СИНТЕЗ ТА АНАЛІЗ БІОЛОГІЧНО АКТИВНИХ РЕЧОВИН The synthesis, spectral properties and the biological activity of 7-arenesulfonyl-3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-dione derivatives

Aim. To synthesize the series of 7-arenesulfonyl-3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-dione, to study their spectral properties and antibacterial activity. Materials and methods. The methods of organic synthesis, instrumental methods of organic compounds analy-sis, as well as the agar diffusion method were used. Results and discussion. By the interaction of 3-arymethyl-1,3,7-triazaspiro[4.4]nonane-2,4-diones with arenesulfonyl cholrides in the presence of triethylamine the series of 7-arenesulfonyl-3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-dione was obtained. For the compounds containing the fragments of 1-sulfonylamido-(2,4)- and 3,4-difluorobenzene the 1 H- 1 H coupling constants in their 1 Н{ 19 F}-NMR fluorine decoupled spectra, as well as the 19 F- 19 F coupling constants in the 19 F{ 1 Н}-NMR proton decoupled spectra were measured. The antimicrobial activity screening showed that the growth of such bacterial strains as Staphylococcus aureus and Bacillus subtilis was inhibited by the compounds of the series obtained. Conclusions. It has been found that the interaction of 3-arymethyl-1,3,7-triazaspiro[4.4]nonane-2,4-diones with arenesulfonyl cholrides is an effective way for the synthesis of 7-arenesulfonyl-3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-diones with the promising biological activity against the strains of gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis . Among 7-arenesulfonyl-3-arylmethyl-1,3,7-triazaspiro[4.4]nonane-2,4-dione derivatives 3-(3-methylbenzyl)-7-(toluene-4-sulfonyl)-1,3,7-triazaspiro[4.4]nonane-2,4-dione exhibited the highest activity.

In recent years the fluorine-containing molecules are of a great interest to scientists [5][6][7][8] since regardless of its larger atomic radii fluorine causes less steric complications for receptor binding rather than the hydrogen atom [9]. Therefore, obtaining of the novel fluorine-containing compounds is a promising way for developing effective antibacterial drugs, that is why we decided to make the combination of the 3-arymethyl-1,3,7-triazaspiro [4.4] nonane-2,4-dione moiety with the sulfonamide fragment the objects for further studies of spectral characteristics and the antibacterial activity.

Chemical part
All solvents and reagents were obtained from the commercial sources. 1 Н, 13 C and 19 F NMR-spectra were recorded with a Bruker 170 Avance 500 spectrometer at 500 МHz for NMR 1 Н-spectra, at 125 МHz for NMR 13 С-spectra and 376 МHz for NMR 19 F-spectra; the solvent was DMSO-d 6 ; TMS was used as an internal standard for 1 Н, 13 C and CFCl 3 for 19 F-spectra. Chromato-mass spectra were recorded using an Agilent 1100 HPLC device equipped with a diode matrix detector and a massspectrometer (Agilent LC-MSD SL), and the Zorbax SB-C18 column (4.6×15 mm) with atmospheric pressure chemical ionization (APCI) was used for the analyses. The TLC was performed on the aluminium plates covered with Merck, Kiesgel 60 F-254. The melting points were measured with a Kofler melting point apparatus and were not corrected.
The general method for the synthesis of the sub-

Microbiological Experiment
According to the WHO recommendations to assess the activity of the compounds tested the following strains of microorganisms were used: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 2785 3, Proteus vulgaris ATCC 4636, Bacillus subtilis ATCC 6633, Candida albicans ATCC 653/885. The inoculum suspension was prepared using a Densi-La-Meter apparatus (PLIVA-Lachema, Czech Republic; 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" [10][11][12]. The cultures were synchronized under the low temperature conditions (4°С). The density of the inoculum was 107 cells per 1 ml of the medium and was determined by comparing with McFarland standard [13]. The 18-24 hour old culture of microorganisms was used for the test. Mueller-Hinton agar was applied for bacteria. The strain of Candida albicans was cultivated using Sabouraud agar. The experiment was carried out using the agar "well" diffusion method [14]. The compounds studied were introduced as 0.3 ml of the 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. The antibacterial activity was assessed by measuring the growth inhibition zones for each microorganism.
In the 1 Н and 19 F NMR-spectra of the fluorine-containing samples (3.2, 3.5, 3.7, 3.12) obtained the multiplicity of the proton signals for difluorobenzenesulfonyl amide fragment were unclear to be interpreted correctly. Such situation was caused by the presence of two magnetically and chemically non-equivalent fluorine atoms, which impeded the spectra because of splitting the protons signals. For resolution of the spectra for the compounds with 2,4and 3,4-difluorobenzenesulfonyl amide fragments (3.2 and 3.5, respectively) the proton or fluorine decoupled spectra 1 Н{ 19 F} and 19 F{ 1 Н} were measured for the adequate interpretation of the proton spectral signals.
In the 1 Н{ 19 F}NMR-spectrum of compound 3.2 with the fluorine decoupling three signals in the region of aromatic proton resonance with the equal integral intensity were observed. In the spectra the proton signal in position 5 was observed as a doublet of doublets (dd) at 7.35 ppm due to the interaction with the protons in posi- The successful interpretation and the coupling constants measurement for the compound with the 3,4-difluorobenzenesulfon amide fragment 3.5 was achieved by detection of 1 Н{ 19 F} and 19 F{ 1 Н} NMR-spectra. The normal 1 Н and 19 F NMR-spectra of compound 3.5 contained the unclear picture of multiplet signals.
In the 1 Н{ 19 F} NMR-spectrum there was the doublet of doublets at 7.73 ppm produced by the proton in position 6, for this signal the first coupling constant was 8.7 Hz; it was caused by the interaction with the proton in position 5, while the second constant (J = 1.9 Hz) resulted from the interaction with the proton in position 2. The doublet at 7.77 ppm (J = 8.7 Hz) was a signal of the proton in position 5, and the doublet at 7.98 (J = 1.9 Hz) was the signal of the proton in position 2. The characteristic difference in intensity of the doublet components (the "roof effect") indicates the magnetic similarity of protons in positions 5 and 6.
The fluorine atoms in the 3,4-difluorobenzenesulfon amide fragment gave two doublets with the spin-spin coupling constant of 21.8 Hz, being 9.3 Hz more than the constant for the 2,4-difluorosubstituted fragment.
Concerning 13 С NMR-spectra of compounds 3.2 and 3.5 the signals of carbon atoms were also split because of the presence of the fluorine atoms in the aromatic ring. Thus, using the NMR-spectroscopic experiential techniques the coupling constants for 1 Н-1 Н and 19 F-19 F were Scheme measured, and it helped to interpret properly the spectra of compounds 3.2 and 3.5 containing 1-sulfonylamido-(2,4)-and 3,4-difluorobenzene fragments, being typical for many biologically active substances [15][16][17]. The data obtained may be used for interpretation of the similar spectra of more complex molecules. The 1 Н, 13 С, 19 F NMR, LC-MS-spectral data for compounds 3 and the melting points are listed in Tab. 1.