2, 3-dihydrobenzofuran derivative and application thereof in preparation of drugs for treating cardiovascular and cerebrovascular diseases

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to 2, 3-dihydrobenzofuran derivatives and application thereof in preparation of drugs for treating cardiovascular and cerebrovascular diseases. The 2, 3-dihydrobenzofuran derivative disclosed by the invention has a protective effect on H9c2 rat myocardial cell injury caused by H2O2, and can improve the cell survival rate and reduce the LDH release rate; meanwhile, the area of myocardial infarction of mice after myocardial infarction can be remarkably reduced, and the compound has an obvious myocardial protection effect, can be developed into drugs for resisting cardiovascular and cerebrovascular diseases and has a good application prospect. When the 2, 3-dihydrobenzofuran derivative and the luteolin are combined for use, a synergistic effect on treatment of cardiovascular and cerebrovascular diseases is achieved, the synergistic effect of the 2, 3-dihydrobenzofuran derivative and the luteolin can improve the protection effect on the heart, and after the 2, 3-dihydrobenzofuran derivative and the luteolin are combined for use, compared with a single drug, the damage of H2O2 to cells is obviously reduced, and the treatment effect on cardiovascular and cerebrovascular diseases is improved. The cell survival rate is obviously increased, and the LDH release rate is obviously reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a class of 2,3-dihydrobenzofuran derivatives and their application in the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases. Background Art

[0002] Cardiovascular-related diseases have become major diseases worldwide due to their high incidence and high mortality. Among them, ischemic heart disease (IHD) is the main cause of global death and disability. Although percutaneous coronary intervention for myocardial ischemia is crucial for reducing myocardial injury, the restoration of blood flow may lead to myocardial ischemia-reperfusion injury (MIR), thus restricting the therapeutic effect of coronary recanalization.

[0003] Therefore, finding new drugs for treating cardiovascular-related diseases such as anti-myocardial injury is an urgent need in current clinical practice.

[0004] 2,3-Dihydrobenzofuran is a class of important benzheterocyclic compounds, which exist in many drugs and natural products with different biological or medicinal activities. In recent years, with the continuous improvement of the synthesis methods of five-membered benzheterocyclic compounds, more and more 2,3-dihydrobenzofuran derivatives have been developed, and their medicinal value has also received extensive attention. Through literature retrieval and practical exploration, the inventors found that some 2,3-dihydrobenzofuran derivatives have good functions for treating cardiovascular and cerebrovascular diseases.

[0005] Based on this, the present invention aims to study the use of 2,3-dihydrobenzofuran derivatives in the treatment of cardiovascular and cerebrovascular diseases, providing new ideas for the treatment of cardiovascular and cerebrovascular diseases. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the first object of the present invention is to obtain a plurality of 2,3-dihydrobenzofuran derivatives and study the application of 2,3-dihydrobenzofuran derivatives in the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases.

[0007] Another object of the present invention is to provide a drug for treating cardiovascular and cerebrovascular diseases.

[0008] In order to achieve the above technical objects, the present invention adopts the following technical solutions:

[0009] The present invention first provides the application of 2,3-dihydrobenzofuran derivatives alone or 2,3-dihydrobenzofuran derivatives combined with luteolin (LUT) or propranolol (Pro) in the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases.

[0010] Specifically, the cardiovascular and cerebrovascular diseases include myocardial injury or myocardial infarction.

[0011] Specifically, the myocardial injury includes H9c2 rat myocardial cells being damaged by H 2 O 2 Inducing cardiomyocyte damage, etc.

[0012] Specifically, when the 2,3-dihydrobenzofuran derivative and luteolin or propranolol are used in combination, the 2,3-dihydrobenzofuran derivative and luteolin or propranolol can be used in a mixed manner or separately.

[0013] Specifically, when the 2,3-dihydrobenzofuran derivative and luteolin or propranolol are used in combination, the 2,3-dihydrobenzofuran derivative and luteolin or propranolol are added once or continuously in multiple times.

[0014] Specifically, when 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are added and used at one time, the molar ratio of 2,3-dihydrobenzofuran derivatives and luteolin or propranolol is (0.25-1):1, and the treatment time is 12-72 hours (preferably 12-24 hours); when 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are added and used continuously in multiple times, the molar ratio of 2,3-dihydrobenzofuran derivatives and luteolin or propranolol added each time is (0.25-1):1, and they are added continuously for 7-14 times in total.

[0015] Preferably, when 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are added and used at one time, the molar ratio of 2,3-dihydrobenzofuran derivatives and luteolin or propranolol is 1:1, and the treatment time is 12-24 hours (preferably 24 hours); when 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are added and used continuously in multiple times, the molar ratio of 2,3-dihydrobenzofuran derivatives and luteolin or propranolol added each time is 1:1, and a total of 7 consecutive additions are made.

[0016] Specifically, when used alone, 2,3-dihydrobenzofuran derivatives at a concentration of 2.5-10 μM and a treatment time of 24-72 hours can inhibit H 2 O 2 It has a protective effect on myocardial cell damage caused by H 2 O 2 It has a therapeutic effect on myocardial infarction caused by it.

[0017] Preferably, when used alone, the 2,3-dihydrobenzofuran derivatives at a concentration of 5 μM or 10 μM and a treatment time of 24 h can inhibit H 2 O2 has a protective effect on cardiomyocyte injury caused, or has a therapeutic effect on myocardial infarction caused by H 2 O 2 has a therapeutic effect on myocardial infarction caused by H

[0018] Specifically, when used in combination, the 2,3-dihydrobenzofuran derivative at a concentration of 2.5 - 10 μM and a treatment time of 24 - 72 h, and luteolin or propranolol at a concentration of 1 - 10 μM and a treatment time of 24 - 72 h can have a protective effect on cardiomyocyte injury caused by H 2 O 2 has a protective effect on cardiomyocyte injury caused, or has a therapeutic effect on myocardial infarction caused by H 2 O 2 has a therapeutic effect on myocardial infarction caused by H

[0019] Preferably, when used in combination, the 2,3-dihydrobenzofuran derivative at a concentration of 2.5 μM, 5 μM, or 10 μM and a treatment time of 24 h, 48 h, or 72 h; and luteolin or propranolol at a concentration of 1 μM, 5 μM, or 10 μM and a treatment time of 24 h, 48 h, or 72 h can have a protective effect on cardiomyocyte injury caused by H 2 O 2 has a protective effect on cardiomyocyte injury caused, or has a therapeutic effect on myocardial infarction caused by H 2 O 2 has a therapeutic effect on myocardial infarction caused by H

[0020] More preferably, when used in combination, the 2,3-dihydrobenzofuran derivative at a concentration of 10 μM and a treatment time of 24 h, and luteolin or propranolol at a concentration of 10 μM and a treatment time of 24 h can have a protective effect on cardiomyocyte injury caused by H 2 O 2 has a protective effect on cardiomyocyte injury caused, or has a therapeutic effect on myocardial infarction caused by H 2 O 2 has a therapeutic effect on myocardial infarction caused by H

[0021] Furthermore, the present invention also provides the use of the 2,3-dihydrobenzofuran derivative alone or in combination with luteolin or propranolol in the preparation of a therapeutic drug for a human or murine cardiomyocyte injury model.

[0022] Specifically, the cardiomyocyte injury includes cardiomyocyte injury or myocardial infarction caused by H 2 O 2 has a therapeutic effect on myocardial infarction caused by H

[0023] Specifically, when the dose of the 2,3-dihydrobenzofuran derivative is 2 - 10 mg / kg / day and the treatment time is 2 - 14 days, it can have a protective effect on cardiomyocyte injury caused by H 2 O 2 has a protective effect on cardiomyocyte injury caused, or has a therapeutic effect on myocardial infarction caused by H2 O 2 has a therapeutic effect on myocardial infarction caused.

[0024] Preferably, when the dose of the 2,3-dihydrobenzofuran derivative is 2.5 mg / kg / day, 5 mg / kg / day, or 10 mg / kg / day and the treatment time is 7 days, it can have a protective effect on myocardial cell damage caused by H 2 O 2 or has a therapeutic effect on myocardial infarction caused by H 2 O 2 caused.

[0025] Specifically, the mouse source is C57BL / 6 mice.

[0026] Furthermore, the present invention also provides a preparation method of the 2,3-dihydrobenzofuran derivative, specifically: reacting o-hydroxyphenyl p-quinone with sulfonium ylide containing trifluoromethyl imino and toluene at 60-90 °C, and then obtaining it through steps such as detection by thin layer chromatography, concentration of the reaction mixture, and purification.

[0027] Specifically, the structure of the 2,3-dihydrobenzofuran derivative is shown in Formula I:

[0028]

[0029] wherein, R 1 is selected from any one of trifluoromethyl, pentafluoroethyl, heptafluoropropyl, and heptafluoroisopropyl;

[0030] R 2 is selected from any one of phenyl, 4-methylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-nitrophenyl, 3-methylphenyl, 3-bromophenyl, 2-fluorophenyl, 2-methylphenyl, 2-methoxyphenyl, 2-naphthyl, and benzyl;

[0031] R 3 is selected from any one of 4-methyl, 4-methoxy, 4-fluoro, 4-chloro, 4-bromo, 5-methyl, 5-methoxy, 5-fluoro, 5-chloro, 5-bromo, 6-methyl, 6-methoxy, 6-fluoro, 6-chloro, 6-bromo, 7-methyl, 7-methoxy, and 7-bromo.

[0032] Specifically, t Bu is tert-butyl.

[0033] Specifically, the 2,3-dihydrobenzofuran derivative is preferably a compound with the following structure:

[0034]

[0035]

[0036] Furthermore, the present invention also provides a drug for preventing and treating cardiovascular and cerebrovascular diseases, which comprises an active ingredient and a pharmaceutically acceptable excipient. The active ingredient comprises a 2,3-dihydrobenzofuran derivative or a pharmaceutically acceptable salt thereof, or a mixture of a 2,3-dihydrobenzofuran derivative or a pharmaceutically acceptable salt thereof with luteolin or propranolol.

[0037] Specifically, the excipient can be appropriately selected according to the dosage form and actual situation. For example, common excipients include starch, low-substituted hydroxypropyl cellulose, microcrystalline silica, magnesium stearate, starch paste, sucrose, dextrin, sodium carboxymethyl starch, talc, polysorbate, polyethylene glycol, soybean phospholipid for injection, and glycerol for injection. When preparing various dosage forms of the drug using the active ingredient provided by the present invention, it can be prepared according to the conventional production methods in the pharmaceutical field.

[0038] Specifically, the drug for preventing and treating cardiovascular and cerebrovascular diseases can be any dosage form in pharmacy, including tablets, capsules, soft capsules, gels, oral preparations, suspensions, granules, patches, ointments, pills, powders, injections, infusions, freeze-dried injections, intravenous emulsions, liposome injections, suppositories, sustained-release preparations or controlled-release preparations.

[0039] Furthermore, the dosage form of the drug for preventing and treating cardiovascular and cerebrovascular diseases is preferably an oral dosage form, an injection dosage form or an external administration preparation.

[0040] Furthermore, the present invention also provides a pharmaceutical composition containing a 2,3-dihydrobenzofuran derivative, which comprises a 2,3-dihydrobenzofuran derivative or a salt thereof, and luteolin or propranolol.

[0041] Specifically, in the pharmaceutical composition containing a 2,3-dihydrobenzofuran derivative, the molar ratio of the 2,3-dihydrobenzofuran derivative to luteolin or propranolol is (0.25-1):1.

[0042] Preferably, in the pharmaceutical composition containing a 2,3-dihydrobenzofuran derivative, the molar ratio of the 2,3-dihydrobenzofuran derivative to luteolin or propranolol is 1:1.

[0043] More preferably, in the pharmaceutical composition containing a 2,3-dihydrobenzofuran derivative, when the concentration of the 2,3-dihydrobenzofuran derivative is 5 μM or 10 μM, it can have a protective effect on myocardial cell injury caused by H 2 O 2 or have a therapeutic effect on myocardial infarction caused by H 2 O 2 ​

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] The 2,3-dihydrobenzofuran derivative of the present invention has a protective effect on H 2 O 2 -induced injury of H9c2 rat cardiomyocytes, can improve the cell survival rate, and reduce the release rate of LDH (lactate dehydrogenase); meanwhile, it can significantly reduce the myocardial infarction area of mice after myocardial infarction, has an obvious myocardial protection effect, can be developed into a drug for preventing and treating cardiovascular and cerebrovascular diseases, and has good application prospects.

[0046] In the present invention, when the 2,3-dihydrobenzofuran derivative and luteolin (LUT) are used in combination, they have a synergistic effect on the treatment of cardiovascular and cerebrovascular diseases. Among them, the synergistic effect of the 2,3-dihydrobenzofuran derivative and luteolin can improve the protective effect on the heart. After the two are used in combination, compared with single drug use, the injury to cells caused by H 2 O 2 is significantly reduced, the cell survival rate is significantly increased, and the LDH release rate is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a test result diagram of the cell survival rate and LDH release rate of compound 7 at different concentrations in Example 2, wherein Figure 1 A is the cell survival rate diagram, Figure 1 B is the test result diagram of the LDH release rate;

[0048] Figure 2 It is a morphological diagram of the protective effect of compound 7 on H9c2 rat cardiomyocytes at different concentrations in Example 3;

[0049] Figure 3 It is a morphological diagram of compound 7 enhancing the protective effect of LUT on H9c2 rat cardiomyocytes in Example 4;

[0050] Figure 4 It is a test result diagram of the cell survival rate and LDH release rate of compound 7 when enhancing LUT in Example 4;

[0051] Figure 5 It is a test result diagram of the echocardiogram of the cardiac function of compound 7 at different doses on myocardial infarction mice in Example 5;

[0052] Figure 6 It is a test result diagram of the myocardial infarction area of compound 7 at different doses on myocardial infarction mice by TTC staining in Example 5;

[0053] Figure 7It is a test result diagram of pathological detection of myocardial tissue by HE staining of mice with myocardial infarction under different doses of compound 7 in Example 5;

[0054] Figure 8 It is a cell survival rate diagram under the action of compounds 1-15 in Example 2. Detailed implementation manners

[0055] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to skilled personnel in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention.

[0056] Unless otherwise specified, the instrument and equipment used in the following embodiments are all commercially available conventional instrument and equipment; unless otherwise specified, the reagents, raw materials, etc. used in the following embodiments are all conventional commercially available products and can be obtained from commercial channels.

[0057] The drug manufacturers and specifications used in the present invention are: luteolin (LUT) (Meilunbio, #MB2712-1) and propranolol (Pro) (Aladdin, #318-98-9).

[0058] Example 1: Preparation of Compound 7

[0059] Example 1 provides a preparation method of compound 7, and the specific steps are as follows:

[0060]

[0061] Into a reaction flask, o-hydroxyphenyl-p-benzoquinone (0.20 mmol, 62 mg, 1.0 equiv.), sulfoxonium ylide containing trifluoromethylimino (0.24 mmol, 70 mg, 1.2 equiv.) and toluene (1.0 mL) were successively added. The reaction was stirred at 80 °C until the o-hydroxyphenyl-p-benzoquinone in the reaction system completely disappeared as detected by thin layer chromatography, then the reaction was stopped. The reaction mixture was concentrated, and the resulting residue was purified by flash column chromatography (ethyl acetate / petroleum ether = 1:60) to obtain the purified product (Compound 7) (80 mg, Yield = 71%), and the systematic name is: 2,6-di-tert-butyl-4-(2-(2,2,2-trifluoro-1-((4-(trifluoromethyl)phenyl)imino)ethyl)-2,3-dihydrobenzofuran-3-yl)phenol.

[0062] In the preparation method of Compound 7 in this example, for the method steps not described in detail, refer to the literature (Li C, Zhou M, Chen Z. Base-mediated formal [4+1] annulation of ortho-hydroxyphenylsubstituted para-quinone methides and CF3-imidoyl sulfoxonium ylides to access trifluoroacetimidoyl-substituted 2,3-dihydrobenzofurans [J]. Journal of Fluorine Chemistry, 2024, 277. DOI: 10.1016 / j.jfluchem.2024.110308; Yi-Hui Li; Chuan-Chuan Wang; Meng-Ru Ren; Qing-Long Wang; Ya-Jing Chen. Additive-Free and Diastereoselective Synthesis for Trans-Disubstituted-2,3-Dihydro-Benzofurans via [4+1] Annulation between p-QMs and TFISYs [J]. European Journal of Organic Chemistry, 2024,:e202400946).

[0063] For the experimental methods of Examples 2-5 below, for other steps, precautions, etc. that are not described in detail, please refer to the literature (Zhao G, Ji Z, Duan Y, et al. A novel anti-inflammatory strategy for myocardial ischemia–reperfusion in rats with cinnamamide derivative compound 7[J]. International Immunopharmacology, 2024, 136. DOI: 10.1016 / j.intimp.2024.112370; Yang F F, Liu J J, Xu X L, et al. Discovery of Novel Imidazo[1,2-a]pyridine-Based HDAC6 Inhibitors as an Anticarcinogen with a Cardioprotective Effect[J].[2025-02-17].).

[0064] Example 2: Protective effects of 2,3-dihydrobenzofuran derivatives (compounds 1-15), luteolin (LUT), and propranolol (Pro) on H 2 O 2 induced H9c2 rat cardiomyocyte injury

[0065] The specific experimental methods are as follows:

[0066] (1) Plating: Digest H9c2 cells (purchased from Shanghai Cell Bank) with trypsin, then resuspend them into a single-cell suspension, plate them in 96-well plates, with 5×10 3 cells per well, and place them in an incubator at 37°C for overnight culture;

[0067] (2) Drug preparation: Compounds 7, luteolin (LUT), and propranolol (Pro) are respectively prepared into 10 mM stock solutions with DMSO. When in use, compound 7 is respectively diluted to 2.5 μM, 5 μM, and 10 μM, and LUT and Pro are diluted to 10 μM. 100 μL is added to each well, and there are 6 replicates for each gradient;

[0068] (3) Drug addition: Aspirate the cell supernatant in the 96-well plates, add 100 μL of compound 7 at different concentrations, as well as LUT and Pro to each well, and place them in an incubator at 37°C for 24 h;

[0069] (4) Model establishment: Aspirate the cell supernatant in the 96-well plates, add 100 μL of H 2 O2 (300 μM) complete DMEM medium (DMEM medium: fetal bovine serum = 9:1), act for 1.5 h;

[0070] (5) Cell viability detection: Dilute the stock solution of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg / mL) with serum-free cell medium (Solarbio, #11965) at a ratio of 1:100. Aspirate the medium, add the MTT detection solution, 100 μL per well, and incubate at 37 °C for 4 h; Aspirate the supernatant, add 150 μL of DMSO to each well, shake and dissolve for 10 min. After complete dissolution of the crystals, measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader; The results are as Figure 1 shown in

[0071] (6) Repeat steps (1) to (4). When repeating step (4), set up an LDH (lactate dehydrogenase) release control group, add the LDH release reagent (Beyotime, #C0016) 1 h before the end of the experiment, then centrifuge the 96-well plate at 2000 rpm for 5 min, aspirate 120 μL of the supernatant from each well, and add the prepared LDH detection working solution (Beyotime, #C0016) (INT(10×): INT diluent: enzyme solution: lactate solution = 1:9:10:10) to the corresponding new 96-well plates that have been pre-labeled, 60 μL per well, incubate in the dark for 30 min, and measure the absorbance at 490 nm and 450 nm with an ELISA reader; The results are as Figure 1 shown in

[0072] Experimental results:

[0073] According to Figure 1 it can be seen that as the concentration of compound 7 increases, the cell survival rate increases significantly, and the LDH release rate decreases significantly, indicating that compound 7 has an obvious protective effect on H9c2 cells and has an obvious concentration dependence. In Figure 1 Figure 2 A, compared with the normal control group (CON group), the cell survival rate of the 2 H Figure 1 O 2 group decreased to 34.77%, and in the compound pre-protected groups, as the concentration increased, the cell survival rate increased to 71.26%, 96.80%, and 98.43% in sequence, which was significantly higher than that of the LUT (93.81%) and Pro (39.64%) groups. 2 In

[0074] Example 1 also tested the protective effect of multiple 2,3-dihydrobenzofuran derivatives (Compounds 1-15) on cardiomyocyte injury. The preparation of Compounds 1-15 refers to the method in the reference (Li C, Zhou M, Chen Z. Base-mediated formal [4+1] annulation of ortho-hydroxyphenyl substituted para-quinone methides and CF3-imidoyl sulfoxonium ylides to access trifluoroacetimidoyl-substituted 2,3-dihydrobenzofurans [J]. Journal of Fluorine Chemistry, 2024, 277. DOI: 10.1016 / j.jfluchem.2024.110308; Yi-Hui Li; Chuan-Chuan Wang; Meng-Ru Ren; Qing-Long Wang; Ya-Jing Chen. Additive-Free and Diastereoselective Synthesis for Trans-Disubstituted-2,3-Dihydro-Benzofurans via [4+1] Annulation between p-QMs and TFISYs [J]. European Journal of Organic Chemistry, 2024,: e202400946).

[0075] The structural formulas of the said Compounds 1-15 are as follows:

[0076]

[0077] The specific test method is the same as above. In the test method, in step (2), Compounds 1-15 and LUT are respectively diluted to 10 μM to test the protective effect of the compounds on H 2 O 2 induced H9c2 rat cardiomyocyte injury. The test results are as Figure 8 shown. It can be seen from Figure 8 that after adding multiple 2,3-dihydrobenzofuran derivatives (Compounds 1-15), the cell survival rates all increased significantly, indicating that multiple compounds have obvious protective effects on H9c2 cells.

[0078] Example 3: Protective effect of 2,3-dihydrobenzofuran derivative (Compound 7), luteolin (LUT), and propranolol (Pro) on H 2 O 2 induced myocardial cell injury in H9c2 rats - morphological diagram

[0079] The specific experimental method is as follows:

[0080] (1) Seeding: Digest H9c2 cells (purchased from Shanghai Cell Bank) with trypsin, then resuspend them into a single-cell suspension, seed them in six-well plates, with 3×10 4 cells per well, and place them in an incubator at 37°C overnight;

[0081] (2) Preparing drugs: Compound 7, luteolin (LUT), and propranolol (Pro) are respectively prepared into 10 mM stock solutions with DMSO. When in use, Compound 7 is diluted to 2.5 μM, 5 μM, and 10 μM respectively, and LUT and Pro are diluted to 10 μM, and 2 mL is added to each well;

[0082] (3) Adding drugs: Aspirate the supernatant of the cells in the six-well plates, add 2 mL of different concentrations of Compound 7, as well as LUT and propranolol to each well, and place them in an incubator at 37°C for 24 h;

[0083] (4) Modeling: Aspirate the supernatant of the cells in the 96-well plates, add 100 μL of DMEM medium (Solarbio, #11965) containing H 2 O 2 (300 μM) and act for 1.5 h;

[0084] (5) Photographing: Take pictures under a fluorescence inverted microscope, bar = 500 nm, 200×.

[0085] Experimental results:

[0086] The results are as Figure 2 shown. It can be seen from Figure 2 that the number of cells in the CON group is relatively large, the morphology is spindle-shaped, the size is uniform and plump; compared with the CON group, the number of cells in the H 2 O 2 group is significantly reduced, the morphology shows obvious shrinkage, the size is uneven, and it shows an aggregated distribution, which indicates that the drug administration group has an obvious protective effect and significantly reduces the damage of H 2 O 2 to the cells, and the protective effect is proportional to the concentration of Compound 7.

[0087] Example 4: 2,3-Dihydrobenzofuran derivative (Compound 7) enhances the protective effect of luteolin on myocardial cells

[0088] The specific experimental method is as follows:

[0089] 1. Cell morphology imaging

[0090] (1) Seeding: Digest H9c2 cells (purchased from Shanghai Cell Bank) with trypsin, then resuspend them into a single-cell suspension and seed them in six-well plates, with 3×10 4 cells per well, and incubate them overnight in a 37°C incubator;

[0091] (2) Preparing drugs: Dissolve compound 7 and LUT in DMSO to prepare 10 mM stock solutions respectively. When in use, dilute compound 7 to 5 μM and LUT to 5 μM respectively. Set up 5 groups, namely the CON group, the H 2 O 2 group, the compound 7 group ( Figure 3 in H 2 O 2 +Compound7), the luteolin group ( Figure 3 in H 2 O 2 +LUT), and the compound 7 + luteolin group ( Figure 3 in H 2 O 2 +Compound7+LUT), and add 2 mL to each well;

[0092] (3) Adding drugs: Aspirate the cell supernatant in the six-well plates, and add 2 mL of solutions with different concentrations (including compound 7 solution or luteolin solution) to each well, and incubate them in a 37°C incubator for 24 h;

[0093] (4) Modeling: Aspirate the cell supernatant in the 96-well plates, and add 100 μL of DMEM medium (Solarbio, #11965) containing H 2 O 2 (300 μM) and let it act for 1.5 h;

[0094] (5) Imaging: Take pictures under a fluorescence inverted microscope, bar = 250 nm, 200×.

[0095] Experimental results:

[0096] The test results are as Figure 3 shown. It can be seen from Figure 3 that the number of cells in the CON group is relatively large, and the cell morphology is spindle-shaped, with uniform size and plumpness; compared with the CON group, the number of cells in the H 2 O 2 group is significantly reduced, the cell morphology shows obvious shrinkage, uneven size, and aggregated distribution.

[0097] 2. Determination of cell viability and LDH leakage rate

[0098] (1) Plating the cells: Digest H9c2 cells (purchased from Shanghai Cell Bank) with trypsin, then resuspend them into a single-cell suspension, plate them in 96-well plates at 5×10 3 cells per well, and culture them overnight in an incubator at 37°C;

[0099] (2) Preparing the drugs: Dissolve compound 7 and luteolin in DMSO to prepare 10 mM stock solutions respectively. Dilute compound 7 to 5 μM and luteolin to 5 μM respectively, add 100 μL per well, and set up 6 replicates for each concentration gradient;

[0100] (3) Adding the drugs: Aspirate the supernatant from the 96-well plates, add 100 μL of compound 7 and luteolin at different concentrations to each well, and culture them in an incubator at 37°C for 24 h;

[0101] (4) Modeling: Aspirate the supernatant from the 96-well plates, add 100 μL of DMEM medium (Solarbio, #11965) containing H 2 O 2 (300 μM) to each well and incubate for 1.5 h;

[0102] (5) Cell viability detection: Dilute the MTT stock solution (5 mg / mL) with serum-free cell medium (Solarbio, #11965) at a ratio of 1:100. Aspirate the medium, add the MTT detection solution, 100 μL per well, and incubate at 37°C for 4 h; Aspirate the supernatant, add 150 μL of DMSO to each well, shake and dissolve for 10 min. After complete dissolution of the crystals, measure the absorbance at 490 nm and 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader; The results are shown in the left bar chart of Figure 4 .

[0103] (6) Repeat steps (1) - (4). When repeating step (4), set up an LDH release control group. Add the LDH release reagent (Beyotime, #C0016) 1 h before the end of the experiment, then centrifuge the 96-well plates at 2000 rpm for 5 min. Aspirate 120 μL of the supernatant from each well and add it to the corresponding pre-labeled new 96-well plates. Add the prepared LDH detection working solution (Beyotime, #C0016) (INT(10×): INT diluent: enzyme solution: lactic acid solution = 1:9:10:10), 60 μL per well, incubate in the dark for 30 min, and measure the absorbance at 450 nm using an ELISA reader; The results are shown in the right bar chart of Figure 4 .

[0104] Experimental results:

[0105] As can be seen from Figure 4 , the luteolin administration group had a protective effect, significantly reducing H 2 O 2The damage to cells, and Compound 7 can synergistically enhance the effect with luteolin and play a protective role in the heart. After the combination of luteolin and Compound 7, compared with the sole use of luteolin, the cell survival rate is significantly increased and the LDH release rate is significantly decreased.

[0106] Example 5: Therapeutic effect of Compound 7 on the infarcted heart

[0107] The specific experimental methods are as follows:

[0108] 1. Analysis of echocardiogram

[0109] Establishment of an in vivo myocardial infarction (MI) injury model:

[0110] C57BL / 6 mice (SPF grade) were anesthetized by inhaling 2% isoflurane using an anesthesia device. Myocardial ischemia-reperfusion injury was induced by temporarily "popping out" the heart and passing through a small hole in the fourth intercostal space, while the left coronary artery (3 mm from the origin of the LCA) was sutured with a 6-0 silk suture. When electrocardiogram monitoring showed ST segment elevation, it indicated successful modeling of myocardial infarction (MI). One week after the surgery, drugs were administered by intraperitoneal injection. The next day, the mice were randomly divided into 5 groups, namely the sham operation group (Sham group), the myocardial infarction group (MI group), the MI + Compound 7 (2.5 mg / kg) group, the MI + Compound 7 (5 mg / kg) group, and the MI + Compound 7 (10 mg / kg) group.

[0111] Using a Vevo 2100 echocardiograph (including an in vivo imaging system (VisualSonics Vevo, Toronto, Canada) available for echocardiographic imaging), under anesthesia, the chest hair of the mice was shaved, and two-dimensional long-axis images were collected for end-diastolic and end-systolic volume measurements; the echocardiogram system automatically calculated and recorded the left ventricular ejection fraction (EF, %) and the fractional shortening (FS, %), and the results are as Figure 5 shown.

[0112] Experimental results:

[0113] As Figure 5 can be seen, Compound 7 has a protective effect on acute myocardial injury caused by in vivo myocardial infarction. Through the summary of echocardiogram data, compared with the Sham group, the EF and FS of the MI group decreased to 50.38% and 26.90% respectively. After administration of Compound 7, with the increase of the dose, EF and FS can increase respectively. As the administration dose of EF increases, it can increase to 54.32%, 76.14%, 81.19%. As the administration dose of EF increases, it can increase to 28.98%, 44.87%, 48.88%, indicating that Compound 7 has an obvious myocardial protective effect and improves the systolic and diastolic functions of the myocardium.

[0114] 2. Determination of myocardial infarction area

[0115] The determination of the myocardial infarction area mainly measures the infarct area. During dissection, the heart was separated and then quickly frozen at -20 °C for 30 min before sectioning. All sections (including the Sham group, MI group, MI + Compound 7 (2.5 mg / kg) group, MI + Compound 7 (5 mg / kg) group, MI + Compound 7 (10 mg / kg) group, MI + LUT (10 mg / kg) group, Sham + Compound 7 (10 mg / kg) group) were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) buffer (pH 7.4) in the dark for 10 min to determine the unstained necrotic area. These areas were segmented and numerically evaluated using Image-Pro Plus 6.0. The myocardial infarction area was white or pale, expressed as the ratio of infarcted myocardium to left ventricular area × 100%; the results are as Figure 6 shown.

[0116] Experimental results:

[0117] As Figure 6 can be seen, through the TTC staining experiment, the infarct area ratio in the MI group increased significantly to 60.71%. As the administration dose increased, the infarct area gradually decreased to 55.43%, 40.08%, 27.94%, indicating that Compound 7 can significantly reduce the myocardial infarction area in mice after myocardial infarction and has an obvious myocardial protective effect.

[0118] 3. Histological changes in the myocardium

[0119] (1) Paraffin embedding

[0120] a. Dehydration: The heart tissue was successively soaked in ethanol with increasing concentrations, generally (volume fraction) 75%, 85%, 95%, and then replaced with absolute ethanol, with each soaking lasting 2 h;

[0121] b. Clearing: The clearing agent used was xylene. The heart tissue was first soaked in a mixture of equal volumes of absolute ethanol and xylene for 30 min, and then in pure xylene for 30 min;

[0122] c. Tissue infiltration with wax (50 - 60 °C) and embedding: After 6 h of wax infiltration, it was placed in an embedding machine for embedding and cooled on a -20 °C freezing table.

[0123] (2) Paraffin sectioning

[0124] a. Sectioning: A microtome was used for sectioning, and the thickness was adjusted to approximately 4 μm. The sections were floated in a 40 °C water bath and then dried in a 60 °C oven to remove moisture;

[0125] b. Dewaxing: Immerse the sections in xylene twice, 10 min each time → twice in absolute ethanol, 5 min each time → 95% ethanol for 5 min → 85% ethanol for 5 min → 75% ethanol for 5 min → wash with distilled water for 5 min.

[0126] (3) Hematoxylin-eosin staining (HE staining)

[0127] a. Stain the cell nucleus with hematoxylin and the cytoplasm with eosin: Incubate the sections with hematoxylin for 3 - 8 min, wash, differentiate with 1% hydrochloric acid ethanol solution for several seconds, wash again, wash the sections with 0.6% ammonia water until blue returns, rinse again, and place the sections in eosin staining solution for 3 min;

[0128] b. Mounting and photographing: Immerse the sections in 95% (volume fraction), 85%, and 75% ethanol for 5 min in sequence, then transfer them to xylene and soak for 5 min each until transparent, then air-dry the sections, seal with neutral gum, and finally collect and photograph images under the microscope.

[0129] Experimental results:

[0130] Test result solution Figure 7 As shown, from Figure 7 it can be seen that by evaluating the pathological changes of mouse tissues through HE staining, the myofibril stripes of the rats in the Sham group were clear, continuous, with normal structure, no obvious necrosis or edema, and no inflammatory cell infiltration; in the MI model mice, there were vacuoles and edema in the myocardial cell stroma, disordered and broken fibrotic tissue structure, loose arrangement, a large number of inflammatory cell infiltrations, and nuclear aggregation; meanwhile, pre-protection with compound 7 significantly improved the myocardial tissue abnormalities induced by MI in a dose-dependent manner.

[0131] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Other embodiments can also be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 2,3-dihydrobenzofuran derivatives alone or in combination with luteolin or propranolol in the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases.

2. The use according to claim 1, characterized in that: When used alone, 2,3-dihydrobenzofuran derivatives can protect myocardial cell damage caused by H2O2 or have a therapeutic effect on myocardial infarction caused by H2O2 at a concentration of 2.5-10 μM and a treatment time of 24-72 hours.

3. The use according to claim 1, characterized in that: When 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are used in combination, when the concentration of 2,3-dihydrobenzofuran derivatives is 2.5-10μM and the treatment time is 24-72h, and when the concentration of luteolin or propranolol is 1-10μM and the treatment time is 24-72h, they can have a protective effect on myocardial cell damage caused by H2O2, or have a therapeutic effect on myocardial infarction caused by H2O2.

4. The use according to claim 1, characterized in that: When 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are used in combination, 2,3-dihydrobenzofuran derivatives and luteolin or propranolol are added at one time or added continuously for multiple times.

5. The use according to claim 4, characterized in that: When the 2,3-dihydrobenzofuran derivative and luteolin or propranolol are added at one time, the molar ratio of the 2,3-dihydrobenzofuran derivative and luteolin or propranolol is (0.25-1):1, and the treatment time is 12-72 hours.

6. The use according to claim 1, characterized in that: The use of 2,3-dihydrobenzofuran derivatives alone or in combination with luteolin or propranolol in the preparation of therapeutic drugs for human or mouse myocardial cell injury models.

7. The use according to claim 6, characterized in that: When the dosage of 2,3-dihydrobenzofuran derivatives is 2-10 mg / kg / day and the treatment time is 2-14 days, it can have a protective effect on myocardial cell damage caused by H2O2, or have a therapeutic effect on myocardial infarction caused by H2O2.

8. The use according to claim 1, characterized in that: The 2,3-dihydrobenzofuran derivative is a compound with the following structure:

9. An anti-cardiovascular and cerebrovascular disease drug, characterized in that: The invention comprises an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient comprises a 2,3-dihydrobenzofuran derivative or a pharmaceutically acceptable salt thereof, or a mixture of a 2,3-dihydrobenzofuran derivative or a pharmaceutically acceptable salt thereof and luteolin or propranolol; The auxiliary material is any one of starch, low-substituted hydroxypropyl cellulose, micro-powder silica gel, magnesium stearate, starch slurry, sucrose, dextrin, sodium carboxymethyl starch, talc, polysorbate, polyethylene glycol, soybean lecithin for injection and glycerol for injection.

10. The anti-cardiovascular and cerebrovascular disease drug according to claim 9, characterized in that: The anti-cardiovascular and cerebrovascular disease drug is a pharmaceutical composition containing 2,3-dihydrobenzofuran derivatives. In the pharmaceutical composition containing 2,3-dihydrobenzofuran derivatives, the molar ratio of 2,3-dihydrobenzofuran derivatives to luteolin or propranolol is (0.25-1):1.

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