Use of phenylpyrimidinone compound

Abstract

Use of a phenylpyrimidinone compound. The present invention specifically relates to use of a phenylpyrimidinone compound represented by formula (I) or a tautomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, or an isotopically labelled compound thereof in the preparation of a drug for ameliorating or treating cognitive impairment, and / or ameliorating or treating cerebrovascular diseases. Studies using cell and animal models have shown that compared to the commercially available clinical drug donepezil and the investigational clinical drug mirodenafil, the compound has demonstrated significant therapeutic characteristics and substantial technical advancements in terms of protecting nerve cells, ameliorating cognitive impairment, ameliorating cerebrovascular diseases, and ameliorating degenerative diseases such as Parkinson's disease.

Description

Uses of a phenylpyrimidinone compound Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to the use of a phenylpyrimidinone compound. Background Technology

[0002] With my country rapidly entering an aging society and its average life expectancy increasing, the rapid rise in age-related disabilities and diseases is placing a heavy burden on society and families. Among these, cognitive impairment and cerebrovascular disease are among the most common causes of loss of daily living abilities in the elderly, seriously affecting public health and sustainable social development in my country. Therefore, strengthening the prevention and treatment of cognitive impairment and cerebrovascular disease, improving or treating these conditions, and alleviating the burden on society and families are urgent public health issues and important components of the national strategy to proactively address population aging.

[0003] Cognitive impairment refers to cognitive dysfunction of varying degrees caused by various factors, ranging from mild cognitive impairment to dementia. Mild cognitive impairment (MCI) and its subtypes refer to memory impairment and / or mild other cognitive impairments, but the individual's social, occupational, or daily living functions are not affected, and cannot be explained by known medical or neuropsychiatric diseases. It is a clinical state between normal aging and mild dementia. Dementia refers to a group of severe cognitive deficits or declines caused by organic diseases, such as progressive thinking, memory, behavioral, and personality disorders, which may be accompanied by mental and motor symptoms, and the impairment reaches a level that affects occupational, social, or daily living abilities. There are multiple classification criteria for dementia. The most common is etiological classification, which can be divided into degenerative and non-degenerative diseases. Alzheimer's disease (AD) is the most common degenerative dementia, accounting for 50-70% of all types of dementia; vascular dementia (VD) is the most common non-degenerative dementia, accounting for 15-20% of dementia patients. The coexistence of degenerative dementia and vascular dementia is called mixed dementia, which is classicly characterized by a rapidly onset new cognitive impairment in AD patients after a stroke. Other types of dementia, such as frontotemporal dementia (FTD), Lewy body dementia (DLB), and Parkinson's disease dementia (PDD), also pose serious threats to patients' health. Currently, cholinesterase inhibitors, such as donepezil, are used as first-line drugs to relieve symptoms, but they have varying degrees of cholinergic side effects. Therefore, there is an urgent clinical need to develop drugs to improve or treat cognitive impairment.

[0004] Cerebrovascular diseases are a group of diseases that occur in the blood vessels of the brain, causing brain tissue damage due to impaired intracranial blood circulation. Stroke includes two main categories: ischemic stroke and hemorrhagic stroke. For example, cerebral thrombosis, cerebral embolism, and lacunar infarction belong to ischemic stroke, while what is commonly referred to as cerebral hemorrhage belongs to hemorrhagic stroke. Ischemic stroke accounts for 75% to 90% of all strokes. Cerebral small vessel disease (CSVD) refers to a series of clinical, imaging, and pathological syndromes caused by lesions in intracranial small arteries, arterioles, capillaries, venules, and small veins. Studies have found that lacunar infarctions caused by CSVD account for 25% to 50% of ischemic strokes in my country. CSVD is closely related to post-stroke depression, cognitive impairment, low quality of life, early Parkinson's disease symptoms, and hematoma expansion in cerebral hemorrhage. Currently, the main drugs used in clinical practice to treat ischemic cerebrovascular disease include anticoagulants such as clopidogrel, aspirin, and dabigatran etexilate, as well as statins to lower blood lipids. However, these drugs may increase the risk of cerebral hemorrhage. Therefore, there is a clinical need to develop new drugs for the prevention and treatment of cerebrovascular diseases.

[0005] Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta and the abnormal aggregation of α-synuclein. Globally, there are over 10 million patients, with a prevalence of 1-2% in people over 65 years of age. With increasing aging, the disease burden is projected to double by 2040, necessitating the development of new treatments. Summary of the Invention

[0006] During their research on phenylpyrimidinone compounds, the inventors discovered that they have neuroprotective effects in cell models, effects in improving or treating cognitive impairment in animal models, and effects in improving or treating cerebrovascular diseases. Based on this, the present invention was proposed.

[0007] Purpose of the invention

[0008] One object of the present invention is to provide the use of a phenylpyrimidinone compound in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease.

[0009] Another object of the present invention is to provide the use of a pharmaceutical composition comprising the phenylpyrimidinone compound in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease.

[0010] Another object of the present invention is to provide a phenylpyrimidinone compound for improving or treating cognitive impairment, or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease.

[0011] Another object of the present invention is to provide a method for improving or treating cognitive impairment, and / or improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease.

[0012] Technical solution

[0013] According to one aspect of the present invention, there is a use of a phenylpyrimidinone compound of formula (I) or its tautomer, pharmaceutically acceptable salt, solvate, or isotopic label thereof in the preparation of a medicament for improving or treating cognitive impairment, and / or improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease:

[0014] in,

[0015] R 1 and R 2 Each is independently selected from halogens and C1-C6 alkyl groups, preferably from bromine, methyl, ethyl, propyl and isopropyl;

[0016] R 3 Selected from C1-C6 alkyl groups, preferably ethyl and propyl;

[0017] R 4 For -LR 5 L represents -SO2-, -CO-, or -NHCOCH2-.

[0018] R 5 Selected from the following substituents:

[0019] And hydroxy C1-C6 alkylamino groups (e.g., hydroxypropylamino).

[0020] According to one embodiment of the present invention, the phenylpyrimidinone compound is selected from the following compounds:

[0021] According to the present invention, the salt of the phenylpyrimidinone compound represented by formula (I) may be selected from one or more of the following: hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, malate, citrate, succinate, maleate, fumarate, and oxalate.

[0022] Preferably, the phenylpyrimidinone compound salt represented by formula (I) may contain 0.5-3 molecules of water of crystallization; more preferably, it contains 1-2 molecules of water of crystallization; and most preferably, it contains 1 molecule of water of crystallization.

[0023] According to the present invention, a pharmaceutical composition comprising the phenylpyrimidinone compound or its tautomer, a pharmaceutically acceptable salt, a solvate, or an isotope-labeled compound is provided for use in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease. The pharmaceutical composition comprises a therapeutically effective amount of the compound or its tautomer, a pharmaceutically acceptable salt, a solvate, or an isotope-labeled compound as described above, and a pharmaceutically acceptable carrier. The carrier includes: a diluent, an excipient such as water; a binder such as a cellulose derivative, gelatin, or polyvinylpyrrolidone; a filler such as starch; a disintegrant such as calcium carbonate or sodium bicarbonate; and a lubricant such as calcium stearate or magnesium stearate. Additionally, other excipients such as flavoring agents and sweeteners may be added to the pharmaceutical composition.

[0024] Furthermore, the pharmaceutical composition is an oral pharmaceutical composition, a topical pharmaceutical composition, or an injectable pharmaceutical composition. Various dosage forms of the pharmaceutical compositions of the present invention are prepared according to conventional methods in the art, wherein the content of the active ingredient is 0.1% to 99.5% (by weight).

[0025] When the pharmaceutical composition is an oral pharmaceutical composition, the pharmaceutical composition is a solid dosage form, preferably selected from tablets, powders, oral disintegrating films, or capsules.

[0026] When the pharmaceutical composition is a topical pharmaceutical composition, the dosage form of the pharmaceutical composition is selected from sprays, liniments, ointments, and patches.

[0027] When the pharmaceutical composition is an injectable pharmaceutical composition, the dosage form of the pharmaceutical composition is selected from injection solution and injectable dry powder; preferably, the content of active ingredient in the injectable composition is 0.1% to 99.5% (by weight).

[0028] According to the present invention, the cognitive impairment is selected from Alzheimer's disease, vascular dementia, mild cognitive impairment, mixed dementia, frontotemporal dementia, Lewy body dementia, Parkinson's disease dementia and other types of dementia.

[0029] Preferably, the cognitive impairment is Alzheimer's disease, vascular dementia, or mixed dementia.

[0030] According to the present invention, the cerebrovascular diseases are selected from ischemic cerebrovascular diseases, cerebral small vessel diseases, atherosclerotic stenosis or occlusion of the head and neck arteries (not leading to cerebral infarction), hypertensive encephalopathy, abnormal vascular network syndrome at the base of the brain (Moyamoya disease), head and neck artery dissection, reversible cerebral vasoconstriction syndrome, primary central nervous system vasculitis, intracranial venous system thrombosis, cerebrovascular diseases without acute focal involvement and functional loss, sequelae of stroke, and other cerebrovascular diseases.

[0031] Preferably, the cerebrovascular disease is ischemic stroke or cerebral small vessel disease.

[0032] According to another aspect of the present invention, there is a use of a phenylpyrimidinone compound of formula (I) or its tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopic label thereof, in combination with other active ingredients, in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, improving or treating degenerative diseases such as Parkinson's disease; wherein the other active ingredients are selected from one or more medicaments for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, improving or treating Parkinson's disease.

[0033] Preferably, it provides the use of a phenylpyrimidinone compound of formula (I) or its tautomer, pharmaceutically acceptable salt, solvate, or isotopic label in combination with other active ingredients in the preparation of a medicament for improving or treating cognitive impairment; wherein the other active ingredients are selected from one or more of donepezil, galantamine, bengalantamine, rivastigmine, memantine, lencanemab, donepemab, mannostatin, edaravone, remternetug, huperzine A, VG-3927, AXS-05, butylphthalide, tenecteplase, alteplase, tirofiban, abexicillin, rivaroxaban, apixaban, dabigatran etexilate, clopidogrel, aspirin, pramipexole, ropinirole, rotigotine, safamide, opipiccapone, levodopa, dopasilidine, carbidopa, etc.

[0034] Preferably, it provides the use of a phenylpyrimidinone compound of formula (I) or its tautomer, pharmaceutically acceptable salt, solvate, or isotopic label in combination with other active ingredients in the preparation of a medicament for improving or treating cerebrovascular diseases; wherein the other active ingredients are selected from one or more of edaravone, butylphthalide, tenecteplase, alteplase, tirofiban, abexisumab, rivaroxaban, apixaban, dabigatran etexilate, clopidogrel, aspirin, etc.

[0035] Preferably, it provides the use of a phenylpyrimidinone compound of formula (I) or its tautomer, pharmaceutically acceptable salt, solvate, or isotope label in combination with other active ingredients in the preparation of a medicament for improving or treating degenerative diseases such as Parkinson's disease; wherein the other active ingredients are selected from one or more of pramipexole, ropinirole, rotigotine, safamide, opipicolane, levodopa, dopasilidine, carbidopa, etc.

[0036] According to some embodiments of the present invention, the medicaments for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases contain the other active ingredients.

[0037] According to another aspect of the invention, a pharmaceutical composition is provided comprising a phenylpyrimidinone compound of formula (I) above, or a tautomer thereof, a pharmaceutically acceptable salt, a solvate, or an isotope label,

[0038] in,

[0039] R 1 To R 5 The definitions of L and L are as described above.

[0040] as well as,

[0041] Other active ingredients, as described above.

[0042] According to another aspect of the invention, a method for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease is provided, the method comprising administering to a subject a therapeutically effective amount of the phenylpyrimidinone compound of formula (I) above, or a tautomer thereof, a pharmaceutically acceptable salt, a solvate, or an isotope label thereof, and optionally other active ingredients described above. The phenylpyrimidinone compound of formula (I) and the other active ingredients are described as described above.

[0043] According to another aspect of the invention, it provides a phenylpyrimidinone compound of formula (I) or its tautomers, pharmaceutically acceptable salts, solvates, or isotopic labels for improving or treating cognitive impairment and / or for improving or treating cerebrovascular diseases.

[0044] The definitions of each substituent are as described above.

[0045] According to another aspect of the invention, a method for improving or treating cognitive impairment and / or improving or treating cerebrovascular disease is provided, the method comprising administering to a subject a therapeutically effective amount of a phenylpyrimidinone compound of formula (I) described above or a tautomer thereof, a pharmaceutically acceptable salt, a solvate, or an isotopic label thereof, or a pharmaceutical composition comprising the thereof. Beneficial effects

[0046] This invention provides a novel use for phenylpyrimidinone compounds, with the following beneficial effects:

[0047] 1. The pyrimidinone phenyl derivatives described in this invention have a significant effect in improving or treating cognitive impairment.

[0048] 2. In terms of protecting nerve cells, the compounds of the present invention have a more potent effect in enhancing cell activity and neuroprotection.

[0049] 3. In terms of improving cognitive function, the compounds of the present invention can significantly improve memory impairment, enhance memory discrimination ability, and have a significant effect on improving working memory and spatial memory.

[0050] 4. When the compounds of this invention are used in combination with other active ingredients, they can enhance the effects of other active ingredients, such as enhancing the effect of donepezil in improving working memory and spatial memory.

[0051] 5. The compounds of the present invention can improve vascular dementia.

[0052] 6. The compounds of this invention can improve stroke.

[0053] 7. Through experiments using different cell and animal models, compared with existing clinical drugs donepezil, pramipexole, and the clinical investigational drug milonafil, the compounds of this invention have significant therapeutic characteristics and substantial technological advancements in protecting nerve cells and improving cognitive dysfunction. Attached Figure Description

[0054] Figure 1 is a bar chart showing the relative values ​​of cell activity in different groups of Aβ cell models using different compounds.

[0055] Figure 2 is a schematic diagram of the research time of the animal experiment in Example 2 of this application.

[0056] Figure 3 is a diagram of the experimental paradigm for new object recognition in the embodiments of this application.

[0057] Figure 4 is a bar chart of the resolution percentage of the new object recognition experiment in Embodiment 2 of this application.

[0058] Figure 5 is a bar chart of the resolution index of the new object recognition experiment in Embodiment 2 of this application.

[0059] Figure 6 is a bar chart of the correct selection rate of the Y-maze in Embodiment 2 of this application.

[0060] Figures 7 and 8 are the results of the water maze test experiment of Embodiment 2 of this application.

[0061] Figure 9 is a schematic diagram of the research time of the animal experiment in Example 3 of this application.

[0062] Figure 10 is a bar chart showing the central area movement distance, time, and number of wall climbs in the open field test of Embodiment 3 of this application.

[0063] Figure 11 is a bar chart of the resolution ratio of the new object recognition experiment in Embodiment 3 of this application.

[0064] Figure 12 is a bar chart of the alternation ratio of the Y-maze in Embodiment 3 of this application.

[0065] Figures 13 and 14 are the results of the water maze test experiment of Embodiment 3 of this application.

[0066] Figure 15 is a schematic diagram of the research time of the animal experiment in Example 4 of this application.

[0067] Figures 16 and 17 are the results of the water maze test experiment of Embodiment 4 of this application.

[0068] Figure 18 is a schematic diagram of the research time of the animal experiment in Example 5 of this application.

[0069] Figures 19 and 20 are the results of the water maze test experiment of Embodiment 5 of this application.

[0070] Figure 21 is a schematic diagram of the research time of the animal experiment in Example 6 of this application.

[0071] Figure 22 is a diagram showing the results of an animal experiment in Example 6 of this application.

[0072] Figure 23 is a bar chart showing the relative values ​​of cell activity in each group of neurons in the 6-OHDA-induced injury model of Example 7 of this application. Detailed Implementation

[0073] The present invention will be described in more detail below through embodiments. These embodiments are only used to illustrate the invention in more detail, and the scope of the invention is not limited to these embodiments, as will be apparent to those skilled in the art to which this invention pertains.

[0074] Unless otherwise specified, the raw materials, reagents, equipment, methods, etc. used in this invention are all conventionally available in the art.

[0075] Preparation of compounds: The compounds represented by formula (I) of this invention can be prepared by referring to prior art known to those skilled in the art or according to WO2010066111A1. Compounds 1, 2, 5, and 6 used in the embodiments of this invention were prepared according to the method disclosed in patent WO2010066111A1.

[0076] Two-way ANOVA analysis was performed on the acquired data. Data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001, ns, no significance, no statistical difference).

[0077] Example 1: Protective effect of the compound of the present invention on neurons at the in vitro cellular level.

[0078] (1) Cell culture and neuronal directional differentiation

[0079] SH-SY5Y (human neuroblastoma cell line, Procell, CL-0208) cells were cultured under the following conditions: complete medium: DMEM / F12 (1:1) medium, 10% FBS and 1% pen / strep, in a 37°C, 5% CO2 incubator. After 48 hours of culture in complete medium, the medium was changed to induce differentiation into neuronal cells. The medium was then changed to Neurobasal medium (containing B27 supplement and GlutaMAX) and 10 μM all-trans-retinoic acid (ATRA), with a half-medium change every 48 hours. Maintaining these conditions for 5 days yielded cultured and differentiated neuronal-like cells.

[0080] (2) Experiment on the oligomerization of Amyloidbeta(Aβ)1-42

[0081] Amyloid beta(Aβ)1-42 protein was dissolved in serum-free DMEM / F12 medium to prepare a 100 μM system, and then placed in a 4°C refrigerator for more than 24 hours to form Amyloid beta(Aβ)1-42 oligomers.

[0082] (3) Amyloid beta(Aβ)1-42 oligomer treatment induced cell damage model

[0083] The Amyloid beta(Aβ)1-42 oligomer prepared in (2) was added to the differentiated cells in (1) at a final concentration of 10 μM. It was added alone or together with the treatment compounds (in this example, the control drugs donepezil and mirtazafil, as well as compounds 1, 2, 5 and 6 of this application) for treatment. CCK8 detection was performed after 48 hours (see step 4).

[0084] (4) CCK8 treatment experiment

[0085] After mixing cck-8 with culture medium (phenol red-free DMEM / F12 + 10% FBS) at a ratio of 1:10, add 100 μL to each well and incubate for 4 h. Then, measure the absorbance (OD) at 450 nm using a microplate reader.

[0086] (5) Data Analysis

[0087] The Amyloid beta(Aβ)1-42 oligomer treatment group was used as the model group. Its CCK8 activity data were normalized. The results of all compound treatments were compared with the model group data using unpaired t-tests and Mann-Whitney tests. Data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001). Results are shown in Figure 1 and Table 1 below.

[0088] Table 1: Relative values ​​and statistical differences of increased cell viability in each group in the Aβ cell model

[0089] As shown in Figure 1 and Table 1 above, treatment of neuronal differentiated cells with Aβ1-42 oligomers damages nerve cells and affects their survival activity. By normalizing the cell survival value of the treated group to 1.0, and treating other compounds using the same method to obtain relative cell activity values ​​compared to the treated group, differences between different groups can be compared uniformly. Experimental results show that donepezil, as a positive control for AD, can enhance cell activity after Aβ1-42 oligomer treatment, and Mirodenafil also enhances cell activity. In comparison, various phenylpyrimidinone derivatives have stronger cell activity enhancement and neuroprotective effects. At the same dosage, the proportion of cell activity enhancement is higher than that of the donepezil and Mirodenafil groups. Therefore, the various phenylpyrimidinone derivatives in this patent have highly effective neuroprotective effects, especially against cell damage caused by Alzheimer's disease-related toxic proteins.

[0090] Example 2: The effect of the compound of the present invention on dementia in an STZ animal model.

[0091] (1) Animal model

[0092] C57BL / 6j (male, 6 months old) was purchased from a certified animal supplier (Vitollife) and fed for one week prior to the experiment. The animals were housed under standard conditions: room temperature 21-23°C, relative humidity 30-70%, and a 12h:12h light-dark cycle. Food and water were provided freely.

[0093] The surgical modeling experiment simulating sporadic Alzheimer's disease (AD) in animals was conducted by stereotactic injection of streptozotocin (STZ) into the brain (STZ-ICV). The specific method is as follows:

[0094] The equipment used includes: stereotaxic instrument, skull drill, 10ul micro-injection needle, surgical scissors, surgical forceps, sutures (5-0), needle holder, razor, petri dish, cotton swabs, iodine tincture, 75% alcohol, physiological saline, STZ, and tribromoethanol.

[0095] Animal preparation was as follows: Six-month-old male C57 / 6j mice were selected. They were grouped according to their weight (ensuring minimal weight difference within each group; animals with a weight deviation greater than ±20% were excluded), with 4 animals per cage, and the cage tags indicating the group corresponding to each cage.

[0096] Preoperative preparation: Sterilize laboratory instruments and surgical equipment (with outer packaging required), and use disposable sterile supplies if necessary.

[0097] Clean and disinfect the operating environment (space, countertops, etc.).

[0098] Anesthesia and Fixation: Mice were anesthetized by intraperitoneal injection of tribromoethanol (20 ml / kg). The animals entered a state of deep anesthesia approximately 5 minutes later. The hair on the skull was shaved clean, and the mice were fixed to the adapter. Eye ointment was applied to the eyes. Both ear rods were fixed to the external auditory canals of the mice, finely adjusted to ensure the scale on both ear rods was aligned, and the screws were tightened to center the mouse's head. The mouse's incisors were fixed to the incisor retainer on the adapter; the incisor clamps should not be tightened too much.

[0099] Rat brain balancing: Exposing the skull: Disinfect the mouse scalp with iodine tincture, then cut a 2cm long incision in the scalp with scissors, and use a dry cotton ball or hydrogen peroxide to corrode and wipe away the periosteum on the surface of the skull to fully expose the skull.

[0100] Positioning the anterior fontanelle: Move the injection needle tip to the anterior part of the midline, contacting the skull surface. Using the anterior fontanelle (bregma point) as a reference point, stop the needle as soon as it touches the surface, and reset the X, Y, and Z axis coordinate readings of the digital display to zero. Observe the needle tip position while inserting the needle to avoid breaking it!

[0101] Left-right leveling: Lift the syringe slightly upwards to avoid touching the skull surface. Then, using the bregma as the midpoint, move it left and right by the same distance (3.0mm / 2.5mm / 2mm recommended) and lower the syringe to contact the skull surface. Read the Z-coordinate of the corresponding point. Use the Z-coordinate values ​​to determine if the left and right sides are level. If the readings differ by less than 0.03mm, the mouse head is considered level. Level the left and right sides by adjusting the height of the ear rods on both sides. During the adjustment process, the bregma zero point must be repositioned after moving the ear rods. Anterior-posterior leveling: Level the anterior and posterior fontanelles using the same method as left-right leveling.

[0102] STZ Injection: Craniotomy: After leveling, use a digital display to locate your target brain region. Taking ICV coordinates as an example, AP (Y-axis): -0.92-0.96mm, ML (X-axis): ±0.22-0.28mm, DV (Z-axis): -2.35mm. Move the Y-axis backward by 0.96mm and the X-axis to the right by 0.26mm. Insert the needle and stop the needle the instant it touches the skull surface. Observe the skull surface features and remember or mark them. Lift the needle to a higher position and then use a cranial drill to drill a hole at the target site to expose the brain tissue.

[0103] To draw up STZ: Turn on the control pump, set the required volume, speed and mode, immerse the needle tip below the liquid surface, and press start to begin drawing up the liquid. The entire process should be performed in the dark.

[0104] Injection: Move the syringe to the target brain region and begin needle insertion according to the coordinates. The insertion process must be slow enough to reach the target brain region depth. Set the volume, speed (generally 1 μL / min), and mode, then press start to inject STZ. After injection, stop the needle for 5 minutes, then slowly lift the needle. The entire process must be performed in the dark.

[0105] Postoperative care: Suture the head skin with medical needles and sutures and disinfect. Place the animal on a heating pad to keep it warm until it is fully awake, then return it to its cage.

[0106] (2) Research time

[0107] The study timeline is shown in Figure 2. Six-month-old male C57BL6J mice were used for the modeling experiment. During the environmental adaptation period, animals were grouped uniformly according to their weight, with the grouping date marked as day 0. After uniform grouping, the animals were immobilized using a stereotactic injection device for modeling, and an equal volume of physiological saline or streptozotocin was injected into the lateral ventricle. The same nursing procedures were followed post-surgery, and the animals were housed in the same environment. Drug administration was administered once daily for 30 consecutive days. Behavioral experiments were then conducted to verify the results. The experiment ended at the completion of all behavioral experiments, at which point the animals were euthanized, and serum, cerebrospinal fluid, and tissue samples were collected.

[0108] (3) Animal grouping

[0109] po: oral

[0110] sc: subcutaneous

[0111] (4) Compound information and configuration

[0112] Streptozotocin (STZ) preparation method: Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0113] Donepezil preparation method: Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0114] Preparation method of mirtazafil: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0115] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0116] (5) Research process

[0117] Weight measurement: Monitor and record weight daily during the first week, and then record weight information twice a week (Monday / Thursday).

[0118] Test compound treatment: once daily.

[0119] New object recognition experiments: NOR, Y-maze experiment, Morris water maze experiment:

[0120] Behavioral tests were conducted one month after the model was established and the tests were conducted one hour after administration.

[0121] The procedure for the New Object Recognition (NOR) experiment is as follows:

[0122] 1) One hour before the test, allow the designated mice to enter the laboratory to acclimatize to the environment;

[0123] 2) Fix identical toy objects in an open space;

[0124] 3) Allow each experimental mouse to explore freely in the open space box for 10 minutes and record the mouse's condition;

[0125] 4) At the end of the experiment, remove the mice from the open field box and return them to their cages;

[0126] 5) Replace one of two identical toy objects with a new object and record the result for 10 minutes;

[0127] 6) Repeat this process until all animals have completed exploring the new object;

[0128] 7) Experimental analysis.

[0129] Figure 3 is a diagram of the experimental paradigm for the new object recognition.

[0130] The results of the resolution percentage and statistical differences in the new object recognition experiment are shown in Figure 4 and Table 2 below.

[0131] Table 2: Resolution ratio and statistical differences in the new object recognition experiment

[0132] The results of the resolution index ratio and statistical differences in the new object recognition experiment are shown in Figure 5 and Table 3 below.

[0133] Table 3: Resolution Index Ratio and Statistical Differences in New Object Recognition Experiments

[0134] The novel object recognition experimental paradigm leverages the natural preference of mice for novel objects, training them to recognize and remember different objects, thereby demonstrating their learning, memory, and discrimination abilities. Inducing models in animals with impaired memory or dementia significantly reduces their ability to remember and distinguish between new and old objects. This experiment used STZ stereotactic injection to create a model. Animals with successful models exhibited typical memory and cognitive impairment phenotypes, showing a significantly reduced ability to distinguish new objects compared to the control group. The animals' memory and discrimination abilities for new and old objects were quantified using the discrimination ratio (DR) and discrimination index (DI). The discrimination ratio (DR) was calculated as: (New object exploration time / (New object exploration time + Old object exploration time)) * 100; the discrimination index (DI) was calculated as: (New object exploration time - Old object exploration time) / (New object exploration time + Old object exploration time) * 100. The resolution ratio and resolution index results in the new object recognition experiment revealed that the STZ-induced model group animals exhibited a significant memory impairment phenotype. Donepezil, a positive drug for Alzheimer's disease, showed some effect in alleviating memory impairment in the animals, but this improvement trend was not statistically significant. In contrast, meronafil, compound 1, significantly improved STZ-induced memory impairment in the animals, enhancing their memory discrimination ability.

[0135] The Y-maze process is as follows:

[0136] 1) One hour before the test, allow the designated mice to enter the laboratory to acclimatize to the environment;

[0137] 2) Label the three arms of the Y maze as arm I, arm II, and arm III. Place the mouse at the distal end of arm I, with its back to the center of the maze.

[0138] 3) Allow the animal to explore the maze undisturbed for 5 minutes and record the results;

[0139] 4) After the experiment, remove the animals from the maze and put them back in their cages;

[0140] 5) After all is completed, conduct behavioral trajectory analysis.

[0141] The results of the correct selection rate and statistical differences in the Y-maze experiment are shown in Figure 6 and Table 4 below.

[0142] Table 4: Correct selection rate and statistical differences of animals in the Y-maze

[0143] Compared with the control group, the STZ-induced model group showed significant memory impairment, manifested as a marked decrease in accuracy in autonomous path selection within the Y-maze, a statistically significant difference, consistent with the conclusion that STZ stereotactic injection can induce dementia and memory impairment in animals. Using donepezil as a positive control, the results showed that the donepezil treatment group exhibited a certain improvement in the correct selection rate in the Y-maze, with the improvement approaching statistical significance. The mirtazafil treatment group showed only an upward trend in the correct selection rate in the Y-maze, but no statistically significant difference. Compared with these two treatment groups, compound 1 significantly improved the correct selection rate in the Y-maze, with a statistically significant difference. These results suggest that compound 1 has a significant effect on improving working memory and spatial memory in animals.

[0144] The Morris water maze experiment procedure is as follows:

[0145] Training phase:

[0146] Day 1: Randomly place the animal in one of the other three quadrants, at the midpoint of the pool wall, facing the wall. Let the animal swim freely in the pool. If the animal finds a platform within one minute and stays on it for 10 seconds, release the animal; otherwise, use a glass or plastic rod to guide the animal to the platform, let it stay for 10 seconds, and then release it. Dry the animal with a paper towel and a heat lamp, then return it to its cage. Start with the next animal. Repeat the same process.

[0147] After the last animal in the current round is finished, start again from the first animal. Train each animal twice a day, and repeat the operation of day 1 for days 2-5.

[0148] Testing Phase (Day 6)

[0149] Disassemble the platform and place the animal in the 3rd quadrant of the pool. Allow the mouse to freely explore the maze for 1 minute.

[0150] After drying the animal with paper towels and a heat lamp, put it back in its cage. Move on to the next animal. Repeat the same process.

[0151] The results of the water maze experiment are shown in Figure 7-8 and Table 5-9 below.

[0152] Table 5: Platform escape time and statistical differences of animals during water maze training.

[0153] Table 6: Time and statistical differences in animals reaching the platform during the water maze test period

[0154] Table 7: Number of times animals crossed platforms and statistical differences during the water maze test period.

[0155] Table 8: Distances traversed by animals in the target quadrant and statistical differences during the water maze test period.

[0156] Table 9: Time spent in the target quadrant and statistical differences of animals during the water maze test period

[0157] The Morris water maze test was used to examine the spatial memory abilities of the animals in each group. The results showed that the STZ model group exhibited significant spatial memory impairment. Specifically, during the water maze learning phase, these animals were unable to identify and remember spatial paths, and the time taken to find the hidden underwater platform did not decrease with further learning. Donepezil, an Alzheimer's disease treatment drug, was a positive result in this group, effectively alleviating the spatial memory impairment induced by the STZ model, showing a statistically significant difference compared to the model group at the fifth day of testing. The mirtazafil treatment group showed a certain trend of improvement, but the difference compared to the model group was not statistically significant. Animals treated with compound 1 showed a significant improvement in spatial memory. Specifically, with prolonged training time, the time taken to find the underwater platform gradually decreased, and at the fifth day of testing, the difference in platform finding time between the compound 1 treatment group and the model group was statistically significant.

[0158] Example 3: The effect of the compound of the present invention on dementia improvement in a 5X FAD animal model.

[0159] (1) Animal model

[0160] 5XFAD (male, 4 months old) were purchased from a certified animal supplier (Cyagen Biosciences) and fed for one week prior to the experiment. The animals were housed under standard conditions: room temperature 21-23°C, relative humidity 30-70%, and a 12h:12h light-dark cycle. Food and water were readily available.

[0161] (2) Research time

[0162] The study timeline is shown in Figure 9. Four-month-old male 5XFAD mice were used in the experiment. During the environmental adaptation period, animals were grouped evenly according to their body weight, with the grouping date marked as day 0. After even grouping, the drug administration was administered once daily for two consecutive months. Behavioral experiments were then conducted to validate the results. The experiment ended after all behavioral experiments were completed, at which point the animals were euthanized, and serum, cerebrospinal fluid, and other tissue samples were collected.

[0163] (3) Animal grouping

[0164] po: oral

[0165] sc: subcutaneous

[0166] (4) Compound information and configuration

[0167] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0168] (5) Research process

[0169] Weight measurement: Monitor and record weight daily during the first week, and then record weight information once a week (Monday).

[0170] Test compound treatment: once daily.

[0171] Behavioral tests: Open Field Test (OFT), Novel Object Recognition Test (NOR), Y-maze Test, Morris Water Maze Test (MWM):

[0172] Behavioral tests were conducted 2 months after administration, and 1 hour after administration.

[0173] Open Field Test (OFT) Procedure and Results:

[0174] 1) One hour before the test, allow the designated mice to enter the laboratory to acclimatize to the environment;

[0175] 2) Clean the open field box area with 70% ethanol and paper towels before testing;

[0176] 3) Remove the experimental mice from the cage and place them in the center of the open field box. Experimenters should leave the area around the open field box to avoid disturbing the mice.

[0177] 4) Allow each experimental mouse to move freely in the open field box for 10 minutes;

[0178] 5) At the end of the experiment, remove the mice from the open field box and return them to their cages;

[0179] 6) Before conducting the experiment on the next animal, clean the entire open field box area with 70% ethanol and paper towels or cloths.

[0180] 7) After all the steps are completed, conduct experimental analysis.

[0181] The results of the central area movement distance, time, number of wall climbs, and statistical differences in the open field test are shown in Figure 10 and Table 10-12.

[0182] Table 10: Distance traveled in the central region of the open field test and statistical differences

[0183] Table 11: Dwell Time and Statistical Differences in the Central Region of the Open Field Test

[0184] Table 12: Number of wall climbing attempts and statistical differences in the open field test

[0185] The open field test paradigm utilizes the natural tendency of mice to explore by walking along walls in open areas. By observing the distance and time mice travel in the central zone within a fixed period, and the number of times they climb walls, the cognitive exploration ability of the animals is assessed. Animals exhibiting cognitive impairment phenotypes show a significant decrease in their cognitive exploration ability in unfamiliar environments. This experiment used transgenic model animals with typical cognitive impairment phenotypes, manifested as a significant decrease in their cognitive exploration ability compared to the control group. The animals' cognitive exploration ability was quantified using "Distance in the Central Zone," "Time in the Central Zone," and "Rearing Number." The results of the central zone distance, time, and number of wall climbs in the open field test revealed a clear cognitive impairment phenotype in the model group animals. In contrast, compound 1 significantly improved STZ-induced memory impairment in animals and enhanced their cognitive exploration ability.

[0186] Novel Object Recognition (NOR) Experiment Procedure and Results:

[0187] The experimental procedure is the same as in Example 2 above. Figure 3 is a diagram of the new object recognition experimental paradigm.

[0188] The results of the resolution percentage and statistical differences in the new object recognition experiment are shown in Figure 11 and Table 13.

[0189] Table 13: Resolution ratio and statistical differences in the new object recognition experiment

[0190] The novel object recognition experiment paradigm leverages the natural preference of mice for novel objects, training them to recognize and remember different objects, thereby demonstrating their learning, memory, and discrimination abilities. Animals exhibiting cognitive impairment phenotypes show a significant decline in their ability to remember and distinguish between new and old objects. This experiment utilizes transgenic model animals with typical memory and cognitive impairment phenotypes, manifested in a significantly reduced ability to distinguish novel objects compared to the control group. The animals' memory and discrimination abilities for new and old objects are quantified using the "discrimination ratio (DR)" and "discrimination index (DI)". The discrimination ratio (DR) is calculated as: (New object exploration time / (New object exploration time + Old object exploration time) * 100), and the discrimination index (DI) is calculated as: (New object exploration time - Old object exploration time) / (New object exploration time + Old object exploration time) * 100. The results of the discrimination ratio and discrimination index in the novel object recognition experiment reveal a clear memory impairment phenotype in the model group animals. In comparison, compound 1 has a certain tendency to improve STZ-induced memory impairment in animals and enhance their memory discrimination ability.

[0191] Y-maze process and results:

[0192] The experimental procedure was the same as in Example 2 above. The results of the correct selection rate and statistical differences in the Y-maze experiment are shown in Figure 12 and Table 14.

[0193] Table 14: Correct selection rate and statistical differences of animals in the Y-maze

[0194] Compared with the control group, the model group animals showed significant memory impairment, manifested as a statistically significant decrease in the accuracy of their path selection in the Y-maze, consistent with the conclusion that transgenic model animals can exhibit dementia-related memory impairment. Compared with the model group, compound 1 significantly improved the correct selection rate in the Y-maze in the experimental animals, with a statistically significant difference. These results suggest that compound 1 has a significant effect on improving working memory and spatial memory in animals.

[0195] Morris water maze experiment procedure and results:

[0196] Training phase:

[0197] The training phase is the same as in Example 2 above.

[0198] Testing phase (Day 5)

[0199] Disassemble the platform and place the animal in the 3rd quadrant of the pool. Allow the mouse to freely explore the maze for 1 minute.

[0200] After drying the animal with paper towels and a heat lamp, put it back in its cage. Move on to the next animal. Repeat the same process.

[0201] The results and statistical differences of the water maze experiment are shown in Figures 13-14 and Tables 15-18.

[0202] Table 15: Platform escape time and statistical differences of animals during water maze training.

[0203] Table 16: Time and statistical differences in animals reaching the platform during the water maze test period

[0204] Table 17: Number of times animals crossed platforms and statistical differences during the water maze test period.

[0205] Table 18: Time spent in the target quadrant and statistical differences of animals during the water maze test period

[0206] The Morris water maze test was used to examine the spatial memory abilities of the animals in each group. The results showed that the 5XFAD model group exhibited significant spatial memory impairment. Specifically, during the water maze training phase, these animals were unable to identify and remember spatial paths, and the time taken to find the hidden underwater platform did not decrease with further learning. Animals treated with Compound 1 showed significant improvement in spatial memory. Specifically, the time taken to find the underwater platform gradually decreased with prolonged training. From the second day onwards, the difference in platform-finding time between the Compound 1-treated group and the model group was statistically significant. During the test period, the results for platform arrival time, number of platform crossings, and time to the target quadrant were all statistically significant between the Compound 1-treated group and the model group.

[0207] Example 4: The effect of the compound of the present invention on improving vascular dementia in an animal model.

[0208] (1) Animal model

[0209] Male SD rats (250-280g) were purchased from a certified animal supplier (Vitol Dermatology) and fed for one week prior to the experiment. Animals were housed under standard conditions: room temperature 21-23°C, relative humidity 30-70%, and a 12h:12h light-dark cycle. Food and water were provided freely.

[0210] An animal model of ischemic dementia was induced by ligation of both common carotid arteries (CCA). The specific method is as follows:

[0211] The equipment used included: surgical scissors, surgical forceps, ophthalmic scissors, ophthalmic forceps, sutures (4-0), ligation sutures (5-0), needle holders, hemostatic forceps, arterial clamps, suture plugs, and a razor. A surgical light (25W) and a rat operating table were also provided. Isoflurane or a similar anesthetic, an anesthesia machine, and an anesthesia mask were also available. Petri dishes, cotton swabs, cotton balls, iodine tincture, 75% alcohol, and physiological saline were also included.

[0212] Animal preparation was as follows: Male SD rats weighing 250-280g were selected. They were grouped according to body weight (ensuring minimal weight variation within each group; animals with a weight deviation greater than ±20% were excluded), with 4 animals per cage. The cage tags were labeled with the corresponding group for each rat in the cage.

[0213] Preoperative preparation: Sterilize laboratory instruments and surgical equipment (with outer packaging required), and use disposable sterile supplies if necessary.

[0214] Clean and disinfect the operating environment (space, countertops, etc.).

[0215] Anesthesia and Fixation: The animal was weighed and anesthetized. After anesthesia, it was fixed in a supine position on the operating table. Routine skin preparation and disinfection were performed, and the animal was kept warm throughout the surgery. The rat's incisors were hooked and fixed with sutures to stretch the neck and facilitate subsequent surgical procedures.

[0216] Using standard ophthalmic scissors or a scalpel, an incision is made in the middle of the neck. Forceps are used to bluntly separate the cervical gland tissue and fascia, exposing and separating the bilateral common carotid arteries (CCA).

[0217] The bilateral common carotid arteries were ligated using sutures.

[0218] After ligation and disinfection, the subcutaneous tissue and skin are sutured layer by layer. Once the animal has recovered from anesthesia, it can be returned to its cage.

[0219] The steps for making the surgical model are the same as before, but the blood vessels are not ligated. After ligation and disinfection, the subcutaneous tissue and skin are sutured.

[0220] Postoperative care: First, place the animal on a heating pad to keep it warm, and then put it back in its cage after it has fully recovered.

[0221] (2) Research time

[0222] The study timeline is shown in Figure 15. Male SD rats weighing 250-280g were used for the modeling experiment. During the environmental adaptation period, animals were grouped evenly according to their weight, with the grouping date marked as day 0. After even grouping, the animals underwent modeling, and bilateral common carotid arteries were ligated. Post-surgery, the same nursing procedures were followed, and the animals were housed in the same environment. Drug administration was administered once daily for 30 consecutive days. Behavioral experiments were then conducted to verify the results. The experiment ended at the completion of all behavioral experiments, at which point the animals were euthanized, and serum, cerebrospinal fluid, and other tissue samples were collected.

[0223] (3) Animal grouping

[0224] IP: Abdominal cavity

[0225] (4) Compound information and configuration

[0226] Preparation method of mirtazafil: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0227] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0228] (5) Research process

[0229] Weight measurement: Monitor and record weight daily during the first week, and then record weight information once a week (Monday).

[0230] Test compound treatment: once daily.

[0231] Morris water maze experiment:

[0232] Behavioral tests were conducted one month after the model was established and the tests were conducted one hour after administration.

[0233] Morris water maze experiment procedure and results:

[0234] The experimental procedure is the same as in Example 2 above.

[0235] The results and statistical differences of the water maze experiment are shown in Figure 16-17 and Table 19-21.

[0236] Table 19: Platform escape time and statistical differences of animals during water maze training.

[0237] Table 20: Time and statistical differences in animal arrival at the platform during the water maze test period

[0238] Table 21: Number of times animals crossed platforms during the water maze test and statistical differences

[0239] The Morris water maze test was used to examine the spatial memory abilities of the animals in each group. The results showed that the model group animals exhibited significant spatial memory impairment after vascular dementia modeling. Specifically, during the water maze training phase, these animals were unable to identify spatial paths, and the time taken to find the hidden underwater platform did not decrease with further learning. The positive control drug mirtazafil showed significant improvement. Animals treated with compound 1 showed some improvement in spatial memory; specifically, the time taken to find the underwater platform gradually decreased with prolonged training. At the fourth day, the difference in platform-finding time between the compound 1 group and the model group was statistically significant. During the test period, the model group animals showed significant spatial memory impairment based on platform arrival time and number of platform crossings. Compared to the model group, both mirtazafil and compound 1 significantly improved the memory impairment in the modeled animals.

[0240] Example 5: The effect of the compound of the present invention in combination with donepezil and other substances on the improvement of dementia in an animal model.

[0241] (1) Animal model

[0242] C57BL / 6j (male, 6 months old) was purchased from a certified animal supplier (Vitollife) and fed for one week prior to the experiment. The animals were housed under standard conditions: room temperature 21-23°C, relative humidity 30-70%, and a 12h:12h light-dark cycle. Food and water were provided freely.

[0243] The surgical modeling experiment simulating sporadic Alzheimer's disease (AD) in animals was conducted by stereotactic injection of streptozotocin (STZ) into the brain (STZ-ICV). The specific method is as follows:

[0244] The equipment used includes: stereotaxic instrument, skull drill, 10ul micro-injection needle, surgical scissors, surgical forceps, sutures (5-0), needle holder, razor, petri dish, cotton swabs, iodine tincture, 75% alcohol, physiological saline, STZ, and tribromoethanol.

[0245] Animal preparation was as follows: Six-month-old male C57 / 6j mice were selected. They were grouped according to their weight (ensuring minimal weight difference within each group; animals with a weight deviation greater than ±20% were excluded), with 4 animals per cage, and the cage tags indicating the group corresponding to each cage.

[0246] Preoperative preparation: Sterilize laboratory instruments and surgical equipment (with outer packaging required), and use disposable sterile supplies if necessary.

[0247] Clean and disinfect the operating environment (space, countertops, etc.).

[0248] Anesthesia and Fixation: Mice were anesthetized by intraperitoneal injection of tribromoethanol (20 ml / kg). The animals entered a state of deep anesthesia approximately 5 minutes later. The hair on the skull was shaved clean, and the mice were fixed to the adapter. Eye ointment was applied to the eyes. Both ear rods were fixed to the external auditory canals of the mice, finely adjusted to ensure the scale on both ear rods was aligned, and the screws were tightened to center the mouse's head. The mouse's incisors were fixed to the incisor retainer on the adapter; the incisor clamps should not be tightened too much.

[0249] Rat brain balancing: Exposing the skull: Disinfect the mouse scalp with iodine tincture, then cut a 2cm long incision in the scalp with scissors, and use a dry cotton ball or hydrogen peroxide to corrode and wipe away the periosteum on the surface of the skull to fully expose the skull.

[0250] Positioning the anterior fontanelle: Move the injection needle tip to the anterior part of the midline, contacting the skull surface. Using the anterior fontanelle (bregma point) as a reference point, stop the needle as soon as it touches the surface, and reset the X, Y, and Z axis coordinate readings of the digital display to zero. Observe the needle tip position while inserting the needle to avoid breaking it!

[0251] Left-right leveling: Lift the syringe slightly upwards to avoid touching the skull surface. Then, using the bregma as the midpoint, move it left and right by the same distance (3.0mm / 2.5mm / 2mm recommended) and lower the syringe to contact the skull surface. Read the Z-coordinate of the corresponding point. Use the Z-coordinate values ​​to determine if the left and right sides are level. If the readings differ by less than 0.03mm, the mouse head is considered level. Level the left and right sides by adjusting the height of the ear rods on both sides. During the adjustment process, the bregma zero point must be repositioned after moving the ear rods. Anterior-posterior leveling: Level the anterior and posterior fontanelles using the same method as left-right leveling.

[0252] STZ Injection: Craniotomy: After leveling, use a digital display to locate your target brain region. Taking ICV coordinates as an example, AP (Y-axis): -0.92-0.96mm, ML (X-axis): ±0.22-0.28mm, DV (Z-axis): -2.35mm. Move the Y-axis backward by 0.96mm and the X-axis to the right by 0.26mm. Insert the needle and stop the needle the instant it touches the skull surface. Observe the skull surface features and remember or mark them. Lift the needle to a higher position and then use a cranial drill to drill a hole at the target site to expose the brain tissue.

[0253] To draw up STZ: Turn on the control pump, set the required volume, speed and mode, immerse the needle tip below the liquid surface, and press start to begin drawing up the liquid. The entire process should be performed in the dark.

[0254] Injection: Move the syringe to the target brain region and begin needle insertion according to the coordinates. The insertion process must be slow enough to reach the target brain region depth. Set the volume, speed (generally 1 μL / min), and mode, then press start to inject STZ. After injection, stop the needle for 5 minutes, then slowly lift the needle. The entire process must be performed in the dark.

[0255] Postoperative care: Suture the head skin with medical needles and sutures and disinfect. Place the animal on a heating pad to keep it warm until it is fully awake, then return it to its cage.

[0256] (2) Research time

[0257] The study timeline is shown in Figure 18. Six-month-old male C57BL6J mice were used for the modeling experiment. During the environmental adaptation period, animals were grouped uniformly according to their weight, with the grouping date marked as day 0. After uniform grouping, the animals were immobilized using a stereotactic injection device to establish the model, and an equal volume of physiological saline or streptozotocin was injected into the lateral ventricle. The same nursing procedures were followed post-surgery, and the animals were housed in the same environment. Drug administration was administered once daily for 30 consecutive days. Behavioral experiments were then conducted to validate the results. The experiment ended at the completion of all behavioral experiments, at which point the animals were euthanized, and serum, cerebrospinal fluid, and other tissue samples were collected.

[0258] (3) Animal grouping

[0259] po: oral

[0260] sc: subcutaneous

[0261] (4) Compound information and configuration

[0262] Streptozotocin (STZ) preparation method: Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0263] Donepezil preparation method: Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0264] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0265] (5) Research process

[0266] Weight measurement: Monitor and record weight daily during the first week, and record weight information once a week thereafter.

[0267] Test compound treatment: once daily.

[0268] Morris water maze experiment:

[0269] Behavioral tests were conducted one month after the model was established and the tests were conducted one hour after administration.

[0270] The Morris water maze experiment procedure is as follows:

[0271] Training phase:

[0272] Day 1: Randomly place the animal in one of the other three quadrants, at the midpoint of the pool wall, facing the wall. Let the animal swim freely in the pool. If the animal finds a platform within one minute and stays on it for 10 seconds, release the animal; otherwise, use a glass or plastic rod to guide the animal to the platform, let it stay for 10 seconds, and then release it. Dry the animal with paper towels and a heat lamp, then return it to its cage. Start with the next animal. Repeat the same process.

[0273] After the last animal in the current round is finished, start again from the first animal. Train each animal twice a day, and repeat the operation of day 1 for days 2-5.

[0274] Testing Phase (Day 6)

[0275] Disassemble the platform and place the animal in the 3rd quadrant of the pool. Allow the mouse to freely explore the maze for 1 minute.

[0276] After drying the animal with paper towels and a heat lamp, put it back in its cage. Move on to the next animal. Repeat the same process.

[0277] The results and statistical differences of the water maze experiment are shown in Figures 19-20 and Tables 22-26.

[0278] Table 22: Platform escape time and statistical differences of animals during water maze training.

[0279] Table 23: Time and statistical differences in animals reaching the platform during the water maze test period

[0280] Table 24: Number of times animals crossed platforms and statistical differences during the water maze test period.

[0281] Table 25: Distances traversed by animals in the target quadrant and statistical differences during the water maze test period.

[0282] Table 26: Time spent in the target quadrant and statistical differences of animals during the water maze test period

[0283] The Morris water maze experiment was used to examine the spatial memory abilities of the animals in each group. The results showed that the model group exhibited significant spatial memory impairment, specifically, during the water maze learning phase, these animals were unable to identify and remember spatial paths, and the time taken to find the hidden underwater platform did not decrease with further learning. Donepezil, an Alzheimer's disease treatment drug, was a positive result in this group, effectively alleviating the spatial memory impairment induced by the STZ model, showing a statistically significant difference compared to the model group from the fourth day's testing point. Animals treated with a combination of donepezil and compound 1 showed significant improvement in spatial memory; specifically, the time taken to find the underwater platform gradually decreased with prolonged training time, and the difference in platform finding time between the compound 1-treated group and the model group was statistically significant at the second day's testing point. During the test period, the escape latency time, number of platform crossings, target quadrant movement distance, and dwell time all indicated significant spatial memory impairment in the model group. Compared with the model group, both donepezil group and combination drug group significantly improved memory impairment in animals after modeling, and the combination drug group was superior to donepezil group.

[0284] Example 6: The effect of the compound of the present invention on stroke improvement in an animal model.

[0285] (1) Animal model

[0286] Male SD rats (250-280g) were purchased from a certified animal supplier (Vitol Dermatology) and fed for one week prior to the experiment. Animals were housed under standard conditions: room temperature 21-23°C, relative humidity 30-70%, and a 12h:12h light-dark cycle. Food and water were provided freely.

[0287] The surgical modeling experiment of simulated stroke in animals induced by ischemia-reperfusion injury (MCAO) is as follows:

[0288] Surgical scissors, surgical forceps, ophthalmic scissors, ophthalmic forceps, sutures (4-0), ligation sutures (5-0), needle holders, hemostatic forceps, arterial clamps, suture plugs, razor. Surgical lamp (25W), rat operating table. Isoflurane or similar anesthetic, anesthesia machine and anesthesia mask. Petri dishes, cotton swabs, cotton balls, iodine tincture, 75% alcohol, physiological saline. Animal preparation: Select male SD rats weighing 250-280g. Group according to weight (ensure minimal weight difference within each group; animals with a weight deviation greater than ±20% are excluded), with 4 animals per cage, and the cage tag indicating the group corresponding to the rat in that cage.

[0289] Preoperative preparation: Sterilize laboratory instruments and surgical equipment (with outer packaging required), and use disposable sterile supplies if necessary.

[0290] Clean and disinfect the operating environment (space, countertops, etc.).

[0291] Anesthesia and Fixation: The animal was weighed and anesthetized. After anesthesia, it was fixed in a supine position on the operating table. Routine skin preparation and disinfection were performed, and the animal was kept warm throughout the surgery. The rat's incisors were hooked and fixed with sutures to stretch the neck and facilitate subsequent surgical procedures.

[0292] Using standard ophthalmic scissors or a scalpel, an incision is made along the midline of the neck. Forceps are used to bluntly dissect the cervical glandular tissue and fascia, exposing and separating the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Both common carotid arteries are then ligated with sutures.

[0293] Isolate the ICA and its extracranial branch, the pterygopalatine artery, and ligate the pterygopalatine artery by tying a slipknot with sutures, or directly clamp the pterygopalatine artery with vascular clamps to prevent suture embolism from entering the pterygopalatine artery, thus keeping the ICA as the only open branch of the CCA.

[0294] Separate the ECA trunk and ligate the distal and proximal ends of the external carotid artery (leaving a long suture end), spaced 5mm apart. Cut the artery between the two ligation points. Then gently pull the proximal suture end to align this section of artery with the course of the common carotid artery.

[0295] Use micro-arterial clamps to clamp the proximal end of the bifurcation of the common carotid artery and external carotid artery. Then, make a small incision in the remaining segment of the external carotid artery and insert a pre-prepared suture up to the beginning of the middle cerebral artery, inserting the suture 17-18mm.

[0296] After 90 minutes, reperfusion is performed, the suture is removed, and the head is moved to the external carotid artery, allowing blood flow from the common carotid artery to reperfuse into the middle cerebral artery.

[0297] After ligation and disinfection, the subcutaneous tissue and skin are sutured layer by layer. Once the animal has recovered from anesthesia, it can be returned to its cage.

[0298] The steps for making the surgical model are the same as before, but after separating the blood vessels, no suture plug is inserted. After ligation and disinfection, the subcutaneous tissue and skin are sutured.

[0299] (2) Research time

[0300] The study timeline is shown in Figure 21. Male SD rats weighing 250-280g were used for the modeling experiment. Following surgery, all animals received the same nursing care and were housed in the same environment. Postoperatively, animals were grouped according to their appearance scores. The drug administration experiment was conducted once daily for 14 consecutive days. Afterward, the animals were euthanized, and brain sections were collected for TTC staining.

[0301] (3) Animal grouping

[0302] IP: Abdominal cavity

[0303] (4) Compound information and configuration

[0304] Edaravone preparation method: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0305] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0306] (5) Research process

[0307] Neurological and behavioral assessments were performed on the animals after they regained consciousness following ischemic surgery.

[0308] ① No neurological deficits: 0 points;

[0309] ②The forepaw on the paralyzed side cannot be fully extended: 1 point;

[0310] ③ When walking, turn in circles towards the paralyzed side: 2 points;

[0311] ④ When walking, lean towards the paralyzed side: 3 points;

[0312] ⑤ Unable to walk automatically, exhibiting signs of loss of consciousness: 4 points.

[0313] The higher the score, the more severe the behavioral disorder in the animal. Criteria for successful establishment of the rat model: the rat exhibits symptoms such as hemiplegia, contralateral forelimb drooping, and unsteady gait.

[0314] Test compound treatment: once daily.

[0315] TTC staining:

[0316] The rat brain was quickly removed, rinsed with cold saline, and placed in a -20°C freezer for 10 minutes until it hardened slightly. The olfactory bulb, pituitary gland, and lower brainstem were then removed, and the brain was sectioned coronally from anterior to posterior. The sections were evenly cut into 2mm thick slices, each slice divided into 5 equal parts, and placed in a 2% TTC solution. The slices were incubated at 37°C in the dark for 30 minutes, turning the brain approximately every 5 minutes. TTC reacts with the dehydrogenase system in normal tissue and is reduced to rose-red, thus staining normal tissue rose-red and infarcted tissue white.

[0317] Brain slices were taken after staining, and the infarct area was calculated using Imagej software.

[0318] See Figure 22 and Table 27 for results and statistical differences.

[0319] Table 27: Infarct area and statistical differences in TTC staining of animal brain slices

[0320] Compared with the control group, the model group showed a significant increase in infarct area. Compared with the model group,

[0321] Both edaravone and compound 1 can improve the infarct area in animals after modeling.

[0322] Example 7: Protective effect of the compound of the present invention on neurons in an in vitro cellular model of 6-OHDA-induced injury.

[0323] (1) Cell culture and neuronal directional differentiation

[0324] SH-SY5Y (human neuroblastoma cell line, Procell, CL-0208) cells were cultured under the following conditions: complete medium: DMEM / F12 (1:1) medium, 10% FBS and 1% pen / strep, in a 37°C, 5% CO2 incubator. After 48 hours of culture in complete medium, the medium was changed to induce differentiation into neuronal cells. The medium was then changed to Neurobasal medium (containing B27 supplement and GlutaMAX) and 10 μM all-trans-retinoic acid (ATRA), with a half-medium change every 24 hours. Maintaining these conditions for 7 days yielded cultured and differentiated neuron-like cells.

[0325] (2) Model establishment and compound preparation

[0326] Ascorbic acid was dissolved in physiological saline to prepare a 0.1% solution. 6-OHDA was then prepared as a 100 mM stock solution using the 0.1% ascorbic acid solution and stored in the dark. 6-OHDA was diluted to 40 μM using neurobasal medium to establish a cell damage model. The compound was then prepared to the desired final molar concentration using a medium containing 40 μM 6-OHDA and incubated in a cell culture incubator for 18 hours.

[0327] (3) CellTiter-Lumi luminescence assay for cell viability detection

[0328] Add 100 μL of CTL and complete culture medium to each well at a 1:1 ratio and incubate for 10 min. Centrifuge at 1000 rpm for 1 min at room temperature, collect the fluorescence signal using a microplate reader, and then perform data processing.

[0329] (4) Data Analysis

[0330] The 6-OHDA treatment group was used as the model group, and its CTL activity data were normalized. The results of all compound treatments were compared with the model group data using unpaired t-test and Mann-Whitney test. Data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ****p<0.0001). The results are shown in Figure 23 and Table 28 below.

[0331] Table 28: Relative values ​​and statistical differences of the proportion of increased cell viability in each group in the 6-OHDA-damaged cell model

[0332] Compared with the blank control group, pramipexole, compounds 1 and 2 all enhanced cell activity damaged by 6-OHDA, and the phenylpyrimidinone derivatives exhibited a stronger effect in enhancing cell activity and neuroprotection. Therefore, the various phenylpyrimidinone derivatives in this patent have a protective effect against Parkinson's disease-related cell damage.

Claims

1. Use of a phenylpyrimidinone compound of formula (I) or its tautomer, pharmaceutically acceptable salt, solvate, or isotopic label thereof in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases: wherein, R 1 and R 2 each independently is selected from the group consisting of halogen and Ci-C6-alkyl, preferably from the group consisting of bromine, methyl, ethyl, propyl and isopropyl; R 3 selected from the group consisting of C1-C6-alkyl, preferably selected from the group consisting of ethyl and propyl; R 4 -L-R 5 ; L is -SO2-, -CO- or -NHCOCH2-; R 5 selected from the group consisting of: and hydroxy C1-C6 alkylamino.

2. Use according to claim 1, characterized in that, The phenylpyrimidinone compound is selected from the following compounds:

3. Use according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salt is selected from the group consisting of hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, malate, citrate, succinate, maleate, fumarate and oxalate.

4. Use according to claim 3, characterized in that: The pharmaceutically acceptable salt further contains 0.5-3 molecules of crystal water; preferably, 1-2 molecules of crystal water, and more preferably, 1 molecule of crystal water.

5. Use of a pharmaceutical composition in the preparation of a medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular disease, and / or for improving or treating degenerative disease, the pharmaceutical composition comprising a therapeutically effective amount of a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled material thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

6. Use according to claim 5, characterized in that: The pharmaceutical composition is an oral pharmaceutical composition or a topical pharmaceutical composition or an injectable pharmaceutical composition; in particular, when used orally, the pharmaceutical composition is a tablet, a powder, a mouth dissolving film or a capsule; when used topically, the pharmaceutical composition is a spray, a liniment, a paste, a patch; when used for injection, the pharmaceutical composition is an injection solution, wherein the content of the active ingredient is 0.1% to 99.5% (by weight).

7. Use according to claim 1 or 5, characterized in that: The cognitive impairment is selected from the group consisting of Alzheimer's disease, vascular dementia, mild cognitive impairment, mixed dementia, frontotemporal dementia, Lewy body dementia, Parkinson's disease dementia and other types of dementia.

8. Use according to claim 1 or 5, characterized in that: The cognitive impairment is selected from the group consisting of Alzheimer's disease, vascular dementia and mixed dementia.

9. Use according to claim 1 or 5, characterized in that: The cerebrovascular disease is selected from the group consisting of ischemic cerebrovascular disease, cerebral small vessel disease, stenosis or occlusion of the head and neck arteries (not resulting in cerebral infarction), hypertensive encephalopathy, cerebral arterial malformations (moyamoya disease), dissection of the head and neck arteries, reversible cerebral vasoconstriction syndrome, primary central nervous system vasculitis, intracranial venous thrombosis, acute focal, functional loss-free cerebrovascular disease, post-stroke sequelae, and other cerebrovascular diseases.

10. Use according to claim 9, characterized in that: The cerebrovascular disease is ischemic stroke or cerebral small vessel disease.

11. Use according to claim 1 or 5, characterized in that: The degenerative disease is Parkinson's disease.

12. Use according to claim 1 or 5, characterized in that: The medicament for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular disease, and / or for improving or treating degenerative disease further comprises other active ingredients selected from one or more of the group consisting of medicaments for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular disease, and / or for improving or treating Parkinson's disease.

13. Use according to claim 12, wherein, The other active ingredient is selected from one or more of donepezil, galantamine, benzylgalantamine, rivastigmine, memantine, lanepitant, donapant, manntria, edaravone, Remternetug, huperzine A, VG-3927, AXS-05, butylphthalide, tenecteplase, alteplase, tirofiban, abicimab, rivaroxaban, apixaban, dabigatran, clopidogrel, aspirin, pramipexole, ropinirole, rotigotine, safinamide, opicapone, levodopa, madopar, carbidopa, and the like.

14. A pharmaceutical combination comprising a phenylpyrimidinone compound of Formula (I) or a tautomer, a pharmaceutically acceptable salt, solvate, or isotopically-labeled material thereof, wherein, R 1 to R 5 and L are as defined in claim 1, and, The other active ingredient is selected from one or more of donepezil, galantamine, benzylgalantamine, rivastigmine, memantine, lanepitant, donapant, manntria, edaravone, Remternetug, huperzine A, VG-3927, AXS-05, butylphthalide, tenecteplase, alteplase, tirofiban, abicimab, rivaroxaban, apixaban, dabigatran, clopidogrel, aspirin, pramipexole, ropinirole, rotigotine, safinamide, opicapone, levodopa, madopar, carbidopa, and the like.

15. The pharmaceutical combination of claim 14, wherein, The other active ingredient is selected from one or more of donepezil, galantamine, benzylgalantamine, rivastigmine, memantine, lanepitant, donapant, manntria, edaravone, Remternetug, huperzine A, VG-3927, AXS-05, butylphthalide, tenecteplase, alteplase, tirofiban, abicimab, rivaroxaban, apixaban, dabigatran, clopidogrel, aspirin, pramipexole, ropinirole, rotigotine, safinamide, opicapone, levodopa, madopar, carbidopa, and the like.

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