Small molecule compound DHN and preparation method and application thereof

Abstract

The invention discloses a small molecule compound DHN as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The compound DHN can trigger opening of mitochondrial membrane permeability conversion pores (mPTP), promote mitochondrial DNA (mtDNA) to be released and enter cytoplasm, activate a cGAS-STING signal channel, and induce melanoma cells to generate pyroptotic death by shearing pyroptosis executive protein GSDME, thereby providing a new target and a lead compound for treatment of melanoma.

Description

Technical Field

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

[0002] Melanoma is the most invasive and dangerous skin cancer developed from transformed melanocytes and is regarded as the "king of cancer". Melanoma has shown a clear upward trend in recent years, accounting for 90% of all skin cancer deaths. At present, the incidence of melanoma is also showing an upward trend year by year. The 5-year disease-free survival (DFS) rate of patients is only 12.3%, and the median overall survival (OS) of patients with advanced melanoma is only 20 months (Tian Huichun et al. Journal of Practical Oncology, 2023, 38, 509-512). People with a family history of skin cancer are more likely to develop melanoma; people with atypical nevi (also known as familial atypical multiple nevus melanoma syndrome) are more likely to develop melanoma (Garbe, C et al. Eur J Cancer, 2010, 46, 270-283). Metastatic melanoma is usually apoptosis-related gene expression loss or mutation, which also makes it extremely insensitive to cell apoptosis induced by traditional chemotherapy and radiotherapy. For example, about 40% of melanomas have Apaf-1 expression loss, and about 30% to 70% of melanoma patients have p16INK / p14ARF gene mutations, resulting in a response rate of only 7.5% to 12.2% to dacarbazine, the leading chemotherapy drug for the treatment of advanced melanoma (Avril, MF et al. J Clin Oncol, 2004, 22, 1118-1125). In the past decade, the clinical diagnosis and treatment of molecular targeted therapies (such as BRAF and MEK inhibitors) and immunotherapies (such as CTLA-4 and PD1 / PDL1 antibodies) have made great progress (Specenier, P et al. Expert Rev Anticancer Ther, 2016, 16, 811-826). However, due to the heterogeneity of melanoma and its extremely strong proliferation and metastasis ability, these new therapies still have many limitations in clinical treatment. Therefore, finding new melanoma treatment pathways and screening broader-spectrum and more efficient anti-melanoma lead compounds have important significance and potential application prospects for clinical treatment.

[0003] The cGAS-STING signaling pathway is one of the important pathways for sensing pathogenic DNA and plays an important role in innate immunity. In addition, the cGAS-STING signaling pathway is also involved in a variety of cellular life processes, including autophagy, translation, metabolic regulation, endoplasmic reticulum stress, aging and programmed cell death (Karl, P. H et al. NAT REV MOL CELL BIO, 2020, 21, 501-521). Studies have shown that STING can activate TBK1 and regulate pro-apoptotic programs through IRF-3 and p53 (Kate McArthur et al. Science, 2018, 359, eaao604). Apoptotic caspases can also inhibit the production of type I interferons by cleaving cGAS, MAVS and IRF3 (Xiaohan Ning et al. Mol Cell, 2019, 74, 19-31). These studies show that there is a complex relationship between STING-mediated innate immunity and cell death. However, there are few reports on the regulation of STING on cell pyroptosis, which needs further exploration.

[0004] The main types of STING agonists are cGAMP mimetics, including cyclic dinucleotides (CDNs) and non-CDN derivatives. However, the chemical properties and instability of CDNs limit their application in systemic immunotherapy. At present, 2', 3'-cGAMP, which can be synthesized autonomously in the human body, is the main activator for studying the biological function of STING in vivo (Zhang Q et al. Curr OpinChem Biol, 2022, 69, 102170). Several other CDN derivatives simulate the binding of cGAMP to STING in cells or in vivo, thereby activating immune responses through STING. However, these STING agonists only play a regulatory role in immune responses and do not play a role in cell pyroptosis. Given the unique role of inducing pyroptotic cell death in melanoma, the development of small molecule compounds to induce pyroptotic death in melanoma is of great significance. Summary of the invention

[0005] The purpose of the present invention is to solve the above problems in the prior art and to provide a small molecule compound 6-hydroxy-2-dodecyl naphthoate (DHN) and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A small molecule compound DHN, the structural formula is as follows:

[0008] .

[0009] The preparation method of the small molecule compound DHN has the following synthesis route:

[0010] .

[0011] The method for preparing the small molecule compound DHN comprises the following steps:

[0012] 1) First, compound 1, i.e., 6-methoxy-2-naphthoic acid and 1-hydroxybenzotriazole HOBT, were added to anhydrous dichloromethane DCM, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI was added under ice bath conditions, and the mixture was stirred thoroughly. Then, 1-dodecanol and triethylamine Et3N were added, and the mixture was heated and stirred to obtain compound 2;

[0013] 2) Compound 2 was dissolved in dichloromethane (DCM), vacuum argon protection, boron tribromide (BBr3) was added, and the mixture was stirred in an ice bath to obtain compound 3 DHN.

[0014] In step 1), the heating temperature is 40~60℃.

[0015] The application of the small molecule compound DHN is used in the preparation of anti-melanoma drugs.

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

[0017] The medicine also includes pharmaceutically acceptable carriers or excipients.

[0018] The medicine also includes other active pharmaceutical ingredients that can be used clinically to fight melanoma.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] The present invention relates to a compound DHN that promotes the opening of the mitochondrial membrane permeability transition pore (mPTP), promotes the release of mitochondrial DNA (mtDNA) into the cytoplasm, activates the cGAS-STING signaling pathway, and promotes GSDME shearing, thereby inducing pyroptotic death of melanoma cells, and ultimately achieving the purpose of treating melanoma. The small molecule compound DHN of the present invention can be used as an agonist of the cGAS-STING signaling pathway and as a pyroptosis inducer, thereby achieving the purpose of treating melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The opening level of mitochondrial permeability transition pore was detected after treating melanoma A375 cells with small molecule compound DHN for 12 hours. The results showed that DHN can significantly promote the opening of mitochondrial permeability transition pore in melanoma A375 cells.

[0022] Figure 2The amount of mitochondrial DNA (mtDNA) in the cytoplasm was detected after the melanoma A375 cells were treated with the small molecule compound DHN for 12 hours. The results showed that DHN can significantly promote the release of mtDNA from mitochondria to the cytoplasm, thereby promoting the damage of cell mitochondria.

[0023] Figure 3 After treating melanoma A375 cells with the small molecule compound DHN for 12 hours, the changes of intracellular cGAS were detected by immunofluorescence. The results showed that DHN could significantly induce cGAS to aggregate in dots, and 1,6-hexanediol could block the cGAS aggregation induced by DHN. The results showed that the small molecule compound DHN could induce phase separation of cGAS.

[0024] Figure 4 After treating melanoma A375 cells with the small molecule compound DHN for 12 hours, the changes of intracellular STING were detected by immunofluorescence. The results showed that DHN could obviously induce STING to aggregate in dots, but 1,6-hexanediol could not block the DHN-induced STING aggregation in dots. The results showed that the small molecule compound DHN could induce STING to aggregate in dots, but the dots were not phase-separated structures.

[0025] Figure 5 The small molecule compound DHN was used to treat melanoma A375 cells for different time periods to observe cell morphological changes, GSDME cleavage and LDH release. The results showed that DHN could induce cell bubbling, and GSDME protein cleavage and large-scale LDH release were observed. The results showed that the small molecule compound DHN could induce pyroptotic death of melanoma cells accompanied by GSDME cleavage and LDH release.

[0026] Figure 6 In the nude mouse transplant tumor model, melanoma A375 cells, A375 cells with knockdown of STING gene, and A375 cells with knockdown of GSDME gene were implanted in the flanks of nude mice. The control group and the experimental group were injected with Saline and DHN once every two days, and the tumor size was measured. After 21 days, the mice were killed and the tumors were collected and weighed. Figure 6 Above: Tumors are arranged from left to right according to weight, with the tumor with the largest weight on the left and the tumor with the smallest weight on the right. Photos are taken for record. Figure 6 Middle panel: Tumor volume was recorded every two days and plotted as a tumor growth curve. Figure 6The figure below: The tumor was weighed after being collected and plotted into a tumor weight statistical chart. The results showed that: in the A375 cell inoculation group, DHN can significantly inhibit tumor growth; in the A375 cell inoculation group with knockdown of the STING gene, the effect of DHN on inhibiting tumor growth was significantly inhibited; in the A375 cell inoculation group with knockdown of the GSDME gene, the effect of DHN on inhibiting tumor growth was significantly inhibited. The results show that the small molecule compound DHN inhibits the growth of nude mouse melanoma cell transplants dependent on STING and GSDME proteins.

[0027] Figure 7 The small molecule compound DHN and its derivatives were used to treat melanoma A375 cells for 24 hours and observed the changes in cell morphology and LDH release. The results showed that DHN could induce cell bubbling and a large amount of LDH release, while its derivatives did not have obvious ability to induce cell pyroptosis. The results showed that the activity of the small molecule compound DHN in inducing cell pyroptosis was better than that of its derivatives. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1

[0030] Synthesis of 6-hydroxy-2-naphthoic acid dodecyl ester (DHN).

[0031] The experimental method is as follows:

[0032] 1. Dissolve compound 1, 6-methoxy-2-naphthoic acid (404 mg, 2.0 mmol) and 1-hydroxybenzotriazole HOBT (297 mg, 2.2 mmol) in anhydrous dichloromethane DCM (10 mL). Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI (421.7 mg, 2.2 mmol) under ice bath conditions and stir thoroughly for 1 hour.

[0033] 2. Then add 1-dodecanol (445 μL, 2.0 mmol) and triethylamine Et3N (306 μL, 2.2 mmol), heat to 50 °C, and stir for 5 hours. After confirming the completion of the reaction with a thin layer plate, add a small amount of water to quench the reaction, wash with sodium bicarbonate and saturated sodium chloride solution in turn, dry with anhydrous magnesium sulfate, and concentrate the solvent in vacuo. The obtained liquid was separated and purified by petroleum ether and ethyl acetate column chromatography to obtain compound 2 (622 mg, 84%) as a white solid. 1 H NMR (600 MHz, CDCl3) : δppm 8.51 - 8.54 (m, 1 H) 8.03 (dd, J = 8.5, 1.7 Hz, 1 H) 7.84 (d, J = 8.8 Hz, 1H) 7.75 (d, J = 8.6 Hz, 1 H) 7.19 (dd, J = 9.0, 2.6 Hz, 1 H) 7.15 (d, J = 2.4 Hz, 1 H) 4.36 (t, J = 6.7 Hz, 2 H) 3.94 (s, 3 H) 1.76 - 1.84 (m, 2 H) 1.44 -1.51 (m, 2 H) 1.35 - 1.41 (m, 2 H) 1.22 - 1.34 (m, 14 H) 0.88 (t, J = 7.0 Hz,3 H). 13 C NMR (150 MHz, CDCl3) : δ ppm 167.0, 159.5, 137.1, 130.9, 130.7, 127.9,126.8, 126.0, 125.6, 119.6, 105.7, 65.2, 55.4, 31.9, 29.7, 29.6, 29.6, 29.6,29.4, 29.3, 28.8, 26.1, 22.7, 14.1. MS (ESI): m / z Theoretical value C 24 H 34 O3[M+H+] 371.3, measured value 371.3.

[0034] 3. Take compound 2 (370 mg, 1.0 mmol) solution in dichloromethane DCM (6 mL), vacuum argon protection, add boron tribromide BBr3 (2.5 mL, 10.0 mmol), stir at -5°C for 2 hours, pour the mixture into water, and extract with ethyl acetate. Wash the diluted solution with water (2× 30 mL) and saturated sodium chloride solution (3× 30 mL), then dry with anhydrous sodium sulfate, and concentrate the solvent in vacuo. Separate and purify with petroleum ether and ethyl acetate (PE / EA = 30:1) column chromatography to obtain compound 3 DHN (271 mg, 76%) as a white solid.

[0035] Structural characterization of compound 3 DHN. 1 H NMR (600 MHz, CDCl3): δ8.51 (s, 1H), 7.99 (dd, J = 8.6, 1.6 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.14 (dd, J = 8.8, 2.4 Hz, 1H), 5.49 (br s, 1H), 4.34 (t, J = 6.7Hz, 2H), 1.83 – 1.75 (m, 2H), 1.49 – 1.41 (m, 2H), 1.40 – 1.32 (m, 2H), 1.26 (dd, J = 17.6, 9.5 Hz, 14H), 0.85 (t, J = 7.0 Hz, 3H). 13 C NMR (150 MHz, CDCl3): δ 167.11 , 155.60 , 137.08 , 131.46 , 130.94 , 127.81 , 126.43 , 126.01 ,125.51 , 118.65 , 109.46 , 65.24 , 31.92 , 31.50 , 30.12 , 29.64 , 29.64 ,29.35 , 29.35 , 28.78 , 26.08 , 22.69 , 14.13 . HRMS (ESI): m / z Theoretical value C 23 H 32 O3[MH] - 355.2273, measured value 355.2271.

[0036] Example 2

[0037] DHN induces the opening of the mPTP pore in mitochondria.

[0038] The experimental method is as follows:

[0039] 1. Take melanoma A375 cells in the logarithmic growth phase and inoculate 3×10 5 cells in 6-well culture plates.

[0040] 2. After overnight culture, the medium was replaced with 2% serum and treated with DHN (15 μM) for 12 hours. The cells were then harvested for mitochondrial mPTP pore opening detection.

[0041] 3. Mitochondrial mPTP pore opening detection:

[0042] 1) Digest the cells with trypsin and resuspend them in culture medium. Use the suspended cells directly, count them, take an appropriate amount of cells and centrifuge them (1000× g, 5 minutes, room temperature), discard the supernatant and resuspend them.

[0043] 2) Incubate the cells with Calcein AM (1 μM) staining solution and cobalt chloride (CoCl2, 5 mM) in a 37 °C incubator in the dark for 30 min.

[0044] 3) After incubation, centrifuge at 1000× g for 5 minutes at room temperature to collect the cells. Add 1 mL of detection buffer to each sample, gently resuspend, and centrifuge at 1000× g for 5 minutes at room temperature to collect the cells.

[0045] 4) Resuspend cells in 400 μL of assay buffer.

[0046] 5) Use a sample of cells without staining and containing only detection buffer for negative control settings on the flow cytometer. Calcein has an excitation maximum wavelength of 494 nm and an emission maximum wavelength of 517 nm. Use flow cytometry to assess the fluorescence intensity emitted by the cells to assess the degree of mPTP opening.

[0047] The experimental results are as follows Figure 1 The results showed that the small molecule compound DHN can significantly reduce the fluorescence of Calcein in cell mitochondria, indicating that the small molecule compound DHN can induce the opening of the MPTP pore in cell mitochondria.

[0048] Example 3

[0049] DHN induces the release of mitochondrial DNA.

[0050] The experimental method is as follows:

[0051] 1. Take human melanoma A375 cells in logarithmic growth phase and inoculate 3×10 6 cells in a 10 cm culture plate.

[0052] 2. After overnight culture, the medium was replaced with 2% serum and treated with DHN (15 μM) for 12 hours. The cells were then collected for detection of cytoplasmic mitochondrial DNA (mtDNA).

[0053] 3. Separation of cell components:

[0054] 1) Taking a 10 cm dish as an example, after the cells are treated with drugs, remove the culture medium and add 2 mL of pre-cooled PBS to wash away the residual culture medium, then remove the PBS.

[0055] 2) Add 2 mL of pre-cooled PBS to a 10 cm dish, scrape the cells with a cell scraper and transfer them to a 10 mL tube, centrifuge at 3000 rpm for 3 minutes at 4 °C, and discard the supernatant.

[0056] 3) Add 1 mL of pre-cooled PBS to the 10 mL tube to resuspend the cells, and take 80 μL of the suspension and place it in a new 1.5 mL EP tube as a standby for total protein. Transfer the remaining 900 μL of cells to the new EP tube and centrifuge at 3000 rpm for 3 minutes at 4 ℃. Wash once more with PBS.

[0057] 4) After washing, discard the supernatant, add 100 μL of Lysis Buffer (75 mM sodium chloride, 1 mM sodium dihydrogen phosphate, 8 mM sodium dihydrogen phosphate, 250 mM sucrose) without digitonin and the protease inhibitor phenylmethanesulfonyl fluoride (PMSF), and use a pipette to blow the cells apart and resuspend them thoroughly.

[0058] 5) Add 100 μL of Lysis Buffer (75 mM sodium chloride, 1 mM sodium dihydrogen phosphate, 8 mM sodium dihydrogen phosphate, 250 mM sucrose, 380 mg / mL digitonin) containing 380 mg / ml digitonin, and then flick the EP tube 8-10 times to mix the mixture thoroughly. Let stand on ice for 5 minutes. Digitonin is a weak membrane permeabilization agent, and the membrane permeabilization time needs to be controlled.

[0059] 6) After standing, centrifuge at 12,000 × g for 5 minutes at 4°C. Transfer the supernatant to a new 1.5 mL EP tube and place on ice as the cytosolic fraction and the precipitate as the fraction containing mitochondria.

[0060] 4. Genomic DNA Extraction

[0061] Genomic DNA was extracted using MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (Cat# 9765, TaKaRa).

[0062] 5. Dilute the extracted DNA 5 times and use it as a template. Prepare the PCR reaction system as follows: 5 μL SYBRmix, 1 μL 2 μM primer, 4 μL cDNA template. Use Bio-rad CFX96 to perform fluorescence quantitative PCR detection.

[0063] The experimental results are as follows Figure 2 The results showed that after treatment with the small molecule compound DHN, mitochondrial genes CO2, ND1 and ATP6 were significantly enriched in the cytoplasm, indicating that the small molecule compound DHN can induce the release of mitochondrial DNA into the cytoplasm.

[0064] Example 4

[0065] DHN induces phase separation of cGAS.

[0066] The experimental method is as follows:

[0067] 1. Take human melanoma A375 cells in logarithmic growth phase and inoculate 3×10 5 The cells were cultured in a 6-well culture plate with glass slides placed in advance.

[0068] 2. After overnight culture, the medium was replaced with 2% serum and treated with DHN (15 μM) for 12 hours.

[0069] 3. Treat the cells with 1,6-hexanediol (5%, 1,6-HD) for 5 minutes and collect the cells for cGAS immunofluorescence detection.

[0070] 4. Immunofluorescence detection

[0071] 1) After drug treatment, add 200 μL of formaldehyde solution (final concentration is 4%) to each well of the culture plate. Shake and fix for 10 minutes at room temperature. Adding formaldehyde directly to the culture medium can maintain the state of the cells when the drug is added.

[0072] 2) After the cells are fixed with formaldehyde, discard the fixative and wash with PBS three times, 5 min each time.

[0073] 3) After washing, add 2 mL of blocking buffer to each well and shake on a shaker at room temperature for 20 minutes. The blocking buffer is prepared by dissolving Triton X-100 (0.2%) and BSA (3%) in PBS and filtering with a 0.22 μM filter to remove large particles.

[0074] 4) After blocking, incubate with the primary antibody cGAS in a 4°C refrigerator overnight. The primary antibody is prepared in blocking solution.

[0075] 6) After primary antibody incubation, wash the cells 5 times with washing buffer, 10-15 minutes each time. The washing buffer is prepared by dissolving Triton X-100 (0.05%) and BSA (0.2%) in PBS and filtering with a 0.22 μM filter to remove large particles.

[0076] 7) After washing, incubate with the corresponding fluorescent secondary antibody at room temperature in the dark for 1 to 1.5 hours. The incubation time of the secondary antibody should not be too long. The secondary antibody is prepared in the blocking solution.

[0077] 8) After incubation with the secondary antibody, wash the cells three times with washing solution, each time for 10-15 minutes.

[0078] 9) After washing with the cleaning solution, wash once with PBS. Add 2 mL of PBS containing DAPI (50 mg / mL) dye to stain the cell nucleus for about 15 minutes at room temperature, protect from light, and incubate on a shaker. After staining, wash the cells 3 times with PBS, each time for 5 to 10 minutes.

[0079] 10) After sealing, observe and photograph under a confocal microscope.

[0080] The experimental results are as follows Figure 3 The results showed that cGAS aggregated in spots after treatment with the small molecule compound DHN, and incubation with 1,6-hexanediol (1,6-HD) could block the cGAS aggregation induced by DHN, indicating that the small molecule compound DHN can induce cGAS aggregation, and this aggregation is a phase separation structure.

[0081] Example 5

[0082] DHN induces the aggregation and activation of STING.

[0083] The experimental method is as follows:

[0084] 1. Take human melanoma A375 cells in logarithmic growth phase and inoculate 3×10 5 The cells were cultured in a 6-well culture plate with glass slides placed in advance.

[0085] 2. After overnight culture, the medium was replaced with 2% serum and treated with DHN (15 μM) for 12 hours.

[0086] 3. Treat the cells with 1,6-hexanediol (5%, 1,6-HD) for 5 minutes and collect the cells for STING immunofluorescence detection.

[0087] 4. Immunofluorescence detection.

[0088] 1) Remove the cell slides and place them in pre-chilled acetone for fixation for 1 minute.

[0089] 2) After the cells are fixed with acetone, discard the fixative and wash with PBS three times, 5 minutes each time.

[0090] 3) After washing, add 2 mL of blocking buffer to each well and shake on a shaker at room temperature for 20 minutes. The blocking buffer is prepared by dissolving Triton X-100 (0.2%) and BSA (3%) in PBS and filtering with a 0.22 μM filter to remove large particles.

[0091] 4) After blocking, incubate with the primary antibody STING in a 4°C refrigerator overnight. The primary antibody is prepared in blocking solution.

[0092] 5) After primary antibody incubation, wash the cells 5 times with washing buffer, each time for 10-15 minutes. The washing buffer is prepared as follows: Dissolve Triton X-100 (0.05%) and BSA (0.2%) in PBS, and filter with a 0.22 μM filter to remove large particles.

[0093] 6) After washing, incubate with the corresponding fluorescent secondary antibody at room temperature in the dark for 1 to 1.5 hours. The incubation time of the secondary antibody should not be too long. The secondary antibody is prepared in the blocking solution.

[0094] 7) After incubation with the secondary antibody, wash the cells three times with washing solution, each time for 10 to 15 minutes.

[0095] 8) After washing with the cleaning solution, wash once with PBS. Add 2 mL of PBS containing DAPI (50 mg / mL) dye to stain the cell nucleus for about 15 minutes at room temperature, protect from light, and incubate on a shaker. After staining, wash the cells 3 times with PBS, each time for 5 to 10 minutes.

[0096] 9) After sealing, observe and photograph under a confocal microscope.

[0097] The experimental results are as follows Figure 4 The results showed that after treatment with the small molecule compound DHN, STING aggregated in spots, but 1,6-HD could not block the DHN-induced STING aggregation, indicating that the small molecule compound DHN can induce STING aggregation but this aggregation is not a phase separation structure.

[0098] Example 6

[0099] DHN induces pyroptosis of melanoma cells accompanied by GSDME protein cleavage and LDH release.

[0100] The experimental method is as follows:

[0101] 1. Cell inoculation is the same as in Example 2.

[0102] 2. After overnight culture, the cells were replaced with 2% serum medium and treated with DHN (15 μM) for 8, 12, 16, 20, and 24 hours. The cells were photographed and lactate dehydrogenase (LDH) release was detected. Protein samples were collected and subjected to immunoblotting.

[0103] 3. LDH release detection:

[0104] 1) After 24 hours of drug treatment, aspirate 200 μL of culture medium from each well and add 200 μL of 20% Triton x-100 aqueous solution to lyse the cells and release total intracellular LDH.

[0105] 2) Collect the cell supernatant of each well with a 2 mL EP tube and centrifuge at 3000 rpm for 3 minutes at room temperature. Transfer 50 μL of supernatant culture medium to each EP tube and transfer to a 96-well plate. Also transfer 50 μL of 2% FBS DMEM culture medium as a blank control group (because serum contains LDH, which will react with the test solution, so 2% FBS DMEM is used as a blank control). Add 50 μL of test solution to each well and react with the LDH enzyme in the culture solution for 10 minutes in the dark.

[0106] 3) After the reaction is completed, add 50 μL of stop solution to each well to terminate the reaction.

[0107] 4) Use an enzyme-labeled instrument to measure the absorbance (490 nm). The absorbance value represents the amount of LDH.

[0108] 4. Western Blot Detection:

[0109] 1) Discard the culture medium, wash three times with PBS, and add 300 μL of cell lysis buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 1% NP-40, 1 mM PMSF and phosphatase inhibitor cocktail).

[0110] 2) Disrupt the cells using an ultrasonic disruptor and centrifuge (13,000 rpm, 10 min, 4°C).

[0111] 3) Aspirate the supernatant, add an equal volume 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.

[0112] 4) Protein SDS-PAGE electrophoresis: The protein loading amount was 10 μg, and the sample was electrophoresed in SDS-PAGE gel with a voltage of 100 V for the stacking gel. After the sample entered the separation gel, the voltage was adjusted to 150 V.

[0113] 5) Transfer: After electrophoresis, cut off the concentrated gel, assemble the transfer sandwich with PVDF membrane and filter paper pretreated with ethanol immersed in electrotransfer solution, remove all bubbles, and perform electrotransfer at 4 °C (100 V, 60 min).

[0114] 6) Antigen-antibody reaction:

[0115] A. Blocking: Block with 5% skim milk or BSA for 1 hour;

[0116] B. Primary antibody reaction: Add diluted primary antibody and incubate at room temperature for 1.5 hours;

[0117] 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.

[0118] 7) 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.

[0119] The experimental results are as follows Figure 5 The results showed that the small molecule compound DHN could induce cell blistering in melanoma cells after 12 hours of treatment, accompanied by the release of lactate dehydrogenase (LDH) and the cleavage of GSDME protein in a time gradient, indicating that the small molecule compound DHN can induce pyroptotic death of melanoma cells accompanied by GSDME cleavage and LDH release.

[0120] Example 7

[0121] DHN inhibits the growth of nude mouse melanoma cell transplants in a manner that is dependent on STING and GSDME proteins.

[0122] The experimental method is as follows:

[0123] 1. Select 60 male BALB / c nude mice (7-8 weeks old, weighing 18-22 g) and divide them into three groups. 6 A375 cells, 2×10 6 A375 cells with STING gene knockdown, 2×10 6 A375 cells with GSDME gene knockdown were suspended in 100 μL of DMEM and injected subcutaneously into the flank of mice to establish a xenograft tumor model.

[0124] After 2 and 4 days, all three groups of mice were equally divided into two groups and administered with blank control Saline (10% DMSO in saline, 100 μL / mouse) and DHN treatment (20 mg / kg; DHN was dissolved in DMSO and then diluted with saline) by intraperitoneal injection every other day for 2 weeks.

[0125] 3. Seven days after inoculation, the tumor size was measured every two days using a vernier caliper.

[0126] 4. 21 days after inoculation, the mice were euthanized and their tumor weights were recorded.

[0127] The experimental results are as follows Figure 6 As shown. The results showed that mice inoculated with melanoma cells in the control group formed obvious tumors after 21 days. In mice inoculated with A375 cells, DHN treatment significantly reduced tumor size and tumor weight, indicating that the small molecule compound DHN can effectively inhibit the growth of nude mouse melanoma cell transplants. In mice inoculated with melanoma cells in which the STING and GSDME genes were knocked down, DHN could not inhibit tumor growth, indicating that the small molecule compound DHN inhibited the growth of nude mouse melanoma cell transplants dependent on STING and GSDME proteins.

[0128] Example 8

[0129] The activity of DHN in inducing cell pyroptosis is better than that of its derivatives.

[0130] The experimental method is as follows:

[0131] 1. Cell inoculation is the same as in Example 2.

[0132] 2. After overnight culture, the cells were replaced with 2% serum medium and treated with 15 μM DHN and its derivatives 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 for 24 hours. The cells were photographed and lactate dehydrogenase (LDH) release was detected.

[0133] 3. LDH release detection is the same as in Example 6.

[0134] The structures of DHN and its derivatives 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 are as follows:

[0135]

[0136] The experimental results are as follows Figure 7The results showed that the small molecule compound DHN could induce cell blistering of melanoma cells after 24 hours of treatment, accompanied by the release of lactate dehydrogenase (LDH), while its derivatives did not observe cell blistering and LDH release, indicating that the ability of the small molecule compound DHN to induce pyroptotic death of melanoma cells is better than that of its derivatives.

Claims

1. A small molecule compound DHN, characterized in that: The structural formula is as follows: 。 2. The method for preparing a small molecule compound DHN according to claim 1, characterized in that: The synthetic route is as follows: 。 3. The method for preparing a small molecule compound DHN according to claim 2, characterized in that: The following steps are involved: 1) First, compound 1, i.e., 6-methoxy-2-naphthoic acid and 1-hydroxybenzotriazole HOBT, were added to anhydrous dichloromethane DCM, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI was added under ice bath conditions, and the mixture was stirred thoroughly. Then, 1-dodecanol and triethylamine Et3N were added, and the mixture was heated and stirred to obtain compound 2; 2) Compound 2 was dissolved in dichloromethane (DCM), vacuum argon protection, boron tribromide (BBr3) was added, and the mixture was stirred in an ice bath to obtain compound 3 DHN.

4. The method for preparing a small molecule compound DHN according to claim 3, characterized in that: In step 1), the heating temperature is 40~60℃.

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

6. A drug, characterized in that: Contains the small molecule compound DHN according to claim 1.

7. The drug according to claim 6, characterized in that: It also includes pharmaceutically acceptable carriers or excipients.

8. The drug according to claim 7, characterized in that: It also includes other active pharmaceutical ingredients that can be used clinically to fight melanoma.

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