Alcohol amine derivatives, preparation method therefor and use thereof, and drug for treating organ fibrosis or pulmonary inflammations caused by acute injury

Abstract

Provided are alcohol amine derivatives, a preparation method therefor and the use thereof, and a drug for treating organ fibrosis or pulmonary inflammations caused by acute injury, belonging to the technical field of medicine. The alcohol amine derivatives have structures represented by formula R-1 or formula R-2 and are R-configuration compounds, and exhibit good therapeutic effects on organ fibrosis or pulmonary inflammations caused by acute injury. The results in the pharmacological embodiments show that the alcohol amine derivatives have excellent pharmaceutical effects both on pulmonary inflammations caused by acute injury and various types of organ fibrosis models, the pharmaceutical effects thereof being overall superior to that of S-configuration compounds and positive drugs Nintedanib and Pirfenidone.

Description

Alkylamine derivatives, their preparation methods and applications; drugs for treating lung inflammation caused by organ fibrosis or acute injury.

[0001] This application claims priority to Chinese Patent Application No. CN202410979370.8, filed on July 24, 2024, entitled "Alcoholamine Derivatives and Their Preparation Methods and Applications, and a Drug for Treating Lung Inflammation Caused by Organ Fibrosis or Acute Injury", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of pharmaceutical technology, specifically to alcohol amine derivatives and their preparation methods and applications, and drugs for treating lung inflammation caused by organ fibrosis or acute injury. Background Technology

[0003] Alkylamines are an important class of organic compounds that can be used as intermediates or adjuvants in the synthesis of various natural products and drugs, as well as beneficial adjuvants in the treatment of many human diseases. Their derivatives also have unique properties that can be used in medicine, materials, and organic synthesis. For example, vanilla amine-propanolamine derivatives have adrenaline alpha / beta receptor blocking and vasodilatory properties; ethylpropanolamine derivatives are a class of plant growth regulators.

[0004] Fibrosis can occur in various organs (such as the lungs, kidneys, liver, skin, or heart). The main pathological change is the excessive proliferation and activation of parenchymal cells within the organ tissues, leading to excessive secretion of extracellular matrix. Continued progression can result in organ structural damage and functional decline, even failure, seriously threatening human health and life. Pulmonary fibrosis (PF) is a disease characterized by persistent alveolar damage, leading to varying degrees of inflammation and fibrosis in the alveoli and pulmonary interstitium, ultimately resulting in lung structural destruction and respiratory failure; therefore, it is also called interstitial lung disease (ILD). The causes of pulmonary fibrosis include known causes such as physical, chemical, and biological factors, as well as pulmonary fibrosis of unknown etiology. Pulmonary fibrosis of known causes includes connective tissue disease-related interstitial lung diseases, such as rheumatoid arthritis-related interstitial lung disease (RA-ILD), systemic sclerosis-related interstitial lung disease (SSc-ILD), and myositis-related interstitial lung disease (PM-ILD). Other known causes include occupational exposure-related fibrosis (such as silicosis) and fibrosis that can develop into fibrosis in the later stages of acute lung injury caused by infection. The pathogenesis and mechanism of these causes are mainly due to an excessive immune response to self- or foreign antigens, leading to the activation of various immune cells, including pulmonary lymphocytes, which then secrete large amounts of cytokines, mediating the immune response and causing pulmonary fibrosis. Pulmonary fibrosis of unknown etiology includes idiopathic pulmonary fibrosis (IPF). IPF belongs to the idiopathic interstitial pneumonia (IIP) group within the interstitial lung disease (ILD) family and is one of the most common and severe chronic interstitial lung diseases of unknown etiology. Interstitial lung disease (IPF) originates from abnormal repair following recurrent or persistent alveolar epithelial injury. Under the combined and persistent damage from multiple known or unknown endogenous and exogenous damaging factors, damaged lung epithelial cells initiate damage repair mechanisms. However, epithelial regeneration and repair are insufficient, and surviving cells undergo mesenchymal-like transformation, exhibiting a pro-fibrotic phenotype and secreting large amounts of pro-fibrotic factors. This leads to abnormal activation and proliferation of fibroblasts, producing excessive extracellular matrix, resulting in fibrotic scar formation, destruction of alveolar structure, and irreversible and continuous decline in lung function, ultimately leading to respiratory failure and death. Clinically, IPF manifests as progressive dyspnea accompanied by an irritating dry cough. The disease often progresses continuously, with a median survival of approximately 2.8 years and a 5-year survival rate of less than 50%. Most patients die from respiratory failure and secondary pulmonary infections.

[0005] Currently, only pirfenidone and nintedanib are approved drugs for the treatment of pulmonary fibrosis. Exploring new potential drugs for the treatment of pulmonary fibrosis and fibrosis of other organs has important social and medical significance. Summary of the Invention

[0006] The purpose of this application is to provide an alcohol amine derivative, its preparation method and application, and a drug for treating lung inflammation caused by organ fibrosis or acute injury. The alcohol amine derivative provided in this application has good efficacy for lung inflammation caused by organ fibrosis or acute injury.

[0007] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0008] This application provides alcoholamine derivatives having the structure shown in formula R-1 or formula R-2:

[0009] Preferably, the space group of the alcoholamine derivative having the structure shown in Formula R-1 is P 212121; the unit cell parameters are: α = 90°, β = 90°, γ = 90°; unit cell volume is Z = 8.

[0010] Preferably, the space group of the alcoholamine derivative having the structure shown in formula R-2 is P 21; the unit cell parameters are: α=90°, β=91.239(8)°, γ=90°; Z=4.

[0011] This application provides a method for preparing the alcoholamine derivatives described in the above technical solution, comprising the following steps:

[0012] Compound A, an alcoholic amine starting compound, a chiral ligand, and an organic solvent are mixed and subjected to an addition reaction to obtain an alcoholic amine derivative having the structure shown in formula R-1 or formula R-2.

[0013] The alcohol amine raw material compound is 3-methylamino-1-propanol or 2-methylaminoethanol;

[0014] The structural formula of compound A is shown below:

[0015] Preferably, the chiral ligand is any one of the following compounds:

[0016] Preferably, the molar ratio of compound A, the alkanolamine raw material compound, and the chiral ligand is 1:0.8-10:0.1-3.

[0017] Preferably, the organic solvent includes acetonitrile, methanol, or ethyl acetate; the ratio of compound A to organic solvent is 4 mmol: 4–24 mL.

[0018] Preferably, the addition reaction is carried out at a temperature of 0–100°C for a time of 2–24 hours.

[0019] Preferably, the addition reaction further includes: concentrating the product system obtained from the addition reaction under reduced pressure to remove the solvent, and then purifying it by silica gel rapid column chromatography.

[0020] Preferably, the eluent used for purification is a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol in the eluent is 15:1.

[0021] This application provides the use of the alcoholamine derivatives described above in the preparation of medicaments for treating lung inflammation caused by organ fibrosis or acute injury.

[0022] Preferably, the organ fibrosis includes pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, or skin fibrosis.

[0023] Preferably, the pulmonary fibrosis includes idiopathic pulmonary fibrosis, pulmonary fibrosis caused by occupational exposure, or pulmonary fibrosis caused by autoimmune diseases.

[0024] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is the alcoholamine derivative.

[0025] Preferably, the drug is administered orally, by inhalation, or by injection.

[0026] Preferably, the dosage form of the drug includes capsules, tablets, oral solutions, inhalers, or injections.

[0027] This application provides a medicament for treating lung inflammation caused by organ fibrosis or acute injury, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is an alcohol amine derivative as described in the above technical solution.

[0028] The present invention provides a method for treating lung inflammation caused by organ fibrosis or acute injury, comprising administering an effective amount of the alcohol amine derivative described above to a patient suffering from lung inflammation caused by organ fibrosis or acute injury.

[0029] Preferably, the alkanolamine derivative is administered to the patient orally, by inhalation, or by injection.

[0030] This application provides alcoholamine derivatives having the structures shown in Formula R-1 or Formula R-2, namely propanolamine derivatives and ethanolamine derivatives, both of which are R-configuration compounds, specifically, the carbon at position 3 is in the R configuration. Taking the propanolamine derivative as an example, the structural formula is shown below:

[0031] The alcohol amine derivatives provided in this application have good therapeutic effects on lung inflammation caused by organ fibrosis or acute injury. Results from pharmacological examples show that the alcohol amine derivatives in this application have excellent efficacy against lung inflammation caused by acute injury and various organ fibrosis models, and are generally superior to S-configuration compounds and the positive control drugs Nintedanib and Pirfenidone. Attached Figure Description

[0032] Figure 1 is an ellipsoid diagram of the propanolamine derivative in Example 1;

[0033] Figure 2 is an ellipsoid diagram of the ethanolamine derivative in Example 2. Detailed Implementation

[0034] This application provides alcoholamine derivatives having the structure shown in formula R-1 or formula R-2:

[0035] In this application, the space group of the alcoholamine derivative having the structure shown in formula R-1 (i.e., the propanolamine derivative, denoted as compound R-1) is P 212121; the unit cell parameters are: α = 90°, β = 90°, γ = 90°; unit cell volume is Z = 8. In this application, the relevant crystal data for the propanolamine derivative are detailed in Table 1 of the embodiments, and will not be repeated here.

[0036] In this application, the space group of the alcoholamine derivative having the structure shown in formula R-2 (i.e., the ethanolamine derivative, denoted as compound R-2) is P 21; the unit cell parameters are: α = 90°, β = 91.239(8)°, γ = 90°; Z = 4. In this application, the relevant crystal data of the ethanolamine derivative are detailed in Table 2 of the examples, and will not be repeated here.

[0037] This application provides a method for preparing the alcoholamine derivatives described in the above technical solution, comprising the following steps:

[0038] Compound A, an alcoholic amine starting compound, a chiral ligand, and an organic solvent are mixed and subjected to an addition reaction to obtain an alcoholic amine derivative having the structure shown in formula R-1 or formula R-2.

[0039] The alcohol amine raw material compound is 3-methylamino-1-propanol or 2-methylaminoethanol;

[0040] The structural formula of compound A is shown below:

[0041] Unless otherwise specified, all raw materials used in this application are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0042] In this application, the chiral ligand is preferably any one of the following compounds:

[0043] In this application, the molar ratio of compound A, the alkanolamine raw material compound, and the chiral ligand is preferably 1:0.8–10:0.1–3, more preferably 1:2.5–5:0.5–2, and even more preferably 1:3.2:1. In this application, the organic solvent preferably includes acetonitrile, methanol, or ethyl acetate, more preferably acetonitrile; the molar ratio of compound A to the organic solvent is preferably 4 mmol:4–24 mL, more preferably 4 mmol:10–16 mL, and even more preferably 4 mmol:12 mL.

[0044] In this application, compound A is preferably dissolved in an organic solvent, and then an alcoholic amine starting compound and a chiral ligand are added to carry out an addition reaction. In this application, the temperature of the addition reaction is preferably 0–100°C, more preferably 30–70°C, and even more preferably 50°C; the time is preferably 2–24 h, more preferably 4–10 h, and even more preferably 6 h. In this application, the addition reaction is preferably carried out under stirring conditions. This application does not have a specific limitation on the stirring rate, as long as it ensures the addition reaction proceeds smoothly. After the addition reaction, in this application, the obtained product system is preferably concentrated under reduced pressure to remove the solvent, and then purified by silica gel rapid column chromatography to obtain an alcoholic amine derivative having the structure shown in formula R-1 or formula R-2, i.e., compounds R-1 and R-2. In this application, the eluent used for purification is preferably a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the eluent is preferably 15:1.

[0045] This application demonstrates that when using compound A and an alcoholamine starting compound as raw materials, different chiral alcoholamine derivatives are obtained by preparing them under conditions where chiral ligands are not used or different types of chiral ligands are used. This application specifically illustrates the influence of eight different types of chiral ligands on the configuration of alcoholamine derivatives. The chiral ligands are (1R,2R)-1N,1N,2N,2N-tetramethyl-1,2-cyclohexanediamine (compound L1), (R)-N,N',N,N'-tetramethylbinaphthylamine (compound L2), (1R,2R)-N,N'-dimethyl-N,N'-bis(3,3-dimethylbutyl)cyclohexane-1,2-diamine (compound L3), and (R)-4-(9-anthrayl)-3-tert-butyl-2,3-dihydro-1,3-benzoxoxophosphate. Heterocyclopentadiene (compound L4), (1S,2S)-N1,N1,N,N2-tetramethylcyclohexane-1,2-diamine (compound L5), (S)-N,N',N,N'-tetramethylbinaphthylamine (compound L6), (1S,2S)-N,N'-dimethyl-N,N'-bis(3,3-dimethylbutyl)cyclohexane-1,2-diamine (compound L7), and (S)-4-(9-anthrayl)-3-tert-butyl-2,3-dihydro-1,3-benzoxoxophosphazene heterocyclopentadiene (compound L8) are all included. Each alcoholamine derivative specifically comprises two R-configuration compounds (i.e., compounds R-1 and R-2 as described in this application) and two S-configuration compounds (denoted as compounds S-1 and S-2, respectively). The structural formulas of the eight chiral ligands and four alcoholamine derivatives are shown below:

[0046] In this application, specifically, when the starting compound for the alkanolamine is 3-methylamino-1-propanol (i.e., to prepare alkanolamine derivative compounds R-1 and S-1), without a chiral ligand, the molar ratio of the obtained compound R-1:S-1 is 1:1; when the chiral ligand is compound L1, the ratio of the obtained compound R-1:S-1 is >99:1; when the chiral ligand is compound L2, the ratio of the obtained compound R-1:S-1 is >50:1; and when the chiral ligand is compound L3, the ratio of the obtained compound... R-1:S-1 = 20:1; when the chiral ligand is compound L4, the resulting compound R-1:S-1 = 2.5:1; when the chiral ligand is compound L5, the resulting compound R-1:S-1 < 1:99; when the chiral ligand is compound L6, the resulting compound R-1:S-1 = 1:10; when the chiral ligand is compound L7, the resulting compound R-1:S-1 = 1:3; when the chiral ligand is compound L8, the resulting compound R-1:S-1 = 1:6.

[0047] In summary, when no chiral ligand is used, the resulting alcoholamine derivative is a mixture of diastereomers. When compound L1 is used as a chiral ligand, the resulting alcoholamine derivative is compound R-1. Therefore, in this application, the preferred chiral ligand for preparing compound R-1 is compound L1. When compound L5 is used as a chiral ligand, the resulting alcoholamine derivative is compound S-1.

[0048] In this application, specifically, when the alcoholamine starting material is 2-methylaminoethanol (i.e., to prepare alcoholamine derivative compounds R-2 and S-2), without a chiral ligand, the ratio of compound R-2 to S-2 is 1:1; when the chiral ligand is compound L1, the ratio of compound R-2 to S-2 is >99:1; when the chiral ligand is compound L2, the ratio of compound R-2 to S-2 is >20:1; and when the chiral ligand is compound L3, the ratio of compound R-2 to S-2 is <20:1. The ratio of R-2 to S-2 is 5:1; when the chiral ligand is compound L4, the ratio of R-2 to S-2 is 2:1; when the chiral ligand is compound L5, the ratio of R-2 to S-2 is <1:99; when the chiral ligand is compound L6, the ratio of R-2 to S-2 is 1:12; when the chiral ligand is compound L7, the ratio of R-2 to S-2 is 1:2; when the chiral ligand is compound L8, the ratio of R-2 to S-2 is 1:4.

[0049] In summary, when no chiral ligand is used, the resulting alcoholamine derivative is a mixture of diastereomers. When compound L1 is used as the chiral ligand, the resulting alcoholamine derivative is compound R-2. Therefore, in this application, the preferred chiral ligand for preparing compound R-2 is compound L1. When compound L5 is used as the chiral ligand, the resulting alcoholamine derivative is compound S-2.

[0050] This application provides the use of the above-described alcoholamine derivatives in the preparation of medicaments for treating lung inflammation caused by organ fibrosis or acute injury. In this application, the organ fibrosis preferably includes pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, or skin fibrosis. The pulmonary fibrosis in this application preferably includes idiopathic pulmonary fibrosis, pulmonary fibrosis caused by occupational exposure, or pulmonary fibrosis caused by autoimmune diseases. In the embodiments of this application, the efficacy of the alcohol amine derivatives described in this application is specifically verified by using bleomycin-induced pulmonary fibrosis, silica suspension-induced silicosis, lipopolysaccharide (LPS)-induced acute lung injury, bleomycin-induced systemic sclerosis-associated interstitial lung disease (SSc-ILD), sodium hypochlorite-induced SSc-ILD, spontaneous systemic lupus erythematosus-associated interstitial lung disease (SLE-ILD) in MRL / Lpr mice, imiquimod-induced SLE-ILD, skeletal muscle homogenate-induced myositis-associated interstitial lung disease (PM-ILD), collagen immunity and bleomycin-induced rheumatoid arthritis-associated interstitial lung disease (RA-ILD), yeast polysaccharide-induced RA-ILD, unilateral ureteral ligation (UUO)-induced renal fibrosis, folic acid-induced renal fibrosis, carbon tetrachloride-induced liver fibrosis, bile duct ligation-induced liver fibrosis, and isoproterenol-induced cardiac fibrosis.

[0051] In this application, the drug specifically includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is an alcohol amine derivative as described in the above technical solution, and the content of the active ingredient in the drug is preferably a unit dose of a therapeutically effective amount.

[0052] In this application, the dosage form of the drug preferably includes capsules, tablets, oral solutions, inhalers, or injections; the administration method of the drug preferably includes oral, inhalation, or injection, and the injection is preferably intravenous injection.

[0053] This application provides a medicament for treating lung inflammation caused by organ fibrosis or acute injury, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is an alcohol amine derivative as described in the above-described technical solution. The composition, dosage form, and administration method of the medicament described in this application are preferably consistent with the above-described technical solution, and will not be repeated here.

[0054] The technical solutions of this application will be clearly and completely described below with reference to the embodiments therein. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0055] Example 1

[0056] Compound R-1 was prepared according to the following reaction formula:

[0057] The structural formula of the ligand used is as follows:

[0058] Using ligand L1: Compound A (1.0575 g, 4.00 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1410 g, 12.8 mmol) and ligand compound L1 (0.6832 g, 4.01 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give propanolamine derivative R-1 (white solid, 1.3957 g, yield 98.7%, dr > 99:1).

[0059] Using ligand L2: Compound A (1.0566 g, 4.00 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1522 g, 12.9 mmol) and ligand compound L2 (1.3641 g, 4.01 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-1 (white solid, 1.3312 g, yield 94.2%, dr > 50:1).

[0060] Using ligand L3: Compound A (1.0629 g, 4.02 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1633 g, 13.1 mmol) and ligand compound L3 (1.2429 g, 4.00 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-1 (white solid, 1.3468 g, yield 94.8%, dr = 20:1).

[0061] Using ligand L4: Compound A (1.0588 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1449 g, 12.8 mmol) and ligand compound L4 (1.4807 g, 4.00 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-1 (white solid, 1.2893 g, yield 91.1%, dr = 2.5:1).

[0062] Without ligands: Compound A (1.0602 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1453 g, 12.8 mmol) was added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give a mixture of diastereomers (white solid, 1.3483 g, yield 95.1%, dr = 1:1).

[0063] The English name of the propanolamine derivative R-1 is: (3R,3aR,4aR,5R,6R,8aR,9aR)-6-Hydroxy-3-(((3-hydroxypropyl)(methyl)amino)methyl)-8a-methyldecahydro-2H-spiro[naphtho[2,3-b]furan-5,2'-oxiran]-2-one.

[0064] The crystal data of the propanolamine derivatives are shown in Table 1, and the ellipsoid diagram is shown in Figure 1.

[0065] Table 1 Crystal data of propanolamine derivatives

[0066] Example 2

[0067] The reaction formula for preparing compound R-2 is shown below:

[0068] The structural formula of the ligand used is as follows:

[0069] Using ligand L1: Compound A (1.0612 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9652 g, 12.9 mmol) and ligand L1 (0.6823 g, 4.01 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-2 (white solid, 1.1967 g, yield 87.8%, dr > 99:1).

[0070] Using ligand L2: Compound A (1.0587 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9678 g, 12.9 mmol) and ligand compound L2 (1.3652 g, 4.01 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-2 (white solid, 1.1495 g, yield 84.6%, dr > 20:1).

[0071] Using ligand L3: Compound A (1.0633 g, 4.02 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9573 g, 12.7 mmol) and ligand compound L3 (1.2497 g, 4.02 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-2 (white solid, 1.1894 g, yield 87.1%, dr = 5:1).

[0072] Using ligand L4: Compound A (1.0598 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9644 g, 12.8 mmol) and ligand compound L4 (1.4822 g, 4.00 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound R-2 (white solid, 1.1063 g, yield 81.3%, dr = 2:1).

[0073] Without ligands: Compound A (1.0609 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9598 g, 12.8 mmol) was added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give a mixture of diastereomers (white solid, 1.1166 g, yield 82.0%, dr = 1:1).

[0074] The English name of the ethanolamine derivative R-2 is: (3R,3aR,4aR,5R,6R,8aR,9aR)-6-Hydroxy-3-(((2-hydroxyethyl)(methyl)amino)methyl)-8a-methyldecahydro-2H-spiro[naphtho[2,3-b]furan-5,2'-oxiran]-2-one.

[0075] The crystal data of the ethanolamine derivatives are shown in Table 2, and the ellipsoid diagram is shown in Figure 2.

[0076] Table 2 Crystal data of ethanolamine derivatives

[0077] Preparation Example 1

[0078] The structural formula of compound S-1 in the following pharmacological examples is shown below:

[0079] The preparation method of compound S-1 is as follows:

[0080] The ligand structure used is as follows:

[0081] Using ligand L5: Compound A (1.0526 g, 3.98 mmol) was dissolved in acetonitrile (12 mL), then compound B (3-methylamino-1-propanol, 1.1410 g, 12.8 mmol) and ligand compound L5 (0.6855 g, 4.03 mmol) were added, and the mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-1 (white solid, 1.3177 g, yield 93.6%, dr > 99:1).

[0082] Using ligand L6: Compound A (1.0618 g, 4.02 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1522 g, 12.9 mmol) and ligand compound L6 (1.3609 g, 4.00 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-1 (white solid, 1.2876 g, yield 90.7%, dr = 10:1).

[0083] Using ligand L7: Compound A (1.0588 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1633 g, 13.1 mmol) and ligand compound L7 (1.2397 g, 3.99 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-1 (white solid, 1.3217 g, yield 93.3%, dr = 3:1).

[0084] Using ligand L8: Compound A (1.0657 g, 4.03 mmol) was dissolved in acetonitrile (12 mL), and then compound B (3-methylamino-1-propanol, 1.1449 g, 12.8 mmol) and ligand compound L8 (1.4855 g, 4.01 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-1 (white solid, 1.3353 g, yield 93.7%, dr = 6:1).

[0085] Preparation Example 2

[0086] The structural formula of compound S-2 in the following pharmacological examples is shown below:

[0087] The preparation method of compound S-2 is as follows:

[0088] The ligand structure used is as follows:

[0089] Using ligand L5: Compound A (1.0687 g, 4.04 mmol) was dissolved in acetonitrile (12 mL). Compound C (2-methylaminoethanol, 0.9652 g, 12.9 mmol) and ligand L5 (0.6799 g, 3.99 mmol) were added to the system, and the mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, yielding a crude product. The crude product was then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 20:1 by volume) to give compound S-2 (white solid, 1.2242 g, yield 89.2%, dr > 99:1).

[0090] Using ligand L6: Compound A (1.0607 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9678 g, 12.9 mmol) and ligand compound L6 (1.3721 g, 4.03 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-2 (white solid, 1.1018 g, yield 80.9%, dr = 12:1).

[0091] Using ligand L7: Compound A (1.0599 g, 4.01 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9573 g, 12.7 mmol) and ligand compound L7 (1.2513 g, 4.03 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-2 (white solid, 1.1394 g, yield 83.7%, dr = 2:1).

[0092] Using ligand L8: Compound A (1.0628 g, 4.02 mmol) was dissolved in acetonitrile (12 mL), and then compound C (2-methylaminoethanol, 0.9644 g, 12.8 mmol) and ligand compound L8 (1.4835 g, 4.00 mmol) were added. The mixture was stirred at 50 °C for 6 h. After the reaction was completed, the resulting product system was concentrated under reduced pressure to remove acetonitrile, and then purified by silica gel rapid column chromatography (the eluent used was dichloromethane:methanol = 15:1 by volume) to give compound S-2 (white solid, 1.1315 g, yield 82.9%, dr = 4:1).

[0093] Unless otherwise specified, all test results in the following pharmacological examples are analyzed using one-way ANOVA, and the results are expressed as "mean ± standard error"; compounds R-1 and R-2 used in the following pharmacological examples are compounds with a dr value > 99:1 prepared using ligand L1, and compounds S-1 and S-2 used are compounds with a dr value > 99:1 prepared using ligand L5; among them, compound X-1 used in pharmacological examples 12-22 is a mixture of diastereomers prepared in Example 1 without the use of ligands, and its structural formula is shown below:

[0094] Pharmacological Example 1: Treatment of Bleomycin-Induced Pulmonary Fibrosis in Mice

[0095] 1. Experimental Methods

[0096] Male C57BL / 6 mice were used to establish a pulmonary fibrosis model via intratracheal injection of bleomycin (2 U / kg). Day 1 was the day of model establishment, and mice were weighed daily. On day 7, mice were regrouped based on their rate of weight loss, with 8 mice in each group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began on day 8, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 14 of treatment.

[0097] 2. Experimental Results

[0098] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, and pulmonary fibrosis area of ​​the mice in each group were measured. The results are as follows:

[0099] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 3 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The positive control drug Pirfenidone (P > 0.05) had no significant effect on FVC, while Nintedanib (P < 0.01) improved the decrease in FVC. R-1 (P < 0.0001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0100] Table 3. FVC results of bleomycin model mice for each compound.

[0101] 2.2 Hydroxyproline content determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were sacrificed, the right lung was isolated, placed in a 5 mL ampoule, dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was then converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). As shown in Table 4, compared with the control group, the HYP content in the whole right lung of the model group was significantly increased; the positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) significantly reduced the HYP content. R-1 (P < 0.0001) significantly reduced HYP levels, exhibiting the best efficacy; R-1 showed better inhibitory efficacy against HYP than S-1 (P < 0.0001); R-2 (P < 0.0001) also showed significantly better inhibitory efficacy against HYP than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.01) was slightly more effective than the positive control drug Pirfenidone, but slightly less effective than the positive control drug Nintedanib.

[0102] Table 4. Hydroxyproline content in the lungs of bleomycin-induced model mice for each compound.

[0103] 2.3 Pulmonary fibrosis area statistics: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 5, the positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) both significantly alleviated pulmonary fibrosis. R-1 (P < 0.0001) significantly reduced the pulmonary fibrosis area and had the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0104] Table 5. Statistics on pulmonary fibrosis area in bleomycin model mice for each compound.

[0105] Pharmacological Example 2: Treatment of bleomycin-induced early pulmonary fibrosis in mice

[0106] 1. Experimental Methods

[0107] Male C57BL / 6 mice were used to establish a pulmonary fibrosis model via endotracheal injection of bleomycin (2 U / kg). On the day of model establishment, mice were divided into groups of eight based on body weight: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Day 1 was the day of model establishment, and mouse body weight was measured daily. Treatment began on day 2, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 8 of treatment.

[0108] 2. Experimental Results

[0109] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, pulmonary fibrosis area, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in BALF were measured in each group of mice. The results are as follows:

[0110] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the lung function data of mice measured using the AniRes2005 animal lung function analysis system. Table 6 shows that compared with the control group, the model group showed a significant decrease in FVC. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P>0.05) did not significantly alleviate FVC in mice. R-1 (P<0.01) significantly improved the decrease in FVC, showing the best efficacy; R-1 was more effective than S-1 (P<0.05); R-2 (P<0.05) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P>0.05) was slightly better than the positive control drugs Pirfenidone and Nintedanib.

[0111] Table 6. FVC results of bleomycin model mice for each compound.

[0112] 2.2 Hydroxyproline content determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were sacrificed, the right lung was isolated, placed in a 5 mL ampoule, dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). As shown in Table 7, compared with the control group, the HYP content in the whole right lung of the model group was significantly increased; the positive control drug Pirfenidone (P > 0.05) had no significant inhibitory effect on the HYP content in mice, while the positive control drug Nintedanib (P < 0.05) could reduce the HYP content in mice. R-1 (P < 0.001) significantly reduced HYP levels, showing the best efficacy; R-1 was more effective at inhibiting HYP than S-1 (P < 0.01); R-2 (P < 0.05) was slightly more effective at inhibiting HYP than the positive control drug Pirfenidone, and comparable to the positive control drug Nintedanib; S-2 (P > 0.05) was as effective as the positive control drug Pirfenidone, but slightly less effective than the positive control drug Nintedanib.

[0113] Table 7. Hydroxyproline content in the lungs of bleomycin-induced model mice for each compound.

[0114] 2.3 Statistical Analysis of Pulmonary Fibrosis Area: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 8, compared with the control group, the pulmonary fibrosis area in the model group was increased. The positive control drug Pirfenidone (P>0.05) had no significant effect on improving pulmonary fibrosis, while the positive control drug Nintedanib (P<0.05) showed some improvement. R-1 (P<0.001) significantly reduced the pulmonary fibrosis area in mice, showing the best efficacy; R-1 was more effective than S-1 (P<0.01); R-2 (P<0.05) was also significantly more effective than the positive control drug Pirfenidone and comparable to the positive control drug Nintedanib; S-2 (P<0.05) was slightly more effective than the positive control drug Pirfenidone but slightly less effective than Nintedanib.

[0115] Table 8. Statistics on pulmonary fibrosis area in bleomycin model mice for each compound.

[0116] 2.4 Total Cell Count in Bronchoalveolar Lavage Fluid: After drug administration, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was used for H&E staining. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. The cell count was counted using a cell counter, as shown in Table 9. Compared with the control group, the total cell count in the model group BALF was significantly increased; the positive control drugs Pirfenidone (P < 0.001) and Nintedanib (P < 0.001) significantly reduced the cell count in BALF. R-1 (P < 0.0001) significantly reduced the total number of BALF cells, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.001) also significantly reduced the total number of BALF cells, slightly better than the positive control drug Nintedanib, but slightly worse than the positive control drug Pirfenidone.

[0117] Table 9 Total BALF cell counts in bleomycin-treated mouse models of each compound.

[0118] 2.5 Expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After administration, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table. The indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea with a syringe to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected by ELISA. As shown in Table 10, compared with the control group, the protein content of pro-inflammatory cytokines TGF-β, TNF-α, IL-6 and IL-1β in the model group was significantly increased. (1) Pro-inflammatory cytokine TGF-β: The positive control drugs Pirfenidone (P<0.001) and Nintedanib (P<0.0001) significantly inhibited the expression of TGF-β protein. R-1 (P < 0.0001) significantly inhibited TGF-β protein expression in BALF, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced TGF-β protein expression in BALF, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0119] (2) Pro-inflammatory cytokine TNF-α: The positive control drugs Pirfenidone (P<0.01) and Nintedanib (P<0.001) significantly inhibited the expression of TNF-α protein. R-1 (P<0.0001) significantly inhibited the protein expression of TNF-α in BALF, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the protein expression of TNF-α in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0120] (3) Pro-inflammatory cytokine IL-6: The positive control drugs Pirfenidone (P<0.01) and Nintedanib (P<0.0001) significantly inhibited the expression of IL-6 protein. R-1 (P<0.0001) significantly inhibited the expression of IL-6 protein in BALF, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the expression of IL-6 protein in BALF, which was significantly better than the positive control drug Pirfenidone, but slightly less effective than Nintedanib; S-2 (P<0.001) was slightly more effective than the positive control drug Pirfenidone.

[0121] (4) Pro-inflammatory cytokine IL-1β: The positive control drugs Pirfenidone (P<0.001) and Nintedanib (P<0.01) significantly inhibited the expression of IL-1β protein. R-1 (P<0.0001) significantly inhibited the protein expression of IL-1β in BALF, with the best efficacy; R-1 was more effective than S-1 (P<0.001); R-2 (P<0.01) also significantly reduced the protein expression of IL-1β in BALF, with slightly worse efficacy than the positive control drug Pirfenidone, and comparable efficacy to Nintedanib.

[0122] Table 10. BALF inflammatory cytokine levels of each compound in bleomycin-induced model mice.

[0123] Pharmacological Example 3: Treatment of a mouse model of silicosis induced by silica suspension

[0124] 1. Experimental Methods

[0125] Male C57BL / 6 mice were used to establish a silicosis model via intratracheal injection of silica suspension (200 mg / kg). Day 1 was the day of model establishment, and mouse weight was measured daily. On day 20, mice were regrouped according to their weight loss rate, with eight mice per group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began on day 21, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until day 30.

[0126] 2. Experimental Results

[0127] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, pulmonary fibrosis area, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in BALF were measured in each group of mice. The results are as follows:

[0128] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the lung function data of mice measured using the AniRes2005 animal lung function analysis system. Table 11 shows that compared with the control group, the model group had a significantly decreased FVC. The positive control drug Pirfenidone (P>0.05) had no significant improvement on FVC, while Nintedanib (P<0.01) showed some improvement. R-1 (P<0.0001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0129] Table 11. FVC results of Silica-induced pulmonary fibrosis model mice for each compound.

[0130] 2.2 Hydroxyproline content determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were sacrificed, the right lung was isolated, placed in a 5 mL ampoule, dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was then converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). Table 12 shows that compared with the control group, the HYP content in the whole right lung of the model group was significantly increased; the positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) could improve the HYP content. R-1 (P < 0.0001) significantly reduced HYP levels, exhibiting the best efficacy; R-1 showed better inhibitory efficacy against HYP than S-1 (P < 0.0001); R-2 (P < 0.0001) also showed significantly better inhibitory efficacy against HYP than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) had efficacy comparable to the positive control drug Nintedanib, and slightly better than the positive control drug Pirfenidone.

[0131] Table 12 Hydroxyproline content of each compound in Silica-induced pulmonary fibrosis model mice

[0132] 2.3 Statistical analysis of pulmonary fibrosis area: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. Compared with the control group, fibrosis appeared in the lung tissue of the model group. Further statistical analysis of the fibrosis area of ​​lung tissue sections in each group was performed using ImageJ software, as shown in Table 13. The positive control drug Pirfenidone (P>0.05) did not significantly improve the pulmonary fibrosis area, while Nintedanib (P<0.01) significantly alleviated the pulmonary fibrosis area. R-1 (P < 0.0001) significantly reduced the area of ​​pulmonary fibrosis and had the best efficacy; R-1 was more effective than S-1 (P < 0.001); R-2 (P < 0.001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.01) was as effective as the positive control drug Nintedanib and slightly better than Pirfenidone.

[0133] Table 13. Statistics on the pulmonary fibrosis area in mice modeled by Silica-induced pulmonary fibrosis for each compound.

[0134] 2.4 Total Cell Count in Bronchoalveolar Lavage Fluid: After drug administration, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was stained with H&E. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. Table 14 shows that, compared with the control group, the total cell count in the bronchoalveolar lavage fluid (BALF) of the model group was significantly increased. The positive control drug Pirfenidone (P < 0.001) significantly reduced the total cell count in BALF, while the positive control drug Nintedanib (P > 0.05) had no significant effect on reducing the total cell count in BALF. R-1 (P < 0.0001) significantly reduced the total cell count in BALF, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001). R-2 (P < 0.01) also significantly reduced the total cell count in BALF, with significantly better efficacy than the positive control drug Nintedanib, but slightly less effective than Pirfenidone; S-2 (P < 0.01) was more effective than the positive control drug Nintedanib.

[0135] Table 14. Total BALF cell count in mice with Silica-induced pulmonary fibrosis model for each compound.

[0136] 2.5 Expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After administration, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table. The indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea with a syringe to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected by ELISA. As shown in Table 15, compared with the control group, the protein content of pro-inflammatory cytokines TGF-β, TNF-α, IL-6 and IL-1β in the model group was significantly increased. (1) Pro-inflammatory cytokine TGF-β: The positive control drugs Pirfenidone (P<0.001) and Nintedanib (P<0.001) significantly inhibited the expression of TGF-β protein. R-1 (P < 0.0001) significantly inhibited TGF-β protein expression in BALF, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced TGF-β protein expression in BALF, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) was slightly less effective than the positive control drugs Pirfenidone and Nintedanib.

[0137] (2) Pro-inflammatory cytokine TNF-α: The positive control drugs Pirfenidone (P<0.01) and Nintedanib (P<0.001) significantly inhibited the expression of TNF-α protein. R-1 (P<0.0001) significantly inhibited the protein expression of TNF-α in BALF, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the protein expression of TNF-α in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0138] (3) Pro-inflammatory cytokine IL-6: The positive control drugs Pirfenidone (P<0.01) and Nintedanib (P<0.0001) significantly inhibited the expression of IL-6 protein. R-1 (P<0.0001) significantly inhibited the expression of IL-6 protein in BALF, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.001) also significantly reduced the expression of IL-6 protein in BALF, which was significantly better than the positive control drug Pirfenidone, and its efficacy was comparable to that of the positive control drug Nintedanib; S-2 (P<0.01) was slightly more effective than the positive control drug Pirfenidone.

[0139] (4) Pro-inflammatory cytokine IL-1β: The positive control drugs Pirfenidone (P<0.001) and Nintedanib (P<0.0001) significantly inhibited the expression of IL-1β protein. R-1 (P<0.0001) was the most effective at significantly inhibiting the expression of IL-1β protein in BALF; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the expression of IL-1β protein in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0140] Table 15. BALF inflammatory cytokine levels of each compound in Silica-induced pulmonary fibrosis model mice.

[0141] Pharmacological Example 4: Prevention of LPS-induced acute lung injury in mice

[0142] 1. Experimental Methods

[0143] An acute lung injury model was established in male C57BL / 6 mice using lipopolysaccharide (LPS) (3 mg / kg) administered intratracheally. The mice were divided into four groups of eight mice each, based on body weight, one day prior to modeling: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 100 mg / kg qd), S-1 group (S-1 100 mg / kg qd), R-2 group (R-2 100 mg / kg qd), and S-2 group (S-2 100 mg / kg qd). Each group received the prescribed dose via gavage once the day before modeling and once 1 hour before modeling, for a total of two administrations. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon. Mice were euthanized and sampled 24 hours after modeling.

[0144] 2. Experimental Results

[0145] After the experiment, the forced vital capacity, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in each group of mice were measured. The results are as follows:

[0146] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the lung function data of mice measured using the AniRes2005 animal lung function analysis system. Table 16 shows that compared with the control group, the model group showed a decrease in FVC. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P>0.05) did not significantly improve the decrease in FVC. R-1 (P<0.0001) significantly improved the decrease in FVC caused by LPS, exhibiting the best efficacy. R-1 was more effective than S-1 (P<0.0001). R-2 (P<0.001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib. S-2 (P<0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0147] Table 16. FVC results of LPS-induced acute lung injury model mice for each compound.

[0148] 2.2 Total Cell Count in Bronchoalveolar Lavage Fluid: After the experiment, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysis buffer was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was used for H&E staining. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. Cell counts were performed using a cell counter. Table 17 shows that, compared with the control group, the total cell count in the bronchoalveolar lavage fluid (BALF) of the model group was significantly higher. The positive control drugs Pirfenidone (P < 0.001) and Nintedanib (P < 0.001) could alleviate the LPS-induced increase in the total cell count in BALF. R-1 (P < 0.0001) significantly reduced the total cell count in BALF, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001). R-2 (P < 0.0001) also significantly reduced the total cell count in BALF, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib. S-2 (P < 0.001) was slightly more effective than the positive control drug Nintedanib, but slightly less effective than Pirfenidone.

[0149] Table 17. Total BALF cell count in mice with LPS-induced acute lung injury model for each compound.

[0150] 2.3 Content of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After the experiment, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table. The indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea with a syringe to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected by ELISA. As shown in Table 18, compared with the control group, the protein content of cytokines TGF-β, TNF-α, IL-6 and IL-1β in the model group was significantly increased. (1) TGF-β: The positive control drugs Pirfenidone (P<0.0001) and Nintedanib (P<0.0001) significantly inhibited the expression of TGF-β protein. R-1 (P < 0.0001) significantly inhibited TGF-β protein expression in BALF, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced TGF-β protein expression in BALF, which was significantly better than the positive control drug Nintedanib, and its efficacy was equal to that of Pirfenidone.

[0151] (2) TNF-α: The positive control drugs Pirfenidone (P<0.001) and Nintedanib (P<0.01) significantly inhibited the expression of TNF-α protein. R-1 (P<0.0001) was the most effective at significantly inhibiting the expression of TNF-α protein in BALF; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the expression of TNF-α protein in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0152] (3) IL-6: The positive control drugs Pirfenidone (P<0.0001) and Nintedanib (P<0.05) significantly inhibited the expression of IL-6 protein. R-1 (P<0.0001) significantly inhibited the expression of IL-6 protein in BALF, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the expression of IL-6 protein in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.001) had an efficacy comparable to the positive control drug Pirfenidone, and was slightly better than Nintedanib.

[0153] (4) IL-1β: The positive control drugs Pirfenidone (P<0.0001) and Nintedanib (P<0.001) significantly inhibited the expression of IL-1β protein. R-1 (P<0.0001) was the most effective at significantly inhibiting the expression of IL-1β protein in BALF; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.0001) also significantly reduced the expression of IL-1β protein in BALF, which was significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0154] Table 18. Levels of inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) of each compound in a mouse model of LPS-induced acute lung injury.

[0155] Pharmacological Example 5: Treatment of Bleomycin-Induced SSc-ILD

[0156] 1. Experimental Methods

[0157] Male C57BL / 6 mice were used to establish an SSc-ILD model by subcutaneous injection of bleomycin (1.5 U / mouse / day) for 28 consecutive days. Day 1 was the day of model establishment, and mice were weighed daily. Fourteen days after model establishment, mice were regrouped based on their rate of weight loss, with eight mice in each group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Mice were given medication starting on day 14. Each group was administered the medication by gavage according to the dosage. The Ctl group and Model group were administered sterile water by gavage as a control. The general condition of the mice was observed twice a day, once in the morning and once in the afternoon, until day 28.

[0158] 2. Experimental Results

[0159] After the experiment, the forced vital capacity, dermal thickness, degree of pulmonary fibrosis, and hydroxyproline content in the skin and lung tissue of each group of mice were measured. The results are as follows:

[0160] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 19 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The positive control drug Pirfenidone (P > 0.05) showed no significant improvement in FVC, while Nintedanib (P < 0.05) showed some improvement. R-1 (P < 0.0001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P < 0.001); R-2 (P < 0.01) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.05) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0161] Table 19. FVC results of SSc-ILD model mice for each compound.

[0162] 2.2 Dermal Thickness Detection: Specifically, skin samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5μm thick sections, and stained with H&E. As shown in Table 20, compared with the control group, the dermal layer of the model group was significantly thicker; the positive control drug Pirfenidone (P>0.05) group did not show a significant decrease compared to the model group, while the Nintedanib group (P<0.01) showed a more significant decrease. R-1 (P<0.0001) significantly reduced the dermal thickness of mice, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.001); R-2 (P<0.001) also significantly reduced the dermal thickness of mice, significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.01) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0163] Table 20: Statistical analysis of dermal thickness in SSc-ILD model mice for each compound.

[0164] 2.3 Statistical Analysis of Pulmonary Fibrosis Degree: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 21, compared with the control group, the model group showed obvious fibrosis at the edge of the lung tissue. The positive control drug Pirfenidone (P>0.05) did not significantly improve the pulmonary fibrosis area, while Nintedanib (P<0.001) significantly alleviated the pulmonary fibrosis area. R-1 (P<0.0001) significantly reduced the pulmonary fibrosis area and had the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.01) was significantly more effective than the positive control drug Pirfenidone, but slightly less effective than Nintedanib.

[0165] Table 21. Statistics on pulmonary fibrosis area in SSc-ILD model mice for each compound.

[0166] 2.4 Hydroxyproline content determination: The determination of skin or lung collagen content is the determination of hydroxyproline content. Specifically, after euthanizing mice, the skin tissue or right lung of the mice is separated and placed in a 5 mL ampoule. The ampoule is dried in an oven at 120℃, hydrolyzed in hydrochloric acid, and the pH value is adjusted to 6.5-8.0. The residue is filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced physiological saline) is added to adjust the total volume to 10 mL. 50 μL of the sample is taken, 350 μL of deionized water is added, 200 μL of chloramine T solution is added and incubated at room temperature for 20 min, 200 μL of perchloric acid is added and incubated at room temperature for 5 min, and 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) is added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume), thus obtaining the hydroxyproline content. Similarly, the amount of hydroxyproline in the skin was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g), thus obtaining the hydroxyproline content. In skin tissue, as shown in Table 22, compared with the control group, the HYP content per g of skin was significantly increased in the model group. The positive control drug Pirfenidone (P > 0.05) did not significantly reduce the skin HYP content, while Nintedanib (P < 0.01) significantly reduced the skin HYP content. R-1 (P < 0.0001) significantly reduced HYP content in the skin, showing the best efficacy; R-1 was more effective than S-1 (P < 0.001) in inhibiting HYP in the skin; R-2 (P < 0.01) was also significantly more effective than the positive control drug Pirfenidone in inhibiting HYP, but slightly less effective than the positive control drug Nintedanib; S-2 (P > 0.05) was slightly more effective than the positive control drug Pirfenidone.

[0167] In lung tissue, as shown in Table 23, compared with the control group, the HYP content in the right lung of the model group was significantly increased. The positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) significantly reduced HYP content. R-1 (P < 0.0001) significantly reduced HYP content, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001) in inhibiting HYP; R-2 (P < 0.01) was also significantly more effective than the positive control drug Pirfenidone in inhibiting HYP, but slightly less effective than the positive control drug Nintedanib; S-2 (P < 0.01) was slightly more effective than the positive control drug Pirfenidone.

[0168] Table 22 Hydroxyproline content in the skin of SSc-ILD model mice for each compound

[0169] Table 23 shows the hydroxyproline content in the lungs of SSc-ILD model mice for each compound.

[0170] Pharmacological Example 6: Treatment of Spontaneous SLE-ILD in MRL / Lpr Mice

[0171] 1. Experimental Methods

[0172] Male MRL / Lpr mice, eight per group, were fed until 16 weeks of age when spontaneous SLE-ILD developed. Treatment was initiated at each of the following groups: control (Ctl), model (Model), positive control pirfenidone group (200 mg / kg qd), positive control nintedanib group (60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began at week 16, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until week 18.

[0173] 2. Experimental Results

[0174] After the experiment, the forced vital capacity, dermal thickness, spleen coefficient, total BALF cell count, and peripheral blood leukocyte count of mice in each group were measured. The results are as follows:

[0175] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 24 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P>0.05) had no significant effect on FVC relief. R-1 (P<0.001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.05); R-2 (P<0.05) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P>0.05) was slightly more effective than the positive control drug Pirfenidone but slightly less effective than Nintedanib.

[0176] Table 24. FVC results of each compound in SLE-ILD model mice.

[0177] 2.2 Dermal Thickness Detection: Specifically, skin samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5μm thick sections, and stained with H&E. Table 25 shows that compared to the control group, the dermal layer of the model group was significantly thicker. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P>0.05) did not significantly reduce dermal thickness in mice. R-1 (P<0.0001) significantly reduced dermal thickness in mice, showing the best efficacy; R-1 was more effective than S-1 (P<0.001). R-2 (P<0.001) also significantly reduced dermal thickness in mice, significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.05) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0178] Table 25. Statistics on dermal thickness of each compound in SLE-ILD model mice.

[0179] 2.3 Spleen Coefficient: The spleen coefficient was determined by weighing the spleen of mice after necropsy and dividing the spleen weight by the body weight. Table 26 shows that the spleen coefficient in the model group was significantly higher than that in the control group. The positive control drug Pirfenidone (P>0.05) showed no significant reduction in this index, while Nintedanib (P<0.001) significantly reduced the spleen coefficient. R-1 (P<0.0001) significantly reduced the mouse spleen coefficient, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.0001). R-2 (P<0.0001) also significantly reduced the mouse spleen coefficient, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.0001) was slightly more effective than both Pirfenidone and Nintedanib.

[0180] Table 26 Spleen coefficient statistics for each compound in SLE-ILD model mice

[0181] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a control table, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This determined the effect of the analyte on the number of inflammatory cells in the BALF. As shown in Table 27, compared with the control group, the total BALF cell count in the model group was significantly increased. The positive control drug Pirfenidone (P > 0.05) had no significant effect on this indicator, while Nintedanib (P < 0.001) significantly reduced the total BALF cell count. R-1 (P < 0.0001) significantly reduced the total number of BALF cells, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.001) also significantly reduced the total number of BALF cells, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) was slightly more effective than the positive control drug Pirfenidone, but slightly less effective than Nintedanib.

[0182] Table 27. Total BALF cell count in SLE-ILD model mice for each compound.

[0183] 2.5 Serum White Blood Cell Count: Peripheral blood white blood cell count was specifically determined by collecting approximately 500 μL of peripheral blood from the eyeballs after mouse sacrifice, and immediately analyzing the total white blood cell count using a blood biochemistry analyzer. As shown in Table 28, compared to the control group, the model group showed a significant increase in peripheral blood white blood cell count; the positive control drugs Pirfenidone (P > 0.05) and Nintedanib (P > 0.05) had no significant effect on this indicator. R-1 (P < 0.0001) significantly reduced white blood cell count, exhibiting the best efficacy; R-1 was more effective than S-1 (P < 0.001); R-2 (P < 0.01) also significantly reduced white blood cell count, with significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.05) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0184] Table 28 Peripheral blood leukocyte counts in SLE-ILD model mice for each compound

[0185] Pharmacological Example 7: Treatment of skeletal muscle homogenate-induced PM-ILD

[0186] 1. Experimental Methods

[0187] Female BALB / c mice were induced to develop a rat skeletal muscle homogenate model by subcutaneous injection on days 0, 7, 14, 21, and 28. Pertussis toxin (2 μg / mouse) was administered intraperitoneally on days 0 and 7 to boost immunization. Day 0 was the day of model initiation, and mice were weighed daily. On day 21 of model initiation, mice were regrouped according to their weight loss rate, with 8 mice in each group: control group (Ctl), model group (Model), positive control pirfenidone group (Pirfenidone 200 mg / kg qd), positive control nintedanib group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Mice were given treatment starting on day 21. Each group was administered the drug by gavage according to the dosage. The Ctl group and Model group were given sterile water as a control by gavage. The general condition of the mice was observed twice a day, once in the morning and once in the afternoon, until day 35.

[0188] 2. Experimental Results

[0189] The degree of myositis was scored throughout the experiment. After the experiment, the forced vital capacity, degree of pulmonary fibrosis, and spleen coefficient of each group of mice were measured. The results are as follows:

[0190] 2.1 Myositis Score: The myositis score refers to the clinical symptoms observed in mice after immunization and scored accordingly. The clinical symptom score followed the Lennon scoring method: 0 points, no obvious muscle weakness; 1 point, animal unable to vocalize or bite; 2 points, hunched posture at rest, head drooping, trembling gait; 3 points, severe muscle weakness, significant weight loss, even muscle atrophy, no vocalization, labored breathing, near death (moderate performance was scored as 0.5, 1.5, or 2.5). Myositis scores were assessed on day 35 (D35) of modeling. The results are shown in Table 29. Compared with the control group, the myositis score in the D35 model group was significantly higher; the positive control drug Nintedanib (P>0.05) did not significantly reduce the myositis score, while Pirfenidone (P<0.01) significantly reduced the myositis score. R-1 (P < 0.0001) significantly reduced myositis scores, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced myositis scores, showing a marked improvement over the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0191] Table 29 Score of myositis severity at D35 in PM-ILD model mice for each compound.

[0192] 2.2 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 30 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P>0.05) had no effect on FVC relief. R-1 (P<0.001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.01); R-2 (P<0.01) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P>0.05) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0193] Table 30: FVC results of PM-ILD model mice for each compound.

[0194] 2.3 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 31, the model group showed obvious pulmonary fibrosis. The positive control drug Pirfenidone (P>0.05) did not significantly improve the pulmonary fibrosis area, while Nintedanib (P<0.01) significantly reduced the pulmonary fibrosis area. R-1 (P<0.0001) could significantly reduce the pulmonary fibrosis area and had the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0195] Table 31. Statistics on pulmonary fibrosis area in PM-ILD model mice for each compound.

[0196] 2.4 Spleen Coefficient: The spleen coefficient was determined by weighing the spleen of mice after necropsy and dividing the spleen weight by the body weight. Table 32 shows that the spleen coefficient in the model group was significantly higher than that in the control group. The positive control drugs Pirfenidone (P < 0.001) and Nintedanib (P < 0.0001) significantly reduced the spleen coefficient. R-1 (P < 0.0001) significantly reduced the spleen coefficient in mice, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001). R-2 (P < 0.0001) also significantly reduced the spleen coefficient in mice, with significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0197] Table 32 Spleen coefficient statistics for PM-ILD model mice of each compound

[0198] Pharmacological Example 8: Treatment of Collagen Immunotherapy and Bleomycin-Induced RA-ILD

[0199] 1. Experimental Methods

[0200] Male DBA1 / J wild-type mice (6-8 weeks old) were used to establish the pulmonary fibrosis model by intradermal injection of type II collagen emulsified with an adjuvant at the base of the tail. Mice were immunized twice, with a booster immunization given on day 21 after the initial immunization. Control mice received the same amount of adjuvant at the same time. Day 0 was the day of model establishment, and mice were weighed daily. On day 35, mice were regrouped according to their rate of weight loss, with six mice in each group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Pulmonary fibrosis was induced by intratracheal injection of bleomycin. Mice were given treatment starting on day 35, with each group receiving the prescribed dose via gavage. The Ctl and Model groups were given sterile water as a control. The mice were observed in their general condition twice a day, once in the morning and once in the afternoon, until day 50.

[0201] 2. Experimental Results

[0202] The degree of RA was scored throughout the experiment; after the experiment, the forced vital capacity, degree of pulmonary fibrosis, and total number of BALF cells in each group of mice were measured. The results are as follows:

[0203] 2.1 RA Score: The mouse arthritis index was determined by scoring the mice every three days, starting from the second booster immunization on day 21, until day 50. Each paw of each mouse was scored, and the scores of all four limbs were summed to obtain the mouse's arthritis index. The scoring criteria were as follows: 0 = no erythema or swelling; 1 = mild erythema or swelling of one toe; 2 = erythema or swelling of more than one toe; 3 = erythema and swelling of the ankle or wrist; 4 = severe erythema and severe edema of the toes and ankles or fingers and wrists, with the ankle or wrist unable to bend normally. The range of the mouse arthritis index was 0–16. Table 33 shows that compared with the control group, the RA score in the model group was significantly higher; the positive control drug Nintedanib (P > 0.05) did not significantly reduce the RA score, while Pirfenidone (P < 0.01) significantly reduced the RA score. R-1 (P < 0.0001) significantly reduced RA scores, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced RA scores, with efficacy significantly better than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0204] Table 33. D50 RA severity scores of RA-ILD model mice for each compound.

[0205] 2.2 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 34 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The two positive control drugs, Pirfenidone (P>0.05) and Nintedanib (P>0.05), had no significant effect on FVC relief. R-1 (P<0.0001) significantly improved the decrease in FVC, exhibiting the best efficacy; R-1 was more effective than S-1 (P<0.001); R-2 (P<0.01) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.01) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0206] Table 34. FVC results of each compound in RA-ILD model mice.

[0207] 2.3 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 35, compared with the control group, the model group showed obvious fibrosis. The positive control drugs Pirfenidone (P < 0.001) and Nintedanib (P < 0.0001) significantly reduced the pulmonary fibrosis area. R-1 (P < 0.0001) significantly reduced the pulmonary fibrosis area and had the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) was also significantly more effective than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0208] Table 35. Statistics on pulmonary fibrosis area in RA-ILD model mice for each compound.

[0209] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a control table, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 36 shows that compared to the control group, the total BALF cell count in the model group was significantly increased; the positive control drugs Pirfenidone (P < 0.001) and Nintedanib (P < 0.001) significantly reduced the total BALF cell count. R-1 (P < 0.0001) significantly reduced the total number of BALF cells, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) also significantly reduced the total number of BALF cells, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P < 0.0001) was slightly more effective than the positive control drugs Pirfenidone and Nintedanib.

[0210] Table 36. Total BALF cell count in RA-ILD model mice for each compound.

[0211] Pharmacological Example 9: Treatment of UUO-induced renal fibrosis

[0212] 1. Experimental Methods

[0213] Male C57BL / 6 mice were used to establish a renal fibrosis model using unilateral ureteral ligation (UUO). Day 0 was the day of model establishment, and mice were weighed daily. Fourteen days after model establishment, mice were regrouped based on their weight loss rate, with eight mice in each group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began on day 14, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until day 28 of treatment.

[0214] 2. Experimental Results

[0215] After the experiment, the renal hydroxyproline content, serum urea nitrogen, and serum creatinine content of mice in each group were measured. The results are as follows:

[0216] 2.1 Renal Collagen Content: The renal collagen content was determined by detecting hydroxyproline content. Specifically, after euthanizing mice, the kidney tissue was separated and weighed, placed in 5 mL ampoules, dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was transferred to a 96-well plate, and the absorbance at 570 nm was measured. A standard curve was plotted using the standard readings, and the hydroxyproline concentration (Cs) of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline per gram of kidney tissue was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / kidney tissue mass. The hydroxyproline content was then obtained, and the specific results are shown in Table 37. Compared with the control group, the model group showed a significant increase in renal collagen content. The positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) significantly reduced renal collagen content. R-1 (P < 0.0001) significantly reduced renal collagen content, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001). R-2 (P < 0.001) also significantly reduced renal collagen content, with efficacy comparable to Nintedanib, and significantly better than the positive control drug Pirfenidone; S-2 (P < 0.01) was slightly more effective than the positive control drug Pirfenidone.

[0217] Table 37 Collagen content in the kidney tissue of mice with renal fibrosis model for each compound.

[0218] 2.2 Serum Urea Nitrogen Content: Serum urea nitrogen content was measured by collecting blood from the eyes of euthanized mice, allowing them to stand for 30 minutes, and then centrifuging at 3000 rpm for 15 minutes to collect serum samples. The urea nitrogen content in the serum was then determined using ELISA. The specific results are shown in Table 38. Compared with the control group, the model group showed a significant increase in serum urea nitrogen. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P<0.01) significantly reduced serum urea nitrogen content. R-1 (P<0.0001) significantly reduced serum urea nitrogen content, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001). R-2 (P<0.001) and S-2 (P<0.01) also significantly reduced serum urea nitrogen content, with significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib.

[0219] Table 38 Serum urea nitrogen levels in mice with renal fibrosis model for each compound.

[0220] 2.3 Serum Creatinine Level: Serum creatinine level detection involved collecting blood from the eyes of euthanized mice, allowing them to stand for 30 minutes, centrifuging at 3000 rpm for 15 minutes to collect serum samples, and then measuring the serum creatinine level using ELISA. Specific results are shown in Table 39. Compared with the control group, the model group showed a significant increase in serum creatinine. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P<0.01) significantly reduced serum creatinine levels. R-1 (P<0.0001) significantly reduced serum creatinine levels, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001). R-2 (P<0.001) significantly reduced serum creatinine levels, showing significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib; S-2 (P<0.01) also significantly reduced serum urea nitrogen levels, showing significantly better efficacy than the positive control drug Pirfenidone.

[0221] Table 39 Serum creatinine levels in mice with renal fibrosis model for each compound

[0222] Pharmacological Example 10: Treatment of Carbon Tetrachloride-Induced Liver Fibrosis

[0223] 1. Experimental Methods

[0224] Male C57BL / 6 mice were used to establish a liver fibrosis model by continuous intraperitoneal injection of carbon tetrachloride for 8 weeks. Day 0 was the day of model establishment, and mice were weighed daily. At week 9, mice were regrouped based on their rate of weight loss, with 8 mice in each group: control group (Ctl), model group (Model), positive control group (pirfenidone 200 mg / kg qd), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began at week 9, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received saline as a control. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until week 10.

[0225] 2. Experimental Results

[0226] After the experiment, the liver hydroxyproline content and blood alanine aminotransferase content of mice in each group were measured. The results are as follows:

[0227] 2.1 Hepatic Hydroxyproline Content: After euthanizing mice, liver tissue was isolated and weighed, placed in 5 mL ampoules, and dried in a 120℃ oven. After hydrolysis with hydrochloric acid, the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. 200 μL of perchloric acid was added and incubated at room temperature for 5 min. 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was transferred to a 96-well plate, and the absorbance at 570 nm was measured. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline per gram of liver tissue was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / liver tissue weight. The hydroxyproline content was then obtained, and the specific results are shown in Table 40. Table 40 shows that the liver collagen content in the model group was significantly increased; the positive control drugs Pirfenidone (P>0.05) and Nintedanib (P<0.001) reduced liver collagen content. R-1 (P<0.0001) significantly reduced liver collagen content, showing the best efficacy; R-1 was more effective than S-1 (P<0.0001); R-2 (P<0.01) and S-2 (P<0.01) also significantly reduced liver collagen content, with significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib.

[0228] Table 40 shows the collagen content in the liver tissue of mice with liver fibrosis model for each compound.

[0229] 2.2 Serum Alanine Aminotransferase (ALT) Levels: Serum ALT levels were measured at 37℃ and pH 7.4. ALT catalyzes the amino-transfer reaction between alanine and α-ketoglutarate, producing pyruvate and glutamic acid. After the reaction time, phenylhydrazine is added, reacting with pyruvate to form phenylhydrazone. Phenyrohydrazone is reddish-brown under alkaline conditions. ALT levels can be quantified by detecting absorbance. Specific results are shown in Table 41. Table 41 shows that serum ALT levels were significantly increased in the model group; the positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.01) significantly reduced serum ALT levels. R-1 (P < 0.0001) significantly reduced serum alanine aminotransferase (ALT) levels, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) and S-2 (P < 0.001) also significantly reduced serum ALT levels, with efficacy significantly superior to the positive control drugs Pirfenidone and Nintedanib.

[0230] Table 41 Serum alanine aminotransferase levels in mice with liver fibrosis model for each compound.

[0231] Pharmacological Example 11: Treatment of Isoproterenol-Induced Cardiac Fibrosis in Mice

[0232] 1. Experimental Methods

[0233] Male C57BL / 6 mice were subcutaneously injected with 10 mg / kg isoproterenol daily for 7 consecutive days; control mice were subcutaneously injected with an equal volume of physiological saline. Day 0 was the day of model establishment. Mice were weighed daily, with 8 mice per group. The specific groupings were as follows: control group (Ctl), model group (Model), positive control group (Pirfenidone 200 mg / kg qd), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), S-1 group (S-1 50 mg / kg bid), R-2 group (R-2 50 mg / kg bid), and S-2 group (S-2 50 mg / kg bid). Treatment began on Day 1, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received physiological saline as a control. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 7 of treatment.

[0234] 2. Experimental Results

[0235] After the experiment, the heart-to-body ratio, heart-to-tibia ratio, and cardiac fibrosis area were measured in each group of mice. The results are as follows:

[0236] 2.1 Heart-to-body ratio: The body weight (M) of the mice was measured. After anesthesia, the thoracic cavity was opened, and residual blood was rinsed with pre-cooled PBS. The water around the heart was dried with filter paper, and the heart weight (m) was recorded. The heart-to-body ratio = m / M. Specific results are shown in Table 42. The heart-to-body ratio in the model group was significantly increased. The positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) could reduce the heart-to-body ratio. R-1 (P < 0.0001) significantly reduced the heart-to-body ratio, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.001) also significantly reduced the heart-to-body ratio, with efficacy significantly better than the positive control drug Pirfenidone and equivalent to Nintedanib.

[0237] Table 42 Heart-to-body ratio in mouse models of cardiac fibrosis for each compound

[0238] 2.2 Heart-to-Tibia Ratio: After anesthetizing the mice, the thoracic cavity was opened, residual blood was rinsed with pre-cooled PBS, and the water around the heart was dried with filter paper. The heart weight (m) was weighed and recorded. The right tibia of the mouse was removed, and the tibia length (l) was measured with calipers. The heart-to-tibia ratio was calculated as m / l. Specific results are shown in Table 43. The heart-to-tibia ratio was significantly increased in the model group. The positive control drugs Pirfenidone (P < 0.05) and Nintedanib (P < 0.001) could reduce the heart-to-tibia ratio. R-1 (P < 0.0001) significantly reduced the heart-to-tibia ratio, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.001) also significantly reduced the heart-to-tibia ratio, with significantly better efficacy than the positive control drugs Pirfenidone and Nintedanib.

[0239] Table 43 Cardiac tibia ratio in mice with cardiac fibrosis model of each compound

[0240] 2.3 Percentage of Cardiac Fibrosis Area: Mice were euthanized after anesthesia, and tissue samples were collected. The mouse heart was cut open at the midpoint of its short axis, and the base of the heart was fixed in 4% neutral formaldehyde. The heart tissue was then immersed in paraffin for 2 hours to obtain embedded tissue blocks. The tissue blocks were then sectioned to a thickness of 5 μm. The heart tissue sections were sequentially immersed in the following reagents: xylene for 10 min, 3 times; 100% ethanol for 3 min, 2 times; 95% ethanol for 3 min, 2 times; 80% ethanol for 3 min, 1 time; 70% ethanol for 3 min, 1 time; and finally washed 3 times with double-distilled water. The heart tissue sections were blotted dry with absorbent paper and stained in saturated picric acid-sirius red staining solution for 8 minutes. The sections were then rinsed in anhydrous ethanol for several minutes. After stained sections were dried in a 60°C oven, cleared with xylene for 5 minutes, and mounted with neutral resin. Microscopic examination was performed, and images of the sections were acquired and analyzed. The fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of cardiac fibrosis area was calculated as: (cardiac fibrosis area / total cardiac section area) × 100%. Specific results are shown in Table 44. The cardiac fibrosis area was significantly increased in the model group. The positive control drugs Pirfenidone (P>0.05) and Nintedanib (P<0.01) reduced the cardiac fibrosis area. R-1 (P < 0.0001) significantly reduced the area of ​​cardiac fibrosis, showing the best efficacy; R-1 was more effective than S-1 (P < 0.0001); R-2 (P < 0.0001) and S-2 (P < 0.001) also significantly reduced the area of ​​cardiac fibrosis, with efficacy significantly superior to the positive control drugs Pirfenidone and Nintedanib.

[0241] Table 44 shows the area of ​​cardiac fibrosis in mouse models of cardiac fibrosis for each compound.

[0242] Pharmacological Example 12: Treatment of bleomycin-induced pulmonary fibrosis in aging mice

[0243] 1. Experimental Methods

[0244] C57BL / 6 aging male mice (12 months old) were used to establish a pulmonary fibrosis model using bleomycin (2 U / kg) via intratracheal injection. Day 1 was the day of model establishment, and mice were weighed daily. On day 7, mice were regrouped based on their rate of weight loss, with 8 mice in each group: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 8, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 14 of treatment.

[0245] 2. Experimental Results

[0246] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, and pulmonary fibrosis area of ​​the mice in each group were measured. The results are as follows:

[0247] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 45 shows that compared to the control group, the model group exhibited a significant decrease in FVC. R-1 significantly improved the decrease in FVC (P < 0.001), while X-1 and S-1 also significantly improved FVC (P < 0.01), but slightly less effectively than R-1. These three compounds showed better efficacy than the positive control drug Nintedanib (P < 0.05), with R-1 showing the best effect.

[0248] Table 45. FVC results of bleomycin-based mouse models for each compound.

[0249] 2.2 Hydroxyproline Content Determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were euthanized, and the right lung was isolated and placed in a 5 mL ampoule. The ampoule was dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). As shown in Table 46, compared with the control group, the HYP content in the whole right lung of the model group was significantly increased. R-1 and X-1 significantly reduced the HYP content (P < 0.001), and S-1 also significantly reduced the HYP content (P < 0.01), but slightly less than R-1 and X-1. The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P < 0.05), with R-1 being the most effective.

[0250] Table 46. Hydroxyproline content in the lungs of bleomycin-induced model mice for each compound.

[0251] 2.3 Statistical analysis of pulmonary fibrosis area: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 47, compared with the control group, the pulmonary fibrosis area in the model group was significantly increased. R-1 and X-1 significantly reduced the pulmonary fibrosis area (P < 0.0001), and S-1 also significantly reduced the pulmonary fibrosis area (P < 0.001), slightly worse than R-1 and X-1. The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P < 0.05), with R-1 being the best.

[0252] Table 47. Statistics on pulmonary fibrosis area in bleomycin model mice for each compound.

[0253] Pharmacological Example 13: Treatment of early pulmonary fibrosis in bleomycin-induced aging mice

[0254] 1. Experimental Methods

[0255] C57BL / 6 aging male mice (12 months old) were used to establish a pulmonary fibrosis model using bleomycin (2 U / kg) administered intratracheally. On the day of model establishment, mice were divided into groups of eight based on body weight: control group (Ctl), model group (Model), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Day 1 was the day of model establishment, and mouse body weight was measured daily. Treatment began on day 2, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as a control. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 8 of treatment.

[0256] 2. Experimental Results

[0257] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, pulmonary fibrosis area, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in BALF were measured in each group of mice. The results are as follows:

[0258] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the determination of lung function data in mice using the AniRes2005 animal lung function analysis system. Table 48 shows that compared to the control group, the model group showed a significant decrease in FVC. R-1 significantly improved the decrease in FVC (P < 0.01), and X-1 and S-1 also significantly improved the decrease in FVC (P < 0.05), but slightly less effectively than R-1. The efficacy of these three compounds was superior to the positive control drug Nintedanib (P > 0.05), with R-1 showing the best effect.

[0259] Table 48. FVC results of bleomycin model mice for each compound.

[0260] 2.2 Hydroxyproline Content Determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were euthanized, and the right lung was isolated and placed in a 5 mL ampoule. The ampoule was dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). As shown in Table 49, compared with the control group, the HYP content in the whole right lung of the model group was significantly increased; R-1 significantly reduced the HYP content (P < 0.001), and X-1 (P < 0.01) and S-1 (P < 0.05) also significantly improved the decrease in FVC, but were slightly worse than R-1. The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P < 0.05), with R-1 being the best.

[0261] Table 49. Hydroxyproline content in the lungs of bleomycin-induced model mice for each compound.

[0262] 2.3 Statistical analysis of pulmonary fibrosis area: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 50, compared with the control group, the pulmonary fibrosis area in the model group was significantly increased. R-1 and X-1 significantly reduced the pulmonary fibrosis area (P < 0.0001), and S-1 also significantly reduced the pulmonary fibrosis area (P < 0.001), but was slightly worse than R-1 and X-1. The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P < 0.001), with R-1 being the best.

[0263] Table 50: Statistics on pulmonary fibrosis area in bleomycin-induced mouse models of various compounds.

[0264] 2.4 Total Cell Count in Bronchoalveolar Lavage Fluid: After drug administration, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was stained with H&E. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. The cell count was determined using a cell counter, as shown in Table 51. Compared with the control group, the total number of BALF cells in the model group was significantly increased. R-1, X-1, and S-1 all significantly reduced the total number of BALF cells (P < 0.0001). The efficacy of these three compounds was superior to that of the positive control drug Nintedanib (P < 0.001), with R-1 being the most effective.

[0265] Table 51 Total BALF cell counts in bleomycin-treated mouse models of each compound

[0266] 2.5 Expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After drug administration, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table, and an indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected using the ELISA method. As shown in Table 52, compared with the control group, the protein content of pro-inflammatory cytokines TGF-β, TNF-α, IL-6, and IL-1β in the model group was significantly increased.

[0267] (1) Pro-inflammatory cytokine TGF-β: R-1, X-1 and S-1 can significantly inhibit the protein expression of TGF-β in BALF (P<0.0001). The efficacy of these three compounds is better than that of the positive control drug Nintedanib (P<0.0001), with R-1 being the best.

[0268] (2) Pro-inflammatory cytokine TNF-α: R-1 significantly inhibited the protein expression of TNF-α in BALF (P<0.001), X-1 and S-1 also significantly inhibited the protein expression of TNF-α in BALF but the significance was slightly worse (P<0.01). The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P<0.05), among which R-1 was the best.

[0269] (3) Pro-inflammatory cytokine IL-6: R-1 significantly inhibited the protein expression of IL-6 in BALF (P<0.001), X-1 and S-1 also significantly inhibited the protein expression of IL-6 in BALF but the significance was slightly worse (P<0.01). The efficacy of these three compounds was better than that of the positive control drug Nintedanib (P<0.05), with R-1 being the best.

[0270] (4) Pro-inflammatory cytokine IL-1β: R-1 and X-1 can significantly inhibit the protein expression of IL-1β in BALF (P<0.001), and S-1 can also significantly inhibit the protein expression of IL-1β in BALF, but the significance is slightly worse (P<0.01). The efficacy of these three compounds is better than that of the positive control drug Nintedanib (P<0.05), among which R-1 is the best.

[0271] Table 52. Bleomycin-induced inflammatory cytokine levels in BALF of mouse model mice.

[0272] Pharmacological Example 14: Treatment of pulmonary fibrosis induced by silica suspension in aging mice

[0273] 1. Experimental Methods

[0274] Male C57BL / 6 mice (12 months old) were used to establish a pulmonary fibrosis model via intratracheal injection of silica suspension (200 mg / kg). Day 1 was the day of model establishment, and mouse weight was measured daily. On day 20, mice were regrouped according to their weight loss rate, with 8 mice in each group: control group (Ctl), model group (Model), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 21, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as a control. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until day 30.

[0275] 2. Experimental Results

[0276] After the experiment, the forced vital capacity, lung tissue hydroxyproline content, pulmonary fibrosis area, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in BALF were measured in each group of mice. The results are as follows:

[0277] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the lung function data of mice measured using the AniRes2005 animal lung function analysis system. Table 53 shows that compared with the control group, the FVC in the model group was significantly decreased. R-1 significantly improved the decrease in FVC (P < 0.0001), X-1 also significantly improved the decrease in FVC but with slightly lower significance (P < 0.001), S-1 also significantly improved the decrease in FVC but with further reduced significance (P < 0.01), and Nintedanib also improved the decrease in FVC (P < 0.05), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the most effective.

[0278] Table 53. FVC results of Silica-induced pulmonary fibrosis model mice for each compound.

[0279] 2.2 Hydroxyproline Content Determination: After drug administration, the hydroxyproline (HYP) content in mouse lung tissue was determined. Mice were euthanized, and the right lung was isolated and placed in a 5 mL ampoule. The ampoule was dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was converted using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume). Table 54 shows that compared with the control group, the HYP content in the whole right lung of the model group was significantly increased; R-1 and X-1 significantly reduced the HYP content in lung tissue (P < 0.0001), and S-1 also significantly reduced the HYP content in lung tissue (P < 0.001), but the significance was slightly weaker. Nintedanib also significantly reduced the HYP content (P < 0.01), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the best.

[0280] Table 54 Hydroxyproline content of each compound in Silica-induced pulmonary fibrosis model mice

[0281] 2.3 Statistical analysis of pulmonary fibrosis area: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 55, compared with the control group, the pulmonary fibrosis area in the model group was significantly increased. R-1 and X-1 significantly reduced the pulmonary fibrosis area (P < 0.0001), and S-1 also significantly reduced the pulmonary fibrosis area, but the significance was slightly weaker (P < 0.001). Nintedanib also improved the pulmonary fibrosis area (P < 0.01), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the best.

[0282] Table 55. Statistics on the pulmonary fibrosis area in mice modeled by Silica-induced pulmonary fibrosis for each compound.

[0283] 2.4 Total Cell Count in Bronchoalveolar Lavage Fluid: After drug administration, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was stained with H&E. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. As shown in Table 56, compared with the control group, the total number of cells in the bronchoalveolar lavage fluid of the model group was significantly increased; R-1 significantly reduced the total number of cells in BALF (P<0.0001), X-1 also significantly reduced the total number of cells in BALF but the significance was slightly worse (P<0.001), and S-1 also significantly reduced the total number of cells in BALF but the significance was further reduced (P<0.01); Nintedanib did not reduce the total number of cells in BALF (P>0.05), and its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0284] Table 56. Total BALF cell count in mice with Silica-induced pulmonary fibrosis model for each compound.

[0285] 2.5 Expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After drug administration, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table, and an indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected using the ELISA method. As shown in Table 57, compared with the control group, the protein content of pro-inflammatory cytokines TGF-β, TNF-α, IL-6, and IL-1β in the model group was significantly increased.

[0286] (1) Pro-inflammatory cytokine TGF-β: R-1 and X-1 can significantly inhibit the protein expression of TGF-β in BALF (P<0.0001), and S-1 can also significantly inhibit the protein expression of TGF-β in BALF, but the significance is slightly worse (P<0.001); Nintedanib can also significantly inhibit the protein expression of TGF-β in BALF (P<0.01), but its efficacy is worse than R-1, X-1 and S-1, among which R-1 is the best.

[0287] (2) Pro-inflammatory cytokine TNF-α: R-1 significantly inhibited the protein expression of TNF-α in BALF (P<0.0001), X-1 also significantly inhibited the protein expression of TNF-α in BALF but the significance was slightly worse (P<0.001), S-1 also significantly inhibited the protein expression of TNF-α in BALF but the significance was further reduced (P<0.01); Nintedanib also significantly inhibited the protein expression of TNF-α in BALF (P<0.05), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0288] (3) Pro-inflammatory cytokine IL-6: R-1 and X-1 can significantly inhibit the protein expression of IL-6 in BALF (P<0.0001), and S-1 can also significantly inhibit the protein expression of IL-6 in BALF, but the significance is slightly worse (P<0.01); Nintedanib can also significantly inhibit the protein expression of IL-6 in BALF (P<0.01), but its efficacy is worse than R-1, X-1 and S-1, among which R-1 is the best.

[0289] (4) Pro-inflammatory cytokine IL-1β: R-1 significantly inhibited the protein expression of IL-1β in BALF (P<0.0001), X-1 also significantly inhibited the protein expression of IL-1β in BALF but the significance was slightly worse (P<0.001), S-1 also significantly inhibited the protein expression of IL-1β in BALF but the significance was further reduced (P<0.01); Nintedanib also significantly inhibited the protein expression of IL-1β in BALF (P<0.05), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0290] Table 57. BALF inflammatory cytokine levels of each compound in Silica-induced pulmonary fibrosis model mice.

[0291] Pharmacological Example 15: Treatment of LPS-induced acute lung injury in aging mice

[0292] 1. Experimental Methods

[0293] Male C57BL / 6 mice (12 months old) were used to establish an acute lung injury model using lipopolysaccharide (LPS) (3 mg / kg) administered intratracheally. The mice were divided into four groups of eight mice each, based on body weight, one day prior to modeling: control group (Ctl), model group (Model), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Each group received the prescribed dose via gavage one day before and one day prior to modeling. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon. Mice were euthanized and sampled 24 hours after modeling.

[0294] 2. Experimental Results

[0295] After the experiment, the forced vital capacity, total cell count in bronchoalveolar lavage fluid (BALF), and expression of pro-inflammatory cytokines in each group of mice were measured. The results are as follows:

[0296] 2.1 Forced Vital Capacity (FVC): Forced vital capacity was measured in mice after anesthesia. FVC measurement refers to the lung function data of mice measured using the AniRes2005 animal lung function analysis system. Table 58 shows that compared with the control group, the FVC in the model group was significantly decreased. R-1 significantly improved the decrease in FVC (P < 0.0001), X-1 also significantly improved the decrease in FVC but with slightly lower significance (P < 0.001), S-1 also significantly improved the decrease in FVC but with further reduced significance (P < 0.01), and Nintedanib also improved the decrease in FVC (P < 0.05), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the most effective.

[0297] Table 58. FVC results of LPS-induced acute lung injury model mice for each compound.

[0298] 2.2 Total Cell Count in Bronchoalveolar Lavage Fluid: After the experiment, the total cell count in the bronchoalveolar lavage fluid was counted. Mice were anesthetized and fixed on the operating table. An indwelling needle was inserted into the trachea, and 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the lavage fluid was collected. The lavage fluid was centrifuged, and the cell pellet was resuspended in red blood cell lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysis buffer was removed. The cells were resuspended in 200 μL of PBS, and 150 μL of the lavage fluid was used for H&E staining. 50 μL of the cell suspension was used to detect the number of viable cells in a live cell counter (three fields of view were randomly selected for each sample) to obtain the cell count in the bronchoalveolar lavage fluid. The cell count was determined using a cell counter. As shown in Table 59, the total cell count in the bronchoalveolar lavage fluid of the model group was significantly higher than that of the control group. R-1 and X-1 significantly reduced the total cell count in BALF (P < 0.0001), and S-1 also significantly reduced the total cell count in BALF, but the significance was slightly weaker (P < 0.001). Nintedanib also significantly reduced the total cell count in BALF (P < 0.05), but its efficacy was worse than that of R-1, X-1, and S-1, with R-1 being the most effective.

[0299] Table 59. Total BALF cell count in mice with LPS-induced acute lung injury model for each compound.

[0300] 2.3 Content of pro-inflammatory cytokines in bronchoalveolar lavage fluid: After the experiment, the protein content of pro-inflammatory cytokines in the bronchoalveolar lavage fluid of mice was detected. Mice were anesthetized and fixed on the operating table, and an indwelling needle cannula was inserted into the trachea. Then, 1 mL of PBS was injected into the lungs of the mice through the trachea to completely lavage the lungs. This was repeated three times, and the bronchoalveolar lavage fluid was collected. The protein content in the bronchoalveolar lavage fluid was quantitatively detected using the ELISA method. As shown in Table 60, compared with the control group, the protein content of pro-inflammatory cytokines TGF-β, TNF-α, IL-6, and IL-1β in the model group was significantly increased.

[0301] (1) Pro-inflammatory cytokine TGF-β: R-1 significantly inhibited TGF-β protein expression in BALF (P<0.0001), X-1 also significantly inhibited TGF-β protein expression in BALF but the significance was slightly worse (P<0.001), S-1 also significantly inhibited TGF-β protein expression in BALF but the significance was further reduced (P<0.01); Nintedanib also significantly inhibited TGF-β protein expression in BALF (P<0.05), and its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0302] (2) Pro-inflammatory cytokine TNF-α: R-1 significantly inhibited the protein expression of TNF-α in BALF (P<0.001), X-1 and S-1 also significantly inhibited the protein expression of TNF-α in BALF but the significance was slightly worse (P<0.01); Nintedanib also significantly inhibited the protein expression of TNF-α in BALF (P<0.05), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0303] (3) Pro-inflammatory cytokine IL-6: R-1 and X-1 can significantly inhibit the protein expression of IL-6 in BALF (P<0.0001), and S-1 can also significantly inhibit the protein expression of IL-6 in BALF, but the significance is slightly worse (P<0.001); Nintedanib can also significantly inhibit the protein expression of IL-6 in BALF (P<0.05), but its efficacy is worse than R-1, X-1 and S-1, among which R-1 is the best.

[0304] (4) Pro-inflammatory cytokine IL-1β: R-1 significantly inhibited the protein expression of IL-1β in BALF (P<0.0001), X-1 also significantly inhibited the protein expression of IL-1β in BALF but the significance was slightly worse (P<0.001), S-1 also significantly inhibited the protein expression of IL-1β in BALF but the significance was further reduced (P<0.01); Nintedanib also significantly inhibited the protein expression of IL-1β in BALF (P<0.05), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0305] Table 60. Levels of inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) of each compound in a mouse model of LPS-induced acute lung injury.

[0306] Pharmacological Example 16: Treatment of Sodium Hypochlorite-Induced SSc-ILD

[0307] 1. Experimental Methods

[0308] Male C57BL / 6 mice (6–8 weeks old) were used to establish an SSc-ILD model by subcutaneous injection of sodium hypochlorite (0.08%, 200 μL / mouse / day) for 84 consecutive days. Day 1 was the day of model establishment, and mice were weighed daily. After 56 days of model establishment, mice were regrouped based on their weight loss rate, with 8 mice per group: control group (Ctl), model group (Model), positive control group (nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 56, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 84 of treatment.

[0309] 2. Experimental Results

[0310] After the experiment, the forced vital capacity, dermal thickness, degree of pulmonary fibrosis, and hydroxyproline content in the skin and lung tissue of each group of mice were measured. The results are as follows:

[0311] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 61 shows that the FVC in the model group was significantly lower than that in the control group. R-1, X-1, and S-1 all significantly improved the decrease in FVC (P < 0.0001). Nintedanib also improved the decrease in FVC (P < 0.05), but its efficacy was worse than R-1, X-1, and S-1, with R-1 showing the best effect.

[0312] Table 61. FVC results of SSc-ILD model mice for each compound.

[0313] 2.2 Dermal thickness detection: The dermal fibrosis thickness was specifically detected by fixing skin samples with 10% formalin, dehydrating, embedding in paraffin, cutting into 5μm thick sections, and performing H&E staining. As shown in Table 62, compared with the control group, the dermal thickness of the model group was significantly increased; R-1 and X-1 significantly improved the dermal thickness of mice (P<0.0001), and S-1 also significantly improved the dermal thickening of mice (P<0.001), but was slightly worse than R-1 and X-1. Nintedanib also reduced the dermal thickness of mice (P<0.05), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the best.

[0314] Table 62. Statistics on dermal thickness of each compound in SSc-ILD model mice.

[0315] 2.3 Statistical analysis of pulmonary fibrosis degree: After drug administration, H&E staining was performed on lung tissue sections of mice. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 63, compared with the control group, obvious fibrosis appeared at the edge of the lung tissue in the model group; R-1 significantly reduced the pulmonary fibrosis area (P < 0.0001), X-1 also significantly reduced the pulmonary fibrosis area but the significance was slightly worse (P < 0.001), and S-1 also significantly reduced the pulmonary fibrosis area but the significance was further reduced (P < 0.01); Nintedanib also improved the pulmonary fibrosis area (P < 0.05), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0316] Table 63. Statistics on pulmonary fibrosis area in SSc-ILD model mice for each compound.

[0317] 2.4 Hydroxyproline content determination: The determination of skin or lung collagen content is the determination of hydroxyproline content. Specifically, after euthanizing mice, the skin tissue or right lung of the mouse is separated and placed in a 5mL ampoule. The ampoule is dried in an oven at 120℃, hydrolyzed in hydrochloric acid, and the pH value is adjusted to 6.5-8.0. The residue is filtered through a 0.5μm filter membrane, and PBS (phosphate balanced saline) is added to adjust the total volume to 10mL. 50μL of the sample is taken, 350μL of deionized water is added, 200μL of chloramine T solution is added and incubated at room temperature for 20min, 200μL of perchloric acid is added and incubated at room temperature for 5min, and 200μL of p-dimethylaminobenzaldehyde (P-DMAB) is added and incubated at 65℃ for 20min. 200 μL of the sample was added to a 96-well plate, and the absorbance was measured at 570 nm. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline in the entire right lung (by mass percentage) was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume), thus obtaining the hydroxyproline content. The amount of hydroxyproline in the skin was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / 0.01 (skin weight, g), thus obtaining the hydroxyproline content. In skin tissue, as shown in Table 64, compared with the control group, the HYP content per gram of skin in the model group was significantly increased. R-1 and X-1 could significantly reduce the HYP content of skin (P < 0.0001), and S-1 could also significantly reduce the HYP content of skin (P < 0.001), but it was slightly worse than R-1 and X-1. Nintedanib could also significantly reduce the HYP content of skin (P < 0.01), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best. In lung tissue, as shown in Table 65, compared with the control group, the HYP content in the right whole lung of the model group was significantly increased; R-1 significantly reduced the HYP content in lung tissue (P < 0.0001), X-1 also significantly reduced the HYP content in lung tissue but the significance was slightly lower (P < 0.001), S-1 also significantly reduced the HYP content in lung tissue (P < 0.01) but the significance was even lower, and Nintedanib also significantly reduced the HYP content (P < 0.01), but its efficacy was worse than R-1, X-1 and S-1, among which R-1 was the best.

[0318] Table 64 shows the hydroxyproline content in the skin of SSc-ILD model mice for each compound.

[0319] Tables 23 and 65 show the hydroxyproline content in the lungs of SSc-ILD model mice for each compound.

[0320] Pharmacological Example 17: Treatment of Imiquimod-induced SLE-ILD in Mice

[0321] 1. Experimental Methods

[0322] Female C57BL / 6 mice (6–8 weeks old), 8 mice per group. SLE-ILD was induced by imiquimod ear application (40 mg / mouse, 3 times / week) for 4 weeks. In week 4, mice were regrouped according to body weight and drug administration began: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Drug administration began in week 4, with each group receiving the drug via gavage. The Ctl and Model groups were administered sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until week 8.

[0323] 2. Experimental Results

[0324] After the experiment, the forced vital capacity, dermal thickness, spleen coefficient, total BALF cell count, and peripheral blood leukocyte count of mice in each group were measured. The results are as follows:

[0325] 2.1 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 66 shows that the model group exhibited a significant decrease in FVC compared to the control group. R-1 and X-1 significantly improved the FVC decrease (P < 0.0001), and S-1 also significantly improved FVC (P < 0.001), but was slightly less effective than R-1 and X-1. Nintedanib also improved FVC (P < 0.05), but its efficacy was inferior to R-1, X-1, and S-1, with R-1 showing the best effect.

[0326] Table 66. FVC results of each compound in SLE-ILD model mice.

[0327] 2.2 Dermal thickness detection: Specifically, skin samples were fixed in 10% formalin, dehydrated, embedded in paraffin, cut into 5 μm thick sections, and stained with H&E. Table 67 shows that compared to the control group, the dermal layer of the model group was significantly thicker; R-1, X-1, S-1, and Nintedanib all significantly reduced dermal thickness (P < 0.0001), with R-1 showing the best effect.

[0328] Table 67. Statistics on dermal thickness of each compound in SLE-ILD model mice.

[0329] 2.3 Spleen Coefficient: The spleen coefficient was determined by weighing the spleen of mice after necropsy and dividing the spleen weight by the body weight. Table 68 shows that the spleen coefficient in the model group was significantly higher than that in the control group. R-1, X-1, S-1, and the positive control drug Nintedanib all significantly reduced the spleen coefficient (P < 0.0001), with R-1 showing the best efficacy.

[0330] Table 68 Spleen coefficient statistics for each compound in SLE-ILD model mice

[0331] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a control table, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the effect of the test substance on the number of inflammatory cells in the BALF. As shown in Table 69, compared with the control group, the total BALF cell count in the model group was significantly increased. R-1, X-1, and S-1 all significantly reduced the total BALF cell count (P < 0.0001). The positive control drug Nintedanib also significantly reduced the total BALF cell count, but the significance was low (P < 0.01). Among these, R-1 showed the best efficacy.

[0332] Table 69. Total BALF cell count in SLE-ILD model mice for each compound.

[0333] 2.5 Serum White Blood Cell Count: Peripheral blood white blood cell count detection specifically refers to obtaining approximately 500 μL of peripheral blood through ocular sampling after mouse sacrifice, and immediately detecting the total white blood cell count using a blood biochemistry analyzer. As shown in Table 70, compared with the control group, the model group showed a significant increase in peripheral blood white blood cell count; R-1 significantly reduced the white blood cell count (P < 0.001), X-1 also significantly reduced the white blood cell count but with slightly lower significance (P < 0.01), and S-1 also significantly reduced the white blood cell count but with further decreased significance (P < 0.05); Nintedanib did not significantly reduce the peripheral blood white blood cell count (P > 0.05), and its efficacy was inferior to R-1, X-1, and S-1, with R-1 showing the best effect.

[0334] Table 70 Peripheral blood leukocyte counts in SLE-ILD model mice for each compound

[0335] Pharmacological Example 18: Treatment of skeletal muscle homogenate-induced PM-ILD

[0336] 1. Experimental Methods

[0337] Female BALB / c mice (6–8 weeks old) were used to induce a rat model on days D0, 7, 14, 21, and 28 by intraperitoneal injection of rat skeletal muscle homogenate (15 mg / mouse). Day D0 was the day of model initiation, and mice were weighed daily. On day 28, mice were regrouped according to their weight loss rate, with 8 mice per group: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 28, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until day 56.

[0338] 2. Experimental Results

[0339] The degree of myositis was scored throughout the experiment. After the experiment, the forced vital capacity, degree of pulmonary fibrosis, and spleen coefficient of each group of mice were measured. The results are as follows:

[0340] 2.1 Myositis Score: The myositis score refers to the observation and scoring of clinical symptoms in mice after immunization. The clinical symptom score is based on the Lennon scoring method: 0 points, no obvious muscle weakness; 1 point, the animal is unable to cry out or bite; 2 points, the body is raised at rest, the head is drooping, and the gait is shaky; 3 points, the muscle weakness is more severe, the weight loss is significant, and even the muscles are atrophied, the animal does not cry out, breathes with difficulty, and is close to death (those with moderate symptoms are scored as 0.5, 1.5, or 2.5 points). On day 56 of modeling (D56), the myositis scores of mice were assessed. The results are shown in Table 71. Compared with the control group, the myositis scores of the model group were significantly increased on day 56. The positive control drug Nintedanib could not significantly reduce the myositis scores (p>0.05). R-1, X-1, and S-1 could all significantly reduce the myositis scores of mice, and were significantly better than the positive control drug Nintedanib. R-1 (p<0.0001) had the best efficacy, which was better than X-1 (p<0.0001). X-1 was more effective than S-1 (p<0.001).

[0341] Table 71 Score of myositis severity at D56 in PM-ILD model mice for each compound.

[0342] 2.2 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 72 shows that compared to the control group, the model group exhibited a significant decrease in FVC. The positive control drug Nintedanib (P > 0.05) had no effect on FVC relief. R-1, X-1, and S-1 all significantly alleviated FVC, showing significantly better efficacy than the positive control drug Nintedanib. R-1 (P < 0.001) showed the best efficacy, superior to X-1 (P < 0.01); X-1 was more effective than S-1 (P < 0.01).

[0343] Table 72. FVC results of PM-ILD model mice for each compound.

[0344] 2.3 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 73, the model group showed obvious pulmonary fibrosis. The positive control drug Nintedanib (P < 0.01) significantly reduced the pulmonary fibrosis area. R-1, X-1, and S-1 all significantly reduced the pulmonary fibrosis area, and their efficacy was better than that of the positive control drug Nintedanib. R-1 (P < 0.0001) had the best efficacy, which was better than X-1 (P < 0.0001). X-1 was more effective than S-1 (P < 0.0001).

[0345] Table 73. Statistics on pulmonary fibrosis area in PM-ILD model mice for each compound.

[0346] 2.4 Spleen Coefficient: The spleen coefficient was determined by weighing the spleen of mice after necropsy and dividing the spleen weight by the body weight. Table 74 shows that the spleen coefficient in the model group was significantly higher than that in the control group; the positive control drug Nintedanib (P < 0.01) significantly reduced the spleen coefficient. R-1, X-1, and S-1 all significantly reduced the spleen coefficient in mice, with better efficacy than the positive control drug Nintedanib; R-1 (P < 0.0001) showed the best efficacy, superior to X-1 (P < 0.0001); X-1 was more effective than S-1 (P < 0.0001).

[0347] Table 74 Spleen coefficient statistics for PM-ILD model mice of each compound

[0348] Pharmacological Example 19: Treatment of yeast polysaccharide-induced RA-ILD

[0349] 1. Experimental Methods

[0350] Male SKG mice (8-10 weeks old) were used to establish a RA-ILD model by intraperitoneal injection of yeast polysaccharide (5 mg, 500 μL / mouse). Control mice received an equal volume of saline intraperitoneally at the same time. Day 0 (D0) was the day of model establishment, and mice were weighed daily. On day 28 (D28), mice were regrouped according to their weight loss rate, with 8 mice per group: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 28, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received sterile water via gavage as a control. The mice's general condition was observed twice daily, once in the morning and once in the afternoon, until day 42.

[0351] 2. Experimental Results

[0352] The degree of RA was scored throughout the experiment; after the experiment, the forced vital capacity, degree of pulmonary fibrosis, and total number of BALF cells in each group of mice were measured. The results are as follows:

[0353] 2.1 RA Score: The mouse arthritis index was determined by scoring the mice every three days, starting from day 28, until day 42. Each paw of each mouse was scored, and the scores of all four limbs were summed to obtain the mouse's arthritis index. The scoring criteria were as follows: 0 = no erythema or swelling; 1 = mild erythema or swelling of one toe; 2 = erythema or swelling of more than one toe; 3 = erythema and swelling of the ankle or wrist; 4 = severe erythema and severe edema of the toes and ankles or fingers and wrists, with the ankle or wrist unable to bend normally. The mouse arthritis index ranged from 0 to 16. Table 75 shows that compared with the control group, the model group showed significant joint swelling and an increased RA score; the positive control drug Nintedanib (P > 0.05) did not significantly reduce the RA score. R-1, X-1, and S-1 all significantly reduced RA scores, showing superior efficacy compared to the positive control drug Nintedanib; R-1 (P < 0.0001) significantly reduced RA scores, exhibiting the best efficacy, and was superior to X-1 (P < 0.0001); X-1 was more effective than S-1 (P < 0.001).

[0354] Table 75. RA severity scores of RA-ILD model mice at D42 for each compound.

[0355] 2.2 Forced Vital Capacity (FVC): Forced vital capacity (FVC) was measured in mice using the AniRes2005 animal lung function analysis system. Table 76 shows that the FVC in the model group was significantly lower than that in the control group; the positive control drug Nintedanib (P > 0.05) had no significant effect on FVC relief. R-1, X-1, and S-1 all significantly alleviated the decrease in FVC, showing superiority over the positive control drug Nintedanib; R-1 (P < 0.0001) significantly improved the decrease in FVC, exhibiting the best efficacy, superior to X-1 (P < 0.001); X-1 was more effective than S-1 (P < 0.01).

[0356] Table 76. FVC results of each compound in RA-ILD model mice.

[0357] 2.3 Degree of pulmonary fibrosis: After drug administration, H&E staining was performed on mouse lung tissue sections. After image acquisition, the pulmonary fibrosis area in the sections was calculated using Image-Pro Plus software. The percentage of pulmonary fibrosis area = pulmonary fibrosis area / total lung section area × 100%. As shown in Table 77, compared with the control group, the model group showed obvious fibrosis; the positive control drug Nintedanib (P < 0.01) significantly reduced the pulmonary fibrosis area. R-1, X-1, and S-1 all significantly reduced the pulmonary fibrosis area, and were superior to the positive control drug Nintedanib; R-1 (P < 0.0001) significantly reduced the pulmonary fibrosis area, with the best efficacy, and was superior to X-1 (P < 0.001); X-1 was more effective than S-1 (P < 0.01).

[0358] Table 77. Statistics on pulmonary fibrosis area in RA-ILD model mice for each compound.

[0359] 2.4 Total Cell Count in BALF: The detection of inflammatory cell count in bronchoalveolar lavage fluid (BALF) involved anesthetizing and fixing mice on a control table, inserting an indwelling needle into the trachea, and then injecting 1 mL of PBS into the lungs via the trachea to completely lavage the lungs. This process was repeated three times. The lavage fluid was collected, centrifuged, and the cell pellet was resuspended in erythrocyte lysis buffer. The cells were lysed at room temperature for 10 min, centrifuged again, and the lysate was discarded. 20 μL of the cell suspension was then resuspended in 100 μL of PBS, and the number of viable cells was detected using a live cell counter (three fields of view were randomly selected for each sample). This yielded the inflammatory cell count in the BALF. Table 78 shows that compared to the control group, the total BALF cell count in the model group was significantly increased; the positive control drug Nintedanib (P < 0.01) significantly reduced the total BALF cell count. R-1, X-1, and S-1 all significantly reduced the total number of BALF cells, which was superior to the positive control drug Nintedanib; R-1 (P < 0.0001) significantly reduced the total number of BALF cells, showing the best efficacy, which was superior to X-1 (P < 0.0001); X-1 was more effective than S-1 (P < 0.0001).

[0360] Table 78. Total BALF cell count in RA-ILD model mice for each compound.

[0361] Pharmacological Example 20: Treatment of Folic Acid-Induced Renal Fibrosis

[0362] 1. Experimental Methods

[0363] Male C57BL / 6 mice (8 weeks old) were used to establish a renal fibrosis model using a single intraperitoneal injection of folic acid. Folic acid was dissolved in 0.3M sodium bicarbonate and injected intraperitoneally at a dose of 250 mg / kg. Control mice were injected with sodium bicarbonate. Day 0 was the day of model establishment. Mice were divided into groups of 8 mice each according to their body weight at model establishment: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 14, with each group receiving the dose via gavage. The Ctl and Model groups received sterile water via gavage as controls. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 28.

[0364] 2. Experimental Results

[0365] After the experiment, the renal hydroxyproline content, serum urea nitrogen, and serum creatinine content of mice in each group were measured. The results are as follows:

[0366] 2.1 Renal Collagen Content: The renal collagen content was determined by detecting hydroxyproline content. Specifically, after euthanizing mice, the kidney tissue was separated and weighed, placed in 5 mL ampoules, dried in a 120℃ oven, hydrolyzed with hydrochloric acid, and the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. Then, 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. Next, 200 μL of perchloric acid was added and incubated at room temperature for 5 min. Finally, 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was transferred to a 96-well plate, and the absorbance at 570 nm was measured. A standard curve was plotted using the standard readings, and the hydroxyproline concentration (Cs) of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline per gram of kidney tissue was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / kidney tissue mass. The hydroxyproline content was then obtained, and the specific results are shown in Table 79. Compared with the control group, the model group showed a significant increase in kidney collagen content; R-1 and X-1 significantly reduced kidney collagen content (P < 0.0001), and S-1 also significantly reduced kidney collagen content (P < 0.001), but slightly less effectively than R-1 and X-1. The efficacy of these three compounds was significantly better than that of the positive control drug Nintedanib (P < 0.01), with R-1 showing the best effect.

[0367] Table 79 shows the collagen content in the renal tissue of mice with renal fibrosis model for each compound.

[0368] 2.2 Serum Urea Nitrogen Content: Serum urea nitrogen content was measured by collecting blood from the eyes of euthanized mice, allowing them to stand for 30 minutes, centrifuging at 3000 rpm for 15 minutes, and then measuring the serum urea nitrogen content using ELISA. The specific results are shown in Table 80. Compared with the control group, the serum urea nitrogen in the model group was significantly increased; R-1 and X-1 (P < 0.0001) significantly reduced serum urea nitrogen content, and S-1 also significantly reduced serum urea nitrogen content (P < 0.001), but slightly less than R-1 and X-1. The efficacy of these three compounds was significantly better than that of the positive control drug Nintedanib (P < 0.05), with R-1 being the best.

[0369] Table 80 Serum urea nitrogen levels in mice with renal fibrosis model for each compound.

[0370] 2.3 Serum Creatinine Level: Serum creatinine level detection involved collecting blood from the eyes of euthanized mice, allowing them to stand for 30 minutes, centrifuging at 3000 rpm for 15 minutes to collect serum samples, and then measuring the serum creatinine level using ELISA. The specific results are shown in Table 81. Compared with the control group, the model group showed a significant increase in serum creatinine. R-1 (P < 0.0001) significantly reduced serum creatinine levels. X-1 and S-1 (P < 0.001) also significantly reduced serum creatinine levels, but were slightly less effective than R-1. The efficacy of these three compounds was significantly better than that of the positive control drug Nintedanib (P > 0.01), with R-1 showing the best effect.

[0371] Table 81 Serum creatinine levels in mice with renal fibrosis model for each compound.

[0372] Pharmacological Example 21: Treatment of Biliary Duct Ligation-Induced Liver Fibrosis

[0373] 1. Experimental Methods

[0374] Male C57BL / 6 mice (10 weeks old) were used to establish a liver fibrosis model by double ligation of the common bile duct. Day 0 was the day of model establishment, and mice were weighed daily. On day 14, mice were regrouped based on their rate of weight loss, with 8 mice in each group: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 14, with each group receiving the prescribed dose via gavage. The Ctl and Model groups were administered saline as a control. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 28.

[0375] 2. Experimental Results

[0376] After the experiment, the liver hydroxyproline content and blood alanine aminotransferase content of mice in each group were measured. The results are as follows:

[0377] 2.1 Hepatic Hydroxyproline Content: After euthanizing mice, liver tissue was isolated and weighed, placed in 5 mL ampoules, and dried in a 120℃ oven. After hydrolysis with hydrochloric acid, the pH was adjusted to 6.5–8.0. The residue was filtered through a 0.5 μm filter membrane, and PBS (phosphate-balanced saline) was added to adjust the total volume to 10 mL. 50 μL of the sample was taken, and 350 μL of deionized water was added. 200 μL of chloramine T solution was added and incubated at room temperature for 20 min. 200 μL of perchloric acid was added and incubated at room temperature for 5 min. 200 μL of p-dimethylaminobenzaldehyde (P-DMAB) was added and incubated at 65℃ for 20 min. 200 μL of the sample was transferred to a 96-well plate, and the absorbance at 570 nm was measured. A standard curve was plotted using the standard readings, and the hydroxyproline concentration Cs of the sample was calculated using the formula derived from the standard curve. The amount of hydroxyproline per gram of liver tissue was calculated using the following formula: Cs × 8 (dilution factor of the sample) × 10 (total sample volume) / liver tissue weight. The hydroxyproline content was then obtained, and the specific results are shown in Table 82. Table 82 shows that the liver collagen content in the model group was significantly increased; the positive control drug Nintedanib (P > 0.05) did not reduce liver collagen content. R-1, X-1, and S-1 all significantly reduced liver collagen content in mice, showing significantly better efficacy than the positive control drug Nintedanib; R-1 (p < 0.0001) had the best efficacy, superior to X-1 (p < 0.001); X-1 was more effective than S-1 (p < 0.01).

[0378] Table 82. Collagen content in liver tissue of mice with liver fibrosis model for each compound.

[0379] 2.2 Serum Alanine Aminotransferase (ALT) Levels: Serum ALT levels were detected at 37℃ and pH 7.4. ALT catalyzes the transamination reaction between alanine and α-ketoglutarate, producing pyruvate and glutamic acid. After the reaction time, phenylhydrazine is added, reacting with pyruvate to form phenylhydrazone. Phenylhydrazone is reddish-brown under alkaline conditions. ALT levels can be quantified by detecting absorbance. Specific results are shown in Table 83. Table 83 shows that serum ALT levels were significantly increased in the model group; the positive control drug Nintedanib (P > 0.05) did not reduce serum ALT levels. R-1, X-1, and S-1 all significantly reduced serum alanine aminotransferase levels, showing significantly better efficacy than the positive control drug Nintedanib. R-1 (p < 0.0001) was the most effective, superior to X-1 (p < 0.0001), while X-1 was more effective than S-1 (p < 0.0001).

[0380] Table 83 Serum alanine aminotransferase levels in mice with liver fibrosis model for each compound.

[0381] 2.3 Sirius Red Staining of Liver Tissue: After drug administration, liver tissue sections from mice were stained with Sirius red. After image acquisition, the collagen fiber area in the sections was calculated using Image-Pro Plus software. The percentage of collagen fiber area = collagen fiber area / total liver section area × 100%. Table 84 shows that compared with the control group, the model group exhibited significant fibrosis. The positive control drug Nintedanib (P < 0.05) significantly reduced the area of ​​liver fibrosis. R-1, X-1, and S-1 all significantly reduced the area of ​​liver fibrosis, superior to the positive control drug Nintedanib; R-1 (P < 0.0001) significantly reduced the area of ​​liver fibrosis, showing the best efficacy, superior to X-1 (P < 0.0001); X-1 was more effective than S-1 (P < 0.001).

[0382] Table 84. Statistics on liver fibrosis area in mice modeling liver fibrosis for each compound.

[0383] Pharmacological Example 22: Treatment of Isoproterenol-Induced Cardiac Fibrosis in Mice

[0384] 1. Experimental Methods

[0385] Male C57BL / 6 mice (8–10 weeks old) were subcutaneously injected with 10 mg / kg isoproterenol daily for 14 consecutive days; control mice were subcutaneously injected with an equal volume of physiological saline. Day 0 was the day of model establishment. Mice were weighed daily, with 8 mice per group. The specific groupings were as follows: control group (Ctl), model group (Model), positive control group (Nintedanib 60 mg / kg qd), R-1 group (R-1 50 mg / kg bid), X-1 group (X-1 50 mg / kg bid), and S-1 group (S-1 50 mg / kg bid). Treatment began on day 7, with each group receiving the prescribed dose via gavage. The Ctl and Model groups received physiological saline as a control. The general condition of the mice was observed twice daily, once in the morning and once in the afternoon, until day 14.

[0386] 2. Experimental Results

[0387] After the experiment, the heart-to-body ratio, heart-to-tibia ratio, and cardiac fibrosis area were measured in each group of mice. The results are as follows:

[0388] 2.1 Heart-to-body ratio: The body weight (M) of the mice was measured. After anesthesia, the thoracic cavity was opened, and residual blood was rinsed with pre-cooled PBS. The water around the heart was dried with filter paper, and the heart weight (m) was recorded. The heart-to-body ratio = m / M. The specific results are shown in Table 85. The heart-to-body ratio of the model group was significantly increased. R-1 and X-1 could significantly reduce the heart-to-body ratio (P < 0.0001). S-1 could also significantly reduce the heart-to-body ratio, but the significance was slightly lower (P < 0.001). Nintedanib could also significantly reduce the heart-to-body ratio (P < 0.05), but its efficacy was worse than that of R-1, X-1, and S-1. Among them, R-1 was the best.

[0389] Table 85 Heart-to-body ratio in mouse models of cardiac fibrosis for each compound

[0390] 2.2 Heart-to-Tibia Ratio: After anesthetizing the mice, the thoracic cavity was opened, residual blood was rinsed with pre-cooled PBS, and the water around the heart was dried with filter paper. The heart weight (m) was weighed and recorded. The right tibia of the mouse was removed, and the tibia length (l) was measured with calipers. The heart-to-tibia ratio was calculated as m / l. Specific results are shown in Table 86. The heart-to-tibia ratio was significantly increased in the model group. R-1 significantly reduced the heart-to-tibia ratio (P < 0.0001), X-1 also significantly reduced the heart-to-tibia ratio but with a slightly lower significance (P < 0.01), and S-1 also significantly reduced the heart-to-tibia ratio but with a further decrease in significance (P < 0.05). Nintedanib did not reduce the heart-to-tibia ratio (P > 0.05), and its efficacy was inferior to R-1, X-1, and S-1, with R-1 showing the best effect.

[0391] Table 86. Heart-to-tibia ratio in mouse models of cardiac fibrosis for each compound.

[0392] 2.3 Percentage of Cardiac Fibrosis Area: Mice were euthanized after anesthesia, and tissue samples were collected. The mouse heart was cut open at the midpoint of its short axis, and the base of the heart was fixed in 4% neutral formaldehyde. The heart tissue was then immersed in paraffin for 2 hours to obtain embedded tissue blocks. The tissue blocks were then sectioned to a thickness of 5 μm. The heart tissue sections were sequentially immersed in the following reagents: xylene for 10 min, 3 times; 100% ethanol for 3 min, 2 times; 95% ethanol for 3 min, 2 times; 80% ethanol for 3 min, 1 time; 70% ethanol for 3 min, 1 time; and finally washed 3 times with double-distilled water. The heart tissue sections were blotted dry with absorbent paper and stained in saturated picric acid-sirius red staining solution for 8 minutes. The sections were then rinsed in anhydrous ethanol for several minutes. After staining, the sections were dried in a 60°C oven, cleared with xylene for 5 minutes, and then mounted with neutral resin. Microscopic examination was performed, and images of the sections were acquired and analyzed. The fibrotic area in the sections was calculated using Image-Pro Plus software. The percentage of cardiac fibrosis area was calculated as: (Cardiofibrosis area / Total area of ​​heart section) × 100%. Specific results are shown in Table 87. The cardiac fibrosis area in the model group was significantly increased. R-1 significantly reduced the cardiac fibrosis area (P < 0.0001), X-1 also significantly reduced the cardiac fibrosis area but with a slightly lower significance (P < 0.001), and S-1 also significantly reduced the cardiac fibrosis area but with a further decrease in significance (P < 0.01). Nintedanib also significantly reduced the cardiac fibrosis area (P < 0.05), but its efficacy was worse than R-1, X-1, and S-1, with R-1 being the most effective.

[0393] Table 87 shows the area of ​​cardiac fibrosis in mouse models of cardiac fibrosis for each compound.

[0394] Pharmacological Example 23: Acute Toxicity Study in Mice

[0395] 1. Experimental Methods

[0396] C57BL / 6 mice (6-8 weeks old), 8 mice per group, half male and half female, were divided into two groups: a control group (Ctl) and an R-1 group (R-1 2000 mg / kg). Mice were treated with a single dose via gavage, with the Ctl group receiving saline as a control. After administration, the mice's general condition was observed, with survival assessed every 30 minutes for 24 hours. Key behavioral indicators included central nervous system indicators (activity level, gait, tremors and convulsions, mental state, etc.) and autonomic nervous system indicators (salivation, lacrimation, urinary incontinence, diarrhea, piloerection, heart rate, and respiratory rate). Other indicators included changes in body surface appearance, body temperature, food intake, and water intake. After 24 hours, the mice were euthanized, and the morphology of the heart, liver, spleen, lungs, and kidneys was observed, followed by pathological examination.

[0397] 2. Experimental Results

[0398] After the experiment, the mice were dissected and observed. The results are as follows:

[0399] 2.1 Behavioral observation of mice: Mice were observed continuously for 24 hours after administration. Compared with the control group, mice in the R-1 group showed no abnormal behavior and no obvious toxic reactions were observed.

[0400] 2.2 Observation of major organs of mice: After dissection of the mice, the major organs (heart, liver, spleen, lung and kidney) were observed. Compared with the control group, no obvious color or morphological abnormalities were found in the organs of R-1 group, and no abnormalities were found in pathological examination.

[0401] No mice died in the toxicity experiment of a single dose of 2000 mg / kg in the R-1 group. These results indicate that no significant toxic reactions were observed in the single-dose toxicity experiment of R-1 in mice, demonstrating that R-1 has extremely high safety, with an LD50 value greater than 2000 mg / kg.

[0402] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. An alcoholamine derivative, characterized in that, It has the structure shown in formula R-1 or formula R-2:

2. The alcoholamine derivative according to claim 1, characterized in that, The space group of the alcoholamine derivative having the structure shown in Formula R-1 is P 212121; the unit cell parameters are: α = 90°, β = 90°, γ = 90°; unit cell volume is Z = 8.

3. The alcoholamine derivative according to claim 1, characterized in that, The space group of the alcoholamine derivative having the structure shown in formula R-2 is P 21; the unit cell parameters are: α=90°, β=91.239(8)°, γ=90°; Z=4.

4. The method for preparing the alcoholamine derivative according to any one of claims 1 to 3, characterized in that, Includes the following steps: Compound A, an alcoholic amine starting compound, a chiral ligand, and an organic solvent are mixed and subjected to an addition reaction to obtain an alcoholic amine derivative having the structure shown in formula R-1 or formula R-2. The alcohol amine raw material compound is 3-methylamino-1-propanol or 2-methylaminoethanol; The structural formula of compound A is shown below:

5. The preparation method according to claim 4, characterized in that, The chiral ligand is any one of the following compounds:

6. The preparation method according to claim 4 or 5, characterized in that, The molar ratio of compound A, the alcohol amine raw material compound, and the chiral ligand is 1:0.8-10:0.1-3.

7. The preparation method according to claim 4 or 5, characterized in that, The organic solvent includes acetonitrile, methanol, or ethyl acetate; the ratio of compound A to organic solvent is 4 mmol: 4–24 mL.

8. The preparation method according to claim 4 or 5, characterized in that, The addition reaction is carried out at a temperature of 0–100°C for a time of 2–24 hours.

9. The preparation method according to claim 4 or 5, characterized in that, The addition reaction further includes: concentrating the product system obtained from the addition reaction under reduced pressure to remove the solvent, and then purifying it by silica gel rapid column chromatography.

10. The preparation method according to claim 9, characterized in that, The eluent used for purification is a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol in the eluent is 15:

1.

11. Use of the alcoholamine derivative according to any one of claims 1 to 3 in the preparation of a medicament for treating lung inflammation caused by organ fibrosis or acute injury.

12. The application according to claim 11, characterized in that, The organ fibrosis includes pulmonary fibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, or skin fibrosis.

13. The application according to claim 12, characterized in that, The pulmonary fibrosis includes idiopathic pulmonary fibrosis, pulmonary fibrosis caused by occupational exposure, or pulmonary fibrosis caused by autoimmune diseases.

14. The application according to any one of claims 11 to 13, characterized in that, The drug comprises an active ingredient and a pharmaceutically acceptable carrier; the active ingredient is the alcohol amine derivative.

15. The application according to any one of claims 11 to 13, characterized in that, The drug can be administered orally, by inhalation, or by injection.

16. The application according to claim 15, characterized in that, The dosage forms of the drug include capsules, tablets, oral solutions, inhalers, or injections.

17. A medicament for treating lung inflammation caused by organ fibrosis or acute injury, comprising an active ingredient and a pharmaceutically acceptable carrier, said active ingredient being an alcohol amine derivative according to any one of claims 1 to 3.

18. A method for treating lung inflammation caused by organ fibrosis or acute injury, comprising administering to a patient suffering from lung inflammation caused by organ fibrosis or acute injury an effective amount of the alcoholamine derivative of any one of claims 1 to 3.

19. The method according to claim 18, characterized in that, The patient may be given the alcohol amine derivative by oral, inhalation, or injection.

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