WO2016173400A1 - 蛋白激酶抑制剂在制备治疗纤维化疾病的药物中的用途 - Google Patents

蛋白激酶抑制剂在制备治疗纤维化疾病的药物中的用途 Download PDF

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WO2016173400A1
WO2016173400A1 PCT/CN2016/079282 CN2016079282W WO2016173400A1 WO 2016173400 A1 WO2016173400 A1 WO 2016173400A1 CN 2016079282 W CN2016079282 W CN 2016079282W WO 2016173400 A1 WO2016173400 A1 WO 2016173400A1
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formula
mice
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pulmonary fibrosis
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孙飘扬
张连山
杨昌永
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Jiangsu Hengrui Medicine Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
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    • 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
    • A61K31/395—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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/395—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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline

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  • the present invention relates to the use of two protein kinase inhibitors in the manufacture of a medicament for the treatment of fibrotic diseases.
  • Tissue cell damage can lead to degeneration, necrosis and inflammatory reaction of tissue cells. If the damage is small, the normal parenchyma cells around the damaged cells will undergo proliferative repair, and this repair can completely restore normal structure and function. However, if the damage is large or the repeated damage exceeds the regenerative capacity of the parenchymal cells surrounding the injury, the interstitial fibrous connective tissue (extracellular matrix) will repair a large amount of hyperplasia to the defect tissue, that is, pathological changes of fibrosis occur. Therefore, fibrosis is essentially a repair reaction after tissue damage to protect the relative integrity of tissues and organs. Although the proliferating fibrous connective tissue repairs the defect, it does not have the structure and function of the original organ parenchymal cells.
  • Fibrosis refers to the pathological process in which the parenchymal cells of the organ are necrotic due to inflammation, and the extracellular matrix in the tissue is abnormally increased and excessively deposited. The light becomes fibrosis, and the severe one causes structural damage and organ hardening.
  • Fibrosis can occur in all tissues, but is especially prevalent in the lungs, skin, digestive tract, kidneys, and liver that are often subject to chemical and biological damage. Fibrosis often severely jeopardizes the normal functioning of organs. In fact, many fibrotic diseases are life-threatening, such as idiopathic pulmonary fibrosis (IPF), cirrhosis, scleroderma, or renal fibrosis.
  • IPF idiopathic pulmonary fibrosis
  • cirrhosis cirrhosis
  • scleroderma scleroderma
  • Pulmonary fibrosis is an unexplained diffuse lung disease. Mainly manifested as diffuse alveolitis, alveolar unit structure disorder and pulmonary fibrosis, which ultimately leads to severe destruction of lung structure and function. Its pathology mainly manifests as early diffuse alveolitis and a large amount of interstitial cell proliferation and progressive accumulation of matrix collagen. As a result, it replaces the normal lung tissue structure, which seriously affects the ventilation and ventilation function of the lungs, and eventually leads to respiratory failure and death. The pathogenesis has not yet been fully elucidated. At present, there is no effective treatment in clinical practice and the mortality rate is very high.
  • the interstitial tissue of the lung is composed of collagen, elastin and proteoglycan.
  • Collagen is the main extracellular matrix (ECM) protein of lung tissue.
  • ECM extracellular matrix
  • the collagen in the lung and other types of ECM components form a three-dimensional network structure. It becomes the main skeleton of lung tissue structure.
  • These protein components maintain the integrity of lung tissue structure and play an important role in maintaining lung epithelial and endothelial cell differentiation.
  • lung fibroblasts are damaged by chemical (such as bleomycin, metamorphic sources) or physical (such as dust, radiation) collagen is secreted to repair the interstitial tissue of the lungs, which in turn causes pulmonary fibrosis.
  • IPF interstitial lung.
  • the basic process of IPF is: alveolitis after early lung injury, serous and cellular components in the alveoli, and a large number of mononuclear cells, lymphocytes, plasma cells, alveolar macrophages and other infiltrating inflammatory immune cells in the interstitial lung.
  • Cytokine secretion at this time the alveolar structure is still intact; with the progress of inflammatory immune response, increased fibrogenic factor production or anti-fibrotic factor production is relatively insufficient, inflammation and abnormal repair lead to pulmonary interstitial cell proliferation, leading to pulmonary fibrosis
  • the abnormal ECM metabolism produces a large amount of ECM protein; in the advanced stage, the alveolar structure is replaced by solid collagen, the alveolar wall, airway and blood vessels are finally fibrotic, and ECM of fibroblasts, collagen and the like are distributed in the interstitial lung, and alveolar epithelialization The squamous epithelium is formed, and the fibrotic lung is finally formed.
  • the clinical manifestation is the patient's breathing. Difficulties, death from respiratory failure.
  • glucocorticoids such as prednisone
  • immunosuppressants such as cyclophosphamide
  • glucocorticoids such as prednisone
  • immunosuppressants such as cyclophosphamide
  • CN103382206A discloses a class of protein kinase inhibitors comprising a compound of the following formula (I) having the chemical name 6-(4-((1-aminocyclopropyl)methoxy)-1,2-di. Hydrogenated [3,2-f]quinolin-9-yloxy)-N-methyl-1-naphthalenecarboxamide and disclosed for its potential use in the treatment of various types of tumors.
  • CN101007815A discloses another protein kinase inhibitor comprising a compound of the following formula (II) having the chemical name 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]pyridine-4 a ketone and discloses its potential to treat tumors.
  • formula (II) having the chemical name 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]pyridine-4 a ketone and discloses its potential to treat tumors.
  • the present invention provides the use of any one or both selected from two known protein kinase inhibitors, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating a fibrotic disease, the structural formula of which is Formula (I), formula (II):
  • the fibrotic disease may be selected from the group consisting of pulmonary fibrosis, cirrhosis, scleroderma or renal fibrosis.
  • the fibrotic disease is idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • the compounds of the formula (I), (II) in the present invention are preferably administered in the form of their pharmaceutically acceptable salts, and in a particularly preferred embodiment, the compound of the formula (I) is present in the form of the hydrochloride salt.
  • the compound of formula (II) is present in the form of malate.
  • the compound of the formula (I), (II) or a pharmaceutically acceptable salt thereof may be formulated together with a pharmaceutically acceptable carrier in the form of a composition well known in the art, such as a tablet, a capsule, an injection or the like.
  • a composition comprising a compound selected from the group consisting of a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a fibrotic disease as described above.
  • Figure 7 shows the effect of each compound on the pathological changes of lung tissue in lung fibrosis mice (H&E, 100 ⁇ ), where A: blank group; B: model group; C: SHR0583 6 mg/kg; D: SHR0583 30 mg/ 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: blank group; B: model group; C: SHR0583 6 mg/kg; D: SHR0583 30 mg/kg E: SHR1020 3mg/kg; F: SHR1020 15mg/kg.
  • Example 1 Evaluation of the effects of a compound of formula (I) and a compound of formula (II) on bleomycin-induced idiopathic pulmonary fibrosis in mice
  • ICR mice 90 females, purchased from Zhejiang Experimental Animal Center, animal certificate number: 0012209, license number SCXK (Zhejiang) 2014-0001. After purchase, it was raised in the IVC system of Zhejiang University Experimental Animal Center, temperature: 20 ⁇ 26 ° C, humidity 40 ⁇ 70%; free access to drinking water.
  • Bleomycin hydrochloride for injection 15 mg/bottle, batch number 140372, Nippon Kayaku Co., Ltd.
  • mice were anesthetized with ether, placed in the supine position, the neck skin was cut, the trachea was separated, and 3 mg/kg of bleomycin (administered volume 10 ⁇ l/10 g body weight) was sprayed into the trachea with a micro-injector; the skin was sutured and disinfected. .
  • mice were divided into 6 groups, 12 in each group (10 in the blank group), which were: blank group, model group, compound (I) compound 6 mg/kg group, formula (I) compound 30 mg/kg group, formula (II) Compound 3 mg/kg group, compound of formula (II) 15 mg/kg group.
  • Normal saline was sprayed into the airway of the blank group.
  • Each group of mice was intragastrically administered after modeling, once a day for 21 consecutive days, and the vehicle was administered to the model group and the blank group.
  • mice 24 hours after the last administration, the mice were sacrificed, the trachea was isolated, tracheal intubation was performed, the left upper lobe was ligated, alveolar lavage was performed, the lavage fluid was collected for white blood cell counting, centrifuged at 1500 rpm/min for 10 min, and the supernatant was stored at -80.
  • the refrigerator is used to measure hydroxyproline, and the precipitate is used for smear for classification and counting.
  • the upper part of the left upper lobe was frozen in liquid nitrogen and stored in a -80 degree refrigerator for the determination of cytokines.
  • the lower part was fixed with formalin, embedded in paraffin, sectioned, H&E stained and Masson's stained, analyzed by Image Pro software.
  • the collagen optical density value was semi-quantitatively analyzed.
  • mice The airway of mice was instilled with 3 mg/kg bleomycin, and the total number of white blood cells and neutrophils in the alveolar lavage fluid after 21 days. Lymphocytes and macrophages increased significantly (p ⁇ 0.001); compounds of formula (I) 6 and 30 mg/kg dose-dependently inhibited neutrophils, lymphocytes and macrophages in alveolar lavage fluid, and both had Statistical differences (p ⁇ 0.05-0.001); Compounds of formula (II) 3 and 15 mg/kg showed a total dose-reducing leukocyte count, and there was a statistically significant difference at high doses (p ⁇ 0.01); formula (II) compound 15 mg/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 showed a downward trend, but there was no statistical difference. See Table 1 and
  • KC 3 mg/kg bleomycin was injected into the airway of mice. After 21 days, the expression of KC mRNA in lung tissue was significantly higher than that in the blank group (p ⁇ 0.01). The mice were given oral compound (I) 6 and 30 mg/kg. The expression of KC mRNA in lung tissue was significantly decreased in a dose-dependent manner (p ⁇ 0.001). The compound of formula (II) 3 and 15 mg/kg decreased the expression of KC mRNA in lung tissue in a dose-dependent manner (p ⁇ 0.05-0.01). ). See Figure 2.
  • IL-1 ⁇ 3 mg/kg bleomycin was injected into the airway of mice. After 21 days, the expression of IL-1 ⁇ mRNA in lung tissue was significantly higher than that in the blank group (p ⁇ 0.001).
  • the mice were orally administered with compound (I) 6 and 30 mg. /kg, dose-dependently decreased IL-1 ⁇ mRNA expression in lung tissue (p ⁇ 0.05-0.01); compound (II) 3 and 15 mg/kg group dose-dependently decreased IL-1 ⁇ mRNA expression in lung tissue, and statistically Difference (p ⁇ 0.01 to 0.001). See Figure 3.
  • IL-6 3 mg/kg bleomycin was injected into the airway of mice. After 21 days, the expression of IL-6 mRNA in lung tissue was significantly higher than that in the blank group (p ⁇ 0.001); the mice were orally administered with compound (I) 6 and 30 mg. /kg, can significantly reduce the expression of IL-6 mRNA in lung tissue (p ⁇ 0.05-0.01); compound (II) compound 3 and 15mg/kg group dose-dependently reduce the expression of IL-6 mRNA in lung tissue, high-dose group statistically significant (p ⁇ 0.01). See Figure 4.
  • TIMP-1 3 mg/kg bleomycin was injected into the airway of mice. After 21 days, the expression of TIMP-1 mRNA in lung tissue was significantly higher than that in the blank group (p ⁇ 0.001). The mice were orally administered with compound (I) 6 and 30 mg. /kg, can significantly reduce TIMP-1 mRNA expression in lung tissue (p ⁇ 0.01 ⁇ 0.001). See Figure 5.
  • TGF- ⁇ 3 mg/kg bleomycin was injected into the airway of mice. After 21 days, the expression of TGF- ⁇ mRNA in lung tissue was significantly higher than that in the blank group (p ⁇ 0.05).
  • the mice were orally administered with compound (I) 6 and 30 mg. /kg, dose-dependently decreased TGF- ⁇ mRNA expression in lung tissue, and was statistically different (p ⁇ 0.001); mice oral administration of compound (II) 3 and 15 mg/kg dose-dependently reduced TGF- in lung tissue. mRNAmRNA expression was statistically significant (p ⁇ 0.05-0.01). See Figure 6.
  • the compound of formula (I) and the compound of formula (II) have a good therapeutic effect on idiopathic pulmonary fibrosis in mice.
  • the strength of action is: compound of formula (I) 30 mg/kg> compound of formula (I) 6 mg/kg compound of formula (II) 15 mg/kg > compound of formula (II) 3 mg/kg.

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Abstract

蛋白激酶抑制剂6-(4-((1-氨基环丙基)甲氧基)-1,2-二氢呋喃并[3,2-f]喹啉-9-基氧基)-N-甲基-1-萘甲酰胺或5-(2-二乙胺基-乙基)-2-(5-氟-2-氧代-1,2-二氢-吲哚-3-亚基-甲基)-3-甲基-1,5,6,7-四氢-吡咯[3,2-c]吡啶-4-酮，或者它们的可药用盐中的一种或几种在制备治疗纤维化疾病的药物中的用途。

Description

蛋白激酶抑制剂在制备治疗纤维化疾病的药物中的用途 技术领域
本发明涉及两种蛋白激酶抑制剂在制备治疗纤维化疾病的药物中的用途。
背景技术
组织细胞损伤后可导致组织细胞发生变性、坏死和炎症反应，如果损伤很小，损伤细胞周边正常实质细胞将发生增生修复，这种修复可完全恢复正常的结构和功能。然而如果损伤较大或反复损伤超出了损伤周围实质细胞的再生能力时，间质纤维结缔组织(细胞外基质)将大量增生对缺损组织进行修复，即发生纤维化的病理改变。因此本质上纤维化是组织遭受损伤后的修复反应，以保护组织器官的相对完整性。增生的纤维结缔组织虽然修复了缺损，但却不具备原来器官实质细胞的结构和功能。如果这种修复反应过度、过强和失控时，就会引起器官的纤维化和导致器官的功能下降。纤维化是指由于炎症导致器官实质细胞发生坏死，组织内细胞外基质异常增多和过度沉积的病理过程。轻者成为纤维化，重者引起组织结构破坏而发生器官硬化。
纤维化可发生于所有组织，但是在经常受到化学和生物损伤的包括肺、皮肤、消化道、肾和肝中尤其普遍。纤维化常常严重危害器官的正常功能，事实上，很多纤维化疾病是危及生命，例如特发性肺纤维化(IPF)、肝硬化、硬皮病或者肾纤维化。
肺纤维化尤其是特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)是一种原因不明的弥漫性肺疾病。主要表现为弥漫性肺泡炎、肺泡单位结构紊乱和肺纤维化，最终导致肺脏结构和功能的严重破坏，其病理主要表现为早期弥漫性肺泡炎及后期大量间质细胞增生、基质胶原进行性聚集以致取代正常的肺组织结构，严重影响肺的通气和换气功能，最终导致呼吸功能衰竭而死亡。其发病机理尚未完全阐明，目前临床上缺乏有效的治疗手段，死亡率很高。
肺脏间质组织由胶原蛋白、弹性素及蛋白聚糖类构成，胶原蛋白是肺组织的主要细胞外基质(extracellular matrix，ECM)蛋白，肺脏中的胶原蛋白与其他类型ECM成分构成三维网状结构，成为肺组织结构的主要骨架，这些蛋白成分保持肺组织结构的完整性，并对维持肺上皮及内皮细胞分化起着十分重要的作用。当肺纤维母细胞受到化学性(如博来霉素、变态源)或物理性(如粉尘、放射线)伤害时，会分泌胶原蛋白进行肺间质组织的修补，进而造成肺脏纤维化。IPF的基本过程是：肺脏早期损伤之后有肺泡炎，肺泡内有浆液和细胞成分，肺间质内有大量单核细胞、淋巴细胞、浆细胞、肺泡巨噬细胞等炎症免疫细胞浸润，多种细胞因子分泌，此时肺泡结构尚完整；随着炎症免疫反应的进展，促纤维化因子生成增多或抗纤维化因子产生相对不足，炎症和异常修复导致肺间质细胞增殖，导致肺纤维化过程的ECM代谢异常，产生大量的ECM蛋白；进入晚期，肺泡结构为坚实的胶原代替，肺泡壁、气道和血管最终纤维化，成纤维细胞、胶原等ECM分布在肺间质中，肺泡上皮化生为鳞状上皮，纤维化肺最终形成，临床表现为患者呼吸 困难，呼吸衰竭而死亡。肺间质纤维化的形成中有炎症免疫反应参与，因此糖皮质激素(如泼尼松)和免疫抑制剂(如环磷酰胺)是传统治疗肺纤维化的药物，可以抑制炎症反应和免疫过程，减轻肺泡炎症，从而延缓肺纤维化的进程，但是由于其不良反应，不能长期使用。因此急需寻找到新的有效药物，用于治疗纤维化相关疾病，例如肺纤维化，尤其是特发性肺纤维化。
CN103382206A公开了一类蛋白激酶抑制剂，其中包括下式(I)所示的化合物，其化学名为6-(4-((1-氨基环丙基)甲氧基)-1,2-二氢呋喃并[3,2-f]喹啉-9-基氧基)-N-甲基-1-萘甲酰胺，并公开了其可能用于治疗各类肿瘤。
Figure PCTCN2016079282-appb-000001
CN101007815A公开了另一种蛋白激酶抑制剂，其中包括如下式(II)所示的化合物，其化学名为5-(2-二乙胺基-乙基)-2-(5-氟-2-氧代-1,2-二氢-吲哚-3-亚基-甲基)-3-甲基--1,5,6,7-四氢-吡咯[3,2-c]吡啶-4-酮，并公开了其具有治疗肿瘤的潜力。
Figure PCTCN2016079282-appb-000002
然而这些文献均没有公开这些化合物具有治疗纤维化相关疾病，例如肺纤维化，尤其是特发性肺纤维化的相关作用。已经发现，这两个化合物在治疗前述的纤维化疾病方面表现出了惊人的效果，从而完成了本发明。
发明内容
本发明提供了选自下述两种已知的蛋白激酶抑制剂或其药学上可接受的盐中的任意一种或两种在制备治疗纤维化疾病的药物中的用途，它们的结构式分别如式(I)、式(II)所示：
Figure PCTCN2016079282-appb-000003
6-(4-((1-氨基环丙基)甲氧基)-1,2-二氢呋喃并[3,2-f]喹啉-9-基氧基)-N-甲基-1-萘甲酰胺
Figure PCTCN2016079282-appb-000004
5-(2-二乙胺基-乙基)-2-(5-氟-2-氧代-1,2-二氢-吲哚-3-亚基-甲基)-3-甲基-1,5,6,7-四氢-吡咯[3,2-c]吡啶-4-酮。
具体的，所述的纤维化疾病可以选自肺纤维化、肝硬化、硬皮病或者肾纤维化。
在本发明优选的实施方案中，所述的纤维化疾病是特发性肺纤维化(IPF)。
本发明中的式(I)、(II)化合物优选以其药学上可接受的盐的形式施用，在特别优选的实施方案中，所述的式(I)化合物以盐酸盐的形式存在，式(II)化合物以苹果酸盐的形式存在。
式(I)、(II)化合物或其药学上可接受的盐可以与药学上可接受的载体一起制成本领域熟知的组合物形式，如片剂、胶囊、注射剂等。本发明还涉及含有选自式(I)或式(II)化合物或者它们的药学上可接受的盐的组合物在制备治疗前述的纤维化疾病的药物中的用途。
附图说明
图1和图1’示出了各化合物对肺纤维化小鼠肺泡灌洗液中炎症细胞的影响(Mean±SEM，n＝7～12)，其中与空白组组比较，##p<0.01、###p<0.001；与模型组组比较，*p<0.05、**p<0.01、***p<0.001。
图2示出各化合物对肺纤维化小鼠肺组织中KC mRNA表达的影响(Mean±SEM，n＝6)，其中与空白组组比较，##p<0.01；与模型组比较，*p<0.05、**p<0.01、***p<0.001。
图3示出各化合物对肺纤维化小鼠肺组织中IL-1βmRNA表达的影响(Mean±SEM，n＝6)，其中与空白组组比较，###p<0.001；与模型组比较，**p<0.01、***p<0.001。
图4示出各化合物对肺纤维化小鼠肺组织中IL-6mRNA表达的影响(Mean±SEM，n＝6)，其中与空白组组比较，###p<0.001；与模型组比较，*p<0.05、**p<0.01。
图5示出各化合物对肺纤维化小鼠肺组织中TIMP-1mRNA表达的影响(Mean±SEM，n＝6)；其中与空白组组比较，###p<0.001；与模型组比较，**p<0.01、***p<0.001。
图6示出各化合物对肺纤维化小鼠肺组织中TGF-βmRNA表达的影响(Mean±SEM，n＝6)，其中与空白组组比较，#p<0.05；与模型组比较，*p<0.05、**p<0.01、***p<0.001。
图7示出各化合物对肺纤维化小鼠肺组织病理结构改变的影响(H&E，100×)，其中，A：空白组；B：模型组；C：SHR0583 6mg/kg；D：SHR0583 30mg/kg；E：SHR1020 3mg/kg；F：SHR1020 15mg/kg。
图8示出各化合物对肺纤维化小鼠肺组织胶原沉积的影响(Masson’s，100×)，其中，A：空白组；B：模型组；C：SHR0583 6mg/kg；D：SHR0583 30mg/kg；E：SHR1020 3mg/kg；F：SHR1020 15mg/kg。
图9示出各化合物对肺纤维化小鼠肺组织胶原沉积的影响(Mean±SEM，n＝7～12)，其中与空白组组比较，###p<0.001；与模型组比较，*p<0.05、**p<0.01。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
实施例1：评价式(I)化合物和式(II)化合物对博莱霉素诱导的小鼠特发性肺纤维化的作用
1、材料
1.1动物：
ICR小鼠，90只，雌性，购于浙江省实验动物中心，动物合格证编号：0012209，许可证号码SCXK(浙)2014-0001。购入后饲养于浙江大学实验动物中心IVC系统，温度：20～26℃，湿度40～70％；自由摄食饮水。
1.2受试化合物：
式(I)化合物：批号SHR140583-013-04，根据CN103382206 A公开的方法合成，附图中代号SHR0583。
式(II)化合物：批号685130501，根据CN101007815A公开的方法合成，附图中代号SHR1020。
1.3仪器：
Mettler Toledo AG245天平；FLUKO/F6高速匀浆机；Bio-Rad荧光定量PCR仪；Olympus BX51显微镜；Eppendorf Centrifuge 5804R离心机；Leica切片机包埋机。
1.4试剂：
(1)注射用盐酸博莱霉素：15mg/瓶，批号140372，日本化药株式会社。
(2)SYBR Green、Oligo d(T)、反转录酶、RNA酶抑制剂均购于北京康为试剂生物有限 公司。
(3)引物：由生工生物工程(上海)股份有限公司合成。
(4)羟脯氨酸测定试剂盒：购于南京建成。
2、方法：
2.1小鼠特发性肺纤维化模型建立
乙醚麻醉小鼠，仰卧位，切开颈部皮肤，分离气管，用微量进样器向气管内喷入3mg/kg的博莱霉素(给药体积为10μl/10g体重)；缝合皮肤，消毒。
2.2分组给药
小鼠分6组，每组12只(空白组10只)，分别为：空白组、模型组、式(I)化合物6mg/kg组、式(I)化合物30mg/kg组、式(II)化合物3mg/kg组、式(II)化合物15mg/kg组。空白组气道内喷入生理盐水。各组小鼠于造模后开始灌胃给药，每天1次，连续21天，模型组和空白组给予溶媒。
2.3指标测定
末次给药后24小时，处死小鼠，分离气管，行气管插管，结扎左肺上叶，行肺泡灌洗，收集灌洗液做白细胞计数，1500rpm/min离心10min，上清保存于-80度冰箱用于测定羟脯氨酸，沉淀用于涂片做分类计数。左肺上叶上半部分液氮速冻后保存于-80度冰箱用于测定细胞因子，下半部分福尔马林固定，石蜡包埋、切片，做H&E染色和Masson’s染色，采用Image Pro软件分析胶原光密度值，做半定量分析。
3、结果：
3.1各化合物对博莱霉素诱导的肺纤维化小鼠肺泡灌洗液中炎症的影响：
小鼠气道滴入3mg/kg博莱霉素，21天后肺泡灌洗液中白细胞总数、中性粒细胞。淋巴细胞和巨噬细胞明显上升(p<0.001)；式(I)化合物6和30mg/kg组呈剂量依赖抑制肺泡灌洗液中的中性粒细胞、淋巴细胞和巨噬细胞，且均具有统计学差异(p<0.05～0.001)；式(II)化合物3和15mg/kg组呈剂量抑制白细胞总数，且高剂量时具有统计学差异(p<0.01)；式(II)化合物15mg/kg组能明显抑制淋巴细胞和巨噬细胞(p<0.05)，有降低中性粒细胞数的趋势，但无统计学差异。3mg/kg组白细胞总数、中性粒细胞、淋巴细胞和巨噬细胞有下降趋势，但无统计学差异。见表1和图1、图1’。
表1.各化合物对肺纤维化小鼠肺泡灌洗液中炎症细胞的影响(Mean±SEM)
Figure PCTCN2016079282-appb-000005
Figure PCTCN2016079282-appb-000006
注：式(II)化合物3mg/kg组有3只小鼠灌洗时肺破裂，灌洗液无法回收，因此剔除。与空白组组比较，##p<0.01、###p<0.001；与模型组组比较，*p<0.05、**p<0.01、***p<0.001。
3.2各化合物对肺纤维化小鼠肺组织中细胞因子表达的影响(每组取6个动物测定)
KC：小鼠气道喷入3mg/kg博莱霉素，21天后，肺组织中KC mRNA表达较空白组明显上升(p<0.01)；小鼠口服式(I)化合物6和30mg/kg，呈剂量依赖性明显降低肺组织中KC mRNA表达(p<0.001)；式(II)化合物3和15mg/kg组呈剂量依赖降低肺组织KC mRNA表达，且具有统计学差异(p<0.05～0.01)。见图2。
IL-1β：小鼠气道喷入3mg/kg博莱霉素，21天后，肺组织中IL-1βmRNA表达较空白组明显上升(p<0.001)；小鼠口服式(I)化合物6和30mg/kg，呈剂量依赖性明显降低肺组织中IL-1βmRNA表达(p<0.05～0.01)；式(II)化合物3和15mg/kg组呈剂量依赖降低肺组织IL-1βmRNA表达，且具有统计学差异(p<0.01～0.001)。见图3。
IL-6：小鼠气道喷入3mg/kg博莱霉素，21天后，肺组织中IL-6mRNA表达较空白组明显上升(p<0.001)；小鼠口服式(I)化合物6和30mg/kg，能明显降低肺组织中IL-6mRNA表达(p<0.05～0.01)；式(II)化合物3和15mg/kg组呈剂量依赖降低肺组织IL-6mRNA表达，高剂量组有统计学差异(p<0.01)。见图4。
TIMP-1：小鼠气道喷入3mg/kg博莱霉素，21天后，肺组织中TIMP-1mRNA表达较空白组明显上升(p<0.001)；小鼠口服式(I)化合物6和30mg/kg，均能明显降低肺组织中TIMP-1mRNA表达(p<0.01～0.001)。见图5。
TGF-β：小鼠气道喷入3mg/kg博莱霉素，21天后，肺组织中TGF-βmRNA表达较空白组明显上升(p<0.05)；小鼠口服式(I)化合物6和30mg/kg，呈剂量依赖性降低肺组织中TGF-βmRNA表达，且具有统计学差异(p<0.001)；小鼠口服式(II)化合物3和15mg/kg呈剂量依赖性降低肺组织中TGF-βmRNA表达，且具有统计学差异(p<0.05～0.01)。见图6。
3.3各化合物对肺纤维化小鼠肺泡灌洗液中羟脯氨酸含量的影响
说明：取小鼠肺泡灌洗液0.8ml，根据试剂盒说明书操作，发现肺泡灌洗液中羟脯氨酸含量低于试剂盒的定量限，由于小鼠肺组织已用于病理和荧光定量PCR检测，无剩余的肺组织用于匀浆测定羟脯氨酸含量，因此本试验中无羟脯氨酸的数据。
3.4各化合物对肺纤维化小鼠肺组织病理结构改变的影响
小鼠气道喷入博莱霉素后，肺泡闭合，肺泡间质增生；口服式(I)化合物6、30mg/kg、式(II)化合物3、15mg/kg 21天，小鼠肺泡间质增生明显减轻，肺泡形态趋于完整，见图7。Masson’s染色可见，小鼠气道喷入博莱霉素后，肺组织中胶原沉积，半定量分析，口服式(I)化合物6、30mg/kg以及式(II)化合物3、15mg/kg呈剂量依赖性降低胶原沉积(p<0.05～0.01)见图8和9。
4、结论：
式(I)化合物和式(II)化合物对小鼠特发性肺纤维化都具有较好的治疗作用。作用强度为：式(I)化合物30mg/kg>式(I)化合物6mg/kg式(II)化合物15mg/kg>式(II)化合物3mg/kg。

Claims (6)

  1. 选自式(I)或式(II)所示化合物，或它们的药学上可接受的盐中的任意一种或两种在制备治疗纤维化疾病的药物中的用途，
    Figure PCTCN2016079282-appb-100001
  2. 根据权利要求1所述的用途，其中所述的纤维化疾病选自肺纤维化、肝硬化、硬皮病或者肾纤维化。
  3. 根据权利要求2所述的用途，其中所述的纤维化疾病是特发性肺纤维化。
  4. 根据权利要求1所述的用途，其中式(I)化合物的药学上可接受的盐是盐酸盐。
  5. 根据权利要求1所述的用途，其中式(II)化合物的药学上可接受的盐是苹果酸盐。
  6. 含有式(I)或式(II)所示化合物，或它们的药学上可接受的盐中的任意一种或两种的组合物在制备治疗权利要求1至3任一项所述的疾病的药物中的用途，其中所述的组合物还含有药学上可接受的载体。
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