A compound for treating myocardial ischemia-reperfusion injury, a pharmaceutical composition and its application

Abstract

The present invention provides a compound, a pharmaceutical composition and its application for treating myocardial ischemia-reperfusion injury. Specifically disclosed is a small molecule compound or a pharmaceutically acceptable salt thereof. The compound and the pharmaceutical composition provided by the present invention have a good effect of protecting myocardial cells and can be used to treat 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, has multiple pharmacological effects, such as laxative, anti-obesity, hypoglycemic, liver protection, anti-fibrosis, anti-inflammatory, anti-tumor, antibacterial, antifungal, antiviral and anti-neurodegenerative, 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 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;

[0009]

[0010] in, are independently a single bond or a double bond;

[0011] R1 is C 1-3alkyl;

[0012] R2 is halogen;

[0013] X1 and X2 are independently CH or a heteroatom, wherein the heteroatom is C, N or O.

[0014] In the present invention, the C 1-3 The alkyl group may be methyl, ethyl, n-propyl or isopropyl.

[0015] In the present invention, the halogen may be F or Cl, such as Cl.

[0016] In the present invention, the compound represented by formula I is

[0017] 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.

[0018] In the present invention, the pharmaceutical composition may further comprise sennoside A.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] The present invention provides an application of sennoside A in preparing a medicine for treating and / or preventing myocardial ischemia-reperfusion injury.

[0023] Explanation of terms

[0024] The term "pharmaceutically acceptable salt" refers to those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and which are commensurate with a reasonable benefit / risk ratio, within the scope of sound 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The reagents and raw materials used in the present invention are commercially available.

[0029] 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

[0030] Figure 1 This is an immunofluorescence image of H9C2 cardiomyocytes after staining.

[0031] Figure 2 This is the skeleton of cardiac troponin distribution after staining in H9C2 cardiomyocytes.

[0032] Figure 3 These are images of TTC and Evans staining of the heart after ischemia-reperfusion.

[0033] Figure 4 H&E staining of cardiac tissue after ischemia-reperfusion.

[0034] Figure 5 Masson staining of cardiac tissue after ischemia-reperfusion.

[0035] Figure 6 This is a picture of Sirius red staining of cardiac tissue after ischemia-reperfusion. DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1 Compound 1

[0038]

[0039] Compound I-1 (0.1 mol, preparation method can refer to CN101143845A) and 900 ml of anhydrous acetonitrile are stirred to dissolve, K2CO3 (0.4 mol) is added, and compound I-2 (0.15 mol, preparation method can refer to CN113248518A) dissolved in 100 ml of anhydrous acetonitrile solution is slowly added dropwise at 40°C. After the addition is completed, the mixture is reacted for 3 hours, filtered, and purified by column chromatography (DCM / methanol = 20:1) to obtain compound I-3 (75 mmol) with a yield of 75%.

[0040] LC-MS (ESI): m / z = 574.2 [M+H]+ .

[0041]

[0042] Take guanidine (1 mol, CAS No: 113-00-8) and add it to 35 ml of isopropanol. Add a suspension of compound I-3 (0.05 mol) and isopropanol (60 ml) 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%.

[0043] LC-MS (ESI): m / z = 587.2 [M+H] + .

[0044] 1 H NMR (400MHz, CD3OD): δ2.21(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(1 H,dd,J=8.3,1.9Hz),7.97(1H,dt,J=8.3,0.5Hz),8.10(1H,s),8.62(1H,dd,J=1.9,0.4Hz),9.10(1H,d,J=0.5Hz).

[0045] Example 2

[0046]

[0047] Compound I-1 (0.1 mol, preparation method can refer to CN101143845A) and 900 ml of anhydrous acetonitrile are stirred to dissolve, K2CO3 (0.4 mol) is added, and compound II-2 (0.15 mol, preparation method can refer to CN113248518A) dissolved in 100 ml of anhydrous acetonitrile solution is slowly added dropwise at 40°C. After the addition is completed, the mixture is reacted for 2 h, filtered, and purified by column chromatography (DCM / methanol = 20:1) to obtain compound II-3 (0.07 mol) with a yield of 70%.

[0048] LC-MS (ESI): m / z = 573.2 [M+H] + .

[0049]

[0050] Take guanidine (1 mol, CAS No: 113-00-8) and add it to 40 ml of isopropanol. Add a suspension of compound II-3 (0.05 mol) and isopropanol (60 ml) 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 liquids and 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 2 (white solid, 30 mmol), with a yield of 60%.

[0051] LC-MS (ESI): m / z = 588.2 [M+H] + .

[0052] 1 H NMR (400MHz, CD3OD): δ2.27(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(1 H,d,J=8.8Hz),7.69-7.81(2H,m),8.13(1H,dt,J=8.3,0.5Hz),8.62(1H,dd,J=1.9,0.4Hz),9.17(1H,d,J=0.5Hz).

[0053] Example 3

[0054]

[0055] Referring to the reaction steps of Example 1, compound I-2 was replaced by compound III-2 (compound III-2 was purchased from Hefei Xingpeptide) to obtain compound III-3 (0.06 mol) with a yield of 60%.

[0056] LC-MS (ESI): m / z = 517.2 [M+H] + .

[0057]

[0058] With reference to Example 1, I-3 was replaced by the above step III-3 to obtain compound 3 (32 mmol) with a yield of 64%.

[0059] LC-MS (ESI): m / z = 530.2 [M+H] + .

[0060] 1H NMR (400MHz, CD3OD): δ2.24(3H,s),2.51-2.70(4H,m),3.4-3.60(4H,m),4.10(2H,s),6.00(1H,d,J=5.2Hz),7.01 -7.15(4H,m),7.64-7.81(2H,m),8.05(1H,dt,J=8.3,0.5Hz),8.62(1H,dd,J=1.9,0.4Hz),9.10(1H,d,J=0.5Hz).

[0061] Effect Test Example 1 Effect of Sennoside A on Hypoxia-Reoxygenation H9C2 Cells

[0062] H9C2 cardiomyocytes (purchased from Saios) in logarithmic phase growth were cultured in DMEM (containing 1.5 g / L NaHCO) + 10% fetal bovine serum + 1% double antibody, and 6000 cells / well were seeded in a 96-well culture plate overnight. After observing that the cells grew well, the culture medium was replaced with a medium containing culture medium, 5 μM sennoside A, and 10 μM sennoside A (purchased from Sinopharm Reagent Company, the solvent was DMEM medium). After anaerobic culture for 2 hours (the control group was not cultured anaerobic), the cells were cultured under normal conditions for 2 hours, the cells were lysed, the cell proteins were separated, and the protein was stained and detected by western blotting: (1) After 2 hours of hypoxia and 2 hours of reoxygenation, the cell morphology 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 (5 μM) is the 5 μM sennoside A group, SEN (10 μM) is the 10 μM sennoside A, green is Troponin T (abbreviated as TnT) cardiac troponin, blue is DAPI stain, Blue: DAPI, Green: Troponin T), the results show that sennoside A improves the cell morphology induced by hypoxia and reoxygenation; (2) through protein immunoblotting experiments, it was found that sennoside A improved the reduced expression of P130cas and Paxilin phosphorylated proteins in myocardial cells induced by hypoxia and reoxygenation, indicating that sennoside A can alleviate the damage of cell migration and proliferation caused by hypoxia and reoxygenation, and protect myocardial cells from the damage induced by hypoxia and reoxygenation.

[0063] Effect test example 2

[0064] Thirty 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; the second group was a vehicle 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.

[0065] 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, SEN (10 mg / kg) was the 10 mg / kg sennoside A group), and the remaining mice were sacrificed for heart tissue sections, which were stained with 1% H&E, Masson, and Sirius red staining kits (see the attached results for the 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 indices 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.

[0066] 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.

[0067] Effect Test Example 3 Evaluation of the Protective Effect of Compounds on Hypoxia-Reoxygenation H9C2 Cells

[0068] H9C2 cardiomyocytes (purchased from Saios) in logarithmic phase growth were taken, and the culture medium was DMEM (containing 1.5 g / L NaHCO) + 10% fetal bovine serum + 1% double antibody. They were inoculated in a 96-well culture plate at 6000 / well. After observing that the cells grew well overnight, 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: 1 μM, the high-dose group: 5 μM, the solvent was DMEM culture medium, and sennoside A was purchased from Sinopharm Reagent Company). 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 of culture (the blank group was not subjected to anaerobic culture), then 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 present application can significantly inhibit the decline in viability of hypoxic and reoxygenated cells.

[0069] Table 1

[0070]

[0071]

[0072] The experimental conclusions 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.

[0073] Effect Test Example 4 NHE1 Inhibitory Activity

[0074] Refer to the method of Rosskopf et al. (Hypertens. 1991, 9(3): 231-237). Take 6 mL of blood from the fundus of each rat and add 0.3 mL of ACD solution. Centrifuge at 150 g for 15 min 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 was replaced with cariporide (CAS: 159138-80-4), dissolved in DMSO, and diluted with standard medium. The final DMSO content was less than 10 -4mol / 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.

[0075] 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 'Compared with the percentage rate constant k%, the k% value of the test drug at different concentrations was used as the Y axis and the corresponding concentration was used as the X axis to perform nonlinear regression analysis (Graphpad Prism software) to fit the S curve, and the curve was calculated to obtain IC50, 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.

[0076] Table 2

[0077] Grouping <![CDATA[IC 50 (μmol / L) <!-- 7 -->]]> Control group 0.3 Group A 5 Group B 2.2 Group C 4 Group D ＞20 Group E 2.0 Group F 3.4

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The compound is 2. A pharmaceutical composition, characterized in that It comprises the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, characterized in that It further comprises sennoside A.

4. The pharmaceutical composition according to claim 3, characterized in that The molar ratio of the compound or its pharmaceutically acceptable salt to the sennoside A is 1:(0.1-10).

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating and / or preventing myocardial ischemia-reperfusion injury.

6. Use of sennoside A in the preparation of a drug for treating and / or preventing myocardial ischemia-reperfusion injury.

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