Epigenetic drugs targeting lncrna-psf interaction, compositions thereof and uses thereof

Abstract

This invention discloses an epigenetic drug targeting lncRNA-PSF interaction, its composition, and its application. First, this invention synthesizes an epigenetic drug targeting lncRNA-PSF interaction as shown in formula (I) or formula (II). Compared with existing technologies, the epigenetic drugs shown in formulas (I) and (II) can inhibit lncRNA-PSF interaction, alter the cell cycle by increasing the expression of p27 and p53 proteins, arrest cancer cells in the G2 / M phase, and thus exert an anti-cancer effect. When the epigenetic drugs shown in formulas (I) and (II) are used in combination with immunosuppressants, they can enhance the effect of immunosuppressants, increase antigen presentation by cancer cells, and reduce their immune escape, thus becoming a novel anti-cancer sensitization strategy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical manufacturing technology, specifically providing epigenetic drugs targeting lncRNA-PSF interactions, their compositions, and their applications. Background Technology

[0002] Epigenetic dysregulation is a common feature of many diseases, especially cancer. Since the introduction of epigenetics in the 1940s, scientific research has demonstrated the link between epigenetic factors and tumorigenesis. Compared to genetic changes, epigenetic alterations are largely reversible and regulated by enzymes or chromatin-binding proteins. Therefore, epigenetic therapy has emerged as a novel strategy in the fight against cancer.

[0003] Cancer epigenetics and cancer immunology are both rapidly developing fields, attracting a large number of researchers. Immune checkpoint inhibitors are considered a major breakthrough in cancer treatment, with antibodies specifically blocking programmed cell death protein 1 / programmed cell death 1 ligand 1 (PD-1 / PD-L1) or cytotoxic T-cell-associated protein-4 being the most promising options. Although they exhibit promising anti-tumor effects, the reactivation of the immune system is often hindered by the immunosuppressive microenvironment within the tumor; alterations at any step in the immune cycle can lead to immune escape by tumor cells, resulting in resistance to immunotherapy.

[0004] Through literature review, we discovered an epigenetic transcriptional switch complex related to tumor growth and development: long noncoding RNA-polypyrimidine bundle-binding protein associated splicing factors (LncRNA-PSF). This specific regulatory pattern has the potential to be used as a cancer biomarker and therapeutic target. In the tumor microenvironment, PSF, as an RNA-binding protein, can not only reduce interferon-γ (IFN-γ) expression in Th1 cells by splicing heterogeneous nuclear RNA of interferon regulator 1, but also affect CD45 expression by binding to the thyroid hormone receptor-associated protein TRAP150. Furthermore, PSF may influence the expression of major histocompatibility complex II (MHC-II) and PD-L1 through SIN3 transcriptional regulatory protein family members A / histone deacetylase and Janus kinase / signal transduction and transcriptional activating proteins, respectively. Therefore, PSF is related to tumor immunogenicity and immune escape, and further development of combination therapies may be more effective than single-agent therapy.

[0005] In order to find new targets for cancer treatment and to address the problem that immunosuppressants alone are not effective in combating immune escape from tumor cells, combining epigenetic drugs with immune checkpoint inhibitors may enhance anti-tumor immune responses, reverse drug resistance, and exert anti-cancer sensitization effects, thus becoming a promising treatment strategy. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention provides an epigenetic drug targeting lncRNA-PSF interaction and its application. This invention first synthesizes an epigenetic drug (new compound) targeting lncRNA-PSF interaction as shown in formula (I) or formula (II). Compared with the prior art, the epigenetic drugs shown in formulas (I) and (II) can inhibit lncRNA-PSF interaction, arresting cancer cells in the G2 / M phase, thereby exerting an anti-cancer effect. When the epigenetic drugs shown in formulas (I) and (II) are used in combination with immunosuppressants, they can enhance the effect of immunosuppressants, increase antigen presentation by cancer cells, and reduce their immune escape, becoming a new anti-cancer sensitization strategy with great potential for widespread application.

[0007] The first object of the present invention is to provide an epigenetic drug targeting lncRNA-PSF interaction as shown in formula (I) or formula (II); wherein compound I of formula (I) is named 4-(4'-tert-butylbenzyl)-7,8-dihydroxycoumarin; and compound II of formula (II) is named 4-(3'-trifluoromethylbenzyl)-7,8-dihydroxycoumarin.

[0008]

[0009] The aforementioned epigenetic drugs can also be pharmaceutically usable salts of the compounds shown in formulas (I) and (II).

[0010] The epigenetic drug synthesis routes shown in formulas (I) and (II) are as follows:

[0011]

[0012] Wherein, a: oxaloyl chloride, DMF, DCM, 37-40℃; b: Miescherichic acid (cyclo(isopropyl)malonide), triethylamine, DCM, 0℃-rt; c: ethanol, 80℃; d: trifluoroacetic acid, sulfuric acid, DCM, 0℃-rt.

[0013] The compounds shown in formulas (I) and (II) are synthesized as follows: 4-tert-butylphenylacetic acid (or 3-trifluoromethylphenylacetic acid) is used as the starting material, and the mixture undergoes acyl chloride reaction in sequence. Then, cycloisopropyl malonate and triethylamine are added to generate heterocycles. After reflux with ethanol to open the ring, the mixture is cyclized with pyrogallol under acidic conditions to obtain the target compound.

[0014] The applicant discovered that the epigenetic drugs shown in formulas (I) and (II) can inhibit lncRNA-PSF interaction and thus inhibit the growth of cancer cells, and therefore can be used to prepare drugs for treating cancer.

[0015] The cancer is preferably colon cancer.

[0016] Preferably, the epigenetic drug is used to increase the expression of p27 and p53 proteins to alter the cell cycle, arrest cancer cells in the G2 / M phase, and affect RNA and protein synthesis.

[0017] A second objective of this invention is to provide the use of the above-mentioned epigenetic drugs in combination with immunosuppressants to enhance the antitumor effect of immunosuppressants.

[0018] Preferably, when the epigenetic drug is used in combination with an immunosuppressant in a Jurkat T / HCT116 co-culture system, it can significantly reduce the IC50 of the immunosuppressant (CA-170). 50 This leads to better anti-tumor effects.

[0019] Preferably, the epigenetic drug is used to increase the expression of MHC-II and reduce the upregulation of PD-L1, thereby increasing the antigen presentation of tumor cells, reducing immune escape, and thus increasing the killing ability of immune cells against tumor cells.

[0020] A third objective of this invention is to provide the sensitizing effect of epigenetic drugs or their pharmaceutically usable salts, as shown in formulas (I) and (II), on existing anticancer drugs. The anticancer drugs may be targeted inhibitors such as palbociclib, abexilib, ribociclib, enzalutamide, apalutamide, apatamide, and neratinib, or chemotherapeutic drugs such as irinotecan, nedaplatin, and pemetrexed disodium.

[0021] A further technical objective of the present invention is to provide an anticancer composition, wherein the active ingredient of the anticancer composition includes the above-mentioned epigenetic drugs.

[0022] A further technical objective of the present invention is to provide an anticancer sensitizing composition, wherein the active ingredient of the anticancer sensitizing composition includes the above-mentioned epigenetic drugs.

[0023] Preferably, the aforementioned anticancer composition or anticancer sensitizing composition is used to treat tumors or tumor-related diseases. These compounds possess broad-spectrum antitumor activity, and examples of cancers they can treat include, but are not limited to, gastric cancer, colon cancer, breast cancer, liver cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, endometrial cancer, skin cancer, esophageal cancer, and central nervous system cancers. The epigenetic drugs of this invention have potential antitumor activity, particularly anti-colon cancer activity, and show potential application prospects in the treatment of cancer and cancer-related diseases.

[0024] The anticancer composition or anticancer sensitizing composition further includes one or more pharmaceutically acceptable carriers. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., commonly used in the pharmaceutical field.

[0025] The anticancer composition or anticancer sensitizing composition can be used orally or non-orally, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as muscle, intradermal, subcutaneous, vein, mucosal tissue; or by being mixed or encapsulated with other substances before being introduced into the body.

[0026] When used orally, it can be formulated into conventional solid dosage forms, such as tablets, powders, granules, capsules, ointments, creams, etc.; or into liquid dosage forms, such as water or oil suspensions or other liquid preparations, such as oral liquids. When used for parenteral administration, it can be formulated into solutions for injection, water or oil suspensions, etc.

[0027] Compared with existing technologies, the beneficial effects of the epigenetic drugs targeting lncRNA-PSF interactions and their applications of the present invention are as follows:

[0028] (i) Epigenetic drugs have good PSF protein inhibitory activity and can inhibit lncRNA-PSF interaction, which is a new targeted therapy.

[0029] (ii) Epigenetic drugs alter the cell cycle by increasing the expression of p27 and p53 proteins in HCT116 cells, arresting cancer cells in the G2 / M phase, affecting RNA and protein synthesis, and inducing apoptosis in cancer cells.

[0030] (III) In the Jurkat T / HCT116 co-culture system, the combination of epigenetic drugs and immunosuppressants (CA-170) significantly reduced the IC50 of CA-170. 50 It has a better anti-tumor effect;

[0031] (iv) Epigenetic drugs increase the expression of MHC-II in HCT116 cells and reduce the upregulation of PD-L1 in HCT116 cells, thereby increasing the antigen presentation of tumor cells, reducing immune escape, and thus increasing the killing ability of immune cells against tumor cells. Attached Figure Description

[0032] Figure 1 It is the compound shown in formula (Ⅰ) 1 H NMR spectrum;

[0033] Figure 2 It is the compound shown in formula (Ⅰ) 13 C NMR spectrum;

[0034] Figure 3 This is the HR-MS spectrum of the compound shown in formula (Ⅰ);

[0035] Figure 4 It is the compound shown in formula (II) 1 H NMR spectrum;

[0036] Figure 5 It is the compound shown in formula (II) 13 C NMR spectrum;

[0037] Figure 6 The HR-MS spectrum of the compound shown in formula (II) is shown below.

[0038] Figure 7 This is a docking diagram of the lead compound 10-3 with the PSF protein; the left image is a 3D docking diagram, and the right image is a 2D docking diagram.

[0039] Figure 8This is a docking diagram of compound (Ⅰ) with PSF protein; the left diagram is a 3D docking diagram, and the right diagram is a 2D docking diagram.

[0040] Figure 9 This is a docking diagram of compound (II) with PSF protein; the left diagram is a 3D docking diagram, and the right diagram is a 2D docking diagram.

[0041] Figure 10 The effects of compounds shown in formulas (I) and (II) and CA-170 on the survival rate of Jurkat T cells;

[0042] Figure 11 The effect of different concentrations of CA-170 on the survival rate of HCT116 cells is shown in Figure a, where figure a represents single culture and figure b represents combined culture.

[0043] Figure 12 These are the results of an RNA-pulldown experiment; the left image is a Western blot protein band diagram, and the right image is a protein quantification diagram.

[0044] Figure 13 The effects of compounds shown in formulas (I) and (II) and 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.

[0045] Figure 14 The effects of compounds shown in formulas (I) and (II) and lead compound 10-3 on p27, p53, bax, p21 and MHC-II proteins in HCT116 cells are shown; the left figure is a Western blot protein band diagram and the right figure is a protein quantification diagram.

[0046] Figure 15 The effects of compounds shown in formulas (I) and (II) and lead compound 10-3 on PD-L1 protein in HCT116 cells induced by IFN-γ are shown in the figure on Western blot protein bands and the figure on the right is protein quantification. Detailed Implementation

[0047] 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.

[0048] Unless otherwise specified, the contents of each component used below are weight percentages; all experimental methods used are conventional methods; and all reagents and biological materials used are commercially available.

[0049] The structural formula of the lead compound 10-3 (4-(4'-methoxyphenyl)-7,8-dihydroxycoumarin) used in this invention is shown below:

[0050]

[0051] The structural formula of CA-170 (PD-L1 small molecule inhibitor) used in this invention is shown below:

[0052]

[0053] Example 1: Preparation of epigenetic drugs (I) and (II) The specific preparation process of compound I (4-(4'-tert-butylbenzyl)-7,8-dihydroxycoumarin) shown in formula (I) is as follows:

[0054] In a 100 mL single-necked reaction flask, add 5 mmol (1 eq) of 4-tert-butylphenylacetic acid, 30 mmol (6 eq) of oxaloyl chloride, 5-6 drops of DMF, and 50 mL of anhydrous DCM. Place the flask in a heated magnetic stirrer and reflux at 37-40 °C for 2 h. After the reaction is complete, evaporate to dryness to obtain crude 4-tert-butylphenylacetyl chloride, which can be directly used for the next step.

[0055] At 0 °C, cycloisopropyl malonate (5 mmol, 1 eq), triethylamine (12.5 mmol, 2.5 eq), and 30 mL of anhydrous DCM were placed in a 100 mL single-necked reaction flask and stirred for 10 min. Then, an anhydrous DCM solution of 4-tert-butylphenylacetyl chloride was slowly added dropwise. After the addition was complete, the temperature was slowly raised to room temperature. The reaction was allowed to proceed overnight. The triethylamine was removed by extraction with 0.1 mol / L citric acid solution. The organic phase was dried and then evaporated to dryness. 60 mL of ethanol was added, and the mixture was refluxed at 80 °C for 4 h. After the reaction was monitored by TLC, the crude ethyl acetate of 4-tert-butylphenylacetyl was obtained by evaporation to dryness. The crude product was purified by column chromatography (packing material: silica gel powder) (eluent: petroleum ether: ethyl acetate = 15:1 by volume), and then concentrated under reduced pressure to obtain an oily pure product (1.16 g).

[0056] At 0 °C, 2 mmol (2 eq) of H₂SO₄ and 2 mmol (2 eq) of CF₃COOH were stirred and mixed in a 100 mL single-necked reaction flask. Then, 30 mL of anhydrous DCM, 1 mmol (1 eq) of ethyl 4-tert-butylphenylacetoacetate, and 1.2 mmol (1.2 eq) of pyrogallol were added sequentially. The temperature was slowly raised to room temperature, and the reaction was continued with stirring. After 1 h, 1-2 mL of methanol was added to increase the solubility of the system. After 4-5 h of reaction, the reaction was monitored by TLC until complete. The reaction solution was extracted with saturated NaHCO₃ solution, and the organic phase was dried by rotary evaporation. Recrystallization was performed using ethyl acetate / petroleum ether (volume ratio 1:5). After the solid precipitate formed, it was cooled and allowed to stand, then filtered. The filter cake was dried in a vacuum drying oven to obtain 0.22 g of pure 4-(4'-tert-butylbenzyl)-7,8-dihydroxycoumarin, with a yield of 67.9%.

[0057] (II) The specific preparation process of the compound shown in formula (II) (4-(3'-trifluoromethylbenzyl)-7,8-dihydroxycoumarin) is as follows:

[0058] In a 100 mL single-necked reaction flask, add 5 mmol (1 eq) of 3-trifluoromethylphenylacetic acid, 30 mmol (6 eq) of oxaloyl chloride, 5-6 drops of DMF, and 50 mL of anhydrous DCM. Place the flask in a heated magnetic stirrer and reflux at 37-40 °C. After 2 h, the reaction is complete, and the crude 3-trifluoromethylphenylacetyl chloride is obtained by rotary evaporation and directly proceeded to the next step.

[0059] At 0 °C, cycloisopropyl malonate (5 mmol, 1 eq), triethylamine (12.5 mmol, 2.5 eq), and 30 mL of anhydrous DCM were placed in a 100 mL single-necked reaction flask and stirred for 10 min. Then, an anhydrous DCM solution of 3-trifluoromethylphenylacetyl chloride was slowly added dropwise. After the addition was complete, the temperature was slowly raised to room temperature. The reaction was allowed to proceed overnight. The triethylamine was removed by extraction with 0.1 mol / L citric acid solution. The organic phase was dried and then evaporated to dryness. 60 mL of ethanol was added, and the mixture was refluxed at 80 °C for 4 h. After the reaction was monitored by TLC, the crude ethyl acetate of 3-trifluoromethylphenylacetyl was obtained by evaporation to dryness. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 15:1) and then concentrated under reduced pressure to obtain an oily pure product (1.26 g).

[0060] At 0 °C, 2 mmol (2 eq) of H₂SO₄ and 2 mmol (2 eq) of CF₃COOH were stirred and mixed in a 100 mL single-necked reaction flask. Then, 30 mL of anhydrous DCM, 1 mmol (1 eq) of ethyl 3-trifluoromethylphenylacetoacetate, and 1.2 mmol (1.2 eq) of pyrogallol were added sequentially. The temperature was slowly raised to room temperature, and the reaction was continued with stirring. After 1 h, 1-2 mL of methanol was added to increase the solubility of the system. After 4-5 h of reaction, the reaction was monitored by TLC until complete. The reaction solution was extracted with saturated NaHCO₃ solution, and the organic phase was dried by rotary evaporation. Recrystallization was performed using ethyl acetate / petroleum ether (volume ratio 1:5). After the solid precipitate formed, it was cooled and allowed to stand, then filtered. The filter cake was dried in a vacuum drying oven to obtain 0.24 g of pure 4-(3'-trifluoromethylbenzyl)-7,8-dihydroxycoumarin, with a yield of 71.4%.

[0061] Example 2: Structural Identification

[0062] The structure of the compound was identified using HPLC-MS and BRUKER 800M NMR. The results are as follows:

[0063] Spectrum and data of the compound shown in formula (Ⅰ) (4-(4'-tert-butylbenzyl)-7,8-dihydroxycoumarin):

[0064] 1 H NMR (800MHz, DMSO-d6) δ7.35–7.31(m,2H),7.24–7.21(m,2H),7.18(d,J=9.0Hz ,1H),6.77(d,J=10.0Hz,1H),5.97(s,1H),4.05(s,2H),1.24(d,J=3.4Hz,9H).

[0065] 13 C NMR(201MHz,DMSO-d6)δ160.37,156.29,149.50,148.98,143.62,134.36,13 2.38,128.58,125.36,115.77,112.18,111.86,110.41,36.53,34.14,31.13.

[0066] MS: m / z (%) [M+H] + 325.1385.

[0067] Spectrum and data of the compound shown in formula (II) (4-(3'-trifluoromethylbenzyl)-7,8-dihydroxycoumarin):

[0068] 1 H NMR (800MHz, DMSO-d6) δ7.74(s,1H),7.65–7.60(m,2H),7.56(t,J=8.3Hz,1H),7.18(d,J=8.9Hz,1H),6.78(d,J=8.9Hz,1H),5.99(s,1H),4.24(s,2H).

[0069] 13 C NMR(201MHz,DMSO-d6)δ160.24,155.38,149.54,143.61,133.14,132.39,129.67,129.40,129 .24,129.08,125.53,125.51,123.56,123.54,123.52,115.71,112.20,111.71,110.82,36.46.

[0070] MS: m / z (%) [M+H] + 337.0644.

[0071] Example 3: Molecular docking research

[0072] Using Discovery Studio (DS), the compounds shown in formulas (I) and (II) were docked with the PSF protein, and compared with the lead compound 10-3 (4-(4'-methoxyphenyl)-7,8-dihydroxycoumarin).

[0073] 1. Experimental procedure:

[0074] (1) In the DS software, open a new molecule window and import the compound. Under the Small Molecules section, prepare the small molecule ligands by clicking Tools-Prepare or Filter Ligands-Prepare ligands to open the parameter settings panel. Select the compound to be prepared in Input Ligands, leave the other parameters as default, and click Run to perform the calculation. Under the Job section, click View Result to view the results.

[0075] (2) Protein preparation: In the DS software, open File-Open URL, enter the ID of the desired protein (PSF:4WII), and import the target protein. Under the Macromolecules section, open Tools-Prepare Protein-Clean Protein, remove the existing ligands and water molecules, then click Prepare Protein to open the parameter settings panel. In Input Protein, select the target protein All, leave the other parameters as default, and click Run to perform the calculation. Under the Job section, click View Results to view the results.

[0076] (3) Lib dock molecular docking: Under the Receptor-Ligand Interactions section, click Tools-Define and Edit Binding Site-Define Site-From Current Selection. Based on reported docking studies (select A:LYS516, A:ASP517, B:TYR490), define the protein active site. Then, open Dock-Ligands-Libdock to access the parameter settings panel. In Input Receptor, select the protein with the defined active site; in Input Ligands, select the prepared compound. Leave the other parameters as default and click Run to perform the calculation. Under the Job section, click View Results to view the results. Simultaneously, use PyMOL to plot the interaction between the small molecule and the protein.

[0077] 2. Results and Analysis

[0078] The docking results of lead compound 10-3 with PSF (PBD:4WII) are as follows: Figure 7 As shown in Table 1, its docking score is 95.134. The stereostructure reveals that 10⁻³ fits well into the cavity formed by the protein RNA recognition motif 2 (RRM2) and the terminal coiled domain. The bisphenol hydroxyl group of compound 10⁻³ provides a hydrogen bond donor, forming strong hydrogen bond interactions with amino acid residues A:GLU520, B:TYP490, and A:LYS516, respectively. The coumarin ring and its 4-position aromatic ring form Pi-Pi stacking and Pi-alkyl interactions with amino acid residues such as B:TRP494, A:LYS516, and A:LYS513, respectively. In addition, 10⁻³ exhibits other interactions with PSF protein. These results provide supplementary references for 10⁻³'s inhibition of LncRNA and PSF binding, and also provide theoretical basis and guidance for 10⁻³ as a lead compound.

[0079] The docking results of the compound shown in formula (Ⅰ) with PSF (PBD:4WII) are as follows: Figure 8 As shown in Table 1, its docking score is 103.354, which is significantly higher than that of the lead compound (10⁻³). From the stereostructure, it can be seen that the compound shown in formula (I) can be embedded in the RRM2 cavity. The three benzene rings in its 4-benzylcoumarin structural framework form Pi-cation, Pi-Pi T-shaped and other interactions with the amino acid residues around the cavity, respectively. At the same time, the hydroxycoumarin structure retained by the compound shown in formula (I) can form hydrogen bond interactions with A:LYS516, B:GLU501 and B:ASP498.

[0080] The docking results of the compound shown in formula (II) with PSF (PBD:4WII) are as follows: Figure 9 As shown in Table 1, its docking score is 108.099, which is significantly higher than that of the lead compound 10⁻³. From the stereostructure, it can be seen that, compared with the lead compound 10⁻³, the substituent on the benzyl group at position 4 in the compound of formula (II) can be embedded deep into the cavity of RRM2 and can form hydrogen bond interactions with the amino acid residue (A:LYS516) of the terminal coiled domain. The hydroxycoumarin structure retained by the compound of formula (II) can form hydrogen bond interactions with the amino acid residues (A:ASN377, A:PRO442) of RRM2.

[0081] Table 1: Docking scores of compounds with PSF protein

[0082]

[0083] Example 4: Cell viability assay

[0084] 1. Cell Culture

[0085] HCT116 (colon cancer cells) were selected and cultured in DMEM medium containing 10% serum.

[0086] Jurkat T cells (human T lymphocyte leukemia cells) were selected and cultured in RPMI 1640 medium with 10% serum.

[0087] All cells were purchased from Jinan Boshan Company.

[0088] 2. Cytotoxicity test

[0089] 2.1 HCT116 Cytotoxicity Assay

[0090] HCT116 cells were seeded at a rate of 5000-7000 cells / 100 μL / well in 96-well plates. After 12 h of incubation, once cell attachment was achieved, 100 μL of different concentrations of drug-treated medium (DMEM containing 10% serum) was added to each well, and the cells were treated for another 48 h. After 48 h, 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the dark for 4 h. The supernatant was then discarded, and 150 μL of DMSO was added to each well, with shaking for 5-10 min to dissolve the purple crystals. The absorbance at 490 nm was measured using a microplate reader, and the inhibitory rate of the target compound on the cells was calculated using the formula: Cell viability (100%) = (Average OD value of the drug group / Average OD value of the normal group) * 100%.

[0091] 2.2 Jurkat cytotoxicity test

[0092] Jurkat cells were grown at a rate of 1-2 × 10⁻⁶. 4 Cells were seeded at 100 μL / well in 96-well plates, with 100 μL of a specific concentration of drug-treated medium (RPMI 1640 medium containing 10% serum and 10 μM of the compound) added to each well. The plates were incubated for 48 h. After 48 h, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated in the dark for 4 h. The supernatant was then discarded, and 150 μL of LDMSO was added to each well, with shaking for 5-10 min to dissolve the purple crystals. The absorbance at 490 nm was measured using a microplate reader, and the inhibition rate of the target compound on cells was calculated using the formula: Cell viability (100%) = (Average OD value of the drug group / Average OD value of the normal group) * 100%.

[0093] 2.3 Jurkat T cell co-culture test with HCT116 cells

[0094] Experimental groups: (1) Blank control group: HCT116 cells not induced by IFN-γ; (2) PHA-P pretreatment group: HCT116 cells not induced by IFN-γ and Jurkat T cells stimulated by PHA-P; (3) Cell co-culture group: HCT116 cells induced by IFN-γ and Jurkat T cells stimulated by PHA-P; (4) Single drug administration group: Cell co-culture group + different concentrations of CA-170; (5) Combined drug administration group: Cell co-culture group + different concentrations of CA-170 and the compound shown in formula (I) or (II).

[0095] When Jurkat T cells reached the logarithmic growth phase, the cells were collected and counted using a cell counting chamber. The cells were then resuspended in 6 mL of RPMI 1640 medium (containing 10% serum) with a final concentration of 2 μg / mL PHA-P added. These cells were seeded into culture flasks and cultured in a temperature- and humidity-controlled incubator for 48 h. Simultaneously, HCT116 cancer cells that had reached 80% confluence were collected, counted using a cell counting chamber, and resuspended in DMEM medium (containing 10% serum and 10 ng / mL IFN-γ). These cells were seeded into 96-well plates at a density of 10,000 cells per well and cultured in a cell culture incubator for 24 h.

[0096] At a Jurkat T cell:HCT116 cell ratio of 2:1, activated Jurkat T cells were seeded into 96-well plates of IFN-γ-pretreated HCT116 cells. Different final concentrations of the target compound were added to the single-drug and combined-drug groups (the compound was dissolved in cell-grade DMSO, ensuring the final DMSO concentration was less than 0.1%), and the cells were cultured for 48 h. The supernatant was removed, and the cells were washed four times with PBS to completely remove the Jurkat T cells. 10 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the dark for 4 h. The supernatant was then discarded, and 150 μL of DMSO was added to each well, with shaking for 5–10 min to dissolve the purple crystals. The absorbance at 490 nm was measured using a microplate reader, and the inhibitory rate of the drug on the cells was calculated using the formula.

[0097] 3. Results Analysis

[0098] 3.1 Data on the effect on HCT116 colon cancer cells

[0099] When the compound shown in formula (Ⅰ) is used alone, IC 50 =2.995±0.540μM;

[0100] When the compound shown in formula (II) is used alone, IC 50 =4.015±0.719μM.

[0101] 3.2 Data on co-culture of Jurkat T cells and HCT116 cells

[0102] A 10 μM concentration of the compound was selected and applied to Jurkat T cells. The results were analyzed after 48 hours. Figure 10 As shown, the survival rate of Jurkat T cells treated with the compound of formula (II) and CA-170 was greater than 90%, while the survival rate of Jurkat T cells treated with the compound of formula (I) was greater than 75%. It can be seen that the compounds of formulas (I) and (II) have low toxicity to Jurkat T cells.

[0103] Furthermore, we found that treating HCT116 cells with different concentrations of CA-170 (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) for 48 h resulted in cell viability greater than 90%. Figure 11 (a) Studies have shown that CA-170 itself is not highly toxic to HCT116 cells. Furthermore, as a PD-L1 inhibitor, CA-170 needs to reduce immune escape and increase T cell killing activity while inhibiting PD-L1. Therefore, we selected a co-culture system of Jurkat T cells and HCT116 cells for our experiments.

[0104] like Figure 11 As shown in Figure a, we found that the survival rate of HCT116 cells in the blank control group (100%) was > that in the co-culture group (94.49%) > that in the PHA-P pretreatment group (76.63%), indicating that Jurkat T cells kill some cancer cells. Compared with the PHA-P pretreatment group, the co-culture group exhibited immune escape, which is because the PD-L1 continuously expressed by IFN-γ-induced HCT116 cells interacts with the PD-1 continuously expressed by PHA-P-treated Jurkat T cells. Simultaneously, we examined the effect of different concentrations of CA-170 on the survival rate of HCT116 cells in the co-culture system, such as... Figure 11 As shown in Figure b, with the co-culture group as the blank control, it can be clearly seen that CA-170 can play a role in reducing the immune escape of cancer cells in the co-culture system.

[0105] When the CA-170 is used alone, the CA-170 IC... 50 =15.053±1.784μM;

[0106] When CA-170 is used in combination with the compound shown in formula (Ⅰ) (at a concentration of 5 μM):

[0107] IC50 of the compound shown in formula (Ⅰ) with CA-170+5μM 50 =0.459±0.011μM;

[0108] When CA-170 is used in combination with the compound shown in formula (Ⅰ) (at a concentration of 1 μM):

[0109] IC50 of the compound shown in formula (Ⅰ) with CA-170+1μM 50 =7.663±1.184μM;

[0110] When CA-170 is used in combination with the compound shown in formula (II) (at a concentration of 5 μM):

[0111] IC50 of the compound shown in formula (II) with CA-170+5μM 50 =2.616±0.014μM;

[0112] When CA-170 is used in combination with the compound shown in formula (II) (at a concentration of 1 μM):

[0113] IC50 of the compound shown in formula (II) with CA-170+1μM 50 =10.129±1.127μM.

[0114] The results showed that the combined use of the compound shown in formula (I) and the compound shown in formula (II) with CA-170 reduced the immune escape of cancer cells and had a better anti-cancer effect.

[0115] Example 5: RNA-pulldown experiment

[0116] Using Pierce TM The Magnetic RNA Protein Pull-Down Kit was used to verify the mechanism of RNA pull-down assays. This kit provides streptavidin magnetic beads and reagents for effectively enriching RNA-binding proteins. In the RNA pull-down assay, in vitro transcribed RNA is first bound to magnetic beads. The RNA-magnetic bead complex is then incubated with cell lysis buffer, causing proteins that bind to the target RNA to adsorb onto the beads as well. After eluting the RNA-protein complex from the magnetic beads with elution buffer, specific binding proteins are detected using Western blotting.

[0117] 1. Experimental Design

[0118] Binding of magnetic beads to labeled RNA: Resuspend 50 μL of magnetic beads in a sterile, enzyme-free 1.5 mL centrifuge tube, place it on a magnetic separator, and discard the supernatant; wash the magnetic beads twice with 50 μL of 20 mM Tris; resuspend the magnetic beads in 50 μL of 1×RNA Capture buffer, then add 50-100 pmol of avidin-labeled RNA (purchased from Gene Pharma), mix gently, and incubate with shaking at room temperature for 30 min.

[0119] Binding of labeled RNA to PSF protein: Place the centrifuge tubes on a magnetic separator rack, collect the magnetic beads from the side, and discard the supernatant; wash the magnetic beads twice with 50 μL of 20 mM Tris; dilute 10× protein-RNA binding buffer to 1× with ultrapure water, and resuspend the magnetic beads in 100 μL of 1× protein-RNA binding buffer; prepare the premix for the RNA-protein binding reaction according to the table below (Table 2); collect the magnetic beads from the side of the magnetic separator rack, discard the supernatant, add 100 μL of the premix for the RNA-protein binding reaction to the magnetic beads, and incubate at 4℃ with shaking for 60 min. Drug administration group: The protein lysis buffer was incubated with the compound (1 nM) at room temperature with shaking for 1 h beforehand.

[0120] Table 2 Premix formulations for RNA-protein binding reactions

[0121]

[0122] Elution of RNA-protein binding complex: Place the centrifuge tube on a magnetic separator and collect the beads from the side. Aspirate the supernatant for analysis. Wash the magnetic beads twice with 100 μL of 1×Wash buffer and collect the supernatant (for analysis). Resuspend the magnetic beads in 50 μL of water, mix gently, and incubate at 37°C with shaking for 30 min. Place the centrifuge tube on the magnetic separator and collect the supernatant. In the final step, add an equal volume of 2×loading buffer (purchased from Solarbio) to the elution buffer supernatant, mix thoroughly, and incubate in a 95°C metal bath for 10-15 min. Detect the PSF protein in the eluent using Western blot.

[0123] 2. Results Analysis

[0124] according to Figure 12 It can be seen that the compounds shown in formula (I) and formula (II) have an ability to inhibit the binding of lncRNA and PSF protein by about 70% and 90%, respectively, which is significantly better than the lead compound 10-3 (control in the figure is a blank control).

[0125] Example 6: Cell cycle detection

[0126] 1. Experimental Design

[0127] Cell sample preparation: When HCT116 cells reached approximately 80% confluence, they were seeded into six-well plates. After cell attachment, a different compound (25 μM) was added to each well, and the cells were cultured for 48 hours. The culture medium was removed, and the cells were washed once with PBS. 300 μL of trypsin was added to each well for approximately 1 minute to digest the cells. The digestion was then stopped, and the cells were collected. The cells were centrifuged at 1000 rpm for 3-5 minutes, and the supernatant was discarded. The cells were washed twice with 2 mL of pre-chilled PBS, and centrifuged again, discarding the supernatant. The cells were resuspended in 250 μL of cold PSB, and 750 μL of pre-chilled anhydrous ethanol was added dropwise. After gentle mixing, the cells were fixed overnight at 4°C.

[0128] Staining: Prepare staining working solution in the dark. After cell fixation, centrifuge the cells at 1000 rpm for 3-5 min, remove the fixative, wash the cells with PBS, centrifuge again and discard the supernatant. Add 500 μL of the prepared staining working solution, mix gently, and incubate at 37°C in the dark for 30 min.

[0129] Flow cytometry: Red fluorescence was detected at an excitation wavelength of 488 nm using a flow cytometer, while light scattering was also detected.

[0130] 2. Results Analysis

[0131] like Figure 13 As shown, compared with the blank control group, the proportion of cells in the G0 / G1 phase decreased and the proportion of cells in the G2 / M phase increased in the compounds shown in formulas (I) and (II). Compared with the lead compound, compounds I and II significantly reduced the number of HCT116 cells in the G0 / G1 phase and significantly increased the number of cells in the G2 / M phase. The decrease in the proportion of cells in the G0 / G1 phase and the increase in the proportion of cells in the G2 / M phase indicate that the treated group was able to arrest cells in the G2 / M phase and affect RNA and protein synthesis.

[0132] Example 7: Western Blot

[0133] 1. Experimental Design

[0134] Preparation of cellular protein samples: When HCT116 cells reached approximately 80% confluence, they were seeded into six-well plates. After cell adhesion, a different compound (25 μM) was added to each well, and the cells were cultured for 48 hours. Cells were observed under a microscope. The culture medium in wells showing minimal cell morphology was discarded, and the cells were washed once with 1 mL of PBS. Then, 150-250 μL of RIPA lysis buffer (PMSF was added to the lysis buffer a few minutes prior to the cell lysis, bringing the final concentration to 1 mM) was added to each well. The cells were gently agitated to ensure adequate contact between the lysis buffer and the cells, and lysis was performed on ice for 5-10 minutes. The lysed cells were scraped off and transferred to centrifuge tubes. The cells were centrifuged at 12000 rpm for 5-10 minutes at 4°C, and the supernatant was collected. The culture medium in wells with a high number of dead cells was retained in centrifuge tubes. After centrifugation, the supernatant was discarded, and RIPA lysis buffer (containing 1 mM PMSF) was added. The mixture was gently mixed, and the cells were lysed on ice. The supernatant was then collected after centrifugation. Protein concentration was determined using a BCA protein concentration assay kit. An equal volume of 2× protein loading buffer was added, mixed thoroughly, and boiled in a 95°C metal bath for 10-15 minutes. The sample was then stored at -20°C for later use.

[0135] Western blot analysis of target protein expression levels: 20 μg of protein was loaded, and the voltage was adjusted to 80 V. Electrophoresis was stopped when the bromophenol blue just emerged from the bottom of the separating gel. The PAGE gel was then placed in a transfer apparatus (BioRad, USA) and transferred at a constant current of 200 mA for 1 h. Blocking with 5% milk powder was performed for 2 h, followed by washing three times with TBST buffer for 10 min each time. Primary antibodies diluted with antibody dilution buffer (β-actin antibody, 1:20000; PD-L1 antibody, 1:6000; p21 antibody, 1:1000; p27 antibody, 1:1000; p53 antibody, 1:5000; Bax antibody, 1:5000; MHC-II antibody, 1:1000) were added and incubated overnight at 4°C. After washing with TBST, horseradish peroxidase-labeled secondary antibody (HRP-IgG, 1:4000) was added and incubated at room temperature for 2 hours. After washing with TBST, ECL developing solution was added, and the gel was exposed and developed using a chemiluminescence imaging system (Thermo Fisher Scientific). The grayscale value was analyzed using the gel image processing system (as shown in the attached figure).

[0136] 2. Results Analysis

[0137] like Figure 14As shown, compared with the blank control group, the expression levels of p27 and p53 proteins of the compound shown in formula (I) were significantly increased, and the expression level of p27 protein of the compound shown in formula (II) was significantly increased (the expression level of p53 protein was slightly increased, but not significantly). Compared with the blank control group, the expression levels of p21 and bax proteins of compounds I and II decreased, but the difference in decrease was not very significant. These changes 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). In addition, we found that compared with the blank control group, the expression of MHC-II protein of the compound shown in formula (II) was significantly increased, while the expression level of MHC-II protein of the compound shown in formula (I) was slightly increased, but the change was not significant. This can prove that the drug-treated group increased the antigen presentation of tumor cells, providing support for the above combined anti-cancer experiment.

[0138] like Figure 15 As shown, we evaluated whether the compounds could improve the upregulation of PD-L1 expression induced by IFN-γ. The results showed that the compounds shown in formula (I) and (II) could reduce the upregulation of PD-L1 expression induced by IFN-γ, which can indicate that these compounds can reduce immune escape and further support the above-mentioned combined anticancer experiments.

Claims

1. A compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof; ， Equation (I), Equation (II).

2. Use of the compound of claim 1 or a pharmaceutically usable salt thereof in the preparation of a medicament for treating cancer, wherein the cancer is colon cancer.

3. An anticancer composition, characterized in that, It contains the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The anticancer composition according to claim 3, characterized in that, The anticancer composition also includes one or more of the immunosuppressants CA-170, palbociclib, abeciclib, ribociclib, enzalutamide, apalutamide, apatamide, neratinib, irinotecan, nedaplatin, and pemetrexed disodium.

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