Galectin-3 inhibitor and therapeutic effect thereof on Alzheimer disease

Abstract

The invention provides novel application of the compound shown in the formula (I), and the compound shown in the formula (I) has excellent anti-inflammatory activity, can effectively inhibit Galectin-3 activity, reduce neuroinflammatory response, improve cognitive function, reduce excessive activation of microglial cells, reduce deposition of A beta and relieve synaptic degeneration, and is used for treating Alzheimer's disease.

Description

Technical Field

[0001] The present application belongs to the field of medicine, and specifically relates to a galectin-3 inhibitor, a preparation method thereof, and its therapeutic effect on Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD), a neurodegenerative disorder characterized by progressive cognitive decline, has become the most common type of dementia among the elderly worldwide. With the advent of an aging society, the incidence of AD has increased significantly, placing a heavy burden on patients, their families, and society. While a variety of therapeutic drugs are currently available, most only alleviate symptoms and lack effective treatments that address the root causes of the disease.

[0003] Galectins are a family of 15 proteins that share significant sequence similarity in their carbohydrate recognition domains and exhibit affinity for β-galactosidase. Galectin-3, as an endogenous ligand for triggering receptor expressed on myeloid cells, receptor 2 (TREM2), is a key regulator of microglial activity and neuroinflammation. The interaction between Galectin-3 and TREM2 plays a crucial role in regulating the inflammatory response during AD progression, particularly in modulating the effects of microglia on Aβ plaques. Upregulation of Galectin-3 is widely recognized as a hallmark of microglial activation; Galectin-3 can trigger microglial activation and promote the production and release of proinflammatory cytokines such as IL-6, IL-8, and TNF-α. Excessive activation of Galectin-3 may also lead to memory impairment.

[0004] Studies have shown that Galectin-3 can promote the aggregation and toxicity of Aβ after injection of Aβ into the hippocampus. Interestingly, aggregated Aβ can feedback stimulate the expression of Galectin-3, indicating that there is a mutually reinforcing relationship between Aβ and Galectin-3. The applicant's recent studies have shown that microglia respond to fibrillar Aβ stimulation with a surge in Galectin-3 expression. In APP / PS1 transgenic mice, Galectin-3 levels and endogenous Aβ oligomer levels increase with age. Studies have shown that Galectin-3 is an important molecule for microglial activation in 5×FAD mice. Knocking out Galectin-3 can attenuate Aβ-mediated inflammatory responses and improve cognitive behavior in 5×FAD mice.

[0005] Galectin-3 is increasingly being recognized as an important marker of aging and neuroinflammatory responses. Considering the importance of Galectin-3 in neurodegenerative diseases and its interaction with Aβ, interventions that regulate Galectin-3 may better treat or alleviate the pathological process of Aβ. In the applicant's previous research, the compound (E)-2-(3,4-dihydroxyphenylvinyl)-3-hydroxy-4H-pyran-4-one (D30) was synthesized and found to be able to improve scopolamine-induced cognitive deficits in mice, downregulate Galectin-3 expression, and alleviate neuroinflammation and cognitive impairment in the AD mouse model induced by fibrillar Aβ.

[0006] Therefore, there is an urgent need for more active compounds that have Galectin-3 inhibitory activity and can be used to treat Alzheimer's disease. Summary of the Invention

[0007] In order to improve the above technical problems, the present invention provides the use of the compound of formula (I), its stereoisomers, tautomers, isotope-labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, and prodrugs:

[0008]

[0009] wherein R1 is selected from H, C1-6 alkyl, C1-6 alkoxy, cyano, halogen, hydroxyl, and nitro;

[0010] R2 is selected from H, C1-6 alkyl, C1-6 alkoxy, cyano, halogen, hydroxyl, nitro;

[0011] m is selected from 0, 1, 2, 3, 4, 5, 6, 7;

[0012] n is selected from 0, 1, 2, 3, 4, 5;

[0013] The application is selected from any one, two or more of the following:

[0014] (i) Use in the preparation of a drug for inhibiting the release of TNF-α from BV-2 cells;

[0015] (ii) use in the preparation of anti-inflammatory drugs;

[0016] (iii) use in the preparation of a drug for inhibiting Galectin-3 activity, or in the preparation of a Galectin-3 inhibitor;

[0017] (iv) use in the preparation of a medicament for inhibiting the interaction between Galectin-3 and TREM2 (triggering receptor expressed on myeloid cells 2), or in the preparation of an inhibitor of the interaction between Galectin-3 and TREM2;

[0018] (v) Use in the preparation of drugs for inhibiting microglial activity;

[0019] (vi) use in the preparation of drugs for preventing and / or treating neuroinflammation (e.g., alleviating inflammatory responses by inhibiting proinflammatory factors and / or inflammatory signaling pathways (e.g., IL-1β, TLR4, and NF-κB));

[0020] (vii) use in the preparation of a medicament for preventing and / or treating Alzheimer's disease;

[0021] (viii) Application in the preparation of drugs for improving cognitive function, reducing excessive activation of microglia, reducing Aβ deposition, and alleviating synaptic degeneration.

[0022] According to an embodiment of the present invention, R1 is selected from H, C1-3 alkyl, C1-3 alkoxy, cyano, halogen, hydroxyl, nitro; preferably, R1 is selected from H, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, cyano, halogen, hydroxyl, nitro; illustratively, R1 is selected from H, halogen.

[0023] According to an embodiment of the present invention, R2 is selected from H, C1-3 alkyl, C1-3 alkoxy, cyano, halogen, hydroxyl, nitro; preferably, R2 is selected from H, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, cyano, halogen, hydroxyl, nitro; illustratively, R2 is selected from H, halogen, methoxy.

[0024] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following specific compounds:

[0025]

[0026] The present invention also provides a pharmaceutical composition comprising one, two or more of the compound represented by the above formula (I), its stereoisomers, tautomers, isotope-labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, and prodrugs.

[0027] According to an embodiment of the present invention, the pharmaceutical composition may further optionally comprise at least one pharmaceutically acceptable excipient. According to an embodiment of the present invention, pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, excipients, flocculants and deflocculating agents, filter aids, and release retardants.

[0028] According to an embodiment of the present invention, the pharmaceutical composition may optionally further comprise at least one additional active ingredient; specifically, the pharmaceutical composition may further comprise one or more active ingredients in addition to the above-mentioned compound, its stereoisomers, tautomers, isotopically labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, and prodrugs. In the pharmaceutical composition, the dosage of the above-mentioned compound, its pharmaceutically acceptable salts, solvates, polymorphs, metabolites, stereoisomers, tautomers, isotopically labeled substances, nitrogen oxides, esters, and prodrugs may be a therapeutically effective amount.

[0029] According to an embodiment of the present invention, the pharmaceutical composition of the present invention can be prepared into a dosage form suitable for administration by methods known in the art. According to an embodiment of the present invention, the preparation (or pharmaceutical composition) includes: an oral preparation and a parenteral preparation. According to an embodiment of the present invention, the preparation includes: a powder, a granule, a capsule, an injection, an inhalant, a tincture, an oral liquid, a tablet, a lozenge, or a pill.

[0030] According to an embodiment of the present invention, the pharmaceutical composition is used for:

[0031] (i) inhibiting the release of TNF-α from BV-2 cells; and / or,

[0032] (ii) anti-inflammatory; and / or,

[0033] (iii) inhibiting the activity of Galectin-3; and / or,

[0034] (iv) inhibiting the interaction between Galectin-3 and TREM2; and / or,

[0035] (v) inhibiting microglial activity; and / or,

[0036] (vi) preventing and / or treating neuroinflammation; and / or,

[0037] (vii) preventing and / or treating Alzheimer's disease; and / or,

[0038] (viii) Improve cognitive function, reduce excessive activation of microglia, reduce Aβ deposition, and alleviate synaptic degeneration.

[0039] The present invention also provides a method comprising administering to a subject a therapeutically effective amount of one, two or more of the compound represented by formula (I), its stereoisomers, tautomers, isotope-labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, prodrugs, or the pharmaceutical compositions thereof;

[0040] The method is selected from any one, two or more of the following:

[0041] (i) inhibiting the release of TNF-α from BV-2 cells;

[0042] (ii) anti-inflammatory;

[0043] (iii) inhibiting the activity of Galectin-3;

[0044] (iv) inhibiting the interaction between Galectin-3 and TREM2;

[0045] (v) inhibiting the activity of microglia;

[0046] (vi) preventing and / or treating neuroinflammation;

[0047] (vii) prevention and / or treatment of Alzheimer's disease;

[0048] (viii) Improve cognitive function, reduce excessive activation of microglia, reduce Aβ deposition, and alleviate synaptic degeneration.

[0049] The present invention also provides a method for preparing the compound represented by the above formula (I), which comprises the following steps:

[0050] (1) Compound SM1 reacts with thionyl chloride and methanol to obtain compound 2A;

[0051] (2) Compound 2A reacts with a compound of formula (I-1) to obtain a compound of formula (I-2);

[0052] (3) hydrolyzing the compound of formula (I-2) to obtain a compound of formula (I-3);

[0053] (4) reacting the compound of formula (I-3) with the compound of formula (I-4) to obtain the compound of formula (I);

[0054] The reaction formula is as follows:

[0055]

[0056] According to an embodiment of the present invention, in step (1), the reaction temperature is 60°C-90°C, for example, 70°C. According to an embodiment of the present invention, in step (1), the molar ratio of compound SM1 to thionyl chloride is 1:1-2, for example, 1:1.2. According to an embodiment of the present invention, in step (1), the mass volume ratio of compound SM1 to methanol is 5-10g:10mL, for example, 8g:10mL. According to an embodiment of the present invention, after the reaction of step (1) is completed, the reaction solution is concentrated and dried, dissolved with dichloromethane, washed with sodium bicarbonate aqueous solution, separated, and purified by column chromatography to obtain compound 2A. Preferably, after adjusting the pH of the aqueous phase to 7-8, separation is performed. Preferably, the eluting reagent for column chromatography is petroleum ether / ethyl acetate (10:1 to 1:1).

[0057] According to an embodiment of the present invention, in step (2), the reaction is carried out in the presence of a base; preferably, the base is potassium carbonate or sodium hydroxide. According to an embodiment of the present invention, in step (2), the reaction is carried out in a solvent; preferably, the solvent is an organic solvent, such as acetonitrile. According to an embodiment of the present invention, in step (2), the molar ratio of compound 2A to the compound of formula (I-1) is 1:1-2, for example, 1:1.5. According to an embodiment of the present invention, in step (2), the molar ratio of compound 2A to the base is 1:1-3, for example, 1:2. According to an embodiment of the present invention, in step (2), the mass volume ratio of compound 2A to the solvent is 5-10:10, for example, 8:10 (g:mL). According to an embodiment of the present invention, in step (2), the reaction temperature is 10°C-35°C, for example, 25°C. According to an embodiment of the present invention, after the reaction of step (2) is completed, the reaction solution is filtered, the filter cake is washed with a solvent, concentrated and dried, and purified by column chromatography to obtain a compound of formula (I-2). Preferably, the elution reagent for column chromatography is petroleum ether / ethyl acetate (10:1 to 1:1).

[0058] According to an embodiment of the present invention, in step (3), the reaction is carried out in the presence of a base; preferably, the base is sodium hydroxide or potassium carbonate. According to an embodiment of the present invention, in step (3), the reaction is carried out in a solvent; preferably, the solvent is a mixed solvent of water and an organic solvent, such as a mixed solvent of water and tetrahydrofuran (volume ratio, for example, 1:3). According to an embodiment of the present invention, in step (3), the molar ratio of the compound of formula (I-2) to the base is 1:1-3, for example, 1:1.2. According to an embodiment of the present invention, in step (3), the mass volume ratio of the compound of formula (I-2) to the solvent is 3-10:10, for example, 7:10 (g:mL). According to an embodiment of the present invention, in step (3), the reaction temperature is 10°C-35°C, for example, 25°C. According to an embodiment of the present invention, after the reaction of step (3) is completed, the reaction solution is concentrated and dried, and then the pH is adjusted to 7-8 with hydrochloric acid, stirred, filtered, and dried to obtain a compound of formula (I-3).

[0059] According to an embodiment of the present invention, in step (4), the reaction is carried out in the presence of a base; preferably, the base is N,N-diisopropylethylamine. According to an embodiment of the present invention, in step (4), the reaction is carried out in a solvent; preferably, the solvent is dichloromethane. According to an embodiment of the present invention, in step (4), the reaction is carried out in the presence of a condensation reagent; preferably, the condensation reagent is selected from HATU (O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate). According to an embodiment of the present invention, in step (4), the molar ratio of the compound of formula (I-3) to the compound of formula (I-4) is 1:1-2, for example, 1:1.2. According to an embodiment of the present invention, in step (4), the molar ratio of the compound of formula (I-3) to the base is 1:1-5, for example, 1:3. According to an embodiment of the present invention, in step (4), the molar ratio of the compound of formula (I-3) to the condensation reagent is 1:1-3, for example, 1:1.2. According to an embodiment of the present invention, in step (4), the mass volume ratio of the compound of formula (I-3) to the solvent is 3-8:20, for example 6:20 (g:mL). According to an embodiment of the present invention, in step (4), the reaction temperature is 10°C-35°C, for example 25°C. According to an embodiment of the present invention, after the reaction in step (4) is completed, the reaction solution is washed with sodium hydrogen hydrochloride aqueous solution and water in sequence, concentrated and dried; then slurried with ethyl acetate, filtered and dried to obtain the compound shown in formula (I).

[0060] Beneficial effects

[0061] The present invention provides a new application of the compound represented by formula (I). The compound represented by formula (I) has excellent anti-inflammatory activity, can effectively inhibit Galectin-3 activity, reduce neuroinflammatory response, improve cognitive function, reduce excessive activation of microglia, reduce Aβ deposition, alleviate synaptic degeneration, and is used to treat Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 : Evaluation of the effect of compound 1887 on Galectin-3. (A) Schematic diagram of the FRET experimental design for Galectin-3 and TREM2. (B) Interference of 1887 on the interaction between Galectin-3 and TREM2. (C) Affinity measurement between 1887 and Galectin-3. (D) Fluorescence staining confirms that 1887 inhibits the levels of Galectin-3 and TREM2 in the hippocampus of 5xFAD mice.

[0063] Figure 2 : Molecular docking diagram of compound 1887 and Galectin-3 (PDB:8ITZ).

[0064] Figure 3 In vivo safety evaluation of compound 1887.

[0065] Figure 4 Effects of compound 1887 on cognitive function and galectin-3 content in 5×FAD mice. (A) Experimental design: 14-month-old C57 mice and 5×FAD mice were randomly divided into two groups: an experimental group (treated with compound 1887) and a control group (treated with an equal volume of solvent). All mice were treated for 4 weeks. (B) Schematic diagram of the Y-maze: Shows the layout of the Y-maze test, used to assess working memory and spatial memory in mice. (C) Discrimination index: Shows the discrimination index of 5×FAD mice in the Y-maze test, comparing cognitive function between the control group and the 1887-treated group. (D, E) Typical Y-maze tracks: Shows typical movement tracks of 5×FAD mice in the Y-maze test for the control group (D) and the 1887-treated group (E). (F) Immunoblotting analysis: Immunoblotting was used to examine the expression levels of galectin-3 (Gal-3) and IBA1 proteins in the brain tissue of 5×FAD mice. (G) Quantitative analysis of Gal-3 is shown. (H) Quantitative analysis of IBA1. (I) Immunofluorescence staining: After 1887 treatment, 14-month-old 5×FAD mice showed decreased expression of Galectin-3 and IBA1 in the hippocampus.

[0066] Figure 5Effects of the 1887 compound on neuroinflammation-related molecules in the hippocampus of 14-month-old 5×FAD mice. (A) Immunofluorescence staining observation: After 1887 treatment, the expression levels of IBA1 and CD68 in the hippocampus of 14-month-old 5×FAD mice were significantly reduced, suggesting that 1887 can alleviate neuroinflammatory responses in mice. (B) Immunofluorescence staining observation: After 1887 treatment, the expression of C1q and iNOS in the hippocampus of 5×FAD mice was also significantly reduced, further indicating that 1887 has a regulatory effect on the mouse immune system and may improve cognitive function by alleviating inflammatory responses. (C) Immunoblotting analysis: The results of the immunoblotting experiment showed that compared with the control group, the expression of IL-1β and p-NF-κB in the hippocampus of 5×FAD mice in the 1887 treatment group was significantly reduced, suggesting that 1887 may alleviate neuroinflammatory responses by inhibiting the expression of these inflammatory factors. (D) Quantitative analysis of IL-1β: Quantitative analysis showed that IL-1β protein expression in the hippocampus of 5×FAD mice in the 1887 treatment group was significantly lower than that in the control group. (E) Quantitative analysis of TLR4: TLR4 expression in the hippocampus of the 1887 treatment group was significantly decreased, suggesting that 1887 may exert its anti-inflammatory effects by inhibiting TLR4 activation. (F) Quantitative analysis of p-NF-κB in the hippocampus: Quantitative analysis of p-NF-κB in the 1887 treatment group showed that p-NF-κB expression was significantly decreased, suggesting that 1887 exerts its anti-inflammatory effects by inhibiting the activation of the NF-κB signaling pathway. (G) Quantitative analysis of the p-NF-κB / NF-κB ratio: The ratio of p-NF-κB to NF-κB in the hippocampus of mice in the 1887 treatment group was significantly lower than that in the control group, further supporting its role in alleviating inflammatory responses by regulating the NF-κB pathway.

[0067] Figure 6 Effects of compound 1887 on microglial activation and Aβ loading in the hippocampus of 14-month-old 5×FAD mice. (A) Immunofluorescence staining: Immunofluorescence staining was performed on the hippocampus of 14-month-old C57 and 5×FAD mice following 1887 treatment. The figure shows the expression of the microglial marker IBA1 (purple) and the Aβ marker 6E10 (red). Compared with untreated 5×FAD mice, the fluorescent signals of IBA1 and 6E10 were significantly reduced in 5×FAD mice treated with compound 1887, indicating that 1887 can inhibit excessive microglial activation and reduce Aβ deposition. DAPI (blue) was used to label cell nuclei.

[0068] Figure 7: Effects of compound 1887 on the synaptic markers PSD95 and Syn in the hippocampus of 14-month-old 5×FAD mice. (A) Immunofluorescence staining observation: After 1887 treatment, the expression level of PSD95 (red) in the hippocampus of 14-month-old 5×FAD mice was significantly increased, and DAPI (blue) was used to label cell nuclei. Compared with the control group, the PSD95 signal in the 1887-treated group was significantly enhanced, indicating that 1887 may combat Alzheimer's disease by improving synaptic function. (B) Immunofluorescence staining observation (Thy1-EGFP mice): In 14-month-old 5×FAD mice labeled with Thy1-EGFP, the expression of PSD95 (red) was significantly increased after 1887 treatment, and green fluorescence indicates neurons. The merged image (Merge) shows that the expression of the synaptic marker PSD95 in neurons was significantly enhanced after 1887 treatment. (C) Immunoblotting analysis: Immunoblotting results showed that the expression levels of synaptic markers Syn and PSD95 in the hippocampus of 5×FAD mice were increased in the 1887-treated group compared to the control group, suggesting that 1887 has a potential role in improving synaptic function. (D) Syn quantitative analysis: Quantitative analysis of Syn protein expression by immunoblotting showed that Syn expression was significantly higher in the 1887-treated group than in the control group. (E) PSD95 quantitative analysis: Quantitative analysis of PSD95 protein expression by immunoblotting showed that PSD95 expression was significantly higher in the 1887-treated group than in the control group. DETAILED DESCRIPTION

[0069] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0070] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0071] Example 1 Synthesis of Compound 1887

[0072]

[0073] (1) Synthesis of compound 2A: SM1 (8.0 g) and methanol (10 mL) were placed in a 250 mL three-necked flask, thionyl chloride (1.2 equivalents of SM1) was added dropwise, and then heated to 70°C and reacted at 70°C for 1 hour, monitored by TLC. After the reaction, the temperature was lowered to 20°C and concentrated to dryness. After dissolving in dichloromethane (20 mL), sodium bicarbonate aqueous solution (adjusted to pH 7-8) was added for separation. The organic phase after separation was purified by column chromatography using petroleum ether / ethyl acetate (10:1 to 1:1) for elution to obtain 7 g of a white solid with a yield of 81%.

[0074] (2) Synthesis of Compound 2B: Compound 2A (7.0 g), potassium carbonate (2 equivalents), and acetonitrile (10 volumes) were placed in a 250 mL three-necked flask. SM2 (1.5 equivalents) was added dropwise and stirred at 25°C for 1 hour. The reaction was monitored by TLC. After completion of the reaction, the reaction solution was filtered and the filter cake was rinsed with acetonitrile (5 volumes), concentrated to dryness, and purified by column chromatography using petroleum ether / ethyl acetate (10:1 to 1:1) as the eluent to obtain 9 g of a white solid (yield: 77%).

[0075] (3) Synthesis of Compound 2C: Compound 2B (9.0 g), water (5 volumes), tetrahydrofuran (15 volumes), and sodium hydroxide (1.2 equivalents) were placed in a 250 ml three-necked flask and stirred overnight at 25°C with TLC monitoring. After the reaction, the THF was concentrated until no fraction was distilled off. The pH was then adjusted to 7-8 with hydrochloric acid. The mixture was stirred for 1 hour, filtered, and dried to obtain 8.1 g of a white solid (yield: 95%).

[0076] (4) Synthesis of compound 1887: Compound 2C (6.0 g), SM3 (1.2 equivalents), N,N-diisopropylethylamine (3.0 equivalents) and dichloromethane (20 volumes) were placed in a 250 ml three-necked flask. O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (1.2 equivalents) was then added to the three-necked flask and stirred at 25°C for 5 hours. TLC monitoring was performed. After the reaction was completed, the organic phase was washed twice with sodium bicarbonate aqueous solution (3 volumes) and once with water (3 volumes), and then concentrated to dryness. The mixture was slurried with ethyl acetate (3 volumes) for 1 hour, filtered and dried to obtain 6.1 g of a white solid with a yield of 70%. The characterization data of compound 1887 (N-(2-([1,2,4]Triazol-3-yl)-4-methoxyphenyl)naphthalen-1-carboxamide) are as follows: 1H NMR(600MHz,DMSO-d6)δ10.61(s,1H),8.50(s,1H),8.42-8.41(m,1H),8.19-8.16(m,1H),7.98-7.61(m,1H),7.90-7.89(m,1H),7.85- 7.84(m,2H),7.81-7.79(m,1H),7.48-7.45(m,1H),7.43-7.40(m,1H),7.35-7.33(m,2H),6.91-6.88(m,2H),5.96(s,2H),3.68(s,3H). 13 C NMR(101MHz,DMSO-d6)δ165.82,159.58,146.99,137.05,134.62,133.76,132.83,130.57,129.87,128.72,128 .09,127.96,126.94,125.41,123.88,121.45,119.67,117.27,114.67,111.58,55.56,51.19.HRMS(ESI+)calcd for C 25 H 20 N4O2Na[M+Na] + :431.1484; found:431.1461. HPLC detection showed that the purity of compound 1 was 98.08%.

[0077] Study on the anti-inflammatory activity of the compound of Example 2

[0078] 1. Experimental Materials

[0079] Compound 1887.

[0080] 2. Experimental Methods

[0081] In this experiment, mouse microglial cells (BV-2) were used as a model. The MTT assay was first used to test whether the compound was cytotoxic to BV-2 cells. The interference of the compound on cell growth activity was eliminated. The drug concentration at which the cell viability was >90% was taken. The NO content in the culture medium was determined by the Griess assay, and the interleukin-6 and tumor necrosis factor levels in the culture medium were determined by the ELISA assay.

[0082] (1) MTT assay to detect the effects of compounds on BV-2 cell viability

[0083] 100 μL of BV-2 cell suspension (cell density of 5×10 3Each well was incubated at 37°C, 5% CO2 for 24 hours before administration. Three replicate wells were set for the background wells, test groups, and blank groups. Sample solutions of varying concentrations were added to the test groups (DMEM medium was added to the blank group). After a further 24 hours of culture, 20 μL of MTT solution (5 mg / mL) was added to each well. After incubation in an incubator for 4 hours, crystals formed. The supernatant was aspirated, and 150 μL of DMSO was added to each well. The cells were shaken at low speed on a shaker for 15 minutes to fully dissolve the crystals. The absorbance of each well was measured at 490 nm on a microplate reader, and the cell viability was calculated as follows.

[0084] Cell viability = (OD s -OD r ) / (OD0-OD r )×100%

[0085] OD s : absorbance of sample group; OD r : absorbance of the background group; OD0: absorbance of the blank group.

[0086] (2) Griess assay to detect the effects of compounds on LPS-induced NO release in BV-2 cells

[0087] 100 μL of BV-2 cell suspension (cell density of 5×10 4 Cells were cultured for 24 hours. LPS (1 μg / mL) was added for 1 hour before administration. After another 24 hours of culture, 50 μL of the cell supernatant was transferred to another 96-well plate. 50 μL of Griess A and B reagents were added. The plates were incubated in the dark for 10 minutes. The OD value was measured at 540 nm. The NO concentration was calculated using a standard curve prepared using NaNO2.

[0088] The experiment was set up with a blank group (addition of DMEM culture medium, without LPS stimulation), an LPS model group (addition of DMEM culture medium, with LPS stimulation), and a test group (drug, with LPS stimulation), each with 3 replicates, and 3 independent experiments.

[0089] (3) ELISA assay to detect the effect of compounds on LPS-induced tumor necrosis factor release in BV-2 cells

[0090] The release of interleukin-6 and tumor necrosis factor from BV-2 cells induced by LPS at different concentrations was determined according to the experimental steps of the Thermo Fisher tumor necrosis factor detection kit, and the IC values were calculated using GraphPad Prism8 software. 50 .

[0091] The experiment was set up with a blank group (addition of DMEM culture medium, without LPS stimulation), an LPS model group (addition of DMEM culture medium, with LPS stimulation), and a test group (drug, with LPS stimulation), each with 3 replicates, and 3 independent experiments.

[0092] 3. Experimental Results

[0093] (1) MTT assay to detect the effects of compounds on BV-2 cell viability

[0094] The MTT assay determined that when the concentration of compound 1887 was 20 μM, the cell survival rate was greater than 90%, so the next step of NO determination was performed under this condition.

[0095] (2) Griess assay to detect the effects of compounds on LPS-induced NO release in BV-2 cells

[0096] The experimental results showed that compound 1887 inhibited NO production with IC 50 The value was 10.95±3.05 μM, indicating that compound 1887 had excellent activity in inhibiting the release of NO from BV-2 cells.

[0097] (3) ELISA assay for the effects of compounds on LPS-induced TNF-α in BV-2 cells

[0098] The experimental results showed that compound 1887 inhibited the production of TNF-α with an IC 50 The value was 4.36±0.96 μM, indicating that compound 1887 had excellent performance in inhibiting the release of tumor necrosis factor from BV-2 cells.

[0099] Example 3: Test of compound 1887 inhibiting Galectin-3

[0100] 1. Experimental Materials

[0101] Compound 1887.

[0102] 2. Experimental Methods

[0103] Fluorescence resonance energy transfer (FRET) assay was used to evaluate the inhibitory effect of compound 1887 on Galectin-3.

[0104] Using the pEGFP-N1 plasmid backbone, the green fluorescent protein (GFP) tag was first changed to mGold (yellow) and moxCerulean3 (moxc, blue) fluorescent protein tags, respectively. Fluorescent fusion protein plasmids expressing mGold-human soluble TREM2 (17-174aa) and moxc-human Gal-3 were then constructed. These plasmids were co-transfected into HEK293t cells, and the proteins expressed in the cells interacted with each other. The mGold-fused TREM2 served as the receptor for the FRET experiment, and the moxc-fused Galectin-3 served as the donor for the FRET experiment. FRET imaging was performed using a laser confocal microscope. The above cells were treated with compound 1887, which inhibited the interaction between Galectin-3 and TREM2, resulting in a decrease in the FRET signal.

[0105] 3. Experimental Results

[0106] like Figure 1 A, Schematic diagram of the FRET mechanism constructed to evaluate the interaction between mGOLD-TREM2 and moxc-Galectin-3. The presence of FRET indicates that the distance between Galectin-3 and TREM2 is very close (<10nm), indicating that the two are bound (FRET on). Conversely, when Galectin-3 is bound by other molecules, the binding of Galectin-3 to TREM2 is disrupted, and the distance between mGOLD-TREM2 and moxc-Galectin-3 increases (>10nm), resulting in a loss of FRET (FRET off).

[0107] Fluorescence imaging FRET analysis evaluated the interaction between moxc-Galectin-3 (blue) and mGOLD-TREM2 (green) under control (Ctrl) and compound 1887 treatment conditions. The FRET signal (red) represents the interaction between Galectin-3 and TREM2. Compared with the control group, the FRET signal was weakened after compound 1887 treatment, indicating that the interaction between Galectin-3 and TREM2 was disrupted by compound 1887. Figure 1 B.

[0108] Example 4: Affinity test of compound 1887 and Galectin-3

[0109] 1. Experimental Materials

[0110] Compound 1887.

[0111] 2. Experimental Methods

[0112] An SA biosensor chip (Streptavidin biosensor chip) was used and initialized on the Octet system. Biotinylated Galectin-3 (100 μg / mL, PBS buffer) was immobilized on the sensor surface to ensure uniform coverage and stable binding. Compound 1887 was diluted to different concentrations (0.1 μM, 1 μM, 10 μM, 25 μM, 50 μM, 100 μM) and added to the reaction wells sequentially. Each concentration was tested in a volume of 200 μL. PBS (pH 7.4) was used as the reaction buffer.

[0113] The experiment was conducted at 37°C and divided into two phases: association and dissociation. During the association phase, the sensor probe was immersed in the compound solution for 300 seconds to measure the binding response; during the dissociation phase, the sensor was transferred to PBS buffer for an additional 300 seconds to monitor the reversibility of the binding.

[0114] 3. Experimental Results

[0115] The binding affinity of compound 1887 to Galectin-3 was determined using the Octet system. Steady-state response values were analyzed using GraphPad Prism, and nonlinear regression analysis was performed using the one-site specific binding model to calculate the dissociation constant (Kd). Figure 1 C, The Kd value of compound 1887 is 7.734 μM, indicating that it has moderate affinity for Galectin-3, suggesting its potential application value in the treatment of Galectin-3-related pathological conditions.

[0116] Example 5: Inhibitory effect of compound 1887 on Galectin-3 and TREM2 in the hippocampus of 5×FAD mice

[0117] 1. Experimental Materials

[0118] Compound 1887.

[0119] 2. Experimental Methods

[0120] Animal Model: 14-month-old C57 mice and 5×FAD mice were divided into three groups: a C57 control group, a 5×FAD control group, and a 5×FAD experimental group (1887 compound-treated group). The experimental and control groups were treated for four weeks. The experimental group mice were given 20 mg / kg of 1887 compound daily, while the control group mice were given the same volume of solvent.

[0121] Sample collection: After the experiment, mouse hippocampal tissue samples were taken and fixed.

[0122] Tissue Sectioning and Staining: Hippocampal tissue was fixed with 4% paraformaldehyde. Tissue sections were cut at a thickness of 20 μm and incubated with specific primary antibodies against Galectin-3 and TREM2, followed by treatment with corresponding fluorescently labeled secondary antibodies. Cell nuclei were stained with DAPI.

[0123] Fluorescence microscopy: Observe the staining results using a fluorescence microscope, capturing fluorescence images of Galectin-3 and TREM2. Analyze the staining intensity based on the images to assess Galectin-3 and TREM2 expression levels.

[0124] 3. Experimental Results

[0125] Compound 1887 significantly inhibited the expression levels of Galectin-3 and TREM2 in the hippocampus of 5×FAD mice, and the IBA1 staining intensity of mice in the experimental group changed compared with the control group. Based on these experimental results, it can be speculated that compound 1887 may inhibit the expression of Galectin-3 and TREM2 in 5×FAD mice, thereby affecting the activity of microglia and neuroinflammatory response, thereby having a positive effect on the treatment of Alzheimer's disease. Figure 1 D.

[0126] Example 6: Molecular docking of compound 1887 and Galectin-3

[0127] 1. Experimental Materials

[0128] Compound 1887.

[0129] Galectin-3 crystal structure (PDB ID: 8ITZ).

[0130] 2. Experimental Methods

[0131] use The Glide module within the software suite performed molecular docking on compound 1887 and Galectin-3 (PDB ID: 8ITZ). First, the crystal structure of Galectin-3 was obtained from the PDB database and preprocessed using Maestro software, including removal of water molecules, addition of hydrogen atoms, and optimization of the protonation state. The three-dimensional structure of compound 1887 was constructed using ChemDraw and optimized using the LigPrep module. In Glide, Galectin-3 was used as the receptor, and the binding pocket was defined as the sugar-binding region in the Galectin-3 crystal structure. Docking parameters, including grid size and center coordinates, were set. After running the docking calculation, the docking conformation with the lowest binding energy was selected for further analysis.

[0132] 3. Experimental Results

[0133] The molecular docking results showed that ( Figure 2 Compound 1887 is located within the carbohydrate-binding pocket of galectin-3. Compound 1887 forms hydrogen bonds and π-π interactions with several key amino acid residues of galectin-3, such as ARG-144, HIS-158, and TRP-181. These interactions are crucial for the binding affinity of compound 1887, suggesting its potential application in inhibiting galectin-3 function.

[0134] Example 7: Safety Evaluation of Compound 1887

[0135] Compound 1887 was administered orally to mice at a single dose of 500 mg / kg. Fourteen days after administration, the animals were sacrificed and macroscopic examinations of the heart, brain, liver, and kidneys were performed. The tissues were then defatted and stained with hematoxylin, and images were collected under a Nikon Eclipse E10 microscope.

[0136] The experimental results showed that compound 1887 had good safety in animal models at a single dose of 500 mg / kg. No significant morphological changes were observed in the heart, brain, liver, and kidney tissues, indicating that compound 1887 did not cause significant acute toxicity in these organs ( Figure 3 ).

[0137] Example 8: Effects of Compound 1887 on Cognitive Function and Gal-3 Content in 5×FAD Mice

[0138] 1. Experimental Materials

[0139] 14-month-old C57BL / 6J mice and 5×FAD transgenic mice (5×FAD mice, purchased from Jax).

[0140] Compound 1887 was dissolved in DMSO and diluted with an aqueous solution of 0.5% hydroxypropyl methylcellulose (HPMC) + 1% polysorbate, and then administered orally at 20 mg / kg.

[0141] Antibodies: Galectin-3 (Gal-3) antibody, IBA1 antibody, TREM2 antibody.

[0142] 2. Experimental Methods

[0143] Experimental Design: All mice were randomly divided into three groups: 14-month-old C57 mice, a 14-month-old 5×FAD control group, and a 5×FAD 1887-treated group. The experimental groups were gavaged daily with 1887 (20 mg / kg), while the control group received an equal volume of solvent. Treatment lasted for four weeks.

[0144] The Y-maze test is used to assess working memory and spatial memory in mice. Each mouse was tested in the Y-maze before and after the experiment. During the test, the mouse explored the three arms of the Y-maze, measuring its preference for exploring the novel arm versus the previously explored arm. The discrimination index was calculated to assess memory function.

[0145] Immunoblotting Analysis: After the experiment, hippocampal tissues were removed from the mice, and proteins were extracted using RIPA lysis buffer and quantified using BCA. Galectin-3 (Gal-3) and IBA1 protein expression in hippocampal tissues was assessed by immunoblotting. Gal-3 and IBA1 were incubated with corresponding antibodies, and signals were detected by ECL luminescence, and quantitatively analyzed using ImageJ software.

[0146] Immunofluorescence staining: Mouse brain tissue was sectioned and immunostained with Galectin-3, anti-TREM2, and IBA1. Corresponding secondary antibodies were used for fluorescent labeling, and the staining results of the hippocampus of each group of mice were observed under a fluorescence microscope.

[0147] 3. Experimental Results

[0148] The Y-maze test showed that the discrimination index of the 5×FAD control group was significantly lower than that of the C57 group, indicating a significant decline in cognitive function in 5×FAD mice. After four weeks of treatment with compound 1887, the discrimination index of the 5×FAD group significantly improved, approaching that of the C57 group, suggesting that 1887 can improve cognitive function in 5×FAD mice.

[0149] Immunoblotting results showed that Galectin-3 expression was significantly increased in 5×FAD mice, as was IBA1 expression, indicating a significant neuroinflammatory response in the hippocampus of 5×FAD mice. Following 1887 treatment, the expression levels of both Galectin-3 and IBA1 in the hippocampus of 5×FAD mice were significantly reduced, indicating that 1887 intervention effectively inhibited Galectin-3-mediated neuroinflammatory responses.

[0150] Immunofluorescence staining results showed that the expression of Galectin-3 and IBA1 was high in the 5xFAD mouse control group, while the expression of Galectin-3 and IBA1 was significantly reduced in the 5xFAD mice treated with 1887. In particular, the 1887-treated group showed significantly reduced Galectin-3 and IBA1 fluorescence signals in the hippocampus, suggesting that 1887 can effectively inhibit the expression of Galectin-3 and IBA1, thereby reducing neuroinflammation ( Figure 4 ).

[0151] Example 9: Effects of Compound 1887 on Neuroinflammation-Related Molecules in the Hippocampus of 14-Month-Old 5×FAD Mice

[0152] 1. Experimental Materials

[0153] Animals: Same as Example 8.

[0154] Reagents: Compound 1887, other biochemical reagents and antibodies (IBA1, CD68, C1q, iNOS, IL-1β, TLR4 antibodies, NF-κB and p-NF-κB).

[0155] 2. Experimental Methods

[0156] Grouping and processing: same as Example 8.

[0157] Immunofluorescence staining: Mouse hippocampal tissue was fixed with 4% paraformaldehyde, sectioned, and then immunostained using antibodies against IBA1, CD68, C1q, and iNOS to analyze the expression and distribution of neuroinflammation-related molecules. Observation was performed under a confocal microscope.

[0158] Immunoblotting analysis: Mouse hippocampal tissue was lysed and subjected to SDS-PAGE electrophoresis. After transfer to a membrane, immunoblotting was performed using antibodies against IL-1β, NF-κB, and p-NF-κB. Protein expression was quantitatively analyzed using image analysis software.

[0159] Immunoblotting analysis: Total protein was extracted from mouse hippocampal tissue and quantified using the BCA assay, followed by SDS-PAGE electrophoresis. After transfer to a membrane, immunoblotting was performed using PSD95 and Syn antibodies, respectively, and protein signals were detected using ECL luminescence. Grayscale values of protein bands were quantified using ImageJ software, and the expression levels of IL-1β, p-NF-κB, NF-κB, and TLR4 were compared among the groups.

[0160] 3. Experimental Results

[0161] Immunofluorescence staining: Immunofluorescence staining results showed that the expression levels of IBA1 and CD68 in the hippocampus of mice treated with compound 1887 were significantly lower than those in the control group ( Figure 5 A), the expression of C1q and iNOS was also significantly decreased ( Figure 5 B), suggesting that 1887 may exert its effects by alleviating neuroinflammatory responses.

[0162] Immunoblotting analysis: Immunoblotting results showed that the expression of IL-1β in the hippocampus of 5×FAD mice in the compound 1887 treatment group was significantly reduced ( Figure 5 CD), which indicates that compound 1887 alleviates neuroinflammatory response by reducing the expression of proinflammatory factor IL-1β. In addition, quantitative analysis of TLR4 ( Figure 5 E) showed that the expression of TLR4 in the hippocampus of the compound 1887-treated group was significantly reduced, suggesting that compound 1887 may exert its anti-inflammatory effect by inhibiting the activation of TLR4. The expression of p-NF-κB and its ratio to NF-κB were significantly reduced in the compound 1887-treated group ( Figure 5 FG), indicating that compound 1887 may alleviate inflammatory response by regulating the NF-κB signaling pathway.

[0163] Therefore, compound 1887 significantly reduced the expression of neuroinflammation-related molecules in the hippocampus of 14-month-old 5×FAD mice, suggesting that it may alleviate neuroinflammatory responses by inhibiting proinflammatory factors and inflammatory signaling pathways (such as IL-1β, TLR4, and NF-κB). These results support further research on compound 1887 as a potential candidate for the treatment of Alzheimer's disease.

[0164] Example 10: Effects of compound 1887 on microglial activation and Aβ load in the hippocampus of 14-month-old 5×FAD mice.

[0165] 1. Experimental Materials

[0166] Animals: Same as Example 8

[0167] Reagents: Compound 1887, Antibodies: IBA1 antibody, 6E10 antibody, DAPI.

[0168] 2. Experimental Methods

[0169] Grouping and processing: same as Example 8.

[0170] Immunofluorescence staining: After treatment, mice were anesthetized and hippocampal tissue was removed and fixed with 4% paraformaldehyde for 24 hours. The fixed tissue was sectioned at 20 μm thickness and incubated with IBA1 and 6E10 antibodies, respectively, to label microglia and Aβ plaques. DAPI was used to label cell nuclei. Fluorescence images were captured using a confocal microscope.

[0171] 3. Experimental Results

[0172] Microglial activation: Immunofluorescence results ( Figure 6 A) shows that compared with the control 5×FAD mice, the fluorescence signal of IBA1 in the hippocampus of 5×FAD mice treated with compound 1887 was significantly reduced, indicating that compound 1887 can effectively inhibit the excessive activation of microglia. This suggests that compound 1887 has potential anti-inflammatory effects in Alzheimer's disease models, reducing neuroinflammation by reducing microglial activation.

[0173] Aβ Load (6E10): Immunofluorescence staining results also showed that the 6E10 fluorescence signal in the hippocampus of 5×FAD mice treated with Compound 1887 was significantly lower than that in the control group, indicating that Compound 1887 significantly reduces Aβ deposition. Aβ deposition is a key pathological feature of Alzheimer's disease, and reducing Aβ load may help alleviate neurotoxicity and improve cognitive function.

[0174] Conclusion: Compound 1887 effectively reduced microglial hyperactivation and Aβ deposition in 5×FAD mice, suggesting that it may exert neuroprotective effects by alleviating neuroinflammation and reducing Aβ load. These results support further investigation of compound 1887 as a potential candidate for the treatment of Alzheimer's disease.

[0175] Example 11: Effects of Compound 1887 on Synaptic Markers PSD95 and Syn in the Hippocampus of 14-month-old 5×FAD Mice 1. Experimental Materials

[0176] Grouping and treatment: Same as Example 8. In addition, 14-month-old 5×FAD + / Thy1-EGFP + Mice were divided into solvent group and 1887 group. 1887 was administered orally at a dose of 20 mg / kg for 4 weeks.

[0177] Compound: Compound 1887.

[0178] Antibodies: PSD95 antibody, Synaptophysin (Syn) antibody,

[0179] 2. Experimental Methods

[0180] Immunofluorescence staining: After treatment, mice were anesthetized and hippocampal tissue was removed and fixed in 4% paraformaldehyde for 24 hours. The fixed tissue was sectioned at 20 μm thickness and incubated with a PSD95 antibody to label the postsynaptic density. Cell nuclei were labeled with DAPI staining, and fluorescence images were captured using a confocal microscope. Fluorescence signals were quantified using ImageJ software.

[0181] Immunoblotting analysis: Total protein was extracted from mouse hippocampal tissue, quantified using the BCA assay, and then subjected to SDS-PAGE electrophoresis. After transfer to a membrane, immunoblotting was performed using antibodies against PSD95 and Syn, respectively, and protein signals were detected using ECL luminescence. ImageJ software was used to quantify the grayscale values of protein bands and compare the expression levels of Syn and PSD95 among the groups.

[0182] 3. Experimental Results

[0183] Immunofluorescence results showed that the expression level of PSD95 in the hippocampus of 5×FAD mice in the compound 1887 treatment group was significantly increased ( Figure 7 A). Especially in Thy1-EGFP-labeled 5×FAD mice, the expression of PSD95 in neurons of the 1887-treated group was significantly enhanced ( Figure 7 B), suggesting that compound 1887 plays a protective role in the postsynaptic region and helps improve the structural integrity of the synapse.

[0184] The results of immunoblotting experiments showed that the expression of presynaptic protein Syn and postsynaptic density protein PSD95 in the hippocampus of mice treated with 1887 was significantly increased ( Figure 7 C). Among them, the quantitative analysis of Syn ( Figure 7 D) showed that the expression of 1887-treated group was significantly higher than that of the control group; quantitative analysis of PSD95 ( Figure 7 E) also showed that the expression of PSD95 was significantly increased in the 1887-treated group.

[0185] Conclusion: Compound 1887 significantly increased the expression of synaptic markers Syn and PSD95 in the hippocampus of 5×FAD mice, indicating its important role in enhancing synaptic function and protecting synaptic structure. These results support the potential of Compound 1887 as a potential therapeutic for Alzheimer's disease, potentially mitigating synaptic degeneration and cognitive impairment.

[0186] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of the compound of formula (I), its stereoisomers, tautomers, isotope-labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, and prodrugs: in, R1 is selected from H, C1-6 alkyl, C1-6 alkoxy, cyano, halogen, hydroxyl, nitro; R2 is selected from H, C1-6 alkyl, C1-6 alkoxy, cyano, halogen, hydroxyl, nitro; m is selected from 0, 1, 2, 3, 4, 5, 6, 7; n is selected from 0, 1, 2, 3, 4, 5; The application is selected from any one, two or more of the following: (i) Use in the preparation of a drug for inhibiting the release of TNF-α from BV-2 cells; (ii) use in the preparation of anti-inflammatory drugs; (iii) use in the preparation of a drug for inhibiting Galectin-3 activity, or in the preparation of a Galectin-3 inhibitor; (iv) use in the preparation of a medicament for inhibiting the interaction between Galectin-3 and TREM2, or in the preparation of an inhibitor of the interaction between Galectin-3 and TREM2; (v) Use in the preparation of drugs for inhibiting microglial activity; (vi) use in the preparation of a medicament for preventing and / or treating neuroinflammation; (vii) use in the preparation of a medicament for preventing and / or treating Alzheimer's disease; (viii) Application in the preparation of drugs for improving cognitive function, reducing excessive activation of microglia, reducing Aβ deposition, and alleviating synaptic degeneration.

2. The use according to claim 1, characterized in that R1 is selected from H, C1-3 alkyl, C1-3 alkoxy, cyano, halogen, hydroxyl, and nitro.

3. The use according to claim 1 or 2, characterized in that R2 is selected from H, C1-3 alkyl, C1-3 alkoxy, cyano, halogen, hydroxyl, and nitro.

4. The use according to any one of claims 1 to 3, characterized in that The compound represented by formula (I) is selected from the following specific compounds:

5. A pharmaceutical composition comprising one, two or more of the compound of formula (I) according to any one of claims 1 to 4, its stereoisomers, tautomers, isotope-labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, and prodrugs.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition may optionally further comprise at least one pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition may optionally further comprise at least one additional active ingredient.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that The pharmaceutical composition is used for: (i) inhibiting the release of TNF-α from BV-2 cells; and / or, (ii) anti-inflammatory; and / or, (iii) inhibiting the activity of Galectin-3; and / or, (iv) inhibiting the interaction between Galectin-3 and TREM2; and / or, (v) inhibiting microglial activity; and / or, (vi) preventing and / or treating neuroinflammation; and / or, (vii) preventing and / or treating Alzheimer's disease; and / or, (viii) Improve cognitive function, reduce excessive activation of microglia, reduce Aβ deposition, and alleviate synaptic degeneration.

8. A method comprising administering to a subject a therapeutically effective amount of one, two or more of the compound of formula (I) according to any one of claims 1 to 4, its stereoisomers, tautomers, isotopically labeled substances, nitrogen oxides, solvates, polymorphs, metabolites, esters, pharmaceutically acceptable salts, prodrugs, or the pharmaceutical composition according to any one of claims 5 to 7; The method is selected from any one, two or more of the following: (i) inhibiting the release of TNF-α from BV-2 cells; (ii) anti-inflammatory; (iii) inhibiting the activity of Galectin-3; (iv) inhibiting the interaction between Galectin-3 and TREM2; (v) inhibiting the activity of microglia; (vi) preventing and / or treating neuroinflammation; (vii) prevention and / or treatment of Alzheimer's disease; (viii) Improve cognitive function, reduce excessive activation of microglia, reduce Aβ deposition, and alleviate synaptic degeneration.

9. A method for preparing the compound of formula (I) according to any one of claims 1 to 4, comprising the following steps: (1) Compound SM1 reacts with thionyl chloride and methanol to obtain compound 2A; (2) Compound 2A reacts with a compound of formula (I-1) to obtain a compound of formula (I-2); (3) hydrolyzing the compound of formula (I-2) to obtain a compound of formula (I-3); (4) reacting the compound of formula (I-3) with the compound of formula (I-4) to obtain the compound of formula (I); The reaction formula is as follows:

10. The preparation method according to claim 9, characterized in that In step (1), the reaction temperature is 60°C-90°C; And / or, in step (1), the molar ratio of compound SM1 to thionyl chloride is 1:1-2; And / or, in step (1), the mass volume ratio of compound SM1 to methanol is 5-10:10; And / or, after the reaction in step (1) is completed, the reaction solution is concentrated to dryness, dissolved in dichloromethane, washed with aqueous sodium bicarbonate solution, separated, and purified by column chromatography to obtain compound 2A; And / or, in step (2), the reaction is carried out in the presence of a base; preferably, the base is potassium carbonate or sodium hydroxide; And / or, in step (2), the reaction is carried out in a solvent; preferably, the solvent is an organic solvent; and / or, in step (2), the molar ratio of compound 2A to the compound of formula (I-1) is 1:1-2; and / or, in step (2), the molar ratio of compound 2A to the base is 1:1-3; And / or, in step (2), the mass volume ratio of compound 2A to solvent is 5-10:10; and / or, in step (2), the reaction temperature is 10° C.-35° C.; And / or, after the reaction of step (2) is completed, the reaction solution is filtered, the filter cake is washed with a solvent, concentrated and dried, and purified by column chromatography to obtain a compound of formula (I-2); And / or, in step (3), the reaction is carried out in the presence of a base; preferably, the base is sodium hydroxide or potassium carbonate; And / or, in step (3), the reaction is carried out in a solvent; preferably, the solvent is a mixed solvent of water and an organic solvent; and / or, in step (3), the molar ratio of the compound of formula (I-2) to the base is 1:1-3; and / or, in step (3), the mass volume ratio of the compound of formula (I-2) to the solvent is 5-10:10; and / or, in step (3), the reaction temperature is 10° C.-35° C.; And / or, after the reaction in step (3) is completed, the reaction solution is concentrated and dried, and then the pH is adjusted to 7-8 with hydrochloric acid, stirred, filtered, and dried to obtain a compound of formula (I-3); And / or, in step (4), the reaction is carried out in the presence of a base; preferably, the base is N,N-diisopropylethylamine; And / or, in step (4), the reaction is carried out in a solvent; preferably, the solvent is dichloromethane; And / or, in step (4), the reaction is carried out in the presence of a condensation reagent; preferably, the condensation reagent is selected from HATU; and / or, in step (4), the molar ratio of the compound of formula (I-3) to the compound of formula (I-4) is 1:1-2; and / or, in step (4), the molar ratio of the compound of formula (I-3) to the base is 1:1-5; and / or, in step (4), the molar ratio of the compound of formula (I-3) to the condensation reagent is 1:1-3; and / or, in step (4), the mass volume ratio of the compound of formula (I-3) to the solvent is 3-8:20; and / or, in step (4), the reaction temperature is 10° C.-35° C.; And / or, after the reaction of step (4) is completed, the reaction solution is washed with sodium hydrogen hydrochloride aqueous solution and water in sequence, concentrated and dried; then slurried with ethyl acetate, filtered and dried to obtain the compound represented by formula (I).

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