Ceramidase specific inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a ceramidase specific inhibitor as well as a preparation method and application thereof. The structural formula of the ceramidase specific inhibitor is as follows: # imgabs0 #. The ceramidase specific inhibitor provided by the invention can inhibit the survival of tumor cells by inhibiting the gene expression level of alkaline ceramidase 3, and a new idea is provided for treating primary liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a ceramidase specific inhibitor and a preparation method and application thereof. Background Art

[0002] Primary liver cancer (PLC), including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is one of the most common fatal malignant diseases. In recent years, the risk factors of PLC, including chronic viral hepatitis, chemical carcinogens, and fatty liver hepatitis, have been gradually identified. PLC has also made much progress in targeted therapy and immunotherapy. However, the pathological molecular mechanism of PLC is still not fully understood, and there is still a lack of effective anti-PLC drugs in clinical treatment. PLC treatment is a major problem that urgently needs to be studied in the field of liver disease.

[0003] Disturbance of sphingolipid homeostasis is a characteristic of many cancers and metabolic diseases. However, the intrinsic complexity of the lipidome makes it challenging to study lipid metabolism. Ceramide (CER) is one of the core biomolecules of sphingolipid metabolism, and metabolic disorders of CER exist in PLC. 18-carbon monounsaturated ceramide (C18:1-ceramide, C18:1-CER) is one of the ceramides present in the body. The plasma level of C18:1-CER is related to the progression of hepatitis C fibrosis and the response rate of antiviral treatment. Its downstream metabolite sphingosine-1-phosphate (Sphingosine-1-phosphate, S1P) can inhibit oxidative stress caused by ischemia-reperfusion in non-alcoholic fatty liver disease, improve ischemia-reperfusion injury, and has a potential hepatoprotective effect in the progression of liver cancer mediated by chronic liver damage.

[0004] Alkaline ceramidase 3 (ACER3) is a ceramidase that specifically degrades C18:1-CER (ACER3 / C18:1-CER metabolism). ACER3 can specifically degrade C18:1-CER to generate sphingosine (SPH) and 18-carbon monounsaturated fatty acids (C18:1-FA). SPH can be phosphorylated by its kinase to generate S1P. ACER3-related metabolic pathways can affect the progression of multiple diseases by regulating cell death, inflammatory response, fibrosis, and oxidative stress. Overexpression of ACER3 can promote cancer in leukemia, hepatocellular carcinoma, and cervical cancer by activating survival signaling pathways, while inhibiting the expression of ACER3 can play a tumor suppressor role in related cancers. Understanding these pathways and developing treatment methods requires selective inhibitory molecules. In recent years, the field of drug intervention aimed at molecular control of ceramide levels has matured. In the case of inhibiting ACER3, research on enzyme inhibition has focused on gene inhibition or lipid analogs. Many enzymes involved in sphingomyelin metabolism are integral membrane proteins, but specific small molecule inhibitors targeting these enzymes are still to be developed. The study of ceramide enzyme inhibitors has become a technical bottleneck that must be overcome in the development of ceramide treatment for liver cancer. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a ceramidase-specific inhibitor and its preparation method and application. The ceramidase-specific inhibitor provided by the present invention can inhibit the survival of tumor cells by inhibiting the gene expression level of alkaline ceramidase 3, providing a new idea for the treatment of primary liver cancer.

[0006] The technical solution of the present invention is:

[0007] A ceramidase-specific inhibitor, the structural formula of the ceramidase-specific inhibitor is as follows:

[0008]

[0009] The present invention also provides a method for preparing the ceramidase-specific inhibitor, comprising the following steps:

[0010] (1) adding 2-methylvaleric acid dropwise to a solution containing dichloromethane and thionyl chloride to obtain a reaction mixture A;

[0011] (2) stirring the reaction mixture A, concentrating the reaction mixture A to obtain a residual liquid;

[0012] (3) adding the remaining liquid dropwise to a solution containing 4-chloro-3-trifluoromethylaniline, N,N-diisopropylethylamine and dichloromethane to obtain a reaction mixture B;

[0013] (4) stirring the reaction mixture B to obtain a reaction mixture C;

[0014] (5) The reaction mixture C is subsequently treated to obtain a ceramidase-specific inhibitor.

[0015] Furthermore, the step (2) is carried out under N2 protection and stirring at 15-30°C for 1-3 hours.

[0016] Furthermore, the step (4) is carried out by stirring at 0-20° C. for 12-20 hours.

[0017] Furthermore, the specific operation of the subsequent treatment of the reaction mixture C in step (5) is: pouring the reaction mixture C into water, extracting with an extractant, drying the organic layer, filtering and concentrating under reduced pressure to obtain a residue; and grinding the residue with petroleum ether.

[0018] Another object of the present invention is to provide the use of the ceramidase-specific inhibitor or the ceramidase-specific inhibitor prepared according to the above-mentioned method for preparing the ceramidase-specific inhibitor in the preparation of drugs for treating primary liver cancer and liver fibrosis.

[0019] Furthermore, the primary liver cancer includes one or both of hepatocellular carcinoma and cholangiocarcinoma.

[0020] Furthermore, the primary liver cancer is liver cancer cell HepG2, liver cancer cell Hep3B, hepatobiliary cancer cell RBE, and bile duct epithelial cancer cell HuCCT1.

[0021] Furthermore, the drug comprises an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is a ceramidase-specific inhibitor.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention utilizes an inhibitor to reduce the gene expression level of alkaline ceramidase 3, and uses diethylnitrosamine (DEN) to induce the process of primary liver cancer, thus providing a new idea for treating primary liver cancer.

[0024] (1) For the cellular disease model of primary liver cancer, the inhibitor can inhibit the survival of tumor cells by inhibiting the gene expression of alkaline ceramidase 3;

[0025] (2) Animal disease models of primary liver cancer:

[0026] ① The preparation can not only stabilize the weight changes of mice and reduce the mortality rate during the modeling of primary liver cancer induced by diethylnitrosamine in mice by knocking down the ACER3 gene, but also significantly reduce the number and volume of tumor lesions observed in the liver of mice modeling diethylnitrosamine chemically induced liver cancer. By pathological methods, H&E staining of liver tissue was performed, and it was observed that the malignant changes in the histology of hepatocytes and bile duct cells were significantly improved after ACER3 gene knockdown.

[0027] ② The preparation can significantly reduce the degree of inflammatory response in the liver tissue of mice modeling diethylnitrosamine chemically induced liver cancer by knocking down the ACER3 gene. The inflammatory factors, oxidative stress factors, and apoptosis factors in the liver tissue were detected by RT-PCR. It was found that after ACER3 gene knockdown, the liver inflammatory response was significantly alleviated, the oxidative stress response was alleviated, and the apoptosis response was promoted.

[0028] ③ The preparation can significantly reduce the collagen infiltration in the liver and the degree of fibrosis in the liver portal area of ​​mice with diethylnitrosamine chemical-induced liver cancer model; fibrosis-related markers in liver tissue were evaluated by RT-PCR detection, immunoblotting and immunohistochemical staining, and it was found that after ACER3 gene knockdown, the collagen infiltration in the liver was significantly reduced and the degree of fibrosis in the liver portal area was significantly weakened.

[0029] (3) The preparation method of the ceramidase-specific inhibitor provided by the present invention has readily available raw materials, is simple to operate, and is easy to industrialize and mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthetic route of the ceramidase-specific inhibitor of Example 3 of the present invention;

[0031] Figure 2 This is the mass spectrum of the ceramidase-specific inhibitor ES_ACR 13 prepared in Example 3 of the present invention;

[0032] Figure 3 The changes in cell viability under time gradient after the hepatoma cell lines HepG2 and Hep3B and the cholangiocarcinoma cell lines RBE and HuCCT1 were treated with the ceramidase-specific inhibitor ES_ACR 13 prepared in Example 3 of the present invention;

[0033] Figure 4 The figure shows the changes in body weight of Acer3 knockout mice and wild-type mice during the modeling period of primary liver cancer animal model induced by diethylnitrosamine (DEN).

[0034] Figure 5This is a survival analysis diagram of Acer3 gene knockout mice and wild-type mice during the modeling period of diethylnitrosamine (DEN) chemically induced primary liver cancer animal model;

[0035] Figure 6 This is a gross image of the livers of Acer3 knockout mice and wild-type mice in the animal model of primary liver cancer chemically induced by diethylnitrosamine (DEN);

[0036] Figure 7 H&E staining of liver tissues (including tumor tissues) of Acer3 knockout mice and wild-type mice;

[0037] Figure 8 This is the result of detecting the mRNA levels of hepatocellular carcinoma and cholangiocarcinoma markers in liver tissues of Acer3 knockout and wild-type mice;

[0038] Fig. 9 The results of immunohistochemistry of hepatocellular carcinoma marker AFP in liver tissue of Acer3 knockout and wild-type mice;

[0039] Fig.10 The immunohistochemical results of CK19, a marker for cholangiocarcinoma, in liver tissue of Acer3 knockout and wild-type mice;

[0040] Fig.11 The results of Western Blot are the results of detecting the protein expression levels of hepatocellular carcinoma marker AFP and cholangiocarcinoma marker CK19 in liver tissues of Acer3 gene knockout and wild-type mice;

[0041] Fig.12 The results of the mRNA level detection of inflammatory factors in liver tissue of Acer3 gene knockout and wild-type mice;

[0042] Fig.13 This is the result of detecting liver tissue fibrosis level in Acer3 gene knockout and wild-type mice. DETAILED DESCRIPTION

[0043] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0044] Example 1: A method for preparing a specific inhibitor of ceramidase

[0045] The preparation method of the ceramidase specific inhibitor comprises the following steps:

[0046] (1) Compound 1 (2-methylpentanoic acid) (15.0 g, 129 mmol, 1.00 Eq) was added dropwise to a solution containing dichloromethane (DCM, 150 mL) and thionyl chloride (SOCl2, 46.1 g, 387 mmol, 28.1 mL, 3.00 Eq) to obtain a reaction mixture A;

[0047] (2) stirring the reaction mixture A under N2 protection at 15°C for 3 hours, concentrating the reaction mixture A to obtain a residual liquid;

[0048] (3) The remaining solution was added dropwise to a solution containing compound 2 (4-chloro-3-trifluoromethylaniline) (20.2 g, 103 mmol, 0.80 Eq), N,N-diisopropylethylamine (DIEA, 50.1 g, 387 mmol, 67.5 mL, 3.00 Eq) and dichloromethane (DCM, 350 mL) at 0°C to obtain a reaction mixture B;

[0049] (4) stirring the reaction mixture B at 0° C. for 20 hours to obtain a reaction mixture C;

[0050] (5) The reaction mixture C was poured into 200 mL of H2O and extracted three times with 50.0 mL of DCM;

[0051] (6) The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue;

[0052] (7) The residue was triturated with petroleum ether (15 mL) at 20° C. for 10 minutes to obtain the ceramidase-specific inhibitor ES_ACR 13. The obtained ES_ACR 13 was a white solid.

[0053] Example 2: A method for preparing a specific inhibitor of ceramidase

[0054] The preparation method of the ceramidase specific inhibitor comprises the following steps:

[0055] (1) Compound 1 (2-methylpentanoic acid) (15.0 g, 129 mmol, 1.00 Eq) was added dropwise to a solution containing dichloromethane (DCM, 150 mL) and thionyl chloride (SOCl2, 46.1 g, 387 mmol, 28.1 mL, 3.00 Eq) to obtain a reaction mixture A;

[0056] (2) stirring the reaction mixture A under N2 protection at 30°C for 1 hour, concentrating the reaction mixture A to obtain a residual liquid;

[0057] (3) The remaining solution was added dropwise to a solution containing compound 2 (4-chloro-3-trifluoromethylaniline) (20.2 g, 103 mmol, 0.80 Eq), N,N-diisopropylethylamine (DIEA, 50.1 g, 387 mmol, 67.5 mL, 3.00 Eq) and dichloromethane (DCM, 350 mL) at 0°C to obtain a reaction mixture B;

[0058] (4) stirring the reaction mixture B at 20° C. for 12 hours to obtain a reaction mixture C;

[0059] (5) The reaction mixture C was poured into 200 mL of H2O and extracted three times with 50.0 mL of DCM;

[0060] (6) The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue;

[0061] (7) The residue was triturated with petroleum ether (15 mL) at 20° C. for 10 minutes to obtain the ceramidase-specific inhibitor ES_ACR 13. The obtained ES_ACR 13 was a white solid.

[0062] Example 3: A method for preparing a specific inhibitor of ceramidase

[0063] The preparation method of the ceramidase specific inhibitor comprises the following steps:

[0064] (1) Compound 1 (2-methylpentanoic acid) (15.0 g, 129 mmol, 1.00 Eq) was added dropwise to a solution containing dichloromethane (DCM, 150 mL) and thionyl chloride (SOCl2, 46.1 g, 387 mmol, 28.1 mL, 3.00 Eq) to obtain a reaction mixture A;

[0065] (2) stirring the reaction mixture A under N2 protection at 20°C for 2 hours, concentrating the reaction mixture A to obtain a residual liquid;

[0066] (3) The remaining solution was added dropwise to a solution containing compound 2 (4-chloro-3-trifluoromethylaniline) (20.2 g, 103 mmol, 0.80 Eq), N,N-diisopropylethylamine (DIEA, 50.1 g, 387 mmol, 67.5 mL, 3.00 Eq) and dichloromethane (DCM, 350 mL) at 0°C to obtain a reaction mixture B;

[0067] (4) stirring the reaction mixture B at 20° C. for 16 hours to obtain a reaction mixture C;

[0068] (5) The reaction mixture C was poured into 200 mL of H2O and extracted three times with 50.0 mL of DCM;

[0069] (6) The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a residue;

[0070] (7) The residue was ground with petroleum ether (15 mL) at 20° C. for 10 minutes to obtain the ceramidase-specific inhibitor ES_ACR 13 (the synthetic route of the ceramidase-specific inhibitor in Example 3 of the present invention is as follows: Figure 1 ES_ACR 13 was obtained as a white solid.

[0071] The mass spectrum of the ceramidase specific inhibitor ES_ACR 13 prepared in Example 3 of the present invention is as follows: Figure 2 As shown. Figure 2 It can be concluded that the molecular formula of the compound is C 13 H 15 ClF3NO, with a relative molecular mass of 293.08, is consistent with the predicted ceramidase-specific inhibitor ES_ACR 13 compound, indicating that the synthesis is effective.

[0072] Experimental Example 1: Study on the inhibitory activity of ES_ACR 13, a specific inhibitor of ceramidase, on hepatocellular carcinoma and cholangiocarcinoma

[0073] The ceramidase-specific inhibitor ES_ACR 13 prepared in Example 3 of the present invention was used to treat four common human liver cancer cell lines (liver cancer cell lines HepG2, Hep3B and cholangiocarcinoma cell lines RBE and HuCCT1), and the changes in their activity and growth were detected by MTT assay.

[0074] The specific steps are as follows:

[0075] A preliminary experiment was designed to determine the optimal ES_ACR 13 concentration for each cell line.

[0076] Day 1:

[0077] 1. Digest, centrifuge and count the cells in the logarithmic phase that were cultured the day before.

[0078] 2. Seed the cells on a 12-well plate and incubate at 37°C, 5% CO2, and 90% humidity for 24 hours.

[0079] Day 2:

[0080] 1. Observe the cell growth status and density under a microscope, and select wells with good growth status, uniform cell distribution and density for experiments.

[0081] 2. Three replicate wells of each group of experiments were added to a 12-well plate. The inoculated cell culture plate was placed in an incubator and cultured until the cell monolayer covered the bottom of the well. An appropriate concentration of ES_ACR 13 solution was added and cultured for an appropriate length of time at 37°C, 5% CO2, and 90% humidity. For example, 1, 3, 5, or 6 days. The experiment set up a control group and an experimental group. The control group (Vehicle) was treated with a solvent, and the experimental group was treated with the ceramidase-specific inhibitor ES_ACR 13 (abbreviated as ACR 13). The other factors remained the same.

[0082] Days 3-8 (1-6 days after dosing):

[0083] 1. Thaw and centrifuge MTT at room temperature before use. Before the experiment, add solvent to MTT powder to prepare 5 mg / mL MTT solvent, and dilute it with PBS buffer to 0.5 mg / mL MTT working solution.

[0084] 2. Use an inverted microscope to detect the cell growth status, and use 4X / 10X to take photos of wells with good growth status and uniform cell distribution and density.

[0085] 3. After the cells have been cultured for a period of time, discard the culture medium, rinse with sterile PBS buffer three times, remove the residual culture medium, add 1 mL of MTT working solution to each well to completely cover the cells, and incubate in a humidified incubator at 37°C, 5% CO2, 1% O2 for 2 hours.

[0086] 4. Add an equal volume of MTT lysis buffer to each well and shake at 70 rpm for 30 minutes at room temperature.

[0087] 5. Take 200 μL from each well and transfer it to a 96-well plate. Use a multifunctional microplate reader to detect the absorbance of the liquid in each well at a wavelength of 590 nm.

[0088] 6. Process and analyze the results using Excel and Graphpad Prism.

[0089] After the hepatoma cell lines HepG2, Hep3B and the cholangiocarcinoma cell lines RBE and HuCCT1 were treated with the ceramidase-specific inhibitor ES_ACR 13 prepared in Example 3 of the present invention, the changes in cell viability under time gradient were as follows: Figure 3 As shown. Figure 3It can be seen that the cell activity of the HepG2 group, Hep3B group, RBE group, and HUCCT1 group cell lines began to show a trend of difference from the third day after administration, and there was a significant difference in the cell activity between the control group and the experimental group on the fifth and sixth days after administration. This shows that the ceramidase-specific inhibitor ES_ACR 13 (abbreviated as ACR 13) prepared in Example 3 of the present invention can significantly inhibit the growth of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) cells, and both have a certain time dependence. Experimental Example 2: Effect of changes in the expression of ACER3 on the biological function of liver cancer

[0090] To explore the effect of altered ACER3 expression on the biological function of liver cancer, this experiment was divided into wild-type mice and ACER3 (Acer3) knockout mice. The DEN treatment group was treated with intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 35 mg / kg per week from 6 weeks of age until the end of modeling (30 weeks after injection); the end time of the solvent treatment group (CON) was the same as the DEN treatment time, and the solvent treatment group was used as a normal control. The number and weight of mice were recorded every week during the modeling process. After the modeling was completed, the samples could be taken for subsequent experiments. The DEN treatment group modeled successfully in this experiment, that is, hepatocellular carcinoma occurred. After the modeling was completed, the samples were taken, and the gross changes and microscopic pathological changes of the liver tissue were evaluated. If it met the pathological characteristics of hepatocellular carcinoma, the modeling was successful, and the mice with successful modeling were subjected to subsequent experiments. The liver tissue was taken to observe the gross changes of the liver tissue, and after fixation, paraffin embedding, and slice preparation, the liver tissue of the two groups of mice was stained with H&E to observe its pathological changes.

[0091] In the animal model of primary liver cancer induced by diethylnitrosamine (DEN), the weight changes of Acer3 knockout mice and wild-type mice were as follows: Figure 4 As shown in the figure; During the modeling period of the animal model of primary liver cancer induced by diethylnitrosamine (DEN), the survival analysis of Acer3 gene knockout mice and wild-type mice is shown in the figure Figure 5 A DEN-induced primary liver cancer animal model was constructed. During the modeling process, the weight monitoring of mice showed that although Acer3 gene knockout inhibited weight gain, it also stabilized the weight changes of mice. In the early and middle stages of modeling, the weight of wild-type mice increased to a certain extent, but began to drop sharply at 23 weeks, accompanied by a sharp drop in survival rate (see Figure 4 , Figure 5 ).

[0092] In the animal model of primary liver cancer induced by diethylnitrosamine (DEN), the gross images of livers of Acer3 knockout mice and wild-type mice are shown in the figure below. Figure 6 As shown. Figure 6It can be seen that by analyzing the gross images of the two groups of liver tissues, it can be found that in the Acer3+ / + group, that is, the Acer3 genome is normally expressed (wild-type mice), the surface fibrosis of the liver tissue is obvious, more tumor nodules are visible, and it is difficult to distinguish the normal liver appearance; in the Acer3- / - group, that is, the Acer3 gene knockout group, the surface fibrosis of the liver tissue is relatively mild, no obvious tumor nodules are seen, and the appearance is no different from that of the normal mouse liver.

[0093] H&E staining of liver tissue (including tumor tissue) of Acer3 knockout mice and wild-type mice Figure 7 shown. Figure 7 “Hepatocellular carcinogenesis” refers to the carcinogenesis of hepatocellular carcinoma and the occurrence of hepatocellular carcinoma; “Cholangiocarcinogenesis” refers to the carcinogenesis of cholangiocarcinoma and the occurrence of cholangiocarcinoma. Figure 7 Analysis of liver tissue pathology results showed that Acer3 gene knockout could significantly inhibit the occurrence of PLC, including obvious improvement of the malignant changes of hepatocytes and bile duct cells histology.

[0094] From the above, it can be seen that Acer3 gene knockout can inhibit the occurrence of PLC chemically induced by diethylnitrosamine, improve the general condition of mice, and increase the survival rate.

[0095] The results of mRNA level detection of hepatocellular carcinoma and cholangiocarcinoma markers in liver tissue of Acer3 gene knockout and wild-type mice are shown in the figure. Figure 8 shown. Figure 8 CON stands for control treatment (i.e. solvent treatment group, which in this experiment refers to mice raised normally without DEN injection); DEN stands for diethylnitrosamine treatment group (starting from the age of 6 weeks, DEN was intraperitoneally injected at a dose of 35 mg / kg every week until the model was established). Figure 8 It can be seen that Acer3 gene knockout can significantly inhibit the upregulation of mRNA expression of HCC and CCA markers, including AFP, GPRC5A, GABRP, ENF, DMBT1, CLDN7, SEMA3C, and CK19, in the liver tissue of PLC modeling mice.

[0096] Using the principle of antigen-antibody binding, the expression and distribution of hepatocellular carcinoma marker AFP protein in the liver tissue of Acer3 knockout and wild-type mice were detected by immunohistochemistry. The immunohistochemical results of hepatocellular carcinoma marker AFP in the liver tissue of Acer3 knockout and wild-type mice are shown in the figure. Fig. 9 shown. Fig. 9 In Chinese, "AFP staining" means AFP immunohistochemical staining. Fig. 9It can be seen that in the liver tissue of Acer3 gene knockout mice, the AFP-positive area (brown area in the figure) is less than that in the wild-type liver tissue, that is, there are fewer hepatocellular carcinoma foci in the liver tissue of Acer3 gene knockout mice.

[0097] The expression and distribution of CK19 protein, a marker of cholangiocarcinoma, in the liver tissue of Acer3 knockout and wild-type mice were detected by immunohistochemistry using the principle of antigen-antibody binding. The immunohistochemical results of CK19, a marker of cholangiocarcinoma, in the liver tissue of Acer3 knockout and wild-type mice are shown in the figure. Fig.10 shown. Fig.10 In Chinese, "CK19staining" means CK19 immunohistochemical staining. Fig.10 It can be seen that in the liver tissue of Acer3 gene knockout mice, the CK19-positive area (brown area in the figure) is less than that in the wild-type liver tissue, that is, there are fewer cholangiocarcinoma foci in the liver tissue of Acer3 gene knockout mice.

[0098] The results of Western Blot analysis of the expression levels of AFP, a marker for hepatocellular carcinoma, and CK19, a marker for cholangiocarcinoma, in the liver tissues of Acer3 knockout and wild-type mice are shown in the figure below. Fig.11 As shown. Fig.11 It can be seen that at the protein level, Acer3 gene knockout can significantly inhibit the upregulation of HCC marker AFP and CCA marker CK19.

[0099] From the above, we can see that Acer3 gene knockout has an inhibitory effect on the occurrence of HCC and CCA.

[0100] The results of mRNA level detection of inflammatory factors in liver tissue of Acer3 gene knockout and wild-type mice are shown in the figure Fig.12 As shown. Fig.12 It can be seen that Acer3 gene knockout significantly inhibited the upregulation of mRNA expression of proinflammatory factors induced by DEN treatment, including IL-1, IL-6, and TNF-a. In terms of anti-inflammatory factors, Acer3 gene knockout can reverse the downregulation of IL-10 by diethylnitrosamine, and significantly upregulate IL-10 after diethylnitrosamine treatment. Acer3 gene knockout can inhibit the upregulation of TGF-β and Collagen1 mRNA expression of diethylnitrosamine liver fibrosis factors.

[0101] The results of liver fibrosis level detection in Acer3 knockout and wild-type mice are shown in the figure Fig.13 shown. Fig.13 The Chinese meaning of “a-SMA staining” is a-SMA (α-smooth muscle actin) immunohistochemical staining, and its level can reflect the degree of fibrosis. Fig.13The results showed that Acer3 gene knockout could inhibit the upregulation of a-SMA, a marker of stellate cell activation, after diethylnitrosamine treatment; Sirius red collagen staining showed that diethylnitrosamine treatment could significantly induce liver fibrosis, while Acer3 gene knockout could significantly improve diethylnitrosamine-induced liver fibrosis.

[0102] In summary, gene knockout of Acer3 has a significant anti-inflammatory effect in liver tissue modeling of primary liver cancer, inhibits pro-inflammatory responses, enhances anti-inflammatory responses, inhibits stellate cell activation, and improves liver fibrosis.

Claims

1. A ceramidase-specific inhibitor, characterized in that: The structural formula of the ceramidase specific inhibitor is as follows:

2. The method for preparing a ceramidase-specific inhibitor according to claim 1, characterized in that: The following steps are involved: (1) adding 2-methylvaleric acid dropwise to a solution containing dichloromethane and thionyl chloride to obtain a reaction mixture A; (2) stirring the reaction mixture A, concentrating the reaction mixture A to obtain a residual liquid; (3) adding the remaining liquid dropwise to a solution containing 4-chloro-3-trifluoromethylaniline, N,N-diisopropylethylamine and dichloromethane to obtain a reaction mixture B; (4) stirring the reaction mixture B to obtain a reaction mixture C; (5) The reaction mixture C is subsequently treated to obtain a ceramidase-specific inhibitor.

3. The method for preparing a ceramidase-specific inhibitor according to claim 2, characterized in that: The step (2) is carried out under N2 protection and stirring at 15-30°C for 1-3 hours.

4. The method for preparing a ceramidase-specific inhibitor according to claim 2, characterized in that: The step (4) is carried out by stirring at 0-20° C. for 12-20 hours.

5. The method for preparing a ceramidase-specific inhibitor according to claim 2, characterized in that: The specific operation of the subsequent treatment of the reaction mixture C in step (5) is: pouring the reaction mixture C into water, extracting with an extractant, drying the organic layer, filtering and concentrating under reduced pressure to obtain a residue; and grinding the residue with petroleum ether.

6. Use of the ceramidase-specific inhibitor according to claim 1 or the ceramidase-specific inhibitor prepared according to the preparation method of any one of claims 2 to 5 in the preparation of drugs for treating primary liver cancer and liver fibrosis.

7. The use according to claim 6, characterized in that: The primary liver cancer includes one or both of hepatocellular carcinoma and cholangiocarcinoma.

8. The use according to claim 6, characterized in that: The primary liver cancer is liver cancer cell HepG2, liver cancer cell Hep3B, hepatobiliary cancer cell RBE, and bile duct epithelial cancer cell HuCCT1.

9. The use according to claim 6, characterized in that: The medicine comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a ceramidase-specific inhibitor.

Images