Small molecule compound for inhibiting RBM45 and application thereof

Abstract

The invention relates to a small molecule compound for inhibiting RBM45, the small molecule compound is 6484-0109, the chemical formula of the small molecule compound is C21H14FN3O3, and the structural formula of the small molecule compound is shown in the specification. The invention discloses a new application of a small molecule compound 6484-0109, which can be combined with RBM45 protein in a targeting manner, act on tumor cells and down-regulate expression of ASCT2 protein in cancer cells, and can remarkably inhibit clone formation capability of hepatocellular carcinoma cells and inhibit proliferation of tumor cells, thereby achieving an anti-tumor effect. After 6484-0109 administration, the IC50 of hepatocellular carcinoma cell strains BEL-7402, SMMC-7721, HepG2, Hep3B, SK-HEP-1 and HCC-LM3 are respectively 5.01 [mu] M, 4.387 [mu] M, 4.662 [mu] M, 2.423 [mu] M, 2.519 [mu] M and 2.409 [mu] M, and the compound has a remarkable effect of inhibiting tumor proliferation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of targeted inhibitors, in particular to a small molecule compound for inhibiting RBM45 and application thereof. BACKGROUND

[0002] RBM45 is an RNA-binding protein, mainly located in the nucleus, and can also shuttle between the nucleus and the cytoplasm, mainly involved in the regulation of gene expression in cells by binding to RNA molecules. RBM45 has been identified as an RNA splicing factor that actively promotes liver lipid accumulation by regulating the expression of key lipogenic enzymes. Currently, only a dozen RNA-binding proteins are considered to be major cancer drivers, and the role of RNA-binding proteins in the entire process of cancer occurrence and development needs further exploration. RBM45 is overexpressed in diffuse large B-cell lymphoma, cholangiocarcinoma and hepatocellular carcinoma. At the same time, the literature reports that RBM45 is closely related to the survival period of clinical patients. In various cancers (such as lung cancer, colorectal cancer), the expression level of RBM45 is abnormally elevated, and is related to the proliferation, invasion and chemoresistance of tumor cells, RBM45 can promote the unlimited proliferation of cancer cells by regulating the mRNA splicing of cell cycle proteins (such as Cyclin D1) or oncogenes (such as c-Myc). There are reports that silencing RBM45 in vitro can reduce the lipid accumulation of human hepatoma cell HepG2 cells and regulate the expression of key lipogenic enzymes, suggesting that RBM45 affects cell metabolism. Metabolic reprogramming is an important reason for the rapid proliferation of tumor cells, and research has found that knocking down RBM45 can inhibit the proliferation of hepatocellular carcinoma cells and affect glutamine metabolism, reduce the expression level of glutamine protein, that is, inhibiting RBM45 can induce cancer cell cycle arrest or apoptosis.

[0003] Current research on RBM45 expression inhibition mainly focuses on gene editing, RNA interference, small molecule inhibitors and protein interaction regulation. Gene editing or shRNA can permanently or long-term inhibit the expression of RBM45, which may cause irreversible physiological effects (such as abnormal function of hematopoietic stem cells). Compared with other inhibition methods, small molecule inhibitors have reversibility, small molecule inhibitors do not directly interfere with the genome DNA, avoiding the risk of chromosomal breakage, insertion mutation and other risks caused by gene editing, and are safer. Therefore, targeting RBM45 and developing inhibitors can be used as a new strategy for cancer treatment. SUMMARY

[0004] The present application aims to provide a small molecule compound for inhibiting RBM45. The small molecule compound can inhibit the protein expression level of glutamine transporter, and at the same time inhibit the proliferation and growth of tumor cells by causing cell cycle arrest, so as to play an anti-tumor role.

[0005] The application aims to provide the application of the small molecule compound.

[0006] The application aims to provide the application of the small molecule compound. The application aims to provide the application of the small molecule compound. 21 H 14 FN3O3, and the structural formula is as follows: .

[0007] Further, the application of the small molecule compound in the preparation of an RBM45 inhibiting drug is specific to target binding to RBM45 and hinder the combination with ASCT2.

[0008] The small molecule compound can dose-dependently down-regulate the expression of ASCT2 protein in hepatocellular carcinoma cells, thereby playing an anti-hepatoma role.

[0009] Further, the application of the small molecule compound in the preparation of an anti-tumor drug.

[0010] Further, the application of the small molecule compound in the preparation of an anti-tumor drug is characterized in that the tumor is a hepatoma tumor, and the hepatoma tumor includes but is not limited to any one of BEL-7402, SMMC-7721, HepG2, Hep3B, SK-HEP-1 and HCC-LM3.

[0011] The application has the following technical effects: The application discloses a new use of the small molecule compound 6484-0109, which can target binding to RBM45 protein, act on tumor cells, down-regulate the expression of ASCT2 protein in cancer cells, significantly inhibit the clone formation ability of hepatocellular carcinoma cells, inhibit the proliferation of tumor cells, thereby achieving an anti-tumor effect, and the IC 50 of the hepatocellular carcinoma cell strains BEL-7402, SMMC-7721, HepG2, Hep3B, SK-HEP-1 and HCC-LM3 after administration of 6484-0109 is 5.01 muM, 4.387 muM, 4.662 muM, 2.423 muM, 2.519 muM and 2.409 muM respectively, and the effect of inhibiting tumor proliferation is significant. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 ZK0109 interacts with RBM45 protein at GLY-110, HIS-112, SER-106 and ARG-27 amino acid sites.

[0013] Figure 2: The results of cell thermal shift experiment of ZK0109 on cancer cell HCC-LM3.

[0014] Figure 3 : ZK0109 hindered the interaction of RBM45-ASCT2 protein.

[0015] Figure 4 : The effect of ZK0109 on the expression of ASCT2 protein in liver cancer cells.

[0016] Figure 5 : The inhibition rate curve of ZK0109 on different liver cancer cells.

[0017] Figure 6 : The growth curve of liver cancer cells SK-HEP-1 and HCC-LM3 under the action of different concentrations of ZK0109.

[0018] Figure 7 : The results of clone experiment of liver cancer cells SK-HEP-1 and HCC-LM3 under the action of different concentrations of ZK0109.

[0019] Figure 8 : The effect of different concentrations of ZK0109 on the cycle arrest of liver cancer cells SK-HEP-1 and HCC-LM3.

[0020] Figure 9 : The effect of different concentrations of ZK0109 on the apoptosis of liver cancer cells SK-HEP-1 and HCC-LM3.

[0021] Figure 10 : The change of body weight of nude mice xenograft tumor model treated with ZK0109.

[0022] Figure 11 : The change of tumor volume of nude mice xenograft tumor model treated with ZK0109.

[0023] Figure 12 : The change of tumor volume and relative tumor proliferation rate of mice xenograft tumor model in different drug groups after 21 days.

[0024] Figure 13 : The results of H&E staining, Ki67 staining and ASCT2 staining of different drug groups.

[0025] Figure 14 : The change of relative expression amount of Ki67 and ASCT2 of different drug groups. DETAILED DESCRIPTION

[0026] The application will be described in detail below by way of examples. It is necessary to point out here that the following examples are only used to further illustrate the application and cannot be understood as limiting the scope of protection of the application. Those skilled in the art can make some non-essential improvements and adjustments to the application according to the above description of the application.

[0027] Example 1 Screening and preliminary verification of small molecule compounds inhibiting RBM45 Screening: Homology modeling: Since there is no crystal structure of full-length RBM45 protein in Protein Data Bank, the known structure homologous protein is found by homology analysis using Modeller 9.15 software, and a structural model is established for RBM45 using the template. Based on the Ramachandran plot evaluation, further optimization is made using Amber12 software. Based on Schrödinger and other software, a program is designed to learn by itself for high-throughput computer virtual screening.

[0028] (1) Protein preparation: the protein structure obtained by homology modeling is used as the protein for docking, and the Protein Preparation Wizard module of Schrödinger software is used to process and optimize the protein: first, the missing side chains in the crystal are filled with Prime, hydrogen atoms are added, and the crystallization water molecules in the system are deleted; the whole system is optimized based on the OPLS-2005 force field until the RMSD value is less than 0.3 Å. After processing the protein, the Receptor Grid Generation module is used to generate a docking grid with the bound small molecule in the protein crystal as the reference center; (2) Ligand preparation: obtain the two-dimensional molecular structure of the candidate drug from the ZINC database, and use the LigPrep module to convert the molecule to a three-dimensional structure and generate different conformations; (3) Molecule docking screening: based on Schrödinger and other software, further programming is made for molecule docking screening, and the lowest energy molecule docking conformation is scored according to the binding energy of the candidate compound and RBM45 protein, and the final result is obtained.

[0029] The compounds in the ChemDiv database are used for computer virtual molecule docking with the RBM45 protein structure, and 23 potential compounds with the lowest drug-protein binding free energy are screened for further analysis. The relevant information of the 23 compounds is shown in Table 1.

[0030] Table 1:

[0031] Among the 23 screened compounds, 6484-0109 (hereinafter referred to as ZK0109) has a significant inhibitory effect on liver cancer cells SK-HEP-1 and AMMC-7721, and therefore, subsequent pharmacodynamic verification is carried out.

[0032] The results, as shown in Figure 1 ZK0109 interacts with RBM45 protein at GLY-110, HIS-112, SER-106, and ARG-27 amino acid sites.

[0033] Preliminary verification: I. Cell thermal shift experiment (1) Take the cells HCC-LM3 in good condition and in the logarithmic growth phase and inoculate them in a 10 cm culture dish. When the cell confluence reaches more than 90%, collect the cells; (2) Cell collection: discard the supernatant in the cell culture dish, wash once with 1xPBS, add 1 mL of 0.25% trypsin (containing EDTA) for digestion, terminate the digestion with complete culture medium, blow off the cells and transfer them to a 1.5 mL sterile culture dish, centrifuge at 800 rpm for 5 min, discard the supernatant, collect the cell precipitate and wash once with pre-cooled 1xPBS; (3) After collecting the cell precipitate, add proteinase inhibitor-containing protein lysis buffer RIPA lysis buffer for resuspension, freeze-thaw repeatedly with liquid nitrogen and a 37°C water bath for 3 times, centrifuge at 17000 g at 4°C for 20 min, collect the cell supernatant (lysate); (4) Divide the cell supernatant equally into 2 parts into two 1.5 mL centrifuge tubes, and incubate with solvent control DMSO and drug ZK0109 (10 μM) at 4°C in a rotary mixer for 3 h, respectively; (5) After incubation, transfer the above mixture to 200 μL doff tubes, about 100 μL per tube, and place in a PCR instrument, and incubate at 50, 50.6, 52.0, 54.5, 57.9, 62.1, 67.1, 71.5, 74.8, 77.7 and 79.2°C for 4 min, and then cool at room temperature for 3 min; (6) After constant temperature heating, centrifuge the mixture at 17000 g at 4°C for 20 min, and collect the cell supernatant; (7) Add 5x protein loading buffer, heat at 100°C metal bath for 10 min, and then perform Western Blot experiment.

[0034] The results of the cell thermal shift experiment are as follows Figure 2As shown, ZK0109 (10 μM) can enhance the thermal stability of RBM45 protein in the lysate of hepatocellular carcinoma cells HCC-LM3, which preliminarily verifies that ZK0109 targets RBM45 to play an anti-hepatocellular carcinoma effect.

[0035] II. Western Blot experiment (1) Take well-conditioned hepatocellular carcinoma cells SK-HEP-1 and HCC-LM3 in the logarithmic growth phase, respectively, count after digestion, blowing and transferring to a sterile centrifuge tube, resuspend after centrifugation, take the required cell volume, resuspend in complete culture medium, blow evenly, inoculate 1.8 mL per well, 1000 cells per well in a 6-well plate, shake the 6-well plate evenly, and place it in a 37°C constant temperature, 5% CO2 incubator for 24 h; (2) After 24 h of culture, randomly divide into 5 groups: negative control group, drug administration group (low, medium and high dose), positive control group, 200 μL per well, continue in the incubator, drug action for 48 hours; take out the 6-well plate, discard the supernatant, wash twice with 1×PBS solution, add 100 μL of protein lysate (containing protease inhibitor and phosphatase inhibitor) per well, lyse on ice for 15 min; after lysis, transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 12000 r, 4°C for 20 min, collect the protein supernatant, and quantitate the protein by BCA method; (3) BCA protein quantification: prepare protein standard BSA protein solution with concentration gradient of 0, 1, 2, 4, 6, 8, 10 mg / mL. Prepare an enzyme-labeled plate, add 2 μL of protein standard solution or sample protein solution and 18 μL of 1×PBS solution per well, set 3 replicate wells for each sample, and finally add 180 μL of BCA working solution (BCA working solution: V A液 :V B液 =50:1). After incubating the enzyme-labeled plate in a 60°C oven for 15 min, measure the OD value at an excitation wavelength of 562 nm on a full-wavelength enzyme-labeled instrument. According to the protein standard concentration and absorbance, draw a protein concentration-absorbance standard curve to calculate the concentration of the protein sample. The sample volume is 10 ug, and the sample volume is calculated; add 5× protein loading buffer to the protein sample, heat at 100°C metal bath for 10 min, and store at -20°C.

[0036] (4) PAGE gel electrophoresis ① Determine the molecular weight of the target protein and select PAGE separation gels of different concentrations. Wash the glass plate with clean water, fix it on the gel rack, and test for leaks with distilled water. Check for leaks, pour out the distilled water, add the pre-prepared separation gel and stacking gel, and insert the comb. After about 10-15 minutes, the gel will solidify. After removing the comb, electrophoresis can be performed. The electrophoresis program is 80 V for the upper gel, about 30 minutes (marker enters the separation gel); 120 V for the lower gel, 90 minutes. The separation range of PAGE separation gels with different concentrations is shown in Table 2; Table 2:

[0037] (5) Rapid wet transfer: After electrophoresis, remove the glass plate and clean the residual electrophoresis solution on the surface with distilled water, then immerse it in rapid wet transfer solution. After the PVDF membrane is completely immersed in methanol for activation for 30 seconds, pry open the glass plate and transfer the gel completely to the PVDF membrane. Clamp the wet transfer clip in the order of positive electrode (white) - sponge - filter paper - PVDF membrane - gel - filter paper - sponge - negative electrode (black). Place the wet transfer clip in the wet transfer tank in the positive and negative directions of the electrodes, pour in the rapid wet transfer solution, and transfer the membrane at a constant current of 400 mA at room temperature.

[0038] (6) Blocking: After wet transfer, take out the PVDF membrane and place it in a protein incubation box. Add protein-free rapid blocking solution to completely submerge the PVDF membrane and place it on a shaker and shake slowly for 10 minutes (60 rpm / min).

[0039] (7) Primary antibody incubation: After blocking, remove the PVDF membrane, wash the residual blocking solution on the membrane surface with TBST, cut the target protein band according to the position of the protein marker, place it in the antibody incubation box, add the primary antibody diluent, and incubate at 4°C for 16 hours.

[0040] (8) Secondary antibody incubation: After the primary antibody incubation is completed, the primary antibody is recovered and 1×TBST solution is added. The plate is shaken at 120 rpm / min and washed three times for 10 minutes each time. The TBST solution is poured out and the secondary antibody diluent is added. The plate is shaken at 60 rpm / min and incubated at room temperature for 1 hour.

[0041] (9) Chemiluminescence imaging: After the secondary antibody incubation is complete, discard the secondary antibody and add 1×TBST solution. Wash three times on a shaker at 120 rpm / min, each time for 10 min. Prepare ECL luminescent solution (Solution A:Solution B = 1:1). After washing, remove the strips and place them in an automatic gel imaging instrument. Add luminescent solution to the strips and expose them. After exposure, save the template and data.

[0042] like Figure 3As shown, the small molecule compound can be targeted to bind to RBM45, hinder the binding of RBM45 to ASCT2, affect the regulation of RBM45 to ASCT2, and thus down-regulate the protein level of ASCT2. The small molecule compound does not affect the expression of RBM45 protein, but affects its function. ZK0109 can dose-dependently regulate the expression of ASCT2 protein in hepatocellular carcinoma cells, as shown in Figure 4 .

[0043] Example 2 Verification of the inhibitory effect of the small molecule compound ZK0109 on the proliferation of cancer cells in vitro CCK-8 experiment The HCC cell strain in good condition in the logarithmic growth phase was washed once with sterile PBS, 2.5% trypsin (containing EDTA) was added for digestion, and complete culture medium was used to terminate digestion. The cells were blown off and the cell suspension was transferred to a clean sterile centrifuge tube. Centrifugation was performed at 800 rpm for 5 min, the supernatant was discarded, 1 mL of complete culture medium was added to suspend the cell pellet, and the cells were counted and diluted to (2-4) x 10 4 The cell dilution suspension was taken with a syringe, inoculated in a 96-well plate at 180 μL / well, and incubated in a 37°C constant temperature, 5% CO2 incubator. After 24 h of cell inoculation, different concentrations of the test drug were added at 20 μL / well, 3 replicate wells were set for each concentration, and incubation was continued for 96 h. The supernatant was discarded, 100 μL of 10% CCK-8 solution was added to each well, and incubation was performed in the incubator for 1 h. The OD value at 450 nm wavelength was measured in the enzyme marker instrument, and the cell inhibition rate of each drug concentration was calculated with the solvent hole as the control. The inhibition rate curve was drawn, and the IC 50 value was calculated. The cell inhibition rate calculation formula is as follows:

[0044] The inhibitory effect of ZK0109 on each hepatocellular carcinoma cell is shown in Figure 5 A-F, and the IC 50 of the hepatocellular carcinoma cell strains BEL-7402, SMMC-7721, HepG2, Hep3B, SK-HEP-1, and HCC-LM3 after administration of ZK0109 was 5.01 μM, 4.387 μM, 4.662 μM, 2.423 μM, 2.519 μM, and 2.409 μM, respectively.

[0045] Growth curve experiment: Take the state of good, in the logarithmic phase of hepatocellular carcinoma cells, after digestion blow transfer to a sterile centrifuge tube after centrifugation resuspended count, take the required cell volume resuspended in complete medium, blow even with 180 μL per well, 2000-3000 cells per well, 96-well plate, placed in 37℃ constant temperature, 5% CO2 incubator for 24 h.

[0046] Set six groups: negative control group, drug group (low, medium and high dose), paclitaxel (Tax) positive control group. OD value was measured by CCK-8 kit, once every 24 h, for 6 consecutive days. Growth curve was drawn by OD value.

[0047] The growth curves of SK-HEP-1 cells and HCC-LM3 cells are shown in Figure 6 Compared with the control group, the growth inhibition rates of SK-HEP-1 cells treated with ZK0109 at 1.25 μM, 2.5 μM, 5 μM and 10 μM were 8%, 24%, 55% and 56%, respectively, and the growth inhibition rate of paclitaxel at 1 μM was 76%. Compared with the control group, the growth inhibition rates of HCC-LM3 cells treated with ZK0109 at 1.25 μM, 2.5 μM, 5 μM and 10 μM were 5%, 28%, 55% and 57%, respectively, and the growth inhibition rate of paclitaxel at 1 μM was 71%. The above results showed that ZK0109 at a certain concentration could significantly inhibit the growth of hepatocellular carcinoma cells.

[0048] Plate colony formation experiment: (1) Take the state of good, in the logarithmic phase of hepatocellular carcinoma cells, after digestion blow transfer to a sterile centrifuge tube after centrifugation resuspended count, take the required cell volume resuspended in complete medium, blow even with 1.8 mL per well, 1000 cells per well, 6-well plate, shake the six-well plate evenly, placed in 37℃ constant temperature, 5% CO2 incubator for 24 h; (2) After 24 h of culture, randomly divided into 5 groups: negative control group, drug group (low, medium and high dose), positive control group, 200 μL per well, continue to culture in the incubator, replace the culture medium every 2-3 days, until the single cell mass grows to 50-100 cells, terminate the culture; (3) Take out the 6-well plate, discard the supernatant, wash twice with 1×PBS solution, add 1 mL 4% paraformaldehyde per well, fix at room temperature for 30 minutes, discard the fixing solution, add 1 mL 0.05% crystal violet solution per well, avoid light staining for 30 minutes, discard the crystal violet solution, wash the dye in the 6-well plate with water, dry at room temperature and take pictures.

[0049] The results are shown in Figure 7As shown, compared with the control group, the growth inhibition rates of SK-HEP-1 cells in ZK0109 administration were 33%, 56%, and 100% at 1.25 μM, 2.5 μM, and 5 μM, respectively, and the growth inhibition rate of the positive drug paclitaxel was 100% at 1 μM; compared with the control group, the growth inhibition rates of HCC-LM3 cells in ZK0109 administration were 40%, 60%, and 100% at 1.25 μM, 2.5 μM, and 5 μM, respectively, and the growth inhibition rate of paclitaxel was 100% at 1 μM. The above results show that ZK0109 can significantly inhibit the clonogenic ability of hepatocellular carcinoma cells.

[0050] Cell cycle detection experiment (1) Take the well-conditioned hepatocellular carcinoma cells in the logarithmic growth phase, digest and transfer to a sterile centrifuge tube, resuspend and count after centrifugation, take the required cell volume, resuspend in complete culture medium, blow evenly, 1.8 mL per well, 200,000 cells per well, inoculate in a 6-well plate, shake the 6-well plate evenly, and place it in a 37°C constant temperature, 5% CO2 incubator for 24 h; (2) After 24 h of culture, randomly divide into 5 groups: negative control group, drug administration group (low, medium, and high dose), and positive control group, 200 μL per well, continue to culture in the incubator for 48 h, and terminate the culture; (3) After 48 h of drug treatment, take out the 6-well plate, discard the supernatant, wash twice with pre-cooled 1xPBS solution, add 2.5% trypsin (containing EDTA) for digestion, terminate the digestion with complete serum culture medium, blow off the cells and transfer the cell suspension to a clean sterile centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant; resuspend the cell pellet with pre-cooled 1xPBS solution, centrifuge at 800 rpm for 5 min, discard the supernatant, add 300 μL of pre-cooled 1xPBS to each tube, and then add 700 μL of pre-cooled anhydrous ethanol to make the final ethanol content 70%, gently blow the cells apart, and place them in a 4°C refrigerator for 12 h of fixation.

[0051] (4) Centrifuge the fixed 12 h cell sample at 800 rpm for 5 min, gently aspirate the supernatant, add 1 mL of pre-cooled 1xPBS for washing twice, and gently blow the cells apart with a pipette; add 500 μL of PI / RNaseA staining working solution (PI:RNaseA = 9:1) to each sample, stain at room temperature for 30 min in the dark; after mixing evenly, detect by flow cytometry.

[0052] Results are as follows Figure 8As shown in the data, compared with the control group, the proportion of G2 / M phase cells of SK-HEP-1 cells increased by 9.66%, 38.19% and 43.41% when ZK0109 was administered at 2.5μM, 5μM and 10μM, respectively; compared with the control group, the proportion of G2 / M phase cells of HCC-LM3 cells increased by 15.74%, 22.39% and 28.93% when ZK0109 was administered at 2.5μM, 5μM and 10μM, respectively. It can be seen that ZK0109 can significantly inhibit the proliferation of liver cancer cells.

[0053] Cell apoptosis detection experiment (1) Take hepatocellular carcinoma cells in good condition and in the logarithmic growth phase, digest them, transfer them to a sterile centrifuge tube, centrifuge and resuspend them for counting. Take the required cell volume and resuspend it in complete culture medium. After blowing evenly, inoculate 200,000 cells per well in a 6-well plate at 1.8 mL per well. Shake the 6-well plate up and down and left and right to evenly mix. Place it in a 37°C incubator with 5% CO2 and culture for 24 hours. (2) After 24 hours of culture, the cells were randomly divided into five groups: negative control group, drug-treated group (low, medium, and high doses), and positive control group. Each well was given 200 μL of the drug and cultured in the dressing box for another 48 hours before terminating the culture. (3) After 48 hours of drug treatment, the 6-well plate was removed, the supernatant was discarded, and the plate was washed twice with pre-cooled 1× PBS solution. 2.5% trypsin (containing EDTA) was added for digestion, and the digestion was terminated with a culture medium containing complete serum. The cells were blown off and the cell suspension was transferred to a clean sterilized centrifuge tube. The tube was centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. The cell pellet was resuspended in pre-cooled 1× PBS solution and centrifuged at 800 rpm for 5 minutes. 500 μL of the buffer solution in the apoptosis detection kit was added to the cell pellet. 5 μL of PI dye and 5 μL of Annexin V dye were added to each sample. The cells were incubated in the dark for 10 minutes and then detected by flow cytometry.

[0054] The results are as follows Figure 9 As shown, ZK0109 failed to induce apoptosis in hepatocellular carcinoma cells after administration, indicating that the anti-hepatocellular carcinoma effect of ZK0109 is not related to the induction of cell apoptosis.

[0055] Example 3 Validation of the small molecule compound ZK0109 in inhibiting the proliferation of hepatocellular carcinoma cells in a nude mouse xenograft tumor model Construction of nude mouse xenograft tumor model (1) Modeling and drug administration methods HCC-LM3 cells in the logarithmic growth phase were taken and 5.0×10 6100 μL cell suspension was inoculated subcutaneously in the right axilla of nude mice. The diameter of the xenograft tumor in nude mice was measured with a vernier caliper. When the tumor grew to 80-100 mm 3 The animals were then randomly divided into groups. Nude mouse body weights and tumor volumes were recorded every two days, and the antitumor effects of the test compound, ZK0109, were dynamically observed. ZK0109 was administered intraperitoneally every other day, while paclitaxel (Tax) was administered via tail vein injection twice weekly. Mice were sacrificed after 21 days of dosing. ZK0109 was diluted with 45% PEG 300, 5% Tween 80, and 55% saline for injection, while paclitaxel was diluted with injectable saline for injection.

[0056] (2) Tumor detection indicators and calculation methods ①Tumor volume (TV): V (mm 3 )=1 / 2ab 2 , where a is the length of the tumor (mm) and b is the width of the tumor (mm); ②Relative tumor volume (RTV): RTV=V t / V0, where V t is the tumor volume obtained at each measurement, V0 is the initial tumor volume at the time of grouping; ③Relative tumor proliferation rate (%): T / C=T RTV / C RTV ×100% (T RTV :RTV in treatment group; C RTV : Negative control group RTV. ) Figure 10 AB results showed that after ZK0109 administration, the body weight of nude mice in the low-dose group (25 mg / kg) and the medium-dose group (50 mg / kg) did not change significantly, while the body weight of nude mice in the high-dose group (100 mg / kg) first decreased and then increased, but ultimately there was no significant difference. Figure 11 、 Figure 12 The results showed that compared with the control group, after ZK0109 administration, the volume of transplanted tumors in nude mice showed a downward trend, and the relative tumor volume and relative tumor proliferation rate showed a significant downward trend.

[0057] like Figure 13As shown, HE staining results show that after administration of ZK0109, tumor cells lose polarity, cell arrangement is disordered, volume is larger than that of normal cells, and shape is irregular; IHC Ki67 staining results show that under the action of the low-dose administration group (25 mg / kg), the medium-dose administration group (50 mg / kg), and the high-dose administration group (100 mg / kg) of ZK0109, different degrees of tumor proliferation inhibition are produced on hepatocellular carcinoma xenograft tumors, and the protein expression level of ASCT2 in tumor cells is down-regulated in a dose-dependent manner, specifically as shown in Figure 14 FIG. 6.

Claims

1. A small molecule compound that inhibits RBM45, characterized in that: The small molecule compound is 6484-0109, whose chemical formula is C 21 H 14 FN3O3, whose structural formula is: 。 2. Use of the small molecule compound of claim 1 in the preparation of a medicament for inhibiting the binding of RBM45 to ASCT2.

3. Use of the small molecule compound of claim 1 in the preparation of an anti-tumor medicament.

4. The use of the small molecule compound according to claim 3 for the preparation of an antitumor medicament, characterized in that: The tumor is a liver cancer tumor, and the liver cancer tumor includes, but is not limited to, any one of BEL-7402, SMMC-7721, HepG2, Hep3B, SK-HEP-1, and HCC-LM3.

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