CN108314652A - Edaravone derivative and application thereof - Google Patents

Abstract

The invention belongs to pharmaceutical field, a kind of edaravone derivative and application thereof is specifically disclosed.The derivative has the therapeutic effect of enhancing and significantly reduced cytotoxicity compared with Edaravone.

Description

Edaravone derivative and application thereof

Technical field

The invention belongs to pharmaceutical fields.Specifically, the present invention relates to edaravone derivative, and utilize the derivative Treat human oxidized property stress relevant disease, especially neurodegenerative disease or cerebrovascular disease purposes.

Background technology

Free radical is the frequent consequence of normal cell aerobic metabolism.The built-in antioxidant system of human body is preventing because of free radical Decisive role is played in terms of caused damage.However, the unbalance defense mechanism of antioxidant, free radical it is excessive production or Introducing life system from environment causes to lead to neurodegenerative serious hindrance.Nerve cell turns in neurodegenerative disease by function Or the forfeiture of sense organ.In addition to several environment or inherent cause, oxidative stress (OS) causes free radical to attack nerve cell, gives Neurodegenerative contribution is catastrophic.Although oxygen is essential to life, metabolic imbalance and excess activity oxygen (ROS) Generation is the Etiological of global chronic disease and degenerative disease such as aging and other degenerative diseases, these degenerative diseases are such as Alzheimer disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), the sclerosis lateralis amyotrophica of the mankind (ALS), atherosclerosis, cancer, diabetes, rheumatoid arthritis (RA), ischemic perfusion after damage, myocardial infarction, Cardiovascular disease, chronic inflammation, apoplexy and infectious shock.According to the report of the World Health Organization (WHO), caused for 10 kinds before the whole world Dead property disease is ischemic heart disease, apoplexy, chronic obstructive pulmonary disease (COPD), lower respiratory tract infection, tracheae/bronchus/lung Cancer, HIV/AIDS, diarrhea, diabetes, hypertension and pulmonary tuberculosis (preceding 1-10 descendings arrangement).The whole world has 28,800,000 patients to exist 2012 because of these disease deaths (mainly by sorting caused by preceding 4 fatal disease).Ischemic heart disease accounts for all dead Nearly 25.7% died, apoplexy accounts for nearly 23.3%, and COPD and lower respiratory tract infection account for nearly the 10.7% of total death respectively.It is various to grind Study carefully show oxidative stress be most of diseases, especially ischemic heart disease in preceding 10 kinds of fatal diseases, apoplexy, COPD, The main cause of HIV/AIDS and diabetes.World market with oxidative stress relevant disease is at 200,000,000,000 dollars or more.

Since oxidative stress is many chronic, denaturation and fatal disease basic places, there is an urgent need to anti-for this prompt Oxidant is horizontal to help ROS in control volume.Therefore, as strong and potential free radical scavenger or antioxidant, it is known that Edaravone (edaravone hereafter writes a Chinese character in simplified form into " EDA ") can play weight in terms of reducing ROS levels and reducing oxidative stress It acts on.

EDA is also known as MCI-186, chemical name：3-methyl-1-phenyl-2-pyrazolin-5-one, molecular formula C₁₀H₁₀N₂O, point Son amount 174.19, structural formula：

EDA is that strong synthesis oxygen free radical scavenger to reduce oxidative stress, and is passed through with antioxidation By non-enzymatic lipid peroxidation and lipoxygenase pathway anti-lipid peroxidation.In addition to this, EDA is also in inflammation, matrix metal Protease, nitric oxide generate and Apoptosis aspect shows advantageous effect.Mitsubishi Tanabe Pharm Corp. (Osaka, Japan) develops EDA first, and is listed with Radicut in 2001, becomes first neural blood vessel protection in the world Drug.2001, Japanese public health and Department of Welfare ratified the medicine for treating cerebral infarction, acute ischemic stroke (AIS) patient. From that time, EDA is not only usually used in treating AIS, is additionally operable to treatment ROS relevant diseases, such as cardiovascular disease and apoplexy.Although EDA is common prescription drug in Japan, India and China, but approval is not yet received in the U.S. and other western countries in it, this master If since its toxicity and shortage clinical research in liver and kidney supports the advantageous effect of EDA.

EDA is designed to phenol sample compound, and phenol is one of the functional group of all phenol antioxidant, and wherein phenols is anti-oxidant Agent is made of the hydroxyl (- OH) being connect with aromatic rings, and is responsible for anti-oxidation characteristics.By contributing a hydrogen ion to freely Base, then becomes a group, and free radical is quenched in phenols.However, electronics goes to localize by aromatic rings via resonating electron on phenol And stablize, and activity declines.But due to toxicity and corrosivity, even if showing radicals scavenging effect and having turned out to be potential Antioxidant, phenols is not particularly suited for pharmaceutical applications.

On the other hand, it is particularly present aromatic hydroxy group, EDA is expected have activity identical with phenols, and shows similar anti- Oxidation and radicals scavenging effect.EDA can be classified as three kinds of different tautomeric forms, i.e. amine shape, ketone shape and enol shape. Aromatic hydroxy group is generated via ketoenol tautomerization.However, with phenols on the contrary, EDA does not have the toxic effect of phenols, this is EDA has more one of the cause of advantage than phenols.

The pKa of EDA is 7.0, therefore under the conditions of physiological pH, and about 50% EDA is ionization, and with anionic form In the presence of.The anionic form of EDA is also more reactive form, is easy to react with ROS in the brain, is generated anti-oxidant Effect.EDA is compared to the advantage of other free radical scavengers (such as Idebenone, baicalein and catechol), readily penetrates through Blood-brain barrier has dissolution characteristics this is because EDA is the lipophilic molecule of low molecular weight in water and lipid.Therefore, it can be very Blood-brain barrier is easily passed through, brain point of impact on target is arrived at and plays a role.The ratio between blood plasma level and cerebrospinal fluid levels of EDA are expected 50-65%.These characteristics may be EDA has a neuroprotection in cerebral infarction, and the not effect of other antioxidants Reason.

Alzheimer disease (AD) be more than the most common type dementia type in 60 years old crowd at the age.Recent decades are Flood tide resource has been put into for AD Therapy studies.On the one hand, certain progress is achieved to symptom treatment, however it is disease modifying developing Also there are several failures (Mangialasche and Solomon etc., 2010) in terms for the treatment of.Only approval several drugs are controlled for AD at present It treats, including four kinds of anticholinesterases (Tacrine, donepezil, Rivastigmine, galanthamine) and N- methyl Ds-asparagus fern ammonia Acid (NMDA) receptor AD antagonists (Memantine (memantine)) (Hyde and Peters etc., 2013).Go out on AD drug developments Many failures are showed, small molecule and immunotherapy fail to show the difference of drug/placebo or have unacceptable poison Property (Cummings and Morstorf etc., 2014).These unsuccessfully cause pair it is appreciated that in the pathogenetic latent defects of AD With in the exploitation of drug candidate potential trap discuss.So far, still there are many clinical and experimental researches In progress, but it must be admitted that by people, the unlikely single therapy medicine that can be found to alzheimer disease, and drug development Method and concept needs are rethought.

Understanding pathogenetic to AD at present includes three kinds of main Types, and " cholinergic hypothesis " is the first for explaining AD Theory.This hypothesis has become guiding treatment slightly to the basic theory of the drug development of moderate dementia.According to the theory, The function of cholinergic neuron and the forfeiture of cholinergic activity, people in Meynert basal nucleis (NBM) are usually observed in AD brains It is in class and non-human primates research shows that acetylcholine plays key effect in learning and memory.These datas are aobvious Show, if blocking central cholinergic system activity with hyoscine, young subjects will show memory impairment this typical AD mark Will, and this damage can be by being treated by rescue (Bartus, 1978) with cholinergic agonist eserine.Second The theory of mainstream is " amyloid protein hypothesis ", by soluble A β segments be considered cynapse toxicity and be in AD pathology Patch formed, the cause (Musiek and Holtzman, 2015) being subsequently mutated of the peroxophosphoric acid of tau and signal transduction path. According to the key effect proposed by " amyloid protein hypothesis ", a variety of therapeutic agents are developed, with by inhibiting enzyme such as β- Secretase reduces the generation (Jonsson of A β by for example anti-A β immunotherapies of strategy for existing patch in removing brain With Atwal etc., 2012).Tau hypothesis are the popular theories of the another kind of AD mechanism.Research confirms that tau oligomer is neurotoxicity , many clinical symptoms are closely related with tau pathology, and the peroxophosphoric acid of tau constitutes the common final of many other dementias Path, therefore tau theories become increasingly to be received.In this theory, abnormal signal passes through a variety of paths such as fyn kinases Path causes tau peroxophosphoric acid.Tau modifications cause its oligomerization to act on and the generation of NFT, cause abnormal transmitter loss, micro- The collapsing of pipe basal cell's skeleton, follow-up neuronal death, and eventually lead to neuronal degeneration it is progressive spread through sex intercourse (Wolfe, 2012)。

Oxidative stress is one of the mark of Alzheimer disease (AD), the free radical and starch induced by oxidative stress The production of crossing of sample β (A β) albumen is connected with the super phosphorylation of Protein tau.The abnormal accumulation of amyloid beta protein and σ albumen is then sharp It is also unbalance in oxygen, and vicious circle is eventually led to, promote the overall development (Zhao and Zhao, 2013) of AD diseases.

It is proved that a variety of main pathologic processes of oxidative stress and AD, such as A beta-aggregations and deposition, tau phosphorylations, Cynapse collapse with metal unstability, extensively be connected.The production of crossing of active oxygen (ROS) may be abnormal through mitochondria disability or transition metal Perhaps, accumulation induction is caused by the synergistic effect of A β accumulations and the super phosphorylation of Protein tau, eventually leads to oxidative stress (Guo With Sun etc., 2013).On the other hand, oxidative stress is responsible for mediating by the neurotoxicity of A β and Protein tau induction, can promote A β Thus production and tau phosphorylations and polymerization further enhance a variety of neurotoxic events, including ROS productions, are conducive to be formed The vicious circle (Zhao and Zhao, 2013) of AD development.

Therefore, oxidative stress has become the most critical risks and assumptions of AD occurrence and development.Inhibit or prevent ROS, passes through Number of mechanisms can postpone the breaking-out of AD or slow down the development of AD.Based on above-mentioned theory, is prevented by antioxidant or treatment AD is Method full of hope, this is because antioxidant can target the signal pathway of numerous different participation AD pathology.

Although in the past few decades, having achieved rapid progress to the understanding of AD pathogenesis, AD management is still lacked Available effective therapy (Holtzman and Morris etc., 2011).Numerous drugs also developed, and by immunization therapy or divide Enzyme inhibitor is secreted, targets A β, tau or oxidative stress, but in clinical test, the cognitive defect of patient is improved slightly, and By failure (Wang, 2014).In view of these examples, the drug for targeting single creature marker or approach will not be to this complexity Disease works.The previous achievement in research of inventor shows that more targeting strategies of AD treatments can be a kind of novel approach.

EDA with potent Scavenger of ROS (ROS) and can inhibit pro-inflammatory (Watanabe and Tahara after cerebral ischemia Deng 2008).Clinical data shows that EDA can be effectively improved inflammation after ischemic, and inflammation is likely to result in brain water after ischemic Swollen and cerebral infarction.Other than anti-stroke acts on, EDA also shows the oxidative damage that can prevent to a variety of karyopyknosis.Nearest Zooscopy confirms that EDA can the potent a variety of key point objects alleviated in AD pathology.It is significantly dropped by drug administration by injection EDA Low A beta-aggregations, Tau peroxophosphoric acid and the cognitive ability (Jiao and Yao etc., 2015) for improving animal.Have revealed that EDA is being controlled Treat AD in terms of several mechanism, EDA can combine and with aβ protein direct interaction, to adjust downstream signal transduction and press down AD pathogenic cascades beta induced A processed.In addition, structural advantage allows EDA to break through blood-brain barrier and transports to (Watanabe in brain With Tahara etc., 2008).

However, the safety of the medicine is troubling.The experimental data that do not deliver is shown, under high concentration, EDA inducing cytotoxics Property, MTT the result shows that, when handling SY5Y with 30 μM or higher concentration, cells viability declines.Apoptosis assay is also shown Similar result.Therefore it needs to synthesize the noval chemical compound based on EDA, it is contemplated that the compound can improve the therapeutic effect of EDA, simultaneously Possess much lower toxicity.

Summary of the invention

The present invention prepares and characterizes EDA derivative BE, and passes through experiment test EDA and its derivative BE, systematicness The influence that both compound on intracellular survivals, Apoptosis, A beta-aggregations, BACE1 expression and neural process are grown is compared, is obtained Inspirer result：BE is to inhibiting AD diseases and inoxidizability that stress show stronger conduct.By with EDA ratios Compared with further acknowledging that the cytotoxicity of BE is lower, therefore BE will be clinically better drug candidate.

The present invention compares the therapeutic effect of EDA and BE, and it has surprisingly been found that BE not only retains, but also More than EDA in terms of many treatments, including neurotoxicity that A is beta induced, BACE1 are expressed, A beta-aggregations, acetylcholine esterase active, together When also meet security requirement, this is beyong contemplation.

The present invention provides a kind of edaravone derivative, as shown in following formula (I)：

Wherein X is connector, is selected from-C (O) O- ,-C (O) S- ,-C (O) NH- ,-C (O)-,-NH- or-CH₂, preferably-C (O) O-.The connector is known as isostere each other, has similar physics and chemical property, will produce substantially similar Bioactivity.

According to the present invention, shown in edaravone derivative such as following formula (Ia)：

Hereinafter referred BE.

The invention further relates to the disease treatment method of following edaravone derivative and pharmaceutical applications：

The present invention provides edaravone derivative for treating and oxidative stress relevant disease.

The present invention provides edaravone derivative answering in preparing the drug for treating oxidative stress relevant disease With.

According to the present invention, the oxidative stress relevant disease include senile/senile disease (arthritis, diabetes, Osteoarthritis, cataract, macular degeneration, prostatosis), cardiovascular disease (arteriosclerosis, heart failure, heart disease, renal failure, high blood Pressure, apoplexy, poor blood circulation, cholesterol and patch formed, reperfusion injury), cancer (prostate cancer, breast cancer, lung cancer, Intestinal cancer, carcinoma of urinary bladder, uterine cancer, oophoroma, lymthoma, cutaneum carcinoma, gastric cancer, liver cancer and other deelines), neurodegeneration disease Sick (Alzheimer disease (AD, also known as senile dementia), Parkinson's disease (PD), multiple sclerosis (MS), schizophrenia Disease, dementia, Huntington's chorea), hepatopathy (toxic hepatitis, virus hepatitis (A, B and C), chronic hepatitis), tuberculosis (asthma Pulmonary emphysema, pneumonia, (acute and chronic) bronchitis, cystic fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), Cheng Renhu Inhale Distress syndrome (ARDS)), disease of digestive tract (inflammatory bowel disease, ulcerative enteritis, Crohn disease, gastritis, gastric cancer, gastric ulcer, Pancreatitis), (oxidative stress caused by renal failure, kidney poison, dialysis), infectious diseases and immunological disease are (viral thoroughly for renal failure and kidney Infected by HIV and AIDS (AIDS), toxic hepatitis and hepatic sclerosis, virus hepatitis (A, B and C), bleb, flu, bacterium sense Dye, chronic fatigue syndrome, autoimmune disorders), skin disease (psoriasis, eczema, systemic loupus erythematosus (SLE), blood Guan Yan, polymyositis, mycosis fungoides, chorionitis, pemphigoid, allergic dermatitis, contact dermatitis, seborrhea, bleb Sample dermatitis, acne conglobata, acne vulgaris, UV irradiate skin injury), diseases of the five sense organs (cataract, macular degeneration, Hearing loss, ear infection, nasosinusitis, periodontosis, nose, oral cavity and throat (upper respiratory tract) disease), gestation, lactation and childbirth Relevant disease (preeclampsia, eclampsia, hypertension, diabetes), motion sickness (syndrome of overtraining and its related oxidized property Stress), andrological diseases (hyperplasia of prostate, prostate cancer, alopecia, male sterility), female infertility, arthropathy and chronic inflammation Disease is especially selected from AD, amyotrophic lateral sclerosis (ALS), PD, ischemic heart disease, cerebrovascular disease (including ischemic brain blood Pipe disease, cerebral infarction/apoplexy/cerebral apoplexy), COPD, HIV/AIDS and diabetes.

Material therefor of the present invention and method are as follows：

(1) animal

The program of the related to animal of the present invention obtain Nan'ao pathology animal ethics committee (Adelaide, ) and the approval of the University of South Australia animal welfare committee (Adelaide, Australia) Australia.All programs are all in accordance with Australia The guide of the big Leah national healthcare health research committee carries out.All animals maintain 12 hours day night cycles, and freely Close to food and water.C57 newborn mices are raised by Reid animal office workers.

(2) cell culture

The human neuroblastoma SH-SY5Y695-app cell lines of the present invention are obtained from American type culture collection (ATCC,Rockville,MD,USA).By cell culture containing 5% fetal calf serum (FBS), penicillin (50IU/ml), streptomysin (50 μ g/ml) and L-Glutamine (2mM) Dahl Burke Improved Eagle Medium (DMEM, Life Technology, VIC, Australia) in.All cells, which maintain, is supplemented with 95%O₂And 5%CO₂Humidification 37 DEG C of insulating boxs in.

The 0th day (P0) to the 1st day (P1) is anaesthetized on ice water slurry after the birth of C57 wild type newborn mices, and decaptitating is killed. After removing meninx, the cortex of two hemisphere of each mouse and brain other parts are separated, and is transferred to containing the cold PBS's of 2-3ml In 15ml centrifuge tubes, centrifuged 3 minutes with 1,500rpm at 4 DEG C.Remove PBS, tissue with 4ml I containing 0.1%DNase (NEB, Ipswich, MA, USA) 37 DEG C of 0.25% trypsase at digest 20 minutes, concussion in every 5 minutes is primary.It is (dense eventually that FBS is added It spends 15%), to determine trypsinized.Centrifuge tube remains stationary 5 minutes allows and digests tissue gravitational settling.It is broken in a organized way After piece sedimentation completely, supernatant is transferred to new pipe, 4 DEG C are centrifuged 2 minutes with 2,000rpm.After centrifugation, supernatant is abandoned, and will be thin Born of the same parents' pellet resuspended 1ml cortical neuron culture base (additive containing B27 (2%), L-Glutamine (2mM) and penicillin/ The Neurobasal medium (Life Technology, VIC, Australia) and beta -mercaptoethanol of streptomysin (100IU/ml) (0.1mM)) in.

Then cell count (Bio-rad, TC20 are carried out to neuron^TMAutomated Cell Counter), it inoculates To tissue culturing plate.To Western blotting or for immunocytochemical assay, by cortical cell (with 2 × 10⁶Concentration) It is seeded in poly- D-Lys-and the coated 6 orifice plates of Laminin ELISA-, and (with 2.5 × 10⁴Concentration) it is seeded to 4- or 24- The poly- D-Lys-and Laminin ELISA-of orifice plate (Thermo Fisher Scientific, Rockford, IL, USA) are coated On coverslip (Thermo Fisher Scientific, Rockford, IL, USA).

(3) A beta oligomers

A β 42 are purchased from American Peptide companies with 1mg/ bottles.42 peptides of A β are dissolved in 1,1 with the concentration of 1mg/ml, In 1,3,3,3- hexafluoro -2- propyl alcohol (HFIP, Sigma), Eppendorf microcentrifugal tubes are added in equal portions, and air is dry in ventilating kitchen Dry at least 2 hours, then -80 DEG C of long-term preservations.When experiment, aliquot A β 42 is dissolved in DMEM, and incubates 24-48 at 4 DEG C Hour, to generate 42 monomers of toxicity oligomer A β.

(4) MTT is analyzed

In order to investigate EDA and BE to CuSO₄, H₂O₂With the antagonism of 42 in vitro toxicities of A β, using human neuroblastoma SH-SY5Y695-app cell lines.Cell is detached with T75 flasks by 0.25% trypsase, then respectively with 0.5 μM CuSO₄, 50 μM of H₂O₂, 1 μM of 42 monomer of A β, 1,3 and 10 μM of EDA and BE are seeded to together in 96 orifice plates.It incubates 19 hours Afterwards, it carries out MTT [3- (4,5- dimethylthiazole -2- bases) -2,5- diphenyltetrazolium bromides] to analyze, 25 μ l is added in every hole MTT (PBS of Sigma-Aldrich, USA, 5mg/ml), and incubate 2 hours.Dimethyl sulfoxide (DMSO) (DMSO) 200 μ L are added, with Insoluble purple formazan is dissolved, colored solutions are generated.Using at porous scanning spectrophotometer 570nm wavelength to optical density (OD) Reading.

(5) thioflavin T (ThT) fluorescence analysis

In order to test the inhibition of EDA and BE to A beta-aggregations, first by 1 μM of 42 monomer (American of A β Peptide 10 days) are incubated with the EDA and BE of a series of concentration at 37 DEG C in DMEM, then with 5 μM of ThT solution incubations.For The pre-formed A β fibrils of depolymerization, 37 DEG C of 42 monomers of pre-formed 1 μM of A β then separately incubate fibril and EDA and BE 3 days.It incubates At the end of, 3ml 5M ThT solution (50mM phosphate buffers, pH6.0, Sigma) is added in reaction solution, measures sample.Work as ThT When being combined with the beta sheet aggregate structure of amyloid fibril, the fluorescent emission displacement of ThT.Pass through luminescence spectrophotometer PE- LS50B (PerkinElmer) monitors fluorescence intensity, excitation and transmitted bandwidth with excitation wavelength 450nm and launch wavelength 482nm It is 5nm.Each experiment is triplicate, in triplicate.

(6) neural process grows analysis

Neural process grows analysis and is carried out on the cortical neuron for being isolated from C57 mouse.Cortical neuron is isolated from the 1st day The brain of C57 mouse, and pass through 0.25% trypsin digestion.By cerebral cortex and hippocampal cell culture in neural basal culture In base, the culture medium contains 2%B27 (Invitrogen, #17504001) and 2%FBS (Invitrogen).Cell inoculation It is incubated 48 hours in the 50 poly- D-Lys of μ g/ml (PDL) coated 6 orifice plates, and at 37 DEG C.Neuron and 1 μM of A β are few Aggressiveness carries the EDA/BE or no EDA/BE of various concentration, incubates 24 hours together.Then cell is fixed on 4% formal Lin Zhong, and be incubated overnight at 4 DEG C of rabbit-anti-MAP-2 antibody.Thereafter, small with two level anti-rabbit Alexa488 antibody and DAPI incubations 1 When.Image is acquired from the microscopical 5 longest neural processes of each visual field Olympus (CX40), and analyzes the number in 6 visuals field of each group According to.Representative Neuronal images are obtained using confocal (LSM710) microscopes of Zeiss.The length of dendron is measured by image J softwares Degree, data are indicated with mean value ± SD.

(7) western blot analysis of the EDA and BE to A beta-aggregation inhibiting effect

The EDA and BE of 1 μM of 42 monomer of A β and a series of concentration are incubated 10 days in DMEM at 37 DEG C, acquired solution and 5 × sample-loading buffer of no reducing agent mixes, then electrophoresis.Non- incubation A β samples (A beta oligomers) are used as control.All samples In loading to 15% (wt/vol) Acrylamide gradient gels, the A β of full gel separation are transferred on nitrocellulose filter.Trace MOAB-2 mouse monoclonal antibodies (Biosensis M-1586-100) to be specific to amyloid beta (A β 40/42) are detected.

(8) Apoptosis assay

SH-SY5Y cells are collected from 24 orifice plates, and are washed in PBS, supernatant is abandoned, is then fixed with cold 70% ethyl alcohol Cell 30 minutes.Twice with PBS washings cell precipitation, 1500rpm centrifugations abandon careful loss cell when supernatant.It is added 200 μ l propidium iodides (PI, 50 μ g/ml) 10 minutes twice with PBS washings cell avoid false positive from dyeing.Then cell is passed through Flow cytometer (Beckman Coulter Gallios, Detmold Family Cytometry Facility) scans, often A sample cell analysis 2 × 10⁴A cell.All tests are at least repeated 3 times.

(9) acetylcholinesteraseanalysis analysis

AchE analyses are carried out using AchE kits (Abcam, ab138871).With the EDA of various concentration and BE in 24 holes Cell is handled on plate.Processing collects cell lysate after 20 hours.Experiment is operated according to manufacturers instruction.

(10) real-time PCR

According to standardization program, BACE1mRNA expressions are determined.Using RNAeasy mini kits (Qiagen, Doncaster, Vic, Australia), the SY5Y695 Cell extraction total serum IgEs handled from EDA and BE by various concentration.It adopts The concentration and purity of RNA are determined with NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). 500ng total serum IgEs are raw using the first chains of Superscript III synthesis system (Life Technology, VIC, Australia) At the first chain cDNA.PCR uses GoTaq Green Master Mix (Promega, Madison, WI, USA), BACE1 (SEQ ID NO:5) forward primer：5’-ACCGACGAAG AGTCGGAGGAG-3’(SEQ ID NO:1), reverse primer：5’- CACAATGCTCTTGTCATAG-3’(SEQ ID NO:2).Using house-keeping gene GAPDH as standard, forward primer：5’- AACATCATCCCTGCATCCAC-3’(SEQ ID NO:3), reverse primer：5’-TTGAAGTCTCAGGAGACAAC-3’(SEQ ID NO:4).GAPDH(SEQ ID NO:6) it is used to calibrate BACE1 mRNA expression.

It was found that and developing a kind of research that new drug needs more than 1,000,000,000 dollars, and may consume up to 10 years.Drug Development process can be divided into four different phases：Early detection, late period find, are preclinical and clinical.Inventor has screened several The anti-AD activity of kind noval chemical compound, this early detection research will pave the way for following drug development.Screening process it Afterwards, the present invention identifies compound BE, is based on chemical constitution, belongs to a kind of new derivative of EDA.Inventor thinks that BE has There are stronger antioxidation and less cytotoxicity.It is an object of the present invention to study BE whether be capable of providing it is beta induced to A The protective effect of the pathological raisings of AD, and detect cytotoxicity of the compound compared with the EDA of its source.In order to fully understand The therapeutic effect and cytotoxicity of BE, the present invention devise a comprehensive study plan.The present invention uses cell culture, albumen Matter trace, flow cytometry and ThT, AchE activity analysis are ground with evaluating the EDA and BE of various concentration from functional and mechanism Study carefully experiment and collects data.

After the noval chemical compound with interested biological property enters the program, simultaneously designed for processing by experience Solve a large amount of tests of many challenges.During the program, new chemical entities may be terminated for many reasons Exploitation, two of which main cause is drug effect and toxicity.To the preclinical animal safety research of new drug candidate and facing The unexpected side effect and toxicity observed during bed experiment are one of the ultimate challenges that current pharmaceuticals industry faces.In order to It solves these problems, vitro cytotoxicity analysis is used as tool to understand the hypothesis about drug toxicity mechanism by recent decades The problem of driving (McKim, 2010).The present invention analyzes the toxicity for detecting and comparing EDA and BE by MTT first, with wide model Enclose EDA and BE the processing SH-SY5Y cells of 0.3-100 μM of concentration.According to as a result, both EDA and BE increase at lower doses Add cell survival rate.However, EDA shows toxicity under 10 μM, and cause the notable suppression to SY5Y cells under higher doses System.On the other hand, BE all shows reliable protective effect up to 30 μM, and has a small amount of reduction under 100 μM of concentration, this As a result it is also consistent with data before, and has obtained duplicate acknowledgment in the following experiments.

Later, the present invention uses hydrogen peroxide (H₂O₂), copper ion (CuSO₄) and 42 oligomer of A β to detect EDA's and BE Neuroprotection.In the present invention, SH-SY5Y human neuroblastoma cells are used as external model, SY5Y cells are frequent It is studied with differentiation for the neuronal function in alzheimer disease and Parkinson's disease research, APP plasmid transfections is thin into this Born of the same parents system.The analysis of 3- (4,5- dimethylthiazole -2- bases) -2,5- diphenyltetrazolium bromides (MTT) is carried out to evaluate EDA and BE Protective effect to the poisonous substance of SY5Y cells.The analysis is based on living cells and the MTT of dissolving is converted to insoluble formazan Ability.Therefore, the amount of generated formazan and the quantity of living cells are directly proportional.Briefly, with poisonous substance and EDA or BE processing Afterwards, MTT is carried out to cell, then adds DMSO with Rong Xie formazans, OD values is measured by microplate reader.

In MTT experiment, by hydrogen peroxide (H₂O₂) for inducing the oxidative stress to SY5Y cells.H₂O₂It is a kind of Strong oxidizer is referred to as cytotoxicity and disorganization's agent, can under certain condition with various kinds of cell component reaction.By H₂O₂Caused by damage mainly by the mechanism of several keys, such as film lipid peroxidation and protein and DNA Hydroxylation (Watt and Proudfoot etc., 2004).The present invention determines H first₂O₂To the IC of SY5Y cells₅₀Concentration.Root According to this as a result, as cytotoxic H₂O₂Cell death under 50 μM induction of 50% selects the concentration for following realities It tests.In MTT analyses, both EDA and BE are shown with dosage-dependent manner to H₂O₂The protection of the cell death of induction is made With.Due to the antioxidant properties of EDA, the increase of survival rate is identified under 1,3 μM.However, decline is shown in the case where 10 μM of EDA, Prompt EDA toxic to cell at higher concentrations.The result is consistent with first screening MTT experiment.On the other hand, in 1,3 and Under 10 μM, compared with EDA, BE more strongly increases cell survival rate (P<0.05), it should be noted that do not shown under 30 μM Go out significant inhibition, shows that BE has the antioxidation improved and to the lower toxicity of cell.The present invention be also evaluated EDA, BE is to CuSO₄The cytotoxicity of induction and the protective effect of Apoptosis.Metal ion such as iron (Fe), zinc (Zn), copper (Cu) It plays a significant role in terms of biological process and neuronal function, the unbalance cytotoxicity and apparent of may causing of metal ion Genetic disease (Spinello and Bonsignore etc., 2016).According to as a result, in terms of inhibiting cytotoxicity and Apoptosis, BE is comprehensively more than EDA.In Apoptosis assay, PI positive cells are even higher than CuSO in 10 μM of groups of EDA₄Negative control group, Show in higher concentrations, EDA can't be protected promotes Apoptosis instead.This discovery is consistent with MTT data, shows that EDA exists It is toxic to SY5Y cells under higher than 10 μM.Later, the present invention handles cell using 42 oligomer of A β together with EDA or BE.For The highest toxicity of realization, by 4 DEG C of A β 42 peptides in DMEM culture mediums precincubation 48 hours to form oligomer.Due to invention People reported before EDA can combine and with A β direct interactions, therefore unquestionably EDA is inhibited and is lured by 42 oligomer of A β The cytotoxicity led.Obtain in this experiment similar as a result, compared with negative group, 3 μM of EDA increase cell survival rate To nearly 80%.BE also increases cell survival rate under same dose, and finds more significantly to increase in 10 μM of groups.

Thioflavin T (ThT) dye fluorescence is the common tool for measuring signal density, and the signal density instruction exists The formation and inhibition (Hudson and Ecroyd etc., 2009) of amyloid fibril in the presence of anti-amyloid binding compound. The present invention has detected the effect of the inhibition that EDA and BE form A β fibrinogens and the dissociation to pre-formed A β fibrinogens.This hair It is bright to find that concentration is enough to interfere and 42 relevant ThT fluorescence of threadiness A β down to 1 μM of EDA.Two analyses show EDA and BE The signal strength of ThT can be reduced with dosage-dependent manner, the shape of A β fibrinogens can effectively be inhibited by showing two kinds of compounds At and dissolve pre-formed A β fibrinogens.It is promising as a result, BE in each concentration level more than EDA.

Streptozotocin (STZ) is widely used as developing the experimental tool of sporadic AD animal models.Genetic modification at present Mouse model is not most suitably adapted for simulating the sporadic AD occurred in 95% patient.Nearest report is shown, uses drug The rodent of streptozotocin processing produces the most of typical brain group observed in sporadic alzheimer disease Variation is knitted, therefore will be a kind of useful model to prevent or reversing alzheimer disease disease with the drug candidate for evaluating exploitation Effect in patient on these tissue changes.In the laboratory of applicant, STZ is used to create small based on AMY transgenosis The novel animal model of mouse, with the AD preventive and therapeutic actions of the candidate alzheimer disease disease medicament of detection.

Acetylcholinesterase (AChE) is a kind of enzyme of hydrolysis of acetylcholine, inhibits AChE that can be effectively increased second in brain The level of phatidylcholine.Therefore developed some AChE inhibitor medicaments such as Tacrine, donepezil and Rivastigmine with Improve the cholinergic function in AD patient.AChE activity has become a key index for evaluating anti-AD drugs.In the present invention, Acetylcholine ester enzymatic determination is carried out to the SH-SY5Y cells handled with STZ, to detect whether EDA and BE have inhibition to make AChE With.Experiment display, compared with STZ groups, AchE activity is down to 35% by BE, and the inhibiting effect ratio EDA (45%) of BE is more By force.The result shows that both EDA and BE have strong effect to the inhibition of AChE, this can increase the level of acetylcholine in brain, To potentially increase the cholinergic function in AD patient.

In work before, inventor has found EDA rescue human neuroblastoma SH-SY5Y cells and primary nerve The neurite collapse induced by A beta oligomers in member shows the neurite collapse that EDA inhibits A beta induced.Therefore the present invention using A β and Both STZ are as poisonous substance to detect the protective effect of EDA and BE to them.As a result it shows and analyzes consistent knot with MTT, AchE By BE has lower toxicity, this is to be significantly improved to EDA, and EDA has the neuron of growth in vitro in higher concentrations Potential neurotoxicity.Based on previous studies as a result, EDA protects neuronal cell by multi-signal pathway, from work( Can be for Journal of Sex Research, the present invention draws a conclusion, and BE will be worked by similar mechanism, but with lower toxicity and higher Therapeutic effect.

In the Mechanism Study carried out before, from the animal tissue and cell pyrolysis liquid that EDA processing groups are collected, inventor It was found that EDA inhibits the expression of BACE1, sAPP β and CTF β, show that the interaction between EDA and A β can block A β to drive The positivity feed-forward loop (Jiao and Yao etc., 2015) that the amyloid protein that BACE1- is mediated generates.Due to oxidative stress and A β two Person is the positive modulators for driving BACE1 to raise by activating JNK and GSK3 β respectively, it is therefore more likely that EDA is to pass through decrease Oxidative stress and the GSK3 β phosphorylations of A β-induction and inhibit BACE1 expression and amyloid protein generate.

The present invention has also checked for the expression of p25/p35 in the cell pyrolysis liquid that EDA and BE is handled.P25 is the calcium of p35 Dependence catabolite, and be the main activator of cell cycle protein dependent kinase 5 (Cdk5).Many researchs confirmations, The unconventionality expression of Cdk5 can significantly raise Tau, and facilitate AD pathology (Patrick and Zukerberg etc., 1999).Therefore, The variation of p25/p35 ratios has become the important indicator (Giese, 2014) of a variety of diseases.In AD, p25 has been found to send out Important function is waved, and participates in neurodegeneration and BACE1 adjustings (Wen and Planel etc., 2008) (referring to Figure 25).

Figure 25 shows the model of p25 dysregulations in alzheimer disease.The expression for being frequently found p25 is increased in AD Add.This expression increases the increase for the Ca2+ oscillations conduction for being attributed to amyloid protein oligomer, eventually leads to p35 to p25's Cracking is raised.In addition, the overactivity of Cdk5 and be dislocatedly distributed be as p25 expression increase caused by, this leads to abnormal tau Peroxophosphoric acid, this is that neurofibrillary tangles are formed and neurodegenerative prerequisite.The present invention shows that p25/p35 ratios reduce, And with the BACE1 levels in the SH-SY5Y human neuroblastoma cells with EDA and BE processing with dosage-dependent manner phase It closes, the BACE1 levels and p25/p35 ratios of the cell reduce, and less p25 is generated from p35 albumen after showing processing.

Based on current discovery, the present invention passes through a variety of analyses, including functional study MTT, Apoptosis, axon growth With ThT analyses and Mechanism Study Western blotting, obtain promising as a result, i.e. new E DA derivatives BE has obviously The therapeutic effect of lower cytotoxicity and raising.

Description of the drawings

In order to more clearly describe technical scheme of the present invention, briefly introduce below in conjunction with attached drawing.It is clear that this A little attached drawings are only some specific implementation modes that the application records.The present invention includes but not limited to these attached drawings.

Fig. 1 is the spectrogram of the compounds of this invention 8；

Fig. 2 is the spectrogram of the compounds of this invention 9；

Fig. 3 is the spectrogram of the compounds of this invention 10；

Fig. 4 is the spectrogram of the compounds of this invention 12 (i.e. EDA derivative BE)；

Fig. 5 is the HPLC spectrograms of BE；

Fig. 6 shows to analyze the neurotoxicity of EDA and BE by MTT；

Fig. 7 shows H₂O₂To the cytotoxicity of SY5Y；

Fig. 8 shows EDA and BE rescues by H₂O₂The cytotoxicity of induction；

Fig. 9 shows CuSO₄Cytotoxicity；

Figure 10 shows EDA and BE rescues by CuSO₄The cytotoxicity of induction；

Figure 11 shows that EDA and BE protect cells from CuSO₄The Apoptosis of induction；

Figure 12 shows that EDA and BE saves the cytotoxicity induced by 42 oligomer of A β；

Figure 13 shows to analyze the effect that EDA and BE forms A β fibrinogens by ThT；

Figure 14 shows to analyze the effect that EDA and BE dissociates A β fibrinogens by ThT；

Figure 15 shows the dissociation to A β fibrinogens by western blot analysis EDA and BE；

Figure 16 shows the influence of EDA and BE to axon growth；

Figure 17 shows that EDA and BE restores to be damaged by axon growth beta induced A；

Figure 18 is photo, shows that the EDA and BE of various concentration restore to be damaged by axon growth beta induced A；

Figure 19 shows influence of the streptozotocin (STZ) of various concentration to axon growth；

Figure 20 shows that EDA and BE saves the neurite collapse induced by STZ；

Figure 21 shows the standard curve of AChE analyses；

Figure 22 shows the inhibiting effect of EDA and BE to AChE；

Figure 23 shows the influence of EDA and BE to BACE1 and downstream signal transduction；

Figure 24 shows the protective effect to cynapse by western blot analysis EDA and BE；

Figure 25 shows the model of p25 dysregulations in Alzheimer disease；And

Figure 26 is analyzed by MTT, is being with or without 0.5 μM of CuSO₄Under the conditions of, compare different compounds, including EDA, The mixture and myrrh sterone of BE, borneol, EDA and borneol, to the survival rate of SY5Y695 cells.**p<0.01.Red line is described There is 0.5 μM of CuSO₄When cell survival rate foundation level.

Specific implementation mode

For a further understanding of the present invention, technical scheme of the present invention is described below in conjunction with embodiment.These Description is merely illustrative the feature and advantage of edaravone derivative, the protection domain being not intended to limit the present invention.

Embodiment 1：The preparation of BE

The preparation process of BE is as follows：

The preparation of compound 8

Weigh Compound 7 (21.8g, 0.13mol) and compound 5 (15.5g, 0.1mol), DCC (41.2g, 0.2mol) and In DMAP (24.44g, 0.2mol) to anhydrous DCM (1L), reaction at room temperature is stayed overnight.Filtering removes not molten solid, then by filtrate Concentration, and through column purification (PE:EA=80:1) product Compound 8 (10.3g, yield 33.1%), is obtained.

¹H NMR (400MHz, DMSO)=8.86 (s, 1H), 8.43 (d, J=4Hz, 2H), 7.67 (s, 1H), 5.25 (m, 1H), 3.39 (s, 2H), 2.47-2.36 (m, 1H), 2.09 (ddd, J=4.4,9.2,13.1Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m, 2H), 1.07 (dd, J=3.5,13.7Hz, 1H), 0.90 (s, 3H), 0.85 (s, 6H).

The spectrogram of compound 8 is referring to Fig. 1.

The preparation of compound 9

300mL THF, Pd/C containing compound 8 (9.56g, 3.3mmol) and H₂Round-bottomed flask, stir 5 at room temperature Hour.Filtering removes not molten solid, then concentrates filtrate, obtains product Compound 9 (8.6g, yield 99.76%).

¹H NMR (400MHz, DMSO)=7.60 (s, 1H), 7.54 (d, J=4Hz, 2H), 7.44 (s, 1H), 7.26 (d, J =7.8Hz, 1H), 5.0 (m, 2H), 3.39 (s, 2H), 2.47-2.36 (m, 1H), 2.09 (ddd, J=4.4,9.2,13.1Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m, 2H), 1.07 (dd, J=3.5,13.7Hz, 1H), 0.90 (s, 3H), 0.85 (s,6H)。

The spectrogram of compound 9 is referring to Fig. 2.

The preparation of compound 10

NaNO is added at 0 DEG C into 50mL 6N HCl (aq) solution of compound 8 (3.36g, 12.3mmol)₂ (0.849g, 12.3mmol).The mixture is stirred 30 minutes at room temperature, the 10mL of SnCl2 (7g, 36.9mmol) is then added 12N HCl(aq).Continue stirring 1 hour at room temperature, then filters mixture.20mL water is added in solid, and water layer is adjusted to pH= 8.It is subsequently added into 20ml DCM.Mixture is filtered, filter cake is washed with DCM.Separating filtrate, concentration of organic layers obtain product chemical combination Object 10 (3.035g, yield 85.76%).

¹H NMR (400MHz, DMSO)=7.42 (s, 1H), 7.18 (d, J=4Hz, 2H), 6.99 (s, 1H), 4.98 (d, J =7.8Hz, 1H), 4.05 (m, 2H), 5.06 (td, J=2.8,9.5Hz, 1H), 3.39 (s, 2H), 2.47-2.36 (m, 1H), 2.09 (ddd, J=4.4,9.2,13.1Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m, 2H), 1.07 (dd, J= 3.5,13.7Hz,1H),0.90(s,3H),0.85(s,6H)。

The spectrogram of compound 10 is referring to Fig. 3.

The preparation of compound 12 (BE)

Weigh Compound 10 (3.693g, 12.81mmol) and compound 11 (1.667g, 12.81mmol) to anhydrous EtOH In (100mL).The reaction mixture is evaporated, and through column purification (PE:EtOAc=8:1), obtaining product Compound 12, (1g is received Rate 22%).

¹H NMR (400MHz, CHLOROFORM-d) 8.54-8.43 (m, 1H), 8.03 (dd, J=1.1,8.2Hz, 1H), 7.79 (d, J=7.8Hz, 1H), 7.48-7.35 (m, 1H), 5.06 (td, J=2.8,9.5Hz, 1H), 3.39 (s, 2H), 2.47- 2.36 (m, 1H), 2.09 (ddd, J=4.4,9.2,13.1Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m, 2H), 1.07 (dd, J=3.5,13.7Hz, 1H), 0.90 (s, 3H), 0.85 (s, 6H).

Calculated mass：354.1, measured value+1=355.2.

The spectrogram of compound 12 (i.e. BE) is referring to Fig. 4, and HPLC is referring to Fig. 5.

Embodiment 2：The neurotoxicity of EDA and BE

According to previous data, it has been proven that EDA has strong nerve to cortical neuron and SH-SY5Y695 cell lines Protective effect (Jiao and Yao etc., 2015).However in the result that do not deliver, inventor has found that EDA of the concentration more than 30 μM drops Low cell survival rate, it is toxic to show EDA at higher concentrations, it is necessary to is administered with the dosage range of safety.In this feelings Under condition, the present invention is based on EDA to have synthesized a kind of new compound BE, it is contemplated that this new compound is not only used more in anti-AD By force, and to human cell there is low toxicity.

In order to study the neuroprotection and neurotoxicity of EDA and BE, by human neuroblastoma cell system SH- SY5Y695 cells are with 1 × 10⁴The density of a cells/well is seeded in 96 orifice plates overnight.Second day, by cell and wide dosage model The EDA and BE enclosed is incubated 24 hours.MTT analysis shows that, EDA can't increase cell survival rate, but under 10 μM and high concentration Inhibit cell survival rate, and BE dramatically increases cell survival rate under 3,10 μM of concentration, and inhibition is shown since 30 μM.Root Compare according to this, drug candidate seemingly safer BE (referring to Fig. 6).

Fig. 6 shows that EDA and BE are dissolved in 100% ethyl alcohol, and vehicle-control contains 0.1% in DMEM cell culture mediums (final concentration) ethyl alcohol.EDA treatment groups are not observed significant change, but cell survival rate since 10 μM forward reduce. In BE groups, cell survival rate is dramatically increased since 3 μM, is increased again up to 10% at 10 μM.Cell toxicant is identified in 30 μM of BE Property, cell survival rate is drastically reduced at 100 μM.By cell inoculation in 96 orifice plates, n=6, P are measured every time<0.05, Dan Yin Plain variance analysis, error bar, SD, GraphPad Prism.

Embodiment 3：The cytotoxicity that EDA and BE rescue cells are induced from oxidant

As free radical scavenger, EDA has been shown to have stronger inoxidizability stress, and BE is the derivative of EDA Object, it is contemplated that both compounds will save the cytotoxicity that cell is induced from oxidative stress.Hydrogen peroxide is A kind of physiological compositions of living cells are continuously generated by a variety of different cellular pathways.According to previous research, cell inner stablity The H of state₂O₂, when concentration is higher than 1 μM, it is considered the oxidative stress (Antunes for causing induced growth to be stagnated with cell death And Cadenas, 2001).In common experimental model, in order to study cell oxidative stress reaction or antioxidant it is thin Born of the same parents protect, and the cell of culture is usually exposed to the hydrogen peroxide being added in culture medium different time point and concentration (Antunes and Cadenas, 2001).H₂O₂For detecting whether BE can save H₂O₂To the toxic effect of SY5Y cells, Also EDA groups are set up in the present invention.First, with the H of various concentration₂O₂It handles cell 24 hours, IC is obtained under 50 μM of concentration₅₀。 Therefore, for following experiment, 50 μM are used.In the MTT analyses that EDA and BE is combined, present invention discover that when with 3 and 10 μM When BE processing, cell survival rate dramatically increases, under 10 μM, cell survival rate ratio H₂O₂Group is high by 20%, and cell survival rate is at 30 μM It is not reduced under BE.Obtain consistent for EDA as a result, it shows the toxicity slight to cell under 10 and 30 μM, this Show that EDA inhibits cell growth in higher concentrations (referring to Fig. 7 and Fig. 8).

Fig. 7 display tests IC₅₀.By cell with 1 × 10⁴The density of a cells/well is seeded in 96 orifice plates overnight.Second It, by cell and 25-250 μM of H₂O₂It incubates, and is incubated 24 hours at 37 DEG C.Later, MTT analyses are carried out to cell, it will be thin Born of the same parents' survival rate is normalized relative to control group.It measures every time, n=6, P<0.05, one-way analysis of variance, error bar, SD, GraphPad Prism。

Fig. 8 is shown, by cell with or without EDA and BE and H₂O₂It handles together, EDA and BE are with 0.3,1 and 3 μM of concentration Addition.By cell with 1 × 10⁴The density of a cells/well is seeded in 96 orifice plates, n=6, P<0.05, one-way analysis of variance, Error bar, SD, GraphPad Prism.

Embodiment 4：The cytotoxicity and Apoptosis that EDA and BE rescue cells are induced from metal ion

It has been widely reported metal ion such as copper, iron and zinc and has played key effect in AD pathogenesis. Identify in the A β plaque block of AD brains be rich in metal ion, this cause neuronal viability integrally reduces (Spinello with Bonsignore etc., 2016).According to research before, due to higher neurotoxicity, heavy metal can promote disease to develop.Separately Outside, the change of certain metal ion levels may influence the degradation of A β enzymes in brain, increase A β and tau aggregations.Exotic metallic ion It is frequently used for neuronal cell toxicity detection and functional study.In the present invention, CuSO₄Nerve for detecting EDA and BE Protective effect.

By the CuSO for adding a certain concentration range into cell culture medium₄24 hours and detect CuSO₄Genotoxic potential, IC₅₀It is confirmed as 0.5 μM.Therefore, for following experiment, 0.5 μM is used in EDA and BE combination MTT analyses.According to MTT As a result, present invention discover that cell survival rate increases when with 1,3 μM of EDA and BE processing, BE groups significantly increase at 3 and 10 μM Add, significant change do not occur for 10,30 μM of processing groups, and shows cytotoxicity (referring to Fig. 9 in 30 μM of EDA And Figure 10).

Fig. 9 display detections IC₅₀.0.5 μM of selection is for EDA and BE MTT analyses.It is tested using identical scheme.With 0.25-1 μM of CuSO₄Incubate cells, and incubated 24 hours at 37 DEG C.Later, MTT analyses are carried out to cell, cell is deposited Motility rate is normalized relative to control group.It measures every time, n=6, P<0.05, one-way analysis of variance, error bar, SD, GraphPad Prism。

Figure 10 is shown, by cell with or without EDA and BE and CuSO₄It handles together, EDA and BE are dense with 0.3,1 and 3 μM Degree addition.By cell with 1 × 10⁴The density of a cells/well is seeded in 96 orifice plates, n=6, P<0.05, single factor test variance point Analysis, error bar, SD, GraphPad Prism.

The present invention also uses CuSO₄To detect whether EDA and BE can protect cells from the Apoptosis of metal inducement.Root According to as a result, CuSO₄1.6 times of Apoptosis, data is only induced to normalize both with respect to control group for SY5Y cells.It is handling In group, 1 and 3 μM of EDA inhibits Apoptosis, and increased under 10 μM, indicates that the EDA inducing cells of high concentration are dead It dies, this discovery is consistent with the MTT results that cell survival rate reduces when the concentration of EDA is higher than 10 μM.It is found that in BE groups It is promising as a result, its Apoptosis in a dose-dependent manner be suppressed (referring to Figure 11).

Figure 11 is shown, is collected SH-SY5Y cells from 24 orifice plates, is then carried out flow cytometer scanning, each sample to cell Quality control analysis 8 × 10³A cell.All detections are at least repeated 3 times.N=3, P<0.05, one-way analysis of variance, error bar, SD,GraphPad Prism。

Embodiment 5：EDA and BE rescue cells are from the cytotoxicity that is induced by 42 oligomer of A β

In order to compare the neuroprotection for the cytotoxicity that EDA and BE in vitro induce 42 monomers of A β, it is single to prepare A β 42 Body (referring to material and method).42 monomers of A β are dissolved in DMEM, are incubated at 4 DEG C 48 hours until processing.Respectively use EDA and BE with A β 42 handle the cell being seeded on 96 orifice plates together.Processing 24 hours then carries out MTT analyses to cell.The results show that EDA and BE both protects cells from the neurotoxicity induced by 3 μM of A β 42.10 μM of BE dramatically increases cell survival rate, And significant increase is not observed in the EDA groups of same concentrations (referring to Figure 12).

Figure 12 is shown, handles cell with or without EDA and BE and A β 42, EDA and BE are added with 0.3,1,3 and 10 μM of concentration Add.By cell with 1 × 10⁴The density of a cells/well is seeded in 96 orifice plates, n=6, P<0.05, one-way analysis of variance, accidentally Poor stick, SD, GraphPad Prism.

Embodiment 6：The effect that EDA and BE assembles A β fibrinogens and dissociates

Whether can inhibit A β fibril formations to study EDA and BE and promote the dissociation of pre-formed A β fibrinogens, it is right The synthesis A β 42 bought from American peptide carry out ThT analyses.In the present invention, 42 peptides of A β are dissolved in 1mg/ml In 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma), and it is divided in the Eppendorf of 200 μ L sizes with equal portions Guan Zhong.So that HFIP is evaporated in draught cupboard, the peptide hyaline membrane of generation is dried under vacuum overnight.A β 42 (2.5 μ g) is dissolved And it is incubated with the EDA or BE of 20 μM of final concentration and three kinds of various concentrations.Same amount of A β 42, without incubate, be used as pair According to.The sample retained in pipe is run into glue on Tris-Tricine gels, by western blot analysis, utilizes MOAB-2 antibody A β are visualized.

Using the method detection EDA and BE described before to the effect (Wang of 42 fibrinogens of A β formation and fibrinogen depolymerization With Wang etc., 2011).In order to inhibit A β fibrillations, by 25 μM of A β 42 in DMEM respectively with it is warm at 37 DEG C of EDA and BE It educates 7 days.For the pre-formed A β fibrinogens of depolymerization, pre-formed fibrinogen is then incubated other 3 at 37 DEG C with EDA and BE It.For incubating twice, A β 42 are individually incubated under the same conditions with EDA, BE, are used as compareing together with experiment.It incubates At the end of, by reaction solution be added to 3ml 5 μM of thioflavin T (ThT) solution (50mM phosphate buffers, pH 6.0, Sigma in), sample is measured.When ThT is bound to β-lamella aggregate structure of amyloid fibril, ThT's is glimmering Light emitting generates offset (LeVine, 1993).450nm excitations are monitored by Fluorescence Spectrometer PE-LS50B (PerkinElmer) The fluorescence intensity of wavelength and 482nm launch wavelengths, both excitation and transmitted bandwidth are 5nm.Reading is to be measured by time sweep Three values average value subtract by dissociate ThT contribution fluorescence result.Each experiment is triplicate.

In experiment before, inventor has confirmed that EDA inhibits A β fibrillations and promotes to dissociate.Based on these researchs As a result, the present invention speculates, as the derivative of EDA, BE should have stronger effect to these processes.In ThT fluorescence analyses In, present invention discover that when A beta monomers and EDA and BE are incubated, the formation of A β fibrinogens reduces in a dose-dependent manner.In BE 10 μM when identify and drastically reduce, there was only 10% compared to fluorescence intensity with A β controls, and EDA be 20% under same concentrations (referring to Figure 13).For dissociation test, when with pre-formed A β fibrinogens incubation, both EDA and BE are shown in a dose-dependent manner Writing reduces fluorescence intensity, and BE groups show higher effect than EDA under each concentration level (referring to Figure 14).

Western blotting also shows that similar as a result, BE almost dissociates all A β fibrinogens under 3 μM.As Figure 15 is clear What Chu showed, EDA and BE both inhibit the formation of A β fibrinogens.In the 3 μM of holes BE, only identified in the bottom in loading hole Very fuzzy band, this clearly shows that A β fibrinogens are dissociated and are displaced downwardly to separation gel, and in EDA groups, it is remained in 3 μM of holes See band.

Figure 15 shows that, for A β fibrinogen control wells, since albumen is oversized, band rests on the bottom in loading hole, It is not moved to gel；For the fresh A β samples not incubated in advance, oligomers are main A β, and in loading Faint fibrinogen is only observed in hole.

Embodiment 7：EDA and BE restores the axon growth damage in mouse cortex neuron

The aixs cylinder induced by A beta oligomers in EDA rescue human neuroblastoma's SH-SY5Y cells has been displayed before to collapse It falls into (Jiao and Yao etc., 2015).The present invention uses Primary cortical neurons as model, to compare the nerve between EDA and BE Protective effect.The present invention uses the EDA and BE of three kinds of various doses first, is used only whether the compound can promote with detection It is grown into dendron.Then, the present invention detects whether EDA and BE can restore to be induced by 42 oligomer of A β and streptozotocin (STZ) Axon growth damage.

Cell is detached from newborn C57WT first, is then seeded on the coverslip that PDL is coated in 24 orifice plates 2 days.Later, The EDA and BE of cell and various concentration are incubated, after incubating 24 hours, the cells are fixed and is carried out by MAP2 and DAPI antibody Cell surface dyes.The image that 5 longest aixs cylinders are intercepted from each visual field, analyzes the data in every group of 6 visuals field. Representative Neuronal images are obtained using Laser Scanning Confocal Microscope.Dendron length is measured by Image J softwares, data are with average value ± SD is indicated.All data are normalized both with respect to positive control.

According to as a result, for most of EDA groups, very small effect is only identified, in 1,3 μM of BE processing groups It is found that similar as a result, being dramatically increased under 10 μM, dendron length shows that BE promotes cortex neural significantly more than control group Axon growth in member (referring to Figure 16).

Figure 16 shows from newborn C57 selective separatings neuron, and it is small to be seeded in 24 orifice plates of PDL coatings 48 with low-density When so that preceding cell confluency degree is being incubated to 30-50% with EDA and BE.EDA and BE are added in culture medium, with cell temperature It educates 24 hours, it is then fixed.10 μM of BE dramatically increases dendron length, however, EDA groups do not identify variation, shows in curative effect And secure context, BE ratios EDA have higher clinical potentials.It is dyed by MAP2 and DAPI antibody on cell, passes through Image J Software measures dendron length, and data are indicated with average value ± SD.

When be seeded in 24 orifice plates on coverslip 48 it is small when after, 1 μM of A β 42 is added together with EDA or BE respectively, the present invention It was found that the neurite collapse in 24 hours in the beta induced cortical neurons of A.The average length of A β negative controls (NC) group is more right than positive According to (untreated to cell) small 50% (p<0.01) the axon growth damage that EDA and BE significantly restore A β-induction, is incubated at the same time Hinder (p<0.01).According to as a result, 1 μM of EDA cannot restore axon growth damage completely, and 1 μM of BE has as 3 μM of EDA Effect, and 3 and 10 μM BE processing group also by by the beta induced neurite collapses of A be restored to control group almost 80% (referring to Figure 17 And Figure 18).

Figure 17 and Figure 18 are shown, 24 holes are seeded in from newborn C57 selective separatings neuron, and with low-density (30-50%) 48 hours on the coverslip that PDL is coated in plate.EDA and BE are added in culture medium, it is then solid with cell culture 24 hours It is fixed.42 oligomer of A β induces the dendron length significantly reduced, and both EDA and BE all show the reliable action to neurite collapse. It is worth noting that, dendron length is increased to compared with negative control almost 80% by 10 μM of BE.

Streptozotocin (STZ) is a kind of glucose being derived from streptomyces chromogenes (Streptomyces achromogenes) The form (Kamat and Kalani etc., 2016) of amine-nitroso-urea compounds.STZ is widely used as experimental tool, and exploitation is used for The animal model of sporadic AD pathological researches.Before studies have shown that when not change the glucose level in brain but influence When serious neuronal function sub- is caused in diabetes dosage injection brain, STZ cause neurodegeneration and neuroinflamation (Chen and Liang etc., 2014).It has been reported and claims STZ injection of brain that working memory and spatial memory can be caused to damage, the insulin in brain Signal transduction, glucose metabolism damage and cholinergic are insufficient and oxidative stress increases and neuroinflamation (Kamat and Kalani etc., 2016).

In the present invention, if the work that STZ tests EDA and BE to (including axon growth) the effect of them for dry analysis With, AchE analysis and In vivo study.The present invention detects effects of the STZ to axon growth of various concentration first, as a result shows The length of dendron is reduced with dosage-dependent manner, and in 30 μM of processing groups, length is almost the half of control group.Based on the hair It is existing, it selects to handle cell together with 30 μM of STZ and EDA and BE.According to as a result, the EDA and BE of low dosage do not save aixs cylinder collapses It falls into, but identifies a small amount of increase in 10 μM of groups of BE (referring to Figure 20).

Axon growth is analyzed：With STZ 0,10,30,100uM processing C57WT neurons 24 hours (referring to Figure 19).

Figure 19 and Figure 20 shows that STZ inducing axonals collapse.Figure 20 is shown with 30 μM of STZ respectively together with 1,3,10 μM of BE Handle cell 24 hours.By on the cell inoculation coverslip that PDL is coated in 24 orifice plates, cell confluency degree is 30- before treatment 50%, P<0.05, one-way analysis of variance, error bar, SD, GraphPad Prism.

Embodiment 8：Acetylcholinesteraseanalysis analysis

Low-level acetylcholine is one of pathological marks of AD in brain.The study found that acetylcholine esterase inhibition (AChE) (a kind of enzyme of hydrolysis of acetylcholine) activity can be effectively increased acetylcholine in brain level (Murray and Faraoni etc., 2013).Therefore, some drugs have been developed for improving cholinergic function in AD patient, for example, he gram Woods (tacrine), donepezil (donezepil) and Rivastigmine (rivastigmine), all of which are AChE inhibitor (Chopra and Misra etc., 2011).

It measures AChE activity and has become a kind of useful tool of the anti-AD drugs of evaluation.In the present invention, using from Whether the acetylcholine ester enzyme detection kit of Abcam is inhibited to AChE to detect EDA and BE.With STZ respectively with EDA or BE handles SH-SY5Y cells together, and cell pyrolysis liquid is collected after 24 hours.It is tested according to the specification of manufacturer. As shown in figure 22, STZ individually induces the increase of AChE, but the activity of AChE is significantly inhibited with EDA and BE coprocessing groups, as a result Show that both EDA and BE have strong inhibiting effect to AChE.In addition, compared with EDA, BE shows stronger effect, knot Fruit meets expection.

Figure 21 is the standard curve of AChE analyses.Using 1000mU/ml acetylcholinesterase standard items, serial dilution at 300,100,30,10,3,1 and 0mU/ml acetylcholinesterase standard items.

By cell inoculation in 24 orifice plates, handled together with the EDA and BE with 30 μM of STZ with 3 μM.After 24 hours incubate, receive Collection cell pyrolysis liquid simultaneously carries out AChE analyses, and the OD values under 140nm are measured by microplate reader.N=3, P<0.05, single factor test variance Analysis, error bar, SD, GraphPad Prism.

Embodiment 9：Influences of the EDA and BE to BACE1 and downstream signal transduction in SY5Y695 cell lines

Since the production of neurotoxicity beta-amyloid protein (A β) peptide especially needs β-site amyloid protein precursor protein Lyases 1 (BACE1) therefore all thinks that always BACE1 in the pathology of alzheimer disease there is conclusive early stage to make With (Vassar, 2014).As a result, BACE1 has become the primary drug targets of AD treatments, and there is an urgent need to develop BACE1 Inhibitor.Present invention has demonstrated that EDA and BE can both reduce the expression of BACE1 in SY5Y695 cells.According to albumen Matter trace, BACE1 expression are reduced with dosage-dependent manner.

Autophagy is characterized as being a kind of lysosomal degradation process, and circulating cells waste simultaneously has from cytoplasm removing is potential The organelle and protein aggregate of the damage of poison.These cytoprotection functions have all played considerable work in many aspects With, the evidence from numerous pathogenicity results all illustrate autophagy dysregulation participate in neurodegeneration obstacle (Nixon and Yang, 2011).In alzheimer disease, still inducing self-body can be stimulated to swallow.However it has been found that autophagy object in lysosome Degradation be suppressed, this may cause autophagic vacuole largely accumulate in the aixs cylinder of the serious swelling of impacted neuron (Wolfe and Lee etc., 2013).The expression of this huge autophagy markers of LC3B in SY5Y cell lines before and after the processing is also evaluated in the present invention. Use the EDA and BE of various concentration.The results show that LC3B is relatively low in untreated samples, but in BE processing groups, LC3B is with agent Amount dependence mode increases, and a small amount of increase is also identified in 10 μM of groups of EDA.The result shows that the BE and EDA of high concentration promotes Autophagy process, this is very crucial to maintaining normal cell homeostasis, thereby promotes the cell under unfavorable conditions Survival.

Nearest evidence is related to calcium/calpain/Cdk5 kinsase signaling pathways in AD pathogenesis, shows that it may The BACE1 of A β 42- inductions is increased and is responsible for.A β 42 increase the cellular calcium in neuron, this has activated calpain to crack Cdk5 adjusts subunit p35 to p25 (27).The overexpression of p25 subunits causes cytoskeletal disruption, modified phoshorylation pattern change and god Through toxicity.It is reported that the raised mouse BACE1 levels of brain Cdk5/p25 activity and A β are generated and are increased.These statistics indicate that Between AD in Cdk5 dysregulations and the mankind and mouse exist association, Cdk5 inhibitor be used as potential AD therapeutic agents into Row exploitation.

Have extensive according to the report, albumen p25 contributes to the morbidity of alzheimer disease (AD).P25 is a kind of calcium of p35 Dependence catabolite, and be the main activator of cell cycle protein dependent kinase 5 (Cdk5), p25/p35 ratios Change (Giese, 2014) usually related to various diseases.In AD, p25 has involved neurodegeneration and BACE1 is adjusted, prompt pair The therapeutic Cdk5 of AD inhibits (Wen and Planel etc., 2008).The present invention shows that p25/p35 ratios reduce, and and SH-SY5Y BACE1 in human neuroblastoma cell is horizontal related.The cell with EDA and BE processing after, BACE1 levels and P25/p35 ratios reduce, and less p25 is generated from p35 albumen after prompting BE to handle (referring to Figure 23).

Figure 23 shows western blot analysis of the EDA and BE to SY5Y695 cytosiies.By cell inoculation in 6 orifice plates, It is handled 24 hours with the EDA and BE of various concentration, Western blotting is carried out to cell pyrolysis liquid.

In addition, the present invention also has detected is steeping relevant memebrane protein -2 with EDA and BE processing post-synapse vacuolar proteins (VAMP2) expression.Fully research shows that with the cognitive impairment and neopallium and edge in alzheimer patient Synaptic loss in system is closely related.Present invention discover that after with EDA and BE processing, the VAMP2 albumen in SY5Y695 cells It is raised, prompts EDA and BE that there is neuroprotection to cynapse (referring to Figure 24).

Figure 24 shows western blot analysis of the EDA and BE to the effect of the neurotoxicity induced by A β 42.By SY5Y695 Cell inoculation is used in combination A β 42 to be handled 24 hours with the EDA of various concentration or BE respectively, cell pyrolysis liquid is carried out in 6 orifice plates Western blotting.

Embodiment 10：Comparison of the different compounds to SY5Y695 cell survival rates

Referring to Figure 26, MTT to SY5Y695 cells analysis shows that, with 0.5 μM of CuSO₄Under incubation, cell survival rate (OD) it significantly reduces.10 μM of EDA can significantly inhibit CuSO₄Effect, and increase cell survival rate.1,3 and 10 μM of Edaravones Derivative BE dramatically increases cell survival rate, as shown in OD value increases.In contrast, borneol does not individually have shadow to cell survival rate It rings, also without further toxicity.On the other hand, the mixture of Edaravone and borneol, when same concentrations, to the survival rate of cell There is no remarkable effect, and myrrh sterone (guggulsterone) inhibits the survival rate of cell in a manner of dose-dependent.These realities It tests and shows compared with its parent molecules, BE has new synergistic effect to cell survival rate, this is wonderful.In view of this As a result clearly prove, BE have the function of it is beyong contemplation, the present invention remaining experiment in only by comparing Edaravone and BE Test other functions.

The explanation of above example is only intended to help to understand core of the invention thought.It should be pointed out that for this field Those of ordinary skill for, without departing from the principle of the present invention, if can also to the present invention EDA derivatives carry out Dry improvement and modification, but these improvement and modification are also fallen into the range of the claims in the present invention are claimed.

Bibliography

Antunes,F.and E.Cadenas(2001)."Cellular titration of apoptosis with steady state concentrations of H(2)O(2):submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state."Free Radic Biol Med 30(9):1008-1018.

Chen,Y.,Z.Liang,Z.Tian,J.Blanchard,C.L.Dai,S.Chalbot,K.Iqbal,F.Liu and C.X.Gong(2014)."Intracerebroventricular streptozotocin exacerbates Alzheimer-like changes of 3xTg-AD mice."Mol Neurobiol 49(1):547-562.

Chopra,K.,S.Misra and A.Kuhad(2011)."Current perspectives on pharmacotherapy of Alzheimer's disease."Expert Opin Pharmacother 12(3):335- 350.

Cummings,J.L.,T.Morstorf and K.Zhong(2014)."Alzheimer's disease drug- development pipeline:few candidates,frequent failures."Alzheimers Res Ther 6 (4):37.

E.S.Musiek,D.M.Holtzman,Three dimensions of the amyloid hypothesis: time,space and'wingmen',Nat Neurosci 18(2015)800-806.

Giese,K.P.(2014)."Generation of the Cdk5 activator p25 is a memory mechanism that is affected in early Alzheimer's disease."Front Mol Neurosci 7:36.

Guo,C.,L.Sun,X.Chen and D.Zhang(2013)."Oxidative stress,mitochondrial damage and neurodegenerative diseases."Neural Regen Res 8(21):2003-2014.

Holtzman,D.M.,J.C.Morris and A.M.Goate(2011)."Alzheimer's disease:the challenge of the second century."Sci Transl Med 3(77):77sr71.

Hudson,S.A.,H.Ecroyd,T.W.Kee and J.A.Carver(2009)."The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds."FEBS J 276(20):5960-5972.

Hyde,C.,J.Peters,M.Bond,G.Rogers,M.Hoyle,R.Anderson,M.Jeffreys, S.Davis,P.Thokala and T.Moxham(2013)."Evolution of the evidence on the effectiveness and cost-effectiveness of acetylcholinesterase inhibitors and memantine for Alzheimer's disease:systematic review and economic model."Age Ageing 42(1):14-20.

Jiao,S.S.,X.Q.Yao,Y.H.Liu,Q.H.Wang,F.Zeng,J.J.Lu,J.Liu,C.Zhu, L.L.Shen,C.H.Liu,Y.R.Wang,G.H.Zeng,A.Parikh,J.Chen,C.R.Liang,Y.Xiang,X.L.Bu, J.Deng,J.Li,J.Xu,Y.Q.Zeng,X.Xu,H.W.Xu,J.H.Zhong,H.D.Zhou,X.F.Zhou and Y.J.Wang(2015)."Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits."Proc Natl Acad Sci U S A 112(16):5225-5230.

Kamat,P.K.,A.Kalani,S.Rai,S.K.Tota,A.Kumar and A.S.Ahmad(2016)." Streptozotocin Intracerebroventricular-Induced Neurotoxicity and Brain Insulin Resistance:a Therapeutic Intervention for Treatment of Sporadic Alzheimer's Disease(sAD)-Like Pathology."Mol Neurobiol 53(7):4548- 4562.LeVine,H.,3rd(1993)."Thioflavine T interaction with synthetic Alzheimer' s disease beta-amyloid peptides:detection of amyloid aggregation in solution."Protein Sci 2(3):404-410.

Mangialasche,F.,A.Solomon,B.Winblad,P.Mecocci and M.Kivipelto(2010)." Alzheimer's disease:clinical trials and drug development."Lancet Neurol 9(7): 702-716.

McKim,J.M.,Jr.(2010)."Building a tiered approach to in vitro predictive toxicity screening:a focus on assays with in vivo relevance."Comb Chem High Throughput Screen 13(2):188-206.

M.S.Wolfe,The role of tau in neurodegenerative diseases and its potential as a therapeutic target,Scientifica(Cairo)2012(2012)796024.

Murray,A.P.,M.B.Faraoni,M.J.Castro,N.P.Alza and V.Cavallaro(2013)." Natural AChE Inhibitors from Plants and their Contribution to Alzheimer's Disease Therapy."Curr Neuropharmacol 11(4):388-413.

Nixon,R.A.and D.S.Yang(2011)."Autophagy failure in Alzheimer's disease--locating the primary defect."Neurobiol Dis 43(1):38-45.

Patrick,G.N.,L.Zukerberg,M.Nikolic,S.de la Monte,P.Dikkes and L.H.Tsai(1999)."Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration."Nature 402(6762):615-622.

R.T.Bartus,Evidence for a direct cholinergic involvement in the scopolamine-induced amnesia in monkeys:effects of concurrent administration of physostigmine and methylphenidate with scopolamine,Pharmacol Biochem Behav 9(1978)833-836.

Spinello,A.,R.Bonsignore,G.Barone,B.K.Keppler and A.Terenzi(2016)." Metal Ions and Metal Complexes in Alzheimer's Disease."Curr Pharm Des 22(26): 3996-4010.

T.Jonsson,J.K.Atwal,S.Steinberg,J.Snaedal,P.V.Jonsson,S.Bjornsson, H.Stefansson,P.Sulem,D.Gudbjartsson,J.Maloney,K.Hoyte,A.Gustafson,Y.Liu,Y.Lu, T.Bhangale,R.R.Graham,J.Huttenlocher,G.Bjornsdottir,O.A.Andreassen, E.G.Jonsson,A.Palotie,T.W.Behrens,O.T.Magnusson,A.Kong,U.Thorsteinsdottir, R.J.Watts,K.Stefansson,A mutation in APP protects against Alzheimer's disease and age-related cognitive decline,Nature 488(2012)96-99.

Vassar,R.(2014)."BACE1 inhibitor drugs in clinical trials for Alzheimer's disease."Alzheimers Res Ther 6(9):89.

Wang,Y.J.(2014)."Alzheimer disease:Lessons from immunotherapy for Alzheimer disease."Nat Rev Neurol 10(4):188-189.

Wang,Y.J.,X.Wang,J.J.Lu,Q.X.Li,C.Y.Gao,X.H.Liu,Y.Sun,M.Yang,Y.Lim, G.Evin,J.H.Zhong,C.Masters and X.F.Zhou(2011)."p75NTR regulates Abeta deposition by increasing Abeta production but inhibiting Abeta aggregation with its extracellular domain."J Neurosci 31(6):2292-2304.

Watanabe,T.,M.Tahara and S.Todo(2008)."The novel antioxidant edaravone:from bench to bedside."Cardiovasc Ther 26(2):101-114.

Watt,B.E.,A.T.Proudfoot and J.A.Vale(2004)."Hydrogen peroxide poisoning."Toxicol Rev 23(1):51-57.

Wen,Y.,E.Planel,M.Herman,H.Y.Figueroa,L.Wang,L.Liu,L.F.Lau,W.H.Yu and K.E.Duff(2008)."Interplay between cyclin-dependent kinase 5 and glycogen synthase kinase 3 beta mediated by neuregulin signaling leads to differential effects on tau phosphorylation and amyloid precursor protein processing."J Neurosci 28(10):2624-2632.

Wolfe,D.M.,J.H.Lee,A.Kumar,S.Lee,S.J.Orenstein and R.A.Nixon(2013)." Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification."Eur J Neurosci 37(12):1949-1961.

Zhao,Y.and B.Zhao(2013)."Oxidative stress and the pathogenesis of Alzheimer's disease."Oxid Med Cell Longev 2013:316523.

Sequence table

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Claims (7)

1. a kind of edaravone derivative, as shown in following formula (I)：

Wherein X is connector, is selected from-C (O) O- ,-C (O) S- ,-C (O) NH- ,-C (O)-,-NH- or-CH₂-。

2. the edaravone derivative of claim 1, wherein X are selected from-C (O) O-.

3. the edaravone derivative of claim 1, as shown in following formula (Ia)：

4. the edaravone derivative of any one of claim 1-3 is preparing the drug for treating oxidative stress relevant disease In application.

5. the application of claim 4, wherein the oxidative stress relevant disease is senile disease, cardiovascular disease, cancer, god Saturating, infectious diseases and immunological disease, skin disease, ENT dept.'s disease through degenerative disease, hepatopathy, tuberculosis, disease of digestive tract, renal failure and kidney Disease, gestation, lactation and childbirth relevant disease, motion sickness, andrological diseases, female infertility, arthropathy or chronic inflammation.

6. the application of claim 4 or 5, wherein the oxidative stress relevant disease is selected from Alzheimer disease, amyotrophic lateral sclerosis Lateral schlerosis, Parkinson's disease, ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease, AIDS and diabetes.

7. the application of claim 6, wherein the cerebrovascular disease is ischemic cerebrovascular disease or cerebral infarction.