阅读说明：本技术 黄烷衍生物及其制备和应用 (Flavane derivative and preparation and application thereof ) 是由 宋少江 刘庆博 史绍春 王雨奚 侯子琳 姚国栋 黄肖霄 于 2020-04-30 设计创作，主要内容包括：本发明属于药物化学领域,涉及黄烷衍生物及其制备方法和应用,具体涉及黄烷衍生物及其制备方法和在制备抗老年性痴呆药物中的应用。本发明提供通式(I)、(II)、(III)或(IV)所示的黄烷衍生物、其立体异构体或药学上可接受的盐,其中,R-(1)-R-(6)如权利要求书和说明书所述。本发明所述的黄烷衍生物、其立体异构体或药学上可接受的盐或其药物组合物具有明显的Aβ蛋白,乙酰胆碱酯酶,丁酰胆碱酯酶抑制活性,可以用于制备抗老年性痴呆药物。(The invention belongs to the field of medicinal chemistry, relates to a flavane derivative, a preparation method and application thereof, and particularly relates to the flavane derivative, the preparation method thereof and the application thereof in preparing an anti-senile dementia medicament. The invention provides flavane derivatives represented by general formula (I), (II), (III) or (IV), stereoisomers or pharmaceutically acceptable salts thereof, wherein R 1 ‑R 6 As described in the claims and specification. The flavane derivative, the stereoisomer or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof has obvious A beta protein, acetylcholinesterase and butyrylcholinesterase inhibition activities,can be used for preparing medicine for treating senile dementia.)

1. A flavan derivative represented by the general formula (I), (II), (III) or (IV), a stereoisomer or a pharmaceutically acceptable salt thereof:

in the formula I, R₁、R₂Independently H, OH, halogen, nitro, amino, cyano, C_1-8Alkoxy radical, C₁-C₆Alkyl, halo C₁-C₆Alkyl, halo C₁-C₆Alkoxy, unsubstituted or substituted benzyl or C_1-8Alkanoyl, benzoyl or biphenylacyl; or R₁And R₂Together form a 5-6 membered heterocyclic ring;

in the formula II, R₃Is H, halogen, nitro, halogeno C₁-C₆Alkyl, halo C₁-C₆Alkoxy, substituted or unsubstituted phenyl, benzyl or naphthalene ring, the substituents being halogen, nitro, amino, cyano, C₁-C₆Alkyl radical, C_1-6An alkoxy group;

in the formula III, R₄、R₅Is H, halogen, nitro, amino, cyano, C₁-C₆Alkoxy radical, C₁-C₆An alkyl group;

in the formula IV, R₆Is H, halogen, C₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkyl radical, C_1-6Alkoxy, substituted or unsubstituted 5-to 10-membered aryl,R₇，R₈Is halogen, C₁-C₆Alkyl, halo C₁-C₆An alkyl group.

2. The flavan derivative of claim 1 represented by the general formula (I), (II), (III) or (IV), a stereoisomer or a pharmaceutically acceptable salt thereof:

wherein, in the formula I, R₁、R₂Independently H, OH, halogen, nitro, amino, cyano, C₁-C₄Alkoxy radical, C₁-C₄Alkyl, halo C₁-C₄Alkyl, halo C₁-C₄An alkoxy group; or R₁And R₂Together form a dioxymethylene group;

in the formula II, R₃Is H, halogen, nitro, halogeno-C₁-C₄Alkyl, halo C₁-C₄An alkoxy group;

in the formula III, R₄、R₅Is H, halogen, nitro, amino, cyano, C₁-C₄Alkoxy radical, C₁-C₄An alkyl group;

in the formula IV, R₆Is C₁-C₆Alkyl radical, C_1-6Alkoxy radical,R₇、R₈Is H, halogen, C₁-C₄Alkyl, halo C₁-C₄An alkyl group.

3. A flavan derivative, a stereoisomer or a pharmaceutically acceptable salt thereof as follows:

4. the process for preparing a flavan derivative according to claim 1, a stereoisomer or a pharmaceutically acceptable salt thereof, characterized by comprising the steps of:

5. a pharmaceutical composition comprising a flavan derivative according to any one of claims 1 to 3, a stereoisomer or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

6. Use of the flavan derivative according to any one of claims 1 to 3, a stereoisomer or pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 5 for the preparation of an a β protease inhibitor.

7. Use of the flavan derivative of any one of claims 1 to 3, a stereoisomer or pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 5 for the preparation of an acetylcholinesterase inhibitor.

8. Use of the flavan derivative of any one of claims 1 to 3, a stereoisomer or pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 5 for the preparation of a butyrylcholinesterase inhibitor.

9. Use of a flavan derivative according to any one of claims 1 to 3, a stereoisomer or pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 5 for the preparation of an anti-alzheimer's disease medicament.

Technical Field

The invention belongs to the field of medicinal chemistry, relates to a flavane derivative, a preparation method and application thereof, and particularly relates to the flavane derivative, the preparation method thereof and the application thereof in preparing an anti-senile dementia medicament.

Background

Alzheimer's disease, the so-called senile dementia. Is a lethal neurodegenerative disease, and has clinical manifestations of continuously deteriorated cognitive and memory functions, progressive decline of daily life capacity, and various neuropsychiatric symptoms and behavioral disorders.

To date, there is no uniform definitive understanding of the pathogenesis of alzheimer's disease. With the increasing younger patients with alzheimer's disease and the higher pursuit of quality of life, alzheimer's disease has become the second largest disease threatening human health following tumors. Secondly, the pathology of alzheimer's disease is mainly characterized by neuritic plaques, neuronal death and neurofibrillary tangles, and belongs to a chronic progressive degenerative change of the central nervous system. The main disease population of alzheimer disease is the elderly over 60 years old, and the patients have memory disorder at the early stage of the disease, and are mainly characterized in that the patients easily forget about the recent events and suffer from memory impairment, along with the progressive worsening of the disease condition of the patients, the patients have aphasia, behavior disorder, cognitive disorder, memory loss and the like, and have anxiety apathy and aggressive behaviors in mood and behavior.

Alzheimer's disease is primarily characterized clinically by extensive death of brain cells, particularly in the basal ganglia region. Normally, fibers from the basal ganglia project to the cortex of the brain associated with memory and cognition, which releases acetylcholine. The formation of short-term memory must involve acetylcholine, and the content of acetylcholine transferase in patients is reduced by 90% compared with normal people.

At present, clinical researches on relevant pathogenesis of the alzheimer disease mainly include the following theories, namely a cholinergic theory, a tau protein hypothesis, a neurovascular theory, an oxidative stress theory, a beta-amyloid protein waterfall theory and the like. Although the pathogenesis and treatment of AD remains a debate in the medical community, relatively mature acetylcholinesterase inhibitors have been studied. Acetylcholine is an important neurotransmitter and has a close relationship with the formation and storage of human memory and cholinergic systems. Plays an important role in the damage of cholinergic neurons caused by various factors and the occurrence of a series of clinical Alzheimer's disease phenomena caused by the damage of cholinergic neurotransmission at relevant parts such as cortical hippocampus.

The traditional medicinal plant resources in China are very rich, the physiological active substances of the medicinal plant resources are lead compounds for researching and discovering new medicines, the medicinal plant resources are natural treasury for developing new medicines, the active substances are extracted from natural products, and the medicinal plant resources are developed into new medicines with good safety. The flavane is one of the effective components of the pithecellobium clypearia, the content is about 0.1 percent, and the structural formula is shown as follows. A large number of documents report that flavane compounds have remarkable antioxidant and free radical scavenging activities, and a large number of documents report in recent years that a series of derivatives with remarkable inhibitory activity on acetylcholine esterase, butyrylcholinesterase, monoamine oxidase and other neuroprotective activities are designed and synthesized based on the principle that a flavone skeleton is taken as a mother nucleus and based on the combination of active fragments, and the derivatives are different from currently known cholinesterase inhibitors or agonists and antagonists of M receptors, have wide effects although the single-target therapeutic activity of the derivatives is limited, and are multifunctional Alzheimer disease target inhibitors.

Disclosure of Invention

The invention aims to provide a series of flavane derivatives.

The invention is realized by the following technical scheme:

the flavane derivative has the following structural general formula:

the present invention provides flavane derivatives represented by general formula (I), (II), (III) or (IV), stereoisomers or pharmaceutically acceptable salts thereof:

in the formula I, R₁、R₂Independently H, OH, halogen, nitro, amino, cyano, C_1-8Alkoxy radical, C₁-C₆Alkyl, halo C₁-C₆Alkyl, halo C₁-C₆Alkoxy, unsubstituted or substituted benzyl or C_1-8Alkanoyl, benzoyl or biphenyloyl, or R₁And R₂Together form a 5-6 membered heterocyclic ring;

in the formula II, R₃Is H, halogen, nitro, halogeno C₁-C₆Alkyl, halo C₁-C₆Alkoxy, substituted or unsubstituted phenyl, benzyl or naphthalene ring, the substituents being halogen, nitro, amino, cyano, C₁-C₆Alkyl radical, C_1-6An alkoxy group;

in the formula III, R₄、R₅Is H, halogen, nitro, amino, cyano, C₁-C₆Alkoxy radical, C₁-C₆An alkyl group;

in the formula IV, R₆Is H, halogen, C₁-C₆Alkyl, halo C₁-C₆Alkyl radical, C₁-C₆Alkyl radical, C_1-6Alkoxy, substituted or unsubstituted 5-to 10-membered aryl,R₇，R₈Is halogen, C₁-C₆Alkyl, halo C₁-C₆An alkyl group;

further, preferred flavan derivatives of the following structure, stereoisomers or pharmaceutically acceptable salts thereof, are preferred in the present invention:

in the formula I, R₁、R₂Independently H, OH, halogen, nitro, amino, cyano, C₁-C₄Alkoxy radical, C₁-C₄Alkyl, halo C₁-C₄Alkyl, halo C₁-C₄An alkoxy group;

or R₁And R₂Together form a dioxymethylene group;

in the formula II, R₃Is H, halogen, nitro, halogeno-C₁-C₄Alkyl, halo C₁-C₄An alkoxy group;

in the formula III, R₄、R₅Is H, halogen, nitro, amino, cyano, C₁-C₄Alkoxy radical, C₁-C₄An alkyl group;

in the formula IV, R₆Is C₁-C₆Alkyl radical, C_1-6Alkoxy radical,R₇、R₈Is H, halogen, C₁-C₄Alkyl, halo C₁-C₄An alkyl group.

Further, the present invention prefers flavan derivatives, stereoisomers or pharmaceutically acceptable salts thereof as follows:

the invention also provides a preparation method of the flavane derivative or the pharmaceutically acceptable salt thereof.

The method comprises the following steps:

(1) adding flavane mother nucleus, potassium carbonate and dimethyl sulfate into dry acetone, heating to 60 deg.C, and reacting under reflux. After the reaction is finished, adding ammonia water, stirring, extracting by ethyl acetate, loading on a silica gel column, flushing impurities by petroleum ether, flushing the petroleum ether and the ethyl acetate (5:1) to obtain an intermediate 1;

(2) the intermediate 1(GTF-1) obtained in the above step, a 10% NaOH solution, was added to THF and reacted at room temperature. After the reaction is finished, 2.0mol/l hydrochloric acid is dripped to regulate the pH value to be 5-6, ethyl acetate is used for extraction, a silica gel column is used, petroleum ether is used for elution to remove impurities, and petroleum ether and ethyl acetate (2:1) are used for washing to obtain an intermediate 2;

(3) adding the intermediate 2(GTF-2) obtained in (2), cinnamic acid containing various substituents on benzene ring, EDCI and DMAP into CH₂Cl₂And reacting at room temperature. After the reaction is finished, extracting with ethyl acetate, concentrating under reduced pressure, filtering with a microporous filter membrane, and separating by high performance liquid chromatography to obtain a compound of formula I;

(4) adding intermediate 2(GTF-2) obtained in (2), benzoic acid containing various substituents on the benzene ring, EDCI and DMAP to CH₂Cl₂And reacting at room temperature. After the reaction is finished, extracting with ethyl acetate, concentrating under reduced pressure, filtering with a microporous filter membrane, and separating by high performance liquid chromatography to obtain a compound of formula II;

(5) adding the intermediate 2(GTF-2) obtained in the step (2), potassium carbonate and benzene sulfonyl chloride containing various substituents on a benzene ring into dry acetone, and carrying out reflux reaction at 60 ℃. After the reaction is finished, extracting with ethyl acetate, concentrating under reduced pressure, filtering with a microporous membrane, and separating with high performance liquid chromatography to obtain a compound of formula III;

(6) adding the intermediate 2(GTF-2) obtained in the step (2), potassium carbonate and bromopropyne into dry acetone, and carrying out reflux reaction at 60 ℃. Loading on silica gel column, washing with petroleum ether and ethyl acetate (2:1) to obtain key intermediate 3 (GTF-3);

(7) adding the intermediate 3(GTF-3) obtained in the step (6), sodium ascorbate, copper sulfate pentahydrate and alkyl bromide into acetonitrile and water in a volume ratio of 1:1, at room temperature. After the reaction is finished, ethyl acetate is used for extraction, decompression and concentration are carried out, the microporous membrane is used for filtration, and high performance liquid chromatography separation is carried out, so as to obtain the compound shown in the formula IV.

The invention provides a pharmaceutical composition, which comprises the flavane derivative, the stereoisomer or the pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

The flavane derivative, the stereoisomer or the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof has obvious A beta protein, acetylcholinesterase and butyrylcholinesterase inhibition activities, and can be used for preparing the anti-senile dementia drugs.

Drawings

FIG. 1 shows the results of acetylcholinesterase and butyrylcholinesterase tests;

a: kinetic interaction pattern of compound a15 with acetylcholinesterase; b: a graph of the slope of the acetylcholinesterase kinetic curve as a function of substrate concentration; c: kinetic interaction pattern of compound C4 with butyrylcholinesterase; d: butyrylcholinesterase kinetic curve slope as a function of substrate concentration.

FIG. 2 shows the protective activity test of SY5Y nerve cells induced by hydrogen peroxide and the cytotoxicity test result of normal SY5Y nerve cells;

a: results of cytotoxic activity of compounds a15 and C4 against SY5Y nerve cells; b: the protective activity of the compounds A15 and C4 on hydrogen peroxide-induced SY5Y nerve cells is shown.

Detailed Description

EXAMPLE 1 preparation of key intermediate 1 of flavan

(1) A50 ml eggplant-shaped bottle was charged with flavan mother nucleus (GTF) (2g,2.30mmol), potassium carbonate (6.24g,46.0mmol), dimethyl sulfate (6.24g,46.0mmol), dried acetone (25 ml), and reacted at 60 ℃ under reflux for 10 hours. After the reaction is finished, adding ammonia water, stirring, extracting for three times by ethyl acetate, loading on a silica gel column, flushing impurities by petroleum ether, flushing the key intermediate 1 by petroleum ether and ethyl acetate (5:1), wherein the structural identification data is as follows:

¹H NMR(600MHz,CDCl₃):δ7.44(s,2H,2”,6”-H),6.65(s,2H,2'，6'-H),6.45 (d,J＝2.11Hz,1H,6-H),6.32(d,J＝2.11Hz,1H,8-H),4.94(dd,J＝10.58,1.67Hz, 1H，2-H),3.94(s,9H,3”，4”，5”-OCH₃),3.88(s,6H,3'，5'-OCH₃),3.85(s,3H, 4'-OCH₃),3.83(s,3H,5-OCH₃),2.86(m,1H,4-H),2.70(m,1H，4-H),2.23(m,1H， 3-H),2.02(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ165.1,158.4,156.2,153.5 (2C),153.1(2C),150.3,143.0,137.8,137.3,108.7,107.4,103.3(2C),97.2,78.2, 61.1,61.0,56.5(2C)，56.3(2C),55.8，29.1,19.7.HR-ESI-MS:563.1888[M+Na]⁺, (calcd for C₂₉H₃₂O₁₀Na,563.1891).

example 2 preparation of key intermediate 2 of flavan

(2) A50 ml eggplant type flask was charged with intermediate 1(GTF-1) (1.5g,4.40mmol), THF 20ml, 10ml of 10% aqueous NaOH solution, and reacted at room temperature for 10 hours. After the reaction is finished, 2.0mol/l hydrochloric acid is dripped to regulate the pH value to be 5-6, ethyl acetate is extracted for three times, the mixture is put on a silica gel column and eluted by petroleum ether to remove impurities, the key intermediate 2 is obtained by washing the petroleum ether and the ethyl acetate (2:1), and the structural identification data is as follows:

¹H NMR(600MHz,CDCl₃):δ6.64(s,2H,2',6'-H),6.06(d,J＝2.28Hz,1H, 6-H),6.04(d,J＝2.29Hz,1H,8-H),4.88(dd,J＝10.59,1.99Hz,1H,2-H),3.87(s, 6H,3',5'-OCH3),3.85(s,3H,4'-OCH3),3.79(s,3H,5-OCH3),2.77(ddd,J＝16.71, 5.70,2.57Hz,1H,4-H),2.62(ddd,J＝16.83,10.12,6.25Hz,1H,4-H),2.18(m,1H, 3-H),1.98(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ159.2,156.7,155.7,153.8 (2C),138.0,137.8,103.8,103.7,96.5,92.0,78.5,61.3,56.6(2C),56.0,30.2,19.9. HR-ESI-MS:369.1309[M+Na]⁺,(calcd for C₁₉H₂₂O₆Na,369.1321).

example 3 preparation of flavan benzoate derivatives:

(1) a10 ml eggplant-type bottle was charged with intermediate 2(GTF-2) (10mg, 27.5. mu. mol), 4-nitrobenzoic acid (9.2mg, 55.0. mu. mol), EDCI (10.54mg, 55.0. mu. mol), DMAP (3.4mg, 27.5. mu. mol), CH₂Cl₂6ml, react at room temperature for 8 h. After the reaction is finished, extracting for three times by ethyl acetate, decompressing and concentrating, filtering by a microporous membrane, separating by high performance liquid chromatography, RP-HPLC (MeCN-H)₂O,65:35,3.0ml/min,t_R＝25min)。

The structural identification data of the 4' -nitrobenzoate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ8.36(m,4H,2”,3”,5”,6”-H),6.65(s,2H,2',6'-H), 6.48(d,J＝2.17Hz,1H,8-H),6.33(d,J＝2.17Hz,1H,6-H),4.94(dd,J＝10.69, 1.85Hz,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.84(s,3H, 5-OCH₃),2.86(m,1H,4-H),2.70(m,1H,4-H),2.23(m,1H,3-H),2.03(m,1H,3-H). ¹³C NMR(150MHz,CDCl₃):δ163.6,158.6,156.3,153.5(2C),151.0,150.5,149.8, 137.7,137.1,135.1,131.4(2C),123.8,109.3,103.2(2C),103.1,96.6,78.2,61.0, 56.3(2C),55.8,29.6,19.9.HR-ESI-MS:518.1462[M+Na]⁺,(calcd for C₂₆H₂₅NO₉Na, 518.1422).HPLC purity:97.76％,retention time:9.46min.

the structural identification data of the 4' -fluorobenzoate derivative are as follows:

¹H NMR(600MHz,CDCl3):δ8.21(dd,J＝8.8,5.4Hz,2H,2”,6”-H),7.18(t,J ＝8.6Hz,2H,3”,5”-H),6.65(s,2H,2',6'-H),6.46(d,J＝2.0Hz,1H,8-H),6.32(d,J ＝2.0Hz,1H,6-H),4.94(m,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s,3H, 4'-OCH₃),3.83(s,3H,5-OCH₃),2.85(m,1H,4-H),2.69(m,1H,4-H),2.23(m,1H, 3-H),2.02(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ164.3,158.5,156.3,153.5 (2C),150.0,137.7,137.2,135.2(q,J_C-F＝32.2Hz),130.7(2C),125.8(q,J_C-F＝3.6 Hz),125.7,123.7(q,J_C-F＝272.7Hz),109.1,103.2(2C),96.7,78.2,70.0,56.3(2C), 55.8,29.6,20.0.HR-ESI-MS:491.1481[M+Na]⁺,(calcd for C₂₆H₂₅FO₇Na,491.1477). HPLC purity:95.38％.

the structural identification data of the 4' -trifluoromethyl benzoate derivative are as follows:

¹H NMR(600MHz,CDCl3):δ8.31(d,J＝8.1Hz,2H,2”,6”-H),7.78(d,J＝8.2 Hz,2H,3”,5”-H),6.65(s,2H,2',6'-H),6.47(d,J＝1.9Hz,1H,8-H),6.33(d,J＝1.9 Hz,1H,6-H),4.94(m,1H,2-H),3.88(s,6H,3',5'-OCH3),3.85(s,3H,4'-OCH3), 3.84(s,3H,5-OCH3),2.86(dd,J＝17.1,3.1Hz,1H,4-H),2.70(m,1H,4-H),2.23 (m,1H,3-H),2.03(m,1H,3-H).¹³C NMR(150MHz,CDCl3):δ164.3,158.5,156.3, 153.5(2C),150.0,137.7,137.2,135.2(q,J_C-F＝32.2Hz),130.7(2C),125.8(q,J_C-F＝ 3.6Hz),125.7,123.7(q,J_C-F＝272.7Hz),109.1,103.2(2C),96.7,78.2,70.0,56.3 (2C),55.8,29.6,20.0.HR-ESI-MS:541.1449[M+Na]⁺,(calcd for C₂₇H₂₅F₃O₇, 541.1445).HPLC purity:96.53％.

example 4 preparation of flavan cinnamate derivatives:

(1) a10 ml eggplant type bottle was charged with intermediate 2(GTF-2) (10mg, 27.5. mu. mol), 4-trifluoromethylcinnamic acid (11.0mg, 55.0. mu. mol), EDCI (11.54mg, 55.0. mu. mol), DMAP (3.4mg, 27.5. mu. mol)), CH₂Cl₂6ml, react at room temperature for 8 h. After the reaction is finished, extracting for three times by ethyl acetate, decompressing and concentrating, filtering by a microporous membrane, separating by high performance liquid chromatography, and performing RP-HPLC (MeCN-H)₂O,65:35,3.0ml/min,t_R＝38 min)。

The structural identification data of the 4' -trifluoromethyl cinnamate derivative is as follows:

¹H NMR(600MHz,CDCl₃)：δ7.87(d,J＝16.02Hz,1H,7”-H),7.68(m,4H, 2”,3”,5”,6”-H),6.69(d,J＝16.02Hz,1H,8”-H),6.65(s,2H,2',6'-H),6.43(d,J＝2.15 Hz,1H,8-H),6.29(d,J＝2.15Hz,1H,6-H),4.92(dd,J＝10.71,1.88Hz,1H,2-H), 3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.83(s,3H,5-OCH₃),2.85(m,1H, 4-H),2.68(m,1H,4-H),2.22(m,1H,3-H),2.02(m,1H,3-H).¹³C NMR(150MHz, CDCl₃):δ165.1,158.4,156.2,153.5(2C),150.0,144.7,137.7,137.2,132.4(q,J_C-F＝ 32.86Hz),128.5,126.1(2C,q,J_C-F＝3.61Hz),108.8,103.2(2C),96.8,78.2,56.3 (2C),55.8,29.6,19.9.HR-ESI-MS:567.1636[M+Na]⁺,(calcd for C₂₉H₂₇F₃O₇Na, 567.1601)，HPLC purity:95.13％,retention time:11.97min.

the structural identification data of the 4' -fluorocinnamate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ7.82(d,J＝16.0Hz,1H,7”-H),7.59(d,J＝5.5 Hz,1H,6”-H),7.58(d,J＝5.4Hz,1H,2”-H),7.11(m,2H,3”-H),6.65(s,2H,2', 6'-H),6.54(d,J＝16.0Hz,1H,8”-H),6.42(d,J＝2.0Hz,1H,8-H),6.28(d,J＝2.0 Hz,1H,6-H),4.92(dd,J＝10.6,1.4Hz,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s, 3H,4'-OCH₃),3.82(s,3H,5-OCH₃),2.85(dd,J＝17.1,3.2Hz,1H,4-H),2.67(m, 1H,4-H),2.02(m,2H,3-H).¹³C NMR(150MHz,CDCl₃):δ165.6,163.5,158.4, 156.2,153.5(2C),150.1,145.4,137.7,137.3,130.4,130.3(2C),117.2,103.2(2C), 96.9,78.2,70.0,56.3(2C),55.8,29.7,20.0.HR-ESI-MS:517.1636[M+Na]⁺,(calcd for C₂₈H₂₇FO₇Na,517.1633).HPLC purity:95.77％.

the structural identification data of the 3' -trifluoromethyl cinnamate derivative is as follows:

¹H NMR(600MHz,CDCl₃):δ7.87(d,J＝16.1Hz,1H,7”-H),7.82(d,J＝7.7Hz, 1H,6”-H),7.76(d,J＝7.9Hz,1H,2”-H),7.68(d,J＝7.7Hz,1H,4”-H),7.56(d,J＝ 7.8Hz,1H,5”-H),6.69(d,J＝16.0Hz,1H,8”-H),6.65(s,2H,2',6'-H),6.43(d,J＝ 2.1Hz,1H,8-H),6.29(d,J＝2.1Hz,1H,6-H),4.93(dd,J＝10.6,1.5Hz,1H,2-H), 3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.83(s,3H,5-OCH₃),2.85(dd,J＝ 17.3,3.3Hz,1H,4-H),2.69(m,1H,4-H),2.02(m,2H,3-H).¹³C NMR(150MHz, CDCl₃):δ165.2,158.5,156.2,153.5(2C),150.0,144.8,137.7,135.1,131.4,129.7 (2C),127.2,125.0,119.5,108.9,103.2(2C),96.8,78.2,70.0,56.3(2C),55.8,29.7, 20.0.HR-ESI-MS:567.1601[M+Na]⁺,(calcd for C₂₉H₂₇F₃O₇Na,567.1598).HPLC purity:95.17％.

the structural identification data for the 3 ", 4" -methylenedioxycinnamate derivative is as follows:

¹H NMR(600MHz,CDCl₃):δ7.76(d,J＝15.9Hz,1H,7”-H),7.06(m,2H,2”, 6”-H),6.84(d,J＝8.0Hz,1H,5”-H),6.65(s,2H,2',6'-H),6.43(d,J＝15.9Hz,1H, 8”-H),6.41(d,J＝2.1Hz,1H,8-H),6.28(d,J＝2.1Hz,1H,6-H),6.02(s,2H, 10”-CH₂),4.92(dd,J＝10.7,1.8Hz,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s,3H, 4'-OCH₃),3.82(s,3H,5-OCH₃),2.84(m,1H,4-H),2.68(m,1H,4-H),2.02(m,1H, 3-H),2.01(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ165.9,158.4,156.1,153.5 (2C),150.2,150.1,148.6,146.4,137.6,137.3,128.7,125.02,115.2,108.8,108.6, 103.3,103.2,101.8,96.9,78.1,61.0,56.3(2C),55.8,29.7,19.9.HR-ESI-MS: 543.1655[M+Na]⁺,(calcd for C₂₉H₂₈O₉Na,543.1626).HPLC purity:95.06％.

the structural identification data of the 4' -N, N-dimethyl cinnamate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ7.79(d,J＝15.8Hz,1H,7”-H),7.47(d,J＝8.9 Hz,2H,2”,6”-H),6.69(d,J＝8.9Hz,2H,3”,5”-H),6.65(s,2H,2',6'-H),6.42(d,J ＝2.1Hz,1H,8-H),6.38(d,J＝15.8Hz,1H,8”-H),6.28(d,J＝2.1Hz,1H,6-H), 4.92(dd,J＝10.7,1.8Hz,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃), 3.81(s,3H,5-OCH₃),2.84(m,1H,4-H),2.67(m,1H,4-H),2.21(m,1H,3-H),2.01 (m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ166.6,158.3,156.1,153.4(2C),152.1, 150.5,147.3,137.6,137.4,130.2(2C),125.1,111.9,108.4,103.4,103.2(2C),101.8, 97.2,78.1,60.1,56.3(2C),55.7,29.7,19.9.HR-ESI-MS:520.2300[M+H]⁺,(calcd for C₃₀H₃₄NO₇,520.2330).HPLC purity:95.08％.

example 5 preparation of flavansulfonate derivatives:

(1) a10 ml eggplant type bottle was charged with intermediate 2(GTF-2) (10mg, 27.5. mu. mol), potassium carbonate (8.0mg, 50. mu. mol), benzenesulfonyl chloride (9.0mg, 50. mu. mol), 6ml of dried acetone, and reacted at 60 ℃ under reflux for 8 hours. After the reaction is finished, extracting for three times by ethyl acetate, decompressing and concentrating, filtering by a microporous membrane, separating by high performance liquid chromatography, and performing RP-HPLC (MeCN-H)₂O,65:35,3.0ml/min,t_R＝38min)。

The structural identification data of the benzene sulfonate derivatives are as follows:

¹H NMR(600MHz,CDCl₃):δ7.90(m,2H,3”,7”-H),7.67(t,J＝7.48Hz,1H, 5”-H),7.54(t,J＝7.77Hz,2H,4”,6”-H),6.59(s,2H,2',6'-H),6.22(d,J＝2.16Hz, 1H,8-H),6.11(d,J＝2.17Hz,1H,6-H),4.86(dd,J＝10.50,1.73Hz,1H,2-H),3.87 (s,6H,3',5'-OCH₃),3.84(s,3H,4'-OCH₃),3.69(s,3H,5-OCH₃),2.77(m,1H,4-H), 2.62(m,1H,3-H),2.18(m,1H,3-H),1.97(m,1H,3-H).¹³C NMR(150MHz, CDCl₃):δ158.3,156.0,153.5(2C),148.8,137.8,136.9,135.9,134.3,129.3,128.6, 110.1,103.8,103.2(2C),97.5,78.2,61.0,56.3(2C),55.8,29.3,19.8.HR-ESI-MS: 509.1242[M+Na]⁺,(calcd for C₂₅H₂₆O₈SNa,509.1241).HPLC purity:95.19％, retention time:6.02min.

the structural identification data of the 4' -nitrobenzenesulfonate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ8.38(d,J＝8.8Hz,2H,3”,5”-H),8.09(d,J＝8.9 Hz,2H,2”,6”-H),6.58(s,2H,2',6'-H),6.21(d,J＝2.2Hz,1H,8-H),6.15(d,J＝2.2 Hz,1H,6-H),4.85(dd,J＝10.7,1.9Hz,1H,2-H),3.86(s,6H,3',5'-OCH₃),3.84(s, 3H,4'-OCH₃),3.75(s,3H,5-OCH₃),2.80(ddd,J＝17.3,5.5,2.3Hz,1H,4-H),2.64 (m,1H,4-H),2.19(m,1H,3-H),1.97(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ 158.6,156.2,153.6(2C),151.1,148.4，141.4,138.0,136.6,130.0(2C),124.5,110.6, 103.4,103.3,97.2,78.4,70.0,56.3(2C),55.9,29.2,19.8.HR-ESI-MS:554.1096 [M+Na]⁺,(calcd for C₂₅H₂₅NO₁₀SNa,554.1091).HPLC purity:96.96％.

the structural identification data of the 4' -cyanobenzenesulfonate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ8.01(d,J＝8.5Hz,2H,3”,5”-H),7.84(d,J＝ 8.5Hz,2H,2”,6”-H),6.59(s,2H,2',6'-H),6.17(d,J＝2.3Hz,1H,8-H),6.16(d,J＝ 2.2Hz,1H,6-H),4.85(dd,J＝10.7,1.9Hz,1H,2-H),3.86(s,6H,3',5'-OCH₃),3.84 (s,3H,4'-OCH₃),3.74(s,3H,5-OCH₃),2.79(ddd,J＝17.2,5.5,2.4Hz,1H,4-H), 2.63(m,1H,4-H),2.19(m,1H,3-H),1.98(m,1H,3-H).¹³C NMR(150MHz, CDCl₃):δ158.5,156.2,153.5(2C),148.4,139.9,137.9,136.6,129.3,118.0,117.0, 110.6,103.3(2C),97.2,78.4,70.0,56.3(2C),55.9,29.1,19.8.HR-ESI-MS: 534.1194[M+Na]⁺,(calcd for C₂₅H₂₅NO₁₀SNa 534.1193).HPLC purity:95.12％.

the structural identification data of the 2 ', 4' -difluorobenzenesulfonate derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ7.87(dd,J＝14.3,8.1Hz,1H,3”-H),7.02(m,2H, 5”,6”-H),6.59(s,2H,2',6'-H),6.31(d,J＝1.9Hz,1H,8-H),6.29(d,J＝1.9Hz,1H, 6-H),4.86(d,J＝9.4Hz,1H,2-H),3.87(s,6H,3',5'-OCH₃),3.84(s,3H,4'-OCH₃), 3.77(s,3H,5-OCH₃),2.78(m,1H,4-H),2.62(m,1H,4-H),1.98(m,1H,3-H),1.97 (m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ167.0(dd,J_C-F＝259.9,11.2Hz),160.5 (dd,J_C-F＝262.9,13.0Hz),158.5,156.1,153.5(2C),148.4,137.8,136.7,133.4(d, J_C-F＝10.80Hz)120.5(dd,J＝14.0,3.8Hz),112.3(dd,J_C-F＝22.1,3.7Hz),110.4, 106.2(t,J_C-F＝24.0Hz),103.3,103.2,97.2,70.0,56.3(2C),55.9,29.6,19.8. HR-ESI-MS:545.1054[M+Na]⁺,(calcd for C₂₅H₂₄F₂O₈SNa,545.1052).HPLC purity: 95.22％.

example 6 preparation of flavantriazole derivatives:

(1) a50 ml eggplant type bottle was charged with intermediate 2(GTF-2) (1.0g,2.75mmol), potassium carbonate (690.0mg,5.5 mmol), bromopropyne (654.5mg, 5.5mmol), dried acetone (20 ml), and reacted at 60 ℃ for 8 hours under reflux. Loading on silica gel column, washing with petroleum ether and ethyl acetate (2:1) to obtain key intermediate 3, and identifying the structure as follows:

¹H NMR(600MHz,CDCl₃):δ6.65(s,2H,2',6'-H),6.21(d,J＝2.28Hz,1H,8-H), 6.21(d,J＝2.27Hz,1H,6-H),4.89(dd,J＝10.70,1.80Hz,1H,2-H),4.64(d,J＝ 2.35Hz,2H,1”-CH₂),3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.80(s,3H, 5-OCH₃),2.79(ddd,J＝16.74,5.59,2.32Hz,1H,4-H),2.64(m,1H,4-H),2.51(t,J ＝2.35Hz,1H,3”-H),2.19(m,1H,3-H),2.00(m,1H,3-H).HR-ESI-MS:407.1469 [M+H]+,(calcd for C₂₂H₂₄O₆,407.1465).

(2) intermediate 3(GTF-3) (18mg, 47.0. mu. mol) was added to a 50ml eggplant-shaped bottle, and dissolved in a mixed solution (6ml) of acetonitrile and water at a volume ratio of 1:1, followed by addition of sodium ascorbate (9.2mg, 47.0. mu. mol), copper sulfate pentahydrate (11.8mg, 47.0. mu. mol), and finally alkyl bromide (190.0. mu. mol). Reflux reacting for 6H, extracting with ethyl acetate for three times, concentrating under reduced pressure, filtering with microporous membrane, separating with high performance liquid chromatography, and RP-HPLC (MeCN-H)₂O,65:35,3.0ml/min,t_R＝38min)。

The structural identification data of the 4' -trifluoromethyl-1, 2, 3-triazole derivative are as follows:

¹HNMR(600MHz,CDCl₃)：δ7.63(s,1H,3”-H),6.65(s,2H,2',6'-H),6.24(d, J＝2.27Hz,1H,8-H),6.17(d,J＝2.27Hz,1H,H-6),5.16(s,2H,6”-CH₂),4.89(dd, J＝10.62,1.51Hz,1H,2-H),4.38(t,J＝6.54Hz,2H,4”-CH₂),3.88(s,6H, 3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.79(s,3H,5-OCH₃),2.79(ddd,J＝16.64,5.50, 2.19Hz,1H,4-H),2.64(m,1H,4-H),2.19(m,1H,3-H),2.01(m,1H,3-H),1.91(dt, J＝14.40,7.12Hz,2H,5”-CH₂),1.37(dq,J＝14.69,7.35Hz,1H,6”-CH₂),0.96(t,J＝ 7.35Hz,3H,7”-CH₃).¹³C NMR(150MHz,CDCl₃):δ158.8,158.2,156.3,153.5 (2C),144.3,137.7,137.4,122.7,104.0,103.3(2),94.7,92.2,78.3,62.3,61.0,56.3 (2C),55.7,50.3,32.4,29.8,19.9,19.6,13.6.HR-ESI-MS:484.2441[M+H]⁺,(calcd for C₂₆H₃₄N₃O₆484.2442).HPLC purity:96.35％,retention time:5.70min.

the structural identification data of the 4' -methyl-1, 2, 3-triazole derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ7.51(s,1H,3”-H),7.18(brs,4H,6”,7”,9”, 10”-H),6.64(s,2H,2',6'-H),6.21(d,J＝2.2Hz,1H,8-H),6.13(d,J＝2.2Hz,1H, 6-H),5.48(s,2H,4”-CH₂),5.10(s,2H,1”-CH₂),4.88(dd,J＝10.7,1.8Hz,1H,2-H), 3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.77(s,3H,5-OCH₃),2.77(ddd,J＝ 16.7,5.6,2.3Hz,1H,4-H),2.63(m,1H,4-H),2.35(s,3H,12”-H),2.18(m,1H,3-H), 2.00(m,3-H).¹³C NMR(150MHz,CDCl₃):δ158.7,158.1,156.3,153.5(2C),144.6, 138.9,137.7,137.4,131.5,129.9,128.4,122.6,104.0,103.3(2C),94.6,92.2,78.3, 62.3,61.0,56.3(2C),55.6,54.2,29.8,21.3,19.6.HR-ESI-MS:532.2443[M+H]⁺, (calcd for C₃₀H₃₄N₃O₆,532.2442).HPLC purity:96.73％.

the structure identification data of the 2' -chlorine-1, 2, 3-triazole derivative are as follows:

¹H NMR(600MHz,CDCl₃):δ7.65(s,1H,3”-H),7.44(d,J＝7.9Hz,1H,8”-H), 7.32(td,J＝7.7,1.5Hz,1H,7”-H),7.28(dd,J＝7.6,0.9Hz,1H,5”-H),7.21(dd,J＝ 7.59,1.1Hz,1H,6”-H),6.65(s,2H,2',6'-H),6.22(d,J＝2.1Hz,1H,8-H),6.17(d,J ＝2.1Hz,1H,6-H),5.68(s,2H,4”-CH₂),5.13(s,2H,1”-CH₂),4.88(dd,J＝10.6,1.4 Hz,1H,2-H),3.88(s,6H,3',5'-OCH₃),3.85(s,3H,4'-OCH₃),3.78(s,3H,5-OCH₃), 2.78(m,1H,4-H),2.63(m,1H,4-H),2.18(m,1H,3-H),2.00(m,1H,3-H).¹³C NMR(150MHz,CDCl₃):δ158.7,158.1,156.3,153.5(2C),144.6,137.7,137.4, 133.7,132.5,130.6,130.5,130.1,127.8,123.1,104.1,103.3(2C),94.7,92.2,78.3, 62.3,61.0,56.3(2C),55.7,51.6,29.8,19.7.HR-ESI-MS:552.1900[M+H]⁺,(calcd for C₂₉H₃₁ClN₃O₆,552.1896).HPLC purity:95.24％.

the structural identification data of the 4' -butyl-1, 2, 3-triazole derivative are as follows:

¹HNMR(600MHz,CDCl₃):δ7.63(s,1H,3”-H),6.65(s,2H,2',6'-H),6.24(d,J ＝2.3Hz,1H,8-H),6.17(d,J＝2.3Hz,1H,6-H),5.16(s,2H,6”-CH₂),4.89(dd,J＝ 10.6,1.5Hz,1H,2-H),4.38(t,J＝6.5Hz,2H,4”-CH₂),3.88(s,6H,3',5'-OCH₃), 3.85(s,3H,4'-OCH₃),3.79(s,3H,5-OCH₃),2.79(ddd,J＝16.6,5.5,2.2Hz,1H, 4-H),2.64(m,1H,4-H),2.19(m,1H,3-H),2.01(m,1H,3-H),1.91(dt,J＝14.4,7.1 Hz,2H,5”-CH₂),1.37(dq,J＝14.7,7.4Hz,1H,6”-CH₂),0.96(t,J＝7.4Hz,3H, 7”-CH₃).¹³C NMR(150MHz,CDCl₃):δ158.8,158.2,156.3,153.5(2C),144.3, 137.7,137.4,122.7,104.0,103.3(2),94.7,92.2,78.3,62.3,61.0,56.3(2C),55.7, 50.3,32.4,29.8,19.9,19.6,13.6.HR-ESI-MS:484.2441[M+H]⁺,(calcd for C₂₆H₃₄N₃O₆484.2442).HPLC purity:96.35％.

example 7 in vitro anti-a β amyloid aggregation activity assay of flavan derivatives:

1. experimental Material

Amyloid beta (Abeta, Sigma), hexafluoroisopropanol (HFIP, Sigma), Thioflavin T (Th-T, Sigma), Curcumin (Curcumin, Sigma), Potassium dihydrogen phosphate (KH)₂PO₄) Sodium hydroxide (NaOH), DMSO 2, laboratory instruments

A constant temperature incubator, a Varoska Flash enzyme labeling instrument, a Dragon manual adjustable pipettor, a Costar 96-hole transparent flat-bottom plate and a vortex mixer

3 Experimental methods

3.1 pretreatment of Abeta

A β previously stored in a refrigerator at-80 ℃ was taken out and dissolved in HFIP at a concentration of 1 mg/mL. Standing at room temperature for 30min until its secondary structure is completely removed, vacuum freeze drying to remove HFIP, and storing in-20 deg.C refrigerator.

3.2 phosphate buffer (PBS, pH 7.4,0.1M) preparation

Preparation of buffer (PBS, buffer): the pH was 7.4 and the concentration was 0.1 mol/mL. According to the appendix of the second part of the 2010 edition of the pharmacopoeia of the people's republic of China, taking 1.36g of monopotassium phosphate, adding 79mL of 0.l mol/L sodium hydroxide solution, diluting with water to 200mL, and correcting by using a precise pH acidimeter to obtain the potassium phosphate.

3.3 measurement method

Group setting:

(1) blank control group: PBS 60. mu.L, Abeta.10. mu.L, PBS + DMSO 10. mu.L

(2) Sample inhibition group: PBS 60 μ L, Abeta 10 μ L, drug 10 μ L

(3) Blank background group: PBS 60. mu.L, PBS 10. mu.L, PBS + DMSO 10. mu.L

(4) Background group of samples: PBS 60 μ L, PBS 10 μ L, drug 10 μ L

The above-mentioned solutions were added to a 96-well plate, and the plate was incubated at 37 ℃ for 24 hours, then 80. mu.L of Th-T solution was added to each group, incubated at 37 ℃ for 5 minutes in a incubator, and the fluorescence of each group was measured with a microplate reader (λ ex ═ 450 nm; λ em ═ 485 nm). Each group was repeated 3 times.

Fluorescence at 485nm measured with Th-T after incubation of A β alone was used as a control: to avoid interference of the results by the fluorescence the compounds themselves have, the background was subtracted from the fluorescence measured by Th-T for the individual test compounds.

Example 8 flavan derivatives in vitro cholinesterase inhibition activity assay:

1. experimental Material

Acetylcholine iodide (AchI, Sigma), acetylcholinesterase (AchE, Sigma), dithio-dinitrobenzoic acid (DTNB, Sigma), potassium dihydrogen phosphate (KH)₂PO₄) Sodium hydroxide (NaOH), DMSO

2. Laboratory apparatus

A constant temperature incubator, a Varoska Flash enzyme labeling instrument, a Dragon manual adjustable pipettor, a Costar 96-hole transparent flat-bottom plate and a vortex mixer

3. Experimental methods

3.1 preparation of enzyme reaction solution

The AChE freeze-dried powder is prepared into 0.22U/mL enzyme solution by using 0.1mol/mLPBS with the pH value of 7.4, and is frozen and stored at the temperature of-20 ℃ for later use.

3.2 substrate and developer configuration

Separately, 0.4338g of AchI powder and 0.0793g of DTNB powder were weighed precisely, and prepared into 15mM AchI working solution and 2mM DNTB working solution with 0.1mol/mL of PBS having a pH of 7.4, and stored at 4 ℃ until use.

3.3 measurement method

Group setting:

(1) blank control group: PBS 40. mu.L, PBS + DMSO 20. mu.L, AchE 20. mu.L

(2) Sample inhibition group: 60 μ L of PBS, 20 μ L of drug, and 20 μ L of AchE

(3) Blank background group: PBS 60. mu.L, PBS + DMSO 20. mu.L, PBS 20. mu.L

(4) Background group of samples: 60 μ L of PBS, 20 μ L of drug, 20 μ L of PBS

Adding the solutions into a 96-well plate, incubating at 4 deg.C for 10min, adding 20 μ LACHI solution and 100 μ LDNTB solution, incubating at 37 deg.C for 20min, and measuring OD at 412nm with enzyme-labeling instrument. Each group was repeated 3 times.

3.4 analysis of results:

the OD 412nm determined by DTNB after incubation of AChE and AchI alone was used as a control; to avoid interference of the compound itself with the results, the OD values determined by AChE with DTNB were subtracted as background.

Example 9 hydrogen peroxide-induced SY5Y nerve cell protective activity test of flavan derivatives and cytotoxicity test of normal SY5Y nerve cells

1. Cell culture

Neuroblastoma SH-SY5Y cell line (purchased from American model culture Collection ATCC, Manassas, USA) was cultured in DMEM medium containing 10% FBS (purchased from Rockwell Hakkrong Hyclone, Logan, USA) at 37 deg.C and 5% CO₂Culturing in the incubator, adhering the cells after 24h, and discarding the old culture solution.

2. Grouping of cells

Blank group: without any drug, only DMEM complete medium was used.

Model group: after the cells were cultured in DMEM complete medium for 4 hours, 1mM MPP was added⁺The cultivation was continued for 36 hours.

Group A15: after culturing the cells in DMEM complete medium for 4 hours, A15 compounds with different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) were added and cultured for 1 hour, and 1mM MPP was added⁺The culture was continued for 36 hours.

Group C4: after culturing the cells in DMEM complete medium for 4h, C4 compounds were added at different concentrations (12.5. mu.M, 25. mu.M, 50. mu.M) and cultured for 1 hour, followed by addition of 1mM MPP⁺The culture was continued for 36 hours.

3. MTT assay

3.1 Add MTT solution (0.5mg/mL) to 20. mu.L wells and incubate at 37 ℃ for an additional 4 h. 2) The waste liquid is discarded, DMSO is added into the waste liquid at a concentration of 150. mu.L/well, and the mixture is shaken on a constant temperature shaker for 10 min.

3.2 microplate reader (Thermo Scientific Multiskan MK3, Shanghai, China), absorbance value was measured at 490nm for each well.

4. Statistical treatment

All results and data were confirmed in at least three independent experiments. All data results obtained are expressed as mean ± sdOne-way analysis of variance, P, was performed on each set of data using GraphPadPrism 7.0 (california, usa) software<0.05 was considered statistically significant.

5. Results of the experiment

The results of the experiment are shown in FIG. 2, comparing MPP with blank group⁺Decreased survival of treated cells (P)<0.001). 50 μ M of A15 compound and C4 compound versus MPP compared to model group⁺Induced SH-SY5Y cell injury has protective effect, and cell survival rate is respectively improved by 5.7% and 4.1%.

Table 1 shows the results of the activity test of the compounds

^aAβ_1-42Both the protein and test compound were 20 μ M.

^bIC of Compounds on eeAChE and eqBuChE₅₀The value is obtained.

No test

Table 2 shows the protection test results of compounds A15 and C4 on SH-SY5Y cells induced by hydrogen peroxide

400μM H₂O₂ for 4h。