Synthesis And Biological Activities Of 3,4-Pyranocoumarins

With the aging of the global population,the patients suffering Alzheimer’s disease（AD）is increasing,it has become one of the more serious health problems.Therefore,it is emergent to develop economical,safe and efficient new drugs against AD.The etiology of AD is relatively complex,including cholinergic dysfunction,β-amyloid oligomerization,oxidative stress,Tau protein hyperphosphorylation,etc.Currently,multi-target directed ligand strategy（MTDLs）is mainly used to develop new drugs,which has high development potential.In this study,a series of 3,4-pyranocoumarins compounds were designed by combining the coumarin parent nucleus and pyran ring using the active group splicing strategy.Based on MTDLs,their anti-cholinesterase activities,antioxidant activities and neuroprotective effects on oxidative stress in PC12 cells were measured.The results were as follows:（1）Synthesis of the target compounds:Using substituted phenols as the starting materials,a series of substituted 4-hydroxycoumarins were synthesized by esterification,Fries rearrangement and cyclization reactions.Then they reacted with substituted aromatic aldehydes and malononitrile to successfully prepare forty-four kinds of 3,4-pyranocoumarins with enamine nitrile motifs by Knoevenagel condensation and Michael addition reactions.Their structures were confirmed by ESI-MS,¹H NMR,¹³C NMR.Thirty-two of them were found to be new compounds.（2）In vitro anticholinesterase activity:The Ellman method was selected to screen the in vitro anti-acetylcholinesterase（ACh E）and anti-butyrylcholinesterase（Bu Ch E）activities of these compounds at 0.4 m M.The results showed that all target compounds had different degrees of inhibitory activities against the tested enzymes.The inhibitory activities against Bu Ch E were relatively higher.The IC₅₀ values of nine compounds with higher activity were further determined.E10 and E11 had the best activities with IC₅₀ values of 31.26 and 36.60μM,respectively,which was very close to the IC₅₀ value（21.86μM）of positive control galantamine.（3）The structure-activity relationship of anti-cholinesterase:According to the results of their anti-cholinesterase activity,it can be inferred that the anti-Bu Ch E activity of E10 was the highest.Those compounds with methyl group at 6 position of coumarin and chlorine on D ring owned higher activity.When chlorine is meta,the activity of the corresponding compound E10 was higher than that of para-isomer E11.（4）The mechanism of anti-cholinesterase:As far as E10,the best activity against Bu Ch E,was concerned,its enzyme kinetic test and molecular docking experiment were carried out.The objective was to explore the mechanism of E10 inhibiting Bu Ch E.Enzyme kinetic test showed that E10 was a competitive inhibitor of Bu Ch E.Molecular docking experiment showed that the hydrogen bond,repulsion ofπ-Alkyl,andπ-πstacking interactions were formed between E10 and Trp82,Asp70,His438.Thus E10 tightly matched with the catalytic center of Bu Ch E and inhibited the activity of Bu Ch E.（5）In vitro antioxidant activity:The antioxidant activity of all compounds was screened by DPPH and ABTS⁺method.The scavenging rates of the target compounds for ABTS⁺were higher than those of DPPH.E8,E17,E26,E35 and I7 had higher free radical scavenging ability,the hydroxyl and methoxy groups of D ring with could improve their antioxidant activity.The activity order of them was E35>E17>E26>E8>I7,consequently,the antioxidant order of the substituents on coumarins was 6-OCH₃>6-CH₃>7-CH₃>H>Ph.（6）Neuroprotective activity:E10&E11 with the higher activity against Bu Ch E and E17&E35 with the higher antioxidant activity were chosen to screen the neuroprotective activities against PC12 cells.The results of MTT assay showed that they could significantly reduce the damage of H₂O₂ on PC12 cells.At the concentration of 12.5–100μM,E35 had the highest neuroprotective effect,therefore it should be a potent neuroprotective agent.In conclusion,we have successfully screened two kinds of selective inhibitors of Bu Ch E and two types of antioxidants with neuroprotective effects,which laid the theoretical foundation for the development of anti-AD drugs with multitarget.