Application of 9-O-succinic acid monoethyl ester berberine derivatives in the preparation of drugs for treating cardiac fibrosis

Abstract

This application relates to the field of biomedical technology, and specifically discloses the use of a 9-O-monosuccinate ethyl berberrubine derivative in the preparation of a drug for treating cardiac fibrosis, wherein the 9-O-monosuccinate ethyl berberrubine derivative has a molecular structure shown in the following formula (I): #imgabs0#X ‑ is Cl ‑ 、Br ‑ 、I ‑ 、SO4 2‑ 、CO3 2‑ 、PO4 3‑ 、citrate or mesylate. The 9-O-monosuccinate ethyl berberrubine derivative in this application can significantly reduce the area of cardiac fibrosis after cardiac injury and can be used to prepare a drug for treating cardiac fibrosis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of a 9-O-succinic acid monoethyl ester berberrubine derivative in the preparation of a drug for treating cardiac fibrosis. Background Art

[0002] Myocardial infarction is a serious cardiovascular disease. It is a pathological process in which the corresponding myocardium undergoes ischemic necrosis due to a sharp reduction or interruption of coronary blood supply. During myocardial infarction, a large number of cytokines are released, causing an inflammatory response, and the necrosis of myocardial cells can lead to myocardial defects, an increase in compensatory extracellular matrix, the formation of scar tissue, myocardial fibrosis, and degradation of cardiac systolic and diastolic function. Myocardial infarction can affect the function and structure of the heart, causing complications such as myocardial remodeling, heart failure, and arrhythmia.

[0003] Drugs used clinically, such as renin-angiotensin system inhibitors, nitrates, beta-blockers, etc., all have serious adverse reactions, such as bronchospasm, cough, hyperkalemia, hypotension, reflex tachycardia, etc. Therefore, finding safe and effective anti-myocardial infarction drugs is of great significance for the treatment of clinical cardiovascular diseases. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide an application of a 9-O-succinic acid monoethyl ester berberine derivative in the preparation of a drug for the treatment of cardiac fibrosis, aiming to solve the problem that existing drugs for the treatment of myocardial infarction may cause adverse reactions to patients.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a 9-O-succinic acid monoethyl ester berberyrin derivative is provided for use in the preparation of a drug for treating cardiac fibrosis, wherein the 9-O-succinic acid monoethyl ester berberyrin derivative has a molecular structure as shown in the following formula (I):

[0007]

[0008] X - Cl - Br - ,I - 、SO4 2- 、CO3 2- PO4 3- , citrate or methanesulfonate.

[0009] Optionally, the 9-O-succinic acid monoethyl ester berberrubine derivative can reduce the area of ​​cardiac fibrosis after cardiac injury.

[0010] Optionally, the disease causing said heart damage comprises myocardial infarction.

[0011] In a second aspect of the present application, a pharmaceutical composition for treating cardiac fibrosis is provided, comprising the above-mentioned 9-O-succinic acid monoethyl ester berberrubine derivative.

[0012] Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0013] Optionally, the auxiliary material includes at least one of a pharmaceutical carrier, a diluent, an adjuvant, and an excipient.

[0014] Optionally, the pharmaceutical composition is one of capsules, tablets, powders, granules or injections.

[0015] Compared with the prior art, this application has the following advantages:

[0016] (1) The 9-O-succinic acid monoethyl ester berberine derivatives in this application can significantly reduce the area of ​​cardiac fibrosis after cardiac injury and can be used to prepare drugs for treating cardiac fibrosis. The 9-O-succinic acid monoethyl ester berberine derivatives in this application have no obvious toxic side effects and can be used orally, which is simple and convenient.

[0017] (2) The pharmaceutical composition containing the 9-O-succinic acid monoethyl ester berberrubine derivative of the present application has strong drugability. Compared with other imported anti-myocardial infarction drugs, it is cheap, cost-effective, and easy for patients to accept; it has stable properties and is easy to transport and store. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 The 1HNMR spectrum of 9-O-succinic acid monoethyl ester berberyrin hydrochloride provided in the examples of the present application;

[0020] Figure 2 The 13CNMR spectrum of 9-O-succinic acid monoethyl ester berberyrosine hydrochloride provided in the examples of the present application;

[0021] Figure 3 Mouse cardiac function assessment diagram provided in the examples of this application:

[0022] A is the ejection fraction analysis diagram; B is the short axis shortening rate analysis diagram; C is the left ventricular end-systolic diameter analysis diagram; D is the right ventricular end-systolic diameter analysis diagram;

[0023] Figure 4 This is a graph of the area analysis of cardiac fibrosis in mice provided in the examples of this application;

[0024] Figure 5 Analysis chart of myocardial fibrosis related indicators provided in the embodiment of this application:

[0025] A is the mRNA expression analysis graph of gene Col1a1; B is the mRNA expression analysis graph of gene Col3a1; C is the mRNA expression analysis graph of gene CTGF; D is the mRNA expression analysis graph of gene Fn1; E is the mRNA expression analysis graph of gene α-SMA. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0027] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0028] A first aspect of the embodiments of the present application provides a use of a 9-O-succinic acid monoethyl ester berberrubine derivative in the preparation of a drug for treating cardiac fibrosis.

[0029] Among them, the 9-O-succinic acid monoethyl ester berberine derivative has a molecular structure shown in the following formula (I):

[0030]

[0031] X - Can be Cl - Br - ,I - 、SO4 2- 、CO3 2- PO4 3- , citrate, methanesulfonate.

[0032] It should be noted that the 9-O-succinic acid monoethyl ester berberyrin derivative can be 9-O-succinic acid monoethyl ester berberyrin and its pharmaceutically acceptable salt, such as hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, methanesulfonate, glucoheptonate, lactobionate and lauryl sulfonate, etc.

[0033] In some embodiments, the synthesis route of the 9-O-succinic acid monoethyl ester berberyrin derivative is as follows, and 9-O-succinic acid monoethyl ester berberyrin hydrochloride is synthesized:

[0034]

[0035] In some embodiments, the 9-O-succinic acid monoethyl ester berberrubine derivative is capable of reducing the area of ​​cardiac fibrosis after cardiac injury.

[0036] In some embodiments, the disease causing cardiac damage comprises myocardial infarction.

[0037] A second aspect of the embodiments of the present application provides a pharmaceutical composition for treating cardiac fibrosis, comprising the 9-O-succinic acid monoethyl ester berberrubine derivative in the embodiments of the present application.

[0038] In some embodiments, the pharmaceutical composition for treating cardiac fibrosis comprises a pharmaceutically acceptable excipient.

[0039] In some embodiments, the auxiliary material includes but is not limited to at least one of a pharmaceutically acceptable carrier, a diluent, an adjuvant, and an excipient.

[0040] In some embodiments, the pharmaceutical composition is in a pharmaceutical dosage form that can be taken.

[0041] In some embodiments, the pharmaceutical composition is a capsule, a tablet, a powder, a granule, or an injection.

[0042] The following is further described by means of specific examples.

[0043] Example 1

[0044] Take 1g of succinic anhydride and 3.5ml of anhydrous ethanol and react them under heating reflux conditions for 3 hours to obtain a clear and transparent liquid. After the reaction is completed, the ethanol is dried by spin drying to obtain an oily substance, monoethyl succinate;

[0045] Take the above monoethyl succinate and 2.5 ml of thionyl chloride and reflux for 2 hours to obtain a green transparent product, and spin dry the thionyl chloride to obtain monoethyl succinate chloride;

[0046] Take 1.33 g of berberine and 0.62 ml of triethylamine and dissolve them in 10 ml of anhydrous dichloromethane. Add ethyl succinate chloride dropwise at 0°C and react at room temperature for 3 hours. Spot the plate with dichloromethane:methanol=10:1.

[0047] After all the blue fluorescence flows out and the eluent is basically colorless, the developing agent ratio is changed to 20:1. Due to the adsorption of impurities, there is no significant stratification at this time. The eluent is detected by spot plate. When the effluent is green and there are no impurities on the spot plate, the eluent is connected. The spot plate detection is stopped until the red berberyrin with a higher polarity appears. The product is then dried and further purified overnight. Recrystallization is performed to obtain 9-O-succinic acid monoethyl ester berberyrin hydrochloride with a higher purity.

[0048] The prepared 9-O-succinic acid monoethyl ester berberine hydrochloride was subjected to H NMR and C NMR analysis. The results are as follows: Figure 1 and Figure 2 The above results confirm that compound B2 has a molecular structure as shown in formula (I), molecular formula: C 23 H 24 ClNO7, molecular weight: 485.9136.

[0049]

[0050] Example 2

[0051] 1 Experimental Materials

[0052] Experimental animals: SPF-grade C57BL / 6 male mice weighing 18-22 g were selected.

[0053] Test substance: 9-O-succinic acid monoethyl ester berberine hydrochloride (B2).

[0054] 2 Experimental Principle

[0055] By ligating the left anterior descending coronary artery to establish a mouse model and then administering the test drug to the mice, the effect of the test substance on cardiac fibrosis can be detected, and the effect of the test substance on the liver function of mice with cardiac fibrosis can be determined.

[0056] 3 Experimental methods

[0057] 3.1 Animal grouping

[0058] Random grouping: After receiving the animals, they were adaptively fed for 3 days. After the adaptation period, the MI model was constructed and the animals were randomly divided into 6 groups, namely, sham operation group, myocardial infarction model group, B2 low-dose group, B2 medium-dose group, B2 high-dose group, and fosinopril sodium group.

[0059] 3.2 Establishment of myocardial infarction model

[0060] The mouse myocardial ischemia model was established by ligating the left anterior descending branch of the mouse coronary artery. Healthy male C57BL / 6 mice (20±2g) were anesthetized with intraperitoneal injection of aflototin (0.2g / kg), and the anesthetized mice were fixed in a supine position on the mouse operating table and connected to a ventilator. An oblique incision of about 1.5-2.0cm was made on the left chest skin from the upper left to the lower right, the pectoralis major and serratus anterior muscles were separated, the intercostal muscles were bluntly separated between the 4th and 5th ribs, the heart was gently pushed out, and a 7 / 0 suture was inserted 1-2mm from the lower edge of the left atrial appendage through the left anterior descending branch of the coronary artery to ligate the left anterior descending branch of the coronary artery. After ligation, the color of the apex of the heart turned white, and obvious ST segment elevation was seen in the electrocardiogram.

[0061] 3.3 Medication and modeling period:

[0062] The SPF-level C57BL / 6 mice after adaptive feeding were randomly weighed and divided into 6 groups, namely, sham operation group, myocardial infarction model group, B2 low-dose group, B2 medium-dose group, B2 high-dose group, and fosinopril sodium group. After modeling, B2, fosinopril sodium tablets and blank solvent were given by gavage for 28 consecutive days. The cardiac function of each group of mice was detected on the 28th day, and the heart weight to body weight ratio and fibrosis area were statistically analyzed.

[0063] 3.4 Observation period

[0064] General vital signs were observed during the experiment.

[0065] 3.5 Main testing indicators

[0066] (1) Weight measurement

[0067] The body weight of each mouse was measured once a week using an electronic balance.

[0068] (2) Mouse cardiac function test

[0069] The 2100 high-resolution small animal ultrasound imaging system and MicroScan MS2500206 probe were used to detect the cardiac hemodynamic parameters of mice. The experimental mice were anesthetized and fixed in the supine position. The chest area was depilated and ultrasound gel was applied. The probe was placed on the left chest of the mouse, and the long axis section of the left ventricle was displayed by 2D ultrasound. The M-mode ultrasound was used under two-dimensional (2D) guidance to record the left ventricular movement, and the ejection fraction (EF) and short axis shortening rate (FS) were measured. The left ventricular function of each group of animals was evaluated by this index.

[0070] (3) Determination of heart-to-body weight ratio in mice

[0071] Take mice from each group, open the abdominal cavity, take out the heart and wash it with saline. Cut off the excess tissue, absorb the residual saline with filter paper, and weigh it.

[0072] 3.6 Masson staining

[0073] Mice were anesthetized as described above, and the intact hearts of mice were taken after the ischemia-reperfusion model was established and placed in a 4% paraformaldehyde solution. After the tissue block was fixed for 24 hours, the tissue with the injured part was cut along the longitudinal axis of the heart and placed in a tissue embedding box, and placed in an automatic dehydrator for dehydration. After 20 hours, the tissue block was taken out. Melt the paraffin and embed the tissue block using an automatic embedding machine. When preparing the sample, the tissue wax block was fixed vertically on the machine, and the blade was fixed to prepare paraffin sections, 4μm / slice, put in warm water to remove wrinkles, flatly stick on the slide, and put in a 55℃ oven overnight for use. The tissue slices were placed in a basket for dewaxing, immersed in a xylene solution so that the solution covered the tissue, immersed twice, 5 minutes each time, and then placed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 70% ethanol in sequence for 2 minutes each. After complete dewaxing, the sample was immersed in distilled water for 2 minutes. After staining in hematoxylin for 10 minutes, rinse with tap water for 10 minutes; after staining in Biebrich red solution for 10 minutes, rinse with tap water for 1 minute; differentiate in phosphomolybdic acid and phosphotungstic acid mixed solution for 15 minutes; after staining in aniline blue solution for 10 minutes, rinse with tap water for 1 minute; after differentiation in acetic acid solution for 4 minutes, rinse with tap water for 30 seconds. Finally, dehydrate, put into 95% ethanol, 100% ethanol, xylene I and xylene II in order, soak for 5 minutes each, seal the cover glass with neutral resin sealing agent, and observe under an optical microscope after drying.

[0074] 3.7 Determination of cardiac fibrosis-related protein levels.

[0075] Heart tissue or cells were homogenized with RIPA lysis buffer (Roche, Switzerland) and supplemented with protease inhibitors 1% protease inhibitor and 10% phosphatase inhibitor. Protein samples were subjected to SDS-PAGE using the Bio-Rad protein mini gel system, using 10%, 12% or 15% (w / v) acrylamide dissolving gels depending on the size of the protein being blotted. Proteins in nucleic acid research gels were transferred to nitrocellulose membranes using the Bio-Rad protean II wet transfer system at 300 mA for 2 hours. After blocking, the membranes were incubated with antibodies overnight at 4°C and the membranes were incubated with secondary antibodies at room temperature for 60 minutes. Dye western blots were imaged using the Odyssey imaging system and analyzed using Image Studio Version software.

[0076] 4 Experimental data and results

[0077] 4.1 Data processing

[0078] Quantitative data were expressed as mean ± standard deviation (mean ± SEM), and the data were statistically analyzed using Graphpad 8.0 software. The t test was used for comparison between 2 groups; one-way analysis of variance (ANOVA) was used for comparison between 3 or more groups, followed by Turkey's multiple comparison test. P < 0.05 was considered statistically significant.

[0079] 4.2 Experimental Results

[0080] The results are as follows Figure 3 As shown, 28 days after the left anterior descending branch of the coronary artery was ligated in mice, compared with the sham operation group, the ejection fraction (EF) and short axis shortening rate (FS) of the myocardial infarction group (***P<0.001vs. sham operation group) were significantly reduced, and the left ventricular end-systolic diameter (LVID:S) and left ventricular end-diastolic diameter (LVID:D) were also significantly reduced, while the B2 group could reverse the above phenomenon (###P<0.001vs. myocardial infarction group, ##P<0.01vs. myocardial infarction group, #P<0.05vs. myocardial infarction group). Figure 4 As shown in the figure, compared with the sham operation group, the myocardial fibrosis area in the myocardial infarction model group increased (***P<0.001vs. sham operation group), while B2 could reduce the increase in cardiac fibrosis area in mice caused by myocardial infarction (###P<0.001vs. sham operation group). Figure 5 As shown, compared with the sham operation group, the myocardial fibrosis-related indicators in the myocardial infarction model group, including Col1a1, Col3a1, α-SMA, CTGF, and Fn1 (***P<0.001vs. sham operation group, **P<0.01vs. sham operation group) were increased, while B2 could reduce the increased degree of cardiac fibrosis in mice caused by myocardial infarction (###P<0.001vs. myocardial infarction group, ##P<0.01vs. myocardial infarction group, #P<0.05vs. myocardial infarction group).

[0081] Those skilled in the art can understand that, since 9-O-succinic acid monoethyl ester berberyrubine has a certain instability and can form salts, 9-O-succinic acid monoethyl ester berberyrubine and its pharmaceutically acceptable salts (i.e., 9-O-succinic acid monoethyl ester berberyrubine derivatives with a molecular structure as shown in formula (I)) also have the same or similar effects as 9-O-succinic acid monoethyl ester berberyrubine hydrochloride, and can significantly reduce the area of ​​cardiac fibrosis after heart damage, and can be used to prepare drugs for treating cardiac fibrosis.

[0082] In summary, the 9-O-succinic acid monoethyl ester berberyrin derivatives in this application can significantly reduce the area of ​​cardiac fibrosis after cardiac injury, and can be used to prepare drugs for the treatment of cardiac fibrosis. The 9-O-succinic acid monoethyl ester berberyrin derivatives in this application have no obvious toxic side effects and can be used orally, which is simple and convenient. The pharmaceutical composition containing the 9-O-succinic acid monoethyl ester berberyrin derivatives in this application has strong drugability. Compared with other imported anti-myocardial infarction drugs, it is cheap, cost-effective, and easy for patients to accept; it has stable properties and is easy to transport and store.

[0083] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. Use of 9-O-succinic acid monoethyl ester berberine derivatives in the preparation of drugs for treating cardiac fibrosis, wherein the 9-O-succinic acid monoethyl ester berberine derivatives have a molecular structure as shown in the following formula (I): X - Cl - Br - ,I - 、SO4 2- 、CO3 2- PO4 3- , citrate or methanesulfonate.

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