Benzofuran compound, preparation method thereof and gout medicine

Abstract

This invention discloses a benzofuran compound and its application in lowering uric acid activity. Compounds 1-3 were isolated from the fermentation products of the endophytic fungus *Septoriella phragmitis*, with compound 1 being a novel compound. The fermentation products were purified using mixed solvent extraction, normal silica gel column chromatography, and high-performance liquid chromatography to obtain compounds 1-3. The inhibitory activity of compounds 1-3 on xanthine oxidase, a target related to gout symptoms, was tested. Experimental results showed that the novel compound 1 exhibited good inhibitory activity against xanthine oxidase. Molecular docking experiments also demonstrated that compound 1 had a good binding energy to the target protein. Therefore, compound 1 can be used to prepare a natural lead compound for delaying or treating diseases caused by xanthine oxidase, providing a potential therapeutic agent for hyperuricemia and gout.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the isolation, purification and application of benzofuran compounds in the fermentation broth of the endophytic fungus Septoriella phragmitis. Specifically, it relates to the isolation, purification and preparation methods of a new compound 1, as well as the inhibitory activity of compound 1 on xanthine oxidase. Background Technology

[0002] Xanthine oxidase (XO) is a key rate-limiting enzyme in the purine metabolism pathway. It irreversibly catalyzes the oxidation of hypoxanthine to xanthine, which in turn oxidizes xanthine to uric acid. This process is accompanied by the generation of reactive oxygen species. Abnormally elevated XO activity is a core contributing factor to hyperuricemia and gout, and it is also closely related to complications such as cardiovascular disease and chronic kidney disease. Therefore, it has become a core target for the treatment of related diseases.

[0003] Currently, the most commonly used xanthine oxidase inhibitors in clinical practice are chemically synthesized drugs such as allopurinol and febuxostat. Although they can effectively inhibit enzyme activity and reduce blood uric acid levels, they have obvious drawbacks: allopurinol is prone to causing hypersensitivity reactions, febuxostat has potential cardiovascular risks, and long-term use may lead to liver and kidney damage, making it difficult to meet the needs of long-term safe clinical use. Therefore, it is urgent to develop new, highly effective, and low-toxicity xanthine oxidase inhibitors.

[0004] Natural products have diverse structures and broad biological activities, and have advantages such as low toxicity, good biocompatibility, and abundant sources. Among them, natural components such as polyphenols and flavonoids have been proven to have good xanthine oxidase inhibitory activity, and are an important resource for the development of novel inhibitors.

[0005] Huazelan ( Eupatorium chinense *Sophora chinensis*, a plant belonging to the genus *Sophora* in the family Asteraceae, is named "Duoxugong" (meaning "many-rooted man") because of its fibrous roots. In Guangdong, it is called "Tu Niuxi" (meaning "earth achyranthes"). *Sophora chinensis* is widely distributed in many parts of my country and is a traditional medicinal plant used by ethnic minorities in Hunan and Hubei provinces. The chemical composition of *Sophora chinensis* encompasses various categories, including benzofurans, flavonoids, terpenes, and phenols. This invention isolated three compounds (1-3) from the fermentation products of the endophytic fungus *S. phragmitis*, with compound 1 being a novel compound. In vitro xanthine oxidase inhibitory activity experiments showed that compounds 1-3 all exhibited good inhibitory activity, with compound 1 showing superior inhibitory activity compared to the positive control drug allopurinol. Molecular docking experiments also indicated that compound 1 has a good binding energy to xanthine oxidase. The good xanthine oxidase inhibitory activity of these three compounds provides a safe and effective natural lead molecule for the treatment of hyperuricemia and gout or gout complications. Summary of the Invention

[0006] The first objective of this invention is to provide a novel benzofuran compound 1 from the fermentation product extract of the endophytic fungus *Euphorbia huazellaria* and a method for its preparation. The second objective is to provide the use of compounds 1-3 in the preparation of medicaments for the prevention or treatment of xanthine oxidase-mediated diseases.

[0007] Endophytic fungi of Zephyranthes huazelan Septoriella phragmitis It was deposited at the China Center for Type Culture Collection on September 13, 2022, with accession number CCTCC NO: M 20221413, and the deposit address is Wuhan University, Wuhan, Hubei Province.

[0008] The compound having the function of inhibiting xanthine oxidase activity has any one of the following structural formulas:

[0009] .

[0010] The technical solution of this invention is the application of benzofuran compounds in the fermentation products of the endophytic fungus *Eupatorium fortunei* in drugs for delaying or treating xanthine oxidase-mediated diseases.

[0011] The method for preparing benzofuran compound 1 from the fermentation product of the endophytic fungus *Eupatorium fortunei* includes the following steps: A1. Solvent extraction: The fermentation culture of the endophytic fungus Septoriella phragmitis was extracted with solvent to obtain the crude fermentation extract a; B1. Normal phase silica gel column chromatography separation: Take extract a, dissolve it in dichloromethane, mix it with silica gel, soak it in petroleum ether overnight, pack it into the column, rinse it, load it dry, and elute it with petroleum ether and dichloromethane / methanol system, and collect the eluent. C1. The eluent was subjected to Sephadex LH-20 gel column chromatography to separate the separated products; D1. Compound 1 was obtained by semi-preparative HPLC of the separated product.

[0012] The fermentation culture mentioned in step A1 is obtained by inoculating the endophytic fungus Septoriella phragmitis into a solid culture medium and allowing it to ferment statically; the extraction solvent used for solvent extraction is selected from dichloromethane, methanol, or a mixture thereof.

[0013] In step B1, a gradient elution was performed using a petroleum ether and dichloromethane-methanol system. The elution volume ratios were as follows: petroleum ether, dichloromethane-methanol 100:0, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 1:1, 0:100. The collected target compound eluent was an eluent with a dichloromethane to methanol volume ratio of (10:1) to (9:1).

[0014] The eluent from step C1 was separated by gel column chromatography using a Sephadex LH-20 with a mixed solvent of dichloromethane and methanol to obtain the separated product.

[0015] In the semi-preparative HPLC described in step D1, the mobile phase is a mixed solvent of acetonitrile and water, and the flow rate is 1-2 mL / min, to prepare compound 1.

[0016] A pharmaceutical composition comprising said compound 1 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0017] This invention relates to the use of the aforementioned furan compounds or their pharmaceutically acceptable salts, or the aforementioned pharmaceutical compositions, in the preparation of medicaments for the prevention or treatment of xanthine oxidase-mediated diseases.

[0018] In some preferred embodiments, the preparation method includes the following steps: A1. Solvent Extraction: The activated strain was aseptically inoculated into PDB liquid medium and cultured at 150 rpm and 28℃ for 2-3 days with shaking until a small number of uniform mycelial balls were formed, yielding a seed culture. 1 mL of the seed culture was transferred to corn solid medium (20 g corn + 35 mL deionized water) and cultured statically at 28℃ for 30 days. The fermentation product was dried at 45℃ to constant weight, and a 1:1 (v / v) dichloromethane-methanol mixture was added. The mixture was sealed and soaked overnight. The extraction was repeated three times, and the extracts were combined and concentrated under reduced pressure to obtain the crude fermentation extract a.

[0019] B1. Normal-phase silica gel column chromatography: Dissolve 56.6 g of extract a in dichloromethane and mix with 100 g of silica gel. Soak 1800 g of normal-phase silica gel in petroleum ether overnight, pack into a column, rinse, and load the sample using a dry method. Elute using a petroleum ether and dichloromethane / methanol system, and collect the eluent.

[0020] C1. The eluent was subjected to Sephadex LH-20 gel column chromatography to separate the product.

[0021] D1. The isolated products were prepared by semi-preparative HPLC to obtain compounds 1-3.

[0022] In step B1, a gradient elution is performed using a petroleum ether and dichloromethane-methanol system, with the following elution ratios: petroleum ether, dichloromethane-methanol (100:0, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 1:1, 0:100). The eluent from the dichloromethane-methanol (10:1) fraction or the dichloromethane-methanol (9:1) fraction is collected.

[0023] The eluent from step C1 was separated by gel column chromatography using Sephadex LH-20 with dichloromethane-methanol at a volume fraction of 50:50 to obtain the separated product.

[0024] In the semi-preparative HPLC described in step D1, the mobile phase was acetonitrile-water 55:45, the flow rate was 2 mL / min, and compound 1 was prepared.

[0025] The present invention also provides a medicament for treating gout or gouty arthritis or gout complications, and hyperuricemia, the medicament comprising compounds 1 to 3 prepared therefrom.

[0026] A compound that inhibits xanthine oxidase activity, said compound comprising compounds 1-3 prepared above.

[0027] A medicament for treating hyperuricemia and gout or gouty arthritis or gout complications by inhibiting the activity of xanthine oxidase, the medicament comprising compounds 1-3 prepared therefrom.

[0028] Compound 1 forms a site containing hydrophobic interactions and hydrogen bond interactions by binding to the amino acid of the active site of xanthine oxidase protein. The compounds 1-3 or the drug of the present invention also include pharmaceutically acceptable excipients.

[0029] Compared with the prior art, this application has at least one of the following beneficial effects: 1. Compounds 1-3 of this application have significant xanthine oxidase inhibitory activity, and compound 1 has higher activity than the positive control drug allopurinol. There are no publicly available reports on the xanthine oxidase inhibitory activity of the compounds and their application in related diseases.

[0030] 2. First discovery of endophytic fungi from Zephyranthes chinensis Septoriella phragmitis A novel compound 1, isolated from fermentation broth, exhibits inhibitory activity against xanthine oxidase. It shows broad application potential as a drug for the prevention or treatment of xanthine oxidase-mediated diseases such as gout, gouty arthritis, gout complications, and hyperuricemia. Attached Figure Description

[0031] Figure 1 The chemical structure diagram and two-dimensional key correlation diagram of compound 1 are shown.

[0032] Figure 2 The ECD calculation spectrum of compound 1 is shown.

[0033] Figure 3 This is the X-ray structure diagram of compound 1.

[0034] Figure 4 For compound 1 1 H-NMR spectrum.

[0035] Figure 5 For compound 1 13 C-NMR spectrum.

[0036] Figure 6 The image shows the DEPT135 spectrum of compound 1.

[0037] Figure 7 The image shows the HSQC spectrum of compound 1.

[0038] Figure 8 For compound 1 1 H- 1 H COSY spectrum.

[0039] Figure 9 The image shows the HMBC spectrum of compound 1.

[0040] Figure 10 The NOESY spectrum of compound 1 is shown.

[0041] Figure 11 The diagram shows the interaction analysis between compound 1 and xanthine oxidase protein (where A: 3D binding mode of compound 1 with xanthine oxidase protein; B: amino acid residue binding mode of compound 1 with xanthine oxidase protein; C: 2D binding mode of compound 1 with xanthine oxidase protein). Detailed Implementation

[0042] The following embodiments are used to further explain and illustrate the present invention. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0043] Example 1: Isolation, preparation, and structural analysis of benzofuran compound 1 from fermentation products of the endophytic fungus *Euphorbia huazelan*. (1) The separation and preparation method includes the following steps: Step A, Solvent Extraction: The activated strain was aseptically inoculated into PDB liquid medium and cultured at 150 rpm and 28℃ for 2-3 days with shaking until a small number of uniform mycelial balls were formed, yielding a seed culture. 1 mL of the seed culture was transferred to corn solid medium (20 g corn + 35 mL deionized water) and cultured statically at 28℃ for 30 days. The fermentation product was dried at 45℃ to constant weight, and a 1:1 (v / v) dichloromethane-methanol mixture was added. The mixture was sealed and soaked overnight. The extraction was repeated three times, and the extracts were combined and concentrated under reduced pressure to obtain crude fermentation extract a.

[0044] Step B, Column Chromatography Crude Fraction: Take 56.6 g of the above extract a, dissolve it in methanol, and mix it with 100 g of silica gel for sample pretreatment. Take 1800 g of normal phase silica gel, soak it in petroleum ether overnight, and pack it into a column with a column volume of 4.0 L. Equilibrate the column bed with 3 column volumes of petroleum ether. After the liquid level stabilizes, retain about 6 cm of liquid layer. Load the sample dry and use silica gel and absorbent cotton as a protective layer. The elution process starts with petroleum ether as the eluent, and then uses a dichloromethane-methanol system with different ratio gradients: 100:0, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 1:1, and 0:100. Collect 13 fractions (Fr. 1-13).

[0045] Thin-layer chromatography, high-performance liquid chromatography, and HPLC-DAD scanning analysis showed that the eluent in the dichloromethane-methanol (10:1) fraction or the dichloromethane-methanol (9:1) fraction was the component containing the target compound 1.

[0046] Step C: Take the dichloromethane-methanol (9:1) fraction eluent and perform isogradient elution using Sephadex LH-20 gel column chromatography (dichloromethane / methanol=50:50) to obtain 20 fractions (Fr.3.1-3.20).

[0047] Step D: Take the 8th fragment Fr.3.8 from step C and separate it by semi-preparative HPLC (acetonitrile-water 55:45, 2 mL / min, YMC C 18 (Column), to obtain compound 1 (3.9 mg).

[0048] (2) Structural analysis of new compound 1: Crystalline form (methanol). Optical rotation data: [α]25D +4.6° (c 0.10, methanol), UV (methanol) λmax = 227.6, 252.0, 302.4 nm; Its molecular formula was determined to be C by high-resolution mass spectrometry HR-ESI-MS m / z: 235.0973 [M+H]+ (C13H15O4, calcd. 235.0970). 13 H 14 O4, with an unsaturation degree of 7. Compound 1... 1 In the 1H-NMR spectrum (Table 1), compound 1 showed two olefin proton signals δH 7.41 (1H, d, J=7.6 Hz, H-4) and 7.31 (1H, d, J=7.6 Hz, H-5), and two methyl signals δH 1.13 (3H, s, H-10) and 1.17 (3H, s, H-11). Compound 1... 13The C-NMR and DEPT135 spectra show 13 carbon signals, including two methyl carbon signals at δC 26.1 (C-10) and 25.5 (C-11), and three oxygen-bonded carbon signals at δC 91.1 (C-2), 70.6 (C-9), and 67.7 (C-8). The DEPT135 spectrum shows two methylene carbon signals at δC 67.7 (C-8) and 30.3 (C-3), one quaternary carbon signal with an oxygen atom at δC 91.1 (C-2), one carbonyl carbon signal at δC 170.9 (C-6), and six olefin carbon signals at δC 154.1 (C-8b), 134.9 (C-5a), 127.1 (C-8a), 126.2 (C-3a), 126.0 (C-4), and 117.3 (C-5). Correlation was observed between H-5 and C-4, C-5a, between H-2 and C-3, C-9, between H-10, H-11 and C-9, and between H-8 and C-8a, C-5a in the HMBC spectrum; correlation was observed between H-4 and H-5, and between H-2 and H-3 in the 1H-1HCOSY spectrum. Based on comprehensive analysis of the two-dimensional spectra of the compound, the planar structure of compound 1 was determined (Figure 1).

[0049] To further elucidate its stereostructure, the CD spectrum of the compound was tested (Figure 2). The compound exhibits a positive Cotton effect at 210 nm, and it was determined that the chirality at the C-2 position is related to the Cotton effect at 210 nm. A single crystal of compound 1 was obtained (Figure 3). Cu-target X-ray single-crystal diffraction analysis revealed its Flack and Hooft parameters to be -0.04 (4) and -0.03 (2), respectively, confirming that the C-2 position is S-configuration. A search revealed that this is a novel compound.

[0050] Table 1. Compound 1 1 H-NMR, 13 C-NMR data

[0051] Example 2: Test of the inhibitory activity of compounds 1-3 on xanthine oxidase (1) Experimental materials Xanthine, xanthine oxidase, allopurinol, potassium dihydrogen phosphate, disodium hydrogen phosphate, etc.

[0052] (2) Experimental methods Xanthine oxidase can catalyze the production of uric acid and superoxide anions from xanthine and hypoxanthine. Uric acid has a characteristic absorption peak at 290 nm. Therefore, in vitro enzymatic reactions were used to evaluate the in vitro activity of Vanisanghuang extract and its various extract fractions.

[0053] Preparation of phosphate buffer (pH 7.4): Dissolve 0.2 g potassium dihydrogen phosphate, 2.89 g disodium hydrogen phosphate, 8.0 g sodium chloride, and 0.2 g potassium chloride in 1000 mL of ultrapure water. Substrate solution: Dissolve 15.2 mg xanthine in an appropriate amount of phosphate buffer, add 1 mL of 1 mol / L sodium hydroxide to aid dissolution, add 1 mol / L hydrochloric acid to adjust the pH to 7.4, sonicate, and add phosphate buffer to bring the volume to 100 mL to obtain a substrate solution with a concentration of 100 μm.

[0054] Enzyme solution: Dissolve 1000 U xanthine oxidase in 2 mL phosphate buffer to obtain a 500 U / mL enzyme stock solution. Aliquot the solution under an ice-water bath and store at -80℃. Dilute to a working concentration of 100 U / L before use and store in an ice-water bath.

[0055] Sample solution: Dissolve each part of the sample in DMSO, store at 4℃, and dilute to the working concentration of 500 μg / mL before use.

[0056] Allopurinol solution: Dissolve 200 mg of allopurinol in an appropriate amount of phosphate buffer, bring the phosphate buffer to a final volume of 100 mL, store at 4°C, and dilute to different concentrations before use.

[0057] The specific experimental steps are as follows: Using a 200 μL final volume reaction system in a 96-well plate, prepare the sample, enzyme, and substrate solutions as described above and dilute to the working concentration. Prepare the enzyme solution fresh for each use. During the experiment, keep the enzyme solution in an ice-water bath to prevent enzyme inactivation. Add 50 μL of enzyme solution, sample solution, allopurinol solution, and the corresponding phosphate buffer according to the groups in Table 2. Incubate at 37°C for 10 min. Finally, add 100 μL of 100 μM substrate to start the reaction and incubate at 37°C for 30 min. Measure the OD value at 290 nm using a microplate reader and calculate the inhibition rate.

[0058]

[0059] Table 2. Experimental methods for XOD inhibition in vitro

[0060] (3) Experimental results Table 3. Results of the inhibitory activity of compounds 1-3 on xanthine oxidase ( n =3, ±SEM)

[0061] As can be seen from Table 3, compounds 1-3 all showed good inhibitory activity against xanthine oxidase, and compound 1 showed better inhibitory activity than the positive control drug allopurinol.

[0062] Example 3: Docking Compound 1 with a protein target molecule (1) Experimental methods Compound 1, used in this docking experiment, was constructed using ChemDraw, then imported into Chem3D software for optimization and energy minimization using the MM2 module, and saved as an SDF file as the ligand molecule for molecular docking. It was then imported into Pymol and Autodock software for further optimization and exported as a PDBQT file. The xanthine oxidase (PDB ID: 1FIQ) protein structure was obtained from the RCSB database (…). https: / / www.rcsb.org / The protein structure was processed using Pymol and Autodock platforms, including water molecule removal, ligand removal, and hydrogenation. Energy minimization and geometric optimization were performed on the protein, and the result was exported as a PDBQT file. Molecular docking was handled and optimized using the Grid module in Autodock software. The PDBQT file was imported into the software, the bounding box fully enclosed the protein, and then molecular docking was performed. Additionally, the protein-small molecule complexes were visualized and analyzed using Pymol.

[0063] (2) Docking results The molecular docking results are shown in the table below: Table 4. Docking results of compound 1 with target protein 1 FIQ

[0064] Compound-protein interaction analysis: In this experiment, compound 1 and the positive control drug allopurinol were molecularly docked with the xanthine oxidase target protein. The docking results showed that the compound and the target protein had a good binding interaction and a high degree of matching (Table 4), with a binding energy of less than -5 kcal / mol. The complex formed by the docked compound and protein was visualized using Pymol 2.1 software to obtain the binding pattern. Based on the binding pattern, it can be clearly seen that compound 1 forms hydrophobic interactions with amino acids ILE-353, VAL-259, LEU-257, and LEU-287 at the active site of xanthine oxidase protein, and forms multiple hydrogen bonds with amino acids SER-347 and ALA-301. Figure 11 ); The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A benzofuran compound, characterized in that, Endophytic fungi from the roots of *Erigeron huazei* Septoriella phragmitis It was isolated from fermentation products and has the following structural formula: Endophytic fungi in the roots of Zephyranthes cusia Septoriella phragmitis It was deposited at the China Center for Type Culture Collection on September 13, 2022, with accession number CCTCC NO: M 20221413, and the deposit address is Wuhan University, Wuhan, Hubei Province.

2. The method for separating and extracting benzofuran compounds according to claim 1, characterized in that, Includes the following steps: A1. Solvent extraction: The fermentation culture of the endophytic fungus Septoriella phragmitis was extracted with solvent to obtain the crude fermentation extract a; B1. Normal phase silica gel column chromatography separation: Take extract a, dissolve it in dichloromethane, mix it with silica gel, soak it in petroleum ether overnight, pack it into the column, rinse it, load it dry, and elute it with petroleum ether and dichloromethane / methanol system, and collect the eluent. C1. The eluent was subjected to Sephadex LH-20 gel column chromatography to separate the separated products; D1. Compound 1 was obtained by semi-preparative HPLC from the separated product. The structural formula of compound 1 is as follows: 。 3. The method for separating and extracting benzofuran compounds according to claim 2, characterized in that, The fermentation culture mentioned in step A1 is obtained by inoculating the endophytic fungus Septoriella phragmitis into a solid culture medium and allowing it to ferment statically; the extraction solvent used for solvent extraction is selected from dichloromethane, methanol, or a mixture thereof.

4. The method for separating and extracting benzofuran compounds according to claim 2, characterized in that, In step B1, a gradient elution was performed using a petroleum ether and dichloromethane-methanol system. The elution volume ratios were as follows: petroleum ether, dichloromethane-methanol 100:0, 50:1, 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 1:1, 0:

100. The collected target compound eluent was an eluent with a dichloromethane to methanol volume ratio of (10:1) to (9:1).

5. The method for separating and extracting benzofuran compounds according to claim 2, characterized in that, The eluent from step C1 was separated by gel column chromatography using Sephadex LH-20 in a mixed solvent of dichloromethane and methanol to obtain the separated product.

6. The method for separating and extracting benzofuran compounds according to claim 2, characterized in that, In the semi-preparative HPLC described in step D1, the mobile phase is a mixed solvent of acetonitrile and water, and the flow rate is 1-2 mL / min, to prepare compound 1.

7. A drug for inhibiting xanthine oxidase activity, characterized in that, The drug comprises the benzofuran compound of claim 1 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

8. A drug for treating hyperuricemia, gout, gouty arthritis, or gout complications by inhibiting the activity of xanthine oxidase, characterized in that, The drug includes the benzofuran compound of claim 1 or the drug of claim 7.

9. The drug according to claim 7 or 8, characterized in that, The benzofuran compound of claim 1 or the drug of claim 7 forms a site containing hydrophobic interactions and hydrogen bond interactions by binding to the amino acid of the active site of xanthine oxidase protein.

10. The use of the benzofuran compound of claim 1 or the drug of claim 7 in the preparation of a medicament for the prevention or treatment of xanthine oxidase-mediated diseases.

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