Apigenin derivatives, processes for their preparation and uses thereof

Abstract

The application belongs to the field of medicine and organic synthesis, and relates to an apioin derivative, a preparation method and application thereof, a structural formula of the apioin derivative is as follows: wherein R is or the apioin derivative provided by the application has excellent inhibitory effect on xanthine oxidase, and can be applied to the preparation of a drug with xanthine oxidase as a target. The application provides a potential selection for the research and development of a drug for treating hyperuricemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine and organic synthesis, and relates to apigenin derivatives, and a preparation method and application thereof. BACKGROUND

[0002] Hyperuricemia (HUA) is a kind of purine metabolism disorder syndrome, which is caused by excessive production or excretion disorder of uric acid in the body, and is also the fundamental cause of gout. Long-term hyperuricemia can cause a series of complications such as hypertension, hyperlipidemia, cardiovascular and cerebrovascular diseases, chronic kidney disease and diabetes. In recent years, with the increase of the intake of high purine, high protein and high calorie food in the dietary structure, the number of patients with hyperuricemia continues to increase, and the average age tends to be younger. At present, hyperuricemia has become the second metabolic disease after diabetes.

[0003] Regarding the pathogenesis of HUA, there are currently a variety of different hypotheses, among which xanthine oxidase (XO) is considered to be the most important target. XO widely exists in liver, kidney and vascular endothelium, etc., and is a key enzyme for the last step of uric acid synthesis, which converts hypoxanthine and xanthine into uric acid. The drugs currently used in clinical practice to inhibit XO include allopurinol, febuxostat and topiroxostat, and their structures are as follows, respectively:

[0004]

[0005] However, clinical practice has found that long-term use of these drugs can produce a series of side effects, including fever, abdominal pain, diarrhea, allergic skin rash, liver and kidney damage, leukopenia and thrombocytopenia, etc. Therefore, it is necessary to develop safe and effective new XO inhibitors.

[0006] In recent years, in view of the potential advantages of safety and structural diversity of natural products, many researchers have focused on discovering XO inhibitors from natural products. It has been found that apigenin has the ability to inhibit XO activity and scavenge free radicals, and acute HUA rat in vivo studies have also confirmed that apigenin can reduce the level of blood uric acid in vivo. However, the activity of apigenin in inhibiting XO is not high, and its IC 50 (μM) value is as high as 4.794±0.193, which is still far from the actual clinical application. Therefore, if the structure of apigenin can be modified on the basis of its structure, new apigenin derivatives can be provided and the activity of apigenin in inhibiting XO can be significantly improved, which will have a positive significance for the further development of hyperuricemia drugs. SUMMARY

[0007] The application aims at overcoming the defects of the prior art, providing apigenin derivatives, and a preparation method and application thereof, so as to effectively improve the activity of apigenin in inhibiting xanthine oxidase, and provide more choices for the development of hyperuricemia drugs.

[0008] To achieve the above-mentioned application purposes, the technical solutions adopted by the application are as follows.

[0009] The apigenin derivative has a structural formula as shown in formula (I):

[0010]

[0011] In formula (I), R is or

[0012] The apigenin derivative provided by the application specifically refers to three compounds with the following structural formula:

[0013]

[0014] The application also provides pharmaceutically acceptable salts of the above-mentioned apigenin derivatives, for example, addition salts of the compound shown in formula (I) and hydrochloric acid, hydrobromic acid, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

[0015] The term "pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, within the scope of sound medical judgment, and are directly or indirectly providing the compounds of the present application or prodrugs of the compounds.

[0016] The application also provides a preparation method of the above-mentioned apigenin derivative, and the synthesis route and steps are as follows:

[0017]

[0018] (1) apigenin and anhydrous K2CO3 are added into a reaction container, inert gas is filled into the reaction container to exclude air, then anhydrous N,N-dimethylformamide is added, and after stirring under ice bath until the apigenin is dissolved, chloromethyl methyl ether is added dropwise into the reaction container, after the dropwise addition is completed, the reaction is carried out at room temperature for 4-6 hours; the obtained reaction product is subjected to solid-liquid separation, water is added into the obtained liquid phase, and the pH is adjusted to neutral by using dilute hydrochloric acid, then the obtained liquid phase is extracted by using ethyl acetate, the obtained organic layer is sequentially washed by using saturated NaCl solution and water, then water is removed, filtration is carried out, the solvent in the obtained liquid phase is removed by distillation under reduced pressure, and the crude product of intermediate 1 is obtained; the crude product of intermediate 1 is purified to obtain intermediate 1;

[0019] In this step, the molar ratio of apigenin to anhydrous K2CO3 is controlled to be 1:(1-1.2), and chloromethyl methyl ether is added dropwise in a proportion of 84-90 μL per 1 mmol of apigenin;

[0020] (2) Intermediate 1, anhydrous K2CO3 and KI are added into a reaction vessel, anhydrous N,N-dimethylformamide is added under ice-bath, then RBr is added, and the reaction is carried out at room temperature for 8-12 h. After the reaction is completed, the reaction is quenched with ice water, and the obtained reaction solution is extracted with ethyl acetate. The organic layer obtained by extraction is sequentially washed with saturated NaCl solution and water, then water is removed, filtered, and the solvent in the liquid phase obtained by filtration is removed by reduced pressure distillation to obtain a crude product of intermediate 2. The crude product of intermediate 2 is purified to obtain intermediate 2.

[0021] In this step, the molar ratio of intermediate 1, anhydrous K2CO3, KI and RBr is controlled to be 1:(1-1.2):(0.3-0.4):(1-1.2), and the RBr is 3-bromopropyne, 3-bromopropene or 1-bromo-2-methylbutane.

[0022] (3) Intermediate 2 is dissolved in a mixed solvent of Et2O and CH2Cl2 in a reaction vessel, and hydrochloric acid solution is added into the reaction vessel under ice-bath to adjust the pH value to 3-3.2, and the reaction is carried out at room temperature for 4-8 h. After the reaction is completed, the reaction is quenched with ice water, and the obtained reaction solution is extracted with ethyl acetate. The organic layer obtained by extraction is sequentially washed with saturated NaCl solution and water, then water is removed, filtered, and the solvent in the liquid phase obtained by filtration is removed by reduced pressure distillation to obtain a crude product of apigenin derivative. The crude product of apigenin derivative is purified to obtain the apigenin derivative.

[0023] In this step, the volume ratio of Et2O to CH2Cl2 in the mixed solvent of Et2O and CH2Cl2 is 1:(0.8-1.2).

[0024] In the technical scheme of the above preparation method of apigenin derivative, the operation of purifying the crude product of intermediate 1 in step (1) is as follows: a silica gel chromatographic column is used for separation, and petroleum ether-ethyl acetate mixed liquid with a volume ratio of (3-5):1 of petroleum ether to ethyl acetate is used as eluent for elution to obtain intermediate 1.

[0025] In the technical scheme of the above preparation method of apigenin derivative, the operation of purifying the crude product of intermediate 2 in step (2) is as follows: a silica gel chromatographic column is used for separation, and petroleum ether-ethyl acetate mixed liquid with a volume ratio of (8-3):1 of petroleum ether to ethyl acetate is used as eluent for gradient elution to obtain intermediate 2.

[0026] The technical scheme of the preparation method of the apigenin derivative is characterized in that, in step (3), the crude product of the apigenin derivative is purified by using a silica gel chromatographic column, and a petroleum ether-ethyl acetate mixed solution with a volume ratio of petroleum ether to ethyl acetate of (6-3):1 is used as an eluent for gradient elution during separation to obtain the apigenin derivative.

[0027] The technical scheme of the preparation method of the apigenin derivative is characterized in that, in step (1), the addition amount of anhydrous N,N-dimethylformamide is controlled to make the concentration of apigenin be 0.3-0.4 mol / L, in step (2), the addition amount of anhydrous N,N-dimethylformamide is controlled to make the concentration of the intermediate 1 be 0.1-0.2 mol / L, and in step (3), the addition amount of the mixed solvent of Et2O and CH2Cl2 is controlled to make the concentration of the intermediate 2 be 0.1-0.2 mol / L.

[0028] The technical scheme of the preparation method of the apigenin derivative is characterized in that, the water removal can be generally performed by drying with anhydrous Na2SO4 to remove water.

[0029] The present application tests the apigenin derivatives provided by the present application and the existing apigenin and allopurinol in inhibiting the activity of XO in vitro by using an XO inhibitor screening model through experiments, and the results show that the three apigenin derivatives provided by the present application with the structural formula have excellent inhibitory effect on XO, and the IC 50 values of the three apigenin derivatives in inhibiting the activity of XO in vitro are 0.178±0.024 μM, 0.197±0.035 μM and 0.399±0.095 μM, respectively.

[0030]

[0031] The IC 50 values of the three apigenin derivatives in inhibiting the activity of XO in vitro are 0.178±0.024 μM, 0.197±0.035 μM and 0.399±0.095 μM, respectively.

[0032] Based on the above experimental results, the present application further provides the use of the apigenin derivative or the pharmaceutically acceptable salt of the apigenin derivative in the preparation of a drug targeting xanthine oxidase. Further, the drug targeting xanthine oxidase includes a drug for treating hyperuricemia.

[0033] Compared with the prior art, the present application has the following beneficial technical effects:

[0034] 1.The application provides apigenin derivatives with excellent inhibitory effect on xanthine oxidase (XO) in vitro, and the application proves through experiments that the apigenin derivatives provided by the application have an activity of inhibiting xanthine oxidase (XO) in vitro with an IC 50 (μM) value, which is 12.01-26.93 times that of a modified base compound apigenin and 8.33-18.67 times that of an existing clinical drug allopurinol, and the apigenin derivatives provided by the application have an activity of inhibiting xanthine oxidase (XO) in vitro with an IC 50 (μM) value, which is as low as (0.178±0.024)-(0.399±0.095) μM, and can be applied to the preparation of drugs with xanthine oxidase as a target, and the application provides a new potential choice for the research and development of drugs for treating hyperuricemia.

[0035] 2.The application also provides a preparation method of the apigenin derivatives, which has simple process, mild reaction conditions, low cost, is suitable for industrial production, and is beneficial to popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 is a logarithmic plot of drug concentration versus inhibition rate of compounds 3a-3f on XO, wherein the A-F graphs represent the test results of compounds 3a-3f, respectively. DETAILED DESCRIPTION

[0037] The following further describes the apigenin derivatives, the preparation method and the application thereof according to the application through examples. It is necessary to point out that the following examples are only used for further describing the application and cannot be understood as limiting the protection scope of the application, and the skilled in the art can make some non-essential improvements and adjustments to the application according to the above description, which still belongs to the protection scope of the application.

[0038] The following prepares apigenin derivatives, i.e., compounds 3a-3f, through examples 1-13, and then tests the activity of the compounds 3a-3f in inhibiting xanthine oxidase (XO) in vitro through example 14.

[0039] Example 1

[0040] In this example, the intermediate l is synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0041]

[0042] Apigenin (2.7 g, 10 mmol) was taken in a 100 mL round bottom flask, then anhydrous K2CO3 (1.38 g, 10 mmol) was added, the round bottom flask was charged with inert gas Ar, 30 mL of anhydrous DMF was added, stirred for 15 min under ice bath conditions, then 840 μL of chloromethyl methyl ether was added dropwise to the round bottom flask, then the round bottom flask was placed at room temperature for 4 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the resulting reaction product was filtered to remove K2CO3, 50 mL of deionized water was added, the pH was adjusted to neutral with dilute hydrochloric acid, 100 mL of ethyl acetate was extracted three times, the organic layer was washed with saturated NaCl solution three times, then washed with water and dried over anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. 100-200 mesh silica gel was used to dry the sample, a 300-400 mesh silica gel chromatographic column was used for separation, and petroleum ether-ethyl acetate mixture (volume ratio of petroleum ether and ethyl acetate 4:1) was used as the eluent for elution to obtain intermediate 1 (light yellow solid, yield 59.6%).

[0043] Intermediate 1 was subjected to 1 H NMR and 13 C NMR detection, the results are as follows:

[0044] 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.39 (s, 1H), 7.96 (d, J = 9.2 Hz, 2H), 6.93 (d, J = 9.2 Hz, 2H), 6.86 (s, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 5.32 (s, 2H), 3.41 (s, 3H).

[0045] 13 C NMR (100 MHz, DMSO-d6) δ = 182.1, 164.3, 162.5, 161.4, 161.2, 157.0, 128.7, 121.1, 116.1, 105.3, 103.1, 99.4, 94.6, 94.0, 56.1 ppm.

[0046] Example 2

[0047] In this example, intermediate 2a was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0048]

[0049] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 1-bromo-2-methylpropane, the round bottom flask was kept at room temperature for 12 h. During the reaction at room temperature, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice cold water, the resulting reaction mixture was extracted with ethyl acetate, the organic layer was washed with saturated NaCl solution, water, dried over anhydrous Na2SO4, filtered and concentrated on rotavapor to get the crude product. The crude product was dry loaded on to 100-200 mesh silica gel, separated on silica gel column using pet ether-ethyl acetate mixture (gradient elution) as an eluent, to get the intermediate 2a (pale yellow solid, 68.1% yield).

[0050] Intermediate 2a was subjected to 1 H NMR detection, the results were as follows:

[0051] 1 H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 8.06 (d, J = 9.2 Hz, 2H), 7.11 (d, J = 9.2 Hz, 2H), 6.95 (s, 1H), 6.85 (J = 2.0 Hz,), 6.45 (J = 2.0 Hz,), 5.33 (s, 1H), 3.86 (d, J = 6.8 Hz, 2H), 3.42 (s, 1H), 2.06 - 1.94 (m, 1H), 0.97 (s, 3H), 0.95 (s, 3H).

[0052] Example 3

[0053] In this example, intermediate 2b was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0054]

[0055] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 1-bromo-3-methylbutane, the round bottom flask was kept at room temperature for 12 h. During the reaction at room temperature, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice cold water, the resulting reaction mixture was extracted with ethyl acetate, the organic layer was washed successively with saturated NaCl solution, water and dried over anhydrous Na2SO4, filtered and evaporated to dryness on a rotary evaporator to obtain the crude product. The crude product was dry packed with 100-200 mesh silica gel and purified by silica gel column chromatography using a gradient elution with petroleum ether-ethyl acetate mixture (gradient elution was carried out using petroleum ether and ethyl acetate in the ratio of 8:1 to 3:1) as eluent to obtain the intermediate 2b (pale yellow solid, 67.9% yield).

[0056] Intermediate 2b was subjected to 1 H NMR detection, the results are as follows:

[0057] 1 H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 8.05 (d, J = 9.2 Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H), 6.95 (s, 1H), 6.85 (d, J = 2.0 Hz, 2H), 6.45 (d, J = 2.4 Hz, 2H), 5.33 (s, 2H), 4.11 (t, J = 6.8 Hz, 2H), 3.42 (s, 3H), 1.80 (dp, J = 19.4, 6.8 Hz, 1H), 1.65 (q, J = 6.8 Hz, 2H), 0.95 (d, J = 6.8 Hz, 6H).

[0058] Example 4

[0059] In this example, intermediate 2c was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0060]

[0061] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 3-bromopropyne, the round bottom flask was kept at room temperature for 12 h. During the reaction at room temperature, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice cold water, the resulting reaction mixture was extracted with ethyl acetate, the organic layer was washed with saturated NaCl solution, water, dried over anhydrous Na2SO4, filtered and concentrated on a rotary evaporator to get the crude product. The crude product was dry loaded on to 100-200 mesh silica gel, separated on a silica gel column using silica gel chromatography, eluting with a gradient of petroleum ether-ethyl acetate (8:1 to 3:1) as eluent to get the intermediate 2c (pale yellow solid, 61.8% yield).

[0062] Intermediate 2c was subjected to 1 H NMR detection, the results are as follows:

[0063] 1 H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.07 (d, J = 9.2 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 6.95 (s, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H), 4.93 (d, J = 2.4 Hz, 2H), 3.63 (t, J = 2.4 Hz, 1H), 3.41 (s, 3H).

[0064] Example 5

[0065] In this example, intermediate 2d was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0066]

[0067] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 2-bromo-butane, the round bottom flask was kept at room temperature for 12 h. During the reaction at room temperature, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice cold water, the resulting reaction mixture was extracted with ethyl acetate, the organic layer was washed successively with saturated NaCl solution, water and dried over anhydrous Na2SO4, filtered and evaporated to dryness on a rotary evaporator to get the crude product. The crude product was dry packed with 100-200 mesh silica gel and purified by silica gel column chromatography using a gradient elution with petroleum ether-ethyl acetate mixture (gradient elution from 8:1 to 3:1 of petroleum ether to ethyl acetate) as eluent to get the intermediate 2d (pale yellow solid, 63.6% yield).

[0068] Intermediate 2d was subjected to 1 H NMR detection and the results were as follows:

[0069] 1 H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 9.2 Hz, 2H), 6.89 (s, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 5.33 (s, 2H), 4.54 (h, J = 6.0 Hz, 1H), 3.44 (s, 3H), 1.72 - 1.59 (m, 2H), 1.27 (d, J = 6.4 Hz, 3H), 0.94 (t, J = 7.6 Hz, 3H).

[0070] Example 6

[0071] In this example, intermediate 2e was synthesized, the synthetic route is shown below and the specific steps are as follows:

[0072]

[0073] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 3-bromopropene, the round bottom flask was kept at room temperature for 12 h. During the reaction at room temperature, the progress of the reaction was monitored by TLC. After completion of the reaction, the reaction was quenched with ice cold water, the resulting reaction mixture was extracted with ethyl acetate, the organic layer was washed successively with saturated NaCl solution, water and dried over anhydrous Na2SO4, filtered and evaporated to dryness on a rotary evaporator to get the crude product. The crude product was dry packed with 100-200 mesh silica gel and purified by silica gel column chromatography using a gradient elution with petroleum ether-ethyl acetate mixture (gradient elution from 8:1 to 3:1 of petroleum ether to ethyl acetate) as eluent to get the intermediate 2e (pale yellow solid, 65.8% yield).

[0074] Intermediate 2e was subjected to 1 H NMR detection, the results are as follows:

[0075] 1 H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H), 8.03 (d, J = 9.2 Hz, 2H), 7.11 (d, J = 9.2 Hz, 2H), 6.91 (s, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 6.07 (ddt, J = 17.2, 10.4, 5.2 Hz, 1H), 5.44 (dp, J = 17.2, 1.6 Hz, 1H), 5.32 (s, 2H), 4.68 (dt, J = 5.2, 1.6 Hz, 2H), 3.43 (s, 3H).

[0076] Example 7

[0077] In this example, intermediate 2f was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0078]

[0079] Intermediate 1 (1 mmol) was taken in a 50 mL round bottom flask, anhydrous K2CO3 (1.2 mmol), KI (0.3 mmol) were added, 10 mL of anhydrous DMF was added under ice bath condition, followed by addition of 1 mmol of 1-bromo-2-methylbutane, the round bottom flask was taken to room temperature for 12 h. The progress of the reaction was monitored by TLC during the reaction at room temperature. After completion of the reaction, the reaction mixture was quenched with ice cold water and the resulting reaction mixture was extracted with ethyl acetate, the organic layers were washed successively with water, saturated NaCl solution and dried over anhydrous Na2SO4, filtered and concentrated under vacuum to get the crude product. The crude product was purified by silica gel column chromatography using 100-200 mesh silica gel, eluting with petroleum ether-ethyl acetate mixture (gradient elution, starting with 8:1 to 3:1 of petroleum ether and ethyl acetate) as eluent to get the intermediate 2f (pale yellow solid, 65.5% yield).

[0080] Intermediate 2f was subjected to 1 H NMR detection, the results are as follows:

[0081] 1 H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 8.02 (d, J = 9.2 Hz, 2H), 7.09 (d, J = 9.2 Hz, 2H), 6.91 (s, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 2.4 Hz, 1H), 5.33 (s, 2H), 3.89 (ddd, J = 31.6, 9.6, 6.4 Hz, 2H), 3.43 (s, 3H), 1.82 (dq, J = 13.2, 6.8 Hz, 1H), 1.58 - 1.19 (m, 2H), 0.99 (d, J = 6.8 Hz, 3H), 0.92 (t, J = 7.6 Hz, 3H).

[0082] Example 8

[0083] In this example, compound 3a was synthesized, the synthetic route is shown in the following formula, and the specific steps are as follows:

[0084]

[0085] To a 50 mL round bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent (volume ratio of Et2O to CH2Cl2was 1:1) was added, then intermediate 2a (1 mmol) was added and dissolved, 10 wt% hydrochloric acid solution was added to adjust pH value to 3 under ice bath condition, then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, the obtained reaction liquid was extracted with ethyl acetate for three times, the organic layer was washed with saturated NaCl solution, washed with water, then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry mixed, and the silica gel chromatographic column was separated, and the gradient elution was carried out with petroleum ether-ethyl acetate mixed liquid (volume ratio of petroleum ether to ethyl acetate was 6:1 to 3:1 gradient elution) as eluent to obtain compound 3a (white solid, yield was 84.4%).

[0086] Compound 3a was subjected to 1 H NMR and 13 C NMR detection, high resolution electrospray mass spectrometry detection and purity determination, and the results were as follows:

[0087] 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.87 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.10 (d, J = 9.2 Hz, 2H), 6.86 (s, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 3.85 (d, J = 6.8 Hz, 2H), 2.10-1.99 (m, 1H), 1.01 (s, 3H), 0.99 (s, 3H).

[0088] 13 C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.7, 163.8, 162.4, 162.0, 157.8, 128., 123.1, 115.5, 104.7, 103.9, 99.4, 94.5, 74.5, 40.6, 40.4, 40.2, 40.0, 39.8, 39.5, 39.3, 28.2, 19.5 ppm.

[0089] HR-MS (ESI) m / z calcd. for C 19 H 18 O5[M-H] - 325.1154, found 325.1081.

[0090] The purity of compound 3a was calculated as a percentage of peak area, and by analyzing the HPLC spectrum, the retention time of compound 3a was 21.6 min, and the HPLC purity was 98.8%.

[0091] Example 9

[0092] In this example, compound 3b was synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0093]

[0094] Into a 50 mL round-bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent with a volume ratio of Et2O to CH2Cl2of 1:1 was added, then intermediate 2b (1 mmol) was added and dissolved, and 10 wt% hydrochloric acid solution was added under ice bath conditions to adjust the pH value to 3, and then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, and the obtained reaction liquid was extracted with ethyl acetate three times, and the organic layer was washed with saturated NaCl solution, washed with water, and then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry mixed, and the silica gel chromatographic column was separated, and gradient elution was performed with petroleum ether-ethyl acetate mixed liquid (petroleum ether and ethyl acetate with a volume ratio of 6:1 to 3:1) as the eluent during separation. Compound 3b (white solid, yield 89.3%) was obtained.

[0095] Compound 3b was subjected to 1 H NMR and 13 C NMR detection, high-resolution electrospray mass spectrometry detection, and purity determination, and the results were as follows:

[0096] 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 10.85 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 9.2 Hz, 2H), 6.84 (s, 1H), 6.50 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 4.08 (t, J = 6.8 Hz, 2H), 1.78 (dq, J = 13.2, 6.8 Hz, 1H), 1.64 (q, J = 6.8 Hz, 2H), 0.94 (d, J = 6.4 Hz, 6H).

[0097] 13C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.7, 163.8, 162.3, 162.0, 157.9, 128.8, 123.2, 104.3, 104.0, 99.4, 94.5, 66.9, 37.8, 25.1, 22.9 ppm.

[0098] HR-MS (ESI) m / z calcd. for C 20 H 20 O5[M-H] - 339.1311, found 339.1234.

[0099] The purity of compound 3b was calculated by peak area percentage, and the retention time of compound 3b was 22.4 min by analyzing the HPLC spectrum. HPLC purity: 97.4%.

[0100] Example 10

[0101] In this example, compound 3c was synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0102]

[0103] Into a 50 mL round-bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent with a volume ratio of 1:1 of Et2O and CH2Cl2was added, then intermediate 2c (1 mmol) was added and dissolved, and 10 wt% hydrochloric acid solution was added under ice bath condition to adjust the pH value to 3, then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, and the obtained reaction liquid was extracted with ethyl acetate for three times, and the organic layer was washed with saturated NaCl solution, washed with water, then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry mixed, and the silica gel chromatographic column was separated, and gradient elution was carried out with petroleum ether-ethyl acetate mixed liquid (the volume ratio of petroleum ether to ethyl acetate was 6:1 to 3:1) as eluent.

[0104] Compound 3c was detected by H NMR and 1 H NMR and 13 C NMR, high resolution electrospray mass spectrometry and purity determination, and the results are as follows:

[0105] 1H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 10.86 (s, 1H), 8.05 (d, J = 8.8 Hz, 2H), 7.17 (d, J = 9.2 Hz, 2H), 6.88 (s, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.22 (d, J = 2.0 Hz, 1H), 4.94 (d, J = 2.4 Hz, 2H), 3.38 (s, 1H).

[0106] 13 C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.8, 163.6, 162.0, 160.7, 157.9, 128.8, 124.1, 115.9, 104.3, 99.4, 94.6, 79.3, 60.3, 56.3, 55.4, 21.3, 14.6 ppm.

[0107] HR-MS (ESI) m / z calcd. for C 18 H 12 O5[M-H] - 307.0685, found 307.0611.

[0108] The purity of compound 3c was calculated by peak area percentage, and the retention time of compound 3c was 17.9 min by analyzing the HPLC spectrum, HPLC purity: 97.3%.

[0109] Example 11

[0110] In this example, compound 3d was synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0111]

[0112] To a 50 mL round bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent with volume ratio of 1:1 was added, then intermediate 2d (1 mmol) was added and dissolved, 10 wt% hydrochloric acid solution was added to adjust pH value to 3 under ice bath condition, then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, the obtained reaction liquid was extracted with ethyl acetate for three times, the organic layer was washed with saturated NaCl solution, washed with water, then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry blended, and the silica gel chromatographic column was separated, and gradient elution was carried out with petroleum ether-ethyl acetate mixed liquid (volume ratio of petroleum ether to ethyl acetate was 6:1 to 3:1) as eluent to obtain compound 3d (white solid, yield was 84.8%).

[0113] Compound 3d was subjected to 1 H NMR and 13 C NMR detection, high resolution electrospray mass spectrometry detection and purity determination, and the results were as follows:

[0114] 1 H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 2H), 10.86 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 6.85 (s, 1H), 6.51 (d, J = 2.2 Hz, 1H), 6.21 (d, J = 2.2 Hz, 1H), 4.55 (q, J = 5.6 Hz, 1H), 1.66 (ddq, J = 26.8, 13.8, 6.8 Hz, 2H), 1.27 (d, J = 6.0 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H).

[0115] 13 C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.8, 163.9, 162.0, 161.5, 157.9, 128.9, 122.9, 116.4, 104.3, 103.9, 99.4, 94.6, 75.1, 29.0, 19.4, 10.0 ppm.

[0116] HR-MS (ESI) m / z calcd. for C 19 H 18 O5[M-H] - 325.1154, found 325.1083.

[0117] The purity of compound 3d was calculated by peak area percentage, and the retention time of compound 3d was 21.0 min by analyzing the HPLC spectrum. HPLC purity: 98.4%.

[0118] Example 12

[0119] In this example, compound 3e was synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0120]

[0121] Into a 50 mL round-bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent with a volume ratio of 1:1 of Et2O and CH2Cl2was added, then intermediate 2e (1 mmol) was added and dissolved, and 10 wt% hydrochloric acid solution was added to adjust the pH value to 3 under ice bath condition, then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, and the obtained reaction liquid was extracted with ethyl acetate for three times, the organic layer was washed with saturated NaCl solution, washed with water, then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry mixed, and the silica gel chromatographic column was separated, and gradient elution was carried out with petroleum ether-ethyl acetate mixed liquid (the volume ratio of petroleum ether to ethyl acetate was 6:1 to 3:1 gradient elution) as eluent during separation to obtain compound 3e (white solid, yield 85.7%).

[0122] Compound 3e was subjected to 1 H NMR and 13 C NMR detection, high resolution electrospray mass spectrometry detection and purity determination, and the results are as follows:

[0123] 1 H NMR (400 MHz,) δ 12.94 (s, 1H), 10.86 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H), 6.85 (s, 1H), 6.50 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 6.07 (ddt, J = 17.2, 10.4 5.2 Hz, 1H), 5.48-5.26 (m, 2H), 4.68 (d, J = 5.2 Hz, 2H).

[0124] 13C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.8, 163.7, 162.0, 161.7, 157.9, 133.7, 128.8, 123.5, 118.42, 115.7, 104.3, 104.1, 99.4, 94.5, 69.0 ppm.

[0125] HR-MS (ESI) m / z calcd. for C 18 H 14 O5[M-H] - 309.0841, found 309.0773.

[0126] The purity of compound 3e was calculated by peak area percentage, and the retention time of compound 3e was 19.8 min by analyzing the HPLC spectrum, and the HPLC purity was 97.6%.

[0127] Example 13

[0128] In this example, compound 3f was synthesized, and the synthetic route is shown in the following formula, and the specific steps are as follows:

[0129]

[0130] Into a 50 mL round-bottom flask, 10 mL of Et2O-CH2Cl2mixed solvent with a volume ratio of 1:1 of Et2O and CH2Cl2was added, then intermediate 2f (1 mmol) was added and dissolved, 10 wt% hydrochloric acid solution was added under ice bath condition to adjust the pH value to 3, then reacted at room temperature for 4-8 h. During the reaction at room temperature, the progress of the reaction was monitored by TCL spotting. After the reaction was completed, the reaction was quenched with ice water, and the obtained reaction liquid was extracted with ethyl acetate for three times, the organic layer was washed with saturated NaCl solution, washed with water, then dried with anhydrous Na2SO4, filtered, and rotary evaporated to dryness to obtain the crude product. The 100-200 mesh silica gel was dry mixed, and the silica gel chromatographic column was separated, and gradient elution was carried out with petroleum ether and ethyl acetate (the volume ratio of petroleum ether to ethyl acetate was 6:1 to 3:1) as eluent. Compound 3f (white solid, yield 89.3%) was obtained.

[0131] Compound 3f was detected by 1 H NMR and 13 C NMR, high resolution electrospray mass spectrometry and purity determination, and the results were as follows:

[0132] 1H NMR (400 MHz, DMSO-d6) δ 12.94 (s, 1H), 7.99 (d, J = 8.8 Hz, 2H), 7.08 (d, J = 9.2 Hz, 2H), 6.83 (s, 1H), 6.50 (d, J = 2.0 Hz, 1H), 6.21 (d, J = 2.0 Hz, 1H), 3.88 (ddd, J = 31.6, 9.6, 6.4 Hz, 2H), 1.83 (dq, J = 13.0, 6.4 Hz, 1H), 1.58 - 1.18 (m, 2H), 0.98 (d, J = 6.8 Hz, 3H), 0.92 (t, J = 7.6 Hz, 3H).

[0133] 13 C NMR (100 MHz, DMSO-d6) δ = 182.3, 164.8, 163.8, 162.4, 162.0, 157.9, 128.8, 123.2, 115.5, 104.3, 104.0, 99.4, 94.5, 73.1, 34.5, 26.0, 16.7, 11.6 ppm.

[0134] HR-MS (ESI) m / z calcd. for C 20 H 20 O5[M-H] - 339.1311, found 339.1237.

[0135] The purity of compound 3f was calculated by peak area percentage, and the retention time of compound 3f was 22.5 min by analyzing the HPLC spectrum, and the HPLC purity was 98.6%.

[0136] Example 14

[0137] In this example, the XO inhibitor screening model was used to detect the in vitro XO activity of apigenin derivatives (i.e. compounds 3a-3f), apigenin and allopurinol, and the specific operation was as follows:

[0138] (1) Preparation of pH 7.4 phosphate buffer solution (PBS solution), weigh Na2HPO4·12H2O 15.6 g, add deionized water to 1000 mL to dissolve, obtain Na2HPO4 solution; weigh NaH2PO4·2H2O 17.8 g, add deionized water to 1000 mL to dissolve, obtain NaH2PO4 solution; take 19 mL NaH2PO4 solution and 81 mL Na2HPO4 solution, mix thoroughly, adjust pH to 7.4, obtain 0.1 mol·L-1 PBS solution, store at room temperature. -1

[0139] ​(2) Preparation of the substrate xanthine: Accurately weigh 3.8 mg of the substrate xanthine and place it in a 50 mL volumetric flask. Dissolve it in 1 mL of 0.1 mol·L⁻¹ solution. -1 The NaOH solution was adjusted to pH 7.4 with dilute hydrochloric acid, and PBS solution was added to bring the volume to 50 mL to obtain 0.5 mmol·L⁻¹. -1 xanthine solution.

[0140] (3) XO solution preparation: Dilute XO with PBS solution to the required concentration, store at 2-8℃, prepare and use immediately, and avoid repeated freeze-thaw cycles.

[0141] (4) Preparation of XO inhibitor solution: Accurately weigh compounds 3a to 3f, dissolve them in DMSO to prepare a high-concentration stock solution, store at -20℃, and dilute with PBS solution to the required concentration when needed. Prepare and use immediately.

[0142] (5) Inhibition experiment of compounds 3a-3f on XO

[0143] Add 50 μL of XO (final concentration 0.025 U·mL) to a 200 μL reaction system. -1 100 μL of XO inhibitor solutions of different concentrations (i.e., solutions of compounds 3a–3f) were added, and the two solutions were incubated at 37 °C for 30 min to equilibrate. Then, 50 μL of 0.5 mmol·L⁻¹ XO inhibitor solution was added. -1 The experiment was conducted using xanthine as a substrate. The absorbance of the mixture was measured every 15 seconds at a wavelength of 295 nm, for a total measurement time of 5 min. Allopurinol and apigenin were used as positive controls, and blank PBS solution was used as a negative control. Each group was measured in triplicate.

[0144] The half-maximum inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0. 50 The XO enzyme activity inhibition rate is calculated using the following formula.

[0145] Inhibition rate (%) = (1 – A / B) × 100%

[0146] In the above formula, A and B are the enzyme reaction rates of the sample and the blank group, respectively.

[0147] The results of in vitro inhibitory activity tests of compounds 3a-3f, allopurinol, and apigenin on XO are shown in Table 1. The logarithm of the drug concentration of compounds 3a-3f versus the inhibition rate of XO are shown in the figure below. Figure 1 As shown.

[0148] Table 1. The in vitro inhibitory activity of the compounds against XO.

[0149]

[0150] In the above table,a Inhibitory activity representing XO was expressed as IC 50 (μM) and IC 50 Values are means ± SD of three independent experiments.

Claims

1. Use of apigenin derivatives or pharmaceutically acceptable salts of apigenin derivatives in the preparation of a drug targeting xanthine oxidase, the structural formula of the apigenin derivatives being shown as formula (I): ###0001### Formula (I) wherein R1, R2, R3, R4 and R5 are the same or different and each is independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, alkylthio, alkylsulfonyl, alkylsulfinyl, alkylsulfonyloxy, alkylsulfinyloxy, alkylamino, dialkylamino, alkylcarbonyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonylamino, alkylcarbonylaminosulfonyl, alkylaminosulfonyl, alkylsulfonylamino, alkylsulfony In formula (I), R is or 2. Use according to claim 1, characterized in that, ​

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