An N-substituted 2-(2,5-dioxoimidazolidin-4-yl)acetamide compound, a synthesis method and application thereof

Abstract

The application discloses an N-substituted 2-(2,5-dioxoimidazolidin-4-yl) acetamide compound and a synthesis method and application thereof, and belongs to the technical field of tumor immunotherapy drug development.The N-substituted 2-(2,5-dioxoimidazolidin-4-yl) acetamide compound disclosed by the application is a small-molecule ITIM motif inhibitor, the action mechanism of which is to combine with the ITIM motif of tyrosine phosphorylation in the tumor cell membrane, thereby blocking the combination of the ITIM of tyrosine phosphorylation and SH2, making SHP2 always in an inhibition state, simultaneously blocking multiple inhibitory receptor pathways including PECAM-1, PD-1, BTLA, KIR, CD3 and SIRP alpha, and finally improving the anti-tumor activity of immune cells.The experimental data show that the N-substituted 2-(2,5-dioxoimidazolidin-4-yl) acetamide compound can effectively enhance the killing of tumor cells by immune cells, inhibit the growth of tumors, and fully support the application of the compound in tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy drug development technology, specifically relating to an N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound, its synthesis method, and its application in the preparation of tumor immunotherapy drugs. Background Technology

[0002] Malignant tumors are the second leading cause of death and a major threat to human health. Tumor immunotherapy has become an important treatment for tumors, following surgery, chemotherapy, radiotherapy, and targeted therapy. Among these, immune checkpoint blockade therapy (ICB) is currently a hot research topic. To date, the overall response rate of ICB treatment is only 20%-30%, and some patients develop drug resistance after treatment. Therefore, immunotherapies targeting only one specific target have limited effectiveness. Finding universal immune checkpoint inhibitors that target common sites of different immune checkpoints may be a new strategy to improve the efficacy of tumor immunotherapy.

[0003] The most prominent feature of many known inhibitory receptors (PECAM-1, PD-1, BTLA, KIR, CD3, and SIRPα, etc.) is the presence of one or more immunoreceptor tyrosine-based inhibition motifs (ITIMs) in their cytoplasmic regions. When an ITIM-containing receptor binds to its corresponding ligand, the ITIM undergoes tyrosine phosphorylation (usually catalyzed by Src family kinases), thereby recruiting and activating the signaling molecules SHP1 / SHP2. SHP1 / SHP2 then leads to the dephosphorylation of downstream cell signaling molecules, thus mediating immunosuppression. The tyrosine phosphorylation sites of many inhibitory receptors, such as PD-1 and BTLA, have been shown to bind to the N-terminal and C-terminal domains of protein tyrosine phosphatase 2 (SH2 domain-containing inositol phosphatase-2, SHP2), making them important functional regions for inhibition. Therefore, ITIMs are a common domain for the inhibitory effects of many immune checkpoints and a key target for drug design targeting immune checkpoints.

[0004] Most existing immunotherapies target only one immunosuppressive molecule. Due to the characteristics of tumors, the complex immune signaling pathways, and the involvement and interaction of immune cells, cytokines, and immune adjuvants in the tumor microenvironment, the therapeutic effect of immunotherapy targeting only one immunosuppressive molecule is limited. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide an N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound (hereinafter referred to as Y041-C), its synthesis method and application.

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

[0007] This invention discloses an N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound, including the compound itself and its R, S configuration isomers and racemic mixtures;

[0008] The structural formula of the compound is shown below:

[0009]

[0010] This invention also discloses a method for synthesizing the above-mentioned N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds, comprising the following steps:

[0011] 1) Using compound 1 as a raw material, compound 2 was prepared by reacting it at 40–100 °C for 2–15 hours in the presence of ethanol and acid solution.

[0012] 2) Compound 3 and compound 2 were reacted in a reaction solvent at 25–50 °C for 5–24 hours to obtain compound 4;

[0013] 3) Compound 4 was hydrolyzed in a dilute alkaline solution to obtain compound 5. The hydrolysis reaction temperature was 25-50℃ and the reaction time was 10-180 minutes.

[0014] 4) Compound 5 and compound 6 were reacted in a reaction solvent at 25–50 °C for 5–24 hours to obtain compound 4, thus obtaining an N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound;

[0015] The structural formulas of compounds 1, 2, 3, 4, 5, and 6 are shown below:

[0016]

[0017] Preferably, in step 1), the acid solution is one of HCl, sulfuric acid, or phosphoric acid.

[0018] Preferably, the reaction solvents used in steps 2) and 4) are DMF, toluene, or N-methylpyrrolidone.

[0019] Preferably, the dilute alkaline solution in step 3) is a lithium hydroxide solution, a sodium hydroxide solution, or a potassium hydroxide solution.

[0020] Preferably, in step 2), the molar ratio of compound 3 to compound 2 is (1.0 to 1.4):1.

[0021] Preferably, in step 4), the molar ratio of compound 5 to compound 6 is (1.0 to 1.4):1.

[0022] The present invention also discloses the application of the above-mentioned N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds in the preparation of tumor immunotherapy drugs.

[0023] Preferably, the drug is a drug that binds to the tyrosine-phosphorylated ITIM motif within the tumor cell membrane, thereby blocking the binding of tyrosine-phosphorylated ITIM to SH2.

[0024] More preferably, the drug is a CD8 enhancer. + Drugs that activate the killing activity of T lymphocytes, NK cells, and macrophages to kill tumor cells.

[0025] Preferably, the cellular administration dose of the N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound is 10 μM, administered in 5 μL doses, for two days. The mouse administration dose is 20 mg / kg, administered for 14 days, every two days.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention discloses an N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound (Y041-C), a small-molecule ITIM motif inhibitor. Its mechanism of action involves binding to the tyrosine-phosphorylated ITIM motif within the tumor cell membrane, thereby blocking the binding of tyrosine-phosphorylated ITIM to SH2, thus maintaining SHP2 in a state of constant inhibition. Simultaneously, it blocks multiple inhibitory receptor pathways, including PECAM-1, PD-1, BTLA, KIR, CD3, and SIRPα, ultimately enhancing the anti-tumor activity of immune cells. Experimental data disclosed in this invention demonstrate that compound Y041-C can effectively enhance the killing effect of immune cells on tumor cells, inhibit the downstream ITIM motifs of all immunosuppressive molecules, suppress tumor growth, and exhibit better broad-spectrum activity. This fully supports the application of Y041-C in tumor immunotherapy, avoiding the toxic side effects and increased treatment costs associated with various combination therapies. Attached Figure Description

[0028] Figure 1 The HPLC peak chromatogram of Y041-C after dissolution in DMF (1 mg / mL);

[0029] Figure 2The HPLC peak chromatogram (1 mg / mL) of Y041-C after salt formation and dissolution in physiological saline is shown.

[0030] Figure 3 The CotoTox96 assay was used to detect how Y041-C promotes NK92 cell killing of K562 cells;

[0031] Figure 4 The CCK-8 assay was used to detect how Y041-C promotes the killing of Hepa1-6 cells by mouse OT-1 cells;

[0032] Figure 5 To detect the cytotoxic activity of NK92 cells against 7404 cells using the RTCA method;

[0033] Figure 6 Image and statistical results of orthotopic hepatocellular carcinoma in mice after Y041-C treatment; where A represents imaging results and B represents statistical results.

[0034] Figure 7 Images and tumor weight statistics of subcutaneous tumors in MC38 mice after Y041-C treatment; where A is the tumor image and B is the tumor weight statistics.

[0035] Figure 8 Images of subcutaneous tumors in Hepa1-6 mice after Y041-C treatment, along with statistical results on tumor volume and weight; where A represents tumor volume, B represents tumor weight, and C represents tumor images. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0037] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0038] The present invention will now be described in further detail with reference to the accompanying drawings:

[0039] The structure of compound Y041-C used in this invention is as follows:

[0040]

[0041] 1. Preparation process of compound Y041-C

[0042] The synthesis route is as follows:

[0043]

[0044] 1.1 Synthesis of Intermediate 2

[0045] In a 500 mL single-necked flask, compound 1 (7.9 g, 50 mmol), ethanol (250 mL), and concentrated sulfuric acid (2.5 mL) were added sequentially. After the addition was complete, the temperature was gradually raised to 90 °C and the reaction was allowed to proceed overnight. Heating was then stopped, and the ethanol was evaporated to dryness. The mixture was then dissolved in ethyl acetate (250 mL), washed successively with saturated sodium bicarbonate solution (50 mL) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and evaporated to dryness to give 8.7 g of white solid 2, with a yield of 94%. 1 HNMR(400MHz,Chloroform-d)δ8.97(s,1H),6.55(s,1H),4.41(d,J=9.6Hz,1H),4.20(q,J=7 .1Hz,2H),3.01(dd,J=17.4,2.9Hz,1H),2.67(dd,J=17.4,9.9Hz,1H),1.28(t,J=7.1Hz,3H); 13 C NMR (101MHz, Chloroform-d) δ173.5,170.3,157.3,61.6,55.0,36.2,14.1.

[0046] 1.2 Synthesis of Intermediate 4

[0047] In a 100 mL single-necked flask, compound 2 (7.4 g, 40 mmol), piperobenzyl bromobenzyl 3 (10.3 g, 48 mmol), potassium carbonate (16.6 g, 120 mmol), and DMF (50 mL) were added sequentially. After the addition was complete, the mixture was reacted overnight at room temperature. The DMF was evaporated to dryness under reduced pressure, dissolved in dichloromethane, washed successively with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and evaporated to dryness to give the crude product. Column chromatography (ethyl acetate: petroleum ether = 1:2) gave 5.2 g of white solid 4, yield 41%. 1 H NMR(400MHz,Chloroform-d)δ6.97–6.85(m,2H),6.76–6.66(m,1H),5.94(s,2H),5.89(s,1H),4.57–4.56(m,2H),4.32(dd,J =10.3,1.5Hz,1H),4.17(q,J=7.1Hz,2H),3.02(dd,J=17.5,3.0Hz,1H),2.55(dd,J=17.4,10.4Hz,1H),1.26(t,J=7.1Hz,3H); 13 C NMR (101MHz, Chloroform-d) δ172.2,170.3,156.5,147.8,147.4,129.6,122.4,109.3,108.3,101.1,61.6,53.5,42.2,36.4,14.1.

[0048] 1.3 Synthesis of Intermediate 5

[0049] In a 100 mL single-necked flask, compound 4 (5.2 g, 16.4 mmol), potassium hydroxide (1.9 g, 49.2 mmol), and a mixed solution of tetrahydrofuran and water (4:1) (50 mL) were added sequentially. After the addition was complete, the temperature was gradually raised to 50 °C and the reaction was allowed to proceed for 3 hours. Heating was then stopped, and tetrahydrofuran was removed under reduced pressure. The pH was then adjusted to 1 with 3 M hydrochloric acid, and the reaction was heated overnight. After cooling to room temperature, the mixture was placed in a refrigerator, and 1.5 g of white solid 5 precipitated, with a yield of 31.3%. 1 H NMR(400MHz,DMSO-d6)δ8.22(s,1H),6.89–6.82(m,2H),6.76(d,J=8.1Hz,1H ),5.98(s,2H),4.53–4.37(m,2H),4.32(t,J=5.1Hz,1H),2.74–2.64(m,2H); 13C NMR (101MHz, DMSO-d6) δ173.9,170.8,157.0,147.1,146.3,130.3,120.7,108.0,107.9,100.8,52.9,40.9,34.6.

[0050] Synthesis of 1.5Y041-C

[0051] In a 100 mL single-necked flask, compound 5 (438 mg, 1.5 mmol), HOBt (230 mg, 1.7 mmol), DCC (413 mg, 2 mmol), and N-methylpyrrolidone (25 mL) were added sequentially. After stirring for 10 minutes, pyridine ethylamine 6 (122 mg, 1 mmol) was added. After the addition was complete, the mixture was reacted overnight at room temperature. The N-methylpyrrolidone was evaporated under reduced pressure to give the crude product. Column chromatography (dichloromethane:methanol = 1:10) gave 257 mg of white solid Y041-C, with a yield of 65%. 1 H NMR (400MHz, DMSO-d6) δ8.46(d,J=5.1Hz,2H),8.21(s,1H),8.09(t,J=5.6Hz,1H),7.24(d,J=5.1Hz,2H),6.91–6.81(m,2H),6.76(d, J=8.0Hz,1H),5.98(s,2H),4.57–4.36(m,2H),4.32(t,J=5.6Hz,1H),3.30(d,J=6.8Hz,2H),2.73(t,J=7.1Hz,2H),2.61–2.35(m,2H); 13 C NMR (101MHz, DMSO-d6) δ173.9,168.1,156.7,149.4,148.3,147.2,146.4,130.6,124.2,120.8,108.0,100.9,53.5,40.9,36.6,34.1.

[0052] 2. Solubility of compound Y041-C

[0053] 2.1 Experimental Procedure

[0054] Y041-C, this compound has extremely poor water solubility. Direct dissolution of the powder with water, followed by stirring, sonication, and heating to aid dissolution, yields a white emulsion that settles to the bottom of the container upon standing. Initially, this compound was dissolved using a cyclodextrin inclusion method, as detailed below:

[0055] 1. Dissolve 10 mg of Y041-C in 2 mL of dimethylformamide (DMF), and weigh 500 mg (approximately 7 times the molar amount of the compound) of hydroxypropyl-β-cyclodextrin (HP-β-CD) and dissolve it in 2 mL of DMF; 2. Mix the two solutions from the above steps into a 15 mL centrifuge tube; 3. Sonicate the mixture using an ultrasonic instrument, ensuring the liquid level in the ultrasonic chamber is higher than the liquid level in the centrifuge tube, at a power of 300 W for 4 hours; 4. Freeze-dry the ultrasonically treated mixture to remove DMF; 5. Dissolve the freeze-dried powder in physiological saline to obtain a clear liquid.

[0056] Since the inclusion method of cyclodextrin is relatively complex, it is considered to improve its solubility by forming a salt of the compound, which may make the dissolution process more convenient. The specific method is as follows: 1. Weigh 7.9 mg of Y041-C and dissolve it in 2 mL of pure water to obtain an emulsion; 2. Add 5 μL of 4M hydrochloric acid to the emulsion to obtain a clear liquid; 3. Freeze-dry the liquid to obtain a powder; 4. Dissolve the powder in physiological saline to obtain a clear liquid. After filtering through a 0.22 μm filter membrane, perform HPLC to detect the content and purity of Y041-C in the solution.

[0057] 2.2 Experimental Results

[0058] According to the HPLC results, after Y041-C was dissolved in DMF, there were two main peaks on the HPLC. The left main peak was the DMF solvent peak, and the right main peak was the Y041-C compound peak. Figure 1 When Y041-C is treated with hydrochloric acid and forms a salt, HPLC analysis after dissolving it in physiological saline or PBS yields only one main peak. Furthermore, when the compound concentration is the same in both dissolution methods, the HPLC peak area is also approximately the same. Figure 2 This demonstrates that Y041-C, after salt formation treatment, can be completely dissolved in an aqueous solvent.

[0059] 3. In vitro validation of Y041-C activity and dose dependence.

[0060] 3.1 Identification of Y041-C activity using the CotoTox96 assay

[0061] 3.1.1 Experimental Methods

[0062] Effector cells (NK92 cells) and target cells (7404 cells and K562 cells) were collected separately, and different effector-to-target ratios were set according to experimental requirements. The cytotoxicity assay kit (Promega#CTB163) was used, and six types of wells were set up (spontaneous LDH release from effector cells, experimental wells, spontaneous LDH release from target cells, maximum LDH release from target cells, volume correction control wells, and culture medium background wells). Each type of well had three replicates, with a final volume of 100 μL / well. The cells were centrifuged at 250 × g for 4 min at room temperature. The assay plate was incubated at 37°C in a 5% CO2 cell culture incubator for 4–6 h. 45 min before the end of incubation, 10 μL of Lysis Solution (10×) was added to the maximum LDH release well and the volume correction control well, respectively. After co-culture, the cells were centrifuged at 250 × g for 4 min at room temperature. Transfer 50 μL of supernatant from each well to a new 96-well flat-bottom (enzyme analysis) plate; add 50 μL of prepared substrate to each well and incubate at room temperature in the dark for 30 min; add 50 μL of Stop Solution to each well; measure the absorbance at 490 nm or 492 nm using a microplate reader.

[0063] Finally, the data was processed according to the reagent instructions, and the different killing efficiencies were calculated using the following formula.

[0064]

[0065] 3.1.2 Experimental Results

[0066] The results are as follows Figure 3 As shown, compound Y041-C was applied at five concentrations (1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) to treat NK92 cells. This effectively improved the killing efficiency of NK92 cells against K562 cells, and the killing effect on NK92 cells increased with increasing concentration, exhibiting a dose-dependent effect. Figure 3 Meanwhile, the same experiment also demonstrated that Y041-C can enhance the killing activity of NK92 against 7404 cells.

[0067] 3.2 CCK-8 assay for identifying Y041-C activity in OT-1 system

[0068] 3.2.1 Experimental Methods

[0069] OT-1 cells were constructed and incubated for 24 h, followed by incubation with Y041-C for another 48 h to prepare a Hepa1-6 cell suspension. 100 μL of this suspension was seeded into each well of a 96-well plate, with three replicates. The plates were pre-cultured in an incubator (37℃, 5% CO2) for 24 h until complete cell adhesion. OT-1 and Hepa1-6 cells were co-cultured for 48 h or 72 h, followed by incubation with 10 μL of CCK-8 reagent added to each well. The incubation time was determined based on the standard curve. Finally, the absorbance at 450 nm was measured using a microplate reader.

[0070] 3.2.2 Experimental Results

[0071] The results are shown in the figure. Compound Y041-C can promote the killing of OVA-Hepa1-6 cells by mouse OT-1 cells. Figure 4 This indicates that Y041-C can promote the killing of tumor cells by mouse T cells in vitro in a dose-dependent manner.

[0072] 3.3 Real-time label-free cell assay (RTCA) for Y041-C activity

[0073] 3.3.1 Experimental Methods

[0074] NK92 cells in the logarithmic growth phase were divided into different treatment groups (control group, peptide treatment group, and compound treatment group) with multiple drug concentration gradients and pretreated for 48 hours. 50 μL of culture medium was added to an E-Plate detection plate, and background impedance was measured. 7404 cells in the logarithmic growth phase were collected and counted. The cell suspension concentration was adjusted to 1.5 × 10⁴ cells / mL. 4 100 μL of 7404 cells per well was seeded into an E-Plate assay plate. Three concentration gradients were set up: 7404 cells alone, a control group, and groups treated with peptides and compounds (2.5, 5, 10 μM). Each experimental group had three replicates. The RTCA assay plate was returned to the RTCA analyzer and incubated at 37°C in a 5% CO2 incubator for 12 h. After 12 h of 7404 cell culture, 100 μL of the supernatant was aspirated from each well, and 7.5 × 10⁶ pretreated NK92 cells were added to each well according to the respective group. 3 Cells / well (100 μL); Place the E-Plate detection plate back into the RTCA instrument, set it to monitor and record cell index (CI) values ​​every 5 minutes, and monitor continuously for 40 hours to obtain the compound-mediated cell effect curve and IC50 values ​​at different time points. The calculation formula is: Cell killing rate % = (Control group CI value - Experimental group CI value) / Control group CI value × 100%.

[0075] 3.3.2 Experimental Results

[0076] Y041-C significantly enhanced the killing activity of NK92 cells against 7404 cells. When the concentration of Y041-C was set at 2.5 μM, 5 μM, and 10 μM, it was observed that the killing efficiency of NK92 cells against 7404 cells increased with increasing compound concentration. Figure 5 It is dose-dependent.

[0077] 4. Validation of the antitumor activity of Y041-C in mice.

[0078] 4.1 Y041-C inhibits the growth of orthotopic hepatocellular carcinoma in mouse Hepa1-6 cells.

[0079] 4.1.1 Experimental Methods:

[0080] ① Construction of a mouse Hepa1-6 orthotopic hepatocellular carcinoma model:

[0081] Male C57BL / 6 mice aged 6-8 weeks were selected to construct an orthotopic hepatocellular carcinoma model. Before tumor inoculation, hair was removed from the inoculation site below the xiphoid process to facilitate postoperative suturing. Hepa1-6 cells were digested with trypsin for 2 min, digestion was terminated with complete culture medium, and the cells were centrifuged at 1000-1300 rpm for 3 min. After centrifugation, the supernatant was discarded, and the cell base was placed on ice and resuspended in pre-cooled RPMI 1640 medium and Matrigel gel at the calculated concentration. Mice were anesthetized with 3% sodium pentobarbital, and a 1-2 cm incision was made below the xiphoid process. The liver was carefully extracted, and each mouse was injected with 3 × 10⁻⁶ cells. 6 Inject approximately 20 μL of cells slowly to prevent leakage. After injection, press the injection site for 1 minute, then suture the peritoneum and epidermis separately and attach an ear tag.

[0082] ② Compound administration:

[0083] Twenty-four male C57BL / 6 mice aged 6-8 weeks were selected and modeled according to the method described above. In vivo imaging of the mice was performed on postoperative day 3. Based on the imaging results and the size of the orthotopic hepatocellular carcinoma tumor, the mice were stratified and randomly divided into four groups of six mice each. In vivo imaging was performed on postoperative days 7, 14, and 21, and the results were collected and analyzed. Intraperitoneal administration began on postoperative day 5, administered every two days. Each group received an intraperitoneal injection of 20 mg HP-β-CD, 125 μg PD-1 antibody, 200 μg Y041-C, and 400 μg Y041-C, respectively. After administration, the hepatocellular carcinoma status was detected using a small animal imaging system, and the fluorescence values ​​in the imaging were recorded.

[0084] 4.1.2 Experimental Results

[0085] The results are shown in the figure. It can be seen that treatment with 125 μg / animal PD-1 antibody and 400 μg / animal Y041-C can significantly inhibit the growth of Hepa1-6 orthotopic hepatocellular carcinoma. Figure 6 In addition, the body weight of mice in each group before and after drug administration was recorded. The results showed that there was no significant difference in body weight between the different treatment groups before and after drug administration, indicating that compound Y041-C itself has no toxic side effects on mice.

[0086] 4.2Y041-C inhibits the growth of MC38 subcutaneous tumors in mice.

[0087] 4.2.1 Experimental Methods

[0088] ① Establishment of a mouse MC38 xenograft model:

[0089] A xenograft tumor model was constructed using 6-8 week old female C57BL / 6 mice. Before tumor inoculation, hair was removed from the inoculation area on the back of the mice to observe the tumor cell inoculation and clearly identify any missed tumor cells. Well-cultured MC38 cells were digested with trypsin for 2 minutes, followed by termination of digestion with complete culture medium, and cell counts were performed. The suspension was transferred to 15 mL centrifuge tubes and centrifuged at 1000-1300 rpm for 3 minutes. After centrifugation, the cells were washed once with physiological saline, the supernatant was discarded, and the cell base was stored on ice for later use. The cell base was resuspended in a 1×10⁻⁶ volume mixture of pre-cooled physiological saline and Matrigel. 7 / mL (this process should be performed at low temperature to avoid Matrigel coagulation), 0.2 mL of MC38 cell suspension was subcutaneously injected into the back of each C57BL / 6 mouse.

[0090] ② Compound administration:

[0091] Forty-eight female C57BL / 6 mice aged 6-8 weeks were selected. Tumor modeling was performed using the method described above. After 10 days, once obvious subcutaneous tumor formation was observed, the mice were ear-tagged and tumor volume was measured. The tumor volume was calculated using the formula: V = L × S 2 ×0.5, where L is the longitudinal diameter of the tumor and S is the transverse diameter. Mice were stratified and randomly assigned to eight groups of six mice each, based on tumor size. Drug administration began on day 14. The control group received intraperitoneal injections of saline and HP-β-CD, while the drug-treated groups received intraperitoneal injections of 200 μg PD-1 antibody, 5 μg peptide, 10 μg peptide, 100 μg Y041-C, 200 μg Y041-C, and 400 μg Y041-C, respectively. Mice were euthanized after day 28, and the tumors were dissected. Tumor weight and volume were measured for subsequent analysis.

[0092] 4.2.2 Experimental Results

[0093] The results are shown in the figure. It can be seen that treatment with 125 μg / animal PD-1 antibody, 5 μg / animal and 10 μg / animal peptides, and 200 μg / animal and 400 μg / animal Y041-C significantly inhibited the growth of MC38 subcutaneous tumors. Figure 7 In addition, the body weight of mice in each group before and after drug administration was recorded. The results showed that there was no significant difference in body weight between the different treatment groups before and after drug administration, indicating that compound Y041-C itself has no toxic side effects on mice.

[0094] 4.3Y041-C inhibits the growth of subcutaneous hepatocellular carcinoma tumors in mice with Hepa1-6 liver cancer.

[0095] 4.3.1 Experimental Methods

[0096] ① Establishment of a mouse Hepa1-6 subcutaneous tumor model:

[0097] A xenograft tumor model was constructed using 6-8 week old female C57BL / 6 mice. Before inoculation, hair was removed from the dorsal region of the mice to allow observation of tumor cell inoculation and to clearly identify any missed tumor cells. Well-cultured Hepa1-6 cells were digested with trypsin for 2 minutes, followed by termination of digestion with complete culture medium, and cell counts were performed. The suspension was transferred to 15 mL centrifuge tubes and centrifuged at 1000-1300 rpm for 3 minutes. After centrifugation, the cells were washed once with physiological saline, the supernatant was discarded, and the cell base was stored on ice for later use. The cell base was resuspended in a mixture of pre-chilled PBS and Matrigel at a 1:1 ratio to a final volume of 2.5 × 10⁻⁶. 7 / mL (this process should be performed at low temperature to avoid Matrigel coagulation), 0.2 mL of Hepa1-6 cell suspension was subcutaneously injected into the back of each C57BL / 6 mouse.

[0098] ② Compound administration:

[0099] Fifty female C57BL / 6 mice aged 6-8 weeks were selected and modeled according to the method described above. After 7 days, when obvious subcutaneous tumor formation was observed, the mice were ear-tagged and the tumor volume was measured. The tumor volume was calculated using the formula: V = L × S 2 ×0.5, where L is the longitudinal diameter of the tumor and S is the transverse diameter. Mice were stratified and randomly divided into 5 groups of 10 mice each, based on tumor size. Drug administration began on day 7. The control group received an intraperitoneal injection of 200 μL of saline. The treatment groups received intraperitoneal injections of 125 μg PD-1 antibody, 200 μg Y041-C, 400 μg Y041-C, and 600 μg Y041-C, respectively. PD-1 antibody was administered every 3 days, while the other treatment groups received it every 2 days. After 16 days, the mice were euthanized, the tumors were dissected, and tumor weight and volume were measured for subsequent analysis.

[0100] 4.3.2 Experimental Results

[0101] The results are shown in the figure. Treatment with 125 μg / animal PD-1 antibody, 200 μg / animal, 400 μg / animal, and 600 μg / animal Y041-C significantly inhibited the growth of Hepa1-6 subcutaneous tumors. Figure 8 In addition, the body weight of mice in each group before and after administration was recorded. The results showed that only the mice in the 600 μg / mouse Y041-C group had a slight decrease in body weight compared to the control group, indicating that 200 μg-400 μg / mouse Y041-C had no significant toxic side effects on mice.

[0102] This invention discloses the application of compound Y041-C in promoting the killing of tumors by immune cells, belonging to the field of tumor immunotherapy. First, the compound is salted by treatment with 4M HCl, then lyophilized and reconstituted to obtain the dissolved compound. At the cellular level, treatment with 1.25 μM to 20 μM Y041-C for 48 hours significantly enhances the killing activity of immune cells such as NK cells and T cells against various tumor cells in a dose-dependent manner. At the animal level, dosages of 100 μg / mouse to 400 μg / mouse significantly inhibit the growth of orthotopic hepatocellular carcinoma, subcutaneous tumors in Hepa1-6 mice, and subcutaneous tumors in MC38 mice. Furthermore, no significant difference in body weight was observed in mice treated with doses up to 400 μg / mouse in the untreated group throughout the treatment process. Therefore, the experimental data disclosed in this invention demonstrate that compound Y041-C can effectively enhance the killing of tumor cells by immune cells and inhibit tumor growth, fully supporting the application of Y041-C in tumor immunotherapy.

[0103] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds, characterized in that, Includes the following steps: 1) Using compound 1 as a raw material, compound 2 was prepared by reacting it at 40-100°C for 2-15 hours in the presence of ethanol and acid solution. 2) Compound 3 and compound 2 were reacted in a reaction solvent at 25-50°C for 5-24 hours to obtain compound 4; 3) Compound 4 was hydrolyzed in a dilute alkaline solution to obtain compound 5. The hydrolysis reaction temperature was 25~50℃ and the reaction time was 10~180 minutes. 4) Compound 5 and compound 6 were reacted in a reaction solvent at 25-50°C for 5-24 hours to prepare N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds; The structural formulas of compounds 1, 2, 3, 4, 5, and 6 are shown below: The structural formulas of N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamides are shown below: 。 2. The method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds according to claim 1, characterized in that, In step 1), the acid solution is one of HCl, sulfuric acid, or phosphoric acid.

3. The method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds according to claim 1, characterized in that, The reaction solvents used in steps 2) and 4) are DMF, toluene, or N-methylpyrrolidone.

4. The method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds according to claim 1, characterized in that, Step 3) The dilute alkaline solution is a lithium hydroxide solution, a sodium hydroxide solution, or a potassium hydroxide solution.

5. The method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds according to claim 1, characterized in that, In step 2), the molar ratio of compound 3 to compound 2 is (1.0~1.4):

1.

6. The method for synthesizing N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds according to claim 1, characterized in that, In step 4), the molar ratio of compound 5 to compound 6 is (1.0~1.4):

1.

7. The use of N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compounds in the preparation of tumor immunotherapy drugs, characterized in that, The structural formula of the N-substituted 2-(2,5-dioxoimidazolidine-4-yl)acetamide compound is shown below: 。 8. The application as described in claim 7, characterized in that, The drug described is one that binds to the tyrosine-phosphorylated ITIM motif within the tumor cell membrane, thereby blocking the binding of tyrosine-phosphorylated ITIM to SH2.

9. The application as described in claim 8, characterized in that, The drug mentioned is a CD8 enhancer. + Drugs that activate the killing activity of T lymphocytes, NK cells, and macrophages to kill tumor cells.

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