# Synthesis and Biological Studies of New 2-Benzoxazolinone Derivatives as Antibacterial Agents
## 3 Results and Discussion
### Biology  
#### 3.2.1 Evaluation of the Antibacterial Activity by the Determination of MIC and MBC Values  
The synthesized benzoxazolinione derivatives **1-26** were evaluated for their antibacterial activity. The bacteria strains of the Gram-positive cocci _Staphylococcus aureus_ (ATCC 9144), Gram-positive sporogenic rods _Bacillus subtilis_ (ATCC 6051), Gram-negative rodsEscherichia coli_ (ATCC 8739), and _Salmonella_ Enteritidis (ATCC 13076) were used for the study of antibacterial properties, employing the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) and the calculation of the ratio of MBC/MIC, respectively. MIC was evaluated by the broth dilution method [47; 48], and MBC was evaluated by plating [49]. Ciprofloxacin (CFN) was used as the standard antibiotic for all bacteria strains. The tests were carried out twice. The antibacterial activity of the selected derivates **4**, **7**, **12**, **22**, and **23** was evaluated and compared via the disc diffusion and agar well diffusion methods. The results of the antibacterial activity of the prepared compounds are shown in Table 1.  
The evaluation of the antibacterial activity of the synthesized derivates was performed according to the synthesis scheme and by comparing the bacteriostatic and bactericidal effects of the compounds and their derivatives, employing the determination of MIC and MB values and inhibition zones. The MIC and MBC values of the synthesized derivates are shown in Table 1.  
At the beginning, according to Scheme 1, \"Synthesis of benzoxazolin-2-ones **2-23**\", the MIC and MBC values of compound 1 and its derivates **2**, **4**, and **5-7** were determined and compared. The inhibitory effect of the synthesized derivates **2** and **4-7** of carboxylic acid **1** for the tested bacteria was distinguished by its diversity. The same 125 \(\upmu\)g/mL concentrations of carboxylic acid **1** and its methyl ester **2** and benzimidazolees **5-7** inhibited the growth of _S. aureus_, while only its amide **4** showed a two-fold lower bacteriostatic concentration of 62.5 \(\upmu\)g/mL. The comparison of the susceptibility \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{3}{*}{**Compounds**} & \multicolumn{4}{c}{**Gram-Positive Bacteria Strains**} & \multicolumn{4}{c}{**Gram-Negative Bacteria Strains**} \\ \cline{2-9}  & \multicolumn{2}{c}{_S. aureus_} & \multicolumn{2}{c}{_B. subtilis_} & \multicolumn{2}{c}{_E. coli_} & \multicolumn{2}{c}{_S. enteritidis_} \\ \cline{2-9}  & **MIC** & **MBC** & **MIC** & **MBC** & **MIC** & **MBC** & **MIC** & **MBC** \\ \hline \multicolumn{9}{c}{**ug/mL**} \\ \hline  
FN** & 15.6 & 15.6 & 62.5 & 62.5 & 15.6 & 15.6 & 15.6 & 15.6 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The MIC and MBC values of compounds **1–26**. of _S. aureus_ and _B. subtilis_ to different compounds showed that the tested bacillus strain was inhibited by higher and lower, but not the same, minimum concentrations. The minimum bacteriostatic concentrations for the Bacillus subtilis strain were as follows: 250 ug/mL of carboxylic acid **1**, its methyl ester **2**, and unsubstituted benzimidazole **5**; 5-fluorobenzimidazole **6** required 500 ug/mL, while amide **4** and 5-chlorobenzimidazole **7** inhibited at 62.5 ug/mL. The inhibition of the growth of Gram-negative bacteria was achieved by minimum concentrations of 125 ug/mL of acid **1** and its derivatives **2**, **5-7**, while amide **4** showed the same inhibition only for _S. enteritidis_. Notably, amide **4** showed the highest inhibition efficiency against the E. coli strain, with the lowest MIC value of 62.5 ug/mL among acid **1** and its derivatives.