Application of chalcone analogs as active substances in the preparation of anti-tumor drugs

Abstract

The present invention discloses a new medical use of chalcone analogs, namely, the use of chalcone analogs as active substances in the preparation of anti-tumor drugs. The contents disclosed in the present invention include the structure of chalcone analogs, the synthesis method of chalcone analogs, and the tumor cell inhibition activity of chalcone analogs. The results of in vitro anti-tumor activity screening of the chalcone analogs of the present invention show that they exhibit strong inhibitory activity against human cervical cancer cells (HeLa), human colon cancer cells (HCT-116) and human gastric cancer cells (MGC-803), and the inhibitory activity of some compounds is higher than that of the current clinical drug cisplatin, so the compounds of the present invention can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to the application of chalcone analogs as active substances in the preparation of anti-tumor drugs. Background Art

[0002] In the past few decades, the incidence of cancer has risen sharply due to various reasons such as environmental pollution, unhealthy lifestyles and high stress. According to the latest statistics from the International Agency for Research on Cancer (IARC), there are an estimated 19,292,789 new cancer cases and 9,958,133 deaths worldwide in 2020. Among them, the number of deaths caused by colorectal cancer and gastric cancer is second only to lung cancer, at 9.4% and 7.7% respectively. The most commonly diagnosed cancer in women is cervical cancer, and its incidence rate is increasing year by year, which is attracting more and more attention.

[0003] Chemotherapy is currently the basic means of treating cancer, and its clinical development depends on the research and development of more anticancer drugs. However, most chemotherapy drugs are limited in their clinical application to a certain extent due to serious side effects, drug resistance, and disease metastasis and invasion. Therefore, it is extremely urgent to find new high-efficiency, low-toxic anti-tumor drugs. Since natural products have good biological activity and weak toxic side effects, their derivatization as an effective strategy for discovering new tumor chemotherapy drugs has attracted widespread attention.

[0004] Tumor cells acquire the ability to form colonies through unlimited proliferation, while highly differentiated and mature cells do not have this proliferation ability. Therefore, the difference in the efficiency of tumor cell colony formation reflects the strength of the tumor cells' independent survival ability in vitro.

[0005] Cell migration is one of the basic physiological functions of maintaining growth and development. Compared with normal cells, tumor cells usually have a higher migration ability, which is also the main reason why tumor cells can spread and proliferate rapidly. The cell wound healing experiment mainly simulates the actual migration of cells in the healing process after being wounded in the body, and can more realistically measure the migration movement and repair ability of cells.

[0006] The cell cycle is one of the basic processes of cell life activities, which is used to maintain normal cell proliferation and development. It can be divided into two stages: interphase and mitosis (M phase); the interphase includes the period from the completion of mitosis to DNA replication (G1 phase), the DNA replication period (S phase), and the time from DNA replication to the preparation for mitosis (G2 phase). However, tumor cells usually show that the cycle regulation is blocked, and the cell differentiation and apoptosis processes are destroyed, so they can proliferate indefinitely.

[0007] Tubulin inhibitors play an important role in tumor treatment. They can organize and destroy tumor blood vessels and quickly cut off the blood supply to the tumor. Representative drugs include colchicine. In addition, CA-4 (Combretastain A-4) is a highly efficient tubulin inhibitor with colchicine binding sites. It binds to tubulin in a reversible manner and significantly reduces the polymerization efficiency between tubulin dimers, thereby blocking the cell cycle at the G2 / M phase. Its effective dose is 1 / 10 of its maximum tolerated dose, which can effectively reduce the toxicity of clinical drugs. Podophyllotoxin and its analogs are also used to treat malignant tumors. Studies have shown that etoposide has a good effect in the treatment of bronchial small cell carcinoma, choriocarcinoma, etc. ZD6126 is a water-soluble prodrug of N-acetylcolchinol (NAC), which is mainly used to treat metastatic renal cell carcinoma. Its metabolite NAC can selectively destroy the endothelial skeleton of tumor cells to achieve the purpose of cutting off the blood supply of the tumor. Comparison of the basic chemical structures of these classic tubulin inhibitors revealed that trimethoxyphenyl is a common chemical structure among them. These results further indicate that trimethoxyphenyl is the pharmacophore of tubulin inhibitors that exert anti-tumor activity.

[0008] Flavonoids are secondary metabolites widely distributed in higher plants, and generally refer to a series of C6-C3-C6 compounds with two benzene rings connected by three carbons. Studies have found that flavonoids have a variety of biological effects, including anti-cancer, cardioprotective, antioxidant, anti-inflammatory, antiviral, antifungal, neurite outgrowth stimulation, and prevention of cardiovascular diseases. Due to the structural diversity of flavonoids, they have low toxicity and a wide range of biological activities, which makes them extremely important in the synthesis of new drugs and become a current research hotspot.

[0009] Chalcone compounds (1,3-diaryl-2-propen-1-ones) are precursors for the biosynthesis of flavonoids. Compared with the pyrone connection between the A / B rings of flavonoids, the A / B rings of chalcone are connected by propenone, which has greater flexibility in molecular structure, can bind to different receptors, has a wide range of target effects, and exhibits obvious biological activity. Summary of the invention

[0010] The purpose of the present invention is to provide a new medical use of a chalcone analog, namely, use of the chalcone analog as an active substance in the preparation of an anti-tumor drug.

[0011] In view of the wide range of target effects exhibited by chalcone compounds due to their structural flexibility, the present invention, based on the understanding of the relationship between chalcone compounds and anti-tumor activity, introduces a trimethoxyphenyl pharmacophore with tubulin polymerization inhibition activity into the A ring of the chalcone compound structure based on the pharmacophore splicing principle. At the same time, substituents with different electronegativity are introduced into the B ring, and it is expected that the anti-tumor activity of the drug can be improved through this structural optimization method, and chalcone compounds with good anti-tumor activity can be screened.

[0012] 1. Structure of chalcone analogs:

[0013] The chalcone analogues of the present invention have the following structural formula:

[0014]

[0015] Wherein, the substituent R is selected from one or more of trifluoromethyl, imidazolyl, chloride ion, fluoride ion, methoxy, methyl, pyridyl, quinolyl, morpholinyl, tetrahydropyrrolyl and 4-methylpiperazinyl.

[0016] 2. Synthesis of chalcone analogs:

[0017] 1.2 mmol potassium hydroxide and benzaldehyde compounds (2.1 mmol) were mixed and filled with inert gas protection. 2.2 mmol 2,3,4-trimethoxy-6-hydroxyacetophenone was dissolved in 10 mL anhydrous ethanol solution and slowly added dropwise to the mixed solution of potassium hydroxide and benzaldehyde compounds. The mixture was placed in an electric heating mantle and heated with stirring at 80°C for 2 hours. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and the excess solvent was removed by evaporation under reduced pressure. The residue was dissolved in anhydrous ethanol and eluted and purified by an eluent petroleum ether / ethyl acetate (v / v=10:1) system to obtain a chalcone product.

[0018] In order to further realize the present invention, the substituents of the above-mentioned benzaldehyde compounds are one or more of trifluoromethyl, imidazolyl, chloride ion, fluoride ion, methoxyl, methyl, pyridyl, quinolyl, morpholinyl, tetrahydropyrrolyl and 4-methylpiperazinyl.

[0019] 3. Inhibitory activity of chalcone analogs on tumor cells:

[0020] The results of in vitro anti-tumor activity screening of the chalcone analogs of the present invention show that they exhibit strong inhibitory activity against human cervical cancer cells (HeLa), human colon cancer cells (HCT-116) and human gastric cancer cells (MGC-803), and the inhibitory activity of some compounds is higher than that of the current clinical drug cisplatin, so the compounds of the present invention can be used to prepare anti-tumor drugs. The chalcone compounds of the present invention have novel structures, cheap and readily available raw materials, simple synthesis processes, high product purity, and exhibit strong inhibitory effects on tumor cells, and have excellent application prospects.

[0021] The present invention determines the cell proliferation ability of chalcone compounds on HGC-27 cell colony detection by plate cell cloning technology, and determines the influence of chalcone compounds on the proliferation ability of MGC-803 cells by colony experiment, and finds that with the continuous increase of the added drug concentration, the area of ​​crystal violet in the culture dish becomes smaller and smaller, and the color gradually becomes lighter until the crystal violet almost completely disappears. The results show that chalcone compounds can significantly inhibit the in vitro growth of gastric cancer MGC-803 cells in a dose-dependent manner.

[0022] The present invention determines the influence of chalcone compounds on the migration and repair ability of MGC-803 cells through a cell wound healing experiment, and finds that on the one hand, the degree of scratch healing in the drug-added group is significantly lower than that in the blank control group without drug addition, and on the other hand, it is found that with the increase of drug concentration, the cell migration rate gradually decreases. The results show that the inhibitory effect of chalcone compounds on the migration and repair ability of MGC-803 cells is dose-dependent.

[0023] The present invention uses flow cytometry to determine the changes of chalcone compounds on the MGC-803 cell cycle, and finds that with the increase in the concentration of chalcone compounds, the proportion of MGC-803 cells distributed in the G2 phase gradually increases. The results show that chalcone compounds can block MGC-803 cells in the G2 / M phase in a dose-dependent manner, thereby having anti-tumor activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The inhibition of colony formation of MGC-803 cells by the synthetic product Ic of chalcone analogues;

[0025] Figure 2 shows the inhibition of the migration of MGC-803 cells by the synthetic product Ic of the chalcone analog:

[0026] (A) Inverted microscopic imaging of the extent of wound healing at different drug concentrations;

[0027] (B) Statistics of the healing rate of scratches from three independent wounds are shown as mean ± SD, ***P < 0.001;

[0028] Figure 3 shows the 48 h cycle distribution of MGC-803 cells treated with chalcone analog synthetic product Ic:

[0029] (A) Flow cytometry analysis showed the cell cycle distribution of PI-stained cells, and FlowJo was used to count the number of cells in different stages of the cell cycle.

[0030] (B) Statistics are mean ± SD of the distribution ratios from three independent cycles, *P < 0.05, **P < 0.01 compared with the control. DETAILED DESCRIPTION

[0031] The synthesis of the chalcone compounds of the present invention and their tumor cell inhibition activity are further described below in conjunction with specific examples and drawings. It should be understood that these examples are used to illustrate the present invention, but do not limit the scope of the present invention.

[0032] 1. Structure and Synthesis of Chalcone Analogues

[0033] In Examples 1-13, the structures of the chalcone analogs are as follows:

[0034]

[0035] The synthesis method is to carry out condensation reaction of 2,3,4-trimethoxy-6-hydroxyacetophenone with different substituted benzaldehyde compounds in the presence of an organic solvent and a base to obtain the target compound according to different configurations. The reaction formula is as follows:

[0036]

[0037] The specific method is as follows: benzaldehyde compounds are mixed with potassium hydroxide, and the mixture is slowly added dropwise to an anhydrous ethanol solution containing 2,3,4-trimethoxy-6-hydroxyacetophenone under nitrogen protection, and the mixture is placed in an electric heating mantle and heated at 80°C with stirring for 2 hours. The reaction progress is detected by TLC. After the reaction is completed, the mixture is cooled to room temperature, and the excess solvent is removed by evaporation under reduced pressure. The residue is dissolved in anhydrous ethanol, and the mixture is eluted and purified by an eluent petroleum ether / ethyl acetate system to obtain a chalcone product.

[0038] The substituents of the benzaldehyde compound are one or more of trifluoromethyl, imidazole, chloride ion, fluoride ion, methoxy, methyl, pyridyl, quinolyl, morpholinyl, tetrahydropyrrolyl and 4-methylpiperazinyl.

[0039] Example 1. Synthesis of 2-trifluoromethyl-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ia)

[0040] The benzaldehyde compound in this embodiment adopts 2-trifluoromethylbenzaldehyde.

[0041] Synthesis steps: 1.2 mmol potassium hydroxide and 2.1 mmol 2-trifluoromethylbenzaldehyde are placed in a two-necked flask with a reflux condenser and filled with inert gas for protection. 2.2 mmol 2,3,4-trimethoxy-6-hydroxyacetophenone is dissolved in anhydrous ethanol and added dropwise to the two-necked flask, and then placed in a warm water bath at 80°C and stirred to react and reflux for 2 hours. The reaction progress is monitored by TLC. After the reaction is completed, it is cooled to room temperature, and the excess solvent is removed by evaporation under reduced pressure. The residue is dissolved in anhydrous ethanol and eluted and purified by an eluent petroleum ether / ethyl acetate (v / v=10:1) system to obtain the target compound 2-trifluoromethyl-2´-hydroxy-4´,5´,6´-trimethoxychalcone. The structural formula of the product is as follows:

[0042]

[0043] The reaction formula is as follows:

[0044]

[0045] The test data of the product are as follows:

[0046] Yield: 75%; Melting point: 128-129 ℃;

[0047] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.53 (s, 1H), 8.00 (d, J = 15.6 Hz,1H), 7.79 (d, J = 16 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 6.29 (s, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.83 (s, 3H).

[0048] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.5, 162.8, 160.6, 154.9, 140.7,138.8, 135.3, 131.7, 131.4, 129.0, 128.4, 125.9, 125.9, 108.6, 96.6, 61.9,61.3, 56.2.

[0049] HRMS, calculated: C 19 H 17F3NaO5: 405.0920 [M + Na] + ; Detected value: 405.0916 [M +Na] + .

[0050] Example 2. Synthesis of 4-(1H-imidazol-1-yl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ib)

[0051] The benzaldehyde compound in this embodiment adopts 4-(1H-imidazol-1-yl)phenyl.

[0052] The synthesis steps were the same as those in Example 1, except that 4-(1H-imidazol-1-yl)phenyl was used instead of 2-trifluoromethylphenyl.

[0053] The structural formula of the product is as follows:

[0054]

[0055] The test data of the product are as follows:

[0056] Yield: 62%, melting point: 146-147 °C;

[0057] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.59 (s, 1H), 7.92 (d, J = 6.8 Hz,2H), 7.80 (d, J = 16.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4Hz, 2H), 7.31 (s, 1H), 7.21 (s, 1H), 6.28 (s, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.82 (s, 3H).;

[0058] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.5, 162.7, 160.4, 154.9, 141.2,135.3, 135.3, 134.5, 129.8, 121.4, 117.8, 108.6, 96.6, 61.9, 61.3, 56.1.;

[0059] HRMS, calculated: C 21 H21 N2O5: 381.1445 [M + H] + ; Detected value: 381.1446 [M + H] + .

[0060] Example 3. Synthesis of 2,6-dichloro-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ic)

[0061] The benzaldehyde compound in this embodiment adopts 2, 6-dichlorophenyl.

[0062] The synthesis steps were the same as those in Example 1, except that 2,6-dichlorophenyl was used instead of 2-trifluoromethylphenyl.

[0063] The structural formula of the product is as follows:

[0064]

[0065] The test data of the product are as follows:

[0066] Yield: 51%, melting point: 129-130 ℃;

[0067] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.56 (s, 1H), 8.05 (dd, J =48 16.0Hz, 1H), 7.39 (d, J =8.0 Hz, 1H), 7.33 (d, J =8.0 Hz, 1H), 7.21-7.16 (m, 2H), 6.29 (s, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.80 (s, 3H).

[0068] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.5, 162.9, 160.6, 155.1, 136.4,135.3, 134.5, 129.8, 129.7, 128.9, 128.5, 108.8, 96.5, 62.1, 61.2, 60.2.

[0069] HRMS, calculated: C 18 H 16 Cl2NaO5: 405.0267 [M + Na] +; Detected value: 405.0276 [M +Na] + ..

[0070] Example 4. Synthesis of 3-fluoro-4-chloro-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Id)

[0071] The benzaldehyde compound in this embodiment adopts 3-fluoro-4-chlorophenyl.

[0072] The synthesis steps were the same as those in Example 1, except that 3-fluoro-4-chlorophenyl was used instead of 2-trifluoromethylphenyl.

[0073] The structural formula of the product is as follows:

[0074]

[0075] The test data of the product are as follows:

[0076] Yield: 53%, melting point: 135-136 °C;

[0077] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.52 (s, 1H), 7.90 (d, J =15.2 Hz,1H), 7.69 (d, J =15.6 Hz, 1H), 7.44 (d, J =8.0 Hz, 1H), 7.40 (d, J =9.6 Hz,1H), 7.34 (d, J =8.4 Hz, 1H), 6.29 (s, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.83(s, 3H).

[0078] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.4, 162.8, 160.6, 154.9, 136.1,136.0, 135.3, 131.1, 128.3, 125.0, 124.9, 115.5, 115.3, 108.6, 96.6, 61.9,61.3, 56.2.

[0079] HRMS, calculated: C 18 H 16 ClFNaO5: 389.0563 [M + Na]+ ; Detected value: 389.0582 [M +Na] + ..

[0080] Example 5. Synthesis of 3-fluoro-4-methoxy-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ie)

[0081] The benzaldehyde compound in this embodiment adopts 3-fluoro-4-methoxyphenyl.

[0082] The synthesis steps were the same as those in Example 1, except that 3-fluoro-4-methoxyphenyl was used instead of 2-trifluoromethylphenyl.

[0083] The structural formula of the product is as follows:

[0084]

[0085] The test data of the product are as follows:

[0086] Yield: 83%, melting point: 127-128 °C;

[0087] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.67 (s, 1H), 7.83 (dd, J =33.2 15.6Hz, 1H), 7.41 (dd, J =12.4 2.4 Hz, 1H), 7.35 (d, J =8.4 Hz, 1H), 6.98 (t, J =8.8 Hz, 1H), 6.28 (s, 1H), 3.93 (s, 6H), 3.90 (s, 3H), 3.83 (s, 3H).

[0088] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.6, 162.7, 160.2, 155.0, 153.7,151.2, 142.0, 135.3, 128.8, 126.0, 125.4, 114.8, 113.2, 108.7, 96.6, 61.9,61.3, 56.3, 56.1.

[0089] HRMS, calculated: C 19 H 20 FO6: 363.1238 [M + H]+ ; Detected value: 363.1230 [M + H] + ..

[0090] Example 6. Synthesis of 3,4-dimethyl-2´-hydroxy-4´,5´,6´-trimethoxychalcone (If)

[0091] The benzaldehyde compound in this embodiment adopts 3,4-dimethylphenyl.

[0092] The synthesis steps were the same as those in Example 1, except that 3,4-dimethylphenyl was used instead of 2-trifluoromethylphenyl.

[0093] The structural formula of the product is as follows:

[0094]

[0095] The test data of the product are as follows:

[0096] Yield: 84%, melting point: 125-126 °C;

[0097] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.73 (s, 1H), 7.93 (dd, J =43.6 15.6Hz, 2H), 7.40 (d, J =7.6 Hz, 2H), 7.18 (d, J =7.6 Hz, 1H), 6.30 (s, 1H), 3.93(s, 3H), 3.90 (s, 3H), 3.84 (s, 3H), 2.30 (d, J =2.8 Hz, 1H).

[0098] 13 C NMR (100 MHz, CDCl3): d (ppm) 193.0, 162.7, 160.0, 155.0, 143.7,139.7, 137.2, 135.3, 133.0, 130.3, 129.8, 126.1, 125.3, 108.8, 96.6, 62.0,61.3, 56.1, 19.9, 19.8.

[0099] HRMS, calculated: C 20 H 23 O5: 343.1540 [M + H] +; Detected value: 343.1530 [M + H] + ..

[0100] Example 7. Synthesis of 4-(2-pyridyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ig)

[0101] The benzaldehyde compound in this embodiment adopts 4-(2-pyridyl)phenyl.

[0102] The synthesis steps were the same as those in Example 1, except that 4-(2-pyridyl)phenyl was used instead of 2-trifluoromethylphenyl.

[0103] The structural formula of the product is as follows:

[0104]

[0105] The test data of the product are as follows:

[0106] Yield: 61%, melting point: 145-146 °C;

[0107] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.61 (s, 1H), 8.65 (d, J =5.2 Hz,1H), 8.01 (d, J =8.0 Hz, 2H), 7.96 (d, J =15.6 Hz, 1H), 7.82 (d, J =15.6 Hz,1H), 7.71-7.67 (m, 4H), 7.19 (s, 1H), 6.23 (s, 1H), 3.88 (s, 3H), 3.84 (s,3H), 3.78 (s, 3H).

[0108] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.9, 162.8, 160.3, 156.5, 155.0,149.9, 142.6, 141.1, 136.9, 135.9, 135.4, 128.9, 127.4, 127.0, 122.6, 120.6,108.8, 96.6, 62.0, 61.4, 56.2.

[0109] HRMS, calculated: C 23 H 22NO5: 392.1492 [M + H] + ; Detected value: 392.1496 [M + H] + ..

[0110] Example 8. Synthesis of 6-quinoline-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ih)

[0111] The benzaldehyde compound in this embodiment adopts 6-quinolinephenyl.

[0112] The synthesis steps are the same as those in Example 1, except that 6-quinolinylphenyl is used instead of 2-trifluoromethylphenyl.

[0113] The structural formula of the product is as follows:

[0114]

[0115] The test data of the product are as follows:

[0116] Yield: 60%, melting point: 150-151 ℃;

[0117] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.64 (s, 1H), 8.90 (dd, J =4.0 1.6Hz, 1H), 8.17 (d, J =8.0 Hz, 1H), 8.08 (t, J =8.8 Hz, 1H), 8.02-7.98 (m, 2H),7.95-7,94 (d, J =2.8 Hz 1H), 7.42 (dd, J =8.0 4.0 Hz, 1H), 6.27 (s, 1H), 3.94(s, 3H), 3.88 (s, 3H), 3.83 (s, 3H).

[0118] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.6, 162.7, 160.3, 154.9, 151.2,149.0, 142.0, 136.4, 135.3, 133.5, 130.2, 129.8, 128.3, 127.7, 127.3, 121.8,108.7, 96.5, 61.9, 61.3, 56.1.

[0119] HRMS, calculated: C 21 H 20 NO5: 366.1336 [M + H] + ; Detected value: 366.1330 [M + H] + ..

[0120] Example 9. Synthesis of 4-(4-morpholinyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ii)

[0121] The benzaldehyde compound in this embodiment adopts 4-(4-morpholinyl)phenyl.

[0122] The synthesis steps were the same as those in Example 1, except that 4-(4-morpholinyl)phenyl was used instead of 2-trifluoromethylphenyl.

[0123] The structural formula of the product is as follows:

[0124]

[0125] The test data of the product are as follows:

[0126] Yield: 73%, melting point: 151-152 °C;

[0127] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.85 (s, 1H), 7.83 (s, 2H), 7.58 (d, J =8.8 Hz, 2H), 6.91 (d, J =9.2 Hz, 2H), 6.29 (s, 1H), 3.92 (s, 3H), 3.90 (s,3H), 3.88 (t, J =4.8 Hz, 4H), 3.83 (s, 3H), 3.28 (t, J =4.8 Hz, 4H).

[0128] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.7, 162.6, 159.8, 155.0, 152.7,143.9, 135.3, 130.2, 126.3, 122.9, 114.7, 108.9, 96.6, 66.7, 62.0, 61.3,56.1, 48.0.

[0129] HRMS, calculated: C 22 H 26 NO6: 400.1755 [M + H] + ; Detected value: 400.1747 [M + H] + ..

[0130] Example 10. Synthesis of 4-(1-tetrahydropyrrolyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ij)

[0131] The benzaldehyde compound in this embodiment adopts 4-(1-tetrahydropyrrolyl)phenyl.

[0132] The synthesis steps were the same as those in Example 1, except that 4-(1-tetrahydropyrrolyl)phenyl was used instead of 2-trifluoromethylphenyl.

[0133] The structural formula of the product is as follows:

[0134]

[0135] The test data of the product are as follows:

[0136] Yield: 52%, melting point: 147-148 °C;

[0137] 1 H NMR (400 MHz, CDCl3): d (ppm) 14.06 (s, 1H), 7.91 (d, J =15.2 Hz,1H), 7.80 (d, J =15.6 Hz, 1H), 7.56 (d, J =8.4 Hz, 2H), 6.57 (d, J =8.4 Hz, 2H), 6.29 (s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.84 (s, 3H), 3.36 (s, 4H), 2.03 (s, 4H).

[0138] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.5, 162.5, 159.4, 154.9, 149.6,145.4, 135.2, 130.8, 122.5, 120.1, 111.8, 108.9, 96.6, 61.9, 61.3, 56.0,47.6, 25.5.

[0139] HRMS, calculated: C 22 H 26 NO5: 384.1805 [M + H] + ; Detected value: 384.1807 [M + H] + ..

[0140] Example 11. Synthesis of 4-(4-methylpiperazine-1-yl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Ik)

[0141] The benzaldehyde compound in this embodiment adopts 4-(1-tetrahydropyrrolyl)phenyl.

[0142] The synthesis steps were the same as those in Example 1, except that 4-(1-tetrahydropyrrolyl)phenyl was used instead of 2-trifluoromethylphenyl.

[0143] The structural formula of the product is as follows:

[0144]

[0145] The test data of the product are as follows:

[0146] Yield: 50%, melting point: 156-157 °C;

[0147] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.88 (s, 1H), 9.77 (s, 1H), 7.82 (s,1H), 7.75 (d, J =8.8 Hz, 1H), 6.92-6.89 (m, 2H), 6.28 (s, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H), 3.40 (t, J =5.2 Hz, 2H), 3.33 (t, J =5.2Hz, 2H),2.57 (dd, J =11.2, 6.0 Hz, 2H), 2.34 (s, 1H).

[0148] 13 C NMR (100 MHz, CDCl3): d(ppm) 190.4, 162.6, 159.7, 155.0, 144.1,135.3, 131.8, 130.2, 127.1, 125.7, 122.5, 114.8, 113.5, 108.9, 96.6, 61.9,61.31, 56.1, 54.8, 54.7, 47.7, 47.1, 46.2.

[0149] HRMS, calculated: C 23 H 29 N2O5: 413.2071 [M + H] + ; Detected value: 413.2088 [M + H] + ..

[0150] Example 12 Synthesis of 2-fluoro-4-methoxy-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Il)

[0151] The benzaldehyde compound in this embodiment adopts 2-fluoro-4-methoxyphenyl.

[0152] The synthesis steps were the same as those in Example 1, except that 2-fluoro-4-methoxyphenyl was used instead of 2-trifluoromethylphenyl.

[0153] The structural formula of the product is as follows:

[0154]

[0155] The test data of the product are as follows:

[0156] Yield: 68%, melting point: 124-125 ℃;

[0157] 1 H NMR (400 MHz, CDCl3): d (ppm) 12.34 (s, 1H), 7.81 (t, J =8.8 Hz,1H), 7.67 (dd, J =28.8 16 Hz, 2H), 7.00 (dd, J =13.2 2.4 Hz, 1H), 6.91 (dd, J =8.8 2.8 Hz, 1H), 6.39 (s, 1H), 3.85 (d, J =2.8 Hz, 3H), 3.70 (s, 3H), 3.34(s, 3H), 2.51 (s, 1H).

[0158] 13 C NMR (100 MHz, CDCl3): d (ppm) 192.6, 162.7, 160.2, 155.0, 153.7,151.2, 142.0, 135.3, 128.8, 126.0, 125.4, 114.8, 113.2, 108.7, 96.6, 61.9,61.3, 56.3, 56.1.

[0159] HRMS, calculated: C 19 H 19 FNaO6: 385.1058 [M + Na] + ; Detected value: 385.1005 [M +Na] + ..

[0160] Example 13 Synthesis of 2-fluoro-2´-hydroxy-4´,5´,6´-trimethoxychalcone (Im)

[0161] The benzaldehyde compound in this embodiment adopts 2-fluorophenyl.

[0162] The synthesis steps are the same as those in Example 1, except that 2-fluorophenyl is used instead of 2-trifluoromethylphenyl.

[0163] The structural formula of the product is as follows:

[0164]

[0165] The test data of the product are as follows:

[0166] Yield: 60%, melting point: 117-118 ℃;

[0167] 1 H NMR (400 MHz, CDCl3): d (ppm) 13.63 (s, 1H), 7.89 (dd, J =38.4,15.6 Hz, 2H), 7.63 (dd, J =8.8, 5.6 Hz, 2H), 7.12 (t, J =8.8 Hz, 2H), 6.29(s, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H).

[0168] 13 C NMR (100 MHz, CDCl3): d(ppm) 192.7, 165.2, 162.7, 160.3, 155.0,141.9, 135.3, 131.6, 130.3, 126.3, 116.2, 108.7, 96.6, 61.9, 61.3, 56.1.

[0169] HRMS, calculated: C 18 H 17 FNaO5: 355.0952 [M + Na] + ; Detected value: 355.0884 [M +Na] + ..

[0170] 2. Antitumor Activity Experiment of Chalcone Analogues

[0171] 1. Examples 14-26 are experiments for detecting the toxicity of chalcone analogs to HeLa cells, MGC-803 cells and HCT-116 cells using the MTT method.

[0172] MTT colorimetric method was used in the experiment. The culture medium used for culturing tumor cells: HeLa cells and HCT-116 cells used DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin mixture; MGC-803 cells used RPMI-1640 culture medium containing 10% FBS and 1% penicillin-streptomycin mixture.

[0173] Tumor cells were inoculated into 96-well plates, with 3-5×10 3 cells, add different concentrations of the chalcone analog synthetic product solution to be tested. After 48 hours of culture, add 20 μL of MTT test solution to each well. The MTT test solution is slightly yellow. Be careful to avoid light, and then put it in the incubator for 4 hours. Then take out the 96-well plate, aspirate the liquid in the well, add 150 μL of DMSO solution to each well to dissolve the crystals, place the 96-well plate on a micro-oscillator and shake it for 10 minutes to observe whether the crystals are completely dissolved. After dissolution, place it on an enzyme reader to detect absorbance. The wavelength of the enzyme reader is set to 570nm, and the OD value is measured.

[0174] Finally, the inhibition rate was calculated: inhibition rate % = 1-(drug group-blank well OD average) / (control group OD value-blank well OD average) × 100%.

[0175] All experiments were set up in three parallel groups or repeated three times.

[0176] The results of the cytotoxicity experiments of the synthetic products of chalcone analogs Ⅰa-Ⅰm are shown in Table 1.

[0177] .

[0178] In vitro experiments show that the chalcone analogs Ia-Im synthesized by the present invention exhibit good inhibitory activity against human cervical cancer cells (HeLa), human gastric cancer cells (MGC-803), and human colon cancer cells (HCT-116). Most of the compounds have higher anti-tumor activity than the control drugs CA-4 and cis-Platin, especially Ic and Im, which show higher inhibitory activity against the three tested cell lines, showing good application prospects.

[0179] 2. Example 27 is to detect the effect of chalcone analog Ic on MGC-803 cell colony formation by plate cell cloning experiment

[0180] The cells were washed with PBS, digested with trypsin, terminated with fresh complete medium, centrifuged and resuspended, counted with a cell counting plate, and then MGC-803 cells were inoculated in a 6-well culture plate at a cell density of 1500 cells per well, and an appropriate amount of fresh culture medium was added, and then incubated in an incubator at 37°C and 5% CO2 for 24 h; the next day, fresh culture medium containing different concentrations of compound Ic (0.25μM, 0.5 μM, 1 μM, 2 μM) was replaced, and a blank group and a fresh culture medium containing only 0.1% DMSO were set as the DMSO control group, and incubated in a 37°C, 5% CO2 cell culture incubator. The culture medium was replaced every three days, and the incubation was 9 days in total. When visible colonies appeared at the bottom of the culture dish, the culture medium was discarded, and the dish was washed twice with 1 mL PBS. The dish was fixed with 4% paraformaldehyde (POM) for 15 minutes, and then stained with 0.1% crystal violet for 15 minutes at room temperature. The dish was then washed again with PBS for several times. After the colonies were dried, they were photographed with a camera under good lighting conditions.

[0181] The effect of chalcone analog Ic on the proliferation ability of MGC-803 cells was successfully determined by plate cell cloning experiment. Figure 1 As shown, the different concentrations of chalcone analog synthesis product Ic (0.25μM, 0.5μM, 1μM, 2μM) in the drug-added group were compared with the blank group and the DMSO control group. It was found that with the continuous increase in the drug concentration, the area of ​​crystal violet in the culture dish became smaller and smaller, and the color gradually became lighter. Even when the drug concentration of chalcone analog synthesis product Ic was 2 μM, the crystal violet almost completely disappeared. The results show that chalcone analog synthesis product Ic can significantly inhibit the in vitro growth of gastric cancer MGC-803 cells in a dose-dependent manner.

[0182] 3. Example 28 shows the inhibitory effect of chalcone analogue synthetic product Ic on the migration and repair ability of MGC-803 cells

[0183] MGC-803 cells that were in good growth condition and in the logarithmic growth phase were plated. The cells were washed with PBS, trypsinized, terminated with fresh complete medium, centrifuged and resuspended, and then MGC-803 cells were plated at 2×10 5 Each group of cells was plated in a 6-well culture plate with 3 replicate wells. After culturing until a monolayer of cells was formed, the culture medium was changed to serum-free medium and starved for 4 h in a cell culture incubator at 37°C and 5% CO2. A 10 μL sterile pipette tip was used to make an I-shaped scratch along the middle of the bottom of the culture plate. The floating matter was washed with PBS to remove the floating matter. The culture medium containing different concentrations of chalcone analog synthetic product Ic (0.5 μM, 1 μM, 2 μM, 4 μM) was replaced. After adding the drug, the culture was continued for 48 h. The degree of healing of the scratched area was observed under a microscope, and photos were taken to calculate the cell migration rate.

[0184] The effect of chalcone analog Ic on the migration and repair ability of MGC-803 cells was successfully determined by cell wound healing experiments. The results are shown in Figure 2. On the one hand, the intuitive inverted microscopic imaging Figure 2A It is obvious from observation that the degree of wound healing in the drug-added group is significantly lower than that in the blank control group without drug addition. Figure 2B As shown in the figure, by comparing the difference in scratch width between 0 h and 48 h of traumatized MGC-803 cells, the cell migration rate of each drug concentration was accurately calculated (blank group: 34.38%; DMSO control group: 37.57%; drug-added group: 0.5 μM: 20.14%, 1 μM: 14.78%, 2 μM: 6.32%, 4 μM: 3.02%). With the increase of drug concentration, the cell migration rate gradually decreased. This shows that the inhibitory effect of the synthetic product Ic of chalcone analogs on the migration and repair ability of MGC-803 cells is dose-dependent.

[0185] 4. Example 29: Synthesis of chalcone analog Ic blocks MGC-803 cell cycle

[0186] Flow cytometry was used to detect whether the synthetic product of chalcone analog Ic could induce apoptosis of MGC-803 cells and block the tumor cell cycle.

[0187] Exponentially growing MGC-803 cells were plated. The cells were washed with PBS, trypsinized, and terminated with fresh complete medium. The cells were centrifuged and resuspended, and counted with a cell counting plate. The counted cells were then diluted to an appropriate concentration, with a concentration of about 1×10 5The cell concentration was 2 mL of cell suspension injected into a 6-well plate, and remember to shake it left and right to make the cells evenly dispersed and then placed in a cell culture incubator overnight. After 24 h, replace the fresh culture medium containing different concentrations of chalcone analog synthetic product Ic (0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM), set up a drug-free control group, and culture in a 37 ℃, 5% CO2 cell culture incubator for 48 h. The cell number was controlled at 1×10 5 -1×10 6 Carefully remove the cell culture medium, digest the cells with trypsin, and prepare a single cell suspension. Centrifuge at 1000 g for 5 min to pellet the cells, discard the supernatant, rinse the cells once with 1 mL of pre-cooled PBS, and collect the cells by centrifugation. Gently mix the cell pellet with 1 mL of pre-cooled 70% ethanol and fix at 4 ℃ for more than 2 h or overnight. Next, centrifuge at 1000 g for 5 min to pellet the cells, and resuspend them in 1 mL of pre-cooled PBS. Then centrifuge at 1000 g for 5 min again to pellet the cells. Add 10 μL of propidium iodide stock solution (40301-B) and 10 μL of RNaseA (40301-A) solution to 0.5 mL of staining buffer (40301-C), mix well and set aside. Add 0.5 mL of the prepared propidium iodide staining solution to each cell sample, gently mix and resuspend the cells. Incubate at 37 ℃ in the dark for 30 min, and then you can perform flow cytometry. Flow cytometry is best completed within 5 h. The prepared PI staining solution can be stored at 4 °C for a short period of time and should be used on the same day. The cells were filtered through a 400-mesh screen and detected by flow cytometry at an excitation wavelength of 488 nm and light scattering. Analysis software was used to analyze the cell DNA content and light scattering. All experimental groups were performed in parallel 3 times.

[0188] The results are shown in Figure 3, where the DNA in the cell nucleus is stained by binding to PI, and PI shows fluorescence after being stimulated by light of a certain wavelength, and its intensity is positively correlated with the DNA content. Figure 3A MGC-803 cells were treated with different concentrations of chalcone analogue synthetic product Ic (0, 0.5, 1, 2, 4, 8 μM) for 48 h (0 μM in Figure 3A The blank group is marked in the middle), the actual cell cycle distribution at different stages; Figure 3BIt is an intuitive statistical chart of the cell cycle distribution ratio, representing the blank group (G1: 74.18%, S: 20.94%, G2: 4.88%), when the drug concentration is 0.5 μM, (G1: 65.29%, S: 24.69%, G2: 10.02%); when the drug concentration is 1 μM, (G1: 52.18%, S: 32.15%, G2: 15.67%); when the drug concentration is 2 μM, (G1: 48.72%, S: 33.26%, G2: 18.01%); when the drug concentration is 4 μM, (G1: 43.66%, S: 39.26%, G2: 17.08%); when the drug concentration is 8 μM, (G1: 43.22%, S: 35.05%, G2: 21.73%). Comparison revealed that as the concentration of the chalcone analog synthetic product Ic increased, the proportion of MGC-803 cells distributed in the G2 phase gradually increased, indicating that the chalcone analog synthetic product Ic could block MGC-803 cells in the G2 / M phase in a dose-dependent manner, thereby possessing anti-tumor activity.

Claims

1. Use of chalcone analogs as active substances in the preparation of anti-tumor drugs, characterized in that: The chalcone analogs are selected from the following compounds: 2-trifluoromethyl-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 4-(1H-imidazol-1-yl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 2, 6-dichloro-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 3-fluoro-4-chloro-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 3-fluoro-4-methoxy-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 3,4-dimethyl-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 4-(2-pyridyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 6-quinoline-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 4-(4-morpholinyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 4-(1-tetrahydropyrrolyl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 4-(4-methylpiperazin-1-yl)-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 2-fluoro-4-methoxy-2´-hydroxy-4´,5´,6´-trimethoxychalcone, 2-fluoro-2´-hydroxy-4´,5´,6´-trimethoxychalcone; the tumor is human cervical cancer, human colon cancer or human gastric cancer.

2. The use of the chalcone analogue as an active substance in the preparation of an anti-tumor drug according to claim 1, characterized in that: The analog is used for preparing medicine for treating human cervical cancer.

3. The use of the chalcone analogue as an active substance in the preparation of an anti-tumor drug according to claim 1, characterized in that: The analog is used for preparing medicine for treating human colon cancer.

4. The use of the chalcone analogue as an active substance in the preparation of an anti-tumor drug according to claim 1, characterized in that: The analog is used for preparing medicine for treating human gastric cancer.

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