Design,synthesis,and Biological Evaluation Of Novel（4-（1,2,4-oxadiazol-5-yl）phenyl）-2-aminoacetamide Derivatives

Alzheimer’s disease（AD）is a type of senile dementia.It is characterized by a progressive and irreversible decline in memory and cognitive functions due to neuronal dysfunction.At present,the exact pathogenesis of the disease remains unknown,but several hypotheses have been proposed,including the cholinergic hypothesis,neuroinflammation,oxidative stress,aggregation of neurotoxic amyloid beta（Aβ）,etc.Among them,the cholinergic hypothesis is currently the most prominent hypothesis in AD,and the inhibition of cholinesterase（Ch Es）has been proved to be a key target for the effective treatment of AD,including acetylcholinesterase（ACh E）and butyrocholinesterase（BuChE）.Studies have found that in healthy brains,ACh E hydrolyzes the main component of acetylcholine（ACh）,but in brains affected by AD,the level of ACh E is significantly reduced and BuChE activity increases,thereby aggravating the toxicity of Aβ.In addition,selective ACh E inhibitors have some undesirable side effects that limit their doses for clinical use.Therefore,the development of selective BuChE inhibitors is an important way to treat AD and the main side effects of ACh E inhibitors would not be reproduced.Moreover,due to the complex mechanisms of AD,it is difficult to reduce to a single therapeutic factor,involving multiple relevant etiological targets that will affect a wide range of pathologies and symptoms.A method for rationally designing new drug candidates,the multi-target directed ligand（MTDLs）strategy,plays an important role in the effective treatment of multifactorial disease progression such as AD.The MTDLs obtained according to this method can simultaneously modulate multiple biological targets with higher efficacy,better safety,and simpler administration.In this study,a novel series of（4-（1,2,4-oxadiazol-5-yl）phenyl）-2-aminoacetamide derivatives were synthesized and evaluated as multifunctional ligands for the treatment of AD.Biological evaluations indicated that the derivatives can be used as anti-AD drugs that have multifunctional properties,including inhibiting BuChE activity,inhibiting neuroinflammation,and exhibiting neuroprotective effects.Among them,f9 is considered to be the most potential compound,it can selectively inhibit the activity of BuChE(IC₅₀:1.21±0.19μM),is a mixed-type inhibition（the dissociation constant Ki is 0.61μM）.It also has anti-neuroinflammatory effect,can significantly inhibited the production of NO,IL-1βand TNF-αin LPS-induced BV2 cells with IC₅₀ of 0.67±0.14,1.61±0.21,4.15±0.44μM,respectively.In neuroprotection experiments,the compound f9 showed the most significant neuroprotective effect（66.4±1.8%）on H₂O₂-stimulated PC12 cells at 20μM.Next,we found that compound f9 could inhibit Aβself-aggregation（52.19±2.24%）by thioflavin T（Th T）fluorescence analysis,and the inhibitory activity of the compound on Aβaggregation was further analyzed by transmission electron microscopy（TEM）.In addition,preliminary anti-inflammatory mechanism studies indicated that the representative compound f9 blocked the activation of the NF-κB signaling pathway in a concentration-dependent manner,and could reduce the expressions of inflammatory factors COX-2 and i NOS in LPS-induced BV2 cells.Moreover,f9 exhibited 1,1-Diphenyl-2-picrylhydrazyl（DPPH）radical scavenging effect,and inhibited H₂O₂-stimulated intracellular reactive oxygen species（ROS）production in SY5Y and PC12cells.The reduction in maximum absorption intensity at 271 nm and the appearance of a shoulder at 315 nm upon the addition of Cu Cl₂ solution to the compound f9 solution,which indicates that it can form a chelate with Cu²⁺.In the bi-directional transport assay,f9 displayed proper blood-brain barrier（BBB）permeability.Finally,in vivo experiments,the title compound improved memory and cognitive functions in a mouse model induced by scopolamine.Hence,the compound f9 can be considered as a promising lead compound for further investigation in the treatment of AD.