# 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
Structure-activity analysis (SAR) for acid **1** and its derivatives **2**, **4**, and **5-7** demonstrated that the introduction of electron-withdrawing chlorine into the benzimidazole molecule (compound **7**), as well as the incorporation of a 4-aminobenzenesulfonamide moiety in the structure of the compound (amide **4**), selectively increased the bacteriostatic effect of the compounds. Interestingly, the formed unsubstituted benzimidazole moiety did not affect the inhibition activity in comparison with the initial acid **1**, while the introduction of a highly electronegative fluorine at the fifth position of benzimidazole strongly diminished the inhibition of the _B. subtilis_ but did not affect other strains tested in comparison with the action of acid **1**. The bactericidal activity of acid **1** and its derivatives **2**, **4**, and **5-7** did not change for _S. aureus_ and _S. enteritidis_ and was found to be 500 ug/mL for the tested _Staphylococcus_ sp. and _Salmonella_ sp. Most of these chemicals were bactericidal against both _B. subtilis_ and _E. coli_ strains at a concentration of 500 ug/mL and exhibited a selective bactericidal effect. However, some compounds were selectively the best regarding the killing of bacteria strains. 5-Chlorobenzimidazole **7** and amide **4** appeared to be bactericidal for the tested Gram-positive _Bacillus subtilis_ at a concentration of 125 ug/mL, with a difference of 2 log\({}_{2}\) dilution, and methyl ester **2** killed \(\geq\)99.9% (\(\geq\)3 log\({}_{10}\) reduction) of bacteria _E. coli_ at a concentration of 250 ug/mL, with a difference of 1 log\({}_{2}\) dilution. Thus, as in the MIC case, the chlorine in the benzimidazole (comp. **7**) and the 4-aminobenzenesulfonamide moiety in the molecule (comp. **4**) selectively improved the bactericidal potency of these compounds. Interestingly, SAR analysis showed that replacing the carboxyl group with an ester group did not affect the inhibitory effect of the compounds, while the bactericidal effect of the ester against _E. coli_ was doubled. The best results of the MBC evaluation among compounds **1**, **2**, **4**, and **5-7** were obtained against the _B. subtilis_ strain, where 5-chlorobenzimidazole **7** and amide **4** showed the lowest MIC of 125 ug/mL. The comparison of the antibacterial activity of ethyl ester **2** and its hydrazide **3** is shown in Table 1. So, ester **2** and hydrazide **3** identically inhibited the growth (MICs, 125 ug/mL) and killed (MBCs, 500 ug/mL) the tested _Staphylococcus_ and _Salmonella_ strains. However, it is worth noting that hydrazide **3** showed antibacterial activity against _B. subtilis_, with 1 log\({}_{2}\) lower dilution compared with ester **2**. As for the _E. coli_ strain, methyl ester **2** killed \(\geq\)99.9% (\(\geq\)3 log\({}_{10}\) reduction) of _E. coli_ after treatment with a two-fold lower concentration of 250 ug/mL when compared to the action of hydrazide **3**. Subsequently, the antibacterial activity of three series of hydrazide derivatives **8-19**, **20-23**, and **24-26** was determined and compared. The comparative evaluation of the antibacterial activity of hydrazide **3** and the first series of hydrazones **8-19** showed identical or close (the difference \(\pm\)1 log\({}_{2}\) dilution) MIC and MBC values for all of the tested bacteria strains. The MIC values of hydrazide **3** and its hydrazones **8-19** for _S. enteritidis_ were identical and equal to 125 ug/mL. Similar results were achieved with _S. aureus_ and _E. coli_, except for hydrazone **12**, with the incorporated chlorine at the third position of the additional phenyl ring, which showed a two-fold higher inhibition efficacy. The higher range (62.5-250 ug/mL) of MIC values was revealed when treating the _B. subtilis_ strain.