Gentiopicroside nitroxide radical derivative, and preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a gentiopicroside nitroxide radical derivative as well as a preparation method and application thereof. A series of gentiopicroside nitroxide radical derivatives with specific structures are prepared and synthesized through a chemical process. The derivatives exhibit good in-vitro COX-2 enzyme inhibition activity and in-vitro ROS scavenging activity. In-vivo and in-vitro experimental results show that each derivative can effectively inhibit the release of inflammatory mediators and oxidative stress during inflammation, thereby more effectively inhibiting the progress of inflammation and protecting tissues, and the derivatives have a wide application prospect in the preparation of drugs with the efficacy of preventing, relieving or treating inflammation.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and specifically relates to a gentiopicroside nitric oxide free radical derivative, its preparation method, and its application. Background Technology

[0002] Inflammation plays a crucial role in the development and progression of chronic inflammatory diseases such as cardiovascular disease, asthma, diabetes, and obesity. It is also closely associated with various progressive diseases, including cancer and neurodegenerative diseases, placing a heavy burden on global public health systems. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, celecoxib, and meloxicam, remain the most widely used anti-inflammatory treatments globally.

[0003] However, first-generation non-selective NSAIDs (such as aspirin) can easily cause adverse reactions such as gastrointestinal damage due to the simultaneous inhibition of COX-1 and COX-2. While second-generation selective COX-2 inhibitors (such as celecoxib) significantly reduce gastrointestinal risks, they still pose potential cardiovascular risks. It is noteworthy that excessive generation of reactive oxygen species (ROS) often accompanies inflammatory responses, which is one of the key mechanisms by which inflammation leads to tissue damage. Studies have shown that simultaneously achieving ROS clearance and COX-2 inhibition holds promise for producing a synergistic effect in inflammatory regulation, thereby more effectively inhibiting inflammation progression and reducing tissue damage, providing a promising research pathway for developing next-generation, highly effective, and safe anti-inflammatory drugs.

[0004] The active ingredients of traditional Chinese medicine have become an indispensable source for modern innovative drug development. Gentiopiroside (GPS) is an enyl ether glycoside compound with anti-inflammatory and analgesic pharmacological activities isolated from traditional Chinese medicines such as gentian and gentiana macrophylla. In previous studies, the inventors' research group discovered a series of gentiopicroside nitroxide radical derivatives with enhanced anti-inflammatory activity obtained through structural modification. Preliminary mechanistic studies showed that their action involves selective COX-2 inhibition, suggesting that gentiopicroside can serve as a lead compound for anti-inflammatory drug design (Chin. J. Nat. Med., 2022, 20, 309; Drug Des., Dev. Ther., 2023, 17, 919). The design and synthesis of novel gentiopicroside nitroxide radical derivatives have promising applications in anti-inflammatory drugs, but a key problem lies in their poor ROS scavenging activity, which limits the full realization of their overall efficacy. Nitrogen oxide radical compounds, due to their stable NO· radical units, exhibit strong free radical scavenging capabilities and can effectively neutralize reactive oxygen species such as ·OH, H2O2, and O2·-, making them a promising class of antioxidants. Furthermore, they can continuously mimic the activity of superoxide dismutase through a "catalytic cycle" mechanism, thereby protecting cells from oxidative damage.

[0005] Therefore, introducing the strong antioxidant unit of nitric oxide free radical into the structure of gentiopicroside to develop gentiopicroside nitric oxide free radical derivatives that simultaneously target inflammation and oxidative stress is of great research value and urgency. Summary of the Invention

[0006] To address the above technical problems, this invention proposes a gentiopicroside nitric oxide radical derivative, its preparation method, and its application.

[0007] The technical solution of this invention is:

[0008] A gentiopicroside nitric oxide radical derivative is provided, the general formula of which is shown in Formula I below: ; Formula I;

[0009] Where n is independently selected from any natural number from 0 to 10;

[0010] R1 is independently selected from any one of hydrogen, methyl, ethyl, and cyclohexyl;

[0011] X is independently selected from either ether bonds or imine bonds;

[0012] R2 is independently selected from any one of the structures shown in A-1, A-2, A-3, and A-4. .

[0013] Preferably, the gentiopicroside nitric oxide radical derivative has a structure as shown in any one of GPS-1 to GPS-10, and the structural formulas of GPS-1 to GPS-10 are shown below: , , , , , , , , , .

[0014] A second aspect of the present invention is to provide a method for preparing the gentiopicroside nitric oxide radical derivative, comprising the following steps:

[0015] (1) The compound shown in structural formula II reacts with the compound shown in structural formula III in the presence of an organic solvent, a base and a condensing agent to give the alkyne compound shown in structural formula IV;

[0016] ;

[0017] (2) The alkyne compound shown in structural formula IV reacts with the gentiopicroside azide derivative shown in structural formula V in the presence of an organic solvent, a base and a catalyst to obtain the gentiopicroside nitric oxide free radical derivative. ;

[0018] In (1) and (2), n is independently selected from any natural number from 0 to 10; R1 is independently selected from any one of hydrogen, methyl, ethyl, and cyclohexyl; X is independently selected from any one of ether bond and imine bond; Y is independently selected from any one of hydroxyl, amino, chlorine atom, and bromine atom; R2 is independently selected from any one of the structures shown in A-1, A-2, A-3, and A-4. .

[0019] In the above preparation method, preferably, in (1), the base is selected from at least one of triethylamine, N,N-diisopropylethylamine, pyridine, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 4-dimethylaminopyridine, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate, barium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide;

[0020] Preferably, the condensing agent described in (1) is selected from at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and O-benzotriazole-tetramethylurea hexafluorophosphate.

[0021] Preferably, the organic solvent described in (1) is selected from at least one of tetrahydrofuran, acetonitrile, dichloromethane, benzene, toluene, xylene, chlorobenzene, chloroform, methanol, ethanol, petroleum ether, N,N-dimethylformamide, and dimethyl sulfoxide.

[0022] Preferably, the base mentioned in (2) is at least one of triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate, barium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0023] Preferably, the organic solvent described in (2) is selected from at least one of tetrahydrofuran, acetonitrile, dichloromethane, benzene, toluene, xylene, chlorobenzene, chloroform, methanol, ethanol, petroleum ether, N,N-dimethylformamide, and dimethyl sulfoxide.

[0024] Preferably, the catalyst described in (2) is selected from at least one of cuprous iodide, cuprous bromide, cuprous chloride, copper sulfate, and copper acetate.

[0025] A third aspect of the invention is the application of gentiopicroside nitric oxide radical derivatives having the structure described above, particularly in the preparation of medicaments for the prevention, relief, and treatment of inflammation.

[0026] In the above applications, preferably, the inflammation includes skin inflammation, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, thrombophlebitis, systemic juvenile idiopathic arthritis, neuroinflammation, viral hepatitis, alcoholic hepatitis, fatty liver disease, gingivitis, periodontitis, myocarditis, cystitis, and prostatitis.

[0027] The present invention has the following advantages and effects compared with the prior art:

[0028] (1) This invention provides a series of gentiopicroside nitric oxide radical derivatives with specific structures. These derivatives exhibit good in vitro COX-2 enzyme inhibitory activity and in vitro ROS scavenging activity. Experimental results show that the gentiopicroside nitric oxide radical derivatives prepared in this invention have a ROS scavenging rate of 33.16% to 55.94% and a COX-2 enzyme inhibition rate of up to 69.43%. Each derivative can effectively inhibit the release of inflammatory mediators and oxidative stress response during the inflammatory process, thereby more effectively inhibiting the progression of inflammation and protecting tissues. They have advantages such as novel structure, good antioxidant and anti-inflammatory activity, and have broad application prospects in the preparation of drugs with the effects of preventing, relieving or treating inflammation.

[0029] (2) The present invention also provides a preparation process for gentiopicroside nitric oxide radical derivatives having the above-described structure. The process is simple to operate and can obtain the derivatives quickly and efficiently in a short time, providing theoretical and technical support for the industrial preparation of gentiopicroside nitric oxide radical derivatives. Attached Figure Description

[0030] Figure 1 High-resolution mass spectrum of GPS-1, a nitrogen oxide radical derivative of gentiopicroside;

[0031] Figure 2 High-resolution mass spectrum of GPS-2, a nitrogen oxide radical derivative of gentiopicroside;

[0032] Figure 3 High-resolution mass spectrum of GPS-3, a nitrogen oxide radical derivative of gentiopicroside;

[0033] Figure 4 High-resolution mass spectrum of GPS-4, a nitrogen oxide radical derivative of gentiopicroside;

[0034] Figure 5 High-resolution mass spectrum of GPS-5, a nitrogen-oxygen free radical derivative of gentiopicroside;

[0035] Figure 6 High-resolution mass spectrum of GPS-6, a nitrogen oxide radical derivative of gentiopicroside;

[0036] Figure 7 High-resolution mass spectrum of GPS-7, a nitrogen oxide radical derivative of gentiopicroside;

[0037] Figure 8 High-resolution mass spectrum of GPS-8, a nitrogen-oxygen radical derivative of gentiopicroside;

[0038] Figure 9 High-resolution mass spectrum of GPS-9, a nitrogen-oxygen free radical derivative of gentiopicroside;

[0039] Figure 10 High-resolution mass spectrum of GPS-10, a nitrogen oxide radical derivative of gentiopicroside;

[0040] Figure 11 The inhibitory effect of gentiopicroside nitric oxide radical derivative GPS-10 on LPS-induced cellular inflammatory factors;

[0041] Figure 12 The in vivo anti-inflammatory effect of GPS-10, a nitrogen oxide free radical derivative of gentiopicroside. Detailed Implementation

[0042] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0043] Example 1

[0044] The synthetic route for the preparation of GPS-1, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0045] The specific steps are as follows:

[0046] (1) Nitrogen oxide radical 1a (0.50 mmol) and compound 1b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane (10 mL). The mixture was stirred until fully dissolved. Subsequently, condensing agents N,N'-dicyclohexylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added sequentially to the reaction system. The reaction was carried out at room temperature for 12 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 1c. MALDI-TOF-MS (C 14 H 22 NO3) Theoretical value m / z = 252.160, measured value: 252.159.

[0047] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 1c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added sequentially to the reaction system. The reaction was carried out at room temperature for 5 hours. After the reaction was completed, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography to obtain the gentiopicroside nitric oxide radical derivative GPS-1. ESI-HRMS(C 30 H 41 N4O 11 Na), theoretical value m / z = 656.2664, measured value: 656.2666, high-resolution mass spectrum of compound GPS-1 is as follows. Figure 1 As shown.

[0048] Example 2

[0049] The synthetic route for the preparation of gentiopicroside nitric oxide radical derivative GPS-2 is as follows: .

[0050] The specific steps are as follows:

[0051] (1) Nitrogen oxide radical 1a (0.50 mmol) and compound 2b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added sequentially to the reaction system. The reaction solution was stirred at room temperature, concentrated under reduced pressure, and separated by column chromatography to obtain alkyne compound 2c. MALDI-TOF-MS (C 18 H 30 NO3) Theoretical value m / z = 308.223, measured value: 308.221.

[0052] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 2c (0.7 mmol) prepared in (1) were dissolved in dichloromethane. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added sequentially to the reaction system. The reaction was carried out at room temperature. After the reaction was completed, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain the gentiopicroside nitric oxide radical derivative GPS-2. ESI-HRMS (C 34 H 49 N4O 11 Na), theoretical value m / z = 712.3290, measured value: 712.3267, high-resolution mass spectrum of compound GPS-2 is as follows. Figure 2 As shown.

[0053] Example 3

[0054] The synthetic route for the preparation of GPS-3, a nitrogen-oxygen radical derivative of gentiopicroside, is as follows: .

[0055] The specific steps are as follows:

[0056] (1) Nitrogen oxide radical 1a (0.50 mmol) and compound 3b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.80 mmol) and N,N-diisopropylethylamine (0.8 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 3c. MALDI-TOF-MS (C 15 H 25 The theoretical value of N2O2 is m / z = 265.191, and the measured value is 265.190.

[0057] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 3c (0.9 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.9 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain the gentiopicroside nitric oxide radical derivative GPS-3. ESI-HRMS(C 31 H 44 N5O 10 Na), theoretical value m / z = 669.2986, measured value: 669.2967, high-resolution mass spectrum of compound GPS-3 is as follows. Figure 3 As shown.

[0058] Example 4

[0059] The synthetic route for the preparation of GPS-4, a nitrogen-oxygen radical derivative of gentiopicroside, is as follows: .

[0060] The specific steps are as follows:

[0061] (1) Nitrogen oxide radical 1a (0.5 mmol) and compound 4b (1.0 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents N,N'-dicyclohexylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.8 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 4c. MALDI-TOF-MS (C10 ... 19 H30 NO3), theoretical value m / z = 320.222, measured value: 320.221, high-resolution mass spectrum of compound GPS-4 is as follows. Figure 4 As shown.

[0062] (2) Gentianoside azide derivative (0.5 mmol) and the alkyne compound 4c (0.7 mmol) prepared in (1) were dissolved in acetonitrile. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain gentianoside nitric oxide radical derivative GPS-4. ESI-HRMS (C 35 H 49 N4O 11 Na), theoretical value m / z = 724.3290, measured value: 724.3296, high-resolution mass spectrum of compound GPS-4 is as follows. Figure 4 As shown.

[0063] Example 5

[0064] The synthetic route for the preparation of GPS-5, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0065] The specific steps are as follows:

[0066] (1) Nitrogen oxide radical 2a (0.50 mmol) and compound 5b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents N,N'-dicyclohexylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 5c. MALDI-TOF-MS (C10 ... 13 H 20 NO3), theoretical value m / z = 238.144, measured value: 238.144.

[0067] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 5c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous bromide (0.05 mmol) and triethylamine (0.75 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain the gentiopicroside nitric oxide radical derivative GPS-5. ESI-HRMS (C 29 H 39 N4O 11Na), theoretical value m / z = 642.2508, measured value: 642.2486, high-resolution mass spectrum of compound GPS-5 as follows: Figure 5 As shown.

[0068] Example 6

[0069] The synthetic route for the preparation of GPS-6, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0070] The specific steps are as follows:

[0071] (1) Nitrogen oxide radical 2a (0.50 mmol) and compound 6b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents N,N'-dicyclohexylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 6c. MALDI-TOF-MS (C10 ... 21 H 36 NO3), theoretical value m / z = 350.270, measured value: 350.270.

[0072] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 6c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and potassium carbonate (0.75 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain the gentiopicroside nitroxide radical derivative GPS-6. ESI-HRMS (C 37 H 55 N4O 11 Na), theoretical value m / z = 754.3760, measured value: 754.3772, high-resolution mass spectrum of compound GPS-6 is as follows. Figure 6 As shown.

[0073] Example 7

[0074] The synthetic route for the preparation of GPS-7, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0075] The specific steps are as follows:

[0076] (1) Nitrogen oxide radical 2a (0.50 mmol) and compound 7b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.90 mmol) and N,N-diisopropylethylamine (1.0 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 7c. MALDI-TOF-MS (C10 ... 12 H 19 N2O2), theoretical value m / z = 223.144, measured value: 223.143.

[0077] (2) The gentiopicroside azide derivative (0.5 mmol) and the alkyne compound 7c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain the gentiopicroside nitric oxide radical derivative GPS-7. ESI-HRMS (C 28 H 39 N5O 10 The theoretical value m / z = 605.2691, the measured value: 605.2698, and the high-resolution mass spectrum of compound GPS-7 are shown below. Figure 7 As shown.

[0078] Example 8

[0079] The synthetic route for the preparation of GPS-8, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0080] The specific steps are as follows:

[0081] (1) Nitrogen oxide radical 2a (0.50 mmol) and compound 8b (0.60 mmol) were added to a round-bottom flask and dissolved in dichloromethane. The mixture was stirred until fully dissolved. Subsequently, condensing agents N,N'-dicyclohexylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 8c. MALDI-TOF-MS (C10 ... 13 H 20 NO3), theoretical value m / z = 238.144, measured value: 238.144.

[0082] (2) Gentianoside azide derivative (0.5 mmol) and the alkyne compound 8c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain gentianoside nitric oxide radical derivative GPS-8. ESI-HRMS (C 29 H 39 N4O 11 Na), theoretical value m / z = 642.2508, measured value: 642.2485, high-resolution mass spectrum of compound GPS-8 is as follows. Figure 8 As shown. Specific Implementation Example 9

[0084] The synthetic route for the preparation of GPS-9, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0085] The specific steps are as follows:

[0086] (1) Nitrogen oxide radical 3a (0.50 mmol) and compound 9b (0.60 mmol) were added to a round-bottom flask and dissolved in chloroform. The mixture was stirred until fully dissolved. Subsequently, the condensing agent N,N'-diisopropylcarbodiimide (0.60 mmol) and 4-dimethylaminopyridine (0.5 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain alkyne compound 9c. MALDI-TOF-MS (C 13 H 19 The theoretical value of N2O2 (m / z) is 235.144, and the measured value is 235.143.

[0087] (2) Gentianoside azide derivative (0.5 mmol) and the alkyne compound 9c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added to the reaction system sequentially. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain gentianoside nitric oxide radical derivative GPS-9. ESI-HRMS(C 29 H 38 N5O 10 Na), theoretical value m / z = 639.2511, measured value: 639.2520, high-resolution mass spectrum of compound GPS-9 is as follows. Figure 9 As shown.

[0088] Example 10

[0089] The synthetic route for the preparation of GPS-10, a nitric oxide radical derivative of gentiopicroside, is as follows: .

[0090] The specific steps are as follows:

[0091] (1) Nitrogen oxide radical 10a (0.50 mmol) and compound 10b (0.60 mmol) were added to a round-bottom flask and dissolved in N,N-dimethylformamide. The mixture was stirred until fully dissolved. Then, sodium hydride (0.8 mmol) was added to the reaction system. After the reaction was completed at room temperature, water was added to quench the reaction, and the mixture was concentrated under reduced pressure. The alkyne compound 10c was obtained by column chromatography. MALDI-TOF-MS (C600000) was used to separate the alkyne compound 10c. 13 H 22 NO2), theoretical value m / z = 210.149, measured value: 210.147.

[0092] (2) Gentianoside azide derivative (0.5 mmol) and the alkyne compound 1c (0.7 mmol) prepared in (1) were dissolved in methanol. Cuprous iodide (0.05 mmol) and triethylamine (0.75 mmol) were added sequentially to the reaction system. After the reaction was completed at room temperature, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain gentianoside nitric oxide radical derivative GPS-10. ESI-HRMS (C 28 H 39 N4O 10 Na), theoretical value m / z = 614.2558, measured value: 614.2555, high-resolution mass spectrum of compound GPS-10 is as follows. Figure 10 As shown.

[0093] Application Example 1

[0094] Verification of the in vitro COX-2 enzyme inhibitory activity of gentiopicroside nitric oxide radical derivatives GPS-1~GPS-10.

[0095] In this invention, the in vitro COX-2 enzyme inhibitory activity assay was performed using a human COX-2 inhibitor screening kit, with celecoxib, a widely used COX-2 inhibitor in clinical practice, as a positive control drug.

[0096] The gentiopicroside nitric oxide radical derivatives GPS-1~GPS-10 prepared in Examples 1-10 and the control sample were prepared into DMSO solutions. Equal portions of these solutions, along with a blank DMSO control, were added to the corresponding reaction mixtures. Subsequently, according to the instructions, the assay buffer, cofactor, working solution, probe, and substrate were added sequentially, to a final compound concentration of 1 μM. After addition, the mixture was shaken for 10 seconds to ensure homogeneity. The reaction mixture was then incubated at 37°C in the dark for 10 minutes. The fluorescence intensity was measured using a microplate reader to calculate the COX-2 inhibition rate. The COX-2 inhibition rates of the gentiopicroside nitric oxide radical derivatives GPS-1~GPS-10 and celecoxib are shown in Table 1.

[0097] Table 1. In vitro COX-2 enzyme inhibitory activity of gentiopicroside nitric oxide radical derivatives serial number COX-2 enzyme inhibition rate (%) GPS-1 55.93 GPS-2 65.57 GPS-3 42.47 GPS-4 68.50 GPS-5 17.87 GPS-6 13.68 GPS-7 15.88 GPS-8 41.23 GPS-9 55.29 GPS-10 69.43 Celecoxib 79.55

[0098] Table 1 shows that celecoxib, a widely used COX-2 inhibitor in clinical practice, exhibits strong COX-2 enzyme inhibitory activity. The gentiopicroside nitric oxide radical derivatives GPS-1 to GPS-10 provided in this application all show certain COX-2 inhibitory activity at a concentration of 1 μM. Therefore, the gentiopicroside nitric oxide radical derivatives provided in this invention can be used in the preparation of anti-inflammatory drugs.

[0099] Application Example 2

[0100] In vitro ROS scavenging activity of gentiopicroside N-oxygen radical derivatives GPS-1~GPS-10 was verified.

[0101] In this invention, the in vitro ROS scavenging activity was determined using the DPPH method. The test compound dissolved in DMSO was added to 200 µL of DPPH solution (final DMSO concentration 0.5%) to form the test reaction mixture, with a final compound concentration of 30 µM. In the control group, an equal volume of DMSO was added to 200 µL of DPPH solution to prepare a DPPH control mixture, and an equal volume of DMSO was added to 200 µL of methanol to prepare a blank control mixture. All mixtures were incubated at room temperature in the dark for 30 minutes. The optical density (OD) of each well was then measured using a microplate reader to calculate the ROS scavenging activity. The results are shown in Table 2.

[0102] Table 2. ROS scavenging rate of gentiopicroside nitric oxide radical derivatives serial number Clearance rate (%) GPS-1 33.16 GPS-2 44.76 GPS-3 41.80 GPS-4 33.21 GPS-5 42.91 GPS-6 36.55 GPS-7 40.25 GPS-8 43.01 GPS-9 37.52 GPS-10 55.94

[0103] The results in Table 2 show that the gentiopicroside nitric oxide radical derivatives GPS-1 to GPS-10 provided by the present invention all exhibit certain ROS scavenging activity at a concentration of 30 μM. Therefore, the gentiopicroside nitric oxide radical derivatives provided by the present invention can be used to scavenge ROS to improve their anti-inflammatory activity.

[0104] Application Example 3

[0105] Validation of the in vitro anti-inflammatory effect of gentiopicroside nitric oxide radical derivative GPS-10.

[0106] In this invention, the in vitro anti-inflammatory effect of gentiopicroside nitric oxide radical derivative GPS-10 was demonstrated using mouse mononuclear macrophage leukemia cells (RAW 264.7 cells) as a model, with celecoxib, a widely used COX-2 inhibitor, as a positive control. RAW 264.7 cells were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Subsequently, the cells were pretreated with different concentrations of GPS-10 or celecoxib for 4 hours, followed by stimulation with LPS (1 μg / mL) for 24 hours. After incubation, the levels of inflammatory factors nitric oxide, TNF-α, and IL-6 in the culture medium were measured to assess the anti-inflammatory effect. The results are as follows: Figure 11 As shown in the figure, the positive control drug celecoxib can effectively inhibit the levels of inflammatory factors nitric oxide, TNF-α, and IL-6. Meanwhile, the gentiopicroside nitric oxide radical derivative GPS-10 provided by this invention exhibits an anti-inflammatory effect almost equivalent to that of the positive control drug celecoxib at the same concentration. Therefore, the gentiopicroside nitric oxide radical derivative provided by this invention can be used in the preparation of anti-inflammatory drugs.

[0107] Application Example 4

[0108] Verification of the in vivo anti-inflammatory effect of gentiopicroside nitric oxide radical derivative GPS-10.

[0109] The in vivo anti-inflammatory effect of the gentiopicroside nitric oxide radical derivative GPS-10 in this invention was investigated using a mouse ear edema model induced by 2-O-tetradecanoylphorbol-13-ethyl ester (TPA), with celecoxib, a widely used COX-2 inhibitor, as a positive control. TPA solution was applied to the dorsal and ventral surfaces of the mouse ears, followed by the application of GPS-10 or celecoxib to the same areas. Six hours after administration, the mice were euthanized, and ear tissue samples were taken using a 6 mm punch, weighed, and the thickness of the ear tissue was measured. The results are as follows: Figure 12 As shown, Figure 12As can be seen, the positive control drug celecoxib can reduce the thickness and weight of ear swelling in mice. The gentiopicroside nitric oxide radical derivative GPS-10 provided by this invention exhibits an anti-inflammatory effect comparable to that of the positive control drug celecoxib at the same concentration. All the above evidence demonstrates that the gentiopicroside nitric oxide radical derivative provided by this invention can be used in the preparation of anti-inflammatory drugs. The above are merely preferred embodiments of this invention and do not limit the scope of the patent. All equivalent changes and modifications made within the scope of this invention should still fall within the scope of this invention.

Claims

1. A gentiopicroside nitric oxide radical derivative, characterized in that, The general formula of the gentiopicroside nitric oxide radical derivative is shown in Formula I below: ； Formula I; Where n is independently selected from any natural number from 0 to 10; R1 is independently selected from any one of hydrogen, methyl, ethyl, and cyclohexyl; X is independently selected from either ether bonds or imine bonds; R2 is independently selected from any one of the structures shown in A-1, A-2, A-3, and A-4. .

2. The method for preparing the gentiopicroside nitric oxide radical derivative as described in claim 1, characterized in that, The steps include the following: (1) The compound shown in structural formula II reacts with the compound shown in structural formula III in the presence of an organic solvent, a base and a condensing agent to give the alkyne compound shown in structural formula IV; ； (2) The alkyne compound shown in structural formula IV reacts with the gentiopicroside azide derivative shown in structural formula V in the presence of an organic solvent, a base and a catalyst to obtain the gentiopicroside nitric oxide free radical derivative. ； In (1) or (2), n is independently selected from any natural number from 0 to 10; R1 is independently selected from any one of hydrogen, methyl, ethyl, and cyclohexyl; X is independently selected from any one of ether bond and imine bond; Y is independently selected from any one of hydroxyl, amino, chlorine atom, and bromine atom; R2 is independently selected from any one of the structures shown in A-1, A-2, A-3, and A-4. .

3. The preparation method according to claim 2, characterized in that, In (1), the base is selected from at least one of triethylamine, N,N-diisopropylethylamine, pyridine, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, 4-dimethylaminopyridine, sodium hydride, potassium hydride, sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate, barium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide; The condensing agent is selected from at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and O-benzotriazole-tetramethylurea hexafluorophosphate. The organic solvent is selected from at least one of tetrahydrofuran, acetonitrile, dichloromethane, benzene, toluene, xylene, chlorobenzene, chloroform, methanol, ethanol, petroleum ether, N,N-dimethylformamide, and dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that, (2) The base is at least one of triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate, barium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide; The organic solvent is selected from at least one of tetrahydrofuran, acetonitrile, dichloromethane, benzene, toluene, xylene, chlorobenzene, chloroform, methanol, ethanol, petroleum ether, N,N-dimethylformamide, and dimethyl sulfoxide; The catalyst is selected from at least one of cuprous iodide, cuprous bromide, cuprous chloride, copper sulfate, and copper acetate.

5. The use of the gentiopicroside nitric oxide radical derivative as described in claim 1 in the preparation of a medicament for the prevention, relief, and treatment of inflammation.

6. The application as described in claim 5, characterized in that, The inflammations mentioned include skin inflammation, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, thrombophlebitis, systemic juvenile idiopathic arthritis, neurological inflammation, viral hepatitis, alcoholic hepatitis, fatty liver disease, gingivitis, periodontitis, myocarditis, cystitis, and prostatitis.

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