Targeted colchicine binding site tubulin inhibitor and application thereof

Abstract

The invention discloses a colchicine binding site-targeted tubulin inhibitor, the structural general formula of which is selected from one of the following structures: the compound prepared by the invention is the colchicine binding site-targeted tubulin inhibitor, has obvious in-vitro anti-proliferative activity on MCF-7, MDA-MB-231, A549 and Hela tumor cells, and can obviously inhibit the invasion of 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 compound provided by the invention can also enable the mitosis cycle of cells to stop at a G2 / M phase, and induce apoptosis of tumor cells. 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] This invention belongs to the field of pharmaceutical technology, specifically, it relates to a microtubule inhibitor that targets the colchicine binding site and its application. Background Technology

[0002] Microtubules play a crucial role in the cytoskeleton, participating in various physiological processes such as maintaining cell morphology, cell division, spindle formation, and intracellular transport. Disruption of the microtubule polymerization homeostasis leads to cell cycle arrest in the G2-M phase, the formation of abnormal mitotic spindles, and ultimately, apoptosis. Given their central role in mitosis, microtubules have become an important target for anticancer drug research. In recent years, scientists have extensively studied various natural and synthetic compounds with vincristine, colchicine, and paclitaxel as binding sites. Compared to drugs targeting the paclitaxel and vincristine binding sites, drugs targeting the colchicine binding site can overcome P-gp-mediated multidrug resistance and inhibit angiogenesis. Currently, various colchicine binding site inhibitors (CBSIs) have been discovered, but due to their narrow therapeutic window, significant toxicity, and side effects such as multi-organ dysfunction, none have yet received FDA approval for cancer treatment. Therefore, further research is needed on novel microtubule destabilizers targeting colchicine binding sites to overcome these limitations and provide more potential active lead compounds for the development of anticancer drugs. Summary of the Invention

[0003] The purpose of this invention is to provide a microtubule inhibitor that targets the colchicine binding site.

[0004] A second objective of this invention is to provide the application of the aforementioned microtubule inhibitor targeting the colchicine binding site in the preparation of antitumor drugs.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a microtubule inhibitor targeting the colchicine binding site, the general structural formula of which is selected from one of the following structures:

[0007]

[0008] R1 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0009] R2 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0010] R3 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile.

[0011] R4 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0012] R5 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0013] R6 is selected from one of the following structures: C3~C8 cycloalkyl,

[0014] ;

[0015] R8 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0016] R9 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0017] R 10 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0018] R 11 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0019] R 12 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups;

[0020] R 13 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0021] R 14 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0022] R 15 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0023] R 16 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0024] R 17 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0025] R 18 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0026] R 19 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0027] R 20 Selected from hydrogen and halogens (fluorine, chlorine, bromine, iodine);

[0028] Alternatively, R8, R9, and C form a six-membered ring;

[0029] Or, R 10 R 11 It forms three- to six-membered rings with C and N;

[0030] R7 is selected from one of the following structures:

[0031] ;

[0032] R 21 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0033] R 22 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0034] R 23 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0035] R 24 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0036] R 25 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0037] Or, R 24 R 25 It forms three to eight-membered rings with C and N;

[0038] R 26 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0039] R 27Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0040] R 28 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0041] R 29 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogens (fluorine, chlorine, bromine, iodine);

[0042] R 30 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen (fluorine, chlorine, bromine, iodine).

[0043] More preferably, in the microtubule inhibitor targeting the colchicine binding site,

[0044] R1 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0045] R2 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0046] R3 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0047] R4 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0048] R5 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0049] R6 is selected from one of the following structures:

[0050] ;

[0051] R8 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile.

[0052] R9 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile.

[0053] R 10 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile;

[0054] R 11 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile;

[0055] R 12 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile;

[0056] R 13 Selected from hydrogen, fluorine, and chlorine;

[0057] R 14 Selected from hydrogen, fluorine, and chlorine;

[0058] R 15 Selected from hydrogen, fluorine, and chlorine;

[0059] R 16 Selected from hydrogen, fluorine, and chlorine;

[0060] R 17 Selected from hydrogen, fluorine, and chlorine;

[0061] R 18 Selected from hydrogen, fluorine, and chlorine;

[0062] R 19 Selected from hydrogen, fluorine, and chlorine;

[0063] R 20 Selected from hydrogen, fluorine, and chlorine;

[0064] Alternatively, R8, R9, and C can be combined. The dashed line indicates the connection point.

[0065] Or, R 10 R 11 Composed of C and N The dashed line indicates the connection point.

[0066] R 31 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl;

[0067] R7 is selected from one of the following structures:

[0068] ;

[0069] R 21 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0070] R 22 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0071] R 23 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0072] R 24 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0073] R 25 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0074] Or, R 24 R 25 Composed of C and N ;

[0075] R 32 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl;

[0076] R 33 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl;

[0077] R 26 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0078] R 27 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0079] R 28 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0080] R 29 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine;

[0081] R 30 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine.

[0082] Most preferably, the structure of the microtubule inhibitor targeting the colchicine binding site is selected from one of the following structures:

[0083]

[0084]

[0085] In a second aspect, the present invention provides the use of the aforementioned colchicine-targeting microtubule inhibitor or its pharmaceutical salt in the preparation of an antitumor drug.

[0086] The tumors were selected from breast cancer, non-small cell lung cancer, melanoma, and cervical cancer.

[0087] The breast cancer mentioned refers to human breast cancer cells MDA-MB-231 and MCF-7.

[0088] The compounds of this invention can significantly inhibit the proliferation and migration of tumor cells, have good anti-tumor activity, and can be used as anti-tumor drugs.

[0089] The compounds of this invention can significantly inhibit the growth of tumor cells in mice, exhibiting good anti-tumor activity and can be used as anti-tumor drugs.

[0090] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0091] The colchicine-binding microtubule inhibitors prepared in this invention were demonstrated through pharmacological experiments to significantly inhibit the proliferation of MCF-7 tumor cells at a drug concentration of 50 μM. Compounds A1, A4-A6, A8, and A9 showed particularly strong inhibitory effects. Compound A6 significantly inhibited the proliferation of MCF-7, MDA-MB-231, A549, HeLa, and B16-F10 cells. At a concentration of 25 μM, compound A6 significantly inhibited microtubule polymerization. Scratch and Transwell assays also demonstrated that the compound inhibited tumor cell proliferation and migration. Immunofluorescence experiments confirmed that the compound could induce the collapse of the microtubule fiber network. Inhibition of cell colony formation further corroborated the compound's ability to significantly inhibit tumor cell proliferation. In vivo activity evaluation also showed that compound A6 could inhibit tumor growth in mice.

[0092] The compounds prepared in this invention are microtubule inhibitors targeting the colchicine binding site, exhibiting significant in vitro anti-proliferative activity against MCF-7, MDA-MB-231, A549, and HeLa tumor cells, and significantly inhibiting the invasion of MCF-7 cells. Furthermore, these compounds inhibit microtubule polymerization and disrupt the microtubule network of MCF-7 cells in vitro. Moreover, they can arrest the cell cycle at the G2 / M phase, inducing tumor cell apoptosis. These compounds also significantly inhibit tumor growth in mice with minimal toxic side effects. Attached Figure Description

[0093] Figure 1 This is a schematic diagram showing the inhibitory activity of the compound prepared in this invention at a concentration of 50 μM on MCF-7 cells.

[0094] Figure 2 This is a schematic diagram showing the effect of compound A6 on the in vitro polymerization of tubulin.

[0095] Figure 3 This is a schematic diagram showing the inhibitory effect of compound A6 on microtubules in MCF-7 cells.

[0096] Figure 4 This is a schematic diagram showing the results of scratching MCF-7 cells with compound A6.

[0097] Figure 5 This is a schematic diagram showing the effect of compound A6 on the migration of MCF-7 cells.

[0098] Figure 6 This is a schematic diagram showing the results of compound A6 inhibiting the formation of MCF-7 cell clones.

[0099] Figure 7This is a schematic diagram showing the effect of compound A6 on the cell cycle of MCF-7 cells.

[0100] Figure 8 This is a schematic diagram showing the effect of compound A6 on apoptosis in MCF-7 cells.

[0101] Figure 9 This is a schematic diagram showing the in vivo activity evaluation results of compound A6 in inhibiting melanoma.

[0102] Figure 10 This is a schematic diagram showing the effects of compound A6 on the liver and kidneys in vivo. Detailed Implementation

[0103] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0104] Example 1

[0105] The compounds A1~A11 of the present invention are prepared as follows:

[0106]

[0107] Step 1: Preparation of intermediate a1

[0108] A 50 mL pear-shaped flask was prepared under argon protection. 5 mL of 1,4-dioxane, 3-bromo-1,2-phenylenediamine (100 mg, 0.53 mmol), 3-trifluoromethylbenzaldehyde (111 mg, 0.67 mmol), and p-toluenesulfonic acid (14 mg, 0.08 mmol) were added sequentially. The flask was placed in an oil bath at 95 °C and reacted for 6 hours, monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure, extracted with 20 mL of ethyl acetate, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography to obtain 70 mg of intermediate a1 as a white solid, with a yield of 38.9%.

[0109] Step 2: Preparation of compound A1

[0110] A 50 mL round-bottom flask was prepared under argon protection. 2.5 mL of N,N-dimethylformamide, 0.5 mL of water, intermediate a1 (70 mg, 0.26 mmol), 3,4,5-trimethoxyphenylboronic acid (67 mg, 0.32 mmol), anhydrous potassium carbonate (108 mg, 0.78 mmol), and bis(triphenylphosphine)palladium dichloride (18 mg, 0.03 mmol) were added sequentially. The flask was placed in an oil bath at 80 °C and reacted for 24 hours. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched with 20 mL of water. The mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 88 mg of compound A1 as a white solid, with a yield of 79.3%.

[0111] The preparation methods of compounds a2~a11 and A2~A11 are the same as above, except that the raw material 3-trifluoromethylbenzaldehyde in the first step is replaced with 2,6-dimethylbenzaldehyde, 2,6-dimethoxybenzaldehyde, 2,4-dimethoxybenzaldehyde, 4-tert-butylbenzaldehyde, 4-methoxybenzaldehyde, 4-nitrobenzaldehyde, 2-methylbenzaldehyde, 3-methoxybenzaldehyde, 4-hydroxybenzaldehyde, and 4-cyanobenzaldehyde, respectively. The rest is the same as in Example 1, and compounds a2~a11 and A2~A11 are obtained respectively.

[0112] Example 2

[0113]

[0114] Step 1: Preparation of intermediate b

[0115] Take a 50 mL pear-shaped flask, and under argon protection, add 8 mL THF, 2 mL water, 2-amino-3-bromobenzamide (200.1 mg, 0.93 mmol), 3,4,5-trimethoxyphenylboronic acid (219.6 mg, 1.03 mmol), potassium carbonate (386.3 mg, 2.79 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (17.7 mg, 0.02 mmol). Place the flask in an oil bath at 75 °C and react for 1.5 hours. After the reaction is complete, cool to room temperature, extract with ethyl acetate, wash with saturated sodium chloride aqueous solution, dry to anhydrous sodium sulfate, filter, concentrate under reduced pressure, and perform silica gel column chromatography (PE:EA = 2:1, v:v) to give 107.2 mg of intermediate b, a yellow solid, in 38.2% yield.

[0116] Step 2: Preparation of target compound B1

[0117] Take a 20 mL reaction flask and add 8 mL of dimethyl sulfoxide, 2-amino-3',4',5'-trimethoxy-[1,1'-biphenyl]-3-carboxamide (150.3 mg, 0.50 mmol), and benzaldehyde (64.4 mg, 0.60 mmol) sequentially. The mixture is reacted in a microwave-assisted reaction at 120 °C for 6 hours. After the reaction is complete, cool to room temperature, extract with ethyl acetate, dry to anhydrous sodium sulfate, filter, concentrate under reduced pressure, and perform silica gel column chromatography (PE:EA = 1:1, v:v) to give 57.1 mg of compound B1 as a yellow solid, with a yield of 29.4%.

[0118] The preparation methods for compounds B2 to B20 are the same as above, except that the benzaldehyde raw material in the second step is replaced with 4-pyridinecarboxaldehyde, 4-nitrobenzaldehyde, 4-cyanobenzaldehyde, 2-trifluoromethylbenzaldehyde, 2-methylbenzaldehyde, 2,6-dimethoxybenzaldehyde, 1-naphthaldehyde, 2-methoxy-1-naphthaldehyde, cyclopropyl, 4-methoxybenzaldehyde, 4-tert-butylbenzaldehyde, cyclobutyl, cyclohexyl, 2-fluoronicotinaldehyde, 1H-pyrazole-4-carboxaldehyde, 1-methyl-1H-indole-5-carboxaldehyde, oxazol-4-carboxaldehyde, 5-thiazolecarboxaldehyde, 4-imidazolaldehyde, and the rest is the same as in Example 2, to obtain compounds B2 to B20 respectively.

[0119] Example 3

[0120] The compounds C1-C12 of this invention are prepared as follows:

[0121]

[0122] Step 1: Preparation of intermediate C1

[0123] A 100 mL pear-shaped flask was prepared under argon protection. 8 mL of tetrahydrofuran, 2 mL of water, 2-chloro-4-iodonicotinonitrile (300 mg, 1.13 mmol), 3,4,5-trimethoxyphenylboronic acid (265 mg, 1.25 mmol), potassium carbonate (469 mg, 3.39 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (19 mg, 0.02 mmol) were added sequentially. The mixture was placed in an oil bath at 75 °C and reacted for 5 hours. After the reaction was complete, the mixture was cooled to room temperature, extracted with ethyl acetate, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give 309 mg of intermediate C1, a yellow-green solid, in 89.8% yield.

[0124] Step 2: Preparation of intermediate C2

[0125] Take a 50 mL round-bottom flask, add 5 mL of N,N-dimethylformamide and intermediate C1 (200 mg, 0.66 mmol), maintain the temperature at -5℃, add methyl mercaptoacetate (80 mg, 0.66 mmol), place in an ice-water bath, and slowly add 0.2 mL of potassium hydroxide aqueous solution (1 mmol / mL). React for 5 hours. After the reaction is complete, pour the reaction solution into 20 mL of ice water, extract with dichloromethane, wash the organic phase with saturated sodium chloride aqueous solution, dry to anhydrous sodium sulfate, filter, concentrate under reduced pressure, and precipitate by silica gel column chromatography to obtain 180 mg of intermediate C2, a yellow solid, with a yield of 73.2%.

[0126] Step 3: Preparation of intermediate C3

[0127] Take a 25 mL round-bottom flask, add 5 mL of anhydrous ethanol, intermediate C2 (50 mg, 0.13 mmol), and 0.2 mL of sodium hydroxide aqueous solution (0.8 mmol / L), and place it in a 90 °C oil bath for 3 hours. After the reaction is complete, add 10 mL of ice water to the reaction system, stir at room temperature for 5 min, add hydrochloric acid aqueous solution to adjust the pH to 4-5, filter, collect the filter cake, dry, and obtain 44 mg of intermediate C3, a yellow solid, with a yield of 94.0%.

[0128] Step 4: Preparation of target compound C1

[0129] A 25 mL round-bottom flask was used. Under argon protection, 2 mL of N,N-dimethylformamide, intermediate C3 (100 mg, 0.28 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (139 mg, 0.37 mmol), and N,N-diisopropylethylamine (109 mg, 0.84 mmol) were added sequentially. The reaction was carried out at room temperature for 1 hour. Then, aniline (135 mg, 0.83 mmol) was added, and the reaction was carried out for 4 hours. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to give 81 mg of compound C1 as an orange-yellow solid, with a yield of 50.3%.

[0130] The preparation method for compounds C2 to C12 is the same as above, except that the aniline in step 4 above is replaced with p-methylaniline, p-methoxyaniline, 4-fluoroaniline, 1-methyl-4-amino-1H-indole, 1-methyl-4-amino-1H-indazole, 5-aminoquinoline, 1-phenylpiperazine, 1-(p-tolyl)piperazine, 1-(p-methoxyphenyl)piperazine, 1-(4-fluorophenyl)piperazine, or 1-(4-cyanophenyl)piperazine to obtain compounds C2 to C12.

[0131] Table 1. Yields, 1H NMR, 1C NMR, and high-resolution mass spectrometry data of the compounds of this invention.

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] Example 3

[0140] Pharmacological experiments of compounds prepared in this invention

[0141] I. Cytotoxicity Tests of Compounds

[0142] 1. The cytotoxicity of the target compound was determined using the CCK-8 assay.

[0143] The cytotoxicity of the compounds prepared in this invention against human breast cancer cells MCF-7, human non-small cell lung cancer cells A549, human breast cancer cells MDA-MB-231, mouse melanoma cells B16-F10, and human cervical cancer cells HeLa was determined using the CCK-8 assay. Cells were seeded at a density of 5000 cells / well in 96-well plates. After removing the culture medium, 100 μL of culture medium containing different concentrations of the compound was added to each well, and the plates were incubated at 37°C with 5% CO2 for 48 hours. The old culture medium was aspirated, CCK-8 reagent was added, and the plates were incubated for 1 hour. The absorbance was then measured at 450 nm using a microplate reader. The IC50 was calculated using nonlinear regression analysis with GraphPadPrism. 50 All experiments should be repeated at least three times.

[0144] 2. Experimental Results

[0145] The preliminary screening results of the cytotoxicity of the compound prepared in this invention against MCF-7 cells are shown in the figure. Figure 1 As shown, Figure 1This diagram illustrates the inhibitory activity of the compounds prepared in this invention at a concentration of 50 μM against MCF-7 cells. As can be seen from the diagram, at a test concentration of 50 μM, the A-series compounds (A1-A11) with a 1H-benzo[d]imidazole skeleton exhibited the best inhibitory effect on MCF-7 cell proliferation. Among them, compounds A1, A4-A6, A8, and A9 showed excellent antitumor activity, with significantly higher inhibition rates than other compounds. In contrast, the B-series compounds (B1-B12), with a thieno[2,3-b]pyridine-3-amine skeleton and amide bonds as linking chains, showed weaker antitumor proliferation activity. The C-series compounds (C1-C12) showed weak inhibitory activity against MCF-7 cells.

[0146] Based on the initial screening results, compounds A1, A4-A6, and A8-A11 with relatively good activity from the A series were selected, with colchicine as a positive control. The IC50 values ​​of these compounds against MCF-7, MDA-MB-231, A549, HeLa, and B16-F10 were further determined. 50 The results are shown in Table 2. As can be seen from the table, compound A1 exhibited strong inhibitory activity against MCF-7, A549, HeLa, and B16-F10 cells, with an IC50 concentration of [missing value]. 50 The concentrations were 3.8 μM, 2.4 μM, 3.6 μM, and 0.8 μM, respectively; compound A5 showed better inhibitory effects on B16-F10 cells, with an IC50 concentration of 3.8 μM, 2.4 μM, 3.6 μM, and 0.8 μM. 50 The concentration was 0.7 μM; compound A6 exhibited significant anti-proliferative activity in all five tumor cell lines, with an IC50 concentration of 0.7 μM. 50 The compounds A8 and A9 were MCF-7 (1.4 μM), MDA-MB-231 (2.5 μM), A549 (1.8 μM), HeLa (1.8 μM), and B16-F10 (0.4 μM), respectively, with particularly outstanding broad-spectrum inhibitory effects. Compounds A8 and A9 also showed good inhibitory activity against MCF-7, HeLa, and B16-F10 cells. Among them, compound A8 showed an IC50 concentration of 100 mg / L against MCF-7, HeLa, and B16-F10 cells. 50 The IC50 values ​​of compound A9 against MCF-7, HeLa, and B16-F10 cells were 4.2 μM, 4.4 μM, and 3.1 μM, respectively. 50 The concentrations were 3.6 μM, 3.6 μM, and 2.1 μM, respectively. Further structure-activity relationship analysis revealed that introducing alkyl or methoxy groups onto R1 of the A-series compounds significantly enhanced their antitumor activity. Compound A6 (4-methoxy) exhibited excellent inhibitory effects on all four tested tumor cell lines, suggesting that this modification strategy may help improve the broad-spectrum antitumor activity of the compounds.

[0147] Table 2. IC50 of some compounds on five types of tumor cells 50 value.

[0148]

[0149] II. Effects of Compound A6 on the In Vitro Polymerization of Tubulin

[0150] 1. Experimental Methods

[0151] Porcine brain tubulin was dissolved in EM buffer containing 5% glycerol (80 mM PIPES, 2 mM GTP, 2 mM MgCl2, and 0.5 mM EGTA (pH 6.9)). The compound was diluted with EM buffer to 50, 25, 10, 5, and 2.5 μM, added to 384-well plates, and pre-incubated at 37 °C. 14 μL of the tubulin-GTP mixture was added to the compound in the 384-well plates. Detection was performed at 340 nm every 1 minute for 60 minutes. Colchicine was used as a positive control, and paclitaxel and 0.1% DMSO were used as negative controls; the detection was repeated three times.

[0152] 2. Experimental Results

[0153] See results Figure 2 As shown, Figure 2 This is a schematic diagram showing the effect of compound A6 on in vitro tubulin polymerization. As can be seen from the figure, compound A6 exhibits good in vitro proliferation inhibitory activity against MCF-7, MDA-MB-231, A549, and HeLa cells. The mechanism of action of compound A6 was verified through in vitro tubulin polymerization experiments. The results showed that a compound concentration of 50 μM significantly inhibited in vitro tubulin polymerization.

[0154] III. Immunofluorescence detection

[0155] 1. Experimental Methods

[0156] MCF-7 cells were seeded in glass dishes and cultured for 24 hours. Compound A6 (0.1, 1, and 10 μM) was added for 24 hours, with 0.1% DMSO as a negative control. The old culture medium was discarded, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde for 20 minutes, followed by permeabilization with Triton X-100 PBS. After washing three times with PBS, the cells were blocked with 100 μL of goat serum at room temperature for 30 minutes. Cells were incubated overnight with β-tubulin at 4°C. After washing three times with PBST, the cells were incubated with Cy3-conjugated fluorescent secondary antibody at room temperature for 1 hour. After washing three times with PBST, the cell nuclei were labeled with 4',6-diamidinyl-2-phenylindole dihydrochloride (DAPI). Finally, the cells were observed using a fluorescence microscope.

[0157] 2. Experimental Results

[0158] The inhibitory effect of compound A6 on microtubules in MCF-7 cells was further investigated by immunofluorescence staining. The results are shown in [Figure number missing]. Figure 3 As shown, Figure 3 This is a schematic diagram illustrating the inhibitory effect of compound A6 on microtubules in MCF-7 cells. As can be seen from the figure, in the control group, the microtubule network exhibits a normal arrangement, with fine, fibrous microtubules (red) surrounding the cell nucleus (blue). However, after cells were exposed to compound A6 (0.1, 1, and 10 μM) for 24 hours, the microtubule network in the cytoplasm was disrupted. These results indicate that compound A6 can induce the disintegration of the microtubule network in tumor cells.

[0159] IV. Cell scratch healing experiment

[0160] 1. Experimental Methods

[0161] MCF-7 cells were seeded and incubated overnight in 6-well plates. A 200 μL pipette tip was used to make a small incision on the confluent monolayer of cells. The incision was then washed twice with culture medium to remove debris and plucked cells. Different concentrations of compound A6 (0, 0.1, 1, and 10 μM) were added. Images were acquired using a phase-contrast microscope at 0 and 24 hours, and the migration distance of cells in the incision area was manually measured.

[0162] 2. Experimental Results

[0163] Tubulin and microtubules play important roles in cell migration and motility. Therefore, this invention performed a cell scratch healing assay to confirm the effect of compound A6 on cell migration. MCF-7 cells were treated with 0, 0.1, 1, and 10 μM of compound A6 for 24 hours, respectively. The results of wound healing are shown in [Figure number missing]. Figure 4 As shown, Figure 4 This is a schematic diagram showing the results of compound A6 on MCF-7 cells after scratching. As can be seen from the figure, control group cells were able to migrate into the wound channels, recovering 16.66±2.20% of the area. After treatment with 0.1, 1, or 10 μM compound A6, only 14.19±4.93%, 7.70±3.16%, and 4.83±2.11% of the wound area, respectively, migrated. These results indicate that compound A6 can inhibit tumor cell migration.

[0164] V. Transwell Experiment

[0165] 1. Experimental Methods

[0166] MCF-7 cells were loaded at 5 × 10 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated at 37°C for 24 hours. Different concentrations of compound A6 (0, 0.1, 1, and 10 μM) were then added for 24 hours. After digestion, single-cell suspensions were prepared. The serum-free cell suspension was added to the upper chamber of a Transwell chamber, while serum-containing normal culture medium was added to the lower chamber. The chambers were incubated for 48 hours. The culture medium in the upper chamber was removed, and the cells were gently washed three times with PBS. Cells were then fixed with methanol and stained with crystal violet. Unmigrated cells in the upper chamber were wiped away with cotton swabs, and the number of cells that migrated to the membrane was observed and counted under a microscope. Finally, the number of migrating cells in the experimental and control groups was compared.

[0167] 2. Experimental Results

[0168] The effect of compound A6 on tumor cell invasion was analyzed using Transwell assays. Results are shown below. Figure 5 As shown, Figure 5 This is a schematic diagram illustrating the effect of compound A6 on the migration of MCF-7 cells. As can be seen from the figure, the control group showed more cells on the lower surface of the Transwell chambers. In contrast, the compound A6 group showed fewer cells invading the lower surface of the Transwell chambers, indicating that treatment with compound A6 inhibited the invasion of MCF-7 cells. This further demonstrates that compound A6 possesses anti-invasion and anti-migration properties against MCF-7 cells.

[0169] VI. Cell Cloning Experiment

[0170] 1. Experimental Methods

[0171] MCF-7 cells (1000 cells / well) were counted and seeded into 6-well plates. After incubation at 37°C for 24 hours, different concentrations of compound A6 (0, 0.1, 1, and 10 μM) were added. After 24 hours of treatment, the culture medium was replaced with normal medium. After 10–14 days of incubation, the old medium was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes, followed by three washes with PBS, and then stained with 0.1% crystal violet for 15 minutes. Finally, the colonies were washed with PBS and photographed.

[0172] 2. Experimental Results

[0173] The long-term growth-inhibiting effect of compound A6 was tested using a cell colony formation assay. Results are shown below. Figure 6 As shown, Figure 6 This is a schematic diagram showing the effect of compound A6 in inhibiting MCF-7 cell colony formation. As can be seen from the figure, after 14 days of treatment with compound A6 (at concentrations of 1 and 10 μM), cell colony formation was significantly reduced.

[0174] VII. Cell Cycle Analysis

[0175] 1. Experimental Methods

[0176] MCF-7 cells were loaded at 5 × 10 5 Cells were seeded at a density of cells / well in 6-well plates and cultured overnight. Cells were treated with compound A6 (0.1, 1, and 10 μM) for 48 hours. Cells were collected and fixed overnight at 4°C with pre-chilled 70% ethanol. After fixation, cells were washed once with PBS, and 0.5 mL of propidium iodide (PI) staining solution was added to each cell sample tube. The cell pellet was slowly and thoroughly resuspended and incubated at 37°C in the dark for 30 minutes. Subsequently, red fluorescence was detected at an excitation wavelength of 488 nm using flow cytometry, and light scattering was also assessed.

[0177] 2. Experimental Results

[0178] This invention employs flow cytometry analysis to detect the effect of compound A6 on the MCF-7 cell cycle; the results are shown below. Figure 7 As shown, Figure 7 This is a schematic diagram illustrating the effect of compound A6 on the cell cycle of MCF-7 cells. As can be seen from the figure, compared to the blank control group (11.20% of cells arrested in the G2 / M phase), after treating MCF-7 cells with different concentrations of compound A6 (0.1 μM, 1 μM, and 10 μM) for 48 hours, the percentage of cells arrested in the G2 / M phase increased from 12.77% to 84.47%. These results are consistent with the basic characteristics of microtubule inhibitors targeting the colchicine binding site.

[0179] VIII. Apoptosis Analysis

[0180] 1. Experimental Methods

[0181] MCF-7 cells were loaded at 5 × 10 5 Cells were seeded at the specified density in 6-well plates and cultured overnight. Cells were treated with compound A6 (0.1, 1, and 10 μM) for 48 hours. Cells were collected, washed once with PBS, and gently resuspended in Annexin-FITC binding buffer. Annexin-FITC and propidium iodide (PI) staining solutions were added to sample tubes, and the cells were incubated at room temperature in the dark for 10–20 minutes before analysis.

[0182] 2. Experimental Results

[0183] Mitotic arrest in tumor cells induced by microtubule polymerization inhibitors is usually associated with apoptosis. Therefore, Annexin V FITC and propidium iodide (PI) assays were performed on MCF-7 cells to assess whether compound A6 could induce apoptosis in cancer cells. The results are shown in [Figure number missing]. Figure 8 As shown, Figure 8This is a schematic diagram illustrating the effect of compound A6 on apoptosis in MCF-7 cells. As shown in the figure, after treating MCF-7 cells with different concentrations (0.1 μM, 1 μM, and 10 μM) of the compound for 48 hours, the proportion of total apoptotic cells increased from 20.99% (control group) to 31.96% (0.1 μM), 33.28% (1 μM), and 79.80% (10 μM). These data indicate that compound A6 can effectively induce tumor cell apoptosis and inhibit tumor cell growth.

[0184] IX. In vivo activity evaluation

[0185] 1. Experimental Methods

[0186] Female BALB / c mice aged 6 to 8 weeks were purchased from Beijing Regen Biotech Co., Ltd. to study the inhibitory effect of compound A6 on a subcutaneous melanoma cell transplantation model. Prior to injection into the mice, B16-F10 cells (4 × 10⁻⁶) in the logarithmic growth phase were used. 6 The suspension was prepared in PBS (containing 5 × 10⁶ cells / mL). Each mouse was subcutaneously injected with 200 μL (containing 5 × 10⁶ cells / mL). 5 A tumor model was established using a suspension of individual cells. Mice were randomly assigned to either a control group or a treatment group (n=8). Compound A6 or paclitaxel was dissolved in a 1:1 PEG300 / PBS solution to prepare the desired concentration. The carrier control solution was prepared using only equal volumes of PEG300 and PBS. The treatment doses were paclitaxel: 10 mg / kg; compound A6: 20 and 50 mg / kg, administered intraperitoneally every two days for 12 days. Tumor inhibition rate (TGI) = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%.

[0187] 2. Experimental Results

[0188] The results are as follows Figure 9 As shown. Figure 9This is a schematic diagram illustrating the in vivo activity evaluation results of compound A6 in inhibiting melanoma. A shows the tumor size in mice 12 days after treatment; B shows the weight change of mice during treatment; C shows the weight distribution of excised tumors in each group; and D shows the tumor volume change in mice during treatment. From A, it can be seen that at different doses, compound A6 resulted in significantly smaller tumors in mice compared to the control group, indicating that compound A6 has good antitumor activity, but its activity is slightly weaker than paclitaxel. From B, it can be seen that compound A6 at doses of 20 mg / kg and 50 mg / kg did not cause a significant decrease in body weight compared to paclitaxel, indicating that this compound has better tolerability than paclitaxel. From C, it can be seen that at different doses, compound A6 resulted in a significant decrease in tumor weight in mice compared to the control group, indicating that compound A6 has good antitumor activity, but its activity is slightly weaker than paclitaxel. From D, it can be seen that at different doses, compound A6 resulted in smaller tumor volumes in mice compared to the control group, indicating that compound A6 has antitumor growth activity, but its antitumor activity is slightly weaker than paclitaxel.

[0189] The results of hematoxylin-eosin (H&E) staining of the liver and kidneys are as follows: Figure 10 As shown, Figure 10 This is a schematic diagram showing the effects of compound A6 on the liver and kidneys in vivo. The diagram shows that compound A6 did not have significant toxic effects on the major organs of mice.

[0190] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A microtubule inhibitor targeting the colchicine binding site, characterized in that, The general structural formula is selected from one of the following structures: ； R1 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R2 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R3 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile. R4 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R5 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R6 is selected from one of the following structures: C3~C8 cycloalkyl, ； R8 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R9 is selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R 10 selected from the group consisting of hydrogen, C1-C30alkyl, C1-C30alkoxy, -CF3, -OCF3, nitro, hydroxy, nitrile; R 11 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R 12 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile groups; R 13 Selected from hydrogen and halogens; R 14 Selected from hydrogen and halogens; R 15 Selected from hydrogen and halogens; R 16 Selected from hydrogen and halogens; R 17 Selected from hydrogen and halogens; R 18 Selected from hydrogen and halogens; R 19 Selected from hydrogen and halogens; R 20 Selected from hydrogen and halogens; Alternatively, R8, R9, and C form a six-membered ring; Or, R 10 R 11 It forms three- to six-membered rings with C and N; R7 is selected from one of the following structures: ； R 21 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 22 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 23 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 24 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 25 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; Or, R 24 R 25 It forms three to eight-membered rings with C and N; R 26 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 27 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 28 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 29 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen; R 30 Selected from hydrogen, C1~C30 alkyl, C1~C30 alkoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, and halogen.

2. The microtubule inhibitor targeting the colchicine binding site according to claim 1, characterized in that, Among the microtubule inhibitors targeting the colchicine binding site... R1 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R2 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R3 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R4 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R5 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R6 is selected from one of the following structures: ； R8 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, and nitrile. R9 is selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, and nitrile. R 10 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile; R 11 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile; R 12 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxy, nitrile; R 13 Selected from hydrogen, fluorine, and chlorine; R 14 Selected from hydrogen, fluorine, and chlorine; R 15 Selected from hydrogen, fluorine, and chlorine; R 16 Selected from hydrogen, fluorine, and chlorine; R 17 Selected from hydrogen, fluorine, and chlorine; R 18 Selected from hydrogen, fluorine, and chlorine; R 19 Selected from hydrogen, fluorine, and chlorine; R 20 Selected from hydrogen, fluorine, and chlorine; Alternatively, R8, R9, and C can be combined. The dashed line indicates the connection point. Or, R 10 R 11 Composed of C and N The dashed line indicates the connection point. R 31 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl; R7 is selected from one of the following structures: ； R 21 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 22 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 23 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 24 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 25 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; Or, R 24 R 25 Composed of C and N ; R 32 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl; R 33 Selected from methyl, ethyl, n-propyl, n-butyl, and tert-butyl; R 26 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 27 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 28 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 29 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine; R 30 Selected from hydrogen, methyl, ethyl, n-propyl, n-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, n-butoxy, tert-butoxy, -CF3, -OCF3, nitro, hydroxyl, nitrile, fluorine, and chlorine.

3. The microtubule inhibitor targeting the colchicine binding site according to claim 2, characterized in that, The structure of the microtubule inhibitor targeting the colchicine binding site is selected from one of the following structures: ； 。 4. The use of a microtubule inhibitor targeting the colchicine binding site as described in any one of claims 1 to 3, or a pharmaceutical salt thereof, in the preparation of an antitumor drug.

5. The application according to claim 4, characterized in that, The tumors were selected from breast cancer, non-small cell lung cancer, melanoma, and cervical cancer.

6. The application according to claim 5, characterized in that, The breast cancer mentioned refers to human breast cancer cells MDA-MB-231 and MCF-7.

Images