Luteolin derivative as well as preparation method and application thereof

Abstract

The invention discloses a luteolin derivative as well as a preparation method and application thereof. The luteolin derivative provided by the invention can obviously inhibit the expression of macrophage inflammatory factors and improve the activity of macrophages in an in-vitro experiment. In an in-vivo experiment, the LPS-induced acute lung injury of the mouse can be remarkably prevented, expression of peripheral blood and lung tissue inflammatory factors is inhibited, injury of the lung tissue of the mouse is improved, and the normal physiological structure of the lung tissue of the mouse is recovered. Therefore, the luteolin derivative can effectively prevent the acute lung injury, has a protection effect on injured lung tissues, can be used as a medicine for treating, relieving or preventing the acute lung injury, and also provides a new thought and theoretical basis for the development of the medicine for treating the acute lung injury.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and specifically relates to a luteolin derivative and a preparation method and application thereof. Background Art

[0002] Acute lung injury (ALI) is a lung disease characterized by alveolar inflammatory cell infiltration. Various immune cells, including neutrophils, rapidly infiltrate cells, trigger a cytokine storm, and damage the epithelial and endothelial cell layers of the lungs. This often further develops into acute respiratory distress syndrome (ARDS). ALI and ARDS most often occur during pneumonia, sepsis, aspiration of gastric contents, or severe trauma. Both ALI and ARDS are life-threatening diseases that show severe inflammatory responses and damage to alveolar structures and lung wall structures. Lipopolysaccharides (LPS), a component of the outer wall of the cell wall of Gram-negative bacteria, can trigger a rapid and strong inflammatory response, impair the normal function of immune cells, and then recruit inflammatory cells to the lungs, leading to ALI. In clinical practice, anti-inflammatory drugs commonly used to treat ALI, such as glucocorticoids, can cause symptoms such as skin atrophy, decreased bone density, and gastrointestinal discomfort. Although strategies for treating ALI are constantly improving, there is still a lack of specific drugs, and poor prognosis remains a major challenge in supporting patient health. Current pharmacological studies have shown that luteolin has a protective effect on ALI caused by LPS. However, there are no reports on the application of luteolin derivatives in the research of drugs for the prevention and treatment of ALI. Summary of the invention

[0003] One of the purposes of the present invention is to provide a luteolin derivative, and another purpose of the present invention is to provide a preparation method and application of the luteolin derivative.

[0004] One of the purposes of the present invention is achieved by providing a luteolin derivative having a molecular formula of C 29 H 29 N3O8S, molecular weight is 579.624, and its structural formula is shown in formula (I): (I).

[0005] Another object of the present invention is achieved by the method for preparing the luteolin derivative, the synthesis route of which is shown in formula (II): (II); Wherein, the operation of step a is as follows: after mixing luteolin with potassium carbonate and DMF, 1,2-dibromoethane is added to react to obtain compound 1; The operation of step b is as follows: Compound 1 is mixed with 1,2-dibromoethane and reacted to obtain compound 2; The operation of step c is as follows: Compound 2 is mixed with 4-(N-BOC-amino)piperidine and DMF, and then Et3N is added to react to obtain compound 3; The operation of step d is as follows: dissolving compound 3 in a mixed solution of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacting at room temperature to obtain compound 4; The operation of step e is as follows: compound 4 is dissolved in a mixed solution of DCM and Et3N, and pyridine-3-sulfonyl chloride is added to react to obtain compound 5J, i.e., the target luteolin derivative.

[0006] The use of the luteolin derivative in preparing a drug for treating and / or alleviating and / or preventing acute lung injury.

[0007] The beneficial effects of the present invention are as follows: the luteolin derivatives provided by the present invention can significantly inhibit the expression of macrophage inflammatory factors in in vitro experiments and improve the activity of macrophages. In in vivo experiments, it can significantly prevent LPS-induced acute lung injury in mice, inhibit the expression of inflammatory factors in peripheral blood and lung tissue, improve the damage to mouse lung tissue, and restore the normal physiological structure of mouse lung tissue. This shows that the luteolin derivatives of the present invention can effectively prevent acute lung injury, have a protective effect on damaged lung tissue, can be used as a drug for treating, alleviating or preventing acute lung injury, and also provide a new idea and theoretical basis for the development of drugs for acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of cell toxicity test of different concentrations of the luteolin derivative 5J prepared in Example 1 (cell survival rate is expressed as a percentage of cell viability of the control group); Figure 2 This is a graph showing the effect of the luteolin derivative 5J prepared in Example 1 on the expression of inflammatory factors in RAW264.7 cells induced by LPS; Figure 3 H&E staining images of lung tissues of mice in each group; Figure 4 The results of the determination of IL-6, TNF-α, and IL-1β levels in the serum of mice in each group, where A is the IL-6 level of mice in each group, B is the TNF-α level of mice in each group, and C is the IL-1β level of mice in each group; Figure 5The results of the determination of IL-6, TNF-α, and IL-1β levels in balf of mice in each group, where A is the IL-6 level of mice in each group, B is the TNF-α level of mice in each group, and C is the IL-1β level of mice in each group; Figure 6 is the H NMR spectrum of the compound luteolin derivative 5J; Figure 7 This is the carbon NMR spectrum of the compound luteolin derivative 5J. DETAILED DESCRIPTION

[0009] The present invention is further described below in conjunction with the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0010] The present invention discloses a luteolin derivative, the molecular formula of which is C 29 H 29 N3O8S, molecular weight is 579.624, and its structural formula is shown in formula (I): (I).

[0011] The present invention also provides a method for preparing the luteolin derivative, and the synthetic route thereof is shown in formula (II): (II); Wherein, the operation of step a is as follows: after mixing luteolin with potassium carbonate and DMF, 1,2-dibromoethane is added to react to obtain compound 1; The operation of step b is as follows: Compound 1 is mixed with 1,2-dibromoethane and reacted to obtain compound 2; The operation of step c is as follows: Compound 2 is mixed with 4-(N-BOC-amino)piperidine and DMF, and then Et3N is added to react to obtain compound 3; The operation of step d is as follows: dissolving compound 3 in a mixed solution of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacting at room temperature to obtain compound 4; The operation of step e is as follows: compound 4 is dissolved in a mixed solution of DCM and Et3N, and pyridine-3-sulfonyl chloride is added to react to obtain compound 5J, i.e., the target luteolin derivative.

[0012] In step a, the molar ratio of luteolin to 1,2-dibromoethane is 1:2.

[0013] In step b, the molar ratio of compound 1 to 1,2-dibromoethane is 1:27.

[0014] In step c, the molar ratio of compound 2 to Et3N is 1:2.

[0015] In step d, the molar ratio of compound 3, dichloromethane and trifluoroacetic acid is 4:1:1.

[0016] In step e, the molar ratio of compound 4 to pyridine-3-sulfonyl chloride is 2:1.

[0017] The present invention also provides the use of the luteolin derivative in preparing a drug for treating and / or alleviating and / or preventing acute lung injury.

[0018] The acute lung injury is LPS-induced acute lung injury.

[0019] Example 1 Synthesis of compound 1: Luteolin (2.86 g, 10 mmol) and potassium carbonate (0.7 g, 5 mmol) were placed in a 100 mL round-bottom flask, and 50 mL of N,N-dimethylformamide (DMF) was added. 1,2-dibromoethane (1.5 mL, 20 mmol) was added to the reaction system, and the reaction was allowed to proceed overnight at 80°C. After the reaction was completed by thin layer chromatography (TLC), the reaction system was cooled to room temperature, and ice water was added dropwise to the reaction mixture until a yellow solid precipitated. The solid was filtered under reduced pressure, and the filter cake was washed with water (20 mL×3), dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane (DCM)) to obtain compound 1.

[0020] Synthesis of compound 2: Compound 1 (0.64 g, 2.05 mmol) and potassium carbonate (0.28 g, 2.05 mmol) were weighed into a 100 mL round-bottom flask, 40 mL of DMF was added, 1,2-dibromoethane (4.2 mL) was added to the reaction system, and the reaction was carried out at 120°C for 2 h. After the reaction was completed by TLC detection, the reaction system was cooled to room temperature, the reaction solution was extracted with dichloromethane (50 mL×3), combined and washed with water (100 mL×3), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo, and the residue was purified by silica gel column chromatography (eluent: DCM) to obtain compound 2.

[0021] Synthesis of compound 3: Compound 2 (1.7 g, 4.1 mmol) and 4-(N-BOC-amino)piperidine (1.63 g, 8.2 mmol) were weighed into a 100 mL round-bottom flask, 30 mL of DMF was added, 1 mL of Et3N (8.2 mmol) was added to the reaction system, and the reaction was carried out at 120°C for 2 h. After the reaction was completed by TLC detection, the reaction system was cooled to room temperature, ice water was added dropwise to the reaction mixture until a yellow solid precipitated, and the mixture was filtered under reduced pressure, the filter cake was washed with water, dried over anhydrous sodium sulfate, and concentrated to obtain compound 3.

[0022] Synthesis of compound 4: Compound 3 (2.0 g, 4.0 mmol) was weighed into a 100 mL round-bottom flask, and 1 mol of DCM / CF3COOH = 1 / 1 (V / V) was added, and the mixture was reacted at room temperature for 3 h. After the reaction was completed by TLC, the reaction mixture was directly concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH / Et3N = 100 / 1 / 0.5, V / V / V) to obtain compound 4.

[0023] Synthesis of compound 5J: Compound 4 (50 mg, 0.114 mmol) was weighed into a 25 mL round-bottom flask, 5 mL of DCM and Et3N (0.23 mmol) were added, and then the corresponding sulfonyl chloride (0.17 mmol) was added to the reaction system, and the reaction was allowed to react overnight at room temperature. After TLC detection, the reaction solution was extracted with dichloromethane (5 mL×3), combined and washed with water (10 mL×3), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: DCM / MeOH / Et3N=100 / 1 / 0.2, V / V / V) to obtain compound 5J.

[0024] The molecular formula of the luteolin derivative obtained in Example 1 is C 29 H 29 N3O8S, with a molecular weight of 579.624, was named luteolin derivative 5J. 1 H NMR (400 MHz, Chloroform-d) δ 12.68 (s, 1H), 9.04 (d, J = 2.3Hz, 1H), 8.74 (d, J = 2.3 Hz, 1H), 8.11 (dt, J = 2.3 Hz, 1H), 7.41 (dd, J = 2.3 Hz,1H), 7.35 – 7.26 (m, 2H),6.89 (dd, J= 8.5, 2.3 Hz, 1H,), 6.48 (d, J = 1.2 Hz,1H), 6.39 (s, 1H), 6.23 (d, J = 2.3 Hz, 1H), 4.31–4.21 (m, 4H), 4.05 (d, J = 5.2Hz, 2H), 3.20 (s, 1H), 2.83 (d, J = 11.4 Hz, 2H), 2.74 (d, J = 5.6 Hz, 2H), 2.15(d, J = 12.9 Hz, 3H), 1.93 (d, J = 6.0 Hz, 1H), 1.81–1.72 (m, 2H). 13 C NMR (100MHz, Chloroform-d) δ 63.63, 63.21, 55.52, 51.31, 49.68, 31.89, 28.67, 28.30, 118.91, 116.95, 114.45,104.61,103.60, 97.50, 92.09, 64.42, 63.63, 63.21, 55.52, 51.31, 49.68, 31.89, 28.67, 28.30.

[0025] Experimental Example 1 Cell viability detection of luteolin derivative 5J 1. Experimental methods RAW264.7 cells in good growth state were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and inoculated into 96-well cell culture plates at a cell density of 5×10 4 / mL. After culturing the 96-well plate in an incubator at 37°C and 5% CO2 for 24 hours, fresh culture medium containing the luteolin derivative 5J prepared in Example 1 was added respectively, and 6 concentration gradients were set, namely 100 μM, 80 μM, 60 μM, 40 μM, 20 μM, and 10 μM. At the same time, a control group without drug was set up, and each group had 6 parallels. After culturing at 37°C for 24 hours, the toxicity to RAW264.7 cells was detected according to the steps recommended in the instructions of the Cellcountingkit-8 kit (CCK-8, C0038, Beyotime, China). Calculate the cell survival rate, cell survival rate = [(OD 450 -Blank control OD 450 ) / (control cell OD 450 -Blank control OD 450 )] × 100%. If the cell survival rate is > 80%, it is selected as the maximum safe concentration of the drug for subsequent experiments.

[0026] Experimental results: After calculation, the maximum safe concentration of luteolin derivative 5J determined by CCK-8 method was 100 μM ( Figure 1 ).

[0027] Experimental Example 2 Effects of luteolin derivative 5J on inflammatory factors produced by LPS-induced RAW264.7 cells 1. Experimental methods RAW264.7 cells were collected at 5×10 4 The cells were inoculated at a density of 100 μg / mL in a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 h. Then they were divided into a normal group (without any reagent), a model group (without any reagent), a dexamethasone group (5 μM), a luteolin derivative 5J low-dose group (25 μM), a 5J medium-dose group (50 μM), and a 5J high-dose group (100 μM). After culturing for 2 h, LPS was added to all the groups except the normal group to make the final LPS concentration 400 ng / mL. After culturing for 24 h, the cell supernatant was collected, and the IL-6 and TNF-α in the cell supernatant were detected using ELISA kits.

[0028] 2. Experimental results from Figure 2 It can be seen that the production of inflammatory factors IL-6, TNF-α, and IL-1β in RAW264.7 cells after LPS induction was significantly increased compared with the normal group, indicating that the cell inflammation model was successfully established. After administration, the levels of IL-6, TNF-α, and IL-1β decreased, especially in the high-dose group of luteolin derivative 5J. The sharp decrease in inflammatory factors indicates that luteolin derivative 5J can alleviate inflammatory responses.

[0029] Experimental Example 3 Detection of the protective effect of luteolin derivative 5J on acute lung injury in mice 1. Experimental Methods C57BL / 6J mice were randomly divided into 6 groups: control group (CON), model group (LPS), positive drug group (DEX), and Example 1 luteolin derivative 5J low, medium, and high dose (5J-L, 5J-M, 5J-H) (20 mg / kg, 40 mg / kg, 80 mg / kg) groups. Mice were given preventive gavage for 3 consecutive days. The control group was given normal saline by gavage, the model group was given normal saline by gavage, the positive drug group was given 5 mg / kg by gavage, and the drug groups were given drugs of different concentrations. One hour after the last day of administration, except for the control group, all other groups were intraperitoneally injected with LPS 10 mg / kg to establish a mouse acute lung injury model.

[0030] After 6 h of modeling, samples were collected and the protective effect of luteolin derivative 5J on acute lung injury was evaluated by the following method.

[0031] 1. Pathological examination of lung tissue by H&E staining; 2. ELISA method to detect IL-6, TNF-α, and IL-1β levels in serum and bronchoalveolar lavage fluid 2. Experimental Results (1) Pathological examination of lung tissue: Figure 3 As shown, H&E staining observation revealed that the lung tissue structure of mice in the CON group was intact, the alveolar structure was intact, and the structure was thin-walled. The mice in the LPS group showed lung injury, including alveolar wall thickening, inflammatory cell infiltration, and alveolar structure destruction, showing symptoms similar to acute lung injury. After administration, the drug group can effectively reduce the infiltration of inflammatory cells, weaken the thickening of the alveolar wall, and restore the structure of the alveoli to a certain extent. This shows that the high-dose group of the luteolin derivative 5J of the present invention can significantly reduce lung pathological changes.

[0032] (2) Effects on inflammatory factors in serum and bronchoalveolar lavage fluid of mice in each group: Figure 4-5 It can be seen that the luteolin derivative 5J of the present invention can down-regulate the levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β, indicating that the luteolin derivative 5J can inhibit the inflammatory response by down-regulating pro-inflammatory factors.

Claims

1. A luteolin derivative, the molecular formula of which is C 29 H 29 N3O8S, molecular weight is 579.624, and its structural formula is shown in formula (I): （I）。 2. The method for preparing the luteolin derivative according to claim 1, characterized in that: The synthesis route of the luteolin derivative is shown in formula (II): (II); Wherein, the operation of step a is as follows: after mixing luteolin with potassium carbonate and DMF, 1,2-dibromoethane is added to react to obtain compound 1; The operation of step b is as follows: Compound 1 is mixed with 1,2-dibromoethane and reacted to obtain compound 2; Step c is as follows: Compound 2 is mixed with 4-(N-BOC-amino)piperidine and DMF, and then Et3N is added to react to obtain to compound 3; The operation of step d is as follows: dissolving compound 3 in a mixed solution of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacting at room temperature to obtain compound 4; The operation of step e is as follows: compound 4 is dissolved in a mixed solution of DCM and Et3N, and pyridine-3-sulfonyl chloride is added to react to obtain compound 5J, i.e., the target luteolin derivative.

3. The method for preparing the luteolin derivative according to claim 2, characterized in that: In step a, the molar ratio of luteolin to 1,2-dibromoethane is 1:

2.

4. The method for preparing the luteolin derivative according to claim 2, characterized in that: In step b, the molar ratio of compound 1 to 1,2-dibromoethane is 1:

27.

5. The method for preparing the luteolin derivative according to claim 2, characterized in that: In step c, the molar ratio of compound 2 to Et3N is 1:

2.

6. The method for preparing the luteolin derivative according to claim 2, characterized in that: In step d, the molar ratio of compound 3, dichloromethane and trifluoroacetic acid is 4:1:

1.

7. The method for preparing the luteolin derivative according to claim 2, characterized in that: In step e, the molar ratio of compound 4 to pyridine-3-sulfonyl chloride is 2:

1.

8. Use of the luteolin derivative according to claim 1 in the preparation of a drug for treating and / or alleviating and / or preventing acute lung injury.

9. The use according to claim 3, characterized in that: The acute lung injury is LPS-induced acute lung injury.

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