Polymethoxy homoisoflavone derivative as well as pharmaceutical composition and application thereof

Abstract

The invention relates to a polymethoxy homoisoflavone derivative or a pharmaceutically acceptable salt thereof, which is characterized in that the structural formula of the derivative is as shown in a general formula (I) or a general formula (II), and the invention also relates to a corresponding pharmaceutical composition and application thereof. The polymethoxy homoisoflavone compound which accords with the general formula and is high in anti-tumor activity is synthesized by the method.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, particularly to the field of pharmaceutical chemical synthesis, and specifically refers to a polymethoxy high isoflavone derivative, its pharmaceutical composition and its application. Background Art

[0002] Currently, the main means of treating tumors clinically are surgery, radiotherapy and chemotherapy. Although it can extend the survival period of patients to a certain extent, its toxic and side effects make the quality of life of patients relatively poor. Traditional Chinese medicine has a unique advantage in anti-tumor because it can regulate the body's own immunity and has few adverse reactions.

[0003] Citrus has the effects of regulating qi and strengthening the spleen, and drying dampness and resolving phlegm. Citrus flavonoids (polymethoxyflavones) are mainly present in the outer skin of citrus fruits and have multiple biological functions, such as anti-inflammatory and antibacterial, antioxidant, hypoglycemic, hypolipidemic, anti-tumor and immune enhancement. It is safe and non-toxic and has been widely used in medicine, food processing and other aspects. And more and more studies show that citrus flavonoids have good anti-tumor activity, including lymphocytic leukemia, liver cancer, gastric cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, pancreatic cancer, etc. The anti-tumor mechanism mainly includes directly inhibiting the proliferation of tumor cells, inducing apoptosis of tumor cells, inhibiting the metastasis of cancer cells, inhibiting angiogenesis, etc.

[0004] Polymethoxyflavones (PMFs) are a class of flavonoid components with multiple methoxy groups, low polarity, planar structure and strong biological activity. In 1991, the European Committee for Standardization defined PMFs as a class of flavonoids with a methoxy group number ≥ 4. The currently discovered PMFs components mainly include tangeretin, nobiletin, sinensetin, etc. and their derivatives. The structural formulas of tangeretin, nobiletin and sinensetin are shown as follows:

[0005]

[0006] Naturally occurring polymethoxyflavonoid compounds have certain disadvantages in anti-tumor: lack of strong specificity, generally low activity, etc. Taking the molecular skeleton of polymethoxyflavonoid compounds as a structural model, through structural modification and transformation, highly active molecules have attracted the attention of many researchers. The polymethoxy A-ring structure of citrus flavonoids is relatively unique among natural product flavonoids. Currently, only this type of compound has this structure. Therefore, the A-ring polymethoxy structure type is retained in the later new drug design.

[0007] Homoisoflavonoids are a subclass of flavonoid natural products. Compared with flavones and isoflavones, the number of homoisoflavonoid natural products is relatively small. The 16-carbon skeleton in their molecular structure has one more carbon (C9) than that of flavones and isoflavones, and includes two aromatic rings and one oxygen-containing heterocyclic ring. The structural formulas of flavones, isoflavones, and homoisoflavones are as follows:

[0008]

[0009] The structures of such natural products are not planar structures, and their water solubility is greater than that of flavonoid compounds. Summary of the Invention

[0010] The main object of the present invention is to provide a polymethoxy homoisoflavone derivative with high anti-tumor activity, its pharmaceutical composition, and its application, aiming at the above existing problems.

[0011] To achieve the above object, in a first aspect of the present invention, there is provided a polymethoxy homoisoflavone derivative or a pharmaceutically acceptable salt thereof, wherein the structural formula of the derivative is shown as general formula (I) or general formula (II),

[0012]

[0013] wherein, R1 and R2 are each independently selected from methoxy and hydrogen, provided that R1 and R2 are not methoxy at the same time;

[0014] R3 is hydrogen, methoxy, -CH2-R 10 ;

[0015] R4 and R5 are each independently selected from hydrogen, substituted or unsubstituted methoxy, hydroxyl, halogen atom, amino, nitro, or R4 and R5 form a five-membered ring

[0016] R6 is hydrogen, substituted or unsubstituted methoxy, hydroxyl, halogen atom, amino, nitro, -CH2-R 10 ;

[0017] R7, R8, and R9 are each independently selected from hydrogen, substituted or unsubstituted methoxy, hydroxyl, halogen atom, amino, nitro, or R7 and R8 form a five-membered ring or, R8 and R9 form a five-membered ring

[0018] The substituent of the substituted methoxy is a halogen;

[0019] If R5 is methoxy, R4 is hydroxyl, and R3 is -CH2-R 10 ;

[0020] If R4 is methoxy, R5 is hydroxyl, and R6 is -CH2-R 10 ;

[0021] R 10 is a substituted secondary amine, pyrrolidinyl, morpholinyl, piperidinyl, a substituted or 4-substituted piperazinyl, a piperidinyl substituted by a hydroxyl group or a C1-C5 hydroxy-substituted alkyl group, and the substituent of the substituted secondary amine is selected from C1-C4 alkyl groups, substituted or unsubstituted benzyl groups. The substituent at the 4-position of the substituted piperazinyl is selected from C1-C12 alkyl groups, C1-C12 hydroxy-substituted alkyl groups, substituted or unsubstituted benzyl groups, and tert-butoxycarbonyl groups. The C1-C5 positions on the benzene ring of the substituted benzyl group have 1 to 5 substituents, and the 1 to 5 substituents are each independently selected from methoxy, hydroxyl, halogen atoms, amino groups, nitro groups, trifluoromethoxy groups, or two adjacent substituents among the 1 to 5 substituents form a five-membered ring

[0022] Preferably, the pharmaceutically acceptable salt is selected from the acid salts of polymethoxy isoflavone derivatives.

[0023] Preferably, the pharmaceutically acceptable salt is selected from the organic acid salts or inorganic acid salts of polymethoxy isoflavone derivatives.

[0024] Preferably, the inorganic acid salts are selected from the hydrochloride, hydrobromide, sulfate, and phosphate of polymethoxy isoflavone derivatives; the organic acid salts are selected from the oxalate, acetate, citrate, maleate, benzoate, malate, toluenesulfonate, fumarate, or tartrate of polymethoxy isoflavone derivatives.

[0025] Preferably, the derivatives are selected from the following compounds:

[0026] Compound 1-1

[0027]

[0028] Compound 1-2

[0029]

[0030] Compound 1-3

[0031]

[0032] Compound 1-4

[0033]

[0034] Compound 1-5

[0035]

[0036] Compound 1-6

[0037]

[0038] Compound 1-7

[0039]

[0040] Compound 1-8

[0041]

[0042] Compound 1-9

[0043]

[0044] Compound 1-10

[0045]

[0046] Compound 1-11

[0047]

[0048] Compound 2-1

[0049]

[0050] Compound 2-2

[0051]

[0052] Compound 2-3

[0053]

[0054] Compound 2-4

[0055]

[0056] Compound 2-5

[0057]

[0058] Compound 2-6

[0059]

[0060] The second aspect of the present invention provides a pharmaceutical composition comprising the polymethoxy isoflavone derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and / or excipient.

[0061] Preferably, the dosage form of the pharmaceutical composition is selected from capsules, granules, injections, tablets, aerosols, oral liquids or sustained release agents.

[0062] The third aspect of the present invention provides the use of the polymethoxy high isoflavone derivative or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of an antitumor drug.

[0063] Preferably, the tumor is: leukemia, skin cancer, breast cancer, lung cancer, liver cancer, colorectal cancer.

[0064] Preferably, the tumor is melanoma.

[0065] Melanoma accounts for less than 5% of all skin cancers, but is the main cause of skin cancer deaths. Melanoma has attracted increasing attention. However, the uncertainty of its targets has led to the inability of its therapeutic drugs to delay the progression of the disease, and only symptomatic treatment is available. After a large number of literature investigations, citrus flavonoids have the characteristics of simple synthesis and good activity and can be used as multi-target drugs, which have attracted our research interest. The B ring of polymethoxy citrus flavonoids has various polymethoxy forms. Among them, in order to simplify the synthesis and save costs, we selected the flavone structure with 6,7,8-trimethoxy structure as the lead compound for structure optimization, hoping to obtain flavonoid compounds with novel structures and further improved activities. According to the synthesis of three compound libraries and the activity tests of 48 compounds, the polymethoxy high isoflavone structure has strong activity in inhibiting melanoma A375 cells. Through cell cycle experiments, it was found that such compounds have obvious arrest on the G2 / M phase of the melanoma cell cycle. Animal experiments showed that compound FD-A6 could significantly inhibit the growth of tumors in tumor-bearing mice. Finally, it can be seen from the results of the tubulin polymerization experiment that polymethoxy high isoflavone compounds inhibit tubulin polymerization. Based on the above experiments, the high isoflavone FD-A6 compound showed the most prominent performance. Description of the Drawings

[0066] Figures 1 to 4 It is a graph showing the experimental results of CCK-8 cell proliferation activity determination in Example 4 of the present invention. Specifically, Figure 1 It is a bar graph of the cell survival rate of compound library A; Figure 2 It is a bar graph of the cell survival rate of compound library B; Figure 3 It is a bar graph of the cell survival rate of compound library C; Figure 4 It is the survival rate and IC of A375 cells treated with high isoflavone FD-A6 for 24 h and 48 h 50 Result graph.

[0067] Figure 5 It is a graph showing the experimental results of the cell cycle in Example 5 of the present invention. Figure 6 It is a bar graph of the A375 cell cycle.

[0068] Figure 7 A to Figure 7 D are graphs showing the experimental results of animal experiments in Example 6 of the present invention.

[0069] Figure 8 This is the experimental result graph of tubulin polymerization in Example 7 of the present invention. Detailed implementation mode

[0070] In order to more clearly understand the technical content of the present invention, the following specific examples are given for detailed description.

[0071] Unless otherwise specified, the reagents and methods involved in the examples are common reagents and methods in the art.

[0072] Example 1

[0073] The structural information of the A series of compounds in the polymethoxy isoflavone derivative library is shown in Table 1 below.

[0074] Table 1 A series of compounds in the polymethoxy isoflavone derivative library

[0075]

[0076] The synthesis methods of compounds FD-A1 to FD-A12 are as follows:

[0077]

[0078] (1) Weigh 10 mmol of compound 3 and put it into a 100 mL round-bottom flask, add 25 mL of toluene to dissolve it, dropwise add DMF-DMA (1.56 mL, 12 mmol), then heat it to reflux for 10 h. First cool it to room temperature and then place it in an ice bath, and then slowly drop 2 mL of concentrated HCl, stir for 15 min, remove the ice bath and then heat it to 50 °C for 4 h. Extract it with ethyl acetate three times, combine the solvents, and remove the solvent under reduced pressure to obtain the crude product of compound 10. Without purification, it can be directly used for the next step of the reaction.

[0079] (2) Dissolve 10 mmol of compound 10 in 25 mL of alcohol, add a catalytic amount of 10% Pd / C (50 mg / mmol) and 3 times the amount of ammonium formate (1.89 g) at room temperature, and then slowly heat it to reflux for 4 h. After the reaction is complete, filter it, add an appropriate amount of water, extract it with ethyl acetate three times, combine the solvents, and purify it through a silica gel column to obtain compound 11. The yield of the two steps is 83%.

[0080] (3) Weigh 1 mmol of compound 11 and dissolve it in 25 mL of benzene, add a catalytic amount of p-TsOH (50 mg / mmol) and the corresponding benzaldehyde (1.1 mmol) at room temperature, and then slowly heat it to reflux for 6 h. After the reaction is complete, add an appropriate amount of water, extract it with ethyl acetate three times, combine the solvents, and purify it through a silica gel column to obtain FD-A1 to FD-A7, FD-A9, FD-A11 to FD-A12.

[0081] (4) In a dry reaction flask, add reducing iron powder Fe (0.115 g, 2 mmol), then add a mixed solution of 3.5 mL ethanol and water (EtOH:H2O = 2.5:1, V:V), and finally add FD-A7 (0.2 g, 0.5 mmol) and 0.2 mL of 37% concentrated hydrochloric acid. Heat under reflux slowly for 4 h. After the reaction is complete, extract with ethyl acetate three times and purify by silica gel column to obtain the product FD-A8.

[0082] Compound FD-A1: Yellow solid; Yield 64% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.48 - 7.40 (m, 1H), 7.32 (d, J = 7.1 Hz, 1H), 7.25 (d, J = 13.1 Hz, 1H), 5.38 (s, 1H), 4.01 (s, 3H), 3.89 (s, 6H). HRMS (ESI): Calcd for C 19 H 18 O5 [M + H] + 327.1232; Found: 327.1237.

[0083] Compound FD-A2: Yellow solid; Yield 65% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.25 (s, 1H), 6.96 - 6.89 (m, 2H), 6.87 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H). HRMS (ESI): Calcd for C 21 H 22 O7 [M + H] + 387.1444; Found: 387.1447.

[0084] Compound FD-A3: Yellow solid; Yield 60% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (d, J = 4.4 Hz, 1H), 6.97 - 6.76 (m, 3H), 6.04 (s, 2H), 5.38 (s, 2H), 4.01 (s, 3H), 3.89 (s, 6H). HRMS (ESI): Calcd for C 20 H 18 O7 [M + H] + 371.1131; Found: 371.1130.

[0085] Compound FD-A4: Yellow solid; yield 63% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.27 (d, J = 12.0 Hz, 1H), 6.54 (s, 2H), 5.44 (s, 2H), 4.02 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H), 3.90 (s, 9H). HRMS (ESI): Calcd for C 22 H 24 O8[M + H] + 416.1471; Found: 416.1475.

[0086] Compound FD-A5: Yellow solid; yield 59% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.27 (s, 1H), 6.99 (d, J = 8.6 Hz, 1H), 6.86 (d, J = 7.2 Hz, 2H), 5.96 (s, 1H), 5.43 (s, 2H), 4.01 (s, 3H), 3.93 (s, 3H), 3.90 (s, 6H). HRMS (ESI): Calcd for C 20 H 20 O7[M + H] + 373.1287; Found: 373.1285.

[0087] Compound FD-A6: Yellow solid; yield 60% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.27 (s, 1H), 6.92 (dd, J = 4.9, 3.3 Hz, 2H), 6.87 (dd, J = 8.7, 1.7 Hz, 1H), 5.74 (s, 1H), 5.41 (s, 2H), 4.01 (s, 3H), 3.95 (s, 3H), 3.89 (s, 6H). HRMS (ESI): Calcd for C 20 H 20 O7[M + H] + 373.1287; Found: 373.1288.

[0088] Compound FD-A7: Yellow solid; yield 57% (three-step reaction); 11H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 2.1 Hz, 1H), 7.76 (s, 1H), 7.53 (dd, J = 8.7, 2.2 Hz, 1H), 7.25 (s, 1H), 7.18 (d, J = 8.7 Hz, 1H), 5.37 (s, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 3.90 (s, 6H). HRMS (ESI): Calcd for C 20 H 19 NO8[M + H] + 402.1189; Found: 402.1188.

[0089] Compound FD-A8: Yellow solid; Yield 25% (four-step reaction); 1 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.80 - 6.75 (m, 1H), 6.74 - 6.70 (m, 2H), 5.42 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.89 (s, 6H). HRMS (ESI): Calcd for C 20 H 21 NO6[M + H] + 372.1447; Found: 372.1449.

[0090] Compound FD-A9: Yellow solid; Yield 56% (three-step reaction); 1 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.85 (s, 1H), 7.74 (s, 2H), 7.26 (s, 1H), 5.32 (s, 2H), 4.03 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H). HRMS (ESI): Calcd for C 21 H 16 F6O5[M + H] + 463.0980; Found: 463.0984.

[0091] Compound FD-A10: Yellow solid; Yield 30% (four-step reaction); 1 1H NMR (400 MHz, MeOD) δ 7.69 (s, 1H), 7.23 (s, 1H), 6.84 (dt, J = 15.3, 5.0 Hz, 3H), 5.42 (s, 2H), 3.94 (s, 3H), 3.86 (s, 6H). HRMS (ESI): Calcd for C 19 H18 O7[M + H] + 359.1131; Found: 359.1136.

[0092] Compound FD-A11: Yellow solid; Yield 63% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.25 (s, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.86 (d, J = 7.0 Hz, 1H), 6.60 (t, J = 73.1 Hz, 1H), 5.74 (s, 1H), 5.36 (s, 2H), 4.02 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H). HRMS (ESI): Calcd for C 19 H 18 F2O7[M + H] + 397.1099; Found: 397.1104.

[0093] Compound FD-A12: Yellow solid; Yield 60% (three-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 7.40 (s, 1H), 7.24 (s, 1H), 7.18 (d, J = 1.3 Hz, 2H), 5.30 (s, 2H), 4.02 (s, 3H), 3.89 (s, 6H). HRMS (ESI): Calcd for C 19 H 16 Cl2O5[M + H] + 395.0453; Found: 395.0456.

[0094] Example 2

[0095] The structural information of the polymethoxy isoflavone derivative library B series compounds is shown in Table 2 below.

[0096] Table 2 Structural information of the polymethoxy isoflavone derivative library B series compounds

[0097]

[0098] The synthesis methods of FD-B1 to FD-B4 and FD-B6 to FD-B14 are as follows:

[0099]

[0100] Weigh 3 times the amount of paraformaldehyde, add 3 times the amount of 4-hydroxypiperidine, and add 15 mL of ethanol at room temperature to form a suspension. Then slowly heat to reflux and react for 0.5 h until the solution becomes clear. Add 1 time the amount of FD-A5 to the reaction system and continue heating under reflux for 5 h. Dilute with an appropriate amount of water, extract 3 times with EA, combine the solvents, remove the solvents under reduced pressure, and purify by silica gel column chromatography to obtain FD-B1 to FD-B4 and FD-B6 to FD-B14.

[0101] The synthesis method of FD-B5 is as follows:

[0102] Dissolve FD-B8 (1 mmol) obtained from the previous step in 15 mL of dichloromethane, then add 2 equ. of HCl (1 M in Diox), stir and react at room temperature for 0.5 h, then adjust the pH = 9 with saturated NaHCO3 solution, extract 3 times with DCM, combine the solvents, remove the solvents under reduced pressure, and purify by silica gel column chromatography to obtain the derivative FD-B5 with a yield of 85%.

[0103] Compound FD-B1: Yellow solid; Yield 81%; 1 H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.27 (s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 5.45 (s, 2H), 4.01 (s, 2H), 3.91 (s, 2H), 3.90 (s, 2H), 3.90 (s, 2H), 3.83 (s, 2H), 3.49 (s, 1H), 2.67 (q, J = 7.2 Hz, 4H), 1.15 (t, J = 7.2 Hz, 6H). HRMS (ESI): Calcd for C 25 H 31 NO7[M + H] + 458.2179; Found: 458.2184.

[0104] Compound FD-B2: Yellow solid; Yield 53%; 1 H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.27 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.45 (s, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.74 (s, 2H), 2.34 (s, 1H), 2.32 (s, 1H), 2.26 (s, 3H), 0.98 (d, J = 6.5 Hz, 6H). HRMS (ESI): Calcd for C 25 H 31 NO7[M + H] + 458.2179; Found: 458.2182.

[0105] Compound FD-B3: yellow solid; yield 63%; 1 H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.26 (s, 1H), 6.82 (d, J = 26.3 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 5.45 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 8H), 2.71 (s, 4H), 1.89 (s, 4H). HRMS (ESI): Calcd for C 25 H 29 NO7 [M + H] + 456.2022; Found: 456.2025.

[0106] Compound FD-B4: yellow solid; yield 65%; 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (s, 1H), 6.77 (s, 1H), 6.61 (s, 1H), 5.45 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H), 3.90 (s, 3H), 3.73 (s, 2H), 1.71 - 1.63 (m, 4H), 1.44 - 1.23 (m, 6H). HRMS (ESI): Calcd for C 26 H 31 NO7 [M + H] + 470.2179; Found: 470.2185.

[0107] Compound FD-B5: yellow solid; yield 49% (two-step reaction); 1 H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 7.24 (s, 1H), 6.84 (s, 1H), 6.75 (s, 1H), 5.42 (s, 2H), 4.00 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.29 (s, 2H), 2.98 (s, 4H), 2.60 (s, 4H). HRMS (ESI): Calcd for C 25 H 30 N2O7 [M + H] + 471.2131; Found: 471.2133.

[0108] Compound FD-B6: yellow solid; yield 70%; 11H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (s, 1H), 6.79 (s, 1H), 6.63 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.78 (s, 3H), 2.64 (s, 8H), 2.32 (s, 3H); 13 13C NMR (100 MHz, CDCl3) δ 181.1, 150.2, 149.2, 149.0, 148.2, 148.0, 141.8, 137.6, 128.3, 125.4, 123.5, 121.2, 117.2, 113.0, 103.3, 68.4, 61.5, 61.3, 60.9, 56.2, 56.0, 54.6, 52.2, 45.7, 29.7. HRMS (ESI): Calcd for C 26 H 32 N2O7 [M + H] + 485.2288; Found: 485.2290.

[0109] Compound FD - B7: Yellow solid; Yield 72%; 1 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (s, 1H), 6.79 (s, 1H), 6.63 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 6H), 3.78 (s, 2H), 2.66 (s, 8H), 2.46 (dd, J = 14.1, 7.0 Hz, 2H), 1.10 (t, J = 7.1 Hz, 3H). HRMS (ESI): Calcdfor C 27 H 34 N2O7 [M + H] + 499.2344; Found: 499.2345.

[0110] Compound FD - B8: Yellow solid; Yield 75%; 1 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.27 (s, 1H), 6.80 (s, 1H), 6.63 (s, 1H), 5.43 (s, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.77 (s, 2H), 3.52 (s, 4H), 2.56 (s, 4H), 1.47 (s, 9H). HRMS (ESI): Calcd for C 30 H38 N2O9[M+H] + 571.2655; Found: 571.2660.

[0111] Compound FD-B9: Yellow solid; Yield 76%; 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (s, 1H), 6.80 (s, 1H), 6.64 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.90 (s, 6H), 3.77 (s, 6H), 2.62 (s, 4H). HRMS (ESI): Calcd for C 25 H 29 NO8[M+H] + 472.1981; Found: 472.1984.

[0112] Compound FD-B10: Yellow solid; Yield 67%; 1 H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.26 (s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 6H), 3.77 (s, 2H), 3.52 (d, J = 6.4 Hz, 2H), 3.08 (d, J = 11.2 Hz, 2H), 2.18 (t, J = 10.9 Hz, 2H), 1.83 (d, J = 12.9 Hz, 2H), 1.61 (s, 1H), 1.29 (s, 1H). HRMS (ESI): Calcd for C 26 H 31 NO8[M+H] + 486.2128; Found: 486.2130.

[0113] Compound FD-B11: Yellow solid; Yield 66%; 11H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.79 (s, 1H), 6.62 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 3.90 (s, 6H), 3.77 (d, J = 7.0 Hz, 2H), 2.93 (s, 1H), 2.71 (s, 1H), 2.32 (s, 2H), 1.88 (d, J = 9.6 Hz, 2H), 1.62 (d, J = 12.6 Hz, 1H), 1.44 (d, J = 9.9 Hz, 1H), 1.29 (s, 1H), 1.26 (s, 1H). HRMS (ESI): Calcd for C 26 H 31 NO8[M + H] + 486.2128; Found: 486.2133.

[0114] Compound FD - B12: Yellow solid; Yield 63%; 1 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.26 (s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 5.44 (s, 2H), 4.01 (s, 3H), 3.90 (s, 9H), 3.75 (s, 2H), 3.54 (d, J = 24.4 Hz, 2H), 3.00 (d, J = 62.1 Hz, 2H), 2.10 (d, J = 46.7 Hz, 2H), 1.90 (s, 1H), 1.74 (t, J = 26.0 Hz, 4H), 0.86 (d, J = 7.0 Hz, 1H). HRMS (ESI): Calcd for C 27 H 33 NO8[M + H] + 500.2284; Found: 500.2285.

[0115] Compound FD - B13: Yellow solid; Yield 70%; 1 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.27 (d, J = 3.1 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 8.3 Hz, 1H), 5.22 (d, J = 1.2 Hz, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.82 (s, 2H), 3.62 (t, J = 5.3 Hz, 2H), 2.63 (s, 8H), 2.59 - 2.55 (m, 2H); 1313C NMR (150 MHz, CDCl3) δ 181.5, 150.7, 149.4, 149.3, 148.2, 148.1, 141.8, 135.8, 131.0, 125.7, 120.5, 120.1, 117.1, 109.9, 103.2, 68.3, 61.5, 61.4, 59.1, 57.7, 57.5, 56.3, 55.9, 52.6, 52.4. HRMS (ESI): Calcd for C 27 H 34 N2O8 [M+H] + 515.2393; Found: 515.2394.

[0116] Compound FD-B14: Yellow solid; Yield 55%; 1 1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.39 - 7.29 (m, 5H), 7.27 (d, J = 3.1 Hz, 1H), 6.79 (s, 1H), 6.64 (s, 1H), 5.44 (d, J = 1.3 Hz, 2H), 4.01 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.81 (s, 2H), 3.67 (s, 2H), 2.28 (s, 3H). HRMS (ESI): Calcd for C 29 H 31 NO7 [M+H] + 506.2179; Found: 506.2181.

[0117] Example 3

[0118] The structural information of the polymethoxy high isoflavone derivative library C series compounds is shown in Table 3 below.

[0119] Table 3 Structural Information of the Polymethoxy High Isoflavone Derivative Library C Series Compounds

[0120]

[0121]

[0122] The synthesis methods of FD-C1 to FD-C7 are as follows:

[0123]

[0124] (1) Weigh 1 mmol of Compound 12 and dissolve it in 20 mL of THF. Add 4 equ. amounts of NaH (4 mmol, 0.096 g) and the corresponding methyl substituted benzoates 15a - 15g (2.5 mmol) at room temperature, then heat to reflux and react for 7 h. After detecting the completion of the reaction by TLC, add an appropriate amount of water, then acidify with an HCl aqueous solution with pH = 6, extract 3 times with EA, combine the solvents, remove the solvents under reduced pressure, and purify by silica gel column to obtain 16a - 16h.

[0125] (2) The synthesis methods of FD - C1 to FD - C5 and FD - C8 are the same as those of Compound 10 in Example 1.

[0126] (3) Synthesis method of FD - C6 - FD - C7. Dissolve 1 mmol of high - isoflavone FD - C4 or FD - C5 in 15 mL of anhydrous dichloromethane, slowly drop 5 mL of BCl3 (1 M in DCM) under ice - bath and stirring conditions, continue to react for 0.5 h, remove the ice - bath, and react at room temperature for 2 h. After detecting the completion of the reaction by TLC, add 15 mL of ice - water and continue to stir for 2.5 h. Then remove dichloromethane under reduced pressure, extract 3 times with ethyl acetate, 15 mL each time, combine the solvents, remove the solvents under reduced pressure, and purify by silica gel column to obtain the final products FD - C6 or FD - C7.

[0127] Compound FD - C1: Pale yellow - white solid; yield 44% (two - step reaction); 1 H NMR(400MHz,CDCl3)δ8.31(s,1H),7.86(d,J = 7.3Hz,2H),7.59(t,J = 7.4Hz,1H),7.47(t,J = 7.7Hz,2H),7.41(s,1H),4.06(s,3H),4.05(s,3H),3.95(s,3H); 13 C NMR(100MHz,CDCl3)δ191.00,172.92,156.95,150.87,146.77,144.71,140.97,136.19,132.44,128.56,127.37,123.44,119.78,99.51,61.13,60.48,55.27.HRMS(ESI):Calcd for C 19 H 16 O6[M + H] + 341.1025; Found:341.1027.

[0128] Compound FD - C2: Pale yellow - white solid; yield 48% (two - step reaction); 11H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 7.40 (s, 1H), 6.99 (d, J = 2.2 Hz, 2H), 6.68 (t, J = 2.2 Hz, 1H), 4.05 (s, 6H), 3.95 (s, 3H), 3.82 (s, 6H). HRMS (ESI): Calcd for C 21 H 20 O8[M + H] + 401.1236; Found: 401.1239.

[0129] Compound FD-C3: Pale yellowish-white solid; yield 49% (two-step reaction); 1 1H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.44–7.40 (m, 2H), 6.87 (d, J = 8.4 Hz, 1H), 4.06 (s, 6H), 3.96 (s, 3H), 3.96 (s, 3H), 3.95 (s, 3H). HRMS (ESI): Calcd for C 21 H 20 O8[M + H] + 401.1236; Found: 401.1238.

[0130] Compound FD-C4: Pale yellowish-white solid; yield 45% (two-step reaction); 1 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 10.9, 6.4 Hz, 4H), 7.38–7.32 (m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 5.23 (s, 2H), 4.05 (s, 3H), 4.05 (s, 3H), 3.96 (s, 3H), 3.95 (s, 3H). HRMS (ESI): Calcd for C 27 H 24 O8[M + H] + 477.1549; Found: 477.1547.

[0131] Compound FD-C5: Pale yellowish-white solid; yield 46% (two-step reaction); 11H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.48–7.44 (m, 3H), 7.41 (s, 1H), 7.39–7.30 (m, 3H), 6.89 (d, J = 8.5 Hz, 1H), 5.19 (s, 2H), 4.06 (s, 6H), 3.96 (s, 3H), 3.94 (s, 3H). HRMS (ESI): Calcd for C 27 H 24 O8[M + H] + 477.1549; Found: 477.1550.

[0132] Compound FD-C6: pale yellowish-white solid; yield 32% (three-step reaction); 1 1H NMR (400 MHz, MeOD) δ 8.41 (s, 1H), 7.39 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.31 (dd, J = 8.3, 2.1 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 4.03 (s, 3H), 4.01 (s, 3H), 3.94 (s, 3H). HRMS (ESI): Calcd for C 20 H 18 O8[M + H] + 387.1080; Found: 387.1084.

[0133] Compound FD-C7: pale yellowish-white solid; yield 30% (three-step reaction); 1 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 2H), 7.48–7.43 (m, 2H), 7.41 (s, 1H), 6.89 (d, J = 8.1 Hz, 1H), 5.74 (s, 1H), 4.05 (s, 6H), 3.96 (s, 3H), 3.95 (s, 3H). HRMS (ESI): Calcd for C 20 H 18 O8[M + H] + 387.1080; Found: 387.1082.

[0134] Compound FD-C8: pale yellowish-white solid; yield 48% (two-step reaction); 11H NMR(400MHz,CDCl3)δ8.29–8.23(m,2H),7.75(ddd,J=8.7,7.2,1.6Hz,1H),7.54(d,J=8.3Hz,1H),7.52–7.45(m,1H),7.00(d,J=2.3Hz,2H),6.68(t,J=2.3Hz,1H),3.82(s,6H).HRMS(ESI):Calcd for C 18 H 14 O5[M+H] + 311.0919;Found:311.0910.

[0135] Example 4

[0136] Cell proliferation inhibition test

[0137] (1) Reagent materials

[0138] A375 (human malignant melanoma cell line): CL-0014, Wuhan Pusaisi Life Science Co., Ltd. DMEM: Gibico, USA; Fetal bovine serum: Hangzhou Sijiqing Biological Technology Co., Ltd.; Trypsin: Shanghai Macklin Biochemical Co., Ltd.; 10 mg / mL RNase (ChemeGen); PBS solution: Shanghai Macklin Biochemical Co., Ltd.; Dimethyl sulfoxide (DMSO): Shanghai Jingxi Chemical Industry Co., Ltd.

[0139] (2) Experimental instruments

[0140] CO2 incubator: Thermo, USA; SW-CJ-2FD ultra-clean workbench: Shanghai Boxun Industry Co., Ltd. Medical Equipment Factory; XSP-17C inverted biological microscope: Shanghai Changfang Optical Instrument Co., Ltd.; Multifunctional microplate reader: Thermo, USA; FA2004A electronic balance: Shanghai Precision Scientific Instrument Co., Ltd.

[0141] (3) Preparation of main solutions

[0142] 1) A375 cell culture medium: Mix the required volume of DMEM medium and an appropriate amount of double antibody (penicillin-streptomycin mixed solution) evenly, and add an appropriate volume of fetal bovine serum (FBS) that has been inactivated in a 56 °C water bath for 30 minutes before use. Finally, it is DMEM containing 10% FBS.

[0143] (4) Experimental method

[0144] Primary screening of the activity of high isoflavone derivatives A, B, and C libraries against melanoma cell A375

[0145] 1. Cell digestion: Discard the original cell culture medium; add 1 mL of PBS to the culture dish and ensure that the PBS completely covers the cell layer. Discard the PBS solution; add 1 mL of trypsin to completely cover the cell layer; place it in the cell culture incubator for 30 s and then remove the trypsin.

[0146] 2. Cell counting: Add 3 mL of DMEM culture medium to the culture dish to terminate digestion; transfer the cells to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; resuspend the cell pellet in 2 mL of complete culture medium, pipette 10 μL of the culture medium into the counting chamber, and use a cell counter to detect the cell density.

[0147] 3. Seeding: Adjust the cell suspension to an appropriate concentration and seed it in a 96-well culture plate at a density of 1×10 4 cells / well.

[0148] 4. After 24 h, add different test compounds at a final concentration of 10 μM and place them in the cell culture incubator for culture.

[0149] 5. After 48 h, discard the culture medium, add 100 μL of 10% CCK-8 solution to each well, and incubate at 37 °C for 1 hour.

[0150] 6. Measure the absorbance at a wavelength of 450 nm using a microplate reader and calculate the cell viability of each compound on A375. Cell viability = (average OD value of the test group / average OD value of the DMSO group) × 100%

[0151] Determination of the half-maximal inhibitory concentration IC 50 of the high isoflavone derivative FD-A6 against melanoma cell A375:

[0152] 1. Seeding: Seed in a 96-well culture plate at a density of 1×10 4 cells / well and place it in the cell culture incubator for culture.

[0153] 2. Treat cells with high isoflavone FD-A6: After 24 h, add different concentrations (0.064 μM, 0.32 μM, 1.6 μM, 8 μM, and 40 μM) of FD-A6 to the 96-well plate and culture it in a cell culture incubator at 37 °C with 5% CO2.

[0154] 3. After 24 h or 48 h, discard the culture medium, add 100 μL of 10% CCK-8 solution to each well, and incubate at 37 °C for 1 hour.

[0155] 4. Measure the absorbance at a wavelength of 450 nm using a microplate reader and calculate the cell viability of FD-A6 at different concentrations on A375 and the IC 50 value. Among them, IC 50= The compound concentration when tumor growth is inhibited by 50%. The above data are the averages of at least three independent repeated experiments.

[0156] (5) Experimental results

[0157] 1) The results of the inhibition of the proliferation of melanoma cell line A375 by the high isoflavone derivative A, B, and C libraries are shown in Tables 4, 5, 6 and Figures 1 to 3 as follows.

[0158] Table 4 Cell viability of compounds in the high isoflavone derivative A library after acting on A375

[0159] Library of Highly Isoflavone Derivative A Compounds (Concentration: 10 μM) Survival Rate of A375 Cells (%) DMSO 100.1267±3.99 FD-A1 47.4095±2.18 FD-A2 38.9048±2.50 FD-A3 56.4641±1.38 FD-A4 46.4191±2.23 FD-A5 20.9774±3.65 FD-A6 5.8750±4.71 FD-A7 47.0315±1.78 FD-A8 59.1289±2.21 FD-A9 46.1409±4.00 FD-A10 15.7333±3.12 FD-A11 18.6941±5.64 FD-A12 32.5905±0.62

[0160] Under the experimental conditions, the conclusion is:

[0161] Figure 1 And the results in Table 4 show that when the compounds FD-A1, FD-A2, FD-A3, FD-A4, FD-A5, FD-A6, FD-A7, FD-A8, FD-A9, FD-A10, FD-A11, FD-A12 in the high isoflavone derivative A library of the present invention act on A375 cells, the cell viability of most compounds is below 50% at a concentration of 10 μM. Only the cell viabilities of compounds FD-A3 and FD-A8 are slightly greater than 50%, indicating that such a high isoflavone compound skeleton has significant anti-tumor activity, and among them, compound FD-A6 has the best activity.

[0162] Meanwhile, the present invention uses a known isoflavone compound (cas: 73083-18-8, 2,3-Dihydro-3-[(3-hydroxy-4-methoxyphenyl)methylene]-4H-1-benzopyran-4-one) as an active control. Its cell viability is 78.3621 ± 1.25 (%), and its anti-tumor activity is not significant and far inferior to the anti-cell proliferation activity of compound FD-A6.

[0163] Table 5 Cell viability of compounds in the high isoflavone derivative B library after acting on A375 cells

[0164]

[0165]

[0166] Under the experimental conditions, the conclusion is:

[0167] Figure 2And the results in Table 5 show that when compounds FD-B1, FD-B2, FD-B3, FD-B4, FD-B5, FD-B6, FD-B7, FD-B8, FD-B9, FD-B10, FD-B11, FD-B12, FD-B13, FD-B14 in the high isoflavone derivative B library of the present invention act on A375 cells, the cell survival rates of some compounds are all above 50% at a concentration of 10 μM. Only the cell survival rates of compounds FD-A6 and FD-B14 are less than 50%, indicating that this type of high isoflavone compound skeleton containing Mannich base does not have obvious anti-tumor activity, and among them, compound FD-A6 still has the best activity.

[0168] Table 6 Survival rates of compounds in the high isoflavone derivative C library acting on A375 cells

[0169] Library of Highly Isoflavone Derivative C Compounds (Concentration: 10 μM) Survival Rate of A375 Cells (%) DMSO 99.9963±6.94 FD-A6 7.0612±1.57 FD-C1 76.9293±0.88 FD-C2 56.5416±3.07 FD-C3 50.5195±7.21 FD-C4 64.9460±6.04 FD-C5 57.5793±7.27 FD-C6 38.4877±5.05 FD-C7 48.3393±9.25

[0170] Under the conditions of this experiment, the conclusion obtained is:

[0171] Figure 3 And the results in Table 6 show that when compounds FD-C1, FD-C2, FD-C3, FD-C4, FD-C5, FD-C6, FD-C7 in the high isoflavone derivative C library of the present invention act on A375 cells, the cell survival rates of most compounds are above 50% at a concentration of 10 μM. Only the cell survival rates of compounds FD-A6, FD-C6 and FD-C7 are less than 50%, indicating that this 9-oxo isoflavone compound skeleton does not have obvious anti-tumor activity, and among them, compound FD-A6 still has the best activity.

[0172] 2) Results of the activity assay of high isoflavone derivative FD-A6 inhibiting melanoma cell A375:

[0173] The inhibition rates of high isoflavone derivative FD-A6 on tumor cell A375 at 0.064 μM, 0.32 μM, 1.6 μM, 8 μM and 40 μM were detected by the CCK-8 method, and the half-maximal inhibitory concentration was calculated. The results are shown in Table 8, Table 9 and Figure 4 as shown.

[0174] Table 7 IC 50 value of the growth inhibitory activity of high isoflavone derivative FD-A6 on A375 cells after 24 h of treatment

[0175]

[0176]

[0177] Table 8 IC 50Value

[0178] Concentration of Highly Isoflavone Derivative FD-A6 (μM) Survival Rate of A375 Cells (%) DMSO 100.00±7.26 0.064 71.87±6.09 0.32 22.47±1.36 1.6 17.35±0.10 8 5.5±1.12％ 40 0.88±0.20 <![CDATA[IC 50 > 0.13 ± 0.02 μM

[0179] Under the conditions of this experiment, the conclusion obtained is:

[0180] The high isoflavone derivative FD-A6 of the present invention has a significant inhibitory effect on the proliferation of A375 melanoma cells, showing a dose-dependent manner.

[0181] Example 5

[0182] The experiment on the cell cycle detection of polymethoxy isoflavones for melanoma cells is as follows:

[0183] Cell cycle detection

[0184] (1) Reagent materials

[0185] A375 cells, DMEM, fetal bovine serum, trypsin, 10 mg / mL RNase (ChemeGen), etc. are the same as those used in the CCK-8 experiment; PI dye (Wuhan Sevier Biochemical Technology Co., Ltd.); ethanol: Shanghai Macklin Biochemical Co., Ltd.; dimethyl sulfoxide (DMSO): Shanghai Jingxi Chemical Co., Ltd.

[0186] (2) Experimental instruments

[0187] Except for the flow cytometer: Agilent Technologies Co., Ltd., the other instruments used are the same as those in the CCK-8 experiment.

[0188] (3) Preparation of main solutions

[0189] The preparation of the A375 cell culture medium is the same as that in the CCK-8 experiment.

[0190] (4) Experimental methods

[0191] 1) The method of cell culture is the same as the operation in Example 4.

[0192] 2) After 24 h, the treatment concentrations of the high isoflavone FD-A6 are set to be 0.2 μM, 0.4 μM, 0.8 μM, and 1.6 μM respectively. Calculate the volumes of the 0.8 mM high isoflavone FD-A6 stock solution added at each concentration to be 0.5 μL, 1 μL, 2 μL, and 4 μL. Add the corresponding volumes of DMSO and the high isoflavone FD-A6 stock solution to the corresponding wells. Then place the cell plate after adding the drug into a cell culture incubator at 37 °C containing 5% CO2 and culture for 24 h.

[0193] 3) Take out A375 cells and observe the cell morphology and cell viability under a microscope. Use a pipette to add 500 μL of PBS solution to the culture dish from which the culture medium has been aspirated for washing. After shaking well, use a pipette to remove the PBS. Add 300 μL of trypsin solution to the culture medium, shake it left and right for 3 - 4 circles, and then place the culture medium in a cell incubator at 37 °C with 5% CO2 for digestion for 30 s. Use a pipette to add 500 μL of PBS solution to the culture dish from which the culture medium has been aspirated for washing. After shaking well, use a pipette to remove the PBS. Add 300 μL of trypsin solution to the culture medium, shake it left and right for 3 - 4 circles, and then place the culture medium in a cell incubator at 37 °C with 5% CO2 for digestion for 30 s. Add 300 μL of PBS solution and use a pipette to pipette the cell pellet into a single-cell suspension. Then place each centrifuge tube on a vortex mixer and add 700 μL of ethanol dropwise while hanging in the air. Place the centrifuge tube flat in a -20 °C refrigerator for fixation for 24 hours.

[0194] 4) Turn on the centrifuge in advance and pre-cool it to 4 °C. Take out the cells fixed at -20 °C for 24 hours and centrifuge them at 500 g for 5 min. After centrifugation is completed, use a pipette to aspirate the supernatant, add 1 mL of PBS for resuspension, centrifuge at 500 g for 3 min, aspirate the supernatant after centrifugation, add another 500 μL of PBS for resuspension, and store it on ice. Use a pipette to add 2.5 μL of RNase (RNA enzyme) to each centrifuge tube respectively, mix well and then place it in an incubator at 37 °C for incubation for 15 min. Subsequently, add 25 μL of propidium iodide (PI) respectively, mix well, and incubate in the dark at room temperature for 15 min. After filtering through a microporous filter membrane, transfer it to a flow cytometry sample loading tube, place it on ice and store it in the dark, and use a flow cytometer to detect the cell cycle distribution.

[0195] (5) The experimental results are as Figure 5 、 Figure 6 and Table 9 below show.

[0196] Table 9 Data of the number of cells in each cycle after treating A375 cells with different concentrations

[0197]

[0198] The results showed that compared with DMSO, the proportions of A375 cells in G1 phase after treatment with 200 nM, 400 nM, 800 nM, and 1600 nM FD-A6 were 47.55 ± 1.56%, 42.95 ± 1.65%, 40.65 ± 1.80%, and 31.06 ± 1.83% respectively, the proportions of cells in S phase were 27.33 ± 1.40%, 29.23 ± 1.25%, 30.13 ± 1.25%, and 28.98 ± 3.61% respectively, and the proportions of cells in G2 / M phase were 24.76 ± 0.35%, 27.69 ± 0.47%, 29.35 ± 0.70%, and 39.74 ± 2.01% respectively. This indicated that when A375 cells were treated with FD-A6, the cells were arrested in the G2 / M phase, and the higher the concentration of FD-A6, the more obvious the effect of G2 / M phase arrest.

[0199] Example 6

[0200] In vivo activity test

[0201] (1) Reagent materials

[0202] 6-week-old female C57 / BL6J mice: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0203] (2) Experimental instruments

[0204] The experimental instruments used were the same as those in Example 4.

[0205] (3) Experimental method

[0206] After 5 days of adaptive cultivation of 6-week-old female C57 / BL6J mice, they were subcutaneously injected with a suspension of melanoma A375 cells (8 × 10 5 / 100 μL) prepared with PBS. When the tumor grew to 40 - 50 mm 3 , they were intraperitoneally injected with 20 mg / kg polymethoxyisoflavone FD-A6 every day, and the control group was injected with an equal amount of solvent (normal saline) for 12 consecutive days. The change in tumor volume was measured every 2 - 4 days, and the change in the body weight of the mice was monitored simultaneously. Attached Figure 7 is a graph showing the relationship between the body weight and tumor volume of the mice and the number of days of drug administration.

[0207] (4) Experimental results

[0208] As Figure 7 shown in Figure 7As shown in D, compared with the control group, there was no significant change in the body weight of mice in the polymethoxy isoflavone FD-A6 administration group; FD-A6 significantly inhibited the growth of tumors in tumor-bearing mice. After 8 days of re-administration, there was a significant difference in the tumor volume of mice in the FD-A6 administration group compared with the control group (*p < 0.05). This indicates that the polymethoxy isoflavone FD-A6 disclosed in the present invention can significantly inhibit the growth of tumors in tumor-bearing mice; while exerting an anti-tumor effect, it has no obvious effect on the body weight of tumor-bearing mice.

[0209] Example 7

[0210] In vivo activity inhibition test for isoflavones

[0211] A. Tubulin polymerization inhibition experiment

[0212] (1) Reagent materials

[0213] Tubulin Polymerization Assay Kit (Cat.#BK011P)

[0214] (2) Experimental instruments

[0215] SpectroFluor Plus fluorometer: TECAN GmbH

[0216] (3) Experimental methods

[0217] Operate according to the tubulin kit instructions, and immediately mix the experimental components (select standard, inhibitor or enhancer conditions).

[0218] ① Start the polymerization reaction: Add 5 μL of the blank control group, combretastatin positive control group (100 μM), and FD-A5 (50 μM), FD-A6 (10 μM), FD-A6 (50 μM), FD-B14 (500 μM), FD-C6 (500 μM), FD-C7 (500 μM) (both set with 2 replicates) to a 96-well plate preheated at 37°C. Place the plate back into the warm plate reader for 1 minute, but not more than 1 minute, because the 5 μL volume will evaporate rapidly.

[0219] ③ Then add 50 μL of the tubulin reaction mixture to the corresponding wells to start the polymerization reaction, and immediately place the 96-well plate into the set microplate reader to start counting. Pay attention to operating quickly, use a medium pipetting speed and place the pipette tip on the well wall. This technique avoids bubble formation, which will interfere with the absorbance reading.

[0220] ④ The fluorometer parameters are as shown in Table 10 below.

[0221] Table 10 Fluorometer parameters

[0222]

[0223] (5) Experimental results:

[0224] As Figure 8 shown, this method verified the effect of the designed and synthesized polymethoxyisoflavone compounds on microtubule polymerization. It can be seen from the polymerization curve results that both FD-A5 and FD-A6 significantly inhibited microtubule polymerization, and had a significant impact on the formation of the spindle and the final cell division process.

[0225] In this specification, the present invention has been described with reference to its specific embodiments. However, it is obvious that various modifications and variations can still be made without departing from the spirit and scope of the present invention. Therefore, the specification should be regarded as illustrative rather than restrictive.

Claims

1. A polymethoxy isoflavone derivative or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the said derivative is as shown in general formula (I) or general formula (II), wherein, R1 and R2 are each independently selected from methoxy and hydrogen, provided that R1 and R2 are not both methoxy at the same time; R3 is hydrogen, methoxy, -CH2-R 10 ; R4 and R5 are each independently selected from hydrogen, substituted or unsubstituted methoxy, hydroxy, halogen atom, amino, nitro, or R4 and R5 form a five-membered ring R6 is hydrogen, a substituted or unsubstituted methoxy group, a hydroxyl group, a halogen atom, an amino group, a nitro group, -CH2-R 10 ; R7, R8, and R9 are each independently selected from hydrogen, substituted or unsubstituted methoxy, hydroxy, halogen atom, amino, nitro, or, R7 and R8 form a five-membered ring Alternatively, R8 and R9 form a five-membered ring The substituent of the substituted methoxy is halogen; If R5 is methoxy, R4 is hydroxyl, and R3 is -CH2-R 10 ; If R4 is a methoxy group, R5 is a hydroxyl group, and R6 is -CH2-R 10 ; R 10 It is a substituted secondary amine, pyrrolidinyl, morpholinyl, piperidinyl, a substituted or 4-position substituted piperazinyl, a piperidinyl substituted by a hydroxyl group or a C1-C5 hydroxy-substituted alkyl group. The substituents of the substituted secondary amine are selected from C1-C4 alkyl groups, substituted or unsubstituted benzyl groups. The substituents at the 4-position of the substituted piperazinyl are selected from C1-C12 alkyl groups, C1-C12 hydroxy-substituted alkyl groups, substituted or unsubstituted benzyl groups, tert-butoxycarbonyl groups. The C1-C5 positions on the benzene ring of the substituted benzyl group have 1 to 5 substituents, and the 1 to 5 substituents are each independently selected from methoxy groups, hydroxyl groups, halogen atoms, amino groups, nitro groups, trifluoromethoxy groups, or two adjacent substituents among the 1 to 5 substituents form a five-membered ring 2. The polymethoxylated isoflavone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The said pharmaceutically acceptable salt is selected from the acid salts of polymethoxyisoflavone derivatives.

3. The polymethoxy isoflavone derivative or a pharmaceutically acceptable salt thereof according to claim 2, wherein The said pharmaceutically acceptable salt is selected from the organic acid salts or inorganic acid salts of polymethoxyisoflavone derivatives.

4. The polymethoxy isoflavone derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The said inorganic acid salts are selected from hydrochloride salts, hydrobromide salts, sulfate salts, and phosphate salts of polymethoxyisoflavone derivatives; the said organic acid salts are selected from oxalate salts, acetate salts, citrate salts, maleate salts, benzoate salts, malate salts, toluenesulfonate salts, fumarate salts, or tartrate salts of polymethoxyisoflavone derivatives.

5. The polymethoxy isoflavone derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The said derivative is selected from the following compounds: Compound 1-1 Compound 1-2 Compound 1-3 Compound 1-4 Compound 1-5 Compound 1-6 Compound 1-7 Compound 1-8 Compound 1-9 Compound 1-10 Compound 1-11 Compound 2-1 Compound 2-2 Compound 2-3 Compound 2-4 Compound 2-5 Compound 2-6 6. A pharmaceutical composition, characterized in that, Comprising the polymethoxyisoflavone derivative or its pharmaceutically acceptable salt as described in any one of claims 1 to 5, and a pharmaceutically acceptable carrier and / or excipient.

7. The pharmaceutical composition according to claim 6, wherein The dosage form of the said pharmaceutical composition is selected from capsules, granules, injections, tablets, aerosols, oral liquids, or sustained-release agents.

8. Use of the polymethoxyisoflavone derivative or its pharmaceutically acceptable salt as described in any one of claims 1 to 5, or the pharmaceutical composition as described in claim 6 in the preparation of an anti-tumor drug.

9. The application according to claim 8, wherein The said tumors are: leukemia, skin cancer, breast cancer, lung cancer, liver cancer, colorectal cancer.

10. The application according to claim 8, wherein, The said tumor is melanoma.

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