Use of pyrrolotriazine compounds for the preparation of a medicament for the prevention and / or treatment of fibrosis or a related disease thereof

Abstract

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

Description

[0001] This application claims priority to the prior application filed on November 3, 2023, with the China National Intellectual Property Office, Patent Application No. 202311469656.3, entitled "Application of Pyrrolotriazine Compounds in the Preparation of Drugs for Preventing and / or Treating Fibrosis or Related Diseases". The entire contents of the said application are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and specifically relates to the application of pyrrolotriazine compounds in the preparation of drugs for preventing and / or treating fibrosis or related diseases. BACKGROUND

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

[0004] In response to tissue injury, myofibroblasts derived from a variety of sources, including resident fibroblasts, mesenchymal cells, circulating fibroblasts, and transdifferentiation of other cell types, can initiate a wound healing response by remodeling the extracellular environment to restore tissue integrity and promote parenchymal cell replacement. Typically, this pro-fibrotic program is turned off when the tissue heals. However, persistent injury and damage can lead to a dysregulation of this process, resulting in excessive deposition of ECM proteins in a pathological manner, accompanied by an upregulation of myofibroblast activity in a chronic inflammatory environment infiltrated by macrophages and immune cells. In this cellular environment, cytokines and growth factors are abundantly released, ultimately leading to the 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 progresses, the structure of the stroma changes and becomes hard.

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

[0006] Pulmonary fibrosis can be roughly classified into idiopathic, primary, immunological, drug-induced, and physicochemical factors according to etiology. Idiopathic pulmonary fibrosis is the highest proportion among them.

[0007] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown etiology that occurs primarily in the elderly population, characterized by dyspnea and progressive worsening of pulmonary function. The abnormal accumulation of fibrotic tissue in the lung parenchyma seriously affects the respiratory function of the human body, manifested as dry cough, progressive dyspnea (feeling that the air is not enough), and with the aggravation of the disease and lung damage, the respiratory function of the patient is continuously deteriorated.

[0008] The etiology and mechanism of IPF are still unclear, and the treatment goal is to relieve symptoms, improve quality of life, slow down or stop disease progression, and improve survival rate. Prednisone, azathioprine and N-acetylcysteine (NAC) have been used to treat related symptoms of IPF, but generally do not significantly increase life expectancy.

[0009] Non-alcoholic steatohepatitis (NASH) is a disease mainly characterized by inflammation and fibrosis, which can develop into cirrhosis, liver failure, etc. Today, NASH is rapidly becoming the primary reason for liver transplantation.

[0010] WO2021098691A1 discloses a class of pyrrolotriazine compounds as MNK inhibitors. Further research of the present invention has found that this class of compounds is expected to be applied to the treatment of fibrotic diseases. SUMMARY

[0011] The present application provides a use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating fibrosis or a related disease or disorder thereof,

[0012]

[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 by 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 piperidyl, wherein the cyclopentyl, cyclohexyl and piperidyl are optionally substituted by 1, 2 or 3 R a ;

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

[0017] R4 is H, F, Cl, Br or C1-3 alkyl;

[0018] R5and R6are each independently H, F, Cl, Br, I, or C 1-3 alkyl;

[0019] R7is pyrrolidinyl, wherein the pyrrolidinyl is optionally substituted with 1, 2, or 3 R b substituted;

[0020] each R b is 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 application, each R a is independently H, F, Cl, Br, -CH3, or -CH2CH3, and other variables are as defined in the application.

[0023] In some embodiments of the application, R2and R3are each independently H, -CH3, or -CH2CH3, and other variables are as defined in the application.

[0024] In some embodiments of the application, R2and R3are taken together with the carbon atom to which they are attached to form R a and other variables are as defined in the application.

[0025] In some embodiments of the application, R2and R3are taken together with the carbon atom to which they are attached to form and other variables are as defined in the application.

[0026] In some embodiments of the application, the structural unit is R1, R a and other variables are as defined in the application.

[0027] In some embodiments of the application, the structural unit is and other variables are as defined in the application.

[0028] In some embodiments of the application, R1is C 1-3 alkyl, such as methyl.

[0029] In some embodiments of the present application, R2and R3, together with the carbon atom to which they are attached, join to form In some embodiments of the present application, R4is C 1-3 alkyl, such as methyl.

[0030] In some embodiments of the present application, R5and R6are each independently H or methyl; n is 2;

[0031] In some embodiments of the present application, is

[0032] In some embodiments of the present application, R7is such as

[0033] In some embodiments of the present application, the above-mentioned compound has a structure represented by any one of formulae (I-1) to (I-4):

[0034]

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

[0036] In some embodiments of the present application, each of the above-mentioned R b is independently H, F, Cl, Br, I, other variables are as defined in the present application.

[0037] In some embodiments of the present application, the above-mentioned R7is wherein the is optionally substituted with 1 or 2 R b is H, F, Cl, Br, I, b and other variables are as defined in the present application.

[0038] In some embodiments of the present application, the above-mentioned R7is R b and other variables are as defined in the present application.

[0039] In some embodiments of the present application, the above-mentioned R7is other variables are as defined in the present application.

[0040] In some embodiments of the present application, the above-mentioned R4is H or -CH3, and other variables are as defined in the present application.

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

[0042]

[0043]

[0044] wherein R1, R5, R6, R a and R b as defined herein.

[0045] In some embodiments of the present application, R1 is H, F, Cl or other variables are as defined herein.

[0046] In some embodiments of the present application, R5 and R6 are each independently H or other variables are as defined herein. Some embodiments of the present application are also combinations of any of the above variables.

[0047] According to embodiments of the present application, the compound of formula (I) is selected from the following structures:

[0048]

[0049]

[0050] According to embodiments of the present application, the pharmaceutically acceptable salt is a salt of the compound of formula (I) with an inorganic acid, including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, bisulfuric acid, hydroiodic acid, phosphorous acid, and the like; and an organic acid salt, including, for example, 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, methanesulfonic acid, and the like; also including salts of amino acids (e.g., arginine and the like), and salts of organic acids such as glucuronic acid; preferably a hydrochloride salt or a p-toluenesulfonic acid salt of the compound of formula (I).

[0051] According to embodiments of the present application, the fibrosis or a disease or condition associated therewith is selected from the group consisting of pulmonary fibrosis, renal fibrosis, bone marrow fibrosis, cystic fibrosis, oral mucosal fibrosis, liver fibrosis, biliary fibrosis, myocardial fibrosis, skin fibrosis, ocular fibrosis, pancreatic fibrosis.

[0052] In some embodiments, the fibrosis or a disease or condition associated therewith is selected from the group consisting of inflammatory diseases, for example, selected from the group consisting of lung inflammation, liver inflammation, kidney inflammation, myocardial inflammation, pancreas inflammation.

[0053] In some embodiments, the fibrosis or a disease or condition associated therewith is selected from the group consisting of liver-related diseases, for example, liver inflammation, liver cirrhosis, liver injury, or liver failure.

[0054] In some embodiments, the fibrosis or its related disease or disorder is selected from non-alcoholic fatty liver disease or non-alcoholic steatohepatitis (NASH).

[0055] In some embodiments, the fibrosis or its related disease or disorder is selected from progressive fibrotic interstitial lung disease (PF-ILD), in particular a disease with a manifestation 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 fibrotic lung disease, idiopathic pneumonia with autoimmune features (IPAF), and sarcoidosis.

[0056] In some embodiments, the fibrosis or its related disease or disorder is selected from muscular dystrophy, fibromatosis and myelofibrosis, preferably from Duchenne muscular dystrophy, Dupuytren's contracture and primary myelofibrosis (PMF).

[0057] The present application also provides a method for preventing and / or treating fibrosis or its related disease or disorder, comprising administering to a patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0058] In preparing the medicament of the present application, the active compound is combined or formulated with an appropriate pharmaceutically acceptable carrier, diluent or excipient to prepare, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres and aerosols. The mode of administration can include oral, intraperitoneal, transdermal, subcutaneous, intravenous or intramuscular injection, inhalation, topical, intralesional, infusion; liposome-mediated delivery; local, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or aural delivery, or any other method known in the art, to achieve prevention and / or treatment of fibrosis or its related disease or disorder. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 Schematic diagram of HE, Masson staining of lung tissue;

[0060] Figure 2 Schematic diagram of fibrosis degree score of mouse lung tissue (* represents compared with normal control group; # represents compared with solvent treatment group);

[0061] Figure 3 Schematic diagram of TG detection results of physiological group, NASH group, 60 nM group of p-toluenesulfonic acid salt of compound 12;

[0062] Figure 4 Results of inflammatory factor detection of each group;

[0063] Figure 5 Results of H&E staining, Sirius red staining, COL1 and a-SMA immunofluorescence staining of each group;

[0064] Figure 6 Ratio of fatty lesion area to total staining area of each group after H&E staining;

[0065] Figure 7 Ratio of fibrosis area to total staining area of each group after Sirius red staining. DETAILED DESCRIPTION

[0066] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0067] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0068] Compound preparation example

[0069] In some aspects of the present application, the compound of formula (I) includes the following structure. These compounds can be prepared according to the method disclosed in WO2021098691A1 or the following examples.

[0070]

[0071]

[0072] Example 1

[0073]

[0074] Synthetic route:

[0075]

[0076] First step

[0077] Compound 11d trifluoroacetate salt (90 mg, 219 μmol), compound 7a (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL), then cesium carbonate (250 mg, 766 μmol) and methane sulfonic acid (2-dicyclohexylphosphino)-3,6-dimethoxy-2,4,6-triisopropyl-1,1- biphenyl (20 mg, 21.9 μmol) were added, and the reaction solution was stirred at 105 °C for 12 hours under nitrogen protection. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (10:1, dichloromethane / methanol, Rf = 0.3). The crude product was added to a mixture of methanol and ethanol (4 / 1, 10 mL), stirred at 20 °C for 16 hours, filtered, the filter cake was washed with methanol (2 mL x 2), washed with water (2 mL x 2), and dried to obtain compound 12.

[0078] MS-ESI calculated value [M+H] + 514, found 514. 1 H NMR (400 MHz, 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 = 5.6 Hz, 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).

[0079] Compound 12 (2 g, 3.89 μmol) was stirred and mixed with hexafluoroisopropanol (40 mL), and p-toluenesulfonic acid monohydrate (814.89 mg, 4.28 mmol) was added to the solution, and 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-toluenesulfonic acid salt corresponding to compound 12. 1H NMR (400 MHz, 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 = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 4.31 (br d, J = 4.4 Hz, 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 [M+H] + 514, found 514.

[0080] Biological activity test

[0081] Biological Example 1: Evaluation of the therapeutic effect of the compound of the present application on bleomycin-induced pulmonary fibrosis

[0082] 1.1 Establishment of a bleomycin-induced pulmonary fibrosis model

[0083] Experimental preparation: C57BL / 6 mice, 8-10 weeks old, female, body weight about 18-22 g. Bleomycin. 1% sodium pentobarbital, sterile saline, insulin needle.

[0084] Drug: p-toluenesulfonate salt of compound 12 (10 mg / ml). The specific drug preparation method is as follows: weigh 282.87 mg of p-toluenesulfonate salt of compound 12 powder, add 1.050 ml of DMSO solution (preheated to 45°C), ultrasonic 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), ultrasonic again until completely dissolved, to get a working solution with a concentration of 10 mg / ml. 1, after the mice start to adapt to the environment, start the experiment, divide the experimental animals into five groups, normal control group, modeling group (bleomycin group), small dose of drug administration group (25 mg / kg), large dose of drug administration group (50 mg / kg), solvent treatment group, the modeling group is used to detect whether the modeling is successful in the normal group of mice, and the solvent treatment group is used to reflect the influence of the solvent on the pulmonary fibrosis of the mice, to exclude interference. Each group of 10, during which the state is observed and the material is taken for subsequent experiments.

[0085] 2, half an hour before the operation, the mice were given intraperitoneal injection of 1% sodium pentobarbital (solvent: saline), 50 mg / kg, 18 g-22 g mice each 90 μl-110 μl. After the mice were anesthetized, the mice were fixed and disinfected.

[0086] 3. Bleomycin modeling at 3 mg / kg dose, 50 μl per mouse, intratracheal injection, pay attention to maintain the mouse mouth breathing during the process, to ensure that the mouse inhale the liquid, to establish the bleomycin-induced pulmonary fibrosis model in mice.

[0087] 4. After the injection of liquid, the mouse is straight, and the drug is evenly distributed in the trachea, bronchus and whole lung. Keep the airway unobstructed and wait for the mouse to recover naturally.

[0088] 5. The model has a total of 1 endpoint, 28 days. According to the time point, the mice are sacrificed, the materials are taken, and the subsequent experiments are carried out. The day of modeling is recorded as the first day.

[0089] 1.2 Lung inflammation score

[0090] 1. Take fresh mouse tissue and soak it in 4% paraformaldehyde solution for fixation. After 48 hours, place it in an embedding box and rinse it under running tap water overnight.

[0091] 2. Place in 75% alcohol for 30 minutes.

[0092] 3. Place in 85% alcohol for 30 minutes

[0093] 4. Place in 95% alcohol twice for a total of one hour

[0094] 5. Place in 100% alcohol three times for a total of one hour

[0095] 6. After taking out, place in xylene twice for a total of one hour

[0096] 7. Paraffin immersion, twice for a total of one hour, after immersion, place the embedding box in new wax for embedding.

[0097] 8. After embedding, place the cooled wax block on ice, and after complete cooling, start slicing on the microtome. First use an old blade to trim the surface of the wax block, then use a new blade to slice the wax block, with a thickness of about 3-5 μm. Then float the slice, control the temperature at 42 degrees Celsius during floating, and after the tissue is unfolded, select a glass slide for spreading, control the spreading temperature at 65 degrees Celsius, and after the paraffin on the slice is baked dry, take out the slice for the next step.

[0098] Slice hydration

[0099] 1. Place the slice on the slice holder and bake for 2 hours at about 65 degrees Celsius. After the paraffin on the slice is melted and baked, take it out.

[0100] 2. Place the slice in xylene twice for a total of one hour.

[0101] 3. Transfer the sections to 100% alcohol, twice, two minutes each.

[0102] 4. Transfer the sections to 95% alcohol, once, two minutes each

[0103] 5. Transfer the sections to 85% alcohol, once, two minutes each

[0104] 6. Transfer the sections to 75% alcohol, once, two minutes each

[0105] 7. After the above steps, transfer the sections to distilled water for rinsing, two minutes.

[0106] HE Staining

[0107] 1. After the sections are hydrated, stain with Mayer's hematoxylin for one minute

[0108] 2. Rinse in tap water, twice, three minutes each

[0109] 3. Place in 75% hydrochloric acid alcohol for about two minutes

[0110] 4. Rinse in tap water for one minute

[0111] 5. Counterstain with eosin for about one minute

[0112] 6. Rinse in tap water for one minute

[0113] 7. 85% alcohol for about two minutes

[0114] 8. 95% alcohol for about two minutes

[0115] 9. 100% alcohol for about two minutes

[0116] 10. After the above steps, place in xylene for 5-10 minutes.

[0117] 11. Mount with neutral resin.

[0118] After scanning the HE-stained 5 μm thick lung tissue paraffin sections, at least two independent scorers selected five representative fields, and scored the degree of fibrosis according to the scoring criteria shown in Table 1, and finally took the average value as the Szapiel Score of the sample. The results are shown in Figure 1 .

[0119] As Figure 2As shown, we compared the degree of lung tissue fibrosis Szapiel Score of each group of mice, and found that compared with the normal control group, the score of the modeling group (bleomycin group) was significantly increased, with significant difference (p value <0.05), indicating that the modeling was successful. Compared with the solvent treatment group, the score of the large dose group (50mg / kg) of the p-toluenesulfonic acid salt of compound 12 was significantly lower, and the difference was statistically significant (p value <0.05).

[0120]

[0121] 1.3 Lung fibrosis grading score

[0122] Ashcroft Scoring System was invented by T Ashcroft in 1988 and is widely used for grading the degree of fibrosis of tissues in animals and humans (e.g., Lancet Respir Med, 2020, PMID: 32061334 & Eur Respir J, 2009, PMID: 19460787).

[0123] Masson staining

[0124] 1. The section after hydration is placed in the Masson composite neutral dyeing solution for 5 minutes.

[0125] 2. Rinse in 0.2% acetic acid aqueous solution for about two minutes.

[0126] 3. Soak in 8% phosphotungstic acid for about 10 minutes.

[0127] 4. Rinse in 0.2% acetic acid aqueous solution for about two minutes.

[0128] 5. 0.2% aniline blue solution for 5 minutes.

[0129] 6. Rinse in 0.2% acetic acid aqueous solution for about two minutes, twice.

[0130] 7. 85% alcohol, about two minutes

[0131] 8. 95% alcohol, about two minutes

[0132] 9. 100% alcohol, about two minutes

[0133] 10. After the above steps are completed, place in xylene for 5-10 minutes.

[0134] 11. Neutral gum is used for mounting.

[0135] After scanning the 5 μm thickness lung tissue paraffin sections after Masson staining, at least 2 independent scorers, 5 representative fields (10 or 20 times magnification) were selected, and the degree of fibrosis was scored according to the scoring criteria shown in Table 1, and the average value was taken as the Ashcroft Score of the sample. The scoring results are shown in Figure G.

[0136] As shown in Figure 2 , we compared the Ashcroft Score of the degree of fibrosis of the lung tissues of the mice in each group, and found that compared with the normal control group, the score of the modeling group (bleomycin group) was significantly increased, with a significant difference (p value < 0.05), indicating that the modeling was successful. Compared with the solvent treatment group, the scores of the small dose group (25 mg / kg) and the large dose group (50 mg / kg) of the p-toluenesulfonic acid salt of compound 12 were significantly lower, and the differences were statistically significant (p value < 0.05).

[0137]

[0138]

[0139] Biological Example 2: Efficacy evaluation of the compound of the present application in NASH model and fibrosis

[0140] 2.1 Modeling of in vitro 3D liver model (reference: CN115386533A)

[0141] 2.1.1 D-2~D0 modeling:

[0142] Four kinds of human primary cells were used for modeling, including human primary hepatocytes (PHH), human primary liver sinus endothelial cells (LSEC), human primary hepatic stellate cells (HSC), and human primary Kupffer cells (KC), and the total number of cells in each model was 3000.

[0143] (2) Incubation is completed, mix the liver parenchymal cells carrying NAC-Linker A and the intrahepatic non-parenchymal cells with NAC-Linker B uniformly, use a pipette to make 20-30 μl droplets on the culture plate cover, add PBS solution and liver physiological culture medium (Peking Hengbo (Shanghai) Biomedicine Co., Ltd. Liver Physiological Culture Medium (Human), Item No. M0001) to the culture plate hole, turn over the prepared culture plate cover to make a hanging droplet on the culture plate, and place the inverted culture plate with the hanging droplet in a 37°C incubator for 12-24 hours. Under the action of the surface curvature and gravity of the hanging droplet, the DNA in the NAC-Linker is complementary to each other and connected to form a 3D liver organ structure.

[0144] 2.1.2 Test grouping, drug treatment

[0145] After successful modeling on Day 0, according to different treatment methods, 5 groups were divided, each group with 9 samples. Among them, the physiological group continues to use the liver physiological culture medium for treatment; the NASH group uses the NASH induction culture medium (Peking Hengbo (Shanghai) Biomedicine Co., Ltd. NASH Induction Culture Medium (Human), Item No. MI001) for treatment; the small dose group of p-toluenesulfonic acid salt of compound 12 uses the NASH induction culture medium and 10 nM of p-toluenesulfonic acid salt of compound 12 for treatment; the medium dose group of p-toluenesulfonic acid salt of compound 12 uses the NASH induction culture medium and 30 nM of p-toluenesulfonic acid salt of compound 12 for treatment; the large dose group of p-toluenesulfonic acid salt of compound 12 uses the NASH induction culture medium and 60 nM of p-toluenesulfonic acid salt of compound 12 for treatment.

[0146] Drug administration: starting from Day 0, the small dose group, the medium dose group, and the large dose group of p-toluenesulfonic acid salt of compound 12 were administered, and the culture conditions remained unchanged. Full liquid change was performed every 2 days, and new culture medium corresponding to the group and the same concentration of drug were replaced.

[0147] 2.1.3 Quality inspection

[0148] The model was detected for quality control at Day 0, Day 5 and Day 9, respectively. Day 0: HE staining, to determine that when the model is completed, the cells are not necrotic, and the model has no pathological changes. Day 5: HE staining and Sirius red (SR) staining: to determine the degree of fibrosis of the model at Day 5. Day 9: HE staining and SR staining: to determine the degree of fatty lesion of the NASH model at Day 9.

[0149] 2.2 Test detection and results

[0150] 2.2.1 Quality control test results

[0151] Day 0, the HE staining result shows that the cell state is normal, there is no obvious necrotic area, the model has no pathological changes, and the subsequent experiment can be carried out.

[0152] Day 5, the HE staining result shows that the cell state of the physiological group is normal, and the NASH group can see ballooning degeneration, showing a tendency of fatty lesion; the SR staining result shows that the NASH group is in a state of fibrosis accumulation.

[0153] Day 9, the HE staining result shows that the cell state is normal, and the NASH group can see obvious ballooning degeneration, and the fatty lesion is significant, and the subsequent experiment can be carried out; the SR staining result shows that the model fibrosis lesion is normally expressed.

[0154] 2.2.2 TG detection

[0155] TG detection was performed on the 10th day, after grinding the liver microspheres in the cell lysis solution using a grinding rod, centrifugation, and taking the supernatant for enzymatic determination. As shown in Figure 3 , the TG level of the 60nM group of the p-toluenesulfonic acid salt of compound 12 has a significant (p<0.05) difference from the NASH group.

[0156] 2.2.3 Inflammatory factor detection (IL-6)

[0157] On the 6th day, the culture medium supernatant was collected, and the culture medium supernatant was detected according to the use instruction of the IL-6 detection kit, combined with the enzyme marker, and the result is shown in Figure 4 . The inflammatory factor level of the 60nM group of the p-toluenesulfonic acid salt of compound 12 is improved compared with the NASH group, and the data of the 60nM group of the p-toluenesulfonic acid salt of compound 12 has a significant difference (p<0.05) from the NASH group.

[0158] 2.2.4 H&E staining

[0159] The liver microspheres were paraffin sectioned, and H&E staining was performed, and the staining result is shown in Figure 5 .

[0160] The fat lesion area of the H&E stained slice was counted by Image J software, and the ratio of the fat lesion area to the total area of each slice was calculated (results shown in Figure 6 ).

[0161] The H&E staining results showed that the 10 nM dose of p-toluenesulfonate of compound 12 improved the fat degeneration and ballooning degeneration of hepatocytes in the drug group compared with the NASH group. At the 30 nM and 60 nM doses of the drug group, the H&E staining results showed significant histological improvement, specifically, the fat degeneration in hepatocytes was significantly reduced and the number of ballooning degeneration hepatocytes was significantly reduced. This result indicated that the p-toluenesulfonate of compound 12 had a significant therapeutic effect on inhibiting the pathological progression of NASH.

[0162] 2.2.5 Sirius red staining

[0163] The liver microspheres were paraffin-embedded, stained with Sirius red, mounted, and photographed, and the results are shown in Figure 5 .

[0164] The fibrosis area of the Sirius red stained slice was counted by Image J software, and the ratio of the fibrosis area to the total area of each slice was calculated ( Figure 7 ).

[0165] Sirius red staining was used to evaluate the effect of the drug on liver fibrosis, and the results showed that the 10 nM dose of p-toluenesulfonate of compound 12 had a tendency to improve liver fibrosis compared with the physiological group. In the staining results of the 30 nM and 60 nM doses of p-toluenesulfonate of compound 12, the fibrosis area in the liver microspheres of each group showed significant differences compared with the NASH group, indicating that the aforementioned two groups showed significant therapeutic effects on the liver fibrosis caused by NASH, and the greater the dose of p-toluenesulfonate of compound 12, the better the therapeutic effect.

[0166] 2.2.6 Immunofluorescence staining

[0167] The liver microspheres were paraffin-embedded, immunofluorescence stained, mounted, and photographed under a fluorescence microscope, and the results are shown in Figure 5 .

[0168] The immunofluorescence staining results of COL1 and a-SMA showed that the p-toluenesulfonate of compound 12 had a therapeutic effect on liver fibrosis, and the effect was dose-dependent.

[0169] The above has exemplarily described the embodiments of the technical scheme of the present application. It should be understood that the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a compound represented by the following formula or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prevention and / or treatment of fibrosis or a disease associated therewith, wherein the fibrosis or the disease associated therewith is selected from the group consisting of liver fibrosis, non-alcoholic steatohepatitis, and idiopathic pulmonary fibrosis.

2. Use according to claim 1, characterized in that, the pharmaceutically acceptable salt is a salt of the compound with an inorganic acid or an organic acid.

3. Use according to claim 2, characterized in that, the inorganic acid is selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonic acid, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, and phosphorous acid; and the organic acid is selected from the group consisting of acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzene sulfonic acid, p-toluene sulfonic acid, citric acid, tartaric acid, methanesulfonic acid, amino acid, and glucuronic acid.

4. Use according to claim 2, characterized in that, the organic acid is arginine.

5. Use according to claim 2, characterized in that, the pharmaceutically acceptable salt is hydrochloride or p-toluenesulfonate of the compound.

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