THE SYNTHESIS AND THE ANTIMICROBIAL ACTIVITY OF THE SUBSTITUTED ARYL AMIDES OF 3-ARYLMETHYL-2 , 4-DIOXO-1 , 3 , 7-TRIAZASPIRO [ 4 . 4 ] NONANE-7-CARBOXYLIC ACIDS

By the interaction of the substituted aryl isocyanates with the series of 3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-diones in the propanol-2 medium the series of the substituted aryl amides of 3-arylmethyl-2,4-dioxo-1,3,7-triazaspiro[4.4]nonane-7-carboxylic acids have been obtained. The structure of the products obtained has been confirmed by the instrumental methods of the analysis, such as 1Н, 13С NMRand chromato-mass spectrometry. The 1Н NMR-spectra of all of the substances obtained contain the signal of the methylene group of the arylmethyl fragment at 4.5 ppm as a singlet; the number and splitting of the signal in the range of the aromatic protons resonance well corresponds with the substitution in the aromatic part of the molecule; the protons of the NHCO urea fragment with the signal of the NH at position 1 are observed as two singlet signals at 7.4-8.2 and 8.9 ppm. The 13С NMR-spectra of all compounds obtained contain four signals at 35, 45, 54 and 65 ppm of the pyrrolidine cycle of the molecules; three signals of the carbonyl groups carbon atoms are observed at 154, 155 and 174 ppm. The LC-MS spectra of the target compounds contain the peaks of [M+H]+ ions, which masses are in good accordance with the structures proposed. The results of the antimicrobial activity screening show the ability of the compounds synthesized to inhibit the growth of the Candida albicans fungi strain. The gram-positive microorganisms such as the strains of Staphylococcus aureus and Bacillus subtilis are found to be sensitive to the compounds containing the methyl groups as substituents in the arylmethyl fragments of the molecule.


Experimental Part Chemical Part
All solvents and reagents were obtained from the commercial sources.The NMR-spectra were recorded with a Bruker 170 Avance 500 spectrometer at 500 МHz for NMR 1 Н-spectra and at 125 МHz for NMR 13 С-spectra, the solvent was DMSO-d 6 ; TMS was used as an internal standard.Chromato-mass spectra were recorded using an Agilent 1100 HPLC device equipped with a diode matrix detector and a mass-spectrometer (Agilent LC-MSD SL); a Zorbax SB-C18 column (4.6×15 mm) and an atmospheric pressure chemical ionization (APCI) were used for the analyses.The TLC was performed on the alu-minium plates covered with a silica gel (Merck, Kiesgel 60 F-254).The melting points were measured with a Kofler melting point apparatus and were not corrected.

Microbiological Experiment
The microbiological experiment was performed by the Microorganism Biochemistry and Nutrient Media Laboratory of the Mechnikov Institute of Microbiology and Immunology of the NAMS of Ukraine.According to the WHO recommendations to estimate the activity of the compounds tested the following strains of microorganisms were used: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 4636, Bacillus subtilis ATCC 6633, Candida albicans ATCC653/885.The inoculum suspension was prepared using a Densi-La-Meter apparatus (made by PLIVA-Lachema, Czech Republic; with the wavelength of 540 nm).The suspension was prepared according to the instruction for the apparatus and the Information letter about innovation in the healthcare system No.163-2006 "Standardization of microbial suspension preparation" (Kyiv).The cultures were synchronized using low temperature conditions (4°С).The inoculum density was 107 cells in 1 ml of the medium and was determined by comparing with McFarland standard [8].The 18 to 24-hour old culture of the microorganism was used for the test.Mueller-Hinton agar was used for bacteria.The strain of Candida albicans was cultivated using Sabouraud agar.The experiment was performed using the agar "well" diffusion method [7].The compounds studied were introduced as 0.3 ml DMSO solution (with the concentration of 100 μg/ml) aliquots.The standards were introduced as the solution in DMSO (30 µg/lm) for Metronidazole and as the water solution (30 µg/lm) for Synthomycine.

Results and Discussion
The synthesis of the compounds for the antimicrobial activity screening was performed according to Scheme.The starting secondary amines 1 [6] were introduced into the reaction with aryl isocyanates in the 2-propanol medium.The products 2 were precipitated as white amorphous solids.
The structures of the compounds obtained were confirmed using 1 H, 13 C NMR-and chromato-mass spectrometric methods.The data of the instrumental analyses and the melting points for compounds 2 are listed in Tab. 1.
Table 1 The data of 1 Н, 13 С NMR-, chromato-mass spectra and the melting points for the substituted aryl amides of 3-arylmethyl-2,4-dioxo-1,3,7-triazospiro [4.4]The study of the antimicrobial activity for the compounds synthesized was performed using the agar "well" diffusion method.The standard test-strains of gram-positive and gram-negative bacteria, as well as fungi were used according to the international standards [1, 2].The results of the antibacterial activity screening are presented in Tab. 2.
As it is presented in Tab. 2, almost all compounds tested exhibit the antimicrobial activity against the strains of gram-positive and gram-negative bacteria, as well as against fungi.The fungi of Candida albicans appeared to be the most sensitive for compounds 2, the diameters of the growth inhibition zones in some cases exceeded 20 mm.It should be also mentioned that methyl substitution in the arylmethyl fragment increases the antimicrobial activity against gram-positive bacteria, and it is typical for compounds 2.3 and 2.4.
CONCLUSIONS Using the methodology of the liquid phase combinatorial synthesis the chemical diversity of the available 3-arylmethyl-1,3,7-triazaspiro [4.4]nionane-2,4-diones has been enlarged by the synthesis of the series of novel aryl amides of 3-arylmethyl-2,4-dioxo-1,3,7-triazaspiro [4.4]nonane-7-carboxylic acids.The antimicrobial activity of the compounds obtained has been studied.According to the results of the microbiological screening it has been found that they mostly inhibited the growth of the Candida albicans fungi strain.