Structural Modification Of Dual Binding Site AChE Inhibitors Based On Complex Crystal Structures

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by symptoms like progressive memory loss, mental decline, language disorders, behavioral disorders and even loss of consciousness. The specifical pathogenesis of Alzheimer’s disease is still unclear, but ’β-amyloid (Aβ) deposition’ and ’abnormal phosphorylation of tau protein’ are considered to be two important mechanisms leading to the onset of AD, and ’cholinergic nerve damage’ is regarded as the major pathological factor of AD patients’ cognitive and behavioral disorders.Currently most drugs used for the treatment of Alzheimer’s disease are acetylcholinesterase (AChE) inhibitors. AChE inhibitors can only relieve the symptoms instead of completely curing or altering the course of AD pathology. Current research of AChE inhibitors focuses on safer or multifunctional AChE inhibitors.This study was based on a dual binding site AChE Inhibitor XQ509, which showed poor oral bioavailability and relatively low Aβ aggregation inhibiting activity. Three series of target compounds were designed and synthesized based on the crystal structure information of XQ509-AChE complex with the help of computer-aided drug design software. The first series of compounds replace all-carbon-substituted linking chain of XQ509 with a polar group or a heteroatom in order to improve the oral bioavailability. The second and third series of compounds connect meptazinol and donepezil indanone structure or phthalimide structure with different length chains in order to improve Aβ aggregation inhibiting activity.Target compounds were initially tested the inhibitory activity of AChE. Results of the first series of compounds indicated that substitution of a polar group or a heteroatom on the linking chain has a great influence on the activity. The linker may need flexibility to achieve optimal binding conformation, so heteroatom substituted at certain locations may cause disturbance of the molecules to be reversed to the optimal conformation and lead to decrease in the activity. Compounds 1b, 1e of the first series showed the best AChE inhibitory activity (IC50= 4.4 nM and 4.3 nM, respectively), which was at the same level as XQ509 (IC50= 5.7 nM). Results of the second series of compounds indicated that the activity of indanone compounds was closely related to their linking chain length, and the optimum length was 5-6. Compounds 2d,2e of the second series had the best AChE inhibitory activity (IC50= 12 nM and 14 nM, respectively), which was superior to donepezil (IC50= 55 nM). Results of the third series of compounds indicated that phthalimide compounds were less active.The active compounds (1b, 1e,2d,2e) for AChE inhibition were submitted to AChE-induced Aβ aggregation tests. No compound showed higher activity in inhibiting Aβ aggregation than XQ.509. Molecular docking studies and conformational analysis revealed the binding modes of compounds in peripheral active site (PAS) varied greatly and their interactios were not strong enough. The most potent compound 1b showed an inhibition rate of 44.8% against Aβ aggregation at the concentration of 50 μM, which was lower than XQ509 (58.4%).Target compounds were calculated ADME/T profiles using CADD software, which indicated good solubility, absorption and BBB permeability. However, in vivo oral bioavailability tests in rats demonstrated that no improvement in oral absorption was found by compounds (1b, 1e,2d,2e). Most of them showed reduced bioavailability, especially for the indanone compound 2d. The most bioavailable compound 1b had an absolute oral bioavailability of 5.54%, slightly lower than that of XQ509 (F= 8.40%). Further tests for the liver microsome stability showed that these compounds were relatively metabolically unstable. This might be another reason for their relative low bioavailabilities.To be concluded, 1b was the most potential target compound, which showed very similar in vitro activities of AChE inhibition, Aβ aggregation inhibition, metabolic stability, and even oral bioavailability, as compared with XQ509. The substitution of a polar hydroxyl group might improve the drug distribution, therefore 1b deserved further studies.