A scutellarein aglycone-7-amino acid carbamate-4'-substituted aminopropyl ether derivative, preparation and use thereof

Abstract

This application relates to the field of medicinal chemistry, specifically to a derivative of ligustilide-7-aminocarbamate-4'-substituted aminopropyl ether, its preparation method, and its application. Addressing the shortcomings of ligustilide in AD treatment, this application utilizes previous studies to demonstrate strong eeAChE and huACh inhibitory activity, strong inhibition of self-induction, and Cu... 2+ Induced Aβ 1‑42 Aggregation activity, significantly reduced Aβ 25‑35 The lead compound, scutellarin aglycone-4′-L-amino acid carbamate, which induces tau hyperphosphorylation and significantly improves learning and memory impairment in scopolamine-induced AD model mice, is used to introduce histamine H at the 7-position of its structure. 3 By using the key structural fragment (3-methylpiperidinyl) or cycloheptane group of the receptor antagonist, a cholinesterase inhibitor and histamine H2 receptor antagonist can be obtained. 3 Multi-targeted ligand derivatives with better anti-AD properties through receptor antagonistic synergistic mechanism.

Description

A derivative containing scutellarin-7-aminocarbamate-4'-substituted aminopropyl ether, its preparation and application Technical Field

[0001] This application relates to the field of medicinal chemistry technology, specifically to a derivative of scutellarin-7-aminocarbamate-4'-substituted aminopropyl ether, its preparation, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a chronic, irreversible neurodegenerative disease characterized by severe memory loss, language impairment, and other cognitive impairments, which can further develop into depression and ultimately lead to death. According to the World Health Organization (WHO), Alzheimer's disease (AD) accounts for approximately 50-60% of all dementia cases in people over the age of 65. It is estimated that there are currently more than 47.5 million people with Alzheimer's disease worldwide, and this number is projected to reach 132 million by 2050 (Sorbi S. et al. Physiol Rev. 2021, 101(3):1047-1081).

[0003] To date, the FDA has approved several drugs for the treatment of Alzheimer's disease (AD), including cholinesterase inhibitors rivastigmine, galantamine, and donepezil, the N-methylaspartate receptor antagonist memantine, and the combination of memantine and donepezil. However, in clinical applications, these drugs can only improve AD symptoms and slow the disease progression to some extent, but cannot reverse or stop the development of AD symptoms (Benek O. et al. Trends Pharmacol Sci. 2020, 41(7):434-445). Therefore, further in-depth analysis of the etiological mechanisms of AD and its related targets is of extremely important medical research value for the creation of novel drugs with AD prevention and treatment properties.

[0004] *Erigeron breviscapus* (Vant.) Hand.-Mazz., also known as *Dengzhanhua*, is included in the 2015 edition of the *Chinese Pharmacopoeia*. It possesses the effects of promoting blood circulation, relieving pain, dispelling wind and cold, and is used for stroke hemiplegia, chest pain, and rheumatic pain. Scutellarin is the most important flavonoid active ingredient in *Erigeron breviscapus*, and scutellarin aglycone is the main metabolic and active form of scutellarin. Recent studies on the mechanism of scutellarin and its aglycone in preventing and treating Alzheimer's disease (AD) have shown that it can protect against Aβ-induced learning and memory impairment in rats by stimulating the translation of nicotinic acetylcholine receptor (nAChR) protein and regulating cholinesterase activity (Guo L Let al. Acta Pharmacol Sin. 2011, 32(12):1446-1453). In APP / PS1 transgenic mice, ligustrazine and its aglycone can alleviate Aβ-induced learning and memory impairment by promoting Aβ aggregation into fibrils or protofibrils while reducing the production of soluble, highly toxic Aβ oligomers (Zhang S. et al. Biomed Pharmacother. 2020, 121, 109682). Although ligustrazine has a definite therapeutic effect, ligustrazine and its aglycone have similar problems such as poor water solubility, poor stability, short elimination half-life, and poor oral bioavailability (Zhang Haiyan et al. Acta Pharmaceutica Sinica. 2005, 40(6): 563-7; Cao F. et al. Eur J PharmSci. 2006(29): 385-93).

[0005] Previous research by our group has shown that scutellarin-7-L-amino acid carbamate derivatives obtained by introducing L-amino acid carbamate structural fragments into the scutellarin aglycone structure exhibit good inhibitory activity against eeAChE and huAChE, with most compounds showing stronger inhibitory activity than the positive control rivastigmine. Furthermore, these target compounds not only inhibit self-induced and Cu... 2+ Induced Aβ 1-42 The aggregation can also depolymerize itself and Cu 2+ Induced Aβ 1-42 Aggregation was observed, with effects superior to the positive control curcumin; furthermore, these compounds could significantly reduce Aβ. 25-35Induced tau hyperphosphorylation. Furthermore, compared to scutellarin and its aglycone, the aforementioned target compounds significantly improved PC12 cell survival, reduced LDH leakage and ROS production, weakened the expression levels of Caspase-3 and Bax proteins, and increased the expression level of Bcl-2 protein, thus protecting cells from oxidative damage in multiple ways. In vivo experiments showed that after administration of the optimized compound, the escape latency of scopolamine-induced memory impairment AD mice was significantly shortened, the number of platform crossings increased, and cognitive function was improved. In addition, the target compound could improve learning and memory impairment in mice by reducing acetylcholinesterase activity, increasing acetylcholine levels, and alleviating hippocampal tissue cell pathological damage. HE staining results of the CA1 and CA3 regions of the hippocampus and the cerebral cortex of the test animals showed that the designed target compound could significantly improve the phenomenon of loosened boundaries and irregular arrangement of hippocampal and cerebral cortex cells in scopolamine-induced AD mice (Luo K Ketal. Bioorg Chem 2023,138,106596; Wu DR et al. Bioorg Chem 2022,122,105760; Li T. et al. Bioorg Chem 2020,101,103980).

[0006] However, the aforementioned ligustrazine aglycone derivatives still cannot exert a synergistic effect with histamine H3 receptor antagonists in the treatment of AD. Therefore, further modification of the aforementioned ligustrazine aglycone derivatives in order to obtain a multi-targeted ligand derivative with better anti-AD properties that combines cholinesterase inhibition and histamine H3 receptor antagonism is currently the focus of research. Summary of the Invention

[0007] To address the aforementioned technical problems in the prior art, this application provides a derivative containing ligustilide-7-aminocarbamate-4'-substituted aminopropyl ether, its preparation method, and its application, as detailed below:

[0008] A scutellarin-7-aminocarbamate-4'-substituted aminopropyl ether derivative, comprising the compound and its salts, salt hydrates, and salt solvates, is represented by the following general formula (I):

[0009]

[0010] In the general formula (I), R1 is H or -CH3; n is 1 or 2; R2 is a hydrocarbon group with 1-4 carbon atoms.

[0011] Furthermore, the amino acid carbamate is any one of phenylalanine carbamate, isoleucine carbamate, leucine carbamate, and valine carbamate.

[0012] Furthermore, the substituted aminopropyl ether is any one of (3-methylpiperidinyl)propyl ether, (4-methylpiperidinyl)propyl ether, and (cycloheptylamino)propyl ether.

[0013] The above-mentioned compounds are specifically:

[0014] Lactobacillus aglycone-7-phenylalanine carbamate-4'-(3-methylpiperidinyl)propyl ether;

[0015] Lactobacillus aglycone-7-isoleucine carbamate-4'-(3-methylpiperidinyl)propyl ether;

[0016] Lactobacillus aglycone-7-leucine carbamate-4'-(3-methylpiperidinyl)propyl ether;

[0017] Lactobacillus aglycone-7-valine carbamate-4'-(3-methylpiperidinyl)propyl ether;

[0018] Lactobacillus aglycone-7-phenylalanine carbamate-4'-(4-methylpiperidinyl)propyl ether;

[0019] Lactobacillus aglycone-7-isoleucine carbamate-4'-(4-methylpiperidinyl)propyl ether;

[0020] Lactobacillus aglycone-7-leucine carbamate-4'-(4-methylpiperidinyl)propyl ether;

[0021] Lactobacillus aglycone-7-valine carbamate-4'-(4-methylpiperidinyl)propyl ether;

[0022] Lactobacillus aglycone-7-phenylalanine carbamate-4'-(cycloheptanoyl)propyl ether;

[0023] Lactobacillus aglycone-7-isoleucine carbamate-4'-(cycloheptamino)propyl ether;

[0024] Lactobacillus aglycone-7-leucine carbamate-4'-(cycloheptaneamino)propyl ether;

[0025] Lamphenicol aglycone-7-valine carbamate-4'-(cycloheptamino)propyl ether.

[0026] The application of the ligustilide-7-aminocarbamate-4'-substituted aminopropyl ether derivative as a pharmaceutical ingredient / in preparation.

[0027] Furthermore, the drug is a treatment for Alzheimer's disease. Specifically, the drug for treating Alzheimer's disease is a drug used in synergistic treatment with a histamine H3 receptor antagonist.

[0028] The preparation method of the ligustilide-7-aminocarbamate-4'-substituted aminopropyl ether derivative includes the following steps:

[0029] (1) Preparation of N-3-chloropropyl substituted chain amines (dimethylamine, diethylamine) or substituted cyclic amines (pyrrole, piperidine, morpholine, N-methylpiperazine, cycloheptylamine);

[0030] (2) Preparation of scutellarin-4'-4'-chain amine or cyclic amino-1-propyl ether;

[0031] (3) Prepare scutellarin-7-amino acid carbamate-4'-chain amine or cyclic amine-1-propyl ether.

[0032] Furthermore, the chain amine or cyclic amine is specifically 3(4)-methylpiperidine or cycloheptamine.

[0033] Furthermore, the method specifically includes the following steps:

[0034] (1) Preparation of N-3-chloropropyl-substituted 3(4)-methylpiperidine or cycloheptamine:

[0035] 3-bromo-1-propanol was reacted with 3(4)methylpiperidine or cycloheptamine at room temperature for more than 24 h in the presence of K2CO3 and KI to obtain N-3-hydroxypropyl-substituted 3(4)methylpiperidine or cycloheptamine. The resulting products were then dissolved in a small amount of toluene, and excess thionyl chloride was slowly added dropwise at 0 °C. After maintaining the low temperature for 10 min, the system temperature was raised to 60 °C and reacted for 8 h to obtain N-3-chloropropyl-substituted 3(4)methylpiperidine or cycloheptamine.

[0036] (2) Preparation of scutellarin aglycone-4'-4'-(3-methylpiperidinyl) or cycloheptamino-1-propyl ether:

[0037] Using scutellarin as raw material, crude scutellarin aglycone was obtained by reflux at 80°C for 20 h under sulfuric acid and ethanol conditions. The hydroxyl groups at the 6 and 7 positions of the aglycone were protected by condensation of dichlorodiphenylmethane in diethylene glycol dimethyl ether (DEME). After reacting with different N-3-chloropropyl-substituted 3(4)-methylpiperidine or cycloheptylamine, the diphenyl ketal was removed in an ethyl acetate system saturated with hydrogen chloride to obtain the intermediate scutellarin aglycone-4'-N,N-cyclohydroamino1-propyl ether.

[0038] (3) Preparation of scutellarin aglycone-7-amino acid carbamate-4'-(3-methylpiperidinyl) or cycloheptamino-1-propyl ether:

[0039] In a dry reaction flask, L-amino acid tert-butyl hydrochloride was added and stirred with dichloromethane to dissolve it. Pyridine was added, and the system was stirred at -10°C for 15 min. Then, dichloromethane containing triphosgene was added, and the reaction was maintained at this temperature for 3 h. After the reaction was completed, the mixture was quickly washed twice each with 0.5 M hydrochloric acid solution with crushed ice and saturated sodium chloride solution. Anhydrous magnesium sulfate was added to the organic layer to remove water. The mixture was filtered, and the filtrate was evaporated under reduced pressure to obtain a yellow oily substance, tert-butyl protected L-amino acid isocyanate. Dissolve 4'-N,N-cyclohydroamino-1-propyl ether in N,N-dimethylformamide solution, then add tert-butyl-protected L-amino acid isocyanate. Add triethylamine under stirring at 50°C and react for 12 h. After the reaction is complete as monitored by TLC, add dichloromethane. Wash the solution twice with 0.5M HCl and twice with saturated NaCl solution. Dry with anhydrous sodium sulfate, filter, and distill under reduced pressure until a yellow solid is obtained. Purify by silica gel column chromatography to obtain the target compound.

[0040] Further, in step (2), the ratio of 3bromo-1-propanol to 3(4)methylpiperidine or cycloheptamine is 1.0-5.5:1 mol, and the reaction is carried out at 10-35°C for more than 12-48 h to obtain N-3-hydroxypropyl-substituted 3(4)methylpiperidine or cycloheptamine. The resulting products are then dissolved in a small amount of toluene, and excess thionyl chloride is slowly added dropwise at -20-0°C. After maintaining the low temperature for 10 min, the system temperature is raised to 30-60°C and the reaction is carried out for 4-24 h to obtain N-3-chloropropyl-substituted 3(4)methylpiperidine or cycloheptamine.

[0041] Further, step (3) is as follows: the ratio of 4'-N,N-disubstituted carbamate of scutellarin to N-3-chloropropyl-substituted 3(4)methylpiperidine or cycloheptylamine is 1.0-3.5:1 mol, potassium carbonate / 4-dimethylaminopyridine is used as the condensing agent, and anhydrous N,N-dimethylformamide is used as the solvent. The reaction is carried out at 40-80℃ for 12-48 h to obtain scutellarin-7-amino acid carbamate-4'-(3-methylpiperidinyl) or cycloheptylaminopropyl ether.

[0042] Furthermore, in step (3), the ratio of 4'-N,N-disubstituted carbamate to N-3-chloropropyl-substituted 3(4)methylpiperidine or cycloheptylamine is 1.0-2.0:1 mol. Potassium carbonate / 4-dimethylaminopyridine is used as the condensing agent, and anhydrous N,N-dimethylformamide is used as the solvent. The reaction is carried out at 50-70°C for 24-36 h to obtain 4'-(3-methylpiperidinyl) carbamate or cycloheptylaminopropyl ether.

[0043] Compared with the prior art, the technical effects created by this application are reflected in:

[0044] (1) This application addresses the critical issue that the design of multi-target guided ligands (MTDLs) derivatives targeting scutellarin aglycone has not yet been reported using a histamine H3 receptor antagonistic synergistic target design strategy, which may lead to insufficient activity of the obtained target compounds for AD treatment. Based on previous studies, scutellarin aglycone-4′-L-amino acid carbamate derivatives were obtained as lead compounds. Combined with reports on the pharmacodynamic group analysis of non-imidazolium compounds with AChE inhibition / H3R antagonistic synergistic effects, the alkylaminoalkyloxyphenol ether skeleton revealed by the analysis of the pharmacodynamic group of these compounds is a key structural feature that gives these compounds high H3R receptor binding and antagonistic effects as well as AChE inhibitory activity. Scutellarin aglycone-7-L-amino acid carbamate-4′-(3-methylpiperidinyl) or cycloheptaminopropyl ether derivatives were designed and synthesized.

[0045] (2) This application addresses the shortcomings of ligustilide in the treatment of AD, and based on previous studies, it has been found to have strong eEAChE and huACh inhibitory activity, strong inhibition of self-induction and Cu 2+ Induced Aβ 1-42 Aggregation activity, significantly reduced Aβ 25-35 Using scutellarin aglycone-4'-L-amino acid carbamate, which induces tau hyperphosphorylation and significantly improves learning and memory impairment in scopolamine-induced AD model mice, as a lead compound, a key structural fragment (3-methylpiperidinyl) or a cycloheptylamino group of histamine H3 receptor antagonist was introduced into its structure at position 7 to obtain a multi-targeted ligand derivative with better anti-AD properties, which has both cholinesterase inhibition and histamine H3 receptor antagonism synergistic mechanism.

[0046] (3) This application utilizes the L-amino acid carbamate fragment in the structure of scutellarin-7-L amino acid carbamate-4'-(3-methylpiperidinyl) or cycloheptaminopropyl ether derivative to improve its oral absorption bioavailability, and utilizes the (3-methylpiperidinyl) or cycloheptaminopropyl ether fragment in the structure to exert the antagonistic activity of the compound against histamine H3 receptor, and finally discovers a multi-targeted ligand derivative with better anti-AD properties that has both cholinesterase inhibition and histamine H3 receptor antagonistic synergistic mechanism.

[0047] (4) This application addresses the shortcomings of poor drug-like properties and unsatisfactory treatment effect of scutellarin in clinical applications, thus laying an important theoretical and practical foundation for the creation of novel anti-AD scutellarin multi-target guided ligand derivatives (MTDLs).

[0048] (5) This application addresses the shortcomings of existing marketed or clinically investigated prodrugs containing scutellarin aglycone, which exhibit poor drug-like properties and unsatisfactory therapeutic effects in treating Alzheimer's disease (AD). It designs a scutellarin aglycone-7-L amino acid carbamate-4'-(3-methylpiperidinyl) or cycloheptaminopropyl ether derivative containing an endogenous carrier to improve oral absorption and bioavailability, while simultaneously increasing the release of neurotransmitters related to learning and attention in the brain by antagonizing histamine H3 receptors and increasing choline levels in the brain by inhibiting cholinesterase, thus achieving better therapeutic effects. The implementation of this application is of significant value for the research and development of novel anti-AD scutellarin aglycone drugs with independent intellectual property rights. Attached Figure Description

[0049] Figure 1 shows the Lineweave-Burk plot (A) of the hydrolysis of eeAChE by the test compound 14l in Example 13 of the present invention; and the relationship between the reaction rate of 14l and the concentration of eeAChE (B).

[0050] Figure 2 shows the UV spectrum (A) of the test compound 14l (30 μM) in methanol, alone or in combination with FeSO4·7H2O, AlCl3, ZnCl2, and CuCl2·2H2O (30 μM); the molar ratio of 14l-Cu was measured. 2+ The complexation ratio (B).

[0051] Figure 3 shows the inhibition of Cu by the test compound 14l in Example 15 of the present invention. 2+ Induced Aβ 1–42 Transmission electron microscopy image of aggregates (A); 14l depolymerized Cu 2+ Induced Aβ 1–42 Image of the aggregated structure (B) using transmission electron microscopy.

[0052] Figure 4 shows the effect of test compound 14l on Aβ in Example 16 of the present invention. 25-35 Inhibitory ability to induce tau hyperphosphorylation in PC12 cells. Western blots of Total-tau and p-tau(Thr 181) (A); relative protein expression of p-Tau(Thr181) / Total-Tau (B).

[0053] Figure 5 shows the effect of the test compound 14l on the expression of pro-inflammatory factors in BV-2 microglia induced by lipopolysaccharide in Example 17 of the present invention. Cytotoxicity of compound 14l (A); Cells were pretreated with 5, 10, and 20 μM of 14l for 1 h, then treated with LPS (100 ng / mL) for 24 h, and the release of IL-6 was measured by ELISA (B); Cells were pretreated with 5, 10, and 20 μM of 14l for 1 h, then treated with LPS (100 ng / mL) for 24 h, and the release of TNF-α was measured by ELISA (C).

[0054] Figure 6 shows the time required for each group to find the hidden platform during the 5-day positioning and navigation test in Embodiment 18 of the present invention (A); the escape latency period in the space search experiment (B); the number of times the virtual platform was traversed (C); the time spent in the target quadrant (D); and the typical trajectory diagram of each group in the positioning and navigation experiment (n=15) (E).

[0055] Figure 7 shows the detection of AChE content in mouse hippocampus tissue in Example 18 of the present invention (A); and the detection of ACh content in mouse hippocampus tissue (n=5) (B).

[0056] Figure 8 is a representative image of HE staining of hippocampal tissue from AD model mice in Example 18 of this invention (n=3). Detailed Implementation

[0057] The technical solution of this application will be further defined below with reference to specific implementation methods, but the scope of protection is not limited to the description.

[0058] Example 1

[0059] Preparation of scutellarin-7-phenylalanine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether (compound 14a)

[0060] (1) Preparation of 3-(3-methylpiperidin-1-yl)-1-propanol

[0061] 400 μl of 3-bromo-1-propanol (4.42 mmol, 1 eq) was added to a 250 mL dry reaction flask, followed by 80 mL of THF and 20 mL of anhydrous ethanol, and the mixture was thoroughly mixed. 733.62 mg of potassium carbonate (5.31 mmol, 1.2 eq) and a catalyst amount of KI were added at room temperature. After stirring thoroughly at room temperature, 575.8 μl of 3-methylpiperidine (4.86 mmol, 1.1 eq) was added, and the reaction was carried out at room temperature for more than 24 h. After most of the starting material had reacted, THF and anhydrous ethanol were evaporated to dryness under reduced pressure. The mixture was then purified by silica gel column chromatography with methanol:dichloromethane = 1:50 (V / V) as the eluent, yielding a white snowflake-like solid or a pale yellow oily liquid, with a yield of 80.7%.

[0062] (2) Preparation of 3-(3-methylpiperidin-1-yl)-1-chloropropane

[0063] In a 250 mL dry reaction flask, a solution of 560 mg (3.56 mmol) of 3-(3-methylpiperidin-1-yl)-1-propanol in dichloromethane was added. A small amount of toluene was used as a solvent. 1.30 mL (17.80 mmol, 5 eq) of thionyl chloride was slowly added dropwise in an ice bath. After the solid was completely melted, the reaction was continued at a low temperature for 10 min. The system temperature was then raised to 60 °C and the reaction was carried out for 8 h. After the reaction was completed by TLC monitoring, the solvent in the system was evaporated under reduced pressure and used directly in the next reaction step.

[0064] (3) Synthesis of 6,7-diphenyl ketal-protected scutellarin aglycone-4'-(3-methyl-1-piperidinyl)propyl ether

[0065] Add 300 mg (0.67 mmol, 1 eq) of scutellarin aglycone protected by benzoyl ketal to a 250 mL dry reaction flask, and dissolve it completely in an appropriate amount of DMF. Add 110.5 mg (0.80 mmol, 1.2 eq) of potassium carbonate at 0 °C, and maintain the temperature to allow the system to cool for 10 min. Add 8.1 mg (0.07 mmol, 0.1 eq) of DMAP, and slowly add dropwise a solution of 128.3 mg (0.73 mmol, 1.1 eq) of 1-(3-chloropropyl)-3-methylpiperidine completely dissolved in a small amount of DMF. After the addition is complete, maintain the temperature for 10 min, remove the flask, and heat the system to 60 °C for 36 h. After the reaction is complete as monitored by TLC, add 100 mL of CH2Cl2 and use 100 mL of 0.5 M... The sample was washed twice with HCl, twice with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure until a yellow solid was obtained. The solid was then purified by silica gel column chromatography with methanol:dichloromethane = 1:40 (V / V) as the eluent, yielding 155.7 mg of yellow solid, with a yield of 45.6%.

[0066] (3) Synthesis of scutellarin aglycone-4'-(3-methyl-1-piperidinyl)propyl ether

[0067] Take a 250mL dry reaction flask, add 30mL of ethyl acetate to a 250mL round-bottom flask, stir at -5℃, add 3mL of methanol and 3mL of acetyl chloride, and maintain the low temperature for 3h to prepare a hydrogen chloride-saturated ethyl acetate solution. Add the above reaction solution to a reaction flask containing 300mg of scutellarin-4'-(3-methyl-1-piperidinyl)propyl ether protected by 6,7-diphenyl ketal, maintain the low temperature for 2h, then raise the temperature to room temperature and react for 12h. When a yellow precipitate forms in the reaction system and adheres to the wall and the precipitate no longer increases, evaporate the reaction solution under reduced pressure to dryness, wash repeatedly with petroleum ether to obtain a yellow powder with a yield of 66.7%-78.4%.

[0068] (4) Preparation of L-phenylalanine isocyanate tert-butyl ester

[0069] Take a 250 mL dry reaction flask, add 800 mg (3.58 mmol) of L-phenylalanine tert-butyl hydrochloride and 100 mL of dichloromethane to dissolve it, add 1.2 mL of pyridine, place the system at -10 °C and stir for 15 min, add 1 g of triphosgene, keep the reaction at this temperature for 3 h, after the reaction is complete, wash twice each with 0.5 M hydrochloric acid solution with crushed ice and saturated sodium chloride solution, add anhydrous sodium sulfate to remove water from the organic layer, filter, evaporate the filtrate under reduced pressure to obtain a yellow oily substance with a yield of 50%-65%.

[0070] (5) Preparation of scutellarin-7-phenylalanine tert-butyl ester carbamate-4'-(3-methyl-1-piperidinyl)propyl ether

[0071] Add 100 mg (0.23 mmol) of scutellarin-4′-3-methyl-1-propoxypiperidine to each of three 100 mL dry reaction flasks. After adding DMF and sonicating until the solid is completely dissolved, add 150 mg (0.61 mmol, 3 eq) of tert-butyl-protected L-amino acid isocyanate. Add 49 μL (0.35 mmol) of triethylamine while stirring at 50 °C. After reacting for 12 h, monitor the reaction for completeness by TLC. Add 100 mL of CH2Cl2, wash twice with 100 mL of 0.5 M HCl, wash twice with saturated NaCl solution, dry with anhydrous sodium sulfate, filter, and distill under reduced pressure to obtain a yellow solid. Purify by silica gel column chromatography with methanol:dichloromethane:glacial acetic acid = 1:30:1 / 1000 (V / V) as eluent to obtain 85.7 mg of dark green solid, with a yield of 28.6%-35.9%.

[0072] (6) Preparation of scutellarin-7-phenylalanine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether (compound 14a)

[0073] Dissolve scutellarin-7-phenylalanine tert-butyl ester carbamate-4'-(3-methyl-1-piperidinyl)propyl ether in 5 mL of trifluoroacetic acid in a 100 mL dry reaction flask. React in an ice bath for 2 h. After TLC monitoring showed no starting material, the solvent and trifluoroacetic acid were removed by vacuum distillation to obtain the target compound with a yield of 29.1%.

[0074] Example 2

[0075] Preparation of scutellarin aglycone-7-isoleucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether (compound 14b)

[0076] Using scutellarin, L-isoleucine tert-butyl hydrochloride, 3-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-isoleucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 23.4%.

[0077] Example 3

[0078] Preparation of scutellarin aglycone-7-leucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether (compound 14c)

[0079] Using scutellarin, L-leucine tert-butyl hydrochloride, 3-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-leucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 25.8%.

[0080] Example 4

[0081] Preparation of scutellarin aglycone-7-valine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether (compound 14d)

[0082] Using scutellarin, L-valine tert-butyl hydrochloride, 3-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-valine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 21.6%.

[0083] Example 5

[0084] Preparation of scutellarin-7-phenylalanine carbamate-4'-(4-methyl-1-piperidinyl)propyl ether (compound 14e)

[0085] Using scutellarin, tert-butyl L-phenylpropionate hydrochloride, 4-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-phenylalanine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 29.7%.

[0086] Example 6

[0087] Preparation of scutellarin aglycone-7-isoleucine carbamate-4'-(4-methyl-1-piperidinyl)propyl ether (compound 14f)

[0088] Using scutellarin, L-isoleucic acid tert-butyl hydrochloride, 4-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-isoleucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 23.9%.

[0089] Example 7

[0090] Preparation of scutellarin aglycone-7-leucine carbamate-4'-(4-methyl-1-piperidinyl)propyl ether (compound 14g)

[0091] Using scutellarin, L-leucine tert-butyl hydrochloride, 4-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-leucine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 26.2%.

[0092] Example 8

[0093] Preparation of scutellarin aglycone-7-valine carbamate-4'-(4-methyl-1-piperidinyl)propyl ether (compound 14h)

[0094] Using scutellarin, L-valine tert-butyl hydrochloride, 4-methylpiperidine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-valine carbamate-4'-(3-methyl-1-piperidinyl)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 22.8%.

[0095] Example 9

[0096] Preparation of scutellarin aglycone-7-phenylalanine carbamate-4'-(1-cycloheptamino)propyl ether (compound 14i)

[0097] Using scutellarin, L-phenylalanine tert-butyl hydrochloride, cycloheptylamine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-phenylalanine carbamate-4'-(1-cycloheptylamino)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 22.8%.

[0098] Example 10

[0099] Preparation of scutellarin aglycone-7-isoleucine carbamate-4'-(1-cycloheptamino)propyl ether (compound 14j)

[0100] Using scutellarin, L-phenylalanine tert-butyl hydrochloride, cycloheptylamine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-isoleucine carbamate-4'-(1-cycloheptylamino)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 22.8%.

[0101] Example 11

[0102] Preparation of scutellarin aglycone-7-leucine carbamate-4'-(1-cycloheptamino)propyl ether (compound 14k)

[0103] Using scutellarin, L-phenylalanine tert-butyl hydrochloride, cycloheptylamine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-leucine carbamate-4'-(1-cycloheptylamino)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 24.7%.

[0104] Example 12

[0105] Preparation of scutellarin aglycone-7-valine carbamate-4'-(1-cycloheptamino)propyl ether (compound 14l)

[0106] Using scutellarin, L-phenylalanine tert-butyl hydrochloride, cycloheptanylamine, and triphosgene as reaction substrates, the target compound scutellarin aglycone-7-valine carbamate-4'-(1-cycloheptanylamino)propyl ether was prepared according to a method similar to that in Example 1, with a yield of 27.3%.

[0107] Example 13

[0108] In vitro cholinesterase inhibition experiment

[0109] 13.1 Experimental Materials and Instruments

[0110] (1) Electric eel acetylcholinesterase (eeAChE), damalactone cholinesterase (eqBuChE) (Sigma-Aldrich, USA), thioacetylcholine iodide (Shanghai Adamas Reagent Co., Ltd.), thiobutyryl iodide (Shanghai Yuanye Biotechnology Co., Ltd.), rivastigmine tartrate (Shanghai Adamas Reagent Co., Ltd.), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (Shanghai Maclean Biochemical Technology Co., Ltd.), and compounds 14a-14l were prepared in the laboratory, i.e., the compounds obtained in Examples 1-12, with a purity >95%.

[0111] (2) Main instruments: KZ-20L ultrapure water system (Shanghai Keqia Environmental Protection Equipment Co., Ltd.); benchtop constant temperature shaker (Shanghai Yuejin Medical Instrument Co., Ltd.); TGL-16.5M low temperature high speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); Varioskan LUX multifunctional microplate reader (Thermo Scientific, USA).

[0112] 13.2 Test Methods

[0113] 13.2.1 Preparation of Reagent Solutions

[0114] AChE solution: Dissolve 500 U of AChE in 50 mL of Tris-HCl buffer to a concentration of 10 U / mL, aliquot and freeze at -80°C. Dilute with buffer to the required concentration before use.

[0115] BuChE solution: Dissolve 100 U of BuChE in 10 mL of Tris-HCl buffer to a concentration of 10 U / mL, aliquot and freeze at -80°C. Dilute the buffer to the required concentration before use.

[0116] ATCI solution: Weigh 10.5 mg of ATCI into a centrifuge tube and add 5 mL of PBS to dissolve it to obtain a 7.5 mM ATCI solution.

[0117] Thiobutyryl choline iodide solution (BTCI): Weigh 7.2 mg of BTCI into a centrifuge tube, add 3 mL of PBS to dissolve and obtain a 7.5 mM BTCI solution.

[0118] DTNB solution: Weigh 60 mg of DTNB, add 15 mL of PBS to dissolve it to obtain a 10 mM DTNB solution, and store it at 4°C protected from light for later use.

[0119] Compound solution: The compound was dissolved in DMSO to prepare a 10mM stock solution, which was then diluted with PBS to the required concentration before use.

[0120] 13.2.2 Experimental Procedure

[0121] (1) Detection of cholinesterase inhibitory activity

[0122] Add 140 μL of PBS buffer, 20 μL of PBS (blank group) or 20 μL of different concentrations of the compound (test group) to a 96-well plate sequentially, along with 20 μL of AChE / BuChE. Simultaneously, set up a blank background group and an experimental background group (using PBS instead of cholinesterase). After adding the buffer and mixing, incubate at 37°C for 20 min. Immediately add 10 μL of DTNB solution and 10 μL of ATCI or BTCI. Incubate at 37°C for 10 min, then measure the absorbance of each well at 412 nm using a microplate reader. Calculate the inhibition rate of the compound against cholinesterase using the formula: Enzyme inhibition rate = 1 - (OD test group - OD test background group) / (OD blank group - OD blank background group) × 100%. Each experiment was independently repeated three times, with seven concentrations for each compound. GraphPadprism 8.0 was used to calculate the IC50 of the compound inhibiting cholinesterase. 50 .

[0123] (2) Enzyme kinetics studies

[0124] Take a 96-well plate and add 140 μL of PBS buffer to each well. Add 20 μL of PBS buffer to the blank group and 20 μL of different concentrations (0, 15, 30, 45 μM) of compound to the test group. Add 20 μL of LChE enzyme (0.075 U / L). Set up a blank background group (with enzyme) and a test local group (with PBS instead of enzyme). Mix well and incubate at 37°C for 20 min. Immediately after incubation, add 10 μL of LTNB solution and 10 μL of different concentrations (0.5, 1, 1.5, 2 mM) of ATCI solution. Mix well and incubate at 37°C for 10 min. Immediately, the OD value of each well at 412 nm was measured using a microplate reader. The enzyme reaction rate at different substrate concentrations was calculated, and a double reciprocal plot of rate versus substrate was plotted to determine the type of inhibition. With ATCI concentration set at 7.5 mmol / L, the initial reaction rate was measured at four different compound concentrations (0, 15, 30, and 45 μM / L for 14L) for different AChE enzyme activities. A graph was plotted with enzyme activity (U / mL) on the x-axis and initial reaction rate V0 [μmol / (L·min)] on the y-axis. The experiment was independently repeated three times, with three replicates per group.

[0125] 13.3 Experimental Results

[0126] Table 1. Inhibitory activity of the target compounds against eeAChE and eqBuChE

[0127]

[0128]

[0129] a Selectivity Index = IC 50 (BuChE) / (AChE).

[0130] b eeAChE fromelectrophorus electricusc.

[0131] c eqBuChE from equine serum.

[0132] d Values ​​are expressed as the mean±standarddeviation ofthemean ofthreeindependent

[0133] experiments.

[0134] e NT = nottest.

[0135] 13.4 Experimental Conclusions

[0136] (1) Results of study on cholinesterase inhibitory activity

[0137] The inhibitory activities of 12 target compounds (14a-14l) against AChE and BuChE were detected using the Ellman assay. AChE is derived from acetylcholinesterase (eeAChE) from electric eels, and BuChE is martinocholinesterase (eqBChE). The results are shown in Table 1. Based on these results, we found that the compounds in the 14a-14d series exhibited moderate inhibitory activity against cholinesterase compared to the positive control drug rivastigmine; 14i-14l were superior to the positive control drug rivastigmine, with 14i showing the strongest activity (IC50). 50 =7.04 μM). Derivative 14i showed the strongest inhibitory activity against eqBuChE (IC50). 50=13.4 μM), which is the strongest BuChE inhibitor among the 12 compounds. 14a-14h are inactive against eqBuCh. Compared with scutellarin aglycone and scutellarin, we observed a clear relationship between ligand structure and cholinesterase activity. Among all derivatives, the anticholinesterase activity is related to the presence of the phenoxyalkylamine fragment in the derivative. Comparing the analogs of 14a-14d and 14e-14h, it can be seen that the derivatives of 14a-14d generally have stronger acetylcholinesterase inhibitory activity than 14e-14h. These results indicate that the position of the substituent on the cycloalkylamine in the compound has a significant impact on the selectivity of the cholinesterase ligand. The acetylcholinesterase inhibitory activity of the compound (14a-14d) with the methyl group at the 3-position of the piperidine ring is higher than that of the compound (14e-14h) with the methyl group at the 4-position of the piperidine ring. The activity of the derivatives of 14i-14l against AChE (IC50) is... 50 The N-heptahydrate concentration (7.04-9.59 μM) was significantly higher than that of the other compounds. At the same time, the compound showed inhibitory activity against butyrylcholinesterase, indicating that the difference in the N-heterocycle has a significant impact on its selectivity with the enzyme. When the cycloheptahydrate fragment is introduced into the compound structure, it exhibits strong enzyme inhibitory activity.

[0138] (2) Based on the above results, compound 14l was selected for eeAChE kinetic studies. The results are shown in Figure 1A. When the concentration of 14l was 0, 15, 30, and 45 μM / L, the reaction rate was significantly lower than that of the group without inhibitor. As the concentration of 14l increased, the reaction rate became slower. Moreover, the four straight lines intersected at the same point in the second quadrant of the Lineweaver-Burker plot, i.e., V max Reduce, K m The increase indicates that the compound's inhibitory mechanism on eeAChE is a mixed inhibition. As shown in Figure 1B, when the concentration of 14l is 0, 15, 30, and 45 μM / L, the obtained straight lines all pass through the origin, and the higher the concentration of the compound, the lower the slope of the corresponding straight line. This result indicates that the compound's inhibitory mechanism on eeAChE is reversible inhibition.

[0139] Example 14

[0140] Study on metal ion chelation ability

[0141] 14.1 Experimental Apparatus and Reagents

[0142] (1) Experimental apparatus

[0143] In addition to the UV-2600 UV-Vis spectrophotometer (Shimadzu Corporation, Japan); KZ-20L ultrapure water system (Shanghai Keqia Environmental Protection Equipment Co., Ltd.); benchtop constant temperature shaker (Shanghai Yuejin Medical Instrument Co., Ltd.); TGL-16.5M low temperature high speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); Varioskan LUX multi-functional microplate reader (Thermo Scientific, USA).

[0144] (2) Experimental reagents

[0145] FeSO4·7H2O, AlCl3, ZnCl2 (Shanghai Maclean Biochemical Technology Co., Ltd.); CuCl2·2H2O (Tianjin Xiens Biochemical Technology Co., Ltd.), DMSO (Aibisin Biotechnology Co., Ltd.).

[0146] 14.2 Experimental Methods

[0147] 14.2.1 Metal ion chelation ability test

[0148] (1) Solution preparation:

[0149] FeSO4·7H2O, AlCl3, ZnCl2, and CuCl2·2H2O were dissolved in pure water to prepare a 10 mM solution containing Fe. 2+ Al 3+ Zn 2+ Cu 2+ The mother liquor is prepared by taking 6 μL of the mother liquor and diluting it with 994 μL of methanol to 60 μM before use.

[0150] The compound was prepared as a 10 mM masterbatch with DMSO and diluted to 60 μM with methanol immediately before use.

[0151] (2) Experimental steps:

[0152] Add 400 μL of metal ion solution (60 μM) and 400 μL of compound solution (60 μM) to a 1.5 ml centrifuge tube, mix well, and let stand at room temperature for 30 min. After baseline correction with methanol (containing 6% DMSO), take 250 μL of the mixture and add it to a micro-quartz cuvette. Observe its ultraviolet absorption spectrum in the wavelength range of 200-600 nm. The control group is methanol + test compound. Observe and record the changes in the maximum absorption wavelength after mixing the test compound with different metal ions.

[0153] 14.2.2 Compound complexation of Cu 2+ Proportion test

[0154] Using the molar ratio method, based on the above metal ion complexation ability test results, Cu, which has a more obvious complexation with the compound, was selected. 2+Further chelation ratio tests were conducted with compound 14l. Cu, which undergoes complexation with compound 14l, was then subjected to further chelation ratio testing. 2+ Ions were prepared into a concentration gradient of 0.1 to 2.5 eq (eq: the ratio of metal ion concentration to compound concentration) mixed solutions. 400 μL of the analyte methanol solution (60 μmol / L) was taken, and 400 μL of different concentrations of metal ion methanol solutions were added to each. After mixing, the solution was allowed to stand for 40 min, and the absorbance was immediately measured using a UV-Vis spectrophotometer. The baseline was adjusted beforehand using methanol solution. The absorbance was plotted to obtain the concentrations of 14L and Cu. 2+ The complexation ratio of ions.

[0155] 14.3 Experimental Results

[0156] See Figure 2 for details.

[0157] 14.4 Experimental Conclusions

[0158] 14.4.1 Metal ion chelation ability test

[0159] To investigate whether the compound possesses metal chelating ability, this paper employs ultraviolet-visible spectrophotometry to analyze the reaction between the synthesized target compound and metal ions (Cu). 2+ Fe 2+ Zn 2+ Al 3+ The strength of the chelating properties was evaluated by comparing the compound with the compound containing the metal, using methanol as a blank control. As shown in Figure 2(A), the compound with Zn... 2+ Afterwards, the maximum absorption wavelength did not change significantly, but the addition of three other metal ions resulted in varying degrees of red shift in the maximum absorption wavelength; among them, compound 14l and Cu... 2+ The changes after chelation are more pronounced, with the maximum absorption wavelength shifting from 234 nm to 383 nm, indicating that compound 14l can selectively bind with Cu. 2+ It forms metal ion complexes.

[0160] 14.4.2 Compound complexation of Cu 2+ Proportion test

[0161] Based on the above experiments and using the molar ratio method, we further investigated Cu. 2+ The chelation ratio with compound 14l, as shown in Figure 2(B), at a wavelength of 383 nm, increases with Cu 2+ As the concentration of Cu increases, the absorbance initially increases linearly, but when Cu... 2+ After reaching a certain concentration, continue to increase Cu 2+ The concentration and absorbance increased slowly. After fitting the two lines, they intersected at 0.98, indicating that compound 14l reacted with Cu. 2+ Complex in a 1:1 ratio.

[0162] Example 15

[0163] Compound inhibits / depolymerizes Aβ 1-42 Aggregation activity study

[0164] 15.1 Experimental Apparatus and Reagents

[0165] (1) Experimental apparatus

[0166] In addition to the JEM-1400FLASH transmission electron microscope (JEOL Corporation, Japan); KZ-20L ultrapure water system (Shanghai Keqia Environmental Protection Equipment Co., Ltd.); benchtop constant temperature shaker (Shanghai Yuejin Medical Instrument Co., Ltd.); TGL-16.5M low temperature high speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); Varioskan LUX multifunctional microplate reader (Thermo Scientific, USA).

[0167] (2) Experimental reagents

[0168] humanAβ 1-42 (Beyotime); Thioflavin T (ThT) (Shanghai Maclean Biochemical Technology Co., Ltd.); Curcumin (Shanghai Yuanye Biotechnology Co., Ltd.); 2% Phosphotungstic acid (Henan Ruixin Experimental Supplies Co., Ltd.); DMSO (Aibisin Biotechnology Co., Ltd.).

[0169] 15.2 Experimental Methods

[0170] 15.2.1 Thiamine T-method for detecting compounds inhibiting Aβ 1-42 Self-induction experiment

[0171] Take a black, light-shielded 96-well plate, and add (20 μL Aβ) to the test group sequentially. 1-42 The test blank group was treated with (20 μL of compound solution + 20 μL of PBS buffer), and the control group was treated with (20 μL of Aβ solution + 20 μL of PBS buffer). 1-42 The test compound solution was added to 20 μL PBS buffer, and the control group was added to 20 μL PBS buffer + 20 μL PBS buffer. Considering that the solution would evaporate, it was sealed with a layer of plastic wrap and then incubated in a 37°C incubator for 24 h. In the system, the test compound solution and Aβ were added. 1-42 The final concentration of all solutions was 25 μM. After incubation, 160 μL of thioflavin T solution was added to the system, and the mixture was shaken in the dark for 1 min. Immediately afterward, the fluorescence intensity was detected using a multi-functional microplate reader at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. The inhibition rate of the compounds was calculated using a formula.

[0172] The calculation formula is: 100 - (IF) A -IF B ) / (IFC -IF D )×100; where IF A For Aβ 1-42 (Test group) Fluorescence value, IF B For Aβ 1-42 Fluorescence value of (test blank group), IF C For Aβ 1-42 (Control group) fluorescence value, IF D The fluorescence value was obtained using only the buffer solution (control blank group). Each experiment was repeated three times independently, with three replicates per group.

[0173] 15.2.2 Compounds on Aβ 1-42 Self-induced aggregation de-aggregation ability

[0174] Take a black, light-proof 96-well plate and add 20 μL of LAβ to each well. 1-42 The solution was sealed with plastic wrap and then covered, and incubated in a 37°C incubator for 24 hours. After incubation, 20 μL of the compound solution was added to the test group, 20 μL of PBS buffer was added to the blank test group, 20 μL of PBS buffer was added to the control group, and 20 μL of PBS buffer + 20 μL of PBS buffer was added to the blank control group. The solution was then sealed with plastic wrap and incubated in a 37°C incubator for another 24 hours. The test compound solution and Aβ... 1-42 The final concentration for all tests was 25 μM. After incubation, 160 μL of thioflavone T solution was added, and the mixture was shaken in the dark for 2 min. Immediately afterward, the fluorescence value was detected using a multi-functional microplate reader (excitation wavelength 450 nm, emission wavelength 485 nm). The depolymerization rate of the compounds was calculated using a formula.

[0175] The calculation formula is: 100 - (IF) A -IF B ) / (IF C -IF D )×100; where IF A For Aβ 1-42 (Test group) fluorescence value, IF B For Aβ 1-42 Fluorescence value of (test blank group), IF C For Aβ 1-42 (Control group) fluorescence value, IF D The fluorescence value was obtained using only the buffer solution (control blank group). Each experiment was independently repeated three times, with three replicates for each sample.

[0176] 15.2.3 Thiamine T-method for detecting compounds inhibiting Cu 2+ Induced Aβ 1-42 Aggregation experiment

[0177] Take a black, light-proof 96-well plate, and add (20 μL of compound solution + 20 μL of Cu) to the test group sequentially. 2+ +20μLAβ 1-42 (solution), the test blank group was prepared by adding (20 μL of compound solution + 20 μL of Cu). 2+ +20 μL HEPES buffer), control group added (20 μL Aβ) 1-42 Solution + 20 μL HEPES buffer + 20 μL Cu 2+ ), control group added (20 μL HEPES buffer + 20 μL HEPES buffer + 20 μL Cu) 2+ Considering the possibility of solution evaporation, the solution was sealed with plastic wrap and then incubated at 37°C for 24 hours. After incubation, 140 μL of thioflavone T solution was added, and the mixture was shaken in the dark for 2 minutes. Immediately afterward, the fluorescence intensity was measured using a multi-functional microplate reader at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. The inhibition rate of the compound was calculated using a formula.

[0178] The calculation formula is: 100 - (IF) A -IF B ) / (IF C -IF D )×100; where IF A For Aβ 1-42 (Test group) fluorescence value, IF B For Aβ 1-42 Fluorescence value of (test blank group), IF C For Aβ 1-42 (Control group) fluorescence value, IF D The fluorescence values ​​were obtained using only the buffer solution (control blank group). Each experiment was independently repeated three times, and the concentration of each compound was measured in three replicates.

[0179] 15.2.4 Thiamine T Method for Detection of Compound Depolymerization Cu 2+ Induced Aβ 1-42 Aggregation experiment

[0180] Take a black, light-proof 96-well plate and add 20 μL of LAβ to each well. 1-42 Solution + 20 μL Cu 2+After sealing with plastic wrap, the samples were incubated at 37°C for 24 hours. After incubation, 20 μL of the compound solution was added to the test group, 20 μL of HEPES buffer to the blank test group, 20 μL of HEPES buffer to the control group, and 20 μL of HEPES buffer + 20 μL of HEPES buffer to the blank control group. The samples were then sealed with plastic wrap and incubated at 37°C for another 24 hours. After incubation, 140 μL of thioflavone T solution was added, and the samples were shaken in the dark for 2 minutes. Immediately afterward, the fluorescence intensity was measured using a multi-mode microplate reader at an excitation wavelength of 450 nm and an emission wavelength of 485 nm. The depolymerization rate was calculated using a formula.

[0181] The calculation formula is: 1-(IF) A -IF B ) / (IF C -IF D )×100; where IF A For Aβ 1-42 (Test group) fluorescence value, IF B For Aβ 1-42 Fluorescence value of (test blank group), IF C For Aβ 1-42 (Control group) fluorescence value, IF D The fluorescence values ​​were obtained using only the buffer solution (control blank group). Each experiment was independently repeated three times, and the concentration of each compound was measured in three replicates.

[0182] 15.3 Experimental Results

[0183] 15.3.1 Compound Inhibition / Depolymerization of Aβ 1-42 Results of aggregation ability study

[0184] Table 2. Compounds and Positive Drugs Inhibit / Depolymerize Themselves and Cu 2+ Induced Aβ 1-42 Gathering

[0185]

[0186]

[0187] a For inhibition / disaggregation ofAβ 1–42 aggregates, the thioflavin-Tfluorescencemethod was used. Data represented as the mean ± SD of three independent experiments.

[0188] b Inhibition of self-induced Aβ 1–42 aggregation by 25 μM of the test compound.

[0189] c Inhibition of Cu 2+ -inducedAβ 1–42 aggregationwith concentrations ofthetestcompound,Cu 2+ andAβ 1–42 of 25μM.

[0190] d Disaggregation of self-inducedAβ 1–42 aggregates with concentrationsofthetest compoundandAβ 1–42 of 25μM.

[0191] e Disaggregation of self-inducedAβ 1–42 aggregates with concentrationsofthetest compoundandAβ 1–42 of 25μM.

[0192] f Disaggregation of Cu 2+ -inducedAβ 1–42 aggregates with concentrationsof the test compound,Cu 2+ andAβ 1–42 of 25μM.

[0193] g nt = nottested.

[0194] 15.3.2 TEM observation of 14l inhibited / depolymerized Cu 2+ Inducing Aβ 1-42 Results of aggregation ability study

[0195] See Figure 3 for details.

[0196] 15.3 Experimental Conclusions

[0197] The effect of the compound on Aβ was studied using the thiosulfate T fluorescence method. 1-42 Inhibitory effect. As shown in Table 2, all compounds exhibited good inhibition of Aβ. 1-42 The aggregation effect, and all compounds inhibit Aβ. 1-42 The aggregation effect was far greater than that of scutellarin and curcumin. Among them, compounds 14b, 14h, and 14l showed inhibition rates exceeding 85%. Therefore, we selected compounds 14b, 14h, and 14l to further investigate their inhibition of Cu. 2+ Induced Aβ 1-42 Aggregation, and self-induced deaggregation and Cu 2+ The induced aggregation results are shown in Table 2. The results show that 14b, 14h, and 14l inhibited Cu aggregation. 2+ Induced Aβ 1-42 Aggregation, and self-induced deaggregation and Cu 2+ The induced aggregations were superior to those of scutellarin and curcumin, with compound 14L inhibiting self-induced aggregation and Cu aggregation. 2+ The induced inhibition rates were 94.58% and 88.63%; 14L depolymerization was self-induced and Cu 2+ The induced depolymerization rates were 80.16% and 89.30%, respectively, both far superior to those of scutellarin and curcumin.

[0198] To further observe the effect of compound 14l on Cu 2+ Induced Aβ 1-42 The aggregation inhibition / deaggregation ability was investigated using transmission electron microscopy (TEM), and the results are shown in Figure 3(A). Unincubated Aβ... 1-42 Under an electron microscope, they appear as relatively dispersed spherical particles, and when alone with Cu... 2+ After 24 hours of incubation, the fibers aggregated into irregular fibers. The addition of Curcumin reduced fiber aggregation, but the reduction in fiber aggregation after adding compound 14l was greater than that after adding Curcumin, indicating that compound 14l effectively inhibited Cu aggregation. 2+ Induced Aβ 1-42 Gathering.

[0199] Furthermore, as shown in Figure 3(B), Cu alone 2+ and Aβ 1-42 After 48 hours of incubation, Aβ 1-42 The fiber aggregation was greater after incubation with 14L or Curcumin for 24 hours than after incubation with 14L. Further incubation with 14L or Curcumin for 24 hours reduced fiber aggregation, indicating that 14L can depolymerize Cu. 2+ Mediated Aβ 1-42 The aggregation of fibrils indicates that compound 14l can effectively depolymerize Cu. 2+ Induced Aβ 1-42Aggregation. The results from transmission electron microscopy are consistent with those obtained using the thiosulfate T method.

[0200] Example 16 Compound 14l inhibits tau hyperphosphorylation

[0201] 16.1 Experimental Instruments and Reagents

[0202] (1) Experimental apparatus

[0203] Canto-II plusL flow cytometer (Bio-Rad Laboratories, USA); electrophoresis solution and transfer system (Bio-Rad Laboratories, USA); KZ-20L ultrapure water system (Shanghai Keqia Environmental Protection Equipment Co., Ltd.); benchtop constant temperature shaker (Shanghai Yuejin Medical Instrument Co., Ltd.); TGL-16.5M low temperature high speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0204] (2) Experimental reagents

[0205] T-Tau and P-Tau (Thr 181) antibodies (Abcom, UK); fetal bovine serum, trypsin, and horse serum (Gbico, USA); penicillin antibodies (Wuhan Pronosai Life Science Technology Co., Ltd.); protein loading buffer, primary antibody diluent, and secondary antibody diluent (Haimen Biyuntian Biotechnology Co., Ltd.).

[0206] 16.2 Experimental Methods

[0207] First, establish Aβ 25-35 In the PC12 cell injury model, PC12 cells were added at a rate of 1×10⁻⁶ per well, with 100 μL per well. 5 Aβ cells / mL were seeded into 96-well plates and incubated for 24 hours in an incubator with 5% CO2 at 37°C, divided into two groups (control group and model group). After incubation, the supernatant was removed, culture medium was added to the control group, and different concentrations of Aβ were added to the model group. 25-35 Cells were incubated at concentrations of 25, 30, 35, 40, 45, 50, and 55 μM for 24 hours. The supernatant was removed, and the cells were washed twice with PBS. 100 μL of 10% CCK8 serum-free DMEM solution was added to each well, and the cells were incubated for another 1.5 hours. The absorbance was measured at 450 nm. Cell viability was calculated. The experiment was independently repeated three times, with five replicates per well.

[0208] Cell viability (%) = ((mean OD value of sample group) / (mean OD value of control group)) × 100

[0209] In Aβ 25-35 At a concentration of 40 μM, cell damage was 50.72%, therefore this concentration was selected as the membrane-forming concentration. PC12 cells were added to each well at a concentration of 1.5 × 10⁶ mL. 6Aβ was seeded at a concentration of 1 / mL in petri dishes and divided into control, model, and compound groups. After incubation for 24 hours in an incubator with 5% CO2 at 37°C, the supernatant was removed. Culture medium was added to the control group, while 40 μM Aβ was added to the model group. 25-35 The compound group was supplemented with 40 μM Aβ. 25-35 Different concentrations (5, 10, 20 μM) of the compound were incubated in an incubator for 24 h. The supernatant was removed, and the cells were washed twice with PBS. An appropriate amount of RIPA protein lysis buffer (containing PMSF) and cell lysis buffer were added. Cells were scraped off and collected in 1.5 mL centrifuge tubes. The cells were lysed on ice for 30 min, vortexed for 30 seconds, and centrifuged at 4 °C for 30 min. The supernatant was used to detect the protein concentration of the sample according to the BCA protein concentration kit.

[0210] After removing the supernatant, add 5× loading buffer and mix well. Heat at 100℃ for 10 min to denature, then cool before loading. Aliquot the remaining proteins and store at -20℃. Following the instructions of the SDS-PAGE gel preparation kit, prepare gels according to the protein molecular weight (10% separating gel, 5% stacking gel). Place the prepared SDS-PAGE gels in the electrophoresis tank, add 1× electrophoresis buffer, and add the marker reagent and prepared protein samples sequentially to the wells. Turn on the electrophoresis and adjust the voltage to (80V, 20 min). After the bromophenol blue reaches the separating gel region, set the voltage to (120V, 70 min) until the bromophenol blue reaches the bottom of the separating gel. Stop electrophoresis and prepare for membrane transfer. Cut the PVDF membrane and immerse it in methanol for 30 seconds. Then, cut the target protein and place it in a transfer apparatus in the following order from bottom to top: negative electrode - filter paper - PVDF membrane - gel - filter paper - positive electrode. Add transfer buffer and transfer for 30 minutes. Cut the PVDF membrane and place it in an incubation box. Add rapid blocking buffer and block on a shaker at 4°C for 20 minutes. Then add primary antibody Total-Tau (1:1000 dilution) and p-tau (Thr181) (1:800 dilution) and incubate at 4°C overnight.

[0211] After overnight incubation with the primary antibody, the membrane was recovered and washed 5 times (5 min each time) with 1×TBST buffer. The secondary antibody (1:5000 dilution) was incubated at 4℃ for 2 h and then washed 5 times (5 min each time) with 1×TBST buffer. 200 μL of luminescent solution (a 1:1 mixture of solution A and solution B, stored in the dark, and prepared fresh) was dropped onto the PVDF membrane, exposed to light for 1 min, and then exposed to light.

[0212] Annexin V-FITC staining analysis

[0213] PC12 cells in logarithmic growth phase were selected, digested with 0.25% trypsin, and counted under a microscope. 6 mL of the resulting solution was then seeded into culture dishes (density 1.0 × 10⁶ cells / mL). 6 Cells were incubated in a 5% CO2 incubator at 37°C, divided into three groups (control group, model group, and drug group). After 24 hours, the supernatant was removed. For the normal control and model groups, 6 mL of serum-containing DMEM culture medium was added per dish. For the drug group, Scu and Trolox were prepared at a concentration of 15 μM using serum-containing DMEM medium, and 14l was prepared at different concentrations (5, 10, and 15 μM) using DMEM medium (containing serum). 6 mL of each drug group was added, and after 24 hours, the supernatant was removed. Cells were washed twice with PBS buffer. The control group received 6 mL of serum-free DMEM culture medium, while the other groups received 6 mL of serum-free DMEM solution (containing 800 μM H2O2). After 3 hours in the incubator, the supernatant was collected, and 2 mL of EDTA-free trypsin digestion solution was added. After 1 minute, 2 mL of serum-containing culture medium was added, and the cells were collected. The cells were centrifuged, and the supernatant was removed. The cells were resuspended in the collected supernatant and diluted to 1 × 10⁻⁶ cells using 1x buffer. 6 Each sample was then incubated with 5 μL FITC Annexin V and 5 μL LPI, protected from light for 15 min, and analyzed by flow cytometry (BD FACSC anto II).

[0214] 16.3 Experimental Results

[0215] See Figure 4 for details.

[0216] 16.4 Experimental Conclusions

[0217] The main pathological features of Alzheimer's disease (AD) are senile plaques formed by extraneuronal Aβ aggregation and neurofibrillary tangles formed by abnormal intraneuronal p-Tau accumulation. Therefore, aberrant phosphorylation of tau protein is extremely important in the occurrence and development of AD. Thus, we used western blotting to test the effect of compound 14l on Aβ... 25-35 The effect of induced tau phosphorylation in PC12 cells. As shown in Figures 4(A) and 4(B), Aβ... 25-35 Following induction, the expression of p-Tau(Thr181) / Total-tau protein increased in PC12 cells. However, this increase was reversed upon treatment with compound 14l, donepezil, or Scu. Compound 14l at 10 μM exhibited inhibitory activity comparable to donepezil but stronger than scutellarein (Scu); at 20 μM, its inhibitory activity was stronger than both donepezil and Scu. Therefore, these results indicate that 14l can inhibit aberrant tau phosphorylation and exert anti-AD activity.

[0218] Example 1714l Study on inhibiting LPS-induced release of inflammatory factors in BV-2 cells

[0219] 17.1 Experimental Apparatus and Reagents

[0220] (1) Experimental apparatus

[0221] Varioskan LUX Multifunctional Microplate Reader (Thermo Scientific, USA); KZ-20L Ultrapure Water System (Shanghai Keqia Environmental Protection Equipment Co., Ltd.); Tabletop Constant Temperature Shaker (Shanghai Yuejin Medical Instrument Co., Ltd.); TGL-16.5M Low Temperature High Speed ​​Centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.).

[0222] (2) Experimental reagents

[0223] BV-2 cells (American Type Culture Collection, ATCC, USA); donepezil (Shanghai Adamas Reagent Co., Ltd.); IL-6 and TNF-α (ELISA) detection kit (Wuhan Pronosei Life Science Technology Co., Ltd.).

[0224] 17.2 Experimental Methods

[0225] 17.2.1 Investigation of the safe concentration range of compound 14l in BV-2 cells

[0226] Select PC12 cells in the logarithmic growth phase, and administer at a dose of 1.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 / ml in 96-well plates and incubated in a 5% CO2 incubator at 37°C. Two groups (control and drug groups) were incubated for 24 hours. The control group received 100 μl of serum-containing medium, while the drug group received 100 mM of drug diluted to different concentrations (5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM) in serum-containing DMEM medium. 100 μL of the drug was added to each well and incubated at 37°C for another 24 hours. After incubation, the supernatant was removed, and the cells were washed twice with PBS. Serum-free CCK8 medium (10% CCK8) was added, and the cells were incubated at 37°C for 1.5 hours. The absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated. Each group had 5 replicates, repeated 3 times.

[0227] 17.2.2 Compound 14L inhibits LPS-induced release of inflammatory factors from BV-2 cells.

[0228] PC12 cells in the logarithmic growth phase were selected, digested with 0.25% trypsin, and counted under a microscope (density 1.15 × 10⁻⁶). 52 mL of serum-containing DMEM medium was inoculated into each well of a 6-well plate and incubated for 24 hours in an incubator with 5% CO2 and 37°C. The plates were divided into three groups (control group, model group, and drug group). After incubation, the supernatant was removed, and 2 mL of serum-containing DMEM medium was added to each well (control group and model group). For the drug group, Scu and Donepezil were prepared to a concentration of 10 μM in serum-containing DMEM medium, and compound 14l was prepared to different concentrations (5, 10, 20 μM) in DMEM medium (containing serum). 2 mL of the drug was added to each well. After 1 hour, 20 μL of 10 μg / mL LPS (final concentration 0.1 μg / mL) was added to each well of the model group and drug group. After culturing for another 24 hours, the supernatant was collected, centrifuged, and the contents of IL-6 or TNF-α were measured by enzyme-linked immunosorbent assay (ELISA).

[0229] 17.3 Experimental Results

[0230] See Figure 5 for details.

[0231] 17.4 Experimental Conclusions

[0232] Neuroinflammation is a hallmark of Alzheimer's disease (AD) pathogenesis. Therefore, inhibiting the neuroinflammatory process may have good potential as an anti-AD treatment. Thus, the in vitro anti-inflammatory effect of compound 14l was evaluated. Cell viability assays were performed to determine the cytotoxicity of 14l against BV-2 cells. The results showed that 14l (0-100 μM) exhibited mild toxicity to BV-2 cells over a 24-hour culture period (Figure 5A), with a cell viability of 77.90% at a 100 μM concentration. After pretreating BV-2 cells with 14l, Scu, and Don for 1 hour, followed by simultaneous pretreatment with lipopolysaccharide (LPS) for 24 hours, the production of interleukin-6 (IL-6) and TNF-α was measured using an ELISA kit. As shown in Figures 5B and 5C, LPS induced a significant increase in the pro-inflammatory factors IL-6 and TNF-α (p<0.05), while 14l significantly inhibited the production of IL-6 and TNF-α in LPS-stimulated BV-2 microglia. Donepezil did not show anti-inflammatory effects.

[0233] Example 18.1 Study on the improvement of cognitive function in scopolamine-induced AD model mice 18.1 Experimental instruments and reagents

[0234] (1) Laboratory animals

[0235] Male Kunming mice, 6 - 8 weeks old, weighing (18 - 25 g), of SPF grade, were purchased from Beijing Spepharm Biotechnology Co., Ltd. with the license number: SCXK(Beijing)-2019-0010. According to the regulations of the Animal Laboratory of the Key Laboratory of Pharmaceutical Preparations of Guizhou Medical University, all animal operations were strictly carried out in accordance with the regulations. The animal rearing environment was at a temperature of 23 ± 2 °C, a humidity of 40 - 60%, with a 12 h light and 12 h dark cycle. Every 15 mice were housed in a large cage, and the animals were allowed to acclimatize to the environment for one week before the experiment.

[0236] (2) Experimental instruments

[0237] Water maze system (Nanjing Calvin Biotechnology Co., Ltd.); JJ-12 dehydrator, JB-P5 embedding machine, JB-L5 freezing table (Wuhan Junjie Electronics Co., Ltd.); RM2016 pathological slicer (Shanghai Leica Instruments Co., Ltd.).

[0238] (3) Experimental reagents

[0239] Scopolamine hydrobromide (Guangzhou CATO Co., Ltd.); Rivastigmine (Shanghai Adamas Reagent Co., Ltd.); Paraformaldehyde (Wuhan Junjie Electronics Co., Ltd.); HE staining solution, blueing solution (Wuhan Sevier Biotechnology Co., Ltd.); AChE kit (Nanjing Jiancheng Bioengineering Institute).

[0240] 18.2 Test methods

[0241] 18.2.1 Establishment of animal model and grouped administration

[0242] The mice were randomly divided into 6 groups (15 mice / group), namely the control group, the model group, the rivastigmine group (1.8 mg / kg), the low-dose group (1.25 mg / kg), the medium-dose group (2.5 mg / kg), and the high-dose group (5 mg / kg). After one week of acclimatization in the environment, the 6 groups were intraperitoneally injected continuously at the same time for 26 days. The control group and the model group were injected with the solvent (DMF:PEG-400:saline = 1:6:3), and the rivastigmine group and the drug groups were injected after dissolving the drugs with the solvent. After the administration ended on the 21st day, each mouse was allowed to swim freely in the pool for 60 s to adapt to the pool environment. One hour after the daily administration ended on the 22nd day, except for the control group which was still injected with the solvent, the other groups were injected with scopolamine (1.25 mg / kg) for 5 consecutive days (from the 22nd to the 26th day) to construct a memory impairment model. After the last administration on the 26th day, it was detected whether the mice were successfully modeled, and then a spatial search test was carried out; on the 27th day, the platform was removed and a spatial search test was carried out.

[0243] 18.2.2 Adaptive training

[0244] On day 21, each mouse was placed in a pool (with the platform removed) and allowed to swim freely for 1 minute to acclimatize to the environment. Placing the mice face down in the pool and allowing them to swim freely can reduce the interference of some emergency factors on the formal experiment. After each mouse finished swimming, it was dried and placed back in its cage.

[0245] 18.2.3 Positioning and navigation test

[0246] A five-day escape navigation experiment was conducted from days 22 to 26. This training tested the animals' spatial memory, assessing their ability to locate the underwater platform more quickly using environmental cues as the training days progressed. At the start of the experiment, a 3-second latency period was set. Mice were placed in the water at the midpoint of each quadrant. Timing began after 3 seconds, and a tracking device on the pool recorded the mice's swimming trajectory. Timing stopped if the mouse found the platform within 60 seconds. If the mouse did not find the platform after 60 seconds, it was placed on the platform for 20 seconds to demonstrate the existence of an escape platform. Mice were then placed in the remaining three quadrants for a total of five days. Changes in escape latency were observed in both the control group and the model group. If the time it took for the control group to find the platform decreased with increasing training days, while the model group showed no change, the model was considered successfully constructed.

[0247] 18.2.4 Determination of acetylcholinesterase (AChE) and cholinesterase (ACh) in hippocampal tissue

[0248] After the space exploration experiment, five mice from each group were randomly selected, anesthetized with urethane, decapitated, and had their brains removed. Hippocampal tissue was isolated, washed briefly with physiological saline, and then homogenized with nine times its weight of physiological saline. The homogenate was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was collected, and protein concentration was determined using a BCA protein quantification kit. The AChE and ACh levels in the hippocampal tissue were then determined according to the AChE kit and the ACh kit instructions.

[0249] 18.2.5 HE staining of hippocampal tissue

[0250] After the water maze experiment, three mice were randomly selected and anesthetized with urethane. 30 mL of physiological saline was perfused into the heart via the apex, followed by 10 mL of 4% paraformaldehyde fixative. Perfusion was stopped after the liver turned white. The intact brain, heart, liver, spleen, and kidney were quickly collected and placed in sample vials, then fixed with 4% paraformaldehyde fixative for at least one day. The tissues were dehydrated with different concentrations of alcohol, placed in paraffin embedding frames, cooled, and sectioned (3 μm thick). The sections were then baked in a 60°C oven on glass slides. The sections were dewaxed in xylene for 40 min, then immersed in anhydrous ethanol, and then immersed in hematoxylin staining solution for 3-5 min. After rinsing with running water, the sections were differentiated with differentiation solution, rinsed with running water, and then immersed in blueing solution for blueing, followed by rinsing with running water. The sections were dehydrated with 85% and 95% alcohol, stained, dehydrated, mounted, and then mounted with neutral resin.

[0251] 18.3 Test Results

[0252] See Figure 6-8 for details.

[0253] 18.4 Experimental Conclusion

[0254] When an AD mouse model was established by intraperitoneal injection of scopolamine, a five-day orientation and navigation experiment and a one-day spatial search experiment were conducted to assess the mice's memory. Comparisons were made during the five-day orientation and navigation experiment. Except for the model group, which showed no significant changes, the escape latency of all other groups was shortened. After training, the swimming routes of the model group mice remained disordered, indicating the successful establishment of the scopolamine-induced AD mouse model. Administration of rivastigmine and various concentrations of 14L improved the escape latency of the model mice, especially at a 14L concentration of 5 mg / kg, where the improvement was similar to that of 1 mg / kg rivastigmine in scopolamine-induced AD mice (Figures 6A-E). This confirms that compound 14L can improve the memory ability of scopolamine-induced AD model mice.

[0255] After the experiment, AChE activity and ACh content in the hippocampus of mouse brain tissue were detected. According to the test results of the kit, the AChE content in the model group mice was significantly increased and the ACh content was decreased compared with the blank group. However, the rivastigmine group and compound 14l group could reduce AChE content and increase ACh level (Figure 7A, B), indicating that the compound can inhibit AChE activity and increase ACh content in the brain.

[0256] Three mice were randomly selected from each group, and HE staining was used to further observe the pathological changes in the hippocampus tissue. The results are shown in Figure 20. In the normal group, the hippocampal cells were neatly arranged and showed intact morphology. In the model group, the hippocampal cells were loosely and disorderly arranged and the number of cells was reduced. Compared with the model group, the different doses of rivastigmine and 14l reduced the relevant pathological damage to hippocampal cells to varying degrees, making the cells more compact and morphologically intact. At 14l (5 mg / kg), the hippocampal cells were similar to those in the normal group (Figure 8), indicating that 14l can effectively improve the pathological morphological changes of neuronal cells induced by scopolamine.

[0257] Example 19: Antagonistic Activity of Target Compound (14a-14l) on Histamine H3 Receptor 19.1 Experimental Materials and Instruments

[0258] (1) HEK-293 cells stably expressing human H3R (provided by Shanghai Chempartner) were preserved in DMEM containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 μg / mL streptomycin and revived in a 5% CO2 environment at 37°C.

[0259] (2) LANCE Ultra cAMP kit (PerkinElmer, USA), Clobenpropion (Sigma-Aldrich, USA), 4X Eu-cAMP tracer solution (Sigma-Aldrich, USA), ULight TM - Anti-cAMP working solution (PerkinElmer, USA), ProxiPlate-384 Plus plate (PerkinElmer, USA)

[0260] (3) EnSpire microplate reader (PerkinElmer, USA), benchtop constant temperature shaker (Thermo Scientific, USA), TGL-16.5M low-temperature high-speed centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.) 19.2 Experimental Methods

[0261] Intracellular cAMP accumulation was measured using the LANCE Ultra cAMP kit (Perkin-Elmer) and HEK293 cells stably expressing hH3R, employing a homogeneous TR-FRET immunoassay. Antagonist dose-response experiments were performed in 20 μL ProxiPlate-384 Plus plates using 3 × 10⁻⁶ plates. 3Cells / well. Target compound 14a-l and Clobenpropit were dissolved in 0.1% DMSO-HBSS to prepare solutions with concentrations ranging from 0.1 nM to 10 μM, and simultaneously added to the cell suspension. Cell stimulation was performed at room temperature for 30 minutes. After the incubation period, 5 μL of 4X Eu-cAMP tracer solution and 5 μL of Light were added. TM Mix the anti-cAMP working solution and incubate for 1 hour. Read the TR-FRET signal using an EnSpire microplate reader (Perkin-Elmer). Based on the cAMP standard curve, convert the measured TR-FRET signal into the actual amount of cAMP produced, and calculate the inhibition rate and IC50. 50 value.

[0262] 13.3 Experimental Results

[0263] Table 3. Antagonistic activity of target compounds 14a-14l against histamine H3 receptor (hH3R).

[0264] compound hH3RIC 50 (nM)14a118.46±5.1414b81.63±3.2714c92.88±1.0714d21.71±1.9314e207.39±4.7714f196.62±2.4114g122. 59±0.8714h32.44±1.0414i17.55±0.6214j18.96±0.8914k11.71±0.1714l1.07±0.15Clobenpropit1.39±0.24 surface

[0265] As shown in Table 3, the target compounds exhibited antagonistic activity against the hH3 receptor at the nM level (1.07–207.39 nM), with compound 14l showing the best activity and its IC50 value being [missing value]. 50The concentration reached 1.07 nM, stronger than the positive control Clobenpropit. This result indicates that the introduction of an alkylaminohydroxyphenol ether fragment at the 7-position in the design of the target compound is the key pharmacophore for its H3R antagonistic activity. Preliminary structure-activity relationship studies revealed that the cyclic alkylamino ether moiety introduced at the 4'-position in the target compound structure has a significant impact on the compound's hH3R antagonistic activity. When it is cycloheptanine, the compound's histamine H3R antagonistic activity is significantly stronger than when the 4'-position is a derivative of methylpiperidine. Furthermore, when the 4'-cyclic alkylamino group in the compound structure is the same, further comparison of the effect of the 7-position L-amino acid carbamate on hH3R antagonistic activity revealed that increased steric hindrance of the amino acid hydrocarbon moiety in the compound structure may reduce its receptor antagonistic activity. When the L-amino acid carbamate moiety uses L-valine, which has less steric hindrance, the target compound exhibits better hH3R antagonistic activity.

[0266] This invention addresses the critical issue of insufficient therapeutic activity of target compounds for Alzheimer's disease (AD) due to the lack of reported histamine H3 receptor antagonistic synergistic target design strategies for the design of scutellarin aglycone multi-target ligands (MTDLs). Using scutellarin aglycone-4′-L-amino acid carbamate derivatives obtained in previous studies as lead compounds, and combining this with reports on the alkylaminoalkyloxyphenolic ether skeleton revealed by pharmacophore analysis of non-imidazolium compounds with AChE inhibition / H3R antagonistic synergistic mechanisms, which is a key structural feature for these compounds to exhibit high H3R receptor binding and antagonistic activity as well as AChE inhibitory activity, this invention designs and synthesizes scutellarin aglycone-7-L-amino acid carbamate-4′-(3-methylpiperidinyl) or cycloheptaminopropyl ether derivatives.

[0267] This invention utilizes the L-amino acid carbamate fragment in the structure of scutellarin-7-L-amino acid carbamate-4'-(3-methylpiperidinyl) or cycloheptaminopropyl ether derivatives to endow the compound with active transport properties and improved oral bioavailability. It also utilizes the (3-methylpiperidinyl) or cycloheptaminopropyl ether fragments to endow the compound with histamine H3 receptor antagonism, thereby promoting the central neurotransmitter properties related to learning and memory. The aim is to discover a multi-targeted ligand derivative with better anti-AD properties that combines the synergistic mechanism of cholinesterase inhibition and histamine H3 receptor antagonism.

[0268] This invention addresses the shortcomings of poor drug-likeness and unsatisfactory therapeutic effects of scutellarin in clinical applications, thus laying an important theoretical and practical foundation for the creation of novel anti-AD scutellarin multi-target guided ligand derivatives (MTDLs) based on cholinesterase inhibition and histamine H3 receptor antagonism.

[0269] This invention addresses the shortcomings of existing marketed or clinically investigated prodrugs containing scutellarin aglycone, which exhibit poor drug-like properties and unsatisfactory efficacy in treating Alzheimer's disease (AD). It designs a target compound containing an endogenous carrier to improve oral absorption and bioavailability, while simultaneously increasing the release of neurotransmitters related to learning and attention in the brain by antagonizing histamine H3 receptors and increasing choline levels in the brain by inhibiting cholinesterase, thus achieving better therapeutic effects. The implementation of this patent is of significant value for the research and development of novel anti-AD scutellarin aglycone drugs with independent intellectual property rights.

[0270] Finally, it should be noted that the above embodiments are merely representative examples of this application. Obviously, the technical solutions of this application are not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this application should be considered within the scope of protection of this application.

Claims

1. A compound containing scutellarin-7-L-amino acid carbamate-4'-substituted aminopropyl ether and its salt, characterized in that, The compound is represented by the following general formula (I): Formula (I), in which the amino acid carbamate It is any one of phenylalanine carbamate, isoleucine carbamate, leucine carbamate, and valine carbamate; the substituted aminopropyl ether It is (3-methylpiperidinyl)propyl ether, (4-methylpiperidinyl)propyl ether, and (cycloheptylamino)propyl ether. Any one of them.

2. The method for preparing the compound of scutellarin-7-L-amino acid carbamate-4'-substituted aminopropyl ether according to claim 1, characterized in that, The process includes the following steps: (1) preparing N-3-chloropropyl-substituted cyclic amines; (2) preparing scutellarin-4'-N,N-cyclic alkylamino-1-propyl ether; (3) Preparation of scutellarin aglycone-7-L-amino acid carbamate-4'-cyclic amine-1-propyl ether; wherein the cyclic amine is 3-methylpiperidine, 4-methylpiperidine, or cycloheptylamine. 。 3. The preparation method according to claim 2, characterized in that, Specifically, the steps include: (1) Preparation of N-3-chloropropyl-substituted cyclic amines: 3-bromo-1-propanol reacts with 3-methylpiperidine, 4-methylpiperidine, or cycloheptylamine at room temperature for more than 24 hours in the presence of K2CO3 and KI to obtain N-3-hydroxypropyl-substituted 3-methylpiperidine, 4-methylpiperidine, or cycloheptylamine. The resulting products are then dissolved in a small amount of toluene, and excess thionyl chloride is slowly added dropwise at 0°C. After maintaining the low temperature for 10 minutes, the system temperature is raised to 60°C and reacted for 8 hours to obtain N-3-chloropropyl-substituted cyclic amines. -Chloropropyl-substituted 3-methylpiperidine, 4-methylpiperidine or cycloheptanamine; (2) Preparation of scutellarin aglycone-4'-N,N-cyclohydroamino1-propyl ether: Using scutellarin as raw material, reflux at 80°C for 20 h under sulfuric acid ethanol conditions to obtain crude scutellarin aglycone. After protecting the 6 and 7 hydroxyl groups of the aglycone by condensation of dichlorodiphenylmethane in diethylene glycol dimethyl ether (DEME), it reacts with different N-3-chloropropyl-substituted 3-methylpiperidine, 4-methylpiperidine or cycloheptanamine and then reacts with ethyl acetate saturated with hydrogen chloride. The intermediate scutigane-4'-N,N-cyclic alkylamino-1-propyl ether was obtained by removing diphenyl ketal from the system; (3) Preparation of scutigane-7-L-amino acid carbamate-4'-cyclic amine-1-propyl ether: In a dry reaction flask, L-amino acid tert-butyl hydrochloride was added and stirred with dichloromethane to dissolve it. Pyridine was added, and the system was placed at -10℃ and stirred for 15 min. Then, dichloromethane containing triphosgene was added and the reaction was kept at the temperature for 3 h. After the reaction was completed, 0.5 M of water with crushed ice was added. The organic layer was rapidly washed twice each with hydrochloric acid solution and saturated sodium chloride solution. Anhydrous magnesium sulfate was added to remove water, and the mixture was filtered. The filtrate was evaporated under reduced pressure to obtain a yellow oily substance, tert-butyl-protected L-amino acid isocyanate. L-amino acid isocyanate 4'-N,N-cyclohydroamino-1-propyl ether was dissolved in N,N-dimethylformamide solution, and then tert-butyl-protected L-amino acid isocyanate was added. Triethylamine was added under stirring at 50°C, and the reaction was allowed to proceed for 12 hours. After the reaction was complete as monitored by TLC, dichloromethane was added. The solution was washed twice with 0.5M HCl and twice with saturated NaCl solution. The solution was dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure until a yellow solid was obtained. The solid was then purified by silica gel column chromatography to obtain the target compound.

4. The preparation method according to claim 3, characterized in that, In step (1), the ratio of 3-bromo-1-propanol to 3-methylpiperidine, 4-methylpiperidine, or cycloheptamine is 1.0–5.5:1 mol. The reaction is carried out at 10–35 °C for more than 12–48 h to obtain N-3-hydroxypropyl-substituted 3-methylpiperidine, 4-methylpiperidine, or cycloheptamine. The resulting products are then dissolved in a small amount of toluene, and excess thionyl chloride is slowly added dropwise at -20–0 °C. After maintaining the low temperature for 10 min, the system temperature is raised to 30–60 °C and the reaction is carried out for 4–24 h to obtain N-3-chloropropyl-substituted 3-methylpiperidine, 4-methylpiperidine, or cycloheptamine.

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