Application of compound D25

Abstract

The invention belongs to the technical field of biological medicine, and relates to application of a compound D25, in particular to application of a small molecule compound D25 in preparation of a product for preventing or treating Alzheimer's disease (AD), and the structural formula of the compound is shown in the specification. Experiments prove that the small molecule compound D25 can pass through a blood brain barrier, can induce autophagy, can relieve pathological characteristics of an AD mouse model and improve cognitive impairment of the AD mouse model, can reduce the proportion of disease-related microglial cells by recovering the morphology of the microglial cells and improving the A beta phagocytosis capacity of the microglial cells, has the potential of promoting neuronal growth and increasing the number of protrusions, and can be used for preparing a medicine for treating the disease-related microglial cells. Therefore, AD can be effectively treated, the safety is high, the effect is achieved, and the application prospect is great.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of compound D25, particularly the application of small molecule compound D25 in products for the prevention and / or treatment of neurodegenerative diseases. Background Technology

[0002] Neurodegenerative diseases are a class of chronic diseases characterized by progressive degeneration and apoptosis of neurons in the central or peripheral nervous system, ultimately leading to irreversible loss of neurological function. The lesions often involve neurons and synaptic connections in the brain, spinal cord, and peripheral nerves. The disease progresses slowly and can gradually cause severe damage to motor, cognitive, sensory, and autonomic nervous functions. At present, most neurodegenerative diseases are incurable, and treatment mainly aims to slow the progression of the disease and improve clinical symptoms. Common neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD), multiple system atrophy (MSA), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), among which Alzheimer's disease and Parkinson's disease are the most common in clinical practice. Although the affected targets and clinical manifestations of different diseases vary, they share several core pathological mechanisms, mainly including (1) protein misfolding and abnormal aggregation: such as Aβ and tau protein (AD), α-synuclein (PD, MSA), TDP-43 (ALS, FTD), etc. These misfolded proteins cannot be degraded normally, forming inclusion bodies in the cell, damaging neuronal function and leading to apoptosis. (2) Progressive neuronal degeneration and apoptosis: Neuronal structure and function are gradually lost, accompanied by cell death. (3) Neuroinflammation: Microglia and astrocytes are abnormally activated, releasing pro-inflammatory factors and exacerbating neuronal damage. (4) Mitochondrial dysfunction: Neuronal energy metabolism is abnormal, reactive oxygen species production increases, triggering oxidative stress and further promoting neuronal apoptosis. (5) Synaptic plasticity abnormality: Manifested as loss of synaptic connections and disordered release of neurotransmitters (such as reduced dopamine and acetylcholine), resulting in impaired nerve signal transmission.

[0003] Alzheimer's disease (AD), commonly known as senile dementia, is the most common primary neurodegenerative disease in the elderly, accounting for 60-80% of all dementia cases. Due to the accelerating aging of the global population, the number of patients is constantly increasing, and AD has reached epidemic levels. As the problem of global population aging continues to worsen, statistics as of 2024 show that more than 55 million people worldwide suffer from AD or AD-related dementia, and the number of patients doubles every 20 years. Clearly, it has become the most serious health problem among the elderly worldwide, placing a significant economic and psychological burden on families and society.

[0004] Clinical symptoms of Alzheimer's disease (AD) include progressive memory loss, impaired executive function, and difficulty with daily activities. Early symptoms include changes in thinking or unconscious behavior, memory impairment for new information, and alterations in language and speech function. In addition, 20-30% of early-stage AD patients exhibit significant depressive symptoms and mood changes. Late-stage AD patients experience severe memory loss, hallucinations, disorientation, and lack of self-satisfaction, ultimately dying from respiratory syndrome, infection, or fasting. The most obvious pathological feature of AD is brain atrophy. Its main pathological features include senile plaques formed by extracellular β-amyloid (Aβ) deposition, neurofibrillary tangles (NFTs) caused by hyperphosphorylation of microtubule-associated protein tau (MAPT), glial cell proliferation, and neuronal loss, accompanied by cerebrovascular amyloidosis, neuroinflammation, and synaptic loss.

[0005] Since the discovery that tau and Aβ are components of NFTs and senile plaques, respectively, research on the pathogenesis of Alzheimer's disease (AD) has primarily focused on their toxic effects. Aβ accumulation can cause synaptic damage and lead to cognitive and electrophysiological deficits. Similarly, although tau protein has been identified as a microtubule-associated protein, its hyperphosphorylation and aggregation have been shown to produce neurotoxicity. Currently, therapeutic research targeting these two proteins mainly focuses on preventing their aggregation, inhibiting their production, or promoting their clearance. Despite promising results in preclinical studies, clinical trials have not shown significant therapeutic effects in AD patients. Although a large number of drugs are now available for clinical use in combating AD, only five drugs were approved by the FDA for the treatment of AD before 2020. These drugs include four cholinesterase inhibitors—tacrine, donepezil, rivastigmine, and galantamine—and one non-competitive NMDA receptor modulator—memantine. They are primarily based on the cholinergic theory but only alleviate AD symptoms, without slowing (let alone stopping or reversing) the progression of the disease. During a period of stagnation in Alzheimer's disease (AD) drug development, Aducanumab emerged in 2021, becoming the first new anti-AD drug approved by the FDA in nearly 20 years. Lecanemab was subsequently approved in 2023 and in China in 2024. Further clinical trials are needed to verify whether these two anti-Aβ monoclonal antibodies can alleviate mild cognitive impairment in AD. In 2019, the team led by Professor Geng Meiyu at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, discovered the role of gut microbiota dysbiosis-promoted neuroinflammation in AD progression. They proposed a new strategy for treating AD by reshaping the gut microbiota and mentioned GV-971, a low-molecular-weight acidic oligosaccharide compound prepared from marine brown algae extract, which has therapeutic effects on AD. Whether it can become a specific drug for AD remains to be clinically verified. The advent of this original drug has laid a more solid foundation and boosted confidence in China's anti-AD drug development, and also demonstrated the great pharmaceutical potential of natural small molecule compounds.

[0006] Reducing or eliminating abnormally accumulated pathogenic proteins such as Aβ in nerve cells can help slow the progression of Alzheimer's disease (AD) and is of great significance for the prevention and treatment of AD. Autophagy, a dynamic self-digestion pathway of cells, is widely present in normal cells. Compared with other cells, long-lived, non-mitotic nerve cells rely more heavily on autophagy for survival. Recent pathological studies of the brains of AD patients have further confirmed that dysfunction of the autophagy-lysosome system plays an important role in the pathogenesis of AD, and defects in multiple stages of the autophagy pathway can promote the pathological process of AD. In recent years, researchers have focused on regulating the autophagy-lysosome system to explore treatments for AD and have made significant progress. In conclusion, regulating the autophagy-lysosome system shows important application prospects in the treatment of AD.

[0007] Compound D25, structural formula This compound, a jatropha-type diterpenoid found in the latex of Euphorbia peplus, is one of the 12 major diterpenoids identified in the plant's latex and has been reported as a natural insecticide. A search revealed no studies on its neuroprotective and AD-related applications. Summary of the Invention

[0008] In view of this, the present invention provides the use of the small molecule compound D25 in products for the prevention and / or treatment of neurodegenerative diseases.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0010] This invention protects the use of the small molecule compound D25 in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases, wherein the structural formula of the small molecule compound D25 is: .

[0011] Furthermore, the neurodegenerative disease is Alzheimer's disease (AD).

[0012] Furthermore, the drug is used to improve learning and cognitive impairments caused by Alzheimer's disease.

[0013] The present invention also protects a pharmaceutical composition for the prevention and / or treatment of Alzheimer's disease, comprising a therapeutically effective amount of the small molecule compound D25 and a pharmaceutically acceptable carrier.

[0014] Furthermore, the dosage form of the drug is an injection.

[0015] Furthermore, the route of administration for the injection is intraperitoneal injection.

[0016] Furthermore, the pharmaceutical composition can cross the blood-brain barrier and activate the autophagy-lysosome system.

[0017] Furthermore, the pharmaceutical composition can improve the autophagy-lysosome function of microglia and restore the morphology and function of microglia, enhancing their ability to phagocytose Aβ.

[0018] Furthermore, the pharmaceutical composition is used to reduce the level of Aβ42 protein in the brain and / or the Aβ42 / Aβ40 ratio.

[0019] Furthermore, the pharmaceutical composition is used to reduce the protein level of β-secretase (BACE1) in the brain.

[0020] This invention provides the use of the small molecule compound D25 in the preparation of medicaments for the prevention and / or treatment of Alzheimer's disease (AD). Compound D25 can cross the blood-brain barrier, improve microglial morphology and function by activating the autophagy-lysosomal system, reduce the pathological burden of Aβ in the brain, thereby improving cognitive impairment in AD patients, and has a good safety profile. Attached Figure Description

[0021] Figure 1 The abundance and signal appearance time of D25 compound in mouse brain tissue contents at different time points after D25 administration were determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Figure 1 The peak elution time of A at 15 min, 30 min, 1 h and 2 h after D25 administration was 6.52 min, which was consistent with the time of D25 standard. Figure 1 B represents the information obtained after D25 standard was processed by HPLC-MS / MS. Figure 2 Open-field behavioral results for WT mice, APP / PS1-treated or untreated mice, among which Figure 2 A is a schematic diagram of an open-field experiment; Figure 2 B indicates that within 15 minutes, there was no significant difference in the movement distance of mice in the WT, APP / PS1 administration group, or the non-administration group, indicating that there was no significant difference in their movement ability. Figure 2 C represents the percentage of time spent exploring the central region of the open field in mice in the WT, APP / PS1-treated, or no-treated groups. D25 administration increased the percentage of time spent in the central region in APP / PS1 mice, improving their anxiety behavior. (ns, no significant difference;**) P < 0.01; ***, P < 0.001; Figure 3 Results of water maze training and short-term / long-term memory in WT mice, APP / PS1-treated or untreated mice, among which Figure 3 A represents the average distance WT mice, APP / PS1-treated or untreated mice learned to find the underwater platform during a 7-day training period; Figure 3 B represents the average time spent by WT mice, APP / PS1-treated or untreated mice, during a 7-day training period to learn to locate underwater platforms. Figure 3 C and Figure 3D represents the average distance swam by WT mice and mice given or not given APP / PS1, as well as the percentage of time spent searching for and staying in the original quadrant (% of time in Quadrant NW), 4 hours after the end of the last training session when the underwater platform was removed. Figure 3 E and Figure 3 F represents the average distance swam by WT mice and mice treated with or without APP / PS1, 72 hours after the last training session ended and the underwater platform was removed, and the percentage of time spent searching for and staying in the original platform quadrant (NW). P < 0.05;**, P < 0.01; ****, P < 0.0001; Figure 4 The changes in the contents of soluble and insoluble Aβ40 and Aβ42, the Aβ42 / Aβ40 ratio, and the Aβ-related proteins APP and BACE1 in the cortex and hippocampus of WT mice, APP / PS1-treated or untreated mice were studied. Figure 4 A represents the soluble and insoluble Aβ40 and Aβ42 contents in the cortex and hippocampus of WT mice, APP / PS1-treated or untreated mice obtained by ELISA kit testing, and the corresponding changes in the Aβ42 / Aβ40 ratio were calculated. Figure 4 B represents the changes and quantification of the content of amyloid precursor protein APP, the key enzyme BACE1 that cleaves APP protein to generate Aβ42, and Aβ42 protein in the cortical tissue of WT mice, APP / PS1-treated or untreated mice; Figure 4 C represents the changes and quantification of amyloid precursor protein APP, and the key enzymes BACE1 and Aβ42 protein (which cleave APP protein to generate Aβ42) in the hippocampus of WT mice, APP / PS1-treated mice, and mice without APP / PS1 treatment; ns, no significant difference; *, P <0.05; **, P <0.01; ***, P <0.001; ****, P <0.0001.

[0022] Figure 5 Changes in the autophagy-lysosome system in the cortex and hippocampus of WT mice, APP / PS1-treated or untreated mice, among which... Figure 5 A and Figure 5 B represents the changes in autophagy-related proteins SQSTM1 and LC3 in the cortex and hippocampus of WT mice, APP / PS1-treated or untreated mice, respectively. Figure 5 C and Figure 5 D represents the expression and quantification of lysosomal membrane protein LAMP1 in microglia surrounding plaques in WT mice, APP / PS1-treated mice, and mice without plaque treatment. * P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Figure 6 Single-cell nuclear sequencing results from hippocampal tissues of WT mice, APP / PS1-treated mice, and mice without APP / PS1 treatment. Figure 6 A flowchart of the single-cell nucleus extraction and sequencing experiment; Figure 6 B is a single-cell UMAP map of mouse hippocampus tissue; Figure 6 C shows the expression of marker genes in each cell group in Figure B; Figure 6 D consists of microglia, which can be mainly divided into two groups; Figure 6 E represents the change in the proportion of two groups of microglia in the three groups of mice; Figure 6 The expression of Apoe+ in F microglia; Figure 6 G represents the staining results of microglia morphology around the plaque, with a scale bar of 5 μm; Figure 6 H represents the quantitative morphological results of microglia in Figure G. P <0.01; ****, P < 0.0001; Figure 7 HE staining results of paraffin sections of liver tissue from WT mice, APP / PS1-treated or untreated mice one month after D25 administration. Each group shows HE staining images of livers from three mice. Scale bar: 100 μm. Figure 8 The results suggest that D25 has the potential to provide neurotrophic benefits, among which... Figure 8 A represents the result of D25 increasing the level of neurotrophic and synapse-related mRNA at the RNA level; Figure 8 B is an immunofluorescence image of neurons stained and photographed 7 days after D25 treatment of mouse neural stem cells; Figure 8 C is a quantitative diagram of the number of neuronal branches and processes. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The methods involved in the embodiments are all methods commonly used in the art. Unless otherwise specified, the materials and reagents involved are commercially available.

[0024] This invention, based on the AD animal model APP / PS1ΔE9 mouse (hereinafter referred to as APP / PS1 mouse), uses a variety of modern neuroscience techniques to study the alleviation of AD symptoms by small molecule compounds, elucidates the molecular mechanism by which the compounds alleviate AD, and finds that they play a role in improving and treating AD by improving autophagy in AD mice and restoring the ability of microglia to phagocytose Aβ.

[0025] Effects of compound D25 on APP / PS1 mice (Example)

[0026] Source of experimental materials Small molecule compound D25: A natural product extracted from Euphorbia milii by Professor Hao Xiaojiang's research group at the Kunming Institute of Botany, Chinese Academy of Sciences. Its chemical structural formula is as follows: .

[0027] Laboratory animals: APP / PS1 mice were purchased from the Institute of Model Animals, Nanjing University, and bred and housed at the Laboratory Animal Center of the Kunming Institute of Zoology, Chinese Academy of Sciences. They were provided with a 23℃-25℃ environment with a 12-hour light / dark cycle and free access to food and water. All experimental methods complied with the requirements of the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences, minimizing harm and suffering to the mice.

[0028] Experimental instruments and equipment were obtained from the Large Instrument Center of Kunming Institute of Zoology, Chinese Academy of Sciences; behavioral equipment was purchased from Reward, tracking and analysis software was purchased from Panlab HARVARD, MA, USA, and various antibodies were purchased from Abcam and Cell Signaling Technology. The Aβ ELISA kit was purchased from Wuhan Yilairuit Biotechnology Co., Ltd.

[0029] Experimental methods 1. Mouse administration: The administration concentrations were 1 mg / kg and 5 mg / kg, administered via intraperitoneal injection, once every two days for a total of 15 administrations over a period of one month.

[0030] 2. Mouse behavior: (1) Open field: to detect the motor ability and anxiety of mice. Mice were placed in an open field of 40×40×40 cm, and the distance the mice moved and the exploration time in the central area of ​​the open field were recorded within 10 min; (2) Recognition of new and old objects: The number of times and the time spent by mice around new and old objects were detected to determine their cognitive ability to recognize new objects. Two identical objects were placed in the same 40×40×40 cm open field to allow the mice to adapt to the objects. After 24 h, one of the old objects was replaced with a new object with a different shape and color. (3) Three-box social interaction: to test the social recognition and social memory abilities of mice. The experiment consists of three stages: mice are allowed to explore the three-box equipment freely for 5 minutes; a non-experimental unfamiliar mouse 1 is placed in Cage 1, and Cage 2 is kept empty, allowing the mice to explore freely for 10 minutes, and the interaction time between the experimental mouse and unfamiliar mouse 1 / empty cage is calculated; Cage 1 is kept in its original state, and unfamiliar mouse 2 is placed in Cage 2, allowing the mice to explore freely for 10 minutes, and the interaction time between the experimental mouse and unfamiliar mouse 1 / unfamiliar mouse 2 is calculated. (4) Water Maze: An experiment designed to teach mice to find platforms hidden in water, primarily used to test the learning and memory abilities of experimental animals regarding spatial location and orientation. A cylindrical water maze with a diameter of 120 cm is divided into four quadrants. A survival platform is placed in one quadrant, and the maze is filled with tap water one day in advance, with the water level 1 cm above the survival platform. A layer of white, odorless, and non-toxic plastic granules is used to cover the water surface. Mice are trained for 7 consecutive days, with the starting position randomly changing between the four quadrants. They are allowed to explore freely for 1 minute, and the time required for the mouse to find the survival platform (latency) is measured. If the mouse finds the platform within 1 minute, it stays on the platform for 20 seconds, allowing it to remember the location of the survival platform based on surrounding landmarks. If the mouse cannot find the survival platform within 1 minute, the inventor carefully guides the mouse to the platform and allows it to stay for 20 seconds. The training interval for each mouse is 90 minutes per day. Each day, mice are randomly placed in three different quadrants of the water maze to search for the survival platform. On day 7, 4 hours and 72 hours after the final training session, the rescue platform in the water maze was removed, and the mice were placed in the water. The time and distance the mice traveled around the rescue platform within 1 minute were measured to assess their short-term memory (4 hours) and long-term memory (72 hours). Throughout the experiment, SMART 3.0 software (Panlab HARVARD, MA, USA) was used to track and record the mice's behavior. Behavioral parameters (speed, distance traveled, time required to reach the rescue platform, percentage of time in each quadrant, percentage of distance traveled in each quadrant) were automatically calculated by SMART 3.0 software based on the mice's behavior.

[0031] 3. Western blotting (protein immunoblotting) (1) Protein extraction: Protein extraction was performed by adding Beyotime Western blot containing protease inhibitor (PMSF) and IP lysis buffer to the sample; (2) Protein concentration determination: The protein concentration was quantified using the Beyotime BCA protein quantitative kit; (3) Protein denaturation: Take 20 μg of protein, add PBS to make up to 16 μL, add 5× Loading buffer denaturant, denature at 95°C for 5 min, and place on ice for 2 min. (4) Gel running: Spot the denatured protein sample into a 12% SDS-PAGE gel and start the electrophoresis run; (5) Transfer: The PVDF membrane was activated with methanol and transferred for 2 h in an ice bath at 100 v 200 mA. (6) Sealing: Sealing with 5% skim milk at room temperature for 2 hours; (7) Incubation of primary antibody: Add the corresponding primary antibody and incubate overnight on a shaker at 4°C; (8) Incubation of secondary antibody: Incubate the secondary antibody according to the species of the primary antibody, and incubate for 1 h at room temperature; (9) Development: Add developer evenly and develop using a Bio-Rad fluorescence image analyzer.

[0032] 4. Immunofluorescence staining of mouse brain tissue (1) Anesthesia and perfusion sampling: 60 mg / kg pentobar was injected into the peritoneum of mice. After complete anesthesia, the peritoneum and thoracic cavity were opened, the right atrial appendage was cut open, and 20 mL of pre-cooled PBS was slowly injected from the apex of the heart for perfusion until there was no obvious blood in the mouse liver. The mouse brain tissue was then dissected on ice. (2) Fixation of tissue: The detached brain tissue was placed in 4% PFA solution for 24 h, and then replaced with new PFA and fixed for another 24 h. (3) Dehydration: The brain tissue was placed in a gradient of alcohols for dehydration. 30% ethanol for 30 min; 50% ethanol for 30 min; 75% ethanol for 4 h; 85% ethanol for 2 h; 90% ethanol for 2 h; 95% ethanol for 1 h; 100% anhydrous ethanol for 30 min; 100% anhydrous ethanol for 30 min; (4) Transparent wax impregnation: benzene for 10 min; xylene for 10 min; xylene for 10 min; melt paraffin at 65℃ for 1 h; melt paraffin at 65℃ for 1 h; (5) Paraffin embedding: First, cover the bottom of the embedding box with a layer of melted paraffin. Then, take out the dehydrated and transparent paraffin-impregnated tissue and place it into the embedding box. Then, fill the embedding box with paraffin, attach the corresponding label, and place it on a -20℃ cold table to cool. After the paraffin block solidifies, remove the embedded tissue from the embedding box for preservation. (6) Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4 μm. Transfer the section to a 40°C warm water plate to flatten the tissue. Use a pre-labeled glass slide to pick up the tissue and attach it to the slide. Bake the slide in a 60°C oven for 3-5 hours. After the water has dried, remove it and store it at room temperature for later use. (7) Dewaxing: Place the slides in a 65℃ oven for about 2-3 hours, then place them in tissue clearing solution at room temperature for 10 minutes; repeat twice. (8) Rehydration: Place the paraffin sections in 100% anhydrous ethanol for 5 min, 100% anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, 50% ethanol for 5 min, and distilled water for 5 min in sequence. (9) Antigen retrieval: Place the rehydrated slides in sodium citrate retrieval solution at pH=6, cover the antigen retrieval box with aluminum foil and place it in a microwave oven. Heat on medium-high for 8 min, let stand at room temperature for 8 min, heat on medium for 8 min, and then let stand at room temperature until the antigen retrieval solution returns to room temperature. Wash three times with PBST for 5 min each time. (10) Blocking: Draw a water-blocking ring around the tissue using a histochemical pen, and add 5% BSA to block at room temperature for 1 h; (11) Incubation of primary antibody: Aspirate the blocking solution in the water-blocking ring, dilute the primary antibody with 5% BSA, mix well, and drop it into the water-blocking ring. Incubate overnight at 4°C, and wash three times with PBST for 5 min each time. (12) Incubation of secondary antibody: Dilute the secondary antibody of the corresponding species with 5% BSA, mix well, drop into the water-blocking ring, incubate at room temperature for 1 h, wash three times with PBST, 5 min each time; (13) Nucleus staining: Dilute DAPI with PBST, add it to the water-blocking zone, incubate at room temperature for 20 min, wash three times with PBST for 5 min each time; (14) Mounting observation: Add 200 μL of anti-fluorescence quencher, cover with a coverslip, and let stand at room temperature until the mounting medium solidifies. Observe the stained sample and obtain the results under a laser confocal microscope.

[0033] 5. Enzyme-linked immunosorbent assay (ELISA) The Aβ content in brain tissue was detected using an ELISA kit (Elabscience). The specific experimental procedures were performed according to the ELISA kit instructions.

[0034] 6. HE staining (1) Dewaxing paraffin sections to water: The sections were placed in environmentally friendly dewaxing solution I for 15 min, environmentally friendly dewaxing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min in sequence, and then rinsed with tap water; (2) Pretreatment: The sections were immersed in high-resolution constant staining pretreatment solution for 1 min; (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 min, wash with tap water, differentiate with differentiation solution for 3-5 s, wash with tap water, blue back solution for 3-6 s, and rinse with running water. (4) Eosin staining: The sections were dehydrated in 95% alcohol for 1 min, and then stained in eosin staining solution for 15 s; (5) Dehydration and mounting: The sections were sequentially immersed in anhydrous ethanol I for 1 min - anhydrous ethanol II for 1 min - anhydrous ethanol III for 1 min - n-butanol I for 1 min - n-butanol II for 1 min - xylene I for 1 min - xylene II for 1 min, and then cleared and mounted with neutral resin; (6) Microscopic examination, image acquisition and analysis.

[0035] Experimental results 1. Compound D25 can cross the blood-brain barrier and improve anxiety and depression-like symptoms in AD mice. To investigate whether compound D25 can cross the blood-brain barrier, mice were intraperitoneally injected with 5 mg / kg D25. After 15 min, 30 min, 1 h, and 2 h following administration, the mice were thoroughly perfused with PBS, and the brain tissue was dissected, homogenized, and extracted using sonication and methanol precipitation. The contents of the brain tissue were then concentrated in methanol to form crystals, and resuspended in 500 μL of methanol to obtain the analyte. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the D25 standard to obtain standard information for this compound. Figure 1 B), followed by testing of the analyte, data showed that compound D25 could cross the blood-brain barrier (B). Figure 1 A).

[0036] Progressive cognitive decline and increased anxiety are two important clinical features of Alzheimer's disease (AD). To investigate whether D25 could alleviate anxiety symptoms in APP / PS1 mice, an open-field assay was used for further testing. Figure 2 A). The results showed no significant difference in kinetic ability between WT and APP / PS1 mice that were treated or not treated (A). Figure 2 B), but the administration of the drug alleviated the anxiety-like symptoms in APP / PS1 mice, and their activity time in the central area of ​​the open field was significantly increased. Figure 2 C).

[0037] 2. D25 can alleviate cognitive and learning impairments in APP / PS1 mice. To investigate whether D25 could alleviate learning and cognitive impairment symptoms in APP / PS1 mice, 10-month-old APP / PS1 mice were administered the drug intraperitoneally for one month before undergoing Morris water maze learning and testing. After 7 days of training, the vast majority of WT and APP / PS1 mice could find the rescue platform. However, compared to WT mice, APP / PS1 mice took longer to find the platform and swam a longer average distance, indicating significant learning impairment in APP / PS1 mice. Compared to the control group (APP / PS1-Control), the learning ability of APP / PS1 mice after D25 administration was significantly improved, as evidenced by less escape lantency and shorter path length. Figure 3 AB).

[0038] Short-term and long-term memory abilities in mice were assessed 4 h and 72 h after the last training session, respectively. Compared to the control group, APP / PS1 mice showed significantly improved short-term memory after D25 administration. Figure 3 CD) and long-term memory Figure 3 Both EF (experimental and experiential) abilities were significantly improved; specifically, compared to the APP / PS1-Control mice, the APP / PS1 mice showed a significant increase in the percentage time spent in the target quadrant and a shorter mean distance traversed the platform area after drug administration. These results suggest that D25 can effectively improve learning and cognitive impairments in APP / PS1ΔE9 mice.

[0039] 3. D25 can alleviate the pathological characteristics of APP / PS1 mice. To further investigate whether D25 could alleviate the pathological characteristics of APP / PS1 mice, brain tissue samples were collected from APP / PS1 mice that had completed behavioral tests. Proteins were extracted from the cortex and hippocampus, and ELISA and Western blot experiments were then performed to detect Aβ and related proteins. ELISA results showed that, compared with the control group, although the Aβ40 content in the hippocampus and cortex of APP / PS1ΔE9 mice after D25 administration was not significantly changed, the Aβ42 / Aβ40 ratio was significantly reduced. Figure 4 A). Western blot results further showed that, compared with the control group, after administration, the cortex of APP / PS1ΔE9 mice ( Figure 4 B) and seahorse ( Figure 4In C), Aβ42 protein levels were significantly reduced. Furthermore, when the enzyme involved in cleaving the amyloid precursor protein APP to generate Aβ42 was detected, D25 was found to decrease β-secretase (BACE1). These results suggest that D25 can improve the Aβ pathological characteristics in APP / PS1 mice.

[0040] 4. D25 can improve the autophagy-lysosomal system in APP / PS1 brain tissue and enhance the phagocytic capacity of microglia. To further investigate whether the decreased Aβ pathological features in the brain tissue of APP / PS1 mice induced by D25 administration are related to autophagy, the expression levels of autophagy marker proteins SQSTM1 and LC3B were detected in the hippocampus and cortex of APP / PS1 mice after D25 administration, as well as in control APP / PS1 and WT mice. Western blot results showed that, compared with WT mice, control APP / PS1 mice exhibited significant autophagy impairment in the cortex and hippocampus. D25 administration alleviated the autophagy abnormalities in APP / PS1 mice, restoring autophagic flux. Figure 5 AB). Given the crucial role of microglia in phagocytosis and clearance of Aβ, and the key role played by their autophagy-lysosomal system, co-staining was performed on the microglia marker IBA1 and the lysosomal marker LAMP1 around the plaque. This revealed significant accumulation of lysosomal signals in microglia in the brains of APP / PS1 mice, indicating significant abnormal activation of autophagy in the APP / PS1 mouse brain, leading to impaired autophagy and significant lysosomal accumulation. This phenomenon was significantly improved after administration of D25, improving autophagy impairment and restoring microglia function. Figure 5 CD).

[0041] 5. D25 can improve the ratio of normal microglia to disease-associated microglia in the brains of APP / PS1 mice and restore the morphology of microglia. To investigate whether D25 possesses any neuronal specificity, single-cell nuclear sequencing technology was used to observe and analyze drug administration changes at single-cell resolution. Figure 6 A). After analyzing each cell group ( Figure 6The results showed that microglia in the brains of APP / PS1 mice could be clearly divided into two groups, and these two groups had different differentially expressed genes. One group of cells highly expressed APOE, which could be preliminarily identified as disease-related microglia. In the brains of APP / PS1 mice, DAM had the highest proportion of all microglia, accounting for approximately 90% of the total number of microglia. In WT mice, the proportions of the two microglia showed the opposite trend. After administration of D25, the proportions of these two cell groups in APP / PS1 mice were improved to some extent, and the proportion of DAM was reduced, bringing the proportions of these two cell groups in APP / PS1 mice closer to those in the WT group ( Figure 6 DF). Disease-associated microglia exhibit enlarged cell bodies and atrophied branching, characteristic of amoebae. To verify whether D25 administration improved DAM in APP / PS1 mice, microglia were stained with IBA1 and co-stained with 6E10-labeled plaques. The results showed that compared to WT mice, microglia in the brains of APP / PS1 mice exhibited typical DAM morphology. D25 administration significantly improved microglia morphology, such as increased branching (…). Figure 6 GH).

[0042] 6. No significant toxic effects were observed in the liver of mice after continuous administration of D25 for one month. To determine the toxicity of D25, fixed liver tissue was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). Figure 7 As shown, the staining results indicated that there were no obvious pathological changes in the liver tissue of the drug-treated group and the control group, meaning that continuous administration of D25 for one month did not produce significant hepatotoxicity in mice.

[0043] 7. D25 has the potential to promote neuronal growth and increase the number of processes, among other neurotrophic effects. like Figure 8 RNA extraction and sequencing analysis of U251-APP cells treated with compound D25 revealed that D25 can induce the expression of neurotrophic mRNAs such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and nerve growth factor receptor (NGFR), as well as synapse-related mRNAs such as postsynaptic density protein 95 (PSD95) and synaptophysin (SYP). Figure 8A) suggests that compound D25 has neurotrophic potential. Subsequently, a co-culture experiment was conducted with D25 and mouse neural stem cells. After 7 days of D25 co-treatment, the cells were fixed, stained, and subjected to immunofluorescence experiments. Laser confocal microscopy images showed that D25 may have the potential to promote neuronal growth and increase the number of processes. Figure 8 BC).

Claims

1. The use of the small molecule compound D25 in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases, characterized in that, The structural formula of the small molecule compound D25 is: .

2. The application according to claim 1, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

3. The application according to claim 2, characterized in that, The drug is used to improve learning and cognitive impairments caused by Alzheimer's disease.

4. A pharmaceutical composition for the prevention and / or treatment of Alzheimer's disease, characterized in that, The small molecule compound D25 of claim 1 comprises a therapeutically effective amount and a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The drug is in the form of an injection.

6. The application according to claim 5, characterized in that, The injection is administered via intraperitoneal injection.

7. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition described above can cross the blood-brain barrier and activate the autophagy-lysosome system.

8. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition described herein can improve the function of the autophagy-lysosome system of microglia and restore the morphology and function of microglia, thereby enhancing their ability to phagocytose Aβ.

9. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is used to reduce the level of Aβ42 protein in the brain and / or the Aβ42 / Aβ40 ratio.

10. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition described herein is used to reduce the protein level of β-secretase (BACE1) in the brain.

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