The synthesis, antimicrobial activity and docking studies of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin- 4(3H)-ones with acetamide and 1,2,4-oxadiazol-5-ylmethyl substituents

1 National University of Pharmacy of the Ministry of Health of Ukraine, Ukraine 53, Pushkinska str., Kharkiv, 61002, Ukraine. E-mail: sergiy.vlasov@gmail.com 2 Institute of Organic Chemistry of the National Academy of Sciences of Ukraine, Ukraine 3 Enamine Ltd., Ukraine 4 V. N. Karazin Kharkiv National University, Ukraine 5 Mechnikov Institute of Microbiology and Immunology of the National Academy of Medical Sciences of Ukraine, Ukraine


Results and discussion
Taking into account the positive results of our previous research on the regioselectivity of the alkylation of 6-(1H-benzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-one with benzyl chlorides [3], as well as some successful experience of the transfer of the method developed for benzyl chlorides to chloroacetamides for the similar systems with the fragment of 5-methylthieno [2,3-d]pyrimidin-4(3H)-one [4] we decided to do the same for benzimidazole containing derivatives. The starting compound 1 obtained according to the method previously reported [3] was treated with either N-arylchloroacetamides or 3-aryl-5-(chloromethyl)-1,2,4-oxadiazoles in the DMF media using the equimolar amount of potassium carbonate to promote the reaction (Scheme).
The reaction was carried out for 5 -8 hours at 60 °C. After cooling the reaction mixture was quenched with water, and the crystalline products were filtered. The properties of compounds 2 and 3 synthesized are given in the Experimental part. If required, products 2 and 3 can be additionally purified by boiling in ethanol.
In the 1 H NMR spectra of compounds 2 the signals of the acetamide CH 2 group protons were observed in the region of 4.83 -4.86 ppm, while for compounds 3 the signals of CH 2 were in the region of 5.61 -5.63 ppm. All compounds 2 and 3 had the signal of benzimidazole NH at 12.63 -12.68 ppm; for compounds 2 the signal of the acetamide NH proton was observed in the region of 10.33 -10.70 ppm. The signal of the methyl group in position 5 of the thieno[2,3-d]pyrimidine system was found as a singlet at 2.86 -2.88 ppm ( Table 1).
To elucidate the mechanism of action of the 3-(Narylacetamido/1,2,4-oxadiazol-5-ylmethyl)-6-(1Hbenzimidazol-2-yl)-5-methylthieno[2,3-d]pyrimidin-4(3H)-ones synthesized the docking studies were conducted. For the antimicrobial activity screening experiment Streptomycin was used as a reference drug. It is widely used in clinics and can effectively tackle many dangerous strains of pathogenic bacteria [10 -12]. On the other hand, for now there are many resistant strains towards this antibiotic [13], and it encourages the search for new antimicrobials with a possibly similar mechanism of action. As for most of aminoglycoside antibiotics [14,15] the complexes of Streptomycin with its molecular target 16S subunit of ribosomal RNA were isolated and studied in details [16]. The structures of these complexes are available as pdb files (1NTB and 1NTA), which represent the structures with different metal cations. For our calculations we chose the model containing a Magnesium cation (1NTB). The results of the docking studies showed that none of the target molecules appeared to be suitable as a ligand for the active site. According to the docking results it is very unlikely for 3-(N-arylacetamido/1,2,4- pyrimidin-4(3H)-ones to have the mechanism of the antimicrobial action similar to aminoglycoside antibiotics. Among the recently discovered molecular targets, which can be applied for the search of new antibiotics, there is tRNA (Guanine37-N 1 )-methyltransferase (TrmD) known as the enzyme important for survival of different bacteria under stress [17]. It has been proven that its inhibitors are effective antimicrobials [17 -20]. Therefore, we tried this protein for the docking calculations with compounds 2 and 3 as ligands.
The calculation performed showed that compared to the known inhibitors the molecules of derivatives 2 and 3 were unable to interact with all of the amino acids of the active site. The best binding results were obtained for compound 2.2, which also showed the best result in the antimicrobial activity assay against P. vulgaris (ATCC 4636) and P. aeruginosa (АТСС 27853) ( Table 2). Its activity was even higher than that one for the reference drug Streptomycin. The obvious correlation between the ability to bind the active site of tRNA (Guanine37-N 1 )-methyltransferase and the antimicrobial activity screening results can be the evidence for the possible impact of compounds 2 and 3 on the activity of the enzyme. On the other hand, the results of the docking studies were shown no Table 1 The results of the antimicrobial activity screening for 3-  evidence for them to have the antimicrobial activity mechanism being similar to Streptomycin.

Chemical part
All solvents and conventional reagents were obtained from the commercial sources or prepared by the well known methods. 1 Н NMR spectra were recorded on a Varian Mercury-200 device (200 MHz) in DMSO-d 6 solution; TMS was used as an internal standard; the spectral chemical shift scale was presented as δ (ppm). The elemental analysis was performed on a EuroVector EA-3000 instrument. Melting points were determined on a Kofler bench.
After the reaction mixture is cooled, it is quenched with water, then the precipitate formed is filtered and dried at 70 °C. If required, compounds 2 and 3 can be purified by boiling in ethanol.

Table 2
The results of the computer docking study of the interaction of compounds

Biological and in silico studies
The study of the antimicrobial activity of compounds 2 and 3 was performed by the agar well diffusion method [21,22]. The concentration of microbial cells was determined by the McFarland standard [23]; the value was 10 7 cells in 1 mL of the media. The 18 -24 hour culture of microorganisms was used for tests. For the bacteria cultivation, Müller-Hinton agar was used, Sabouraud agar was applied for C. albicans cultivation. The compounds were tested as the DMSO solution with the concentration of 100 µg/mL; the volume of the solution was 0.3 mL, the same volume was used for Streptomycin (the concentration 30 µg/mL). Each experiment was repeated thrice. The antibacterial activity was estimated by the growth inhibition zone diameter for each microorganism.
The docking studies were performed using Autodock Vina [24]. They were performed for flexible ligands and rigid models of proteins. Crystallographic data for complexes of Streptomycin with the 16S subunit of ribosomal RNA (1NTB) with its active site [25] and tRNA (Guanine37-N 1 )-methyltransferase (EC 2.1.1.228; TrmD) (5ZHN) with its active site [26] were obtained from the Protein Data Bank.
Conflict of interests: the authors have no conflict of interests to declare.