## 2 Results and Discussion
### In Vitro Biological Evaluation  
#### 2.2.1 Antimicrobial Activity  
The vast majority of synthesized compounds were tested for their antimicrobial activity against _Candida albicans_, _Escherichia coli_, _Staphylococcus aureus_, and _Mycobacterium smegmatis_. It was shown that most of the studied substances demonstrated antimicrobial activity against two or more microbial species from different groups. In our study, 10 compounds were found to have high activity against _C. albicans_, while 22 compounds demonstrated a moderate level of activity (see Supplementary Materials). The most active compounds are indolylbenzo[_d_]imidazoles **3 ad**, **3 ag**, and **3 aq** (Table 1). Previously, it was shown that some derivatives of benzimidazoles exhibit antimyotic activity [30; 31; 32]. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Compounds**} & \multicolumn{2}{c}{_C.a._ **10231 b**} & \multicolumn{2}{c}{_M. s._ **70084 c**} & \multicolumn{2}{c}{_E. c._ **25922 d**} & \multicolumn{2}{c}{_E. c. c._ **8739 e**} & \multicolumn{2}{c}{_S. a._ **25923 f**} & \multicolumn{2}{c}{**MRSA \$} \\ \cline{2-13}  & **MIC** & **MFC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** & **MIC** \\ \hline
**3a** & 15.6 & 15.6 & 125 & 250 & - h & - & 500 & 1000 & 125 & 125 & 125 & 125 \\
**3b** & 125 & 125 & 62.5 & 125 & \(>\)500 & \(>\)500 & \(>\)500 & 31.3 & 31.3 & 62.5 & 62.5 \\  
** & 15.6 & 15.6 & 62.5 & 250 & - & - & - & - & 15.6 & 250 & 31.2 & 125 \\ Cefotaxime & n.d. i & n.d. & n.d. & n.d. & 0.038 & 0.038 & 0.038 & 0.31 & 0.61 & 19.53 & 39.06 \\ Cefazolin & n.d. & n.d. & n.d. & n.d. & 2.44 & 2.44 & 2.44 & 0.15 & 0.61 & 9.77 & 39.06 \\ Amikacin & n.d. & n.d. & n.d. & n.d. & 19.53 & 19.53 & 19.53 & 4.88 & 9.77 & 9.77 & 9.77 \\ Fluconazoleide & 1.94 & 7.8 & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. & n.d. \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Selected antimicrobial data (MIC and MBC/MFC, \(\mu\)g/mL) for the indolylbenzimidazole derivatives **3** and their analogs a. None of the compounds showed high activity against the Gram-negative bacterium _E. coli_. Some of the tested compounds, **3q**, **3r**, **3w**, **3w**, and **3 af**, showed moderate activity (MIC 125 \(\mathrm{\SIUnitSymbolMicro}\)/mL) against this microorganism. Benzimidazoles **3z** and **3 at** showed an MIC of 250 \(\mathrm{\SIUnitSymbolMicro}\)/mL. At the same time, 2-(furan-2-yl)-1\(H\)-benzo[\(d\)]imidazole (**3 at**) was only bacteriostatic but not bactericidal against _E. coli_. Most of the compounds that have shown antibacterial activity against _E. coli_ are the 5-substituted benzimidazoles **3q**, **3r**, **3w**, and **3w**. The low activity of benzimidazoles against Gram-negative bacteria was also observed in other studies [26; 30]. This is probably due to the structure of the cell wall of this group of bacteria, which contains an outer membrane. The outer membrane effectively prevents the transport of hydrophobic compounds into the cell. Nine compounds from the library showed high and twenty-two showed moderate inhibitory effects against Gram-positive _S. aureus_ (MIC \(<\) 16 \(\mathrm{\SIUnitSymbolMicro}\)/mL and 17-125 \(\mathrm{\SIUnitSymbolMicro}\)/mL, respectively). The most active substances **3 ao** and **3 aa** showed an MIC \(<\) 1 \(\mathrm{\SIUnitSymbolMicro}\)/mL against staphylococci, including MRSA. The MIC against _S. aureus_ for compounds **3 aa** and **3 ad** did not exceed 7.8 \(\mathrm{\SIUnitSymbolMicro}\)/mL, which was a promising result. Activity against staphylococci was found in most tested compounds. This result deserves attention since benzimidazole derivatives were previously thought to have predominantly antifungal activity. The target of benzimidazole binding in most eukaryotic parasites is tubulin, which is involved in the formation of the cytoskeleton [33; 34; 35; 36]. Upon exposure of eukaryotic cells to benzimidazoles, the process of cell division is damaged.