CN104257661B - Application of azaazulene alkaloids topreparing medicine for preventing or treating pulmonary fibrosis - Google Patents

Abstract

The invention discloses the use of the alkaloid with the structural formula I in the preparation of medicines for preventing and/or treating pulmonary fibrosis. It has the alkaloid shown in structural formula I, which can significantly reduce the degree of inflammation in diseased lung tissue, reduce the content of pro-fibrosis factor TGF-β1 in diseased lung tissue, reduce the excessive deposition of collagen in diseased lung tissue, and have a significant effect on pulmonary fibrosis. prevention and treatment of .

Description

A kind of azazulene alkaloid in the preparation of prevention or treatment of pulmonary fibrosis medicine application

technical field

The present invention relates to an azazulene alkaloid with anti-pulmonary fibrosis activity, in particular to the application of the alkaloid represented by structural formula I in the preparation of a pharmaceutical composition for preventing or treating pulmonary fibrosis.

Background technique

Idiopathic pulmonary fibrosis (IPF) is a diffuse lung disease of unknown etiology, characterized by rapidly progressive lung parenchymal damage and fibrosis, which eventually diffuses to the entire lung, resulting in severe damage to lung structure and function. The clinical manifestations are progressive dyspnea, dry cough, progressive reduction of lung function, hypoxemia, and eventually respiratory failure and death. The typical pathological changes of IPF are fibroblastic focal lesions composed of fibroblasts and myofibroblasts. In active fibrous foci, myofibroblasts occupy a dominant position and are important effector cells in the process of fibrosis, which can cause abnormal deposition of extracellular matrix and are an important pathological sign of IPF entering into irreversible fibrosis. In recent years, due to the aggravation of environmental pollution, its incidence has been increasing year by year.

Due to the unclear etiology and pathogenesis, the treatment of pulmonary fibrosis has always been one of the difficult problems in the medical field. Although new drugs are constantly being developed, there is still no effective treatment plan and professional treatment drugs. The prognosis is extremely poor, and the average survival period is only 3 years, the 5-year survival rate is less than 50%, and it is called "cancer" that is not cancer. Inflammatory immune response is involved in the formation of pulmonary interstitial fibrosis, so glucocorticoids (such as prednisone) and immunosuppressants (such as cyclophosphamide) are traditional drugs for the treatment of pulmonary fibrosis, but their therapeutic effects are limited and long-term Use will also aggravate and accelerate the process of pulmonary fibrosis, so finding new anti-pulmonary fibrosis drugs has always been an important topic in pharmaceutical research.

In the process of pulmonary fibrosis, TGF-β1 is a recognized pro-fibrosis marker and the mediator most closely related to the occurrence and formation of pulmonary fibrosis, so it has become the main target of anti-fibrosis. Therefore, by measuring the content of hydroxyproline in lung tissue, the content of collagen in the tissue can be determined; by measuring the changes in the expression level of TGF-β1, the possible mechanism of drug intervention in pulmonary fibrosis can be explained, that is, the drug is not anti-inflammatory but anti-inflammatory. Inhibit the TGF-β1/Smad pathway by reducing the level of TGF-β1, thereby improving the degree of pulmonary fibrosis. For example, pirfenidone (pirfenidone), which has just been launched in Japan, India and the European Union, can inhibit TGF-β1 and other fibrogenic factors, but it still has many side effects such as photosensitivity in clinical practice.

Cough is a common symptom of respiratory diseases. Like many respiratory diseases, cough symptoms also appear in the early stage of pulmonary fibrosis. Cough and pulmonary fibrosis, while somewhat related, are completely different. Strictly speaking, coughing is a manifestation of many respiratory diseases, but it is not a real disease. Although coughing may also occur in the early stage and during the development of pulmonary fibrosis, cough medicines can relieve symptoms such as coughing and wheezing caused by early pulmonary fibrosis, but when the disease develops seriously, cough medicines are ineffective, and only hormones, Antibiotics to relieve symptoms. All of the above are palliatives, not root causes, and cannot fundamentally treat or prevent pulmonary fibrosis. Long-term use of hormones and antibiotics may even aggravate and accelerate the process of pulmonary fibrosis.

The traditional Chinese medicine Baibu is the dry root tuber of Stemonaceae (Stemonaceae) plant, which has the effects of moistening lung and lowering qi, relieving cough, killing insects and lice, etc. It is used for chronic cough, tuberculosis cough, and whooping cough. Pharmacological activity studies have shown that the alkaloids of babubu are the main active ingredients of babubu, which have very good insecticidal activity and the effect of eliminating phlegm and relieving cough. The researches on Bamboo are mainly focused on the separation of chemical constituents, except for the insecticidal and antitussive activities, there are few other activities. Although one of the symptoms of pulmonary fibrosis is cough, so far, there are no research reports that cough medicine can improve or treat pulmonary fibrosis.

Azazulene type alkaloids as shown in structural formula I, such as protostemonine etc., are the compound that is isolated from Libulum erectus at first, have significant antitussive effect to the cough model guinea pig that citric acid induces, in addition, have not yet See other active reports. There are not many patents on alkaloids of basil, and most of them are about the preparation method of total alkaloids or the antitussive and insecticidal activities. So far, no patent has been retrieved for the azazune-type alkaloids shown in structural formula I, and there have been no literature reports that this type of alkaloids has the effect of preventing or treating pulmonary fibrosis or its role in drugs for the treatment of pulmonary fibrosis. application.

Contents of the invention

Aiming at the lack of effective drugs for preventing or treating pulmonary fibrosis, the present invention discloses the use of alkaloids represented by structural formula I, which can effectively prevent or treat pulmonary fibrosis diseases.

The technical scheme of the present invention is: the application of a compound in the preparation of a pharmaceutical composition for preventing and/or treating pulmonary fibrosis, the compound having the structural formula I

in:

R is selected from α or β hydrogen, α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α or β α _- methyl-γ- butyrolactone group;

R2 is selected from α or β _hydrogen , α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α or β α-methyl-γ- butyrolactone group;

Or R ₁ and R ₂ are as follows:

Wherein: X is selected from oxygen, sulfur;

_R is selected from hydrogen, alpha or beta alkyl, alpha or beta hydroxyl, alpha or beta carboxy, alpha or beta halo, alpha or beta alkoxy;

_R4 is selected from alpha or beta hydrogen, alpha or beta hydroxyl, alpha or beta carboxyl, alpha or beta halo, alpha or beta alkoxy, alpha or beta alkyl, alpha-methyl-gamma-butyrolactone base;

R _is selected from α or β hydrogen, α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α-methyl-γ-butyrolactone base;

Or R ₄ and R ₅ are as follows:

Wherein: Y is selected from oxygen, sulfur, or the following formula:

Wherein: _R9 is selected from methoxy, hydrogen;

R is selected from alpha or beta hydrogen, alpha or beta hydroxyl _;

_R7 is selected from alpha or beta hydrogen, alpha or beta hydroxyl;

_R8 is selected from alpha or beta hydrogen, alpha or beta hydroxyl.

The application of a compound in the preparation of a pharmaceutical composition for preventing and/or treating pulmonary fibrosis is characterized in that the compound has the structural formula Ia.

in:

R is selected from α or β hydrogen, α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α or β α _- methyl-γ- butyrolactone group;

R2 is selected from α or β _hydrogen , α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α or β α-methyl-γ- butyrolactone group;

Or R ₁ and R ₂ are as follows:

Wherein: X is selected from oxygen, sulfur;

_R is selected from hydrogen, alpha or beta alkyl, alpha or beta hydroxyl, alpha or beta carboxy, alpha or beta halo, alpha or beta alkoxy;

_R4 is selected from alpha or beta hydrogen, alpha or beta hydroxyl, alpha or beta carboxyl, alpha or beta halo, alpha or beta alkoxy, alpha or beta alkyl, alpha-methyl-gamma-butyrolactone base;

R _is selected from α or β hydrogen, α or β hydroxyl, α or β carboxyl, α or β halo, α or β alkoxy, α or β alkyl, α-methyl-γ-butyrolactone base;

Or R ₄ and R ₅ are as follows:

Wherein: Y is selected from oxygen, sulfur, or the following formula:

Wherein: R ₆ is selected from methoxy, hydrogen.

The application is characterized in that the compound is an alkaloid represented by structural formula I or a pharmaceutically acceptable derivative thereof.

Said application is characterized in that said compound pharmaceutical derivative is a salt or ester of alkaloid shown in structural formula I.

The application is characterized in that the pharmaceutical composition comprises at least one active ingredient such as the alkaloid shown in structural formula I and a pharmaceutical carrier.

The application is characterized in that the pharmaceutical composition comprises 0.01%-99% by mass of the alkaloid shown in structural formula I and 0.01%-99% by mass of the pharmaceutical carrier.

The "pulmonary fibrosis" mentioned in the present invention refers to the pulmonary fibrosis of humans or animals caused by various reasons, which is characterized by the pathological changes of idiopathic pulmonary fibrosis.

The "prevention" refers to preventing or reducing the occurrence of pulmonary fibrosis after use in the presence of possible factors that cause pulmonary fibrosis; the "treatment" refers to reducing the degree of pulmonary fibrosis, or curing Pulmonary fibrosis makes it normal, or slows down the progression of pulmonary fibrosis.

Salts or esters of alkaloids refer to pharmaceutically acceptable salts, esters and the like.

The pharmaceutical composition includes at least one alkaloid active ingredient as shown in structural formula I and a pharmaceutical carrier. The so-called "active ingredient" refers to the function of preventing or treating pulmonary fibrosis. In the composition, the alkaloid can be used alone or together with other compounds as the active ingredient. The pharmaceutical carrier includes pharmaceutically acceptable excipients, fillers, diluents and the like. In the pharmaceutical composition, the content of the alkaloid with the structure shown in formula 1 is 0.01%-99%, the content of the pharmaceutical carrier is 0.01%-99%, and the percentages are mass percentages.

The dosage form of the pharmaceutical combination is not particularly limited, it may be in solid, semi-solid or liquid form, it may be in aqueous solution, non-aqueous solution or suspension, and it may be administered orally, intravenously, intramuscularly, intradermally or subcutaneously, or Administration through sublingual, rectal, transdermal absorption and respiratory nebulization. The daily dose of the pharmaceutical composition is about 0.01-1000 mg, preferably 1-500 mg.

When preventing or treating pulmonary fibrosis, the pharmaceutical composition of alkaloids such as structural formula I can be used alone or in combination with other drugs.

The alkaloids with structural formula I described in the present invention are prepared and separated from plants of the family Centipede (purity greater than 98%), and the rest of the raw materials or reagents are commercially available.

Beneficial effect

1. The present invention provides a new compound for preventing or treating pulmonary fibrosis such as the azazulene type alkaloid shown in structural formula I, proves that this type of alkaloid is for bleomycin-induced mouse lung fibrosis The chemical has significant preventive and therapeutic effects, can significantly reduce the degree of pulmonary fibrosis in experimental animals, and can be used for preparing medicines for treating pulmonary fibrotic diseases. It also shows that common cough medicines have no effect on pulmonary fibrosis. The present invention discovers for the first time that a variety of azazune-type alkaloids (I-IV) shown in structural formula I have anti-pulmonary fibrosis effects, which has not been reported in the literature before.

Specifically:

The results of the present invention show that the compound with the core shown in structural formula I, such as four alkaloids such as compounds I-IV, has significant preventive and/or therapeutic effects on pulmonary fibrosis (Example 3). The activity study of compound I further demonstrates that the structure shown in structural formula I has obvious anti-pulmonary fibrosis effect. The results of multi-index animal experiments show that the effect of this type of compound on improving pulmonary fibrosis is better than or equivalent to that of the positive control drug pirfenidone newly listed in Japan, and can significantly reduce the effect of bleomycin-induced pulmonary fibrosis in mice. death, significantly reduce the lung coefficient of model mice, improve the degree of pulmonary fibrosis of model mice, and significantly reduce the content of Hyp and pro-fibrosis factor TGF-β1 in lung tissue, indicating that the alkaloid with the structure shown in structural formula I can alleviate Inflammation of the lungs of mice induced by bleomycin model reduces collagen deposition in the lungs. The results of the present invention provide a scientific basis for the application of the azazulene-type alkaloids with the structure shown in structural formula I in the pharmaceutical composition for preventing and/or treating pulmonary fibrosis.

2. The experimental materials involved in the present invention come from a variety of medicinal materials, mainly stem stems, stem stems and stem stems, which are cheap and readily available.

Description of drawings

Fig. 1 Effects of four alkaloids and Kebiqing on mice with pulmonary fibrosis induced by bleomycin. A: Hydroxyproline content; B: HE-stained pathological section; C: Masson-stained pathological section. Among them, 1-sham operation group, 2-model group, 3-kebetidine, 4-pirfenidone, 5-compound I, 6-compound II, 7-compound III, 8-compound IV. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

Fig. 2 Effect of compound I on lung coefficient of mice with bleomycin-induced pulmonary fibrosis. Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

Fig. 3 Effect of compound I on pathological changes of lung histopathology in mice with bleomycin-induced pulmonary fibrosis (HE staining). Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group.

Fig. 4 Effect of compound I on inflammatory score of lung tissue in mice with bleomycin-induced pulmonary fibrosis (HE staining). Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

Fig. 5 Effect of compound I on pathological changes of lung histopathology in mice with bleomycin-induced pulmonary fibrosis (Masson staining). Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group.

Fig. 6 Effect of compound I on collagen content in lung tissue of mice with bleomycin-induced pulmonary fibrosis (average optical density value statistics). Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

Fig. 7 Effect of compound I on hydroxyproline content in lung tissue of mice with bleomycin-induced pulmonary fibrosis. Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

Fig. 8 Effect of compound I on TGFβ1 content in lung tissue of mice with bleomycin-induced pulmonary fibrosis (ELISA). Wherein, A: prevention group, B: treatment group; 1-sham operation group, 2-model group, 3-compound I low dose (30mg/kg) group, 4-compound I high dose (60mg/kg) group, 5 - Pirfenidone (300 mg/kg) group. Compared with the sham operation group, ^# P<0.05, ^## P<0.01; compared with the model group, ^* P<0.05, ^** P<0.01.

detailed description

Example 1 Preparation and structure identification of alkaloids

1. Medicinal materials and reagents

The medicinal material is the dried root of Stemona sessilifolia (Miq.) Miq., which was purchased from Bozhou Medicinal Materials Market in Anhui. Reagents such as ethanol, dichloromethane, and methanol were all analytically pure.

2. Extraction and separation

3 times of reflux extraction with 95% ethanol, concentrated under reduced pressure until there is no alcohol smell, acidified with 5% dilute hydrochloric acid to pH 1-2, filtered, the filtrate was alkalized with concentrated ammonia water to pH 10, extracted with chloroform, recovered chloroform to obtain the total biological base site. The total alkaloids were eluted by silica gel column chromatography, dichloromethane-methanol gradient (100:0～100:5), and the same fractions were combined according to the TLC results, and then subjected to repeated silica gel column chromatography and separated by Sephadex LH-20 column chromatography. , Compound II (Isoprotostemonine) and Compound I (Protostemonine, former 100-butine) were separated from dichloromethane-methanol (100:2) elution fraction; Compound IV (Stemoamide); Compound III (Neostemonine) was isolated from the elution fraction of dichloromethane-methanol (100:4); the above four compounds were analyzed by HPLC with a purity of over 98%.

3. Structural identification

Compound I (Protostemonine, former Protostemonine): white crystal (chloroform), positive for bismuth potassium iodide reaction, ESI-MS (m/z: 418[M+H] ⁺ ). ¹ H NMR (CDCl ₃ , 300MHz) δ: 1.92, 1.55 (2H, m, H-1), 1.89, 1.48 (2H, m, H-2), 3.27 (1H, m, H-3), 3.48, 2.92 (2H, dd, J=4.0, 15.5Hz; dd, J=7.1, 15.2Hz, H-5), 1.50, 1.65 (2H, m, H-6), 2.32, 1.50 (2H, m, H- 7), 4.08 (1H, ddd, J=10.4, 3.4, 14.3Hz, H-8), 2.19 (1H, ddd, J=10.4, 4.1, 9.5Hz, H-9), 3.73 (1H, m, H -9a), 2.89(1H, m, H-10), 2.04(3H, s, H-16), 1.41(3H, d, J=6.6Hz, H-17), 4.15(1H, ddd, J= 11.1, 5.5, 5.4Hz, H-18), 2.35, 1.52 (2H, m, H-19), 2.60 (1H, m, H-20), 1.23 (3H, d, J=7.0Hz, H-22 ), 4.12 (3H, s, H-23). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 26.8 (C-1), 27.6 (C-2), 64.1 (C-3), 46.4 (C-5), 20.2 (C-6), 34.2 (C- 7), 84.3(C-8), 56.0(C-9), 58.5(C-9α), 39.5(C-10), 149.2(C-11), 124.4(C-12), 163.2(C-13 ), 97.4(C-14), 170.1(C-15), 20.8(C-16), 9.2(C-17), 83.4(C-18), 33.8(C-19), 34.9(C-20) , 179.4 (C-21), 14.9 (C-22), 58.8 (C-23).

Compound II (Isoprotostemonine): Pale yellow crystals (methanol), positive for bismuth potassium iodide reaction, EI-MS (m/z: 418[M+H] ⁺ ). ¹ H NMR (CDCl ₃ , 300MHz) δ: 1.89, 1.55 (2H, m, H-1), 1.87, 1.48 (2H, m, H-2), 3.24 (1H, ddd, J=7.3, 7.5, 11.5 Hz, H-3), 3.50, 2.89 (2H, dd, J=14.8, 4.8Hz; dd, J=11.2, 14.8Hz, H-5), 1.50, 1.65 (2H, m, H-6), 2.32 , 1.50 (2H, m, H-7), 4.18 (1H, m, H-8), 2.12 (1H, ddd, J=10.4, 10.3, 5.3Hz, H-9), 3.69 (1H, ddd, J =5.6, 10.7, 10.6Hz, H-9a), 3.01 (1H, dq, J=6.7, 10.5, H-10), 2.01 (3H, s, H-16), 1.32 (3H, d, J=6.7 Hz, H-17), 4.14 (1H, m, H-18), 2.36, 1.52 (2H, m, 19), 2.58 (1H, m, H-20), 1.24 (3H, d, J=6.9Hz , H-22), 4.10 (3H, s, H-23). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 26.5 (C-1), 27.2 (C-2), 64.4 (C-3), 46.6 (C-5), 20.2 (C-6), 34.3 (C- 7), 82.5(C-8), 54.2(C-9), 58.4(C-9α), 41.7(C-10), 150.8(C-11), 125.7(C-12), 163.8(C-13 ), 98.3(C-14), 168.5(C-15), 18.1(C-16), 8.5(C-17), 83.3(C-18), 34.3(C-19), 34.8(C-20) , 179.2 (C-21), 14.9 (C-22), 59.5 (C-23).

Compound III (Neostemonine): colorless needle crystals (methanol), positive for modified bismuth potassium iodide reaction, FAB-MS (m/z: 320[M+H] ⁺ ). ¹ H NMR (CDCl ₃ , 300MHz) δ: 2.24, 1.85 (2H, m, H-1), 2.25, 2.05 (2H, m, H-2), 3.67, 3.10 (2H, m; ddd, J=15.8 , 3.0, 6.4Hz, H-3), 3.35 (2H, m, H-5), 2.14, 1.85 (2H, m, H-6), 1.62, 2.57 (2H, m, H-7), 4.18 ( 1H, ddd, J=10.8, 10.7, 3.7Hz, H-8), 2.22 (1H, m, H-9), 4.27 (1H, m, H-9a), 2.91 (1H, s, H-10) , 2.05 (3H, s, H-16), 1.40 (3H, d, J=6.8Hz, H-17), 4.10 (3H, s, H-18). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 23.4 (C-1), 26.8 (C-2), 52.4 (C-3), 49.8 (C-5), 17.5 (C-6), 32.6 (C- 7), 81.6(C-8), 52.1(C-9), 60.5(C-9α), 40.1(C-10), 146.9(C-11), 125.1(C-12), 163.0(C-13 ), 97.4 (C-14), 170.0 (C-15), 19.8 (C-16), 9.1 (C-17), 60.5 (C-18).

Compound IV (Stemoamide): white powder (methanol), positive for bismuth potassium iodide reaction, EI-MS (m/z: 224[M+H] ⁺ ). ¹ H NMR (CDCl ₃ , 300MHz) δ: 1.93, 1.61 (2H, m, H-1), 2.32 (2H, m, H-2), 2.55 (1H, ddd, J=14.2, 12.5, 1.5Hz, H-5α), 4.01 (1H, ddd, J=14.2, 4.7, 2.1Hz, H-5β), 1.75, 1.41 (2H, m, H-6), 1.62 (2H, m, H-7), 4.09 (1H, ddd, J=2.9, 10.2, 11.1Hz, H-8), 2.29 (1H, ddd, J=11.1, 12.4, 6.4Hz, H-9), 3.88 (1H, ddd, J=6.4, 11.1 , 6.3Hz, H-9a), 2.48 (1H, dq, J=12.4, 6.7Hz, H-10), 1.18 (3H, d, J=6.7Hz, H-12). ¹³ C NMR (CDCl ₃ , 75MHz) δ: 30.4 (C-1), 34.8 (C-2), 173.8 (C-3), 40.0 (C-5), 22.3 (C-6), 25.5 (C- 7), 77.5 (C-8), 52.5 (C-9), 55.8 (C-9α), 37.1 (C-10), 177.2 (C-11), 13.9 (C-12).

Example 2 Preparation of an animal model of pulmonary fibrosis

1. Main reagents and experimental animals

The bleomycin used in the experiment was purchased from Nippon Kayaku Co., Ltd., batch number 730342.

The SPF grade C57BL/6 mice (female, 8 weeks old) used in the experiment were purchased from the Comparative Medicine Center of Yangzhou University.

2. Model Preparation

Bleomycin-induced pulmonary fibrosis in mice is an internationally recognized animal model for screening anti-pulmonary fibrosis drugs. Acute lung injury caused by bleomycin modeling, mainly manifested as inflammation at the initial stage, fibrosis began to appear in about 10 days, and a large amount of collagen concentrated in the lungs, these pathological changes are very similar to those of idiopathic pulmonary fibrosis in clinical practice resemblance. Although the pathogenesis and mechanism of idiopathic pulmonary fibrosis are not clear, both bleomycin-induced pulmonary fibrosis and idiopathic pulmonary fibrosis are caused by damage to lung tissue leading to immune and inflammatory responses, which in turn lead to fibrotic pathology Therefore, the bleomycin-induced pulmonary fibrosis model can represent idiopathic pulmonary fibrosis and a series of pulmonary fibrosis diseases with similar pathological changes caused by different pathogenic factors.

Pulmonary fibrosis model preparation method: female C57BL/6 mice (8 weeks old) started modeling after 7 days of adaptation to the environment. The mice were fasted overnight, anesthetized with 3% chloral hydrate (10ml/kg, i.p.), fixed the mice, disinfected the neck of the mice, cut the skin of the neck longitudinally with as little trauma as possible, and separated the fascia and muscles longitudinally with tweezers , expose the trachea, inject about 35 μl (3.5 mg/kg) bleomycin into the trachea with a micro-syringe, then quickly stand upright and rotate the mouse for 3 to 5 minutes, so that the bleomycin can enter the left and right lung lobes evenly, observe the small For the respiratory condition of the rats, disinfect the neck wounds with 75% alcohol cotton, suture them up, drop 1-2 drops of penicillin injection on the sutures, put them back into a dry and clean cage to rest, and feed them normally after waking up. Surgical operations are carried out on the operating table at about 60°C. The same volume of normal saline for injection was injected into the trachea of the sham operation group.

Example 3 Identification of four alkaloids and Kebiqing anti-pulmonary fibrosis activity in mice

This example aims to study the anti-pulmonary fibrosis activity of the alkaloids obtained in Example 1 to identify whether they have an anti-pulmonary fibrosis effect. Since these alkaloids are the active components of cough suppressant in Baibu, a traditional Chinese medicine, in order to know whether the cough suppressant can resist pulmonary fibrosis, this example chooses a commonly used cough suppressant in clinical practice——pentoxyverine, pentoxyverine citrate tablet, cough suppressant Positive control drugs commonly used in pharmacological experiments) were simultaneously screened for anti-pulmonary fibrosis activity.

1. Main reagents and experimental animals

Compounds I-IV were obtained according to Example 1, and the purity was >98%. Kebiqing was purchased from Sinopharm Rongsheng Pharmaceutical Co., Ltd., batch number 13110221. The bleomycin used in the experiment was purchased from Nippon Kayaku Co., Ltd., batch number 730342. Pirfenidone (prifenidone, positive drug) was purchased from Dalian Meilun Biology, with a purity of >99%. Pirfenidone is a new drug for the treatment of pulmonary fibrosis developed by Marnac Corporation of the United States, and has authorized the development rights of Japan, Taiwan, and South Korea to Shionogi Corporation of Japan. On October 17, 2008, the company was the first to go public in Japan.

SPF grade C57BL/6 mice (female, 8 weeks old) used in the experiment were purchased from the Comparative Medicine Center of Yangzhou University.

2. Experimental method

The experimental mice were randomly divided into 8 groups, the model group and the Kebiqing group each had 10 mice, and the other groups had 5 mice. The first group is the sham operation group, the second group is the model group, the third group is the Kebiqing group, the fourth group is the positive drug pirfenidone group, and the fifth to eighth groups are the compound I~IV administration groups respectively. Groups 2 to 8 were modeled with bleomycin, and the method was the same as in Example 2. From the first day after modeling, the third group was given 30 mg/kg Kebiqing by intragastric administration every day, the fourth group was given 300 mg/kg pirfenidone by intragastric administration every day, and the 5th to 8th groups were given 30 mg/kg by intragastric administration every day. kg of compound I-IV, the sham operation group and the model group were given the same dose of solvent until the end of the 21st day. After the last administration, the mice were sacrificed, and the lung tissues were taken out. The right lobe was used to determine the content of hydroxyproline, and the left lobe was used to make pathological sections.

3. Experimental results

It can be seen from Figure 1-A that compared with the sham operation group, the content of hydroxyproline in the lung tissue of the mice in the model group was significantly increased, indicating that the collagen in the lung tissue of the mice in the model group was significantly increased, and the fibrosis was serious; 4 After administration of azazulene-type alkaloid monomers, they can significantly reduce the increase in hydroxyproline content caused by mouse modeling, and the effects of compounds I and II are better than the positive control pirfenidone, and the effects of compounds III and IV It was equivalent to pirfenidone; however, the Hyp content after Kebiqing administration was not significantly different from that of the model group, but was significantly higher than that of the sham operation group. It can be seen from Figure 1-B and 1-C that after 21 days of modeling, the structure of the lung lobules of the mice in the sham operation group was normal, the alveolar walls were intact, and no pathological changes of inflammation and fibrosis were seen; the lungs of the mice in the model group showed obvious inflammatory damage and Tissue clumps, normal lung tissue disappeared, alveolar occlusion, and obvious collagen deposition; after administration of the four azazulene-type alkaloids, all of them could significantly reduce the lung inflammation caused by bleomycin in mice, and significantly improve the degree of lung injury , Compounds I, II, and III can basically restore the normal structure of the lungs of model mice, and the effect of compound I is particularly obvious, which is almost equivalent to that of the blank group; Kebiqing can not improve the degree of lung tissue damage and fibrosis in model mice, The tissue sections were similar to those of the model group, with severe inflammatory damage, alveolar occlusion, and obvious collagen deposition. The result of this experiment shows that the alkaloid with the core of structural formula I can significantly inhibit and improve the pulmonary fibrosis of the bleomycin model mice, while the cough medicine can not improve the pulmonary fibrosis of the model mice.

Example 4 Effect of Compound I on Prevention and Treatment of Pulmonary Fibrosis

1. Experimental materials and methods

(1) Main reagents and experimental animals

Compound I was obtained according to Example 1, with a purity >98%. Pirfenidone (prifenidone, positive drug) was purchased from Dalian Meilun Biology, with a purity of >99%. Pirfenidone is a new drug for the treatment of pulmonary fibrosis developed by Marnac Corporation of the United States, and has authorized the development rights of Japan, Taiwan, and South Korea to Shionogi Corporation of Japan. On October 17, 2008, the company was the first to go public in Japan.

The experimental animals were model mice prepared according to Example 2, and the day of modeling was 0 day.

(2) Experimental method

The model animals were administered in groups according to prevention and treatment. According to the international general drug administration method of this model, the prophylactic administration starts from the second day of modeling and ends on the 14th day, and is administered continuously by intragastric administration; the therapeutic administration starts from the 8th day after modeling and ends on the 21st day, Continuous intragastric administration. The grouping and drug administration are shown in Table 1.

Table 1. Grouping and dosing regimen of mice with pulmonary fibrosis after modeling

2. Determination of Mouse Mortality

Preventive administration starts from day 0 to day 14 on the day of modeling, and therapeutic administration starts from day 8 to day 21 of modeling. The death of animals in each group of preventive administration is counted every day, and the survival of animals in each group is calculated. The results are shown in Table 2.

It can be seen from Table 2 that compared with the sham operation group, the mortality rate of the bleomycin-induced mice was 20% at 14 days and 33% at 21 days. Compared with model group, compound I has obvious protective effect to model mice, and low dosage group (30mg/kg) prevents and treats administration mouse death rate is respectively 0% and 17%, high dosage group (60mg/kg ) preventive and therapeutic administration of mouse mortality were 20% and 17%. The results of this experiment also showed that the protective effect of the low-dose group on the model mice was slightly better than that of the low-dose group regardless of the prophylactic or therapeutic administration. Overall, the effect of compound I on reducing the mortality of mice was significantly stronger than that of the positive control pirfenidone.

Table 2. The death situation of mice in each group of prophylactic and therapeutic administration

3. Detection of mouse lung coefficient

After the last administration, the mice were sacrificed, the lungs of the mice were peeled off and the wet weight was weighed, and the lung weight (mg) was divided by the mouse body weight (g) to obtain the lung coefficient (Figure 2).

It can be seen from Figure 2 that compared with the sham operation group, the lung coefficients of the model group were significantly increased. Regardless of prophylactic administration or therapeutic administration, the two dose groups (30, 60 mg/kg) of compound I can significantly reduce the lung coefficient of model mice, and the effect of the low dose group is better than that of the high dose group. Overall, the effect of compound I on reducing the lung coefficient of model mice was slightly better than that of the positive control pirfenidone.

4. HE staining pathological evaluation and inflammation score

After the last administration, the mice were anesthetized and sacrificed, and the lung tissues of the mice were taken out. The left lobular lung was immersed in 10% formalin, fixed in paraffin, embedded in paraffin, sectioned, and its pathological changes were observed by HE staining. It can be seen from Figure 3 that the structure of the lung lobule in the mice in the sham operation group was normal, the alveolar wall was intact, and no obvious pathological changes of inflammation and fibrosis were seen. In the model group, obvious inflammatory damage and tissue mass appeared in the lungs of the mice, the normal lung tissue disappeared, and the alveoli were occluded. Whether it is prophylactic administration or therapeutic administration, compound I can significantly reduce the lung inflammation caused by bleomycin, improve lung injury, and restore the normal structure of the lung, and the effect of the low-dose group (30mg/kg) is slightly better than At high doses, the lung inflammation basically disappeared, and the alveolar structure was clear.

Semi-quantitative statistical analysis was performed on the inflammatory grade of HE staining results. Grade 0: Normal tissue or minimal inflammatory changes; Grade 1 (+): Mild to moderate inflammatory changes without obvious lung tissue destruction; Grade 2 (++): Moderate to severe inflammatory damage, alveolar septum thickening , the formation of tissue clumps, or localized pneumonia areas lead to structural damage to the lung tissue; Grade 3 (+++): severe inflammatory injury, severe damage to the local area of the lung tissue structure causing lumen closure, etc. The inflammatory score results of each group of prophylactic administration and therapeutic administration are shown in Fig. 4 . Compared with the sham operation group, significant inflammation occurred in the lungs of bleomycin-induced mice; both dose groups (30, 60 mg/kg) of compound I could significantly reduce the lung inflammation caused by bleomycin, The effect of low dose was slightly better than that of high dose; overall, it was slightly better than the positive control pirfenidone.

5. Masson staining pathological evaluation and imaging analysis

Masson staining is a method of specifically staining fibrous collagen. After the last administration, the mice were anesthetized and killed, and the lung tissues of the mice were taken out. The left lobular lung was immersed in 10% formalin, fixed in paraffin, embedded in paraffin, sectioned, and observed by Masson staining for the deposition of collagen fibers. Preventive administration and therapeutic administration groups The results of Masson staining are shown in Figure 5, respectively. Use Image-Pro Plus6.0 for semi-quantitative analysis, measure the integrated optical density (IOD) value (IOD) of collagen in each visual field after Masson staining, analyze 5 specimens in each group, randomly select 5 visual fields for each specimen, and take the mean value as each group The relative content of collagen was analyzed statistically. The analysis results of the prophylactic and therapeutic administration groups are shown in Figure 6, respectively.

It can be seen from Figure 5 and Figure 6 that there was no obvious Masson collagen deposition in the lungs of mice in the sham operation group; after 14 days and 21 days of administration of bleomycin, obvious collagen accumulation appeared in the lungs of mice, and a large amount of fibrotic tissue was formed. 14 days is more serious. The two dose groups of compound I (30, 60 mg/kg) can significantly reduce the collagen deposition caused by bleomycin, and the effect of improving pulmonary fibrosis is extremely obvious. Among them, the effect of the low-dose group for prophylaxis was better than that of the high-dose group, and the effect of the high-dose group for treatment was slightly better than that of the low-dose group. Overall, compound I was better than the positive control pirfenidone in improving collagen deposition in model mice.

6. Determination of hydroxyproline content

Hydroxyproline mainly exists in collagen, and the content in elastin is very small, and it does not exist in other proteins. Therefore, the determination of hydroxyproline content can indicate the level of collagen content, so as to evaluate the degree of pulmonary fibrosis. Hydroxyproline was determined using a kit (Nanjing Jiancheng Biotechnology Co., Ltd.) and carried out according to the instructions of the kit. The results are shown in Figure 7.

It can be seen from Figure 7 that compared with the sham operation group, the content of hydroxyproline in the lung tissue of the model group mice was significantly increased, indicating that the lung tissue fibrosis of the model group mice was serious. Whether it is preventive administration or therapeutic administration, the high and low dose groups (30, 60mg/kg) of compound I can effectively reduce the content of hydroxyproline in model mice, indicating that compound I can significantly improve the effect of bleomycin Modeling-induced pulmonary fibrosis in mice.

7. Elisa content determination of TGF-β1

TGF-β1 is recognized as a powerful fibrosis factor, which can stimulate cells to synthesize and secrete extracellular matrix components, and can also change the activity of matrix degrading enzyme components, directly aggravating the deposition of ECM. Reducing the content of TGF-β1 in lung tissue can slow down the process of pulmonary fibrosis. The content of TGF-β1 in the lung tissue of mice was determined by Elisa kit (Shanghai Yikesai Biological Products Co., Ltd.), according to the instructions of the kit, and the results are shown in FIG. 8 .

It can be seen from Fig. 8 that compared with the sham operation group, the TGF-β1 content in the lung tissue of mice administered with bleomycin was significantly increased. Compared with the model group, both high and low doses (30, 60 mg/kg) of Compound I can significantly reduce the level of TGF-β1 and significantly improve the degree of pulmonary fibrosis, whether it is preventive administration or therapeutic administration. (30mg/kg) the effect is more significant.

discuss

The results of the present invention show that the compounds with the core shown in structural formula I, such as compounds I-IV and other four azazune alkaloids, have significant preventive and/or therapeutic effects on pulmonary fibrosis (Example 3). The activity study of compound I further demonstrates that the structure shown in structural formula I has obvious anti-pulmonary fibrosis effect. The results of multi-index animal experiments show that the effect of this type of azazulene-type alkaloid on improving pulmonary fibrosis is better than or equivalent to that of the positive control drug pirfenidone newly launched in Japan, and can significantly reduce the bleomycin-induced The death of the pulmonary fibrosis mouse significantly reduces the lung coefficient of the model mouse, improves the degree of pulmonary fibrosis of the model mouse, and significantly reduces the content of Hyp and pro-fibrosis factor TGF-β1 in the lung tissue, indicating that it has the structure shown in structural formula I The compound can reduce the lung inflammation of mice induced by bleomycin and reduce the deposition of collagen in the lungs.

The results of the present invention provide a scientific basis for the application of the azazulenes alkaloids having the structure shown in structural formula I in the pharmaceutical composition for preventing and/or treating pulmonary fibrosis.

Claims (3)

1. application in preparation prevention or treatment pulmonary fibrosis medicine for a kind of compound, described compound is following structural formula Middle one kind：

2. a kind of application in preparation prevention or treatment pulmonary fibrosis medicine for compositionss is it is characterised in that described medicine group Compound at least includes a kind of active component compound as claimed in claim 1 and a kind of pharmaceutical carrier.

3. application in preparation prevention or treatment pulmonary fibrosis medicine for a kind of compositionss as described in claim 2, it is special Levy and be, described pharmaceutical composition include compound described in the claim 1 that mass percent is 0.01%～99% and Mass percent is 0.01%～99% pharmaceutical carrier.