*2.4. Molecular Docking Studies*

Inhibition of NO overproduction is usually the result of inhibition of iNOS enzyme expression or activity [27,28]. In order to investigate the inhibitory mechanism of compound **2** on NO production, the interaction and binding mode between compound **2** and iNOS (PDB: 3E6T) [29], molecular docking study was carried out using AUTODOCK 4.2.6 modeling software. Docking procedure was validated by docking of ligand indomethacin (positive drug) in the active site of iNOS, and root-mean square deviation (RMSD) of 0.12 Å to the X-ray structure. The results revealed that the lowest energy of compound **2** (−7.49 Kcal/mol) was lower than that of positive drug indomethacin (−7.45 Kcal/mol), and the lowest energy of compound **1** (−7.36 Kcal/mol) was higher than that of positive drug. Further observations showed indomethacin formed a hydrogen bond with key amino acid residues GLU-371, two hydrogen bonds with amino acid residues ARG-260, and a hydrogen bond with GLN-257 in the iNOS active pocket (Figure 6A). Compound **2** formed a hydrogen bond with the key amino acid residue GLU-371 through the methoxy group, two hydrogen bonds with the residue ASP-379 and ARG-382 by the hydroxyl group in the iNOS active pocket (Figure 6B), respectively. Notably, for compound **1**, while the carbonyl group in compound **2** at the 4' position changed to the hydroxyl group, the optimized conformation of **1** was different from that of compound **2** and could not enter into the iNOS active pocket to form hydrogen bonds with the key amino acid residues (Figure 6C). As a result, compound **1** had no production inhibition activity (Table 3).

**Figure 6.** (**A**) Docking results of the binding pose of positive drug indomethacin in iNOS. (**B**) Predicted binding mode of compound **2** docked into iNOS (PDB: 3E6T). (**C**) Predicted binding mode of compound **1** docked into iNOS. (PDB: 3E6T) (indomethacin is in green stick; compound **2** is in purple stick; compound **1** is in yellow stick; red dashed lines represent H-bonds; the amino acids involved in hydrogen bond interactions are in blue stick).
