Compound for treating myocardial ischemia-reperfusion injury, pharmaceutical composition and application thereof

Abstract

The invention provides a compound for treating myocardial ischemia reperfusion injury, a pharmaceutical composition and application thereof. Specifically disclosed is a small molecule compound or a pharmaceutically acceptable salt thereof. The compound and the pharmaceutical composition provided by the invention have a good myocardial cell protection effect, and can be used for treating myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a compound, a pharmaceutical composition and application thereof for treating myocardial ischemia-reperfusion injury. Background Art

[0002] Myocardial ischemia / reperfusion (I / R) refers to the process of restoring blood supply to the ischemic myocardium in heart diseases such as acute myocardial infarction (AMI). In this process, the restoration of blood supply after tissue ischemia may cause additional heart damage and complications. This phenomenon is called myocardial ischemia reperfusion injury (MIRI).

[0003] In order to reduce myocardial ischemia-reperfusion injury, the academic community has discovered some treatments and drugs targeting the existing mechanisms of MIRI, such as ischemic preconditioning (IPC) or the use of atorvastatin. However, these methods are often accompanied by surgical risks and may be accompanied by liver and gastrointestinal disorders.

[0004] In the present invention, sennoside A (CAS No: 81-27-6) is a natural dianthrone glycoside extracted from senna leaves, which has multiple pharmacological effects, such as laxative, anti-obesity, hypoglycemic, liver protection, anti-fibrosis, anti-inflammatory, anti-tumor, antibacterial, antifungal, antiviral and anti-neurodegenerative effects, and is a class of compounds with great development value and clinical application prospects.

[0005]

[0006] However, there are no reports on the use of sennoside A in the treatment of myocardial ischemia-reperfusion injury. And no breakthrough has been made in the treatment of myocardial ischemia-reperfusion injury. Therefore, exploring new drugs for the treatment of myocardial ischemia-reperfusion injury has important clinical value. Summary of the invention

[0007] In view of the above technical problems, the present invention provides a new compound, a pharmaceutical composition and its application, which can be used to prevent or treat myocardial ischemia-reperfusion injury.

[0008] The present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof; ,in, are independently single bonds or double bonds; R1 is C 1-3 R2 is halogen; X1 and X2 are independently CH or a heteroatom, and the heteroatom is C, N or O.

[0009] In the present invention, the C1-3 The alkyl group may be methyl, ethyl, n-propyl or isopropyl.

[0010] In the present invention, the halogen may be F or Cl, such as Cl.

[0011] In the present invention, the compound represented by formula I is or

[0012] The present invention provides a pharmaceutical composition, which comprises the compound represented by the above formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0013] In the present invention, the pharmaceutical composition may further comprise sennoside A.

[0014] In the present invention, in the pharmaceutical composition, the molar ratio of the compound represented by formula I or a pharmaceutically acceptable salt thereof to the sennoside A is 1:(0.1-10), for example, 2:3.

[0015] In the present invention, in the pharmaceutical composition, the content of sennoside A can be conventional in the art, for example, 1%-50%, and can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%, wherein the percentage is the mass content of sennoside A.

[0016] The present invention provides a use of the compound represented by the above formula I or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition in the preparation of a drug for treating and / or preventing myocardial ischemia-reperfusion injury.

[0017] The present invention provides an application of sennoside A in preparing a medicine for treating and / or preventing myocardial ischemia-reperfusion injury.

[0018] Explanation of terms: The term "pharmaceutically acceptable salt" refers to salts that are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or with organic acids (acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid) or by using other methods known in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Alkyl)4 - Salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, as appropriate.

[0019] The term "excipient" includes inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients, such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavorings and fragrances may also be present in the composition.

[0020] The term "pharmaceutically acceptable" means suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and lower animals without undue toxicity, irritation or allergic response.

[0021] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0022] The reagents and raw materials used in the present invention are commercially available.

[0023] In the present invention, the composition, compound or pharmaceutically acceptable salt thereof can better protect the activity of myocardial cells, can effectively act on myocardial ischemia-reperfusion injury, and has good medicinal prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is an immunofluorescence image of H9C2 cardiomyocytes after staining.

[0025] Figure 2 This is the skeleton of cardiac troponin distribution after staining in H9C2 cardiomyocytes.

[0026] Figure 3 These are images of TTC and Evans staining of the heart after ischemia-reperfusion.

[0027] Figure 4 H&E staining of cardiac tissue after ischemia-reperfusion.

[0028] Figure 5 Masson staining of cardiac tissue after ischemia-reperfusion.

[0029] Figure 6 This is a picture of Sirius red staining of cardiac tissue after ischemia-reperfusion. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0031] Example 1: Compound 1:

[0032] Compound I-1 (0.1 mol, preparation method can refer to CN101143845A) and 900 ml of anhydrous acetonitrile were stirred and dissolved, K2CO3 (0.4 mol) was added, and compound I-2 (0.15 mol, preparation method can refer to CN113248518A) dissolved in 100 ml of anhydrous acetonitrile solution was slowly added dropwise at 40°C. After the addition was completed, the reaction was allowed to proceed for 3 hours, filtered, and purified by column chromatography (DCM / methanol = 20:1) to obtain compound I-3 (750 mmol) with a yield of 75%.

[0033] LC-MS (ESI): m / z = 574.2 [M+H] + .

[0034]

[0035] Take guanidine (1 mol, CAS No: 113-00-8) and add it to 35 ml of isopropanol. Add compound I-3 (0.05 mol) and isopropanol (60 ml) suspension dropwise at room temperature and stir to react for 4 hours. Pour the reaction solution into 300 ml of a mixed solution of ethyl acetate and water (v / v=1.5 / 1) under ice bath while stirring. Separate the liquids, extract the water layer with ethyl acetate three times, 170 ml each time, combine the ester layers, wash with saturated sodium chloride aqueous solution, dry over anhydrous sodium sulfate, filter, and vacuum dry to obtain compound 1 (white solid, 25 mmol), with a yield of 50%.

[0036] LC-MS (ESI): m / z = 587.2 [M+H] + .

[0037] 1 H NMR (400MHz, CD3OD): δ 2.21 (3H, s), 2.31-2.45 (4H, m), 3.73-3.86(4H, m), 4.22 (2H, s), 7.01-7.22 (4H, m), 7.81 (1H, dd, J = 8.3, 1.9 Hz), 7.97 (1H, dt, J = 8.3, 0.5 Hz), 8.10 (1H, s), 8.62 (1H, dd, J = 1.9, 0.4 Hz), 9.10 (1H, d, J = 0.5 Hz).

[0038] Embodiment 2:

[0039] Compound I-1 (0.1 mol, preparation method can refer to CN101143845A) and 900 ml of anhydrous acetonitrile were stirred and dissolved, K2CO3 (0.4 mol) was added, and compound II-2 (0.15 mol, preparation method can refer to CN113248518A) dissolved in 100 ml of anhydrous acetonitrile solution was slowly added dropwise at 40°C. After the addition was completed, the reaction was allowed to proceed for 2 h, filtered, and purified by column chromatography (DCM / methanol = 20:1) to obtain compound II-3 (0.7 mol) with a yield of 70%.

[0040] LC-MS (ESI): m / z = 573.2 [M+H] + .

[0041]

[0042] Take guanidine (1 mol, CAS No: 113-00-8) and add it to 40 ml of isopropanol. Add compound II-3 (0.05 mol) and isopropanol (60 ml) suspension dropwise at room temperature and stir to react for 3 hours. Pour the reaction solution into 300 ml of a mixed solution of ethyl acetate and water (v / v=1.5 / 1) under ice bath while stirring. Separate the liquid, extract the water layer with ethyl acetate three times, 170 ml each time, combine the ester layers, wash with saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, filter, and vacuum dry to obtain compound 2 (white solid, 30 mmol), with a yield of 60%.

[0043] LC-MS (ESI): m / z = 588.2 [M+H] + .

[0044] 1 H NMR(400MHz, CD3OD): δ 2.27 (3H, s), 2.26-2.45(4H, m), 3.33-3.54 (4H,m), 4.02 (2H, s), 7.05-7.22 (4H, m), 7.40 (1H, d, J = 8.8 Hz), 7.69-7.81 (2H,m), 8.13 (1H, dt, J =8.3, 0.5 Hz), 8.62 (1H, dd, J = 1.9, 0.4 Hz), 9.17 (1H,d, J = 0.5 Hz).

[0045] Embodiment 3:

[0046] Referring to the reaction steps of Example 1, compound I-2 was replaced by compound III-2 (compound III-2 was purchased from Hefei Xingtide) to obtain compound III-3 (0.6 mol) with a yield of 60%.

[0047] LC-MS (ESI): m / z = 517.2 [M+H] + .

[0048]

[0049] Referring to Example 1, I-3 was replaced by the above step III-3 to obtain compound 3 (32 mmol) with a yield of 64%.

[0050] LC-MS (ESI): m / z = 530.2 [M+H] + .

[0051] 1 H NMR (400MHz, CD3OD): δ 2.24 (3H, s), 2.51-2.70 (4H, m), 3.4-3.60 (4H,m), 4.10 (2H, s), 6.00 (1H, d, J = 5.2 Hz), 7.01-7.15 (4m), 7.64-7.81 (2H,m), 8.05 (1H, dt, J = 8.3, 0.5 Hz), 8.62 (1H, dd, J = 1.9, 0.4 Hz), 9.10 (1H, d, J = 0.5 Hz).

[0052] Effect test example 1: Effect of sennoside A on hypoxia-reoxygenation H9C2 cells: H9C2 cardiomyocytes (purchased from Saios) in logarithmic phase were taken, and the culture medium was DMEM (containing 1.5g / L NaHCO3) + 10% fetal bovine serum + 1% double antibody. They were seeded in a 96-well culture plate at 6000 / well. After observing that the cells grew well overnight, they were replaced with culture medium containing culture medium, 5μM sennoside A, and 10μM sennoside A (purchased from Sinopharm Reagent Company, the solvent was DMEM culture medium). After 2 hours of anaerobic culture (the control group was not cultured anaerobic), they were cultured under normal conditions for 2 hours, the cells were lysed, the cell proteins were separated, and the staining and detection were performed by western blotting: (1) After 2 hours of hypoxia and 2 hours of reoxygenation, the morphology of the cells was detected by immunofluorescence detection technology, as shown in the attached Figure 1 , Attachment Figure 2 As shown in the figure (Con is the control group, H / R is the culture medium group, SEN (5μM) is the 5μM sennoside A group, SEN (10μM) is the 10μM sennoside A, green is Troponin T (abbreviated as TnT) cardiac troponin, blue is DAPI stain, Blue: DAPI, Green: Troponin T), the results showed that sennoside A improved the cell morphology induced by hypoxia and reoxygenation; (2) Western blotting experiments found that sennoside A improved the decreased expression of P130cas and Paxilin phosphorylated proteins in myocardial cells induced by hypoxia and reoxygenation, indicating that sennoside A can alleviate the damage to cell migration and proliferation caused by hypoxia and reoxygenation, and protect myocardial cells from the damage induced by hypoxia and reoxygenation.

[0053] Effect test example 2: 30 SD rats, half male and half female, were randomly divided into 6 groups, one of which was a blank control group (Con), in which the ischemia-reperfusion model was not established, that is, only puncture was performed without ligation during ligation; the second group was a solvent control group (Vehicle, the solvent was DMSO); the third to sixth groups were sennoside A groups (the dosage was 1.25 mg, 2.5 mg, 5 mg, and 10 mg of sennoside A per kg (rat body weight), respectively.

[0054] Anesthesia was performed by intraperitoneal injection of 20% urethane 5ml / kg. After successful anesthesia, a normal electrocardiogram (ECG) was recorded. Tracheotomy was performed on the rats, and endotracheal intubation and positive pressure artificial respiration were performed. Thoracotomy was performed by cutting the 3rd to 5th ribs on the left side of the rat's sternum. After entering the pleural cavity, the pericardium was opened, and the thread was threaded at the left anterior descending branch of the coronary artery (LAD) between the arterial cone and the left atrial appendage. The ligature was tightened under the LAD so that the silicone tube compressed the LAD to cause blood flow blockage (the ST segment of ECG II lead was elevated by 0.1mV or the T wave was tall, and the myocardial color turned dark red, which was a sign of success). After 30 minutes, the ligature was loosened to restore blood reperfusion to the LAD. Except for the control group injected with 10ml / kg of normal saline and the solvent group intravenously injected with 10ml / kg of blank solvent as a control, the other groups were injected with the corresponding experimental drugs through the femoral vein, and then blood was perfused for 4 hours. Two mice in each group were intravenously injected with 1% TTC dye / Evans, stained, and then counterstained (the experimental results are shown in the attached Figure 3 Con was the control group, Vehicle was the culture medium group, SEN (5 mg / kg) was the 5 mg / kg sennoside A group, and SEN (10 mg / kg) was the 10 mg / kg sennoside A group. The remaining mice were sacrificed and heart tissue sections were sliced ​​and stained with 1% H&E, Masson, and Sirius red staining kits, respectively (see the attached experimental results). Figure 4 , Attachment Figure 5 , Attachment Figure 6 Con was the control group, Vehicle was the culture medium group, SEN (1.25 mg / kg) was the 1.25 mg / kg sennoside A group, and SEN (2.5 mg / kg) was the 2.5 mg / kg sennoside A group). At the same time, conventional physiological instruments were used to detect the following physiological indicators of each experimental animal group before ischemia, 1 mouse after ischemia, and 4 hours after reperfusion: serum CK, serum LDH, serum cTnT, myocardial oxidation (MDA), myocardial antioxidant (SOD), etc.

[0055] The results showed that after half an hour of myocardial ischemia and 4 hours of reperfusion, TTC staining was used to detect cardiac ischemia, confirming the role of sennoside A in improving cardiac injury induced by ischemia-reperfusion. After half an hour of myocardial ischemia and 4 hours of reperfusion, H&E, Masson, and Sirius red staining kits were used to detect myocardial cell damage and fibrosis, confirming the role of sennoside A in improving cardiac injury induced by ischemia-reperfusion.

[0056] Effect Test Example 3: Evaluation of the protective effect of the compound on hypoxia-reoxygenation H9C2 cells: H9C2 cardiomyocytes (purchased from Saios) in logarithmic phase growth were taken, and the culture medium was DMEM (containing 1.5g / L NaHCO3) + 10% fetal bovine serum + 1% double antibody. They were inoculated in a 96-well culture plate at 6000 / well, overnight, and after observing that the cells grew well, they were divided into 64 experimental groups, of which every 4 groups were parallel controls. Each experimental group used grouped drugs containing different liquid concentrations (the grouping is shown in Table 1 below, the low-dose group in the drug group: 1μM, the high-dose group: 5μM, the solvent was DMEM culture medium, and sennoside A was purchased from Sinopharm Reagent Company), and 2,3'-dibromo-4,5,6'-trihydroxybenzophenone (abbreviated as M49, the preparation method can refer to CN 102423308) was added to the positive control group. B) (low-dose group: 1 μM, high-dose group: 10 μM) culture medium, placed in an anaerobic device for 2 hours (blank group was not subjected to anaerobic culture), replaced with completely normal DMEM culture medium, cultured for 6 hours, added 100L of 0.5mg / mL MTT solution, continued to culture for 4 hours, discarded the culture medium, added 150L of DMSO, oscillated and mixed, placed on a microplate reader, and measured the absorbance value (wavelength 490mm). As shown in the table below, the drug of the application can significantly inhibit the decline in the viability of hypoxic and reoxygenated cells.

[0057]

[0058] The experimental conclusion showed that the blank group was 100%, in the high-dose group, the cell activity of the control group was 75%, and the activities of the AF group were 74%, 82%, 76%, 20%, 85% and 77% respectively; in the low-dose group, the cell activity of the control group was 55%, and the activities of the AF group were 42%, 48%, 40%, 18%, 57% and 47% respectively. The compounds of the present invention have good activity.

[0059] Effect test example 4: NHE1 inhibitory activity: refer to the method of Rosskopf et al. (Hypertens. 1991, 9 (3): 231-237). Take 6 mL of fundus blood from each rat and add 0.3 mL of ACD solution. Centrifuge at 150g for 15 minutes at room temperature and take the upper two-thirds of the plasma. The grouping of the tested drugs is shown in Table 1 above. The control group is replaced with cariporide (CAS: 159138-80-4), dissolved in DMSO, and diluted with standard medium. The final DMSO content is less than 10 -4 mol / L. Each drug was tested in 4 groups of concentrations, and each group was measured twice. The optical density was measured by a multi-scanning spectrophotometer. 175 μL of sodium propionate medium and 25 μL of the test sample and standard medium were added to a 96-well plate, preheated to 37°C, and then 50 μL of the above plasma was added. The OD value change at 550 nm was measured, and one point was recorded every 7.5 seconds for a total of 2 minutes.

[0060] The calculation method is based on the method of Rosskopf et al. (Hypertens. 1991, 9(3): 231-237); the natural logarithm of the measured OD value is taken, and a scatter plot is made with lnOD as the Y axis and time as the X axis. The first 50 seconds are taken for linear regression, and the absolute value of the slope is k. min k' and k max The percentage rate constant k% was obtained by comparison. The k% value of the test drug at different concentrations was taken as the Y axis and the corresponding concentration was taken as the X axis to perform nonlinear regression analysis (Graphpad Prism software) to fit the S curve, and the IC 50 , that is, the concentration that reduces platelet swelling by 50%. The results are shown in Table 2. The drug of the present invention has good activity.

[0061]

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof; ,in, are independently single bonds or double bonds; R1 is C 1-3 R2 is halogen; X1 and X2 are independently CH or a heteroatom, and the heteroatom is C, N or O.

2. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The C 1-3 The alkyl group is methyl, ethyl, n-propyl or isopropyl.

3. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The halogen is F or Cl.

4. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The halogen is Cl.

5. The compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The compound is: or .

6. A pharmaceutical composition, characterized in that It comprises a compound represented by formula I as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

7. The pharmaceutical composition according to claim 6, characterized in that It further comprises sennoside A.

8. The pharmaceutical composition according to claim 7, characterized in that The molar ratio of the compound represented by formula I or a pharmaceutically acceptable salt thereof to the sennoside A is 1:(0.1-10).

9. Use of the compound of formula I as described in any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in any one of claims 6 to 8, in the preparation of a drug for treating and / or preventing myocardial ischemia-reperfusion injury.

10. Use of sennoside A in preparing a drug for treating and / or preventing myocardial ischemia-reperfusion injury.

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