## 2 Results and Discussion
### In Vitro Biological Evaluation
#### 2.2.2 Antibioticfilm Activity
The results of our molecular docking analysis revealed that the majority of the compounds were capable of binding to the active sites of (p)ppGpp synthetases (Table 3). Among them, the compound **3 bd** demonstrated the most favorable binding energy of \(-\)6.101 kcal/mol with _E. coli_ RelA (Figure 3a). This value was slightly less favorable than the binding energy of the reference ligand relacin (\(-\)6.757 kcal/mol), which has been demonstrated to inhibit this enzyme in vitro [23]. Compound **3u** displayed the most favorable binding energy, measuring \(-\)4.719 kcal/mol, in its interaction with _S. aureus_ Rel (Figure 3b). Furthermore, for _M. smegmatis_ Rel, compound **3b** exhibited a more favorable binding energy of \(-\)5.615 kcal/mol (Figure 3c) compared to the reference ligand DMNP (\(-\)3.309 kcal/mol), which has been shown to inhibit this target in vitro [44]. The compounds **3** exhibited an average binding energy of \(-\)4.57 kcal/mol for _E. coli_ RelA, \(-\)4.01 kcal/mol for _S. aureus_ Rel, and \(-\)4.54 kcal/mol for _M. smegmatis_ Rel. The results indicate the possibility to use compounds **3** to inhibit the (p)ppGpp-synthesizing activity of RSH proteins. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Compounds**} & \multirow{2}{*}{\begin{tabular}{c} _E. coli_ \\ **RelA (AF)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} _S. aureus_ \\ **Rel (AF)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} _M._ \\ **_smegmatis_ \\ **Rel (AF)** \\ \end{tabular} } & \multirow{2}{*}{**Compounds**} & \multirow{2}{*}{\begin{tabular}{c} _E. coli_ \\ **RelA (AF)** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} _S. aureus_ \\ **Rel (AF)** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} _M._ \\ **_smegmatis_ \\ **Rel (AF)** \\ \end{tabular} } \\ \hline  
** & \(-\)4.619 & \(-\)4.234 & \(-\)5.299 & relacin a  & \(-\)6.757 & \(-\)6.880 & \(-\)7.457 \\ DMNP a,b  & \(-\)3.898 & \(-\)4.612 & \(-\)3.309 & carboxyl acid a  & \(-\)5.461 & \(-\)5.883 & \(-\)5.283 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The docking results for interaction of compounds **3** with (p)ppGpp synthetases/hydrolases from different bacterial species presented as docking scores (kcal/mol). In attempts to provide explanation for the observed antibacterial activity of some investigated compounds, we conducted molecular docking on essential proteins FtsZ and pyruvate kinase. The protein structures of _E. coli_, _S. aureus_, and _M. singematis_ FtsZ were obtained from AlphaFold (P0A9A6, P0A031, A0A8B4R473). Compounds lacking substantial antibacterial or antifungal activity (MIC \(\geq\) 250 mg/mL) or lacking activity data were excluded from this analysis. In order to evaluate the ability of benzimidazoles to bind to the FtsZ protein of _S. aureus_ (crystal structure PDB 4 DXD), we selected its allosteric site specific to a known inhibitor PC190723. [45]. However, the synthesized compounds **3** were unable to fit in this binding pocket via this mechanism. Thus, we decided to investigate the possibility of interaction in the active site. The GTP-binding site was located using the structure of _S. aureus_ FtsZ (PDB 3 WGN) [46]. The results demonstrated a strong interaction of benzimidazoles and their analogs **3** in the GTP-binding site of FtsZ proteins from all three species (Table 4). The native substrate GTP possessed favorable low binding energies in its binding site (\(-7.500\) kcal/mol). The lowest binding energy to _E. coli_ FtsZ was demonstrated by **3u** (\(-6.546\) kcal/mol) (Figure 3d). For both _S. aureus_ and _M. singematis_ FtsZ, the strongest binding was observed for **3 ag** (\(-5.756\) and \(-6.139\) kcal/mol) (Figure 3e,f). The tested compounds **3** demonstrated an average binding energy of \(-5.66\) kcal/mol in interaction with _E. coli_ FtsZ, \(-4.97\) kcal/mol with _S. aureus_ FtsZ, and \(-5.46\) kcal/mol with _M. singematis_ FtsZ.