Compound for treating cardiovascular and cerebrovascular diseases, pharmaceutical composition, and use of compound or pharmaceutical composition

Abstract

The present invention relates to the technical field of medicine. Disclosed are a compound for treating cardiovascular and cerebrovascular diseases, a pharmaceutical composition, and a use of the compound or the pharmaceutical composition. The compound or the pharmaceutical composition thereof provided by the present invention can significantly reduce the infarct area in mouse and rat models of ischemic stroke, and can be used for treating cerebrovascular diseases. In addition, the compound or the pharmaceutical composition thereof has a significant protective effect on the myocardium in mouse models of ischemic myocardial infarction and heart failure induced by the myocardial infarction, and can be used for treating cardiovascular diseases.

Description

Compounds, pharmaceutical compositions and their applications for treating cardiovascular and cerebrovascular diseases Technical Field

[0001] This invention belongs to the field of pharmaceutical science, and more specifically, relates to compounds, pharmaceutical compositions and their applications in the treatment of cardiovascular and cerebrovascular diseases. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are a collective term for diseases of the heart, blood vessels, and cerebral blood vessels. They are a group of diseases caused by insufficient blood supply due to blood vessel blockage, resulting in tissue damage. They include myocardial infarction, chronic heart failure, coronary heart disease, ischemic stroke, peripheral arterial vascular disease, deep vein thrombosis, and pulmonary embolism, and are the leading cause of death worldwide.

[0003] Stroke, also known as cerebrovascular accident, is an acute cerebrovascular disease caused by the sudden rupture or blockage of blood vessels in the brain, leading to brain tissue damage. It includes ischemic and hemorrhagic strokes, with ischemic strokes accounting for 85% of cases. Current treatment strategies for ischemic stroke primarily focus on two aspects: first, improving cerebral blood circulation, including intravenous thrombolytic enzymes, arterial thrombectomy, and anticoagulation; second, using neuroprotective agents such as edaravone (ED) and butylphthalide (NBP) to protect nerve tissue by scavenging free radicals and preventing oxidative damage. Despite these current treatment strategies, the mortality rate of ischemic stroke remains high, necessitating better treatment options. Only by rapidly restoring blood supply to the ischemic area can the neurological deficit after ischemia be minimized. While thrombolytic therapy can quickly restore blood supply to the ischemic area, its narrow time window and numerous contraindications significantly limit its clinical application. Therefore, promoting the establishment of collateral circulation and angiogenesis is crucial for the recovery of neurological function. Early collateral circulation in ischemic stroke depends on the opening of the original vascular network, which varies from person to person and is difficult to intervene in. Later collateral circulation establishment depends on angiogenesis. Angiogenesis helps blood flow bypass the blocked segment to reach brain tissue, improving blood perfusion in the ischemic area and reducing infarct volume. It also promotes neuronal remodeling and functional recovery. Therefore, we develop novel compounds based on mechanisms that promote angiogenesis. In addition, the ability of drug molecules to cross the blood-brain barrier is also a crucial factor in alleviating cerebrovascular lesions in cerebrovascular diseases. Therefore, developing drug molecules that can more easily cross the blood-brain barrier is a common aspiration in this field.

[0004] Ischemic myocardial infarction (MI) refers to acute, severe, and persistent ischemic necrosis of the corresponding myocardial region due to a sudden reduction or interruption of coronary blood supply. It is one of the most common causes of heart failure (HF). After a myocardial infarction, the heart undergoes a series of compensatory and pathological remodelings, including myocardial fibrosis, inflammatory responses, and ventricular dilation. These pathological changes are the core mechanisms of the development and progression of heart failure. Although modern medical treatments have significantly reduced the acute-phase mortality rate of myocardial infarction, the incidence of post-infarction heart failure remains high, with approximately 20% of patients progressing to heart failure within five years. This high incidence, along with the complex course, high recurrence rate, and severe impact on patients' quality of life of heart failure itself, constitutes a significant socioeconomic and public health burden.

[0005] Currently, drug treatment for ischemic myocardial infarction and its resulting heart failure mainly includes antiplatelet and anticoagulant drugs, beta-blockers, and aldosterone inhibitors. These drugs can reduce myocardial oxygen consumption, delay ventricular remodeling, and improve long-term prognosis to some extent, but their main effects are still focused on symptom control and long-term prognosis improvement, with limited effects on repairing existing myocardial damage and microcirculatory disorders.

[0006] Against this backdrop, promoting angiogenesis and alleviating ischemic damage have become important directions in treatment. The core pathological process after myocardial infarction is irreversible necrosis of cardiomyocytes caused by ischemia and the resulting ventricular remodeling. By promoting angiogenesis, local blood perfusion in the ischemic area can be enhanced, microcirculatory disturbances can be improved, and thus the necessary oxygen and nutrient support can be provided to cardiomyocytes. In addition, the formation of new blood vessels not only directly improves blood supply, but also creates a more suitable microenvironment for myocardial repair, which helps promote the survival and functional recovery of cardiomyocytes. Therefore, treatment strategies targeting angiogenesis and the mechanisms of ischemic damage relief not only have the potential to overcome the limitations of existing drug therapies, but also provide new possible avenues for improving the overall treatment outcomes of ischemic myocardial infarction and heart failure. Summary of the Invention

[0007] The primary objective of this invention is to provide a new class of compounds that have a high brain / blood distribution coefficient and are capable of resisting ischemic-like injury, promoting angiogenesis, and having neuroprotective effects.

[0008] A second objective of the present invention is to provide a pharmaceutical composition containing the above-mentioned compounds, which has a high brain / blood distribution coefficient and is capable of resisting ischemic-like injury, promoting angiogenesis, and having neuroprotective effects.

[0009] A third objective of the present invention is to provide the use of the above-mentioned compound or pharmaceutical composition for the prevention, treatment or improvement of cerebrovascular diseases. By applying an effective amount of the above-mentioned compound or pharmaceutical composition, blood perfusion in the ischemic area can be improved and infarct volume reduced, thereby achieving the purpose of preventing, treating or improving cerebrovascular diseases, especially transient or permanent ischemic stroke.

[0010] The fourth objective of this invention is to provide the use of the above-mentioned compounds or pharmaceutical compositions for the prevention, treatment or improvement of cardiovascular diseases. By applying an effective amount of the above-mentioned compounds or pharmaceutical compositions, cardiac contractile function can be improved and myocardial ischemia can be alleviated, thereby achieving the purpose of preventing, treating or improving cardiovascular diseases, especially ischemia-induced myocardial infarction and chronic heart failure.

[0011] Based on the above objectives, the core problem that this invention aims to solve is: addressing the unsatisfactory effects of existing drug treatments.

[0012] [1. Compound]

[0013] The first aspect of this invention provides a compound or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, wherein the structure of the compound is shown in Formula I below:

[0014] R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas:

[0015] R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups;

[0016] n is selected from any integer from 1 to 5;

[0017] R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups.

[0018] According to any embodiment of the first aspect of the invention, a compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or a tautomer or a prodrug or mixture thereof, wherein n = 1 and R3 is hydrogen;

[0019] R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas:

[0020] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains.

[0021] According to any embodiment of the first aspect of the invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof, wherein R1 and R2 are each independently selected from hydrogen or a group having one of the following formulas:

[0022] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids.

[0023] In particular, a first aspect of the present invention provides a compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or a tautomer thereof or a prodrug thereof or a mixture thereof, said compound being any one of formula II-a, II-b, II-c or II-d:

[0024] Alternatively, the prodrug of the compound is of formula II-d;

[0025] Cellular experiments have shown that the compounds of general formula I of the present invention, especially those of formula II-a, II-b, II-c, or II-d, can simultaneously achieve anti-ischemic injury, promote angiogenesis, and provide neuroprotection, and have a high brain / blood distribution coefficient, making them suitable for the prevention, treatment, and improvement of cardiovascular and cerebrovascular diseases.

[0026] In the process of occurrence, development, treatment and prognosis of cerebrovascular diseases, especially ischemic stroke, the compounds represented by general formula I of this invention, especially those of formula II-a, II-b, II-c or II-d, can achieve the purpose of preventing, treating and improving cerebrovascular diseases, especially ischemic stroke, by promoting angiogenesis and neuroprotection synergistically, combined with their high brain / blood distribution coefficient.

[0027] The present invention further discovers that, in the occurrence, development, treatment and prognosis of cardiovascular diseases, especially chronic heart failure, the compounds represented by general formula I of the present invention, especially the compounds of formula II-a, II-b, II-c or II-d, achieve the purpose of preventing, treating and improving cardiovascular diseases, especially myocardial infarction and its induced chronic heart failure, through the synergistic effect of promoting angiogenesis and protecting against OGD / R-induced damage.

[0028] Compared to apigenin, compounds such as those shown in formula II-a and II-b have a stronger ability to cross the blood-brain barrier, making them ideal drugs for the treatment of cerebrovascular diseases, especially stroke.

[0029] This study found that some flavonoids not only promote angiogenesis, but also have a protective effect against OGD / R-induced damage at almost the same level as stroke drugs, such as apigenin (CAS: 520-36-5).

[0030] Further research in this invention has revealed that compounds represented by formulas II-a, II-b, II-c, or II-d have good protective effects against OGD / R-induced damage and promote angiogenesis, making them ideal drugs for the prevention and / or treatment of cerebrovascular diseases, especially stroke, and even more so ischemic stroke.

[0031] [2. Pharmaceutical Composition]

[0032] A second aspect of the present invention provides a pharmaceutical composition comprising a compound of formula I as described in any one of the first aspects of the present invention, or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof or a mixture thereof (hereinafter referred to as: active ingredient), and a pharmaceutically acceptable carrier or diluent.

[0033] A pharmaceutical composition refers to a composition in which the active ingredient is a compound or a pharmaceutically acceptable equivalent (a pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and a mixture thereof) described in any embodiment of the first or second aspect of the present invention, and which contains one or more pharmaceutically acceptable carriers or diluents.

[0034] The term "pharmaceutically acceptable carrier or diluent" as used in this invention refers to excipients, additives, or solvents commonly used in pharmaceutical preparations, including but not limited to lactose, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, lower alkyl ethers of cellulose, corn starch, potato starch, gums, fatty acids, fatty acid amines, glyceryl monostearate or glyceryl distearate, phospholipids, olive oil, peanut oil, syrups, colorants, flavoring agents, preservatives, water, ethanol, propanol, physiological saline, and glucose solution.

[0035] In particular, a second aspect of the present invention provides a pharmaceutical composition comprising a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0036] In particular, a second aspect of the present invention provides another pharmaceutical composition comprising a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0037] In particular, a second aspect of the present invention provides another pharmaceutical composition comprising a compound of formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0038] In particular, a second aspect of the present invention provides another pharmaceutical composition comprising a prodrug represented by formula II-d;

[0039] According to any embodiment of the second aspect of the present invention, the pharmaceutical composition is in a single-dose form, wherein the single-dose form contains 0.03 mg to 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer or a prodrug or mixture thereof, and a pharmaceutically acceptable carrier or diluent.

[0040] According to any embodiment of the second aspect of the present invention, the pharmaceutical composition thereof, or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, is administered as a single dose selected from any of the following numerical ranges: 0.03-400 mg, 0.03-300 mg, 0.05-280 mg, 0.08-250 mg, 0.1-220 mg, 0.15-200 mg, 0.2-180 mg, 0.25-150 mg, 0.3-100 mg, 0.35-80 mg, 0.4-50 mg, 0.45-30 mg, 0.5-20 mg, 0.8-20 mg, 1-20 mg, 1.5-20 mg, 2-10 mg, 2-8 mg, 2-6 mg, 2.5-5 mg, 2.5-4 mg.

[0041] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0042] As stated herein, “single dose” as used herein refers to a dose of medicine suitable for a single administration to a subject.

[0043] According to any embodiment of the second aspect of the present invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof is administered at a dose selected from any of the following numerical ranges: 0.01-20 mg / Kg, 0.01-10 mg / Kg, 0.01-5 mg / Kg, 0.01-4 mg / Kg, 0.01-3 mg / Kg, 0.01-2 mg / Kg, 0.01-1 mg / Kg, 0.01-0.5 mg / Kg, 0.01-0.2 mg / Kg, 0.01-0.1 mg / Kg.

[0044] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0045] According to any embodiment of the second aspect of the present invention, the pharmaceutical composition thereof, wherein the compound thereof or a pharmaceutically acceptable salt, ester, hydrate, solvate thereof or a tautomer thereof or a prodrug thereof or a mixture thereof, is administered to a mammal, particularly a human, at a dose of 0.02 to 20 mg / kg / day / person.

[0046] Preferably, the medication is administered to the patient at a dose of 0.05–5 mg / kg / day / person.

[0047] Preferably, the medication is administered to the patient at a dose of 0.2–2 mg / kg / day / person.

[0048] When administered within the above dosage range, the pharmaceutical composition did not exhibit any toxic side effects.

[0049] According to any embodiment of the second aspect of the present invention, the pharmaceutical composition wherein the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof comprises 1 to 99 wt% by mass.

[0050] According to any embodiment of the second aspect of the present invention, the drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the drug composition is 0.01 to 10 mg / mL.

[0051] The pharmaceutical composition according to any embodiment of the second aspect of the present invention further includes an agent selected from at least one of the following:

[0052] Combination medications other than those for the treatment of cerebrovascular diseases or cardiovascular diseases;

[0053] Other treatments for cerebrovascular diseases;

[0054] Other cardiovascular disease treatments.

[0055] According to any embodiment of the pharmaceutical composition of the second aspect of the present invention, the other cerebrovascular disease treatment agent is selected from any one, two or more of the following:

[0056] A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating inflammation in the brain;

[0057] A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating the volume of cerebral infarction;

[0058] Treatment agents for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating reduced blood flow to the brain;

[0059] A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or relieving cerebral blood flow interruption;

[0060] A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating cerebral hemorrhage.

[0061] According to any embodiment of the second aspect of the present invention, the combination drug other than the cerebrovascular disease treatment agent or the cardiovascular disease treatment agent is selected from any one, two or more of the following:

[0062] Combination drugs with anti-cerebral thrombosis effects;

[0063] Combination drugs with antiplatelet activity;

[0064] Combination drugs with thrombolytic effects;

[0065] Combination drugs with antioxidant effects;

[0066] Combination drugs with anticoagulant effects;

[0067] Combination drugs that have cholesterol-lowering effects.

[0068] According to any embodiment of the second aspect of the present invention, the combination drug other than the cerebrovascular disease treatment agent or the cardiovascular disease treatment agent, or other cerebrovascular disease treatment agents or cardiovascular disease treatment agents, includes any one, two or more of butylphthalide, edaravone, dextroborneol, aspirin, clopidogrel, dipyridamole, prasugrel, ticagrelor, heparin, warfarin, dabigatran, apixaban, rivaroxaban and atorvastatin.

[0069] The compounds of the present invention, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers thereof, or their prodrugs and mixtures thereof, can be used in combination with drugs, such as agents for the treatment of diabetes, agents for the treatment of diabetic complications, agents for the treatment of hyperlipidemia, agents for the treatment of arteriosclerosis, agents for the treatment of hypertension, agents for the treatment of obesity, diuretics, agents for the treatment of gout, agents for the treatment of thrombosis, anti-inflammatory agents, chemotherapeutic agents, immunotherapeutic agents, agents for the treatment of osteoporosis, agents for the treatment of dementia, agents for the treatment of erectile dysfunction, agents for the treatment of urinary incontinence / frequency, agents for the treatment of dysuria, etc. (hereinafter referred to as combination drugs). These combination drugs can be low molecular weight compounds, high molecular weight proteins, peptides, antibodies, vaccines, etc.

[0070] There are no restrictions on the timing of administration of the above-mentioned combined drugs. The compounds of the present invention, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers, or their prodrugs, mixtures, or pharmaceutical compositions thereof, can be administered to the subject simultaneously or at different times. The dosage of the combined drugs can be based on clinical dosage and can be appropriately determined according to the subject, route of administration, disease, combination, etc. The above-mentioned combined drugs can be a combination of two or more in suitable proportions.

[0071] Examples of the above-mentioned administration modes include the following: (1) administering a single formulation obtained by simultaneously processing the compound or pharmaceutical composition of the present invention and a combination drug; (2) administering two formulations of the compound or pharmaceutical composition of the present invention and a combination drug prepared separately via the same route of administration; (3) administering two formulations of the compound or pharmaceutical composition of the present invention and a combination drug prepared separately via the same route of administration in an alternating manner; (4) administering two formulations of the compound or pharmaceutical composition of the present invention and a combination drug prepared separately via different routes of administration; (5) administering two formulations of the compound or pharmaceutical composition of the present invention and a combination drug prepared separately via different routes of administration in an alternating manner (e.g., administering in the order of the compound or pharmaceutical composition of the present invention and the combination drug, or in the reverse order), etc.

[0072] According to any embodiment of the second aspect of the present invention, the dosage form of the pharmaceutical composition is selected from one or more of powder, granule, tablet, pill, capsule, sustained-release, controlled-release, injection, infusion or suspension.

[0073] According to any embodiment of the second aspect of the present invention, the pharmaceutical composition is in the form of a tablet; the tablet is a sublingual tablet; or

[0074] The dosage form of the pharmaceutical composition is an injection, and the injection further contains a solvent.

[0075] According to any embodiment of the second aspect of the present invention, the solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and aqueous cyclodextrin solutions;

[0076] Preferably, the alcohol solvent includes one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol;

[0077] The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether;

[0078] The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone;

[0079] The sulfur-containing organic solvent includes dimethyl sulfoxide;

[0080] The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution.

[0081] A second aspect of the invention also provides a medicine box comprising one or more single-dose units of any compound of any one aspect of the invention, or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, or a pharmaceutical composition comprising one or more single-dose units of any one aspect of the invention, and instructions for use in treating a disease.

[0082] Preferably, the medicine box further includes a medicine selected from at least one of the following:

[0083] Combination medications other than those for the treatment of cerebrovascular diseases or cardiovascular diseases;

[0084] Other treatments for cerebrovascular diseases;

[0085] Other cardiovascular disease treatments.

[0086] Preferably, the pharmaceutical agent comprises any one, two or more of butylphthalide, edaravone, dextroborneol, aspirin, clopidogrel, dipyridamole, prasugrel, ticagrelor, heparin, warfarin, dabigatran, apixaban, rivaroxaban and atorvastatin.

[0087] As mentioned in this article, "cardiovascular and cerebrovascular diseases" refers to a general term for diseases of the heart and brain blood vessels, broadly encompassing ischemic or hemorrhagic diseases of the heart, brain, and other tissues caused by conditions such as hyperlipidemia, high blood viscosity, atherosclerosis, and hypertension. Specifically, it includes cardiovascular diseases or cerebrovascular diseases.

[0088] As discussed in this article, "cerebrovascular disease" refers to brain dysfunction caused by cerebrovascular lesions or blood flow disorders due to various reasons, including neurological dysfunction caused by vascular occlusion, vascular rupture, vascular wall damage, or abnormal blood components. Examples include asymptomatic cerebrovascular diseases, transient ischemic attacks, stroke, cerebrovascular dementia, hypertensive encephalopathy, cerebral infarction, etc.

[0089] As mentioned in this article, "stroke" is a general term for acute cerebrovascular diseases of the brain. Stroke is also known as "cerebrovascular accident" or "stroke", and specifically includes hemorrhagic stroke and ischemic stroke.

[0090] As described in this article, "ischemic stroke" refers to a stroke caused by insufficient blood supply to neurons due to vascular blockage, also known as "cerebral infarction," "cerebral infarction," or "cerebral stroke."

[0091] As discussed in this article, "cardiovascular disease" refers to diseases of the heart or blood vessels, also known as circulatory system diseases, which are a series of diseases involving the circulatory system. Specifically, these include chronic heart failure, myocardial infarction, coronary heart disease, ischemic stroke, peripheral arterial vascular disease, deep vein thrombosis, and pulmonary embolism.

[0092] As described in this article, "chronic heart failure" also refers to the gradual appearance and persistence of symptoms and signs of heart failure (such as shortness of breath, fatigue, and edema of the limbs) on the basis of pre-existing chronic heart disease.

[0093] As mentioned in this article, "myocardial infarction," "myocardial infarction," or "myocardial rupture" refers to a serious coronary heart disease caused by acute blockage of the coronary arteries (the arteries that supply blood to the heart), resulting in ischemic necrosis of part of the myocardium. It is common in the elderly and manifests as severe chest pain.

[0094] As mentioned in this article, "coronary heart disease" generally refers to coronary atherosclerotic heart disease. Coronary heart disease is a heart disease caused by atherosclerotic lesions in the coronary arteries, leading to narrowing or blockage of the blood vessel lumen, resulting in myocardial ischemia, hypoxia, or necrosis.

[0095] As described in this article, "peripheral arterial vascular disease" is a chronic ischemic disease of the limbs, characterized by narrowing and blockage of antegrade blood flow in major systemic arteries other than the brain and coronary circulation system.

[0096] The term "prevention" as used herein refers to administering the compounds or pharmaceutical compositions of the present invention to a subject before the onset of the disease or symptoms, in order to avoid the occurrence of the disease or symptoms or to reduce the risk of the occurrence of the disease or symptoms.

[0097] The “reduction in the risk of disease or symptoms” described herein refers to a subject’s lower likelihood of developing a disease or symptom than an equivalent control individual, for example, a subject given the compound or pharmaceutical composition of the present invention while the control did not receive treatment or medication.

[0098] As used herein, the term "treatment" refers to the relief of symptoms or complications by suppressing, alleviating, or eradicating a disease state or its symptoms, to delaying disease progression, and / or to curing or eliminating the disease. Patients wishing to be treated are preferably mammals, particularly humans.

[0099] As described herein, the term "therapeutic and / or preventative effective amount" for the pharmaceutical compositions of the present invention refers to an amount sufficient to cure, alleviate, or partially prevent the clinical manifestations of a given disease and its complications in a therapeutic intervention including administration of the composition. An amount sufficient to achieve the above is defined as a "therapeutic and / or preventative effective amount." The effective amount for each purpose will depend on the severity of the disease or lesion and the subject's weight and general condition. However, it should be recognized that the total daily dosage of the pharmaceutical compositions of the present invention must be determined by the attending physician within the bounds of reliable medical judgment. For any specific patient, the specific therapeutically effective dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific pharmaceutical composition used; the specific pharmaceutical composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific pharmaceutical composition used; the duration of treatment; other drugs used in combination with or concurrently with the pharmaceutical composition used; and similar factors known in the medical field. For example, it is practiced in the art to start the dose of the pharmaceutical composition below the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0100] The term “treatment of disease” refers to reducing the frequency or severity of at least one symptom or sign of a disease or condition experienced by a subject.

[0101] The term "pharmaceutically acceptable salt" refers to an acidic or basic salt of a compound of the present invention, which has the desired pharmaceutical activity and is biologically and otherwise undesirable. Acidic salts include inorganic acid salts and organic acid salts. Inorganic acids include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, and methanesulfonic acid; organic acids include, but are not limited to, acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid, and tartaric acid.

[0102] Preferably, the pharmaceutically acceptable salt is a hydrochloride salt.

[0103] Preferably, the pharmaceutically acceptable salt is selected from one or more of aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganese salts, manganese salts, potassium salts, sodium salts, or zinc salts.

[0104] As described herein, when a compound of Formula I or a compound of Formula II-a or II-b or II-c or II-d has isomers, such as optical isomers, stereoisomers, positional isomers, rotational isomers, etc., any isomers and mixtures of isomers are included within the scope of a compound of Formula I or a compound of Formula II. For example, when a compound of Formula I or a compound of Formula II-a or II-b or II-c or II-d has an optical isomer, the optical isomer separated from the racemic mixture is also included within the scope of a compound of Formula I or a compound of Formula II-a or II-b or II-c or II-d. These isomers can be obtained as individual products by synthetic or separation methods (e.g., concentration, solvent extraction, column chromatography, recrystallization, etc.).

[0105] Compounds of Formula I, II-a, II-b, II-c, and II-d may be in crystalline or amorphous form. When the compound is crystalline, both single crystals and crystalline mixtures are included within the scope of the compound. Crystals may be prepared according to crystallization methods known in the art.

[0106] Compounds of Formula I, II-a, II-b, II-c, and II-d can be pharmaceutically acceptable cocrystals or cocrystal salts. In this document, a cocrystal or cocrystal salt refers to a crystalline substance composed of two or more specific solids, each possessing different physical properties (e.g., structure, melting point, heat of fusion, etc.) at room temperature. Cocrystals and cocrystal salts can be prepared using co-crystallization methods known in the art.

[0107] Compounds of Formula I, II-a, II-b, II-c, and II-d can be solvates (e.g., hydrates, etc.) or non-solvates, both of which are included within the scope of compounds of Formula I or II-a or II-b.

[0108] Isotopes can be used (e.g.) 2 H, 3 H, 14 Compounds of formula I, II-a, II-b, II-c, and II-d are designated as such.

[0109] Deuterated compounds (of which) 1 H has been transformed 2H(D) is also included in the range of compounds of formula I, II-a, II-b, II-c, and II-d.

[0110] As described herein, "prodrugs," also known as prodrug precursors, are compounds that may have weak or no activity on their own, but which, after administration, are converted under physiological conditions (e.g., through metabolism, solvation, or other means) into a biologically active form of compound of general formula I, II-a, II-b, II-c, or II-d. In one context, a prodrug refers to a prodrug that, upon metabolism in vivo, produces compound of general formula I, II-a, II-b, II-c, or II-d.

[0111] [3. Therapeutic applications of the compound in cerebrovascular diseases]

[0112] In a third aspect, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases.

[0113] R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas:

[0114] R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups;

[0115] n is selected from any integer from 1 to 5;

[0116] R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups.

[0117] According to any embodiment of the third aspect of the present invention, in the compound of formula I:

[0118] The n = 1 and R3 is hydrogen;

[0119] R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas:

[0120] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains.

[0121] According to any embodiment of the third aspect of the present invention, in the compound of formula I:

[0122] R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas:

[0123] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids.

[0124] In particular, a third aspect of the invention provides the use of compounds of formula II-a or pharmaceutically acceptable salts, esters, hydrates, solvates or tautomers thereof or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cerebrovascular diseases.

[0125] Experiments have shown that compounds of formula II-a, or pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers thereof, and mixtures thereof, when used to treat cerebrovascular diseases, can simultaneously promote angiogenesis and provide neuroprotection.

[0126] In particular, a third aspect of the invention provides the use of compounds of formula II-b or pharmaceutically acceptable salts, esters, hydrates, solvates or tautomers thereof or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cerebrovascular diseases.

[0127] Experiments have shown that compounds of formula II-b, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers, and mixtures thereof, when used to treat cerebrovascular diseases, can simultaneously promote angiogenesis and provide neuroprotection. In the occurrence, development, treatment, and prognosis of cerebrovascular diseases, particularly ischemic stroke, compounds of formula II-b, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers, and mixtures thereof, through the synergistic effect of promoting angiogenesis and providing neuroprotection, can improve blood perfusion in ischemic areas and reduce infarct volume, thereby achieving the goal of preventing, treating, or improving cerebrovascular diseases, especially transient or permanent ischemic stroke.

[0128] In particular, the use of compounds of formula II-c or their pharmaceutically acceptable salts, esters, hydrates, solvates or their tautomers or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cerebrovascular diseases;

[0129] In particular, the prodrug shown in Formula II-d is used for the preparation of drugs for the prevention or treatment of cerebrovascular diseases;

[0130] According to any embodiment of the third aspect of the present invention, the compound described in any embodiment of the first aspect of the present invention, or its pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, and mixtures thereof, are also used for the prevention and inhibition of the gradual development of secondary diseases, such as cerebrovascular diseases (e.g., stroke).

[0131] A third aspect of the present invention also provides the use of a pharmaceutical composition for preparing a medicament for the prevention or treatment of cerebrovascular diseases, the pharmaceutical composition comprising a compound of general formula I as described in any embodiment of the first aspect of the present invention, or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof, or a prodrug thereof, and a mixture thereof, and a pharmaceutically acceptable carrier or diluent.

[0132] Specifically, a third aspect of the present invention provides the use of a pharmaceutical composition for preparing a drug for the prevention or treatment of cerebrovascular diseases, the pharmaceutical composition comprising a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0133] Wherein, the compound represented by Formula II-a or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug or mixture thereof may be the sole active ingredient in the pharmaceutical composition or one of the active ingredients in the pharmaceutical composition.

[0134] Specifically, a third aspect of the present invention provides the use of another pharmaceutical composition for preparing a drug for the prevention or treatment of cerebrovascular diseases, said pharmaceutical composition comprising a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0135] Wherein, the compound represented by Formula II-b or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug or mixture thereof may be the sole active ingredient in the pharmaceutical composition or one of the active ingredients in the pharmaceutical composition.

[0136] In particular, a third aspect of the present invention provides the use of another pharmaceutical composition for preparing a medicament for the prevention or treatment of cerebrovascular diseases, said pharmaceutical composition comprising a compound represented by formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent.

[0137] In particular, a third aspect of the present invention provides the use of another pharmaceutical composition for preparing a medicament for the prevention or treatment of cerebrovascular diseases, said pharmaceutical composition comprising a prodrug represented by formula II-d;

[0138] According to any embodiment of the third aspect of the present invention, the cerebrovascular disease includes:

[0139] Cerebrovascular diseases characterized by deficits in brain neurological function; or

[0140] Cerebrovascular diseases characterized by increased inflammation in the brain; or

[0141] Cerebrovascular diseases characterized by an increase in the volume of cerebral infarction; or

[0142] Cerebrovascular diseases characterized by reduced or interrupted blood flow to the brain; or

[0143] Cerebrovascular diseases characterized by rupture of cerebral blood vessels; or

[0144] Cerebrovascular diseases characterized by hemorrhage caused by ruptured blood vessels in the brain.

[0145] Existing research has found that cerebrovascular diseases are typically characterized by brain neurological deficits, increased brain inflammation, increased infarct volume, reduced or interrupted cerebral blood flow, ruptured cerebral blood vessels, and hemorrhage caused by ruptured cerebral blood vessels. Cell experiments of this invention show that the compounds of formula I, especially compounds of formula II-a or II-b, formula II-c, and formula II-d, have anti-ischemic injury, angiogenesis promotion, and neuroprotective effects. Among them, animal experiments show that compounds of formula II-b or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers thereof, as well as mixtures thereof, have a significant effect on improving and treating cerebral infarction volume in mouse models.

[0146] According to any embodiment of the third aspect of the present invention, the cerebrovascular disease includes:

[0147] Ischemic cerebrovascular disease; or

[0148] Hemorrhagic cerebrovascular disease; or

[0149] Atherosclerosis, stenosis, or occlusion of the arteries in the head and neck.

[0150] According to any embodiment of the third aspect of the present invention, the cerebrovascular disease includes any one, two or more of the following: transient ischemic attack, cerebral infarction, stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, chronic cerebral ischemia, cerebral hemorrhage, cerebral hemorrhage, and subarachnoid hemorrhage.

[0151] According to any embodiment of the third aspect of the present invention, the stroke is an ischemic stroke.

[0152] According to any embodiment of the third aspect of the present invention, the ischemic stroke is selected from one or more of transient ischemic stroke, permanent ischemic stroke, acute ischemic stroke, and acute cerebral infarction.

[0153] According to any embodiment of the third aspect of the present invention, the single-dose form of the medicament for the prevention or treatment of cerebrovascular diseases contains between 0.03 mg and 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer or prodrug or mixture thereof.

[0154] According to any embodiment of the third aspect of the present invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof (active ingredient) is administered as a single dose selected from any of the following numerical ranges: 0.03-400 mg, 0.03-300 mg, 0.05-280 mg, 0.08-250 mg, 0.1-220 mg, 0.15-200 mg, 0.2-180 mg, 0.25-150 mg, 0.3-100 mg, 0.35-80 mg, 0.4-50 mg, 0.45-30 mg, 0.5-20 mg, 0.8-20 mg, 1-20 mg, 1.5-20 mg, 2-10 mg, 2-8 mg, 2-6 mg, 2.5-5 mg, 2.5-4 mg.

[0155] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0156] As stated herein, “single dose” as used herein refers to a dose of medicine suitable for a single administration to a subject.

[0157] According to any embodiment of the third aspect of the invention, the active ingredient is administered at a dose selected from any of the following numerical ranges: 0.01-20 mg / Kg, 0.01-15 mg / Kg, 0.01-10 mg / Kg, 0.01-5 mg / Kg, 0.01-4 mg / Kg, 0.01-3 mg / Kg, 0.01-2 mg / Kg, 0.01-1 mg / Kg, 0.01-0.5 mg / Kg, 0.01-0.2 mg / Kg, 0.01-0.1 mg / Kg.

[0158] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0159] According to any embodiment of the third aspect of the invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof is administered to a mammal, particularly a human, at a dose of 0.02 to 20 mg / kg / day / person.

[0160] Preferably, the medication is administered to the patient at a dose of 0.05–5 mg / kg / day / person.

[0161] Preferably, the medication is administered to the patient at a dose of 0.2–2 mg / kg / day / person.

[0162] When administered within the above dosage range, the pharmaceutical composition did not exhibit any toxic side effects.

[0163] According to any embodiment of the third aspect of the present invention, the mass percentage of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the pharmaceutical composition is 1 to 99 wt%.

[0164] According to any embodiment of the third aspect of the present invention, the pharmaceutical composition wherein the mass percentage of the active ingredient in the pharmaceutical composition is selected from any of the following numerical ranges: 1–99 wt%, 30–99 wt%, 30–95 wt%, 30–90 wt%, 30–85 wt%, 30–80 wt%, 30–75 wt%, 30–70 wt%, 30–65 wt%, 30–60 wt%, 30–55 wt%, 30–50 wt%, 40–99 wt%, 40–95 wt%, 40–90 wt%, 40–85 wt%, 40–80 wt%, 40–75 wt%, 40–70 wt%, 40–65 wt%, 40–60 wt%, 40–55 wt%, 40–50 wt%. 50~99wt%, 50~95wt%, 50~90wt%, 50~85wt%, 50~80wt%, 50~75wt%, 50~70wt%, 50~6 5wt%, 50~60wt%, 50~55wt%, 60~99wt%, 60~95wt%, 60~90wt%, 60~85wt%, 60~80wt%, 60~75wt%, 60~70wt%, 60~65wt%, 70~99wt%, 70~95wt%, 70~90wt%, 70~85wt%, 70~8 0wt%, 70~75wt%, 80~99wt%, 80~95wt%, 80~90wt%, 80~85wt%, 90~99wt%, 90~95wt%.

[0165] According to any embodiment of the third aspect of the present invention, the drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the drug for the prevention or treatment of cerebrovascular diseases is 0.01 to 10 mg / mL.

[0166] As a preferred embodiment of any of the third aspects of the present invention, the concentration (drug loading concentration) of the active ingredient in the pharmaceutical composition is selected from any of the following numerical ranges: 0.01-10 mg / mL, 0.01-8 mg / mL, 0.01-5 mg / mL, 0.01-3 mg / mL, 0.01-2 mg / mL, 0.01-1 mg / mL, 0.1-10 mg / mL, 0.1-8 mg / mL, 0.1-5 mg / mL, 0.1- 3mg / mL, 0.1-2mg / mL, 0.1-1mg / mL, 1-10mg / mL, 1-8mg / mL, 1-5mg / mL, 1-3mg / mL, 1-2mg / mL, 2-10mg / mL , 2-8mg / mL, 2-5mg / mL, 2-3mg / mL, 4-10mg / mL, 4-8mg / mL, 4-5mg / mL, 6-10mg / mL, 6-8mg / mL, 8-10mg / mL.

[0167] According to any embodiment of the third aspect of the present invention, the medicament for the prevention or treatment of cerebrovascular diseases further comprises a pharmaceutically acceptable carrier or diluent.

[0168] According to any embodiment of the third aspect of the present invention, the dosage form of the drug for preventing or treating cerebrovascular diseases is selected from one or more of powder, granule, tablet, pill, capsule, sustained-release, controlled-release, injection, infusion or suspension.

[0169] According to any embodiment of the third aspect of the present invention,

[0170] The dosage form of the drug for the prevention or treatment of cerebrovascular diseases is a tablet; the tablet is a sublingual tablet; or

[0171] The dosage form of the drug for the prevention or treatment of cerebrovascular diseases is an injection, and the injection also contains a solvent.

[0172] According to any embodiment of the third aspect of the present invention, the solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and aqueous cyclodextrin solutions;

[0173] Preferably, the alcohol solvent includes one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol;

[0174] The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether;

[0175] The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone;

[0176] The sulfur-containing organic solvent includes dimethyl sulfoxide;

[0177] The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution.

[0178] In a third aspect, the present invention also provides the use of the compound described in any of the above embodiments, or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, for the treatment of cerebrovascular diseases.

[0179] [4. Therapeutic applications of the compound in cardiovascular diseases]

[0180] In a fourth aspect, the present invention provides the use of a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases.

[0181] R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas:

[0182] R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups;

[0183] n is selected from any integer from 1 to 5;

[0184] R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups.

[0185] According to any embodiment of the fourth aspect of the present invention, in the compound of formula I:

[0186] The n=1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas:

[0187] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains.

[0188] According to any embodiment of the fourth aspect of the present invention, in the compound of formula I:

[0189] R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas:

[0190] R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids.

[0191] In particular, the fourth aspect of the invention provides the use of compounds of formula II-a or pharmaceutically acceptable salts, esters, hydrates, solvates or tautomers thereof or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cardiovascular diseases.

[0192] In particular, the fourth aspect of the invention provides the use of compounds of formula II-b or pharmaceutically acceptable salts, esters, hydrates, solvates or tautomers thereof or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cardiovascular diseases.

[0193] The compound developed in this invention exhibits unique advantages by alleviating ischemic damage and promoting angiogenesis, thereby protecting cardiomyocytes and inhibiting the development of cardiovascular diseases, especially heart failure. Compared with traditional drugs, this compound fundamentally alleviates the pathological process of myocardial ischemia by promoting angiogenesis and assisting in vascular reconstruction. Simultaneously, it inhibits apoptosis, reduces ischemic damage, and buys valuable time for vascular reconstruction. Its multi-target effect synergistically improves cardiac function, providing new potential for comprehensive intervention in cardiovascular diseases, especially after myocardial infarction. The discovery of this compound provides a novel and promising treatment for cardiovascular diseases, particularly ischemic myocardial infarction and its resulting heart failure. With its multi-target mechanism of action, excellent pharmacokinetic properties, and low toxicity, this small molecule compound shows great application potential in improving cardiovascular diseases, especially ischemic myocardial injury, and alleviating heart failure.

[0194] Experiments have shown that compounds of formula II-b, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers, and mixtures thereof, when used to treat cardiovascular diseases, can simultaneously promote angiogenesis and provide neuroprotection. In the occurrence, development, treatment, and prognosis of cardiovascular diseases, particularly chronic heart failure, compounds of formula II-b, or their pharmaceutically acceptable salts, esters, hydrates, solvates, or tautomers, and mixtures thereof, through the synergistic effects of promoting angiogenesis and providing neuroprotection, can improve cardiac systolic function and alleviate myocardial ischemia, thereby achieving the goal of preventing, treating, or improving cardiovascular diseases, especially chronic heart failure.

[0195] In particular, the use of compounds of formula II-c or their pharmaceutically acceptable salts, esters, hydrates, solvates or their tautomers or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cardiovascular diseases;

[0196] In particular, the prodrug shown in Formula II-d is used for the preparation of drugs for the prevention or treatment of cardiovascular diseases;

[0197] The fourth aspect of the present invention also provides the use of a pharmaceutical composition for preparing a medicament for the prevention or treatment of cardiovascular diseases, the pharmaceutical composition comprising a compound of general formula I as described in any embodiment of the first aspect of the present invention, or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof, or a prodrug thereof, and a mixture thereof, and a pharmaceutically acceptable carrier or diluent.

[0198] Specifically, the fourth aspect of the present invention provides the use of a pharmaceutical composition for preparing a drug for the prevention or treatment of cardiovascular diseases, the pharmaceutical composition comprising a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0199] Wherein, the compound represented by Formula II-a or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug or mixture thereof may be the sole active ingredient in the pharmaceutical composition or one of the active ingredients in the pharmaceutical composition.

[0200] Specifically, the fourth aspect of the present invention provides the use of another pharmaceutical composition for preparing a medicament for the prevention or treatment of cardiovascular diseases, said pharmaceutical composition comprising a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent;

[0201] Wherein, the compound represented by Formula II-b or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug or mixture thereof may be the sole active ingredient in the pharmaceutical composition or one of the active ingredients in the pharmaceutical composition.

[0202] In particular, a fourth aspect of the present invention provides the use of another pharmaceutical composition for preparing a medicament for the prevention or treatment of cardiovascular diseases, said pharmaceutical composition comprising a compound represented by formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent.

[0203] In particular, a fourth aspect of the present invention provides the use of another pharmaceutical composition for preparing a medicament for the prevention or treatment of cardiovascular diseases, said pharmaceutical composition comprising a prodrug represented by formula II-d;

[0204] According to any embodiment of the fourth aspect of the present invention, the cardiovascular disease includes:

[0205] Cardiovascular diseases characterized by impaired myocardial function; or

[0206] Cardiovascular diseases characterized by angina pectoris; or

[0207] Cardiovascular diseases characterized by myocardial infarction; or

[0208] Cardiovascular diseases characterized by ischemic cardiomyopathy; or

[0209] Cardiovascular disease characterized by chronic heart failure.

[0210] According to any embodiment of the fourth aspect of the present invention, the compounds of the present invention can be effectively used as agents to inhibit or improve the gradual development and deterioration of cardiovascular diseases, such as cardiac hypertrophy, acute heart failure, chronic heart failure, congestive heart failure, weakened vasodilation, cardiomyopathy, angina pectoris, myocarditis, atrial fibrillation, arrhythmia, tachycardia, myocardial infarction, etc.

[0211] According to any embodiment of the fourth aspect of the present invention, the cardiovascular disease is selected from chronic heart failure, myocardial infarction, angina pectoris, coronary heart disease, and peripheral arterial vascular disease.

[0212] Preferably, the cardiovascular disease is ischemic heart failure.

[0213] According to any embodiment of the fourth aspect of the present invention, the single-dose form of the medicament for the prevention or treatment of cardiovascular diseases contains between 0.03 mg and 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer or prodrug or mixture thereof.

[0214] According to any embodiment of the fourth aspect of the present invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof (active ingredient) is administered as a single dose selected from any of the following numerical ranges: 0.03-400 mg, 0.03-300 mg, 0.05-280 mg, 0.08-250 mg, 0.1-220 mg, 0.15-200 mg, 0.2-180 mg, 0.25-150 mg, 0.3-100 mg, 0.35-80 mg, 0.4-50 mg, 0.45-30 mg, 0.5-20 mg, 0.8-20 mg, 1-20 mg, 1.5-20 mg, 2-10 mg, 2-8 mg, 2-6 mg, 2.5-5 mg, 2.5-4 mg.

[0215] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0216] As stated herein, “single dose” as used herein refers to a dose of medicine suitable for a single administration to a subject.

[0217] According to any embodiment of the fourth aspect of the present invention, the active ingredient is administered at a dose selected from any of the following numerical ranges: 0.01-20 mg / Kg, 0.01-15 mg / Kg, 0.01-10 mg / Kg, 0.01-5 mg / Kg, 0.01-4 mg / Kg, 0.01-3 mg / Kg, 0.01-2 mg / Kg, 0.01-1 mg / Kg, 0.01-0.5 mg / Kg, 0.01-0.2 mg / Kg, 0.01-0.1 mg / Kg.

[0218] Satisfactory results are obtained when administered in a single dose as described above. It is preferable to administer the dose 1, 2, or 3 times daily, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0219] According to any embodiment of the fourth aspect of the invention, the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug or mixture thereof is administered to a mammal, particularly a human, at a dose of 0.02 to 20 mg / kg / day / person.

[0220] Preferably, the medication is administered to the patient at a dose of 0.05–5 mg / kg / day / person.

[0221] Preferably, the medication is administered to the patient at a dose of 0.2–2 mg / kg / day / person.

[0222] When administered within the above dosage range, the pharmaceutical composition did not exhibit any toxic side effects.

[0223] According to any embodiment of the fourth aspect of the present invention, the mass percentage of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixtures thereof in the pharmaceutical composition is 1 to 99 wt%.

[0224] According to any embodiment of the fourth aspect of the present invention, the mass percentage of the active ingredient in the pharmaceutical composition is selected from any of the following numerical ranges: 1–99 wt%, 30–99 wt%, 30–95 wt%, 30–90 wt%, 30–85 wt%, 30–80 wt%, 30–75 wt%, 30–70 wt%, 30–65 wt%, 30–60 wt%, 30–55 wt%, 30–50 wt%, 40–99 wt%, 40–95 wt%, 40–90 wt%, 40–85 wt%, 40–80 wt%, 40–75 wt%, 40–70 wt%, 40–65 wt%, 40–60 wt%, 40–55 wt%, 40–50 wt%. 50~99wt%, 50~95wt%, 50~90wt%, 50~85wt%, 50~80wt%, 50~75wt%, 50~70wt%, 50~6 5wt%, 50~60wt%, 50~55wt%, 60~99wt%, 60~95wt%, 60~90wt%, 60~85wt%, 60~80wt%, 60~75wt%, 60~70wt%, 60~65wt%, 70~99wt%, 70~95wt%, 70~90wt%, 70~85wt%, 70~8 0wt%, 70~75wt%, 80~99wt%, 80~95wt%, 80~90wt%, 80~85wt%, 90~99wt%, 90~95wt%.

[0225] According to any embodiment of the fourth aspect of the present invention, the drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the medicament for the prevention or treatment of cardiovascular diseases is 0.01 to 10 mg / mL.

[0226] As a preferred embodiment of any aspect of the fourth aspect of the present invention, the concentration (drug loading concentration) of the active ingredient in the pharmaceutical composition is selected from any of the following numerical ranges: 0.01-10 mg / mL, 0.01-8 mg / mL, 0.01-5 mg / mL, 0.01-3 mg / mL, 0.01-2 mg / mL, 0.01-1 mg / mL, 0.1-10 mg / mL, 0.1-8 mg / mL, 0.1-5 mg / mL, 0.1- 3mg / mL, 0.1-2mg / mL, 0.1-1mg / mL, 1-10mg / mL, 1-8mg / mL, 1-5mg / mL, 1-3mg / mL, 1-2mg / mL, 2-10mg / mL , 2-8mg / mL, 2-5mg / mL, 2-3mg / mL, 4-10mg / mL, 4-8mg / mL, 4-5mg / mL, 6-10mg / mL, 6-8mg / mL, 8-10mg / mL.

[0227] According to any embodiment of the fourth aspect of the present invention, the medicament for the prevention or treatment of cardiovascular diseases further comprises a pharmaceutically acceptable carrier or diluent.

[0228] According to any embodiment of the fourth aspect of the present invention, the dosage form of the drug for preventing or treating cardiovascular diseases is selected from one or more of powders, granules, tablets, pills, capsules, sustained-release agents, controlled-release agents, injections, infusions, or suspensions.

[0229] Preferably, the dosage form of the drug for preventing or treating cardiovascular diseases is a tablet; the tablet is a sublingual tablet; or

[0230] The dosage form of the drug for the prevention or treatment of cardiovascular diseases is an injection, and the injection also contains a solvent.

[0231] Preferably, the solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and aqueous cyclodextrin solutions;

[0232] Preferably, the alcohol solvent includes one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol;

[0233] The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether;

[0234] The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone;

[0235] The sulfur-containing organic solvent includes dimethyl sulfoxide;

[0236] The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution.

[0237] According to any embodiment of the fourth aspect of the invention, the pharmaceutical composition can be specifically formulated as a formulation for administration via a suitable route of administration, such as oral, rectal, nasal, pulmonary, intraperitoneal, vaginal, and parenteral administration (including subcutaneous, intramuscular, intravenous, and intradermal administration), preferably by injection. It should be understood that the preferred route of administration depends on the overall condition and age of the treated subject, the nature of the disease or symptom being treated, and the selected active ingredient.

[0238] When the compounds of the present invention are administered parenterally, they are generally given in the form of liquid formulations (e.g., injections).

[0239] As an injectable preparation, it can be administered intravenously, subcutaneously, intradermally, intramuscularly, or by infusion. As a sustained-release formulation, it can be administered via iontophoresis. This injectable preparation is prepared using methods known in the prior art, namely, by dissolving, suspending, or emulsifying a compound of formula I or formula II-a, II-b, II-c, or II-d in a sterilized aqueous solution or oily liquid.

[0240] Furthermore, aqueous solutions for injection can be physiological saline, isotonic solutions containing glucose or other adjuvants (such as D-sorbitol, D-mannitol, sodium chloride, etc.), etc., and can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80), etc. As oily liquids, sesame oil, soybean oil, etc., can be used, and can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Additionally, buffer solutions (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol, etc.) can be mixed with it. The prepared injections are generally filled into vials.

[0241] In a fourth aspect, the present invention also provides the use of the compound described in any of the above embodiments, or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, for the treatment of cardiovascular diseases.

[0242] The definitions of “cardiovascular disease,” “cerebrovascular disease,” “stroke,” “ischemic stroke,” “chronic heart failure,” “myocardial infarction,” “coronary heart disease,” and “peripheral arterial vascular disease” used in this article are the same as those described above.

[0243] [definition]

[0244] As used herein, the term "solvent" refers to a compound that carries solvent molecules, such as a hydrate.

[0245] In this invention, the term "comprising" or "containing" indicates that various ingredients may be used together in the composition of this invention. Therefore, the terms "consistent with..." and "composed of..." are included in the term "comprising" or "containing".

[0246] In this invention, a "pharmaceuticalally acceptable" ingredient is a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., has a reasonable benefit / risk ratio.

[0247] The actual dosage level and route of administration of the active ingredient (compound of formula I or compound of formula II-a or II-b or II-c or II-d, or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or prodrug or mixture thereof) in the pharmaceutical composition of the present invention can be modified so that the resulting amount of active ingredient can effectively achieve the desired therapeutic response in a specific patient. The dosage level must be selected based on the activity of the specific active ingredient, the route of administration, the severity of the condition being treated, and the condition and medical history of the patient to be treated. However, it is the practice in the art to start the dose of the active ingredient below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0248] The definition of "treatment" as described here is the same as that mentioned above. 3. Beneficial effects

[0249] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0250] (1) Existing positive drugs mostly prevent or treat cardiovascular and cerebrovascular diseases such as ischemic stroke by inhibiting nerve cell death through neuroprotective and anti-inflammatory effects. This invention discovers a new compound within the general formula range of Formula I, especially compounds of Formula II-a, Formula II-b, Formula II-c and Formula II-d. Cell experiments show that it has neuroprotective effects, as well as promoting angiogenesis and protecting against ischemic-like damage. The anti-ischemic damage and the promotion of angiogenesis work synergistically to achieve the purpose of preventing or treating cardiovascular and cerebrovascular diseases such as ischemic stroke.

[0251] (2) Animal experiments further demonstrate that, in a mouse tMCAO stroke model, the application of compound II-b of this invention can significantly reduce the percentage of cerebral infarction volume in mice with cerebral ischemia; compound II-b provides an effective solution for the prevention and treatment of stroke. In a rat pMCAO ischemic stroke model, compound II-b of this invention can significantly reduce the cerebral infarction volume in rats.

[0252] (3) Animal experiments have further shown that the application of compound II-b of the present invention can significantly improve myocardial function in mouse models of ischemic myocardial infarction and its induced chronic heart failure. Compound II-b provides an effective solution for the prevention and treatment of ischemic myocardial infarction and its induced chronic heart failure.

[0253] (4) Flavonoids such as apigenin not only promote angiogenesis, but also have a better protective effect against OGD / R-induced damage than stroke drugs. However, because apigenin has difficulty crossing the blood-brain barrier, its brain / blood distribution coefficient is small. In contrast, compounds such as those shown in Formula II-b have a stronger ability to cross the blood-brain barrier than apigenin and existing positive drugs (such as butylphthalide), making them ideal drugs for the treatment of cardiovascular and cerebrovascular diseases, especially stroke.

[0254] (5) Cell experiments in the embodiments of the present invention show that, in the OGD / R injury model, the compound shown in Formula II-c has the same protective effect on vascular endothelial cells (HUVEC) and the effect of alleviating damage caused by oxygen and glucose deprivation as the compound shown in Formula II-b.

[0255] (6) Cell experiments in the embodiments of the present invention show that, in the OGD / R injury model, the compound shown in Formula II-d has the same effect as the compound shown in Formula II-b in promoting epithelial cell proliferation, protecting vascular endothelial cells (HUVEC), protecting myoblasts (H9c2), and alleviating damage caused by oxygen and glucose deprivation.

[0256] (6) Based on the effects of compounds within the general formula range of Formula I, especially those shown in Formula II-a, Formula II-b, Formula II-c and Formula II-d, on promoting angiogenesis and protecting against ischemic-like injury, the compounds shown in Formula I and pharmaceutical compositions containing the compounds shown in Formula I, especially those containing the compounds shown in Formula II-a, Formula II-b, Formula II-c and Formula II-d, are beneficial in improving collateral circulation, restoring blood oxygen supply, achieving blood supply, and thus inhibiting heart and brain injury, providing a synergistic prevention and / or treatment solution for cardiovascular and cerebrovascular diseases, especially chronic heart failure, myocardial infarction and stroke. Attached Figure Description

[0257] Figures 1 and 2 show the effects of apigenin (Api), butylphthalide (NBP), and edaravone-dexborneol (ED) on angiogenesis and anti-ischemic injury, among which:

[0258] Figure 1 shows the effects of the normal control group (labeled Ctrl), different concentrations of butylphthalide (1 μM and 3 μM, labeled NBP-1 and NBP-3, respectively), different concentrations of edaravone-dextromethorphanol (30 μM and 100 μM, labeled ED-30 and ED-100, respectively), and apigenin (0.3 μM, 1 μM, and 3 μM, labeled Api-0.3, Api-1, and Api-3, respectively) on the proliferation of human umbilical vein endothelial cells (HUVECs).

[0259] Figure 2 shows the effects of the normal control group (labeled Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and different concentrations of butylphthalide (1 μM and 3 μM, labeled NBP-1 and NBP-3, respectively), different concentrations of edaravone-dextropine (30 μM and 100 μM, labeled ED-30 and ED-100, respectively) under OGD / R injury on vascular endothelial cell survival (anti-ischemic injury).

[0260] Figures 3-5 illustrate the effects of EW266 (Formula II-a) on three aspects: anti-ischemic injury, angiogenesis, and neuronal proliferation.

[0261] Figure 3 shows the effects of different concentrations of EW266 (0.3 μM, 1 μM, 3 μM) on vascular endothelial cell survival in the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and the OGD / R injury group (anti-ischemic injury).

[0262] Figure 4 shows the effects of the blank control group (Ctrl) and different concentrations of EW266 (0.3 μM, 1 μM, 3 μM) on the proliferation of human umbilical vein endothelial cells (HUVEC).

[0263] Figure 5 shows the effects of the blank control group (Ctrl) and the EW266 group (0.3μM, 1μM, 3μM, 10μM) on promoting the proliferation of neural stem cells.

[0264] Figures 6-15 illustrate the effects of EW238 (Formula II-b) on four aspects: anti-ischemic injury, angiogenesis, neuronal cell proliferation, and synaptic growth.

[0265] Figure 6 shows the effects of normal control group (Ctrl), oxygen-glucose deprivation injury group (OGD / R), different concentrations of EW238 under OGD / R injury (0.3 μM, 1 μM, 3 μM), and apigenin group under OGD / R injury (Api, 3 μM) on vascular endothelial cell survival (anti-ischemic injury).

[0266] Figure 7 shows the effects of the blank control group (Ctrl), different concentrations of EW238 (0.3 μM, 1 μM, 3 μM), and apigenin group (Api, 3 μM) on the proliferation of human umbilical vein endothelial cells (HUVEC).

[0267] Figure 8 shows the effects of the blank control group (Ctrl), the EW238 group (3 μM), and the apigenin group (3 μM) on the proliferation of human brain microvascular endothelial cells (HBMEC).

[0268] Figure 9 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3 μM, and positive control group (VEGF) 20 ng / mL on the formation of tubular structures in vascular endothelial cells: tubular structures under a fluorescence microscope.

[0269] Figure 10 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: total length of tubular structures.

[0270] Figure 11 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: number of lumens.

[0271] Figure 12 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: number of branches.

[0272] Figure 13 shows the effects of the blank control group (Ctrl) and the EW238 group (1μM, 3μM, 10μM) on promoting the proliferation of neural stem cells.

[0273] Figure 14 shows representative microscopic images of the effects of the blank control group (Ctrl), the RA group (5 μM), and the EW238 group (5 μM) on promoting synaptic growth (all images were taken using an Olympus IX73 inverted microscope at 20x).

[0274] Figure 15 shows the effects of the blank control group (Ctrl), the RA group (retinoic acid, 5 μM), and the EW238 group (5 μM) on promoting synaptic growth.

[0275] Figures 16-17 show the effects of EW238 (Formula II-b) on a mouse model of transient middle cerebral artery embolism (tMCAO), where:

[0276] Figure 16 is a schematic diagram of the drug administration process for tMCAO model mice.

[0277] Figure 17 shows the representative and statistical graphs of cerebral infarction area after intravenous injection once a day for 3 consecutive days in the blank control group (Veh), the EW238 treatment group (2mg / kg / time, 6mg / kg / time) or the butylphthalide treatment group (NBP, 6mg / kg / time) under the tMCAO model.

[0278] Figures 18-22 show the effects of EW238 (Formula II-b) on cardiac function in mice with ischemic myocardial infarction and its induced chronic heart failure model (LAD ligation model), where:

[0279] Figure 18 is a schematic diagram of the drug administration process in LAD ligation model mice.

[0280] Figure 19 shows the effect of EW238 (Formula II-b) on the survival of mice in the LAD ligation model. Veh: model group, n=6; EW238: EW238 group, n=6.

[0281] Figure 20 shows the effect of EW238 (Formula II-b) on cardiac function in LAD ligation model mice (3 days post-surgery). Veh: model group, n=6; EW238: EW238 group, n=6. Data are expressed as mean ± SEM. Compared with Veh: **P<0.01.

[0282] Figure 21 shows the effect of EW238 (Formula II-b) on left ventricular ejection fraction (EF) (3 days post-surgery) in LAD ligation model mice. Veh: model group, n=6; EW238: EW238 group, n=6. Data are expressed as mean ± SEM. Compared with Veh: **P<0.01.

[0283] Figure 22 shows the effect of EW238 (Formula II-b) on the left ventricular fractional shortening (FS) value (3 days post-surgery) in LAD ligation model mice. Veh: model group, n=6; EW238: EW238 group, n=6. Data are expressed as mean ± SEM. Compared with Veh: **P<0.01.

[0284] Figure 23 shows the effect of EW238 (Formula II-b) on rats with permanent middle cerebral artery embolism (pMCAO model). Specifically, under the pMCAO model, the blank control group (Model), the EW238 treatment group (6 mg / kg / time), and the butylphthalide treatment group (NBP, 4 mg / kg / time) were injected intravenously twice a day for 7 consecutive days. The infarct area is represented by a graph (A) and a statistical graph (B).

[0285] Figures 24-25 show EW238. 1 H NMR and 13 C NMR.

[0286] Figure 24 shows EW238. 1 H NMR.

[0287] Figure 25 shows EW238. 13 C NMR.

[0288] Figure 26 shows the effects of the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and different concentrations of EW254 (0.3 μM, 1 μM, 3 μM) and EW238 (3 μM) under OGD / R injury on vascular endothelial cell survival (anti-ischemic injury).

[0289] Figures 27-29 illustrate the effects of EW274 (Formula II-d) on three aspects: promoting epithelial cell proliferation, protecting HUVECs against ischemic-like injury, and protecting H9c2s against ischemic injury.

[0290] Figure 27 shows the effects of the blank control group (Ctrl), different concentrations of EW274 (1 μM, 3 μM, 10 μM), and EW238 (3 μM) on the proliferation of human umbilical vein endothelial cells (HUVEC).

[0291] Figure 28 shows the effects of the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and different concentrations of EW274 (1 μM, 3 μM, 10 μM) and EW238 (3 μM) under OGD / R injury on vascular endothelial cell survival (anti-ischemic injury).

[0292] Figure 29 shows the effects of the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and different concentrations of EW274 (3μM, 10μM, 30μM) and EW238 (10μM) under OGD / R injury on the survival of H9c2 cells (anti-ischemic injury).

[0293] Figure 30 shows EW254. 1 H NMR.

[0294] Figure 31 shows EW274. 1H NMR. Detailed Implementation

[0295] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0296] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0297] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0298] Concentration, content, percentage content, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0299] Butylphthalide, purchased from WuXi AppTec, 1g specification.

[0300] Edaravone-dexborneol, wherein the molar ratio of edaravone to dexborneol is 4:1; edaravone was purchased from MCE, catalog number: HY-B0099; dexborneol was purchased from Sigma, catalog number: 420247; the concentration of the edaravone-dexborneol group in the examples is the concentration of edaravone.

[0301] RA (retinoic acid), purchased from MCE, product number: HY-14649.

[0302] Api, supplied by MCE, with a purity of ≥99.5%.

[0303] The compound shown in Formula II-a (EW266) has a purity ≥98%. It is prepared from DMSO to the required concentration for cell experiments.

[0304] The compound shown in Formula II-b (EW238) has a purity ≥98%. For cell experiments, it is prepared at the required concentration using DMSO.

[0305] The compound shown in Formula II-c (EW254) has a purity ≥98%. For cell experiments, it is prepared at the required concentration using DMSO.

[0306] The compound shown in Formula II-d (EW274) has a purity ≥98%. For cell experiments, it is prepared at the required concentration using DMSO.

[0307] The present invention will be further described below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0308] Example 1

[0309] Preparation of EW238

[0310] This invention synthesizes the target compound through the above steps:

[0311] That is, EW238.

[0312] Compound preparation examples

[0313] 1. Synthesis of M1

[0314] Under an argon atmosphere, SM (10.20 g, 60.0 mmol) and potassium carbonate (49.72 g, 360 mmol, 6.0 eq) were placed in a 500 mL three-necked flask. Acetone (200 mL) was added to dissolve the mixture. Dimethyl sulfate (34 mL, 360 mmol, 6.0 eq) was slowly added dropwise to the solution under an ice bath and stirred for 0.5 h. The ice bath was then removed, and the mixture was heated under reflux with stirring for 3 h until the starting material disappeared (monitored by TLC). The reaction solution was cooled to room temperature and quenched in a saturated sodium bicarbonate solution (500 mL). The combined organic phases were extracted with ethyl acetate (3 × 100 mL) and dried over anhydrous sodium sulfate (approximately 90 g). After filtration and solvent removal, the crude product was a yellow oily liquid. LC-MS: 227.1, [M+H] + After confirmation by mass spectrometry, it was directly used in the next reaction.

[0315] 2. Synthesis of M2

[0316] Under an argon atmosphere, M1 (approximately 13 g of crude product) was placed in a 500 mL three-necked flask and dissolved in dichloromethane (190 mL). A boron trichloride dichloromethane solution (75 mL, 75 mmol) was slowly added dropwise to the solution at -78 °C. The mixture was stirred for 15 min, then the ice bath was removed, and the temperature was raised to room temperature (22 °C). Stirring continued for 2 h until the starting material disappeared (monitored by TLC). The reaction mixture was cooled again to -78 °C, and then methanol (50 mL) was injected. The reaction mixture was raised to room temperature, stirred for 30 min, and then quenched in water. Extraction was performed with dichloromethane (1 × 200 mL). The organic phase was dried over anhydrous sodium sulfate (approximately 50 g), filtered, and the solvent was evaporated. The crude product was slurried with 15% ethyl acetate / cyclohexane for 1 h, filtered, and the filter cake was collected to give a yellow solid (7.15 g, 60% yield in two steps).

[0317] LC-MS: 213.1, [M+H] + .

[0318] 1 H NMR (500MHz, Chloroform-d) δ 12.06 (s, 1H), 6.14 (d, J = 2.3Hz, 1H), 5.99 (d, J = 2.3Hz, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H).

[0319] 13 C NMR (126MHz, Chloroform-d) δ 171.69, 165.99, 165.37, 162.17, 96.58, 93.45, 91.59, 56.09, 55.48, 52.20.3, Synthesis of M3

[0320] Under an argon atmosphere, M2 (7.12 g, 34 mmol) was placed in a 1 L three-necked flask and dissolved in dichloromethane (350 mL). Triethylamine (15.8 mL, 117 mmol, 3.5 eq.) and trifluoromethanesulfonic anhydride (7.3 mL, 50 mmol, 1.5 eq.) were slowly added dropwise to the solution at -20 °C. After the addition was complete, the mixture was stirred for 30 min, then heated to room temperature (22 °C) and stirred for 2 h until the starting material disappeared (monitored by TLC). The solution was then quenched in water and extracted with dichloromethane (2 × 200 mL). The combined organic phase was dried over anhydrous sodium sulfate (approximately 50 g). The mixture was filtered, and the solvent was evaporated. The crude product was purified by neutral alumina chromatography (eluent: 20% EA / PE) to give a yellow solid (10.23 g, yield 89%).

[0321] LC-MS: 345.0, [M+H] + .

[0322] 1 H NMR (500MHz, Chloroform-d) δ6.47 (d, J = 2.2 Hz, 1H), 6.42 (d, J = 2.2 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H).

[0323] 13 C NMR (126MHz, Chloroform-d) δ163.41,162.56,159.69,148.26,119.79,117.24,109.90,99.06,98.46,56.47,55.89,52.55.

[0324] 4. Synthesis of M4

[0325] Under an argon atmosphere, M3 (7.30 g, 21.2 mmol) was placed in a 500 mL three-necked flask, and DMF (200 mL) and triethylamine (50 mL) were added and stirred to dissolve. Argon gas was bubbled into the flask for 15 min using a balloon and a long needle in an ultrasonic bath. Then, under argon protection, cuprous iodide (410.5 mg, 2.12 mmol, 0.1 eq.) and bis(triphenylphosphine) palladium chloride (751.0 mg, 1.06 mmol, 0.05 eq.) were added. The reaction solution rapidly turned brown. Bubbling continued for 15 min; then phenylacetylene (3.6 mL, 31.8 mmol, 1.5 eq.) was added, followed by another 5 min of bubbling. The mixture was heated to 60 °C and stirred overnight. The solution was then quenched in water and extracted with ethyl acetate (3 × 300 mL). The organic phase was dried over anhydrous sodium sulfate (approximately 100 g). The solvent was removed by filtration and rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: 20% EA / PE) to give a yellow solid (2.10 g, yield 33%, containing some unseparated phenylacetylene).

[0326] LC-MS: 297.1, [M+H] + .

[0327] 5. Synthesis of M5

[0328] Under an argon atmosphere, M4 (1.50 g, 5.06 mmol) was placed in a 250 mL three-necked flask and dissolved in methanol (190 mL). Lithium hydroxide (4.25 g, 101 mmol, 20.0 eq. 10 mL aqueous solution) was added under ice bath conditions. The mixture was heated to room temperature and stirred overnight. The reaction solution was acidified to pH 2 with dilute hydrochloric acid (1 M) and extracted with ethyl acetate (3 × 200 mL). The combined organic phases were dried over anhydrous sodium sulfate (approximately 50 g). After filtration and solvent drying, the crude product was used directly in the next reaction step.

[0329] LC-MS: 283.1, [M+H] + .

[0330] 6. Synthesis of M6

[0331] Under an argon atmosphere, M5 (1.13 g, 4.00 mmol) was placed in a 250 mL three-necked flask, and trifluoromethanesulfonic acid (30 mL) was added to dissolve it. The mixture was heated to 80 °C and stirred for 2 h until the starting material disappeared (monitored by TLC). The reaction solution was cooled to room temperature and quenched with an ice-water mixture. Extraction was performed with ethyl acetate (2 × 100 mL), and the organic phase was dried over anhydrous sodium sulfate (approximately 50 g). After filtration and solvent removal, the crude product was purified by silica gel column chromatography (eluent: 20% EA / PE) to give a yellow solid (325.0 mg, yield 29%).

[0332] 1 H NMR (600MHz, CDCl3) δ7.77–7.74(m,2H),7.36–7.28(m,3H),7.15(s,1H),6.68(s,1H),6.36(d,J=2.6Hz,2H),3.87(d,J=4.5Hz,3H),3.81(s,3H).

[0333] 13 C NMR(151MHz, CDCl3)δ165.43(s),163.36(s),154.35(s),142.26(s),131.9 2(s),129.98(s),128.74(s),125.39(s),101.94(s),100.48(s),98.81(s).

[0334] 7. Synthesis of PD (EW238)

[0335] Under an argon atmosphere, M6 (623.0 mg, 2.21 mmol) was placed in a 100 mL three-necked flask. Dichloromethane (25 mL) was added to dissolve the product. Boron tribromide (1.4 mL, 13.2 mmol, 6.0 eq.) was slowly added dropwise to the solution at -78 °C and stirred for 15 min. The ice bath was then removed, and the mixture was heated to room temperature (22 °C) and stirred for 16 h until the starting material disappeared (monitored by TLC). The reaction solution was cooled again to -30 °C, and methanol (20 mL) was injected. The reaction solution was then heated to room temperature and stirred for 30 min before being poured into water to quench the reaction. The product was extracted from the aqueous phase with dichloromethane (3 × 200 mL). The organic phases were combined and dried with anhydrous sodium sulfate (approximately 50 g). After filtration and solvent removal, the crude product was purified by silica gel column chromatography (eluent: 10-20% EA / PE) to obtain a colorless solid (420.0 mg, yield 75%). EW238 1 H NMR and 13 The C NMR spectra are shown in Figures 24 and 25.

[0336] LC-MS: 255.1, [M+H] + .

[0337] LC-MS: 277.0, [M+Na] + .

[0338] Example 2

[0339] Preparation of EW266

[0340] This invention synthesizes the target compound EW266 through the above steps:

[0341] 1. Synthesis of intermediate 6

[0342] Palladium dichloride (55 mg, 0.31 mmol), triphenylphosphine (162 mg, 0.62 mmol), cuprous iodide (118 mg, 0.62 mmol), and triethylamine (1.1 g, 10.89 mmol) were added to a solution of compound 4 (1 g, 3.11 mmol) and compound 5 (616 mg, 4.67 mmol) in acetonitrile (20 mL). The reaction was stirred at room temperature for 16 hours under nitrogen protection. After cooling, the reaction solution was extracted with water and ethyl acetate (30 mL × 3). The combined organic phases were washed with 1N hydrochloric acid solution (20 mL × 2), saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel rapid chromatography (PE / EA = 5 / 1) to give compound 6 (0.9 g brown solid, yield: 89.1%).

[0343] LCMS: 327.1 (M+H)+ .

[0344] 2. Synthesis of Intermediate 7

[0345] Trifluoromethanesulfonic acid (0.83 g, 5.52 mmol) was added to a 20 mL solution of compound 6 (0.9 g, 2.76 mmol) in acetonitrile. The reaction mixture was stirred at 85°C for 3 hours. After cooling, water was added, and the mixture was extracted with ethyl acetate (30 mL × 2). The combined organic phases were washed with saturated sodium bicarbonate solution (30 mL × 3) and saturated brine solution (30 mL × 1), respectively. The mixture was dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel rapid chromatography (PE / EA = 1 / 1) to give compound 7 (0.58 g yellow solid, yield: 67.4%).

[0346] LCMS: 313.0 (M+H) + .

[0347] 3. Synthesis of compound EW266

[0348] Boron tribromide (8.5 mL, 18.60 mmol) was added dropwise to a 10 mL solution of compound 7 (530 mg, 1.86 mmol) in dichloromethane at 0°C, and the reaction was stirred at room temperature for 16 hours. The reaction was quenched with methanol (20 mL) at -70°C, and the reaction solution was evaporated to dryness. The crude product was purified by silica gel rapid chromatography (PE / EA = 3 / 1) to give compound EW266 (255 mg white solid, yield: 55.6%).

[0349] 1 H NMR (400MHz, DMSO-d6): δ10.94(s,1H),10.87(s,1H),10.05(s,1H),7.72(d,J=8.8Hz, 2H), 7.19 (s, 1H), 6.89 (d, J = 8.8Hz, 2H), 6.47 (d, J = 2.0Hz, 1H), 6.32 (d, J = 2.0Hz, 1H).

[0350] LCMS: 269.0 (MH) + .

[0351] Example 3

[0352] Specific experimental methods

[0353] Vascular endothelial cell proliferation experiment

[0354] HUVEC or HBMEC cells were seeded at 3000 cells / well in 96-well plates and cultured for 24 h. Subsequently, the culture medium was replaced with Ham's F-12K containing 0.2% FBS + 50 ng / mL heparin sodium for 6 h of starvation. After 6 h, the test compound was added and the cells were cultured for another 48 h. After 48 h, cell proliferation was assessed using the CTG method (Cell Titer-Glo Luminescent Assay, DD1101-02, Novizan).

[0355] OGD / R (Oxygen-glucose deprivation / reperfusion) induced vascular endothelial cell injury experiment

[0356] HUVEC cells were seeded at a rate of 4000 cells / well in 96-well plates and cultured normally for 24 hours. After 24 hours, the culture medium was replaced with sugar-free DMEM (11966025, Gibco) containing the test compound, and the plates were placed in an anaerobic incubator. After 4 hours, the culture medium was replaced with normal culture medium containing the test compound, and the plates were placed in a normal incubator for another 24 hours. Cell viability was assessed using the CTG method.

[0357] Alternatively, H9c2 cells were seeded at 4000 cells / well in 96-well plates and cultured normally for 24 hours. After 24 hours, the culture medium was replaced with sugar-free DMEM (11966025, Gibco) containing the test compound, and the plates were placed in an anaerobic incubator. After 12 hours, the culture medium was replaced with normal culture medium containing the test compound, and the plates were placed in a normal incubator for another 12 hours. Cell viability was then assessed using the CTG method.

[0358] Tubular structure formation experiment

[0359] The culture medium for HUVEC cells was replaced with Ham's F-12K medium containing 1% FBS and 50 ng / mL heparin sodium, and the cells were starved for 3 hours.

[0360] Adhesive application: Apply Matrigel (354230, Corning) at 50 μL per well of the 96-well plate and place at 37°C for 30 min to allow the adhesive to solidify.

[0361] Preparation of the test compound: Prepare the test compound at a 10-fold concentration, adding 11 μL of 10× test compound to each well.

[0362] After digesting and starving HUVEC cells, centrifuge at 1000 rpm for 5 minutes, resuspend in Ham's F-12K medium containing 1% FBS and 50 ng / mL heparin sodium, and count the cells.

[0363] Seed cells at a density of 10,000 cells per well, with 100 μL of cell suspension per well, and incubate at 37°C for 6 hours.

[0364] Six hours later, Calcein AM (C2012, Beyotime) was added for staining, and the images were taken using an Olympus IX73 microscope (4× objective).

[0365] Neural stem cell proliferation experiment

[0366] First, 3500 cells / well were seeded into 96-well plates. The following day, the test compound was added for treatment. After 48 hours of treatment, the degree of cell proliferation was detected using the CTG method.

[0367] Neural synapse growth

[0368] SHSY-5Y neural cells were seeded at 50,000 cells / well in 10% FBS MEM / F12 medium in 24-well plates and cultured for 24 hours. The following day, the cells were treated with the test compound in 1% FBS MEM / F12 medium. After 24 hours of compound treatment, cell morphology was observed under an inverted microscope, and images were taken at random from 10 fields of view per well. To determine neurite growth, the lengths of the five longest neurites in each well were calculated using ImageJ plugins, and the average neurite length was statistically analyzed from data obtained from at least three independent replicate experiments. The establishment of a mouse transient middle cerebral artery occlusion (tMCAO) model was also discussed.

[0369] After modeling, C57BL / 6J mice were randomly divided into four groups: the Veh group (solvent 5% NMP + 50% PEG400 + 45% PG), the EW238 group (2.0 mg / kg, 6.0 mg / kg), and the NBP (positive control drug, butylphthalide) group (6.0 mg / kg). The administration was via tail vein injection, once daily for three consecutive days. Brain samples were then collected for TTC staining.

[0370] Establishment of a mouse model of transient middle cerebral artery embolism (tMCAO model):

[0371] (1) Before the tMCAO surgery, the mice were fasted for 12 hours but allowed to drink water. Then they were anesthetized with isoflurane inhalation (4% for induction, 2% for maintenance), fixed in a supine position, and a midline incision was made in the neck to expose the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA).

[0372] (2) Ligate the proximal ends of the ECA and CCA with sutures, and keep the distal end of the CCA with sutures. Temporarily clamp the ICA with a mini arterial clamp.

[0373] (3) Make a small incision 3 mm from the bifurcation of the ECA, insert a 2 mm silicone-coated suture plug through the incision, and gently push the suture plug (Huayang, 14-3003). Open the arterial clamp on the ICA, allowing the suture plug to enter the middle cerebral artery (MCA) along the ICA. Secure the suture with a spare suture. The distance from the bifurcation of the CCA to the point of resistance (the origin of the middle cerebral artery) is approximately 8 mm.

[0374] (4) Use the spare suture at the distal end of the CCA to tie and fix the suture plug and the CCA, and then suture.

[0375] (5) During reperfusion, mice were anesthetized with isoflurane (4% for induction, 2% for maintenance), the cervical suture incision was opened, the suture plug was gradually pulled out until it was completely removed from the ECA, the ECA stump was electrocoagulated with an electrocoagulator, the cervical incision was sutured again, and povidone-iodine was applied for disinfection. After surgery, the animals were kept at body temperature by irradiating with a heater for 4 hours, and were kept in individual cages for 24 hours without food but without water.

[0376] Intravenous administration began 2 hours after modeling, once daily for 3 days. On the 4th day, the brain was harvested for TTC staining.

[0377] Measurement of cerebral infarction area

[0378] The intact brain tissue was frozen at -20℃ for 20 minutes, and coronal slices were cut from the forebrain into 6 slices, each slice being 2mm thick.

[0379] Brain slices were placed in six-well plates containing 0.2% TTC solution (prepared with physiological saline) and incubated at 37°C in the dark for 15 minutes. The plates were then removed, and the brain slices were gently turned over with forceps to ensure even contact with the staining solution. After 15 minutes, the TTC staining solution was aspirated, and the brain slices were fixed with 4% paraformaldehyde solution. Images were taken after 30 minutes, and the images were analyzed using Image Pro Plus image analysis software to determine the area of ​​cerebral infarction. Normal brain tissue areas were shown in red, while infarcted and surrounding areas were shown in white. Raw data required included the area of ​​the healthy hemisphere, the area of ​​normal tissue in the affected hemisphere, and the infarct area for each brain slice. The percentage of cerebral infarction area (%) = infarct area / total brain tissue area.

[0380] Preparation of mouse models of ischemic myocardial infarction and its induced heart failure

[0381] A model of ischemic myocardial infarction and its induced heart failure was established by ligation of the left anterior descending coronary artery. Two groups were established: a model group (Veh) and an EW238 group. EW238 was administered to mice half an hour before surgery. After administration, the behavioral signs and survival status of mice in each group were recorded daily for 4 weeks. The administration was intraperitoneal injection, 2 mg / kg / time, twice daily, using 0.5% DMSO + 8% PEG400 + 8% β-cyclodextrin as the solvent. Left ventricular ejection fraction (EF) and left ventricular short axis shortening rate (FS) were continuously observed using echocardiography to evaluate the protective effect of EW238 on the mouse model.

[0382] To evaluate cardiac contractile function in mice and the effect of EW238, the survival rate of mice was recorded at 3, 7, 14 and 21 days after surgery. Echocardiography was performed on mice in each group on the 3rd day after surgery.

[0383] Blood brain concentration test method

[0384] Pharmacokinetic Experiment

[0385] Experimental animals: C57BL / 6J mice, male, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0386] Administration: 2-3 male C57BL / 6J mice were administered intravenously to each group after fasting overnight. The dosage of apigenin was 6 mg / kg and EW238 was 10 mg / kg, respectively, using 5% NMP + 50% PEG400 + 45% PG as a solvent. The administration volume was 2 mL / kg.

[0387] Sample collection: Blood collection: Blood will be collected from the inferior vena cava or heart of each animal (approximately 0.1 mL at each time point). The collected blood will be placed in pre-cooled EDTA-K2 tubes and kept on ice until centrifugation.

[0388] Plasma preparation: Blood samples were centrifuged at approximately 4°C and 3,200 g for 10 minutes to prepare plasma. Plasma was collected separately and transferred to pre-labeled 96-well plates or polypropylene tubes, rapidly frozen with dry ice, and stored at -60°C or below until LC-MS / MS analysis.

[0389] Brain tissue collection: After perfusion with physiological saline, the brain was rinsed with physiological saline and placed on soft absorbent paper to drain any remaining fluid. The brain was then weighed and transferred to pre-labeled test tubes, and homogenized under ice-cold conditions with PBS*1 at a ratio of 1:3 (1 gram of tissue to 3 ml of buffer). After final homogenization, all homogenates were stored at approximately -80°C until LC-MS / MS analysis was performed.

[0390] Sample processing and testing:

[0391] 1) Add 20uL of plasma sample to 400uL of acetonitrile or methanol containing internal standard (100ng / mL) for precipitation, then vortex mix at 800 rpm for 10 minutes, and centrifuge at 4℃ and 3220×g for 15 minutes.

[0392] 2) Add 400 μL of acetonitrile or methanol containing internal standard (100 ng / mL) to 40 μL of brain homogenate sample for precipitation, then vortex mix at 800 rpm for 10 minutes, and centrifuge at 4℃ and 3220×g for 15 minutes.

[0393] 3) Take 50 μL of the supernatant solution after treatment 1) or 2) and transfer it to a clean 96-well plate. Centrifuge at 4℃ and 3220×g for 5 minutes. Then inject the supernatant directly for LC-MS / MS analysis.

[0394] Analysis and detection were performed using LC-MS / MS-DS_TQ7500 or LC-MS / MS_CT_Triple Quad 6500plus instruments.

[0395] Statistical analysis

[0396] All data are presented as mean ± SEM. All charts were created using GraphPad Prism 8.4.0. Left ventricular ejection fraction and left ventricular fractional shortening were analyzed using t-tests; animal survival rates were analyzed using Kaplan-Meier tests; other data were analyzed using one-way ANOVA combined with Turkey's post-hoc test. A p-value < 0.05 was considered statistically significant.

[0397] Experimental results

[0398] Figures 1 and 2 show the effects of apigenin (Api), butylphthalide (NBP), and edaravone-dexborneol (ED) on angiogenesis and anti-ischemic injury. Furthermore, among them:

[0399] Figure 1 shows the effects of the blank control group, different concentrations of butylphthalide (1 μM, 3 μM), different concentrations of edaravone-dexborneol (30 μM, 100 μM), and apigenin (0.3 μM, 1 μM, 3 μM) on the proliferation of human umbilical vein endothelial cells (HUVECs). As can be seen from Figure 1, compared with the blank control group, apigenin promoted the proliferation of vascular endothelial cells, while butylphthalide and edaravone-dexborneol did not.

[0400] Figure 2 shows the effects of different concentrations of butylphthalide (1 μM, 3 μM), edaravone-dexborneol (30 μM, 100 μM), and apigenin (0.3 μM, 1 μM, 3 μM) on vascular endothelial cell survival (anti-ischemic injury) in the normal control group, the oxygen-glucose deprivation injury (OGD / R) group, and under OGD / R injury. As can be seen from Figure 2, in the OGD / R injury model, apigenin has a better protective effect on vascular endothelial cells and alleviates the damage caused by oxygen-glucose deprivation compared to butylphthalide and edaravone-dexborneol.

[0401] Figures 3-5 illustrate the effects of EW266 (Formula II-a) on three aspects: anti-ischemic injury, angiogenesis, and neuronal proliferation.

[0402] Figure 3 shows the effects of different concentrations of EW266 (0.3 μM, 1 μM, and 3 μM) on vascular endothelial cell survival in the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and the OGD / R injury group (anti-ischemic injury). As can be seen from Figure 3, in the OGD / R injury model, compared with the blank group, EW266 has a protective effect on vascular endothelial cells and alleviates the damage caused by oxygen-glucose deprivation.

[0403] Figure 4 shows the effects of the blank control group (Ctrl) and different concentrations of EW266 (0.3 μM, 1 μM, 3 μM) on the proliferation of human umbilical vein endothelial cells (HUVECs). As can be seen from Figure 4, EW266 promoted the proliferation of vascular endothelial cells compared to the blank control group.

[0404] Figure 5 shows the effects of the blank control group (Ctrl) and the EW266 group (0.3μM, 1μM, 3μM, 10μM) on promoting neural stem cell proliferation. As can be seen from Figure 5, EW266 has the effect of promoting neural stem cell proliferation compared with the blank control group.

[0405] Figures 6-12 illustrate the effects of EW238 (Formula II-b) on four aspects: anti-ischemic injury, angiogenesis, neuronal cell proliferation, and synaptic growth.

[0406] Figure 6 shows the effects of the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), different concentrations of EW238 (0.3 μM, 1 μM, 3 μM) under OGD / R injury, and the apigenin group (Api, 3 μM) under OGD / R injury on vascular endothelial cell survival (anti-ischemia-like injury). As can be seen from Figure 6, in the OGD / R injury model, EW238, compared with apigenin, has the same degree of protective effect on vascular endothelial cells and alleviates the damage caused by oxygen-glucose deprivation.

[0407] Figure 7 shows the effects of the blank control group (Ctrl), different concentrations of EW238 (0.3 μM, 1 μM, 3 μM), and apigenin (Api, 3 μM) on the proliferation of human umbilical vein endothelial cells (HUVECs). As can be seen from Figure 7, compared with the blank control group, EW238 promoted the proliferation of vascular endothelial cells, and the effect of EW238 on promoting vascular endothelial cell proliferation was more significant compared with the apigenin group.

[0408] Figure 8 shows the effects of the blank control group (Ctrl), the EW238 group (3 μM), and the apigenin group (3 μM) on the proliferation of human brain microvascular endothelial cells (HBMEC). As can be seen from Figure 8, compared with the blank control group, EW238 promoted the proliferation of human brain microvascular endothelial cells, and the effect of EW238 on the proliferation of human brain microvascular endothelial cells was more significant compared with the apigenin group.

[0409] Figure 9 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3 μM, and positive control group (VEGF) 20 ng / mL on the formation of tubular structures in vascular endothelial cells: tubular structures under a fluorescence microscope.

[0410] Figure 10 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: total length of tubular structures.

[0411] Figure 11 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: number of lumens.

[0412] Figure 12 shows the effects of blank control group (Ctrl), EW238 (Formula II-b) 3μM, and positive control group (VEGF) 20ng / mL on the formation of tubular structures in vascular endothelial cells: number of branches.

[0413] As shown in Figures 9-12, compared with the blank control group, the positive control VEGF can significantly promote the formation of tubular structures by vascular endothelial cells, and EW238 can also promote the formation of tubular structures by vascular endothelial cells, as evidenced by a significant increase in the total length (Figure 10), tube number (Figure 11), and branch points (Figure 12) of the tubular structures.

[0414] Figure 13 shows the effects of the blank control group (Ctrl) and the EW238 group (1μM, 3μM, 10μM) on promoting the proliferation of neural stem cells. As can be seen from Figure 13, EW238 has the effect of promoting the proliferation of neural stem cells compared with the blank control group.

[0415] Figures 14 and 15 show the effects of the blank control group (Ctrl or Vehicle), the RA group (retinoic acid, 5 μM), and the EW238 group (5 μM) on promoting synaptic growth. As can be seen from Figures 14 and 15, compared with the blank control group, the positive control RA significantly promoted the growth of synapses of nerve cells, and EW238 also showed the effect of promoting the growth of synapses of nerve cells.

[0416] Figures 16-17 show the effects of EW238 (Formula II-b) on a mouse model of transient middle cerebral artery embolism (tMCAO), where:

[0417] Figure 16 shows the tMCAO model and the dosing regimen.

[0418] Figure 17 shows the representative and statistical graphs of cerebral infarction area after three consecutive days of intravenous injection, administered once daily, to the blank control group (Veh), EW238 treatment group (2 mg / kg, 6 mg / kg), and NBP treatment group (6 mg / kg) under the tMCAO model. As can be seen from Figure 17, compared with the Veh group, EW238 significantly reduced the infarct area in the ischemic region. At 6 mg / kg, EW238 showed a superior reduction in infarct area compared with butylphthalide (NBP).

[0419] Figures 18-22 show the effects of EW238 (Formula II-b) on cardiac function in mice with ischemic myocardial infarction and its induced chronic heart failure model (LAD ligation model), where:

[0420] Figure 18 shows the LAD ligation model and drug administration regimen.

[0421] Figure 19 shows the effect of EW238 on the survival of mice in the LAD ligation model.

[0422] As shown in Figure 19, the survival rate of the Veh group decreased significantly in the early stage of myocardial ischemia, with a survival rate of only 16.67% on day 6, while EW238 could significantly prolong the survival time of mice, with a survival rate of 100% as of Day 21.

[0423] In addition, the mice were carefully observed in the experiment. It was found that the mice in the Veh group had dark fur, consumed less food and water, and had relatively less activity. The mice also experienced difficulty breathing. However, after treatment with EW238, the mice showed a significant increase in activity, had neat and shiny fur, did not experience difficulty breathing, and remained aggressive at night.

[0424] Figure 20 shows the effect of EW238 on cardiac function (3 days post-surgery) in LAD ligation model mice, as a representative figure.

[0425] Figure 21 shows the effect of EW238 on left ventricular ejection fraction (EF, 3 days post-surgery) in mice with LAD ligation model.

[0426] Figure 22 shows the effect of EW238 on the left ventricular short axis shortening rate (FS, 3 days post-surgery) in LAD ligation model mice.

[0427] As shown in Figures 20-22, EW238 treatment can rapidly improve cardiac contractile function in model mice, with significant increases in EF and FS values ​​on day 3.

[0428] As shown in Figures 18-22, EW238 has a significant cardioprotective effect on mice with ischemic myocardial infarction and its induced heart failure.

[0429] As shown in Tables 1 and 2, the plasma concentration of apigenin was much higher than that in brain homogenate, indicating that relatively little apigenin entered the brain. In contrast, the concentration of the modified compound EW238 in brain homogenate was much higher than that in plasma, indicating that its ability to enter the brain was greatly improved.

[0430] Table 1. Blood brain concentration of apigenin (6 mg / kg) after intravenous injection (n=3)

[0431] Table 2. Blood brain concentrations of EW238 (10 mg / kg) after intravenous injection (n=2)

[0432] Figures 26-29 show the effects of EW254 (II-c) and EW274 (Formula II-d) on promoting epithelial cell proliferation, or on HUVEC's resistance to ischemic-like injury, or on H9c2's resistance to ischemic injury.

[0433] Figure 26 shows the effects of different concentrations of EW254 (0.3 μM, 1 μM, 3 μM) and EW238 (3 μM) on vascular endothelial cell survival (anti-ischemic injury) in the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and OGD / R injury (0.3 μM, 1 μM, 3 μM and 3 μM). As can be seen from Figure 26, in the OGD / R injury model, compared with the control group, EW254 has a protective effect on vascular endothelial cells and alleviates the damage caused by oxygen-glucose deprivation.

[0434] Figure 27 shows the effects of the blank control group (Ctrl), different concentrations of EW274 (1 μM, 3 μM, 10 μM), and EW238 (3 μM) on the proliferation of human umbilical vein endothelial cells (HUVECs). As can be seen from Figure 27, compared with the blank control group, EW238 and EW274 promoted the proliferation of vascular endothelial cells, and the effect of EW274 on promoting vascular endothelial cell proliferation was more significant compared with the EW238 group.

[0435] Figure 28 shows the effects of the normal control group (Ctrl), the oxygen-glucose deprivation injury group (OGD / R), and different concentrations of EW274 (1 μM, 3 μM, 10 μM) and EW238 (3 μM) on vascular endothelial cell survival under OGD / R injury (anti-ischemic-like injury). As can be seen from Figure 28, in the OGD / R injury model, compared with the control group, EW274 has a protective effect on vascular endothelial cells and alleviates damage caused by oxygen-glucose deprivation.

[0436] Figure 29 shows the effects of different concentrations of EW274 (3 μM, 10 μM, 30 μM) and EW238 (10 μM) on H9c2 cell survival under OGD / R injury (anti-ischemia-like injury). As can be seen from Figure 29, in the OGD / R injury model, compared with the control group, EW274 has a protective effect on H9c2 cells and alleviates the damage caused by oxygen-glucose deprivation.

[0437] Example 4

[0438] Preparation of EW254

[0439] This invention synthesizes the target compound through the above steps:

[0440] The specific steps for preparing the compound are as follows:

[0441] 1. Synthesis of M1

[0442] SM (60.74 g, 352 mmol, 1.0 eq.) was added to a 2 L three-necked round-bottom flask, followed by 880 mL of bottled acetone. Argon gas was purged three times. Potassium carbonate (345.81 g, 2.47 mol, 7.0 eq.) was added at room temperature, followed by cooling to 0 °C and dropwise addition of dimethyl sulfate (200 mL, 2.12 mol, 6.0 eq.) (the system became a grayish-white suspension). After the reaction was complete, the mixture was refluxed in an oil bath at 65 °C for 5 h (the system became a grayish-white suspension). The reaction endpoint was determined by TLC (20% EA / PE; Rf = 0.3). After the starting material was confirmed to have disappeared, the reaction mixture was cooled to room temperature, filtered through a diatomaceous earth layer, and the solid was washed with EA (3 × 200 mL). The filtrate was concentrated and 500 mL of ether was added. Dissolve the crude product in EA and pour it into 1L of water to separate the organic phase. Extract the aqueous phase with EA (3×300mL), dry and concentrate the organic phase to obtain compound M1 (88.60g, pale orange powder solid, 100% yield).

[0443] 1 H NMR(500MHz,Chloroform-d)δ6.10(s,1H),3.96(s,1H),3.88(s,1H),3.82(s,1H),3.80(s,2H).

[0444] 13 C NMR (126MHz, CDCl3) δ166.99,162.56,158.63,105.96,90.61,58.57,56.00,55.45,52.30.

[0445] HRMS (ESI-MS): Calculated for C 11 H 14 O5[M+H] + =227.0919, found:227.0915.

[0446] 2. Synthesis of M2

[0447] M1 (10.24 g, 44.2 mmol, 1.0 eq.) was added to a 500 mL three-necked round-bottom flask and dissolved in 150 mL of dry DCM. The mixture was stirred at -78 °C under argon protection, and the reaction solution was white and turbid. Boron trichloride (58 mL, 57.5 mmol, 1.3 eq.) was added, and the reaction solution became grayish-white and turbid. The reaction was then slowly brought back to room temperature and allowed to proceed for 4 h. The reaction endpoint was determined by TLC (20% EA / PE; Rf = 0.8). The reaction was cooled to 0℃, and 30 mL of methanol was slowly added to quench the reaction. The reaction was directly evaporated to dryness, dissolved in 100 mL of DCM, and 200 mL of water was added. The organic phase was separated, and the aqueous phase was extracted again with DCM (2 × 100 mL). The organic phases were combined and washed with 200 mL of saturated NaCl. The organic phase was filtered and dried, and the resulting brownish-gray solid crude product was slurried with 100 mL of PE, ultrasonically dispersed, filtered and collected. The solid was then dried by an oil pump to obtain compound M2 (5.83 g, brownish-gray solid, 62% yield).

[0448] 1 H NMR (600MHz, Chloroform-d) δ12.03 (s, 1H), 6.12 (d, J = 2.4Hz, 1H), 5.97 (d, J = 2.4Hz, 1H), 3.92 (s, 3H), 3.82 (d, J = 10.5Hz, 6H).

[0449] 13 C NMR (151MHz, CDCl3) δ171.68,165.98,165.37,162.16,96.58,93.45,91.58,56.08,55.46,52.19.

[0450] HRMS (ESI-MS): Calculated for C 10 H 12 O5[M+H] + =213.0763, found:213.0757.

[0451] 3. Synthesis of M3

[0452] In a 500 mL three-necked round-bottom flask, M2 (10.25 g, 47.1 mmol, 1.0 eq.) was dissolved in 190 mL of dry DCM. The system was clear and purplish-gray. Argon gas was purped three times. Triethylamine (17 mL, 118 mmol, 2.5 eq.) was added while stirring at -20 °C. The reaction solution was clear and yellow. Trifluoromethanesulfonic anhydride (8.1 mL, 56.6 mmol, 1.2 eq.) was added while stirring at -20 °C. The system turned clear and brownish-black. After the addition was complete, the mixture was moved to room temperature and reacted for 3 h. The reaction endpoint was determined by TLC (25% EA / PE; Rf = 0.4). After the reaction, the reaction solution was poured into 200 mL of ice water to separate the organic phase. The aqueous phase was extracted with DCM (3 × 200 mL). The organic phases were combined, washed with 300 mL of saturated brine, dried over approximately 40 g of anhydrous sodium sulfate, concentrated, and purified with neutral alumina (100-200 mesh, 25% EA / PE) to obtain a crude brownish-yellow oily substance. The crude substance was frozen at -78 °C for 5 min, and the oily substance turned into a grayish-white solid. 50 mL of PE was added, and the solid was ultrasonically dispersed. The mixture was stirred at -78 °C for 5 min, filtered while cold, and the solid was collected, dried, and stored at low temperature for later use to obtain compound M3 (10.48 g, white powder solid, 65% yield).

[0453] 1 H NMR(500MHz,Chloroform-d)δ6.48(d,J=2.2Hz,1H),6.42(d,J=2.2Hz,1H),3.91(s,3H),3.85(d,J=8.7Hz,6H).

[0454] 13 C NMR (126MHz, CDCl3) δ163.41,162.56,159.68,148.26,119.78,117.23,109.89,99.06,98.45,56.46,55.89,52.54.

[0455] 4. Synthesis of M4-1

[0456] SM1 (5.14 g, 22.7 mmol, 1.0 eq.) was dissolved in 120 mL of dry DCM in a 250 mL three-necked round-bottom flask. The mixture was purged with argon three times, and the reaction solution was a clear gray liquid. PPTS (582.3 mg, 2.3 mmol, 0.1 eq.) was added at 0 °C, followed by the slow addition of DHP (2.94 g, 34.1 mmol, 1.5 eq.). After the addition was complete, the mixture was moved to room temperature and stirred for 17 h. The reaction endpoint was determined by TLC (10% EA / PE; Rf = 0.8). After the reaction was complete, the reaction solution was added to 200 mL of water, and the organic phase was separated. The aqueous phase was extracted with DCM (3 × 80 mL). The organic phases were combined, washed with 300 mL of saturated brine, dried over approximately 40 g of anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (100-200 mesh, 2% EA / PE) to obtain compound M4-1 (6.24 g, white solid, 90% yield).

[0457] 1 H NMR(500MHz,Chloroform-d)δ7.59-7.49(m,2H),6.87-6.74(m,2H),5.37(t,J=3.2Hz,1H),3.86(ddd,J=11.4,9.8,3.1Hz ,1H),3.59(dtd,J=11.4,4.1,1.4Hz,1H),1.98(dddd,J=14.2,8.3,7.0,3.6Hz,1H),1.89-1.77(m,2H),1.73-1.49(m,4H).

[0458] 13 C NMR (126MHz, CDCl3) δ156.95,138.19,118.87,96.35,83.93,61.98,30.23,25.13,18.62.

[0459] 5. Synthesis of M4-2

[0460] Add M4-1 (8.03 g, 26.3 mmol, 1.0 eq.) to a 1 L three-necked round-bottom flask, dissolve in DMF (130 mL), purge with argon three times, then add triethylamine (15 mL, 105.2 mmol, 4.0 eq.), and degas using an argon balloon under ice bath conditions for 20 min using ultrasonic bubbling. Then add CuI (516.4 mg, 2.6 mmol, 0.1 eq.) and PdCl2(PPh3)2 (386.8 mg, ... 0.54 mmol (0.02 eq.) was added, and the mixture was purged with argon gas three times. The mixture was then degassed by sonication for 20 min using an argon balloon. Trimethylsilylacetylene (3.87 g, 36.8 mmol, 1.4 eq.) was added, and the mixture was degassed by sonication for 10 min using an argon balloon. The mixture was heated to 85°C under argon protection and reacted for 24 h. The reaction endpoint was determined by TLC (1% EA / PE; Rf = 0.2). After the reaction, the mixture was filtered through a diatomaceous earth filter. The filter cake was then filtered using 100 mL of diatomaceous earth. Wash with EA, pour the filtrate into 500 mL of water to separate the organic layer, then extract with 30% EA / PE (3 × 100 mL), combine the organic phases and wash with saturated brine (1 × 300 mL), dry with about 80 g of anhydrous sodium sulfate, concentrate the dry solvent to obtain crude product M4-2 (9.74 g, brownish-black oily liquid containing DMF, 134% yield (due to the presence of DMF, the yield exceeds 100%)).

[0461] 1 H NMR(500MHz,Chloroform-d)δ7.43-7.39(m,2H),7.01-6.96(m,2H),5.44(t,J=3.3Hz,1H),3.89(ddd,J=11.5,9.7,3.1Hz,1H ),3.62(dtd,J=11.3,4.1,1.3Hz,1H),2.09-1.94(m,1H),1.88(ddd,J=8.6,4.8,3.3Hz,2H),1.79-1.53(m,4H),0.26(s,9H).

[0462] 13 C NMR (126MHz, CDCl3) δ157.17,133.28,116.15,116.02,105.15,96.15,92.43,61.96,30.17,25.06,18.59,-0.00.

[0463] 6. Synthesis of M4

[0464] M4-2 (7.21 g, 26.3 mmol, 1.0 eq.) was dissolved in 130 mL of MeOH in a 250 mL three-necked round-bottom flask. The reaction solution was a clear, brownish-black liquid. Potassium carbonate (10.91 g, 78.9 mmol, 3.0 eq.) was added at room temperature. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction was considered complete when the LC-MS peak times of the starting material M4-2 and the product M4 were not the same. After the reaction was complete, the reaction mixture was filtered, and the solid was washed with DCM (3 × 50 mL). The filtrate was evaporated to dryness and purified by silica gel column chromatography (100-200 mesh, 2% EA / PE) to obtain compound M4 (4.27 g, brownish-yellow oil, 80% yield).

[0465] 1 H NMR(500MHz,Chloroform-d)δ7.48-7.32(m,2H),7.04-6.92(m,2H),5.43(t,J=3.3Hz,1H),3.87(ddd,J=11.4,9.7,3.1Hz,1H),3.6 0(dtd,J=11.4,4.1,1.4Hz,1H),2.99(s,1H),2.10-1.94(m,1H),1.87-1.83(m,2H),1.73-1.64(m,2H),1.60(dt,J=7.3,2.9Hz,1H).

[0466] 13 C NMR (126MHz, CDCl3) δ157.45,133.50,116.34,114.98,96.21,83.70,77.29,77.03,76.78,75.83,62.03,30.23,29.72,25.13,18.64.

[0467] 7. Synthesis of M5

[0468] Add M3 (6.20 g, 18.0 mmol, 1.0 eq.) to a 1 L three-necked round-bottom flask, dissolve in DMF (90 mL), and purge with argon gas three times. Then add triethylamine (10 mL, 72.0 mmol, 4.0 eq.), purge with argon gas three times, and degas using an argon balloon under ice bath conditions for 20 min using ultrasonic bubbling. Then add CuI (367.2 mg, 1.8 mmol, 0.1 eq.) and PdCl2(PPh3)2 (257.5 mg, 0.1 eq.). M4 (4.24 g, 21.6 mmol, 1.1 eq.) was added, and the mixture was purged with argon gas three times. The mixture was then degassed by sonication for 20 min using an argon balloon. M5 (6.73 g, yellow oily liquid, 94% yield) was added and the mixture was further degassed by sonication for 10 min using an argon balloon. The mixture was heated to 85°C under argon protection and reacted for 27 h. The reaction endpoint was determined by TLC (15% EA / PE; Rf = 0.3). After the reaction, the reaction mixture was filtered through a layer of diatomaceous earth and the filtrate was collected. The filter cake was washed with 100 mL of EA. The filtrate was added to 500 mL of water, and the organic layer was separated. The organic layer was then extracted with EA (3 × 100 mL). The combined organic phases were washed with saturated brine (1 × 500 mL), dried over approximately 100 g of anhydrous sodium sulfate, and the solvent was concentrated. The solution was purified by silica gel column chromatography (100-200 mesh, 10% EA / PE) to obtain M5 (6.73 g, yellow oily liquid, 94% yield).

[0469] 1 H NMR(500MHz,Chloroform-d)δ7.42(d,J=2.1Hz,1H),7.41(d,J=2.1Hz,1H),7. 08-6.96(m,2H),6.64(d,J=2.2Hz,1H),6.45(d,J=2.3Hz,1H),5.44(t,J=3.2H z,1H),3.93(s,3H),3.91(s,1H),3.86(d,J=3.1Hz,1H),3.83(d,J=4.3Hz,6H) ,2.34-2.26(m,1H),2.05-1.95(m,1H),1.91-1.79(m,2H),1.72-1.67(m,2H).

[0470] 13C NMR (126MHz, CDCl3) δ167.38,161.36,157.99,157.43,133.09,123.74,118.79,116.41,115.69,108.22,107.71,99.44, 98.46,96.23,92.88,85.58,64.32,62.06,56.47,56.02,55.89,55.60,52.55,52.32,35.83,31.95,30.23,25.13,18.64.

[0471] HRMS (ESI-MS): Calculated for C 23 H 24 O6[M+H] + =397.1651,found:397.1660.

[0472] 8. Preparation of M6

[0473] In a 250 mL three-necked flask, add M5 (6.20 g, 16 mmol, 1.0 eq.), dissolve in 105 mL of methanol:H2O (20:1), and cool to 0 °C under argon protection with stirring. Add barium hydroxide octahydrate (25.36 g, 78.2 mmol, 5.0 eq.), and after the addition is complete, raise the temperature to no more than 45 °C and stir for 36 h under argon protection. Determine the reaction endpoint by TLC (40% EA / PE; Rf = 0.2) (a small amount of raw material remains in the reaction; excessive time will produce other byproducts). After the reaction is complete, cool to room temperature and pour into 300 mL of water. Add 150 mL of EA, cool to 0 °C, and adjust the pH to approximately 6-7 with 1 M HCl. Separate the organic phase, and extract the aqueous phase again with EA (4 × 150 mL). Combine the organic phases, wash with 300 mL of saturated NaCl, dry and concentrate the organic phase, and add 50 mL of the crude solid product. EA was dissolved, PE was added to precipitate a viscous solid, which was filtered, the solid was collected, and dried to obtain compound M6 (4.36 g, yellow powder solid, 73% yield).

[0474] 1 H NMR(500MHz,DMSO-d6)δ7.49-7.30(m,2H),7.10-7.03(m,2H),6.70-6.59(m,2H),5. 53(t,J=3.3Hz,1H),3.81(s,3H),3.79(s,3H),1.89-1.70(m,4H),1.68-1.48(m,5H).

[0475] HRMS (ESI-MS): Calcd. For C 22 H 22 O6[M+H] + =383.1495,found:383.1488.

[0476] 9. Preparation of compound M7

[0477] Take a 100 mL three-necked flask, add M6 (2.64 g, 6.8 mmol, 1.0 eq.), and dissolve it in redistilled trifluoromethanesulfonic acid (20 mL) at room temperature (the reaction solution quickly turns dark brown). Stir at room temperature for 3 h under argon protection (to obtain a brownish-yellow solution). Determine the reaction endpoint by TLC (40% EA / PE; Rf = 0.4). After the reaction is complete, pour the reaction solution into 200 mL of ice water. Adjust the pH to 6-7 with saturated sodium bicarbonate solution. Extract the aqueous phase with EA (4 × 100 mL). Dry with about 40 g of anhydrous sodium sulfate and concentrate to obtain the crude product. Purify by column chromatography (stationary phase: 200-300 mesh silica gel, mobile phase: 40-50% EA / PE (v / v)) to obtain compound M7 (926.4 mg, brownish-yellow solid, 46% yield).

[0478] 1 H NMR(500MHz,DMSO-d6)δ10.02(s,1H),7.75-7.57(m,2H),7.08(s,1H),6.91-6. 86(m,2H),6.69(d,J=2.3Hz,1H),6.59(d,J=2.3Hz,1H),3.89(d,J=7.2Hz,6H).

[0479] 13 C NMR (126MHz, DMSO) δ165.65,163.29,159.77,157.76,154.05,142.76,128. 79,127.11,122.81,116.26,102.10,101.35,100.07,98.80,56.56,56.22.

[0480] HRMS (ESI-MS): Calcd. For C 17 H 14 O5[M+H] + =299.0919, found:299.0916.

[0481] 10. Preparation of compound M8

[0482] Take a 50 mL three-necked flask, add M7-1 (1.89 g, 4.0 mmol, 1.5 eq.) and CsF (3.26 g, 21.5 mmol, 8.0 eq.) under nitrogen protection. Use phosphorus pentoxide as a desiccant to react the mixture. The reaction is carried out at 140 °C under reduced pressure for 4 h (resulting in a grayish-white solid). Cool the system to room temperature, and add this solid to another flask containing M7 (796.4 mg, 2.7 mmol, 1.0 eq.). Purge the flask three times with argon gas, and then add 18 mL of dry toluene. Argon gas was replaced three times (grayish-white turbidity; note: M7 was treated with 2×30mL of dry THF with water, and 3×30mL of dry benzene with water). The reaction was heated to reflux for 38 hours (the system was brown and turbid). The reaction was cooled to room temperature, filtered, and the solid was washed with toluene (3×30mL). The filtrate was concentrated and purified by column chromatography (stationary phase: 200-300 mesh silica gel, mobile phase: 30-40% EA / PE (v / v)) to obtain compound M8 (506.7mg, grayish-white solid, 63% yield).

[0483] 1 H NMR (500MHz, DMSO-d6) δ7.96-7.84(m,2H),7.41-7.30(m,2H),7.28(s,1H),6.73(d,J=2.3Hz,1H),6.65(d,J=2.3Hz,1H),3.91(d,J=5.9Hz,6H).

[0484] 13 C NMR (126MHz, DMSO) δ165.74,163.34,162.50,157.45,152.51,142.12,128.60, 127.77,127.70,116.61,116.44,102.42,102.37,101.90,99.39,56.63,56.27.

[0485] HRMS (ESI-MS): Calcd. For C 17 H 13 FO4[M+H] + =301.0876, found:301.0870.

[0486] 11. Preparation of EW254

[0487] Add M8 (456.8 mg, 1.5 mmol, 1.0 eq.) to a 50 mL three-necked flask, then add 3 mL of anhydrous DCM. Replace the flask with argon gas and cool to -78 °C, stirring for 15 min. Dissolve boron tribromide (2 mL, 22.8 mmol, 15 eq.) in 3 mL of dry DCM and slowly add it (the reaction solution turns brown and cloudy). Return to room temperature and stir for 16 h. The reaction endpoint was determined by TLC (40% EA / PE; Rf = 0.6). After the reaction was completed, the mixture was poured into ice water and extracted with EA (3 × 80 mL). The extract was dried and concentrated, then purified by column chromatography (stationary phase: 100-200 mesh silica gel, mobile phase: 30% EA / PE (V / V)). The crude product was concentrated to dryness to obtain 324.6 mg of crude product. The solid was added to 10 mL of DCM and 50 mL of PE and stirred at room temperature for 30 min. After filtration, the filter cake was collected and freeze-dried to obtain EW254 (324.6 mg, white powder solid, 78% yield). The 1H NMR of EW254 is shown in Figure 30.

[0488] 1 H NMR (500MHz, DMSO-d6) δ10.88(s,2H),8.00-7.81(m,2H),7.40-7.23(m,3H),6.51(d,J=2.2Hz,1H),6.36(d,J=2.1Hz,1H).

[0489] 13 C NMR (126MHz, DMSO) δ165.71,164.57,164.06,162.68,162.08,151.18,139.46,1 27.91,127.88,127.39,127.32,116.16,115.99,103.82,102.94,102.15,98.33.

[0490] HRMS (ESI-MS): Calcd. For C 15 H9FO4[MH] - =271.0412,found:271.0356.

[0491] Example 5

[0492] Preparation of EW274

[0493] This invention synthesizes the target compound through the above steps:

[0494] That is, EW274.

[0495] Compound preparation examples

[0496] Tetrazazole (242 mg, 3.46 mmol, 306 μL, 1.10 eq) and Cpd.1 (1.20 g, 3.46 mmol, 1.16 mL, 1.10 eq) were added to a solution of EW238 (800 mg, 3.15 mmol, 1.00 eq) in acetonitrile (MeCN, 16 mL). The reaction was stirred at 20 °C for 12 h. Tert-butanol peroxide (1.22 g, 9.44 mmol, 1.29 mL, 70% purity, 3.00 eq) was added to the reaction mixture and stirred at 20 °C for 5 min. LC-MS (ET99403-1-P1A2) showed 23% of the starting material remaining and 66% of Cpd.2 was detected. The reaction system was purified by water (20 mL) at 20 °C and extracted with ethyl acetate (20 mL * 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product, which was purified by column chromatography (SiO2, Commercial hexanes / Ethyl acetate = 19 / 1 to 4 / 1). The obtained Cpd.2 (900 mg, 1.66 mmol, 52.8% yield, 95.0% purity) was a white solid.

[0497] Taking EW238 as an example, it means that 1V (1 volume) of 1g substrate is 1mL, and the 20V volume of 800mg EW238 is 16mL, as stated below.

[0498] HNMR: (400MHz, CDCl3) δ10.99(s,1H),7.72-7.76(m,2H),7.37-7.40(m,3H),7.27(s, 10H), 6.73 (s, 1H), 6.68 (d, J = 1.43Hz, 1H), 6.58-6.60 (m, 1H), 5.08 (d, J = 9.18Hz, 4H).

[0499] To a solution of Cpd.2 (600 mg, 1.11 mmol, 1.00 eq, 95.0% purity) in tetrahydrofuran (120 mL) and ethyl acetate (120 mL) was added Pd / C (235 mg, 221 μmol, 10% purity, 0.200 eq). Then the gas in the system was evacuated and replaced with hydrogen three times. Finally, it was stirred at 20 °C for 3 hours under a hydrogen atmosphere of 15 Psi. LC-MS (ET99403-3-P1E5) showed that Cpd.2 was completely consumed and a new main peak with the product ms was monitored. The reaction mixture was filtered and the filtrate was concentrated. The crude product was recrystallized from ethyl acetate (5 ml) at 20 °C. The recrystallized product was purified by high performance liquid chromatography (column: Waters Xbridge BEH C18 100*25 mm*10 um; mobile phase: [H2O(10 mM NH4HCO3)-ACN]; gradient: 1%-35% B over 8.0 min). The resulting EW274 (101 mg, 299 μmol, 27.0% yield, 99.1% purity) was a white solid. The 1 1H NMR is shown in Figure 31.

[0500] 1H NMR: (400 MHz, MeOD) δ 7.90-7.90 (m, 1H), 7.91 (dd, J = 8.25, 1.38 Hz, 1H), 7.43-7.54 (m, 3H), 7.23 (s, 1H), 7.02 (d, J = 1.88 Hz, 1H), 6.86 (d, J = 1.63 Hz, 1H).

[0501] Example 6

[0502] 1. Experimental Materials and Methods

[0503] Experimental animals: Male Sprague-Dawley rats, 6-8 weeks old, weighing about 260-280 g. The rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. Animal production license number: SCXK(Zhe)2019-0001. Before the experiment, the animals were adaptively fed in the facility for 7 days, and 3 animals were housed in each cage during the adaptation period. The feeding environment was at room temperature of 23-26 °C, with a 12 / 12 h light / dark cycle, the light on time in the feeding room was 7:00-19:00, and the light off time was 19:00-next day 7:00.

[0504] Table 3 Animal Grouping Information and Drug Administration Scheme

[0505] In the table: BID*7D means continuous drug administration at a frequency of twice a day for a total of 7 days

[0506] 2. Compound preparation information:

[0507] NBP preparation: Weigh 10g of hydroxypropyl-β-cyclodextrin, add 100mL of purified water, and sonicate to dissolve, thus obtaining a 10% hydroxypropyl-β-cyclodextrin aqueous solution. Weigh an appropriate amount of NBP compound, add the corresponding amount of 10% hydroxypropyl-β-cyclodextrin aqueous solution, stir until completely dissolved, filter using a 0.22μm PTFE filter membrane, prepare and use immediately, and store at 4℃ protected from light.

[0508] Preparation of EW238: Accurately weigh 100 mg of compound EW238, dissolve it in 1 mL of DMSO (100 mg / mL), then add 3.5 mL of HS-15 (Solutol HS-15, CAS: 61909-81-7), vortex to mix, and dispense into 108 μL tubes, storing at -20°C. Separately, add 1 mL of PEG400 to 12 mL of physiological saline, vortex to mix, and use this to dilute the stock solution. For the working solution, remove the stock solution, add 1092 μL of a pre-mixed 1:12 PEG400-physiological saline solution, vortex to mix, and administer via tail vein injection at a volume of 3 mL / kg. Prepare and use immediately.

[0509] Solvent: HS-15 is solid at room temperature and needs to be melted in a 45°C water bath (5 min) beforehand. Sequentially pipette 2% DMSO + 7% HS-15 + 7% PEG400 + 84% physiological saline into a 50 mL centrifuge tube and vortex to mix.

[0510] 3. Experimental Design

[0511] (1) Adaptation phase: Animals adapt to the experimental environment for 7 days; animal weight is recorded before the experiment and they are randomly divided into 6 groups.

[0512] (2) Pre-experiment preparation stage: Disinfection of surgical instruments and experimental environment was carried out 2 days before the experiment;

[0513] (3) pMCAO modeling method:

[0514] Skin preparation: On the day of the experiment, rats were anesthetized with acetaminophen (3 mL / kg). After anesthesia, the neck was prepared by opening the neck tissue layer by layer to expose and free the common carotid artery and experimental blood vessels.

[0515] A suture is applied proximally to the external carotid artery (ECA), and two ligations are made distally, 2-5 mm apart. The vessel is then cut between the two distal ligation points. A slipknot is applied proximally to the common carotid artery (CCA). The ECA is pulled downwards until it is nearly parallel to the CCA. Blood flow is blocked distally to the internal carotid artery (ICA) using a microscopic hemostatic clip. A small incision is made before the ECA ligation point, and a silicone-tipped suture plug (product model: 2634A5, head diameter 0.34±0.02 mm, purchased from Beijing Xinong Technology Co., Ltd.) is inserted along the CCA to the microscopic hemostatic clip. The hemostatic clip is then opened, and the suture is slowly inserted upwards along the ICA.

[0516] Slowly insert the suture until the suture mark reaches the intersection of ECA and CCA, then stop inserting the suture and ligate the ECA at the upper end of the ligation site. Cut off any excess suture. At the same time, loosen the carotid artery live knot and suture directly. Then place the mouse on a warming pad.

[0517] (4) Administration of the test substance:

[0518] Groups 1 to 4: The first dose was administered 2 hours after ischemia, followed by a second dose 6 hours later (Day 0). Subsequently, the dose was administered daily at 8-hour intervals in a BID pattern for a total of 7 consecutive days, with the last dose on Day 6.

[0519] 4. Sampling at the endpoint

[0520] TTC staining for cerebral infarction:

[0521] a) Cardiac perfusion for brain harvesting: The rat's thoracic cavity was fully opened to expose the heart. Surrounding fatty tissue was cleared using ophthalmic and anatomical forceps to expose the aorta. An injection needle was inserted into the left ventricle near the apex. Upon successful insertion, blood return was observed with each heartbeat. The auricle was opened, and pre-cooled saline was infused using a peristaltic pump at a rate of approximately 20 mL / min for a total of 7 minutes. Continuous blood flow from the right atrium and gradual blood loss from the liver were observed.

[0522] b) Brain harvesting: Open the skull, observe the internal carotid artery at the suture insertion site for bleeding, and take photographs for documentation. Animals with brain hemorrhage should be removed from the group.

[0523] c) Staining: The brain was placed in a -80°C freezer for about 3 minutes. Using a rat brain mold and a scalpel, 6 coronal sections were cut from the olfactory bulb to the back, with a thickness of about 2 mm. The sections were collected in cryovials and thoroughly mixed with 2% TTC staining solution. The mixture was then stained in a 37°C water bath for about 10 minutes. After staining, the brain slices were removed and photographed.

[0524] d) Infarct Area Analysis: ImageJ software was used to calculate the infarct area, providing images of the infarcted tissue region. The raw data should include the area of ​​the healthy hemisphere, the area of ​​normal tissue in the affected hemisphere, and the infarct area for each brain tissue segment. Infarct area percentage (%) = Infarcted area / Total brain tissue area.

[0525] 5. Experimental Data Processing

[0526] All raw data were entered into Excel and statistical analysis was performed using GraphPad Prism 10.2. The results are expressed as Mean ± SEM and statistical differences between groups were analyzed using one-way ANOVA.

[0527] 6. Inspection Results

[0528] Figure 23 shows the effect of EW238 (Formula II-b) on rats with a permanent middle cerebral artery occlusion model (pMCAO model), where:

[0529] Figure 23 shows the cerebral infarction area in the model group, the positive drug group (NBP, 4 mg / kg), and the EW238 group (6 mg / kg): A. TTC staining reflects the ameliorative effect of different drug administration groups on the cerebral infarction area of ​​pMCAO model rats. B. Statistical analysis of cerebral infarction area in different drug administration groups. The results show that the infarction area in the model group was significantly increased, while treatment with NBP or EW238 significantly reduced the cerebral infarction area in rats (p<0.01). Compared with the NBP group, the EW238 group showed a better trend in reducing the cerebral infarction area, which indicates that the test compound EW238 has a potential therapeutic effect in improving ischemic stroke.

[0530] The above description provides an illustrative overview of the present invention and its embodiments. This description is not restrictive, and the embodiments shown are merely one example of the invention's implementation. Actual implementations are not limited to these examples. Therefore, if those skilled in the art are inspired by this description and design similar implementations and examples without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

Claims

A compound or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug thereof, and mixtures thereof, characterized in that, The structure of the compound is shown in the following formula: R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups; n is selected from any integer from 1 to 5; R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups. The compound according to claim 1, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug thereof, or mixture thereof, is characterized in that, The n = 1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains. The compound according to claim 2, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug thereof, and mixtures thereof, is characterized in that, R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids. A compound or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug thereof, and mixtures thereof, characterized in that, The compound is any one of formula II-a, II-b, or II-c: The compound according to claim 4, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug thereof, and mixtures thereof, is characterized in that, The prodrug of the compound is the compound shown in formula II-d: A pharmaceutical composition comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition comprising a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and mixtures thereof, and a pharmaceutically acceptable carrier or diluent; A pharmaceutical composition comprising a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and mixtures thereof, and a pharmaceutically acceptable carrier or diluent; A pharmaceutical composition comprising a compound of formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and a mixture thereof, and a pharmaceutically acceptable carrier or diluent; A pharmaceutical composition comprising a compound of formula II-d or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and mixtures thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition according to any one of claims 6 to 10 is characterized in that, The pharmaceutical composition is in a single-dose form, wherein the single-dose form contains 0.03 mg to 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof and mixtures thereof, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition according to any one of claims 6 to 11 is characterized in that, The mass percentage of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, or prodrug and mixture thereof in the pharmaceutical composition is 1 to 99 wt%. The pharmaceutical composition according to any one of claims 6 to 12 is characterized in that, The drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the pharmaceutical composition is 0.01 to 10 mg / mL. The pharmaceutical composition according to any one of claims 6 to 13 is characterized in that, It also includes agents selected from at least one of the following: Combination medications other than those for the treatment of cerebrovascular diseases or cardiovascular diseases; Other treatments for cerebrovascular diseases; Other cardiovascular disease treatments. The pharmaceutical composition according to claim 14 is characterized in that, The other cerebrovascular disease treatment agents mentioned are selected from any one or more of the following: A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating inflammation in the brain; A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating the volume of cerebral infarction; Treatment agents for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating reduced blood flow to the brain; A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or relieving cerebral blood flow interruption; A therapeutic agent for cerebrovascular diseases characterized by inhibiting, reducing, or alleviating cerebral hemorrhage. The pharmaceutical composition according to claim 14 or 15 is characterized in that, The combination drugs, excluding cerebrovascular disease treatment agents or cardiovascular disease treatment agents, are selected from any one or more of the following: Combination drugs with anti-cerebral thrombosis effects; Combination drugs with antiplatelet activity; Combination drugs with thrombolytic effects; Combination drugs with antioxidant effects; Combination drugs with anticoagulant effects; Combination drugs that have cholesterol-lowering effects. The pharmaceutical composition according to any one of claims 14 to 16 is characterized in that, The combination drugs other than cerebrovascular disease treatment agents or cardiovascular disease treatment agents, or other cerebrovascular disease treatment agents or other cardiovascular disease treatment agents, include any one or more of butylphthalide, edaravone, dextroborneol, aspirin, clopidogrel, dipyridamole, prasugrel, ticagrelor, heparin, warfarin, dabigatran, apixaban, rivaroxaban, and atorvastatin. The pharmaceutical composition according to any one of claims 6 to 17 is characterized in that, The dosage form of the pharmaceutical composition is selected from one or more of the following: powder, granule, tablet, pill, capsule, sustained-release, controlled-release, injection, infusion, or suspension. The pharmaceutical composition according to claim 18 is characterized in that, The dosage form of the pharmaceutical composition is a tablet, wherein the tablet is a sublingual tablet; or, The dosage form of the pharmaceutical composition is an injection, and the injection further contains a solvent. The pharmaceutical composition according to claim 19 is characterized in that, The solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and cyclodextrin aqueous solutions; Preferably, The alcohol solvents include one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol; The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether; The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone; The sulfur-containing organic solvent includes dimethyl sulfoxide; The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution. A medicine box comprising one or more single-dose units of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, hydrate, solvate, or tautomer thereof, or a prodrug thereof, or a mixture thereof, or a pharmaceutical composition comprising one or more single-dose units according to any one of claims 6 to 20, and instructions for use in treating a disease. The medicine box according to claim 21 is characterized in that, The medicine box also includes a medicine selected from at least one of the following: Combination medications other than those for the treatment of cerebrovascular diseases or cardiovascular diseases; Other treatments for cerebrovascular diseases; Other cardiovascular disease treatments. The medicine box according to claim 22 is characterized in that, The drug comprises any one or more of butylphthalide, edaravone, dextroborneol, aspirin, clopidogrel, dipyridamole, prasugrel, ticagrelor, heparin, warfarin, dabigatran, apixaban, rivaroxaban, and atorvastatin. The use of a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases. R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas: in, R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups; n is selected from any integer from 1 to 5; R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups. The use according to claim 24 is characterized in that, In the compound of formula I: The n = 1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains. The use according to claim 25 is characterized in that, In the compound of formula I: R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids. The use of a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases. The use of a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases. The use of compounds of formula II-c or their pharmaceutically acceptable salts, esters, hydrates, solvates or their tautomers or their prodrugs and mixtures thereof for the preparation of medicaments for the prevention or treatment of cerebrovascular diseases. The use of a compound of formula II-d or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases. The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cerebrovascular diseases, wherein the pharmaceutical composition comprises a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas: in, R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups; n is selected from any integer from 1 to 5; R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups. The use according to claim 31 is characterized in that, In the compound of formula I: The n = 1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains. The use according to claim 31 is characterized in that, In the compound of formula I: R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids. The pharmaceutical composition is used for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cerebrovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used for the preparation of a medicament for the prevention or treatment of cerebrovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-d or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof, or mixture thereof, and a pharmaceutically acceptable carrier or diluent; The use according to any one of claims 24 to 37 is characterized in that, The cerebrovascular diseases mentioned include: Cerebrovascular diseases characterized by deficits in brain neurological function; or Cerebrovascular diseases characterized by increased inflammation in the brain; or Cerebrovascular diseases characterized by an increase in the volume of cerebral infarction; or Cerebrovascular diseases characterized by reduced or interrupted blood flow to the brain; or Cerebrovascular diseases characterized by rupture of cerebral blood vessels; or Cerebrovascular diseases characterized by hemorrhage caused by ruptured blood vessels in the brain. The cerebrovascular disease according to any one of claims 24 to 38 includes: Ischemic cerebrovascular disease; or Hemorrhagic cerebrovascular disease; or Atherosclerosis, stenosis, or occlusion of the arteries in the head and neck. The use according to claim 39 is characterized in that, The cerebrovascular diseases include any one or more of the following: transient ischemic attack, cerebral infarction, stroke, cerebral infarction, cerebral thrombosis, cerebral embolism, chronic cerebral ischemia, cerebral hemorrhage, cerebral hemorrhage, and subarachnoid hemorrhage. The use according to claim 40 is characterized in that, The stroke mentioned is ischemic stroke. The use according to claim 41 is characterized in that, The ischemic stroke is selected from one or more of transient ischemic stroke, permanent ischemic stroke, acute ischemic stroke, and acute cerebral infarction. The use according to any one of claims 24 to 42 is characterized in that, The single-dose form of the drug for the prevention or treatment of cerebrovascular diseases contains 0.03 mg to 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer or prodrug or mixture thereof. The use according to any one of claims 24 to 42 is characterized in that, The drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the drug for the prevention or treatment of cerebrovascular diseases is 0.01 to 10 mg / mL. The use according to any one of claims 24 to 44 is characterized in that, The drugs for the prevention or treatment of cerebrovascular diseases also contain pharmaceutically acceptable carriers or diluents. The use according to any one of claims 24 to 45 is characterized in that, The dosage form of the drug for the prevention or treatment of cerebrovascular diseases is selected from one or more of the following: powder, granule, tablet, pill, capsule, sustained-release, controlled-release, injection, infusion, or suspension. The use according to claim 46 is characterized in that, The dosage form of the drug for the prevention or treatment of cerebrovascular diseases is a tablet; the tablet is a sublingual tablet; or The dosage form of the drug for preventing or treating cerebrovascular diseases is an injection, and the injection also contains a solvent. The use according to claim 47 is characterized in that, The solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and cyclodextrin aqueous solutions; Preferably, The alcohol solvents include one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol; The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether; The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone; The sulfur-containing organic solvent includes dimethyl sulfoxide; The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution. The use of a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases. R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas: in, R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups; n is selected from any integer from 1 to 5; R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups. The use according to claim 49 is characterized in that, In the compound of formula I: The n = 1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains. The use according to claim 50 is characterized in that, In the compound of formula I: R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids. The use of a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases. The use of a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases. The use of a compound of formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases. The use of a compound of formula II-d or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer thereof or a prodrug thereof and mixtures thereof for the preparation of a medicament for the prevention or treatment of cardiovascular diseases. The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cardiovascular diseases, wherein the pharmaceutical composition comprises a compound of formula I or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; R1 and R2 are each independently selected from hydrogen, deuterium, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted fatty acid acyl, substituted or unsubstituted arylformyl, Fmoc-aminoacyl, aminoacyl, or a group having one of the following formulas: in, R4 and R5 are each independently selected from hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl groups; n is selected from any integer from 1 to 5; R3 represents one or more substituents at any substituted position on the benzene ring, each R3 being independently selected from hydrogen, deuterium, alkyl, halogen, nitro, cyano, hydroxyl, amino, mercapto, formyl, carboxyl, carbamoyl, alkoxy, alkylthio, alkylamino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclic, aryl, and heterocyclic groups. The use according to claim 56 is characterized in that, The n = 1 and R3 is hydrogen; R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from H, substituted or unsubstituted aliphatic side chains, or substituted or unsubstituted aromatic side chains. The use according to claim 56 is characterized in that, R1 and R2 are each independently selected from hydrogen or groups having one of the following formulas: R4 and R5 are each independently selected from hydrogen, deuterium, or CH3; R6 is selected from the α-side chain of one of the natural amino acids. The pharmaceutical composition is used for the preparation of a medicament for the prevention or treatment of cardiovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-a or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cardiovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-b or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cardiovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-c or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The pharmaceutical composition is used to prepare a medicament for the prevention or treatment of cardiovascular diseases, wherein the pharmaceutical composition comprises a compound of formula II-d or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer thereof or a prodrug thereof or a mixture thereof, and a pharmaceutically acceptable carrier or diluent; The use according to any one of claims 49 to 62 is characterized in that, The cardiovascular diseases mentioned include: Cardiovascular diseases characterized by impaired myocardial function; or Cardiovascular diseases characterized by angina pectoris; or Cardiovascular diseases characterized by myocardial infarction; or Cardiovascular diseases characterized by ischemic cardiomyopathy; or Cardiovascular disease characterized by chronic heart failure. The use according to any one of claims 49 to 63 is characterized in that, The cardiovascular diseases mentioned are selected from chronic heart failure, myocardial infarction, angina pectoris, coronary heart disease, and peripheral arterial vascular diseases. The use according to any one of claims 49 to 64 is characterized in that, The cardiovascular disease mentioned is ischemic heart failure. The use according to any one of claims 49 to 65 is characterized in that, The single-dose form of the drug for the prevention or treatment of cardiovascular diseases contains between 0.03 mg and 500 mg of the compound or a pharmaceutically acceptable salt, ester, hydrate, solvate or tautomer or prodrug or mixture thereof. The use according to any one of claims 49 to 66 is characterized in that, The drug loading concentration of the compound or its pharmaceutically acceptable salt, ester, hydrate, solvate or its tautomer or its prodrug and mixture thereof in the drug for the prevention or treatment of cardiovascular diseases is 0.01 to 10 mg / mL. The use according to any one of claims 49 to 67 is characterized in that, The drugs for the prevention or treatment of cardiovascular diseases also contain pharmaceutically acceptable carriers or diluents. The use according to any one of claims 49 to 68 is characterized in that, The dosage form of the drug for the prevention or treatment of cardiovascular diseases is selected from one or more of the following: powder, granule, tablet, pill, capsule, sustained-release, controlled-release, injection, infusion, or suspension. The use according to claim 69 is characterized in that, The dosage form of the drug for the prevention or treatment of cardiovascular diseases is a tablet; the tablet is a sublingual tablet; or The dosage form of the drug for the prevention or treatment of cardiovascular diseases is an injection, and the injection also contains a solvent. The use according to claim 70 is characterized in that, The solvent is selected from one or more of alcohol solvents, ether solvents, ketone solvents, sulfur-containing organic solvents, and cyclodextrin aqueous solutions; Preferably, the alcohol solvent includes one or more of ethanol, isopropanol, ethylene glycol, propylene glycol, and polyethylene glycol; The ether solvents include one or more of ethylene glycol monoethyl ether and ethylene glycol monobutyl ether; The ketone solvents include one or more of acetone and N-methyl-2-pyrrolidone; The sulfur-containing organic solvent includes dimethyl sulfoxide; The cyclodextrin aqueous solution includes one or more of β-cyclodextrin aqueous solution, dimethyl-β-cyclodextrin aqueous solution, sulfobutyl-β-cyclodextrin aqueous solution, hydroxypropyl-β-cyclodextrin aqueous solution, and carboxymethyl-β-cyclodextrin aqueous solution.

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