A small molecule compound DHN and a preparation method and application thereof

Abstract

A small molecule compound DHN and a preparation method and application thereof relate to the technical field of biological medicine.The compound DHN can induce the opening of a mitochondrial membrane permeability transition pore (mPTP), promote the release of mitochondrial DNA (mtDNA) into the cytoplasm, activate a cGAS-STING signal pathway, cut pyroptosis execution protein GSDME, induce pyroptosis death of melanoma cells, and provide a new target and a lead compound 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 DHN and a preparation method and application thereof. Background Art

[0002] Melanoma, the most aggressive and dangerous skin cancer that develops from transformed melanocytes, is considered the "king of cancers." Melanoma has shown a significant upward trend in recent years, accounting for 90% of all skin cancer deaths. Melanoma incidence is also increasing annually, with the 5-year disease-free survival (DFS) rate remaining at only 12.3%, and the median overall survival (OS) for patients with advanced melanoma being only 20 months (Tian Huichun et al., Journal of Practical Oncology, 2023, 38, 509-512). People with a family history of skin cancer are at increased risk for developing melanoma, and those with atypical nevi (also known as familial atypical multiple nevus melanoma syndrome) are even more likely to develop melanoma (Garbe, C et al., Eur J Cancer, 2010, 46, 270-283). Metastatic melanomas often display loss of expression or mutations in apoptosis-related genes, making them extremely insensitive to apoptosis induced by conventional chemoradiotherapy. For example, approximately 40% of melanomas lack Apaf-1 expression, and approximately 30%–70% of melanoma patients harbor p16INK / p14ARF gene mutations, resulting in a response rate of only 7.5%–12.2% to dacarbazine, the leading chemotherapy drug for advanced melanoma (Avril, MF et al., J Clin Oncol, 2004, 22, 1118–1125). Over the past decade, significant progress has been made in the clinical diagnosis and treatment of molecularly targeted therapies (such as BRAF and MEK inhibitors) and immunotherapies (such as CTLA-4 and PD1 / PDL1 antibodies) (Specenier, P et al., Expert Rev Anticancer Ther, 2016, 16, 811–826). However, due to the heterogeneity of melanoma and its strong proliferative and metastatic potential, these novel therapies still have many limitations in clinical treatment. Therefore, finding new melanoma treatment approaches 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 a key pathway for sensing pathogenic DNA and plays a crucial role in innate immunity. Furthermore, the cGAS-STING pathway is involved in numerous cellular processes, including autophagy, translation, metabolic regulation, endoplasmic reticulum stress, senescence, and programmed cell death (Karl, P. H. et al. Nat Rev Mol Cell Biol, 2020, 21, 501-521). Studies have shown that STING can activate TBK1, regulating pro-apoptotic programs through IRF-3 and p53 (Kate McArthur et al. Science, 2018, 359, eaao604). Apoptotic caspases can also inhibit type I interferon production by cleaving cGAS, MAVS, and IRF3 (Xiaohan Ning et al. Mol Cell, 2019, 74, 19-31). These studies suggest a complex relationship between STING-mediated innate immunity and cell death. However, STING's regulation of pyroptosis is less well-documented, warranting further investigation.

[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. Currently, 2',3'-cGAMP, which is synthesized autonomously in the human body, is the primary activator for studying the biological functions of STING in vivo (Zhang Q et al., Curr Opin Chem Biol, 2022, 69, 102-170). Several other CDN derivatives mimic the binding of cGAMP to STING in cells or in vivo, thereby activating immune responses through STING. However, these STING agonists only exert regulatory functions on immune responses and do not induce pyroptosis. Given the unique role of pyroptotic cell death in melanoma, the development of small molecule compounds to induce pyroptotic cell 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 provide a small molecule compound 6-hydroxy-2-dodecyl naphthoate (DHN) and its preparation method and application.

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

[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 synthetic route:

[0010] .

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

[0012] 1) Compound 1, 6-methoxy-2-naphthoic acid, and 1-hydroxybenzotriazole (HOBT), were added to anhydrous dichloromethane (DCM). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was added under ice-cooling conditions and stirred thoroughly. 1-Dodecanol and triethylamine (Et3N) were then added, and the mixture was heated with stirring to obtain compound 2.

[0013] 2) Compound 2 was dissolved in dichloromethane (DCM), vacuum-protected with argon, and boron tribromide (BBr3) was added. 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 of 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 treat 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 called DHN, which promotes the opening of the mitochondrial permeability transition pore (mPTP), the release of mitochondrial DNA (mtDNA) into the cytoplasm, and activates the cGAS-STING signaling pathway, promoting GSDME cleavage, thereby inducing pyroptosis in melanoma cells, ultimately achieving the goal of treating melanoma. The small molecule compound DHN of the present invention can act as both an agonist of the cGAS-STING signaling pathway and a pyroptosis inducer, thereby achieving the goal of treating melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0023] Figure 3 Immunofluorescence analysis of cGAS in melanoma A375 cells following 12-hour treatment with the small molecule compound DHN showed that DHN significantly induced cGAS punctate aggregation, and 1,6-hexanediol blocked DHN-induced cGAS punctate aggregation. These results suggest that the small molecule compound DHN can induce phase separation of cGAS.

[0024] Figure 4 Immunofluorescence analysis of STING activity in melanoma A375 cells following 12-hour treatment with the small molecule compound DHN showed that DHN significantly induced STING punctate aggregation, but 1,6-hexanediol was unable to block DHN-induced STING punctate aggregation. These results suggest that DHN can induce STING punctate aggregation, but these punctate aggregations do not form phase-separated structures.

[0025] Figure 5 Melanoma A375 cells were treated with the small molecule compound DHN for various periods of time to observe changes in cell morphology, GSDME cleavage, and LDH release. The results showed that DHN triggered cell blebbing, GSDME cleavage, and significant LDH release. These results suggest that the small molecule compound DHN can induce pyroptosis in melanoma cells, accompanied by GSDME cleavage and LDH release.

[0026] Figure 6 In a nude mouse xenograft model, melanoma A375 cells, STING knockdown A375 cells, and GSDME knockdown A375 cells were implanted in the flanks of nude mice. Saline and DHN were injected every two days in the control and experimental groups, respectively, and tumor size was measured. Mice were sacrificed 21 days later, and tumors were harvested and weighed. Figure 6 Above: Tumors are arranged from left to right according to weight, with the left side showing the largest tumor and the right side showing the smallest tumor. Photos were taken for record. Figure 6 Middle panel: Tumor volume was recorded every two days and plotted as a tumor growth curve. Figure 6Figure 2: Tumors were harvested, weighed, and plotted as a tumor weight chart. Results show that DHN significantly inhibited tumor growth in the A375 cell-inoculated group. This inhibitory effect was significantly suppressed in the STING knockdown A375 cell-inoculated group, and even more so in the GSDME knockdown A375 cell-inoculated group. These results demonstrate that the small molecule compound DHN's ability to inhibit the growth of nude mouse melanoma xenografts is dependent on STING and GSDME proteins.

[0027] Figure 7 Melanoma A375 cells were treated with the small molecule compound DHN and its derivatives for 24 hours and observed for changes in cell morphology and LDH release. The results showed that DHN triggered cell blebbing and significant LDH release, while its derivatives did not significantly induce pyroptosis. These results suggest that the small molecule compound DHN exhibits superior pyroptosis-inducing activity compared to 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 with reference to the accompanying drawings and embodiments.

[0029] Example 1

[0030] Synthesis of 6-hydroxy-2-dodecyl naphthoate (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) in an ice bath 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 reaction completion using a thin layer plate, quench the reaction with a small amount of water, wash with sodium bicarbonate and saturated sodium chloride solution, dry over anhydrous magnesium sulfate, and concentrate the solvent in vacuo. The resulting liquid was purified by column chromatography using petroleum ether and ethyl acetate to yield 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. Dissolve compound 2 (370 mg, 1.0 mmol) in dichloromethane (DCM) (6 mL) under vacuum with argon. Add boron tribromide (BBr3) (2.5 mL, 10.0 mmol) and 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 over anhydrous sodium sulfate and concentrate the solvent in vacuo. Purify by column chromatography with petroleum ether and ethyl acetate (PE / EA = 30:1) 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 assay.

[0041] 3. Mitochondrial mPTP pore opening detection:

[0042] 1) Trypsinize the cells and resuspend them in culture medium. Use the suspended cells directly. Count the cells and centrifuge an appropriate amount of cells (1000 × g, 5 minutes, room temperature). Discard the supernatant and resuspend the cells.

[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 minutes.

[0044] 3) After incubation, collect the cells by centrifugation at 1000 × g for 5 minutes at room temperature. Add 1 mL of assay 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 an unstained cell sample containing only assay buffer as a negative control on the flow cytometer. Calcein has an excitation maximum of 494 nm and an emission maximum of 517 nm. Assess the fluorescence intensity of the cells using flow cytometry to assess the extent 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 harvested 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-chilled PBS to wash away the remaining culture medium. Then remove the PBS.

[0055] 2) Add 2 mL of pre-chilled 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) Resuspend the cells in 1 mL of pre-chilled PBS to the 10 mL tube. Transfer 80 μL of the suspension to a new 1.5 mL EP tube 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°C. Wash once more with PBS.

[0057] 4) After washing, discard the supernatant and add 100 μL of digitonin-free Lysis Buffer (75 mM sodium chloride, 1 mM sodium dihydrogen phosphate, 8 mM sodium dihydrogen phosphate, 250 mM sucrose) and the protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Use a pipette to disperse the cells 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. Gently flick the EP tube 8-10 times to mix thoroughly. Incubate on ice for 5 minutes. Digitonin is a weak permeabilizing agent, so the permeabilization time must be controlled.

[0059] 6) After the rest period, 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. This will be the cytosolic fraction, and the pellet will be the mitochondrial fraction.

[0060] 4. Genomic DNA Extraction

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

[0062] 5. Dilute the extracted DNA 5-fold and use it as a template. Prepare the PCR reaction system as follows: 5 μL SYBRMix, 1 μL 2 μM primer, and 4 μL cDNA template. Perform fluorescence quantitative PCR using a Bio-rad CFX96.

[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 cell 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 a glass slide placed in advance.

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

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

[0070] 4. Immunofluorescence detection

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

[0072] 2) After fixing the cells with formaldehyde, discard the fixative and wash them three times with PBS, each time for 5 minutes.

[0073] 3) After washing, add 2 mL of blocking buffer to each well and shake on a shaker at room temperature for 20 minutes. To prepare the blocking buffer, dissolve Triton X-100 (0.2%) and BSA (3%) in PBS and filter through 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 five times with washing buffer for 10–15 minutes each time. Prepare the washing buffer by dissolving Triton X-100 (0.05%) and BSA (0.2%) in PBS and filtering through 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 1-1.5 hours. The secondary antibody incubation time should not be too long. The secondary antibody should be prepared in blocking solution.

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

[0078] 9) After washing with the cleaning solution, wash once with PBS. Add 2 mL of DAPI (50 mg / mL) in PBS to stain the cell nuclei for approximately 15 minutes at room temperature, protected from light, and incubate on a rocking platform. After staining, wash the cells three times with PBS, each for 5–10 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 a punctate manner after treatment with the small molecule compound DHN, and that incubation with 1,6-hexanediol (1,6-HD) could block the DHN-induced cGAS aggregation, indicating that the small molecule compound DHN can induce cGAS aggregation, and that this aggregation is a phase-separated 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 a glass slide placed in advance.

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

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

[0087] 4. Immunofluorescence detection.

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

[0089] 2) After fixing the cells with acetone, discard the fixative and wash them 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. To prepare the blocking buffer, dissolve Triton X-100 (0.2%) and BSA (3%) in PBS and filter through 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 should be prepared in blocking solution.

[0092] 5) After primary antibody incubation, wash cells five times with washing buffer for 10–15 minutes each time. Prepare the washing buffer by dissolving Triton X-100 (0.05%) and BSA (0.2%) in PBS and filtering through 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 1-1.5 hours. The secondary antibody incubation time should not be too long. The secondary antibody should be prepared in blocking solution.

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

[0095] 8) After washing with the cleaning solution, wash once with PBS. Add 2 mL of DAPI (50 mg / mL) in PBS to stain the cell nuclei for approximately 15 minutes at room temperature, protected from light, and incubate on a rocking platform. After staining, wash the cells three times with PBS for 5–10 minutes each time.

[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 formed punctate aggregation, but 1,6-HD could not block the DHN-induced STING punctate 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 was 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 analyzed by 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 an aqueous solution containing 20% ​​Triton X-100 to lyse the cells and release total intracellular LDH.

[0105] 2) Collect the cell supernatant from each well using a 2 mL EP tube and centrifuge at 3000 rpm for 3 minutes at room temperature. Transfer 50 μL of supernatant to each EP tube and transfer to a 96-well plate. Also, transfer 50 μL of 2% FBS-containing DMEM to serve as a blank control (since serum contains LDH, which reacts with the assay solution, 2% FBS-containing DMEM serves as a blank control). Add 50 μL of assay solution to each well and allow the reaction with the LDH enzyme in the culture medium to proceed 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) Measure the absorbance (490 nm) using a microplate reader. The absorbance value indicates the LDH content.

[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 a 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 and 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: Load 10 μg of protein on an SDS-PAGE gel at 100 V for stacking gel electrophoresis. Once the sample enters the separating gel, adjust the voltage to 150 V.

[0113] 5) Membrane transfer: After electrophoresis, remove the concentrated gel and assemble a transfer sandwich using ethanol-pretreated PVDF membrane and filter paper immersed in electrotransfer buffer. 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 for 5 minutes each time. Add the corresponding species-specific secondary antibody and incubate at room temperature for 1.5 hours.

[0118] 7) ECL Detection: Wash three times with TBST for 10 minutes each. Mix ECL Solution A and Solution B in a 1:1 ratio and dropwise apply to the PVDF membrane. Develop using a chemiluminescence imager.

[0119] The experimental results are as follows Figure 5 The results showed that 12 hours after treatment with the small molecule compound DHN, a compound that is used to treat melanoma cells, induced cell blebbing, accompanied by the time-dependent release of lactate dehydrogenase (LDH) and the cleavage of GSDME protein. This indicates 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 depends 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 knockdown of STING gene, 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 normal saline, 100 μL / mouse) and DHN treatment (20 mg / kg; DHN was dissolved in DMSO and then diluted with normal saline) by intraperitoneal injection once 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 developed obvious tumors after 21 days. In mice inoculated with A375 cells, DHN treatment significantly reduced tumor size and weight, indicating that the small molecule compound DHN can effectively inhibit the growth of melanoma cell transplants in nude mice. In mice inoculated with melanoma cells in which the STING and GSDME genes were knocked down, DHN failed to inhibit tumor growth, indicating that the small molecule compound DHN's inhibition of nude mouse melanoma cell transplant growth depends on the STING and GSDME proteins.

[0128] Example 8

[0129] DHN has better activity in inducing cell pyroptosis than its derivatives.

[0130] The experimental method is as follows:

[0131] 1. Cell inoculation was 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, and 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 in melanoma cells after 24 hours of treatment, accompanied by the release of lactate dehydrogenase (LDH), while its derivatives did not show cell blistering and LDH release, indicating that the small molecule compound DHN is more capable of inducing pyroptotic death in melanoma cells than 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, wherein: The synthetic route is as follows: Wherein, EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

3. The method for preparing a small molecule compound DHN according to claim 2, wherein: The following steps are involved: 1) Compound 1, i.e., 6-methoxy-2-naphthoic acid and 1-hydroxybenzotriazole (HOBT), were added to anhydrous dichloromethane (DCM). 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was added under ice-cooling conditions and stirred thoroughly. 1-dodecanol and triethylamine (Et3N) were then added, and the mixture was heated with stirring to obtain compound 2. 2) Compound 2 was dissolved in dichloromethane (DCM), vacuum-protected with argon, and boron tribromide (BBr3) was added. The mixture was stirred in an ice bath to obtain compound 3DHN.

4. The method for preparing a small molecule compound DHN according to claim 3, wherein: In step 1), the heating temperature is 40-60°C.

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

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

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

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

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