Application of N-(3-cyano-tetrahydro-1-benzothiophene-2-yl)-2-(morpholine-4-yl) acetamide to treatment of Alzheimer disease

Abstract

The invention provides an application of N-(3-cyano-tetrahydro-1-benzothiophene-2-yl)-2-(morpholine-4-yl) acetamide in the treatment of an Alzheimer's disease. The CN-0928 compound and the analogue CN-0929 of the CN-0928 compound can be used for reducing BACE1 protein expression by regulating and controlling a biomolecular aggregate, so that the A beta plaque load is reduced; in addition, mitochondrial structure change can be recovered, so that AD pathology is further relieved. The treatment based on the intracellular RNPs particles provides a new thought for developing drugs for preventing or treating neurological diseases such as Alzheimer's disease and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to the prevention or treatment of Alzheimer's disease, in particular to the application of N-(3-cyano-tetrahydro-1-benzothiophene-2-yl)-2-(morpholin-4-yl)acetamide in the treatment of Alzheimer's disease. BACKGROUND

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of senile dementia. Alzheimer's disease is characterized by cognitive impairment, behavioral and psychological symptoms, and gradual loss of daily living ability, which brings heavy burden to patients' families and society ("2024 Alzheimer's disease facts and figures," 2024).

[0003] The main pathological features of AD include senile plaques (SP) formed by the deposition of beta-amyloid (Aβ) in the brain and neurofibrillary tangles (NFTs) formed by the overphosphorylation of tau protein. These pathological changes lead to neuronal and synaptic dysfunction and loss, ultimately causing brain atrophy.

[0004] Studies have found that abnormal aggregation and dysfunction of ribonucleoprotein granules (RNPs) often occur in the brains of AD patients, which can lead to RNA metabolism disorders, protein synthesis disorders, and abnormal cell stress responses in neurons. The abnormality of RNPs granules is related to the pathological aggregation of tau protein. Studies have found that PCBP2 granules (a type of RNPs granule) also affect the expression of BACE1 by regulating the mRNA degradation of BACE1, a key rate-limiting enzyme in the Aβ pathway, thereby participating in the regulation of the Aβ pathway. Therefore, drugs developed based on RNPs granules are expected to provide new ideas and targets for the development of AD treatment strategies.

[0005] At the same time, mitochondrial dysfunction also plays an important role in the occurrence and development of AD. Mitochondria, as the center of cellular energy metabolism, abnormal function can lead to increased oxidative stress, reduced ATP production, and calcium ion homeostasis imbalance. Studies have found that after the formation of a large number of pathological RNPs granules, the mitochondrial structure of cells is significantly damaged and accompanied by significant functional decline. Therefore, drugs developed based on RNPs granules may have better efficacy than single-target molecule drugs.

[0006] Currently, the drugs used in the clinic for the treatment of Alzheimer's disease mainly include: acetylcholinesterase inhibitors, such as: donepezil, galantamine and rivastigmine; NMDA receptor antagonists: memantine. These drugs mainly improve the cognitive function of patients by increasing the content of neurotransmitters or reducing neuronal damage. However, these treatment methods mainly aim at symptom relief and cannot effectively prevent or reverse the progression of the disease. At the same time, there are also new treatment strategies targeting Aβ pathology, such as Lecanemab (Leqembi) and donanemab (Kisunla), etc., but some patients have serious side effects such as brain edema or cerebral hemorrhage during the drug process, so it is necessary to develop new drugs for the treatment of AD. SUMMARY

[0007] In order to solve the problems in the prior art, the application provides the application of N-(3-cyano-tetrahydro-1-benzothiophene-2-yl)-2-(morpholine-4-yl)acetamide in the treatment of Alzheimer's disease.

[0008] The application adopts the following technical solutions:

[0009] The application of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating Alzheimer's disease; the compound of formula I has the chemical structure shown below:

[0010]

[0011] wherein Y is N or O.

[0012] When Y is O, formula I is CN-0928; when Y is N, formula I is CN-0929. The structural formulas of formulas CN-0928 and CN-0929 are as follows:

[0013]

[0014] The above-mentioned pharmaceutically acceptable salt is a salt formed by the compound of formula I and an acid selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydrobromic acid, tartaric acid, fumaric acid, hydroiodic acid, maleic acid, pyrosulfuric acid, phosphoric acid, nitric acid, ethanesulfonic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, cinnamic acid, cyclopentane propionic acid, dodecylsulfuric acid, 2-naphthalenesulfonic acid, citric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid or aspartic acid.

[0015] The application relates to the use of a compound of formula I (compound of formula CN-0928 or CN-0929) or a pharmaceutically acceptable salt thereof in the manufacture of a BACE1 inhibitor. The compound of formula I of the application can alleviate and improve Alzheimer's disease: specifically, the compound of formula I can alleviate and improve Alzheimer's disease by reducing the expression of BACE1; more specifically, the compound of formula I can reduce the formation of PCBP2 particles, thereby reducing the expression of BACE1 protein, and further reducing the deposition of A beta in the brain; and meanwhile, the structure of mitochondria in cells can be restored to a certain extent, so that the purposes of preventing and treating Alzheimer's disease are achieved. The application relates to Alzheimer's disease (AD).

[0016] The application relates to the use of a compound of formula I (compound of formula CN-0928 or CN-0929) in the manufacture of a drug for preventing or treating Alzheimer's disease. The application specifically relates to the use of the compound of formula I (compound of formula CN-0928 or CN-0929) in the manufacture of a drug for preventing or treating Alzheimer's disease; in the manufacture of a drug for preventing or treating language use disorders; in the manufacture of a drug for preventing or treating visual space disorders; in the manufacture of a drug for preventing or treating attention disorders; and in the manufacture of a drug for preventing or treating reasoning and abstract thinking ability disorders.

[0017] The application specifically relates to the use of the compound of formula I (compound of formula CN-0928 or CN-0929) in the manufacture of a drug for preventing or treating Alzheimer's disease; in the manufacture of a drug for preventing or treating spatial and associative learning and memory disorders; and in the manufacture of a drug for preventing or treating hippocampus-dependent learning and memory disorders.

[0018] The application further provides a preparation method of the compound of formula CN-0928 or CN-0929.

[0019] The application provides a preparation method of the compound of formula CN-0928, characterized in that compound 1 and compound 2 are reacted to obtain compound 3, and compound 3 and morpholine (compound 4b) are reacted to obtain the compound of formula CN-0928. The synthesis route is as follows:

[0020] The application provides a preparation method of the compound of formula CN-0929, characterized in that compound 1 and compound 2 are reacted to obtain compound 3, compound 3 and piperazine-1-carboxylic acid tert-butyl ester (compound 4a) are reacted to obtain compound 5a, and then the protecting group is removed to obtain the compound of formula CN-0929. The synthesis route is as follows:

[0021]

[0022] The present application also provides a method of treating Alzheimer's disease in a patient comprising administering to a patient in need of such treatment an effective amount of a CN-0928 or CN-0929 compound.

[0023] The present application further provides a method of preventing mild cognitive impairment from developing into Alzheimer's disease in a patient comprising administering to a patient in need of such treatment an effective amount of a CN-0928 or CN-0929 compound.

[0024] The present application also provides a method of inhibiting amyloid Aβ in a patient comprising administering to a patient in need of such treatment an effective amount of a CN-0928 or CN-0929 compound.

[0025] The CN-0928 or CN-0929 used in the present application is preferably at least 99.0% pure by weight.

[0026] Mild cognitive impairment is defined as the potential prodromal stage of dementia associated with Alzheimer's disease based on clinical presentation and the progression of patients exhibiting mild cognitive impairment over time to Alzheimer's dementia. The term "preventing mild cognitive impairment from developing into Alzheimer's disease" includes slowing, arresting or reversing the development of mild cognitive impairment in a patient into Alzheimer's disease.

[0027] The term "treatment" as used herein includes limiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.

[0028] The term "patient" as used herein means a human.

[0029] The term "effective amount" as used herein means the quantity or dose of a CN-0928 or CN-0929 compound of the present application, or a pharmaceutically acceptable salt thereof, which, when administered to a patient in a single or multiple dose, is effective to provide the intended effect on the patient being diagnosed or treated. As a person of ordinary skill in the art, a diagnostician can readily determine an effective amount using known techniques and taking into account the results obtained in similar situations. In determining the effective amount for a patient, a diagnostician will consider a number of factors, including but not limited to: the species of patient; age and general health of the patient; the specific disease or condition involved; the degree of or involvement or the severity of the disease or condition; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; concomitant medications; and other relevant circumstances.

[0030] Preferably, the CN-0928 and CN-0929 compounds of the present application are formulated into pharmaceutical compositions and administered by any route capable of resulting in bioavailability of the compound, including oral, transdermal, and parenteral routes. Most preferably, such compositions are administered orally or transdermally, with oral administration being especially preferred. Such pharmaceutical compositions and methods for their preparation are well known in the art, e.g., tablets, troches, powders, granules, capsules and the like; injectables such as injectable powders, injectable lyophilized powders and the like, all of which can be prepared following conventional methods. The above dosage forms are preferably oral capsules, tablets and injectables.

[0031] As a form of a preparation or a pharmaceutical composition which contains the compound of the present application as an effective ingredient, there are no particular limitations and examples of the preparation include tablets, powders, granules, capsules, oral solutions, emulsions, elixirs, lemonades, suspensions, syrups, buccal tablets, oral sprays, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nose drops, ear drops, patches, external solutions, and the like. In the formulation, commonly used excipients, binders, lubricants, coloring agents, taste- and odor-correcting agents, and, as needed, stabilizers, emulsifiers, absorption accelerators, surfactants, pH adjustors, preservatives, antioxidants, and the like, which are used as raw materials for pharmaceutical preparations, can be used in combination, and the formulation can be prepared by a conventional method. For example, in the production of oral preparations, the crystalline or non-crystalline compound of the present application and an excipient are added, and, as needed, a binder, a disintegrant, a lubricant, a coloring agent, a taste- and odor-correcting agent, and the like are further added as additives, and then, powders, fine granules, granules, tablets, coated tablets, capsules, and the like are prepared by a conventional method. As the additives, examples include: soybean oil, beef tallow, synthetic glycerides, and the like, animal and plant oils; liquid paraffin, squalane, solid paraffin, and the like, hydrocarbons; octyldodecyl myristate, isopropyl myristate, and the like, ester oils; cetyl stearyl alcohol, behenyl alcohol, and the like, higher alcohols; silicone resins; silicone oils; polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardened castor oil, polyoxyethylene polyoxypropylene block copolymers, and the like, surfactants; hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinylpyrrolidone, methyl cellulose, and the like, water-soluble polymers; ethanol, isopropyl alcohol, and the like, lower alcohols; glycerin, propylene glycol, dipropylene glycol, sorbitol, and the like, polyhydric alcohols; glucose, sucrose, and the like, sugars; anhydrous silicic acid, magnesium aluminum silicate, aluminum silicate, and the like, inorganic powders, purified water, and the like. As the excipients, examples include lactose, corn starch, white sugar, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide, and the like, and as the binders, examples include polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, acacia, tragacanth gum, gelatin, gellan gum, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polypropylene glycol·polyoxyethylene·block polymer, meglumine, and the like, and as the disintegrants, examples include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, carboxymethyl cellulose·calcium, and the like, and as the lubricants, examples include magnesium stearate, talc, polyethylene glycol, silicon dioxide, hardened vegetable oil, and the like, and as the coloring agents, coloring agents which are permitted to be added to pharmaceutical products can be used, and as the taste- and odor-correcting agents, examples include cocoa powder, menthol, aromatic sprays, peppermint oil, borneol, cassia powder, and the like. In the production of tablets or granules, sugar coating can be performed, and, as needed, appropriate coating can be performed.Furthermore, when manufacturing liquid preparations such as syrups, emulsions, elixirs, lemonades, suspensions, and injectable formulations, pH adjusters, solubilizers, emulsifiers, dispersants, isotonic agents, cosolvents, and stabilizers can be added as additives as needed, and the preparation can be formulated using conventional methods. The method for manufacturing topical preparations is not limited and can be carried out using conventional methods. That is, various raw materials commonly used in pharmaceuticals, quasi-drugs, and cosmetics can be used as the base raw materials used in formulation. Specifically, examples of base raw materials include, for instance, animal and vegetable oils, mineral oils, ester oils, waxes, higher alcohols, fatty acids, silicone oils, surfactants, phospholipids, alcohols, polyols, water-soluble polymers, clay minerals, resins, plastics, rubber, and other non-water-soluble natural or synthetic polymers, purified water, etc. Furthermore, pH adjusters, antioxidants, chelating agents, preservatives, antifungal agents, colorants, fragrances, etc., can be added as needed, but the base raw materials for the topical preparations of this invention are not limited to these. In addition, it can be combined with ingredients that induce differentiation, blood flow promoters, bactericides, anti-inflammatory agents, cell activators, vitamins, amino acids, moisturizers, keratolytic agents, etc., as needed. Furthermore, the amount of the above-mentioned base ingredients added is to achieve the concentration set during the manufacturing of typical topical preparations.

[0032] Beneficial effects

[0033] Studies have found that CN-0928(N-(3-cyano-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)-2-(morpholin-4-yl)acetamide (PubChem CID 826287) has no inhibitory effect on JNK2 and JNK3 kinases (Angell et al., 2007).

[0034] Surprisingly, the inventors of this invention discovered that compound CN-0928 can reduce BACE1 protein expression by regulating biomolecular condensates, thereby alleviating Aβ plaque burden; furthermore, it can restore mitochondrial structural changes, thus further alleviating AD pathology. This treatment based on intracellular RNP particles provides a new approach for developing drugs to prevent or treat neurological diseases such as Alzheimer's disease. CN-0928 targets INTS1 to regulate PCBP2 protein expression levels, thereby regulating the biomolecular condensates formed by PCBP2 and influencing multiple pathological processes in AD, demonstrating significant potential for development and application.

[0035] Animal experiments have shown that compound CN-0928 and its analogue CN-0929 can significantly improve spatial learning and memory abilities and associative learning and memory abilities in AD model mice. Attached Figure Description

[0036] Figure 1is the 1H NMR (600 MHz, DMSO-d6) spectrum of compound 5b.

[0037] Figure 2 is the 1H NMR (600 MHz, DMSO-d6) spectrum of compound CN-0929-HCL.

[0038] Figure 3 is the result graph of CN-0928 reducing BACE1 expression and its downstream products in human neuroblastoma cells (SH-SY5Y cells); (A) WB was used to detect the protein levels of PCBP2, BACE1, APP and ADAM10, B is the statistical graph; (C) is the live cell fluorescence imaging of SH-SY5Y cells stably expressing mCherry-PCBP2 after 48 hours of CN-0928 (1 μM) intervention, D is the statistical graph; scale bar: 10 μm; (E) is the level of amyloid pathway degradation product α / β-CTFs of APP detected using WB, F is the statistical graph; (G) is the level of intracellular (in) and extracellular (ex) Aβ40 and Aβ42 detected by ELISA after SH-SY5Y cells were treated with DMSO or CN-0928 (1 μM) for 48 hours; ns: not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001.

[0039] Figure 4 is the graph of CN-0928 improving the structure of damaged mitochondria in SH-SY5Y-PCBP2 cells: (A) Transmission electron microscopy was used to observe mitochondria in SH-SY5Y-mCherry-PCBP2 cells; the results show that after the addition of CN-0928, the morphology and number of mitochondria in cells have been restored to a certain extent, B and C are the statistical graphs of the number and length of mitochondria, respectively; *P < 0.05, ***P < 0.001.

[0040] Figure 5Figure 1 is a result graph of CN-0928 regulating the expression level of PCBP2 and BACE1 through its target INTS1: (A-D) SH-SY5Y cells were transfected with candidate target siINTS1 (A) or simH2A1 (C) for 24 hours, then treated with CN-0928 (1 mM) for 48 hours, Western blotting was used to detect the expression of PCBP2 and BACE1, B, D are the statistical graphs of A, C, respectively; (E) is the molecular docking diagram of CN-0928 and INTS1, showing that the amino acid at position ARG-1404 contributes the most in the binding process; (F) is the pull-down WB diagram of biotin-labeled CN-0928 and wild-type INTS1-flag, mutant R1404A-INTS1-flag and mutant R1404L-INTS1-flag; ns: not statistically significant, *P<0.05, **P<0.01, ***P<0.001; the results show that INTS1 (but not mH2A1) is the target of CN-0928 and mediates the effect of CN-0928.

[0041] Figure 6 Figure 2 is a result graph of CN-0928 reducing the Aβ plaque load of 5xFAD mice: (A-D) The hippocampal (A) and cortical (C) tissues of WT mice and 5xFAD mice treated with NS or CN-0928 (3.5 mg / kg) once every other day for 1 month were extracted, and the protein levels of PCBP2, BACE1 and APP were detected by WB; B, D are the statistical graphs of A, C, respectively; (E) is the fluorescence picture of the hippocampal region of WT mice and 5xFAD mice after NS or CN-0928 intervention, where blue marks DAPI, red marks PCBP2, and F is the statistical graph, scale: 10 pm; (G-J) The hippocampal (G) and cortical (I) tissues of WT mice and 5xFAD mice treated with NS or CN-0928 were extracted, and the levels of amyloidogenic degradation products a / β-CTFs of APP were detected by WB; H, J are the statistical graphs of G, I, respectively; (K) is the fluorescence picture of the hippocampal region of 5xFAD mice after NS or CN-0928 intervention, where blue marks DAPI, red marks BACE1, and green marks Aβ plaques, plaque area (L) and plaque number (M) statistical graphs, scale: 200 pm; (N) ELISA was used to detect the levels of soluble / insoluble Aβ in the hippocampal tissues of two groups of mice (5xFAD-NS and 5xFAD-CN-0928); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0042] Figure 7Figure 5 is a graph showing the results of CN-0928 improving cognitive function in 5xFAD mice; (A-F) 5xFAD mice were intraperitoneally injected with NS or CN-0928 (3.5 mg / kg) every other day for 1 month, and the same age WT mice intervened with NS were used as the complete blank control; (A-C) are the typical trajectory graphs of WT-NS group (A), 5xFAD-NS group (B), and 5xFAD-CN-0928 (C) mice in the spatial exploration experiment, respectively; (D) is the time used by the mice to reach the platform in the hidden platform test; (E-F) are the number of times the mice crossed the original hidden platform position (E) and the exploration time of the mice in the original platform quadrant (F) in the spatial exploration test, respectively; *P<0.05, **P<0.01.

[0043] Figure 8 Figure 6 is a graph showing the results of CN-0929-HCl reducing the Aβ plaque load in 5xFAD mice; (A-D) 5xFAD mice were intraperitoneally injected with NS or CN-0929-HCl (3.5 mg / kg) every other day for 1 month, and the same age WT mice intervened with NS were used as the complete blank control; In order to ensure the consistency of the experiment, the CN-0929-HCl administration experiment was carried out at the same time as the CN-0928 administration experiment, so the control group mice at this time were the same batch of mice; (A-D) are the tissue proteins of the hippocampus (A) and the cortex (C) of WT mice and 5xFAD mice after intervention, and the protein levels of PCBP2, BACE1, and APP were detected by WB; B and D are the statistical graphs of A and C, respectively; (E-H) are the tissue proteins of the hippocampus (E) and the cortex (G) of WT mice and 5xFAD mice after intervention, and the levels of amyloidogenic degradation products α / β-CTFs of APP were detected by WB; F and H are the statistical graphs of E and G, respectively; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0044] Figure 9Figure is the result of CN-0929-HCl improving the cognitive function of 5xFAD mice; (A-F) 5xFAD mice after intraperitoneal injection of NS or CN-0929-HCl (3.5 mg / kg) once every other day for 1 month, the same age WT mice intervened by NS as the complete blank control; In order to ensure the consistency of the experiment, the CN-0929-HCl administration experiment was carried out at the same time as the CN-0928 administration experiment, so the control group mice at this time were the same batch of mice; (A-C) are the typical trajectory diagrams of WT-NS group (A), 5xFAD-NS group (B), and 5xFAD-CN-0929-HCl group (C) mice in the spatial exploration experiment; (D) is the time used by the mice to reach the platform in the hidden platform test; (E-F) are the number of times the mice crossed the original hidden platform position (E) and the time the mice explored the original platform quadrant (F) in the spatial exploration test; *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0045] The application will be described in detail below through specific examples, it is pointed out here that the following examples are only used to further illustrate the application and cannot be understood as limiting the protection scope of the application, and those skilled in the art can make some non-essential improvements and adjustments to the application according to the above description.

[0046] DMSO (Dimethyl sulfoxide) was purchased from Solabio Technology Co., Ltd. in Beijing, China. SH-SY5Y cells were from the Chinese Academy of Sciences Cell Bank. The antibodies used in the present application are as follows: ADAM10 (polyclonal antibody, Abeam, Catalog No. ab1997); APP / CTF (polyclonal antibody, Sigma, Catalog No. A8717); β-amyloid, 1-16 [6E10] (monoclonal antibody, BioLegend, Catalog No. 803014); BACE1 (polyclonal antibody, Abeam, Catalog No. ab2077); BACE1 [EPR3956] (monoclonal antibody, Abeam, Catalog No. ab108394); BACE1 [EPR19523] (monoclonal antibody, Abeam, Catalog No. ab183612); PCBP2 [EPR14858] (monoclonal antibody, Abeam, Catalog No. ab184962); INTS1 (polyclonal antibody, Proteintech, Catalog No. 31428-1-AP); mH2A.1 (monoclonal antibody, Invitrogen, Catalog No. MA5-24696); Flag [DYKDDDDK tag] (monoclonal antibody, Proteintech, Catalog No. 66008-4-Ig); GAPDH (monoclonal antibody, Proteintech, Catalog No. 60004-1-Ig). ECL chemiluminescence solution was purchased from Themro Company in the United States. siRNA was purchased from Genomed Technology Co., Ltd. in China. Plasmids were purchased from Yubao Company in China. Medium, fetal bovine serum and Opti-MEM were purchased from Gibco Company (USA). ELISA kit Aβ1-42 (Elabscience, Catalog No. E-EL-H0543) and Aβ1-40 (Elabscience, Catalog No. E-EL-H0542).

[0047] CN-0928 (N-(3-cyano-4, 5, 6, 7-tetrahydro-1-benzothiophene-2-yl)-2-(morpholine-4-yl) acetamide (PubChem CID 826287)) and CN-0929-HCl (hydrochloride salt of compound CN-0929) in the present application can be obtained by Example 1.

[0048] Example 1 Preparation of compound of formula 1 (CN-0928 and CN-0929-HCl) The preparation process route of CN-0928 is as follows:

[0049]

[0050] The preparation process route of CN-0929-HCl is as follows:

[0051]

[0052] Procedure:

[0053] Synthesis of 2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (Compound 3)

[0054] Dissolve 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (Compound 1, 5.0 g, 28.0 mmol, 1 eq.) in 1,4-dioxane (20.0 mL) and then slowly add chloroacetyl chloride (Compound 2, 6.3 g, 56.0 mmol, 2 eq.) dropwise through a dropping funnel over a period of 10 min at 0 °C. After allowing the reaction mixture to warm to room temperature, continue stirring for 20 min. Monitor the progress of the reaction by thin layer chromatography. After completion of the reaction, pour the reaction mixture into ice-cold water. Collect the desired product by filtration and further wash with a mixture of diethyl ether and hexane (80:20, v / v) to obtain Compound 3 (6.2 g, 87%) as a light white solid.

[0055] Procedure for synthesis of compounds (5a-b)

[0056] Dissolve Compound 3 (0.78 mmol, 1 eq.) and triethylamine (2.35 mmol, 3 eq.) in 1,4-dioxane (3 mL) and stir for 10 min at room temperature, then add Compound 4a-4b (0.78 mmol, 2 eq.). Next, heat the reaction mixture to 70 °C and continue stirring until the reaction is complete. After completion of the reaction, cool to room temperature and pour the reaction mixture into ice-cold water. Collect the precipitate by vacuum filtration and dry. Purify the crude product by washing with diethyl ether to obtain 5a-5b as a light white solid. 4a = tert-butyl piperazine-1-carboxylate, 4b = morpholine.

[0057] tert-Butyl 4-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)piperazine-1-carboxylate (5a, CN-0928 compound): yield 63%, light white solid.

[0058] N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-morpholinoacetamide (5b): yield 50%, light white solid, NMR as shown in Figure 1

[0059] Synthesis of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(piperazin-1-yl)acetamide

[0060] ​Compound 5a (150 mg, 0.37 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (5.0 mL), then HCl (4M solution in 1,4-dioxane, 0.46 mL) was added. The reaction mixture was stirred at room temperature for 3 hours. The progress of the reaction was monitored by thin layer chromatography. After the reaction was completed, the mixture was concentrated under vacuum to obtain a light white powder CN-0929-HCl, whose nuclear magnetic resonance is shown in Figure 2 .

[0061] Example 2 Cell Experiment Drug Cell Intervention Experiment of CN-0928

[0062] Under a microscope, human neuroblastoma cells SH-SY5Y cells were observed, and when the fusion rate was about 85%-90%, the cells were digested and centrifuged, and the cells were plated in a 6-well plate at a density of 4x105 cells / well. When the cell fusion rate of the cells was about 70%, CN-0928 (1 μM) was added for 48 h of treatment, and then protein extraction was performed. The experimental results showed that CN-0928 1 μM concentration was safe, stable and effective, and 1 μM was selected as the drug treatment concentration in subsequent experiments.

[0063] Cell Protein Extraction

[0064] The protein lysis solution was prepared as needed in a ratio of RIPA: protease inhibitor (100x): phosphatase inhibitor (100x) = 100:1:1, mixed well, and placed on ice for standby. The cells treated with drugs for 48 hours were taken out of the incubator, the cell culture medium was discarded, and the cells were rinsed with pre-cooled PBS for 2-3 times. According to the cell amount, an appropriate amount of prepared lysis solution was added, and the cells were scraped with a cell scraper. The lysis solution was sucked into the corresponding EP tube labeled. Lysis for 15 min on ice, then centrifuged at 4°C, 14000g for 10 min, then the supernatant was transferred to a new EP tube. The BCA concentration determination kit was used according to the instructions, and the sample protein concentration was determined. According to the sample protein concentration calculation, an appropriate amount of SDS Loading Buffer was added to balance the sample. After denaturation at 95°C metal bath for 7 min, the sample was stored at -20°C.

[0065] Western Blot (WB)

[0066] The 3 μl protein pre-stained Marker and 10 μl protein sample were added into the corresponding lane respectively using 8% separation gel and 4% concentration gel (Tricine gel was used for the detection of α / β-CTFs, and electrophoresis was performed according to the kit instructions). First, 70V was used for about 20 min to reach the separation gel, then 110V electrophoresis was used for about 50 min, and the electrophoresis was stopped after the bromophenol blue reached the bottom of the separation gel, then 400mA constant current was used for 40 min for membrane transfer, after the membrane transfer was completed, the membrane was blocked with 5% skimmed milk powder at room temperature for 1 h. After blocking, the membrane was washed with TBST three times, then placed in the corresponding primary antibody dilution solution prepared in advance, and slowly incubated at 4°C with shaking. After 16-20 hours, the membrane was washed with TBST three times, then placed in the secondary antibody diluted with blocking solution at 1:5000, and slowly incubated at room temperature with shaking for 1 h. Then the membrane was washed with TBST three times, ECL chemiluminescence solution A and B were mixed at a ratio of 1:1, and evenly dropped onto the membrane, then scanned and analyzed by Fusion imaging system, and the scale value of each band was analyzed and counted by ImageJ software. The results showed that CN-0928 1 μM could significantly reduce the protein levels of PCBP2 and BACE1 in SH-SY5Y cells, and reduce the level of BACE1 enzyme cut product α / β-CTFs Figure 3 ).

[0067] Cell ELISA (Enzyme linked immunosorbent assay)

[0068] Sample preparation: For cell samples, collect intracellular and culture medium samples according to the instructions of the ELISA kit.

[0069] Detection step: ELISA kit is balanced to room temperature. Protein standard is centrifuged at 10000xg for 1 min at room temperature, 1 mL standard diluent is added to obtain 1000 pg / mL standard working solution, the standard working solution is diluted according to the instructions to obtain the corresponding concentration gradient diluted standard. Add 100 μl of each concentration standard or sample to be tested to each well, set 3 replicate wells for each sample. Cover the film, place in a 37°C incubator for 1.5 h. Take out the well plate, shake off the liquid, add 100 μL / well of biotinylated antibody working solution, cover the film, place in a 37°C incubator for 1 h. Take out the well plate, shake off the liquid, add 350 μL / well of washing solution, soak for 1 min, pat dry, repeat 3 times. After washing the plate, pat dry the well plate, immediately add 100 μl / well of HRP enzyme conjugate working solution, cover the film, and place in a 37°C incubator for 30 min. Wash the plate 5 times. Add 90 μL / well of substrate solution, cover the film, and place in a 37°C incubator for about 15 min. Then add 50 μl / well of stop solution to stop the reaction. Set the wavelength of the microplate reader to 450 nm to measure the OD value of each well. According to the standard value, draw a standard curve to calculate the concentration. The results show that CN-0928 1 μM can significantly reduce the level of Aβ in SH-SY5Y cells Figure 3 ).

[0070] Cellular immunofluorescence

[0071] Inoculate cells on a confocal dish, and perform drug intervention as before. Prepare 4% (w / v) sucrose-containing paraformaldehyde cell fixation solution with 4% paraformaldehyde in sucrose. Take out the intervened cells in the incubator, discard the culture medium, wash the cells with preheated PBS 3 times, and aspirate the liquid. Add cell fixation solution to immerse the cells, and place in a 37°C oven for 30 min. Discard the fixation solution, and wash with PBS 3 times as above, aspirate the liquid, add 0.3% (v / v) Triton membrane breaker prepared with PBS to immerse the cells, and place at room temperature for 10 min. Wash with PBS 3 times as above, add blocking horse serum (10%) to immerse the cells, and block in a 37°C oven for 30 min. Dilute the primary antibody with PBS, aspirate the horse serum, and add the primary antibody to the cell surface, making sure it evenly covers the cells, and incubate at 4°C overnight. Take out the cells after 16-20 h, reheat at room temperature for 10 min, wash with PBS 3 times as above, aspirate the liquid, dilute the green anti-rabbit fluorescent secondary antibody (1:200) with PBS, add the secondary antibody to the cell surface, making sure it evenly covers the cells, and incubate at 37°C for 1 h in the dark. Wash with PBS 3 times in the dark, aspirate the liquid, and mount with anti-fluorescent quenching mounting medium containing DAPI, and store at -20°C in the dark. Observe and take color pictures with a laser confocal microscope or a fluorescence microscope Figure 3 ). The results show that CN-0928 1 μM can significantly reduce the level of PCBP2 granules in SH-SY5Y cells.

[0072] Transmission electron microscope experiment

[0073] Cells treated with CN-0928 were taken out of the incubator and placed on a clean bench, and then washed twice with preheated PBS, and then pre-fixed with 2.5% glutaraldehyde solution at room temperature for 20 minutes. After pre-fixing, the cells were scraped and collected, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was obtained. Slowly add 2.5% glutaraldehyde solution along the wall of the tube, and fix at 4°C overnight, and contact the company for sectioning and transmission electron microscope shooting. Then use ImageJ to measure the length of mitochondria. The results show that CN-0928 1 μM can improve the damaged morphology of mitochondria to a certain extent. Figure 4

[0074] Example 3 Target experiment

[0075] Target pull-down identification

[0076] In order to explore the target of CN-0928 drug, the inventors use CN-0928 with biotin label (hereinafter referred to as CN-0928-biotin) to pull down the cell lysate protein, and complete the mass spectrometry analysis to identify the combined protein. The specific operation steps are as follows: SH-SY5Y cells are lysed in NETN buffer (0.1% NP-40, 0.5mM EDTA, 20mM Tris-HCl, 150mM NaCl2, protease inhibitor cocktail, pH=8.0). Pre-incubation: group 1 (CN-0928 group) and group 2 (blank control group): the extracted protein (2mg) is placed at 4°C overnight; group 3 (competitive binding group): the extracted protein (2mg) is first pre-incubated at 4°C overnight in the presence of excess free CN-0928 (100mM). Solidification: CN-0928-biotin is solidified on streptavidin-containing magnetic beads according to the requirements of the instructions. Pull-down: CN-0928 solidified magnetic beads are added to group 1 and group 3; group 2 is added to magnetic beads without CN-0928 solidification, and incubated at 4°C for another 4 hours. Then, the beads are washed with NETN for 3 times, and then the magnetic beads are sent for sample, and target identification is carried out by LC-MS / MS. The results show that according to the mass spectrometry identification results of the target, combined with the expression distribution and basic molecular function of each protein molecule, two protein molecules (INTS1 and mH2A1) are finally confirmed as candidate target molecules of CN-0928.

[0077] Target knockdown verification experiment

[0078] ​To further explore the target of CN-0928, the inventors knocked down the target and then added CN-0928 intervention to observe whether the regulation of CN-0928 on PCBP2 and BACE1 still exists. The specific operation steps are as follows: the SH-SY5Y cells were plated in advance for one day, and when the confluence rate was about 70%, the transfection reagent RNAiMAX was used to transfect according to the instruction manual. Transfection system: RNAiMAX: 20 μL / well plate, siRNA concentration: 50 nM. According to the amount calculated in advance, RNAiMAX and OPTI-MEM were mixed, and siRNA and OPTI-MEM were mixed. Mix well and stand at room temperature for 5 min. Mix the above two liquids containing RNAiMAX and siRNA, and stand at room temperature for 5 min. Discard the old culture medium in the well plate, add the calculated culture medium containing serum but not containing antibiotics, and add the mixed liquid of RNAiMAX and siRNA to each well according to the planned grouping, and culture in the cell incubator. After transfection for 24 h, replace the liquid and add CN-0928 (1 μM) for continuous culture for 48 h. After the intervention was completed, the cells were lysed and the protein was extracted as described above, and then WB detection was performed. The results show that INTS1 (but not mH2A1) is the target of CN-0928 and mediates the effect of CN-0928 Figure 5 ).

[0079] Target mutation verification experiment

[0080] To determine the binding site of CN-0928 and INTS1, the inventors used molecular docking to predict the binding site and found that the interaction between CN-0928 and INTS1 may occur through the Arg-1404 site. To further verify this binding site, the inventors mutated the INTS1-Arg-1404 site and added a flag tag, and then performed flag pull-down to verify the binding site. The specific operation steps are as follows: plate the SH-SY5Y cells, and when the confluence rate is about 70%, transfect the empty vector, INTS1-flag, and INTS1 mutant plasmids R1404A-flag and R1404L-flag plasmids according to the instruction manual of transfection reagent Lipofectamine3000. After transfection for 48 h, the cell lysate was collected, and the pull-down was performed according to the target pull-down experiment steps. The results show that the Arg-1404 site of INTS1 is the target of CN-0928 Figure 5 ).

[0081] Example 4 animal experiment

[0082] Experimental animals: 5xFAD (APPSwFlLon, PSEN1*M146L*L286V, B6SJL, #034840-JAX) mice, littermate wild-type mice, the parents of mice were purchased from the Jackson Laboratory, USA, and were purchased from GENE AND PEACE Co., Ltd. Male mice were used in all experiments. The mice were raised in the SPF mouse breeding room of Chongqing Medical University, and all animal feeding processes were in accordance with the "Regulations on the Management of Experimental Animals in China". All animal experiments were approved by the Ethics Committee of Chongqing Medical University, and all operations were carried out in accordance with the requirements of animal ethics.

[0083] Experimental mouse grouping: WT mouse normal saline intervention group (WT-NS); 5xFAD mouse normal saline intervention group (5xFAD-NS); 5xFAD mouse CN-0928 intervention group (5xFAD-CN-0928); 5xFAD mouse CN-0929-HCl intervention group (5xFAD-CN-0929-HCl).

[0084] Drug intervention scheme: The above four groups of mice started drug intervention at 5 months of age, and 3.5 mg / kg CN-0928 or 3.5 mg / kg CN-0929-HCl was injected intraperitoneally once every other day at the same time. The control group of mice was injected with normal saline. Continuous injection for 1 month.

[0085] Animal behavior: Morris water maze

[0086] The experiment contains two parts of 5 days of hidden platform test and 1 day of space exploration test. 3 days before the end of drug intervention, the mice were transferred from the SPF generation room to the behavior laboratory to adapt to the new environment, so as to avoid the interference caused by environmental changes. The water maze pool (diameter 1.2 meters) is filled with water, and the water temperature of the water maze is set to 22℃. Since the mice are black, the water is dyed white with white food coloring to facilitate tracking by the video system. A day before the formal experiment, the swimming ability of the mice was tested, and the mice with swimming ability defects were removed. At the same time every day, the mice were placed in the water maze pool from the four quadrants in order, and the water maze tracking system was used to record the time required for the mice to climb onto the hidden platform from entering the water. The exploration time was set to 60s (more than 60s, recorded as 60s). If the mouse fails to climb onto the platform within 60s, guide it to find the hidden platform and make it stay on the platform for 15s. A total of four rounds were performed in four quadrants per day, with an interval of more than 30 min, for 5 consecutive days. 24 hours after completing the hidden platform test, the platform was removed and the space exploration test was performed. The second quadrant was used as the mouse entry quadrant, and the mice were placed in order as above, and the tracking system recorded. The software analysis, statistics of each mouse's platform latency in the hidden platform test, and the mouse's exploration time, exploration distance and crossing of the original platform in the fourth quadrant in the space exploration test. The results show that compared with 5xFAD-NS, the time required for 5xFAD-CN-0928 and 5xFAD-CN-0929-HCl group mice to climb onto the hidden platform from entering the water gradually shortened, and the time spent in the platform quadrant was significantly longer, and the number of times through the platform was more Figure 7 and Figure 9 ). These results suggest that the spatial learning and memory ability of 5xFAD mice after drug treatment is improved.

[0087] Mouse hippocampus / cortex tissue protein extraction and WB detection

[0088] Prepare the protein lysis solution in the same proportion as needed, mix well, and place on ice for standby. Take the mouse hippocampus tissue out of the liquid nitrogen and place it in a labeled EP tube, weigh and add an appropriate amount of lysis solution (100 mg / mL) according to the tissue weight. Use the tissue homogenizer to homogenize the tissue on ice, and after homogenization, place it on ice for 30 min, then centrifuge at 4℃, 14000g for 10 min, then transfer the supernatant to a new EP tube. Measure the concentration, balance, and denature as above, and store at -80℃. WB detection is carried out according to the cell WB detection method to detect the protein expression level. The results show that compared with 5xFAD-NS, the expression levels of BACE1, PCBP2 and downstream product α / β-CTFs in 5xFAD-CN-0928 and 5xFAD-CN-0929-HCl group mice are significantly decreased Figure 6 and Figure 8 ).

[0089] tissue immunofluorescence

[0090] Observe the paraffin sections under a microscope, selecting sections with intact tissue and appropriate locations. Bake the paraffin sections in a 56℃ oven for 1-2 hours. Then, place the paraffin sections in xylene I and xylene II for 15 minutes each for dewaxing. Next, immerse the sections sequentially in anhydrous ethanol for 7 minutes, then in 95% ethanol, 80% ethanol, 70% ethanol, and tap water for 5 minutes each for hydration. Place the sections in freshly prepared PBS and wash them on a shaker at 90-110 rpm for 5 minutes, 3 times. Antigen retrieval: Place the sections in freshly prepared sodium citrate solution and heat in a microwave oven on high to boiling. After boiling, maintain high heat for 5 minutes, then on low heat for 15 minutes. Washing: After the sodium citrate solution cools naturally to room temperature, remove the sections and wash them 3 times with PBS as above. Blocking endogenous peroxidase: Place the sections in a humidified chamber with the tissue side up, and drop 3% hydrogen peroxide onto the tissue, completely and evenly covering it. Cover the chamber and incubate at room temperature for 10 minutes. Wash three times with PBS as above. Subsequent blocking, application of primary and secondary antibodies, and immunofluorescence were performed as before. Results showed that compared with 5×FAD-NS, mice in the 5×FAD-CN-0928 group had fewer PCBP2 particles and fewer Aβ plaques. Figure 6 ).

[0091] ELISA detection of mouse brain tissue

[0092] Sample preparation: For cell samples, collect intracellular and culture medium samples according to the ELISA kit instructions. For tissue samples, soluble proteins are extracted using the method described above, and the supernatant is collected; further, add 20 μl of 70% formic acid to the remaining precipitate after centrifugation, vortex intermittently, incubate at 4°C for 1 h, add 380 μL Tris-HCl (pH 8.0), and centrifuge again at 4°C, 16000 rpm for 1.5 h, and collect the supernatant as insoluble protein.

[0093] Detection step: the ELISA kit is balanced to room temperature. The protein standard is centrifuged at 10000xg for 1 min at room temperature, 1 mL of standard diluent is added to obtain a standard working solution of 1000 pg / mL, the standard working solution is diluted according to the instructions to obtain a gradient dilution of the corresponding concentration standard. Add 100 μl of each concentration standard or sample to be tested to each well, set up 3 replicate wells for each sample. Cover the film, place in 37℃, incubate for 1.5h. Take out the hole plate, shake off the liquid, add 100 μL / well of biotinylated antibody working solution, cover the film, place in 37℃, incubate for 1h. Take out the hole plate, shake off the liquid, add 350 μL / well of washing solution, soak for 1 min, pat dry, repeat 3 times. After washing the plate, pat the plate dry, immediately add 100 μl / well of HRP enzyme conjugate working solution, cover the film, and place in 37℃ for 30 min. Wash the plate 5 times. Add 90 μL / well of substrate solution, cover the film, and place in 37℃ for about 15 min. Then add 50 μl / well of stop solution to terminate the reaction. Set the wavelength of the enzyme labeler to 450 nm, measure the OD value of each well. And according to the standard value, draw a standard curve, calculate the concentration. The results show that compared with 5xFAD-NS, the level of Aβ in 5xFAD-CN-0928 group of mice is reduced Figure 6

[0094] Statistical analysis of experimental data

[0095] All experiments were repeated three times or more independently, and statistical analysis was performed by GraphPad Prism (version 9.0) software. The statistical comparison between two groups used Student's t test. Comparison among multiple groups used one-way ANOVA for testing. p<0.05 was considered statistically significant.​

Claims

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of Alzheimer's disease; said compound of formula I having the following chemical structure: Where Y is N or O.

2. The application as described in claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by a compound of formula I and an acid selected from hydrochloric acid, sulfuric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, hydrobromic acid, tartaric acid, fumaric acid, hydroiodic acid, maleic acid, pyrosulfonic acid, phosphoric acid, nitric acid, ethanesulfonic acid, acetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, hexanoic acid, benzoic acid, salicylic acid, cinnamic acid, cyclopentanepropionic acid, dodecyl sulfuric acid, 2-naphthalenesulfonic acid, citric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, or aspartic acid.

3. The application as described in claim 1, characterized in that, The compound of formula I is CN-0928, and its structural formula is as follows:

4. The application as described in claim 1, characterized in that, The compound of formula I is CN-0929, and its structural formula is as follows:

5. The application as described in any one of claims 1-4, characterized in that, The use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of BACE1 inhibitors.

6. The application as described in claim 5, characterized in that, Compound I alleviates and improves Alzheimer's disease by reducing the expression of BACE1.

7. The application as described in claim 5, characterized in that, Compound I reduces PCBP2 particle formation, thereby reducing BACE1 protein expression and alleviating Aβ deposition in the brain; or / and compound I restores intracellular mitochondrial structure to prevent or treat Alzheimer's disease.

8. The application as described in claim 5, characterized in that, The compound of Formula I may be used in the preparation of drugs for the prevention or treatment of language use disorders; or in the preparation of drugs for the prevention or treatment of visuospatial disorders; or in the preparation of drugs for the prevention or treatment of attention disorders; or in the preparation of drugs for the prevention or treatment of reasoning and abstract thinking disorders; or in the preparation of drugs for the prevention or treatment of spatial and associative learning and memory disorders; or in the preparation of drugs for the prevention or treatment of hippocampal-dependent learning and memory disorders.

9. A method for preparing compound CN-0928, characterized in that, Compound 1 and compound 2 react to give compound 3, and compound 3 reacts with morpholine (compound 4b) to give compound CN-0928; the synthetic route is as follows: .

10. A method for preparing compound CN-0929, characterized in that, Compound 1 and compound 2 react to give compound 3, and compound 3 reacts with piperazine-1-carboxylic acid tert-butyl ester (compound 4a) to give compound 5a. Then, the protecting group is removed to give compound CN-0929. The synthetic route is as follows: 。

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