Medicine for treating Alzheimer's disease and preparation method thereof

Abstract

The invention relates to the field of medicines, in particular to a medicine for treating Alzheimer's disease and a preparation method thereof. The invention provides a compound with a structure as shown in a formula (1), application of the compound as an active ingredient or a medicine finished product for treating Alzheimer's disease, and a preparation method of the compound. Tests prove that the compound has an excellent effect of treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a medicine for treating Alzheimer's disease and a preparation method thereof. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. It usually occurs in middle to late adulthood. Patients will experience brain atrophy and clinical manifestations include a series of mental and cognitive disorders, such as memory loss and behavioral changes, which seriously affect their ability to live a normal life.

[0003] Currently, there are limited types of drugs for treating Alzheimer's disease (AD), and their efficacy varies; therefore, there is a need to develop a drug with stable efficacy for treating Alzheimer's disease (AD). Summary of the Invention

[0004] In view of the above technical problems, the technical content of the present invention is proposed, and the present invention discloses a drug for treating Alzheimer's disease and a preparation method thereof.

[0005] The present invention provides a compound having a structure as shown in formula (A):

[0006]

[0007] In formula (A), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R3 is

[0008] The present invention also provides a compound having a structure as shown in formula (B):

[0009]

[0010] In formula (B), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3.

[0011] Furthermore, the above compound may be a compound having a structure as shown in formula (1).

[0012]

[0013] Furthermore, the above compounds have activity in treating Alzheimer's disease.

[0014] The present invention also provides use of the above compound as a drug for treating Alzheimer's disease.

[0015] The present invention provides a drug for treating Alzheimer's disease, the structure of which is as shown in formula (1):

[0016]

[0017] The present invention provides a method for preparing the drug, comprising the following steps:

[0018] Step 1: Prepare product 1-2; the structural formula of product 1-2 is as shown in formula (2):

[0019]

[0020] Step 2: Prepare product 1-3; the structural formula of product 1-3 is as shown in formula (3):

[0021]

[0022] Step 3: Prepare product 1-4; the structural formula of product 1-4 is as shown in formula (4):

[0023]

[0024] Step 4: Use the drug represented by the product formula (1).

[0025] The preparation method comprises the following steps:

[0026] Step 1: dissolve methyl 2-amino-4,6-dimethoxybenzoate in a solvent, add benzaldehyde, acetic acid and NaBH3CN to react, add water to mix, extract with an extractant, concentrate the extract, and dry to obtain product 1-2;

[0027] Step 2: dissolving the product 1-2 and a bromine-containing reagent in a solvent, reacting, then adding water, extracting with an extractant, concentrating, and purifying to obtain the product 1-3;

[0028] Step 3: Dissolve product 1-3 and ammonium acetate (NH4OAc) in a solvent, react, concentrate, and obtain a thick substance, which is purified to obtain a white solid; then dissolve the white solid in a solvent, stir, and add boron tribromide (BBr3), react, then add water, extract with an extractant, and concentrate to obtain product 1-4;

[0029] Step 4: dissolving the product 1-4 and 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-yl)-1,3,2-dioxaborolane in a solvent, adding a base and a catalyst for reaction, extracting, concentrating, and purifying to obtain a drug for treating Alzheimer's disease represented by formula (1).

[0030] The synthesis process route of the above steps is:

[0031]

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] The present invention provides a compound having a structure of formula (A), (B), and (1), which is used as an active drug or medicine for treating Alzheimer's disease, and provides a method for preparing the compound. Tests conducted in the present invention have verified that the compound has excellent therapeutic effects on Alzheimer's disease.

[0034] The study confirmed the successful establishment of the mouse AD model through behavioral tests such as the water maze, and found that NB798 and huperzine A compound-mediated treatment could improve AD symptoms such as memory loss in mice to a certain extent.

[0035] By detecting the GFAP level in mouse serum, it was found that the GFAP level in the AD group mice was significantly higher than that in the Control group, and each drug-treated group was able to partially downregulate the increase in GFAP caused by the drug, thereby intervening in the AD process.

[0036] In addition, compared with the control group, the brain inflammation of the AD group mice increased and stress and other responses induced APOE4 expression, aggravating AD brain damage; after treatment mediated by the drug NB798, the expression of TNF and APOE4 in brain tissue decreased significantly, indicating that drug intervention can protect the neurons of AD mice and reduce brain tissue damage.

[0037] Pathological examination of the hippocampus region of the mouse brain revealed that a large number of loosely structured neurons were swollen in the hippocampus region of the AD group mice, accompanied by a significant increase in the expression of NFT and phosphorylated tau protein, as well as a large number of activated microglia. Treatment with the drug NB798 had a certain therapeutic effect on these phenomena, suggesting that the NB798 compound can alleviate brain nerve damage in AD mice, thereby affecting the disease progression of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : H NMR spectrum of product 1.

[0039] Figure 2 : Mass spectrum of product 1.

[0040] Figure 3 : H NMR spectrum of product 1-2.

[0041] Figure 4 : Mass spectrum of product 1-2.

[0042] Figure 5 : H NMR spectrum of product 1-3.

[0043] Figure 6 : Mass spectrum of product 1-3.

[0044] Figure 7 : H NMR spectrum of product 1-4.

[0045] Figure 8 : Mass spectrum of product 1-4.

[0046] Figure 9 : Open field test results.

[0047] Figure 10 : Movement trajectory of mice in water maze experiment.

[0048] Figure 11 : The number of times mice passed the platform in the water maze experiment.

[0049] Figure 12 : Movement trajectory of mice in conditioned fear experiment.

[0050] Figure 13 : Number of freezing times in conditioned fear experiment.

[0051] Figure 14 :qPCR test.

[0052] Figure 15 :ELISA test.

[0053] Figure 16 : HE staining test.

[0054] Figure 17 :IHC-APP (app-like precursor protein) expression test.

[0055] Figure 18 : IHC-P-tau (phosphorylated tau protein) expression test.

[0056] Figure 19 : IHC-NeuN (neuron) expression level test.

[0057] Figure 20 : IF-Iba1 (microglia) expression level test.

[0058] Figure 21 : IF-CD68 (microglia) expression test. DETAILED DESCRIPTION

[0059] The present invention is described and illustrated in detail by providing specific embodiments.

[0060] The present invention provides a compound having a structure as shown in formula (A):

[0061]

[0062] In formula (A), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R3 is

[0063] The present invention also provides a compound having a structure as shown in formula (B):

[0064]

[0065] In formula (B), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3.

[0066] Furthermore, the above compound may be a compound having a structure as shown in formula (1).

[0067]

[0068] The above compounds have activity in treating Alzheimer's disease.

[0069] The present invention also provides use of the compound as a drug for treating Alzheimer's disease.

[0070] The present invention provides a drug for treating Alzheimer's disease, the structure of which is as shown in formula (1):

[0071]

[0072] The present invention provides a method for preparing the drug, comprising the following steps:

[0073] Step 1: Prepare product 1-2; the structural formula of product 1-2 is as shown in formula (2):

[0074]

[0075] Step 2: Prepare product 1-3; the structural formula of product 1-3 is as shown in formula (3):

[0076]

[0077] Step 3: Prepare product 1-4; the structural formula of product 1-4 is as shown in formula (4):

[0078]

[0079] Step 4: Use the drug represented by the product formula (1).

[0080] The preparation method comprises the following steps:

[0081] Step 1: dissolving methyl 2-amino-4,6-dimethoxybenzoate in a solvent, adding benzaldehyde, acetic acid and NaBH3CN to react, adding water to mix, extracting with an extractant, concentrating the extract, and drying to obtain product 1-2 (methyl 2-benzylamino-4,6-dimethoxybenzoate as a white solid);

[0082] Step 2: dissolving the product 1-2 and a bromine-containing reagent in a solvent, reacting, then adding water, extracting with an extractant, concentrating, and purifying to obtain the product 1-3 (white solid 6-benzylamino-3-bromo-2,4-dimethoxybenzoic acid methyl ester);

[0083] Step 3: dissolving the product 1-3 and ammonium acetate NH4OAc in a solvent, reacting, concentrating to obtain a thick substance, purifying to obtain a white solid (1-benzyl-6-bromo-5,7-dimethoxyquinazolin-4-one); then dissolving the white solid in a solvent, stirring and adding boron tribromide BBr3, reacting, then adding water, extracting with an extractant, and concentrating to obtain the product 1-4 (white solid 1-benzyl-6-bromo-5-hydroxy-7-methoxyquinazolin-4-one);

[0084] Step 4: Dissolve the product 1-4 and 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-yl)-1,3,2-dioxaborolane in a solvent, add a base and a catalyst for reaction, extract, concentrate, and purify to obtain a drug for treating Alzheimer's disease (Formula (1), white solid, 1-benzyl-5-hydroxy-7-methoxy-6-(3-methylbut-2-en-1-yl)quinazolin-4-one).

[0085] The synthesis process route of the above steps is:

[0086]

[0087] In each step, the extractant includes ethyl acetate.

[0088] In each step, purification was performed by column chromatography.

[0089] In step 2, high performance liquid column chromatography is used for purification.

[0090] In step 4, purification is performed by silica gel column chromatography.

[0091] The above-mentioned bromine-containing reagent includes N-bromosuccinimide NBS.

[0092] The base used above includes at least one of carbonate or bicarbonate.

[0093] The catalyst used above includes Pd(dtbpf)Cl2.

[0094] The solvent in step 1 includes methanol.

[0095] The solvent in step 2 includes dimethylformamide.

[0096] The solvent in step 3 includes at least one of diethoxymethoxyethane and dichloromethane.

[0097] The solvent in step 4 includes 1,4-dioxane.

[0098] In step 1, the preparation method of methyl 2-amino-4,6-dimethoxybenzoate comprises: stirring 2-amino-4,6-dimethoxybenzoic acid, potassium carbonate, dimethylformamide, and iodomethane to react, diluting with water, extracting with ethyl acetate, and concentrating under reduced pressure to obtain a white solid, namely methyl 2-amino-4,6-dimethoxybenzoate. The reaction temperature can be room temperature, and the reaction time can be 2-10 hours.

[0099] In step 1, the ratio of 2-amino-4,6-dimethoxybenzoic acid, potassium carbonate, dimethylformamide and iodomethane is 130-175 g: 200-240 g: 1000-3000 mL: 90-130 g.

[0100] In step 1, the ratio of methyl 2-amino-4,6-dimethoxybenzoate: solvent: benzaldehyde: acetic acid: NaBH3CN is 90-120 g: 800-2000 mL: 150-170 g: 30-45 g: 90-110 g. The reaction temperature of the raw materials can be room temperature, and the reaction time can be 5-30 hours.

[0101] In step 2, the ratio of product 1-2: bromine-containing reagent: solvent is 100-130 g: 50-60 g: 800-2000 mL. The reaction temperature in step 2 can be room temperature, and the reaction time can be 1-10 hours.

[0102] In step 3, product 1-3 and ammonium acetate NH4OAc are dissolved in a solvent, reacted, concentrated to obtain a thick material, and purified to obtain a white solid. In the process, the ratio of product 1-3: ammonium acetate NH4OAc: solvent is 80-105 g: 40-80 g: 300-1200 mL. The reaction temperature can be room temperature, and the reaction time can be 15-50 hours.

[0103] In step 3, the white solid is dissolved in a solvent, stirred, and boron tribromide (BBr3) is added for reaction, followed by addition of water, extraction with an extractant, and concentration to obtain product 1-4. In this process, the ratio of white solid: solvent: boron tribromide (BBr3) is 50-70 g: 300-1200 mL: 500-1000 mL. The stirring (reaction) temperature is -90 to -70°C, and the reaction time can be 0.5 to 5 hours.

[0104] In step 4, the ratio of product 1-4: 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-yl)-1,3,2-dioxaborolane: solvent: base: catalyst is 20-40 g: 40-55 g: 200-1200 mL: 30-45 g: 4-6 g. The reaction temperature can be room temperature and the reaction time can be 8-20 hours.

[0105] Synthesis steps:

[0106] Step 1: To a mixture of 2-amino-4,6-dimethoxybenzoic acid (product 1-1, 150 g, 0.76 mol) and potassium carbonate (210.2 g, 1.52 mol) in dimethylformamide (1500 mL) was added iodomethane (107.9 g, 0.76 mol) at 20°C under nitrogen. The mixture was stirred at room temperature for 3 hours. The reaction mixture was then diluted with 5 L of water and extracted three times with ethyl acetate (5 L each). The extract was concentrated under reduced pressure to obtain methyl 2-amino-4,6-dimethoxybenzoate (107 g) as a white solid.

[0107] Methyl 2-amino-4,6-dimethoxybenzoate (107 g, 506.58 mmol) was dissolved in 1 L of methanol solvent and stirred. Benzaldehyde (161.2 g, 1.52 mol) and acetic acid (37.4 g, 623.10 mmol) were added, followed by NaBH3CN (95.5 g, 1.52 mol). The mixture was allowed to react overnight at room temperature for 12 hours. After the reaction, 2 L of water was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (2 L each time). The extract was concentrated under reduced pressure to obtain methyl 2-benzylamino-4,6-dimethoxybenzoate (product 1-2, 114 g, yield 74.6%) as a white solid.

[0108] Step 2: Dissolve 1-2 (114 g, 378.30 mmol) and NBS (53.8 g, 302.64 mmol) in 1.1 L of dimethylformamide and stir at room temperature for 2 hours. After the reaction, add 3 L of water to the reaction mixture and extract three times with 3 L of ethyl acetate. The extract is concentrated under reduced pressure to obtain a yellow viscous product. This viscous product is purified by preparative HPLC column chromatography (C18 column; mobile phase: acetonitrile / water, machine setting: 30% to 50% gradient elution; elution time: 10 minutes; UV detection: 254 nm detection wavelength) to obtain methyl 6-benzylamino-3-bromo-2,4-dimethoxybenzoate (product 1-3, 96.5 g, yield 67.1%) as a white solid.

[0109] Step 3: Dissolve 1-3 (95 g, 249.84 mmol) and NH4OAc (57.7 g, 749.53 mmol) in 400 mL of diethoxymethoxyethane and stir at room temperature for 24 hours. After completion of the reaction, the reaction solution was concentrated to obtain a viscous product, which was then purified by silica gel column chromatography using a CH2Cl2 / MeOH gradient elution setting of 20:1 to 10:1 to obtain 1-benzyl-6-bromo-5,7-dimethoxyquinazolin-4-one (60 g) as a white solid.

[0110] 1-Benzyl-6-bromo-5,7-dimethoxyquinazolin-4-one (60 g, 159.90 mmol) was dissolved in 400 mL of dichloromethane. BBr (640 mL, 639.62 mmol) was added with stirring at -78°C and allowed to react for 2 hours. After the reaction, 400 mL of water was added, and the mixture was extracted three times with ethyl acetate (400 mL each). The extract was concentrated under reduced pressure to obtain 1-benzyl-6-bromo-5-hydroxy-7-methoxyquinazolin-4-one (product 1-4, 30 g, yield 51.9%) as a white solid.

[0111] Step 4: Dissolve 1-4 (30 g, 83.05 mmol) and 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-yl)-1,3,2-dioxaborolane (48.8 g, 249.17 mmol) in 300 mL of 1,4-dioxane solvent. Add K2CO3 (34.44 g, 249.17 mmol) and Pd(dtbpf)Cl2 (5.4 g, 8.31 mmol) with stirring and allow to react overnight at room temperature (12 hours). After completion of the reaction, extract the reactant with ethyl acetate three times (200 mL each time), and concentrate the extract under reduced pressure to obtain a brown viscous product. The obtained brown viscous material was purified by silica gel column chromatography with a petroleum ether / ethyl acetate gradient elution (1:1, 0:1) to give 1-benzyl-5-hydroxy-7-methoxy-6-(3-methylbut-2-en-1-yl)quinazolin-4-one as a white solid (product 1, 10.05 g, yield 33.9%).

[0112] The above products 1, 1-2, 1-3 and 1-4 were tested by nuclear magnetic resonance hydrogen spectrum and mass spectrum. The spectra obtained by the test are shown in the following order: Figures 1-8 .

[0113] The product 1 prepared above was tested.

[0114] To investigate the therapeutic effect of product 1 (hereinafter referred to as compound NB798) on Alzheimer's disease in mice.

[0115] Grouping of AD mouse models:

[0116] The mice were divided into 4 groups, with 6 mice in each group. The specific groups were:

[0117] ①Administration groups: NB798 group and Huperzine A group.

[0118] ②AD model group: AD mice without drug treatment.

[0119] ③Blank control group: mice without any treatment.

[0120] Establishment of AD mouse model:

[0121] After the mice were acclimated to feeding, all but the control group received intraperitoneal injections of D-galactose (80 mg / kg / day) and AlCl₃ aqueous solution (5 mg / kg / day) for 40 days. Following this, D-galactose (80 mg / kg / day), AlCl₃ aqueous solution (5 mg / kg / day), and sodium nitrite (45 mg / kg / day) were injected for 20 consecutive days to establish AD modeling. Following the completion of dosing, the mice were sampled.

[0122] Drug administration in AD mouse model:

[0123] The drug-treated group was gavaged with NB798 (200 mg / kg) and Huperzine A (30 ug / kg) at noon every day for 60 days, and the AD group was gavaged with the solvent for 60 days.

[0124] Behavioral testing

[0125] Open field:

[0126] Place the mouse in an open field box and record its behavior for 5 minutes. Clean the open field box with alcohol after each mouse test.

[0127] Water maze:

[0128] A circular swimming pool was filled with water and a platform was placed somewhere underwater. The pool was divided into four sectors of equal area. A mouse was placed in the pool and allowed to swim freely for 1 minute to search for the platform. If the mouse found the platform, it was immediately removed and the number of times it passed the platform was recorded. If the mouse did not find the platform after 1 minute, it was placed on the platform for 10 seconds. This was repeated for four days. On the fifth day, the platform was removed and the mouse was allowed to search for the platform in the pool. The number of times the mouse passed the platform within 1 minute was recorded.

[0129] Conditioned fear:

[0130] Mice were placed in a fear conditioning chamber and subjected to a specific stimulus (electrical stimulation: 0.5 mA, 1 second, acoustic stimulation: 70 dB, 5000 Hz, 30 seconds, six cycles) for three days. On the fourth day, the electrical stimulation was removed, leaving only the acoustic stimulation, and the freezing time of the mice was recorded for 5 minutes.

[0131] IHC staining:

[0132] 1) Embedding and Sectioning: Dehydrate the fixed mouse brain tissue in the following order: soak in 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour, anhydrous ethanol for 1 hour, xylene for 1 hour, and xylene for 1 hour. Next, soak the dehydrated tissue in melted paraffin for 1 hour, then replace with new paraffin and soak for 2 hours to complete embedding. Place the embedded tissue into a microtome and slice it, with each slice being 5 μm thick. Then, bake the sliced ​​tissue at 60°C for 2 hours before use.

[0133] 2) Antigen retrieval: Soak the sliced ​​tissue in EDTA buffer and microwave on medium heat for 5 minutes. Place the slices at room temperature for 10 minutes, then microwave on medium-low heat for 2 minutes. Cool the slices at room temperature and then rinse the slices three times in PBS for 5 minutes each.

[0134] 3) Primary Antibody: Remove the sections from the PBS, wipe off any excess water, and draw a circle around the tissue section with a histochemical pen. Dilute the primary antibody 1:50 with 5% BSA. Carefully apply the diluted primary antibody dropwise to the tissue section and incubate in a 37°C oven for 2 hours. Next, remove the sections and wash them three times with PBS for 5 minutes each.

[0135] 4) Secondary Antibody: Remove the sections from the PBS and wipe away any excess water. Dilute the secondary antibody with 5% BSA at a 1:1000 dilution ratio. Carefully apply the diluted secondary antibody dropwise to the tissue sections and incubate in a 37°C oven for 1 hour. Next, remove the sections and wash them three times with PBS for 5 minutes each.

[0136] 5) Color Development: Mix DAB (1:1) and dropwise apply to the sections. Once the sections turn yellow, rinse with PBS. Next, soak the sections in hematoxylin solution for 8 minutes to stain the nuclei. After staining, wash the sections three times with PBS for 5 minutes each. Next, add 1% hydrochloric acid alcohol dropwise to the sections, rinse after 2 seconds, then use ammonia solution to reverse the blueing for 2 seconds, and rinse again with PBS.

[0137] 6) Dehydration: Dehydrate the sections in the order of 50% alcohol for 5 min, 75% alcohol for 5 min, 85% alcohol for 5 min, 95% alcohol for 5 min, and 100% alcohol for 5 min. Finally, add a drop of neutral resin to seal the sections. Observe and photograph using an upright microscope.

[0138] HE staining:

[0139] 1) Embedding and Sectioning: Dehydrate the fixed mouse brain tissue using a gradient process. Then, soak the dehydrated tissue in melted paraffin for 1 hour. Then, replace the paraffin with fresh paraffin and soak for 2 hours to complete the embedding process. Place the embedded tissue in a microtome and slice it to a thickness of 5 μm. Then, bake the sliced ​​tissue at 60°C for 2 hours until ready for use.

[0140] 2) HE staining: Dewax and rehydrate the baked tissue, place it in hematoxylin solution for several minutes, then soak it in acid water and ammonia water for several seconds, rinse it with running water for 10 minutes, and stain it with eosin solution for 3 minutes.

[0141] 3) Dehydration: Dehydrate the sections in the order of 50% alcohol for 5 min, 75% alcohol for 5 min, 85% alcohol for 5 min, 95% alcohol for 5 min, and 100% alcohol for 5 min. Finally, add a drop of neutral resin to seal the sections. Observe and photograph using an upright microscope.

[0142] qPCR:

[0143] 1) Collection of RNA samples

[0144] Collection and lysis of mouse brain tissue: One day after behavioral testing, mice were sacrificed with an intraperitoneal injection of 3% sodium pentobarbital. The mice were dissected along the ventral line and perfused transcardially with 0.9% saline. The mice were then flipped over, the skin around the brain cut open, and the brain removed. 500 μl of RNA lysis buffer was added to the tissue, and the tissue was lysed using a tissue disruptor (55 Hz, 10 sec, 7 times). After complete lysis, the tissue was centrifuged at 12,000 × g for 5 minutes to remove impurities.

[0145] 2) Extraction of RNA samples

[0146] Use a pipette to transfer the supernatant to gDNA-Filter Columns and centrifuge at 12,000 x g for 2 minutes to filter out the DNA. Mix the supernatant with 1.6 times the volume of Buffer RL2 and transfer it to the RNAPure Columns. Centrifuge at 12,000 x g for 1 minute and discard the supernatant. Add 500 μl of Buffer RW1 solution to the RNAPure Columns and centrifuge at 12,000 x g for 1 minute. Discard the waste liquid. Then, add 700 μl of Buffer RW2 solution to the RNAPure Columns and centrifuge at 12,000 x g for 1 minute. Discard the liquid. Repeat the centrifugation twice and then centrifuge at 12,000 x g for 2 minutes to dry the column. Place the column in a new centrifuge tube and add 50 μl of preheated RNAse-free ddH2O to the center of the column. Let it sit at room temperature for 2 minutes. Centrifuge at 12,000 × g for 1 minute to elute the RNA. Finally, measure the RNA concentration and immediately store the RNA at -80°C.

[0147] 3) Reverse transcription of cDNA

[0148] Mix 200 ng of RNA sample with 4 μl of 4×g DNA wiper mix. Add RNAse-free ddH₂O to a final volume of 16 μl. Mix thoroughly and heat in a 42°C water bath for 2 minutes. Next, add 4 μl of 5× HiScript III qRT Super Mix and mix thoroughly. Finally, perform reverse transcription using a PCR amplifier using the following protocol: 37°C for 15 minutes; 85°C for 5 seconds. After reverse transcription, measure the cDNA concentration and immediately store the cDNA at -20°C.

[0149] 4) qRT-PCR

[0150] This study used a dye-based qRT-PCR method. 0.4 μl of each designed upstream and downstream primers were mixed with 10 μl of 2× ChamQ Univegsal SYBG qPCR Master Mix, followed by 2 μl of cDNA sample. RNAse-free ddH2O was then added to bring the solution volume to 20 μl. The mixture was mixed thoroughly using a low-speed centrifuge, and assays (TNF-α, APOE4) were performed using a fluorescent quantitative gene amplification instrument.

[0151] Information on all primers used is shown in Table 1.

[0152] Table 1: Primer information

[0153]

[0154] IF staining:

[0155] 1) Embedding and Sectioning: Dehydrate the fixed mouse brain tissue in the following order: soak in 70% ethanol for 1 hour, 80% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol for 1 hour, anhydrous ethanol for 1 hour, xylene for 1 hour, and xylene for 1 hour. Next, soak the dehydrated tissue in melted paraffin for 1 hour, then replace with new paraffin and soak for 2 hours to complete embedding. Place the embedded tissue into a microtome and slice it, with each slice being 5 μm thick. Then, bake the sliced ​​tissue at 60°C for 2 hours before use.

[0156] 2) Antigen retrieval: Soak the sliced ​​tissue in EDTA buffer and microwave on medium heat for 5 minutes. Place the slices at room temperature for 10 minutes, then microwave on medium-low heat for 2 minutes. Cool the slices at room temperature and then rinse the slices three times in PBS for 5 minutes each.

[0157] 3) Primary Antibody: Remove the sections from the PBS, wipe off any excess water, and draw a circle around the tissue section with a histochemical pen. Dilute the primary antibody in 5% BSA according to the dilution ratio specified in the antibody instructions. Carefully apply the diluted primary antibody dropwise to the tissue section and incubate in a 37°C oven for 2 hours. Next, remove the sections and wash them three times with PBS for 5 minutes each.

[0158] 4) Secondary Antibody: Remove the sections from the PBS and wipe off any excess water. Dilute the secondary antibody with 5% BSA according to the dilution ratio specified in the antibody instructions. Carefully apply the diluted secondary antibody dropwise to the tissue sections and incubate in a 37°C oven for 1 hour. Next, remove the sections and wash them three times with PBS for 5 minutes each.

[0159] 5) Nuclear staining: DAPI diluted in 5% FBS was added dropwise to the tissue sections and incubated at 37°C for 10 minutes. Next, the sections were washed three times with PBS for 5 minutes each, and the slides were dried. Finally, neutral resin was added to the sections for mounting. The sections were observed and photographed using an upright microscope.

[0160] ELISA:

[0161] After collecting mouse serum, GFAP was detected by ELISA kit according to the kit instructions.

[0162] Reagents and consumables

[0163] Experimental animals:

[0164] Twenty-five Kunming mice, 12 months old, were purchased from the Guangdong Provincial Laboratory Animal Center.

[0165] Reagents and consumables are shown in Table 2.

[0166] Table 2: Experimental reagents and consumables

[0167]

[0168]

[0169] The instruments used in the experiment are shown in Table 3.

[0170] Table 3: Experimental instruments

[0171]

[0172] Test results

[0173] Open field test:

[0174] After drug treatment, open field test was performed. Mice were placed in an unfamiliar environment and the number of times, time and walking distance of mice passing through the center within 5 minutes were recorded. The test results are shown in Figure 9 .

[0175] Figure 9 Figure 2, A: the number of times mice passed through the center position, B: the time mice stayed in the center position, C: the proportion of distance mice moved in the center position, *P<0.05, **P<0.01; compared with the Control group, the number of times mice passed through the center position and the time they stayed in the center position were shortened, the proportion of distance they moved in the center position was reduced, and their mobility and autonomous exploration abilities were weakened; after treatment with different drugs, the number of times mice passed through the center position and the time they stayed in the center position increased, and the proportion of distance they moved in the center position increased, indicating that the mobility and autonomous exploration abilities of mice were restored to varying degrees.

[0176] Water maze experiment:

[0177] After drug treatment, a water maze test was performed. The mice were placed in a circular pool (large circle in the figure below) and the number of times the mice passed the platform position (small circle in the figure below) within 1 minute was recorded. The test results are shown in Figure 10 and Figure 11 .

[0178] in, Figure 10 The green irregular line in the middle is the trajectory of the mouse, the red dot is the starting position of the mouse, and the blue dot is the ending position of the mouse.

[0179] Figure 10-11The results showed that compared with the control group, the number of times the mice in the AD group passed through the platform was significantly reduced. Compared with the AD group, the number of times the mice in the NB798 group and the Huperzine A group passed through the platform was increased, but there was no significant difference.

[0180] Fear conditioning experiment:

[0181] After drug treatment, a conditioned fear test was conducted. Mice were placed in a conditioned fear box and given different stimuli (sound, light, electricity). The number of times the mice froze within 5 minutes was recorded. The test results are shown in Figure 12-13 .

[0182] Figure 12 In the figure, the green irregular line is the trajectory of the mouse, the red dot is the starting position of the mouse, and the blue dot is the position of the mouse at the end of the experiment.

[0183] Figure 12-13 The results showed that compared with the control group, the AD group had impaired memory; and after treatment with different drugs, the number of stiffness episodes in mice increased to varying degrees.

[0184] qPCR assay:

[0185] Test results see Figure 14 and Table 4. The tests showed that compared with the control group, AD mice had increased TNF-α mRNA levels and APOE4 mRNA expression in the hippocampus. However, after treatment with different drugs, both TNF-α mRNA and APOE4 mRNA levels in brain tissue were significantly reduced. This suggests that NB798 intervention can control overall brain inflammation, reduce the increase in APOE4 mRNA expression induced by stress in the mice brain, and mitigate brain damage in AD mice, thereby protecting brain neurons and slowing the disease progression in AD mice.

[0186] Table 4: mRNA expression of TNF and APOE4 in the hippocampus of mice

[0187]

[0188] Compared with the blank control group: ####P<0.0001; compared with the model group: *P<0.05, ***P<0.001, ****P<0.0001

[0189] ELISA assay:

[0190] GFAP is an important marker of Alzheimer's disease. The expression of GFAP in mouse serum was detected. Figure 15As shown in Table 5, GFAP expression was significantly increased in the AD group compared to the control group, while GFAP expression was significantly decreased after NB798 treatment, indicating that NB798 has a clearing effect on GFAP, a key marker in AD mice.

[0191] Table 5: Detection of GFAP levels in mouse serum

[0192]

[0193] Compared with the blank control group: ####P<0.0001; compared with the model group: ***P<0.001

[0194] HE staining:

[0195] HE staining test Figure 16 Compared to the control group, AD mice showed a large number of loosely structured neuronal swellings in the hippocampus, accompanied by NFTs (neurofibrillary tangles), indicating damage to the hippocampus. Compared to the AD group, all treatment groups showed a certain recovery in hippocampal neuronal structure, indicating that compounds NB798 and Huperzine A were able to effectively protect neurons.

[0196] IHC-APP (protein-like precursor protein) expression:

[0197] During the development of AD, APP expression in the hippocampus increased significantly. Figure 17 ) found that compared with the control group, APP expression in the hippocampus of AD mice was significantly increased, suggesting the development of AD. APP levels decreased to varying degrees in all treatment groups compared with the AD group, indicating that compounds NB798 and Huperzine A can effectively reduce APP expression in the hippocampus of AD mice.

[0198] IHC-P-tau (phosphorylated tau protein) expression:

[0199] During the development of AD, tau protein accumulates in large quantities. P-tau in the hippocampus of mice was detected (see the results). Figure 18 ) found that compared with the control group, the expression of phosphorylated tau protein (red arrow) in the hippocampus of AD mice was significantly increased. Compared with the AD group, P-tau levels in all treatment groups decreased to varying degrees, indicating that the compounds NB798 and Huperzine A can effectively reduce P-tau expression in the hippocampus of AD mice.

[0200] IHC-NeuN (neuron) expression:

[0201] During the development of AD, a large number of hippocampal neurons undergo apoptosis. IHC was used to detect neurons in the hippocampus of mice (see the results). Figure 19 ) found that NeuN expression in the hippocampus of AD mice was significantly reduced compared to the control group. Compared to the AD group, NeuN levels increased to varying degrees in all treatment groups, indicating that compounds NB798 and Huperzine A can effectively protect hippocampal neurons.

[0202] IF-Iba1 (microglia) expression:

[0203] Microglial cell expression is closely related to inflammatory response. Immunofluorescence was used to detect microglial cell expression in the hippocampus of mice. The results are shown in Figure 20 The results showed that compared with the control group, the expression of Iba1 in the hippocampus of the AD mice was significantly increased, indicating a large increase in microglia. Compared with the AD group, the expression of Iba1 in each treatment group decreased to varying degrees, indicating that the compounds NB798 and huperzine A can effectively reduce the activation of microglia in the brains of AD mice.

[0204] IF-CD68 (microglia) expression:

[0205] Immunofluorescence was used to detect the activation of microglia in the hippocampus of mice. Figure 21 The results showed that compared with the control group, the expression of CD68 in the hippocampus of the AD mice was significantly increased, indicating the activation of microglia. Compared with the AD group, the expression of CD68 in each treatment group decreased to varying degrees, indicating that the compounds NB798 and huperzine A can effectively reduce the transformation of brain microglia of AD mice to MI type.

[0206] Summarize:

[0207] The study confirmed the successful establishment of the mouse AD model through behavioral tests such as the water maze, and found that NB798 and huperzine A compound-mediated treatment could improve AD symptoms such as memory loss in mice to a certain extent.

[0208] Testing of mouse serum GFAP levels revealed significantly higher GFAP levels in the AD group compared to the control group. Each drug-treated group was able to partially downregulate the drug-induced GFAP increase, thereby intervening in the progression of AD. Furthermore, compared to the control group, AD mice showed increased brain inflammation and stress-induced APOE4 expression, exacerbating AD brain damage. However, treatment with the drug NB798 significantly decreased both TNF and APOE4 expression in brain tissue, suggesting that drug intervention can protect neurons in AD mice and reduce brain damage.

[0209] Pathological examination of the hippocampus region of the mouse brain revealed that a large number of loosely structured neurons were swollen in the hippocampus region of the AD group mice, accompanied by a significant increase in the expression of NFT and phosphorylated tau protein, as well as a large number of activated microglia. Treatment with the drug NB798 had a certain therapeutic effect on these phenomena, suggesting that the NB798 compound can alleviate brain nerve damage in AD mice, thereby affecting the disease progression of AD.

Claims

1. A compound having activity in treating Alzheimer's disease, characterized in that: Its structure is as shown in formula (A): In formula (A), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R3 is Or, its structure is as formula (B): In formula (B), R1 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3; R2 is -OH, -CH3, -OCH3, -OCH2CH3 or -OCH2CH2CH3.

2. A compound having activity in treating Alzheimer's disease, characterized in that: Its structure is as shown in formula (1):

3. Use of the compound according to claim 1 or 2 as a drug for treating Alzheimer's disease.

4. A drug for treating Alzheimer's disease, characterized in that: Its structure is as shown in formula (1):

5. A method for preparing the drug according to claim 4, characterized in that: The steps include: Step 1: Prepare product 1-2; the structural formula of product 1-2 is as shown in formula (2): Step 2: Prepare product 1-3; the structural formula of product 1-3 is as shown in formula (3): Step 3: Prepare product 1-4; the structural formula of product 1-4 is as shown in formula (4): Step 4: Use the drug represented by the product formula (1).

6. The preparation method according to claim 5, characterized in that The steps include: Step 1: dissolving methyl 2-amino-4,6-dimethoxybenzoate in a solvent, adding benzaldehyde, acetic acid and NaBH3CN to react, adding water to the reactants, extracting with an extractant, concentrating the extract, and drying to obtain product 1-2; Step 2: Dissolve product 1-2 and a bromine-containing reagent in a solvent, react, then add water and mix, extract with an extractant, concentrate, and purify to obtain product 1-3 Step 3: Dissolve product 1-3 and ammonium acetate (NH4OAc) in a solvent, react, concentrate, and obtain a thick substance, which is purified to obtain a white solid; then dissolve the white solid in a solvent, stir, and add boron tribromide (BBr3), react, then add water and mix, extract with an extractant, and concentrate to obtain product 1-4; Step 4: dissolving the product 1-4 and 4,4,5,5-tetramethyl-2-(3-methylbut-2-en-1-yl)-1,3,2-dioxaborolane in a solvent, adding a base and a catalyst for reaction, extracting, concentrating, and purifying to obtain a drug for treating Alzheimer's disease represented by formula (1).

7. The preparation method according to claim 6, characterized in that The extractant includes ethyl acetate.

8. The preparation method according to claim 6, characterized in that Purification was performed by column chromatography.

9. The preparation method according to claim 6, characterized in that The bromine-containing reagent includes N-bromosuccinimide NBS; or, the base includes at least one of carbonate or bicarbonate; or, the catalyst includes Pd(dtbpf)Cl2.

10. The preparation method according to claim 6, characterized in that The solvent in step 1 includes methanol; or the solvent in step 2 includes dimethylformamide; or the solvent in step 3 includes at least one of diethoxymethoxyethane and dichloromethane; or the solvent in step 4 includes 1,4-dioxane.

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