Application of pyrrolotriazine compound in preparation of medicine for preventing and / or treating fibrosis or related diseases thereof

Abstract

The invention relates to application of a compound shown in a formula (I) or pharmaceutically acceptable salt thereof in preparation of medicines for preventing and / or treating fibrosis or related diseases thereof. # imgabs0 #

Description

[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 and invention name “Application of pyrrolotriazine compounds in the preparation of drugs for the prevention and / or treatment of fibrosis or its related diseases”. The entire text of the application is incorporated into this application by reference. Technical Field

[0002] The present invention belongs to the field of medicine, and specifically 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 transdifferentiation of resident fibroblasts, mesenchymal cells, circulating fibroblasts, and other cell types) can initiate a wound healing response by remodeling the extracellular environment to restore tissue integrity and promote replacement of parenchymal cells. Normally, this profibrotic program is shut down as tissues heal. However, continued injury and damage can lead to dysregulation of this process, resulting in pathological excessive deposition of ECM proteins, accompanied by upregulation of myofibroblast activity, creating a chronic inflammatory environment with infiltration of 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 functions to further enhance myofibroblast differentiation and the production and secretion of ECM proteins (including collagen, laminin, and fibronectin). As excessive ECM deposition proceeds, the structure of the matrix changes and becomes stiff.

[0005] Fibrosis is a tumor-like lesion between benign and malignant. Fibrosis 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 and cause cancer, seriously endangering life and health.

[0006] Pulmonary fibrosis can be roughly divided into idiopathic, primary, immune, drug-induced, physical and chemical factors, etc. Idiopathic pulmonary fibrosis accounts for the highest proportion among them.

[0007] Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown etiology that occurs mainly in the elderly and is characterized by dyspnea and progressive deterioration of lung function. The abnormal accumulation of fibrotic tissue in the lung parenchyma seriously affects the respiratory function of the human body, manifesting as dry cough and progressive dyspnea (feeling that there is not enough air), and as the disease and lung damage worsen, the patient's respiratory function continues to deteriorate.

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

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

[0010] WO2021098691A1 discloses a class of pyrrolotriazine compounds as MNK inhibitors. The present invention further studies and finds that such compounds are expected to be used in the treatment of fibrotic diseases. Summary of the invention

[0011] 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 a disease or condition related thereto.

[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 The alkyl group 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 by 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 C1-3 alkyl;

[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 The alkyl group 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, each of the above R a independently represents H, F, Cl, Br, -CH3 or -CH2CH3, and the other variables are as defined herein.

[0023] In some embodiments of the present invention, the above 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 connected together with the carbon atom 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 connected together with the carbon atom to which they are attached to form Other variables are as defined herein.

[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 herein.

[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 above compound has a structure represented by any one of formulas (I-1) to (I-4):

[0035]

[0036] Among them, R1, R4, R5, R6, R7, R a and n are as defined in the present invention.

[0037] In some embodiments of the present invention, each of the above R b Independently H, F, Cl, Br, I, Other variables are as defined herein.

[0038] In some embodiments of the present invention, the above R7 is It is stated Optional 1 or 2 R b Replaced by R b and other variables are as defined in the present invention.

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

[0040] In some embodiments of the present invention, the above R7 is Other variables are as defined herein.

[0041] In some embodiments of the present invention, the above R4 is H or -CH3, and other variables are as defined in the present invention.

[0042] In some embodiments of the present invention, the above compound has a structure represented by any one of formula (I-5) to formula (I-9):

[0043]

[0044]

[0045] Among them, R1, R5, R6, R a and R b As defined in the present invention.

[0046] In some embodiments of the present invention, the above R1 is H, F, Cl or Other variables are as defined herein.

[0047] In some embodiments of the present invention, R5 and R6 are each independently H or Other variables are as defined in the present invention. Some other schemes of the present invention are obtained by any combination of the above variables.

[0048] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following structures:

[0049]

[0050]

[0051] According to an embodiment of the present invention, the pharmaceutically acceptable salt is a salt formed by the compound represented by formula (I) and an inorganic acid, the inorganic acid includes, for example, 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 an organic acid salt, the organic acid includes 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 includes 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).

[0052] 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.

[0053] 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.

[0054] In some embodiments, the fibrosis or a disease or disorder related thereto is selected from a liver-related disease, such as hepatitis, cirrhosis, liver damage, or liver failure.

[0055] 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).

[0056] In some embodiments, the fibrosis or its related diseases or disorders are selected from progressive fibrosing interstitial lung diseases (PF-ILDs), 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 diseases, idiopathic pneumonia with autoimmune features (IPAF) and sarcoidosis.

[0057] 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).

[0058] 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.

[0059] When preparing the medicament described in the present invention, the active compound is combined or formulated with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and can be formulated into a solid, semi-solid, liquid or gaseous preparation, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. The administration method includes 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 ear 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

[0060] Figure 1 Schematic diagram of HE and Masson staining in lung tissue;

[0061] Figure 2 Schematic diagram of the fibrosis degree score of mouse lung tissue (* represents comparison with the normal control group; # represents comparison with the solvent treatment group);

[0062] Figure 3TG test results of physiological group, NASH group, and 60 nM group of p-toluenesulfonate of compound 12 are shown;

[0063] Figure 4 Schematic diagram of the inflammatory factor detection results of the physiological group, NASH group, and 60 nM group of the p-toluenesulfonate salt of compound 12;

[0064] Figure 5 Schematic diagram of the results of H&E staining, picrosirius red staining, and COL1 and α-SMA immunofluorescence staining in each group;

[0065] Figure 6 It shows the ratio of the fat lesion area to the total stained area in the H&E stained film;

[0066] Figure 7 It shows the ratio of the fibrosis area to the total stained area after Sirius red staining. DETAILED DESCRIPTION

[0067] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope that the present invention is intended to protect.

[0068] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0069] Compound Preparation Example

[0070] 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.

[0071]

[0072]

[0073] Example 1

[0074]

[0075] Synthesis route:

[0076]

[0077] first step

[0078] The trifluoroacetate of compound 11d (90 mg, 219 μmol) and compound 7a (72 mg, 241 μmol) were dissolved in anhydrous dioxane (2 mL), and then cesium carbonate (250 mg, 766 μmol) and methanesulfonic acid (2-dicyclohexylphosphine)-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 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 purified by column chromatography (10:1, dichloromethane / methanol, Rf=0.3) to obtain a 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), water (2 mL×2), and dried to obtain compound 12.

[0079] 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).

[0080] Compound 12 (2 g, 3.89 μmol) and hexafluoroisopropanol (40 mL) were stirred and mixed, 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 into isopropanol (160 mL), filtered, and the filter cake was vacuum dried to obtain the p-toluenesulfonate corresponding to compound 12. 1H 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.

[0081] Biological activity test

[0082] Biological Example 1: Evaluation of the efficacy of the compounds of the present invention in bleomycin-induced pulmonary fibrosis

[0083] 1.1 Establishment of bleomycin-induced pulmonary fibrosis model

[0084] Experimental preparation: C57BL / 6 mice, 8-10 weeks old, female, weighing about 18-22 g. Bleomycin. 1% sodium pentobarbital, sterile saline, insulin needle.

[0085] 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 begin to adapt to the environment, the experiment is started. The experimental animals are divided into five groups: normal control group, modeling group (bleomycin group), low-dose administration group (25 mg / kg), high-dose administration group (50 mg / kg), and solvent treatment group. The modeling group is used to control the normal group of mice to detect whether the modeling is successful, and the solvent treatment group is 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 and samples were taken for subsequent experiments.

[0086] 2. Half an hour before the operation, give the mouse an intraperitoneal injection of 1% sodium pentobarbital (solvent is normal saline), 50 mg / kg, 90 μl-110 μl for each 18g-22g mouse. After the mouse is anesthetized, fix the mouse and disinfect it.

[0087] 3. The model was established according to the 3 mg / kg dose of bleomycin, 50 μl per mouse, injected through the mouse's oral trachea. During the period, attention was paid to maintaining the mouse's mouth breathing to ensure that the mouse inhaled liquid, and the bleomycin-induced mouse pulmonary fibrosis model was established.

[0088] 4. After injecting the liquid, place the mouse upright to evenly distribute the drug to the trachea, bronchi and lungs. Keep the breathing unobstructed and wait for the mouse to recover naturally.

[0089] 5. This model has one endpoint, 28 days. According to the time point, the mice were killed, the samples were collected, and the subsequent experiments were carried out. The day of modeling was recorded as the first day.

[0090] 1.2 Pulmonary inflammation score

[0091] 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.

[0092] 2. Place in 75% alcohol for 30 minutes.

[0093] 3. Place in 85% alcohol for 30 minutes

[0094] 4. Place in 95% alcohol twice for a total of one hour

[0095] 5. Place in 100% alcohol three times for a total of one hour

[0096] 6. After taking it out, place it in xylene twice for a total of one hour

[0097] 7. Soak in paraffin wax twice for a total of one hour. After soaking, place the embedding box in new wax for embedding.

[0098] 8. After embedding, place the cooled wax block on ice. After it is completely cooled, start slicing on a microtome. First, use an old blade to trim the surface of the wax block, and then use a new blade to slice the wax block to a thickness of about 3-5μm. Then, bleach the slices 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. After the paraffin on the slices is dried, take out the slices for the next step.

[0099] Slice hydration

[0100] 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.

[0101] 2. Place the sections in xylene twice for a total of one hour.

[0102] 3. Transfer the sections to 100% alcohol, twice, two minutes each time.

[0103] 4. Transfer the slices to 95% alcohol, once for two minutes each time

[0104] 5. Transfer the slices to 85% alcohol, once for two minutes each time

[0105] 6. Transfer the slices to 75% alcohol, once for two minutes each time

[0106] 7. After completing the above steps, transfer the slices to distilled water and rinse for two minutes.

[0107] HE staining

[0108] 1. After the hydration, the slices were stained with Mayer's hematoxylin for one minute.

[0109] 2. Rinse in tap water twice, three minutes each time

[0110] 3. Place in 75% hydrochloric acid alcohol for about 2 minutes

[0111] 4. Rinse with tap water for one minute

[0112] 5. Re-stain with red for about one minute

[0113] 6. Rinse with tap water for one minute

[0114] 7. 85% alcohol, about two minutes

[0115] 8. 95% alcohol, about two minutes

[0116] 9. 100% alcohol, about two minutes

[0117] 10. After the above steps are completed, place in xylene for 5-10 minutes.

[0118] 11. Seal the slides with neutral gum.

[0119] After scanning the 5 μm thick paraffin sections of lung tissue stained with HE, at least two independent scorers selected 5 representative fields and scored the degree of fibrosis according to the scoring criteria shown in Table 1. Finally, the average value was taken as the Szapiel Score of the sample. Figure 1 .

[0120] like Figure 2As shown in the figure, we compared and analyzed the Szapiel Score of the lung tissue fibrosis degree of each group of mice. The results showed 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 high-dose group (50 mg / kg) of p-toluenesulfonate of compound 12 had a significantly lower score, and the difference was statistically significant (p value <0.05).

[0121]

[0122] 1.3 Pulmonary fibrosis grading score

[0123] 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 (eg, Lancet Respir Med, 2020, PMID: 32061334 & Eur Respir J, 2009, PMID: 19460787).

[0124] Masson staining

[0125] 1. Place the hydrated sections in Masson's complex neutral staining solution for 5 minutes;

[0126] 2. Rinse in 0.2% acetic acid solution for about two minutes;

[0127] 3. Soak in 8% phosphotungstic acid for about 10 minutes;

[0128] 4. Rinse in 0.2% acetic acid solution for about two minutes;

[0129] 5. 0.2% aniline blue solution for 5 minutes;

[0130] 6. Rinse in 0.2% acetic acid solution for about two minutes, twice;

[0131] 7. 85% alcohol, about two minutes

[0132] 8. 95% alcohol, about two minutes

[0133] 9. 100% alcohol, about two minutes

[0134] 10. After the above steps are completed, place in xylene for 5-10 minutes.

[0135] 11. Seal the slides with neutral gum.

[0136] After scanning the 5 μm thick paraffin sections of the lung tissue after Masson staining, 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. Finally, the average value was taken as the Ashcroft Score of the sample. The scoring results are shown in Figure G.

[0137] like Figure 2 As shown in the figure, we compared and analyzed the Ashcroft Score of lung tissue fibrosis in each group of mice. The results showed 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 low-dose group (25 mg / kg) and the high-dose group (50 mg / kg) of p-toluenesulfonate of compound 12 were significantly lower, and the differences were statistically significant (p value <0.05).

[0138]

[0139]

[0140] Biological Example 2: Evaluation of the efficacy of the compounds of the present invention in NASH models and fibrosis

[0141] 2.1 In vitro 3D liver model modeling (reference: CN115386533A)

[0142] 2.1.1D-2~D0 modeling:

[0143] Four types of human primary cells, including primary human hepatocytes (PHH), primary human sinusoidal endothelial cells (LSEC), primary human hepatic stellate cells (HSC), and primary human Kupffer cells (KC), were used for modeling, with a total of 3000 cells in each model.

[0144] The cell connector NAC-Linker A was mixed with the hepatic parenchymal cells and incubated at room temperature for 30 minutes to fix NAC-Linker A on the cell membrane surface. Meanwhile, the cell connector NAC-Linker B was mixed with the mixture of the intrahepatic 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, the hepatic parenchymal cells carrying NAC-Linker A and the intrahepatic non-parenchymal cells carrying NAC-Linker B were mixed evenly. A 20-30 μl droplet was made on the cover of the culture plate using a pipette. PBS solution and liver physiological culture medium (Liver Physiological Culture Medium (Human), Product No. M0001, Puheng Bomai (Shanghai) Biopharmaceutical Co., Ltd.) were added to the wells of the culture plate. The prepared culture plate cover was flipped over 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.

[0145] 2.1.2 Trial Grouping and Drug Treatment

[0146] After the modeling was successful on Day 0, the samples were divided into 5 groups according to different treatment methods, with 9 samples in each group. 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) of Park Heng Biomedicine (Shanghai) Co., Ltd., catalog number MI001); 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; the high-dose group of p-toluenesulfonate of compound 12 was treated with NASH induction culture medium and 60nM p-toluenesulfonate of compound 12.

[0147] Drug administration: Starting from Day 0, the low-dose group, medium-dose group, and high-dose group of p-toluenesulfonate of compound 12 were treated with drugs respectively. The culture conditions remained unchanged, and the whole medium was replaced every 2 days with new culture medium of the corresponding group and drugs of the same concentration.

[0148] 2.1.3 Quality Inspection

[0149] The quality control of the model was tested on Day 0, Day 5, and Day 9. Day 0: HE staining to confirm that there was no cell necrosis and no pathological changes in the model when the model was completed. Day 5: HE staining and Sirius red (SR) staining: to determine the degree of fibrosis in the model on Day 5. Day 9: HE staining and SR staining: to determine the degree of fat lesions in the NASH model on Day 9.

[0150] 2.2 Test and results

[0151] 2.2.1 Quality control test results

[0152] Day 0, HE staining results showed that the cells were in normal condition, there was no obvious necrotic area, and the model had no pathological changes, so subsequent experiments could be carried out.

[0153] On Day 5, HE staining results showed that the cells in the physiological group were normal, ballooning changes were observed in the NASH group, and there was a trend of fatty lesions; SR staining results showed that the NASH group showed a state of fiber accumulation.

[0154] Day 9, HE staining results showed that the cells were in normal state, obvious ballooning degeneration and significant fatty lesions were observed in the NASH group, and subsequent experiments could be performed; SR staining results showed that the model fibrotic lesions were expressed normally.

[0155] 2.2.2TG detection

[0156] On the 10th day, TG test was performed. The liver microspheres were ground in the cell lysate using a grinding rod, centrifuged, and the supernatant was taken for enzymatic assay. Figure 3 As shown, the TG level of the 60 nM group of the p-toluenesulfonate salt of compound 12 was significantly different from that of the NASH group (p<0.05).

[0157] 2.2.3 Detection of inflammatory factors (IL-6)

[0158] On the 6th day, the culture supernatant was collected and tested according to the instructions of the IL-6 detection kit using an ELISA instrument. The results were as follows: Figure 4 As shown. The inflammatory factor level of the 60 nM group of the p-toluenesulfonate of compound 12 was improved compared with that of the NASH group, and the data of the 60 nM group of the p-toluenesulfonate of compound 12 was significantly different from that of the NASH group (p<0.05).

[0159] 2.2.4 H&E staining

[0160] The liver microspheres were paraffin sectioned and stained with H&E. The staining results were as follows Figure 5 shown.

[0161] Image J software was used to count the fat lesion area of ​​the H&E-stained films, and the ratio of the fat lesion area to the total area of ​​each film was calculated (see the results). Figure 6 ).

[0162] The results of H&E staining showed that the fatty degeneration and ballooning degeneration in hepatocytes in the 10nM dose group of the p-toluenesulfonate of compound 12 were improved compared with the NASH group. In the 30nM and 60nM dose groups, the H&E staining results showed significant histological improvement, specifically, the fatty degeneration in hepatocytes was significantly reduced and the number of ballooning hepatocytes was significantly reduced. This result shows that the p-toluenesulfonate of compound 12 has a significant therapeutic effect in inhibiting the pathological progression of NASH.

[0163] 2.2.5 Sirius red staining

[0164] The liver microspheres were paraffin sectioned, stained with picrosirius red, sealed, and photographed. Figure 5 shown.

[0165] Image J software was used to count the fibrosis area of ​​the slides after Sirius red staining, and the ratio of the fibrosis area to the total area of ​​each slide was calculated ( Figure 7 ).

[0166] Sirius red staining was used to evaluate the effect of the drug on liver fibrosis, and the results showed that the degree of liver fibrosis in the p-toluenesulfonate 10 nM group of compound 12 showed an improved trend compared with the physiological group. In the staining results of the p-toluenesulfonate 30 nM group of compound 12 and the p-toluenesulfonate 60 nM group 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 liver fibrosis caused by NASH, and the larger the dose of p-toluenesulfonate of compound 12, the better the therapeutic effect.

[0167] 2.2.6 Immunofluorescence staining

[0168] The liver microspheres were paraffin sectioned, immunofluorescence stained, sealed and photographed under a fluorescence microscope. Figure 5 Shown

[0169] 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.

[0170] The above is an exemplary description of the implementation of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above implementation. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention should be included in the protection scope 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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