Use of pyrrolidone derivative in preparation of GPI small-molecule inhibitor

Abstract

Provided is the use of a pyrrolidone derivative in the preparation of a GPI small-molecule inhibitor. The pyrrolidone derivative has a chemical structural formula as represented by formula (I): formula (I). The pyrrolidone derivative binds to GPI with high affinity and specifically inhibits GPI activity, thereby modulating the metabolism, particularly lactate accumulation, in a tumor microenvironment. The pyrrolidone derivative not only significantly enhances the killing effects of immune cells against tumor cells, but also potentiates the efficacy of an immunotherapeutic agent as an adjuvant drug, thereby enhancing the efficacy of tumor immunotherapy. The pyrrolidone derivative can be widely used in the field of tumor immunotherapy.

Description

Application of pyrrolidone derivatives in preparation of GPI small molecule inhibitors TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, more particularly, to application of pyrrolidone derivatives in preparation of GPI small molecule inhibitors. BACKGROUND

[0002] At present, the traditional means of tumor treatment mainly include surgery, radiotherapy, chemotherapy, etc., but these therapies are usually accompanied by large side effects and recurrence risk. In recent years, the development of immunotherapy has brought a major breakthrough to cancer treatment, especially the immunological checkpoint inhibitor (ICIs) therapy represented by anti-PD-1 and anti-CTLA-4, which significantly improves the survival period of some cancer patients. When tumor cells proliferate or invade, the immune system can recognize tumor-specific antigens on the surface of tumor cells, activate humoral immune response and cellular immune response, and thus eliminate tumor cells. However, the immunosuppressive mechanism in the tumor microenvironment makes some patients resistant or non-responsive to these immunotherapies. Therefore, how to enhance the effect of immunotherapy and improve the recognition and killing ability of tumor cells has become the focus of current research.

[0003] Tumor immunotherapy activates the immune system through exogenous intervention, maintains tumor-immune circulation, and thus improves the body's anti-tumor immune response. This therapy has high specificity, can specifically kill tumor cells, and has relatively small side effects. However, immunotherapy also has certain limitations, for example, as the treatment progresses, some patients may develop immune escape or resistance to immune checkpoint inhibitors. In addition, immunotherapy is ineffective for some patients, and may even accelerate tumor progression. This indicates that single dependence on immune checkpoint inhibitors cannot effectively solve the challenges of tumor immunotherapy, and new adjuvant therapies are urgently needed to overcome these obstacles.

[0004] A large number of studies have shown (for example, KRAS mutations enhance lactate production by promoting glycolysis, inhibit immune responses in the tumor microenvironment

PMID:39348506

PMID:38417442

PMID:37327788

[0005] 6-phosphogluconate isomerase (GPI) is a key glycolytic enzyme that can catalyze the reversible conversion between 6-phosphogluconate and 6-phosphofructose. In various cancers, high expression of GPI is closely related to the invasiveness, metastatic ability and poor prognosis of tumors. Studies have found that GPI not only promotes tumor metabolism, but also enhances the immune escape ability of tumor cells by regulating lactate levels in the tumor microenvironment. In malignant tumors such as lung cancer and intestinal cancer, GPI activity is significantly increased and is closely related to the diagnosis and prognosis of patients. Therefore, GPI has broad application prospects as a target for anti-cancer therapy.

[0006] Currently, although the role of GPI in cancer has been extensively studied, there is no GPI inhibitor approved for marketing. Developing GPI inhibitors, especially those that can significantly inhibit tumor cell metabolism, reduce lactate accumulation and enhance the effectiveness of immunotherapy, has important clinical value.

[0007] SUMMARY

[0008] The primary object of the present application is to overcome the problems existing in the prior art, and to provide the application of pyrrolidone derivatives in the preparation of GPI small molecule inhibitors. The present application provides the application of pyrrolidone derivatives in the preparation of GPI small molecule inhibitors, which binds to and specifically inhibits the activity of GPI with high affinity, controls the metabolic pathways in the tumor microenvironment, especially the accumulation of lactate, thereby not only significantly enhancing the killing effect of immune cells on tumor cells, but also enhancing the effectiveness of immunotherapeutic agents in the form of an adjuvant drug, thereby enhancing the effectiveness of tumor immunotherapy, and can be widely used in the field of tumor immunotherapy. In addition, the pyrrolidone derivative exhibits good pharmacokinetic properties and safety to normal cells.

[0009] The above object of the present application is achieved by the following technical solutions.

[0010] Application of pyrrolidone derivatives in preparation of GPI (6-phosphoglucose isomerase) small molecule inhibitors, wherein the chemical structure of the pyrrolidone derivatives is shown in formula (I):

[0011] wherein R1, R2, R3 and R4 are independently H, halogen, -CN, -NO2, -OH, -NH2, C 1～6 alkyl, C 1～6 alkoxy, aryl or heteroaryl.

[0012] Preferably, the halogen is at least one of chlorine, bromine, iodine or fluorine.

[0013] Preferably, the C 1～6 alkyl includes but is not limited to at least one of methyl, ethyl, propyl or butyl, pentyl or hexyl.

[0014] Preferably, the C 1～6 alkoxy includes but is not limited to at least one of methoxy, ethoxy, propoxy or butoxy, pentoxy or hexyloxy.

[0015] Preferably, the aryl is at least one of phenyl, tolyl, naphthyl or xylyl.

[0016] Preferably, R1 is H, R2 is methoxy, R3 is -NH2 and R4 is H.

[0017] Preferably, the GPI small molecule inhibitor is a drug for treating / preventing tumors.

[0018] More preferably, the drug is a drug for enhancing the killing effect of immune cells on tumor cells.

[0019] Further preferably, the immune cells are T cells.

[0020] Further preferably, the T cells are CD8+ T cells.

[0021] More preferably, the tumor is at least one of cancer or benign tumor.

[0022] Further preferably, the cancer is at least one of intestinal cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, kidney cancer or melanoma.

[0023] More preferably, the drug is an auxiliary drug for enhancing the therapeutic effect of tumor immunotherapy.

[0024] Further preferably, the immunotherapeutic agent used in the tumor immunotherapy is at least one of an anti-PD-1 antibody or an anti-CTLA-4 antibody.

[0025] The pyrrolidone derivative in combination with the immunotherapeutic agent shows good synergistic anti-tumor effect.

[0026] Further preferably, the anti-PD-1 antibody is at least one of pembrolizumab, nivolumab, atezolizumab, durvalumab or avelumab.

[0027] Further preferably, the anti-CTLA-4 antibody is at least one of ipilimumab or tremelimumab.

[0028] More preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0029] Further preferably, the pharmaceutically acceptable excipient is at least one of a pharmaceutically acceptable carrier, diluent or excipient.

[0030] More preferably, the dosage form of the medicine is an injection, a capsule, a tablet, a pill or a granule.

[0031] More preferably, the medicine further comprises a pharmaceutically acceptable salt or solvate of the pyrrolidone derivative.

[0032] Specifically, the pharmaceutically acceptable salt includes but is not limited to: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate (mesylate), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate; or an ammonium salt (such as a primary, secondary, tertiary, or quaternary ammonium salt), a metal salt (such as a sodium, potassium, calcium, magnesium, manganese, iron, zinc, copper, lithium, or aluminum salt).

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] This invention provides the application of pyrrolidone derivatives in the preparation of small molecule GPI inhibitors. These pyrrolidone derivatives bind with high affinity and specifically inhibit GPI activity, controlling metabolic pathways in the tumor microenvironment, particularly lactate accumulation. This not only significantly enhances the killing effect of immune cells on tumor cells but also enhances the efficacy of immunotherapeutic agents as adjuvant drugs, thereby strengthening the effect of tumor immunotherapy. This invention has wide applications in the field of tumor immunotherapy. Furthermore, these pyrrolidone derivatives exhibit good pharmacokinetic properties and safety in normal cells. Attached Figure Description

[0035] Figure 1 is a schematic diagram of the interaction between compound GW002 of Example 1 and the GPI protein through virtual docking. The figure shows the key binding sites of compound GW002 and GPI and their binding mode.

[0036] Figure 2 shows the surface plasmon resonance (SPR) results of the interaction between compound GW002 and GPI protein in Example 1.

[0037] Figure 3 shows the experimental results of the effect of compound GW002 from Example 2 on the proliferation of colon cancer cell lines SW620 (Figure 3A) and SW4800 (Figure 3B).

[0038] Figure 4 shows the experimental results of the effect of compound GW002 from Example 2 on the proliferation of normal colon cells NCM460.

[0039] Figure 5 shows the experimental results of the effect of compound GW002 in Example 2 on the co-incubation model of MC38 colon cancer cells expressing OVA and OT1 mouse T cells.

[0040] Figure 6 shows the experimental results of the effect of compound GW002 from Example 2 in a co-incubation model of human peripheral blood mononuclear cells (PBMCs) and colon cancer cells SW620.

[0041] Figure 7 shows the experimental results of the inhibitory effect of compound GW002 from Example 3 on tumor growth in a mouse subcutaneous xenograft model of intestinal cancer.

[0042] Figure 8 shows the experimental results of the effect of compound GW002 from Example 3 on tumor volume changes in a mouse subcutaneous xenograft model of intestinal cancer.

[0043] Figure 9 shows the experimental results of the effect of compound GW002 from Example 3 on tumor weight in a mouse subcutaneous xenograft model of intestinal cancer.

[0044] Figure 10 shows the experimental results of the effect of compound GW002 from Example 3 on the intratumoral lactate level in a mouse subcutaneous xenograft model of intestinal cancer.

[0045] Figure 11A shows the injection sequence and number of injections in each group of the immunotherapy adjuvant effect verification experiment of compound GW002 in Example 3; Figures 11B and C show the experimental results of compound GW002, anti-PD-1 antibody alone and in combination in Example 3. Detailed Implementation

[0046] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0047] Example 1: Study on the specific targeting characteristics of pyrrolidone derivatives to GPI

[0048] The pyrrolidone derivative of this embodiment is 5-amino-4-(1,3-benzothiazol-2-yl)-1-(2-methoxybenzyl)-1,2-dihydro-3H-pyrrol-3-one (denoted as compound GW002), with PubChem CID 136021791, and its chemical structural formula is as follows:

[0049] (1) Virtual docking study of small molecules and GPI protein using computer software (MOE software)

[0050] 1.1.1 Obtaining the structure of GPI protein

[0051] The crystal structure of glucose-6-phosphate isomerase (GPI) protein was downloaded from the PDB database and preliminarily processed in MOE software to ensure that the protein structure was intact and without any inactivated parts.

[0052] 1.1.2 Obtaining the structure of compound GW002

[0053] Download the three-dimensional structure file (SDF format) of compound GW002 from the SciFinder database and import it into MOE software for small molecule structure preprocessing, including geometry optimization and energy minimization.

[0054] 1.1.3 Pretreatment of Proteins and Small Molecules

[0055] In the MOE software, the processed GPI protein structure and the small molecule structure of compound GW002 were imported, respectively. MOE's QuickPrep function was used to preprocess the protein and small molecule to ensure that the protein structure was free of water molecules and had an appropriate charge distribution. Simultaneously, energy minimization was performed on compound GW002 to achieve its optimal conformation.

[0056] 1.1.4 Virtual docking operation

[0057] In the Compute module of the MOE software, select the Dock function, import compound GW002 as the ligand and GPI protein as the receptor, and set the binding pocket to the known active site region. Click Run to start the virtual docking simulation. The system will calculate the binding fraction based on the interaction energy between compound GW002 and GPI.

[0058] 1.1.5 Analysis of Virtual Docking Results

[0059] The docking results are shown in Figure 1. Compound GW002 forms a stable binding port with the GPI protein. Compound GW002 can insert into the binding port of GPI, forming hydrogen bonds and hydrophobic interactions with key residues. The virtual docking score is -7.06, indicating that compound GW002 has a high binding affinity to GPI, meeting the requirements for further experimental verification.

[0060] (2) Interaction experiment between compound GW002 and GPI protein

[0061] 1.2.1 GPI protein preparation

[0062] The purchased GPI protein (Abcam, catalog number ab87625) underwent preliminary purification. The GPI protein solution was concentrated using a 3k ultrafiltration tube. After adding 500 μL of ultrapure water, the solution was centrifuged at 14000 x g at 4°C for 30 minutes. This step was repeated three times to further remove impurities. The ultrafiltration tube was then inverted into a clean collection tube and centrifuged at 1000 x g for 2 minutes to collect the concentrated GPI protein solution for subsequent experiments.

[0063] 1.2.2 Buffer Preparation

[0064] ① 1x PBS (phosphate) solution: Pour 1 packet of PBS powder into 2L of ultrapure water, mix well, and then filter to obtain the solution.

[0065] ② 5% DMSO in PBS: Add 25 mL of DMSO solution to 500 mL of 1x PBS solution and mix well.

[0066] ③ 4.5% DMSO in PBS: Add 0.45 mL of DMSO solution to 9.5 mL of 1x PBS solution and mix well.

[0067] ④ 5.8% DMSO in PBS: Add 0.58 mL of DMSO solution to 9.5 mL of 1x PBS solution and mix well.

[0068] ⑤ Prepare a 5% DMSO concentration calibration curve by mixing 4.5% DMSO and 5.8% DMSO according to the table below.

[0069] Table 1

[0070] 1.2.3 Small molecule sample preparation

[0071] Take 10 mM of the stock solution of compound GW002 (purchased from TargetMol, L5600-Mini Scaffold Library, ID: F187-0104), and dilute it 20-fold with 1x PBS to obtain a 500 μM solution with 5% DMSO. Continue to dilute the sample with 5% DMSO in PBS buffer to obtain gradient solutions of different concentrations. The highest injection concentration was set at 50 μM, and the concentrations were halved downwards at the following gradients: 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.56 μM. A repeat concentration was set at intervals during the dilution process to ensure the reproducibility and reliability of the data.

[0072] 1.2.4 SPR Experiment Procedure

[0073] Place the prepared samples sequentially into the Biacore S200 biomolecular interaction system according to their location numbers. Set the experimental parameters, including sample flow rate, binding time, and dissociation time, ensuring all experimental conditions are consistent. Start the experiment and monitor the interaction between compound GW002 and the GPI protein.

[0074] 1.2.5 Data Analysis

[0075] After the experiment, data analysis was performed using Biacore S200 Evaluation Software. The Kinetics model was selected in the Evaluation module to fit the data and calculate the binding affinity between compound GW002 and the GPI protein.

[0076] As shown in Figure 2, the fitting results indicate that the affinity constant (KD value) between compound GW002 and GPI protein is 5.63 × 10⁻⁶. 6 M. This result indicates that compound GW002 forms a non-covalent bond with GPI protein and has a high binding affinity.

[0077] Example 2: Effects of pyrrolidone derivatives on cancer cells

[0078] The pyrrolidone derivative in this embodiment is compound GW002 from Example 1.

[0079] The colon cancer cell lines SW620, SW480, MC38, and CT26, as well as the normal colon cell line NCM460, were all purchased from ATCC. These cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics in a 37°C, 5% CO2 incubator. Human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin antibiotics, also at 37°C, 5% CO2. All cells were maintained in logarithmic growth phase through routine passages before the experiment to ensure cell viability and proliferative capacity.

[0080] (1) Colon cancer cell proliferation experiment

[0081] 2.1.1 Cell Seeding

[0082] SW480, SW620 and NCM460 cells were collected from the culture, counted, and then seeded 3000 cells into each well of a 96-well plate, ensuring that the cell count in each well was consistent.

[0083] 2.1.2 Drug treatment

[0084] The seeded cells were cultured overnight in a cell culture incubator at 37°C and 5% CO₂ to allow for cell adhesion and recovery of growth. Next, for colon cancer cells, 0.1% DMSO (control group) or 5 μM compound GW002 (experimental group) was added to each well, and incubation was continued under the same conditions for 48 hours. For normal colon cells, 0.1% DMSO (control group), 5 μM compound GW002 (experimental group), or 10 μM compound GW002 (experimental group) was added to each well, and incubation was continued under the same conditions for 48 hours.

[0085] 2.1.3 Cell proliferation detection

[0086] After incubation, the cells in the 96-well plates were digested with trypsin. Cells in each well were counted using a cell counter, and cell proliferation in different treatment groups was recorded. To ensure the reliability of the results, each experiment was repeated three times, and the results were statistically analyzed.

[0087] 2.1.4 Experimental Results

[0088] The experimental results are shown in Figure 3. After 48 hours of treatment, compared with the 0.1% DMSO control group, compound GW002 did not significantly inhibit the proliferation of colon cancer cells SW480 and SW620. The results indicate that compound GW002 does not directly inhibit or kill the proliferation of colon cancer cells. Therefore, the mechanism of action of compound GW002 on cancer cells is not directly through cytotoxicity, but may depend on the regulation of the tumor microenvironment or the mediation of immune cells. Furthermore, as shown in Figure 4, compound GW002 also did not significantly inhibit the proliferation of normal colon cells NCM460, indicating that it has good safety (low toxicity).

[0089] (2) Study on the tumor-killing effect of T cells in co-culture

[0090] 2.2.1 Isolation and extraction of CD8+ T cells from OT1 mice

[0091] a) Splenic separation

[0092] Under aseptic conditions, the spleens of OT1 transgenic mice were dissected and isolated, then rinsed twice in ice-cold PBS solution to remove surface blood and impurities. The spleens were then transferred to a cell sieve.

[0093] b) Tissue grinding

[0094] Gently grind the spleen tissue using a sterile tissue grinding rod until no obvious red lumps remain and the tissue is fully dissociated into a cell suspension.

[0095] c) Cell collection and centrifugation

[0096] Rinse the cell filter with 15 mL of ice-cold PBS and collect the rinsing solution in a 15 mL centrifuge tube. Centrifuge at 300 × g for 5 minutes, discard the supernatant, and retain the cell pellet.

[0097] c) Red blood cell lysis

[0098] Add 2 mL of 1× erythrocyte lysis buffer to the cell pellet (operate at room temperature), resuspend the cells, and lyse for 5 minutes. After lysis, immediately add 10 mL of PBS to neutralize, centrifuge at 300×g for 5 minutes, and discard the supernatant.

[0099] d) Cell resuspension and filtration

[0100] Spleen cells were resuspended in PBS containing 2% FBS (fetal bovine serum), and then the cell suspension was filtered again through a 200-mesh sieve. The filtered cells were counted, and the cell concentration was adjusted to 1×10⁻⁶. 8 cells / mL.

[0101] e) CD8+ T cell isolation

[0102] CD8+ T cells were isolated using the Stemcell mouse CD8+ T cell isolation kit. 50 μL of mouse serum was added to the cell suspension and mixed thoroughly. 50 μL of separation mixture was added, mixed thoroughly, and incubated at room temperature for 10 minutes. 125 μL of magnetic beads was added to the cell suspension, mixed thoroughly, and incubated at room temperature for 5 minutes.

[0103] 2.2.2 MC38 cells express OVA antigen

[0104] a) MC38 cell infection

[0105] Lentiviral fluid carrying the OVA antigen gene and polyethyleneimine (PEI) were added to the MC38 cell culture medium and incubated for 48 hours to induce MC38 cells to overexpress the OVA antigen.

[0106] b) Screening for stable expression cell lines

[0107] Infected MC38 cells were selected using 2 μg / mL puromycin. After 48 hours, the viral load was removed, fresh culture medium was added, and 2 μg / mL puromycin was added again. The culture medium was changed every 24 hours, and this treatment was repeated three times to obtain MC38 cell lines stably expressing OVA antigen.

[0108] c) MC38-OVA cell seeding

[0109] The selected MC38-OVA cells were seeded in 96-well plates at a density of 20,000 cells / well and cultured overnight at 37°C and 5% CO2 to ensure cell adhesion and stable growth.

[0110] 2.2.3 Co-culture experiment of MC38-OVA cells and CD8+ T cells

[0111] a) CD8+ T cell activation

[0112] The isolated CD8+ T cells were activated and cultured in 96-well plates for 24 hours using anti-CD3 (400 ng / mL) and anti-CD28 (400 ng / mL) antibodies in combination with IL-2 (50 IU / mL). These stimulating factors promoted the proliferation and activation of CD8+ T cells through a signaling pathway mimicking the T cell receptor (TCR), preparing the cells for subsequent co-culture experiments.

[0113] b) Co-culture of CD8+ T cells and MC38 OVA cells

[0114] Overnight activated CD8+ T cells were added at a concentration of 100,000 cells / well to 96-well plates pre-platened with MC38 OVA cells (20,000 cells / well). MC38 OVA cells express OVA antigen to mimic specific T cell-mediated tumor killing.

[0115] c) Drug treatment

[0116] In the co-culture system, DMSO (control group) or compound GW002 (experimental group) were added for drug treatment. Low (1 μM) and high (5 μM) concentrations of compound GW002 were used as predetermined gradients. Cells were then incubated at 37°C and 5% CO₂ for 48 hours to evaluate the effect of compound GW002 on CD8+ T cell-mediated tumor cell killing.

[0117] d) Cell killing detection

[0118] To evaluate the killing effect of CD8+ T cells on MC38 OVA tumor cells, the release of lactate dehydrogenase (LDH) in the culture medium was measured. LDH is an intracellular stabilizing enzyme; when cells undergo apoptosis or necrosis, cell membrane damage leads to the release of LDH into the culture medium. A commercial LDH detection kit (Promega's CytoTox) was used. The Non-Radioactive Cytotoxicity Assay reflects the relative amount of LDH content in the culture medium, indicating the relative death of MC38 OVA cells caused by CD8+ T cells.

[0119] e) Experimental Results

[0120] The experimental results are shown in Figure 5. The GW002 treatment groups (including low-dose and high-dose groups) showed a significant increase in the killing ability of CD8+ T cells against MC38 OVA tumor cells compared to the DMSO control group. The tumor-killing effect of T cells increased with increasing GW002 dosage. This indicates that GW002 enhances the killing effect of immune cells on tumor cells and promotes the activation of the tumor immune response.

[0121] 2.2.4 Isolation of PBMCs (Peripheral Blood Mononuclear Cells)

[0122] a) Dilution of peripheral blood

[0123] Take 30 mL of human peripheral blood sample (blood from a healthy person provided by a volunteer) and pour it into an equal volume of 60 mL of sterile PBS buffer. Mix gently and dilute.

[0124] b) Density gradient centrifugation

[0125] Add 10 mL of Ficoll solution to a 50 mL centrifuge tube. Carefully add the diluted peripheral blood (approximately 20 mL) slowly to the top layer of the Ficoll solution using a dropper. Ensure clear liquid separation during the procedure to avoid mixing and facilitate PBMC separation.

[0126] c) Density gradient centrifugation

[0127] Place the centrifuge tubes in a horizontal rotor centrifuge and centrifuge at 300xg for 30 minutes at room temperature for density gradient centrifugation. After centrifugation, four layers are formed in the tube: the top layer is plasma and PBS, the middle floating flocculent layer is the target PBMCs, and the bottom layer is red blood cells and other densely packed cellular components.

[0128] d) Collection of PBMCs

[0129] Carefully aspirate the PBMC cells from the middle flocculent layer using a sterile dropper and transfer them to a new 50 mL centrifuge tube. Dilute with 3 volumes of RPMI-1640 medium. Centrifuge at 1500 rpm for 10 minutes, discard the supernatant, and retain the PBMC cell pellet.

[0130] e) Cell washing and resuspension

[0131] PBMC cells were resuspended in RPMI-1640 medium and washed twice to ensure the removal of Ficoll and plasma residues. The cells were then resuspended in an appropriate amount of medium, counted, and adjusted to a suitable concentration for subsequent experiments.

[0132] f) Activation of PBMC

[0133] To enhance the activity of T cells in PBMCs, CD3 (400 ng / mL) and CD28 (400 ng / mL) antibodies were added to resuspended PBMCs, and T cells were activated in combination with IL-2 (50 IU / mL). The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours to ensure that the T cells were fully activated and ready for subsequent experiments.

[0134] 3.2.5 Co-culture experiment of PBMC cells and SW620 colon cancer cells

[0135] a) SW620 cell plating

[0136] SW620 colon cancer cells were seeded into 96-well plates at a density of 20,000 cells / well and cultured overnight at 37°C in a 5% CO2 incubator to ensure cell adhesion and stable growth.

[0137] b) Co-culture of PBMCs and SW620 cells

[0138] After 24 hours of activation, PBMC cells were added at a ratio of 100,000 cells / well to 96-well plates pre-coated with SW620 cells, ensuring uniform distribution of PBMC cells in each well. This co-culture system was used to simulate the killing effect of immune cells (especially T cells) in PBMCs on tumor cells.

[0139] c) Drug treatment

[0140] DMSO (control group) or compound GW002 (experimental group) were added to the co-culture system, and low-dose (1 μM) and high-dose (5 μM) of compound GW002 were set according to the experimental design. The cells were incubated at 37°C and 5% CO2 for 48 hours to evaluate the effect of compound GW002 on the killing ability of PBMC cells mediated by SW620 tumor cells.

[0141] d) Cell killing detection

[0142] To evaluate the killing effect of PBMC cells on SW620 colon cancer cells, the LDH content in the culture medium was measured. The amount of LDH released in the culture medium reflects the killing effect of PBMC cells on SW620 tumor cells.

[0143] e) Experimental Results

[0144] The experimental results are shown in Figure 6. After 48 hours of co-culture, compared with the DMSO control group, both the low-dose and high-dose groups of compound GW002 significantly enhanced the killing ability of PBMC cells against SW620 colon cancer cells. This indicates that compound GW002 can promote the immune killing effect of PBMC cells against tumor cells, demonstrating that compound GW002 can regulate the anti-tumor response activity of immune cells.

[0145] Example 3: Study on the inhibitory effect of pyrrolidone derivatives on tumor growth in a mouse tumor model.

[0146] The pyrrolidone derivative in this embodiment is compound GW002 from Example 1.

[0147] (1) Effects of compound GW002 on mouse subcutaneous intestinal cancer xenograft model

[0148] 3.1.1 Preparation of CT26 colon cancer cells

[0149] CT26 mouse colon cancer cells in logarithmic growth phase were harvested, digested with trypsin, and then the cells were counted and resuspended in ice-cold PBS to prepare a cell suspension. The final concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6Cells / mL were collected, and the cell suspension was kept on ice for later use in the construction of a mouse subcutaneous tumor model.

[0150] 3.1.2 Construction of a mouse subcutaneous xenograft model

[0151] Select 3-5 week old Balb / c mice, and subcutaneously inject 100 μL of CT26 cell suspension into each mouse on both sides of the abdomen and back (total injection 1×10⁶ cells). 6 (cells). After injection, the mice were placed in a normal feeding environment to observe tumor growth.

[0152] 3.1.3 Tumor volume monitoring and grouping

[0153] When the subcutaneous tumor reaches a size of approximately 50 mm 3 At time (usually 7-10 days after inoculation), mice were randomly divided into 3 groups, each containing at least 6 mice, as follows: control group, injected with an equal volume of physiological saline or DMSO solution; low-dose GW002 group, with a dose of 5 mg / kg, injected intraperitoneally every three days; high-dose GW002 group, with a dose of 20 mg / kg, injected intraperitoneally every three days.

[0154] In verifying the adjuvant effect of immunotherapy agents, CT26 cells were transplanted into the cecum in situ, and the specific groups were as follows: control group, injected with an equal volume of physiological saline or DMSO solution; compound GW002 administration group, dose of 20 mg / kg, intraperitoneal injection every two days; immunotherapy agent anti-PD-1 antibody (#BP0273, BioXCell) administration group, dose of 200 μg / kg, intraperitoneal injection every three days; combination administration group, compound GW002 (20 mg / kg) + anti-PD-1 antibody (200 μg / kg), the injection sequence and number of times are shown in Figure 11A.

[0155] 3.1.4 Tumor growth and weight monitoring

[0156] During the 20-day continuous administration period, the size of the subcutaneous tumors in mice was measured every three days using calipers, and the tumor volume was calculated (formula: tumor volume = length × width). 2 (×0.5). Simultaneously, mouse body weight was recorded, and a weight growth curve was plotted to assess the effects of compound GW002 on mouse health.

[0157] 3.1.5 Tumor tissue dissection and weighing

[0158] Twenty days after administration, the mice were sacrificed and dissected. The tumor tissue under the skin of the mice was removed and weighed on a balance. The tumor weight of each group of mice was recorded, and the tumor inhibition rate was calculated (formula: tumor inhibition rate = (tumor weight of control group - tumor weight of experimental group) / tumor weight of control group × 100%) to evaluate the inhibitory effect of compound GW002 on tumor growth.

[0159] (2) Detection of lactic acid content in mouse tumor tissue

[0160] 3.2.1 Tumor tissue preparation

[0161] Weigh 10 mg of mouse tumor tissue and wash it with ice-cold PBS solution to remove blood and impurities. After washing, transfer the tumor tissue to a tissue homogenate tube.

[0162] 3.2.2 Preparation of tissue homogenate

[0163] Add 200 μL of detection buffer to the tumor tissue and homogenize it using a tissue homogenizer until no obvious particles are visible. Ensure thorough homogenization to release intracellular lactate.

[0164] 3.2.3 Centrifuge and collect the supernatant

[0165] The homogenized sample was placed in a centrifuge and centrifuged at 12,000 × g at 4°C for 10 minutes. After centrifugation, the supernatant was carefully collected for lactic acid content determination.

[0166] 3.2.4 Preparation of Samples and Standards

[0167] Add 50 μL of distilled water to the blank wells of the 96-well plate, add 50 μL of standard (a lactic acid standard solution of known concentration) to the standard tube, and add 50 μL of sample supernatant to each test well.

[0168] 3.2.5 Add detection reagents and incubate.

[0169] Add 200 μL of the mixed detection reagent (prepared according to the lactate detection kit instructions) to each well and incubate at 37°C in the dark for 30 minutes. After incubation, measure the absorbance of each well at a wavelength of 570 nm.

[0170] 3.2.6 Calculate the absorbance difference

[0171] ΔA measurement = A measurement tube - A blank tube

[0172] ΔA standard = A standard tube - A blank tube

[0173] The lactic acid content in the sample is assessed by calculating the absorbance difference.

[0174] 3.2.7 Plotting the Standard Curve

[0175] A standard curve was plotted with the concentration of each standard solution as the x-axis and the corresponding absorbance difference (ΔA standard) as the y-axis, yielding the standard equation y = kx + b. The measured ΔA value of the sample was then substituted into the equation to calculate the lactic acid concentration x (μmol / mL) in the sample.

[0176] 3.2.8 Calculation of lactic acid content

[0177] Lactic acid content (μmol / g tissue mass) = x × 50 μL ÷ (W × V supernatant ÷ total volume)

[0178] Where W is the mass of the tumor tissue and Vsupernatant is the volume of the supernatant. The formula can be simplified to:

[0179] Lactic acid content = 1.1875 × x ÷ W, to finally calculate the concentration of lactic acid content.

[0180] (3) Analysis of experimental results

[0181] Figure 7 shows the appearance of the dissected subcutaneous xenograft of intestinal cancer 20 days after administration. The results indicate that the high-dose group of compound GW002 significantly inhibited tumor growth in mice, while the tumor size of the control and low-dose groups showed no significant change.

[0182] Figure 8 shows the changes in tumor volume in mice during drug administration. The results indicate that the high-dose group of compound GW002 significantly inhibited tumor growth, while the low-dose group had no significant effect on tumor volume.

[0183] Figure 9 shows the final weight of tumors in each group of mice. The results indicate that the high-dose group of compound GW002 significantly reduced tumor weight, while the low-dose group did not show a significant reduction effect.

[0184] Figure 10 shows the changes in total lactate levels in mouse tumor tissues after 20 days of administration. The results indicate that the high-dose compound GW002 significantly reduced lactate levels in tumor tissues. This suggests that compound GW002 can effectively reduce lactate accumulation in the tumor microenvironment, thereby alleviating lactate-mediated immunosuppression and enhancing the killing ability of immune cells (especially CD8+ T cells) against tumor cells.

[0185] Figures 11B and C show that GW002 alone or anti-PD-1 antibody can significantly inhibit tumor growth, while the combined treatment group has a more significant inhibitory effect. Specifically, the combined treatment group showed a significant reduction in the proliferation of orthotopic colon tumors in mice and the most significant decrease in tumor weight, indicating that GW002 produces a synergistic anti-tumor effect by enhancing the anti-PD-1 immunotherapy.

[0186] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of pyrrolidone derivatives in the preparation of small molecule GPI inhibitors, characterized in that, The chemical structural formula of the pyrrolidone derivatives is shown in formula (Ⅰ): Among them, R1, R2, R3, and R4 are independently H, halogen, -CN, -NO2, -OH, -NH2, and C. 1～6 Alkyl, C 1～6 Alkyl, aryl, or heteroaryl.

2. The application according to claim 1, characterized in that, R1 is H, R2 is methoxy, R3 is -NH2, and R4 is H.

3. The application according to claim 1, characterized in that, The GPI small molecule inhibitor is a drug for treating and / or preventing tumors.

4. The application according to claim 3, characterized in that, The drug is a drug that enhances the killing effect of immune cells on tumor cells.

5. The application according to claim 4, characterized in that, The immune cells mentioned are T cells.

6. The application according to claim 3, characterized in that, The tumor is at least one of cancer or benign tumor.

7. The application according to claim 6, characterized in that, The cancer is at least one of the following: colorectal cancer, liver cancer, pancreatic cancer, stomach cancer, lung cancer, kidney cancer, or melanoma.

8. The application according to claim 3, characterized in that, The drug is an adjuvant medication used to enhance the therapeutic effect of tumor immunotherapy.

9. The application according to claim 7, characterized in that, The immunotherapy agent used in the tumor immunotherapy is at least one of anti-PD-1 antibody or anti-CTLA-4 antibody.

10. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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