Synthesis and Antimicrobial Assessment of Some New 2-(Thiazol-5-yl)-1,3,4-oxadiazoles

CATALIN ARANICIU1, SMARANDA DAFINA ONIGA1*, CRISTINA IOANA STOICA1, MARIANA CARMEN CHIFIRIUC2,3, MARCELA POPA2,3, LAURIAN VLASE1, MARIANA PALAGE1, OVIDIU ONIGA1 1 Iuliu Haieganu University of Medicine and Pharmacy, Faculty of Pharmacy, 12 Ion Creanga Str., 400010, Cluj-Napoca, Romania 2 University of Bucharest, Faculty of Biology, Microbiology Department,1-3 Portocalelor Lane, 60101, Bucharest, Romania 3 Research Institute of the University of Bucharest (ICUB), 91-95 Independenei Str., Bucharest, Romania

Developing new antimicrobial drugs is a key concern as the emergence of multi-drug, extended-drug and pan-drug resistance among bacterial and fungal pathogens is a worldwide health threat [1].

Experimental part Chemistry
All chemical reagents and solvents used in the synthesis, isolation and purification process were of analytical grade purity and were purchased from Alfa Aesar (Karlsruhe, Germany). Initial reaction progress monitoring and initial purity assessment were performed using Silica Gel thin layer chromatography sheets and UV visualization. The melting points are uncorrected and were obtained by using an Electrothermal 9100 melting point apparatus. The 1 H-NMR were recorded on a Bruker Avance NMR spectrometer, operating at 500MHz, in DMSO-d6 as solvent.
Chemical shift values are reported in δ units, relative to * email: smaranda.oniga@umfcluj.ro; Phone: 0723 577 124 TMS as internal standard. Elemental analyses were performed by an Elemental Analyser Systeme GmbH VarioEL. MS spectra were recorded on a LC-MSD-Trop-VL mass spectrophotometer.

Synthesis of ethyl 4-methyl-2-(pyridin-3-yl)-thiazole-5carboxylate (A)
To a solution of thiobenzamide (13.7 g, 0.1 mol) in 40 mL ethanol an equimolar quantity of ethyl-2chloroacetoacetate (16.45 g, 0.1 mol) was added and it was refluxed for 5 h. The reaction mixture was cooled to room temperature and neutralized with sodium hydrogen carbonate. The solid obtained was filtered, washed with water and then with acetone and recrystallised from ethanol [25,26].

Synthesis of the 4-methyl-2-phenylthiazole-5-carbohydrazide (B)
The solution obtained by dissolving 10 mM of the corresponding ester in 4 mL ethanol was treated with 5 mL hydrazine hydrate and refluxed for 5h. The resulting mixture was allowed to cool overnight and then poured over ice water. The resulting solid was filtered and washed with water in order to yield the pure compound [25,26].
General procedure for the synthesis of the 2-(4-methyl-2phenylthiazol-5-yl)-5-aryl-1,3,4-oxadiazoles (C1-8) A mixture of the 4-methyl-2-phenylthiazole-5carbohydrazide (0.001 M) and the appropriate aromatic acid (0.001 M) was refluxed in 3 mL phosphorus oxychloride for 8 h. The reaction mixture was allowed to cool down and then slowly poured over crushed ice. The resulting mixture was neutralized with solid sodium hydrogen carbonate and kept overnight. The solid that separated was filtered, washed with cold water and recrystallised from methanol.
Biological Assays: The in vitro qualitative screening of the antimicrobial activity The in vitro qualitative screening of the antimicrobial activity was carried out by an adapted agar disk diffusion technique using a bacterial suspension of 0. 5  The antimicrobial activity was tested on Mueller-Hinton Agar (MHA) medium, while yeast peptone glucose (YPG) medium was used in the case of Candida spp.. The compounds or standards (ciprofloxacin, fluconazole) were solubilised in dimethylsulfoxide to a final concentration of 10 mg/mL. A volume of 5µL of each tested compound solution was distributed directly on the solid medium previously seeded with the microbial inocula. The inoculated plates were incubated for 24 h at 37 °C. Antimicrobial activity was assessed by measuring the growth inhibition zones diameters expressed in mm [21,22,27].

Results and discussions Chemistry
The 3 step reaction path used proved very efficient as it was characterized by high yields. The first step of the synthesis was a Hantzsch condensation between thiobenzamide and the alpha-keto carbonyl component represented by ethyl-2-chloroacetoacetate. This led to the formation of a new thiazole ring with an ethyl carboxylate substituent in the 5 position. The ester derivative (A) was then transformed in the corresponding 5-carbohydrazide (B) by reflux with hydrazine hydrate. The structural variety of the 2-(thiazol-5-yl)-1,3,4-oxadiazole final products was due to the various aromatic carboxylic acids used in the last condensation reaction, as shown in figure 1.
All new compounds presented spectral data consistent with the proposed structure and elemental microanalysis within 0.4% of the theoretical values, as described in the following paragraphs.  The 1 H-NMR spectra of the compounds show an abundance of signals in the interval δ 7.5-8.7 corresponding to the numerous protons from the aromatic rings. A common element of all the spectra is represented by the signal of the 3 protons in the thiazole 4-methyl group at δ 2.84. Characteristic signals can be found in C2, due to a multitude of aliphatic protons, and in C7, due to the methoxy group that provides a singlet 3H at δ 3.75.

Biological evaluation
The in vitro qualitative screening of the antimicrobial activity showed an overall modest activity for the tested compounds, as can be seen from table 1. Antibacterial activity was varied with the most promising being that of C1 against B. subtilis. Generally the tested molecules performed better against Gram-positive strains than against Gram-negative strains.
The weakest activity was observed against the fungal strains, with practically no activity against C. parapsilopsis.
Although most of the tested compounds showed a certain degree of antimicrobial action it was modest in comparison with the activity of the standard substances used. This may indicate that the new molecules could be active only at higher concentrations. Also it is imperative to analyze the antimicrobial data from these findings and corroborate them with our previous results in order to highlight some structure-activity relationships that can eventually lead to better agents.

Conclusions
A series of 8 new thiazolyl-oxadiazoles were synthesized and characterized via 1 H-NMR, mass spectrometry and elemental analyses. The structures of all new compounds were verified and confirmed. Also, a preliminary biological evaluation was performed under the form of an in vitro qualitative antimicrobial activity screening. Despite their structural similarities with other active compound the new molecule seem to have limited antimicrobial effect. Further molecular modeling must be undertaken in order to generate new molecules with improved antimicrobial effect.