Application of Psammatlysene D and derivative thereof in preparation of medicine for resisting chronic liver diseases

Abstract

The invention discloses application of Psammatlysene D and a derivative thereof in preparation of a medicine for resisting chronic liver diseases, and belongs to the technical field of medicines. The technical scheme of the invention comprises the application of the Psammaglyene D in preparation of a product for resisting chronic liver diseases. The Psammatlyene D can inhibit the growth of LX2 cells, reduce liver function indexes ALT and AST, and relieve the degree of hepatic fibrosis in a mouse body; the growth of liver cancer cells can be inhibited in vivo and in vitro, and a synergistic effect can be generated with sorafenib; meanwhile, the anti-proliferation ability is also shown in sorafenib drug-resistant liver cancer. The derivative B12 of the Psammatlysene D has a stronger in-vitro anti-hepatic fibrosis effect, and the C11 has a very weak inhibition capability on hERG (human endothelial growth factor) and has no potential cardiotoxicity. The results show that the Psammaglyene D has relatively strong capabilities of resisting hepatic fibrosis, inhibiting liver cancer development and relieving sorafenib drug-resistant liver cancer, and the Psammaglyene D and C11 can be used as potential lead compounds for the development of anti-hepatic fibrosis and anti-liver cancer drugs, and are finally applied to the treatment of chronic liver diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to application of Psammaplysene D and derivatives thereof in preparation of medicines for resisting chronic liver diseases. BACKGROUND

[0002] Liver fibrosis is a wound healing response of chronic liver injury, which can be caused by hepatitis virus infection, alcoholism, cholestasis, autoimmunity, drugs, toxins and nonalcoholic steatohepatitis (NASH), and can develop into cirrhosis in the end stage, which is characterized by excessive deposition of extracellular matrix (ECM) in the liver, and insufficient degradation activity, which destroys the normal liver lobule structure and affects the metabolic function of the liver. In the process of chronic liver injury, hepatic stellate cells (HSCs) can be continuously activated by factors such as TGF-β1 and transformed into myofibroblasts, which are the main source of ECM and the main cell type of liver fibrosis. The continuous activation of hepatic stellate cells will lead to excessive matrix accumulation and liver fibrosis, which is a necessary process for various chronic liver diseases to develop into cirrhosis. Cirrhosis is currently considered to be irreversible, but liver fibrosis has the possibility of reversal, and there is still a lack of safe and effective treatment methods and drugs in the clinic. Activated hepatic stellate cells are a key factor in the process of liver fibrosis, so finding drugs to eliminate activated hepatic stellate cells can provide an important strategy for the clinical treatment of liver fibrosis.

[0003] The Psammaplysene family is a kind of marine alkaloids derived from the Psammaplysilla genus of sponges, which has attracted much attention due to its unique structure and diverse biological activities. The compounds of the family are connected head-to-tail by amide bonds between two dibromotyrosine subunits and contain an α,β-unsaturated double bond. Since 2005, the compounds of the Psammaplysene family have shown various biological activities. Psammaplysene D (PD) was first extracted and discovered in 2007. PD is reported to be able to inhibit the proliferation of human epidermoid carcinoma cell lines (IC 50 = 0.7 μM). It can also inhibit the activity of acetylcholinesterase, with an IC 50 = 1.3 μM. There is no application of the compounds of the Psammaplysene family in preparation of medicines for resisting chronic liver diseases. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide application of Psammaplysene D and derivatives thereof in preparation of medicines for resisting chronic liver diseases.

[0005] To solve the technical problem, the technical scheme adopted by the present application is: In one aspect, the present application provides an application of Psammaplysene D in preparing a product for resisting chronic liver disease, wherein the Psammaplysene D has the following structural formula: .

[0006] Preferably, the chronic liver disease includes hepatitis, liver fibrosis, liver cirrhosis or liver cancer.

[0007] In a second aspect of the present application, a medicine for resisting chronic liver disease is provided, wherein the effective component of the medicine for resisting chronic liver disease includes the Psammaplysene D.

[0008] Preferably, the medicine for resisting chronic liver disease has at least one of the following effects: 1) inhibiting the growth of LX2 cells; 2) reducing the liver function indexes ALT and AST; 3) reducing the inflammatory cell infiltration at the damaged site of the liver; 4) inhibiting the growth of liver cancer cells; 5) combining with the FGFR4 protein and entering the active pocket to form hydrogen bonds.

[0009] In a third aspect of the present application, the application of the Psammaplysene D in preparing an FGFR4 inhibitor medicine is provided.

[0010] In a fourth aspect of the present application, the application of the Psammaplysene D in preparing a medicine for resisting liver cancer together with sorafenib is provided.

[0011] In a fifth aspect of the present application, a medicine for resisting liver cancer is provided, wherein the dose ratio of sorafenib to the Psammaplysene D is 4:1.

[0012] In a sixth aspect of the present application, the application of the Psammaplysene D in preparing a medicine for resisting sorafenib-resistant liver cancer is provided.

[0013] In a seventh aspect of the present application, an application of a Psammaplysene D derivative in preparing a product for resisting chronic liver disease is provided, wherein the Psammaplysene D derivative includes four series of derivatives A, B, C and D, which are named as A1-A27, B1-B33, C1-C25 and D1-D24, and have the following structural formulas, respectively:

[0014]

[0015]

[0016]

[0017]

[0018] .

[0019] As preferred, the Psammaplysene D derivatives are B12 and C11.

[0020] Compared with the prior art, the present application has the beneficial effects that: The present application provides application of Psammaplysene D in preparation of products against chronic liver disease, Psammaplysene D can inhibit the growth of LX2 cells, reduce liver function indexes ALT and AST, and reduce the degree of liver fibrosis in mice; and can inhibit the growth of liver cancer cells in vivo and in vitro, and can produce a synergistic effect with sorafenib; and also shows anti-proliferation ability in sorafenib-resistant liver cancer. The derivative B12 of Psammaplysene D has stronger anti-liver fibrosis effect in vitro, and C11 has very weak inhibition ability on hERG, without potential cardiotoxicity. These results show that Psammaplysene D has strong anti-liver fibrosis, inhibition of liver cancer development, and ability to relieve sorafenib-resistant liver cancer, and Psammaplysene D and C11 can be used as potential lead compounds for development of anti-liver fibrosis and anti-liver cancer drugs, and finally applied in treatment of chronic liver disease. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The present application provides Psammaplysene D to LX2 cells and CCl4-induced liver fibrosis model; wherein, Figure 1 A is a structural schematic diagram of Psammaplysene D; Figure 1 B is a curve graph of MTT method for detecting the growth inhibition of Psammaplysene D on LX2 cells; Figure 1 C is a Western-Blot method for detecting the influence of Psammaplysene D on the expression level of COL1A1 and α-SMA protein in LX2 cells; Figure 1 D is the influence of Psammaplysene D on ALT in CCl4 model; Figure 1E is the effect of Psammaplysene D on AST in CCl4 model; Figure 1 F is the schematic diagram of HE staining of liver tissue in CCl4 model; Figure 1 G is the schematic diagram of Masson staining (upper) and Sirius red staining (lower) of liver tissue in CCl4 model; Figure 2 The result graph of Psammaplysene D provided by the embodiment of the present application is shown in the following figure, wherein, Figure 2 A is the curve graph of MTT method for detecting the growth inhibition of Psammaplysene D on HepG2 cells; Figure 2 B is the curve graph of MTT method for detecting the growth inhibition of Psammaplysene D on Huh7 cells; Figure 2 C is the result of MTT method for detecting the growth inhibition of Psammaplysene D combined with sorafenib on HepG2 cells (left) and the result graph of CI value of Psammaplysene D combined with sorafenib (right); Figure 2 D is the result of MTT method for detecting the growth inhibition of Psammaplysene D combined with sorafenib on Huh7 cells (left) and the result graph of CI value of Psammaplysene D combined with sorafenib (right); Figure 2 E is the schematic diagram of HE staining (upper), Masson staining (middle) and Sirius red staining (lower) of liver tissue in DEN-CCl4 induced mouse hepatoma model; Figure 2 F is the tumor volume line graph of mouse HepG2 cell transplantation tumor model; Figure 2 G is the tumor weight statistical graph of mouse HepG2 cell transplantation tumor model; Figure 3 The result graph of Psammaplysene D provided by the embodiment of the present application is shown in the following figure, wherein, Figure 3 A is the curve graph of MTT method for detecting the growth inhibition of Psammaplysene D on HepG2-SR cells; Figure 3 B is the curve graph of MTT method for detecting the growth inhibition of Psammaplysene D on Huh7-SR cells; Figure 3 C is the tumor volume line graph of mouse HepG2-SR cell transplantation tumor model; Figure 3 D is the tumor weight statistical graph of mouse HepG2-SR cell transplantation tumor model; Figure 4 The target test result graph of Psammaplysene D provided by the embodiment of the present application is shown in the following figure, wherein, Figure 4 A is the data table of reverse targeting; Figure 4B is a graph of the binding ability of Psammaplysene D to FGFR4 detected by SPR method; Figure 4 C is the KD value of the binding ability of Psammaplysene D to FGFR4 detected by SPR method; Figure 4 D is the effect of Psammaplysene D on the thermal stability of FGFR4 detected by CETSA method; Figure 4 E is a schematic diagram of the binding site of Psammaplysene D to FGFR4 detected by molecular docking method; Figure 5 A1-A15 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 6 A16-A27 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 7 B1-B18 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 8 B19-B33 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 9 C1-C12 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 10 C13-C25 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 11 D1-D12 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 12 D13-D24 are structural schematic diagrams of derivatives of Psammaplysene D provided by the embodiments of the present application; Figure 13 B12 is the effect of derivative of Psammaplysene D on LX2 cells and CCl4-induced liver fibrosis model provided by the embodiments of the present application; wherein, Figure 13 A is a structural schematic diagram of derivative B12; Figure 13 B is a curve graph of derivative B12 on the growth inhibition of LX2 cells detected by MTT method; Figure 13 C is a schematic diagram of HE staining (top), Masson staining (middle) and Sirius red staining (bottom) of liver tissue in CCl4-induced mouse liver fibrosis model; Figure 14The influence of the derivative C11 of Psammaplysene D provided by the embodiment of the present application on LX2 cells and a CCl4-induced liver fibrosis model and toxicity detection; wherein, Figure 14 A is a structural schematic diagram of the derivative C11; Figure 14 B is a curve diagram of the MTT method for detecting the growth inhibition of derivative C11 on LX2 cells; Figure 14 C is a result diagram of the inhibition ability of C11 on hERG; Figure 14 D is a schematic diagram of HE staining (top), Masson staining (middle) and Sirius red staining (bottom) of liver tissues in a CCl4-induced mouse liver fibrosis model. DETAILED DESCRIPTION

[0022] The technical solutions in the specific embodiments of the present application will be described in detail and completely below with reference to the drawings. Obviously, the described embodiments are only part of the specific embodiments of the general technical solution of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the general concept of the present application fall within the scope of protection of the present application.

[0023] In one aspect, the present application provides use of Psammaplysene D in the preparation of a product for treating chronic liver disease, wherein Psammaplysene D has the following structural formula (I): Figure 1 A): .

[0024] In a preferred embodiment, the chronic liver disease includes hepatitis, liver fibrosis, liver cirrhosis or liver cancer.

[0025] In this study, we first used continuously activated LX2 cells and a CCl4-induced mouse liver fibrosis model to explore whether the marine-derived compound Psammaplysene D (PD) has the ability to resist liver fibrosis. The results showed that PD can inhibit the growth of LX2 cells, reduce liver function indicators ALT and AST, alleviate the cell vacuoles caused by CCl4, and at the same time reduce the inflammatory cell infiltration at the damage site; HE, Masson and Sirius red staining showed that PD can reduce the degree of liver fibrosis in mice.

[0026] It should be noted that if the development of liver fibrosis is not intervened, it will gradually progress to liver cirrhosis and finally evolve into liver cancer. Therefore, next, we verified the anti-liver cancer ability of PD. The results showed that PD can inhibit the growth of liver cancer cells in vitro and in vivo.

[0027] In another aspect, the present application provides an anti-chronic liver disease drug, wherein the effective component of the anti-chronic liver disease drug includes the above-mentioned Psammaplysene D.

[0028] The dosage form of the drug is selected from one of a tablet, a powder, a granule, a capsule, an oral liquid, a needle, or a sustained-release agent.

[0029] In a preferred embodiment, the drug for treating chronic liver disease has at least one of the following effects: 1) inhibiting the growth of LX2 cells; 2) reducing liver function indicators ALT and AST; 3) reducing inflammatory cell infiltration at the site of liver damage; 4) inhibiting the growth of liver cancer cells; 5) binding to FGFR4 protein and forming hydrogen bonds in its active pocket.

[0030] In another aspect of the present application, the use of the above-mentioned Psammaplysene D in the preparation of an FGFR4 inhibitor drug is provided.

[0031] In terms of target points, we use chemical biology reverse targeting, SPR, CETSA and molecular docking technology to determine that FGFR4 is the target point of PD, PD can form hydrogen bond interaction with amino acid sites such as Cys552 of FGFR4, and can enhance the thermal stability of FGFR4.

[0032] In another aspect of the present application, the use of the above-mentioned Psammaplysene D in the preparation of an anti-liver cancer drug with sorafenib is provided.

[0033] In another aspect of the present application, an anti-liver cancer drug is provided, and the dose ratio of sorafenib to the above-mentioned Psammaplysene D in the anti-liver cancer drug is 4:1.

[0034] The present application further explores whether PD can produce a synergistic effect with sorafenib in the process of resisting liver cancer. As shown in Figure 2 C and D, when the dose ratio of sorafenib to PD is 4:1, the two drugs produce a synergistic effect in inhibiting the growth of liver cancer cell lines. This shows that PD has an anti-liver cancer effect in vitro and can produce a synergistic effect with sorafenib.

[0035] In another aspect of the present application, the use of the above-mentioned Psammaplysene D in the preparation of an anti-liver cancer drug resistant to sorafenib is provided.

[0036] It should be noted that sorafenib as a first-line anti-cancer drug has been showing resistance in recent years, and there is also a phenomenon of sorafenib resistance in liver cancer patients. Next, we verified whether PD can inhibit the growth of sorafenib-resistant liver cancer cells.

[0037] The present application uses a mouse subcutaneous xenograft tumor model to verify the in vivo efficacy, and the results are as followsFigure 3 As shown in C and D, sorafenib did not exhibit a significant antitumor effect, indicating that HepG2-SR cells remain resistant in vivo. PD significantly inhibited the growth of sorafenib-resistant tumors, with comparable effects at 5 mg / kg and 10 mg / kg. There was no significant difference in tumor volume and weight between the two PD treatment groups. These results demonstrate that PD possesses good anti-sorafenib-resistant liver cancer capabilities both in vitro and in vivo.

[0038] In another aspect, this invention provides the application of the above-mentioned Psammaplysene D derivatives in the preparation of products for treating chronic liver diseases. The Psammaplysene D derivatives include four series of derivatives: A, B, C, and D, named A1-A27 (e.g., ...). Figure 5 and 6 As shown), B1-B33 (as shown) Figure 7 and 8 As shown), C1-C25 (as shown) Figure 9 and 10 (as shown) and D1-D24 (as shown) Figure 11 and 12 (As shown).

[0039] After synthesizing four series of derivatives, their in vitro anti-liver fibrosis ability was further verified. LX2 cells were treated with 0.00084 μM, 0.0025 μM, 0.0076 μM, 0.023 μM, 0.069 μM, 0.206 μM, 0.617 μM, 1.85 μM, 5.56 μM, 16.67 μM, 50 μM, and 150 μM of the derivatives for 24 h, respectively. Cell viability was detected by the MTT assay, and the results are shown in Table 1. All four series of derivatives showed in vitro anti-liver fibrosis ability.

[0040] In a preferred embodiment, the Psammaplysene D derivative is B12 and C11.

[0041] Derivatives B12 and C11 are the most representative, with B12 exhibiting the strongest in vitro activity at 0.055 μM. Compared to PD, derivative C11 shows very weak inhibition of hERG and no potential cardiotoxicity. Both derivatives B12 and C11 demonstrated anti-liver fibrosis activity in in vivo models. These results indicate that PD possesses strong anti-liver fibrosis properties, inhibits liver cancer development, and can alleviate sorafenib-resistant liver cancer. Its derivative C11 has no cardiotoxicity. PD and C11 can serve as promising lead compounds for the development of anti-liver fibrosis and anti-liver cancer drugs, ultimately for the treatment of chronic liver diseases.

[0042] In order to more clearly and specifically introduce the application of Psammaplysene D and its derivatives in the preparation of drugs for resisting chronic liver diseases, the following will be described in combination with specific examples.

[0043] Example 1: Test of the anti-hepatic fibrosis ability of Psammaplysene D 1. Establishment of an in-vitro hepatic fibrosis activity screening model: LX2 cells were continuously stimulated by TGF-β1 to obtain continuously activated LX2 cells. The specific method was as follows: 10 ng / mL of TGF-β1 was used to continuously stimulate LX2 cells for 1 week, and the mRNA levels of COL1A1 and ACTA2 were detected by RT-qPCR experiments. When the levels were significantly increased (fold>2), it was indicated that the construction was successful.

[0044] 2. Detection of the in-vitro activity of PD by MTT method: LX2 cells were treated with PD at concentrations of 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM and 160 μM for 24 h. The results are shown in Figure 1 B, the IC 50 of PD on LX2 cells was 2.75 μM.

[0045] 3. Effect of PD on fibrosis markers in LX2 cells: The effect of PD on fibrosis markers in LX2 cells was detected by western blot experiments. The results are shown in Figure 1 C, PD significantly inhibited the expression of Collagen Type I Alpha 1 Chain (COL1A1) and Alpha Smooth Muscle Actin (α-SMA) at doses of 5 μM and 10 μM.

[0046] 4. Test of the in-vivo anti-hepatic fibrosis ability of PD: After verifying the in-vitro efficacy of PD, the in-vivo anti-hepatic fibrosis effect of PD was detected by using a CCl4-induced mouse hepatic fibrosis model.

[0047] 6-week-old C57BL / 6 male mice were selected, and CCl4 (25%) dissolved in olive oil was injected intraperitoneally, twice a week, for six weeks. The control group was injected with the same dose of control solvent. Seven mice were selected in each group, and each test data was obtained by randomly selecting 5 mice in each group.

[0048] Treatment (2.5 and 5 mg / kg) was started four weeks after injection and was given intraperitoneally once a day until the end of the experiment. As Figure 1 D and E, the liver function indicators ALT and AST decreased after treatment. As Figure 1 F, the HE results showed that PD (2.5 and 5 mg / kg) treatment alleviated the cell vacuoles caused by CCl4 and reduced the inflammatory cell infiltration at the damage site. MASSON and Sirius red were used to stain the collagen deposited in the liver. The results showed that the CCl4 model group had more collagen deposition and the bridging between blood vessels was very obvious. PD (2.5 and 5 mg / kg) treatment reduced collagen deposition and significantly reduced the degree of bridging (as shown in Figure 1 F and G). These results showed that PD had strong anti-liver fibrosis ability.

[0049] Example 2: Test of the anti-liver cancer ability of Psammaplysene D 1. Test of the inhibitory activity of PD on liver cancer cell lines HepG2 and Huh7 cells by MTT method: Liver cancer cell lines HepG2 and Huh7 cells were treated with PD at concentrations of 0.023 μM, 0.069 μM, 0.206 μM, 0.617 μM, 1.85 μM, 5.56 μM, 16.67 μM, 50 μM and 150 μM, respectively, for 48 h. The results are shown in Figure 2 A and B. PD can inhibit the growth of the two cells. The IC 50 of PD on HepG2 cells was 1.36 μM and the IC 50 of PD on Huh7 cells was 0.64 μM. This showed that PD had certain anti-liver cancer ability in vitro.

[0050] 2. Test of the synergistic effect of PD and sorafenib in the process of anti-liver cancer: As shown in Figure 2C and D, when the cells were treated with sorafenib and PD at a dose ratio of 4:1 for 48 hours, the dose of PD used for HepG2 cells was 1 μM, 2 μM, 3 μM and 4 μM, the dose of sorafenib was 4 μM, 8 μM, 12 μM and 16 μM, the dose of PD used for Huh7 cells was 0.5 μM, 1 μM, 1.5 μM and 2 μM, the dose of sorafenib was 2 μM, 4 μM, 6 μM and 8 μM, the results showed that, compared with the use of the two compounds alone, the combined use significantly inhibited the tumor cell viability. Combination index (CI) was analyzed by Chou-Talalay algorithm method. The CI of the evaluated combination showed that synergy, additivity and antagonism were defined as CI <1, CI =1 and CI >1, respectively. At the same time, the CI value was calculated by Chou-Talalay method and CompuSyn software (CI <1 represents synergy, CI =1 represents additivity, and CI >1 represents antagonism), and the results showed that, when PD and sorafenib were used in combination, there was a concentration ratio of CI <1, indicating that PD combined with sorafenib had synergistic effect on inhibiting tumor cell viability. The two drugs produced a synergistic effect in inhibiting the growth of hepatocarcinoma cell lines. This indicates that PD has an anti-hepatocarcinoma effect in vitro and can produce a synergistic effect with sorafenib.

[0051] 3. Test of the anti-hepatocarcinoma ability of PD in vivo: A chronic hepatocarcinoma model was used: 2-week-old C57BL / 6 male mice were injected intraperitoneally with DEN (25 mg / kg) once, and at the age of 6 weeks, CCl4 (25%) was injected intraperitoneally twice a week, continuously until the age of 18 weeks. 7 mice were selected in each group, and the test data were obtained by randomly selecting 5 mice in each group.

[0052] Drug treatment was started at the age of 14 weeks, and PD (10 mg / kg) and sorafenib (10 mg / kg) were injected intraperitoneally, respectively. The results are shown in Figure 2 E, it can be found from the HE results that PD significantly inhibited the development of tumors on the liver. The results of MASSON and picro-sirius red staining showed that collagen deposition was reduced after PD administration.

[0053] In addition, we also used a mouse subcutaneous xenograft tumor model for verification, and HepG2 cells in good growth condition were injected subcutaneously into 6-week-old BALB / c nude mice at a concentration of 5×10 6 The mice were divided into groups according to the tumor size, and 7 mice were selected in each group. The test data were obtained by randomly selecting 6 mice in each group.

[0054] A blank group, a sorafenib 10 mg / kg group, a PD-5 mg / kg group and a PD-10 mg / kg group were set up, respectively.

[0055] Results are shown in Figure 2 F and G, PD at 10 mg / kg can significantly inhibit the growth of tumor, and is equivalent to the effect of sorafenib at 10 mg / kg. This shows that PD can inhibit the growth of liver cancer cells in vivo.

[0056] Example 3: Test of Psammaplysene D's ability to resist sorafenib-resistant liver cancer 1. In vitro test: The in vitro sorafenib-resistant cell lines HepG2 and Huh7 are selected, which are referred to as HepG2-SR and Huh7-SR, respectively. The construction method is to use a concentration gradient (0.5 μM, 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM and 10 μM) of sorafenib to culture for three months to obtain sorafenib-resistant strains. The two resistant cells are treated with PD at concentrations of 0.023 μM, 0.069 μM, 0.206 μM, 0.617 μM, 1.85 μM, 5.56 μM, 16.67 μM, 50 μM and 150 μM for 48 h.

[0057] Results are shown in Figure 3 A and B, PD can inhibit the growth of two sorafenib-resistant liver cancer cells, the IC 50 of HepG2-SR cells is 5.11 μM, and the IC 50 of Huh7-SR cells is 1.75 μM. Then the in vivo efficacy is verified by using a mouse subcutaneous xenograft tumor model. The well-grown HepG2-SR cells are injected subcutaneously into 6-week-old BALB / c nude mice at a concentration of 5×10 6 The mice are divided into groups according to the tumor size. Each group has 7 mice, and each test data is obtained by randomly selecting 6 mice from each group.

[0058] The blank group, sorafenib 10 mg / kg group, PD-5 mg / kg group and PD-10 mg / kg group are set up. The results are shown in Figure 3 C and D, sorafenib does not show obvious anti-tumor effect, which shows that HepG2-SR cells still have drug resistance in vivo, PD can significantly inhibit the growth of sorafenib-resistant tumors, and the effects of 5 mg / kg and 10 mg / kg are equivalent. There is no significant difference in tumor volume and weight between the two PD treatment groups. The above results show that PD has good anti-sorafenib-resistant liver cancer ability in vivo and in vitro.

[0059] Example 4: Test of Psammaplysene D's target 1. The method for capturing the protein combined with PD by using biochemical reverse targeting: the biotin-labeled PD compound is fixed on the streptomycin-labeled magnetic beads as a stationary phase, and the total protein of the activated hepatic stellate cell line LX2 is used as a mobile phase to realize the reverse capture of the compound target protein, and the mass spectrometry-based proteomics analysis technology is used to confirm the target protein of PD in LX2 cells.

[0060] The results are shown in Figure 4 A, the proteins capable of being combined with PD include MDM4, GDF6, FGFR4, TGFβ1 and BMP7, etc.

[0061] Among them, FGFR4 and TGFβ1 are closely related to the occurrence and development of liver fibrosis, and because the score of FGFR4 is higher than that of TGFβ1, it is further verified whether PD can be combined with FGFR4.

[0062] 2. SPR experiment: The SPR experiment is used to verify the combination of PD and FGFR4, and 0.78 μM, 1.56 μM, 3.12 μM, 6.25 μM, 12.5 μM and 25 μM of PD are used to flow through the CM5 chip embedded with FGFR4 protein, and the instrument is detected and analyzed.

[0063] The results are shown in Figure 4 B and C, PD can be combined with FGFR4, and the KD value of the combination is 1.229 x 10 -5 M.

[0064] 3. CETSA experiment: Next, the CETSA experiment is used to verify the influence of PD on the thermal stability of FGFR4 protein, the LX2 cells in good growth condition are collected into a 1.5 mL centrifuge tube, resuspended with PBS, rapidly frozen in liquid nitrogen, then melted in a 37°C water bath, and the freezing and thawing is repeated for three times, the supernatant is centrifuged and divided into two parts, one part is added with 20 μM of PD, and the other part is added with the same volume of DMSO, and is placed in a 37°C incubator for two hours. After two hours, the PD group and the DMSO group are divided into six parts, respectively, and heated by using a PCR instrument, and the temperature gradient is set to 45°C, 52.6°C, 60.9°C, 66°C, 72°C and 80.9°C, and heated for three minutes, then rapidly placed on ice for cooling, and after cooling, the supernatant is centrifuged and added with loading buffer for western blot detection.

[0065] The results are shown in Figure 4 D, PD can enhance the thermal stability of FGFR4, which indicates that it can be combined with FGFR4. Next, the MOE software is used for molecular docking to simulate the binding site of PD and FGFR4 (PDB: 6jpe). The results are shown in Figure 4As shown in E, PD can form hydrogen bond interactions with Val481, Cys552, Gly556, Asn557 and Asp630 residues of the FGFR4 active pocket. The above binding description shows that PD can bind to FGFR4 and enter its active pocket to form hydrogen bonds.

[0066] Example 5: Test of anti-hepatic fibrosis ability of Psammaplysene D derivatives 1. Synthesis of derivatives and test of anti-hepatic fibrosis ability in vitro: After the in vivo and in vitro efficacy and target of PD are disclosed, we design and synthesize derivatives based on the structure of PD (A5), and synthesize four series of derivatives A, B, C and D, including 109 in total. Among them, there are 27 in series A, named A1-A27 (as shown in Figure 5 and 6 ). There are 33 derivatives in series B, named B1-B33 (as shown in Figure 7 and 8 ). The derivatives in series C include C1-C25 (as shown in Figure 9 and 10 ). The derivatives in series D are named D1-D24, a total of 24 (as shown in Figure 11 and 12 ).

[0067] After the four series of derivatives are synthesized, the in vitro anti-hepatic fibrosis ability of these derivatives is further verified. LX2 cells are treated with 0.00084 μM, 0.0025 μM, 0.0076 μM, 0.023 μM, 0.069 μM, 0.206 μM, 0.617 μM, 1.85 μM, 5.56 μM, 16.67 μM, 50 μM and 150 μM of derivatives for 24 h, and the cell survival is detected by MTT method, and the results are shown in Table 1. The four series of derivatives all have in vitro anti-hepatic fibrosis ability.

[0068] Table 1 Inhibitory effect of different derivatives on LX2 cells

[0069] 2. Performance test of representative derivatives B12 and C11: 2.1 Test of in vitro anti-hepatic fibrosis ability of derivative B12: We selected two representative compounds B12 and C11 from the 109 PD derivatives to further verify the in vivo and in vitro anti-hepatic fibrosis ability. First, we detected B12 (as shown in Figure 13The in vitro and in vivo efficacy of A) was tested by treating LX2 cells with 0.00084 μM, 0.0025 μM, 0.0076 μM, 0.023 μM, 0.069 μM, 0.206 μM, 0.617 μM, 1.85 μM, 5.56 μM, 16.67 μM, 50 μM and 150 μM of B12 for 24 h, and the results are shown in Figure 13 B. B12 was able to inhibit the proliferation of LX2 cells with an IC 50 of 0.055 μM.

[0070] Next, the in vivo anti-hepatic fibrosis ability of B12 was tested in a CCl4-induced mouse model of hepatic fibrosis, and the model was constructed in the same way as in Figure 1 the method for verifying the in vivo efficacy of PD. The results are shown in Figure 13 C. After B12 (5 and 10 mg / kg) administration, the inflammatory cell infiltration at the injury site was reduced. By MASSON and Sirius red staining of the collagen deposited in the liver, it was found that after B12 (5 and 10 mg / kg) administration, the collagen deposition was reduced. These results indicate that B12 has a strong anti-hepatic fibrosis ability.

[0071] 2.2 In vitro anti-hepatic fibrosis ability test of derivative C11: LX2 cells were treated with 0.046 μM, 0.137 μM, 0.412 μM, 1.23 μM, 3.7 μM, 11.1 μM, 33.3 μM, 100 μM and 300 μM of C11 for 24 h, and the results are shown in Figure 14 B. C11 was able to inhibit the proliferation of LX2 cells with an IC 50 of 12.61 μM. Before verifying the in vivo efficacy, we tested the hERG toxicity of PD and C11 using electrophysiological patch clamp technology with terfenadine as a positive reference. The results are shown in Figure 14 C. The IC 50 of hERG of terfenadine at 1 μM was 87.56%, the IC 50 of hERG of PD at 50 μM was 92.81%, and the IC 50 of hERG of C11 at 100 μM was 44.32%, which indicates that C11 has no potential cardiotoxicity.

[0072] Next, the in vivo anti-hepatic fibrosis ability of C11 was tested in a CCl4-induced mouse model of hepatic fibrosis, and the model was constructed in the same way as in Figure 1 the method for verifying the in vivo efficacy of PD, and PD and sorafenib were used as control drugs. The results are shown in Figure 14As shown in FIG. D, C11 (5 and 10 mg / kg) treatment reduced inflammatory cell infiltration at the injury site. MASSON and picrosirius staining of collagen deposition in the liver showed that C11 (5 and 10 mg / kg) treatment reduced collagen deposition, and the effect of C11 at 10 mg / kg was comparable to that of PD. These results indicate that C11 has the ability to resist liver fibrosis and has no cardiotoxicity.

[0073] These results show that PD not only can inhibit the development of liver fibrosis and liver cancer, but also has the ability to relieve liver cancer with sorafenib resistance. Its derivative B12 has a stronger in vitro anti-liver fibrosis effect, and can still effectively relieve liver fibrosis in vivo. Compared with PD, the derivative C11 has very weak inhibition on hERG and has no potential cardiotoxicity. PD and C11 can be used as potential lead compounds for the development of anti-liver fibrosis and anti-liver cancer drugs, and ultimately applied to the treatment of chronic liver disease.

Claims

1. Use of psammaplysene D for the preparation of a product against chronic liver disease, characterized in that, The Psammaplysene D has the following structural formula: 。 2. Use according to claim 1, characterized in that, The chronic liver disease includes hepatitis, liver fibrosis, liver cirrhosis or liver cancer.

3. A medicament for the treatment of chronic liver disease, characterized in that, The effective component of the drug for resisting chronic liver disease includes the Psammaplysene D of claim 1.

4. The medicament according to claim 3, characterized in that, The drug for resisting chronic liver disease has at least one of the following effects: 1) Inhibiting the growth of LX2 cells; 2) Reducing the liver function indexes ALT and AST; 3) Reducing the inflammatory cell infiltration in the damaged part of liver; 4) Inhibiting the growth of liver cancer cells; 5) Binding with FGFR4 protein and entering the active pocket to form hydrogen bond.

5. The use of the Psammaplysene D of claim 1 in the preparation of FGFR4 inhibitor drug.

6. The use of the Psammaplysene D of claim 1 in the preparation of anti-liver cancer drug together with sorafenib.

7. A medicament for preventing liver cancer, characterized by comprising the compound according to claim 1. In the anti-liver cancer drug, the dose ratio of sorafenib to the Psammaplysene D is 4:

1.

8. The use of the Psammaplysene D of claim 1 in the preparation of drug for resisting sorafenib-resistant liver cancer.

9. Use of the Psammaplysene D derivative according to claim 1 for the preparation of a product against chronic liver diseases, characterized in that, The Psammaplysene D derivatives include four series of derivatives A, B, C and D, named A1-A27, B1-B33, C1-C25 and D1-D24, respectively having the following structural formula: 。 10. Use according to claim 9, characterized in that, The Psammaplysene D derivative is B12 and C11.

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