Design And Synthesis Of Tacrine-TPA Derivatives,Evaluation Of Their Anti-AD Activity And Fluorescence Imaging Analysis

Objective:Alzheimer’s disease（AD）is a multifactorial progressive neurological disease that occurs in the elderly,seriously impairs the health and quality of life of the elderly,and increases a huge burden on families and society.The pathogenesis of AD is not clear,and it has been reported that loss of cholinergic transmission is one of the most important causes of AD.Most of the drugs currently on the market are anticholinesterase inhibitors,such as donepezil,rivastigmine,tacrine,and galantamine.As an effective anticholinesterase drug,tacrine was introduced to the market in 1945,which increases the level of acetylcholine in the cerebral cortex by inhibiting the activity of acetylcholinesterase.However,tacrineis limited by its various side effects due to its hepatotoxicity and has now been withdrawn from the market.However,there are still a large number of studies on tacrine derivatives in recent years,and attempts have been made to modify and modify tacrine to achieve the purpose of reducing tacrine toxicity,while maintaining or even improving its anti-AD activity.At the same time,most of the research reports have been devoted to improving the therapeutic effect of tacrine,while the exploration of its mechanism of action has rarely been reported.As one of the essential tools to study biomolecules,signaling pathways and biological reactions,fluorescence imaging can track the physiological and biochemical processes of organisms in real time,non-invasively and dynamically.In addition to its large steric hindrance,triphenylamine（TPA）is widely used in molecular recognition,molecular imaging and other fields because of its superconjugate electron effect and aggregation-induced luminescence（AIE）characteristics.In view of this,we envision starting from tacrine,a series of tacrine-triphenylamine（Tacrine-TPA）derivatives with both AD therapeutic efficacy and fluorescence imaging function were synthesized by connecting the triphenylamine backbone.Therefore,this thesis focuses on the design and synthesis of Tacrine-TPA derivatives,anti-AD activity evaluation and fluorescence imaging to carry out related work,laying the foundation forthe subsequent development of AD drugs with tracerfunction.Methods:Intermediate 1（tacrine）was synthesized from o-aminobenzonitrile and cyclohexanone,and intermediate 2 was obtained after acylation.Intermediate 3 was obtained by formylation with triphenylamine and phosphorus oxychloride,then brominated with N-bromosuccinimide to obtain intermediate 4,and then Suzuki coupling reaction with 2-chloro-4-pyridylboronic acid to obtain intermediate 5.Starting from intermediates 4 and 5,respectively,intermediates 6-9 and 10-13 were obtained by Suzuki coupling reaction.Intermediates 6,8 and 9 were stripped of Boc group by trifluoroacetic acid under acidic conditions,and then reacted with intermediate 2 under the action of triethylamine to obtain target compounds P1,P18 and P19.Intermediate 10,12,13 and intermediate 2give P28,P29,P27,respectively.Intermediate 7 and 11 react with intermediate 2 under the action of potassium carbonate to obtain target compounds P3 and P26,respectively.The synthesis process of the above compounds was monitored by thin layer chromatography（TLC）,then separated and purified by column chromatography,and finally their structures were characterized and determined by nuclear magnetic resonance（NMR）and mass spectrometry（MS）.After preparing the above target compounds,Ellman spectrophotometry was used to detect the effects of the target compounds on the activities of AChE and Bu Ch E,and then cell culture and CCK-8 experiments were used to investigate the biosafety and neuroprotective effects of the target compounds,and finallythe photophysical properties and cellular imaging ability of the target compounds were determined by means of fluorescencespectroscopy andconfocal imaging.Results:（1）In the design and synthesis of Tacrine-TPA derivatives:Eight target compounds have been prepared,which are P1,P3,P18,P19,P26,P27,P28,P29,and the corresponding structural characterization and determination have been completed;（2）In the evaluation of anti-AD activity of Tacrine-TPAderivatives:（1）The results of cytotoxicity and neuroprotection experiments showed that Tacrine-TPAderivatives P1,P3,P26,and P28 had good biosafety,and also showed protective effects against H₂O₂-induced neural PC12 cell injury,in which Tacrine-TPA derivatives P1,P3,P19,P26,P27,and P29 alleviated H₂O₂-induced neural PC12 cell injury at certain concentrations;（2）The results of cytotoxicity and neuroprotection showed that Tacrine-TPAderivatives P1,P3,P26 and P28had good biosafety,and also showed inhibitory effect on ROS secretion in H₂O₂-induced PC12 injury model of neural cells,in which Tacrine-TPA derivatives P1,P3,P19,P26,P27 and P29 could down-regulate ROS levels at certain concentrations;（3）The fluorescence characteristic analysis showed that these 8 target compounds had good fluorescence properties,and P1,P26 and P28selected by anti-AD activity evaluation could change fluorescencebefore and afterrecognizing AChE,and had good fluorescenceimaging ability.Conclusion:（1）Tacrine-TPA derivatives P1,P19 and P26 showed better anti-AChE activity from the anti-AD activity index,while Tacrine-TPA derivative P28 had dual inhibition of AChE and Bu Ch E;（2）According to the results of cytotoxicity and neuroprotection experiments,P1 and P26 have good biosafety and reduce H₂O₂-induced neural PC12 cell injury,and can perform fluorescence imaging of neural PC12 cells.In summary,we believe that Tacrine-TPAderivatives P1,P26,and P28 have great potential inthe treatment and tracing of AD.