A tumor cell inhibitor and its preparation method and application

Abstract

The application provides a tumor cell inhibitor and a preparation method and application thereof. The preparation method of the tumor cell inhibitor comprises the following steps: dissolving an intermediate compound, 3, 4, 5-trimethoxy aniline, in 1, 4-dioxane, adding p-toluenesulfonamide to carry out a reaction, and obtaining a reaction solution; mixing the reaction solution with silica gel, spinning dry, and carrying out column chromatography purification to obtain the tumor cell inhibitor. The tumor cell inhibitor has a significant inhibitory effect on the proliferation, clone formation, migration and invasion of tumor cells, and has a good anti-tumor effect.

Description

Technical Field

[0001] This application relates to the field of tumor cell inhibitor technology, and in particular to a tumor cell inhibitor, its preparation method, and its application. Background Technology

[0002] Generally, thyroid cancer is classified into different subtypes based on its morphological characteristics and pathological tissue origin. Among these, 95% are differentiated thyroid cancers (DTC) originating from thyroid follicular epithelial cells, including papillary carcinoma (PTC) and follicular carcinoma (FTC). PTC is the most common histological subtype, accounting for approximately 90% of differentiated thyroid cancers. Medullary thyroid carcinoma (MTC) originates from parafollicular C cells of the thyroid gland and belongs to the neuroendocrine tumors of the thyroid gland, exhibiting a certain familial genetic predisposition. Undifferentiated thyroid cancer (ATC), also known as anaplastic thyroid cancer, is one of the most malignant and deadliest malignant tumors currently known, with a median survival of only 3-6 months. ATC is a rare subtype of thyroid cancer, accounting for 1%-2% of all malignant thyroid tumors. It can develop from follicular cells or dedifferentiate from DTC.

[0003] Currently, traditional treatment strategies mainly consist of three steps: surgery, radioactive iodine (I-I), and radioactive iodine (I-I). 131 I) Treatment, including thyroid-stimulating hormone (TSH) suppression therapy. Most patients with diabetic thyroid cancer (DTC) have a relatively good overall prognosis after surgery, radioactive iodine therapy, and TSH suppression, with a 10-year survival rate exceeding 90%. However, a small number of patients experience disease progression. During follow-up, it was observed that 20% of patients relapsed within 10 years, and approximately 10% developed cervical lymph node metastasis or distant metastasis. Radioactive iodine resistance is one of the main characteristics of thyroid cancer progression. It refers to the inability of papillary thyroid carcinoma cells to take up radioactive iodine during the first or subsequent radioactive iodine therapy in PTC patients. The presence of iodine resistance often indicates a poor prognosis; the 10-year survival rate for these patients is less than 10%, and treatment options are limited. The disease progresses rapidly, posing significant challenges to clinicians. Furthermore, due to the dedifferentiated phenotype and high invasiveness of acute thyroid cancer (ATC), many patients are no longer candidates for surgical resection, and radiotherapy and chemotherapy are often ineffective. 131 The efficacy of I is not ideal, and its treatment poses a great challenge to clinicians.

[0004] Therefore, there is an urgent need to find new treatment strategies for patients with advanced thyroid cancer in order to improve the overall prognosis of these patients. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a tumor cell inhibitor, its preparation method and application.

[0006] Based on the above purpose, the first aspect of the present application provides a tumor cell inhibitor, which has the structural formula as shown in the following:

[0007] .

[0008] The second aspect of the present application provides a preparation method of a tumor cell inhibitor, which comprises:

[0009] The intermediate compound and 3,4,5-trimethoxyaniline are dissolved in a first solvent, and p-toluenesulfonamide is added for reaction to obtain a reaction liquid; the first solvent comprises at least one of 1,4-dioxane, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide and pyridine;

[0010] The reaction liquid is mixed with silica gel, dried by spinning, and purified by column chromatography to obtain the tumor cell inhibitor;

[0011] The intermediate compound has the structural formula as shown in the following:

[0012] .

[0013] Optionally, the molar ratio of the intermediate compound, 3,4,5-trimethoxyaniline and p-toluenesulfonamide is 1:1.2-3:0.8-3.

[0014] Optionally, the preparation method of the intermediate compound comprises:

[0015] The initial compound is dissolved in a second solvent, N,N-diisopropylethylamine is added, stirred, and 2,4-dichloropyrimidine is added for reflux reaction for a period of time to obtain an initial reaction liquid; the second solvent comprises at least one of anhydrous ethanol, n-butanol, acetonitrile, dioxane, dimethyl sulfoxide, N,N-dimethylformamide and pyridine;

[0016] The initial reaction liquid is mixed with silica gel, dried by spinning, and purified by column chromatography to obtain the intermediate compound;

[0017] The initial compound has the structural formula as shown in the following:

[0018] .

[0019] Optionally, the molar ratio of the initial compound, N,N-diisopropylethylamine and 2,4-dichloropyrimidine is 1:1.2-3:0.8-2.

[0020] Optionally, the preparation method of the initial compound comprises:

[0021] The p-phenylenediamine is dissolved in a third solvent pre-cooled, triethylamine is added, p-methoxybenzenesulfonyl chloride is added dropwise, the reaction is stopped after stirring in an ice bath for a period of time, and a reaction liquid is obtained; the third solvent includes at least one of dichloromethane, 1,2-dichloroethane, chloroform, diethyl ether, tetrahydrofuran, methyl tert-butyl ether, dimethyl sulfoxide, N,N-dimethylformamide and pyridine;

[0022] After the reaction liquid is mixed with glue and dried, the initial compound is obtained after purification.

[0023] Optionally, the molar ratio of the p-phenylenediamine, triethylamine and p-methoxybenzenesulfonyl chloride is 0.8-2.02:1:0.8-1.

[0024] The third aspect of the present application provides an anti-cancer composition comprising the tumor cell inhibitor of the first aspect or the tumor cell inhibitor prepared by any one of the second aspect, and / or a pharmaceutically acceptable excipient.

[0025] The fourth aspect of the present application provides an application, the tumor cell inhibitor of the first aspect, the tumor cell inhibitor prepared by any one of the second aspect or the anti-cancer composition of the third aspect in the preparation of anti-cancer drugs.

[0026] Optionally, the anti-cancer drug can be at least one of a medical drug, a reagent or a food.

[0027] Optionally, the tumor cell inhibitor is used to inhibit the proliferation, migration and / or invasion of tumor cells.

[0028] As can be seen from the above, the tumor cell inhibitor, its preparation method and application provided by the present application have a significant inhibitory effect on the proliferation, clonal formation, migration and invasion of tumor cells, and have good anti-tumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present application or related art, the drawings needed in the following embodiment or related art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0030] Figure 1 Chemical structural formula of Fam20C inhibitor FL-1607 and compound 3r disclosed in related art;

[0031] Figure 2 Preparation flowchart of the tumor cell inhibitor of the embodiment of the present application;

[0032] Figure 3 The ESI-MS spectrum of the tumor cell inhibitor of the present application is shown in Figure 3. 1 H NMR spectrum;

[0033] Figure 4 The ESI-MS spectrum of the tumor cell inhibitor of the present application is shown in Figure 3. 13 C NMR spectrum;

[0034] Figure 5 The ESI-MS spectrum of the tumor cell inhibitor of the present application is shown in Figure 3.

[0035] Figure 6 The experimental results of the proliferation inhibition experiment of compound 3 and compound 3r on four thyroid cancer cells of the present application are shown in Figure 1.

[0036] Figure 7 The experimental results of the colony formation inhibition experiment of compound 3 and compound 3r on two thyroid cancer cells of the present application are shown in Figure 2.

[0037] Figure 8a The experimental results of the migration and invasion inhibition experiment of compound 3 and compound 3r on BCPAP thyroid cancer cells of the present application are shown in Figure 4.

[0038] Figure 8b The experimental results of the migration and invasion inhibition experiment of compound 3 and compound 3r on BHT101 thyroid cancer cells of the present application are shown in Figure 5.

[0039] In the above figures, represents p < 0.01, represents p < 0.001, represents p < 0.0001. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below in combination with specific embodiments.

[0041] It should be noted that, unless otherwise defined, the technical terms used in the following embodiments have the same meanings as generally understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following embodiments are all conventional biochemical reagents unless otherwise specified; and the experimental methods described are all conventional methods unless otherwise specified.

[0042] As described in the background, there is an urgent need to find new treatment strategies for patients with advanced thyroid cancer to improve the overall prognosis of thyroid cancer patients. With the progress of tumor molecular biology research and the in-depth study of the pathogenesis of thyroid cancer, molecular targeted therapy has become a hot research topic. Multi-target receptor tyrosine drugs can inhibit multiple signaling pathways of thyroid cancer and also can resist angiogenesis, greatly improving the plight of patients with advanced and late-stage thyroid cancer who have no available drugs. With the in-depth study of the molecular mechanisms of thyroid cancer, more and more cancer proteins are expected to become potential targets for thyroid cancer treatment, providing new candidate drugs.

[0043] It has been reported in the related art that many sequence similarity 20 family protein C (Fam20C) substrates are related to tumor cell apoptosis and metastasis, including insulin-like growth factor binding proteins (IGFBPs), osteopontin (OPN), several extracellular proteases and serine protease inhibitors. Fam20 plays an important role in the secretion pathway of phosphorylated proteins and proteoglycans.

[0044] The human genome encodes three Fam20 paralogs: Fam20A, Fam20B, and Fam20C. Among them, Fam20A lacks the active site residues critical for kinase activity, binds ATP in a catalytically inactive manner, and is therefore a pseudokinase. Fam20B phosphorylates xylose residues to regulate proteoglycan synthesis. Fam20C is a true Golgi tyrosine kinase and a secreted protein with kinase activity that can phosphorylate a series of secreted proteins involved in biomineralization, lipid homeostasis, wound healing, cell adhesion, and migration. Its structure and function have been extensively studied in recent years, and it can phosphorylate the Ser-X-Glu / pSer motif. Studies have shown that Fam20C plays an important role in the occurrence and development of malignant tumors such as breast cancer, lung cancer, and glioma, and inhibition of Fam20C can effectively inhibit the progression of triple-negative breast cancer. Related art verified the inhibitory effect of FL-1607 (the structural formula thereof is shown in Figure 1 a) on Fam20C and the proliferation inhibitory activity on breast cancer cells in 2016. In 2021, a class of Fam20C inhibitors was reported in the related art, among which compound 3r (the structural formula thereof is shown in Figure 1 b) has the best activity, which exhibits proliferation inhibitory activity on three breast cancer cells with IC 50 values between 5.986 μM and 10.335 μM.

[0045] In addition to high expression in breast cancer cells, the inventors' previous studies found that Fam20C is also highly expressed in thyroid cancer and is closely related to malignant biology. Further biological experiments showed that knocking down Fam20C can significantly inhibit the proliferation and invasion of thyroid cancer cells, which also suggests that it can be a potential target for thyroid cancer treatment.

[0046] To verify the above guess, in the previous experiment, the inventors used Fam20C inhibitor 3r to treat DTC cell lines, and found that it can significantly inhibit the progression of thyroid cancer through in vivo and in vitro experiments. However, when using 3r to treat ATC cells with higher malignancy, it was found that the anti-tumor effect was not good.

[0047] Therefore, the present application provides a tumor cell inhibitor and its preparation method and application, which has a high inhibitory effect on ATC cells with higher malignancy, and has a good anti-tumor effect.

[0048] The present disclosure will be further described in detail below in combination with specific examples.

[0049] Example 1 Synthesis of initial compound

[0050] .

[0051] A certain amount of p-phenylenediamine (0.8 mol) was dissolved in at least one solvent (40 ml) selected from the group consisting of pre-cooled dichloromethane, 1,2-dichloroethane, chloroform, diethyl ether, tetrahydrofuran, methyl tert-butyl ether, dimethyl sulfoxide, N,N-dimethylformamide and pyridine, then triethylamine (1.0 mol) was added, and finally p-methoxybenzenesulfonyl chloride (0.8 mol) was added to the reaction system at a rate of 1 drop / s using a constant pressure dropping funnel. The reaction solution was stirred in an ice bath for 2-4 h and then the reaction was stopped. The reaction solution was mixed with an equal amount of 200-300 mesh silica gel and then rotary evaporated to dryness. The product was obtained as a yellow solid by silica gel column purification (DCM:EA = 10:1-3:1, V / V), and the yield was 88%. Figure 2 The initial compound was characterized as follows:

[0052] The initial compound was characterized as follows:

[0053] 1 H NMR (400 MHz, DMSO-d6), δ (ppm): 9.29 (1H, s), 7.55 (2H, d, J = 8.68 Hz), 7.02 (2H, d, J = 8.64 Hz), 6.67 (2H, d, J= 8.36 Hz), 6.38 (2H, d, J = 8.32 Hz), 4.92 (2H, s), 3.78 (3H, s);

[0054] 13 C NMR (100 MHz, DMSO-d6), δ (ppm): 162.0, 146.3, 131.4, 128.8, 125.6, 124.4, 114.0, 113.9, 55.5;

[0055] ESI-MS (Electrospray Mass Spectrum): 279.1 [M+H] + .

[0056] Synthesis of intermediate compound in Example 2

[0057] .

[0058] The starting compound (1.0 mol) was dissolved in at least one of the solvents of anhydrous ethanol, n-butanol, acetonitrile, dioxane, dimethyl sulfoxide, N,N-dimethylformamide and pyridine at room temperature, then N,N-diisopropyl ethylamine (1.2 mol) was added and stirred for 10-30 min, and finally 2,4-dichloropyrimidine (0.8 mol) was added and refluxed for 22-48 h. The reaction solution was mixed with an equal amount of 200-300 mesh silica gel and rotary dried, and then purified by silica gel column (DCM: MeOH = 80:1-70:1, V / V) to obtain yellow solid, which was the intermediate compound (i.e. Compound 2) shown in the formula, and the yield was 93%. Figure 2

[0059] The characterization results of the intermediate compound are as follows:

[0060] 1 H NMR (400 MHz, DMSO-d6), δ (ppm): 10.05 (1H, s), 9.92 (1H, s), 8.11 (1H, d, J = 5.92 Hz), 7.68 (2H, d, J = 8.88 Hz), 7.43 (2H, d, J = 8.60 Hz), 7.06 (4H, m), 6.67 (1H, d, J = 5.88 Hz), 3.79 (3H, s);

[0061] 13 ​C NMR (100 MHz, DMSO-d6), δ (ppm): 162.3, 161.3, 159.3, 157.0, 134.8, 133.3, 131.1, 128.8, 121.3, 121.2, 114.3, 105.5, 55.5;

[0062] ESI-MS: 391.0 [M+H] + .

[0063] Example 3. Synthesis of tumor cell inhibitors

[0064] .

[0065] The intermediate compound (1.0 mol) and 3,4,5-trimethoxyaniline (1.2 mol) were dissolved in at least one of 1,4-dioxane, acetonitrile, dimethyl sulfoxide, N,N- dimethylformamide and pyridine, then p-toluenesulfonamide (0.8 mol) was added, and the reaction was carried out at 80°C for 22-48 h. The reaction solution was mixed with an equal amount of 200-300 mesh silica gel and rotary dried, and then purified by silica gel column (DCM:MeOH = 100:1-30:1, V / V) to obtain the crude product of compound 3. The crude product of compound 3 was dissolved in methanol and purified by semi-preparative-HPLC (the mobile phase was methanol:water = 8:2, V / V) to obtain the tumor cell inhibitor (i.e. compound 3 as shown in Figure 2 , with a yield of 18.57%.

[0066] The characterization results of the tumor cell inhibitor are as follows:

[0067] 1 H NMR (400 MHz, DMSO-d6), δ (ppm): 9.89 (1H, s), 9.26 (1H, s), 8.95 (1H, s), 7.97 (1H, d, J = 5.76 Hz), 7.65 (2H, d, J = 8.92 Hz), 7.61 (2H, d, J = 8.80 Hz), 7.08 (2H, s), 7.05 (2H, d, J = 8.88 Hz), 6.99 (2H, d, J = 8.84 Hz), 6.14 (1H, d, J = 5.76 Hz), 3.78 (3H, s), 3.67 (6H, s), 3.62 (3H, s), as shown in Figure 3 .

[0068] 13 C NMR (100 MHz, DMSO-d6), δ (ppm): 162.2, 160.1, 159.5, 155.7, 152.5, 136.92, 136.65, 132.2, 132.0, 131.5, 128.7, 121.5, 120.1, 114.2, 98.7, 97.3, 62.7, 60.0, 55.6, 55.5, see Figure 4

[0069] ESI-MS: 538.2 [M+H] + , see Figure 5

[0070] Example 4. Cell proliferation experiment (CCK-8)

[0071] I. Experimental operation:

[0072] (1) In this experiment, four thyroid cancer cell lines, BCPAP, BHT101, K1 and KTC, were selected. All cell lines were purchased from American Type Culture Collection (ATCC) and the culture generation was within 20 generations. If the cell culture dish was observed and the cell growth was fused to 70-80% and the growth state was good, further processing could be carried out. The supernatant culture medium was carefully aspirated with a pipette, and an appropriate amount of PBS buffer was added to the culture dish to gently shake off the residual culture medium. The trypsin was added and observed under a microscope. When most of the cells were digested to the state of floating away from the wall, the digestion was terminated.

[0073] (2) The cells after termination of digestion were moved to a centrifuge tube, the centrifuge speed was set to 800 rpm for 5 min, the supernatant was discarded and the bottom cell precipitate was reserved. The prepared culture medium was added again, among which 1640 culture medium was used for BCPAP, K1 and KTC, and DMEM culture medium was used for BHT101. The cells were suspended by uniform blowing. The whole process should not be blown out too much air bubbles to cause cell breakage. After blowing, the cell concentration in the cell suspension was counted and diluted to an appropriate concentration for plating.

[0074] (3) A sterile 96-well plate was taken and 100 μL of cell suspension was added to the plate. Each cell line was divided into three groups, namely blank control group, compound 3 (tumor cell inhibitor) group and compound 3r (compound shown in Figure 1

[0075] ​​​(4) After culturing for 24 hours, observe the 96-well plate under a microscope. If the cells adhere well, proceed to the next step. Prepare culture media with different drug concentration gradients according to experimental needs (the concentration gradients of compound 3r group and compound 3 group are 0, 2.5, 5, 10, and 20 μM, respectively). Pipette out the original culture medium of the 96-well plate, add an appropriate amount of PBS to wash away the residual culture medium, add the experimental drug culture medium, and then place it in an incubator for further culture.

[0076] (5) After the culture is completed, remove the experimental drug culture medium and add the prepared reagent (100 μL serum-free culture medium + 10 μL CCK8 reagent per well) to each well. Place the cells in an incubator and culture for 1-4 h. Take out the 96-well plate and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value and record the results.

[0077] II. Experimental Results:

[0078] like Figure 6 As shown, compounds 3 and 3r exhibited inhibitory effects on the proliferation of all four cell lines. In BCPAP, KTC, and BHT101 cells, compound 3 at a concentration of 20 μM showed significantly better inhibitory effects on proliferation than compound 3r, with an inhibition efficiency of approximately 80% (p<0.001).

[0079] Example 5: Cloning Experiment

[0080] I. Experimental Procedure:

[0081] (1) In this experiment, two cell lines, BCPAP and BHT101, were selected. Cell culture dishes were observed. If the cells grew to 70%-80% and were in good condition, further treatment could be carried out.

[0082] (2) Carefully remove the supernatant culture medium with a pipette, add an appropriate amount of PBS buffer to the culture dish and gently shake to remove the culture medium residue. After adding trypsin, observe quietly under a microscope. The digestion can be stopped when most of the cells have been digested to the point of floating off the wall.

[0083] (3) Transfer the cells after digestion to centrifuge tubes, set the centrifuge speed to 800 rpm for 5 min, discard the supernatant and keep the cell pellet at the bottom, add the prepared culture medium again, and evenly pipette to suspend the cells, then dilute them to 400 cells / ml and set aside for plating.

[0084] (4) Take out a disposable sterile 24-well plate and place it in a laminar flow hood for later use. Add 500 μL of cell suspension to the plate to ensure 200 cells per well. Each cell line is divided into 3 groups: blank control group, compound 3 group, and compound 3r group. Each group has 3 replicates. After adding the samples, place the plate in an incubator for culture.

[0085] (5) Put the 24-well plate under the microscope to observe, if the cells adhere well, the next step can be performed. According to the experimental requirements, configure different drug culture medium, pipette out the original 24-well plate culture medium, add appropriate PBS to wash the residual culture medium, add experimental drug culture medium, and then put it into the incubator for continuous culture for about 10 days.

[0086] (6) Suck out the culture medium in the hole, wash the residual culture medium with normal saline for 2-3 times, add 4% paraformaldehyde to cover the hole bottom, and fix it at room temperature for half an hour.

[0087] (7) Pour out the paraformaldehyde, add crystal violet staining, wash the crystal violet with tap water after 20 minutes, invert the 24-well plate to dry and count.

[0088] II. Experimental results:

[0089] As shown in Figure 7 , compound 3 and compound 3r have inhibitory effects on the clone formation of BCPAP and BHT101 cell lines, and the inhibitory effect of compound 3 on the clone formation of BCPAP and BHT101 cells at a concentration of 5 μM is significantly better than that of compound 3r (p < 0.001), and the inhibition efficiency is about 60% (p < 0.001).

[0090] Example 6 Cell migration and invasion experiment

[0091] I. Experimental principle:

[0092] The filter membrane is placed between the upper and lower chambers on the transwell chamber, and the tumor cells pass through the pores of the filter membrane by deformation movement, and the size of the tumor cell movement and invasion ability can be evaluated by this method. During the experiment, blank control group, compound 3 group and compound 3r group are set up, and the size of the compound 3 inhibiting the chemotactic migration ability of thyroid cancer cells is compared and studied, and the strength of the compound 3 inhibiting the metastasis activity of tumor cells is further evaluated.

[0093] II. Experimental steps:

[0094] (I) Cell migration experiment:

[0095] (1) In this experiment, BCPAP and BHT101 cell lines are selected, and tumor cells are added to DMSO, compound 3 and compound 3r culture one day in advance, and then trypsinized, centrifuged at 850 rpm for 5 min, and the cell pellet is resuspended with serum-free medium, mixed thoroughly by blowing, and then counted. Dilute the cells to the desired concentration for standby.

[0096] (2) Each cell line is divided into 3 groups, namely blank control group, compound 3 group and compound 3r group. 8 μm transwell chamber is taken and placed in a 24-well plate. 600 μL of culture medium containing 10% FBS is added to the lower chamber, and 200 μL of serum-free medium is added to the upper chamber. The cells are placed in an incubator for further culture.

[0097] (3) The transwell chamber is taken out, and the inside and outside of the chamber are washed with PBS. The cells in the upper chamber of the transwell are gently wiped with a cotton swab. 4% paraformaldehyde is added to the upper and lower chambers for at least 30 min of fixation, and finally 200 μL and 500 μL of crystal violet are added to the upper and lower chambers for 20 min of staining.

[0098] (4) The chamber is washed, and the crystal violet dye not combined in the upper chamber is gently wiped with a cotton swab. Under a microscope, observation and photography are performed, and 3 fields of view are randomly selected from each chamber. The number of cells passing through each field of view is counted and averaged.

[0099] (II) Cell invasion experiment:

[0100] (1) The basement glue (Matrigel glue) is transferred from a -20 ℃ refrigerator to a 4 ℃ refrigerator for melting. The gun head used for coating, 24-well plate and transwell chamber are pre-cooled in a 4 ℃ refrigerator. 100 μL of Matrigel is taken and added to 300 μL of pre-cooled serum-free medium, and mixed well. 25 μL of the above diluted Matrigel is added to the upper chamber of the transwell plate to cover the entire polycarbonate membrane. The Matrigel is aggregated into glue at 37 ℃ for 30 min.

[0101] (2) The tumor cells are added to DMSO, compound 3 and compound 3r one day in advance. After the tumor cells are grown, they are trypsinized and centrifuged at 850 rpm for 5 min. The cell pellet is resuspended with serum-free medium, mixed well by blowing, and then counted. The cells are diluted to the desired concentration for standby.

[0102] (3) Each cell line is divided into 3 groups, namely blank control group, compound 3 group and compound 3r group. 8 μm transwell chamber is taken and placed in a 24-well plate. 600 μL of culture medium containing 10% FBS is added to the lower chamber, and 200 μL of serum-free medium is added to the upper chamber. The cells are placed in an incubator for further culture.

[0103] (4) The transwell chamber is taken out, and the inside and outside of the chamber are washed with PBS. The cells in the upper chamber of the transwell are gently wiped with a cotton swab. 4% paraformaldehyde is added to the upper and lower chambers for at least 30 min of fixation, and finally 200 μL and 500 μL of crystal violet are added to the upper and lower chambers for 20 min of staining.

[0104] (5) Wash the chamber and gently wipe off the unbound crystal violet dye with a cotton swab. Observe and take photos under a microscope. Randomly select 3 fields of view from each chamber, count the number of cells passing through each field of view, and take the average value.

[0105] III. Experimental results

[0106] As shown in Table 1, compounds 3 and 3r have inhibitory effects on the migration and invasion of BCPAP and BHT101 cell lines. In the BCPAP cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 70%, and the invasion inhibitory efficiency is about 75% (p < 0.001). In the BHT101 cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 60%, and the invasion inhibitory efficiency is about 70% (p < 0.001). Figure 8a Figure 8b As shown in Table 1, compounds 3 and 3r have inhibitory effects on the migration and invasion of BCPAP and BHT101 cell lines. In the BCPAP cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 70%, and the invasion inhibitory efficiency is about 75% (p < 0.001). In the BHT101 cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 60%, and the invasion inhibitory efficiency is about 70% (p < 0.001).

[0107] As shown in Table 1, compounds 3 and 3r have inhibitory effects on the migration and invasion of BCPAP and BHT101 cell lines. In the BCPAP cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 70%, and the invasion inhibitory efficiency is about 75% (p < 0.001). In the BHT101 cell line, the migration inhibitory effect of compound 3 at a concentration of 5 μM is significantly better than that of compound 3r, with an inhibition efficiency of about 60%, and the invasion inhibitory efficiency is about 70% (p < 0.001).

[0108] It should be understood by those of ordinary skill in the art that the above discussion of any of the embodiments is merely exemplary and is not intended to suggest that the scope of the disclosure (including the claims) is limited to these examples; under the concept of the present disclosure, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other changes to the different aspects of the embodiments of the present disclosure as described above. In order to be brief, they are not provided in detail.

[0109] The embodiments of the present disclosure are intended to cover all such alternatives, modifications, and variations as fall within the broad scope of the appended claims. Accordingly, any and all such modifications, alternatives, variations and equivalents are intended to be encompassed by the claims of the present disclosure.​

Claims

1. A tumor cell inhibitor, characterized by, The tumor cell inhibitor has a structural formula as shown in the following: 。 2. A method of preparing the tumor cell inhibitor of claim 1, wherein, The preparation method comprises the following steps: The intermediate compound, 3,4,5-trimethoxyaniline and p-toluenesulfonamide are dissolved in a first solvent to obtain a reaction liquid; the first solvent comprises at least one of 1,4-dioxane, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide and pyridine; The reaction liquid is mixed with silica gel and then dried by spinning, and column chromatography purification is performed to obtain the tumor cell inhibitor; The intermediate compound has a structural formula as shown in the following: 。 3. The preparation method according to claim 2, characterized in that, The molar ratio of the intermediate compound, 3,4,5-trimethoxyaniline and p-toluenesulfonamide is 1:1.2-3:0.8-3.

4. The production method according to claim 2, characterized by, The preparation method of the intermediate compound comprises the following steps: The initial compound is dissolved in a second solvent, N,N-diisopropylethylamine is added, stirring is performed, 2,4-dichloropyrimidine is added, and reflux reaction is performed for a period of time to obtain an initial reaction liquid; the second solvent comprises at least one of anhydrous ethanol, n-butanol, acetonitrile, dioxane, dimethyl sulfoxide, N,N-dimethylformamide and pyridine; The initial reaction liquid is mixed with silica gel and then dried by spinning, and column chromatography purification is performed to obtain the intermediate compound; The initial compound has a structural formula as shown in the following: 。 5. The preparation method according to claim 4, characterized in that, The molar ratio of the initial compound, N,N-diisopropylethylamine and 2,4-dichloropyrimidine is 1:1.2-3:0.8-2.

6. The preparation method according to claim 4, characterized in that, The preparation method of the initial compound comprises the following steps: The p-phenylenediamine is dissolved in a pre-cooled third solvent, triethylamine is added, p-methoxybenzenesulfonyl chloride is added dropwise, stirring is performed for a period of time under ice bath, and then the reaction is stopped to obtain a reaction liquid; the third solvent comprises at least one of dichloromethane, 1,2-dichloroethane, chloroform, diethyl ether, tetrahydrofuran, methyl tert-butyl ether, dimethyl sulfoxide, N,N-dimethylformamide and pyridine; The reaction liquid is mixed with silica gel and then dried by spinning, and purification is performed to obtain the initial compound.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the p-phenylenediamine, triethylamine and p-methoxybenzenesulfonyl chloride is 0.8-2.02:1:0.8-1.

8. An anticancer composition, characterized by, The preparation method comprises the following steps:

9. The tumor cell inhibitor of claim 1, the tumor cell inhibitor prepared by any one of claims 2-7 or the anti-cancer composition of claim 8 in the preparation of an anti-cancer drug, wherein the cancer is thyroid cancer.

10. Use according to claim 9, characterized in that, The tumor cell inhibitor is used for inhibiting proliferation, migration and / or invasion of tumor cells, and the tumor cells are thyroid cancer cells. The tumor cell inhibitor is used for inhibiting proliferation, migration and / or invasion of tumor cells, and the tumor cells are thyroid cancer cells.

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