A luteolin derivative and its preparation method and application

Abstract

The present invention discloses a luteolin derivative, a preparation method, and an application thereof. The luteolin derivative provided by the present invention can significantly inhibit the expression of macrophage inflammatory factors and improve the activity of macrophages in in vitro experiments. 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 the mouse lung tissue, and restore the normal physiological structure of the mouse lung tissue. This shows that the luteolin derivative of the present invention can effectively prevent acute lung injury and has a protective effect on damaged lung tissue. It can be used as a drug for treating, alleviating or preventing acute lung injury, and also provides a new idea and theoretical basis for the development of acute lung injury drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical synthesis, and particularly 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 the alveoli, triggering a cytokine storm and damaging the epithelial and endothelial cell linings of the lungs. This often progresses to acute respiratory distress syndrome (ARDS). ALI and ARDS most commonly occur during pneumonia, sepsis, gastric aspiration, or severe trauma. Both ALI and ARDS are life-threatening conditions characterized by severe inflammation and damage to alveolar and lung wall structures. Lipopolysaccharides (LPS), a component of the outer cell wall of Gram-negative bacteria, trigger a rapid and intense inflammatory response, impairing the normal function of immune cells and subsequently recruiting 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. Despite continuous improvement in ALI treatment strategies, specific drugs are still lacking, and the poor prognosis remains a major challenge in supporting patient health. Current pharmacological studies have shown that luteolin has a protective effect against LPS-induced ALI. However, there are currently no reports on the application of luteolin derivatives in the prevention and treatment of ALI. Summary of the Invention

[0003] One of the objects of the present invention is to provide a luteolin derivative, and another object 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, with a molecular weight of 579.624, has a structural formula as shown in formula (I):

[0005]

[0006] (I).

[0007] Another object of the present invention is achieved by the method for preparing the luteolin derivative, the synthetic route of which is shown in formula (II):

[0008]

[0009] (II);

[0010] The operation of step a is as follows: luteolin is mixed with potassium carbonate and DMF, and 1,2-dibromoethane is added to react to obtain compound 1;

[0011] Step b is as follows: Compound 1 is mixed with 1,2-dibromoethane and reacted to obtain Compound 2;

[0012] 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;

[0013] The operation of step d is as follows: compound 3 is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid, the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacted at room temperature to obtain compound 4;

[0014] 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, the target luteolin derivative.

[0015] The use of the luteolin derivative in preparing a drug for treating and / or alleviating and / or preventing acute lung injury.

[0016] 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 and improve macrophage activity in in vitro experiments. In in vivo experiments, they can significantly prevent LPS-induced acute lung injury in mice, inhibit the expression of inflammatory factors in peripheral blood and lung tissue, improve lung tissue damage in mice, 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 and have a protective effect on damaged lung tissue. They can be used as drugs to treat, alleviate, or prevent acute lung injury, and also provide new ideas and theoretical basis for the development of drugs for acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of cytotoxicity test of different concentrations of the luteolin derivative 5J prepared in Example 1 (cell viability is expressed as a percentage of cell viability in the control group);

[0018] 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;

[0019] Figure 3 H&E staining images of lung tissues of mice in each group;

[0020] Figure 4The results of the measurement 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;

[0021] Figure 5 The 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;

[0022] Figure 6 is the H NMR spectrum of the compound luteolin derivative 5J;

[0023] Figure 7 This is the carbon NMR spectrum of the compound luteolin derivative 5J. DETAILED DESCRIPTION

[0024] The present invention is further described below with reference to 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 fall within the scope of protection of the present invention.

[0025] The present invention provides a luteolin derivative, the molecular formula of which is C 29 H 29 N3O8S, with a molecular weight of 579.624, has a structural formula as shown in formula (I):

[0026]

[0027] (I).

[0028] The present invention also provides a method for preparing the luteolin derivative, and the synthetic route thereof is shown in formula (II):

[0029]

[0030] (II);

[0031] The operation of step a is as follows: luteolin is mixed with potassium carbonate and DMF, and 1,2-dibromoethane is added to react to obtain compound 1;

[0032] Step b is as follows: Compound 1 is mixed with 1,2-dibromoethane and reacted to obtain Compound 2;

[0033] 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;

[0034] The operation of step d is as follows: compound 3 is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid, the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacted at room temperature to obtain compound 4;

[0035] 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, the target luteolin derivative.

[0036] In step a, the molar ratio of luteolin to 1,2-dibromoethane is 1:2.

[0037] In step b, the molar ratio of compound 1 to 1,2-dibromoethane is 1:27.

[0038] In step c, the molar ratio of compound 2 to Et3N is 1:2.

[0039] In step d, the molar ratio of compound 3, dichloromethane and trifluoroacetic acid is 4:1:1.

[0040] In step e, the molar ratio of compound 4 to pyridine-3-sulfonyl chloride is 2:1.

[0041] 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.

[0042] The acute lung injury is LPS-induced acute lung injury.

[0043] Example 1

[0044] Synthesis of Compound 1: Luteolin (2.86 g, 10 mmol) and potassium carbonate (0.7 g, 5 mmol) were dissolved in a 100 mL round-bottom flask. 50 mL of N,N-dimethylformamide (DMF) was added, and 1,2-dibromoethane (1.5 mL, 20 mmol) was added to the reaction system. The mixture was allowed to react overnight at 80°C. After completion of the reaction as determined by thin-layer chromatography (TLC), the reaction system was cooled to room temperature. Ice water was added dropwise to the reaction mixture until a yellow solid precipitated. The solid was then filtered under reduced pressure, and the filter cake was washed with water (20 mL x 3), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane (DCM)) to obtain Compound 1.

[0045] 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, and 1,2-dibromoethane (4.2 mL) was added to the reaction system. The reaction was allowed to react at 120°C for 2 h. After TLC analysis, the reaction system was cooled to room temperature. The reaction mixture was extracted with dichloromethane (50 mL x 3). The combined extracts were washed with water (100 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: DCM) to obtain Compound 2.

[0046] 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, and 1 mL of Et3N (8.2 mmol) was added to the reaction system. The mixture was reacted at 120°C for 2 h. After TLC, the reaction system was cooled to room temperature. Ice water was added dropwise to the reaction mixture until a yellow solid precipitated. The solid was filtered under reduced pressure, washed with water, dried over anhydrous sodium sulfate, and concentrated to yield Compound 3.

[0047] Synthesis of Compound 4: Compound 3 (2.0 g, 4.0 mmol) was weighed into a 100 mL round-bottom flask. DCM and CF3COOH (1 / 1, v / v) (1 mol each) were added and allowed to react at room temperature for 3 h. After completion of the reaction, the reaction mixture was concentrated and purified by silica gel column chromatography (eluent: DCM / MeOH / Et3N (100 / 1 / 0.5, v / v / v) to yield Compound 4.

[0048] 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. The corresponding sulfonyl chloride (0.17 mmol) was then added to the reaction system and allowed to react overnight at room temperature. After TLC, the reaction mixture was extracted with dichloromethane (5 mL x 3). The combined solution was washed with water (10 mL x 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 yield Compound 5J.

[0049] 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. 1H 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 .31, 49.68, 31.89, 28.67, 28.30, 118.91, 116.95, 114.45,104.61,103.60, 97.50, 92.09, 65.42, 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,

[0050] Experimental Example 1 Cell viability assay of luteolin derivative 5J

[0051] 1. Experimental methods

[0052] RAW264.7 cells in good growth state were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and seeded into 96-well cell culture plates at a cell density of 5×10 4 / mL. After culturing the 96-well plate in a 37°C, 5% CO2 incubator for 24 hours, fresh culture medium containing the luteolin derivative 5J prepared in Example 1 was added, 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, with 6 parallels in each group. 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 Cellcounting kit-8 kit (CCK-8, C0038, Beyotime, China). The cell survival rate was calculated, and the 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 and used in subsequent experiments.

[0053] Experimental results: The maximum safe concentration of luteolin derivative 5J was calculated to be 100 μM using the CCK-8 method ( Figure 1 ).

[0054] Experimental Example 2 Effects of luteolin derivative 5J on LPS-induced inflammatory cytokine production in RAW264.7 cells

[0055] 1. Experimental methods

[0056] RAW264.7 cells were cultured at 5 × 10 4 The cells were seeded at a density of 1000 cells / mL in a 96-well plate and cultured in an incubator at 37°C with 5% CO2 for 24 h. Then, the cells 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. The cells were cultured for 24 h, and the cell supernatants were collected. ELISA kits were used to detect IL-6 and TNF-α in the cell supernatants.

[0057] 2. Experimental results

[0058] from Figure 2As shown, LPS-induced RAW264.7 cells produced significantly more inflammatory factors, including IL-6, TNF-α, and IL-1β, compared to the normal control group, indicating a successful cellular inflammation model. Following administration, IL-6, TNF-α, and IL-1β levels decreased, particularly in the high-dose luteolin derivative 5J group. This dramatic decrease in inflammatory factors suggests that luteolin derivative 5J can alleviate inflammatory responses.

[0059] Experimental Example 3 Detection of the protective effect of luteolin derivative 5J on acute lung injury in mice

[0060] 1. Experimental Methods

[0061] C57BL / 6J mice were randomly divided into six groups: a control group (CON), a model group (LPS), a positive drug group (DEX), and low-, medium-, and high-dose (5J-L, 5J-M, and 5J-H) groups (20 mg / kg, 40 mg / kg, and 80 mg / kg) of the luteolin derivative 5J from Example 1. Mice were administered prophylactic drugs by gavage for three consecutive days: the control group received normal saline, the model group received normal saline, and the positive drug group received 5 mg / kg. The drug-treated groups were also given drugs at varying concentrations. One hour after the final day of drug administration, all groups except the control group received an intraperitoneal injection of 10 mg / kg of LPS to establish an acute lung injury model in mice.

[0062] Six hours after modeling, samples were collected, and the protective effect of luteolin derivative 5J on acute lung injury was evaluated using the following method.

[0063] 1. Pathological examination of lung tissue by H&E staining;

[0064] 2. ELISA method to detect IL-6, TNF-α, and IL-1β levels in serum and bronchoalveolar lavage fluid

[0065] 2. Experimental Results

[0066] (1) Pathological examination of lung tissue: Figure 3 As shown, H&E staining revealed that the lung tissue structure of mice in the CON group was intact, with intact alveolar structures and a thin-walled structure. Mice in the LPS group showed signs of lung injury, including alveolar wall thickening, inflammatory cell infiltration, and alveolar structural destruction, exhibiting symptoms resembling acute lung injury. Following administration, the drug group was able to effectively reduce inflammatory cell infiltration, attenuate alveolar wall thickening, and restore alveolar structure to a certain extent. This suggests that the high-dose group of the present luteolin derivative 5J significantly alleviated lung pathological changes.

[0067] (2) Effects on inflammatory factors in serum and bronchoalveolar lavage fluid of mice in each group: Figure 4-5It can be seen that the luteolin derivative 5J of the present invention can downregulate the levels of proinflammatory factors IL-6, TNF-α, and IL-1β, indicating that the luteolin derivative 5J can inhibit the inflammatory response by downregulating proinflammatory factors.

Claims

1. A luteolin derivative, the molecular formula of which is C 29 H 29 N3O8S, with a molecular weight of 579.624, has a structural formula as shown in formula (I): （I）。 2. The method for preparing the luteolin derivative according to claim 1, characterized in that: The synthetic route of the luteolin derivative is shown in formula (II): (II); The operation of step a is as follows: luteolin is mixed with potassium carbonate and DMF, and 1,2-dibromoethane is added to react to obtain compound 1; 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 Compound 3; The operation of step d is as follows: compound 3 is dissolved in a mixed solution of dichloromethane and trifluoroacetic acid, the volume ratio of dichloromethane to trifluoroacetic acid is 1:1, and reacted 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, 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 application according to claim 8, characterized in that: The acute lung injury is LPS-induced acute lung injury.

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