Application of compound with general formula

Abstract

The invention belongs to the technical field of medicines, provides novel application of a compound with a general formula, and particularly provides application of the compound with the general formula in prevention or / and treatment of pulmonary fibrosis and acute lung injury. The compound with the general formula can be used for effectively preventing and treating bleomycin (BLM)-induced pulmonary fibrosis (PF) in mice. In the aspect of the drug effect of preventing pulmonary fibrosis, the compound with the general formula has the same effect as the pulmonary fibrosis drug nintedanib; compared with the pulmonary fibrosis medicine pirfenidone, the compound with the general formula has the advantages of smaller dosage and better in-vivo safety in the aspect of pulmonary fibrosis treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of a general formula compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine or food for preventing, treating or alleviating at least one disease selected from pulmonary fibrosis and acute lung injury. Background Art

[0002] Pulmonary fibrosis (PF) is a serious respiratory disease with a high mortality rate. Infection with pathogenic microorganisms such as bacteria and viruses, drug induction, and aging are all high-risk factors for PF. Among them, idiopathic pulmonary fibrosis (IPF) is a disease of unknown cause characterized by diffuse alveolitis and alveolar structural disorder that eventually leads to pulmonary interstitial fibrosis. Its median survival period is only 2 to 3 years, and the five-year survival rate is about 20%. It is also called a tumor-like disease, and there is no specific treatment. With the invasion of pathogens such as environmental pollution, respiratory viruses (such as SARS-CoV-2 or seasonal influenza viruses), and bacteria, pulmonary fibrosis caused by inflammation of the lower respiratory tract has also become a human health problem that cannot be ignored.

[0003] At present, anti-fibrotic drugs are widely used in clinical practice to treat PF. China has launched two anti-pulmonary fibrosis drugs - Pirfenidone and Nintedanib. These two drugs are effective for mild and moderate pulmonary fibrosis and can delay the decline of lung function, but cannot completely control or reverse the damaged lung function. Among them, Pirfenidone has side effects such as photosensitivity, fatigue, rash, stomach discomfort and anorexia. Nintedanib is a multi-target tyrosine kinase inhibitor that can inhibit platelet-derived growth factor receptors, vascular endothelial growth factor receptors and fibroblast growth factor receptors, but it can only alleviate the disease process to a certain extent, and there are adverse reactions of liver damage and kidney damage in clinical applications. The treatment of pulmonary fibrosis needs to be improved urgently, and safer and more effective therapeutic drugs are still needed in clinical practice.

[0004] In order to solve the adverse reactions of existing drugs, finding functional ingredients or drugs to prevent / treat pulmonary fibrosis and lung injury has always been a hot topic in the research and development of anti-pulmonary fibrosis and anti-lung injury drugs. Summary of the invention

[0005] The present invention aims to solve one of the technical problems existing in the prior art at least to a certain extent. To this end, the present invention provides a compound of a general formula and application thereof.

[0006] According to one aspect of the present invention, there is provided a compound of formula I or a pharmaceutically acceptable salt thereof for use in preparing a drug or food for preventing, treating or alleviating at least one disease selected from pulmonary fibrosis and acute lung injury.

[0007] Wherein, R is selected from one of H and alcoholic hydroxyl group.

[0008] Preferably, the compound represented by formula I is selected from compound I, compound II, compound III, and compound IV.

[0009]

[0010] Preferably, the pulmonary fibrosis disease includes at least one of primary pulmonary fibrosis, idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, and interstitial pulmonary fibrosis.

[0011] Preferably, the lung injury disease includes direct lung injury and indirect lung injury. The direct lung injury is induced lung injury, including at least one of lung infection caused by bacteria, viruses, etc., aspiration of gastric contents, lung contusion, inhalation of toxic gases, and oxygen poisoning; the indirect lung injury includes at least one of lung injury caused by severe non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, extracorporeal circulation, and disseminated intravascular coagulation.

[0012] According to another aspect of the present invention, there is provided a use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug or food for preventing, treating or alleviating at least one of the following symptoms: i) weight loss of an individual; ii) worsening of lung imaging; iii) alveolar structural lesions; iv) abnormal infiltration of inflammatory cells in the lungs, wherein the inflammatory cells include at least one of total leukocytes, lymphocytes and macrophages; v) pulmonary fibrosis; vi) abnormal collagen deposition in lung tissue; vii) abnormal hydroxyproline content in lung tissue; viii) overexpression of fibrosis-related proteins in lung tissue, wherein the fibrosis-related proteins include N-cadherin, Vimentin, Fibronectin, Collagen; I and smooth muscle actin α-SMA; overexpression of TGFBRII receptor protein and phosphorylated P-Smad2 protein in lung tissue; ix) abnormal total leukocyte count in bronchoalveolar lavage fluid; x) abnormal expression of proinflammatory factors in bronchoalveolar lavage fluid, wherein the proinflammatory factors include at least one of TNF-α, IL-1β, and IL-6; xi) abnormal myeloperoxidase (MPO) content in bronchoalveolar lavage fluid; xii) abnormal levels of neutrophils and macrophages in lung tissue; xiii) abnormal total protein concentration in bronchoalveolar lavage fluid and abnormal expression of tight junction protein ZO-1 in lung tissue;

[0013] Wherein, R is selected from one of H and alcoholic hydroxyl group.

[0014] Preferably, the compound represented by formula I is selected from compound I, compound II, compound III, and compound IV.

[0015]

[0016] Preferably, the dosage form of the drug is one or more of tablets, capsules, granules, liquids, oral dissolving tablets, oral suspensions, oral emulsions, pills, injections, aerosols, powders, wine preparations, dew preparations, and films.

[0017] Preferably, the administration method of the drug for treating pulmonary fibrosis and lung injury disease includes one or more of oral administration, injection, spray, inhalation, implantation, and external application.

[0018] According to another aspect of the present invention, a medicine is provided, which comprises the above-mentioned compound.

[0019] According to yet another aspect of the present invention, a food is provided, comprising the above-mentioned compound.

[0020] Although the prior art has reported that the ginsenoside composition mainly composed of primary ginsenosides, the primary ginsenoside monomer components Rg1 and Rd, and the secondary ginsenoside monomers Rg5 and Rg3 have different degrees of efficacy in preventing bleomycin (BLM)-induced pulmonary fibrosis (PF) in mice or rats, the prior art uses the PF prevention model of immediate administration after BLM modeling to evaluate the anti-pulmonary fibrosis effect of ginsenoside components.

[0021] However, in vivo metabolism studies have shown that primary ginsenosides have a large molecular weight and molecular polarity, and their absorption rate in the human body is extremely low via oral administration. The active ingredients that are truly absorbed by the human body are secondary ginsenosides or a small amount of metabolites such as aglycones produced by metabolism in the stomach (acidic environment) and intestinal bacteria. After metabolism in the gastrointestinal tract, the secondary ginsenoside components or aglycones (protopanaxatriol PPT, or / and protopanaxadiol PPD) are absorbed into the portal vein by the mesentery. Compared to primary ginsenosides and secondary ginsenosides, the general formula compounds of the present invention are stable in the acidic environment of the stomach, have a small molecular weight, and have a molecular polarity that is more suitable for rapid intestinal absorption.

[0022] In the present invention, the term "primary ginsenoside" refers to a triterpene glycoside compound that exists in large quantities in plants of the genus Panax in the Araliaceae family. Primary ginsenosides have similar basic triterpene aglycone structures, all of which contain a dammarane-type tetracyclic triterpene mother nucleus composed of 30 carbon atoms. Primary ginsenosides can be divided into protopanaxatriol (PPT) primary ginsenosides and protopanaxadiol (PPD) primary ginsenosides, and both PPT-type primary ginsenosides and PPD-type primary ginsenosides contain the following 20 (S) configuration, i.e., sugar or sugar chain at C-20, and in addition, PPT-type primary ginsenosides also contain sugar or sugar chain at C-6, while PPD-type primary ginsenosides also contain sugar or sugar chain at C-3.

[0023]

[0024] In the present invention, the term "secondary ginsenoside" refers to the secondary metabolites of primary ginsenosides, which are less or non-existent in plants of the genus Panax in the Araliaceae family. Generally, secondary metabolites include secondary ginsenoside types that only hydrolyze sugars at the C-20 position, secondary metabolites produced by retaining sugars at the C-20 position and only hydrolyzing sugars at the C-6 position (PPT type) and sugars at the C-3 position (PPD type) (such as PPT-type ginsenoside F1 and PPD-type ginsenoside M1), and various other types of metabolites. In the present invention, secondary ginsenosides mainly refer to secondary ginsenoside types that only hydrolyze sugars at the C-20 position. PPT-type secondary ginsenosides refer to the products of PPT-type primary ginsenosides that only hydrolyze sugars at the C-20 position. PPD-type secondary ginsenosides refer to the products of PPD-type primary ginsenosides that only hydrolyze sugars at the C-20 position.

[0025] The general formula compound of the present invention can significantly improve the survival rate of PF mice at a 10mg / kg intraperitoneal injection dosage, significantly improve the weight of PF mice, and can effectively prevent and protect bleomycin-induced mouse pulmonary fibrosis. The general formula compound of the present invention can effectively prevent and protect bleomycin-induced mouse pulmonary fibrosis at a 10mg / kg intragastric administration dosage, and its effect is comparable to the positive drug nintedanib (clinically effective dose 30mg / kg), and has a smaller dosage than nintedanib. In the intragastric administration treatment mode of pulmonary fibrosis, compared with the clinical effective dose (200mg / kg) of the positive drug pirfenidone, the general formula compound of the present invention is administered intragastrically (20mg / kg) small, and the efficacy and biosafety of treating pulmonary fibrosis are significantly better than pirfenidone (200mg / kg). Compared with existing listed drugs, the general formula compound is safer and more efficient, and can not only be used as a drug for the treatment of pulmonary fibrosis, but also can be used as the main functional ingredient in the health food for preventing pulmonary fibrosis and lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 This is a schematic diagram of the experimental process of preventing pulmonary fibrosis by intraperitoneal injection of the compound of the general formula in Example 1 of the present invention.

[0028] Figure 2 This is the curve of the survival rate and body weight change of mice in each experimental group administered by intraperitoneal injection in Example 1 of the present invention; in this figure, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001vs sham operation group; # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001vs model group.

[0029] Figure 3 The Micro-CT scan images of the lung tissues of mice in each experimental group administered by intraperitoneal injection in Example 1 of the present invention, the statistical results of the grayscale values ​​of the lung cross-sections, and the photographs of the lungs of mice; in this figure, ***p<0.001vs the sham operation group; # p<0.05, ## p<0.01vs model group.

[0030] Figure 4 This is the H&E staining image of the lung tissue of mice in each experimental group administered by intraperitoneal injection in Example 1 of the present invention; in this figure, the scale bar is 50 microns.

[0031] Figure 5 The statistical results of the number of total leukocytes, lymphocytes and macrophages in the bronchoalveolar lavage fluid of mice in each experimental group administered by intraperitoneal injection in Example 1 of the present invention; in this figure, ***p<0.001, ****p<0.0001vs sham operation group; # p<0.05, ## p<0.01vs model group.

[0032] Figure 6 The Masson staining diagram and the statistical results of the collagen fiber positive area ratio of the lung tissue of mice in each experimental group of the intraperitoneal injection administration method in Example 1 of the present invention are shown; in the Masson staining diagram, the scale bar is 50 microns; in the statistical results of the collagen fiber positive area ratio, ***p<0.001vs sham operation group; # p<0.05, ## p<0.01vs model group.

[0033] Figure 7 The results of the detection of hydroxyproline content in the lung tissue of mice in each experimental group administered by intraperitoneal injection in Example 1 of the present invention; in this figure, **p<0.01vs sham operation group; # p<0.05, ## p<0.01vs model group.

[0034] Figure 8 The results of Western blotting of N-Cadherin, α-SMA and Vimentin expressed in lung tissue of mice in the experimental groups TSA1 and TSA2 administered by intraperitoneal injection in Example 1 of the present invention; in this figure, * p<0.05, ** p<0.01, *** p<0.001vs sham operation group; # p<0.05, ## p<0.01, ### p<0.001vs model group.

[0035] Fig. 9 The results of Western blotting of N-Cadherin, α-SMA and Vimentin expressed in lung tissue of mice in the experimental groups DSA1 and DSA2 administered by intraperitoneal injection in Example 1 of the present invention; in this figure, * p<0.05, ** p<0.01vs sham operation group; # p<0.05vs model group.

[0036] Fig.10 This is a schematic diagram of the experimental process of preventing pulmonary fibrosis by oral administration of the general formula compound TSA2 in Example 1 of the present invention.

[0037] Fig.11 The Micro-CT scan images of the lung tissues of mice in each experimental group of the intragastric administration method in Example 1 of the present invention and the statistical results of the grayscale values ​​of the lung cross-sections and the photographs of the mouse lungs are shown; in the figure, **** p<0.0001vs sham operation group; # p<0.05vs model group.

[0038] Fig.12 is the lung weight index of mice in each experimental group administered by oral gavage in Example 1 of the present invention; in this figure, ** p<0.01vs sham operation group; # p<0.05vs model group.

[0039] Fig.13This is the H&E staining image of the lung tissue of mice in each experimental group administered by oral gavage in Example 1 of the present invention; in this figure, the scale bar is 50 microns.

[0040] Fig.14 The statistical results of the total leukocytes and lymphocytes in the bronchoalveolar lavage fluid of mice in each experimental group administered by intragastric administration in Example 1 of the present invention; in this figure, ***p<0.001vs sham operation group; # p<0.05vs model group.

[0041] Fig.15 The Masson staining diagram and the statistical results of the collagen fiber positive area ratio of the lung tissue of mice in each experimental group of the oral administration method in Example 1 of the present invention are shown; in the Masson staining diagram, the scale bar is 50 microns; in the statistical results of the collagen fiber positive area ratio, ***p<0.001vs the sham operation group; # p<0.05vs model group.

[0042] Fig.16 The results of the detection of hydroxyproline content in the lung tissue of mice in each experimental group administered by intragastric administration in Example 1 of the present invention; in this figure, **p<0.01vs sham operation group; # p<0.05vs model group.

[0043] Fig.17 The results of Western blotting of N-Cadherin, Vimentin, α-SMA, Fibronection and COL1A1 expressed in the lung tissues of mice in each experimental group administered by oral gavage in Example 1 of the present invention; in this figure, * p<0.05, ** p<0.01, **** p<0.0001vs sham operation group; # p<0.05, ## p<0.01, ### p<0.001vs model group.

[0044] Fig.18 The results of Western blotting of the receptor protein TGFBRII and pathway protein Smad2 expressed in the lung tissues of mice in each experimental group administered by oral gavage in Example 1 of the present invention; in this figure, ** p<0.01, *** p<0.001vs sham operation group; ## p<0.01, ### p<0.001vs model group.

[0045] Fig.19This is a schematic diagram of the experimental process of treating pulmonary fibrosis by intragastric administration of the general formula compound TSA2 in Example 2 of the present invention.

[0046] Fig. 20 The figure is a graph of the lung tissue pathology and hydroxyproline content determination results of each group of mice in Example 2 of the present invention; in the figure, A is the H&E staining picture; B is the Masson staining picture; C is the statistical result of the collagen fiber positive area ratio in the Masson staining picture; D is the hydroxyproline content detection result in the lung tissue of mice in each experimental group. In Figures C and D, **** p<0.0001vs sham operation group; # p<0.05, ## p<0.01, #### p<0.0001vs model group.

[0047] Fig.21 These are H&E staining images of the heart, liver, spleen and kidney of mice in each experimental group in Example 2 of the present invention; in this figure, the scale bar is 50 microns.

[0048] Fig. 22 Schematic diagram of the experimental process of preventing acute lung injury by intraperitoneal injection of the general formula compound TSA2 in Example 3 of the present invention.

[0049] Fig.23 This is the H&E staining image of the lung tissue of mice in each experimental group in Example 3 of the present invention; in this figure, the scale bar is 50 microns.

[0050] Fig.24 The total leukocyte determination results in the bronchoalveolar lavage fluid of mice in each experimental group in Example 3 of the present invention; in this figure, ***p<0.001vs control group; # p<0.05, ## p<0.01, ### p<0.001vs model group.

[0051] Fig.25 The results of the determination of the contents of MPO and proinflammatory factors TNF-α, IL-6, and IL-1β in the bronchoalveolar lavage fluid of mice in each experimental group in Example 3 of the present invention; in this figure, ***p<0.001, ****p<0.0001vs the control group; # p<0.05, ## p<0.01, #### p<0.0001vs model group.

[0052] Fig.26The immunohistochemical detection results and positive area percentage statistical results of neutrophils (LY-6G) and macrophages (F4 / 80) in the lung tissues of mice in each experimental group in Example 3 of the present invention are shown; in the immunohistochemical result graph, the scale bar is 50 microns; in the positive area percentage statistical result graph, **p<0.01, ***p<0.001vs control group; # p<0.05, ## p<0.01, ### p<0.001vs model group.

[0053] Fig. 27 The results of the determination of protein concentration in the bronchoalveolar lavage fluid of mice in each experimental group in Example 3 of the present invention; in this figure, ****p<0.0001vs control group; # p<0.05, ## p<0.01vs model group.

[0054] Fig.28 The results of Western blotting of tight junction protein ZO-1 expressed in lung tissues of mice in each experimental group in Example 3 of the present invention; in this figure, **p<0.01vs control group; # p<0.05vs model group. DETAILED DESCRIPTION

[0055] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. The raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.

[0056] Explanation of terms

[0057] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0058] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0059] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0060] The term "or more" used in the present invention includes the number itself. For example, "two or more" includes two, three, four, etc.

[0061] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0062] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0063] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0064] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0065] In the present invention, “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0066] In the present invention, "optionally", "optional", "optionally", "optionally", "optional", "optional", "optional" means optional, that is, it means to be selected from any one of the two parallel schemes of "yes" or "no". If multiple "optional" or "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0067] In the present invention, the term "pulmonary fibrosis" refers to pulmonary fibrosis, abbreviated as PF, which is a terminal manifestation of interstitial lung disease. This disease is characterized by fibroblast proliferation and a large amount of extracellular matrix deposition, leading to the destruction of lung tissue structure.

[0068] In the present invention, the term "acute lung injury" refers to acute lung injury, abbreviated as ALI, which is caused by various direct and indirect injury factors that damage alveolar epithelial cells and capillary endothelial cells, resulting in diffuse pulmonary interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency. This injury usually occurs due to a systemic inflammatory response caused by severe diseases or injuries, such as infection, trauma, burns, drowning, etc. The pathophysiological characteristics are reduced lung volume, reduced lung compliance, and ventilation / perfusion imbalance. Clinically, it manifests as progressive hypoxemia and respiratory distress, and lung imaging shows non-uniform exudative lesions. Acute lung injury develops to a severe stage, namely Acute Respiratory Distress Syndrome (ARDS), which is characterized by extensive damage to the alveoli and microvessels, resulting in severe lung function impairment, and usually requires important supportive treatments such as mechanical ventilation.

[0069] The present invention found that the compound shown in formula I has significant effects in preventing and treating BLM-induced pulmonary fibrosis and LPS-induced acute lung injury. Moreover, the compound can not only prevent pulmonary fibrosis (the prior art adopts a dosing mode for preventing pulmonary fibrosis, i.e., dosing starts on the second day of modeling), but also treat pulmonary fibrosis (modeling for 7 days, from lung inflammation to pulmonary fibrosis, dosing starts after pulmonary fibrosis, and the therapeutic efficacy is examined).

[0070] The present invention finds that the general formula compound can prevent and treat bleomycin (BLM)-induced pulmonary fibrosis (PF) in mice, which is specifically manifested in significantly improving the survival rate of PF mice, increasing the weight of PF mice, improving the lung imaging changes of PF mice, reducing the lung weight index of PF mice, reducing the pathological damage of lung tissue, and reducing the degree of lung inflammation and fibrosis. At the same time, the general formula compound can also reduce the lung inflammatory cell infiltration of mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS), reduce the permeability of the lungs and the expression of proinflammatory factors in bronchoalveolar lavage fluid (BALF), reduce the level of neutrophils and macrophages in lung tissue, and provide alveolar vascular endothelial barrier protection. In terms of the efficacy of preventing pulmonary fibrosis, the general formula compound has the same efficacy as the pulmonary fibrosis drug nintedanib; in terms of the efficacy of treating pulmonary fibrosis, the general formula compound has better efficacy and in vivo safety than the pulmonary fibrosis drug pirfenidone.

[0071] Preparation and structural characterization of compounds of general formula

[0072] The compound of the present invention is synthesized by the following method, wherein: 1 H-NMR and 13 C-NMR was measured on a Bruker Ascend 600 NMR spectrometer. Coupling constants are expressed in Hz, tetramethylsilane is used as the internal standard, and chemical shift values ​​are expressed in ppm. High-resolution mass spectra (HRMS) were measured on an Agilent 6230ESI-TOF-MS (electrospray ionization time of flight) mass spectrometer in positive ion mode:

[0073]

[0074] 20(S)-protopanaxantriol (1000 mg, 2.1 mmol, CAS: 34080-08-5, Shanghai Yaogu Pharmaceutical Co., Ltd.) was dissolved in methanol, Shi epoxidation diketal catalyst (2166 mg, 8.4 mmol, Sigma-Aldrich) was dissolved in acetonitrile (HPLC grade), Oxone (7743 mg, 12.6 mmol) and sodium bicarbonate (3174 mg, 37.8 mmol) were added to the methanol solution, and acetonitrile containing Shi epoxidation diketal catalyst was added dropwise, and the reaction solution was stirred at room temperature. TLC was used to monitor the reaction progress, and 10% sulfuric acid ethanol was used for color development. After the reaction, the reaction solution was spin-dried, the concentrate was extracted with water (Milli-Q) and ethyl acetate, the organic phases were combined, anhydrous sodium sulfate was added to remove water, and then concentrated under reduced pressure, and finally separated and purified by silica gel column chromatography (dichloromethane: ethyl acetate = 1:2) to obtain TSA1 (white solid, 491.4 mg, yield 47.6%) and TSA2 (white solid, 434 mg, yield 42%).

[0075]

[0076] 20(S)-protopanaxadiol (1000 mg, 2.2 mmol, CAS: 30636-90-9, Shanghai Yaogu Pharmaceutical Co., Ltd.) was dissolved in methanol, Shi epoxidation diketal catalyst (2244 mg, 8.7 mmol, CAS: 18422-53-2) was dissolved in acetonitrile, (8001 mg, 13.3 mmol, CAS: 70693-62-8) and sodium bicarbonate (3285 mg, 39.1 mmol, CAS: 144-55-8) were added to the methanol solution, and acetonitrile containing Shi epoxidation diketal catalyst was added dropwise, and the reaction solution was stirred at room temperature. TLC monitored the reaction progress and 10% sulfuric acid ethanol was used for color development. After the reaction, the reaction solution was spin-dried, the concentrate was extracted with water and ethyl acetate, the organic phases were combined, anhydrous sodium sulfate was added to remove water, and then concentrated under reduced pressure, and finally separated and purified by silica gel column chromatography (dichloromethane: ethyl acetate = 1:2) to obtain DSA1 (white solid, 370.8 mg, yield 36%) and DSA2 (white solid, 432 mg, yield 41.4%).

[0077]

[0078] Structural elucidation data of TSA1: 1H NMR(600MHz, CDCl3)δ:4.09(dt,J=3.6,10.8Hz,1H),3.83(dd,J=6.6,9.0Hz,1H),3.49(dt,J=4.8,1 0.8Hz,1H),3.16(dd,J=4.8,11.4Hz,1H),2.17-2.21(m,1H),1.82-2.05(m,5H),1.55-1.71(m,7H), 1.47-1.50(m,2H),1.41(s,1H),1.30(s,3H),1.27(s,3H),1.26(s,3H),1.12-1.15(m,1H),1.09(s, 3H),1.06(s,3H),0.99-1.04(m,1H),0.97(s,3H),0.93(s,3H),0.91(s,3H),0.87(d,J=10.8Hz,1H). 13 C NMR(150MHz, CDCl3)δ:86.50,85.41,78.57,75.04,74.27,70.90,70.23,68.71,62.84,61.31,60.54,51.85,49.98,48.98,47.89,46.98,40.90, 39.29,39.11,38.74,32.62,31.20,30.81,28.60,27.90,27.55,27.05, 26.76,26.10,24.98,24.90,18.13,17.42,16.93,15.43; HRMS(ESI):m / z calcd for C 30 H 53 O5 + ([M+H] + ):493.3893,found 493.3888.

[0079] Structural elucidation data of TSA2: 1H NMR (600MHz, CDCl3) δ: 4.09 (dt, J=3.6, 10.8Hz, 1H), 3.86 (dd, J=5.4, 10.8Hz, 1H), 3.51 (dt ,J=4.8,10.2Hz,1H),3.17(dd,J=4.8,11.4Hz,1H),2.23(dt,J=4.2,10.2Hz,1H),1.79-2.0 6(m,6H),1.49-1.73(m,10H),1.31(s,3H),1.27(s,3H),1.23(s,3H),1.12-1.16(m,1H),1. 09(s,3H),1.10(s,3H),1.02-106(m,1H),0.98(s,3H),0.94(s,3H),0.89(d,J=10.8Hz,1H). 13 C NMR (150MHz, CDCl3) δ: 87.45, 87.13, 78.54, 70.46, 70.07, 68.72, 61.28, 51.98, 49.70, 48.88, 48.41, 46.95, 40.91, 39.29, 39. 15,38.70,32.19,31.64,31.52,30.83,28.86,28.54,28.02,27.07,25.06,24.22,17.73,17.36,17.01,15.45; HRMS(ESI):m / z calcd for C 30 H 53 O5 + ([M+H] + ):493.3893,found 493.3887.

[0080] Structural elucidation data of DSA1: 1 H NMR (600MHz, CDCl3) δ: 3.83 (dd, J=6.6, 9.0Hz, 1H), 3.48 (dt, J=4.8, 10.8Hz, 1H), 3.17 (dd, J=4.8,12.0Hz,1H),2.16-2.20(m,1H),1.95-2.07(m,2H),1.84-1.91(m,3H),1.54-1.72(m, 7H),1.42-1.46(m,3H),1.29-1.31(m,2H),1.27(s,3H),1.26(s,3H),1.10-1.13(m,1H),1. 09(s,3H),0.96(d,J=9.0Hz,7H),0.89(s,3H),0.85(s,3H),0.77(s,3H),0.71-0.73(m,1H).13 C NMR(150MHz, CDCl3)δ:86.53,85.45,78.89,71.02,70.10,56.00,52.05,50.53,49.43,47.99,39.76,38.97,38.94,37.17,34. 85,32.64,31.35,31.22,28.61,28.01,27.94,27.63,27.47,26.15,25.02,18.30,18.19,16.34,15.40,15.28; HRMS(ESI):m / z calcd for C 30 H 53 O4 + ([M+H] + ):477.3944,found477.3948.

[0081] Structural elucidation data of DSA2: 1 H NMR (600MHz, CDCl3) δ: 3.86 (dd, J=5.4, 11.4Hz, 1H), 3.50 (dt, J=4.8, 10.2Hz, 1H), 3. 18(dd,J=4.2,12.0Hz,1H),2.23(dt,J=4.2,10.2Hz,1H),1.83-2.07(m,6H),1.49-1.7 8(m,10H),1.29-1.32(m,2H),1.27(s,3H),1.23(s,3H),1.12-1.16(m,2H),1.10(s,3H ),1.01(s,3H),0.97(s,3H),0.91(s,3H),088(s,3H),0.78(s,3H),0.73-0.75(m,1H). 13 C NMR(150MHz, CDCl3)δ:87.40,87.17,78.88,70.55,70.03,55.95,52.19,50.25,48.94,48.84,39.75,38.94,38.92,37.19,34. 78,32.23,31.72,31.63,28.90,28.57,28.08,28.01,27.47,25.05,24.25,18.32,17.80,16.30,15.48,15.31; HRMS(ESI):m / z calcd for C 30 H 53 O4 + ([M+H] + ):477.3944,found 477.3949.

[0082] application

[0083] The present invention provides use of the general compound described in any of the aforementioned embodiments in the preparation of medicines or foods, wherein the medicines or foods are used to prevent, treat or alleviate at least one disease selected from pulmonary fibrosis and acute lung injury.

[0084] In some embodiments, the drug or food is used to prevent, treat or alleviate at least one of the following symptoms: i) improve the survival rate of an individual and slow down the weight loss of an individual; ii) improve lung imaging; iii) reduce lung weight index; iv) reduce the degree of alveolar structural lesions; v) reduce the degree of lung inflammatory cell infiltration; vi) reduce the degree of lung fibrosis; vii) reduce collagen deposition in lung tissue; viii) reduce the hydroxyproline content in lung tissue; ix) reduce the expression of fibrosis-related proteins in lung tissue and reduce the expression of related proteins in the TGFβ / Smad signaling pathway; x) reduce the total number of leukocytes in bronchoalveolar lavage fluid; xi) reduce the expression of proinflammatory factors in bronchoalveolar lavage fluid; xii) reduce the content of myeloperoxidase (MPO) in bronchoalveolar lavage fluid; xiii) reduce the level of neutrophils and macrophages in lung tissue; xiv) reduce the total protein concentration in bronchoalveolar lavage fluid, increase the expression of tight junction protein ZO-1 in lung tissue, and provide alveolar vascular endothelial barrier protection.

[0085] In some embodiments, the inflammatory cells include at least one of total leukocytes, lymphocytes, and macrophages.

[0086] In some embodiments, the fibrosis-related protein includes at least one of N-cadherin, Vimentin, Fibronectin, Collagen I and smooth muscle actin α-SMA.

[0087] In some embodiments, the proteins related to the TGFβ / Smad signaling pathway include at least one of a TGFBRII receptor protein and a Smad2 protein.

[0088] In some embodiments, the pro-inflammatory factor includes at least one of TNF-α, IL-1β, and IL-6.

[0089] In some embodiments, the inflammatory cells include at least one of neutrophils and macrophages.

[0090] In some embodiments, the dosage form of the drug is an injection dosage form.

[0091] In some embodiments, the dosage form of the drug is an oral dosage form.

[0092] In some embodiments, the drug further comprises at least one of the following components: (1) excipients: ingredients used to fix, disperse, improve the taste or increase the stability of the drug, such as fillers, adhesives, dispersants, etc.; (2) flavoring agents: ingredients used to improve the taste of the drug or mask the unpleasant taste of the drug, such as sweeteners, flavors, etc.; (3) coating agents: ingredients used to encapsulate the drug, protect the drug from damage by gastric acid, etc., delay the release rate of the drug or control the release rate, such as capsule shells, tablet coatings, etc.; (4) dissolution aids: ingredients used to enhance the solubility of the drug in the gastrointestinal tract and improve the bioavailability of the drug, such as surfactants, solubilizers, etc.; (5) fillers: ingredients used to adjust the drug dosage and improve the physical properties or volume of the oral dosage form, such as cellulose, starch, etc.

[0093] In some embodiments, the drug or food is administered to a mammal.

[0094] In some embodiments, the subjects of administration of the drug or food include but are not limited to: humans, primates (such as apes, apes and gorillas), canines (such as dogs, wolves, etc.), felines (such as cats, leopards, lions, etc.), rodents (such as mice, hamsters, etc.), even-toed ungulates (such as cattle, sheep, pigs, etc.), and odd-toed ungulates (such as horses, etc.).

[0095] The present invention also provides a medicine comprising the compound of the general formula described in any of the aforementioned embodiments or a composition thereof.

[0096] The present invention also provides a food, which comprises the compound of the general formula or a composition thereof according to any of the aforementioned embodiments.

[0097] The present invention also provides a method for preventing, treating or alleviating a disease, wherein the disease includes at least one selected from pulmonary fibrosis and acute lung injury, and the method comprises: using the general formula compound, drug and / or food described in any of the aforementioned embodiments.

[0098] Example

[0099] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0100] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0101] Example 1 Compounds of the general formula prevent and protect against bleomycin (BLM)-induced pulmonary fibrosis in mice

[0102] Intraperitoneal injection: Sixty male C57BL / 6J mice (8-10 weeks old) were selected and randomly divided into 6 groups (n=10), respectively named as sham operation group, model group (bleomycin), TSA1 group (bleomycin + 10 mg / kg TSA1), TSA2 group (bleomycin + 10 mg / kg TSA2), DSA1 group (bleomycin + 10 mg / kg DSA1) and DSA2 group (bleomycin + 10 mg / kg DSA2), as shown in Table 1.

[0103] Table 1

[0104]

[0105] like Figure 1 As shown, during the experiment, according to the dosage requirements of each group, the corresponding general formula compound was dissolved in a solution of dimethyl sulfoxide: Tween 80: double distilled water = 5:5:90 (blank solvent), and each group of mice was administered by intraperitoneal injection. On day 0, the sham operation group was instilled with an equal amount of 0.9% sterile saline once through the trachea, and the model group and each drug group were instilled with 2.5 mg / kg bleomycin (Nippon Kayaku Co., Ltd., production batch number 600700) once through the trachea for modeling. Starting from the first day of modeling, the sham operation group and the model group were intraperitoneally injected with an equal amount of blank solvent every day, and each drug group was intraperitoneally injected according to the set dose once a day for 21 consecutive days. The weight of the mice was weighed and recorded every day, and the survival of the mice was observed and recorded at the same time. On the 22nd day of modeling, the mice in each group were anesthetized and the mouse lungs were imaged using a Micro-CT scanner. Finally, samples were collected from the mice, and the degree of pulmonary fibrosis in the model animals was evaluated according to experimental methods known in the art, including: H&E and Masson staining of mouse lung tissue, determination of hydroxyproline (HYP) content in mouse lung tissue, determination of the number of total leukocytes, lymphocytes and macrophages in mouse bronchoalveolar lavage fluid (BALF), and Western blot analysis to detect pulmonary fibrosis-related proteins in mouse lung tissue.

[0106] During the experiment, the survival time and weight changes of mice in each group were recorded and counted. Figure 2. The survival rate of mice in the model group was 60%, and the survival rates of mice in the TSA1, TSA2, DSA1 and DSA2 groups were 80%, 100% (completely overlapped with the sham operation group in the figure), 90% and 100% (completely overlapped with the sham operation group in the figure). Compared with the model group, TSA2 and DSA2 significantly improved the survival rate of PF mice (p<0.05). Compared with the sham operation group, the body weight of mice in the model group decreased significantly, and the body weight of mice dropped to the lowest level on the 9th day of modeling. Compared with the model group, TSA1, TSA2, DSA1 and DSA2 can significantly increase the body weight of PF mice (p<0.05). At the end of the experiment, the body weight of mice in the DSA2 group had returned to the level of the sham operation group.

[0107] Micro-CT scanning image results of mouse lung tissue ( Figure 3 ) showed that there were a large number of dense fibrous foci (high-density shadows) in the lungs of mice in the model group, indicating that bleomycin can induce imaging changes of pulmonary fibrosis in mice. TSA1, TSA2, DSA1 and DSA2 can significantly reduce the imaging changes of pulmonary fibrosis in mice. The lung images of mice in the TSA2 and DSA2 groups are closer to those in the sham operation group. Image J software was used to perform statistical analysis on the average gray value of the lung cross section. Compared with the sham operation group, the average gray value of the lung cross section of mice in the model group was significantly increased (p<0.001), and TSA1, TSA2, DSA1 and DSA2 all significantly reduced the average gray value of the lung cross section (p<0.05 or p<0.01).

[0108] Based on macroscopic observation of lung tissue ( Figure 3 ), the lung tissue of mice in the sham operation group was pink, soft, smooth and full. The lungs of mice in the model group were dark red, hard, with poor elasticity, and bleeding spots and scar-like textures were visible to the naked eye. Compared with the model group, bleeding spots were occasionally seen in the lungs of mice in the TSA1 and DSA1 groups, and the degree of pulmonary fibrosis was significantly improved. The lung tissues of mice in the TSA2 and DSA2 groups were light red, without obvious fibrous lines, which was close to that of mice in the sham operation group. This shows that the general formula compound has a significant preventive and protective effect on mouse pulmonary fibrosis.

[0109] H&E staining of mouse lung tissues showed Figure 4 As shown in the figure, the alveolar structure of the lung tissue of mice in the sham operation group was normal, without inflammatory cell infiltration; the local alveolar septa of the lung tissue of mice in the model group were significantly thickened, a large number of alveolar cavities were significantly reduced or disappeared, and a large number of inflammatory cells infiltrated the lung tissue interstitium. Compared with the lung tissue of mice in the model group, after intervention with the general formula compound, the thickening of the alveolar wall of the mouse lung tissue was significantly improved and the infiltration of inflammatory cells was significantly reduced. The performance of the TSA2 and DSA2 groups was close to that of the sham operation group.

[0110] Results of total leukocytes, lymphocytes and macrophages in mouse bronchoalveolar lavage fluid ( Figure 5) showed that compared with the sham operation group, the number of inflammatory cells in the model group mice increased significantly (p<0.0001). Compared with the model group, TSA2 and DSA2 significantly reduced the number of total leukocytes, lymphocytes and macrophages in the bronchoalveolar lavage fluid of PF mice (p<0.05 or p<0.01). After TSA1 and DSA1 intervention, the inflammatory cells in the bronchoalveolar lavage fluid of PF mice showed a downward trend.

[0111] Masson staining of mouse lung tissues was performed. Figure 6 As shown in the figure, there was no collagen fibril protein deposition in the lung tissue of mice in the sham operation group; a large amount of blue collagen fiber deposition appeared in the lung tissue of mice in the model group. Compared with the lung tissue of mice in the model group, the collagen fiber deposition in the lung tissue of mice in the TSA1 (p<0.05), TSA2 (p<0.01), DSA1 (p<0.05) and DSA2 (p<0.01) groups was significantly reduced. The TSA2 and DSA2 groups were close to the level of the sham operation group.

[0112] Results of hydroxyproline content detection in mouse lung tissue ( Figure 7 ) showed that the hydroxyproline content in the lung tissue of mice in the model group was significantly increased compared with that in the sham operation group (p<0.01). Compared with the model group, TSA1 (p<0.05), TSA2 (p<0.01), DSA1 (p<0.05) and DSA2 (p<0.01) could significantly reduce the hydroxyproline content in the lung tissue of PF mice.

[0113] The results of immunoblotting analysis of N-cadherin, vimentin and α-SMA in lung tissues of mice in TSA1 and TSA2 groups ( Figure 8 ) showed that compared with the sham operation group, the levels of N-Cadherin (p<0.05), Vimentin (p<0.001) and α-SMA (p<0.01) in the lung tissue of mice in the model group were significantly increased. Compared with the model group, TSA1 significantly reduced the levels of N-Cadherin (p<0.05), α-SMA (p<0.01) and Vimentin (p<0.001) proteins in the lung tissue of PF mice; TSA2 significantly reduced the content of α-SMA protein in the lung tissue of PF mice (p<0.01), and had a decreasing trend for N-Cadherin and Vimentin proteins.

[0114] The results of immunoblotting analysis of N-cadherin, vimentin and smooth muscle actin α-SMA in lung tissues of mice in the DSA1 and DSA2 groups ( Fig. 9) showed that compared with the model group, DSA2 significantly reduced the content of N-Cadherin and α-SMA proteins in the lung tissue of PF mice (p<0.05), and had a decreasing trend for Vimentin protein; DSA1 significantly reduced the content of N-Cadherin protein in the lung tissue of PF mice (p<0.05), and had a decreasing trend for α-SMA and Vimentin proteins.

[0115] Intragastric administration: Male C57BL / 6J mice (8-10 weeks old) were selected and randomly divided into 7 groups, named as sham operation group, model group (bleomycin), nintedanib group (bleomycin + 30 mg / kg nintedanib), TSA2 low-dose group (bleomycin + 10 mg / kg TSA2), TSA2 high-dose group (bleomycin + 30 mg / kg TSA2), PPT group (bleomycin + 30 mg / kg 20(S)-protopanaxatriol) and Rg1 group (bleomycin + 30 mg / kg native ginsenoside Rg1), as shown in Table 2.

[0116] Table 2

[0117]

[0118]

[0119] like Fig.10 As shown, 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 each group of mice was administered by gavage. On day 0, the sham operation group was instilled with an equal amount of 0.9% sterile saline once through the trachea, and the model group and each drug group were instilled with 2.5 mg / kg bleomycin (Nippon Kayaku Co., Ltd., production batch number 600700) once through the trachea for modeling. Starting from the first day of modeling, the sham operation group and the model group were gavaged with an equal amount of 0.5% CMC-Na aqueous solution every day, the positive drug control group was gavaged with 30 mg / kg nintedanib (McLean) every day, and the other drug groups and TSA2 drug groups were gavaged according to the set dose every day, once a day, for 21 consecutive days. And on the 22nd day of modeling, after anesthetizing the mice in each group, the mouse lungs were imaged using a Micro-CT scanner. Finally, samples were collected from the mice, and the degree of pulmonary fibrosis in the model animals was evaluated according to experimental methods known in the art, including: lung weight index of mice (ratio of lung weight to body weight, mg / g), H&E and Masson staining of mouse lung tissue, determination of hydroxyproline (HYP) content in mouse lung tissue, determination of total leukocyte and lymphocyte counts in mouse bronchoalveolar lavage fluid (BALF), and immunoblotting analysis to detect pulmonary fibrosis-related proteins and pathway-related proteins in mouse lung tissue.

[0120] Micro-CT scanning image results of mouse lung tissue ( Fig.11 ) showed that there were a large number of dense fibrous foci (high-density images) in the lungs of the model group mice. Compared with the model group, in the oral administration mode for the prevention of pulmonary fibrosis, low-dose TSA2 (10 mg / kg) and high-dose TSA2 (30 mg / kg) can significantly reduce the area of ​​fibrous foci in the lungs of PF mice, indicating that TSA2 can significantly improve the imaging changes of lung tissue in PF mice by oral administration. PPT (30 mg / kg) and Rg1 (30 mg / kg) have a certain degree of improvement on the lung images of model animals. Image J software was used to perform statistical analysis on the average gray value of the lung cross section. Compared with the sham operation group, the average gray value of the lung cross section of the mice in the model group was significantly increased (p<0.0001). Both low-dose TSA2 (10 mg / kg) and high-dose TSA2 (30 mg / kg) significantly reduced the average gray value of the lung cross section (p<0.05). PPT (30 mg / kg) and Rg1 (30 mg / kg) had a decreasing trend on the average gray value of the lung cross section of the model animals, but there was no statistical significance.

[0121] Based on macroscopic observation of lung tissue ( Fig.11 ), compared with the sham operation group, the lung tissue of the model group mice was dark red, and scar-like texture was visible to the naked eye. Compared with the model group, the lung tissue of the mice in the two TSA2 dosage groups was light pink, with no obvious bleeding spots and no obvious fibrous texture, which was comparable to the positive control nintedanib group. This shows that TSA2 has a significant preventive and protective effect on pulmonary fibrosis in mice by oral administration.

[0122] Lung weight index can reflect the degree of animal lung fibrosis to a certain extent. Fig.12 As shown, low dose of TSA2 (10 mg / kg) significantly reduced the lung weight index of model animals (p<0.05), high dose of TSA2 (30 mg / kg) also reduced the lung weight index of model animals (p=0.0678), PPT (30 mg / kg) and Rg1 (30 mg / kg) had a trend of reducing the lung weight index of model animals, but there was no statistical significance.

[0123] H&E staining of mouse lung tissues showed Fig.13As shown in the figure, compared with the sham operation group, the alveolar septa of the lung tissue of the model group mice were significantly thickened, a large number of alveolar cavities were significantly reduced or disappeared, and a large number of inflammatory cells infiltrated. Compared with the model group, low-dose TSA2 (10 mg / kg) and high-dose TSA2 (30 mg / kg) significantly reduced the degree of inflammation in the lung tissue of BLM-induced pulmonary fibrosis mice, and significantly improved the alveolar wall thickness, alveolar cavity volume and inflammatory cell infiltration, which was comparable to the positive drug nintedanib (30 mg / kg). PPT (30 mg / kg) and Rg1 (30 mg / kg) had a certain degree of improvement on the degree of inflammation in the lung tissue of the model animals, but the effect was not as good as TSA2.

[0124] Results of total leukocyte and lymphocyte counts in mouse bronchoalveolar lavage fluid ( Fig.14 ) showed that compared with the sham operation group, the number of inflammatory cells in the model group mice increased significantly (p<0.001). Compared with the model group, the low-dose TSA2 (10 mg / kg) group significantly reduced the number of total leukocytes and lymphocytes in the bronchoalveolar lavage fluid of PF mice (p<0.05), and the high-dose TSA2 (30 mg / kg) group significantly reduced the number of total leukocytes in the bronchoalveolar lavage fluid of model animals (p<0.05), and there was a decreasing trend for lymphocytes, indicating that TSA2 can reduce the number of inflammatory cells in the bronchoalveolar lavage fluid of PF mice by oral administration, and can significantly improve the lung inflammation of PF mice. PPT (30 mg / kg) and Rg1 (30 mg / kg) have a decreasing trend for the total leukocytes and lymphocytes in the bronchoalveolar lavage fluid of model animals, but there is no statistical significance. TSA2 is significantly better than native ginsenoside Rg1 and PPT in improving lung inflammation in model animals.

[0125] Masson staining of mouse lung tissues was performed. Fig.15 As shown in the figure, compared with the sham operation group, the alveolar wall structure of the lung tissue of the mice in the model group was significantly destroyed, and a large amount of blue collagen fibril protein deposition was observed. Compared with the lung tissue of the mice in the model group, the collagen fiber deposition in the lung tissue of the mice in the TSA2 (10 mg / kg) and TSA2 (30 mg / kg) groups was significantly reduced, and the degree of pulmonary fibrosis was significantly alleviated (p<0.05). The effect of two doses of TSA2 in inhibiting pulmonary fibrosis in mice was comparable to that of the positive drug nintedanib. PPT (30 mg / kg) and Rg1 (30 mg / kg) had a decreasing trend on collagen fibril protein in the lung tissue of the model animals, but there was no statistical significance.

[0126] Results of hydroxyproline content detection in mouse lung tissue ( Fig.16) showed that compared with the sham operation group, the hydroxyproline content in the lung tissue of the model group mice increased significantly. Compared with the model group, TSA2 (10mg / kg) and the positive drug nintedanib (30mg / kg) could significantly reduce the hydroxyproline content in the lung tissue of PF mice; TSA2 (30mg / kg), PPT (30mg / kg) and Rg1 (30mg / kg) had a decreasing trend on the hydroxyproline content in the lung tissue of PF mice.

[0127] The results of immunoblotting analysis of N-cadherin, Vimentin, α-SMA, Fibronection and COL1A1 in mouse lung tissues ( Fig.17 ) showed that compared with the sham operation group, the levels of N-Cadherin (p<0.05), Vimentin (p<0.01), α-SMA (p<0.0001), Fibronection (p<0.01) and COL1A1 (p<0.0001) in the lung tissue of mice in the model group were significantly increased. Compared with the model group, both the low-dose and high-dose TSA2 groups significantly reduced the levels of Vimentin (p<0.05), α-SMA (p<0.05 or p<0.001) and COL1A1 (p<0.001) proteins in the lung tissue of PF mice; the low-dose TSA2 group significantly reduced the content of Fibronection protein in the lung tissue of PF mice (p<0.05); the high-dose TSA2 group had a decreasing trend on the N-Cadherin protein in the lung tissue of PF mice.

[0128] Immunoblotting analysis results of receptor protein TGFBRII and pathway protein Smad2 in mouse lung tissue ( Fig.18 ) showed that compared with the sham operation group, the TGFBRII receptor protein (p<0.01) and phosphorylated Smad2 protein (p<0.001) in the lung tissue of the model group mice were significantly increased. Compared with the model group, TSA2 (10mg / kg) and TSA2 (30mg / kg) significantly reduced the expression of TGFBRII receptor protein (p<0.01) and phosphorylated P-Smad2 protein (p<0.001) in the lung tissue of PF mice. This suggests that the molecular mechanism of TSA2 in preventing and treating PF is related to the TGFβ / Smad signaling pathway.

[0129] From the results of Example 1 above, it can be seen that whether it is intraperitoneal injection or oral administration, the general formula compound can effectively prevent and protect bleomycin-induced pulmonary fibrosis in mice, which is manifested by improved survival rate of PF mice, significantly improved body weight of PF mice, reduced high-density shadows of lung tissue, reduced lung weight index, reduced alveolar structural damage, reduced inflammatory cell infiltration, reduced fibrosis, significantly reduced hydroxyproline content, and significantly reduced expression of fibrosis-related proteins (cadherin N-Cadherin, vimentin Vimentin, fibronectin Fibronectin, collagen COL1A1 and smooth muscle actin α-SMA). In the prevention of PF mode by oral administration, TSA2 (10 mg / kg) and TSA2 (30 mg / kg) have the same efficacy in preventing pulmonary fibrosis as the positive drug nintedanib (30 mg / kg). Molecular mechanism studies showed that TSA2 significantly reduced the expression of TGFBRII receptor protein and phosphorylated P-Smad2 protein in the lung tissue of PF mice, suggesting that TSA2 prevents and alleviates pulmonary fibrosis through the TGFβ / Smad signaling pathway.

[0130] Example 2 Treatment of BLM-induced pulmonary fibrosis in mice using TSA2 as a representative compound of the general formula

[0131] Male C57BL / 6J mice (8-10 weeks old) were selected and raised for one week before modeling. On day 0, a single instillation of 2.5 mg / kg bleomycin was given to mice via the trachea to establish an animal model of bleomycin-induced pulmonary fibrosis; mice in the sham group were given a single instillation of an equal amount of 0.9% sterilized saline (NS) via the trachea. Grouping and administration began on the 7th day after bleomycin modeling, and the mice were divided into a sham operation group (Sham), a model group (bleomycin), a pirfenidone group (bleomycin + 200 mg / kg pirfenidone) and a TSA2 group (bleomycin + 20 mg / kg TSA2), as shown in Table 3.

[0132] Table 3

[0133]

[0134] like Fig.19As shown, according to the dosage requirements of each group, the corresponding drugs were dissolved in 0.5% sodium carboxymethyl cellulose (0.5% CMC-Na) aqueous solution. Starting from the 7th day after modeling, the sham operation group and the model group were gavaged with an equal amount of 0.5% CMC-Na aqueous solution every day, the positive drug control group was gavaged with 200 mg / kg pirfenidone (McLean) every day, and the TSA2 group was gavaged with 20 mg / kg TSA2 every day, once a day, for 14 consecutive days. On the 21st day of modeling, the mice were sampled to evaluate the degree of pulmonary fibrosis, including: pathological detection of mouse lung tissue (H&E staining and Masson staining), and determination of hydroxyproline (HYP) content in mouse lung tissue. At the same time, the pathological conditions of the heart, liver, spleen and kidney of each group of mice were evaluated (H&E staining).

[0135] H&E staining of mouse lung tissues showed Fig. 20 As shown in A, the alveolar structure of the lung tissue of mice in the sham operation group was normal, without inflammatory cell infiltration; the alveolar septa of the lung tissue of mice in the model group were significantly thickened, a large number of alveolar cavities were significantly reduced or disappeared, and a large number of inflammatory cells infiltrated the lung tissue interstitium; compared with the model group, TSA2 and pirfenidone significantly reduced the degree of inflammation in the lung tissue of BLM-induced pulmonary fibrosis mice, and significantly improved the alveolar wall thickness, alveolar cavity volume and inflammatory cell infiltration. The therapeutic effect of TSA2 is equivalent to that of the positive drug pirfenidone.

[0136] Masson staining of mouse lung tissues was performed. Fig. 20 As shown in B and 20C, compared with the sham operation group, a large amount of blue collagen fibril protein deposition was observed in the lung tissue of the model group mice (p<0.0001). Compared with the lung tissue of the model group mice, the collagen fiber deposition in the lung tissue of the TSA2 group (20 mg / kg) mice was significantly reduced, and the degree of pulmonary fibrosis was significantly alleviated (p<0.0001), which was comparable to the positive control drug pirfenidone group.

[0137] Results of hydroxyproline content detection in mouse lung tissue ( Fig. 20 D) shows that compared with the sham operation group, the hydroxyproline content in the lung tissue of the model group mice increased significantly (p<0.0001). Compared with the model group, TSA2 (20 mg / kg) significantly reduced the hydroxyproline content in the lung tissue of PF mice (p<0.01), which significantly improved the excessive deposition of collagen in the lung tissue of PF mice.

[0138] Pathological examination results of the heart, liver, spleen and kidney of mice ( Fig.21) showed that compared with the sham-operated mice, TSA2 (20 mg / kg) did not observe obvious pathological changes in the heart, liver, spleen and kidney of mice, indicating that TSA2 (20 mg / kg) was biosafe under this experimental condition. However, pirfenidone (200 mg / kg) caused significant pathological changes in the spleen of mice, indicating that pirfenidone has a certain spleen toxicity to mice.

[0139] From the results of Examples 1 and 2 above, it can be seen that the general formula compound can effectively prevent and treat pulmonary fibrosis, as manifested by reduced damage to the alveolar structure of mice, reduced inflammatory cell infiltration, reduced degree of fibrosis, and significantly reduced hydroxyproline content and expression of fibrosis-related proteins.

[0140] The efficacy of the general formula compound in treating pulmonary fibrosis is better than that of the marketed drug pirfenidone, and at an effective dose for treating pulmonary fibrosis, the in vivo safety of TSA2 (20 mg / kg) is also better than that of pirfenidone (200 mg / kg).

[0141] Example 3 Compounds of Formula Alleviate LPS-Induced Acute Lung Injury

[0142] Lipopolysaccharide (LPS) is the main component of endotoxin and the main substance of acute lung injury induced by sepsis caused by Gram-negative bacteria. It can destroy the vascular endothelial barrier and cause edema in the lung tissue space and alveoli. It is widely used by researchers. The present invention adopts an animal model of acute lung injury in mice induced by a one-time tracheal instillation of LPS. The model is established by instilling LPS (5 mg / kg, Sigma) through the mouse trachea 1 hour after the intraperitoneal injection, and further verifies the protective effect of the general formula compound on acute lung injury.

[0143] Balb / c male mice (6-8 weeks old) were randomly divided into a control group, a model group (LPS group), a dexamethasone group (LPS+5 mg / kg dexamethasone), a TSA2 low-dose group (LPS+5 mg / kg TSA2) and a TSA2 high-dose group (LPS+10 mg / kg TSA2) after one week of adaptive feeding, with 6-9 mice in each group, as shown in Table 4.

[0144] Table 4

[0145]

[0146] like Fig. 22As shown, according to the dosage requirements of each group, the corresponding drugs were dissolved in dimethyl sulfoxide-Tween 80-double distilled water (volume ratio 5:5:90, blank solvent) solution. The control group and model group mice were intraperitoneally injected with blank solvent, and dexamethasone and TSA2 were intraperitoneally injected according to the doses set for each group. One hour after the end of the administration, after the mice were anesthetized, LPS (5 mg / kg, Sigma) saline solution was instilled through the trachea to model acute lung injury (ALI) in mice. 6 hours after the tracheal instillation of LPS, the mice were sampled; only the mice in the control group were sampled. The detection indicators include: taking the right alveolar lavage fluid (BALF) of mice, centrifuging it, and measuring the total white blood cell count; using enzyme-linked immunosorbent assay (ELISA) to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and myeloperoxidase (MPO) in the BALF supernatant to evaluate inflammatory mediators; and measuring the total protein concentration in the BALF supernatant to investigate the degree of alveolar capillary endothelial barrier damage in mice. The left lung of mice was fixed with paraformaldehyde, and H&E pathological analysis and immunohistochemical analysis of neutrophils (LY-6G) and macrophages (F4 / 80) were performed.

[0147] The experimental results are as follows:

[0148] H&E staining of mouse lung tissue ( Fig.23 ) The results showed that compared with the control group, the model group had obvious inflammatory cell infiltration in the lung tissue of mice; compared with the model group, TSA2 (5 mg / kg) and TSA2 (10 mg / kg) significantly reduced the degree of inflammatory cell infiltration in the lung tissue of mice, which was comparable to the effect of the positive drug dexamethasone.

[0149] Total leukocyte count in mouse bronchoalveolar lavage fluid (BALF) Fig.24 ) The results showed that compared with the control group, the total leukocyte count in the BALF of the model group mice was significantly increased (p<0.001). TSA2 (5mg / kg) and TSA2 (10mg / kg) significantly reduced (p<0.01 and p<0.001) the total leukocyte count in the BALF of ALI mice in a dose-dependent manner.

[0150] Myeloperoxidase (MPO) is mainly present in neutrophils. During inflammatory reactions, it is released into the extracellular fluid. The large-scale aggregation of neutrophils can cause changes in tissue and cell permeability and cause tissue damage. The results of this experiment ( Fig.25) showed that compared with the control group, the MPO content in the bronchoalveolar lavage fluid of the model group mice was significantly increased (p<0.001). After TSA2 intervention, the MPO content in the bronchoalveolar lavage fluid of mice decreased significantly in a dose-dependent manner (p<0.05 or p<0.01). In addition, TNF-α, IL-1β, and IL-6 are typical proinflammatory factors in various inflammatory diseases. In order to further evaluate the anti-inflammatory activity of TSA2, the contents of TNF-α, IL-1β, and IL-6 in the bronchoalveolar lavage fluid of mice were detected according to the ELISA kit operating instructions. The results are as follows Fig.25 As shown: After LPS tracheal instillation modeling, the levels of TNF-α, IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice were significantly increased (p<0.0001). After TSA2 (10 mg / kg) intervention, the levels of TNF-α, IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice were significantly decreased (p<0.01); TSA2 (5 mg / kg) also significantly reduced the levels of IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice (p<0.05).

[0151] Immunohistochemical detection of the expression of neutrophils (LY-6G) and macrophages (F4 / 80) in the lung tissue of ALI mice. Fig.26 As shown in the figure, compared with the control group, the neutrophils and macrophages in the lung tissue of mice in the ALI model group were significantly increased (p<0.01). Compared with the model group, TSA2 (5 mg / kg) and TSA2 (10 mg / kg) significantly reduced the levels of neutrophils and macrophages in the lung tissue of ALI mice (p<0.05).

[0152] Total protein concentration in mouse bronchoalveolar lavage fluid ( Fig. 27 ) The results showed that compared with the control group, the total protein concentration in the bronchoalveolar lavage fluid of the model group mice was significantly increased (p<0.0001). Both low-dose TSA2 (p<0.05) and high-dose TSA2 (p<0.01) significantly reduced the total protein concentration in the bronchoalveolar lavage fluid of mice.

[0153] According to the protein immunoblotting detection of tight junction protein ZO-1 expressed in mouse lung tissue ( Fig.28 ) The results showed that compared with the control group, the tight junction protein ZO-1 in the lung tissue of the model group mice decreased significantly (p<0.01), indicating that the tight junction of the alveolar vascular endothelial barrier of the model group mice was severely damaged. Compared with the model group, after TSA2 (10mg / kg) intervention, the expression level of tight junction protein ZO-1 in the lung tissue of mice was significantly upregulated (p<0.05), indicating that TSA2 can promote the tight junction of the alveolar vascular endothelial barrier and provide lung barrier protection.

[0154] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug or food for preventing, treating or alleviating at least one disease selected from pulmonary fibrosis and acute lung injury, in, R is selected from one of H and alcoholic hydroxyl group.

2. The use according to claim 1, characterized in that: The compound represented by formula I is selected from compound I, compound II, compound III, and compound IV, 3. The use according to claim 1, characterized in that: The pulmonary fibrosis disease includes at least one of primary pulmonary fibrosis, idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, and interstitial pulmonary fibrosis.

4. The use according to claim 1, characterized in that: The lung injury diseases include direct lung injury and indirect lung injury. The direct lung injury is induced lung injury, including at least one of lung infection caused by bacteria, viruses, etc., aspiration of gastric contents, lung contusion, inhalation of toxic gases, and oxygen poisoning. The indirect lung injury includes at least one of severe non-thoracic trauma, acute severe pancreatitis, massive blood transfusion, extracorporeal circulation, and lung injury caused by disseminated intravascular coagulation.

5. Use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug or food for preventing, treating or alleviating at least one of the following symptoms in, R is selected from one of H and alcohol hydroxyl group; i) weight loss of the individual; ii) worsening of lung imaging; iii) alveolar structural lesions; iv) abnormal infiltration of inflammatory cells in the lungs, wherein the inflammatory cells include at least one of total leukocytes, lymphocytes and macrophages; v) pulmonary fibrosis; vi) abnormal collagen deposition in lung tissue; vii) abnormal hydroxyproline content in lung tissue; viiii) overexpression of fibrosis-related proteins in lung tissue, wherein the fibrosis-related proteins include N-cadherin, Vimentin, Fibronectin, Collagen I and smooth muscle actin α-SMA; overexpression of TGFBRII receptor protein and phosphorylated P-Smad2 protein in lung tissue; ix) abnormal total leukocyte count in bronchoalveolar lavage fluid; x) abnormal expression of proinflammatory factors in bronchoalveolar lavage fluid, wherein the proinflammatory factors include at least one of TNF-α, IL-1β, and IL-6; xi) abnormal myeloperoxidase (MPO) content in bronchoalveolar lavage fluid; xii) abnormal levels of neutrophils and macrophages in lung tissue; xiii) abnormal total protein concentration in bronchoalveolar lavage fluid and abnormal expression of tight junction protein ZO-1 in lung tissue.

6. The use according to claim 5, characterized in that: The compound represented by formula I is selected from compound I, compound II, compound III, and compound IV, 7. The use according to claim 1 or 5, characterized in that: The dosage form of the drug is one or more of tablets, capsules, granules, liquids, oral dissolving tablets, oral suspensions, oral emulsions, pills, injections, aerosols, powders, wine preparations, dew preparations, and films.

8. The use according to claim 1, characterized in that: The administration method of the drug for treating pulmonary fibrosis and lung injury diseases includes one or more of oral administration, injection, spraying, inhalation, implantation and external application.

9. A drug, characterized in that The invention comprises the compound described in claim 1.

10. A food, characterized in that Comprising the compound according to claim 1.

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