Coumarin compound as well as preparation method and medicinal composition thereof

Abstract

The invention discloses a coumarin compound as well as a preparation method and a medicinal composition thereof. Compared with the prior art, the coumarin compound shown in the formula (I) can inhibit the interaction of lncRNA-PSF and increase the expression of p53 protein so as to change the cell cycle, has a sensitization effect on an immune checkpoint inhibitor, improves the treatment effect of the immune checkpoint inhibitor on tolerant tumors, and has very good popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine production, and particularly provides a coumarin compound, a preparation method therefor and a pharmaceutical composition. BACKGROUND

[0002] Colorectal cancer (CRC) is a highly prevalent malignant tumor. At present, the clinical treatment of colorectal cancer is still mainly surgery, chemotherapy and radiotherapy, but the prognosis of patients with advanced and metastatic CRC is poor, and traditional therapy is often accompanied by obvious toxic side effects. In recent years, targeted therapy and immunotherapy have made important breakthroughs in this field, and a large number of studies have focused on targeted therapy for driver gene mutations (such as EGFR, VEGF, BRAF, KRAS, MEK) and immunotherapy checkpoint inhibitors and their combination therapy strategies. However, the gene mutations of different signaling pathways in tumor cells and the immune escape mechanisms mediated thereby often lead to recurrence, metastasis and treatment resistance of colorectal cancer. Therefore, in-depth elucidation of the molecular mechanisms of drug resistance and development of new targeted drugs or immunomodulators that can effectively overcome drug resistance, improve efficacy and reduce toxic side effects have become the core direction of future research on colorectal cancer.

[0003] Immune checkpoint inhibitors (ICIs), such as PD-1 / PD-L1 inhibitors, have made a breakthrough in the treatment of tumors, but their application in the treatment of colorectal cancer (CRC) still has obvious limitations. At present, the efficacy of ICIs is mainly concentrated in a small number of CRC patients with specific biomarkers (such as microsatellite instability / high mismatch repair deficiency, i.e. MSI-H / dMMR), while the vast majority of microsatellite stable / mismatch repair normal (MSS / pMMR) patients have poor response to them, which constitutes the main bottleneck of current clinical application. Behind this difference in efficacy is the complex tumor immune microenvironment of MSS type colorectal cancer, which is usually characterized by insufficient T cell infiltration and coexistence of multiple immunosuppression mechanisms, leading to it becoming a "cold tumor" that is not sensitive to immunotherapy.

[0004] In addition to the limited efficiency, immune checkpoint inhibitors also face the challenge of primary or secondary drug resistance. Even in some patients who initially respond, the tumor may develop secondary drug resistance through upregulation of other inhibitory checkpoints (such as LAG-3, TIM-3) or changes in antigen presentation mechanisms, etc. In addition, immune-related adverse events are also a problem that cannot be ignored. These toxicities caused by excessive activation of the immune system can involve multiple organ systems, and can be life-threatening in severe cases, and the use of high doses of immunosuppressive agents to deal with some refractory toxicities can also affect the anti-tumor efficacy.

[0005] As the guardian of the genome, p53 plays a central role in maintaining cellular homeostasis and preventing malignant transformation. In colorectal cancer, the mutation rate of TP53 (the gene encoding p53 protein) is extremely high, especially in the stage of late adenoma to carcinoma transformation, accounting for about 50-60% of cases. Most p53 mutations are missense mutations, which not only lose the tumor suppressor function of wild-type p53, but also often acquire new oncogenic functions, i.e. gain-of-function mutations. This mutant p53 can promote tumor cell proliferation, invasion, metastasis, and resistance to conventional chemotherapy and radiotherapy.

[0006] In recent years, studies have further revealed the close relationship between p53 status and tumor immune microenvironment. Wild-type p53 can enhance anti-tumor immunity by up-regulating the expression of multiple immune-related molecules. However, mutant p53 not only loses these functions, but also actively shapes an inhibitory immune microenvironment. For example, it can help tumor cells achieve immune escape by up-regulating PD-L1 expression, recruiting myeloid-derived suppressor cells, etc. This makes p53 mutant tumors often exhibit the characteristics of "immune cold tumors", i.e. lack of T cell infiltration, which directly explains why such tumors are not sensitive to single-agent treatment with immune checkpoint inhibitors.

[0007] Targeting the p53 pathway, especially the oncogenic function of mutant p53, has become a promising direction in the field of colorectal cancer treatment. Therefore, it is urgent to find a way to enhance the role of p53 to overcome the resistance of immune checkpoint inhibitors and enhance the sensitivity of colorectal cancer to them. SUMMARY

[0008] The present application is aimed at the deficiencies of the prior art, and provides a coumarin compound capable of inhibiting lncRNA-PSF interaction and activating p53.

[0009] The technical scheme adopted by the present application to solve its technical problems is: a coumarin compound represented by structural formula (I) or a pharmaceutical salt thereof, .

[0010] The compound represented by structural formula (I) is named: 4-((4-(4-(tert-butyl) benzoyl) piperazin-1-yl) methyl)-7,8-dihydroxy-2H-chromen-2-one.

[0011] The present application further provides a preparation method of the coumarin compound represented by structural formula (I).

[0012] The synthesis route of the coumarin compound represented by structural formula (I) of the present application is: .

[0013] As preferred, the preparation method of the coumarin compound of the present application comprises: S1. taking pyrogallic acid and ethyl 4-chloroacetoacetate as raw materials, and obtaining intermediate compound 1a through Pechmann condensation reaction; S2. taking 1-Boc-piperazine and 4-tert-butyl benzoyl chloride as raw materials, and obtaining intermediate compound 2a through nucleophilic substitution reaction, and obtaining intermediate compound 3a after removing Boc; S3. obtaining the target compound shown in formula (I) through nucleophilic substitution reaction of intermediate compound 1a and intermediate compound 3a.

[0014] As preferred, step S1 comprises: S11. mixing appropriate amount of H2SO4 and CF3COOH at -5℃ to 1℃, and stirring uniformly; S12. adding anhydrous dichloromethane, ethyl 4-chloroacetoacetate and pyrogallic acid into the reaction system in sequence, and increasing the temperature to room temperature; S13. after reacting for 0.5 to 2 hours, adding methanol, and continuing to react until the reaction is completed; S14. obtaining the reaction product through purification to obtain intermediate compound 1a.

[0015] As preferred, step S2 comprises: S21. dissolving 1-Boc-piperazine and triethylamine in dichloromethane; S22. adding appropriate amount of 4-tert-butyl benzoyl chloride drop by drop under N2 protection at -5℃ to 1℃; S23. after the reaction is completed at room temperature, obtaining the reaction product through purification to obtain intermediate compound 2a; S24. adding appropriate amount of trifluoroacetic acid into the dichloromethane solution of intermediate compound 2a at -5℃ to 1℃, and reacting until the reaction is completed at room temperature; S25. obtaining the reaction product through purification to obtain intermediate compound 3a.

[0016] As preferred, step S3 comprises: S31. adding appropriate amount of triethylamine into the acetone solution of intermediate compound 3a under N2 protection; S32. cooling to -5℃ to 1℃, and adding the acetone solution of intermediate compound 1a into the reaction system under N2 protection; S33. increasing the temperature to 45℃ to 55℃, and reacting until the reaction is completed under stirring; S34. obtaining the target compound shown in formula (I) through purification of the reaction product.

[0017] Still further technical task of the present application is to provide an epigenetic drug capable of inhibiting LncRNA-PSF interaction, comprising a coumarin compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof.

[0018] Still further technical task of the present application is to provide a pharmaceutical composition for synergistically treating cancer, characterized by comprising an immune checkpoint inhibitor and a coumarin compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof, which can improve the anti-tumor effect.

[0019] As a preferred, the immune checkpoint inhibitor is a PD-L1 inhibitor, such as CA-170.

[0020]

[0021] As a preferred, the molar ratio of the immune checkpoint inhibitor to the coumarin compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof is 1:1-1:3, for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, etc. In particular, when the pharmaceutical composition is used for preparing a drug for treating colorectal cancer, the molar ratio of the immune checkpoint inhibitor to the coumarin compound represented by structural formula (I) or a pharmaceutically acceptable salt thereof is preferably 1:2-1:3.

[0022] The epigenetic drug and the pharmaceutical composition for synergistically treating cancer of the present application further comprises a pharmaceutically acceptable carrier. The carrier comprises diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. which are conventional in the pharmaceutical field.

[0023] The epigenetic drug and the pharmaceutical composition for synergistically treating cancer can be used in oral or non-oral forms, such as being introduced into the body by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as intramuscular, intradermal, subcutaneous, intravenous, mucosal tissue; or being mixed or wrapped with other substances and then introduced into the body.

[0024] For oral use, it can be made into conventional solid preparations, such as tablets, powders, granules, capsules, ointments, creams, etc.; or liquid preparations, such as aqueous or oily suspensions or other liquid preparations, such as oral liquids, etc. For parenteral administration, it can be made into injection solutions, aqueous or oily suspensions, etc.

[0025] Compared with the prior art, the coumarin compound, the preparation method and the pharmaceutical composition of the present application have the following outstanding advantages: (1) The coumarin compound of the present application can inhibit the interaction of lncRNA-PSF, increase the expression of p53 protein to change the cell cycle, block the cancer cells in the G2 / M phase, and affect the synthesis of RNA and protein, which can be used for preparing a drug for treating cancer; (ii) The coumarin compounds of the present invention have a sensitizing effect on immune checkpoint inhibitors, significantly increasing the sensitivity of immune checkpoint inhibitors to tumors and enhancing the therapeutic effect of immune checkpoint inhibitors on resistant tumors. In colorectal cancer, they can significantly reduce the IC50 of immune checkpoint inhibitors. 50 This leads to better anti-tumor effects. Attached Figure Description

[0026] Appendix Figure 1 It is the compound of formula (I) obtained in Example 1. 1 H NMR spectrum; Appendix Figure 2 It is the compound of formula (I) obtained in Example 1. 13 C NMR spectrum; Appendix Figure 3 This is the HR-MS spectrum of the compound of formula (I) obtained in Example 1; Appendix Figure 4 These are the results of an RNA-pulldown experiment: Western blot protein band diagram. Appendix Figure 5 The effects of the compound shown in formula (I) and its lead compound 10-3 on the cell cycle of HCT116 cells; where blue represents G0 / G1 phase, green represents S phase, and red represents G2 / M phase. Appendix Figure 6 The effect of the compound shown in formula (I) and the lead compound 10-3 on the expression level of p53 in HCT116 cells; Appendix Figure 7 This is a diagram illustrating the antitumor effects of the compound shown in formula (I), immune checkpoint inhibitors, and combination therapy. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the present invention.

[0028] Unless otherwise specified, the contents of each component used below are by mass percentage; all experimental methods used are conventional methods; and all reagents and biological materials used are commercially available.

[0029]

Preparation Examples

[0030] The synthetic route of the coumarin compounds shown in structural formula (I) in this embodiment:

[0031] wherein: (a): H2SO4, CF3COOH, DCM, 0 °C - rt, 6 h; (b): Triethylamine, N2, DCM, rt, overnight; (c): DCM, TFA, rt, 1 h; (d): Triethylamine, Acetone, N2, 0 °C - 50 °C, overnight.

[0032] The specific preparation method is as follows: a. 1.2 g (12.2 mmol) of H2SO4 and 1.39 g (12.2 mmol) of CF3COOH were respectively added into 100 mL single-mouth bottles at 0 °C and stirred uniformly. Then, 50 mL of anhydrous dichloromethane, 1.0 g (6.1 mmol) of 4-chloroacetoacetate and 923 mg (7.32 mmol) of pyrogallic acid were sequentially added into the reaction system, and slowly warmed to room temperature. After 1 hour of reaction, 11 mL of methanol was added. The reaction was continuously reacted for 5 hours, and TLC was used to monitor the completion of the reaction. The solvent was evaporated under reduced pressure, and the residue was slowly poured into 500 mL of distilled water, and a solid was precipitated. Filtration and drying were performed to obtain 1.34 g of a pink solid (intermediate compound 1a), with a yield of 97%, which can be directly used for the next step; b. In a 100 mL three-necked round-bottom flask, 1.0 g (5.4 mmol) of 1-Boc-piperazine and 655 mg (6.48 mmol) of triethylamine were dissolved in 20 mL of dichloromethane. The flask was cooled to 0 °C, and 942 mg (5.94 mmol) of 4-tert-butylbenzoyl chloride was added dropwise under N2 protection. The resulting mixture was stirred at room temperature overnight. TLC was used to monitor the completion of the reaction, and then water was added, and the product was extracted with dichloromethane (3×15 mL). The organic layers were combined, washed with saturated aqueous sodium chloride solution (2×45 mL), dried over anhydrous sodium sulfate (Na2SO4), and then the solvent was removed by rotary evaporation. The concentrate was purified by column chromatography (eluent: ethyl acetate / petroleum ether = 1:5, v / v) to obtain 1.5 g of a white solid (intermediate compound 2a), with a yield of 90.1%; c. 1.0 g (3.25 mol) of intermediate compound 2a was dissolved in 15 mL of DCM, 5 mL of trifluoroacetic acid (TFA) was added at 0 °C under stirring, and stirred at room temperature for 1 hour. TLC monitoring showed that the reaction was complete, the solvent was evaporated under reduced pressure, the oily residue was dissolved in 10 mL of DCM, and treated with saturated aqueous NaHCO3 solution, the organic layer was separated, and the aqueous phase was extracted with DCM (2 x 20 mL). The combined extract was washed with saturated aqueous sodium chloride solution (2 x 50 mL), dried over anhydrous sodium sulfate (Na2SO4), and concentrated under vacuum to obtain 558 mg of yellow oily product (intermediate compound 3a), with a yield of 83%; d. In a 100 mL three-necked round-bottom flask, 558 mg (2.68 mmol) of intermediate compound 3a was dissolved in 10 mL of acetone, protected by N2, and added with 813 mg (8.04 mmol) of triethylamine under stirring. The flask was cooled to 0 °C, and a solution of 668 mg (2.95 mmol) of intermediate compound 1a in 10 mL of acetone was added dropwise slowly under N2 protection. After the addition was completed, the reaction liquid was warmed to 50 °C and stirred overnight. TLC monitoring showed that the reaction was complete, the reaction liquid was concentrated by rotary evaporation, the residue was dissolved in 10 mL of ethyl acetate, extracted with water, the organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 x 20 mL). The combined extract was washed with saturated aqueous sodium chloride solution (2 x 50 mL), dried over anhydrous sodium sulfate (Na2SO4), and concentrated under vacuum. The concentrate was purified by column chromatography (eluent: dichloromethane / methanol = 1:40, v / v) to obtain 651 mg of white solid product (coumarin compound shown in structural formula (I)), with a yield of 61%; e. The white solid product compound (4-((4-(4-(tert-butyl)benzoyl)piperazin-1-yl)methyl)-7,8-dihydroxy-2H-chromen-2-one) obtained in step d was identified by HPLC-MS and BRUKER 800M nuclear magnetic resonance.

[0033] The results are as follows: 1H NMR (800 MHz, Chloroform-d) δ ( Figure 1 ): 7.45 (d, J = 8.4 Hz, 2H), 7.32(d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.25(s, 1H), 3.71 – 3.55 (m, 4H), 3.38 (s, 2H), 2.49 (d, J = 58.7 Hz, 4H), 1.29(s, 9H). 13C NMR (201 MHz, CDCl3) δ( Figure 2 ): 169.20, 160.61, 153.10, 152.35,149.95, 143.84, 133.19, 132.57, 127.10, 125.35, 115.67, 112.36, 111.60,109.88, 57.84, 47.44, 41.81, 34.72, 31.19. MS( Figure 3 ): m / z (%) [M + H]+437.2079. Test Example 1 - Test Sample Preparation

[0034] Test Sample: Coumarin compound of structural formula (I) prepared by the method described in Preparation Example; Immune checkpoint inhibitor: PD-L1 inhibitor (CA-170) with the following structural formula: ; Lead compound 10-3: 4-(4'-methoxyphenyl)-7,8-dihydroxycoumarin with the following structural formula: .

[0035] Test Example 2 - Sensitization activity determination 1. Cell culture Cell selection: HCT116, cultured in DMEM medium with 10% fetal bovine serum.

[0036] 2. Cell toxicity test HCT116 cells were plated in 96-well plates at 5000-7000 cells / 100 μL per well and incubated in a cell incubator overnight after the cells adhered. Then 100 μL of different concentrations of drug-containing medium was added for further processing for 48 h. The concentration gradient of CA-170 was 0.78, 1.56, 3.125, 6.25, 12.5, 25 μM, respectively. The concentration of the coumarin compound of structural formula (I) was 1-10 μM. After 48 h, 10 μL of MTT was added to each well, and incubated at 37°C for 4 h, protected from light. The culture medium was discarded, and 150 μL of DMSO was added to each well. Shake for 10 min until the blue-purple crystals were completely dissolved. The absorbance value at 490 nm was determined by an enzyme-labeled analyzer. Cell activity (100%) = (average OD value of drug group / average OD value of normal group)*100%.

[0037] Results analysis: CA-170 single drug application: IC50=16.61±0.4962 μM; The coumarin compound represented by structural formula (I) alone: IC50= 2.012 ± 0.3888 μM; CA-170 in combination with the coumarin compound represented by structural formula (I) (5 μM): IC50= 1.107 ± 0.3853 μM.

[0038] Test Example 3 - RNA-pulldown experiment An RNA pulldown experiment was performed to verify the mechanism using the Pierce™ Magnetic RNA Protein Pulldown Kit. The kit provides streptavidin magnetic beads and reagents for efficient enrichment of RNA-binding proteins. In the RNA pulldown experiment, in vitro transcribed RNA is first bound to magnetic beads, and then the RNA-bead complex is incubated with cell lysate, so that the proteins bound to the target RNA are adsorbed on the magnetic beads. After the RNA-protein complex is eluted from the magnetic beads with an eluent, Western Blot is used to detect specific binding proteins.

[0039] 1. Experimental protocol Binding of magnetic beads to labeled RNA: 50 μL of magnetic beads were resuspended in a sterile 1.5 mL centrifuge tube without enzymes, placed in a magnetic separation rack, and the supernatant was aspirated. The magnetic beads were washed twice with 50 μL of 20 mM Tris. 50 μL of 1x RNA Capture buffer was added to resuspend the magnetic beads, and 50-100 pmol of biotin-labeled RNA (purchased from Gene Pharma) was added, mixed gently, and incubated at room temperature for 30 min with shaking.

[0040] Binding of labeled RNA to PSF protein: The above centrifuge tube was placed in a magnetic separation rack, the magnetic beads were collected on the side, and the supernatant was discarded. The magnetic beads were washed twice with 50 μL of 20 mM Tris. 100 μL of 1x protein-RNA Binding buffer was used to resuspend the magnetic beads. The premix for the RNA-protein binding reaction was prepared according to the following table (Table 2). The magnetic beads were collected on the side of the magnetic separation rack, the supernatant was discarded, and 100 μL of the RNA-protein binding reaction premix was added to the magnetic beads, which were incubated at 4°C for 60 min with shaking. The drug group: the protein lysate was pre-incubated with the compound (the coumarin compound represented by structural formula (I) or lead compound 10-3) (1 nM) at room temperature for 1 h with shaking.

[0041] Table 1 Formula of RNA-protein binding reaction premix Reagent name Amount 10x protein-RNA Binding buffer 10 μL 50% Glycerol 20 μL Protein lysis solution 50-200µg Nuclease-free water q.s. to 100 μL Elution of RNA-protein binding complex: Place the centrifuge tube on the magnetic separation rack, collect the beads on the side, and aspirate the supernatant for analysis; wash the magnetic beads twice with 100 μL of 1x Wash buffer, and retain the supernatant (for analysis); resuspend the magnetic beads with 50 μL, mix gently, and incubate at 37°C for 30 min with vibration; place the centrifuge tube on the magnetic separation rack, and retain the supernatant. Add an equal volume of 2x loading buffer (purchased from Solarbio) to the supernatant of the last step Elution buffer, mix well, and boil at 95°C for 10-15 min. Detect the pulled-down PSF protein by Western blot.

[0042] 2. Result analysis As Figure 4 can be seen, the coumarin compound represented by structural formula (I) and the lead compound 10-3 can both significantly inhibit the binding of lncRNA to PSF protein, and the inhibitory effect of the coumarin compound represented by structural formula (I) is obviously better than that of the lead compound 10-3.

[0043] Test Example 4 - Cell cycle detection 1. Experimental scheme Preparation of cell samples: when growing to about 80%, HCT116 cells were inoculated in a six-well plate, and after the cells adhered, different compounds (5 μM, coumarin compound represented by structural formula (I) and lead compound 10-3) were added to each well, and the cells were further cultured in a cell incubator for 48 h. The culture medium was removed, washed once with PBS, and each well was added with 300 μL of trypsin for digestion for about 1 min, and the cells were collected after termination of digestion, centrifuged at 1000 rpm for 3-5 min, and the supernatant was discarded; the cells were washed twice with 2 mL of pre-cooled PBS, and the supernatant was again discarded after centrifugation. The cells were resuspended in 250 μL of cold PSB, 750 μL of pre-cooled anhydrous ethanol was added dropwise, and after gentle mixing, the cells were placed at 4°C for overnight fixation.

[0044] Staining: prepare the staining working solution in the dark. After the cells were fixed, the cells were centrifuged at 1000 rpm for 3-5 min, the fixing solution was removed, the cells were washed with PBS, and the supernatant was again discarded after centrifugation. Add 500 μL of the prepared staining working solution, mix gently, and incubate at 37°C for 30 min in the dark.

[0045] Flow detection: detect the red fluorescence at an excitation wavelength of 488 nm using a flow cytometer, and simultaneously detect the light scattering.

[0046] 2. Result analysis As Figure 5As shown, compared with the blank control group, the coumarin compounds represented by structural formula (I) and the lead compound 10-3 significantly reduced the number of HCT116 cells in the G0 / G1 phase, and the coumarin compounds represented by structural formula (I) were particularly effective, and the number of cells in the G2 / M phase was significantly increased. This means that the administration group can block the cells in the G2 / M phase and affect the synthesis of RNA and protein.

[0047] Test Example 5 - Mechanism Study 1. Experimental Scheme Preparation of cell protein samples: when the HCT116 cells grow to about 80%, the HCT116 cells are inoculated in a six-well plate, and after the cells adhere, the test sample (5 μM) is added to each well, and the cells are further cultured in the cell incubator for 48 h. Under the microscope, the culture medium of the well with little change in cell state is discarded, and 1 mL of PBS is added for washing once, and then 150-250 μL of RIPA lysis buffer (PMSF is added to the lysis buffer a few minutes ago to make the final concentration 1 mM) is added to each well, and the lysis buffer and cells are gently shaken to make full contact, and lysed on ice for 5-10 min. The lysed cells are scraped with a scraper and transferred to a centrifuge tube, which is centrifuged at 4°C, 12000 r / min for 5-10 min, and the supernatant is retained. The culture medium of the well with a large number of cell deaths is retained in the centrifuge tube, the supernatant is discarded after centrifugation, and RIPA lysis buffer (containing 1 mM PMSF) is added, gently mixed, and lysed on ice before centrifugation to obtain the supernatant. The protein concentration is determined by BCA protein concentration determination kit. Add an equal volume of 2x protein loading buffer, mix well, and boil in a 95°C metal bath for 10-15 min. The sample is stored in a -20°C refrigerator for standby.

[0048] Western blot detection of target protein expression level: 20 μg of protein is loaded, and the voltage is adjusted to 80 V. The electrophoresis is terminated when the bromophenol blue just runs out of the bottom of the separation gel. Then the PAGE gel is placed in a transmembrane instrument (BioRad, USA) under the condition of constant current 200 mA for 1 h, 5% milk is blocked for 2 h, and TBST buffer is washed for 3 times, 10 min each time. The diluted primary antibody (p53 antibody, 1: 5000) is added, and incubated at 4°C overnight. After TBST washing, the horseradish peroxidase labeled secondary antibody (HRP-IgG, 1:4000) is added, and incubated at room temperature for 2 h. After TBST washing, ECL developing solution is added, and exposed and developed in a chemiluminescence imaging system (Thermo), and the gray value is analyzed by using the gel image processing system.

[0049] 2. Results Analysis As Figure 6As shown, compared with the blank control group, the p53 protein expression level of the coumarin compounds shown in structural formula (I) was significantly increased. This change in the expression of cell cycle-related proteins may be the reason for the above cell cycle experiment results (significantly reduced number of cells in G0 / G1 phase and significantly increased number of cells in G2 / M phase).

[0050] Test Example 6 — In vivo animal experiment 1. Experimental Methods HCT116 cells (1×10) 6 A colon cancer animal model was established by subcutaneous injection of PSF-lncRNA interaction inhibitors (coumarin compounds shown in structural formula (I)) into the forelimbs of 4-6 week old male mice. One week later, the mice were randomly divided into 4 groups: a PSF-lncRNA interaction inhibitor group (coumarin compound shown in structural formula (I)), an immune checkpoint inhibitor group (CA-170) group, a PSF-lncRNA interaction inhibitor + immune checkpoint inhibitor group, and a control group, with 5 mice in each group.

[0051] Control group: injected with the same volume of physiological saline.

[0052] The tumor volume was measured with calipers every 3 days, and the mice were sacrificed on the last day to collect and weigh the tumor.

[0053] The formula for calculating tumor volume is: Tumor volume = 1 / 2 × Tumor long axis × Tumor short axis 2 2. Results Analysis like Figure 7 As shown, after two weeks of treatment, the group treated with only immune checkpoint inhibitors showed only a slight difference compared to the control group, indicating that the tumor model was insensitive to immune checkpoint inhibitor treatment. However, the combination of the coumarin compound shown in structural formula (I) and the immune checkpoint inhibitor significantly inhibited tumor growth, leading to a reduction in tumor volume and weight, indicating that the coumarin compound shown in structural formula (I) can enhance the killing effect of immune checkpoint inhibitors on its drug-resistant tumor model. Furthermore, the tumor inhibition rate of the combined effect of the coumarin compound shown in structural formula (I) and the immune checkpoint inhibitor was greater than the sum of the tumor inhibition rates of the coumarin compound shown in structural formula (I) and the immune checkpoint inhibitor, indicating that the coumarin compound shown in structural formula (I) and the immune checkpoint inhibitor exerted a synergistic effect.

Claims

1. The coumarin compound shown in structural formula (I) or its pharmaceutical salt, 。 2. Preparation method of the coumarin compounds shown in structural formula (I): S1. Using pyrogallol and ethyl 4-chloroacetoacetate as raw materials, intermediate compound 1a was obtained by Pechmann condensation reaction; S2. Using 1-Boc-piperazine and 4-tert-butylbenzoyl chloride as raw materials, intermediate compound 2a is generated by nucleophilic substitution reaction, and intermediate compound 3a is obtained after removing Boc. S3. Intermediate compound 1a and intermediate compound 3a undergo a nucleophilic substitution reaction to obtain the target compound shown in formula (I). ， ， ， 。 3. The preparation method according to claim 2, characterized in that, Step S1 includes: S11. Mix appropriate amounts of H2SO4 and CF3COOH at -5℃ to 1℃ and stir until homogeneous; S12. Add anhydrous dichloromethane, ethyl 4-chloroacetoacetate and pyrogallic acid sequentially to the reaction system, and heat to room temperature; S13. After reacting for 0.5 to 2 hours, add methanol and continue the reaction until it is complete; S14. The reaction product was purified to obtain intermediate compound 1a.

4. The preparation method according to claim 2, characterized in that, Step S2 includes: S21. Dissolve 1-Boc-piperazine and triethylamine in dichloromethane; S22. Under N2 protection at -5℃ to 1℃, add an appropriate amount of 4-tert-butylbenzoyl chloride dropwise; S23. After the reaction is completed at room temperature, the reaction product is purified to obtain intermediate compound 2a; S24. At -5℃ to 1℃, add an appropriate amount of trifluoroacetic acid to a dichloromethane solution of intermediate compound 2a and react at room temperature until the reaction is complete. S25. The reaction product was purified to obtain intermediate compound 3a.

5. The preparation method according to claim 2, characterized in that, Step S3 includes: S31. Under N2 protection, add an appropriate amount of triethylamine to an acetone solution of intermediate compound 3a; S32. Cool to -5℃~1℃, and under N2 protection, add an acetone solution of intermediate compound 1a to the reaction system; S33. Heat to 45℃~55℃ and react under stirring until the reaction is complete; S34. The reaction product was purified to obtain the target compound shown in formula (I).

6. Epigenetic drugs capable of inhibiting LncRNA-PSF interactions, including coumarin compounds of structural formula (I) or their pharmaceutically acceptable salts. 。 7. A pharmaceutical composition for synergistic treatment of cancer, comprising an immune checkpoint inhibitor and a coumarin compound of structural formula (I) or a pharmaceutically acceptable salt thereof. 。 8. The pharmaceutical composition for synergistic cancer treatment according to claim 7, characterized in that, The immune checkpoint inhibitor is a PD-L1 inhibitor.

9. The pharmaceutical composition for synergistic cancer treatment according to claim 7, characterized in that, The molar ratio of the immune checkpoint inhibitor to the coumarin compound or its pharmaceutical salt shown in structural formula (I) is 1:1 to 1:3.

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