Use of compound d753-0027 in the manufacture of a medicament for treating myocardial fibrosis

Abstract

The application discloses use of a compound D753-0027 in preparation of a drug for treating myocardial fibrosis, and relates to the technical field of biological medicines.The use of the compound D753-0027 in preparation of the drug for treating myocardial fibrosis, and a chemical structural formula of the compound D753-0027 is as follows: The D753-0027 compound of the application is started from blocking protein-protein interaction of TGF-beta and its receptor, then blocking TGF-beta signal transduction, and the protein-protein interaction inhibitor of TGF-beta and its receptor can reach the target point without passing through a cell membrane, therefore, it is more likely to become a kind of inhibitor with high specificity but low side effect.The D753-0027 compound screened by the application is subjected to cell proliferation and toxicity detection, no obvious cytotoxicity is found, and cell proliferation is not affected.The target specificity of the D753-0027 compound of the application is superior to that of a reported kinase inhibitor.In a cardiac fibrosis animal model, fibrosis protein levels are significantly reduced, and animal cardiac function is improved, and the D753-0027 compound has good application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of a compound D753-0027 in the preparation of a drug for treating myocardial fibrosis. Background Technology

[0002] Myocardial fibrosis is a pathological change caused by excessive collagen deposition in the myocardial tissue after cardiac damage. It manifests as cardiac structural remodeling and decreased elasticity, and may lead to serious consequences such as arrhythmia and heart failure. It is commonly seen in diseases such as myocardial infarction, hypertension, and myocarditis, and requires diagnosis through imaging and biopsy. Treatment focuses on controlling the primary disease and slowing the progression of fibrosis.

[0003] Myocardial fibrosis is a process of cardiac interstitial remodeling, characterized by abnormal proliferation of cardiac interstitial fibroblasts, excessive accumulation of collagen fibers, a significant increase in collagen concentration and volume fraction per unit mass of myocardium, and an imbalance and abnormal distribution of different types of collagen. Myocardial fibrosis is present to varying degrees in some common cardiovascular diseases, such as diabetic cardiomyopathy, viral myocarditis, ischemic cardiomyopathy, and hypertensive heart disease.

[0004] Currently available medications for treating myocardial fibrosis include:

[0005] (1) ACEI / ARB drugs (such as enalapril and valsartan) can inhibit the renin-angiotensin system and reduce collagen production;

[0006] (2) Aldosterone receptor antagonists (such as spironolactone) can delay the fibrosis process;

[0007] (3) SGLT-2 inhibitors (such as dapagliflozin) have been shown to improve cardiac remodeling in recent years.

[0008] Transforming growth factor-β (TGF-β) plays a central and multifaceted driving role in fibrosis, serving as a key pathological mediator in almost all organ system fibrotic diseases (such as pulmonary fibrosis, liver fibrosis, kidney fibrosis, myocardial fibrosis, and scleroderma). TGF-β1 is the most prevalent subtype involved in fibrosis. TGF-β binds to type II receptors on the cell surface, recruits and phosphorylates type I receptors (mainly ALK5 / TGFβRI), initiating downstream signaling and subsequently directly regulating the expression of numerous pro-fibrotic target genes, leading to fibrosis. Given TGF-β's central role, it has become a major target for anti-fibrotic drug development. However, currently, targeted drugs that inhibit TGF-β are still in clinical trials.

[0009] Current TGF-β inhibitors have the following drawbacks: some inhibitors lack specificity, leading to adverse reactions and thus affecting treatment efficacy; some small molecule inhibitors suffer from poor water solubility, low bioavailability, and metabolic instability, limiting their clinical application. Therefore, there is an urgent need to develop new TGF-β inhibitors for the treatment of myocardial fibrosis. Summary of the Invention

[0010] To address the technical problems existing in the prior art, this invention provides the use of compound D753-0027 in the preparation of a drug for treating myocardial fibrosis. The technical solution is as follows:

[0011] The use of compound D753-0027 in the preparation of a medicament for treating myocardial fibrosis, the chemical structural formula of which is as follows:

[0012] .

[0013] Optionally, the compound D753-0027 is a TGF-β inhibitor.

[0014] Optionally, the compound D753-0027 blocks TGF-β signal transduction by blocking the protein-protein interaction between TGF-β and its receptor, reduces the expression of fibrosis markers type I collagen, fibronectin and α-smooth muscle actin, and improves myocardial fibrosis and cardiac function.

[0015] A medicament or pharmaceutical composition for treating myocardial fibrosis, said medicament or pharmaceutical composition comprising compound D753-0027, wherein the chemical structural formula of said compound D753-0027 is as follows:

[0016] .

[0017] Optionally, the compound D753-0027 is a TGF-β inhibitor.

[0018] Optionally, the compound D753-0027 blocks TGF-β signal transduction by blocking the protein-protein interaction between TGF-β and its receptor, reduces the expression of fibrosis markers type I collagen, fibronectin and α-smooth muscle actin, and improves myocardial fibrosis and cardiac function.

[0019] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0020] The D753-0027 compound screened in this invention initiates the blocking of protein-protein interactions between TGF-β and its receptor (molecular docking technology screens compounds that block protein-protein interactions), thereby blocking TGF-β signal transduction. Inhibitors that block TGF-β and its receptor's protein-protein interactions do not need to cross the cell membrane to reach their target; therefore, they are more likely to become inhibitors with high specificity and low side effects. The D753-0027 compound screened in this invention was subjected to cell proliferation and toxicity tests, and no significant cytotoxicity was found, nor did it affect cell proliferation. The target specificity of the D753-0027 compound of this invention is superior to currently reported kinase inhibitors. In animal models of cardiac fibrosis, it significantly reduces fibrosis protein levels and improves cardiac function, demonstrating significant application value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a Western Blot result of the downstream signaling pathways of phosphorylated SMAD2 (p-SMAD2) and fibrosis markers type I collagen (COL1) and fibronectin in H9C2 cells treated with the top 4 compounds in Table 1 provided in Example 2 of this invention.

[0023] Figure 2 This is a graph showing the cell proliferation and toxicity detection of compound D753-0027 provided in Example 3 of this invention;

[0024] Figure 3A This is an echocardiogram of a mouse cardiac fibrosis model after administration of D753-0027, provided in Example 4 of this invention. In this model, "mock" refers to the control group, "ISO" refers to the isoproterenol treatment group (model group), and "ISO+D7" refers to the group treated with both isoproterenol and D753-0027 (administration group). D753-0027 in... Figure 3A The winning bidder is D7; Figure 3B This is a graph showing the results of detecting fibrosis markers in a mouse cardiac fibrosis model provided in Example 4 of this invention after administration of D753-0027. In this graph, "mock" refers to the control group, "ISO" refers to the isoproterenol treatment group (model group), and "ISO+D7" refers to the group treated with both isoproterenol and D753-0027 (administration group). D753-0027 in... Figure 3BThe winning bidder is D7. Detailed Implementation

[0025] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0026] In this invention, the following abbreviations have the following meanings:

[0027] HW / BW: Heart weight / body weight ratio; EF: Ejection fraction; FS: Left ventricular fractional shortening;

[0028] LVIDs: Left ventricular end-systolic diameter; LVIDd: Left ventricular end-diastolic diameter;

[0029] COL1A2: Collagen Type I Alpha 2 Chain;

[0030] FN1: Fibronectin;

[0031] SMA: α-smooth muscle actin.

[0032] This invention utilizes molecular docking technology, based on the three-dimensional structure of TGF-β, to automatically match small molecules from a compound database at the binding site. Then, it calculates the binding energy of possible binding modes using a molecular force field-based scoring function, ultimately obtaining a ranking of the compounds by energy. Subsequently, we selected the top four compounds for cell experiments and found that one of them (D753-0027) effectively inhibited TGF-β-induced fibrosis in cardiomyocytes. Subsequent animal experiments confirmed that this compound could improve ISO-induced cardiac dysfunction.

[0033] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0034] Example 1: High-throughput virtual screening

[0035] Experimental steps:

[0036] This study uses Autodock Vina 1.2.3 for high-throughput virtual screening. In performance tests of interface tools, Autodock Vina's screening capabilities are considered comparable to, or even superior to, mainstream commercial software. Therefore, this study uses Autodock 1.2.3 for batch screening.

[0037] Use the raccoon applet in the Autodock suite to batch convert small molecule structures into pdbqt format.

[0038] The screening library was obtained from the Chemdiv compound library. The SmartTM Library and the MCE-18 Trends in Medicinal Chemistry library were selected and mixed together to form the small molecule library for this virtual screening.

[0039] Discovery Studio was used to perform drug-like five-rule screening (preliminary screening) on ​​the small molecule library for this screening, eliminating compounds that did not meet the RO5 attribute of the lead compound, and finally obtaining a total of 85,494 small molecules in the effective screening library for virtual screening.

[0040] Using molecular docking technology, based on the three-dimensional structure of TGF-β, batch screening was performed using the Autodock 1.2.3 tool. Conformational ranking was based on docking scores, ultimately yielding an energy ranking of the compounds.

[0041] Experimental results:

[0042] The top 10 compounds after screening are shown in Table 1. The names of the top 10 compounds are represented by their IDs in the Chemdiv compound library (referred to as ChemDiv IDs). Information on specific compounds can be found in the Chemdiv compound library.

[0043] Table 1. The top 10 small molecules by binding energy in this screening and their corresponding binding energies.

[0044]

[0045] For detailed information on D753-0027, please refer to the Chemdiv compound library, for example, the following webpage: https: / / www.chemdiv.com / catalog / screening-compounds / compound-D753-0027 / . The chemical structural formula of D753-0027 is as follows:

[0046] ;

[0047] Chemical formula: C 27 H 28 N4O2;

[0048] Name: N-{4-[(1-oxo-1,3-dihydro-2H-isoindol-2-yl)methyl]cyclohexyl}-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide;

[0049] Molecular weight: 440.54.

[0050] The chemical structural formula of L856-0042 is as follows:

[0051] ;

[0052] Chemical formula: C 25 H 24 FN5O3S;

[0053] Name: 3-[5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl]-1-methyl-5-(5,6,7,8-tetrahydronaphthalene-2-sulfonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine;

[0054] Molecular weight: 493.56.

[0055] The chemical structural formula of S022-1367 is as follows:

[0056] ;

[0057] Chemical formula: C 24 H 18 ClN5O2;

[0058] Name: 2-(2-chlorophenyl)-7-(1H-indole-5-carbonyl)-5,6,7,8-tetrahydropyrazolo[1,5-a]pyrido[4,3-d]pyrimidine-9(1H)-one;

[0059] Molecular weight: 443.89.

[0060] The chemical structural formula of L303-2457 is as follows:

[0061] ;

[0062] Chemical formula: C 26 H 30 N4O4S;

[0063] Name: 1-(4-{5-[(4-methylphenyl)methyl]-1,2,4-oxadiazol-3-yl}benzene-1-sulfonyl)piperidin-4-yl ketone;

[0064] Molecular weight: 494.61.

[0065] Example 2 Cell Screening Experiment

[0066] Materials and methods:

[0067] 1. Cell culture:

[0068] H9C2 (2-1) (rat cardiomyocytes, purchased from Beijing Zhong Sheng Aobang Biotechnology Co., Ltd.), growth medium: DMEM medium + 10% FBS (fetal bovine serum, purchased from Suzhou Yikesai Biotechnology Co., Ltd.) + 1% P / S (penicillin-streptomycin solution). Culture conditions: CO2, 5%, temperature: 37℃.

[0069] 2. TGF-β treatment of rat cardiomyocytes

[0070] TGF-β (MCE, HY-P7118) at 10 ng / ml was added and treated for one day, followed by Western blotting.

[0071] 3. H9C2 cells (rat cardiomyocytes) were treated with the four compounds obtained in Example 1 (purchased from ChemDiv) (final concentration 10µM).

[0072] Subsequently, the cells were lysed to detect downstream signaling pathways such as phosphorylated SMAD2 (p-SMAD2) and fibrosis markers type I collagen (COL1) and fibronectin. The specific steps are as follows:

[0073] (1) Cell lysis

[0074] 1) Wash twice with PBS (1 ml each time);

[0075] 2) Add 100-200 μL of pre-cooled RIPA cell lysis buffer containing protease inhibitors (purchased from Solarbio), pipette, and place in a 1.5 ml centrifuge tube;

[0076] 3) Place on ice for 15 minutes, shake for about 10 seconds every 5 minutes, shake 3 times to fully lyse rat cardiomyocytes (H9c2 cells).

[0077] 4) Centrifuge at 12000 g for 10 min (4℃), and transfer the supernatant to a new centrifuge tube.

[0078] (2) Running the gel and developing

[0079] 1) Add 4X SDS loading buffer (purchased from Beijing Zhong Sheng Aobang Biotechnology Co., Ltd.) to the supernatant of the first step to a final concentration of 1X, then put it in a metal bath and boil for 5 minutes, and centrifuge at 6000 rpm for 1 minute at 4℃ for subsequent experiments;

[0080] 2) Proteins are separated using SDS-PAGE gel electrophoresis. Typically, the voltage in the stacking gel is 80V, and the voltage in the separating gel is 180V.

[0081] 3) When the bromophenol blue reaches the specific position, stop the electrophoresis. Following the order of filter paper-gel-NC membrane-filter paper, place the clamp into the electroporation tank and electroporate at 200mA for 2 hours on ice. The electroporation time can be appropriately extended or shortened depending on the size of the protein.

[0082] 4) After electroporation, place the NC membrane in Ponceau S staining solution, cut the target band according to the protein size, and then elute with TBST until no red liquid precipitates.

[0083] 5) Seal with 5% skim milk for 1 hour at room temperature;

[0084] 6) Incubate overnight at 4°C with primary antibodies (Phospho-SMAD2, CST; Collagen I, abcam; Fibronectin, Proteintech), recover the primary antibodies, and wash the membrane three times with TBST for 10 minutes each time.

[0085] 7) Add secondary antibodies (Anti-rabbit IgG, HRP-linked Antibody and Anti-mouse IgG, HRP-linked Antibody, CST, respectively), incubate at room temperature for 1 hour, then wash the membrane 3 times with TBST for 10 minutes each time;

[0086] 8) Add the luminescent solution, develop the solution using a developer, and analyze the Western blot results.

[0087] Experimental results:

[0088] The inventors selected the top four compounds from Table 1 and added them to H9C2 cells (rat cardiomyocytes) while simultaneously inducing cardiomyocyte fibrosis with TGF-β1 stimulation. The experimental results are shown below. Figure 1 .

[0089] Figure 1 This is a Western blot diagram of the downstream signaling pathways of phosphorylated SMAD2 (p-SMAD2) and the fibrosis markers COL1 and fibronectin in H9C2 pre-cells treated with the compound ranked 4th in Table 1. Figure 1 As can be seen, only D753-0027 reduced both p-SMAD2 and the fibrosis markers COL1 and fibronectin.

[0090] from Figure 1 The results showed that treatment with compound D753-0027 significantly reduced downstream signaling pathways and the degree of cardiomyocyte fibrosis, indicating that compound D753-0027 antagonized the activation of TGF-β signaling.

[0091] Example 3 Toxicity Detection

[0092] The cell proliferation and toxicity of D753-0027 in H9C2 cells (rat cardiomyocytes) were analyzed using a CCK-8 assay kit (purchased from Beijing Zhong Sheng Aobang Biotechnology Co., Ltd.). The specific steps are as follows:

[0093] 1. Cell seeding: H9C2 cell suspension (100 μL / well) was seeded into 96-well plates, and D753-0027 was added to a final concentration of 5 / 10 / 20 µM. The plates were then incubated at 37°C and 5% CO2 for 12 hours to allow the cells to attach.

[0094] 2. Add CCK-8 reagent: Add 10 μL of CCK-8 solution to each well and mix gently, avoiding air bubbles.

[0095] 3. Incubation: Incubate at 37℃ and 5% CO2 for 2 hours.

[0096] 4. Measure absorbance: Use an ELISA reader to measure absorbance at 450 nm.

[0097] Experimental results:

[0098] Figure 2 This is a graph showing the cell proliferation and toxicity assays for compound D753-0027. From... Figure 2 As can be seen, the concentrations of D753-0027 at 5 / 10 / 20 µM did not significantly affect the proliferation level of H9c2 cells.

[0099] Figure 2 The above results demonstrate that, using CCK-8 to analyze cell proliferation and toxicity, D753-0027 showed no significant cytotoxicity in H9C2 cells (rat cardiomyocytes) and did not affect cell proliferation.

[0100] Example 4 Animal Experiment

[0101] Experimental steps:

[0102] 1) C57BL / 6J mice (purchased from Spifor (Beijing) Biotechnology Co., Ltd.), male, 8 weeks old.

[0103] 2) Isoprenaline (ISO) was injected subcutaneously into mice to induce cardiac fibrosis. The injection was given once a day for 7 days at a dose of 5 mg / kg.

[0104] 3) Intraperitoneal injection of D753-0027 (purchased from ChemDiv, the compound and ISO were injected on the same day), three times a week, each dose of 25 mg / kg.

[0105] 4) Mouse echocardiography to assess cardiac function, including: ejection fraction, fractional shortening, end-systolic volume, end-diastolic volume, left ventricular end-systolic dimension, and end-diastolic dimension. The detection methods are as follows:

[0106] The cardiac function of the mice was tested using a small animal ultrasound system (VINNO 6, Vinno Corporation). The specific steps are as follows:

[0107] (1) Hair was removed from the chest of mice with depilatory cream. After anesthesia with isoflurane at a flow rate of 1.5 ml / min, the mice were placed on an electrode platform at 37°C and the heart rate was controlled between 450-650 bp with continuous administration of isoflurane.

[0108] (2) Apply coupling agent to the upper chest, use a 23MHz ultrasound probe to take the long axis section of the left ventricle, and record the heart function of the mouse in B-Mode and M-Mode respectively. Then rotate the probe 90 degrees and take the short axis section of the left ventricle. Record the heart function of the mouse in B-Mode and M-Mode respectively.

[0109] 5) Sacrifice the mice, isolate the mouse hearts, extract RNA for qPCR, and detect the mRNA levels of COL1A2, fibronectin, and α-SMA.

[0110] RNA extraction was performed using an animal tissue / cell total RNA extraction kit purchased from Nanjing Novizan Biotechnology Co., Ltd. qPCR was used to detect the mRNA levels of COL1A2, fibronectin, and α-SMA using THUNDERBIRD. ® The Next SYBR qPCR Mix kit was purchased from TOYOBO. Refer to the corresponding kit instruction manual for detailed operating procedures.

[0111] Experimental results:

[0112] See the experimental results. Figures 3A to 3B .

[0113] in, Figure 3A This is an echocardiogram of a mouse model of cardiac fibrosis after administration of D753-0027 (labeled D7 in the image). Figure 3AAs can be seen from echocardiography, administration of D753-0027 to the mouse cardiac fibrosis model significantly improved cardiac function indicators such as heart weight / body weight ratio, ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd). Specifically, the heart weight / body weight ratio was 5.54 mg / g in the control group, increasing to 8.12 mg / g in the cardiac fibrosis model group, before returning to 5.32 mg / g after D753-0027 administration. The ejection fraction (EF) was 70.67% in the control group, decreasing to 47.33% in the cardiac fibrosis model group, but increasing to 69.33% after D753-0027 administration. The left ventricular fractional shortening (FS) was 38.33% in the control group, decreasing to 21.67% in the cardiac fibrosis model group, but increasing to 38.67% after D753-0027 administration. The left ventricular end-systolic diameter (LVIDs) was 2.24 mm in the control group, dilated to 2.98 mm in the cardiac fibrosis model group, and returned to 2.20 mm after administration of D753-0027. The left ventricular end-diastolic diameter (LVIDd) was 3.28 mm in the control group, dilated to 4.20 mm in the cardiac fibrosis model group, and returned to 3.42 mm after administration of D753-0027.

[0114] Figure 3A The above results demonstrate that D753-0027 can significantly improve cardiac fibrosis in mice and significantly improve cardiac function (heart weight / body weight ratio, ejection fraction, fractional shortening, end-systolic volume, end-diastolic volume, left ventricular end-systolic dimension, and end-diastolic dimension).

[0115] Figure 3B This is a graph showing the results of detecting fibrosis markers after administering D753-0027 in a mouse model of cardiac fibrosis. From... Figure 3B The results showed that the mRNA levels of COL1A2, fibronectin, and α-SMA were significantly reduced. Specifically, the COL1A2 level in the cardiac fibrosis model group was 12.17 times that of the control, but decreased to 3.97 times after administration of D753-0027. The fibronectin level in the cardiac fibrosis model group was 5.13 times that of the control, but decreased to 1.50 times after administration of D753-0027. The α-SMA level in the cardiac fibrosis model group was 8.80 times that of the control, but decreased to 3.17 times after administration of D753-0027.

[0116] Figure 3B The above results demonstrate that D753-0027 can significantly improve cardiac fibrosis in mice, and the mRNA levels of cardiac fibrosis markers COL1A2, fibronectin, and α-SMA in mice are significantly reduced.

[0117] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of compound D753-0027 in the preparation of a medicament for treating myocardial fibrosis, characterized in that, The chemical structural formula of compound D753-0027 is as follows: 。 2. The use according to claim 1, characterized in that, The compound D753-0027 is a TGF-β inhibitor.

3. The use according to claim 1, characterized in that, The compound D753-0027 blocks TGF-β signal transduction by blocking the protein-protein interaction between TGF-β and its receptor, reduces the expression of fibrosis markers type I collagen, fibronectin and α-smooth muscle actin, and improves myocardial fibrosis and cardiac function.

4. A medicament or pharmaceutical composition for treating myocardial fibrosis, characterized in that, The drug or drug composition comprises compound D753-0027, wherein the chemical structural formula of compound D753-0027 is as follows: 。 5. The drug or drug composition according to claim 4, characterized in that, The compound D753-0027 is a TGF-β inhibitor.

6. The drug or drug composition according to claim 4, characterized in that, The compound D753-0027 blocks TGF-β signal transduction by blocking the protein-protein interaction between TGF-β and its receptor, reduces the expression of fibrosis markers type I collagen, fibronectin and α-smooth muscle actin, and improves myocardial fibrosis and cardiac function.

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