CN112409335B - 1,2,3, 4-tetrahydroacridine-9-amine compound and preparation method and application thereof - Google Patents

1,2,3, 4-tetrahydroacridine-9-amine compound and preparation method and application thereof Download PDF

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CN112409335B
CN112409335B CN202011216802.8A CN202011216802A CN112409335B CN 112409335 B CN112409335 B CN 112409335B CN 202011216802 A CN202011216802 A CN 202011216802A CN 112409335 B CN112409335 B CN 112409335B
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章彬
崔巍
何山
张盼盼
王宁
邹佳美
刘志文
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Abstract

The invention discloses a1, 2,3, 4-tetrahydroacridine-9-amine compound and a preparation method and application thereof, which are characterized in that the compound takes tacrine as a mother nucleus and has structural formulas shown as formulas I and II or pharmaceutically acceptable salts, esters or solvates of the compounds shown as formulas I and II, the compound has the application in the aspects of preparing medicaments for resisting Alzheimer diseases, preparing medicaments for delaying neurodegenerative diseases, preparing medicaments for repairing cholinergic neurons in vitro, preparing medicaments for resisting oxidative stress injury in vitro, preparing medicaments for relieving beta-amyloid (Abeta) neurotoxicity in vitro and in vivo and improving cognitive disorders through CREB-ERK channels or reducing phosphorylation of Tau protein, and has the advantage of excellent activity in the aspect of resisting Alzheimer diseases.

Description

1,2,3, 4-tetrahydroacridine-9-amine compound and preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and particularly relates to a1, 2,3, 4-tetrahydroacridine-9-amine compound, a preparation method thereof and application thereof in resisting Alzheimer disease.
Background
Neurodegenerative diseases are a class of diseases characterized primarily by progressive loss of brain and spinal cord neurons. Alzheimer's Disease (AD) is the most common neurodegenerative Disease in the elderly population. At present, the number of AD patients in the world exceeds 5000 million, and the estimated world health organization estimates that the number of AD patients in the world will reach 8200 million in 2030 and 1.52 million in 2050. At the same time, AD will be second only to cardiovascular disease, the second highest mortality. According to the Alzheimer's society, the lifetime care cost of the AD patients in 2018 reaches to $ 350174, and serious economic burden is brought to social families.
There are numerous hypotheses about the pathogenic mechanism of AD, among which the "cholinergic hypothesis" and the "amyloid-beta hypothesis" have high acceptance. The "cholinergic hypothesis" was the first hypothesis of relevance, based on the observation that cholinergic fibers are lost and acetylcholine levels are reduced in the brain of patients. Acetylcholine is an important neurotransmitter, and its loss can seriously affect neuronal function and signal transmission. beta-Amyloid (β -Amyloid, a β) is a major component of senile plaques and is considered to be the most important marker in the pathological process of AD. In fact, A.beta.is a peptide consisting of 40 or 42 amino acids, formed by cleavage of the APP precursor protein by beta-or gamma-secretase, and can be aggregated into oligomers, polymers or fibers. Among them, oligomers are considered as the most toxic fragments, which can destroy synaptic structure and function, and thus are considered as the main toxic substances causing AD.
Pharmacotherapy is currently the mainstay of AD treatment. The clinical medicines comprise memantine, donepezil, galantamine, rivastigmine and the like, and the medicines have certain curative effect on patients with early AD. However, these drugs have single target and cannot adapt to the complex pathological environment of AD. Therefore, multi-target drug design is considered as a new direction for anti-AD drug development.
Disclosure of Invention
The invention aims to solve the technical problem of providing a1, 2,3, 4-tetrahydroacridine-9-amine compound with excellent activity in the aspect of resisting Alzheimer disease, and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: a1, 2,3, 4-tetrahydroacridine-9-amine compound takes tacrine as a mother nucleus and has a structural formula shown in formulas I and II or pharmaceutically acceptable salts, esters or solvates of the compounds shown in the formulas I and II, and the specific formula is as follows:
Figure BDA0002760675850000021
wherein R is1Is H, CH3、OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or straight-chain alkanes having 1-5 carbon atoms, R2And R3Is H,
Figure BDA0002760675850000022
Figure BDA0002760675850000023
Figure BDA0002760675850000024
n ═ 1,2,3,4 or 5, m ═ 1,2,3,4 or 5, and the salt is an inorganic acid salt or an organic acid salt; the inorganic acid salt is formed by hydrochloric acid, sulfuric acid or phosphoric acid; the organic acid salt is formed by acetic acid, trifluoroacetic acid, malonic acid, citric acid or p-toluenesulfonic acid.
The preparation method of the 1,2,3, 4-tetrahydroacridine-9-amine compound comprises the following steps:
(1) reacting a compound of formula III with sodium hydroxide in aqueous methanol to obtain a compound of formula IV;
(2) reacting a compound of formula IV with a compound of formula V in phosphorus oxychloride to obtain a compound of formula VI;
(3) reacting a compound of formula VI with a compound of formula VII in phenol to obtain a compound of formula VIII;
(4) reacting the compound shown in the formula IX with the compound shown in the formula X under the protection of nitrogen to obtain a compound shown in the formula XI;
(5) reacting a compound of formula XI with sodium hydroxide in aqueous methanol to obtain a compound of formula XII;
(6) reacting a compound shown in a formula XII, a compound shown in a formula VIII and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) in N, N-Dimethylformamide (DMF), and then adding N, N-Diisopropylethylamine (DIPEA) for reaction to obtain a compound shown in a formula I;
(7) reacting a compound of formula VI with a compound of formula X in phenol or N, N-Dimethylformamide (DMF) to obtain a compound of formula II, or reacting a compound of formula VI with a compound of XIII in phenol or N, N-Dimethylformamide (DMF) to obtain a compound of formula II,
Figure BDA0002760675850000031
wherein R is1Is H, CH3、OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or straight-chain alkanes having 1-5 carbon atoms, R2，R3Is H,
Figure BDA0002760675850000032
Figure BDA0002760675850000033
Figure BDA0002760675850000034
n ═ 1,2,3,4 or 5, m ═ 1,2,3,4 or 5, pharmaceutically acceptable salts, esters or solvates of the compounds of formulae i and ii above, wherein the salts are salts of inorganic or organic acids; the inorganic acid salt is formed by hydrochloric acid, sulfuric acid or phosphoric acid; the organic acid salt is acetic acidTrifluoroacetic acid, malonic acid, citric acid or p-toluenesulfonic acid.
The step (1) is specifically as follows: dissolving the compound shown in the formula III and sodium hydroxide in a methanol aqueous solution mixed in an equal volume ratio according to a molar ratio of 1:1.3 at 40 ℃ for reacting for 6 hours to obtain a compound shown in a formula IV;
the step (2) is specifically as follows: adding the compound shown in the formula IV and the compound shown in the formula V into phosphorus oxychloride at the temperature of 0 ℃ according to the molar ratio of 1:1.2, transferring the mixture to the temperature of 110 ℃ and reacting for 6 hours to obtain the compound shown in the formula VI;
the step (3) is specifically as follows: adding the compound shown in the formula VI and the compound shown in the formula VII into phenol according to a molar ratio of 1:2.1 at 170 ℃ by taking potassium iodide as a catalyst to react for 4 hours to obtain a compound shown in the formula VIII;
the step (4) is specifically as follows: adding a compound shown in a formula IX and a compound shown in a formula X into DMF according to a molar ratio of 1.1:1 at 60 ℃ by using potassium iodide as a catalyst and triethylamine as a base, and reacting overnight under the protection of nitrogen to obtain a compound shown in a formula XI;
the step (5) is specifically as follows: dissolving the compound shown in the formula XI and sodium hydroxide in a methanol aqueous solution mixed according to a volume ratio of 4:1 at 50 ℃ according to a molar ratio of 1:1.5, and reacting overnight to obtain a compound shown in the formula XII;
the step (6) is specifically as follows: reacting a compound of formula XII, a compound of formula VIII, and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) at room temperature in the following ratio of 1: 1:1.5 mol ratio, adding the mixture into DMF to react for 20 minutes, adding N, N-Diisopropylethylamine (DIPEA) with 2 times of mol ratio of the compound shown in formula XII to react for 6 to 8 hours to obtain a compound shown in formula I;
the step (7) is specifically as follows: mixing the compound of formula VI and the compound of formula X or the compound of formula VI and the compound of formula XIII according to the molar ratio of 1 (1.1-1.5) at 90-170 ℃ by using potassium iodide as a catalyst and triethylamine as a base, and reacting in phenol or DMF for 4-17 hours under the protection of nitrogen to obtain the compound of formula II.
The 1,2,3, 4-tetrahydroacridine-9-amine compound is applied to the preparation of medicines for resisting Alzheimer disease.
The 1,2,3, 4-tetrahydroacridine-9-amine compound is applied to the preparation of medicines for delaying neurodegenerative diseases.
The 1,2,3, 4-tetrahydroacridine-9-amine compound is applied to the preparation of the medicine for repairing cholinergic neurons in vitro.
The 1,2,3, 4-tetrahydroacridine-9-amine compound is applied to the preparation of in vitro anti-oxidative stress injury active drugs.
The 1,2,3, 4-tetrahydroacridine-9-amine compound is applied to the preparation of medicaments for reducing beta-amyloid (A beta) neurotoxicity in vitro and in vivo.
The use of the above 1,2,3, 4-tetrahydroacridine-9-amine compounds for improving cognitive disorders through the CREB-ERK pathway or for improving cognitive disorders by reducing phosphorylation of Tau protein.
Compared with the prior art, the invention has the advantages that: the compound is a potential AD-resistant multi-target medicine, can effectively inhibit AChE, can weaken A beta neurotoxicity in vitro and in vivo, and shows a multi-target synergistic anti-AD effect. The compound provided by the invention is proved to be a potential anti-AD active medicament through in-vitro inhibition AChE activity determination and action mode exploration experiments, dot printing experiments, molecular docking experiments, enzyme-linked immunosorbent assays, MTT method drug toxicity detection experiments, real-time polymerase chain reaction experiments, ThT fluorescence experiments, Y maze and Morris water maze experiments. The compound provided by the invention has the advantages of easily available raw materials and simple preparation method, a series of novel tacrine compounds are designed and synthesized, and experiments prove that the tacrine compounds have good activity and good application prospect in the field of design and research of anti-Alzheimer disease drugs.
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FIG. 1 shows the results of AChE inhibition in vitro of Compound 3 in the examples of the present invention;
FIG. 2 is a Lineweaver-Burk double reciprocal plot of the in vitro inhibition of AChE by Compound 3 in the examples of the present invention;
FIG. 3 is a graph of the slope of a double reciprocal line as a function of concentration of Compound 3 in an example of the present invention;
figure 4 is a graph of the effect of compound 3 in inhibiting a β oligomerization in vitro in the examples of the invention: (A) inhibition of Α β oligomer formation by compound 3 at 5mM, 10mM (quantification B);
FIG. 5 shows the docking of compound 3 with A.beta.molecules in accordance with an embodiment of the present invention;
FIG. 6 shows the effect of compound 3 on IL-1 β (A) and TNF- α (B) expression in vivo in mouse hippocampus in examples of the present invention;
FIG. 7 shows the results of in vitro cytotoxicity tests of Compound 1, Compound 2, Compound 3, and Compound 4 in examples of the present invention;
FIG. 8 shows the in vitro inhibition of nNOS enzyme activity of Compound 3 in accordance with an embodiment of the present invention;
fig. 9 is a result of the effect of compound 3 on a β oligomer-induced cognitive impairment in mice in examples of the present invention; (A) the method comprises the following steps of (1) line crossing times of an open field experiment mouse, (B) standing times of the open field experiment mouse, (C) percentage of time of a Y labyrinth experiment mouse entering a new forearm to total time, (D) cognition index of the mouse in a new object cognition experiment, (E) time of the mouse on a platform in a training period of a Morris water labyrinth experiment, (F) percentage of time of the mouse in a quadrant where the platform is located in the Morris water labyrinth experiment to total time, (G) times of the mouse on the platform in the Morris water labyrinth experiment, (H) movement track of the mouse in the Morris water labyrinth experiment;
FIG. 10 shows the extent of ERK phosphorylation and quantification in hippocampus of mice of different treatment groups; the experimental groups included: control group, Abeta +0.2 μ M Compound 3 group, Abeta +0.4 μ M Compound 3 group, and Abeta +0.4 μ M tacrine group;
FIG. 11 shows the extent of CREB phosphorylation in hippocampus of mice of different treatment groups and quantification results; control group, Abeta +0.2 μ M Compound 3 group, Abeta +0.4 μ M Compound 3 group, and Abeta +0.4 μ M tacrine group;
FIG. 12 shows the extent of phosphorylation of Tau in hippocampus of mice of different treatment groups and the quantification thereof. Control group, Abeta + 0.2. mu.M Compound 3 group, Abeta + 0.4. mu.M Compound 3 group, and Abeta + 0.4. mu.M tacrine group.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. Herein, a "compound of formula N" is also sometimes referred to herein as "compound N", where N is any integer from 1 to 14, e.g., "compound of formula 2" may also be referred to herein as "compound 2".
Detailed description of the preferred embodiment
A novel 1,2,3, 4-tetrahydroacridine-9-amine compound is a compound with structural formulas shown in formulas I and II, or a pharmaceutically acceptable salt, ester or solvate of the compound with the structural formulas shown in formulas I and II,
Figure BDA0002760675850000061
wherein R is1Is H, CH3、OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or straight-chain alkanes having 1-5 carbon atoms, R2，R3Is H,
Figure BDA0002760675850000062
Figure BDA0002760675850000063
Figure BDA0002760675850000064
n is 1,2,3,4 or 5, m is 1,2,3,4 or 5, a pharmaceutically acceptable salt, ester or solvate of the compound of formula I or II, wherein the salt is an inorganic acid salt orAn organic acid salt; the inorganic acid salt is selected from salts formed by any one of the following inorganic acids: hydrochloric acid, sulfuric acid and phosphoric acid; the organic acid is selected from salts formed by any one of the following organic acids: acetic acid, trifluoroacetic acid, malonic acid, citric acid and p-toluenesulfonic acid.
The novel 1,2,3, 4-tetrahydroacridine-9-amine compounds represented by the above formulas I and II are preferably any one of the following compounds:
Figure BDA0002760675850000071
the compound can effectively inhibit AChE, can weaken A beta neurotoxicity in vivo and in vitro, and shows a multi-target synergistic anti-AD effect, so that the compound has anti-AD potential. The compound provided by the invention is proved to be a potential anti-AD active medicament through in vitro inhibition AchE activity determination and action mode exploration experiments, dot printing experiments, molecular docking experiments, enzyme-linked immunosorbent assays, MTT method drug toxicity detection experiments, real-time polymerase chain reaction experiments, ThT fluorescence experiments, Y-maze and Morris water maze experiments.
Detailed description of the invention
The preparation method of the novel 1,2,3, 4-tetrahydroacridine-9-amine compound of the above embodiment comprises the following steps:
(1) reacting a compound of formula III with sodium hydroxide in aqueous methanol to obtain a compound of formula IV; specifically, dissolving a compound shown in a formula III and sodium hydroxide in a methanol aqueous solution mixed in an equal volume ratio according to a molar ratio of 1:1.3 at 40 ℃ for reacting for 6 hours to obtain a compound shown in a formula IV; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(2) reacting a compound of formula IV with a compound of formula V in phosphorus oxychloride to obtain a compound of formula VI; specifically, at 0 ℃, adding a compound shown in a formula IV and a compound shown in a formula V into phosphorus oxychloride according to a molar ratio of 1:1.2, transferring to 110 ℃, and reacting for 6 hours to obtain a compound shown in a formula VI; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(3) reacting a compound of formula VI with a compound of formula VII in phenol to obtain a compound of formula VIII; specifically, at 170 ℃, potassium iodide is used as a catalyst, and a compound shown as a formula VI and a compound shown as a formula VII are added into phenol according to a molar ratio of 1:2.1 to react for 4 hours to obtain a compound shown as a formula VIII; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(4) reacting the compound shown in the formula IX with the compound shown in the formula X under the protection of nitrogen to obtain a compound shown in the formula XI; the method specifically comprises the following steps: at 60 ℃, taking potassium iodide as a catalyst and triethylamine as a base, and reacting a compound shown in a formula IX with a compound shown in a formula X in DMF according to a molar ratio of 1.1:1, taking potassium iodide as a catalyst and triethylamine as a base under the protection of nitrogen overnight to obtain a compound shown in a formula XI; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(5) reacting a compound of formula XI with sodium hydroxide in aqueous methanol to obtain a compound of formula XII; the method specifically comprises the following steps: dissolving the compound shown in the formula XI and sodium hydroxide in a methanol aqueous solution mixed according to a volume ratio of 4:1 at 50 ℃ according to a molar ratio of 1:1.5, and reacting overnight to obtain a compound shown in the formula XII; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(6) reacting a compound shown in a formula XII, a compound shown in a formula VIII and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) in DMF, and then adding N, N-Diisopropylethylamine (DIPEA) for reaction to obtain a compound shown in a formula I; the method specifically comprises the following steps: reacting a compound of formula XII, a compound of formula VIII, and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) at room temperature in the following ratio of 1: 1: adding 1.5 mol ratio of N, N-Diisopropylethylamine (DIPEA) into DMF, reacting for 20 min, adding 2 times mol ratio of N, N-Diisopropylethylamine (DIPEA) into the compound shown in formula XII, and reacting for 6-8 h to obtain the compound shown in formula I; therefore, the method is beneficial to improving the reaction efficiency, reducing side reactions and improving the yield;
(7) reacting a compound of formula VI with a compound of formula X, or with a compound of formula VI XIII in phenol or DMF to obtain a compound of formula ii: mixing the compound of formula VI and the compound of formula X or the compound of formula VI and the compound of formula XIII according to the molar ratio of 1 (1.1-1.5) at 90-170 ℃ by using potassium iodide as a catalyst and triethylamine as a base, and reacting in phenol or DMF for 4-17 hours under the protection of nitrogen to obtain the compound of formula II.
Figure BDA0002760675850000091
Wherein R is1Is H, CH3、OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or straight-chain alkanes having 1-5 carbon atoms, R2，R3Is H,
Figure BDA0002760675850000092
Figure BDA0002760675850000093
Figure BDA0002760675850000094
n is 1,2,3,4 or 5, and m is 1,2,3,4 or 5. The compound can be quickly and effectively prepared and obtained by the preparation method, and the method is simple to operate, convenient and quick, and suitable for large-scale production.
Example 1: compound 1
Figure BDA0002760675850000095
Preparation of
1. Preparation of 2-aminobenzoic acid
Figure BDA0002760675850000096
Ethyl 2-aminobenzoate (6.06mmol) and sodium hydroxide (7.878mmol) were stirred in methanol (5mL) and water (5mL), and the mixture was stirred at 40 ℃ for 6 hours. After the reaction was complete, the methanol was removed by rotary evaporation and the resulting solution was acidified with hydrochloric acid to pH 3-4. The resulting suspension was stirred for 10 minutes, and the precipitate was filtered and dried to give a white solid powder in 95% yield with compound structure data characterized as:1H NMR(600MHz,CDCl3):7.69(d,J＝7.8,1H),6.93(t,J＝7.6,1H),6.45(d,J＝8.1,1H),6.37(t,J＝6.4,1H),3.17(s,1H)。
2. preparation of 9-chloro-1, 2,3, 4-tetrahydroacridine
Figure BDA0002760675850000101
Intermediate 2-aminobenzoic acid (3.6mmol) and cyclohexanone (4.32mmol) obtained in the previous step were added to phosphorus oxychloride (10mL) at 0 deg.C, and then reacted at 110 deg.C under reflux for 6 hours. After cooling to room temperature, the sodium carbonate solution was slowly added to the reaction system with rapid stirring under ice bath conditions, and the PH was adjusted to 8. Methylene chloride (40mL) was added to the reaction solution. The organic layer was washed with water (30 mL. times.3), dried over magnesium sulfate, and the dichloromethane was removed under reduced pressure to give a crude product. The crude product was dissolved in hot acetone (5mL), then left overnight at 4 ℃ and filtered to give a yellow solid powder with a yield of 74.3%, melting point 64-65 ℃ and compound structure data characterized as:1H NMR(600MHz,CDCl3):δ8.89(d,J＝8.6Hz,1H),8.30(dd,J＝8.5,1.2Hz,1H),7.92(ddd,J＝8.4,6.9,1.3Hz,1H),7.80(ddd,J＝8.3,7.0,1.1Hz,1H),3.63(t,J＝6.0Hz,2H),3.07(t,J＝6.0Hz,2H),1.98(dt,J＝8.8,5.4Hz,4H)。
3. preparation of N1- (1,2,3, 4-tetrahydroacridin-9-yl) propane-1, 3-diamine
Figure BDA0002760675850000102
The intermediate 9-chloro-1, 2,3, 4-tetrahydroacridine (1.15mmol) obtained in the previous step, 1, 3-propanediamine (2.415mmol) and KI (catalytic amount) were added to phenol (10.35mmol), followed by reaction at 170 ℃ for 4 hours under reflux. The phenol was neutralized by slowly adding potassium hydroxide solution to the reaction solution. The mixture was partitioned between dichloromethane (30mL) and water (30 mL). The organic extract was washed with water (30 mL. times.3), dried (magnesium sulfate), and the dichloromethane was removed under reduced pressure to give the crude product. Petroleum ether/ethyl acetate was used as eluent in column chromatography to give a brown oily liquid, yield: 90.5%, compound structure data are characterized as:1H NMR(600MHz,DMSO-d6)δ8.17(dd,J＝8.6,1.3Hz,1H),7.71(dd,J＝8.4,1.3Hz,1H),7.52(ddd,J＝8.2,6.8,1.4Hz,1H),7.34(ddd,J＝8.3,6.8,1.4Hz,1H),3.47(t,J＝6.9Hz,2H),2.90(t,J＝6.5Hz,2H),2.72(dt,J＝13.5,6.7Hz,4H),1.83(m,2H),1.81(m,2H),1.76(m,2H)。
4. preparation of
Figure BDA0002760675850000103
Methyl 3-bromopropionate (3.531mmol), lutidine amine (3.21mmol), catalytic amounts of potassium iodide and triethylamine (0.1mL) were added to DMF (10 mL). The reaction was stirred at 60 ℃ overnight under nitrogen. After cooling to room temperature, the mixture was partitioned between dichloromethane (30mL) and water (30 mL). The organic extract was washed with water (30 mL. times.5), dried (magnesium sulfate), and the dichloromethane was removed under reduced pressure to give the crude product. Petroleum ether/ethyl acetate was used as eluent in column chromatography to give a yellow oily liquid in 43.2% yield, and the compound structural data characterize:1H NMR(500MHz,CDCl3)δ8.55–8.50(m,2H),7.67(td,J＝7.7,1.8Hz,2H),7.50(d,J＝7.8Hz,2H),7.19–7.15(m,2H),3.85(s,4H),3.63(s,3H),2.96–2.91(m,2H),2.57(t,J＝7.1Hz,2H)。
5. preparation of
Figure BDA0002760675850000111
The intermediate is reacted with a catalyst
Figure BDA0002760675850000112
(2.24mmol), sodium hydroxide (3.36mmol) were added to methanol (8mL) and water (2 mL). The mixture was stirred at 50 ℃ overnight. After completion of the reaction, dichloromethane (10mL) was added to the reaction solution, and extracted 1-2 times to remove some impurities. The resulting solution was acidified to pH 7 with hydrochloric acid and water was removed under reduced pressure to give the crude product. The residue was dissolved in methanol and filtered to remove inorganic salts. The methanol was removed under reduced pressure to give the product, which was carried on to the next reaction without further purification.
6. Preparation of Compound 1
Figure BDA0002760675850000113
Reacting intermediate N1- (1,2,3, 4-tetrahydroacridin-9-yl) propane-1, 3-diamine (1.85mmol), intermediate
Figure BDA0002760675850000114
(1.85mmol) and 2- (7-azabenzotriazol-1-yl) -N, N' -tetramethyluronium Hexafluorophosphate (HATU) (2.775mmol) in dimethylformamide (5mL) were stirred at room temperature for 20 minutes, then N, N-diisopropylethylamine (DIPEA, 3.65mmol) was added and the reaction stirred at room temperature for 6-8 hours until the thin layer chromatography (ethyl acetate/triethylamine ═ 10/1) reaction was complete, and the mixture was partitioned between dimethylformamide (40mL) and water (40 mL). The organic extract was washed with water (40mL × 5), dried (magnesium sulfate), and the organic layer was treated to give a residue, which was eluted with ethyl acetate/petroleum ether/triethylamine and purified by column chromatography to give the target compound 1 as a tan viscous solid in 63.5% yield and characterized by the compound structure data:1H NMR(600MHz,CDCl3):δ8.50(dddd,J＝13.2,4.9,1.9,0.9Hz,2H),8.00(ddd,J＝13.3,8.5,1.3Hz,1H),7.92(m,1H),7.63(td,J＝7.6,1.8Hz,1H),7.56(m,2H),7.50(m,1H),7.35(m,2H),7.12(dddd,J＝13.7,7.5,4.8,1.2Hz,2H),3.82(s,2H),3.77(s,2H),3.50(m,2H),3.46(m,2H),3.06(q,J＝9.7,7.6Hz,2H),2.92(d,J＝31.1Hz,2H),2.74(dd,J＝12.8,6.1Hz,2H),2.60(m,2H),2.28(td,J＝8.0,7.5,6.1Hz,2H),1.87(m,2H),1.83(m,2H).13C NMR(151MHz,CDCl3)δ23.2,23.8,25.2,29.8,31.2,31.7,34.5,36.6,54.0,60.3,60.6,122.0,122.2,122.6,122.7,122.8,123.1,123.5,124.0,124.1,124.1,128.6,136.4,136.5,149.1,149.2,159.4,160.0,162.0,174.5.HR-MS(ESI):C31H36N6O,Calcd for[M+H]+509.3029；Found:509.3021。
example 2: compound 2
Figure BDA0002760675850000121
Preparation of
Prepared according to the procedure of example 1, except that: 1, 3-propanediamine in step 3 of example 1 was replaced with 1, 4-propanediamine for reaction to give the title compound 2 as a tan viscous solid in a yield of 70.3% and having a compound structureAnd (3) data characterization:1H NMR(400MHz,CDCl3)δ8.56(d,J＝4.8Hz,2H),8.11(d,J＝8.6Hz,1H),7.65(td,J＝7.7,1.9Hz,2H),7.43(s,1H),7.36(d,J＝7.8Hz,2H),7.21(s,1H),7.18(m,2H),6.85(d,J＝8.0Hz,1H),3.98(s,4H),3.48(dt,J＝11.1,5.4Hz,2H),3.29(d,J＝6.2Hz,2H),2.75(t,J＝6.0Hz,2H),2.51(t,J＝8.1Hz,2H),2.18(dt,J＝33.3,7.6Hz,4H),2.05(m,2H),2.00(m,2H),1.90(m,2H),1.86(m,2H).13C NMR(151MHz,CDCl3)δ21.7,22.5,24.5,26.9,27.1,28.6,29.8,37.6,39.0,48.5,48.5,54.9,122.1,122.5,123.8,123.8,124.5,124.7,124.7,130.0,130.7,135.2,136.7,136.9,149.4,152.4,153.6,154.6,159.7,161.8,170.8.HR-MS(ESI):C32H38N6O,Calcd for[M+H]+523.3185；Found:523.3168。
example 3: compound 3
Figure BDA0002760675850000122
Preparation of
Prepared according to the procedure of example 1, except that: the methyl 3-bromopropionate in step 4 in example 1 was replaced by methyl 4-bromobutyrate to react, and finally the target compound 3 was obtained as a tan viscous solid with a yield of 62.1%, and the structural data of the compound are characterized in that:1H NMR(600MHz,DMSO-d6)δ8.48–8.42(m,2H),8.25(d,J＝8.6Hz,1H),7.91(t,J＝5.9Hz,1H),7.79(d,J＝8.2Hz,1H),7.73(td,J＝7.6,1.8Hz,2H),7.49(d,J＝7.8Hz,2H),7.46(t,J＝7.6Hz,1H),7.21(ddd,J＝7.5,4.8,1.1Hz,2H),3.69(s,4H),3.64–3.56(m,2H),3.09(q,J＝6.3Hz,2H),2.95(t,J＝5.6Hz,2H),2.67(d,J＝5.7Hz,2H),2.41(t,J＝7.1Hz,2H),2.05(t,J＝7.4Hz,2H),1.81(d,J＝5.5Hz,4H),1.71(dt,J＝14.9,7.1Hz,4H).13C NMR(151MHz,CDCl3)δ22.8,24.9,27.2,29.0,34.1,37.9,39.1,48.9,53.9,54.0,60.3,60.5,116.5,120.4,122.1,122.8,123.0,123.4,123.8,128.4,128.7,136.4,147.4,149.1,150.6,158.6,159.4,159.9,161.4,164.6,173.5.HR-MS(ESI):C32H38N6O,Calcd for[M+H]+523.3185；Found:523.3165。
example 4: compound 4
Figure BDA0002760675850000131
Preparation of
Prepared according to the procedure of example 1, except that: the 1, 3-propanediamine in step 3 of example 1 was replaced by 1, 4-propanediamine, and the methyl 3-bromopropionate in step 4 was replaced by methyl 4-bromobutyrate, to give the title compound 4 as a tan viscous solid in 68.4% yield, as characterized by the structural data:1H NMR(600MHz,CDCl3)δ8.26(d,J＝1.6Hz,1H),8.09(d,J＝8.7Hz,2H),7.94(t,J＝8.4Hz,4H),7.57(ddd,J＝8.4,6.7,1.5Hz,2H),7.38(dd,J＝8.6,6.9Hz,2H),3.64(m,4H),3.46(d,J＝6.3Hz,2H),3.40(d,J＝6.2Hz,4H),3.07(m,6H),2.74(m,6H),2.04(s,4H).13C NMR(151MHz,CDCl3)δ22.1,22.8,23.3,24.8,29.8,31.3,31.5,31.7,35.3,36.5,44.8,44.9,46.6,114.6,118.7,122.0,123.1,123.2,123.4,124.3,124.5,125.1,125.7,129.9,130.1,135.2,144.1,153.0,155.7,156.2,162.6,171.9.HR-MS(ESI):C33H40N6O,Calcd for[M+H]+537.3342；Found:537.3327。
example 5: compound 5
Figure BDA0002760675850000132
Preparation of
Prepared according to the procedure of example 1, except that: the 1, 3-propanediamine of step 3 in example 1 was replaced with 1, 5-propanediamine to give the title compound 5 as a tan viscous solid in 74.9% yield, characterized by the compound structure data:1H NMR(600MHz,DMSO-d6)δ8.45(d,J＝4.5Hz,2H),8.10(d,J＝8.4Hz,1H),7.73–7.67(m,3H),7.52(t,J＝7.6Hz,1H),7.47(d,J＝7.8Hz,2H),7.33(t,J＝7.6Hz,1H),7.20(dd,J＝7.1,5.2Hz,2H),3.71(s,4H),3.01–2.98(m,2H),2.88(s,2H),2.72–2.66(m,4H),2.53(d,J＝6.9Hz,2H),2.30(t,J＝7.0Hz,2H),1.79(dt,J＝12.6,5.5Hz,4H),1.55(p,J＝7.5Hz,2H),1.36(p,J＝7.1Hz,2H),1.27(q,J＝8.2Hz,2H).13C NMR(126MHz,CDCl3)δ172.4,158.4,149.2,149.0,136.6,136.4,130.9,124.5,124.0,123.4,123.0,122.3,122.0,120.8,117.3,112.9,60.2,59.7,50.5,50.1,48.9,38.7,33.4,30.5,29.4,24.1,23.8,22.2,21.4.HR-MS(ESI):C33H40N6O,Calcd for[M+H]+537.3342；Found:537.3336。
example 6: compound 6
Figure BDA0002760675850000141
Preparation of
Prepared according to the procedure of example 1, except that: the 1, 3-propanediamine in step 3 of example 1 was replaced with 1, 5-propanediamine, and the methyl 3-bromopropionate in step 4 was replaced with methyl 4-bromobutyrate, to give the title compound 6 as a tan viscous solid in a yield of 70.1%, and the compound structural data showed that:1H NMR(600MHz,DMSO-d6)δ8.47–8.44(m,1H),8.25(d,J＝8.5Hz,0H),7.77–7.72(m,1H),7.69(q,J＝8.4,6.9Hz,1H),7.50(d,J＝7.8Hz,1H),7.45(t,J＝7.4Hz,0H),7.24–7.20(m,1H),3.70(s,1H),3.64–3.57(m,1H),2.99–2.95(m,1H),2.94(t,J＝6.6Hz,1H),2.67(t,J＝5.5Hz,1H),2.41(t,J＝7.1Hz,1H),1.81(d,J＝5.5Hz,1H),1.70(p,J＝7.4Hz,1H),1.63(p,J＝7.5Hz,1H),1.34(p,J＝7.0Hz,1H),1.27(q,J＝8.0Hz,1H).13C NMR(126MHz,CDCl3)δ173.7,165.1,162.6,159.2,155.0,152.0,148.8,140.1,136.6,131.7,124.9,124.4,123.4,122.1,121.4,116.4,112.0,77.3,60.1,53.8,48.6,38.8,36.5,34.1,30.2,29.2,29.1,23.8,23.5,23.3,22.0,21.0.HR-MS(ESI):C34H42N6O,Calcd for[M+H]+551.3498；Found:551.3478。
example 7: compound 7
Figure BDA0002760675850000142
Preparation of
Prepared according to the procedure of example 1, except that: the reaction was carried out by replacing methyl 3-bromopropionate in step 4 of example 1 with methyl 5-bromovalerate to finally obtain the target compound 7 as a tan viscous solid in 56.2% yield, characterized by the compound structural data:1H NMR(600MHz,DMSO-d6)δ8.48–8.44(m,1H),8.19(d,J＝8.5Hz,0H),7.84(t,J＝5.7Hz,0H),7.73(td,J＝7.6,1.7Hz,1H),7.63(t,J＝7.5Hz,0H),7.48(d,J＝7.8Hz,1H),7.40(t,J＝7.3Hz,0H),7.24–7.20(m,1H),3.69(s,1H),3.54(q,J＝6.2Hz,1H),3.11(q,J＝6.4Hz,1H),2.92(t,J＝6.0Hz,1H),2.67(d,J＝5.7Hz,1H),2.41(s,1H),1.99(t,J＝6.4Hz,1H),1.84–1.75(m,2H),1.74–1.67(m,1H),1.44(s,1H).13C NMR(126MHz,CDCl3)δ175.5,172.8,162.6,156.3,150.5,148.8,138.3,137.1,132.6,125.3,124.4,123.5,122.7,119.9,115.6,111.5,59.5,53.7,46.3,44.0,35.7,35.6,30.8,29.7,28.3,25.3,23.4,21.9,20.6.HR-MS(ESI):C33H40N6O,Calcd for[M+H]+537.3342；Found:537.3326。
example 8: compound 8
Figure BDA0002760675850000151
Preparation of
Prepared according to the procedure of example 1, except that: the reaction was carried out by replacing 1, 3-propanediamine in step 3 of example 1 with 1, 4-propanediamine and replacing methyl 3-bromopropionate in step 4 with methyl 5-bromovalerate to give the title compound 8 as a tan viscous solid in 61.5% yield, characterized by the structural data of the compound:1H NMR(600MHz,DMSO-d6)δ8.48–8.44(m,1H),8.28(d,J＝8.6Hz,0H),7.78–7.69(m,2H),7.48(d,J＝7.8Hz,1H),7.25–7.20(m,1H),3.68(s,1H),3.02(q,J＝6.7Hz,1H),2.94(t,J＝5.5Hz,1H),2.66(d,J＝5.7Hz,1H),2.40(t,J＝6.4Hz,1H),1.96(t,J＝6.6Hz,1H),1.81(d,J＝5.4Hz,1H),1.64(p,J＝7.4Hz,1H),1.43(q,J＝8.6,8.0Hz,3H).13C NMR(126MHz,CDCl3)δ173.9,162.5,159.2,155.4,151.3,148.8,139.3,136.7,132.1,125.0,124.5,123.4,122.2,120.6,116.0,111.8,60.0,53.6,48.1,38.5,35.9,29.7,28.8,27.8,26.6,25.7,23.4,23.4,21.8,20.8.HR-MS(ESI):C34H42N6O,Calcd for[M+H]+551.3498；Found:551.3487。
example 9: compound 9
Figure BDA0002760675850000152
Preparation of
Prepared according to the procedure of example 1, except that: the 1, 3-propanediamine in step 3 of example 1 was replaced with 1, 5-propanediamine, and the methyl 3-bromopropionate in step 4 was replaced with5-bromomethyl valerate is reacted to finally obtain the target compound 9 as a tan viscous solid with the yield of 57.6%, and the structural data of the compound are characterized in that:1H NMR(600MHz,DMSO-d6)δ8.49–8.43(m,1H),8.14(d,J＝8.4Hz,0H),7.77–7.69(m,1H),7.67(t,J＝5.5Hz,0H),7.56(t,J＝7.4Hz,0H),7.49(d,J＝7.8Hz,1H),7.36(t,J＝7.5Hz,0H),7.25–7.19(m,1H),3.69(s,1H),3.44(q,J＝6.6Hz,1H),2.97(q,J＝6.7Hz,1H),2.91(t,J＝6.2Hz,1H),2.68(t,J＝6.0Hz,1H),2.42(t,J＝6.2Hz,1H),1.96(t,J＝6.5Hz,1H),1.80(dt,J＝10.8,5.9Hz,2H),1.57(p,J＝7.5Hz,1H),1.48–1.40(m,2H),1.34(q,J＝7.1Hz,1H),1.26(p,J＝8.0Hz,1H).13C NMR(126MHz,CDCl3)δ173.7,168.2,159.2,155.5,150.9,148.7,139.0,136.7,132.1,125.0,124.6,123.3,122.1,120.2,115.8,111.2,59.9,53.6,48.4,38.7,36.0,30.1,29.0,28.3,25.7,23.8,23.5,23.4,21.8,20.7.HR-MS(ESI):C35H44N6O,Calcd for[M+H]+565.3655；Found:565.3649。
example 10: compound 10
Figure BDA0002760675850000161
Preparation of
Prepared according to the procedure of example 1, except that: the methyl 3-bromopropionate in step 4 of example 1 was replaced by methyl 6-bromohexanoate to react, and finally the target compound 10 was obtained as a tan viscous solid with a yield of 66.7%, and the structural data of the compound are characterized:1H NMR(500MHz,CDCl3)δ8.48(d,J＝4.8Hz,2H),8.15(d,J＝8.6Hz,1H),7.97(d,J＝8.5Hz,1H),7.65(t,J＝7.6Hz,2H),7.58(t,J＝7.7Hz,1H),7.51(d,J＝7.8Hz,2H),7.42(t,J＝7.8Hz,1H),7.15–7.11(m,2H),6.76(t,J＝6.1Hz,1H),3.78(s,6H),3.42(q,J＝6.0Hz,2H),3.07(t,J＝5.9Hz,2H),2.70(t,J＝5.8Hz,2H),2.52(t,J＝7.2Hz,2H),2.23(t,J＝7.5Hz,2H),1.95–1.87(m,4H),1.87–1.82(m,2H),1.56(dt,J＝21.1,7.4Hz,4H),1.32–1.28(m,2H).13C NMR(126MHz,CDCl3)δ175.4,159.7,155.4,151.7,148.8,139.6,136.5,131.8,125.0,124.0,123.1,122.0,121.2,116.3,112.1,60.3,54.2,44.0,36.4,35.8,30.9,29.7,29.0,26.8,26.6,25.6,23.9,22.1,20.9.HR-MS(ESI):C34H42N6O,Calcd for[M+H]+551.3498；Found:551.3486。
example 11: compound 11
Figure BDA0002760675850000171
Preparation of
Prepared according to the procedure of example 1, except that: the reaction was carried out by replacing 1, 3-propanediamine in step 3 of example 1 with 1, 4-propanediamine and replacing methyl 3-bromopropionate in step 4 with methyl 6-bromohexanoate, to finally obtain the target compound 11 as a tan viscous solid in a yield of 62.8%, and the structural data of the compound are characterized in that:1H NMR(600MHz,DMSO-d6)δ8.46(ddd,J＝4.8,1.7,0.9Hz,2H),8.24(d,J＝8.5Hz,1H),7.78–7.71(m,4H),7.69(t,J＝7.5Hz,1H),7.49(d,J＝7.8Hz,2H),7.45(t,J＝7.6Hz,1H),7.22(ddd,J＝7.4,4.9,1.0Hz,2H),3.69(s,4H),3.66–3.56(m,2H),3.01(q,J＝6.7Hz,2H),2.93(t,J＝5.9Hz,2H),2.66(t,J＝5.6Hz,2H),2.39(t,J＝7.2Hz,2H),1.96(t,J＝7.4Hz,2H),1.81(d,J＝5.4Hz,4H),1.61(p,J＝7.4Hz,2H),1.43(dq,J＝15.7,7.4Hz,4H),1.36(q,J＝7.5Hz,2H),1.16(q,J＝8.1Hz,2H).13C NMR(126MHz,CDCl3)δ173.8,164.9,162.5,159.7,154.5,152.6,148.8,140.7,136.5,131.3,124.8,124.2,123.0,122.0,116.8,112.4,77.3,60.3,54.2,48.2,38.6,36.4,31.4,29.7,28.0,26.8,26.7,26.6,25.5,23.8,22.1,21.1.HR-MS(ESI):C35H44N6O,Calcd for[M+H]+565.3655；Found:565.3654。
example 12: compound 12
Figure BDA0002760675850000172
Preparation of
Prepared according to the procedure of example 1, except that: the reaction was carried out by replacing 1, 3-propanediamine in step 3 of example 1 with 1, 5-propanediamine and replacing methyl 3-bromopropionate in step 4 with methyl 6-bromohexanoate, to finally obtain the target compound 12 as a tan viscous solid in a yield of 68.4%, and the structural data of the compound are characterized in that:1H NMR(600MHz,DMSO-d6)δ8.47–8.44(m,2H),8.15(d,J＝8.5Hz,1H),7.77–7.70(m,3H),7.67(t,J＝5.5Hz,1H),7.58(t,J＝7.4Hz,1H),7.49(d,J＝7.8Hz,2H),7.37(t,J＝7.5Hz,1H),7.22(ddd,J＝7.4,4.9,1.0Hz,2H),3.69(s,4H),3.49–3.43(m,2H),2.97(q,J＝6.7Hz,2H),2.91(d,J＝6.0Hz,2H),2.68(t,J＝6.0Hz,2H),2.43–2.37(m,2H),1.97(t,J＝7.4Hz,2H),1.80(dt,J＝11.9,7.1Hz,4H),1.57(p,J＝7.5Hz,2H),1.45(p,J＝7.4Hz,2H),1.36(ddd,J＝18.0,14.8,7.3Hz,4H),1.26(dt,J＝12.7,6.2Hz,3H),1.17(p,J＝7.6Hz,2H).13C NMR(126MHz,CDCl3)δ173.7,162.6,158.9,155.7,150.7,148.7,138.8,136.8,132.3,125.1,124.8,123.2,122.2,120.0,115.7,111.3,77.3,60.1,54.3,48.4,38.7,36.5,36.3,30.0,28.9,28.4,26.7,26.4,25.5,23.7,23.2,21.8,20.6.HR-MS(ESI):C36H46N6O,Calcd for[M+H]+579.3811；Found:579.3801。
example 13: compound 13
Figure BDA0002760675850000181
Preparation of
9-chloro-1, 2,3, 4-tetrahydroacridine (2.30mmol), N-lutidine (3.46mmol), a catalytic amount of potassium iodide and triethylamine (0.1mL) were added to phenol (23mmol), and the reaction was stirred at 170 ℃ for 4h under nitrogen. After cooling to room temperature, a potassium hydroxide solution was added to the reaction system to remove the remaining phenol. The mixture was partitioned between dichloromethane (30mL) and water (30 mL). The organic extract was washed with water (30 mL. times.3), dried (magnesium sulfate), and the dichloromethane was removed under reduced pressure to give the crude product. The crude product was purified by column chromatography to afford the title compound 13 as a brown viscous solid in 45.5% yield, and compound structure data characterisation:1H NMR(400MHz,DMSO-d6)δ8.40(d,J＝4.1Hz,2H),7.96(d,J＝8.3Hz,1H),7.76(d,J＝8.2Hz,1H),7.61(td,J＝7.7,1.6Hz,2H),7.49(t,J＝7.2Hz,1H),7.34(t,J＝7.3Hz,1H),7.20–7.13(m,4H),4.38(s,4H),2.86(t,J＝6.7Hz,2H),2.44(d,J＝6.2Hz,2H),1.67(p,J＝6.5Hz,2H),1.45(q,J＝5.8Hz,2H).13C NMR(126MHz,CDCl3)δ165.3,162.6,159.5,158.1,149.4,136.5,129.3,128.1,125.6,125.3,124.6,123.2,122.4,58.8,32.4,26.7,22.5,22.2.HR-MS(ESI):C25H24N4,Calcd for[M+H]+381.2079；Found:381.2059。
example 14: compound 14
Figure BDA0002760675850000182
Preparation of
9-chloro-1, 2,3, 4-tetrahydroacridine (2.30mmol), tryptamine (2.53mmol), a catalytic amount of potassium iodide and triethylamine (0.1mmol) were added to dimethylformamide (5mL) and the reaction was stirred at 90 ℃ for 17 hours under nitrogen. After cooling to room temperature, the mixture was partitioned between dichloromethane (30mL) and water (30 mL). The organic extract was washed with water (30 mL. times.3), dried (magnesium sulfate), and the dichloromethane was removed under reduced pressure to give the crude product. The crude product was purified by column chromatography to afford target compound 14 as a brown viscous solid in 49.2% yield, and compound structure data characterisation:1H NMR(600MHz,DMSO-d6)δ8.10(dd,J＝8.5,1.3Hz,1H),7.71(dd,J＝8.4,1.2Hz,1H),7.52(ddd,J＝8.2,6.7,1.3Hz,1H),7.49(d,J＝7.9Hz,1H),7.33(d,J＝8.4Hz,1H),7.31(s,0H),7.14(d,J＝2.3Hz,1H),7.06(ddd,J＝8.1,6.9,1.1Hz,1H),6.95(ddd,J＝8.0,6.9,1.0Hz,1H),3.76–3.69(m,2H),2.98(t,J＝7.5Hz,2H),2.89(t,J＝6.4Hz,2H),2.63(t,J＝6.3Hz,2H),1.84–1.78(m,2H),1.78–1.72(m,1H).13C NMR(151MHz,dmso)δ157.76,150.14,146.87,136.25,128.23,127.86,127.10,124.82,123.13,122.95,120.94,120.09,118.23,118.15,115.64,111.38,111.29,48.72,33.49,26.53,24.80,22.69,22.39.HR-MS(ESI):C23H23N3,Calcd for[M+H]+342.1970；Found:342.1955。
detailed description of the preferred embodiment
Use of a compound prepared as in example one or embodiment two above in the preparation of a medicament. The medicine is a potential anti-AD multi-target medicine, can effectively inhibit AChE, can weaken A beta neurotoxicity in vitro and in vivo, and shows a multi-target synergistic anti-AD effect.
The present invention uses mice as an animal model of alzheimer's disease for the testing of the therapeutic effects of novel tacrine compounds.
The variance was analyzed by comparison between treatment groups using Tukey-Kramer separation test to determine its statistical significance. The allowable significant difference is p < 0.05.
It is to be noted that the above-mentioned drug of the present invention can be introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or can be mixed or coated with other materials and introduced into body. If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field. In addition, the medicine of the invention can be prepared into various forms such as injection, tablets, powder, granules, capsules, oral liquid, ointment, cream and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.
Example 1
In vitro inhibition of AChE Activity assay
Mice were sacrificed and the brains of the mice were removed for the experiment. Adding 10 times of volume of lysis solution according to the tissue mass, fully mixing, performing ice-bath ultrasonic homogenization, centrifuging at 3000rpm and 4 ℃ for 15 minutes, and collecting supernatant. Mixing the supernatant with 0.1M Na2HPO4(pH 7.5), adding Compound 3 to react for 5 minutes, adding the substrate iodoacetylthiocholine (AChI, from Sigma), and incubating at 37 ℃ for 30 minutes. Dithiobis (2-nitrobenzoic acid) (DTNB, available from Life Science) was added to cause a color reaction with the reaction product, resulting in a yellow substance. Absorbance at 412nm was measured using a Varioskan LUX multimode microplate reader (Thermo Fisher scientific, Waltham, MA, USA). As shown in FIG. 1, the semi-inhibitory dose (IC) of Compound 3 on AChE activity50) It was 7.13 nM. IC of positive control donepezil under the same conditions50Was 15 nM. The results show that compound 3 (at 10 μ M concentration) inhibits AChE with high efficiency and more than donepezil.
Example 2
Investigation of mode of action of Compound 3 with AChE
In the AChE activity determination system, the substrate AChI concentration is fixed, the AChE concentration is changed, and the influence of different concentrations of the compound 3 on the enzyme activity is determined. As shown in fig. 2, Lineweaver Burk bipolarity mapping results indicate that compound 3 non-competitively inhibits AChE. As shown in fig. 3, Km is 1.37 and the inhibitor constant Ki is 2.7.
Example 3
In vitro inhibition of Abeta oligomerization experiments (dot blot)
Adding 2 μ L of A β oligomer of compound 3 with different concentrations and A β oligomer blank control, sequentially spotting on PDVF membrane, air drying, blocking with 5% BSA solution for 30 min, and then using anti-oligomer A11 antibody or anti-A β1-17Monomeric 6E10 antibody was incubated for 1 hour. The strips were washed 3 times with 1% TBST solution, incubated with secondary antibody for one hour, and washed 3 times with 1 wt% TBST. And (3) uniformly dropwise adding the ECL developing solution on the surface of the strip, exposing by using an exposure machine, and statistically analyzing the obtained data by using Image J gray scale. As shown in fig. 4, the experimental results showed that the ability of compound 3 to prevent a β oligomer formation gradually increased with increasing concentration(s) ((r))***p<0.001，**p<0.01(ANOVA and Tukey’s Test))。
Example 4
Butt joint experiment of compound 3 and Abeta molecules
Molecular docking was performed using SYBYL software. Small molecule modeling is optimized using the Powell method of the software itself. The Surflex-Dock program was used to mimic ligand-receptor docking. When the method is used for simulation, the receptor position is relatively fixed, and the receptor can be freely changed. The higher the corresponding Score (Surflex-Dock Score) obtained after the end of the simulation, indicating a stronger predicted affinity between the ligand receptors. As shown in FIG. 5, the binding fraction of compound 3 and A beta is-7.08, and the inhibition rate is as high as 71.08, which indicates that compound 3 may have an inhibition effect on A beta. Due to beta folding, a 'valley' penetrating through five monomers exists in the molecular structure of the A beta pentamer, and the A beta pentamer is easy to combine with a small molecular compound to form more stable A beta, so that the A beta pentamer is inhibited from further oligomerization. The molecular docking experiment results show that: compound 3 can enter the a β trough. Where compound 3 interacts with Leu at positions a β 17 and 34, this suggests that compound 3 will advantageously bind to the amino acid residues described above, longitudinally to the surface of one molecular assembly "valley". Thus, compound 3 can act on a β through numerous hydrophobic interactions with the hydrophobic pocket (Leu residues at positions 17 and 34), thereby acting to alter a β assembly and thereby stabilize and inhibit its oligomerization.
Example 5
Enzyme linked immunosorbent assay (ELISA) for detecting expression of inflammatory factors in mouse brain
Mouse brain tissue was homogenized by adding 0.5mL of lysate (10mM HEPES (pH 7.5), 1mM EGTA,1mM EDTA, 0.5% Triton X-100and 150mM NaCl) in ice bath. After centrifugation at 13400rpm for 30 minutes at 4 ℃ the supernatant was taken. The expression levels of IL-1. beta. and TNF-. alpha.were measured using an ELISA kit. As shown in FIG. 6, compound 3 can significantly reduce the expression of inflammatory factors such as IL-1 beta, TNF-alpha and the like in the hippocampus of mice (II) ((III))***p<0.001，**p<0.01(ANOVA and Tukey’s Test))。
Example 6
MTT assay for drug toxicity
Cell viability was assessed by 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT) assay. Mu. M A beta oligo and A beta oligo to which Compound 3 was added were added to a 96-well plate, incubated at 37 ℃ for 24 hours, and then 10. mu.L of MTT was added. After 4 hours, 100 μ L of 10% SDS was added. After 16 hours, the absorbance of the sample was measured at a wavelength of 570nm and 655nm using a Varioskan LUX multimode microplate reader (Thermo Fisher). As shown in fig. 7, 2mM compound 3 did not itself affect cell proliferation nor did it exhibit cytotoxic effects during the course of the reaction at the experimental concentrations.
Example 7
Real-time polymerase chain reaction (RT-PCR) detection of iNOS expression
After sacrifice, hippocampal tissue mRNA was extracted using Trizol reagent (purchased from Sigma). It is then transcribed into cDNA using reverse transcriptase. Quantitative PCR (qpcr) was performed using Mx3005P multiplex quantitative PCR system (agent Stratagene, USA). The primer sequences of Inducible Nitric Oxide Synthase (iNOS) are: f: GTTCTCAG CCCAACACATAATACAAGA, R: GTGGACGGGTCGATGTCAC are provided. As shown in FIG. 8, RT-PCR experiments showed that Compound 3 reduced the expression level of iNOS in vitro, suggesting that it may reduce neuroinflammation(***p<0.001，**p<0.01(ANOVA and Tukey’s Test))。
Example 8
Experiment of Effect of Compound 3 on A beta oligomer-induced cognitive impairment mouse behavior
We found that compound 3 did not impair mouse motor ability by the open field test (fig. 9A-B). Through the Y maze detection, the compound 3 is found to improve the three-dimensional space cognitive ability of the mouse with cognitive impairment induced by the Abeta oligomer. Compared with the Abeta group, the cognitive ability of the mice is not improved by injecting 0.08mg of tacrine into the hippocampus, and the three-dimensional space cognitive ability of the mice with cognitive impairment is obviously improved by injecting 0.1mg of compound 3(p <0.05) and 0.2mg of compound 3(p <0.05) into the hippocampus (figure 9C). In a novel object cognition assay, compound 3 increased the cognition index in a β oligomer-induced cognitive impairment mice. Compared with the Α β group, the hippocampal injection tacrine group did not improve the cognitive index of mice, and the hippocampal injection of 0.1mg of compound 3(p <0.05) and 0.2mg of compound 3(p <0.01) significantly improved the cognitive index of cognitive impaired mice (fig. 9D). Hippocampal injection of 0.08mg tacrine group (p <0.01) significantly reduced the time to platform search and increased the residence time of mice in the platform quadrant during exploration compared to Α β group, with 0.1mg compound 3(p <0.01) and 0.2mg compound 3(p <0.01) producing better effects (fig. 9E-H).
Example 9
Protein expression experiment in mouse hippocampus by Western blotting
The mouse takes the brain by perfusion, and the protein is extracted by adding the lysis solution of RIPA-PIC-phosphatase inhibitor. Proteins were separated by SDS-PAGE gel electrophoresis and the separated proteins were transferred to PDVF membranes. After adding 5% skimmed milk powder and sealing for 1h, adding CREB, ERK and Tau-resistant protein, and incubating overnight at 4 ℃. After washing 4 times with TBST, the corresponding secondary antibody was added and incubated for 2h, and then washed 4 times with TBST. And detecting the protein band by using an ECL kit, putting the protein band into an exposure machine for exposure, analyzing the optical density value of the band, calculating the protein expression level, and averaging the three results. As shown in FIGS. 10-12, compound 3 can significantly inhibit phosphorylation of CREB protein, ERK protein and Tau protein in mouse hippocampus.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims (8)

1.1, 2,3, 4-tetrahydroacridine-9-amine compounds, characterized in that: the 1,2,3, 4-tetrahydroacridine-9-amine compound takes tacrine as a mother nucleus and has a structural formula shown in a formula I, and the structural formula is as follows:
Figure DEST_PATH_IMAGE002
formula I
Wherein R is1Is H, OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or a linear alkane having 1 to 5 carbon atoms, n =1, 2,3,4 or 5, m =1, 2,3,4 or 5.
2. A method for producing the 1,2,3, 4-tetrahydroacridine-9-amine compound according to claim 1, characterized by comprising the steps of:
(1) reacting a compound of formula III with sodium hydroxide in aqueous methanol to obtain a compound of formula IV;
(2) reacting a compound of formula IV with a compound of formula V in phosphorus oxychloride to obtain a compound of formula VI;
(3) reacting a compound of formula VI with a compound of formula VII in phenol to obtain a compound of formula VIII;
(4) reacting the compound shown in the formula IX with the compound shown in the formula X under the protection of nitrogen to obtain a compound shown in the formula XI;
(5) reacting a compound of formula XI with sodium hydroxide in aqueous methanol to obtain a compound of formula XI
Figure DEST_PATH_IMAGE004
A compound;
(6) reacting a compound of formula XII, a compound of formula VIII and 2- (7-azabenzeneAnd reacting triazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate in N, N-dimethylformamide, and adding N, N-diisopropylethylamine to react to obtain the compound
Figure DEST_PATH_IMAGE006
A compound which is a mixture of a compound having a structure,
Figure DEST_PATH_IMAGE008
Figure DEST_PATH_IMAGE002A
formula I
Wherein R is1Is H, OCH3、OCH2CH3、Cl、Br、CF3、NO2、NH2Or a linear alkane having 1 to 5 carbon atoms, n =1, 2,3,4 or 5, m =1, 2,3,4 or 5.
3. The method for preparing the 1,2,3, 4-tetrahydroacridine-9-amine compound according to claim 2, characterized in that the step (1) is specifically as follows: dissolving the compound shown in the formula III and sodium hydroxide in a methanol aqueous solution mixed in an equal volume ratio according to a molar ratio of 1:1.3 at 40 ℃ for reacting for 6 hours to obtain a compound shown in a formula IV;
the step (2) is specifically as follows: adding the compound shown in the formula IV and the compound shown in the formula V into phosphorus oxychloride at the temperature of 0 ℃ according to the molar ratio of 1:1.2, transferring the mixture to the temperature of 110 ℃ and reacting for 6 hours to obtain the compound shown in the formula VI;
the step (3) is specifically as follows: adding the compound shown in the formula VI and the compound shown in the formula VII into phenol according to a molar ratio of 1:2.1 at 170 ℃ by taking potassium iodide as a catalyst to react for 4 hours to obtain a compound shown in the formula VIII;
the step (4) is specifically as follows: adding a compound shown in a formula IX and a compound shown in a formula X into DMF according to a molar ratio of 1.1:1 at 60 ℃ by using potassium iodide as a catalyst and triethylamine as a base, and reacting overnight under the protection of nitrogen to obtain a compound shown in a formula XI;
the step (5) is specifically as follows: dissolving the compound shown in the formula XI and sodium hydroxide in a methanol aqueous solution mixed according to a volume ratio of 4:1 at 50 ℃ according to a molar ratio of 1:1.5, and reacting overnight to obtain a compound shown in the formula XII;
the step (6) is specifically as follows: reacting a compound of formula XII, a compound of formula VIII, and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate at room temperature in the following ratio of 1: 1:1.5 mol ratio, adding into DMF to react for 20 minutes, adding N, N-diisopropylethylamine with 2 times mol ratio of the compound shown in formula XII, reacting for 6-8 hours to obtain the compound shown in formula
Figure 839749DEST_PATH_IMAGE006
The compounds shown.
4. The use of 1,2,3, 4-tetrahydroacridine-9-amine compounds as claimed in claim 1 for the preparation of anti-alzheimer's disease drugs.
5. Use of 1,2,3, 4-tetrahydroacridine-9-amines as claimed in claim 1 for the preparation of medicaments for the delay of neurodegenerative diseases.
6. Use of the 1,2,3, 4-tetrahydroacridine-9-amine compounds as claimed in claim 1 for the preparation of medicaments for the in vitro repair of cholinergic neurons.
7. Use of 1,2,3, 4-tetrahydroacridine-9-amines as defined in claim 1 for the preparation of medicaments active against oxidative stress damage in vitro.
8. Use of 1,2,3, 4-tetrahydroacridine-9-amines as defined in claim 1 for the preparation of medicaments for the alleviation of β -amyloid neurotoxicity in vitro and in vivo.
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