Apigenin derivative and its preparation method and application

Abstract

The present invention discloses an apigenin derivative, its preparation method, and application. The apigenin derivative has the following molecular structure: it is prepared by attaching apigenin to a thiophene pyrimidine structure and has good application as a uric acid-lowering drug; #imgabs0#

Description

Technical Field

[0001] The present invention relates to an apigenin modified structure, in particular to an apigenin derivative and a preparation method and application thereof. Background Art

[0002] Uric acid (UA) is a metabolite of purines, which are major components of cellular energy storage, such as ATP, DNA, and RNA. Disturbances in endogenous purine metabolism or excessive exogenous purine intake can easily lead to hyperuricemia (HUA), a well-known risk factor for gout. Studies have shown a concentration-dependent correlation between serum UA levels and gout incidence. The incidence and prevalence of gout have been increasing in recent decades, and research on drugs to lower uric acid levels has garnered increasing attention.

[0003] Currently, drugs for treating hyperuricemia are divided into the following categories: Uric acid production inhibitors, primarily xanthine oxidase inhibitors, represented by allopurinol, febuxostat, and topiroxetine; uricosuric agents, such as probenecid and benzbromarone; and uricosuric agents, such as rasburicase and pegolactone. However, all known drugs may produce varying degrees of side effects after human administration, including adverse gastrointestinal reactions, renal impairment, and an increased risk of cardiovascular disease.

[0004] There are currently multiple hypotheses regarding the pathogenesis of HUA, with xanthine oxidase (XO) being considered the most important target. XO is widely present in the liver, kidneys, and vascular endothelium. It is a key enzyme in the final step of uric acid synthesis, converting hypoxanthine and xanthine to uric acid. In recent years, given the potential safety and structural diversity of natural products, many researchers have focused on discovering XO inhibitors from natural products. Studies have shown that apigenin (4',5,7-trihydroxyflavone) has the ability to inhibit XO activity and scavenge free radicals. In vivo studies in rats with acute HUA have also demonstrated that apigenin can reduce serum uric acid levels. However, apigenin's XO inhibitory activity is low, placing it far from practical clinical application. Therefore, if apigenin's structure can be modified to provide new apigenin derivatives with significantly enhanced XO inhibitory activity, this would have significant implications for the further development of drugs for hyperuricemia. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an apigenin derivative and a preparation method and application thereof.

[0006] The present invention first provides an apigenin derivative having the following molecular structure expression:

[0007]

[0008] The present invention also provides a method for preparing the apigenin derivative as described above, comprising the following steps:

[0009]

[0010] 1) 4-amino-2-chlorothiophene[3,2-D]pyrimidine and thiourea are mixed and dissolved, and reacted under reflux to produce compound 2;

[0011] 2) Compound 2 and methyl 4-bromobutyrate are mixed and dissolved, and then fully reacted in the presence of a base catalyst to produce compound 3;

[0012] 3) Compound 3 and apigenin are mixed and dissolved, an alkali catalyst is added, and the mixture is refluxed to generate compound 5, i.e., an apigenin derivative.

[0013] As a preferred embodiment of the preparation method of the present invention, it is characterized in that, in step 1), the molar ratio of 4-amino-2-chlorothienyl[3,2-D]pyrimidine to thiourea is 1:(2-5), for example, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, etc.; and / or,

[0014] In step 1), the reaction solvent is one or more of n-butanol, tetrahydrofuran, ethyl acetate, dichloromethane, and chloroform; and / or,

[0015] The reaction time in step 1) is 3-18 h, for example, 5 h, 8 h, 10 h, 13 h, 15 h, etc.; and / or,

[0016] After the reaction in step 1) is completed, the mixture is cooled to room temperature, an alkaline aqueous solution is added and stirred for 30-60 minutes; the organic layer is separated and purified to obtain compound 2.

[0017] The alkaline aqueous solution is preferably an aqueous solution of sodium hydroxide and / or potassium hydroxide.

[0018] As a preferred embodiment of the preparation method of the present invention, in step 2), the molar ratio of compound 2 to methyl 4-bromobutyrate is 1:(1.2-1.5), for example, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, etc.; and / or,

[0019] In step 2), the base catalyst is selected from one or more of potassium carbonate, sodium carbonate, potassium acetate, and sodium acetate; preferably, the amount of the base catalyst is 0.02-0.06 times the molar amount of compound 2, for example, 0.03 times, 0.04 times, 0.05 times, etc.

[0020] As a preferred embodiment of the preparation method of the present invention, the reaction conditions in step 2) are: reaction at room temperature for 4-10 hours, for example, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, etc.; and / or,

[0021] The reaction solvent in step 2) is one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and toluene.

[0022] As a preferred embodiment of the preparation method of the present invention, after the reaction in step 2) is completed, water is added to dilute the reaction solution, and the organic layer is separated after extraction with an organic solvent, and then purified to obtain compound 3.

[0023] As a preferred embodiment of the preparation method of the present invention, in step 3), the molar ratio of compound 3 to apigenin is 1:(1.1-1.3), for example, 1:1.15, 1:1.2, 1:1.25, etc.; and / or,

[0024] In step 3), the base catalyst is selected from one or more of potassium carbonate, sodium carbonate, potassium acetate, and sodium acetate; preferably, the amount of the base catalyst is 0.02-0.06 times the molar amount of compound 3, for example, 0.03 times, 0.04 times, 0.05 times, etc.

[0025] As a preferred embodiment of the preparation method of the present invention, the reaction conditions in step 3) are: reflux stirring reaction for 8-12 hours, for example, 9 hours, 10 hours, 11 hours, etc.; and / or,

[0026] The reaction solvent in step 3) is one or more of acetonitrile, ethanol, and tetrahydrofuran.

[0027] As a preferred embodiment of the preparation method of the present invention, after the reaction in step 3) is completed, the mixture is cooled to room temperature, saturated sodium bicarbonate solution is added to adjust the pH to 5-7, extraction is performed with an organic solvent, the organic layer is separated, and the mixture is purified to obtain compound 5.

[0028] The present invention does not impose any restrictions on the purification method of the product in steps 1), 2), and 3), which can be any one or more of distillation, water washing, recrystallization, column chromatography, or a conventional combination of these with known separation and purification methods (such as extraction, rotary evaporation, etc.).

[0029] The present invention also provides a use of the apigenin derivative as described above or the apigenin derivative prepared by the method described above in a uric acid-lowering drug or preparation.

[0030] Apigenin, also known as apigenin, is a natural flavonoid compound widely found in warm and tropical vegetables and fruits, with celery being particularly abundant. It is highly biocompatible. The apigenin derivatives provided by this invention not only significantly enhance the uric acid-lowering effects of apigenin, but also have minimal effects on blood glucose, serum total cholesterol, and serum total protein in experimental mice. They are promising biocompatible potential treatments for hyperuricemia and related renal damage. DETAILED DESCRIPTION

[0031] The present invention will be further described below through specific examples. The examples of the present invention are only for illustration of the present invention and do not limit the scope of the present invention.

[0032] In the following examples and comparative examples of the present invention, the main raw materials were purchased from commercial sources.

[0033] The preparation processes of the apigenin derivatives in the following Examples 1-3 all adopt the following synthetic routes:

[0034]

[0035] [Example 1]

[0036] A method for preparing an apigenin derivative comprises the following steps:

[0037] 1) 4-Amino-2-chlorothienyl[3,2-D]pyrimidine and thiourea were dissolved in a 1:3 molar ratio in n-butanol, heated to 115°C, and stirred under reflux for 15 hours. The reaction solution was cooled to room temperature, and 1 mol / L NaOH solution was added and stirred for 30 minutes to remove excess thiourea. The organic layer was then separated and washed three times with saturated brine. Excess water was removed from the organic phase with anhydrous sodium sulfate and dried in vacuo. The mixture was then purified by column chromatography to obtain compound 2 (yield 79%).

[0038] 1 H NMR (500MHz, Chloroform-d): δ12.15(s,1H),7.57(d,J=5.3Hz,1H),7.26–7.20(m,2H),7.16(d,J=7.5Hz,1H).

[0039] 2) Compound 2 and methyl 4-bromobutyrate were dissolved in DMF in a molar ratio of 1:1.5, and 0.05 times the molar amount of compound 2 as a potassium carbonate catalyst was added. The reaction was stirred at room temperature for 10 hours. After the reaction was completed, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic phase was then washed with saturated brine, dried, and purified by column chromatography to obtain compound 3 (yield 72%).

[0040] 1H NMR (500MHz, Chloroform-d): δ7.69(d,J=7.5Hz,1H),7.64(d,J=7.5Hz,1H),7.57(d,J=5.3Hz,1H),7. 38(d,J=5.3Hz,1H),3.64(s,3H),3.27(t,J=8.3Hz,2H),2.48(t,J=8.1Hz,2H),2.06(q,J=8.2Hz,2H).

[0041] 3) Compound 3 and apigenin were mixed and dissolved in acetonitrile in a molar ratio of 1:1.2. Potassium carbonate catalyst (0.05 times the molar amount of compound 3) was added, the temperature was raised to 80°C, and the reaction was carried out under reflux for 8 hours. After the reaction, the mixture was cooled to room temperature, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the mixed solution was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 5, an apigenin derivative (yield 81%).

[0042] 1 H NMR (500MHz, Chloroform-d): δ8.90 (s, 1H), δ7.97 (d, J = 9.5Hz, 1H), δ7.93–7.87 ( m,2H),7.69(d,J=7.5Hz,1H),7.64(d,J=7.5Hz,1H),7.57(d,J=5.3Hz,1H),7.38( d,J=5.3Hz,1H),7.25–7.19(m,2H),6.75(s,1H),6.52(d,J=1.8Hz,1H),6.25(d,J =1.8Hz,1H),3.28(t,J=8.3Hz,2H),2.61(d,J=16.0Hz,2H),2.10(p,J=8.2Hz,2H).

[0043] [Example 2]

[0044] A method for preparing an apigenin derivative comprises the following steps:

[0045] 1) 4-Amino-2-chlorothienyl[3,2-D]pyrimidine and thiourea were dissolved in a 1:2 molar ratio of ethyl acetate, heated to 75°C, and stirred under reflux for 18 hours. The reaction solution was cooled to room temperature, and 1 mol / L KOH solution was added and stirred for 30 minutes to remove excess thiourea. The organic layer was then separated and washed three times with saturated brine. Excess water was removed from the organic phase with anhydrous sodium sulfate and dried in vacuo. The mixture was then purified by column chromatography to obtain compound 2 (yield 75%).

[0046] 2) Compound 2 and methyl 4-bromobutyrate were dissolved in N,N-dimethylacetamide in a molar ratio of 1:1.2, and 0.03 times the molar amount of compound 2 as a sodium carbonate catalyst was added. The reaction was stirred at room temperature for 5 hours. After the reaction was completed, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic phase was then washed with saturated brine, dried, and purified by column chromatography to obtain compound 3 (yield 73%).

[0047] 3) Compound 3 and apigenin were mixed and dissolved in ethanol at a molar ratio of 1:1.1. A sodium carbonate catalyst (0.06 times the molar amount of compound 3) was added, and the temperature was raised to 90° C. and the reaction was allowed to reflux for 10 hours. After the reaction, the mixture was cooled to room temperature, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the mixed solution was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 5, an apigenin derivative (yield 85%).

[0048] [Example 3]

[0049] A method for preparing an apigenin derivative comprises the following steps:

[0050] 1) 4-Amino-2-chlorothiopheno[3,2-D]pyrimidine and thiourea were dissolved in a 1:5 molar ratio of chloroform, heated to 65°C, and stirred under reflux for 10 hours. The reaction solution was cooled to room temperature, and 1 mol / L NaOH solution was added and stirred for 30 minutes to remove excess thiourea. The organic layer was then separated and washed three times with saturated brine. Excess water was removed from the organic phase with anhydrous sodium sulfate and dried in vacuo. The mixture was then purified by column chromatography to obtain compound 2 (yield 78%).

[0051] 2) Compound 2 and methyl 4-bromobutyrate were dissolved in DMF in a molar ratio of 1:1.4, and 0.06 times the molar amount of compound 2 as a potassium carbonate catalyst was added. The reaction was stirred at room temperature for 8 hours. After the reaction was completed, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic phase was then washed with saturated brine, dried, and purified by column chromatography to obtain compound 3 (yield 76%).

[0052] 3) Compound 3 and apigenin were mixed and dissolved in tetrahydrofuran in a molar ratio of 1:1.3. Potassium carbonate catalyst (0.04 times the molar amount of compound 3) was added, the temperature was raised to 65°C, and the reaction was carried out under reflux for 12 hours. After the reaction, the mixture was cooled to room temperature, the pH was adjusted to 7 with saturated sodium bicarbonate solution, and the mixed solution was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 5, an apigenin derivative (yield 80%).

[0053] Application Examples

[0054] 1. Cytotoxicity test:

[0055] The cytotoxicity test of PC12 cells was performed using the CCK-8 method, wherein the experimental group was a culture medium containing the apigenin derivative prepared in Example 1; the control group was a culture medium containing apigenin. The results showed that at a drug concentration of 20-80 μmol / L, there was no significant change in the OD values ​​at 450 nm of the experimental group and the control group at the same concentration.

[0056] 2. In vivo experiments in rats

[0057] (1) Animal Preparation: Adult male Sprague-Dawley rats weighing 150–170 g were provided by Yangzhou University. During the experiment, rats were maintained at 21 ± 2°C with free access to food and water and a 12-h light / dark cycle. Animal welfare and experimental procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize animal suffering.

[0058] (2) Animal grouping and feeding: The rats were divided into 7 groups, namely blank group, model group, low-dose experimental group, medium-dose experimental group, high-dose experimental group, positive control group, and raw material control group; each group consisted of 10 rats.

[0059] (3) Drug preparation: First, a 0.8 wt% CMC-Na solution was prepared, and the apigenin derivative prepared in Example 1 was dissolved in the CMC-Na solution to prepare a low-dose experimental group solution, a medium-dose experimental group solution, and a high-dose experimental group solution with drug concentrations of 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively; then, benzbromarone was dissolved in the CMC-Na solution to prepare a positive control group solution with a drug concentration of 0.5 mg / mL; and apigenin was dissolved in the CMC-Na solution to prepare a raw material control group solution with a drug concentration of 1 mg / mL.

[0060] (4) Drug administration experiment: Rats were intragastrically administered with a dose of 10 mL / kg per day according to their body weight for 7 consecutive days; the specific drug administration method was as follows: the blank group and the model group were both given 0.8 wt% CMC-Na solution; the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group were given experimental group drug solutions with concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively; the positive control group was given a positive control group drug solution with a concentration of 0.5 mg / mL; and the raw material control group was given a raw material control group drug solution with a concentration of 0.5 mg / mL.

[0061] (5) Sample Processing and Sampling: 30 minutes after the last administration on day 7, all groups except the blank group were intraperitoneally injected with 50 mg / ml hypoxanthine solution (solvent: 0.8 wt% CMC-Na solution) to establish a hyperuricemia model. The dosage was 0.1 ml / 10 g. 30 minutes after administration, all experimental rats were immediately tested for blood uric acid, blood glucose, total cholesterol, and total protein levels. The specific test methods and results are as follows:

[0062] ≤ Blood uric acid level test>

[0063] Blood was collected from rats by enucleation of the eyeballs. After serum exuded, the blood was centrifuged at 15,000 rpm for 4 minutes. The upper serum layer was aspirated and placed in a capped sample tube. The tube was then made up to the required volume with normal saline. The blood uric acid content was measured on a Beckman LX20 fully automatic biochemical analyzer. The data are expressed as mean ± standard deviation. The test results are shown in Table 1.

[0064] Table 1. Blood uric acid content test results

[0065] Serum uric acid level (μmol / L) Blank group 128.42±20.32 Model Group 670.38±30.18 Low-dose experimental group 564.30±29.42 Medium dose experimental group 501.28±22.43 High-dose experimental group 438.46±24.62 Positive control group 582.36±30.52 Raw material control group 633.24±26.51

[0066] <Blood sugar level test>

[0067] Blood was collected from the tail vein of each mouse (10 μl / time). The blood sample was centrifuged at 3000 rpm for 5 minutes in a low-temperature centrifuge. 2 μl of the supernatant was accurately collected with a quantitative pipette and placed at the base of the vertebral column of a test tube. Blood glucose levels were determined using the glucose oxidase method. Data are expressed as mean ± standard deviation. The test results are shown in Table 2.

[0068] Table 2. Blood glucose test results

[0069] Blood glucose level (mmmmol / l) Blank group 7.82±1.32 Model Group 11.24±1.82 Low-dose experimental group 9.24±1.36 Medium dose experimental group 8.67±1.42 High-dose experimental group 7.90±1.34 Positive control group 9.16±1.42 Raw material control group 10.25±1.56

[0070] <Serum total cholesterol level test>

[0071] Blood was collected from the rat tail vein, allowed to clot naturally for 20 minutes at room temperature, centrifuged at 2000 rpm for 20 minutes, and the supernatant was collected. Serum total cholesterol (TC) levels were quantitatively determined by ELISA. Data are expressed as mean ± standard deviation. The test results are shown in Table 3.

[0072] Table 3. Serum total cholesterol test results

[0073] Serum total cholesterol level (mmmmol / l) Blank group 4.18±0.42 Model Group 4.29±0.36 Low-dose experimental group 4.16±±0.28 Medium dose experimental group 3.94±0.22 High-dose experimental group 3.71±0.26 Positive control group 3.92±0.32 Raw material control group 4.22±0.26

[0074] <Serum total protein level test>

[0075] Blood was collected from the rat tail vein, allowed to clot naturally at room temperature for 20 minutes, centrifuged at 2000 rpm for 20 minutes, and the supernatant was collected. Serum total protein (TP) levels were quantitatively determined by ELISA. Data are expressed as mean ± standard deviation. The test results are shown in Table 4.

[0076] Table 4. Serum total protein content test results

[0077] Serum total protein level (g / L) Blank group 50.62±1.98 Model Group 38.66±1.58 Low-dose experimental group 40.83±1.88 Medium dose experimental group 45.26±1.47 High-dose experimental group 50.33±1.42 Positive control group 48.56±1.79 Raw material control group 39.75±1.54

[0078] It can be seen from the performance test results in Tables 1-4 that the apigenin derivatives provided by the present invention can exert the effect of lowering uric acid in a short period of time at a dosage of 5-20 mg / kg, and have almost zero biological toxicity; and the uric acid-lowering level at a lower dosage is significantly better than that of the control group using apigenin alone, which has produced unexpected effect changes compared to known technologies, and has obvious technical advantages. In addition, low, medium, and high doses of apigenin derivatives have no adverse effects on the blood sugar, total cholesterol, and total protein content of hyperuricemia mice, and there is an application trend of lowering blood sugar and total cholesterol levels and compensating for total protein loss, and it is expected to have good clinical applicability.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be considered within the scope of protection of the present invention.

Claims

1. An apigenin derivative, characterized in that It has the following molecular structure expression: 。 2. A method for preparing the apigenin derivative according to claim 1, characterized in that: The following steps are involved: ； ； 1) 4-amino-2-chlorothiophene[3,2-D]pyrimidine and thiourea were mixed and dissolved, and reacted under reflux to produce compound 2; 2) Compound 2 and methyl 4-bromobutyrate are mixed and dissolved, and then fully reacted in the presence of a base catalyst to produce compound 3; 3) Compound 3 and apigenin are mixed and dissolved, an alkali catalyst is added, and the mixture is refluxed to generate compound 5, which is an apigenin derivative.

3. The method for preparing the apigenin derivative according to claim 2, wherein In step 1), the molar ratio of 4-amino-2-chlorothienyl[3,2-D]pyrimidine to thiourea is 1:(2-5); and / or, In step 1), the reaction solvent is one or more of n-butanol, tetrahydrofuran, ethyl acetate, dichloromethane, and chloroform; and / or, The reaction time in step 1) is 3-18 hours; and / or, After the reaction in step 1) is completed, the mixture is cooled to room temperature, and an alkaline aqueous solution is added and stirred for 30-60 minutes; the organic layer is separated and purified to obtain compound 2.

4. The method for preparing the apigenin derivative according to claim 2 or 3, wherein: In step 2), the molar ratio of compound 2 to methyl 4-bromobutyrate is 1:(1.2-1.5); and / or, In step 2), the alkaline catalyst is selected from one or more of potassium carbonate, sodium carbonate, potassium acetate, and sodium acetate.

5. The method for preparing the apigenin derivative according to claim 4, wherein In step 2), the amount of the base catalyst used is 0.02-0.06 times the molar amount of compound 2.

6. The method for preparing the apigenin derivative according to claim 4, wherein: The reaction conditions in step 2) are: reaction at room temperature for 4-10 hours; and / or, The reaction solvent in step 2) is one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and toluene.

7. The method for preparing the apigenin derivative according to claim 6, wherein: After the reaction in step 2) is completed, water is added to dilute the reaction solution, and the organic layer is separated after extraction with an organic solvent, and then purified to obtain compound 3.

8. The method for preparing the apigenin derivative according to any one of claims 2 to 3, characterized in that: In step 3), the molar ratio of compound 3 to apigenin is 1:(1.1-1.3); and / or, In step 3), the alkaline catalyst is selected from one or more of potassium carbonate, sodium carbonate, potassium acetate, and sodium acetate.

9. The method for preparing the apigenin derivative according to claim 8, characterized in that: The amount of the base catalyst used is 0.02-0.06 times the molar amount of compound 3.

10. The method for preparing the apigenin derivative according to claim 8, characterized in that: In step 3), the reaction conditions are: stirring under reflux for 8-12 hours; and / or, The reaction solvent in step 3) is one or more of acetonitrile, ethanol, and tetrahydrofuran.

11. The method for preparing the apigenin derivative according to claim 10, characterized in that: After the reaction in step 3) is completed, the mixture is cooled to room temperature, saturated sodium bicarbonate solution is added to adjust the pH to 5-7, and extraction is performed with an organic solvent. The organic layer is separated and purified to obtain compound 5.

12. Use of the apigenin derivative according to claim 1 in a uric acid-lowering drug or preparation.

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