Cardiovascular drugs and their use

Abstract

The present invention provides cardio-cerebrovascular drugs and their use.Specifically, the compound of the present invention has the structure represented by formula (I), where each group is defined as described in the specification.The present invention also discloses the use of said compound in treating thrombotic disease, anti-inflammation and treating neurological injury. [Formula 1] JPEG2024545920000054.jpg4242

Description

[Technical Field]

[0001] This invention relates to the field of pharmaceuticals, and more specifically to cardiovascular drugs and their use. [Background technology]

[0002] In China, the incidence of cardiovascular disease is on the rise, with an estimated 330 million people currently suffering from the condition. Stroke incidence is also on the rise in Japan, with new cases accounting for approximately 40% of global cases and deaths accounting for approximately 30% of global cases. With 13 million stroke patients surviving with chronic conditions, Japan already has the highest lifelong risk of stroke and the heaviest burden of the disease. Over the next 10 years, the incidence of coronary heart disease in China is projected to continue rising, and the incidence of acute myocardial infarction is expected to increase sharply. After 2030, there are projected to be over 20 million patients with acute myocardial infarction in China. Acute pulmonary embolism is the third most common acute cardiovascular syndrome worldwide, after myocardial infarction and stroke, with an annual incidence rate of (39-115) cases per 100,000 people, and has been increasing in recent years.

[0003] When thrombotic disorders occur, clinical treatment primarily involves surgical thrombectomy, thrombolysis, and antithrombotic therapy. Commonly used thrombolytic agents include urokinase, streptokinase, alteplase, and leteplase. Commonly used antithrombotic agents include antiplatelet agents and anticoagulants. While thrombolytic agents and orally administered antithrombotic agents offer good preventive and therapeutic effects, a drawback is that they all carry a risk of bleeding, requiring strict monitoring of the blood coagulation mechanism during their use.

[0004] During the course of a stroke, vascular occlusion occurs in the brain, cutting off blood supply and damaging neuronal cells. Therefore, clinically, it is necessary to treat the damaged neuronal cells.

[0005] Edaravone is a drug developed and marketed in Japan to treat acute stroke attacks and improve neurological symptoms, daily living activities, and functional impairments caused by acute stroke. It has been approved for market sale in China, but is not approved in major countries in Europe and the United States. Some positive results were obtained in a Phase III clinical study of the edaravone composition (edaravone-dexborneol), and it has been approved for market sale in China. However, because the design of its clinical protocol does not align with the requirements of current recognized clinical research guidelines for neuroprotective agents outside of China, it has not received approval or guideline recommendations from the US FDA.

[0006] In China's diagnostic and treatment guidelines for ischemic stroke, only a Grade II recommendation is made for butylphthalide as an agent to improve cerebral microcirculation, and there are currently no neuroprotective agents that can be recommended in international clinical guidelines. The field of mainstream, medically recognized neuroprotective agents worldwide remains largely unexplored.

[0007] Therefore, the inventors have conceived of developing a new therapeutic agent for stroke nerve injury that can effectively perform thrombolytic therapy for thrombotic diseases while simultaneously protecting the brain and nerves. This would fill a gap in the field of neuroprotective agents, meet unmet needs in the clinical field of cardiovascular diseases, and have significant clinical significance and practical value. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a cardiovascular drug and its use. [Means for solving the problem]

[0009] In a first aspect of the present invention, the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug composition or formulation, wherein the drug composition or formulation comprises a compound of formula I as an active ingredient, and the drug composition or formulation is (a) Prevention and / or treatment of thrombotic disorders, (b) Anti-inflammatory treatment, and / or (c) Treatment of neuron damage caused by surgery for cerebral infarction, cerebral trauma, cerebral hemorrhage or brain tumor for use.

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0010] In another preferred embodiment, the pharmaceutical composition is used for preventing thrombosis, thrombolytic therapy, or preventing and / or treating cerebral ischemia-reperfusion injury, inhibiting ferroptosis of nerve cells induced by cerebral ischemia-reperfusion, or eliminating vascular inflammation. In another preferred embodiment, the thrombus is selected from the group consisting of vascular endothelial injury-induced thrombus, arteriovenous shunt thrombus, cerebral ischemia-reperfusion injury, or a combination thereof.

[0011] In another preferred embodiment, the cerebral ischemia-reperfusion injury comprises cerebral infarction, cerebral edema and / or ferroptosis of nerve cells. In another preferred embodiment, the anti-inflammatory treatment is anti-vascular inflammation treatment. In another preferred embodiment, the vascular inflammation comprises vascular inflammation caused by high glucose and high lipid.

[0012] In another preferred embodiment, the pharmaceutical composition or preparation is used for preventing and / or treating thrombus-related diseases. In another preferred embodiment, the thrombus-related disease is selected from the group consisting of cerebral thrombosis, cerebral infarction (acute ischemic stroke) and neuronal damage in nerve tissue caused thereby, cerebral edema, myocardial infarction, pulmonary embolism, or a combination thereof. In another preferred embodiment, the thrombus-related disease comprises an infarction-related disease.

[0013] In another preferred embodiment, the pharmaceutical composition or preparation is used for anti-inflammatory treatment. In another preferred embodiment, the pharmaceutical composition or preparation is used for treating neuronal damage caused by surgery for cerebral infarction, cerebral trauma, cerebral hemorrhage or brain tumor.

[0014] In another preferred embodiment, R1 is selected from the group consisting of a hydroxy group, a methoxy group,

Chemical Formula

[0015] In another suitable example, m=1. In another preferred example, R4 is Na + That is the case. In another preferred example, R5 is CH3. In another preferred example, n is 6, 7, or 8.

[0016] In another preferred example, R1 is a hydroxyl group, a methoxy group, [ka] It is selected from the group consisting of the following. In another preferred example, R2 and R3 are independently H, a methyl group, a hydroxypropyl group, a hydroxyethyl group, or a carboxymethyl group.

[0017] In another preferred example, the compound of formula I is selected from the group consisting of methylcyclodextrin, carboxymethylcyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl-β-cyclodextrin. In another preferred example, the parent compound of formula I is a cyclodextrin.

[0018] In another preferred example, the compound of formula I is [ka] That is the case.

[0019] In another preferred example, the compound of formula I is selected from the following group: [ka]

[0020] In another preferred example, the drug composition further comprises another active ingredient. In another preferred example, the other active ingredient is selected from the group consisting of butylphthalide, urokinase, edaravone, or a combination thereof. In another preferred example, the drug composition or formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0021] In another preferred example, the dosage form of the drug composition or formulation is selected from the group consisting of injection solutions, lyophilized powder injections, sustained-release, controlled-release, enteric-coated tablets or capsules, and granules. In another preferred example, the drug composition or formulation contains, by weight of the composition, 0.001–99 wt%, preferably 0.1–90 wt%, and more preferably 1–80 wt%, of the compound of formula I or a pharmaceutically acceptable salt thereof as the active ingredient.

[0022] A second aspect of the present invention provides the use of a composition of compound I and urokinase for the manufacture of a drug for treating thrombosis-related diseases. A third aspect of the present invention provides the use of a composition of compound I and butylphthalide for the manufacture of a drug for treating nerve damage caused by cerebral infarction.

[0023] A fourth aspect of the present invention is a drug composition or formulation comprising (a) an active ingredient comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, (1) Prevention and / or treatment of thrombotic disorders, (2) Anti-inflammatory treatment, and / or (3) Treatment of neuronal damage resulting from surgery for cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumor To provide a drug composition or formulation for use in this purpose.

[0024] In another preferred example, if the active ingredient comprises two components, the weight ratio of the two components is 1:20 to 20:1, preferably 1:10 to 10:1, and more preferably 1:5 to 5:1.

[0025] A fifth aspect of the present invention provides a kit characterized by comprising the following: (1) A first container, and a first drug composition comprising a first compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, contained within the container; (2) An nth drug composition comprising an nth container, the nth compound or a pharmaceutically acceptable salt thereof contained in the container, and a pharmaceutically acceptable carrier; where n is any positive integer between 2 and 8; Here, both the first compound and the nth compound are compound I or a pharmaceutically acceptable salt thereof, or the first compound is compound I or a pharmaceutically acceptable salt thereof, and at least one of the nth compounds is another active substance, where compound I is as defined in claim 1; (3) an optional instruction manual.

[0026] In a sixth aspect of the present invention, the present invention provides a method for treating a disease, wherein the disease is as described in claim 1, and the method comprises the step of applying a compound of formula I or a pharmaceutically acceptable salt thereof to a target subject, wherein the compound of formula I is as defined in claim 1. In another preferred example, the subject is a mammal. In another preferred example, the subject is a human being.

[0027] Of course, within the scope of the present invention, it is understood that the above-mentioned technical features of the present invention and the technical features specifically described below (for example, in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, a detailed explanation will not be provided here. [Modes for carrying out the invention]

[0028] Specific Embodiments Through extensive and in-depth research, the inventors have unexpectedly discovered that compounds represented by the structure of formula I have remarkable effects in the prevention and / or treatment of thrombotic diseases, anti-inflammatory treatment, and treatment of neuronal damage resulting from surgery for cerebral infarction, traumatic brain injury, cerebral hemorrhage, or brain tumors. Experiments have shown that the aforementioned compound of formula I has a good therapeutic effect on brain nerve damage caused by stroke. The compound of formula I of the present invention can be used for the prevention and thrombolysis of thrombotic diseases, anti-inflammatory treatment, and the like. Based on this, the present invention has been completed.

[0029] term: The term "halogen" refers to F, Cl, Br, and I. The term "C1-C6 alkyl group" refers to a linear or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, neopentyl, t-pentyl, or similar groups.

[0030] The term "C2-C6 alkenyl group" refers to a linear or branched alkenyl group having 2-6 carbon atoms and containing one double bond, including, but not limited to, vinyl, propenyl, butenyl, isobutenyl, pentenyl, and hexenyl groups. The term "C2-C6 alkynyl group" refers to a linear or branched alkynyl group having 2-6 carbon atoms and containing one triple bond, including, but not limited to, ethynyl, propagyl, butynyl, isobutynyl, pentynyl, and hexynyl groups.

[0031] The term "C1-C6 hydroxyalkyl group" refers to a linear or branched alkyl group having 1 to 6 carbon atoms and containing one hydroxyl group, including but not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl, and hydroxybutyl groups, with C1-C3 hydroxyalkyl groups being preferred. The term "C3-C8 cycloalkyl group" refers to a cyclic alkyl group having 3 to 8 carbon atoms in its ring, and includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.

[0032] The term "C1-C6 alkoxy group" refers to a linear or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy, and butoxy groups. C1-C4 alkoxy groups are preferred. The terms "aromatic ring" and "aryl group" have the same meaning, and "C6-C10 aryl group" is preferred. The term "C6-C10 aryl group" refers to an aromatic ring group that does not contain heteroatoms and has 6-10 carbon atoms, such as the phenyl group and the naphthyl group.

[0033] The term "heteroaryl group" refers to a heteroaromatic system containing one to four heteroatoms, where the heteroatoms are nitrogen, oxygen, or S(O)r (where r is an integer 0, 1, or 2). For example, a 4-8 membered heteroaryl group is a heteroaromatic system containing 4-8 ring atoms, and a 4-10 membered heteroaryl group is a heteroaromatic system containing 4-10 ring atoms. These include, but are not limited to, pyrrolyl, furyl, thienyl, pyrazolyl, thiazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridyl, pyranyl, pyridazyl, pyrimidinyl, pyrazinyl, benzimidazolyl, and triazolyl groups.

[0034] Unless otherwise specifically described as "substituted or unsubstituted," any of the groups described in the present invention may be substituted with substituents selected from the group consisting of halogens, acyloxy groups, cyano groups, amino groups, nitro groups, carboxyl groups, amide groups, carboxymethyl groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, C1-C6 haloalkyl groups, C2-C6 alkenyl groups, C2-C6 haloalkenyl groups, C2-C6 alkynyl groups, C1-C6 haloalkynyl groups, hydroxyl groups, C3-C6 cycloalkyl groups, C3-C6 halocycloalkyl groups, hydroxyC1-C4 alkyl groups, C5-C7 cycloalkenyl groups, phenyl groups, and naphthyl groups. [ka] This indicates the bonding position of the base.

[0035] active substance As used herein, the terms “compound of the present invention” and “active ingredient of the present invention” are interchangeable and refer to compound I. [ka]

[0036] The present invention further includes pharmaceutically acceptable salts of the compound of formula I. The term "pharmaceutically acceptable salt" refers to a salt suitable as a drug, formed with the compound of the present invention and an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. One preferred salt is a salt formed with the compound of the present invention and an acid. Acids suitable for salt formation include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propanoic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0037] The compound of formula I of the present invention can be produced by methods well known to those skilled in the art in the prior art, and the reaction parameters of each step are not particularly limited. Furthermore, exemplary compounds of the present invention can also be obtained as commercial products. As used herein, when a chiral carbon atom exists in the compound of formula I, the chiral carbon atom may be in R configuration, may be in S configuration, or may be a mixture of both.

[0038] In the present invention, the active ingredient described above is the compound of formula I.

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

Chemical Formula

[0039] In one embodiment, R1 is a hydroxyl group, a methoxy group, [ka] It is selected from the group consisting of the following.

[0040] In another embodiment, m=1. In another embodiment, R4 is Na + That is the case. In another embodiment, R5 is CH3. In another embodiment, n is 6, 7, or 8.

[0041] In another embodiment, R1 is a hydroxyl group, a methoxy group, [ka] It is selected from the group consisting of the following. In another embodiment, R2 and R3 are independently H, a methyl group, a hydroxypropyl group, a hydroxyethyl group, or a carboxymethyl group.

[0042] In another embodiment, the compound of formula I is selected from the group consisting of methylcyclodextrin, carboxymethylcyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl-β-cyclodextrin. In another embodiment, the core of the compound of formula I is a cyclodextrin.

[0043] In another embodiment, the compound of formula I is [ka] That is the case. In another embodiment, the compound of formula I is selected from the following group: [ka]

[0044] Drug composition and method of administration Furthermore, the present invention provides a drug composition comprising a pharmaceutically acceptable carrier and one or more safely effective amounts of a compound according to the present invention. Because the compounds of the present invention possess excellent antithrombotic activity, the compounds of the present invention and their various crystalline forms, pharmaceutically acceptable inorganic and organic salts, hydrates or solvates, and drug compositions containing the compounds of the present invention as the main active ingredient are useful for the treatment, prevention, and alleviation of embolism-related diseases.

[0045] The drug composition of the present invention comprises the compound of the present invention or a pharmaceutically acceptable salt in a safe effective amount, and a pharmaceutically acceptable excipient or carrier. Here, "safe effective amount" means an amount of the compound that is sufficient for a significant improvement of the disease condition without causing serious side effects. Typically, the drug composition contains the compound of the present invention in an amount of 1-2000 mg / formulation, preferably 10-1000 mg / formulation. Preferably, the "formulation" is a capsule or a tablet.

[0046] "Pharmacochemically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are applicable to humans and must be of sufficient purity and sufficiently low toxicity. "Compatible" means that each component in the composition can be compounded with and with the compounds of the present invention without significantly reducing the effect of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., twinol). R These include humectants (e.g., sodium dodecyl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, and distilled water from which pyrogenic substances have been removed.

[0047] The aforementioned drug composition is in the form of an injection, capsule, tablet, pill, powder, or granule. The mode of administration of the drug composition of the present invention is not particularly limited, but typical modes of administration include, but are not limited to, oral administration, intratumoral, rectal, extragastrointestinal (intravenous, intramuscular, or subcutaneous) administration, and local administration.

[0048] Solid dosage forms used for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is usually mixed with at least one inactive excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) a filler or phase solvent, such as starch, lactose, sucrose, glucose, mannitol, or silicic acid; (b) a binder, such as hydromethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, or gum arabic; (c) a humectant, such as glycerin; or (d) a disintegrant, such as agar or carbonate. It is mixed with ingredients such as (e) solution retarders, such as paraffin, (f) absorption enhancers, such as ammonium compounds, (g) wetting agents, such as cetanol and glycerin monostearate, (h) adsorbents, such as kaolin, and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may include buffers.

[0049] Solid dosage forms, such as tablets, pills, capsules, rounds, and granules, can be manufactured with coatings and shells, such as venous coatings and other materials known in the art. Opaque agents may be included, and in such compositions, the release of the active substance or compound may be delayed and released in a portion of the gastrointestinal tract. Examples of usable embedding components include polymers and waxy substances. If necessary, the active compound may also be formed in the form of microcapsules with one or more of the excipients.

[0050] Liquid dosage forms used for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage forms may also include inert diluents commonly used in this art, such as water or other solvents, phase solvents, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, or mixtures thereof.

[0051] In addition to these inert diluents, the composition may also contain auxiliary agents, such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, and fragrances. In addition to the active compound, the suspension may also contain suspending agents, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol or sorbitan ester, microcrystalline cellulose, methoxyaluminum or agar, or mixtures thereof.

[0052] Compositions for extra-gastrointestinal injection include physiologically acceptable sterile water-containing or water-free solutions, dispersions, suspensions, and emulsions, as well as sterile powders for redissolving into sterile, injectable solutions or dispersions. Suitable water-containing or water-non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyhydric alcohols, and suitable mixtures thereof. Dosage forms of the compounds of the present invention for topical administration include ointments, powders, poultices, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, an excipient.

[0053] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (for example, antithrombotic agents). The therapeutic method of the present invention may be administered alone or in combination with other therapeutic means or therapeutic agents. When using the drug composition, a safe and effective amount of the compound of the present invention should be administered to a mammal in need of treatment (e.g., human), and the dosage should be a pharmaceutically effective dose. For a human weighing 60 kg, the daily dose is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dose should take into account factors such as the mode of administration and the patient's health condition, all of which should be within the skill range of a qualified physician.

[0054] The main advantages of this invention are as follows: (a) Compound I of the present invention has a beneficial therapeutic effect on brain nerve damage caused by stroke. (b) The compound of formula I of the present invention can be used for the prevention and thrombolysis of thrombosis in thrombotic diseases and does not lead to bleeding. (c) Compound I of the present invention has an anti-inflammatory effect. (d) Of the compounds of formula I of the present invention, compound 1 has excellent safety and low toxicity and side effects.

[0055] The present invention will be further described below with reference to specific examples. These examples are used solely for the purpose of illustrating the present invention and are not intended to limit the scope of the invention. Experimental methods in the following examples where specific conditions are not given will generally follow standard conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or the manufacturer's recommended conditions. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight.

[0056] Example 1: Experiment to evaluate the preventive effect of the sample against vascular endothelial damage thrombosis. Experimental method: 420 5 dpf wild-type AB zebrafish were randomly placed in a 6-well plate, with 30 fish in each well (experimental group). Compounds (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), and (11) (see Table 1 for structural formulas) were administered by injection at a dose of 4.00 ng / fish. Acetylsalicylic acid, a positive control drug, was administered in aqueous solution at a concentration of 30.0 μg / mL. Simultaneously, a normal control group (i.e., zebrafish treated with standard diluted water) and a model control group were established, with each well (experimental group) having a volume of 3 mL. Except for the normal control group, all other experimental groups were administered ponatinib in aqueous solution to induce a zebrafish endothelial thrombosis model. Under incubation conditions of 28°C, the samples were treated with ponatinib for a set period of time, and then stained with o-dianisidine.

[0057] After staining, 10 zebrafish were randomly selected from each group, photographed under a microscope, and the staining intensity of red blood cells in the zebrafish hearts was analyzed using NIS-Elements D 3.20 advanced image processing software. The prophylactic effect of the samples against endothelial thrombosis was evaluated based on the statistical results of the staining intensity of red blood cells in the hearts.

[0058] The formula for calculating the thrombosis-preventive effect is as follows:

number

[0059] Experimental results: [Table 1-1] [Table 1-2]

[0060] Experimental results: The staining intensities of cardiac red blood cells in zebrafish administered 4.00 ng / fish for compound (1), compound (2), compound (3), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), and compound (11) were 4397, 3655, 3045, 4300, 4340, 4938, 4584, 4580, 4709, 4326, and 4749 pixels, respectively.

[0061] Compared to the model control group, the thrombopreventive effect of compound (3) at a dose of 4.00 ng / animal was 17% with p > 0.05, while the thrombopreventive effects of compound (1), compound (2), compound (4), compound (5), compound (6), compound (7), compound (8), compound (9), compound (10), and compound (11) at a dose of 4.00 ng / animal were 66%, 39%, 63%, 64%, 86%, 73%, 73%, 78%, 64%, and 79%, respectively, in the groups receiving p < 0.01, p < 0.05, p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, and p < 0.001.

[0062] Experimental conclusion: Compounds (1), (2), (4), (5), (6), (7), (8), (9), (10), and (11) have a clear prophylactic effect on ponatinib-induced endothelial thrombosis in zebrafish under the conditions of this experiment. In particular, compounds (6), (11), (9), (7), (8), and (1) had a very significant preventive effect against ponatinib-induced endothelial thrombosis in zebrafish at a dose of 4.00 ng / fish, with a thrombopreventive effect (%) index of 66%-86%, compared to 101% for acetylsalicylic acid at 30 μg / ml.

[0063] Example 2: Evaluation of allergy risk using samples Experimental method: Two dpf wild-type AB zebrafish were randomly selected and placed in a 24-well plate. Six duplicate wells were created in each group, and 10 zebrafish were treated in each well. The sample was administered intravenously at a dose of 500 ng / fish, and the concentration of the positive control "C48 / 80" was 1.5 μg / mL. A normal control group was also established, with each well containing 1 mL of sample. In each experimental group, BAPNA was administered in aqueous solution. After treatment at 28°C for 1 day, the liquid was transferred to a 96-well plate, and the relative absorbance (OD) value of the tryptase expression level in each experimental group was detected using a microplate reader at a rate of 200 μL / well. The allergy risk of the samples was evaluated based on the results of statistical analysis of the OD values. Statistical results are expressed as mean ± standard error.

number

[0064] [Table 2]

[0065] Experimental results: Even under conditions where the dosage was increased 125 times from 4 ng / animal in Example 1 (i.e., 500 ng / animal), the tested compounds of the present invention showed a low risk of allergies. Representatively, as shown in Table 2, all tested compounds (1), (2), and (10) of the present invention showed significantly lower allergy risks than the positive control. Of these, "Compound (2)" and "Compound (1)" had little to no allergy risk, while "Compound (10)" had a low allergy risk.

[0066] Experimental conclusion: Combining the experimental results of Examples 1 and 2, that is, taking into account safety and efficacy, compound (1) was selected as the preferred compound for subsequent experiments in this invention.

[0067] Example 3 Evaluation of the preventive effect of Compound 1 against vascular endothelial damage thrombosis Experimental Method: A zebrafish endothelial thrombosis model was constructed by treating wild-type AB zebrafish with 4 μg / mL ponatinib for 18 hours. 150 5 dpf wild-type AB zebrafish were randomly placed in a 6-well plate, with 30 fish in each well (experimental group). Compound (1) was administered by injection at doses of 2.06 and 6.18 ng / fish. Acetylsalicylic acid, a positive control drug, was administered in aqueous solution at a concentration of 30 μg / mL. Simultaneously, a normal control group (i.e., zebrafish treated with standard diluted water) and a model control group were established, with each well (experimental group) having a volume of 3 mL. Except for the normal control group, all other experimental groups were administered ponatinib in aqueous solution to induce the zebrafish endothelial thrombosis model. Under incubation conditions of 28°C, compound (1) was treated with ponatinib for a set period of time, followed by staining with o-dianisidine. After staining, 10 zebrafish were randomly selected from each group and photographed under a microscope. The staining intensity of red blood cells in the hearts of the zebrafish was analyzed using NIS-Elements D 3.10 advanced image processing software, and the prophylactic effect of compound (I) against endothelial thrombosis was evaluated based on the statistical results of the staining intensity of red blood cells in the hearts. The formula for calculating the thromboprophylactic effect is as follows.

number

[0068] [Table 3]

[0069] Experimental results: In the groups administered compound (1) at doses of 2.06 ng / fish and 6.18 ng / fish, the zebrafish cardiac erythrocyte staining intensity was 4601 and 5044 pixels, respectively. Compared to the model control group, the p < 0.001 effect indicated a thrombopreventive effect of 83% and 99%, respectively. Experimental conclusion: Compound (1) has a significant prophylactic effect against ponatinib-induced endothelial thrombosis in zebrafish, and this prophylactic effect is somewhat dose-dependent. Compound (1) has a better prophylactic effect against endothelial thrombosis than the positive control acetylsalicylic acid.

[0070] Example 4: Effect of compound (1) on rat arteriovenous shunt thrombosis Experimental method: Grouping: The animals were divided into 5 groups of 10 animals each. The model control group was administered the solvent, while the test drug compound (1) and the positive control group were administered intravenously for 3 consecutive days. The model was constructed once daily, 30 minutes after the last dose. Model construction: 50 male SD rats were anesthetized by ip with pentobarbital tonate sodium 60 mg / kg. The rats were fixed in a supine position, an incision was made in the middle of the neck, the fascia and muscle tissue were separated, and the left common carotid artery and right external jugular vein were freed. Arteriovenous (AV) bypass surgery was performed using a polyethylene tube with a thread embedded in it. The proximal part of the carotid artery was clamped with an arterial clip, and one end of the polyethylene tube was inserted into the artery and the other into the vein to complete the bypass surgery. 30 minutes after the last dose, the arterial clip was opened, and blood flowed from the left common carotid artery through the bypass tube to the right external jugular vein, creating a shunt circulation. The thrombus was removed 15 minutes later.

[0071] Detection criteria: After 15 minutes, blood flow was released, the thread was removed, residual fluid was aspirated, the weight of the thread with the thrombus attached was measured, and the wet weight of the thrombus was calculated. The thread was heated and dried in a 50°C oven, and the dry weight of the thrombus was calculated. The thrombus formation inhibition rate was calculated. After the end of the test, the tail was cut and the bleeding time was detected. [Table 4]

[0072] Experimental results: Compared to the model control group, intravenous injection of compound (1) at 10 mg / kg, 20 mg / kg, and 40 mg / kg suppressed rat arteriovenous shunt thrombus formation to varying degrees. The suppression rates of wet weight and dry weight of thrombus were 19.4% (P>0.005), 15.9% (P>0.005), 21.9% (P<0.001), 26.8% (P<0.001), 34.3% (P<0.001), and 37.8% (P<0.001), respectively. Intravenous administration of ozagrel at 16 mg / kg resulted in suppression rates of wet weight and dry weight of thrombus of 34% (P<0.001) and 34.1% (P<0.01), respectively. Experimental conclusion: The antithrombotic effect of compound (1) is dose-dependent, and its antithrombotic effect is equivalent to or better than that of ozagrel.

[0073] [Table 5]

[0074] Experimental results: Compared to the model control group, intravenous administration of different doses of compound (1) had no significant effect on rat bleeding time. In the positive control group, intravenous administration of ozagrel significantly prolonged the bleeding time in rats (P<0.01). Experimental conclusion: Bleeding experiments with compound (1) showed non-dose-dependent effects, thus proving that compound (1) does not pose a significant bleeding risk.

[0075] Example 5: Effects of prophylactic administration of compound (1) on cerebral ischemia-reperfusion injury in rats Experimental Method: SD rats were randomly divided into six groups: a sham surgery group, a model control group, a positive control drug ozagrel (6 mg / kg) group, and groups receiving high (67.5 mg / kg), medium (22.5 mg / kg), and low (7.5 mg / kg) doses of the test drug compound (1). In the treatment groups, the drug was administered by injection via the tail vein beforehand. In the sham surgery group and the model control group, an equal volume of physiological saline was administered once daily for three consecutive days. Ten minutes after the last dose, a rat MCAO / R model was constructed, and reperfusion was performed two hours after ischemia. Twenty-four hours after MCAO reperfusion, the rats were scored on a neurobehavioral scale to measure their neurological function. The incidence of cerebral infarction and cerebral water content in rats were measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

[0076] [Table 6]

[0077] [Table 7]

[0078] [Table 8]

[0079] Experimental results: Each dose group of compound (1) and the positive drug ozagrel group were able to reduce the neurological function score, stroke rate, and cerebral water content in MCAO / R rats. Experimental conclusion: Prophylactic administration of compound (1) can reduce the incidence of cerebral infarction and cerebral water content in rats with cerebral ischemia-reperfusion injury and improve rat neuromotor function.

[0080] Example 6 Effects of combination of compound (1) and urokinase (iv) on autologous thrombosis and thromboplastin-induced ischemic brain injury in rats Experimental Method: An SD rat cerebral ischemia injury model was constructed by injecting autologous thrombus and thromboplastin into the internal carotid artery via the external carotid artery to embolize the arteries in the rat cerebrum. The rats were randomly divided into five groups based on behavioral scores: a sham surgery group (external carotid artery isolation only), a model control group (both groups were administered equal volumes of saline), a urokinase (5000 U / kg) group, and a compound (1) (22.5 mg / kg) group. Each group consisted of 20 rats (10 used for detecting cerebral infarction rates and 10 for pathological detection). Two hours after model construction, a single dose was administered by slow injection (1 mL / min) into the tail vein. After thromboplastin injection, the decrease in blood flow was monitored at 120 minutes post-administration. After 24 hours, the rats were scored on their neurobehavioral levels to measure their neurological function. The incidence of cerebral infarction and cerebral water content in rats were measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and pathological damage to the rat brain 2 hours after administration following cerebral ischemia was observed by hematoxylin-eosin (HE) staining.

[0081] Experimental results: [Table 9]

[0082] (The data is presented as mean ± standard deviation, and n=10.) [Table 10]

[0083] [Table 11]

[0084] [Table 12]

[0085] After constructing models of cerebral ischemia injury induced by autologous thrombosis and thromboplastin, intravenous administration of compound (1), urokinase, and compound (1) + urokinase significantly reduced the percentage decrease in cerebral blood flow, the cerebral infarction area and cerebral water content in model rats, and improved pathological injury and behavioral changes. Among these, compound (1) + urokinase showed the most significant improvement in cerebral ischemia injury induced by autologous thrombosis and thromboplastin.

[0086] Experimental conclusion: Compound (1)(iv) and its combination with urokinase reduced the rate of cerebral infarction, cerebral water content, and pathological damage in rats with cerebral ischemia caused by autologous thrombosis and thromboplastin, and improved neuromotor function in rats, demonstrating a good anti-ischemic brain injury effect.

[0087] Example 7 Effects of therapeutic administration of compound (1) on cerebral ischemia-reperfusion injury in rats Experimental method: SD rats were randomly divided into 11 groups: a sham surgery group, a model control group, a group receiving intravenous injection of the positive control drug edaravone (6 mg / kg, iv), a group receiving intravenous injection of the positive control butylphthalide solution (5 mg / kg, iv), a group receiving high-dose intravenous injection of compound (1) (24 mg / kg, iv), a group receiving medium-dose intravenous injection of compound (1) (12 mg / kg, iv), a group receiving low-dose intravenous injection of compound (1) (6 mg / kg, iv), a group receiving intragastric administration of the positive drug butylphthalide soft capsules (60 mg / kg, ig), a group receiving high-dose intragastric administration of compound (1) (60 mg / kg, ig), a group receiving medium-dose intragastric administration of compound (1) (30 mg / kg, ig), and a group receiving low-dose intragastric administration of compound (1) (15 mg / kg, ig). A middle cerebral artery occlusion (MCAO) cerebral ischemia-reperfusion model was constructed in male rats using internal carotid fissure, and reperfusion was performed after 90 minutes of ischemia. One hour after reperfusion, the drug was administered via tail vein and orally. After 24 hours, the rats were scored on a neurobehavioral scale to measure their neurological function. The incidence of cerebral infarction and cerebral water content in rats were measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining.

[0088] [Table 13]

[0089] [Table 14]

[0090] [Table 15]

[0091] Experimental results: Each dose group of compound (1) and each positive drug group were able to reduce the neurological function score, stroke rate, and cerebral water content of MCAO / R rats to varying degrees. Among these, the compound (1) (24 mg / kg, iv) group and the compound (1) (60 mg / kg, ig) group showed the most significant effects. Experimental conclusion: Therapeutic administration of compound (1) can reduce the stroke rate and cerebral water content in rats with cerebral ischemia-reperfusion injury, and improve rat neuromotor function.

[0092] Example 8: Study on the suppression of neuronal ferroptosis during cerebral ischemia-reperfusion by compounds 1, 3, and 5. Experimental method: Primary neuron cells were incubated for 24 hours in a medium containing elastin (final concentration 50 μM), followed by 24 hours of incubation in a medium containing different concentrations of the test drug and elastin (final concentration 50 μM). After incubation, GPX4 mRNA expression in each cell group was detected by real-time PCR, cell vitality was detected by CCK8 experiment, intracellular lipid ROS levels were detected by C11-BODIPY probe method, and intracellular iron content was detected by PGSK probe method. Experimental data are presented as mean ± SD. Between-group statistical analysis was performed using one-way ANOVA and Tukey's test, with a p-value < 0.05 considered statistically significant.

[0093] Experimental results: [Table 16]

[0094] After incubation, the cellular vitality of the cells in each group was detected by the CCK8 experiment. Results are expressed as mean ± SD. Compared to the model control group, ***p < 0.0001. Compared to the model control group, # p<0.05, ## p < 0.01. As can be seen from Table 16, in the model group, cell vitality was significantly lower than in the control group (p<0.0001), when the concentration of compound 3 was greater than 10 μM, cell vitality was clearly improved compared to the model group (p<0.05), and when the concentrations of compound 5 and compound 1 were greater than 5 μM, cell vitality was clearly increased compared to the model group (p<0.05).

[0095] [Table 17]

[0096] After incubation, intracellular lipid ROS levels were detected in each group using the C11-BODIPY probe method. Results are expressed as mean ± SD. Compared to the control group, p<0.0001, and compared to the model group, # p<0.05, ## p < 0.01. As can be seen from Table 17, in the model group, intracellular lipid ROS levels were significantly elevated compared to the control group (p<0.0001), when the concentration of compound 3 was greater than 10 μM, intracellular lipid ROS levels were clearly lower compared to the model group (p<0.05), and when the concentrations of compound 5 and compound 1 were greater than 5 μM, intracellular lipid ROS levels were significantly lower compared to the model group (p<0.01).

[0097] [Table 18]

[0098] After incubation, intracellular iron content was detected using the PGSK probe method. Results are expressed as mean ± SD. Compared to the control group, p<0.0001, and compared to the model group, # p<0.05, ## p < 0.01. As can be seen from Table 18, in the model group, the fluorescence intensity of the cellular PGSK probe was clearly reduced compared to the control group, and the iron content was significantly increased (p<0.0001). When the concentration of compound 3 was greater than 10 μM, the intracellular iron content was clearly reduced compared to the model group (p<0.05). When the concentrations of compound 5 and compound 1 were greater than 5 μM, the intracellular iron content was significantly reduced compared to the model group (p<0.05).

[0099] [Table 19]

[0100] After incubation, GPX4 mRNA expression in the cells of each group was detected by real-time PCR. Results are expressed as mean ± SD. Compared to the control group, *** When p<0.0001, compare with the group of models. # p<0.05, ## p < 0.01. As can be seen from Table 19, in the model group, cellular GPX4 mRNA expression was significantly reduced compared to the control group (p<0.0001), when the concentration of compound 3 was greater than 5 μM, the cellular GPX4 mRNA expression level was clearly increased compared to the model group (p<0.05), and when the concentrations of compound 5 and compound 1 were greater than 2.5 μM, the cellular GPX4 mRNA expression level was significantly increased compared to the model group (p<0.01).

[0101] Experimental conclusions: Compounds 1, 3, and 5 all significantly increased the cellular vitality and GPX4 mRNA expression of elastin-induced rat neurons, and significantly decreased intracellular lipid ROS levels and iron content. These results indicate that compounds 1, 3, and 5 can all suppress elastin-induced ferroptosis in rat neurons.

[0102] Example 9 Evaluation of the anti-inflammatory effects of Compound 1, Compound 3, and Compound 5 on vascular inflammation Experimental Method: 360 5 dpf macrophage fluorescent zebrafish were randomly selected and placed in beakers. Compounds (1), (3), and (5) were administered intravenously to 30 zebrafish in each beaker. The positive control group received atorvastatin calcium in aqueous solution at a concentration of 30 μg / mL. Simultaneously, a normal control group (zebrafish treated with rearing water) and a model control group were established, with each beaker (experimental group) having a volume of 25 mL. All groups except the normal control group were given a high-sugar, high-fat diet in aqueous solution. Compounds (1), (3), and (5) were treated with the high-sugar, high-fat diet for 30 hours to reach 9 dpf. Ten zebrafish were randomly selected from each experimental group, and the fluorescence intensity of macrophages in and around the tail vein of the zebrafish was detected using a fluorescence microscope. The reductive effects of compounds (1), (3), and (5) on vascular inflammation in high-sugar, high-fat model zebrafish were evaluated based on the results of statistical analysis.

number

[0103] Experimental results: [Table 20]

[0104] Experimental conclusion: Under the dosage conditions of this experiment, compounds (1), (3), and (5) have a significant anti-inflammatory effect on vascular inflammation in high-sugar, high-lipid zebrafish models.

[0105] Example 10 Preparation of Injectable Solution [Table 21]

[0106] 80% of the total volume of sterile water for injection was taken, and the prescribed amounts of sodium chloride, sodium citrate dihydrate, glycerin, and compound (1) were added in order. The mixture was stirred to dissolve, and the pH of the solution was adjusted to 5.0-6.5 with 0.1M hydrochloric acid or sodium hydroxide. The solution was then diluted to the specified volume with sterile water for injection. The solution was divided into 10 ml glass bottles, sterilized by heating and pressurizing, and an injectable solution of the compound was obtained. Each bottle contained 20 mg of compound (1).

[0107] Example 11 Preparation of lyophilized powder injection [Table 22]

[0108] Compound (1) was added to 80% of the amount of sterile water for injection, stirred to dissolve, and then sterile water for injection was added until the desired amount was reached. Activated carbon (0.1-0.5%) was added to the above solution and stirred for several tens of minutes. The solution was decolorized with activated carbon and filtered to remove the carbon. The pH of the solution was adjusted with lithium hydroxide solution (or hydrochloric acid solution). A sample was taken to detect the semi-finished product. The solution was filtered again using a 0.22 μm filter and bottled. The solution was freeze-dried according to the specified freeze-drying parameters, pressed, sealed with a lid, and packaged.

[0109] All documents relating to the present invention are cited herein by reference, so that each document may be cited independently. Furthermore, after reading the above, those skilled in the art will understand that various variations and modifications of the present invention may be made, and that equivalent forms thereof are included within the scope of the claims of the present invention.

Claims

1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a drug composition or formulation, wherein the drug composition or formulation comprises a compound of formula I as an active ingredient, and the drug composition or formulation is It is used for the prevention and / or treatment of thrombotic disorders, where thrombotic disorders are selected from the group consisting of cerebral thrombosis, cerebral infarction and resulting neuronal damage to nerve tissue, cerebral edema, myocardial infarction, pulmonary embolism, or combinations thereof. The aforementioned use, characterized by the above. 【Chemistry 1】 (In the formula, R １ teeth, 【Chemistry 2】 That is the case. Here, m is a positive integer between 1 and 6. 【Transformation 3】 in 【Chemistry 4】 The carbon atoms are chiral carbon atoms, and the aforementioned chirality is, 【Transformation 5】 They are selected from among them. R ４ Na ＋ _K ＋ Li ＋ or Cs ＋ It is a metal ion selected from the group consisting of the following. R ５ These are C1-C6 alkyl groups or C3-C8 cycloalkyl groups. R ２ and R ３ are each independently H or a C1-C6 alkyl group. n is a positive integer between 6 and 12.

2. R １ but, 【Transformation 6】 The use according to claim 1, characterized in that it is selected from the group consisting of the following.

3. The use according to claim 1, characterized in that n is 6, 7, or 8.

4. R ２ and R ３ The use according to claim 1, characterized in that each is independently H.

5. Compound I of formula is as follows: 【Transformation 7】 The use according to claim 1, characterized in that it is selected from the group consisting of the following.

6. The use according to claim 1, characterized in that the thrombus is selected from the group consisting of vascular endothelial damage thrombus, arteriovenous shunt thrombus, cerebral ischemia-reperfusion injury, or a combination thereof.

7. The use according to claim 6, characterized in that the cerebral ischemia-reperfusion injury is selected from the group consisting of cerebral infarction, cerebral edema, and / or neuronal ferroptosis.

8. The use according to claim 1, characterized in that thrombosis-related diseases include infarction-related diseases.

9. The use according to claim 1, characterized in that the dosage form of the drug composition or formulation is selected from the group consisting of injection solution, lyophilized powder injection, sustained-release, controlled-release, enteric-coated tablet, capsule, and granule formulation.

10. The use of a combination of a compound of formula I and a urokinase composition, used in the manufacture of a drug for treating thrombotic disorders, wherein the thrombotic disorder is selected from the group consisting of cerebral thrombosis, cerebral infarction and resulting neuronal damage to nerve tissue, cerebral edema, myocardial infarction, pulmonary embolism, or a combination thereof. 【Transformation 8】 (In the formula, R １ teeth, 【Chemistry 9】 That is the case. Here, m is a positive integer between 1 and 6. 【Chemistry 10】 in 【Chemistry 11】 The carbon atoms are chiral carbon atoms, and the aforementioned chirality is, 【Chemistry 12】 They are selected from among them. R ４ Na ＋ _K ＋ Li ＋ or Cs ＋ It is a metal ion selected from the group consisting of the following. R ５ These are C1-C6 alkyl groups or C3-C8 cycloalkyl groups. R ２ and R ３ Each of these is independently either H or a C1-C6 alkyl group. n is a positive integer between 6 and 12.

11. A drug composition or formulation comprising (a) an active ingredient comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier, It is used for the prevention and / or treatment of thrombotic disorders, where thrombotic disorders are selected from the group consisting of cerebral thrombosis, cerebral infarction and resulting neuronal damage to nerve tissue, cerebral edema, myocardial infarction, pulmonary embolism, or combinations thereof. The drug composition or formulation characterized by the above. 【Chemistry 13】 (In the formula, R １ teeth, 【Chemistry 14】 That is the case. Here, m is a positive integer between 1 and 6. 【Chemistry 15】 in 【Chemistry 16】 The carbon atoms are chiral carbon atoms, and the aforementioned chirality is, 【Chemistry 17】 They are selected from among them. R ４ Na ＋ _K ＋ Li ＋ or Cs ＋ It is a metal ion selected from the group consisting of the following. R ５ These are C1-C6 alkyl groups or C3-C8 cycloalkyl groups. R ２ and R ３ Each of these is independently either H or a C1-C6 alkyl group. n is a positive integer between 6 and 12.

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