Synthesis and Antimicrobial Evaluation of New Series of 1,3,4-Oxadiazole Containing Cinnamic Acid Derivatives

Background: New drugs must be designed and synthesized for combating resistant pathogens. In this study, antibacterial and antifungal activities of 4 new derivatives of 1,3,4-oxadiazole were assessed against 8 bacterial and 2 fungal pathogens. Methods: To this end, the cinnamic acid derivatives were dissolved in acetonitrile solvent and N-iso-cianoimino-triphenyl-phosphorane was added to the above-mentioned solution, followed by applying Petroleum ether and Ethyl acetate as solvent and base. Then, antimicrobial susceptibility tests were used to determine inhibition zone diameter, minimum inhibitory concentration, the minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values. Results: The chemical structure of all compounds was characterized with infrared spectra, 1H-NMR, and 13C-NMR. A variety of inhibitory effects were observed by the synthesized compounds. Methoxyphenyl derivative (3c) affected bacterial strains, especially Streptococcus mutans. Other compounds also had antibacterial properties. Additionally, compound 3c showed the greatest effect on fungal samples, especially Aspergillus flavus. Conclusions: In general, our new derivatives of 1,3,4-oxadiazole are able to destroy Gram-positive bacteria. In addition, developing new derivatives of 1,3,4-oxadiazole in future research can improve therapeutic properties. It seems that with the addition of other functional groups and increasing the destructive power of compounds, inhibitory effects on fungal samples can also be observed.

of N-iso-ciano-imino-triphenyl-phosphorane is attacked by the anion of cinnamic acid derivatives ( Figure 3) and an intermediate is created [6]. The intermediate [6] reacts with the intramolecular Wittig to form product [3] together with triphenylphosphine oxide [4]. It is noteworthy that these structures were synthesized for the first time.   (11). The results were reported as the mean of three independent experiments.

Inhibition Zone
After preparing the suspension of bacteria and fungi (separately) in the tube with distilled water and adjusting the turbidity of the suspensions to match 0.5 McFarland standard (1.5 × 10 6 CFU/mL), some of each suspension was removed with sterile cotton swabs and was cultivated as grass in Mueller-Hinton agar medium for bacterial culture and Sabouraud dextrose agar for fungal culture. Then, wells were made in the plate by Pasteur pipette. Then, 10 μL of solutions prepared from oxadiazole derivatives (at concentrations of 0.5 mg/mL), ciprofloxacin (positive control for bacteria), and clotrimazole (positive control for fungi) was injected into the wells. Positive control samples were used to compare their growth areas with new compounds. Finally, the plates were closed and they were incubated for 24 hours at 37°C for bacterial samples and 48 hours at 35°C for fungal samples. After the time mentioned, the plates were examined for the presence of growth inhibition zone (15).

Minimum Inhibitory Concentration Experiment
MIC of the compounds was determined using the two-fold serial dilution method (bacterial-fungal). Concentrations of 1000, 500, 250, 125, 62.50, 31.25, and 15.62 µg/mL were prepared in sterile molten Mueller-Hinton agar from the stock solution. The microplates were inoculated with 1.5×10 6 CFU/mL of bacterial suspension at 37°C for 24 hours. The MIC was defined as the lowest concentration of the compounds that prevented visible growth of microorganisms after 24 hours of incubation. The antibacterial activity of the compounds was compared with ciprofloxacin as standard antibacterial agents. Next, the fungal suspension was made and adjusted to turbidity equivalent to a 0.5 McFarland standard (1.5×10 6 CFU/mL). The inoculated agar was poured into the assay plate and allowed to cool down on a leveled surface. Once the medium solidified, wells were created on the agar, and 70 µL of the antifungal agents was poured into each well. After adding the synthesized compounds, the plate was incubated at 35 o C for 48 hours (16). Antifungal susceptibility test was performed by the agar dilution method. Stock solutions of 1,3,4-oxadiazole derivatives were prepared at a concentration of 0.5 mg/ mL in dimethyl sulfoxide (0.001 g of each derivative in first time. An Overview of Single-Stage Synthesis of 1,3,4-Oxadiazole   Overview of Single-Stage Synthesis of 1,3,4-Oxadiazole   2 mL of DMSO). Next, 1.6 mL of molten Sabouraud dextrose agar was poured into sterile microplates and allowed to cool to 50°C. Then, 0.4 mL of dilutions prepared from the stock solutions of the clotrimazole was added in descending order of concentration. In addition, 10 μL of the standardized fungal inoculum was added to all microplates except for the sterility control. The microplates were incubated at 35°C for 7 days and visualized for growth. The lowest concentration that inhibited the growth of fungi was defined as the MIC. All experiments were performed in duplicate and results were reported as mean ± standard deviation.

MBC and MFC Experiments
To determine the MBC and the MFC of the synthesized compounds, a loopful was taken from the MIC tubes and streaked on Mueller-Hinton agar for bacterial culture and Sabouraud dextrose agar for fungal culture. Growth was observed after incubation at 37ºC for 24 hours. The lowest concentration at which no growth was observed was determined as the MIC and MFC (17).

Chemicals
Structures, Infrared, C-NMR, and H-NMR of all compounds were obtained ( Figure 4).

Determination of the In Vitro Antibacterial and Antifungal Properties
Antibacterial and antifungal properties of new derivatives of 1,3,4-oxadiazole (3a-3d) were evaluated in terms of their structures (Figure 4). The inhibition zone diameters of the synthesized compounds against the tested bacteria and fungi are presented in Tables 1 and 2. As shown in Table 1, the greatest effect of compound 3a was observed on S. mutans with IZ=31.33 ± 0.50 mm, MIC=125 mg/mL, and MBC=250 mg/mL. This result can be due to the presence of chlorophenyl group in the main compound (18). The greatest effect of compound 3b was observed on S. aureus with IZ=24.66 ± 0.50 mm, MIC=500 mg/mL, and MBC=1000 mg/mL. This result can be due to the presence of fluorophenyl group in the main compound (19). The greatest effect of compound 3c was observed on S. mutans with IZ=39.33 ± 0.50 mm, MIC=62.50 mg/mL, and MBC=125 mg/mL. This result can be due to the presence of methoxyphenyl group in the main compound (9). The greatest effect of compound 3d was observed on S. epidermidis with IZ=28.66 ± 0.50 mm, MIC=250 mg/mL, and MBC=500 mg/mL. This result can be due to the presence of bromophenyl group in the main compound (20).
As shown in Table 2, the greatest effect is related to compound 3c on A. flavus with IZ=33.33 ± 0.50 mm, MIC=250 mg/mL, and MFC=500 mg/mL. This result can be due to the presence of methoxyphenyl group in the main compound.
As shown in Tables 1 and 2, the significant results obtained in this study displayed that most of the synthesized compounds performed very well against the gram-positive samples; however, compound 3C showed acceptable inhibitory effects with high IZ, MIC, and MBC among all.

Conclusions
In general, 1,3,4-oxadiazoles are multi-functionalized compounds with a variety of biological properties; therefore, synthesis of their new derivatives is of great importance. This study evaluated the antibacterial and antifungal effects of all 4 synthesized 1,3,4-oxadiazole derivatives on pathogenic bacteria and fungi. These  Note. IZ (mm): inhibition zone; MIC (μg/mL -1 ): minimum inhibitory concentration; MFC (μg/mL -1 ); minimum fungicidal concentration compounds, chiefly methoxyphenyl, showed relatively acceptable antibacterial effects on Gram-positive strains such as S. mutans and S. epidermidis, as well as Gramnegative strains such as P. mirabilis. In addition, good to excellent antifungal activities were observed for compound 3c. Our results show that 1,3,4-oxadiazole derivatives would be helpful structures for the possible development of new drugs; however, this result needs to be confirmed by other extensive clinical trials that will be part of our future plans. Furthermore, the easy workup procedure, high yield, and short reaction times make the method a useful addition for preparing modern pharmaceutical synthetics. These derivatives were synthesized for the first time and the relevant tests were done to ensure the biological properties of the compounds. This study was done to provide new structures and confirm the existence of antibacterial and antifungal activity of the compounds. In future research, several concentrations will be used for other resistant bacteria and cell lines as well as MTT assay and so on.