A fusidic acid derivative, its preparation method and application

Abstract

The present invention discloses a fusidic acid derivative, and the molecular formula of the fusidic acid derivative is: C 40 H 55 N3O6, and the molecular weight is: 673.40909. By adopting the above-mentioned fusidic acid derivative, the present invention effectively inhibits the inflammatory response of macrophages induced by LPS, and can be used in the field of preparation of anti-inflammatory drugs. By connecting phenyl-1,2,3-triazole to the carboxyl group at the C-21 position of fusidic acid, it is found that the introduction of phenyl-1,2,3-triazole can improve the anti-inflammatory activity of fusidic acid. Another object of the present invention is to provide a preparation method and application of the fusidic acid derivative.

Description

Technical Field

[0001] The present invention relates to the technical field of compound synthesis, and in particular to a fusidic acid derivative, a preparation method thereof and an application thereof. Background Art

[0002] Inflammation is a gradual physiological response, usually resulting from chemical, mechanical or innocuous reperfusion caused by irritant pathogens, injury, allergens and harmful agents. Inflammation enables immune system cells to migrate to the stimulation target through a series of steps such as promoting the production of cytokines, chemokines and acute-phase proteins and coordinating transportation. On the one hand, the rapid inflammatory response can protect the body and remove the harmful stimuli to gradually restore the damaged area to its original state. On the other hand, excessive inflammatory response and chronic inflammation can lead to tissue damage and fibrosis. Inflammation is associated with complex medical conditions such as diabetes, neurodegenerative diseases, tumors and related diseases. Inflammation is a complex phenomenon involving enzymes and biochemical mediators, and fluid flow leads to edema, cell swelling and damage. Macrophages play a crucial role in the inflammatory response by triggering the production of stimulatory factors such as prostaglandin E2 (PGE2), nitric oxide (NO) and different cytokines such as IL-1β, IL-6, IL-10 and TNF-α.

[0003] A large number of non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat pain, fever, inflammatory diseases and rheumatoid arthritis. The pharmacological activity of non-steroidal anti-inflammatory drugs is related to the inhibition of arachidonic acid synthesis of prostaglandins by inhibiting prostaglandin endoperoxidase (commonly known as cyclooxygenase and 5-lipoxygenase). Long-term use of non-steroidal anti-inflammatory drugs is associated with indigestion, gastrointestinal ulcers and nephrotoxicity. The damage of non-steroidal anti-inflammatory drugs to the gastrointestinal tract is usually attributed to two factors: the local irritation (local action) of the common carboxylic acid part of most non-steroidal anti-inflammatory drugs, and the reduction of tissue prostaglandin production, which determines the physiological role of cytoprotective prostaglandins in maintaining gastrointestinal health and homeostasis. Non-steroidal anti-inflammatory drugs are the most widely used drugs worldwide and are usually used to treat the fever, pain and inflammation of diseases. Although non-steroidal anti-inflammatory drugs have powerful anti-inflammatory, analgesic and antipyretic effects, they also pose a greater risk of side effects such as gastrointestinal complications, renal failure and heart failure. Therefore, it is crucial to find anti-inflammatory drugs without ulcer side effects. Since many chemically synthesized drugs have strong toxic and side effects, people have begun to focus on developing anti-inflammatory drugs with less side effects from natural products.

[0004] Previous studies have shown that fusidic acid has anti-inflammatory activity, but the anti-inflammatory activity is not strong enough. Therefore, it is necessary to further carry out chemical modification and expand the research in this field to develop new and effective anti-inflammatory drugs. Summary of the Invention

[0005] The object of the present invention is to provide a fusidic acid derivative which can effectively inhibit the inflammatory response of macrophages induced by LPS and can be used in the field of preparation of anti-inflammatory drugs. A phenyl-1,2,3-triazole is connected to the carboxyl group at the C-21 position of fusidic acid, and it is found that the introduction of phenyl-1,2,3-triazole can improve the anti-inflammatory activity of fusidic acid. Another object of the present invention is to provide a preparation method and application of a fusidic acid derivative.

[0006] To achieve the above object, the present invention provides a fusidic acid derivative, and the structural formula of the fusidic acid derivative is as follows:

[0007]

[0008] The molecular formula of the fusidic acid derivative is: C 40 H 55 N 3 O 6 , and the molecular weight is: 673.40909.

[0009] A preparation method of a fusidic acid derivative comprises the following steps:

[0010] (1) Take a 250 mL round-bottom flask, add aniline, add 10 - 20 mL of 10% hydrochloric acid aqueous solution, place it in an ice bath and stir. Dissolve sodium nitrite in 50 - 100 mL of water, and slowly add it dropwise to the reaction solution through a constant pressure dropping funnel. After 30 min, dissolve sodium azide in water and slowly add it dropwise to the reaction solution. Stir and react at 0 °C. After 2 - 4 h, the reaction is complete. After monitoring the reaction is complete by TLC, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter with a Buchner funnel, and concentrate the filtrate under reduced pressure to obtain a yellow oily substance A, which is directly used for the next step of the reaction;

[0011] (2) Take a single-neck round-bottom flask, add the substance A obtained in step (1), then pour in the solvent, add sodium ascorbate and anhydrous copper sulfate, stir at room temperature, add propargyl alcohol under stirring, and react at room temperature for 24 h. After monitoring by TLC and showing that the reaction is complete, extract with ethyl acetate three times, combine the organic phases, wash with H 2 O, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter and concentrate under reduced pressure. The crude product is purified by silica gel column chromatography, eluted with a gradient of petroleum ether and ethyl acetate, or recrystallized with an ethyl acetate / petroleum ether system to obtain intermediate B;

[0012] (3) Take 0.2 mmol of intermediate B in a 50 mL single-necked flask, dissolve it with sufficient dichloromethane, add sufficient thionyl chloride, react at room temperature for 8 h, monitor the reaction by TLC. After the reaction is complete, evaporate the solvent, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, concentrate the filtrate under reduced pressure, crystallize the residue with an ethyl acetate / petroleum ether system, and filter to obtain intermediate C;

[0013] (4) Take a certain amount of fusidic acid and intermediate C, add K 2 CO 3 , DMF, acetonitrile or acetone (5 - 10 mL), place all in a 25 mL round-bottom flask, react overnight under vigorous stirring at 60 °C. After testing the reaction to be complete by TLC, extract with ethyl acetate three times, wash the organic phase twice with saturated sodium bicarbonate solution and brine, dry with an appropriate amount of anhydrous sodium sulfate or anhydrous magnesium sulfate, filter under reduced pressure and rotary evaporate to obtain the crude product, and purify it by silica gel chromatography to obtain compound D, which is the fusidic acid derivative.

[0014] Preferably, in the step (1), the dosage of aniline is: 0.01 - 0.02 mol, the dosage of sodium nitrite is: 0.012 - 0.024 mol, and the dosage of sodium azide is: 0.02 - 0.04 mol.

[0015] Preferably, in the step (2), the dosage of substance A is: 5 - 10 mmol, the dosage of sodium ascorbate is: 0.5 - 1.0 mmol, the dosage of anhydrous copper sulfate is: 0.25 - 0.50 mmol, and the dosage of propargyl alcohol is: 5.5 - 11.0 mmol.

[0016] Preferably, in the step (2), the solvent is prepared by mixing tert-butanol and water in a volume ratio of 1:1.

[0017] Preferably, in the step (4), the dosage of fusidic acid is: 0.12 - 0.24 mmol, the dosage of intermediate C is: 0.14 - 0.28 mmol, and the dosage of K 2 CO 3 is: 0.30 - 0.60 mmol.

[0018] An application of a fusidic acid derivative, and the application is to prepare an anti-inflammatory drug.

[0019] The advantages and positive effects of a fusidic acid derivative, its preparation method and application according to the present invention are:

[0020] 1. The present invention provides a new compound, fusidic acid derivative. In vitro toxicity experiments show that the compound has no toxicity at 60 μM.

[0021] 2. The compounds of the present invention significantly reduced the release of IL-6 and TNF-α from LPS-induced RAW264.7 cells at 10 μM, and the inhibitory ability was significantly better than that of the lead compound fusidic acid. It can effectively inhibit the inflammatory response of macrophages induced by LPS and is used in the field of anti-inflammatory drug preparation.

[0022] 3. The preparation method of the compounds of the present invention has a simple reaction process operation, mild reaction conditions, and the reagents used are cheap and easily available.

[0023] The technical solution of the present invention will be further described in detail below through the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a graph showing the effect of fusidic acid (FA) and compound D of the present invention on the cell viability of mouse macrophages (RAW264.7) at a concentration of 60 μM;

[0025] Figure 2 It is a graph showing the inhibitory effect of fusidic acid (FA) and compound D of the present invention on the release of IL-6 and TNF-α from LPS-induced RAW264.7 cells;

[0026] Figure 3 It is a structural formula diagram of a fusidic acid derivative of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The technical solution of the present invention will be further described below through the accompanying drawings and examples.

[0028] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning as understood by those of ordinary skill in the field to which the present invention belongs.

[0029] A fusidic acid derivative, and the structural formula of the fusidic acid derivative is as follows:

[0030]

[0031] The molecular formula of the fusidic acid derivative is: C 40 H 55 N 3 O 6 , and the molecular weight is: 673.40909.

[0032] The name of the fusidic acid derivative of the present invention is:

[0033] (1-phenyl-1H-1,2,3-triazol-4-yl)methyl (Z)-2-((3R,4S,5S,8S,10S,11R,13R,14S,16S)-16-acetoxy-3,11-dihydroxy-4,8,10,14-tetramethylhexadecahydro-17H-cyclopenta[a]phenanthren-17-ylidene)-6-methylhept-5-enoate, hereinafter referred to as D.

[0034] Use of a fusidic acid derivative, the use being for preparing an anti-inflammatory drug.

[0035] Example 1

[0036] A method for preparing a fusidic acid derivative, comprising the following steps:

[0037] (1) Take a 250 mL round-bottom flask, add 0.01 mol of aniline, add 10 - 20 mL of 10% hydrochloric acid aqueous solution, place it in an ice bath and stir. Dissolve 0.012 mol of sodium nitrite in 50 - 100 mL of water, and slowly add it dropwise to the reaction solution through a constant pressure dropping funnel. After 30 min, dissolve 0.02 mol of sodium azide in water and slowly add it dropwise to the reaction solution. Stir and react at 0 °C. After 2 - 4 h, the reaction is complete. After monitoring the reaction is complete by TLC, extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, and concentrate the filtrate under reduced pressure to obtain a yellow oily substance A, which is directly used for the next step of the reaction;

[0038] (2) Take a single-neck round-bottom flask, add 5 mmol of the substance A obtained in step (1), then pour 20 mL of a solvent prepared by mixing tert-butanol and water in a volume ratio of 1:1, add 0.5 mmol of sodium ascorbate, 0.25 mmol of anhydrous copper sulfate, stir at room temperature, add 5.5 mmol of propargyl alcohol under stirring, and react at room temperature for 24 h. After monitoring by TLC and showing that the reaction is complete, extract with ethyl acetate 3 times, combine the organic phases, wash with H 2 O, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter and concentrate under reduced pressure. The crude product is purified by silica gel column chromatography, eluted with a gradient of petroleum ether and ethyl acetate, or recrystallized with an ethyl acetate / petroleum ether system to obtain intermediate B;

[0039] (3) Take 0.2 mmol of intermediate B in a 50 mL single-necked flask, dissolve it with sufficient dichloromethane, add sufficient thionyl chloride, react at room temperature for 8 h, monitor the reaction by TLC. After the reaction is complete, evaporate the solvent, extract with ethyl acetate three times, combine the organic phases, dry over anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, concentrate the filtrate under reduced pressure, crystallize the residue with an ethyl acetate / petroleum ether system, and filter to obtain intermediate C;

[0040] (4) Take 0.12 mmol of fusidic acid and 0.14 mmol of intermediate C, add 0.30 mmol of K 2 CO 3 , DMF, acetonitrile or acetone (5 - 10 mL), place all in a 25 mL round-bottom flask, react overnight under vigorous stirring at 60 °C. After testing the reaction to be complete by TLC, extract with ethyl acetate three times, wash the organic phase twice with saturated sodium bicarbonate solution and brine, dry with an appropriate amount of anhydrous sodium sulfate or anhydrous magnesium sulfate, filter under reduced pressure and rotary evaporate to obtain the crude product, and purify by silica gel chromatography to obtain compound D, which is the fusidic acid derivative.

[0041] The reaction formula for the above preparation process is as follows:

[0042]

[0043] The nuclear magnetic resonance data of compound D are as follows:

[0044] White powder; m.p. 94 - 96 °C; Yield: 86%. 1 H-NMR (300 MHz, CDCl 3 ) δ: 8.15 (s, 1H, triazole-H), 7.78 (d, J = 9 Hz, 2H, Ar-H), 7.55 (t, J = 9 Hz, 2H, Ar-H), 7.47 (t, J = 9 Hz, 1H, Ar-H), 5.91 (d, J = 6 Hz, 1H, C 16 -H), 5.35 (d, J = 15 Hz, 1H, -COO-CH 2 -), 5.18 (d, J = 12 Hz, 1H, -COO-CH 2 -), 5.07 (t, J = 9 Hz, 1H, C 24 -H), 4.35 (s, 1H, C 11 -H), 3.77 (s, 1H, C 3 -H), 3.06 (d, J = 9 Hz, 1H, C 13 -H), 2.51 - 2.44 (m, 2H, C 22-H), 2.35 - 2.28 (m, 1H), 2.22 - 2.05 (m, 4H), 1.96 (s, 3H, CH 3 COO-), 1.90 - 1.72 (m, 4H), 1.64 - 1.54 (m, 12H), 1.38 (s, 3H, -CH 3 ), 1.30 (d, J=15Hz, 2H), 1.19 - 1.10 (m, 2H), 0.99 - 0.87 (m, 9H). 13 C-NMR(75MHz, CDCl 3 ) δ: 170.58, 169.91, 149.10, 143.29, 136.94, 132.65, 130.06, 129.77, 128.88, 122.92, 122.37, 120.63, 74.40, 71.37, 68.22, 57.95, 49.20, 48.71, 44.15, 39.46, 39.07, 37.02, 36.24, 36.09, 35.56, 32.35, 30.22, 29.96, 28.99, 28.32, 25.68, 24.10, 22.79, 21.00, 20.77, 17.88, 17.71, 15.94. ESI-HRMS calcd for C 39 H 56 NO 7 + ([M + H] + ): 674.41636; found: 674.41437.

[0045] Example 2

[0046] A method for preparing a fusidic acid derivative, comprising the following steps:

[0047] (1) Take a 250 mL round-bottom flask, add 0.015 mol of aniline, add 10 - 20 mL of 10% aqueous hydrochloric acid solution, place it in an ice bath and stir. Dissolve 0.018 mol of sodium nitrite in 50 - 100 mL of water, and slowly add it dropwise to the reaction solution through a constant pressure dropping funnel. After 30 min, dissolve 0.03 mol of sodium azide in water and slowly add it dropwise to the reaction solution. Stir and react at 0 °C. After 2 - 4 h, the reaction is complete. After monitoring the completion of the reaction by TLC, extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, and concentrate the filtrate under reduced pressure to obtain a yellow oily substance A, which is directly used for the next step of the reaction;

[0048] (2) Take a single-necked round-bottom flask, add 7.5 mmol of substance A obtained in step (1), then pour in 20 mL of a solvent prepared by mixing tert-butanol and water in a volume ratio of 1:1. Then add 0.75 mmol of sodium ascorbate and 0.40 mmol of anhydrous copper sulfate. Stir at room temperature. While stirring, add 8.0 mmol of propargyl alcohol and react at room temperature for 24 h. Monitor the reaction by TLC. After the reaction is complete as shown by TLC, extract with ethyl acetate three times, combine the organic phases, wash with H 2 O, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography, elute with a gradient of petroleum ether and ethyl acetate, or recrystallize with an ethyl acetate / petroleum ether system to obtain intermediate B;

[0049] (3) Take 0.2 mmol of intermediate B in a 50 mL single-necked flask, dissolve it in sufficient dichloromethane, add sufficient thionyl chloride, and react at room temperature for 8 h. Monitor the reaction by TLC. After the reaction is complete, evaporate the solvent, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter with a Buchner funnel, concentrate the filtrate under reduced pressure, crystallize the residue with an ethyl acetate / petroleum ether system, and filter to obtain intermediate C;

[0050] (4) Take 0.18 mmol of fusidic acid and 0.21 mmol of intermediate C, add 0.45 mmol of K 2 CO 3 , DMF, acetonitrile or acetone (5 - 10 mL), place all in a 25 mL round-bottom flask, react overnight with vigorous stirring at 60 °C. After testing the reaction to be complete by TLC, extract with ethyl acetate three times, wash the organic phase twice with saturated sodium bicarbonate solution and brine, dry with an appropriate amount of anhydrous sodium sulfate or anhydrous magnesium sulfate, filter under reduced pressure and rotary evaporate to obtain the crude product, and purify by silica gel chromatography to obtain compound D, which is the fusidic acid derivative.

[0051] Example 3

[0052] A method for preparing a fusidic acid derivative, comprising the following steps:

[0053] (1) Take a 250 mL round-bottom flask, add 0.02 mol of aniline, add 10 - 20 mL of 10% hydrochloric acid aqueous solution, place it in an ice bath and stir. Dissolve 0.024 mol of sodium nitrite in 50 - 100 mL of water, and slowly drip it into the reaction solution through a constant pressure dropping funnel. After 30 min, dissolve 0.04 mol of sodium azide in water and slowly drip it into the reaction solution. Stir and react at 0 °C. After 2 - 4 h, the reaction is complete. After monitoring the reaction to be complete by TLC, extract with ethyl acetate three times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter with a Buchner funnel, concentrate the filtrate under reduced pressure to obtain a yellow oily substance A, which is directly used for the next step of the reaction;

[0054] (2) Take a single-necked round-bottom flask, add 10 mmol of substance A obtained in step (1), then pour in 20 mL of a solvent prepared by mixing tert-butanol and water in a volume ratio of 1:1. Then add 1.0 mmol of sodium ascorbate and 0.50 mmol of anhydrous copper sulfate. Stir at room temperature. While stirring, add 11.0 mmol of propargyl alcohol and react at room temperature for 24 h. Monitor by TLC. After the reaction is complete as shown by TLC, extract with ethyl acetate three times. Combine the organic phases, wash with H 2 O, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography, elute with a gradient of petroleum ether and ethyl acetate, or recrystallize with an ethyl acetate / petroleum ether system to obtain intermediate B;

[0055] (3) Take 0.2 mmol of intermediate B in a 50 mL single-necked flask, dissolve it completely with dichloromethane, add an excess of thionyl chloride, and react at room temperature for 8 h. Monitor the reaction by TLC. After the reaction is complete, evaporate the solvent, extract with ethyl acetate three times. Combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter with a Buchner funnel, concentrate the filtrate under reduced pressure, crystallize the residue with an ethyl acetate / petroleum ether system, and filter to obtain intermediate C;

[0056] (4) Take 0.24 mmol of fusidic acid and 0.28 mmol of intermediate C, add 0.60 mmol of K 2 CO 3 , DMF, acetonitrile or acetone (5 - 10 mL), and place all in a 25 mL round-bottom flask. React overnight with vigorous stirring at 60 °C. After testing the reaction to be complete by TLC, extract with ethyl acetate three times. Wash the organic phase twice with saturated sodium bicarbonate solution and brine, dry with an appropriate amount of anhydrous sodium sulfate or anhydrous magnesium sulfate, filter under reduced pressure and rotary evaporate to obtain the crude product. Purify by silica gel chromatography to obtain compound D, which is the fusidic acid derivative.

[0057] The anti-inflammatory ability of the fusidic acid derivative of the present invention is further verified by the following examples. The experimental materials and equipment in the following examples are all commercially available.

[0058] Example 4

[0059] The cytotoxicity of fusidic acid (FA) and compound D on RAW264.7 cells at a concentration of 60 μM was evaluated by observing cell viability analysis using the MTT method.

[0060] Experimental materials: incubator, mouse macrophage (RAW264.7) cell line, DMEM medium, fetal bovine serum (FBS), penicillin, streptomycin, MTT.

[0061] Experimental method: Seed RAW264.7 macrophages at 1×104 The cells were seeded at a density of [number of cells] / well in complete medium in a 96-well plate. After the cells adhered, they were pretreated with fusidic acid and compound D (60 μM) for 24 h respectively. Then, the culture medium was discarded, MTT solution was added at a final concentration of 2 mg / mL, and the cells were incubated for another 4 h in the dark. The MTT solution was aspirated, 150 μL of DMSO was added to dissolve the formazan crystals, and the plate was shaken on a shaker for 10 min. The optical density (OD) was read on an ELISA reader at a wavelength of 492 nm, and then the data was analyzed.

[0062] Blank control group: RAW264.7 macrophages were seeded at a density of 1×10 4 The cells were seeded at a density of [number of cells] / well in complete medium in a 96-well plate. After the cells adhered, MTT solution was added at a final concentration of 2 mg / mL, and the cells were incubated for another 4 h in the dark. The MTT solution was aspirated, 150 μL of DMSO was added to dissolve the formazan crystals, and the plate was shaken on a shaker for 10 min. The optical density (OD) was read on an ELISA reader at a wavelength of 492 nm.

[0063] The experimental results are as Figure 1 shown. At a concentration of 60 μM, fusidic acid and compound D had no obvious cytotoxicity.

[0064] Example 5

[0065] By constructing an LPS-induced RAW264.7 cell model, the anti-inflammatory activity of compound D was evaluated by detecting the effects of the compound on the release of TNF-α and IL-6 using ELISA method.

[0066] Experimental materials: incubator, mouse macrophage (RAW264.7) cell line, DMEM medium, fetal bovine serum (FBS), penicillin, streptomycin, ELISA kit.

[0067] Experimental method: RAW264.7 macrophages were seeded at a density of 1×10 4 cells in a 96-well plate, cultured for 24 h, and treated with 10 μM fusidic acid and compound D for 1 h respectively, and then stimulated with LPS (1 μg / mL) for 24 h. The cell culture supernatant was centrifuged at 4 °C for 10 min to remove insoluble matter, and the supernatant was collected and stored at -20 °C until cytokine detection. Secreted TNF-α and IL-6 were measured in the cell culture supernatant using a commercially available ELISA kit according to the manufacturer's instructions. The absorbance (450 nm) of each sample was analyzed using an ELISA reader, and the values were determined using a standard curve.

[0068] Blank control: RAW264.7 macrophages were seeded at a density of 1×10 4Cells were seeded in 96-well plates and cultured for 24 h. The cell culture supernatant was centrifuged at 4 °C for 10 min to remove insolubles, and the supernatant was collected and stored at -20 °C until cytokine detection. Secreted TNF-α and IL-6 were measured in the cell culture supernatant using a commercially available ELISA kit according to the manufacturer's instructions. The absorbance (450 nm) of each sample was analyzed using a microplate reader, and values were determined using a standard curve.

[0069] LPS-induced cell model: RAW264.7 macrophages were seeded at 1×10 4 cells in 96-well plates and cultured for 24 h, then stimulated with LPS (1 μg / mL) for 24 h. The cell culture supernatant was centrifuged at 4 °C for 10 min to remove insolubles, and the supernatant was collected and stored at -20 °C until cytokine detection. Secreted TNF-α and IL-6 were measured in the cell culture supernatant using a commercially available ELISA kit according to the manufacturer's instructions. The absorbance (450 nm) of each sample was analyzed using a microplate reader, and values were determined using a standard curve.

[0070] The experimental results are as Figure 2 shown: Compound D could significantly reduce the release of TNF-α and IL-6 from LPS-induced RAW264.7 cells. Compared with the lead compound fusidic acid (FA) at the same concentration (10 μM), the inhibitory ability of Compound D was significantly enhanced, indicating that Compound D could effectively inhibit the inflammatory response of macrophages induced by LPS.

[0071] The above experiments showed that the present invention modified fusidic acid at the C-21 position with a phenyl-1,2,3-triazole heterocycle to obtain fusidic acid derivative D with improved anti-inflammatory activity.

[0072] Therefore, the present invention adopts the above-mentioned fusidic acid derivative and its preparation method and application, effectively inhibits the inflammatory response of macrophages induced by LPS, can be used in the field of anti-inflammatory drug preparation, connects phenyl-1,2,3-triazole to the carboxyl group at the C-21 position of fusidic acid, and the introduction of phenyl-1,2,3-triazole can improve the anti-inflammatory activity of fusidic acid.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A fusidic acid derivative, characterized in that, the structural formula of the fusidic acid derivative is as follows: The molecular formula of the fusidic acid derivative is: C 40 H 55 N 3 O 6 , and the molecular weight is: 673.40909.

2. A method for preparing the fusidic acid derivative according to claim 1, characterized in that, it comprises the following steps: (1) Take a 250 mL round-bottom flask, add aniline, add 10 - 20 mL of 10% hydrochloric acid aqueous solution, place it in an ice bath and stir. Dissolve sodium nitrite in 50 - 100 mL of water, and slowly add it dropwise to the reaction solution through a constant pressure dropping funnel. After 30 min, dissolve sodium azide in water and then slowly add it dropwise to the reaction solution. Stir and react at 0 °C. After 2 - 4 h, the reaction is complete. After monitoring the completion of the reaction by TLC, extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, and concentrate the filtrate under reduced pressure to obtain a yellow oily substance A, which is directly used for the next step of the reaction. The structural formula of the yellow oily substance A is as follows: (2) Take a single-necked round-bottom flask, add the substance A obtained in step (1), then pour in the solvent, and then add sodium ascorbate and anhydrous copper sulfate. Stir at room temperature, add propargyl alcohol under stirring, and react at room temperature for 24 h. Monitor by TLC. After it is shown that the reaction is complete, extract with ethyl acetate three times, combine the organic phases, wash with H 2 2O, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography, elute with a gradient of petroleum ether and ethyl acetate, or recrystallize with an ethyl acetate / petroleum ether system to obtain intermediate B. The structural formula of intermediate B is as follows: (3) Take 0.2 mmol of intermediate B in a 50 mL single-necked flask, dissolve it with sufficient dichloromethane, add sufficient thionyl chloride, and react at room temperature for 8 h. Monitor the reaction by TLC. After the reaction is complete, evaporate the solvent, extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate or anhydrous magnesium sulfate, filter by Buchner funnel, and concentrate the filtrate under reduced pressure. Crystallize the residue with an ethyl acetate / petroleum ether system, and filter to obtain intermediate C. The structural formula of intermediate C is as follows: (4) Take a certain amount of fusidic acid and intermediate C, add K 2 CO 3 , DMF, acetonitrile or acetone (5 - 10 mL), place all in a 25 mL round-bottom flask, react overnight under vigorous stirring at 60 °C. After testing that the reaction is complete by TLC, extract with ethyl acetate three times. Wash the organic phase twice with saturated sodium bicarbonate solution and brine, dry with an appropriate amount of anhydrous sodium sulfate or anhydrous magnesium sulfate, filter under reduced pressure and rotary evaporate to obtain the crude product. Purify by silica gel chromatography to obtain compound D, which is the fusidic acid derivative.

3. The method for preparing a fusidic acid derivative according to claim 2, characterized in that: in the step (1), the dosage of aniline is: 0.01 - 0.02 mol, the dosage of sodium nitrite is: 0.012 - 0.024 mol, and the dosage of sodium azide is: 0.02 - 0.04 mol.

4. The method for preparing a fusidic acid derivative according to claim 2, characterized in that: in the step (2), the dosage of substance A is: 5 - 10 mmol, the dosage of sodium ascorbate is: 0.5 - 1.0 mmol, the dosage of anhydrous copper sulfate is: 0.25 - 0.50 mmol, and the dosage of propargyl alcohol is: 5.5 - 11.0 mmol.

5. The method for preparing a fusidic acid derivative according to claim 2, characterized in that: in the step (2), the solvent is prepared by mixing tert-butanol and water in a volume ratio of 1:

1.

6. The method for preparing a fusidic acid derivative according to claim 2, characterized in that: In the step (4), the dosage of fusidic acid is: 0.12 - 0.24 mmol, the dosage of intermediate C is: 0.14 - 0.28 mmol, and the dosage of K 2 CO 3 is: 0.30 - 0.60 mmol.

7. An application of the fusidic acid derivative according to claim 1, characterized in that, the application is for preparing an anti-inflammatory drug.

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