Synthesis and Antimicrobial Activity of 3-(2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol-4-yl)-2H-chromen-2-ones

The aim of this work is to study methods of 3-(2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol-4-yl)-2H-chromen-2-ones preparation and their antimicrobial activity.<br><br>Materials and methods. 1Н NMR spectra were recorded on Varian Mercury-200 (200 MHz), 13C NMR spectra were acquired on Bruker Avance 500 1H NMR (500 MHz) and 13C NMR (125 MHz) in DMSO-d6 and CDCl3. LC-MS analysis of compounds was performed on an Agilent 1100 HPLC instrument with chemical ionization at atmospheric pressure (APCI). The study of antimicrobial activity of compounds was performed by agar well diffusion method. The docking studies were performed using Autodock Vina.<br><br>Results and discussion. The interaction of 3-(2-bromopropanoyl)-2H-chromen-2-ones with N-substituted thioureas produced novel derivatives of 3-(2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol-4-yl)chromen-2-ones. The study of antimicrobial activity of the obtained compounds allowed to identify active samples against E. сoli and P. aeruginosa strains. Among the tested compounds, 8-methoxy-3-{2-[(2-methoxyphenyl)amino]-5-methyl-1,3-thiazol-4-yl}-2H-chromen-2-one showed higher activity than the reference drug Streptomycin against E. coli strain. Some compounds showed high activity against P. aeruginosa. Docking studies of the synthesized compounds indicated that they can bind in the active site to bacterial tRNA (guanine37-N1)-methyltransferase.<br><br>Conclusions. Novel derivatives of 2H-chromen-2-ones with 2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol moiety at the position 3 were obtained by the Hantzsch thiazole synthesis starting from 3-(2-bromopropanoyl)-2H-chromen-2-ones. Studies of antimicrobial activity allowed to identify new 2H-chromen-2-one derivatives as equipotent antimicrobial agents to the reference drug Streptomycin or even more potent. The docking studies revealed that the synthesized compounds may be inhibitors of tRNA (guanine37-N1)-methyltransferase, which is a crucial enzyme for survival of different bacteria, e.g. P. aeruginosa during stress conditions<br>


Materials and methods
Chemical experiments. 1 Н NMR spectra were recorded on Varian Mercury-200 (200 MHz), 13 C NMR spectra were acquired on Bruker Avance 500 1 H NMR (500 MHz) and 13 C NMR (125 MHz) in DMSO-d 6 and CDCl 3 . TMS was used as internal standard. LC-MS analysis of compounds was performed on an Agilent

Materials and methods
Chemical experiments. 1 Н NMR spectra were recorded on Varian Mercury-200 (200 MHz), 13 C NMR spectra were acquired on Bruker Avance 500 1 H NMR (500 MHz) and 13 C NMR (125 MHz) in DMSO-d 6 and CDCl 3 . TMS was used as internal standard. LC-MS analysis of compounds was performed on an Agilent 1100 HPLC instrument with diode array and massselective detectors (Agilent LC-MSD SL), Zorbax SB-C18 column (4.6×15 mm) with chemical ionization at atmospheric pressure (APCI). Elemental analysis was performed on a EuroVector EA-3000 instrument. Melting points were determined on a Kofler bench. All solvents and reagents were commercially available. A study of antimicrobial activity of compounds 4 was performed by agar well diffusion method [18,19]. The concentration of microbial cells was determined by McFarland standard [20]; the value was 10 7 cells in 1 ml of the media. The 18-24 hours culture of microorganisms was used for tests. For the bacteria cultivation, Müller-Hinton agar was used and Sabouraud agar was applied for C. albicans cultivation. The compounds were tested as the DMSO solution (concentration 100 µg per mL); the volume of the solution was 0.3 mL (the same as Streptomycin). Each experiment was repeated thrice. The antibacterial activity was estimated by the growth inhibition zone diameter for each microorganism [18].

3-(2-Bromopropanoyl)-2H-chromen-2-ones (general method).
3-Propanoyl-2H-chromen-2-one 1 (0.1 mole) was dissolved in 160 ml of chloroform. The bromine solution (0.1 mole, 5.2 ml in 16 ml of chloroform) was added dropwise to the solution of 1 stirring at room temperature. The reaction mixture was stirred and slightly heated (to 40-50 °C) until hydrogen bromide evolution was complete and absorbed by 10 % water solution of NaOH. Then, 70 % of the solvent volume was evaporated and allowed to cool. Crystals, which were formed were collected by filtration and gently washed with small portions of chloroform and ethanol.

3-(2-N-R-amino-5-alkyl-1,3-thiazol-4-yl)-2Hchromen-2-ones 4 (general method).
To the solution of 3-(2-bromopropanoyl)-2H-chromen-2-one 2 (1 mmole) in the minimal amount of 2-propanol, the solution of thiourea 3 (1 mmole) in the same solvent was added. Almost immediately after addition, the formation of clear solution was observed and after 4-10 minutes, the product precipitated. The mixture was additionally stirred at reflux for 1 hour and then cooled down to room temperature. The reaction mixture was neutralized with ammonium hydroxide solution. The precipitate was collected by filtration and dried.
For construction of the thiazole cycle of 3-(2-N-Ramino-1,3-thiazol-4-yl)-2H-chromen-2-ones 3, the reaction of 2 with substituted thioureas 3 was performed (Fig. 1). The synthesis was carried out in 2-propanol media at reflux. The target compounds 4 were isolated after the treatment of the reaction mixture with aqueous solution of ammonia. The obtained 3-(2-N-R-amino-5-alkyl-1,3thiazol-4-yl)-2H-chromen-2-ones 4 in 1 H NMR spectra have the signals of amino-group protons in the region 6.85 -6.97 ppm for the derivatives 4a,b while for the aryl-substituted compounds 4d,f the signal of NH-group is shifted downfield to 9.38 -10.07 ppm and for the acetamides 4c,d the NH-group signal is observed in the region 11.80 -12.12 ppm. The antimicrobial activity of the obtained compounds 4 was studied by agar well diffusion method using the standard strains of microorganisms [18]. It was found that compounds 4b, 4c, 4e inhibit the growth of the S. aureus strain. As far as compounds 4b and 4e are concerned, they also inhibit growth of E. coli, but their activity is smaller than it is for the reference drug Streptomycin ( Table 1).
The compounds 4b, 4e and 4f showed similar activity against P. aeruginosa. The antimicrobial activity against the E. coli strain of the compound 4f appeared to be higher than the activity of the reference drug Streptomycin.
The obtained results of antimicrobial activity screening revealed the growth-inhibitory activity of the most of compounds 4 against the P. aeruginosa bacterial strain, which is known to be highly resistant for a variety of modern antibiotics. They do not have bactericidal activity for this microorganism which quickly adapts for a new antibiotic [23]. Therefore we decided to perform the computer docking study of the obtained compounds with the aim to see whether they could be selective inhib-itors of tRNA (guanine37-N1)-methyltransferase (EC2.1.1.228; TrmD), which is known to be the crucial enzyme for survival of bacterial (also Pseudomonas aeruginosa) at a moment of stress [24].   The computer docking results for binding of compounds 4 with PaTrmDc in comparison with its known selective inhibitor showed that all of them are able to bind the active site of the enzyme ( Table 2). On the other hand the decrease in polarity of the substituent at position 2 of the thiazole increases the affinity of the ligand towards the target.

Discussion
The previously reported results [10] show that the derivatives of 8-ethoxy-2H-chromen-2-one bearing the fragment of thiazole at position 3 can inhibit the growth of B. bronchiseptica ATCC 4617 and B. pumilus ATCC 14884 better than the reference drug Ampicillin. The promising antibacterial inhibitory concentrations were reported for the compounds with the similar structures bearing 5-methoxy-2H-chromen-2-one fragment and 2bromophenylamino or 3,4-dichlorophenyl in 2aminothiazole moiety (60-73 µM) [9]. The other paper [11] presents the research where 3-(thiazol-4-yl)-2Hchromen-2-ones inhibited the growth of S. aureus ATCC 25923 and H. influenzae ATCC 10211 strains at higher level than Tetracycline and were even able to inhibit M. tuberculosis H37Rv ATCC 27294. Although the inhibitory concentration for the tested compounds was tenfold higher than those of Isoniazid.
The results of our chemical experiments show the successful bromination of 3-propanoyl-2H-chromen-2ones, and also perfect results in the Hantzsch thiazole synthesis as the key-step for preparation of the novel target 3-(2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol-4yl)-2H-chromen-2-ones. Therefore, our research revealed the possibility of synthesis of 3-(thiazol-4-yl)-2Hchromen-2-ones' analogues with the methyl group at position 5 of thiazole. It has been also shown that the synthesized compounds can effectively inhibit the growth of S. aureus АТСС 25923, E. coli АТСС 25922 and P. aeruginosa АТСС 27853 and are also able to show better inhibitory results than the reference drug Streptomycin.
The results of the docking studies of the target compounds as the ligands for bacterial tRNA (gua-nine37-N1)-methyltransferase obviously show that the introduction of the substituted phenyl substituents to amino-group of the thiazole moiety in the core 3-(2amino-5-methyl-1,3-thiazol-4-yl)-2H-chromen-2-one is favourable for interaction with the enzyme. It also correlates with the results of agar-well diffusion screening results because the compounds 4e and 4f, which display promising docking results, also have the largest growth inhibition zones. Therefore, the docking modelling of interaction of the 3-(2-amino-5-methyl-1,3-thiazol-4-yl)-2H-chromen-2-one derivatives with tRNA (guanine37-N1)-methyltransferase could be useful at the stage of prescreening filtration of virtual libraries of the compounds planned for further antimicrobial activity search.
Study limitations. As far as this research contains only primary screening results on the activity of 3-(2amino-5-methyl-1,3-thiazol-4-yl)-2H-chromen-2-ones against the standard strains of microorganisms the clear obstacle for the research expand is the availability of the clinical strains of the corresponding bacteria.

Conclusions
By the Hantzsch thiazole synthesis, the derivatives of 2H-chromen-2-ones having the fragment of 2-N-(aryl,acyl)amino-5-methyl-1,3-thiazol at the position 3 were obtained. The study of antimicrobial activity of the synthesized compounds allowed for identification of the 2H-chromen-2-ones derivatives as compounds of similar of better activity than the reference drug Streptomycin. The docking studies revealed that the synthesized compound may be inhibitors of tRNA (guanine37-N1)methyltransferase, which is a crucial enzyme for survival of different bacteria e.g. P. aeruginosa during stress conditions.