SYNTHESIS AND THE STUDY OF THE ANTIMICROBIAL ACTIVITY OF 3-AMINO-5-METHYL-2-( ALKYLTHIO )-4-OXON-ARYL-3 , 4-DIHYDROTHIENO [ 2 , 3-d ] PYRIMIDINE-6-CARBOXAMIDES

By alkylation of 3-amino-5-methyl-4-oxo-N-aryl-2-thioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine6-carboxamides with substituted benzylchlorides and chloroacetic acid the series of novel derivatives of 3-amino-5-methyl-2-(alkylthio)-4-oxo-N-aryl-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamides have been obtained. The structures of these compounds have been confirmed by 1H NMR spectral data and the elemental analysis. For all of the products obtained the 1H NMR spectra contain the signals of the amino group as the sharp singlet in the region of 5.75-5.84 ppm, the signal of the methyl group at position 5 of the thieno[2,3-d]pyrimidine system (2.68-2.75 ppm); the signal of the carboxamide NH group, which position varies from 9.67 ppm to 10.61 ppm depending on the structure of the substituent of the amide aromatic cycle, is also observed. In the spectra the signals of the benzyl CH2 group protons are located in the region of 4.23-4.29 ppm, while the same signal for the derivative of thioacetic acid is observed at 3.82 ppm. The antimicrobial activity screening for the compounds obtained has been performed by the agar well diffusion method. In general, it has been found that all of the compounds tested appeared to be more active than the reference drugs against the strains of both Proteus vulgaris and Pseudomonas aeruginosa; 3-amino-2-(benzylthio)-5-methylN-(4-methylphenyl)-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxamide was the most active compound, which moderately inhibited the growth of all test-strains of microorganisms, and showed even higher activity than the reference drugs streptomycin and metronidazole against Bacillus subtilis and Candida albicans fungi.

It was reported that some derivatives of thieno [2,3-d] pyrimidin-4(1H)-one showed the antimicrobial activity [1-4, 6, 8-10].The compounds with modified amino and thioxo groups at positions 2 and 3 of the core heterocyclic system [3], as well as the derivatives containing these groups included into the other fused heterocyclic system [1,6,9] are known among them.Our special attention was paid to the derivatives containing the carboxamide group at α-position of the thiophene ring.Earlier we reported about the triheterocyclic systems containing similar heterocyclic fragments as the effective antimicrobials [1,9].However, the simpler compounds with the free amino group at position 3 were not prepared previously and were not used for antimicrobial trials.Therefore, the synthesis and study of the antimicrobial activity of derivatives of 3-amino-5-methyl-2-(alkylthio)-4-oxo-N-aryl-3,4-dihydrothieno [2,3-d]pyrimidine-6-carboxamide were the aim of our research work.

Chemical Part
All of the solvents and reagents were received from the commercial sources.Melting points (°С) were de-termined with a Kofler (Hotbench) melting point apparatus. 1H NMR spectra were recorded using a Varian Mercury (200 MHz) spectrometer in DMSO-d 6 with TMS as an internal standard.Chemical shifts (δ) are reported in ppm.Elemental analysis was performed by Kjeldahl method.

The general method for the synthesis of 3-amino-5-methyl-2-(alkylthio)-4-oxo-N-aryl-3,4-dihydrothieno [2,3-d]pyrimidine-6-carboxamides (2)
To the suspension of 0.00045 mol of the starting compound 1 in dimethylformamide add 0.0005 mol of triethylamine and 0.0005 mol of the corresponding chloroderivative.Heat the reaction mixture (70°С) with stirring for 5-8 hours.After cooling the reaction mixture dilute it with water.Filter the precipitate of compound 2 formed and wash with water.Purify the products by boiling in 2-propanol.

The study of the antimicrobial activity
The microbiological experiment was performed at the premises of the laboratory of Biochemistry of Mic-ISSN 1562-7241 roorganisms and Nutrient Media at the Institute of Microbiology and Immunology named after I.I.Mechnikov at NAMS of Ukraine.According to the WHO recommendations [5,7] such test-strains as Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 4636, Bacillus subtilis ATCC 6633, Candida albicans ATCC653/885 were used.Bacterial concentration was 10 7 CFU/ml (determined by McFarland standard).Overnight cultures kept for 18-24 h at 36ºC±1ºC were used.The bacterial suspension was inoculated onto the entire surface of a Mueller-Hinton agar (Dagestan Research Institute of Nutrient Media).The compounds were introduced to the wells in the form of DMSO solution in concentrations of 100 μg/ml; the open wells were filled with 0.3 ml of the solution.
The structure of the compounds obtained was confirmed by1 H NMR spectroscopic method (Table 2).For all of the compounds 2 their 1 H NMR spectra contain the signals of the amino group as the sharp singlet in the region of 5.75-5.84ppm, the signal of the methyl group at position 5 of the thieno[2,3-d]pyrimidine sys- As to the spectrum and efficacy of antimicrobial properties, the highest activity in the range of compounds 2 (Table 3) was revealed by compound 2d, which moderately inhibited the growth of all test-strains of microorganisms and showed even higher activity than the reference drugs against Bacillus subtilis and Candida albicans fungi.All of the 4-methylphenyl amides 2e and 2f tested also exhibited a moderate antimicrobial activity.Its noteworthy that most of the compounds studied, namely 2a, 2d-2g and 2i appeared to be more active than the reference drugs against Proteus vulgaris and Pseudomonas aeruginosa; it agreed with the previously reported typical manner of thieno [2,3-d]pyrimidines [10].

Table 1 Physico
tem (2.68-2.75ppm); the signal of the carboxamide NH group, which position varies from 9.67 ppm to 10.61 ppm depending on the structure of the substituent of the amide aromatic cycle, is also observed.In the spectra the signals of benzyl CH 2 group protons are located in the region of 4.23-4.29 ppm, while the same signal for the derivative of thioacetic acid is observed at 3.82 ppm.