TWI715570B - Use of protein kinase inhibitors in the preparation of a medicament for the treatment of fibrotic diseases - Google Patents

Abstract

The invention relates to use of protein kinase inhibitors in the preparation of a medicament for the treatment of fibrotic diseases. Specifically, The invention relates to use of 6-(4-((1-aminocyclopropyl)methoxy)-1,2-dihydrofuro[3,2-f]quinolin-9-yloxy)-N-methyl-1-naphthamide or 5-(2-Diethylamino-ethyl)-2-(5-fluoro-2-oxo-1,2-dihydro-indol -3-yldenemethyl)-3-methyl-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridine-4-one or their pharmaceutical salts in the preparation of a medicament for the treatment of fibrotic diseases.

Description

Use of protein kinase inhibitor in preparing medicine for treating fibrotic diseases

The invention relates to the use of two protein kinase inhibitors in the preparation of drugs for treating fibrotic diseases.

Tissue cell injury can cause tissue cell degeneration, necrosis, and inflammation. If the damage is small, normal parenchymal cells around the damaged cell will undergo proliferation and repair, and this repair can completely restore normal structure and function. However, if the damage is large or the repeated damage exceeds the regeneration capacity of the surrounding parenchymal cells, the interstitial fibrous connective tissue (extracellular matrix) will proliferate in a large amount to repair the defect tissue, that is, the pathological changes of fibrosis will occur. Therefore, in essence, fibrosis is a repair response after tissue damage to protect the relative integrity of tissues and organs. Although the hyperplastic fibrous connective tissue repairs the defect, it does not have the structure and function of the original organ parenchymal cells. If this repair response is excessive, strong and out of control, it will cause organ fibrosis and lead to a decline in organ function. Fibrosis refers to a pathological process in which inflammation leads to necrosis of organ parenchymal cells, abnormal increase of extracellular matrix in the tissue and excessive deposition. The lighter ones become fibrosis, and the more severe ones cause damage to the organization structure. Organ sclerosis.

Fibrosis can occur in all tissues, but it is especially common in lungs, skin, digestive tract, kidneys, and liver, which are frequently subjected to chemical and biological damage. Fibrosis often seriously harms the normal function of organs. In fact, many fibrotic diseases are life-threatening, such as idiopathic pulmonary fibrosis (IPF), liver cirrhosis, scleroderma or renal fibrosis.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), is a diffuse lung disease of unknown cause. Mainly manifested as diffuse alveolitis, structural disorder of alveolar units, and pulmonary fibrosis, which ultimately lead to severe damage to lung structure and function. Its pathology is mainly manifested as early diffuse alveolitis and a large number of interstitial cell proliferation in the later stage, and progressive accumulation of matrix collagen As a result, it replaces the normal lung tissue structure and seriously affects the ventilation and ventilation function of the lungs, and eventually leads to respiratory failure and death. Its pathogenesis has not yet been fully elucidated, and there is currently a lack of effective clinical treatments, and the mortality rate is high.

Lung interstitial tissue is composed of collagen, elastin, and proteoglycans. Collagen is the main extracellular matrix (ECM) protein of lung tissue. Collagen in the lung and other types of ECM components form a three-dimensional network structure. Become the main skeleton of lung tissue structure. These protein components maintain the integrity of lung tissue structure and play a very important role in maintaining lung epithelial and endothelial cell differentiation. When lung fibroblasts are damaged chemically (such as bleomycin, allergy sources) or physical (such as dust, radiation), they secrete collagen to repair the lung interstitial tissue, which in turn causes lung fibrosis. The basic process of IPF is: alveolitis after early lung injury, there are serous and cellular components in the alveoli, and a large number of mononuclei in the lung interstitium Cells, lymphocytes, plasma cells, alveolar macrophages and other inflammatory immune cells infiltrate and secrete a variety of cytokines. At this time, the alveolar structure is still intact; as the inflammatory immune response progresses, the generation of pro-fibrosis factors or anti-fibrosis factors increases Relatively insufficient, inflammation and abnormal repair lead to the proliferation of pulmonary interstitial cells, resulting in abnormal ECM metabolism in the process of pulmonary fibrosis, producing a large amount of ECM protein; in the advanced stage, the alveolar structure is replaced by solid collagen, and the alveolar walls, airways and blood vessels finally fibrosis , ECM such as fibroblasts and collagen are distributed in the lung interstitium, alveolar epithelial metaplasia into squamous epithelium, fibrotic lung eventually forms, the clinical manifestation is that the patient has difficulty breathing and died of respiratory failure. The formation of pulmonary fibrosis involves inflammation and immune response. Therefore, glucocorticoids (such as prednisone) and immunosuppressive agents (such as cyclophosphamide) are traditional drugs for the treatment of pulmonary fibrosis, which can inhibit inflammation and immunity Process, reduce alveolar inflammation, thereby delaying the process of pulmonary fibrosis, but due to its adverse reactions, it cannot be used for a long time. Therefore, there is an urgent need to find new effective drugs for the treatment of fibrosis-related diseases, such as pulmonary fibrosis, especially idiopathic pulmonary fibrosis.

CN103382206 A discloses a class of protein kinase inhibitors, including the compound represented by the following formula (I), whose chemical name is 6-(4-((1-aminocyclopropyl)methoxy)-1,2- Dihydrofuro[3,2-f]quinolin-9-yloxy) -N -methyl-1-naphthamide, and disclosed its possible use in the treatment of various tumors.

CN101007815A discloses another protein kinase inhibitor, which includes a compound represented by the following formula (II), whose chemical name is 5-(2-diethylamino-ethyl)-2-(5-fluoro-2-side Oxy-1,2-dihydro-indol-3-ylidene-methyl)-3-methyl-1,5,6,7-tetrahydro-pyrrole[3,2-c]pyridine-4- Ketone, and disclosed its potential to treat tumors.

However, none of these documents disclose that these compounds have the related effects of treating fibrosis-related diseases, such as pulmonary fibrosis, especially idiopathic pulmonary fibrosis. It has been found that these two compounds have shown surprising effects in treating the aforementioned fibrotic diseases, thus completing the present invention.

The present invention provides the use of any one or two of the following two known protein kinase inhibitors or their pharmaceutically acceptable salts in the preparation of drugs for the treatment of fibrotic diseases, and their structural formulas are as follows: Formula (I) and Formula (II) are shown in:

6-(4-((1-aminocyclopropyl)methoxy)-1,2-dihydrofuro[3,2-f]quinolin-9-yloxy) -N -methyl- 1-naphthamide

5-(2-Diethylamino-ethyl)-2-(5-fluoro-2-oxo-1,2-dihydro-indol-3-ylidene-methyl)-3-methyl -1,5,6,7-Tetrahydro-pyrrole [3,2-c]pyridin-4-one.

Specifically, the fibrotic disease may be selected from pulmonary fibrosis, liver cirrhosis, scleroderma or renal fibrosis.

In a preferred embodiment of the present invention, the fibrotic disease is idiopathic pulmonary fibrosis (IPF).

The compounds of formula (I) and (II) in the present invention are preferably administered in the form of their pharmaceutically acceptable salts. In a particularly preferred embodiment, the compound of formula (I) exists in the form of hydrochloride, (II) The compound exists in the form of malate.

The compounds of formula (I) and (II) or their pharmaceutically acceptable salts can be made into the form of compositions well known in the art together with pharmaceutically acceptable carriers, such as tablets, capsules, injections and the like. The present invention also relates to the use of a composition containing a compound selected from formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of the aforementioned fibrotic diseases.

Figure 1 and Figure 1'show the effect of each compound on inflammatory cells in the alveolar lavage fluid of mice with pulmonary fibrosis (Mean±SEM, n=7~12), where Compared with the control group, ##p<0.01, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

Figure 2 shows the effect of each compound on the expression of KC mRNA in the lung tissue of pulmonary fibrosis mice (Mean±SEM, n=6), where compared with the control group, ##p<0.01; compared with the model group, *p <0.05, **p<0.01, ***p<0.001.

Figure 3 shows the effect of each compound on the expression of IL-1β mRNA in the lung tissue of pulmonary fibrosis mice (Mean±SEM, n=6), where compared with the control group, ###p<0.001; compared with the model group , **P<0.01, ***p<0.001.

Figure 4 shows the effect of each compound on the expression of IL-6mRNA in the lung tissue of pulmonary fibrosis mice (Mean±SEM, n=6), which is compared with the control group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01.

Figure 5 shows the effect of each compound on the expression of TIMP-1 mRNA in the lung tissue of pulmonary fibrosis mice (Mean±SEM, n=6); among them, compared with the control group, ###p<0.001; compared with the model group , **P<0.01, ***p<0.001.

Figure 6 shows the effect of each compound on the expression of TGF-β mRNA in the lung tissue of pulmonary fibrosis mice (Mean±SEM, n=6), which is compared with the control group, #p<0.05; compared with the model group, * p<0.05, **p<0.01, ***p<0.001.

Figure 7 shows the effect of each compound on the pathological structure of lung tissue in mice with pulmonary fibrosis (H&E, 100×), where A: control group; B: model group; C: SHR0583 6mg/kg; D: SHR0583 30mg /kg; E: SHR1020 3mg/kg; F: SHR1020 15mg/kg.

Figure 8 shows the effect of each compound on collagen deposition in lung tissue of mice with pulmonary fibrosis (Masson’s, 100×), where A: control group; B: model Group; C: SHR0583 6mg/kg; D: SHR0583 30mg/kg; E: SHR1020 3mg/kg; F: SHR1020 15mg/kg.

Figure 9 shows the effect of each compound on the deposition of collagen in lung tissue of mice with pulmonary fibrosis (Mean±SEM, n=7~12). Compared with the control group, ###p<0.001; compared with the model group, * p<0.05, **p<0.01.

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.

Example 1 : Evaluation of the effects of compounds of formula (I) and formula (II) on bleomycin-induced idiopathic pulmonary fibrosis in mice

1. Material 1.1 Animals :

ICR mice, 90 females, were purchased from Zhejiang Laboratory Animal Center, animal certificate number: 0012209, license number SCXK (Zhejiang) 2014-0001. After purchase, they will be kept in the IVC system of Zhejiang University Experimental Animal Center, temperature: 20~26℃, humidity 40~70%; free food and water.

1.2 Test compound :

Compound of formula (I): batch number SHR140583-013-04, synthesized according to the method disclosed in CN103382206 A, code SHR0583 in the diagram.

Compound of formula (II): batch number 685130501, synthesized according to the method disclosed in CN101007815A, code name SHR1020 in the diagram.

1.3 Instrument :

Mettler Toledo AG245 balance; FLUKO/F6 high-speed homogenizer; Bio-Rad fluorescent quantitative PCR instrument; Olympus BX51 microscope; Eppendorf Centrifuge 5804R centrifuge; Leica slicer embedding machine.

1.4 Reagents :

(1) Bleomycin hydrochloride for injection: 15 mg/bottle, batch number 140372, Nippon Kayaku Co., Ltd.

(2) SYBR Green, Oligo d(T), reverse transcriptase, and RNase inhibitors were all purchased from Beijing Kangwei Reagent Biology Co., Ltd.

(3) Primers: synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.

(4) Hydroxyproline determination kit: purchased in Nanjing and built.

2. Method : 2.1 Establishment of mouse idiopathic pulmonary fibrosis model

Mice were anesthetized with ether, supine, the neck skin was cut, the trachea was separated, 3mg/kg bleomycin (administration volume was 10μl/10g body weight) was sprayed into the trachea with a microinjector; the skin was sutured and disinfected.

2.2 Group administration

The mice were divided into 6 groups, each with 12 (10 in the control group), namely: control group, model group, 6 mg/kg compound of formula (I) group, 30 mg/kg compound of formula (I) group, and formula (II) Compound 3mg/kg group, formula (II) compound 15mg/kg group. Normal saline was sprayed into the airway of the control group. After modeling, mice in each group were given intragastric administration once a day for 21 consecutive days. The model group and the control group were given vehicle.

2.3 Index measurement

Twenty-four hours after the last administration, the mice were sacrificed, the trachea was separated, the trachea was intubated, the left upper lobe was ligated, the alveolar lavage was performed, the lavage fluid was collected for white blood cell count, centrifuged at 1500rpm/min for 10min, and the supernatant was stored at -80 ℃ refrigerator is used to determine hydroxyproline, and precipitation is used for smears for classification and counting. The upper part of the upper lobe of the left lung was quick-frozen in liquid nitrogen and stored in a refrigerator at -80 degrees for cytokine determination. The lower part was fixed with formalin, embedded in paraffin, sectioned, H&E stained and Masson's stained, and analyzed by Image Pro software. The density value, do semi-quantitative analysis

3. Results : 3.1 The effects of each compound on inflammation in the alveolar lavage fluid of mice with bleomycin-induced pulmonary fibrosis :

3 mg/kg bleomycin was instilled into the airway of mice, and the total number of white blood cells and neutrophils in the alveolar lavage fluid 21 days later. Lymphocytes and macrophages increased significantly ( p<0.001 ); Compound 6 of formula (I) and 30mg/kg group showed a dose-dependent inhibition of neutrophils, lymphocytes and macrophages in alveolar lavage fluid, and all had Statistical difference ( p<0.05~0.001 ); Formula (II) compound 3 and 15mg/kg group showed a dose to inhibit the total number of white blood cells, and there was a statistical difference at high dose ( p<0.01 ); Formula (II) compound 15mg/kg The group can significantly inhibit lymphocytes and macrophages ( p<0.05 ), and there is a tendency to reduce the number of neutrophils, but there is no statistical difference. The total number of white blood cells, neutrophils, lymphocytes and macrophages in the 3 mg/kg group had a downward trend, but there was no statistical difference. See Table 1 and Figure 1, Figure 1'.

3.2 The effect of each compound on cytokine expression in lung tissue of mice with pulmonary fibrosis (6 animals in each group for determination)

KC : 3mg/kg bleomycin was sprayed into the airway of mice. After 21 days, the expression of KC mRNA in lung tissue was significantly higher than that in the control group ( p<0.01 ); mice were orally administered formula (I) compound 6 and 30mg/kg, The expression of KC mRNA in the lung tissue was significantly reduced in a dose-dependent manner ( p<0.001 ); the compound 3 of formula (II) and the 15mg/kg group showed a dose-dependent reduction in the expression of KC mRNA in the lung tissue, and there was a statistical difference ( p<0.05~0.01) ). See figure 2.

IL-1β : The mouse airway was sprayed with 3mg/kg bleomycin. After 21 days, the expression of IL-1β mRNA in the lung tissue was significantly higher than that in the control group ( p<0.001 ); the mice were orally administered with formula (I) compound 6 and 30mg/kg, significantly reduced the expression of IL-1β mRNA in the lung tissue in a dose-dependent manner ( p<0.05~0.01 ); the compound 3 of formula (II) and the 15mg/kg group showed a dose-dependent reduction in the expression of IL-1β mRNA in the lung tissue, and There is a statistical difference ( p<0.01~0.001 ). See figure 3.

IL-6 : 3mg/kg bleomycin was sprayed into the airway of mice. After 21 days, the expression of IL-6 mRNA in the lung tissue was significantly higher than that in the control group ( p<0.001 ); the mice were orally administered formula (I) compound 6 and 30mg/kg, can significantly reduce the expression of IL-6 mRNA in the lung tissue ( p<0.05~0.01 ); the compound 3 of formula (II) and the 15mg/kg group showed a dose-dependent reduction in the expression of IL-6 mRNA in the lung tissue. The high-dose group has Statistical difference ( p<0.01 ). See figure 4.

TIMP-1 : The mouse airway was sprayed with 3mg/kg bleomycin. After 21 days, the expression of TIMP-1 mRNA in the lung tissue was significantly higher than that in the control group ( p<0.001 ); the mice were orally administered the compound 6 of formula (I) and 30mg/kg, can significantly reduce the expression of TIMP-1 mRNA in lung tissue ( p<0.01~0.001 ). See figure 5.

TGF-β : The mouse airway was sprayed with 3mg/kg bleomycin. After 21 days, the expression of TGF-β mRNA in the lung tissue was significantly higher than that in the control group ( p<0.05 ); the mice were orally administered with compound 6 of formula (I) and 30mg/kg, decreased the expression of TGF-β mRNA in lung tissue in a dose-dependent manner, and there was a statistical difference ( p<0.001 ); oral administration of compound 3 of formula (II) and 15mg/kg in mice showed a dose-dependent decrease in lung tissue The expression of TGF-β mRNA was statistically different ( p<0.05~0.01 ). See figure 6.

3.3 The effect of each compound on the content of hydroxyproline in the alveolar lavage fluid of mice with pulmonary fibrosis

Note: Take 0.8ml of mouse alveolar lavage fluid and operate according to the kit instructions. It is found that the content of hydroxyproline in the alveolar lavage fluid is lower than the quantitative limit of the kit, because mouse lung tissue has been used for pathology and fluorescence quantification PCR detection, no remaining lung tissue was used for homogenization to determine the content of hydroxyproline, so there is no data on hydroxyproline in this test.

3.4 The effect of each compound on the pathological structure of lung tissue in mice with pulmonary fibrosis

After the mouse airway was sprayed with bleomycin, the alveoli closed and the alveolar interstitial hyperplasia; oral administration of formula (I) compound 6, 30 mg/kg, formula (II) compound 3, 15 mg/kg for 21 days, the mouse alveolar interstitial hyperplasia Significantly reduced, the alveolar morphology tends to be complete, see Figure 7. Masson's staining showed that after the mouse airway was sprayed with bleomycin, collagen deposition in the lung tissue, semi-quantitative analysis, oral administration of formula (1) compound 6, 30mg/kg and formula (II) compound 3, 15mg/kg were dose-dependent Sexually reduces collagen deposition ( p<0.05~0.01 ), see Figure 8 and Figure 9.

4. Conclusion :

Both the compound of formula (I) and the compound of formula (II) have good therapeutic effects on mice with idiopathic pulmonary fibrosis. The action intensity is: 30 mg/kg of the compound of formula (I)> 6 mg/kg of the compound of formula (I) 15 mg/kg of the compound of formula (II)> 3 mg/kg of the compound of formula (II).

Claims (4)

A use of the compound represented by the formula (II) or its pharmaceutically acceptable salt in the preparation of a medicine for treating fibrotic diseases, wherein the fibrotic disease is pulmonary fibrosis,

The use as described in item 1 of the scope of patent application, wherein the fibrotic disease is idiopathic pulmonary fibrosis. The use as described in item 1 of the scope of patent application, wherein the pharmaceutically acceptable salt of the compound of formula (II) is malate. A use of a pharmaceutical composition, which contains a compound represented by formula (II), or a pharmaceutically acceptable salt thereof, for the preparation of a medicine for treating the diseases described in item 1 or 2 of the scope of patent application, wherein The pharmaceutical composition also contains a pharmaceutically acceptable carrier.

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