Application of small molecule compound for inhibiting degradation of HNF4alpha in preparation of medicine for treating liver cancer

Abstract

The invention discloses application of a small molecule compound for inhibiting degradation of HNF4alpha in preparation of a medicine for treating liver cancer. The structural general formula of the small molecule compound for inhibiting the degradation of the HNF4alpha is as follows: # imgabs0 #, the small molecule compound for inhibiting degradation of the HNF4alpha can play the biological effects of correcting liver glycolipid metabolism disorder, relieving liver fibrosis and inhibiting liver cancer malignant phenotypes, and is applied to clinical treatment of chronic liver diseases and liver cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of a small molecule compound that inhibits the degradation of HNF4α in the preparation of a medicament for treating liver cancer. Background Art

[0002] Hepatocyte nuclear factor-4α (HNF4α) is one of the highly conserved ligand-dependent transcription factors in the nuclear receptor superfamily, and is expressed in tissues such as the liver, kidney, pancreas, and intestine, but is mainly expressed in mature hepatocytes. In mature hepatocytes, HNF4α can bind to the promoters of approximately 12% of the genes in the cell, and is involved in maintaining important functions such as hepatocyte lipid metabolism, albumin synthesis, drug detoxification, energy metabolism, and bile acid synthesis, playing a key regulatory role in hepatocyte differentiation and maintaining hepatocyte function. HNF4α also regulates the development of kidney and intestinal tissues, and regulates the production of insulin in pancreatic tissues. Previous studies have shown that the expression of HNF4α decreases in various epithelial-derived tumors such as liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), pancreatic cancer, colorectal cancer, and kidney cancer. Overexpression of HNF4α can inhibit the proliferation and metastasis of tumor cells and promote tumor cell apoptosis, suggesting that HNF4α is a potential target for tumor treatment. The overexpression of HNF4α in liver cancer cells mediated by an adenovirus vector has been reported, and the therapeutic effect of HNF4α on tumors has been clarified. At the same time, previous studies have also shown that the expression of HNF4α decreases significantly in various chronic liver diseases including fatty liver and liver fibrosis. In animal experiments, upregulating the expression of HNF4α in damaged hepatocytes during fatty liver and liver fibrosis by genetic engineering means can promote hepatocyte function and alleviate the progression of hepatocyte lipid deposition and liver fibrosis, indicating that HNF4α has important therapeutic value for chronic liver diseases. These studies have clarified that increasing the expression of HNF4α has a therapeutic effect on chronic liver diseases and liver cancer.

[0003] The tripartite motif (TRIM) protein family is one of the subfamilies of E3 ubiquitin ligases, and participates in various biological processes such as intracellular signal transduction, apoptosis, autophagy, and immunity by regulating the ubiquitination of target proteins. Among them, the TRIM47 protein is closely related to the occurrence and development of chronic liver diseases and liver cancer. It has been found that TRIM47, as an E3 ubiquitin ligase, can interact with HNF4α through the SPRY domain and mediate its ubiquitination and degradation, thereby reducing the level of HNF4α. Therefore, blocking the interaction between TRIM47 and HNF4α to stabilize HNF4α is an important direction for developing drugs against chronic liver diseases and liver cancer based on HNF4α. Therefore, screening small molecule compounds against the three-dimensional conformation of the binding region between TRIM47 and HNF4α is expected to inhibit the ubiquitination and degradation of HNF4α by TRIM47.

[0004] So far, there has been no research report on the application of blockers or inhibitors that affect the binding of TRIM47 to the HNF4α protein. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide an application of a small molecule compound that inhibits the degradation of HNF4α in the preparation of drugs for treating liver cancer.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided an application of a small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt in the preparation of drugs for treating liver cancer. The general structural formula of the small molecule compound that inhibits the degradation of HNF4α is shown as follows:

[0008]

[0009] Among them, R1 is selected from C1-C20 alkyl, ;

[0010] R2 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0011] R3 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0012] R4 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0013] R5 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0014] R6 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0015] R7 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0016] R8 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0017] R9 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0018] R 10 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0019] R 11 is selected from hydrogen, C1-C20 alkyl, and C1-C20 alkoxy;

[0020] R 12 is selected from hydrogen, C1-C20 alkyl;

[0021] Or, R4 and R5 form a five-membered or six-membered ring with carbon and oxygen (e.g., , where the dotted line indicates the connection to the benzene ring).

[0022] More preferably, in the small molecule compound that inhibits HNF4α degradation,

[0023] R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl,

[0024]

[0025] R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0026] R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0027] R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0028] R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0029] R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0030] Or, the five-membered ring formed by R4, R5 and carbon, oxygen ;

[0031] Or, the six-membered ring formed by R4, R5 and carbon, oxygen , the dashed line indicates the connection to the benzene ring.

[0032] Most preferably, the small molecule compound that inhibits the degradation of HNF4α is selected from one of the following structures:

[0033] The small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt in the application is used as the sole active ingredient.

[0034] In the second aspect of the present invention, there is provided an application of a small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt in the preparation of a drug for treating chronic liver disease.

[0035] The general structural formula of the small molecule compound that inhibits the degradation of HNF4α is shown as follows:

[0036]

[0037] Wherein, R1 is selected from C1-C20 alkyl, ;

[0038] R2 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0039] R3 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0040] R4 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0041] R5 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0042] R6 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen (fluorine, chlorine, bromine, iodine), ;

[0043] R7 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0044] R8 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0045] R9 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0046] R 10 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0047] R 11 selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy;

[0048] R 12 selected from hydrogen, C1-C20 alkyl;

[0049] or, R4 and R5 together with carbon and oxygen form a five-membered or six-membered ring (such as: , the dotted line indicates the connection to the benzene ring).

[0050] More preferably, in the small molecule compound that inhibits the degradation of HNF4α,

[0051] R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl,

[0052]

[0053] R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0054] R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0055] R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0056] R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0057] R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ;

[0058] or, the five-membered ring formed by R4, R5 and carbon, oxygen ;

[0059] or, the six-membered ring formed by R4, R5 and carbon, oxygen , the dotted line indicates the connection to the benzene ring.

[0060] Most preferably, the small molecule compound that inhibits the degradation of HNF4α is selected from one of the following structures:

[0061] .

[0062] The chronic liver disease is selected from non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver failure.

[0063] In the application, the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt is used as the sole active ingredient.

[0064] In the third aspect of the present invention, a pharmaceutical preparation is provided, which is made of the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt and a medically acceptable excipient.

[0065] The dosage form of the pharmaceutical preparation is injection, capsule, tablet, granule, pill, microcapsule preparation, microsphere preparation, nano-preparation, etc.

[0066] In the fourth aspect of the present invention, a pharmaceutical composition is provided, which is made of the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt, a pharmaceutically acceptable carrier, and a drug for improving chronic liver disease or a drug for treating liver cancer.

[0067] The drug for improving chronic liver disease is selected from ursodeoxycholic acid, polyene phosphatidylcholine, glutathione, etc.

[0068] The drug for treating liver cancer is selected from lenvatinib, donafenib, regorafenib, atezolizumab, nivolumab, cisplatin, paclitaxel, etc.

[0069] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0070] The present invention discovers that the small molecule compound that inhibits the degradation of HNF4α directly targets the intracellular HNF4α protein, promotes the transcriptional activity of the HNF4α protein, and up-regulates the protein level of HNF4α in liver cancer cells.

[0071] The present invention uses a series of protein function research techniques such as protein interaction, docking, and site mutation, and combines in vitro and in vivo experiments to determine that TRIM47 is an E3 ubiquitin ligase that binds to HNF4α and promotes its ubiquitination and degradation, and clarifies the key amino acid sites that affect the binding of TRIM47 to the HNF4α protein, and further screens out small molecule drugs that can inhibit the binding of TRIM47-HNF4α and maintain the stability of the HNF4α protein.

[0072] The small molecule compounds provided by the present invention for inhibiting the degradation of HNF4α can exert biological effects of correcting liver glycolipid metabolism disorders, reducing liver fibrosis, and inhibiting the malignant phenotypes of liver cancer, and can be applied to the clinical treatment of chronic liver diseases and liver cancer.

[0073] The present invention for the first time discovers that the TRIM47 protein can directly bind to the HNF4α protein and mediate the degradation of the HNF4α protein, thereby promoting the progression of liver cancer. Through a series of protein interaction techniques, the key amino acid sites affecting the binding of TRIM47 and the HNF4α protein are identified, which are His534 / His600 of TRIM47 and Trp349 / Glu353 / Pro342 of HNF4α respectively. Mutating the relevant sites of the two proteins can significantly inhibit the binding of TRIM47 and HNF4α and promote the stability and transcriptional activity of the HNF4ɑ protein. It is predicted by molecular docking experiments and the MOE-Site Finder software that the compound CZ2401 can bind to HNF4α. Further, the compound CZ2401 and its analogs CZ-2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9, and CZ2401-10 can upregulate the activity of the HNF4α reporter gene and increase the expression level of the HNF4α protein in liver cancer cells. Pharmacological activity experiments find that the preferred compound CZ2401 can inhibit lipid deposition in hepatocytes, reduce the progression of CCl4-induced liver fibrosis in mice, significantly inhibit the proliferation, migration, invasion, and colony formation ability of liver cancer cells, and inhibit the growth of subcutaneous implanted tumors of liver cancer cells in mice.

[0074] The present invention discovers that a series of compounds such as the small molecule CZ2401 can upregulate the protein level of HNF4α by inhibiting the interaction between TRIM47 and HNF4α. Specifically, the compound CZ2401 directly binds to HNF4α to block the interaction between HNF4α and TRIM47, thereby inhibiting the ubiquitination and degradation of HNF4α. Adding the compound CZ2401 during the culture of liver cancer cells can inhibit the malignant phenotypes of liver cancer cells, promote the transcriptional activity of HNF4α, and upregulate the expression of its downstream genes. Further in vivo experiments show that the compound CZ2401 can inhibit the growth of subcutaneous implanted tumors of liver cancer cells in mice, indicating that the compound CZ2401 has potential tumor treatment effects. On the other hand, the compound CZ2401 can also inhibit lipid deposition in hepatocytes and reduce liver fibrosis in mice by upregulating HNF4α, indicating that the compound CZ2401 also has potential therapeutic effects on chronic liver diseases. These research results show that CZ-2401 and its analogs have potential therapeutic effects on chronic liver diseases and liver cancer and have very good clinical application prospects. Description of the Drawings

[0075] Figure 1 Schematic diagram showing that knockdown of TRIM47 protein significantly inhibits the degradation of HNF4α protein and promotes the expression of its downstream liver function-related genes.

[0076] Figure 2 Schematic diagram of the binding results between TRIM47 and HNF4α proteins.

[0077] Figure 3 Schematic diagram of screening potential amino acid sites affecting the binding of TRIM47 and HNF4α proteins by molecular docking technology.

[0078] Figure 4 Schematic diagram of mutating relevant sites of TRIM47 to inhibit its binding to HNF4α protein.

[0079] Figure 5 Schematic diagram of mutating relevant sites of HNF4α to inhibit its binding to TRIM47 protein.

[0080] Figure 6 Schematic diagram of the predicted ligand-binding pocket of HNF4α.

[0081] Figure 7 Schematic diagram of the results of detecting the enhancing effect of compounds on the transcriptional activity of HNF4α by the dual-luciferase reporter gene system.

[0082] Figure 8 Schematic diagram of the results of detecting the increasing effect of compounds on the protein level of HNF4α by Western blotting.

[0083] Figure 9 Schematic diagram of the binding results between the selected compound CZ2401 and HNF4α protein detected by microscale thermophoresis (MST).

[0084] Figure 10 Schematic diagram of the result that compound CZ2401 significantly inhibits the binding of TRIM47 and HNF4α proteins.

[0085] Figure 11 Schematic diagram of the results of detecting the enhancing effect of structural analogs of compound CZ2401 on the transcriptional activity of HNF4α by the luciferase reporter gene system.

[0086] Figure 12 Schematic diagram of the results of detecting the increasing effect of compound CZ2401 and its structural analogs on the protein level of HNF4α by Western blotting.

[0087] Figure 13 Schematic diagram of the result that compound CZ2401 significantly inhibits oleic acid (OA)-induced hepatocyte lipotoxicity.

[0088] Figure 14 Schematic diagram of the result that compound CZ2401 can significantly alleviate the progression of CCl4-induced liver fibrosis in mice.

[0089] Figure 15 Schematic diagram of the result that compound CZ2401 can significantly inhibit the proliferation, migration, invasion and colony formation of Hep3B hepatoma cells.

[0090] Figure 16 Schematic diagram of the result that compound CZ2401 can significantly inhibit the tumor progression in a subcutaneous xenograft tumor model of Hep3B hepatoma cells in mice. Detailed implementation manners

[0091] To illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0092] Compound CZ2401 (Compound ID G856-9858), CZ2401-1 (Compound ID G856-9872), CZ2401-2 (Compound ID G856-9887), CZ2401-3 (Compound ID G856-9873), CZ2401-4 (Compound ID G856-9856), CZ2401-5 (Compound ID G856-9857), CZ2401-6 (Compound IDG856-9861), CZ2401-7 (Compound ID G856-9859), CZ2401-8 (Compound ID G856-9860), CZ2401-9 (Compound ID G856-9837) and CZ2401-10 (Compound ID G856-9921), all of the above compounds are purchased from Chemdiv INC.

[0093] Example 1

[0094] Wild-type Hep3B hepatoma cells were seeded in a 60 mm culture dish at a density of 5×10 5 , transfected with small interfering TRIM47 to knockdown the expression of TRIM47, after 48 hours, proteins were harvested using a protein lysate, and immunoblotting was performed after high-temperature denaturation; at the same time, cells were lysed using TRIZOL to extract RNA, and the expression levels of HNF4α downstream genes were detected by qRT-PCR. Figure 1Schematic diagram showing that TRIM47 protein knockdown significantly inhibits the degradation of HNF4α protein and promotes the expression of its downstream liver function-related genes. Among them, A is the schematic diagram of the immunoblotting experiment result, and B is the schematic diagram of the qRT-PCR result. As can be seen from the figure, TRIM47 protein knockdown significantly upregulates the protein level of HNF4α ( Figure 1 as shown in A) and promotes the expression of its downstream liver function-related genes ( Figure 1 as shown in B).

[0095] Example 2

[0096] Transfect the Flag-tagged TRIM47 overexpression plasmid and the V5-tagged HNF4α overexpression plasmid into Huh7 liver cancer cells. After 24 hours, add MG132 and treat for 6 - 8 hours. When collecting samples, add IP lysis buffer (containing phosphatase and protease inhibitors) and lyse on ice for 30 minutes, then extract the protein. After centrifugation at 12000 rpm for 15 minutes, take the supernatant for protein quantification. Add anti-Flag or anti-V5 affinity gel respectively, and incubate with rotation at 4℃ overnight. The next day, wash 3 times with 100 mM Wash Buffer, denature at high temperature, and then perform immunoblotting experiment. The results are as Figure 2 shown, Figure 2 which is the schematic diagram of the binding result between TRIM47 and HNF4α proteins. As can be seen from the figure, TRIM47 protein can bind to HNF4α protein mutually.

[0097] Example 3

[0098] Perform protein-protein molecular docking experiment using the HNF4α-LBD protein domain (PDB: 4IQR) and the predicted three-dimensional structure of TRIM47 by Alphafold. The results are as Figure 3 shown, Figure 3 which is the schematic diagram of screening potential amino acid sites affecting the binding between TRIM47 and HNF4α proteins by molecular docking technology. As can be seen from the figure, HNF4α protein and TRIM47 protein form strong interactions in the complex. The key amino acids of HNF4α are Pro342, Trp349, and Glu353, and the key amino acids of TRIM47 are His534 and His600.

[0099] Example 4

[0100] Construct mutant TRIM47 plasmids expressing with Flag tags respectively (including TRIM47-K534A, TRIM47-K600A and double mutant TRIM47-2A(K534A / K600A)). Co-transfect the HNF4α overexpression plasmid with V5 tag and the wild-type or mutant TRIM47 plasmids with Flag tag into 293T cells respectively. Lyse the cells and harvest the proteins 48 hours later. Add anti-Flag affinity gel and incubate with rotation at 4°C overnight, wash three times with 100 mM Wash Buffer (20 mM Tris-HCL, 4 mM NaCl, 10% TritonX-100, NP40, glycerol, with dosages of 1 mL, 1.25 mL, 2.5 mL, 2.5 mL, 6.25 mL respectively, and 36.5 mL of ultrapure water), and perform immunoblotting after high-temperature denaturation. The results are as Figure 4 shown, Figure 4 which is a schematic diagram of the inhibition of the binding of mutant TRIM47-related sites to HNF4α protein. It can be seen from the figure that the TRIM47-2A mutation can significantly inhibit the binding of TRIM47 to HNF4α.

[0101] Example 5

[0102] Construct mutant HNF4α plasmids, including overexpression plasmids of K342A, W349A, E353A and HNF4α-3A (K342A / W349A / E353A). Co-transfect the wild-type TRIM47 plasmid with Flag tag and the wild-type or mutant HNF4α plasmids with V5 tag into 293T cells respectively. Lyse the cells and harvest the proteins 48 hours later. Add anti-Flag affinity gel and incubate with rotation at 4°C overnight, wash three times with 100 mM Wash Buffer, and perform immunoblotting after high-temperature denaturation. The results are as Figure 5 shown, Figure 5 which is a schematic diagram of the inhibition of the binding of mutant HNF4α-related sites to TRIM47 protein. It can be seen from the figure that the HNF4α-3A mutation can significantly inhibit the binding of HNF4α to TRIM47.

[0103] Example 6

[0104] Molecular docking of HNF4α and TRIM47 shows that Pro342, Trp349 and Glu353 of HNF4α are important binding sites, and the MOE-Site Finder software is further used for binding pocket prediction. The results are as Figure 6 shown, Figure 6Schematic diagram of the predicted HNF4α ligand-binding pocket. Among them, the left figure is the schematic diagram of the top 5 candidate pockets before prediction, and the right figure is the partial schematic diagram of the preferred binding pocket Pocket5. It can be seen from the figure that among the top 5 candidate binding pockets in the prediction results (shown in the left figure), the preferred binding pocket is Pocket5, which contains Pro342, Gln345, Ser346, Trp349, Gln350 and Glu353, covering 3 key amino acids. Subsequently, virtual screening of compounds was carried out based on this pocket (shown in the right figure).

[0105] Example 7

[0106] Hep3B liver cancer cells were seeded into 96-well plates at 8×10 3 / well, transfected with pGL3-NINJ1-9p (HNF4α reporter gene) and pRL-SV40 plasmids, and the medium was changed after 8 hours. Twenty TRIM47-HNF4α binding inhibitory candidate compounds (CZ2401 to CZ2420, working concentration 10 µM) were added respectively and treated for 24 hours. Samples were collected using the Dual-Luciferase Reporter kit, and the OD135nm readings were measured with a TECAN infinite F200 microplate reader to read the values related to firefly luciferase and Renilla luciferase respectively. The results are as Figure 7 shown Figure 7 Schematic diagram of the results of the dual-luciferase reporter system for detecting the enhancing effect of compounds on the transcriptional activity of HNF4α (*P<0.05, **P<0.01, ***P<0.001). It can be seen from the figure that compound CZ2401 significantly promoted the transcriptional activity of HNF4α protein.

[0107] The structure of compound CZ2401 is as follows:

[0108]

[0109] Example 8

[0110] Hep3B liver cancer cells were seeded into 6-well plates at 3×10 5 cells / well, and the medium was changed after the cells adhered. Twenty TRIM47-HNF4α binding inhibitory candidate compounds (CZ2401 to CZ2420, working concentration 10 µM) were added respectively and treated for 24 hours. Proteins were extracted using a protein lysate (containing PMSF), denatured at high temperature, and then subjected to immunoblotting experiments to detect the protein expression level of HNF4α. The results are as Figure 8 shown Figure 8Schematic diagram of the results of detecting the effect of compounds on the elevated level of HNF4α protein by Western blotting. As can be seen from the figure, compound CZ2401 significantly increased the level of HNF4α protein.

[0111] Example 9

[0112] The binding ability of the preferred compound CZ2401 and HNF4α protein was detected by microscale thermophoresis (MST) technology. The small molecule of compound CZ2401 was used as a ligand, and the HNF4α protein was used as a target. The protein was labeled with a RED-NHS protein labeling kit. The small molecule reaction buffer was serially diluted 16-fold (50 mM HEPES buffer [pH = 7.4], containing 0.05% Tween20). After equal-volume mixing, it was placed at room temperature for 20 minutes. The thermophoretic signal was measured using Monolith NT.115 (Nano Temper), and the Kd value was calculated with Nano Temper analysis software. The results are as Figure 9 shown. Figure 9 Schematic diagram of the binding results of the preferred compound CZ2401 and HNF4α protein detected by microscale thermophoresis (MST). Among them, A is the schematic diagram of the binding of HNF4α and CZ2401, and B is the schematic diagram of the MST experimental results of HNF4α and CZ2401; as can be seen from the figure, the binding constant of compound CZ2401 and HNF4α is Kd = 5.17 ± 0.57 µM.

[0113] Example 10

[0114] In Hep3B liver cancer cells, the HNF4α overexpression plasmid with a V5 tag was transfected. After treating the cells with different concentrations of compound CZ2401 (1 μM and 10 μM) for 6 - 8 hours, the cells were lysed to collect proteins. Anti-V5 affinity gel was added, and the mixture was incubated with rotation at 4°C overnight, washed 3 times with 100 mM Wash Buffer, and subjected to immunoblotting after high-temperature denaturation. The results are as Figure 10 shown. Figure 10 Schematic diagram of the results that compound CZ2401 significantly inhibited the binding of TRIM47 and HNF4α protein. As can be seen from the figure, compound CZ2401 can inhibit the binding of HNF4α and TRIM47 in a dose-dependent manner.

[0115] Example 11

[0116] Seed Hep3B liver cancer cells at 8×10 3Inoculate into 96-well plates by holes, transfect with pGL3-NINJ1-9p (HNF4α reporter gene) and pRL-SV40 plasmids, and change the medium 8 hours later. Add compound CZ2401 and its analogs (CZ2401-1 to CZ2401-10, working concentration is 10 μM) respectively and treat for 24 hours. Use the Dual-Luciferase Reporter kit to collect samples, and read the values at OD135nm with a TECAN infinite F200 microplate reader, and read the relevant values of firefly luciferase and Renilla luciferase respectively. The results are as Figure 11 shown, Figure 11 It is a schematic diagram of the results of the luciferase reporter gene system detecting the enhancing effect of the structural analogs of compound CZ2401 on the transcriptional activity of HNF4α (*P<0.05, **P<0.01, ***P<0.001). It can be seen from the figure that compound CZ2401 and its structural analogs CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9 and CZ2401-10 significantly promoted the transcriptional activity of HNF4α protein.

[0117] The structures of compound CZ2401 and its structural analogs CZ2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9 and CZ2401-10 are as follows:

[0118] .

[0119] Example 12

[0120] Seed Hep3B liver cancer cells into 6-well plates at 3×10 5 cells / well, and change the medium after the cells adhere. Add the preferred compound CZ2401 and its analogs (CZ2401-1 to CZ2401-10, working concentration is 10 μM) respectively and treat for 24 hours. Extract proteins using a protein lysate (containing PMSF), perform immunoblotting experiments after high-temperature denaturation, and detect the protein expression level of HNF4α. The results are as Figure 12 shown, Figure 12Schematic diagram of the results of detecting the effect of compound CZ2401 and its structural analogs on the elevated level of HNF4α protein by Western blotting method. As can be seen from the figure, compound CZ2401 and its structural analogs CZ2401-1, CZ2401-2, CZ2401-3, CZ2401-4, CZ2401-5, CZ2401-6, CZ2401-7, CZ2401-8, CZ2401-9 and CZ2401-10 significantly increased the level of HNF4α protein.

[0121] Example 13

[0122] HepG2 liver cancer cells were seeded in 6-well plates at a density of 3 × 10 5 . After the cells adhered, the medium was changed, and the cells were stimulated with 0.8 mM oleic acid (OA) solution for 24 hours. After treatment with compound CZ2401 (10 µM) or DMSO for 48 hours, the cells were fixed with 4% PFA (paraformaldehyde) for Oil Red O staining. The results are as Figure 13 shown. Figure 13 Schematic diagram of the results that compound CZ2401 significantly inhibited oleic acid (OA)-induced hepatocyte lipotoxicity. As can be seen from the figure, compound CZ2401 can significantly inhibit oleic acid (OA)-induced cell lipotoxicity. It is suggested that CZ2401 can inhibit hepatocyte fat deposition and may have the effect of reducing fatty liver.

[0123] Example 14

[0124] Five- to six-week-old male C57BL / 6 mice were adaptively fed in a SPF-class animal room for one week and randomly divided into a control group (Control), a model group (CCl4 + DMSO), and a drug administration group (CCl4 + CZ2401), with 3 mice in each group. The model group and the drug administration group were intraperitoneally injected with 10% CCl4 to establish a liver fibrosis model, which was injected twice a week. Four weeks after CCl4 injection for modeling, the mice in the drug administration group were intraperitoneally injected with compound CZ2401 (8 mg / kg) once a day, while the mice in the model control group were given normal saline. During the drug administration period, CCl4 continued to be used for modeling ( Figure 14 as shown in A). After continuous drug administration for 4 weeks, the experiment was terminated and the mice were sacrificed. The liver tissues were isolated, weighed and photographed ( Figure 14 as shown in B and C). The results are as Figure 14 shown. Figure 14Schematic diagram of the results showing that compound CZ2401 can significantly alleviate the progression of CCl4-induced liver fibrosis in mice. Among them, A is the schematic diagram of mouse modeling; B is the schematic diagram of the liver tissue photo of the modeled mice; C is the schematic diagram of mouse liver weight; D is the schematic diagram of the ratio of mouse liver weight to body weight; E is the schematic diagram of detecting the level of alanine aminotransferase (ALT) in mouse serum; F is the schematic diagram of detecting the level of aspartate aminotransferase (AST) in mouse serum; G is the schematic diagram of detecting the α-SMA gene level in liver tissue by qRT-PCR; H is the schematic diagram of detecting the COL1A1 gene level in liver tissue by qRT-PCR; I is the schematic diagram of detecting the fibrosis degree in liver tissues of mice in different groups by H&E staining, Sirius red staining, and immunohistochemistry (*P<0.05, **P<0.01, ***P<0.001). The results showed that CZ2401 reduced the liver enlargement caused by CCl4, and the liver weight / body weight ratio of the mice decreased significantly ( Figure 14 as shown in D). Detection of the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum by a small animal biochemical analyzer showed that the levels of ALT and AST in the CZ2401 treatment group were lower than those in the model control group, indicating that CZ2401 could inhibit the liver inflammation caused by CCl4 ( Figure 14 as shown in E and F); qRT-PCR experiments showed that CZ2401 inhibited the expression of the activated hepatic stellate cell markers α-SMA and collagen COL1A1 genes in fibrotic liver tissue ( Figure 14 as shown in G and H), H&E staining and Sirius red staining showed that CZ2401 reduced the collagen deposition in fibrotic livers, and immunohistochemistry also showed that CZ2401 reduced the expression of the fibrosis index α-SMA ( Figure 14 as shown in I). These results indicate that CZ2401 can significantly alleviate the progression of CCl4-induced liver fibrosis in mice.

[0125] Example 15

[0126] Hep3B liver cancer cells were seeded in 96-well plates at a density of 8×10 3 cells / well. After the cells were completely adherent, compound CZ2401 (10 μM) or DMSO was added to treat the cells, and the complete medium containing the drug was changed every 2 days. When detecting, according to the instructions of the CellCounting Kit-8 kit, the medium in the wells to be detected was aspirated, and the wells were washed once with PBS buffer. The CCK8 working solution was prepared by mixing serum-free DMEM medium and CCK8 stock solution at a volume ratio of 10:1. 100 µL / well of the CCK8 working solution was added to the wells to be detected, and the plates were incubated in a 37°C cell culture incubator for 1 hour. The OD450nm was detected with a multifunctional microplate reader, and the cell growth curve was plotted after continuous detection for 7 days. The results are as Figure 15 shown.Figure 15 Schematic diagram of the results showing that compound CZ2401 can significantly inhibit the proliferation, migration, invasion and colony formation of Hep3B liver cancer cells. Among them, A is the schematic diagram of the inhibitory effect of CZ2401 on the proliferation of liver cancer cells detected by CCK8; B is the schematic diagram of the inhibitory effect of CZ2401 on the migration and invasion of liver cancer cells detected by Transwell chamber; C and D are the schematic diagrams of the statistical results of migration and invasion; E is the schematic diagram of the effect of CZ2401 on the colony formation ability of liver cancer cells verified by colony formation experiment; F is the schematic diagram of the statistical results of colony formation (*P<0.05, **P<0.01). The results are as Figure 15 shown in A, indicating that compound CZ2401 can significantly inhibit the proliferation of Hep3B liver cancer cells.

[0127] After digesting Hep3B liver cancer cells, resuspend them to 2×10 5 / mL, take 200 μL and add it into the Transwell chamber or the Transwell chamber with Matrigel added dropwise. Place the chamber in a 24-well plate, and add 500 μL of DMEM complete culture medium containing 10% FBS to the lower layer of the chamber. Add compound CZ2401 (10 μM) or DMSO, and set 3 replicates for each treatment. Incubate in a 37°C cell culture incubator for 72 hours, and then perform cell migration or invasion detection. When detecting, aspirate the culture medium inside the chamber, fix the chamber in 4% PFA for 20 minutes, and gently wash the chamber 3 times with PBS buffer. Gently wipe off the cells on the inner side of the polycarbonate membrane of the chamber with a cotton swab, then place it in a new 24-well plate, and add 500 μL of crystal violet staining solution to stain for 30 minutes. After washing the chamber, observe it under a microscope, take pictures and record in five fields of view of the upper, middle, lower, left and right, and use Image J software to statistically analyze the proportion of the positive staining area of migrating cells. Figure 15 B in it is a representative schematic diagram, Figure 15 C and D in it show that compound CZ2401 can significantly inhibit the migration and invasion of Hep3B liver cancer cells.

[0128] Seed Hep3B liver cancer cells at a density of 1×10 3Cells were seeded at a density of Figure 15 per well in a 6-well plate. After overnight cell attachment, compound CZ2401 (10 µM) or DMSO was added, and each treatment was set up with 3 replicates. The cells were cultured in a 37 °C cell incubator for about 2 weeks, and the complete medium containing the drug was changed every 3 days. During the culture process, the size and number of cell colonies formed were continuously observed. When detecting, the cells were washed twice with PBS buffer, 2 mL / well of 4% PFA was added to fix for 20 minutes, and then the residual PFA was washed away with PBS buffer. 2 mL / well of crystal violet staining solution was added to stain for 30 minutes, and the excess crystal violet staining solution was repeatedly rinsed off with PBS buffer. After drying in an oven, photos were taken and recorded, and the number of cell colonies formed in each well was counted using Image J software. The colony formation situation is as shown in Figure 15 E in

[0129] As shown in E and F in

[0130] Example 16

[0131] Male BALB / c nude mice at 4 - 5 weeks of age were subcutaneously injected with Hep3B hepatoma cells (4×10 6 cells / mouse) into the right axillary subcutaneous area to establish a subcutaneous xenograft tumor model. The size of the subcutaneous xenograft tumor was observed every 2 days, and the long and short diameters of the tumor were measured. When the average volume of the xenograft tumor reached about 100 mm 3 , the nude mice were randomly divided into a model control group (VEH) and a drug administration group (CZ2401), with 7 mice in each group. The mice in the drug administration group were intraperitoneally injected with compound CZ2401 (8 mg / kg) once a day, while the mice in the model control group were given normal saline ( Figure 16 as shown in A in Figure 16 . The tumor volume was measured once a day after drug administration, and a tumor growth curve was plotted (

[0132] as shown in B in Figure 16 . After 14 consecutive days of drug administration, the experiment was terminated, and the mice were sacrificed. The subcutaneous tumors were isolated, weighed, and photographed (as shown in C and D in Figure 16). Figure 16Schematic diagram of the results showing that compound CZ2401 can significantly inhibit the tumor progression in a subcutaneous xenograft tumor model of Hep3B liver cancer cells. Among them, A is the schematic diagram of model establishment; B is the schematic diagram of measuring the tumor volume every two days and plotting the tumor growth curve; C is the schematic diagram of the gross photograph of the subcutaneous tumor in mice; D is the schematic diagram of the tumor weight in mice; E is the schematic diagram of the results of detecting the expression of HNF4α protein by Western blotting; F is the schematic diagram of the results of statistically analyzing the protein expression level using Image J software; G is the schematic diagram of the results of immunohistochemical detection of the expression levels of HNF4α and proliferation index Ki67 in liver cancer tissues; H is the schematic diagram of the statistical results of Ki-67 (*P<0.05).

[0133] The results showed that compound CZ2401 inhibited the growth of subcutaneous xenograft tumors of liver cancer cells in mice. After extracting proteins from part of the tumor tissues, the protein level of HNF4α was detected by Western blotting ( Figure 16 as shown in E), and the relative expression level was statistically analyzed using Image J software ( Figure 16 as shown in F). The results showed that compound CZ2401 could significantly up-regulate the expression of HNF4α in the liver cancer xenograft tumor tissues. Further, the expression levels of HNF4α and proliferation index Ki67 in liver cancer tissues were detected by immunohistochemical staining ( Figure 16 as shown in G), and the detection results were statistically analyzed ( Figure 16 as shown in H). The results further indicated that compound CZ2401 could up-regulate the expression of HNF4α in the liver cancer xenograft tumor tissues and inhibit the proliferation ability of liver cancer cells. The above results showed that compound CZ2401 could significantly inhibit the tumor progression in the subcutaneous xenograft tumor model mice.

[0134] From the above experimental results, through a series of protein function research techniques such as protein interaction, docking, site mutation, and combined with in vitro and in vivo biochemical experiments, it was determined that the TRIM47 protein is a key molecule that binds to HNF4α and inhibits its transcriptional activity, and the key amino acid sites that affect the binding of TRIM47 to HNF4α protein and the function of HNF4α protein were identified. Further, a small molecule drug that can inhibit the binding of TRIM47-HNF4α and protect the activity of HNF4α protein including the key sites was screened out. This small molecule drug plays a biological role in correcting liver glycolipid metabolism disorders, reducing liver fibrosis, and inhibiting the malignant phenotype of liver cancer in vitro and in vivo. The present invention lays a foundation for the development of small molecule drugs for the treatment of chronic liver diseases and liver cancer and is widely used in adjuvant treatment.

[0135] The above are only the preferred embodiments of the present invention, and there is no restriction on the present invention in any form. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, may make some changes or modifications using the technical content prompted above to form equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention's solution.

Claims

1. Use of a small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt in the preparation of a medicament for treating liver cancer, characterized in that, The general structural formula of the small molecule compound that inhibits the degradation of HNF4α is as follows: ； wherein, R1 is selected from C1-C20 alkyl, ; R2 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R3 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R4 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R5 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R6 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R7 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R8 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R9 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R 10 selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R 11 selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R 12 selected from hydrogen, C1-C20 alkyl groups; Or, R4, R5 and carbon, oxygen form a five-membered or six-membered ring.

2. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that, In the small molecule compound that inhibits the degradation of HNF4α, R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, ； R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; Or, a five-membered ring formed by R4, R5, carbon, and oxygen ; Or, a six-membered ring formed by R4, R5 and carbon and oxygen , where the dashed line indicates the connection to the benzene ring.

3. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 2 in the preparation of a medicament for treating liver cancer, characterized in that, The small molecule compound that inhibits the degradation of HNF4α is selected from one of the following structures: 。 4. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 1 in the preparation of a medicament for treating liver cancer, characterized in that In the application, the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt is used as the sole active ingredient.

5. Use of a small molecule compound or its pharmaceutically acceptable salt that inhibits the degradation of HNF4α in the preparation of a drug for treating chronic liver diseases, characterized in that, The general structural formula of the small molecule compound that inhibits the degradation of HNF4α is as follows: ； wherein, R1 is selected from C1-C20 alkyl, ; R2 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R3 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R4 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R5 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R6 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy, halogen, ; R7 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R8 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R9 is selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R 10 selected from hydrogen, C1-C20 alkyl, and C1-C20 alkoxy; R 11 selected from hydrogen, C1-C20 alkyl, C1-C20 alkoxy; R 12 selected from hydrogen, C1-C20 alkyl; Or, R4, R5 and carbon, oxygen form a five-membered or six-membered ring.

6. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 5 in the preparation of a medicament for treating chronic liver diseases, characterized in that, In the small molecule compound that inhibits the degradation of HNF4α, R1 is selected from methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, ； R2 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R3 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R4 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R5 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; R6 is selected from hydrogen, methyl, ethyl, isopropyl, n-propyl, n-butyl, tert-butyl, n-pentyl, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, fluorine, chlorine, bromine, ; Or, a five-membered ring formed by R4, R5, carbon, and oxygen ; Or, a six-membered ring formed by R4, R5 and carbon and oxygen , and the dashed line indicates the connection to the benzene ring.

7. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 6 in the preparation of a medicament for treating chronic liver diseases, characterized in that, The small molecule compound that inhibits the degradation of HNF4α is selected from one of the following structures: 。 8. Use of the small molecule compound or its pharmaceutically acceptable salt for inhibiting HNF4α degradation according to claim 5 in the preparation of a medicament for treating chronic liver diseases, characterized in that, The chronic liver diseases are selected from non-alcoholic steatohepatitis, liver fibrosis, liver cirrhosis, liver failure.

9. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation is made of the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt according to any one of claims 1 to 3 and a medically acceptable excipient.

10. A pharmaceutical composition, characterized in that, It is made of the small molecule compound that inhibits the degradation of HNF4α or its pharmaceutically acceptable salt according to any one of claims 1 to 3, a pharmaceutically acceptable carrier, and a drug for improving chronic liver diseases or a drug for treating liver cancer.

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