3-hydroxy-2-oxindole compound and application thereof in preparation of antitumor drugs

Abstract

The application discloses a 3-hydroxy-2-oxindole compound and application thereof in preparation of antitumor drugs. The 3-hydroxy-2-oxindole compound has strong EZH2 binding effect. The application proves by experiments at a cell level that the 3-hydroxy-2-oxindole compound can lower the expression level of EZH2, thereby inhibiting the proliferation of tumor cells and achieving the effect of inhibiting tumors, so that the compound has good prospects in preparation of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a 3-hydroxy-2-oxoindole compound and its application in the preparation of antitumor drugs. Background Technology

[0002] Polycomb repressive complex 2 (PRC2) possesses histone methyltransferase activity, catalyzing the trimethylation of lysine 27 at histone H3 to silence downstream target genes. EZH2, the core catalytic subunit of PRC2, is an ideal target for directly shutting down PRC2 activity. EZH2 can act directly on certain factors independently of PRC2, activating downstream genes and inducing carcinogenesis. Furthermore, EZH2 is frequently associated with cancer cell proliferation, tumor drug resistance, and poor prognosis.

[0003] In 2020, the EZH2-targeting agent Tazemetostat was approved. Its robust pharmacokinetic profiles in Phase I and II clinical trials for epithelioid sarcoma and solid tumors confirmed the feasibility of EZH2 as an anti-tumor target. Domestic and international reports also indicate the development of unc1999, which exhibits high selectivity for both EZH2 and EZH1 and is orally absorbed, showing greater potential in diseases where both EZH1 and EZH2 play a significant role. In various tumors, combining different anticancer drugs with EZH2 inhibitors can enhance efficacy.

[0004] EZH2 is highly expressed in liver cancer, cholangiocarcinoma, and colon cancer. Colon cancer and liver cancer are common malignant tumors of the digestive tract, second only to stomach cancer in mortality rate, with poor prognosis and a high recurrence rate. Cholangiocarcinoma, due to its poor response to interventional therapy and the lack of targeted drugs, is difficult to treat effectively. Targeted inhibitors have shown good efficacy in treating these tumors and improving cancer prognosis. In conclusion, as an anti-tumor target, finding novel and highly effective targeted agents for EZH2 has extremely high research value and significance. Summary of the Invention

[0005] The purpose of this invention is to provide a 3-hydroxy-2-oxoindole compound and its application in the preparation of antitumor drugs. The 3-hydroxy-2-oxoindole compound can bind to EZH2, thereby inhibiting EZH2 expression and achieving the effect of inhibiting the proliferation of various tumor cells.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a 3-hydroxy-2-oxoindole compound with the following general structural formula:

[0008]

[0009] Wherein, R1 is selected from a straight-chain alkyl group consisting of 1-5 carbon atoms or a cycloalkyl group consisting of 3-6 carbon atoms or a cycloalkylmethyl group; R2 is selected from a benzene ring containing 1-3 substituents, thiophene, benzothiophene or benzothiazole; wherein the substituent is a straight-chain alkyl group consisting of 1-3 carbon atoms or a methoxy group.

[0010] Furthermore, the general structural formula of the 3-hydroxy-2-oxoindole compounds is as follows:

[0011]

[0012] Wherein, R1 is selected from or R2 is selected from or

[0013] Furthermore, the 3-hydroxy-2-oxoindole compounds specifically refer to compounds 1-13, whose structural formulas are as follows:

[0014]

[0015] This invention also provides the application of the aforementioned 3-hydroxy-2-oxoindole compounds in the preparation of EZH2 targeting agents.

[0016] The present invention also provides the application of the aforementioned 3-hydroxy-2-oxoindole compounds in the preparation of antitumor drugs.

[0017] Furthermore, in the antitumor drug, the concentration of the 3-hydroxy-2-oxoindole compound is 0.5 μM-10 μM.

[0018] Furthermore, the reaction time of the 3-hydroxy-2-oxoindole compound is 18-48 h.

[0019] Furthermore, the 3-hydroxy-2-oxoindole compounds inhibit tumor cell proliferation by significantly downregulating EZH2 expression.

[0020] Furthermore, the tumors include liver cancer, bile duct cancer, and colon cancer.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] This invention screened various compounds that inhibit EZH2 activity using a combination of molecular docking methods and binding free energy calculations. Based on the 3-hydroxy-2-oxoindole core structure, a variety of compounds were designed, all exhibiting strong EZH2 binding activity. At the cellular level, experiments have confirmed that 3-hydroxy-2-oxoindole compounds can downregulate EZH2 expression levels, thereby inhibiting tumor cell proliferation and achieving tumor suppression. Therefore, these compounds show promising potential in the preparation of antitumor drugs. Attached Figure Description

[0023] Figure 1 The diagram shows the binding conformation of active compound 1 in the EZH2 binding pocket; the active compound is shown as a stick, EZH2 is shown as the electrostatic surface of the protein, the short dashed line on the right represents hydrogen bonding, and the long dashed line on the left represents pi-pi interaction.

[0024] Figure 2 The diagram shows the interaction between active compound 1 and EZH2; the left diagram shows the relative positions of key amino acids and active compound 1 in its three-dimensional structure; the right diagram shows the two-dimensional interaction.

[0025] Figure 3 This is a diagram showing the interaction between a protein and a representative inhibitor.

[0026] Figure 4 The expression of EZH2 in human hepatocellular carcinoma cell lines, human normal hepatocytes, human cholangiocarcinoma cell lines, human normal colonic epithelial cells, and human colorectal carcinoma cell lines is shown.

[0027] Figure 5 shows that compound 1 significantly downregulated the expression level of EZH2 in human hepatocellular carcinoma cell lines PLC / PRF / 5, colon cancer cell line HT9, and cholangiocarcinoma cell line QBC939.

[0028] Figure 6 Figure 1 shows the survival rates of cholangiocarcinoma cells QBC939, colon cancer cells HT29, and liver cancer cells PLC / PRF / 5 under concentration- and time-dependent conditions of compound 1. Figure A shows the cell survival rates of human colon cancer cell line HT29 after 48 h of culture at different concentrations of compound 1, and the time-dependent curves of the optimal compound concentration of 5 μM selected at 72 h. Figure B shows the cell survival rates of human cholangiocarcinoma cell line QBC939 after 48 h of culture at different concentrations of compound 1, and the time-dependent curves of the optimal compound concentration of 10 μM selected at 72 h. Figure C shows the cell survival rates of liver cancer cells PLC / PRF / 5 after 48 h of culture at different concentrations of compound 1, and the time-dependent curves of the optimal compound concentration of 5 μM selected at 72 h. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail with reference to the following specific examples.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0031] Example 1: Binding mode of the compound to EZH2

[0032] A combination of molecular docking and binding free energy calculations was employed: the crystal structure of the EZH2-small molecule inhibitor complex (PDBID: 5LS6) was selected as the starting structure. AutoDock Tools were used to sequentially remove all components except the protein, followed by hydrogenation, charge addition, protonation state confirmation, and energy optimization of the protein. The processed structure was used for subsequent virtual screening studies based on molecular docking. The grid points for the docking process were centered on the small molecule inhibitor, with side lengths of [missing information]. A cube-shaped box, where the length of each grid point is... Approximately 50,000 compounds were preprocessed through hydrogenation, charge addition, ensuring proper protonation states, and energy optimization. Their three-dimensional structures were then aligned with the generated EZH2 mesh file. During alignment, only one conformation of each compound was retained. AutoGrid 4 was used to acquire and normalize all atomic coordinates during alignment, while Autodock 4 was used to align small molecule compounds into binding pockets and perform scoring and sorting.

[0033] The top 500 compounds in docking scores were selected and re-doped into the binding pocket of EZH2. In the second round of molecular docking, three optimal binding conformations were retained for each compound to facilitate analysis of binding modes and structure-activity relationships. The binding modes of the top 100 compounds in docking scores were observed visually to analyze intramolecular tension, and the well-bound compounds underwent activity testing.

[0034] Among the compounds tested for inhibitory activity, compound 1 was determined to have a docking score of -8.5 with EZH2, and a binding free energy of -70.63 kcal / mol. The binding mode of compound 1 with EZH2 is as follows: Figure 1-3As shown, compound 1 binds tightly to EZH2 through a shape-matched binding pocket, primarily via hydrophobic and hydrogen-bonded interactions. The oxidized indole ring of compound 1 forms a pi-pi interaction with the benzene ring side chain of Phe686 in EZH2, while the benzene ring at the other end forms a side-to-side pi-pi interaction with the aromatic rings of Phe665 and Tyr111. Simultaneously, the indole ketone group of compound 1 forms a rich hydrogen bond network with the NH group of the Trp624 backbone of EZH2, the substituted hydroxyl group with the carbonyl group of the Ile109 backbone, and the carbonyl group attached to it with the NH group of the Tyr111 backbone. Therefore, compound 1 can inhibit EZH2, reducing its expression level and thus suppressing tumor proliferation.

[0035] The structural formula of compound 1 is as follows:

[0036]

[0037] Example 2: Cytotoxicity of Compound 1 against EZH2-overexpressing liver cancer, colon cancer, and cholangiocarcinoma, as well as immunoblotting and proliferation assays.

[0038] I. Cell-level experimental methods

[0039] 1. Cell resuscitation

[0040] Human hepatocellular carcinoma cell lines (HepG2, Hep3B, PLC / PRF / 5), human normal hepatocytes (L02), human cholangiocarcinoma cell lines (Hucct1, QBC939), human normal colonic epithelial cells (NCM460), and human colorectal carcinoma cell lines (HT29, Sw480) were all adherent cells, and all were cultured in DMEM complete medium. After rapid thawing from liquid nitrogen or a -80°C freezer, the cell suspension was transferred to 15mL EP tubes, 2mL of DMEM complete medium was added, and the tubes were sealed and centrifuged at 800rpm for 5min. The supernatant was discarded, and the cells were resuspended thoroughly in prepared DMEM complete medium. The resuspended cells were then added to cell culture flasks, gently agitated to ensure even distribution, and then cultured in a cell culture incubator (37°C, 5% CO2).

[0041] 2. Cell passage culture

[0042] 1) Take out cells that are in good condition and have grown to 80% of their bottom surface, discard the original culture medium, add an appropriate amount of PBS to wash them, and then pour out the PBS. Repeat this step three times.

[0043] 2) Add an appropriate amount of EDTA-trypsin digestion solution to the cleaned cell culture flask. After digestion, add an appropriate amount of complete culture medium, blow off the cells attached to the wall, and transfer them to a 15mL EP tube. Wrap the tube cap and place it in a centrifuge. Centrifuge at 800rpm for 5min, discard the supernatant, and use a pipette to remove the residual liquid. Add complete culture medium, use a dropper or pipette to blow off the precipitate, mix well, and transfer it to several cell culture flasks. Place them in a cell culture incubator for culture and observe their growth in real time.

[0044] 3. Cytotoxicity and proliferation experiments

[0045] 1) Pre-coating: To determine the optimal cell seeding concentration, before the formal experiment, a cell culture flask containing well-grown cells was used. The cells were digested with EDTA-trypsin and centrifuged. The supernatant was discarded, and the cells were resuspended in 1 ml of DMEM complete medium and resuspended. 200 μl of the resuspended cells were then added to 800 μl of DMEM complete medium and resuspended again. The stock solution was diluted 5-, 25-, 125-, and 625-fold. The diluted solutions and the stock solution were seeded into labeled 96-well plates according to these dilution ratios, and then placed in a cell culture incubator. Timing was started at 24, 48, and 72 hours after cell adhesion, and the optimal seeding concentration was observed at each time point to determine the final seeding concentration.

[0046] 2) Seeding: Seedling is carried out according to the concentration corresponding to the selected culture time of the pre-seeded plate.

[0047] 3) Treatment: After cell adhesion, the cells are treated to meet the experimental requirements.

[0048] 4) Prepare 1×MTT: Dilute 5×MTT to 1×MTT according to the kit from KGI Biotech and store at 4 degrees Celsius in the dark for later use.

[0049] 5) MTT reaction: After the treatment time is reached, the 96-well plate is removed, 50 μl of 1×MTT is added to each well in the dark, and then the plate is placed in an incubator and incubated for 4 h to carry out the formazan formation reaction.

[0050] 6) Color development: After 4 hours, the crystals formed by MTT and live cells adhere to the bottom of the 96-well plate. Discard the liquid in the 96-well plate, add 150 μl / well of DMSO, and place it in a 37°C constant temperature shaker in the dark for 15 minutes to dissolve and develop the color.

[0051] 7) Measure absorbance: Adjust the microplate reader to a wavelength of 490 nm and measure the absorbance.

[0052] 8) Data analysis: Survival rate = (absorbance of treatment group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) * 100%.

[0053] II. Protein Level Related Experiments

[0054] 1. Extraction of total cell protein

[0055] 1) Remove the cultured cells from the 6-well plate, discard the culture medium, and wash with PBS. Scrape off as many cells as possible and transfer them to EP tubes. Centrifuge at 800g and 4°C for 5 minutes using a high-speed refrigerated centrifuge.

[0056] 2) Discard the supernatant, add an appropriate amount of lysis buffer (1 mL lysis buffer = 950 mL RIPA + 40 mL PPI + 10 mL PMSF) to the precipitate, mix well, lyse on ice for 30 min, then centrifuge at 13000 r for 15 min, and collect the supernatant in a centrifuge tube to obtain the protein.

[0057] 3) Use the Beyotime BCA kit and strictly follow the instructions to determine the protein concentration.

[0058] 2. Western Blot Experimental Method

[0059] 1) Sample preparation: Take out the extracted protein sample, add 5× protein loading buffer. Therefore, the protein concentration needs to be multiplied by 0.8 to obtain the protein loading concentration. Boil the sample at 100℃ for 5 minutes.

[0060] 2) Electrophoresis: Place an 8% separating gel and a 5% stacking gel in the electrophoresis tank, add 1× electrophoresis buffer to cover both plates, remove the comb to load the samples, and then place the tank in an ice box. First, set the electrophoresis apparatus to 80V for 40 minutes. When the proteins are compressed into a single line and the stacking gel has finished running, switch to 120V until the proteins reach the appropriate position.

[0061] 4) Transfer: Take out the cut PVDF membrane, activate it in anhydrous methanol for 10 seconds, prepare the recovered electrophoresis buffer, pry open the glass plate in the recovered electrophoresis buffer, scrape off the stacking gel, and gently transfer the separating gel portion to the prepared clamp-sponge-filter paper. Mark the activated PVDF membrane and carefully attach it to the gel. The gel after electrophoresis will show the color of the marker. Use a small roller to remove air bubbles between the gel and the membrane. Attach the filter paper and sponge to form a "sandwich" structure. Clamp the plate and place it in the transfer tank. Pour in the transfer buffer to cover the membrane and place it in an ice box. While closing the lid, check the positive and negative electrodes. After covering the transfer tank with ice and closing the lid, adjust the electrophoresis apparatus to 100V for 90 minutes.

[0062] 5) Blocking: Place two PVDF membranes containing the transferred protein back-to-back into the same container filled with blocking solution and incubate for 1 hour. Discard the blocking solution and wash with 1×TBST for 10 minutes each time, 3 times.

[0063] 6) Incubation with primary antibody: Cut the membrane according to the molecular weight and incubate with the corresponding primary antibody at 4°C overnight. Recover the primary antibody and wash with 1×TBST for 10 min × 3 times.

[0064] 7) Incubation of secondary antibody: Select a secondary antibody according to the type of primary antibody and incubate at room temperature for 1 hour. Recover the secondary antibody and wash with 1×TBST for 10 minutes × 3 times.

[0065] 8) Development: Prepare the developing solution, coat it evenly on the membrane, and perform chemical development. Preserve the bands for subsequent analysis.

[0066] III. Experimental Results

[0067] 1. For example Figure 4 As shown, EZH2 is expressed in multiple cell lines, and it is highly expressed in hepatocellular carcinoma cells PLC / PRF / 5, cholangiocarcinoma cells QBC939 and Hucct1, and colon cancer cells HT29 and Sw480.

[0068] 2. The results are shown in Figure 5. Compound 1 clearly downregulated EZH2 in hepatocellular carcinoma cells PLC / PRF / 5, cholangiocarcinoma cells QBC939, and colon cancer cells HT29.

[0069] 2. A small concentration gradient (10 μM, 5 μM, 1 μM, 0.5 μM) was selected for a 48-hour MTT assay on colon cancer cells HT29, bile duct cancer cells QBC939, and liver cancer cells PLC / PRF / 5. The results are as follows. Figure 6 As shown, compared to cholangiocarcinoma cells QBC939, colon cancer cells HT29 and liver cancer cells PLC / PRF / 5 were significantly more sensitive to compound 1. Based on this, time-dependent (4, 18, 24, 48, 72 h) cell proliferation experiments were conducted on compound 1 at concentrations of 5 μM or 10 μM. The results showed that compound 1 achieved its best effect in HT29 cells at approximately 48 h, in QBC939 cells at approximately 18 h, and in PLC / PRF / 5 cells at approximately 48 h. This indicates that compound 1 has high safety and can inhibit tumor cell proliferation.

[0070] Example 3: Based on the 3-hydroxy-2-oxoindole core structure, several compounds were rationally designed, all exhibiting strong EZH2 binding.

[0071] The core structure of 3-hydroxy-2-oxoindole is shown below:

[0072]

[0073]

[0074]

[0075]

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A 3-hydroxy-2-oxoindole compound, characterized in that, The 3-hydroxy-2-oxoindole compound is: 。 2. The use of the 3-hydroxy-2-oxoindole compound of claim 1 in the preparation of EZH2 inhibitors.

3. The use of the 3-hydroxy-2-oxoindole compound according to claim 1 in the preparation of antitumor drugs.

4. The application according to claim 3, characterized in that, In antitumor drugs, the concentration of the 3-hydroxy-2-oxoindole compound is 0.5 μM-10 μM.

5. The application according to claim 3, characterized in that, The reaction time of the 3-hydroxy-2-oxoindole compound is 18-48 h.

6. The application according to claim 3, characterized in that, The 3-hydroxy-2-oxoindole compounds inhibit tumor cell proliferation by downregulating EZH2 expression.

7. The application according to claim 4, characterized in that, The tumor is liver cancer, bile duct cancer, or colon cancer.

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