Alzheimer's disease (AD) is a common neurodegenerative disorder among the elderly, and BuChE has emerged as a potential therapeutic target. In this study, we reported the development of compound 8e, a selective reversible BuChE inhibitor (eqBuChE IC₅₀ = 0.049 μmol/L, huBuChE IC₅₀ = 0.066 μmol/L), identified through extensive virtual screening and lead optimization. Compound 8e demonstrated favorable blood-brain barrier permeability, good drug-likeness property and pronounced neuroprotective efficacy. Additionally, 8e exhibited significant therapeutic effects in zebrafish AD models and scopolamine-induced cognitive impairments in mice. Further, 8e significantly improved cognitive function in APP/PS1 transgenic mice. Proteomics analysis demonstrated that 8e markedly elevated the expression levels of very low-density lipoprotein receptor (VLDLR), offering valuable insights into its potential modulation of the Reelin-mediated signaling pathway. Thus, compound 8e emerges as a novel and potent BuChE inhibitor for the treatment of AD, with significant implications for further exploration into its mechanisms of action and therapeutic applications.

OBJECTIVE： To design and synthesize N-aroyl substituted indoline-3-acetic acid derivatives and evaluate their in vitro hypoglycemic activity. METHODS： Using indoline derivative 2-[5-（benzyloxy）-1-（4-chlorobenzoyl）-2-methyl-1H-inclol-3-yl]acetic acid （GY3） as leading compound， 4-（benzyloxy）phenyl hydrazine hydrochloride and methyl 4-oxopentanoate as raw material， 8 kinds of N-aroyl （3-hydroxybenzoyl， 3-cyanobenzoyl， 4-nitrobenzoyl， 4-methylsulfonylbenzoyl， 4-acetamidobenzoyl， 3-acetylaminobenzoyl， isoniacyl and pyridine-2-formyl） substituted indoline-3-acetic acid derivatives were synthesized via 4 steps reactions： Fischer indole cyclization， reduction， amidation and hydrolyzation. The human hepatoma HepG2 cell lines were used to investigate the glucose consumption activity of the target compounds. RESULTS： Totally 8 various N-aroyl substituted indoline-3-acetic acids were synthesized and their structures were confirmed by mass spectrum（MS）， nuclear magnetic resonance 1H-NMR and 13C spectrum. Under the condition of 1.0 μmol/L， the percentage of glucose- promoting consumption of the synthesized compounds on HepG2 cells was 5.4％-9.1％. 2-[（2R， 3S）-5-benzyloxy-2-methyl-1-（4-methylsulfonyl benzoyl）-2，3-dihydro-indole-3-yl] acetic acid showed the best hypoglycemic activity. The percentage of glucose- promoting consumption was （9.1±1.81）％， which was close to that of positive control metformin [（10.58±1.68）％]， but less potent than that of leading compound GY3[（12.15±0.78）％]. CONCLUSIONS： Different electron-withdrawing substituents are introduced into N-aroyl aromatic rings of dihydroindole compounds， such as cyano， nitro， methyl sulfonyl； hypoglycemic activity decreases in varying degrees and is weaker than halogen substituents.

OBJECTIVE： To compare plasma protein binding rate of cajanonic acid A with different species of plasma. METHODS：Using UPLC-MS/MS as the detection means. Plasma protein binding rate of low， medium and high concentrations of cajanonic acid A （2.5， 5， 20 μg/mL） with rats， rabbits and human plasma were determined by ultrafiltration method. The chromatographic conditions included that Waters BEH C18 as chromatographic column， WatersVanGuard BEH C18 as guard column， mobile phase consisted of ultrapure water solution containing 0.01％ formic acid （solvent A） and acetonitrile solution of 0.01％ formic acid （solvent B） gradient elution， at the flow rate of 0.15 mL/min， column temperature of 30 ℃， sample size of 2 μL. Mass spectrum condition included that ESI， negative ion mode acquisition， capillary voltage of 1.5 kV， cone voltage of 30 V， ion source temperature of 100 ℃， desolvent gas temperature of 400 ℃， cone gas flow of 50 L/h， desolvent gas flow of 800 L/h， scanning range of m/z 50→1 200. RESULTS： At the concentration of 2.5， 5 and 20 μg/mL， the plasma protein binding rates of cajanonic acid A were （75.63±0.90）％， （98.30±0.03）％ and （99.42±0.01）％ in the rats plasma； （79.61±1.08）％， （98.48±0.10）％ and （99.42±0.03）％ in rabbits plasma （n＝3）； （76.74±1.22）％， （97.99±0.11）％ and （99.37±0.01）％ in human plasma （n＝3）. At the concentration of 2.5 μg/mL， plasma protein binding rates of cajanonic acid A in plasma of rats and human were significantly lower than that in plasma of rabbits （P＜0.05）. CONCLUSIONS： The plasma protein binding rate of 5，20 μg/mL cajanonic acid A with rats， rabbits and human plasma are higher than that of 2.5 μg/mL cajanonic acid A. There is significant difference in plasma protein binding rate of 2.5 μg/mL cajanonic acid A with different species of plasma，and there is no significant difference in plasma protein binding rate of 5， 20 μg/mL cajanonic acid A with different species of plasma.

OBJECTIVE： To establish a determination method for the concentration of cajanonic acid A （CAA） in liver microsome incubation system， and to compare the metabolism characteristics of it in different species of liver microsomes. METHODS： CAA was dissolved in liver microsome incubation system of rat， Beagle dog and human initiated by reduced nicotinamide adenine dinucleotide phosphate （NADPH）， and was incubated in water at 37 ℃. The reaction was terminated with acetonitrile at 0， 5， 10， 15， 30， 45 and 60 min， respectively. Using genistein as internal standard， the concentration of CAA in  different incubation systems was determined by UPLC-MS/MS. The determination was performed on Waters BEH C18 column with mobile phase consisted of water （containing 0.1％ formic acid）-acetonitrile （containing 0.1％ formic acid） （45 ∶ 55， V/V） at the flow rate of 0.25 mL/min. The column temperature was 30 ℃， and the sample size was 2 μL. The electrospray ionization source was used to the select reaction monitoring mode for negative ion scanning. The ion pairs for quantitative analysis were m/z 353.14→309.11 （CAA）， m/z 269.86→224.11 （internal standard） respectively. The residual percentage and enzymatic kinetic parameters of CAA in different incubation systems were calculated according to the mass concentration of CAA at 0 min. RESULTS： The linear range of CAA was 0.05-20 μg/mL； the limit of quanti- tation was 0.05 μg/mL， and the lowest detection limit was 0.01 μg/mL. RSDs of intra-day and inter-day were lower than 10％； relative errors ranged －4.83％-8.94％； extraction method and matrix effect did not affect the determination of the substance to be measured. At 60 min of incubation， residual percentages of CAA in rat， Beagle dog and human liver microsomes were（62.79±9.99）％，（64.07±11.59）％，（96.66±5.71）％， respectively. The half-life period （72.19， 68.61 min） of CAA in rat and Beagle dog liver microsomes were significantly shorter than human liver microsome （364.74 min）. The clearance rates [0.019 2， 0.020 2 mL/（min·mg）] were significantly higher than human liver microsome [0.003 8 mL/（min·mg）] （P＜0.05）. CONCLUSIONS： Established UPLC-MS/MS method is simple， rapid， specific and sensitive， and can be used for the determination of CAA concentration in liver microsome incubation system and the study of metabolism stability in vitro. The stability of CAA metabolism in rat and Beagle dog liver microsomes are poorer than human liver microsome.