Use of pyrimidinotriazinedione derivatives for the preparation of a medicament for inhibiting the activity of the human protease caspase-1

Abstract

The application discloses application of a pyrimidinotriazine diketone derivative in preparation of a medicine for inhibiting activity of human protease caspase-1, and belongs to the technical field of medicines. In view of defects of existing caspase-1 inhibitors, the compound has the characteristics of high affinity binding to caspase-1 (K D = 8.11 μM; 8.78 μM) and strong inhibition on the activity (IC 50 = 11.90 nM-14.47 nM). The compound is used for preparation of a medicine for inhibiting the activity of caspase-1 and a medicine for treating inflammation-related diseases such as sepsis-related lung injury, acute respiratory distress syndrome or acute pancreatitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology. More specifically, this invention relates to the use of a pyrimidine triazine dione derivative in the preparation of a medicament that inhibits the activity of the human protease caspase-1. Background Technology

[0002] Inflammation is a defensive response of the body to harmful stimuli such as injury or infection. It has beneficial functions such as clearing harmful stimuli, initiating tissue repair, and restoring tissue function. However, excessive or persistent inflammation can cause tissue damage and dysfunction, leading to various inflammation-related diseases. Therefore, inhibiting excessive or persistent inflammatory responses is an important treatment strategy for preventing inflammatory pathological damage and improving the symptoms of inflammation-related diseases.

[0003] Caspase-1, a cysteine-aspartic protease, is a key protein mediating intracellular inflammatory responses. When intracellular inflammasomes sense stimulation from exogenous pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs), caspase-1 is activated through the formation of a multi-protein complex. Activated caspase-1, acting as an executive protein, induces pyroptosis and promotes the release of the inflammatory cytokine interleukin-1 (IL-1β), thereby further triggering a cascade of inflammatory responses in the body. Caspase-1 has been confirmed to be involved in the occurrence and progression of various inflammatory diseases, including acute inflammatory diseases such as acute pancreatitis, acute peritonitis, acute respiratory distress syndrome, and sepsis-related organ damage, as well as chronic inflammatory diseases such as gout, non-alcoholic fatty liver disease, atherosclerosis, neurodegenerative diseases, diabetes, inflammatory bowel disease, and autoimmune diseases. Therefore, finding safe and effective caspase-1 inhibitors is of great significance for the treatment of these inflammatory diseases.

[0004] Currently reported caspase-1 inhibitors mainly include synthetic small molecule compounds and peptides. Small molecule inhibitors such as VX-765 are the most widely studied caspase-1 inhibitors, currently in Phase II clinical trials; however, studies have found that long-term use can cause significant hepatotoxicity. Sennoside A is a caspase-1 inhibitor that effectively inhibits caspase-1 enzymatic activity in vitro, but its mechanism of anti-inflammatory effect remains to be elucidated, and it has not yet entered the clinical trial stage. Ac-YVAD-CMK is a peptide inhibitor that irreversibly inhibits caspase-1 activity by mimicking the substrate sequence (YVAD) of caspase-1 and introducing a chloromethyl ketone (CMK) group, effectively inhibiting the production of active IL-1β and IL-18. However, Ac-YVAD-CMK also has many drawbacks, such as poor stability, low bioavailability, short biological half-life, and susceptibility to degradation by intestinal proteolytic enzymes, leading to loss of efficacy. Therefore, the search for new caspase-1 inhibitors has a very broad prospect for translational applications. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] To achieve these objectives and other advantages according to the present invention, the use of pyrimidine triazine dione derivatives in the preparation of medicaments that inhibit the activity of human protease caspase-1 is provided, wherein the chemical formula of the pyrimidine triazine dione derivative is shown in Formula III:

[0007] Formula III,

[0008] In Formula III, R1 is a benzene ring group, as shown in Formula I, or a pyridine ring group, as shown in Formula II.

[0009] Formula I, Formula II.

[0010] Preferably, the use of pyrimidine triazine dione derivatives in the preparation of medicaments for treating inflammation-related diseases, including sepsis-related lung injury.

[0011] The present invention has at least the following beneficial effects:

[0012] First, it provides a highly efficient detection system based on fluorescent substrates, demonstrating that the compound can significantly inhibit caspase-1 enzyme activity at nanomolar concentrations and achieve high selectivity without complex structural modifications, thus solving the problems of insufficient efficacy or poor selectivity of traditional inhibitors.

[0013] Second, for the first time, surface plasmon resonance technology was used to reveal the unique binding mode of the compound with caspase-1—fast binding and slow dissociation kinetics, indicating that it can form a stable complex. This elucidates the high-affinity inhibition mechanism at the molecular interaction level and provides a new strategy for designing long-acting inhibitors.

[0014] Third, in a macrophage inflammation model, the compound can penetrate the cell membrane and effectively block caspase-1 activation, with an inhibitory efficiency superior to the positive control drug. This effect was verified by both flow cytometry and laser confocal microscopy, highlighting its intracellular delivery capability and target specificity.

[0015] Fourth, the breakthrough discovery compound inhibits caspase-1, simultaneously downregulates the expression of GSDMD-NT, a key pyroptosis executive protein, and significantly reduces IL-1β release and LDH leakage. This is the first time that the compound has achieved synergistic regulation of multiple indicators of this pathway, overcoming the limitation of existing drugs that only block a single inflammatory factor.

[0016] Fifth, in a sepsis-associated lung injury model, intraperitoneal administration of the compound can effectively reduce pathological damage to lung tissue. The mechanism is to inhibit the expression of caspase-1 active fragment p10, and simultaneously reduce neutrophil infiltration and IL-1β secretion, confirming that it has both anti-inflammatory and organ-protective effects, providing a new option with strong efficacy and low toxicity for clinical translation.

[0017] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0018] Figure 1 The chemical formula of a pyrimidine triazine dione derivative;

[0019] Figure 2 A diagram illustrating the binding affinity of compounds I and II to the target protein caspase-1;

[0020] Figure 3 The diagram shows the inhibitory effects of compounds I and II on the intracellular activity of caspase-1 and its mediated pyroptosis.

[0021] Figure 4 The diagram shows the protective effects of compounds I and II against LPS-induced acute lung injury in mice. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] like Figures 1-4 As shown, this invention provides two compounds that, through drug screening, can significantly inhibit caspase-1 (cysteine ​​aspartate protease-1) activity, and can be used as anti-inflammatory drugs to treat sepsis-related lung injury and other inflammatory diseases. Compound I and Compound II were purchased from Shanghai Taoshu Biotechnology Co., Ltd., and their chemical names are 1,6-dimethyl-3-phenylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione and 1,6-dimethyl-3-(pyridin-4-yl)pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione, respectively. Both belong to the pyrimidotriazine dione derivatives.

[0025] The chemical formulas of compounds I and II are shown in Formula I and Formula II, respectively.

[0026] Formula I, Formula II.

[0027] This invention tested the binding affinity of compounds I and II to caspase-1, demonstrating that both compounds I and II exhibit strong binding affinity to caspase-1, with the binding signal increasing with increasing molar concentration of compounds I and II. This invention also provides the application of compounds I and II in the preparation of medicaments for treating or preventing inflammatory diseases.

[0028] Furthermore, treatment of immune cells with bacterial lipopolysaccharide (LPS) and nigericin significantly activated intracellular caspase-1, leading to pyroptosis, manifested by significantly increased release of interleukin-1β (IL-1β) and lactate dehydrogenase (LDH), and significant upregulation of the membrane porin GSDMD-NT. Compounds I and II significantly inhibited caspase-1-mediated pyroptosis, characterized by decreased caspase-1 activity, reduced release of IL-1β and LDH, and downregulation of GSDMD-NT expression. In mice induced with LPS-induced acute lung injury, injection of compounds I and II significantly reduced inflammatory pathological damage in lung tissue, decreased inflammatory cell infiltration, inhibited caspase-1 activation, and reduced IL-1β secretion.

[0029] <Example 1>

[0030] Determination of the inhibitory activity of compounds (compound I and compound II) against caspase-1 enzyme using a fluorescent substrate method

[0031] Experimental methods:

[0032] 1. Preparation of samples and reagents for the kit:

[0033] (1) Sample buffer: Mix 2 mL of sample buffer (5×) with 7.9 mL of water and 100 µL of DTT (1M) reagent provided in the kit to make 10 mL of sample buffer (1×).

[0034] (2) Compound solution: Weigh the specified amount of the compound and dissolve it in the diluted sample buffer solution at a concentration of 4 times the required final detection concentration.

[0035] (3) Caspase-1 substrate (Ac-YVAD-AFC) solution: Mix 20 µL of caspase-1 substrate (Ac-YVAD-AFC) with 3.98 mL of sample buffer (1×). Ac-YVAD-AFC is a fluorescent substrate used to detect Caspase-1 activity.

[0036] (4) Caspase-1 enzyme solution: Thaw the caspase-1 enzyme (recombinant human) on ice and mix before diluting. Mix 16 µL of caspase-1 enzyme solution with 1.984 mL of sample buffer (1×).

[0037] 2. Caspase-1 Activity Assay: In a 96-well plate, add 10 μL of sample buffer to each well, followed by 5 μL of aqueous solutions of compounds (I) and (II) at different concentrations (0, 5, 10, 25, 50, and 100 nM), and 5 μL of recombinant human caspase-1 enzyme solution. Three additional wells were treated with 15 μL of sample buffer and 5 μL of recombinant human caspase-1 enzyme solution, respectively, as 100% initial enzyme activity controls. The 96-well plate was incubated at 37°C for 10 min, and then 10 μL of caspase-1 substrate solution was added to all wells to initiate the reaction. The plate was sealed with an aluminum foil cover and incubated at room temperature for 2 hours. The cover was then removed, and the fluorescence at an excitation wavelength of 400 nm and an emission wavelength of 505 nm was read using a microplate reader. The inhibition rate of the compounds against caspase-1 enzyme at different concentrations was calculated based on the absorbance values. The measured concentrations of the compounds and their corresponding inhibition rates were substituted into GraphPad Prism 8.0.2 (data analysis software) to obtain the half-maximal inhibitory concentration (IC50) of the compounds against trypsin. The inhibition rates of compounds I and II against caspase-1 at different concentrations are shown in Table 1.

[0038] Table 1. Inhibition rates of compounds I and II on caspase-1 enzyme at different concentrations.

[0039]

[0040] The calculated IC50 values ​​for compound I against caspase-1 were 11.90 nM and for compound II against caspase-1 were 14.47 nM.

[0041] <Example 2>

[0042] Surface plasmon resonance (SPR) assay for the binding affinity of compounds (compound I and compound II) to caspase-1

[0043] Experimental methods:

[0044] (1) Chip preparation: Before injection, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (400 mM) and NHS (N-hydroxysuccinimide) (100 mM) were mixed to prepare an activator. The mixture was then used to activate the CM5 sensor chip for 7 minutes at a constant flow rate (10 μL / min). CM5 refers to a carboxymethyl dextran-modified gold chip.

[0045] (2) Ligand immobilization: Caspase-1 was diluted to 40 μg / mL in immobilization buffer and then injected into the sample channel (Fc2) at a flow rate of 10 μL / min. The immobilization level was typically 12800 RU. The reference channel (Fc1) did not require a ligand immobilization step. The chip was inactivated with 1 M ethanolamine hydrochloride at a flow rate of 10 μL / min for 7 minutes.

[0046] (3) Analytical cycles were performed using the multi-cycle method: The compound was diluted to six concentrations with analytical buffer. The compound was injected into the Fc1-Fc2 channels at a flow rate of 30 μL / min, with binding for 120 seconds and dissociation for 300 seconds. Both binding and dissociation processes were carried out in analytical buffer. Six cycles of analyte were repeated according to the analyte concentration from high to low. Binding curves were fitted, and relevant kinetic parameters were calculated.

[0047] Experimental results: such as Figure 2 As shown, both compounds I and II bind to caspase-1 in a concentration-dependent manner, exhibiting a fast binding and slow dissociation pattern, with relatively high binding strength. The equilibrium dissociation constant K of compound I is also shown. D The equilibrium dissociation constant K of compound II is 8.11 µM. D It is 8.78 µM.

[0048] <Example 3>

[0049] Flow cytometry was used to detect the inhibitory effects of compounds (compound I and compound II) on intracellular caspase-1 activity.

[0050] Experimental methods:

[0051] (1) Cell Culture: Transfer the THP-1 (human monocytic leukemia cell) cell suspension to a culture flask or dish, add an appropriate amount of pre-warmed RPMI 1640 complete medium at 37℃, adjust the seeding density as needed, and mix thoroughly with a cross-shaped shaker. Place the seeded cells in a cell culture incubator at 37℃ and 5% CO2. After 24 hours, observe the cell status to confirm cell growth and health. If necessary, change the culture medium every 2-3 days to remove metabolic products and dead cells.

[0052] (2) Drug treatment: Replace with fresh RPMI 1640 complete culture medium without PMA (phorbol 12-tetradecanoate 13-acetate). According to the experimental groups, the blank control group was not treated with drugs, while the experimental groups were treated with positive control drug VX-765 (small molecule caspase-1 inhibitor) and compounds I and II, respectively. After 1 hour, bacterial lipopolysaccharide LPS was added for stimulation and mixed evenly. After another 4 hours, Nigericin was added for stimulation. After 1 hour, subsequent experimental procedures were carried out.

[0053] (3) FLICA (fluorescently labeled caspase inhibitor) cell staining and treatment: Under strict light-protected conditions, 50 µL of DMSO (dimethyl sulfoxide) was added to the lyophilized FLICA powder vial to obtain a 150× FLICA stock solution. The stock solution was aliquoted into opaque brown EP tubes at a ratio of 10 µL per tube, and 40 µL of buffer was added to each EP tube to obtain a 30× FLICA solution. After drug treatment, the cells were removed, and the freshly prepared 30× FLICA solution was added at a ratio of 1:30. After mixing, the cells were incubated in a 37℃ cell culture incubator in the dark for 30-60 minutes. During this period, the wells were removed and gently shaken every 15-20 minutes to ensure that the reagents were mixed evenly and that the staining was thorough. After staining, aspirate the cell culture medium containing FLICA, add an appropriate amount of 1× apoptosis washing buffer to each well, incubate at 37°C for 10 minutes, then replace with fresh apoptosis washing buffer to allow excess FLICA reagent that has not bound to the cells to diffuse out of the cells. Repeat the above washing steps 2-3 times.

[0054] (4) Cell collection: Add an appropriate amount of trypsin to each well and place the adherent cells in a 37°C cell culture incubator to digest the cells. After the cells detach (3 min), add an appropriate amount of fresh culture medium to stop the digestion. Collect the cell suspension into centrifuge tubes and centrifuge at 200-300 rpm for 5-10 minutes at room temperature to pellet the cells. Resuspend the cells with apoptosis washing buffer, then centrifuge again, repeating the washing twice. Discard the cell supernatant, resuspend the cells with apoptosis washing buffer, and place them on ice. Adjust the concentration and volume of the cell suspension by cell counting. Add fixative to each well at a ratio of 1:5-10 and incubate at room temperature in the dark for 15-20 minutes.

[0055] (5) Flow cytometry: FLICA-stained cells were quantified using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 530 nm to detect the fluorescence signal of FLICA labeling.

[0056] Experimental results: such as Figure 3 As shown in Figure a, the percentage of cells stained with FLICA in the LPS+Nigericin treatment group was 12.5%, significantly higher than the 1.31% in the control group, indicating that caspase-1 was induced and activated. Treatment with the positive control drug VX-765 significantly reduced the percentage of cells stained with FLICA (3.44%), while treatment with compounds I and II showed even lower FLICA staining positivity rates (2.23% and 1.34%, respectively), indicating that compounds I and II have a significant inhibitory effect on intracellular caspase-1 activation, and their inhibitory activity is superior to that of VX-765.

[0057] <Example 4>

[0058] Laser confocal microscopy was used to observe the inhibitory effects of compounds (compound I and compound II) on the activity of intracellular caspase-1.

[0059] Experimental methods:

[0060] (1) Cell Culture: Transfer the THP-1 (human monocytic leukemia cell) cell suspension to a culture flask or dish, add an appropriate amount of pre-warmed RPMI 1640 complete medium at 37℃, adjust the seeding density as needed, and mix thoroughly with a cross-shaped shaker. Place the seeded cells in a cell culture incubator at 37℃ and 5% CO2. After 24 hours, observe the cell status to confirm cell growth and health. If necessary, change the culture medium every 2-3 days to remove metabolic products and dead cells.

[0061] (2) Preparation of cell smears: Place the cell smears in a sterile 24-well plate, add 2 mL of PBS buffer to each well to wash the cell smears, repeat the washing 2-3 times, then aspirate dry and sterilize with UV for 15-20 minutes.

[0062] (3) Cell seeding: Select cells in the logarithmic growth phase and at a suitable density for seeding. The number of cells seeded on each slide should be appropriate (e.g., 2 × 10⁻⁶). 5 Up to 5×10 5 (1 cell / slice), ensuring the cells are evenly distributed on the slide. Add PMA to the cell culture medium and culture for 36-48 hours to induce cell differentiation into adherent macrophages.

[0063] (4) Drug treatment: Replace with fresh RPMI 1640 complete culture medium without PMA. According to the experimental groups, no drugs were added to the blank control group, and positive control drug VX-765 and compounds I and II were added to the experimental groups, respectively. LPS was added for stimulation after 1 hour and mixed well. Nigericin was added for stimulation after another 4 hours. Subsequent experimental procedures were carried out after 1 hour.

[0064] (5) FLICA cell staining and treatment: Add freshly prepared 30×FLICA solution to a 24-well plate at a ratio of 1:30 to achieve a final FLICA concentration of 1×. After mixing, incubate at 37°C in the dark for 30-60 minutes. During this period, remove the plate every 15-20 minutes and gently shake it to ensure thorough mixing and staining. After staining, discard the cell culture medium containing FLICA. Add an appropriate amount of apoptosis washing buffer to each well and incubate at 37°C for 10 minutes. Replace with fresh buffer to allow any excess FLICA reagent that has not bound to the cells to diffuse out. Repeat the washing steps 2-3 times. Add 0.5% DAPI (4',6-diamidinyl-2-phenylindole) staining solution to each well and incubate at 37°C in the dark for 15-20 minutes. Then, add fixative to each well at a ratio of 1:5-10, incubate at room temperature in the dark for 15-20 minutes, then add an appropriate amount of 1× apoptosis wash buffer to each well and incubate at 37°C for 10 minutes. Replace with fresh apoptosis wash buffer and repeat the above washing steps 2-3 times. Place an appropriate amount of anti-fluorescence attenuation mounting medium on a clean glass slide, gently remove the cell smear with tweezers and invert it onto the slide, avoiding air bubbles. Air dry at room temperature in the dark for 15 minutes.

[0065] (6) Observation under laser confocal microscope: The mounted slide was observed under a laser confocal microscope. The excitation wavelength was set to 488 nm and the emission wavelength to 530 nm to detect the fluorescence signal of the FLICA label.

[0066] Experimental results: such as Figure 3As shown in b, caspase-1 activation induced by intracellular inflammasome activation can be detected in real time by the fluorescent probe FLICA. Laser confocal microscopy revealed that, compared to the control group, cells exhibited significant green fluorescence upon stimulation with LPS+Nigericin, indicating the generation of active caspase-1 within the cells. No significant green fluorescence was observed after treatment with compounds I and II, a result consistent with treatment with the caspase-1 inhibitor VX-765, indicating that compounds I and II significantly inhibit intracellular caspase-1 activation.

[0067] <Example 5>

[0068] Inhibitory effects of compounds (compound I and compound II) on intracellular caspase-1 activity and pyroptosis

[0069] Experimental methods:

[0070] (1) Cell Culture: Transfer the THP-1 (human monocytic leukemia cell) cell suspension to a culture flask or dish, add an appropriate amount of pre-warmed RPMI 1640 complete medium at 37℃, adjust the seeding density as needed, and mix thoroughly with a cross-shaped shaker. Place the seeded cells in a cell culture incubator at 37℃ and 5% CO2. After 24 hours, observe the cell status to confirm cell growth and health. If necessary, change the culture medium every 2-3 days to remove metabolic products and dead cells.

[0071] (2) Construction of cellular inflammation model and drug treatment: THP-1 cells treated with PMA (100 ng / mL) were cultured for 36-48 hours to induce differentiation into adherent macrophages. The culture medium was replaced with RPMI 1640 complete medium without PMA. THP-1 cells were co-incubated with the given concentrations of compound I, compound II solution or positive control drug VX-765 for 1 hour, then treated with LPS (1 μg / mL) for 4 hours, and then treated with Nigericin (20 μM) for 1 hour.

[0072] (3) Sample collection: Cell supernatant was collected and the concentration of the inflammatory factor IL-1β was detected using an ELISA (enzyme-linked immunosorbent assay) kit. The activity of total lactate dehydrogenase (LDH) in cells was determined by colorimetric method. Cell proteins were extracted and the expression of Gasdermin D active fragment (GSDMD-NT, gasdermin D active fragment (N-terminal domain)) was detected by Western blotting.

[0073] Experimental results: such as Figure 3As shown in Figure c, compared with the control group, the concentration of IL-1β in the cell supernatant was significantly increased after LPS+Nigericin combined stimulation, while the positive control drug VX-765 significantly reduced the concentration of IL-1β. Compounds I and II significantly reduced the concentration of IL-1β and showed a clear dose-response effect. Figure 3 The results showed that LPS+Nigericin-induced pyroptosis led to the release of large amounts of LDH (lactate dehydrogenase), and compounds I and II, along with the positive control VX-765, significantly reduced LDH release. GSDMD-NT is a product of intracellular caspase-1 activation and degradation, which can form pores in the cell membrane, leading to pyroptosis. Figure 3 The results showed that compounds I and II, along with the positive control drug VX-765, significantly downregulated the expression of GSDMD-NT, demonstrating their ability to inhibit pyroptosis.

[0074] <Example 6>

[0075] Protective effects of compounds (compound I and compound II) on LPS-induced acute lung injury in mice

[0076] Experimental methods:

[0077] Male Balb / c mice, weighing approximately 20 g and aged 6-8 weeks, were used. Mice were divided into five experimental groups (n=6): a sham-operated control group (saline group), a mouse acute lung injury inflammation model group (LPS group), a positive control group (VX-765 group), a compound I administration group, and a compound II administration group. Each drug treatment group received an intraperitoneal injection of 50 mg / kg. One hour after drug administration, mice were anesthetized with 1% sodium pentobarbital, and an indwelling needle (22G) was inserted into the trachea. LPS was then infused through the indwelling needle, allowing it to enter the lungs with normal respiration. The sham-operated control group received an equal dose of sterile saline. Mice in the drug treatment groups underwent a repeat administration one hour after model establishment. Twelve hours after tracheal infusion of LPS, the mice were sacrificed and dissected. Lung tissue was fixed with 4% paraformaldehyde, routinely dehydrated with alcohol, embedded in paraffin, and cut into 6 μm sections for HE (hematoxylin-eosin) staining. The prepared HE-stained pathological sections were observed under an optical microscope to detect pathological changes in the lung tissue of mice in each group; bronchoalveolar lavage fluid was collected and the degree of neutrophil recruitment was detected by Wright-Gymsa staining; the concentration of IL-1β in bronchoalveolar lavage fluid was detected by ELISA to assess the degree of inflammatory response in lung tissue; lung tissue proteins were extracted and the expression of caspase-1 active fragment (p10, molecular weight 10 kDa) was detected by Western Blot.

[0078] Experimental results: such as Figure 4As shown in Figure a, the lung tissue structure in the saline group was normal. There was no significant thickening or connective tissue hyperplasia of the pleura, and the morphology of the bronchial and alveolar epithelial cells was normal, without degeneration or necrosis. No obvious inflammatory cell infiltration or fibrosis was observed in the interstitium, and the overall lung tissue appeared healthy. In the LPS group, inflammatory cell infiltration, hemorrhage, congestion, and alveolar epithelial cell necrosis were observed in the lung tissue. The VX-765 group showed milder inflammatory infiltration in the lung tissue compared to the LPS group, mainly manifested as neutrophil infiltration and a small number of lymphocytes. Damage to alveolar epithelial cells was relatively mild, and atelectasis was less pronounced than in the LPS group. In the compound I group, mild inflammatory reactions and neutrophil infiltration were observed in the alveolar epithelial cells and interstitium, with mild alveolar necrosis and vascular congestion in some tissues, but the overall inflammatory response was improved compared to the LPS group. In the compound II group, lung tissue lesions were relatively mild, with only a small amount of inflammatory cell infiltration and mild alveolar necrosis. The results suggest that compounds I and II can alleviate LPS-induced acute lung tissue damage in mice. Figure 4 The results showed that, compared with the control group, the number of neutrophils in the lung tissue of the LPS treatment group was significantly increased, while the number of neutrophils in the compound I and compound II administration groups was significantly reduced. Figure 4 The results showed that, compared with the control group, the concentration of IL-1β in the bronchoalveolar lavage supernatant of the model group mice was significantly increased, and the release of IL-1β in the compound I and compound II administration groups was significantly reduced. Figure 4 The results showed that the expression of caspase-1 active fragment p10 was significantly upregulated in the model group, while the expression of caspase-1 active fragment p10 was significantly downregulated after administration of compound I and compound II.

[0079] This invention confirms that compounds I and II are potent caspase-1 inhibitors, inhibiting caspase-1 activity through binding. In an LPS+Nigericin-induced pyroptosis model, compounds I and II suppressed pyroptosis by inhibiting caspase-1 activity; in an LPS-induced acute lung injury mouse model, compounds I and II effectively alleviated inflammation-induced lung injury by inhibiting caspase-1 activity. LPS-induced lung injury is a common and serious complication of sepsis, and these experimental results directly demonstrate the anti-inflammatory activity of compounds I and II, providing data support for their future clinical application.

[0080] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. The application of pyrimidine triazine dione derivatives in the preparation of drugs that inhibit the activity of human protease caspase-1, among which, The chemical formulas of pyrimidine triazine dione derivatives are shown in Formula III: Formula III, In Formula III, R1 is a benzene ring group, as shown in Formula I, or a pyridine ring group, as shown in Formula II. Formula I, Formula II; The drug is used to treat inflammation-related diseases, including sepsis-associated lung injury.

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