## 3 Results and Discussion
### Molecular Docking Analysis  
For the molecular docking studies, the compounds with the best in vitro results 2d and 3d were selected in order to gain insight of their interactions with the AChE receptor and explain their biological results. AutoDock Vina was selected to perform the in silico analysis, using the crystallographic structure of the Pacific electric ray _Torpedo californica_ AChE (TcAChE) available from Protein Data Bank (PDB ID:1 EVE) as its similarity with human AChE allows for employing it in docking studies [58]. Having selected 1 EVE, a docking grid was generated in the active site region defined by the co-crystallized receptor's ligand donepezil. To assure effectiveness of the analysis, it was necessary to validate the method; for this, a re-docking of the co-crystallized donepezil was realized with the same parameters intended to use for our ligands. We compared the best result pose for donepezil with the co-crystallized one obtaining a RMSD of 1.1, which, being lower than 1.5-2 A, is considered to be a successful analysis, suitable for predicting ligand poses [59]. In Table 3, the resulting scores of the docking for compounds 2d and 3d with the inhibition constants can be observed, which AutoDock calculates by the formula:  
\[\text{Ki}=\exp(\Delta\text{G}\times 1000\text{ Rcal}\times\text{TK}\,) \tag{5}\]  
\(\Delta\text{G}\) is the docking binding energy, Rcal value is 1.98719, and T\({}_{\text{K}}\) value is 298.15. In the in vitro results, chalcone 2d was the most active synthesized compound with an IC\({}_{50}\) of 21.5 ug/mL, followed by the flavone 3d (26.8 ug/mL); accordingly, the docking gave a better score for 2d (\(-\)10.2 kcal/mol) against 3d (\(-\)9.78 kcal/mol). The more negative the docking score (binding energy value), the better affinity the ligand has for the receptor. Analysis of the poses generated showed us that our compounds interact with AChE mostly by \(\pi\)-\(\pi\) interactions with the active site residues. The active site of AChE consists in a cavity or gorge in the receptor surface that is approximately 20 A deep; besides the catalytic triad of the enzyme (Ser200, His440, and Glu327), which is located at the bottom of the gorge, several residues have been identified as important zones for the interaction of ligands, such as Trp84, Phe330, Glu199, and Gly441 that make the central anionic site (CAS), and \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Compound**} & **Binding Energy** & **Inhibition Constant** & **Principal Residues** \\  & **(kcal/mol)** & **(nM)** & **Interactions** \\ \hline
**2d** & \(-\)10.2 & 33.37 & Trp84, Tyr334, Tyr130 \\
**3d** & \(-\)9.78 & 67.79 & Trp84, Tyr334, Tyr130 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Docking scores for compounds 2d and 3d. Trp279, Tyr34, Tyr121, and Tyr70 at the peripheral anionic site (PAS) [60; 61]. Both **2d** and **3d** poses are observed nearer to the CAS region, as seen in the Figure 3.  
The best pose generated for **2d** does not form hydrogen bonds to the nearest residues; although, one oxygen of the nitro group is near the hydrogen of Tyr130, at only 2.7 A, the angle between them does not allow a proper hydrogen bond. A small turn of the nitro or hydroxyl groups would be needed for this to happen, but a weak interaction between them might be helping the interaction of **2d** with this residue. The clearer interactions for **2d** appear with Trp84, a residue that has been reported as an important one for ligand interactions (especially with quaternary groups from acetylcholine and other compounds) [58; 62]. In this case, Trp84 is near the positively charged nitrogen of the nitro group, although its position appears to be almost over the B ring of the chalcone, allowing for \(\pi\)-\(\pi\) stacking at 3.7 A. Another \(\pi\)-\(\pi\) stacking can be observed between the A ring of **2d** and Tyr334 in the PAS region, with 4 A among the aromatic rings (Figure 4).