Flavonoid compound with neuroprotective effect and preparation method and application thereof

Abstract

The application discloses flavonoid compounds with neuroprotective activity, and a preparation method and application thereof, and particularly relates to four specific ester derivatives of formononetin, namely, an allyl carbonate derivative (compound A), a 2-chlorobenzoic acid ester derivative (compound B), a cinnamic acid ester derivative (compound C), and a propionic acid ester derivative (compound D). The application significantly improves the defects of the parent compound, such as poor liposolubility, low blood-brain barrier permeability and low bioavailability, through specific ester group modification. Experiments show that the lipid-water partition coefficients and bioavailability of the four types of derivatives are significantly improved compared with the parent compound. In addition, the four types of derivatives show better neuroprotective activity than the parent compound and a positive control drug in Alzheimer's disease and Parkinson's disease models, and show a synergistic effect between the parent compound and the modification group. The application further provides a preparation method of the above compounds and application of the above compounds in preparation of drugs for treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a flavonoid compound with neuroprotective effects, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease) are a class of chronic diseases characterized by the progressive loss of neurons, and currently there are no effective cures in clinical practice. The natural isoflavone compound 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one (also known as formononetin) is considered a potential neuroprotective agent due to its multiple pharmacological activities, including antioxidant, anti-inflammatory, and regulation of amyloid protein metabolism.

[0003] However, the translation of gentianin into clinical applications faces the following challenges: Physicochemical defects: The molecule contains free phenolic hydroxyl groups, which are highly polar and have poor lipid solubility (low logP value), making it difficult to penetrate the blood-brain barrier (BBB) ​​and resulting in extremely low concentrations in brain tissue.

[0004] Pharmacokinetic defects: poor oral absorption, significant first-pass effect, short in vivo half-life, and extremely low absolute bioavailability (usually less than 5%).

[0005] Limitations of existing modification techniques: Current techniques for modifying the parent nucleus mainly focus on methylation, glycosylation, or simple alkylation. Although some modifications can improve solubility, they often lead to loss of activity or only limited improvement.

[0006] Therefore, developing a strychnosine derivative that can significantly improve lipid solubility and bioavailability while maintaining or even enhancing neuroprotective activity is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a flavonoid compound with neuroprotective effects, its preparation method, and its applications. Through in-depth research, this invention unexpectedly discovered that by introducing a specific aromatic acyl group (o-chlorobenzoyl or cinnamyl) at the 7-position of argentin, not only can the lipid-water partition coefficient of the molecule be significantly improved, greatly enhancing blood-brain barrier permeability and oral bioavailability, but it can also produce a neuroprotective effect in vivo superior to that of the parent nucleus.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: This invention provides a flavonoid compound with neuroprotective effects, wherein the compound is selected from one or more of the following compounds; Compound A: allyl carbonate-7-yl-3-(4-methoxyphenyl)-4H-chromen-4-one (allyl), with the following structural formula: ; Compound B: 2-chlorobenzoic acid-7-yl-3-(4-methoxyphenyl)-4H-chromen-4-ester 3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl 2-chlorobenzoate, with the following structural formula: ; Compound C: cinnamic acid-7-yl-3-(4-methoxyphenyl)-4H-chromen-4-ester 3-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl cinnamate, with the following structural formula: ; Compound D: 3-(4-methoxyphenyl)-4H-chromen-4-yl propionate, with the following structural formula: ; Preferably, the compound is selected from compound B or compound C.

[0009] The present invention also provides a method for preparing the flavonoid compound, comprising the following steps: Starting from 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one, esterification reaction is carried out with the corresponding acylation reagent in an organic solvent in the presence of a phase transfer catalyst or an organic base. in: In preparing compound A, the acylation reagent is allyl chloroformate; In preparing compound B, the acylation reagent is o-chlorobenzoyl chloride; In preparing compound C, the acylation reagent is cinnamyl chloride; When preparing compound D, the acylation reagent is propionic anhydride; After the reaction was completed, the target compound was obtained through post-processing and purification.

[0010] Preferably, the phase transfer catalyst is selected from tetrabutylammonium bromide, triethylbenzylammonium chloride, polyethylene glycol-400 or Aliquat 336; The organic base is selected from triethylamine, pyridine, or N,N-diisopropylethylamine.

[0011] Preferably, the organic solvent is selected from acetone, acetonitrile, dichloromethane, ethyl acetate, or tetrahydrofuran.

[0012] Preferably, the molar ratio of 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one to the acylation reagent is 1:1-3.

[0013] Preferably, the reaction temperature is 10℃~40℃ and the reaction time is 6-20 hours.

[0014] The present invention also provides the use of the compound in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases.

[0015] Preferably, the neurodegenerative disease is selected from Alzheimer's disease (AD) or Parkinson's disease (PD).

[0016] As a further description of the above scheme: the compound exerts its neuroprotective effect by increasing blood-brain barrier permeability, activating the Nrf2 / HO-1 antioxidant pathway, inhibiting the NF-κB inflammatory pathway, regulating the PI3K / Akt signaling pathway, or inhibiting monoamine oxidase B (MAO-B) activity.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Drugs for neurodegenerative diseases need to cross the blood-brain barrier to exert their pharmacological effects in vivo. However, the original nucleus 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one has poor lipid solubility and weak ability to cross the blood-brain barrier, thus limiting its in vivo efficacy. This invention designs a targeted esterification modification, introducing different ester substituents (allyloxycarbonyl, o-chlorobenzoyl, cinnamyl, propionyl) at the 7-hydroxy site of the original nucleus. By utilizing the hydrophobicity of the ester group to optimize the lipid-water partition coefficient of the molecule, this improves the absorption efficiency and bioavailability of the compound in vivo, and enhances its ability to cross the blood-brain barrier, laying a physicochemical foundation for its neuroprotective and anti-AD / PD activities in vivo.

[0018] One of the core advantages of natural flavonoids is their low in vivo toxicity. In this invention, the structural modification design only involves esterification of the 7-hydroxyl site of the parent nucleus, without altering its core skeletal structure. This aims to preserve the low toxicity of the parent nucleus and avoid introducing toxic groups due to excessive structural modification. Simultaneously, esterification improves its physicochemical properties and pharmacological activity, achieving the dual goals of "low toxicity + high activity," thus enhancing the clinical safety and translational potential of the candidate drug. In an H2O2-induced neuronal injury model, compound C showed a protection rate as high as 93.9%, significantly superior to the parent nucleus (64%) and the clinically positive drug edaravone (86.4%). Compound C also demonstrated excellent therapeutic effects in animal models of Alzheimer's disease (AD) and Parkinson's disease (PD), simultaneously improving cognitive and motor coordination impairments, showing broad-spectrum neuroprotective potential.

[0019] In its synthetic method design, this invention abandons the drawbacks of traditional flavonoid modification methods, which require high temperatures, high pressures, and harsh reaction conditions. Instead, it designs a phase-transfer catalytic reaction system at room temperature: for compounds A to C, one drop of Aliquat 336 is added as a phase-transfer catalyst to improve the reaction efficiency between the aqueous and organic phases; for compound D, triethylamine is used as a base catalyst to simplify the reaction system. Furthermore, the synthesis of all compounds employs universal silica gel column chromatography and petroleum ether recrystallization purification, resulting in mild reaction conditions, simple operation, and stable yields. This reduces the operational difficulty of laboratory research and lays a technological foundation for the subsequent large-scale preparation and development of these compounds. Attached Figure Description

[0020] Figure 1 This is the mass spectrum of compound A; Figure 2 The hydrogen NMR spectrum of compound A; Figure 3 Here is the carbon NMR spectrum of compound A; Figure 4 This is the mass spectrum of compound B; Figure 5 The hydrogen NMR spectrum of compound B; Figure 6 This is the carbon NMR spectrum of compound B; Figure 7 This is the mass spectrum of compound C; Figure 8 The hydrogen NMR spectrum of compound C; Figure 9 This is the carbon NMR spectrum of compound C; Figure 10 This is the mass spectrum of compound D; Figure 11 The hydrogen NMR spectrum of compound D; Figure 12 The carbon NMR spectrum of compound D; Figure 13 For Example 7, the escape latency (A), number of platform crossings (B), and percentage of time spent in the original platform quadrant were compared among the mice in each group (Mean ± SD, n=8). Compared with the model group, * P <0.05,** P <0.01; Figure 14 This example compares the levels of AChE(A), ACh(B), ChAT(C), Aβ40(D), Aβ42(E), and p-Tau(F) in the brain tissue of mice in each group (Mean ± SD, n=3). Compared with the model group, ** P <0.01; Figure 15 This is a diagram showing the pathological changes of neurons in the hippocampus of mice in Example 7. Figure 16 Example 7: Expression of PI3K / Akt signaling pathway-related proteins in mouse brain tissue of each group; A: Protein electrophoresis (1: blank group; 2: model group; 3: low-dose group; 4: medium-dose group; 5: high-dose group; 6: donepezil group); B: Relative gray value of PI3K protein; C: Relative gray value of Akt protein; (Mean ± SD, n=3), compared with the model group, **P<0.01; Figure 17 For the comparison of pole climbing time (A) and rotational dwell time (B) of mice in each group in Example 8; (x ± s, n=8), compared with the model group, * P <0.05,** P <0.01; Figure 18 This example compares the levels of MDA(A), SOD(B), and GSH-PX(C) in the substantia nigra and striatum of mice in different groups (x±s, n=8). Compared with the model group, ** P <0.01; Figure 19 The images show the pathological changes of substantia nigra neurons in each group of mice in Example 8. Figure 20 Example 8: Expression of Nrf2, HO-1, and NQO1 proteins in the substantia nigra and striatum of mice in each group; A: Protein electrophoresis (1: blank group; 2: model group; 3: low-dose group; 4: medium-dose group; 5: high-dose group; 6: levodopa group); B: Relative expression level of Nrf2 protein; C: Relative expression level of HO-1 protein; D: Relative expression level of NQO1 protein; (x ± s, n=3), compared with the model group, ** P <0.01. Detailed Implementation

[0021] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions. Unless otherwise specified, all reagents used in the present invention are purchased from reagent companies.

[0022] Example 1: Preparation method of compound A

[0023] 7-Hydroxy-3-(4-methoxyphenyl)benzopyran-4-one (1340 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature (generally 15-25℃), and NaOH (3M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Allyl chloroformate (960 mg, 8 mmol, 1.6 eq) was then added dropwise, and the reaction was allowed to proceed overnight. The reaction mixture was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum, and the mixture was separated by rapid silica gel column chromatography. Recrystallization from PE yielded a white solid, compound A (0.936 g, yield: 53.1%). The structure of compound A was identified as follows. Figures 1-3 As shown. ESI-HRMS: m / z [M + Na] + calcd for C 20 H 16 NaO6: 375.0845; found: 375.0848. 1 H NMR (500 MHz, CDCl3) δ 8.35 (d, J = 8.8 Hz, 1H), 8.00 (s, 1H), 7.58-7.47 (m, 2H), 7.41 (d, J = 2.2 Hz, 1H), 7.28 (dd, J = 8.6, 2.4 Hz, 1H), 7.08-6.92 (m, 2H), 6.15-5.93(m, 1H), 5.48 (dq, J = 17.2, 1.4 Hz, 1H), 5.39 (dq, J = 10.3, 1.2 Hz, 1H), 4.80 (dt, J = 5.9, 1.3 Hz, 2H), 3.86 (d, J = 1.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 175.71, 159.74, 156.62, 154.58, 152.68, 152.50, 130.73, 130.73,130.13, 128.03, 125.23, 123.73, 122.43, 120.08, 118.79, 114.04, 114.04,110.31, 69.67, 55.36.

[0024] Example 2: Preparation method of compound B

[0025] 7-Hydroxy-3-(4-methoxyphenyl)benzopyran-4-one (1340 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Then, o-chlorobenzoyl chloride (1225 mg, 7 mmol, 1.4 eq) was added dropwise, and the reaction was allowed to proceed overnight. The reaction mixture was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum. The mixture was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid compound B (1.047 g, yield: 51.5%) was collected. The structure of compound B was identified as follows. Figures 4-6 As shown. EI-HRMS: m / z [M + Na] + calcd for C 23 H 15 ClNaO5:429.0506; found:429.0508. 1 H NMR (500 MHz, CDCl3) δ 8.39(d, J = 8.6 Hz, 1H), 8.09 (dd, J = 7.9, 1.5 Hz, 1H), 8.01 (d, J = 4.0 Hz,1H), 7.62-7.40 (m, 6H), 7.33 (dd, J = 8.7, 2.3 Hz, 1H), 7.07-6.94 (m, 2H), 3.85 (s, 3H). 13C NMR (125MHz, CDCl3) δ 175.81, 163.14, 159.76, 156.71,154.35, 152.69, 134.77, 133.77, 132.13, 131.60, 130.18, 130.16, 128.42,128.04, 126.92, 125.27, 123.79, 122.56, 119.47, 114.06, 114.06, 111.09,55.38.

[0026] Example 3: Preparation method of compound C

[0027] 7-Hydroxy-3-(4-methoxyphenyl)benzopyran-4-one (1340 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Cinnamyl chloride (1162 mg, 7 mmol, 1.4 eq) was then added dropwise, and the reaction was allowed to proceed overnight. The reaction mixture was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum. The mixture was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid compound C (1.147 g, yield: 57.6%) was collected. The structure of compound C was identified as follows. Figures 7-9 As shown. EI-HRMS: m / z [M + Na] + calcd forC 25 H 18 NaO5:421.1052; found: 421.1056. 1 H NMR (500 MHz, CDCl3) δ 8.37 (d, J = 8.7Hz, 1H), 8.01 (s, 1H), 7.94 (d, J = 16.0 Hz, 1H), 7.69-7.58 (m, 2H), 7.57-7.51 (m, 2H), 7.47 (dd, J = 5.1, 1.9 Hz, 3H), 7.41 (d, J = 2.2 Hz, 1H), 7.29-7.26 (m, 1H), 7.04-6.95 (m, 2H), 6.67 (d, J = 16.0 Hz, 1H), 3.87 (s, 3H). 13CNMR (125 MHz, CDCl3) δ 175.84, 164.56, 159.73, 156.71, 154.63, 152.63,147.80, 133.89, 131.12, 130.16, 130.16, 129.12, 129.12, 128.48, 127.86,125.18, 123.86, 122.29, 119.54, 116.44, 114.05, 114.05, 110.97, 91.94, 55.38.

[0028] Example 4: Preparation method of compound D

[0029] 7-Hydroxy-3-(4-methoxyphenyl)benzopyran-4-one (1340 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature, and triethylamine (1515 mg, 15 mmol, 3.0 eq) and propionic anhydride (910 mg, 7 mmol, 1.4 eq) were added. The reaction was carried out overnight. The solvent was evaporated to dryness under vacuum, and the mixture was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid compound D (0.985 g, yield: 60.7%) was collected. The structure of compound D was identified as follows. Figures 10-12 As shown. EI-HRMS: m / z [M + Na] + calcd for C 19 H 16 NaO5:347.0895; found:347.0899. 1 H NMR (500MHz, CDCl3) δ 8.32 (d, J = 8.7 Hz, 1H), 7.97 (s, 1H), 7.52-7.47 (m, 2H), 7.30 (d, J = 2.1 Hz, 1H), 7.16 (dd, J = 8.7, 2.2 Hz, 1H), 7.00-6.95 (m, 2H), 3.84 (s, 3H), 2.65 (q, J =7.5 Hz, 2H), 1.30 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 175.82,172.16, 159.72, 156.68, 154.56, 152.62, 130.14, 130.14, 127.83, 125.17,123.83, 122.24, 119.46, 114.04, 114.04, 110.89, 55.37, 27.81, 8.95.

[0030] Example 5: Determination of lipid solubility (logP value), blood-brain barrier permeability, and bioavailability

[0031] 1. Determination of lipid solubility (logP value) To verify the effect of esterification modification on improving the lipophilicity of the parent nucleus, the relationship between structural modification and lipophilicity was clarified.

[0032] This study used the shake-flask method to determine the partition coefficient of the target compound in the n-octanol / water system and calculated the logP value. Three parallel duplicates were set for each sample to ensure the reliability of the experimental results. The results are shown in Table 1.

[0033] Table 1. Results of AD and logP values ​​of compound AD and its parent nucleus (Mean ± SD, n=3)

[0034] The parent substrate has a logP value of only 0.9, indicating strong hydrophilicity and difficulty in crossing the blood-brain barrier. Compounds A–D, by introducing hydrophobic aromatic acyl groups, achieved logP values ​​of 3.6, 3.9, 4.1, and 4.9, respectively, all falling within the ideal lipophilic window (logP 2–5) for centrally acting drugs. This qualitative change in physicochemical properties forms the basis for subsequent efficacy enhancements, and the significant differences in the degree of enhancement brought about by different acyl groups demonstrate the specificity of structural selection.

[0035] 2. Evaluation of the in vitro blood-brain barrier permeability of compound AD and its parent nucleus (PAMPA-BBB method) This study assessed the effect of esterification modification on the transmembrane permeability of the parent nucleus by simulating the lipid bilayer of the blood-brain barrier, thereby evaluating its brain penetration potential. Using the PAMPA-BBB kit, following the manufacturer's instructions, the bottom of the donor plate wells was coated with PBLE and allowed to air dry at room temperature to form a biomimetic lipid bilayer, which was then stored at 4°C for later use. The parent nucleus and compound AD were accurately weighed and dissolved in a small amount of 5% DMSO by sonication (200W, 5 min, 30 s interval). The solutions were then serially diluted to 50 μmol / L with pre-chilled sterile pH 7.4 PBS, with three replicates per sample. The solutions were filtered through a 0.22 μm filter and stored on ice in the dark. The donor and recipient plates and reagents were equilibrated at room temperature for 30 min. 300 μL of sample was added to the donor plate, and 200 μL of pH 7.4 PBS was added to the recipient plate, avoiding air bubbles. The two plates were stacked and incubated at 25°C in the dark for 4 h. After incubation, 100 μL of sample was taken and the concentration was determined by HPLC-MS / MS. Blank controls and QC samples were included. Effective permeability coefficient was determined. P e =(V A ×C A ) / (A× C D ×t） ;parameter: V A =2×10 -4 cm 3 , A =0.33cm 2 , C D =5×10 -5 μmol / cm 3 t=14400s, C A The values ​​represent the measured concentrations on the receptor side. The experimental results are shown in Table 2.

[0036] Table 2. Evaluation results of in vitro blood-brain barrier permeability of compound AD and its parent nucleus (Mean ± SD, n=3)

[0037] Effective permeability coefficient of parent substrate in PAMPA-BBB model P e Only 0.3×10 -6 The blood-brain barrier has extremely low permeability (cm / s), making passive diffusion across the cerebral microvascular endothelial cell layer difficult. Compounds A–D, obtained through esterification modification, exhibit significantly improved effective permeability coefficients of 2.8, 3.5, 4.4, and 4.9 × 10⁻⁶, respectively. -6The permeability grade improved from "very low" to moderate to excellent. The results indicate that aromatic acyl esterification modification significantly improves the transmembrane permeability of the parent compound in biomimetic lipid membranes, greatly enhancing its brain penetration potential. Furthermore, different acyl structures show a clear structure-dependent effect on the improvement of BBB permeability.

[0038] 3. Pharmacokinetics and Bioavailability Studies in Rats To evaluate the in vivo absorption efficiency and bioavailability of compound AD relative to its parent substrate, and to clarify the effect of esterification modification on the in vivo pharmacokinetic properties of the compound, healthy SD rats were randomly divided into 5 groups (n=8 per group). Each group was administered the parent substrate and compound AD by gavage, respectively, at a dose of 20 mg / kg. After administration, the drug concentrations in rat plasma at different time points were measured using LC-MS / MS to analyze and evaluate the in vivo absorption and utilization of each compound. The results are shown in Table 3.

[0039] Table 3. Results of pharmacokinetic and bioavailability studies in rats (Mean ± SD, n=8)

[0040] The oral bioavailability of compound AD is as high as 34.1%–52.9%, which is 8.12–12.59 times that of the parent core, confirming that esterification modification effectively overcomes the first-pass effect and absorption barrier. Esterification modification improves the lipophilicity and membrane permeability of the parent core, reduces gastrointestinal metabolic degradation, and promotes its absorption in the intestine, thereby significantly improving the oral bioavailability of the compound. This lays a good pharmacokinetic foundation for its subsequent in vivo efficacy. Moreover, different aromatic acyl modifications have different degrees of improvement in bioavailability, further demonstrating the specificity of structural modification.

[0041] Example 6: Study on the neuroprotective activity of compound AD This experiment used HT22 hippocampal neurons as the research object, and 1×10⁻⁶ of them were used. 4Cells were seeded per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator until adherence and confluence. This experiment included a control group, a model group, a compound AD group, a strychnine nucleus control group, and an edaravone positive control group. The compound AD group, the strychnine nucleus control group, and the edaravone positive control group were each prepared with three concentration gradients of 1 μmol / L, 5 μmol / L, and 10 μmol / L to systematically investigate the cell protective activity of each compound at different concentrations. After cell adhesion, the drug-treated groups were given the corresponding drug concentration, while the control and model groups were pretreated with an equal volume of culture medium for 8 h. Subsequently, except for the control group, all other groups were treated with a final concentration of 200 μmol / L H2O2 solution to establish the cell model and cultured for another 24 h. Cell viability was detected using the CCK-8 assay, and the absorbance of each well was measured at 450 nm using a microplate reader to calculate the relative viability.

[0042] The experimental results showed that the cell survival rate in the model group was 45.432%, which was significantly lower than the 100% survival rate in the control group. P The cell survival rate was <0.01%, confirming the successful construction of the H2O2-induced HT22 cell damage model. The cell survival rate of the edaravone positive control group increased dose-dependently with increasing drug concentration, verifying the reliability of this experimental system. The survival rate of the formononetin nucleus at all concentrations was lower than that of all esterified compounds AD and the positive control drug edaravone, indicating that esterification modification can significantly enhance the cell-protective activity of formononetin. Compound C showed the best protective effect; its protective effect was optimal at a concentration of 10 μmol / L, with a cell survival rate of 93.855%, significantly better than edaravone at the same concentration (86.356%), suggesting that compound C has a potent protective effect against H2O2-induced HT22 cell damage. The results are shown in Table 4.

[0043] Table 4. Cell viability in each group (Mean ± SD, n=6)

[0044] Example 7: Therapeutic effect on Alzheimer's disease (AD) model mice Forty-eight 11-week-old SPF-grade healthy male BALB / c mice, weighing 20–25 g, were randomly divided into four groups (n=8 per group): a blank control group, a model group, a donepezil hydrochloride group, and low-, medium-, and high-dose groups of compound C. Except for the blank control group, all other groups received subcutaneous injections of D-galactose (120 mg / kg) and gavage with aluminum chloride (20 mg / kg) once daily for 60 days to establish an AD model. From day 31, mice in the low-, medium-, and high-dose groups of compound C were administered compound C at doses of 20, 30, and 40 mg / kg via gavage, respectively. Mice in the donepezil hydrochloride group received a tail vein injection of donepezil hydrochloride at a dose of 1.0 mg / kg. Mice in the blank control and model groups were administered an equal volume of physiological saline via gavage once daily for 30 days. After the experiment, the Morris water maze test was conducted first, and the mice were trained for 5 consecutive days. On the 6th day, the escape latency, number of times the mice crossed the platform, and the time spent in the target quadrant were measured to assess their learning and memory abilities. After the behavioral experiment, all mice were anesthetized with 1% sodium pentobarbital and then sacrificed. Brain tissue was dissected, a portion of which was fixed in 4% paraformaldehyde solution, and the remaining portion was blotted dry with filter paper, weighed, and shredded. The shredded brain tissue was mixed with PBS and ground under ice bath conditions. The homogenate was centrifuged at 3000 r / min for 10 min at 4 ℃, and the supernatant was collected. The contents of AChE, ACh, ChAT, Aβ (Aβ40, Aβ42), and p-Tau were detected by an ELISA reader.

[0045] The water maze test results showed that, compared with the control group, the escape latency of mice in the model group was significantly prolonged, the number of times they crossed the original platform was significantly reduced, and the proportion of time spent in the original platform quadrant was significantly decreased, suggesting that their spatial learning and memory abilities were significantly impaired. Intervention with compound C significantly shortened the escape latency, increased the number of times they crossed the original platform, and prolonged the time spent in the original platform quadrant. The intervention effect of compound C in the high-dose group was superior to that of the positive control donepezil hydrochloride. Figure 13 HE staining results showed that the hippocampal neurons in the model group mice were disordered, pyknotized, and had widened intercellular spaces, accompanied by extensive neuronal necrosis, indicating significant damage to the hippocampal neurons in the model group mice. Figure 15 Compound C intervention can improve the pathological damage of hippocampal neurons. ELISA results showed that the levels of AChE, Aβ40, Aβ42, and p-Tau in the brain tissue of the model group mice were significantly increased, while the levels of ACh and ChAT were significantly decreased, indicating that the AD model was successfully established; while compound C intervention can significantly reduce the levels of AChE, Aβ40, Aβ42, and p-Tau in the mouse brain tissue, and increase the levels of ACh and ChAT. Figure 14This suggests that compound C has a protective effect on hippocampal neurons and can improve cognitive function. Furthermore, compound C can increase the phosphorylation levels of PI3K and Akt proteins. Figure 16 The results suggest that the anti-AD effect of compound C may be related to the activation of the PI3K / Akt signaling pathway. In summary, compound C has a good anti-AD effect, and its mechanism may be related to the activation of the PI3K / Akt signaling pathway, improvement of the cholinergic system, reduction of Aβ deposition, and inhibition of neuronal apoptosis by Tau protein hyperphosphorylation.

[0046] Example 8: Therapeutic effect on Parkinson's disease (PD) model mice Experimental grouping: 48 mice were randomly divided into 6 groups: control group, PD model group (PD group), positive control group, low-dose intervention group of compound C, medium-dose intervention group of compound C, and high-dose intervention group of compound C, with 8 mice in each group; PD model establishment: Except for the control group, the PD group, positive control group, and each dose group of compound C were administered 25 mg / kg of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) solution (30 mL / kg) via intraperitoneal injection for 7 consecutive days. The control group was injected intraperitoneally with an equal volume of physiological saline as a blank control. Experimental drug intervention: The low, medium, and high dose groups of compound C were injected intraperitoneally with 20, 40, and 60 mg / kg of compound C, respectively. The positive control group was injected intraperitoneally with levodopa (100 mg / kg). All drug administration groups were administered for 21 consecutive days. The PD group and the control group were injected intraperitoneally with an equal volume of physiological saline during the same period. Behavioral tests were performed 24 hours after the last administration.

[0047] Behavioral testing methods: 1) Pole climbing test: A wooden pole with a diameter of 1.0 cm and a length of 55 cm, wrapped in coarse gauze, was used (with a 2.0 cm rubber ball placed at one end away from the ground). The time taken for mice to climb from the top of the rubber ball to the ground was measured at the same time interval. After adaptive training, the test was performed 3 times and the average value was taken. 2) Rotation test: After mice underwent adaptive exercise on a low-speed rotating wheel, they were placed on a 75 r / min roller. The duration of continuous movement was recorded at the same time interval, and the average value was taken at 3 times. After the behavioral tests, the mice were intraperitoneally anesthetized with 1% pentobarbital. Whole brain tissue was taken. One sample was fixed in paraformaldehyde at 4℃ for 24 h for HE staining; the other sample was used for oxidative stress markers and Western blot detection.

[0048] This study used pole climbing and rotation tests to evaluate the motor function of mice. The results showed that the pole climbing time was significantly prolonged and the rotation dwell time was significantly shortened in Parkinson's disease (PD) model mice. Intervention with compound C reversed these abnormalities, suggesting that it can effectively improve the motor function of PD mice. Figure 17 Meanwhile, this study found that the neurons in the blank group mice had normal morphology, while the neurons in the model group mice showed significant loss and degeneration, indicating successful modeling. In the compound C group, the number and morphology of neurons gradually improved with increasing dosage. The neuroprotective effect of the high-dose compound C group was comparable to that of the positive control drug, Levodopa, demonstrating that the drug has a dose-dependent neuroprotective effect. Figure 19 Furthermore, malondialdehyde (MDA) levels were significantly elevated in the substantia nigra striatum of PD model mice, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were significantly reduced. Compound C could reverse these abnormal changes in oxidative stress-related indicators. Figure 18 This indicates that it can improve nerve damage by regulating the oxidative stress state in the substantia nigra and striatum of PD mice. Further research revealed that the protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and quinone oxidoreductase 1 (NQO1) in the substantia nigra and striatum of PD model mice were all significantly downregulated, and compound C could reverse this abnormal expression. Figure 20 This indicates that it can protect dopaminergic (DA) neurons in the substantia nigra from oxidative stress damage by activating the Nrf2 / HO-1 / NQO1 signaling pathway, thereby improving motor dysfunction caused by PD.

[0049] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. A flavonoid compound with neuroprotective effects, characterized in that, The compound is selected from one or more of the following compounds; Compound A: Allyl carbonate-7-yl-3-(4-methoxyphenyl)-4H-chromen-4-one, its structural formula is: ； Compound B: 2-Chlorobenzoic acid-7-yl-3-(4-methoxyphenyl)-4H-chromene-4-ester, its structural formula is: ； Compound C: 7-yl-3-(4-methoxyphenyl)-4H-chromene-4-ester of cinnamic acid, with the following structural formula: ； Compound D: 7-yl-3-(4-methoxyphenyl)-4H-chromene-4-ester propionate, with the following structural formula: 。 2. The flavonoid compound according to claim 1, characterized in that, The compound is selected from compound B or compound C.

3. A method for preparing the flavonoid compound as described in claim 1, characterized in that, Includes the following steps: Starting from 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one, esterification reaction is carried out with the corresponding acylation reagent in an organic solvent in the presence of a phase transfer catalyst or an organic base. in: In preparing compound A, the acylation reagent is allyl chloroformate; In preparing compound B, the acylation reagent is o-chlorobenzoyl chloride; In preparing compound C, the acylation reagent is cinnamyl chloride; When preparing compound D, the acylation reagent is propionic anhydride; After the reaction was completed, the target compound was obtained through post-processing and purification.

4. The preparation method according to claim 3, characterized in that, The phase transfer catalyst is selected from tetrabutylammonium bromide, triethylbenzylammonium chloride, polyethylene glycol-400 or Aliquat 336; The organic base is selected from triethylamine, pyridine, or N,N-diisopropylethylamine.

5. The preparation method according to claim 3, characterized in that, The organic solvent is selected from acetone, acetonitrile, dichloromethane, ethyl acetate, or tetrahydrofuran.

6. The preparation method according to claim 3, characterized in that, The molar ratio of 7-hydroxy-3-(4-methoxyphenyl)benzopyran-4-one to the acylation reagent is 1:1-3.

7. The preparation method according to claim 3, characterized in that, The reaction temperature is 10℃~40℃, and the reaction time is 6-20 hours.

8. Use of the compound of claim 1 or 2 in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that, The neurodegenerative disease mentioned is selected from Alzheimer's disease or Parkinson's disease.

10. The application according to claim 8, characterized in that, The compound exerts its neuroprotective effects by increasing blood-brain barrier permeability, activating the Nrf2 / HO-1 antioxidant pathway, inhibiting the NF-κB inflammatory pathway, regulating the PI3K / Akt signaling pathway, or inhibiting monoamine oxidase B activity.

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