Synthesis and Characterization of New Heterocyclic Condensed Systems with Bridgehead Nitrogen from Thiazolo[3,2-b][1,2,4]triazol-6(5 H )-ones Class with Potential Antimicrobial Activity

In this study, the synthesis and characterization of new thiazolo[3,2-b][1,2,4]triazol-6(5H)-ones 3-5 are described starting from 5-[4-(4-X-phenylsulfonyl)phenyl]-4H-1,2,4-triazole-3-thioles 2 . The treatment of 1,2,4-triazole-3-thioles 2 with chloroacetic acid and substituted or non-substituted benzaldehydes in the presence of sodium acetate, acetic acid and acetic anhydride gave fused ring compounds 3-5 . The structures of thiazolo-triazolones 3-5 have been elucidate by elemental analysis and IR, UV, 1 H-NMR, 13 C-NMR, MS spectra. Some of the newly synthesized products were tested for their antimicrobial effects.


Experimental part
The melting points of the new compounds were determined with a Böetius apparatus and are not corrected.The IR spectra were registered in KBr pellets with a spectrophotometer with Fourier transform FTS-135 BIORAD, at 4000-400 cm -1 .The UV spectra were acquired with a spectrophotometer SPECORD 40 Analytik Jena, within the range 200-600 nm.The NMR spectra were registered with a Varian Gemini 300BB apparatus, at 300 MHz for 1 H-NMR and 75 MHz for 13 C-NMR, using tetramethylsilane (TMS) as internal standard.DMSO-d 6 or CDCl 3 and TFA were used as solvents with a minimum grade of deuteration of 99%.The mass spectra of the * email: stefaniafelicia_barbuceanu@yahoo.comcompounds 3a-c were registered with a triple quadrupole mass spectrometer Varian 1200 L/MS/MS coupled with a high performance liquid chromatograph with Varian ProStar 240 pump and a Varian ProStar 410 automatic injector.An atmospheric pressure chemical ionization interface (APCI) was used in order to obtain the ions.The liquid chromatography was performed on a Hypersil Gold (Thermo) column with pre-column, and the mobile phase was 30% water and 70% methanol.
The mass spectra of compounds 4a and 5c have been acquired with a hybrid triple quadrupole -time of flight (QqTOF) high resolution mass spectrometer model API QStar Pulsar produced by Applied Biosystems/SCIEX.The instrument was operated in positive ions mode, using an atmospheric pressure pneumatically assisted electrospray ionization interface (ESI, AB model Turboionspray).The voltage of the mass spectrometer (MS) source was set at 5000V.Molecular ions were detected in full scan over an adequate mass range.Stock solutions were prepared at 1mg/mL in DMSO.The sample solution (2µg/mL in water/ methanol 1/1, v/v) was introduced in the MS interface by direct infusion, at a flow rate of 20 µL/min, with the help of the built-in Harvard syringe pump.
A mixture of 2 mmol 1,2,4-triazole 2, 3 mmol chloroacetic acid, 2 mmol aromatic aldehyde and 0,27 g of anhydrous sodium acetate was refluxed in 4 mL of acetic anhydride and 5 mL of acetic acid for 6-8 h.The mixture was poured into ice-water.The formed precipitate was separated by filtration and dissolved in CH 2 Cl 2 .The organic layer was washed with NaHCO 3 6% and dried over Na 2 SO 4 anhydrous.The new compound was obtained by solvent evaporation under reduced pressure or by precipitation with petroleum ether and was recrystalized from benzene.
The spectral data IR (KBr; cm -1 ) for all the newly compounds are presented in table 1.

Biological activity
The in vitro antimicrobial activity of the compounds was tested using the microdilution method for determination of minimum inhibitory concentration (MIC).For this, all tested compounds were dissolved in DMSO at the concentration of 2048µg/mL and afterwards, serial dilutions in liquid broth (1024-2µg/mL) were prepared in 96-well plates.It was determined that the solvent had no antimicrobial activity against any of the tested microorganisms.
The compounds were tested for their in vitro growth inhibitory activity against the following Gram- The cultures were obtained in Mueller-Hinton broth (Merck) for all the bacteria after 18-24 h of incubation at 37°C and Sabouraud dextrose broth (Merck) for the yeast strain after an incubation at 25°C for 48 h.

Results and discussions Chemistry
The proposed mechanism of reaction implies, in the first stage, the alkylation of 1,2,4-triazole 2 with chloroacetic acid, in basic media, with formation of the alkylated intermediary at the sulphur atom A (non-isolation), followed by cyclisation to intermediary B and, finally, crotonic condensation with aromatic aldehydes in acidic media.This latter stage occurs by electrophilic attack of the aromatic aldehyde C protonated to the carbon atom at the double bond of the enolic tautomer B' (fig.2).
In the IR spectra, all new compounds 3-5 displayed a strong absorption band in the range 1739-1753 cm -1 due to the valence vibration of C=O (table 1).In the region 1492-1611 cm -1 characteristic bands for C=C and C=N groups valence vibration are found.The presence of new structural elements is indicated in compounds 4a-c by the absorption bands ν asym CH 3 -O-C ar and ν sym CH 3 -O-C ar which appear at 1263-1266 cm -1 and 1071 cm -1 , respectively.Also, the IR spectra of compounds 5a-c showed two absorption bands due to the valence vibration of NO 2 group: ~ 1534 cm -1 (ν asym NO 2 ) and ~ 1356 cm -1 (ν sym NO 2 ), respectively.
In the compounds 4a-c the methoxy group protons resonated as a singlet at δ = ~3.93ppm integrating for three protons.
In the 1 H-NMR spectra of these new compounds, the signals of the NH or SH protons from the 1,2,4-triazoles 2ac, are not found [26].
The 13 C-NMR spectra of the new compounds show characteristic signals attributed to the carbonyl group at 166.34-167.56ppm and to the methine group at 140-145 ppm [14].
A new signal resonating at 160.49-162.00ppm corresponds to C-5 carbon atom from the thiazole ring which is equivalent with C-3a carbon atom (table 3).
The signal of the -OCH 3 group from compounds 4a-c has been observed at δ~ 56 ppm.
In the mass spectra, molecular ion [M+H] + peak which appears at different intensities in positive ionisation mode confirmed the molecular weights of the examined compounds (3a, 3b, 3c, 4a, 5c).The isotopic distribution in the molecular ion region confirms the presence of one bromine atom in the compounds 3c and 5c.Also, compound 3b heaving a chlorine atom in the molecule shows in its mass spectrum the characteristic peaks for 35 Cl and 37 Cl isotopes.

Antimicrobial activity
The MIC of each tested substance was determined by macroscopic observations of microbial growth and corresponds to the well with the lowest concentration of the tested substance where microbial growth was clearly inhibited.
The MIC values (µg/mL) of newly prepared compounds 3a,b; 4b; 5a,b were compared with the MIC value of the starting triazole 2a,b and the results of antimicrobial activity screening were summarized in the table 4.
None of the synthesized compounds showed an important antimicrobial activity.
The antibacterial screening indicated that the tested compounds exhibited some inhibitory activity against Acinetobacter baumannii ATCC 19606, the most active being 4b (MIC=128µg/mL).In this case, the inhibitory activity was probably enhanced by the presence of chloro atom and para-methoxy group.The compounds 2a, 3a, 4a have shown a weak to moderate activity against Staphylococcus aureus ATCC 12600, in contrast to 2b, 3b and 4b, which had a chloro atom on para position in diphenylsulfone moiety and were inactive.
The structures of new compounds were determined by spectral data and elemental analysis.Some of these newly synthesized compounds were tested for their antimicrobial activity.Only compound 4b exhibits good inhibitory activity against Acinetobacter baumannii (MIC=128µg/mL).