Pharmaceutical use of azvudine drug

Abstract

Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or an isotopically labeled compound thereof in the preparation of a drug for preventing or treating neurodegenerative diseases, autoimmune and systemic inflammatory diseases, metabolic and cardiovascular diseases, respiratory diseases, ophthalmic diseases, or kidney diseases.

Description

Pharmaceutical uses of azvudine Technical Field

[0001] This invention relates to the use of compounds of formula (I) or pharmaceutically acceptable salts thereof, or isotopically labeled compounds thereof, in the preparation of medicaments for the prevention or treatment of the neurodegenerative disease Alzheimer's disease (AD). Background Technology

[0002] The NLRP3 inflammasome, a key sensor in the innate immune system, plays a central role in various diseases. Its activation mediates the release of pro-inflammatory factors such as IL-1β and IL-18, driving chronic aseptic inflammatory responses and widely participating in pathological processes including neurodegenerative diseases, autoimmune diseases, metabolic disorders, and cardiovascular and renal damage. Abnormally activated NLRP3 has been shown to be pathogenic in diseases such as Alzheimer's disease, gout, systemic lupus erythematosus, NASH, and atherosclerosis, making it a popular target for inhibition.

[0003] For example, Alzheimer's disease is a chronic neurodegenerative disease that leads to cognitive decline, mood swings, irreversible memory loss, disorientation, language impairment, and loss of self-protection abilities (Hardy J, et al. Science 2002, 297, 353-356). Alzheimer's disease typically begins to slowly progress and gradually worsens over time, accounting for 60% to 70% of dementia cases. Alzheimer's patients gradually withdraw from their families and society, becoming increasingly dependent on assistance, eventually leading to death. Alzheimer's disease has an insidious onset, a long course, and is extremely difficult to reverse once it occurs. Undoubtedly, Alzheimer's disease is a complex disease involving multiple factors. Currently, research into its pathogenesis and prevention mechanisms is in full swing in the medical community; however, its true causes remain unclear. Progress in clinical trials for treating Alzheimer's disease, whether with new candidate drugs or existing treatments, has been very limited. Finding new drug targets and new treatments for the neurodegenerative disease Alzheimer's is a very urgent task.

[0004] Azvudine (FNC) has been approved for marketing as a treatment for HIV and COVID-19, and its safety has been guaranteed.

[0005] Azvding Summary of the Invention

[0006] The inventors tested the effect of azvudine on inhibiting the production of IL-1β pro-inflammatory factors by activated NLRP3 inflammasomes. The results showed that azvudine significantly inhibited IL-1β release driven by NLRP3 inflammasomes. Furthermore, the inventors also tested the effect of azvudine-like drugs on the SAMP8 spatial learning and memory abilities of model mice. The results showed that azvudine-like drugs significantly improved the SAMP8 spatial learning and memory abilities.

[0007] In view of this, this document provides, on one hand, the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isotopically labeled compound thereof, in the preparation of a medicament for inhibiting NLRP3 inflammasome activation. On another hand, this document provides a method for inhibiting NLRP3 inflammasome activation, comprising administering to a patient in need a therapeutically or preventively effective amount of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isotopically labeled compound thereof; on yet another hand, this document provides the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isotopically labeled compound thereof, for inhibiting NLRP3 inflammasome activation; and on yet another hand, this document provides a pharmaceutical composition comprising the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isotopically labeled compound thereof, and optional excipients, said pharmaceutical composition for inhibiting NLRP3 inflammasome activation.

[0008] This article, in another aspect, provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or isotopically labeled compounds thereof, in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases or autoimmune and systemic inflammatory diseases or metabolic and cardiovascular diseases or respiratory diseases or ophthalmic diseases or kidney diseases; this article, in another aspect, provides a method for the prevention or treatment of neurodegenerative diseases or autoimmune and systemic inflammatory diseases or metabolic and cardiovascular diseases or respiratory diseases or ophthalmic diseases or kidney diseases, comprising administering to a patient in need a therapeutically or preventively effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or isotopically labeled compounds thereof. The present invention provides, in another aspect, compounds of formula (I) or pharmaceutically acceptable salts thereof, or isotopically labeled compounds thereof, for the prevention or treatment of neurodegenerative diseases or autoimmune and systemic inflammatory diseases or metabolic and cardiovascular diseases or respiratory diseases or ophthalmic diseases or kidney diseases; and in another aspect, a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, or isotopically labeled compounds thereof, and optional excipients, said pharmaceutical composition for the prevention or treatment of neurodegenerative diseases or autoimmune and systemic inflammatory diseases or metabolic and cardiovascular diseases or respiratory diseases or ophthalmic diseases or kidney diseases.

[0009] Optionally, the neurodegenerative diseases include Alzheimer's disease (AD), Lewy body dementia (LBD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), depression, and stroke; preferably Alzheimer's disease.

[0010] Optionally, autoimmune and systemic inflammatory diseases, including gout / hyperuricemia, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), cryopyrin-associated periodic syndrome (CAPS), ankylosing spondylitis, psoriasis, and hidradenitis suppurativa;

[0011] Optionally, metabolic and cardiovascular diseases, including type 2 diabetes and its complications, non-alcoholic steatohepatitis (NASH / MASH), obesity-related metabolic syndrome, atherosclerosis, and hypertension;

[0012] Optionally, respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma;

[0013] Optionally, ophthalmic diseases, including age-related macular degeneration (AMD) and diabetic retinopathy;

[0014] Optionally, kidney disease, including chronic kidney disease (CKD) / renal interstitial fibrosis, lupus nephritis;

[0015] The structural formula of equation (I) is as follows:

[0016] In formula (I),

[0017] R 1 It can be: H, azide, C1-C6 alkyl (e.g., methyl, ethyl), C1-C6 alkoxy (e.g., methoxy, ethoxy), C2-C6 alkynyl (e.g., ethynyl), C2-C6 alkenyl (e.g., vinyl), or halo-C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl);

[0018] R 2 It can be H, OH, halogen (e.g., F), C1-C6 alkyl (e.g., methyl, ethyl), or C1-C6 alkoxy (e.g., methoxy, ethoxy);

[0019] B is selected from:

[0020] Where X1 is -OH, -NH2, R 3 CONH-, R 3 COO- or R 3 O(C=O)NH-;

[0021] X2 represents OH, SH, NH2, and R. 3 COO-, R 3 COS-, R 3 CONH2- or R 3 O(C=O)NH-;

[0022] X3 is H, F, OH or NH2;

[0023] Y is either CH or N;

[0024] Z is H, OH, or F;

[0025] Each R 3 Each is independently selected from H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, isopropyl), C2-C6 ynyl (e.g., ethynyl), C2-C6 alkenyl (e.g., vinyl), halo-C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl), optionally C 1-6 Alkyl, C 1-6 Alkyl, CN, N3, OH, NH2, halogen-substituted (e.g., F, Cl, Br, I) phenyl groups, optionally C-substituted 1-6 Alkyl, C 1-6 Naphthyl groups substituted with alkoxy groups, CN, N3, OH, NH2, or halogens (e.g., F, Cl, Br, I).

[0026] Preferably, in formula (I),

[0027] R 1 It can be H, azide, or C2-C6 ynyl (e.g., ethynyl).

[0028] Preferably, in formula (I),

[0029] R 2 It can be H, OH, or a halogen (e.g., F).

[0030] Preferably, in formula (I),

[0031] B is selected from:

[0032] Where X1 is -OH or -NH2;

[0033] X2 is OH or NH2;

[0034] X3 is H, F, OH or NH2;

[0035] Y is either CH or N;

[0036] Z is H, OH, or F;

[0037] Optionally, Z is F.

[0038] Preferably, in formula (I),

[0039] Each R 3 Each is independently selected from H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, isopropyl), and halogenated C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl).

[0040] The following compounds, or pharmaceutically acceptable salts thereof, or isotopically labeled compounds thereof, are preferred: or

[0041] Preferably, the isotope-labeled compound is a deuterated compound.

[0042] Pharmaceutically acceptable salts of the compounds represented by formula (I) include, but are not limited to, salts formed by the compounds represented by formula (I) with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine, etc.

[0043] Compounds of formula (I) can be purchased commercially or prepared by known methods.

[0044] The administration route can be oral or parenteral. The dosage form of the drug can be immediate-release, sustained-release, or controlled-release. Specific dosage forms can be various conventional dosage forms in the art; for example, oral preparations can include tablets, hard capsules, soft capsules, aqueous or oily suspensions, granules, emulsions, syrups, or elixirs. Injectable preparations can include injection solutions and powder for injection. Attached Figure Description

[0045] Figure 1 is a bar chart showing the total activity distance, central area activity distance, and peripheral area activity distance of mice in each group in the mine experiment of Example 1.

[0046] Figure 2 is a bar chart showing the activity time in the central region and the activity time in the periphery of mice in each group of the mine experiment in Example 1.

[0047] Figure 3 shows a schematic diagram of the Morris water maze in Example 1.

[0048] Figure 4 is a bar chart showing the number of times mice in each group crossed the platform within 1 minute in the Morris water maze experiment of Example 1.

[0049] Figure 5 is a bar chart showing the latency period for the first crossing of the platform in each group of mice in the Morris water maze experiment of Example 1.

[0050] Figure 6 is a bar chart showing the total distance traveled by mice in each group during the Morris water maze experiment in Example 1, the distance traveled within the quadrant where the platform is located, and the distance traveled in the area surrounding the platform.

[0051] Figure 7 is a sample analysis mode diagram of the FLOWJO V10 software in Example 1.

[0052] Figure 8 is a bar chart of the proportions of different immune cell populations in the blood of mice in Example 1 (# indicates statistical analysis between the model control group and the normal control group; * indicates statistical analysis between the drug treatment group (FNC 1 mg / kg, FNC 0.5 mg / kg) and the model control group).

[0053] Figure 9 is a columnar section of TUNEL staining of hippocampal tissue from SAMP8 mice in each group of Example 1.

[0054] Figure 10 is a representative image of TUNEL staining of hippocampal tissue from SAMP8 mice in each group of Example 1 (red (bright) represents apoptosis, blue (dark) represents nuclear staining).

[0055] Figure 11 shows the inhibitory effect of compounds FNC and MCC950 on IL-1β release from THP-1 cells in Example 2.

[0056] Figure 12 shows the cytotoxicity results of compounds FNC and MCC950 on THP-1 cells in Example 2. Detailed Implementation

[0057] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0058] Example 1: Effect of the test drug on memory impairment in SAMP8 mice

[0059] Experimental materials

[0060] The test drug, Azvudine, was sourced from Henan Zhenshi Biotechnology Co., Ltd.

[0061] SAMR1 mice and SAMP8 mice (SPF grade) were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (SAMR1 mice) and Beijing Huafukang Biotechnology Co., Ltd. (SAMP8 mice), respectively. Experimental equipment...

[0062] Experimental methods

[0063] 1.1 Animal grouping

[0064] Mice were purchased and acclimatized. Once the SAMR1 and SAMP8 mice reached 6 months of age, they were stratified and randomly grouped according to body weight. The SAMP8 mice were divided into 3 groups, with the number of animals in each group shown in Table 1 below. Thirteen SAMR1 mice were selected as a normal control group. Specific dosages, administration methods, frequencies, and cycles are shown in Table 1 below.

[0065] Table 1

[0066] 1.2 Drug Preparation

[0067] Table 2

[0068] Note: The test drug FNC should be prepared every 3 days and stored at 4°C.

[0069] 1.3 Test Methods and Items

[0070] After grouping, mice in each group were administered the drug orally via gavage according to their body weight, once daily for two consecutive months. The first day of administration was designated as D1. Body weight was monitored once weekly during the continuous administration period. At the end of the administration period, spontaneous activity was first assessed; subsequently, the Morris water maze test was used to assess memory function. After all behavioral tests were completed, the animals were weighed, deeply anesthetized with CO2, and blood was collected from the heart. The blood was placed in K2-EDTA anticoagulant tubes (for the detection of T cells and MDSCs cell populations). After blood collection, brain tissue was harvested, and the brain was divided into left and right hemispheres in the sagittal plane. One hemisphere was paraffin-embedded for TUNEL staining, and the remaining hemisphere was cryopreserved for later analysis.

[0071] 1.3.1 Autonomous Activity Test (Mine Experiment)

[0072] The mine experiment is a method for evaluating the autonomous and exploratory behaviors of laboratory animals in novel environments.

[0073] The experimental setup consists of two parts: a mine reaction chamber and an automatic data acquisition and processing system. The experiment was conducted in a quiet environment.

[0074] Each group of animals was given the test substance. One hour later, the mice were placed in home cages (one mouse per cage). After acclimatization for 5 minutes, the automatic video recording program was started. The test lasted for 10 minutes. The Noldus video analysis system monitored and analyzed the animals' activities, including multiple sets of data such as the distance traveled per unit time, the time spent in the central area, and the distance traveled in the peripheral area. After each mouse was tested, the cage was wiped clean with 75% ethanol to avoid the odor affecting the correct testing of the next mouse.

[0075] The results of the mine experiment are shown in Figure 1 and Figure 2.

[0076] The results of the open field experiment showed that after two months of continuous oral administration, the total activity distance of mice in each group was similar and at a relatively high level, indicating that the spontaneous activity ability of SAMP8 mice of this age was not significantly weakened, and that 0.5 mg / kg and 1 mg / kg of FNC had little effect on them.

[0077] The results of the spontaneous activity test showed that, compared with the normal control group, the mice in the model control group had reduced activity distance and activity time in the central area, and correspondingly increased activity and activity time in the peripheral area, indicating that the exploration ability of the mice in the model group was reduced. Compared with the model group, 0.5 mg / kg and 1 mg / kg of FNC had a weaker effect on total activity distance and exploration ability.

[0078] 1.3.2 Morris Water Maze Test

[0079] Figure 3 is a schematic diagram of the Morris water maze quadrants. Training method: On the first day, before training in each quadrant, the mouse was placed on a platform for 10 seconds. Then, the mouse was placed in the water in quadrant I facing the wall and asked to find the platform, swimming for 60 seconds. If the mouse could not find the platform within 60 seconds, it was placed on the platform and stayed there for 10 seconds. Then, the training of the next mouse began. After training in quadrant I, the mouse was placed in quadrant II to swim and find the platform, following the same process. The mice were then placed in quadrants III and IV in turn. On the second day, the quadrant training sequence was G2-G3-G4-G1, and so on. This drug administration and swimming training process was repeated once a day for 4 consecutive days. On the fifth day, the platform removal experiment was conducted: the platform in quadrant II was removed, and the mouse was placed in the water from any quadrant of the pool, swimming along the wall, to find the platform for 1 minute. Morris water maze statistical software will record the number of times each animal crosses the platform, the time when it first finds the platform (the incubation period for the first crossing), the distance it travels in each of the four quadrants, and the distance it travels in the area surrounding the platform, and perform statistical processing.

[0080] Data statistics

[0081] Experimental data are expressed as Mean ± SD, and images are expressed as Mean ± SEM. Measurements deviating from the mean by more than two standard deviations were excluded. One-way ANOVA was used for statistical analysis. If the ANOVA was statistically significant (P ≤ 0.05) and the variances were homogeneous, the Tukey test was used for inter-group comparisons. If the variances were heterogeneous, Dunnett's T3 test was used for inter-group comparisons. A P < 0.05 was considered statistically significant.

[0082] Morris water maze experiment results and discussion

[0083] The results of the Morris water maze experiment are shown in Figures 4, 5, and 6.

[0084] Figure 4 is a bar chart showing the number of platform crossings by mice in each group within 1 minute. The results showed that compared with the normal control group SAMR1 mice, the number of platform crossings by the model control group SAMP8 mice was significantly reduced. The number of platform crossings by the two FNC dosage groups was significantly increased compared with the model control group, with the FNC 0.5 mg / kg group showing the most significant increase.

[0085] Figure 5 is a bar chart showing the latency period for the first crossing of the platform in each group of mice. The results showed that the latency period for the first crossing of the platform was significantly increased in the model control group SAMP8 mice compared with the normal control group SAMR1 mice; the latency period for the first crossing of the platform was significantly reduced in both FNC dose groups compared with the model control group, and the recovery of the animals in the FNC 0.5 mg / kg group was comparable to that of the normal control group.

[0086] Figure 6 shows a bar chart of the total activity distance, activity distance within the quadrant of the platform, and activity distance around the platform for each group of mice. The results indicate that compared to the normal control group (SAMR1 mice), the activity distance around the platform in the model control group (SAMP8 mice) was significantly reduced, suggesting a significant decrease in spatial memory ability. The FNC 0.5 mg / kg group showed a significant increase in the distance around the platform compared to the model control group, and the distance within the platform quadrant showed an increasing trend in both FNC dosage groups. The total activity distance in the model group was significantly reduced compared to the normal control group, suggesting poor activity endurance in SAMP8 mice at the end of the experiment (approximately 8 months of age). The total activity distance in both FNC dosage groups showed an increasing trend, with the FNC 0.5 mg / kg group showing a significant increase compared to the model control group, and comparable to the normal control group.

[0087] The results in summary indicate that FNC has a significant ameliorative effect on spontaneous Alzheimer's disease in SAMP8 mice.

[0088] 1.3.3 Detection of T cells and MDSCs cell populations in whole blood

[0089] Dosing was performed according to the administration regimen described in Table 1. Blood samples from each group of animals were then collected for flow cytometry analysis. Approximately 200 μL of whole blood was collected from mice and placed in anticoagulant tubes containing K2-EDTA. All samples were collected on ice and transferred to the flow cytometer within 2 hours. Subsequent procedures followed the standard operating procedures outlined below:

[0090] 1.3.3.1 Preparation of experimental reagents

[0091] 1) Preparation of 1×RPMI 1640 + 10% fetal bovine serum (10% HI-FBS)

[0092] Heat-inactivated fetal bovine serum was diluted with 1×RPMI 1640 medium to prepare 1×RPMI 1640+10% fetal bovine serum, stored at 4°C, and used within one week.

[0093] 2) Preparation of 1×Pharm Lyse™ lysis buffer

[0094] Dilute 10×BD Pharm Lyse™ lysis buffer with ultrapure water to 1× and use within 2 hours.

[0095] 3) Preparation of Transcription Factor Kit Buffers

[0096] Fixation / Permeabilization Concentrate (4×) is diluted with Fixation / Permeabilization Diluent to 1×Fix / Perm Buffer.

[0097] 1.3.3.2 Experimental Procedure

[0098] Blood sample single-cell suspension acquisition

[0099] 1) Take out the peripheral blood sample and place it in a 15mL centrifuge tube. Add 1×Pharm Lyse™ lysis buffer (v / v = 1:9), mix gently, and place at room temperature for 10 minutes.

[0100] 2) Centrifuge at 250×g for 5 minutes at room temperature and remove the supernatant.

[0101] 3) After resuspending the cells in 5 mL of RPMI 1640 + 10% HI FBS, centrifuge at 350×g for 5 minutes and discard the supernatant.

[0102] 4) Resuspend the cells in 5 mL of RPMI 1640 + 10% HI FBS.

[0103] Single-cell suspension staining with live and dead dyes

[0104] 1) The obtained blood single-cell suspension was plated in a 96-V well plate and centrifuged at 350×g for 5 minutes at 4℃.

[0105] 2) Prepare a live / dead staining solution using 1×DPBS, and add 100μL to each well to resuspend the cells.

[0106] Add 1 drop of ArC Amine Reactive Compensation Beads (negative, reactive beads), 1 drop of ArC Amine Reactive Compensation Beads (positive, reactive beads), and 0.1 μL of the active or inactive dye to each well of the single-staining dye.

[0107] 3) Incubate at 4℃ in the dark for 20 minutes.

[0108] 4) Add 100 μL of 1×DPBS to wash, centrifuge at 350×g for 5 minutes at 4℃, and remove the supernatant.

[0109] 5) After resuspending in 200 μL of staining buffer, centrifuge at 350 × g for 5 minutes at 4 °C and remove the supernatant.

[0110] Single-cell suspension antibody staining (extracellular staining)

[0111] 1) Add 50 μL of Fc mixture (45 μL of stain buffer and 5 μL of hFc Block) to each well and resuspend. Incubate at 4°C in the dark for 10 minutes.

[0112] 2) Prepare a 2× antibody mixture using stain buffer plus (10 μL / test) and stain buffer.

[0113] Add 50 μL of the 2× extracellular antibody mixture to each well.

[0114] (50 μL of Fc mixture has been added to each well, meaning the final extracellular antibody content per well is 1×).

[0115] Except for well A5, add 1 drop of UltraComp eBeads to each well, and add 1 μL of antibody to the corresponding UltraComp eBeads well to form a single-stained well. Add 1 drop of ArC Amine Reactive Compensation Beads (negative, reactive beads) and 1 drop of ArC Amine Reactive Compensation Beads (positive, reactive beads) to the "Unstained Control" well. For the remainder of the steps, treat the beads with the same buffer as the cell wells, unless otherwise noted. After CST calibration, apply the compensation settings and calculations via LSR Tortessa; compensation is generally completed without any warnings.

[0116] 3) Incubate at 4℃ in the dark for 30 minutes.

[0117] 4) Add 100 μL of stain buffer, centrifuge at 350 × g for 5 minutes at 4°C, and remove the supernatant.

[0118] 5) Resuspend in 200 μL of stain buffer, centrifuge at 350 × g for 5 minutes at 4 °C, and remove the supernatant.

[0119] Resuspend the cells in 200 μL of 1×Fix Buffer and incubate overnight at 4°C in the dark.

[0120] 6) On the second day (within 18 hours), centrifuge at 500×g, 4℃ for 5 minutes, and resuspend the cells in 200μL of stain buffer. Repeat twice.

[0121] 7) After the final wash, add 200 μL of stain buffer to each well to resuspend the cells, transfer to flow cytometry tubes, incubate at 4°C in the dark, and analyze with FACSFortessa within 48 hours.

[0122] 1.3.3.3 Data Collection, Statistical Analysis, and Results

[0123] All FCS format streaming test files were analyzed using FLOWJO V10 software according to the pattern shown in Figure 7, and relevant data were collected.

[0124] All data are expressed as Mean ± SEM. One-way ANOVA was used to assess differences. If the data met the homogeneity of variance requirement, Dunnett's t-test was used for multiple comparisons; if the data did not meet the homogeneity of variance requirement, Tamhane's T2 was used for multiple comparisons. A p-value < 0.05 was considered statistically significant.

[0125] Figure 8 is a bar chart showing the proportions of different immune cell populations in mouse blood. As shown in Figure 8, the CD8+T and CD4+T cells in the FNC 0.5 mg / kg and 1 mg / kg groups were significantly lower than those in the model control group, while the proportion of myeloid cells was significantly increased, especially PMN-MDSC, with the highest increase observed at the low dose of 0.5 mg / kg. This result may be related to the pharmacological effect of FNC in improving memory impairment.

[0126] 1.3.4 Effect of the test drug FNC on Tunel staining of hippocampal tissue in SAMP8 mice

[0127] [Experimental Principle]

[0128] DNA fragments in apoptotic cells are detected using fragment end labeling (FragEL™). During apoptosis, double-strand or single-strand breaks in chromosomal DNA produce numerous sticky 3'-OH ends. These ends can be labeled with fluorescein-labeled and unlabeled deoxyribonucleotides by terminal deoxyribonucleotide transferase (TdT), thus detecting the DNA fragments from apoptotic cells. Normal or proliferating cells have almost no DNA breaks and therefore do not form 3'-OH ends, making them rarely stained.

[0129] [Reagents and Materials]

[0130] 1. Reagents:

[0131] (1) Apoptosis kit name: In situ cell death detection kit; Catalog number: 12156792910; Manufacturer: Roche;

[0132] (2) TBS buffer

[0133] (3) Xylene

[0134] (4) Ethanol

[0135] (5) Proteinase K

[0136] (6) Gold Antifade Reagent with DAPI, Product No.: P-36931, Manufacturer: invitrogen

[0137] 2. Materials: Paraffin sections of brain tissue from SAMP8 mice

[0138] 3. Equipment: Semi-automatic dehydrator, Manufacturer: Leica, Model: TP1020

[0139] Fully automatic paraffin embedding machine, manufacturer: Leica, model: EG1150

[0140] Fully automatic paraffin slicer, manufacturer: Leica, model: RM2235

[0141] Nikon H500S electron microscope image processing software for photomicrography

[0142] NIS-Element Image Analysis Software

[0143] Microscope: Nikon Eclipse 50i, Model: H550S

[0144] Digital camera: Nikon model: DS-FiI

[0145] Image Pro Plus 6.0, a professional image analysis software.

[0146]

Operating Procedure

[0147] 1. Dewaxing and hydration

[0148] (1) Immerse the glass slide in xylene for 10 min × 3.

[0149] (2) 100% ethanol, 5 min × 2.

[0150] (3) 90%, 80%, and 70% ethanol for 3 min each.

[0151] (4) Rinse with running water for 5 minutes.

[0152] (5) Rinse with TBS for 5 min × 3, and carefully dry the glass slide around the sample.

[0153] Sample permeability treatment

[0154] (1) Incubate 100ul of 20ug / ml proteinase K at room temperature for 20 minutes.

[0155] (2) TBS rinse, 5 min × 3.

[0156] 3. Inactivate endogenous peroxidase

[0157] (1) Incubate 100ul of 3% H2O2 at room temperature for 5 minutes.

[0158] (2) TBS rinse, 5 min × 3.

[0159] 4. Labeling reaction

[0160] (1) Add 1 part of enzyme solution (vial 1) to 9 parts of labeling solution to obtain TUNEL reaction mixture.

[0161] (2) Add 50 μL of TUNEL reaction mixture to the sample.

[0162] Note: Add 50 μL of labeling solution to the negative control. Ensure the TUNEL reaction mixture is evenly distributed on the monolayer of cells to avoid evaporation loss. Cover the sample with Parafilm or a coverslip during incubation.

[0163] (3) Place the glass slide in a humidified box and incubate at 37°C for 1 hour.

[0164] 5. Testing

[0165] (1) Wash three times with PBS, 5 min each time.

[0166] (2) Add DAPI reagent to the slide, cover with a coverslip, and observe under a fluorescence microscope.

[0167] Tunel staining, by fluorescently labeling the 3'-OH exposed during genomic DNA breakage, detected apoptosis in hippocampal cells of mice in each group. The results are shown in Table 5, Figure 9, and Figure 10. As shown in Figure 10, under the microscope, red fluorescence (bright color) indicates positive expression, and blue fluorescence (dark color) indicates cell nuclei. The more and stronger the red fluorescence expression, the more severe the apoptosis; conversely, the degree of apoptosis is inhibited. The results showed that the intensity of Tunel positive expression in the granular layer cells of the hippocampus of the SAMP8 mouse model control group was higher than that of the normal control group SAMR1 mice, suggesting that there was significant apoptosis and necrosis in the neurons of the SAMP8 mouse brain. The tested drug FNC (0.5, 1 mg / kg) showed a trend of reducing apoptosis and necrosis in the neurons of the SAMP8 mouse brain, and the low dose of 0.5 mg / kg had a statistically significant difference in reducing neuronal apoptosis and necrosis (P < 0.01).

[0168] Table 5. Effects of continuous oral gavage administration of the test drug for 2 months on Tunel staining of hippocampal tissue in SAMP8 mice (Mean±SD, n=5) Note: **P < 0.01 vs. model control group.

[0169] Example 2: Effect of the test drug on THP-1NLRP3 inflammasome activation

[0170] 1. Experimental Principle:

[0171] After THP-1 induces differentiation into macrophages, LPS and ATP are used as the first and second signal stimuli, respectively, to activate NLRP3 and produce IL-1β cytokine, thereby causing IL-1β to be released into the extracellular space.

[0172] 2. Testing the efficacy of the test drug:

[0173] Following PMA-induced differentiation of THP-1 cells into macrophages, the NLRP3 inflammasome was stimulated by LPS / ATP. The test compound inhibited the production of IL-1β cytokine by activated NLRP3 inflammasomes, and the inhibitory effect was determined by detecting the amount of IL-1β released. MCC950 was used as a positive control compound.

[0174] 3. Experimental Methods and Materials

[0175] 3.1 Experimental Instruments and Reagents

[0176] Table 6 Experimental Instruments

[0177] Table 7 Reagents and Consumables

[0178] 3.2 Test Compound Information

[0179] Table 8 Compound Information Table

[0180] 3.3 Experimental Procedure

[0181] 3.3.1 Preparation of compound dilution solution

[0182] Dissolve 5.0 mg of MCC950 or 8.7 mg of FNC in DMSO, and add 410.5 μL (MCC950) and 759.9 μL (FNC) of DMSO respectively until completely dissolved. After shaking and mixing, the stock solutions prepared have a concentration of 30 mM for MCC950 and 40 mM for FNC.

[0183] FNC and MCC950 were diluted from 40 mM and 30 mM stock solutions to 10 mM and 1 mM, respectively. Then, they were serially diluted 4-fold with DMSO to: 9.766 nM, 2.441 nM, 0.610 nM, 0.153 nM, and 0.038 nM for FNC, and 1000 nM, 250 nM, 62.5 nM, 15.63 nM, 3.91 nM, 0.98 nM, 0.24 nM, 0.06 nM, and 0.02 nM for MCC950. For each concentration gradient, 4 μL was added to 196 μL of assay buffer (DMEM medium containing 1% FBS) and mixed to prepare intermediate dilutions. Then, for each concentration gradient, 30 μL of the intermediate dilution was added to 270 μL of assay buffer and mixed to prepare compound dilutions for later use.

[0184] 3.3.2 NLRP3 activation in THP-1 cells

[0185] THP-1 cells were passaged and cultured using 1640 complete medium (1640 + 10% FBS + 1% Penicilline / Streptomycin).

[0186] After subculture, THP-1 was induced to differentiate for 6 h with 1640 complete medium containing 400 ng / mL PMA.

[0187] After induced differentiation, THP-1 cells were collected into 15 mL centrifuge tubes and centrifuged at 1200 rpm for 5 minutes to remove the culture medium supernatant.

[0188] After centrifugation, discard the supernatant, resuspend the cell pellet in 1640 complete culture medium, count the cell density, and adjust the density to 5 × 10⁻⁶ cells / mL. 5 / mL; then the cells were transferred to 96-well plates at 100 μL / well, with a cell count of 5 × 10⁶ cells / well. 4 Each cell was placed in a 37°C, 5% CO2 cell culture incubator and cultured overnight.

[0189] After overnight culture, remove the culture medium and add 100 μL of assay buffer (1% FBS in DMEM medium) containing 500 ng / mL LPS, and incubate for 4 h.

[0190] Centrifuge for 4 hours to remove the culture medium; according to the layout diagram, add 80 μL of the compound dilution prepared in step 3.3.1 above to each well, add the control well to the assay buffer containing 0.1% DMSO, and continue incubation for 1 hour.

[0191] One hour later, 80 μL of assay buffer containing 20 mM ATP (the working concentration of ATP is 10 mM) was added to each well in the experimental group and the positive control group, and 80 μL of assay buffer without ATP was added to each well in the blank group. The mixture was then incubated in an incubator for 1 hour.

[0192] One hour later, the supernatant was collected by centrifugation and used to detect IL-1β and LDH.

[0193] 3.3.3 IL-1β ELISA Detection

[0194] 1) Add 100 μL of antibody dilution buffer to each well of a 96-well microplate. Seal the microplate and incubate it overnight at 4°C.

[0195] 2) After coating, wash the coated wells three times with washing buffer. After the last wash, invert the plate and blot dry on absorbent paper to remove any residual buffer.

[0196] 3) Block the microplate with 200 μL / well of sample dilution buffer, and then incubate at room temperature for 1 h.

[0197] 4) After the sealing is complete, wash the ELISA plate as in step 2).

[0198] 5) Add 100 μL of each concentration of standard and sample to each well of the ELISA plate and incubate at room temperature for 2 h.

[0199] 6) After incubation, wash the ELISA plate as in step 2).

[0200] 7) Add 100 μL of diluted detection antibody to each well of the ELISA plate and incubate at room temperature for 1 h.

[0201] 8) After incubation, wash the ELISA plate as in step 2).

[0202] 9) Add 100 μL of diluted HRP to each well of the microplate and incubate at room temperature in the dark for 0.5 h.

[0203] 10) After incubation, wash the ELISA plate 7 times as per step 2).

[0204] 11) Add 100 μL of TMB substrate to each well of the microplate, develop the color in the dark until the desired color is reached, then add 50 μL of stop solution to each well. Use a microplate reader with a wavelength of 450 nm to read the OD value.

[0205] 3.3.4 LDH detection in supernatant

[0206] Add 50 μL of sample or control supernatant to each well of a 96-well microplate.

[0207] Add 50 μL of CytoTox to each well of the above-mentioned microplate. LDH reagent, shake to mix; incubate at room temperature for 30 minutes in the dark.

[0208] After 30 minutes of incubation, 50 μL of stop solution was added to each well; then, the OD value at 490 nm wavelength was measured using a microplate reader.

[0209] 3.4 Data Analysis

[0210] IL-1β was detected using ELISA. ELISACalc software was used to perform 4-parameter curve fitting on the OD values ​​of the standards, and the relative IL-1β concentration was calculated based on the sample OD values. Statistical significance analysis was performed using a one-way ANOVA method.

[0211] 4. Experimental Results

[0212] As shown in Figure 11 and Table 9, LPS+ATP stimulation of differentiated THP-1 cells activated the NLRP3 inflammasome to produce IL-1β cytokine. The positive control compound MCC950 inhibited NLRP3 inflammasome-driven IL-1β release from THP-1 cells, indicating the successful establishment of the model group. Compared with the model group, FNC significantly inhibited NLRP3 inflammasome-driven IL-1β release from THP-1 cells within the range of 0.038 nM to 9.766 nM. As shown in Figure 12 and Table 10, neither compound exhibited significant cytotoxicity at any concentration.

[0213] Table 9. Inhibitory effects of compounds on IL-1β release from THP-1 cells.

[0214] Table 10. Cytotoxicity of compounds to THP-1 cells

Claims

1. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or isotopically labeled compounds thereof, in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases or autoimmune and systemic inflammatory diseases or metabolic and cardiovascular diseases or respiratory diseases or ophthalmic diseases or renal diseases, Optionally, the neurodegenerative diseases include Alzheimer's disease (AD), Lewy body dementia (LBD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), depression, and stroke; preferably Alzheimer's disease. Optionally, autoimmune and systemic inflammatory diseases, including gout / hyperuricemia, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), cryopyrin-associated periodic syndrome (CAPS), ankylosing spondylitis, psoriasis, and hidradenitis suppurativa; Optionally, metabolic and cardiovascular diseases, including type 2 diabetes and its complications, non-alcoholic steatohepatitis (NASH / MASH), obesity-related metabolic syndrome, atherosclerosis, and hypertension; Optionally, respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma; Optionally, ophthalmic diseases, including age-related macular degeneration (AMD) and diabetic retinopathy; Optionally, kidney diseases, including chronic kidney disease (CKD) / renal interstitial fibrosis, lupus nephritis; In formula (I), R 1 It can be: H, azide, C1-C6 alkyl (e.g., methyl, ethyl), C1-C6 alkoxy (e.g., methoxy, ethoxy), C2-C6 alkynyl (e.g., ethynyl), C2-C6 alkenyl (e.g., vinyl), or halo-C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl); R 2 is H, OH, halogen (e.g. F), Ci-C6alkyl (e.g. methyl, ethyl), or Ci-C6alkoxy (e.g. methoxy, ethoxy); B is selected from: wherein X1is -OH, -NH2, R 3 CONH-, R 3 COO- or R 3 O(C=O)NH-; X2is OH, SH, NH2, R 3 COO-, R 3 COS-, R 3 CONH2-, or R 3 O(C=O)NH-; X3 is H, F, OH or NH2; Y is either CH or N; Z is H, OH, or F; Each R 3 Each is independently selected from H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, isopropyl), C2-C6 ynyl (e.g., ethynyl), C2-C6 alkenyl (e.g., vinyl), halo-C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl), optionally C 1-6 Alkyl, C 1-6 Alkyl, CN, N3, OH, NH2, halogen-substituted (e.g., F, Cl, Br, I) phenyl groups, optionally C-substituted 1-6 Alkyl, C 1-6 Naphthyl groups substituted with alkoxy groups, CN, N3, OH, NH2, or halogens (e.g., F, Cl, Br, I).

2. The use according to claim 1, wherein in formula (I), R 1 Rais H, azido, or C2-C6alkynyl (e.g., ethynyl); Optionally, R 2 is H, OH, or halo (e.g., F). Optionally, B is selected from: wherein X1 is -OH or -NH2; X2 is OH or NH2; X3 is H, F, OH or NH2; Y is either CH or N; Z is H, OH, or F; Optionally, Z is F; Optionally, each R 3 each independently is selected from H, C1-C6alkyl (e.g., methyl, ethyl, propyl, isopropyl), haloC1-C6alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl).

3. The use according to claim 1, wherein the compound represented by formula (I) is one of the following compounds: or 4. The use according to any one of claims 1-3, wherein the isotope-labeled compound is a deuterated compound; Optionally, a pharmaceutically acceptable salt of the compound represented by formula (I) includes salts formed by the compound represented by formula (I) with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

5. The use according to any one of claims 1-4, wherein the dosage form of the drug is an immediate-release dosage form, a sustained-release dosage form, or a controlled-release dosage form; Optionally, the dosage form of the drug is tablets, hard capsules, soft capsules, aqueous or oily suspensions, granules, emulsions, syrups, elixirs, injections, or powder for injection.

6. Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isotopically-labeled compound thereof, in the manufacture of a medicament for inhibiting NLRP3 inflammasome activation; In formula (I), R 1 R is H, azido, C1-C6alkyl (e.g., methyl, ethyl), C1-C6alkoxy (e.g., methoxy, ethoxy), C2-C6alkynyl (e.g., ethynyl), C2-C6alkenyl (e.g., ethenyl), or haloC1-C6alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl); R 2 is H, OH, halogen (e.g. F), Ci-C6alkyl (e.g. methyl, ethyl), or Ci-C6alkoxy (e.g. methoxy, ethoxy); B is selected from: wherein X1is -OH, -NH2, R 3 CONH-, R 3 COO- or R 3 O(C=O)NH-; X2is OH, SH, NH2, R 3 COO-, R 3 COS-, R 3 CONH2-, or R 3 O(C=O)NH-; X3 is H, F, OH or NH2; Y is either CH or N; Z is H, OH, or F; Each R 3 Each is independently selected from H, C1-C6 alkyl (e.g., methyl, ethyl, propyl, isopropyl), C2-C6 ynyl (e.g., ethynyl), C2-C6 alkenyl (e.g., vinyl), halo-C1-C6 alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl), optionally C 1-6 Alkyl, C 1-6 Alkyl, CN, N3, OH, NH2, halogen-substituted (e.g., F, Cl, Br, I) phenyl groups, optionally C-substituted 1-6 Alkyl, C 1-6 Naphthyl groups substituted with alkoxy groups, CN, N3, OH, NH2, or halogens (e.g., F, Cl, Br, I).

7. The use according to claim 1, R 1 is H, azido, or C2-C6alkynyl (e.g., ethynyl); Optionally, R 2 is H, OH, or halogen (e.g., F); Optionally, B is selected from: wherein X1 is -OH or -NH2; X2 is OH or NH2; X3 is H, F, OH or NH2; Y is either CH or N; Z is H, OH, or F; Optionally, Z is F. Optionally, each R 3 each independently is selected from H, C1-C6alkyl (e.g., methyl, ethyl, propyl, isopropyl), haloC1-C6alkyl (e.g., 2-chloroethyl, 2-fluoroethyl, trifluoroethyl).

8. The use according to claim 1, wherein the compound represented by formula (I) is one of the following compounds: or 9. The use according to any one of claims 6-8, wherein the isotope-labeled compound is a deuterated compound; Optionally, a pharmaceutically acceptable salt of the compound represented by formula (I) includes salts formed by the compound represented by formula (I) with the following acids: hydrochloric acid, hydrobromic acid, aminosulfonic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, propionic acid, oxalic acid, glycolic acid, malonic acid, benzoic acid, lactic acid, gluconic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, mandelic acid, malic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, ascorbic acid palmitic acid, salicylic acid, sulfosalicylic acid, 2-hydroxy-3-naphthoic acid, phthalic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, or leucine.

10. The use according to any one of claims 6-9, wherein the dosage form of the drug is an immediate-release dosage form, a sustained-release dosage form, or a controlled-release dosage form; Optionally, the dosage form of the drug is tablets, hard capsules, soft capsules, aqueous or oily suspensions, granules, emulsions, syrups, elixirs, injections, or powder injections.

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