## 3 Results and Discussion
### In Vitro Tubulin Inhibition Assay  
Benzimidazole antelmintics (such as albendazole and mebendazole) are known to inhibit tubulin polymerization in parasitic worms, causing their immobilization, cessation of reproduction, and death [7]. These agents bind to nematode tubulin with 250-400 times greater affinity than to mammalian tubulin, indicating selective toxicity to the parasites. In view of establishing the potential mechanism of anti-trichinella action, the ability of the synthesized compounds **4a**, **4b** and **6a-c** to interfere with tubulin polymerization was assayed in vitro. The effect of the compounds was tested on porcine tubulin at a 10 \(\mathrm{\SIUnitSymbolMicro m}\) concentration and compared to paclitaxel, albendazole and nocodazole as reference drugs (Figure 3 and Supplementary Materials). The measured kinetic curves were approximated to sigmoidal or bi-sigmoidal curves and fitted by Boltzmann equations (Table 2). The spontaneous tubulin polymerization was characterized with a half-time of aggregation \(\chi_{0}\) of 17.6 min (Table 2), while in the presence of paclitaxel the half-time of aggregation was considerably shorter--of 7.4 min. In contrast, the presence of both nocodazole and albendazole, which are tubulin polymerization inhibitors, resulted in increased half-time of aggregation--32.4 and 24.1 min, respectively. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multirow{2}{*}{**Substituents**} & \multicolumn{2}{c}{**Cencentration**} & \multicolumn{2}{c}{**Efficacy (\%)**} & \multicolumn{2}{c}{**Efficacy (\%)**} \\  & & **50 (100) \(\mathrm{\SIUnitSymbolMicro g}\)/mL in \(\mathrm{\SIUnitSymbolMicro m}\)** & \multicolumn{2}{c}{**After 24 h**} & \multicolumn{2}{c}{**After 48 h**} \\ \hline  & **R** & **Het** & & **50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL** & **100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL** & **50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL** & **100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL** \\ \hline
**4a** & H & thiophen-2-yl & 0.220 (0.440) & 54.5 \(\pm\) 0.52 \(\mathrm{\SIUnitSymbolMicro g}\) & 68.9 \(\pm\) 0.53 & 71.5 \(\pm\) 3.21 \(\mathrm{\SIUnitSymbolMicro s}\) & 80.5 \(\pm\) 0.33 \(\mathrm{\SIUnitSymbolMicro d}\) \\
**4b** & CH\({}_{3}\) & thiophen-2-yl & 0.207 (0.414) & 50.9 \(\pm\) 0.35 & 63.8 \(\pm\) 1.82 & 68.6 \(\pm\) 1.03 & 74.0 \(\pm\) 0.48 \(\mathrm{\SIUnitSymbolMicro d}\) \\
**4c** & CH\({}_{2}\)H\({}_{5}\) & thiophen-2-yl & 0.196 (0.392) & 48.4 \(\pm\) 1.57 & 61.7 \(\pm\) 0.51 & 58.9 \(\pm\) 0.55 & 73.4 \(\pm\) 0.63 \(\mathrm{\SIUnitSymbolMicro c}\) \\
**6a** & H & thiophen-2-yl & 0.206 (0.413) & 25.4 \(\pm\) 0.58 \(\mathrm{\SIUnitSymbolMicro c}\) & 28.9 \(\pm\) 0.39 & 53.8 \(\pm\) 0.34 & 57.5 \(\pm\) 1.02 \\
**6b** & H & furan-2-yl & 0.221 (0.442) & 20.4\(\pm\) 0.45 & 28.0 \(\pm\) 0.67 & 22.8 \(\pm\) 0.05 & 38.4 \(\pm\) 0.67 \\
**6c** & H & pyridin-4-yl & 0.210 (0.421) & 11.4 \(\pm\) 2.48 & 13.5 \(\pm\) 1.11 & 18.5 \(\pm\) 0.57 & 25.5 \(\pm\) 1.61 \(\mathrm{\SIUnitSymbolMicro b}\) \\
**Albendazole** & & & 0.188 (0.377) & 10.5 \(\pm\) 0.62 & 10.7 \(\pm\) 0.32 & 14.5 \(\pm\) 0.26 & 15.7 \(\pm\) 0.34 \\
**Ivermectin** & & & 0.057 (0.114) & 45.5 \(\pm\) 3.96 & 48.9 \(\pm\) 0.01 & 62.1 \(\pm\) 0.04 & 78.3 \(\pm\) 0.86 \(\mathrm{\SIUnitSymbolDegree}\) \\ \hline \hline \end{tabular}  
* Positive Control (untreated)-97.6 \(\pm\) 1.14 viable parasitic larvae after 24 h, 95.8 \(\pm\) 1.48 viable parasitic larvae after 48 h; \(\mathrm{\SIUnitSymbolDegree}\) \(p<\) 0.05; \(\mathrm{\SIUnitSymbolDegree}\) \(p<\) 0.001; \(\mathrm{\SIUnitSymbolDegree}\) \(p<\) 0.0001.  
\end{table} 
Table 1: In vitro activity of tested compounds against _Trichinella spiralis_ muscle larvae \(\mathrm{\SIUnitSymbolDegree}\)a. Four of the tested compounds--**4a**, **4b**, **6a**, and **6b**--slightly promoted the tubulin aggregation, showing half-times of 12-16 min (Table 2). Hydrazzone **6c** showed a two-step polymerization curve (Figure 3f).