THE SYNTHESIS, REACTIVITY AND THE ANTIMICROBIAL ACTIVITY OF SUBSTITUTED THIENO[2,3-d]PYRIMIDINE-4(3H)-THIO(SELENO)NES

A new series of R1,R2-thieno[2,3-d]pyrimidine-4(3H)-one, thione and selenone derivatives have been synthesized; the reaction of alkylation of the compounds obtained has been studied. Their structures have been confirmed by the NMR 1 H and mass spectra, and elemental analysis. The antibacterial and antifungal activities in vitro against three bacterial and two fungal pathogens have been revealed using the stiff plate agar diffusion method and the serial dilution method. The minimal bactericidal, fungicidal and bacteriostatic concentrations have been obtained. The pharmacological screening has shown that some of the compounds obtained possess a good antimicrobial activity. The culture of S.aureus. appeared to be the most sensitive to compound 10a. The best fungistatic indicators against A.niger have been found for compounds 4a and 9a.

Thieno [2,3-d]pyrimidines are of great interest in pharmaceutical industry due to the potent physiological properties such as blood platelet aggregation inhibition, the antiviral and anti-inflammatory activity [1][2][3]. Molecular docking also shows that thieno [2,3-d] pyrimidines reveal the increased activity as inhibitors for PARP-1 (poly(ADP-ribose)-polymerase-1) [4]. Although the synthesis of thieno [2,3-d]pyrimidines has attracted much attention in organic synthesis, so far only few methods have been reported for the synthesis of benzothieno [2,3-d]pyrimidines [5,6]. Moreover, thieno [2,3-d]pyrimidines containing active functional groups are especially of great interest. The presence of different groups (R=O, S, Se) in position 4 of thieno [2,3-d]pyrimidine causes the possibility of further structural modification by different reagents. Thus, as part of our ongoing research in discovery of new biologically active thienopyrimidine deriva-tives the aim of this work was to develop several simple and highly efficient methods of the synthesis of R 1 ,R 2 -thieno [2,3-d]pyrimidines 3a-c and 4a-c, the study their alkylation and antibacterial properties depending on atom in position 4 of the heterocycle.
The presence of different reaction centres in thieno- [2,3-d]pyrimidines 1a-c and 3,4a-c made possible to obtain a number of new potential biologically active compounds. The alkylation of the starting compounds 1a-c was studied at 50-70°C in the presence of alkali (Scheme 2). The acidity of the NH group under these conditions provided the easy formation of the anion, which underwent alkylation.
Since substance 3а-с can exist in the form of thiole and thione, the formation of both S-and N-derivatives could be expected depending on the reaction conditions. Thus, when pyrimidine-4(3H)-thiones 3a-c are refluxed with NaOH and the corresponding derivatives of chloroacetic acid in ethanol, S-substituted derivatives are supposed to be obtained. However, the use of NaHCO 3 and DMF as a solvent did not lead to formation of N-substituted derivatives as it was expected. It was found that the alkylation occurred at the more nucleophilic sulphur atom in both cases (Scheme 3). All the final products were isolated as crystalline substances with a high melting point ( Table 4). The structures of the compounds obtained were confirmed by IR-and NMR 1 H spectroscopy ( Table 5).
The formation of the energetically preferable aromatic system was confirmed by the chemical shift of the signal of protons of the -CH group (8.7-8.9 ppm) in 1 H NMR spectra compared to the basic values. In the case of N-alkylation such an effect could not be observed.
All the compounds synthesized were screened for their antimicrobial and antifungal activities. The results of the antimicrobial screening by the stiff plate agar diffusion method are given in Table 1. The results of the antimicrobial and antifungal screening by the serial dilution method are presented in Table 2 and 3. Substances that did not reveal the antimicrobial and antifungal activity were not included in Tab-les. According to the stiff plate agar diffusion method compounds 4a and 4c revealed a significant activity against E. coli, S. aureus, and M. luteum appeared to be sensitive only to compound 10a.
The best fungistatic indicators against A. niger were estimated for substances 4a and 9a. The diameter of the inhibition zones of the microbial growth for compound 4a was 22.0 and 12.0 mm in the concentrations of 0.5 and 0.1%, respectively. MIC of 4a and 9a were 31.2 µg/mL.

Table 1
The antimicrobial activity of the compounds synthesized (the stiff plate agar diffusion method) Compound a) Сoncentration, % The inhibition zones of the compounds studied (mm) Compounds 1a, 4b, 5a-7b, 8a-12b in the concentration of 0.5 and 1.0 % did not inhibit bacteria and fungi selected; b/a -bacteriostatic action; f/a -fungistatic action; -have not been found.

Table 2
The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the compounds synthesized (serial dilution method) Compounds 1a-b, 2a-4b, 5a-b, 6a-b, 7a-b, 8a-9c, 10b-c, 11b-12b did not reveal the antibacterial activity in the concentrations tested; b) + The antibacterial activity was not observed in the concentrations tested; c) * The antibacterial activity was not determined in the concentrations tested. Table 3 The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of the compounds synthesized (serial dilution method) Compounds 1a-b, 2a-3b, 4b-c, 5a, 6a-b, 7a-c, 8b-c, 9b-c, 10a-12b did not reveal the antifungal activity in the concentrations tested; b) * The antifungal activity was not determined in the concentrations tested; c) + The antifungal activity was not observed in the concentrations tested.  Table 5 The data of IR and 1 H NMR spectral of alkylation compounds 5a-12b According to the serial dilution method the growth of S. aureus was mostly inhibited by compounds 10a and 11a in the concentrations of 31.2 μg/mL. E. coli appeared to be almost insensitive to the compounds synthesized in the concentrations tested. Only compounds 4c revealed a weak inhibitory activity against it (MIC = 500.0 µg/mL). Compounds 7c and 10a had the moderate antibacterial activity against M. luteum (MIC = 31.2 µg/mL).

Chemical Part
The 1 H NMR spectra were recorded in DMSO-d 6 on a Varian Mercury VX-200 instrument (200 MHz). The internal standard for the NMR spectra was TMS. IR spectra (4000-600 cm -1 ) were recorded on a Bruker ALPHA FTIR spectrometer using a module for measuring attenuated total reflection (ATR) on a Bruker Tensor 27 spectrometer (KBr) and expressed in cm -1 . The mass spectra were recorded on a Varian 1200L instrument with direct injection of the sample at 250°C and 70eV ionizing electrons. Elemental analysis was performed on an LECO CHNS-900 instrument. Melting points were determined in open capillary tubes and were uncorrected. The reaction and the purity of the compounds obtained were monitored by TLC on Merck Silicagel 60 F-254 plates with 10:1 CHCl 3 -2-PrOH as an eluent.
Compounds 1a-c and 2a-c were synthesized by the method described in the literature [7,8] 4.47; N 15.54. The general procedure of the synthesis of compounds 2a-c: Dissolve compounds 1a-c (12 g) in phosphorus oxychloride, and then add gradually dimethylaniline 14 ml (0.11 Mol). Heat the reaction mixture for 4 h, evaporate the excess of phosphorous oxychloride under reduced pressure, neutralize the solid residue was with 5% aq Na 2 CO 3 solution. Filter the precipitate formed and recrystallize from toluene.  H 4.11;N 14.25. C8H8N2S2. Calculated,%: C 48.95;H 4.11;N 14.27.

4-Chloro-5,6-dimethylthieno[2,3-d]pyrimidi-
The general procedure of the synthesis of compound 4a-c: Add selenium (0.0012 Mol) gradually to absolute ethanol (50 mL) with stirring on an ice bath. After that gradually add sodium hydroborate 0.4 g (0.0032 Mol) to the solution for 15 min. Stir the resulting mixture for 1.5 h until selenium is completely dissolved. Dissolve compounds 2a-c in ethanol, cool and add to the solution of sodium hydroselenide.
The general procedure of alkylation of compounds 3a-c -method A: Heat the mixture of compounds 3a-c (0.0044 Mol) and potassium carbonate 0.8 g (0.0061 Mol) in DMF (5 ml) at 40°C for 5 min. Then add the corresponding alkylhalogenide (0.0044 Mol). Stir the reaction mixture at 40°C for 24 h and pour into water. Filter the precipitate formed, dry and recrystallize from EtOH.
Method B: Add while stirring the solution of compounds 3a-c (0.0044 Mol) in ethanol (5 ml) to 0.2 g (0.0057 Mol) of the solution of sodium hydroxide in ethanol (3 ml). Then add the corresponding alkylhalogenide (0.0044 Mol) and reflux at 70°С for 30 min. Cool the reaction mixture, filter the precipitate formed, dry and recrystallize from CH 3 CN.
The general procedure of alkylation of compounds 4a-b: Add while stirring the solution of compounds 4a-c (0.0044 Mol) in ethanol (5 ml) to the solution of sodium hydroxide (0.0057 Mol) in ethanol (3 ml). Then add the corresponding alkylhalogenide (0.0044 Mol) and reflux at 70°С for 30 min. Cool the reaction mixture, filter the precipitate formed, dry and recrystallize from CH 3 CN.

Biological Assay
Antimicrobial and antifungal test 1) Stiff plate agar diffusion method. The study of the antimicrobial and antifungal activity of the compounds synthesized was carried out using the stiff plate agar diffusion method against E. coli, S. aureus, M. luteum, C. tenuis and A. niger [6]. The amount of microbial cells was 109 c.f.u./mL. The incubation period for bacteria was 24 h at 35°C, for fungi -48-72 h at 28-30°C. Antibiotics vancomicin, oxacillin, nystatin were used as standards. The bacterial cultures, standards and the substances obtained were applied across grooves in the concentration of 5 mg/mL, and then allowed to diffuse in the agar nutrient plate. The antimicrobial effect and the degree of activity of the compounds tested were evaluated by measuring of diameters of the inhibition zones (insensitive: 11-15 mm; sensitive: 16-25 mm; highly sensitive >25 mm). All experiments were repeated three times.
2) Serial dilution method. The minimal inhibitory concentration (MIC) was determined using serial dilutions in the liquid nutrient medium by the classical method [7]. Innoculations were made in the meat infusion broth at pH 7.0 for bacteria or in the wort agar for fungi with different concentrations of the compounds tested. The amount of microbial cells was 109 c.f.u./mL.
Results were calculated after storing the control and test tubes for 24-72 h (at 37°C for bacteria and at 30°C for fungi) by determining the presence or absence of microbial growth in the medium containing various dilutions of the compound tested. The last tube with growth inhibition (transparent broth) was considered to be the MIC of the compound against the given strain.
In the test tubes 0.02 mL of the transparent medium was innoculated in the sterilized meat infusion broth (for bacteria) or the wort agar (for fungi) in a stiff plate. The incubation period for bacteria was 24 h at 35°C, for fungi -48-72 h at 28-30°C. The minimal bactericidal concentration (MBC) and the minimal fungicidal concentration (MFC) were calculated by the absence of the colony growth. All experiments were repeated three times.

Conclusions
The aim of the present study was to synthesize and investigate the antibacterial and antifungal activities of some new thienopyrimidine derivatives. The starting material 1 (R 1 ,R 2 -thieno [2,3-d]pyrimidine-4(3H)-one) was used to synthesize the corresponding thiones and selenones. The reaction of alkylation of the compounds obtained was studied. In the case of thieno [2,3-d]pyrimidine-4(3H)-ones the products of N-alkylation were obtained. When thieno [2,3-d] pyrimidine-4(3H)-thio(seleno)nes were alkylated, S-and Se-derivatives were obtained. Changing of the reaction conditions did not lead to N-alkylated products as it was expected.
The antibacterial and antifungal activities of the compounds obtained in vitro have been screened using the stiff plate agar diffusion method and the serial dilution method. According to the stiff plate agar diffusion method compounds 4a and 4c have revealed a significant activity against Escherichia coli, S. aureus, and M. luteum appeared to be the most sensitive to compound 10a in the concentration of 0.5%, it has also been confirmed by the serial dilution method (MIC 1=31.2 µg/mL; MBC =125 µg/mL for M. luteum). The best fungistatic indicators against A.niger have been found for compounds 4a and 9a (MIC = 31.2 µg/mL).