# Synthesis of Novel Chalcones as Acetylcholinesteraseerase Inhibitors
## 2 Materials and Methods
### Molecular Modeling Study  
In an attempt to propose a binding mode for benzylaminochalcones to their target enzyme, molecular modeling was carried out by software packages including Molecular Operating Environment (MOE) [27], BiosolveIT LeadIT 2.1.1 [28] and Sybyl X-2.0 [29].  
The Protein Data Bank crystallographic structure of _Tc_AChE (\(-\))-Galanthamine complex (PDB code ID: 1 DX6) [30; 31] was used as a receptor model in this study. The 3D structure of the crystallographic complex was rendered by BioSolveIT LeadIT. The active site was defined as all the important amino acid residues enclosed within 6.5 A radius sphere centered by the bound ligand, Galanthamine. All unbound water molecules were eliminated and the structures of amino acid residues were checked before re-establishing the active site of the enzyme.  
The 2D and 3D chemical structures of 10 benzylaminochalcones were built by ChemBioOffice 2008 [32]. The structures of molecules were optimized using the energy minimization and molecular dynamic modules in SYBYL-X 2.0. In the energy minimization process, Conj Grad method was chosen and the structures of molecules were optimized until converged to a minimum energy change of 0.001 kcal.mol\({}^{-1}\). Gasteiger-Huckel charges were assigned to the structure atoms and the maximum number of iterations to perform during minimization was set to 10,000. Molecular dynamic process was proceeded to obtain conformations with the minimum global energy. The method used in this process was Simulated Annealing. In this method, the molecules were heated at 700 K in a period of 1000 femtoseconds, then they were cooled down to 200 K in another period of 1000 femtoseconds to come to the stable states from which their final conformations were obtained. The process was run in five cycles to figure out different necessary structures. Finally, the energy minimization process was performed one more time and the steric energy of final conformations was specified [33; 34].  
Initially, docking was performed with the co-crystallized AChE: (\(-\))-Galanthamine.which was re-prepared in SYBYL-X 2.0. The RMSD value between re-docked conformation and the original structure, which was \(\leqslant\) 1.5A, indicated the reliability of the binding ability prediction of new ligands. Docking process was utilized BioSolveIT LeadIT with the following options: The method in which base fragment placed in binding pocket was Triangle Matching; the maximum number of solutions per iteration was set to 1000; the maximum number of solutions per fragmentation was set to 200; the  
\begin{table}
\begin{tabular}{l c c c c c} \hline \hline \multirow{2}{*}{**Samples**} & \multicolumn{6}{c}{**Compositions**} \\ \cline{2-6}  & **ATCI** & **DTNB** & **Tris-buffer pH8** & **Chalcon** & **AChE** \\ \hline Control blank sample with enzyme & + & + & + & - & + \\ Blank sample & + & + & + & - & - \\ Test sample & + & + & + & + & + \\ Blank test sample & + & + & + & + & - \\ \hline \hline \end{tabular} (+): present; (-): absent.  
\end{table}
Table 1: Compositions of sample solutions.  
number of poses to keep for further analysis of interaction was set to 10. The best conformation is the one that has the most minus docking score [33; 34; 35].  
The interactions between chalcone molecules and their target were rendered and analyzed in MOE 2008.10 such as hydrogen bonds, \(\pi\)-\(\pi\) interaction, cation-\(\pi\) interaction, ionic interaction. Van der Waals surface interactions were detected by the contact of hydrophilic and lipophilic surfaces of the ligands with those of binding points.