WO2018133621A1

WO2018133621A1 - Edaravone derivative and use thereof

Info

Publication number: WO2018133621A1
Application number: PCT/CN2017/117914
Authority: WO
Inventors: 周乐乐; 周新富; 单正南
Original assignee: 周意
Priority date: 2017-01-18
Filing date: 2017-12-22
Publication date: 2018-07-26
Also published as: CN108314652A

Abstract

An edaravone derivative and a use thereof. As compared with edaravone, the derivative has enhanced treatment effects and significantly reduced cytotoxicity.

Description

Edaravone derivative and its use

Technical field

The invention belongs to the field of pharmacy. In particular, the present invention relates to edaravone derivatives, and the use of the derivatives to treat human oxidative stress-related diseases, particularly neurodegenerative diseases or cerebrovascular diseases.

Background technique

Free radicals are a common result of aerobic metabolism in normal cells. The body's built-in antioxidant system plays a decisive role in preventing damage caused by free radicals. However, the imbalanced defense mechanisms of antioxidants, the excessive production of free radicals or the introduction of living systems from the environment lead to serious obstacles to neurodegeneration. Neuronal degeneration of the neurodegenerative disease suffers from loss of function or senses. In addition to several environmental or genetic factors, oxidative stress (OS) causes free radicals to attack nerve cells, and the contribution to neurodegeneration is catastrophic. Although oxygen is essential to life, metabolic imbalances and excessive reactive oxygen species (ROS) production are major causes of chronic and degenerative diseases such as aging and other degenerative diseases worldwide, such as human Alzheimer's disease ( AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), atherosclerosis, cancer, diabetes, rheumatoid arthritis (RA), ischemia Post-infusion injury, myocardial infarction, cardiovascular disease, chronic inflammation, stroke and septic shock. According to the World Health Organization (WHO), the world's top 10 lethal diseases are ischemic heart disease, stroke, chronic obstructive pulmonary disease (COPD), lower respiratory tract infection, tracheal/bronchial/lung cancer, HIV/AIDS, diarrhea. , diabetes, high blood pressure and tuberculosis (first 1-10 descending order). There are 28.8 million patients worldwide who died from these diseases in 2012 (mainly caused by the top 4 fatal diseases). Ischemic heart disease accounts for nearly 25.7% of all deaths, stroke accounts for nearly 23.3%, and COPD and lower respiratory tract infections account for nearly 10.7% of total deaths, respectively. Various studies have shown that oxidative stress is the leading cause of most diseases in the top 10 lethal diseases, especially ischemic heart disease, stroke, COPD, HIV/AIDS and diabetes. The global market for diseases related to oxidative stress is over $200 billion.

Since oxidative stress is fundamental to many chronic, degenerative, and lethal diseases, this suggests that antioxidants are urgently needed to help control ROS levels in the body. Therefore, as a strong and potential free radical scavenger or antioxidant, edaravone (hereinafter abbreviated as "EDA") is known to play an important role in reducing ROS levels and reducing oxidative stress.

EDA is also known as MCI-186, chemical name: 3-methyl-1-phenyl-2-pyrazoline-5-one, molecular formula C ₁₀ H ₁₀ N ₂ O, molecular weight 174.19, structural formula:

EDA is a potent synthetic oxygen free radical scavenger with antioxidant effects to reduce oxidative stress and inhibit lipid peroxidation via non-enzymatic lipid peroxidation and lipoxygenase pathways. In addition, EDA also shows beneficial effects in inflammation, matrix metalloproteinases, nitric oxide production, and apoptosis. Mitsubishi Tanabe Pharm Corp. (Osaka, Japan) first developed EDA and was listed as Radicut in 2001, becoming the world's first neurovascular protective drug. In 2001, the Japanese Ministry of Public Health and Welfare approved the drug for the treatment of patients with cerebral infarction and acute ischemic stroke (AIS). Since then, EDA has not only been used to treat AIS, but also to treat ROS-related diseases such as cardiovascular disease and stroke. Although EDA is a commonly prescribed drug in Japan, India, and China, it has not been approved in the United States and other Western countries, mainly due to its toxicity in the liver and kidneys and the lack of clinical research to support the beneficial effects of EDA.

EDA is designed as a phenol-like compound, and phenol is one of the functional groups of all phenolic antioxidants, wherein the phenolic antioxidant consists of a hydroxyl group (-OH) attached to an aromatic ring and is responsible for antioxidant properties. By donating a hydrogen ion to the free radical, which in turn becomes a group, the phenol quenches the free radical. However, electrons on the phenol are stabilized by localization of the aromatic ring via resonance electrons, and the activity is lowered. However, due to toxicity and corrosivity, phenols are not suitable for pharmaceutical use even though they exhibit free radical scavenging effects and have proven to be potential antioxidants.

On the other hand, in particular, aromatic hydroxyl groups are present, and EDA is expected to have the same activity as phenols and exhibits similar antioxidant and free radical scavenging effects. EDA can be classified into three different tautomeric forms, namely amine, keto and enol. The aromatic hydroxy group is produced via keto-enol tautomerization. However, in contrast to phenols, EDA has no toxic effects of phenols, which is one of the reasons why EDA is more advantageous than phenols.

EDA has a pKa of 7.0, so about 50% of EDA is ionized under physiological pH conditions and exists in an anionic form. The anionic form of EDA is also a more reactive form that readily reacts with ROS in the brain to produce an antioxidant effect. The advantage of EDA over other free radical scavengers (such as idebenone, baicalein and catechol) is that it easily crosses the blood-brain barrier because EDA is a low molecular weight lipophilic molecule in water and lipids. There are solubility characteristics in all. Therefore, it can easily cross the blood-brain barrier and reach the target in the brain. The ratio of plasma levels of EDA to cerebrospinal fluid levels is expected to be 50-65%. These characteristics may be due to the neuroprotective effects of EDA in cerebral infarctions, while other antioxidants do not.

Alzheimer's disease (AD) is the most common type of dementia in people over the age of 60. Thousands of resources have been invested in AD treatment research for decades. On the one hand, there has been some progress in the treatment of symptoms, but there have also been several failures in the development of disease-modifying treatment (Mangialasche and Solomon et al., 2010). Only a few drugs are currently approved for AD treatment, including four cholinesterase inhibitors (tacrine, donepezil, lissamine, galantamine) and N-methyl-D-aspartate ( NMDA) Receptor AD antagonist (memantine) (Hyde and Peters et al, 2013). There have been many failures in the development of AD drugs, and both small molecules and immunotherapy have failed to show drug/placebo differences or have unacceptable toxicity (Cummings and Morstorf et al., 2014). These failures lead to potential defects in the understanding of the pathogenesis of AD and potential pitfalls in the development of drug candidates. To date, many clinical and experimental studies are still underway, but one must acknowledge that it is unlikely that a single therapeutic drug for Alzheimer's disease will be discovered and that the methods and concepts of drug development need to be reconsidered.

The current understanding of the pathogenesis of AD includes three main types, and the "choline energy hypothesis" is the first theory used to explain AD. This hypothesis has become the basic theory guiding drug development for the treatment of mild to moderate dementia. According to this theory, the function of cholinergic neurons and the loss of cholinergic activity in the Meynert basal ganglia (NBM) are usually observed in AD brains. Studies in humans and non-human primates have shown that acetylcholine is in learning and memory. Play a key role. These studies show that if scopolamine blocks central cholinergic activity, young subjects will exhibit a typical AD marker of memory deficit, and this injury can be treated with a choline agonist physostigmine Salvation (Bartus, 1978). The second mainstream theory is the "amyloid hypothesis," which considers soluble A[beta] fragments to be synaptotoxic and is responsible for plaque formation, tau hyperphosphorylation, and subsequent mutations in signaling pathways in AD pathology ( Musiek and Holtzman, 2015). According to the key role proposed by the "amyloid hypothesis", various therapeutic agents have been developed to inhibit enzymes such as beta-secretase or by strategies aimed at clearing existing plaques in the brain, such as anti-A beta immunotherapy. Reduce the production of Aβ (Jonsson and Atwal et al., 2012). The Tau hypothesis is another popular theory of the AD mechanism. Studies have shown that tau oligomers are neurotoxic, many clinical symptoms are closely related to tau pathology, and tau hyperphosphorylation constitutes a common final pathway for many other dementias, so tau theory is becoming more and more accepted. In this theory, abnormal signals cause tau hyperphosphorylation through a variety of pathways such as the fyn kinase pathway. Tau modification results in its oligomerization and NFT, causing abnormal intracellular trafficking, microtubule-based cytoskeletal collapse, subsequent neuronal death, and ultimately progressive neuronal degeneration (Wolfe, 2012).

Oxidative stress is one of the hallmarks of Alzheimer's disease (AD), and free radicals induced by oxidative stress are linked to overproduction of amyloid β (Aβ) protein and hyperphosphorylation of tau. Abnormal accumulation of amyloid beta protein and sigma protein, in turn, facilitates imbalance of oxygen and ultimately leads to a vicious circle that promotes the overall development of AD disease (Zhao and Zhao, 2013).

Oxidative stress has been shown to be widely associated with a variety of major pathological processes of AD, such as A[beta] aggregation and deposition, tau phosphorylation, synaptic collapse, and metal instability. Overproduction of reactive oxygen species (ROS) may be induced by mitochondrial disability or abnormal accumulation of transition metals, perhaps by accumulation of Aβ The synergistic effect of hyperphosphorylation with tau protein leads to oxidative stress (Guo and Sun et al., 2013). On the other hand, oxidative stress is responsible for mediating neurotoxicity induced by Aβ and tau proteins, which promotes Aβ production and tau phosphorylation and polymerization, thereby further enhancing a variety of neurotoxic events, including ROS production, thereby forming a benefit for AD. The vicious circle of development (Zhao and Zhao, 2013).

Therefore, oxidative stress has become the most critical risk factor for the development and progression of AD. Inhibition or prevention of ROS can delay the onset of AD or slow the progression of AD through a variety of mechanisms. Based on the above theory, prevention or treatment of AD by antioxidants is a promising approach because antioxidants are capable of targeting a number of different signaling pathways involved in AD pathology.

Despite significant advances in the understanding of the pathogenesis of AD over the past few decades, there is still a lack of effective therapies available for AD management (Holtzman and Morris et al., 2011). Numerous drugs have also been developed to target Aβ, tau, or oxidative stress through immunotherapy or secretase inhibitors, but in clinical trials, cognitive impairment has been slightly improved, and both have failed (Wang, 2014). Given these examples, drugs that target a single biomarker or pathway will not work for this complex disease. Earlier research by the inventors showed that multi-targeting strategies for AD treatment would be a novel approach.

EDA potently removes reactive oxygen species (ROS) and inhibits pro-inflammatory responses following cerebral ischemia (Watanabe and Tahara et al., 2008). Clinical studies have shown that EDA can effectively improve post-ischemic inflammation, which may lead to cerebral edema and cerebral infarction. In addition to its anti-stroke effect, EDA has been shown to prevent oxidative damage to a variety of extracerebral organs. Recent animal studies have demonstrated that EDA can potently alleviate multiple key markers in AD pathology. Administration of EDA by injection significantly reduced Aβ aggregation, Tau hyperphosphorylation, and improved cognitive performance in animals (Jiao and Yao et al., 2015). Several mechanisms of EDA in the treatment of AD have been revealed, EDA is capable of binding and interacting directly with A[beta] proteins, thereby modulating downstream signaling and inhibiting A[beta]-induced AD pathology cascades. In addition, structural advantages allow EDA to break through the blood-brain barrier and transport it to the brain (Watanabe and Tahara et al., 2008).

However, the safety of the drug is worrying. Unpublished experimental data showed that EDA induced cytotoxicity at high concentrations, and MTT results showed that cell viability decreased when SY5Y was treated at a concentration of 30 μM or higher. Apoptosis analysis also showed similar results. There is therefore a need to synthesize new EDA-based compounds that are expected to improve the therapeutic effects of EDA while having much lower toxicity.

Summary of invention

The present invention prepares and characterizes the EDA derivative BE, and experimentally tests EDA and its derivative BE, and systematically compares the cell survival, apoptosis, Aβ aggregation, BACE1 expression and neurite outgrowth of the two compounds. The impact, with encouraging results: BE showed a stronger role in inhibiting AD disease and antioxidant stress. By comparing with EDA, it was also confirmed that BE is less cytotoxic, so BE would be a clinically better candidate.

The present invention compares the therapeutic effects of EDA and BE, and surprisingly finds that BE not only retains, but also exceeds EDA in many therapeutic aspects, including Aβ-induced neurotoxicity, BACE1 expression, Aβ aggregation, and acetylcholinesterase activity, while It also meets safety requirements, which is unexpected.

The present invention provides an edaravone derivative which is represented by the following formula (I):

Wherein X is a linker selected from the group consisting of -C(O)O-, -C(O)S-, -C(O)NH-, -C(O)-, -NH- or -CH ₂ -, preferably -C (O)O-. The linkers are known to each other as isosteres, which have similar physical and chemical properties and which produce substantially similar biological activities.

According to the invention, the edaravone derivative is represented by the following formula (Ia):

Hereinafter referred to as BE.

The invention also relates to the treatment of diseases and pharmaceutical uses of the following edaravone derivatives:

The present invention provides edaravone derivatives for use in the treatment of diseases associated with oxidative stress.

The present invention provides the use of an edaravone derivative for the preparation of a medicament for the treatment of an oxidative stress-related disease.

According to the present invention, the oxidative stress-related diseases include senile/aging diseases (arthritis, diabetes, osteoarthritis, cataract, macular degeneration, prostate disease), cardiovascular diseases (atherosclerosis, heart failure, heart) Disease, renal failure, hypertension, stroke, poor blood circulation, cholesterol and plaque formation, reperfusion injury), cancer (prostate cancer, breast cancer, lung cancer, colon cancer, bladder cancer, uterine cancer, ovarian cancer, lymphoma) , skin cancer, stomach cancer, liver cancer and other wasting diseases), neurodegenerative diseases (Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), spirit Schizophrenia, dementia, Huntington's disease), liver disease (toxic hepatitis, viral hepatitis (A, B and C), chronic hepatitis), lung disease (asthmatic emphysema, pneumonia, (acute and chronic) bronchitis, cystic fiber , pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), digestive tract disease (inflammatory bowel disease, ulcerative enteritis, Crohn's disease, gastritis, stomach cancer, stomach ulcer, pancreas Inflammation), renal failure and kidney penetration (renal failure, kidney , oxidative stress caused by dialysis), infectious diseases and immune diseases (viral infections of HIV and AIDS (AIDS), toxic hepatitis and cirrhosis, viral hepatitis (A, B and C), herpes, colds, bacteria Infection, chronic fatigue syndrome, autoimmune dysfunction), skin disease (psoriasis, eczema, systemic lupus erythematosus (SLE), vasculitis, polymyositis, mycosis fungoides, scleroderma, pemphigoid, allergies Dermatitis, contact dermatitis, seborrheic dermatitis, herpes-like dermatitis, polymeric acne, common acne, UV-irradiated skin damage), ENT disease (white Internal cataract, glaucoma, macular degeneration, hearing loss, ear infection, sinusitis, periodontal disease, nasal, oral and throat (upper respiratory tract) diseases, pregnancy, breastfeeding and childbirth related diseases (pre-eclampsia, eclampsia, high Blood pressure, diabetes), sports disease (training syndrome and related oxidative stress), male diseases (prostatic hyperplasia, prostate cancer, alopecia, male infertility), female infertility, arthropathy and chronic inflammation, Especially selected from AD, amyotrophic lateral sclerosis (ALS), PD, ischemic heart disease, cerebrovascular disease (including ischemic cerebrovascular disease, cerebral infarction/stroke/stroke), COPD, HIV/AIDS And diabetes.

The materials and methods used in the present invention are as follows:

(1) Animals

All procedures relating to animals of the present invention were approved by the South Australian Pathology Animal Ethics Committee (Adelaide, Australia) and the University of South Australia Animal Welfare Committee (Adelaide, Australia). All procedures are performed in accordance with the guidelines of the Australian National Health Research Council. All animals were maintained on a 12 hour day/night cycle and were free to access food and water. C57 newborn mice were raised by Reid animal staff.

(2) Cell culture

The human neuroblastoma SH-SY5Y695-app cell line of the invention was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS), penicillin (50 IU/ml), streptomycin (50 μg/ml) and L-glutamine (2 mM). , Life Technology, VIC, Australia). All cells were maintained in a humidified 37 ° C incubator supplemented with 95% O ₂ and 5% CO ₂ .

C57 wild-type newborn mice were anesthetized on ice water slurry from day 0 (P0) to day 1 (P1) after birth and decapitated. After removal of the meninges, the cortex of both hemispheres of each mouse was separated from the rest of the brain and transferred to a 15 ml centrifuge tube containing 2-3 ml of cold PBS, and centrifuged at 1,500 rpm for 3 minutes at 4 °C. The PBS was removed and the tissue was digested with 4 ml of 0.2% trypsin containing 0.1% DNase I (NEB, Ipswich, MA, USA) for 20 minutes at 37 ° C and shaken every 5 minutes. FBS (final concentration 15%) was added to determine trypsinization. Centrifugal tube protection Hold the quiescent for 5 minutes and allow the digestive tissue to settle by gravity. After all tissue debris had completely settled, the supernatant was transferred to a new tube and centrifuged at 2,000 rpm for 2 minutes at 4 °C. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in 1 ml of cortical neuronal medium (containing B27 additive (2%), L-glutamine (2 mM) and penicillin/streptomycin (100 IU/ml). In basal basal medium (Life Technology, VIC, Australia) and β-mercaptoethanol (0.1 mM).

Then neuronal cell counts ^{(Bio-rad, TC20 TM Automated} Cell Counter), and then inoculated into tissue culture plate. For Western blotting or for immunocytochemical analysis, cortical cells (at a concentration of 2×10 ⁶ ) were seeded into poly-D-lysine- and laminin-coated 6-well plates, and (to 2.5 ×10 ⁴ concentration) poly-D-lysine- and layer-mucin-coated coverslips (Thermo Fisher Scientific, inoculated into 4- or 24-well plates (Thermo Fisher Scientific, Rockford, IL, USA)) Rockford, IL, USA).

(3) Aβ oligomers

Aβ42 was purchased from American Peptide at 1 mg/bottle. The Aβ42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) at a concentration of 1 mg/ml, and aliquots were added to an Eppendorf microcentrifuge tube and air dried in a ventilated kitchen. Store for at least 2 hours and then -80 ° C for long periods of time. During the experiment, aliquots of A[beta]42 were dissolved in DMEM and incubated at 4[deg.] C. for 24-48 hours to generate toxic oligomer A[beta]42 monomers.

(4) MTT analysis

To investigate the antagonism of EDA and BE on the in vitro toxicity of CuSO ₄ , H ₂ O ₂ and Aβ42, a human neuroblastoma SH-SY5Y695-app cell line was used. The cells were separated from the T75 flask by 0.25% trypsin and then seeded into 96-well plates with 0.5 μM CuSO ₄ , 50 μM H ₂ O ₂ , 1 μM Aβ42 monomer, 1, 3 and 10 μM EDA and BE, respectively. After 19 hours of incubation, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was analyzed and 25 μl of MTT was added to each well (Sigma- Aldrich, USA, 5 mg/ml in PBS) and incubated for 2 hours. 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the insoluble purple formazan to form a colored solution. Optical density (OD) readings were taken at 570 nm wavelength using a multi-well scanning spectrophotometer.

(5) Thioflavin T (ThT) fluorescence analysis

To test the inhibitory effect of EDA and BE on A[beta] aggregation, 1 [mu]M A[beta]42 monomer (American Peptide) was first incubated with a range of concentrations of EDA and BE in DMEM for 10 days at 37[deg.] C. and then incubated with 5 [mu]M ThT solution. To understand poly-preformed A[beta] fibrils, 1 [mu]M A[beta]42 monomer was pre-formed at 37[deg.] C., followed by incubation of the filaments with EDA and BE for another 3 days. At the end of the incubation, the reaction solution was added to 3 ml of 5 M ThT solution (50 mM phosphate buffer, pH 6.0, Sigma), and the sample was measured. When ThT binds to the β-sheet aggregate structure of amyloid fibrils, the fluorescence emission shift of ThT. The fluorescence intensity was monitored by an illuminating spectrophotometer PE-LS50B (PerkinElmer) at an excitation wavelength of 450 nm and an emission wavelength of 482 nm with an excitation and emission bandwidth of 5 nm. Each experiment was performed in triplicate and repeated three times.

(6) Analysis of neurite outgrowth

The neurite outgrowth analysis was performed on cortical neurons isolated from C57 mice. Cortical neurons were isolated from the brain of day 1 C57 mice and digested by 0.25% trypsin. The cerebral cortex and hippocampal cells were cultured in a neural basal medium containing 2% B27 (Invitrogen, #17504001) and 2% FBS (Invitrogen). The cells were seeded in 6-well plates coated with 50 μg/ml poly-D-lysine (PDL) and incubated at 37 ° C for 48 hours. Neurons were incubated with 1 [mu]M A[beta] oligomers with different concentrations of EDA/BE or without EDA/BE for 24 hours. The cells were then fixed in 4% formalin and incubated overnight with rabbit anti-MAP-2 antibody at 4 °C. Thereafter, it was incubated with secondary anti-rabbit Alexa488 antibody and DAPI for 1 hour. Images were taken from the 5 longest neurites of each field of Olympus (CX40) microscope and the data for each of the six fields of view was analyzed. Representative neuron images were obtained using a Zeiss confocal (LSM710) microscope. The length of the dendrites was measured by Image J software, and the data was expressed as mean ± SD.

(7) Western blot analysis of inhibition of Aβ aggregation by EDA and BE

1 μM Aβ42 monomer was incubated with a series of concentrations of EDA and BE in DMEM for 10 days at 37 ° C, and the resulting solution was mixed with 5× loading buffer without reducing agent, followed by electrophoresis. Non-incubated A[beta] samples (A[beta] oligomers) were used as controls. All samples were loaded onto a 15% (wt/vol) acrylamide gradient gel and the fully gelled A[beta] was transferred to a nitrocellulose membrane. The blot was probed with MOAB-2 mouse monoclonal antibody (Biosensis M-1586-100) specific for amyloid beta peptide (Aβ40/42).

(8) Analysis of apoptosis

SH-SY5Y cells were collected from 24-well plates and washed in PBS, the supernatant was discarded, and the cells were fixed with cold 70% ethanol for 30 minutes. The cell pellet was washed twice with PBS, centrifuged at 1500 rpm, and cell loss was carefully observed when the supernatant was discarded. 200 μl of propidium iodide (PI, 50 μg/ml) was added for 10 minutes, and the cells were washed twice with PBS to avoid false positive staining. The cells were then scanned by flow cytometry (Beckman Coulter Gallios, Detmold Family Cytometry Facility), and 2 x 10 ⁴ cells were analyzed per sample tube. All tests were repeated at least 3 times.

(9) Acetylcholinesterase analysis

AchE analysis was performed using an AchE kit (Abeam, ab138871). Cells were treated in 24-well plates at different concentrations of EDA and BE. Cell lysates were collected after 20 hours of treatment. The experiment was performed according to the manufacturer's instructions.

(10) Real-time PCR

BACE1 mRNA expression levels were determined according to standard procedures. Total RNA was extracted from SY5Y695 cells treated with different concentrations of EDA and BE using the RNAeasy mini kit (Qiagen, Doncaster, Vic, Australia). The concentration and purity of the RNA was determined using a NanoDrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). 500 ng total RNA The first strand cDNA was generated using Superscript III First Strand Synthesis System (Life Technology, VIC, Australia). PCR using GoTaq Green Master Mix (Promega, Madison, WI, USA), forward primer of BACE1 (SEQ ID NO: 5): 5'-ACCGACGAAG AGTCGGAGGAG-3' (SEQ ID NO: 1), reverse primer: 5 '-CACAATGCTCTTGTCATAG-3' (SEQ ID NO: 2). Taking the housekeeping gene GAPDH as a standard, its forward primer: 5'-AACATCATCCCTGCATCCAC-3' (SEQ ID NO: 3), reverse primer: 5'-TTGAAGTCTCAGGAGACAAC-3' (SEQ ID NO: 4). GAPDH (SEQ ID NO: 6) was used to calibrate BACE1 mRNA expression.

The discovery and development of a new drug requires more than $1 billion and can consume up to 10 years of research. The drug development process can be divided into four distinct phases: early detection, late detection, preclinical, and clinical. The inventors screened the anti-AD activity of several new compounds, and this early discovery study will pave the way for future drug development. Following the screening process, the present invention identified the compound BE, which is a novel derivative of EDA based on chemical structure. The inventors believe that BE has a stronger antioxidant effect and less cytotoxicity. The goal of the present invention was to investigate whether BE can provide enhanced protection against A[beta]-induced AD pathology and to detect cytotoxicity of this compound compared to its source EDA. In order to fully understand the therapeutic effects and cytotoxicity of BE, the present invention has devised a comprehensive research program. The present invention uses cell culture, Western blot, flow cytometry, and ThT, AchE activity assays to evaluate different concentrations of EDA and BE, and collects data from functional and mechanistic study experiments.

When a new compound with biological properties of interest enters the program, it will undergo extensive testing designed to handle and solve many complex problems. During this procedure, new chemical entities may be discontinued for a number of reasons, two of which are efficacy and toxicity. Unexpected side effects and toxicity observed during preclinical animal safety studies and clinical trials of new drug candidates are among the biggest challenges facing the pharmaceutical industry today. To address these issues, in vitro cytotoxicity assays have been used as tools for decades to understand the hypothesis-driven problem with drug toxicity mechanisms (McKim, 2010). The present invention first detects and compares the toxicity of EDA and BE by MTT assay, and treats SH-SY5Y cells with a wide range of concentrations of 0.3-100 μM EDA and BE. According to the results, both EDA and BE increased cell viability at lower doses. However, EDA showed toxicity at 10 μM and caused significant inhibition of SY5Y cells at higher doses. On the other hand, BE showed reliable protection up to 30 μM and a small decrease at a concentration of 100 μM, which was consistent with the previous data and was confirmed repeatedly in the following experiments.

Thereafter, the present invention employs hydrogen peroxide (H ₂ O ₂ ), copper ions (CuSO ₄ ), and Aβ42 oligomers to detect the neuroprotective effects of EDA and BE. In the present invention, SH-SY5Y human neuroblastoma cells are used as an in vitro model, and SY5Y cells are frequently used for neuronal function and differentiation studies in Alzheimer's disease and Parkinson's disease research, transfecting APP plasmids into This cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis was performed to evaluate the protective effects of EDA and BE on the toxicants of SY5Y cells. This analysis is based on the ability of living cells to convert dissolved MTT to insoluble formazan. Therefore, the amount of formazan produced is proportional to the number of living cells. Briefly, after treatment with toxicants and EDA or BE, the cells were subjected to MTT, followed by the addition of DMSO to dissolve the formazan, and the OD value was measured by a microplate reader.

In the MTT assay, hydrogen peroxide (H ₂ O ₂ ) was used to induce oxidative stress on SY5Y cells. H ₂ O ₂ is a strong oxidant called cytotoxicity and tissue disrupting agent that can react with a variety of cellular components under certain conditions. The damage caused by H ₂ O ₂ is mainly through several key mechanisms such as lipid peroxidation of membranes and hydroxylation of proteins and DNA (Watt and Proudfoot et al., 2004). The present invention first determines the concentration of ₅₀ pair of IC H _₂ O ₂ SY5Y cells. According to the results, H ₂ O ₂ as a cytotoxin induced 50% of cell death at 50 μM, and this concentration was selected for the following experiment. In the MTT assay, both EDA and BE showed protection against H ₂ O ₂ -induced cell death in a dose-dependent manner. Due to the antioxidant properties of EDA, an increase in survival was identified at I, 3 μM. However, a decrease was shown at 10 [mu]M EDA, suggesting that EDA is toxic to cells at higher concentrations. This result is consistent with the first screening MTT experiment. On the other hand, at 1, 3 and 10 μM, BE increased cell viability more strongly than EDA (P < 0.05), notably showing no significant inhibition at 30 μM, indicating that BE has improved Antioxidant and less toxic to cells. The present invention also evaluated EDA, BE protective effect of toxicity and apoptosis of cells induced CuSO _4. Metal ions such as iron (Fe), zinc (Zn), and copper (Cu) play important roles in biological processes and neuronal functions. Metal ion imbalances can lead to cytotoxicity and epigenetic diseases (Spinello and Bonsignore et al., 2016). ). According to the results, BE completely exceeded EDA in terms of inhibition of cytotoxicity and apoptosis. In the apoptosis analysis, PI-positive cells in the EDA 10 μM group were even higher than the CuSO ₄ negative control group, indicating that EDA did not protect but promoted apoptosis at high concentrations. This finding is consistent with MTT data, indicating that EDA is toxic to SY5Y cells above 10 [mu]M. Thereafter, the present invention treats cells with EDA or BE using A[beta]42 oligomers. To achieve the highest toxicity, the A[beta]42 peptide was pre-incubated in DMEM medium for 48 hours at 4[deg.] C. to form oligomers. Since the inventors previously reported that EDA can bind and interact directly with A[beta], there is no doubt that EDA inhibits the cytotoxicity induced by A[beta]42 oligomers. Similar results were obtained in this experiment, with 3 μM EDA increasing cell viability to nearly 80% compared to the negative group. BE also increased cell viability at the same dose and a more significant increase was found in the 10 [mu]M group.

Thioflavin T (ThT) dye fluorescence is a commonly used tool for measuring signal density indicating the formation and inhibition of amyloid fibrils in the presence of anti-amyloid compounds (Hudson and Ecroyd et al, 2009). The present invention detects the inhibition of A[beta] fibril formation by EDA and BE and the dissociation of pre-formed A[beta] fibrils. The present inventors have found that EDA at concentrations as low as 1 [mu]M is sufficient to interfere with ThT fluorescence associated with fibrous A[beta]42. Both analyses showed that EDA and BE reduced the signal intensity of ThT in a dose-dependent manner, indicating that both compounds are effective in inhibiting the formation of Aβ fibrils and dissolving preformed Aβ fibrils. The promising result is that BE exceeds EDA at each concentration level.

Streptozotocin (STZ) is widely used as an experimental tool for the development of animal models of sporadic AD. The current genetically modified mouse model is not optimal for simulating sporadic AD in 95% of patients. Recent reports have shown that rodents treated with the drug streptozotocin produce most of the typical brain tissue changes observed in sporadic Alzheimer's disease and will therefore be a useful model to evaluate development. The role of drug candidates in preventing or reversing these tissue changes in patients with Alzheimer's disease. In the Applicant's laboratory, STZ was used to create a novel animal model based on AMY transgenic mice to detect AD preventive and therapeutic effects of drugs for candidate Alzheimer's disease.

Acetylcholinesterase (AChE) is an enzyme that catalyzes the hydrolysis of acetylcholine, inhibiting AChE It can effectively increase the level of acetylcholine in the brain. Therefore, some AChE inhibitor drugs such as tacrine, donepezil and lissamine have been developed to improve cholinergic function in AD patients. AChE activity has become a key indicator for evaluating anti-AD drugs. In the present invention, SHZ-treated SH-SY5Y cells are subjected to acetylcholinesterase assay to detect whether EDA and BE have an inhibitory effect on AChE. The experiment showed that BE reduced AchE activity to 35% compared to the STZ group, and the inhibition of BE was stronger than that of EDA (45%). The results indicate that both EDA and BE have a strong effect on the inhibition of AChE, which can increase the level of acetylcholine in the brain, thereby potentially increasing cholinergic function in AD patients.

In previous work, the inventors found that EDA rescued axon collapse induced by Aβ oligomers in human neuroblastoma SH-SY5Y cells and primary neurons, indicating that EDA inhibits Aβ-induced axonal collapse. The present invention therefore uses both A[beta] and STZ as toxicants to detect their protective effects by EDA and BE. The results show consistent results with MTT, AchE analysis, BE has lower toxicity, which is a significant improvement for EDA, while EDA is potentially neurotoxic to neurons grown in vitro at high concentrations. Based on previous research results, EDA protects neuronal cells through a variety of signaling pathways. From a functional study, the present invention concludes that BE will act through a similar mechanism, but with lower toxicity and higher treatment effect.

In a previous mechanism study, in the animal tissues and cell lysates collected from the EDA-treated group, the inventors found that EDA inhibits the expression of BACE1, sAPPβ, and CTFβ, indicating that the interaction between EDA and Aβ can block Aβ-driven. BACE1-mediated Positive Feedforward Loop of Amyloid Gene Production (Jiao and Yao et al., 2015). Since both oxidative stress and Aβ are positive regulators that drive up-regulation of BACE1 by activating JNK and GSK3β, respectively, it is likely that EDA inhibits BACE1 expression by attenuating oxidative stress and Aβ-induced GSK3β phosphorylation. Amyloid production.

The present invention also examined the expression of β25/p35 in EDA and BE treated cell lysates. P25 is a calcium-dependent degradation product of p35 and is a major activator of cyclin-dependent kinase 5 (Cdk5). Many studies have confirmed that abnormal expression of Cdk5 can be revealed Tau is up-regulated and contributes to AD pathology (Patrick and Zukerberg et al., 1999). Therefore, changes in the p25/p35 ratio have become important indicators of many diseases (Giese, 2014). In AD, p25 has been shown to play an important role and is involved in neurodegeneration and BACE1 regulation (Wen and Planel et al., 2008) (see Figure 25).

Figure 25 shows a model of p25 dysregulation in Alzheimer's disease. It is often found that the expression of p25 is increased in AD. This increase in expression is attributed to an increase in calcium signaling by amyloid oligomers, which ultimately leads to upregulation of p35 to p25 cleavage. Furthermore, over-activation and misplacement of Cdk5 is caused by increased expression of p25, which results in abnormal tau hyperphosphorylation, which is a prerequisite for neurofibrillary tangles and neurodegeneration. The present invention shows a decrease in the p25/p35 ratio and is associated with BACE1 levels in SH-SY5Y human neuroblastoma cells treated with EDA and BE in a dose-dependent manner, with decreased BACE1 levels and p25/p35 ratios, This indicated that less p25 was produced from the p35 protein after treatment.

Based on current findings, the present invention has obtained promising results by various analyses, including functional studies of MTT, apoptosis, axonal growth and ThT analysis, and mechanism studies of Western blots, ie, the novel EDA derivative BE has significant Lower cytotoxicity and improved therapeutic effect.

DRAWINGS

In order to more clearly describe the technical solution of the present invention, a brief description will be made below with reference to the accompanying drawings. It is apparent that these drawings are only some of the specific embodiments described herein. The invention includes, but is not limited to, the drawings.

Figure 1 is a spectrum of the compound 8 of the present invention;

Figure 2 is a spectrum of the compound 9 of the present invention;

Figure 3 is a spectrum of the compound 10 of the present invention;

Figure 4 is a spectrum of Compound 12 (i.e., EDA derivative BE) of the present invention;

Figure 5 is an HPLC chromatogram of BE;

Figure 6 shows the analysis of neurotoxicity of EDA and BE by MTT;

Figure 7 shows the cytotoxicity of H ₂ O ₂ to SY5Y;

Figure 8 shows that EDA and BE rescue cytotoxicity induced by H ₂ O ₂ ;

Figure 9 shows the cytotoxicity of CuSO ₄ ;

Figure 10 shows that EDA and BE rescue cytotoxicity induced by CuSO ₄ ;

11 illustrates BE EDA and protect cells from apoptosis CuSO ₄ induction;

Figure 12 shows that EDA and BE rescue cytotoxicity induced by Aβ42 oligomers;

Figure 13 shows the effect of EDA and BE on the formation of Aβ fibrils by ThT analysis;

Figure 14 shows the effect of EDA and BE on the dissociation of Aβ fibrils by ThT analysis;

Figure 15 shows the dissociation of Aβ fibrils by EDA and BE by Western blot analysis;

Figure 16 shows the effect of EDA and BE on axon growth;

Figure 17 shows that EDA and BE restore axonal growth damage induced by Aβ;

Figure 18 is a photograph showing that different concentrations of EDA and BE restore axonal growth damage induced by Aβ;

Figure 19 shows the effect of different concentrations of streptozotocin (STZ) on axon growth;

Figure 20 shows that EDA and BE rescue axon collapse induced by STZ;

Figure 21 shows a standard curve of the AChE analysis;

Figure 22 shows the inhibition of AChE by EDA and BE;

Figure 23 shows the effect of EDA and BE on BACE1 and downstream signaling;

Figure 24 shows the protection of synapses by EDA and BE by Western blot analysis;

Figure 25 shows a model of p25 dysregulation in Alzheimer's disease;

26 through FIG MTT assay, at 0.5μM CuSO ₄ with or without the conditions, compare the different compounds, including a mixture of EDA, BE, borneol, EDA and myrrh borneol and aldosterone, SY5Y695 viability of the cells. **p<0.01. The red line depicts the basal level of cell viability at 0.5 μM CuSO ₄ .

detailed description

In order to further understand the present invention, the technical solutions of the present invention will be described below in conjunction with the embodiments. These descriptions are merely illustrative of the features and advantages of the edaravone derivatives and are not intended to limit the scope of the invention.

Example 1: Preparation of BE

The preparation process of BE is as follows:

Preparation of Compound 8

Compound 7 (21.8 g, 0.13 mol) and compound 5 (15.5 g, 0.1 mol), DCC (41.2 g, 0.2 mol) and DMAP (24.44 g, 0.2 mol) were weighed into anhydrous DCM (1 L) at room temperature The reaction was overnight. The undissolved solid was removed by filtration, and the filtrate was concentrated and purified by column (PE: EA = 80:1) to give product compound 8 (10.3 g, yield: 33.1%).

^{1 H 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, 13.1 Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m,, 2H), 1.07 (dd, J = 3.5, 13.7 Hz, 1H), 0.90 (s, 3H), 0.85 (s, 6H).

See Figure 1 for the spectrum of Compound 8.

Preparation of compound 9

A round bottom flask containing Compound 8 (9.56 g, 3.3 mmol) in 300 mL of THF, Pd/C and H ₂ was stirred at room temperature for 5 hours. The undissolved solid was removed by filtration, and the filtrate was concentrated to give the product compound 9 (8.6 g, yield: 99.76%).

^{1 H 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, 13.1 Hz, 1H), 1.79-1.62 (m, 2H), 1.43-1.20 (m, 2H), 1.07 (dd, J = 3.5, 13.7 Hz, 1H), 0.90 (s, 3H), 0.85 (s, 6H).

See Figure 2 for the spectrum of Compound 9.

Preparation of Compound 10

Solution of compound 8 (3.36g, 12.3mmol) in 50mL 6N HCl (aq) was added NaNO ₂ (0.849g, 12.3mmol) at 0 ℃. The mixture was stirred at room temperature for 30 minutes, then SnCl 2 (7 g, 36.9 mmol), 10 mL of 12N HCl (aq). Stirring was continued for 1 hour at room temperature and then the mixture was filtered. The solid was added to 20 mL of water and the aqueous layer was adjusted to pH = 8. Then 20 ml of DCM was added. The mixture was filtered and the filter cake was washed with DCM. The filtrate was separated, and the organic layer was concentrated to afford product compound 10 (3.035 g, yield: 85.76%).

^{1 H 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, 9.5 Hz, 1H), 3.39 (s, 2H), 2.47-2.36 (m, 1H), 2.09 (ddd, J = 4.4, 9.2, 13.1 Hz, 1H), 1.79 -1.62 (m, 2H), 1.43-1.20 (m,, 2H), 1.07 (dd, J = 3.5, 13.7 Hz, 1H), 0.90 (s, 3H), 0.85 (s, 6H).

See Figure 3 for the spectrum of Compound 10.

Preparation of Compound 12 (BE)

Compound 10 (3.693 g, 12.81 mmol) and Compound 11 (1.667 g, 12.81 mmol) to dry EtOH (100 mL). The reaction mixture was evaporated and purified EtOAcqqqqqqqq

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

Mass calculated value: 354.1, measured value +1 = 355.2.

See Figure 4 for the spectrum of Compound 12 (i.e., BE) and Figure 5 for HPLC.

Example 2: Neurotoxicity of EDA and BE

According to earlier data, EDA has been shown to have strong neuroprotective effects on cortical neurons and SH-SY5Y695 cell lines (Jiao and Yao et al., 2015). However, in unpublished results, the inventors found that EDA at concentrations greater than 30 [mu]M reduced cell viability, indicating that EDA is toxic at higher concentrations and must be administered in a safe dosage range. In this case, the present invention synthesizes a novel compound BE based on EDA, and it is expected that this new compound is not only stronger in anti-AD action. It is also less toxic to human cells.

To investigate the neuroprotective effects and neurotoxicity of EDA and BE, human neuroblastoma cell line SH-SY5Y695 cells were seeded at a density of 1 x 10 ⁴ cells/well in 96-well plates overnight. The next day, cells were incubated with a wide range of EDA and BE for 24 hours. MTT analysis showed that EDA did not increase cell viability, but inhibited cell viability at 10 μM and high concentrations, while BE significantly increased cell viability at 3 and 10 μM concentrations and showed inhibition from 30 μM. Based on this comparison, BE appears to be a safer drug candidate (see Figure 6).

Figure 6 shows that EDA and BE were dissolved in 100% ethanol and the vehicle control contained 0.1% (final concentration) ethanol in DMEM cell culture medium. No significant changes were observed in the EDA-treated group, but cell viability decreased from 10 μM forward. In the BE group, cell viability increased significantly from 3 μM and increased by 10% at 10 μM. At 30μM BE Given cytotoxicity, cell viability decreased dramatically at 100 μM. Cells were seeded in 96-well plates for n=6, P < 0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 3: EDA and BE rescue cells from oxidant-induced cytotoxicity

As a free radical scavenger, EDA has been shown to have a strong antioxidant stress, and BE is a derivative of EDA, which is expected to rescue cells from oxidative stress-induced cytotoxicity. Hydrogen peroxide is a physiological component of living cells that is continuously produced by a variety of different cellular pathways. According to earlier studies, intracellular stable H ₂ O ₂ , at concentrations above 1 μM, is thought to cause oxidative stress that induces growth arrest and cell death (Antunes and Cadenas, 2001). In a common experimental model, in order to study the oxidative stress response of cells or the cytoprotection of antioxidants, cultured cells are typically exposed to different time points and concentrations of hydrogen peroxide added to the medium (Antunes and Cadenas, 2001). H ₂ O _{2 was} used to detect whether BE can rescue the toxic effects of H ₂ O ₂ on SY5Y cells, and an EDA group was also established in the present invention. First, H _₂ O ₂ for 24 h with different concentrations of cells at a concentration of 50μM to obtain IC _50. Therefore, for the following experiment, 50 μM was used. In the MTT assay of the combination of EDA and BE, the present inventors found that when treated with 3 and 10 μM of BE, the cell viability was significantly increased, and at 10 μM, the cell viability was 20% higher than that of the H ₂ O ₂ group, and the cell survival rate was It does not decrease under 30μM BE. Consistent results were obtained for EDA, which showed slight toxicity to cells at 10 and 30 [mu]M, indicating that EDA inhibited cell growth at high concentrations (see Figures 7 and 8).

Figure 7 shows the test IC ₅₀ . The cells were seeded at a density of 1 x 10 ⁴ cells/well in 96-well plates overnight. The next day, the cells were incubated with 25-250 μM H ₂ O ₂ and incubated at 37 ° C for 24 hours. Thereafter, the cells were subjected to MTT analysis, and the cell survival rate was normalized with respect to the control group. For each determination, n=6, P<0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Figure 8 shows, the cells were treated with or without EDA and BE and with H _₂ O ₂ treatment, EDA and 0.3, and BE added at a concentration of 3μM. Cells were seeded at a density of 1 x 10 ⁴ cells/well in 96-well plates, n=6, P < 0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 4: EDA and BE rescue cells from metal ion-induced cytotoxicity and apoptosis

It has been widely reported that metal ions such as copper, iron and zinc play a key role in the pathogenesis of AD. It has been identified that A[beta] plaques in AD brain are enriched with metal ions, which results in overall reduced neuronal viability (Spinello and Bonsignore et al, 2016). According to previous studies, heavy metals can promote disease progression due to high neurotoxicity. In addition, changes in the levels of certain metal ions in the brain may affect Aβ enzyme degradation and increase Aβ and tau aggregation. Exogenous metal ions are often used for neuronal cytotoxicity testing and functional studies. In the present invention, CuSO _{4 is} used to detect the neuroprotective effects of EDA and BE.

The potential toxicity of CuSO ₄ was determined by adding a concentration range of CuSO ₄ to the cell culture medium for 24 hours, and the IC ₅₀ was determined to be 0.5 μM. Therefore, for the following experiments, 0.5 μM was used in the EDT and BE combined MTT assay. Based on the results of MTT, the present inventors found that cell viability increased when treated with 1, 3 μM EDA and BE, and significantly increased in the BE group at 3 and 10 μM, and did not change significantly in the 10, 30 μM treatment group, but Cytotoxicity was shown in 30 μM EDA (see Figures 9 and 10).

Figure 9 shows detection IC _50. 0.5 μM was selected for EDA and BE MTT analysis. Experiment with the same protocol. The cells were incubated with 0.25-1 μM CuSO ₄ and incubated at 37 ° C for 24 hours. Thereafter, the cells were subjected to MTT analysis, and the cell survival rate was normalized with respect to the control group. For each determination, n=6, P<0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Figure 10 shows the EDA and BE cells with CuSO _4, together with or without treatment, and EDA was added at a concentration of 0.3, 1 and BE of 3μM. Cells at a density of 1 × 10 ⁴ cells / well were seeded in 96 well plates, n = 6, P <0.05 , ANOVA, error bars, SD, GraphPad Prism.

The present invention is further used to detect whether EDA CuSO ₄ and BE can protect cells from apoptosis induced by metal. According to the results, CuSO ₄ induced only 1.6-fold apoptosis in SY5Y cells, and the data were normalized to the control group. In the treatment group, 1 and 3 μM of EDA inhibited apoptosis, but increased at 10 μM, indicating that high concentrations of EDA induced cell death, a finding that decreased cell viability when EDA concentrations were above 10 μM. The MTT results are consistent. Promising results were found in the BE group, and apoptosis was inhibited in a dose-dependent manner (see Figure 11).

Figure 11 shows that SH-SY5Y cells were collected from 24-well plates, and then the cells were subjected to flow cytometry scanning, and 8 x 10 ³ cells were analyzed per sample tube. All tests were repeated at least 3 times. n=3, P<0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 5: EDA and BE rescue cells from cytotoxicity induced by A[beta]42 oligomers

In order to compare the neuroprotective effects of EDA and BE on Aβ42 monomer-induced cytotoxicity in vitro, Aβ42 monomer was prepared (see Materials and Methods). The A?42 monomer was dissolved in DMEM and incubated at 4 °C for 48 hours until treatment. Cells seeded in 96-well plates were treated with EDA and BE with Aβ42, respectively. After 24 hours of treatment, the cells were subsequently subjected to MTT assay. The results showed that both EDA and BE protected the cells from neurotoxicity induced by 3 μM Aβ42. 10 μM BE significantly increased cell viability, while no significant increase was observed in the same concentration of EDA groups (see Figure 12).

Figure 12 shows that cells were treated with or without EDA and BE and A[beta]42, EDA and BE were added at concentrations of 0.3, 1, 3 and 10 [mu]M. Cells were seeded at a density of 1 x 10 ⁴ cells/well in 96-well plates, n=6, P<0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 6: Effect of EDA and BE on aggregation and dissociation of Aβ fibrils

To investigate whether EDA and BE inhibit Aβ fibril formation and promote preformation The dissociation of Aβ fibrils was subjected to ThT analysis of synthetic Aβ42 purchased from American peptide. In the present invention, the Aβ42 peptide is dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) at 1 mg/ml, and is placed in an aliquot at 200 μL. The size of the Eppendorf tube. The HFIP was evaporated in a fume hood and the resulting peptide transparent film was dried under vacuum overnight. Aβ42 (2.5 μg) was dissolved and incubated with three different concentrations of EDA or BE at a final concentration of 20 μM. The same amount of A[beta]42 was used as a control without incubation. The samples retained in the tubes were run on a Tris-Tricine gel and visualized by Western blot analysis using AMOAB-2 antibody.

The effects of EDA and BE on Aβ42 fibril formation and fibril depolymerization were examined using the previously described method (Wang and Wang et al., 2011). To inhibit A[beta] fibrillation, 25 [mu]M A[beta]42 was incubated in DMEM with EDA and BE at 37[deg.] C. for 7 days, respectively. To understand the poly preformed A[beta] fibrils, the preformed fibrils were then incubated with EDA and BE for an additional 3 days at 37 °C. For both incubations, A[beta]42 was incubated alone with EDA and BE under the same conditions and used as a control together with the experiment. At the end of the incubation, the reaction solution was added to 3 ml of a 5 μM Thioflavin T (ThT) solution (50 mM phosphate buffer, pH 6.0, Sigma), and the sample was measured. When ThT binds to the β-sheet agglomerate structure of amyloid fibrils, the fluorescence emission of ThT shifts (LeVine, 1993). The fluorescence intensity at 450 nm excitation wavelength and 482 nm emission wavelength was monitored by a fluorescence spectrometer PE-LS50B (PerkinElmer) with both excitation and emission bandwidths of 5 nm. The reading is the result of subtracting the fluorescence contributed by free ThT from the average of the three values determined by the time sweep. Each experiment was performed in triplicate.

In previous experiments, the inventors have demonstrated that EDA inhibits A[beta] fibrillation and promotes dissociation. Based on these findings, the present invention speculates that BE, as a derivative of EDA, should have a stronger effect on these processes. In ThT fluorescence analysis, the present inventors found that when A[beta] monomers were incubated with EDA and BE, the formation of A[beta] fibrils decreased in a dose dependent manner. A sharp decrease was identified at BE 10 [mu]M, with a fluorescence intensity of only 10% compared to the A[beta] control and an EDA of 20% at the same concentration (see Figure 13). For the dissociation test, both EDA and BE significantly reduced fluorescence in a dose-dependent manner when incubated with pre-formed Aβ fibrils. For light intensity, the BE group showed a higher effect than EDA at each concentration level (see Figure 14).

Western blots also showed similar results, with BE dissociating almost all A[beta] fibrils at 3 [mu]M. As clearly shown in Figure 15, both EDA and BE inhibit the formation of A[beta] fibrils. In the BE 3 μM well, only very blurred bands were identified at the bottom of the well, which clearly showed that the Aβ fibrils were dissociated and moved down to the separation gel, while in the EDA group, still visible in the 3 μM well. Bands.

Figure 15 shows that for Aβ fibril control wells, due to excessive protein size, the band stays at the bottom of the well and does not move to the gel; for fresh Aβ samples not previously incubated, soluble oligomers are predominant Aβ, and only weak fibrils were observed in the wells.

Example 7: EDA and BE restore axonal growth damage in mouse cortical neurons

EDA has previously been shown to rescue axonal collapse induced by A[beta] oligomers in human neuroblastoma SH-SY5Y cells (Jiao and Yao et al, 2015). The present invention uses primary cortical neurons as a model to compare the neuroprotective effects between EDA and BE. The present invention first uses three different doses of EDA and BE to detect whether the use of only the compound promotes dendritic growth. The present invention then detects whether EDA and BE are capable of restoring axonal growth damage induced by A[beta]42 oligomers and streptozotocin (STZ).

Cells were first isolated from fresh C57 WT and then plated on PDL coated coverslips in 24-well plates for 2 days. Thereafter, the cells were incubated with different concentrations of EDA and BE, and after 24 hours of incubation, the cells were fixed and subjected to cell surface staining by MAP2 and DAPI antibodies. The images of the five longest axons were taken from each field of view and the data for each of the six fields of view was analyzed. Representative neuron images were obtained using confocal microscopy. The dendritic length was measured by Image J software and the data was expressed as mean ± SD. All data were normalized to the positive control.

Based on the results, only a very small effect was identified for most EDA groups, and similar results were found in the 1,3 μM BE treatment group, with a significant increase at 10 μM, and the dendritic length significantly exceeded the control group, indicating BE. Promotes axonal growth in cortical neurons (see Figure 16).

Figure 16 shows the isolation of cortical neurons from neonatal C57, seeded in PDL-coated 24-well plates at low density for 48 hours, allowing cell confluence to 30-50% prior to incubation with EDA and BE. EDA and BE were added to the medium, incubated with the cells for 24 hours, and then fixed. 10 μM of BE significantly increased dendritic length. However, no changes were identified in the EDA group, indicating that BE has higher clinical potential than EDA in terms of efficacy and safety. Cells were stained with MAP2 and DAPI antibodies and dendritic lengths were measured by Image J software and the data are expressed as mean ± SD.

After 48 hours of inoculation on a coverslip in a 24-well plate, 1 μM Aβ42 was added with EDA or BE, respectively, and the present invention found that Aβ induced axonal collapse in cortical neurons within 24 hours. The mean length of the Aβ negative control (NC) group was 50% smaller than the positive control (untreated cells) (p<0.01), while the simultaneous incubation of EDA and BE significantly restored Aβ-induced axonal growth injury (p<0.01). ). According to the results, 1 μM EDA could not completely restore axonal growth damage, while 1 μM BE was as effective as 3 μM EDA, and the 3 and 10 μM BE treatment groups also restored Aβ-induced axonal collapse to almost 80% of the control group (see Figure 17). And Figure 18).

Figures 17 and 18 show that cortical neurons were isolated from neonatal C57 and plated on PDL-coated coverslips in 24-well plates at low density (30-50%) for 48 hours. EDA and BE were added to the medium, incubated with the cells for 24 hours, and then fixed. Aβ42 oligomers induced a significantly reduced dendritic length, and both EDA and BE showed a reliable effect on axonal collapse. It is worth noting that 10 μM BE increased the dendritic length to almost 80% compared to the negative control.

Streptozotocin (STZ) is a kind of Streptomyces originating from Streptomyces Achromogenes) forms of glucosamine-nitrosourea compounds (Kamat and Kalani et al., 2016). STZ is widely used as an experimental tool to develop animal models for sporadic AD pathology studies. Previous studies have shown that STZ causes neurodegeneration and neuroinflammation when injected into the brain with sub-diabetic doses that do not alter glucose levels in the brain but affect severe neuronal function (Chen and Liang et al., 2014). It has been reported that STZ brain injection can cause working memory and spatial memory damage, insulin signaling in the brain, glucose metabolism damage and cholinergic deficit, as well as increased oxidative stress and neuroinflammation (Kamat and Kalani et al., 2016).

In the present invention, STZ is used for several analyses to test the effects of EDA and BE on their efficacy (including axonal growth), AchE analysis, and in vivo studies. The present invention first detects the effect of different concentrations of STZ on axon growth, and the results show that the length of dendrites is reduced in a dose-dependent manner, and in the 30 μM treatment group, the length is almost half of that of the control group. Based on this finding, 30 μM of STZ was selected to treat cells with EDA and BE. According to the results, low doses of EDA and BE did not rescue axonal collapse, but a small increase was identified in the BE 10 μM group (see Figure 20).

Axonal growth analysis: C57 WT neurons were treated with

STZ

0, 10, 30, 100 uM for 24 hours (see Figure 19).

Figures 19 and 20 show STZ-induced axonal collapse. Figure 20 shows that cells were treated with 30 μM STZ with 1, 3, 10 μM BE for 24 hours, respectively. Cells were seeded on PDL-coated coverslips in 24-well plates with cell confluence of 30-50%, P < 0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 8: Acetylcholinesterase analysis

Low levels of acetylcholine in the brain are one of the hallmarks of AD pathology. Studies have shown that inhibition of acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine, is effective in increasing the level of acetylcholine in the brain (Murray and Faraoni et al., 2013). Therefore, some drugs have been developed to improve cholinergic function in patients with AD, such as tacrine. (tacrine), donepezil and rivastigmine, all of which are AChE inhibitors (Chopra and Misra et al., 2011).

Measuring AChE activity has become a useful tool for evaluating anti-AD drugs. In the present invention, an acetylcholinesterase detection kit from Abcam is used to detect whether EDA and BE have an inhibitory effect on AChE. SH-SY5Y cells were treated with EDA or BE, respectively, in STZ, and cell lysates were collected 24 hours later. Experiment according to the manufacturer's instructions. As shown in Figure 22, STZ alone induced an increase in AChE, but co-treatment with EDA and BE significantly inhibited the activity of AChE. The results showed that both EDA and BE had a strong inhibitory effect on AChE. In addition, BE showed a stronger effect than EDA, and the results were in line with expectations.

Figure 21 is a standard curve of the AChE analysis. Serial dilutions were made at 300, 100, 30, 10, 3, 1 and 0 mU/ml acetylcholinesterase standards using 1000 mU/ml acetylcholinesterase standards.

Cells were seeded in 24-well plates and treated with STZ 30 [mu]M with 3 [mu]M EDA and BE. After 24 hours of incubation, cell lysates were collected and subjected to AChE analysis, and the OD value at 140 nm was measured by a microplate reader. n=3, P<0.05, one-way ANOVA, error bars, SD, GraphPad Prism.

Example 9: Effect of EDA and BE on BACE1 and downstream signaling in SY5Y695 cell line

Since the production of neurotoxic β-amyloid (Aβ) peptide specifically requires β-site amyloid proprotein cleavage enzyme 1 (BACE1), it has been considered that BACE1 has a pathology in Alzheimer's disease. A decisive early role (Vassar, 2014). As a result, BACE1 has become the primary drug target for AD treatment, and there is an urgent need to develop BACE1 inhibitors. The present invention has demonstrated that both EDA and BE are capable of reducing the expression of BACE1 in SY5Y695 cells. According to Western blotting, BACE1 expression was reduced in a dose-dependent manner.

Autophagy is characterized by a lysosomal degradation process that circulates cellular waste and removes potentially toxic damaged organelles and protein aggregates from the cytoplasm. These cytoprotective functions play a significant role in many aspects, and evidence from numerous pathogenic outcomes indicates that autophagy dysregulation is involved in neurodegenerative disorders (Nixon and Yang, 2011). In Alzheimer's disease, it is still possible to stimulate the induction of autophagy. However, it was found that degradation of autophages in lysosomes was inhibited, which may result in a large accumulation of autophagic vacuoles in the heavily swollen axons of affected neurons (Wolfe and Lee et al., 2013). The present invention also evaluated the expression of the macroautophagy marker of LC3B in the SY5Y cell line before and after treatment. Different concentrations of EDA and BE were used. The results showed that LC3B was lower in the untreated samples, but in the BE treated group, LC3B was increased in a dose-dependent manner, and a small increase was also identified in the EDA 10 μM group. The results indicate that BE and high concentrations of EDA promote autophagy, which is critical for maintaining normal cell homeostasis, thereby promoting cell survival under adverse conditions.

Recent evidence relates to the calcium/calpain/Cdk5 kinase signaling pathway in the pathogenesis of AD, suggesting that it may be responsible for Aβ42-induced BACE1 increase. Aβ42 increases intracellular calcium in neurons, which activates calpain to cleave the Cdk5 regulatory subunit p35 to p25 (27). Overexpression of the p25 subunit causes cytoskeletal destruction, altered phosphorylation patterns, and neurotoxicity. It has been reported that BACE1 levels and Aβ production are increased in mice with elevated brain Cdk5/p25 activity. These data indicate a link between Cdk5 dysregulation and AD in humans and mice, and Cdk5 inhibitors are being developed as potential AD therapeutics.

It has been widely reported that the protein p25 contributes to the onset of Alzheimer's disease (AD). P25 is a calcium-dependent degradation product of p35 and is a major activator of cyclin-dependent kinase 5 (Cdk5), and changes in the p25/p35 ratio are often associated with various diseases (Giese, 2014). In AD, p25 has been implicated in neurodegeneration and BACE1 regulation, suggesting therapeutic Cdk5 inhibition of AD (Wen and Planel et al., 2008). The present invention shows a decrease in the p25/p35 ratio and is associated with BACE1 levels in SH-SY5Y human neuroblastoma cells. After treatment of the cells with EDA and BE, their BACE1 levels and p25/p35 The decrease in ratio suggests that less p25 is produced from the p35 protein after BE treatment (see Figure 23).

Figure 23 shows Western blot analysis of the effects of EDA and BE on SY5Y695 cells. The cells were seeded in 6-well plates and treated with different concentrations of EDA and BE for 24 hours, and the cell lysate was subjected to Western blotting.

In addition, the present invention also examined the expression of membrane protein-2 (VAMP2) associated with synaptic vesicles after treatment with EDA and BE. Adequate studies have shown that cognitive impairment in patients with Alzheimer's disease is closely related to synaptic loss in the neocortex and limbic systems. The present inventors found that VAMP2 protein in SY5Y695 cells was up-regulated after treatment with EDA and BE, suggesting that EDA and BE have neuroprotective effects on synapses (see Figure 24).

Figure 24 shows Western blot analysis of the effects of EDA and BE on Aβ42-induced neurotoxicity. SY5Y695 cells were seeded in 6-well plates and treated with different concentrations of EDA or BE for 24 hours with Aβ42, respectively, and the cell lysates were subjected to Western blotting.

Example 10: Comparison of survival rates of different compounds against SY5Y695 cells

Referring to Figure 26, MTT analysis of SY5Y695 cells showed a significant decrease in cell viability (OD) under incubation with 0.5 μM CuSO ₄ . 10 μM EDA significantly inhibited the action of CuSO ₄ and increased cell viability. 1, 3 and 10 μM edaravone derivatives BE significantly increased cell viability as indicated by an increase in OD value. In contrast, borneol alone had no effect on cell viability and no further toxicity. On the other hand, a mixture of edaravone and borneol had no significant effect on cell viability at the same concentration, whereas gugulsterone inhibited cell viability in a dose-dependent manner. These experiments show that it is surprising that BE has a new synergistic effect on cell viability compared to its parental molecule. In view of the fact that the results have clearly demonstrated that BE has unexpected functions, the present invention tested other functions only by comparing edaravone with BE in the remaining experiments.

The above description of the embodiments is merely for helping to understand the core idea of the present invention. It should be noted that those skilled in the art, without departing from the principles of the invention, It is also possible to make several modifications and modifications to the EDA derivatives of the present invention, but such modifications and modifications are also within the scope of the claims of the present invention.

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Claims

An edaravone derivative which is represented by the following formula (I):

Wherein X is a linker selected from -C (O) O -, - C (O) S -, - C (O) NH -, - C (O) -, - NH- or -CH 2 -.
The edaravone derivative of claim 1, wherein X is selected from the group consisting of -C(O)O-.
The edaravone derivative of claim 1, which is represented by the following formula (Ia):
Use of an edaravone derivative according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of an oxidative stress-related disease.
The use according to claim 4, wherein said oxidative stress-related diseases are aging diseases, cardiovascular diseases, cancers, neurodegenerative diseases, liver diseases, lung diseases, digestive tract diseases, renal failure and renal permeation, infectious diseases, and immune diseases. , skin diseases, ENT diseases, pregnancy, breastfeeding and childbirth related diseases, sports diseases, male diseases, female infertility, joint diseases or chronic inflammation.
The use according to claim 4 or 5, wherein said oxidative stress-related disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, ischemic heart disease, cerebrovascular disease, chronic obstructive pulmonary disease, AIDS and diabetes.
The use of claim 6, wherein the cerebrovascular disease is ischemic cerebrovascular disease or cerebral infarction.