Application of phenyl pyrimidone compound

Abstract

The invention relates to application of a phenyl pyrimidone compound, in particular to application of the phenyl pyrimidone compound shown in a formula (I) or tautomers, pharmaceutically acceptable salts, solvates or isotope labels of the phenyl pyrimidone compound in preparation of drugs for improving or treating cognitive impairment and / or improving or treating brain vascular diseases. Through cell model and animal model test research, compared with a clinically available drug donepezil on the market and a clinically researched drug mironafil, the compound disclosed by the invention has remarkable treatment characteristics and substantive technical progress in the aspects of protecting nerve cells, improving cognitive impairment, improving brain vascular diseases, improving degenerative diseases such as Parkinson's disease and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of a phenylpyrimidinone compound. BACKGROUND

[0002] With China's rapid entry into an aging society and the improvement of average life expectancy, the rapid increase of disability and disease related to aging has brought a heavy burden to society and family. Among them, cognitive impairment and cerebrovascular disease are one of the most common diseases that lead to the loss of daily life ability of the elderly, seriously affecting the public health and sustainable development of society in China. Therefore, strengthening the prevention and treatment of cognitive impairment and cerebrovascular disease, improving or treating cognitive impairment and cerebrovascular disease, and reducing the burden on society and family are urgent public health problems and important contents of the national strategy to actively respond to population aging.

[0003] Cognitive impairment refers to cognitive impairment of various degrees caused by various reasons, from mild cognitive impairment to dementia. Mild cognitive impairment (MCI) and its subtypes refer to memory impairment and / or mild other cognitive impairment, but the individual's social occupation or daily life function is not affected, and it 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 clinical syndromes of severe cognitive impairment or decline caused by organic diseases, such as progressive thinking, memory, behavior and personality disorders, which can be accompanied by mental and motor function symptoms, and damage reaches the degree of affecting occupation, social function or daily life ability. There are various subtypes of dementia. The most common is etiological classification, which can be divided into degenerative disease and non-degenerative disease. Alzheimer's disease (AD) is the most common degenerative disease dementia with the highest incidence, accounting for 50-70% of all types of dementia; vascular dementia (VD) is the most common non-degenerative disease dementia, accounting for 15-20% of dementia patients. Degenerative disease dementia and vascular dementia coexist at the same time, which is called mixed dementia (Mixed Dementia). The classic performance is that AD patients develop cerebral infarction and new cognitive impairment with rapid onset. In addition, there are also frontotemporal dementia (FTD), dementia with Lewy bodies (DLB), Parkinson's disease dementia (PDD), etc. which also pose a serious threat to the health of patients. At present, the cholinesterase inhibitor represented by donepezil is used as a first-line drug to relieve symptoms in clinical practice, but there are varying degrees of cholinergic side effects. Therefore, there is an urgent need for the development of drugs to improve or treat cognitive impairment in clinical practice.

[0004] Cerebral vascular disease is a group of diseases that occur in the brain blood vessels, caused by intracranial blood circulation disorders and brain tissue damage. Cerebral stroke includes two major categories of ischemic stroke and hemorrhagic stroke, such as cerebral thrombosis, cerebral embolism, lacunar infarction, etc. belong to ischemic stroke, and the brain hemorrhage commonly said is belong to hemorrhagic stroke. Among them, ischemic stroke accounts for 75% to 90% of all cerebral stroke. Cerebral small vessel disease (CSVD) refers to a series of clinical, imaging and pathological syndromes caused by intracranial small artery, arteriole, capillary, venule and small vein lesions. Research has found that the number of lacunar infarction caused by CSVD accounts for 25% to 50% of ischemic stroke. CSVD is closely related to post-stroke depression, cognitive impairment, low quality of life, early Parkinson's disease symptoms, and brain hemorrhage hematoma enlargement. At present, the drugs for treating ischemic cerebrovascular disease in clinical mainly include anticoagulants such as clopidogrel, aspirin, dabigatran and statins for lipid regulation, but these may increase the risk of brain hemorrhage. Therefore, new drugs for preventing and treating cerebrovascular diseases need to be developed in clinical.

[0005] Parkinson's disease (PD) is a progressive neurodegenerative disease, with selective loss of dopaminergic neurons in the substantia nigra compacta and abnormal aggregation of alpha-synuclein (α-synuclein) as the core pathological features. There are more than 10 million patients worldwide, and the prevalence rate of people over 65 years old is 1-2%. With the intensification of aging, the disease burden is expected to double by 2040, and new treatment methods are urgently needed. SUMMARY

[0006] The inventors found that the phenylpyrimidinone compounds have neuroprotective effects in cell models, can improve or treat cognitive dysfunction in animal models, and can improve or treat cerebrovascular diseases on the basis of which the present application is proposed.

[0007] OBJECTIVE

[0008] An object of the present application is to provide the use of a phenylpyrimidinone compound in the preparation of a medicament for improving or treating cognitive dysfunction, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating Parkinson's disease and other degenerative diseases.

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

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

[0011] Another object of the present application is to provide a method for improving or treating cognitive dysfunction, and / or for 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 application, there is provided a use of a phenylpyrimidinone compound represented by Formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled material thereof, in the manufacture of a medicament for improving or treating cognitive dysfunction, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases such as Parkinson's disease:

[0014]

[0015] wherein,

[0016] R 1 and R 2 each independently is selected from the group consisting of halogen and C1-C6 alkyl, preferably from the group consisting of bromine, methyl, ethyl, propyl and isopropyl;

[0017] R 3 is selected from the group consisting of C1-C6 alkyl, preferably from the group consisting of ethyl and propyl;

[0018] R 4 is -L-R 5 ; L is -SO2-, -CO-, or -NHCOCH2-;

[0019] R 5 is selected from the group consisting of:

[0020] and hydroxy C1-C6 alkyl amino (e.g. hydroxypropylamino).

[0021] According to one embodiment of the present application, the phenylpyrimidinone compound is selected from the group consisting of:

[0022]

[0023] According to the present application, the salt of the phenylpyrimidinone compound represented by Formula (I) can be selected from one or more of hydrochloride, hydrobromide, sulfate, phosphate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, tartrate, malate, citrate, succinate, maleate, fumarate, and oxalate.

[0024] Preferably, the phenylpyrimidinone compound of formula (I) can contain 0.5-3 molecules of crystal water; more preferably, 1-2 molecules of crystal water, and most preferably, 1 molecule of crystal water.

[0025] According to the present application, there is provided a use of a pharmaceutical composition comprising the phenylpyrimidinone compound or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled form thereof, for the manufacture of a medicament for ameliorating or treating cognitive impairment, and / or for ameliorating or treating cerebrovascular disease, and / or for ameliorating or treating Parkinson's disease and other degenerative diseases, the pharmaceutical composition comprising a therapeutically effective amount of the compound or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled form thereof, and a pharmaceutically acceptable carrier; the carrier includes a diluent, an excipient such as water; a binder such as a cellulose derivative, gelatin, polyvinylpyrrolidone; a filler such as starch; a disintegrant such as calcium carbonate, sodium bicarbonate; a lubricant such as calcium stearate or magnesium stearate. In addition, other auxiliary agents such as a flavoring agent and a sweetening agent can be further added to the pharmaceutical composition.

[0026] Further, the pharmaceutical composition is an oral pharmaceutical composition or a topical pharmaceutical composition or an injectable pharmaceutical composition. The various dosage forms of the pharmaceutical composition according to the present application are prepared according to the conventional methods in the art, wherein the content of the active ingredient is 0.1% to 99.5% by weight.

[0027] When the pharmaceutical composition is an oral pharmaceutical composition, the pharmaceutical composition is a solid preparation, and preferably, the solid preparation is selected from a tablet, a powder, a mouth dissolving film, or a capsule.

[0028] When the pharmaceutical composition is a topical pharmaceutical composition, the dosage form of the pharmaceutical composition is selected from a spray, a liniment, a paste, a patch.

[0029] When the pharmaceutical composition is an injectable pharmaceutical composition, the dosage form of the pharmaceutical composition is selected from an injection solution, a dry powder for injection; preferably, the content of the active ingredient in the injectable composition is 0.1% to 99.5% by weight.

[0030] According to the present application, 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.

[0031] Preferably, the cognitive impairment is Alzheimer's disease, vascular dementia, and mixed dementia.

[0032] According to the present application, the brain vascular disease is selected from the group consisting of ischemic cerebrovascular disease, cerebral small vessel disease, head and neck atherosclerotic stenosis or occlusion (not leading to cerebral infarction), hypertensive encephalopathy, cerebral basal abnormal vascular network disease (moyamoya disease), head and neck arterial dissection, reversible cerebral vasoconstriction syndrome, primary central nervous system vasculitis, intracranial venous system thrombosis, non-acute focal, functional loss of cerebrovascular disease, sequelae of stroke, and other cerebrovascular diseases.

[0033] Preferably, the brain vascular disease is ischemic stroke or cerebral small vessel disease.

[0034] According to another aspect of the present application, it provides a use of a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled material thereof in combination with other active ingredients for the manufacture of a medicament for improving or treating cognitive dysfunction, and / or for improving or treating brain vascular disease, improving or treating Parkinson's disease, and the like; wherein the other active ingredients are selected from one or more of the drugs for improving or treating cognitive dysfunction, and / or for improving or treating brain vascular disease, improving or treating Parkinson's disease indications.

[0035] Preferably, it provides a use of a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled material thereof in combination with other active ingredients for the manufacture of a medicament for improving or treating cognitive dysfunction; wherein the other active ingredients are selected from one or more of the group consisting of donepezil, galantamine, benzyl galantamine, rivastigmine, memantine, lanicemast, donanemab, glysopentine, 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.

[0036] Preferably, it provides a use of a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically labeled material thereof in combination with other active ingredients for the manufacture of a medicament for improving or treating brain vascular disease; wherein the other active ingredients are selected from one or more of the group consisting of edaravone, butylphthalide, tenecteplase, alteplase, tirofiban, abicimab, rivaroxaban, apixaban, dabigatran, clopidogrel, aspirin, and the like.

[0037] Preferably, it provides a use of a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically-labeled material thereof in combination with other active ingredients for the manufacture of a medicament for ameliorating or treating a cognitive dysfunction, and / or for ameliorating or treating a cerebrovascular disease, and / or for ameliorating or treating a degenerative disease; wherein the other active ingredients are selected from one or more of pramipexole, ropinirole, rotigotine, safinamide, opicapone, levodopa, madopar, carbidopa, and the like.

[0038] According to some embodiments of the present application, the medicament for ameliorating or treating a cognitive dysfunction, and / or for ameliorating or treating a cerebrovascular disease, and / or for ameliorating or treating a degenerative disease comprises the other active ingredients.

[0039] According to another aspect of the present application, it provides a pharmaceutical combination comprising a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically-labeled material thereof,

[0040]

[0041] wherein,

[0042] R 1 to R 5 and L are defined as described above, respectively,

[0043] and,

[0044] other active ingredients are described above, respectively.

[0045] According to another aspect of the present application, it provides a therapeutic method for ameliorating or treating a cognitive dysfunction, and / or for ameliorating or treating a cerebrovascular disease, and / or for ameliorating or treating a degenerative disease such as Parkinson's disease, the method comprising administering to a subject 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, and optionally other active ingredients described above. The phenylpyrimidinone compound represented by formula (I) and other active ingredients are described above, respectively.

[0046] According to another aspect of the present application, it provides a phenylpyrimidinone compound represented by formula (I) or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically-labeled material thereof for use in ameliorating or treating a cognitive dysfunction, and / or for ameliorating or treating a cerebrovascular disease,

[0047]

[0048] each substituent is described above.

[0049] According to another aspect of the present application, there is provided a method for improving or treating cognitive dysfunction, and / or improving or treating cerebrovascular disease, the method comprising administering to a subject a therapeutically effective amount of a phenylpyrimidinone compound represented by formula (I) as described above, or a tautomer, a pharmaceutically acceptable salt, a solvate, or an isotopically-labeled version thereof, or a pharmaceutical composition comprising the same.

[0050] Advantages

[0051] The present application provides a novel use of phenylpyrimidinone compounds, which has the following advantages:

[0052] 1. The pyrimidinone phenyl derivatives described in the present application have a significant effect of improving or treating cognitive dysfunction.

[0053] 2. In terms of protecting nerve cells, the compounds of the present application have a stronger effect of improving cell activity and neuroprotection.

[0054] 3. In terms of improving cognitive function, the compounds of the present application can significantly improve memory impairment, improve memory discrimination, and have a significant effect of improving working memory and spatial memory.

[0055] 4. The compounds of the present application can enhance the effect of other active ingredients when used in combination with other active ingredients, such as enhancing the effect of donepezil in improving working memory and spatial memory.

[0056] 5. The compounds of the present application can improve vascular dementia.

[0057] 6. The compounds of the present application can improve stroke.

[0058] 7. Compared with the existing marketed drugs donepezil, pramipexole, and the clinical drug candidate miroanafil, the compounds of the present application have significant therapeutic characteristics and substantial technical progress in protecting nerve cells and improving cognitive dysfunction, as shown by different cell models and animal models. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 is a column chart of the relative values of cell activity in each group in different Aβ cell models of different compounds.

[0060] Figure 2 is a research time schematic diagram of the animal experiment of Example 2 of the present application.

[0061] Figure 3 is a new object recognition experiment paradigm diagram in the present application.

[0062] Figure 4 is a column chart of the percentage of discrimination in the new object recognition experiment of Example 2 of the present application.

[0063] Figure 5 is a bar chart of the resolution index of the new object recognition experiment of Example 2 of the present application.

[0064] Figure 6 is a bar chart of the Y-maze correct selection rate of Example 2 of the present application.

[0065] Figure 7 and 8 is a result graph of the water maze test experiment of Example 2 of the present application.

[0066] Figure 9 is a research time schematic diagram of the animal experiment of Example 3 of the present application.

[0067] Figure 10 is a bar chart of the central area movement distance, time and wall climbing times of the open field test of Example 3 of the present application.

[0068] Figure 11 is a bar chart of the resolution rate of the new object recognition experiment of Example 3 of the present application.

[0069] Figure 12 is a bar chart of the alternation rate of the Y-maze of Example 3 of the present application.

[0070] Figure 13 , 14 is a result graph of the water maze test experiment of Example 3 of the present application.

[0071] Figure 15 is a research time schematic diagram of the animal experiment of Example 4 of the present application.

[0072] Figure 16 , 17 is a result graph of the water maze test experiment of Example 4 of the present application.

[0073] Figure 18 is a research time schematic diagram of the animal experiment of Example 5 of the present application.

[0074] Figure 19 , 20 is a result graph of the water maze test experiment of Example 5 of the present application.

[0075] Figure 21 is a research time schematic diagram of the animal experiment of Example 6 of the present application.

[0076] Figure 22 is a result graph of the animal experiment of Example 6 of the present application.

[0077] Figure 23 is a bar chart of the relative values of the cell activity of each group on the neurons of the 6-OHDA-induced damage model of Example 7 of the present application. DETAILED DESCRIPTION

[0078] The present application is described in more detail by the following examples. Such examples are only for illustrating the present application in more detail, and the scope of the present application is not limited by these examples, which will be apparent to those skilled in the art, according to the gist of the present application.

[0079] Unless otherwise specified, the raw materials, reagents, apparatuses, methods, etc. used in the present application are conventionally available in the art.

[0080] Preparation of compounds: The compounds of formula (I) of the present application can be prepared according to the prior art known to those skilled in the art or according to WO2010066111A1. The compounds 1, 2, 5, 6 used in the examples of the present application are prepared according to the method disclosed in WO2010066111A1.

[0081] The data obtained were analyzed by Two-way ANOVA. Data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001, ns, no significance).

[0082] Example 1 Protective effect of the compounds of the present application on neurons at the in vitro cell level

[0083] (1) Cell culture and neuron directional differentiation

[0084] SH-SY5Y (human neuroblastoma cell line, Procell, CL-0208) cells were cultured in the following conditions: complete medium: DMEM / F12 (1:1) medium, 10% FBS and 1% pen / strep, in a 37-degree, 5% carbon dioxide incubator. After the cells were cultured in the complete medium for 48 hours, the medium was changed to induce differentiation into neuron cells. The medium was changed to Neurobasal medium (containing B27 supplement and GlutaMAX) and 10 μM all-trans-retinoic acid (ATRA), and half of the medium was changed every 48 hours. The differentiated neuron-like cells were obtained after 5 days of culture under the above conditions.

[0085] (2) Amyloid beta (Aβ) 1-42 oligomerization experiment

[0086] Amyloid beta (Aβ) 1-42 protein was dissolved in serum-free DMEM / F12 medium, configured into a 100 μM system, and placed in a 4-degree refrigerator for 24 hours or more, thereby forming Amyloid beta (Aβ) 1-42 oligomers.

[0087] (3) Amyloid beta (Aβ) 1-42 oligomer-treated cell-induced damage model

[0088] Amyloid beta (Aβ) 1-42 oligomers prepared in (2) were added to the differentiated cells in (1), with a final concentration of 10 μM, or added alone or with treatment of processing compounds (including control drugs donepezil, mirodenafil, and compounds 1, 2, 5, and 6 of the present application in this example), and CCK8 detection was performed after 48 hours (see Step 4 below).

[0089] (4) CCK8 treatment experiment

[0090] After cck-8 was mixed with the culture medium (phenol red-free DMEM / F12 + 10% FBS) at a ratio of 1:10, 100 ul was added to each well, incubated for 4 hours, and then the absorbance value (OD) at 450 nm was measured using an enzyme marker.

[0091] (5) Data analysis

[0092] The Amyloid beta (Aβ) 1-42 oligomer-treated group was used as the model group, and the CCK8 activity data were normalized and converted. The results of compound treatment were compared with the data of the model group, and unpaired t-test and Mann-Whitney test were used for analysis. The data were expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001). The results are shown in Figure 1 and Table 1 below.

[0093] Table 1: Relative values of cell activity improvement ratio and statistical differences in each group in the Aβ cell model

[0094]

[0095] From Figure 1As can be seen from Table 1 above, the treatment of neuron differentiated cells with Aβ1-42 oligomer can cause damage to the nerve cells and affect the survival activity thereof. The survival value of the cells in the treatment group is set to 1.0 by the homogenization method, and the relative values of the cell activity of the other compounds relative to the treatment group are obtained by the same method after treatment, and then the differences between different groups can be uniformly compared. The experimental results show that donepezil, as a positive drug for treating AD, can improve the cell activity after treatment with Aβ1-42 oligomer, and mirodenafil also has the effect of improving the cell activity. Compared with them, the various phenyl pyrimidinone derivatives have stronger effects of improving the cell activity and nerve protection, and the proportion of the improvement of the cell activity is higher than that of the donepezil group and the mirodenafil group under the same dosage. Therefore, the various pyrimidinone phenyl derivatives in the present patent have high nerve protection effects, especially for the cell damage caused by toxic proteins related to Alzheimer's disease.

[0096] Example 2: Improvement effect of the compound of the present application on dementia in STZ animal model

[0097] (1) Animal model

[0098] C57BL / 6j (male, 6 months old) was purchased from a certified animal supplier (Vivant Lab Animal) and was raised for 1 week before the experiment. The animals were raised under standard conditions, with room temperature of 21-23℃, relative humidity of 30-70%, and 12h:12h light-dark cycle. Food and water were available at will.

[0099] The animal model was induced by stereotactic injection of streptozotocin (STZ-ICV) to simulate the dementia model of sporadic AD patients, and the specific method was as follows:

[0100] The equipment used included a brain stereotaxic apparatus, a skull drill, a 10ul microsyringe, surgical scissors, surgical forceps, suture thread (5-0), needle holders, a shaving knife, a petri dish, a cotton swab, iodine tincture, 75% alcohol, normal saline, STZ, and tribromoethanol.

[0101] The animals were prepared as follows: 6-month-old male C57 / 6j mice were selected. According to the body weight, the animals were divided into groups (to ensure that the body weight difference in each group is not large, and animals with individual body weight deviation greater than ±20% were excluded), with 4 animals per cage, and the corresponding group of mice was marked on the cage label.

[0102] Preoperative preparation: The vessels, surgical equipment, etc. used in the experiment were sterilized (with an outer package), and disposable sterile supplies were used when necessary.

[0103] Clean and disinfect the operating environment (space, table top, etc.).

[0104] Anesthesia and fixation: Anesthetize the mouse with tribromoethanol (20 ml / kg) by intraperitoneal injection. After about 5 minutes, the animal enters a deep anesthetic state. Shave the skull with a shaver and fix the mouse on the adapter. Apply ophthalmic ointment to the eyes. Fix the bilateral ear bars in the mouse's external auditory canal. Fine-tune the left and right ear bar scales to be consistent and tighten the screws to make the mouse's head in the middle. Fix the mouse's incisors on the adapter incisor fixer. Do not tighten the incisor clamp too much.

[0105] Mouse brain leveling: Expose the skull: Disinfect the mouse's scalp with iodine tincture, then cut the scalp with scissors to make a 2 cm long incision. Use dry cotton balls or hydrogen peroxide to erode the galea on the surface of the skull to completely expose the skull.

[0106] Bregma fixation: Move the needle tip to the anterior part of the midline, touch the surface of the skull, and use the bregma point as the reference point. When the needle just touches the surface, stop the needle and set the X, Y, Z axis coordinate readings of the digital display instrument to zero. When lowering the needle, observe the position of the needle tip to avoid breaking the needle!

[0107] Left-right leveling: Lift the syringe slightly to avoid touching the surface of the skull, then move it left and right by the same distance (recommended 3.0 mm / 2.5 mm / 2 mm) with bregma as the midpoint and lower the needle to touch the surface of the skull. Read the corresponding Z coordinates respectively, and determine whether the left and right are level by the numerical value of the Z coordinates. If the readings differ by less than 0.03 mm, it is considered that the mouse's head is level left and right. Adjust the height of the ear bars to level left and right. During the adjustment process, the ear bars must be repositioned after moving.

[0108] STZ injection: Craniotomy: After leveling, start positioning your target brain area through the digital display instrument. For example, ICV coordinates: AP (Y axis): -0.92-0.96 mm ML (X axis): ±0.22-0.28 mm DV (Z axis): -2.35 mm, Y axis moves 0.96 mm backward, X axis moves 0.26 mm to the right, lower the needle, stop the needle at the moment of touching the surface of the skull, observe the characteristics of the skull surface and remember or make a mark, then raise the needle to a higher position, and use the skull drill to drill a hole at the target site to expose the brain tissue.

[0109] STZ suction: Turn on the control pump, set the required volume, speed and mode, immerse the needle tip below the liquid surface, press start to begin liquid suction, and avoid light during the entire process.

[0110] 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.

[0111] 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 the cage.

[0112] (2) Research time

[0113] Research time such as Figure 2 As shown. Six-month-old male C57BL6J mice were used for the modeling experiment. During the environmental adaptation period, the animals were divided into groups based on their weight, and the grouping time was marked as day 0. After uniform grouping, the animals were fixed in place 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 after surgery, and the animals were kept in the same environment. The drug administration experiment was conducted once a day for 30 consecutive days. After the drug administration was carried out, behavioral experiments were performed to verify the results. The animals were euthanized after all behavioral experiments were completed, and serum, cerebrospinal fluid, and various tissue samples were collected.

[0114] (3) Animal grouping

[0115]

[0116] po: oral

[0117] sc: subcutaneous

[0118] (4) Compound information and configuration

[0119] Preparation method of streptozotocin (STZ): Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0120] Donepezil preparation method: Weigh the required amount, add physiological saline, and vortex sonicate to obtain a homogeneous solution.

[0121] Preparation method of mirtazafil: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0122] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0123] (5) Research process

[0124] Body weight measurement: Body weight was monitored daily for the first week and then twice a week (Monday / Thursday) thereafter.

[0125] Test compound treatment: Test compound was administered once daily.

[0126] New object recognition experiment (NOR), Y-maze test, Morris water maze test:

[0127] Behavioral tests were performed one month after the administration of the model and 1 hour after the administration of the test compound.

[0128] The procedure for the new object recognition experiment (NOR) was as follows:

[0129] 1) One hour before the test, the designated mice were allowed to acclimate to the laboratory environment;

[0130] 2) The same toy object was fixed in the open field;

[0131] 3) Each test mouse was allowed to freely explore the open field box for 10 minutes, and the mouse was recorded;

[0132] 4) At the end of the test, the mouse was removed from the open field box and returned to the cage;

[0133] 5) One of the two identical toy objects was replaced with a new object, and the mouse was recorded for 10 minutes;

[0134] 6) This cycle was repeated until all animals completed the new object exploration;

[0135] 7) Experimental analysis.

[0136] Figure 3 is a diagram of the new object recognition experiment paradigm.

[0137] The results of the discrimination percentage and statistical differences in the new object recognition experiment are shown in Figure 4 and Table 2 below.

[0138] Table 2: Discrimination percentage and statistical differences in the new object recognition experiment

[0139]

[0140] The results of the discrimination index ratio and statistical differences in the new object recognition experiment are shown in Figure 5 and Table 3 below.

[0141] Table 3: Discrimination index ratio and statistical differences in the new object recognition experiment

[0142]

[0143] The new object recognition experiment paradigm takes advantage of the natural tendency of mice to prefer new objects. By training mice to recognize and remember different objects, the learning and memory and discrimination abilities of animals can be demonstrated. By modeling and inducing memory impairment or dementia in animals, their ability to remember and distinguish new and old objects will be significantly reduced. In this experiment, STZ was injected into the brain by stereotactic positioning to model the animals. The animals with typical memory and cognitive impairment phenotype showed a significant decrease in their ability to distinguish new objects compared to the control group. The memory and discrimination ability of animals for new and old objects was quantified by the "discrimination ratio" and "discrimination index" indices. The discrimination ratio (DR) calculation formula = new object exploration time / (new object exploration time + old object exploration time) * 100, and the discrimination index (DI) = (new object exploration time - old object exploration time) / (new object exploration time + old object exploration time) * 100. Through the discrimination ratio and discrimination index results in the new object recognition experiment, it can be found that the STZ modeling group of animals showed a significant memory impairment phenotype. The Alzheimer's disease positive drug donepezil has a certain degree of effect on alleviating animal memory impairment, but the improvement trend has no statistically significant difference. In contrast, mirodenafil and compound 1 can significantly improve the memory impairment of STZ-induced animals and improve their memory discrimination ability.

[0144] The Y-maze procedure is as follows:

[0145] 1) One hour before the test, let the designated mice adapt to the laboratory environment;

[0146] 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, facing away from the center of the maze;

[0147] 3) Let the animal explore the maze without interference for 5 minutes, and record;

[0148] 4) At the end of the test, remove the animal from the maze and return it to the cage;

[0149] 5) After completing all the behavior trajectory analysis.

[0150] The correct selection rate and statistical difference of the Y-maze experiment are shown in Figure 6 and Table 4 below.

[0151] Table 4: Correct selection rate of animals in Y-maze and statistical difference value

[0152]

[0153] Compared with the control group animals, the STZ model group animals showed obvious memory impairment, which was manifested as a significant decrease in the correct rate in the Y maze self-selection path, with a statistically significant difference, which was consistent with the conclusion that STZ brain stereotactic injection could induce animal dementia and memory impairment. Using donepezil as a treatment positive drug, the experimental results showed that the correct selection rate of the donepezil treatment group in the Y maze was improved to a certain extent, and the improvement degree was close to a statistically significant difference. The correct selection rate of the mirodenafil treatment group in the Y maze only showed an upward trend, but there was no statistically significant difference. Compared with the treatment effects of the two groups, the correct selection rate of the experimental animals in the Y maze was significantly improved after the administration of compound 1, and the improvement had a statistically significant difference. The experimental results suggested that compound 1 had a significant effect of improving the working memory and spatial memory of animals.

[0154] The procedure of the water maze experiment (Morris) is as follows:

[0155] Training stage:

[0156] Day 1: The animals were randomly placed in the other three quadrants, and the midpoint of the pool wall was faced. The animals were allowed to swim freely in the pool. If the animal found the platform within 1 minute and stayed on the platform for 10 seconds, the animal was put back; if not, a glass rod or plastic rod was used to guide the animal to swim to the platform, and the animal was put back after staying on the platform for 10 seconds. After the animal was wiped with a paper towel and dried with a heating lamp, it was put back into the cage. The next animal was started. The same process was repeated.

[0157] After the last animal in the current round was finished, the first animal was started again, and each animal was trained twice a day, and the operation of day 1 was repeated every day from day 2 to day 5.

[0158] Test stage (day 6)

[0159] Remove the platform and place the animal in the 3th quadrant of the pool. Let the mouse explore the maze freely for 1 minute.

[0160] After the animal was wiped with a paper towel and dried with a heating lamp, it was put back into the cage. The next animal was started. The same process was repeated.

[0161] The results of the water maze experiment are shown in Figures 7-8 and Tables 5-9 below.

[0162] Table 5: Platform escape time of animals in the water maze training period and statistical difference value

[0163]

[0164] Table 6: Time of animals reaching the platform in the water maze test period and statistical difference value

[0165]

[0166] Table 7: Number of times animals crossed the platform during the water maze test period and statistical difference values

[0167]

[0168] Table 8: Distance moved in the target quadrant by animals during the water maze test period and statistical difference values

[0169]

[0170] Table 9: Time spent in the target quadrant by animals during the water maze test period and statistical difference values

[0171]

[0172] The spatial memory ability of animals in each group was tested by the Morris water maze experiment, and the results showed that the animals in the STZ modeling group exhibited a significant spatial memory impairment, which was manifested by the fact that during the water maze learning stage, the animals in this group could not recognize the spatial path of memory and the time taken to find the hidden platform under water did not decrease with the progress of learning. The Alzheimer's disease treatment drug donepezil, as the positive drug in this group, could alleviate the spatial memory impairment of animals caused by STZ modeling, and there was a significant statistical difference compared with the model group at the fifth detection time point. The milonafil group had a certain alleviating trend, but the difference from the model group was not statistically significant. The animals treated with compound 1 had a significant improvement in spatial memory, which was manifested by the fact that the time taken by animals to find the platform under water gradually shortened as the training and learning time was prolonged, and at the fifth detection time point, the difference in the time taken by animals in the compound 1 administration group to find the platform compared with the model group was statistically significant.

[0173] Example 3 Improvement effect of the compound of the present application on dementia in 5 X FAD animal model

[0174] (1) Animal model

[0175] 5XFAD (male, 4 months old) were purchased from a certified animal supplier (Saiye Biotechnology Co., Ltd.) and were raised for 1 week before the experiment. The animals were raised under standard conditions, with room temperature of 21-23°C, relative humidity of 30-70%, and 12h:12h light-dark cycle. Food and water were available at will.

[0176] (2) Study time

[0177] The study time was as follows: Figure 9The experiment was performed using 4-month-old male 5XFAD mice. The animals were grouped during the acclimation period, and the animals were evenly grouped according to the body weight, and the time of grouping was marked as day 0. After the animals were evenly grouped, the drug administration experiment was arranged once a day, and the behavioral experiment was verified after 2 months of continuous administration. After all the behavioral experiments were completed, the experiment endpoint was set, and the animals were euthanized, and the serum, cerebrospinal fluid and tissue samples were reserved.

[0178] (3) Animal grouping

[0179]

[0180] p.o.: oral

[0181] s.c.: subcutaneous

[0182] (4) Compound information and configuration

[0183] Compound 1 preparation method: the required amount was weighed, and a mother liquor was prepared with normal saline at 2 mpk, and then diluted by a factor of 2.

[0184] (5) Research process

[0185] Body weight measurement: the body weight was monitored and recorded every day in the first week, and then the body weight information was recorded once a week (Monday).

[0186] Test compound treatment: the test compound was treated once a day.

[0187] Behavioral test: open field test (OFT), novel object recognition test (NOR), Y maze test (Y-maze), Morris water maze test (MWM):

[0188] Behavioral test after 2 months of drug administration: test 1 hour after drug administration.

[0189] Open field test (OFT) procedure and results:

[0190] 1) One hour before the test, the designated mice were allowed to adapt to the laboratory environment;

[0191] 2) The open field box area was cleaned with 70% ethanol and paper towels before the test;

[0192] 3) The experimental mice were taken out of the mouse cage and placed in the center area of the open field box, and the experimenter should leave the open field box to avoid disturbing the mice;

[0193] 4) Each test mouse was allowed to freely move in the open field box for 10 minutes;

[0194] 5) At the end of the test, the mice were removed from the open field box and returned to the mouse cage;

[0195] 6) Clean the entire open field box area with 70% ethanol and paper towel or cloth piece before proceeding to the next animal experiment;

[0196] 7) After all the procedures, perform the experiment analysis.

[0197] The results of distance in central zone, time in central zone and rearing number in open field test and statistical difference are shown in Figure 10 and Tables 10-12.

[0198] Table 10: Distance in central zone in open field test and statistical difference

[0199]

[0200] Table 11: Time in central zone in open field test and statistical difference

[0201]

[0202] Table 12: Rearing number in open field test and statistical difference

[0203]

[0204] The open field test paradigm takes advantage of the natural tendency of mice to prefer to walk along the walls of an open area. By observing the distance and time in central zone and rearing number of mice in a fixed time, the cognitive exploration ability of animals can be reflected. Animals with cognitive impairment phenotypes will have a significant decrease in cognitive exploration ability in unfamiliar environments. This experiment uses transgenic model animals with typical cognitive impairment phenotypes, which are characterized by a significant decrease in cognitive exploration ability compared to control animals. The cognitive exploration ability of animals is quantified by "distance in central zone", "time in central zone" and "rearing number". Through the results of distance in central zone, time in central zone and rearing number in open field test, it can be found that the model animals have obvious cognitive impairment phenotype. In contrast, compound 1 can significantly improve the memory impairment of STZ-induced animals and improve their cognitive exploration ability.

[0205] The procedure and results of the novel object recognition experiment (NOR) are as follows:

[0206] The experimental procedure is the same as in Example 2 above. Figure 3 is a novel object recognition experiment paradigm.

[0207] The results of the discrimination percentage and statistical difference of the novel object recognition experiment are shown in Figure 11 and Table 13.

[0208] Table 13: Discrimination ratio and statistical difference of new object recognition experiment

[0209]

[0210] The new object recognition experiment paradigm takes advantage of the natural instinct of mice to like new objects, and can reflect the learning and memory and discrimination ability of animals by training mice to recognize and remember different objects. Animals with cognitive impairment phenotypes will have a significant decline in their ability to remember and distinguish new and old objects. This experiment uses transgenic model animals with typical memory and cognitive impairment phenotypes, which are manifested as a significant decline in their ability to distinguish new objects compared to control animals. The memory and discrimination ability of animals for new and old objects is quantified by the "discrimination ratio (DR)" and "discrimination index (DI)" indices. The discrimination ratio (DR) calculation formula is new object exploration time / (new object exploration time + old object exploration time) * 100, and the discrimination index (DI) is (new object exploration time - old object exploration time) / (new object exploration time + old object exploration time) * 100. The discrimination ratio and discrimination index results in the new object recognition experiment can reveal that the model group animals have a significant memory impairment phenotype. In comparison, compound 1 can have a certain trend to improve the memory impairment of STZ-induced animals, and improve their memory discrimination ability.

[0211] Y-maze procedure and results:

[0212] The experimental procedure is the same as in Example 2 above. The results of the correct selection rate and statistical difference of the Y-maze experiment are shown in Table 14. Figure 12 and Table 14.

[0213] Table 14: Correct selection rate and statistical difference value of animals in Y-maze

[0214]

[0215] Compared with control animals, model animals have a significant memory impairment phenomenon, which is manifested as a significant decline in the correct rate of self-selection path in Y-maze, with a statistical difference, which is consistent with the conclusion that transgenic model animals can exhibit dementia and memory impairment. Compared with the model group, compound 1 can significantly improve the correct selection rate of experimental animals in the Y-maze after administration, and this improvement has a significant statistical difference. The experimental results suggest that compound 1 has a significant effect on improving the working memory and spatial memory of animals.

[0216] Morris water maze procedure and results:

[0217] Training phase:

[0218] The training phase was the same as in Example 2 above.

[0219] Test phase (day 5)

[0220] Remove the platform and place the animal in the pool in the 3rd quadrant. Allow the mouse to freely explore the maze for 1 minute.

[0221] After the animal is dried with a paper towel and a heat lamp, place it back in the cage. Start the next animal. Repeat the same process.

[0222] The results of the water maze experiment and statistical differences are shown in Tables 15-18. Figures 13-14 and Tables 15-18.

[0223] Table 15: Time to find the platform and statistical difference values of animals in the platform escape phase of the water maze training period

[0224]

[0225] Table 16: Time to reach the platform and statistical difference values of animals in the water maze test period

[0226]

[0227] Table 17: Number of times to cross the platform and statistical difference values of animals in the water maze test period

[0228]

[0229] Table 18: Time spent in the target quadrant and statistical difference values of animals in the water maze test period

[0230]

[0231] The spatial memory ability of animals in each group was tested by the Morris water maze experiment, and the results showed that the 5XFAD model group animals showed significant spatial memory impairment, which was manifested as the inability of the animals in this group to recognize and remember the spatial path during the training and learning phase of the water maze, and the time to find the hidden platform under water did not decrease with the progress of learning. The animals treated with Compound 1 had significant improvement in spatial memory, which was manifested as the time for the animals to find the platform under water gradually shortened with the extension of the training and learning time. From the second day of detection, the difference in the time to find the platform between the animals in the Compound 1 administration group and the model group was statistically significant. In the test period, from the time to reach the platform, the number of times to cross the platform, and the time spent in the target quadrant, the results of the animals in the Compound 1 administration group were different from those of the model group, and the differences were statistically significant.

[0232] Example 4: Improvement effect of the compound of the present application on vascular dementia in an animal model

[0233] (1) Animal model

[0234] SD rats (male, 250-280 g) were purchased from a certified animal supplier (Vivant) and raised for 1 week before the experiment. The animals will be raised under standard conditions, room temperature 21-23℃, relative humidity 30-70%, 12h:12h light-dark cycle. Food and water can be taken at will.

[0235] The animal model was induced by ligating the bilateral common carotid artery (CCA) to simulate the ischemic dementia model operation, and the specific method was as follows:

[0236] The equipment used included: surgical scissors, surgical forceps, ophthalmic scissors, ophthalmic forceps, suture (4-0), ligature (5-0), needle holder, hemostatic forceps, artery clamp, thread plug, shaving knife. Operating lamp (25W illuminating lamp), rat operating table. Isoflurane or similar anesthetics, anesthetic machine and anesthesia mask. Petri dish, cotton swab, cotton ball, iodine tincture, 75% alcohol, physiological saline.

[0237] The animals were prepared as follows: 250-280 g male SD rats were selected. According to the body weight, they were divided into groups (ensure that the body weight difference in each group is not large, and animals with individual body weight deviation greater than ±20% are excluded), 4 animals per cage, and the cage label indicates the corresponding group of mice in the cage.

[0238] Preoperative preparation: The vessels, surgical equipment, etc. used in the experiment were sterilized (with outer packaging), and disposable sterile supplies were used when necessary.

[0239] Clean and disinfect the operating environment (space, table surface, etc.).

[0240] Anesthesia and fixation: The animals were weighed and anesthetized, and after anesthesia, they were fixed in a supine position on the operating table. Routine skin preparation and disinfection were performed, and the whole operation was kept warm. The rat's incisors were hooked and fixed with suture, so that the neck was stretched, making it convenient for the subsequent operation.

[0241] A conventional ophthalmic scissors or surgical knife was used to make a midline incision on the neck, and the gland tissue and fascia of the neck were bluntly separated with forceps to expose and separate the bilateral common carotid artery (CCA).

[0242] The bilateral common carotid artery was ligated with suture.

[0243] After ligation and disinfection, the subcutaneous tissue and skin were sutured layer by layer, and after the animal woke up from anesthesia, it was put back into the cage.

[0244] The control operation model was made according to the same steps as before, but the blood vessels were not ligated. After ligation and disinfection, the subcutaneous tissue and skin were sutured.

[0245] 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.

[0246] (2) Research time

[0247] Research time such as Figure 15 As shown. 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 time marked as day 0. After even grouping, the animals underwent modeling, and bilateral common carotid arteries were ligated. Postoperatively, 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.

[0248] (3) Animal grouping

[0249]

[0250] IP: Abdominal cavity

[0251] (4) Compound information and configuration

[0252] Preparation method of mirtazafil: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0253] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0254] (5) Research process

[0255] Weight measurement: Monitor and record weight daily during the first week, and then record weight information once a week (Monday).

[0256] Test compound treatment: once daily.

[0257] Morris water maze experiment:

[0258] Behavioral tests were conducted one month after the model was established and the tests were conducted one hour after administration.

[0259] Morris water maze experiment procedure and results:

[0260] The experimental procedure is the same as in Example 2 above.

[0261] For the results and statistical differences of the water maze experiment, see [link to relevant documentation]. Figures 16-17 See Tables 19-21.

[0262] Table 19: Platform escape time and statistical differences of animals during water maze training.

[0263]

[0264] Table 20: Time taken by animals to reach the platform during the water maze test period and statistical difference values

[0265]

[0266] Table 21: Number of times animals crossed the platform during the water maze test period and statistical difference values

[0267]

[0268] The spatial memory ability of animals in each group was tested by the Morris water maze experiment, and the results showed that after modeling of vascular dementia, the model group of animals showed significant spatial memory impairment, which was manifested as that during the water maze training and learning stage, the animals in this group could not recognize the memory space path, and the time to find the hidden platform under water did not decrease with the learning progress. The improvement effect of the positive drug mirodenafil was obvious. The animals treated with compound 1 had a certain improvement effect on spatial memory, which was manifested as that with the extension of the training and learning time, the time for the animals to find the platform under water gradually shortened, and at the fourth day of the detection time point, the difference in the time to find the platform between the animals in the compound 1 administration group and the model group had statistical significance. During the test period, from the results of the time to reach the platform and the number of times to cross the platform, the model group of animals showed significant spatial memory impairment. Compared with the model group, mirodenafil and compound 1 could significantly improve the memory impairment of animals after modeling.

[0269] Example 5 Improvement effect of the compound of the present application combined with donepezil, etc. on dementia in animal models (1) Animal model

[0270] C57BL / 6j (male, 6 months old) was purchased from a certified animal supplier (Vivantia) and fed for 1 week before the experiment. The animals will be fed under standard conditions, room temperature 21-23℃, relative humidity 30-70%, 12h:12h light-dark cycle. Food and water can be taken at will.

[0271] The animal model simulating the dementia of sporadic AD patients was induced by stereotactic injection of streptozotocin (STZ) (STZ-ICV), and the specific method was as follows:

[0272] The equipment used includes: brain stereotaxic apparatus, skull drill, 10ul microsyringe, surgical scissors, surgical forceps, suture (5-0), needle holder, shaving knife, petri dish, cotton swab, iodine tincture, 75% alcohol, normal saline, STZ, tribromoethanol.

[0273] Animal preparation: 6-month-old male C57 / 6j mice were selected. According to the body weight, the mice were grouped (to ensure that the body weight difference in each group is small, and animals with individual body weight deviation greater than ± 20% are excluded), and 4 mice were placed in each cage. The corresponding group of mice in the cage was marked on the cage.

[0274] Preoperative preparation: The experimental vessels, surgical instruments, etc. were sterilized (with outer packaging), and disposable sterile supplies were used when necessary.

[0275] Clean and disinfect the operating environment (space, table top, etc.).

[0276] Anesthesia and fixation: The mice were anesthetized by intraperitoneal injection of tribromoethanol (20 ml / kg). After about 5 minutes, the animals entered a deep anesthetic state. The hair on the skull was shaved clean with a hair clipper, and the mice were fixed on the adapter. The eyes were smeared with eye ointment. The bilateral ear bars were fixed in the mouse's external auditory canal, and the left and right ear bar scales were adjusted to be consistent and the screws were tightened, so that the mouse's head was in the middle. The mouse's incisors were fixed on the adapter incisor fixator, and the incisor clamp should not be tightened too tightly.

[0277] Mouse brain leveling: expose the skull: iodine tincture disinfect the mouse scalp, then cut the scalp with scissors to make a 2 cm long incision, and use dry cotton balls or hydrogen peroxide to erode and wipe off the bone membrane on the surface of the skull to completely expose the skull.

[0278] Bregma determination: move the needle tip to the anterior part of the midline, touch the surface of the skull, and use the bregma point as the reference point. When the surface is just touched, stop the needle and set the X, Y, Z axis coordinate readings of the digital display instrument to zero. When the needle is lowered, observe the position of the needle tip to avoid breaking the needle!

[0279] Left and right leveling: slightly raise the sample injector to avoid touching the skull surface, then move the bregma as the midpoint to the left and right by the same distance (recommended 3.0 mm / 2.5 mm / 2 mm) and lower the needle to touch the skull surface. Read the corresponding Z coordinate respectively, and determine whether the left and right are level by the Z coordinate value. If the readings differ by 0.03 mm or less, it is considered that the mouse's head is level left and right. Adjust the height of the ear bars to level left and right. During the adjustment process, the bregma zero point must be repositioned after moving the ear bars.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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 the cage.

[0284] (2) Research time

[0285] Research time such as Figure 18 As shown. Six-month-old male C57BL6J mice were used for the modeling experiment. During the environmental adaptation period, the animals were divided into groups based on their weight, and the grouping time was marked as day 0. After uniform grouping, the animals were fixed in place 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 after surgery, and the animals were kept in the same environment. The drug administration experiment was conducted once a day for 30 consecutive days. After the drug administration was carried out, behavioral experiments were performed to verify the results. The animals were euthanized after all behavioral experiments were completed, and serum, cerebrospinal fluid, and various tissue samples were collected.

[0286] (3) Animal grouping

[0287]

[0288] po: oral

[0289] sc: subcutaneous

[0290] (4) Compound information and configuration

[0291] Streptozotocin (STZ) Preparation: Weigh the required amount, add normal saline, and vortex to obtain a uniform solution.

[0292] Donepezil Preparation: Weigh the required amount, add normal saline, and vortex to obtain a uniform solution.

[0293] Compound 1 Preparation: Weigh the required amount, and prepare a mother liquor with normal saline at 2 mpk, then dilute by a factor of 2.

[0294] (5) Study Process

[0295] Body weight measurement: Body weight was monitored and recorded daily in the first week, and then once a week.

[0296] Test compound treatment: Test compound was administered once daily.

[0297] Morris water maze experiment:

[0298] Behavioral testing was performed one month after model administration, and testing was performed 1 hour after administration.

[0299] The procedure for the Morris water maze experiment is as follows:

[0300] Training phase:

[0301] Day 1: The animals were randomly placed in the other three quadrants, at the midpoint of the pool wall, facing the pool wall. The animals were allowed to swim freely in the pool. If the animal found the platform within 1 minute and stayed on the platform for 10 seconds, the animal was returned; if not, a glass or plastic rod was used to guide the animal to the platform, and the animal was allowed to stay on the platform for 10 seconds before being returned. After the animal was dried with a paper towel and a heating lamp, it was returned to the cage. The next animal was started. The same process was repeated.

[0302] After the last animal in the current round was finished, the first animal was started again, and each animal was trained twice a day, with the same procedure repeated on days 2-5.

[0303] Testing phase (day 6)

[0304] The platform was removed, and the animal was placed in the 3th quadrant of the pool. The mouse was allowed to freely explore the maze for 1 minute.

[0305] After the animal was dried with a paper towel and a heating lamp, it was returned to the cage. The next animal was started. The same process was repeated.

[0306] The results of the water maze experiment and statistical differences are shown in Figures 19-20 and Tables 22-26.

[0307] Table 22: Platform escape period and statistical difference values for animals in the water maze training period

[0308]

[0309] Table 23: Time taken by animals to reach the platform during the water maze test period and statistical difference values

[0310]

[0311] Table 24: Number of times the platform was crossed by animals during the water maze test period and statistical difference values

[0312]

[0313] Table 25: Distance moved in the target quadrant by animals during the water maze test period and statistical difference values

[0314]

[0315] Table 26: Time spent in the target quadrant by animals during the water maze test period and statistical difference values

[0316]

[0317] The spatial memory ability of animals in each group was tested by the Morris water maze experiment, and the results showed that the model group animals showed significant spatial memory impairment, which was manifested as the inability of the animals in this group to recognize the spatial path during the learning stage of the water maze, and the time taken to find the hidden platform under water did not decrease with the progress of learning. The Alzheimer's disease treatment drug donepezil, as a positive drug in this group, could alleviate the spatial memory impairment of animals caused by STZ modeling, and had a significant statistical difference compared with the model group from the fourth detection time point. The animals treated with the combination of donepezil and compound 1 had a significant improvement in spatial memory, which was manifested as the time taken by animals to find the platform under water gradually shortened with the extension of training and learning time. At the second detection time point, the difference in the time taken by animals in the compound 1 administration group to find the platform compared with the model group was statistically significant. During the test period, the results of escape latency time, platform crossing times, target quadrant moving distance and residence time showed that the model group animals had significant spatial memory impairment. Compared with the model group, the donepezil group and the combination drug group could significantly improve the memory impairment of animals after modeling, and the combination drug group was superior to the donepezil group.

[0318] Example 6 Improvement effect of compound of the present application on stroke in animal model

[0319] (1) Animal model

[0320] SD rats (male, 250-280 g) were purchased from certified animal suppliers (Vantianlihua) and raised for 1 week before the experiment. The animals will be raised under standard conditions, room temperature 21-23℃, relative humidity 30-70%, 12h:12h light-dark cycle. Food and water can be taken at will.

[0321] The animal model was induced by ischemia-reperfusion (MCAO) to simulate the operation of the stroke model. The specific method is as follows:

[0322] Surgical scissors, surgical forceps, ophthalmic scissors, ophthalmic forceps, suture (4-0), ligature (5-0), needle holder, hemostatic forceps, artery clamp, thread plug, shaving knife. Operating lamp (25W illuminating lamp), rat operating table. Isoflurane or similar anesthetics, anesthetic machine and anesthetic mask. Petri dish, cotton swab, cotton ball, iodine tincture, 75% alcohol, physiological saline. Animal preparation as follows: 250-280g male SD rats were selected. According to the body weight (ensure that the body weight difference of each group is not large, and the animals with individual body weight deviation greater than ±20% are excluded), the number of animals in each cage is 4, and the cage label is marked with the corresponding group of mice in the cage.

[0323] Preoperative preparation: the vessels, surgical instruments and other instruments used in the experiment were sterilized (with outer packaging), and disposable sterile supplies were used when necessary.

[0324] Clean and disinfect the operating environment (space, table surface, etc.).

[0325] Anesthesia and fixation: the animals were weighed and anesthetized, and then fixed in a supine position on the operating table after anesthesia. Routine skin preparation and disinfection, and keep warm during the whole operation. The rat's incisors were hooked and fixed with suture, so that the neck was stretched, making it convenient for the following operation.

[0326] Make a midline incision on the neck with a conventional ophthalmic scissors or surgical knife, and use forceps to bluntly separate the glandular tissue and fascia of the neck, exposing and separating the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). Use suture to tie a slipknot to ligate the bilateral common carotid artery.

[0327] Separate the ICA and its extracranial branch pterygopalatine artery, tie a slipknot with suture, ligate the pterygopalatine artery, or directly clamp the pterygopalatine artery with a vascular clamp, avoid inserting the thread plug into the pterygopalatine artery, and keep the only open branch of the CCA as the ICA.

[0328] Separate the ECA main stem, ligate the distal and proximal ends of the external carotid artery (leave long thread ends), and cut the artery 5mm apart. Then gently pull the proximal end of the thread to make the cut artery consistent with the shape of the common carotid artery.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] (2) Research time

[0334] Research time such as Figure 21 As shown. Male SD rats weighing 250-280g were used for the modeling experiment. After surgery, the same nursing procedures were followed, and the animals were housed in the same environment. Postoperatively, they were grouped according to their appearance scores. The drug administration experiment was conducted once daily for 14 consecutive days. Afterwards, the animals were euthanized, and brain sections were collected for TTC staining.

[0335] (3) Animal grouping

[0336]

[0337] IP: Abdominal cavity

[0338] (4) Compound information and configuration

[0339] Edaravone preparation method: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0340] Preparation method of compound 1: Weigh the required amount, prepare a stock solution with physiological saline at 2 mpk, and then dilute it serially.

[0341] (5) Research process

[0342] Neurological and behavioral assessments were performed on the animals after they regained consciousness following ischemic surgery.

[0343] ① No neurological deficits: 0 points;

[0344] ②The forepaw on the paralyzed side cannot be fully extended: 1 point;

[0345] ③ When walking, turn in circles towards the paralyzed side: 2 points;

[0346] ④ When walking, lean towards the paralyzed side: 3 points;

[0347] (5) unable to walk automatically, with loss of consciousness: 4 points.

[0348] The higher the score, the more serious the animal behavior disorder. The standard for successful construction of the model rats: rats show symptoms such as hemiplegia, drooping of the contralateral forelimb and unstable standing.

[0349] Test compound treatment: once a day test compound treatment.

[0350] TTC staining:

[0351] The rat brain was quickly removed, washed with cold saline, and then quickly placed in a -20°C refrigerator for 10 minutes. After the brain tissue was slightly hard, the olfactory bulb, pituitary gland and low brain stem were removed, and the brain was cut into 2mm thick slices from front to back, and then divided into 5 equal parts and placed in 2% TTC solution at 37°C for 30 minutes in the dark, and turned over every 5 minutes. TTC can react with the dehydrogenase system in normal tissue to be reduced to rose red, so that the normal tissue is stained rose red, and the infarct tissue is white.

[0352] After staining, the brain slices were photographed, and the infarct area was calculated using Imagej software.

[0353] Results and statistical differences are shown in Figure 22 and Table 27.

[0354] Table 27: Infarct area of animal brain slices after TTC staining and statistical difference values

[0355]

[0356] Compared with the control group animals, the model group animals showed a significant increase in infarct area. Compared with the model group,

[0357] Both edaravone and compound 1 can improve the infarct area of animals after modeling.

[0358] Example 7 Protective effect of the compound of the present application on neurons in a 6-OHDA-induced injury model at the in vitro cell level

[0359] (1) Cell culture and neuron directional differentiation

[0360] SH-SY5Y (human neuroblastoma cell line, Procell, CL-0208) cells were cultured in complete medium: DMEM / F12 (1:1) medium, 10% FBS and 1% pen / strep, in a 37-degree, 5% carbon dioxide constant temperature cell incubator. After the cells were cultured in complete medium for 48 hours, the medium was changed to induce differentiation into neuron cells. The medium was changed to Neurobasal medium (containing B27 supplement and GlutaMAX) and 10 μM all-trans-retinoic acid (ATRA), and half of the medium was changed every 24 hours. The cells were cultured in this condition for 7 days to obtain cultured differentiated neuron-like cells.

[0361] (2) Model establishment and compound configuration

[0362] Ascorbic acid was prepared into a 0.1% solution with normal saline, and 6-OHDA was configured into a 100 mM stock solution with 0.1% ascorbic acid solution, which was stored in the dark. 6-OHDA was diluted to 40 uM with Neurobasal medium to establish a cell damage model, and the compound was configured into the required final molar concentration with the medium containing 40 uM 6-OHDA, and was incubated in a cell incubator for 18 hours.

[0363] (3) CellTiter-Lumi luminescence method for cell viability detection

[0364] CTL was mixed with complete medium at a ratio of 1:1, 100 ul was added to each well, and incubated for 10 min, and then centrifuged at 1000 rpm for 1 min at room temperature. The fluorescence signal of the Luminometer was collected by an enzyme marker, and subsequent data processing was performed.

[0365] (4) Data analysis

[0366] The 6-OHDA treatment group was used as the model group, and the CTL activity data was normalized and converted. The compound treatment results were compared with the data of the model group, and unpaired t-test and Mann-Whitney test were used for analysis. The data were expressed as mean ± standard deviation (*p<0.05, **p<0.01, ****p<0.0001). The results are shown in Table 28. Figure 23

[0367] Table 28: Relative value of cell activity improvement ratio and statistical difference in 6-OHDA damaged cell model

[0368] ​

[0369] Compared with the blank control group, pramipexole, compounds 1 and 2 can all improve the cell activity due to 6-OHDA damage, and the phenyl pyrimidone derivatives have stronger effects of improving cell activity and neuroprotection. Therefore, the various pyrimidone phenyl derivatives in this patent have protective effects on cell damage related to Parkinson's disease.

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: in, 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 represents -SO2-, -CO-, or -NHCOCH2-; R 5 selected from the group consisting of: and hydroxyCi-C6alkylamino.

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 salts are selected from 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 also contains 0.5-3 molecules of water of crystallization; preferably, it contains 1-2 molecules of water of crystallization, more preferably, it contains 1 molecule of water of crystallization.

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 diseases, and / or for improving or treating degenerative diseases, said pharmaceutical composition comprising a therapeutically effective amount of a phenylpyrimidinone compound of formula (I) according to any one of claims 1 to 4 or a tautomer thereof, a pharmaceutically acceptable salt, a solvate, or an isotopic label thereof, and a pharmaceutically acceptable carrier.

6. Use according to claim 5, characterized in that: The pharmaceutical composition is an oral pharmaceutical composition, a topical pharmaceutical composition, or an injectable pharmaceutical composition; in particular, when used orally, the pharmaceutical composition is a tablet, powder, orally disintegrating film, or capsule; when used topically, the pharmaceutical composition is a spray, liniment, ointment, or 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 mentioned 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.

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

9. Use according to claim 1 or 5, characterized in that: The cerebrovascular diseases mentioned 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 thrombosis, cerebrovascular diseases without acute focal involvement and functional loss, sequelae of stroke, and other cerebrovascular diseases.

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

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

12. Use according to claim 1 or 5, characterized in that: The medicaments for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and / or for improving or treating degenerative diseases, further contain other active ingredients selected from one or more medicaments for improving or treating cognitive impairment, and / or for improving or treating cerebrovascular diseases, and 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 represented by 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, an other active ingredient selected from one or more of a drug for improving or treating cognitive dysfunction, and / or for improving or treating cerebrovascular disease, improving or treating Parkinson's disease indication.

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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