Use of pyrrolotriazine compound in preparation of drug for preventing and / or treating fibrosis or related disease thereof

Abstract

The present invention relates to use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating fibrosis or a related disease thereof.

Description

Application of pyrrolotriazine compounds in the preparation of drugs for preventing and / or treating fibrosis or related diseases

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on November 3, 2023, with patent application number 202311469656.3, entitled “Application of pyrrolotriazine compounds in the preparation of medicaments for the prevention and / or treatment of fibrosis or its related diseases.” The entire text of that application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of medicine, and particularly relates to the use of pyrrolotriazine compounds in the preparation of drugs for preventing and / or treating fibrosis or related diseases. Background Art

[0003] Fibrosis is scarring and tissue hardening caused by excessive deposition of extracellular matrix (ECM) proteins by myofibroblasts in response to chronic inflammation. A variety of noxious stimuli, including toxins, infectious pathogens, autoimmune reactions, and mechanical stress, can induce fibrotic cellular responses.

[0004] In response to tissue injury, myofibroblasts derived from a variety of sources (including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and transdifferentiated cell types) initiate a wound healing response by remodeling the extracellular environment to restore tissue integrity and promote the replacement of parenchymal cells. Normally, this pro-fibrotic program is shut down during tissue healing. However, persistent injury and damage can lead to dysregulation of this process, resulting in pathologically excessive deposition of ECM proteins, accompanied by upregulation of myofibroblast activity, and a chronic inflammatory environment infiltrated by macrophages and immune cells. In this cellular environment, cytokines and growth factors are released in large quantities, ultimately leading to upregulation of target gene expression, which further enhances myofibroblast differentiation and the production and secretion of ECM proteins (including collagen, laminin, and fibronectin). As excessive ECM deposition proceeds, the matrix architecture changes and becomes stiffer.

[0005] Fibrosis is a tumor-like lesion between benign and malignant. It often occurs in organs and tissues such as the liver, kidneys, heart, lungs, and bone marrow. It can affect almost every organ and eventually induce multiple organ failure, cause cancer, and seriously endanger life and health.

[0006] Pulmonary fibrosis can be broadly classified by etiology into idiopathic, primary, immune, drug-induced, and physical and chemical factors. Idiopathic pulmonary fibrosis accounts for the largest proportion of these types.

[0007] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown etiology that primarily occurs in the elderly and is characterized by dyspnea and progressive deterioration of lung function. The abnormal accumulation of fibrotic tissue in the lung parenchyma severely impairs respiratory function, manifesting as a dry cough and progressive dyspnea (a feeling of shortness of breath). As the disease and lung damage worsen, respiratory function deteriorates.

[0008] The cause and mechanism of IPF remain unclear. Treatment goals are to relieve symptoms, improve quality of life, slow or halt disease progression, and increase survival. Prednisone, azathioprine, and N-acetylcysteine ​​(NAC) have been used to treat IPF symptoms, but they generally do not significantly increase life expectancy.

[0009] Nonalcoholic steatohepatitis (NASH) is a disease characterized by inflammation and fibrosis that can progress to cirrhosis and liver failure. Currently, NASH is rapidly becoming the leading reason for liver transplantation.

[0010] WO2021098691A1 discloses a class of pyrrolotriazine compounds as MNK inhibitors. Further research by the present inventors has found that these compounds are promising for the treatment of fibrotic diseases.

[0011] Summary of the Invention

[0012] The present invention provides a use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating fibrosis or related diseases or conditions.

[0013] Wherein, R1 is H, F, Cl, Br or C 1-3 alkyl;

[0014] R2 and R3 are each independently H or C 1-3 Alkyl, wherein the C 1-3 Alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br or I;

[0015] or R2 and R3 are linked together with the carbon atom to which they are attached to form a cyclopentyl, cyclohexyl or piperidinyl group, wherein the cyclopentyl, cyclohexyl and piperidinyl groups are optionally substituted with 1, 2 or 3 R a replaced by;

[0016] Each R a are independently H, F, Cl, Br or C 1-3 alkyl;

[0017] R4 is H, F, Cl, Br or C 1-3alkyl;

[0018] R5 and R6 are each independently H, F, Cl, Br, I or C 1-3 alkyl;

[0019] R7 is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted by 1, 2 or 3 R b replaced by;

[0020] Each R b are independently H, F, Cl, Br, I or C 1-3 Alkyl, wherein the C 1-3 Alkyl is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br or I;

[0021] n is 1 or 2.

[0022] In some embodiments of the present invention, the above R a are independently H, F, Cl, Br, -CH3 or -CH2CH3, and other variables are as defined herein.

[0023] In some embodiments of the present invention, R2 and R3 are each independently H, -CH3 or -CH2CH3, and other variables are as defined in the present invention.

[0024] In some embodiments of the present invention, the above R2 and R3 are linked together with the carbon atoms to which they are attached to form R a and other variables are as defined in the present invention.

[0025] In some embodiments of the present invention, the above R2 and R3 are linked together with the carbon atoms to which they are attached to form Other variables are as defined in the present invention.

[0026] In some embodiments of the present invention, the above structural unit for R1, R a and other variables are as defined in the present invention.

[0027] In some embodiments of the present invention, the above structural unit for

[0028] Other variables are as defined in the present invention.

[0029] In some embodiments of the present invention, R1 is C 1-3 Alkyl groups, such as methyl.

[0030] In some embodiments of the present invention, R2 and R3 are linked together with the carbon atoms to which they are attached to form In some embodiments of the present invention, R4 is C 1-3 Alkyl groups, such as methyl.

[0031] In some embodiments of the present invention, R5 and R6 are each independently H or methyl; n is 2;

[0032] In some embodiments of the present invention, for

[0033] In some embodiments of the present invention, R7 is substituted by 1, 2 or 3 H, F, Cl or methyl. For example,

[0034] In some embodiments of the present invention, the compound has a structure represented by any one of formulas (I-1) to (I-4):

[0035] Among them, R1, R4, R5, R6, R7, R a and n are as defined in the present invention.

[0036] In some embodiments of the present invention, the above R b Independently H, F, Cl, Br, I, Other variables are as defined in the present invention.

[0037] In some embodiments of the present invention, the above R7 is wherein Optional 1 or 2 R b Replaced by R b and other variables are as defined in the present invention.

[0038] In some embodiments of the present invention, the above R7 is R b and other variables are as defined in the present invention.

[0039] In some embodiments of the present invention, the above R7 is Other variables are as defined in the present invention.

[0040] In some embodiments of the present invention, the above R4 is H or -CH3, and the other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, the compound has a structure represented by any one of formulas (I-5) to (I-9):

[0042] Among them, R1, R5, R6, R a and R b As defined in the present invention.

[0043] In some embodiments of the present invention, the above R1 is H, F, Cl or Other variables are as defined in the present invention.

[0044] In some embodiments of the present invention, R5 and R6 are each independently H or Other variables are as defined in the present invention.

[0045] Some other solutions of the present invention are obtained by any combination of the above variables.

[0046] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0047] According to an embodiment of the present invention, the pharmaceutically acceptable salt is a salt of the compound represented by formula (I) and an inorganic acid, the inorganic acid including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, etc.; and an organic acid salt, the organic acid including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like; also including salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid; preferably, the hydrochloride and p-toluenesulfonate of the compound represented by formula (I).

[0048] According to an embodiment of the present invention, the fibrosis or its related diseases or conditions are selected from pulmonary fibrosis, renal fibrosis, myelofibrosis, cystic fibrosis, oral mucosal fibrosis, liver fibrosis, biliary fibrosis, myocardial fibrosis, skin fibrosis, eye fibrosis, and pancreatic fibrosis.

[0049] In some embodiments, the fibrosis or its related disease or disorder is selected from an inflammatory disease, for example, selected from pneumonia, hepatitis, nephritis, myocarditis, pancreatitis.

[0050] In some embodiments, the fibrosis or a disease or disorder related thereto is selected from a liver-related disease, such as hepatitis, cirrhosis, liver injury, or liver failure.

[0051] In some embodiments, the fibrosis or a disease or disorder associated therewith is selected from non-alcoholic fatty liver disease or non-alcoholic steatohepatitis (NASH).

[0052] In some embodiments, the fibrosis or its related disease or condition is selected from progressive fibrosing interstitial lung disease (PF-ILD), in particular diseases with manifestations of pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF), systemic sclerosis-associated ILD (SSc-ILD), connective tissue disease-associated ILD (CTD-ILD), rheumatoid arthritis-associated ILD (RA-ILD), chronic fibrosing hypersensitivity pneumonitis (HP), idiopathic nonspecific interstitial pneumonia (iNSIP), unclassifiable idiopathic interstitial pneumonia (IIP), environmental / occupational fibrosing lung disease, idiopathic pneumonia with autoimmune features (IPAF) and sarcoidosis.

[0053] In some embodiments, the fibrosis or its related disease or disorder is selected from muscular dystrophy, fibromatosis, and myelofibrosis, preferably selected from Duchenne muscular dystrophy, Dupuytren's contracture, and primary myelofibrosis (PMF).

[0054] The present invention also provides a method for preventing and / or treating fibrosis or related diseases or conditions, which comprises administering to a patient a therapeutically effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0055] When preparing the medicaments described herein, the active compounds are combined or formulated with appropriate pharmaceutically acceptable carriers, diluents, or excipients, and can be formulated into solid, semisolid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Administration routes include oral, intraperitoneal, transdermal, subcutaneous, intravenous or intramuscular injection, inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular, or aural delivery, or any other method known in the art, all of which can achieve the prevention and / or treatment of fibrosis or its related diseases or conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows HE and Masson staining images of lung tissue;

[0057] FIG2 shows the fibrosis degree scores of mouse lung tissue (* represents comparison with the normal control group; # represents comparison with the solvent treatment group);

[0058] FIG3 shows the TG test results of the physiological group, NASH group, and 60 nM group of the p-toluenesulfonate salt of compound 12;

[0059] Figure 4 shows the inflammatory factor detection results of the physiological group, NASH group, and 60 nM group of the p-toluenesulfonate salt of compound 12;

[0060] Figure 5 shows the results of H&E staining, picrosirius red staining, and COL1 and α-SMA immunofluorescence staining of each group;

[0061] FIG6 shows the ratio of the fat lesion area to the total stained area on the H&E stained slide;

[0062] FIG7 shows the ratio of the fibrosis area to the total area of ​​the stained region after Sirius red staining. DETAILED DESCRIPTION

[0063] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0064] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0065] Compound Preparation Examples

[0066] In some embodiments of the present invention, the compound of formula (I) includes the following structures. These compounds can be prepared according to the method disclosed in WO2021098691A1 or the following examples.

[0067] Example 1

[0068] Synthesis route:

[0069] first step

[0070] The trifluoroacetate salt of compound 11d (90 mg, 219 μmol) and compound 7a (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL). Cesium carbonate (250 mg, 766 μmol) and methanesulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium(II) (20 mg, 21.9 μmol) were then added. The reaction mixture was stirred at 105°C under nitrogen for 12 hours. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (10:1, dichloromethane / methanol, Rf = 0.3) to obtain the crude compound. A mixed solution of methanol and ethanol (4 / 1, 10 mL) was added to the crude product, stirred at 20°C for 16 hours, filtered, and the filter cake was washed with methanol (2 mL×2), washed with water (2 mL×2), and dried to obtain compound 12.

[0071] MS-ESI calculated value [M+H] + 514, measured value 514. 1 H NMR (400MHz, DMSO-d6) δ = 10.00 (s, 1H), 8.84 (s, 1H), 8.64 (s, 1H), 8.08 (s, 1H), 7.70 (s, 1H), 4.08 (t, J = 5.6Hz, 2H), 3.00 (t, J = 13.5 Hz, 2H), 2.91-2.78 (m, 6H), 2.47 (s, 3H), 2.46 (s, 3H), 2.31-2.18 (m, 2H), 2.04-1.92 (m, 2H), 1.91-1.78 (m, 2H), 1.76-1.62 (m, 2H).

[0072] Compound 12 (2 g, 3.89 μmol) was stirred and mixed with hexafluoroisopropanol (40 mL). To the solution was added p-toluenesulfonic acid monohydrate (814.89 mg, 4.28 mmol). The reaction solution was stirred at 40°C for 3 hours. The reaction solution was added dropwise to isopropanol (160 mL), filtered, and the filter cake was dried under vacuum to obtain the p-toluenesulfonate corresponding to compound 12. 1 H NMR (400MHz, DMSO-d6) δ = 10.02 (s, 1H), 8.86 (br s, 1H), 8.64 (s, 1H), 8.11 (s, 1H), 7.78 (s, 1H), 7.48 (d, J = 8.0Hz, 2H), 7.10 (d, J = 8.0Hz, 2H), 4.31 (br d, J=4.4Hz, 2H), 4.08-3.62(m, 6H), 2.89-2.78(m, 2H), 2.72-2.57(m, 2H), 2.51(br s, 3H), 2.46 (s, 3H), 2.28 (s, 3H), 1.95-1.97 (m, 2H), 1.89-1.79 (m, 2H), 1.73-1.64 (m, 2H). MS-ESI calculated value [M+H] + 514, measured value 514.

[0073] Biological activity test

[0074] Biological Example 1: Evaluation of the efficacy of the compounds of the present invention in bleomycin-induced pulmonary fibrosis

[0075] 1.1 Establishment of bleomycin-induced pulmonary fibrosis model

[0076] Experimental preparation: C57BL / 6 mice, 8-10 weeks old, female, weighing approximately 18-22 g. Bleomycin, 1% sodium pentobarbital, sterile saline, and insulin syringes were used.

[0077] Drug: p-toluenesulfonate of compound 12 (10 mg / ml). The specific drug preparation method is: weigh 282.87 mg of p-toluenesulfonate powder of compound 12, add 1.050 ml of DMSO solution (preheated to 45 ° C in advance), and ultrasonicate in a water bath until completely dissolved. Then add 19.95 ml of mixed solvent (Solutol 1.995 ml + hydroxypropyl-β-cyclodextrin 1.995 g + water 15.96 ml), and ultrasonicate again until completely dissolved to obtain a working solution with a concentration of 10 mg / ml. 1. After the mice began to adapt to the environment, the experiment was started. The experimental animals were divided into five groups: normal control group, modeling group (bleomycin group), low-dose group (25 mg / kg), high-dose group (50 mg / kg), and solvent treatment group. The modeling group was used to control the normal group mice to detect whether the modeling was successful, and the solvent treatment group was used to reflect the effect of the solvent on mouse pulmonary fibrosis and eliminate interference. There were 10 mice in each group. Their conditions were observed during the period and samples were collected for subsequent experiments.

[0078] 2. Half an hour before surgery, intraperitoneally inject the mouse with 1% sodium pentobarbital (solvent: normal saline) at 50 mg / kg. For each 18-22 g mouse, administer 90-110 μl. After the mouse is anesthetized, secure it and disinfect it.

[0079] 3. The model was established with a dose of 3 mg / kg bleomycin, 50 μl per mouse, injected through the mouse's oral trachea. During the injection, care was taken to maintain oral breathing of the mouse to ensure that the mouse inhaled liquid, thus establishing a bleomycin-induced mouse pulmonary fibrosis model.

[0080] 4. After injecting the liquid, place the mouse upright to evenly distribute the drug throughout the trachea, bronchi, and lungs. Maintain unobstructed breathing and wait for the mouse to recover naturally.

[0081] 5. This model has one endpoint, 28 days. At each time point, mice were sacrificed, samples were collected, and subsequent experiments were conducted. The day of model establishment was recorded as the first day.

[0082] 1.2 Pulmonary inflammation score

[0083] 1. Take fresh mouse tissue and immerse it in 4% paraformaldehyde solution for fixation. After 48 hours, place it in an embedding box and rinse it under running tap water overnight.

[0084] 2. Place in 75% alcohol for 30 minutes.

[0085] 3. Place in 85% alcohol for 30 minutes

[0086] 4. Place in 95% alcohol twice for a total of one hour

[0087] 5. Place in 100% alcohol three times for a total of one hour

[0088] 6. After taking it out, place it in xylene twice for a total of one hour

[0089] 7. Soak in paraffin wax twice for a total of one hour. After soaking, place the embedding box in new wax for embedding.

[0090] 8. After embedding, place the cooled wax block on ice. Once completely cooled, begin slicing on a microtome. First, use an old blade to trim the surface of the wax block, then use a new blade to slice the wax block to a thickness of approximately 3-5 μm. Then, perform bleaching at a temperature of 42 degrees Celsius. After the tissue is unfolded, use a glass slide to spread the slices at a temperature of 65 degrees Celsius. Once the paraffin on the slices is dried, remove the slices and proceed to the next step.

[0091] Slice hydration

[0092] 1. Place the slices on a slice rack and bake at about 65 degrees Celsius for 2 hours. Take them out after the paraffin on the slices melts and dries.

[0093] 2. Place the sections in xylene twice for a total of one hour.

[0094] 3. Transfer the sections to 100% alcohol, twice, for two minutes each.

[0095] 4. Transfer the slices to 95% alcohol, once for two minutes each time

[0096] 5. Transfer the slices to 85% alcohol, once for two minutes each time

[0097] 6. Transfer the slices to 75% alcohol, once for two minutes each time

[0098] 7. After the above steps are completed, transfer the slices to distilled water and rinse for two minutes.

[0099] HE staining

[0100] 1. After hydration, the sections were stained with Mayer's hematoxylin for one minute.

[0101] 2. Rinse in tap water twice, three minutes each time

[0102] 3. Place in 75% hydrochloric acid alcohol for about 2 minutes

[0103] 4. Rinse with tap water for one minute

[0104] 5. Re-stain with red for about one minute

[0105] 6. Rinse with tap water for one minute

[0106] 7. 85% alcohol, about two minutes

[0107] 8. 95% alcohol, about two minutes

[0108] 9. 100% alcohol, about two minutes

[0109] 10. After the above steps are completed, place it in xylene for 5-10 minutes.

[0110] 11. Seal the slides with neutral gum.

[0111] After scanning 5 μm thick paraffin sections of lung tissue stained with hematoxylin and eosin (HE), at least two independent scorers selected five representative fields and scored the degree of fibrosis according to the scoring criteria shown in Table 1. The average value was used as the Szapiel score for the sample. The results are shown in Figure 1.

[0112] As shown in Figure 2, we compared the Szapiel Score of lung fibrosis in each group of mice. The results showed that the Szapiel Score in the modeling group (bleomycin group) was significantly higher than that in the normal control group (p < 0.05), indicating successful modeling. The high-dose p-toluenesulfonate salt group (50 mg / kg) of compound 12 had a significantly lower Szapiel Score than the solvent-treated group (p < 0.05).

[0113] 1.3 Pulmonary fibrosis grading score

[0114] The Ashcroft Scoring System was invented by T Ashcroft in 1988 and has been widely used to grade the degree of fibrosis in animal and human tissues (e.g., Lancet Respir Med, 2020, PMID: 32061334 & Eur Respir J, 2009, PMID: 19460787).

[0115] Masson staining

[0116] 1. Place the hydrated sections in Masson's complex neutral stain for 5 minutes;

[0117] 2. Rinse in 0.2% acetic acid solution for about two minutes;

[0118] 3. Soak in 8% phosphotungstic acid for about 10 minutes;

[0119] 4. Rinse in 0.2% acetic acid solution for about two minutes;

[0120] 5. 0.2% aniline blue solution for 5 minutes;

[0121] 6. Rinse in 0.2% acetic acid solution for about two minutes, twice;

[0122] 7. 85% alcohol, about two minutes

[0123] 8. 95% alcohol, about two minutes

[0124] 9. 100% alcohol, about two minutes

[0125] 10. After the above steps are completed, place it in xylene for 5-10 minutes.

[0126] 11. Seal the slides with neutral gum.

[0127] [Corrected 08 / 11 / 2024 according to Rule 91] Masson-stained, 5-μm-thick paraffin sections of lung tissue were scanned. At least two independent scorers selected five representative fields (10x or 20x magnification) and scored the degree of fibrosis according to the scoring criteria shown in Table 1. The average score was used as the Ashcroft score for the sample. The scoring results are shown in Figure 2.

[0128] As shown in Figure 2, we compared the Ashcroft Score of lung fibrosis in each group of mice. The results showed that the Ashcroft Score in the modeling group (bleomycin group) was significantly higher than that in the normal control group (p < 0.05), indicating successful modeling. Compared with the solvent-treated group, the scores in the low-dose (25 mg / kg) and high-dose (50 mg / kg) p-toluenesulfonate groups of compound 12 were significantly lower, with statistically significant differences (p < 0.05).

[0129] Biological Example 2: Evaluation of the efficacy of the compounds of the present invention in NASH models and fibrosis

[0130] 2.1 In vitro 3D liver model modeling (reference: CN115386533A)

[0131] 2.1.1 D-2~D0 modeling:

[0132] Four types of human primary cells, including primary human hepatocytes (PHH), primary human liver sinusoidal endothelial cells (LSEC), primary human hepatic stellate cells (HSC), and primary human Kupffer cells (KC), were used for modeling, with a total of 3,000 cells in each model.

[0133] The cell connector NAC-Linker A was mixed with the liver parenchymal cells and incubated at room temperature for 30 minutes to fix NAC-Linker A on the cell membrane surface. At the same time, the cell connector NAC-Linker B was mixed with the mixture of the liver non-parenchymal cells and incubated at room temperature for 30 minutes to fix NAC-Linker B on the cell membrane surface. (2) After the incubation was completed, the liver parenchymal cells carrying NAC-Linker A and the liver non-parenchymal cells carrying NAC-Linker B were mixed evenly. A 20-30 μl droplet was made on the culture plate cover with a pipette. PBS solution and liver physiological culture medium (liver physiological culture medium (human) of Park Heng Bo Mai (Shanghai) Biopharmaceutical Co., Ltd., product number M0001) were added to the wells of the culture plate. The prepared culture plate cover was flipped and buckled onto the culture plate to make a hanging drop. The culture plate with the hanging drop was inverted and placed in a 37°C incubator for 12-24 hours. Under the action of the curvature of the lower surface of the hanging drop and gravity, the DNA complementary pairing in the NAC-Linker connected to form a 3D liver organ structure.

[0134] 2.1.2 Trial Grouping and Drug Treatment

[0135] After successful modeling on Day 0, the samples were divided into 5 groups according to different treatment methods, with 9 samples in each group. Among them, the physiological group continued to be treated with liver physiological culture medium; the NASH group was treated with NASH induction culture medium (NASH induction culture medium (human), product number MI001, produced by Park Heng Biomedicine (Shanghai) Co., Ltd.); the low-dose group of p-toluenesulfonate of compound 12 was treated with NASH induction culture medium and 10nM p-toluenesulfonate of compound 12; the medium-dose group of p-toluenesulfonate of compound 12 was treated with NASH induction culture medium and 30nM p-toluenesulfonate of compound 12; and the high-dose group of p-toluenesulfonate of compound 12 was treated with NASH induction culture medium and 60nM p-toluenesulfonate of compound 12.

[0136] Drug administration: Starting from Day 0, the low-dose group, medium-dose group, and high-dose group of the p-toluenesulfonate salt of compound 12 were treated with drugs. The culture conditions remained unchanged, and the whole medium was replaced every 2 days with new culture medium of the corresponding group and the same concentration of drugs.

[0137] 2.1.3 Quality Inspection

[0138] Quality control tests were performed on the models on Day 0, Day 5, and Day 9. Day 0: HE staining was performed to confirm the absence of cell necrosis and pathological changes upon model construction. Day 5: HE and Sirius Red (SR) staining were performed to determine the degree of fibrosis in the model on Day 5. Day 9: HE and SR staining were performed to determine the extent of fatty lesions in the NASH model on Day 9.

[0139] 2.2 Test and results

[0140] 2.2.1 Quality control test results

[0141] Day 0, HE staining results showed that the cells were in normal condition, with no obvious necrotic areas, and the model had no pathological changes, so subsequent experiments could be carried out.

[0142] On Day 5, HE staining results showed that the cells in the physiological group were normal, while ballooning changes were observed in the NASH group, showing a trend towards fatty lesions; SR staining results showed that the NASH group showed a state of fiber accumulation.

[0143] On Day 9, HE staining results showed that the cells were in normal condition. In the NASH group, obvious ballooning and fatty lesions were observed, and subsequent experiments were possible. SR staining results showed that the fibrotic lesions in the model were expressed normally.

[0144] 2.2.2 TG detection

[0145] On day 10, TG was measured by grinding the liver microspheres in cell lysate using a grinding rod, centrifuging, and collecting the supernatant for enzymatic analysis. As shown in Figure 3, the TG level in the 60 nM p-toluenesulfonic acid salt group of compound 12 was significantly higher than that in the NASH group (p < 0.05).

[0146] 2.2.3 Inflammatory factor detection (IL-6)

[0147] On day 6, the culture supernatant was collected and assayed using a microplate reader according to the instructions of the IL-6 assay kit. The results are shown in Figure 4. The inflammatory factor levels in the 60 nM p-toluenesulfonate salt group of Compound 12 were improved compared to those in the NASH group, and the differences were significant (p < 0.05).

[0148] 2.2.4 H&E staining

[0149] The liver microspheres were paraffin-sectioned and stained with H&E. The staining results are shown in Figure 5 .

[0150] The fat lesion area of ​​the H&E-stained slides was counted using Image J software, and the ratio of the fat lesion area to the total area of ​​each slide was calculated (see Figure 6 for the results).

[0151] H&E staining revealed that the p-toluenesulfonate salt of compound 12 at a 10 nM dose improved hepatic steatosis and ballooning compared to the NASH group. In the 30 nM and 60 nM dose groups, H&E staining revealed significant histological improvement, demonstrated by a significant decrease in hepatic steatosis and the number of ballooning hepatocytes. These results suggest that the p-toluenesulfonate salt of compound 12 has a significant therapeutic effect in inhibiting the pathological progression of NASH.

[0152] 2.2.5 Sirius red staining

[0153] The liver microspheres were paraffin sectioned, stained with picrosirius red, sealed, and photographed. The results are shown in FIG5 .

[0154] The fibrosis area of ​​the slides after Sirius red staining was counted using Image J software, and the ratio of the fibrosis area to the total area of ​​each slide was calculated ( Figure 7 ).

[0155] Sirius red staining was used to assess the drug's effect on liver fibrosis. Results showed that the degree of liver fibrosis in the 10 nM p-toluenesulfonate group of Compound 12 showed an improved trend compared to the physiological group. The staining results for the 30 nM and 60 nM p-toluenesulfonate groups of Compound 12 showed significant differences in the fibrosis area within the liver microspheres compared to the NASH group, demonstrating that both groups exhibited significant therapeutic effects on NASH-induced liver fibrosis, with higher doses of Compound 12 p-toluenesulfonate associated with greater efficacy.

[0156] 2.2.6 Immunofluorescence staining

[0157] The liver microspheres were paraffin sectioned, immunofluorescence stained, sealed and photographed under a fluorescence microscope. The results are shown in Figure 5.

[0158] The results of COL1 and α-SMA immunofluorescence staining showed that the p-toluenesulfonate salt of compound 12 had a therapeutic effect on liver fibrosis in a dose-dependent manner.

[0159] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating fibrosis or diseases related thereto, in, R1 is H, F, Cl, Br or C 1-3 alkyl; R2 and R3 are each independently H or C 1-3 Alkyl, wherein the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br or I; or R2 and R3 are linked together with the carbon atom to which they are attached to form a cyclopentyl, cyclohexyl or piperidinyl group, wherein the cyclopentyl, cyclohexyl and piperidinyl groups are optionally substituted by 1, 2 or 3 R a replaced by; Each R a are independently H, F, Cl, Br or C 1-3 alkyl; R4 is H, F, Cl, Br or C 1-3 alkyl; R5 and R6 are each independently H, F, Cl, Br, I or C 1-3 alkyl; R7 is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted by 1, 2 or 3 R b replaced by; Each R b are independently H, F, Cl, Br, I or C 1-3 Alkyl, wherein the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from F, Cl, Br or I; n is 1 or 2.

2. The use according to claim 1, characterized in that: The R a are independently H, F, Cl, Br, -CH3 or -CH2CH3; Preferably, R2 and R3 are each independently H, -CH3 or -CH2CH3; Preferably, the R2 and R3 are linked together with the carbon atoms to which they are attached to form Preferably, the R2 and R3 are linked together with the carbon atoms to which they are attached to form Preferably, the for Preferably, the for Preferably, the compound of formula (I) has a structure represented by any one of formulas (I-1) to (I-4): Among them, R1, R4, R5, R6, R7, R a and n is as defined above or in claim 1; Preferably, each R b Independently H, F, Cl, Br, I, Preferably, said R7 is It is stated Optional 1 or 2 R b replaced by; Preferably, said R7 is Preferably, said R7 is Preferably, said R4 is H or -CH3; Preferably, the compound of formula (I) has a structure represented by any one of formulas (I-5) to (I-9): Among them, R1, R5, R6, R a and R b As defined above or in claim 1; Preferably, R1 is H, F, Cl or Preferably, R5 and R6 are each independently H or Preferably, R1 is C 1-3 Alkyl groups, such as methyl; Preferably, R2 and R3 are linked together with the carbon atoms to which they are attached to form Preferably, R4 is C 1-3 Alkyl groups, such as methyl; Preferably, R5 and R6 are each independently H or methyl; n is 2; Preferably, for Preferably, R7 is substituted by 1, 2 or 3 H, F, Cl or methyl. For example Preferably, the compound represented by formula (I) is selected from the following structures:

3. The use according to claim 1 or 2, characterized in that: The pharmaceutically acceptable salts are salts of the compound represented by formula (I) and inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and the like; and also include salts of amino acids (such as arginine, etc.) and salts of organic acids such as glucuronic acid; preferably, the hydrochloride and p-toluenesulfonate of the compound represented by formula (I).

4. The use according to any one of claims 1 to 3, characterized in that: The fibrosis or its related diseases or conditions are selected from pulmonary fibrosis, renal fibrosis, myelofibrosis, cystic fibrosis, oral mucosal fibrosis, liver fibrosis, biliary fibrosis, myocardial fibrosis, skin fibrosis, eye fibrosis, and pancreatic fibrosis.

5. The use according to any one of claims 1 to 3, characterized in that: The fibrosis or its related diseases or disorders are selected from inflammatory diseases, for example, selected from pneumonia, hepatitis, nephritis, myocarditis, pancreatitis.

6. The use according to any one of claims 1 to 3, characterized in that: The fibrosis or its related disease or disorder is selected from liver related diseases, such as hepatitis, cirrhosis, liver damage or liver failure.

7. The use according to any one of claims 1 to 3, characterized in that: Selected from non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

8. The use according to any one of claims 1 to 3, characterized in that: The fibrosis or its associated disease or condition is selected from progressive fibrosing interstitial lung disease (PF-ILD), particularly a disease with pulmonary fibrosis manifestations, such as idiopathic pulmonary fibrosis (IPF), systemic sclerosis-related ILD (SSc-ILD), connective tissue disease-related ILD (CTD-ILD), rheumatoid arthritis-related ILD (RA-ILD), chronic fibrosing hypersensitivity pneumonitis (HP), idiopathic nonspecific interstitial pneumonia (iNSIP), unclassifiable idiopathic interstitial pneumonia (IIP), environmental / occupational fibrosing lung disease, idiopathic pneumonia (IPAF) and sarcoidosis with autoimmune characteristics; or the fibrosis or its associated disease or condition is selected from muscular dystrophy, fibromatosis and myelofibrosis, preferably selected from Duchenne muscular dystrophy, Dupuytren's contracture and primary myelofibrosis (PMF).

9. A method for preventing and / or treating fibrosis or a disease or condition related thereto, the method comprising administering to a patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

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