Indometacin puerarin ester as well as synthesis method and application thereof

Abstract

The invention relates to indometacin puerarin ester as well as a synthesis method and application thereof, and belongs to the technical field of pharmaceutical chemistry synthesis. In order to solve the problem that side effects are large when indomethacin is used for a long time, puerarin with a multi-target protection effect and indomethacin are coupled through ester bonds on the basis of an innovative twin drug design strategy, and the novel compound indomethacin puerarin ester is constructed. The indometacin puerarin ester synthesized by the invention has uric acid reducing activity, can inhibit XOD activity and reduce uric acid generation, and promotes uric acid excretion by up-regulating ABCG2 protein expression quantity and down-regulating GLUT9 and URAT1 protein expression quantity. In addition, the indometacin puerarin ester also has good anti-inflammatory activity, and the activity is superior to that of puerarin, indometacin and combined application of the puerarin and the indometacin. The indometacin puerarin ester not only reduces the adverse reaction of indometacin, but also can improve the compliance of patients, and has wide clinical application prospects in the field of gout resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical synthesis, and in particular relates to indomethacin puerarin ester and a synthesis method and application thereof. Background Art

[0002] Indometacin has anti-inflammatory, antipyretic, and analgesic properties and is primarily used to treat soft tissue injuries and inflammation. It inhibits prostaglandin synthesis and the effects of substances like bradykinin, providing a strong analgesic effect for mild to moderate pain. Therefore, indometacin is also used to relieve the pain and inflammation of acute gouty arthritis, quickly alleviating symptoms such as joint redness, swelling, heat, and pain caused by gout, helping patients alleviate pain and improve joint function. Although recent literature reports suggest that indometacin has some xanthine oxidase inhibitory activity, reducing uric acid production, this activity is very low and cannot correct hyperuricemia. Long-term use can cause kidney damage and gastrointestinal adverse reactions, making it unsuitable for the long-term treatment of chronic gout.

[0003] The combined use of diuretics can reduce the kidney damage caused by indomethacin. For example, hydrochlorothiazide can help excrete excess water and salt from the body, while potassium-sparing diuretics such as spironolactone can prevent excessive loss of potassium ions and maintain electrolyte balance. The combined use of proton pump inhibitors such as omeprazole or H2 receptor blockers such as ranitidine can effectively reduce gastrointestinal discomfort caused by indomethacin and protect the gastric mucosa. The combined use of gastric mucosal protective agents such as sucralfate and bismuth potassium citrate can form a protective layer on the gastric mucosa, reducing the direct irritation of indomethacin on the gastric mucosa.

[0004] Although this combination therapy can alleviate the side effects of long-term indomethacin use to a certain extent, it also has a number of limitations in practical applications. Combination therapy requires patients to take multiple medications simultaneously, increasing the complexity of medication and the financial burden on patients, thereby reducing medication compliance. Summary of the Invention

[0005] In order to solve the problem of severe side effects of long-term use of indomethacin, the present invention provides indomethacin puerarin ester and a synthesis method and application thereof.

[0006] The technical solution of the present invention:

[0007] Indomethacin puerarin ester, the chemical structure is: C 40 H 34 ClNO 12 ; Its molecular structure is shown in Formula I:

[0008]

[0009] Formula I.

[0010] A method for synthesizing indomethacin puerarin ester comprises the following steps:

[0011] Step 1: completely dissolve puerarin in anhydrous pyridine, and add 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 4-dimethylaminopyridine and indomethacin in sequence, stirring until completely dissolved and then starting the reaction at a certain temperature. After the reaction is completed, concentrate under reduced pressure to obtain a solid product;

[0012] Step 2: The solid product obtained in step 1 is washed, dried, dissolved in methanol, and purified by chromatography to obtain a powdery solid product, which is indomethacin puerarin ester.

[0013] Furthermore, in step 1, the mass volume ratio of puerarin to anhydrous pyridine is 500 mg: (15 mL~20 mL).

[0014] Furthermore, in step 1, the molar ratio of puerarin, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 4-dimethylaminopyridine and indomethacin is 5:5:1:5.

[0015] Furthermore, the reaction temperature in step 1 is 40-60° C., and the reaction time is 8-10 h.

[0016] Furthermore, the chromatography in step 2 is silica gel column chromatography, and the mobile phase of the chromatography is a mixture of dichloromethane and methanol.

[0017] Furthermore, the volume ratio of dichloromethane to methanol is 10:1.

[0018] Disclosed is an application of indomethacin puerarin ester in the preparation of a medicine for treating hyperuricemia combined with gout.

[0019] Disclosed is an application of indomethacin-puerarin ester in the preparation of a medicine for treating hyperuricemia.

[0020] Disclosed is an application of indomethacin-puerarin ester in the preparation of a medicine for treating gout.

[0021] Beneficial effects of the present invention:

[0022] The present invention successfully synthesized indomethacin puerarin ester by introducing the puerarin structure. This novel compound significantly reduces its adverse reactions while retaining the original anti-inflammatory and analgesic effects of indomethacin. At the same time, the indomethacin puerarin ester synthesized by the present invention also has uric acid-lowering activity, can inhibit xanthine oxidase activity, reduce uric acid production, and promote uric acid excretion by increasing ABCG2 protein expression and decreasing GLUT9 and URAT1 protein expression. In addition, the anti-inflammatory activity of indomethacin puerarin ester is superior to the effect of puerarin, indomethacin, and the combination of the two, providing theoretical support for the treatment of hyperuricemia combined with gout.

[0023] The synthesis method of the present invention is simple and low-cost, making the production and popularization of the drug possible. Indomethacin puerarin ester not only reduces the adverse reactions of indomethacin, but also improves patient compliance, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the HPLC spectrum of indomethacin puerarin ester I-Pue prepared in Example 1;

[0025] Figure 2 This is the high-resolution mass spectrum of indomethacin puerarin ester I-Pue prepared in Example 1 in positive ion mode;

[0026] Figure 3 Indomethacin puerarin ester I-Pue prepared in Example 1 1 H-NMR nuclear magnetic resonance spectrum;

[0027] Figure 4 Indomethacin puerarin ester I-Pue prepared in Example 1 13 C-NMR nuclear magnetic resonance spectrum;

[0028] Figure 5 This is a comparison of the survival rates of HK-2 cells at different drug concentrations in each drug administration group in Example 5, A is the indomethacin group, B is the puerarin group, C is the indomethacin and puerarin combined group, and D is the indomethacin puerarin ester group;

[0029] Figure 6 This is a comparison of the survival rates of RAW24.7 cells at different drug concentrations in each administration group in Example 5, A is the indomethacin group, B is the puerarin group, C is the indomethacin and puerarin combined group, and D is the indomethacin puerarin ester group;

[0030] Figure 7 This is a comparison chart of the test results of the HK-2 cell hyperuricemia model in Example 5, where A represents the uric acid content, B represents the electrophoresis band of the uric acid transporter, and C represents the expression level of the uric acid transporter;

[0031] Figure 8 This is a comparison of uric acid content in the supernatant of HK-2 cells, a model of cellular hyperuricemia, at different drug concentrations in each dosing group in Example 5;

[0032] Figure 9 This is a comparison chart of XOD content in the supernatant of HK-2 cells, a model of hyperuricemia, at different drug concentrations in each dosing group in Example 5;

[0033] Figure 10 The WB images of urate transporter in HK-2 cells, a model of hyperuricemia, at different drug concentrations in each dosing group in Example 5;

[0034] Figure 11 This is a comparison of the expression levels of uric acid transporters in the HK-2 cell model of hyperuricemia at different drug concentrations in each dosing group in Example 5, A represents ABCG2, B represents URAT1, and C represents GLUT9;

[0035] Figure 12 This is a comparison chart of NO release levels in the inflammatory cell model RAW264.7 cells at different drug concentrations in each dosing group in Example 5. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0037] Example 1

[0038] This example provides indomethacin puerarin ester (I-Pue) and its synthesis method.

[0039] The molecular structure of indomethacin puerarin ester I-Pue in this embodiment is shown in Formula I:

[0040]

[0041] Formula I.

[0042] The synthetic route for preparing indomethacin-puerarin ester from puerarin and indomethacin is as follows:

[0043]

[0044] The specific steps of the synthesis method of indomethacin puerarin ester in this embodiment are as follows:

[0045] Step 1: Place puerarin in a reaction flask and completely dissolve it in anhydrous pyridine. Add puerarin, 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), and indomethacin in a molar ratio of 5:5:1:5. Stir until completely dissolved and then start the reaction. The reaction temperature is 60°C. The reaction is monitored by TLC (methanol to dichloromethane volume ratio is 10:1). After reacting for 8 hours, the solvent is recovered under reduced pressure.

[0046] Step 2: The solid product obtained in step 1 was rinsed with distilled water 3 to 5 times, dried, dissolved in a small amount of methanol, and purified by silica gel column chromatography. The mobile phase was a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 10:1 to obtain a light yellow powdery solid product, which was indomethacin puerarin ester I-Pue.

[0047] The compound structural formula of I-Pue obtained in this embodiment is C 40 H 34 ClNO 12 , the molecular weight is 755.1802, and the purity, mass spectrometry and nuclear magnetic resonance spectrum detection are carried out as follows:

[0048] (1) Purity test: Weigh a certain amount of I-Pue and dissolve it in methanol. After filtering through a filter membrane (0.22 µm), the sample was automatically injected into the HPLC (10 µL). The purity of the complex was determined by normalizing the chromatographic peak area.

[0049] Chromatographic conditions: A Thermo C18 column (4.6 mm × 250 mm, 5 µm) was used at 25°C. The mobile phase consisted of a methanol-water gradient (methanol:water = 0:100 to 100:0 (v / v)) at a flow rate of 1.0 mL / min. Detection wavelengths for F-Pue and I-Pue were 239 nm and 248 nm, respectively.

[0050] HPLC chromatogram Figure 1 As shown, the test results show that the purity of I-Pue obtained in this example is 97.67%.

[0051] The yield calculation formula was applied: Yield (%) = actual yield / theoretical yield × 100%, and the yield of I-Pue in this example was calculated to be 55.30%.

[0052] (2) Mass spectrometry (MS): Weigh about 2 mg of I-Pue sample and dissolve it in anhydrous methanol. After filtering through a filter membrane (0.22 μm), perform high-resolution mass spectrometry detection. The results of high-resolution mass spectrometry detection in positive ion mode are as follows: Figure 2 shown.

[0053] (3) Nuclear magnetic resonance spectrum test: The I-Pue sample to be tested was dissolved in DMSO-d6, filtered through a filter membrane (0.22 μm), and then transferred to a nuclear magnetic resonance tube for 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) test, the results are as follows Figure 3 and Figure 4 shown.

[0054] 1 H-NMR (600 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.22 (d, J = 76.5 Hz, 1H), 7.79 (d, J = 9.8 Hz, 1H), 7.56 (d, J = 72.7 Hz, 4H), 7.42-7.28 (m, 2H), 6.91(d, J = 9.4 Hz, 1H), 6.82 (dd, J = 30.6, 8.3 Hz, 3H), 6.68 (d, J = 11.9 Hz,2H), 5.64 (dt, J = 90.2, 9.6 Hz, 1H), 5.38 (s, 1H), 5.22 (s, 1H), 4.97 (d, J= 10.1 Hz, 1H), 4.57 (s, 1H), 3.77 (td, J = 15.0, 7.4 Hz, 1H), 3.68 (s, 3H), 3.58 (s, 2H), 3.48-3.30 (m, 5H), 1.95-1.79 (m, 3H).

[0055] 13 C-NMR (150 MHz, DMSO-d6) δ 175.05, 169.66, 168.15, 157.59, 157.02,155.94, 152.87, 138.01, 135.51, 134.65, 131.51, 130.77, 130.39, 129.45,127.13, 123.48, 122.90, 115.40, 115.03, 112.79, 111.77, 110.81, 110.32,101.68, 82.61, 76.51, 73.52, 72.64, 71.08, 61.96, 55.77, 40.50, 29.50, 13.50.MS m / z calcd for C 40 H 34 ClNO 12 [M+H]+ 756.18, found 756.1802.

[0056] Example 2

[0057] This embodiment provides a method for synthesizing indomethacin puerarin ester, and the specific steps are as follows:

[0058] Step 1: Place puerarin in a reaction flask and completely dissolve it in anhydrous pyridine. Add puerarin, 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), and indomethacin in a molar ratio of 5:5:1:5. Stir until completely dissolved and then start the reaction. The reaction temperature is 60°C. The reaction is monitored by TLC (the volume ratio of methanol to dichloromethane is 10:1). After reacting for 10 hours, the solvent is recovered under reduced pressure.

[0059] Step 2: The solid product obtained in Step 1 was rinsed several times with water, dried, dissolved in a small amount of methanol, and purified by silica gel column chromatography (dichloromethane:methanol volume ratio of 10:1) to obtain a pale yellow solid product, indomethacin puerarin ester I-Pue. Testing showed that the yield of I-Pue in this example was 51.88%.

[0060] Example 3

[0061] This embodiment provides a method for synthesizing indomethacin puerarin ester, and the specific steps are as follows:

[0062] Step 1: Place puerarin in a reaction flask and completely dissolve it in anhydrous pyridine. Add puerarin, 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP) and indomethacin in a molar ratio of 5:5:1:5. Stir until completely dissolved and then start the reaction. The reaction temperature is 40°C. The reaction is monitored by TLC (the volume ratio of methanol to dichloromethane is 10:1). After reacting for 10 hours, the solvent is recovered under reduced pressure.

[0063] Step 2: The solid product obtained in Step 1 was rinsed several times with water, dried, dissolved in a small amount of methanol, and purified by silica gel column chromatography (dichloromethane:methanol volume ratio of 10:1) to obtain a pale yellow solid product, indomethacin puerarin ester I-Pue. Testing showed that the yield of I-Pue in this example was 48.23%.

[0064] Example 4

[0065] This embodiment provides a method for synthesizing indomethacin puerarin ester, and the specific steps are as follows:

[0066] Step 1: Place puerarin in a reaction flask and completely dissolve it in anhydrous pyridine. Add puerarin, 1-ethyl-3(3-dimethylpropylamine)carbodiimide (EDCI), 4-dimethylaminopyridine (DMAP), and indomethacin in a molar ratio of 5:5:1:5. Stir until completely dissolved and then start the reaction. The reaction temperature is 40°C. The reaction is monitored by TLC (the volume ratio of methanol to dichloromethane is 10:1). After reacting for 8 hours, the solvent is recovered under reduced pressure.

[0067] Step 2: Rinse the solid product obtained in Step 1 several times with water, dry it, dissolve it in a small amount of methanol, and purify it by silica gel column chromatography (dichloromethane:methanol volume ratio of 10:1) to obtain a pale yellow solid product, indomethacin puerarin ester I-Pue. Testing showed that the yield of I-Pue in this example was 45.54%.

[0068] Example 5

[0069] This example investigates the uric acid-lowering activity and anti-inflammatory effect of indomethacin puerarin ester.

[0070] This example investigates the effects of I-Pue on uric acid production and excretion in an adenosine / XOD-induced HK-2 cell hyperuricemia model. Furthermore, the LPS-induced RAW264.7 cell inflammation model is used to evaluate I-Pue's regulatory effects on inflammatory cytokine expression. The indomethacin puerarin ester used in this experiment was the I-Pue prepared in Example 1.

[0071] The HK-2 cells used in this example are human renal proximal tubular epithelial cell lines purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; and the RAW264.7 cells are mouse macrophages purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0072] Depending on the cell growth conditions, HK-2 cells were grown in DMEM / F12 medium, while RAW264.7 cells were grown in DMEM medium. Complete culture medium was prepared as required, as shown in Table 1.

[0073] Table 1

[0074]

[0075] The cell recovery method of this embodiment is:

[0076] Before cell culture, prewarm complete DMEM / F12 (for HK-2 cells) and DMEM (for RAW264.7 cells) at 37°C. After removing frozen cells from -80°C, quickly thaw them in a 37°C water bath and harvest them by centrifugation at 3000 rpm for 5 minutes. Resuspend the pellet in 2 mL of the corresponding complete medium and inoculate it into a T25 flask containing 3 mL of culture medium. Incubate at 37°C with 5% CO2.

[0077] The cell passage method of this embodiment is:

[0078] (1) HK-2 cell passage

[0079] HK-2 cells should be subcultured when the cell density reaches approximately 80%. PBS, complete culture medium, and trypsin solution should be preheated to 37°C before use. The specific steps are as follows: After discarding the old culture medium, wash the cells twice with 3 mL of PBS; add 1 mL of trypsin and digest for 1-2 minutes, observing under a microscope until most cells become round and fall off; after digestion, divide the cell suspension into new culture flasks at a ratio of 1:3, and add 4 mL of complete culture medium to each flask. After subculture, confirm the cell status by microscopic examination and continue culturing in a 37°C, 5% CO2 incubator. During the operation, care should be taken to avoid over-digestion and mechanical damage to the cells.

[0080] (2) RAW264.7 cell passaging

[0081] RAW264.7 cells have a semi-adherent growth pattern, are round, and readily differentiate. They can be passaged when the cell density reaches 80%. Given their sensitivity to trypsin, mechanical passaging should be used: first, discard the culture medium and gently wash twice with 3 mL of PBS. Then, add 3 mL of PBS and gently pipette to detach the cells. Collect the suspension and centrifuge at 3000 rpm for 5 minutes, then resuspend in 3 mL of complete culture medium. Aliquot the suspension into new culture flasks at a 1:3 ratio, add 4 mL of culture medium to each flask, mix thoroughly, and incubate in a 37°C, 5% CO2 incubator. Exercise gentleness throughout the procedure to avoid cell damage.

[0082] The cell freezing method of this embodiment is:

[0083] When cells are confluent in the culture flask and in good condition, trypsinize HK-2 cells and mechanically pipette RAW264.7 cells to obtain a cell suspension. Centrifuge the cell suspension at 3000 rpm for 10 minutes, discard the supernatant, and resuspend in an appropriate amount of serum-free freezing buffer. Aliquot into cryovials and label with cell name, passage number, freezing date, and operator information. Seal the cryovials and transfer to a -80°C freezer for long-term storage.

[0084] The cell counting method of this embodiment is:

[0085] First, disinfect the counting slide with 75% alcohol and install a coverslip. Slowly pipette 10 μL of cell suspension along the edge of the coverslip, avoiding bubbles. Observe and count under a microscope. Count cells from the top, not the bottom, and from the left, not the right.

[0086] Counting formula: number of cells / mL = sum of 4 large grids / 4×10 4 × dilution factor

[0087] The preparation methods of Ino, Pue, Ino+Pue and I-Pue solutions in this embodiment are as follows:

[0088] Preparation of test sample solutions: Weigh indomethacin (Ino), puerarin (Pue), an equimolar mixture of indomethacin and puerarin (Ino+Pue), and indomethacin-puerarin ester (I-Pue). Dissolve thoroughly in DMSO to prepare stock solutions of defined concentrations. Refrigerate and store until ready for use. Dilute the stock solutions with DMEM / F12 and DMEM basal media to concentrations of 300, 100, 30, 10, 3, and 1 mg / L, respectively (maximum final DMSO concentration of 1‰). Sterilize by filtration through a 0.22 µm filter prior to use.

[0089] This example investigates the safe dosing concentration of indomethacin puerarin ester, and the method for detecting cytotoxic activity using the CCK-8 method is:

[0090] (1) HK-2 cytotoxic activity assay

[0091] The experiment was divided into four groups:

[0092] Blank control group: only DMEM / F12 medium, no cells inoculated;

[0093] Normal control group: cells were inoculated with DMEM / F12 medium only;

[0094] DMSO control group: cells were inoculated and the culture medium contained DMSO at the same amount as the highest drug concentration;

[0095] Drug-treated group: cells were inoculated and the culture medium contained different concentrations of drugs.

[0096] Cell plating and drug administration: Take HK-2 cell suspension in the logarithmic growth phase and adjust the cell density to 5×10 4Cells were seeded at 100 μL per well in a 96-well plate, with 100 μL of cell suspension added to each well. Six replicates were set up for each group. To minimize edge effects, PBS buffer was added to the periphery of the wells. The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to fully adhere and grow. After 24 hours of incubation, the remaining medium was aspirated and 100 μL of the corresponding sample solution was added to each group: indomethacin (Ino), puerarin (Pue), an equimolar mixture of indomethacin and puerarin (Ino+Pue), and indomethacin-puerarin ester (I-Pue). The sample concentrations were 300, 100, 30, 10, 3, and 1 mg / L.

[0097] After 24 and 48 hours of incubation, 10 µL of CCK-8 solution was added to each well and incubated at 37°C for 2 hours. The absorbance (OD) of each well at 450 nm was measured using a microplate reader and the data were recorded and analyzed.

[0098] (3) Calculation of cell viability

[0099] According to the toxicity test results of HK-2 and RAW264.7 cells, the experimental data were substituted into the following formula to calculate the cell viability:

[0100] Cell viability: CV% = (As-Ab) / (Ac-Ab) × 100%

[0101] As: OD values ​​of different concentrations of drug groups

[0102] Ac: OD value of normal control group

[0103] Ab: OD value of blank control group

[0104] After calculating the cell viability of each group, the survival rates of HK-2 cells at different drug concentrations in each group are shown in Table 2 and Figure 5 shown.

[0105] Table 2

[0106]

[0107] As shown in Table 2 and Figure 5 Comparison of the data revealed that 0.1% DMSO had no significant effect on HK-2 cell viability. Pue ​​exhibited no cytotoxicity within the 1-100 mg / L concentration range, with cell viability still exceeding 90% at 300 mg / L. Ino, the combination of Ino and Pue, and I-Pue exhibited cytostatic activity above 100 mg / L, but showed no cytotoxicity within the 1-30 mg / L concentration range.

[0108] Therefore, 3, 10, and 30 mg / L were selected as safe dosing concentrations in subsequent experiments.

[0109] (2) The experimental method for cytotoxic activity detection of RAW264.7 cells was the same as that for (1) HK-2 cytotoxic activity detection, but RAW264.7 cells were cultured in DMEM medium. The survival rates of RAW264.7 cells at different drug concentrations in each group were shown in Tables 3 and Figure 6 shown.

[0110] Table 3

[0111]

[0112] As shown in Table 3 and Figure 6 Comparison of the data revealed that 0.1% DMSO had no significant effect on RAW264.7 cell viability. The experimental results showed that Pue exhibited no cytotoxicity within the 1-300 mg / L concentration range; Ino+Pue and I-Pue exhibited no cytotoxicity within the 1-100 mg / L concentration range; and indomethacin exhibited a cytostatic effect at concentrations greater than 100 mg / L, with no effect on cell viability within the 1-30 mg / L range.

[0113] Based on the above results, this experiment determined 3, 10, and 30 mg / L as the safe dosing concentration ranges for subsequent experiments.

[0114] The method for establishing the HK-2 hyperuricemia cell model in this example is as follows:

[0115] In this example, a conventional method was used to establish a hyperuricemia model in HK-2 cells, and a blank group (basal culture medium) and a negative control group were set up: 2.5 mmol / L adenosine + 0.005 U / mL XOD (xanthine oxidase, purchased from Dalian Meilun Biotechnology Co., Ltd.).

[0116] The density of HK-2 cells in the logarithmic growth phase was adjusted to 5×10 5 Cells were plated in 24-well plates (1 mL / well) and cultured at 37°C, 5% CO2 for 24 h. The original culture medium was discarded, washed three times with PBS, and replaced with fresh DMEM / F12 basal medium for continued culture. The negative control group was first treated with adenosine for 24 h and then with XOD for 8 h. The uric acid content in the cell supernatant was determined by Elisa. The results are as follows: Figure 7 As shown in the figure, the uric acid level in the negative control group (NC) was significantly higher than that in the blank group (N) (P<0.01).

[0117] Further testing of uric acid transporter content showed the following results: Figure 7As shown in the results, compared with the blank group, the expression levels of GLUT9 and URAT1 in the negative control group were significantly decreased (P < 0.01), and the expression level of ABCG2 was significantly increased (P < 0.01), which proved that the high uric acid cell model was successfully established.

[0118] The method for determining the uric acid-lowering activity of indomethacin puerarin ester in this example is as follows:

[0119] Experimental groups: The groups were blank group (N), negative control group (NC), allopurinol group (Allo), and drug-treated groups (Ino, Pue, I-Pue, Ino+Pue). The groups were divided into low-dose, medium-dose, and high-dose groups (3, 10, and 30 mg / L) according to the dose.

[0120] Cell plating: Take HK-2 cell suspension in logarithmic growth phase and dilute it to a certain multiple to make the cell density 10 5 Cells were plated on a 24-well plate at a volume of 1 mL per well, with six replicates per group. The plate was gently shaken to evenly distribute the cells on the bottom and incubated in an incubator for 24 h.

[0121] Cell administration: When the cells grew to about 80%, the culture medium was aspirated and 1 mL of culture medium containing 2.5 mmol / L adenosine and 0.005 U / mL XOD was added to the negative control group and the drug administration group. After further incubation for 24 h, the supernatant was aspirated and the cells were washed twice with PBS buffer. 1 mL of drug-containing culture medium at concentrations of 3, 10, and 30 mg / L was added to the drug administration group.

[0122] After incubation for 24 h, the uric acid content in the supernatant of each well was detected using a uric acid kit. The uric acid content in the supernatant of each group of cells is shown in Table 4 and Figure 8 shown.

[0123] Table 4

[0124]

[0125] As shown in Table 4 and Figure 8Comparison of the data revealed that compared with the negative control group, the low, medium, and high doses of indomethacin (3, 10, and 30 mg / L) failed to reduce uric acid levels in HK-2 cells (P>0.05). Low-dose puerarin, low-dose I+Pue, and low-dose I-Pue (3 mg / L) had no significant effect on uric acid levels in HK-2 cells (P>0.05). However, the medium and high doses (10 and 30 mg / L) significantly reduced uric acid levels in HK-2 cells (P<0.05, P<0.01). Compared with the puerarin group, the high-dose I-Pue group showed a significant decrease in uric acid levels (P<0.05). Comparison of the different dose groups revealed that the uric acid-lowering effects of puerarin, puerarin+indomethacin, and I-Pue were dose-dependent. I-Pue exhibited a uric acid-lowering effect, with superior activity to puerarin, indomethacin, and their combination.

[0126] This example investigates the uric acid-lowering mechanism of indomethacin puerarin ester, and the specific method is as follows:

[0127] (1) Impact on XOD content

[0128] The experimental grouping, plating and drug administration methods were the same as those for the uric acid-lowering activity assay.

[0129] After incubation for 24 h, the XOD content in the supernatant of each well was detected by Elisa. The XOD content of each group of cells is shown in Table 5 and Figure 9 shown.

[0130] Table 5

[0131]

[0132] As shown in Table 5 and Figure 9 Comparison of the data revealed that the XOD content in the negative control group was significantly increased compared with the normal group (P < 0.01), suggesting that adenosine modeling may promote uric acid production by activating XOD activity. Compared with the negative control group, XOD content was significantly decreased in the medium-dose puerarin, medium-dose puerarin + indomethacin, and medium-dose I-Pue groups (P < 0.05). XOD content was significantly decreased in the allopurinol, high-dose puerarin, high-dose puerarin + indomethacin, and high-dose I-Pue groups (P < 0.01). Compared with the puerarin group, XOD content was significantly decreased in the high-dose I-Pue group (P < 0.01). Comparison of the different dose groups revealed that the inhibitory activity of puerarin, puerarin + indomethacin, and I-Pue on XOD was dose-dependent. In summary, I-Pue can inhibit XOD activity and reduce uric acid production, and its activity is superior to that of puerarin.

[0133] (2) Effect on uric acid transporter expression

[0134] Cell grouping: According to the control principle, the experimental groups were set up as blank group, negative control group, puerarin group (Pue), indomethacin group (Ino), Ino and Pue combination group, low-dose I-Pue group, medium-dose I-Pue group, and high-dose I-Pue group. The doses were divided into low, medium, and high-dose groups (3, 10, and 30 mg / L).

[0135] Cell administration: Take HK-2 cell suspension in logarithmic growth phase and dilute to 10 5 Cells were transferred to a T25 culture flask at a concentration of 100 μg / mL. The cells were gently shaken to evenly distribute them on the bottom and cultured at 37°C, 5% CO₂ for 24 h to allow the cells to adhere. The culture medium was discarded, and 4 mL of basal culture medium containing 2.5 mmol / L adenosine was added to the negative control and treatment groups. After incubation for 1 h, XOD (final concentration 0.005 U / mL) was added and cultured for a further 23 h. After washing twice with PBS, 4 mL of drug-containing culture medium was added to the treatment groups and cultured for a further 24 h.

[0136] Protein extraction: After discarding the culture supernatant, wash the cells twice with PBS, mechanically scrape the cells, centrifuge at 3000 rpm for 10 min, discard the supernatant, add 150 μl of a well-mixed lysis buffer (RIPA:PMSF = 100:1), fully lyse on ice for 15 min, centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant and store in aliquots.

[0137] The method for detecting the expression of uric acid transporter is as follows:

[0138] (1) Preparation of solution:

[0139] Running buffer: 3.03 g Tris + 14.4 g glycine + 0.37 g SDS, dilute to 1 L with ddH2O (store at 4°C).

[0140] Transfer buffer: 3.0 g Tris + 14.4 g glycine + 200 mL methanol, dilute to 1 L with ddH2O (store at 4°C).

[0141] TBST: 8.0 g NaCl + 2.4 g Tris + 1 mL Tween-20, dilute to 1 L with ddH2O.

[0142] 10% APS: 2.0 g ammonium persulfate + 18 mL ddH2O (store at 4°C).

[0143] 10% SDS: 2.0 g SDS+18 mL ddH2O.

[0144] Blocking solution: 1.0 g skim milk powder + 19 mL TBST (prepared and used immediately).

[0145] (2) Protein denaturation:

[0146] The protein sample was mixed with 5× SDS-PAGE loading buffer at a ratio of 4:1, boiled at 100°C for 10 min to denature the protein, and stored at −20°C until use.

[0147] (3) Gel configuration:

[0148] Based on the molecular weights of the target proteins (ABCG2: 75 kDa; GLUT9: 59 kDa; URAT1: 60 kDa), SDS-PAGE was performed using a 10% separating gel and a 5% stacking gel system (see Table 6 for the recipe). Before preparing the gel, check the electrophoresis tank for tightness. After polymerization, seal the separating gel with anhydrous ethanol. After separation, remove the ethanol and aspirate the remaining liquid. Prepare the stacking gel immediately before use, and avoid introducing bubbles when inserting the sample comb. The amount of TEMED used can be adjusted based on the polymerization rate.

[0149] Table 6

[0150]

[0151] Note: APS is ammonium persulfate, SDS is sodium dodecyl sulfate, Tris-HCl is tris(hydroxymethyl)aminomethane hydrochloride, and TEMED is N,N,N',N'-tetramethylethylenediamine.

[0152] (4) SDS-PAGE electrophoresis

[0153] Mount the prepared gel in a vertical electrophoresis tank. After adding an appropriate amount of running buffer, load the prestained protein marker and denatured protein sample in sequence. Run the gel stacking step at 80 V for 30 minutes and the separation step at 120 V for 90 minutes. Terminate the electrophoresis when the bromophenol blue indicator migrates to approximately 1 cm from the bottom of the gel. Immediately transfer the sample to the membrane after the electrophoresis is complete.

[0154] (5) Transfer

[0155] Transfer experiments were performed using a standard wet transfer method. First, assemble the SDS-PAGE gel and preactivated PVDF membrane in the following order: With the black side facing down, place a transfer cassette with a sponge soaked in transfer buffer, filter paper, target protein gel, PVDF membrane, filter paper, and sponge, ensuring close contact and no air bubbles. Place the transfer cassette in an ice bath, add pre-chilled transfer buffer, and transfer the membrane at a constant current of 200 mA for 90 minutes. After transfer, proceed immediately to the subsequent blocking and antibody incubation steps.

[0156] (6) Blocking and antibody incubation

[0157] After transfer, the PVDF membrane was first incubated in 5% skim milk powder / TBST blocking buffer at room temperature for 2 hours with shaking to block nonspecific sites. After extensive washing with TBST (3 × 10 min), the membrane was transferred to the primary antibody incubation buffer and incubated overnight at 4°C. The following day, the membrane was washed three times with TBST (3 × 10 min) and incubated with HRP-conjugated secondary antibody at room temperature for 1.5 hours. Excess antibody was then removed with TBST (3 × 10 min).

[0158] (7) ECL chemiluminescence

[0159] ECL luminescent reagent (A:B=1:1) was used for color development, chemiluminescence imaging system was used to collect signals, and Image J software was used for analysis.

[0160] The expression of GLUT9, ABCG2 and URAT1 proteins in cells is shown in Tables 7 and Figure 10 shown.

[0161] Table 7

[0162]

[0163] As shown in Table 7 and Figure 10 、 Figure 11 Data comparison showed that compared with the normal group, the expression of ABCG2 protein in the negative control group was significantly downregulated (P<0.01), while the expression of GLUT9 and URAT1 proteins was significantly upregulated (P<0.01).

[0164] Compared with the negative control group, no significant differences were observed in the expression levels of GLUT9, ABCG2, and URAT1 proteins in the indomethacin and low-dose I-Pue groups (P>0.05). The expression of ABCG2 protein in the puerarin group and the indomethacin and puerarin combination group was significantly increased (P<0.05), while the expression levels of URAT1 and GLUT9 proteins were significantly decreased (P<0.05). The expression of ABCG2 protein in the medium- and high-dose I-Pue groups was significantly increased (P<0.01), while the expression levels of URAT1 and GLUT9 proteins were significantly decreased (P<0.05). Compared with the puerarin group, the expression of ABCG2 in the high-dose I-Pue group was significantly increased (P<0.01), while the expression levels of URAT1 and GLUT9 proteins were significantly decreased (P<0.01).

[0165] In summary, indomethacin alone cannot regulate urate transporter expression; puerarin, the combination of indomethacin and puerarin, and I-Pue all affect urate transporter expression. The mechanism by which urate transporters promote urate excretion may be through upregulating ABCG2 and downregulating GLUT9 and URAT1 protein expression, thereby promoting urate excretion.

[0166] This example investigates the anti-inflammatory activity of indomethacin puerarin ester.

[0167] The method for establishing the RAW264.7 inflammatory cell model is as follows:

[0168] In this example, a conventional method in the art was used to establish a RAW264.7 cell inflammation model by LPS induction. 4 RAW264.7 cells were seeded into 96-well plates at 100 μL / well and incubated at 37°C, 5% CO₂ for 24 hours until the cells adhered. After 24 hours of stimulation with 100 μL of LPS solution (1 μg / mL), NO levels in the supernatant were measured by the Griess method. A significant increase in NO levels in the negative control group compared to the blank group (P < 0.01) confirmed the successful establishment of the inflammatory model.

[0169] The Griess method for measuring NO levels is as follows:

[0170] NO is a pro-inflammatory factor produced when cells undergo inflammatory response and is closely related to the inflammatory response. Its content can be used as an indicator to judge the degree of inflammation, so the Griess method is used for detection.

[0171] Experimental groups: normal group, model group, dexamethasone group and different concentrations of drug treatment groups

[0172] Cell plating: RAW264.7 cells were seeded into 96-well plates, following the same procedure as above.

[0173] Cellular Dosing: Adherent RAW264.7 cells cultured for 24 hours were treated with discarded culture medium and various concentrations of the test drug and dexamethasone (10 μM) added, with six replicates per well. After incubation at 37°C, 5% CO₂ for 1 hour, LPS solution (final concentration 1 μg / mL) was added to the experimental and positive control groups. An equal volume of culture medium was added to the negative control group. An LPS model group (final concentration 1 μg / mL) was also established, and incubation continued for 23 hours.

[0174] Griess method was used to determine NO content: 50 μL of cell supernatant was transferred to a new 96-well plate, and 50 μL of Griess Reagent I and 50 μL of Griess Reagent II (equilibrated at room temperature) were added in sequence. After mixing, the plate was allowed to stand for 10 min. The absorbance at 540 nm was measured using a microplate reader to calculate the NO release. The NO release levels of cells in each group are shown in Table 8 and Figure 12 shown.

[0175] Table 8

[0176]

[0177] As shown in Table 8 and Figure 12Comparison of the data revealed that compared with the blank control group, the NO content in the model group was significantly increased (P < 0.01), indicating that LPS successfully induced an inflammatory response in RAW264.7 cells. Compared with the model group, the NO content in the dexamethasone group was significantly decreased (P < 0.01). The NO content in the high-dose puerarin, medium-dose indomethacin, and medium-dose puerarin plus indomethacin groups was significantly decreased (P < 0.05). The NO content in the high-dose indomethacin, high-dose puerarin plus indomethacin, and medium- and high-dose I-Pue groups was significantly decreased (P < 0.01). Compared with puerarin, the NO content in the high-dose I-Pue group was significantly decreased (P < 0.01). Comparison of the different dose groups revealed that the NO content in puerarin, indomethacin, puerarin plus indomethacin, and I-Pue showed a dose-dependent trend. These results indicate that I-Pue exhibits potent anti-inflammatory activity and is more active than puerarin, indomethacin, or their combination.

[0178] Statistical analysis

[0179] The experimental data in this example are presented as mean ± standard deviation (±S) and processed using SPSS 27.0 statistical analysis software. One-way analysis of variance (ANOVA) was used to compare differences between groups, supplemented by the least significant difference (LSD) method for post hoc multiple comparisons. P < 0.05 indicated a statistically significant difference, and P < 0.01 indicated a significant difference.

Claims

1. Indomethacin puerarin ester, characterized in that The compound structural formula is: C 40 H 34 ClNO 12 ; Its molecular structure is shown in Formula I: Formula I.

2. A method for synthesizing indomethacin puerarin ester according to claim 1, characterized in that: The steps include: Step 1: completely dissolve puerarin in anhydrous pyridine, and add 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 4-dimethylaminopyridine and indomethacin in sequence, stirring until completely dissolved and then starting the reaction at a certain temperature. After the reaction is completed, concentrate under reduced pressure to obtain a solid product; Step 2: The solid product obtained in step 1 is washed, dried, dissolved in methanol, and purified by chromatography to obtain a powdery solid product, which is indomethacin puerarin ester.

3. The method for synthesizing indomethacin puerarin ester according to claim 2, wherein: In step 1, the mass volume ratio of puerarin to anhydrous pyridine is 500 mg: (15 mL~20 mL).

4. The method for synthesizing indomethacin puerarin ester according to claim 2 or 3, characterized in that: In step 1, the molar ratio of puerarin, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide, 4-dimethylaminopyridine and indomethacin is 5:5:1:

5.

5. The method for synthesizing indomethacin puerarin ester according to claim 4, characterized in that: The reaction temperature in step 1 is 40-60°C, and the reaction time is 8-10 h.

6. The method for synthesizing indomethacin puerarin ester according to claim 5, characterized in that: The chromatography in step 2 is silica gel column chromatography, and the mobile phase of the chromatography is a mixture of dichloromethane and methanol.

7. The method for synthesizing indomethacin puerarin ester according to claim 6, characterized in that: The volume ratio of dichloromethane to methanol is 10:

1.

8. Use of the indomethacin puerarin ester according to claim 1 in the preparation of a medicament for treating hyperuricemia combined with gout.

9. Use of the indomethacin puerarin ester according to claim 1 in the preparation of a medicament for treating hyperuricemia.

10. Use of the indomethacin puerarin ester according to claim 1 in the preparation of a medicament for treating gout.

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