Apigenin derivative and its application in anti-tumor

Abstract

This invention belongs to the field of pharmaceutical technology and specifically relates to the use of apigenin derivatives in the preparation of anti-tumor drugs. The structure of the apigenin derivative is shown below: wherein the groups are defined as described in the specification. The compounds of this invention effectively inhibit the proliferation of renal cancer Caki-1 cells, human gastric cancer cells SNU-5, lung cancer cells, and human colon cancer cells HCT 116 in vitro. They also exhibit good inhibitory activity against renal cancer Caki-1 cells in vivo, demonstrating promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to an apigenin derivative, a preparation method thereof, and an application thereof in the preparation of anti-tumor drugs. Background Art

[0002] Apigenin (APG) is an important flavonoid compound found in large quantities in medicinal plants such as Polygonum cuspidatum, Polygonum cuspidatum, Plantago asiatica, and Trachelospermum officinale. It exhibits multiple biological activities, including anti-inflammatory, antioxidant, and tumor growth inhibition, and is particularly advantageous in anti-tumor settings. Numerous studies have demonstrated that apigenin can effectively delay or prevent the proliferation of various malignant tumor cells, including liver cancer, lung cancer, prostate cancer, breast cancer, and colorectal cancer. It exerts its anti-tumor effects through multiple pathways, including cell cycle arrest, apoptosis induction, anti-angiogenesis, and inhibition of cell metastasis.

[0003] To date, a variety of different chemotherapy drugs have been developed for different cancers, and important progress has been made in the treatment of cancer. However, due to the shortcomings of conventional chemotherapy drugs such as non-specific targeting, low bioavailability, low therapeutic index and high dosage requirements, they often cause serious side effects, including neurological problems, hair loss, weight changes, sexual dysfunction and anemia. There is still a lack of safe and effective therapeutic drugs. Therefore, the development of new anti-cancer drugs that can selectively act on targets without side effects has become the primary goal of medicinal chemists. Compared with other natural products with similar structural types, apigenin is less toxic to normal cells, can inhibit the activity of tumor cells in various ways and is non-mutagenic, and is considered an ideal lead compound.

[0004] Renal cell carcinoma (RCC) is a major form of kidney cancer and one of the most common cancers worldwide. Approximately 400,000 patients are diagnosed with kidney cancer each year worldwide, resulting in approximately 170,000 deaths. Approximately 65% ​​of RCC patients have localized tumors, which can usually be successfully controlled with surgery. However, those who experience disease recurrence after surgery, and the remaining 35% who have metastatic disease at the time of initial diagnosis, require systemic treatment and have a poor prognosis. It is estimated that the five-year survival rate for patients with localized disease is 71.0%, while the five-year survival rate for patients with metastatic disease is 13.9%. RCC is not sensitive to traditional chemotherapy and radiotherapy.

[0005] Prior art CN 115160277 A discloses a novel class of apigenin derivatives and pharmaceutical compositions containing such compounds and their use in treating renal cancer. These compounds are compounds represented by formula (I) or stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof of the compounds represented by formula (I).

[0006]

[0007] However, the anti-proliferative activity of the apigenin derivatives of this invention on renal cancer Caki-1 cells still needs to be improved. Summary of the Invention

[0008] The present invention aims to provide an apigenin derivative and application thereof in the preparation of anti-tumor drugs.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] An apigenin derivative, which is a compound represented by formula (I) or a stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof of the compound represented by formula (I),

[0011]

[0012] Wherein, R1 is selected from: CONH-R 3 , five-membered nitrogen-containing heterocycle, six-membered nitrogen-containing heterocycle, NR 4 R 5 、COR 7 , C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, C3-C8 heterocyclyl, substituted C3-C8 heterocyclyl; the substituents in the substituted C1-C6 alkyl, substituted C3-C8 cycloalkyl, substituted C3-C8 heterocyclyl may be independently and optionally selected from one or more hydrogen, D, F, Cl, Br, I, -OH, -NH2, -NO2, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl or C6-C10 aryl;

[0013] R 3 Selected from: H, D, C1-C6 alkyl, substituted C1-C6 alkyl, C3-C8 cycloalkyl, substituted C3-C8 cycloalkyl, C3-C8 heterocyclyl, substituted C3-C8 heterocyclyl; the substituents in the substituted C1-C6 alkyl, substituted C3-C8 cycloalkyl, substituted C3-C8 heterocyclyl may be independently and optionally selected from one or more hydrogen, D, F, Cl, Br, I, -OH, -NH2, -NO2, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl or C6-C10 aryl;

[0014] R 4 、R 5Each independently selected from: H, D, C1-C6 alkyl, COR 6 ;

[0015] R 6 Selected from: H, D, C1-C6 alkyl;

[0016] R 7 Selected from: H, D, C1-C6 alkyl, COR 8 ;

[0017] R 8 Selected from: H, D, C1-C6 alkyl;

[0018] R 2 Selected from: C1-C8 alkylene or substituted C1-C8 alkylene; the substituents may be independently and optionally selected from one or more hydrogen, D, F, Cl, Br, I, -OH, -NH2, -NO2, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C3-C8 cycloalkyl or C6-C10 aryl.

[0019] Preferably, R 3 Selected from: H, D, C1-C3 alkyl, cyclopropane, cyclobutane, cyclopentane.

[0020] Preferably, the five-membered nitrogen-containing heterocycle includes: Imidazole, pyrazole, thiazole.

[0021] Preferably, the six-membered nitrogen-containing heterocycle includes: Pyridine, pyrazine, pyrimidine, pyridazine.

[0022] Preferably, R 4 、R 5 、R 6 、R 7 、R 8 Each independently selected from: C1-C3 alkyl.

[0023] Preferably, R 2 Selected from: C1-C5 alkylene.

[0024] Preferably, the apigenin derivatives include:

[0025]

[0026]

[0027] Based on a unified approach, the present invention also claims protection for the synthetic route of the apigenin derivative, which is:

[0028] (1) Synthesis of intermediate products:

[0029]

[0030] Methylation of 2,4,6-trihydroxyacetophenone gave intermediate 18;

[0031] Intermediate 18 reacts with iodine to give compound 19, which is then converted to chalcone intermediate 20 via aldol condensation.

[0032] Intermediate 20 is cyclized to give compound 21; then Suzuki coupling reaction is performed to generate 22a-q, and finally deprotection reaction is performed to generate intermediate 23a-q;

[0033] (2) Synthesis of products:

[0034]

[0035] Phenylthioacetic acid 29 was catalyzed by hydrogen peroxide and fuming nitric acid to generate 3,4-diphenylsulfonylfuran 30; 3,4-diphenylsulfonylfuran 30 was linked to pentanediol, and the linker arm was oxidized to give 32;

[0036] 32 was ligated with the 4'-OH site of intermediate 23a-q to obtain NO-donating apigenin derivatives 33a-p.

[0037] Preferably, the synthetic route comprises: starting from 2,4,6-trihydroxyacetophenone, methylating the intermediate 18 with dimethyl sulfate. Compound 18 reacts with elemental iodine in the presence of iodic acid to produce compound 19, which is then reacted with 4-benzyloxybenzaldehyde in the presence of a base to produce chalcone intermediate 20. Oxidation with cerium sulfate tetrahydrate at reflux at 110°C in DMSO results in a cyclization reaction to produce compound 21, which undergoes a Suzuki coupling reaction in the presence of tetrakistriphenylphosphine palladium to produce a C-8 phenylapigenin derivative. Finally, deprotection with aluminum chloride in anhydrous acetonitrile produces 17 target products 23a-q.

[0038] Phenylthioacetic acid 29 was used as the starting material to generate 3,4-diphenylsulfonylfuran 30 under the catalysis of hydrogen peroxide and fuming nitric acid, which was then linked with pentanediol under alkaline conditions, and the linker arm was further oxidized to generate 32;

[0039] Finally, under the catalysis of DMAP and DCC, they were respectively spliced ​​with the 4'-OH site of the C-8 phenyl apigenin derivative 23a-p to obtain 16 NO-donating apigenin derivatives 33a-p.

[0040] Based on the unified idea, the present invention also claims protection for the use of the apigenin derivatives in the preparation of anti-tumor drugs.

[0041] Preferably, the tumor includes: kidney cancer, gastric cancer, lung cancer, and colon cancer.

[0042] Compared with the prior art, the present invention has the following beneficial effects: the compound of the present invention can effectively inhibit the proliferation of renal cancer Caki-1 cells, human gastric cancer cells SNU-5, lung cancer cells (NCI-H441, NCI-H1975, A549), and human colon cancer cells HCT116 in vitro, and also has good inhibitory activity against renal cancer Caki-1 cells in vivo, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The compounds of the present invention are used to evaluate the therapeutic effect of Caki-1 against renal cancer in vivo;

[0044] Figure 2 This is the result of the compounds of the present invention being used to inhibit the phosphorylation of AKT and ERK in Caki-1 cells;

[0045] Figure 3 The present invention relates to the effects of the compounds of the present invention on c-Met in Caki-1 cells. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0047] Example 1

[0048] The derivatives of the present invention can be prepared by the following methods:

[0049] 1. Synthesis of Class I Compounds 23a-q

[0050]

[0051] 2. Synthesis of Class II Compounds 33a-p

[0052]

[0053] Starting from 2,4,6-trihydroxyacetophenone, intermediate 18 was obtained by methylation with dimethyl sulfate. Compound 18 reacted with iodine in the presence of iodic acid to give compound 19, which then underwent base-catalyzed aldol condensation with 4-benzyloxybenzaldehyde to produce chalcone intermediate 20. Compound 21 was cyclized by oxidation with cerium sulfate tetrahydrate at reflux at 110°C in DMSO. Suzuki coupling catalyzed by tetrakistriphenylphosphine palladium gave C-8 phenylapigenin derivatives. Finally, deprotection with aluminum chloride in anhydrous acetonitrile gave 17 target products 23a-q.

[0054] Phenylthioacetic acid 29 was used as the starting material to generate 3,4-diphenylsulfonylfuran 30 under the catalysis of hydrogen peroxide and fuming nitric acid. It was then linked with pentanediol under alkaline conditions, and the linker arm was further oxidized to generate 32. Finally, under the catalysis of DMAP and DCC, it was respectively combined with the 4'-OH site of the C-8 phenyl apigenin derivative 23a-p to obtain 16 NO-donating apigenin derivatives 33a-p.

[0055] The synthesis process is described in detail using the compound of the following formula as an example:

[0056]

[0057] Step 1: Weigh 2,4,6-trihydroxyacetophenone (25.2 g, 0.15 mol, 1 eq) and anhydrous potassium carbonate (0.225 mol, 1.5 eq) into a flask. Add 240 mL of acetone and slowly add dimethyl sulfate (28.4 mL, 0.3 mmol, 2 eq) dropwise through a dropping funnel under reflux and stirring. Continue the reaction for 3 h. After the reaction is complete, cool the reaction solution, filter it, collect the filtrate, evaporate the solvent under reduced pressure, and add 5% hydrochloric acid to the concentrate. After the precipitate forms, filter it, wash the filter cake with water, and recrystallize the residue from methanol to obtain 18 (27 g), a white solid powder with a yield of 91.7%.

[0058] Step 2: 2-Hydroxy-4,6-dimethoxyacetophenone 18 (25 g, 0.13 mol, 1 eq) and iodine (14.9 g, 0.059 mol, 0.45 eq) were weighed and placed in a round-bottom flask. 200 mL of ethanol was added and stirred to dissolve. Iodic acid (3.5 g, 0.02 mol, 0.15 eq) was then weighed and dissolved in 7 mL of water. The mixture was added dropwise to the reaction system and stirred at room temperature for 3 h. After TLC monitoring, the starting material spot disappeared, and the mixture was filtered to collect the filter cake. The filtrate was concentrated by evaporation under reduced pressure, and a precipitate formed again. The precipitate was filtered and the filter cakes were combined to obtain the white solid product 19 (39 g) in a 93.3% yield.

[0059] Step 3: Weigh 2-hydroxy-3-iodo-4,6-dimethoxyacetophenone 19 (35 g, 0.11 mol, 1 eq) and 4-benzyloxybenzaldehyde (23 g, 0.11 mol, 1 eq), add 350 mL of ethanol, and slowly add 100 mL of 30% potassium hydroxide aqueous solution dropwise. After the addition is complete, stir at room temperature for 48 hours. After the reaction is completed, add 10% hydrochloric acid solution to adjust the pH to 5, during which a yellow solid precipitates. After filtration, collect the filter cake, wash it with cold water until neutral, and then recrystallize it from methanol to obtain a yellow solid 20 (51 g) with a yield of 90%.

[0060] Step 4: Weigh compound 20 (45 g, 0.09 mol, 1 eq) and cerium sulfate tetrahydrate (121.6 g, 0.3 mol, 2.5 eq) into a round-bottom flask, add 400 mL of dimethyl sulfoxide, and react at 110 ° C. After the reaction is completed, cool the reaction system to room temperature and pour it into ice water. After a yellow solid precipitates, filter it, wash the filter residue with ice water, collect the filter cake, and recrystallize it from ethyl acetate to obtain a yellow solid 21 (40 g) with a yield of 86%.

[0061] Step 5: Compound 21 (400 mg, 0.78 mmol, 1 eq), 4-methylaminophenylboronic acid (167.5 mg, 0.94 mmol, 1.2 eq), tetrakistriphenylphosphine palladium (92.4 mg, 0.08 mmol, 0.2 eq), and anhydrous cesium carbonate (508.3 mg, 1.56 mmol, 2 eq) were weighed and placed in a round-bottom flask. DMF-H2O (5 mL, 9:1, v / v) was added and reacted at 110°C overnight under nitrogen. Heating was stopped after the reaction. The reaction solution was cooled to room temperature and extracted three times with water and ethyl acetate (3 × 30 mL). The organic layer was extracted with saturated sodium chloride, collected, dried over anhydrous sodium sulfate, and concentrated by evaporation under reduced pressure. Purification by silica gel column chromatography (PE / EA = 2:1) gave 350 mg of a yellow solid 22a in an 86.2% yield.

[0062] Step 6: 22a (250 mg, 0.48 mmol, 1 eq) was weighed and placed in a round-bottom flask. 4 mL of anhydrous acetonitrile and anhydrous aluminum chloride (448 mg, 3.36 mmol, 7 eq) were added and refluxed under nitrogen overnight. After the reaction was completed, the mixture was cooled to room temperature, 1 mL of 30% hydrochloric acid solution was added, and the reflux reaction was continued for 2 h. After stopping the reaction, the reaction solution was poured into ice water, where a precipitate was formed. The filter cake was collected by filtration and purified by silica gel column chromatography (DCM / MeOH = 50:1, v / v) to obtain 176 mg of a yellow solid in 85% yield.

[0063] Step 7: Phenylthioacetic acid (6.73 g, 0.04 mol) was weighed into a 100 mL round-bottom flask, and 30 mL of glacial acetic acid was added. With stirring at room temperature, 7.8 mL of 30% hydrogen peroxide was added, and the mixture was refluxed at 60-70°C for 6 h. After completion of the reaction, the solvent was evaporated under reduced pressure to obtain compound 29-1. Next, compound 29-1 (5 g, 24.9 mmol) was weighed into a round-bottom flask, and 8 mL of glacial acetic acid was added. With stirring at 0°C, 15 mL of fuming nitric acid was added dropwise to the aqueous solution of benzenesulfonylacetic acid in glacial acetic acid. After the addition was complete, the mixture was stirred at room temperature for 5 minutes, and then refluxed at 110-140°C for 1.5 h. After 15 minutes, ice water was added to precipitate the precipitate, which was filtered, washed with water, and the filter cake was collected and dried to obtain compound 30.

[0064] Step 8: 1,5-Pentanediol (11.4 mmol, 4.22 eq) was weighed into a round-bottom flask and tetrahydrofuran (THF, 10 mL) was added. Compound 30 (2.7 mmol, 1 eq) was then added dropwise. Aqueous sodium hydroxide (2.5 N, 1 mL) was slowly added dropwise at 5°C and stirred for 30 minutes. The reaction was monitored by TLC. After completion of the reaction, the mixture was concentrated and extracted three times with EA. The organic layer was washed with saturated NaCl and the solvent was evaporated under reduced pressure. Finally, the compound was purified by silica gel column chromatography (PE / EA = 6 / 1, v / v) to afford 31 as a white solid (600 mg, 71% yield).

[0065] Step 9: Compound 31 (1.5 mmol, 1 eq) was weighed into a round-bottom flask, acetone was added, and Jones reagent was slowly added dropwise with stirring at 0-5°C. Stirring was continued for 5 minutes after the addition was complete, and then stirred at room temperature. After the reaction was completed, the solvent was mostly removed by vacuum concentration, and the mixture was extracted with EA (3 × 10 mL). The organic layer was collected, dried, and concentrated to obtain the crude product. Finally, the product was purified by silica gel column chromatography (PE / EA = 4 / 1, v / v) to obtain 32 as a white solid (480 mg, 99% yield).

[0066] Step 10: Compound 23a (70 mg, 0.16 mmol, 1 eq), compound 32 (82 mg, 0.24 mmol, 1.5 eq), and 4-dimethylaminopyridine (0.016 mmol, 0.1 eq) were weighed into a round-bottom flask, and anhydrous DMF was added. Then, DCC (0.32 mmol, 2 eq) was weighed into the round-bottom flask and stirred at room temperature overnight. The mixture was extracted with ethyl acetate, and the organic layer was collected and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (DCM) to give 33a as a yellow solid (95 mg, yield 78.5%). NMR (500MHz, CDCl3) δ8.22(d,J=8.3Hz,1H,NH),8.07(d,J=7.9Hz,2H,Ar-H),7.82(d,J=7.8Hz,2H,Ar-H ),7.75-7.72(m,2H,Ar-H),7.58(tt,J=15.8,7.8Hz,4H,Ar-H),7.12-7.07(m,2H,Ar-H),6.72(s,1H,Ar- H),6.22(s,1H,Ar-H),4.54-4.44(m,2H,OCH2),4.08(s,3H,OCH3),3.87(s,3H,OCH3),3.06(d,J=4.8Hz, 3H,NCH3),2.71(t,J=7.2Hz,2H,CH2),2.03(dt,J=11.1,6.2Hz,2H,CH2),1.96(p,J=7.4Hz,2H,CH2).13C NMR (125MHz, CDCl3) δ180.74,171.43,168.08,165.04,164.53,159.32,158 .93,151.13,147.52,137.98,135.71,133.99,133.38,132.37,132.14,130 .67,129.74,128.51,126.44,122.09,121.42,110.47,109.62,106.63,104 .92,90.08,71.04,56.47,56.40,33.62,27.79,26.94,21.14.HRMS(ESI)m / z calcd for[C38H33N3O12S+H]+,756.1858,found756.1833.

[0067] Example 2

[0068]

[0069] Step 1: 4-Methylaminophenylboronic acid in Example 1 was replaced with 4-formamidophenylboronic acid (0.94 mmol, 1.2 eq). The remaining steps were followed by the synthesis method of Example 1 to prepare compound 22b as a yellow solid (320 mg) in 80% yield.

[0070] Step 2: 22a in Example 1 was replaced with 22b (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23b as a yellow solid in an 86% yield.

[0071] Step 3: Compound 33b (8 mg) was prepared by replacing compound 23a in Example 1 with compound 23b according to the synthesis method of Example 1. The yield was 76.9%. 1 H NMR(400MHz,DMSO-d6)δ8.07(s,1H,NH),8.00(t,J=8.4Hz,4H,Ar-H),7.84(dd,J=13.6,7.9Hz,3H, Ar-H),7.72(t,J=7.8Hz,2H,Ar-H),7.66(d,J=8.0Hz,2H,Ar-H),7.44(s,1H,NH),7.17(d,J=8.2Hz, 2H, Ar-H), 6.73 (s, 1H, C=CH), 6.58 (s, 1H, Ar-H), 4.44 (t, J=5.9Hz, 2H, OCH2), 4.02 (s, 3H, OCH3), 3. 96(s,3H,OCH3),2.69(t,J=7.3Hz,2H,CH2),1.86(p,J=6.4Hz,2H,CH2),1.75(p,J=7.6Hz,2H,CH2). 13 C NMR(100MHz,DMSO-d6)δ179.69,171.96,168.21,165.58,164.33,159.45,159.34,151.50,147.56,137.66,136.60,134.06,133.60,132.35,130 .69,130.49,130.31,128.77,127.54,122.85,110.95,108.93,106.39, 104.34,92.02,71.60,57.50,56.91,33.25,27.62,20.96.HRMS(ESI)m / z calcd for[C 37 H 31 N3O 12 S+H] + ,742.1701,found 742.1699.

[0072] Example 3

[0073]

[0074] Step 1: 4-Methylaminophenylboronic acid (0.94 mmol, 1.2 eq) was substituted for 4-morpholinophenylboronic acid in Example 1. The remaining steps were followed by the synthesis method of Example 1 to obtain compound 22c as a yellow solid (351 mg) in 82% yield.

[0075] Step 2: 22a in Example 1 was replaced with 22c (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23c as a yellow solid in an 84% yield.

[0076] Step 3: Compound 33c was prepared as a yellow solid (95 mg) in a yield of 75.8% by replacing 23a in Example 1 with 23c according to the synthesis method of Example 1. 1 H NMR (500MHz, CDCl3) δ8.05 (d, J=7.9Hz, 2H, Ar-H), 7.78 (dd, J=8.7, 2.3Hz, 2H, Ar-H), 7.75-7.68 (m, 1H, Ar-H), 7.59 (t ,J=8.0,2.1Hz,2H,Ar-H),7.52(d,J=8.8,2H,Ar-H),7.07(d,J=8.6,2H,Ar-H),7.01(d,J=9.6Hz,2H,Ar-H),6.71(s,1 H,C=CH),6.25(s,1H,Ar-H),4.48(t,J=6.0,2.1Hz,2H,CH2),4.05(s,3H,OCH3),3.92(s,3H,OCH3),3.92-3.85(m,4H, OCH2), 3.25 (p, J = 2.6Hz, 4H, NCH2), 2.70 (t, J = 7.2Hz, 2H, CH2), 2.01 (p, J = 6.4Hz, 3H, CH2), 1.95 (p, J = 7.5Hz, 2H, CH2). 13C NMR (125MHz, CDCl3) δ181.13,171.35,165.26,164.67,158.94,158.54,15 0.98,150.45,147.77,138.04,135.67,132.26,131.33,130.52,129.71,12 8.53,121.92,121.69,114.83,110.47,109.28,107.69,105.01,90.13,71 .01,66.92,56.50,56.35,49.04,33.64,27.80,21.15.HRMS(ESI)m / zcalcd for[C 40 H 37 N3O 12 S+H] + ,784.2171,found 784.2166.

[0077] Example 4

[0078]

[0079] Step 1: Substituting 4-(4-methyl-1-piperazinyl)phenylboronic acid (0.94 mmol, 1.2 eq) for 4-methylaminophenylboronic acid in Example 1, the remaining steps were carried out according to the synthesis method of Example 1 to obtain compound 22d as a yellow solid (346 mg) in a yield of 79%.

[0080] Step 2: 22a in Example 1 was replaced with 22d (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to prepare 173 mg of compound 23d as a yellow solid in an 82% yield.

[0081] Step 3: Compound 33d was prepared as a yellow solid (8 mg) in 76.9% yield by replacing 23a in Example 1 with 23d according to the synthesis method of Example 1. 1H NMR (500MHz, CDCl3) δ8.05(d,J=7.8Hz,2H),7.77(d,J=7.7Hz,2H),7.72(t,J=7.5Hz,1H) ,7.59(t,J=7.2Hz,2H),7.49(d,J=11.4Hz,2H),7.14-7.04(m,2H),7.04-6.98(m,2H),6.7 1(s,1H),6.24(s,1H),4.48(t,J=5.9Hz,2H),4.04(s,3H),3.92(s,3H),3.36-3.27(m,4H) ,2.68(dt,J=18.2,6.0Hz,5H),2.40(s,3H),2.02(p,J=6.4Hz,2H),1.95(q,J=7.6Hz,2H). 13 C NMR (125MHz, CDCl3) δ181.12,171.31,165.26,164.68,158.94,158.51,15 0.98,150.38,147.79,138.08,135.65,132.24,131.26,130.53,129.71,12 8.52,121.91,121.44,115.27,110.46,109.25,107.79,105.02,90.16,71. 02,56.48,56.35,54.99,48.68,45.99,33.63,27.80,21.13.HRMS(ESI)m / z calcd for[C 41 H 40 N4O 11 S+H] + ,797.2487,found 797.2489.

[0082] Example 5

[0083]

[0084] Step 1: 4-dimethylaminophenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22e as a yellow solid (346 mg) in 85% yield.

[0085] Step 2: 22a in Example 1 was replaced with 22e (0.48 mmol, 1 eq) and the compound 23e was prepared according to the synthesis method of Example 1 as a yellow solid (173 mg) in a yield of 84%.

[0086] Step 3: Compound 33e was prepared as a yellow solid (98 mg) in 76.9% yield by replacing 23a in Example 1 with 23e according to the synthesis method of Example 1. 1 H NMR (500MHz, CDCl3) δ8.06 (d, J=7.5Hz, 2H, Ar-H), 7.81 (d, J=8.6Hz, 2H, Ar-H), 7.72 (td, J=7.4, 1.3Hz, 1H ,Ar-H),7.59(t,J=7.9Hz,2H,Ar-H),7.49(d,J=8.6Hz,2H,Ar-H),7.08(dp,J=124.4,8.8,8.6Hz,2H,Ar-H ),6.83(d,J=8.4Hz,2H,Ar-H),6.72(s,1H,C=CH),6.25(s,1H,Ar-H),4.48(t,J=6.0Hz,2H,C=CH),4.05(s ,3H,OCH3),3.93(s,3H,OCH3),2.69(t,J=7.2Hz,2H,CH2),2.07-1.99(m,2H,CH2),1.99-1.91(m,2H,CH2). 13 C NMR (125MHz, CDCl3) δ181.30,171.34,165.29,164.65,158.95,158.25,150.92,149.83,147.88,138.03,135.68,132.30,131.20,130.63,129 .72,128.53,121.89,118.04,111.90,110.46,109.17,108.17,105.01, 90.09,71.00,56.47,56.33,40.59,33.64,27.80,21.16.HRMS(ESI)m / z calcd for[C 38 H 35 N3O 11 S+H] + ,742.2065,found742.2067.

[0087] Example 6

[0088]

[0089] Step 1: Substituting 4-(N-tetrahydropyrrolyl)phenylboronic acid (0.94 mmol, 1.2 eq) for 4-methylaminophenylboronic acid in Example 1, the remaining steps were followed by the synthesis method of Example 1 to obtain compound 22f as a yellow solid (346 mg) in a yield of 75%.

[0090] Step 2: 22a in Example 1 was replaced with 22f (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23f as a yellow solid in an 85% yield.

[0091] Step 3: Substituting 23f for 23a in Example 1, compound 33f was prepared according to the synthesis method of Example 1 as a yellow solid (80 mg) in a yield of 65%. 1 H NMR (500MHz, CDCl3) δ8.07(d,J=7.9Hz,2H,Ar-H),7.84(d,J=8.3Hz,2H,Ar-H),7.74(t,J=7.5Hz,1H,Ar-H),7. 61(t,J=7.7Hz,2H,Ar-H),7.49(d,J=8.2Hz,2H,Ar-H),7.10(d,J=8.3Hz,2H,Ar-H),6.73(s,1H,C=CH),6.68(d, J=8.3Hz,2H,Ar-H),6.26(s,1H,Ar-H),4.50(t,J=6.0Hz,2H,OCH2),4.07(s,3H,OCH3),3.94(s,3H,OCH3),3.3 7(d,J=6.2Hz,4H,CH2),2.71(t,J=7.2Hz,2H,CH2),2.04(q,J=9.1,7.8Hz,6H,CH2),1.97(q,J=7.5Hz,2H,CH2). 13 C NMR (125MHz, CDCl3) δ181.37,171.36,165.33,164.58,158.94,158.11,1 50.89,147.91,147.21,138.01,135.70,132.31,131.27,130.65,129.73 ,128.52,121.90,116.78,111.09,110.46,109.11,108.46,104.98,90.1 0,71.01,56.46,56.31,47.66,33.64,27.79,25.55,21.15.HRMS(ESI)m / z calcd for[C 40 H 37 N3O 11 S+H] + ,768.2222,found 768.2206.

[0092] Example 7

[0093]

[0094] Step 1: 4-acetylphenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22 g as a yellow solid (346 mg) with a yield of 85%.

[0095] Step 2: Substitute 22g (0.48mmol, 1eq) for 22a in Example 1 and prepare compound 23g as a yellow solid (173mg) by referring to the synthesis method of Example 1. The yield is 85%.

[0096] Step 3: Substituting 23a in Example 1 with 23g, compound 33g was prepared according to the synthesis method of Example 1 as a yellow solid (100 mg). The yield was 84%. 1 H NMR (500MHz, CDCl3) δ8.06(dd,J=8.0,5.4Hz,4H,Ar-H),7.72(dd,J=16.0,8.2Hz,5H,Ar-H),7.59(t,J=7.7Hz,2H,Ar-H),7.07(d,J=8.3Hz,2 H,Ar-H),6.74(s,1H,C=CH),6.27(s,1H,Ar-H),4.48(t,J=6.1Hz,2H,OCH2),4.07(s,3H,OCH3),3.95(s,3H,OCH3),2.68(d,J=12.9Hz,5H,CH 2, , CH3), 2.02 (dq, J=11.2, 6.3Hz, 2H, CH2), 1.94 (dq, J=14.4, 7.2Hz, 2H, CH2). 13 C NMR (125MHz, CDCl3) δ197.88,180.73,171.33,165.11,164.72,159.55,158. 94,151.18,147.49,138.05,136.06,135.97,135.66,132.22,130.81,130.23 ,129.71,128.53,128.50,127.98,122.06,110.46,109.89,106.71,105.12,9 0.14,71.02,56.58,56.48,40.97,33.61,27.80,26.74,21.12.HRMS(ESI)m / z calcd for[C 38 H 32 N2O 12 S+H] + ,741.1749,found 741.1732.

[0097] Example 8

[0098]

[0099] Step 1: 4-isopropylphenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22h as a yellow solid (346 mg) in 85% yield.

[0100] Step 2: Substituting 22a in Example 1 with 22h (0.48 mmol, 1 eq), compound 23h was prepared according to the synthesis method of Example 1 as a yellow solid (173 mg) in an 86% yield.

[0101] Step 3: Substituting 23h for 23a in Example 1, compound 33h was prepared according to the synthesis method of Example 1 as a yellow solid (89 mg) in a yield of 75%. 1 H NMR (500MHz, CDCl3) δ8.05(d,J=8.0,2H,Ar-H),7.77(d,J=8.5Hz,2H,Ar-H),7.72(t,J=7.5Hz,1H),7.59(t,J= 7.8Hz,2H,Ar-H),7.53(d,J=7.9Hz,2H),7.34(d,J=7.9Hz,2H),7.05(d,J=8.5Hz,2H,Ar-H),6.72(s,1H,C=CH), 6.26(s,1H,Ar-H),4.48(t,J=6.0Hz,2H,OCH2),4.06(s,3H,OCH3),3.94(s,3H,OCH3),2.99(hept,J=7.0Hz,1H, CH),2.70(t,J=7.1Hz,2H,CH2),2.09-1.99(m,2H,CH2),1.95(q,J=7.5Hz,2H,CH2),1.32(d,J=6.9Hz,6H,CH3). 13 C NMR (125MHz, CDCl3) δ181.10,171.32,165.22,164.78,158.95,158.80,1 51.01,148.22,147.75,138.05,135.66,132.32,130.50,130.44,129.72 ,128.53,127.92,126.07,121.86,110.47,109.38,107.85,105.01,90.0 7,71.00,56.49,56.38,33.96,33.63,27.80,24.06,21.14.HRMS(ESI)m / z calcd for[C39 H 36 N2O 11 S+H] + ,741.2113,found 741.2095.

[0102] Example 10

[0103]

[0104] Step 1: 4-Methoxycarbonylphenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22j as a yellow solid (346 mg) in a yield of 74%.

[0105] Step 2: 22a in Example 1 was replaced with 22j (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23j as a yellow solid in an 85% yield.

[0106] Step 3: Compound 33j was prepared by replacing compound 23a in Example 1 with compound 23j according to the synthesis method of Example 1 as a yellow solid (95 mg) with a yield of 78.5%. 1 H NMR (400MHz, CDCl3) δ8.14 (d, J=8.0Hz, 2H, Ar-H), 8.08-8.04 (m, 2H, Ar-H), 7.74 (dd, J=9.5, 2.6Hz, 2H, Ar- H),7.69(dd,J=7.3,5.7Hz,2H,Ar-H),7.62-7.56(m,2H,Ar-H),7.10-7.05(d,J=8.5Hz,2H,Ar-H),6.74(s,1 H,C=CH),6.27(s,1H,Ar-H),4.48(t,J=6.0Hz,2H,OCH2),4.07(s,3H,OCH3),3.96(s,3H,OCH3),3.94(s,4H ,OCH3),2.69(t,J=7.1Hz,2H,CH2),2.01(qd,J=6.4,5.8,4.2Hz,2H,CH2),1.94(qd,J=7.0,3.5Hz,2H,CH2). 13C NMR (100MHz, CDCl3) δ180.75,171.30,167.02,165.12,164.71,159.49,1 58.94,151.17,147.50,138.07,135.82,135.65,132.22,130.61,130.26, 129.71,129.19,129.06,128.53,122.06,110.46,109.85,106.82,105.1 1,90.13,71.01,56.57,56.47,52.21,33.61,27.81,21.12.HRMS(ESI)m / z calcd for[C 38 H 32 N2O 13 S+H] + ,757.1698,found757.1691.

[0107] Example 11

[0108]

[0109] Step 1: 4-Acetamidophenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22k as a yellow solid (346 mg) in 85% yield.

[0110] Step 2: 22a in Example 1 was replaced with 22k (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23k as a yellow solid in an 80% yield.

[0111] Step 3: Compound 33k was prepared as a yellow solid (83 mg) in 68.6% yield by replacing 23a in Example 1 with 23k according to the synthesis method of Example 1. 1H NMR(500MHz,DMSO-d6)δ10.05(s,1H),8.02(d,J=7.8Hz,2H),7.85(td,J=11.5,8.6,5 .2Hz,3H),7.74-7.68(m,4H),7.50(dd,J=8.3,3.0Hz,2H),7.13(d,J=7.6Hz,2H),6.73 (d,J=3.1Hz,1H),6.57(d,J=3.1Hz,1H),4.44(q,J=5.1Hz,2H),4.01(s,3H),3.95(s, 3H), 2.69 (d, J = 8.0Hz, 2H), 2.07 (s, 3H), 1.86 (q, J = 7.0Hz, 2H), 1.76 (d, J = 7.9Hz, 3H). 13 C NMR (125MHz, DMSO-d6) δ179.80,171.90,168.80,165.63,164.27,159.34,158.92,151.42,147.68,138.97,137.70,136.57,132.35,131.17,13 0.47,128.76,125.41,122.68,118.98,110.95,108.66,107.01,104.30 ,92.02,71.61,57.40,56.85,33.29,27.63,24.50,20.99.HRMS(ESI)m / z calcd for[C 38 H 33 N3O 12 S+H] + ,756.1858,found 756.1849.

[0112] Example 12

[0113]

[0114] Step 1: 4-(Cyclopropylcarbamoyl)phenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 221 as a yellow solid (346 mg) in 85% yield.

[0115] Step 2: 22a in Example 1 was replaced with 22l (0.48 mmol, 1 eq) and the compound 23l was prepared according to the synthesis method of Example 1 to obtain 173 mg of a yellow solid in an 82% yield.

[0116] Step 3: Compound 331 was prepared as a yellow solid (87 mg) in a yield of 69.6% by replacing compound 23a in Example 1 with compound 231 according to the synthesis method of Example 1. 1 H NMR (500MHz, CDCl3) δ8.06(d,J=7.9Hz,2H,Ar-H),7.84(d,J=8.1Hz,2H,Ar-H),7.73(dd,J=8.1,4.9Hz,3H,Ar-H),7.64(d,J=8. 0Hz,2H,Ar-H),7.60(t,J=7.8Hz,2H,Ar-H),7.08(d,J=8.4Hz,2H,Ar-H),6.74(s,1H,C=CH),6.25(s,1H,Ar-H),4.48(dt,J=15. 6,6.1Hz,2H,OCH2),4.07(s,3H,OCH3),3.92(s,3H,OCH3),2.94(tt,J=7.3,3.7Hz,1H,NCH),2.70(t,J=7.2Hz,2H,CH2),2.03(d q,J=11.2,6.2Hz,2H,CH2),1.95(dq,J=14.9,7.3Hz,2H,CH2),0.89(p,J=7.5,7.0Hz,2H,CH2),0.67(dd,J=6.5,4.1Hz,2H,CH2). 13 C NMR (125MHz, CDCl3) δ180.80,171.37,168.64,165.09,164.67,159.39,1 58.94,151.14,147.52,138.03,135.67,133.21,132.21,130.75,130.26 ,129.72,128.54,126.50,122.09,110.47,109.85,106.77,105.09,90.0 6,71.03,56.53,56.46,33.62,27.81,23.22,21.15,6.85.HRMS(ESI)m / z calcd for[C 40 H 35 N3O 12 S+H] + ,782.2014,found 782.2008.

[0117] Example 14

[0118]

[0119] Step 1: 4-ethylphenylboronic acid (0.94 mmol, 1.2 eq) was used instead of 4-methylaminophenylboronic acid in Example 1. The remaining steps were carried out according to the synthesis method of Example 1 to prepare compound 22n as a yellow solid (346 mg) with a yield of 87%.

[0120] Step 2: Substituting 22a in Example 1 with 22n (0.48 mmol, 1 eq), the compound 23n was prepared according to the synthesis method of Example 1 to obtain 173 mg of a yellow solid in an 82% yield.

[0121] Step 3: Compound 33n was prepared as a yellow solid (86 mg) in 74% yield by replacing 23a in Example 1 with 23n according to the synthesis method of Example 1. 1 H NMR (500MHz, CDCl3) δ8.01(d,J=7.9Hz,2H,Ar-H),7.73(d,J=8.2Hz,2H,Ar-H),7.68(t,J=7.5Hz,1H,Ar-H),7. 55(t,J=7.8Hz,2H,Ar-H),7.47(d,J=7.7Hz,2H,Ar-H),7.27(d,J=7.7Hz,2H,Ar-H),7.02(d,J=8.2Hz,2H,Ar-H ),6.67(s,1H,C=CH),6.22(s,1H,Ar-H),4.44(t,J=6.2Hz,2H,OCH2),4.01(s,3H,OCH3),3.89(s,3H,OCH3),2. 70-2.64(m,4H,CH2),1.97(t,J=7.4Hz,2H,CH2),1.90(d,J=7.9Hz,2H,CH2),1.26(td,J=7.6,2.1Hz,3H,CH3). 13 C NMR (125MHz, CDCl3) δ181.10,171.35,165.27,164.73,158.95,158.80,1 51.01,147.75,143.63,138.01,135.70,132.29,130.48,130.45,129.73 ,128.52,127.79,127.52,121.91,110.47,109.37,107.82,104.97,90.0 8,71.03,56.51,56.37,33.63,28.74,27.79,21.13,15.58.HRMS(ESI)m / z calcd for[C 38 H 34 N2O 11 S+H] +,727.1956,found 727.1949.

[0122] Example 15

[0123]

[0124] Step 1: 4-Methylaminophenylboronic acid in Example 1 was replaced with 4-pentylphenylboronic acid (0.94 mmol, 1.2 eq). The remaining steps were followed by the synthesis method of Example 1 to obtain compound 22o as a yellow solid (346 mg) in 87% yield.

[0125] Step 2: 22a in Example 1 was replaced with 22o (0.48 mmol, 1 eq) and the synthetic method of Example 1 was followed to obtain 173 mg of compound 23o as a yellow solid in an 86% yield.

[0126] Step 3: Compound 33o was prepared by replacing 23a in Example 1 with 23o according to the synthesis method of Example 1 as a yellow solid (91 mg) in a yield of 74%. 1 H NMR (500MHz, DMSO-d6) δ8.02(d,J=7.9Hz,2H,Ar-H),7.85(t,J=7.5Hz,1H,Ar-H),7.80(d,J=8.3Hz,2H,Ar-H),7.71(t,J=7.7Hz,2H,Ar-H ),7.41(d,J=7.7Hz,2H,Ar-H),7.24(d,J=7.8Hz,2H,Ar-H),7.09(d,J=8.3Hz,2H,Ar-H),6.69(s,1H,C=CH),6.51(s,1H,Ar-H),4.43(t,J= 6.1Hz,2H,OCH2),3.99(s,3H,OCH3),3.95(s,3H,OCH3),2.67(t,J=7.3Hz,2H,CH2),2.58(t,J=7.6Hz,2H,CH2),1.85(dt,J=11.9,6.1Hz,2 H,CH2),1.75(p,J=7.6Hz,2H,CH2),1.58(p,J=7.4Hz,2H,CH2),1.27(dtd,J=14.2,10.4,9.7,4.3Hz,4H,CH2),0.83(t,J=6.8Hz,3H,CH3). 13C NMR(125MHz,DMSO-d6)δ208.70,207.88,184.46,182.23,179.77,171.84,165.47,164.24 ,159.32,158.90,153.44,151.33,147.66,141.92,137.68,136.56,132.34,130.69,130. 45,130.42,128.76,128.26,128.16,122.59,110.92,108.56,107.09,104.22,91.80,71. 56,57.25,56.74,35.36,33.26,31.26,31.11,27.60,22.47,21.02,14.34.HRMS(ESI)m / z calcd for[C 41 H 40 N2O 11 S+H] + ,769.2426,found 769.2435. Example 17 Biological Experiment

[0127] The compounds were evaluated for their inhibitory effects on the proliferation of human renal cancer cell lines Caki-1, human gastric cancer cell lines SNU-5, human lung cancer cells (NCI-H441, NCI-H1975, A549), human colon cancer cell lines HCT 116, and human normal liver cell lines L-02.

[0128] Purpose of the experiment:

[0129] The CCK-8 assay was used to determine the inhibitory effect of the compounds on the proliferation of human renal cancer cells Caki-1, human gastric cancer cells SNU-5, human lung cancer cells (NCI-H441, NCI-H1975, A549), human colon cancer cells HCT 116, and human normal liver cells L-02.

[0130] Experimental methods:

[0131] In vitro antitumor activity screening was performed using the CCK-8 assay. Cells in the logarithmic growth phase were obtained and washed twice with PBS to remove dead cells and residual culture medium. 1 mL of trypsin was added for digestion. After digestion, 2 mL of complete culture medium for the corresponding cell type was added to terminate digestion and resuspend the cells. The cells were transferred to a 15-mL centrifuge tube and centrifuged at 1000 r / min for 3 minutes. The supernatant was removed, and 4 mL of complete culture medium was added to mix thoroughly by pipetting. After counting, the cell density was adjusted to the appropriate concentration (HepG2: 6000 / well, Caki-1: 6000 / well, SNU-5: 7000 / well, NCI-H441: 7000 / well, NCI-H1975: 6000 / well, A549: 7000 / well, L-02: 7000 / well, HCT 116: 6000 / well) and seeded into 96-well plates at 100 μL per well. The cells were incubated overnight in a cell culture incubator at 37°C and 5% CO2. The culture medium was then replaced with a medium containing the target compound at the desired concentration and cultured in a cell culture incubator at 37°C and 5% CO2 for 72 hours. CCK-8 solution was then added at a final concentration of 10% (v / v) and incubated at 37°C and 5% CO2 for 0.5-2 hours. The absorbance of each well was then measured at a wavelength of 450 nm using a microplate reader. The half inhibition rate (IC) was calculated. 50 .

[0132] Data processing:

[0133] The cell proliferation inhibition rate (IR) of each compound was calculated based on the measured absorbance value. The calculation formula is as follows:

[0134] IR%=[AC-AD] / [AC-AB]×100%

[0135] AB: blank group, absorbance of wells containing 0.5% DMSO, CCK-8, and cell-free culture medium;

[0136] AD: experimental group, absorbance of wells containing 0.5% DMSO, CCK-8, drugs, and culture medium with cells;

[0137] The experimental group included two positive control groups, and the positive drugs were APG and 15c.

[0138] The structural formulas of APG and 15c are

[0139] AC: control group, absorbance of wells containing 0.5% DMSO, CCK-8, and culture medium with cells;

[0140] Graph Pad Prism 5 software was used to analyze the data and plot the graphs, and the half-maximal inhibitory concentration (IC 50 ). The experimental results are shown in Table 1.

[0141] Table 1 Cytotoxicity of compounds 33a-p

[0142]

[0143] Data are expressed as mean ± SD, n = 3, drug group vs blank group

[0144] Pharmacological experiments demonstrated that the target derivatives of the present invention exhibited excellent antitumor activity, particularly against renal cancer Caki-1 cells and colon cancer cells, demonstrating significantly superior antiproliferative activity to APG and 15c. They also exhibited selectivity for tumor cells over normal cells and could be used to further prepare antitumor drugs. In human gastric cancer cells SNU-5, compounds 33a, 33b, 33c, 33d, 33f, 33g, 33k, 33l, and 33o exhibited superior antiproliferative activity to APG. In human lung cancer cells NCI-H441, compounds 33a, 33b, 33c, 33d, 33e, 33f, 33g, 33h, 33j, 33k, 33l, 33n, and 33o exhibited superior antiproliferative activity to APG. In NCI-H1975 cells, compounds 33b and 33c exhibited superior antiproliferative activity to APG. In A549 cells, compound 33a exhibited superior antiproliferative activity to APG.

[0145] Example 18 Transplanted Tumor Experiment

[0146] Based on the in vitro cytotoxicity evaluation, compound 33d was selected for further evaluation of its therapeutic effect against renal cancer Caki-1 in vivo.

[0147] Experimental methods

[0148] Caki-1 cells were cultured at a rate of 1×10 7 The number of cells / mouse was suspended in 50 μL of Matrigel and 50 μL of DMEM serum-free medium and inoculated into the subcutaneous tissue of the right back of nude mice by subcutaneous injection. 3 The animals were randomly divided into four groups. Each mouse was intraperitoneally injected with 15 mg / kg and 5 mg / kg of APG (15 mg / kg) and the same volume of vehicle (5% DMSO and 20% PEG 400 in normal saline) once a day for 33 days. The tumor volume and body weight of the nude mice were measured every other day. The formula 1 / 2ab was used to calculate the nude mouse tumor volume and body weight. 2 (a: tumor long diameter, mm; b: tumor short diameter, mm) Tumor volume was calculated. After 18 consecutive days of administration, the tumors were removed, photographed, and weighed.

[0149] The results are as follows Figure 1 As shown, Figure 1 In A, B and C, compared with the vehicle control group, 15 mg / kg of 33d significantly inhibited tumor growth, while 5 mg / kg of 33d and 15 mg / kg of APG had no significant inhibitory effect. Figure 1 D, 33d (15 mg / kg) showed significant antitumor activity with an inhibition rate of 58.3% (w / w), which was much higher than 5 mg / kg 33d (8.82%) and 15 mg / kg APG (13.68%). On the other hand, there was no significant change in the body weight of mice treated with 5 mg / kg 33d and 15 mg / kg APG, while 33d at a dose of 15 mg / kg caused a very slight and insignificant change in the body weight of nude mice (e.g. Figure 1 E), but weight returned to normal after drug cessation. Therefore, it can be concluded that compound 33d has an effective therapeutic effect on Caki-1 renal cancer tumors in vivo. This indicates that the compound of the present invention has excellent antitumor activity and good potential for treating renal cancer.

[0150] Example 19 33d inhibits the phosphorylation of AKT and ERK in Caki-1 cells

[0151] In order to elucidate the mechanism by which 33d inhibits the proliferation and migration of Caki-1 cells and induces apoptosis of Caki-1 cells, 33d was selected to detect its regulatory effect on AKT and ERK signaling in Caki-1 cells.

[0152] Experimental methods

[0153] Caki-1 cells in the logarithmic growth phase were collected and plated at 2×10 5Cells were seeded into 6-well plates at a density of 100 cells / well and cultured overnight in an incubator at 37°C with 5% CO2. The medium was then discarded, the cells were washed once with PBS, and complete medium containing the target concentration of 33d or a DMSO vehicle control was added. The cells were incubated in an incubator for 24 hours. The medium was discarded, the cells were washed with PBS, and the cells were rapidly lysed with lysis buffer. The cells were centrifuged at 4°C for 20 minutes, and the supernatant was transferred to a fresh EP tube. Protein content was then determined using a BCA assay, with 30 μg of protein per well being quantified. SDS-PAGE was performed at 100 V for 110 minutes. The gel was then transferred to a polyvinylidene membrane at 110 V and blocked with 5% BSA / PBST. After 1 hour, the membrane was incubated with primary antibody diluted in 5% BSA / PBST overnight at 4°C. The primary antibody was recovered and the cells were washed three times with PBST every 10 minutes. The membrane was then incubated with secondary antibodies at room temperature for 1 hour and washed three times with PBST solution every 10 minutes. The membrane was then developed and photographed using a developer under a chemiluminescence system, and the grayscale value was quantified using Image J.

[0154] The results are as follows Figure 2 The results showed that although 33d did not change the expression levels of AKT and ERK in Caki-1 cells, it could downregulate the phosphorylation levels of AKT and ERK in a dose-dependent manner, further indicating that 33d may exert anti-proliferation, migration and induce cell apoptosis by affecting Akt and Erk.

[0155] Example 20 Effect of 33d on c-Met in Caki-1 cells

[0156] To verify whether the structurally modified NO-donating apigenin derivatives exert their anti-Caki-1 effect by inhibiting the phosphorylation of MET, we studied the effect of 33d on MET protein expression by western blotting experiments.

[0157] Experimental methods

[0158] Caki-1 cells in the logarithmic growth phase were collected and plated at 2×10 5Cells were seeded into 6-well plates at a density of 100 cells / well and cultured overnight in an incubator at 37°C with 5% CO2. The medium was then discarded, the cells were washed once with PBS, and complete medium containing the target concentration of 33d or a DMSO vehicle control was added. The cells were incubated in an incubator for 24 hours. The medium was discarded, the cells were washed with PBS, and the cells were rapidly lysed with lysis buffer. The cells were centrifuged at 4°C for 20 minutes, and the supernatant was transferred to a fresh EP tube. Protein content was then determined using a BCA assay, with 30 μg of protein per well being quantified. SDS-PAGE was performed at 100 V for 110 minutes. The gel was then transferred to a polyvinylidene membrane at 110 V and blocked with 5% BSA / PBST. After 1 hour, the membrane was incubated with primary antibody diluted in 5% BSA / PBST overnight at 4°C. The primary antibody was recovered and the cells were washed three times with PBST every 10 minutes. The membrane was then incubated with secondary antibodies at room temperature for 1 hour and washed three times with PBST solution every 10 minutes. The membrane was then developed and photographed using a developer under a chemiluminescence system, and the grayscale value was quantified using Image J.

[0159] The experimental results are as follows Figure 3 The results showed that the total MET level in Caki-1 cells treated for 33 days did not change significantly, but the phosphorylation level of MET was downregulated, indicating that 33 days may have an inhibitory effect on the phosphorylation level of MET.

[0160] The contents described in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications to the embodiments made by those skilled in the art fall within the scope defined by the claims attached to the present invention.

Claims

1. An apigenin derivative, characterized in that The structure is as follows: Wherein, R1 is selected from: CONH-R3, five-membered nitrogen-containing heterocycle, six-membered nitrogen-containing heterocycle, NR4R5, COR7, C1-C6 alkyl; R3 is selected from the group consisting of: H, D, C1-C3 alkyl, cyclopropane, cyclobutane, cyclopentane; R4 and R5 are independently selected from: H, D, C1-C3 alkyl, COR6; R6 is selected from the group consisting of: H, D, C1-C3 alkyl; R7 is selected from: H, D, C1-C3 alkyl; R2 is selected from: C1-C5 alkylene; The five-membered nitrogen-containing heterocyclic ring is The six-membered nitrogen-containing heterocyclic ring is 2. An apigenin derivative, characterized in that The apigenin derivative has the following structure:

3. The synthetic route of the apigenin derivative according to claim 1 or 2, characterized in that: The synthetic route comprises: (1) Synthesis of intermediate products: Methylation of 2,4,6-trihydroxyacetophenone gave intermediate 18; Intermediate 18 reacts with iodine to give compound 19, which is then converted to chalcone intermediate 20 via aldol condensation. Intermediate 20 is cyclized to give compound 21; then Suzuki coupling reaction is performed to generate 22a-q, and finally deprotection reaction is performed to generate intermediate 23a-q; (2) Synthesis of products: Phenylthioacetic acid 29 was catalyzed by hydrogen peroxide and fuming nitric acid to generate 3,4-diphenylsulfonylfuran 30; 3,4-diphenylsulfonylfuran 30 was linked to pentanediol, and the linker arm was oxidized to give 32; 32 was ligated with the 4′-OH site of intermediate 23a-q to obtain NO-donating apigenin derivatives 33a-p.

4. The use of the apigenin derivative according to claim 1 or 2 in the preparation of anti-tumor drugs, characterized in that: The tumors are kidney cancer and lung cancer.

5. Use of an apigenin derivative in the preparation of an anti-gastric cancer drug, characterized in that: The structure of the apigenin derivative is shown below: wherein R1 is selected from the group consisting of: CONH-R3, a five-membered nitrogen-containing heterocycle, a six-membered nitrogen-containing heterocycle, NR4R5, and COR7; R3 is selected from the group consisting of: H, D, C1-C3 alkyl, cyclopropane, cyclobutane, cyclopentane; R4 and R5 are independently selected from: H, D, COR6; R6 is selected from the group consisting of: H, D, C1-C3 alkyl; R7 is selected from the group consisting of: H, D, C1-C3 alkyl; R2 is selected from: C1-C5 alkylene; The five-membered nitrogen-containing heterocyclic ring is The six-membered nitrogen-containing heterocyclic ring is 6. Use of an apigenin derivative in the preparation of an anti-gastric cancer drug, characterized in that: The structure of the apigenin derivative is shown below:

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