Application of F919 compounds and salts and stereoisomers thereof in preparation of anti-hepatoma drugs and anti-cancer drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of F919 compounds and salts and stereoisomers thereof in preparation of anti-hepatoma drugs and the anti-hepatoma drugs. The compounds can efficiently inhibit the proliferation capacity of human hepatoma cells, induce arrest and apoptosis of the hepatoma cells in the G0 / G1 period, inhibit the proliferation of the human hepatoma cells and inhibit the proliferation of the human hepatoma cells. The obvious tumor inhibition activity on nude mouse in-situ tumors of liver cancer cells is realized. The invention provides a new medicine for treating the liver cancer, provides an effective method for developing a targeted treatment medicine for treating the liver cancer, and is of great significance for preparing the medicine for treating the liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of F919 compounds and salts and stereoisomers thereof in the preparation of anti-liver cancer drugs and anti-cancer drugs. Background Art

[0002] Liver cancer is one of the causes of malignant tumor-related death. Due to the relatively hidden location of the liver, and generally, the early symptoms of liver cancer are only manifested as common digestive system disorders such as loss of appetite, fatigue, and nausea, so it is often easily overlooked by people. At present, early liver cancer screening mainly relies on serum alpha-fetoprotein and abdominal ultrasound examination. In the present invention, serum alpha-fetoprotein is Alpha fetoprotein in English, abbreviated as AFP, but the sensitivity, specificity and predictive value of AFP detection are still controversial in the academic community, and asymptomatic patients will not take the initiative to seek medical treatment for ultrasound examination under normal circumstances. In addition, liver cancer has a high degree of malignancy, easy metastasis, easy recurrence and other characteristics, resulting in most patients being in the middle and late stages of liver cancer once discovered, and basically need drug treatment. However, the currently marketed anti-liver cancer drugs have poor therapeutic effects, and most systemic therapeutic drugs will produce drug resistance after use, resulting in extremely poor prognosis of liver cancer patients, causing drug treatment of liver cancer patients to become a major clinical problem. Therefore, the development of new anti-liver cancer drugs has important theoretical significance and application value for improving the prognosis of liver cancer patients.

[0003] The goal of new drug research and development is to develop high-quality drugs that can prevent, diagnose, treat and / or relieve symptoms more effectively than existing therapies. In recent years, with the rapid development of new research methods such as high-throughput sequencing technology, researchers have begun to focus on the use of tumor biomarkers to guide the early clinical development of anti-tumor drug candidates, thereby reducing the risk of drug development failure. The successful launch of epidermal growth factor receptor inhibitors, tyrosine kinase inhibitors, and immune checkpoint inhibitors in emerging immunotherapy are all based on the results of in-depth research on tumor biomarkers in the field of basic research. The research on the application of amides in liver cancer is in the development stage. Currently, there are few types of amides used for liver cancer, and the role of F919 compounds as amides in liver cancer has not been studied. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of F919 compounds and their salts and stereoisomers in the preparation of anti-liver cancer drugs and anti-cancer drugs.

[0005] The use of F919 compounds in the preparation of anti-liver cancer drugs, the structural formula of the F919 compounds is as follows:

[0006]

[0007] Wherein, R1 is C1-C6 alkyl or halogen;

[0008] R2 is thiophene, a benzene ring, a halogen-substituted benzene ring or an alkyl-substituted benzene ring.

[0009] Preferably, the structural formula of the F919 compound is as follows:

[0010]

[0011] Preferably, the anti-liver cancer drug comprises one or more compounds having the structural formula of the formula (1), the formula (2), the formula (3) and the formula (4).

[0012] Preferably, the anti-liver cancer drug is a drug that inhibits the proliferation ability of liver cancer cells and reduces the clone-forming ability of liver cancer cells.

[0013] Preferably, the anti-liver cancer drug is a drug that induces cell cycle arrest and apoptosis of liver cancer cells.

[0014] Preferably, the anti-liver cancer drug is a drug that affects the expression of proteins related to the cell cycle progression of liver cancer cells.

[0015] Preferably, the anti-liver cancer drug is a drug that has tumor inhibition activity on nude mouse in situ tumors of liver cancer cells.

[0016] An anti-liver cancer drug comprises any one of the compounds, or a salt thereof, or a stereoisomer thereof.

[0017] Preferably, the medicine also includes pharmaceutically acceptable excipients.

[0018] Preferably, the pharmaceutically acceptable excipient is sterile water for injection or sodium chloride injection.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The compound of the present invention can effectively inhibit the proliferation ability of human liver cancer cells, induce G0 / G1 phase arrest and apoptosis of liver cancer cells, and has significant tumor inhibition activity on nude mouse in situ tumors of liver cancer cells. The present invention provides a new class of drugs for liver cancer treatment, provides an effective method for developing targeted therapeutic drugs for liver cancer treatment, and is of great significance for the preparation of drugs for liver cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Figure 1 is a graph showing the effects of different compounds on the viability of L02 cells and MHCC97-H cells; A is the effect of F919-1131 on the viability of L02 cells and MHCC97-H cells detected by CCK-8 method; B is the effect of F919-1171 on the viability of L02 cells and MHCC97-H cells detected by CCK-8 method; C is the effect of F919-1187 on the viability of L02 cells and MHCC97-H cells detected by CCK-8 method; D is the effect of F919-1172 on the viability of L02 cells and MHCC97-H cells detected by CCK-8 method; E is the effect of sorafenib on the viability of L02 cells and MHCC97-H cells detected by CCK-8 method;

[0022] Figure 2 The figure shows the effect of F919-1172 on the clone-forming ability and apoptosis of MHCC97-H cells; A is the plate cloning experiment to detect the effect of F919-1172 on the clone-forming ability of MHCC97-H cells; B is the flow cytometry to detect the effect of F919-1172 on the apoptosis of MHCC97-H cells;

[0023] Figure 3 Figure 1 is the effect of F919-1172 on the cell cycle of MHCC97-H cells; A is the effect of F919-1172 on the cell cycle of MHCC97-H cells detected by flow cytometry; B is the effect of F919-1172 on the expression of G1 / S cell cycle checkpoint-related proteins in MHCC97-H cells detected by Western blot; C is the statistical analysis of the expression levels of different G1 / S cell cycle checkpoint-related proteins in MHCC97-H cells;

[0024] Figure 4 Figure 1 is the effect of F919-1172 on the proliferation of nude mouse liver cancer orthotopic implanted tumors; A is a schematic diagram of nude mouse liver cancer orthotopic implanted tumor experiment; 5-6 week old female nude mice were constructed with MHCC97-H-Luc cell liver cancer orthotopic implanted tumor model, and the tumor size was measured and grouped using in vivo imaging technology on the 7th day after modeling. High and low doses of F919-1172 or sorafenib were intraperitoneally injected once every 2 days and weighed; nude mice were anesthetized and killed on the 22nd day; B is the bioluminescence image of nude mouse liver cancer orthotopic implanted tumors; C is the statistics of fluorescence intensity of nude mouse liver cancer orthotopic tumors; D is the image of nude mouse liver and intrahepatic tumors; E is the expression of proliferation marker Ki-67 protein in liver tissues of each group of nude mice observed by immunohistochemical staining, the left side of the dotted line in the figure is adjacent to the cancer tissue, and the right side is the tumor tissue; F is the statistics of nude mouse weight;

[0025] Figure 5 This is a diagram of the effect of F919-1172 on the heart, spleen, lung, kidney and brain tissues of nude mice observed by HE staining. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0027] In the present invention, the F919 compounds of different concentrations are prepared into solutions using sterile water for injection as solvent.

[0028] Example 1: Cell culture: Human hepatoma cell line MHCC97-H was cultured in high-glucose DMEM complete medium, and human normal liver cell line L02 was cultured in RPMI 1640 complete medium. The temperature of the incubator was kept constant at 37°C, and the CO2 concentration was kept constant at 5% by volume. The high-glucose DMEM complete medium contained 10% by volume fetal bovine serum and 1% by volume penicillin / streptomycin, and the RPMI 1640 complete medium contained 10% by volume fetal bovine serum and 1% by volume penicillin / streptomycin.

[0029] Example 2: Detection of the effect of F919-type small molecule compounds on the proliferation ability of human liver cancer cells and normal liver cells

[0030] The structures of the F919 small molecule compounds of the present invention are shown in formula (1), formula (2), formula (3) and formula (4):

[0031]

[0032] Formula (1) is denoted as F919-1131; formula (2) is denoted as F919-1171; formula (3) is denoted as F919-1187; and formula (4) is denoted as F919-1172.

[0033] The experimental cells included human liver cancer cells MHCC97-H and normal human liver cells L02. After various cells were treated with different concentrations of F919 compounds and positive drug sorafenib for 48 hours, the original culture medium was discarded, 90 μL of culture medium and 10 μL of CCK-8 were added to each well, and the cells were incubated in an incubator for 40 minutes, and the OD value was detected at 450 nm using an enzyme reader.

[0034] The results of CCK-8 assay are as follows Figure 1 As shown, the results showed that compared with the solvent group, F919 compounds could significantly inhibit the proliferation of human liver cancer MHCC97-H cells, but had a smaller inhibitory effect on the proliferation of normal liver cells L02.

[0035] Example 3: Detection of the effect of F919-1172 on the cloning ability of human liver cancer cells

[0036] The cancer cells used in the experiment were human liver cancer cells MHCC97-H. After the cells were treated with the F919-1172 compound for 14 days, the original culture medium was discarded, and the culture medium containing F919-1172 was replaced every 72 hours within 14 days. The cells were washed three times with PBS, 1 mL of 4% paraformaldehyde was added to each well, and fixed for 40 minutes; washed three times with PBS, 1 mL of crystal violet dye was added to each well, and the cells were stained for 10 minutes; washed three times with PBS, dried, and the entire six-well plate and each well were photographed separately with a digital camera.

[0037] It should be noted that similar effects can be achieved by fixing the cells with 4% paraformaldehyde for 30 to 60 minutes or staining the cells with crystal violet stain for 10 to 20 minutes.

[0038] The results of the cell plate clone formation experiment are as follows Figure 2 As shown in A, the results showed that compared with the solvent group, the number of human liver cancer MHCC97-H cell clones in the F919-1172-treated group was significantly reduced, indicating that F919-1172 can inhibit the cell clone formation ability of liver cancer cell lines.

[0039] Example 4: Detection of apoptosis level of human liver cancer cells after treatment with F919-1172

[0040] The cancer cells used in the experiment were human liver cancer cells MHCC97-H.

[0041] Flow cytometry was used to detect the level of cell apoptosis: cells were inoculated in a 6-well plate and treated with different concentrations of F919-1172. After 48 hours, the cell supernatant was aspirated and collected in a centrifuge tube. The cells were washed three times with PBS, digested with trypsin, and collected in an EP tube containing the supernatant. The cells were centrifuged at 4°C, 1000 rpm for 10 min, the supernatant was aspirated, 500 μL of binding buffer was added, and the cells were gently blown to make a cell suspension. The cell count was adjusted to 5 × 10 5 / mL; aspirate 195μL of cell resuspension and transfer to a 1.5mL EP tube; add 5μL Annexin V-FITC dye to each group, mix well and then add 10μL PI dye; protect from light, let stand for 15min, and detect on the machine.

[0042] The experimental results are as follows Figure 2As shown in B, in MHCC97-H cells, the percentage of apoptotic cells in the solvent control group was 6.79% ± 0.35%, the percentage of apoptotic cells in the F919-11725 μM group was 9.31% ± 0.21%, the percentage of apoptotic cells in the F919-117210 μM group was 13.03% ± 0.33%, and the percentage of apoptotic cells in the F919-117220 μM group was 26.07% ± 0.38%, all of which were significantly higher than the solvent control group. The above results indicate that F919-1172 can induce apoptosis in MHCC97-H cells.

[0043] Example 5: Detection of the cell cycle and cell cycle-related protein expression in human liver cancer cells after treatment with F919-1172

[0044] The cancer cells used in the experiment were human liver cancer cells MHCC97-H.

[0045] 1. Flow cytometry for cell cycle detection: cells were inoculated in 6-well plates and treated with different concentrations of F919-1172. After 48 hours, the cell supernatant was collected, and the cells were digested with trypsin and collected in 1.5 mL EP tubes. Centrifuge at 4°C, 1000 rpm for 10 min, discard the supernatant, and wash the cells twice with PBS; add 1 mL of pre-cooled 70% ethanol to each tube, blow the cells apart, and fix at 4°C for 24 hours; centrifuge at 1000 rpm for 5 min, discard the supernatant; add 1 mL of pre-cooled PBS to wash the cells twice, and discard the supernatant; add 500 μL of propidium iodide staining solution to each tube to resuspend the cells, and incubate at 37°C in the dark for 30 min; use a flow cytometer to detect fluorescence and light scattering at a wavelength of 488 nm.

[0046] The experimental results are as follows Figure 3As shown in A, in MHCC97-H cells, the proportion of G0 / G1 phase cells in the solvent control group was 46.06%±1.02%, while the proportion of G0 / G1 phase cells in the F919-117210μM group was 59.63%±2.61%, and the proportion of G0 / G1 phase cells in the F919-117220μM group was 63.78%±0.71%, which were significantly higher than those in the solvent control group; the proportion of S phase cells in the solvent control group was 30.60%±0.80%, and the proportion of S phase cells in the F919-117210μM group was 63.78%±0.71%, which were significantly higher than those in the solvent control group. The percentage of cells in the G2 / M phase of the solvent control group was 13.14%±1.06%, the percentage of cells in the G2 / M phase of the F919-117210μM group was 13.71%±0.73%, and the percentage of cells in the G2 / M phase of the F919-117220μM group was 12.55%±1.16%, which were not significantly different from those in the solvent control group. The above results indicate that F919-1172 can inhibit the transition of MHCC97-H cells from G0 / G1 phase to S phase and block the cell cycle process.

[0047] 2. Western Blotting experiment to detect cell cycle related proteins: cells were inoculated on 60 mm 2 Different concentrations of F919-1172 were treated in culture dishes, and cells were collected 48 hours later. Western Blotting experiments were performed according to the following method.

[0048] Western Blotting experiments were performed as follows:

[0049] 1. Protein extraction

[0050] (1) Cell preparation: Use a cell scraper to scrape the cells and transfer the cells with the culture medium to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 10 min to collect the cell precipitate. Wash and resuspend the cells twice with PBS. Add an appropriate amount of RIPA cell lysis buffer and transfer to a 1.5 mL EP tube. The RIPA cell lysis buffer contains protease inhibitors and phosphatase inhibitors.

[0051] (2) Cell disruption: Use a cell ultrasonic disruptor at 15% power to disrupt cells on ice for 3 seconds, cool for 3 seconds, and repeat 3 times; after lysis, let stand on ice for 30 minutes, then centrifuge at 4°C, 12,000 rpm, for 20 minutes; carefully aspirate the supernatant into a clean, pre-cooled new EP tube.

[0052] 2. Protein quantification, referred to as BCA method

[0053] (1) Preparation of standard curve: Use RIPA cell lysis buffer to prepare 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, and 0 mg / mL BSA standard solutions.

[0054] (2) Preparation of color developing solution: Use the Thermo Fisher BCA quantitative kit to prepare the color developing solution at a ratio of A:B = 50:1.

[0055] (3) Sample addition: Add 10 μL of sample and 10 μL of standard of different concentrations to each well of a 96-well plate. Immediately add 200 μL of AB colorimetric solution and incubate at 37°C for 30 min.

[0056] (4) Concentration determination: The protein concentration was measured at a wavelength of 562 nm using an ELISA reader. Based on the measured absorbance value of the standard, RIPA cell lysis buffer and 5× loading buffer were added to adjust the cell concentration to a consistent level.

[0057] (5) Protein denaturation: Heat in a metal bath for 10 min to fully denature the protein and store in a -20°C refrigerator.

[0058] 3. Glue preparation: According to the different molecular weights of proteins, a lower separation gel with a volume concentration of 6% to 15% and an upper concentration gel with a volume concentration of 5% are required.

[0059] (1) Install the glass plate: The glass plate should be cleaned in advance and dried in the oven. When installing, make sure the lower end is aligned on a flat surface, otherwise the glue will leak. Fix the glass plate on the bracket and clamp it for later use.

[0060] (2) Prepare the lower separation gel: Prepare the required gel according to the recipe, quickly pour the mixed gel into the groove of the glass plate, add 2 mL of deionized water, flatten the separation gel, and let it stand for 40 min.

[0061] (3) Prepare the upper layer of condensation glue: After the lower layer of glue solidifies, pour out the deionized water, drain the liquid, gently absorb the remaining liquid with filter paper, pour the upper layer of glue, insert a comb between the two glass plates, and let it stand for 30 minutes.

[0062] (4) Protein loading: Take the protein sample out from -20°C and immediately place it on ice. Determine the loading volume according to the protein concentration, so that the loading amount is generally 20 μg.

[0063] (5) Electrophoresis: When running the upper gel, keep the voltage constant at 80 V and run the electrophoresis for 30 min. When running the lower gel, keep the voltage constant at 120 V and run the electrophoresis for 60 min, so that the marker bands corresponding to the target bands are fully separated. It should be noted that similar effects can be achieved by running the electrophoresis for 60 min to 80 min.

[0064] (6) Transfer: Activate the PVDF membrane in methanol for 30 seconds, cut off the upper layer of gel, install the transfer device, blackboard (-) → porous padding sponge → second layer of filter paper → gel → PVDF membrane → second layer of filter paper → porous padding sponge → whiteboard (+), carefully remove the bubbles between each layer, install the transfer device into the transfer tank, add transfer solution, put an ice box in the empty space in the transfer tank, cover the lid, put the transfer tank into the foam box, fill it with ice and water mixture, turn on the power, keep the voltage at 100V, and transfer for 1 hour. It should be noted that transferring for 1 hour to 2 hours can achieve similar results.

[0065] (7) Blocking: Prepare a 5% blocking solution with TBST and skimmed milk powder and block for 1.5 h.

[0066] (8) Primary antibody incubation: Remove the PVDF membrane, wash the blocking solution thoroughly with TBST, and then add the diluted target protein antibody. The primary antibodies are: cyclin D1, manufactured by CST, with a dilution ratio of 1:1000; cyclin E1, manufactured by CST, with a dilution ratio of 1:1000; CDK 2, manufactured by CST, with a dilution ratio of 1:1000; CDK4, manufactured by CST, with a dilution ratio of 1:1000; P21, manufactured by CST, with a dilution ratio of 1:1000; P27, manufactured by CST, with a dilution ratio of 1:1000; β-actin, manufactured by Proteintech, with a dilution ratio of 1:5000, and incubate at 4°C overnight. Remove the PVDF membrane and wash it three times with room temperature TBST on a shaker for 10 min each time.

[0067] (9) Secondary antibody incubation: Place the PVDF membrane in an antibody incubation box and add the secondary antibody corresponding to the target protein antibody. The secondary antibodies include Anti-Mouse IgG, manufactured by Zhuangzhi Bio, at a dilution ratio of 1:5000; Anti-Rabbit IgG, manufactured by Zhuangzhi Bio, at a dilution ratio of 1:5000. Incubate on a shaker at room temperature for 1.5 h. Remove the PVDF membrane and wash it three times on a shaker with room temperature TBST, each time for 10 min.

[0068] (10) Gel imaging luminescence: Prepare luminescence solution with A:B=1:1, mix well, and protect from light. Remove the residual liquid with filter paper, soak the PVDF membrane in a black box, and make sure it is fully in contact for imaging.

[0069] The experimental results are as follows Figure 3As shown in B and C, after treatment with F919-1172, the expression of cyclin D1, cyclin E1, CDK2 and CDK4 proteins in MHCC97-H cells was significantly reduced, while the expression levels of P21 and P27 proteins were significantly increased. These results indicate that F919-1172 can inhibit the proliferation of human liver cancer cell MHCC97-H by affecting the expression of G1 / S cell cycle checkpoint-related proteins in MHCC97-H cells and blocking the cell cycle process.

[0070] Example 6: Investigation of the tumor-suppressing ability of F919-1172 in nude mice

[0071] The cancer cells in the experiment were human liver cancer cells MHCC97-H. The experimental animals were 30 5-6 week old female BALB / c nude mice, and the experimental days were 25 days. The content of the investigation was the ability of F919-1172 to inhibit the growth of human liver cancer cells MHCC97-H in situ tumors in nude mice and its toxic effects on nude mice.

[0072] 1. Nude mouse liver in situ tumorigenesis experiment: When the cell healing degree reached 80%, the cells were collected. It should be noted that the cells can be collected when the cell healing degree reached 80%-90%. The cells were washed three times with pre-cooled PBS to remove the residual serum in the cells. After complete digestion with warm trypsin, the digestion was terminated with complete medium. The cells were centrifuged at 1000 rpm for 10 min and the supernatant was discarded. The matrix gel and serum-free medium were mixed at a ratio of 1:1, and the cells were resuspended with the mixed solution to adjust the concentration of the cell suspension to 1×10 8Cells / mL. Nude mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. During anesthesia, 10 mg of chloral hydrate was injected per kg of body weight of the mice. The limbs of the nude mice were fixed in the center of the foam board with the abdomen facing up. After disinfection with alcohol cotton balls, a disposable surgical blade was used to transversely cut the abdominal skin and muscle layer below the xiphoid process, and the left liver lobe was carefully highlighted with a cotton swab. A disposable insulin syringe was used to absorb the cell suspension and 20 μL was injected in situ into the liver. After injection, the muscle and skin tissues were sutured with disposable medical sutures, and the nude mice were placed on a 37°C constant temperature heating pad until the nude mice woke up. Seven days after the injection of the cell suspension, D-Luciferin with a concentration of 15 mg / mL was injected intraperitoneally at a volume of 10 μL / g. After 15 minutes, isoflurane gas anesthesia was performed using the gas anesthesia system of the in vivo imaging instrument, and then the mice were placed under the in vivo imaging instrument for imaging, fluorescence was observed, and the tumor size was calculated. According to the tumor size, the mice were divided into 4 groups, with 5 mice in each group. Seven days after the injection of the cell suspension, the experimental group was given intraperitoneal injection once every two days, and the control group was given the same dose of solvent, and in vivo imaging was performed once every 7 days. After 3 weeks, blood was collected from the orbits, and the nude mice were killed by cervical dislocation. The animals were dissected with medical scissors and medical forceps, and the tumor tissue in the liver was observed and photographed. The heart, liver, spleen, lung, kidney and brain were separated and fixed with 4% paraformaldehyde.

[0073] 2. Immunohistochemical staining: dewax and rehydrate the paraffin sections, perform antigen repair, and block with goat serum at room temperature for 1 hour after cooling; place the sections face up on the slide, add 100 μL of primary antibody diluted with goat serum to each section, the primary antibody is Ki-67, the manufacturer is Abcam, the dilution ratio is 1:200, put into a wet box, and incubate at 4°C overnight; place the sections in a washing box, wash three times with PBS, 5 minutes each time; add 100 μL of diluted secondary antibody, the secondary antibody is Anti-Rabbit, the manufacturer is Zhuangzhi Bio, the dilution ratio is 1:100, and incubate at room temperature for 1 hour; place the sections in a washing box, wash three times with PBS, 5 minutes each time; seal the sections with sealing agent, and observe and take pictures under a microscope.

[0074] 3. HE staining: After the tissue is fixed, rinse with running water for 2 hours, dehydrate with 70%, 80%, and 90% ethanol solutions in turn, each for 30 minutes, and then put into 95% and 100% ethanol solutions twice, each for 20 minutes; put into pure ethanol and xylene mixed solution for 15 minutes, xylene I for 15 minutes, and xylene II for 15 minutes, until transparent; put into a 60℃ constant temperature box with a mixture of half xylene and half paraffin for 15 minutes, and then put into paraffin I and paraffin II for 50 minutes each. It should be noted that similar effects can be achieved by putting into paraffin I and paraffin II for 50 minutes to 60 minutes each; use tweezers to pick up the metal paraffin mold, heat it slightly on the alcohol lamp, and put it on a flat table. Take out the wax cup containing pure paraffin from the incubator, pour a little paraffin, and then put the tweezers. Heat the slicer slightly on an alcohol lamp, clamp the material and put it into the wax mold with the cut surface facing down, arrange it neatly, then put on the embedding box, and gently pour in the molten wax for embedding; install the fixed and repaired paraffin block on the clamping table of the slicer, fix the slicer on the knife clamp with the blade facing up; shake and push the spiral to make the paraffin block close to the blade; adjust the angle and position between the paraffin block and the blade, so that the blade and the paraffin slice are about 15 degrees; adjust the thickness regulator to make the slice thickness 5μm; put the wax tape cut into 20cm long flat on the wax tape box, cut a small section of the wax slice with a single-sided blade, put it on the slide, add a drop of water, and observe the slice under a microscope. It should be noted that the wax tape can be cut into 20cm~30cm to achieve a similar effect; take a clean slide, pick up the unfolded slice, so that it is located at 1 / 3 of the slice, mark or label it on the grinded surface, and put it on the slice rack.Adjust the temperature of the water bath to 60°C. When the water temperature is controlled at 60°C, place the slices together with the slice rack into a dry staining jar, put them into the water bath, cover them with a lid, and wait for 30 minutes until the wax melts; sequentially place the slices into xylene for 10 minutes → xylene for 10 minutes → anhydrous ethanol for 5 minutes → anhydrous ethanol for 5 minutes → 95% alcohol by volume for 5 minutes → 90% alcohol by volume for 5 minutes → 80% alcohol by volume for 5 minutes → 70% alcohol by volume for 5 minutes → wash with distilled water; dye the slices with Harris hematoxylin for 5 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia water, and rinse with running water; rinse with running water for about 15 minutes to turn the slices blue; place the slices in 1% hydrochloric acid ethanol solution for about 2 seconds to tens of seconds to fade, and see the slices. The slice turns red and the color is lighter; put the slice into running water to restore the blue color; put the slice into 50% ethanol by volume → 70% ethanol by volume → 80% ethanol by volume for 3 minutes each. It should be noted here that 3 minutes to 5 minutes can achieve similar effects; put the slice into eosin stain for 3 minutes. It should be noted here that 1 minute to 3 minutes can achieve similar effects; put the slice into 95% ethanol by volume to wash away the excess red, and then put it into anhydrous ethanol for 3 minutes. It should be noted here that 3 minutes to 5 minutes can achieve similar effects, and finally use absorbent paper to absorb the excess ethanol; put the slice into xylene I and II for 3 minutes each. It should be noted here that 3 minutes to 5 minutes can achieve similar effects; seal with neutral gum, examine under a microscope, and collect and analyze images.

[0075] The experimental results are as follows Figure 4 As shown, compared with the solvent control group, high and low doses of F919-1172 and sorafenib treatment were able to significantly inhibit the growth of orthotopic transplanted tumors of liver cancer in nude mice. On the 22nd day after modeling, the nude mice were anesthetized and killed, and the livers were removed for observation. The results showed that the livers of nude mice in the solvent control group were enriched with more tumor tissue cells, while only a few tumor tissue cells were visible in the livers of most nude mice in the high and low doses of F919-1172 groups and sorafenib groups. Microscopic observation of the immunohistochemical staining of liver tissues showed that the number of Ki-67 positive cells, a proliferation marker, in the liver tumor tissues of nude mice in the high and low doses of F919-1172 groups and sorafenib groups was significantly less than that in the solvent control group. All experimental nude mice did not die during the experiment, and there was no significant difference in the body weight of nude mice among the groups. The HE staining results are shown in Figure 5 As shown, high and low doses of F919-1172 and sorafenib did not cause obvious pathological changes in important tissues and organs such as the heart, spleen, lung, kidney, and brain of nude mice. The above results indicate that F919-1172 can also exert anti-liver cancer activity in vivo and has good safety.

[0076] Based on the above experiments, it can be seen that the F919-type small molecule compounds provided in the embodiments of the present invention can be used as targeted drugs, showing high efficiency in inhibiting the proliferation, clone formation, cell cycle progression, and nude mouse tumor in situ growth of MHCC97-H cells, and inducing apoptosis of MHCC97-H cells, and thus have the potential to be used in anti-tumor, especially liver cancer treatment.

[0077] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0078] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of F919 compounds and their salts and stereoisomers in the preparation of anti-liver cancer drugs, characterized in that: The structural formula of the F919 compound is as follows: Wherein, R1 is C1-C6 alkyl or halogen; R2 is thiophene, a benzene ring, a halogen-substituted benzene ring or an alkyl-substituted benzene ring.

2. The use according to claim 1, characterized in that: The structural formula of the F919 compound is as follows:

3. The use according to claim 2, characterized in that The anti-liver cancer drug comprises one or more compounds having the structural formula of the formula (1), the formula (2), the formula (3) and the formula (4).

4. The use according to claim 1, characterized in that The anti-liver cancer drug is a drug that inhibits the proliferation ability of liver cancer cells and reduces the cloning ability of liver cancer cells.

5. The use according to claim 1, characterized in that The anti-liver cancer drug is a drug that induces cell cycle arrest and apoptosis of liver cancer cells.

6. The use according to claim 1, characterized in that The anti-liver cancer drug is a drug with tumor inhibition activity on nude mouse in situ tumors of liver cancer cells.

7. An anti-liver cancer drug, characterized in that: The invention comprises the compound described in any one of claim 2, or a salt thereof, or a stereoisomer thereof.

8. The anti-liver cancer drug according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

9. The anti-liver cancer drug according to claim 8, characterized in that: The pharmaceutically acceptable excipient is sterile water for injection or sodium chloride injection.

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