A balasubamide derivative and its application in the preparation of drugs for treating acute lung injury

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to a balasubamide derivative and its application in the preparation of a drug for treating acute lung injury. It has been proven by experiments that the balasubamide derivative can significantly reduce the expression of TNF-α in a macrophage cell line inflammation model, has no obvious toxicity at effective doses, and has high safety; moreover, the balasubamide derivative (+)3C-20 can significantly alleviate acute lung injury induced by sepsis or pulmonary exposure to bacterial endotoxin, and achieve the effect of improving acute lung injury in mice by inhibiting the expression of inflammatory factors in macrophages, significantly increasing the survival rate of mice in the acute lung injury model.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to a balasubamide derivative and its application in the preparation of drugs for treating acute lung injury. Background Art

[0002] Sepsis is a critical illness caused by the invasion of pathogenic microorganisms such as bacteria into the body. Its clinical manifestations are systemic inflammatory response syndrome, accompanied by complications such as septicemia and insufficient organ blood perfusion. Moreover, sepsis can also lead to septic shock, acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), multiple organ failure, and even death. Currently, it is believed that the out-of-control inflammatory response and immune disorder caused by pathogenic microorganism infection of the body are the main causes of sepsis. During sepsis infection, endotoxin and other substances stimulate immune cells to produce a large number of inflammatory mediators, promoting the recruitment and activation of inflammatory response cells, especially macrophages, in tissues, and further producing cytokines, chemokines, oxygen free radicals, etc., forming a cascading reaction, resulting in damage and even failure of organs such as the lungs, and ultimately causing the death of patients.

[0003] Acute lung injury (ALI) is a common and severe clinical disease. ALI often develops into acute respiratory distress syndrome (ARDS), and the latter is characterized by pulmonary edema, decreased lung compliance, and acute hypoxic respiratory failure, ultimately leading to death. According to statistics, there are approximately 150,000 ARDS patients in North America every year. Although the mortality rate has decreased significantly due to auxiliary medical devices such as mechanical ventilation, 40% - 70% of patients still die from ARDS. If accompanied by complications such as sepsis, the mortality rate is as high as 90%. Moreover, sepsis is the most common triggering factor for ALI / ARDS. The pathophysiological mechanism of ALI is often considered to be related to the imbalance of pulmonary inflammation. Its pathological characteristics are an increase in the production of pre-inflammatory factors, an increase in the infiltration of inflammatory cells, and apoptosis of alveolar epithelial cells, resulting in lung damage.

[0004] Balasubamide is an octalactam compound extracted from the leaves of Clausena indica (Dalz.) Oliv, a plant of the Rutaceae Clausena genus in Sri Lanka. After structural modification, it has a significant therapeutic effect on neuroinflammation caused by brain injury ((+)3C-20 (CN110684027A)). Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the high mortality rate of existing diseases such as acute lung injury and the lack of better treatment means, and provide a balasubamide derivative.

[0006] Another object of the present invention is to provide the application of the balasubamide derivative in the preparation of anti-inflammatory drugs.

[0007] Another object of the present invention is to provide the use of balasulamide derivatives in the preparation of drugs for treating acute lung injury.

[0008] The above object of the present invention is achieved by the following technical solutions:

[0009] A balasulamide derivative, the balasulamide derivative having the structure of formula (I):

[0010]

[0011] Wherein, R is hydrogen, halogen, C 1～4 alkyl, C 1～7 alkoxy, benzyloxy or nitro-substituted benzoyloxy;

[0012] R1 is hydrogen or R2 is hydrogen, C 1～4 alkyl, C 2～5 alkenylalkyl; and R, R1, and R2 are not simultaneously hydrogen.

[0013] Preferably, R is hydrogen, halogen, C 1～3 alkyl, C 1～5 alkoxy, benzyloxy or nitro-substituted benzoyloxy; R1 is hydrogen or R2 is hydrogen, C 1～3 alkyl, C 2～3 alkenylalkyl; and R, R1, and R2 are not simultaneously hydrogen.

[0014] More preferably, R is hydrogen, halogen, methyl, ethoxy, benzyloxy or nitro-substituted benzoyloxy; R1 is hydrogen or R2 is hydrogen, methyl, ethyl, propyl, allyl; and R, R1, and R2 are not simultaneously hydrogen.

[0015] In addition, the present invention also provides the use of balasulamide derivatives in the preparation of anti-inflammatory drugs, the balasulamide derivative having the structure of formula (I):

[0016]

[0017] Wherein, R is hydrogen, halogen, C 1～4 alkyl, C 1～7 alkoxy, benzyloxy or nitro-substituted benzoyloxy;

[0018] R1 is hydrogen or R2 is hydrogen, C 1～4 alkyl, C 2～5 alkenylalkyl.

[0019] In addition, the present invention also provides the use of balasulamide derivatives in the preparation of drugs for treating acute lung injury, and the balasulamide derivatives have the structure of formula (I):

[0020]

[0021] Wherein, R is hydrogen, halogen, C 1～4 alkyl, C 1～7 alkoxy, benzyloxy or nitro-substituted benzoyloxy; R1 is hydrogen or R2 is hydrogen, C 1～4 alkyl, C 2～5 alkenylalkyl.

[0022] Furthermore, the acute lung injury is caused by sepsis.

[0023] Even further, the acute lung injury is induced by bacterial endotoxin. Preferably, the bacterial endotoxin is lipopolysaccharide.

[0024] Furthermore, the acute lung injury includes sepsis caused by peripheral inflammation, inflammatory lung injury, bronchial asthma, tracheitis, bronchitis, chronic obstructive pulmonary disease, cor pulmonale, pulmonary fibrosis.

[0025] Furthermore, the balasulamide derivatives treat acute lung injury by inhibiting the expression of inflammatory factors.

[0026] Even further, the inflammatory factors include TNF-α, IL-1β, IL-6, COX-2.

[0027] Furthermore, the drug contains an effective amount of balasulamide derivatives or pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, tautomers thereof.

[0028] Even further, the drug contains an effective amount of balasulamide derivatives and pharmaceutically acceptable excipients.

[0029] Furthermore, the dosage form of the drug is a pulmonary or nasal inhaled nebulizer, metered dose inhaler or dry powder inhaler.

[0030] The present invention has the following beneficial effects:

[0031] It has been experimentally proven in the present invention that balasulamide derivatives can significantly reduce the expression of TNF-α in the macrophage cell line inflammation model, have no obvious toxicity at effective doses, and have high safety; and balasulamide derivative (+)3C-20 can significantly relieve acute lung injury induced by sepsis or pulmonary exposure to bacterial endotoxin, and improve the acute lung injury of mice by inhibiting the expression of inflammatory factors in macrophages, significantly increasing the survival rate of mice in the acute lung injury model. Description of the Drawings

[0032] Figure 1 Statistical chart of the effects of balasamide derivatives with different structures on the expression and release of inflammatory factor TNF-α in lipopolysaccharide-stimulated RAW264.7 macrophages at a concentration of 10 μM.

[0033] Figure 2 Statistical chart of the effects of balasamide derivatives with different structures on the cell viability of RAW264.7 at a concentration of 10 μM.

[0034] Figure 3 Statistical chart of the effects of different concentrations of (+)3C-20 on the expression and release of inflammatory factor TNF-α in lipopolysaccharide-stimulated RAW264.7 macrophages.

[0035] Figure 4 Statistical chart of the effects of different concentrations of (+)3C-20 on the expression levels of inflammatory genes in lipopolysaccharide-stimulated RAW264.7 macrophages.

[0036] Figure 5 Statistical chart of the effects of different concentrations of (+)3C-20 on the expression and release of inflammatory factor TNF-α in primary macrophages BMDMs stimulated by lipopolysaccharide.

[0037] Figure 6 Statistical chart of the effects of (+)3C-20 on the body weight and lung organ coefficient of mice with sepsis complicated with lung injury induced by intraperitoneal injection of lipopolysaccharide.

[0038] Figure 7 Statistical chart of the effects of (+)3C-20 on the expression of inflammatory factor TNF-α in the serum of mice with sepsis complicated with lung injury induced by intraperitoneal injection of lipopolysaccharide and histological section of lung tissue.

[0039] Figure 8 Statistical chart of the effects of (+)3C-20 on the mRNA expression levels of inflammatory genes in the lung tissue of mice with sepsis complicated with lung injury induced by intraperitoneal injection of lipopolysaccharide.

[0040] Figure 9 Statistical chart of the effects of (+)3C-20 on the body weight and lung organ coefficient of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide.

[0041] Figure 10 Statistical chart of the effects of (+)3C-20 on the expression of inflammatory factor TNF-α in the lung tissue of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide and histological section of lung tissue.

[0042] Figure 11Statistical chart of the effect of (+)-3C-20 on the mRNA expression levels of inflammatory genes in the lung tissues of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide.

[0043] Figure 12 Statistical chart of the effect of (+)-3C-20 on the wet / dry weight ratio of the lungs, TNF-α level, total protein, and the ratio of each immune myeloid cell in the bronchoalveolar lavage fluid of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide.

[0044] Figure 13 Statistical chart of the effect of (+)-3C-20 on the survival rate of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide. Detailed implementation manners

[0045] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0046] Lipopolysaccharide (LPS) was purchased from Beyotime Biotechnology Co., Ltd., and other reagents were commercially available analytical pure reagents. Cells: RAW264.7 macrophage cell line was purchased from the China Center for Type Culture Collection. Animals: ICR mice and C57BL / 6J mice were both purchased from Nanjing Huachuang Biotechnology Co., Ltd. Kits: ELISA kit for TNF-α was purchased from Shenzhen Dakewei Biotechnology Co., Ltd., RNA extraction kit and SYBR reagent were both purchased from Novoprotein Scientific Inc., primers were purchased from Genewiz, Inc., and flow antibodies were purchased from Nanjing Fumaisi Biotechnology Co., Ltd.

[0047] Among them, the synthesis of balasubamide derivatives (+)-3C, (+)-3C-20, (±)-3C-20, and (+)-B001 was prepared with reference to Chinese Patent Application CN110684027A.

[0048] The preparation methods for the remaining balasubamide derivatives are as follows:

[0049]

[0050] S1. Weigh 3.53 g (10 mmol) of the compound of formula (+)-(II) and dissolve it in 30 mL of distilled water. Add 30 mL of ethyl acetate. Under an ice bath, adjust the pH to 3 - 4 with 1 M HCl. Let it stand for layering. Extract the aqueous layer with 3 × 10 mL of ethyl acetate. Combine the organic layers and wash them with 15 mL of saturated sodium chloride aqueous solution. Dry, concentrate, add 30 mL of DMF, and add 1.62 g (10 mmol) of CDI under an ice bath. Raise the temperature to room temperature and stir for 1 h. Under stirring, add 3.03 g (30 mmol) of triethylamine and 10 mmol of the compound of formula (III). React at room temperature for 8 h. After the reaction is completed, add 50 mL of distilled water and extract with 3 × 30 mL of ethyl acetate. Combine the organic layers and wash them with 3 × 30 mL of saturated sodium chloride aqueous solution. Dry, concentrate, add 10 mL of n-hexane for pulping, evaporate the solvent under reduced pressure. Recrystallize the obtained solid with ethyl acetate and n-hexane to obtain the compound of formula (IV).

[0051]

[0052] S2. At room temperature, add 6.0 mmol of the compound of formula (IV), 0.744 g (1.2 mmol) of ytterbium trifluoromethanesulfonate and 20 mL of anhydrous acetonitrile to a 50 mL eggplant-shaped flask and stir. React at room temperature for 8 h. Concentrate, dissolve the residue with 30 mL of dichloromethane, wash with 2 × 20 mL of saturated sodium chloride aqueous solution. Dry, concentrate. Purify the residue by column chromatography to obtain the compound of formula (V).

[0053]

[0054] S3. At room temperature, dissolve 3 mmol of the compound of formula (V) in 10 mL of acetonitrile in a 50 mL eggplant-shaped flask. Under stirring, add 0.690 g (3.6 mmol) of EDCI, 81 mg (0.6 mmol) of DMAP and 0.655 g (3 mmol) of the compound of formula (VI) in sequence. Stir and react for 3 h. After the reaction is completed, concentrate, dissolve the residue with 30 mL of dichloromethane, wash with 10 mL of saturated sodium bicarbonate aqueous solution and 10 mL of saturated sodium chloride aqueous solution in sequence. Dry, concentrate. Purify the residue by column chromatography. Recrystallize the obtained solid with V 正己烷 :V 乙酸乙酯 = 1:1 to obtain the compound of formula (I).

[0055] The structures and related data of the obtained balasamide derivatives are as follows:

[0056] 1. (+)-B3a: C 30 H 23 F7N2O3, 592.1597, White powder 1.39 g, yield 78.1%, m.p. 222.9 - 223.4℃, (c 0.5, CH3OH); 1 1H NMR (600 Hz, Chloroform-d) δ 8.17 (s, 1H), 7.56 (d, J = 8.20 Hz, 2H), 7.50 (d, J = 8.13 Hz, 2H), 7.42 (d, J = 8.23 Hz, 2H), 7.19 (d, J = 7.99 Hz, 2H), 7.06 (td, J = 7.98, 4.96 Hz, 1H), 7.00 (d, J = 8.01 Hz, 1H), 6.76 (dd, J = 11.41, 7.76 Hz, 1H), 5.67 (d, J = 11.14 Hz, 1H), 4.68 (d, J = 11.18 Hz, 1H), 3.96 - 3.85 (m, 2H), 3.51 - 3.37 (m, 2H), 2.93 (t, J = 7.72 Hz, 2H), 2.78 - 2.61 (m, 2H).

[0057]

[0058] 2. (+)-B3c: C 31 H 26 F6N2O4, 604.1797, White powder 1.44 g, yield 79.5%, m.p. 272.6 - 274.7 °C, (c 0.5, CH3OH); 1 1H NMR (600 Hz, Chloroform-d) δ 7.96 (s, 1H), 7.53 (d, J = 8.15 Hz, 2H), 7.49 (d, J = 7.91 Hz, 2H), 7.38 (d, J = 8.15 Hz, 2H), 7.17 (d, J = 7.96 Hz, 1H), 7.12 (d, J = 8.73 Hz, 1H), 6.89 (d, J = 2.25 Hz, 1H), 6.83 (dd, J = 8.74, 2.37 Hz, 1H), 5.66 (d, J = 11.16 Hz, 1H), 4.68 (d, J = 11.07 Hz, 1H), 3.98 - 3.94 (m, 1H), 3.86 (s, 3H), 3.52 - 3.42 (m, 2H), 3.40 - 3.35 (m, 1H), 2.91 (t, J = 7.63 Hz, 2H), 2.77 - 2.60 (m, 2H).

[0059]

[0060] 3. (+)-B3d: C 30 H 23F7N2O3, 592.1597, White powder 1.37 g, yield 76.9%, m.p. 147.3 - 149.2 °C, (c 0.5, CH3OH); 1 H NMR(600 Hz, Chloroform - d) δ7.98(s, 1H), 7.59(d, J = 8.21 Hz, 2H), 7.52 - 7.47(m, 3H), 7.39(d, J = 8.11 Hz, 1H), 7.30(d, J = 8.10 Hz, 1H), 7.25 - 7.22(m, 1H), 7.16(d, J = 7.99 Hz, 1H), 6.96 - 6.85(m, 2H), 6.86 - 6.77(m, 1H), 5.70(d, J = 3.95 Hz, 1H), 4.47 - 4.46(m, 1H), 3.54 - 3.28(m, 3H), 2.99(t, J = 7.46Hz, 1H), 2.73 - 2.69(m, 2H), 2.62 - 2.43(m, 2H).

[0061]

[0062] 4. (+)-B3e: C 30 H 23 BrF6N2O3, 652.0796, White powder 1.58 g, yield 80.8%, m.p. 226.1 - 227.3 °C, (c 0.5, CH3OH); 1 H NMR(600 Hz, Chloroform - d) δ8.07(s, 1H), 7.67(d, J = 1.51 Hz, 1H), 7.55(d, J = 7.90 Hz, 2H), 7.37(d, J = 8.20 Hz, 2H), 7.49(d, J = 8.08 Hz, 2H), 7.25(d, J = 1.80 Hz, 1H), 7.18(d, J = 8.01 Hz, 2H), 7.10(d, J = 8.52 Hz, 1H), 5.65(d, J = 11.13 Hz, 1H), 4.69(d, J = 11.15 Hz, 1H), 3.50 - 3.44(m, 2H), 3.39 - 3.36(m, 1H), 2.92(t, J = 7.63 Hz, 2H), 2.78 - 2.61(m, 3H).

[0063]

[0064] 5. (+)-B3f: C 30 H23 ClF6N2O3, 608.1301, White powder, 1.46 g, yield 79.8%, m.p. 301.9 - 302.7 °C, (c 0.5, CH3OH); 1 H NMR (600 Hz, Chloroform - d) δ8.02 (s, 1H), 7.55 (d, J = 8.22 Hz, 2H), 7.49 (d, J = 8.08 Hz, 2H), 7.44 (d, J = 8.49 Hz, 1H), 7.38 (d, J = 8.23 Hz, 2H), 7.22 (d, J = 1.63 Hz, 1H), 7.18 (d, J = 7.98 Hz, 2H), 7.11 (dd, J = 8.52, 1.78 Hz, 1H), 5.66 (d, J = 11.14 Hz, 1H), 4.68 (d, J = 11.15 Hz, 1H), 4.01 - 3.97 (m, 1H), 3.53 - 3.36 (m, 3H), 2.92 (t, J = 7.66 Hz, 2H), 2.78 - 2.61 (m, 2H).

[0065]

[0066] 6. (+)-B3g: C 31 H 26 F6N2O3, 588.1848, White powder, 1.38 g, yield 78.2%, m.p. 189.1 - 190.5 °C, (c 0.5, CH3OH); 1 H NMR (600 Hz, Chloroform - d) δ7.99 (s, 1H), 7.55 (d, J = 8.18 Hz, 2H), 7.49 (d, J = 8.09 Hz, 2H), 7.41 (dd, J = 12.91, 8.20 Hz, 3H), 7.18 (d, J = 8.01 Hz, 2H), 7.07 (t, J = 7.28 Hz, 1H), 6.98 (d, J = 7.10 Hz, 1H), 5.66 (d, J = 11.15 Hz, 1H), 4.75 (d, J = 11.12 Hz, 1H), 3.99 - 3.95 (m, 1H), 3.58 - 3.53 (m, 1H), 3.49 - 3.36 (m, 2H), 2.93 (t, J = 7.69 Hz, 2H), 2.79 - 2.62 (m, 2H), 2.36 (s, 3H).

[0067]

[0068] 7. (+)-B3h: C 37 H 30 F6N2O4, 680.2110, White powder, 1.63 g, yield 79.7%, m.p. 118.9 - 120.1 °C, (c 0.5, CH3OH); 1 1H NMR (600 Hz, Chloroform - d) δ 8.02 (s, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.48 (t, J = 7.7 Hz, 4H), 7.41 - 7.36 (m, 4H), 7.33 (t, J = 7.4 Hz, 1H), 7.16 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 6.91 (dd, J = 8.7, 2.4 Hz, 1H), 5.66 (s, 1H), 5.11 (s, 2H), 4.68 (d, J = 11.1 Hz, 1H), 3.97 - 3.90 (m, 1H), 3.49 - 3.33 (m, 3H), 2.91 (t, J = 7.7 Hz, 2H), 2.76 - 2.60 (m, 2H).

[0069]

[0070] 8. (+)-c1: C 31 H 26 F6N2O3, 588.18, White solid, 78.3% yield, mp. 234 - 236 °C, [α]20D = +13.6 (c 0.5, CH3OH), 1 1H NMR (600 MHz, CDCl3) δ 7.57 (dd, J = 12.1, 8.1 Hz, 3H), 7.51 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 3.8 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.16 (dd, J = 8.0, 4.0 Hz, 1H), 5.87 (d, J = 5.1 Hz, 1H), 5.70 (d, J = 11.2 Hz, 1H), 4.80 (d, J = 11.2 Hz, 1H), 4.00 – 3.92 (m, 1H), 3.66 – 3.57 (m, 1H), 3.51 – 3.39 (m, 5H), 2.95 (t, J = 7.7 Hz, 2H), 2.78 (dt, J = 15.6, 7.7 Hz, 1H), 2.69 – 2.63 (m, 1H).

[0071]

[0072] 9. (+)-c2: C 32 H 28 F6N2O3, 602.20, White solid, 70.6% yield, mp. 245 - 247 °C, [α]20D = +14.2 (c 0.5, CH3OH), 1 1H NMR (600 MHz, CDCl3) δ 7.59 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 5.7 Hz, 1H), 7.22 (dd, J = 9.8, 4.3 Hz, 3H), 7.18–7.14 (m, 1H), 5.83 (d, J = 4.7 Hz, 1H), 5.68 (d, J = 11.1 Hz, 1H), 4.77 (d, J = 11.1 Hz, 1H), 4.02 (dq, J = 14.6, 7.2 Hz, 1H), 3.95 (dd, J = 12.8, 3.8 Hz, 1H), 3.90 (td, J = 14.5, 7.2 Hz, 1H), 3.65–3.56 (m, 1H), 3.4–3.39 (m, 2H), 2.96 (t, J = 7.7 Hz, 2H), 2.79 (dt, J = 15.5, 7.6 Hz, 1H), 2.71–2.64 (m, 1H), 1.03 (t, J = 7.2 Hz, 3H).

[0073]

[0074] 10. (+)-c3: C 33 H 30 F6N2O3, 616.21, White solid, 68.5% yield, mp. 242 - 245 °C, [α]20 D = +17.3 (c 0.5, CH3OH), 11H NMR (600 MHz, CDCl3) δ 7.58 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 7.7 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H), 7.27–7.19 (m, 4H), 7.16 (d, J = 7.4 Hz, 1H), 5.72 (d, J = 5.0 Hz, 1H), 5.67 (d, J = 11.0 Hz, 1H), 4.77 (d, J = 11.0 Hz, 1H), 3.91 (ddd, J = 15.7, 15.1, 7.5 Hz, 2H), 3.79–3.70 (m, 1H), 3.66–3.57 (m, 1H), 3.47–3.37 (m, 2H), 2.96 (t, J = 7.6 Hz, 2H), 2.84–2.76 (m, 1H), 2.72–2.64 (m, 1H), 1.64–1.57 (m, 1H), 1.35 (dd, J = 14.9, 7.8 Hz, 1H), 0.81 (t, J = 7.3 Hz, 3H).

[0075]

[0076] 11. (+)-c4: C 33 H 28 F6N2O3, 614.20, White solid, 67.5% yield, mp. 251 - 253 °C, [α]20 D = +16.2 (c 0.5, CH3OH), 1 1H NMR (600 MHz, CDCl3) δ 7.58 (d, J = 7.8 Hz, 1H), 7.52 (dd, J = 22.0, 8.1 Hz, 4H), 7.38 (d, J = 8.1 Hz, 2H), 7.22–7.14 (m, 5H), 6.16 (s, 1H), 5.66 (d, J = 11.2 Hz, 1H), 5.6–5.55 (m, 1H), 4.75 (d, J = 10.3 Hz, 1H), 4.69 (d, J = 11.2 Hz, 1H), 4.55 (ddd, J = 23.4, 12.8, 10.7 Hz, 1H), 4.48 (d, J = 17.1 Hz, 1H), 4.36–4.26 (m, 1H), 3.98–3.87 (m, 1H), 3.68–3.55 (m, 1H), 3.50–3.38 (m, 2H), 2.93 (t, J = 7.7 Hz, 2H), 2.81–2.59 (m, 2H).

[0077]

[0078] 12. (+)-c5:C 35 H 33 F6N3O5, 689.23, gave 0.12 g of white solid, yield 32.6%, mp. 189 - 192 °C, (c 1, CH3OH), 1 H NMR (600 MHz, CDCl3) δ 8.49 (s, 1H), 7.51 (d, J = 8.6 Hz, 3H), 7.44 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.6 Hz, 2H), 7.17 (d, J = 8.0 Hz, 3H), 7.14–7.11 (m, 2H), 5.87 (s, 1H), 5.76 (s, 1H), 5.55 (d, J = 8.8 Hz, 1H), 4.73 (d, J = 11.0 Hz, 1H), 3.82 (d, J = 10.5 Hz, 1H), 3.48 (dd, J = 13.7, 8.3 Hz, 1H), 3.31 (ddd, J = 23.7, 15.0, 8.1 Hz, 2H), 2.84 (s, 2H), 1.42 (s, 9H).

[0079]

[0080] 13. (+)-D7a:C 32 H 28 F6N2O4, 618.19, 30 mg of white solid, yield 43.5%. m.p. 239.6 - 242.1 °C, (c 0.5, MeOH); 1 H NMR (600 MHz, Chloroform - d) δ 8.16 (s, 1H), 7.56 (dd, J = 23.5, 8.9 Hz, 4H), 7.36 (d, J = 7.6 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 7.08 (d, J = 9.5 Hz, 1H), 6.96 (d, J = 3.5 Hz, 1H), 6.85 (dd, J = 9.9, 1.9 Hz, 1H), 5.60 (d, J = 10.9 Hz, 1H), 4.66 (d, J = 10.3 Hz, 1H), 4.13 (q, J = 8.2 Hz, 2H), 3.54–3.35 (m, 4H), 2.91 (t, J = 6.9 Hz, 2H), 2.74–2.58 (m, 2H), 1.43 (t, J = 6.8 Hz, 3H).

[0081]

[0082] 14. (+)-D7c: 739.62, 94.1 mg of yellow solid, yield 74.9. m.p. 225.2 - 227.9 °C, (c 0.5, CHCl3); 1 1H NMR (600 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.42–8.33 (m, 4H), 7.53 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 2.2 Hz, 1H), 7.11 (dd, J = 13.9, 8.3 Hz, 3H), 6.92 (dd, J = 8.6, 2.2 Hz, 1H), 5.67 (d, J = 11.2 Hz, 1H), 4.71 (d, J = 11.2 Hz, 1H), 3.90 (t, J = 10.4 Hz, 1H), 3.39–3.28 (m, 3H), 2.87 (t, J = 7.8 Hz, 2H), 2.72–2.57 (m, 2H).

[0083]

[0084] 15. (+)-3C: C 20 H 17 F3N2O2, 374.12, (c 0.5, CHCl3) 1 1H NMR (600 Hz, DMSO-d6) δ 10.91 (s, 1H), 7.65 (d, J = 8.27 Hz, 2H), 7.54 (dd, J = 14.17, 8.21 Hz, 3H), 7.34 (dd, J = 6.92, 4.17 Hz, 1H), 7.18 (d, J = 7.94 Hz, 1H), 7.03 - 7.00 (m, 1H), 6.98 - 6.95 (m, 1H), 5.21 (d, J = 9.34 Hz, 1H), 4.89 (t, J = 9.83 Hz, 1H), 4.30 (d, J = 10.25 Hz, 1H), 3.7 - 3.65 (m, 1H), 3.28 - 3.18 (m, 2H).

[0085]

[0086] 16. (+)-B001: C 19 H 17 BrH2O2, 385.25, (c 0.5, CHCl3) 11H NMR (500 MHz, Chloroform-d) δ 9.47 (s, 1H), 7.66 (d, J = 1.5 Hz, 1H), 7.50 (t, J = 7.1 Hz, 1H), 7.36–7.23 (m, 4H), 7.19–7.12 (m, 3H), 5.82 (d, J = 7.7 Hz, 1H), 4.97 (dd, J = 7.7, 7.0 Hz, 1H), 4.54 (dd, J = 7.0, 0.9 Hz, 1H), 3.61–3.47 (m, 2H), 2.93 (td, J = 7.1, 2.2 Hz, 2H).

[0087]

[0088] 17, (±)-3C-20: C 30 H 24 F6N2O3, 574.16, 1 1H NMR (600 Hz, Chloroform-d) δ 7.80 (s, 1H), 7.57 (d, J = 7.75 Hz, 1H), 7.51 (dd, J = 20.54, 7.99 Hz, 4H), 7.36 (d, J = 8.10 Hz, 2H), 7.23 (d, J = 7.72 Hz, 1H), 7.20 - 7.15 (m, 4H), 5.61 (d, J = 11.23 Hz, 1H), 4.63 (d, J = 11.21 Hz, 1H), 3.90 - 3.85 (m, 1H), 3.53 - 3.48 (m, 1H), 3.37 - 3.29 (m, 2H), 2.93 (t, J = 7.71 Hz, 2H), 2.78 - 2.61 (m, 2H).

[0089] 18, (+)-3C-20: C 30 H 24 F6N2O3, 574.16, 1 1H NMR (600 Hz, Chloroform-d) δ 8.22 (s, 1H), 7.55 - 7.52 (m, 3H), 7.48 (d, J = 8.18 Hz, 2H), 7.38 (d, J = 8.18 Hz, 2H), 7.22 (d, J = 7.47 Hz, 1H), 7.18 - 7.12 (m, 4H), 5.67 (d, J = 11.20 Hz, 1H), 4.73 (d, J = 11.15 Hz, 1H), 3.97 - 3.92 (m, 1H), 3.56 - 3.52 (m, 1H), 3.46 - 3.34 (m, 2H), 2.89 (t, J = 7.47 Hz, 2H), 2.75 - 2.58 (m, 2H).

[0090]

[0091] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0092] Example 1 In vitro screening of anti-inflammatory active drugs for macrophage line inflammation model

[0093] 1. Experimental method:

[0094] RAW264.7 cells were seeded into 96-well plates at a density of 1×10^5 cells per well. When the cell density reached 70% - 80%, 18 balasulamide derivatives were separately prepared into 10 mM working solutions with DMSO, and then mouse monocyte macrophages RAW264.7 were pretreated with a final concentration of 10 μM. After 3 h, except for the blank group (Control well), lipopolysaccharide LPS (100 ng / mL) was added and incubated for 2 h. The supernatant was collected for standby, and the expression level of the inflammatory factor TNF-α in the cell supernatant was detected according to the operation instructions of the commercial ELISA kit.

[0095] 2. Experimental results:

[0096] The results are shown in Table 1 and [[ID=ID=19]] Figure 1 .

[0097] Table 1 Anti-inflammatory activities of balasulamide derivatives against macrophage line inflammation model

[0098]

[0099]

[0100] Note: Compared with the blank group, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.05, ### P < 0.001.

[0101] As can be seen from the results in the table and figure, lipopolysaccharide can significantly up-regulate the expression of the inflammatory factor TNF-α in macrophages RAW264.7. Most of the balasulamide derivatives have good anti-inflammatory activities and can well inhibit the expression of inflammatory factors in macrophages. Among them, (+)3C-20 and (±)-3C-20 have better anti-inflammatory activities than other derivatives at a concentration of 10 μM.

[0102] Example 2 Cytotoxicity test of balasulamide derivatives

[0103] 1. Experimental method:

[0104] RAW264.7 cells were seeded into 96-well plates at a density of 1×10^5 cells per well. When the cell density reached 70%-80%, RAW264.7 cells were pretreated with 10 mM working solutions of 18 balasamide derivatives at a final concentration of 10 μM for 24 h, and cell viability was determined by the CCK-8 method.

[0105] 2. Experimental results:

[0106] The results are shown in Table 2 and Figure 2 .

[0107] Table 2 Effects of balasamide derivatives on the viability of RAW264.7 cells

[0108]

[0109]

[0110] As shown in the results of the table and figure, all balasamide derivatives showed no obvious toxicity to RAW264.7 cells at 10 μM.

[0111] Example 3 Determination of the expression of inflammatory factors in macrophage cell lines

[0112] 1. Experimental method:

[0113] RAW264.7 cells were seeded into 96-well plates at a density of 1×10^5 cells per well. When the cell density reached 70%-80%, RAW264.7 cells were pretreated with (+)3C-20 (0.1-80 μM). After 3 h, lipopolysaccharide LPS (100 ng / mL) was added to all wells except the Control wells and incubated for 2 h. The expression level of the inflammatory factor TNF-α in the cell supernatant was detected according to the operation instructions of a commercial ELISA kit; and the cell viability under different concentrations of (+)3C-20 administration was determined according to the operation instructions of the CCK-8 kit.

[0114] 2. Experimental results:

[0115] The results are shown in Table 3 and Figure 3 .

[0116] Table 3 Effects of different concentrations of (+)3C-20 on TNF-α and cell viability in macrophages

[0117] Group Percentage of TNF-α in the Model group (%) Percentage of CCK-8 in the Model group (%) Control 20.36±0.17 110.43±2.47 Model *100±0.43 100±1.53 (+)3C-20 (0.1 μM) 99.83±0.89 108.6±1.49 (+)3C-20 (0.5 μM) 98.13±1.56 106.11±2.44 (+)3C-20 (1 μM) 94.38±0.45 106.01±1.75 (+)3C-20 (2 μM) <![CDATA[88.59±2.07 # > 106.41±3.04 (+)3C-20 (5 μM) <![CDATA[83.73±3.44 ### > 105.55±1.06 (+)3C-20 (10 μM) <![CDATA[51.19±1.33 ### > 96.64±1.45 (+)3C-20 (20 μM) <![CDATA[39.35±3.15 ### > 95.83±2.29

[0118] Note: Compared with the blank group, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0119] As can be seen from the results in the table and figures, lipopolysaccharide can significantly up-regulate the expression of the inflammatory factor TNF-α in macrophages RAW264.7. The (+) balasubamide derivative (+) 3C-20 has good anti-inflammatory activity and can well inhibit the expression of the inflammatory factor TNF-α in macrophages. The effective dose is 2 μM to 20 μM.

[0120] Example 4 Determination of the Expression Level of Inflammatory Genes in Macrophage Lines

[0121] 1. Experimental method:

[0122] Seed RAW264.7 cells at 5×10^5 cells per well in a 24-well plate. When the cell density reaches 70% - 80%, pretreat RAW264.7 with (+) 3C-20 (20 μM). After 3 h, add lipopolysaccharide LPS (100 ng / mL) to all wells except the Control well and incubate for 2 h. Discard the supernatant, and use Trizol (Nanjing Novoprotein, R401-01) to fully lyse the cells at the bottom of the plate. Extract RNA according to its RNA extraction instructions. After reverse transcription into cDNA, perform qPCR using the amplification method of 95 °C for 5 min, (95 °C for 10 s, 60 °C for 30 s) × 40 cycles, and finally perform statistical calculation with ΔCt.

[0123] 2. Experimental results:

[0124] The results are shown in Table 4 and Figure 4 .

[0125] Table 4 Effects of Balasubamide Derivatives on the Expression Level of Inflammatory Genes in Macrophage Lines

[0126] Group Change multiple of TNF-α Change multiple of IL-1β Change multiple of IL-6 Change multiple of COX-2 Control 1±0.07 1±0.06 1±0 1±0.06 Model <![CDATA[14.72±0.7 *** > <![CDATA[97.8±12.78 *** > <![CDATA[43.73±7.27 *** > <![CDATA[11.23±0.43 *** > (+)3C-20 (5 μM) 11.03±0.87 <![CDATA[62.02±7.52 # > <![CDATA[23.17±0.9 # > <![CDATA[4.20±0.16 ## > (+)3C-20 (10 μM) <![CDATA[6.00±1.96 ## > <![CDATA[28.51±0.4 ## > <![CDATA[16.09±0.24 ## > <![CDATA[3.95±0.9 ## > (+)3C-20 (20 μM) <![CDATA[4.49±0.81 ## > <![CDATA[12.58±1.72 ### > <![CDATA[6.53±0.31 ## > <![CDATA[3.49±0.0 8### >

[0127] Note: Compared with the blank group, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0128] As can be seen from the results in the table and figures, lipopolysaccharide can significantly up-regulate the gene expression of the inflammation-related genes TNF-α, IL-1β, IL-6, and COX-2 in macrophages RAW264.7. After administration of the balasubamide derivative (+) 3C-20, this up-regulation trend can be significantly alleviated, and it can dose-dependently inhibit the expression of inflammatory genes, that is, (+) 3C-20 has good anti-inflammatory activity, and the effective doses are 5 μM, 10 μM, and 20 μM.

[0129] Example 5 Determination of the Expression of Inflammatory Factors in Primary Macrophage Lines

[0130] 1. Experimental methods:

[0131] (1) Isolation of primary macrophages (bone marrow derived macrophages, BMDMs): C57BL / 6J mice were sacrificed by cervical dislocation and soaked in 75% ethanol for whole-body disinfection. The mice were fixed on a mouse board with their limbs in a supine position. The skin of the hind limbs was cut open, and the legs were cut at the ankle and femoral joints. The muscles and knee joints were removed. A 1 mL syringe was used to aspirate pre-cooled serum-free DMEM to rinse the bone marrow until the leg bones turned pale. The rinsing solution was filtered through a 200-mesh filter membrane and collected into a 15 mL centrifuge tube. The cells were centrifuged at 500×g for 5 min, and the supernatant was discarded. The cell pellet was resuspended with 1 mL of red blood cell lysate. After 1 min of red blood cell lysis, 5 mL of DMEM was added to terminate the lysis. After centrifugation at 500×g for 5 min, the supernatant was discarded. The cells in the lower layer were resuspended with DMEM containing 10% FBS and incubated overnight. After centrifuging to collect the monocytes, they were resuspended with DMEM medium containing 20% FBS, and M-CSF was added at a final concentration of 25 ng / mL. The cells were seeded into a 96-well plate at a density of 6×10^5 cells per well and cultured in a cell incubator at 37°C and 5% CO2 concentration for 48 h. Then, M-CSF was added again at a final concentration of 25 ng / mL per well and cultured for another 48 h. The monocytes adhered and differentiated into macrophages.

[0132] (2) Determination of inflammatory factor expression in primary macrophages BMDMs: BMDMs were pretreated with (+)3C-20 at final concentrations (0.1 μM, 1 μM, 2.5 μM, 5 μM) for 3 h, and then lipopolysaccharide LPS (100 ng / mL) was added to all wells except the Control well and incubated for 3 h. According to the operation instructions of the commercial ELISA kit, the expression level of the inflammatory factor TNF-α in the cell supernatant was detected. According to the operation instructions of the CCK-8 kit, the cell viability under different concentrations of (+)3C-20 administration was determined.

[0133] 2. Experimental results:

[0134] The results are shown in Table 5 and Figure 5 .

[0135] Table 5 Effects of different concentrations of (+)3C-20 on TNF-α and cell viability in primary macrophages

[0136] Group Percentage of TNF-α in the Model group (%) Percentage of CCK-8 in the Model group (%) Control 14.55±0.3 108.01±2.25 Model <![CDATA[100±2.91 *** > 100±2.13 (+)3C-20 (0.1 μM) 88.17±7.91 100.35±3.74 (+)3C-20 (1 μM) 86.35±7.41 96.71±2.26 (+)3C-20 (2.5 μM) <![CDATA[62.94±8.54 ## > 97.49±1.35 (+)3C-20 (5 μM) <![CDATA[35.12±4.63 ### > 89.87±3.68

[0137] Note: Compared with the blank group, *** P < 0.001; compared with the model group, ## P < 0.05, ### P < 0.001.

[0138] As shown by the results in the table and figures, lipopolysaccharide can significantly upregulate the expression of the inflammatory factor TNF-α in primary murine macrophages BMDMs. The balasumide derivative (+)3C-20 has good anti-inflammatory activity and can effectively inhibit the expression of the inflammatory factor TNF-α in macrophages. The effective doses on BMDMs are 2.5 μM and 5 μM.

[0139] Example 6 Pharmacodynamic evaluation of (+)3C-20 in improving sepsis-induced acute lung injury

[0140] 1. Recording of body weight and lung coefficient

[0141] Grouping of animals for drug administration: The experimental animals were male ICR mice, which were randomly grouped by body weight into a control group, a model group, a positive drug group, and a drug administration group. In the control group and the model group, the solvent (the solvent was prepared by mixing DMSO: Kolliphor HS15: Saline at a ratio of 1:2:7) was injected intraperitoneally. In the positive drug group, 5 mg / kg of dexamethasone (DEX) was injected intraperitoneally. In the drug administration group, 10 mg / kg and 50 mg / kg of (+)3C-20 were injected intraperitoneally. Both dexamethasone and (+)3C-20 were dissolved in the above solvent and were clear and transparent. After continuous drug administration for 3 days, 2 h after the last drug administration, except for the control group which was injected with an equal volume of normal saline intraperitoneally, the other groups were injected with LPS (10 mg / kg) intraperitoneally. Body weight and lung organ coefficient: On the first day, the mice were weighed and randomly grouped, and their weights were measured before continuous drug administration for 3 days to statistically analyze the growth status of the mice. At 2 h after LPS injection intraperitoneally on the 3rd day of drug administration, the mice were dissected 6 h later, and the lungs were taken and weighed and recorded. The results are shown in Table 6 and Figure 6 。

[0142] Table 6 Body weight and lung coefficient of mice

[0143] Group Body weight (Day1) Body weight (Day2) Body weight (Day3) Lung coefficient (%) G1: Control 25.05±0.2 25.86±0.21 26.14±0.37 0.631±0.009 G2: Model 25.58±1.11 25.52±1.13 25.89±1.04 <![CDATA[0.746±0.01 *** > G3: Dexamethasone (5 mg / kg) 25.11±0.52 26.23±0.38 26.97±0.45 <![CDATA[0.649±0.01 ## > G4: (+)3C-20 (10 mg / kg) 25.42±1.1 25.46±1.2 25.77±1.03 0.714±0.02 G5: (+)3C-20 (50 mg / kg) 25.34±0.57 26.16±1.22 27.01±1.08 <![CDATA[0.672±0.014 ## >

[0144] Note: Compared with the blank group, *** P < 0.001; compared with the model group, ## P < 0.01.

[0145] 2. Determination of lung pathology and serum inflammatory factors

[0146] At 6 h after LPS injection intraperitoneally on the 3rd day, whole blood was collected from the orbital cavity, centrifuged to prepare serum, and stored at -80 °C. The lungs were perfused with PBS and fixed with paraformaldehyde. The fixed lungs were dehydrated, embedded in paraffin, sectioned, and then stained with hematoxylin-eosin (H&E). The serum was tested with a commercial TNF-α ELISA kit. The tissue pathological results and data statistics are shown in Figure 7 and Table 7.

[0147] Table 7 Statistical Data of Lung Pathology and Serum Inflammatory Factors

[0148] Group Percentage of alveolar cells in the area (%) TNF-α content in serum (% of Model) G1: Control 29.22±0.87 23.11±0.59 G2: Model <![CDATA[47.24±2.0 * > <![CDATA[100±7.19 *** > G3: Dexamethasone (5 mg / kg) <![CDATA[32.2±0.95 # > <![CDATA[40.09±2.26 ### > G4: (+)3C-20 (10 mg / kg) 38.68±3.73 78.38±11.32 G5: (+)3C-20 (50 mg / kg) <![CDATA[30.43±2.41 # > <![CDATA[55.06±5.96 ### >

[0149] Note: Compared with the blank group, * P < 0.05, *** P < 0.001; compared with the model group, # P < 0.05, ### P < 0.001.

[0150] 3. Determination of the Expression Level of Inflammation-related Genes in Lung Tissue

[0151] Take a small amount of frozen lung tissue, grind it thoroughly, and according to the operation instructions of the RNA extraction kit, use RT-PCR technology to detect the expression of inflammation-related genes in the lung tissue. The experimental results are shown in Table 8 and Figure 8 .

[0152] Table 8 Expression of Inflammation-related Genes in Lung Tissue

[0153] Group Change multiple of TNF-α Change multiple of IL-1β Change multiple of IL-6 Change multiple of iNOS G1: Control 1±0.09 1±0.07 1±0.13 1±0.12 G2: Model <![CDATA[17.14±1.16 *** > <![CDATA[23.27±2.96 *** > <![CDATA[54.28±8.04 *** > <![CDATA[14.31±1.82 *** > G3: Dexamethasone (5 mg / kg) <![CDATA[6.09±0.82 ### > <![CDATA[8.65±1.04 ### > 40.56±7.55 <![CDATA[2.61±0.39 ### > G4: (+)3C-20 (10 mg / kg) <![CDATA[10.58±1.72 ## > 9.68±0.67 47.5±7.38 12.44±1.28 G5: (+)3C-20 (50 mg / kg) <![CDATA[5.55±0.86 ### > <![CDATA[10.13±1.72 ## > <![CDATA[13.73±1.56 ## > <![CDATA[5.42±1.02 ### >

[0154] Note: Compared with the blank group, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0155] 4. Result Analysis

[0156] After administration of each drug group, there was no significant change in the body weight of mice. After 6 hours of intraperitoneal injection of lipopolysaccharide, the lung organ coefficient of mice was significantly up-regulated, TNF-α in serum and lung tissue increased significantly, the lung tissue was significantly damaged, and the expression level of related inflammation genes in the lung tissue increased significantly; compared with the Model group, the 3C-20 (50 mg / kg) administration group could significantly down-regulate the lung coefficient, significantly reduce the expression and release of TNF-α in serum, reduce the expression of related inflammation genes in the lung tissue, and had a significant improvement effect on lung pathological damage.

[0157] Pharmacodynamic Evaluation of Example 7 (+) 3C-20 in Improving Acute Lung Injury Caused by Pulmonary Exposure to Bacterial Endotoxin

[0158] 1. Recording of Body Weight and Lung Organ Coefficient

[0159] Animal grouping, drug administration and model establishment: The experimental animals were male ICR mice, which were randomly grouped by body weight into a control group, a model group, a positive drug group, and a drug administration group. In the control group and the model group, the solvent (prepared by mixing DMSO: Kolliphor HS15: Saline at a ratio of 1:2:7) was intraperitoneally injected. In the positive drug group, 5 mg / kg of dexamethasone was intraperitoneally injected. In the drug administration group, 10 mg / kg and 50 mg / kg of (+)3C-20 were intraperitoneally injected. Both dexamethasone and (+)3C-20 were dissolved in the above-mentioned solvent and were clear and transparent. After continuous drug administration for 3 days, 2 hours after the last drug administration, the mice were anesthetized with 2.5% isoflurane, the neck was disinfected, the neck skin was incised, the trachea was separated. Except for the control group in which 50 μL of Saline was intratracheally instilled (it.), 50 μL of 10 mg / mL LPS (LPS was dissolved in Saline) with a dose of 20 mg / kg was instilled into the tracheas of the other groups, and then the skin was sutured. 6 hours after the LPS instillation, the mice were sacrificed, and the serum and lung tissues were collected for standby. Body weight and lung organ coefficient: On the first day, the mice were weighed and randomly grouped. Before continuous drug administration for 3 days, they were weighed again. 2 hours after the last drug administration, 50 μL of 20 mg / kg LPS was instilled into the trachea. 6 hours later, the mice were dissected, and the lung organs were taken and weighed. The results are shown in Table 9 and Figure 9 。

[0160] Table 9 Body weight and lung coefficient of mice

[0161] Group Body weight (Day1) Body weight (Day2) Body weight (Day3) Lung coefficient (%) G1: Control 27.67±0.47 28.16±0.54 28.11±0.57 0.603±0.011 G2: Model 27.75±0.35 28.33±0.41 29.07±0.38 <![CDATA[0.651±0.01 *** > G3: Dexamethasone (5 mg / kg) 27.51±0.11 28.75±0.17 29.48±0.17 <![CDATA[0.591±0.008 ## > G4: (+)3C-20 (10 mg / kg) 27.67±0.38 27.44±0.43 27.93±0.53 0.656±0.007 G5: (+)3C-20 (50 mg / kg) 27.63±0.48 27.04±0.48 27±0.61 <![CDATA[0.591±0.011 ### >

[0162] Note: ns P > 0.05, *** P < 0.001, vs Control; ## P < 0.01, ### P < 0.001, vs LPS.

[0163] 2. Determination of lung pathology and serum inflammatory factors

[0164] 6 hours after the LPS was instilled into the trachea on the 3rd day, for a part of the mice, the right lobe of the lung was perfused with PBS and fixed with paraformaldehyde, and for the other part of the mice, the lungs were taken after dissection and stored frozen in liquid nitrogen for standby; the fixed right lung was dehydrated, paraffin-embedded and sectioned, and then stained with hematoxylin-eosin (H&E). 40 mg of the frozen lung was taken and added to 300 μL of RIPA lysis buffer, homogenized at a frequency of 60 Hz for 4 cycles with 45 s on and 15 s off, and after sufficient lysis, centrifuged at 12,000 rpm at 4°C, and the supernatant was taken. The protein concentration was calibrated and adjusted with a BCA protein assay kit, and then the level of the inflammatory factor TNF-α in the lung tissue homogenates of each group was measured with a commercial TNF-α ELISA kit. The results are shown in Table 10 and Figure 10 。

[0165] Table 10 Statistical data of lung pathology and serum inflammatory factors

[0166] Group Percentage of alveolar cells in the area (%) TNF-α content in lung tissue homogenate (% of Model) G1: Control 30.882±0.731 27.337±1.481 G2: Model <![CDATA[45.641±1.045 *** > <![CDATA[100±9.29 *** > G3: Dexamethasone (5 mg / kg) <![CDATA[34.322±2.452 ### > <![CDATA[54.882±6.836 ### > G4: (+)3C-20 (10 mg / kg) <![CDATA[36.611±1.64 ### > 90.587±7.374 G5: (+)3C-20 (50 mg / kg) <![CDATA[32.805±0.814 ### > <![CDATA[61.408±6.525 ### >

[0167] Note: Compared with the blank group, *** P < 0.001; compared with the model group, ### P < 0.001.

[0168] 3. Determination of the expression levels of lung tissue inflammation-related genes

[0169] Take a small amount of frozen lung tissue, add Trizol lysis reagent and grind it thoroughly. Extract RNA according to the operation instructions of the RNA extraction kit, and use RT-PCR technology to detect the expression of inflammation-related genes in the lung tissue. The experimental results are shown in Table 11 and Figure 11 .

[0170] Table 11 Expression of inflammation-related genes in lung tissue

[0171] Group Change multiple of TNF-α Change multiple of IL-1β Fold change of IL-6 Fold change of iNOS Fold change of COX-2 G1:Control 1±0.22 1±0.13 1±0.13 1±0.18 1.07±0.12 G2:Model <![CDATA[20.06±1.38 *** > <![CDATA[19.87±2.19 *** > <![CDATA[28.55±2.55 *** > <![CDATA[9.88±1.68 *** > <![CDATA[2.8±0.49 ** > G3:Dexamethasone(5mg / kg) 18.63±2.26 <![CDATA[12.38±1.28 ## > <![CDATA[14.75±2.71 ### > 10.02±1.78 2.75±0.24 G4:(+)3C-20(10mg / kg) <![CDATA[13.32±0.94 # > <![CDATA[13.04±1.24 ## > <![CDATA[17.12±2.28 ## > <![CDATA[4.56±0.4 ## > 2.05±0.38 G5:(+)3C-20(50mg / kg) <![CDATA[8.56±1.21 ### > <![CDATA[11.94±1.05 ## > <![CDATA[15.11±1.58 ### > <![CDATA[3.75±0.72 ## > <![CDATA[1.14±0.24 ## >

[0172] Note: Compared with the blank group, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0173] 4. Result analysis

[0174] After administration of each drug group, there was no significant change in the body weight of mice. After 6 hours of intratracheal instillation of lipopolysaccharide, the lung organ coefficient of mice was significantly up-regulated, the TNF-α level in the lung tissue was significantly increased, the lung tissue was significantly damaged, and the expression levels of related inflammation genes in the lung tissue were significantly increased; compared with the Model group, the (+)3C-20 (50 mg / kg) administration group could significantly down-regulate the lung coefficient, significantly reduce the expression and release of TNF-α in the lung tissue, reduce the expression of related inflammation genes in the lung tissue, and have a significant improvement effect on lung pathological damage.

[0175] Example 8 Improvement effect of (+)3C-20 on acute lung injury caused by intratracheal instillation of lipopolysaccharide

[0176] Animal grouping and drug administration: 50 male ICR mice were randomly divided into 3 groups according to body weight, namely the control group, the model group, and the 50 mg / kg (+) 3C-20 group. The drug preparation, administration, and modeling methods were the same as those in Example 7. After continuous pre-administration for 3 days, 2 hours after the last administration, LPS was instilled into the trachea for modeling. After 6 hours of modeling, some mice were sacrificed directly, and the whole lungs were weighed to obtain the wet weight. After the whole lungs were baked at 60 °C for 24 hours, their weights were measured as the dry weight, and the wet-to-dry weight ratios of each group were statistically obtained. The remaining mice were given bronchoalveolar lavage 6 hours after modeling, and bronchoalveolar lavage fluid (BALF) was collected. The lavage procedure was as follows: after the mice were sacrificed, their four limbs were fixed face up, the neck skin was incised, the trachea was separated, and a small incision was made at an appropriate position. A 16G needle with a flattened tip was inserted to a depth of 1 cm, and the trachea was tied tightly to the needle stem as an indwelling needle. A 1 mL syringe was used to slowly push 400 μL of pre-cooled PBS, aspirated twice, and the above operations were repeated. The recovered BALF was combined, and the recovery efficiency was 70% - 80%. The pooled BALF was centrifuged at 500×g at 4 °C for 5 minutes, and the supernatant was collected. The total protein content level in the supernatant was measured using a BCA kit, and the expression level of the inflammatory factor TNF-α in the BALF supernatant was detected according to the operating instructions of a commercial ELISA kit. The results are shown in Table 12 and Figure 12 .

[0177] Resuspend the bottom precipitate after centrifugation of BALF with 100 μL of ACK Buffer (Thermo A10492 red blood cell lysing agent), mix well and let stand for 1 min. Terminate red blood cell lysis with 1 mL of FACS Buffer (PBS containing 1% FBS), centrifuge at 500×g at 4°C for 5 min, discard the supernatant, add 100 μL of FACS Buffer, add 1 μL of CD16 / CD32 Fc Block antibody (1:100), and incubate in the dark at 4°C for 30 min; add 1 mL of FACS Buffer, centrifuge at 500×g at 4°C for 5 min, discard the supernatant, add 100 μL of FACS Buffer to resuspend, and add each fluorescent dye (mCD45-PerCP-Cy5.5, mCD11b-FITC, mF4 / 80-APC, mLy6C-PE, mLy6G-BV421, FVS-780) at a ratio of 1:300. The single-stained tubes of each fluorescent dye are also prepared according to the above ratio, and incubate in the dark at 4°C for 30 min; add 1 mL of FACS Buffer to resuspend, centrifuge at 500×g at 4°C for 5 min, discard the supernatant, repeat the washing once, centrifuge and discard the supernatant, then add 500 μL of 1% paraformaldehyde to fix in the dark for 30 min, centrifuge at 500×g at 4°C for 5 min, discard the supernatant, add 200 μL of FACS Buffer to resuspend, and store in the dark at 4°C. Before loading, dilute the stained cell suspension with FACS Buffer, transfer it to a flow cytometry tube and perform detection according to the operating procedures of the CytoFlex flow cytometer; Flow cytometry gating strategy: First, adjust the gain according to each single-stained tube and appropriately adjust the compensation, among which CD45 + , CD11b + , F4 / 80 + are macrophages, CD45 + , CD11b + , Ly6G + are neutrophils, CD45 + , CD11b + , Ly6C + are monocytes. The results are shown in Table 12 and Figure 12 .

[0178] Table 12 The improvement effect of (+)3C-20 on acute lung injury induced by intratracheal instillation of lipopolysaccharide

[0179] Group Wet / Dry ratio TNF-α level in BALF Total protein level in BALF Macrophage(%) Neutrophil(%) Monocyte(%) Control 4.3±0.15 11.92±0.34 52.71±3.55 0.29±0.1 2.64±0.85 1.06±0.59 Model <![CDATA[5.04±0.12 *** > <![CDATA[100±0.04 *** > <![CDATA[100±6.12 *** > <![CDATA[2.87±0.48 *** > <![CDATA[24.86±3.28 *** > <![CDATA[5.61±0.25 *** > (+)3C-20(50mg / kg) <![CDATA[4.28±0.06 ### > <![CDATA[43.31±5.19 ### > <![CDATA[73.44±3.09 ## > <![CDATA[0.82±0.27 ## > <![CDATA[10.47±2.13 ## > 4.13±0.66

[0180] Note: *** P < 0.001, vs Control; # P < 0.05, ## P < 0.01, ### P < 0.001, vs Model.

[0181] The experimental results showed that 6 h after intratracheal instillation of lipopolysaccharide, the wet / dry weight ratio of the lungs of mice was significantly up-regulated, and the levels of TNF-α and total protein in bronchoalveolar lavage fluid (BALF) were both significantly increased. The infiltration of macrophages, neutrophils, and monocytes in BALF was also significantly up-regulated. In the (+)3C-20 (50 mg / kg) administration group, compared with the Model group, the wet / dry weight ratio of the lungs of mice was significantly down-regulated, the levels of total protein and TNF-α in BALF were down-regulated, and the infiltration of macrophages, neutrophils, and monocytes was reduced at the same time.

[0182] Example 9 Effect of (+)3C-20 on the survival rate of mice with acute lung injury induced by intratracheal instillation of lipopolysaccharide

[0183] Animal grouping and administration: Forty-five male ICR mice were randomly divided into 5 groups according to body weight, namely the control group, the model group, the positive drug dexamethasone (5 mg / kg) group, and the (+)3C-20 (10 mg / kg, 50 mg / kg) groups, with 9 mice in each group. The drug preparation, administration method, and modeling method were the same as those in Example 7. Taking the time point of LPS intratracheal instillation as 0 h, the survival status of the mice was examined at 4 h, 12 h, 16 h, 20 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and the survival curve was statistically analyzed. The results are shown in Figure 13 (where *** P < 0.001, vs LPS (20 mg / kg)).

[0184] As can be seen from the figure, 50 mg / kg (+)3C-20 can significantly improve the survival rate of mice with an acute lung injury model induced by LPS (20 mg / kg it.).

[0185] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of balasamide derivatives in the preparation of drugs for treating acute lung injury, characterized in that, The balasamide derivative has the structure of formula (I): （I） Among them, R is hydrogen, halogen, C 1~4 alkyl or C 1~7 alkoxy; R1 is hydrogen; R2 is hydrogen, C 1~4 alkyl or allyl.

2. Use of a pharmaceutically acceptable salt or enantiomer of balasamide derivative in the preparation of a drug for treating acute lung injury, characterized in that, The balasamide derivative has the structure of formula (I): （I） Among them, R is hydrogen, halogen, C 1~4 alkyl or C 1~7 alkoxy; R1 is hydrogen; R2 is hydrogen, C 1~4 alkyl or allyl.

3. The application according to claim 1 or 2, characterized in that, The dosage form of the drug is an inhalation aerosol, an aerosol or a dry powder inhaler.

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