Compound with antitumor activity and application thereof

Abstract

The invention discloses a compound with antineoplastic activity, the structural general formula of the compound is shown as follows: the compound prepared by the invention is a targeted tubulin colchicine binding site inhibitor, has obvious in-vitro anti-proliferative activity on MCF-7, MDA-MB-231, A549, Hela, A375 and B16-F10 tumor cells, and can obviously inhibit invasion of the MCF-7 cells. In addition, the compounds of the present invention inhibit tubulin polymerization in vitro and destroy the microtubule network of MCF-7 cells. In addition, the compounds of the present invention can arrest the mitosis cycle of cells in the G2 / M period. The compound disclosed by the invention can also remarkably inhibit tumor growth in a mouse body, and has relatively small toxic and side effects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, in particular to a compound with anti-tumor activity and its application. BACKGROUND

[0002] Malignant tumor (cancer) is one of the most serious and common diseases threatening human health. In 2020, there were about 19.3 million new cancer cases worldwide, and about 9.9 million cancer-related deaths, and the incidence and mortality are expected to continue to rise in the next ten years, which has become a global health problem (CA Cancer J Clin, 2021, 71(3): 209-49.).

[0003] Among the commonly used anti-tumor drugs in clinical practice, plant-derived compounds such as paclitaxel, vinblastine and colchicine bind to tubulin, interfere with microtubule polymerization and dynamics, and block mitosis, thereby inhibiting tumor cell proliferation and inducing apoptosis (Cancer Metastasis Rev, 2024, 43(1): 197-228.). However, these drugs are often accompanied by adverse reactions such as myelosuppression, neurotoxicity and multidrug resistance, and there is an urgent need to develop more efficient and less toxic new microtubule inhibitors.

[0004] Microtubules are dynamic polymers composed of highly conserved α- and β-tubulin heterodimers, which are involved in key life activities such as cell structure support, intracellular transport, mitosis and signal transduction. Among them, the stability of microtubules is regulated by GTP / GDP state, and its abnormal dynamics is closely related to various diseases, especially the sustained proliferation of cancer cells and the formation of drug resistance (Cells, 2019, 8(10.).

[0005] Indole skeleton is an important lead structure for designing new microtubule polymerization inhibitors due to its unique planar aromatic structure and easy chemical modification. In recent years, many studies have reported that indole derivatives can bind to the colchicine binding site, disrupt microtubule dynamics, induce G2 / M phase arrest in cancer cells, and trigger apoptosis, showing good anti-proliferative activity and low toxicity potential (J Enzyme Inhib Med Chem, 2018, 33(1): 1225-38.). On this basis, the introduction of 3,4,5-trimethoxyphenyl side chain can significantly enhance the affinity to the colchicine binding site. For example, ABI-231 series of modified molecules have been confirmed to disrupt microtubule polymerization and inhibit tumor cell proliferation in in vitro experiments (Journal of Medicinal Chemistry, 2019, 62(14): 6734-50.). Similarly, 6-aryl-3-aroylindole analogs also show significant inhibition of multiple human cancer cell lines (Eur J Med Chem, 2024, 263: 115794.). More and more types of indole derivatives have shown excellent activity in anti-microtubule polymerization, and the prior art does not involve the compound with anti-tumor activity of the present application. SUMMARY

[0006] The purpose of the present application is to provide a compound with anti-tumor activity.

[0007] Another purpose of the present application is to provide the use of the compound with anti-tumor activity in the preparation of an anti-tumor drug.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] In a first aspect of the present application, a compound with anti-tumor activity is provided, and the structure general formula is as follows:

[0010]

[0011] wherein R1 is selected from ;

[0012] R2 is selected from ;

[0013] R3 is selected from ;

[0014] R4 is selected from ;

[0015] R5 is selected from hydrogen, CN, -OCF3, halogen (fluorine, chlorine, bromine, iodine), C1-C30 alkyl, -SR 10 , ;

[0016] R6is selected from the group consisting of hydrogen, CN, -OCF3, halogen (fluorine, chlorine, bromine, iodine), C1-C30alkyl, -SR 10 , ;

[0017] R7is selected from the group consisting of hydrogen, CN, -OCF3, halogen (fluorine, chlorine, bromine, iodine), C1-C30alkyl, -SR 10 , ;

[0018] R8is selected from the group consisting of hydrogen, CN, -OCF3, halogen (fluorine, chlorine, bromine, iodine), C1-C30alkyl, -SR 10 , ;

[0019] R9is selected from the group consisting of hydrogen, CN, -OCF3, halogen (fluorine, chlorine, bromine, iodine), C1-C30alkyl, -SR 10 , ;

[0020] R 10 is selected from the group consisting of C1-C30alkyl.

[0021] More preferably, in the compounds having antitumor activity,

[0022] R5is selected from the group consisting of hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ;

[0023] R6is selected from the group consisting of hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ;

[0024] R7is selected from the group consisting of hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ;

[0025] R8is selected from the group consisting of hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ;

[0026] R9is selected from the group consisting of hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, .

[0027] Most preferably, the structure of the compound having antitumor activity is selected from one of the following structures:

[0028]

[0029] In a second aspect of the present application, there is provided a use of the compound having antitumor activity or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

[0030] The tumor is selected from breast cancer, non-small cell lung cancer, cervical cancer, melanoma.

[0031] The breast cancer cell is selected from MCF-7, MDA-MB-231.

[0032] The melanoma cell is selected from A375 cells, B16-F10 cells.

[0033] The compound of the present application has an inhibitory effect on the polymerization of tubulin, inhibits the growth of tumor cells by destroying the dynamic balance of tubulin and collapsing the tubulin network, and can be used as an antitumor drug.

[0034] Due to the above technical solutions, the present application has the following advantages and beneficial effects:

[0035] The pharmacological tests show that the compound prepared by the present application exhibits good in vitro antitumor activity at a drug concentration of 50 μM, and the compounds 3a, 3b, 3c, 3d, 3f, 3g and 3n exhibit good in vitro antitumor activity. The IC 50 values of the compound 3g on MCF-7 cells, MDA-MB-231 cells, A549 cells, Hela cells, A375 cells and B16-F10 cells are 2.94, 1.61, 6.30, 6.10, 0.57 and 1.69 μM, respectively. 50

[0036] ​The results of the in vitro microtubule polymerization experiment of the preferred compound 3g show that the compound 3g can significantly inhibit the in vitro microtubulin polymerization when the concentration of the compound reaches 25 μM. Meanwhile, the results of the cell scratch healing test and the Transwell test show that the compound 3g can effectively inhibit the invasion and migration of tumor cells. In addition, the results of the immunofluorescence test and the cell clone formation test show that the compound 3g can disintegrate the microtubule network of tumor cells and significantly inhibit the clone formation of tumor cells. The apoptosis of tumor cells is also analyzed and detected by flow cytometry, and the results show that the compound 3g (at a concentration of 10 μM) can effectively induce cell apoptosis after treating the cells for 48 hours.

[0037] The compound prepared in the present application is a microtubulin colchicine binding site inhibitor, has significant in vitro anti-proliferation activity on MCF-7, MDA-MB-231, A549, Hela, A375 and B16-F10 tumor cells, and can significantly inhibit the invasion of MCF-7 cells. In addition, the compound of the present application can inhibit the polymerization of microtubulin in vitro and destroy the microtubule network of MCF-7 cells. Moreover, the compound of the present application can also make the cell mitotic cycle arrest at the G2 / M phase. The compound of the present application can also significantly inhibit the growth of tumors in mice in vivo, and has less toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of the in vitro inhibition rate of MCF-7 cells when the concentration of the compound prepared in the present application is 50 μM.

[0039] Figure 2 is a schematic diagram of the inhibition of microtubulin polymerization by compound 3g at different concentrations.

[0040] Figure 3 is a schematic diagram of the effect of compound 3g at different concentrations on the migration of MCF-7 cells.

[0041] Figure 4 is a schematic diagram of the immunofluorescence results of the effect of compound 3g at different concentrations on the microtubules of MCF-7 cells.

[0042] Figure 5 is a schematic diagram of the inhibition of clone formation of MCF-7 cells by compound 3g at different concentrations.

[0043] Figure 6 is a schematic diagram of the anti-invasion ability of MCF-7 cells by compound 3g at different concentrations.

[0044] Figure 7 is a schematic diagram of the effect of compound 3g at different concentrations on the apoptosis of MCF-7 cells.

[0045] Figure 8Figure 1 is a schematic diagram showing the results of the effect of different concentrations of compound 3g on the growth of melanoma tumors in mice.

[0046] Figure 9 Figure 2 is a schematic diagram showing the results of histopathological analysis. DETAILED DESCRIPTION

[0047] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the specific descriptions below are illustrative rather than limiting, and should not limit the scope of protection of the present application.

[0048] Example 1

[0049] The preparation of compounds 3a-3p of the present application is as follows:

[0050]

[0051] First step, preparation of intermediate 2

[0052] Indole-6-boronic acid (500 mg, 3.11 mmol), iodobenzene (633.69 mg, 3.11 mmol), tetrakis triphenylphosphine palladium (717.89 mg, 0.62 mmol), sodium carbonate (658.46 mg, 6.21 mmol), 1,4-dioxane (8 mL) and water (4 mL) were sequentially added to a pear-shaped reaction flask, and reacted at 80°C overnight. The reaction was monitored by TLC, and after the reaction was completed, the organic phase was extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain white solid, i.e., intermediate 2 (272 mg), with a yield of 45.3%.

[0053] Second step, preparation of compound 3a

[0054] Intermediate 2 (78.85 mg, 0.41 mmol), 5-iodo-1,2,3-trimethoxybenzene (100 mg, 0.34 mmol), cuprous iodide (12.57 mg, 0.07 mmol), cesium carbonate (221.58 mg, 0.68 mmol) and N,N-dimethylformamide 6 mL were sequentially added to a 25 mL pear-shaped flask, and reacted at 150°C for 16 h under argon protection. After the reaction was completed, the organic phase was extracted with ethyl acetate three times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain pink solid, i.e., compound 3a (80.9 mg), with a yield of 57.3%.

[0055] The preparation method of compounds 3b-3p is the same as above. The raw material iodobenzene in the first step is replaced by m-iodobenzonitrile, m-iodotoluene, 1-ethyl-2-iodobenzene, 1-ethyl-4-iodobenzene, 1-iodo-2-isopropylbenzene, 5-iodo-2-methylbenzonitrile, 3-(trifluoromethoxy)iodobenzene, 1-n-butyl-4-iodobenzene, 4-iodobenzyl sulfide, 1-iodo-4-isopropylbenzene, o-iodobenzonitrile, p-iodobenzonitrile, 3-fluoro-4-iodobenzonitrile, 4-iododiphenyl ether, 2-iodobenzothiazole, and the others are the same as in Example 1, to obtain compounds 3b-3p, respectively.

[0056] The structure, yield, nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum data of the compound prepared in Example 1 of the present application are shown in Table 1:

[0057] Table 1 Yield, nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum data of the compound prepared in the present application

[0058]

[0059]

[0060]

[0061]

[0062] Example 2

[0063] Pharmacological experiment of the compound prepared in Example 1 of the present application

[0064] I. In vitro anti-tumor activity test of the compound

[0065] 1. Test method

[0066] The CCK-8 method is used to evaluate the inhibitory effect of the compound on tumor cell proliferation. Human breast cancer cells MCF-7, human breast cancer cells MDA-MB-231, human non-small cell lung cancer cells A549, human cervical cancer cells Hela cells, human malignant melanoma cells A375 cells and mouse melanoma cells B16-F10 cells are selected. 100 μL (5×10 3 cells / well) of cell suspension is added to a 96-well cell culture plate, which is placed in a carbon dioxide incubator for 24 hours. The original culture medium is removed, and 100 μL of fresh culture medium containing different concentrations of drugs is added, with 6 replicate wells for each concentration. At the same time, 6 replicate wells of control group (with cells) and blank group (without cells) are set up. The plate is placed in a carbon dioxide incubator for continuous culture for 48 hours. The culture plate is taken out, and 10 μL of CCK-8 reagent is added to each well. The 96-well plate is placed in a carbon dioxide incubator for continuous culture for 90 minutes. The absorbance value is measured by enzyme label instrument at 450 nm, and the inhibition rate of the drug on cell proliferation is calculated.

[0067] Inhibition rate = [(OD 对照孔 - OD 实验孔 ) / (OD 对照孔 - OD 空白孔 )] x 100%.

[0068] Test results: the inhibitory activity of the target compounds on MCF-7 cells is shown in Table 1. Figure 1 Figure 1 is a schematic diagram of the in vitro inhibition rate of MCF-7 cells when the concentration of the compound prepared by the present application is 50 μM. As can be seen from the figure, when the concentration is 50 μM, the compound of the present application has good in vitro anti-proliferation activity on MCF-7 cells. Based on the above screening results, the target compounds 3a, 3b, 3c, 3d, 3f, 3g, 3n with better activity are selected for further research.

[0069] 2. IC 50 (half inhibitory concentration) test of preferred compounds

[0070] MCF-7 cells, MDA-MB-231 cells, A549 cells, Hela cells, A375 cells and B16-F10 cells are selected for testing, and 100, 50, 25, 10, 5, 2.5, 1 μM concentrations are set respectively, 6 replicate wells are set for each concentration, and control groups and blank groups are set for 6 replicate wells. Put in the carbon dioxide incubator and continue to culture for 48 hours. Take out the culture plate, add 10 μL of CCK-8 reagent to each well. Put the 96-well plate in the carbon dioxide incubator and continue to culture for 90 minutes. Measure the absorbance value at 450 nm by enzyme label instrument, and calculate the inhibition rate of the drug on cell proliferation.

[0071] Test results: the IC 50 values of the target compounds 3a, 3b, 3c, 3d, 3f, 3g, 3n on MCF-7 cells, MDA-MB-231 cells, A549 cells, Hela cells, A375 cells and B16-F10 cells are shown in Table 2. By comparing their activities, it is found that compound 3g shows better activity on the selected tumor cell lines. Therefore, compound 3g has the strongest in vitro anti-tumor activity, and the preferred compound 3g is further researched.

[0072] Table 2 IC 50

[0073]

[0074] II. In vitro microtubule polymerization experiment of compound 3g

[0075] ​Experimental Methods: Porcine brain tubulin was dissolved in EM buffer containing 5% glycerol (80 mM PIPES, 2 mM GTP, 2 mM MgCl2, 0.5 mM EGTA, pH 6.9). 3 g of the compound was serially diluted with EM buffer to 50, 25, 10, 5, and 2.5 μM. The negative control was 0.1% DMSO, and the positive controls were colchicine (5 μM) and paclitaxel (5 μM). 14 μL of the tubulin-GTP mixture was added to each well of a 384-well plate along with equal volumes of the compound solutions, with three replicates per concentration. The plates were incubated at 37°C, and absorbance (OD) was measured at 340 nm every minute. 340 ( ), lasting 60 minutes. Absorbance change rate (ΔOD) 340 ( / min) is used to characterize the microtubule polymerization rate.

[0076] Experimental results: The results of the in vitro microtubule polymerization experiment of 3g of compound are as follows Figure 2 As shown, Figure 2 This diagram illustrates the inhibitory effects of different concentrations of compound 3g on tubulin polymerization. As shown, colchicine (the positive control) inhibited tubulin polymerization, while paclitaxel promoted it. 0.1% DMSO had no significant effect on tubulin polymerization. When the concentration of compound 3g was 50 μM, it significantly inhibited in vitro tubulin polymerization in a concentration-dependent manner.

[0077] III. Effects of Compound 3g on MCF-7 Cell Migration

[0078] Experimental Methods: The effect of 3g of the target compound on the migration of MCF-7 cells was determined using a scratch assay. MCF-7 cells cultured to the logarithmic growth phase were collected and their concentration adjusted to a specific level (MCF-7 cell concentration: 1.0 × 10⁻⁶) using high-glucose complete medium containing 10% fetal bovine serum. 3 To prepare a 6-well cell culture plate, add 1 mL of cell suspension and 1 mL of 10% fetal bovine serum high-glucose complete medium to each well. Incubate the plate in a CO2 incubator for 24 hours. Scratch the plate with a 200 μL pipette tip to remove the original medium. Wash twice with sterile PBS to remove cell debris and scraped cells. Add 2 mL of fresh medium containing 0.1, 1, and 10 μM of the drug, with three replicates for each concentration and three replicates for the control group. Obtain images at 0 hours using a fluorescence inverted microscope. Continue incubation in a CO2 incubator, obtaining images at 12 and 24 hours using a fluorescence inverted microscope. Measure the migration distance of cells in the scratched area using ImageJ.

[0079] Experimental results: The effect of compound 3g on MCF-7 cell migration is as follows:Figure 3 As shown, Figure 3 This diagram illustrates the effects of different concentrations of compound 3g on MCF-7 cell migration. Figure A shows the effect of compound 3g on scratch wounds on MCF-7 cells, and Figure B shows the wound healing rate 24 hours after treatment with different concentrations of compound 3g. Figure A shows that, compared to the untreated group, treatment with compound 3g significantly inhibited scratch closure, indicating impaired lateral cell motility. Compound 3g inhibited the migration ability of MCF-7 cells in a dose-dependent manner. Figure B shows that control group cells were able to migrate into the wound channel, restoring 23±1.10% of the area. After treatment with 0.1, 1, or 10 μM of compound 3g, only 7.10±3.54%, 4.70±2.15%, and 1.94±1.20% of MCF-7 cells migrated to the wound area, respectively. These results indicate that compound 3g can inhibit tumor cell migration.

[0080] IV. Immunofluorescence staining analysis

[0081] Experimental Methods: Immunofluorescence staining was used to analyze the effect of 3g of the target compound on microtubule protein in MCF-7 cells. MCF-7 cells cultured to the logarithmic growth phase were collected and their concentration adjusted to a specific level (MCF-7 cell concentration 1.0 × 10⁻⁶) using high-glucose complete medium containing 10% fetal bovine serum. 5 Add 1 mL of cell suspension and 1 mL of high-glucose complete medium containing 10% fetal bovine serum to each well of a confocal culture dish with a glass bottom. Incubate the culture plate in a CO2 incubator for 24 hours. Remove the original medium, wash twice with sterile PBS to remove cell debris, and add 2 mL of fresh medium containing 0.1, 1, and 10 μM of the drug, with 3 replicates for each concentration and 3 replicates for the control group. Incubate in a CO2 incubator for 24 hours. Discard the old medium and wash three times with PBS. Add 1 mL of 4% paraformaldehyde to each dish and fix on a shaker for 20 min. Discard the fixative and wash three times with PBS. Add 1 mL of immunopermeation buffer to each dish and permeate at room temperature for 10 min on a shaker. Discard the permeation buffer and wash three times with PBS. Add goat serum and block slowly on a shaker for 30 min. Wash away the blocking buffer, without washing with PBS, add sufficient primary antibody to each glass dish, and incubate overnight at 4°C. Remove the primary antibody, add PBST, and wash on a shaker for 5 min, repeating three times. Add diluted fluorescent secondary antibody to the glass dish and incubate at room temperature in the dark for 1 h. Remove the fluorescent secondary antibody, add PBST, and wash on a shaker for 5 min, repeating three times. Add DAPI staining solution and incubate at room temperature in the dark for 5–10 min. After incubation, add PBST and wash on a shaker in the dark for 5 min, repeating three times to remove excess DAPI staining solution. Observe and photograph under a microscope.

[0082] Results: The results of the effect of compound 3g on microtubules in MCF-7 cells are shown in Figure 4 Figure 4 Figure 3 is a schematic diagram of the immunofluorescence results of the effect of different concentrations of compound 3g on microtubules in MCF-7 cells. As can be seen from the figure, in the control group, the microtubule network is normally arranged, and the fine fibrous microtubules (red) surround the nucleus (blue). However, after the cells were exposed to compound 3g (0.1, 1 and 10 μM) for 24 hours, the microtubule network in the cytoplasm was destroyed, and the fluorescence signal was weakened. These results indicate that compound 3g can cause the disintegration of the microtubule network of tumor cells.

[0083] V. Effect of compound 3g on the colony formation of tumor cells

[0084] Method: The effect of target compound 3g on the colony formation of MCF-7 cells was determined by a colony experiment. MCF-7 cells cultured to the logarithmic growth phase were collected, and the cell concentration was adjusted to a certain concentration (MCF-7 cell concentration was 1.0 x 10 3 6 cells per well, and 1 mL of 10% fetal bovine serum high-glucose complete culture medium was added. The culture plate was placed in a carbon dioxide incubator for 24 hours. The original culture medium was removed, and the cells were washed twice with sterile PBS to remove cell debris, 2 mL of fresh culture medium containing 0.1, 1 and 10 μM of the drug was added, 3 replicate wells were set for each concentration, and 3 blank wells were set at the same time. After incubation at 37°C in a 5% CO2 incubator for 24 hours, the culture medium was replaced with normal culture medium. After another 10-14 days of culture (the culture medium was replaced every three days), the cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 15 minutes. After washing with PBS, the cells were photographed and quantified using Image J.

[0085] Results: The results of the effect of compound 3g on the colony formation of MCF-7 cells are shown in Figure 5 Figure 5 Figure 4 is a schematic diagram of the results of the inhibitory effect of different concentrations of compound 3g on the colony formation of MCF-7 cells. A is a schematic diagram of the results of the inhibition of MCF-7 cell colony formation by compound 3g, and B is a schematic diagram of the percentage of the inhibition of MCF-7 cell colony formation by different concentrations of compound 3g compared with the control group. As can be seen from Figure A, compound 3g significantly inhibited colony formation at a concentration of 10 μM, indicating that compound 3g can inhibit the formation of cell colonies in a dose-dependent manner. As can be seen from Figure B, as the concentration increases, the number of cell colonies decreases, indicating that compound 3g can inhibit the proliferation of breast cancer cells MCF-7.​​

[0086] Six, the effect of compound 3g on tumor cell invasion

[0087] Test method: Transwell experiment was used to determine the effect of target compound 3g on the invasion ability of MCF-7 cells. MCF-7 cells cultured to the logarithmic growth phase were collected, and the cell concentration was adjusted to a certain concentration (MCF-7 cell concentration was 5x10 5 Each well of a 6-well cell culture plate was added with 1 mL of cell suspension and 1 mL of high-sugar complete culture medium containing 10% fetal bovine serum. The culture plate was placed in a carbon dioxide incubator for 24 hours. The original culture medium was removed, and the cells were washed twice with sterile PBS to remove cell debris, and 2 mL of fresh culture medium containing 0.1, 1 and 10 μM of the drug was added, with 3 replicate wells for each concentration, and 3 blank wells were set up at the same time. Incubate in a 37°C, 5% CO2 incubator for 48 hours.

[0088] After each well of cells was digested separately to form a single cell suspension, the cells were treated with serum-free medium and seeded into the upper chamber of the transwell chamber (8 micron pore size, Millipore), and 600 μL of high-sugar complete culture medium containing 10% fetal bovine serum was added to the bottom chamber, and then placed in an incubator for 48 hours. Remove the culture medium in the upper chamber and wash twice with sterile PBS. Add 200 μL of 4% paraformaldehyde to each well and fix for 20 min. Discard the fixing solution and wash three times with PBS. Add 200 μL of crystal violet staining solution to each well and stain for 10 min. Use a cotton swab to wipe off the cells that have not migrated in the upper chamber, and then observe and count the number of migrated cells under a microscope.

[0089] Test results: Transwell experiment is a classic method for evaluating the migration and invasion ability of tumor cells. The results of the effect of compound 3g on the invasion of MCF-7 cells are shown in Figure 6 . Figure 6 is a schematic diagram of the results of the anti-invasion ability of MCF-7 cells treated with different concentrations of compound 3g. As can be seen from the figure, there are more cells on the lower surface of the transwell chamber in the control group. In contrast, fewer cells of compound 3g invade the lower surface of the transwell chamber, which indicates that the invasion of MCF-7 cells is inhibited after treatment with compound 3g. It is further indicated that compound 3g has anti-invasion and anti-migration ability on breast cancer cells MCF-7.

[0090] Seven, the effect of compound 3g on tumor cell cycle

[0091] Test method: flow cytometry (PI staining method) was used to determine the effect of target compound 3g on MCF-7 cell cycle. MCF-7 cells cultured to the logarithmic growth phase were collected, and the cell concentration was adjusted to a certain concentration (MCF-7 cell concentration was 5 x 10 5 The culture plate was placed in a carbon dioxide incubator for 24 hours. The original culture medium was removed, and the cells were washed twice with sterile PBS to remove cell debris, 2 mL of fresh culture medium containing 0.1, 1 and 10 μM drug was added, 3 replicates for each concentration, and 3 blank groups were set up, and cultured in a 37°C, 5% CO2 incubator for 48 hours. The cell culture solution was aspirated into a centrifuge tube, and the adherent cells were washed once with PBS, 1 mL of trypsin without EDTA was added for 1 minute at room temperature, and the trypsin cell digestion solution was aspirated to avoid excessive digestion. Add the original culture medium and blow the cells, transfer to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells. Add 1 mL of pre-cooled PBS, resuspend the cells, and transfer to a 1.5 mL centrifuge tube. Centrifuge the cells again, carefully aspirate the supernatant to avoid aspirating the cells. Add 1 mL of pre-cooled 70% ethanol, mix gently, and fix overnight at 4°C. The fixed cells were washed once with pre-cooled PBS, and then 0.5 mL of propidium iodide (PI) staining solution was added to each cell sample, and the cell pellet was resuspended slowly and thoroughly using a vortex shaker, and stained at 37°C for 30 minutes in the dark. Then, the red fluorescence signal was detected at an excitation wavelength of 488 nm using a flow cytometer, and the light scattering was also detected.

[0092] Test results: cell cycle experiment is a key method for evaluating the effect of drugs on tumor cell proliferation, which is usually detected by flow cytometry to detect cell cycle distribution (G0 / G1, S, G2 / M phase). The effect of compound 3g on MCF-7 cell cycle is shown in Figure 7 Figure 7 The effect of different concentrations of compound 3g on MCF-7 cell apoptosis is shown in the figure. As can be seen from the figure, compound 3g regulates cell cycle in a concentration-dependent manner, and compound 3g at a concentration of 10 μM shows the most significant effect (52%), and MCF-7 cells accumulate significantly in G2 / M phase, which is significantly higher than that of the control group (18%). Compound 3g at a concentration of 1 μM caused an increase in G2 / M phase (44%), but the effect was weaker than that of compound 3g at a concentration of 10 μM.

[0093] Eight, in vivo anti-tumor activity research

[0094] ​Test method: BALB / c-nu athymic nude mice, male, 16-20 g, SPF level. All animals were formally tested in a pathogen-free environment with a temperature of 21-26°C and humidity of 40-70% for one week to adapt to the growth environment. After adapting to the environment for one week, the tumor model was established by subcutaneously inoculating melanoma cells B16-F10. The specific steps are as follows: B16-F10 cells in the logarithmic growth phase were suspended in PBS at a concentration of 4 x 106cells / mL, and 200 μL (containing 8 x 106cells) was injected into the right axillary of the mice. After inoculation, 48 mice were randomly divided into 4 groups, 12 in each group, and the experimental grouping was as follows: Control group, Paclitaxel group, Compound 3g low-dose group (20 mg / kg), and Compound 3g high-dose group (50 mg / kg). The drug was administered by intraperitoneal injection, and the dose was paclitaxel 10 mg / kg, Compound 3g 20 mg / kg and 50 mg / kg, respectively, once a day, for 14 consecutive days. The body weight of the mice was monitored daily during the experiment to assess acute toxicity. The tumor volume was measured with a vernier caliper, and the tumor volume (mm3) was calculated according to the following formula: Tumor volume (mm3) = a x b x 0.5, where a is the maximum diameter of the tumor and b is the minimum diameter perpendicular to a. The mice were sacrificed on the 14th day after the first administration, and the tumors were peeled off and weighed. The tumor growth inhibition rate (TGI) was calculated according to the following formula: TGI (%) = [1-(Wt / Wc)] x 100%, where Wt is the average tumor weight of the treatment group and Wc is the average tumor weight of the control group. At the same time, the liver and kidney and other major organs were taken and fixed with 4% paraformaldehyde, and the routine paraffin embedding treatment was performed. After HE staining, the histological changes were observed under a microscope to further evaluate the systemic toxicity of the drug. 6 5 3 2 t v t v

[0095] Test results: The in vivo anti-tumor efficacy of Compound 3g was further evaluated in a mouse melanoma model established by subcutaneous injection of B16-F10 cells. The results are shown in Table 1. Figure 8 Figure 8 ​​​​​​​​​is the result diagram of the influence of different concentrations of compound 3g on the growth of melanoma tumors in mice. Among them, A is the tumor size diagram of mice after 12 days of treatment, B is the weight change diagram of mice during treatment, C is the weight distribution diagram of the excised tumors of mice in each group, and D is the tumor volume change diagram of mice during treatment. As can be seen from A, the tumor size of mice using compound 3g at different concentrations is significantly smaller than that of the control group, indicating that compound 3g has good anti-tumor activity, but the activity is slightly worse than that of paclitaxel; as can be seen from B, the use of compound 3g at a concentration of 20 mg / kg and 50 mg / kg does not cause significant weight loss compared with paclitaxel, indicating that the compound has better tolerance than paclitaxel; as can be seen from C, the use of compound 3g at different concentrations causes the tumor weight of mice to decrease significantly compared with the control group, indicating that compound 3g has good anti-tumor activity, but the activity is slightly worse than that of paclitaxel; as can be seen from D, the use of compound 3g at different concentrations causes the tumor volume of mice to be smaller than that of the control group, indicating that compound 3g has anti-tumor growth activity, but the anti-tumor activity is slightly worse than that of paclitaxel.

[0096] The histopathological analysis of the main organs was performed by H&E staining, and the results are shown in Figure 9 . Figure 9 is a result diagram of histopathological analysis. As can be seen from the figure, the liver and kidney of the administered animals do not show obvious toxicological changes, further confirming that compound 3g has good safety at the tested dose.

[0097] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiments, it is not intended to limit the present application, and any skilled person in the art can make some changes or modifications to the above-mentioned technical content as equivalent embodiments with slight changes or modifications without departing from the technical solution of the present application. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the present application.

Claims

1. A compound having antitumor activity, characterized in that, The structural general formula is shown as follows: ； wherein R1is selected from ; R2is selected from ; R3is selected from ; R4is selected from ; R5is selected from hydrogen, CN, -OCF3, halogen, C1-C30alkyl, -SR 10 , ; R6is selected from hydrogen, CN, -OCF3, halogen, C1-C30alkyl, -SR 10 , ; R7is selected from hydrogen, CN, -OCF3, halogen, C1-C30alkyl, -SR 10 , ; R8is selected from hydrogen, CN, -OCF3, halogen, C1-C30alkyl, -SR 10 , ; R9is selected from hydrogen, CN, -OCF3, halogen, C1-C30alkyl, -SR 10 , ; R 10 selected from C1-C30alkyl.

2. The compound according to claim 1, having antitumor activity, characterized by, The compound with antitumor activity is selected from one of the following structures: R5is selected from hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ; R6is selected from hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ; R7is selected from hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ; R8is selected from hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, ; R9is selected from hydrogen, CN, -OCF3, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, -SCH3, -SCH2CH3, .

3. The compound according to claim 2, having antitumor activity, characterized by, The compound with antitumor activity is selected from one of the following structures: 。 4. Use of the compound with antitumor activity or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of an antitumor drug.

5. Use according to claim 4, characterized in that, The tumor is selected from breast cancer, non-small cell lung cancer, cervical cancer, melanoma.

6. Use according to claim 5, characterized in that, The breast cancer cell is selected from MCF-7, MDA-MB-231.

7. Use according to claim 5, characterized in that, The melanoma cell is selected from A375 cells, B16-F10 cells.

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