Flavonoid glycoside compound as well as preparation method and application thereof
By synthesizing flavonoid glycoside compounds, the problems of poor tolerance and toxic side effects of existing hyperuricemia treatment drugs have been solved, and the effect of significantly reducing uric acid is achieved, and the preparation method is simple and environmentally friendly.
Patent Information
- Application Number
- CN202510249195.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-04
- Publication Date
- 2025-04-25
AI Technical Summary
Existing hyperuricemia treatments have problems such as poor tolerance, high cost and can produce various toxic and side effects.
By synthesizing flavonoid compounds, and reacting flavonoid compounds with glycoside ligand compounds under specific conditions, a novel compound with a uric acid reduction effect was prepared.
The prepared flavonoid glycoside compounds can significantly upregulate the expression of the uric acid secreted protein OAT1, promote the excretion of uric acid, reduce the level of uric acid, and have no killing effect on normal cells. The preparation method is simple, low-cost and environmentally friendly.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of chemical medicine, and in particular to a flavonoid glycoside compound and a preparation method and application thereof. Background Art
[0002] Hyperuricemia (HUA) refers to a disease caused by excessive synthesis of uric acid in the liver or excessive excretion of uric acid in the kidneys. At present, hyperuricemia has become the second largest metabolic disease after diabetes, with an increasing prevalence and a younger age, which has caused great harm to the health of people around the world. However, current clinical drugs for the treatment of hyperuricemia have disadvantages such as poor drug tolerance, high prices, and various toxic side effects. Therefore, finding and developing new, safer and more effective anti-hyperuricemia drugs based on natural product skeletons has become a research hotspot for scientific researchers.
[0003] In some examples of using traditional Chinese medicine preparations to treat hyperuricemia, it was found that the active ingredients that play the main therapeutic role are flavonoids, such as apigenin, naringenin, luteolin, kaempferol, etc., which can significantly inhibit the activity of xanthine oxidase (XOD) and thus exert a uric acid-lowering effect. Therefore, the development of anti-hyperuricemia drug molecules using flavonoids as the basic skeleton has broad prospects. Summary of the invention
[0004] The purpose of the present invention is to provide a flavonoid glycoside compound and a preparation method and application thereof, so as to solve the defects of the current drugs for treating hyperuricemia, such as poor tolerance, high price, various toxic side effects, etc., and to provide a new class of compounds prepared with natural compounds as raw materials, in order to provide new candidate compounds for the development of safer and more effective new anti-hyperuricemia drugs.
[0005] To achieve the above object, the present invention provides a flavonoid glycoside compound, the structural formula of which is as follows:
[0006]
[0007] in:
[0008] The synthesis method of the flavonoid glycoside compound as described above comprises the following steps:
[0009] S1, dissolving the flavonoid compound and Ag2CO3 in an organic solvent, then adding the glycoside ligand compound, stirring the reaction at room temperature in the dark, and after the reaction is completed by TLC monitoring, filtering, concentrating and purifying to obtain a solid b;
[0010] S2, under an inert gas atmosphere, dissolving and reacting the solid b obtained in step S1 and a highly active oxidant. After the reaction is completed by TLC monitoring, the solid obtained is filtered, washed with saturated NaHCO3, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure and purified to obtain a solid c;
[0011] S3. In an anhydrous and oxygen-free environment, the solid c obtained in step S2 is dissolved in anhydrous methanol, and then solid sodium methoxide is added. The pH of the reaction solution is adjusted and then reacted at room temperature. The reaction is completed by TLC monitoring. The pH of the solution is adjusted to neutral, filtered, concentrated, recrystallized, and then filtered to obtain flavonoid glycosides.
[0012] Preferably, in step S1, the flavonoid compound is one or more of naringenin, apigenin, kaempferol and luteolin; and the glycoside ligand compound is one or more of peracetylated glucose, peracetylated L-arabinose and peracetylated rhamnose.
[0013] Preferably, in step S1, the mass volume ratio of flavonoid compound: Ag2CO3: organic solvent: glycoside ligand compound is 2.0g:3.0g:50mL:4.5g; the filtration is normal pressure filtration; the concentration is reduced pressure concentration, and the reduced pressure condition is 0.01MPa; the purification is column chromatography purification, and the chromatographic liquid is a mixed liquid with a volume ratio of petroleum ether: ethyl acetate of 1:1.
[0014] Preferably, in step S2, the highly active oxidant is 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; the dissolved solvent is 1,4-dioxane; the mass volume ratio of solid b: highly active oxidant: solvent is 26.0 g:19.0 g:0.4 L; and the reaction is carried out at 110° C. for 15 h.
[0015] Preferably, the purification in step S2 is performed by column chromatography, and the chromatography liquid is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:2.
[0016] Preferably, in step S3, the mass volume ratio of solid c: anhydrous methanol: sodium methoxide is 14.0 g: 1 L: 4.59 g.
[0017] Preferably, the pH of the reaction after adding solid sodium methoxide in step S3 is 9 to 10; after the reaction is completed, Amberlite IR120 (H + ) to adjust the pH; concentration is concentration under reduced pressure; and recrystallization uses anhydrous ethanol.
[0018] The use of the flavonoid glycoside compound as described above in the preparation of a drug for treating hyperuricemia, wherein the drug contains the flavonoid glycoside compound as an effective ingredient.
[0019] Therefore, the flavonoid glycoside compounds and preparation methods and applications provided by the present invention have the following specific technical effects:
[0020] (1) The method provided by the present invention is used to successfully prepare flavonoid glycoside compounds, which can significantly upregulate the expression of uric acid secretion protein OAT1, promote the excretion of uric acid, and thus achieve the purpose of reducing uric acid levels; and the prepared flavonoid glycoside compounds have no killing effect on normal HK-2 cells within a certain concentration range, and can be used in the development of therapeutic drugs for hyperuricemia;
[0021] (2) The preparation method provided by the present invention has high yield and low cost; it is simple and easy to operate, and has low requirements on equipment; it does not use toxic and harmful reagents and materials, conforms to the concept of green environmental protection; and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.
[0023] Figure 1 This is a trend diagram of the cell activity of normal HK-2 cells acted with different concentrations of flavonoid glycoside AGlu in Application Example 1 of the present invention;
[0024] Figure 2 This is a trend diagram of the cell activity of normal HK-2 cells acted with different concentrations of flavonoid glycoside AAr1 in Application Example 2 of the present invention;
[0025] Figure 3 The image (A) and the gray value analysis image (B) of the target band in the protein immunoassay in Application Example 3 of the present invention;
[0026] Figure 4 This is a trend diagram of the cell activity of normal HK-2 cells under different concentrations of flavonoid glycoside NRha in Application Example 4 of the present invention;
[0027] Figure 5 The following are the development diagram (A) and gray value analysis diagram (B) of the target band in the protein immunoassay in Application Example 5 of the present invention. DETAILED DESCRIPTION
[0028] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.
[0029] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.
[0030] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.
[0031] Example 1
[0032] The reaction process for synthesizing flavonoid glycoside AGlu is as follows:
[0033]
[0034] The specific steps are:
[0035] (1) Naringenin (2.0 g, 7.3 mmol, 1.0 equiv.) and Ag2CO3 (3.0 g, 11.0 mmol, 1.5 equiv.) were dissolved in anhydrous acetonitrile (50.0 mL), and the compound peracetylated glucose (4.5 g, 11.0 mmol, 1.5 equiv.) was added, and stirred at room temperature in the dark for 3 h. The reaction was completed by TLC monitoring (the volume ratio of the developing solvent DCM:MeOH was 15:1, R f =0.65), after the reaction was completed, the mixture was filtered at normal pressure, concentrated under reduced pressure, and purified by column chromatography (eluent: PE / EtOAc=1 / 1) to obtain a white foamy solid 1b (3.6 g, yield: 81.0%).
[0036] (2) Under N2 protection, compound 1b (2.6 g, 4.3 mmol, 1.0 equiv.) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.9 g, 8.6 mmol, 2.0 equiv.) were placed in a 100.0 mL three-necked flask, 1,4-dioxane (40.0 mL) was added to dissolve the compound, and the mixture was reacted at 110°C for 15 h. The reaction was completed by TLC monitoring (the developing solvent was a mixed solution of DCM:MeOH with a volume ratio of 15:1, R f =0.61) and then filtered, washed with a large amount of saturated NaHCO3, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent PE / EtOAc=1 / 2) to give a yellow solid 1c (1.5 g, 57.6% yield).
[0037] (3) Under anhydrous and oxygen-free environment, compound 1c (1.4 g, 2.3 mmol, 1.0 equiv.) was dissolved in 100.0 mL of anhydrous methanol, and sodium methoxide (459.0 mg, 8.5 mmol, 3.7 equiv.) was added to adjust the pH value of the reaction solution to 9-10. The reaction was carried out at room temperature for 1 h. The reaction was completed after monitoring by TLC (the developing solvent was a mixed solution of EtOAc:MeOH with a volume ratio of 6:1, R f =0.40), add AmberliteIR120 (H + ) to neutralize the reaction solution to pH 7.4, stop stirring, filter, and concentrate under reduced pressure (the reduced pressure condition is 0.01 MPa), add anhydrous ethanol (15.0 mL) for recrystallization, and filter to obtain an off-white solid AGlu (875.0 mg, yield is 88.0%).
[0038] AGlu 1 H NMR is (400MHz, DMSO-d6) δ12.97(s,1H),10.41(s,1H),7.96(d,J=8.8Hz,2H),6.94(d,J=8.8Hz,2H),6.87(s,1H),6.83(d,J=2.2Hz,1H) ,6.44(d,J=2.2Hz,1H),5.42(s,1H),5.07(d,J=7.3Hz,3H),4.63(s,1H),3.72(d,J=10.3Hz,1H),3.51-3.42(m,2H),3.29-3.15(m,3H). 13 C NMR(101MHz,DMSO-d6)δ182.10,164.34,163.05,161.46,161.20,157.03,128.71,121.12, 116.09,105.42,103.20,99.98,99.61,94.93,77.27,76.53,73.19,69.62,60.68; HRMS(ESI + )calcd m / zfor C 21 H 20 O 10 [M+H] + :433.1135,found:433.1131.
[0039] Application Example 1
[0040] The survival rate of HK-2 cells treated with AGlu prepared in Example 1 was investigated as follows:
[0041] The purchased HK-2 cells were revived and cultured. According to the molecular weight of the compound, 1.69 mg of AGlu was accurately weighed and dissolved in 20.0 μL DMSO to prepare a stock solution. Before the test, DMED medium was added to dilute it to different multiples (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μM, respectively). The cell viability of AGlu was determined using the CCK8 method. The results are as follows: Figure 1 As shown, after normal HK-2 cells were treated with different concentrations of AGlu, it had no killing effect on the cells within the concentration range of 0 to 400 μM.
[0042] Example 2
[0043] Synthesize flavonoid glycoside AArl, the reaction formula is as follows:
[0044]
[0045] The specific synthesis method is different from that in Example 1 in that the peracetyl glucose in step (1) is replaced with the glycoside ligand compound peracetyl L-arabinose (10.8 g, 31.8 mmol, 1.5 equiv.), and the other methods are exactly the same as in Example 1.
[0046] The yield of flavonoid glycoside AAr1 was 61.0%.
[0047] 1 H NMR (400MHz, DMSO-d6) δ12.96 (s, 1H), 10.40 (s, 1H), 7.97 (d, J = 8.8Hz, 2H), 6.94 (d ,J=8.8Hz,2H),6.87(s,1H),6.81(d,J=2.2Hz,1H),6.42(d,J=2.2Hz,1H),5.30(d,J =5.1Hz,1H),5.04(d,J=6.8Hz,1H),4.89(d,J=5.6Hz,1H),4.70(d,J=4.1Hz,1H),3 .76(d,J=8.9Hz,1H),3.71(d,J=11.2Hz,2H),3.65-3.61(m,1H),3.50-3.47(m,1H). 13 CNMR(101MHz,DMSO-d6)δ182.12,164.34,162.90,161.48,161.19,157.08,128.76,121. 12,116.09,105.38,103.16,100.26,99.57,94.73,72.46,70.12,67.60,66.04.HRMS(ESI +)calcd m / z for C 20 H 18 O9[M+Na] + :425.0843,found:425.0834.
[0048] Application Example 2
[0049] The survival rate of HK-2 cells treated with AAR1 prepared in Example 2 was investigated as follows:
[0050] The method of application example 1 was used for testing, and the results were as follows Figure 2 As shown, the prepared AARl exhibited a dose-dependent inhibitory effect on cells in the concentration range of 0-400 μM, but within the concentration range of 0-50 μM, the cell survival rate of AARl was above 90%, which was used as a safe dosing concentration for subsequent experiments.
[0051] Application Example 3
[0052] The regulatory effects of AGlu prepared in Example 1 and AArl prepared in Example 2 on uric acid secretory protein OAT1 were investigated as follows:
[0053] 1) To construct a hyperuricemia model cell, add uric acid with a final concentration of 0.2 mg / mL to 1.0 M sodium hydroxide, shake vigorously to form a suspension, and heat in a water bath at 90-100°C for 3 min. Repeat the steps of vigorous shaking and water bathing until a clear solution is obtained. After the solution is sterilized by a 0.22 μm filter membrane, it is added to the culture medium for culturing HK-2 cells, and the cell sample after modeling is obtained after continuing to culture at room temperature for 24 hours.
[0054] 2) AGlu and AArl at a working concentration of 1.0 μM were used to incubate the model cells respectively, and then lysed with 0.1 mL of 4°C pre-cooled lysis buffer (2.5 mL Lysis Buffer, 25.0 μL phosphatase inhibitor, 2.5 μL protease inhibitor and 12.5 μL 100.0 nM PMSF mixed), and then the total protein was extracted. The protein was quantitatively analyzed using a BCA protein concentration assay kit (Elabscience, E-BC-K318-M), and the loading buffer was added for gel electrophoresis analysis. The loading volume was 5.0 μL, and the uric acid secretion protein OAT1 was selected as the binding antibody. The target band was analyzed and observed after development. The results are as follows: Figure 3 As shown in part A. By gray value analysis Figure 3 The results of Part A are as follows Figure 3 As shown in part B, it was found that AGlu and AArl can significantly regulate the increase of uric acid secretion protein OAT1, promote the excretion of uric acid, and achieve the purpose of lowering uric acid levels.
[0055] Example 3
[0056] Synthesize flavonoid glycoside NRha, the reaction formula is as follows:
[0057]
[0058] The specific synthesis steps are as follows:
[0059] (1) Naringenin (2.2 g, 8.1 mmol, 1.0 equiv.) and Ag2CO3 (2.8 g, 12.2 mmol, 1.5 equiv.) were dissolved in anhydrous acetonitrile (25.0 mL), and the compound peracetyl rhamnose (4.3 g, 12.2 mmol, 1.5 equiv.) was added, and stirred at room temperature in the dark for 3 h. After the reaction, column chromatography was used to purify the white foamy solid 3b (3.0 g, yield 67.9%, eluent DCM / MeOH = 15 / 1, R f =0.61).
[0060] (2) Compound 3b (332.0 mg, 0.6 mmol, 1.0 equiv.) and sodium methoxide (66.0 mg, 1.2 mmol, 2.0 equiv.) were dissolved in anhydrous methanol (10.0 mL) and purified by column chromatography (eluent: EtOAc / MeOH = 30 / 1) to obtain a white solid compound Nrha (113.0 mg, yield: 45.1%, developing solvent: EtOAc / MeOH = 5 / 1, R f =0.42).
[0061] Flavonoid glycoside NRha 1 H The NMR spectrum is: (400MHz, DMSO-d6) δ12.05 (s, 1H), 9.60 (s, 1H), 7.33 (d, J = 8.4Hz, 2H), 6.79(d,J=8.5Hz,2H),6.15(s,2H),5.46(dd,J=6.4,1.9Hz,1H),5.11(s,1H), 5.04(d,J=2.4Hz,1H),4.88(s,1H),4.81(d,J=1.7Hz,1H),4.71(d,J=7.8Hz,1 H),3.78(s,1H),3.59(s,1H),3.27(d,J=3.4Hz,1H),3.17(s,2H),1.14(s,3H). 13CNMR(101MHz,DMSO-d6)δ190.74,170.44,170.20,163.06,157.89,128.76,128.51,115.2 7,91.26,80.61,78.71,73.83,72.67,71.86,71.61,68.32,67.77,42.11,21.06.HRMS(ESI + )calcd m / z forC 21 H 22 O9[M+Na] + :441.1156,found:441.1147.
[0062] Application Example 4
[0063] The survival rate of HK-2 cells treated with the flavonoid glycoside NRha prepared in Example 3 was investigated as follows:
[0064] The method of application example 1 was used for testing, and the results were as follows Figure 4 As shown, the flavonoid glycoside NRha prepared in Example 3 exhibited a dose-dependent inhibitory effect on cells in the concentration range of 0 to 400 μM, but in the concentration range of 0 to 100 μM, the cell survival rate of NRha was above 90%, and this was used as the safe dosing concentration for subsequent experiments.
[0065] Application Example 5
[0066] The regulatory effect of the flavonoid glycoside NRha prepared in Example 3 on the uric acid secretion protein OAT1 was investigated as follows:
[0067] The protein immunoassay was performed using the method of Application Example 3. The results are as follows: Figure 5 As shown in part A. Figure 5 The results of Part A were analyzed and the results were as follows Figure 5 As shown in part B, it was found that NRha can also significantly regulate the increase of uric acid secretion protein OAT1, promote the excretion of uric acid, and achieve the purpose of lowering uric acid levels.
[0068] Therefore, the method provided by the present invention is used to successfully prepare flavonoid glycoside compounds, and the prepared flavonoid glycoside compounds can significantly upregulate the expression of uric acid secretion protein OAT1, promote the excretion of uric acid, and thus achieve the purpose of lowering uric acid levels; and the prepared flavonoid glycoside compounds have no killing effect on normal HK-2 cells within a certain concentration range, and can be applied to the development of therapeutic drugs for hyperuricemia; the provided preparation method has high yield and low cost; it is simple and easy to operate, and has low requirements for equipment; it does not use toxic and harmful reagents and materials, and is in line with the concept of green environmental protection; and it is suitable for popularization and application.
[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.
Claims
1. A flavonoid glycoside compound, characterized in that: The structural formula of the flavonoid glycoside compound is shown below: in:
2. The method for synthesizing the flavonoid glycoside compound as claimed in claim 1, characterized in that: Here are the steps: S1, dissolving the flavonoid compound and Ag2CO3 in an organic solvent, then adding the glycoside ligand compound, stirring the reaction at room temperature in the dark, and after the reaction is completed by TLC monitoring, filtering, concentrating and purifying to obtain a solid b; S2, under an inert gas atmosphere, dissolving and reacting the solid b obtained in step S1 and a highly active oxidant. After the reaction is completed by TLC monitoring, the solid obtained is filtered, washed with saturated NaHCO3, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure and purified to obtain a solid c; S3. Under an anhydrous and oxygen-free environment, the solid c obtained in step S2 is dissolved in anhydrous methanol, and then solid sodium methoxide is added. After adjusting the pH of the reaction solution, the reaction is allowed to react at room temperature. The reaction is completed after monitoring by TLC. The pH of the solution is adjusted to neutral, filtered, concentrated, recrystallized, and then filtered to obtain flavonoid glycosides.
3. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: In step S1, the flavonoid compound is one or more of naringenin, apigenin, kaempferol and luteolin; the glycoside ligand compound is one or more of peracetylated glucose, peracetylated L-arabinose and peracetylated rhamnose.
4. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: In the step S1, the mass volume ratio of flavonoid compound: Ag2CO3: organic solvent: glycoside ligand compound is 2.0g:3.0g:50mL:4.5g; the filtration is normal pressure filtration; the concentration is reduced pressure concentration, and the reduced pressure condition is 0.01MPa; the purification is column chromatography purification, and the chromatographic liquid is a mixed liquid of petroleum ether: ethyl acetate in a volume ratio of 1:
1.
5. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: In step S2, the highly active oxidant is 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; the dissolved solvent is 1,4-dioxane; the mass volume ratio of solid b: highly active oxidant: solvent is 26.0 g:19.0 g:0.4 L; and the reaction is carried out at 110° C. for 15 hours.
6. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: The purification in step S2 is performed by column chromatography, and the chromatography liquid is a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:
2.
7. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: In the step S3, the mass volume ratio of solid c: anhydrous methanol: sodium methoxide is 14.0 g: 1.0 L: 4.59 g.
8. The method for synthesizing a flavonoid glycoside compound according to claim 2, characterized in that: After adding solid sodium methoxide in step S3, the pH of the reaction is 9-10; after the reaction, AmberliteIR120 (H + ) to adjust the pH; concentration is concentration under reduced pressure; and recrystallization uses anhydrous ethanol.
9. Use of the flavonoid glycoside compound as claimed in claim 1 in the preparation of a drug for treating hyperuricemia, characterized in that: The medicine uses the flavonoid glycoside compound as an effective ingredient.