Study On The Design,synthesis And Biological Activities Of Multifunctional Compounds To Combat Alzheimer’s Disease

Alzheimer’s disease（AD）is a typical brain disease which is characterized with cognitive impairment and memory loss.Over time,this disease will turn worsen and remarkably disturb patients’daily life.There exist about 50 million people suffering from AD in the world and the number is estimated to increase to 152 million by 2050,which is three times as much as it is now.Undoubtedly,AD has burdened the social society and families,and it is imperative to develop therapeutical strategies.AD shares multi-pathological features,including metal ion dyshomeostasis,loss of neurons,homeostasis disorder,proteopathy,oxidative stress,and neurotransmitter deficiencies.Usually,these promotors interrelate and interact with each other in the process of AD.Considering the complex pathogenesis,it is considered to be an effective strategy to develop multifunctional compounds that interact with multiple pathological factors at the same time.Besides,in view of the specific heterogeneity of AD,we pursuit to design and discover potential multi-functional compounds to combat with AD,hoping to treat AD at multi-levels and pave a new way for the development of anti-AD drugs.Firstly,we designed and synthesized a series of reactive oxygen species（ROS）responsive prodrugs based on the strategies of multi-target directed ligands（MTDLs）.In vitro cytotoxicity assays showed that all target compounds obtained good biocompatibility and significantly protected neurons against the cytotoxicity induced by H₂O₂.It was noted in the HPLC chromatograms and ¹H-NMR spectra that 2-22could instantaneously release tacrine derivative 2-11 and ibuprofen upon exposure to ROS.The results in transwell assays demonstrated that 2-22 could effectively penetrate the blood brain barrier（BBB）,and easily entered into SH-SY5Y cells and BV-2 cells.Further biological studies revealed that 2-22 exserted little inhibitory activity on ee ACh E（only 47.25%activity inhibition rate of ee ACh E at 10μM）,while the IC₅₀ value of ee ACh E was significantly increased to 39.16 n M upon stimulation of H₂O₂.In addition,the release of 2-22 can effectively consume H₂O₂,reducing intracellular oxidative stress,protecting neurons against cell damage induced by H₂O₂and regulating the expression of mitochondrial related apoptotic proteins（Bax,Bcl-2,cleaved caspase 3）.More importantly,2-22 significantly down-regulated expression of LPS-induced proinflammatory cytokines（IL-1β,TNF-?）in BV-2 cells to normal levels and showed moderate hepatotoxicity as compared with tacrine at the same concentration of 50?M,indicating the safety of 2-22 for AD treatment.Finally,the results of in vivo assays revealed that 2-22 apparently enhanced the learning and memory function in Aβ-induced AD models,which was better than tacrine even at the same dose.Secondly,we constructed donepezil-loaded organic nanoparticles（regadenoson-PEG2000-YC-1-NO donor nanoparticles,Reg NPs）,taking advantages of the regulatory effects of NO donor and YC-1 in SGC/c GMP/p-CREB signaling pathway and the targeted delivery of regadenoson（Reg）in nanomaterials.The results of in vitro cytotoxicity tests showed that Reg-NPs obtained good biocompatibility and promoted proliferation at the concentration ranging from 8 to 50?g/m L.Besides,Reg-NPs significantly protected neurons against oxygen glucose deprivation（OGD）-induced apoptosis at the concentration ranged from 16 to 50μg/m L.In addition,Reg-NPs can permeate BBB via regulation of tight junctions between endothelial cells,and help Reg-NPs deliver drugs to the brain.Further biological mechanisms showed that Reg-NPs were efficiently absorbed by SH-SY5Y cells,resulting in the sustained release of NO and donepezil upon the stimulation of glutathione（3.0 m M）,to protect neurons and improve the level of choline transmitter.Besides,the released NO and YC-1 synergistically regulated soluble guanylate cyclase（s GC）and phosphodiesterase（PDE）and thereafter activated PKG/PI3K and NO/c GMP/PKG/p-CREB signaling pathways,and ultimately promoted cell growth,survival and memory formation.Moreover,Reg-NPs blocked the mitochondrial-related apoptosis induced by Aβand promote cell survival via up-regulation of pro-apoptotic proteins（Bcl-2,cleaved caspase 3/9）and down-regulation of anti-apoptotic protein（Bax）.Finally,the results of in vivo assays revealed that Reg-NPs can enhance the learning function and memory retention in Aβ-induced AD models,which were significantly better than that of donepezil at the same dose.Finally,we designed and synthesized FA-S-S-Sim functionalized iron oxide nanoparticles（FSSIO）,taking the functional advantages of brain-derived neurotrophic factor（BDNF）,ferulic acid（FA）and simvastatin（SIM）on apoptosis,spatial cognitive impairment and memory impairment induced by Aβ.The in vitro cytotoxicity assays showed that FSSIO significantly stimulated the growth of cells at the concentration ranging from 17.5 to 140?g/m L.At the concentration of 35 or 70?g/m L,FSSIO exhibited neuroprotective effects against the cytotoxicity induced by Aβ.In addition,FSSIO efficiently penetrated BBB and entered into nerve cells,resulting the decrease of intracellular ROS accumulation caused by A?.Further immunofluorescence assays showed that FSSIO stimulated the expression of BDNF in PC12 cells,activated PI3K/Akt and Trk B/ERK signaling pathways,and promoted cell survival.Besides,FSSIO efficiently alleviated the inflammatory response induced by Aβand regulated pro-inflammatory factors（IL-1βand TNF-α）to normal levels.Finally,the results of western blot assays demonstrated that FSSIO apparently regulated the expression of apoptosis related proteins（Bax,Bcl-2,cleaved caspase3/9）,resulting in inhibition of mitochondria-related apoptosis pathway and the depolarization of mitochondrial membrane potential,to protect neurons against cell damage caused by Aβ.