## 2 Results
Cell viability values are shown in Table 1. Antiparasitic assays were also performed with the less cytotoxic derivatives, and the analogs **15**-**18** and **20** (presenting low cytotoxicity towards healthy cells) were selected for further testing. The cell death study is shown in Figure 2, which contains the scatter plot density graphs of the cells. Flow cytometry assays with Rho 123 (rhodamine 123) are shown in Figure 3.  
In the flow cytometry assays, the epimastigote forms of _T. cruzi_ were used, and the cells were divided into four cell populations: viable cells, with low levels of labeling for both fluorochromes (un-labeled); necrotic cells, labeled only with 7-AAD (7 AAD+) (7-Aminoacetinomycin D); apoptotic cells, labeled with annexin V-PE only (/AxPE+); and doubly labeled late apoptotic cells (7 AAD+/AxPE+) [16]. Cells were incubated with compounds **15**-**18** and **20** for 24 h at concentrations of equivalent to IC\({}_{50}\) and 2 \(\times\) IC\({}_{50}\). In this assay, a reduction in the percentage of viable cells and an increase in the percentage of 7-AAD-labeled cells were observed, indicating cell death by necrosis (Figure 2). Necrosis results in irreversible cell injury with loss of cell membrane integrity [17]. The increase is also observed in the displacement of cell populations, as shown in Figure 2. \begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**Compound**} & \multirow{2}{*}{\begin{tabular}{c} **Y Strain** \\ **(Trypomastigotesotes)** \\ **(IC\({}_{50}\)\(\mu\)M)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Y Strain** \\ **(Eprimastigotes)** \\ **(IC\({}_{50}\)\(\mu\)M)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Cell Viability** \\ **(LLC-MK2)** \\ **(IC\({}_{50}\)\(\mu\)M)** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **S.I.** \\ **(Trypomastigotesotes)** \\ **(Trypomastigotesotes)** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} **S.I.** \\ **(Amastigotes)** \\ \end{tabular} } \\ \hline  
(8.95\pm 1.98\) & \(4.06\pm 0.63\) & \(3.06\pm 0.53\) & \(>\)630.26 & \(>\)70.42 & 279.17 \\ \hline
**21** & - & - & - & \(305.58\pm 45.63\) & - & - \\ \hline
**22** & - & - & - & \(343.13\pm 121.03\) & - & - \\ \hline
**23** & - & - & - & \(157.78\pm 22.22\) & - & - \\ \hline Benzmidazole & \(161.40\pm 31.80\) & \(115.10\pm 16.32\) & - & \(502.60\pm 57.80\) & 3.11 & - \\ \hline \end{tabular} Legend—the assays were carried out in triplicate (n = 3). All data are reported as mean \(\pm\) standard error mean (SEM). Selectivity index (S.I.) = IC\({}_{50}\) (trypomastigote or amastigotesote forms)/CC\({}_{50}\) (LLC-MK2).  
\end{table} 
Table 1: Cell viability and trypanocidal activity of 3,4,5-trimethoxybenzoic acid derivatives against _Trypamosoma cruzi_ (trypomastigote, epimastigote, and amastigotes forms). Flow cytometry assays with Rho 123 (rhodamine 123) were also performed to evaluate the mitochondrial transmembrane potential of the epimastigotes treated with the derivatives. As shown in Figure 3, treatment with the compounds at both concentrations was able to markedly reduce epimastigote mitochondrial capacity. The derivatives that presented the best reductions were compounds **15**, **17**, and **20** at 36%, 37%, and 43%, respectively. Modeling studies were performed to investigate the possible trypanicidal mechanism of action of compound **17**. Initially, potential targets for the compound were identified using a homology-based target fishing approach. Then, the compound was docked to each of its predicted targets to produce binding hypotheses. The free energies of binding are finally predicted for these protein-compound **17** complexes using an approach that bases on MD simulations. These MD-derived energies of binding are the criteria for prioritizing the possible mechanism of action of the compound.