Small molecule compound DBIC and preparation method and application thereof

Abstract

The invention relates to a small molecule compound DBIC and a preparation method and application thereof, and belongs to the technical field of biological medicine.The small molecule compound DBIC directly binds to and activates an orphan nuclear receptor Nur77 to activate an integrated stress response (ISR), so that the expression level and activity of granzyme B (GZMB) in tumor cells are further activated, then pyroptosis executive protein GSDMC is cut, and the small molecule compound DBIC can be used for preparing a tumor cell model. Finally, melanoma cells are induced to generate pyroptotic death, and a brand new lead compound is provided for 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 type of lytic cell death performed by members of the gasdermin family. Gasdermin protein family members include: GSDMA, GSDMB, GSDMC, GSDMD, GSDME and PVJK, which have highly conserved N-terminal and C-terminal domains. When the gasdermin protein is cleaved, the C-terminal domain no longer inhibits the activity of the N-terminal domain, releasing the N-terminal domain with membrane pore-forming activity. The N-terminal domain then binds to the cell membrane phospholipids and oligomerizes to form a stable pore structure, thereby destroying the integrity of the cell membrane, leading to the release of intracellular substances and the cell absorbing water and bursting, thereby triggering pyroptotic cell death (Shi Jianjin et al. Trends in biochemical sciences, 2017, 42, 245-254). Different gasdermin proteins exhibit a variety of functions in the occurrence and development of tumors and the body's anti-tumor immunity. For example, GSDME has a broad-spectrum inhibitory effect on a variety of tumors, and its expression in tumor cells is usually silenced; however, GSDME is highly expressed in normal tissues, so while inducing GSDME-mediated tumor pyroptosis, it also causes pyroptosis in normal cells and produces toxic side effects (Wang Yupeng et al., Nature, 2017, 547, 99-103). At present, the research on cell pyroptosis mainly focuses on GSDMD and GSDME, while the research on the regulation of GSDMC is still unclear. One of the main reasons is the lack of effective lead compounds that induce GSDMC-mediated pyroptosis.

[0003] Current tumor treatment also faces some difficulties. For example, the use of chemotherapy drugs is a key part of the tumor treatment process. The mechanism of action of most clinical chemotherapy drugs is to inhibit tumors by inducing tumor cell apoptosis. Due to the long-term use of chemotherapy drugs, many tumors have apoptosis resistance. At the same time, some tumors, such as melanoma, are resistant to apoptotic death, which limits the clinical use and treatment effect of chemotherapy drugs (Wang Weijia et al., Nature Chemical Biology, 2014, 10, 133-140). The promotion of immunotherapy is also limited by factors such as the fact that immunotherapy does not work well in some solid tumors and the high cost.

[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 over inducing apoptosis are as follows: 1) Cell contents will not be released during apoptosis, which is an immune-silent death mode. Pyroptosis is a pro-inflammatory death mode. When cells undergo pyroptosis, they release many inflammatory factors, thereby recruiting immune cells to further kill tumor cells, and can amplify the effect by inducing the body's anti-tumor immune response. The research group of Academician Shao Feng found that although only a small number of tumor cells were induced to undergo pyroptosis, the body was able to almost completely eliminate the tumor through immune response (Wang Qinyang et al., Nature, 2020, 579, 421-426). 2) Pyroptosis is faster and more rapid than apoptosis. Therefore, screening drugs (including small molecule compounds) that induce new cell death (such as pyroptotic death) in tumor cells has become a new direction for tumor treatment. Since melanoma highly expresses gasdermin family proteins, especially GSDMC, screening compounds that induce pyroptosis in melanoma is of great significance for 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). Other types of GZMB agonists (such as small molecule compounds) have been less studied. GZMB is mainly highly expressed in immune cells and lowly expressed in tumor cells. The functional research of endogenous GZMB in tumor cells is very unclear, and small molecule compounds that can activate GZMB have basically not been reported.

[0006] Orphan nuclear receptor Nur77 (also known as TR3, NGF1-B and NAK1) is a protein encoded by the early gene NR4A1 and belongs to the steroid / thyroid / retinoic acid receptor superfamily (Hazel, TG, 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 glucose and lipid metabolism, the 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 in the present invention is an in vitro agonist of the orphan nuclear receptor Nur77. At the same time, it can not only serve as an activator of granzyme B (GZMB), but also as an inducer of pyroptosis, and has broad application prospects. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems in the prior art and provide a small molecule compound benzimidazole-5-carboxylic acid dodecyl ester ( dodecyl 1H- b enzo[d] i midazole-5- c The invention discloses a novel deoxyribonucleic acid (DBIC) and its preparation method and application, wherein the DBIC can directly bind to Nur77 and cause activation of 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 scheme:

[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 reacted at room temperature to obtain compound 3;

[0016] 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 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 medicine comprising the small molecule compound DBIC described in the present invention.

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

[0021] The small molecule compound DBIC of the present invention can directly bind to and activate Nur77, promote the activation of ISR, lead to the increase of expression and activity of the shearing enzyme GZMB, thereby shearing GSDMC to induce pyroptotic death of melanoma cells, and finally inhibit the development of melanoma. The small molecule compound DBIC proposed in the present invention is not only an in vitro agonist of the orphan nuclear receptor Nur77, but also can be used as an activator of granzyme B (GZMB) and an inducer of pyroptosis mediated by GSDMC. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This 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 The protein expression level of Nur77 was detected after melanoma A375 cells were treated with the small molecule compound DBIC for 5 hours. The results showed that DBIC could significantly increase the protein expression of Nur77.

[0024] Figure 3 The expression and activity of GZMB protein were detected after treating melanoma A375 cells with the small molecule compound DBIC for 6 hours. "-" refers to the control with DMSO solvent without DBIC, and "+" refers to the addition of DBIC. The results show that DBIC can significantly increase the protein expression level and activity of GZMB. The results show that the small molecule compound DBIC can activate GZMB.

[0025] Figure 4 After treating melanoma A375 cells with the small molecule compound DBIC for 9 hours, the cell morphology, LDH release and GSDMC shearing were observed. The results showed that DBIC could significantly induce the cells to produce "bubbling" pyroptosis morphology, LDH release and GSDMC shearing. The results showed that the small molecule compound DBIC can induce GSDMC-mediated cell pyroptosis in melanoma cells.

[0026] Figure 5 The small molecule compound DBIC inhibits the development of nude mouse transplant tumors derived from melanoma cells A375. Melanoma cells A375 were inoculated on the flanks of nude mice, and DBIC was intraperitoneally administered every other day. Tumor growth was detected every day. The mice were killed after 15 days, and the tumors were collected and weighed. The upper picture shows the morphology of mouse tumors arranged from large to small in the corn oil control, i.e. "Ctrl", and three concentrations of DBIC drugs. The lower left picture is the tumor volume detection picture of the four treatments, and the lower right picture is the statistical picture of the tumor weight of the four treatments. The results show that DBIC can significantly inhibit the growth of transplant tumors.

[0027] Figure 6In the nude mouse transplant tumor model, melanoma A375 cells, A375 cells with Nur77 knockdown, and A375 cells with GSDMC knockdown were planted in the flanks of nude mice, respectively. DBIC was injected every two days and the tumor size was measured. The mice were killed after 30 days, and the tumors were collected and weighed. The upper figure is a tumor morphology arranged from large to small after injection of three types of cells, namely "Ctrl", "Nur77 KD" and "GSDMC KD", and treated with the control corn oil solvent, namely "-" or DBIC; the lower left figure is a tumor volume detection figure of 6 treatments, and the lower right figure is a statistical figure of tumor weight of 6 treatments; the results show that DBIC can significantly inhibit the growth of tumors derived from A375 cells, and when Nur77 or GSDMC in tumor cells is knocked down, the ability of DBIC to inhibit tumor growth is greatly weakened. The results show that the small molecule compound DBIC inhibits the growth of nude mouse melanoma cell transplant tumors 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 being treated with corn oil control, i.e. "Ctrl" and DBIC drugs. The lower left picture is a tumor volume detection picture of the two treatments, and the lower right picture shows the various immune cells in the tumor samples after being treated 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 was also increased. The results showed 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 in conjunction with 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, triethylamine Et3N (2.6 mL, 18.5 mmol) was added dropwise, and the reaction 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, ethyl acetate (30 mL) was used for extraction, and the combined aqueous phase solution was retained, and then the aqueous phase pH was 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 solution was vacuum concentrated 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 one pot, stirred at 0 °C for 1 hour, then dodecanol (2.9 g, 15.4 mmol) and Et3N (2.3mL, 17 mmol) were added to the mixed solution, and the mixture was stirred at room temperature for 12 hours. After the reaction was completed, the mixture was quenched with saturated sodium bicarbonate NaHCO3 solution, the organic phase was dried with anhydrous sodium sulfate Na2SO4 and concentrated under reduced pressure, and purified by column chromatography with petroleum ether and ethyl acetate as eluents to obtain oily compound 3 (5 g, yield: 75%).

[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 the protection of inert argon gas (Ar), and the reaction was stirred at room temperature for 6 hours. The solution after the reaction was poured into ice water, extracted with ethyl acetate, dried over Na2SO4, and evaporated under reduced pressure. Column chromatography was performed with petroleum ether and ethyl acetate (PE / EA = 1:9) to obtain white compound 4 (1.8 g, yield: 47.3%) with a melting point of 110~112 °C.

[0036] 1H NMR (600 MHz, CDCl3) δ 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) δ 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 Nur77.

[0039] The experimental method is as follows:

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

[0041] 2. Obtain crystals by hanging drop vapor diffusion for 3-7 days at 4°C in 100 mM sodium citrate (pH 4.0), 20% glycerol, and 5%-10% PEG4000. Briefly transfer the crystals to crystallization conditions supplemented with 20% ethylene glycol as a cryoprotectant and then plunge them into liquid nitrogen.

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

[0043] The experimental results are as follows Figure 1 The results showed that the co-crystal 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 changed to 0.5% serum medium and treated with DBIC (20 μM) for 0.5 h. The Nur77 protein level was detected by Western blotting.

[0049] 3. Western Blotting Experiment:

[0050] 1) Discard the DBIC-treated cell culture medium, add 200 μL 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: 20 μg of protein was loaded and electrophoresed on SDS-PAGE gel at 100 V until it reached the bottom of the gel.

[0052] 3) Transfer: After electrophoresis, cut off the concentrated gel, assemble and transfer the PVDF membrane and filter paper pretreated with ethanol and immersed in the electrotransfer solution, and transfer at 4 ℃ (100 V, 60 minutes).

[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 and incubate at room temperature overnight (other antibodies should be incubated at room temperature for 1.5 hours);

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

[0057] 5) ECL detection: Wash with TBST 3 times, 10 minutes each time. Mix ECL solution A and solution B in a ratio of 1:1, drop onto the surface of the PVDF membrane, and develop using a chemiluminescence imager.

[0058] The experimental results are as follows Figure 2 The results showed that DBIC could increase the protein expression level of Nur77 and exhibit a concentration gradient effect.

[0059] Example 4

[0060] DBIC increases the expression level and activity of GzmB.

[0061] The experimental method is as follows:

[0062] 1. Take human melanoma A375 cells in logarithmic growth phase and inoculate 1.5×10 5 cells in 12-well plates, and the experimental steps were the same as those in Example 3; when measuring GZMB activity, 10 10 cm dishes were plated in each group, and 3×10 6 cells in a 10 cm dish.

[0063] 2. GZMB activity experiment

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

[0065] 2) Resuspend and lyse the cells in 150 μL Triton X-100 lysis buffer (1% Triton (v / v) in PBS) on ice, and then centrifuge at 12,000 g for 15 min at 4°C.

[0066] 3) Take the supernatant after centrifugation and incubate it with Ac-IEPD-AMC TFA, and read the fluorescence continuously for 1 hour using a microplate reader.

[0067] The experimental results are as follows Figure 3 The results showed that the small molecule compound DBIC can increase the protein expression level of GZMB in tumor cells and has a concentration gradient effect; DBIC can also increase 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 in 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 detection, and protein samples were collected for GSDMC immunoblot detection.

[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 aqueous solution containing 20% ​​Triton x-100 was added to lyse the cells and release the total intracellular LDH.

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

[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 an ELISA reader.

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

[0079] The experimental results are as follows Figure 4 As shown. The results showed that after 9 hours of treatment with the small molecule compound DBIC, the cells showed a "bubbling" pyroptosis morphology, 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 cell pyroptosis. "-" refers to the control with DMSO solvent added instead of 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 was dissolved in 10% DMSO and diluted in corn oil), which were injected intraperitoneally 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 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 method of administration to mice and the detection of tumor size and weight are the same as those in Example 6.

[0092] The experimental results are as follows Figure 6 As shown. The results showed that DBIC effectively inhibited the growth and size of melanoma cell transplants, but the inhibitory effect of DBIC was greatly weakened after knocking down Nur77 or GSDMC. This shows that the small molecule compound DBIC inhibits nude mouse melanoma cell transplants depending on the presence of Nur77 and GSDMC proteins.

[0093] Example 8

[0094] DBIC inhibits melanoma xenograft progression by activating anti-tumor immunity.

[0095] The experimental method is as follows:

[0096] 1. Select 30 male C57BL / 6J (6-7 weeks old, weighing 18-22 g) and place 1×10 5 B16 cells were suspended in 100 μL of DMEM culture medium and injected subcutaneously into the flank of mice to establish a xenograft tumor model. Twenty mice were used to test the inhibitory effect of DBIC on tumors, and 10 mice were used to test the activation of the immune system by DBIC.

[0097] 2. The method of administration to mice and the detection of tumor size and weight are the same as those in Example 6.

[0098] 3. Flow cytometry 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 for 30 minutes in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 5 mg / mL collagenase IV, and 0.5 mg / mL DNase I. The digested tissue was filtered through a 70 μm 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, purified immune cells were washed with FACS buffer (PBS containing 2% heat-inactivated FBS) and blocked with normal mouse serum and CD16 / 32 antibody. The purified immune cells were then divided into two groups: (i) cell surface antibodies or fluorescent dyes; (ii) cell surface markers and intracellular cytokine staining.

[0102] The experimental results are as follows Figure 7 The results showed that DBIC effectively inhibited the growth and size of melanoma cell transplants and increased the proportion of NK cells in the tumor microenvironment. + / CD8 + , IFNγ + / CD8 + , GZMB + / CD8 + , TNF-α + / CD8 + T cells and GZMB +The proportion of NK positive cell populations also increased, indicating that DBIC can activate anti-tumor immunity.

[0103] The small molecule compound DBIC of the present invention is used as an activator of Nur77, GZMB and GSDMC to induce pyroptosis of melanoma cells, thereby providing a lead compound for the development of melanoma therapeutic drugs.

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

[0105] The small molecule compound DBIC can activate the GZMB-GSDMC signaling pathway, acting as both a small molecule compound agonist of GZMB and a GSDMC-mediated pyroptosis inducer. Specifically, DBIC can induce activation of the integrated stress response, leading to increased mRNA and protein levels of GZMB, and then cleaving GSDMC to induce cell pyroptosis death.

[0106] The small molecule compound DBIC can induce pyroptotic death of melanoma cells and can be used as a candidate compound for the treatment of melanoma. Specifically, DBIC can induce the cleavage of the pyroptotic execution protein GSDMC, thereby inducing pyroptotic death of melanoma cells. In the mouse model, DBIC can significantly inhibit the growth of tumor transplants and activate anti-tumor immunity, and can be developed and studied 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 a small molecule compound DBIC according to claim 1, characterized in that: The synthetic route is as follows: 。 3. The method for preparing a small molecule compound DBIC according to claim 2, characterized in that: The following steps are involved: 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. Then, dodecanol was 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 4.

4. The method for preparing a small molecule compound DBIC according to claim 3, characterized in that: In step 1), the heating temperature is 75~105°C.

5. The use of a small molecule compound DBIC according to claim 1, characterized in that: Used in preparing anti-melanoma drugs.

6. A drug, characterized in that: Contains the small molecule compound DBIC as claimed in claim 1.

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