Use of a galectin-3 inhibitor in neurodegenerative diseases

Abstract

The application provides a Galectin-3 inhibitor compound 10 which can penetrate the blood-brain barrier, a preparation method thereof and application thereof in the treatment of neurodegenerative diseases. Compared with compound 1, compound 10 has lower cLogP and AB-MPS values, stronger Galectin-3 binding capacity, higher PAMPA-BBB permeability, and better oral exposure and pharmacokinetic performance; after oral administration, clear exposure can be detected in the hippocampus and cortex of mice. In 8-month-old 5xFAD mice, compound 10 improves recognition memory, spatial learning and memory ability and nesting behavior, and reduces Aβ-related pathology, plaque-related Galectin-3 accumulation and microglia activation. In addition, compound 10 shows good tolerance in acute safety and repeated administration observation, and shows improvement in pS129-alphaSyn and glial cell-related pathological indicators in the exploratory A53T mouse experiment. The above results support that compound 10 is used for preparing a drug for preventing and / or treating Alzheimer's disease, Parkinson's disease and other related neurodegenerative diseases.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, specifically relating to the use of a brain-penetrating galactoglobulin-3 inhibitor or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Background Technology

[0002] Alzheimer's disease is increasingly recognized as a multicellular brain disorder, in which the interaction of amyloid-β (Aβ) deposition, tau protein pathological changes, glial cell activation, synaptic dysfunction, and metabolic disorders drives progressive cognitive decline. While Aβ-targeting therapies have significantly altered the treatment landscape for Alzheimer's disease, concerns about the extent of clinical benefit and safety highlight the necessity of developing complementary disease-modifying strategies. Such strategies should not be limited to clearing existing Aβ plaques but should also address the ongoing neuroinflammatory damage and abnormal remodeling of the brain microenvironment. Phase III clinical trials of anti-Aβ antibodies, such as lecanemab, have shown that reducing Aβ burden in the brain can alleviate cognitive and functional decline to some extent, but its overall benefit, safety profile, and applicable population remain limited.

[0003] Large-scale proteomics, transcriptomics, and metabolomics studies have further revealed synergistic alterations in Alzheimer's disease involving neurons, glial cells, vascular-related components, protein homeostasis networks, immune pathways, and metabolic regulation. Multiscale proteomics studies also show that the progression of Alzheimer's disease involves network interactions between glial cells, neurons, and metabolic modules. These studies suggest that targeting upstream regulators of disease-related inflammatory responses and brain microenvironment remodeling may offer a therapeutic approach to Alzheimer's disease that differs from simply clearing Aβ.

[0004] Parkinson's disease is another common neurodegenerative disease, characterized by progressive damage to dopaminergic neurons in the substantia nigra and abnormal accumulation of α-synuclein (α-Syn). Abnormal phosphorylation of α-Syn at serine 129 (pS129-αSyn) is closely associated with pathological accumulation in Parkinson's disease. In addition to neuronal pathology, abnormal activation of microglia and astrocytes, and the resulting persistent inflammatory microenvironment, also contribute to the spread of α-Syn and neuronal functional impairment. Therefore, drugs that can enter brain tissue and modulate abnormal glial cell activation and related inflammatory signals may also have an interventional effect on the pathological processes associated with Parkinson's disease.

[0005] Neuroinflammation is a crucial pathological link in the deposition of abnormal connective proteins, synaptic dysfunction, and neuronal damage. Whole-brain cell type-specific transcriptomic studies show that aging is accompanied by coordinated remodeling of neurons and glial cell populations; single-cell or mononuclear transcriptomic studies of Alzheimer's disease brain tissue further reveal coordinated cell type-specific alterations in neurons, oligodendrocytes, astrocytes, and microglia. These results support the view that neurodegenerative diseases involve the pathological remodeling of multiple brain cells and their interaction networks, rather than being solely caused by intrinsic neuronal abnormalities.

[0006] Microglia are innate immune cells of the central nervous system, and their response to abnormal protein deposition exhibits significant stage-dependent and context-dependent responses. Plaque-associated microglia (PAMs) can exert a certain tissue protection role by surrounding, isolating, and phagocytosing Aβ; however, continuous exposure to Aβ, abnormal α-syn, age-related stress, and disease-related metabolic signals can also drive microglia into a maladaptive inflammatory state. This state can promote the release of inflammatory mediators, alter phagocytic and lysosomal functions, immune regulation, and lipid metabolism, and further exacerbate synaptic damage and neuronal dysfunction. Disease-associated microglia (DAMs) exhibit characteristics such as immune activation, phagocytic remodeling, and altered lipid metabolism. Therefore, compared to non-selectively inhibiting all microglia function, targeting upstream regulatory molecules that drive maladaptive microglia activation and inflammatory amplification may be more beneficial in suppressing pathological neuroinflammation while preserving necessary immune surveillance and abnormal protein clearance functions. Summary of the Invention

[0007] Gallectin-3 (Gal-3) is a lectin encoded by the LGALS3 gene that recognizes β-galactoside structures. Current Gal-3 inhibitors are primarily developed for tumors, fibrosis, or peripheral inflammatory diseases. However, some compounds suffer from drawbacks such as large molecular weight, high polarity, insufficient oral exposure, or limited blood-brain barrier permeability. For central nervous system diseases such as Alzheimer's and Parkinson's, current technology still lacks small molecule compounds that possess both definitive Gal-3 binding and functional inhibitory activity, good blood-brain barrier permeability, measurable brain exposure, suitable pharmacokinetic properties, and in vivo therapeutic effects.

[0008] Therefore, it is necessary to develop a small molecule compound that can enter brain tissue and effectively inhibit Gal-3 function. By inhibiting the abnormal activation of Gal-3-related glial cells and downstream inflammatory responses, it can improve the pathology, synaptic function and neurobehavioral performance of Aβ or α-Syn-related brain tissues, thereby providing a new drug intervention strategy for Alzheimer's disease, Parkinson's disease and other related neurodegenerative diseases.

[0009] This invention provides for the use of compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0010] Among them, R1 is selected from H and C. 1-6 Alkyl, C 1-6 Alkyl, cyano, halogen, hydroxyl, nitro; R2 is selected from H and C. 1-6 Alkyl, C 1-6 Alkyl, cyano, halogen, hydroxyl, nitro; m is selected from 0, 1, 2, 3, 4, 5; n is selected from 0, 1, 2, 3, 4, and 5; The application is selected from any one, two, or more of the following: (i) Application in the preparation of drugs that inhibit the release of NO or TNF-α from microglia; (ii) Application in the preparation of anti-inflammatory drugs; (iii) Prepare drugs that reduce pathological-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Application in the preparation of drugs that inhibit microglial cell activation; (v) Use in the preparation of medicaments for the prevention and / or treatment of neuroinflammation; (vi) Use in the preparation of medicines for the prevention and / or treatment of neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease); (vii) Application in the preparation of drugs that improve cognitive function and restore synaptic integrity.

[0011] According to an embodiment of the present invention, R1 is selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, cyano, halogen, hydroxy, nitro; preferably, R1 is selected from H, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, cyano, halogen, hydroxy, nitro; exemplaryly, R1 is selected from methyl.

[0012] According to an embodiment of the present invention, R2 is selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy, cyano, halogen, hydroxy, nitro; preferably, R2 is selected from H, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, cyano, halogen, hydroxy, nitro; exemplaryly, R2 is selected from H, halogen, methoxy.

[0013] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the compounds represented by formula (I-1) below: ; Among them, R1 is the same or different, and is independently selected from C. 1-4 alkyl; R2 is selected from C 1-4 Alkyl group.

[0014] According to an embodiment of the present invention, R1 is always methyl. According to an embodiment of the present invention, R2 is methoxy.

[0015] According to an embodiment of the present invention, the compound represented by formula (I) is selected from the following specific compounds: .

[0016] According to embodiments of the present invention, neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0017] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) above or a pharmaceutically acceptable salt thereof.

[0018] According to embodiments of the present invention, the pharmaceutical composition may optionally further comprise at least one pharmaceutically acceptable excipient. According to embodiments of the present invention, pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, excipients, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0019] According to embodiments 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 other than the compounds described above or their pharmaceutically acceptable salts. In the pharmaceutical composition, the dosage of the compounds described above or their pharmaceutically acceptable salts may be a therapeutically effective amount.

[0020] According to embodiments of the present invention, the pharmaceutical compositions of the present invention can be formulated into dosage forms suitable for administration using methods known in the art. According to embodiments of the present invention, the formulations (or pharmaceutical compositions) include: oral formulations and non-oral formulations. According to embodiments of the present invention, the formulations include: powders, granules, capsules, injections, inhalants, tinctures, oral liquids, tablets, lozenges, or drops.

[0021] According to an embodiment of the present invention, the pharmaceutical composition is used for: (i) To prepare drugs that inhibit the release of NO or TNF-α from microglia; (ii) Preparation of anti-inflammatory drugs; (iii) Reduce pathology-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Preparation of drugs to inhibit microglial cell activation; (v) To prepare drugs for the prevention and / or treatment of neuroinflammation; (vi) To prepare medicines for the prevention and / or treatment of neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease); (vii) Prepare drugs that improve cognitive function and restore synaptic integrity.

[0022] According to embodiments of the present invention, neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0023] The present invention also provides a method comprising administering to a therapeutic subject a therapeutically effective amount of the compound represented by formula (I) above or a pharmaceutically acceptable salt thereof, or one, two or more of the above pharmaceutical compositions; The method is selected from any one, two, or more of the following: (i) Inhibit the release of NO or TNF-α from microglia; (ii) Anti-inflammatory; (iii) Use in drugs that reduce pathological-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Inhibit microglia activation; (v) Prevention and / or treatment of neuroinflammation; (vi) Prevention and / or treatment of neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease); (vii) Improve cognitive function and restore synaptic integrity.

[0024] According to embodiments of the present invention, neurodegenerative diseases include Parkinson's disease and Alzheimer's disease.

[0025] This invention also provides a method for preparing the compound represented by formula (I) above or a pharmaceutically acceptable salt thereof, the method comprising the following steps: (1) Compound SM1 reacts with thionyl chloride to give compound 2A; (2) Compound 2A reacts with the compound shown in formula (I-1) to obtain the compound shown in formula (I-2); (3) The compound shown in formula (I-2) undergoes a hydrolysis reaction to obtain the compound shown in formula (I-3); (4) The compound shown in formula (I-3) reacts with the compound shown in formula (I-4) to obtain the compound shown in formula (I); The reaction formula is as follows: ; Wherein, R1, R2, m and n have the aforementioned definitions; X is a leaving group, preferably chlorine, bromine or iodine.

[0026] According to an embodiment of the present invention, the compound shown in formula (I) has the foregoing definition.

[0027] According to an embodiment of the present invention, in step (1), the reaction is carried out in a solvent (e.g., methanol). According to an embodiment of the present invention, in step (1), the reaction temperature is 60–90 °C, for example 70 °C. According to an embodiment of the present invention, in step (1), the reaction time is 0.5–5 h, for example 1 h.

[0028] 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 selected from potassium carbonate, sodium carbonate, or sodium hydroxide, more preferably potassium carbonate. According to an embodiment of the present invention, in step (2), the reaction can also be carried out in the presence of a halide promoter; preferably, the halide promoter is selected from potassium bromide or potassium iodide, more preferably potassium bromide.

[0029] 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 reaction temperature is 10–35 °C, for example, room temperature or 25 °C. According to an embodiment of the present invention, in step (2), the reaction time is 0.5 h–12 h, for example, 1 h.

[0030] According to an embodiment of the present invention, in step (3), the hydrolysis reaction is carried out in the presence of an alkali; preferably, the alkali is selected from sodium hydroxide, potassium hydroxide, or potassium carbonate, more preferably sodium hydroxide. According to an embodiment of the present invention, in step (3), the reaction is carried out in a solvent; preferably, the solvent is an organic solvent; preferably, the organic solvent is selected from any one or a combination of methanol, ethanol, and tetrahydrofuran; more preferably, the reaction solvent includes methanol and tetrahydrofuran; preferably, the volume ratio of tetrahydrofuran to methanol is 1:2.

[0031] According to an embodiment of the present invention, in step (3), the reaction temperature is 10 to 35 °C, for example, room temperature or 25 °C.

[0032] According to an embodiment of the present invention, after the reaction in step (3) is completed, the reaction solution is adjusted to acidity with hydrochloric acid so that the compound shown in formula (I-3) precipitates; preferably, 2 mol / L hydrochloric acid is used to adjust the reaction solution to acidity.

[0033] 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 condensing agent; preferably, the condensing agent is HATU, namely O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate.

[0034] According to an embodiment of the present invention, in step (4), the reaction temperature is 10–35 °C, for example 25 °C. According to an embodiment of the present invention, in step (4), the reaction time is 1–12 h, for example 5 h.

[0035] Beneficial effects This application provides an application of a Galectin-3 inhibitor compound 10 that can penetrate the blood-brain barrier. Compared to the first-generation compound 1, compound 10 maintains the same tPSA value (69.04 Å). 2 Compound 10 exhibits lower cLogP and AB-MPS values; its MST dissociation constant KD with Galectin-3 is 0.51 ± 0.44 μM, lower than that of compound 1 (1.55 ± 0.66 μM). The effective permeability coefficient Pe of compound 10's PAMPA-BBB is 8.89 ± 0.24 × 10⁻⁶. -6 cm·s -1 It is higher than that of compound 1 (4.24 ± 0.08 × 10⁻⁶). -6 cm·s -1 Clear exposure was detected in the hippocampus and cortex after oral administration. In rats, a single oral gavage dose of 15 mg / kg... -1 Afterwards, compound 10 exhibited a higher C value compared to compound 1. max and AUC 0-t It also has a longer terminal half-life, demonstrating superior overall drug efficacy.

[0036] In 8-month-old 5×FAD mice with a clear amyloid pathology, compound 10 was administered at a dose of 10 mg / kg. -1 Following daily gavage administration, it improved cognitive memory, spatial learning and memory abilities, and nesting behavior; reduced Aβ-related pathology, plaque-associated Galectin-3 accumulation, and microglial activation; and restored hippocampal neurogenesis, neurotrophic factors, and synaptic integrity markers such as DCX, BDNF, and PSD95. (20 mg / kg) -1 Compared to compound 1, compound 10 still showed comparable or better behavioral improvement at lower doses.

[0037] In addition, compound 10 showed good tolerability in in vivo safety evaluation.

[0038] In summary, compound 10 exhibits good brain penetration, in vivo exposure, pharmacological activity, and safety, making it a potential candidate drug for the prevention and / or treatment of Alzheimer's disease and other Galectin-3-related neurodegenerative diseases. Attached Figure Description

[0039] Figure 1 Structural and dynamic characterization of the combination of compound 10 and Gal-3. Figure 1 a. The predicted binding pattern of compound 10 in the Gal-3 sugar recognition domain is shown as an electrostatic surface representation, a magnified three-dimensional interaction view, and a two-dimensional ligand-protein interaction map. Key residues involved in ligand recognition include Arg144, His158, Asn160, Arg162, Asn174, and Trp181. Figure 1 b. The RMSD trajectory of the Gal-3 protein backbone during molecular dynamics simulation. Figure 1 c, the RMSD trajectory of compound 10 during the simulation. Figure 1 d, RMSF spectrum of Gal-3 residue levels. Figure 1 e, the change in the solvent-accessible surface area of ​​the complex over simulation time. Figure 1 f, the time-dependent change in the number of hydrogen bonds between compound 10 and Gal-3. Figure 1 g, the change in the radius of gyration of the complex. Figure 1 h, the contribution spectrum of residue-level binding energy. Figure 1 i and Figure 1 j, projected onto the Gibbs free energy landscape of the first two principal components, is displayed as a two-dimensional contour map and a three-dimensional surface map, respectively.

[0040] Figure 2 Compound 10 showed preliminary in vivo safety and improved cognitive and nesting performance in 8-month-old 5×FAD mice. Figure 2 a. ICR mice were administered a single oral dose of 1000 mg / kg -1 Representative H&E staining images of heart, brain, liver, and kidney sections collected 14 days after compound 10 administration. Mice treated with the vector served as controls. Figure 2 b, Changes in body weight in ICR mice treated with the vector and compound 10 during the 14-day acute safety observation period, n=8 mice per group. Figure 2 c. Timeline of in vivo efficacy experiments. Eight-month-old C57BL / 6J and 5×FAD mice were treated as described, and then subjected to open field test, new object recognition test, Morris water maze test and nesting test before sacrifice. Figure 2d. Changes in body weight of 5×FAD mice treated with C57BL / 6J, 5×FAD and compound 10 during repeated administration and behavioral tests, n=10 mice per group. Figure 2 e. Open field experiment shows the central region movement distance of 5×FAD mice treated with C57BL / 6J, 5×FAD and compound 10. The right side is a schematic diagram of the central region analysis. Figure 2 f and Figure 2 g, New object recognition experiment. Figure 2 f. Schematic diagram of the experiment and representative exploration trajectory. Figure 2 g, quantitative identification index, n=10 mice per group. Figure 2 h~ Figure 2 j. Morris water maze experiment. Figure 2 h, the 6-day learning and training phase of the escape latency period. Figure 2 i, a representative swimming path during the probe test. Figure 2 j, the quantification of the number of platform crossings during the probe experiment, with n=10 mice per group. Figure 2 k and Figure 2 l, Nest building experiment. Figure 2 Representative nesting images of 5×FAD mice treated with k, C57BL / 6J, 5×FAD, and compound 10. Figure 2 l, Quantitative determination of nesting material weight, n=10 mice per group. Data are expressed as mean ± SEM. Figure 2 b、 Figure 2 d and Figure 2 Statistical significance was determined using two-way repeated measures ANOVA. Figure 2 e Figure 2 g、 Figure 2 j and Figure 2 Significance was determined using one-way ANOVA combined with appropriate post-hoc multiple comparison tests. If the difference was within ns, it was not statistically significant. * P <0.05; ** P <0.01; *** P <0.001.

[0041] Figure 3 Compound 10 binds to Gal-3 and attenuates Aβ plaque-associated Gal-3 accumulation and microglial activation in the hippocampus of 5×FAD mice. Figure 3 a. The binding affinity of compound 10 to Gal-3 protein determined by micro-thermophoresis (MST). The fitted binding curves showed a concentration-dependent interaction between compound 10 and Gal-3, with an estimated dissociation constant KD of 0.51 ± 0.44 μM, n = 3 independent experiments. Figure 3b. Representative immunofluorescence images of hippocampal sections from 5×FAD mice treated with C57BL / 6J, 5×FAD, and compound 10, stained to identify Aβ plaques (6E10, green), microglia (IBA1, red), Gal-3 (magenta), and nuclei (DAPI, blue). The right side shows the merged image. Figure 3 c~ Figure 3 e, 6E10 in hippocampal slices ( Figure 3 c), IBA1 ( Figure 3 d) and Gal-3 Figure 3 e) Quantification of relative fluorescence intensity, n=6 mice per group. Figure 3 f and Figure 3 g, magnified representative images show C57BL / 6J mice ( Figure 3 f), untreated 5×FAD mice and compound 10-treated 5×FAD mice ( Figure 3 Spatial relationships between Aβ plaques, IBA1-positive microglia, and Gal-3 signaling in g). Figure 3 Representative Western blot images of Gal-3 and Aβ in hippocampal tissue of 5×FAD mice treated with C57BL / 6J, 5×FAD, and compound 10. Actin was used as a loading control. Figure 3 i and Figure 3 j, Gal-3 ( Figure 3 i) and Aβ ( Figure 3 j) Gray-scale quantification of protein levels relative to Actin, n=6 mice per group. Figure 3 Representative immunofluorescence images of microglia from the control group, the fibrillary Aβ stimulation group, and the fibrillary Aβ combined with compound 10 treatment group. Cells are stained with IL-1β (green), iNOS (red), CD68 (magenta), and cell nuclei (DAPI, blue). The right side shows the merged image. Scale bar is 10 μm. Figure 3 l~ Figure 3 n, IL-1β in microglia ( Figure 3 l), iNOS ( Figure 3 m) and CD68 ( Figure 3 The relative fluorescence intensity of n) was quantified, with n=3 independent experiments. Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA combined with appropriate post-hoc multiple comparison tests. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

[0042] Figure 4 Compound 10 restored hippocampal neurogenesis and synaptic plasticity-related markers in 5×FAD mice and attenuated APOE / CD68-related inflammatory microenvironmental alterations. Figure 4 a. Representative immunofluorescence images of DCX staining (green) and DAPI (blue) on the dentate gyrus of the hippocampus in 5×FAD mice treated with C57BL / 6J, 5×FAD, and compound 10. The right side shows the merged image. Scale bar is 25 μm. Figure 4 b. Representative magnified immunofluorescence images of APOE (green), PSD95 (red), CD68 (magenta), and DAPI (blue) in the hippocampus of 5×FAD mice treated with C57BL / 6J, 5×FAD, and compound 10. Scale bar: 10 μm. Figure 4 c~ Figure 4 f, DCX ( Figure 4 c) APOE ( Figure 4 d), PSD95 ( Figure 4 e) and CD68 Figure 4 f) Quantification of relative fluorescence intensity, n=6 mice per group. Figure 4 Representative Western blot images of PSD95 and BDNF in hippocampal tissue of 5×FAD mice treated with g, C57BL / 6J, 5×FAD, and compound 10. Actin was used as a loading control. Figure 4 h and Figure 4 i, PSD95 ( Figure 4 h) and BDNF ( Figure 4 i) Gray-scale quantification of protein levels relative to Actin, n=6 mice per group. Data are expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA combined with appropriate post-hoc multiple comparison tests. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001.

[0043] Figure 5 Compound 10 inhibited Gal-3 / TLR4 / MYD88-related inflammatory signaling in the hippocampus of 5×FAD mice and attenuated microglial cell inflammatory activation. Figure 5a, Representative immunofluorescence images of hippocampal sections from C57BL / 6J mice, 5×FAD mice, and 5×FAD mice treated with Compound 10, with staining labeling MYD88 (green), TLR4 (red), Gal-3 (magenta) and cell nuclei (DAPI, blue). Merged images are shown on the right. Scale bar = 25 μm. Figure 5 b~ Figure 5 d, Figure 5 Quantification of fluorescence intensity for MYD88 in a ( Figure 5 b), TLR4 ( Figure 5 c) and Gal-3 ( Figure 5 d), n=6 mice per group. Figure 5 e, Representative Western blot images of CD68, IL-1β and Actin in hippocampal tissues of untreated 5×FAD mice and 5×FAD mice treated with Compound 10. Actin serves as the loading control. Figure 5 f and Figure 5 g, Grayscale quantification of CD68 ( Figure 5 f) and IL-1β ( Figure 5 g) protein levels relative to Actin, n=6 mice per group. Figure 5 h~ Figure 5 l, qPCR analysis of inflammation and innate immunity-related transcripts in the hippocampus of C57BL / 6J mice, 5×FAD mice and 5×FAD mice treated with Compound 10, including Lgals3 ( Figure 5 h), Tlr4 ( Figure 5 i), Myd88 ( Figure 5 j), Gsdmd ( Figure 5 k) and Il1b ( Figure 5 l), n=6 mice per group. Figure 5 m~ Figure 5 o, qPCR analysis of inflammatory transcripts in BV-2 microglial cells from the control group, Compound 10 treatment group, LPS stimulation group and LPS plus Compound 10 co-treatment group, including Nfkb1 ( Figure 5 m), Il1b ( Figure 5 n) and Tnfa ( Figure 5 o), n=3 independent experiments. Figure 5 p, Representative immunofluorescence images of microglial cells from the control group, fibrillar Aβ stimulation group and fibrillar Aβ plus Compound 10 co-treatment group. Cell staining labels MYD88 (green), TLR4 (red), Gal-3 (magenta) and cell nuclei (DAPI, blue). Merged images are shown on the right. Scale bar = 10 μm. Figure 5 q~ Figure 5 s, Figure 5 quantification of the fluorescence intensity of MYD88 ( Figure 5 q), TLR4 ( Figure 5 r) and Gal-3 ( Figure 5 s) in p, n=3 independent experiments. Data are expressed as mean±SEM. Comparisons of three or four groups were performed using one-way ANOVA combined with an appropriate post hoc multiple comparison test, and unpaired two-tailed Student's t-test was used for two-group Western blot comparisons. ns, no statistically significant difference; * P <0.05; Figure 5 P <0.01; *** P <0.001; **** P <0.0001.

[0044] Figure 6 : The improving effects of Compound 10 and Compound 1 on cognitive function and nesting behavior of 5×FAD mice. Figure 6 a is the body weight change curve of mice in each group during the administration period. Figure 6 b is the escape latency of mice in each group during 6 consecutive days of training in the Morris water maze positioning navigation experiment. Figure 6 c and Figure 6 d are the results of the spatial probe experiment after removing the platform, where Figure 6 c is the number of times mice in each group crossed the original platform position, Figure 6 d is the residence time of mice in each group in the area where the original platform was located. Figure 6 e is the nest material weight of mice in each group in the nesting experiment. The experimental groups include normal control group (Ctrl), 5×FAD model group, 5×FAD combined with Compound 10 treatment group (10 mg / kg) and 5×FAD combined with Compound 1 treatment group (20 mg / kg). There are n=10 mice in each group. Data are expressed as mean ± standard error (mean±SEM).

[0045] Figure 7 : Effects of Compound 10 on pathological α-synuclein phosphorylation and glial cell-related indicators in the reticular part of the substantia nigra of aged A53T mice. Figure 7 a is the representative immunofluorescence images of pS129-αSyn, IBA1, Gal-3 and DAPI; Figure 7 b is the representative immunofluorescence images of GFAP, CD68 and DAPI; Figure 7 c, Figure 7 d, Figure 7e represents the quantification of the average fluorescence intensity of IBA1, pS129-αSyn, and GFAP in the SNr region. Experimental groups included a 19-month-old A53T model control group and an A53T combined with compound 10 treatment group, with n=4 in each group. Compound 10 was administered at 10 mg / kg. -1 The drug was administered once daily by gavage for one month. Data are expressed as mean ± SEM. Unpaired two-tailed Student's t-test was used for comparisons between the two groups. ns indicates no statistically significant difference. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001. Detailed Implementation

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory 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 covered within the scope of protection intended by the present invention.

[0047] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0048] Example 1: Synthesis of the compound The preparation route of compound 10 is as follows: .

[0049] The specific preparation process is as follows: Carboxylic acid compound S4 is first converted into methyl ester compound S5 in methanol using thionyl chloride; compound S5 undergoes N-alkylation with the corresponding substituted benzyl chloride to generate intermediate compound S6, and compound S6 is hydrolyzed to the corresponding carboxylic acid compound S7; finally, compound 10 is obtained by reacting with a specified aromatic amine via HATU / DIPEA-mediated amide coupling.

[0050] Characterization data of compound 10: 1 H NMR (400 MHz, DMSO- d 6) δ 10.26 (d, J = 17.0 Hz, 1H), 8.60 (d, J= 9.3Hz, 1H), 8.24 – 8.05 (m, 1H), 8.02 – 7.90 (m, 1H), 7.67 – 7.44 (m, 2H), 7.42– 7.29 (m, 2H), 7.19 – 7.05 (m, 1H), 7.00 – 6.85 (m, 2H), 5.97 (d, J = 9.8 Hz,2H), 3.72 (s, 3H), 2.22 (dd, J = 11.9, 2.4 Hz, 6H); 13 C NMR (101 MHz, DMSO- d 6) δ 165.9, 160.3, 146.0, 144.2, 137.8, 137.1,134.2, 132.5, 131.0, 130.5, 127.9, 126.7, 122.6, 118.9, 115.0, 60.4, 56.1,20.6,19.8; HRMS (ESI) m / z calcd. for C 23 H 23 N4O2[M+H] + 387.1821, found 387.1816.

[0051] Example 2: Methods for studying the efficacy of compounds Compounds used in biological experiments were prepared as DMSO stock solutions and diluted with the specified detection medium or carrier solution before use. The final DMSO concentration was kept consistent across all treatment groups.

[0052] 1. Molecular docking The interaction between Galectin-3 (PDB ID 8ITZ) and the compound was evaluated via molecular docking. The crystal structure of the Galectin-3 sugar recognition domain was pretreated by appropriately removing water of crystallization molecules, adding hydrogen atoms, and assigning protonated states. The ligands were pretreated after energy minimization and generating appropriate ionization states. Docking within the Galectin-3 sugar recognition domain was performed using an induced-fit docking workflow. The binding conformation was examined to identify polar, electrostatic, hydrophobic, and aromatic interactions between the compound and key residues within the binding pocket.

[0053] 2. Molecular dynamics simulations This study used the GROMACS 2026 software package combined with the AMBER 19SB force field to perform molecular dynamics simulations of the complex. The complex was placed in a dodecahedral periodic boundary water box, and a 10 Å thick water layer was set out from the surface of the complex using the TIP3P water model. Na was added. + and Cl - Ions neutralize the system charge to ensure overall electroneutrality. A 50,000-step energy minimization process using the conjugate gradient method is employed to eliminate interatomic steric conflict. This is followed by a 200 ps NPT ensemble equilibrium simulation: applying 1000 kJ·mol⁻¹ to the protein backbone atoms. -1 ·nm -2 Harmonic constraints were applied, and these constraints were gradually released during the last 50 ps to allow for full relaxation of the system. The production simulation was repeated three times, each run for 100 ns, with an integration time step of 2 fs. The simulation parameters were set as follows: hydrogen bond constraints were handled using the LINCS algorithm; long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method; and the cutoff radius for short-range non-bonded interactions was set to 1.0 nm. Temperature was controlled using the V-rescale method, and pressure was controlled using the C-rescale method.

[0054] Structural stability was assessed using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). Hydrogen bond number was analyzed to reveal dynamic interactions during the simulation. Principal component analysis (PCA) and free energy landscape (FEL) were performed using the gmx covar and gmx anaeig modules, respectively. Binding free energies were calculated using the MMGBSA method with the gmx_MMPBSA tool to quantify the binding strength between the small molecule and the protein. Simulation trajectory visualization was performed using PyMOL software, and data post-processing was performed using DuIvyTools and the Matplotlib library in Python.

[0055] 3. Evaluation of the anti-inflammatory activity of BV-2 microglia Mouse BV-2 microglia were purchased from Wuhan Pronosei Life Science Technology Co., Ltd., and cultured in DMEM medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Newzerum, New Zealand), 100 U / mL penicillin and 100 μg / mL streptomycin (Dalian Meilun Biotechnology), and maintained in a humidified incubator at 37°C and 5% CO2.

[0056] Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dalian Meilun Biotechnology). BV-2 cells were counted at 5 × 10⁶ cells per well. 3Cells were seeded at a density of [number] cells per well in 96-well plates. After 24 h of adhesion, the cells were treated with a 1.5 μM test compound for 24 h, a concentration consistent with the concentration used for subsequent anti-inflammatory activity evaluation. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C for 2 h according to the manufacturer's instructions. Absorbance was measured at 450 nm using a BioTek Epoch microplate reader. Compounds maintaining cell viability above 90% relative to the vector control were included in the subsequent anti-inflammatory evaluation; compounds not meeting this standard were not further evaluated at this screening concentration.

[0057] Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Dalian Meilun Biotechnology). BV-2 cells were counted at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and allowed to adhere for 24 h. For initial cytotoxicity screening, test compounds were treated at a concentration of 20 μM for 24 h. For selected compounds, concentration-dependent cytotoxicity was further evaluated at specified concentrations. Subsequently, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 2 h according to the manufacturer's instructions. Absorbance was measured at 450 nm using a BioTek Epoch microplate reader (Agilent Technologies, USA). Compounds that maintained cell viability above 90% compared to the vector-treated control group were considered to have acceptable cell compatibility and were included in subsequent anti-inflammatory evaluation.

[0058] In the anti-inflammatory activity assessment, BV-2 cells were used at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and allowed to adhere overnight. Cells were pretreated with a specified concentration of the test compound for 1 h, followed by stimulation with lipopolysaccharide (LPS, 1 μg / mL) for 24 h to induce inflammatory activation. Culture supernatant was collected and centrifuged at 300×g for 5 min to clarify. Nitric oxide (NO) production was assessed by measuring nitrite accumulation in the supernatant using the Griess kit (catalog number S0021, Beyotime Biotech, Shanghai) according to the manufacturer's instructions. Tumor necrosis factor-α (TNF-α) levels in the culture supernatant were quantified using a commercial ELISA kit (catalog number ECM008, Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

[0059] The inhibitory effects of the compounds on LPS-induced NO and TNF-α production were tested to evaluate the anti-inflammatory activity of Gal-3 inhibitor analogs and to support structure-activity relationship analysis. For compound 10, concentration-response curves were generated, and the half-maximal inhibitory concentration (IC50) for TNF-α inhibition was calculated using nonlinear regression analysis. 50 )value.

[0060] 4. Micro-volume thermophoresis The binding affinity of compound 10 to Galectin-3 was determined using micro-thermophoresis (MST). HEK293T cells were transiently transfected with the Galectin-3 expression plasmid. Cells were collected and lysed after transfection, and Galectin-3-containing protein extracts were prepared according to standard protein extraction procedures. In the binding assay, the Galectin-3-containing protein sample was incubated with serially diluted compound 10 to form a ligand-protein complex. The mixture was loaded into an MST capillary, and thermophoretic mobility was measured using a Monolith NT.115 system (NanoTemper Technologies, Munich, Germany). Changes in thermophoretic motion induced by compound 10 binding were recorded and analyzed using the instrument's accompanying software. Binding curves were generated by plotting the normalized MST signal against compound concentration. The dissociation constant KD was calculated by fitting the data to a standard unit point binding model. The obtained KD value was used to assess the binding affinity of compound 10 to Galectin-3.

[0061] 5. PAMPA-BBB test Passive blood-brain barrier permeability of compound 10 was assessed using a parallel artificial membrane permeation assay (PAMPA-BBB) based on the previously established protocol. Test solutions were prepared in a methanol-phosphate buffered saline (PBS, pH 7.4) mixture (7:3, v / v) and added to the donor wells. The artificial membrane was coated with porcine brain lipids to mimic the lipid environment of the blood-brain barrier. The donor and recipient plates were then assembled in a sandwich configuration and incubated at 25°C for 8.5 h.

[0062] Following incubation, the concentrations of the compounds in the donor and acceptor pores were determined by UV absorbance or appropriate analytical methods. The effective permeability coefficient Pe was calculated according to the standard PAMPA-BBB equation. Reference compounds with known central nervous system penetration characteristics were included in the experiment, including CNS-positive, CNS-negative, and intermediate controls, to validate the performance of the experimental system.

[0063] According to its P Classification by e-value: P e>4.0×10 -6 cm·s -1 CNS positive, P e<2.0×10 -6 cm·s -1 CNS negative, P e ranges from 2.0 to 4.0 × 10⁻⁶ -6 cm·s -1 The penetration between them is uncertain.

[0064] 6. Brain and plasma distribution of compound 10 Brain and plasma distribution of compound 10 was assessed after a single oral administration. Twelve-week-old male C57BL / 6J mice were orally administered compound 10 at a dose of 20 mg / kg. -1 Blood and brain tissue samples were collected at specified time points after drug administration. Blood samples were centrifuged to obtain plasma, and brain tissue was rapidly dissected to separate the hippocampus and cortex.

[0065] Hippocampal and cortical tissues were weighed and homogenized prior to extraction. The concentrations of compound 10 in plasma, hippocampus, and cortex were quantified using a validated LC-MS / MS method. Brain exposure was assessed based on the concentrations measured in hippocampal and cortical tissues at each time point. The brain / plasma ratio was calculated by dividing the compound concentration in each brain region by the corresponding plasma concentration at the same time point.

[0066] 7. Pharmacokinetic Analysis The plasma pharmacokinetic characteristics of compounds 10 and 1 were evaluated in male Sprague-Dawley rats after a single oral gavage administration of 15 mg / kg. -1 Approximately 0.25 mL of blood was collected from the posterior orbital venous plexus at predetermined time points of 5, 15, 30, 60, 120, 200, 360, 420, 660, 1440, 2160, and 3600 min after drug administration to cover the absorption, distribution, and elimination phases.

[0067] Plasma was separated by centrifugation at 3500 rpm for 10 min and stored in [location missing]. Incubate at 80°C until analysis. Plasma concentrations of the compound were quantified using a validated LC-MS / MS method, with a limit of quantitation of approximately 10 ng / mL. -1 Pharmacokinetic parameters include maximum plasma concentration (Cmax), time to peak concentration (Tmax), and area under the plasma concentration-time curve (AUC) from zero to the last quantifiable concentration. 0-t The terminal elimination half-life t1 / 2 was calculated using a non-compartmental model with WinNonlin 8.1 (Certara) and DAS 3.2.8 software.

[0068] All pharmacokinetic parameters are expressed as mean ± SEM, with data from four animals for each compound. This analysis was used to compare the systemic exposure, absorption characteristics, and elimination properties of compound 10 with those of compound 1.

[0069] 8. Acute toxicity evaluation The acute toxicity of compound 10 was evaluated in 12-week-old male ICR mice. Mice were administered a single oral dose of 1000 mg / kg. -1 Compound 10 was administered at a dose and monitored for 14 days post-administration. Weight, general appearance, behavioral changes, and mortality were recorded during the observation period.

[0070] Mice were sacrificed at the end of the 14-day observation period, and major organs such as the heart, brain, liver, and kidneys were harvested for gross examination and histopathological analysis. Tissue samples were fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histopathological changes were examined under a light microscope.

[0071] 9. 5×FAD Alzheimer's Disease Mouse Model and Behavioral Evaluation Animals and Ethical Approval All animal procedures were performed in accordance with the Fujian Medical University Laboratory Animal Care and Use Guidelines. The animal experimental protocol was reviewed and approved by the Fujian Medical University Institutional Animal Care and Use Committee, approval number IACUC FJMU 2025-0347. All mice and rats used in this study were housed in a temperature and humidity controlled facility free from specific pathogens, maintaining a 12-hour light / 12-hour dark cycle, with free access to food and water. All measures were taken to minimize animal suffering and reduce the number of animals used.

[0072] Animals and Drug Administration Animals and Drug Administration The therapeutic effect of compound 10 was evaluated using 8-month-old heterozygous 5×FAD mice (both male and female). Age-matched wild-type C57BL / 6J mice served as a non-transgenic control. Animals were randomly divided into three groups of 10 each: a C57 vector control group, a 5×FAD vector group, and a 5×FAD group treated with compound 10 (10 mg / kg). -1 Each group has an equal number of males and females, with 5 males and 5 females.

[0073] The carrier consisted of 0.1% hydroxypropyl methylcellulose (HPMC), 0.1% polysorbate 80, and 1% DMSO in sterile distilled water. Compound 10 was first prepared in DMSO at a concentration of 100 mg / mL. -1 The stock solution was then diluted with sterile distilled water containing 0.1% HPMC and 0.1% polysorbate 80. Compound 10 was administered at a concentration of 10 mg / kg. -1 The dosage is administered once daily by gavage, with a volume of 10 mL / kg. -1 The actual dosage volume was adjusted according to body weight. C57BL / 6J and 5×FAD mice treated with the carrier were given an equal volume of carrier. Administration continued for 6 weeks starting from day 1, and tissues were collected on day 42.

[0074] Behavioral tests were initiated 30 days after continuous drug administration: open field test on day 31, new object recognition test on day 32, Morris water maze test on days 33–39, nesting test on day 41, and mice were sacrificed and tissues and blood were collected on day 42. All behavioral tests were conducted in a quiet laboratory at a temperature maintained at 22±2 ℃ during the light period, and the data were recorded and analyzed using ANY-maze software; the experimental apparatus was cleaned with 75% ethanol between each test.

[0075] Open field experiment On day 31, an open field test was conducted to assess motor activity and anxiety-like behavior. Mice were placed in the center of a 50×50×40 cm square arena and allowed to explore freely for 10 minutes. Total distance traveled, distance traveled in the central area, and time spent in the central area were automatically recorded using ANY-maze software. The experimental setup was cleaned with 75% ethanol between trials to eliminate olfactory cues.

[0076] New object recognition experiment On day 32, a two-trial paradigm was used to assess recognition memory. During the familiarization phase, mice were placed in an arena containing two identical objects and allowed to explore for 5 minutes. After a 1-hour retention interval, one familiar object was replaced with a new object of different shape and color but similar size. The mice were then allowed to explore the arena for another 5 minutes, and the time spent exploring the familiar and new objects was recorded. The Recognition Index (RI) was calculated using the following formula:

[0077] in and These represent the exploration time for new objects and familiar objects, respectively.

[0078] Morris water maze experiment Spatial learning and memory abilities were assessed using the Morris water maze from days 33 to 39. The circular pool was 120 cm in diameter and 50 cm high, filled with opaque water at a temperature maintained at 22 ± 1°C. A hidden platform with a diameter of 10 cm was submerged 1 cm below the water surface in the target quadrant.

[0079] For six consecutive days, from days 33 to 38, mice were given four training trials daily, with each trial lasting a maximum of 60 seconds. Mice that failed to find the platform within 60 seconds were gently guided to the platform and allowed to stay there for 30 seconds. The escape latency during the learning phase was recorded and analyzed using ANY-maze software. On day 39, the platform was removed, and a probe test was conducted. The number of platform crossings, the time spent in the target quadrant, the latency to reach the original platform area, and the swimming trajectory were recorded and analyzed.

[0080] Nest building experiment Nesting behavior was assessed on day 41 as an indicator of instinctive behavior and daily living function. Each mouse was placed individually in a clean cage containing 2.5 g of compressed cotton nesting material. After 12 hours, the remaining undisturbed nesting material was weighed. Nesting performance was quantified based on the amount of nesting material used.

[0081] Brain tissue and blood collection On day 42, blood and tissue samples were collected from mice under deep anesthesia. Blood was first collected from the retro-orbital venous plexus into EDTA anticoagulant tubes for plasma metabolomics analysis. Blood samples were placed on ice and centrifuged at 4 °C, 3500 × g for 10 min; the supernatant plasma was collected, aliquoted, and stored. 80 ℃ until analysis.

[0082] After blood collection, mice were perfused with ice-cold 0.9% saline via cardiac perfusion to remove residual blood from the circulatory system. Brain tissue was rapidly removed and processed according to the downstream assay plan. Brain tissue used for immunofluorescence analysis was fixed in 4% paraformaldehyde, post-fixed, cryoprotected, and sectioned for subsequent staining. Hippocampal tissue used for molecular detection and omics analysis was rapidly dissected on ice and used for RNA sequencing, proteomics analysis, real-time quantitative PCR, or Western blotting according to the experimental design. Plasma samples were used for non-targeted metabolomics analysis. Freshly dissected hippocampal tissue was flash-frozen in liquid nitrogen and stored... 80°C until analysis.

[0083] Real-time quantitative PCR analysis Gene expression levels in hippocampal tissue and cultured BV-2 cells were assessed using real-time quantitative PCR (qPCR). BV-2 cell experiments were performed by drug administration when cells reached approximately 70–80% confluence. To assess the effect of compound 10 on mRNA expression, RNA was extracted from BV-2 cells after treatment with 1.5 μM compound 10 for 6 h. In the inflammatory stimulation assay, BV-2 cells were treated according to the specified protocol, and total RNA was collected for downstream analysis.

[0084] Total RNA was extracted from hippocampal tissue or cultured cells using the Hipure Total RNA Mini Kit (Magen, Guangzhou, China) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (ThermoFisher Scientific, USA). Reverse transcription was performed using TransScript one-step gDNA removal and cDNA synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's protocol.

[0085] Real-time quantitative PCR was performed using Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen, Shanghai, China) on a Quantagne q225 real-time PCR system (Kubo Technology, Beijing, China). Each 20 μL reaction volume contained 10 μL SYBR Green Master Mix, 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL cDNA template, and nuclease-free water. The PCR cycling conditions were as follows: 95°C pre-denaturation for 5 min, followed by 40 cycles: 95°C denaturation for 10 s, 60°C annealing / extension for 30 s.

[0086] Relative gene expression was used in 2 - The ΔΔCt method was used for calculation, with Gapdh as the internal reference gene for normalization. Each sample underwent technical triplet analysis.

[0087] Target genes in hippocampal inflammatory pathway analysis include Lgals3 , Tlr4 , MYD88 , Gsdmd and Il1b LPS-stimulated BV-2 cells target genes including Nfkb1 , Il1b and Tnfa Target genes for hippocampal tissue remodeling validation include Notch1 , Hey1 , Wnt5a , Tgfbr1 and Tgfb1 .

[0088] Western blotting of the hippocampus Hippocampal tissue was homogenized in RIPA lysis buffer (Servicebio, Wuhan, China, G2002) supplemented with protease and phosphatase inhibitors (APExBIO, Texas, USA). The lysates were incubated on ice for 30 min and centrifuged at 12,000 × g for 15 min at 4°C. Protein concentration was determined using a BCA protein quantification kit (Meilun Biotechnology, Dalian, China, MA0082).

[0089] Protein samples were mixed with 5× SDS-PAGE loading buffer (Yeasen, Shanghai, China, 20315ES), boiled at 95°C for 5 min, and then subjected to SDS-PAGE electrophoresis using a 5%–15% gel, loading 25 μg of protein per well. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany, ISEQ00010). The membrane was blocked with rapid blocking buffer (Babio, Jinan, China, 200077) at room temperature for 15 min, followed by overnight incubation with primary antibody at 4°C.

[0090] After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Protein bands were developed using an ECL kit (Yeasen, Shanghai, China), and images were acquired using an automated chemiluminescence imaging system (Cytiva, Massachusetts, USA). Band intensities were quantified using ImageJ software and normalized to β-actin or GAPDH. Data are expressed as relative protein expression levels.

[0091] Immunofluorescence staining of brain sections Brain tissue was rapidly dissected and removed, then fixed, cryoprotected, and frozen sectioned to prepare 40 μm thick coronal sections. Sections with clearly visible dorsal dentate gyri were selected for immunofluorescence staining.

[0092] Free sections were permeabilized in PBS containing 0.1% Triton X-100 for 15 min, followed by blocking with immunostaining blocking buffer (Beyond Biotech, Shanghai, P0260, China) at room temperature for 2 h. Sections were incubated overnight at 4°C with primary antibodies against 6E10, IBA1, Galectin-3, DCX, APOE, PSD95, MYD88, TLR4, Notch1, SMAD2 / 3, IL-1β, TGFBR1, TGFBR2, or CD68. After washing three times with PBS for 15 min each time, sections were incubated with secondary antibodies conjugated to the corresponding species-matched fluorescent groups at room temperature for 2 h. After washing three more times with PBS for 15 min each time, sections were mounted with anti-quenching mounting medium containing 5 μM DAPI.

[0093] Images were acquired using a Leica TCS SP8 confocal microscope (Wetzlar, Germany). All samples from the same stained batch were acquired using consistent parameters, including laser intensity, gain, offset, and scan parameters. Representative fields of view of comparable hippocampal regions were selected across groups based on consistent anatomical and image acquisition standards. After background subtraction, fluorescence intensity was quantified using ImageJ / Fiji with consistent threshold parameters in each experiment. Data are expressed as relative fluorescence intensity.

[0094] Aβ-induced inflammatory activation of fibrils and immunofluorescence staining of primary microglia Primary microglia were isolated from newborn C57BL / 6 mice and cultured under the aforementioned standard conditions. Mature microglia in suspension were collected by gentle shaking and centrifugation and seeded onto poly-D-lysine-coated glass coverslips to allow for cell adhesion. After cell adhesion, the medium was replaced with Neurobasal medium supplemented with 2% B27 and 2 mM GlutaMAX.

[0095] To induce inflammatory activation, primary microglia were cultured in serum-free Neurobasal medium containing 2% B27 and 2 mM GlutaMAX with 1 μM fibrillary Aβ. 1-42 Stimulation was performed for 24 h; to evaluate the effect of compound 10, 2 μM of compound 10 was administered concurrently during stimulation. After treatment, the culture medium was removed, and the cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 before immunofluorescence staining.

[0096] After blocking, cells were incubated overnight at 4 °C with primary antibodies against MYD88, TLR4, Galectin-3, IL-1β, TGFBR2, iNOS, or CD68. They were then washed with PBS and incubated at room temperature for 90 min with secondary antibodies conjugated to the corresponding species-matched fluorescent groups. Finally, they were mounted with DAPI-containing anti-quenching mounting medium.

[0097] Images were acquired using confocal microscopy, with consistent acquisition parameters, including exposure time, laser intensity, gain, and offset, for all samples from the same staining batch. Representative fields of view were randomly selected from each coverslip based on consistent image acquisition and cell selection criteria. After background subtraction, fluorescence intensity was quantified using ImageJ / Fiji with consistent threshold parameters in each experiment. Data are expressed as relative fluorescence intensity.

[0098] In the quantitative analysis, each group consisted of n=3 independent primary microglia cultures. Within each independent culture, five fields of view were randomly selected from each coverslip for imaging, and the average value was taken to generate a biological replicate. Statistical analysis used the mean at the culture level as the independent data point, rather than individual cells or fields of view.

[0099] Fluorescence intensity analysis Mean fluorescence intensity of the target channels was quantified using Fiji / ImageJ. Raw immunofluorescence images for all groups were acquired under consistent imaging parameters, including laser power, exposure time, gain, and offset. After importing the images into Fiji, the target fluorescence channels were isolated for analysis. Regions of interest (ROIs) were defined based on anatomical landmarks, DAPI staining, cell morphology, or marker-specific reference channels, with consistent ROI selection criteria used across all experimental groups. Background fluorescence was measured from signal-free regions and subtracted from the measurements for each ROI. The mean gray value of the target channel within each ROI was measured as the mean fluorescence intensity for that region. Values ​​from multiple slices or fields of view for each animal were averaged to generate a biological replicate. The resulting fluorescence intensities were normalized to the control mean and expressed as relative fluorescence intensity. Quantification was performed by researchers unaware of the group assignments.

[0100] 10. Compound 10 intervention and immunofluorescence analysis of the substantia nigra reticularis in A53T Parkinson's disease model mice. The effects of compound 10 on pathological changes in brain tissue related to a Parkinson's disease model were evaluated using 19-month-old A53T transgenic mice. Animals were randomly divided into an A53T model control group and an A53T combined with compound 10 treatment group, with n=4 in each group.

[0101] The carrier consisted of 0.1% hydroxypropyl methylcellulose, 0.2% polysorbate 80, 10% PEG400, and 10% DMSO. Compound 10 was freshly prepared using the above carrier and administered once daily by gavage at a dose of 10 mg / kg, with the volume adjusted according to the mouse's body weight. This administration continued for one month. The A53T model control group received an equal volume of the carrier daily.

[0102] Mice were sacrificed after the last dose and brain tissue was collected. After fixation, cryoprotection and sectioning, coronal sections containing SNr and matching anatomical positions were selected for immunofluorescence staining with pS129-αSyn, IBA1, Gal-3, GFAP and CD68.

[0103] All slides were incubated with the same antibody, and the same conditions were applied for image acquisition and analysis. The mean fluorescence intensity of IBA1 and GFAP in the SNr region was measured using ImageJ / Fiji. Multiple slides or fields of view matching the anatomical location were selected for analysis for each animal, and the average values ​​from each field of view were used as an independent biological replicate for that animal. Data are expressed as mean ± standard error, and unpaired two-tailed Student's t-test was used for comparisons between two groups.

[0104] Example 3: Results of pharmacological studies of the compound 1. Physicochemical properties of the compound and its anti-inflammatory activity against microglia. As shown in Table 1, compounds 1 and 10 were characterized by tPSA, cLogP, and AB-MPS, and their inhibitory effects on LPS-induced NO and TNF-α production were evaluated at a concentration of 1.5 μM. At this concentration and under these experimental conditions, compound 1 did not show detectable inhibition of NO and TNF-α production. Compared to compound 1, compound 10 maintained the same tPSA value (69.04 Å). 2 However, cLogP decreased from 4.8937 to 4.3573, and AB-MPS decreased from 11.8937 to 10.3573. At a concentration of 1.5 μM, compound 10 inhibited NO and TNF-α production by 8.75±5.71% and 54.87±1.79%, respectively, with a more significant inhibitory effect on TNF-α production. These results indicate that, compared with compound 1, compound 10 significantly enhanced the inhibitory effect on microglial inflammatory responses, especially on TNF-α production, while maintaining suitable CNS-directing physicochemical properties.

[0105] Table 1. Physicochemical properties and anti-inflammatory activity against microglia of the compounds. 1 10, Ar represents

[0106] Note: Data are expressed as mean ± SEM, n=3. NO inhibition and TNF-α inhibition represent the inhibition rates of the compounds on LPS-induced NO and TNF-α production, respectively. The inhibition rate is calculated as "100% - relative production level (%)", with higher values ​​indicating stronger inhibition. nd indicates no significant inhibition was detected under the test conditions. tPSA, topological polar surface area; cLogP, calculated octanol / water partition coefficient; AB-MPS, AbbVie multi-parameter score.

[0107] 2. Structural and dynamic simulations support the stable binding of compound 10 to Gal-3. The predicted binding pattern and dynamic stability of compound 10 in the Gal-3 sugar recognition domain were investigated. Molecular docking revealed that compound 10 occupied the Gal-3 binding pocket and formed a cooperative interaction network involving Arg144, His158, Asn160, Arg162, Asn174, and Trp181. Figure 1a). These residues overlap with the anchoring region of FJMU1887 (compound 1), indicating that compound 10 retains the core Gal-3 recognition features while introducing a more compact peripheral substitution pattern. Compared to FJMU1887 (1), compound 10 has a reduced aromatic substituent burden and decreased lipophilicity, which may help improve its CNS-directed physicochemical characteristics while maintaining effective Gal-3 pocket binding.

[0108] Molecular dynamics simulations further support the stability of the Gal-3 / compound 10 complex. During the 100 ns simulation, compound 10 remained bound to the binding pocket, while the protein backbone exhibited limited conformational fluctuations, reflected in the RMSD and RMSF curves. Figure 1 b– Figure 1 d). The solvent-accessible surface area remained relatively stable over time, supporting ligand binding without causing significant conformational instability. Figure 1 e). Hydrogen bond analysis revealed a persistent but dynamically fluctuating interaction between compound 10 and Gal-3, consistent with the persistent pocket occupancy during the simulation. Figure 1 f). A stable radius of gyration indicates that the overall structure of the Gal-3 protein remains compact, without unfolding or significant loosening after ligand binding, strongly supporting the stability of the complex from a global conformational perspective. Figure 1 g).

[0109] Residue-level binding energy analysis identified several pocket residues as major contributors to complex stability, including residues within the anchoring regions of Arg144, Arg162, and Trp181. Figure 1 h). Consistently, Gibbs free energy landscape analysis revealed a distinct low-energy basin, indicating that the Gal-3 / compound 10 complex tended to sample a stable conformational state during simulations. Figure 1 i, Figure 1 j).

[0110] In summary, these structural and kinetic analyses provide a molecular-level basis for the selection of compound 10 and support its further evaluation in Gal-3 binding, blood-brain barrier penetration, pharmacokinetics, and in vivo efficacy studies.

[0111] 3. Compound 10 exhibits good blood-brain barrier penetration and improved pharmacokinetic exposure. The improved physicochemical and anti-inflammatory properties of compound 10 were assessed to determine whether they translated into favorable CNS exposure. Blood-brain barrier penetration was first evaluated using the PAMPA-BBB assay, calibrated with reference compounds covering high, medium, and low CNS penetration. The penetration coefficients of the reference compounds were consistent with their expected CNS classifications, supporting the reliability of the experiment (Table 2). Within this framework, compound 10's... PThe e-value is 8.89 ± 0.24 × 10⁻⁶. -6 cm·s -1 It surpassed CNS + Threshold (4.0 × 10) -6 cm·s -1 ), and is superior to FJMU1887(1) (Pe value 4.24 ± 0.08 × 10 ) -6 cm·s -1 In contrast, the clinically advanced Gal-3 inhibitor TD139 (used for peripheral fibrosis) has lower penetration. P The e-value is 2.90 ± 0.13 × 10⁻⁶. - 6 cm·s -1 Under the same conditions, they are classified as CNS + / - These results support the view that compound 10 is a Gal-3 inhibitor with good predictive power for BBB penetration.

[0112] Table 2. PAMPA-BBB permeability of Compound 10 and the reference compound

[0113] Note: Data are from three independent experiments and are expressed as mean ± SEM. Data from reference: Guo J , et al. N-Propargylamine-hydroxypyridinone hybrids as multitarget agents for the treatment of Alzheimer's disease. Bioorganic Chemistry 113, (2021). Data from the reference: Di L, Kerns EH, Fan K, McConnell OJ, Carter GT. High throughput artificial membrane permeability assay for blood–brain barrier. European Journal of Medicinal Chemistry 38, 223-232 (2003). CNS + Predicted high BBB penetration, Pe > 4.0 × 10⁻⁶ -6 cm / s; CNS - Predicted low BBB penetration, Pe < 2.0 × 10⁻⁶-6 cm / s; CNS + / - The predicted BBB penetration is uncertain, Pe = 2.0–4.0 × 10⁻⁶. -6 cm / s. Pe, effective permeability coefficient.

[0114] To determine whether this permeability translates into measurable brain exposure in vivo, this application administered 20 mg / kg orally to C57 mice. -1 Following the administration of compound 10, the concentrations of compound 10 in plasma, hippocampus, and cortex were quantified. Compound 10 was rapidly absorbed, with peak plasma exposure occurring at 1 hour. max It was 7798.00 ± 1117.00 ng·mL -1 (Table 3). Brain exposure was also detectable at early time points, peaking in both the hippocampus and cortex at 1 hour, reaching 286.10 ± 63.14 ng·mL, respectively. -1 and 475.40 ± 136.60 ng·mL -1 The corresponding 1-hour brain / plasma ratio was approximately 3.7% in the hippocampus and approximately 6.1% in the cortex. Although plasma concentrations decreased after 2 hours, quantifiable levels of compound 10 were still detectable in both brain regions at 4 hours, indicating continued central exposure during the early elimination phase. These data demonstrate that oral administration of compound 10 can achieve measurable exposure in brain tissues associated with Alzheimer's disease pathology.

[0115] Table 3 Plasma and brain concentrations of compound 10 in C57 mice after oral administration.

[0116] Note: Oral administration of 20 mg / kg -1 The concentrations of compound 10 in plasma, hippocampus, and cortex were then measured. Data are expressed as mean ± SEM, n=3.

[0117] This application further describes the single oral administration of 15 mg·kg to rats. -1 Subsequently, the systemic pharmacokinetic characteristics of compound 10 and FJMU1887(1) were compared. Compound 10 was rapidly absorbed, T max The systemic exposure was 0.63 ± 0.07 h, which was significantly improved compared to FJMU1887(1) (Table 4). The C60 of compound 10 was significantly lower than that of FJMU1887(1). max It increased by approximately 41 times (3784.31 ± 1087.06 vs 92.68 ± 15.87 ng·mL). -1 The peak time was earlier (0.63 ± 0.07 vs 2.78 ± 0.72 h), and the AUC was higher. 0-tIt increased by approximately 10-fold (8873.79 ± 2992.41 vs 881.10 ± 113.60 ng·h·mL). -1 The terminal half-life of compound 10 was also longer than that of FJMU1887(1) (91.92 ± 45.88 vs 13.76 ± 1.82 h).

[0118] Combined with its good PAMPA-BBB penetration and proven hippocampal and cortical exposure, these data suggest that compound 10 has significantly improved oral exposure and CNS-guided pharmacokinetic properties compared to compound 1.

[0119] Table 4. Plasma pharmacokinetic parameters of compounds 10 and 1 after gavage administration in rats.

[0120] Note: Administer by gavage at a dose of 15 mg / kg. -1 The values ​​are then expressed as mean ± SEM, n=4. Pharmacokinetic parameters were determined using a non-compartmental model. t1 / 2, terminal half-life; T max Peak time; C max Maximum plasma concentration; AUC 0-t The area under the blood drug concentration-time curve from zero to the last quantifiable time point.

[0121] 4. Compound 10 showed preliminary in vivo safety and improved cognitive and nesting performance in 5×FAD mice. To assess preliminary in vivo safety, ICR mice were administered a single oral dose of 1000 mg / kg. -1 Compound 10 was administered at a dose and monitored for 14 days. Histological examination of heart, brain, liver, and kidney sections showed no significant morphological abnormalities compared to the carrier-treated control group. Figure 2 a). During the observation period, body weight remained stable, and mice in the compound 10 treatment group did not show significant treatment-related weight loss ( Figure 2 (b) These findings support the good preliminary acute safety profile of compound 10 at doses well above the pharmacodynamic test doses.

[0122] The in vivo efficacy of compound 10 was then evaluated in 8-month-old 5×FAD mice—mice at this age with established amyloid pathology and cognitive impairment. According to the experimental timeline, mice were administered compound 10 by gavage, followed by open field tests, new object recognition, Morris water maze, and nesting tests before tissue collection. Figure 2 c). During treatment and behavioral testing, the overall body weight of 5×FAD mice treated with C57, 5×FAD, and compound 10 remained stable, indicating good tolerability of repeated administration of compound 10 under efficacy study conditions. Figure 2 d).

[0123] In the open field test, compared with the C57 control group, 5×FAD mice showed reduced exploration of the central region, while compound 10 did not significantly increase the central movement distance in 5×FAD mice. Figure 2 e). This result suggests that the subsequent cognitive improvement was unlikely to be driven by a general increase in motor or exploratory activity. In the novel object recognition experiment, 5×FAD mice exhibited impaired recognition memory (reduced recognition index). Compared with untreated 5×FAD mice, treatment with compound 10 significantly improved the recognition index, indicating improved recognition memory (e). Figure 2 f, Figure 2 g).

[0124] Spatial learning and memory were further assessed using the Morris water maze. During the 6-day learning training phase, 5×FAD mice showed a prolonged escape latency compared to the C57 control group, while compound 10-treated 5×FAD mice showed a progressively shorter escape latency during training. Figure 2 h). In the probe assay, untreated 5×FAD mice had fewer platform crossings than the C57 control group, while compound 10 treatment significantly increased the number of platform crossings (h). Figure 2 i, Figure 2 (j). These results support the improvement in spatial learning and memory retrieval capabilities following treatment with compound 10.

[0125] Finally, nesting behavior was used to assess instinctive behavioral performance related to daily living functions. Untreated 5×FAD mice showed impaired nesting ability, with significantly reduced nest weight compared to the C57 control group. Compared to untreated 5×FAD mice, compound 10 treatment significantly increased nest weight ( Figure 2 k, Figure 2 In summary, these results indicate that compound 10 demonstrates preliminary in vivo safety and improves recognition and memory, spatial learning and memory abilities, and nesting performance in 8-month-old 5×FAD mice.

[0126] 5. Compound 10 binds to Gal-3 and reduces plaque-associated Gal-3 accumulation and microglial activation in 5×FAD mice. To determine whether compound 10 directly binds to Gal-3, this application used micro-thermophoresis (MST) to determine its binding affinity to the Gal-3 protein. Compound 10 showed concentration-dependent binding to Gal-3, with an estimated dissociation constant KD of 0.51 ± 0.44 μM. Figure 3 (a) This supports its direct target binding and provides a biochemical basis for subsequent in vivo mechanism evaluation.

[0127] Next, we examined whether compound 10 modulated Gal-3-related neuroinflammatory pathology in the hippocampus of 5×FAD mice. Hippocampal sections from C57, 5×FAD, and compound 10-treated 5×FAD mice were stained for Aβ plaques, microglia, and Gal-3. C57 mice showed minimal 6E10 immunoreactivity and low baseline IBA1 and Gal-3 signaling, while 5×FAD mice showed strong 6E10-positive amyloid deposition, increased IBA1-positive microglia activation, and elevated Gal-3 immunoreactivity in the hippocampus. Figure 3 b– Figure 3 e). Compared with untreated 5×FAD mice, compound 10 significantly reduced the fluorescence signals of 6E10, IBA1, and Gal-3, indicating that amyloid-related pathology, microglial activation, and Gal-3 accumulation were all attenuated. Figure 3 c– Figure 3 e).

[0128] High-magnification images further revealed that Gal-3 was enriched around Aβ plaques in 5×FAD mice and spatially co-localized with activated IBA1-positive microglia. This plaque-associated Gal-3-positive microglia phenotype was significantly reduced after treatment with compound 10, while C57 mice showed low Gal-3 signaling and no obvious plaque-associated microglia aggregation. Figure 3 f, Figure 3 g). Consistent with the immunofluorescence results, Western blot analysis showed that the levels of Gal-3 and Aβ proteins in the hippocampus of 5×FAD mice were increased compared with the C57 control group, while treatment with compound 10 decreased both ( Figure 3 h– Figure 3 j).

[0129] To further evaluate whether compound 10 directly inhibits Aβ-induced microglial inflammatory activation, microglia were exposed to fibrillary Aβ with or without compound 10. Fibrillary Aβ significantly increased the immunoreactivity of IL-1β, iNOS, and CD68, indicating strong inflammatory and phagocytic activation in microglia. Figure 3 k– Figure 3 Compound 10 significantly reduced fibrillary Aβ-induced IL-1β, iNOS, and CD68 signaling, supporting its inhibition of Aβ-triggered in vitro microglial inflammatory activation.

[0130] In summary, these findings indicate that compound 10 binds directly to Gal-3 and attenuates plaque-associated Gal-3 accumulation, amyloid-associated pathology, and microglia activation in the 5×FAD hippocampus, while inhibiting fibrillary Aβ-induced microglial inflammatory activation.

[0131] 6. Compound 10 restored hippocampal neurogenesis and synaptic markers in 5×FAD mice. To determine whether the reduced Gal-3-related inflammatory activation is accompanied by hippocampal remodeling, this application examined neurogenesis, synaptic, and plaque-related inflammatory markers. Immunofluorescence staining showed abundant DCX-positive signals in the dentate gyrus of C57 mice, while DCX immunoreactivity was significantly reduced in 5×FAD mice, indicating impaired hippocampal neurogenesis and remodeling. Compared with untreated 5×FAD mice, treatment with compound 10 significantly increased DCX fluorescence intensity, suggesting partial recovery of DCX-related neurogenesis and remodeling. Figure 4 a, Figure 4 c).

[0132] Next, plaque-associated inflammation and the synaptic microenvironment were examined by co-staining APOE, PSD95, and CD68. In C57 mice, APOE and CD68 signals were relatively low, while PSD95 immunoreactivity was preserved. In contrast, 5×FAD mice showed increased APOE and CD68 accumulation, along with decreased PSD95 immunoreactivity, indicating the presence of a disease-associated microenvironment characterized by enrichment of inflammatory markers and loss of synaptic markers. Treatment with compound 10 reduced the fluorescence intensity of APOE and CD68 while restoring PSD95 signal, supporting the coordinated remodeling of inflammation and the synaptic microenvironment. Figure 4 b, Figure 4 d– Figure 4 f).

[0133] Consistent with immunofluorescence results, Western blot analysis showed that the levels of PSD95 and BDNF proteins in the hippocampus of 5×FAD mice were lower than those in the C57 control group. Treatment with compound 10 significantly increased the expression of PSD95 and BDNF, indicating that synaptic integrity and neurotrophic support were restored. Figure 4 g– Figure 4 i).

[0134] In summary, these findings indicate that compound 10 not only attenuates plaque-related inflammatory remodeling but also promotes hippocampal recovery characterized by the restoration of DCX-positive neurogenesis signals, increased PSD95 and BDNF expression, and reduced APOE / CD68-related inflammatory microenvironmental alterations.

[0135] 7. Compound 10 inhibits Gal-3 / TLR4 / MYD88-related inflammatory signaling in 5×FAD hippocampal and microglia. To further clarify the inflammatory pathway regulated by compound 10, this application examined the activity of the Gal-3 / TLR4 / MYD88 axis in the hippocampus of C57, 5×FAD, and compound 10-treated 5×FAD mice. Immunofluorescence staining showed that MYD88, TLR4, and Gal-3 signaling remained at relatively low levels in C57 mice, while all three were significantly increased in the hippocampus of 5×FAD mice. Compared with untreated 5×FAD mice, compound 10 treatment significantly reduced the immunoreactivity of MYD88, TLR4, and Gal-3, indicating that Gal-3-related innate immune signaling was weakened in the 5×FAD hippocampus. Figure 5 a– Figure 5 d).

[0136] Consistent with these findings, Western blot analysis showed that compound 10 reduced the levels of CD68 and IL-1β proteins in the hippocampus of 5×FAD mice. Figure 5 e– Figure 5 g), supporting the inhibition of microglia activation and downstream inflammatory responses in vivo. Further qPCR analysis showed that in the hippocampus of 5×FAD mice... Lgals3 , Tlr4 , MYD88 , Gsdmd and Il1b The mRNA levels of these transcripts were higher than those of the C57 control group, while compound 10 significantly reduced these transcripts. Figure 5 h– Figure 5 These data indicate that compound 10 inhibits Gal-3 / TLR4 / MYD88-related inflammatory transcription in the 5×FAD hippocampus and attenuates GSDMD-related inflammatory activation.

[0137] Next, we examined whether compound 10 directly regulates inflammatory activation in microglia. In LPS-stimulated BV-2 microglia, Nfkb1 , Il1b and Tnfa Expression was significantly induced, while compound 10 significantly attenuated the induction of these inflammatory transcripts. Figure 5 m– Figure 5 o). This application then tested whether compound 10 could inhibit Aβ-related innate immune activation in microglia. In fibrillary Aβ-stimulated microglia, the immunoreactivity of MYD88, TLR4, and Gal-3 was significantly increased compared to control cells, while compound 10 significantly reduced the upregulation of these three markers (o). Figure 5 p– Figure 5 s).

[0138] In summary, these results indicate that compound 10 inhibits Gal-3 / TLR4 / MYD88-related inflammatory signaling in the 5×FAD hippocampus, reduces CD68 and IL-1β protein expression, attenuates inflammation and GSDMD-related transcriptional activation, and directly inhibits LPS and fibrillary Aβ-induced inflammatory responses in microglia.

[0139] Example 4: Evaluation of in vivo efficacy of compound 10 compared to compound 1 To evaluate the in vivo efficacy advantage of compound 10 obtained after structural optimization compared with compound 1, an Alzheimer's disease animal model was established using 8-month-old 5×FAD transgenic mice. Compound 10 (10 mg / kg) or compound 1 (20 mg / kg) was administered by gavage for 35 consecutive days, with n=10 mice in each group.

[0140] During the administration period, the weight change trends of animals in each experimental group were basically the same, and no significant weight loss or abnormal changes were observed. Figure 6 (a) This suggests that both compound 10 and compound 1 exhibit good in vivo tolerability at the experimental doses.

[0141] The Morris water maze test was then used to evaluate the spatial learning and memory abilities of the animals in each group. The results showed that, compared with the normal control group, the escape latency of the 5×FAD model mice was significantly prolonged during training, indicating impaired spatial learning ability. After administration of compound 10, the escape latency of the model mice gradually shortened, showing a significant improvement trend in the later stages of training. Figure 6 b). In the space exploration experiment, treatment with compound 10 increased the number of times 5×FAD mice traversed the original platform location and prolonged the time spent in the original platform area. Figure 6 c and Figure 6 (d) This suggests that compound 10 can improve spatial memory impairment in 5×FAD mice.

[0142] It is noteworthy that, compared with the higher dose of compound 1 (20 mg / kg), compound 10 still showed comparable or better behavioral improvement at a lower dose (10 mg / kg), indicating that the structurally optimized compound 10 has better in vivo efficacy potential.

[0143] Furthermore, nesting experiments showed that the nesting ability of 5×FAD model mice was significantly reduced, while treatment with compound 10 improved nesting indicators, indicating that compound 10 can improve instinctive behavior and daily life-related functions in model mice. Figure 6 e).

[0144] In summary, compared to compound 1, compound 10 was able to improve cognitive function and nesting behavior in 5×FAD mice at lower doses, showing better in vivo efficacy potential and could be a candidate compound for the prevention or treatment of Alzheimer's disease.

[0145] Example 5: The effect of compound 10 on the amelioration of pathological changes in brain tissue of aged A53T Parkinson's disease model mice. To further evaluate the effects of compound 10 on pathological changes associated with other neurodegenerative diseases, in vivo efficacy studies were conducted using 19-month-old A53T transgenic mice (an animal model for studying Parkinson's disease, PD). Animals were randomly divided into an A53T model control group and an A53T combined with compound 10 treatment group, with four mice in each group.

[0146] The carrier consisted of 0.1% hydroxypropyl methylcellulose (HPMC), 0.2% polysorbate 80, 10% PEG400, and 10% DMSO. Compound 10 was freshly prepared using the above carrier and administered once daily by gavage at a dose of 10 mg / kg, with the volume adjusted according to the mouse's body weight, for one month. The A53T model control group received an equal volume of carrier daily. The general condition, activity level, and body weight changes of the mice were monitored regularly during the administration period. Brain tissue was collected after the last administration, and the substantia nigra pars reticulata (SNr) was subjected to immunofluorescence staining and quantitative analysis.

[0147] First, immunofluorescence staining for pS129-αSyn, IBA1, and Gal-3 was used to evaluate pathological α-synuclein phosphorylation, microglia-related changes, and Gal-3 signaling. Compared with the A53T model control group, the compound 10 treatment group showed a decreasing trend in pS129-αSyn and IBA1 signals in the SNr region, and reduced microglia accumulation and activation-like morphology; Gal-3 signaling also showed a decreasing trend. Figure 7 a, Figure 7 d).

[0148] Quantitative results of the mean fluorescence intensity of IBA1 further showed that treatment with compound 10 reduced IBA1 signal in the SNr region of A53T mice, supporting its role in alleviating microglial cell accumulation and abnormal activation. Figure 7 c).

[0149] Further immunofluorescence staining with GFAP and CD68 was used to evaluate changes in astrocyte reactivity and local phagocytosis-lysosome-related signals. Results showed that compared with the A53T model control group, treatment with compound 10 reduced GFAP-positive astrocyte signals, and cell processes changed from a coarse, disordered reactive morphology to a relatively slender morphology, suggesting that abnormal astrocyte activation was alleviated. Figure 7 b). The quantitative results of the average fluorescence intensity of GFAP were consistent with those of the representative images. Figure 7 e).

[0150] It is noteworthy that CD68 signaling did not decrease synchronously with GFAP and IBA1 signaling, suggesting that while compound 10 alleviated excessive glial cell activation, it did not comprehensively inhibit phagocytosis-lysosome-related activities in the SNr region. Figure 7 c and Figure 7 Data for d are expressed as mean ± standard error (mean ± SEM), with n = 4 mice in each group; comparisons between two groups were performed using an unpaired two-tailed Student's t test.

[0151] In summary, under the conditions of this exploratory A53T mouse experiment, compound 10 at 10 mg / kg... -1 After one month of continuous intragastric administration, compound 10 showed improvement in pathological markers related to pS129-αSyn, IBA1, and GFAP in the SNr region. These results provide preliminary experimental support for the use of compound 10 in Parkinson's disease and other α-synuclein-related neurodegenerative diseases.

Claims

1. Use of compounds of formula (I) or pharmaceutically acceptable salts thereof: in, R1 is selected from H and C. 1-6 Alkyl, C 1-6 Alkoxy; R2 is selected from H and C. 1-6 Alkyl, C 1-6 Alkoxy; m is selected from 0, 1, 2, 3, 4, 5; n is selected from 0, 1, 2, 3, 4, and 5; The application is selected from any one, two, or more of the following: (i) Application in the preparation of drugs that inhibit the release of NO or TNF-α from microglia; (ii) Application in the preparation of anti-inflammatory drugs; (iii) Use in the preparation of drugs that reduce pathological-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Application in the preparation of drugs that inhibit microglial cell activation; (v) Use in the preparation of medicaments for the prevention and / or treatment of neuroinflammation; (vi) Use in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases; (vii) Its application in the preparation of drugs that improve cognitive function and restore synaptic integrity; Preferably, the neurodegenerative disease includes Alzheimer's disease and Parkinson's disease.

2. The application according to claim 1, characterized in that, R1 is selected from H and C. 1-4 Alkyl, C 1-4 Alkoxy; Preferably, R2 is selected from H and C. 1-4 Alkyl, C 1-4 Alkyl group.

3. The application according to claim 1 or 2, characterized in that, The compound shown in formula (I) is selected from the compound shown in formula (I-1) below: ； Among them, R1 is the same or different, and is independently selected from C. 1-4 alkyl; R2 is selected from C 1-4 Alkoxy; Preferably, the compound represented by formula (I) is selected from the following specific compounds: 。 4. A pharmaceutical composition comprising a compound of formula (I) according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof; The pharmaceutical composition is used for: (i) To prepare drugs that inhibit the release of NO or TNF-α from microglia; (ii) Preparation of anti-inflammatory drugs; (iii) Prepare drugs that reduce pathological-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Preparation of drugs to inhibit microglial cell activation; (v) To prepare drugs for the prevention and / or treatment of neuroinflammation; (vi) To prepare medicines for the prevention and / or treatment of neurodegenerative diseases; (vii) To prepare drugs that improve cognitive function and restore synaptic integrity; Preferably, the neurodegenerative disease includes Alzheimer's disease and Parkinson's disease.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition optionally includes at least one pharmaceutically acceptable excipient; Preferably, pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, excipients, flocculants and anti-flocculation agents, filter aids, and release inhibitors; Preferably, the pharmaceutical composition is formulated in the following ways: powder, granules, capsules, injections, inhalers, tinctures, oral liquids, tablets, lozenges, or drops.

6. A method comprising administering to a therapeutic subject a therapeutically effective amount of the compound of formula (I) according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 4 or 5; The method is selected from any one, two, or more of the following: (i) Inhibit the release of NO or TNF-α from microglia; (ii) Anti-inflammatory; (iii) Reduce pathology-related Galectin-3 accumulation and / or inhibit Galectin-3 function; (iv) Inhibit microglia activation; (v) Prevention and / or treatment of neuroinflammation; (vi) Prevention and / or treatment of neurodegenerative diseases; (vii) Improve cognitive function and restore synaptic integrity; Preferably, the neurodegenerative disease includes Alzheimer's disease and Parkinson's disease.

7. A method for preparing the compound of formula (I) according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, said preparation method comprising the following steps: (1) Compound SM1 reacts with thionyl chloride to give compound 2A; (2) Compound 2A reacts with compound (I-1) to give compound (I-2); (3) The hydrolysis of compound (I-2) yields compound (I-3); (4) The compound of formula (I-3) reacts with the compound of formula (I-4) to obtain the compound shown in formula (I); The reaction formula is as follows: ； Wherein, R1, R2, m, and n have the definitions described in any one of claims 1-3.

8. The preparation method according to claim 7, characterized in that, In step (1), the reaction is carried out in an organic solvent; preferably, the solvent is methanol; And / or, in step (1), the reaction temperature is 60–90 °C; And / or, in step (1), the reaction time is 0.5 to 5 h; And / or, in step (2), the reaction is carried out in the presence of a base; preferably, the base is selected from potassium carbonate, sodium carbonate or sodium hydroxide; And / or, in step (2), the reaction is carried out in the presence of a halide; preferably, the halide promoter is selected from potassium bromide or potassium iodide; And / or, in step (2), the reaction is carried out in an organic solvent; preferably, the organic solvent is acetonitrile; And / or, in step (2), the reaction temperature is 10–35 °C; And / or, in step (2), the reaction time is 0.5 h to 12 h; And / or, in step (3), the hydrolysis reaction is carried out in the presence of an alkali; preferably, the alkali is selected from sodium hydroxide, potassium hydroxide or potassium carbonate; And / or, in step (3), the reaction is carried out in an organic solvent; preferably, the organic solvent is selected from any one or a combination of methanol, ethanol, tetrahydrofuran; And / or, in step (3), the reaction temperature is 10–35 °C; And / or, after the reaction in step (3) is completed, the reaction solution is adjusted to acidity with hydrochloric acid so that the compound shown in formula (I-3) precipitates; 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 condensing agent; preferably, the condensing agent is HATU; And / or, in step (4), the reaction temperature is 10–35 °C; And / or, in step (4), the reaction time is 1 to 12 h.

Images