CN109761945B

CN109761945B - naringenin-O-alkylamine compound, preparation method and application

Info

Publication number: CN109761945B
Application number: CN201910095990.4A
Authority: CN
Inventors: 桑志培; 王柯人; 柳文敏; 时健; 于林涛; 程新锋; 刘青妹
Original assignee: Nanyang Normal University
Current assignee: Nanyang Normal University
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2020-07-31
Anticipated expiration: 2039-01-31
Also published as: CN109761945A

Abstract

The invention discloses a naringenin-O-alkylamine compound, a preparation method and application thereof, wherein the chemical structural general formula of the compound is shown as (I), and naringenin and dibromoalkane Br (CH)₂)_nBr is taken as a starting material, the reaction is carried out under the first solvent and the first alkaline condition to obtain corresponding 2, 7-dibromo alkoxy naringenin, and then the 2, 7-dibromo alkoxy naringenin reacts with different secondary amines NR under the second solvent and the second alkaline condition₁R₂Reacting to obtain the target compound naringenin-O-alkylamine compound. The naringenin-O-alkylamine compound has multi-target effect,can be widely used for preventing or treating neurodegenerative diseases.

Description

naringenin-O-alkylamine compound, preparation method and application

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to a naringenin-O-alkylamine compound, and a preparation method and application thereof.

Background

Alzheimer's Disease (AD) is a degenerative disease of the central nervous system mainly caused by progressive cognitive impairment and memory impairment, and with the rapid aging of the global population, the health problems of the elderly population have become social importance which needs to be solved urgently. Alzheimer's Disease (AD) is one of the most prevalent diseases in the elderly. The report on global Alzheimer's disease 2015 issued by the International Association for Alzheimer's Disease (ADI) states that over 4600 million people worldwide have dementia 2015, and it is predicted that 1.315 million people worldwide will suffer from dementia 2050, with the incidence of Chinese dementia reaching 6.61%. With the growing life of everyone, the disease has developed into a major burden for society and health care system, and has brought a heavy mental and economic stress to society, patients and family members. Currently approved drugs for the treatment of mild/moderate AD include acetylcholinesterase (AChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists for the treatment of severe AD, but clinical use of these drugs suggests that these drugs can alleviate AD symptoms by increasing acetylcholine levels or inhibiting excitotoxicity of excitatory amino acids in patients, but do not effectively prevent or reverse the course of the disease, and can also cause severe toxic side effects such as hallucinations, chaos of consciousness, dizziness, headache, nausea, hepatotoxicity, poor appetite, and frequent stools, and thus the long-term efficacy is not ideal. Therefore, the development of AD therapeutic drugs with a novel mechanism of action is urgently needed clinically.

The AD belongs to diseases caused by various factors, the pathogenesis is complex, the pathogenesis is not completely clarified so far, but researches show that various factors such as reduction of acetylcholine level in the brain of a patient, excessive generation and deposition of β -amyloid, neurofibrillary tangle caused by tau-protein hyperphosphorylation, generation of a large amount of Reactive Oxygen Species (ROS) and free radicals by oxidative stress, nerve verification reaction and the like play an important role in the pathogenesis of the AD.

Based on the above results, researchers have proposed a strategy of 'multi-target-oriented drugs' (MTD L s) to develop anti-neurodegenerative disease drugs, wherein a single chemical entity acts on multiple targets in a disease network simultaneously, and the effect on each target can produce a synergistic effect, so that the total effect is greater than the sum of single effects, the drugs are also called 'multifunctionality' or 'multipotent' drugs.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks, a first object of the present invention is to provide a naringenin-O-alkylamine compound.

The second purpose of the invention is to provide a preparation method of naringenin-O-alkylamine compounds.

The third purpose of the invention is to provide the application of the naringenin-O-alkylamine compound in preparing the medicines for treating the neurodegenerative diseases.

The fourth purpose of the invention is to provide a pharmaceutical composition for treating neurodegenerative diseases, which comprises naringenin-O-alkylamine compound or its pharmaceutically acceptable salt synthesized with acid.

A naringenin-O-alkylamine compound has a chemical structural general formula shown in (I):

wherein: n is 2-12;

R₁represents H or C₁～C₁₂An alkyl group; r₂Is represented by C₁～C₁₂Alkyl, benzyl, substituted benzyl, 1,2,3, 4-tetrahydroacridin-9-yl, 6-chloro-1, 2,3, 4-tetrahydroacridin-9-yl, 8-chloro-1, 2,3, 4-tetrahydroacridin-9-yl or 6, 8-dichloro-1, 2,3, 4-tetrahydroacridin-9-yl;

or NR₁R₂Represents N-demethyl-galanthamine, tetrahydropyrrolyl, morpholinyl, piperidyl, or a 4-position is substituted by C₁～C₁₂Piperidinyl substituted by alkyl, piperidinyl substituted by benzyl or substituted benzyl in the 4-position, piperazinyl, piperidinyl substituted by C in the 4-position₁～C₁₂Piperazinyl substituted with alkyl, piperazinyl substituted at the 4-position with benzyl or substituted benzyl; 1,2,3, 4-tetrahydroisoquinolinyl, optionally substituted by C₁～C ₁₂1,2,3, 4-tetrahydroisoquinolinyl substituted with alkyl or 1,2,3, 4-tetrahydroisoquinolinyl substituted at an arbitrary position with benzyl or substituted benzyl;

or O (CH)₂)nNR₁R₂To represent

m represents 0-10, R₃Representation H, C₁～C₁₂Alkyl, benzyl or substituted benzyl.

Preferably, said substituted benzyl is benzyl substituted by 1 to 4 groups on the phenyl ring selected from: F. cl, Br, I, C_1-4Alkyl radical, C_1-4Alkoxy, trifluoromethyl, trifluoromethoxy, dimethylamino, nitro, cyano, these substituents being in any possible position of the phenyl ring.

A preparation method of naringenin-O-alkylamine compounds comprises the following steps:

(1) with naringenin and dibromoalkane Br (CH)₂)_nBr is taken as a starting material, and the reaction is carried out under a first solvent and a first alkaline condition to obtain corresponding 2, 7-dibromo alkoxy naringenin;

(2)2, 7-dibromo-alkoxy naringenin reacts with different secondary amines NR in second solvent and second alkaline condition₁R₂Reacting to obtain the target compound naringenin-O-alkylamine compound.

Preferably, the base used in the first alkaline condition or the second alkaline condition is: alkaline earth metal hydroxide, alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate, C_1-8Alkali metal salts of alcohols, organic tertiary amines and quaternary ammonium bases.

Preferably, the first solvent or the second solvent is diethyl ether, tetrahydrofuran, N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, C_3-8Aliphatic ketones, benzene, toluene, acetonitrile and C_5-8One or more alkanes.

Preferably, the naringenin of step (1)): dibromoalkane Br (CH)₂)_nBr: the feeding ratio of the alkali is 1.0: 2.0-10.0: 2.0-10.0, the reaction temperature is 25-150 ℃, and the reaction time is 10-48 h.

Preferably, the first and second electrodes are formed of a metal,the 2, 7-dibromoalkoxynaringenin described in step (2)): secondary amine NR₁R₂: the molar charge ratio of alkali is 1.0: 1.0-10.0: 1.0-10.0, the reaction temperature is 25-150 ℃, and the reaction time is 10-48 h.

The chemical reaction structure general formula of the invention is as follows:

wherein: n, R₁、R₂The definition of the (D) is the same as the chemical structural general formula of the naringenin-O-alkylamine compound (I).

An application of naringenin-O-alkylamine compound in preparing the medicines for treating the neurodegenerative diseases is disclosed.

Preferably, the neurodegenerative disease is alzheimer's disease, vascular dementia, parkinson's disease, huntington's disease, HIV-associated dementia, multiple sclerosis, progressive lateral sclerosis, neuropathic pain or glaucoma.

A pharmaceutical composition for treating neurodegenerative disease comprises naringenin-O-alkylamine compound or its pharmaceutically acceptable salt synthesized with acid.

The pharmaceutical composition comprises one or more naringenin-O-alkylamine compounds (I) or pharmaceutically acceptable salts thereof with a therapeutically effective amount, and the pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers or excipients, wherein the therapeutically effective amount refers to a medicament or a dosage which can cause biological or medical response of tissues, systems or animals aimed by researchers or doctors; the term "composition" refers to a product formed by mixing more than one substance or component; the "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or vehicle, such as: liquid or solid fillers, diluents, excipients, solvents or encapsulating substances, which carry or transport certain chemical substances; the ideal proportion of the pharmaceutical composition provided by the invention is that the naringenin-O-alkylamine compound (I) or the pharmaceutically acceptable salt thereof is taken as an active component and accounts for 2-99.0% of the total weight.

The naringenin-O-alkylamine compound (I) contains amino in the molecule, the amino is alkaline, and pharmaceutically acceptable salt can be prepared by any appropriate acid through a pharmaceutically conventional salifying method.

Preferably, the acid is hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, C_1-6Fatty carboxylic acid, oxalic acid, benzoic acid, salicylic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, C_1-6Alkyl sulfonic acid, camphor sulfonic acid, benzene sulfonic acid or p-toluene sulfonic acid.

The invention has the following positive beneficial effects:

1. the naringenin-O-alkylamine compound of the invention introduces an amine alkoxy fragment on a naringenin skeleton, and is designed and found to have stronger acetylcholinesterase and butyrylcholinesterase resistance, oxidation stress resistance, metal ion complexing activity and AlCl improvement₃Inducing zebrafish AD motor function, H₂O₂The induced PC12 cell injury has obvious neuroprotective function, inhibits the excessive generation and deposition of A β, has balanced activity, and can be widely used for preventing or treating neurodegenerative diseases such as Alzheimer disease, vascular dementia, Parkinson disease, Huntington disease, HIV-related dementia, multiple sclerosis, progressive lateral sclerosis, neuropathic pain or glaucoma.

2. The naringenin-O-alkylamine compound has obvious inhibitory activity on acetylcholinesterase and butyrylcholinesterase, and IC (integrated Circuit) of the naringenin-O-alkylamine compound₅₀0.004-8.1 mu M and 0.003-21.9 mu M, and the naringenin-O-alkylamine compound is used for treating A β under the concentration of 25.0 mu M_1-42The inhibition rate of self-aggregation is more than 46.8 percent for A β_1-42Self-aggregation has obvious inhibitory activity; the anti-oxidation activity of the naringenin-O-alkylamine compound is 1.1-1.6 times of that of Trolox, which shows that the compound has strong anti-oxidation activity; compounds of the invention are described in 10^-5mol/L～10^-6Has obvious protective effect on PC12 cell damage induced by hydrogen peroxide under the concentration of mol/L, and the naringenin-O-alkylamine compounds of the invention have the performanceHas strong complexation effect on metal ions.

3. The compound can obviously improve AlCl₃Inducing the motor function of the zebra fish AD, and comparing the zebra fish AD with a model control group to obtain statistical difference; the compound naringenin-O-alkylamine compound has obvious improvement effect on mouse dysmnesia caused by scopolamine.

Drawings

FIG. 1 shows the naringenin-O-alkylamine compound (I) of the present invention vs H₂O₂A graph of the protective effect assay results of induced PC12 cell damage;

FIG. 2 shows the reaction of naringenin-O-alkylamines (I) to AlCl₃A result chart of the prevention and treatment effect of the zebra fish Alzheimer disease model is induced;

FIG. 3 is a graph showing the results of measurement of the effect of naringenin-O-alkylamines compound (I) of the present invention on mouse memory acquisition disorder caused by scopolamine.

Detailed Description

The invention will be further illustrated with reference to some specific examples.

Example 1

The chemical structural general formula of the naringenin-O-alkylamine compounds of embodiments 1-128 of the invention is as follows:

wherein n and R₁、R₂See table 1.

TABLE 1 naringenin-O-alkylamines of inventive examples 1-128

I-43:Light yellow oil,50.6％yield,98.2％HPLC purity.¹H NMR(400 MHz,CDCl₃)12.02(s,1H,OH),7.52(d,J＝8.0 Hz,1H,Ar-H),7.48(d,J＝7.2 Hz,1H,Ar-H),7.36(d,J＝8.4 Hz,2H,2×Ar-H),7.31-7.24(m,2H,2×Ar-H),6.96(t,J＝7.6 Hz,2H,2×Ar-H),6.91(d,J＝8.8 Hz,2H,2×Ar-H),6.89(d,J＝3.2 Hz,1H,Ar-H),6.87(d,J＝3.2Hz,1H,Ar-H),6.01(dd,J₁＝6.4 Hz,J₂＝2.4 Hz,2H,2×Ar-H),5.36(dd,J₁＝10.0 Hz,J₂＝2.8 Hz,1H,Ar-H),3.99-3.93(m,4H,2×OCH₂),3.87(s,2H,phCH₂),3.84(s,3H,OCH₃),3.83(s,3H,OCH₃),3.81(s,2H,phCH₂),3.09(dd,J₁＝13.2 Hz,J₂＝4.0 Hz,1H,1/2 COCH₂),2.79(dd,J₁＝14.0 Hz,J₂＝3.2 Hz,1H,1/2COCH₂),2.78-2.63(m,8H,4×NCH₂)1.84-1.81(m,4H,2×CH₂),1.78-1.76(m,4H,2×CH₂),1.24-1.16(m,6H,2×CH₃).HR-ESI-MS:Calcd.forC₄₃H₅₄N₂O₇[M+H]⁺:711.3965,found:711.3997。

I-49：Light yellow oil,58.3％yield,98.1％HPLC purity.¹H NMR(400 MHz,CDCl₃)12.03(s,1H,OH),7.35-7.22(m,12H,12×Ar-H),6.92(d,J＝8.4 Hz,2H,2×Ar-H),6.02(dd,J₁＝9.2 Hz,J₂＝2.4 Hz,2H,2×Ar-H),5.33(dd,J₁＝10.4 Hz,J₂＝2.8Hz,1H,Ar-H),3.99-3.94(m,4H,2×OCH₂),3.51(s,2H,phCH₂),3.50(s,2H,phCH₂),3.08(dd,J₁＝12.8 Hz,J₂＝4.4 Hz,1H,1/2 COCH₂),2.76(dd,J1＝14.4Hz,J2＝2.8 Hz,1H,1/2 COCH₂),2.49-2.30(m,20H,10×NCH₂),1.83-1.73(m,4H,2×CH₂),1.71-1.58(m,4H,2×CH₂).HR-ESI-MS:Calcd.for C₄₅H₅₆N₄O₅[M+H]⁺:733.4284,found:733.4314。

I-53:Light yellow oil,63.7％yield,98.0％HPLC purity.¹H NMR(400 MHz,CDCl₃)12.01(s,1H,OH),7.32(d,J＝8.0 Hz,2H,2×Ar-H),6.88(d,J＝8.4 Hz,2H,2×Ar-H),6.04(d,J＝9.6 Hz,2H,2×Ar-H),5.34(dd,J₁＝10.8 Hz,J₂＝2.0 Hz,1H,CH),3.95(t,J＝6.4 Hz,2H,OCH₂),3.40(t,J＝6.8 Hz,2H,BrCH₂),3.08(d,J₁＝5.2 Hz,J₂＝4.0 Hz,1H,1H,1/2 CH₂),2.78(dd,J₁＝14.8 Hz,J₂＝2.4 Hz,1H,1/2CH₂),1.88-1.80(m,2H,CH₂),1.77-1.72(m,2H,CH₂),1.43-1.38(m,4H,2×CH₂),1.29-1.27(m,12H,6×CH₂).HR-ESI-MS:Calcd.for C₄₇H₅₈N₂O₅[M+H]⁺:731.4379,found:731.4403。

I-54:Light yellow oil,68.9％yield,98.1％HPLC purity.¹H NMR(400 MHz,CDCl₃)12.04(s,1H,OH),7.36(d,J＝8.8 Hz,2H,2×Ar-H),7.13-7.11(m,5H,5×Ar-H),7.03-7.01(m,2H,2×Ar-H),6.95(d,J＝8.4 Hz,2H,2×Ar-H),6.06(s,1H,Ar-H),6.03(s,1H,Ar-H),5.35(dd,J₁＝10.8 Hz,J₂＝2.0 Hz,1H,CH),4.03(t,J＝6.0 Hz,2H,OCH₂),4.01(t,J＝6.4 Hz,2H,OCH₂),3.67(s,2H,phCH₂N),3.65(s,2H,phCH₂N),3.10(dd,J₁＝13.2 Hz,J₂＝3.6 Hz,1H,1/2 COCH₂),2.93-2.90(m,4H,2×phCH₂),2.80-2.73(m,5H,1/2 COCH₂+2×NCH₂),2.63-2.56(m,4H,2×NCH₂),1.93-1.69(m,8H,4×CH₂).¹³C NMR(100 MHz,CDCl₃)181.4,167.4,164.1,162.9,159.5,134.2(2C),130.2,128.7(3C),127.8(3C),126.6(3C),126.2(2C),125.7(2C),114.8(3C),103.1,95.5,94.6,79.0,68.2,67.8,57.8,57.7,56.0(2C),50.8(2C),43.2(2C),28.9(2C),27.2(2C),26.9(2C),23.6,23.5.HR-ESI-MS:Calcd.for C₄₁H₄₆N₂O₅[M+H]⁺:647.3440,found:647.3477。

The method for preparing the naringenin-O-alkylamines compound of embodiment 1 includes the following steps:

(1) 5.0mmol of naringenin and 15mmol of the corresponding dibromoalkane Br (CH) are added into a reaction flask₂)_nBr, 10mmol potassium carbonate and 50m L acetonitrile, stirring uniformly, reacting for 24h at 65 ℃, decompressing and evaporating the solvent after the reaction is finished, adding 100m L deionized water into the residual liquid, extracting for three times by using 300m L dichloromethane, merging organic layers, washing by using saturated sodium chloride aqueous solution, decompressing and evaporating the solvent, and purifying the residue by column chromatography (petroleum ether in eluent: acetone is 100: 1v/v) to obtain corresponding 2, 7-dibromo alkoxy naringenin;

(2) 3.0mmol of 2, 7-dibromoalkoxynaringenin and 4.0mmol of the corresponding secondary amine NR were added to the reaction flask₁R₂4.5mmol of potassium carbonate and 30m of L acetonitrile, stirring uniformly, reacting at 65 ℃ for 24 hours, evaporating the solvent to dryness under reduced pressure after the reaction is finished, adding 100m of L deionized water into the residual solution, extracting with 300m of L dichloromethane for three times, combining organic layers, dissolving with saturated sodium chloride waterWashing, vacuum evaporating solvent, purifying residue by column chromatography (50: 1v/v petroleum ether in eluent) to obtain corresponding naringenin-O-alkylamine compound (I), wherein the chemical structure is shown in¹H NMR、¹³C NMR and ESI-MS.

The naringenin-O-alkylamines of examples 2-128 are prepared in substantially the same manner as in example 1, except as set forth in tables 2 and 3, and are not repeated here.

TABLE 2 naringenin-O-alkylamine compound of examples 1-10 of the present invention, one of the parameters of the preparation method

TABLE 3 preparation of naringenin-O-alkylamines according to the invention in examples 1-10

Biological activity assay

1. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of naringenin-O-alkylamines compound (I)

Adding 1.0 mmol/L thioacetylcholine iodide or thiobutyrylcholine (all from Sigma) 30 μ L, PBS buffer solution with pH 8.0 40 μ L, test compound solution 20 μ L (DMSO content is less than 1%) and 10 μ L electrosenstrocholinesterase (EeAChE) or butyrylcholinesterase (equine serum BuChE, equi) (0.045U, all from Sigma) to a 96-well plate in this order, incubating at 37 ℃ for 15min after completion of mixing, adding 5,5' -dithio-bis (2-nitro) benzoic acid (DTNB, from Sigma) 30 μ L in a mass fraction of 0.2% to each well, measuring the blank Optical Density (OD) value of each well at 405nm with a plate reader, comparing with the blank well without the test sample, and calculating the inhibition rate of the test compound on the enzyme [ enzyme inhibition rate (1-sample group OD × 100% ] (1-sample group OD) ×%)](ii) a Selecting five to six concentrations of the compound, measuring the enzyme inhibition rate, performing linear regression by using the negative logarithm of the molar concentration of the compound and the enzyme inhibition rate, and obtaining the molar concentration when the 50% inhibition rate is obtained as the IC of the compound₅₀The results of the tests using Rivastigmine (Rivastigmine) and Donepezil (Donepezil) as positive controls are shown in Table 4.

TABLE 4 cholinesterase inhibitory Activity, antioxidant Activity, auto-induced A β of the Compounds of the invention_1-42Aggregation inhibiting activity and Cu²⁺Induced A β_1-42Self-aggregation inhibitory Activity

The test result shows that the mother nucleus of the compound of the invention, i.e. IC of naringenin for inhibiting acetylcholinesterase and butyrylcholinesterase₅₀Both are more than 500 mu M, the naringenin-O-alkylamine compound has obvious inhibitory activity to acetylcholinesterase and butyrylcholinesterase, and the IC of the naringenin-O-alkylamine compound is IC₅₀0.004-8.1 mu M and 0.003-21.9 mu M, which is equivalent to or better than the positive control Rivastigmine/Donepzil detection result.

2. naringenin-O-alkylamine compound (I) p-A β_1-42Inhibitory Activity of self-aggregation

The determination is carried out according to the method reported in the literature (Sangg, Z.P.et al.Eur.J.Med.chem.2015, 94, 348-366), namely, A β after pretreatment_1-42Stock solutions were prepared with DMSO, and the test compounds were diluted to 50. mu.M with PBS buffer pH7.4 before use, and 2.5mM stock solutions were prepared with DMSO, diluted to the appropriate concentration with PBS buffer pH7.4 before use, and 20. mu. L of A β was taken_1-42Solution + 20. mu. L test Compound solution, 20. mu. L A β_1-42Solutions +20 μ L PBS buffer (containing 2% DMSO), 20 μ L PBS buffer (containing 2% DMSO) +20 μ L PBS buffer (containing 25% DMSO) in black 96-well plates, compounds and A β_1-42Was incubated at 25. mu.M.37 ℃ for 24h, then 160. mu. L of 50mM glycine-NaOH buffer (pH 8.5) containing 5. mu.M thioflavin T was added, shaken for 5s and immediately afterwards used in a Varioskan Flash Multimode Reader multifunctional microplate ReaderMeasurement of fluorescence at excitation wavelength of 446nm and emission wavelength of 490nm A β_1-42+ fluorescence values of test compounds are recorded as IF_i，Aβ_1-42Fluorescence values of + PBS buffer were recorded as IF_cThe fluorescence value of the buffer solution containing only PBS was recorded as IF₀Inhibition of A β by Compounds_1-42The inhibition rate of self-aggregation is calculated by the formula: 100- (IF)_i-IF₀)/(IF_c-IF₀) × 100, two duplicate wells are determined for each concentration of each compound, and curcumin is used as a positive control, and the detection results are shown in Table 4.

The measurement result shows that the inhibition rate of curcumin is 43.1% under the same concentration, and the widely used anti-AD drug donepezil can treat A β under the concentration of 25.0 mu M_1-42The inhibition rate of self-aggregation is less than 20.0%, and the naringenin-O-alkylamine compound provided by the invention can inhibit A β under the concentration of 25.0 mu M_1-42The inhibition rate of self-aggregation is more than 46.8 percent for A β_1-42Self-aggregation has obvious inhibitory activity.

3. Determination of antioxidant Activity of naringenin-O-alkylamines Compound (I) (ORAC-F L method)

Reagents and apparatus 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid (trolox, available from Chiloei chemical industries, Ltd.) was prepared as a 10-80. mu. mol/L solution with 75mM PBS buffer (pH7.4), fluorescein (fluoroscein, available from Chiloei chemical industries, Ltd.) was prepared as a 250 nmol/L solution with 75mM PBS buffer (pH7.4), 2' -azobisisobutylamidine dihydrochloride (AAPH, available from Shaoshan Seiko Chemicals, Ltd.) was prepared as a 40 mmol/L solution with 75mM PBS buffer (pH7.4) before use, and the microplate reader was Varioska filtration MultitereReader (Thermo Scientific).

The experimental method comprises the steps of adding 50 or 10 mu mol/L compound solution 20 mu L and fluorescein solution 120 mu L into a black 96-well plate, uniformly mixing, incubating at 37 ℃ for 15min, adding AAPH solution 60 mu L to make the total volume of each well 200 mu L, uniformly mixing, immediately placing into a Varioskan Flash Multimode Reader instrument, measuring the fluorescence value once per minute at 485nm excitation wavelength and 535nm emission wavelength, continuously measuring for 90min, and automatically calculating the area AUC under the fluorescence attenuation curve by the instrument, wherein Trolox of 1-8 mu mol/L is taken as a standard, a Sample to be measured is not added as a blank, the antioxidant activity result of the compound is expressed as the equivalent of Trolox, the calculation formula is that [ (AUC-Trolox-AUC blank) ] × [ (control of Trolox/control) ], each compound is measured for 3 multiple wells, and the detection result is shown in Table 4.

The determination result shows that the antioxidant activity of the naringenin-O-alkylamine compound (I) is 1.1-1.6 times of that of Trolox, which indicates that the compound has strong antioxidant activity.

4. naringenin-O-alkylamine compound (I) p-H₂O₂Protective effects of induced PC12 cell injury

PC12 cells were cultured in DMEM containing 10% calf serum at 1 × 10⁵Inoculating at density of L/m on 96-well culture plate with volume of L/m, and adding 5% CO₂Culturing in a constant temperature incubator at 37 ℃. After 24 hours of incubation, the corresponding concentration of compound (final concentration 10) was added to the group administered^-5mol/L，10^-6mol/L) 10m L/well, preincubated for 2 hours (10 mu L/well PBS was added to the control group and the injured group respectively to keep the volumes equal), after PC12 cells were incubated for 2 hours, 100 mu m H was added to the administration group and the injured group respectively₂O₂The injury agent is 10 mu L/hole (10 mu L/hole PBS is added in the control group), after 30 minutes, the culture solution of each group is changed into RPMI1640 culture solution without calf serum and is continuously placed in a constant temperature incubator for 24 hours, the volume of the culture solution is 100 mu L/hole, after the culture is continuously carried out for 24 hours, 5mg/m L100 mu L/hole is added in each group, living cells are stained, after 3 hours, 100 percent DMSO termination solution 100 mu L/hole is added in each group, the mixture is fully dissolved and uniformly mixed, the OD value of each group is measured under the wavelength of 490nm, the test result is repeated for 3 times, the number value of each group is counted by a Duncan's test method, the number value of each group is the mean value +/-S.E.M, the number of the control group is 100 percent, and the values of the administration group and the injury group are expressed by the percentage of the control group, and the detection is shown in figure 1.

The determination result shows that the compound of the invention is 10^-5mol/L～10^-6The concentrations of the components are all obvious in the hydrogen peroxide-induced PC12 cell damage under mol/L concentrationThe protective effect of (1).

5. Determination of complexation of naringenin-O-alkylamine compound (I) and metal ion

Dissolving CuCl with methanol₂·2H₂O、ZnCl₂、FeSO₄·7H₂O、AlCl₃And a compound to be detected, preparing a solution of 75 mu mol/L, adding 100 mu L of the solution of the compound to be detected and 100 mu l of a metal ion solution into a 96-well plate, uniformly mixing, standing for 30 minutes at room temperature, recording an ultraviolet absorption curve within the range of 200-600nm on a multifunctional microplate reader, and observing the red shift phenomenon of the maximum absorption peak and the intensity of the maximum absorption peak of the mixed solution of the metal ions and the compound to be detected by taking the 100 mu L of the solution of the compound to be detected and 100 mu L of methanol mixed solution as comparison.

The determination result shows that the naringenin-O-alkylamine compound (I) has strong complexation effect on metal ions.

6. The naringenin-O-alkylamine compound (I) prepared by the invention is opposite to AlCl₃Inducing the prevention and treatment effect of the zebra fish Alzheimer disease model (taking the compound I-43 as an example).

Randomly selecting 3dpf wild AB line zebra fish in a six-hole plate, and adding aluminum trichloride (AlCl)₃) Inducing zebrafish model of Alzheimer's disease (AD zebrafish), respectively dissolving I-43 compound in water at concentrations of 1 μ g/M L, 3 μ g/M L and 10 μ g/M L, and positive control drug donepezil 8 μ M, and setting normal control group (untreated) and model control group (AlCl)₃Group), 30 zebrafish per experimental concentration group. After 3 days of administration, the movement distances of the zebra fish in each experimental group in 3 light and shade periods (namely, the dark 10min and the light 10min alternate 3 periods) within 60min are respectively observed and recorded by a behavior analyzer, the movement distance of the zebra fish in 60min is analyzed, the statistical analysis is carried out by the movement distance and a model control group, the prevention and treatment effects of the compound on the zebra fish Alzheimer's disease model are evaluated in a statistical sense, and the detection result is shown in figure 2.

The test result shows that the medicine is normalCompared with a control group (7303mm), the moving distance of the model control group is 5203mm, which indicates that the model is successfully established; while the positive control group donepezil increased the movement distance to 6358mm (p)<0.01) compared with the positive control donepezil group, the high, medium and base dose groups of the drug I-43 have more movement distances, which shows that the compound I-43 can obviously improve AlCl₃The motor function of the zebra fish AD is induced, and the zebra fish AD has statistical difference compared with a model control group.

7. The influence of the naringenin-O-alkylamine compound (I) on mouse memory acquisition disorder caused by scopolamine (diving platform passive avoidance experiment) (taking the compound I-43 as an example).

SPF grade ICR male mice, 25-30g, randomly divided into: normal group, model group, high (20mg/kg), medium (10mg/kg), low dose group (5.0mg/kg) of test drug, 10 animals per group. The tested medicine is given by one-time intragastric administration, the solvent of 0.5 percent CMC-Na is given to the blank group and the model group, and the administration volumes are both 0.1ml/10 g; injecting normal saline into abdominal cavity of normal group mice 45min after drug administration, and injecting scopolamine 3mg/kg into other groups of animals; and (3) carrying out platform jump training 20min after molding, putting the animals into a reaction box for adaptation for 3min, and immediately introducing 36V alternating current. Training for 5min, and recording the number of shocks (number of errors) to which the mouse receives each time, and thus as the learning achievement. The test was performed 24h later, and each mouse was assayed for 5min, and the number of shocked animals and the latency of the first platform jump and the number of errors within 5min were recorded. The results were statistically analyzed. All data are expressed as mean ± standard error (Stand error, s.e.). Analysis was performed using SPSS11.5 software. One-way ANOVA (One-way ANOVA) was selected for ANOVA. The measurement data is compared by adopting one-factor analysis of variance, the average of each group is compared by adopting t test, and the detection result is shown in figure 3.

The determination result shows that the latency period of the model group diving platform passive avoidance experiment test is obviously shortened (p is less than 0.01) compared with the control group, which indicates that the model is successfully established; compared with a dementia model group, a dosage group and a donepezil group in the medicine have obvious extension trend (p is less than 0.01), and the compound naringenin-O-alkylamine compound has obvious improvement effect on mouse dysmnesia caused by scopolamine.

Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.

Claims

1. A naringenin-O-alkylamine compound is characterized in that the chemical structural general formula is shown as (I):

wherein: n is 2-12;

R₁represents H or C₁～C₁₂An alkyl group; r₂Is represented by C₁～C₁₂Alkyl, benzyl, substituted benzyl, 1,2,3, 4-tetrahydroacridin-9-yl or 6-chloro-1, 2,3, 4-tetrahydroacridin-9-yl;

or NR₁R₂Represents N-demethyl-galanthamine, morpholinyl, piperidinyl, 4-substituted by C₁～C₁₂Piperidinyl substituted by alkyl, piperidinyl substituted by benzyl at the 4-position, piperazinyl substituted by benzyl or substituted benzyl at the 4-position, 1,2,3, 4-tetrahydroisoquinolinyl, 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinolinyl or 1-methyl-1, 2,3, 4-tetrahydroisoquinolinyl;

the substituted benzyl is benzyl substituted by 1 to 4 groups selected from the following groups on a benzene ring: c_1-4Alkyl radical, C_1-4Alkoxy, trifluoromethoxy and dimethylamino, and the substituents can be in any possible positions of the benzene ring.

2. A method for preparing the naringenin-O-alkylamines of claim 1, comprising the steps of:

(1) with naringenin and dibromoalkane Br (CH)₂)_nBr as starting material in the first solventReacting with the first alkaline condition to obtain corresponding 2, 7-dibromo alkoxy naringenin;

(2) reacting 2, 7-dibromo-alkoxy naringenin with compound NHR in second solvent under second alkaline condition₁R₂Reacting to obtain a target compound naringenin-O-alkylamine compound;

the compound NHR₁R₂In R₁Represents H or C₁～C₁₂An alkyl group; r₂Is represented by C₁～C₁₂Alkyl, benzyl, substituted benzyl, 1,2,3, 4-tetrahydroacridin-9-yl or 6-chloro-1, 2,3, 4-tetrahydroacridin-9-yl;

3. The method for preparing naringenin-O-alkylamines as in claim 2, wherein the base used in the first or second alkaline condition is: alkaline earth metal hydroxide, alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate, C_1-8Alkali metal salts of alcohols, organic tertiary amines and quaternary ammonium bases.

4. The method for preparing naringenin-O-alkylamines as in claim 2, wherein said first or second solvent is selected from the group consisting of diethyl ether, tetrahydrofuran, N-dimethylformamide, dimethylsulfoxide, dichloromethane, chloroform, C_3-8Aliphatic ketone, benzeneToluene, acetonitrile and C_5-8One or more alkanes.

5. The process for preparing naringenin-O-alkylamines according to claim 2, wherein the naringenin in step (1)): dibromoalkane Br (CH)₂)_nBr: the feeding ratio of the alkali is 1.0: 2.0-10.0: 2.0-10.0, the reaction temperature is 25-150 ℃, and the reaction time is 10-48 h.

6. The process for preparing naringenin-O-alkylamines according to claim 2, wherein the 2, 7-dibromoalkoxynaringenin in step (2)): compound NHR₁R₂: the molar charge ratio of alkali is 1.0: 1.0-10.0: 1.0-10.0, the reaction temperature is 25-150 ℃, and the reaction time is 10-48 h.

7. An application of the naringenin-O-alkylamine compound in claim 1 in preparing medicine for treating neurodegenerative diseases.

8. The use according to claim 7, wherein the neurodegenerative disease is Alzheimer's disease, vascular dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, progressive lateral sclerosis, neuropathic pain or glaucoma.

9. A pharmaceutical composition for treating neurodegenerative diseases, comprising the naringenin-O-alkylamine compound of claim 1 or a pharmaceutically acceptable salt thereof synthesized with an acid.