Benzopyrone compound, composition and application of benzopyrone compound in preparation of medicine for treating hyperuricemia

Abstract

The invention belongs to the field of medical chemistry, and relates to a benzopyrone compound, a benzopyrone composition and application of the benzopyrone compound and the benzopyrone composition in preparation of a medicine for treating hyperuricemia. The chemical structure is as shown in formula I; in the formula, R1 is fluorine or hydrogen; r2 is fluorine or hydrogen; r3 is hydrogen, nitryl or tertiary butyl; r4 is hydrogen, fluorine or trifluoromethyl; and R1, R2, R3 and R4 are not hydrogen at the same time. The benzopyrone compound provided by the invention has good inhibitory activity on XOD, and can reduce the level of uric acid, thereby being beneficial to treatment of hyperuricemia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and relates to a benzopyranone compound, a composition, and its use in the preparation of a drug for treating hyperuricemia. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Hyperuricemia is a metabolic disease caused by excessive uric acid production or reduced excretion, leading to excessively high blood uric acid levels. Long-term development can lead to complications such as gouty arthritis, uric acid nephropathy, and kidney stones, and it is closely related to the development of chronic diseases such as hypertension, diabetes, and cardiovascular disease. Its pathogenesis is related to factors such as abnormally elevated xanthine oxidase (XOD) activity and obstruction of uric acid excretion pathways.

[0004] Natural products and their derivatives are an important source of new drug development. Benzopyranones (also known as coumarins) are a class of heterocyclic compounds widely found in nature, and their derivatives exhibit a wide variety of biological activities. Previous studies have shown that some benzopyranone structures can exert uric acid-lowering effects by inhibiting xanthocyanin-dependent oxidation (XOD). For example, demethylwedelolactone and its analogues, extracted from plants, have been reported to have significant XOD-inhibiting activity. Current research generally agrees that the key structural feature of these compounds is the presence of multiple phenolic hydroxyl groups on their molecular backbone, particularly the phenolic hydroxyl group at the C-5 position, which is considered the pharmacophore through which they bind to the XOD active site via hydrogen bonding.

[0005] However, the aforementioned compounds that rely on the phenolic hydroxyl group for their effects also have certain limitations. The phenolic hydroxyl group is prone to phase II metabolic reactions such as methylation and glucuronidation in vivo, which may lead to reduced bioavailability or a shortened duration of action. Furthermore, the relatively reactive nature of the phenolic hydroxyl group may affect the chemical stability of the compound or introduce other unintended metabolites. Therefore, exploring novel benzopyranone XOD inhibitors that do not rely on the traditional phenolic hydroxyl pharmacophore is of great significance for the treatment and control of hyperuricemia. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a benzopyranone compound, a composition, and its application in the preparation of a medicament for treating hyperuricemia. The benzopyranone compound provided by the present invention exhibits good inhibitory activity against XOD and can reduce uric acid levels, thereby facilitating the treatment of hyperuricemia.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a benzopyranone compound with the chemical structure shown in Formula I; ; Wherein, R1 is fluorine or hydrogen; R2 is fluorine or hydrogen; R3 is hydrogen, nitro or tert-butyl; R4 is hydrogen, fluorine or trifluoromethyl; and R1, R2, R3 and R4 are not all hydrogen at the same time.

[0008] In a second aspect, a method for preparing the benzopyranone compound described in the first aspect of the present invention includes the step of obtaining the compound shown in Formula I by using the compound shown in Formula II and the compound shown in Formula III as raw materials according to the following reaction route; ; R1, R2, R3, and R4 are the same as R1, R2, R3, and R4 in the benzopyranone compounds described in the first aspect of the present invention.

[0009] Thirdly, a composition comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is a benzopyranone compound as described in the first aspect of the present invention, and the chemical structure of the second active ingredient is shown in Formula V-4. .

[0010] Fourthly, the use of a benzopyranone compound as described in the first aspect of the present invention or a composition as described in the third aspect in the preparation of a medicament for treating hyperuricemia.

[0011] The beneficial effects of this invention are as follows: 1. The benzopyranone compounds provided by this invention have good inhibitory activity against XOD and significantly reduce uric acid in the HK-2 hyperuricemia cell model, showing good potential medicinal value and can be used in the preparation of drugs for the treatment of hyperuricemia.

[0012] 2. The benzopyranone compounds provided by this invention can be used in combination with compound V-4 to synergistically increase XOD inhibition activity and lower uric acid, demonstrating highly effective targeted therapy, thereby achieving synergistic effects, reducing single-drug dosage, and minimizing side effects. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 The 1H NMR spectrum of the benzopyranone compound of formula I-1 prepared in Example 1 of this invention; Figure 2 The carbon NMR spectrum of the benzopyranone compound of formula I-1 obtained in Example 1 of this invention; Figure 3 This is a high-performance liquid chromatogram of the benzopyranone compound of formula I-1 obtained in Example 1 of the present invention; Figure 4 The 1H NMR spectrum of the benzopyranone compounds of formula I-2 obtained in Example 2 of this invention; Figure 5 The carbon NMR spectrum of the benzopyranone compounds of formula I-2 obtained in Example 2 of this invention; Figure 6 The high-performance liquid chromatogram of the benzopyranone compounds of formula I-2 obtained in Example 2 of this invention; Figure 7 The 1H NMR spectrum of the benzopyranone compounds of formula I-3 obtained in Example 3 of this invention; Figure 8 The carbon NMR spectra of the benzopyranone compounds of formula I-3 obtained in Example 3 of this invention; Figure 9 The high-performance liquid chromatograms of the benzopyranone compounds of formula I-3 obtained in Example 3 of this invention are shown below. Figure 10 The above are the 1H NMR spectra of the benzopyranone compounds of formula I-4 obtained in Example 4 of this invention. Figure 11 The carbon NMR spectra of the benzopyranone compounds of formula I-4 obtained in Example 4 of this invention; Figure 12 The high-performance liquid chromatograms of the benzopyranone compounds of formula I-4 obtained in Example 4 of this invention are shown below. Figure 13 The above are the 1H NMR spectra of the benzopyranone compounds of formula I-5 obtained in Example 5 of this invention. Figure 14 The carbon NMR spectra of the benzopyranone compounds of formula I-5 obtained in Example 5 of this invention; Figure 15 The high-performance liquid chromatograms of the benzopyranone compounds of formula I-5 obtained in Example 5 of this invention are shown below. Figure 16 The 1H NMR spectra of the benzopyranone compounds of formula I-6 prepared in Comparative Example 1 of this invention are shown below. Figure 17 The carbon NMR spectra of the benzopyranone compounds of formula I-6 prepared in Comparative Example 1 of this invention are shown below. Figure 18The high-performance liquid chromatograms of the benzopyranone compounds of formula I-6 prepared in Comparative Example 1 of this invention are shown below. Figure 19 The 1H NMR spectrum of compound V-4 of this invention; Figure 20 This is the carbon NMR spectrum of compound V-4 of the present invention; Figure 21 This is a high-performance liquid chromatogram of compound V-4 of the present invention. Detailed Implementation

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0017] A typical embodiment of the present invention provides a benzopyranone compound with the chemical structure shown in Formula I; ; Wherein, R1 is fluorine or hydrogen; R2 is fluorine or hydrogen; R3 is hydrogen, nitro or tert-butyl; R4 is hydrogen, fluorine or trifluoromethyl; and R1, R2, R3 and R4 are not all hydrogen at the same time.

[0018] In some embodiments, the compounds are selected from the following: .

[0019] Another embodiment of the present invention provides a method for preparing the above-mentioned benzopyranone compounds of the present invention, comprising the step of obtaining the compound of formula I by using the compound of formula II and the compound of formula III as raw materials according to the following reaction route; ; R1, R2, R3, and R4 are the same as R1, R2, R3, and R4 in the benzopyranone compounds described above.

[0020] In this invention, the compounds shown in Formula I, Formula II, Formula III, Formula IV, and Formula V can be simply referred to as Compound I, Compound II, Compound III, Compound IV, and Compound V, respectively.

[0021] In some embodiments, compounds II and III undergo a Pulgrin reaction under conditions of acid anhydride and triethylamine to prepare compound IV. Specifically, the acid anhydride is acetic anhydride; wherein the molar ratio of acetic anhydride to compound II is 2.7–3.3:1. Specifically, the reaction temperature is 105–110 °C, and the reaction time is 2–4 h. Specifically, after the Pulgrin reaction, the resulting solution is extracted, distilled under reduced pressure, and purified. The extraction method is as follows: first, extract with ethyl acetate at least twice, collect the organic phase, and then wash the organic phase with saturated brine. The purpose of extracting with saturated brine here is to reduce the solubility of the product in the organic phase (ethyl acetate) in water, thereby reducing the loss of the target compound; at the same time, wash away residual water-soluble impurities in the organic phase, and promote the separation of the organic and aqueous phases through the salting-out effect, thereby improving the efficiency of subsequent drying and purification steps.

[0022] In some embodiments, the molar ratio of compound II to compound III is 1:1 to 2, preferably 1:1.01 to 1.10.

[0023] In some embodiments, a mixed solution of hydrochloric acid and ethanol is added to compound IV to remove the acetyl group, yielding compound V. Specifically, the reaction temperature is 82–88 °C, and the reaction time is 0.5–2 h. The mixed solution of hydrochloric acid and ethanol is generally prepared by mixing commercially available concentrated hydrochloric acid (mass concentration of 36–38%) with ethanol, wherein the volume ratio of commercially available concentrated hydrochloric acid to ethanol is (18:82)–(22:78), which can be denoted as an 18–22% hydrochloric acid / ethanol solution. Specifically, the purification method after the reaction is silica gel column chromatography.

[0024] In some embodiments, compound V is reacted with DDQ (dichlorocyanobenzoquinone) at 108-112°C for 13-17 min under microwave conditions to obtain compound I. Specifically, the molar ratio of compound V to DDQ is 1:1.8-2.2. After the reaction, the crude product is subjected to vacuum distillation and purification. More specifically, the purification method is silica gel column chromatography, using a mixture of petroleum ether and ethyl acetate as the eluent.

[0025] A third embodiment of the present invention provides a composition comprising a first active ingredient and a second active ingredient, wherein the first active ingredient is the above-mentioned benzopyranone compound, and the chemical structure of the second active ingredient is shown in Formula V-4; .

[0026] In some embodiments, the molar ratio of the first active ingredient and the second active ingredient is 1:0.9~1.1.

[0027] A fourth embodiment of the present invention provides the use of the above-mentioned benzopyranone compounds or compositions in the preparation of a medicament for treating hyperuricemia.

[0028] In some embodiments, the drug further includes pharmaceutical excipients. These excipients include, but are not limited to, binders, fillers, disintegrants, lubricants, preservatives, flavoring agents, and fragrances. The pharmaceutical excipients constitute 1-99% of the total weight of the drug.

[0029] In some embodiments, the dosage form of the drug is tablets, granules, capsules, or injections. Different dosage forms of the drug may have appropriate pharmaceutical excipients added as needed.

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0031] The synthesis routes in the following embodiments are as follows: .

[0032] The compounds presented in the following examples and comparative examples are shown below: .

[0033] Example 1 This embodiment provides a benzopyranone compound as shown in Formula I-1; .

[0034] The method for synthesizing the above-mentioned benzopyranone compounds includes the following steps: Step a: Triethylamine (12 mmol, 2.0 eq) and acetic anhydride (18 mmol, 3.0 eq) were added to compound II-1 (4-nitrosalicylic acid, 6 mmol, 1.0 eq) and compound III (6 mmol, 1.0 eq). The mixture was stirred and refluxed at 110 °C for 3 hours. The reaction mixture was quenched with ice water, and the aqueous phase was extracted three times with ethyl acetate. The organic phase was retained, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solution was removed by vacuum distillation. The solution was then purified by silica gel column chromatography (PE / EA = 5:1) to obtain compound IV-1 (1.6 g, yield 86.3%, purity 99.4%), the chemical structure of which is shown below: .

[0035] Step b: Compound IV-1 (3.5 mmol, 1.0 eq) was dissolved in 20 mL of 20% hydrochloric acid / ethanol solution, stirred, and reacted at 85 °C for 1 hour. The reaction was quenched with ice water, and the aqueous phase was extracted three times with ethyl acetate. The organic phase was retained, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solution was removed by vacuum distillation. The solution was then purified by silica gel column chromatography to obtain compound V-1 (914 mg, yield 89.7%, purity 99.8%), whose chemical structure is shown below: .

[0036] Step c: Add DDQ (2.52 mmol, 2.0 eq) to intermediate V-1 (1.26 mmol, 1.0 eq), add 8 g of silica gel, transfer to an experimental microwave oven, and react at 110 °C for 15 min. After sonication with ethyl acetate and silica gel, filter, collect the filtrate, remove the solution by vacuum distillation, and purify by silica gel column chromatography (PE / EA = 10:1) to obtain the target compound I-1 (293 mg, yield 82.1%, purity 99.9%). The 1H NMR spectrum of the target compound is shown below. Figure 1 As shown, the carbon NMR spectrum is as follows: Figure 2 As shown, the high-performance liquid chromatogram is as follows: Figure 3 As shown.

[0037] Examples 2-5 Examples 2-5 provide benzopyranone compounds as shown in Formula I-2 to Formula I-5, respectively.

[0038] The synthesis methods of the benzopyranone compounds shown in Formulas I-2 to I-5 provided in Examples 2 to 5 are similar to those in Example 1, except that 4-nitrosalicylic acid aldehyde in step a is replaced accordingly. The remaining steps are the same as in the examples and will not be repeated here.

[0039] It should be noted that the molecular weight of the target compound was determined by Q-TOF-HRMS, and the nuclear magnetic resonance data and mass spectrometry data of the benzopyranone compounds are shown in Table 1.

[0040] The proton NMR spectrum of compound I-2 (i.e., compound I-2) prepared in Example 2 is shown below. Figure 4 As shown, the carbon NMR spectrum is as follows: Figure 5 As shown, the high-performance liquid chromatogram is as follows: Figure 6 As shown.

[0041] The proton NMR spectrum of compound I-3 (i.e., compound I-3) prepared in Example 3 is shown below. Figure 7 As shown, the carbon NMR spectrum is as follows: Figure 8 As shown, the high-performance liquid chromatogram is as follows: Figure 9 As shown.

[0042] The proton NMR spectrum of compound I-4 (i.e., compound I-4) prepared in Example 4 is shown below. Figure 10 As shown, the carbon NMR spectrum is as follows: Figure 11 As shown, the high-performance liquid chromatogram is as follows: Figure 12 As shown.

[0043] The proton NMR spectrum of compound I-5 (i.e., compound I-5) prepared in Example 5 is shown below. Figure 13 As shown, the carbon NMR spectrum is as follows: Figure 14 As shown, the high-performance liquid chromatogram is as follows: Figure 15 As shown.

[0044] Comparative Example 1 The synthesis method of the benzopyranone compounds shown in Formulas I-6 provided in Comparative Example 1 is similar to that in Example 1, except that the salicylaldehyde in step a is replaced accordingly. The remaining steps are the same as in the examples and will not be repeated here.

[0045] The proton NMR spectrum of compound I-6 (i.e., compound I-6) prepared in Comparative Example 1 is shown below. Figure 16 As shown, the carbon NMR spectrum is as follows: Figure 17 As shown, the high-performance liquid chromatogram is as follows: Figure 18 As shown.

[0046] Compound V-4 (i.e., compound V-4) is an intermediate of compound I-4 prepared in Example 4, and its 1H NMR spectrum is shown below. Figure 19 As shown, the carbon NMR spectrum is as follows: Figure 20 As shown, the high-performance liquid chromatogram is as follows: Figure 21 As shown.

[0047] Table 1. Nuclear magnetic resonance and mass spectrometry data of compounds I-1 to I-6 and compound V-4

[0048] Pharmacological activity experiment XOD inhibitory activity tests of compound I, compound V-4, and norweedrolide: Experimental methods (1) Dissolve compound V-4, compounds I-1~I-6 and desiccated pyridostigmine in DMSO to prepare a 10mM stock solution for later use.

[0049] (2) Use 0.067 M PBS with pH 6.8 to serially dilute the stock solution in step (1) to 5 μM, 10 μM and 25 μM.

[0050] (3) The compounds of different concentrations in step (2) were incubated with 1 μM XOD at 37 °C for 15 minutes (the blank control group was added with an equal amount of PBS). Then, 1 μM substrate xanthine solution was added and pre-incubated at 37 °C for 10 minutes. The system was immediately placed in a microplate reader and the absorbance at 295 nm was detected. PBS solvent was used as a blank control and the measurements were repeated three times in parallel.

[0051] The experimental results are shown in Table 2.

[0052] Table 2. Inhibitory activities of benzopyranone compounds and nordecibelone on XOD

[0053] Table 2 evaluates the inhibitory activity of the compounds against XOD. The results show that compounds I-1–I-6, V-4, and norfedipine all exhibited inhibitory effects on XOD at concentrations ranging from 5 to 25 μM, in a concentration-dependent manner. Compound I-4 showed the strongest activity, achieving an inhibition rate of 74.52% at 25 μM, which was superior to the non-phenolic hydroxyl compound I-6 and the polyhydroxyl compound norfedipine. This indicates that these compounds block uric acid production by directly inhibiting XOD, which is the clear mechanism by which they exert their uric acid-lowering effect.

[0054] XOD inhibitory activity tests were conducted on compound I and nordecibelin in combination with compound V-4: Experimental methods (1) Compounds I-1~I-6 and des-cresting wedelia lactone were combined with compound V-4 at a molar ratio of 1:1, dissolved in DMSO, and prepared into a 10 mM stock solution for later use. (2) Use 0.067 M PBS with a pH of 6.8 to serially dilute the stock solution in step (1) to 5 μM, 10 μM and 25 μM; (3) The compounds of different concentrations in step (2) were incubated with 1 μM XOD at 37 °C for 15 minutes (the blank control group was added with an equal amount of PBS). Then, 1 μM substrate xanthine solution was added and pre-incubated at 37 °C for 10 minutes. The system was immediately placed in a microplate reader and the absorbance at 295 nm was detected. PBS solvent was used as a blank control. The measurements were performed three times in parallel. The results are shown in Table 3.

[0055] Table 3. XOD inhibitory activity of compounds I-1~I-6 and the combination of norfelidomide with compound V-4.

[0056] Table 3 evaluates the effects of the combination of compounds of formula I series and norweedin with compound V-4 on XOD inhibitory activity. The results show that the XOD inhibition rates of compounds I-1 to I-5 in combination with compound V-4 are significantly higher than those of the compounds used alone, and the inhibition rate gradually increases with increasing total concentration. Meanwhile, I-6 and norweedin exhibit antagonistic effects when combined with compound V-4. This indicates a significant synergistic XOD inhibitory effect between compounds I-1 to I-5 and compound V-4, which can reduce the dosage of individual components, decrease potential side effects, and improve drug safety and efficacy through combination therapy.

[0057] Cytotoxicity assay of HK-2 human renal proximal tubular epithelial cells with compounds I, V-4, and norfelidomide: Experimental methods (1) Cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were digested, counted, and resuspended at 3 × 10⁻⁶ cells / mL. 3 Inoculate the cells / wells into 96-well plates and culture them. (2) After culturing for 24 hours, I-1~I-5 (5 μM, 10 μM and 25 μM) and compound V-4 were added to the culture medium. I-6 without phenolic hydroxyl groups and polyhydroxyl-containing desiccated pyrethroids were selected as controls. The culture medium was incubated for 72 hours in a 37 ℃, 5% CO2 incubator. Then, 20 μL of 5 mg / L MTT was added to the culture medium. After culturing for 4 hours in the incubator, the culture medium was removed, and formazan was dissolved in 150 μL of DMSO. The absorbance at 450 nm was measured using an ELISA reader.

[0058] The experimental results are shown in Table 4.

[0059] Table 4. Cytotoxic effects of benzopyranone compounds and nordecibelone on HK-2 cells.

[0060] Table 4 evaluates the toxicity of benzopyranone compounds and norweedin to human renal cortical proximal tubular epithelial cells (HK-2), as the kidney is a vital organ for uric acid excretion, and uric acid-lowering drugs need to act safely in this site. At concentrations of 5–25 μM, none of the compounds significantly affected the activity of HK-2 cells, with cell viability remaining above 89%. Compounds I-1–I-5 were slightly superior to the non-phenolic hydroxyl compound I-6 and the polyhydroxyl compound norweedin. Compound I-4 exhibited the best cell compatibility, with cell viability of 98.02% at 5 μM, 96.27% at 10 μM, and 93.96% even at 25 μM, showing no significant difference in toxicity. This demonstrates the excellent biocompatibility of the compounds in target organ-related cell models, supporting their safety as uric acid-lowering drugs.

[0061] Effects of Compound I, Compound V-4, and Norfedipine on Uric Acid Content in the HK-2 Hyperuricemia Cell Model: Experimental methods (1) After digesting, resuspending, and counting HK-2 cells in the logarithmic growth phase, they were then subjected to 3 × 10⁻⁶ cells / mL. 4 Cells were seeded into 24-well plates at a rate of 1 cell per well and incubated in an incubator at 37 °C and 5% CO2 for 24 hours. The obtained cells were then divided into a blank control group and an experimental group. (2) 1 μM adenosine was added to the blank control group and the experimental group. The experimental group was treated with 5 μM, 10 μM and 25 μM of compounds I-1~I-6, compound V-4 and desiccane lactone. DMSO (<0.3%) was added to the blank group.

[0062] (3) After incubating the cells from each group treated in step (2) in an incubator for 24 hours, the cells were collected into centrifuge tubes, washed twice with 1 mL PBS, cultured in serum-free medium containing 1 μM adenosine for 24 hours, and then cultured in 0.5 μM XOD for 1 hour. The supernatant was collected and the uric acid content was determined according to the kit instructions. The experiment was repeated three times.

[0063] The experimental results are shown in Table 5.

[0064] Table 5. Inhibitory activity of benzopyranone compounds and nordecibelone on uric acid levels.

[0065] Table 5 validates the actual uric acid-lowering effects of the compounds in a hyperuricemia model constructed from HK-2 cells. Data shows that compounds I-1 to I-5 effectively reduced uric acid levels in the cell model at concentrations of 5–25 μM, with the effect being concentration-dependent. Compound I-4 showed the best effect, reducing uric acid levels to 80.56% of the control group at 5 μM, 72.53% at 10 μM, and further to 64.76% at 25 μM. Compound I-4's reduction of uric acid levels in the cell model at concentrations of 5–25 μM was superior to that of compound I-6 (without phenolic hydroxyl groups) and the polyhydroxy compound nordecibelone. This ultimately confirms at the cellular level that these compounds do indeed possess significant uric acid-lowering functions.

[0066] Effects of compound I and norweedrolactone on uric acid levels in the HK-2 hyperuricemia cell model, respectively: Experimental methods (1) After digesting, resuspending, and counting HK-2 cells in the logarithmic growth phase, they were then subjected to 3 × 10⁻⁶ cells / mL. 4 Inoculate the cells / wells into 24-well plates and incubate them at 37 °C in a 5% CO2 incubator for 24 hours; (2) Add 1 μM adenosine, and treat compound V-4 with compounds I-1~I-6 and desiccanetolide at a molar ratio of 1:1 with 5 μM, 10 μM and 25 μM respectively.

[0067] (3) After incubating the cells treated in step (2) in an incubator for 24 hours, collect the cells into centrifuge tubes, wash the cells twice with 1 mL PBS, and culture them in serum-free medium containing 1 μM adenosine for 24 hours. Then add 0.5 μM XOD and culture for 1 hour. Take the supernatant and determine the uric acid content according to the kit instructions. Repeat the experiment three times.

[0068] The experimental results are shown in Table 6.

[0069] Table 6. Inhibitory activity of compounds I-1~I-6 and norfelidomide in combination with compound V-4 on uric acid levels.

[0070] Table 6 validates the actual uric acid-lowering effect of the combination of compound I and compound V-4 in the HK-2 cell hyperuricemia model. Data shows that in the HK-2 hyperuricemia model, the uric acid levels in the combination groups of compounds I-1 to I-5 were lower than those in the single-drug groups and the single-drug group of compound V-4. The combination of compound I-4 and compound V-4 showed the best effect, with uric acid levels at 5 μM being 66.89% of the control group, 59.63% at 10 μM, and decreasing to 53.89% at 25 μM, superior to the effect of any single compound. However, the uric acid levels after the combination of compound I-6 (without phenolic hydroxyl groups) and the polyhydroxy compound terpinene with compound V-4 were slightly higher than those in the single-drug groups. This indicates that the combination of compounds I-1 to I-5 with compound V-4 can synergistically enhance the uric acid-lowering efficacy. Through the synergistic uric acid-lowering effect of the two compounds, the effective therapeutic dose of each single drug is reduced while significantly improving the uric acid-lowering efficacy.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A benzopyranone compound, characterized in that, Its chemical structure is shown in Formula I; ； Wherein, R1 is fluorine or hydrogen; R2 is fluorine or hydrogen; R3 is hydrogen, nitro or tert-butyl; R4 is hydrogen, fluorine or trifluoromethyl; and R1, R2, R3 and R4 are not all hydrogen at the same time.

2. The benzopyranone compound as described in claim 1, characterized in that, Selected from the following compounds: 。 3. A method for preparing the benzopyranone compound according to claim 1, characterized in that, The steps include obtaining the compound of formula I from the compounds shown in formula II and formula III via the following reaction route; ； R1, R2, R3, and R4 are the same as R1, R2, R3, and R4 in the benzopyranone compounds of claim 1.

4. The preparation method according to claim 3, characterized in that, Compounds II and III were subjected to the Pullman reaction under acid anhydride and triethylamine conditions to prepare compound IV.

5. The preparation method according to claim 3, characterized in that, Adding a mixture of hydrochloric acid and ethanol to compound IV removes the acetyl group, yielding compound V.

6. The preparation method according to claim 3, characterized in that, Compound V was reacted with DDQ at 108-112 °C for 13-17 min under microwave conditions to obtain compound I.

7. A composition characterized in that, It includes a first active ingredient and a second active ingredient, wherein the first active ingredient is the above-mentioned benzopyranone compound, and the chemical structure of the second active ingredient is shown in Formula V-4; 。 8. The composition of claim 7, characterized in that, The molar ratio of the first active ingredient to the second active ingredient is 1:0.9~1.

1.

9. Use of a benzopyranone compound of claim 1 or 2 or a composition of claim 7 or 8 in the preparation of a medicament for treating hyperuricemia.

10. The application as described in claim 9, characterized in that, The drug also includes pharmaceutical excipients; Alternatively, the dosage form of the drug may be tablets, granules, capsules, or injections.

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