Benzoheterocyclic compound and use thereof

Abstract

Provided in the present invention are a benzoheterocyclic compound and the use thereof. Specifically, provided in the present invention is a compound represented by formula D, a pharmaceutically acceptable salt thereof, a solvate thereof or a solvate of the pharmaceutically acceptable salt thereof, wherein X2 is O, S or Se and Z2 is F-18 or I-124. The benzoheterocyclic compound of the present invention can inhibit the formation of Aβ polymer, promote the depolymerization of Aβ polymer, inhibit abnormal activation of a microglial cell, and improve memory, thereby realizing the effects of improving cognitive impairment and treating AD. In addition, the benzoheterocyclic compound provided in the present invention is used as Aβ ligands. These ligands have high affinity and can specifically bind to Aβ protein in the brain. Further, these ligands can be labeled with radioisotopes F-18 and Ga-68 and can be used to observe and measure the distribution and accumulation of Aβ protein in the brain by means of the non-invasive PET imaging method.

Description

A benzoheterocyclic compound and its application

[0001] This application claims priority to Chinese patent application No. 202410102966X, filed on January 24, 2024, and priority to Chinese patent application No. 2024102819552, filed on March 12, 2024, and cites the full text of the above-mentioned Chinese patent applications. Technical Field

[0002] The present application relates to a benzoheterocyclic compound and its application. Background Art

[0003] Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease and one of the most common causes of dementia in the elderly. The disease primarily affects brain function, leading to a gradual loss of intellectual functions such as cognition and memory. The main pathological feature of AD is the presence of two abnormal protein deposits in brain tissue: amyloid-β (Aβ) and Tau protein. Aβ protein aggregates in the brain to form amyloid plaques, while Tau protein aggregates in neurons to form neurofibrillary tangles. The accumulation of these abnormal proteins causes neurons to gradually lose function and connectivity, ultimately leading to atrophy of brain tissue. Currently, there is no cure for Alzheimer's disease.

[0004] Aggregation of amyloid β-protein (Aβ) is the most important mechanism underlying the pathogenesis of AD, and preventing Aβ aggregation has been shown to be an effective treatment. Existing studies have demonstrated that soluble Aβ polymers are the primary source of toxicity, playing a crucial role in the development and progression of AD by affecting cell membrane ion channels, generating oxidative stress, and activating glial cells to trigger an inflammatory response. Aβ exists in multiple forms in the human body, primarily Aβ42 and Aβ40. While Aβ42 has been the focus of research due to its greater toxicity and increased aggregation susceptibility, other forms of Aβ, such as N-terminally truncated and modified N3pE, are also believed to be highly relevant to AD pathology. Therefore, targeting Aβ clearance in the brain is the most widely studied mechanism of action for drugs. Anti-Aβ monoclonal antibodies are currently the most extensively tested and the only approved novel therapeutic for AD. Numerous trials have demonstrated that Aβ monoclonal antibodies can clear Aβ plaques and, to a certain extent, mitigate cognitive and functional decline. Although some monoclonal antibodies have been discontinued due to poor blood-brain barrier passage, insignificant efficacy, cerebral edema or microbleeding and other adverse events, there are still more than a dozen Aβ monoclonal antibodies in different stages of clinical trials.

[0005] Various drug development approaches targeting Aβ essentially use Aβ prepared under specific conditions as a drug target. Screening for drug molecules that specifically bind to various Aβ species requires a diverse array of Aβ polymers, representing diverse sequences, aggregation levels, and mixed ratios. Aβ preparation methods can generally be categorized into three types. The first involves extracting and purifying Aβ from in vivo sources. This method maximizes the availability of a wide range of Aβ aggregates that closely resemble the in vivo state. However, extraction from postmortem AD patient brains is clearly impractical and cannot be used for drug screening. Transgenic mouse-derived Aβ, due to genotype constraints, is difficult to reflect the complex Aβ types found in the human brain. Furthermore, the extraction and purification process is complex and laborious, and the quality of the extraction is easily affected by various conditions, making it equally difficult to apply to large-scale drug screening. The second method involves extracting and purifying Aβ expressed in transfected cells. This method is more efficient than the first method in obtaining sufficient Aβ, but requires rigorous sterile biological culture and manipulation, complex plasmid construction, and inefficient simultaneous acquisition of multiple Aβ sequences and their mixed forms. The third approach involves the use of artificially synthesized Aβ. Solid-phase peptide synthesis technology is well established, allowing for rapid, easy, and highly purified production of a wide variety of Aβ sequences. However, there remains a lack of standardized methods for using these Aβ sequences to prepare various Aβ multimers for drug screening. The inventors discovered that numerous methods for preparing Aβ aggregates have been reported in various publications, but most rely on simple buffer or culture medium conditions, lacking systematic comparison and analysis of the morphology and aggregation of Aβ multimers prepared under different conditions. Unlike other common protein targets, Aβ exhibits relatively well-defined binding sites and a defined three-dimensional structure. The polymorphism of Aβ significantly increases the difficulty of studying it as a target. Due to the presence of various Aβ species with varying lengths and terminal modifications in vivo, the aggregation properties of these Aβ species reported in prior art vary significantly. Different incubation conditions can also significantly affect Aβ aggregation behavior. Furthermore, the Aβ aggregation process itself exhibits dynamic aggregation with varying degrees, morphologies, and sizes, resulting in significant heterogeneity. Consequently, the success rate of anti-Aβ drug development is extremely low.

[0006] Some medications and treatments can help relieve symptoms and improve patients' quality of life, so early diagnosis and intervention are crucial for the management of Alzheimer's disease.

[0007] To date, the diagnosis of AD still primarily relies on standard clinical evaluation, brain biopsies, and postmortem tissue studies. However, the development of in vivo diagnostic methods for Alzheimer's disease has involved researchers in multiple fields, including genetic testing, immunoassays, and imaging technologies. In the field of genetic testing, researchers are focused on identifying genetic factors associated with Alzheimer's disease. Variations in specific genes are associated with the risk of developing AD, so genetic testing can assess an individual's potential risk. This genetic testing offers a potential approach for early diagnosis, facilitating early intervention and treatment. Immunoassays, on the other hand, are widely used to detect AD biomarkers in body fluids. By detecting changes in specific proteins, such as Aβ and Tau, in cerebrospinal fluid or blood, AD diagnosis and disease progression can be assessed. These biomarkers have potential value in AD diagnosis and disease monitoring. Furthermore, imaging technologies are also being extensively investigated to achieve non-invasive AD diagnosis. Positron emission tomography (PET) is a commonly used imaging technique that uses specific tracers to label Aβ and Tau proteins in the brain. These imaging methods provide visualization of AD pathological changes, facilitating early diagnosis and disease monitoring.

[0008] Currently, AD is typically diagnosed clinically through tests such as plasma markers, cerebrospinal fluid (CSF) biochemical profiles, cognitive function tests, and medical imaging. Blood tests and brain imaging can exclude other potential causes of dementia symptoms (e.g., cardiovascular and cerebrovascular disease, tumors, or vitamin deficiencies). Cognitive function test results are often associated with Alzheimer's disease but do not directly reflect the severity of the disease. A definitive diagnosis of Alzheimer's disease relies on pathological examination of brain tissue. Aβ levels in cerebrospinal fluid (CSF) are determined by measuring Aβ42 or the ratio of Aβ40 to Aβ42. However, CSF collection requires a puncture, which is difficult for patients to tolerate and is not suitable for clinical diagnosis and screening of AD. Compared to these methods, Aβ-PET imaging is performed using a specific Aβ radiotracer. PET imaging can better assess the extent and location of Aβ deposition and has higher sensitivity and specificity for visualizing Aβ. Importantly, PET imaging has more standardized procedures and is highly reproducible across individuals, which is essential for comparing data across studies and longitudinal changes.

[0009] F-18 labeled fluorodeoxyglucose ( 18 F-FDG) was the first PET tracer used in AD diagnosis. 18The uptake rate of F-FDG by tissues is highly dependent on the metabolic activity level of the tissues. Many tissues also take up glucose under normal physiological conditions, especially the brain, heart, and skeletal muscle. Therefore, a high background signal may be generated in these areas, which cannot accurately reflect the pathological changes of AD. Therefore, this tracer is no longer used as an imaging agent for AD diagnosis. In 2002, in vivo Aβ-PET imaging was first performed on AD patients, in which C-11-labeled Pittsburgh compound B ( 11 C-PIB), an Aβ-PET molecular imaging tracer designed based on the Aβ dye thioflavin-T (ThT). Imaging at injected nanomolar concentrations demonstrated that 11 C-PIB can specifically bind to Aβ, reflecting the location and concentration of Aβ deposition in living brain tissue, indicating that the radioactivity in the frontal, temporal, and parietal lobes of AD patients is significantly increased, which is consistent with the Aβ plaque deposition areas found in autopsy pathology. However, the short half-life of the C-11 isotope (T1 / 2 = 20 min) limits the use of this tracer to PET research centers equipped with cyclotrons. On this basis, molecular imaging tracers labeled with F-18 (T1 / 2 = 110 min) with a longer half-life have been developed for a wider range of applications. Currently, the three Aβ-PET molecular imaging tracers approved by the FDA for early clinical diagnosis of AD are all F-18 radioactively labeled molecular probes, namely 18 F-Florbetapir, 18 F-Florbetaben, and 18 F-Flutemetamol. However, the shortcomings of this type of PET molecular imaging tracer are: poor stability, high white matter uptake, and the need to improve diagnostic sensitivity and specificity. An ideal Aβ-PET molecular imaging tracer should meet the following characteristics: 1. Have a certain degree of lipid solubility, able to cross the blood-brain barrier, reach the brain, and bind to Aβ; 2. Have highly specific binding to Aβ while exhibiting minimal nonspecific binding; 3. Have good in vivo stability, reducing the nonspecific signal of metabolites in the brain; 4. Be simple and efficient to prepare, meeting the needs of clinical application.

[0010] Therefore, the development of new drugs and PET molecular imaging tracers targeting Aβ is of great significance for the specific diagnosis and treatment of AD. Summary of the Invention

[0011] The technical problem to be solved by the present invention is that the Aβ-PET molecular imaging tracer in the prior art has a single structure, few types of AD therapeutic drugs, and poor effects, and provides a benzoheterocyclic compound and its application. The benzoheterocyclic compound of the present invention can inhibit the formation of Aβ polymers, promote the disaggregation of Aβ polymers, inhibit abnormal activation of microglia, improve memory, and achieve the effect of improving cognitive impairment and treating AD. At the same time, the benzoheterocyclic compounds provided in this application serve as Aβ ligands. These ligands have a high affinity and can specifically bind to Aβ proteins in the brain. Furthermore, these ligands can be labeled with radioactive isotopes F-18 and Ga-68, and the distribution and accumulation of Aβ proteins in the brain can be observed and measured by non-invasive PET imaging methods.

[0012] The present invention provides a compound as shown in Formula A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0013] Among them, R consists of a chelating group and a radionuclide;

[0014] The chelating group is unmodified or modified with a membrane-penetrating peptide;

[0015] The radionuclide is a diagnostic radionuclide;

[0016] m is 1 or 2;

[0017] Each X is independently O, S or Se;

[0018] each Y is independently CH or N;

[0019] Each Z is independently H, F, F-18, Cl, Br or I;

[0020] Each n is independently 1, 2, 3 or 4.

[0021] In some embodiments, in the compound represented by Formula A, its pharmaceutically acceptable salt, its solvate, or its pharmaceutically acceptable salt solvate, the definitions of certain groups may be as described below, and the definitions of other groups may be as described in any embodiment of the present invention (hereinafter referred to as "in some embodiments"), X is O or S.

[0022] In some embodiments, Z is F or H.

[0023] In some embodiments, n is 3 or 4.

[0024] In some embodiments, the cell-penetrating peptide is HAIYPRH (SEQ ID NO: 1), THRPPMWSPVWP (SEQ ID NO: 2), TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 3), AGILKRW (SEQ ID NO: 4), CGNKRTR (SEQ ID NO: 5), LRKLRKRLLR (SEQ ID NO: 6), or YGRKKRRQRRR (SEQ ID NO: 7).

[0025] In some embodiments, the chelating group is

[0026] In some embodiments, the radionuclide is selected from Cu-64 and Ga-68, such as Ga-68.

[0027] In some embodiments, the compound of formula A is represented by formula A-1:

[0028] In one embodiment, the compound represented by formula A is selected from any one of the following compounds:

[0029] The present invention also provides a compound as shown in Formula B, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0030] Wherein, the definitions of X, Y, Z, m, and n are as described in any of the previous schemes, Q is a chelating group, and the chelating group is as described in any of the previous schemes.

[0031] In one embodiment, the compound represented by formula B is any one of the following compounds:

[0032] The present invention also provides a compound as shown in Formula C, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0033] Wherein, the definitions of X, Y, Z, m, and n are as described in the previous scheme;

[0034] T consists of a chelating group and a non-radioactive nuclide, wherein the chelating group is as described in any of the previous schemes.

[0035] In some embodiments, the non-radioactive nuclide is Cu or Ga, such as Ga.

[0036] In one embodiment, the compound shown in formula C is

[0037] The present invention also provides a compound as shown in Formula D, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0038] Wherein, X2 is O, S or Se;

[0039] Z2 is F-18 or I-124.

[0040] In some embodiments, X2 is O or S.

[0041] In some embodiments, Z2 is F-18.

[0042] In some embodiments, the compound shown in Formula D is

[0043] In one embodiment, the compound shown in formula D is

[0044] The present invention also provides a compound as shown in Formula E, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof:

[0045] Wherein, the definition of X2 is as described in the previous scheme;

[0046] Z3 is F or I.

[0047] In some embodiments, Z3 is F.

[0048] In some embodiments, the compound shown in Formula E is

[0049] In one embodiment, the compound shown in Formula E is

[0050] The present invention also provides a pharmaceutical composition comprising the compound as described in any of the preceding schemes, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

[0051] The present invention also provides a kit comprising the compound as described in any of the above schemes, its pharmaceutically acceptable salt, its solvate, or its solvate of a pharmaceutically acceptable salt, or the aforementioned pharmaceutical composition, and instructions for use.

[0052] The present invention also provides the use of the compound represented by formula A or the compound represented by formula D in the preparation of a PET molecular probe.

[0053] In some embodiments, the PET molecular probe is used for the diagnosis of Alzheimer's disease.

[0054] In some embodiments, the PET molecular probe is used to image Aβ protein binding.

[0055] The present invention also provides the use of the aforementioned compound, its pharmaceutically acceptable salt, its solvate, or its pharmaceutically acceptable salt solvate in the preparation of a drug for preventing and / or treating Alzheimer's disease. For example, the use of the compound shown in Formula E, its pharmaceutically acceptable salt, its solvate, or its pharmaceutically acceptable salt solvate in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0056] The present invention also provides the use of the aforementioned compound, its pharmaceutically acceptable salt, its solvate, or its pharmaceutically acceptable salt solvate in the preparation of an anti-Aβ drug, wherein the substance V is defined as described in claim 15; preferably, the anti-Aβ drug exerts its effect by disaggregating Aβ polymers or preventing the formation of Aβ polymers.

[0057] The present invention also provides the aforementioned compound, its pharmaceutically acceptable salt, its solvate, or its pharmaceutically acceptable salt solvate, which plays one or more of the following roles: (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; (iii) binding to Aβ polymers.

[0058] Another object of the present invention is to provide a method for preparing Aβ aggregates.

[0059] Another object of the present invention is to provide a target for screening anti-Aβ drugs.

[0060] Another object of the present invention is to provide a method for screening anti-Aβ drugs.

[0061] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing Aβ aggregates, which comprises the following steps:

[0062] S1, dissolving Aβ lyophilized powder and drying to remove the solvent to obtain Aβ peptide film;

[0063] S2, mixing the Aβ peptide film and a buffer to obtain an Aβ solution with a concentration of 0.2-100 μM;

[0064] S3, incubating the Aβ solution to obtain Aβ aggregates; and

[0065] In step S1, the Aβ lyophilized powder is Aβ40, Aβ42, N3pE42, Aβ38 or a combination thereof;

[0066] In step S2, the buffer is selected from at least one of Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12 mM HEPES and 140-160 mM NaCl, artificial cerebrospinal fluid (aCSF), DMEM, FBS, endosome simulation fluid, and lysosome simulation fluid;

[0067] In step S3, the incubation temperature is 30-43° C., the incubation speed is 0-2500 rpm, and the incubation time is not less than one day.

[0068] In some preferred embodiments, in step S1, the solvent is hexafluoroisopropanol.

[0069] In some preferred embodiments, in step S3, the incubation time is 1-7 days.

[0070] In some preferred embodiments, in step S3, the incubation is performed in a constant temperature homogenizer, and the rotation speed of the constant temperature homogenizer is 0-2500 rpm.

[0071] In some preferred embodiments, the concentration of the Aβ solution is 0.5-50 uM, more preferably 1-20 uM.

[0072] In some preferred embodiments, the buffer is Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12 mM HEPES and 140-160 mM NaCl, or artificial cerebrospinal fluid (aCSF).

[0073] In some preferred embodiments, the endosome simulation solution is a mixture of a sodium citrate buffer solution with a pH of 5.5 and a concentration of 18-22 mM, a NaCl solution with a concentration of 75-85 mM, and a KCl solution with a concentration of 18-22 mM.

[0074] In some preferred embodiments, the lysosome simulation solution is a mixture of a sodium citrate buffer solution with a concentration of 18-22 mM and a pH of 4.5, a NaCl solution with a concentration of 45-55 mM, and a KCl solution with a concentration of 45-55 mM.

[0075] In some preferred embodiments, the lyophilized Aβ powder is a combination of Aβ40 and Aβ42, or a combination of Aβ40 and N3pE42, or a combination of Aβ40, Aβ42, and N3pE42. Preferably, the molar percentage of Aβ40 in the combination is not less than 50%. In some preferred embodiments, the molar ratio of Aβ40 to the other Aβ in the combination is (5-9):(1-5).

[0076] In some preferred embodiments, the Aβ lyophilized powder is a combination of Aβ40 and Aβ42, wherein the molar ratio of Aβ40 to Aβ42 is 9:1, 8:2, 7:3, 6:4 or 5:5.

[0077] In some preferred embodiments, the Aβ lyophilized powder is a combination of Aβ40 and N3pE42, wherein the molar ratio of Aβ40 to N3pE42 is 9: 1. In some preferred embodiments, the Aβ lyophilized powder is a combination of Aβ40, Aβ42, and N3pE42, wherein the molar ratio of Aβ40, Aβ42, and N3pE42 is 5:4:1.

[0078] In some preferred embodiments, the incubation temperature is 36-41° C. and the incubation speed is 1500-2500 rpm.

[0079] The second aspect of the present invention provides the use of Aβ aggregates as a target for screening anti-Aβ drugs, wherein the Aβ aggregates are prepared by the method described in the first aspect of the present invention.

[0080] The third aspect of the present invention provides a target for screening anti-Aβ drugs, wherein the target is Aβ aggregates, and the Aβ aggregates are prepared by the method described in the first aspect of the present invention.

[0081] The fourth aspect of the present invention provides an anti-Aβ drug screening method, which comprises the steps of: screening anti-Aβ drugs using the anti-Aβ drug screening target described in the third aspect of the present invention.

[0082] In some preferred embodiments, the method includes the steps of screening for drugs that can disaggregate Aβ aggregates, or can bind to Aβ aggregates, or can inhibit the polymerization of Aβ aggregates, wherein the drug is selected from at least one of antibodies, polypeptides and small molecule compounds.

[0083] The fifth aspect of the present invention provides an anti-Aβ drug, which is obtained by screening using the method described in the fourth aspect of the present invention.

[0084] In some preferred embodiments, the compound is a compound of formula I,

[0085] In a sixth aspect, the present invention provides the use of a compound of formula I for non-therapeutic in vitro (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; and / or (iii) binding to Aβ polymers;

[0086] Compared with the prior art, the present invention has at least the following advantages:

[0087] (1) The screening method provided by the present invention uses different Aβ aggregates prepared by simulating the in vivo environment and conditions as targets to more accurately and effectively screen for anti-Aβ candidate drugs;

[0088] (2) The screening method provided by the present invention involves incubating the aggregation of Aβ targets under different microenvironmental conditions close to those in vivo, without the need for any labeling or modification, and the drug molecules screened also do not need any modification or labeling;

[0089] (3) The screening method provided by the present invention can more intuitively screen and obtain anti-Aβ drug candidates with good activity;

[0090] (4) The screening method provided by the present invention is not limited to a certain type of drug, but can be widely applied to different types of drugs such as antibodies, peptides, and small molecules. It can truly reflect the interaction between the target and these drug molecules, greatly reducing the probability of false positives, false negatives, or incorrect binding patterns.

[0091] The compounds provided by the present invention have the following positive effects: (1) the F-18 radiochemical synthesis labeling rate in the present invention is high, the labeling conditions are mild, and the target compound is easy to prepare; (2) the Ga-68 radiochemical synthesis labeling yield in the present invention is high, the labeling time is short, and the operation is simple. A portable Ge-Ga generator can be used to achieve sufficient dose preparation without relying on a large cyclotron, which is convenient for production and popular use in grassroots hospitals; (3) the new PET imaging agent in the present invention has good in vitro and in vivo biological activity and excellent in vivo kinetic properties, which can improve the shortcomings of current PET imaging agents. (4) the compounds provided by the present invention inhibit the formation of Aβ polymers, which is better than the marketed drug Aricept; (5) the compounds provided by the present invention promote the disaggregation of Aβ polymers, which is better than the marketed drug Aricept; (6) the compounds provided by the present invention have good binding activity with Aβ polymers; (7) the compounds provided by the present invention have high activity in reducing Aβ plaques; (8) the compounds provided by the present application show significant effects in reducing the activation level of microglia in the brains of AD mice and can alleviate neuroinflammation. (9) The compounds provided in this application can improve the learning and memory functions of AD mice and are superior to the marketed drug Aricept.

[0092] the term

[0093] Unless otherwise indicated, the definitions of groups and terms in the present specification and claims, including definitions provided as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the present specification.

[0094] The term "membrane-penetrating peptide" refers to a short peptide that can carry a compound across the blood-brain barrier, and its membrane-penetrating ability does not rely on classical endocytosis. The membrane-penetrating peptide includes but is not limited to HAIYPRH (SEQ ID NO: 1), THRPPMWSPVWP (SEQ ID NO: 2), TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 3), AGILKRW (SEQ ID NO: 4), CGNKRTR (SEQ ID NO: 5), LRKLRKRLLR (SEQ ID NO: 6), YGRKKRRQRRR (SEQ ID NO: 7) or other polypeptides.

[0095] The term "treatment" means administering the compound or formulation described herein to prevent, improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0096] (i) preventing a disease or disease state from occurring in a mammal, particularly where such mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state;

[0097] (ii) inhibiting the disease or disease state, i.e., curbing its development;

[0098] (iii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0099] Those skilled in the art will appreciate that, after knowing the structures of the compounds of the present invention, the compounds of the present invention can be obtained by commercially available or various methods well known in the art, using known starting materials, such as the methods described in the examples of the present invention. These methods are all encompassed by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 shows compound 2" (i.e. [ 69 The ultraviolet absorption peak of the Ga] compound 2) is at 365 nm.

[0101] Figure 2 shows compound 2 (i.e. [ 68 Ga] compound 2) of the radiation signal peak.

[0102] Figure 3 is [ 68 Autoradiographic imaging results of [Ga] compound 2 on APP / PS1 mouse brain sections.

[0103] Figure 4 is [ 68 Quantitative analysis results of the autoradiographic imaging of compound 2.

[0104] Figure 5 is [ 68 PET / CT imaging of the brains of AD model mice (APP / PS1) and normal mice (WT) was performed using Ga] compound 2.

[0105] Figure 6 is [ 68 Quantitative analysis of the uptake of compound 2 in the brains of AD model mice (APP / PS1) and normal mice (WT).

[0106] FIG7 is a representative atomic force microscope (AFM) image of a typical Aβ polymer according to an embodiment of the present invention (left: Aβ42 oligomer; right: Aβ42 plaque);

[0107] FIG8 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0108] FIG9 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 uM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0109] FIG10 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (2 uM, 37° C., 2000 rpm, HBS incubation for 24 h);

[0110] FIG11 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, incubated in HBS for 1 week);

[0111] FIG12 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 μM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0112] FIG13 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0113] FIG14 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (2 uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0114] FIG15 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (50 uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0115] FIG16 is a representative AFM image according to an embodiment of the present invention, left: Aβ40, right: Aβ42 (10 uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0116] FIG17 is a representative AFM image according to an embodiment of the present invention (Aβ40 / 42=5 / 5uM, 37° C., 2000 rpm, aCSF incubation for 24 h);

[0117] FIG18 is a representative AFM image according to an embodiment of the present invention (Aβ40 / 42=5 / 5uM, 37° C., 2000 rpm, aCSF incubation for 1 week);

[0118] FIG19 is a representative example of monitoring Aβ aggregation dynamics by ThT according to an embodiment of the present invention, wherein the Aβ aggregate incubation conditions are 25uM Aβ42+40uM ThT, DPBS, 37° C.;

[0119] FIG20 is an example of ThT binding to Aβ aggregates according to an embodiment of the present invention, wherein the Aβ aggregate incubation conditions are 5uM Aβ42 incubated in DPBS at 37° C. for 78h, 40uM ThT);

[0120] FIG21 is an AFM screening of compounds that can inhibit Aβ polymerization according to an embodiment of the present invention, wherein the Aβ polymer incubation conditions are 10 mM HCl + 150 mM NaCl, 37° C., and incubation for 48 h;

[0121] FIG22 is an AFM screening of compounds that can inhibit Aβ polymerization according to an embodiment of the present invention, wherein the Aβ polymer incubation conditions are DPBS, 37° C., and incubation for 78 h;

[0122] Figure 23 shows the aggregation observed by AFM before and after the addition of compounds according to an embodiment of the present invention, wherein the left side shows 25uM N3pE42 aggregates (DPBS, 70h), and the right side shows 25uM N3pE42 aggregates (DPBS, 70h) + 40uM compound 1', and then incubated for 90h. ；

[0123] FIG24 is a representative AFM image of Aβ38 (10 μM, 37° C., 2000 rpm, incubated in aCSF for 24 h) according to an embodiment of the present invention;

[0124] FIG25 is a representative AFM image of Aβ38 (2 μM, 41° C., 2000 rpm, aCSF incubation for 24 h) according to an embodiment of the present invention;

[0125] FIG26 is a representative AFM image of Aβ38 (2 μM, 41° C., 2000 rpm, incubated in Lysosome-simulating fluid for 24 h) according to an embodiment of the present invention;

[0126] FIG27 is a representative AFM image of Aβ42 (10 μM, 37° C., 0 rpm, incubated in HBS for 24 h) according to an embodiment of the present invention;

[0127] FIG28 is a representative AFM image of Aβ40 (50 μM, 37° C., 0 rpm, incubated in HBS for 24 h) according to an embodiment of the present invention;

[0128] FIG. 29 is a representative AFM image of Aβ40 (50 μM, 37° C., 0 rpm, incubated in HBS for 1 week) according to an embodiment of the present invention.

[0129] FIG30 shows the Aβ aggregation observed by atomic force microscopy (AFM) before and after the addition of the compound.

[0130] FIG31 shows the effects of different drugs on the area of ​​Aβ plaques in the mouse brain.

[0131] FIG32 shows the effects of administering different drugs on the area of ​​activated microglia in the brain of mice.

[0132] FIG33 shows the effects of different drugs on the performance of mice in the water maze test.

[0133] Figure 34 shows the hippocampus and cortex of the treated group mice and the model group [ 18 F] Comparison of AV45 uptake ratio. Figure 35 shows the [ 18 F] Comparison of DPA714 uptake ratios. DETAILED DESCRIPTION

[0134] The animal models used in this application are as follows:

[0135] APP / PS1 mice (i.e., AD mice) were purchased from The Jackson Laboratory, 6 or 16 months old, half male and half female.

[0136] C57BL / 6J mice (i.e., normal control mice / wild mice): Gender matched with mice in the experimental group.

[0137] Example 1 Synthesis of the compound represented by formula D

[0138] Taking the 6-position of hydroxyl group, X=O, Y=C, Z=F-18 as an example, other compounds in the general formula can be synthesized by the same method by replacing the substrate.

[0139] Step (1) Synthesis of 6-[(ethoxymethyl)oxy]-1-benzofuran

[0140] 6-Hydroxybenzofuran (2.7 g, 20.0 mmol) was dissolved in dry THF (30 mL) and cooled in an ice bath. NaH (60% dispersion in kerosene, 1.2 g, 30.0 mmol) was added portionwise. After stirring for 10 min, chloromethyl ether (1.89 g, 20.0 mmol) was added dropwise to the mixture. The mixture was stirred at room temperature for 3 h. The reaction was quenched with saturated aqueous NH4Cl solution and extracted with ethyl acetate. The organic layer was washed with saturated NaCl solution, dried over anhydrous magnesium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by flash chromatography to obtain 6-[(ethoxymethyl)oxy]-1-benzofuran (2.9 g, 75% yield).

[0141] Step (2) Synthesis of {6-[(ethoxymethyl)oxy]-1-benzofuran-2-yl}boronic acid

[0142] 6-[(Ethoxymethyl)oxy]-1-benzofuran (0.96 g, 5.0 mmol) was dissolved in dry THF (20 mL). After cooling to -78°C, a 2.4 M n-butyllithium hexane solution (2.5 mL, 6.0 mmol) was slowly added dropwise. The mixture was stirred at -78°C for 1 hour. Triisopropyl borate (1.4 mL, 6.0 mmol) was then added dropwise to the mixture, and stirring continued at -78°C for 30 minutes. The reaction solution was slowly warmed to room temperature and stirred for 30 minutes. After the reaction was completed, 2N aqueous HCl (6.0 mL) was added dropwise at room temperature. The mixture was then poured into water (20 mL) and extracted with ethyl acetate. The organic layer was washed with saturated NaCl solution, dried over anhydrous magnesium sulfate, filtered, and the solvent removed under reduced pressure. The crude product was purified by flash chromatography to obtain 6-[(ethoxymethyl)oxy]-1-benzofuran-2-yl}boronic acid (0.35 g, 30% yield).

[0143] Step (3) Synthesis of 2-methylpropane-2-yl[(4-{6-[(ethoxymethyl)oxy]-1-benzofuran-2-yl}-2-nitrophenyl)(methyl)amino]methane

[0144] {6-[(Ethoxymethyl)oxy]-1-benzofuran-2-yl}boronic acid (140 mg, 0.59 mmol) was dissolved in ethanol (10 mL), and 2-methylpropan-2-yl [(4-bromo-2-nitrophenyl)(methyl)amino]methane (151 mg, 0.46 mmol), Pd(PPh3)2Cl2 (42 mg, 0.06 mmol), and Et3N (127 μL, 0.91 mmol) were added. The reaction mixture was reacted in a microwave reactor at 100°C for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was diluted with water (15 mL). The mixture was extracted with ethyl acetate, and the organic layer was washed with saturated NaCl solution and dried over anhydrous magnesium sulfate. After filtration, the solvent was removed under reduced pressure, and the crude product was purified by flash chromatography to give N-(4-(6-(ethoxymethoxy)benzofuran-2-yl)-2-nitrophenyl)-N-methylformamide (70 mg, yield: 41%); ESMS: m / z 371.12 [M+1] + ; 1 H NMR(500MHz,Chloroform-d)δ8.58(q,J=0.9Hz,1H),8.40(d,J=2.1Hz,1H),7.98(dd,J=7.4,2.1Hz,1H),7.70–7 .63(m,1H),6.88–6.83(m,1H),5.07(s,1H),3.62(q,J=7.4Hz,1H),3.48(d,J=1.0Hz,2H),1.18(t,J=7.5Hz,2H).

[0145] Step (4) Synthesis of 2-(3-fluoro-4-(methylamino)phenyl)benzofuran-6-ol (Preparation of Compound 1')

[0146] 4.1 Dissolve {6-[(Ethoxymethyl)oxy]-1-benzofuran-2-yl}boronic acid (140 mg, 0.59 mmol) in ethanol (10 mL), add 2-methylpropan-2-yl [(4-bromo-2-fluorophenyl)(methyl)amino]methane (107 mg, 0.46 mmol), Pd(PPh3)2Cl2 (42 mg, 0.06 mmol), and Et3N (127 μL, 0.91 mmol). The reaction mixture is stirred in a microwave reactor at 100°C for 30 minutes. The mixture was concentrated under reduced pressure, and the residue was diluted with water (15 ml). The mixture was extracted with ethyl acetate, and the organic layer was washed with saturated NaCl solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and the crude product was purified by flash chromatography to give N-(4-(6-(ethoxymethoxy)benzofuran-2-yl)-2-fluorophenyl)-N-methylformamide as a light yellow solid (90 mg, yield: 57%).

[0147] 4.2 N-(4-(6-(Ethoxymethoxy)benzofuran-2-yl)-2-fluorophenyl)-N-methylformamide (90 mg, 0.26 mmol) was dissolved in DMF (5.0 mL) and 6M HCl (5.0 mL) and stirred at 100°C for 60 minutes. TLC spot plate analysis indicated the reaction was complete. The reaction was quenched with water, the pH was adjusted to neutral with 1N NaOH, extracted with dichloromethane, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure and the crude product was purified by flash chromatography to afford 2-(3-fluoro-4-(methylamino)phenyl)benzofuran-6-ol as a white solid (64 mg, yield: 96%). ESMS: m / z 258.09 [M+1] + ; 1 H NMR(500MHz,Chloroform-d)δ8.47(s,1H),7.43(dd,J=9.1,2.2Hz,2H),7.32(dd,J=12.1,2.2Hz,1H),7.00(dd,J=9.2,4.7Hz,1 H), 6.93 (d, J = 2.0Hz, 1H), 6.85 (d, J = 2.3Hz, 1H), 6.78 (dd, J = 8.4, 2.2Hz, 1H), 6.63 (m, J = 4.6, 3.5Hz, 1H), 3.01 (d, J = 4.8Hz, 3H).

[0148] Step (5) F-18 radiolabeling (compound wherein X=O in general formula A, compound 1)

[0149] Towards[ 18To an aqueous solution of [F]fluoride (370-740 MBq) was added K2.2.2. (0.01 g, 25 mmol), potassium carbonate (12.5 mg), and acetonitrile (1 mL), and the mixture was dried at 110°C under a nitrogen stream. After drying, the mixture was cooled to room temperature, and a solution of 2-methylpropan-2-yl [(4-{6-[(ethoxymethyl)oxy]-1-benzofuran-2-yl}-2-nitrophenyl)(methyl)amino]methane (3 mg, 0.26 mmol) in DMSO (1.0 mL) was added. The mixture was heated at 85°C for 15 min, cooled to 70°C, and hydrochloric acid (6 M, 0.25 mL) was added. The reaction was continued for 30 min. The reaction solution was diluted with methanol / water (2 mL, V / V = 1 / 1) and loaded onto a semi-preparative HPLC column (Luna 5 μm C18, 250 x 10 mm) and eluted with methanol / water (50:50) at 4.0 mL / min. The product radioactive peak eluting after 18-20 min was collected and evaporated to dryness under a nitrogen purge. 0.5 mL of ethanol and 5.0 mL of 0.9% NaCl solution were added to prepare the product surrogate. The radiochemical yield was 30%-40%. Radiochemical purity (HPLC): 97%.

[0150] Example 2 Synthesis of Compound 2

[0151] Taking the substitution of the 6-hydroxyl group, X=S, Y=N, Z=H, n=4, and the chelating group as DOTA as an example, other compounds in the general formula can be synthesized by the same method by replacing the substrate.

[0152] Step (1) Synthesis of tert-butyl (5-((2-(4-(methylamino)phenyl)benzo[d]thiazol-6-yl)oxy)pentyl)carbamate

[0153] A mixture of 2-(4-(methylamino)phenyl)benzo[d]thiazol-6-ol (118 mg, 0.46 mmol), tert-butyl (5-bromopentyl)carbamate (245 mg, 0.92 mmol), and potassium carbonate (207 mg, 1.5 mmol) was dissolved in DMF (5 mL) and stirred at 90°C for 5 hours. Upon completion of the reaction, the reaction solution was extracted with ethyl acetate and water, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. Silica gel column chromatography afforded a light yellow solid (175 mg, 86% yield). LCMS (m / z): 442.2696 [M+H] + . 1H NMR(500MHz,DMSO)δ8.02–7.95(m,2H),7.91(d,J=8.9Hz,1H),7.81–7.75(m ,1H),7.68(d,J=2.5Hz,1H),7.50–7.43(m,3H),7.11(dd,J=9.0,2.5Hz,1H) ,6.83–6.72(m,1H),4.03(q,J=7.2Hz,2H),3.99(t,J=6.3Hz,1H),3.25(s,3 H),2.95(q,J=6.3Hz,2H),2.84(q,J=6.5Hz,1H),2.42(s,2H),1.44(s,9H).

[0154] Step (2) Synthesis of 4-(6-((5-aminopentyl)oxy)benzo[d]thiazol-2-yl)-N-methylaniline

[0155] Dissolve tert-butyl (4-(6-hydroxybenzo[d]thiazol-2-yl)phenyl)(methyl)carbamate (170 mg, 0.5 mmol) and trifluoroacetic acid (1 mL) in dichloromethane (1 mL). Stir the reaction mixture overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and the residue was purified by semi-preparative HPLC to afford a light yellow solid (119 mg, 70% yield). LCMS (m / z): 342.1655 [M+H] + .

[0156] Step (3) Synthesis of tri-tert-butyl 2,2',2"-(10-(2-((5-((2-(4-(methylamino)phenyl)benzo[d]thiazol-6-yl)oxy)pentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0157] 4-(6-((5-aminopentyl)oxy)benzo[d]thiazol-2-yl)-N-methylaniline (119 mg, 0.35 mmol), (10-{1-[(methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-4,7-bis{2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl-7,10-tetraazacyclododec-1-yl)acetic acid (239 mg, 0.42 mmol), and the HBTU reagent O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylisourea phosphorus hexafluoride (200 mg, 0.52 mmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (116 μL, 0.7 mmol) was added. The reaction solution was stirred at room temperature overnight. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The product was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain a light yellow solid (214 mg, yield 68%). LCMS (m / z): 896.5294 [M+H] + . 1 H NMR (500MHz, CDCl3) δ7.86–7.79(m,3H),7.29(d,J=2.5Hz,1H),6.98(dd,J=8.9,2.5H z,1H),6.67–6.61(m,2H),6.31(t,J=5.9Hz,1H),4.12(q,J=7.1Hz,4H),3.99(t,J=6. 5Hz,2H),3.69(s,2H),3.22(q,J=6.7Hz,3H),2.89(s,5H),2.80(s,2H),2.04(s,6H), 1.84–1.75(m,2H),1.63–1.53(m,3H),1.44(d,J=2.1Hz,27H),1.26(t,J=7.1Hz,8H).

[0158] Step (4) Synthesis of 2,2',2"-(10-(2-((5-((2-(4-(methylamino)phenyl)benzo[d]thiazol-6-yl)oxy)pentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (Compound 2')

[0159] Tri-tert-butyl 2,2',2"-(10-(2-((5-((2-(4-(methylamino)phenyl)benzo[d]thiazol-6-yl)oxy)pentyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (170 mg, 0.18 mmol) and trifluoroacetic acid (1 mL) were dissolved in dichloromethane (1 mL), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified by semi-preparative HPLC to give a light yellow solid (60 mg, 46% yield). LCMS (m / z): 728.3410 [M+H] + . 1 H NMR (500MHz, DMSO) δ8.52(s,1H),7.83–7.73(m,3H),7.59(d,J=2.6Hz,1H),7.04(dd,J=8.9,2.5Hz,1H),6.70–6.62(m,2H),4.60(s,4H),4.11(s,2 H),4.04(t,J=6.4Hz,2H),3.92(s,2H),3.56–3.35(m,8H),3.18–3.05(m, 8H), 2.76 (s, 3H), 1.77 (p, J = 6.7Hz, 2H), 1.56–1.43 (m, 4H), 1.24 (s, 1H).

[0160] Step (5) Synthesis of Compound 2

[0161] 5.1 Use a syringe to draw 6mL of 0.6M hydrochloric acid solution, remove all bubbles and inject it into the germanium gallium generator to prepare the eluent 68 GaCl3, collect 1 mL of eluate per tube, measure the activity of each tube, and select the two tubes with the highest activity for subsequent labeling. (2 mL, approximately 20 mCi)

[0162] 5.2 Take 5.1 to get 68 2 mL of GaCl3 was added, and the pH value was adjusted to about pH 4.0 with 3 M NaOAc solution. 30 μL of reaction precursor (compound 2, 1.0 mg / mL), 10 μg of vitamin C, and 2.0 mL of acetonitrile were added, and the mixture was heated at 95°C for 15 min.

[0163] 5.3 Use C18 column to purify the reaction solution in the previous step.

[0164] (1) Use a syringe to draw 10 mL of anhydrous ethanol to activate the C18 column

[0165] (2) Use a syringe to draw 10 mL of ultrapure water (or saline) to rinse the C18 column for later use

[0166] (3) Use a syringe to slowly push the labeled mixture in step 5.2 into the C18 column

[0167] (4) Use a syringe to draw 10 mL of ultrapure water (or saline) to rinse the C18 column three times

[0168] (5) Use a syringe to draw 0.2-0.3 mL of anhydrous ethanol to elute the C18 column to obtain an ethanol solution of the radiolabeled product.

[0169] 5.3 The ethanol solution obtained in the previous step was diluted with physiological saline to an ethanol concentration of <10%, and detected by radio-HPLC. The chromatographic conditions were as follows:

[0170] Chromatographic column: Luna 5μm, C18, 250*4.6mm

[0171] Mobile phase: A: MeCN containing 0.1% TFA; B: H2O containing 0.1% TFA: 20% A for 0-2 minutes; 20% A to 95% A for 2-8 minutes; 95% A for 8-10 minutes; 20% A for 10-12 minutes. The retention time of the labeled product was 7.9 minutes, and the radiolabeled purity was >95% (see Figures 1 and 2; the HPLC elution time of compound 2 was consistent with that of compound 2").

[0172] Example 3 Synthesis of Compound 3

[0173] Taking hydroxyl 3 as the substitution, X=S, Y=C, Z=H, n=3, and chelating group as NOTA as an example, other compounds in the general formula can be synthesized by replacing the substrate using the same method.

[0174] Step (1) Synthesis of 4-({2-[4-(methylamino)phenyl]benzo[b]thiophen-3-yl}oxy)butan-1-amine

[0175] Dissolve tert-butyl (4-((2-(4-(methylamino)phenyl)benzo[b]thiophen-3-yl)oxy)butyl)carbamate (600 mg, 1.4 mmol) in dichloromethane (2 mL) and trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the solvent was removed under reduced pressure to afford a light yellow oil (530 mg, 86% yield), which was used directly in the next reaction.

[0176] Step (2) Synthesis of [4,7-bis(2-{[4-({2-[4-(methylamino)phenyl]benzo[b]thiophen-3-yl}oxy)butyl]amino}-2-oxyylideneethyl)-1,4,7-triazacyclononan-1-yl]acetic acid (Compound 3')

[0177] 2,2',2"-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (60 mg, 0.2 mmol) was dissolved in DMSO (1 mL), and 4-({2-[4-(methylamino)phenyl]benzo[b]thiophen-3-yl}oxy)butan-1-amine (130 mg, 0.4 mmol), EDC (62 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol) and DIPEA (104 mg, 0.8 mmol) were added. The mixture was stirred at room temperature for 1 h, and the reaction process was monitored by HPLC-MS. The corresponding product was purified by preparative HPLC using a C18 reverse-phase column (10×250 mm, 5 μm) at a flow rate of 4.0 mL / min with a linear gradient of 20-80% solvent B (solvent A: aqueous solution containing 0.1% trifluoroacetic acid; solvent B: acetonitrile solution containing 0.1% trifluoroacetic acid) for 20 minutes, followed by a linear gradient of 80-100% solvent B for 5 minutes, followed by a constant flow of 100% solvent B for 5 minutes. The corresponding product peak was collected, the solvent was removed under reduced pressure, and lyophilized to obtain a white solid product (55 mg, yield: 15%). ESMS m / z: 920.41 (M+1) + ; 1 H NMR (500MHz, DMSO-d6) δ: 9.71 (s, 1H), 8.36 (s, 2H), 7.82 (s, 2H), 7.62 (d, J = 7.9Hz, 2H), 7.56 (s, 4H), 7.35 (s, 2H), 7.28 (t, J = 7.5Hz, 2H), 6.63 (s, 4H),6.08(s,2H),3.85(t,J=6.3Hz,4H),3.53(s,2H),3.22(s,4H),3.11( s,8H),2.83(s,4H),2.68(s,7H),2.60(s,4H),1.68(s,4H),1.60(s,4H).

[0178] Step (3) Synthesis of Compound 3

[0179] Pick 68 GaCl3 solution (1 mL) was adjusted to pH 4.0 with 3M NaOAc solution, and 30 μL of labeled precursor (1.0 mg / mL), 10 μg of vitamin C, and acetonitrile (1.0 mL) were added. The mixture was heated at 95°C for 15 minutes and purified by C18 column with ethanol to obtain 68 Ga labeled probe solution. Preparation method: Refer to step (4) of Example 3

[0180] Example 4 Synthesis of Compound 4

[0181] Taking DOTA modified with THRPPMWSPVWP as an example, where the 6-hydroxyl group is substituted, X=S, Y=N, Z=H, n=4, and the chelating group is THRPPMWSPVWP, other compounds in the general formula can be synthesized using the same method by replacing the substrate.

[0182] Step (1) Synthesis of DOTA-THRPPMWSPVWP

[0183] 2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetic acid (21 mg, 60 μM) and HBTU (O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (23 mg, 60 μM) were dissolved in dimethylformamide (1.0 mL), and diisopropylethylamine (7.8 mg, 60 μM) was added. The mixture was stirred at room temperature for 2 min. The reaction mixture was added to THRPPMWSPVWP (22 mg, 15 μM) and reacted for 3 h. After the reaction was completed by HPLC monitoring, TFA (1.0 mL) was added and stirred at room temperature for 8 h. After the reaction was completed, the reaction solution was purified by semi-preparative high performance liquid chromatography (HPLC) using a 0-100% MeOH + 0.1% TFA gradient within 30 minutes and isolated as a white solid after lyophilization (14 mg, yield: 48%). ESI-MS (m / z) for [M+1] + (calculated):1892.6(1892.19).

[0184] Step (2) Synthesis of DOTA biligand compound (Compound 4')

[0185] DOTA-THRPPMWSPVWP (14 mg, 7.2 μM) and HBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (4 mg, 10 μM) were dissolved in dimethylformamide (1.0 mL), and diisopropylethylamine (2 mg, 10 μM) was added. The mixture was stirred at room temperature for 2 min. The reaction mixture was added to 4-(6-((5-aminopentyl)oxy)benzo[d]thiazol-2-yl)-N-methylaniline (Synthesis of General Formula B, Step 2) (1.0 mg, 3 μM) and reacted for 8 h. The reaction was monitored by HPLC. The reaction solution was purified by semi-preparative high-performance liquid chromatography (HPLC) using a 0–100% MeOH + 0.1% TFA gradient over 30 minutes and isolated as a white solid after lyophilization (2 mg, yield: 30%). ESI–MS (m / z) for [M+1]+ (calculated):2216.6(2216.34).

[0186] Step (3) DOTA-biligand compound 68 Ga labeling (Synthesis of compound 4)

[0187] Pick 68 GaCl3 solution (1 mL) was adjusted to pH 4.0 with 3 M NaOAc solution, 30 μL of labeled precursor (1.0 mg / mL) and 10 μg of vitamin C were added, and the mixture was heated at 80 °C for 15 min. The mixture was purified by C18 column and eluted with ethanol to obtain 68 Ga labeled probe solution. (Preparation method refers to the synthesis step (4) of general formula B)

[0188] Example 5 In vitro Aβ binding test of compounds

[0189] 1) SPR determination of the in vitro Aβ binding ability of standard compounds

[0190] The interaction between the compounds and Aβ was analyzed using a Biacore 8K surface plasmon resonance instrument. The system temperature was set at 25°C, the flow rate was set at 10 μL / min, channel 2 (FC-2) was designated as the coupled protein channel, and channel 1 (FC-1) was designated as the control channel to detect nonspecific binding reactions and to subtract background signals.

[0191] Pretreatment-coupling pH selection: Without activating the chip surface, Aβ samples (Gill Biochemical, Cat#52487) were dissolved in sodium acetate buffer at different pH values ​​(pH = 4.0, 4.5, 5.0, 5.5) to impart different amounts of positive charge. The protein samples were then flowed over the chip surface at a flow rate of 10 μL / min. The appropriate pH conditions were determined based on the integrin binding curve between the chip and the sample.

[0192] Aβ coupling: CM5 chip was selected, and protein coupling was performed according to the ammonia coupling method. The coupling steps were as follows: (1) Activation: NHS / EDC mixture (0.1 M NHS and 0.4 MEDCC, mixed in a volume ratio of 1:1 before use and used immediately) was injected at a flow rate of 10 μL / min to activate the carboxyl groups of the dextran on the chip surface; (2) Coupling: 50 μg / mL Aβ solutions (Aβ42 monomers, Aβ42 oligomers, and Aβ42 fibers) were prepared in buffer solutions with the pH values ​​determined in the pretreatment. The preparation methods of Aβ42 oligomers and Aβ42 fibers were based on Alzheimer's Disease and Frontotemporal Dementia 2010, 670, 13-32. The samples were injected at a flow rate of 10 μL / min until the immobilization level reached about 3000 RU; (3) Blocking: Blocking with 1 M ethanolamine hydrochloride to complete the protein coupling process.

[0193] Binding experiment: The analyte to be tested was dissolved in DMSO and prepared to a concentration of 10 mM, then diluted to the required concentration series with PBS-P+ (Cytiva), injected at a flow rate of 30 μL / min for 5 minutes, and the binding (k on ) for 60s, dissociation (k off ) for 60 s. After solvent correction, the experimental results were analyzed by subtracting the reference channel sensorgram from the sample sensorgram. Analyte concentrations were 3.91, 7.81, 15.63, 31.25, 62.50, and 125.0 μM, respectively. Kinetic parameters were derived assuming a 1:1 binding ratio.

[0194] In an SPR experiment, when the analyte solution flows onto the chip surface, the analyte binds to the protein conjugate on the chip surface, causing a change in the chip surface mass and, consequently, a change in the resonance signal. This interaction is represented by a sensorgram, where the vertical axis represents the binding level of the small molecule to the protein, continuously recorded as the response signal (RU), and the horizontal axis represents time. The dissociation equilibrium constant (KD) obtained through fitting is shown in Table 1 for some experimental compounds.

[0195] Table 1. Results of the ability of compounds to bind to Aβ

[0196] The results showed that the exemplary compounds 1' and 2' provided in this application have good Aβ binding ability, and in the SPR experiment, they exhibited Aβ42 monomer, Aβ42 oligomer and Aβ42 fiber binding abilities comparable to those of the reference compounds Flutemetamol and AZD4694 (i.e., Flutafuranol).

[0197] 2) The compound competes with thioflavin T (ThT) for binding to Aβ aggregates

[0198] Aβ aggregates (including fibrils, plaques, and oligomers) prepared in step 1) were added to a 96-well plate. A 1 mM DMSO solution of the standard compound and an equal volume of DMSO without the standard compound were added under neutral conditions, resulting in an Aβ concentration of 5 μM and a standard compound concentration of 40 μM. Fluorescence values ​​(Ex / Em: 440 nm / 490 nm) were measured using a fluorescence spectrophotometer (BioTek, Cytation3) at 37°C. ThT (final concentration 40 μM) was then added to each well and fluorescence values ​​(Ex / Em: 440 nm / 490 nm) were measured. Three replicates were performed for each group, and statistical analysis was performed using GraphPad Prism.

[0199] In the thioflavin T (ThT) competition binding assay, example compounds 1' and 2' competed with thioflavin T (ThT) for binding to Aβ aggregates. The compounds themselves showed no significant fluorescence under the assay conditions. However, after the compounds were incubated with Aβ aggregates and then ThT was added, the fluorescence was significantly lower than that of the control group without the addition of the compound, indicating that compounds 2' and 1' can compete with ThT for binding to Aβ aggregates.

[0200] Example 6 Radioautographic Experiment of Ga-68 Labeled Aβ PET Imaging Agent (Compound 2) in Transgenic AD Mouse (APP / PS1) Model

[0201] APP / PS1 mice (purchased from The Jackson Laboratory, 16 months old, half male and half female) and normal mice of the same age were anesthetized with isoflurane and the whole brain was removed. After embedding in OCT embedding medium, frozen sections (-20°C, 10μm) were prepared. The surface of the brain slices was first covered with PBS (pH 7.4) and pre-incubated at room temperature for 10 minutes to eliminate endogenous binding. The compound 2 solution (about 60μCi / mL) prepared in step 5.2 of Example 2 was covered on the surface of the brain slices and incubated at room temperature for 60 minutes. Among them, the Block and Self-Block groups added the positive compound Flutemetamol (10μM) and unlabeled compound 2 (10μM) to the incubation solution, respectively. After incubation, the slices were washed twice with PBS (pH 7.4) for 2 minutes each; then washed twice with 40% ethanol aqueous solution for 2 minutes each; finally, washed with deionized water for 10 seconds; and air-dried at room temperature. Cover the slices with plastic wrap and expose them under a phosphor screen at room temperature for 12 hours. The phosphor screen was placed in a phosphor imaging system (Typhoon FLA 9500, GE Healthcare) in a dark environment and scanned at a resolution of 25 μm. Autoradiographic images were quantified using image analysis software (ImageQuant TL, GE Healthcare) to assess the specific binding ability of the probe in the mouse brain. The results are shown in Figures 3 and 4. The experimental results show that compound 2 can specifically bind to Aβ in the brains of APP / PS1 mice, and the binding signal can be blocked by the known Aβ binder flutemetamol as well as compound 2.

[0202] Example 7 PET imaging of compound 2 in transgenic AD mouse (APP / PS1) model

[0203] The day before the experiment, mice (control group, APP / PS1 model group) were fasted, but water was not allowed, and the fasting time did not exceed 18 hours (8 mice per group, a total of 16 mice). The mice were placed in an anesthesia box for isoflurane inhalation anesthesia, and were transferred to the instrument animal table after they were fully anesthetized. Under isoflurane anesthesia, the PET molecular probe solution (~500μCi, 0.1mL) prepared in step 5.2 of Example 2 was injected into the mouse from the tail vein, and PET / CT imaging was performed on it. The imaging time was about 0-60 minutes according to the experimental needs, and inhalation anesthesia was maintained during the imaging process. After imaging, the small animal was transferred out of the instrument and waited for it to wake up naturally. The experimental results showed that the uptake of compound 2 in the brain of the APP / PS1 AD mouse model was higher than that of the control group (Figures 5 and 6), which is consistent with the experimental results of in vitro autoradiography.

[0204] The success rate of developing anti-Aβ drugs in the existing technology is low. The inventors unexpectedly discovered that the morphology, size, and aggregation state of the target Aβ aggregates used will affect the efficiency of the screening method and whether the screened drugs are effective. The incubation conditions used for Aβ aggregates used in the existing technology are different and differ greatly from the in vivo environment. The inventors believe that this factor is one of the reasons for the low success rate of anti-Aβ drug development. Furthermore, the inventors simulated different microenvironments in the human brain to prepare different Aβ aggregates, including DPBS, HBS, aCSF, etc. that simulate the neutral extracellular environment and buffer solutions that simulate the endosome / lysosome internal environment, and simulated common human body temperature, the temperature of superficial parts of the brain, the temperature of deep areas of the brain, etc. at different temperatures such as 37°C, 38.5°C, and 41°C. Different speed conditions such as 0-2000rpm were used to simulate various different conditions such as human rest, movement, and blood flow. Under the above-mentioned different conditions, Aβ aggregates of each single sequence were incubated for binding specificity screening, and aggregates of other Aβ sequences were mixed and incubated with Aβ40 as the main body to simulate complex Aβ aggregates in vivo for broad screening.

[0205] Preparation method of Aβ aggregates

[0206] The present invention relates to a method for preparing Aβ aggregates, comprising the steps of: S1, dissolving Aβ freeze-dried powder in hexafluoroisopropanol, and drying and removing the hexafluoroisopropanol to obtain an Aβ peptide film; S2, mixing the Aβ peptide film and a buffer to obtain an Aβ solution with a concentration of 0.2-100 μM; and S3, incubating the Aβ solution to obtain Aβ aggregates.

[0207] As Aβ lyophilized powder, one or more Aβ subtypes can be selected, such as Aβ40, Aβ42, Aβ38, N3pE42, or a combination thereof. The sequences of these Aβ subtypes are all known and commercially available. For example, it can be an Aβ40 multimer, an Aβ42 multimer, or a multimer formed by any two or all three of Aβ40, Aβ42, Aβ38, and N3pE42. When multiple Aβ subtypes are mixed and incubated, preferably, at least one subtype is Aβ40, and more preferably, Aβ40 is used as the main component. In some embodiments of the present invention, a mixture of Aβ40 and other Aβ is used for co-incubation to simulate different physiological and pathological states in the brain, for example, a mixture of Aβ40 and Aβ42, or a mixture of Aβ40 and N3pE42, or a mixture of Aβ40 and Aβ38, or a mixture of Aβ40, Aβ42, and N3pE42. When multiple Aβ isoforms are mixed and incubated, the molar percentage of Aβ40 is preferably not less than 50%. In some embodiments of the present invention, the molar ratio of Aβ40 to other Aβ is (5-9):(1-5), for example (molar ratios in parentheses): Aβ40 / 42 (9:1), Aβ40 / 42 (8:2), Aβ40 / 42 (7:3), Aβ40 / 42 (6:4), Aβ40 / 42 (5:5), Aβ40 / N3pE42 (9:1), Aβ40 / 42 / N3pE42 (5:4:1), Aβ40 / 42 / N3pE42 (5:4:1), etc.

[0208] As a buffer, it is optionally a buffer that simulates different microenvironments in the human brain, preferably at least one of Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, HBS buffer containing 8-12mM HEPES and 140-160mM NaCl, artificial cerebrospinal fluid (aCSF), DMEM, FBS, endosome simulation fluid and lysosome simulation fluid. In some embodiments of the invention, Dulbecco's phosphate buffered saline (DPBS) without calcium and magnesium elements, artificial cerebrospinal fluid (aCSF) or HBS buffer containing 8-12mM HEPES and 140-160mM NaCl is used as a buffer.

[0209] The incubation temperature is 36-41°C, preferably 37°C, 38.5°C, or 41°C, to simulate normal human body temperature, superficial brain temperature, and deep brain temperature, respectively. The incubation speed is 0-2500 rpm, for example, 0 or 2000 rpm to simulate resting and moving states, respectively.

[0210] In the present invention, the term "Aβ aggregate" refers to a polymer formed by the aggregation of one or more β-amyloid protein monomers.

[0211] Uses of Aβ Aggregates

[0212] The present invention also relates to the use of Aβ aggregates prepared by the above-mentioned method as targets for screening anti-Aβ drugs. Due to the polymorphism of Aβ aggregates, Aβ aggregates prepared by different methods are essentially different, similar to different targets. Compared with Aβ aggregates obtained in classical literature, Aβ aggregates prepared by the present method can be more intuitive and rapid in screening for effective candidate drugs. AFM images show that the screened drugs have a disaggregating effect on Aβ aggregates, a property of candidate drugs that cannot be directly determined by other methods.

[0213] Anti-Aβ drug screening targets

[0214] The present invention also relates to a target for anti-Aβ drug screening, which is the Aβ aggregates prepared by the above-mentioned method. Representative AFM images show that the Aβ aggregates obtained by the present invention are significantly different from typical Aβ monomers, typical Aβ oligomers, typical Aβ fibers, and typical Aβ plaques, representing distinct targets. The terms "typical Aβ monomers," "typical Aβ oligomers," "typical Aβ fibers," and "typical Aβ plaques" refer to typical Aβ monomers, typical Aβ oligomers, typical Aβ fibers, and typical Aβ plaques prepared by the methods described in the comparative examples of the present invention (methods in classic literature).

[0215] Anti-Aβ drug screening method

[0216] The present invention also relates to a method for screening anti-Aβ drugs, comprising the steps of screening for anti-Aβ drugs using the aforementioned anti-Aβ drug screening target. Preferably, the method screens for drugs that can disaggregate Aβ aggregates, bind to Aβ aggregates, or inhibit the aggregation of Aβ aggregates. The types of the aforementioned drugs are not limited and may include antibodies, peptides, and small molecule compounds.

[0217] Anti-Aβ drugs

[0218] The present invention also relates to an anti-Aβ drug screened by the method of the present invention, as shown in the following formula I:

[0219] The present invention also determined through SPR experiments and ThT competitive binding experiments that the compound of formula I can bind to Aβ, and the binding effect is comparable to that of Flutemetamol and AZD4694 (ie, Flutafuranol).

[0220] The present invention also relates to the in vitro non-therapeutic use of the compound of formula I for (i) inhibiting the formation of Aβ polymers; (ii) promoting the disaggregation of Aβ polymers; and / or (iii) binding to Aβ polymers.

[0221] In a preferred embodiment of the present invention, the "anti-Aβ drug" refers to a drug that can disaggregate Aβ polymers or prevent the formation of Aβ polymers.

[0222] Example 8

[0223] In this example, Aβ polymers were prepared. The specific method is as follows:

[0224] (1) Preparation and storage of Aβ peptide film: HFIP (hexafluoroisopropanol) was added to the artificially synthesized Aβ freeze-dried powder Aβ40, Aβ42, and Aβ38 (purchased from Gill Biochemical) to prepare a 1 mM solution, and the solution was incubated at room temperature for 30 minutes. 10 μl of the solution was dispensed into 0.5 ml low-adsorption microcentrifuge tubes, and the tubes were placed in a fume hood overnight to allow HFIP to evaporate. The tubes were then transferred to a vacuum centrifuge (SpeedVac) and dried for 1 hour to remove residual HFIP and moisture to obtain the Aβ peptide film. The tubes were sealed with a desiccant and stored at -20°C for later use.

[0225] (2) Preparation of Aβ DMSO stock solution (i.e., Aβ stock solution)

[0226] The Aβ peptide film was taken out and equilibrated to room temperature, and anhydrous DMSO (dimethyl sulfoxide) was added to prepare a 5 mM stock solution. After vortexing for 30 seconds, it was sonicated for 10 minutes. The stock solution was used as the starting material for preparing various Aβ aggregates.

[0227] (3) Preparation of single-sequence Aβ aggregates under conditions close to those in vivo:

[0228] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 24 hours, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 8 shows the AFM morphology of Aβ40 and Aβ42.

[0229] Example 9

[0230] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0231] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 24 hours, the aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 9 shows the AFM morphology of Aβ40 and Aβ42.

[0232] Example 10

[0233] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0234] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 2 μM with HBS buffer and incubated at 37°C and 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 10 shows the AFM morphology of Aβ40 and Aβ42.

[0235] Example 11

[0236] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0237] The 5 mM Aβ DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and transferred to 37°C and incubated at 2000 rpm. After incubation for 1 week, the aggregate morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 11 shows the AFM morphology of Aβ40 and Aβ42.

[0238] Example 12

[0239] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0240] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 50 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 12 shows the AFM morphology of Aβ40 and Aβ42.

[0241] Example 13

[0242] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0243] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 10 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 13 shows the AFM morphology of Aβ40 and Aβ42.

[0244] Example 14

[0245] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0246] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 2 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 14 shows the AFM morphology of Aβ40 and Aβ42.

[0247] Example 15

[0248] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0249] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 50 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 1 week before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. Figure 15 shows the AFM morphology of Aβ40 and Aβ42.

[0250] Example 16

[0251] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0252] The 5 mM Aβ DMSO stock solution prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 10 μM with aCSF buffer. The solution was transferred to 37°C and incubated at 2000 rpm for 1 week before use. The aggregation morphology was determined by AFM, and Aβ aggregates with corresponding aggregation degrees and morphologies were obtained. Figure 16 shows the AFM morphology of Aβ40 and Aβ42.

[0253] Example 17

[0254] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0255] Using Aβ40 as a background, Aβ42 was mixed with Aβ40 at a specific ratio and added to aCSF to create a 5µM:5µM solution. The solution was then transferred to 37°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, yielding Aβ aggregates of corresponding aggregation degree and morphology. Figure 17 shows the AFM morphology of Aβ40 and Aβ42.

[0256] Example 18

[0257] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0258] Using Aβ40 as a background, Aβ42 was mixed with Aβ40 at a specific ratio and added to aCSF to create a 5µM:5µM solution. The solution was then transferred to 37°C and incubated at 2000 rpm for one week before use. AFM was used to determine the aggregation morphology and obtain Aβ aggregates of the corresponding aggregation degree and morphology. Figure 18 shows the AFM morphology of Aβ40 and Aβ42.

[0259] Example 19

[0260] In this example, Aβ multimers were prepared by referring to the method for forming typical Aβ plaques in classical literature.

[0261] The 5 mM Aβ DMSO stock solution prepared in (2) was added to a mixture of 10 mM HCl + 150 mM NaCl to prepare a 100 μM Aβ solution, which was then diluted to 5 μM with a mixture of 10 mM HCl + 150 mM NaCl. The solution was transferred to 37°C and incubated at 0 rpm for 48 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. The left image of Figure 21 shows the AFM morphology of Aβ42.

[0262] Example 20

[0263] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0264] The 5 mM Aβ DMSO stock solution prepared in (2) was added to DPBS to prepare a 100 μM Aβ solution, which was then diluted to 25 μM with DPBS buffer and incubated at 37°C, 0 rpm. After 78 hours of incubation, the solution was used to determine its aggregation morphology using AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. The left image of Figure 22 shows the AFM morphology of Aβ42.

[0265] Example 21

[0266] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0267] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was taken and added to aCSF to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with aCSF buffer and incubated at 37°C and 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 24 shows the AFM morphology of Aβ38.

[0268] Example 22

[0269] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0270] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was added to aCSF to prepare a 100 μM Aβ solution, which was then diluted to 2 μM with aCSF buffer. The solution was transferred to 41°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. Figure 25 shows the AFM morphology of Aβ38.

[0271] Example 23

[0272] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0273] The 5 mM DMSO stock solution of Aβ38 prepared in (2) was added to a 100 μM Aβ solution prepared by lysosomes, and then diluted to 2 μM with lysosome buffer. The cells were transferred to 41°C and incubated at 2000 rpm for 24 hours before use. The aggregation morphology was determined by AFM, and Aβ aggregates with the corresponding aggregation degree and morphology were obtained. Figure 26 shows the AFM morphology of Aβ38.

[0274] Example 24

[0275] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0276] The 5 mM DMSO stock solution of Aβ42 prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 10 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 24 hours, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 27 shows the AFM morphology of Aβ42.

[0277] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 9 right), the degree of polymerization of the samples incubated at 0 rpm was slightly lower.

[0278] Example 25

[0279] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0280] The 5 mM Aβ40 DMSO stock solution prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 24 hours, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 28 shows the AFM morphology of Aβ40.

[0281] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 8 left), the degree of aggregation of the samples incubated at 0 rpm was significantly lower.

[0282] Example 26

[0283] The method for preparing Aβ multimers in this example is substantially the same as that in Example 8, except that the incubation conditions are partially adjusted.

[0284] The 5 mM DMSO stock solution of Aβ40 prepared in (2) was taken and HBS was added to prepare a 100 μM Aβ solution. The solution was then diluted to 50 μM with HBS buffer and incubated at 37°C and 0 rpm. After incubation for 1 week, the aggregation morphology was determined using AFM to obtain Aβ aggregates with the corresponding aggregation degree and morphology. Figure 28 shows the AFM morphology of Aβ40.

[0285] Compared with the samples incubated at 2000 rpm under the same conditions (Figure 11 left), the degree of polymerization of the samples incubated at 0 rpm was significantly lower.

[0286] The following comparative examples provide methods for preparing Aβ in classical literature.

[0287] Comparative Example 1

[0288] Preparation of typical Aβ monomers

[0289] The Aβ stock solution prepared in Example 8 was added with ice water to a final concentration of 100 μM Aβ, and vortexed for 15 seconds before being used immediately as an Aβ monomer.

[0290] Comparative Example 2

[0291] Preparation of typical Aβ oligomers

[0292] Cold F12 medium was added to the Aβ stock solution to dilute Aβ to 100 μM, vortexed for 15 seconds, and transferred to 4°C for incubation for 24 hours to obtain typical Aβ oligomers, as shown in the left figure of Figure 7.

[0293] Comparative Example 3

[0294] Preparation of typical Aβ fibers

[0295] 10 mM HCl was added to the Aβ stock solution to dilute Aβ to 100 uM, vortexed for 15 seconds, and transferred to 37°C for incubation for 24 hours to obtain typical Aβ fibers. Typical Aβ fibers formed slender and curved fibrous aggregates.

[0296] Comparative Example 4

[0297] Preparation of typical Aβ plaques

[0298] 10 mM HCl + 150 mM NaCl was added to the Aβ stock solution to adjust the Aβ concentration to 100 uM, vortexed for 15 seconds, and transferred to 37°C for incubation for 24 hours to obtain Aβ plaques, as shown in the right figure of Figure 7 .

[0299] As shown in Figures 7, 8-18, 21, 22, and 24-29 above, the morphology of the Aβ aggregates prepared by incubation under the conditions of the extracellular matrix environment (physiological salt concentration, neutral) in the human brain in Examples 8-21 is different from the typical Aβ plaques in the comparative examples. As shown in Figures 8, 9, 11, 12, and 15, the aggregation morphology of Aβ40 tends to form fibrous aggregates, while Aβ42 tends to form small spherical aggregates and irregular clumps formed by the accumulation thereof. The morphology of the Aβ40 and Aβ42 aggregates of the present invention is significantly different from that of their respective comparative examples in the classic literature. The two also have different aggregation rates. References to Aβ40 at 100 μM only occasionally produce short fibers, while at physiological salt concentrations, 50 μM and even 10 μM Aβ40 can form fibrillar aggregates within 24 hours (Figures 8, 9, and 12). The morphology of these fibers is also different from that of Aβ40 and Aβ42 in the literature (The Journal of Biological Chemistry, 2002, 277(35), 32046-32053). Therefore, drug screening using these aggregates separately will lead to different screening results, similar to drug screening for different targets. The screening of Aβ aggregates prepared in a microenvironment close to the in vivo microenvironment used in the present invention has higher research value and application prospects.

[0300] Test Example 1

[0301] The formation of Aβ aggregates was detected by ThT binding assay.

[0302] ThT can bind to Aβ aggregates and thus exhibit fluorescence enhancement. The following experiments were performed to monitor the degree of Aβ aggregation and the binding of ThT to Aβ aggregates.

[0303] a) Monitoring the kinetics of Aβ aggregation: The starting Aβ solution (5-100 μM) prepared according to the method of Example 21 was immediately added to a 96-well plate, and a certain amount of ThT (5-100 μM) was added. The fluorescence value was regularly measured using a fluorescence spectrophotometer (BioTek, Cytation3) to monitor the aggregation process (Ex / Em: 440 nm / 490 nm). Three replicates were set up for each group, and statistical analysis was performed using GraphPad Prism. See Figure 19.

[0304] b) Fluorescence Changes in Aβ Aggregates after Addition of ThT: Aβ aggregates prepared in Example 21 were added to a 96-well plate at an Aβ concentration of 5-100 μM. Fluorescence values ​​were measured using a fluorescence spectrophotometer (BioTek, Cytation3). ThT (final concentration 5-100 μM) was added and incubated for 10-30 minutes. Fluorescence values ​​were again measured (Ex / Em: 440 nm / 490 nm). Three replicates were performed for each group, and statistical analysis was performed using GraphPad Prism. As shown in Figure 21 below, fluorescence values ​​significantly increased after the addition of ThT to Aβ aggregates.

[0305] Test Example 2

[0306] The morphology of each prepared Aβ aggregate was monitored by atomic force microscopy (AFM).

[0307] a) Fill a syringe with ultrapure water and filter through a 0.02 mm filter. Discard the first 1–2 mL and use 0.02 mm filtered water for all subsequent steps.

[0308] b) Each Aβ sample was diluted to 10 uM or samples with a final concentration lower than 10 uM were directly used for preparing test samples on mica.

[0309] c) Use tape to remove the top one to four layers of mica to expose a clean, flat mica surface.

[0310] d) Drop the sample onto mica and let it sit for 30 seconds. Rinse with 200-400 μl of water and blow dry with compressed air.

[0311] e) Place in a clean cabinet at room temperature (cover can be used to prevent dust) until analysis.

[0312] f) Scanning was performed using the intelligent tapping mode (peakforce) of a Bruker FastScan atomic force microscope and an SNL-A probe, with at least four areas scanned to ensure representative sampling.

[0313] g) NanoScope software was used for data processing and image presentation.

[0314] Test Example 3

[0315] Typical Aβ plaques and the Aβ aggregates prepared in the present invention were used to screen active compound 1'.

[0316] Using the typical Aβ plaque preparation method of Comparative Example 4, and under typical Aβ plaque formation conditions (10 mM HCl + 150 mM NaCl, 37°C), even when the Aβ concentration was reduced from 100 uM to 5 uM, incubation with Compound 1' only slightly inhibited Aβ aggregation, as shown in Figure 21. This result indicates that Aβ aggregates prepared under solution conditions that form typical Aβ plaques are not effective targets for screening Compound 1'.

[0317] However, when incubated under conditions closer to the in vivo environment provided in Example 20 of the present invention (DPBS, 37°C, 0rpm), even at a higher concentration (25uM), the same concentration of compound 1' can significantly inhibit the aggregation of Aβ. As shown in the right figure in Figure 22, co-incubation of compound 1' with Aβ can significantly inhibit the formation of Aβ aggregates, indicating that the Aβ aggregates of the present invention can be used as a target for screening to obtain active compound 1, and the results are more intuitive.

[0318] In this test example, the co-incubation of compound 1' and Aβ means that based on the method of comparative example 4 or example 20, compound 1' is added to the Aβ stock solution and incubated together with the Aβ stock solution to make the concentration of compound 1' reach the concentration required for the experiment.

[0319] In addition, the inventors also found that after incubating the Aβ aggregates prepared in the examples with the compounds for a certain period of time and comparing the aggregation before and after the addition of the compounds, AFM observation showed that compound 1 can also significantly promote the disaggregation of Aβ aggregates. Representative implementation results are shown in Figure 23.

[0320] Example 27 Molecular Biological Evaluation of the Therapeutic Effect of Compound 1' on AD Model Mice

[0321] To evaluate the therapeutic efficacy of compound 1' in AD model mice, we conducted systematic molecular biology experiments. Specifically, we used Aβ immunofluorescence staining and microglial immunostaining to assess pathological changes in the brains of AD model mice after four months of treatment. This assessment included detailed analysis of changes in the number of Aβ plaques per unit area in the brain and the area of ​​activated microglia.

[0322] Aβ immunofluorescence staining: Brain sections were obtained from mice anesthetized with Avertin and perfused with PBS. The brains were removed and fixed overnight in 4% PFA. The sections were then dehydrated in 15% and 30% sucrose and embedded. 30-μm thick sections were obtained using a freezing microtome and incubated in blocking buffer (10% bovine serum albumin and 0.3% Triton X-100 in PBS) for 30 minutes at 37°C. The samples were then incubated with the primary antibody (6E10) against Aβ at a dilution of 1:500 in PBS containing 3% bovine serum albumin and 0.3% Triton X-100 at 4°C overnight. After removal from 4°C and allowing to warm for at least 10 minutes, the primary antibody was aspirated and the sections were washed three times with PBS for 10 minutes each. The sections were then incubated with the secondary antibody (mouse-647) at a dilution of 1:1000 in PBS) at 37°C for 1 hour in the dark. Images were acquired by fluorescence microscopy to quantitatively assess the number of Aβ plaques per unit area in the mouse brain. The experimental results showed that after 4 months of treatment with compound 1' (intraperitoneal injection, once a day, 10 mg / kg, solvent is 0.5% CMCNa aqueous solution), the area of ​​Aβ plaques in the brains of Alzheimer's disease (AD) model mice was significantly reduced (see Figure 30 and Table 2-3). In contrast, the number of Aβ plaques in the mice in the positive control group did not change significantly after 7 months of treatment with Aricept (dose of 2 mg / kg, oral, once a day) (see Figure 30 and the table below). This result highlights that compound 1' can effectively reduce the formation of Aβ plaques in a shorter period of time, thereby showing its potential advantages in the treatment of AD.

[0323] Table 2: Number of Aβ plaques per unit area in the brain of 6-month-old APP / PS1 mice 1 to 4 months after administration of the compound

[0324] Table 3: Number of Aβ plaques per unit area in the brain of 6-month-old APP / PS1 mice after 7 months of Aricept administration

[0325] For microglial immunostaining, mice were anesthetized with avertin and perfused with PBS. Brain tissue was removed and fixed overnight in 4% PFA. The tissue was then dehydrated in 15% and 30% sucrose and embedded. 30-μm sections were obtained using a freezing microtome and incubated in blocking buffer (10% bovine serum albumin and 0.3% Triton X-100 in PBS) for 30 minutes at 37°C. The samples were then incubated overnight at 4°C with the primary antibody (Iba1) targeting activated microglia at a dilution of 1:500 in PBS containing 3% bovine serum albumin and 0.3% Triton X-100. After removal from 4°C and allowing the sample to warm for at least 10 minutes, the primary antibody was aspirated and the sample was washed three times with PBS for 10 minutes. The secondary antibody, rabbit-488, was then added at a dilution of 1:1000 in PBS and incubated for 1 hour at 37°C in the dark.

[0326] Fluorescence microscopy observation and analysis were performed to evaluate the number and distribution of activated microglia. The results showed that in Alzheimer's disease (AD) mice treated with compound 1' for 4 months (intraperitoneal injection, once a day, 10 mg / kg, solvent is 0.5% CMCNa aqueous solution), the area ratio of activated microglia in the brain was significantly reduced (as shown in Figure 31 and Table 4). Similarly, in the group treated with the positive drug Aricept (dose of 2 mg / kg, oral, once a day), a significant decrease in the area ratio of activated microglia in the brain was also observed (as shown in Figure 31 and Table 5). This result shows that both compound 1' and Aricept show significant effects in reducing the degree of activation of microglia in the brains of AD mice.

[0327] Table 4: Percentage of activated microglia in the brain of 6-month-old APP / PS1 mice after administration of compounds for 1 to 4 months

[0328] Table 5: Percentage of activated microglia in the brains of 6-month-old APP / PS1 mice after 7 months of Aricept administration

[0329] Example 28 Behavioral evaluation of the therapeutic effect of compound 1' in AD model mice

[0330] The present invention focuses on evaluating the effect of compound 1' on improving cognitive function in Alzheimer's disease (AD) model mice (specifically 6-month-old APP / PS1 mice). We administered the compound systemically for up to 7 months (once a day, intraperitoneal injection, a dose of 10 mg / kg, the solvent was 0.5% CMCNa aqueous solution). At the end of the treatment period, the performance of the compound 1'-treated group and the control group (given the same volume of 0.5% CMCNa aqueous solution) in the water maze test was compared, and the speed and path length of the mice reaching the platform were recorded in detail to analyze the intervention effect of the compound on spatial learning and memory ability. The results showed that after 7 months of treatment, the AD model mice had significant improvements in learning and memory function (as shown in Figure 32 and Tables 6-7). It is worth noting that in the control group treated with Aricept (at a dose of 2 mg / kg, orally, once a day, the solvent was water), the long-term memory ability was reduced compared with the solvent control group. This result highlights the potential superiority of compound 1' in improving AD-related cognitive impairment.

[0331] Table 6: Results of water maze test for 6-month-old APP / PS1 mice administered with compounds for 1 to 7 months

[0332] Table 7: Water maze test results of 6-month-old APP / PS1 mice administered Aricept for 7 months

[0333] Example 29 PET imaging evaluation of the therapeutic effect of compound 1' on AD model mice

[0334] PET imaging technology has shown unique advantages in detecting the therapeutic effect of compound 1' on AD models. As a highly sensitive molecular imaging tool, PET can detect pathological changes in vivo in real time and non-invasively, providing accurate quantitative data for in vivo studies. Using specific radioactive probes such as [ 18 F]AV45 and [ 18 F]DPA714, PET imaging quantified the level of neuroinflammation caused by Aβ plaque deposition and microglial TSPO expression, which are related to the core pathological characteristics of AD. Compared with traditional tissue section or in vitro analysis methods, PET imaging can dynamically evaluate the changes before and after treatment in the same animal, reduce the use of animals, and improve the reliability of experimental results. Therefore, this study combined the use of Aβ probe [ 18 F]AV45 and TSPO probes[ 18 F]DPA714, which evaluates changes in pathological characteristics and inflammatory responses, enabling dynamic tracking of pathological states in living animals.

[0335] Animal model: APP / PS1 double transgenic mice (AD model) and age- and sex-matched C57BL / 6J normal control mice were selected. APP / PS1 mice were randomly divided into a treatment group (treated with compound 1') and a model group (administered with normal saline). Each group of mice was injected with approximately 10 MBq of [ 18 F]AV45 or [ 18 F]DPA714 for PET imaging.

[0336] PET imaging method: injection via tail vein [ 18 F]AV45 (for detection of Aβ deposition) or [ 18 F]DPA714 (for detecting TSPO expression in microglia) was injected, and static PET scans were performed for 15 minutes 45 minutes after injection. Mice were anesthetized with 1-2% isoflurane during the scan.

[0337] Data processing: PET data were reconstructed using 3D OSEM and corrected for attenuation, scattering, normalization, and detector dead time. Pmod software was used to align the PET images with the mouse brain template to accurately locate and segment brain regions. Reference brain regions and data analysis: 18 F]AV45 probe selected cerebellum as the reference brain region, [ 18 The hypothalamus was selected as the reference brain region for the F]DPA714 probe. The SUVR values ​​of different brain regions were calculated to analyze the effects of compound 1' on Aβ deposition and neuroinflammation.

[0338] Evaluation of treatment effect:

[0339] Changes in Aβ deposition: The treatment group mice had 18 The [F]AV45 uptake ratio was significantly lower than that of the model group, indicating that compound 1' effectively reduced Aβ plaque deposition (as shown in Figure 33 and Table 8).

[0340] Table 8

[0341] Neuroinflammation level: The treatment group mice had 18 F] DPA714 uptake was also significantly lower than that in the model group, indicating that compound 1' can reduce microglial activation and alleviate neuroinflammation (as shown in Figure 34 and Table 9).

[0342] Table 9

[0343] Example 30: ([ 18 F] Compound 1 and control drug [ 18 F]Flutemetamol PET imaging comparison

[0344] APP / PS1 mice and normal mice (same age and sex) were injected intravenously with approximately 10 MBq of [ 18 F] Compound 1 and control drug [ 18 F]Flutemetamol. Static PET scans were performed 45 minutes after injection and the scan duration was 15 minutes. During the scan, mice were anesthetized with 1–2% isoflurane. PET data were reconstructed using the 3D OSEM method and corrected for attenuation, scattering, normalization, and scanner dead time. The corrected PET images were co-registered with the CT images. The reconstructed data were aligned to a standardized mouse brain template using Pmod 4.3 software to accurately locate and segment brain regions. The cerebellum was selected as a reference region for comparison [ 18 F] Compound 1 and control drug [ 18 The standard uptake ratio (SUVR) of F]Flutemetamol in the brain of APP / PS1 mice and normal mice is shown in Figure 35 and Table 10. 18 F] Compound 1 had significantly higher SUVR in cortex and hippocampus than the control drug [ 18 F]Flutemetamol, indicating [ 18 F] Compound 1 has advantages over [ 18 Imaging effect of F]Flutemetamol.

[0345] Table 10

Claims

1. A compound of formula D, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof: Among them, X2 is O, S or Se; Z2 is F-18 or I-124.

2. The compound as claimed in claim 1, its pharmaceutically acceptable salt, its solvate, or the solvate of its pharmaceutically acceptable salt; characterized in that, It satisfies one or more of the following conditions: (1) The X2 is O or S; (2) The Z2 is F-18; (3) The compound represented by Formula D is Preferably, the compound represented by Formula D is 3. A compound of formula E, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof: Among them, X2 is defined as described in claim 1 or 2; Z3 is F or I, such as F; Preferably, the compound represented by Formula E is 4. A compound of formula A, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof: Among them, R consists of a chelating group and a radionuclide; The O atom is connected to a connectable site on the benzene ring or five-membered ring; The chelating group is unmodified or modified with a cell-penetrating peptide; The radionuclide is a diagnostic radionuclide; m is 1 or 2; Each X is independently O, S or Se; Each Y is independently CH or N; Each Z is independently H, F, F-18, Cl, Br or I; Each n is independently 1, 2, 3 or 4.

5. The compound, pharmaceutically acceptable salt, solvate thereof, or solvate of the pharmaceutically acceptable salt according to claim 4, characterized in that, It satisfies one or more of the following conditions: (1) The X is O or S; (2) The Z is F or H; (3) The n is 3 or 4; (4) The cell-penetrating peptide is HAIYPRH, THRPPMWSPVWP, TFFYGGSRGKRNNFKTEEY, AGILKRW, CGNKRTR, LRKLRKRLLR or YGRKKRRQRRR; (5) The chelating group is (6) The radionuclide is selected from Cu-64 and Ga-68, such as Ga-68.

6. The compound according to claim 4, its pharmaceutically acceptable salt, its solvate, or the solvate of its pharmaceutically acceptable salt, characterized in that, The compound of formula A is as shown in formula A-1: wherein, the definitions of R, X, Y, Z, and n are as described in any one of claims 4-5; 7. The compound according to claim 4, its pharmaceutically acceptable salt, its solvate, or the solvate of its pharmaceutically acceptable salt, characterized in that, The compound represented by Formula A is selected from any one of the following compounds:

8. A compound of formula B, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof: Among them, X, Y, Z, m, n are defined as described in any one of claims 4-7, Q is a chelating group, and the chelating group is as described in any one of claims 4-7.

9. The compound according to claim 8, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of a pharmaceutically acceptable salt thereof; characterized in that, The compound represented by Formula B is any of the following compounds:

10. A compound of formula C, its pharmaceutically acceptable salts, its solvates, or solvates of its pharmaceutically acceptable salts: Among them, X, Y, Z, m, n are defined as described in any one of claims 4-7; T consists of a chelating group and a non-radionuclide, and the chelating group is as described in any one of claims 4-7.

11. The compound according to claim 10, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt; characterized in that, The non-radionuclide is Cu or Ga, such as Ga; Preferably, the compound shown by Formula C is 12. A pharmaceutical composition, characterized in that, It comprises a compound as described in any one of claims 1-11, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt, and pharmaceutical excipients.

13. A kit, characterized in that, It comprises substance V and an instruction manual; the substance V is a compound as described in any one of claims 1-11, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt or a pharmaceutical composition as described in claim 12.

14. Use of substance W in the preparation of a PET molecular probe, characterized in that, Substance W is a compound of formula A as described in any one of claims 4-7, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt or a compound of formula D as described in claim 1 or 2, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt; Preferably, the PET molecular probe is used for the diagnosis of Alzheimer's disease; more preferably, the PET molecular probe is used for imaging the binding of Aβ protein.

15. Use of substance V in the preparation of a medicament for preventing and / or treating Alzheimer's disease, characterized in that, The substance V is a compound as described in any one of claims 1-11, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt, or a pharmaceutical composition as described in claim 12; preferably, the substance V is a compound as shown in formula D as described in claim 1 or 2, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt, or a pharmaceutical composition comprising a compound as shown in formula D as described in claim 1 or 2, its pharmaceutically acceptable salt, its solvate, or a solvate of its pharmaceutically acceptable salt, and pharmaceutical excipients.

16. Use of substance V in the preparation of an anti-Aβ drug, wherein the definition of substance V is as described in claim 15; preferably, the anti-Aβ drug exerts its effect by depolymerizing Aβ oligomers or preventing the formation of Aβ oligomers.

17. Substance V plays one or more of the following roles: (i) inhibiting the formation of Aβ oligomers; (ii) promoting the depolymerization of Aβ oligomers; (iii) binding to Aβ oligomers; wherein the definition of substance V is as described in claim 15.

18. A method for preparing Aβ aggregates, characterized in that, The method for preparing the Aβ aggregate comprises the steps of: S1, dissolving the Aβ lyophilized powder and drying to remove the solvent to obtain an Aβ peptide film; S2, mixing the Aβ peptide film and a buffer solution to obtain an Aβ solution with a concentration of 0.2-100 μM; S3, incubating the Aβ solution to obtain an Aβ aggregate; and In step S1, the Aβ lyophilized powder is Aβ40, Aβ42, N3pE42, Aβ38, or a combination thereof; In step S2, the buffer solution is selected from at least one of Dulbecco's phosphate buffered saline solution without calcium and magnesium elements, HBS buffer solution containing 8-12 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 140-160 mM NaCl, artificial cerebrospinal fluid, DMEM, FBS, endosome-mimicking solution, and lysosome-mimicking solution; In step S3, the temperature of the incubation is 30-43 °C, the time of the incubation is not less than 1 day, and the rotation speed of the incubation is 0-2500 rpm.

19. The method according to claim 18, wherein In step S3, the time of the incubation is 1-7 days.

20. The method according to claim 18, wherein In step S3, the incubation is carried out in a thermostatic shaker, and the rotation speed of the thermostatic shaker is 0-2500 rpm.

21. The method according to claim 18, characterized in that The buffer solution is Dulbecco's phosphate buffered saline solution (DPBS) without calcium and magnesium elements, HBS buffer solution containing 8-12 mM HEPES and 140-160 mM NaCl, or artificial cerebrospinal fluid (aCSF).

22. A screening target for anti-Aβ drugs, characterized in that, The target is an Aβ aggregate, and the Aβ aggregate is prepared by the method described in any one of claims 18-21. Use of Aβ aggregates as a target for screening anti-Aβ drugs, characterized in that, The Aβ aggregate is prepared by the method described in any one of claims 18-21.

24. A method for screening anti-Aβ drugs, characterized in that, The method comprises the steps of: screening the anti-Aβ drug using the target described in claim 22.

25. The method according to claim 24, wherein Screening for drugs that can depolymerize Aβ aggregates or inhibit the polymerization of Aβ to form aggregates, and the drugs are selected from at least one of antibodies, polypeptides, and small molecule compounds.

26. An anti-Aβ drug, characterized in that, The anti-Aβ drug is obtained by the method as described in claim 24 or 25. Use of the compound of formula I, characterized in that, For non-therapeutically inhibiting (i) the formation of Aβ oligomers; (ii) promoting the disaggregation of Aβ oligomers; and / or (iii) binding to Aβ oligomers in vitro;

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