WO2018177301A1 - 15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物及其在制备抗纤维化药物中的应用 - Google Patents

15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物及其在制备抗纤维化药物中的应用 Download PDF

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WO2018177301A1
WO2018177301A1 PCT/CN2018/080776 CN2018080776W WO2018177301A1 WO 2018177301 A1 WO2018177301 A1 WO 2018177301A1 CN 2018080776 W CN2018080776 W CN 2018080776W WO 2018177301 A1 WO2018177301 A1 WO 2018177301A1
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chlorophenyl
fibrosis
group
cooh
compound
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PCT/CN2018/080776
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French (fr)
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戴桂馥
徐海伟
王亚可
张晓沛
杨卫
吴健
赵进
张颜
关珍贞
沈鹏鹏
尚宁
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郑州大学
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Priority claimed from CN201710214066.4A external-priority patent/CN106946821B/zh
Application filed by 郑州大学 filed Critical 郑州大学
Priority to EP18777705.7A priority Critical patent/EP3608313B1/en
Publication of WO2018177301A1 publication Critical patent/WO2018177301A1/zh
Priority to US16/589,101 priority patent/US11369584B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/58—One oxygen atom, e.g. butenolide
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    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/60—Two oxygen atoms, e.g. succinic anhydride
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    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/06—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
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    • C07D405/10—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention relates to an andrographolide derivative and its use as an anti-fibrotic drug, in particular to a 15-subunit-14-deoxy-11,12-dehydroandrographolide derivative, belonging to the technical field of medicine.
  • Tissue fibrosis is a chronic disease that occurs in the liver, lungs, heart, kidneys and other parts.
  • tissue fibrosis is a chronic disease that occurs in the liver, lungs, heart, kidneys and other parts.
  • tissue is damaged, a series of cellular reactions occur at the injury site, resulting in excessive deposition of the extracellular matrix and tissue fibrosis. Eventually it can lead to organ dysfunction and even death.
  • fibrotic diseases although there are interferon, phosphatidylcholine, verapamil, glucocorticoids and other drugs in the clinic, they are usually accompanied by certain adverse reactions, so anti-fibrotic drugs based on Chinese herbal medicines The research and development market has broad prospects.
  • Hepatic fibrosis is a common pathological process in the development of various chronic liver diseases to cirrhosis. It is generally caused by an increase in the extracellular matrix content of the liver caused by the wound repairing reaction of the body. Factors causing liver fibrosis include viral hepatitis, metabolic diseases, autoimmune liver diseases, and chronic liver diseases caused by alcohol, drugs, and the like. According to existing reports, it is widely believed that the process of liver fibrosis is reversible (Yu Hui, Jiang Feng, Medical Information, 2015.28(7): 334-335), which can treat primary disease, inhibit inflammatory activity, and resist oxidation reduction. Hepatic fibrosis is treated by reaction, reduction of hepatic stellate cell activity and epithelial-mesenchymal transition, but the mechanism of liver fibrosis is complicated, and the regulation mechanism is affected by many factors.
  • Pulmonary fibrosis is the final outcome of the development, evolution, and scarring of many lung diseases, and its causes are diverse.
  • Many chronic lung diseases including asthma, bronchiectasis, chronic obstructive pulmonary disease, tuberculosis, lung cancer, and interstitial lung disease, are associated with pathological changes in fibrosis. Its pathological features are persistent alveolar damage caused by diffuse inflammation of the lungs and repeated destruction, repair, reconstruction and excessive deposition of extracellular matrix (Li Xianwei et al., Chinese Journal of Applied Physiology, 2017.33(1):16-21).
  • lung diseases such as idiopathic pulmonary fibrosis, respiratory distress syndrome, eosinophilic granuloma
  • the degree of fibrosis and fibrosis of the lung tissue determines the clinical recovery of the disease.
  • alveolitis occurs after early lung injury, inflammation and abnormal repair lead to the proliferation of pulmonary interstitial cells, and gradually form fibrotic lungs.
  • the clinical manifestations are patients with dyspnea and respiratory failure.
  • the average survival time after diagnosis is about 3 years, and the 5-year survival rate is 30% to 50%.
  • Renal fibrosis is caused by various pathogenic factors such as trauma, infection, inflammation, blood circulation disorder, and immune reaction. Its intrinsic cells are damaged, and a large amount of collagen deposition and accumulation occurs in the later stage, causing the kidney parenchyma to gradually harden and form scars. Until the kidneys completely lose organ function. The process of cell fibrosis and hardening in the kidney is also the process of renal fibrosis. Many acute and chronic kidney diseases are also closely related to the development of tissue fibrosis, especially the changes of renal fibrosis caused by diabetic nephropathy and hypertension.
  • Myocardial fibrosis is a pathological name proposed on the basis of modern medical research. It refers to the proliferation of cardiac fibroblasts (CFs) in the pathological state, and the extracellular matrix (ECM). (mainly type I and type III collagen fibers) abnormally increased deposition, leading to decreased ventricular compliance, affecting the normal contraction and diastolic function of the heart.
  • MF is a common pathological change in the development of a variety of heart diseases to a certain stage, and is also a key cause of ventricular remodeling.
  • myocardial fibrosis is closely related to many heart diseases, such as atrial fibrillation, myocardial infarction, Chronic heart failure, rheumatic heart valve disease, etc. It is often induced by diseases such as rheumatic heart disease, hypertension, myocardial infarction and heart failure. MF also occurs during aging and coronary atherosclerosis. The main pathological manifestations are increased myocardial stiffness and decreased myocardial contractility. The blood flow reserve is reduced, and even malignant arrhythmia and sudden death are caused.
  • heart diseases such as atrial fibrillation, myocardial infarction, Chronic heart failure, rheumatic heart valve disease, etc. It is often induced by diseases such as rheumatic heart disease, hypertension, myocardial infarction and heart failure. MF also occurs during aging and coronary atherosclerosis. The main pathological manifestations are increased myocardial stiffness and decreased myocardial contractility. The blood flow reserve is reduced, and even malignant arrhythm
  • TGF- ⁇ 1 /Smad signaling pathway Wnt/ ⁇ -catenin signaling pathway
  • PI3K/Akt phosphatidylinositol 3-kinase/protein kinase
  • corklactone down-regulates the production of ⁇ -SMA and collagen by inhibiting the TGF- ⁇ signaling pathway in mouse fibrotic lung tissue (CN 105560255 A).
  • a casein-sulfate significantly inhibits the proliferation of human embryonic lung fibroblasts induced by TGF- ⁇ and its hydroxyproline (HYP) content, thereby exerting pharmacological effects on the degree of pulmonary fibrosis (CN 103919769 A) .
  • PI3K inhibitors acting downstream of TGF- ⁇ are useful in the treatment of pulmonary fibrotic diseases (CN 104093408 A).
  • Ganoderma lucidum D can significantly inhibit TGF- ⁇ 1-induced phosphorylation of Smad3 in renal tubular epithelial cells, and has a therapeutic effect on renal fibrosis (CN 106220643 A).
  • the Third affiliated Hospital of the Third Military Medical University of the Chinese People's Liberation Army found that apigenin mediates the influx of Ca 2+ through TRPV4, activates the AMPK/SITR1 signaling pathway, and inhibits renal fibrosis.
  • Andrographolide one of the main diterpene lactone active ingredients of Andrographis paniculata (Burm.f.) Nees, which is mainly used for detoxification, antibacterial and anti-inflammatory, and anti-fibrosis. It also has a significant effect in blocking the early pathway of fibrosis by inhibiting the NF-excellentB inflammatory pathway (CN106831669A).
  • Andrographolide has a good protective effect on experimental liver injury in a variety of animals. Yao Qing et al found that andrographolide has a protective effect on acute liver damage caused by cocaine. The mechanism of liver protection may be related to inhibiting lipid peroxidation and reducing the production of oxygen free radicals in tissues. Visen PK [J Ethnopharmacol, 1993, 40(2), 131-136] and Handa S [Indian J Med Res, 1990, 92: 284-292] have demonstrated that andrographolide has protective effects against paracetamol-induced liver injury, among which Handa S's study also showed that andrographolide has a protective effect on hepatotoxicity caused by galactosamine.
  • Huang Chengliang et al found that andrographolide can reduce the degree of alveolitis and fibrosis in rats with pulmonary fibrosis induced by bleomycin or pingyangmycin, reduce the expression of PDGF in lung tissue and reduce the content of HYP; Fan Xianming and other studies found that andrographolide can reduce Bo Lungin-induced pulmonary fibrosis in rats with alveolitis and pulmonary fibrosis, decreased the expression of type I and III collagen mRNA in lung tissue, decreased the concentration of TNF- ⁇ and TGF- ⁇ 1 in bronchoalveolar lavage fluid (BALF), and The kidney has no obvious side effects.
  • BALF bronchoalveolar lavage fluid
  • the present inventors obtained a large number of novel andrographolide derivatives in the preliminary study (CN200510107247.4, CN200710053807.1, CN200710053806.7, CN200610017357.6, CN201210358667X), and some derivatives are anti-tumor, anti-inflammatory, Anti-HBV, HCV and acute liver injury protection applications and other applications for patent protection, the present invention further on the 15-subunit-14-deoxy-11,12-dehydroandrographolide derivatives in anti-tissue (organ) fibrosis Activity experiments have been carried out, and no reports on the use of it as an active ingredient for the preparation of anti-fibrotic drugs have been reported.
  • the object of the present invention is to provide 15-subunit-14-deoxy-11,12-dehydro-andrographolide derivatives and their use in the preparation of anti-fibrotic drugs.
  • the structure is a 15-subunit-14-deoxy-11,12-dehydro-andrographolide derivative represented by Formula 1.
  • R 1 , R 2 are each hydrogen or C 1-5 alkyl; R 1 , R 2 are each hydrogen or phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxybenzene Base, 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-chloroph
  • one of R 1 and R 2 is selected from the group consisting of methyl, ethyl or propyl, phenyl, 2-methoxyphenyl, 3- Methoxyphenyl, 4-methoxyphenyl, 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-chlor
  • one of R 1 and R 2 is selected from the group consisting of phenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methyl.
  • one of R 1 and R 2 is selected from the group consisting of methyl, ethyl or propyl, 2-furyl, 2-pyrrolyl, 6-chloro 3-mercapto, 3-indolyl, 5-chloro-3-indolyl, 6-chloro-2-pyridyl, 3-pyridyl, 4-n-propylphenyl, 4-isopropyl Phenyl, 4-isobutylphenyl, 3-fluoro-4-(4-methylpiperazinyl)phenyl; or R 1 and R 2 are bonded to a cyclohexyl group; each of R 3 and R 4 is hydrogen.
  • R 1 H
  • R 2 C 6 H 5
  • R 1 H
  • R 2 2-FC 6 H 4
  • R 1 H
  • R 2 2-Cl-C 6 H 4
  • R 1 H
  • R 2 2-Br-C 6 H 4
  • R 1 H
  • R 2 3-FC 6 H 4
  • R 1 H
  • R 2 3-Cl-C 6 H 4
  • R 1 H
  • R 2 3-Br-C 6 H 4
  • R 1 H
  • R 2 4-Cl-C 6 H 4
  • R 1 H
  • R 2 4-FC 6 H 4
  • R 1 H
  • R 2 4-Br-C 6 H 4
  • R 1 H
  • R 2 2-CH 3 O-4-Cl-C 6 H 3
  • R 2 H
  • R 1 2-Br-C 6 H 4
  • R 2 H
  • R 1 3-Cl-C 6 H 4
  • R 2 H
  • R 1 2-F-4-Cl-C 6 H 3
  • R 2 H
  • R 1 4-FC 6 H 4
  • R 2 H
  • R 1 C 6 H 5
  • R 1 H
  • R 2 3-F-4-Cl-C 6 H 3
  • R 1 H
  • R 2 4-Cl-C 6 H 4
  • R 5 CH 2 CH 2 COOH
  • R 2 H
  • R 1 4-Cl-C 6 H 4
  • R 5 3-pyridyl.
  • the present inventors used the hepatic stellate cell LX-2 to measure the inhibitory activity of the compound of the present invention on cell migration and activation, and evaluated the anti-liver fibrosis activity of the compound; Further, CCl 4 induced SD rat liver fibrosis model, pig serum induced Wistar rat liver fibrosis model and bile duct ligation (BDL) induced SD rat liver fibrosis model as representative, by measuring liver tissue collagen content (Masson The level of expression of ⁇ -SMA of the HSC activation marker molecule was evaluated by staining, and the anti-fibrotic activity of the compound was evaluated.
  • the human lung alveolar type II cell A549 was used to study the inhibitory activity of the compound of the present invention on TGF- ⁇ 1-induced transformation of A549 cells into mesenchymal cells, and the anti-pulmonary fibrosis activity of the compound was evaluated, and the mouse lung induced by bleomycin was further used.
  • a fibrosis model was studied to study the anti-pulmonary fibrosis effect of compounds in vivo.
  • the inhibitory activity of the compound of the present invention on TGF- ⁇ 1-induced HK-2 cell-to-mesenchymal cell transformation was evaluated by using proximal tubular epithelial cell HK-2, and the anti-renal fibrosis activity of the compound was evaluated, and unilateral ureteral ligation induction was further used.
  • the rat renal fibrosis model (UUO) studies the in vivo anti-renal fibrosis activity of the compounds of the invention.
  • the compound of the present invention was evaluated for anti-myocardial fibrosis by using AngII to stimulate primary cardiac fibroblast HCFB and detecting cell proliferation rate.
  • the cis and the reverse structures of the compounds have anti-fibrotic activity, and the compounds are effective pharmaceutical ingredients, or various prodrug forms of the compounds, alone or in combination with other drugs, according to various conventional pharmaceuticals.
  • the method and process requirements are combined with acceptable auxiliary and/or additive ingredients in the pharmaceutical industry to prepare various pharmaceutical dosage forms for anti-fibrotic oral preparations, injection preparations and the like. It is preferred to prepare a medicament for treating or preventing various types of fibrotic diseases such as liver, lung, kidney, and heart.
  • the oral preparation is a tablet, a pill, a capsule, a granule or a syrup; the injectable preparation includes an injection or a lyophilized powder dosage form.
  • the above compounds are determined to have clear anti-organ and/or tissue fibrotic activity by activity screening. It has been experimentally demonstrated that the compounds of the present invention have significantly improved anti-liver, lung, kidney and/or myocardial fibrosis compared to the parent compound, andrographolide (AD). Therefore, the use of such a compound as an active ingredient for preparing various anti-humanized fibrotic drugs provides a new drug route for the treatment and prevention of diseases associated with fibrosis, thereby expanding the range of clinical drug selection and having good Application development prospects.
  • AD andrographolide
  • FIG 1A on human hepatic stellate cells of LX-2 activity, and the ratio of AD AD group and the compound of the present invention (15.00 ⁇ M), # P ⁇ 0.05 ;
  • FIG. 1B on human hepatic stellate cells LX-2 activity the compounds of the AD group than in the AD and the present invention is represented by (30.00 ⁇ M), # P ⁇ 0.05 ;
  • 2A is a compound of the present invention and AD (5.00 m) suppression result (statistical results) human hepatic stellate cells migrate LX-2, and AD group than, # P ⁇ 0.05;
  • FIG 2B is a compound represented by the present invention and AD (1.00 m and 5.00 m) inhibition of human hepatic stellate cells migrate results LX-2 (statistics), and the AD group than, # P ⁇ 0.05.
  • Figure 3 is a graph showing the inhibition of migration of human hepatic stellate cells LX-2 by AD and the compound of the present invention (5.00 ⁇ M) (partial photomicrograph; ⁇ 100 fold);
  • Figure 4 is a graph showing the effect of a representative compound of the present invention on the degree of hepatic fibrosis induced by CCl 4 in liver fibrosis of SD rats (relative collagen area /%), compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with the silybin group, & P ⁇ 0.05;
  • Figure 5 is a graph showing the effect of a representative compound of the present invention on the degree of liver fibrosis in SD rats induced by CCl 4 in liver fibrosis (part of Masson staining; ⁇ 100 times);
  • Figure 6 is a graph showing the effect of some representative compounds on the expression level of ⁇ -smooth muscle actin ( ⁇ -SMA) in liver tissue of rats with liver fibrosis induced by CCl 4 (statistical results), compared with the model group, * P ⁇ 0.05; group than in the AD, # P ⁇ 0.05; compared with silibinin group, & P ⁇ 0.05;
  • Figure 7 is a graph showing the effect of a representative compound of the present invention on the expression level of ⁇ -SMA in liver tissue of SD rats induced by CCl 4 (partial immunohistochemistry; ⁇ 100 times);
  • Figure 8 is a graph showing the effect of a representative compound of the present invention on liver fibrosis induced by porcine serum in Wistar rats (relative collagen area/%), compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with the silybin group, & P ⁇ 0.05;
  • Figure 9 is a graph showing the effect of a representative compound of the present invention on the degree of hepatic fibrosis in Wistar rats induced by porcine serum (Parts of Masson staining; ⁇ 100 times);
  • Figure 10 is a graph showing the effect of a representative compound of the present invention on the expression level of ⁇ -SMA in liver tissue of Wistar rats induced by porcine serum (statistical results), compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with the silybin group, & P ⁇ 0.05;
  • Figure 11 is a graph showing the effect of a representative compound of the present invention on the expression level of ⁇ -SMA in liver tissue of Wistar rats induced by porcine serum (partial immunohistochemistry picture; ⁇ 100 times);
  • FIG. 12 representative compounds of the present invention, part of the bile duct ligation impact fiberization of SD rat liver tissue (relative collagen area /%), compared with model group, * P ⁇ 0.05 0.05; compared with ursodeoxycholic acid group, & P ⁇ 0.05;
  • Figure 13 is a graph showing the effect of a representative compound of the present invention on the degree of hepatic fibrosis in SD rats induced by total bile duct ligation (part of Masson staining; x 100 times);
  • Figure 14 is a graph showing the effect of some representative compounds on the expression of ⁇ -SMA in the liver of SD rats induced by total bile duct ligation (statistical results), compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05 Compared with ursodeoxycholic acid group, & P ⁇ 0.05;
  • Figure 15 is a diagram showing the effect of a representative compound of the present invention on the expression level of ⁇ -SMA in liver tissue of SD rats induced by total bile duct ligation (partial immunohistochemistry picture; ⁇ 100 times);
  • Figure 16 is a graph showing the effect of Compound H on the level of laminin (LN) in the serum of SD rats induced by CCl 4 in the present invention, compared with the model group, * P ⁇ 0.05; compared with the oxymatrine group, & P ⁇ 0.05;
  • Figure 17 is a graph showing the effect of compound H on the level of type I collagen (C-I) in serum of SD rats induced by CCl 4 induced by compound C, compared with model group, * P ⁇ 0.05;
  • Figure 18 is a graph showing the effect of Compound H on the level of procollagen type III (PC-III) in serum of SD rats induced by CCl 4 in the present invention, compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with oxymatrine group, & P ⁇ 0.05;
  • Figure 19 is a graph showing the effect of Compound H on the level of type IV collagen (C-IV) in serum of SD rats induced by CCl 4 in the present invention, compared with the model group, * P ⁇ 0.05; compared with the oxymatrine group, & P ⁇ 0.05;
  • Figure 20 is a graph showing the effect of Compound H on the level of interleukin-6 (IL-6) in serum of SD rats induced by CCl 4 in the present invention, compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with oxymatrine group, & P ⁇ 0.05;
  • Figure 21 is a graph showing the effect of Compound H on the level of tumor necrosis factor- ⁇ (TNF- ⁇ ) in serum of SD rats induced by CCl 4 in the present invention, compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with matrine group, & P ⁇ 0.05;
  • Figure 22 is a graph showing the effect of compound H on the level of superoxide dismutase (SOD) in liver tissue of SD rats induced by CCl 4 in liver fibrosis induced by the present invention, compared with the model group, * P ⁇ 0.05;
  • Figure 23 is a graph showing the effect of compound H on the level of malondialdehyde (MDA) in the liver tissue of SD rats induced by CCl 4 induced by CCl 4 , compared with the model group, * P ⁇ 0.05;
  • Figure 24 is a graph showing the effect of compound H on the inflammatory grade of hepatic histopathological sections of SD rats induced by CCl 4 induced by CCl 4 in the present invention (statistical results), compared with the model group, * P ⁇ 0.05; compared with the AD group, # P ⁇ 0.05; compared with oxymatrine group, & P ⁇ 0.05;
  • Figure 25 is a graph showing the effect of compound H on the inflammatory grade of hepatic histopathological sections of SD rats induced by CCl 4 induced by CCl 4 (H&E staining; ⁇ 100 times);
  • Figure 26A is a compound of the present invention and AD (30.00 ⁇ M) Effect on Type II A549 human alveolar cell-like activity, the ratio of the AD group, # P ⁇ 0.05;
  • FIG 26B is a compound of the present invention and AD Effect A549 human alveolar type II activity like cells, FIG.:. F, g, h, k test concentration 3.00 ⁇ M, the rest of 30.00 ⁇ M, group than in the AD, # P ⁇ 0.05;
  • FIG. 27A Inhibition of TGF- ⁇ 1-induced conversion of human alveolar type II cells A549 to mesenchymal cells by AD and the compounds of the invention (statistical results), low and high levels of compounds AD, N, P, Q, SW and Z
  • the concentrations were 0.63 ⁇ M and 1.25 ⁇ M, respectively.
  • the low and high concentrations of compounds C, EG, IL, O, R, X and Y were 0.31 ⁇ M and 0.63 ⁇ M, respectively, and the low and high concentrations of compounds A, B, D, H and M. concentrations of 0.16 ⁇ M and 0.31-0.33; and TGF- ⁇ added control group, # P ⁇ 0.05; AD and control group, * P ⁇ 0.05;
  • 27B is a compound represented by AD and the present invention inhibit TGF- ⁇ 1-induced
  • Figure 28 is a graph showing the effect of representative compound H of the present invention on the degree of pulmonary fibrosis induced by bleomycin in lung and lung tissues of KM mice (part of Masson staining; x 100 times);
  • Figure 29 is a graph showing the effect of representative compound H on the degree of pulmonary fibrosis induced by bleomycin in KM mice (collagen area/%), compared with the model group, * P ⁇ 0.05, ** P ⁇ 0.01; AD group ratio, ## P ⁇ 0.01;
  • Figure 30 is a graph showing the effect of Compound H on the inflammatory grade of lung tissue pathological sections of KM mice induced by bleomycin (H&E staining; x 100 times);
  • Figure 31 is a graph showing the effect of compound H on the inflammatory grade of lung tissue pathological sections of KM mice induced by bleomycin (statistical results), ** P ⁇ 0.01; compared with AD group, ## P ⁇ 0.01.
  • the pathological criteria for pulmonary fibrosis are: level 0 without alveolitis; level 1 alveolitis area is less than 1/5; level 2 alveolitis area is above 1/5, 1/2 or more; level 3 alveolitis area is 1 /2 or more; compared with the model group, ** P ⁇ 0.01; compared with the AD group, ## P ⁇ 0.01;
  • FIG 32A is a compound of the present invention and AD in human proximal renal epithelial cells HK-2 activity, the concentration of compound 30.00 ⁇ M, than the AD group, # P ⁇ 0.05;
  • FIG 32B is a representative compound of the present invention and AD in human proximal renal epithelial HK-2 cell viability; compound concentration of 30.00 ⁇ M, than the AD group, # P ⁇ 0.05;
  • Figure 33A shows AD and the compounds of the present invention inhibit TGF- ⁇ 1-induced conversion of human proximal tubular epithelial cells HK-2 cells to mesenchymal cells (partial photomicrograph; ⁇ 100 fold), in which: 1. Control; 2. TGF- ⁇ 1; 3. TGF- ⁇ 1+AD (0.63 ⁇ M); 4. TGF- ⁇ 1+T (0.63 ⁇ M); 5. TGF- ⁇ 1+A (0.63 ⁇ M); 6. TGF- ⁇ 1+H ( 0.08 ⁇ M); 7. TGF- ⁇ 1+F (0.63 ⁇ M); 8. TGF- ⁇ 1+J (0.63 ⁇ M); 9. TGF- ⁇ 1+K (0.63 ⁇ M); 10. TGF- ⁇ 1+Z (0.63 ⁇ M) ); 11. TGF- ⁇ 1+Y (0.16 ⁇ M);
  • FIG. 33B shows that AD and a compound represented by the present invention inhibit TGF- ⁇ 1-induced conversion of human proximal tubular epithelial cells HK-2 to mesenchymal cells (partial photomicrograph; ⁇ 100-fold), in which: 1. Control ; 2. TGF- ⁇ 1; 3. TGF- ⁇ 1+AD (0.63 ⁇ M); 4. TGF- ⁇ 1+d (0.31 ⁇ M); 5. TGF- ⁇ 1+e (0.08 ⁇ M); 6. TGF- ⁇ 1+j (0.31 ⁇ M); 7. TGF- ⁇ 1+h (0.08 ⁇ M); 8. TGF- ⁇ 1+i (0.08 ⁇ M); 9. TGF- ⁇ 1+b (0.31 ⁇ M).
  • Figure 34 is a graph showing the effect of representative compound H on the degree of renal fibrosis induced by unilateral ureteral ligation in SD rats (relative collagen area/%), compared with the model group, * P ⁇ 0.05, ** P ⁇ 0.01; AD group ratio, ## P ⁇ 0.01;
  • Figure 35 is a graph showing the effect of representative compound H of the present invention on the degree of renal fibrosis induced by unilateral ureteral ligation in SD rats (part of Masson staining; x 100 times);
  • Figure 36 is a graph showing the effect of compound H on the size of kidney tissue of SD rats induced by unilateral ureteral ligation in the present invention (analysis of kidney tissue after ligation of some ligatures);
  • Figure 37 is a graph showing the effect of compound H on the inflammatory grade of renal tissue pathological changes induced by unilateral ureteral ligation in the present invention (statistical results), compared with the model group, * P ⁇ 0.05, ** P ⁇ 0.01; Ratio, # P ⁇ 0.05.
  • grade 1 is basically normal interstitial, mild tubule degeneration
  • grade 2 is interstitial fibrosis, tubule atrophy ⁇ 20%, scattered in inflammatory cell infiltration
  • grade 3 is inter Fibrosis, tubule atrophy accounted for 30%, scattered and/or diffuse inflammatory cell infiltration
  • fourth grade was interstitial fibrosis, tubule atrophy >50%, scattered and/or diffuse inflammatory cell infiltration.
  • Figure 38 is a graph showing the effect of compound H on the score of renal tissue histopathitis in SD rats induced by unilateral ureteral ligation (statistical results), compared with the model group, * P ⁇ 0.05, ** P ⁇ 0.01; Ratio, # P ⁇ 0.05, Note: According to renal tubular degeneration, necrosis, no, light, medium and severe, respectively, 0, 1, 2, 3 points. The overall score is 0: 0; Level 1: 1 to 4; Level 2: 5 to 8; Level 3: 9 to 12. Compared with model group, * P ⁇ 0.05, ** P ⁇ 0.01; compared with the AD group, # P ⁇ 0.05;
  • Figure 39 is a graph showing the effect of Compound H on the inflammatory grade of renal tissue histopathology of SD rats induced by unilateral ureteral ligation (H&E staining; ⁇ 100 times);
  • Figure 40 is a graph showing the effects of AD and the representative compounds H (0.3, 3.0 and 15.0 ⁇ M) of the present invention on the proliferation of human primary myocardial fibrosis cells HCFB.
  • Figure 41 is a graph showing that AD (0.63 ⁇ M) and the representative compounds H of the present invention (0.08 ⁇ M, 0.16 ⁇ M, 0.31 ⁇ M and 0.63 ⁇ M) inhibit angiotensin II (Ang II)-induced proliferation of human primary myocardial fibrosis cells HCFB, and AngII group ratio, * P ⁇ 0.05, ** P ⁇ 0.01; compared with the AD group, # P ⁇ 0.05, ## P ⁇ 0.01.
  • the invention is illustrated below in conjunction with specific embodiments. It is to be understood that these embodiments are only illustrative of the invention and are not intended to limit the scope of the invention.
  • the compound to which the present invention relates is not limited to the representative structure used in the examples, and a different substituent at the 15-position can be substituted to obtain a compound having anti-fibrotic activity; the present invention can be obtained by various causes of fibrosis as a research object.
  • the compounds have anti-fibrotic effects; various other in vitro and in vivo studies (models) can also be utilized to derive anti-fibrotic effects of the compounds of the invention.
  • Example 1 The compounds of the invention inhibit LX-2 migration in human hepatic stellate cells.
  • hepatic stellate cells migrate to the inflammation site of damaged liver tissue, and then proliferate and activate.
  • ECM components such as collagen are the key to the development of liver fibrosis. Therefore, the anti-liver fibrosis effect of the compound of the present invention was evaluated by the scratch damage method.
  • LX-2 Human hepatic stellate cells LX-2 (provided by Beijing Bei Na Chuanglian Biotechnology Research Institute) were used to study the anti-fibrosis effect of the compounds of the present invention in vitro compared with andrographolide (AD).
  • LX-2 cells were cultured in 10% (V/V) fetal bovine serum, 100 ⁇ g/mL streptomycin, 100 IU/mL penicillin RPMI1640 medium, and placed in a volume fraction of 5% CO2 incubator at saturation humidity, 37 °C. to cultivate.
  • LX-2 cells in logarithmic growth phase were digested with 0.25% (w/v) trypsin and diluted to 3.5 ⁇ 10 5 /mL cell suspension with RPMI1640 medium containing 10% (v/v) fetal bovine serum.
  • the solution was placed in a 96-well plate at 200 ⁇ L/well and cultured in a volume fraction of 5% CO 2 incubator at 37 ° C for 24 h.
  • the medium containing different concentrations of the drug was added, and the final concentration of the drug was up to 30.00 ⁇ M.
  • the holes are repeated.
  • the culture was continued for 48 h, MTT (5 mg/mL) was added, 20 ⁇ L/well, cultured for 4 h, the supernatant was discarded, 150 ⁇ L of DMSO was added, and the mixture was shaken for 10 min, and the absorbance was measured by a microplate reader.
  • the measurement wavelength was 570 nm and the reference wavelength was 450 nm.
  • Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • the log phase LX-2 cells were digested with 0.25% trypsin and diluted into 1.0 ⁇ 10 6 /mL cell suspension in RPMI1640 medium containing 10% fetal bovine serum, and plated in a 96-well plate. 200 ⁇ L per well. After 12 hours of culture, the cells were fused, the original medium was discarded, and the medium with a serum content of 0.5% was added and re-synchronized for 12 hours, then streaked, washed twice with PBS, and 200 ⁇ L of RPMI1640 containing the test compound (5 ⁇ M) was added. Immediately after the medium, photographs were taken under a microscope. Set 3 wells to repeat and set the controls. After 24 hours of culture, the photos were taken under a microscope.
  • Migration inhibition rate 1 - (0h scratch distance of the administration group - 24h scratch distance) / (blank group 0h scratch distance - 24h scratch distance) ⁇ 100%.
  • the results are shown in Figures 2A, 2B and 3. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • Figures 1A and 1B show that the compound of the present invention showed no significant inhibitory effect on the proliferation of human hepatic stellate cells at a concentration of 15 ⁇ M, and the survival rate was significantly higher than that of the parent compound AD.
  • the results show that, compared with AD, the compound of the present invention can significantly inhibit the migration of LX-2 cells at a non-toxic concentration, and compared with AD, on human hepatic stellate cells. Migration has a stronger inhibitory effect and a higher safety index.
  • Example 2 The compound of the present invention significantly reduces the degree of liver fibrosis induced by carbon tetrachloride (CCl 4 ) in SD rats
  • CCl 4 induced rat liver fibrosis model is a classic animal model of liver fibrosis. It has great similarity with human liver fibrosis in many aspects such as morphology and pathophysiology, and it has not only toxicant induction.
  • the liver function abnormalities are similar to those of human liver cirrhosis, and the molecular mechanism of fibrosis, serum markers after injury, and pathological changes of liver tissue are very similar to humans. Therefore, the model of liver fibrosis induced by CCl 4 is widely used to study the pathogenesis of liver fibrosis, the screening of anti-fibrosis drugs, and the mechanism of anti-fibrosis drugs.
  • Andrographolide is produced by Sichuan Shifang Jinxin Biotechnology Co., Ltd. (batch number: 120822), and the purity is greater than 99%; the compound of the present invention is synthesized by the inventor's laboratory, and the purity is more than 99%; the pharmaceutical grade oligomeric carboxymethyl cellulose Sodium (CMC-Na) is produced by Anhui Shanhe Pharmaceutical Excipient Co., Ltd. (batch number: 131114); Kushensu capsule, produced from Zhengda Tianqing Pharmaceutical Co., Ltd. (batch number: Guoyao Zhunzi H20010763). Shuifeiqibin Capsule is produced by Tianjin Tianshili Shengte Pharmaceutical Co., Ltd.
  • CCl 4 is produced by Tianjin Kaiji Chemical Reagent Co., Ltd., and other reagents are commercially available analytical grades.
  • SD rats were fed ad libitum for 3 days, randomly divided into groups according to body weight, and the other rats were subcutaneously injected with 40% CCl 4 soy oil for the first time.
  • the first dose was 4 mL/kg, and the other dose was 2 mL/kg. 2 times / w, the normal group of rats were injected subcutaneously with the corresponding volume of soybean oil. After modeling for 4 weeks, except for the normal group, the remaining rats were grouped according to body weight, 8 rats/group.
  • the dosages of andrographolide, oxymatrine, silybin and the compound A of the present invention were both 20 mg/kg, and the doses of other compounds were the same molar mass as A; the model group and the normal group were given the same amount of 0.5% CMC- Na.
  • the 5th and 6th modeling and administration were carried out simultaneously, and the modeling was stopped at the 7th and 8th, and only the administration was performed. Each gavage was performed on an empty stomach in the morning, and the drug was stopped for one day for 10 days. The rats were replaced with litter 8 hours before the last gavage, and the rats were strictly fasted.
  • liver fibrosis was quickly and completely dissected after blood collection.
  • the collected blood was allowed to stand in an incubator at 37 ° C for 45 min, centrifuged at 3500 rpm for 15 min at 4 ° C, and the upper serum was taken and used.
  • the liver of the lower left part of the rat was fixed in 10 times volume of 4% paraformaldehyde fixative, and the fixative was updated after 24 hours. Pathological sections were performed after fixation, and the degree of liver fibrosis was observed by Masson's three-color staining.
  • Image-Pro Plus software performed semi-quantitative analysis of fibrosis histology on the photographs of Masson stained sections.
  • the relative collagen area was calculated: (average area of the administration group - average area of the normal group) / (average area of the model group - average area of the normal group) ⁇ 100%, and the results are shown in Figs.
  • the expression of ⁇ -SMA (a marker of the degree of HSC activation) in liver tissue was evaluated by immunohistochemistry, and the positive expression was quantified using Image-Pro Plus. The results are shown in Figures 6 and 7. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • the results show that the compounds of the present invention can significantly reduce the degree of fibrosis in liver tissue of animals.
  • the collagen area of the liver tissue of the treatment group of the present invention was significantly reduced as compared with AD.
  • the curative effect is more prominent.
  • the compound of the present invention significantly down-regulated the expression level of ⁇ -SMA in liver tissue, and the difference was significant compared with AD, and the effect was better than that of the positive drug. It is indicated that its anti-fibrosis effect is related to the inhibition of hepatic stellate cell activation.
  • Example 3 Compounds of the Invention Significantly Reduce Porcine Serum Inducing Degree of Hepatic Fibrosis in Wistar Rats
  • Immune hepatic fibrosis is mainly caused by the immune response of the liver itself, and other factors (virus, alcohol, schistosomiasis and certain chemicals) are often accompanied by different degrees of immune response.
  • xenogenic serum or protein porcine serum g, schistosomiasis, human or bovine serum albumin, etc.
  • heterologous serum mainly activates hepatic stem cells through MHCII molecules and inflammatory factors to produce liver damage caused by immune response.
  • liver fibrosis and immune response persist, which can well reproduce other models.
  • the possible processes and mechanisms of liver fibers caused by human autoimmune liver injury make this model a great advantage in the screening and evaluation of immune drugs against liver fibrosis.
  • Wistar rats were randomly divided into groups according to body weight, with 6 rats in each group. Except for the normal group of animals, all rats were intraperitoneally injected with porcine serum, 1 mL / only, 2 times / w, for 6 w, the normal group of animals were injected with the same dose of normal saline. The experiment ends at the end of the 8th. Administered by intragastric administration once a day. Each gavage was performed on an empty stomach in the morning, and the drug was stopped for one day for 10 days. The preventive administration group (H: 2.5 mg/kg) was administered from the day of modeling, silybin (50 mg/kg), andrographolide (20 mg/kg), and 5 mg/kg and 10 mg of the present compound H.
  • the results show that the compounds of the present invention exhibit good anti-fibrosis effects on the pig serum-induced liver fibrosis model. Both prophylactic or therapeutic administration significantly reduced the degree of liver fibrosis in rats. The effect was better than that of the AD and silybin (50 mg/kg) treatment group, and even after administration for 6 weeks, only 2w of the compound of the present invention was administered with good therapeutic effect. At the same time, it was observed that the compound of the present invention significantly down-regulated the expression level of ⁇ -SMA in liver tissue, and the difference was significant compared with AD, and the effect was better than that of the positive drug. It is indicated that its anti-fibrosis effect is related to the inhibition of hepatic stellate cell activation.
  • Example 4 Compounds of the Invention Significantly Reduce the Degree of Hepatic Fibrosis in SD Rats with Total Bile Duct Ligation
  • Ursodeoxycholic acid was produced by Shanghai Xinyi Pharmaceutical Co., Ltd. (batch number: Guoyao Zhunzi H31021875); other test drugs and compounds were the same as in Example 2, and other reagents were commercially available analytically pure; the drug was formulated into 0.5% carboxylate. Sodium methylcellulose (CMC-Na) suspension.
  • SD rats were randomly divided into groups according to body weight: sham operation group, model group, AD control group (5 mg/kg and 20 mg/kg), ursodeoxycholic acid control group (25 mg/kg) and the present invention.
  • sham operation group model group
  • AD control group 5 mg/kg and 20 mg/kg
  • ursodeoxycholic acid control group 25 mg/kg
  • the sham operation group and the model group were intragastrically administered with 0.5% CMC-Na, and the other administration groups were given 0.5% CMC-Na suspension of the corresponding drug, and the administration was completed for 4 weeks.
  • the litter was replaced 12 hours before the operation, and the water was strictly fasted.
  • collagen is an important component of the stromal membrane of the sinus, mainly C-I and C-IV.
  • C-I and C-IV When the liver is damaged, a large amount of collagen and glycoprotein are transcribed, translated and assembled, mainly LN, C-I and C-III.
  • serum LN, C-I, PC-III, and C-IV levels are important indicators for the diagnosis of fibrotic patients.
  • the levels of LN, C-I, PC-III and C-IV in the serum of the animals were determined by ELISA using the compound H of the present invention as a representative.
  • the changes in serum collagen index are shown in Figures 16-19. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • the results show that the compound of the present invention significantly enhances the inhibitory activity against PC-III while maintaining the significant reduction of LN, C-I and C-IV levels of the parent compound AD.
  • the compound of the invention reduces LN, PC-III, C-IV to normal levels at a dose of 20 mg/kg.
  • the degree of down-regulation of PC-III and C-IV by Compound H at the 5 mg/kg dose was comparable to that of AD at the 20 mg/kg dose.
  • Example 6 Effect of the Compound of the Invention on the Levels of Serum IL-6 and TNF- ⁇ in Hepatic Fibrosis Induced by Carbon Tetrachloride (CCl 4 ) in SD Rats
  • Activated HSC can induce macrophages to produce large amounts of TNF- ⁇ , continue to participate in the differentiation of HSC, and also expand the inflammatory response in the liver.
  • IL-6 is one of the pro-fibrotic factors, involved in complex physiological processes such as inflammation, lipid peroxidation, apoptosis and regeneration in liver tissue, and is also an effector molecule downstream of NF- ⁇ B.
  • the level of IL-6 and TNF- ⁇ in the serum of the animal was determined by ELISA using the compound H of the present invention as a representative. The results are shown in Figures 20 and 21. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • Example 7 Effect of the Compound of the Invention on the Levels of SOD and MDA in Liver Tissue of Liver Fibrosis Induced by Carbon Tetrachloride (CCl 4 ) in SD Rats
  • SOD and MDA are important indicators for evaluating lipid peroxidation.
  • SOD is an antioxidant that not only inhibits lipid peroxidation caused by free radical initiation, but also scavenges free radicals to protect the integrity of biofilms. It is a sensitive indicator of antioxidant capacity in the body. Lipid peroxidation produces a large amount of MDA, which is proportional to the degree of damage to peroxidation in the tissue.
  • the level of SOD and MDA in the liver tissue of the animal was determined by the compound H of the present invention. The results are shown in Figures 22 and 23. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • Example 8 The compound of the present invention substantially improved the inflammatory state of liver tissue of liver fibrosis induced by carbon tetrachloride (CCl 4 ) in SD rats
  • the results showed that the liver lobule structure of the normal group was intact, the hepatocyte cords were neatly arranged, no abnormal changes occurred, and the vascular area was normal; no hepatocytes showed deformation, necrosis, and no fibrous connective.
  • the hepatic cell line was disordered, the nuclear-to-plasma ratio was significantly increased and accompanied by visible, flaky and focal necrotic areas. The lymphocyte infiltration was severe, and the hepatic lobular structure was seriously damaged and could not be distinguished.
  • liver damage of the animals in the drug-administered group was improved to different extents, especially in the middle and high-dose treatment groups of the compound H of the present invention, the hepatocytes were only slightly enlarged, and the structure of the hepatic cell cord was normal. It was significantly better than the positive drug Kushen treatment group and AD treatment group.
  • Example 9 The compound of the present invention inhibits TGF- ⁇ 1-induced transformation of human type II alveolar epithelial cells A549 to mesenchymal cells.
  • Type II alveolar epithelial cells present in the alveoli are stimulated by cytokines such as inflammatory mediators and growth factors, and cell morphology. It changes from cobblestone to fusiform, completes epithelial mesenchymal transition (EMT), has the function of interstitial cells, and then synthesizes collagen fibers. The deposition of a large amount of collagen fibers can aggravate the course of interstitial pulmonary fibrosis. Therefore, the compounds of the present invention were evaluated for anti-pulmonary fibrosis by morphological observation. 1 cell culture and drug treatment
  • the anti-pulmonary fibrosis effect of the compound of the present invention was studied in vitro using human type II alveolar epithelial cells A549 in comparison with andrographolide.
  • A549 cells were cultured in RPMI1640 medium containing 10% (v/v) fetal bovine serum, 100 ⁇ g/mL streptomycin, 100 IU/mL penicillin, and placed in a volume fraction of 5% CO 2 incubator at saturation humidity, 37 ° C. to cultivate.
  • the log phase A549 cells were digested with 0.25% (w/v) trypsin and diluted to 2.5 ⁇ 10 4 /mL (verified) cell suspension with RPMI1640 medium containing 10% (v/v) fetal bovine serum.
  • the solution was placed in a 96-well plate at 200 ⁇ L/well, and cultured in a volume fraction of 5% CO 2 incubator at 37 ° C for 24 hours.
  • the drug-containing medium was added to a final concentration of 30.00 ⁇ M, and each well was replicated in 4 wells. Continue to culture for 48 hours. Others are the same as in the first embodiment. The results were averaged as shown in Figures 26A and 26B.
  • the log phase A549 cells were digested with 0.25% (w/v) trypsin and diluted into 2.5 ⁇ 10 4 /mL cell suspension with RPMI1640 medium containing 10% (v/v) fetal bovine serum. In a 96-well plate, 200 ⁇ L per well. After 24 hours of culture, the cells were grown into a fusion state, the original medium was discarded, and the serum-free medium was added and re-synthesized for 24 hours. The medium was discarded, washed twice with PBS, and 200 ⁇ L of TGF- ⁇ 1 (5 ng/mL) was added. Immediately after taking RPMI1640 medium with different concentrations of the test compound, photographs were taken under a microscope (100 ⁇ ).
  • 27A and 27B show that the compound of the present invention can significantly inhibit the epithelial mesenchymal transition of A549 cells at a non-toxic concentration, and has a stronger inhibitory effect on human mesenchymal transition of human type II alveolar epithelial cells than AD.
  • the safety index is higher.
  • Example 10 Compounds of the Invention Significantly Reduce the Degree of Pulmonary Fibrosis Induced by Bleomycin in KM Mice
  • Pulmonary fibrosis is a lung injury caused by a variety of reasons.
  • the pathogenesis of pulmonary fibrosis is complex. Different pathogenic factors initiate inflammation and immune response, involving a variety of cells including vascular endothelial cells, alveolar epithelial cells, fibroblasts and Macrophage, etc., the interaction of various cytokines and inflammatory mediators.
  • Bleomycin is an alkaline glycopeptide anticancer antibiotic.
  • One of the serious toxic adverse reactions of this drug is pulmonary fibrosis. It has been confirmed in animal experiments that the pathological changes of pulmonary fibrosis induced by bleomycin and Human lung fibrosis is very similar and is commonly used as a model for studying pulmonary fibrosis.
  • mice were randomly divided into groups according to body weight: sham operation control group, model group, AD control group (250 mg/kg), bnisin control group (5 mg/kg), and H compound administration group (62.5 mg). /kg) and H compound administration group (250. mg / kg), a total of 6 groups, 15 per group.
  • the mice were anesthetized with intraperitoneal injection of 4% sodium pentobarbital (2 ml/kg). The mice were fixed in the supine position, and the neck hair was shaved. After iodine disinfected the skin, the incision was made about 1 cm down the neck.
  • mice After 0.5 h of administration, the whole blood of the mice was collected by eyeball method. After the blood was collected, the mice were sacrificed by cervical dislocation. The lungs were collected, weighed, and the lung lesions were observed and recorded. After taking the picture, the lungs were fixed in 4% paraformaldehyde fixative. Serum preparation and Masson staining and statistical methods of results were the same as in Example 2, Masson staining and collagen area results are shown in Figures 28 and 29.
  • Figures 28 and 29 show that the compound H of the present invention significantly reduced the lung fibrosis area of pulmonary fibrosis KM mice on a bleomycin-induced mouse pulmonary fibrosis model, and the effect was significantly stronger than AD.
  • Example 11 The compound of the present invention significantly improved the inflammatory state of lung tissue in pulomycin-induced pulmonary fibrosis in KM mice
  • Compound H of the present invention the improvement of the inflammatory state of the lung tissue by the compound of the present invention was observed by H&E staining.
  • the staining results and pathological scores are shown in Figures 30 and 31.
  • Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • the results showed that the lung tissue of the sham-operated group had intact alveolar structure, uniform lung septum thickness, no abnormal changes and no infiltration of inflammatory cells by the naked eye.
  • the alveolar structure of the mice was disordered, the lung interval became thicker, and the pulmonary interstitial fibrosis material segmented the alveolar to form the reconstructed alveoli, and a large number of inflammatory cells infiltrated.
  • the lung tissue damage of the animals in the drug-administered group was improved to some extent, especially in the high-dose treatment group of the compound H of the present invention, the lung tissue of the lung tissue was basically intact and normal, and the inflammatory cell infiltration was in the naked eye. It is difficult to identify, and its efficacy is significantly better than that of the AD treatment group.
  • Example 12 The compound of the present invention inhibits TGF- ⁇ 1-induced conversion of human proximal tubular epithelial cells HK-2 to mesenchymal cells
  • tubular epithelial cells can transdifferentiate to fibroblasts and express their fibroblast-specific protein (FSP1). Renal tubular epithelial-mesenchymal cells are differentiated into interstitial fibers of the kidney.
  • FSP1 fibroblast-specific protein
  • HK-2 Human proximal tubular epithelial cells HK-2 (provided by the China Center for Type Culture Collection) were used to study the anti-renal fibrosis effect of the compounds of the present invention in comparison with andrographolide AD.
  • HK-2 cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (V/V), 100 ⁇ g/mL streptomycin, 100 IU/mL penicillin, and placed in a volume fraction of 5% CO 2 incubator. Incubate at 37 ° C in saturated humidity.
  • HK-2 cells grown to log phase were digested with 0.25% (w/v) trypsin + 0.02% EDTA and diluted to 5.0 ⁇ with DMEM-F12 medium containing 10% (v/v) fetal bovine serum.
  • 10 4 /mL cell suspension was plated in a 96-well plate at 200 ⁇ L per well. After 24 hours of culture, the cells were grown into a single layer, the original medium was discarded, washed twice with 0.01 M PBS, and the serum-free medium was replaced to synchronize. After 24 hours of culture, the serum-free medium was discarded, and 200 ⁇ L of different concentrations were added.
  • Figures 32A and 32B show that the inhibition of HK-2 cell proliferation by the compounds of the present invention is significantly reduced at a concentration of 30.00 ⁇ M compared to AD.
  • Tables 1A, 1B and Figures 33A, 33B show that the compound of the present invention can significantly inhibit TGF- ⁇ 1-induced conversion of human proximal tubular epithelial cells HK-2 to mesenchymal cells at a non-toxic concentration, and with AD In contrast, HK-2 cells have a stronger inhibitory effect on mesenchymal transition and a higher safety index.
  • Table 1A Compounds of the present invention inhibit TGF- ⁇ 1-induced conversion of human proximal tubular epithelial cells HK-2 to mesenchymal cells
  • test concentration is 0.08-1.25 ⁇ M
  • control there is interaction between epithelial cells, the tissue structure is tight, the cells are typical paving stones
  • TGF- ⁇ 1 treatment epithelial cells lose their typical state, the interaction between cells disappears
  • the tissue structure is relatively loose, the cell density is reduced, and the cubic pavement stone-like epithelial cells are transformed into the shape of spindle-shaped fibroblasts
  • extremely strong (inhibition) the cells are almost the same as the control, and the spindle is rarely seen in the field of view.
  • test concentration is 0.08-1.25 ⁇ M
  • control there is interaction between epithelial cells, the tissue structure is tight, the cells are typical paving stones
  • TGF- ⁇ 1 treatment epithelial cells lose their typical state, the interaction between cells disappears
  • the tissue structure is relatively loose, the cell density is reduced, and the cubic pavement stone-like epithelial cells are transformed into the shape of spindle-shaped fibroblasts
  • extremely strong (inhibition) the cells are almost the same as the control, and the spindle is rarely seen in the field of view.
  • Example 13 The compound of the present invention significantly reduced the degree of renal fibrosis induced by unilateral ureteral ligation in SD rats
  • UUO Unilateral ureteral ligation-induced renal fibrosis model
  • SD rats were randomly divided into groups according to body weight: sham operation group, model group, AD control group (0.15 mmol/kg), H compound administration group (0.06 mmol/kg), and H compound administration group ( There were 6 groups of 0.10 mmol/kg) and H compound administration group (0.15 mmol/kg), 4 in each group.
  • Preoperative preparation and anesthesia were the same as in Example 4. After anesthesia, the rats in the left lateral position were fixed, the lower edge of the sternum was shaved to the hind limbs to see the hair, and the surgical cloth and the shaved skin were placed.
  • the results show that on the UUO model, the representative compound H of the present invention significantly reduces the degree of renal fibrosis, improves the structure of the diseased kidney, and has a significantly stronger effect than AD.
  • Example 14 The compound of the present invention significantly improved the inflammatory state of renal tissue in renal fibrosis of SD rats induced by unilateral ureteral ligation
  • the results showed that the surface of the kidney tissue of the sham operation group was moist and lustrous, the glomerular structure was intact, the renal tubules were tight and compact, and no visible lesions were visible.
  • the kidney tissue was swollen and swollen, with a large amount of effusion in the middle and adhesion to the surrounding tissues.
  • the renal interstitial fibrosis material was used to compress the renal tubules. The tubules are severely atrophied and a large number of inflammatory cells infiltrate in the renal interstitium.
  • the renal tissue damage of the animals in the drug-administered group was improved to some extent, especially in the high-dose treatment group of the compound H of the present invention.
  • the surface of the kidney tissue was smooth and moist, non-adhesive, and the kidney was slightly inflated. See effusion, glomerular structure is basically intact, tubular atrophy is not obvious, its efficacy is significantly better than AD treatment group.
  • Example 15 The compound of the present invention inhibits angiotensin II (AngII)-induced proliferation of human primary myocardial fibrosis cells HCFB
  • cardiac fibroblasts are the main effector cells of myocardial fibrosis, and they proliferate when stimulated by active substances such as AngII, and their phenotypes are transformed into myofibroblasts that secrete extracellular matrix. Therefore, the cell inhibition of AngII after stimulation of primary cardiac fibroblast HCFB was examined by MTT assay. The compound H of the present invention was evaluated for anti-myocardial fibrosis.
  • HCFB human primary cardiac fibroblast HCFB (supplied by Mall North Natron Biotech Co., Ltd.).
  • the HCFB cells were cultured in a culture flask containing H-DMEM culture medium containing 10% fetal bovine serum (GIBCO, USA: Cat. No. 302220F), 100 ⁇ g/mL streptomycin, 100 IU/mL penicillin, and placed in a culture flask.
  • the volume fraction was 5% CO 2 incubator (Binder, Germany) and cultured at 37 ° C under saturated humidity.
  • the HCFB cells grown in log phase were digested with 0.25% trypsin + 0.02% EDTA, and diluted into 5.0 ⁇ 10 4 /mL cell suspension in H-DMEM medium containing 10% fetal bovine serum.
  • Orifice plate (Costar, USA), 200 ⁇ L/well, cultured at 37 ° C, volume fraction 5% CO 2 , saturated humidity incubator for 24 h, added medium containing different concentrations of compound AD or H, continue to culture for 48 h, add MTT (5 mg/mL), 20 ⁇ L/well, cultured for 4 h, the supernatant was discarded, 150 ⁇ L of DMSO was added, and the mixture was shaken for 10 min, and the absorbance was measured by a microplate reader.
  • the measurement wavelength was 570 nm and the reference wavelength was 450 nm.
  • HCFB cells in logarithmic growth phase were digested with 0.25% trypsin + 0.02% EDTA, and diluted into 5.0 ⁇ 10 4 /mL cell suspension in H-DMEM medium containing 10% fetal bovine serum. Inside the well plate, 200 ⁇ L per well. After culture for 24 hours, the cells were grown into a single layer, the original medium was discarded, washed twice with 0.01 M PBS, and the serum-free medium was replaced to synchronize. After 24 hours of culture, the serum-free medium was discarded, and 200 ⁇ L of different concentrations were added. The compound and the stimulating factor AngII (10 -7 mol/L) in H-DMEM medium were measured.
  • H-DMEM medium containing 0.5% DMSO was used as a negative control
  • H-DMEM medium containing stimulating factor AngII (10 -7 mol/L) and 0.5% DMSO was used as a positive control.
  • Cell viability was measured by MTT assay after 48 hours of culture. The results are shown in Figure 41. Data were processed and analyzed using SPSS 17.0 statistical software. Data were average ⁇ standard deviation To show; P ⁇ 0.05 difference between the groups was significant.
  • Figure 40 shows that the compound H of the present invention showed no significant inhibitory effect on the proliferation of human primary cardiac fibroblast HCFB cells at a concentration of 15.0 ⁇ M.
  • Figure 41 shows that the compound H of the present invention can significantly inhibit the proliferation of HCFB by AngII at a non-toxic concentration, and has a stronger inhibition effect on human HCFB proliferation and a higher safety index than AD.

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Abstract

本发明属于医药技术领域，涉及15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物及其抗纤维化用途。经实验证明，该类化合物显著抑制肝星状细胞LX-2的迁移、活化。显著降低肝纤维化大鼠肝组织纤维化水平，降低细胞外基质蛋白(ECM)相关组分含量，并有效抑制免疫炎症反应；抑制肝组织中肝星状细胞活化，促进胶原降解。显著抑制TGF-β1诱导的人肺泡Ⅱ型样细胞A549和肾上皮细胞HK-2向间充质细胞转化(EMT)；显著降低博来霉素诱导的小鼠肺纤维化程度和单侧输尿管结扎诱导的大鼠肾纤维化程度；显著抑制血管紧张素Ⅱ(AngⅡ)诱导的人原代心肌成纤维细胞HCFB增殖。将该类化合物作为活性成份用于制备抗纤维化药物，高效低毒，为纤维化相关疾病的治疗和预防提供了新的药物途径，从而扩大了临床用药的可选择范围，具有良好的应用开发前景。

Description

15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物及其在制备抗纤维化药物中的应用 技术领域
本发明涉及穿心莲内酯衍生物及其作为抗纤维化药物的应用，具体涉及15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物，属医药技术领域。
背景技术
组织纤维化是一种慢性疾病，多发于肝、肺、心脏、肾脏等部位。全球有1/3的人死于组织纤维化以及由此产生的器官衰竭。当组织受到损伤后，损伤部位发生一系列细胞反应，导致细胞外基质过度沉积，发生组织纤维化。最终可导致器官功能障碍，甚至死亡。在纤维化疾病的治疗中，临床上虽然有干扰素、磷脂酰胆碱、维拉帕米、糖皮质激素等药物，但是一般会伴随一定的不良反应，因此以中草药为基础的抗纤维化药物研究与开发市场前景广阔。
慢性肝病(chronic liver disease)是严重危害人类健康的一大类疾病。肝纤维化是多种慢性肝病向肝硬化发展的共同病理学过程。一般是因为机体产生创伤修复反应而造成肝脏细胞外基质含量上升过量沉积所致。引起肝纤维化的因素包括病毒性肝炎、代谢性疾病、自身免疫性肝病和乙醇、药物等引起的慢性肝病。根据现有的报道，广泛认为，肝纤维化过程是可逆的(余慧,江峰,医学信息,2015.28(7):334-335)，可通过处理原发病、抑制炎症活动、抗氧化还原反应、降低肝星状细胞活性及上皮间质转化等途径来治疗肝纤维化，但肝纤维化发生机制复杂，调节机制又受多种因素影响。
近年来肺纤维化发病率和致死率呈不断上升趋势。肺纤维化(pulmonary fibrosis，PF)是许多肺部疾病发展、演变、疤痕化的最终结局，其病因多种多样。许多慢性肺疾病，包括哮喘、支气管扩张、慢阻肺、肺结核、肺癌、间质性肺病等，都伴有纤维化病理改变。其病理特点是肺部弥漫性炎症所致肺泡持续性损伤及细胞外基质的反复破坏、修复、重建和过度沉积(李先伟等,中国应用生理学杂 志,2017.33(1):16-21)。对于特发性肺纤维化、呼吸窘迫综合征、嗜酸性肉芽肿等多种肺疾病而言，肺组织纤维增生和纤维化程度决定了该疾病的临床愈后情况。一般肺脏早期损伤之后有肺泡炎，炎症和异常修复导致肺间质细胞增殖，逐渐形成纤维化肺，临床表现为患者呼吸困难、呼吸衰竭。在过去的20年间，特发性肺纤维化发病率总体呈现明显增长的趋势，诊断后平均存活时间约3年，5年生存率30％～50％，愈后极差。
肾纤维化是由于受到创伤、感染、炎症、血循环障碍，以及免疫反应等多种致病因素刺激，其固有细胞受损，发展到后期出现大量胶原沉积和积聚，造成肾实质逐渐硬化，形成瘢痕，直至肾脏完全丧失脏器功能。肾脏内固有细胞纤维化、硬化的过程也就是肾脏纤维化的过程。许多急慢性肾脏疾病也都与组织纤维化发生发展密切相关，特别是糖尿病肾病和高血压引起的肾纤维化改变。
心肌纤维化(myocardial fibrosis，MF)是在现代医学研究基础上提出的病理名称，指在病理状态下心肌间质心肌成纤维细胞(cardiac fibroblasts，CFs)增殖，细胞外基质(extracellular matrix，ECM)(主要是Ⅰ型和Ⅲ型胶原纤维)沉积异常增加，导致心室顺应性下降，影响心脏的正常收缩与舒张功能。MF是多种心脏疾病发展至一定阶段具有的共同病理改变，也是引起心室重塑的关键原因，近年来大量研究表明，心肌纤维化与许多心脏疾病有密切关系，如心房纤颤、心肌梗死、慢性心力衰竭、风湿性心瓣膜病等。常由风湿性心脏病、高血压、心肌梗死及心力衰竭等疾病诱发，在增龄和冠状动脉粥样硬化过程中也会发生MF，主要病理表现有心肌僵硬度增加，心肌收缩力下降，冠脉血流储备降低，甚至引起恶性心律失常和猝死。
目前研究较多的与组织、器官纤维化发生发展相关的信号通路包括TGF-β 1/Smad信号通路、Wnt/β-catenin信号通路、磷脂酰肌醇-3激酶/蛋白激酶(PI3K/Akt)信号通路等。例如，黄柏内酯通过对小鼠纤维化肺组织中TGF-β信号通路的抑制，下调α-SMA和胶原蛋白的生成(CN 105560255 A)。甲蟛蟆菊内酯—硫酸酯可显著抑制TGF-β诱导的人胚肺成纤维细胞的增殖及其羟脯氨酸(HYP)含量从而发挥改善肺纤维化程度的药理作用(CN 103919769 A)。作用于TGF-β下游的PI3K抑制剂可用于治疗肺纤维化疾病(CN 104093408 A)。还有利用土木香中的倍半菇一异土木香内酯类衍生物及其盐治疗肺纤维化(CN 106496243 A) 的专利报道。在抗肾纤维化方面，灵芝内酯D可明显抑制TGF-β1诱导的肾小管上皮细胞Smad3磷酸化，对于肾脏纤维化具有治疗作用(CN 106220643 A)。中国人民解放军第三军医大学第三附属医院发现芹菜素通过TRPV4介导Ca 2+的内流，激活AMPK/SITR1信号通路，抑制肾脏纤维化。
目前，临床上虽然有一些治疗上述纤维化疾病的药物，但是药物效果尚不够理想。穿心莲内酯(andrographolide)，作为爵床科Acanthaceae植物穿心莲Andrographis paniculata(Burm.f.)Nees的主要二萜内酯类活性成分之一，临床上主要用于清热解毒、抗菌消炎，在抗纤维化方面也有明显的效果，可以通过抑制NF-кB炎症通路来阻断纤维化的早期途径(CN106831669A)。另外，现代药理研究表明，穿心莲内酯及其衍生物具有抗炎、抗病毒、抗肿瘤、保肝以及镇痛等活性，临床应用广泛且疗效显著(骆瑜等,中国中药杂志,2017.42(20):3847-3859)。
穿心莲内酯对多种动物实验性肝损伤具有良好的保护作用。姚青等发现穿心莲内酯对可卡因引起的急性肝脏损害有一定的保护作用，其保肝机制可能与抑制脂质过氧化反应，降低组织中氧自由基的生成有关。Visen PK[J Ethnopharmacol,1993,40(2),131-136]与Handa S[Indian J Med Res,1990,92:284-292]皆证明穿心莲内酯对扑热息痛诱导的肝损伤具有保护作用，其中Handa S的研究还显示穿心莲内酯对半乳糖胺引起的肝中毒具有保护作用。Kapil A[Biochem Pharmacol,1993,46(1):182-185]等证明了穿心莲内酯、穿心莲内酯甙和新穿心莲内酯对四氯化碳和叔丁基过氧化氢引起的肝中毒具有保护作用。Singha P[J Ethnopharmacol,2007,111(1):13-21]等的研究显示穿心莲内酯对乙醇引起的小鼠肝肾损伤具有一定的保护作用。Roy DN[Toxicol Appl Pharmacol,2011；250(1):54-68]等的研究证明穿心莲内酯与D-青霉胺联合治疗铜中毒比单一D-青霉胺在抗纤维化及细胞坏死方面效果更佳。宁光等在其申请的专利(CN201010266185.2)中公开了穿心莲内酯作为制备治疗急性肝损伤药物的应用，穿心莲内酯可以显著抑制刀豆素A诱发的肝损伤，抑制刀豆素A引起的肝细胞的凋亡，抑制肝脏的炎症反应。因此，可以用来治疗刀豆素A诱发的肝损伤。黄成亮等发现穿心莲内酯可减轻博来霉素或者平阳霉素致肺纤维化大鼠肺泡炎和纤维化程度，降低肺组织PDGF表达和减少HYP含量；范贤明等研究发现穿心莲内酯可减轻博来霉素致肺纤维化大鼠肺泡炎和肺纤维化程度,降低肺组织I、III型胶原mRNA表达,降低支气管 肺泡灌洗液(BALF)中TNF-α,TGF-β1浓度,且对肝、肾无明显毒副作用。孙景辉等研究发现穿心莲内酯注射液减轻病毒性心肌炎小鼠炎症和纤维化程度，机制可能是能抑制心肌中ST2的表达。聂梦琪等研究发现穿心莲内酯具有一定的抗肾间质纤维化作用，其机制可能与抗炎、抗氧化、降低TGF-β1及IGFBP-3的表达有关。钟星明等研究证明了穿心莲内酯可通过提高钠钾ATP酶和钙镁ATP酶活性，降低羟脯氨酸含量，从而抑制心脏纤维化，起抗心肌肥厚作用。Singha P等研究显示穿心莲内酯对乙醇引起的小鼠肝肾损伤具有一定的保护作用。Roy DN等研究证明穿心莲内酯与D-青霉胺联合治疗铜中毒比单一D-青霉胺在抗纤维化及细胞坏死方面效果更佳。
本发明人在前期研究中(CN200510107247.4、CN200710053807.1、CN200710053806.7、CN200610017357.6、CN201210358667X)获得了大量结构新颖的穿心莲内酯衍生物，并对部分衍生物在抗肿瘤、抗炎、抗HBV、HCV及急性肝损伤保护作用等应用申请了专利保护，本发明进一步对15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物在抗组织(器官)纤维化方面进行了活性实验研究，目前未见将其作为活性成份用于制备抗纤维化药物的相关报道。
发明内容
本发明人在前期研究成果的基础上，通过对所合成化合物的抗肝、肺、肾和心肌纤维化活性筛选，发现通式1所示结构的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物具有显著的预防和治疗人体组织器官纤维化作用，高效低毒，具有开发为抗纤维化药物的潜力。为此，本发明目的在于提供15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物及其在制备抗纤维化药物中的应用。
所述结构如通式1所示的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物。
Figure PCTCN2018080776-appb-000001
Figure PCTCN2018080776-appb-000002
R 1，R 2各自为氢或C 1-5烷基；R 1，R 2各自为氢或苯基、2-甲氧基苯基、3-甲氧基苯基、4-甲氧基苯基、2，3，5-三甲氧基苯基、2-羟基苯基、3-羟基苯基、4-羟基苯基、2-氟苯基、2-氯苯基、2-溴苯基、3-氟苯基、3-氯苯基、3-溴苯基、4-氟苯基、4-氯苯基、4-溴苯基、2-氟-3-甲氧基苯基、3-甲氧基-4-氯苯基、2，4-二氟苯基、2，4-二氯苯基、2，4-二溴苯基、2-氟-4-氯苯基、2-溴-4-氯苯基、3-氟-4-氯苯基、3-溴-4-氯苯基、3，4-二氟苯基、3，4-二氯苯基、3，4二溴苯基、2-氯-4-氟苯基、2-溴-4-氟苯基、3-氯-4-氟苯基、3-溴-4-氟苯基、2-氟-4-溴苯基、2-氯-4-溴苯基、3-氟-4-溴苯基、3-氯-4-溴苯基、2，3，4-三氯苯基、2-甲氧基-4-氯苯基、2-羟基-4-氯苯基、2-羟基-4-甲氧基苯基、3-氨基-4-氯苯基、2-氨基-4-氯苯基，2-硝基-4-氟苯基，2-硝基-4-氯苯基，或被C 1-5烷基取代的苯基、被卤素和吗啉基取代的苯基、被卤素和哌嗪基取代的苯基、N，N-二烷基胺基苯基；或吡啶基、呋喃基、噻吩基、吡咯基、吲哚基或被卤素取代的吡啶基、呋喃基、噻吩基、吡咯基、吲哚基；或苯并呋喃基、苯并咪唑基、苯并噻喃基、苯并噻唑基、苯并四唑基、苯并异唑基、苯并异噻唑基、苯并咪唑啉基、苯并吡唑基、苯并三唑基、苯并噻吩基、苯并恶唑基；或R 1和R 2相连成环己基；R 1，R 2同时相同或不同但不同时为氢；R 3、R 4各自为氢或R 3、R 4分别为甲基磺酰基、三苯甲基、3-吡啶基或R 3、R 4分别为CH 2CH 2COOH或CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种，或R 3、R 4各自为COR 5，R 5为3-吡啶基或CH 2CH 2COOH或CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种。R 3、R 4选相同或不同的取代基团。
优选：当R 1，R 2其中之一为氢时，R 1，R 2其中之一选如下基团：甲基、乙基或丙基、苯基、2-甲氧基苯基、3-甲氧基苯基、4-甲氧基苯基、2-氟苯基、2-氯苯基、2-溴苯基、3-氟苯基、3-氯苯基、3-溴苯基、4-氟苯基、4-氯苯基、4-溴苯基、2-氟-3-甲氧基苯基、3-甲氧基-4-氯苯基、2，4-二氟苯基、2，4-二氯苯基、2，4-二溴苯基、2-氟-4-氯苯基、2-溴-4-氯苯基、3-氟-4-氯苯基、3-溴-4-氯苯基、3，4-二氟苯基、3，4-二氯苯基、3，4-二溴苯基、2-氯-4-氟苯基、2-溴-4-氟苯基、3-氯-4-氟苯基、3-溴-4-氟苯基、2-氟-4-溴苯基、2-氯-4-溴苯基、3-氟-4- 溴苯基、3-氯-4-溴苯基、2-甲氧基-4-氯苯基4-正丙基苯基、4-异丙基苯基、4-异丁基苯基、4-(N,N-二甲胺基)苯基、3-氟-4-(4-吗啉基)苯基、3-氟-4-(4-甲基哌嗪基)苯基、2-呋喃基、2-吡咯基、6-氯-3-吲哚基、3-吲哚基、5-氯-3-吲哚基、6-氯-2-吡啶基、3-吡啶基；或R 1和R 2相连成环己基；R 3、R 4各自为氢或R 3、R 4分别为CH 2CH 2COOH或CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种或者R 3、R 4各自为COR 5，R 5为3-吡啶基或CH 2CH 2COOH，R 3、R 4选相同取代基团。
优选：当R 1，R 2其中之一为氢时，R 1，R 2其中之一选如下基团：苯基、2-甲氧基苯基、3-甲氧基苯基、4-甲氧基苯基、2-氟苯基、2-氯苯基、2-溴苯基、3-氟苯基、3-氯苯基、3-溴苯基、4-氟苯基、4-氯苯基、4-溴苯基、2-氟-3-甲氧基苯基、3-甲氧基-4-氯苯基、2，4-二氟苯基、2，4-二氯苯基、2，4-二溴苯基、2-氟-4-氯苯基、2-溴-4-氯苯基、3-氟-4-氯苯基、3-溴-4-氯苯基、3，4-二氟苯基、3，4-二氯苯基、3，4二溴苯基、2-氯-4-氟苯基、2-溴-4-氟苯基、3-氯-4-氟苯基、3-溴-4-氟苯基、2-氟-4-溴苯基、2-氯-4-溴苯基、3-氟-4-溴苯基、3-氯-4-溴苯基、2-甲氧基-4-氯苯基，；R 3、R 4各自为氢或R 3、R 4分别为CH 2CH 2COOH或CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种或者R 3、R 4各自为COR 5，R 5为CH 2CH 2COOH，R 3、R 4选相同取代基团。
优选：当R 1，R 2其中之一为氢时，R 1，R 2其中之一选如下基团：甲基、乙基或丙基、2-呋喃基、2-吡咯基、6-氯-3-吲哚基、3-吲哚基、5-氯-3-吲哚基、6-氯-2-吡啶基、3-吡啶基、4-正丙基苯基、4-异丙基苯基、4-异丁基苯基、3-氟-4-(4-甲基哌嗪基)苯基；或R 1和R 2相连成环己基；R 3、R 4各自为氢。
更优选如下化合物：
A：R 1＝H，R 2＝C 6H 5，R 3＝R 4＝H；
B：R 1＝H，R 2＝2-F-C 6H 4，R 3＝R 4＝H；
C：R 1＝H，R 2＝2-Cl-C 6H 4，R 3＝R 4＝H；
D：R 1＝H，R 2＝2-Br-C 6H 4，R 3＝R 4＝H；
E：R 1＝H，R 2＝3-F-C 6H 4，R 3＝R 4＝H；
F：R 1＝H，R 2＝3-Cl-C 6H 4，R 3＝R 4＝H；
G：R 1＝H，R 2＝3-Br-C 6H 4，R 3＝R 4＝H；
H：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝H；
I：R 1＝H，R 2＝4-F-C 6H 4，R 3＝R 4＝H；
J：R 1＝H，R 2＝4-Br-C 6H 4，R 3＝R 4＝H；
K：R 1＝H，R 2＝4-CH 3O-C 6H 4，R 3＝R 4＝H；
L：R 1＝H，R 2＝2-CH 3O-4-Cl-C 6H 3，R 3＝R 4＝H；
M：R 2＝H，R 1＝2-Br-C 6H 4，R 3＝R 4＝H；
N：R 2＝H，R 1＝3-Cl-C 6H 4，R 3＝R 4＝H；
O：R 2＝H，R 1＝2-F-4-Cl-C 6H 3，R 3＝R 4＝H；
P：R 2＝H，R 1＝2,4-diCl-C 6H 3，R 3＝R 4＝H；
Q：R 2＝H，R 1＝4-F-C 6H 4，R 3＝R 4＝H；
R：R 2＝H，R 1＝C 6H 5，R 3＝R 4＝H；
S：R 1＝H，R 2＝3-F-4-Cl-C 6H 3，R 3＝R 4＝H；
T：R 1＝H，R 2＝2,4-diF-C 6H 3，R 3＝R 4＝H；
U：R 1＝H，R 2＝3,4-diCl-C 6H 3，R 3＝R 4＝H；
V：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝3-吡啶基；
W：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝CH 2CH 2COOH；
X：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝CH 2CH 2COOH；
Y:R 2＝H，R 1＝4-Cl-C 6H 4，R 3＝R 4＝H；
Z:R 2＝H，R 1＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝3-吡啶基。
a.R 1＝CH 3，R 2＝CH 3，R 3＝R 4＝H；
b.15-环己亚基-14-脱氧-11,12-脱氢-穿心莲内酯；R 3＝R 4＝H；
c.R 1＝H，R 2＝3-F-4-(4-甲基哌嗪基)-C 6H 3，R 3＝R 4＝H；
d.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000003
R 3＝R 4＝H；
e.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000004
R 3＝R 4＝H；
f.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000005
R 3＝R 4＝H；
g.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000006
R 3＝R 4＝H；
h.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000007
R 3＝R 4＝H；
i.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000008
R 3＝R 4＝H；
j.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000009
R 3＝R 4＝H；
k.R 1＝H，R 2＝
Figure PCTCN2018080776-appb-000010
R 3＝R 4＝H；
本发明提出的上述化合物其制备方法已在发明专利CN:200510107247.4中公开。
a:C 23H 32O 4； 1H NMR(400MHz,DMSO-d6)δ7.98(s,1H),6.52(dd,J＝15.7,10.6Hz,1H),6.21(d,J＝15.7Hz,1H),5.50(s,1H),5.12(s,1H),4.30(s,1H),3.94(d,J＝10.9Hz,1H),3.40(d,J＝10.9Hz,1H),3.23(dd,J＝10.9,4.1Hz,1H),2.48(d,J＝10.8Hz,1H),2.01(s,2H),1.95(s,3H),1.94(s，3H)1.68–1.48(m,3H),1.46(s,3H),1.26(dd,J＝10.6,6.2Hz,1H),1.17(td,J＝13.0,3.5Hz,1H),1.08(s,3H),0.80(s,3H). 13C NMR(100MHz,DMSO-d6)δ169.06,144.93,137.67,132.91,132.57,126.13,123.18,122.65,122.32,79.37,62.99,60.16,49.98,42.11,38.28,36.11,27.82,23.71,23.22,22.68,18.84,18.80,15.99.HRMS(ESI):m/z calcd for C23H32NaO4[M+Na] +,395.2198；found,395.2196.
b:C 26H 36O 4； 1H NMR(400MHz,DMSO-d6)δ8.02(s,1H),6.52(dd,J＝15.7,10.6Hz,1H),6.21(d,J＝15.7Hz,1H),5.50(s,1H),5.12(s,1H),4.30(s,1H),3.94(d,J＝10.9Hz,1H),3.40(d,J＝11.1Hz,1H),3.22(dd,J＝10.9,4.1Hz,1H),2.48(d,J＝11.0Hz,1H),2.45–2.36(m,4H),2.01(s,2H),1.59(s,6H),1.57–1.47(m,3H),1.46(s,3H),1.26(dd,J＝10.6,6.3Hz,1H),1.17(td,J＝13.1,3.7Hz,1H),1.08(s,3H),0.79(s,3H). 13C NMR(100MHz,DMSO-d6)δ169.15,142.54,137.70,132.59,132.57,130.84,126.33,122.65,122.33,79.37,62.99,60.16,49.97,42.11,38.28,36.11,28.94,28.72,28.16,27.82,27.49,26.08,23.71,23.22,22.67,15.99.HRMS (ESI):m/z calcd for C26H36NaO4[M+Na] +,435.2511；found,435.2516.
c:C 32H 41FN 2O 4； 1H NMR(400MHz,DMSO-d6)δ7.69(s,1H),7.52(dd,J＝14.9,1.7Hz,1H),7.46(dd,J＝8.6,1.5Hz,1H),7.06(t,J＝9.0Hz,1H),6.58(dd,J＝15.7,10.6Hz,1H),6.29(d,J＝15.7Hz,1H),6.26(s,1H),5.50(s,1H),5.12(d,J＝4.9Hz,1H),4.31(d,J＝5.0Hz,1H),3.94(d,J＝9.5Hz,1H),3.45–3.39(m,1H),3.22(dd,J＝10.4,4.9Hz,1H),3.14–3.06(m,4H),2.51(s,1H),2.49–2.44(m,4H),2.22(s,3H),2.01(s,2H),1.64–1.49(m,3H),1.47(s,3H),1.25(dd,J＝10.5,6.3Hz,1H),1.22–1.13(m,1H),1.08(s,3H),0.80(s,3H). 13C NMR(100MHz,DMSO-d6)δ168.93,154.54(d,J＝243Hz),147.10,140.65(d,J＝9Hz),138.68,137.17,132.47,127.89(d,J＝2Hz),127.33(d,J＝8Hz),125.61,122.75,122.28,119.53,119.49,117.58,117.36,112.47(d,J＝2Hz),79.37,62.99,60.24,54.94,49.95,49.91,46.20,42.12,38.31,36.21,27.82,23.71,23.22,22.70,16.02.HRMS(ESI):m/z calcd for C32H42FN2O4[M+H] +,537.3129；found,537.3129.
d:C 26H 30ClNO 4； 1H NMR(400MHz,DMSO)δ7.96(d,J＝1.6Hz,1H),7.95(s,1H),7.82(s,1H),7.48–7.42(m,1H),6.89(dd,J＝15.8,10.2Hz,1H),6.33(s,1H),6.31(d,J＝16.8Hz,1H),5.05(d,J＝4.5Hz,1H),4.76(s,1H),4.44(s,1H),4.14(d,J＝4.9Hz,1H),3.85(d,J＝10.8Hz,1H),3.32–3.27(m,1H),3.27–3.20(m,1H),2.47(d,J＝10.1Hz,1H),2.38(d,J＝13.6Hz,1H),1.99(dd,J＝12.9,8.4Hz,1H),1.74(d,J＝13.2Hz,1H),1.61(dd,J＝25.5,7.3Hz,2H),1.49–1.30(m,2H),1.24–1.14(m,2H),1.10(s,3H),0.80(s,3H). 13C NMR(101MHz,DMSO)δ168.00,152.75,150.45,148.71,140.47,136.64,128.01,124.09,110.84,78.59,62.64,60.74,53.68,42.39,36.22,27.63,23.15,23.00,15.46.
e:C 26H 31NO 4: 1H NMR(400MHz,DMSO)δ8.83(s,1H),8.52(d,J＝3.6Hz,1H),8.18–
8.164(m,1H),7.79(s,1H),7.48(m,4.8Hz,1H),6.87(dd,J＝16.7,9.2Hz,1H),6.39(s,1H),6.27(d,J＝16.6Hz,1H),5.05(s,1H),4.76(s,1H),4.45(s,1H),4.15(dd,J＝7.4,2.6Hz,1H),3.85(dd,J＝10.9,2.6Hz,1H),δ3.26(m,2H),2.44(d,J＝11.9Hz,1H),2.38(d,12Hz,1H),1.99(t,J＝11.0Hz,1H),1.74(d,J＝13.4Hz,1H),1.58–1.60(m,2H),1.45-1.3(m,2H),1.23-1.18(overlap,1H),1.10(s,3H),0.80(s,3H).
f:C 29H 32ClNO 4； 1H NMR(400MHz,DMSO)δ11.89(s,1H),7.95(s,1H),7.90(d,J＝8.5Hz,1H),7.69(s,1H),7.52(d,J＝1.8Hz,1H),7.17(dd,J＝8.5,1.9Hz,1H),6.77(dd,J＝15.7,10.1Hz,1H),6.70(s,1H),6.25(d,J＝15.7Hz,1H),5.03(d,J＝4.7Hz,1H),4.75(s,1H),4.46(s,1H),4.14(d,J＝4.6Hz,1H),3.86(d,J＝11.1Hz,1H),δ3.29–3.18(m,2H),2.40(t,J＝11.5Hz,2H),1.99(t,J＝10.9Hz,1H),1.74(d,J＝13.4Hz,1H),1.66–1.52(m,2H),1.50–1.30(m,2H),1.18(dd,J＝19.8,7.6Hz,2H).1.11(s,3H),0.79(s,3H).
g:C 29H 33NO 4； 1H NMR(400MHz,DMSO)δ11.84(s,1H),7.94(s,1H),7.89(d,J＝7.6Hz,1H),7.68(s,1H),7.48(d,J＝7.8Hz,1H),7.18(ddd,J＝14.8,13.8,6.6Hz,2H),6.82–6.74(m,1H),6.71(s,1H),6.24(d,J＝15.7Hz,1H),5.04(s,1H),4.75(s,1H),4.47(s,1H),4.15(s,1H),3.88(d,J＝10.9Hz,1H),δ3.33–3.20(m,2H),2.43–2.33(m,2H),1.99(dd,J＝13.0,8.4Hz,1H),1.72(t,J＝13.5Hz,1H),1.60(dd,J＝15.2,10.3Hz,2H),1.49–1.31(m,2H),1.17(dd,J＝18.2,8.9Hz,2H).1.11(s,3H),0.79(s,3H). 13C NMR(101MHz,DMSO)δ168.67,148.99,144.24,136.22,136.04,134.45,129.52,126.20,123.10,122.45,121.83,120.42,118.59,112.17,109.69,106.98,78.66,62.69, 60.86,53.78,42.37,36.26,27.68,23.16,22.99,15.48.
h:C 29H 32ClNO 4； 1H NMR(400MHz,DMSO)δ11.96(s,1H),8.00(d,J＝1.9Hz,1H),7.98(s,1H),7.66(s,1H),7.48(d,J＝8.6Hz,1H),7.20(dd,J＝8.6,2.0Hz,1H),6.83–6.76(m,1H),6.74(s,1H),6.25(d,J＝15.7Hz,1H),5.03(s,1H),4.75(s,1H),4.47(s,1H),4.13(s,1H),3.87(d,J＝10.9Hz,1H),δ3.27(d,J＝18.7Hz,2H),2.39(t,J＝10.1Hz,2H),2.00(t,J＝10.7Hz,1H),1.74(d,J＝12.9Hz,1H),1.60(dd,J＝15.6,10.5Hz,2H),1.41(m,J＝17.2,13.2,9.4Hz,2H),1.17(m,J＝20.4,7.6Hz,2H).1.11(s,3H),0.79(s,3H). 13C NMR(101MHz,DMSO)δ168.58,148.97,144.44,136.15,134.80,134.54,130.85,127.37,125.13,123.54,122.39,121.77,118.27,113.74,109.51,108.15,106.41,78.65,62.67,60.88,53.77,
i:C 25H 30O 5； 1H NMR(400MHz,DMSO)δ7.87(d,J＝1.3Hz,1H),7.70(s,1H),
6.93–6.88(m,1H),6.85–6.77(m,1H),6.69–6.66(m,1H),6.39(s,1H),6.29(dd,J＝23.3,15.8Hz,1H),5.04(d,J＝4.9Hz,1H),4.75(s,1H),4.44(s,1H),4.14(dd,J＝7.4,2.8Hz,1H),3.85(dd,J＝10.9,2.6Hz,1H),δ3.31–3.18(m,2H),2.41(dd,J＝21.6,11.7Hz,2H),1.98(d,J＝11.6Hz,1H),1.73(d,J＝12.9Hz,1H),1.66–1.52(m,2H),1.44(m,J＝14.0,10.3Hz,1H),1.35(m,J＝13.4,2.5Hz,1H),1.17(m,J＝21.2,8.6Hz,2H).1.10(s,3H),0.79(s,2H). 13C NMR(101MHz,DMSO)δ168.25,148.97,148.82,146.56,145.42,145.21,136.76,135.80,128.34,126.00,121.44,114.86,113.19,101.15,78.60,62.65,60.76,53.71,42.38,36.23,27.63,26.31,23.16,22.99,15.45.
j:C 25H 31NO 4； 1H NMR(400MHz,DMSO)δ11.17(s,1H),7.69(s,1H),7.10–6.97(m,1H),6.73(dd,J＝15.8,10.1Hz,1H),6.64(s,1H),6.25(s,1H),6.22(dd,J＝14.2,9.1Hz,2H),5.03(d,J＝4.8Hz,1H),4.75(s,1H),4.45(s,1H),4.13(dd,J＝7.3,2.5Hz,1H),3.86(dd,J＝10.9,2.1Hz,1H),δ3.25(ddd,J＝15.1,10.6,4.0Hz,2H),2.39(t,J＝11.8Hz,2H),1.99(dd,J＝13.1,8.5Hz,1H),1.73(d,J＝13.0Hz,1H),1.59(dd,J＝15.4,10.3Hz,2H),1.50–1.28(m,2H),1.25–1.12(m,2H).1.10(s,3H),0.78(s,3H). 13C NMR(101MHz,DMSO)δ168.58,148.98,143.12,136.08,134.72,126.18,123.37,123.25,121.66,114.79,110.78,108.14,104.83,78.64,62.67,60.80,53.76,48.57,42.38,36.25,27.65,23.16,23.00,15.47.
k:C 31H 40O 4；S 1H NMR(400MHz,DMSO)δ7.73(s,1H),7.68(d,J＝8.5Hz,2H),7.48(d,J＝8.5Hz,2H),6.83(dd,J＝15.8,10.1Hz,1H),6.31(s,1H),6.26(d,J＝15.8Hz,1H),5.05(d,J＝4.4Hz,1H),4.75(s,1H),4.45(s,1H),4.14(d,J＝5.2Hz,1H),3.86(d,J＝10.8Hz,1H),δ3.31–3.18(m,2H),2.41(dd,J＝20.9,11.9Hz,2H),1.99(t,J＝10.6Hz,1H),1.71(t,J＝16.9Hz,1H),1.67–1.51(m,2H),1.49–1.31(m,2H),1.29(s,9H),1.24–1.13(m,2H),1.10(s,3H),0.79(s,3H).
为研究所述化合物在制备抗纤维化药物中的应用效果，本发明人利用肝星状细胞LX-2，测定本发明化合物对细胞迁移和活化的抑制活性，评价化合物的抗肝纤维化活性；进一步以CCl 4诱导的SD大鼠肝纤维化模型、猪血清诱导的Wistar大鼠肝纤维化模型和胆管结扎(BDL)致SD大鼠肝纤维化模型为代表，通过测定肝组织胶原含量(Masson染色)、HSC活化标志分子α-SMA表达水平，评价化合物的抗肝纤维化活性。进一步，通过CCl 4模型，系统研究说明了代表性化 合物发挥抗肝纤维化作用的主要相关作用环节和机制。采用人肺泡Ⅱ型样细胞A549研究本发明化合物对TGF-β1诱导的A549细胞向间充质细胞转化的抑制活性，评价化合物的抗肺纤维化活性，进一步采用博来霉素诱导的小鼠肺纤维化模型，研究化合物的体内抗肺纤维化作用。利用近端肾小管上皮细胞HK-2评价本发明化合物对TGF-β1诱导的HK-2细胞向间充质细胞转化的抑制活性，评价化合物的抗肾纤维化活性，进一步采用单侧输尿管结扎诱导大鼠肾纤维化模型(UUO)研究本发明化合物的体内抗肾纤维化活性。采用AngⅡ刺激原代心肌成纤维细胞HCFB后检测细胞增殖率评价本发明化合物抗心肌纤维化作用。
该类化合物的顺、反结构均具有抗纤维化活性，以该类化合物为有效药用成份，或该类化合物的各类前药形式，单独或与其它药物组合，按目前各种常规的制药方法和工艺要求，与制药中可以接受的辅助和/或添加成份混合后，制成用于抗纤维化的口服型制剂、注射型制剂等各类药物剂型。优选将其制备治疗或预防肝、肺、肾、心脏等各类纤维化疾病的药物。口服型制剂为片剂、丸剂、胶囊、冲剂或糖浆等；注射型制剂包括注射液或冻干粉针剂型等。
本发明优点及创新点：通过活性筛选，确定上述化合物具有明确的抗器官和/或组织纤维化活性。经实验证明，与母体化合物穿心莲内酯(AD)相比，本发明化合物抗肝、肺、肾和/或心肌纤维化作用显著提高。因此，将该类化合物作为活性成份用于制备抗人体各类纤维化药物，为与纤维化相关疾病的治疗和预防提供了新的药物途径，从而扩大了临床用药的可选择范围，具有良好的应用开发前景。
附图说明
图1A为AD和本发明化合物(15.00μM)对人肝星状细胞LX-2活力的影响，与AD组比， #P＜0.05；
图1B为AD和本发明代表的化合物(30.00μM)对人肝星状细胞LX-2活力的影响，与AD组比， #P＜0.05；
图2A为AD和本发明化合物(5.00μM)抑制人肝星状细胞LX-2迁移的结果(统计结果)，与AD组比， #P＜0.05；
图2B为AD和本发明代表的化合物(1.00μM和5.00μM)抑制人肝星状细胞LX-2迁移的结果(统计结果)，与AD组比， #P＜0.05。
图3为AD和本发明化合物(5.00μM)抑制人肝星状细胞LX-2迁移的结果(部分显微图片；×100倍)；
图4为本发明部分代表性化合物对CCl 4诱导的肝纤维化SD大鼠肝组织纤维化程度的影响(相对胶原面积/％)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与水飞蓟宾组比， &P＜0.05；
图5为本发明部分代表性化合物对CCl 4诱导的肝纤维化SD大鼠肝组织纤维化程度的影响(部分Masson染色图片；×100倍)；
图6为本发明部分代表性化合物对CCl 4诱导的肝纤维化SD大鼠肝组织α-平滑肌肌动蛋白(α-SMA)表达水平的影响(统计结果)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与水飞蓟宾组比， &P＜0.05；
图7为本发明部分代表性化合物对CCl 4诱导的肝纤维化SD大鼠肝组织α-SMA表达水平的影响(部分免疫组化图片；×100倍)；
图8为本发明部分代表性化合物对猪血清诱导的肝纤维化Wistar大鼠肝组织纤维化程度的影响(相对胶原面积/％)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与水飞蓟宾组比， &P＜0.05；
图9为本发明部分代表性化合物对猪血清诱导的肝纤维化Wistar大鼠肝组织纤维化程度的影响(部分Masson染色图片；×100倍)；
图10为本发明部分代表性化合物对猪血清诱导的肝纤维化Wistar大鼠肝组织α-SMA表达水平的影响(统计结果)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与水飞蓟宾组比， &P＜0.05；
图11为本发明部分代表性化合物对猪血清诱导的肝纤维化Wistar大鼠肝组织α-SMA表达水平的影响(部分免疫组化图片；×100倍)；
图12为本发明部分代表性化合物对总胆管结扎致SD大鼠肝组织纤维化程度的影响(相对胶原面积/％)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与熊去氧胆酸组比， &P＜0.05；
图13为本发明部分代表性化合物对总胆管结扎致SD大鼠肝组织纤维化程度的影响(部分Masson染色图片；×100倍)；
图14为本发明部分代表性化合物对总胆管结扎致SD大鼠肝组织α-SMA表达水平的影响(统计结果)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与熊去 氧胆酸组比， &P＜0.05；
图15为本发明部分代表性化合物对总胆管结扎致SD大鼠肝组织α-SMA表达水平的影响(部分免疫组化图片；×100倍)；
图16为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中层粘连蛋白(Laminin，LN)水平的影响，与模型组比， *P＜0.05；与苦参素组比， &P＜0.05；
图17为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中I型胶原(C-Ⅰ)水平的影响，与模型组比， *P＜0.05；
图18为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中前III型胶原(PC-Ⅲ)水平的影响，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与苦参素组比， &P＜0.05；
图19为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中Ⅳ型胶原(C-Ⅳ)水平的影响，与模型组比， *P＜0.05；与苦参素组比， &P＜0.05；
图20为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中白介素-6(IL-6)水平的影响，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与苦参素组比， &P＜0.05；
图21为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠血清中肿瘤坏死因子-α(TNF-α)水平的影响，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与苦参素组比， &P＜0.05；
图22为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠肝组织中超氧化物歧化酶(SOD)水平的影响，与模型组比， *P＜0.05；
图23为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠肝组织中丙二醛(MDA)水平的影响,与模型组比， *P＜0.05；
图24为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠肝组织病理切片炎症级别的影响(统计结果)，与模型组比， *P＜0.05；与AD组比， #P＜0.05；与苦参素组比， &P＜0.05；
图25为本发明代表化合物H对CCl 4诱导的肝纤维化SD大鼠肝组织病理切片炎症级别的影响(H&E染色；×100倍)；
图26A.为AD和本发明化合物(30.00μM)对人肺泡Ⅱ型样细胞A549活力的影响，与AD组比， #P＜0.05；
图26B.为AD和本发明化合物对人肺泡Ⅱ型样细胞A549活力的影响，图中：f、g、h、k测试浓度为3.00μM，其余为30.00μM，与AD组比， #P＜0.05；
图27A.为AD和本发明化合物抑制TGF-β1诱导的人肺泡Ⅱ型样细胞A549向间充质细胞转化作用(统计结果)，化合物AD、N、P、Q、S-W和Z的低、高浓度分别为0.63μM和1.25μM，化合物C、E-G、I-L、O、R、X和Y的低、高浓度分别为0.31μM和0.63μM，化合物A、B、D、H和M的低、高浓度分别为0.16μM和0.31μM；与加TGF-β对照组比， #P＜0.05；与AD对照组比， *P＜0.05；图27B为AD和本发明代表的化合物抑制TGF-β1诱导的人肺泡Ⅱ型样细胞A549向间充质细胞转化作用(统计结果)，化合物AD-d、f、g的低、高浓度分别为0.63μM和1.25μM，e，h-k的低、高浓度分别为0.31μM和0.63μM，与加TGF-β1对照组比， #P＜0.05；与AD对照组比， *P＜0.05；
图28为本发明代表性化合物H对博来霉素诱导的KM小鼠肺肺组织纤维化程度的影响(部分Masson染色图片；×100倍)；
图29为本发明代表性化合物H对博来霉素诱导的KM小鼠肺组织纤维化程度的影响(胶原面积/％)，与模型组比， *P＜0.05， **P＜0.01；与AD组比， ##P＜0.01；
图30为本发明代表化合物H对博来霉素诱导的KM小鼠肺组织病理切片炎症级别的影响(H&E染色；×100倍)；
图31为本发明代表化合物H对博来霉素诱导的KM小鼠肺组织病理切片炎症级别的影响(统计结果)，与模型组比， **P＜0.01；与AD组比， ##P＜0.01。注：肺纤维化病理评级标准为：0级无肺泡炎症；1级肺泡炎面积在1/5以下；2级肺泡炎面积在1/5以上，1/2以上；3级肺泡炎面积在1/2以上；与模型组比， **P＜0.01；与AD组比， ##P＜0.01；
图32A为AD和本发明化合物对人近端肾小管上皮细胞HK-2活力的影响，化合物浓度为30.00μM，与AD组比， #P＜0.05；
图32B为AD和本发明代表的化合物对人近端肾小管上皮细胞HK-2活力的影响；化合物浓度为30.00μM，与AD组比， #P＜0.05；
图33A为AD和本发明化合物抑制TGF-β1诱导的人近端肾小管上皮细胞HK-2细胞向间充质细胞转化作用(部分显微图片；×100倍)，图中：1.对照；2.TGF-β1；3.TGF-β1+AD(0.63μM)；4.TGF-β1+T(0.63μM)；5.TGF-β1+A(0.63μM)； 6.TGF-β1+H(0.08μM)；7.TGF-β1+F(0.63μM)；8.TGF-β1+J(0.63μM)；9.TGF-β1+K(0.63μM)；10.TGF-β1+Z(0.63μM)；11.TGF-β1+Y(0.16μM)；
图33B为AD和本发明代表的化合物抑制TGF-β1诱导的人近端肾小管上皮细胞HK-2向间充质细胞转化作用(部分显微图片；×100倍)，图中：1.对照；2.TGF-β1；3.TGF-β1+AD(0.63μM)；4.TGF-β1+d(0.31μM)；5.TGF-β1+e(0.08μM)；6.TGF-β1+j(0.31μM)；7.TGF-β1+h(0.08μM)；8.TGF-β1+i(0.08μM)；9.TGF-β1+b(0.31μM)。
图34为本发明代表性化合物H对单侧输尿管结扎诱导的SD大鼠肾纤维化程度的影响(相对胶原面积/％)，与模型组比， *P＜0.05， **P＜0.01；与AD组比， ##P＜0.01；
图35为本发明代表性化合物H对单侧输尿管结扎诱导的SD大鼠肾纤维化程度的影响(部分Masson染色图片；×100倍)；
图36为本发明代表化合物H对单侧输尿管结扎诱导的SD大鼠肾纤维化肾组织大小的影响(部分给药结束后结扎侧肾组织解剖图片)；
图37为本发明代表化合物H对单侧输尿管结扎诱导的SD大鼠肾组织病理切片炎症级别的影响(统计结果)，与模型组比， *P＜0.05， **P＜0.01；与AD组比， #P＜0.05。注：肾间质纤维化病理分级标准为：1级为间质基本正常，轻度小管变性扩张；2级为间质纤维化，小管萎缩<20％,散在炎性细胞浸润；3级为间质纤维化，小管萎缩占30％，散在和(或)弥漫性炎性细胞浸润；四级为间质纤维化，小管萎缩>50％，散在和(或)弥漫性炎性细胞浸润。与模型组比， *P＜0.05， **P＜0.01；与AD组比， #P＜0.05；
图38为本发明代表化合物H对单侧输尿管结扎诱导的SD大鼠肾组织病理切片炎评分的影响(统计结果)，与模型组比， *P＜0.05， **P＜0.01；与AD组比， #P＜0.05，注：根据肾小管变性、坏死按无、轻、中、重度分别记0、1、2、3分。总体评分为0级：0分；1级：1～4分；2级：5～8分；3级：9～12分。与模型组比， *P＜0.05， **P＜0.01；与AD组比， #P＜0.05；
图39为本发明代表化合物H对单侧输尿管结扎诱导的SD大鼠肾组织病理切片炎症级别的影响(H&E染色；×100倍)；
图40为AD与本发明代表化合物H(0.3、3.0和15.0μM)对人原代心肌纤维化 细胞HCFB增殖作用的影响。
图41为AD(0.63μM)与本发明代表化合物H(0.08μM、0.16μM、0.31μM和0.63μM)抑制血管紧张素Ⅱ(AngⅡ)诱导的人原代心肌纤维化细胞HCFB增殖作用，与加AngII组比， *P＜0.05， **P＜0.01；与AD组比， #P＜0.05, ##P＜0.01。
具体实施方案
下面结合具体实施方案来阐述本发明。应理解这些实施方案仅用于说明本发明而不用于限制本发明的范围。本发明涉及的化合物不限于实施例中使用的代表性结构，可以更换15位的不同取代基，获得具有抗纤维化活性的化合物；可以用各种导致纤维化的原因作为研究对象来得出本发明化合物具有抗纤维化作用；也可以利用其他各种体内外研究方法(模型)来得出本发明化合物具有抗纤维化作用。
实施例1本发明化合物抑制人肝星状细胞LX-2迁移作用。
在各种炎症介质、生长因子等细胞因子的刺激下，肝星状细胞迁移到受损肝组织的炎症部位，进而增殖、活化，合成胶原等ECM成分是肝纤维化发生发展的关键。因此，采用划痕损伤法评价本发明化合物抗肝纤维化作用。
1细胞培养和药物处理
采用人肝星状细胞LX-2(由北京北纳创联生物技术研究院提供)，与穿心莲内酯(AD)比较，研究本发明化合物的体外抗肝纤维化作用。将LX-2细胞培养在含10％(V/V)胎牛血清、100μg/mL链霉素、100IU/mL青霉素RPMI1640培养液中，置体积分数5％CO2培养箱中于饱和湿度、37℃培养。
2MTT法测定细胞毒
将处于对数生长期的LX-2细胞用0.25％(W/V)胰蛋白酶消化后，用含10％(V/V)胎牛血清的RPMI1640培养基稀释成3.5×10 5/mL细胞悬液，铺于96孔板内，200μL/孔，于37℃，体积分数5％CO 2培养箱中培养24h，加入含不同浓度药物的培养基，药物终浓度最高为30.00μM,每个处理4孔重复。继续培养48h，加入MTT(5mg/mL)，20μL/孔，培养4h，弃上清，加入150μL DMSO，震荡l0min，用酶标仪测定吸光值。测定波长为570nm，参考波长为450nm。计算化合物作用后的细胞存活率，存活率(％)＝药物组A值/细胞对照组A值×100％，结果见附图1A和1B。数据利用SPSS 17.0统计学软件进行处理和分析。 数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000011
来表示；P﹤0.05组间差异有显著性意义。
3划痕损伤法观察药物对LX-2细胞迁移的影响
将生长对数期的LX-2细胞用0.25％胰蛋白酶消化后，用含体积分数10％胎牛血清的RPMI1640培养基稀释成1.0×10 6/mL细胞悬液，铺于96孔板内，每孔200μL。培养12h之后细胞长成融合状态，弃去原培养基，加入血清量为0.5％的培养基再同步化培养12h之后划线，用PBS洗两遍，加入200μL含待测化合物(5μM)的RPMI1640培养基后立即在显微镜下拍照。设3孔重复并且设置对照。培养24h后分别在显微镜下拍照测量。迁移抑制率＝1－(给药组0h划痕距离－24h划痕距离)/(空白组0h划痕距离－24h划痕距离)×100％。结果见附图2A、2B和3。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000012
来表示；P﹤0.05组间差异有显著性意义。
4实验结果
附图1A和1B结果表明，本发明化合物在15μM浓度下，对人肝星状细胞增殖未表现出明显的抑制作用，且存活率均显著高于母体化合物AD。
结合附图1A、1B、2A、2B和3，结果表明：与AD比，本发明化合物在无毒浓度下，可显著抑制LX-2细胞的迁移，且与AD比，对人肝星状细胞迁移的抑制作用更强，安全指数更高。
实施例2本发明化合物显著降低四氯化碳(CCl 4)诱导SD大鼠的肝纤维化程度
CCl 4诱导大鼠肝纤维化模型是一种经典的肝纤维化动物模型之一，其在形态学、病理生理学等诸多方面，都与人肝纤维化有极大的相似性，不仅具有毒物诱导的肝纤维化特征，而且还与乙肝病毒感染后的病理特征相似，能很好地模拟人类肝纤维化的病理变化。动物受低剂量的CCl 4长期刺激后，不但表现出的肝功异常与人肝硬化体征十分相似，而且纤维化发生分子机制、损伤后血清标志物、肝组织病理改变与人也非常相似。因此，CCl 4诱导肝纤维化模型现被广泛应用于研究肝纤维化发病机制、抗肝纤维化药物筛选、抗肝纤维化药物的作用机制等。
1材料与方法
1)实验动物
清洁级SD大鼠，雄性，体重200±20g，购于湖南斯莱克景达实验动物有限 公司。其许可证号是SCXK(湘)2011-0003。
2)药品、试剂及其配制
穿心莲内酯由四川什邡市金鑫生物科技有限公司生产(批号：120822)，纯度大于99％；本发明化合物由本发明人所在实验室合成，纯度大于99％；药用级低聚羧甲基纤维素钠(CMC-Na)由安徽山河药用辅料有限公司生产(批号：131114)；苦参素胶囊，产自正大天晴药业公司(批号：国药准字H20010763)。水飞蓟宾胶囊由天津天士力圣特制药有限公司(批号：国药准字H20040299)生产，药物用0.5％CMC-Na配成混悬液。CCl 4由天津凯基化学试剂公司生产，其它试剂均为市售分析纯。
2实验方法
SD大鼠适应性喂养3d，按体重随机分组，分出正常组后，其余大鼠进行背部皮下注射含40％CCl 4的大豆油造模，首次剂量为4mL/kg，其余剂量为2mL/kg，2次/w，正常组大鼠背部皮下注射相应体积的大豆油。造模4w后，除正常组外，其余大鼠按体重进行分组，8只/组。穿心莲内酯、苦参素、水飞蓟宾和本发明化合物A剂量均为20mg/kg，其他化合物剂量为与A相同的摩尔物质量；模型组和正常组动物给予等量的0.5％CMC-Na。第5w、6w造模和给药同时进行，第7w、8w停止造模，只给药。每次灌胃均在早上空腹时进行，每给药10d停药一天。大鼠在末次灌胃前8h更换垫料，严格禁食不禁水。给药后1h腹腔注射3％的戊巴比妥钠(2mL/kg)麻醉剂，采血后迅速完整地剖离肝脏。采集的血液在37℃孵育箱中静置45min后，4℃下3500rpm离心15min，取上层血清，分装备用。取大鼠左叶下半部分肝脏于10倍体积4％多聚甲醛固定液中固定，24h后更新固定液。固定充分后进行病理切片，经Masson三色染观察肝脏纤维化程度，Image-Pro Plus软件对Masson染色切片的拍照结果进行纤维化组织学半定量分析。计算相对胶原面积：(给药组平均面积－正常组平均面积)/(模型组平均面积－正常组平均面积)×100％，结果见附图4、5。利用免疫组织化学法评价肝组织α-SMA(HSC活化程度的标志物)表达情况，使用Image-Pro Plus对阳性表达进行量化分析，结果见附图6、7。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000013
来表示；P﹤0.05组间差异有显著性意义。
3实验结果
结合附图4、5、6、7，结果表明：本发明化合物能显著降低动物肝组织的纤维化程度。与AD比，本发明化合物治疗组肝组织的胶原面积显著减少。与阳性参照水飞蓟宾和苦参素比，疗效更突出。同时观察到本发明化合物显著下调肝组织中α-SMA的表达水平，与AD比，差异显著，且效果优于阳性药。说明其发挥抗肝纤维化作用与抑制肝星状细胞活化有关。
实施例3本发明化合物显著降低猪血清诱导Wistar大鼠的肝纤维化程度
免疫性肝纤维化主要是由肝脏自身出现免疫反应所引起的疾病，而且其他因素(病毒、酒精、血吸虫及某些化学物质等)所致肝纤维化也常伴随不同程度的免疫反应。对实验动物反复腹腔注射异种血清或蛋白(猪血清g、血吸虫血清、人或牛血清白蛋白等)是目前主要的造模方法之一。该法的独特之处在于异种血清主要通过MHCⅡ分子和炎症因子激活肝干细胞产生免疫应答导致的肝脏损伤，在这个过程中肝纤维化与免疫反应持续存在，能很好地重现其他模型所不能呈现的人类自身免疫性肝损伤所致肝纤维可能的过程和机制，使该模型在抗肝纤维化的免疫性药物的筛选和评估方面具有很大优势。
1材料与方法
1)实验动物
清洁级Wistar大鼠，雄性，体重140±20g，购于南疆君科生物工程有限公司，合格证号SCXK(辽)2015-0001。
2)药品、试剂及其配制
水飞蓟宾胶囊由天津天士力圣特制药有限公司(批号：国药准字H20040299)生产；猪血清由广州蕊特生物科技有限公司生产(批号：160608)。其他供试药物、化合物同实施例2，其它试剂均为市售分析纯；药物配成0.5％的羧甲基纤维素钠(CMC-Na)混悬液。
3)实验方法
Wistar大鼠适应性喂养3d后，按照体重随机分组，每组6只。除正常组动物外，全部大鼠均进行腹腔注射猪血清，1mL/只，2次/w，持续6w，正常组动物注射同等剂量的生理盐水。实验至第8w末结束。灌胃给药，1次/d。每次灌胃均在早上空腹时进行，每给药10d停药一天。预防给药组(H：2.5mg/kg)动 物从造模当天开始给药，水飞蓟宾(50mg/kg)、穿心莲内酯(20mg/kg)和本发明化合物H的5mg/kg和10mg/kg剂量组动物均从第5w开始给药，而20mg/kg剂量组动物从第7w开始给药；对照组和模型组灌胃给予0.5％CMC-Na。大鼠肝脏采集和固定、血清制备、病理切片制作、Masson染色剂结果统计同实施例2，结果见附图8、9。利用免疫组织化学法评价肝组织α-SMA(HSC活化程度的标志物)表达情况，使用Image-Pro Plus对阳性表达进行量化分析，结果见附图10、11。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000014
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图8、9、10、11，结果表明：在猪血清诱导的肝纤维化模型上，本发明化合物表现出良好的抗肝纤维化作用。无论预防给药还是治疗给药均显著减轻了大鼠的肝纤维化程度。效果优于AD和水飞蓟宾(50mg/kg)处理组，即使造模6w后给药，仅给药2w本发明化合物也取得了良好的治疗效果。同时观察到本发明化合物显著下调肝组织中α-SMA的表达水平，与AD比，差异显著，且效果优于阳性药。说明其发挥抗肝纤维化作用与抑制肝星状细胞活化有关。
实施例4本发明化合物显著减轻总胆管结扎的SD大鼠肝纤维化程度
1材料与方法
1)实验动物
清洁级SD大鼠，雄性，体重200±20g，购于湖南斯莱克景达实验动物有限公司。其许可证号是SCXK(湘)2016-0002。
2)药品、试剂及其配制
熊去氧胆酸由上海信谊药厂有限公司(批号：国药准字H31021875)生产；其他供试药物、化合物同实施例2，其它试剂均为市售分析纯；药物配成0.5％的羧甲基纤维素钠(CMC-Na)混悬液。
3)实验方法
SD大鼠适应性喂养3d后，按照体重随机分组：假手术对照组、模型组、AD对照组(5mg/kg和20mg/kg)、熊去氧胆酸对照组(25mg/kg)和本发明化合物各组，每组4只。假手术组和模型组灌胃给予0.5％CMC-Na，其余各给药组给予0.5％CMC-Na混悬的相应药物，给药4w结束。术前12h更换垫料，严格 禁食不禁水，灌胃前2h肌肉注射青霉素8万U/只，灌胃后1h腹腔注射3％的戊巴比妥钠(2mL/kg)，麻醉生效后，仰位固定大鼠四肢，剪毛，碘酒消毒皮肤，铺洞巾，沿腹中线开腹，沿胃向下找到并向上牵引十二指肠，游离胆总管，距近肝门部0.5cm处，以4/0号丝线双重结扎并离断胆总管，检查无出血及胆漏情况后，以3/0号丝线连续缝针法逐层关腹，碘酒消毒伤口，术后保暖至清醒，假手术组只进行麻醉、开腹、游离胆总管，不结扎、不离断胆总管。每次灌胃均在早上空腹时进行，每给药10d停药一天。大鼠肝脏采集和固定、血清制备、病理切片制作、Masson染色剂结果统计同实施例2，结果见附图12、13。利用免疫组织化学法评价肝组织α-SMA(HSC活化程度的标志物)表达情况，使用Image-Pro Plus对阳性表达进行量化分析，结果见附图14、15。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000015
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图12、13、14、15，结果表明：在总胆管结扎致大鼠肝纤维化模型上，本发明化合物抗肝纤维化作用均显著强于AD和阳性药处理。同时观察到本发明化合物显著下调肝组织中α-SMA的表达水平，与AD比，差异显著，且效果优于阳性药。说明其发挥抗肝纤维化作用与抑制肝星状细胞活化有关。
实施例5本发明化合物对四氯化碳(CCl 4)诱导SD大鼠肝纤维化动物血清胶原指标的影响
正常肝组织中，胶原物质是窦间隙基质膜的重要组成成分，以C-Ⅰ和C-Ⅳ为主。当肝脏受到损伤后，大量胶原物质和糖蛋白被转录、翻译和组装，以LN、C-Ⅰ和C-Ⅲ为主。临床上，血清LN、C-Ⅰ、PC-Ⅲ和C-Ⅳ水平是纤维化患者诊断的重要指标。
1材料与方法
同实施例2。
以本发明化合物H为代表，采用ELISA方法测定动物血清中LN、C-Ⅰ、PC-Ⅲ和C-Ⅳ水平。血清中胶原指标变化见附图16-19。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000016
来表示；P﹤0.05 组间差异有显著性意义。
2实验结果
结果显示，与正常组相比，模型组中LN与三种胶原含量均大幅提高；各治疗组显著降低肝组织中LN含量，本发明化合物H高剂量组和阳性药苦参组相比有显著性差异；苦参、AD和本发明化合物均显著降低了C-Ⅰ含量，H呈剂量依赖性降低C-Ⅰ水平；H低、高剂量显著降低组织内PC-Ⅲ水平，给药浓度为20mg/kg时，与苦参组和AD组相比有显著性意义；苦参、AD和化合物H显著降低肝组织中C-Ⅳ的含量，H呈剂量依赖性降低血清C-Ⅳ水平。
结合附图16、17、18、19，结果表明：本发明化合物在保持了母体化合物AD的大幅降低LN、C-Ⅰ和C-Ⅳ水平的基础上，显著提高了对PC-III的抑制活性。其中本发明化合物在20mg/kg剂量使LN、PC-Ⅲ、C-Ⅳ降低到正常水平。并且化合物H在5mg/kg剂量对PC-Ⅲ、C-Ⅳ的下调程度与AD在20mg/kg剂量时的相当。
实施例6本发明化合物对四氯化碳(CCl 4)诱导SD大鼠肝纤维化动物血清IL-6和TNF-α水平的影响
活化的HSC可诱导巨噬细胞产生大量的TNF-α，继续参与HSC的分化，同时还会扩大肝脏内炎症反应。IL-6是促纤维化因子之一，参与肝组织内炎症反应、脂质过氧化、细胞凋亡与再生等复杂的生理过程，也是NF-κB下游的效应分子。
1材料与方法
同实施例2。
以本发明化合物H为代表，采用ELISA方法测定动物血清中IL-6和TNF-α水平。结果见附图20、21。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000017
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图20、21，结果表明：三个剂量的化合物H都使肝纤维化大鼠血清中TNF-α和IL-6水平大幅降低，高剂量组效果最为显著，在给药剂量为20mg/kg时血清中IL-6和TNF-α的含量与正常组动物水平相当。结果提示：本发明化合物的抗肝纤维化作用与抑制TNF-α、IL-6的表达密切相关，通过降低TNF-α和IL-6的含量，抑制炎症反应。
实施例7本发明化合物对四氯化碳(CCl 4)诱导SD大鼠肝纤维化动物肝组织中SOD和MDA水平的影响
SOD和MDA是评价脂质过氧化的重要指标。SOD是抗氧化剂，不仅可以抑制因自由基启动导致的脂质过氧化，还可以清除自由基以保护生物膜的完整性，是机体内抗氧化能力的敏感指标。脂质过氧化会产生大量的MDA，它的含量与组织内过氧化的损伤程度成正比。
1材料与方法
同实施例2。
以本发明化合物H为代表，测定动物肝组织中SOD、MDA水平。结果见附图22，23。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000018
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图22、23，结果表明：本发明化合物5、10、20mg/kg三个剂量组SOD水平均大幅升高，并接近正常水平，10、20mg/kg两个剂量组MDA水平均大幅降低，并接近正常值。说明本发明化合物保持了AD的强抗脂质过氧化能力。
实施例8本发明化合物大幅改善四氯化碳(CCl 4)诱导SD大鼠肝纤维化动物肝组织炎症状态
1材料与方法
同实施例2。
以本发明化合物H为代表，通过H&E染色观察分析本发明化合物对肝组织免疫炎症状态的改善情况，结果见附图24、25。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000019
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图24、25，结果表明：正常组大鼠的肝小叶结构完整、肝细胞索排列整齐，没有异变发生且维管区正常；肝细胞未见变形、坏死，无纤维结缔产生。模型组大鼠肝细胞索排列混乱，核质比明显增大并伴随可见点、片状及灶状坏死 区，淋巴细胞浸润严重，肝小叶结构严重受损无法辨别。与模型组相比，给药组动物肝损伤均得到不同程度的改善，尤其是本发明化合物H的中、高剂量治疗组最为显著，肝细胞仅有轻微肿大，肝细胞索的结构正常，明显优于阳性药苦参治疗组和AD治疗组。
实施例9本发明化合物抑制TGF-β1诱导的人Ⅱ型肺泡上皮细胞A549向间充质细胞转化作用存在于肺泡里的Ⅱ型肺泡上皮细胞受到炎症介质、生长因子等细胞因子的刺激，细胞形态由鹅卵石状变为梭状，完成了上皮间充质转化(EMT)，具有了间质细胞的功能，进而合成胶原纤维，大量胶原纤维沉积可加剧间质肺纤维化的病程。因此，采用形态学观察法评价本发明化合物抗肺纤维化作用。1细胞培养和药物处理
采用人Ⅱ型肺泡上皮细胞A549，与穿心莲内酯比较，研究本发明化合物的体外抗肺纤维化作用。将A549细胞培养在含10％(V/V)胎牛血清、100μg/mL链霉素、100IU/mL青霉素的RPMI1640培养液中，置体积分数5％CO 2培养箱中于饱和湿度、37℃培养。
2MTT法测定细胞毒
将生长对数期的A549细胞用0.25％(W/V)胰蛋白酶消化后，用含10％(V/V)胎牛血清的RPMI1640培养基稀释成2.5×10 4/mL(核实)细胞悬液，铺于96孔板内，200μL/孔，于37℃，体积分数5％CO 2培养箱中培养24h，加入含药培养基，药物终浓度最高为30.00μM，每个处理4孔重复，继续培养48h。其它同实施例1。结果取平均值，如附图26A和26B所示。
3形态学观察法检测药物对A549细胞EMT的影响
将生长对数期的A549细胞用0.25％(W/V)胰蛋白酶消化后，用含10％(V/V)胎牛血清的RPMI1640培养基稀释成2.5×10 4/mL细胞悬液，铺于96孔板内，每孔200μL。培养24h之后细胞长成融合状态，弃去原培养基，加入不含血清的培养基再同步化培养24h，弃去培养基，用PBS洗两遍，同时加入200μL含有TGF-β1(5ng/mL)及不同浓度待测化合物的RPMI1640培养基后立即在显微镜下拍照(100×)。设3孔重复并且设置对照。培养48h后分别在显微镜下拍照。每种化合物相同浓度的三孔下共选取5个视野，测量大于100个细胞。利用 photoshopCS6图像软件对图片进行处理，并计算其圆形度(公式e＝4π×S/C 2，其中e代表圆形度，S代表面积，C代表周长)。结果取平均值，见附图27A和27B。
4实验结果
附图26A和26B结果表明，与AD相比，在30.00μM浓度(除了f、g、h、k为3.00μM外)下，本发明化合物对A549细胞的细胞毒活性未见增强。
附图27A和27B结果表明：本发明化合物在无毒浓度下，可显著抑制A549细胞上皮间充质转化，且与AD比，对人Ⅱ型肺泡上皮细胞间充质转化的抑制作用更强，安全指数更高。
实施例10本发明化合物显著降低博来霉素诱导的KM小鼠肺纤维化程度
肺纤维化是由多种原因而引起的肺脏损伤，肺纤维化形成的病理机制复杂，不同致病因子启动炎症、免疫反应，涉及多种细胞包括血管内皮细胞、肺泡上皮细胞、成纤维细胞及巨噬细胞等，多种细胞因子和炎症介质的相互作用。博来霉素属碱性糖肽类抗癌抗生素，该药严重的毒不良反应之一是引起肺纤维化，在动物实验中已证实，博莱霉素所致肺纤维化病理组织学改变与人类肺纤维化非常相似，被普遍作为研究肺纤维化的模型。
1材料与方法
1)实验动物
清洁级KM小鼠，雄性，体重20±2g，购于河南省实验动物中心。许可证号：SCXK(豫)2015-0004。
2)药品、试剂及其配制
注射用盐酸博来霉素由海正辉瑞制药有限公司生产(批号：YBH15562005国药准字：H20055883)；醋酸波尼松片由浙江仙琚制药股份有限公司生产(批号：170410，国药准字，33021207)。其他供试药物、化合物同实施例2；药物配成0.5％的羧甲基纤维素钠(CMC-Na)混悬液。
3)实验方法
KM小鼠适应性喂养3d后，按照体重随机分组：假手术对照组、模型组、AD对照组(250mg/kg)、波尼松对照组(5mg/kg)、H化合物给药组(62.5mg/kg)和H化合物给药组(250.mg/kg)，共6组，每组15只。腹腔注射质量百分比4％的戊巴比妥钠(2ml/kg)对小鼠进行麻醉，仰卧位固定小鼠，剃去颈部毛发，碘 酒消毒皮肤后，沿颈部向下行1cm左右切口，游离支气管，注射博来霉素(2mg/mL)50μL，随即注入150μL空气，快速转动小鼠，使药液均匀分布，然后以4/0号丝线缝合切口，碘伏消毒后，无菌纱布包扎伤口，最后将术后小鼠送至温暖处直至苏醒，其中，假手术组注射同等体积的生理盐水。造模结束24h后，开始定时定点定人灌胃给药，给药方式同实施例2，给药28d后结束实验。小鼠在末次灌胃前12h更换垫料，严格禁食不禁水。给药后0.5h后采用摘眼球法收集小鼠全血，采集完血液后颈椎脱臼法处死小鼠，采集肺脏，称重，观察并记录肺部病变。拍照后将肺固定于4％多聚甲醛固定液中。血清制备和Masson染色及结果统计方法同实施例2，Masson染色及胶原面积结果见附图28和29。
2实验结果
附图28和29结果表明：在博来霉素诱导的小鼠肺纤维化模型上，本发明化合物H显著减少肺纤维化KM小鼠肺组织纤维化面积，且作用显著强于AD。
实施例11本发明化合物显著改善博来霉素诱导KM小鼠肺纤维化动物肺组织炎症状态
1材料与方法
同实施例10。
以本发明化合物H为代表，通过H&E染色观察分析本发明化合物对肺组织炎症状态的改善情况，染色结果和病理评分见附图30和31。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000020
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图30和31，结果表明：假手术组小鼠肺组织肺泡结构完整、肺间隔粗细均匀，没有异变发生且无肉眼可见炎细胞浸润。模型组小鼠肺泡结构紊乱，肺间隔变粗，肺间质纤维化物质分割肺泡形成重构肺泡，大量炎性细胞浸润。与模型组相比，给药组动物肺组织损伤均得到不同程度的改善，尤其是本发明化合物H的高剂量治疗组最为显著，肺组织肺泡结构基本完整趋于正常，炎性细胞浸润情况肉眼难以辨认，其药效明显优于AD治疗组。
实施例12本发明化合物抑制TGF-β1诱导的人近端肾小管上皮细胞HK-2向间充质细胞转化作用
早期研究发现肾小管上皮细胞可以向成纤维细胞转分化并表达其标志蛋白成纤维特异性蛋白(fibroblast-specific protein 1，FSP1)，肾小管上皮细胞－间充质细胞转分化是肾脏间质纤维化的重要发病机制之一。因此，采用TGF-β1刺激后形态学观察法评价本发明化合物抗肾纤维化作用。
1细胞培养和药物处理
采用人近端肾小管上皮细胞HK-2(由中国典型培养物保藏中心提供)，与穿心莲内酯AD比较，研究本发明化合物的体外抗肾纤维化作用。将HK-2细胞培养在含10％胎牛血清(V/V)、100μg/mL链霉素、100IU/mL青霉素的DMEM-F12培养液中，置含体积分数5％CO 2培养箱中，于饱和湿度、37℃培养。
2.MTT法测定细胞毒
将生长对数期的HK-2细胞用0.25％(W/V)胰蛋白酶+0.02％EDTA(W/V)消化后，用含10％(V/V)胎牛血清的DMEM-F12培养基稀释成7.0×10 4/mL细胞悬液，铺于96孔板内，200μL/孔，于37℃，体积分数5％CO 2培养箱中培养24h，更换为含不同浓度药物培养基，药物最高终浓度为30.00μM，每个处理4孔重复，继续培养48h。其它同时实施例1。结果取平均值，如附图32A和32B所示。
3TGF-β1刺激后观察药物对HK-2细胞形态的影响
将生长至对数期的HK-2细胞用0.25％(W/V)胰蛋白酶+0.02％EDTA消化后，用含10％(V/V)胎牛血清的DMEM-F12培养基稀释成5.0×10 4/mL细胞悬液，铺于96孔板内，每孔200μL。培养24h之后细胞长成单层，弃去原培养基，用0.01M PBS清洗两遍，更换无血清培养基以同步化，再培养24h后，吸弃无血清培养基，加入200μL含不同浓度待测化合物与刺激因子TGF-β1(5ng/mL)的DMEM-F12培养基。设3孔重复并且设置对照。培养48h后分别在显微镜下拍照记录。部分本发明化合物作用细胞后的形态学变化见附图33A和33B。
4实验结果
附图32A和32B结果表明，与AD相比，在30.00μM浓度下，本发明化合物对HK-2细胞增殖的抑制作用显著降低。
表1A、1B和附图33A、33B结果表明：本发明化合物在无毒浓度下，可显著抑制TGF-β1诱导的人近端肾小管上皮细胞HK-2向间充质细胞转化，且与AD比，对HK-2细胞向间充质转化的抑制作用更强，安全指数更高。
表1A 本发明化合物抑制TGF-β1诱导的人近端肾小管上皮细胞HK-2向间充质细胞转化作用
Figure PCTCN2018080776-appb-000021
注：测试浓度为0.08-1.25μM；对照：上皮细胞之间具有相互作用，组织结构紧密，细胞呈典型的铺路石状；TGF-β1处理：上皮细胞失去其典型状态，细胞之间相互作用消失，组织结构相对松散，细胞密度变小，立方呈铺路石状上皮细胞转变为纺锤状纤维细胞的形态；极强(抑制作用)：细胞几乎与对照无异，视野下极少见纺锤状，胞间恢复相互作用，形态恢复其典型的铺路石状；强(抑制作用)：抑制了细胞的侵袭性，细胞紧实，细胞状态几乎完全恢复，少见纺锤纤维状细胞；中强(抑制作用)：细胞密度变大，大部分细胞仍呈立方状态。
表1B 本发明化合物抑制TGF-β诱导的人近端肾小管上皮细胞HK-2向间充质细胞转化作用
Figure PCTCN2018080776-appb-000022
注：测试浓度为0.08-1.25μM；对照：上皮细胞之间具有相互作用，组织结构紧密，细胞呈典型的铺路石状；TGF-β1处理：上皮细胞失去其典型状态，细胞之间相互作用消失，组织结构相对松散，细胞密度变小，立方呈铺路石状上皮细胞转变为纺锤状纤维细胞的形态；极强(抑制作用)：细胞几乎与对照无异，视野下极少见纺锤状，胞间恢复相互作用，形态恢复其典型的铺路石状；强(抑制作用)：抑制了细胞的侵袭性，细胞紧实，细胞状态几乎完全恢复，少见纺锤纤维状细胞；中强(抑制作用)：细胞密度变大，大部分细胞仍呈立方状态。
实施例13本发明化合物显著降低单侧输尿管结扎诱导的SD大鼠肾纤维化程度
单侧输尿管结扎诱导大鼠肾纤维化模型(UUO)是研究肾纤维化经典模型之一，该模型特征为肾小管间质中细胞成分积聚、成纤维细胞分化/增殖、ECM沉积增加和肾小管萎缩等，与临床肾脏疾病的发生发展过程相似，极其有助于各种肾小管间质损伤疾病防治的研究，且造模方法简便，成模率100％，病变均一，有较好的重复性，能够在短期内造成纤维化，就研究肾间质纤维化的发病规律及机制探讨而言，是一种较为快速、可靠的动物模型。因此，UUO模型广泛应用于肾间质纤维化机制研究及改善肾间质纤维化的治疗效果的评价等。
1材料与方法
1)实验动物
清洁级SD大鼠，雄性，体重200±20g，购于河南省实验动物中心。许可证号：SCXK(豫)2015-0004。
2)药品、试剂及其配制
穿心莲内酯、羧甲基纤维素钠及本实验化合物的来源和配制同实施例2。
3)实验方法
SD大鼠适应性喂养3d后，按照体重随机分组：假手术对照组、模型组、AD对照组(0.15mmol/kg)、H化合物给药组(0.06mmol/kg)、H化合物给药组(0.10mmol/kg)、H化合物给药组(0.15mmol/kg)共6组，每组4只。术前准备及麻醉同实施例4，麻醉后，左侧卧位固定大鼠，剃去胸骨下缘至后肢见毛发，铺手术布与剃去毛发的皮肤上，碘酒消毒皮肤后，沿胸骨下缘约0.2cm处向下行2cm左右切口，挤出肾脏，游离输尿管，在距离膀胱约1/3输尿管长度处，以4/0号丝线双重结扎并离断输尿管，肾脏送回腹腔后，以4/0号丝线全层关腹，碘伏消毒后，无菌纱布包扎伤口，最后将术后大鼠送至温暖处直至苏醒，其中，假手术组只游离输尿管不结扎、不离段。造模结束24h后，开始定时定点定人灌胃给药，给药方法同实施例2，给药14d后结束实验。大鼠在末次灌胃前12h更换垫料，严格禁食不禁水。给药后0.5h腹腔注射质量百分比3％的戊巴比妥钠(2mL/kg)麻醉剂，采血后迅速完整地剖离左侧肾脏，称量肾重，测定肾大小，拍照后固定于4％多聚甲醛固定液中。血清制备及Masson染色方法和统计处理同实施例2。Masson染色结果及相对胶原面积统计结构如附图34和35。
2实验结果
结合附图34和35，结果表明：在UUO模型上，本发明代表性化合物H显著降低肾纤维化程度，改善病变肾组织结构，且作用显著强于AD。
实施例14本发明化合物显著改善单侧输尿管结扎致SD大鼠肾纤维化动物肾组织炎症状态
1材料与方法
同实施例13。
以本发明化合物H为代表，通过观察解剖肾脏和病理切片H&E染色分析本 发明化合物对肺组织炎症状态的改善情况，染色结果和病理分级和评分见附图36-40。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000023
来表示；P﹤0.05组间差异有显著性意义。
2实验结果
结合附图36、37、38、39，结果表明：假手术组大鼠肾组织表面水润有光泽、肾小球结构完整，肾小管紧致密实，没有肉眼可见病变。模型组大鼠肾组织肿大膨胀，中间有大量积液且与周围组织有黏连，肾小球内有纤维增生样组织且部分坏死脱落，肾间质纤维化物质包饶压迫肾小管致肾小管严重萎缩，肾间质中大量炎性细胞浸润。与模型组相比，给药组动物肾组织损伤均得到不同程度的改善，尤其是本发明化合物H的高剂量治疗组最为显著，肾组织表面光滑水润无黏连，肾脏轻微膨胀，中间未见积液，肾小球结构基本完整，肾小管萎缩不明显，其药效明显优于AD治疗组。
实施例15本发明化合物抑制血管紧张素Ⅱ(AngⅡ)诱导的人原代心肌纤维化细胞HCFB增殖作用
有研究表明，心肌成纤维细胞是心肌纤维化的主要效应细胞，在受到AngⅡ等活性物质刺激后会发生数量增殖，其表型转化为有分泌胞外基质功能的肌成纤维细胞。因此，采用MTT法检测AngⅡ刺激原代心肌成纤维细胞HCFB后的细胞抑制作用评价本发明化合物H抗心肌纤维化作用。
1细胞培养
采用人原代心肌成纤维细胞HCFB(由商城北纳创联生物科技有限公司提供)，与穿心莲内酯比较，研究本发明化合物H的体外抗心肌纤维化作用。将HCFB细胞培养在含H-DMEM培养液的培养瓶中，培养液中含体积分数10％胎牛血清(美国GIBCO，货号：302220F)、100μg/mL链霉素、100IU/mL青霉素，置于体积分数为5％CO 2培养箱(德国Binder公司)中于饱和湿度、37℃培养。
2MTT法测定细胞毒
将生长对数期的HCFB细胞用0.25％胰蛋白酶+0.02％EDTA消化后，用含体积分数10％胎牛血清的H-DMEM培养基稀释成5.0×10 4/mL细胞悬液，铺于96孔板(美国Costar公司)内，200μL/孔，于37℃，体积分数5％CO 2，饱和湿度的培养箱中培养24h，加入含不同浓度化合物AD或H的培养基，继续培养 48h，加入MTT(5mg/mL)，20μL/孔，培养4h，弃上清，加入150μL DMSO，震荡l0min，用酶标仪测定吸光值。测定波长为570nm，参考波长为450nm。计算化合物作用后的细胞存活率，存活率(％)＝药物组A值/细胞对照组A值×100％，结果见附图40。数据利用SPSS 17.0统计学软件进行处理和分析。
3MTT法检测药物对AngⅡ刺激的心肌成纤维细胞HCFB增殖能力的抑制作用
将对数生长期的HCFB细胞用0.25％胰蛋白酶+0.02％EDTA消化后，用含体积分数10％胎牛血清的H-DMEM培养基稀释成5.0×10 4/mL细胞悬液，铺于96孔板内，每孔200μL。培养24h至细胞长成单层，弃去原培养基，用0.01M PBS清洗两遍，更换无血清培养基以同步化，继续培养24h后，吸弃无血清培养基，加入200μL含不同浓度待测化合物与刺激因子AngⅡ(10 -7mol/L)的H-DMEM培养基。设3孔重复，以含0.5％DMSO的H-DMEM培养基为阴性对照，含刺激因子AngⅡ(10 -7mol/L)和0.5％DMSO的H-DMEM培养基为阳性对照。培养48h后MTT法检测细胞存活率。结果见附图41。数据利用SPSS 17.0统计学软件进行处理和分析。数据均采用平均值±标准差
Figure PCTCN2018080776-appb-000024
来表示；P﹤0.05组间差异有显著性意义。
4实验结果
附图40结果表明，本发明化合物H在15.0μM浓度下，对人原代心肌成纤维细胞HCFB细胞增殖未表现出明显的抑制作用。
附图41，结果表明：本发明化合物H在无毒浓度下，可显著抑制AngII对HCFB的增殖作用，且与AD比，对人HCFB增殖抑制作用更强，安全指数更高。

Claims (9)

  1. 结构如式I所示的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物在制备药物中的应用，其特征在于，将其作为活性成份用于制备治疗或预防人体组织或器官纤维化药物：
    Figure PCTCN2018080776-appb-100001
    其中：R 1，R 2各自为氢或C 1-5烷基；R 1，R 2各自为氢或苯基、2-甲氧基苯基、3-甲氧基苯基、4-甲氧基苯基、2，3，5-三甲氧基苯基、2-羟基苯基、3-羟基苯基、4-羟基苯基、2-氟苯基、2-氯苯基、2-溴苯基、3-氟苯基、3-氯苯基、3-溴苯基、4-氟苯基、4-氯苯基、4-溴苯基、2-氟-3-甲氧基苯基、3-甲氧基-4-氯苯基、2，4-二氟苯基、2，4-二氯苯基、2，4-二溴苯基、2-氟-4-氯苯基、2-溴-4-氯苯基、3-氟-4-氯苯基、3-溴-4-氯苯基、3，4-二氟苯基、3，4-二氯苯基、3，4二溴苯基、2-氯-4-氟苯基、2-溴-4-氟苯基、3-氯-4-氟苯基、3-溴-4-氟苯基、2-氟-4-溴苯基、2-氯-4-溴苯基、3-氟-4-溴苯基、3-氯-4-溴苯基、2，3，4-三氯苯基、2-甲氧基-4-氯苯基、2-羟基-4-氯苯基、2-羟基-4-甲氧基苯基、3-氨基-4-氯苯基、2-氨基-4-氯苯基，2-硝基-4-氟苯基，2-硝基-4-氯苯基，或被C 1-5烷基取代的苯基、被卤素和吗啉基取代的苯基、被卤素和甲基哌嗪基取代的苯基、N，N-二烷基胺基苯基；或吡啶基、呋喃基、噻吩基、吡咯基、吲哚基或被卤素取代的吡啶基、呋喃基、噻吩基、吡咯基、吲哚基；或苯并呋喃基、苯并咪唑基、苯并噻喃基、苯并噻唑基、苯并四唑基、苯并异唑基、苯并异噻唑基、苯并咪唑啉基、苯并吡唑基、苯并三唑基、苯并噻吩基、苯并恶唑基；或R 1和R 2相连成环己烷；R 1， R 2同时相同或不同但不同时为氢；R 3、R 4各自为氢或R 3、R 4分别为甲基磺酰基、三苯甲基、3-吡啶基或R 3、R 4分别为CH 2CH 2COOH、CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种，或R 3、R 4各自为COR 5，R 5为3-吡啶基、CH 2CH 2COOH、CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种；R 3、R 4选相同或不同的取代基团。
  2. 如权利要求1所述的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物在制备药物中的应用，其特征在于，当R 1，R 2其中之一为氢时，R 1，R 2其中之一选如下基团：甲基、乙基或丙基、苯基、2-甲氧基苯基、3-甲氧基苯基、4-甲氧基苯基、2-氟苯基、2-氯苯基、2-溴苯基、3-氟苯基、3-氯苯基、3-溴苯基、4-氟苯基、4-氯苯基、4-溴苯基、2-氟-3-甲氧基苯基、3-甲氧基-4-氯苯基、2，4-二氟苯基、2，4-二氯苯基、2，4-二溴苯基、2-氟-4-氯苯基、2-溴-4-氯苯基、3-氟-4-氯苯基、3-溴-4-氯苯基、3，4-二氟苯基、3，4-二氯苯基、3，4-二溴苯基、2-氯-4-氟苯基、2-溴-4-氟苯基、3-氯-4-氟苯基、3-溴-4-氟苯基、2-氟-4-溴苯基、2-氯-4-溴苯基、3-氟-4-溴苯基、3-氯-4-溴苯基、2-甲氧基-4-氯苯基4-正丙基苯基、4-异丙基苯基、4-异丁基苯基、4-(N,N-二甲胺基)苯基、3-氟-4-(4-吗啉基)苯基、3-氟-4-(4-甲基哌嗪基)苯基、2-呋喃基、2-吡咯基、6-氯-3-吲哚基、3-吲哚基、5-氯-3-吲哚基、6-氯-2-吡啶基、3-吡啶基；或R 1和R 2相连成环己基；R 3、R 4各自为氢或R 3、R 4分别为CH 2CH 2COOH或CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2COOH、CH 2CH 2CH 2CH 2CH 2CH 2CH 2COOH中的一种或者R 3、R 4各自为COR 5，R 5为3-吡啶基或CH 2CH 2COOH，R 3、R 4选相同取代基团。
  3. 如权利要求1所述的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物在制备药物中的应用，其特征在于，选如下化合物：
    A：R 1＝H，R 2＝C 6H 5，R 3＝R 4＝H；
    B：R 1＝H，R 2＝2-F-C 6H 4，R 3＝R 4＝H；
    C：R 1＝H，R 2＝2-Cl-C 6H 4，R 3＝R 4＝H；
    D：R 1＝H，R 2＝2-Br-C 6H 4，R 3＝R 4＝H；
    E：R 1＝H，R 2＝3-F-C 6H 4，R 3＝R 4＝H；
    F：R 1＝H，R 2＝3-Cl-C 6H 4，R 3＝R 4＝H；
    G：R 1＝H，R 2＝3-Br-C 6H 4，R 3＝R 4＝H；
    H：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝H；
    I：R 1＝H，R 2＝4-F-C 6H 4，R 3＝R 4＝H；
    J：R 1＝H，R 2＝4-Br-C 6H 4，R 3＝R 4＝H；
    K：R 1＝H，R 2＝4-CH 3O-C 6H 4，R 3＝R 4＝H；
    L：R 1＝H，R 2＝2-CH 3O-4-Cl-C 6H 3，R 3＝R 4＝H；
    M：R 2＝H，R 1＝2-Br-C 6H 4，R 3＝R 4＝H；
    N：R 2＝H，R 1＝3-Cl-C 6H 4，R 3＝R 4＝H；
    O：R 2＝H，R 1＝2-F-4-Cl-C 6H 3，R 3＝R 4＝H；
    P：R 2＝H，R 1＝2,4-diCl-C 6H 3，R 3＝R 4＝H；
    Q：R 2＝H，R 1＝4-F-C 6H 4，R 3＝R 4＝H；
    R：R 2＝H，R 1＝C 6H 5，R 3＝R 4＝H；
    S：R 1＝H，R 2＝3-F-4-Cl-C 6H 3，R 3＝R 4＝H；
    T：R 1＝H，R 2＝2,4-diF-C 6H 3，R 3＝R 4＝H；
    U：R 1＝H，R 2＝3,4-diCl-C 6H 3，R 3＝R 4＝H；
    V：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝3-吡啶基；
    W：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝CH 2CH 2COOH；
    X：R 1＝H，R 2＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝CH 2CH 2COOH；
    Y:R 2＝H，R 1＝4-Cl-C 6H 4，R 3＝R 4＝H；
    Z:R 2＝H，R 1＝4-Cl-C 6H 4，R 3＝R 4＝COR 5，R 5＝3-吡啶基；
    a.R 1＝CH 3，R 2＝CH 3，R 3＝R 4＝H；
    b.15-环己亚基-14-脱氧-11,12-脱氢-穿心莲内酯；R 3＝R 4＝H；
    c.R 1＝H，R 2＝3-F-4-(4-甲基哌嗪基)-C 6H 3，R 3＝R 4＝H；
    d.R 1＝H，
    Figure PCTCN2018080776-appb-100002
    R 3＝R 4＝H；
    e.R 1＝H，
    Figure PCTCN2018080776-appb-100003
    R 3＝R 4＝H；
    f.R 1＝H，
    Figure PCTCN2018080776-appb-100004
    R 3＝R 4＝H；
    g.R 1＝H，
    Figure PCTCN2018080776-appb-100005
    R 3＝R 4＝H；
    h.R 1＝H，
    Figure PCTCN2018080776-appb-100006
    R 3＝R 4＝H；
    i.R 1＝H，
    Figure PCTCN2018080776-appb-100007
    R 3＝R 4＝H；
    j.R 1＝H，
    Figure PCTCN2018080776-appb-100008
    R 3＝R 4＝H；
    k.R 1＝H，
    Figure PCTCN2018080776-appb-100009
    R 3＝R 4＝H。
  4. 如权利要求1-3其中之一所述的15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物在制备药物中的应用，其特征在于，将其作为活性成份用于制备治疗或预防肝纤维化、肺纤维化、肾纤维化或心肌纤维化药物。
  5. 如权利要求4所述的15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物在制备药物中的应用，其特征在于，将其作为活性成份或其各类前药、或与其它药物组合，与制药中可以接受的辅助和/或添加成分混合后，按常规的制药方法和工艺要求，制成口服型制剂或注射型制剂药物。
  6. 如权利要求5所述的15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物在制备药物中的应用，其特征在于，口服型制剂为片剂、丸剂、胶囊、冲剂或糖浆等；注射型制剂为注射液或冻干粉针剂型。
  7. 15-亚基-14-脱氧-11,12-脱氢穿心莲内酯衍生物，其特征在于，结构如通式1所示：
    Figure PCTCN2018080776-appb-100010
    其中：R 1，R 2各自为氢或C 1-5烷基；或吡啶基、呋喃基、噻吩基、吡咯基、吲哚基或被卤素取代的吡啶基、呋喃基、噻吩基、吡咯基、吲哚基；或被C 1-5烷基取代的苯基、被卤素和吗啉基取代的苯基、被卤素和甲基哌嗪基取代的苯基、N，N-二烷基胺基苯基；或苯并呋喃基、苯并咪唑基、苯并噻喃基、苯并噻唑基、苯并四唑基、苯并异唑基、苯并异噻唑基、苯并咪唑啉基、苯并吡唑基、苯并三唑基、苯并噻吩基、苯并恶唑基；或R 1和R 2相连成环己基；R 1，R 2同时相同或不同但不同时为氢；R 3、R 4各自为氢或R 3、R 4分别为甲基磺酰基、三苯甲基、3-吡啶基或R 3、R 4各自为COR 5，R 5为3-吡啶基或CH 2CH 2COOH，R 3、R 4选相同取代基团。
  8. 如权利要求7所述的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物，其特征在于，当R 1，R 2其中之一为氢时，R 1，R 2其中之一选如下基团：甲基、乙基或丙基、2-呋喃基、2-吡咯基、6-氯-3-吲哚基、3-吲哚基、5-氯-3-吲哚基、6-氯-2-吡啶基、3-吡啶基、4-正丙基苯基、4-异丙基苯基、4-异丁基苯基、3-氟-4-(4-甲基哌嗪基)苯基；或R 1和R 2相连成环己基；R 3、R 4各自为氢。
  9. 如权利要求7所述的15-亚基-14-脱氧-11,12-脱氢-穿心莲内酯衍生物，其特征在于，选如下化合物：
    a.R 1＝CH 3，R 2＝CH 3，R 3＝R 4＝H；
    b.15-环己亚基-14-脱氧-11,12-脱氢-穿心莲内酯；R 3＝R 4＝H；
    c.R 1＝H，R 2＝3-F-4-(4-甲基哌嗪基)-C 6H 3，R 3＝R 4＝H；
    d.R 1＝H，
    Figure PCTCN2018080776-appb-100011
    R 3＝R 4＝H；
    e.R 1＝H，
    Figure PCTCN2018080776-appb-100012
    R 3＝R 4＝H；
    f.R 1＝H，
    Figure PCTCN2018080776-appb-100013
    R 3＝R 4＝H；
    g.R 1＝H，
    Figure PCTCN2018080776-appb-100014
    R 3＝R 4＝H；
    h.R 1＝H，
    Figure PCTCN2018080776-appb-100015
    R 3＝R 4＝H；
    i.R 1＝H，
    Figure PCTCN2018080776-appb-100016
    R 3＝R 4＝H；
    j.R 1＝H，
    Figure PCTCN2018080776-appb-100017
    R 3＝R 4＝H；
    k.R 1＝H，
    Figure PCTCN2018080776-appb-100018
    R 3＝R 4＝H。
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