阅读说明：本技术 氧杂蒽酮类化合物及其制备方法和应用 (Xanthone compound and preparation method and application thereof ) 是由 谢赛赛 张志鹏 刘婧 程茂军 郭杰 于 2020-12-08 设计创作，主要内容包括：本发明公开了一系列氧杂蒽酮类化合物及其制备方法和应用,所述氧杂蒽酮类化合物具有下述式4或5所示结构,其中式4化合物由卤代烃中间体和芐胺类化合物以亚甲基偶联所得,式5化合物由式4化合物和异氰酸酯在催化剂存在下反应而得。申请人的试验表明,大部分目标化合物能透过血脑屏障,部分目标化合物具有较强的自由基清除能力且能够有效抑制乙酰胆碱酯酶,体内药效试验表明能够通过提高脑内胆碱酯酶的水平改善认知损伤。所述式4和式5结构如下：其中n＝1-12；R-1为H、Me、Et或正丙基；R-2为H、OH、OCH-3或N(CH-3)-2；R-3表示H、OH、OCH-3或N(CH-3)-2；R-4表示Et、氯乙基或环己基。(The invention discloses a series of xanthone compounds and a preparation method and application thereof, wherein the xanthone compounds have a structure shown in a formula 4 or 5, wherein the compound shown in the formula 4 is obtained by coupling a halogenated hydrocarbon intermediate and a benzylamine compound with methylene, and the compound shown in the formula 5 is obtained by reacting the compound shown in the formula 4 with isocyanate in the presence of a catalyst. The experiments of the applicant show that most target compounds can penetrate through a blood brain barrier, part of target compounds have strong free radical scavenging capacity and can effectively inhibit acetylcholinesterase, and in-vivo efficacy experiments show that cognitive impairment can be improved by improving the level of intracerebral cholinesterase. The structures of the formulas 4 and 5 are as follows: wherein n is 1-12; r 1 Is H, Me, Et or n-propyl; r 2 Is H, OH, OCH 3 Or N (CH) 3 ) 2 ；R 3 Represents H, OH, OCH 3 Or N (CH) 3 ) 2 ；R 4 Represents Et, chloroethyl or cyclohexyl.)

1. Xanthone compounds having a structure represented by the following formula 4 or 5:

wherein n is 1-12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；R₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

2. The compound according to claim 1, wherein the compound is at least one of the following compounds 4a-n and 5 a-f:

3. a process for the preparation of the compounds according to claim 1,

the preparation method of the compound shown in the formula 4 comprises the following steps: taking a halogenated hydrocarbon intermediate shown in the following formula 3 and a benzylamine compound shown in the formula A to react in a first organic solvent under the heating condition, and recovering the solvent after the reaction is finished to obtain a crude compound shown in the formula 4;

in the structures represented by formula 3 and formula a, n is 1 to 12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；

The preparation method of the compound shown in the formula 5 comprises the following steps: taking a compound shown in a formula 4 and isocyanate shown in a formula B to react in a second organic solvent in the presence of a catalyst, and recovering the solvent after the reaction is finished to obtain a crude compound shown in a formula 5; wherein the catalyst is one or the combination of more than two of triethylamine, triethylene diamine, dibutyltin dilaurate and stannous octoate

In the structures represented by formula 4 and formula B, n is 1 to 12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；R₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

4. The process according to claim 3, wherein an acid-binding agent is added before the reaction in the process for producing the compound represented by formula 4.

5. The method according to claim 3 or 4, wherein a catalyst is added before the reaction in the method for preparing the compound represented by formula 4, and the catalyst is sodium iodide and/or potassium iodide.

6. The method according to claim 3 or 4,

in the preparation method of the compound shown in the formula 4, the first organic solvent is one or a combination of more than two of acetonitrile, ethanol, methanol, propanol, tetrahydrofuran, acetone, N-dimethylformamide, 1, 4-dioxane and water;

in the preparation method of the compound shown in the formula 5, the second organic solvent is one or a combination of more than two selected from methanol, ethanol, N-dimethylformamide and tetrahydrofuran.

7. The method according to claim 3 or 4,

the preparation method of the compound shown in the formula 4 further comprises the step of purifying the prepared crude compound shown in the formula 4;

the preparation method of the compound shown in the formula 5 further comprises the step of purifying the prepared crude compound shown in the formula 5.

8. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of an acetylcholinesterase inhibitor.

9. The use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of alzheimer's disease or for the manufacture of a health product for the amelioration of alzheimer's disease.

10. An acetylcholinesterase inhibitor or a drug for treating alzheimer's disease, which comprises a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

Technical Field

The invention relates to the technical field of medicines, in particular to a xanthone compound and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is one of the most severe and prevalent neurodegenerative diseases, affecting about 4600 million people worldwide, causing huge economic losses to families and society. According to the international alzheimer's disease association report, the population of AD will increase to 6600 million by 2030 and 1.15 million by 2050. Clinical evidence suggests that AD is a complex disease characterized by progressive memory loss, severe behavioral abnormalities, cognitive dysfunction and ultimately death. Although the etiology of AD is not completely understood, a variety of factors including acetylcholine (ACh) loss, beta-amyloid (a β) deposition, oxidative stress, metabolic disorders of biological metals, and neuroinflammation have been recognized to play a critical role in the onset and progression of AD. Studies have shown that in the brain of patients with severe AD, acetylcholinesterase (AChE) levels drop to 10-15% of normal levels, while butyrylcholinesterase (BuChE) levels have exceeded normal levels.

Although a number of potential AD treatment strategies have been proposed, only 4 drugs (rivastigmine, galantamine, donepezil and memantine) have been approved by the U.S. Food and Drug Administration (FDA) for clinical treatment to date. Although they remain the most effective drugs for the treatment of AD, they only provide temporary relief from symptoms.

Xanthone is an iso-tricyclic compound, is an important organic synthesis intermediate, and a parent body of the xanthone does not exist in a plant body, but a derivative of the xanthone widely exists in the nature and is one of effective components of medicinal plants. Pharmacological research shows that in some natural Chinese herbal medicines, xanthone compounds contained in the natural Chinese herbal medicines have very wide pharmacological activities including activities such as diuresis, antimicrobial, antiviral, antituberculosis, anticancer, anti-heparin and the like (research progress of the xanthone compounds, Guangdong chemical industry, No. 2 p46-47 in 2014). At present, no new compound formed by coupling xanthone and alkylbenzylamine by a methyl chain and introducing a hydroxyl group at the ortho position of the xanthone and a related report of the application of the new compound in AD treatment are found.

Disclosure of Invention

The invention aims to provide a series of xanthone compounds with novel structures and a preparation method and application thereof.

The xanthone compound disclosed by the invention is a xanthone compound with a structure shown in a formula 4 or a formula 5 or a pharmaceutically acceptable salt thereof:

wherein n is 1-12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；R₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

In the above technical solution, preferably, n is 3 to 8; r₁Represents H, Me or Et; r₂Denotes H, OH or OCH₃；R₃Denotes H, OH or OCH₃；R₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

More preferably, the xanthone compound of the structure represented by formula 4 is specifically at least one compound selected from the group consisting of the following compounds 4a to n, and the xanthone compound of the structure represented by formula 5 is specifically at least one compound selected from the group consisting of the following compounds 5a to f:

in the above technical scheme, the pharmaceutically acceptable salt of the xanthone compound may be specifically a hydrochloride, a maleate, a citrate, a sulfate, a maleate, a trifluoroacetate, a citrate, a tartrate, a benzenesulfonate, an acetate, a propionate, a tartrate, an ethanesulfonate, a benzoate, a p-toluenesulfonate, or the like of the xanthone compound.

The preparation methods of the xanthone compound with the structure shown in the formula 4 or the formula 5 are respectively as follows:

the preparation method of the compound shown in the formula 4 comprises the following steps: taking a halogenated hydrocarbon intermediate shown in the following formula 3 and a benzylamine compound shown in the formula A to react in a first organic solvent under the heating condition, and recovering the solvent after the reaction is finished to obtain a crude compound shown in the formula 4;

in the structures represented by formula 3 and formula a, n is 1 to 12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；

The preparation method of the compound shown in the formula 5 comprises the following steps: taking a compound shown in a formula 4 and isocyanate shown in a formula B to react in a second organic solvent in the presence of a catalyst, and recovering the solvent after the reaction is finished to obtain a crude compound shown in a formula 5; wherein the catalyst is one or the combination of more than two of triethylamine, triethylene diamine, dibutyltin dilaurate and stannous octoate.

The above formulas 4 and 4In the structure shown in B, n is 1-12; r₁Represents H, Me, Et or n-propyl; r₂Represents H, OH, OCH₃Or N (CH)₃)₂；R₃Represents H, OH, OCH₃Or N (CH)₃)₂；R₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

In the preparation process of the compound represented by the formula 4, the halogenated hydrocarbon intermediate represented by the formula 3 can be synthesized by referring to the existing literature (Jie Liu, Cao Zhuang, Huailing Wang, Lei Zhuang, Zhenlei Jiang, Jianrun Zhang, Zhijun Liu, Heru Chen, Inc of nitrile oxide inside 1,3-dioxyxanthones lead to synthetic antibody activity, European Journal of Medicinal Chemistry (151 2018) 158-172), or by self-designed route synthesis. The molar ratio of the halocarbon intermediate of formula 3 to the benzylamine compound of formula a is generally stoichiometric. The first organic solvent is one or a combination of two or more selected from acetonitrile, ethanol, methanol, propanol, tetrahydrofuran, acetone, N-dimethylformamide, 1, 4-dioxane and water, and the amount of the first organic solvent is determined as required, and is usually 20 to 25mL based on 1mmol of the halogenated hydrocarbon intermediate represented by formula 3.

In the method for preparing the compound represented by formula 4, it is preferable to add an acid-binding agent before the reaction in order to accelerate the rate of the reaction. The selection and the dosage of the acid-binding agent are the same as those of the prior art, and specifically, the acid-binding agent can be one or the combination of more than two of sodium hydroxide, potassium carbonate, sodium bicarbonate, triethylamine, pyridine and N, N-diisopropylethylamine, and the addition amount of the acid-binding agent is 1-2 times of that of the halogenated hydrocarbon intermediate substance shown in the formula 3. Further, in order to further increase the reaction rate and increase the reaction yield, it is preferable to add a catalyst, specifically sodium iodide and/or potassium iodide, before the reaction; the amount of the catalyst to be added is preferably 1 to 2 times the amount of the halogenated hydrocarbon intermediate represented by formula 3. In this method, the reaction is preferably carried out at a temperature ranging from 50 ℃ to the boiling point of the first organic solvent, and more preferably at 60 to 80 ℃. The reaction can be followed by thin layer chromatography.

The crude compound of formula 4 is obtained by the above-mentioned preparation method of the compound of formula 4, and can be purified by conventional purification methods to increase the purity of the compound of formula 4. The crude compound of formula 4 is usually subjected to silica gel column chromatography to obtain the purified compound of formula 4. When the crude compound shown in the formula 4 is purified by silica gel column chromatography, a mixed solvent consisting of dichloromethane and methanol is used as an eluent, wherein dichloromethane can be replaced by petroleum ether, chloroform, cyclohexane, acetone or carbon tetrachloride; the methanol can be replaced by n-butanol or water. In the composition of the mixed solvent, the volume ratio of dichloromethane (or petroleum ether, etc.) to methanol (or n-butanol, etc.) is preferably 20 to 50: 1, more preferably 20 to 40: 1, most preferably 30: 1.

in the method for preparing the compound represented by the formula 5, the molar ratio of the compound represented by the formula 4 to the isonitrile acid ester represented by the formula B is usually a stoichiometric ratio. The second organic solvent is one or a combination of two or more selected from methanol, ethanol, N-dimethylformamide and tetrahydrofuran, and the amount of the second organic solvent can be determined according to the need, and is usually 20 to 25mL based on 1mmol of the compound represented by formula 4.

In the method for preparing the compound represented by formula 5, the reaction may be carried out with or without heating. The reaction is preferably carried out without heating, and more preferably carried out at room temperature. The reaction can be followed by thin layer chromatography. The operation of recovering the solvent after the reaction to obtain the crude product of the target product can also be replaced by the following operation: pouring the solution obtained by the reaction into water, stirring, extracting with an extracting agent (such as ethyl acetate and the like), collecting an organic layer, drying with anhydrous sodium sulfate, filtering under reduced pressure, collecting filtrate, and concentrating to obtain a crude product of the target product.

The crude compound of formula 5 is obtained by the above-mentioned preparation method of the compound of formula 5, and can be purified by conventional purification methods to increase the purity of the compound of formula 5. The crude compound of formula 5 is usually subjected to silica gel column chromatography to obtain the purified compound of formula 5. When the crude compound shown in the formula 5 is purified by silica gel column chromatography, a mixed solvent consisting of dichloromethane and methanol is used as an eluent, wherein dichloromethane can be replaced by petroleum ether, chloroform, cyclohexane, acetone or carbon tetrachloride; the methanol can be replaced by n-butanol or water. In the composition of the mixed solvent, the volume ratio of dichloromethane (or petroleum ether, etc.) to methanol (or n-butanol, etc.) is preferably 30 to 80: 1, more preferably 40 to 60: 1, most preferably 50: 1.

the invention also comprises the application of the compound shown in the formula 4 or the formula 5 or the pharmaceutically acceptable salt thereof in pharmacy, which is specifically as follows: the application of the derivative in preparing acetylcholinesterase inhibitors, medicines for treating Alzheimer's disease or health-care products for improving Alzheimer's disease.

The invention also comprises a medicament for inhibiting acetylcholinesterase, which contains a therapeutically effective dose of a compound shown in formula 4 or formula 5 or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials.

The invention further comprises a medicament for treating the Alzheimer disease, which comprises a therapeutically effective dose of a compound shown in formula 4 or formula 5 or pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials.

The dosage form of the medicine can be any pharmaceutically acceptable dosage form, and specifically can be conventional dosage forms such as granules, tablets, pills, capsules or injections.

Compared with the prior art, the invention provides a series of xanthone compounds with novel structures and preparation methods thereof, and the obtained compounds obtain the double-cholinesterase inhibition capability of AChE and BuChE by coupling the fragments of xanthone and alkylbenzylamine through methylene chains; in addition, the structure brings extra antioxidant capacity and metal ion chelating capacity to the obtained compound by introducing hydroxyl at the ortho position of xanthone and forming a six-membered ring by intramolecular hydrogen bonding with the hydroxyl. The test results of the applicant show that most of the xanthone compounds can reach the treatment target through the blood brain barrier, part of the xanthone compounds have strong free radical scavenging capacity and can effectively inhibit acetylcholinesterase, and in-vivo efficacy tests show that the compounds can improve cognitive impairment by improving the level of the intracerebral cholinesterase, have good potential medicinal value and are expected to be used for preparing the medicines for treating diseases such as Alzheimer's disease and the like.

Drawings

FIG. 1 is a graph showing the effect of different doses of Compound 4j on body weight in mice in Experimental example 4.

Fig. 2 is a graph of mouse jump experiment, wherein a represents a histogram of mouse latency and B represents a histogram of mouse error number, in which # P <0.05, # P < 0.001: compared with a control group; p <0.05, P < 0.01: compared to the model set.

Detailed Description

The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.

Example 1

1) Preparation of a compound of formula 2:

adding a proper amount of ZnCl into a single-neck flask₂(15eq), POCl was added₃The solution (25eq) was dissolved and the temperature was raised to 75 ℃. With stirring, an appropriate amount of phloroglucinol (1.2eq) and salicylic acid (1eq) were added and reacted at 75 ℃ for 2 hours. After the reaction is finished, cooling to room temperature, pouring the solution into an ice water bath, stirring for 10 minutes, extracting with ethyl acetate for three times, combining organic layers, drying with anhydrous sodium sulfate, filtering under reduced pressure to obtain a filtrate, mixing the filtrate with a sample, and purifying with a silica gel column (petroleum ether: ethyl acetate: 25: 3, volume ratio) to obtain the target product.

2) Preparation of a compound represented by formula 3:

dissolving the compound (1eq) shown in the formula 2, potassium carbonate (2eq) and dibromoalkanes (50eq) with different lengths in a proper amount of DMF, and stirring for reaction at room temperature for 6 hours. After the reaction, the reaction mixture was filtered, and the filtrate was purified by spin-drying on a silica gel column (petroleum ether: ethyl acetate: 30: 1, volume ratio) to obtain the target products 3a to f, the structures of which are shown in table 1 below.

TABLE 1 concrete Structure of the Compound represented by formula 3

3) Preparation of Compounds represented by formulas 4 a-n:

wherein n is 3-8; r₁Represents H, Me or Et; r₂Denotes H, OH or OCH₃；R₃Denotes H, OH or OCH₃。

Dissolving the compound (1eq) shown in 3a-f in a proper amount of acetonitrile, adding sodium iodide (1.1eq), triethylamine (1.5eq) and corresponding alkylbenzylamine (1.2eq), and heating to reflux for reaction for 9-12 hours. After completion of the reaction, the reaction mixture was cooled, the solvent was evaporated under reduced pressure, and the residue was purified with a silica gel column (dichloromethane: methanol: 30: 1, volume ratio) to obtain the intended product 4a-n, the structure of which is shown in table 2 below.

4) Formulas 5a-f were prepared as follows:

wherein R is₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

Dissolving the compound 4j (1eq) or 4n (1eq) in a proper amount of DMF, adding triethylamine (2eq) and corresponding isocyanate (5eq), and stirring at room temperature for 36-48 hours under the protection of nitrogen. After the reaction, the solution was poured into water, stirred for 5 minutes, extracted twice with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, filtered under reduced pressure to give a filtrate, and then sample-mixed and purified by silica gel column (dichloromethane: methanol 50: 1, volume ratio) to give the target products 5a-f, the structures of which are shown in table 2 below.

TABLE 2 concrete structures of the compounds represented by the formulae 4 and 5

Each target product and its characterization were as follows:

compound 4 a: yield 69%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.72(s,1H),8.17(d,J＝7.8Hz,1H),7.90(t,J＝8.4Hz,1H),7.63(d,J＝8.4Hz,1H),7.51(t,J＝7.8Hz,1H),7.27(s,1H),6.89(d,J＝28.2Hz,3H),6.66(s,1H),6.40(s,1H),4.36(s,1H),4.22(s,3H),3.18(s,2H),2.72(s,3H),2.18(s,2H).¹³C NMR(151MHz,DMSO)δ180.68,165.99,163.08,158.09,157.78,155.96,136.54,130.41,125.82,125.15,121.94,120.33,118.27,117.00,103.74,98.01,93.79,66.31,59.10,55.41,52.51,27.02.HRMS:calcd for C₂₄H₂₄NO₅[M+H]⁺406.1649,found 406.1604.

Compound 4 b: yield 71%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.82(s,1H),9.72(s,1H),8.17(dd,J＝7.8,1.8Hz,1H),7.92–7.89(m,1H),7.63(d,J＝8.4Hz,1H),7.53–7.50(m,1H),7.26(t,J＝7.8Hz,1H),6.93–6.91(m,2H),6.86(dd,J＝8.4,1.8Hz,1H),6.66(d,J＝2.4Hz,1H),6.42(d,J＝2.4Hz,1H),4.33(d,J＝12.0Hz,1H),4.17(t,J＝6.0Hz,3H),3.21(s,1H),3.10–3.09(m,1H),2.69(s,3H),1.88–1.77(m,4H).¹³C NMR(151MHz,DMSO)δ180.60,166.36,163.07,157.93,157.77,155.93,136.44,130.04,125.78,125.07,120.31,118.23,103.57,97.95,93.72,68.64,55.72,48.94,30.57,29.47.HRMS:calcd for C₂₅H₂₆NO₅[M+H]⁺420.1805,found 420.1757.

Compound 4 c: yield 65% as a yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.73(s,1H),8.17(dd,J＝8.4,1.8Hz,1H),7.91–7.88(m,1H),7.63(d,J＝8.3Hz,1H),7.52–7.50(m,1H),7.27(t,J＝7.8Hz,1H),6.93–6.86(m,3H),6.66(d,J＝2.4Hz,1H),6.42(d,J＝2.4Hz,1H),4.32(d,J＝12.0Hz,1H),4.15(t,J＝6.3Hz,3H),3.13–3.09(m,2H),2.65(s,3H),1.81–1.74(m,4H),1.47–1.42(m,2H).¹³C NMR(151MHz,DMSO)δ180.61,166.44,163.09,158.08,157.79,155.94,136.47,131.77,130.44,125.79,125.10,121.98,120.32,118.26,118.23,116.98,103.57,97.94,93.73,68.72,58.94,55.21,46.22,28.23,23.40,23.01.HRMS:calcd for C₂₆H₂₈NO₅[M+H]⁺434.1962,found 434.1902.

Compound 4 d: yield 67%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,J＝7.8,1.8Hz,1H),9.72(s,1H),8.16(dd,J＝7.9,1.6Hz,1H),7.91–7.88(m,1H),7.62(d,J＝8.4Hz,1H),7.52–7.49(m,1H),7.27(t,J＝7.8Hz,1H),6.92–6.86(m,3H),6.65(d,J＝1.8Hz,1H),6.41(d,J＝2.4Hz,1H),4.31(d,J＝11.4Hz,1H),4.14(t,J＝6.0Hz,3H),3.12–3.09(m,1H),3.00(s,1H),2.67(s,3H),1.79–1.69(m,4H),1.47–1.43(m,2H),1.36–1.34(m,2H).¹³C NMR(151MHz,DMSO)δ180.60,166.50,163.09,158.07,157.80,155.94,136.46,131.81,130.44,125.79,125.09,121.95,120.32,118.23,116.96,103.55,97.92,93.70,68.94,58.93,55.25,46.21,28.52,26.07,25.38,23.66.HRMS:calcd for C₂₇H₃₀NO₅[M+H]⁺448.2118,found 448.2050.

Compound 4 e: yield 68% as a yellow solid powder.¹H NMR(600MHz,DMSO)δ12.80(s,1H),9.72(s,1H),8.16(d,J＝7.8Hz,1H),7.89(t,J＝8.4Hz,1H),7.62(d,J＝8.4Hz,1H),7.50(t,J＝7.8Hz,1H),7.26(t,J＝7.2Hz,1H),6.91–6.85(m,3H),6.65(s,1H),6.40(s,1H),4.29(s,1H),4.13(d,J＝6.0Hz,3H),3.04(d,J＝54.6Hz,2H),2.66(s,3H),1.42(d,J＝6.6Hz,2H),1.37–1.22(m,8H).¹³C NMR(151MHz,DMSO)δ180.60,166.53,163.08,158.07,157.79,155.93,136.45,130.43,125.78,125.08,121.92,120.31,118.23,116.93,103.53,97.91,93.69,69.04,58.96,55.32,28.68,28.59,26.34,25.62.HRMS:calcd for C₂₈H₃₂NO₅[M+H]⁺462.2275,found 462.2183.

Compound 4 f: yield 65% as a yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.72(s,1H),8.16(d,J＝6.0Hz,1H),7.89(t,J＝8.4Hz,1H),7.62(d,J＝7.8Hz,1H),7.52–7.49(m,1H),7.27(t,J＝7.8Hz,1H),6.91–6.85(m,3H),6.65(d,J＝1.8Hz,1H),6.40(d,J＝1.2Hz,1H),4.29(s,1H),4.13(t,J＝6.0Hz,3H),3.08(s,1H),2.98(s,1H),2.66(s,3H),1.77–1.67(m,4H),1.43–1.40(m,2H),1.33–1.30(m,6H).¹³C NMR(151MHz,DMSO)δ180.59,166.54,163.08,158.07,157.79,155.93,136.44,131.81,130.43,125.78,125.07,121.93,120.32,118.23,116.94,103.52,97.91,93.69,69.07,58.92,55.29,28.88,28.86,28.78,26.35,25.75,23.69.HRMS:calcd for C₂₉H₃₄NO₅[M+H]⁺476.2431,found 476.2363.

Compound 4 g: yield 61%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.82(s,1H),9.81(s,1H),8.17(d,J＝8.4Hz,1H),7.92–7.89(m,1H),7.64(d,J＝8.4Hz,1H),7.52(d,J＝7.2Hz,1H),7.33(d,J＝7.8Hz,2H),6.84(d,J＝9.0Hz,2H),6.66(s,1H),6.41(s,1H),4.32(s,1H),4.22(d,J＝6.0Hz,3H),3.29(s,2H),2.70(s,3H),2.18(s,2H).¹³C NMR(151MHz,DMSO)δ180.67,165.95,163.07,158.97,157.77,155.95,136.54,133.21,125.81,125.15,120.31,118.27,116.01,103.74,98.00,93.79,66.27,58.79,52.02,46.23,23.85.HRMS:calcd for C₂₄H₂₄NO₅[M+H]⁺406.1649,found 406.1608.

Compound 4 h: yield 59%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.82(s,1H),9.80(s,1H),8.17(dd,J＝7.8,1.2Hz,1H),7.92–7.89(m,1H),7.64(d,J＝8.4Hz,1H),7.52(d,J＝7.2Hz,1H),7.32(d,J＝8.4Hz,2H),6.82(d,J＝8.4Hz,2H),6.67(d,J＝2.4Hz,1H),6.43(d,J＝1.8Hz,1H),4.28(d,J＝12.0Hz,1H),4.18–4.14(m,3H),3.18(s,1H),3.04(s,1H),2.66(s,3H),1.87–1.77(m,4H).¹³C NMR(151MHz,DMSO)δ180.64,166.26,163.09,158.96,157.79,155.95,136.50,133.23,125.80,125.13,120.33,118.25,115.99,103.63,97.98,93.78,68.39,58.63,54.47,46.21,25.91,20.85.HRMS:calcd for C₂₅H₂₆NO₅[M+H]⁺420.1805,found 420.1746.

Compound 4 i: yield 63% as a yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.81(s,1H),8.16(dd,J＝7.8,1.8Hz,1H),7.91–7.88(m,1H),7.62(d,J＝8.4Hz,1H),7.51(d,J＝7.2Hz,1H),7.32(d,J＝8.4Hz,2H),6.83(d,J＝8.4Hz,2H),6.66(d,J＝2.4Hz,1H),6.41(d,J＝1.8Hz,1H),4.28(d,J＝12.6Hz,1H),4.15(d,J＝6.0Hz,2H),4.11(d,J＝9.0Hz,1H),3.10(d,J＝4.8Hz,1H),2.98(s,1H),2.65(s,3H),1.80–1.73(m,4H),1.46–1.42(m,2H).¹³C NMR(151MHz,DMSO)δ180.61,166.44,163.08,158.95,157.79,155.93,136.47,133.20,125.79,125.09,120.31,118.23,116.01,103.56,97.93,93.72,68.73,58.62,54.72,38.97,28.23,23.45,23.03.HRMS:calcd for C₂₆H₂₈NO₅[M+H]⁺434.1962,found 434.1897.

Compound 4 j: yield 70%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.80(s,1H),8.17(dd,J＝7.8,1.8Hz,1H),7.91–7.88(m,1H),7.63(d,J＝8.4Hz,1H),7.51(d,J＝7.8Hz,1H),7.31(d,J＝8.4Hz,2H),6.83(d,J＝8.4Hz,2H),6.66(d,J＝2.4Hz,1H),6.41(d,J＝1.8Hz,1H),4.27(d,J＝12.6Hz,1H),4.15–4.10(m,3H),3.09(s,1H),2.95(s,1H),2.65(s,3H),1.78–1.68(m,4H),1.47–1.42(m,2H),1.37–1.33(m,2H).¹³C NMR(151MHz,DMSO)δ180.61,166.51,163.09,158.94,157.80,155.94,136.47,133.18,125.79,125.09,120.32,118.24,116.01,103.55,97.93,93.71,68.94,58.62,54.79,46.21,28.52,26.09,25.37,23.71.HRMS:calcd for C₂₇H₃₀NO₅[M+H]⁺448.2118,found 448.2060.

Compound 4 k: yield 75% as a yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.79(s,1H),8.17(dd,J＝7.8,1.8Hz,1H),7.91–7.89(m,1H),7.63(d,J＝8.4Hz,1H),7.52–7.50(m,1H),7.31(d,J＝8.4Hz,2H),6.83(d,J＝8.4Hz,2H),6.66(d,J＝1.8Hz,1H),6.41(d,J＝1.8Hz,1H),4.26(d,J＝13.2Hz,1H),4.14(t,J＝6.6Hz,3H),3.00–2.95(m,2H),2.64(s,3H),1.77–1.70(m,4H),1.44–1.41(m,2H),1.37–1.35(m,2H),1.23(t,J＝4.8Hz,2H).¹³C NMR(151MHz,DMSO)δ180.62,166.55,163.09,158.94,157.82,155.94,136.48,133.19,125.79,125.10,120.40,120.33,118.25,116.00,103.55,97.94,93.72,69.06,58.59,54.83,53.15,28.68,28.57,26.33,25.61,25.51.HRMS:calcd for C₂₈H₃₁NO₅[M+H]⁺462.2275,found 462.2193.

Compound 4 l: yield 81%, yellow solid powder.¹H NMR(600MHz,DMSO)δ12.81(s,1H),9.80(s,1H),8.16(dd,J＝7.8,1.8Hz,1H),7.91–7.88(m,1H),7.62(d,J＝8.4Hz,1H),7.51(d,J＝7.8Hz,1H),7.30(d,J＝8.4Hz,2H),6.83(d,J＝8.4Hz,2H),6.65(d,J＝1.8Hz,1H),6.40(d,J＝2.4Hz,1H),4.25(d,J＝12.6Hz,1H),4.14–4.10(m,3H),3.06(s,1H),2.94(s,1H),2.64(s,3H),1.77–1.72(m,2H),1.68–1.64(m,2H),1.43–1.39(m,2H),1.35–1.30(m,6H).¹³C NMR(151MHz,DMSO)δ180.62,166.56,163.10,158.95,157.81,155.95,136.47,133.18,125.79,125.09,120.33,118.25,116.01,103.54,97.93,93.71,69.08,58.62,54.83,46.22,28.88,28.85,28.78,26.36,25.74,23.74.HRMS:calcd for C₂₉H₃₄NO₅[M+H]⁺476.2431,found 476.2357.

Compound 4 m: yield 69%, yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.88(s,1H),8.27(dd,J＝8.4,1.8Hz,1H),7.74–7.71(m,1H),7.44(d,J＝8.4Hz,1H),7.41–7.38(m,1H),7.25(d,J＝8.4Hz,2H),6.88(d,J＝9.0Hz,2H),6.44(d,J＝2.4Hz,1H),6.36(d,J＝2.4Hz,1H),4.06(t,J＝6.0Hz,2H),3.82(s,3H),3.49(s,2H),2.42(t,J＝7.2Hz,2H),2.23(s,3H),1.86–1.82(m,2H),1.62–1.57(m,2H),1.52–1.47(m,2H),1.43–1.40(m,2H).¹³C NMR(151MHz,CDCl₃)δ180.78,166.34,163.52,158.73,157.74,156.04,134.96,130.38,125.87,123.98,120.66,117.58,113.61,103.83,97.46,93.24,68.62,61.51,56.96,55.25,41.93,28.91,27.09,27.05,25.86.HRMS:calcd for C₂₈H₃₂NO₅[M+H]⁺462.2275,found 462.2187.

Compound 4 n: yield 74% yellow solid powder.¹H NMR(600MHz,DMSO)δ12.74(s,1H),9.26(s,1H),8.15(d,J＝7.8Hz,1H),7.88(t,J＝7.8Hz,1H),7.62(d,J＝8.4Hz,1H),7.50(t,J＝7.2Hz,1H),7.07(d,J＝8.4Hz,2H),6.68(d,J＝8.4Hz,2H),6.63(s,1H),6.39(s,1H),4.10(t,J＝6.6Hz,2H),2.42–2.39(m,2H),2.34(t,J＝7.2Hz,2H),1.71(t,J＝7.2Hz,2H),1.42(t,J＝7.2Hz,2H),1.38(t,J＝7.8Hz,2H),1.30(t,J＝6.6Hz,4H),0.95(t,J＝7.2Hz,3H).¹³C NMR(151MHz,DMSO)δ180.58,166.56,163.08,157.79,156.52,155.93,136.40,130.13,125.76,125.03,120.32,118.23,115.25,103.51,97.93,93.67,69.05,57.28,52.52,46.84,28.83,26.97,25.69,22.56,11.98.HRMS:calcd for C₂₈H₃₂NO₅[M+H]⁺462.2275,found 462.2198.

Compound 5 a: yield 48%, yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.88(s,1H),8.26(dd,J＝7.8,1.8Hz,1H),7.74–7.71(m,1H),7.45(d,J＝8.4Hz,1H),7.40–7.38(m,1H),7.30(d,J＝8.4Hz,2H),7.07(d,J＝8.4Hz,2H),6.44(d,J＝1.8Hz,1H),6.36(d,J＝2.4Hz,1H),5.19(s,1H),4.04(t,J＝6.6Hz,2H),3.47(s,2H),3.34–3.29(m,2H),2.37(t,J＝7.8Hz,2H),2.21(s,3H),1.83–1.81(m,2H),1.58–1.53(m,2H),1.47–1.45(m,2H),1.42–1.39(m,2H),1.24–1.21(m,3H).¹³C NMR(151MHz,CDCl₃)δ180.81,166.40,163.45,158.45,157.74,156.05,149.92,134.99,129.81,125.83,123.99,121.29,120.63,117.60,103.81,97.50,93.28,68.68,61.78,59.01,57.00,42.26,36.12,28.91,27.19,26.98,25.79,15.11.HRMS:calcd for C₃₀H₃₅N₂O₆[M+H]⁺519.2489,found 519.2392.

Compound 5 b: yield 45% yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.88(br s,1H),8.27(dd,J＝7.8,1.2Hz,1H),7.74–7.71(m,1H),7.45(d,J＝8.4Hz,1H),7.41–7.36(m,3H),7.12(d,J＝8.4Hz,2H),6.44(d,J＝2.4Hz,1H),6.36(d,J＝1.8Hz,1H),5.09(s,1H),4.05(d,J＝6.0Hz,2H),δ3.70(t,J＝5.4Hz,2H),3.65–3.62(m,3H),3.56(t,J＝6.0Hz,1H),2.47(t,J＝7.2Hz,2H),2.29(s,3H),1.85–1.80(m,2H),1.65–1.60(m,2H),1.50–1.45(m,2H),1.43–1.39(m,2H).¹³C NMR(151MHz,CDCl₃)δ180.80,166.35,163.47,157.74,157.37,156.05,150.11,134.99,130.27,125.86,123.99,121.44,120.64,117.60,103.83,97.49,93.27,68.60,61.36,56.68,43.89,42.99,41.84,28.87,26.89,26.66,25.74.HRMS:calcd for C₃₀H₃₄ClN₂O₆[M+H]⁺553.2099,found 553.1986.

Compound 5 c: yield 44%, yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.89(s,1H),8.27(dd,J＝7.8,1.2Hz,1H),7.74–7.71(m,1H),7.45(d,J＝8.4Hz,1H),7.41–7.38(m,1H),7.30(d,J＝8.4Hz,2H),7.09(d,J＝8.4Hz,2H),6.44(d,J＝2.4Hz,1H),6.37(d,J＝1.8Hz,1H),4.97(d,J＝7.8Hz,1H),4.05(t,J＝6.0Hz,2H),3.59–3.54(m,1H),3.49(s,2H),2.38(t,J＝7.2Hz,2H),2.22(s,3H),2.02(d,J＝9.0Hz,2H),1.85–1.73(m,8H),1.65–1.53(m,4H),1.48–1.46(m,2H),1.42–1.40(m,2H).¹³C NMR(151MHz,CDCl₃)δ180.79,166.39,163.49,157.73,156.05,153.77,150.01,134.96,129.83,125.86,123.97,121.30,120.65,117.59,103.82,97.50,93.27,68.67,61.75,56.96,50.11,42.23,33.26,28.92,27.16,27.00,25.81,25.46,24.76.HRMS:calcd for C₃₄H₄₀N₂O₆[M+H]⁺573.2959,found 573.2837.

Compound 5 d: yield 45% yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.89(s,1H),8.27(dd,J＝7.8,1.2Hz,1H),7.74–7.72(m,1H),7.45(d,J＝7.8Hz,1H),7.39(d,J＝7.8Hz,1H),7.32(d,J＝8.4Hz,2H),7.07(d,J＝7.8Hz,2H),6.44(d,J＝2.4Hz,1H),6.37(d,J＝2.4Hz,1H),5.08(s,1H),4.03(d,J＝6.6Hz,2H),3.55(s,2H),3.33–3.31(m,2H),2.55–2.51(m,2H),2.44(t,J＝7.2Hz,2H),1.81(d,J＝6.6Hz,2H),1.51(t,J＝7.8Hz,2H),1.45–1.42(m,2H),1.39–1.37(m,2H),1.23(t,J＝7.2Hz,3H),1.05(d,J＝7.2Hz,3H).¹³C NMR(151MHz,CDCl₃)δ180.80,166.42,163.48,157.73,156.06,154.62,149.78,134.97,129.58,125.86,123.97,121.22,120.65,117.59,103.81,97.50,93.29,68.69,57.50,52.75,47.27,36.12,28.93,26.99,25.75,15.14,11.72.HRMS:calcd for C₃₁H₃₇N₂O₆[M+H]⁺533.2646,found 533.2527.

Compound 5 e: yield 45% yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.89(br s,1H),8.26(dd,J＝7.8,1.8Hz,1H),7.74–7.71(m,1H),7.45(d,J＝8.4Hz,1H),7.40–7.38(m,3H),7.10(d,J＝8.4Hz,2H),6.44(d,J＝2.4Hz,1H),6.36(d,J＝2.4Hz,1H),5.15(s,1H),4.03(d,J＝6.0Hz,2H),3.70(t,J＝5.4Hz,2H),3.64–3.62(m,3H),3.56(t,J＝6.0Hz,1H),2.61(s,2H),2.51(s,2H),1.82–1.80(m,2H),1.57(s,2H),1.47–1.42(m,2H),1.40–1.37(m,2H),1.11(s,3H).¹³C NMR(151MHz,CDCl₃)δ180.82,166.38,163.48,157.74,157.45,156.06,154.63,135.00,130.01,125.87,124.00,121.35,120.65,117.60,103.83,97.50,93.29,68.63,64.33,57.24,52.51,43.00,42.14,28.89,26.91,25.70,11.30.HRMS:calcd for C₃₁H₃₆ClN₂O₆[M+H]⁺567.2256,found 567.2134.

Compound 5 f: yield 43%, yellow solid powder.¹H NMR(600MHz,CDCl₃)δ12.89(s,1H),8.27(dd,J＝8.4,1.8Hz,1H),7.74–7.72(m,1H),7.45(d,J＝8.4Hz,1H),7.41–7.38(m,1H),7.33(t,J＝7.8Hz,2H),7.07(d,J＝7.8Hz,2H),6.44(d,J＝2.4Hz,1H),6.37(d,J＝2.4Hz,1H),4.98(d,J＝7.8Hz,1H),4.03(t,J＝6.6Hz,2H),3.53(s,1H),3.50(s,2H),2.55–2.51(m,2H),2.45–2.43(m,2H),1.82–1.79(m,2H),1.65–1.60(m,4H),1.54–1.49(m,2H),1.38–1.35(m,6H),1.23(d,J＝10.2Hz,3H),1.07–1.04(m,4H).¹³C NMR(151MHz,CDCl₃)δ180.80,166.41,163.49,157.73,156.76,156.06,149.82,134.96,129.99,129.57,129.41,125.86,123.97,121.21,120.66,117.60,115.08,103.81,97.50,93.29,68.69,57.49,52.75,50.10,47.25,33.96,28.93,27.00,26.85,25.62,24.95,11.84.HRMS:calcd for C₃₅H₄₃N₂O₆[M+H]⁺587.3115,found 587.2982.

Example 2: preparation of Compounds represented by formulas 4 a-n:

wherein n is 3-8; r₁Represents H, Me or Et; r₂Denotes H, OH or OCH₃；R₃Denotes H, OH or OCH₃。

Dissolving a compound (1eq) shown by 3a-f in a proper amount of a first organic solvent (the solvent adopted by a target compound 3a-b is ethanol, the solvent adopted by a target compound 3c is water, the solvent adopted by a target compound 3d is acetone, the solvent adopted by a target compound 3e is tetrahydrofuran, and the solvent adopted by a target compound 3f is DMF), adding potassium iodide (1eq), an acid-binding agent (1.5eq), the acid-binding agent adopted by the target compound 3a-c is potassium carbonate, the acid-binding agent adopted by the target compound 3d is pyridine, the acid-binding agent adopted by the target compound 3e is sodium hydroxide, the acid-binding agent adopted by the target compound 3f is N, N-diisopropyl ethyl amine) and corresponding alkylbenzylamine (1.2eq), and heating to 70 ℃ for reaction to be complete. After the reaction, the reaction mixture was cooled, the solvent was evaporated under reduced pressure, the residue was purified with a silica gel column (dichloromethane: methanol: 30: 1, volume ratio), and the obtained compounds were each characterized by structure (hydrogen nuclear magnetic resonance spectrum, carbon spectrum, high resolution mass spectrum), and were each identified as the target products 4a to n.

Example 3: preparation of a compound of formula 4 a:

the compound 3a (1eq) was dissolved in an appropriate amount of acetonitrile, triethylamine (1.5eq) and 3- [ (methylamino) methyl ] phenol (1.2eq) were added, and the mixture was heated to reflux reaction until completion. After the reaction is finished, the reaction product is cooled, the solvent is evaporated under reduced pressure, and the reaction product is purified by a silica gel column (dichloromethane: methanol: 30: 1, volume ratio) to obtain a yellow solid, and the yellow solid is determined to be the target product 4a through structural characterization (nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high resolution mass spectrum).

Example 4: preparation of formulas 5 a-f:

wherein R is₄Represents Et, CH₃CH₂Cl or Cyclohexyl.

Dissolving a compound 4j (1eq) or 4n (1eq) in a proper amount of a second organic solvent (the solvent adopted by the target compound 5a-b is ethanol, the solvent adopted by the target compound 5c-d is methanol, and the solvent adopted by the target compound 5e-f is tetrahydrofuran), adding a catalyst (2eq, the catalyst adopted by the target compound 5a-b is triethylene diamine, the catalyst adopted by the target compound 5c-d is dibutyltin dilaurate, and the catalyst adopted by the target compound 5e-f is stannous octoate) and corresponding isocyanate (5eq), and stirring at room temperature for 36-48 hours under the protection of nitrogen. After the reaction is finished, pouring the solution into water, stirring for 5 minutes, extracting twice with ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, filtering under reduced pressure to obtain filtrate, mixing with a sample, purifying with a silica gel column (petroleum ether: n-butanol is 50: 1, volume ratio), respectively carrying out structural characterization (nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high resolution mass spectrum) on the obtained compounds, and determining that the obtained compounds are respectively target products 5 a-f.

Experimental example 1: experiment on cholinesterase inhibitory ability of the Compound of the present invention

The methods for testing the inhibitory activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) refer to the Ellman method. Compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted sequentially to 5 desired concentrations. To a 96-well plate, 160. mu.L of a solution of 5, 5' -dithiobis (2-nitrobenzoic acid) (DTNB), 50. mu.L of AChE and 10. mu.L of the corresponding concentration of compound were added sequentially, incubated at 37 ℃ for 5 minutes, followed by rapid addition of 30. mu.L of the acetylcholine iodide substrate. Absorbance was measured at 405nm with a microplate reader for 0,60,120 and 180 seconds. The butyrylcholinesterase was tested as above. The results are shown in Table 3 below.

TABLE 3 inhibitory Activity of the Compounds of the present invention on AChE and BuChE

Note:^aAChE was derived from electric eels, and the results values are expressed as mean ± SD of at least three independent measurements.

^bBuChE was derived from horse serum and the results values are expressed as mean ± SD of at least three independent measurements.

As can be seen from Table 3, the xanthone compounds of the present invention have certain inhibitory activity against both acetylcholinesterase and butyrylcholinesterase. The structure-activity relationship shows that the change of the inhibition ability of cholinesterase changes along with the change of the chain length of methylene of the compound 4 a-l. In particular, methylene chains of compoundsWhen the number of carbon atoms is 3, the inhibition of cholinesterase is weak. When the methylene chain of the compound is extended to 4, the inhibition capacity of cholinesterase is remarkably improved, for example, the inhibition capacity of the compound 4b on acetylcholinesterase is increased by 8.8 times compared with that of the compound 4 a; compared with 4g, the compound 4h has 41-fold increased inhibitory capacity on butyrylcholinesterase. When the length of the methylene chain of the compound is continuously prolonged to 7 carbon atoms, the result shows that the cholinesterase inhibition capability of the compound with even number of the carbon atoms of the methylene chain is better than that of the corresponding odd compound. When the chain length of the methylene is further prolonged to 8 carbon atoms, the inhibitory activity of the compound on cholinesterase is greatly reduced, for example, the inhibitory activity of 4e and 4k on acetylcholinesterase is reduced to 42.6% of the inhibitory rate of the compound under the concentration of 100 mu M, and the inhibitory capacity on butyrylcholinesterase is respectively reduced by 12 times and 5.3 times. When the number of methylene chain carbon atoms of the compound is 6, the compound 4j shows the optimal double-target inhibition capacity, and the inhibition capacities on acetylcholinesterase and butyrylcholinesterase are IC respectively₅₀0.85. mu.M and IC₅₀0.59 μ M. In order to further enhance the cholinesterase inhibition capability of the compound, the terminal benzene ring hydroxyl of the compound 4j is grafted with methyl and carbamate for optimization to obtain compounds 4m and 5 a-f. However, from the results, the inhibitory activity of the carbamate compound on cholinesterase was rather reduced. The applicant speculates that the hydroxyl on the benzene ring at the terminal may interact with cholinesterase to form a hydrogen bond to play an inhibiting role. Therefore, the compound 4j is expected to have better curative effect on mild and severe Alzheimer's disease.

Experimental example 2: antioxidant capacity test of the Compound of the present invention

The DPPH, ABTS, and ORAC methods were used to examine the radical scavenging ability of the target compounds, and the results are shown in Table 4 below.

The DPPH method. DPPH and the target compound are dissolved in DMSO to prepare a stock solution, and the stock solution is diluted with methanol to prepare a working solution with the required concentration. Respectively placing 100 μ L DPPH working solution and 5 samples with different concentrations and 100 μ L in 96-well plate, shaking, mixing, reacting at room temperature in dark place for 30min, and reacting with enzymeThe standard instrument measures the OD value of each hole under the condition of 517nm, and Trolox is used as a positive drug. Two control groups were set up for this experiment, one control group without the target compound (DPPH solution + methanol solution) and one control group without DPPH (methanol solution + sample solution). DPPH free radical clearance%₁-A₂)/A₀Wherein A is₁Is DPPH solution + sample solution, A₂Is methanol solution + sample solution, A₀Is DPPH solution + methanol solution. The experiment was repeated three times and the mean value was taken and the half-maximal clearance concentration (IC) of the compound to DPPH was calculated using GraphPad software₅₀)。

ABTS method. The ABTS method was performed using a petyunsday S0119 kit according to the instructions. The ABTS working mother liquor is prepared by using equal volume of ABTS stock solution and oxidant stock solution to react at room temperature and dark place according to the ratio of 1:1 for 12-16 hours. The target compound was diluted into 5 working solutions of different concentrations. Adding 190 μ L of ABTS working solution into a 96-well plate, adding 10 μ L of 80% ethanol solution into a blank control hole, adding 10 μ L of sample solutions with different concentrations into a sample detection hole, and gently mixing. After incubation for 5 minutes at room temperature, detection was carried out under a microplate reader at 405nm, and Trolox was used as a positive drug. Two sets of controls were set up for this experiment, a control without sample (ABTS solution + ethanol solution) and a control without ABTS (ethanol solution + sample solution). ABTS free radical clearance%₁-A₂)/A₀Wherein A is₁Is sample solution + ABTS working solution, A₀Is ABTS working solution and ethanol solution, A₂The sample solution + ethanol solution. The experiment was repeated three times and the mean value was taken and the half maximal clearance concentration (IC) of the compound to ABTS was calculated using GraphPad software₅₀)。

ORAC method. The free-radical generator 2, 2-azobis (2-methylpropylamidine) dihydrochloride (AAPH) and sodium Fluorescein (FL) were dissolved in 10mM phosphate buffer at pH 7.4 and stored at low temperature in the dark. The target compound is dissolved in DMSO to prepare a stock solution, and the stock solution is diluted into a working solution with the required concentration by using a buffer solution of 50% acetone. Adding 20 μ L of different concentrated sample working solutions into black 96-well plate, incubating 120 μ L of FL working solution at 37 deg.C for 5min, adding 60 μ L of AAPH working solution into each well to start reaction, and rapidly adding enzyme labelAnd (5) detecting in the instrument. The enzyme-linked immunosorbent assay device is set at 37 ℃, the excitation wavelength is 485nm, the emission wavelength is 538nm, the fluorescence intensity is measured every 5min, the fluorescence intensity is measured for 90min, and Trolox is used as a positive drug. ORAC experiment A control group (+ AAPH) of free radical scavenging effect was set up without antioxidant. The absolute fluorescence intensity values at different time points of each microwell were compared with each initial fluorescence intensity and converted into relative fluorescence intensities f to calculate the area under the fluorescence quenching curve (AUC), which was expressed by the formula AUC 0.5 × [2 × (f)₀+f₁+...+f_n-1+f_n)-f₀-f_n]xDeltat, where Deltat indicates a test interval of 5min, f_nRepresents the relative fluorescence intensity at the nth measurement point. The difference between the area under the fluorescence decay curve in the presence of the antioxidant and the area under the fluorescence decay curve in the absence of the antioxidant is the protection area of the antioxidant, i.e., the Net AUC. The antioxidant capacity of the samples is expressed as the equivalent of Trolox, ORAC, e.g.the Net AUC of a 1. mu.M sample corresponds to the Net AUC of 3. mu.M Trolox, and the ORAC value is 3.

TABLE 4 scavenging ability of the compounds of the invention for free radicals

Note:^aDPPH radical clearance at half inhibitory concentration or 1mM drug concentration.

^bABTS free radical clearance at half inhibitory concentration or 100. mu.M drug concentration.

^cTrolox equivalent.

As can be seen from Table 4, the compound has a large fluctuation range in the scavenging ability for DPPH and ABTS radicals, and the compound 4c is the most excellent scavenging ability. The structure-activity relationship shows that the change of the free radical scavenging capacity of the compound is related to the length of a methylene chain of the compound and the position of a hydroxyl group on a terminal benzene ring. E.g., 4a-f and 4g-l, the radical scavenging ability of the compounds increases with the length of the methylene chain, whereas the compounds with a meta hydroxyl group on the terminal phenyl ring are more active than those with a para hydroxyl group, contrary to the above law for cholinesterase activity. When the terminal hydroxyl group is replaced with another group to obtain compounds 5a-f, the compound has a drastically reduced ability to scavenge DPPH and ABTS radicals. Also, the compounds have a broad free radical scavenging capacity as measured by ORAC. Among them, compounds 4a-d,4h,4j,4n and 5e have stronger radical quenching ability than the positive drug Trolox (ORAC >1), and the compound 4b with the strongest quenching ability is close to the strong radical scavenger quercetin. The quenching capacity of the compound structure change on free radicals is basically consistent with the rule measured by DPPH and ABTS methods.

Experimental example 3: blood brain Barrier Transmission test of the Compound of the present invention

The PAMPA-BBB artificial membrane method is adopted for determination. The porcine brain phospholipid construct external membrane model was used. A blood-brain barrier permeability curve was established by testing 9 commercially available drugs with different blood-brain barrier permeabilities.

The specific method comprises the following steps: the PAMPA model experiment was performed in 96-well filter plates, and a 2% solution of porcine brain phospholipid/dodecane (V/V) was prepared and dissolved by sonication until well mixed. mu.L of this solution was applied evenly to the lipophilic filter in each well of the PAMPA donor plate, allowed to stand for 5min, and 5. mu.L of dodecane solution was added to the control wells. To each well of the receiver plate, 300. mu.L of buffer (PBS/EtOH 70:30, pH 7.4) was added, and to each well of the donor plate, 150. mu.L of a pre-prepared commercial drug or target compound (three wells per drug) was added, and the entire dosing process was completed within 10 min. The supply plate was placed on the receiver plate, the lid was closed, and the entire 96-well filter plate assembly was incubated in a 25 ℃ incubator for 16 h. After incubation, the solutions of the donor and receiver plates were collected and the concentrations were determined according to the formula: p_e＝{-V_dV_a/[(V_d+V_a)At]}ln(1-drug_acceptor/drug_equilibrium) Calculating the transmittance P_eIn which V is_dIs donor pore volume (mL), V_aTo receive the pore volume (mL), A is the filter area (cm)²) T is the penetration time(s), drug_acceptorTo receive the concentration of the plate solution at time t, drug_equilibriumThe theoretical equilibrium absorption concentration. By mixing the known standard drugs in the literature P_eValue and measured P_eValue comparison establishmentThe standard curve of (2) is obtained, and the blood brain barrier permeability P is obtained_eThe value range, the permeability of the compound is judged. The blood-brain barrier permeability coefficients of the commercially available 9 drugs and the blood-brain barrier permeability coefficient of the compound of the present invention are shown in tables 5 and 6 below, respectively.

TABLE 5 blood brain Barrier permeability coefficients for 9 drugs on the market

Note:^adata are derived from the document eur.j.med.chem.2003,38: 223-.

^bMean of three independent experiments, expressed as mean ± SD.

TABLE 6 blood brain Barrier permeability coefficient of target Compounds

Note:^amean of three independent experiments, expressed as mean ± SD.

^bCNS + indicates that the compound can penetrate the blood-brain barrier, CNS-indicates that the compound cannot penetrate the blood-brain barrier, and CNS + -indicates that it cannot be determined whether the compound can penetrate the blood-brain barrier.

The experimental result shows that 18 compounds can reach the therapeutic target through the blood brain barrier except that 4f and 5b can not clearly determine whether the compounds can penetrate the blood brain barrier.

Experimental example 4: acute toxicity study of Compound 4j of the present invention

The specific experimental method comprises the following steps: 40 mice of 18-20g of healthy Kunming seeds are randomly divided into 4 groups, each group comprises 10 mice, each half of the male and female mice is provided with 2500mg/Kg, 1250mg/Kg and 625mg/Kg of dose and a solvent group (0.5 percent of sodium carboxymethyl cellulose solution). The patient is fasted for 12 hours before the experiment without water prohibition and is administrated by single intragastric gavage according to 30mL/Kg (maximum intragastric gavage volume). Mice were observed for abnormalities such as vomiting, diarrhea, shaking, tremor or death within 4 hours after dosing. The observation period is 14 days, the weight, defecation, death and the like of the mice are recorded every day, the mice are sacrificed on the 14 th day, the heart, liver, lung, kidney and brain organs are taken, and the pathological changes of the tissue organs are observed.

The effect of different doses of 4j on mouse body weight after oral administration is shown in figure 1.

The experimental results show that the high concentration of compound 4j does not cause abnormal behaviors or death to the mice, and the body weight of the mice in the administration group is basically consistent with that in the vehicle group. The compound 4j is shown to have no acute toxicity, and the tolerated dose reaches 2500 mg/Kg.

Experimental example 5: in vivo efficacy study of Compound 4j of the present invention

The specific experimental method comprises the following steps: 36 mice of healthy male Kunming species, 18-20g, were randomly divided into six groups of 6 mice each. The mouse is placed in the diving platform box to be familiar with the internal environment for 5min, the switch of the device is turned on to be electrified after the current at the bottom of the diving platform box is set to be 0.4mA (24V), the mouse is trained, the insulated platform can be jumped to avoid after the normal mouse is subjected to electric shock, and the mouse which does not jump down the platform in the training period is discarded without use. The experiment was carried out again after 24 hours. The setting dose is 100mg/Kg, 50mg/Kg and 25mg/Kg, the solvent group, the positive drug donepezil group (10mg/Kg), and the model group (3mg/Kg of scopolamine). The CMC-Na solution and the donepezil solution (10mg/kg) of the compound 4j are administered in a mode of oral gavage 1 hour before the experiment, the CMC-Na solution with the same dose is administered in a solvent group, scopolamine hydrobromide injection is injected in an abdominal cavity after 30 minutes for molding, the trained animals are put into a diving platform box again, current stimulation is performed again after familiarizing for 5 minutes, the time (latency) for the mice to jump down the diving platform for the first time within 5 minutes and the frequency (error frequency) for the mice to jump down the platform within 5 minutes are recorded, and the in-vivo efficacy of the medicine is evaluated according to the latency and the error frequency. The results are shown in FIG. 2.

The experimental results show that the model group injected with scopolamine has shorter latency and more error times compared with the vehicle group, which indicates that the memory impairment model is successfully constructed. After treatment with compound 4j, the latency and number of errors reversed in a dose-dependent manner to some extent. For example, the high (100mg/Kg) and medium (50mg/Kg) dose groups extended the latency, approaching that of the positive drug donepezil. The number of errors was better for the high dose group than for donepezil, but the medium and low dose (25mg/Kg) groups were inferior to donepezil. Taken together, compound 4j is able to ameliorate cognitive impairment by increasing brain cholinesterase levels.