Application of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury

Abstract

This invention provides the application of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury. Indolequinolinone compounds significantly improve the survival rate, weight change, lung function, lung tissue pathological damage, and degree of pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis, and reduce the hydroxyproline content in lung tissue. Simultaneously, they can significantly alleviate lung pathological damage and inflammatory cell infiltration in mice with lipopolysaccharide-induced acute lung injury, reduce lung permeability and pulmonary edema, reduce the total white blood cell count and total protein concentration in bronchoalveolar lavage fluid, and reduce the expression and release of inflammatory factors TNF-α, IL-1β, IL-6, and MPO in bronchoalveolar lavage fluid. Indolequinolinone compounds have significant anti-pulmonary fibrosis and anti-lung injury effects, and compared with existing anti-pulmonary fibrosis and lung injury drugs, they possess stronger in vivo anti-pulmonary fibrosis and lung injury effects and better safety, and can be used as the active ingredient of the drug.

Description

Technical Field

[0001] This invention belongs to the technical field of new pharmaceutical applications of indolequinolinone compounds, specifically relating to the application of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury. Background Technology

[0002] Pulmonary fibrosis (PF) is a disease characterized by diffuse pneumonia and alveolar structural disorder, ultimately leading to interstitial fibrosis. The pathogenesis of PF is generally believed to be characterized by persistent damage to alveolar epithelial cells, abnormal activation of fibroblasts (called myofibroblasts), and excessive deposition of extracellular matrix, resulting in varying degrees of inflammation and fibrosis in the alveoli and interstitium, ultimately leading to structural destruction and loss of lung function.

[0003] Idiopathic pulmonary fibrosis (IPF) is the most common and serious chronic interstitial lung disease with unknown etiology. Clinically, it is characterized by progressive dyspnea accompanied by irritating dry cough. The disease often continues to progress, and the median survival is about 3-5 years.

[0004] Currently, the two oral anti-fibrotic drugs approved by the U.S. Food and Drug Administration (FDA) for clinical use are pirfenidone and nintedanib. Pirfenidone (PFD) works by regulating pro-fibrotic cytokines such as transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF), reducing the biological activity of fibroblasts, inhibiting fibroblast proliferation, and decreasing collagen expression and extracellular matrix synthesis and accumulation. Nintedanib is a small-molecule tyrosine kinase inhibitor that simultaneously blocks the signal transduction pathways of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), exhibiting anti-fibrotic and anti-inflammatory effects. While these two marketed drugs can slow the decline in lung function, they cannot reverse disease progression. Furthermore, pirfenidone treatment can cause side effects such as photosensitivity, anorexia, dizziness, and abdominal discomfort; nintedanib commonly causes adverse reactions including diarrhea, nausea, vomiting, elevated liver enzymes, decreased appetite, and hypertension. In addition, both drugs are currently difficult to promote clinically due to their high cost. Besides drug therapy, lung transplantation is currently the only treatment that can prolong the survival of patients with pulmonary fibrosis; however, lung transplantation is expensive, and the overall survival rate after surgery is low, with numerous and complex complications, and available lung resources are scarce. Therefore, developing economical, effective, and relatively safe drugs for pulmonary fibrosis has significant social and medical implications.

[0005] Acute lung injury (ALI) is an acute injury to alveolar epithelial cells and capillary endothelial cells caused by various direct and indirect factors, resulting in diffuse interstitial and alveolar edema. It presents as acute-onset bilateral lung infiltration accompanied by hypoxemia, characterized by decreased lung volume, reduced lung compliance, and ventilation / perfusion mismatch. It is a major cause of death in critically ill patients. Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms such as bacteria and their toxic components (e.g., LPS) into the body. The lungs are the most vulnerable target organ in sepsis; acute lung injury occurs earliest, has a high incidence, is difficult to treat, and has a high mortality rate. Sepsis-induced acute lung injury is characterized by bilateral pneumocytic cell infiltration, vascular permeability destruction, and pulmonary edema. Specifically, in ALI, pathogenic substances such as LPS stimulate macrophages to secrete cytokines and activate the inflammatory response. The resulting inflammatory factors strongly attract and aggregate neutrophils, damaging vascular endothelial and epithelial cells, leading to the disruption of the pulmonary capillary endothelial cell and alveolar epithelial cell barrier function, resulting in the leakage of large amounts of blood cells and proteins, and the accumulation of large amounts of edema fluid in the alveoli and pulmonary interstitium. Current main treatment methods are respiratory support therapy and drug therapy. Respiratory support therapy is further divided into oxygen therapy, invasive mechanical ventilation, and non-invasive mechanical ventilation, but all of these treatments have a stimulating effect on the patient's body and do not substantially promote alveolar repair. However, new drug treatments are constantly emerging, but they are still in the clinical research stage. Currently, there are still no specific preventive or therapeutic drugs for ALI internationally. Therefore, there is an urgent need to develop new drugs for treating ALI.

[0006] In summary, there is currently a lack of cost-effective and relatively safe drugs on the market for treating pulmonary fibrosis and lung injury-related diseases. Summary of the Invention

[0007] The present invention aims to solve the technical problems existing in the prior art. To this end, the present invention proposes an application of indolequinolinone compounds, specifically, the application of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury, which can solve the above-mentioned technical problems.

[0008] This invention provides the use of indolequinolinone compounds in the preparation of medicaments for the prevention and / or treatment of pulmonary fibrosis and lung injury.

[0009] Preferably, the indolequinolinone compound includes compounds with the structure shown in formula (I) and their pharmaceutically acceptable salts:

[0010]

[0011] in,

[0012] R1 is selected from one of phenyl, substituted phenyl, benzyl, naphthyl, thiophene-2-methylene, propargyl, and alkyl;

[0013] R2 is selected from H, halogen, alkyl and alkoxy;

[0014] R3 is selected from H, halogens, and alkyl groups.

[0015] Preferably, the substituent group on the substituted phenyl group includes at least one selected from halogen, oxyacyl, acyl, phenyl, alkyl, and alkoxy groups.

[0016] Preferably, the indolequinolinone compound comprises at least one of the compounds with structures shown in formulas I-3a to I-3ac:

[0017]

[0018]

[0019] Preferably, the indolequinolinone compound is synthesized via the following reaction:

[0020] Under the catalysis of an acid catalyst, the indole-2-amide compound shown in formula (Ia) and the 1,4-benzoquinone compound shown in formula (Ib) react to give the indolequinone compound shown in formula (I):

[0021]

[0022] The acid catalyst includes at least one of trifluoroacetic acid, acetic acid, p-toluenesulfonic acid, and trifluoromethanesulfonic acid.

[0023] Preferably, the molar ratio of the indole-2-amide compound, the 1,4-benzoquinone compound, and the acid catalyst is 1.0:1.1-1.5:0.1-0.5.

[0024] Preferably, the pulmonary fibrosis includes at least one of primary pulmonary fibrosis, idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, and interstitial pulmonary fibrosis.

[0025] Preferably, the lung injury disease includes direct lung injury and indirect lung injury.

[0026] The direct lung injury is induced lung injury, including at least one of bacterial or viral lung infection, aspiration of gastric contents, lung contusion, inhalation of toxic gases, and oxygen poisoning.

[0027] The indirect lung injury includes at least one of the following: severe non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, cardiopulmonary bypass, and disseminated intravascular coagulation.

[0028] Preferably, the dosage form of the drug for treating pulmonary fibrosis and lung injury is one or more of the following: tablets, capsules, pills, aerosols, oral liquid preparations, granules, powders, injections, syrups, tinctures, lotions, and films.

[0029] Preferably, the administration method of the drug for treating pulmonary fibrosis and lung injury includes one or more of oral, injection, spray, inhalation, implantation, and topical application.

[0030] This invention provides the application of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury. Indolequinolinone compounds significantly improve the survival rate, weight change, lung function, lung tissue pathological damage, and degree of pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis, and reduce the hydroxyproline content in lung tissue. Simultaneously, they can significantly alleviate lung pathological damage and inflammatory cell infiltration in mice with lipopolysaccharide-induced acute lung injury, reduce lung permeability and pulmonary edema, reduce the total white blood cell count and total protein concentration in bronchoalveolar lavage fluid, and reduce the expression and release of inflammatory factors TNF-α, IL-1β, IL-6, and MPO in bronchoalveolar lavage fluid. Therefore, indolequinolinone compounds have significant anti-pulmonary fibrosis and anti-lung injury effects, and compared with existing anti-pulmonary fibrosis and lung injury drugs, they possess stronger in vivo anti-pulmonary fibrosis and lung injury effects and better safety, and can be used as the active ingredient of the drug. Attached Figure Description

[0031] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0032] Figure 1 The survival rate (A) and weight change (B) of mice in each drug administration group are shown in Example 1 of the application of this invention.

[0033] Figure 2 Examples of application of this invention include Micro-CT scan images of mouse lung tissue and photographs of mouse lungs in each drug administration group;

[0034] Figure 3 The graph shows the total number of white blood cells (A) in the bronchoalveolar lavage fluid of mice in each treatment group of Example 1 of this invention and the content of hydroxyproline (HYP) in the lung tissue of mice (B).

[0035] Figure 4Example 1 of this invention shows the expression of proteins such as Fibronectin, Collagen I, and α-SMA in the lung tissue of mice in each group, detected by Western blot.

[0036] Figure 5 The graphs show multiple lung function tests (including dynamic resistance Rrs, inspiratory volume IC, dynamic elasticity Ers, and dynamic compliance Crs) of mice in each drug administration group in Example 1 of this invention.

[0037] Figure 6 The images show H&E staining and Masson staining of lung tissue from mice in each drug administration group in Example 1 of this invention.

[0038] Figure 7 Immunohistochemical images of α-SMA, Collagen I, and Fibronectin in the lung tissues of mice in each drug administration group in Example 1 of this invention;

[0039] Figure 8 The mouse safety assessment diagram for Example 1 of the present invention includes mouse weight change (A); mouse organ index (B); H&E staining of mouse heart, liver, spleen, lung and kidney (C); and the levels of aspartate aminotransferase (AST) (D), alanine aminotransferase (ALT) (E) and serum creatinine (CRE) in mouse serum (F).

[0040] Figure 9 This is a graph showing the wet / dry weight ratio of the lungs of mice in each treatment group in Example 2 of the application of this invention.

[0041] Figure 10 H&E staining of lung tissue from mice in each drug administration group in Example 2 of this invention;

[0042] Figure 11 The graph shows the total white blood cell count (A) and total protein concentration (B) in the bronchoalveolar lavage fluid of mice in each treatment group in Example 2 of the present invention.

[0043] Figure 12 The graph shows the levels of inflammatory cytokines IL-6 (A), IL-1β (B), TNF-α (C), and MPO (D) in the bronchoalveolar lavage fluid of mice in each treatment group in Example 2 of this invention.

[0044] Figure 13 This is a diagram illustrating the effect of indolequinolinone derivative 3a (Example 3) on the morphology of TGF-β1-induced human lung epithelial cells A549.

[0045] Figure 14 This is a graph showing the effect of indolequinolinone derivative 3a (Example 3) of the present invention on the expression levels of N-cadherin and E-cadherin protein bands, which are related to EMT, in TGF-β1-induced human lung epithelial cells A549.

[0046] Figure 15 The figure shows the effect of indolequinolinone derivative 3a (Example 3) on the migration ability of TGF-β1-induced human lung epithelial cells A549.

[0047] Figure 16 This is a graph showing the effect of indolequinolinone derivative 3a (Example 4 of this invention) on the expression levels of Fibronectin and α-SMA protein bands in TGF-β1-induced human embryonic lung fibroblasts (HFL1). Detailed Implementation

[0048] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0049] CN114605407A discloses an indolequinoline ketone compound, its synthesis method, and its applications. The examples specifically disclose the preparation processes of 29 indolequinoline ketone compounds corresponding to different chemical formulas. Furthermore, growth inhibition experiments were conducted on tumor cells and normal cells using the indolequinoline ketone compounds obtained in the 29 examples, revealing that the indolequinoline ketone compounds exhibit good inhibitory effects on both in vitro and in vivo tumor cells. This invention further develops upon CN114605407A.

[0050] This invention investigated the effects of 29 indolequinolinone compounds corresponding to the chemical formulas in CN114605407A on pulmonary fibrosis and lung injury. The study found that these 29 indolequinolinone compounds significantly improved the survival rate, body weight change, lung function, lung tissue pathological damage, and degree of pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis, and reduced the hydroxyproline content in lung tissue. Simultaneously, they significantly reduced lung pathological damage and inflammatory cell infiltration, decreased lung permeability and pulmonary edema, reduced the total white blood cell count and total protein concentration in bronchoalveolar lavage fluid, and reduced the expression and release of inflammatory factors TNF-α, IL-1β, IL-6, and MPO in bronchoalveolar lavage fluid in mice with lipopolysaccharide-induced acute lung injury. Indolequinolinone compounds exhibit significant anti-pulmonary fibrosis and anti-lung injury effects, and compared with existing anti-pulmonary fibrosis and lung injury drugs, they possess stronger in vivo anti-pulmonary fibrosis and lung injury effects and better safety, and can be used as active ingredients in the aforementioned drugs.

[0051] It should be noted that this invention uses indolequinolinone derivative 3a, one of the 29 chemical formulas, as an example for illustration. The other 28 chemical formulas corresponding to indolequinolinone compounds can also yield results consistent with indolequinolinone derivative 3a by referring to the experimental method provided in this invention. The chemical formula of indolequinolinone derivative 3a is as follows.

[0052]

[0053] Example 1: Indolequinolinone derivative 3a slows down bleomycin (BLM)-induced pulmonary fibrosis in mice.

[0054] The bleomycin-induced pulmonary fibrosis (IPF) model in mice via intratracheal infusion is currently the most widely used animal model for preclinical IPF experiments. The model's progression can be broadly divided into an inflammatory phase (days 0-7 post-modeling) and a fibrotic phase (days 7-28 post-modeling). To evaluate the preventive and therapeutic effects of compound 3a on IPF, this invention established a mouse IPF model induced by a single intratracheal infusion of bleomycin. Prophylactic administration (once-daily gavage administration from day 1-20 after a single intratracheal infusion of BLM) or therapeutic administration (once-daily gavage administration from day 7-20 after a single intratracheal infusion of BLM) was employed. Samples were collected on day 21 to further verify the anti-fibrotic effect of indolequinone 3a in vivo.

[0055] The specific implementation method is as follows:

[0056] Preventive administration regimen: Seventy-two male C57BL / 6J mice (8-10 weeks old) were randomly divided into six groups (n=12), named as follows: normal control group, model group, pirfenidone group (200 mg / kg), 3a-L low-dose group (15 mg / kg), 3a-M medium-dose group (30 mg / kg), and 3a-H high-dose group (60 mg / kg). During the experiment, according to the dosage requirements of each group, the corresponding compound was dissolved in 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) aqueous solution and administered to the mice in each group by gavage. On day 0, the normal control group received a single intratracheal infusion of an equal volume of 0.9% sterile saline, while the model group and the administration group received a single intratracheal infusion of 2.5 mg / kg bleomycin (Nippon Kayaku Co., Ltd.) to establish the model. Starting from day 1 of modeling, the normal control group and the model group were administered an equal volume of 0.5% CMC-Na aqueous solution by gavage daily. The positive control group was administered 200 mg / kg pirfenidone (Maclean) by gavage daily. The experimental compound administration groups were administered 15 mg / kg, 30 mg / kg, and 60 mg / kg of compound 3a by gavage daily, respectively, for 20 consecutive days. During the experiment, the mice were weighed daily and their survival was observed. On day 21 of modeling, after anesthesia, mice in each group underwent lung imaging using a Micro-CT scanner. Finally, after anesthesia, tissue samples were collected to evaluate the degree of pulmonary fibrosis, including: the determination of hydroxyproline (HYP) content in mouse lung tissue, the count of total white blood cells in mouse bronchoalveolar lavage fluid (BALF), and the expression levels of key markers of pulmonary fibroblast activation in mouse lung tissue, such as smooth muscle actin (α-SMA), type I collagen (collagen I), and fibronectin.

[0057] like Figure 1 Figure 1 shows the survival rate (A) and body weight change of mice in each treatment group (B). Normal represents the normal control group; Model represents the model control group; PFD represents the positive control group (200 mg / kg); 3a-L represents the low-dose group (15 mg / kg); 3a-M represents the medium-dose group (30 mg / kg); and 3a-H represents the high-dose group (60 mg / kg). Compared with the Normal group, ***p < 0.001 and ****p < 0.0001; compared with the Model group, #p < 0.05.

[0058] like Figure 2The images show Micro-CT scans of mouse lung tissue and photographs of mouse lungs in each treatment group. Normal represents the normal control group; Model represents the model control group; PFD represents the positive control group (200 mg / kg); 3a-L represents the low-dose group (15 mg / kg); 3a-M represents the medium-dose group (30 mg / kg); and 3a-H represents the high-dose group (60 mg / kg).

[0059] like Figure 3 The figures show the total white blood cell count (A) in bronchoalveolar lavage fluid and the hydroxyproline (HYP) content (B) in mouse lung tissue in each treatment group. Normal represents the normal control group; Model represents the model control group; PFD represents the positive control group (200 mg / kg); 3a-L represents the low-dose group (15 mg / kg); 3a-M represents the medium-dose group (30 mg / kg); and 3a-H represents the high-dose group (60 mg / kg). Compared with the Normal group, ****p < 0.0001; compared with the Model group, ##p < 0.01, ###p < 0.001, ####p < 0.0001; compared with different dose groups of 3a, &p < 0.05, &&p < 0.01.

[0060] like Figure 4 As shown, Western blot was used to detect the expression of fibrosis-related proteins such as Fibronectin, Collagen I, and α-SMA in the lung tissue of mice in each group, with GAPDH as an internal control. Normal represents the normal control group; Model represents the model control group; PFD represents the positive drug pirfenidone group (200 mg / kg); 3a-L represents the low-dose 3a group (15 mg / kg); 3a-M represents the medium-dose 3a group (30 mg / kg); and 3a-H represents the high-dose 3a group (60 mg / kg).

[0061] Results analysis:

[0062] Survival rate: Survival rate curves of mice in each group ( Figure 1 A) The results showed that the survival rate of mice in the Model group was 50%, and the survival rate of mice in each of the 3a dose groups was 100%, which was significantly higher than that of the positive drug pirfenidone PFD group (survival rate of 67%), indicating that the 3a treatment could significantly reduce the mortality rate of mice.

[0063] Weight change: Weight change curves of mice in each group ( Figure 1B) The results showed that, starting from day 3, the body weight of mice in the Model group was significantly reduced compared to that in the Normal group (p < 0.001), but the body weight of mice in the 3a-H high-dose group (60 mg / kg) was greater than that in the Model group, and the difference was significant starting from day 12 (p < 0.05), indicating that treatment with 3a can significantly improve the change in body weight of mice.

[0064] Imaging analysis: Micro-CT scan results of mouse lung tissue ( Figure 2 The results showed that the Model group mice had a large number of dense fibrotic foci in their lungs, while the fibrosis in the 3a group mice was alleviated. This indicates that bleomycin can induce radiographic changes of pulmonary fibrosis in mice, and 3a can alleviate radiographic changes of pulmonary fibrosis.

[0065] Total white blood cell count in bronchoalveolar lavage fluid (BALF): Results of total white blood cell count in mouse bronchoalveolar lavage fluid (BALF) Figure 3 A) shows that, compared with the Normal group, the total number of white blood cells in the Model group was significantly increased (p < 0.0001). Compared with the Model group, the total number of white blood cells in the bronchoalveolar lavage fluid of mice in each dose group of 3a was significantly reduced (p < 0.0001) and showed a dose-dependent relationship, indicating that 3a can significantly reduce the total number of white blood cells in the bronchoalveolar lavage fluid of mice.

[0066] Hydroxyproline (HYP) content: Results of HYP content detection in mouse lung tissue ( Figure 3 B) shows that, compared with the Normal group, the HYP content in the lung tissue of the Model group mice was significantly increased. Compared with the Model group, the HYP content in the lung tissue of mice in each 3a dose group was significantly decreased, and this was dose-dependent, indicating that 3a can significantly reduce the HYP content in the lung tissue of IPF mice.

[0067] Western blot (WB) analysis: WB results of mouse lung tissue ( Figure 4 The results showed that, compared with the Normal group, the expression of Fibronectin, Collagen I, and α-SMA proteins in the lung tissue of the Model group mice was significantly increased, indicating that the in vivo pulmonary fibrosis model was successfully established. Compared with the Model group, the expression of Fibronectin, Collagen I, and α-SMA proteins in the lung tissue of mice in different doses of 3a and the PFD group were all reduced to varying degrees, with the 3a-H group showing a significant reduction in the expression levels of these proteins. Moreover, the 3a-H group was significantly superior to the PFD group.

[0068] Treatment administration regimen: Seventy-two male C57BL / 6J mice (8-10 weeks old) were randomly divided into six groups (n=12): normal control group, model group, positive control pirfenidone group (200 mg / kg), 3a-L low-dose group (15 mg / kg), 3a-M medium-dose group (30 mg / kg), and 3a-H high-dose group (60 mg / kg). According to the dosage requirements of each group, the corresponding compound was dissolved in 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) aqueous solution. On day 0, the normal control group received a single intratracheal infusion of an equal volume of 0.9% sterile saline, while the model group and the treatment group received a single intratracheal infusion of 2.5 mg / kg bleomycin (Nippon Kayaku Co., Ltd.) to induce the model. Starting on day 7 of modeling, the normal control group and the model group were administered an equal volume of 0.5% CMC-Na aqueous solution by gavage daily. The positive control group was administered 200 mg / kg pirfenidone (Maclean) by gavage daily. The experimental compound administration groups were administered 15 mg / kg, 30 mg / kg, and 60 mg / kg of compound 3a by gavage daily, respectively, for 14 consecutive days. On day 21 of modeling, mice in each group were anesthetized, and multiple lung function tests (n=5 per group) were performed using a pulmonary function testing instrument, mainly including dynamic resistance (Rrs), inspiratory volume (IC), dynamic elasticity (Ers), and dynamic compliance (Crs). The remaining mice in each group were anesthetized, and tissue samples were collected to evaluate the degree of pulmonary fibrosis, including: histopathological examination of mouse lung tissue (HE staining and Masson staining), and immunohistochemical staining of mouse lung tissue to detect the expression levels of pulmonary fibrosis-related proteins such as Fibronectin, Collagen I, and α-SMA.

[0069] like Figure 5 As shown, multiple lung function tests (including dynamic resistance Rrs, inspiratory volume IC, dynamic elasticity Ers, and dynamic compliance Crs) were performed on mice in each treatment group. Normal represents the normal control group; Model represents the model control group; PFD represents the positive control group (200 mg / kg); 3a-H represents the 3a high-dose group (60 mg / kg). Compared with the Normal group, ***p < 0.001, ****p < 0.0001; compared with the Model group, #p < 0.05, ##p < 0.01, ###p < 0.001.

[0070] like Figure 6The images show H&E staining and Masson staining of lung tissue from mice in each treatment group. Normal represents the normal control group; Model represents the model control group; PFD represents the positive drug pirfenidone group (200 mg / kg); 3a-L represents the low-dose 3a group (15 mg / kg); 3a-M represents the medium-dose 3a group (30 mg / kg); and 3a-H represents the high-dose 3a group (60 mg / kg).

[0071] like Figure 7 The image shows immunohistochemical images of α-SMA, Collagen I, and Fibronectin in the lung tissue of mice in each treatment group. Normal represents the normal control group; Model represents the model control group; PFD represents the positive drug pirfenidone group (200 mg / kg); 3a-H represents the 3a high-dose group (60 mg / kg). Compared with the Normal group, **p < 0.01, ***p < 0.001, ****p < 0.0001; compared with the Model group, #p < 0.05, ##p < 0.01, ###p < 0.001.

[0072] Results analysis:

[0073] Lung function tests: Multiple lung function tests were performed on mice using a pulmonary function testing instrument. Figure 5 The results showed that, compared with the Normal group, the Model group mice had significantly lower IC and Crs levels, while Rrs and Ers levels were significantly higher, resulting in a significant downregulation of lung function. 3a (60 mg / kg) could significantly reverse bleomycin-induced lung function damage, specifically by significantly increasing IC and Crs levels and significantly downregulating Rrs and Ers levels.

[0074] Pathological examination: H&E and Masson staining were performed on mouse lung tissue, and the results are as follows: Figure 6 As shown, the alveolar structure of the lung tissue of mice in the Normal group was normal, with no inflammatory cell infiltration and no collagen deposition. In the Model group, the alveolar septa of the lung tissue of mice were significantly thickened, a large number of alveolar cavities were significantly reduced or disappeared, a large number of inflammatory cells were infiltrated in the interstitial lung tissue, and a large amount of collagen was deposited. Compared with the Model group, the lung tissue of mice in the 3a different dose groups showed significantly reduced vacuolar degeneration, significantly improved alveolar wall thickening, significantly improved inflammatory infiltration, and significantly reduced collagen deposition. Moreover, the performance of the 3a-H group was better than that of the pirfenidone group and closer to that of the normal group.

[0075] Immunohistochemical analysis: This invention utilizes immunohistochemical analysis to detect the distribution, localization, and expression levels of pulmonary fibrosis markers in lung tissue sections. Immunohistochemical results ( Figure 7The results showed that the positive expression level of α-SMA protein in the lung tissue of mice in the Normal group was low, while the positive expression level of α-SMA protein in the lung tissue of mice in the Model group was significantly higher than that in the Normal group (p < 0.001). After 3a-H treatment, the positive expression of α-SMA protein was significantly lower than that in the Model group (p < 0.01). Similarly, the positive expression level of Collagen I protein in the lung tissue of mice in the Normal group was low, while the positive expression level of Collagen I protein in the lung tissue of mice in the Model group was significantly higher than that in the Normal group (p < 0.0001). After 3a-H treatment, the positive expression of Collagen I protein was significantly lower than that in the Model group (p < 0.001). Correspondingly, the positive expression level of Fibronectin protein in the lung tissue of mice in the Normal group was low, while the positive expression level of Fibronectin protein in the lung tissue of mice in the Model group was significantly higher than that in the Normal group (p < 0.01). After 3a-H treatment, the positive expression of Fibronectin protein was significantly lower than that in the Model group (p < 0.05).

[0076] Safety Assessment: Twenty-four male C57BL / 6J mice (8-10 weeks old) were randomly divided into two groups (n=12), designated as the normal control group and the 3a (60 mg / kg) treatment group. A 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) aqueous solution was used as the solvent, and the drug was administered via gavage for 14 consecutive days (one dose daily from day 0 to day 13). Mouse weight was measured daily during the experiment, and tissue samples were collected under anesthesia on the day after the last administration, including serum, heart, liver, spleen, lungs, and kidneys. Weight changes during the administration period were plotted, and organ indices were calculated. Organ tissues from both groups were fixed with 0.4% paraformaldehyde and then stained with H&E. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum creatinine (CRE) were measured using a kit to evaluate the drug's effects on liver and kidney function.

[0077] Experimental results are as follows Figure 8 As shown, the mouse's body weight change ( Figure 8 A) showed that there was no significant difference in body weight change between the normal group and the 3a (60 mg / kg) group throughout the administration period; the organ indices of the two groups of mice ( Figure 8 B) indicates that there were no significant differences in organ indices between the normal group and the 3a (60 mg / kg) group of mice. Specifically, the heart index (p = 0.2815), liver index (p = 0.1031), spleen index (p = 0.9304), lung index (p = 0.5816), and kidney index (p = 0.3060) were significantly different. The H&E staining results for each organ in both groups of mice (…) Figure 8 C) showed that the morphology of various organs in the 3a (60 mg / kg) group of mice was not different from that in the normal group; the results of serum AST, ALT and CRE levels in the two groups of mice were as follows: Figure 8 DF) showed that there were no significant differences in serum AST, ALT and CRE between the normal group and the 3a (60mg / kg) group mice, with AST (p=0.4832), ALT (p=0.9892) and CRE (p=0.9966).

[0078] The results from the above examples demonstrate that indolequinolinone compound 3a can effectively prevent and treat pulmonary fibrosis, exhibiting significantly increased survival rate, significant weight gain, marked improvement in lung function, reduced alveolar structural damage, decreased inflammatory cell infiltration, reduced degree of fibrosis, and significantly decreased hydroxyproline content and fibrosis-related protein expression in mice. Furthermore, safety evaluation results show that indolequinolinone compound 3a has good in vivo safety in animals.

[0079] Example 2: Indolequinolinone derivative 3a alleviates lipopolysaccharide (LPS)-induced acute lung injury.

[0080] Lipopolysaccharide (LPS)-induced acute lung injury (ALI) is a widely used model of acute lung injury induced by sepsis. It disrupts the vascular endothelial barrier, causing interstitial lung edema and alveolar edema. LPS is a major component of endotoxins and a key substance in Gram-negative bacterial sepsis-induced ALI. This invention established an animal model of ALI induced by a single intratracheal infusion of LPS and further verified the anti-inflammatory protective effect of indolequinolinone 3a against lung injury by administering LPS 1 hour before the infusion.

[0081] Sixty male Balb / c mice (6-8 weeks old) were randomly divided into four groups (n=12 each) after one week of acclimatization feeding: a normal control group, a model group, a dexamethasone group (DEX 5 mg / kg), a low-dose 3a-L group (10 mg / kg), and a high-dose 3a-H group (20 mg / kg). LPS (Sigma) was dissolved in physiological saline and administered intratracheally at a dose of 5 mg / kg to induce pulmonary edema. One hour prior to LPS administration, mice were given intraperitoneal injection, and 6 hours after intratracheal administration, the mice were anesthetized and lung tissue was collected. The following parameters were measured: whole lung tissue was collected from six mice in each group. The wet weight of the lungs was measured, and the lung tissue was dried at 80°C for 24 hours before weighing the dry weight. The wet weight / dry weight ratio (W / D ratio) was calculated to assess the degree of pulmonary edema. The remaining six mice had bronchoalveolar lavage fluid (BALF) collected. After centrifugation, the expression and release levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and myeloperoxidase (MPO) in the BALF supernatant were detected using enzyme-linked immunosorbent assay (ELISA). Simultaneously, the total leukocyte count and total protein concentration in the BALF were measured to assess inflammatory mediators. Finally, the mouse lung tissue samples were examined for pathological evaluation. The experimental results are as follows:

[0082] like Figure 9 The table shows the lung wet weight / dry weight ratio of mice in each treatment group. Compared with the Norma1 group, **p < 0.01; compared with the Model group, #p < 0.05.

[0083] like Figure 10 As shown, H&E staining of lung tissue from mice in each drug administration group.

[0084] like Figure 11 As shown, the total white blood cell count (A) and total protein concentration (B) in the bronchoalveolar lavage fluid of mice in each treatment group. Compared with the Normal group, ****p<0.0001; compared with the Model group, #p<0.05, ###p<0.001, ####p<0.0001.

[0085] like Figure 12 The table shows the levels of inflammatory cytokines IL-6 (A), IL-1β (B), TNF-α (C), and MPO (D) in the bronchoalveolar lavage fluid of mice in each treatment group. Compared with the Normal group, ****p < 0.0001; compared with the Model group, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001.

[0086] Results analysis:

[0087] Lung edema assessment: Results of lung wet weight / dry weight ratio in mice of each treatment group ( Figure 9 The results showed that, compared with the Normal group, the wet / dry weight ratio of the lungs in the Model group was significantly increased (p < 0.01), while the wet / dry weight ratio of the lungs in the 3a-L and 3a-H groups was significantly decreased (p < 0.05). These results indicate that 3a can reduce lung edema in ALI mice, with an effect comparable to that of DEX.

[0088] Pathological evaluation: H&E staining results of lung tissues from mice in each treatment group ( Figure 10 The results showed that, compared with the Normal group, the Model group mice exhibited significant inflammatory lesions in their lung tissue pathology, including inflammatory cell infiltration, hemorrhage, and alveolar structural destruction. After 3a intervention, the inflammatory response in the mouse lung tissue was weakened, including reduced inflammatory cell infiltration and improved alveolar structural integrity.

[0089] Inflammatory mediator assessment: Based on the total white blood cell count in the bronchoalveolar lavage fluid of mice in each treatment group ( Figure 11 A) and total protein concentration ( Figure 11 B) The results showed that the total white blood cell count in the bronchoalveolar lavage fluid (BALF) of mice in the Normal group was lower than that in the Model group (p < 0.0001). After 3 years (10 mg / kg) of intervention, the total white blood cell count in the BLF of mice was significantly lower (p < 0.05), and after 3 years (20 mg / kg) of intervention, the total white blood cell count in the BLF of mice was significantly lower (p < 0.0001). Similarly, the total protein concentration in the BLF of mice in the Normal group was lower than that in the Model group (p < 0.0001). After 3 years (10 mg / kg) of intervention, the total protein concentration in the BLF of mice was significantly lower (p < 0.001), and after 3 years (20 mg / kg) of intervention, the total protein concentration in the BLF of mice was significantly lower (p < 0.0001). This indicates that 3a reduces lung permeability in LPS-induced ALI mice.

[0090] TNF-α, IL-1β, and IL-6 are typical pro-inflammatory mediators in various inflammatory diseases. To further evaluate the anti-inflammatory properties of compound 3a, the present invention detected the levels of TNF-α, IL-1β, and IL-6 in mouse bronchoalveolar lavage fluid according to the ELISA kit instructions. The results are as follows: Figure 12As shown in AC, after LPS intratracheal instillation model, the IL-6 content in the bronchoalveolar lavage fluid of mice was significantly increased (p < 0.0001). After 3 years of intervention, the IL-6 level in the bronchoalveolar lavage fluid of mice was significantly decreased (p < 0.0001). Similarly, after LPS intratracheal instillation model, the IL-1β content in the bronchoalveolar lavage fluid of mice was significantly increased (p < 0.0001). After 3 years of intervention (10 mg / kg), the IL-1β content in the bronchoalveolar lavage fluid of mice was significantly decreased (p < 0.01). After 3 years of intervention (20 mg / kg), the IL-1β content in the bronchoalveolar lavage fluid of mice was significantly decreased (p < 0.0001). Similarly, after LPS intratracheal instillation modeling, the TNF-α content in the bronchoalveolar lavage fluid of mice was significantly increased (p < 0.0001), and after 3 years of intervention, the TNF-α level in the bronchoalveolar lavage fluid of mice was significantly decreased (p < 0.0001). In particular, myeloperoxidase (MPO) is mainly found in neutrophils. During inflammatory responses, it is released into the extracellular fluid. Large-scale neutrophil aggregation leads to changes in tissue and cell permeability, causing tissue damage. The results of this experiment (…) Figure 12 D) showed that the MPO content in the bronchoalveolar lavage fluid of mice in the Normal group was lower than that in the Model group (p < 0.0001). After 3 years (10 mg / kg) of intervention, the MPO content in the bronchoalveolar lavage fluid of mice decreased significantly (p < 0.05), and after 3 years (20 mg / kg) of intervention, the MPO content in the bronchoalveolar lavage fluid of mice decreased significantly (p < 0.001).

[0091] In summary, administering LPS via intraperitoneal injection 3 years prior to LPS infusion significantly reduced the wet / dry weight ratio of LPS-induced lungs in mice, improved the pathological manifestations of acute lung injury, and decreased the protein content, total white blood cell count, and expression and release levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, MPO) in bronchoalveolar lavage fluid. This approach can alleviate pulmonary edema, reduce pneumonia response, and improve the pathological lesions of acute lung injury.

[0092] Example 3: Indolequinolinone derivative 3a inhibits the epithelial-mesenchymal transition (EMT) process in human type II alveolar epithelial cells A549.

[0093] Type II alveolar epithelial cells, located in the alveoli, undergo a transformation in morphology from their original cobblestone-like epithelial form to a spindle shape upon stimulation by inflammatory mediators, growth factors, and other cytokines. This process completes the epithelial-mesenchymal transition (EMT), giving them the functions of mesenchymal cells and enabling them to synthesize collagen fibers. Excessive collagen fiber deposition can exacerbate the progression of pulmonary fibrosis. This invention establishes an in vitro model of TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells A549. Morphological observation, cell scratch assays, and Western blot experiments were used to evaluate the in vitro anti-pulmonary fibrosis effects of the compounds of this invention.

[0094] The specific implementation method is as follows:

[0095] Morphological observation method: The experiment was divided into a blank control group, a model group, and a drug-treated group. A549 cells in the logarithmic growth phase were collected, and the cell suspension was adjusted to 2×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / 2 well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The medium was then replaced with serum-free RPMI-1640 medium for 24 h of starvation. The medium was then changed. Cells in the blank group were added with complete medium, while cells in the model group were added with complete medium containing TGF-β1 at a final concentration of 5 ng / mL. Cells in the drug-treated group were treated with the drug and TGF-β1 medium at a final concentration of 5 ng / mL for 36 h. During this period, morphological changes were observed using an inverted phase-contrast microscope.

[0096] like Figure 13 The image shows the effect of indolequinolinone derivative 3a on the morphology of TGF-β1-induced human lung epithelial cells A549. Ctrl represents the normal control group; TGF-β1 represents the model group treated with 5 ng / mL TGF-β1; and TGF-β1+3a(10 μM) represents the treatment group co-treated with 3a (10 μM) and TGF-β1 (5 ng / mL).

[0097] Results analysis:

[0098] according to Figure 13 As shown, the cells in the Ctrl group exhibited a typical cobblestone shape; the cells treated with TGF-β1 lost their epithelial cell morphology and transformed into spindle-shaped fibrous cells; the cells treated with 3a (10 μM) rarely showed a spindle shape in the field of view, and their shape returned to the typical cobblestone shape.

[0099] Westem blot experiment: The experiment consisted of a blank control group, a model group, and a drug-treated group. A549 cells in logarithmic growth phase were collected, and the cell suspension was adjusted to 2 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 1 / 2 well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The medium was then replaced with serum-free RPMI-1640 medium for 24 h of starvation. The medium was changed again. Cells in the blank group were added with complete medium, while cells in the model group were added with complete medium containing TGF-β1 at a final concentration of 5 ng / mL. Cells in the drug-treated group were treated with the drug and medium containing TGF-β1 at a final concentration of 5 ng / mL for 36 h. Cell samples were collected after 36 h, and the expression of epithelial marker E-cadherin and mesenchymal marker N-cadherin was detected by Western blotting.

[0100] The results are as follows:

[0101] like Figure 14 The figure shows the effect of indolequinolinone derivative 3a on the expression levels of EMT-related proteins N-cadherin and E-cadherin in TGF-β1-induced human lung epithelial cells A549. In the figure, Ctrl represents the normal control group; TGF-β1 represents the model group treated with 5 ng / mL TGF-β1; and TGF-β1+3a(μM) represents the treatment groups treated with different concentrations of 3a and co-treated with TGF-β1 (5 ng / mL).

[0102] Results analysis: Western blot results ( Figure 14 The results showed that, compared with the expression levels in the Controll group, TGF-β1 treatment significantly increased N-Cadherin protein expression and significantly decreased E-Cadherin protein expression. After intervention with different concentrations of 3a, N-Cadherin protein expression significantly decreased and E-Cadherin protein expression significantly increased, indicating that 3a can improve the EMT transformation process induced by TGF-β1 in human lung epithelial cells A549.

[0103] Cell scratching: A549 cells were scratched at a rate of 3 × 10⁻⁶. 5 Plant cells in 6-well plates at a density of 1 / 2 well. When cell confluence reaches 70-80%, replace with low-serum RPMI-1640 medium. Cross-scrape the cells with a 200 μL pipette tip, wash twice with PBS, treat the blank group with low-serum medium, treat the model group with low-serum medium containing a final concentration of 5 ng / mL TGF-β1, and treat the drug group with the drug and low-serum medium containing a final concentration of 5 ng / mL TGF-β1. Continue culturing for 24 h and observe cell migration.

[0104] The results are as follows Figure 15 The effect of indolequinolinone derivative 3a on the migration ability of TGF-β1-induced human lung epithelial cells A549 is shown.

[0105] Results analysis: EMT facilitates cell migration and invasion, based on the results of cell scratch assays ( Figure 15The results showed that, compared with the Controll group, the migration ability of cells treated with TGF-β1 was significantly increased 24 hours later, while the migration ability of cells treated with different concentrations of 3a was significantly weakened 24 hours later, indicating that 3a can inhibit the migration ability of human lung epithelial cells A549 induced by TGF-β1.

[0106] Example 4: Experiment on the inhibition of human lung fibroblast activation by indolequinolone derivative 3a.

[0107] The accumulation of lung fibroblasts with an activated phenotype (called myofibroblasts) is a key feature of pulmonary fibrosis and is crucial to its pathogenesis. To further explore the therapeutic effect of compound 3a on BLM-induced pulmonary fibrosis, this invention established an in vitro model of TGF-β1-induced activation of human embryonic lung fibroblasts.

[0108] The experiment was divided into a blank control group, a model group, and a drug-treated group. HFL1 cells in the logarithmic growth phase were collected, and the cell suspension was adjusted to 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / 2 well in 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. The medium was then replaced with serum-free HF-12K medium for 24 h of starvation. After the medium was changed, cells in the blank group were added with complete medium, while cells in the model group were added with complete medium containing TGF-β1 at a final concentration of 5 ng / mL. Cells in the drug-treated group were treated with the drug and medium containing TGF-β1 at a final concentration of 5 ng / mL for 36 h. Cell samples were collected after 36 h, and the expression of α-SMA, Fibronectin, etc. was detected by Western blotting.

[0109] The results are as follows:

[0110] like Figure 16 The image shows the effect of indolequinolinone derivative 3a on the expression levels of Fibronectin and α-SMA protein bands in TGF-β1-induced human embryonic lung fibroblasts (HFL1). Ctrl represents the normal control group; TGF-β1 represents the model group treated with 5 ng / mL TGF-β1; and TGF-β1+3a(μM) represents the treatment groups treated with different concentrations of 3a and co-treated with TGF-β1 (5 ng / mL).

[0111] Results analysis:

[0112] α-SMA is an important marker of myofibroblast activation, as shown in Western blot results ( Figure 16 The results showed that, compared with the expression levels in the Ctrl group, the expression of α-SMA and Fibronectin proteins in HFL1 was significantly increased after TGF-β intervention, and the intervention of indolequinone derivative 3a inhibited the increase of TGF-β1-induced α-SMA and Fibronectin protein expression in HFL1.

[0113] The beneficial effects of this invention are as follows:

[0114] The inventors constructed a bleomycin (BLM)-induced mouse pulmonary fibrosis animal model and observed that the indolequinoline ketone derivative 3a of this invention has a significant alleviating effect on bleomycin-induced mouse pulmonary fibrosis, including: improving mouse survival rate and weight changes, reducing the total number of cells in bronchoalveolar lavage fluid, reducing the hydroxyproline content in mouse lung tissue, reducing inflammatory cell infiltration, reducing alveolar septal thickening, and improving symptoms such as fibroblast activation and collagen deposition. Simultaneously, the inventors constructed an LPS-induced mouse acute lung injury animal model and observed that the indolequinoline ketone derivative 3a of this invention can reduce the total protein content in bronchoalveolar lavage fluid (BALF), improve tissue section inflammation, and reduce the expression and release of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and myeloperoxidase (MPO), demonstrating significant therapeutic effects.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that although the present invention has been described above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make several improvements and modifications based on the above-mentioned technical content without departing from the scope of the technical solution of the present invention. These improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of indolequinolinone compounds in the preparation of drugs for the prevention and / or treatment of pulmonary fibrosis and lung injury, wherein the indolequinolinone compound is a compound with the structure shown in formula I-3a. 。 2. The application according to claim 1, characterized in that, The indolequinoline ketone compounds are synthesized via the following reaction: Under the catalysis of an acid catalyst, the indole-2-amide compound shown in formula (Ia) and the 1,4-benzoquinone compound shown in formula (Ib) react to give the indolequinone compound shown in formula (I): ； The acid catalyst includes at least one of trifluoroacetic acid, acetic acid, p-toluenesulfonic acid, and trifluoromethanesulfonic acid.

3. The application according to claim 2, characterized in that, The molar ratio of the indole-2-amide compound, the 1,4-benzoquinone compound, and the acid catalyst is 1.0:1.1-1.5:0.1-0.

5.

4. The application according to claim 1, characterized in that, The pulmonary fibrosis includes at least one of primary pulmonary fibrosis and secondary pulmonary fibrosis.

5. The application according to claim 1, characterized in that, The lung injury diseases mentioned include direct lung injury and indirect lung injury. The direct lung injury is induced lung injury, including at least one of bacterial or viral lung infection, aspiration of gastric contents, lung contusion, inhalation of toxic gases, and oxygen poisoning. The indirect lung injury includes at least one of the following: severe non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, cardiopulmonary bypass, and disseminated intravascular coagulation.

6. The application according to claim 1, characterized in that, The dosage form of the medicine for treating pulmonary fibrosis and lung injury is one or more of the following: tablets, capsules, pills, aerosols, oral liquid preparations, granules, powders, injections, syrups, tinctures, lotions, and films.

7. The application according to claim 1, characterized in that, The administration methods of the drugs for treating pulmonary fibrosis and lung injury include one or more of the following: oral, injection, spray, inhalation, implantation, and topical application.

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