An anthocyanin-memantine conjugate, preparation method and application thereof

Abstract

The application belongs to the technical field of chemical drugs, and particularly relates to a cyanine-memantine conjugate, a preparation method and application. The cyanine-memantine conjugate has a simple synthesis method, mild conditions and high yield. In vitro cell experiments show that the cyanine-memantine conjugate can protect neurons and beta-amyloid protein-induced neuron damage. Animal experiments show that intragastric administration of the cyanine-memantine conjugate can improve the learning and memory abilities of an Alzheimer's disease mouse model, has good tolerance, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug technology, specifically relating to an anthocyanin-memantine conjugate, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease. There are currently no effective treatments for AD, and no complete cure has been reported to date. This is related to the unclear pathogenesis. Several hypotheses exist regarding the pathogenesis of AD, including the cholinergic neuron hypothesis, the β-amyloid (Aβ) toxicity hypothesis, the Tau protein hypothesis, the insulin hypothesis, and the inflammatory damage hypothesis. Current research focuses primarily on Aβ deposition and Tau protein neurotangles in AD.

[0003] Inflammatory responses have received increasing attention in recent years. Inflammation is prevalent in the brains of Alzheimer's disease (AD) patients, persisting from the early clinical stages to the terminal phase. Microglia play a crucial role in this inflammatory response. Widely distributed in the brain, microglia function as "scavenger" cells, maintaining the brain's microenvironment and clearing inflammatory plaques. In the pathological conditions of AD, prolonged inflammatory factors lead to excessive activation of microglia, thereby activating related signaling pathways and causing cells to secrete cytokines such as IL-1, IL-6, and TNF-α, which exert toxic effects on nerve cells and neurons. Furthermore, studies have shown a significant increase in the number of activated microglia in AD models, concentrated in the hippocampus. The significant decline in memory and behavioral abilities in these animal models further confirms that microglia can serve as an important target for targeted therapy in the AD inflammatory response.

[0004] Memantine (MEM; CAS19982-08-2) is an NMDA receptor antagonist approved by the FDA in 2003 for the treatment of moderate to severe Alzheimer's disease (AD). MEM primarily works by antagonizing ionotropic NMDA receptors, blocking neuronal damage caused by pathologically elevated glutamate (GLU) levels. However, the clinical efficacy of memantine in treating AD remains insufficient; simply blocking the neurotoxicity of GLU is not effective in halting AD progression. Some studies have focused on structural modifications of memantine, but these modifications have led to unpredictable changes in its target or activity. For example, CN115819328A discloses a memantine urea derivative whose target is not NMDA but soluble cyclooxygenase. This disclosed memantine urea derivative exhibits inhibitory activity against soluble cyclooxygenase, potentially offering efficacy against soluble cyclooxygenase-mediated diseases. However, only in vitro enzyme inhibition experiments were conducted. Rosini et al. disclosed a ferulic acid-memantine covalent conjugate that shows some activity against NMDA receptors. However, the IC of ferulic acid-memantine covalent conjugate 50 It showed higher activity than memantine, but lower intracellular antioxidant activity than ferulic acid. Furthermore, studies revealed that the hydrocarbon group between the amino groups in the linker arm of its covalently coupled compound required at least a 4-carbon chain to achieve acceptable activity, and it also exhibited some cytotoxicity. The compound with the highest activity was compound 7 (Rosini M, Simoni E, Caporaso R, et al. Merging memantine and ferulic acid to probe connections between NMDA receptors, oxidative stress and amyloid-βpeptide in Alzheimer's disease[J]. European Journal of Medicinal Chemistry, Eur J Med Chem, 2019; PMID:31301562.).

[0005] Anthocyanins (CAS 13306-05-3) and their chlorides (CAS 528-58-5) are water-soluble pigments, mainly found in fruits and vegetables, with a core structure of 2-phenylbenzofuran cations. Geranium pigment, cornflower pigment, delphinidin pigment, and peony pigment are the most common types of anthocyanins. Modern pharmacological studies have shown that anthocyanins have broad-spectrum anti-inflammatory effects and potential therapeutic effects on Alzheimer's disease (AD). Anthocyanins can positively influence the anti-neuroinflammatory function of glial cell lines in BV2 and C8-B4 mice, demonstrating that anthocyanins can, to some extent, reduce the inflammatory induction effects of inflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (Cox-2). These inflammatory proteins are substances that appear in the blood during neuroinflammation; their reduced levels indicate that the inflammatory response has been suppressed, thus demonstrating that anthocyanins possess the ability to inhibit neuroinflammation in terms of their products. Studies have shown that anthocyanin treatment of nerve cells can significantly reduce the production and secretion of NO, IL-1β, and TNF-α. These factors are substances that normally exist in the human body and participate in biochemical reactions, but they can trigger inflammatory responses when the nervous system's secretion is imbalanced. Anthocyanins can effectively protect nerve cells by inhibiting the secretion of inflammatory proteins. Therefore, the anti-inflammatory effects of anthocyanins have potential development value in the treatment of Alzheimer's disease (AD). However, to date, anthocyanins and their derivatives have not been effectively developed and studied in the field of AD treatment drugs. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, this invention covalently conjugates anthocyanins and memantine, and investigates their pharmacodynamic characteristics in an AD model. It was found that an anthocyanin-memantine conjugate exhibits good in vitro and in vivo inhibitory effects on the AD model. Based on this, an anthocyanin-memantine conjugate, its preparation method, and its applications are provided.

[0007] The first aspect of this invention provides an anthocyanin-memantine conjugate and a pharmaceutically acceptable salt, solvate, optical isomer, or polymorph. The structural formula of the anthocyanin-memantine conjugate is shown in formula (I); a salt (i.e., hydrochloride) of the anthocyanin-memantine conjugate is shown in formula (II).

[0008]

[0009] A second aspect of the present invention provides a method for preparing the anthocyanin-memantine conjugate shown in formula (I), the synthetic route of which is as follows:

[0010] .

[0011] Its preparation method includes the following steps:

[0012] S1: Memantine is subjected to a reductive amination reaction with glyoxylic acid or glyoxylic acid hydrate under 10% palladium on carbon catalysis to obtain intermediate 1;

[0013] S2: Intermediate 1 undergoes a condensation reaction with (BOC)2O in the presence of a base, introducing a Boc protecting group onto the N atom of intermediate 1 to obtain intermediate 2.

[0014] S3: Intermediate 2 and anthocyanin undergo an ester condensation reaction at 100°C under NDPANI catalysis to obtain intermediate 3;

[0015] S4: The intermediate 3 is deprotected from the Boc protecting group to obtain the anthocyanin-methionine conjugate shown in formula (I).

[0016] Preferably, the alkali mentioned in step S2 is any one of the organic base triethylamine, the inorganic base NaOH, and the inorganic base NaHCO3. More preferably, the alkali mentioned in step S2 is selected from either triethylamine or NaOH.

[0017] The NDPANI (aluminum doped nano polyaniline) mentioned in step S3 is an aluminum nano-polyaniline dopant, which is one of the commonly used catalysts.

[0018] Preferably, step S1 includes the following steps:

[0019] S11: Add memantine and ethylamine to an aqueous solution containing glyoxylic acid or glyoxylic acid hydrate and stir. Keep the reaction temperature below 40°C and stir for 1 hour.

[0020] S12: Transfer the reactants of S11 to a hydrogenation reaction vessel containing 10% palladium on carbon and hydrogenate for 18 hours. Filter to remove the palladium on carbon catalyst, concentrate the filtrate under reduced pressure, and add 2-propanol.

[0021] S13: Concentrate the 2-propanol mixture from step S12 to obtain a solid residue, collect the solid residue, and add it to 2-propanol and stir.

[0022] S14: Filter the 2-propanol mixture from step S13 to collect the solid, wash with ether, and dry to obtain intermediate 1.

[0023] Preferably, step S2 includes the following steps:

[0024] S21: Add intermediate 1 to an alkaline aqueous solution, add (BOC)2O, and stir for 3 hours;

[0025] S22: Add the reactants from step S21 to an aqueous HCl solution, extract with ethyl acetate, wash the organic layer with water and brine, dry with MgSO4, and concentrate and dry under reduced pressure to obtain intermediate 2.

[0026] Preferably, step S3 includes the following steps:

[0027] S31: Intermediate 2 is mixed with anthocyanins and acetic acid, and NDPANI catalyst is added. The reaction is carried out at 100°C with stirring. The reaction process is monitored by thin-layer chromatography.

[0028] S32: The reactants from step S31 were filtered to remove the NDPANI catalyst, washed with ethyl acetate, and saturated NaHCO3 solution was added to remove excess acetic acid. The mixture was extracted with chloroform, the organic layer was dried with anhydrous Na2SO4, and the product was concentrated under vacuum. The resulting product was placed on a silica gel chromatography column and filtered with hexane-ethyl acetate to obtain intermediate 3.

[0029] More preferably, in step 32, the volume ratio of hexane to ethyl acetate in the hexane-ethyl acetate mixture is 8:2.

[0030] Preferably, step S4 includes the following steps:

[0031] S41: Intermediate 3 was dissolved in dichloromethane, trifluoroacetic acid was added, the mixture was stirred at room temperature for 16 hours, and then concentrated under vacuum to obtain the crude product.

[0032] S42: Dissolve the crude product obtained in step S41 in ethyl acetate, wash with NaOH until pH≥9, extract the organic matter with ethyl acetate, dry with anhydrous Na2SO4, and concentrate under vacuum to obtain the anthocyanin-memantine conjugate shown in formula (I).

[0033] A third aspect of the invention provides the use of the anthocyanin-memantine conjugate of formula (I) and pharmaceutically acceptable salts, solvates, optical isomers or polymorphs in the preparation of a medicament for alleviating neuronal damage caused by β-amyloid.

[0034] A fourth aspect of the invention provides the use of the anthocyanin-memantine conjugate of formula (I) and pharmaceutically acceptable salts, solvates, optical isomers or polymorphs in the preparation of a medicament for alleviating Alzheimer's disease.

[0035] The beneficial effects of this invention are:

[0036] (1) In vitro studies using the HT22 mouse hippocampal neuron cell line showed that the anthocyanin-memantine conjugate represented by formula (I) could inhibit Aβ. 25-35 It has a better anti-inflammatory effect than anthocyanins and memantine on inflammatory damage to HT22 cells.

[0037] (2) In vivo studies using the APP / PS1 double transgenic AD mouse model showed that the anthocyanin-memantine conjugate shown in formula (I) administered by gavage could: significantly reduce the expression levels of p-Tau, Aβ, etc. in the hippocampus of APP / PS1 mice, with an inhibitory effect superior to memantine and anthocyanin alone; significantly reduce the expression levels of IL-1β, IL-6, IL-10 and TNF-α in the hippocampus of AD model mice, and have an anti-inflammatory effect superior to anthocyanin and memantine; significantly inhibit the activation of microglia in the hippocampus of AD model mice; and significantly improve the learning and memory abilities of AD model mice.

[0038] (3) In vivo studies using the APP / PS1 double transgenic AD mouse model showed that no drug-related toxic reactions were observed during the 4-month administration and experimental period, indicating good safety.

[0039] (4) The synthesis method of the anthocyanin-memantine conjugate shown in formula (I) of the present invention is simple, mild, and has a high yield. Attached Figure Description

[0040] Figure 1 This is a diagram showing the results of the HT22 cell viability experiment in Example 5;

[0041] Figure 2 Figure 6 shows the experimental results of IL-1β levels in HT22 cells in Example 6.

[0042] Figure 3 Figure 6 shows the experimental results of TNF-α level in HT22 cells in Example 6;

[0043] Figure 4 This is a graph showing the results of the mouse model recognition index experiment in Example 7;

[0044] Figure 5 This is a graph showing the experimental results of the number of errors in the dark avoidance test of the mouse model in Example 7;

[0045] Figure 6 This is a diagram showing the results of the localization and cruise latency experiment in the mouse model of Example 7;

[0046] Figure 7 This is a graph showing the experimental results of the mouse model's dwell time in the target quadrant in Example 7;

[0047] Figure 8 This is a diagram showing the results of the p-Tau expression experiment in the hippocampus of the mouse model in Example 7;

[0048] Figure 9 This is a diagram showing the experimental results of Aβ deposition area in the hippocampus of the mouse model in Example 7;

[0049] Figure 10This is a diagram showing the experimental results of IL-1β protein expression in the hippocampus of the mouse model in Example 7;

[0050] Figure 11 This is a diagram showing the experimental results of TNF-α protein expression in the hippocampus of the mouse model in Example 7;

[0051] Figure 12 This is a diagram showing the experimental results of IL-6 protein expression in the hippocampus of the mouse model in Example 7;

[0052] Figure 13 This is a diagram showing the experimental results of IL-10 protein expression in the hippocampus of the mouse model in Example 7;

[0053] Figure 14 This is a graph showing the fluorescence density results of the IBA-1 mouse model in Example 7;

[0054] Figure 15 HE staining pathological images of heart, liver, kidney, and spleen tissues from the mouse model in Example 7. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. In the synthesis methods of the embodiments, anthocyanins can be cyanidin (CAS registration number 13306-05-3) or its chloride (CAS registration number 528-58-5); memantine is CAS registration number 19982-08-2. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the instructions of the product (such as reagent kits, testing instruments, etc.). Reagents, instruments, cells, and transgenic animals used without specified manufacturers / suppliers are all conventional products that can be purchased through formal channels. Unless otherwise specified, the experimental methods in the embodiments are conventional methods; unless otherwise specified, the experimental materials / reagents used in the embodiments are all commercially available products.

[0056] The synthetic route of the anthocyanin-memantine conjugate of this invention is as follows:

[0057]

[0058] Methods related to compound structure confirmation in the examples:

[0059] (1) LC-MS analysis conditions:

[0060] Chromatographic detection: Instrument model AB SCIEX LC-AD, column model ACQUITY UPLC BEH C 18(100mm × 2.1mm, 1.7μm); column temperature 40℃; injection volume 5μL; flow rate 0.4mL / min; detection time 15min; mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was pure acetonitrile; elution program: 0-5min: 5%–95% B; 5–11min: 95% B; 11–12min: 95%–5% B; 12–15min: 5% B. Mass spectrometry detection: instrument model AB SCIEX 5600Plus, positive ion detection mode, primary mass spectrometry and secondary mass spectrometry scanning ranges are 100–1200m / z and 50–1200m / z, respectively; nitrogen was used in all gas paths; detailed parameters are shown in Tables 1 and 2.

[0061] Table 1 Positive Ion Mass Spectrometry Conditions

[0062]

[0063] Table 2 Mass Spectrometry Negative Ion Conditions

[0064]

[0065] NMR analysis: The obtained samples were dissolved in deuterated dimethyl sulfoxide (DMSO) and placed in an NMR tube for NMR analysis using a Bruker Avance Core 400MHz NMR spectrometer. 1 H-NMR detection.

[0066] Synthesis of Intermediate 1 in Example 1

[0067] 1 mol of ammonium nitrate and 1.25 mol of ethylamine (100 mL 70% aqueous solution) were added to 700 mL of glyoxylic acid monohydrate (92.05 g, 1 mol) aqueous solution and stirred. The reaction temperature was maintained below 40 °C (35 °C was sufficient here). The mixture was stirred for 1 h, then transferred to a 200 mL pressure hydrogenation flask containing 20 g of 10% palladium on carbon. The mixture was hydrogenated at 50 psi for 18 h. The palladium on carbon catalyst was removed by filtration, and the filtrate was concentrated to half its volume under reduced pressure (temperature <50 °C; 45 °C was sufficient here). 100 mL of 2-propanol was added to the mixture, and the mixture was concentrated to obtain a solid residue. The solid residue was collected and stirred in 2-propanol. The solid was filtered, washed with diethyl ether, and dried to give intermediate 1, yield 65%. MS: ESI-[MH] - 175.1593; 1H NMR(400MHz,DMSO-d6)δ2.20(p,J=4.6Hz,1H),1.81(dd,J=13.0,4.3Hz,1H),1.72-1.57(m,3 H), 1.50 (s, 1H), 1.49-1.39 (m, 3H), 1.33 (dd, J = 13.0, 4.7Hz, 2H), 1.23 (s, 2H), 0.87 (s, 5H).

[0068] Synthesis of Intermediate 2 in Example 2

[0069] Intermediate 1 (1 mmol) was added to 100 mL of an aqueous solution containing triethylamine (4.18 mL, 30.0 mmol), followed by (BOC)₂O (10.0 mmol) of di-tert-butyl dicarbonate, and stirred for 3 h. The reaction mixture was poured into an aqueous solution of HCl (1 IN, 30 mL), and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic layer was washed with distilled water (1 × 100 mL) and brine (2 × 50 mL), filtered, and dried over MgSO₄. Finally, the organic layer was concentrated under reduced pressure and dried to give intermediate 2, with a yield of 80%. MS: ESI-[MH] - 336.2172; 1 H NMR(400MHz,DMSO-d6)δ2.14(tt,J=4.7,4.0Hz,1H),2.06-1.95(m,3H),1.87(dd,J=13.0,4.0Hz ,1H),1.78(d,J=13.0Hz,2H),1.49-1.40(m,2H),1.43(s,12H),1.35-1.14(m,4H),0.90(s,6H).

[0070] Synthesis of Intermediate 3 in Example 3

[0071] Intermediate 2 (1.5 mmol), anthocyanin (1.5 mmol), and acetic acid (3 mmol) were placed in a round-bottom flask. NDPANI catalyst (0.3% of the mass of intermediate 2) was added to the reaction mixture. The mixture was stirred continuously at 100 °C, and the reaction was monitored by thin-layer chromatography. The NDPANI catalyst was removed by filtration, and the reaction mixture was washed with ethyl acetate. Saturated NaHCO3 solution was added to the reaction mixture to remove excess acetic acid. The mixture was extracted with chloroform, and the organic layer was dried over anhydrous Na2SO4. The mixture was then evaporated under vacuum. The crude product was placed on a silica gel column for purification and separation using hexane and ethyl acetate (8:2). The purified product was evaporated to dryness to obtain intermediate 3, with a yield of 75%. MS: ESI-[MH] - 605.2617; 1H NMR(400MHz,DMSO-d6)δ7.98(dd,J=8.2,1.8Hz,1H),7.96(s,1H),7.91(d,J=1.8Hz,1H),7 .38(d,J=8.2Hz,1H),6.84(d,J=2.2Hz,1H),6.75-6.70(m,1H),4.33-4.21(m,1H),2.14(tt ,J=4.7,4.0Hz,1H),2.06-1.95(m,2H),1.87(dd,J=13.0,4.0Hz,1H),1.78(d,J=13.0Hz,1 H), 1.49-1.40 (m, 1H), 1.43 (s, 8H), 1.33 (d, J = 4.6Hz, 1H), 1.32-1.14 (m, 2H), 0.90 (s, 4H).

[0072] Example 4 Synthesis of the anthocyanin-memantine conjugate shown in formula (I)

[0073] TFA (10 mL) was added to a solution of intermediate 3 (1 mmol / L) dissolved in dichloromethane (10 mL). The mixture was stirred at room temperature for 16 h, and the reaction mixture was concentrated under vacuum to obtain a residue. The crude product was dissolved in ethyl acetate (10 mL) and washed with 2 M NaOH until pH ≥ 9. The organic matter was extracted with ethyl acetate (3 × 20 mL). The crude product was placed on a silica gel column for column chromatography and purified and separated with hexane and ethyl acetate (6:2) in 70% yield. The organic matter was dried on anhydrous Na₂SO₄ and concentrated under vacuum to obtain the anthocyanin-memantine conjugate shown in formula (I). MS: ESI-[MH] - 505.2094; 1 HNMR(400MHz,DMSO-d6)δ7.98(dd,J=8.2,1.8Hz,1H),7.96(s,1H),7.91(d,J=1 .7Hz,1H),7.38(d,J=8.2Hz,1H),6.84(d,J=2.2Hz,1H),6.75-6.70(m,1H),2.2 1(hept,J=4.4Hz,1H),1.84-1.74(m,3H),1.64(dd,J=13.0,4.6Hz,3H),1.46(d d, J=13.0, 4.3Hz, 2H), 1.33 (dd, J=13.0, 4.3Hz, 2H), 1.25 (s, 2H), 0.87 (s, 5H).

[0074] Example 5: In vitro neuroprotective pharmacodynamic study

[0075] HT22 mouse neurons were seeded into 96-well plates and cultured for 24 hours in a 5% CO2, 37°C incubator. The control group was then replaced with fresh high-glucose DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 g / L streptomycin, and cultured for another 24 hours. Different concentrations of the anthocyanin-memantine conjugate solution obtained in Example 4, or different concentrations of anthocyanin and memantine solutions, were added to achieve final drug concentrations of 5, 10, 20, 40, and 80 μM (using high-glucose DMEM medium as the solvent). After 2 hours of culture, a final concentration of 20 μmol·L⁻¹ was added. -1 Aβ 25-35 Continue culturing for 24 hours. Replace the culture medium in the control group with fresh medium and continue culturing for 24 hours.

[0076] Two hours after the model group was replaced with fresh culture medium, Aβ was added. 25-35 The solution was prepared to achieve a final concentration of 20 μmol·L⁻¹. -1 Continue culturing for 24 hours. After culturing, add 50 μL of MTT (thiazolyl blue) to each well and continue culturing in a 5% CO2, 37℃ incubator for another 4 hours. Discard the culture medium, add 150 μL of DMSO, mix well on a plate shaker, and read the plate at 495 nm using a microplate reader. Calculate the cell inhibition rate based on the measured absorbance values. Data are expressed as Mean ± SD (n = 6). Results are as follows: Figure 1 As shown.

[0077] Within the concentration range of 5–80 μmol / L (i.e., μM), the anthocyanin-memantine conjugate increases Aβ in a concentration-dependent manner. 25-35 The cell viability of HT22 cells treated with anthocyanin-memantine was significantly increased at a concentration of 80 μM, showing a highly significant difference compared to the model group (p < 0.001). Furthermore, at each experimental concentration, the anthocyanin-memantine conjugate showed effects on Aβ... 25-35 The protective effect against HT22 cell damage was significantly better than that of the control drugs anthocyanins and memantine at the same concentration (p<0.05).

[0078] Example 6: In vitro anti-inflammatory pharmacodynamic study

[0079] HT22 mouse neurons were cultured and modeled according to the method in Example 5. The drugs used for pretreatment before modeling were the anthocyanin-memantine conjugate solution (80 μM) or anthocyanin (80 μM) and memantine solution (80 μM) obtained in Example 4.

[0080] ELISA kits were used to determine the levels of IL-1β (mouse interleukin 1β ELISA kit, D721017) and TNF-α (mouse tumor necrosis factor α ELISA kit, D721217) according to the kit instructions. After culture, 50 μl of culture medium was added to each well, followed by 50 μl of biotin-labeled antibody. The mixture was gently vortexed and incubated at 37°C for 1 h. The plate was washed three times, and 50 μl of streptavidin-HRP was added to each well. The mixture was gently vortexed and incubated at 37°C for 30 min. The plate was washed three times, and 50 μl each of substrates A and B were added to each well. The mixture was gently vortexed and incubated at 37°C for 10 min. Light exposure was avoided. The plate was removed, and 50 μl of stop solution was quickly added. Results were measured immediately after adding the stop solution. The OD value of each well was measured at 450 nm. Results are shown below. Figure 2 , 3 As shown.

[0081] At the experimental concentrations, the levels of IL-1β and TNF-α in the anthocyanin-memantine conjugate pretreatment group were significantly lower than those in the model group (p<0.01), anthocyanin (p<0.05), and memantine (p<0.05), indicating that the anthocyanin-memantine conjugate has a significant effect on Aβ. 25-35 The anti-inflammatory effect of HT22 cells damaged by the drug was significantly better than that of anthocyanins and memantine (p<0.05).

[0082] Example 7: In vivo pharmacodynamic study

[0083] Effects of anthocyanin-memantine conjugate on learning and memory in APP / PS1 AD model mice.

[0084] Five-month-old APP / PS1 double transgenic mice were randomly divided into a model group, a memantine group (5 mg / kg), an anthocyanin group (100 mg / kg), and an anthocyanin-memantine conjugate group (5.6 mg / kg) based on body weight. Litter-negative C57BL / 6J mice served as a control group. Each group consisted of 20 mice, half male and half female. Mice in the drug-treated groups were administered the drugs once daily by gavage (each drug was prepared with double-distilled water) for 4 months. Simultaneously, mice in the model group and wild-type group (i.e., control group) were administered the same volume of double-distilled water by gavage. Behavioral experiments (new object recognition test, darkness avoidance test, and Morris water maze test) were conducted 3 months after drug administration. Drug administration continued until the mice were sacrificed at 4 months of age.

[0085] The experimental results of the new object recognition experiment, the darkness avoidance experiment, and the Morris water maze experiment are shown in [the table]. Figures 4 to 7Compared with the model group, mice in the methimazole group, anthocyanin group, and anthocyanin-methimazole conjugate group showed significantly improved recognition index in the new object recognition test (p<0.01, p<0.05, p<0.01), significantly reduced errors in the dark avoidance test (p<0.05, p<0.05, p<0.01), significantly reduced escape latency in the water maze test (p<0.01, p<0.05, p<0.01), and significantly increased total time spent in the target quadrant (p<0.01, p<0.01, p<0.01). Furthermore, the improvement effect of the anthocyanin-methimazole conjugate group was significantly better than that of the methimazole group and the anthocyanin group (p<0.05).

[0086] After euthanizing the mice, the effects of different therapeutic drugs on the expression of p-Tau and Aβ in the hippocampus of APP / PS1 double transgenic AD mice were evaluated according to the kit instructions, the Western blot method reported by Wang Huping et al., and the immunohistochemical method reported by Zhao Min et al. (Wang Huping, Zhou Jun, Li Mingcheng, et al. Exploring the mechanism of action of Danzhi Xiaoyao San on tau protein phosphorylation level in Alzheimer's disease model rats based on PP2A and GSK-3β [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2023, 29(06):63-70; Zhao Min, Kong Yanying, Yan Huacheng, et al. Cognitive improvement and anti-inflammatory effects of CYP46A1 overexpression in transgenic Alzheimer's disease model mice [J]. PLA Medical Journal, 2018, 43(04):271-277.). The results are shown in the table below. Figure 8 , Figure 9 Anthocyanin-memantine conjugate significantly reduced the expression levels of p-Tau (p<0.05) and Aβ (p<0.05) in the hippocampus of APP / PS1 mice, and the inhibitory effect of the anthocyanin-memantine conjugate group was better than that of the memantine group and the anthocyanin group, while the expression level of Aβ was significantly lower than that of the anthocyanin group (p<0.05).

[0087] Further, referring to the kit instructions and the Western blot analysis reported by Wang Jiawen et al., the effects of different drugs on the expression of IL-1β, IL-6, IL-10 and TNF-α proteins in the hippocampus of APP / PS1 double transgenic AD mice were analyzed (Wang Jiawen, Wu Jun, Wang Chengfei, et al. Aqueous extract of Dendrobium nobile alleviates learning and memory impairment by regulating synaptic plasticity and glial cell activation in APP / PS1 mice [J]. Journal of Chongqing Medical University, 2023, 48(10):1208-1214.). The results are shown in the table below. Figures 10 to 13 .

[0088] All three drug treatments inhibited the expression of IL-1β, IL-6, IL-10, and TNF-α in the hippocampus of AD model mice. Compared with the model group, the levels of TNF-α (p<0.05, p<0.05, p<0.01) and IL-1β (p<0.05, p<0.05, p<0.01) in the memantine group, anthocyanin group, and anthocyanin-memantine conjugate group were significantly reduced, and the levels of IL-6 (p<0.05) and IL-10 (p<0.05) in the anthocyanin-memantine conjugate group were significantly lower than those in the model group. These results indicate that the anti-inflammatory effect of the anthocyanin-memantine conjugate is superior to that of memantine and anthocyanins alone.

[0089] Finally, referring to the kit instructions and the immunofluorescence staining method reported by Yang Jige et al., the effects of different therapeutic drugs on the activation level of hippocampal microglia in APP / PS1 double transgenic AD mice were observed and evaluated (Yang Jige, Li Lixin, Li Zhonghua, et al. Regulation of LSD1 / PSD95 by Cornus officinalis extract on Aβ). 25-35 Study on the protective effect of induced neurological injury in Alzheimer's disease mice [J]. Journal of Beijing University of Chinese Medicine, 2024, 47(03):352-363.). Results are shown in Figure 14 The level of IBA-1 (microglia marker)-labeled microglia in the model group was significantly higher than that in the control group. After drug treatment, the fluorescence intensity of IBA-1 in the memantine group, anthocyanin group, and anthocyanin-memantine conjugate group was significantly decreased (p<0.05, p<0.05, p<0.01), and the fluorescence intensity of IBA-1 in the anthocyanin-memantine conjugate group was significantly lower than that in the memantine group and the anthocyanin group (p<0.05). This indicates that the anthocyanin-memantine conjugate can significantly inhibit the activation of microglia in the hippocampus of AD model mice.

[0090] Gross observation and histopathological examination of the animals (heart, liver, spleen, kidneys) were conducted during the animal experiments. Animals in the anthocyanin-memantine conjugate group had smooth, shiny fur, and their weight gain was comparable to the control group. There were no significant differences in water intake and food consumption compared to the control group. Histopathological examination revealed no drug-related adverse reactions or organ damage (such as...). Figure 15 (As shown).

[0091] The above experimental results demonstrate that the anthocyanin-memantine conjugate of formula (I) of this invention can enhance the viability of Aβ-induced HT22 cells and reduce the level of inflammatory response, thus alleviating neuronal damage caused by β-amyloid protein. Animal experiments have also demonstrated that the anthocyanin-memantine conjugate of formula (I) can significantly improve hippocampal markers and microglial activation in AD model mice, and improve the learning and memory abilities of the model mice. Even at low doses (lower than mol / kg of memantine and anthocyanins), the improvement effect on hippocampal markers, microglial activation markers, and learning and memory abilities in model mice is still significantly better than that of memantine and anthocyanins. Furthermore, the anthocyanin-memantine conjugate can also exert its pharmacological effects orally, making it suitable for convenient administration methods such as oral administration.

[0092] Obviously, without departing from the principles of this invention, those skilled in the art can make various modifications and variations to this invention, such as using pelargonidin, delphindin, peonidin, petunidin, and malvidin to replace the anthocyanins of this invention, and appropriately changing the synthesis method to prepare the corresponding anthocyanin-memantine conjugates. In particular, peonidin differs from the anthocyanins used in this invention only in the alkyl group at the coupling linker position. These modifications and variations fall within the scope of the claims of this invention and their equivalents.

Claims

1. The anthocyanin-memantine conjugate of formula (I) or a pharmaceutically acceptable salt thereof: 。 2. The method for preparing the anthocyanin-memantine conjugate shown in formula (I), characterized in that, Includes the following steps: S1: Memantine reacts with glyoxylic acid or glyoxylic acid hydrate under 10% palladium on carbon catalysis to give intermediate 1, with the following structure: ； S2: Intermediate 1 undergoes a condensation reaction with (BOC)₂O in the presence of a base, introducing a Boc protecting group onto the N atom of intermediate 1 to obtain intermediate 2, with the following structure: ； S3: Intermediate 2 and anthocyanin undergo an ester condensation reaction at 100℃ under NDPANI catalysis to obtain intermediate 3, with the following structure: ； S4: Deprotect the Boc protecting group from intermediate 3 to obtain the anthocyanin-memantine conjugate shown in formula (I), with the following structure: 。 3. The preparation method according to claim 2, characterized in that, The base mentioned in step S2 is selected from either triethylamine or NaOH.

4. The preparation method according to claim 2, characterized in that, Step S1 includes the following steps: S11: Add memantine and ethylamine to an aqueous solution containing glyoxylic acid or glyoxylic acid hydrate and stir. Keep the reaction temperature below 40°C and stir for 1 hour. S12: Transfer the reactants of S11 to a hydrogenation reaction vessel containing 10% palladium on carbon and hydrogenate for 18 hours. Filter to remove the palladium on carbon catalyst, concentrate the filtrate under reduced pressure, and add 2-propanol. S13: Concentrate the 2-propanol mixture from step S12 to obtain a solid residue, collect the solid residue, and add it to 2-propanol and stir. S14: Filter the 2-propanol mixture from step S13 to collect the solid, wash with ether, and dry to obtain intermediate 1.

5. The preparation method according to claim 4, characterized in that, Step S2 includes the following steps: S21: Add intermediate 1 to an alkaline aqueous solution, add (BOC)2O, and stir for 3 hours; S22: Add the reactants from step S21 to an aqueous HCl solution, extract with ethyl acetate, wash the organic layer with water and brine, dry with MgSO4, and concentrate and dry under reduced pressure to obtain intermediate 2.

6. The preparation method according to claim 5, characterized in that, Step S3 includes the following steps: S31: Intermediate 2 is mixed with anthocyanins and acetic acid, and NDPANI catalyst is added. The reaction is carried out at 100°C with stirring. The reaction process is monitored by thin-layer chromatography. S32: The reactants from step S31 were filtered to remove the NDPANI catalyst, washed with ethyl acetate, and excess acetic acid was removed by adding saturated NaHCO3 solution. The mixture was extracted with chloroform, dried with anhydrous Na2SO4, and evaporated under vacuum. The resulting product was placed on a silica gel column for column chromatography with a mixture of hexane and ethyl acetate to obtain intermediate 3.

7. The preparation method according to claim 6, characterized in that, In step 32, the volume ratio of hexane to ethyl acetate in the hexane-ethyl acetate mixture is 8:

2.

8. The preparation method according to claim 7, characterized in that, Step S4 includes the following steps: S41: Intermediate 3 was dissolved in dichloromethane, trifluoroacetic acid was added, the mixture was stirred at room temperature for 16 hours, and then concentrated under vacuum to obtain the crude product. S42: Dissolve the crude product obtained in step S41 in ethyl acetate, wash with NaOH until pH≥9, extract the organic matter with ethyl acetate, dry with anhydrous Na2SO4, and concentrate under vacuum to obtain the anthocyanin-memantine conjugate shown in formula (I).

9. The use of the anthocyanin-memantine conjugate of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a drug for alleviating neuronal damage caused by β-amyloid.

10. The use of the anthocyanin-memantine conjugate of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for alleviating Alzheimer's disease.

Images