Use of apol2 inhibitors in the manufacture of a product for the treatment of liver fibrosis

Abstract

This invention belongs to the field of biomedicine, specifically relating to the application of APOL2 (Apolipoprotein L2) inhibitors in the preparation of products for treating liver fibrosis. The inventors isolated a series of natural tetrodopane-type diterpenes from *Euphorbia pekinensis*, a plant in the Euphorbiaceae family. Anti-liver fibrosis-related activity tests on this series of diterpenes revealed that they significantly inhibited the expression of fibronectin, type I collagen, and α-smooth muscle actin in LX-2 cells. In animal studies, their therapeutic effect was superior to that of pirfenidone, a phase II clinical trial drug for treating liver fibrosis, and they showed no significant toxicity. Mechanistic studies showed that TD1 is an APOL2 inhibitor, and knocking out APOL2 protein in vivo can alleviate the progression of liver fibrosis. In summary, this series of tetrodopane-type diterpenes, especially TD1, shows promise as a candidate drug for treating liver fibrosis and provides a new target for researching novel drugs for treating liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of APOL2 inhibitors in the preparation of products for treating liver fibrosis. Background Technology

[0002] Liver fibrosis is a significant pathological process in the progression of various chronic liver diseases, such as viral hepatitis (hepatitis B and hepatitis C), alcoholic / non-alcoholic steatohepatitis, autoimmune and hereditary liver diseases, ultimately leading to liver dysfunction. Its main characteristics include an imbalance between the production and degradation of extracellular matrix (ECM) proteins in liver tissue, and abnormal proliferation of intrahepatic fibrosis and scar tissue. Without treatment, liver fibrosis can eventually develop into malignant diseases such as portal hypertension, hepatic encephalopathy, cirrhosis, and even hepatocellular carcinoma, seriously threatening human life and health.

[0003] The pathological mechanisms of liver fibrosis are highly complex. Although several drugs with anti-fibrotic effects have entered clinical trials (such as the PPARα / δ agonist Elafibranor, the FXR agonist Obeticholic acid, and the ASK1 inhibitor Selonsertib), these drugs can only alleviate the progression of liver fibrosis to a certain extent, have failed to meet clinical trial endpoints, and most have toxic side effects. The popular anti-fibrotic drug pirfenidone (PFD) is also undergoing a Phase II clinical trial (NCT04099407), but it also carries the risk of acute liver failure, limiting its clinical application. To date, no drugs specifically targeting liver fibrosis have been marketed. Therefore, developing anti-fibrotic drugs with novel targets and mechanisms of action is of significant research and application value.

[0004] APOL2 is a member of the apolipoprotein family, involved in cholesterol biosynthesis and transport, and is thought to mediate interferon-γ or virus-induced cell death. However, there are no reports on APOL2 as a drug target for the treatment of liver fibrosis or its related mechanisms of action.

[0005] Natural products, due to their structural diversity and good biocompatibility, are an important source of innovative drugs. Tigrone-type diterpenes have complex molecular structures with a 5 / 7 / 6 / 3 ring carbon skeleton and contain multiple chiral centers, exhibiting activities such as anti-HIV, anti-tumor, and anti-angiogenic effects. However, to date, there are no reports on the treatment of liver fibrosis with tigrone-type diterpenes. Summary of the Invention

[0006] The purpose of this invention is to overcome the lack of existing drugs and to provide an application of APOL2 as a target in the preparation of tetrodopane-type diterpenoid drugs for the treatment of liver fibrosis.

[0007] The first aspect of the present invention aims to provide a diterpenoid compound of the tetrane type or a pharmaceutically acceptable derivative thereof.

[0008] The second aspect of the present invention is to provide a product.

[0009] A third aspect of the present invention is to provide an application.

[0010] The fourth aspect of this invention is to provide a method for preparing the tetraalkyl-type diterpenoid compound of the first aspect of this invention.

[0011] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0012] A first aspect of the present invention provides a tetraalkyl diterpenoid compound or a pharmaceutically acceptable derivative thereof.

[0013] The general formulas of the tetrane-type diterpenoid compounds are shown in Formula I, Formula II, or Formula III:

[0014]

[0015] In Formula I and Formula II, β-OH is attached to the C-4 position, and in Formula III, α-H is attached to the C-4 position; in Formula I, the C-12 position is methylene, and in Formula II and Formula III, β-OH or β-oxyacyl group is attached to the C-12 position.

[0016] In Formula I: R1 is selected from one of hydrogen, acetyl, crotonyl, isovaleryl, decanyl, dodecanoyl, or tetradecanoyl; R2 is selected from one of hydrogen, acetyl, crotonyl, isovaleryl, decanyl, dodecanoyl, or tetradecanoyl.

[0017] In Formula II: R1 is selected from one of hydrogen, acetyl, crotonyl, isovaleryl, decanyl, dodecanoyl or tetradecanoyl; R2 is selected from one of acetyl, isobutyryl or isovaleryl; R3 is selected from one of hydrogen or acetyl.

[0018] In Formula III: R1 is selected from one of hydrogen, acetyl, crotonyl, isovaleryl, decanyl, dodecanoyl or tetradecanoyl; R2 is selected from one of acetyl, isobutyryl or isovaleryl; R3 is selected from one of hydrogen or acetyl.

[0019] The tetrane-type diterpenoid compound is selected from at least one of TD1 to TD15.

[0020] The structural formulas of TD1 to TD15 are as follows:

[0021]

[0022] Preferably, the tetrane-type diterpenoid compound includes TD1.

[0023] Preferably, the pharmaceutically acceptable derivative includes a pharmaceutically acceptable salt.

[0024] A second aspect of the present invention provides a product comprising a terpene-type diterpene compound or a pharmaceutically acceptable derivative thereof as described in the first aspect of the present invention, and a pharmaceutically acceptable excipient.

[0025] A third aspect of the invention provides the use of the tetrane-type diterpenoid compounds described in the first aspect of the invention or pharmaceutically acceptable derivatives thereof, and / or the products of the second aspect of the invention, in a1) to a2):

[0026] a1) Preparation of APOL2 protein inhibitors;

[0027] a2) Prepare products for the treatment of liver fibrosis-related diseases.

[0028] Preferably, the APOL2 protein inhibitor is a functional inhibitor of the APOL2 protein.

[0029] Preferably, the TD1 is used to prepare a functional inhibitor of the APOL2 protein.

[0030] The sequence of the APOL2 protein is: MNPESSIFIEDYLKYFQDQVSRENLLQLLTDDEAWNGFVA AAELPRDEADELRKALNKLASHMVMKDKNRHDKDQQHRQWFLKEFPRLKRELEDHIRKLRALAEEVEQVHRGTTIANVVSNSVGTTSGILTLLGLGLAPFTEGISFVLLDTGMGLGAAAAVAGITCSVVELVNKLRARAQARNLDQSGTN VAKVMKEFVGGNTPNVLTLVDNWYQVTQGIGRNIRAIRRARANPQLGAYAPPPHVIGRISAEGGEQVERVVEGPAQAMSRGTMIVGAATGGILLLLDVVSLAYESKHLLEGAKSESAEELKKRAQELEGKLNFLTKIHEMLQPGQDQ(SEQ ID NO: 1).

[0031] Preferably, the liver fibrosis-related diseases include at least one of viral hepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, hereditary liver disease, cirrhosis, liver cancer, parasitic liver disease, toxic liver injury, and liver fibrosis.

[0032] A fourth aspect of the present invention provides a method for preparing the tetrane-type diterpenoid compound or a pharmaceutically acceptable derivative thereof as described in the first aspect of the present invention, comprising the following steps:

[0033] 1) The plants of the Euphorbiaceae family were crushed, impregnated with organic solvents, and filtered under reduced pressure to obtain an extract of the plants of the Euphorbiaceae family.

[0034] 2) Disperse the Euphorbiaceae plant extract with water, extract, concentrate under reduced pressure to obtain the extract.

[0035] 3) The diterpenoid compounds of the terpene type are obtained by separation by at least one of normal phase silica gel column chromatography, gel column chromatography, ODS column chromatography or high performance liquid chromatography.

[0036] Preferably, the Euphorbiaceae plants include at least one of the following: Croton tiglium, Euphorbia fischeriana, Euphorbia kansuensis, Sapium sebiferum, Spatholobus suberectus, Spatholobus suberectus, Spatholobus suberectus, Coral Flower, Coral Flower with Cotton Leaf, Eupatorium fortunei, Osmanthus fragrans var. rubra, Spotted Seed, Euphorbia lathyris, Eupatorium fortunei, and Aquilaria sinensis.

[0037] Preferably, the Euphorbiaceae plant is Euphorbia fischeriana.

[0038] Preferably, the organic solvent includes ethanol; and / or

[0039] The extraction reagent includes ethyl acetate;

[0040] Preferably, the volume ratio of the organic solvent to the Euphorbiaceae plant is (2-4):1;

[0041] Preferably, the soaking time is 3 to 30 days;

[0042] Preferably, the impregnation is repeated 2 to 4 times;

[0043] Preferably, the extraction is repeated 2 to 4 times.

[0044] The beneficial effects of this invention are:

[0045] The inventors isolated a series of natural tetrodinane-type diterpenes from *Euphorbia fischeriana*, a plant in the Euphorbiaceae family. Anti-hepatic fibrosis-related activity tests on this series of diterpenes revealed that they significantly inhibited the expression of fibrosis-related factors fibronectin (FN), type I collagen (Collagen I), and α-smooth muscle actin (α-SMA) in hepatic stellate cells (LX-2). In animal studies, their therapeutic effect was superior to that of the phase II clinical trial drug PFD (TD1 at 1 / 10 the dose of PFD was equivalent to its effect), with no significant toxicity. Mechanistic studies showed that TD1 is an APOL2 inhibitor; knocking out APOL2 protein in vivo can alleviate the progression of liver fibrosis. In conclusion, this series of tetrodinane-type diterpenes, especially TD1, shows promise as a candidate drug for anti-hepatic fibrosis and provides a new target for researching novel drugs to treat liver fibrosis. Attached Figure Description

[0046] Figures 1-2 The images show the proton and carbon spectra of compound TD1, respectively.

[0047] Figures 3-4 The images show the proton and carbon spectra of compound TD2, respectively.

[0048] Figures 5-6 The images show the proton and carbon spectra of compound TD3, respectively.

[0049] Figures 7-8 The images show the proton and carbon spectra of compound TD4, respectively.

[0050] Figures 9-10 The images show the proton and carbon spectra of compound TD5, respectively.

[0051] Figures 11-12 The images show the proton and carbon spectra of compound TD6, respectively.

[0052] Figures 13-14 The images show the proton and carbon spectra of compound TD7, respectively.

[0053] Figures 15-16 The images show the proton and carbon spectra of compound TD8, respectively.

[0054] Figures 17-18 The images show the proton and carbon spectra of compound TD9, respectively.

[0055] Figures 19-20 The images show the proton and carbon spectra of compound TD10, respectively.

[0056] Figures 21-22 The images show the proton and carbon spectra of compound TD11, respectively.

[0057] Figures 23-24 The images show the proton and carbon spectra of compound TD12, respectively.

[0058] Figures 25-26 The images show the proton and carbon spectra of compound TD13, respectively.

[0059] Figures 27-28 The images show the proton and carbon spectra of compound TD14, respectively.

[0060] Figures 29-30 The images show the proton and carbon spectra of compound TD15, respectively.

[0061] Figure 31 The results of screening for high-content anti-hepatic fibrosis activity of compounds TD1–TD15 are shown in Figure A. The structures of compounds TD1–TD15 are shown in Figure B. The fluorescence image of FN is shown in Figure C. The quantitative analysis diagram of fluorescence intensity is shown in Figure C.

[0062] Figure 32 The following diagram illustrates the therapeutic effect of oral administration of compound TD1 on liver fibrosis: A is the experimental flowchart; B shows liver images of mice in the blank group, model group, and drug-treated group, as well as Sirus Red, Masson, and α-SMA stained sections; C shows quantitative staining of sections and analysis of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver hydroxyproline (Hyp) levels; D shows Western blot analysis of livers from different groups.

[0063] Figure 33 A) A graph showing the difference in APOL2 expression between fibrotic and healthy livers; B) A graph showing the comparison of APOL2 protein expression levels between the livers of 100 patients with liver fibrosis and 10 healthy livers in tissue microarray; C) A graph showing the linear analysis of APOL2 protein expression levels between disease stage progression (Ishak score F0-F4); D) A graph showing the linear analysis of APOL2 expression levels between α-SMA protein expression levels; E) A graph showing the analysis of APOL2 mRNA levels in animal liver tissues; and E) A graph showing APOL2 protein expression levels in animal liver tissues.

[0064] Figure 34 A is a diagram showing the interaction between compound TD1 and APOL2; A is a diagram showing the cell thermostability experiment; B and C are diagrams showing the cell rescue experiment.

[0065] Figure 35 To illustrate the therapeutic effect of APOL2 knockout on liver fibrosis; A is the experimental flowchart; B shows liver images of mice in the blank group, model group, and Apol2 knockout group, as well as Sirus Red, Masson, and α-SMA stained sections; C shows quantitative staining of sections and analysis of serum ALT, AST, and liver Hyp levels; D shows Western blot analysis of livers from different groups. Detailed Implementation

[0066] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0067] Example 1: Preparation of diterpenoid compounds of the tetraalkyl type

[0068] 1. Equipment and reagents

[0069] NMR spectra were recorded using a Bruker AM-400 / 500 spectrometer with TMS internal standard. Column chromatography silica gel (300–400 mesh): Qingdao Ocean Chemical Plant; GF254 silica gel thin-layer chromatography pre-prepared plates: Qingdao Ocean Chemical Plant; MCI packing material (CHP20P, 75–150 μm): Mitsubishi Corporation, Japan; dextran gel (Sephadex LH-20): GE Healthcare, USA; ODS packing material (12 nm, S-50 μm): YMC Corporation, Japan; other solvents and reagents: analytical grade (AR), Tianjin Baishi Chemical Co., Ltd.

[0070] 2. Preparation method

[0071] Take 20 kg of Euphorbia fischeriana and crush it into coarse powder. Add 3 times its volume of 95% ethanol, soak for 1 week, filter, and recover the ethanol under reduced pressure. Repeat the soaking and extraction steps more than 3 times to finally obtain 2.50 kg of Euphorbia fischeriana ethanol extract. Disperse the Daphne genkwa extract in 3 L of water and extract three times with ethyl acetate. Combine the ethyl acetate extracts and concentrate under reduced pressure to obtain 1.54 kg of ethyl acetate extract.

[0072] The ethyl acetate extract was loaded onto a silica gel column and initially fractionated using a petroleum ether:ethyl acetate and dichloromethane:methanol mixture. Further separation was then performed using ODS, MCI, and Sephadex LH-20 gel chromatography. Finally, semi-preparative high-performance liquid chromatography (HPLC) was used for further purification to obtain 15 monomeric compounds. The specific separation procedure was as follows: The ethyl acetate extract (1.54 kg) was rapidly fractionated into four fractions (Frs.AD) using a silica gel column (PE / EtOAc, 50:1→0:1). Fr.B was fractionated into four fractions (Frs.B1-B4) using a reverse-phase silica gel column (MeOH / H2O, 5:5→10:0). Fr.B3 was fractionated into five fractions (Frs.B3A-B3E) using a silica gel column (PE / EtOAc, 50:1→1:1). Fr.B3A was separated into Fr.B3A1 and Fr.B3A2 using a Sephadex LH-20 (MeOH) gel column. Fr.B3A1 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 70 / 30, 3 mL / min) to obtain TD10 (27.4 mg, t). R 10.3 min), TD9 (41.2 mg, t R 11.8 min) and TD8 (15.7 mg, t R 13.3 min). Fr.B3A2 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 70 / 30, 3 mL / min) to obtain TD5 (32.2 mg, t). R 14.0 min). Fr.B3D was separated into Fr.B3D1 and Fr.B3D2 by Sephadex LH-20 (MeOH) gel column chromatography. Fr.B3D1 was further separated into TD6 (17.7 mg, t) by semi-preparative liquid chromatography (MeCN / H2O, 85 / 15, 3 mL / min). R 19.6 min). Fr.B3D2 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 70 / 30, 3 mL / min) to obtain TD3 (23.5 mg, t). R 14.5 min) and TD4 (18.9 mg, t R 15.8 min). Fr.B3C was subjected to silica gel column chromatography (PE / acetone, 100:1→2:1) to obtain Frs.B3C1-B3C3. Fr.B3C1 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 82 / 18, 3 mL / min) to obtain TD15 (36.0 mg, t). R 16.1 min) and TD7 (22.7 mg, t R 18.6 min). Fr.B3C2 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 85 / 15, 3 mL / min) to obtain TD13 (32.4 mg, t). R16.2 min) and TD14 (24.3 mg, t R 17.6 min). Fr.B3C3 was subjected to semi-preparative liquid chromatography (MeCN / H2O, 88 / 12, 3 mL / min) to obtain TD11 (26.1 mg, t). R 15.3 min) and TD12 (17.4 mg, t R Fr.B3B was reacted with Sephadex LH-20 (MeOH) to obtain Fr.B3B1 and Fr.B3B2. Fr.B3B1 was reacted with semi-preparative liquid phase (MeCN / H2O, 90 / 10, 3 mL / min) to obtain TD1 (856.2 mg, t). R 18.4 min). Fr.B2B2 was processed by semi-preparative liquid chromatography (MeCN / H2O, 65 / 35, 3 mL / min) to obtain TD2 (17.0 mg, t). R 13.7 min).

[0073] 3. Structural characterization

[0074] The structural formula and data of compound TD1 are as follows:

[0075]

[0076] 12-deoxyphorbol-13-palmitate(TD1). 1 H NMR (CDCl3, 400MHz) δ H 7.56(1H,br s,H-1),2.55(1H,d,J=19.0Hz,H2-5a),2.45(1H,d,J=19.0Hz,H2-5b),5.66(1H,d,J=4.2Hz,H-7),3.00(1H,t,J=5.0Hz,H-8),3.24(1H,br s,H-10),1.97(1H,m,H-11),2.05(1H,dd,J=14.2Hz,6.9Hz,H-12a),1.53(1H,d d,J=14.2Hz,11.0Hz,H-12b),0.80(1H,d,J=5.0Hz,H-14),1.17(3H,s,H3-16), 1.05(3H,s,H3-17),0.87(3H,d,J=6.5Hz,H3-18),1.74(3H,d,J=1.7Hz,H3-19) ,4.02(1H,d,J=12.5Hz,H2-20a),3.95(1H,d,J=12.5Hz,H2-20b); 13-OCO(CH2) 14CH3:2.28(2H,t,J=7.5Hz,),1.58(2H,m),1.30~1.24(24H,m),0.87(3H,t,J=7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 161.3(C-1),132.8(C-2),209.4(C-3),73.7(C-4),38.5(C-5),140.0(C-6),130.3(C-7),39.0(C-8),76.1(C-9),55.7(C-10),36.3(C- 11),31.9(C-12),63.3(C-13),32.6(C-14),22.7(C-15),23.2(C-16),15.3(C-17),18.6(C-18),10.1(C-19),68.2(C-20); 13-OCO(CH2) 14 CH3: 176.0, 34.6, 31.8, 29.7~29.6×6, 29.4, 29.3, 29.2, 29.1, 24.8, 22.7, 14.1. The proton and carbon spectra of compound TD1 are as follows... Figure 1 , 2 As shown.

[0077] The structural formula and data of compound TD2 are as follows:

[0078]

[0079] Prostratin (TD2). 1 H NMR (CDCl3, 400MHz) δ H 7.58(1H,br s,H-1),2.52(1H,d,J=19.5Hz,H2-5a),2.45

[0080] (1H,d,J=19.5Hz,H2-5b),5.67(1H,d,J=5.0Hz,H-7),2.99(1H,t,J=5.0Hz,H-8),3.26(1H,br s,H-10),1.97(1H,m,H-11),2.01(1H,dd,J=14.5,7.5Hz,H-12a),1.57(1H,dd,J =14.5Hz,11.5Hz,H-12b),0.85(1H,d,J=5.3Hz,H-14),1.19(3H,s,H3-16),1.06 (3H,s,H3-17),0.88(3H,d,J=6.4Hz,H3-18),1.77(3H,d,J=1.7Hz,H3-19),4.04 (1H,d,J=13.5Hz,H2-20a),3.98(1H,d,J=13.5Hz,H2-20b); 13-OAc:2.06(3H,s); 13 C NMR (CDCl3, 100MHz) δ C 161.3 (C-1), 132.8 (C-2), 209.1 (C-3), 73.7 (C-4), 38.6 (C-5), 139.8 (C-6), 130.2 (C-7), 39.1 (C-8), 76.0 (C-9), 55.7 (C-10), 36.3 (C-11), 31.8 (C-12), 63.6 (C-13), 32.4 (C-14), 22.7 (C-15), 23.2 (C-16), 15.3 (C-17), 18.5 (C-18), 10.1 (C-19), 68.3 (C-20); 13-OAc: 173.2, 21.3. The proton and carbon spectra of compound TD2 are as follows: Figure 3 , 4 As shown.

[0081] The structural formula and data of compound TD3 are as follows:

[0082]

[0083] 4α-deoxyphorbol-12-tiglate-13-isobutyrate(TD3). 1 H NMR (CDCl3, 400MHz) δ H7.05(1H,br s,H-1),2.76(1H,m,H-4α),3.44(1H,m,H2-5a),2.45(1H,dd,J=15.6,5.1Hz,H2-5b),5.11(1H,br s,H-7),1.95(1H,br s,H-8),3.49(1H,m,H-10),1.70(1H,m,H-11),5.49(1H,d,J=10.4Hz,H-12),0.75(1H,d,J=5. 0Hz,H-14),1.16(3H,s,H3-16),1.22(3H,s,H3-17),1.05(3H,d,J=6.8Hz,H3-18),1.77(3H,br s, H3-19), 3.99 (1H, d, J=12.5Hz, H2-20a), 3.87 (1H, d, J=12.5Hz, H2-20b); 9-OH: 5.31 (1H, s); 12-O-tigl: 6.85 (1H, m), 1.85 (3H, br s), 1.81 (3H, dd, J = 7.0, 1.0Hz); 13-O-iBu: 2.51 (1H, m), 1.14 (3H, d, J = 7.0Hz), 1.11 (3H, d, J = 7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 156.2(C-1),143.2(C-2),213.2(C-3),49.5(C-4),25.1(C-5),137.6(C-6),126.3(C -7),40.7(C-8),78.0(C-9),47.3(C-10),43.5(C-11),75.4(C-12),64.8(C-13),37. 1 (C-14), 25.3 (C-15), 24.1 (C-16), 16.5 (C-17), 12.2 (C-18), 10.4 (C-19), 69.2 (C-20); 12-O-tigl: 167.4, 137.6, 128.5, 14.4, 12.2; 13-O-iBu: 179.1, 34.4, 18.6, 18.4. The proton and carbon spectra of compound TD3 are as follows: Figure 5 , 6 As shown.

[0084] The structural formula and data of compound TD4 are as follows:

[0085]

[0086] Crodamoid A(TD4). 1 H NMR (CDCl3, 400MHz) δH 7.05(1H,br s,H-1),2.76(1H,m,H-4α),3.44(1H,m,H2-5a),2.47(1H,dd,J=15.5,5.0Hz,H2-5b),5.11(1H,br s,H-7),1.96(1H,br s,H-8),3.49(1H,m,H-10),1.69(1H,m,H-11),5.46(1H,d,J=10.5Hz,H-12),0.75(1H,d,J=5. 0Hz,H-14),1.16(3H,s,H3-16),1.21(3H,s,H3-17),1.07(3H,d,J=6.5Hz,H3-18),1.78(3H,br s, H3-19), 4.01 (1H, d, J = 12.5Hz, H2-20a), 3.89 (1H, d, J = 12.5Hz, H2-20b); 9-OH: 5.26 (1H, s); 12-O- (2MeBu): 2.42 (1H, m), 1.70 (1H ,m),1.51(1H,m),1.19(3H,d,J=7.0Hz),0.95(3H,t,J=7.5Hz); 13-O-iBu:2.52(1H,m),1.14(3H,d,J=7.0Hz),1.13(3H,d,J=7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 156.1(C-1),143.3(C-2),213.2(C-3),49.6(C-4),25.1(C-5),137.0(C-6),126.3(C -7),40.7(C-8),78.0(C-9),47.3(C-10),43.1(C-11),75.0(C-12),64.8(C-13),37. 0 (C-14), 25.2 (C-15), 24.1 (C-16), 16.5 (C-17), 11.8 (C-18), 10.4 (C-19), 69.3 (C-20); 12-O-(2MeBu): 175.9, 41.9, 26.7, 17.1, 11.8; 13-O-iBu: 179.0, 34.2, 18.5, 18.5. The proton and carbon spectra of compound TD4 are as follows: Figure 7 , 8 As shown.

[0087] The structural formula and data of compound TD5 are as follows:

[0088]

[0089] 12-O-[2'-Methyl-2'-butenoyl]-13-O-[2”-methylbutanoyl]-4-epi-4-deoxyphorbol (TD5). 1 H NMR (CDCl3, 400MHz) δ H 7.06(1H,br s,H-1),2.78(1H,m,H-4α),3.46(1H,m,H2-5a),2.48(1H,dd,J=15.6,5.0Hz,H2-5b),5.12(1H,br s,H-7),1.96(1H,brs,H-8),3.50(1H,m,H-10),1.72(1H,m,H-11),5.52(1H,d,J=10.3Hz,H-12),0.75(1 H,d,J=5.0Hz,H-14),1.24(3H,s,H3-16),1.18(3H,s,H3-17),1.07(3H,d,J=6.5Hz,H3-18),1.78(3H,br s, H3-19), 4.02 (1H, d, J = 12.4Hz, H2-20a), 3.89 (1H, d, J = 12.4Hz, H2-20b); 9-OH: 5.36 (1H, s); 12-O-tigl: 6.87 (1H, m), 1.87 (3H ,s),1.83(3H,d,J=7.0Hz); 13-O-(2MeBu):2.34(1H,m),1.69(1H,m),1.42(1H,m),1.11(3H,d,J=7.0Hz),0.91(3H,t,J=7.5Hz); 13 CNMR (CDCl3, 100MHz)δ C 156.2(C-1),143.3(C-2),213.2(C-3),49.6(C-4),25.1(C-5),136.9(C-6),126.4(C-7 ),40.8(C-8),78.0(C-9),47.4(C-10),43.7(C-11),75.5(C-12),64.8(C-13),37.2(C- 14), 25.4 (C-15), 24.1 (C-16), 16.6 (C-17), 11.6 (C-18), 10.4 (C-19), 69.3 (C-20); 12-O-tigl: 167.4, 137.6, 128.5, 14.5, 12.3; 13-O-(2MeBu): 178.8, 41.2, 26.1, 16.2, 11.6. The proton and carbon spectra of compound TD5 are as follows: Figure 9 , 10 As shown.

[0090] The structural formula and data of compound TD6 are as follows:

[0091]

[0092] 12-O-tetradecanoyl(4-deoxy-4α-phorbol)13,20-diacetate(TD6). 1 H NMR (CDCl3, 400MHz) δ H 6.97(1H,br s,H-1),2.72(1H,m,H-4α),3.35(1H,m,H2-5a),2.45(1H,dd,J=15.5,4.5Hz,H2-5b),5.11(1H,br s,H-7),1.96(1H,br s,H-8),3.45(1H,m,H-10),1.69(1H,m,H-11),5.45(1H,d,J=10.4Hz,H-12),0.79(1H,d,J=5. 0Hz,H-14),1.20(3H,s,H3-16),1.17(3H,s,H3-17),1.05(3H,d,J=6.5Hz,H3-18),1.75(3H,br s, H3-19), 4.45 (1H, d, J = 12.4Hz, H2-20a), 4.32 (1H, d, J = 12.4Hz, H2-20b); 9-OH: 5.15 (1H, s); 12-OCO (CH2) 12 CH3:2.35(2H,m),1.66(2H,m),1.30~1.25(20H,m),0.86(3H,t,J=7.0Hz); 13-OAc:2.11(3H,s); 20-OAc:2.05(3H,s); 13 C NMR (CDCl3, 100MHz) δ C 155.2(C-1),143.3(C-2),211.0(C-3),48.8(C-4),25.2(C-5),132.6(C-6),128.7(C-7),40.8(C-8),77.7(C-9),46.8(C-10),42.8(C- 11),75.2(C-12),65.2(C-13),36.7(C-14),25.0(C-15),24.1(C-16),16.3(C-17),11.9(C-18),10.5(C-19),70.7(C-20); 12-OCO(CH2) 12CH3: 173.5, 34.5, 31.9, 29.6×4, 29.5, 29.3, 29.3, 29.0, 26.3, 22.6, 14.1; 13-OAc: 173.4, 21.1; 20-OAc: 171.0, 21.0. The proton and carbon spectra of compound TD6 are as follows: Figure 11 , 12 As shown.

[0093] The structural formula and data of compound TD7 are as follows:

[0094]

[0095] 4-deoxy(4α)phorbol 12-tetradecanoate-13-acetate(TD7). 1 H NMR (CDCl3, 400MHz) δ H 7.01(1H,br s,H-1),2.74(1H,m,H-4α),3.39(1H,m,H2-5a),2.43(1H,dd,J=15.5,4.5Hz,H2-5b),5.11(1H,br s,H-7),1.92(1H,br s,H-8),3.47(1H,m,H-10),1.64(1H,m,H-11),5.45(1H,d,J=10.4Hz,H-12),0.77(1H,d,J=5. 0Hz,H-14),1.17(3H,s,H3-16),1.14(3H,s,H3-17),1.05(3H,d,J=6.5Hz,H3-18),1.75(3H,br s, H3-19), 3.96 (1H, d, J = 12.5Hz, H2-20a), 3.85 (1H, d, J = 12.5Hz, H2-20b); 9-OH: 5.09 (1H, s); 12-OCO (CH2) 12 CH3:2.33(2H,m),1.64(2H,m),1.30~1.24(20H,m),0.85(3H,t,J=7.0Hz); 13-OAc:2.02(3H,s); 13 C NMR (CDCl3, 100MHz) δ C156.2(C-1),143.4(C-2),213.2(C-3),49.6(C-4),25.3(C-5),137.1(C-6),126.3(C-7),40.7(C-8),78.1(C-9),47.4(C-10),43.1(C- 11),75.4(C-12),65.3(C-13),37.1(C-14),25.1(C-15),24.2(C-16),16.4(C-17),11.9(C-18),10.5(C-19),69.2(C-20); 12-OCO(CH2) 12 CH3: 173.6, 34.6, 32.0, 29.7×4, 29.6, 29.4, 29.4, 29.1, 24.2, 22.8, 14.2; 13-OAc: 173.6, 21.1. The proton and carbon spectra of compound TD7 are as follows: Figure 13 , 14 As shown.

[0096] The structural formula and data of compound TD8 are as follows:

[0097]

[0098] 12-O-tiglyl-4-deoxy-4α-phorbol-13-acetate(TD8). 1 H NMR (CDCl3, 400MHz) δ H7.05(1H,br s,H-1),2.77(1H,m,H-4α),3.42(1H,m,H2-5a),2.46(1H,dd,J=15.5,5.0Hz,H2-5b),5.11(1H,br s,H-7),1.96(1H,br s,H-8),3.49(1H,m,H-10),1.72(1H,m,H-11),5.52(1H,d,J=10.4Hz,H-12),0.79(1H,d,J=5. 0Hz,H-14),1.22(3H,s,H3-16),1.16(3H,s,H3-17),1.06(3H,d,J=6.5Hz,H3-18),1.77(3H,br s,H3-19),3.99(1H,d,J=12.5Hz,H2-20a),3.87(1H,d,J=12.5Hz,H2-20b); 9-OH:5.28(1H,s); 20-OH:5.18(1H,s); 12-O-tigl:6.87(1H,m),1.86(3H,br s),1.82(3H,d,J=7.0Hz); 13-OAc:2.05(3H,s); 13 C NMR (CDCl3, 100MHz) δ C 156.1(C-1),143.3(C-2),213.1(C-3),49.5(C-4),25.1(C-5),137.0(C-6),126. 3(C-7),40.6(C-8),78.0(C-9),47.3(C-10),43.4(C-11),75.4(C-12),65.3(C-1 3), 37.0 (C-14), 25.1 (C-15), 24.1 (C-16), 16.4 (C-17), 11.8 (C-18), 10.4 (C-19), 69.2 (C-20); 12-O-tigl: 167.6, 137.8, 128.4, 14.4, 12.2; 13-OAc: 173.5, 21.0. The proton and carbon spectra of compound TD8 are as follows: Figure 15 , 16 As shown.

[0099] The structural formula and data of compound TD9 are as follows:

[0100]

[0101] 4α-deoxy-phorbol-13-acetate(TD9). 1 H NMR (CDCl3, 400MHz) δ H7.05(1H,br s,H-1),2.74(1H,m,H-4α),3.29(1H,br d,J=15.5Hz,H2-5a),2.44(1H,dd,J=15.5,4.6Hz,H2-5b),5.11(1H,br s,H-7),1.89(1H,br s,H-8),3.47(1H,m,H-10),1.53(1H,m,H-11),4.00(1H,d,J=10.0Hz,H-12),0.74(1H,d,J=5. 0Hz,H-14),1.18(3H,s,H3-16),1.15(3H,s,H3-17),1.24(3H,d,J=6.1Hz,H3-18),1.73(3H,br s, H3-19), 3.94 (1H, d, J = 12.4Hz, H2-20a), 3.85 (1H, d, J = 12.4Hz, H2-20b); 9-OH: 5.27 (1H, s); 12-OH: 3.42 (1H, s); 13-OAc: 2.05 (3H, s); 13 C NMR (CDCl3, 100MHz) δ C 156.8 (C-1), 143.2 (C-2), 213.3 (C-3), 49.5 (C-4), 25.2 (C-5), 136.5 (C-6), 126.8 (C-7), 40.7 (C-8), 78.0 (C-9), 47.6 (C-10), 45.3 (C-11), 75.2 (C-12), 67.8 (C-13), 35.9 (C-14), 25.9 (C-15), 24.1 (C-16), 16.4 (C-17), 12.4 (C-18), 10.5 (C-19), 69.2 (C-20); 13-OAc: 174.0, 21.1. The proton and carbon spectra of compound TD9 are as follows: Figure 17 , 18 As shown.

[0102] The structural formula and data of compound TD10 are as follows:

[0103]

[0104] 13-O-(2-methylbutyryl)-4-deoxy-4α-phorbol (TD10). 1 H NMR (CDCl3, 400MHz) δ H7.05(1H,br s,H-1),2.77(1H,m,H-4α),3.35(1H,br d,J=15.5,H2-5a),2.50(1H,dd,J=15.5,5.0Hz,H2-5b),5.14(1H,br s,H-7),1.94(1H,br s,H-8),3.50(1H,m,H-10),1.55(1H,m,H-11),3.98(1H,d,J=10.0Hz,H-12),0.74(1H,d,J=5. 0Hz,H-14),1.22(3H,s,H3-16),1.19(3H,s,H3-17),1.28(3H,d,J=6.5Hz,H3-18),1.77(3H,br s,H3-19),4.01(1H,d,J=12.5Hz,H2-20a),3.90(1H,d,J=12.55Hz,H2-20b); 13-O-(2MeB u):2.38(1H,m),1.69(1H,m),1.46(1H,m),1.14(3H,d,J=7.0Hz),0.91(3H,t,J=7.5Hz); 13 C NMR (CDCl3, 100MHz) δ C 156.4(C-1),143.3(C-2),213.1(C-3),49.4(C-4),25.1(C-5),136.6(C-6) ,126.8(C-7),40.7(C-8),78.0(C-9),47.5(C-10),45.3(C-11),75.6(C-12) ,67.2(C-13),35.9(C-14),25.8(C-15),24.1(C-16),16.5(C-17),12.4(C-1 8),10.4(C-19),69.3(C-20); 13-O-(2MeBu):179.3,41.2,26.4,16.4,11.7. The proton and carbon spectra of compound TD10 are as follows: Figure 19 , 20 As shown.

[0105] The structural formula and data of compound TD11 are as follows:

[0106]

[0107] 12-O-tigloyl-phorbol-13-decanoate(TD11). 1 H NMR (CDCl3, 400MHz) δ H7.59(1H,br s,H-1),2.59(1H,d,J=19.0Hz,H2-5a),2.48(1H,d,J=19.0Hz,H2-5b),5 .69(1H,brs,H-7),3.26(1H,m,H-8),3.23(1H,m,H-10),2.17(1H,m,H-1 1),5.44(1H,d,J=10.1Hz,H-12),1.05(1H,d,J=5.0Hz,H-14),1.20(3H, s,H3-16),1.26(3H,s,H3-17),0.88(3H,d,J=6.0Hz,H3-18),1.74(3H,br s, H3-19), 4.04 (1H, d, J = 12.8Hz, H2-20a), 3.97 (1H, d, J = 12.8Hz, H2-20b); 9-OH: 5.76 (1H, s); 12-O-tigl: 6.82 (1H, m), 1. 82(3H,s),1.78(3H,d,J=7.0Hz); 13-OCO(CH2)8CH3:2.32(2H,m),1.60(2H,m),1.30~1.23(12H,m),0.86(3H,t,J=7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 161.1(C-1),132.9(C-2),209.3(C-3),73.8(C-4),38.7(C-5),140.6(C-6),129.5(C-7),39.2( C-8),78.5(C-9),56.3(C-10),43.4(C-11),76.9(C-12),65.6(C-13),36.6(C-14),25.9(C-15) ,24.0(C-16),17.0(C-17),14.6(C-18),10.2(C-19),68.2(C-20); 12-O-tigl:167.9,137.6,128.7,14.6,12.4; 13-OCO(CH2)8CH3:176.6,34.5,32.0,29.5,29.4,29.4,29.2,24.7,22.8,14.2. The proton and carbon spectra of compound TD11 are as follows: Figure 21 , 22 As shown.

[0108] The structural formula and data of compound TD12 are as follows:

[0109]

[0110] 12-O-acetylphorbol-13-decanoate(TD12). 1 H NMR (CDCl3, 400MHz) δ H 7.55(1H,br s,H-1),2.58(1H,d,J=19.0Hz,H2-5a),2.47(1H,d,J=19.0Hz,H2-5b),5. 69(1H,d,J=5.0Hz,H-7),3.25(1H,m,H-8),3.24(1H,m,H-10),2.14(1H,m, H-11),5.35(1H,d,J=10.3Hz,H-12),1.03(1H,d,J=5.0Hz,H-14),1.19(3H ,s,H3-16),1.22(3H,s,H3-17),0.86(3H,d,J=6.0Hz,H3-18),1.72(3H,br s,H3-19),4.01(1H,d,J=12.8Hz,H2-20a),3.94(1H,d,J=12.8Hz,H2-20b); OH:5.70(1H,br s),3.21(1H,br s); 12-OAc: 2.05 (3H, s); 13-OCO (CH2) 8CH3: 2.30 (2H, m), 1.59 (2H, m), 1.28 ~ 1.23 (12H, m), 0.86 (3H, t, J = 7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 160.9(C-1),132.9(C-2),209.4(C-3),73.8(C-4),38.5(C-5),140.8(C-6),129.3(C-7), 39.1(C-8),78.4(C-9),56.1(C-10),43.1(C-11),77.2(C-12),65.5(C-13),36.5(C-14), 26.0 (C-15), 24.0 (C-16), 16.9 (C-17), 14.2 (C-18), 10.2 (C-19), 68.1 (C-20); 12-OAc: 171.0, 21.1; 13-OCO(CH2)8CH3: 176.6, 34.5, 31.9, 29.5, 29.4, 29.3, 29.2, 24.6, 22.8, 14.2. The proton and carbon spectra of compound TD12 are as follows: Figure 23 , 24 As shown.

[0111] The structural formula and data of compound TD13 are as follows:

[0112]

[0113] 12-O-tigloyl-phorbol-13-dodecanoate(TD13). 1 H NMR(CDCl3,400MHz)δ H 7.59(1H,br s,H-1),2.58(1H,d,J=19.0Hz,H2-5a),2.48(1H,d,J=19.0Hz,H2-5b),5.69(1H,d,J=5.0Hz,H-7),3.26(1H,m,H-8),3.25(1H,m,H-10),2.18(1H,m,H-11),5.45(1H,d,J=10.5Hz,H-12),1.06(1H,d,J=5.0Hz,H-14),1.20(3H,s,H3-16),1.26(3H,s,H3-17),0.88(3H,d,J=6.0Hz,H3-18),1.75(3H,br s,H3-19),4.04(1H,d,J=12.5Hz,H2-20a),3.98(1H,d,J=12.5Hz,H2-20b);9-OH:5.75(1H,s);12-O-tigl:6.83(1H,m),1.82(3H,s),1.79(3H,d,J=7.0Hz);13-OCO(CH2) 10 CH3:2.33(2H,m),1.61(2H,m),1.30~1.23(16H,m),0.87(3H,t,J=7.0Hz); 13 C NMR(CDCl3,100MHz)δ C 161.1(C-1),133.0(C-2),209.3(C-3),73.8(C-4),38.7(C-5),140.6(C-6),129.5(C-7),39.2(C-8),78.4(C-9),56.3(C-10),43.4(C-11),76.9(C-12),65.6(C-13),36.6(C-14),25.9(C-15),24.0(C-16),17.1(C-17),14.6(C-18),10.2(C-19),68.2(C-20);12-O-tigl:167.9,137.7,128.6,14.6,12.4;13-OCO(CH2) 10CH3: 176.6, 34.5, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 24.7, 22.8, 14.3. The proton and carbon spectra of compound TD13 are as follows... Figure 25 , 26 As shown.

[0114] The structural formula and data of compound TD14 are as follows:

[0115]

[0116] 12-O-(α-methyl)butyryl-phorbol-13-dodecanoate(TD14). 1 H NMR (CDCl3, 400MHz) δ H 7.59(1H,br s,H-1),2.57(1H,d,J=19.0Hz,H2-5a),2.48(1H,d,J=19.0Hz,H2-5b),5.69(1H,br s,H-7),3.25(2H,m,H-8and H-10),2.14(1H,m,H-11),5.41(1H,d,J=10.2Hz,H-12),1.06(1H,d,J=5.0Hz,H-14) ,1.19(3H,s,H3-16),1.25(3H,s,H3-17),0.90(3H,d,J=6.0Hz,H3-18),1.76(3H,br s,H3-19),4.04(1H,d,J=12.8Hz,H2-20a),3.98(1H,d,J=12.8Hz,H2-20b); 9-OH:5.70(1H,s); 12-O-(2M eBu):2.37(1H,m),1.66(1H,m),1.47(1H,m),1.15(3H,d,J=7.0Hz),0.91(3H,t,J=7.5Hz); 13-OCO(CH2) 10 CH3:2.32(2H,m),1.60(2H,m),1.30~1.23(16H,m),0.88(3H,t,J=7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C161.0(C-1),133.0(C-2),209.2(C-3),73.8(C-4),38.7(C-5),140.6(C-6),12 9.4(C-7),39.2(C-8),78.4(C-9),56.3(C-10),43.0(C-11),76.5(C-12),65.5( C-13),36.5(C-14),25.8(C-15),24.0(C-16),17.0(C-17),14.5(C-18),10.2( C-19),68.2(C-20); 12-O-(2MeBu):176.6,42.0,26.9,17.2,11.8; 13-OCO(CH2) 10 CH3: 176.3, 34.5, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 24.6, 22.8, 14.3. The proton and carbon spectra of compound TD14 are as follows... Figure 27 , 28 As shown.

[0117] The structural formula and data of compound TD15 are as follows:

[0118]

[0119] phorbol 12-monomyristate(TD15). 1 H NMR (CDCl3, 400MHz) δ H 7.57(1H,br s,H-1),2.58(1H,d,J=

[0120] 19.0Hz,H2-5a),2.42(1H,d,J=19.0Hz,H2-5b),5.62(1H,br s,H-7),3.13(1H,m,H-8),3.12(1H,m,H-10),2.10(1H,m,H-11),4.83(1H,d,J=9.7Hz,H-12),0.90(1H ,d,J=5.0Hz,H-14),1.02(3H,s,H3-16),1.14(3H,s,H3-17),0.98(3H,d,J=6.0Hz,H3-18),1.74(3H,br s, H3-19), 4.00 (1H, d, J = 12.5Hz, H2-20a), 3.93 (1H, d, J = 12.5Hz, H2-20b); OH: 3.57 (1H, br s); 13-OCO (CH2) 12CH3:2.31(2H,m),1.60(2H,m),1.28~1.23(20H,m),0.86(3H,t,J=7.0Hz); 13 C NMR (CDCl3, 100MHz) δ C 160.7(C-1),133.4(C-2),209.6(C-3),73.6(C-4),38.5(C-5),141.0(C-6),129.6(C-7),39.0(C-8),79.1(C-9),57.3(C-10),43.3(C- 11),87.6(C-12),61.1(C-13),35.4(C-14),27.4(C-15),22.6(C-16),17.3(C-17),16.0(C-18),10.3(C-19),67.9(C-20); 13-OCO(CH2) 12 CH3: 177.0, 34.5, 322.0, 29.8, 29.7×3, 29.6, 29.7×2, 29.3, 25.1, 22.8, 14.2. The proton and carbon spectra of compound TD15 are as follows: Figure 29 , 30 As shown.

[0121] Example 2: Compound TD1-TD15 exhibits anti-liver fibrosis activity.

[0122] 1. Experimental Methods

[0123] Logarithmically growing LX-2 cells (3000 cells / 100 μL / well) were seeded into 96-well plates and treated with 10 ng / mTGF-β1 and 10 μM of the compound (TD1-TD15 prepared in Example 1) for 48 h. After washing the cells with PBS, they were fixed with 4 wt% paraformaldehyde for 10 min at room temperature. After washing the cells three times with PBS, they were incubated with 0.2 v / v% Triton X-100 for 10 min at room temperature. The cells were washed three times again with PBS and then blocked with 5 wt% BSA for 1 h at room temperature. The fixed cells were incubated with FN primary antibody (Proteintech, 15613-1-AP, 1:200 dilution) at 4 °C for 12 h. After washing three times with PBS, the cells were incubated with FITC fluorescent secondary antibody in the dark for 1 h at room temperature. After washing three times with PBS, the cells were stained with DAPI at room temperature. Finally, after washing three times with PBS, images were captured using an ArrayScan VTI 600Plus instrument, and fluorescence intensity was analyzed using Cellomics Cell Health Profiling Assay Software.

[0124] 2. Experimental Results

[0125] The results are as follows Figure 31 As shown, TGF-β1 significantly induced FN expression in LX-2 cells, indicating successful model establishment. Compounds TD1–TD15 significantly inhibited FN expression at a concentration of 10 μM, with compound TD1 exhibiting the best activity, inhibiting TGF-β1-induced FN production in LX-2 cells by 96.7% at a concentration of 10 μM.

[0126] Example 3: In vivo pharmacodynamic evaluation in animals

[0127] 1. Experimental Methods

[0128] Eight-week-old male C57BL / 6J mice were purchased from Guangdong Yaokang Biotechnology Co., Ltd. and housed in an SPF (Self-Protected Free) environment in Area C of the Animal Center on the East Campus of Sun Yat-sen University. Mice were kept in a temperature- and humidity-controlled room with 12 hours of light and 12 hours of darkness, and had free access to food and water. Experiments were conducted after the mice passed quarantine and acclimatized to their new environment. All animal care and experiments were approved by the Laboratory Animal Ethics Committee of Sun Yat-sen University (Approval Nos.: SYSU-IACUC-2023-000618 and SYSU-IACUC-2021-000646). Mice were randomly divided into 6 groups of 8 mice each based on body weight. The normal control group received intraperitoneal injections of corn oil, while the modeling group received intraperitoneal injections of 5 mL of 25v / v% carbon tetrachloride (CCl4) corn oil solution per kg of body weight twice a week. Drug administration was initiated in the third week at the time of model establishment. Mice in the drug-treated group received oral gavage of compound TD1 at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg every two days. The positive control group mice were orally administered 200 mg / kg of PFD (MedChemExpress, HY-B0673) via gavage every two days. The model group was given the same volume of physiological saline containing DMSO. After the experiment, liver tissue was harvested and photographed. Paraffin-embedded sections were then subjected to Sirius Red staining, Masson staining, and α-SMA immunohistochemical staining analysis. Serum AST and ALT levels, as well as liver Hyp (Nanjing Jiancheng Bioengineering Institute, A030-1-1), were detected. Western blot analysis was performed on the expression of FN, collagen I, and α-SMA in the mouse liver.

[0129] 2. Experimental Results

[0130] The mouse modeling process is as follows Figure 32 As shown in Figure A, the therapeutic effects of TD1 on the liver are as follows: Figure 31As shown in Figures B-D, after seven weeks of CCl4 treatment, mouse liver inflammatory cell infiltration significantly increased, central venous wall thickened slightly, fibrosis occurred, liver hydroxyproline content significantly increased, serum ALT and AST levels significantly increased, and the expression of fibrosis markers FN, collagen I, and α-SMA proteins significantly increased, indicating successful model establishment. Compound TD1 can dose-dependently inhibit CCl4-induced pathological changes, and its therapeutic effect at a dose of 20 mg / kg is comparable to that of PFD at a dose of 200 mg / kg.

[0131] Example 4: Differential analysis of APOL2 expression in fibrotic and healthy livers

[0132] 1. Experimental Methods

[0133] Immunohistochemistry was used to analyze the expression level of APOL2 protein in the livers of 100 patients with liver fibrosis and 10 healthy livers in a tissue microarray (Bioaitech, D170Lv01). Quantitative analysis was performed using ImageJ software. Furthermore, PCR and Western blot were used to detect APOL2 expression in mouse fibrotic livers (obtained through the model in Example 3) and healthy livers.

[0134] 2. Experimental Results:

[0135] Immunohistochemical analysis results of livers from 100 patients with liver fibrosis and 10 healthy livers are as follows: Figure 33 As shown in Figures A through C, compared to normal liver, APOL2 protein expression was elevated in fibrotic liver tissue and positively correlated with Ishak scores. Furthermore, the results of APOL2 protein expression level detection in animal liver tissue are as follows: Figure 33 As shown in Figures D and E, compared with normal liver tissue, the mRNA level and protein expression of APOL2 were also increased in animal fibrotic liver tissue. These results indicate that APOL2 is closely related to the occurrence and development of liver fibrosis, and that knocking out APOL2 through gene therapy or using small molecules to inhibit APOL2 function can suppress the progression of liver fibrosis.

[0136] The target of compound TD1 in Example 5 is APOL2.

[0137] 1. Experimental Methods

[0138] For temperature gradient experiments, LX-2 cells were incubated with TD1 or DMSO for 3 h, then digested with trypsin and resuspended in 1 mL of PBS containing protease and phosphatase inhibitors (Roche, 4906845001). The cell suspension was evenly distributed into seven 0.2 mL PCR tubes. Each sample was heated in a 96-well PCR instrument at the specified temperature for 3 min, and then incubated at room temperature for another 3 min. For concentration gradient experiments, LX-2 cells were incubated with different concentrations of TD1 for 3 h, then digested with trypsin and resuspended in 1 mL of PBS containing protease and phosphatase inhibitors. The cell suspension was heated at 55 °C for 3 min, and then incubated at room temperature for another 3 min. The heat-treated samples were then subjected to three freeze-thaw cycles in liquid nitrogen at 25 °C to lyse the cells. The cell lysates were centrifuged at 15000 g at 4 °C, and the supernatant was collected. Western blot analysis was then performed on the samples. For gene rescue experiments, LX-2 cells in logarithmic growth phase (3 × 10⁻⁶ cells) were used. 5 Seeds (1 cell / well) were seeded into 6-well plates, shaken well, and incubated for 24 h. To prepare the transfection solution, for each well, first added 3 μL of Lipo 2000 transfection reagent (Invitrogen, 11668019) to 100 μL of Opti-MEM medium (ThermoFisher Scientific, 31985070) and mixed well, then incubated for 5 min. Next, in another 100 μL of Opti-MEM medium, added 2.5 μL of siRNA (final concentration 80 nM) or pLX304-V5-APOL2 plasmid (final concentration 1 μg) and mixed well, then incubated for 5 min. Finally, the two solutions were mixed and incubated for 15 min. The old culture medium was aspirated from the wells, and the cells were washed with Opti-MEM medium. Then, 800 μL of Opti-MEM was added to each well, followed by 200 μL of transfection mixture. After incubation for 6 h, the medium in the wells was replaced with DMEM medium containing 10% FBS, and the cells were cultured for another 48 h before Western blot analysis. The sequence of siAPOL2-1 is GCAGUGUGGUAGAACUAGUAATT (SEQ ID NO: 2).

[0139] 2. Experimental Results

[0140] like Figure 34 As shown in Figure A, TD1 dose-dependently increased the thermostability of APOL2, indicating that TD1 and APOL2 bind intracellularly. Figure 34As shown in Figures B and C, knockdown of APOL2 inhibits TGF-β1-induced fibrosis. TD1 did not further reduce fibrosis markers in APOL2-knockdown cells, while overexpression of APOL2 promoted fibrosis. Furthermore, TD1 could alleviate the pro-fibrotic effect induced by APOL2 overexpression. These results indicate that TD1 exerts its anti-hepatic fibrosis effect by inhibiting APOL2 function.

[0141] Example 6: Effects of APOL2 knockout on liver fibrosis in animals

[0142] 1. Experimental Methods

[0143] Using CRISPR / Cas9 technology, a frameshift and loss of function in the APOL2 gene protein were induced through non-homologous recombination repair. In short, Cas9 mRNA, gRNA1 (CAGTCTACAGAACTGGAATTTGG, SEQ ID NO: 3), and gRNA2 (AGATCAGTGCGGTAGCTAAGG, SEQ ID NO: 4) were obtained via in vitro transcription, mixed, and injected into fertilized eggs of C57BL / 6J female mice to obtain F0 generation mice. Due to the rapid early cleavage rate of fertilized eggs, the resulting F0 generation mice are chimeras and may not possess stable heritability; passaging is required to obtain stably heritable F1 generation mice. To verify the successful construction of Apol2 knockout mice (Apol2- / -), PCR analysis was performed using primers mApol2-F1 (ACAGACCTCAGAGGCCACA, SEQ ID NO: 5) and mApol2-R1 (AGAATGCTCACTGTGACCCG, SEQ ID NO: 6). Eight-week-old male C57BL / 6J mice were used for the experiment. Liver fibrosis was induced by intraperitoneal injection of 1.25 mL / kg body weight of 25% CCl4 corn oil (twice a week for four weeks). Normal mice and Apol2 mice were tested according to the method described in Example 4. - / - Expression of fibrosis markers in mouse liver.

[0144] 2. Experimental Results

[0145] The results are as follows Figure 35 As shown, Apol2 knockout in vivo exhibited significant resistance to CCl4-induced liver injury and fibrosis, specifically manifested as reduced levels of FN, collagen I, and α-SMA proteins in liver tissue, and decreased levels of liver Hyp, serum ALT, and AST, indicating that Apol2 knockout can alleviate the progression of liver fibrosis.

Claims

1. Use of tetraalkyl diterpenoids or their pharmaceutically acceptable salts in a1)~a2): a1) Preparation of APOL2 protein inhibitors; a2) Prepare products for the treatment of liver fibrosis-related diseases; The general formula of the tetrane-type diterpenoid compound is shown in Formula I: ； In Formula I: R1 is selected from one of acetyl, crotonyl, isovaleryl, decanyl, dodecanoyl or tetradecanoyl; R2 is hydrogen.

2. Application of tetrodopane-type diterpenoids or their pharmaceutically acceptable salts in a1)~a2): a1) Preparation of APOL2 protein inhibitors; a2) Prepare products for the treatment of liver fibrosis-related diseases; The tetrane-type diterpene compound is TD1; The structural formula of TD1 is as follows: 。 3. Use of tetraalkyl diterpenoids or their pharmaceutically acceptable salts in a1)~a2): a1) Preparation of APOL2 protein inhibitors; a2) Prepare products for the treatment of liver fibrosis-related diseases; The tetrane-type diterpene compound is TD2; The structural formula of TD2 is as follows: 。 4. The application according to any one of claims 1 to 3, characterized in that: The APOL2 protein inhibitor is an inhibitor that inhibits the function of the APOL2 protein.

5. The application according to any one of claims 1 to 3, characterized in that: The liver fibrosis-related diseases include at least one of viral hepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, hereditary liver disease, parasitic liver disease, or toxic liver injury.

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