CN109730990B

CN109730990B - Application of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative in anti-fibrosis drugs

Info

Publication number: CN109730990B
Application number: CN201910155493.9A
Authority: CN
Inventors: 戴桂馥; 王亚可; 徐海伟; 赵安琪; 赵进; 张淑秋; 王丙顺
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2018-03-02
Filing date: 2019-03-01
Publication date: 2021-07-02
Anticipated expiration: 2039-03-01
Also published as: CN109730990A

Abstract

The invention belongs to the technical field of medicines, and discloses an application of a 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative in preparing a medicine for preventing and treating human organ or tissue fibrosis. Experiments prove that the compound obviously inhibits the mesenchymal transformation of human alveolar II type epithelial cells A549 induced by TGF-beta 1; obviously inhibiting human renal cortex proximal tubular epithelial cell HK-2 mesenchymal transformation induced by TGF-beta 1; inhibiting human primary myocardial fibrosis cell HCFB proliferation induced by angiotensin II (Ang II). Obviously reduces the degree of pulmonary fibrosis of mice induced by bleomycin, and obviously reduces the degree of renal fibrosis of rats induced by unilateral ureteral ligation and the degree of myocardial fibrosis of Kunming mice caused by isoproterenol ISO. The compound is used as an active ingredient for preparing medicaments for resisting fibrosis of kidney, lung, heart and the like, has high efficiency and low toxicity, and provides a new medicament approach for treating and preventing diseases related to the fibrosis, thereby expanding the selectable range of clinical medicaments and having good application and development prospects.

Description

Application of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative in anti-fibrosis drugs

Technical Field

The invention relates to application of andrographolide derivatives as a medicament for resisting human organ or tissue fibrosis, in particular to 14-deoxy-11, 12-dehydroandrographolide C15 substituted derivatives, and belongs to the technical field of medicines.

Background

Tissue fibrosis is a chronic disease, and is frequently found in the liver, lung, heart, kidney and other parts. 1/3 worldwide die from tissue fibrosis and the resulting organ failure. When tissue is damaged, a series of cellular reactions occur at the damaged part, which leads to excessive deposition of extracellular matrix and tissue fibrosis. Eventually leading to organ dysfunction and even death. Cardiovascular tissue fibrosis plays an important role in cardiovascular tissue remodeling caused by hypertension and heart failure, and is also a major cause of atherosclerosis. Myocardial fibrosis is characterized by the accumulation of extracellular matrix proteins in the intercellular matrix and causes systolic and diastolic dysfunction. Myocardial fibrosis is an important marker of decompensated myocardial hypertrophy and heart failure, and is involved in myocardial remodeling caused by hypertension, hypertrophic cardiomyopathy, heart failure and myocardial infarction.

In recent years, the incidence rate and the fatality rate of pulmonary fibrosis are in an increasing trend. Pulmonary fibrosis is the ultimate outcome of the development, progression, scarring of many pulmonary diseases, and its etiology is diverse. Many chronic lung diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, tuberculosis, lung cancer, interstitial lung disease, and the like, are accompanied by fibrotic pathological changes. The main pathological characteristics of the lung tissue include the proliferation of mesenchymal cells in lung tissue, the proliferation and deposition of extracellular matrix, the reconstruction of lung parenchyma and the like. For various lung diseases such as idiopathic pulmonary fibrosis, respiratory distress syndrome, eosinophilic granuloma and the like, the degree of fibroplasia and fibrosis of lung tissues determines the clinical prognosis of the disease. These diseases progress to an advanced stage, which seriously hampers the normal work and quality of life of the patient and even leads to death of the patient from respiratory failure or cardiac failure. During the last 20 years, the incidence rate of idiopathic pulmonary fibrosis generally shows a trend of obvious increase, the average survival time after diagnosis is about 3 years, the survival rate in 5 years is 30-50%, and the survival rate after recovery is extremely poor.

Renal fibrosis, a pathological process in which extracellular matrix and inappropriate connective tissue accumulate in the kidney, leading to structural changes and impaired function of the kidney, is also a common pathway for almost all renal diseases to progress to end-stage renal failure. Many acute and chronic kidney diseases are also closely related to the development of tissue fibrosis, especially the change of renal fibrosis caused by diabetic nephropathy and hypertension. There are also changes in tissue fibrosis in a variety of immune and autoimmune diseases such as arthritis, systemic sclerosis and systemic lupus erythematosus.

Because the causes of the fibroproliferative diseases of various tissues and organs are numerous, the pathogenesis is complex, the disease course is prolonged for years to decades, the exact pathogenesis of the tissue fibrosis is not completely clarified at present, and no drug which is acknowledged and can really reverse the tissue fibrosis is provided. At present, the medicines for treating the fibrosis diseases are very few, and most medicines only have the auxiliary treatment effect. In the aspect of pulmonary fibrosis, glucocorticoid, immunosuppressive drugs and the like can improve the symptoms of pulmonary fibrosis and delay the development of diseases, but have strong side effects. The only FDA approved drugs in the united states are Esbriet (pirfenidone) developed by interbone, inc. and Nintedanib (Nintedanib) developed by haggaren, germany, but these two drugs currently do not benefit the patient ideally.

The signaling pathway related to the development of fibrosis of tissues and organs, which is currently studied more, includes TGF-beta₁A Smad signal pathway, a Wnt/beta-catenin signal pathway, a phosphatidylinositol-3 kinase/protein kinase (PI3K/Akt) signal pathway and the like. For example, phellodendron lactone down-regulates the production of α -SMA and collagen through inhibition of TGF- β signaling pathway in mouse fibrotic lung tissue (Exclusive)Benefit number: CN 105560255 a). A oviductus Ranae lactone-sulfate can significantly inhibit TGF-beta induced human embryonic lung fibroblast proliferation and Hydroxyproline (HYP) content thereof, thereby playing a pharmacological role in improving pulmonary fibrosis (patent No.: CN 103919769A). PI3K inhibitors acting downstream of TGF-beta are useful for the treatment of pulmonary fibrotic diseases (patent No.: CN 104093408A). There is also a patent report of using sesquialter mushroom-isoalantolactone derivatives and their salts in inula root to treat pulmonary fibrosis (patent No. CN 106496243A). In the aspect of resisting renal fibrosis, the ganodermolide D can obviously inhibit TGF-beta 1-induced phosphorylation of tubular epithelial cells Smad3 and has a therapeutic effect on renal fibrosis (patent number: CN 106220643A). The third subsidiary hospital of the third military medical university of the Chinese people's liberation army finds that apigenin mediates Ca through TRPV4²⁺Activates the AMPK/SITR1 signaling pathway and inhibits renal fibrosis.

Andrographolide is a diterpene lactone compound extracted from Nees of Andrographis paniculata (Burm.f.), and is one of the main effective components of traditional Chinese medicine Andrographis paniculata. Is mainly used for treating upper respiratory tract infection, bacillary dysentery and the like in clinic. In recent years, the application of andrographolide in the aspects of tumor resistance, liver protection, gallbladder benefiting, virus resistance and the like is continuously and deeply researched. The andrographolide is found to relieve pulmonary fibrosis rat alveolitis and fibrosis degree caused by bleomycin or pingyangmycin, reduce PDGF expression of lung tissues and reduce HYP content; studies of Vanxingming and the like find that andrographolide can relieve pulmonary fibrosis pulmonary alveolitis and pulmonary fibrosis degree of rats caused by bleomycin, reduce I, III collagen mRNA expression of lung tissues, reduce TNF-alpha and TGF-beta 1 concentrations in bronchoalveolar lavage fluid (BALF), and has no obvious toxic or side effect on livers and kidneys. Studies such as Sunjiahui and the like find that the andrographolide injection can reduce the inflammation and the fibrosis degree of a mouse with viral myocarditis, and the mechanism of the andrographolide injection can possibly inhibit the expression of ST2 in the myocardium. Neimenqi and other researches find that andrographolide has certain renal interstitial fibrosis resistance, and the mechanism of andrographolide is probably related to inflammation resistance, oxidation resistance and reduction of expression of TGF-beta 1 and IGFBP-3. The studies of the stacchinine and the like prove that the andrographolide can inhibit cardiac fibrosis and has the effect of resisting myocardial hypertrophy by improving the activities of sodium potassium ATPase and calcium magnesium ATPase and reducing the content of hydroxyproline. The research of Singha P and the like shows that andrographolide has a certain protection effect on liver and kidney injury of mice caused by ethanol. Roy DN and other researches prove that the combination of andrographolide and D-penicillamine for treating copper poisoning has better effect on the aspects of anti-fibrosis and anti-necrosis than that of single D-penicillamine.

The inventor obtains a large number of andrographolide derivatives with novel structures in earlier researches (CN 1978437; CN 100999520; CN 100999535; CN101003527), applies patent protection to the application of partial derivatives in the protection of tumor resistance, inflammation resistance, HBV resistance, HCV resistance, acute liver injury and the like, and also protects the application of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives in the preparation of anti-hepatic fibrosis drugs (application number: 201710214066.4). Furthermore, activity test research is carried out on the 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives in the aspect of fibrosis of other tissues (organs) except liver, and no relevant literature report is found at present.

Disclosure of Invention

On the basis of earlier research results, the inventor discovers that the 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative has remarkable effects of preventing and treating fibrosis-related diseases, is high in efficiency and low in toxicity and has the potential of being developed into anti-fibrosis drugs by screening anti-human tissue or organ fibrosis activity of synthesized compounds. Therefore, the invention aims to provide the application of the 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative in preparing the medicines for resisting pulmonary fibrosis, renal fibrosis and cardiac fibrosis.

The 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative has a structure shown in a general formula 1.

General formula 1

Wherein: r₁、R₂Respectively hydrogen or phenyl and one of mono-substituent and multi-substituent of the hydrogen or the phenyl; r₁、R₂Are respectively a ringOne of cyclic substituent groups such as hexyl and cyclopentyl; r₁、R₂May be the same substituent group or different substituent groups. R₃、R₄Are each hydrogen or CH₂CH₂COOHCH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂COOH, etc. or COR₅，R₅The carbon chain is a substituted or unsubstituted aromatic heterocycle such as phenyl, pyridyl, pyrrolyl, furyl or the like, a carbocyclic or heterocyclic structure such as cyclohexyl, cyclopentyl, cyclopropyl, morpholinyl, piperidyl or the like, a saturated or unsaturated carbon chain with the length of C1-18, and the like. R₃、R₄May be the same substituent group or different substituent groups at the same time.

The following compounds are preferred: r₁，R₂Each is hydrogen or phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2, 3, 5-trimethoxyphenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 2-fluoro-3-methoxyphenyl, 3-methoxy-4-chlorophenyl, 2, 4-difluorophenyl, 2, 4-dichlorophenyl, 2, 4-dibromophenyl, 2-fluoro-4-chlorophenyl, 2-bromo-4-chlorophenyl, 3-fluoro-4-chlorophenyl, 2-bromo-4-chlorophenyl, 3-bromo-4-chlorophenyl group, 3, 4-difluorophenyl group, 3, 4-dichlorophenyl group, 3, 4-dibromophenyl group, 2-chloro-4-fluorophenyl group, 2-bromo-4-fluorophenyl group, 3-chloro-4-fluorophenyl group, 3-bromo-4-fluorophenyl group, 2-fluoro-4-bromophenyl group, 2-chloro-4-bromophenyl group, 3-fluoro-4-bromophenyl group, 3-chloro-4-bromophenyl group, 2, 3, 4-trichlorophenyl group, 2-methoxy-4-chlorophenyl group, 2-hydroxy-4-methoxyphenyl group, 3-amino-4-chlorophenyl group, 3, 4-dichlorophenyl group, 3-bromo-4-fluorophenyl group, 3-chloro-4-fluorophenyl group, 2-amino-4-chlorophenyl, 2-nitro-4-fluorophenyl, 2-nitro-4-chlorophenyl, R₁，R₂Simultaneously the same or different but not simultaneously hydrogen; r₃、R₄Each is hydrogen; or R₃、R₄Are respectively CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂COOH, or R₃、R₄Each is COR₅；R₅Is 3-pyridyl or CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂One of COOH, R₃、R₄And optionally identical or different substituents.

Preferred compounds are: r₁，R₂Each is hydrogen or phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2, 3, 5-trimethoxyphenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 2-fluoro-3-methoxyphenyl, 3-methoxy-4-chlorophenyl, 2, 4-difluorophenyl, 2, 4-dichlorophenyl, 2, 4-dibromophenyl, 2-fluoro-4-chlorophenyl, 2-bromo-4-chlorophenyl, 3-fluoro-4-chlorophenyl, 2-bromo-4-chlorophenyl, 3-bromo-4-chlorophenyl group, 3, 4-difluorophenyl group, 3, 4-dichlorophenyl group, 3, 4-dibromophenyl group, 2-chloro-4-fluorophenyl group, 2-bromo-4-fluorophenyl group, 3-chloro-4-fluorophenyl group, 3-bromo-4-fluorophenyl group, 2-fluoro-4-bromophenyl group, 2-chloro-4-bromophenyl group, 3-fluoro-4-bromophenyl group, 3-chloro-4-bromophenyl group, 2, 3, 4-trichlorophenyl group, 2-methoxy-4-chlorophenyl group, 2-hydroxy-4-methoxyphenyl group, 3-amino-4-chlorophenyl group, 3, 4-dichlorophenyl group, 3-bromo-4-fluorophenyl group, 3-chloro-4-fluorophenyl group, 2-amino-4-chlorophenyl, 2-nitro-4-fluorophenyl, 2-nitro-4-chlorophenyl, with the proviso that R₁，R₂Different; r₃、R₄Each is hydrogen; or R₃、R₄Are respectively CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂COOH, or R₃、R₄Each is COR₅，R₅Is 3-pyridyl or CH₂CH₂COOH，R₃、R₄The same substituent groups are selected.

More preferred compounds are: when R is₁，R₂When one of them is hydrogen, R₁，R₂One of them is selected from the following groups: phenyl group, 2-methoxyphenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group, 2-fluorophenyl group, 2-chlorophenyl group, 2-bromophenyl group, 3-fluorophenyl group, 3-chlorophenyl group, 3-bromophenyl group, 4-fluorophenyl group, 4-chlorophenyl group, 4-bromophenyl group, 2-fluoro-3-methoxyphenyl group, 3-methoxy-4-chlorophenyl group, 2, 4-difluorophenyl group, 2, 4-dichlorophenyl group, 2, 4-dibromophenyl group, 2-fluoro-4-chlorophenyl group, 2-bromo-4-chlorophenyl group, 3-fluoro-4-chlorophenyl group, 3-bromo-4-chlorophenyl group, 3, 4-difluorophenyl group, 3, 4-dichlorophenyl group, 3-bromo-4-chlorophenyl group, 3, 4-dichlorophenyl group, and the like, 3, 4-dibromophenyl, 2-chloro-4-fluorophenyl, 2-bromo-4-fluorophenyl, 3-chloro-4-fluorophenyl, 3-bromo-4-fluorophenyl, 2-fluoro-4-bromophenyl, 2-chloro-4-bromophenyl, 3-fluoro-4-bromophenyl, 3-chloro-4-bromophenyl, 2-methoxy-4-chlorophenyl; r₃、R₄Each is hydrogen; or R₃、R₄Are respectively CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂COOH, or R₃、R₄Each is COR₅，R₅Is 3-pyridyl or CH₂CH₂COOH，R₃、R₄The same substituent groups are selected.

More preferred compounds are:

A：R₁＝H，R₂＝C₆H₅，R₃＝R₄＝H；

B：R₁＝H，R₂＝2-F-C₆H₄，R₃＝R₄＝H；

C：R₁＝H，R₂＝2-Cl-C₆H₄，R₃＝R₄＝H；

D：R₁＝H，R₂＝2-Br-C₆H₄，R₃＝R₄＝H；

E：R₁＝H，R₂＝3-F-C₆H₄，R₃＝R₄＝H；

F：R₁＝H，R₂＝3-Cl-C₆H₄，R₃＝R₄＝H；

G：R₁＝H，R₂＝3-Br-C₆H₄，R₃＝R₄＝H；

H：R₁＝H，R₂＝4-Cl-C₆H₄，R₃＝R₄＝H；

I：R₁＝H，R₂＝4-F-C₆H₄，R₃＝R₄＝H；

J：R₁＝H，R₂＝4-Br-C₆H₄，R₃＝R₄＝H；

K：R₁＝H，R₂＝4-CH₃O-C₆H₄，R₃＝R₄＝H；

L：R₁＝H，R₂＝2-CH₃O-4-Cl-C₆H₃，R₃＝R₄＝H；

M：R₂＝H，R₁＝2-Br-C₆H₄，R₃＝R₄＝H；

N：R₂＝H，R₁＝3-Cl-C₆H₄，R₃＝R₄＝H；

O：R₂＝H，R₁＝2-F-4-Cl-C₆H₃，R₃＝R₄＝H；

P：R₂＝H，R ₁2, 4-dichlorophenyl, R₃＝R₄＝H；

Q：R₂＝H，R₁＝4-F-C₆H₄，R₃＝R₄＝H；

R：R₂＝H，R₁＝C₆H₅，R₃＝R₄＝H；

S：R₁＝H，R₂＝3-F-4-Cl-C₆H₃，R₃＝R₄＝H；

T：R₁＝H，R ₂2, 4-difluorophenyl group, R₃＝R₄＝H；

U：R₁＝H，R ₂3, 4-dichlorophenyl, R₃＝R₄＝H；

V：R₁＝H，R₂＝4-Cl-C₆H₄，R₃＝R₄＝COR₅，R₅-3-pyridyl;

W：R₁＝H，R₂＝4-Cl-C₆H₄，R₃＝R₄＝CH₂CH₂COOH；

X：R₁＝H，R₂＝4-Cl-C₆H₄，R₃＝R₄＝COR₅，R₅＝CH₂CH₂COOH；

Y：R₂＝H，R₁＝4-Cl-C₆H₄，R₃＝R₄＝H；

Z：R₂＝H，R₁＝4-Cl-C₆H₄，R₃＝R₄＝COR₅，R₅3-pyridyl.

The preparation method of the compound provided by the invention is disclosed in patent CN: 200510107247.4.

In order to research the application effect of the compound in preparing the anti-pulmonary, kidney and heart fibrosis drugs, the invention utilizes the human alveolar II type epithelial cells A549 to evaluate the inhibition activity of the compound on the mesenchymal transformation of A549 cells induced by TGF-beta 1 and evaluate the anti-pulmonary fibrosis activity of the compound; evaluating the inhibitory activity of the compound on HK-2 cell mesenchymal transition induced by TGF-beta 1 by using human renal cortex proximal tubular epithelial cells HK-2, and evaluating the anti-renal fibrosis activity of the compound; the anti-myocardial fibrosis effect of the compound is evaluated by detecting the cell proliferation condition after primary human myocardial fibroblast HCFB is stimulated by Ang II. Furthermore, the anti-fibrosis effect of the representative compounds of the invention on lung, kidney and cardiac muscle was studied and the anti-fibrosis effect in vivo of some representative structures was evaluated by using a bleomycin-induced pulmonary fibrosis model in mice, a unilateral ureteral ligation-induced renal fibrosis model (UUO) in rats and a Kunming mouse myocardial fibrosis model caused by isoproterenol ISO, respectively.

The compound obviously inhibits the mesenchymal transformation of human alveolar II type epithelial cells A549 induced by TGF-beta 1; obviously inhibiting human renal cortex proximal tubular epithelial cell HK-2 mesenchymal transformation induced by TGF-beta 1; inhibiting human primary myocardial fibrosis cell HCFB proliferation induced by angiotensin II (Ang II). Obviously reducing the pulmonary fibrosis degree of KM mice induced by bleomycin, obviously reducing the renal fibrosis degree of SD rats induced by unilateral ureteral ligation, and obviously reducing the myocardial fibrosis degree of Kunming mice caused by isoproterenol ISO.

The cis-structure and the trans-structure of the compound have anti-fibrosis activity, and the compound is in a molecular form containing no crystal water, various crystal forms or amorphous forms as effective medicinal components, or various prodrug forms of the compound, and the compound is singly combined with other medicaments or mixed with auxiliary and/or additive components acceptable in pharmacy according to various conventional pharmaceutical methods and process requirements to prepare various medicament forms such as anti-fibrosis oral preparations, injection preparations and the like. Preferably, the compound is used for preparing medicaments for treating or preventing various fibrotic diseases such as lung, kidney, heart and the like. The oral preparation is tablet, pill, capsule, granule or syrup; the injection preparation comprises injection or freeze-dried powder injection and the like.

The invention has the advantages and innovation points that: the compound is determined to have specific activity for resisting fibrosis of kidney, lung, heart organ and tissue by activity screening. Experiments prove that compared with a parent compound Andrographolide (AD), the compound provided by the invention has the advantage that the fibrosis of organs such as lung, kidney and heart is obviously improved. Therefore, the compounds are used as active ingredients for preparing medicaments for resisting renal fibrosis, pulmonary fibrosis and cardiac fibrosis, and provide a new medicament approach for treating and preventing diseases related to fibrosis, thereby enlarging the selectable range of clinical medicaments and having good application and development prospects.

Drawings

FIG. 1 is a graph of the effect of AD and a compound of the invention on the viability of human alveolar type II epithelial cells A549 at a compound concentration of 30.00. mu.M, in which 1. AD; 2, A; 3, B; c, 4; 5, D; 6, E; f, 7, F; g, 8; 9. H; 10, I; 11, J; 12, K; l is 13; 14. M; 15. N; o, 16; p is 17; q, 18; r is 19; 20, S; t, 21; 22, U; 23. V; w, 24. W; x, 25. X; y, 26. Y; z, 27;

FIG. 2 shows the inhibition of TGF-. beta.1-induced mesenchymal transition of human alveolar type II epithelial cells A549 by AD and the compounds of the present invention (statistical results), with low and high concentrations of compounds AD, N, P, Q, S-W and Z of 0.63. mu.M and 1.25. mu.M, respectively, low and high concentrations of compounds C, E-G, I-L, O, R, X and Y of 0.31. mu.M and 0.63. mu.M, respectively, and low and high concentrations of compounds A, B, D, H and M of 0.16. mu.M and 0.31. mu.M, respectively; in the figure, 1 is a control; TGF-beta 1; TGF-beta 1+ AD; TGF-beta 1+ A; TGF-beta 1+ B; TGF-beta 1+ C; TGF-. beta.1 + D; TGF-beta 1+ E; TGF-. beta.1 + F; TGF-. beta.1 + G; TGF-. beta.1 + H; TGF-. beta.1 + I; TGF-. beta.1 + J; TGF-. beta.1 + K; TGF-. beta.1 + L; TGF-. beta.1 + M; TGF-. beta.1 + N; TGF-. beta.1 + O; TGF-. beta.1 + P; TGF-. beta.1 + Q; TGF-. beta.1 + R; TGF-. beta.1 + S; TGF- β 1+ T; TGF- β 1+ U; TGF-. beta.1 + V; TGF-. beta.1 + W; TGF-. beta.1 + X; TGF-. beta.1 + Y; TGF- β 1+ Z;

FIG. 3 is a graph showing the effect of AD and a compound of the present invention on the viability of HK-2 human renal cortical proximal tubular epithelial cells at a concentration of 30.00. mu.M, in which: 1, AD; 2, A; 3, B; c, 4; 5, D; 6, E; f, 7, F; g, 8; 9. H; 10, I; 11, J; 12, K; l is 13; 14. M; 15. N; o, 16; p is 17; q, 18; r is 19; 20, S; t, 21; 22, U; 23. V; w, 24. W; x, 25. X; y, 26. Y; z, 27;

FIG. 4 is a graph showing the inhibition of TGF- β 1-induced mesenchymal transition between human renal cortical proximal tubular epithelial cells HK-2 cells by AD and a compound of the present invention (partial micrographs;. times.100), in which: 1. comparison; TGF-beta 1; TGF-. beta.1 + AD (0.63. mu.M); TGF-. beta.1 + T (0.63. mu.M); TGF-. beta.1 + A (0.63. mu.M); TGF-. beta.1 + H (0.08. mu.M); TGF-. beta.1 + F (0.63. mu.M); TGF-. beta.1 + J (0.63. mu.M); TGF-. beta.1 + K (0.63. mu.M); TGF-. beta.1 + Z (0.63. mu.M); TGF-. beta.1 + Y (0.16. mu.M).

FIG. 5 shows the effect of AD on the proliferation of HCFB in primary human cardiac fibroblasts in combination with a representative compound H of the invention (0.3, 3.0 and 15.0. mu.M).

FIG. 6 shows the inhibition of angiotensin II (AngII) -induced proliferation of HCFB in primary human cardiac fibroblasts by AD (0.63. mu.M) and the representative compound of the invention H (0.08. mu.M, 0.16. mu.M, 0.31. mu.M and 0.63. mu.M), compared to AngII,^*P＜0.05，^**p is less than 0.01; compared with the group of AD,^#P＜0.05,^##P＜0.01。

FIG. 7 is a graph showing the effect of a representative compound H of the present invention on the extent of bleomycin-induced fibrosis in lung tissue of KM mice (collagen area/%), compared to model groups,^*P＜0.05，^**p is less than 0.01; compared with the group of AD,^##P＜0.01；

FIG. 8 is a graph showing the effect of a representative compound H of the present invention on the degree of bleomycin-induced lung tissue fibrosis in KM mice (partial Masson staining pictures;. times.100);

FIG. 9 shows the effect of representative compound H of the present invention on the degree of renal fibrosis induced by unilateral ureteral ligation in SD rats (relative collagen area/%), compared to model group,^*P＜0.05，^**p is less than 0.01; compared with the group of AD,^##P＜0.01；

FIG. 10 is a graph showing the effect of a representative compound H of the present invention on the degree of renal fibrosis in SD rats induced by unilateral ureteral ligation (partial Masson staining picture;. times.100).

Fig. 11 is a graph showing the effect of representative compound H of the present invention on the degree of myocardial fibrosis in KM mice caused by isoproterenol ISO (collagen area/%), wherein: 1. normal control; 2.a model group; AD (15 mg/kg); AD (30 mg/kg); h (3.75 mg/kg); h (7.5 mg/kg); h (15 mg/kg); in comparison to the model set,^*P＜0.05，^**p is less than 0.01; compared with the group of AD,^##P＜0.01；

FIG. 12 is a graph of the effect of a representative compound of the invention H on the degree of myocardial fibrosis in KM mice by isoproterenol ISO (partial sirius red staining picture;. times. 100), in which: 1. normal control; 2.a model group; AD (15 mg/kg); AD (30 mg/kg); h (3.75 mg/kg); h (7.5 mg/kg).

Detailed description of the preferred embodiments

The invention is illustrated below with reference to specific embodiments. It should be understood that these embodiments are illustrative of the invention only and are not limiting upon the scope of the invention. The compound related to the invention is not limited to the representative structure used in the examples, and different substituents at the 15 th position can be replaced to obtain the compound with anti-fibrosis activity; various causes of fibrosis can be used as research objects to obtain that the compound has anti-fibrosis effect; other in vivo and in vitro research models (methods) can also be utilized to obtain the compound with the anti-fibrosis effect.

EXAMPLE 1 inhibition of mesenchymal transition in human type II alveolar epithelial cells A549 by Compounds of the invention

The II type alveolus epithelial cells in the alveolus are stimulated by cytokines such as inflammatory mediators, growth factors and the like, the cell morphology is changed from cobblestone shape to fusiform shape, the epithelial mesenchymal transition process (EMT) is completed, the function of interstitial cells is achieved, collagen fibers are further synthesized, and the disease course of the pulmonary interstitial fibrosis can be aggravated by a large amount of collagen fiber deposition. Therefore, compared with andrographolide, the in vitro anti-pulmonary fibrosis effect of the compound of the invention is evaluated by using human type II alveolar epithelial cells A549 by adopting a morphological observation method and a cell scratch (migration) experiment.

1) Cell culture

A549 cells were cultured in RPMI1640 medium containing 10% (V/V) fetal bovine serum, 100. mu.g/mL streptomycin, and 100IU/mL penicillin, with volume fraction of 5% CO₂Culturing in an incubator at 37 deg.C under saturated humidity.

2) Medicaments and agents

Andrographolide, produced by Sichuan Erythrosin 37025, Jinxin Biotech, Inc. (batch No. 120822), has a purity of greater than 99%; the compound of the invention is synthesized by the laboratory of the inventor, and the purity is more than 99 percent, as follows.

3) MTT method for determining cytotoxicity

A549 cells in logarithmic phase of growth were digested with 0.25% (W/V) trypsin and diluted to 5X 10 with RPMI1640 medium containing 10% (V/V) fetal bovine serum⁴The cell suspension was plated in 96-well plates at 200. mu.L/well with a volume fraction of 5% CO at 37 ℃₂Culturing in an incubator for 24h, adding a drug-containing culture medium, wherein the final concentration of the drug is 30.00 mu mol/L at most, repeating each treatment for 4 holes, continuing culturing for 48h, adding MTT (5mg/mL) and 20 mu L/hole, culturing for 4h, discarding the supernatant, adding 150 mu L DMSO, shaking for L0min, and measuring the light absorption value by using a microplate reader. The measurement wavelength was 570nm and the reference wavelength was 450 nm. The cell survival rate after the compound action, the survival rate (%) ═ drug group OD was calculated_570-450Value/control OD_570-450The values are multiplied by 100%, the results are averaged, see FIG. 1.

4) Morphological observation method for detecting influence of drug on A549 cell EMT

A549 cells in logarithmic phase of growth were digested with 0.25% (W/V) trypsin and diluted to 3X 10 with RPMI1640 medium containing 10% (V/V) fetal bovine serum⁴Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. After 24h of culture, when the adherent cells are fused to 80% -90%, discarding the original culture medium, synchronously culturing for 24h in a serum-free culture medium, discarding the culture medium, washing twice with PBS, simultaneously adding 200 mu L of RPMI1640 culture medium containing TGF-beta 1(5ng/mL) and compounds to be detected with different concentrations, and immediately taking a picture under a microscope (100X). Replicate 3 wells and set controls. After 48h incubation, pictures were taken under the microscope. 5 fields were selected for the same concentration of each compound and greater than 100 cells were measured. The pictures were processed using photoshop CS6 software and their circularity was calculated (formula e-4 pi × S/C2, where e represents circularity, S represents area, and C represents circumference). The results were averaged and are shown in FIG. 2.

5) Results of the experiment

The results in FIG. 1 show that the cytotoxic activity of the compounds of the present invention against A549 cells was not enhanced at a concentration of 30.00. mu.M, compared to AD.

Figure 2 the results show that: the compound can obviously inhibit the epithelial mesenchymal transformation of A549 cells under the nontoxic concentration, and has stronger inhibiting effect on the epithelial mesenchymal transformation of human II-type alveolus and higher safety index compared with AD.

EXAMPLE 2 inhibition of TGF-beta 1-induced mesenchymal transformation of HK-2 human renal cortical proximal tubular epithelial cells by Compounds of the invention

Early studies found that tubular epithelial cells can transdifferentiate into fibroblasts and express their marker protein, fibroblast-specific protein (FSP1, fibroblastic protein 1), and that tubular epithelial cell-mesenchymal cell transdifferentiation is one of the important pathogenesis of renal interstitial fibrosis. Therefore, in comparison with andrographolide AD, the in vitro renal fibrosis resisting effect of the compound of the invention is researched by using human renal cortex proximal convoluted tubule epithelial cells HK-2 (provided by China center for type culture Collection) and adopting a TGF-beta 1 stimulation post-morphological observation method and a scratch experiment.

1) Cell culture

HK-2 cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (V/V), 100. mu.g/mL streptomycin, 100IU/mL penicillin, with a volume fraction of 5% CO₂The cells were cultured in an incubator at 37 ℃ under saturated humidity.

2) MTT method for determining cytotoxicity

HK-2 cells in log phase of growth were digested with 0.25% (W/V) trypsin + 0.02% EDTA (W/V) and then diluted to 7.0X 10 with DMEM-F12 medium containing 10% (V/V) fetal bovine serum⁴The cell suspension was plated in 96-well plates at 200. mu.L/well with a volume fraction of 5% CO at 37 ℃₂Culturing in incubator for 24h, changing into culture medium containing different concentrations of drug with maximum final concentration of 30.00 μ M, repeating each treatment for 4 wells, and culturing for 48 h. The rest is the same as example 1. The results were averaged as shown in FIG. 3.

3) Observation of the effects of drugs on HK-2 cell morphology following TGF-beta 1 stimulation

HK-2 cells grown to logarithmic phase were digested with 0.25% (W/V) trypsin + 0.02% EDTA and then diluted to 5.0X 10 with DMEM-F12 medium containing 10% (V/V) fetal bovine serum⁴Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. After 24h of culture, the cells grow into a monolayer, the original culture medium is discarded, the cells are washed twice by 0.01MPBS (multi-point double-walled brazier) and are synchronized by replacing a serum-free culture medium, after 24h of culture, the serum-free culture medium is removed by suction, and 200 mu L of DMEM-F12 culture medium containing the compounds to be detected and the stimulation factor TGF-beta 1(5ng/mL) with different concentrations is added.Replicate 3 wells and set controls. After 48h incubation, the photographs were recorded under a microscope. The morphological changes of some of the compounds of the invention after exposure to cells are shown in FIG. 4.

4) Results of the experiment

The results in FIG. 3 show that the inhibitory effect of the compounds of the present invention on the proliferation of HK-2 cells is significantly reduced at a concentration of 30.00. mu.M, compared to AD.

The results in table 1 and figures 3 and 4 show that: the compound can obviously inhibit the transformation of human proximal tubular epithelial cells HK-2 to mesenchymal cells induced by TGF-beta 1 under the nontoxic concentration, and has stronger inhibiting effect on the transformation of HK-2 to mesenchymal cells and higher safety index compared with AD.

TABLE 1 TGF-beta-induced transformation of human proximal tubular epithelial cells HK-2 into mesenchymal cells by the compounds of the invention

Note: the test concentration is 0.08-1.25 mu M; comparison: epithelial cells have interaction, the tissue structure is compact, and the cells are in a typical paving stone shape; TGF-beta 1 treatment: the epithelial cells lose the typical state, the interaction among the cells disappears, the tissue structure is relatively loose, the cell density is reduced, and the cube is in the form of a paved stone-shaped epithelial cell converted into a spindle-shaped fiber cell; very strong (inhibitory effect): the cells are almost the same as the control, spindle shapes are rarely seen under the visual field, intercellular interaction is recovered, and the shape is recovered to be in the typical paving stone shape; strong (inhibitory effect): the invasiveness of the cells is inhibited, the cells are compact, the cell state is almost completely recovered, and spindle fibrous cells are rarely seen; moderate (inhibitory effect): the cell density becomes higher and most of the cells are still in a cubic state.

EXAMPLE 3 inhibition of angiotensin II (Ang II) -induced proliferation of HCFB in human Primary myocardial fibrosis cells by Compounds of the invention

Research shows that the cardiac fibroblasts are the main effector cells of cardiac fibrosis, and can proliferate quantitatively after being stimulated by active substances such as Ang II and the like, and the phenotype of the cardiac fibroblast is transformed into the myofibroblasts with the function of secreting extracellular matrix. Therefore, the MTT method is adopted to detect the cytostatic effect of Ang II after stimulating HCFB (human cardiac fibroblast cell) of primary human cardiac fibroblasts, and the anti-myocardial fibrosis effect of the compound H is evaluated.

1) Cell culture

The in vitro anti-myocardial fibrosis effect of the compound H of the invention was studied by comparing primary human myocardial fibroblasts HCFB (provided by the Biotech Co., Ltd., North Nawa, Shanghai) with andrographolide. Culturing HCFB cells in culture flask containing H-DMEM culture solution containing 10% by volume fetal bovine serum (GIBCO, cat # 302220F), 100 μ g/mL streptomycin, and 100IU/mL penicillin in 5% by volume CO₂The cells were cultured in an incubator (Binder, Germany) at 37 ℃ under saturated humidity.

2) MTT method for determining cytotoxicity

The HCFB cells in the logarithmic phase of growth were digested with 0.25% trypsin + 0.02% EDTA and diluted to 5.0X 10 in H-DMEM medium containing 10% by volume fetal bovine serum⁴The cell suspension was plated in 96-well plates (Costar, USA) at 200. mu.L/well at 37 ℃ with a volume fraction of 5% CO₂Culturing for 24H in an incubator with saturated humidity, adding culture media containing compounds AD or H with different concentrations, continuing culturing for 48H, adding MTT (5mg/mL) and 20 mu L/hole, culturing for 4H, discarding supernatant, adding 150 mu L DMSO, shaking for L0min, and measuring light absorption value by using a microplate reader. The measurement wavelength was 570nm and the reference wavelength was 450 nm. The cell viability after the compound was measured, and the viability (%) ═ drug group a value/cell control group a value × 100% was calculated, and the results are shown in fig. 5. Data were processed and analyzed using SPSS 17.0 statistical software.

3) MTT method for detecting inhibition effect of drug on Ang II stimulated myocardial fibroblast HCFB proliferation capacity

HCFB cells in logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA, and then diluted to 5.0X 10 in H-DMEM medium containing 10% fetal bovine serum by volume fraction⁴Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. Culturing for 24 hr until the cells grow into monolayer, discarding the original culture medium, washing with 0.01M PBSTwice, replacing serum-free culture medium for synchronization, continuously culturing for 24h, removing the serum-free culture medium, adding 200 μ L of the solution containing different concentrations of the compound to be detected and stimulating factor Ang II (10)^-7mol/L) of H-DMEM medium. 3-well replicates were run in H-DMEM medium containing 0.5% DMSO as a negative control, containing the stimulating factor Ang II (10)^-7mol/L) and 0.5% DMSO in H-DMEM medium as a positive control. And after 48h of culture, the cell survival rate is detected by an MTT method. The results are shown in FIG. 6. Data were processed and analyzed using SPSS 17.0 statistical software. Data are all mean. + -. standard deviation

To represent; the difference between groups in P <0.05 has significant meaning.

4) Results of the experiment

The results in figure 5 show that the compound H of the invention does not show obvious inhibition effect on human primary myocardial fibroblast HCFB cell proliferation at the concentration of 15.00 mu M.

Figure 6 the results show that: the compound H can obviously inhibit the proliferation of HCFB by AngII under the condition of non-toxic concentration, and has stronger proliferation inhibition effect on human HCFB and higher safety index compared with AD.

Example 4 the compounds of the invention significantly reduced the degree of bleomycin-induced pulmonary fibrosis in KM mice

Pulmonary fibrosis is lung injury caused by various reasons, the pathological mechanism of pulmonary fibrosis formation is complex, different pathogenic factors initiate inflammation and immune response, various cells including vascular endothelial cells, alveolar epithelial cells, fibroblasts, macrophages and the like are involved, and the interaction of various cytokines and inflammatory mediators is realized. Bleomycin belongs to alkaline glycopeptide anticancer antibiotics, one of serious toxic adverse reactions of the bleomycin is pulmonary fibrosis, and animal experiments prove that the pathologic histological change of the pulmonary fibrosis caused by the bleomycin is very similar to human pulmonary fibrosis, and the bleomycin is generally used as a model for researching the pulmonary fibrosis.

1 materials and methods

1) Laboratory animal

Clean KM mice, male, weighing 20 + -2 g, were purchased from the center of laboratory animals in Henan province. License number: SCXK (Yu) 2015-.

2) Medicine, reagent and their preparation

Bleomycin hydrochloride for injection is produced by Haizingrui pharmaceutical Co., Ltd (batch No. YBH15562005 national drug Standard: H20055883); boninsong acetate tablets were manufactured by Zhejiang Xianju pharmaceutical Co., Ltd. (batch No. 170410, national Standard, 33021207). Other test drugs and compounds were the same as in example 1; the medicine is prepared into 0.5 percent sodium carboxymethyl cellulose (CMC-Na) suspension. Pharmaceutical grade sodium carboxymethylcellulose (CMC-Na) was produced by Anhui mountain river pharmaceutic adjuvants, Inc. (batch No. 131114).

3) Experimental methods

After 3 days of adaptive feeding of KM mice, randomly grouped by body weight: the sham-operated control group, model group, AD control group (250mg/kg), prednisone control group (5mg/kg), H compound-administered group (62.5mg/kg) and H compound-administered group (250.mg/kg) were 6 groups, and 15 subjects were each group. The method comprises the steps of carrying out intraperitoneal injection on 4% by mass of pentobarbital sodium (2mL/kg) to carry out anesthesia on a mouse, fixing the mouse in a supine position, removing neck hair, disinfecting skin by iodine, making an incision downwards along the neck by about 1cm, dissociating bronchus, injecting 50 mu L of bleomycin (2mg/mL), injecting 150 mu L of air immediately, rotating the mouse rapidly, enabling medicine liquid to be distributed uniformly, then suturing the incision by using No. 4/0 silk thread, wrapping the wound by using sterile gauze after iodophor disinfection, and finally sending the mouse after operation to a warm position until the mouse is awakened, wherein a sham operation group is injected with physiological saline with the same volume. After the molding is finished for 24 hours, the fixed-point intragastric administration is started, wherein CMC-Na with the same volume proportion is administered for 1 time every day in a sham operation group, and the experiment is finished after 28 days of administration. The padding of the mice is replaced 12 hours before the last gavage, and the mice are strictly fasted without water supply. Collecting whole blood of the mouse by an eyeball picking method 0.5h after administration, killing the mouse by a cervical dislocation method after blood collection, collecting lung, weighing, observing and recording lung lesion. After photographing, lungs were fixed in 4% paraformaldehyde fixing solution. After sufficient fixation, pathological sections are taken, the liver fibrosis degree is observed through Masson trichrome staining, and the photographing result of the Masson stained sections is subjected to fibrosis histology semi-quantitative analysis through Image-Pro Plus software. Calculating the relative collagen area: (average area of administration group-average area of normal group)/(average area of model group-average area of normal group) × 100%, the results are shown in FIGS. 7 and 8.

2 results of the experiment

The results of figures 7 and 8 show that: on a bleomycin-induced mouse pulmonary fibrosis model, the compound H provided by the invention can be used for remarkably reducing the pulmonary fibrosis KM mouse pulmonary tissue fibrosis area, and the effect is remarkably stronger than that of AD.

Example 5 Compounds of the invention significantly reduce the degree of unilateral ureteral ligation-induced renal fibrosis in SD rats

A unilateral ureteral ligation induced rat renal fibrosis model (UUO) is one of classic renal fibrosis models, is characterized in that cell components in renal interstitial tissue accumulate, fibroblast differentiates/proliferates, ECM deposition increases, renal tubular atrophy and the like, is similar to the occurrence and development process of clinical renal diseases, is extremely beneficial to the research on prevention and treatment of various renal interstitial injury diseases, has simple and convenient modeling method, 100 percent of modeling rate, uniform lesion and better repeatability, can cause fibrosis in a short time, and is a faster and more reliable animal model in terms of research on the incidence rule and mechanism discussion of renal interstitial fibrosis. Therefore, the UUO model is widely applied to research on the mechanism of renal interstitial fibrosis and evaluation of the therapeutic effect of improving renal interstitial fibrosis.

1 materials and methods

1) Laboratory animal

Clean grade SD rats, male, body weight 200 + -20 g, purchased from the center of laboratory animals in Henan province. License number: SCXK (Yu) 2015-.

2) Medicine, reagent and their preparation

The sources and formulations of andrographolide, sodium carboxymethylcellulose and the compound of this experiment were the same as in example 1 or 4.

3) Experimental methods

After 3 days of adaptive feeding, the SD rats were randomly grouped by body weight: the sham operation control group, model group, AD control group (0.15mmol/kg), H compound administration group (0.06mmol/kg), H compound administration group (0.10mmol/kg), H compound administration group (0.15mmol/kg) were 6 groups in total, and each group had 4 animals. Preoperative preparation and anesthesia as in example 4, after anesthesia, rats were fixed in the left lateral decubitus position, the lower edge of the sternum was shaved off to the hind limb to see hair, a surgical cloth was laid on the skin from which the hair was shaved off, after the skin was sterilized with iodine, an incision was made downward at about 0.2cm along the lower edge of the sternum to about 2cm, the kidney and the free ureter were extruded, the kidney was double-ligated with 4/0 number silk thread and disconnected from the ureter at a distance of about 1/3 ureter length, after the kidney was returned to the abdominal cavity, the abdomen was closed with 4/0 number silk thread in full layer, after sterilization with iodine, the wound was bandaged with sterile gauze, and finally the postoperative rats were sent to a warm place until awakening, wherein only the free ureter was not ligated and. After the molding is finished for 24 hours, the fixed-point human gavage administration is started, the administration method is the same as that of example 4, and the experiment is finished after 14 days of administration. The padding of the rats is changed 12 hours before the last gavage, and the rats are strictly fasted without water prohibition. And 3 percent of sodium pentobarbital (2mL/kg) anesthetic is intraperitoneally injected 0.5h after administration, the left kidney is quickly and completely dissected away after blood collection, the weight of the kidney is weighed, the size of the kidney is measured, and the kidney is fixed in 4 percent paraformaldehyde fixing solution after photographing. Serum preparation and Masson staining methods and statistical treatment were the same as in example 2. The Masson staining results and the relative collagen area statistics are shown in figures 9 and 10.

2 results of the experiment

The results, taken in conjunction with figures 9 and 10, show that: on a UUO model, the representative compound H obviously reduces the degree of renal fibrosis, improves the pathological change renal tissue structure, and has the effect obviously stronger than that of AD.

Example 6 Compounds of the invention significantly reduce the degree of Isoproterenol (ISO) induced myocardial fibrosis in KM mice

1 materials and methods

Test animals: SPF-grade Kunming mice, male, weighing 23 s 2g, were purchased from the center of the laboratory animals in Henan province. The license number is SCXK 2017-

Isoproterenol ISO: sigma is the product; test compounds were formulated as 0.5% sodium carboxymethylcellulose (CMC-Na) suspensions. The sources and formulations of andrographolide, sodium carboxymethylcellulose and the compound of this experiment were the same as in example 1 or 4.

The test method comprises the following steps: after 3 days of adaptive feeding, KM mice were randomly assigned to saline control group, model group, AD 15mg/kg and 30mg/kg dose groups, compound H of the present invention 3.75mg/kg, 7.5mg/kg, 15mg/kg dose groups, and 9 mice per group, according to body weight. The model group and the other administration groups were injected subcutaneously with ISO of 20mg/kg for 7 consecutive days, and the saline group was injected subcutaneously with an equivalent amount of saline. After the drug is injected subcutaneously for 2h, the normal saline group and the model group are intragastrically administered with 0.5% CMC-Na, the other administration groups are administered with the corresponding drug in suspension with 0.5% CMC-Na, and the administration of 2w is finished. The padding of the mice is replaced 12 hours before the last gavage, and the mice are strictly fasted without water supply. Blood was collected from the eye 1h after dosing, after which the heart was quickly dissected away intact and photographed. After blood centrifugation, the supernatant was collected and stored for further use. Mouse heart tissue was fixed in 10-fold volume of 4% (w/v) paraformaldehyde fixing solution, and the fixing solution was refreshed after 24 h. After the fixing is fully performed, pathological sections are taken, the heart fibrosis degree is observed through sirius red staining, and Image-Pro Plus software is used for performing fibrosis histology semi-quantitative analysis on the photographing result of the sirius stained sections. The relative collagen area was calculated and the results are shown in fig. 11 and 12. Data were processed and analyzed using SPSS 17 statistical software. The data are expressed by mean value standard deviation (X Shi S), and the difference between P <0.05 groups has significance.

2 results of the experiment

The results of combining fig. 11 and fig. 12 show that the anti-hepatic fibrosis effect of the compound of the invention is obviously stronger than that of the AD drug treatment group on the isoproterenol induced myocardial fibrosis model.

Claims

1. The application of the 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivative with the structure shown in the general formula 1 in preparing medicaments is characterized in that the derivative is used as an active ingredient for preparing medicaments for resisting renal fibrosis, pulmonary fibrosis or myocardial fibrosis;

general formula 1

R₁，R₂Each is hydrogen or phenyl, 2-methoxyPhenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2, 3, 5-trimethoxyphenyl, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 2-fluoro-3-methoxyphenyl, 3-methoxy-4-chlorophenyl, 2, 4-difluorophenyl, 2, 4-dichlorophenyl, 2, 4-dibromophenyl, 2-fluoro-4-chlorophenyl, 2-bromo-4-chlorophenyl, 3-fluoro-4-chlorophenyl, 3-bromo-4-chlorophenyl, 2-bromo-4-chlorophenyl, etc, 3, 4-difluorophenyl group, 3, 4-dichlorophenyl group, 3, 4-dibromophenyl group, 2-chloro-4-fluorophenyl group, 2-bromo-4-fluorophenyl group, 3-chloro-4-fluorophenyl group, 3-bromo-4-fluorophenyl group, 2-fluoro-4-bromophenyl group, 2-chloro-4-bromophenyl group, 3-fluoro-4-bromophenyl group, 3-chloro-4-bromophenyl group, 2, 3, 4-trichlorophenyl group, 2-methoxy-4-chlorophenyl group, 2-hydroxy-4-methoxyphenyl group, 3-amino-4-chlorophenyl group, 2-amino-4-chlorophenyl group, 2-nitro-4-fluorophenyl, 2-nitro-4-chlorophenyl, except that R₁，R₂Different; r₃、R₄Each is hydrogen; or R₃、R₄Are respectively CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂COOH, or R₃、R₄Each is COR₅，R₅Is 3-pyridyl or CH₂CH₂COOH，R₃、R₄The same substituent groups are selected.

2. The use of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives according to claim 1, wherein R is R₁，R₂When one of them is hydrogen, R₁，R₂One of them is selected from the following groups: phenyl group, 2-methoxyphenyl group, 3-methoxyphenyl group, 4-methoxyphenyl group, 2-fluorophenyl group, 2-chlorophenyl group, 2-bromophenyl group, 3-fluorophenyl group, 3-chlorophenyl group, 3-bromophenyl group, 4-fluorophenyl group, 4-chlorophenyl group, 4-bromophenyl group, 2-fluoro-3-methoxyphenyl group, 3-methoxy-4-chlorophenyl group, 2, 4-difluorophenyl group, 2, 4-dichlorobenzene groupA phenyl group, a 2, 4-dibromophenyl group, a 2-fluoro-4-chlorophenyl group, a 2-bromo-4-chlorophenyl group, a 3-fluoro-4-chlorophenyl group, a 3-bromo-4-chlorophenyl group, a 3, 4-difluorophenyl group, a 3, 4-dichlorophenyl group, a 3, 4-dibromophenyl group, a 2-chloro-4-fluorophenyl group, a 2-bromo-4-fluorophenyl group, a 3-chloro-4-fluorophenyl group, a 3-bromo-4-fluorophenyl group, a 2-fluoro-4-bromophenyl group, a 2-chloro-4-bromophenyl group, a 3-fluoro-4-bromophenyl group, a 3-chloro-4-bromophenyl group, a 2-methoxy-4-chlorophenyl group; r₃、R₄Each is hydrogen; or R₃、R₄Are respectively CH₂CH₂COOH、CH₂CH₂CH₂CH₂COOH、CH₂CHCHCH₂COOH、CH₂CH₂CH₂CH₂CH₂CH₂CH₂One of COOH, or R₃、R₄Each is COR₅，R₅Is 3-pyridyl or CH₂CH₂COOH，R₃、R₄The same substituent groups are selected.

3. The use of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives according to claim 1 for the manufacture of a medicament, selected from the group consisting of:

A：R₁=H，R₂=C₆H₅，R₃= R₄=H；

B：R₁=H，R₂=2-F-C₆H₄，R₃= R₄=H；

C：R₁=H，R₂=2-Cl-C₆H₄，R₃= R₄=H；

D：R₁=H，R₂=2-Br-C₆H₄，R₃= R₄=H；

E：R₁=H，R₂=3-F-C₆H₄，R₃= R₄= H；

F：R₁=H，R₂=3-Cl-C₆H₄，R₃= R₄= H；

G：R₁=H，R₂=3-Br-C₆H₄，R₃= R₄= H；

H：R₁=H，R₂=4-Cl-C₆H₄，R₃= R₄= H；

I：R₁=H，R₂=4-F-C₆H₄，R₃= R₄= H；

J：R₁=H，R₂=4-Br-C₆H₄，R₃= R₄= H；

K：R₁=H，R₂=4-CH₃O-C₆H₄，R₃= R₄= H；

L：R₁=H，R₂=2-CH₃O-4-Cl-C₆H₃，R₃= R₄= H；

M：R₂=H，R₁=2-Br-C₆H₄，R₃= R₄= H；

N：R₂=H，R₁=3-Cl-C₆H₄，R₃= R₄= H；

O：R₂=H，R₁=2-F-4-Cl-C₆H₃，R₃= R₄= H；

P：R₂=H，R₁=2, 4-dichlorophenyl group, R₃= R₄= H；

Q：R₂=H，R₁=4-F-C₆H₄，R₃= R₄= H；

R：R₂=H，R₁=C₆H₅，R₃= R₄= H；

S：R₁=H，R₂=3-F-4-Cl-C₆H₃，R₃= R₄= H；

T：R₁=H，R₂=2, 4-difluorophenyl group, R₃= R₄= H；

U：R₁=H，R₂=3, 4-dichlorophenyl, R₃= R₄= H；

V：R₁=H，R₂=4-Cl-C₆H₄，R₃= R₄= COR₅，R₅= 3-pyridyl;

W：R₁=H，R₂=4-Cl-C₆H₄，R₃= R₄= CH₂CH₂COOH；

X：R₁=H，R₂=4-Cl-C₆H₄，R₃= R₄= COR₅，R₅= CH₂CH₂COOH；

Y: R₂=H，R₁=4-Cl-C₆H₄，R₃= R₄= H；

Z: R₂=H，R₁=4-Cl-C₆H₄，R₃= R₄= COR₅，R₅= 3-pyridyl group.

4. The use of the 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives according to any of claims 1-3 for the manufacture of a medicament for oral or injectable use, according to conventional pharmaceutical methods and process requirements.

5. The use of 15-benzylidene-14-deoxy-11, 12-dehydroandrographolide derivatives according to claim 4 in the manufacture of a medicament, wherein the oral dosage form is a tablet, pill, capsule, granule or syrup; the injection preparation is injection or freeze-dried powder injection.