Design,Synthesis And Activity Evaluation Of Scutellarin And Scutellarein Derivatives For The Treatment Of Alzheimer’s Disease Based On Multiple Mechanisms

Objective:In order to develop multifunctional anti-Alzheimer disease（AD）drugs,this thesis designed and synthesized a series of scutellarin and scutellarein-N,N-disubstituted carbamate-L-amino acid derivatives based on multi-target-directed ligand strategies,and evaluated the anti-AD biological activity for the target compounds.Methods:（1）Scutellarein-7-L-amino acid-4′-N,N-disubstituted carbamate:scutellarin was hydrolyzed into scutellarein,which was then treated with dichlorodiphenylmethane in diethylene glycol dimethyl ether（DEME）to provide the 6,7-dihydroxy ketal protection intermediate of scutellarein,the obtained compound was then coupled with N,N-disubstituted carbamoyl chloride,followed by the removal of 6,7-ketal protecting groups in the ethyl acetate system saturated with hydrogen chloride to obtain the intermediate scutellarein-4′-N,N-disubstituted carbamates.L-amino acid tert-butyl ester hydrochlorides was reacted with triphosgene in presence of pyridine and in anhydrous CH₂C1₂ to produce L-amino acid ester isocyanates,which then coupled with scutellarein-4′-N,N-disubstituted carbamates and finally the tert-butyl was removed in trifluoroacetic acid/dichloromethane system to obtain to scutellarein-7-L-amino acid-4′-N,N-disubstituted carbamates（6a-6l）.Scutellarin-6"-L-amino acid ethyl ester-4′-N,N-disubstituted carbamate:Boc-L-amino acid was reacted with2-bromo-ethanol in N,N’-dicyclohexylcarbodiimide（DCC）and 4-dimethylamino pyridine（DMAP）condition to obtain Boc-L-amino acid ethyl bromide,which was reacted with scutellarin in KI/TEA to obtain Boc-protected scutellarin-6"-L-amino acid ethyl bromide,the latter was coupled with N,N dissubstituted carbamyl chloride in K₂CO₃/DMAP condition,and the protecting group was removed by using aqueous trifluoroacetic acid/dichloromethane as a hydrolysis agent to give the scutellarin-6"-L-amino acid ethyl ester-4′-N,N-disubstituted carbamate（10a-10l）.（2）The IC₅₀ values of compounds inhibiting ACh E/Bu Ch E was determined and the inhibition kinetics of ACh E was studied by Ellman method.The antioxidant activity of the compounds was studied by ORAC-FL method and the 6h chelating ability of metal ions was evaluated by UV-Vis spectrophotometry.The inhibition/disaggregation of Aβ_1-42 self-induced and Cu²⁺induced aggregation were studied by thioflavin T method and transmission electron microscopy.The oxidative damage model of PC12 cells induced by H₂O₂ was established,and the antioxidant mechanism of the compounds was investigated by measuring cell viability,LDH activity,ROS and related apoptotic protein expression levels,the damage of PC12cells was induced by GPX4 inhibitor（RSL3）and the effect of the compound on GPX4 was preliminarily evaluated by cell viability.Uptake of 6h by MDCK cells and h Pep T1-MDCK cells was studied by UPLC-MS.In vitro metabolic stability of 6h was studied by incubation with liver microsomes.（3）Scopolamine was injected intraperitoneally at 4 mg/kg to establish a scopolamine-induced memory impairment model in AD mice,and the cognitive function improvement effect of 6h on AD mice was evaluated by Morris water maze experiment,HE staining,and determination of ACh E activity and ACh level in hippocampus.Results:（1）A total of 24 target compounds including 12 scutellarein-7-L-amino acid-4′-N,N-disubstituted carbamates and 12 scutellarin-6"-L-amino acid ethyl ester-4′-N,N-disubstituted carbamates were synthesized with a total yields of 25.8%-34.2%and27.8%-35.5%,respectively.And the structures of the target compounds were confirmed by ¹H/¹³C nuclear magnetic resonance（NMR）spectroscopy,electrospray ionization-mass spectrometry（MS-ESI）as well as high resolution mass spectrometry（HRMS-ESI）analyses,and spectral data were in accordance with the assigned structures.（2）In vitro activity studies showed that compound（6a-6l）with scutellarein as the parent nucleus had better inhibitory activity against acetylcholinesterase（ACh E）,with compound 6h inhibiting ee ACh E(IC₅₀=6.01±1.66μM)and hu ACh E(IC₅₀=7.91±0.49μM)more strongly than rivastigmine(ee ACh E IC₅₀=19.6±1.23μM and hu ACh E IC₅₀=8.95±0.12μM),the type of 6h inhibition ee ACh E and hu ACh E is mixed inhibition.The compound 6h not only inhibited the self-induced and Cu²⁺induced Aβ_1-42 aggregation（89.17%and 86.19%inhibition,respectively）,but also disaggregation the self-induced and Cu²⁺-induced Aβ_1-42 aggregation（85.37%and82.21%,disaggregation respectively）,which was better than the positive control curcumin,and 6h could selectively chelate Cu²⁺and Al³⁺.In addition,compared with scutellarin,6h can significantly improve the viability of PC12 cells,reduce LDH leakage and ROS production,decreased the expression of caspase-3 and Bax proteins,increase the level of Bcl-2,which protected the cells from oxidative damage in many ways,moreover compound 6h has a protective effect on RSL3-induced damage in PC12 cells.Uptake test of h Pep T1-MDCK cell line and metabolic stability of liver microsomes showed that 6h had the highest uptake（15.2 times of scutellarin）and a long half-life.（3）The results of in vivo experiments showed that after 6h treatment,AD mice showed a significant decrease in escape latency,an increase in the number of platform crossing,and an improvement in cognitive function.Further mechanistic studies revealed that 6h may improve learning memory impairment in mice by decreasing acetylcholinesterase activity,increasing acetylcholine levels,and reducing cytopathic damage in hippocampal tissue.Conclusions:The results of in vitro experiments showed that compound 6h has strong ACh E inhibitory activity and antioxidant activity,and also inhibits/depolymerizes the aggregation of Aβ_1-42,and selectively chelates Cu²⁺and Al³⁺and protects cells from oxidative damage,in addition,6h has high uptake in cells and stable hepatic microsomal metabolism.The results of in vivo experiments showed that 6h significantly improved scopolamine-induced cognitive dysfunction in mice,which was a potential multifunctional anti-AD drug.