Inhibiting the activity of AChE can maintain a proper level of ACh under dysregulated cholinergic conditions. Thus, the inhibitory capability of 1-6 against AChE was investigated by a previously reported fluorescent AChE assay with slight modifications. As summarized in Fig. , the IC50 values of 2-6 were in a micromolar range (IC50 = 1.40-12.34 µM). It should be noted that the IC50 value of 1 could not be obtained due to its negligible AChE inhibition under our experimental conditions. As expected, 4 and 6 that contain three hydroxyl groups on the A ring among our molecules exhibited relatively low IC50 values. The inclusion of three hydroxyl groups on the A ring is critical for controlling the catalytic activity of AChE, as previously reported, whereas the incorporation of a chloro substituent on the A ring, with three hydroxyl groups, can decline such inhibitory activity. The noticeable AChE inhibition by 2 and 3 over 1 illustrates the importance of the position of hydroxyl moieties on the A ring in directing compounds' activity against AChE. Docking studies were further conducted to visualize potential interactions between the flavonoids (1-6) and AChE employing an X-ray crystal structure of electrophorus electricus AChE (PDB 1C2B ). As displayed in Figs. and, the flavonoids can interact with neighboring amino acid residues that lie in the active site of AChE via hydrogen bonding. For example, hydroxyl Hydrogen bonds between compounds and AChE are indicated with dashed lines (2.2-3.5 Å). N, O, H, and C (from Ser203) atoms are depicted in blue, red, white, and green, respectively. groups on the A ring could serve as a hydrogen-bond donor to interact with the backbone amide group between Gly121 and Gly122 (for 1 and 2), the backbone carbonyl moiety of Gly120-Gly121 (for 3 and 6), and the hydroxyl group of Ser203 (for 6) in AChE. In addition, hydrogen bonds mediated by the central O donor atom and the 4-oxo functionality on the C ring with Tyr337/Tyr341 (for 1 and 2), Gly122/Tyr124 (for 3), Tyr124/Ser203 (for 4), Tyr124 (for 5), and Tyr337 (for 6) were also monitored. Anchoring the flavonoids into the active site gorge of AChE could prevent the substrate binding and, ultimately, inhibit the activity of AChE. In particular, hydrogen bonds between Ser203 at the catalytic triad and the 4-oxo functionality on the C ring of 4 or the hydroxyl group on the A ring of 6 were indicated within 3.1 Å and 2.3 Å, respectively. The Ser203 residue is responsible for initiating the hydrolysis of ACh through hydrogen bonding; 51 thus, the intermolecular interaction with Ser203 could dominantly restrict ACh from binding into the active site of AChE. Overall, these observations highlight the pertinent role of some structural features, such as hydroxyl groups on the A ring and the C ring that contains two O donor atoms, in controlling the catalytic activity of AChE. It should be noted that the p-p stacking between the flavonoids' chromone moiety (A and C rings) and amino acid residues at the anionic subsite of AChE could afford the stability for their positioning at the active site gorge of AChE, 36 but our docking studies were not able to visualize such p-p stacking interactions.