NLRP3 inflammasome inhibitor coupled with hydrogen sulfide donor as well as preparation method and application of NLRP3 inflammasome inhibitor

Abstract

The invention provides an NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor and a preparation method and application of the NLRP3 inflammasome inhibitor, all inhibitors can release H2S in vitro, the inhibitor CYT-1 has the most stable H2S release rate, the most appropriate LogP value (3.83) and the lowest cytotoxicity, and the H2S release amount of the inhibitor CYT-1 relative to TBZ is 29.73%. Under the action of body enzyme, CY-09 and a hydrogen sulfide donor are released, so that not only can the activation of NLRP3 inflammasome be inhibited in a targeted manner, but also H2S can be released to inhibit phosphorylation of NF-kB so as to achieve the purpose of synergistically inhibiting pyroptosis of neurons, and the activation of NF-kB and NLRP3 inflammasome can be inhibited at the same time so as to synergistically inhibit pyroptosis of neurons. Furthermore, cognitive impairment of mice with diabetic encephalopathy is improved, and a new thought is provided for drug development and treatment of diabetic encephalopathy.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medicinal chemistry and pharmaceutical therapy, and particularly relates to an NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetic Encephalopathy (DE) is one of the severe complications of diabetes, mainly manifested as cognitive dysfunction. Pyroptosis is a pro-inflammatory programmed cell death. Research shows that NLRP3 inflammasome-mediated neuronal pyroptosis is a key factor in DE-induced cognitive dysfunction. Therefore, inhibiting the over-activation of neuronal pyroptosis is of great significance for improving DE cognitive dysfunction.

[0003] As a gaseous signaling molecule, hydrogen sulfide (H2S) has been found to reduce high-glucose-induced neuroinflammation by inhibiting the NF-κB signal; inhibiting NF-κB can also increase the expression of synaptic proteins in diabetic mice, improve the ultrastructural synaptic plasticity in the hippocampal region, and enhance their cognitive ability. Based on current evidence, supplementing exogenous H2S may be an effective intervention measure to inhibit NF-κB to reduce pyroptosis and improve DE cognitive dysfunction.

[0004] Currently, the activation of the NLRP3 inflammasome is mainly inhibited to reduce pyroptosis. However, since pyroptosis is regulated by multiple pathways, a single NLRP3 inflammasome inhibitor has poor inhibitory effects on pyroptosis. NF-κB is another key regulator of pyroptosis, which mediates the pyroptosis process by regulating the transcription and translation of NLRP3 protein, precursor inflammatory factors Pro-IL-1β and Pro-IL-18. Therefore, simultaneously inhibiting the activation of the NLRP3 inflammasome and NF-κB will better inhibit neuronal pyroptosis to improve the cognitive dysfunction caused by DE. Summary of the Invention

[0005] Based on the existing technology, the objective of the present invention is to provide an NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor. All the inhibitors can release H2S in vitro. Among them, the inhibitor CYT-1 has the most stable H2S release rate, the most appropriate LogP value (3.83), and the lowest cytotoxicity. Its H2S release amount relative to TBZ is 29.73%. Under the action of body enzymes, CY-09 and the hydrogen sulfide donor are released, which can not only target and inhibit the activation of the NLRP3 inflammasome, but also release H2S to inhibit NF-κB phosphorylation, so as to achieve the purpose of synergistically inhibiting neuronal pyroptosis. It has a better therapeutic effect than single administration of TBZ or CY-09, can synergistically inhibit neuronal pyroptosis by simultaneously inhibiting the activation of NF-κB and the NLRP3 inflammasome, and further improve the cognitive dysfunction of diabetic encephalopathy mice, providing new ideas for the drug development and treatment of diabetic encephalopathy.

[0006] The second objective of the present invention is to provide a preparation method of the above NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor.

[0007] The third objective of the present invention is to provide the application of the above NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor in the preparation of drugs for treating diabetic encephalopathy, especially in the preparation of drugs for inhibiting neuronal pyroptosis.

[0008] The technical solution of the present invention is as follows:

[0009] The NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor, and the structural formula of the inhibitor shown by formula CYT is as follows:

[0010]

[0011] Among them,

[0012] A represents a covalent bond, -CH 2 -O-, -CH 2 -CH 2 -O-, -CH 2 -CH 2 -CH 2 -O or -CH 2 -CH 2 -CH 2 -CH 2 -O-.

[0013] In a preferred embodiment, the inhibitor is selected from the following compounds:

[0014]

[0015] In the present invention, for the preparation method of the NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor, the synthetic route is as follows:

[0016] a) When A represents a covalent bond, the synthesis route of the inhibitor shown by formula CYT is as follows:

[0017]

[0018] b) When A does not represent a covalent bond, the synthesis route of the inhibitor shown by formula CYT is as follows:

[0019]

[0020] Among them, n represents an integer from 1 to 4.

[0021] In a preferred embodiment, a method for preparing an NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor includes the following steps:

[0022] In step a):

[0023] In the presence of EDCI and DMAP, compound CY-09 and compound TBZ undergo a chemical reaction to prepare inhibitor CYT-1;

[0024] In step b):

[0025] (1) In the presence of potassium carbonate, compound TBZ reacts with compound M to prepare compound TBZ-Br;

[0026] (2) In the presence of potassium carbonate, compound CY-09 reacts with compound TBZ-Br under the condition of 50-70 °C to prepare inhibitor CYT.

[0027] In the present invention, in step a), the molar ratio of compound CY-09 to compound TBZ is 1:0.8-1.5, which can be but is not limited to 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5. To obtain better effects, the molar ratio of compound CY-09 to compound TBZ is 1:1.0.

[0028] The molar ratio of compound CY-09 to EDCI is 1:1.0-2.0, which can be but is not limited to 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0. To obtain better effects, the molar ratio of compound CY-09 to EDCI is 1:1.5.

[0029] The molar ratio of compound CY-09 to DMAP is 1:0.1 - 0.2, which can be but is not limited to 1:0.1, 1:0.11, 1:0.12, 1:0.13, 1:0.14, 1:0.15, 1:0.16, 1:0.17, 1:0.18, 1:0.19 or 1:0.2. For better results, the molar ratio of compound CY-09 to DMAP is 1:0.15.

[0030] In the present invention, in step b), in step (1), the molar ratio of compound TBZ to compound M is 1:2.5 - 3.5, which can be but is not limited to 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5. For better results, the molar ratio of compound TBZ to compound M is 1:3.0.

[0031] In step (1), the molar ratio of compound TBZ to potassium carbonate is 1:2.5 - 3.5, which can be but is not limited to 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5. For better results, the molar ratio of compound TBZ to potassium carbonate is 1:3.0.

[0032] In step (1), the reaction temperature is 20 - 30 °C.

[0033] In the present invention, in step b), in step (2), the molar ratio of compound CY-09 to compound TBZ-Br is 1:0.8 - 1.5, which can be but is not limited to 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5. For better results, the molar ratio of compound CY-09 to compound TBZ-Br is 1:1.0.

[0034] In step (2), the molar ratio of compound CY-09 to potassium carbonate is 1:2.5 - 3.5, which can be but is not limited to 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4 or 1:3.5. For better results, the molar ratio of compound CY-09 to potassium carbonate is 1:3.0.

[0035] In step (2), the reaction temperature is 60 °C.

[0036] The present invention provides the use of the above-mentioned NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor in the preparation of a medicament for treating diabetic encephalopathy, particularly in the preparation of a medicament for inhibiting neuronal pyroptosis.

[0037] In the present invention, the NLRP3 inflammasome-targeted inhibitor CY-09 is conjugated with the hydrogen sulfide donor TBZ to obtain an inhibitor capable of synergistically inhibiting pyroptosis. Under the action of body enzymes, CY-09 and the hydrogen sulfide donor are released, which can not only targetedly inhibit the activation of the NLRP3 inflammasome, but also release H2S to inhibit NF-κB, so as to achieve the purpose of synergistically inhibiting neuronal pyroptosis.

[0038] To verify its efficacy, the ability of the compound to release H2S in vitro was determined by the methylene blue method, and the cytotoxicity of the compound was detected by CCK-8. According to the results of H2S release ability and cytotoxicity, a compound inhibitor CYT-1 with the best H2S release ability and the highest safety was selected for subsequent pharmacodynamic experiments. At the cellular level, high-glucose-induced cell pyroptosis was used, and the inhibitory effect of the inhibitor on neuronal pyroptosis was verified by experiments such as CCK-8, Western Blot, IF, PI staining, RT-PCR, and ELISA. At the animal level, diabetes was induced in mice by intraperitoneal injection of STZ, and the success of the diabetic encephalopathy mouse model was confirmed by behavioral experiments; the inhibitory effect of the inhibitor on neuronal pyroptosis and the improvement effect on the cognitive dysfunction of diabetic encephalopathy mice were verified by behavioral experiments, Western Blot, Nissl staining, etc., aiming to provide new ideas for the drug development and treatment of diabetic encephalopathy.

[0039] Adopting the technical solution of the present invention, the advantages are as follows:

[0040] The present invention provides an NLRP3 inflammasome inhibitor conjugated with a hydrogen sulfide donor. All inhibitors can release H2S in vitro. Among them, the inhibitor CYT-1 has the most stable H2S release rate, the most appropriate LogP value (3.83), and the lowest cytotoxicity. Its H2S release amount relative to TBZ is 29.73%. Under the action of body enzymes, CY-09 and the hydrogen sulfide donor are released, which can not only targetedly inhibit the activation of the NLRP3 inflammasome, but also release H2S to inhibit NF-κB, so as to achieve the purpose of synergistically inhibiting neuronal pyroptosis. It has a better therapeutic effect than single administration of TBZ or CY-09, can synergistically inhibit neuronal pyroptosis by simultaneously inhibiting the activation of NF-κB and the NLRP3 inflammasome, and further improve the cognitive dysfunction of diabetic encephalopathy mice, providing new ideas for the drug development and treatment of diabetic encephalopathy. Description of the Drawings

[0041] Figure 1 is the hydrogen spectrum of compound CYT-1;

[0042] Figure 2 is the carbon spectrum of compound CYT-1;

[0043] Figure 3 is the hydrogen spectrum of compound CYT-2;

[0044] Figure 4 is the carbon spectrum of compound CYT-2;

[0045] Figure 5 is the hydrogen spectrum of compound CYT-3;

[0046] Figure 6 is the carbon spectrum of compound CYT-3;

[0047] Figure 7 is the hydrogen spectrum of compound CYT-4;

[0048] Figure 8 is the carbon spectrum of compound CYT-4;

[0049] Figure 9 is the linear relationship between the absorbance at 670 nm and the concentration of Na2S in PBS buffer (0 - 120 μM), and the data are presented as mean ± standard error (n = 3);

[0050] Figure 10 is the content of H 2 S released after co-incubation of 200 μM of TBZ, CYT-1, CYT-2, CYT-3, and CYT-4 with 1 mM TCEP respectively, and the data are presented as mean ± standard error (n = 3);

[0051] Figure 11 are the cytotoxicity results of compounds CYT-1, CYT-2, CYT-3, and CYT-4. The OD values were recorded after co-incubating the compounds at different concentrations (0, 5, 10, 20, 40, 80, and 160 μM) with HT-22 cells for 24 hours. The data are presented as mean ± standard error (n = 3), *P < 0.05, **P < 0.01, and ***P < 0.001, compared with Control;

[0052] Figure 12 are the cytotoxicity results, where Figure 12 A is TBZ; Figure 12 B is CY-09 in it; The OD values were recorded after co-incubating TBZ and CY-09 at different concentrations (0, 5, 10, 20, 40, 80, and 160 μM) with HT-22 cells for 24 hours respectively. The data are presented as mean ± standard error (n = 3); *P < 0.05 and **P < 0.01, compared with Control;

[0053] Figure 13 Changes in cell viability after incubation of high glucose with HT-22 cells for 24 hours. Data are expressed as mean ± SEM (n = 3); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with HG group;

[0054] Figure 14 Effect of TBZ, CY-09 and CYT-1 on H 2 S level in high glucose-induced HT-22 cells. Data are expressed as mean ± SEM (n = 3); ***P < 0.001, compared with Control; ##P < 0.01 and P < 0.001, compared with HG group;

[0055] Figure 15 Effect of TBZ, CY-09 and CYT-1 on the expression of p-NF-κB in high glucose-induced HT-22 cells; among them, Figure 15 A shows the expression of p-NF-κB protein analyzed by Western Blot; Figure 15 B shows the expression of p-NF-κB protein analyzed by immunofluorescence; the scale bar is 20 μM, and data are expressed as mean ± SEM (n = 3); **P < 0.01, compared with Control; #P < 0.05, compared with HG group;

[0056] Figure 16 Effect of TBZ, CY-09 and CYT-1 on the expression of NLRP3 in high glucose-induced HT-22 cells; among them, Figure 16 A shows the expression of NLRP3 protein analyzed by Western Blot; Figure 15 B shows the expression of NLRP3 protein analyzed by immunofluorescence; the scale bar is 20 μM, and data are expressed as mean ± SEM (n = 3). ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with HG group;

[0057] Figure 17 Effect of TBZ, CY-09 and CYT-1 on the expression of Caspase-1 in high glucose-induced HT-22 cells; among them, Figure 17 A shows the expression of Caspase-1 protein analyzed by Western Blot; Figure 17B was for analyzing the expression of Caspase-1 protein by immunofluorescence; the scale bar was 20 μM, and the data were expressed as mean ± standard error (n = 3); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the HG group;

[0058] Figure 18 were the effects of TBZ, CY-09 and CYT-1 on the expression of GSDMD-N in high glucose-induced HT-22 cells; among them, Figure 18 A was for analyzing the expression of GSDMD-N protein by Western Blot; Figure 18 B was for analyzing the expression of GSDMD-N protein by immunofluorescence; the scale bar was 20 μM, and the data were expressed as mean ± standard error (n = 3); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the HG group;

[0059] Figure 19 were the effects of TBZ, CY-09 and CYT-1 on the expression of IL-1β and IL-18 in high glucose-induced HT-22 cells; among them, Figure 19 A was for analyzing the level of IL-1β by ELISA; Figure 19 B was for analyzing the level of IL-18B by ELISA; the data were expressed as mean ± standard error (n = 3); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the HG group;

[0060] Figure 20 were the effects of TBZ, CY-09 and CYT-1 on the mRNA expression in high glucose-induced HT-22 cells; among them, Figure 20 A was for analyzing the level of Caspase-1 by reverse transcription polymerase chain reaction (RT-PCR); Figure 20 B was for analyzing the level of GSDMD-N by RT-PCR; Figure 20 C was for analyzing the level of IL-1β by RT-PCR and Figure 20 D was for analyzing the level of mRNA by RT-PCR; the data were expressed as mean ± standard error (n = 3); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the HG group;

[0061] Figure 21 were the inhibitory effects of TBZ, CY-09 and CYT-1 on HG-induced HT-22 cell membrane rupture, the scale bar was 100 μM, (n = 3);

[0062] Figure 22 To detect the improvement effects of TBZ, CY-09 and CYT-1 on cognitive dysfunction in diabetic encephalopathy mice through the step-through test; Figure 22 A is the step-through latency; Figure 22 B is the number of errors; Data are expressed as mean ± standard error (n = 10); **P < 0.01 and ***P < 0.001, compared with Control; #P < 0.05 and ##P < 0.01, compared with the DM group;

[0063] Figure 23 To detect the improvement effects of TBZ, CY-09 and CYT-1 on cognitive dysfunction in diabetic encephalopathy mice through the novel object recognition test; Data are expressed as mean ± standard error (n = 10); **P < 0.01, compared with Control; #P < 0.05, compared with the DM group;

[0064] Figure 24 To detect the improvement effects of TBZ, CY-09 and CYT-1 on cognitive dysfunction in diabetic encephalopathy mice through the Morris water maze test; Among them, Figure 24 A is the escape latency; Figure 24 B is the number of platform crossings; Figure 24 C is the percentage of time in the target quadrant; Figure 24 D is the movement trajectory of the mice in each quadrant; Data are expressed as mean ± standard error (n = 10); **P < 0.01 and ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the DM group;

[0065] Figure 25 Effects of TBZ, CY-09 and CYT-1 on the levels of H 2 S in the serum and hippocampus of diabetic encephalopathy mice; Among them, Figure 25 A is the serum; Figure 25 B is the hippocampus; Data are expressed as mean ± standard error (n = 6); ***P < 0.001, compared with Control; #P < 0.05, ##P < 0.01 and P < 0.001, compared with the DM group;

[0066] Figure 26 Effects of TBZ, CY-09 and CYT-1 on pyroptosis proteins in the hippocampus of diabetic encephalopathy mice; Among them, Figure 26 A is the analysis of p-NF-κB by Western Blot; Figure 26 B is the analysis of NLRP3 by Western Blot; Figure 26C was for analyzing Caspase-1 by Western blot and Figure 26 D was for analyzing the expression of GSDMD-N protein by Western blot; Data were expressed as mean ± SEM (n = 6); ***P < 0.001, compared with Control; ##P < 0.01 and P < 0.001, compared with the DM group;

[0067] Figure 27 were the effects of TBZ, CY-09 and CYT-1 on hippocampal neurons of diabetic encephalopathy mice; Among them, Figure 26 The scale bar in A was 200 μM; Figure 26 The scale bar in B was 20 μM; Figure 26 C was the number of neurons; Data were expressed as mean ± SEM (n = 3); *P < 0.05, compared with Control; #P < 0.05, compared with the DM group. Detailed implementation manners

[0068] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0069] I. Implementation methods

[0070] 1. Materials

[0071] 1.1 Cell lines

[0072] Mouse hippocampal neuron cell line HT-22, Shanghai Gaining Biotechnology Co., Ltd.

[0073] 1.2 Experimental animals

[0074] Adult male ICR mice, Experimental Animal Center of Xuzhou Medical University

[0075] License number: 202210S038

[0076] 1.3 Preparation of relevant solutions required for H 2 2S release

[0077] (1) PBS solution: Dissolve a bag of PBS powder completely in 2 L of double-distilled water to obtain a PBS solution with a concentration of 10 mM.

[0078] (2) Sodium sulfide standard solution: Weigh 24.22 mg of Na 2 2S·9H 2In a 10 mL small beaker, add PBS solution that has been purged with nitrogen in advance, and then transfer it to a 100 mL volumetric flask. Dilute it to the mark with PBS solution to obtain a 1 mM sodium sulfide standard stock solution. The sodium sulfide solution needs to be prepared and used immediately.

[0079] (3) 3,3,3-Phosphinetriyltripropionic acid (TCEP) solution: Weigh 250.19 mg of TCEP, dissolve it with PBS solution that has been purged with nitrogen in advance and transfer it to a 10 mL volumetric flask to dilute to the mark, obtaining a TCEP stock solution with a final concentration of 100 mM.

[0080] (4) Preparation of methylene blue (MB + ) solution: Weigh 97.32 mg of ferric chloride and dissolve it in 20 mL of 1.2 M HCl, with a final concentration of 30 mM; weigh 93.71 mg and dissolve it in 20 mL of 7.2 M HCl, with a final concentration of 20 mM; weigh 200 mg of zinc acetate and dissolve it in 20 mL of double-distilled water, with a concentration of 1% (w / v).

[0081] (5) Preparation of compound solutions: Weigh CYT-1 (111.61 mg), CYT-2 (120.41 mg), CYT-3 (123.22 mg) and CYT-4 (126.02 mg) respectively and dissolve them in 1 mL of DMSO, with a final concentration of 200 mM for all.

[0082] 1.4 Preparation of solutions related to cell experiments

[0083] (1) DMEM complete medium: Take 450 mL in a laminar flow hood, add 50 mL of fetal bovine serum to obtain DMEM complete medium (10%), seal it, and store it in a 4°C refrigerator for later use.

[0084] (2) High-glucose solution: Weigh 1.0796 g of glucose, dissolve it in 100 mL of DMEM medium (25 mM) in a laminar flow hood, and then filter and sterilize it through a 0.22 μM microporous membrane to obtain an 85 mM high-glucose solution. Seal it and store it in a 4°C refrigerator for later use.

[0085] (3) TBST membrane washing solution: Completely dissolve a bag of TBS powder in 2 L of double-distilled water to obtain a 10 mM TBS solution, add 2 mL of Tween-20 to obtain TBST solution. Mix well and store it at room temperature for later use.

[0086] (4) TBZ, CY-09 and CYT-1 solutions: Weigh TBZ (24.51 mg), CY-09 (67.75 mg) and CYT-1 (89.29 mg) respectively and dissolve them in 1 mL of DMSO for cells. The final concentration is 160 mM for all. Seal it and store it in a 4°C refrigerator for later use.

[0087] 1.5 Preparation of solutions related to animal experiments

[0088] STZ solution: Dissolve 75 mg of STZ in 15 mL of pre-cooled 0.01 M citrate buffer (pH = 4.5) to obtain a 5 mg / mL STZ solution. Inject 0.1 mL of STZ per 10 g of mouse body weight. Prepare the STZ solution freshly before use.

[0089] 2. Method

[0090] 2.1 Synthesis of the compound

[0091] 2.1.1 Synthesis of compound CYT-1

[0092] Add CY-09 (2.12 g, 5 mmol), EDCI (1.44 g, 7.5 mmol), DMAP (0.09 g, 0.75 mmol), and 25 mL of dichloromethane to a 100 mL reaction flask. React at 0 °C for 30 min, then add TBZ (0.78 g, 5 mmol) to the resulting mixture and continue the reaction for 4 - 5 h. Monitor the reaction by TLC (dichloromethane:methanol = 20:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by vacuum distillation to obtain the crude product. Separate and purify it by column chromatography (dichloromethane) to obtain the yellow solid CYT-1 (1.63 g, 58.42%). 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.95 (s, 1H), 9.58 (s, 1H), 8.27 (d, J = 8.1, 2H), 8.00 (d, J = 8.6, 2H), 7.96 (s, 1H), 7.87 (d, J = 8.1, 2H), 7.76 (s, 1H), 7.68 - 7.56 (m, 3H), 7.37 (d, J = 8.6, 2H), 5.34 (s, 2H). CYT-1 13 C NMR (101 MHz, DMSO-d 6 ) δ 199.0, 193.5, 167.1, 163.7, 152.7, 138.0, 137.4, 136.2, 131.8, 131.6, 130.9, 130.8, 129.9, 130.8, 129.9, 129.8, 129.9 (d, J = 31.8 Hz), 128.9, 125.6, 124.6 (q, J = 3.7 Hz), 124.1 (q, J = 273.6), 121.4, 55.0, 46.9. 19 F NMR (376 MHz, DMSO-d 6 ) δ -51.82--71.29 (m).

[0093] The relevant hydrogen spectrum and carbon spectrum are asFigure 1 and Figure 2 as shown below. The synthetic route is as follows:

[0094]

[0095] 2.1.2 Synthesis of Compound CYT-2

[0096] (1) Add TBZ (0.78 g, 5 mmol), anhydrous potassium carbonate (2.07 g, 15 mmol), and 30 mL of acetonitrile to a 100 mL reaction flask. Stir the reaction at 20 - 30 °C for 30 min, then add 1,2-dibromoethane (2.82 g, 1.34 mL, 15 mmol) to the resulting mixture and continue the reaction at the same temperature for 48 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 2:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product, which is separated and purified by column chromatography (petroleum ether:ethyl acetate = 20:1, v / v) to obtain a colorless oily liquid compound e (712 mg, 54.78%).

[0097] (2) Add CY-09 (0.85 g, 2 mmol), anhydrous potassium carbonate (0.83 g, 6 mmol), and 20 mL of DMF to a 100 mL reaction flask. Stir the reaction at 60 °C for 30 min, then add compound e (0.52 g, 2 mmol) to the resulting mixture and continue the reaction at the same temperature for 24 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 1:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product, which is separated and purified by column chromatography (petroleum ether:ethyl acetate = 15:1, v / v) to obtain a pale yellow solid CYT-2 (256 mg, 21.36%). 1 H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 9.58 (s, 1H), 8.27 (d, J = 8.1, 2H), 8.00 (d, J = 8.6, 2H), 7.96 (s, 1H), 7.87 (d, J = 8.1, 2H), 7.76 (s, 1H), 7.68 - 7.56 (m, 3H), 7.37 (d, J = 8.6, 2H), 5.34 (s, 2H). 13 C NMR (100 MHz, CDCl3) δ 192.4, 167.6, 165.5, 161.8, 137.7, 135.6, 134.2, 132.5, 131.9, 131.1, 130.7, 130.5, 129.3, 126.1, 125.6, 125.3, 119.1, 115.4, 115.3, 104.8, 66.2, 63.3, 47.1.

[0098] The relevant hydrogen spectrum and carbon spectrum are as Figure 3 and Figure 4 shown, and the synthetic route is as follows.

[0099]

[0100] 2.1.3 Synthesis of Compound CYT-3

[0101] (1) Add TBZ (0.78 g, 5 mmol), anhydrous potassium carbonate (2.07 g, 15 mmol), and 30 mL of acetonitrile to a 100 mL reaction flask. Stir and react at 20 - 30 °C for 30 min. Then add 1,3-dibromopropane (3.03 g, 1.59 mL, 15 mmol) to the resulting mixture and continue the reaction at the same temperature for 48 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 2:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product. Purify it by column chromatography (petroleum ether:ethyl acetate = 20:1, v / v) to obtain a pale yellow oily liquid compound f (933.79 mg, 68.16%).

[0102] (2) Add CY-09 (0.85 g, 2 mmol), anhydrous potassium carbonate (0.83 g, 6 mmol), and 20 mL of DMF to a 100 mL reaction flask. Stir and react at 60 °C for 30 min. Then add compound f (0.55 g, 2 mmol) to the resulting mixture and continue the reaction at the same temperature for 24 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 3:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product. Purify it by column chromatography (petroleum ether:ethyl acetate = 30:1, v / v) to obtain a pale yellow solid CYT-3 (266.30 mg, 21.65%). 1 H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.0, 2H), 7.75 (d, J = 11.2, 2H), 7.66 (d, J = 7.7, 1H), 7.62 - 7.54 (m, 5H), 7.45 (t, J = 7.8, 1H), 7.02 - 6.98 (m, 2H), 5.36 (s, 2H), 4.72 (t, J = 4.6, 2H), 4.36 (t, J = 4.7, 2H). 1313C NMR (100 MHz, CDCl3) δ 192.4, 167.6, 165.5, 161.8, 137.7, 135.6, 134.2, 132.5, 131.9, 131.1, 130.7, 130.5, 129.3, 126.1, 125.6, 125.3, 119.1, 115.4, 115.3, 104.8, 66.2, 63.3, 47.1.

[0103] The related 1H NMR and 13C NMR spectra are as Figure 5 and Figure 6 shown, and the synthetic route is as follows.

[0104]

[0105] 2.1.4 Synthesis of Compound CYT-4

[0106] (1) Add TBZ (0.78 g, 5 mmol), anhydrous potassium carbonate (2.07 g, 15 mmol), and 30 mL of acetonitrile to a 100 mL reaction flask. Stir the reaction at 20 - 30 °C for 30 min, then add 1,4-dibromobutane (3.24 g, 1.80 mL, 15 mmol) to the resulting mixture and continue the reaction at the same temperature for 48 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 4:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product, which is separated and purified by column chromatography (petroleum ether:ethyl acetate = 30:1, v / v) to obtain a pale yellow oily liquid compound g (988.13 mg, 68.162%).

[0107] (2) Add CY-09 (0.85 g, 2 mmol), anhydrous potassium carbonate (0.83 g, 6 mmol), and 20 mL of DMF to a 100 mL reaction flask. Stir the reaction at 60 °C for 30 min, then add compound g (0.55 g, 2 mmol) to the resulting mixture and continue the reaction at the same temperature for 24 h. Monitor the reaction by TLC (petroleum ether:ethyl acetate = 3:1, v / v). After the reaction is completed, extract with ethyl acetate. Dry the organic phase with anhydrous Na2SO4 and remove the solvent by distillation under reduced pressure to obtain the crude product, which is separated and purified by column chromatography (petroleum ether:ethyl acetate = 30:1, v / v) to obtain a pale yellow solid CYT-3 (345.87 mg, 27.45%). 1HNMR(400MHz,CDCl3)δ8.11(dd,J=9.5,3.7,2H),7.75(d,J=10.6,2H),7.66(d,J=8.0,1H),7.57(t,J=8.8,5H),7.45(t,J=7.9,1H),6.94(d,J=8.2,2H),5.35(d,J=10.7,2H),4.55(t,J=6.3,2H),4.18(t,J=6.0,2H),2.33-2.27(m,2H). 13 C NMR(100MHz,CDCl3)δ192.5,191.6,167.6,165.5,162.1,137.5,135.6,134.1,132.5,131.9,131.6,130.5,130.5,129.6,129.5,129.3,126.1,126.0,125.5,125.3,119.2,115.3,64.9,62.1,47.2,28.6.

[0108] The related 1H-NMR and 13C-NMR spectra are as Figure 7 and Figure 8 shown. The synthetic route is as follows:

[0109]

[0110] 2.2 Determination of the in vitro H2S release content of the compound

[0111] Using PBS as the solvent, take 7 2-mL EP tubes and add 0, 20, 40, 60, 80, 100, and 120 μL of Na2S standard solution, 10 μL of TCEP (final concentration 1 mM) respectively. The total volume of the reaction system is 1 mL, and the rest is made up with PBS to obtain Na2S solutions with final concentrations of 0, 20, 40, 60, 80, 100, and 120 μM respectively. Then add MB to the reaction systems respectively + Mixed solution: 30 mM FeCl3 dissolved in 1.2 M HCl (200 μL); 20 mM N,N-dimethyl-1,4-phenylenediamine sulfate in 7.2 M HCl (200 μL); 1% w / v Zn(OAc)2 (100 μL). Then react at 37 °C for 30 min, record the absorbance of each reaction solution at 670 nm, and obtain the standard curve of the Na2S solution.

[0112] Using the same method for preparing the reaction system as above, using PBS as the solvent, add 1 μL of CYT-1, CYT-2, CYT-3, and CYT-4 (final concentrations are all 200 μM) respectively, then add 10 μL of TCEP (final concentration 1 mM), and finally add MB +A mixed solution was prepared, and then the absorbance value of the reaction solution at 670 nm was recorded. The H2S concentration of each sample was calculated according to the calibration curve of Na2S. The H2S release curve was obtained by plotting the H2S concentration against time.

[0113] 2.3 Cell culture

[0114] 2.3.1 Cell resuscitation

[0115] The cryopreservation tube of HT-22 cells was taken out from the liquid nitrogen tank and placed in warm water at 37 °C. It was shaken rapidly in the same direction to quickly thaw the cell cryopreservation solution. Subsequently, after disinfecting the cell cryopreservation tube with 75% alcohol, the cells were transferred to a 15 mL sterile centrifuge tube in a laminar flow hood. 2 mL of high-glucose DMEM complete medium was added, and the cells were gently resuspended with a Pasteur pipette. The tube was sealed and centrifuged (1000 rpm, 3 min). After centrifugation, the supernatant was discarded, and 5 mL of high-glucose DMEM complete medium was added again to resuspend the cells. After resuspending evenly, the cell suspension was transferred to a T25 cell culture flask, and the cell flask was gently shaken in a "cross" shape. Then it was placed in an incubator at 37 °C and 5% CO2 for culture.

[0116] 2.3.2 Cell culture

[0117] HT-22 cells are adherent cells and are generally cultured in high-glucose DMEM complete medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The incubator conditions are 37 °C and 5% CO2. Generally, subculture is performed once every 1-2 days. During culture, avoid excessive cell growth density. All operations are completed in a laminar flow hood.

[0118] 2.3.3 Cell subculture

[0119] Before cell subculture, the cell status needs to be observed through an inverted microscope to ensure that the cells are free from bacterial and fungal contamination and have a good growth state. Then, when the cells grow to 60%-80% (in the logarithmic growth phase), subculture can be carried out. In the laminar flow hood, the medium in the cell flask was discarded, and the cells were washed twice with 2 mL of PBS. 1-2 mL of 0.25% trypsin was added to digest the cells. After digesting for about 60 seconds, the cell flask was gently tapped to suspend the cells (the cell suspension situation can be observed under the microscope). Then, 2 mL of high-glucose DMEM complete medium was added to terminate the digestion. The cell wall was gently blown with a sterile Pasteur pipette, and the cell suspension was transferred to a 15 mL centrifuge tube. The tube was sealed and centrifuged (1000 rpm, 3 min). After centrifugation, the supernatant was discarded, 2 mL of high-glucose DMEM complete medium was added to resuspend the cells. After resuspending evenly, 1 mL of the cell suspension was taken and inoculated into a new culture flask, and 4 mL of high-glucose DMEM complete medium was added. It was placed in an incubator at 37 °C and 5% CO2 for culture. The medium was changed the next day. When the cells grow to 60-80%, subculture can be carried out again.

[0120] 2.3.4 Cell Cryopreservation

[0121] Before cell cryopreservation, ensure that the cells are in good growth condition and the growth density reaches about 80%. Wash, digest, resuspend, and centrifuge the cells according to the subculture procedure. After centrifugation, discard the supernatant, add 1 mL of pre-cooled non-programmed cryopreservation solution to each cryopreservation tube, resuspend the cells, then transfer them to the cryopreservation tube, seal it, and make marks according to the cell type, name, passage number, and cryopreservation date. Place the cells in a -80 °C refrigerator for cryopreservation. Transfer them to a liquid nitrogen tank for storage the next day.

[0122] 2.4 CCK-8 Assay for Detecting the Viability of HT-22 Cells

[0123] 2.4.1 Detection of the Cytotoxicity of Compounds

[0124] The safety of compounds is an important basis for their clinical application. In this invention, the cytotoxicity of compounds CYT-1, CYT-2, CYT-3, and CYT-4 against HT-22 cells was detected by CCK-8 assay. Seed the cells at a density of 5×10 3 cells per well in a 96-well plate, 100 μL per well, and incubate them in a 37 °C, 5% CO 2 2 incubator. After the cells are completely adherent, discard the medium in the 96-well plate, add the above compounds prepared with incomplete medium at different concentrations (0, 5, 10, 20, 40, 80, and 160 μM), and continue to incubate for 24 h. Then discard the medium again, add 100 μL of 10% CCK-8 solution to each well in the dark, incubate in the incubator for 1 h, and record the absorbance (OD) value of the sample at 450 nm wavelength using an enzyme-linked immunosorbent assay reader. 2.4.2 Optimal Dosing Concentrations of TBZ and CY-09

[0125] Determine the dosing concentrations of the control drugs TBZ and CY-09 by CCK-8 assay. Seed the cells in a 96-well plate as in step 2.4.1. After the cells are adherent, incubate them with different concentrations of TBZ and CY-09 (both 0, 5, 10, 20, 40, 80, and 160 μM) for 24 h. After incubation, add 100 μL of 10% CCK-8 solution to each well, and measure the absorbance (OD) value at 450 nm wavelength after 1 h.

[0126] 2.5 Cell Modeling and Grouping

[0127] (1) Control group: Cultured with incomplete medium, and no special treatment is performed for the rest.

[0128] (2) HG group: The cells are incubated with an 85 mM high-glucose solution prepared with incomplete medium for 24 h.

[0129] (3) HG + TBZ group: Cells were pre-incubated with 10 μM TBZ for 2 h, and then incubated with 85 mM high glucose solution for another 24 h.

[0130] (4) HG + CY-09 group: Cells were pre-incubated with 10 μM CY-09 for 2 h, and then incubated with 85 mM high glucose solution for another 24 h.

[0131] (5) HG + CYT-1 group: Cells were pre-incubated with 30 μM CYT-1 for 2 h, and then incubated with 85 mM high glucose solution for another 24 h.

[0132] 2.6 Changes in cell viability of HT-22 cells before and after modeling and drug administration

[0133] The changes in cell viability of HT-22 cells before and after modeling and drug administration were detected by CCK-8. Cells were seeded into 96-well plates as in step 2.4.1. After the cells adhered, modeling and drug administration were performed according to step 2.5 and incubated for 24 h. After incubation, 100 μL of 10% CCK-8 solution was added to each well. After 1 h, the OD value at 450 nm wavelength was measured.

[0134] 2.7 Detection of the expression of pyroptosis-related proteins in HT-22 cells by Western Blot

[0135] 2.7.1 Cell protein extraction

[0136] After modeling and drug administration according to step 2.5, the cells were taken out, the culture medium was discarded, and the remaining culture medium was washed with 2 mL of PBS. Then, 2 mL of PBS was added again, and the cells were scraped off with a cell scraper and transferred to an EP tube. After centrifugation (3000 rpm / min) for 3 min, the supernatant was discarded. 100 μL of lysis buffer (RIPA:PMSF:Na3VO4:NaF = 100:1:1:2) was added, mixed well, and lysed on ice for 0.5 h, vortexing for 10 s every 5 min. After lysis, centrifugation was performed at 4 °C for 15 min (12000 rpm), and the supernatant was collected and the volume was recorded.

[0137] 2.7.2 Detection of protein concentration by BCA method

[0138] (1) Establish a standard curve: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standard at a final concentration of 0.5 mg / mL to a 96-well plate in sequence, and supplement with normal saline to a total volume of 20 μL to obtain standard solutions with final concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL.

[0139] (2) Preparation of sample solution: Dilute the protein sample 10 - 20 times with normal saline (ensure that its OD value is within the OD value range of the standard product), gently vortex, and then add 20 μL of the sample solution to each well. Set 3 replicate wells for each group.

[0140] (3) Prepare the BCA working solution in the ratio of Solution A : Solution B = 50 : 1, then add 200 μL to each well and incubate in an oven at 37 °C for 0.5 h.

[0141] (4) Record the OD value at a wavelength of 550 nm using an enzyme - linked immunosorbent assay (ELISA) reader, and calculate the protein concentration of the sample from the standard curve and dilution factor.

[0142] 2.7.3 Preparation of protein samples

[0143] According to the measured concentration of the protein sample, dilute each group of samples to the same concentration with normal saline, and add protein loading buffer (5×) at one - quarter of the total sample volume. Vortex and mix well, then centrifuge slightly. Perform protein denaturation in a metal bath (100 °C, 15 min). After the denaturation is complete, let it return to room temperature and then store it in a - 20 °C refrigerator for later use.

[0144] 2.7.4 Protein separation

[0145] (1) Preparation of separating gel

[0146] Before preparing the gel, leak detection is required. After installing the gel - making rack, fill the space between the two glass plates with distilled water. After standing for 10 min, if the water level basically does not drop, it indicates good sealing and the next step can be carried out. Otherwise, reinstall the glass plates and perform leak detection again. After leak detection, discard the water and dry the water droplets, and prepare 10% upper and lower layer gels according to the formula in Table 1.

[0147] Table 1 Preparation method of separating gel

[0148]

[0149] (2) Electrophoresis

[0150] After the upper layer gel has solidified, install it into the electrophoresis tank, add electrophoresis buffer (Electrophoresis buffer: 1 L of double - distilled water, 14.4 g of glycine, 3 g of tris (hydroxymethyl) aminomethane (TrisBase) and 1 g of sodium dodecyl sulfate (SDS)), vertically pull out the comb upward (to avoid skewing of the sample loading wells), add 4 μL of prestained protein Marker, and then add each group of protein samples (50 μg) in turn. Then perform electrophoresis, and the electrophoresis program is 200 V, 50 min.

[0151] (3) Transfer

[0152] Prepare the transfer buffer according to the ratio of semi-dry transfer buffer (800 mL distilled water, 3 g tris(hydroxymethyl)aminomethane, and 14.4 g glycine): methanol = 4:1, and soak the pre-prepared NC membrane and thick filter paper of the corresponding size in the transfer buffer. After electrophoresis is completed, cut the target protein at the corresponding position according to the marker. Use a semi-dry transfer instrument to transfer the target protein to the NC membrane, and place it in the order of thick filter paper, NC membrane, gel, and thick filter paper. During the operation, add the transfer buffer dropwise to prevent the gel and NC membrane from drying out. The transfer program can be adjusted appropriately according to the molecular weight of the protein. Generally, keep the voltage at 20 V for 20 - 40 min.

[0153] (4) Blocking: After the transfer is completed, place the strip in a 5% BSA solution and block it at room temperature for 1 - 2 h.

[0154] (5) Incubate with primary antibody: Dilute the primary antibodies of the target proteins p-NF-κB, NF-κB, Caspase-1, GSDMD, and NLRP3 according to the ratio provided in the instruction manual, and then place the transferred NC membrane in the primary antibody and incubate it overnight at 4°C.

[0155] (6) Incubate with secondary antibody: Take out the strip the next day, wash it three times with TBST, 5 min each time. After washing, incubate the secondary antibody at room temperature for 1 - 2 h according to whether the primary antibody is rabbit or mouse. After incubation, wash it three times with TBST, 5 min each time, and store it for development after washing. (Keep it dark throughout the process).

[0156] (7) Development: Scan the strip using an Odyssey infrared fluorescence scanning imaging system, and perform statistical analysis with ImageJ and SPSS 21.0 software.

[0157] 2.8 Immunofluorescence detection of the expression of pyroptosis-related proteins in HT-22 cells

[0158] Place the sterile cell slides in a 12-well plate, inoculate the cells in the well plate for culture. When the cells are completely adherent and grow to an appropriate density, group and administer drugs according to 2.5, and then perform subsequent immunofluorescence staining.

[0159] (1) Cell fixation: After the drug administration is completed, take out the 12-well plate, discard the culture medium, wash the residual culture medium with PBS, and then add 500 μl of 4% paraformaldehyde to each well and fix it at room temperature for 15 min.

[0160] (2) Cell permeabilization: After fixation, discard the paraformaldehyde, add PBS and wash it slowly on a shaker 3 times, 5 min each time. Subsequently, add 500 μL of 0.5% Triton X-100 (VTritonX-100 / VPBS = 0.5) to each well and permeabilize it at room temperature for 5 min.

[0161] (3) Blocking: After the permeabilization is completed, discard Triton X-100, add PBS, and wash by gently shaking on a shaker 3 times, 5 minutes each time. Subsequently, add 500 μl of 5% BSA solution to each well and block at room temperature for 1 h.

[0162] (4) Primary antibody incubation: Discard the BSA solution, and add the diluted primary antibodies (p-NF-κB, GSDMD, Caspase-1, and NLRP3 diluted at 1:200) dropwise onto the coverslips, and incubate overnight at 4 °C.

[0163] (5) Secondary antibody incubation: Recover the primary antibodies, add PBS, and wash by gently shaking on a shaker 3 times, 5 minutes each time. Subsequently, slowly add the FITC-labeled fluorescent secondary antibody (diluted at 1:200) dropwise onto the coverslips in the dark, and incubate at room temperature in the dark for 1 h.

[0164] (6) DAPI counterstaining: After the secondary antibody incubation, discard the secondary antibody, add PBS, and wash by gently shaking on a shaker 3 times, 5 minutes each time. Subsequently, add DAPI dropwise onto the coverslips and counterstain for 10 min (this process is carried out in the dark).

[0165] (7) Mounting: After the counterstaining, discard DAPI, add PBS, and wash by gently shaking on a shaker 3 times, 5 minutes each time. Use a curved ophthalmic forceps to transfer the coverslips to the glass slides, add an anti-fluorescence quencher for mounting, and take pictures under a fluorescence microscope in the upright position.

[0166] 2.9 Detection of endogenous H2S content in cells by methylene blue method

[0167] Extract the proteins in the cells according to the method in 2.7 and determine their concentrations. Measure the H2S content in the samples according to the method in 2.2. After the reaction system reacts at 37 °C for 30 min, record the absorbance at 670 nm. Calculate the H2S concentration of each sample according to the calibration curve of Na2S (0 - 10 μM), and the results are expressed as μM / g protein.

[0168] 2.10 Detection of the content of inflammatory factors in the supernatant of HT-22 cells by ELISA

[0169] The levels of inflammatory factors IL-1β and IL-18 were detected using an ELISA kit. Cells were seeded in culture dishes and grouped and treated with drugs according to 2.5. After the drug treatment was completed, the culture dishes were taken out, the culture medium was collected in EP tubes, and cell debris was removed by centrifugation at 4000 rpm / min for 20 min. Then, the supernatant was collected and stored in a -20°C refrigerator for further measurement. According to the kit instructions, the OD values of IL-1β (0, 5, 10, 20, 40, and 80 ng / L) and IL-18 (0, 7.5, 15, 30, 60, and 120 ng / L) standards at different concentrations were measured, and a standard curve was established with the OD values and the standard concentrations to determine the levels of IL-1β and IL-18 in the samples.

[0170] 2.11 Detection of the expression of pyroptosis-related gene mRNA by RT-PCR

[0171] (1) Cell culture and treatment

[0172] HT-22 cells were seeded in 6-cm-diameter culture dishes and cultured. After the cells grew to an appropriate density (80%), modeling and drug administration were performed according to step 2.5.

[0173] (2) Total RNA extraction

[0174] Nuclease-free pipette tips and EP tubes of various specifications were prepared in advance. New gloves and masks were worn during the experiment to avoid contamination. After the cell treatment was completed, the culture medium was discarded, and the culture dishes were washed twice with PBS. 2 mL of Trizol was added to each culture dish, and it was allowed to stand for about 10 s. The cell layer at the bottom of the dish was gently pipetted with a 1-mL pipette tip to detach the cells and transferred to a 5-mL EP tube, and it was allowed to stand for 5 min. Subsequently, 400 μL of chloroform was added, and the EP tube was inverted 15 times to mix well and left at room temperature for 10 min. Then, it was centrifuged at 12000 rpm at 4°C for 15 min, and the supernatant was aspirated and transferred to a new EP tube. An equal volume of isopropanol was added to the supernatant, and it was shaken well and allowed to stand for 10 min; then centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was discarded. The white crescent at the bottom was RNA. 1 mL of pre-cooled 75% ethanol was added, and the precipitate was washed by vortexing and then centrifuged at 12000 rpm at 4°C for 10 min (washed twice). RNA can be stored in 75% ethanol at -20°C for one year or at 4°C for one week.

[0175] (3) Reverse transcription

[0176] The RNA concentrations of each group were measured using a ultra-micro spectrophotometer, with 1 μg of RNA. The reaction system is shown in Table 2.

[0177] Table 2 Reverse transcription system

[0178]

[0179] DEPC water and total RNA (6.5 μL in total) were mixed. After gentle centrifugation, other reaction components were added. After thorough mixing, centrifugation was performed again, and reverse transcription reaction was carried out in a PCR instrument. The reaction conditions were as follows: 25 °C for 10 min; 55 °C for 15 min; 85 °C for 5 min; 4 °C for 10 min (the obtained cDNA was stored at -20 °C, and for long-term storage, it was placed in a -80 °C refrigerator).

[0180] (4) RT-PCR

[0181] 1) The primers used in this experiment are shown in Table 3:

[0182] Table 3 Gene primer sequences

[0183]

[0184] 2) After mixing and centrifuging each reagent according to Table 4, it was added to a PCR-96 well plate. After sealing the film and centrifuging, it was loaded onto the machine for detection.

[0185] Table 4 PCR reaction system

[0186]

[0187] 3) The PCR program was as follows:

[0188] Activation stage: 95 °C for 2 min; PCR cycle stage: 95 °C for 15 s; 40 cycles; Melting curve stage: 60 °C for 15 s, Cooling stage: 40 °C; 30 s.

[0189] 4) Data processing: Calculate the expression levels of mRNA of each target gene by the 2∧-ΔΔCt method.

[0190] 2.12 PI (Propidium Iodide) staining experiment

[0191] The rupture of the cell membrane was observed by PI staining. The nuclear dye PI cannot penetrate normal cells with intact cell membranes, that is, living cells reject PI dye. Conversely, cells with ruptured cell membranes can be stained by PI. Pyroptosis is accompanied by the rupture of the cell membrane and the release of intracellular inflammatory factors. Therefore, PI can reflect the integrity of the cell membrane. Cells were seeded in 6-well plates and treated with drugs according to step 2.5. After the drug administration was completed, the culture medium was discarded, and the remaining culture medium was washed with PBS. Then, according to the kit instructions, 1 mL of cell staining buffer and 5 μL of PI staining solution were added to each well, and incubated at 4 °C for 30 min. After staining, it was washed once with PBS and then observed under a fluorescence microscope.

[0192] 2.13 Animal model establishment and drug administration

[0193] Fifty adult male ICR mice were selected. Ten mice were randomly selected as the normal group (Control), and the remaining mice were induced with type 1 diabetes by intraperitoneal injection of STZ (50 mg / kg) for 5 consecutive days. At 24 h after the end of STZ administration, tail tip blood of the mice was taken, and their fasting blood glucose was measured with a blood glucose meter. A fasting blood glucose ≥ 11.1 mmol / L indicated successful establishment of the diabetic mouse model. The diabetic mice were randomly divided into a diabetic group (DM), a TBZ administration group (DM+TBZ), a CY-09 administration group (DM+CY-09), and a CYT-1 administration group (DM+CYT-1), with 10 mice in each group. Among them, the TBZ administration dose was 3.6 mg / kg, the CY-09 dose was 10 mg / kg, and the CYT-1 dose was 42 mg / kg. They were intraperitoneally injected once a day for 4 consecutive weeks. The Control and DM groups were injected with the same volume of normal saline. Before and after the start and end of the administration, the fasting blood glucose and body weight of the mice were recorded.

[0194] 2.14 Behavioral experiments

[0195] 2.14.1 Step-through avoidance test

[0196] Avoiding the dark and approaching the light is the habit of mice. This habit was used to detect their learning and memory abilities. One side of the step-through avoidance apparatus was a dark chamber and the other side was a light chamber. The dark and light chambers were connected by a passage. The bottom of the dark chamber had an alternating current of 36 V and 52 Hz. The first day was the training period. The mice were placed in the step-through avoidance apparatus for training for 5 min. When the mice entered the dark chamber and were electroshocked, they would flee to the light chamber. The next day was the formal test, for 5 min. The mice were placed with their backs to the hole in the light chamber. If they entered the dark chamber, they would be electroshocked and recorded by the step-through avoidance apparatus. The number of errors was the number of times the mice entered the dark chamber within 5 min. The step-through latency was the time of the first entry into the dark chamber (if they did not enter the dark chamber within 5 min, the latency was calculated as 5 min).

[0197] 2.14.2 Novel object recognition test

[0198] The novel object recognition experiment utilizes the innate tendency of mice to explore novel objects to detect their learning and memory behaviors in a freely moving state. The experimental apparatus is a light gray-blue box measuring 40×40×45 cm. Three objects, namely A, B, and C, are prepared in advance. Among them, A and B are identical, while C is different. The experiment is divided into three days. On the first day, the mice are allowed to familiarize themselves with the environment. Without placing any objects, the mice are placed in the experimental box and allowed to freely explore for 5 minutes. On the second day, which is the training stage, objects A and B are fixed at the bottom on the same side of the box, 4 cm away from the box wall. The mice are placed into the box from the opposite side with their backs to the objects and allowed to explore the objects for 5 minutes. On the third day, object B is replaced with object C, and the time the mice spend exploring the new and old objects within 5 minutes is recorded. The Recognition Index (RI) = time of new object / (total time of new object + old object). Throughout the experimental process, before placing the next mouse, the experimental box needs to be cleaned with 75% alcohol to minimize olfactory cues as much as possible.

[0199] 2.14.3 Morris Water Maze

[0200] The spatial learning and memory abilities of mice are detected through the Morris water maze test. The test includes four consecutive days of training trials and a detection trial on the fifth day. The test is conducted in a circular pool (120 cm in diameter and 50 cm in height) filled with water (water temperature 25±2 °C). A circular platform with a diameter of 10 cm and a height of 30 cm is placed at the exact center of the first quadrant, with the water surface approximately 1.5 cm above the platform. During the first four days of training, the mice are respectively allowed to enter the water clockwise from the four quadrants to train their ability to find the underwater platform. Each time, the movement trajectory of the mice during the experiment and the time taken to find the platform are recorded. The time is set to 90 s. If the platform is found within the specified time, the time taken is the latency; otherwise, the mice need to be guided to the platform (in this case, the latency is 90 s). On the 5th day, a spatial exploration trial is conducted. The platform is removed, and the mice are allowed to freely move in the pool for 90 s. The time of the first arrival at the platform (escape latency), the number of times passing through the platform, and the percentage of time spent in the target quadrant are recorded.

[0201] 2.15 Western Blot

[0202] 2.15.1 Extraction of hippocampal tissue proteins

[0203] After the mouse behavioral experiment is completed, the mice are anesthetized and sacrificed, and the hippocampal tissue is removed and stored in a -80 °C refrigerator for further testing. The hippocampal tissue is placed in a 2 mL EP tube, and lysis buffer (RIPA:PMSF:Na 3 VO 4:NaF = 100:1:1:2 (1 μg of tissue plus 10 μL). After homogenization using a handheld tissue homogenizer, it was lysed on ice for 0.5 h with vortexing for 10 s every 5 min. The subsequent steps were carried out as in 2.7.2 - 2.7.4.

[0204] 2.16 Detection of H2S Content in Mouse Serum and Hippocampus

[0205] After the mice were anesthetized, blood was collected by eye enucleation. It was centrifuged at 12,000 rpm for 10 min, and the supernatant was taken to obtain serum for detecting the H2S content. Tissue proteins were extracted and their concentrations were determined according to 2.15.1. The H2S content in the samples of each group was measured by the method in 2.9. After the reaction system reacted at 37 °C for 30 min, the absorbance at 670 nm was recorded. The H2S concentration of each sample was calculated according to the calibration curve of Na2S (0 - 10 μM), and the results were expressed as μM / g protein.

[0206] 2.17 Nissl Staining

[0207] 2.17.1 Obtaining Brain Tissue Samples

[0208] After the behavioral experiments were completed, 3 mice were randomly selected from each group for perfusion and then the brains were taken. The mice were anesthetized with 4% chloral hydrate, the chest cavity was opened, a perfusion needle was inserted into the left ventricle, the right atrium was cut open, first rapidly perfused with normal saline until the whole body blood flowed out (clear liquid flowed out from the right atrium), and then slowly perfused with about 300 mL of 4% paraformaldehyde solution, and then the head was cut off to take the brain. The brain tissue was fixed with 4% paraformaldehyde solution in a 4 °C refrigerator for 24 h for standby.

[0209] 2.17.2 Dehydration, Embedding and Sectioning of Brain Tissue

[0210] The brain tissue fixed with 4% paraformaldehyde was dehydrated with a biological tissue dehydrator, embedded with a biological tissue embedding machine, and sectioned with a paraffin slicer (section thickness 5 μm). The cut tissue sections were placed in water at 42 °C for floating, and then transferred to glass slides (2 - 3 tissue sections were placed on each glass slide), spread on the glass slides in a 37 °C oven until there were no water droplets on the glass slides, baked at 60 °C for 40 min, and baked overnight in a 37 °C oven. After taking it out the next day and restoring to room temperature, the subsequent experiments could be carried out. If the next step of the experiment was not carried out in time, it could be temporarily stored in a 4 °C refrigerator.

[0211] 2.17.3 Staining

[0212] (1) Rewarming: Take out the sections from the 4 °C refrigerator and place them at room temperature for 1 h before dewaxing.

[0213] (2) Dewaxing: Immerse the sections in xylene I and II for 10 min each for dewaxing.

[0214] (3) Rehydration: Rehydrate with 100%, 90%, 80%, 70% ethanol, double-distilled water Ⅰ, and Ⅱ for 5 min each.

[0215] (4) Staining: Place the sections in Nissl staining solution and stain for 1 h (preheat the staining solution in an oven at 37 °C for 15 min before use), then soak in distilled water for about 3 s.

[0216] (5) Dehydration: Dehydrate with 80% and 90% ethanol for 2 - 3 s each, and dehydrate with 100% ethanol Ⅰ and Ⅱ for 10 s each.

[0217] (6) Clearing: Clear with xylene Ⅰ for 5 min and xylene Ⅱ for 10 min.

[0218] (7) Mounting: Mount with neutral balsam.

[0219] (8) Photographing: Observe and photograph with an upright microscope.

[0220] (9) Statistics: Select the same part and count the number of Nissl bodies in the same field of view area.

[0221] 2.18 Data processing

[0222] Perform statistical analysis on the data using SPSS 21.0 software, and the results are expressed as mean ± standard error (Mean ± SEM). One-way analysis of variance (one-way ANOVA) with a completely randomized design is used for comparison among multiple groups. For data with normal distribution and homogeneous variance, the LSD method is used for testing; for data with heterogeneous variance, the Dunnett’T3 method is used. The test level α = 0.05, and P < 0.05 indicates statistical significance. Statistical charts are drawn using GraphPad Prism 8.0 software.

[0223] II. Effect verification

[0224] 1. The level of H 2 S released by the compound in vitro

[0225] The present invention detected the ability of the compound to release H2S in vitro. First, measure the OD values corresponding to Na2S standards at different concentrations (0, 20, 40, 60, 80, 100, and 120 μM), and establish a standard curve from the OD values and concentrations (as Figure 9 ), and evaluate the content of H2S released by the compound according to this standard curve. The results are as Figure 10 shown. All compounds can release H2S within a certain time. According to its release curve and Table 5, compared with other compounds, the release rate of CYT-1 is more stable. Predict the LogP value of the compound through the SwissTargetprediction platform, and the results show that CYT-1 has a more suitable LogP value (3.83) and is more likely to cross the blood-brain barrier.

[0226] Table 5 Release amount of H2S and LogP prediction in compounds

[0227]

[0228] 2. Evaluation of compound cytotoxicity

[0229] The cytotoxicity of the synthesized compounds against HT-22 was detected by CCK-8 assay. Compounds CYT-1, CYT-2, CYT-3 and CYT-4

[0230] (at concentrations of 0, 5, 10, 20, 40, 80 and 160 μM) were co-incubated with HT-22 cells for 24 h, and the changes in OD values corresponding to different concentrations were detected. As Figure 11 shown, compared with the Control group, the cell viability of the remaining compounds decreased significantly at 160 μM except for CYT-1, while the cell viability of CYT-1 at 160 μM was still not significantly different from that of the Control group, indicating that CYT-1 has the highest safety. Combining the H2S release rate and LogP value, CYT-1 was selected for subsequent pharmacodynamic experiments.

[0231] 3. Inhibitory effect of CYT-1 on pyroptosis of HT-22 cells

[0232] 3.1 Screening of dosing concentrations of TBZ and CY-09

[0233] The appropriate dosing concentrations of TBZ and CY-09 were determined by CCK-8 assay. As Figure 12 shown, CY-09 had basically no cytotoxicity below 40 μM, while TBZ still had no cytotoxicity at a high concentration of 160 μM. The dosing concentrations of TBZ and CY-09 were determined to be 10 μM, and the dosing concentration of CYT-1 was 30 μM (the H2S release amount of CYT-1 relative to TBZ was 29.73%).

[0234] 3.2 CYT-1 improved the viability of cells induced by high glucose

[0235] In the present invention, 85 mM glucose (Highglucose, HG) solution was used to induce pyroptosis of HT-22 cells. As Figure 13 shown, the cell viability was significantly decreased after incubation with 85 mM high glucose solution for 24 h (P<0.001). Administration of TBZ, CY-09 and CYT-1 all increased the cell viability (P<0.05, P<0.01, P<0.001), but the therapeutic effect of CYT-1 was better.

[0236] 3.3 Changes in endogenous H2S content

[0237] The content of endogenous H2S was determined by methylene blue method. As Figure 14 shown, compared with the Control group, the content of H2S in the HG group decreased significantly (P<0.001); compared with the HG group, the content of H2S increased significantly after administration of TBZ, CY-09 and CYT-1 (P<0.001, P<0.01, P<0.001).

[0238] 3.4 CYT-1 inhibited the expression of pyroptosis-related proteins

[0239] NF-κB, NLRP3, Caspase-1 and GSDMD-N are several key proteins in the process of pyroptosis initiation. We detected the inhibitory effect of CYT-1 on pyroptosis by Western Blot and IF. As Figure 15 shown, TBZ and CYT-1 significantly inhibited the expression of p-NF-κB protein (P<0.05, P<0.05), while CY-09 had no obvious inhibitory effect (P>0.05), indicating that CYT-1 can release H2S and play the role of H2S in inhibiting NF-κB signal. As Figure 16 shown, similarly, TBZ and CYT-1 also inhibited the expression of NLRP3 (P<0.01, P<0.001), which may be due to the inhibition of the activation of NF-κB signal, reducing the transcription and translation of NLRP3 protein.

[0240] As Figure 17 and Figure 18 shown, CY-09 and CYT-1 significantly inhibited the expression of Caspase-1 (P<0.05, P<0.01) and GSDMD-N (P<0.05, P<0.01) proteins, and CYT-1 had a better inhibitory effect, but TBZ had no obvious effect on Caspase-1 and GSDMD-N proteins, indicating that CYT-1 can play the role of simultaneously inhibiting the activation of NF-κB and NLRP3 inflammasome, and has a better therapeutic effect than single administration of TBZ and CY-09.

[0241] 3.5 CYT-1 inhibited the expression of pyroptosis inflammatory factors

[0242] The expression of pyroptosis inflammatory factors IL-1β and IL-18 was detected by ELISA. As Figure 19 shown, compared with the Control group, the contents of pyroptosis inflammatory factors IL-1β and IL-18 in the HG group increased significantly (all P<0.001); administration of TBZ, CY-09 and CYT-1 significantly inhibited the expression of inflammatory factors, but CYT-1 had a better inhibitory effect.

[0243] 3.6 CYT-1 inhibited the expression of pyroptosis-related genes

[0244] The expression of pyroptosis-related gene mRNA was detected by RT-PCR. As Figure 20 shown, compared with the Control group, the expressions of Caspase-1, GSDMD-N, IL-1β and IL-18 mRNA in the HG group were significantly increased (all P<0.001); the administration of TBZ, CY-09 and CYT-1 significantly inhibited their mRNA expressions, but CYT-1 had a better inhibitory effect.

[0245] 3.7 CYT-1 inhibited the damage of high glucose to cell membrane

[0246] The results of PI staining showed ( Figure 21 ) compared with the Control group, the red fluorescence of cells in the HG group was significantly enhanced, indicating that the cell membrane in the HG group was severely ruptured. Compared with the HG group, the red fluorescence was significantly weakened after the administration of CYT-1, indicating that the administration of CYT-1 inhibited the damage of high glucose solution to cell membrane.

[0247] 4 Improvement effect of CYT-1 on cognitive dysfunction in diabetic encephalopathy mice

[0248] 4.1 Effects of CYT-1 on blood glucose and body weight in diabetic encephalopathy mice

[0249] Type 1 diabetes was induced by injecting STZ into mice, and the fasting blood glucose and body weight of mice were recorded before and after the administration. As shown in Table 6, after injecting STZ, the fasting blood glucose of mice increased significantly, all greater than 11.1 mM, showing significant differences compared with the fasting blood glucose of mice in the Control group (all P<0.001); there were no obvious differences in the body weight of all mice before administration. After the administration, there was no significant difference in the fasting blood glucose of mice in the Control group compared with that before administration; the fasting blood glucose of mice in the DM group continued to increase significantly compared with that before administration (P<0.001); there was no significant difference in the fasting blood glucose of mice in the administration group compared with that before administration. After the administration, the body weight of mice in the Control group was significantly higher than that of other groups (all P<0.001); there were no significant differences in the body weight of mice in the DM group and the administration group before and after administration.

[0250] Table 6 Effects of TBZ, CY-09 and CYT-1 on blood glucose and body weight in mice before and after treatment

[0251]

[0252] Data are expressed as mean ± standard error (n = 10). ***P<0.001, compared with Control; #P<0.05 and P<0.001, compared with before treatment after treatment.

[0253] 4.2 CYT-1 improved the cognitive dysfunction in diabetic encephalopathy mice

[0254] 4.2.1 Step-through test

[0255] The cognitive function of mice was detected by the step-through test. The results showed that compared with the Control group, the step-through latency of mice in the DM group was significantly decreased (P<0.001, Figure 22 A); compared with the DM group, after administration of CYT-1, the step-through latency of mice was significantly increased (P<0.01). Compared with the Control group, the number of errors of mice in the DM group was significantly increased (P<0.01, Figure 22 B), while administration of CYT-1 significantly reversed the above situation (P<0.05). The above results indicated that the mouse model of diabetic encephalopathy was successfully established, the mice had cognitive dysfunction, and CYT-1 could significantly improve the cognitive dysfunction of mice.

[0256] 4.2.2 Novel object recognition test

[0257] The results of the novel object recognition test showed ( Figure 23 ), compared with the Control group, the recognition index of mice in the DM group was significantly decreased (P<0.01), and after administration of CYT-1, its recognition index was significantly up-regulated (P<0.05). TBZ and CY-09 could increase the recognition index, but there was no significant difference compared with the DM group.

[0258] 4.2.3 Morris water maze test

[0259] The experimental results showed that compared with the Control group, the escape latency of mice in the DM group was significantly increased (P<0.001, Figure 24 A), the number of times of crossing the platform and the residence time ratio in the target quadrant were significantly decreased (P<0.001, P<0.01, Figure 24 B and Figure 24 C); compared with the DM group, after administration of TBZ, CY-09 and CYT-1, the above situation was significantly reversed, but the effect of CYT-1 was better.

[0260] 4.3 Effect of CYT-1 on H2S in the hippocampus of mice

[0261] The levels of H2S in the serum and hippocampus of mice were measured by methylene blue method. As Figure 25 shown, compared with the Control group, the levels of H2S in the serum and hippocampus of mice in the DM group were significantly decreased (all P<0.001); compared with the DM group, administration of TBZ and CYT-1 significantly restored the level of H2S, but there was no statistical difference between the CY-09 group and the DM group.

[0262] 4.4 Inhibitory effect of CYT-1 on pyroptosis-related proteins in the hippocampus of mice with diabetic encephalopathy

[0263] The changes in the proteins of p-NF-κB, NLRP3, Caspase-1 and GSDMD-N in the hippocampus of mice in each group were detected by Western Blot. As Figure 26 shown, consistent with the cell results trend, compared with the Control group, the protein expressions of p-NF-κB, NLRP3, Caspase-1 and GSDMD-N in the hippocampus of DM group mice were significantly increased (P<0.01, P<0.001, P<0.001, P<0.001). The administration of TBZ, CY-09 and CYT-1 inhibited the expressions of the above proteins, but CYT-1 had a better inhibitory effect.

[0264] 4.5 CYT-1 prevented the loss of neurons in the hippocampus of diabetic encephalopathy mice

[0265] The changes in the number of hippocampal neurons and Nissl bodies in diabetic encephalopathy mice were observed by Nissl staining. As Figure 27 shown, compared with the Control group, the number of neurons in the hippocampus of DM group mice was significantly reduced (P<0.05), and the Nissl body staining was lighter; compared with the DM group, the number of neurons increased significantly after the administration of CYT-1 (P<0.05), and the Nissl body staining was significantly deeper; although TBZ and CY-09 improved the neurons of mice, there was no significant difference compared with the DM group. This indicates that CYT-1 has a better therapeutic effect than single administration of TBZ and CY-09, can prevent neuron loss, and improve the cognitive dysfunction of mice.

[0266] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The NLRP3 inflammasome inhibitor coupled to a hydrogen sulfide donor, the structure of the inhibitor represented by the formula CYT is as follows: in, A represents a covalent bond, -CH2-O-, -CH2-CH2-O-, -CH2-CH2-CH2-O or -CH2-CH2-CH2-CH2-O-.

2. The NLRP3 inflammasome inhibitor coupled to a hydrogen sulfide donor according to claim 1, wherein the inhibitor is selected from the following compounds:

3. A method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 1, characterized in that: The synthetic route is as follows: a) When A represents a covalent bond, the synthetic route of the inhibitor represented by the formula CYT is as follows: b) When A does not represent a covalent bond, the synthetic route of the inhibitor represented by the formula CYT is as follows: Here, n represents an integer from 1 to 4.

4. The method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 3, characterized in that: The steps include: In step a): In the presence of EDCI and DMAP, compound CY-09 and compound TBZ react chemically to prepare the inhibitor CYT-1; In step b): (1) In the presence of potassium carbonate, compound TBZ reacts with compound M to prepare compound TBZ-Br; (2) In the presence of potassium carbonate, compound CY-09 and compound TBZ-Br are chemically reacted at 50-70° C. to prepare inhibitor CYT.

5. The method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 4, characterized in that: In step a), the molar ratio of compound CY-09 to compound TBZ is 1:0.8-1.5; the molar ratio of compound CY-09 to EDCI is 1:1.0-2.0; and the molar ratio of compound CY-09 to DMAP is 1:0.1-0.

2.

6. The method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 5, characterized in that: In step a), the molar ratio of compound CY-09 to compound TBZ is 1:1.0; the molar ratio of compound CY-09 to EDCI is 1:1.5; The molar ratio of compound CY-09 and DMAP was 1:0.

15.

7. The method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 4, characterized in that: In step b), in step (1), the molar ratio of compound TBZ to compound M is 1:2.5-3.5, preferably 1:3.0; the molar ratio of compound TBZ to potassium carbonate is 1:2.5-3.5, preferably 1:3.0, and the reaction temperature is 20-30°C.

8. The method for preparing the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 7, characterized in that: In step b), in step (2), the molar ratio of compound CY-09 to compound TBZ-Br is 1:0.8-1.5, preferably 1:1.0; the molar ratio of compound CY-09 to potassium carbonate is 1:2.5-3.5, preferably 1:3.0; and the reaction temperature is 60°C.

9. Use of the NLRP3 inflammasome inhibitor coupled with a hydrogen sulfide donor according to claim 1 in the preparation of a drug for treating diabetic encephalopathy.

10. The use according to claim 9, wherein the NLRP3 inflammasome inhibitor coupled to a hydrogen sulfide donor is used in the preparation of a drug for inhibiting neuronal pyroptosis.

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