A small molecule compound DBIC, a preparation method and application thereof

Abstract

The application relates to a small molecule compound DBIC, a preparation method and application thereof, and belongs to the technical field of biological medicines.The small molecule compound DBIC directly combines and activates an orphan nuclear receptor Nur77 to activate an integrated stress response (ISR), thereby further activating the expression level and activity of granzyme B (GZMB) in tumor cells, and then causing pyroptosis execution protein GSDMC to be cleaved, so that pyroptosis death of melanoma cells is induced, and a new lead compound is provided for the treatment of melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule compound DBIC and a preparation method and application thereof. Background Art

[0002] Pyroptosis is a lytic cell death caused by members of the gasdermin family. The gasdermin family includes GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and PVJK. These proteins possess highly conserved N-terminal and C-terminal domains. When gasdermin is cleaved, the C-terminal domain no longer inhibits the activity of the N-terminal domain, releasing the membrane-forming N-terminal domain. The N-terminal domain then binds to cell membrane phospholipids and oligomerizes to form stable pores, disrupting the integrity of the cell membrane. This leads to the release of intracellular substances and the rupture of the cell due to water absorption, triggering pyroptosis (Shi Jianjin et al., Trends in biochemical sciences, 2017, 42, 245-254). Different gasdermin proteins exhibit diverse functions in tumor development and anti-tumor immunity. For example, GSDME has a broad-spectrum inhibitory effect on various tumors, and its expression is typically silenced in tumor cells. However, GSDME is highly expressed in normal tissues. Therefore, while inducing GSDME-mediated pyroptosis in tumors, it also causes pyroptosis in normal cells, resulting in toxic side effects (Wang Yupeng et al., Nature, 2017, 547, 99-103). Current research on pyroptosis primarily focuses on GSDMD and GSDME, while research on the regulation of GSDMC remains unclear, primarily due to the lack of effective lead compounds that induce GSDMC-mediated pyroptosis.

[0003] Current cancer treatment faces several challenges. For example, chemotherapy is a key component of cancer treatment. Most clinically, chemotherapy drugs inhibit tumors by inducing apoptosis. Long-term use of chemotherapy drugs can lead to apoptosis resistance in many tumors. Furthermore, some tumors, such as melanoma, are inherently resistant to apoptotic cell death, limiting their clinical use and therapeutic efficacy (Wang Weijia et al., Nature Chemical Biology, 2014, 10, 133-140). Furthermore, immunotherapy's suboptimal efficacy in some solid tumors and its high cost have also hindered its widespread adoption.

[0004] In recent years, research on pyroptosis has also verified the feasibility of treating tumors by inducing pyroptosis. The advantages of inducing tumor cell pyroptosis compared with inducing apoptosis are as follows: 1) During the process of cell apoptosis, the cell contents are not released, which belongs to an immunosuppressive death mode. Cell pyroptosis is a pro-inflammatory death mode, when cells undergo pyroptosis, many inflammatory factors are released, thereby recruiting immune cells to further kill tumor cells, and can amplify the effect by inducing the body's anti-tumor immune response. The academician Shao Feng's research group found that although only a small part of tumor cells were induced to undergo pyroptosis, the body could almost completely clear the tumor through the immune response (Wang Qinyang et al., Nature, 2020, 579, 421-426). 2) Cell pyroptosis is faster and more rapid than cell apoptosis. Therefore, screening drugs (including small molecule compounds) that induce tumor cells to undergo new cell death (such as pyroptosis) has become a new direction of tumor treatment. Since melanoma highly expresses gasdermin family proteins, especially GSDMC, screening compounds that induce melanoma to undergo pyroptosis is of great significance for the drug development and treatment of melanoma.

[0005] Currently known granzyme B (GZMB) agonists are mainly cytokines, such as IL-2 (Jiang Liwei et al., Cell, 2020, 183, 1219-1233), and other types of GZMB agonists (such as small molecule compounds) are less studied. GZMB is mainly highly expressed in immune cells and lowly expressed in tumor cells. The function of endogenous GZMB in tumor cells is not clear, and small molecule compounds capable of activating GZMB have not been reported.

[0006] Orphan nuclear receptor Nur77 (also known as TR3, NGF1-B and NAK1) is a protein encoded by the immediate-early gene NR4A1, and belongs to the steroid / thyroid / retinoid receptor superfamily (Hazel, T.G et al., PNAS, 1988, 85, 8444-8448). Nur77 plays an important role in the development and maturation of immune cells, cell proliferation and differentiation, regulation of glycolipid metabolism, occurrence and development of tumors, and cell death processes such as apoptosis, autophagy and ferroptosis. Nur77 is an orphan nuclear receptor, but its endogenous ligand has not been reported so far. The small molecule compound DBIC discovered by the present application is an in vitro agonist of orphan nuclear receptor Nur77, and it can not only be used as an activator of granzyme B (GZMB), but also as an inducer of pyroptosis, which has broad application prospects. SUMMARY

[0007] The purpose of the present application is to solve the above-mentioned problems in the prior art, and to provide a small molecule compound benzoimidazole-5-carboxylic acid dodecyl ester (DBIC) as a granzyme B (GZMB) agonist and a pyroptosis inducer. dodecyl 1H- b enzo[d] i midazole-5- c The invention discloses a novel deoxyribonucleic acid (DBIC) and its preparation method and application. The DBIC can directly bind to Nur77 and activate the integrated stress response, thereby activating granzyme B to cleave GSDMC and promote pyroptotic death of melanoma cells.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A small molecule compound DBIC, the structural formula is as follows:

[0010] .

[0011] The preparation method of the small molecule compound DBIC has the following synthesis route:

[0012] .

[0013] The method for preparing the small molecule compound DBIC comprises the following steps:

[0014] 1) Compound 1, benzimidazole-5-carboxylic acid and di-tert-butyl dicarbonate were dissolved in N,N-dimethylformamide, triethylamine was added dropwise, and the mixture was heated to react to obtain compound 2;

[0015] 2) Compound 2, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in dichloromethane and reacted in an ice bath. Then, dodecanol was added and the reaction was carried out at room temperature to obtain compound 3;

[0016] 3) Compound 3 was dissolved in dichloromethane (DCM), trifluoroacetic acid was added dropwise under an ice bath and protective atmosphere, and the reaction was carried out at room temperature to obtain compound 4.

[0017] In step 1), the heating temperature is 75-105°C.

[0018] The application of the small molecule compound DBIC is used in the preparation of anti-melanoma drugs.

[0019] A drug comprising the small molecule compound DBIC according to the present invention.

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0021] After the small molecule compound DBIC described in the present application directly binds and activates Nur77, it promotes the activation of ISR, leading to the expression and activity of the GZMB cleavage enzyme being improved, thereby cleaving GSDMC to induce pyroptosis of melanoma cells, and ultimately inhibiting the development process of melanoma. The small molecule compound DBIC proposed in the present application is not only an in vitro agonist of the orphan nuclear receptor Nur77, but also an activator of granzyme B (GZMB) and an inducer of GSDMC-mediated pyroptosis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is the co-crystal structure of the small molecule compound DBIC and Nur77-LBD (ligand binding domain). The results show that DBIC directly binds to the Nur77-LBD domain.

[0023] Figure 2 It is the detection of the protein expression level of Nur77 after the small molecule compound DBIC is used to treat melanoma A375 cells for 5 hours. The results show that DBIC can significantly improve the protein expression level of Nur77.

[0024] Figure 3 It is the detection of the protein expression level and activity of GZMB after the small molecule compound DBIC is used to treat melanoma A375 cells for 6 hours. "-" means no DBIC is added, but DMSO solvent is added as a control, and "+" means DBIC is added. The results show that DBIC can significantly improve the protein expression level and activity of GZMB. The results show that the small molecule compound DBIC can activate GZMB.

[0025] Figure 4 It is the observation of cell morphology, LDH release and GSDMC cleavage after the small molecule compound DBIC is used to treat melanoma A375 cells for 9 hours. The results show that DBIC can significantly induce the pyroptosis morphology of "bubble vomiting" of cells, LDH release and GSDMC cleavage. The results show that the small molecule compound DBIC can induce GSDMC-mediated cell pyroptosis of melanoma cells.

[0026] Figure 5 It is the inhibition of the development of melanoma cell A375-derived transplanted tumors by the small molecule compound DBIC. Melanoma cells A375 are inoculated on the flank of nude mice, and DBIC is administered intraperitoneally every other day. The tumor growth is detected every day, the mice are sacrificed after 15 days, and the tumors are collected and weighed. The upper graph is the tumor morphology of mice treated with corn oil (Ctrl) and three concentrations of DBIC from large to small, the lower left graph is the tumor volume detection graph of the four treatments, and the lower right graph is the tumor weight statistics graph of the four treatments. The results show that DBIC can significantly inhibit the growth of transplanted tumors.

[0027] Figure 6In a nude mouse xenograft model, melanoma A375 cells, Nur77 knockdown A375 cells, and GSDMC knockdown A375 cells were implanted in the flanks of nude mice. DBIC was injected every two days, and tumor size was measured. Mice were sacrificed 30 days later, and tumors were harvested and weighed. The top image shows tumor morphology, arranged from largest to smallest, after injection of three cell types: "Ctrl," "Nur77 KD," and "GSDMC KD," and treated with a corn oil solvent ("-") or DBIC. The bottom left image shows tumor volume measurements for the six treatments, and the bottom right image shows tumor weights for the six treatments. Results show that DBIC significantly inhibits the growth of A375 cell-derived tumors. However, knockdown of Nur77 or GSDMC in the tumor cells significantly weakens DBIC's ability to inhibit tumor growth. These results indicate that the small molecule compound DBIC inhibits the growth of nude mouse melanoma xenografts in a manner dependent on Nur77 and GSDMC proteins.

[0028] Figure 7 B16 cells were inoculated into the flanks of C57BL / 6J mice to establish a transplant tumor model. 20 mice were used to detect the inhibitory effect of DBIC on tumors, and 10 mice were used to detect the activation of the immune system by DBIC. DBIC was injected every two days and the tumor size was measured. The mice were killed after 15 days, and the tumors were collected and weighed. The results showed that DBIC can significantly inhibit the growth of B16-derived transplant tumors. After 24 hours of DBIC treatment of mice, tumor tissues were collected and immune cells in the tumor microenvironment were enriched for flow cytometry detection. The upper picture shows the morphology of mouse tumors arranged from large to small after treatment with corn oil control, i.e. "Ctrl" and DBIC drug. The lower left picture is the tumor volume detection picture of the two treatments, and the lower right picture shows the various immune cells in the tumor samples after treatment with the control solvent corn oil "-" or DBIC. The results showed that DBIC can effectively increase the proportion of NK cells in the tumor microenvironment, PFN (Perforin) + / CD8 + , IFNγ + / CD8 + , GZMB + / CD8 + , TNF-α + / CD8 + Tcells and GZMB + The proportion of NK positive cell populations also increased. These results indicate that DBIC can activate anti-tumor immunity. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Example 1

[0031] Synthesis of DBIC compounds.

[0032] The experimental method is as follows:

[0033] 1. Compound 1, benzimidazole-5-carboxylic acid (2.5 g, 15.4 mmol) and di-tert-butyl dicarbonate Boc2O (4.0 g, 18.5 mmol) were dissolved in N,N-dimethylformamide DMF (20 mL) solution, and triethylamine Et3N (2.6 mL, 18.5 mmol) was added dropwise. The mixture was stirred at 50 °C for 12 hours. After the reaction was completed, the solution was cooled to room temperature, saturated sodium bicarbonate NaHCO3 (30 mL) was added to adjust the pH of the solution to about 8, and ethyl acetate (30 mL) was used for extraction. The combined aqueous phase solution was retained and then adjusted to about 3 with 1N hydrochloric acid solution. Ethyl acetate (20 mL*3) was used for extraction, and Na2SO4 was used for drying. The solvent was concentrated in vacuo to obtain compound 2.

[0034] 2. Compound 2 (4 g, 15.4 mmol), 1-hydroxybenzotriazole (HOBT) (2.6 g, 17 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (3.2 g, 17 mmol) were dissolved in dichloromethane (DCM) (40 mL) in a one-pot reaction. The mixture was stirred at 0°C for 1 hour. Dodecanol (2.9 g, 15.4 mmol) and Et3N (2.3 mL, 17 mmol) were then added to the mixture, and the mixture was stirred at room temperature for 12 hours. After completion, the reaction was quenched with saturated sodium bicarbonate (NaHCO3) solution, and the organic phase was dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure. Purification was performed by column chromatography using petroleum ether and ethyl acetate as eluents to obtain compound 3 (5 g, yield: 75%) as an oil.

[0035] 3. Compound 3 was dissolved in dichloromethane (DCM) (25 mL). Trifluoroacetic acid (TFA) (1.3 mL, 17.4 mmol) was added dropwise at 0°C under inert argon (Ar). The reaction was stirred at room temperature for 6 hours. The resulting solution was poured into ice water and extracted with ethyl acetate. The mixture was dried over Na2SO4 and evaporated under reduced pressure. Purification by column chromatography using petroleum ether and ethyl acetate (PE / EA = 1:9) afforded white compound 4 (1.8 g, yield: 47.3%) with a melting point of 110–112°C.

[0036] 1H NMR (600 MHz, CDCl3) delta 8.96 (s, 1H), 8.47 (s, 1H), 8.14 (dd, J = 8.7,1.2 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 4.32 (t, J = 6.7 Hz, 2H), 1.83 – 1.69 (m,2H), 1.48 – 1.38 (m, 2H), 1.32 (dd, J = 14.5, 7.2 Hz, 2H), 1.29 – 1.19 (m,14H), 0.85 (t, J = 7.0 Hz, 3H). 13 C NMR (150 MHz, CDCl3) delta 166.23, 144.50, 138.40,135.57, 125.62, 124.85, 117.41, 115.38, 65.23, 31.75, 29.48, 29.47, 29.43,29.40, 29.17, 29.12, 28.64, 25.96, 22.56, 14.41. HRMS (ESI): m / z Theoretical value C 20 H 30 N2O2[M+H] + 331.23800, measured value 331.23753.

[0037] Example 2

[0038] DBIC directly binds to Nur77.

[0039] The experimental method is as follows:

[0040] 1. Add DBIC from a 200 mM stock solution to a 6 mg / mL Nur77-LBD (ligand binding domain) protein solution to a final concentration of 1 mM (0.5% DMSO) and incubate on ice for 2 hours.

[0041] Obtain crystals by hanging-drop vapor diffusion in 100 mM sodium citrate (pH 4.0), 20% glycerol, and 5%–10% PEG 4000 at 4°C for 3–7 days. Briefly transfer the crystals to crystallization conditions supplemented with 20% ethylene glycol as a cryoprotectant and then plunge them into liquid nitrogen.

[0042] 3. Diffraction data were collected at 100 K using synchrotron radiation at the Shanghai Synchrotron Radiation Facility BL18U1. Data were processed and scaled using the HKL3000 software package. The structure of the complex of Nur77-LBD and compound DBIC was determined by molecular replacement using the Phaser program, using the LBD structure (Protein Data Bank accession code 3V3E) as the initial search model.

[0043] The experimental results are as follows Figure 1 The results showed that the cocrystal structure showed that DBIC could directly bind to Nur77-LBD (resolution 2.57 Å), and residues L506 and L559, E445 and R563 were important sites for DBIC to bind to Nur77-LBD.

[0044] Example 3

[0045] DBIC increases the protein expression level of Nur77.

[0046] The experimental method is as follows:

[0047] 1. Take human melanoma A375 cells in logarithmic growth phase and inoculate 1.5×10 5 cells in 12-well plates.

[0048] 2. After overnight culture, the medium was replaced with 0.5% serum and the cells were treated with DBIC (20 μM) for 0.5 h. Nur77 protein levels were detected by Western blotting.

[0049] 3. Western Blotting Experiment:

[0050] 1) Discard the DBIC-treated cell culture medium and add 200 μL of 2× loading buffer (100 mM Tris-Cl (pH 6.8), 4% SDS, 20% glycerol, 200 mM β-mercaptoethanol) and boil at 95°C for 10 minutes.

[0051] 2) SDS-PAGE electrophoresis: Load 20 μg of protein on an SDS-PAGE gel at 100 V until the sample reaches the bottom of the gel.

[0052] 3) Membrane transfer: After electrophoresis, cut the stacking gel and assemble the membrane using ethanol-pretreated PVDF membrane and filter paper immersed in electrotransfer solution. Electrotransfer the membrane at 4°C (100 V, 60 min).

[0053] 4) Antigen-antibody reaction:

[0054] A. Blocking: Block with 5% skim milk or BSA for 1 hour;

[0055] B. Primary antibody reaction: add diluted Nur77 primary antibody, incubate overnight at room temperature (incubate other antibodies for 1.5 hours at room temperature);

[0056] C. Secondary antibody reaction: wash 3 times with TBST for 5 minutes each time, add corresponding species secondary antibody, incubate for 1.5 hours at room temperature.

[0057] 5) ECL detection: wash 3 times with TBST for 10 minutes each time. Mix A and B of ECL at 1:1, drop on the surface of the PVDF membrane, and develop using a chemiluminescence imager.

[0058] The experimental results are shown in Figure 2 . The results show that DBIC can improve the protein expression level of Nur77, and shows a concentration gradient effect.

[0059] Example 4

[0060] DBIC improves the expression level and activity of GZMB.

[0061] The experimental method is as follows:

[0062] 1. Take human melanoma A375 cells in the logarithmic growth phase, when detecting the GZMB protein expression level, inoculate 1.5 x 10 5 cells in a 12-well plate, and the experimental steps are the same as in Example 3; when measuring the GZMB activity, 10 10 cm dishes per group, inoculate 3 x 10 6 cells in a 10 cm dish.

[0063] 2. GZMB activity experiment

[0064] 1) Discard the supernatant, add 1 ml of PBS to each dish, scrape and collect in a 15 ml tube, centrifuge at 800 g for 5 minutes.

[0065] 2) Resuspend and lyse the cells with 150 μL Triton X-100 lysis buffer (1% Triton (v / v) in PBS) on ice, then centrifuge at 12000 g for 15 minutes at 4°C.

[0066] 3) Take the supernatant after centrifugation and incubate with Ac-IEPD-AMC TFA, continuously read the fluorescence with an enzyme marker for 1 hour.

[0067] The experimental results are shown in Figure 3 . The results show that the small molecule compound DBIC can improve the protein expression level of GZMB in tumor cells, and has a concentration gradient effect; DBIC can also improve the activity of GZMB.

[0068] Example 5

[0069] DBIC induces GSDMC-mediated pyroptosis in melanoma cells.

[0070] The experimental method is as follows:

[0071] 1. Take human melanoma A375 cells in the logarithmic growth phase and inoculate 1.5×105 cells into a 12-well plate.

[0072] 2. After overnight culture, the cells were replaced with 0.5% serum medium and treated with DBIC (20 μM) for 9 hours. The cell morphology was observed and recorded, the culture supernatant was collected for lactate dehydrogenase (LDH) release assay, and protein samples were collected for GSDMC immunoblotting assay.

[0073] 3. LDH release detection:

[0074] 1) After 9 h of drug treatment, 100 μL of culture medium was aspirated from each well of the control group (wells without drug treatment), and 100 μL of an aqueous solution containing 20% ​​Triton X-100 was added to lyse the cells and release total intracellular LDH.

[0075] 2) Collect the supernatant in a 2 mL EP tube and centrifuge at 3000 rpm for 5 minutes. Add 50 μL of detection solution and 50 μL of supernatant to a transparent 96-well plate and incubate in the dark for 10 minutes.

[0076] 3) After the reaction is completed, add 50 μL of stop solution to each well to terminate the reaction.

[0077] 4) Detect the absorbance (490 nm) using a microplate reader.

[0078] 4. During Western blotting, the dead cells floating in the culture medium need to be collected by centrifugation. The subsequent operations are the same as those in Example 3.

[0079] The experimental results are as follows Figure 4 The results show that after 9 hours of treatment with the small molecule compound DBIC, cells exhibited a pyroptotic morphology characterized by bubbling, accompanied by the release of lactate dehydrogenase (LDH) and the cleavage of GSDMC protein, indicating that the small molecule compound DBIC can induce GSDMC-mediated pyroptosis. "-" indicates a DMSO control without DBIC.

[0080] Example 6

[0081] DBIC inhibits the growth of melanoma cell xenografts in nude mice.

[0082] The experimental method is as follows:

[0083] 1. Select 40 male BALB / c nude mice (7-8 weeks old, weighing 18-22 g) and place 2×10 6 A375 cells were suspended in 100 μL of DMEM and injected subcutaneously into the flank of mice to establish a xenograft tumor model.

[0084] After 2 and 4 days, the mice were divided into two groups: a blank control group (10% DMSO in corn oil, 100 μL / mouse) and a DBIC-treated group (10 / 20 / 30 mg / kg; DBIC dissolved in 10% DMSO and then diluted in corn oil). The mice were intraperitoneally injected every other day, and the tumor size was measured with a vernier caliper.

[0085] 3. 15 days after inoculation, the mice were euthanized and their tumor weights were recorded.

[0086] The experimental results are as follows Figure 5 The results showed that DBIC could effectively inhibit tumor growth and size, and exhibited a concentration gradient effect, indicating that DBIC has the potential to treat melanoma.

[0087] Example 7

[0088] DBIC inhibits the progression of melanoma cell xenografts in nude mice through the Nur77-GSDMC signaling axis.

[0089] The experimental method is as follows:

[0090] 1. Sixty male BALB / c nude mice (7-8 weeks old, weighing 18-22 g) were selected and divided into three groups. A375 control cells (2×10 6 ), A375 cells with Nur77 gene knockdown (2×10 6 ) and A375 cells with GSDMC gene knockdown (2×10 6 ) were suspended in 100 μL of DMEM culture medium and injected subcutaneously into the flank of mice to establish a xenograft tumor model.

[0091] 2. The administration method for mice and the determination of tumor size and weight were the same as those in Example 6.

[0092] The experimental results are as follows Figure 6 The results showed that DBIC effectively inhibited the growth and size of melanoma cell xenografts, but the inhibitory effect of DBIC was greatly weakened after knockdown of Nur77 or GSDMC. This indicates that the inhibition of nude mouse melanoma cell xenografts by the small molecule compound DBIC is dependent on the presence of Nur77 and GSDMC proteins.

[0093] Example 8

[0094] DBIC inhibited the progression of melanoma tumor transplants by activating anti-tumor immunity.

[0095] The experimental method is as follows:

[0096] 1. 30 male C57BL / 6J mice (6-7 weeks old, 18-22 g in weight) were selected, 1x10 5 B16 cells were suspended in 100 μL of DMEM culture solution and injected subcutaneously into the flank of the mice to establish a xenograft tumor model. Among them, 20 mice were used to detect the inhibition of DBIC on tumor; 10 mice were used to detect the activation of DBIC on the immune system.

[0097] 2. The method of administering DBIC to mice and the detection of tumor size and weight were the same as in Example 6.

[0098] 3. Flow cytometry was used to detect the activation of immune cells in the tumor microenvironment

[0099] 1) After 24 hours of DBIC (20 mg / kg) treatment, B16-derived xenograft tumors were collected, cut into small pieces, and incubated in RPMI-1640 culture solution containing 10% heat-inactivated FBS, 5 mg / mL collagenase IV, and 0.5 mg / mL DNase I for 30 minutes. The digested tissue was filtered through a 70-micron filter.

[0100] 2) The collected cell suspension was purified by 40% and 70% Percoll gradient centrifugation to purify immune cells.

[0101] 3) Before detecting tumor-infiltrating immune cells, the purified immune cells were washed with FACS buffer (2% heat-inactivated FBS in PBS) and blocked with normal mouse serum and CD16 / 32 antibodies. Then the purified immune cells were divided into two groups: (i) cell surface antibody or fluorescent dye; (ii) cell surface marker and intracellular cytokine staining.

[0102] The experimental results are shown in Figure 7 . The results show that DBIC effectively inhibits the growth and size of melanoma cell transplants and increases the proportion of NK cells in the tumor microenvironment, PFN (Perforin) + / CD8 + , IFNγ + / CD8 + , GZMB + / CD8 + , TNF-α + / CD8 + T cells and GZMB +The proportion of positive cell groups of NK is also increased, which indicates that DBIC can activate anti-tumor immunity.

[0103] The small molecule compound DBIC can activate Nur77, GZMB and GSDMC as an inducer of pyroptosis of melanoma cells, and provides a lead compound for the development of melanoma treatment drugs.

[0104] The small molecule compound DBIC can activate orphan nuclear receptor Nur77 as a novel in vitro ligand of Nur77. Specifically, DBIC directly binds to Nur77 to induce an increase in the expression level of Nur77 protein.

[0105] The small molecule compound DBIC can activate the GZMB-GSDMC signaling pathway, both as an agonist of the small molecule compound class of GZMB and as an inducer of GSDMC-mediated pyroptosis. Specifically, DBIC can induce the activation of integrated stress response, leading to an increase in the mRNA and protein levels of GZMB, and further cleave GSDMC to induce pyroptosis death of cells.

[0106] The small molecule compound DBIC can induce pyroptosis death of melanoma cells and can be used as a candidate compound for treating melanoma. Specifically, DBIC can induce cleavage of the pyroptosis execution protein GSDMC, thereby inducing pyroptosis death of melanoma cells. DBIC can significantly inhibit the growth of tumor transplanted tumors in a mouse model and activate anti-tumor immunity, and can be developed as a lead compound for the treatment of melanoma.

Claims

1. A small molecule compound DBIC, characterized in that The structural formula is as follows:

2. The method for preparing the small molecule compound DBIC according to claim 1, characterized in that: The synthetic route is as follows: The steps include: 1) Compound 1, benzimidazole-5-carboxylic acid and di-tert-butyl dicarbonate were dissolved in N,N-dimethylformamide, triethylamine was added dropwise, and the mixture was heated to react to obtain compound 2; 2) Compound 2, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in dichloromethane and reacted in an ice bath. Dodecanol was then added and reacted at room temperature to obtain Compound 3; 3) Compound 3 was dissolved in dichloromethane (DCM), trifluoroacetic acid was added dropwise under ice bath conditions and protective atmosphere, and the reaction was carried out at room temperature to obtain compound DBIC.

3. The method for preparing a small molecule compound DBIC according to claim 2, wherein: In step 1), the heating temperature is 75-105°C.

4. The use of the small molecule compound DBIC according to claim 1, characterized in that: Used in the preparation of anti-melanoma drugs.

5. A drug, characterized in that: The invention comprises the small molecule compound DBIC according to claim 1.

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