL in duplicates in order to find active derivatives. Next, to determine MIC values preliminary assays were followed by a dose response test, using 8 double dilution concentrations of the compounds in duplicates.

Discussion
The obtained data indicate that six compounds (5bb, 5fc, 5cd, 5eb, and 5fa) display antimicrobial activity against gram-positive bacteria (S. aureus). The compounds are active in concentration range of 6.2-25.0 μg/ mL, and derivative 5cd appeared to be the most active. It is worth noting that antistaphylococcal activity of the compounds mentioned was similar to the activity of Linezolid and more pronounced than the activity of Cefixime [17,18].
At the same time the compounds tested had no activity against gram-negative bacteria. Only compound 5fa inhibited growth of E. coli strain in concentration of 50.0 g/mL. It was also established that all derivatives are inactive against P. aeruginosa and inferior to the reference drug Cefixime [19,20].
Investigation of antifungal properties of the compounds evidence that four out of nine derivatives (5bb, 5fc, 5cd and 5eb) inhibited the growth of C. albicans in concentrations of 6.2-25.0 g/mL. The most active derivative 5bb is of the same level of activity as antifungal drug Fluconazole [21,22]. The study of antibacterial and antifungal activities of 3-arylaminomethyl-1-(2-oxo-2-arylethyl)-6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepin-1-ium bromides 5 demonstrate that representatives of this class of compounds may have either single (antibacterial or antifungal) or polyvalent (antibacterial and antifungal) activity. The level of inhibiting effect and the antimicrobial spectrum of the compounds synthesized are clear evidences of prospects to create new antimicrobial drugs based on them.
Study 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.
Prospects for further research. The further investigation of antimicrobial properties of the most active derivatives 5bb and 5cd seems to be promising way of finding and creating novel effective antimicrobial drugs.