Apigenin derivative as well as preparation method and application thereof

Abstract

The invention discloses an apigenin derivative as well as a preparation method and application thereof. The apigenin derivative has the following molecular structure expression, and the preparation method comprises the following steps: 1) in the presence of an optional catalyst, reacting p-coumaric acid with ethyl sulfonyl chloride to generate an intermediate 1; and 2) mixing the purified intermediate 1 with oxalyl chloride for reaction, carrying out acylating chlorination, adding apigenin, and carrying out stirring reaction in the presence of an acid-binding agent to obtain the apigenin derivat.The apigenin derivative is novel in structure and has good biological activity for reducing the blood uric acid level in vivo.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of apigenin derivatives, especially a kind of apigenin derivatives and its preparation method and application. BACKGROUND

[0002] Hyperuricemia is a chronic purine metabolism disorder, characterized by abnormal elevation of serum uric acid concentration, 85-90% of hyperuricemia patients are asymptomatic, so it is easy to be forgotten. However, many studies have shown that hyperuricemia is positively correlated with the risk of hypertension, cardiovascular disease, kidney disease, obesity, metabolic disease (such as non-alcoholic fatty liver disease and type 2 diabetes) and many other diseases.

[0003] Existing uric acid-lowering drugs are classified according to different mechanisms of action: drugs that inhibit uric acid production include allopurinol, febuxostat, drugs that promote uric acid excretion include benzbromarone, probenecid, and drugs that promote uric acid decomposition include pegloticase. However, known drugs may have varying degrees of side effects. Allopurinol, although inexpensive, is the first choice for hyperuricemia or gout patients who need long-term medication, but it takes longer to take effect, and there are large fluctuations in the early stages of treatment, and even rebound in a period of time, etc. Unstable conditions, and may cause fever, rash, abnormal liver function, kidney function problems, etc.

[0004] Future research and development of uric acid-lowering drugs should consider safety, efficacy and patient compliance to meet the needs of different patient groups. SUMMARY

[0005] To solve the above technical problems, the present application first proposes a kind of apigenin derivatives. The substance is synthesized on the basis of the molecular structure of natural antioxidant apigenin, which not only has a novel structure, but also has good biological activity in reducing blood uric acid levels in vivo.

[0006] Based on the second aspect of the present application, a preparation method of apigenin derivatives is also proposed. Through the design of synthesis route and process, a simple and feasible implementation scheme for the preparation of the apigenin derivatives is provided.

[0007] Based on the third aspect of the present application, the application of apigenin derivatives is also proposed, especially in the application of uric acid-lowering drugs.

[0008] To achieve the above purpose, the technical solutions adopted by the present application are as follows: First, a kind of apigenin derivatives has the following molecular structure expression: .

[0009] Second, a preparation method of apigenin derivatives includes the following steps:

[0010] 1) reacting p-coumaric acid and ethylsulfonyl chloride in the presence of an optional catalyst to form intermediate 1; 2) mixing and reacting the purified intermediate 1 and oxalyl chloride to perform acyl chloride reaction, then adding apigenin, and stirring the reaction in the presence of an acid binding agent to produce the apigenin derivative.

[0011] As a preferred embodiment, the molar ratio of p-coumaric acid to ethylsulfonyl chloride in step 1) is 1:(0.9-1.2); Preferably, the catalyst is an organic base catalyst, more preferably one or more of triethylamine, pyridine; Preferably, the amount of the catalyst is 1-2 times the molar amount of p-coumaric acid.

[0012] As a preferred embodiment, the reaction solvent in step 1) is one or more of tetrahydrofuran, acetonitrile, dichloromethane, trichloromethane; Preferably, the reaction conditions in step 1) are stirring at 0-5°C for 5-10h.

[0013] As a preferred embodiment, after the reaction in step 1) is completed, the solvent is first spun dry, and then purified by silica gel column chromatography to obtain intermediate 1.

[0014] As a preferred embodiment, in step 2), the molar ratio of intermediate 1 to oxalyl chloride is 1:(3-5); Preferably, the reaction conditions of intermediate 1 and oxalyl chloride are stirring at room temperature for 3-5h; Preferably, the acyl chloride reaction is performed in an optional organic solvent, which is preferably one or more of dichloromethane, toluene, diethyl ether, carbon tetrachloride, dichloroethane.

[0015] As a preferred embodiment, in step 2), the amount of apigenin added is 0.8-1.2 times the molar amount of intermediate 1; Preferably, the acid binding agent is one or more of 4-dimethylaminopyridine, pyridine, triethylamine; Preferably, the amount of the acid binding agent is 2-3 times the molar amount of intermediate 1.

[0016] As a preferred embodiment, in step 2), after the acyl chloride reaction is completed, the reaction raw materials and optional organic solvent are first removed by rotary evaporation, and then 1,4-dioxane is used as a new solvent to add apigenin and the acid binding agent for mixing reaction; Preferably, after the addition of apigenin, the stirring reaction is continued at room temperature for 1-5h to produce the target product.

[0017] As a preferred embodiment, after the last step of the reaction in step 2) is completed, the reaction is quenched by adding a phosphate buffer at pH 7.0, and the product is extracted with dichloromethane to obtain the apigenin derivative.

[0018] Again, the application of the apigenin derivative as described above or prepared by the method described above in a drug for reducing uric acid.

[0019] The apigenin derivative provided by the application has a completely new material structure. By designing and modifying the molecular structure of the natural active substance apigenin, it is unexpectedly found that it not only has significant uric acid-lowering activity, but also has a smooth decrease in uric acid level during the onset process, and has the advantages of better drug efficacy stability and higher safety compared to the positive drug allopurinol. DETAILED DESCRIPTION

[0020] The application will be further described below through specific examples, and the examples described in the application are only used to illustrate the application and do not limit the scope of the application.

[0021] The raw materials and reagents used in the following examples of the application can be purchased through commercial channels unless otherwise specified. Example 1

[0022] The apigenin derivative is prepared by the following method:

[0023] 1) In the presence of triethylamine (20 mmol), p-coumaric acid (10 mmol) and ethyl sulfonyl chloride (10 mmol) are reacted in a solvent dichloromethane, the reaction conditions are stirring at 0℃ for 10h; after the reaction is completed, the solvent is dried by vacuum rotary evaporation, the sample is mixed with silica gel powder and column chromatography is performed with ethyl acetate (EA) and petroleum ether (PE) at a volume ratio of 1:10 as the eluent to obtain intermediate 1; 1 H NMR (500 MHz, Chloroform-d) δ 7.66 – 7.56 (m, 4H), 7.13 – 7.07 (m,2H), 6.49 (d, J = 16.5 Hz, 1H), 3.25 (q, J = 9.4 Hz, 2H), 1.46 (m, J = 9.4Hz, 3H). 2) Dissolve intermediate 1 (1 mmol) in 10 mL of dichloromethane, mix with oxalyl chloride (4.6 mmol), and stir the reaction at room temperature for 3 h to perform acyl chloride reaction; after the end of stirring, the mixed solution is vacuum-rotary evaporated to dry the solvent, then the residue is dissolved with 1,4-dioxane, apigenin (1 mmol) is added, and the reaction is stirred for 1 h in the presence of 4-dimethylaminopyridine (2 mmol); after the reaction is completed, pH 7.0 phosphate buffer is added for quenching, followed by dichloromethane extraction; the obtained organic layer is vacuum-rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain the apigenin derivative.

[0024] 1 H NMR (500 MHz, Chloroform-d) δ 8.96 (s, 1H), 8.42 (s, 1H), 7.85 –7.79 (m, 2H), 7.62 – 7.54 (m, 3H), 7.26 – 7.20 (m, 2H), 7.12 – 7.06 (m, 2H),6.78 (s, 1H), 6.66 (d, J = 16.1 Hz, 1H), 6.42 (d, J = 1.8 Hz, 1H), 6.16 (d, J= 1.8 Hz, 1H), 3.25 (q, J = 9.4 Hz, 2H), 1.43 (d, J = 9.4 Hz, 3H). Example 2

[0025] The apigenin derivative is prepared by the following method:

[0026] 1) In the presence of triethylamine (10 mmol), p-coumaric acid (10 mmol) and ethylsulfonyl chloride (9 mmol) are reacted in the solvent tetrahydrofuran, and the reaction conditions are stirring at 5°C for 5 h; after the reaction is completed, the solvent is vacuum-rotary evaporated to dryness, the sample is mixed with silica gel powder, and column chromatography is performed with ethyl acetate (EA) and petroleum ether (PE) in a volume ratio of 1:10 as the eluent to obtain intermediate 1; 2) Dissolve intermediate 1 (1 mmol) in 10 mL of carbon tetrachloride, mix with oxalyl chloride (3 mmol), stir at room temperature for 4 h to perform acyl chloride reaction; after stirring, the mixed solution is vacuum-rotated to dry the solvent on a rotary evaporator, then the residue is dissolved with 1,4-dioxane, apigenin (1.2 mmol) is added, and stirring is performed in the presence of triethylamine (2.5 mmol) for 5 h; after the reaction is completed, pH 7.0 phosphate buffer is added for quenching, followed by dichloromethane extraction, vacuum-rotation of the obtained organic layer on a rotary evaporator, and silica gel column chromatography to purify to obtain the apigenin derivative. Example 3

[0027] The apigenin derivative is prepared by the following method:

[0028] 1) In the presence of pyridine (15 mmol), p-coumaric acid (10 mmol) and ethyl sulfonyl chloride (12 mmol) are reacted in solvent acetonitrile, the reaction conditions are stirring at 2℃ for 8 h; after the reaction is completed, the solvent is vacuum-rotated to dry on a rotary evaporator, the sample is mixed with silica gel powder and column chromatography is performed with ethyl acetate (EA) and petroleum ether (PE) in a volume ratio of 1:10 as eluent to obtain intermediate 1; 2) Dissolve intermediate 1 (1 mmol) in 10 mL of dichloroethane, mix with oxalyl chloride (4 mmol), stir at room temperature for 5 h to perform acyl chloride reaction; after stirring, the mixed solution is vacuum-rotated to dry the solvent on a rotary evaporator, then the residue is dissolved with 1,4-dioxane, apigenin (0.8 mmol) is added, and stirring is performed in the presence of 4-dimethylaminopyridine (3 mmol) for 3 h; after the reaction is completed, pH 7.0 phosphate buffer is added for quenching, followed by dichloromethane extraction, vacuum-rotation of the obtained organic layer on a rotary evaporator, and silica gel column chromatography to purify to obtain the apigenin derivative.

[0029] Application Example 1. Experimental animal preparation Animal selection: healthy male C57BL / 6 mice aged 6-8 weeks, weighing 20-25 g, are selected.

[0030] Raising conditions: raised in a SPF level environment, temperature 22℃±2℃, humidity 55%±5%, 12-hour light / dark cycle, free access to food and water.

[0031] Adaptive feeding: after adapting to the environment for 7 days, blood is collected through the orbital plexus to determine the blood uric acid (SUA) level as the baseline.

[0032] 2. Hyperuricemia model establishment Modeling method: Except for the control group, the rest of the mice were given a mixed solution of adenine (300 mg / kg) and potassium oxonate (250 mg / kg) by gavage once a day for 14 consecutive days.

[0033] Model verification: On the 14th day, blood was collected through the orbital plexus, and the SUA level was determined. If the SUA level is increased by more than 1.3 times compared with the baseline, the model is successfully established.

[0034] 3. Grouping and administration Grouping: The mice were randomly divided into a control group, a model group, a low-dose experimental group, a high-dose experimental group, and a positive drug group.

[0035] Administration: The low-dose experimental group was administered according to 40 mg / kg of apigenin derivative; the high-dose experimental group was administered according to 80 mg / kg of apigenin derivative; the positive drug group was administered according to 40 mg / kg of allopurinol; the control group and the model group were given the same volume of normal saline; gavage administration was performed every day, and administration was performed for 14 days.

[0036] 4. Sample collection and detection Serum collection: On the 0th day, the 2nd day, the 4th day, the 6th day, the 8th day, the 10th day, the 12th day, and the 14th day of administration, blood was collected through the orbital plexus, and serum was separated by centrifugation for detection of blood uric acid level.

[0037] Urine collection: 24h urine was collected on the 14th day of administration, and the supernatant was obtained by centrifugation to measure the levels of urine uric acid and 24h urine creatinine.

[0038] Tissue collection: At the end of the experiment on the 14th day, the mice were sacrificed by cervical dislocation, and tissues such as liver and kidney were collected for subsequent analysis.

[0039] 5. Data analysis The results of blood uric acid level analysis are shown in Table 1 (unit: mg / dL).

[0040]

[0041] The results of urine analysis are shown in Table 2.

[0042]

[0043] From the above test results, it can be seen that: The apigenin derivative provided by the application can significantly reduce the blood uric acid level, and its therapeutic effect is comparable to that of the positive drug allopurinol. However, the SUA level of allopurinol decreases rapidly after administration, and then rebounds temporarily, which may indicate that it strongly interferes with the uric acid metabolism pathway. The SUA level of the application decreases more stably and does not fluctuate greatly, which is expected to effectively reduce the side effects of the drug.

[0044] (2) The apigenin derivative provided by the application is closer to the control group in terms of urine uric acid and 24h urine creatinine levels, indicating that it has significant advantages in reducing kidney damage and is expected to be safer than allopurinol.

[0045] These results show that the apigenin derivative provided by the application has potential application value in the treatment of hyperuricemia and is worthy of further research and development.

[0046] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the method of the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. An apigenin derivative, characterized in that Has the following molecular structure expression: 。 2. A method for preparing an apigenin derivative, characterized in that: The following steps are involved: ； 1) reacting p-coumaric acid with ethylsulfonyl chloride in the presence of an optional catalyst to produce intermediate 1; 2) The purified intermediate 1 is mixed with oxalyl chloride to perform chlorination, and then apigenin is added and stirred in the presence of an acid-binding agent to react to obtain an apigenin derivative.

3. The method for preparing the apigenin derivative according to claim 2, wherein In step 1), the molar ratio of p-coumaric acid to ethylsulfonyl chloride is 1:(0.9-1.2); Preferably, the catalyst is an organic base catalyst, more preferably one or more of triethylamine and pyridine; Preferably, the amount of the catalyst used is 1-2 times the molar amount of p-coumaric acid.

4. The method for preparing the apigenin derivative according to claim 2 or 3, wherein: In step 1), the reaction solvent is one or more of tetrahydrofuran, acetonitrile, dichloromethane, and chloroform; Preferably, the reaction conditions in step 1) are to keep stirring at 0-5°C for 5-10 hours.

5. The method for preparing the apigenin derivative according to any one of claims 1 to 4, characterized in that: After the reaction in step 1) is completed, the solvent is first dried by spin drying, and then separated and purified by silica gel column chromatography to obtain intermediate 1.

6. The method for preparing the apigenin derivative according to claim 5, characterized in that: In step 2), the molar ratio of intermediate 1 to oxalyl chloride is 1:(3-5); Preferably, the reaction conditions of intermediate 1 and oxalyl chloride are: stirring at room temperature for 3-5 hours; Preferably, the acyl chlorination reaction is carried out in an optional organic solvent, and the organic solvent is preferably one or more of dichloromethane, toluene, ether, carbon tetrachloride, and dichloroethane.

7. The method for preparing the apigenin derivative according to any one of claims 1 to 6, characterized in that: In step 2), the amount of apigenin added is 0.8-1.2 times the amount of 1 mole of the intermediate; Preferably, the acid binding agent is one or more of 4-dimethylaminopyridine, pyridine, and triethylamine; Preferably, the amount of the acid binding agent is 2-3 times the amount of 1 mole of the intermediate.

8. The method for preparing the apigenin derivative according to any one of claims 1 to 7, characterized in that: In step 2), after the acyl chloride reaction is completed, the reaction raw materials and the optional organic solvent are first removed by rotary evaporation, and then 1,4-dioxane is used as a new solvent, and apigenin and an acid-binding agent are added to carry out a mixing reaction; Preferably, after adding apigenin, the reaction is continued with stirring at room temperature for 1-5 to prepare the target product.

9. The method for preparing the apigenin derivative according to any one of claims 1 to 8, characterized in that: After the last step of the reaction in step 2) is completed, a phosphate buffer solution with a pH of 7.0 is added to quench the reaction, and the product is extracted with dichloromethane and purified to obtain the apigenin derivative.

10. Use of the apigenin derivative according to claim 1 or the apigenin derivative prepared by the method according to any one of claims 2 to 9 in a uric acid-lowering drug.

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