WO2020024977A1 - 用于治疗神经系统疾病的化合物及其应用 - Google Patents

用于治疗神经系统疾病的化合物及其应用 Download PDF

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WO2020024977A1
WO2020024977A1 PCT/CN2019/098578 CN2019098578W WO2020024977A1 WO 2020024977 A1 WO2020024977 A1 WO 2020024977A1 CN 2019098578 W CN2019098578 W CN 2019098578W WO 2020024977 A1 WO2020024977 A1 WO 2020024977A1
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compound
disease
group
treating
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王瑞玲
丁伟
付国琴
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陕西麦科奥特科技有限公司
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Priority to JP2020540299A priority Critical patent/JP7106162B2/ja
Priority to US16/977,931 priority patent/US20200390755A1/en
Priority to EP19844234.5A priority patent/EP3831821A4/en
Publication of WO2020024977A1 publication Critical patent/WO2020024977A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/453—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/10—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • the invention relates to the technical field of medicine, in particular to a compound for treating a nervous system disease and its application.
  • Neurological disease is a disease that occurs in the central nervous system, peripheral nervous system, and autonomic nervous system, and is mainly manifested as sensory, motor, consciousness, and autonomic dysfunction. It is also called neuropathy.
  • the main clinical manifestations are motor, sensory, reflex, autonomic and advanced neural activity dysfunction (Oliveira-Giacomelli A, Naaldijk Y, Sarda-Arroyo L, et al. Purinergic Receptors in Neurological Diseases With Motor Torsy Symptoms: Targets, for Therapy [J] .Front Pharmacol, 2018, 9: 325).
  • neurological diseases have unknown etiology and overlapped classification, but they can be roughly classified according to etiology, location, and pathology.
  • the causes of neurological diseases include infections, poisoning, genetic defects, nutritional disorders, immune damage, metabolic disorders, endocrine disorders, congenital malformations, blood circulation disorders, and abnormal proliferation.
  • Common neurological diseases such as cardiovascular and cerebrovascular diseases, craniocerebral injury, and some neurodegenerative diseases, which seriously threaten human health, are epilepsy, Alzheimer's disease, Parkinson's disease, and Huntington's disease. category. With the emergence of neurological diseases, there are often some mental disorders, such as depression and anxiety.
  • Huntington's disease is a rare autosomal dominant genetic disease, also known as chronic progressive dance disease, big dance disease, and Huntington's disease. Patients generally develop middle-aged symptoms with motor, cognitive, and mental symptoms. The clinical symptoms of Huntington's disease are complex and changeable, and the patient's condition is progressively worsening, usually dying 15 to 20 years after onset. The clinical symptoms of Huntington's disease include three aspects, namely dyskinesia, cognitive impairment and mental disorder. HD can be combined with other diseases, and individual patients can develop epilepsy, hereditary ataxia, and migraine.
  • the mutant gene IT-15 which causes Huntington ’s disease, is located on human chromosome 4. It encodes a protein with a relative molecular weight of 3.5 ⁇ 105 and consists of 3144 amino acids. It is named Huntingtin (Htt).
  • a repeating glutamine sequence begins at amino acid residue 17 at the amino terminus, which is a cytosine-adenine-guanine (CAG) repeat at the first exon 1 of the lT-15 gene coding. It has been confirmed that CAG repeat mutations can cause HD to occur.
  • the CAG sequence repeat number in the Huntington gene in the normal population is between 17 and 20. When the repeat number is 40 or more, it can cause disease.
  • the CAG repeat number is 36 to 39.
  • Time is an incomplete penetrance.
  • CAG repeats on the IT-15 gene encode ployQ repeats that lead to their abnormal amplification, which generally results in two direct results.
  • One is the loss of function of wild-type huntingtin (wHtt), and the other is the production of mutant huntingtin. (mutant Htt, mHtt) and thus exert a toxic effect.
  • the wHtt of the normal population is widely expressed in various organs of the body including the central nervous system. It plays a key role in the development of the nervous system and the endocytosis and secretion of cells. It can promote the production of brain-derived neurotrophic factor (BDNF) in neurons. And transport capacity, as well as play a role in inhibiting apoptosis.
  • BDNF brain-derived neurotrophic factor
  • Htt configuration is prone to conformational changes in ⁇ -lamellar structure, resulting in abnormal splicing. It may produce a truncated mRNA, which contains the ployQ region, which encodes only exon1 and is translated into mHtt protein.
  • Htt breaks under the action of protease, which will form the N-terminal fragment of mHtt protein containing mutant ployQ.
  • Post-transcriptional modifications can also produce more toxic fragments through phosphorylation, acetylation, and conformational changes.
  • mHtt can form larger, insoluble protein aggregates through homogeneous or heterodimerization, which can be seen in the cytoplasm or nucleus of the neurocortex and striatum of the brain of HD patients, and cannot be ubiquitin
  • the proteasome system dissolves and can change its function, causing a series of damage to neurons.
  • mHtt interacts with the mitochondrial membrane, interfering with calcium's homeostasis until exposure to mitochondrial imbalance, and areas where mHtt aggregates show high-energy phosphate storage and reduced ATP levels, leading to a metabolic load in Huntington's patients.
  • tetrabenazine For the treatment of HD-related chorea and tardive dyskinesia.
  • the main mechanism of tetrabenazine is to inhibit monoamine vesicle transport.
  • the drug When the drug is used in large amounts, it can significantly reduce Huntington's dance symptoms, but if the drug is stopped, the dance symptoms will become worse.
  • Use of such drugs must pay attention to its side effects, including bradykinesia with tremor, depression and increased suicide rates, as well as Parkinson's disease and neuroleptic Malignant Syndrome.
  • Deuterated tetrabenazine is marketed in the United States. It is used to treat HD-related chorea and tardive dyskinesia. It is slower in metabolism than tetrabenazine, has a longer half-life, and has a lower dose.
  • Ischemic cardio-cerebral vascular disease refers to a disorder of blood supply to the brain based on changes in the blood vessel wall supplying the brain, changes in blood composition, or hemodynamic changes, causing brain tissue in the corresponding blood supply area to appear due to ischemia and hypoxia. Necrosis of the brain tissue and cause transient or long-term local or extensive brain damage, causing a series of symptoms of neurological deficits. The damage caused by ischemia is divided into primary ischemia (Cerebral ischemia, primary PI) and reperfusion injury (CIRI).
  • primary ischemia Cerebral ischemia, primary PI
  • CIRI reperfusion injury
  • Ischemic reperfusion injury is caused by cerebral ischemia or Before the myocardial ischemia caused tissue necrosis, after the occluded blood vessels were reopened, not only the function was not restored, but the damage was aggravated, which was the main pathophysiological process that caused various cardiovascular and cerebrovascular diseases. Among them, stroke is the number one cause of death and disability in China. There are about 2 million new stroke patients in China every year, and the incidence is increasing year by year.
  • drugs to prevent platelet aggregation such as aspirin
  • drugs to improve microcirculation and expand blood volume such as dextran
  • thrombolytic drugs such as urokinase
  • Anticoagulant drugs such as heparin
  • calcium antagonists such as nimodipine
  • the object of the present invention is to provide a novel compound for treating neurological diseases.
  • the compound can activate the TrkB receptor and its downstream signaling pathway.
  • the TrkB receptor is the main signal of brain-derived neurotrophic factors. Receptors, activation of the TrkB receptor and its downstream signaling pathways can improve the symptoms of many neurological diseases.
  • Another object of the present invention is to provide a novel compound for treating neurological diseases, so that the compound has a therapeutic effect on degenerative diseases of the nervous system, and can be specifically applied to treat and improve Huntington's disease and amyotrophic lateral sclerosis , Alzheimer's disease, multiple sclerosis, clinical symptoms caused by Parkinson's disease.
  • Another object of the present invention is to provide a novel compound for treating neurological diseases.
  • the compound has protective and therapeutic effects on central brain injury, and is particularly applicable to the treatment and improvement of clinical symptoms caused by stroke.
  • Another object of the present invention is to provide the application of the above compounds in the preparation of drugs for related neurological diseases, and to provide specific pharmaceutical products.
  • the present invention provides the following technical solutions:
  • a compound for treating a nervous system disease is a compound represented by formula (I), or a stereoisomer, geometric isomer, tautomer, solvate, metabolite of a compound represented by formula (I) , A pharmaceutically acceptable salt, or a prodrug of a compound of formula (I);
  • R 3 is H, D, F, Cl or Br
  • R 4 and R 5 are independently selected from H, D, F, Cl, Br, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy and C 1 -C 6 alkylamino ; M and n are each independently 0, 1, 2, 3, or 4.
  • R 1 and R 2 are independently selected from -OH, -NH 2 , F, C 1 -C 6 hydroxyalkyl, C 1 -C 6 heteroalkyl, C 1 -C 6 alkoxy And C 1 -C 6 alkylamino.
  • R 1 and R 2 are independently selected from -OH, -NH 2 , F, and -OCH 3 ; in some specific embodiments of the present invention, R 1 and R 2 are as follows Choice of one:
  • the R 4 and R 5 are independently selected from H, D, F, Cl, Br, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH ( CH 3 ) 2 , -N (CH 3 ) 2 , -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 CH 3, and -OCH (CH 3 ) 2 .
  • R 5 is -CH 3 , -CH 2 CH 2 CH 3 , -N (CH 3 ) 2 , -OCH 3 , D, or F.
  • each of m and n is independently 0, 1, or 2. In other specific embodiments of the present invention, n is 0, that is, there is no substituent R 4 ; and m is 1 or 2.
  • R 3 is H, D, F, Cl or Br
  • R 4 and R 5 are independently selected from H, D, F, Cl, Br, -CH 3 , -CH 2 CH 3 , -CH 2 CH 2 CH 3 , -CH (CH 3 ) 2 , -N (CH 3 ) 2, -OCH 3, -OCH 2 CH 3, -OCH 2 CH 2 CH 3 and -OCH (CH 3) 2;
  • n and n are each independently 0, 1, or 2.
  • R 5 is -CH 3 , -CH 2 CH 2 CH 3 , -N (CH 3 ) 2 , -OCH 3 , D, or F; n is 0, that is, There is no substituent R 4 ; the m is 0, 1 or 2.
  • the compound represented by formula (I) is one of the following compounds:
  • the compound provided by the present invention can increase the relative strength of p-TrkB / t-TrkB protein in neuronal cells by 2.06-2.34 times, showing a significant difference (P ⁇ 0.05), p-Akt / The relative strength of t-Akt protein was increased by 2.09 to 2.40 times, which was equivalent to BDNF. It is shown that the compounds provided by the present invention can activate the TrkB receptor and its downstream AKT signaling pathway, further indicating that these compounds have a therapeutic effect on neurological diseases and a better protective effect on neurons.
  • the present invention simulates the pathological symptoms of Huntington's disease and stroke through mouse models, and confirms that the compounds provided by the present invention can have neurological effects through testing items such as behavioral, physiological and biochemical indicators.
  • Systemic degenerative lesions and central brain injury have protective and therapeutic effects.
  • the present invention provides a pharmaceutical composition comprising any one of the compounds provided by the present invention. Further, it may also include pharmaceutically acceptable excipients and / or other drugs for treating neurological diseases.
  • pharmaceutically acceptable excipient means a pharmaceutically acceptable material, carrier, excipient, mixture, or vehicle that is related to the consistency of the dosage form or pharmaceutical composition.
  • Each excipient must be compatible with the other ingredients of the pharmaceutical composition when mixed to avoid interactions that would greatly reduce the efficacy of the compounds disclosed herein when administered to a patient and result in mutual interactions that are not pharmaceutically acceptable pharmaceutical compositions effect.
  • each excipient must be pharmaceutically acceptable, for example, of sufficiently high purity. Suitable pharmaceutically acceptable excipients will vary depending on the particular dosage form chosen.
  • pharmaceutically acceptable excipients can be selected based on their specific function in the composition. For example, certain pharmaceutically acceptable excipients can be selected that can help produce a uniform dosage form. Certain pharmaceutically acceptable excipients can be selected to help produce stable dosage forms. Certain pharmaceutically acceptable excipients may be selected to help carry or transport a compound disclosed herein from one organ or part of the body to another organ or part of the body when administered to a patient. Certain pharmaceutically acceptable excipients may be selected to enhance patient compliance.
  • Suitable pharmaceutically acceptable excipients include the following types of excipients: excipients, diluents, fillers, binders, disintegrating agents, lubricants, glidants, granulating agents, coating agents, wetting agents , Solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste-masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, tackifiers, antioxidants, Preservatives, stabilizers, surfactants and buffers.
  • excipients include the following types of excipients: excipients, diluents, fillers, binders, disintegrating agents, lubricants, glidants, granulating agents, coating agents, wetting agents , Solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste-masking agents, colorants, anti-
  • the compounds of the present invention may be administered as a single active agent or may be administered in combination with other neurological disease treatment drugs, including other compounds having the same or similar therapeutic activity and determined to be safe and effective for such combination administration.
  • the invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administering a safe and effective amount of a combination drug comprising a compound disclosed herein and one or more other neurological disease treatment drugs.
  • the other drugs for treating neurological diseases include drugs for treating Huntington's disease, drugs for treating Parkinson's disease, drugs for treating depression, drugs for treating dyskinesia, drugs for treating stroke, Medicine for treating diseases of central nervous system injury, medicine for treating amyotrophic lateral sclerosis, and medicine for treating multiple sclerosis.
  • these other neurological disease treatment drugs are selected from one or more of drugs used to treat ataxia, drugs used to treat Huntington's disease, and drugs used to treat ischemic cerebrovascular disease.
  • the combination drug comprises one or two other neurological disease drugs.
  • Drugs used to treat Huntington's disease include, but are not limited to, tetrabenazine, deuterated tetrabenazine, Pridopidine; drugs used to treat stroke include, but are not limited to, butylphthalide, edaravone; used to treat Drugs for multiple sclerosis are selected from the group consisting of Ocelizumab, Tecfidera, Glatire, Kepasson, Fingolimod, Natalizumab, Avonex, Rebif, Teriflunomide, Plegridy, Betaferon, Ampyra; drugs used to treat depression include, but are not limited to, citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, or verazodone; serotonin-de Norepinephrine reuptake inhibitors, such as divenlafaxine, duloxetine, milnacipran, venlafaxine; norepinephrine and specific serot
  • Drugs used to treat amyotrophic lateral sclerosis include, but are not limited to, IL-6 receptor monoclonal antibody (Tocilizumab), ozanibizumab, BIIB067 (Isis-SOD1Rx).
  • dosage forms include those suitable for the following routes of administration: (1) oral administration, such as tablets, capsules, caplets, pills, tablets, powders, syrups, elixirs, suspensions, Solutions, emulsions, granules and cachets; (2) parenteral administration, such as sterile solutions, suspensions and lyophilized powders; (3) transdermal administration, such as transdermal patches (4) rectal administration, such as suppositories; (5) inhalation, such as aerosols, solutions, and dry powders; and (6) topical administration, such as creams, ointments, lotions, solutions, pastes , Sprays, foams and gels.
  • routes of administration such as tablets, capsules, caplets, pills, tablets, powders, syrups, elixirs, suspensions, Solutions, emulsions, granules and cachets
  • parenteral administration such as sterile solutions, suspensions and lyophilized powders
  • transdermal administration such as transdermal
  • the methods of treatment disclosed herein include administering to a patient in need a safe and effective amount of a compound of the invention or a pharmaceutical composition comprising a compound of the invention.
  • Embodiments of the present invention include methods of treating neurological diseases by administering a safe and effective amount of a compound of the present invention or a pharmaceutical composition comprising the compound of the present invention to a patient in need thereof.
  • the present invention provides the use of the compound and the pharmaceutical composition in the preparation of a medicament for preventing or treating a nervous system disease.
  • the neurological diseases include neuron damage, neuron degradation, brain atrophy-related diseases or conditions, central nervous system damage, stroke (stroke), depression, anxiety, amyotrophic lateral sclerosis, Alzheimer's disease , Autism, Huntington's disease, Rett syndrome, epilepsy, Parkinson's disease, post-traumatic stress disorder, diabetic neuropathy, multiple sclerosis, and peripheral neuropathy.
  • the present invention also provides the use of the compound and the pharmaceutical composition for preparing a medicament for stimulating the TrkB receptor.
  • the present invention provides a method for stimulating a TrkB receptor, comprising administering to a subject or sample in need thereof a therapeutically effective amount of a compound disclosed by the present invention, a compound prepared by the preparation method of the present invention or the present disclosure Pharmaceutical composition of compounds.
  • the present invention provides a method for preventing, treating or alleviating a nervous system disease, which comprises administering a therapeutically effective amount of the compound disclosed by the present invention, the compound prepared by the preparation method of the present invention or the present disclosure to an individual or sample in need Pharmaceutical composition of compounds.
  • the "therapeutically effective amount” or “effective amount” of a compound of the present invention may be 0.1 mg / kg to 100 mg / kg. In some embodiments, the “therapeutically effective amount” or “effective amount” of a compound of the present invention may be 0.5 mg / kg to 50 mg / kg; in other embodiments, the “therapeutically effective amount of a compound of the present invention” "" Can be 0.2 mg / kg to 25 mg / kg.
  • the present invention provides a novel flavonoid derivative having the structure of formula (I) or formula (II), which can activate the TrkB receptor and its downstream AKT signaling pathway, and further explain that the compound has an effect on nerves Systemic diseases have a better therapeutic effect and a better protective effect on neurons, especially for neurodegenerative diseases such as Huntington disease and central brain injuries such as stroke.
  • FIG. 1 shows p-TrkB / t-TrkB and p-AKT / t-AKT electrophoresis pictures and relative protein intensity diagrams of the test compounds provided in Example 1, where A is p-TrkB / t-TrkB protein electrophoresis Figure, B is the statistical result of p-TrkB / t-TrkB protein level, C is the protein electrophoresis chart of p-AKT / t-AKT; D is the statistical result of p-AKT and t-AKT protein level;
  • Figure 2 shows the statistical results of the relative content of p-Akt detected by ELISA for each test compound
  • FIG. 3 shows the results of volume MRI of whole brain, striatum, and neurocortex of the test compound provided in Example 1. Among them, A is a statistical result of whole brain volume, B is a statistical result of striatum volume, and C is Cortical volume statistical results;
  • FIG. 4 shows the experimental results of the relative intensity of the DARPP32 protein of the test compound provided in Example 1.
  • A is the DARPP32 electrophoresis chart
  • B is the relative intensity of the DARPP32 / ⁇ -action protein level
  • FIG. 5 shows a representative image of the neurocortex of the brain tissue mHtt protein aggregates of the test compound provided in Example 1 with EM48 antibody immunostaining
  • Figure 6 shows the results of TTC staining of five rat brain tissue samples from each group.
  • the invention discloses a compound for treating diseases of the nervous system and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention.
  • the compounds described in the present invention and their related applications have been described through preferred embodiments, and the relevant person can obviously modify or appropriately modify and combine the compounds described herein and their related applications without departing from the content, spirit and scope of the present invention. To implement and apply the technology of the present invention.
  • stereoisomers, tautomers, solvates, metabolites, salts and pharmaceutically acceptable prodrugs of the compounds represented by formula (I) or (II) are included within the scope of the present invention .
  • the compounds disclosed herein may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms.
  • the present invention aims to make all stereoisomeric forms of the compounds represented by formula (I) or (II), including but not limited to diastereomers, enantiomers, atropisomers and geometric (or Conformational) isomers, and their mixtures, such as racemic mixtures, form an integral part of the invention.
  • stereochemistry of any particular chiral atom when the stereochemistry of any particular chiral atom is not specified, all stereoisomers of the structure are considered in the present invention and are included in the present invention as the compounds disclosed in the present invention .
  • stereochemistry is indicated by a solid wedge or a dashed line representing a specific configuration, the stereoisomers of the structure are defined and defined accordingly.
  • the compounds represented by formula (I) or (II) may exist in different tautomeric forms, and all these tautomers, such as the tautomers described in the present invention, are included in the scope of the present invention. Inside.
  • the compound represented by the formula (I) or (II) may exist as a salt.
  • the salt refers to a pharmaceutically acceptable salt.
  • the salt may also be used for preparing and / or purifying a compound represented by formula (I) or (II) and / or for isolating a compound represented by formula (I) or (II). Enantiomer intermediate.
  • Stereoisomers refer to compounds that have the same chemical structure, but differ in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotomers), geometric isomers, (cis / trans) isomers, atropisomers, etc. Wait. "Enantiomers” refer to two isomers of a compound that cannot overlap but mirror image each other. "Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high resolution analytical operations such as electrophoresis and chromatography, such as HPLC.
  • tautomers or “tautomeric forms” refers to structural isomers with different energies that can be converted to each other through a low energy barrier. If tautomerization is possible (eg in solution), the chemical equilibrium of the tautomers can be reached.
  • protontautomers also known as prototropic tautomers
  • Valence tautomers include interconversions through the reorganization of some bonding electrons.
  • keto-enol tautomerism is the interconversion of pentane-2,4-dione and 4-hydroxypent-3-en-2-one tautomers.
  • tautomerism is phenol-keto tautomerism.
  • a specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4 (1H) -one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • substituted means that one or more hydrogen atoms that can be substituted in a given structure are replaced with a specific substituent. Unless otherwise indicated, a substituted group may have one substituent substituted at each substitutable position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be substituted at the same or different positions.
  • an optionally substituted heterocyclic group means that an alkyl group may, but does not have to exist, and the description includes a case where the heterocyclic group is substituted with an alkyl group and a case where the heterocyclic group is not substituted with an alkyl group.
  • unsubstituted means that the specified group is unsubstituted.
  • optionally substituted with can be used interchangeably with the term “unsubstituted or substituted with”, ie the structure is unsubstituted or substituted with one or more substituents described herein
  • C 1 -C 6 alkyl refers in particular to independently disclosed methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • alkyl or "alkyl group” used in the present invention means a saturated straight or branched chain monovalent hydrocarbon group containing 1 to 20 carbon atoms, wherein the alkyl group may be optionally Is substituted with one or more substituents described herein. Unless otherwise specified, alkyl groups contain 1-20 carbon atoms.
  • the alkyl group contains 1-12 carbon atoms; in other embodiments, the alkyl group contains 2-12 carbon atoms; in other embodiments, the alkyl group contains 1 -6 carbon atoms; in other embodiments, the alkyl group contains 2-6 carbon atoms; in still other embodiments, the alkyl group contains 1-4 carbon atoms; also in some embodiments The alkyl group contains 1-3 carbon atoms.
  • the alkyl group may be optionally substituted with one or more substituents described herein.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), Isopropyl (i-Pr, -CH (CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH (CH 3 ) 2 ), sec-butyl (s-Bu, -CH (CH 3 ) CH 2 CH 3 ), tert-butyl (t-Bu, -C (CH 3 ) 3 ), and the like.
  • heteroalkyl means that one or more heteroatoms may be inserted into the alkyl group, wherein the alkyl group and the heteroatom have the meaning as described in the present invention. Heteroalkyl groups are connected to other groups via carbon atoms. Unless otherwise specified, a heteroalkyl group contains 1-12 carbon atoms, in other embodiments, the heteroalkyl group contains 1-8 carbon atoms, and in other embodiments, the heteroalkyl group contains 1 -6 carbon atoms, in other embodiments, the heteroalkyl group contains 1-4 carbon atoms, and in other embodiments, the heteroalkyl group contains 1-3 carbon atoms.
  • heteroalkyl examples include, but are not limited to, -CH 2 OCH 3 , -CH 2 CH 2 OCH 3 , -CH 2 SCH 3 , -CH 2 N (CH 3 ) 2 , -CH 2 OCH 2 ( CH 3 ) 2 , -CH 3 CH 2 CH 2 OCH 3 , -CH 3 CH 2 OCH 2 CH 3 , -CH 3 CH 2 CH 2 OCH 2 CH 3 and the like.
  • alkoxy means that an alkyl group is connected to the rest of the molecule through an oxygen atom, wherein the alkyl group has the meaning as described in the present invention. Unless otherwise specified, the alkoxy group contains 1-12 carbon atoms. In some embodiments, the alkoxy group contains 1-6 carbon atoms; in other embodiments, the alkoxy group contains 1-4 carbon atoms; in still other embodiments, the alkoxy group The pellet contains 1-3 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents described herein.
  • haloalkyl means an alkyl, alkenyl or alkoxy group substituted with one or more halogen atoms, such examples include, but are not limited to, Difluoroethyl (-CH 2 CHF 2 , -CF 2 CH 3 , -CHFCH 2 F), trifluoroethyl (-CH 2 CF 3 , -CF 2 CH 2 F, -CFHCHF 2 ), trifluoromethyl (-CF 3 ), trifluoromethoxy (-OCF 3 ), and the like.
  • hydroxyalkyl means that an alkyl group is substituted with one or more hydroxyl groups, and such examples include, but are not limited to, hydroxymethyl (-CH 2 OH), hydroxyethyl (-CH 2 CH 2 OH) )Wait.
  • halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).
  • alkylamino includes “N-alkylamino” and "N, N-dialkylamino", wherein the amino groups are each independently substituted with one or two alkyl groups.
  • the alkylamino group is a lower alkylamino group formed by connecting one or two C 1 -C 12 alkyl groups to a nitrogen atom.
  • two or alkylamino group is a C 1 -C 6 alkyl group attached to the nitrogen atom of the lower alkyl amino groups.
  • two or alkylamino group is a C 1 -C 4 alkyl group attached to the nitrogen atom of the lower alkyl amino groups.
  • two or alkylamino group is a C 1 -C 3 alkyl group attached to the nitrogen atom of the lower alkyl amino groups.
  • Suitable alkylamino groups may be monoalkylamino or dialkylamino.
  • alkylamino include, but are not limited to, N-methylamino (-NH (CH 3 )), N-ethylamino (-NH ( CH 2 CH 3 )), N, N-dimethylamino (-N (CH 3 ) 2 ), N, N-diethylamino (-N (CH 2 CH 3 ) 2 ), N-ethylpropyl- 2-amino and so on.
  • the substituent represents a ring system formed by a bond to the central ring (as shown in formula a), which represents that the substituent is substituted at all substitutable positions on the ring system.
  • formula a represents that the substituent R may optionally replace all positions that can be substituted on the D ring, as shown in formula b to formula d. If there is more than one substituent, each substituent may select a different substitution position or the same substitution position.
  • the wavy lines in the following structural formulas b to d represent the connecting bonds.
  • pharmaceutically acceptable means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or mammal treated with it.
  • the "pharmaceutically acceptable salt" of the present invention can be synthesized from the parent compound, a basic or acidic moiety by conventional chemical methods.
  • such salts can be obtained by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base, such as hydroxide, carbonate, bicarbonate, etc. of Na, Ca, Mg or K, or by These compounds are prepared by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. This type of reaction is usually carried out in water or an organic solvent or a mixture of both.
  • a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is required.
  • a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile.
  • the compounds disclosed in the present invention can also be obtained in the form of their hydrates or in the form of solvents containing them (such as ethanol, DMSO, etc.) for their crystallization.
  • solvents containing them such as ethanol, DMSO, etc.
  • the compounds disclosed herein may form solvates inherently or by design with pharmaceutically acceptable solvents, including water; therefore, the invention is intended to include both solvated and unsolvated forms.
  • the compounds disclosed herein can be administered in prodrug form.
  • the "prodrug" of the compound disclosed in the present invention is a functional derivative that can finally release the compound disclosed in the body when administered to a patient.
  • prodrug used in the present invention means that a compound is converted into a compound represented by formula (I) or formula (II) in vivo. Such transformations are affected by the prodrug's hydrolysis in the blood or the enzyme's conversion into the parent structure in the blood or tissues.
  • the prodrug compound of the present invention may be an ester.
  • the ester may be used as an aromatic ester of a prodrug, an aliphatic (C 1 -C 24 ) ester, an acyloxymethyl ester, or a carbonate. , Carbamates and amino acid esters.
  • a compound in the present invention contains a hydroxyl group, which can be acylated to obtain a compound in the form of a prodrug.
  • Other prodrug forms include phosphate esters.
  • these phosphate ester compounds are obtained by phosphorylation of hydroxy groups on the parent.
  • Methodabolite refers to a product obtained by metabolizing a specific compound or a salt thereof in the body.
  • the metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by experimental methods as described in the present invention.
  • Such a product can be obtained by administering a compound through methods such as oxidation, reduction, hydrolysis, amidolation, deamidation, esterification, degreasing, and enzymatic cleavage.
  • the invention includes metabolites of a compound, including metabolites produced by sufficient contact of a compound of the invention with a mammal for a period of time.
  • the reagents used in the specific embodiments of the present invention were purchased from commercial suppliers such as Aldrich Chemical Company, Arco Chemical Company and Alfa Chemical Company, and were used without further purification unless otherwise indicated.
  • General reagents were purchased from Aladdin reagent, Tianjin Fuchen Chemical Reagent Factory, Wuhan Xinhuayuan Technology Development Co., Ltd., Qingdao Tenglong Chemical Reagent Co., Ltd. and Qingdao Ocean Chemical Plant.
  • Neurobasal-SFM, Leibovitz's, PBS, FBS, GlutaMAX-I (100 ⁇ ) were purchased from Thermo Fisher Scientific; PDL, DNase, Trypsin-EDTA (0.25%), isoflurane, DPBS were purchased from Sigma-Aldrich; Anti-TrkB, Anti-AKT was purchased from Cell signalling.
  • the Western blot experimental device was purchased from Invitrogen, USA.
  • BDNF was purchased from Peprotech.
  • 1 H NMR spectra were recorded using a Bruker 400MHz or 600MHz nuclear magnetic resonance spectrometer.
  • the 1 H NMR spectrum uses CDCl 3 , D 2 O, DMSO-d 6 , CD 3 OD, or acetone-d 6 as a solvent (in ppm), and uses TMS (0 ppm) or chloroform (7.26 ppm) as a reference standard.
  • MS mass spectrometry
  • the compound of the present invention having a structure represented by the formula (1-6) can be prepared by a general synthetic method described in Synthesis Scheme 1.
  • Synthesis Scheme 1 the substituted 2-hydroxyacetophenone compound (1-1) and optionally substituted 4-bromobenzaldehyde (1-2) undergo an aldol condensation reaction under basic conditions to form the compound (1 -3).
  • compound (1-3) is first reacted with iodine to obtain an activated intermediate under high temperature conditions, and then undergoes a ring-closure reaction to form compound (1-4).
  • compound (1-4) and optionally substituted piperidine (1-5) Coupling under the catalysis of Xphos and Pd 2 (dba) 3 to obtain compound (1-6).
  • the compound disclosed in the present invention having the structure shown by formula (2-6) can be prepared by the general synthetic method described in Synthesis Scheme 2.
  • the specific steps can refer to the examples.
  • the substituted 1- (2,3,4-trihydroxy) phenyl ethyl ketone (2-1) reacts with BnBr under basic conditions to obtain a bisbenzyl-protected compound (2-2)
  • the compound (2-2) and the optionally substituted 4-bromobenzaldehyde (1-2) undergo an aldol condensation reaction under basic conditions to form the compound (2-3).
  • compound (2-3) is first reacted with iodine to obtain an activated intermediate under high temperature conditions, and then undergoes a ring-closure reaction to form compound (2-4), compound (2-4) and optionally substituted piperidine ( 1-5) Coupling under the catalysis of Xphos and Pd 2 (dba) 3 to obtain compound (2-5).
  • Compound (2-5) is catalytically hydrogenated to remove the benzyl protecting group to obtain compound (2-6).
  • a compound for treating neurological diseases and its application provided by the present invention are further described below.
  • Example 21 Activation of the TrkB receptor by the compounds of the present invention
  • Number of cells / mL Total number of cells in the four grids / 4 ⁇ 10 4 / mL
  • Total number of cells / mL Total number of cells in the four grids / 4 ⁇ 10 4 / mL ⁇ dilution factor.
  • the cells were diluted with Neurobasal selection medium so that 1 mL of Neurobasal broth contained 0.5 ⁇ 10 6 cells.
  • the PDL solution was heated in a 37 ° C water bath in advance, and 200 ⁇ L of the PDL solution was added to each well of the 6-well plate, and the PDL solution was evenly coated on the cell well plate at 37 ° C overnight.
  • the PDL solution was aspirated and washed twice with PBS. Before use, blot the PBS in a clean bench and dry it with the lid open. 2 mL of cell suspension (37 ° C, 5% CO 2 ) was added to each well. After the cells were adhered, the culture medium was changed and cultured for 3 days to observe the growth of neurons. Change the whole culture medium once every 3 days, and change the medium every time. Culture until the primary cortical neuron cells mature (usually mature until 14 days).
  • a blank control group (Vehicle), a positive control group of BDNF (10ng / mL) and a control compound group (500nM), and a test compound group of 6 groups (500nM) were set. Each dose was set up in three parallel groups. Incubate at 37 ° C for 10 minutes, then discard the culture solution, wash the cells with pre-cooled PBS, add 40 ⁇ L of pre-cooled lysate (RIPA buffer 10 mL and protease and phosphatase inhibitor 100 ⁇ L) to each well, and lyse on ice for 30 min.
  • pre-cooled lysate (RIPA buffer 10 mL and protease and phosphatase inhibitor 100 ⁇ L)
  • the cells were pulverized by an ultrasonic cell pulverizer, centrifuged at 14000 rpm for 20 min at 4 ° C, and the supernatant was aliquoted and transferred to a new 1.5 mL centrifuge tube to extract the protein.
  • Western blotting assay was used to detect (A) p-TrkB and t-TrkB; (B) p-AKT and t-AKT protein levels.
  • Figure 1A is the p-TrkB / t-TrkB protein electrophoresis chart
  • Figure 1C is the p-AKT / t-AKT protein electrophoresis chart
  • Figure 1B is the statistical result of p-TrkB / t-TrkB protein level
  • Figure 1D is the statistical result of p-AKT / t-AKT protein level.
  • Brain-derived neurotrophic factor has a neurotrophic effect on a variety of neuron clusters, including sensory neurons, motor neurons, substantia nigra dopaminergic neurons, and basal forebrain cholinergic neurons, These neurons are involved in several neurological and neuropsychiatric disorders.
  • BDNF Brain-derived neurotrophic factor
  • Preclinical evidence suggests that BDNF may be suitable for the treatment of a variety of neurological and neuropsychiatric disorders; however, the instability of this peptide in vivo and its inability to effectively cross the blood-brain barrier limit its usefulness.
  • pre-cooled lysate (RIPA buffer 10 mL and protease and phosphatase inhibitor 100 ⁇ L) to each well, and lyse on ice for 30 min.
  • the cells were pulverized by an ultrasonic cell pulverizer, centrifuged at 14000 rpm for 20 min at 4 ° C, and the supernatant was aliquoted and transferred to a new 1.5 mL centrifuge tube to extract the protein.
  • p-Akt ELISA assay was used to quantitatively detect the level of p-Akt protein in cells.
  • Phosphate-Akt1 (Ser473) sandwich ELISA kit was purchased from Cell Signaling Corporation (catalog number 7160).
  • the antibodies were coated in 96-well plates at 4 ° C overnight and washed. The coated plate was blocked with 2% bovine serum albumin (BSA), and after thorough washing, 100 ⁇ L of the sample dilution solution (supplied in the kit) was added to each well to dilute the extracted protein, and at 4 ° C Incubate overnight. After washing 4 times with 200 ⁇ L of washing buffer (supplied in the kit), 100 ⁇ L / well of detection antibody was added and incubated at 37 ° C. for 1 hour.
  • BSA bovine serum albumin
  • Optical density was determined by subtracting the reading at 650 nm (the interference of the solution itself) from the reading at 450 nm. Corresponding to the standard curve according to the OD value, the p-Akt concentration in each well was obtained.
  • the activation effect of the compound of the embodiment of the present invention on the downstream signal AKT of the BDNF-TrkB signaling pathway is equivalent to that of BDNF; the activation effect of the compound of the present invention on the AKT is significantly better than that of the control compound.
  • the results of this experiment are consistent with the results of the western blotting experiment of Example 21.
  • Example 23 Experimental analysis of the efficacy of the compounds of the present invention in transgenic mice with Huntington's disease
  • test compound was suspended in a 1% CMC-Na solution and administered orally to the end of the study. Measure body weight once a week. Behavioral tests were performed at 12, 18, and 24 weeks of age to investigate motor function. At 20 weeks, 3 cases of tissues were taken for western blotting and immunohistochemistry experiments. MRI scans were performed at 24 weeks. At the end of the study, the effects of the test compounds on the mortality of mice were counted.
  • the motor function of the mice was evaluated by the rotating rod experiment and three types of balance beam experiments. The shorter the time of crossing the balance beam and the longer the fall latency, the stronger the coordination ability of the mice.
  • mice were trained to cross the beam three times at one-eighth, one-quarter, and full-length distances from the black box. If the mouse stops walking during training, gently press the tail with long tweezers to encourage movement. After the training experiment, the mice were allowed to rest for at least one hour in an undisturbed environment. The formal experiment was started. The maximum time for each mouse to cross the balance beam is up to 60 seconds. If the movement time is greater than 60 seconds, it is recorded as 60 seconds.
  • Rotating stick experiment The maximum time for the mouse fatigue stick tester is set to 5 minutes, the acceleration setting is increased from 4rpm to 40rpm, the mouse is placed on a running stick tester, the timing is started, and the time that the mouse remains on the rod is recorded , Which is the fall latency period, which indicates its ability to coordinate movement.
  • Each mouse was trained for 5 minutes one day before the start of the twiddle experiment, and then rested in a cage after the training session. The official experiment started the next day. The mice were placed on a turntable for three rounds of experiments. After each experiment, they took a 30-minute break and proceeded to the next round of experiments. The results were averaged over three experiments.
  • the HD test compound group showed an excellent improvement on exercise performance. From the results of the three types of balance beam experiments, compared with the HD Vehicle group, the time taken by the HD test compound group to cross the balance beam was significantly shorter in all experiments at the three time points of weeks 12, 18, and 24 (P ⁇ 0.05), which is equivalent to the time spent in the WT Vehicle group.
  • the HD Vehicle group did not show significant dyskinesia at the 12th week, and the motor capacity was lost at the 18th week, but there was no significant difference; but at 24 The weekly exercise capacity decreased significantly (P ⁇ 0.05).
  • the HD test compound group showed an excellent improvement on exercise performance.
  • the fall latency of the mice in the HD test compound group was significantly longer (P ⁇ 0.05); the experiments at the 24th week As a result, the fall latency of the HD administration group was comparable to that of the WT Vehicle group mice.
  • test compounds prepared in the examples of the present invention show excellent effects on improving dyskinesia in HD model mice.
  • mice All mice were measured for body weight once a week. The survival of the mice was monitored daily, the death was recorded, and the survival rate was calculated. If the mouse cannot be returned to its normal position after being placed upside down, and it begins to move after 30 seconds of light stimulation, it can also be regarded as the end of life.
  • mice in the HD vehicle group started to die earlier overall. At about seven months of age (210 days), 6 of the 11 mice died, with a mortality rate of 54.5%. Three died, with a mortality rate of 30%. This shows that the compounds of the present invention can delay the onset time of HD mice.
  • the WT Vehicle group, WT test compound group, HD Vehicle group and HD test compound group were administered to 24 weeks, and 4 mice were taken from each group for in vivo MRI scan and brain volume measurement.
  • a 9.4T magnetic resonance imager was used, equipped with a triaxial gradient and an animal imaging probe. Mice were anesthetized with 1% isoflurane, respiration was monitored, and maintained at a constant temperature throughout the scan. Images were obtained using three-dimensional T2-weighted fast spin echo sequence technology, which has imaging resolution and contrast sufficient to automatically describe the volume of mouse brains and substructures.
  • the instrument contains a powerful linux cluster that can encode and run large deformation diffusion metric maps (LDDMM) to obtain high-intensity standardized images. These transformations encode morphological differences between images and can be analyzed based on deformed morphological measurements (DBM) to detect regional changes in brain volume. The volume of the whole brain (Brain), striatum (Neocortex) and neurocortex (Caudate Putamen) was analyzed.
  • LDMM deformation diffusion metric maps
  • the MRI scanning technology was used to analyze the morphological differences between the subject and the template image, and the LDDMM method was used to calculate and analyze the brain tissue structure of mice.
  • the experimental results are shown in Table 6.
  • the statistical results of the brain volume, striatum volume, and neurocortical volume of the test compound Example 1 are shown in FIG. 3.
  • a in FIG. 3 is the total brain volume statistical result
  • B in FIG. 3 is the striatum.
  • Volume statistical results, C in Figure 3 is the statistical results of neurocortical volume.
  • HD Vehicle mice showed significant atrophy of the whole brain region, striatum, and neurocortex compared to WT Vehicle mice.
  • the test compounds 1 to 5 5 mg / kg / d prepared in the examples of the present invention
  • the degree of atrophy of the entire brain, neurocortex and striatum was significantly reduced (P ⁇ 0.05).
  • the test compound of the present invention can prevent brain atrophy in HD mice.
  • the volume of the entire brain, neurocortex, and striatum of the HD test compound group is equivalent to that of normal WT Vehicle mice.
  • HD pathology is characterized by A large number of MSNs are lost in the striatum, and MSNs can express high levels of DARPP32. Therefore, DARPP32 can be used as a marker of neural damage and neural dysfunction in HD models.
  • the behavioral evaluation of mice in each experimental group was completed. After administration to 25 weeks, 3 mice were taken from each group to isolate hippocampal tissue, striatum, and neurocortical tissue, and to extract total proteins in striatum tissue. Western Blotting experiment was used to detect the protein level of DARPP32.
  • the extraction method of total protein in striatum is as follows: Put the striatum tissue block in 100 ⁇ L lysate (RIPA buffer 10 mL, protease inhibitor 100 ⁇ L, phosphatase inhibitor 100 ⁇ L), lyse on ice for 30 min, and sonicate the cells The machine pulverized the tissue and centrifuged at 14000 rpm for 20 min at 4 ° C. The supernatant was aliquoted and transferred to a new 1.5 mL centrifuge tube, and the protein was extracted. The DARPP 32 protein level was detected by Western blotting experiments.
  • mice The behavioral evaluation of the mice in each experimental group is complete.
  • 3 mice are taken from each group to detect the DARPP 32 protein level.
  • the right brain of the mouse is isolated in 4% paraformaldehyde.
  • the fixed brain tissue was placed in a 30% sucrose solution to the bottom, and then a brain slice with a thickness of 35 ⁇ m was cut out in a coronal slice and stored in DPBS.
  • the primary antibody (anti-Huntingtin, clone EM48, 1:25, mouse, the primary antibody is prepared with blocking solution). Incubate in a 24-well plate and place in a –4 ° C cold room shaker overnight. After washing with PBS, the cells were washed in a 24-well plate (555, anti-mouse) supplemented with a secondary antibody, and incubated in a shaker at room temperature for 2 h. After washing with PBS, the cells were stained in a 24-well plate containing 800 ⁇ L DAPI (prepared with PBS, 1: 10000), and shaken at room temperature for 10 min.
  • DAPI prepared with PBS, 1: 10000
  • the brain slices were taken out and washed once in a 24-well plate containing 800 ⁇ L of PBS, 10 minutes, and shaken at room temperature.
  • the slide was laid flat on the experimental table with the front side facing up, PBS was dropped on the slide, and the brain slide was spread on the PBS. After waiting to dry, the mHtt protein was immunofluorescently stained with anti-Huntingtin antibody and photographed for observation.
  • the cells with mHtt protein aggregation in six visual fields in the right cerebral cortex were counted, and the average value was used for analysis and statistics.
  • the statistical results of the average cell number with mHtt protein aggregation in the cerebral cortex are shown in Table 8.
  • a representative image of the neurocortex immunostained with the EM48 antibody of the test compound Example 1 is shown in FIG. 5.
  • the scale bars are 50 ⁇ m, and the light-colored dots are cells with mHtt protein aggregation.
  • the test compound of the present invention exhibits excellent performance in rescuing motor dysfunction of HD mice in behavioral tests, and significantly improves the motor coordination ability of HD mice.
  • the time for the mice in each HD test compound group to cross the beam in three types of balance beam experiments was equivalent to that of Vehicle-treated HD mice.
  • the drop latency of the HD mice in the administration group was obvious.
  • the growth latency of each HD test compound group was comparable to that of the WT Vehicle group mice.
  • the test compound of the present invention can delay the onset time of HD mice and prolong the onset latency period of HD mice.
  • test compound of the present invention greatly reduces the degree of atrophy of the entire brain, neurocortex and striatum of HD mice.
  • the test compound of the present invention can prevent the loss of DARPP32 in the striatum of HD mice, maintain the level of DARPP32 in the striatum, and improve the function of MSNs in the striatum of HD mice.
  • test compound of the present invention reduces the mHtt protein aggregates in the neurocortex of HD mice, thereby reducing the damage of neurons.
  • test compound of the present invention has significant neuroprotective effects and can be used to treat neurological diseases.
  • Example 24 Protective effect of the compound of the present invention on rats with permanent cerebral ischemia
  • a ligature is reserved at the internal carotid artery bifurcation, and the distal end of the internal carotid artery is temporarily clamped with an arterial clip.
  • a small cut is made at the common carotid artery bifurcation, and a 5 cm long and apex is fired into a spherical shape of 0.3 mm 3 -0 Nylon thread, loosen the arterial clamp, slowly advance the thread plug 18-20mm, stop the thread entry when there is resistance, at this time the nylon thread plug enters the middle artery and blocks blood flow, the ischemic period begins, and the skin is sutured.
  • the sham operation group has the same steps except that no thread plug is inserted.
  • the drugs were given once at 0, 3, 6, and 12 hours after ischemia, and then were administered once a day for 7 consecutive days.
  • the test compound was prepared as a 4 mg / mL solution in a 1% CMC-Na solution, and administered orally by oral administration.
  • the intravenous dose of the edaravone group was 0.078 mg / kg.
  • mice 1 Observe the activity of mice closely after ischemia, record their deaths, and calculate the mortality of mice at 6h, 1d, 2d, 3d, 4d, 5d, 6d, and 7d after ischemia.
  • TTC triphenyltetrazolium chloride
  • Cerebral infarction volume (%) (volume of the contralateral hemisphere of surgery-volume of the uninfarcted part of the surgical hemisphere) / volume of the contralateral hemisphere of surgery ⁇ 100%
  • the neurobehavioral score was scored at the time points of ischemia 6h, 1d, 2d, 3d, 4d, 5d, 6d, and 7d, and the effect of the test compound on neurobehavior of rats with permanent cerebral ischemia was evaluated.
  • the experimental results are shown in the following table 9 shown.
  • n is the initial number of animals in each group of the experiment
  • the Model group and the Sham group showed significant differences, indicating that the modeling was successful.
  • the behavioral symptoms of the model group tended to increase progressively, and then alleviated.
  • the positive control edaravone group showed a difference only on day 7.
  • the neurobehavioral scores on the 3rd and 7th days of the Model group were 3 ⁇ 0.38 and 2.5 ⁇ 0.33, respectively.
  • the percentage of infarct volume in the Model group was 44.4% ⁇ 6.31%, indicating that the model was successfully created.
  • the test compound (20 mg / kg) group prepared in Examples 1 to 5 can reduce the infarct volume.
  • the percentage of infarct volume is 19.58% ⁇ 9.56% to 21.33% ⁇ 7.68%. Therefore, the results of this experiment prove that the test compound examples 1 to 5 (20mg / kg) can significantly reduce the volume of cerebral infarction (P ⁇ 0.05).
  • test compound has a protective effect on permanent cerebral ischemic injury, can reduce behavioral symptoms in rats, and reduces cerebral infarction volume.
  • the compound of the present invention has protective and therapeutic effects on degenerative diseases of the nervous system and central brain injury.

Abstract

本发明具体涉及一类用于治疗神经系统疾病的化合物，该化合物为式(I)所示的化合物或式(I)所示化合物的立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、药学上可接受的盐或所述化合物(I)的前药。本发明还涉及包含这类化合物的药物组合物，以及所述化合物和药物组合物的用途。

Description

用于治疗神经系统疾病的化合物及其应用
本申请要求于2018年08月01日提交中国专利局、申请号为201810865456.2、发明名称为“用于治疗神经系统疾病的化合物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及医药技术领域，具体涉及一种用于治疗神经系统疾病的化合物及其应用。
背景技术
神经系统疾病(Neurological Disease)是发生于中枢神经系统、周围神经系统、植物神经系统的以感觉、运动、意识、植物神经功能障碍为主要表现的疾病，又称神经病。主要临床表现为运动、感觉、反射、自主神经以及高级神经活动功能障碍(Oliveira-Giacomelli A,Naaldijk Y,Sarda-Arroyo L,et al.Purinergic Receptors in Neurological Diseases With Motor Symptoms:Targets for Therapy[J].Front Pharmacol,2018,9:325)。
神经系统疾病中有许多病因不明，分类有重叠现象，但大致可按病因、部位、病理等分类。神经系统疾病的病因包括感染、中毒、遗传缺陷、营养障碍、免疫损伤、代谢紊乱、内分泌紊乱、先天畸形、血液循环障碍和异常增生。常见的神经系统疾病例如严重威胁人类健康的心脑血管疾病，颅脑损伤，和一些神经退行性病变疾病癫痫、阿尔茨海默症、帕金森症和亨廷顿氏舞蹈症等都属于神经系统疾病的范畴。伴随神经系统疾病的出现往往还有一些精神障碍，如抑郁、焦虑等。
亨廷顿舞蹈病(Huntington’s disease,HD)是一种罕见的常染色体显性遗传病，又称慢性进行性舞蹈病、大舞蹈病、亨廷顿病。患者一般在中年发病，出现运动、认知和精神方面的症状。亨廷顿舞蹈病临床症状复杂多变，患者病情呈进行性恶化，通常在发病15～20年后死亡。亨廷顿舞蹈病的临床症状包括三方面，即运动障碍、认知障碍和精神障碍。HD可合并其他疾病，个别患者可发生癫痫、遗传性共济失调和偏头痛等。
导致亨廷顿氏舞蹈症的突变基因IT-15位于人体第4号染色体上，它编码一个相对分子量为3.5×105的蛋白质，由3144个氨基酸组成，被命名为亨廷顿蛋白(Huntingtin,Htt)，从Htt氨基末端第17位氨基酸残基开始有一段重复的谷氨酰胺序列(ployQ)，由lT-15基因第1个外显子(exon1)上的胞嘧啶-腺嘌呤-鸟嘌呤(CAG)重复序列编码。现已确认CAG重复突变可导致HD发生，在正常人群中亨廷顿基因中CAG序列重复数在17到20之间，当重复数在40及其以上者可引起发病，CAG重复数在36到39之间是一种不完全外显性。IT-15基因上的CAG重复编码ployQ重复序列导致其异常扩增一般会产生两个直接结果,其一是野生型亨廷顿蛋白(wild huntingtin,wHtt)的功能缺失,其二是产生突变型亨廷顿蛋白(mutant Htt,mHtt)从而发挥毒性作用。正常人群的wHtt在全身各个器官包括中枢神经系统广泛表达，它在神经系统的发育及 细胞的内吞和分泌中起的关键作用，在神经元中可促进脑源性神经营养因子(BDNF)生产和运输的能力，以及在抑制细胞凋亡中发挥作用。wHtt缺失可能使其功能丧失,对细胞产生致毒性作用，可能会导致亨廷顿疾病。然而多数证据表明，mHtt不管作为单体、中间体、成熟聚集体还是N端片段，均能够表现出毒性。单体是由突变的IT-15产生的全长mHtt,中间体是单体形成聚集体过程中的过渡形式。随着PolyQ的延长，Htt构型容易发生β-片层结构的构象变化导致异常的剪接作用可能产生一个截断的mRNA，其中包含了ployQ区域，它只编码exon 1，被翻译成mHtt蛋白。即使完整的Htt基因被转录和翻译，在蛋白酶的作用下Htt断裂，也会形成含有突变的ployQ的mHtt蛋白N端碎片。转录后修饰也可以通过磷酸化、乙酰化和构象变化来产生更多有毒片段。mHtt可以通过同质或异质二聚化作用形成较大的，不易溶的蛋白质聚集体，这种聚集在HD患者脑部的神经皮层及纹状体的细胞质或细胞核中可见，不能被泛素蛋白酶体系统所溶解，且能改变它的功能，从而对神经元产生一系列损害。另外，mHtt和线粒体膜相互作用，干扰钙的体内平衡直到暴露线粒体失衡，mHtt聚集的区域显示出高能量磷酸盐储存，ATP水平降低，导致在亨廷顿患者中的新陈代谢负荷。
根据US神经学症状和休克国家学会(NINDS)的报道，目前还没有停止或逆转亨廷顿疾病进程的办法，目前尚缺乏特异性的治疗方法，多数情况下，针对不同的HD症状，会选择不同的药物进行对症治疗。一般来说，对于HD患者，运动障碍最容易显现出来，患有舞蹈病的病人会有无意识的舞蹈样动作，它会影响病人的生活质量、运动功能，对此症状被认为与多巴胺能神经元的过度激活有关，因此能够减少多巴胺能神经传递的药物为在临床上使用最多的药物。丁苯那嗪是美国食品药品局(FDA)批准治疗HD舞蹈动作的药物之一。用于治疗HD相关的舞蹈症和迟发性运动障碍。丁苯那嗪的主要机制是抑制单胺囊泡转运。其用药量大时，可以显著减轻亨廷顿的舞蹈症状，但如果停止用药，舞蹈症状会加重。使用此类药物须注意其副作用，包括运动迟缓伴震颤，还需注意抑郁症和自杀率增大，以及引起帕金森病和神经安定药恶性综合征(Neuroleptic Malignant Syndrome)。氘代丁苯那嗪在美国上市，用于治疗HD相关的舞蹈症和迟发性运动障碍，比丁苯那嗪体内代谢更缓慢，半衰期延长，剂量更低。
缺血性心脑血管疾病是指在供应脑的血管壁病变、血液成分改变或血流动力学变化的基础上发生脑部血液供应障碍，导致相应供血区脑组织由于缺血、缺氧而出现的脑组织坏死，并引起短暂或持久的局部或广泛性脑损害，造成一系列神经功能缺损的症状。由缺血造成的损伤分为缺血期原发性损伤(Cerebral ischemia primary injury,CIPI)和缺血期再灌注损伤(Cerebral ischemia reperfusion injury,CIRI)，缺血再灌注损伤是因脑缺血或心肌缺血致组织坏死前，闭塞的血管再通后，不仅功能未得到恢复，反而使损伤加重，是引起多种心脑血管疾病的主要病理生理过程。其中，脑卒中是我国排名第一的致死和致残原因。我国每年约有200万新发脑卒中患者，且发病率有逐年增高的趋势。目前，脑卒中常用药物有：(1)防止血小板凝聚的药物，如阿司匹林，(2)改善微循环、扩充血容量的药物，如右旋糖酐，(3)溶栓药物，如尿激酶，(4)抗凝药物，如肝素，(5)钙离子拮抗剂，如尼莫地平，这些药物都是针对部分脑卒中 患者起效，并且药物作用的机制都不包括对脑神经的保护作用。
综上所述，无论是亨廷顿病还是缺血性脑卒中，现有治疗药物都远不能达到安全性和有效性的治疗预期，需要继续开发具有更优治疗效果的新药。
发明内容
有鉴于此，本发明的目的在于提供一种新型的用于治疗神经系统疾病的化合物，所述化合物可以激活TrkB受体及其下游信号通路，TrkB受体是脑源性神经营养因子的主要信号受体，激活TrkB受体及其下游信号通路可改善多种神经系统疾病的症状。
本发明的另外一个目的在于提供一种新型的用于治疗神经系统疾病的化合物，使所述化合物对神经系统退行性病变具有治疗作用，具体可应用于治疗和改善亨廷顿病、肌萎缩侧索硬化、阿尔茨海默氏病、多发性硬化症、帕金森氏病所导致的临床症状。
本发明的另外一个目的在于提供一种新型的用于治疗神经系统疾病的化合物，所述化合物对中枢性脑损伤具有保护和治疗作用，具体可应用于治疗和改善脑卒中导致的临床症状。
本发明的另外一个目的在于，提供上述化合物在相关神经系统疾病药物制备中的应用，以及提供具体的药物产品。
为了实现上述目的，本发明提供如下技术方案：
一种用于治疗神经系统疾病的化合物，为式(I)所示化合物，或式(I)所示化合物的立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、药学上可接受的盐，或式(I)所示化合物的前药；
Figure PCTCN2019098578-appb-000001
其中，R 1和R 2独立选自-OH、-NH 2、F、Cl、Br、C 1-C 12烷基、C 1-C 12羟基烷基、C 1-C 12杂烷基、C 1-C 12卤代烷基、C 1-C 12烷氧基、C 1-C 12烷氨基、-OC(＝O)R 6和-NHC(＝O)R 7；R 6和R 7独立选自C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氨基；
R 3为H、D、F、Cl或Br；
R 4和R 5独立选自H、D、F、Cl、Br、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基和C 1-C 6烷氨基；m和n各自独立地为0、1、2、3或4。
在一些实施方案中，所述R 1和R 2独立选自-OH、-NH 2、F、C 1-C 6羟基烷基、C 1-C 6 杂烷基、C 1-C 6烷氧基和C 1-C 6烷氨基。在另一些实施方案中，所述R 1和R 2独立选自-OH、-NH 2、F和-OCH 3；在本发明的一些具体实施方案中，所述R 1和R 2有如下之一的选择方案：
(1)R 1＝-OH，R 2＝-OH；
(2)R 1＝-OH，R 2＝-OCH 3；
(3)R 1＝-OCH 3，R 2＝-OCH 3；
(4)R 1＝-OCH 3，R 2＝-OH；
(5)R 1＝-NH 2，R 2＝-NH 2；
(6)R 1＝-OH，R 2＝-F。
在本发明的一些实施方案中，所述R 4和R 5独立选自H、D、F、Cl、Br、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、-N(CH 3) 2、-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3和-OCH(CH 3) 2。在另一些具体实施方案中，所述R 5为-CH 3、-CH 2CH 2CH 3、-N(CH 3) 2、-OCH 3、D或F。
在一些实施方案中，所述m和n各自独立地为0、1或2。在本发明另一些具体实施方案中，所述n为0，即没有取代基R 4；所述m为1或2。
当R 1＝-OH，R 2＝-OH时，式(I)所示化合物具有式(II)所示结构；
Figure PCTCN2019098578-appb-000002
其中，R 3为H、D、F、Cl或Br；
R 4和R 5独立选自H、D、F、Cl、Br、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、-N(CH 3) 2、-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3和-OCH(CH 3) 2；
m和n各自独立地为0、1或2。
在本发明的一些具体实施方式中，所述R 5为-CH 3、-CH 2CH 2CH 3、-N(CH 3) 2、-OCH 3、D或F；所述n为0，即没有取代基R 4；所述m为0、1或2。
在本发明具体实施方式中，式(I)所示化合物为如下之一化合物：
Figure PCTCN2019098578-appb-000003
Figure PCTCN2019098578-appb-000004
与Vehicle组相比，本发明提供的化合物可使神经元细胞中p-TrkB/t-TrkB蛋白相对强度增强2.06～2.34倍，表现出了显著性的差异(P＜0.05)，p-Akt/t-Akt蛋白相对强度增强2.09～2.40倍，与BDNF效果相当。表明本发明提供的化合物可以激活TrkB受体及其下游AKT信号通路，进而说明这些化合物对神经系统疾病有治疗作用以及对神经元有较好地保护作用。
在更加具体的神经系统疾病药效分析中，本发明分别通过小鼠模型模拟亨廷顿疾病和脑卒中的病变症状，通过行为学、生理生化指标等检测项目证实了本发明所提供的化合物可对神经系统退行性病变和中枢性脑损伤具有保护和治疗作用。
基于此，本发明提供了一种药物组合物，其包含本发明所提供的任何一种化合物。进一步地，还可包括药学上可接受辅料和/或其他神经系统疾病治疗药物。本发明所用“药学上可接受的辅料”意指与给药剂型或药物组合物一致性相关的药学上可接受的材料、载体、赋形剂、混合物或溶媒。每种辅料在混合时必须与药物组合物的其它成分相容，以避免对患者给药时会大大降低本发明公开化合物的功效的相互作用和会导致不是药学上可接受的药物组合物的相互作用。此外，每种辅料必须是药学上可接受的，例如，具有足够高的纯度。合适的药学上可接受的辅料会依所选具体剂型而不同。此外，可根据它们在组合物中的特定功能来选择药学上可接受的辅料。例如，可选择能有助于生产均一剂型的某些药学上可接受的辅料。可选择能有助于生产稳定剂型的某些药学上可接受的辅料。可选择对患者给药时有助于携带或运输本发明公开化合物从身体的一个器官或部分到身体的另一个器官或部分的某些药学上可接受的辅料。可选择增强患者依从性的某些药学上可接受的辅料。合适的药学上可接受的辅料包括以下类型的辅料：赋形剂、稀释剂、填充剂、粘合剂、崩解剂、润滑剂、助流剂、造粒剂、包衣剂、润湿剂、溶剂、共溶剂、助悬剂、乳化剂、甜味剂、矫味剂、掩味剂、着色剂、防结块剂、保湿剂、螯合剂、塑化剂、增粘剂、抗氧化剂、防腐剂、稳定剂、表面活性剂和缓冲剂。技术人员可认识到，某些药学上可接受的辅料可提供不 止一种功能，并提供可供选择的功能，这取决于制剂中存在多少该辅料和制剂中存在哪些其他辅料。
本发明化合物可以作为单独的活性试剂给药，或者可以与其它神经系统疾病治疗药物联合给药，包括具有相同或相似治疗活性并且对于此类联合给药确定为安全且有效的其它化合物。一方面，本发明提供治疗、预防或改善疾病或病症的方法，包括给予安全有效量的包含本发明公开化合物与一种或多种其它神经系统疾病治疗药物的联合药物。所述其他神经系统疾病治疗药物包括用于治疗亨廷顿病的药物、用于治疗帕金森病的药物、用于治疗抑郁症的药物、用于治疗运动失调的药物、用于治疗脑卒中的药物、用于治疗中枢神经系统损伤疾病的药物、用于治疗肌萎缩侧索硬化的药物和用于治疗多发性硬化症的药物。在一些实施方案中，这些其它神经系统疾病治疗药物选自用于治疗运动失调的药物、用于治疗亨廷顿病的药物、用于治疗缺血性脑血管疾病的药物中的一种或多种。在一些实施方案中，联合药物包含一种或两种其他神经系统疾病药物。用于治疗亨廷顿病的药物包括但不限于，丁苯那嗪、氘代丁苯那嗪、Pridopidine；用于治疗脑卒中的药物包括但不限于，丁苯酞、依达拉奉；用于治疗多发性硬化症的药物选自Ocrelizumab，Tecfidera，格拉替雷、克帕松，芬戈莫德、那他珠单抗、Avonex、利比(Rebif)、特立氟胺、Plegridy、倍泰龙、Ampyra；用于治疗抑郁症的药物包括但不限于，西酞普兰、依地普仑、氟西汀、氟伏沙明、帕罗西汀、舍曲林、或维拉佐酮；5-羟色胺-去甲肾上腺素再摄取抑制剂，例如地文拉法辛、度洛西汀、米那普仑、文拉法辛；去甲肾上腺素能和特定血清素能抗抑郁剂，例如米安色林和米氮平；去甲肾上腺素再摄取抑制剂，例如阿托西汀、马吲哚、瑞波西汀、维洛沙秦；去甲肾上腺素-多巴胺再摄取抑制剂，例如安非他酮；选择性5-羟色胺再摄取增强剂，例如噻奈普汀和安咪奈丁；去甲肾上腺素-多巴胺去抑制剂，例如阿戈美拉汀；三环类抗抑郁剂，例如阿米替林、氯米帕明、多虑平、丙咪嗪、曲米帕明、去郁敏、去甲替林、普罗替林；单胺氧化酶抑制剂，例如异卡波肼、吗氯贝胺、苯乙肼、司来吉兰、反苯环丙胺。用于治疗肌萎缩侧索硬化的药物包括但不限于，IL-6受体单抗(Tocilizumab)、奥扎尼珠单抗、BIIB067(Isis-SOD1Rx)。
本发明公开的化合物或所述药物组合物通常被配制成适合于通过所需途径对患者给药的剂型。例如，剂型包括那些适合于以下给药途径的剂型：(1)口服给药，例如片剂、胶囊剂、囊片剂、丸剂、含片剂、粉剂、糖浆剂、酏剂、混悬剂、溶液剂、乳剂、颗粒剂和扁囊剂；(2)胃肠外给药，例如无菌溶液剂、混悬剂和冻干粉末剂；(3)透皮给药，例如透皮贴片剂；(4)直肠给药，例如栓剂；(5)吸入，例如气雾剂、溶液剂和干粉剂；和(6)局部给药，例如乳膏剂、油膏剂、洗剂、溶液剂、糊剂、喷雾剂、泡沫剂和凝胶剂。
在一些实施方案中，本发明公开的治疗方法包括对有需要的患者给予安全有效量的本发明化合物或包含本发明化合物的药物组合物。本发明公开的各实施方案包括通过对有需要的患者给予安全有效量的本发明公开化合物或给予包含本发明公开化合物的药物组合物，来治疗神经系统疾病的方法。
根据本发明记载的试验结果，本发明提供了所述的化合物和所述的药物组合物在 制备用于预防或治疗神经系统疾病的药物中的用途。所述神经系统疾病包括神经元损伤、神经元退化、脑萎缩相关疾病或病症、中枢神经系统损伤、脑卒中(中风)、抑郁症、焦虑症、肌萎缩侧索硬化、阿尔茨海默氏病、自闭症、亨廷顿氏病、瑞特综合征、癫痫症、帕金森氏病、创伤后应激障碍、糖尿病性神经病变、多发性硬化症和周围神经病。
根据本发明记载的试验结果，本发明还提供了所述的化合物和所述的药物组合物在制备用于激动TrkB受体的药物中的用途。
还在另一方面，本发明提供一种用于激动TrkB受体的方法，包括向有需要的个体或样本给予治疗有效量的本发明公开的化合物、本发明制备方法制备的化合物或本发明公开的化合物的药物组合物。同时，本发明提供一种用于预防、治疗或减轻神经系统疾病的方法，包括向有需要的个体或样本给予治疗有效量的本发明公开的化合物、本发明制备方法制备的化合物或本发明公开的化合物的药物组合物。
本发明所述化合物的“治疗有效量”或“有效量”可以是0.1mg/kg～100mg/kg。在一些实施例中，本发明所述化合物的“治疗有效量”或“有效量”可以是0.5mg/kg～50mg/kg；在另一些实施例中，本发明所述化合物的“治疗有效量”可以是0.2mg/kg～25mg/kg。
由以上技术方案可知，本发明提供了一种新型的具有式(I)或式(II)结构的黄酮衍生物，其可以激活TrkB受体及其下游AKT信号通路，进而说明所述化合物对神经系统疾病有较好地治疗作用以及对神经元有较好地保护作用，特别是对亨廷顿疾病这种神经系统退行性病变和脑卒中这种中枢性脑损伤具有较佳的治疗作用。
附图说明
图1所示为实施例1提供的受试化合物的p-TrkB/t-TrkB和p-AKT/t-AKT电泳图和蛋白相对强度图，其中，A为p-TrkB/t-TrkB蛋白电泳图，B为p-TrkB/t-TrkB蛋白水平统计结果，C为p-AKT/t-AKT的蛋白电泳图；D为p-AKT和t-AKT的蛋白水平统计结果；
图2所示为各受试化合物的ELISA检测p-Akt相对含量统计结果；
图3所示为实施例1提供的受试化合物的全脑、纹状体及神经皮层的体积MRI检测结果，其中，A为全脑体积统计结果，B为纹状体体积统计结果，C为神经皮层体积统计结果；
图4所示为实施例1提供的受试化合物的DARPP32蛋白相对强度实验结果；其中，A为DARPP32电泳图；B为DARPP 32/β-action蛋白水平相对强度；
图5所示为实施例1提供的受试化合物的脑组织mHtt蛋白聚集体经EM48抗体免疫染色的神经皮层代表性图像；
图6所示为各组5个大鼠脑组织样本TTC染色结果。
具体实施方式
本发明公开了一种用于治疗神经系统疾病的化合物及其应用，本领域技术人员可 以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明中。本发明所述化合物及其相关应用通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的化合物及其相关应用进行改动或适当变更与组合，来实现和应用本发明技术。
除非另作说明，式(I)或(II)所示化合物的立体异构体、互变异构体、溶剂化物、代谢产物、盐和药学上可接受的前药都包含在本发明范围内。
本发明公开化合物可含有不对称或手性中心，因此可以不同的立体异构体形式存在。本发明旨在使式(I)或(II)所示化合物的所有立体异构体形式，包括但不限于非对映异构体、对映异构体、阻转异构体和几何(或构象)异构体，以及它们的混合物如外消旋混合物，成为本发明的组成部分。
在本发明公开的结构中，当任意特定的手性原子的立体化学未指明时，则该结构的所有立体异构体都考虑在本发明之内，并且作为本发明公开化合物包括在本发明中。当立体化学被表示特定构型的实楔形线(solid wedge)或虚线指明时，则该结构的立体异构体就此明确和定义。
式(I)或(II)所示化合物可以以不同的互变异构体形式存在，并且所有这些互变异构体，如本发明所述的互变异构体，都包括在本发明范围内。
式(I)或(II)所示化合物可以以盐的形式存在。在一些实施方案中，所述盐是指药学上可接受的盐。在另一些实施方案中，所述盐还可以是用于制备和/或提纯式(I)或(II)所示化合物和/或用于分离本式(I)或(II)所示化合物的对映体的中间体。
除非另外说明，本发明所使用的所有科技术语具有与本发明所属领域技术人员的通常理解相同的含义。本发明涉及的所有专利和公开出版物通过引用方式整体并入本发明。
“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体、(顺/反)异构体、阻转异构体，等等。“对映异构体”是指一个化合物的两个不能重叠但互成镜像关系的异构体。“非对映异构体”是指有两个或多个手性中心并且其分子不互为镜像的立体异构体。非对映异构体具有不同的物理性质，如熔点、沸点、光谱性质和反应性。非对映异构体混合物可通过高分辨分析操作如电泳和色谱，例如HPLC来分离。
术语“互变异构体”或“互变异构形式”是指具有不同能量的可通过低能垒(low energy barrier)互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(protontautomer)(也称为质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组来进行的互相转化。酮-烯醇互变异构的具体实例是戊烷-2,4-二酮和4-羟基戊-3-烯-2-酮互变异构体的互变。互变异构的另一个实例是酚-酮互变异构。酚-酮互变异构 的一个具体实例是吡啶-4-醇和吡啶-4(1H)-酮互变异构体的互变。除非另外指出，本发明化合物的所有互变异构体形式都在本发明的范围之内。
术语“取代的”表示所给结构中的一个或多个可被取代的氢原子被具体取代基所取代。除非其他方面表明，一个取代的基团可以有一个取代基在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。
术语“任选”或者“任选地”意味着随后所描述的事件或者环境可以但不必发生，该说明包括该事情或者环境发生或者不发生的场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情景和杂环基团不被烷基取代的情景。
术语“未取代的”，表示指定基团不带有取代基。术语“任选地被……所取代”，可以与术语“未取代或被……所取代”交换使用，即所述结构是未取代的或者被一个或多个本发明所述的取代基取代，本发明所述的取代基包括，但不限于D、-OH、-NH 2、F、Cl、Br、C 1-C 12烷基、C 1-C 12卤代烷基、C 1-C 12烷氧基或C 1-C 12烷氨基、-OC(＝O)R 6或-NHC(＝O)R 7，其中R 6和R 7具有本发明所述含义。
在本发明中所采用的描述方式“……独立选自”、“各……独立地为”、“……各自独立地为”和“……独立地为”可以互换，均应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1-C 6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
本发明使用的术语“烷基”或“烷基基团”，表示含有1至20个碳原子，饱和的直链或支链一价烃基基团，其中，所述烷基基团可以任选地被一个或多个本发明描述的取代基所取代。除非另外详细说明，烷基基团含有1-20个碳原子。在一些实施方案中，烷基基团含有1-12个碳原子；在另一些实施方案中，烷基基团含有2-12个碳原子；在另一些实施方案中，烷基基团含有1-6个碳原子；在另一些实施方案中，烷基基团含有2-6个碳原子；在又一些实施方案中，烷基基团含有1-4个碳原子；还在一些实施方案中，烷基基团含有1-3个碳原子。所述烷基基团可以任选地被一个或多个本发明描述的取代基所取代。烷基基团的实例包含，但并不限于，甲基(Me、-CH 3)，乙基(Et、-CH 2CH 3)，正丙基(n-Pr、-CH 2CH 2CH 3)，异丙基(i-Pr、-CH(CH 3) 2)，正丁基(n-Bu、-CH 2CH 2CH 2CH 3)，异丁基(i-Bu、-CH 2CH(CH 3) 2)，仲丁基(s-Bu、-CH(CH 3)CH 2CH 3)，叔丁基(t-Bu、-C(CH 3) 3)，等等。
术语“杂烷基”表示烷基中可以插入一个或多个杂原子，其中烷基和杂原子具有如本发明所述的含义。杂烷基通过碳原子与其他基团相连。除非另外详细说明，杂烷基基团含有1-12个碳原子，另外一些实施方案是，杂烷基基团含有1-8个碳原子，另外一些实施方案是，杂烷基基团含有1-6个碳原子，另外一些实施方案是，杂烷基基团含有1-4个碳原子，另外一些实施方案是，杂烷基基团含有1-3个碳原子。“杂烷基”的实 例包括，但并不限于，-CH 2OCH 3、-CH 2CH 2OCH 3、-CH 2SCH 3、-CH 2N(CH 3) 2、-CH 2OCH 2(CH 3) 2、-CH 3CH 2CH 2OCH 3、-CH 3CH 2OCH 2CH 3、-CH 3CH 2CH 2OCH 2CH 3等。
术语“烷氧基”表示烷基基团通过氧原子与分子其余部分相连，其中烷基基团具有如本发明所述的含义。除非另外详细说明，所述烷氧基基团含有1-12个碳原子。在一些实施方案中，烷氧基基团含有1-6个碳原子；在另一些实施方案中，烷氧基基团含有1-4个碳原子；在又一些实施方案中，烷氧基基团含有1-3个碳原子。所述烷氧基基团可以任选地被一个或多个本发明描述的取代基所取代。
术语“卤代烷基”、“卤代烯基”或“卤代烷氧基”表示烷基，烯基或烷氧基基团被一个或多个卤素原子所取代，这样的实例包含，但并不限于，二氟乙基(-CH 2CHF 2,-CF 2CH 3,-CHFCH 2F)、三氟乙基(-CH 2CF 3,-CF 2CH 2F,-CFHCHF 2)、三氟甲基(-CF 3)、三氟甲氧基(-OCF 3)等。
术语“羟基烷基”表示烷基基团被一个或多个羟基所取代，这样的实例包含，但并不限于，羟甲基(-CH 2OH)、羟乙基(-CH 2CH 2OH)等。
术语“卤素”是指氟(F)、氯(Cl)、溴(Br)或碘(I)。
术语“烷氨基”包括“N-烷氨基”和“N,N-二烷氨基”，其中，氨基基团分别独立地被一个或两个烷基基团所取代。其中，一些实施方案中，烷氨基是一个或两个C 1-C 12烷基连接到氮原子上形成的较低级的烷基氨基基团。在另一些实施方案中，烷氨基是一个或两个C 1-C 6烷基连接到氮原子上形成的较低级的烷基氨基基团。在另一些实施方案中，烷氨基是一个或两个C 1-C 4烷基连接到氮原子上形成的较低级的烷基氨基基团。还在另外一些实施方案中，烷氨基是一个或两个C 1-C 3烷基连接到氮原子上形成的较低级的烷基氨基基团。合适的烷氨基基团可以是单烷基氨基或二烷基氨基，烷氨基的实例包括，但并不限于，N-甲氨基(-NH(CH 3))，N-乙氨基(-NH(CH 2CH 3))，N,N-二甲氨基(-N(CH 3) 2)，N,N-二乙氨基(-N(CH 2CH 3) 2)，N-乙基丙基-2-氨基等等。
像本发明所描述的，取代基画一个键连接到中心的环上形成的环体系(如式a所示)代表取代基在该环体系上所有可取代的位置择一取代。例如，式a代表取代基R可以择一取代D环上所有可以被取代的位置，如式b～式d所示。若该取代基不止一个，则各取代基可以选择不同的取代位置或相同的取代位置。以下结构式b～式d中波浪线表示连接键。
Figure PCTCN2019098578-appb-000005
术语“药学上可接受的”是指物质或组合物必须与包含制剂的其它成分和/或用其治疗的哺乳动物化学上和/或毒理学上相容。
本发明的“药学上可接受的盐”可以用常规化学方法由母体化合物、碱性或酸性部 分来合成。一般而言，该类盐可以通过使这些化合物的游离酸形式与化学计量量的适宜碱(如Na、Ca、Mg或K的氢氧化物、碳酸盐、碳酸氢盐等)反应，或者通过使这些化合物的游离碱形式与化学计量量的适宜酸反应来进行制备。该类反应通常在水或有机溶剂或二者的混合物中进行。一般地，在适当的情况中，需要使用非水性介质如乙醚、乙酸乙酯、乙醇、异丙醇或乙腈。在例如“Remington′s Pharmaceutical Sciences”，第20版，Mack Publishing Company，Easton，Pa.，(1985)；和“药用盐手册：性质、选择和应用(Handbook of Pharmaceutical Salts：Properties，Selection，and Use)”，Stahl and Wermuth(Wiley-VCH，Weinheim，Germany，2002)中可找到另外一些适宜盐的列表。
另外，本发明公开的化合物、包括它们的盐，也可以以它们的水合物形式或包含其溶剂(例如乙醇、DMSO，等等)的形式得到，用于它们的结晶。本发明公开化合物可以与药学上可接受的溶剂(包括水)固有地或通过设计形成溶剂化物；因此，本发明旨在包括溶剂化的和未溶剂化的形式。
此外，本发明公开的化合物可以以前药形式给药。在本发明中，本发明公开化合物的“前药”是对患者给药时，最终能在体内释放出本发明公开化合物的功能性衍生物。
本发明所使用的术语“前药”，代表一个化合物在体内转化为式(I)或式(II)所示的化合物。这样的转化受前体药物在血液中水解或在血液或组织中经酶转化为母体结构的影响。本发明前体药物类化合物可以是酯，在现有的发明中酯可以作为前体药物的芳香酯类，脂肪族(C 1-C 24)酯类，酰氧基甲基酯类，碳酸酯，氨基甲酸酯类和氨基酸酯类。例如本发明里的一个化合物包含羟基，即可以将其酰化得到前体药物形式的化合物。其他的前体药物形式包括磷酸酯，如这些磷酸酯类化合物是经母体上的羟基磷酸化得到的。
“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用实验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。
本发明具体实施方式中采用的试剂购买于商品供应商如Aldrich Chemical Company,Arco Chemical Company and Alfa Chemical Company，使用时都没有经过进一步纯化，除非其他方面表明。一般的试剂从阿拉丁试剂、天津市福晨化学试剂厂、武汉鑫华远科技发展有限公司、青岛腾龙化学试剂有限公司和青岛海洋化工厂购买得到。Neurobasal-SFM、Leibovitz’s、PBS、FBS、GlutaMAX-I(100×)购自Thermo Fisher Scientific；PDL、DNase、Trypsin-EDTA(0.25％)、异氟烷、DPBS购自Sigma-Aldrich；Anti-TrkB、Anti-AKT购自Cell signalling。Western blot实验装置购自美国Invitrogen公司。BDNF购自Peprotech公司。
1H NMR谱使用Bruker 400MHz或600MHz核磁共振谱仪记录。 1H NMR谱以CDCl 3、D 2O、DMSO-d 6、CD 3OD或丙酮-d 6为溶剂(以ppm为单位)，用TMS(0ppm) 或氯仿(7.26ppm)作为参照标准。当出现多重峰的时候，将使用下面的缩写：s(singlet，单峰)、d(doublet，双峰)、t(triplet，三重峰)、m(multiplet，多重峰)、br(broad ened，宽峰)、dd(d oublet of doublets，双二重峰)、ddd(doublet of doublet of doublets，双重双二重峰)、dt(doublet of triplets，双三重峰)、tt(triplet of triplets，三三重峰)。偶合常数J，用赫兹(Hz)表示。
低分辨率质谱(MS)数据的测定条件是：Agilent 6120四级杆HPLC-M(柱子型号：Zorbax SB-C18,2.1×30mm,3.5微米，6min，流速为0.6mL/min。流动相：5％～95％(含0.1％甲酸的CH3CN)在(含0.1％甲酸的H 2O)中的比例)，采用电喷雾电离(ESI)，在210nm/254nm下，用UV检测。
纯的化合物的使用Agilent 1260pre-HPLC或Calesep pump 250pre-HPLC(柱子型号：NOVASEP 50/80mm DAC)，在210nm/254nm用UV检测。
下面简写词的使用贯穿本发明：
BDNF 脑源性神经营养因子
CMC-Na 羧甲基纤维素钠
DARPP-32 多巴胺和cAMP调节的磷蛋白
DPBS 杜氏磷酸缓冲液
EtOAc 乙酸乙酯
FBS 胎牛血清
HRP 辣根过氧化物酶
MCAO 中脑血管阻塞
MSNs 纹状体中等棘状神经元
MRI 磁共振成像
THF 四氢呋喃
TrkB 酪氨酸激酶受体B
Xphos 2-二环己基磷-2’,4’,6’-三异丙基联苯
Pd 2(dba) 3 三(二亚苄基茚丙酮)二钯
Pd/C 钯/碳
PE 石油醚
PBS 磷酸盐缓冲液
WT 野生型
制备本发明公开化合物的典型合成步骤如下面的合成方案所示。除非另外说明，各R 1、R 2、R 3、R 4、R 5、R 6、R 7、n和m具有如本发明所述的定义。
合成方案一:
Figure PCTCN2019098578-appb-000006
具有如式(1-6)所示结构的本发明公开化合物可以通过合成方案1描述的一般合成方法制备得到，具体步骤可参考实施例。合成方案一中，取代的2-羟基苯乙酮类化合物(1-1)与任选取代的4-溴苯甲醛(1-2)在碱性条件下发生羟醛缩合反应，生成化合物(1-3)。接着，化合物(1-3)在高温条件下先与碘反应得到活化的中间体，再经关环反应，生成化合物(1-4)，最后化合物(1-4)和任选取代的哌啶(1-5)在Xphos和Pd 2(dba) 3的催化下偶联，得到化合物(1-6)。
合成方案二：
Figure PCTCN2019098578-appb-000007
Figure PCTCN2019098578-appb-000008
具有如式(2-6)所示结构的本发明公开化合物可以通过合成方案二描述的一般合成方法制备得到，具体步骤可参考实施例。合成方案二中，取代的1-(2,3,4-三羟基)苯基乙酮(2-1)在碱性条件下与BnBr发生反应，得到双苄基保护的化合物(2-2)，化合物(2-2)与任选取代的4-溴苯甲醛(1-2)在碱性条件下发生羟醛缩合反应，生成化合物(2-3)。接着，化合物(2-3)在高温条件下先与碘反应得到活化的中间体，再经关环反应，生成化合物(2-4)，化合物(2-4)和任选取代的哌啶(1-5)在Xphos和Pd 2(dba) 3的催化下偶联，得到化合物(2-5)，化合物(2-5)经催化氢化脱去苄基保护基，就得到化合物(2-6)。
所属领域的专业人员将认识到：本发明所描述的化学反应可以用来合适地制备许多本发明的其他化合物，且用于制备本发明的化合物的其它方法都被认为是在本发明的范围之内。例如，根据本发明那些非例证的化合物的合成可以成功地被所属领域的技术人员通过修饰方法完成，如适当的保护干扰基团，通过利用其他已知的试剂除了本发明所描述的，或将反应条件做一些常规的修改。另外，本发明所公开的反应或已知的反应条件也公认地适用于本发明其他化合物的制备。
以下就本发明所提供的一种用于治疗神经系统疾病的化合物及其应用做进一步说明。
实施例1：7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(1；7,8-dihydroxy-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000009
步骤(1)1-(3,4-二(苄氧基)-2-羟基苯基)乙酮(1b)的合成
向1-(2,3,4-三羟基苯基)乙酮(1a,50.0g,297.4mmol)、溴化苄(101.7g,594.7mmol)和乙腈(1200mL)的混合物中，加入碳酸钾(82.2g,594.7mmol)，所得混合物在80℃下搅拌15小时。反应结束后，冷却至室温，减压抽滤除去不溶物，所得滤液减压浓缩得到残留物。残留物溶于乙醇(1000mL)中，升温至80℃搅拌1小时，冷却至室温，有固体析出，过滤得到滤饼，减压干燥后得黄色固体，即为化合物1-(3,4-二(苄氧基)-2-羟基苯基)乙酮(1b,60.0g,产率58％)。LC-MS(ESI):m/z 348.9[M+H] +.
步骤(2)1-(3,4-二(苄氧基)-2-羟基苯基)-3-(4-溴苯基)丙-2-烯-1-酮(1c)的合成
向1-(3,4-二(苄氧基)-2-羟基苯基)乙酮(1b,60.0g,172.2mmol)、4-溴苯甲醛(31.9g,172.2mmol)、乙醇(300mL)和H 2O(100mL)的混合物中加入氢氧化钾(19.3g,344.4mmol)。所得混合物升温至80℃，搅拌过夜。反应结束后，冷却至室温，然后用1N的盐酸溶液调节至PH＝5，有固体析出。减压抽滤，收集滤饼，减压干燥后得到红色固体75.1g，即为粗产物1-(3,4-二(苄氧基)-2-羟基苯基)-3-(4-溴苯基)丙-2-烯-1-酮(1c)。化合物不需纯化直接用于下一步反应。LC-MS(ESI):m/z 515.1[M+H] +.
步骤(3)7,8-二(苄氧基)-2-(4-溴苯基)-4H-色烯-4-酮(1d)的合成
向1-(3,4-二(苄氧基)-2-羟基苯基)-3-(4-溴苯基)丙-2-烯-1-酮(1c,103.7g,201.2mmol)的DMSO(800mL)溶液中，加入碘单质(5.0g,20.0mmol)，所得混合物升温至130℃～140℃搅拌6小时。混合物冷却至室温，加入EtOAc(300mL)稀释，所得溶液依次用水(100mL×3)、饱和食盐水(100mL×3)洗涤，然后用无水硫酸钠干燥，滤液减压浓缩，得到残留物。所得残留物经硅胶柱层析(洗脱剂：PE/EtOAc(v/v)＝ 3/1～1/1)纯化，得到黄色固体，即为产物7,8-二(苄氧基)-2-(4-溴苯基)-4H-色烯-4-酮(1d,41.0g,两步总产率40％)。LC-MS(ESI):m/z 513.1[M+H] +.
步骤(4)7,8-二(苄氧基)-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮(1e)的合成
在手套箱中，向7,8-二(苄氧基)-2-(4-溴苯基)-4H-色烯-4-酮(1d,41.0g,79.9mmol)、Xphos(7.4g,15.6mmol),Pd 2(dba) 3(7.15g,7.8mmol)和碳酸铯(50.7g,15.6mmol)的混合物中加入1,4-二氧六环(400mL)和哌啶(9.9g,117mmol)。所得混合物升温至110℃搅拌2.5小时。反应结束后，混合物冷却至室温，倾入水(300mL)中，加入EtOAc(200mL×3)萃取，分出有机相，用无水硫酸钠干燥。滤液减压浓缩，所得残留物经硅胶柱层析(洗脱剂：DCM/MeOH(v/v)＝40/1)到10:1)纯化，得到黄色固体，即为产物(1e,21.2g,产率51％)。LC-MS(ESI):m/z 518.2[M+H] +.
步骤(5)7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮(1)的合成
向化合物7,8-二(苄氧基)-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮(1e,21.0g,40.6mmol)、THF(200mL)和甲醇(50mL)的混合物中，加入Pd/C(2.1g,10％含量)，所得混合物抽除空气用氢气填充，重复三次。所得混合物在室温下搅拌过夜。反应结束后，硅藻土过滤，除去催化剂，滤液减压浓缩得到棕色残留物。向残留物中加入MeOH(30mL)和Et 2O(50mL)，升温至50℃搅拌15分钟，有固体析出。减压过滤，滤饼用乙醚洗涤，收集滤饼，减压干燥得到黄色固体，即为产物7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮(1,共11.04g,产率81％)。LC-MS:337.9[M+H] +，98.12％(纯度，UV 214nm)；
1H NMR(400MHz,DMSO-d 6)δ10.23(s,1H),9.37(s,1H),7.96(d,J＝9.2Hz,2H),7.36(d,J＝8.4Hz,1H),7.04(d,J＝8.8Hz,2H),6.91(d,J＝8.8Hz,1H),6.67(s,1H),3.34-3.33(m,4H),1.63-1.53(m,6H).
实施例2：7,8-二甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(2；7,8-dimethoxy-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000010
Figure PCTCN2019098578-appb-000011
步骤(1)3-(4-溴苯基)-1-(2-羟基-3,4-二甲氧基苯基)-丙-2-烯-1-酮(2b)的合成
向1-(2-羟基-3,4-二甲氧基苯基)乙酮(2a,33.7g,172.2mmol)、4-溴苯甲醛(31.9g,172.2mmol)、乙醇(300mL)和H 2O(100mL)的混合物中加入氢氧化钾(19.3g,344.4mmol)。所得混合物升温至80℃，搅拌过夜。反应结束后，冷却至室温，然后用1N的盐酸溶液调节至PH＝5，有固体析出。减压抽滤，收集滤饼，减压干燥后得到红色固体52.9g，即为粗产物3-(4-溴苯基)-1-(2-羟基-3,4-二甲氧基苯基)-丙-2-烯-1-酮(2b)。化合物不需纯化直接用于下一步反应。LC-MS(ESI):m/z 364.1[M+H] +.
步骤(2)2-(4-溴苯基)-7,8-二甲氧基-4H-色烯-4-酮(2c)的合成
向3-(4-溴苯基)-1-(2-羟基-3,4-二甲氧基苯基)-丙-2-烯-1-酮(2b，73.0g,201.2mmol)的DMSO(800mL)溶液中，加入碘单质(5.0g,20.0mmol)，所得混合物升温至130～140℃搅拌6小时。混合物冷却至室温，加入EtOAc(300mL)稀释，所得溶液依次用水(100mL×3)、饱和食盐水(100mL×3)洗涤，然后用无水硫酸钠干燥，滤液减压浓缩，得到残留物。所得残留物经硅胶柱层析(洗脱剂：PE/EtOAc(v/v)＝3/1～1/1)纯化，得到黄色固体，即为产物2-(4-溴苯基)-7,8-二甲氧基-4H-色烯-4-酮(2c,25.2g,两步总产率40％)。LC-MS(ESI):m/z 361.1[M+H] +.
步骤(3)7,8-二甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮(2)的合成
在手套箱中，向2-(4-溴苯基)-7,8-二甲氧基-4H-色烯-4-酮(2c,25.2g,79.9mmol)、Xphos(7.4g,15.6mmol),Pd 2(dba) 3(7.15g,7.8mmol)和碳酸铯(50.7g,15.6mmol)的混合物中加入1,4-二氧六环(400mL)和哌啶(9.9g,117mmol)。所得混合物升温至110℃搅拌2.5小时。反应结束后，混合物冷却至室温，倾入水(300mL)中，加入EtOAc(200mL×3)萃取，分出有机相，用无水硫酸钠干燥。滤液减压浓缩，所得残留物经硅胶柱层析(洗脱剂：DCM/MeOH(v/v)＝30/1)到20/1)纯化，得到黄色固体，即为产物(2,共14.9g,产率51％)。LC-MS(ESI):m/z 366.2[M+H] +.
1H NMR(400MHz,DMSO-d 6)δ7.66(d,J＝9.2Hz,2H),7.26(d,J＝8.4Hz,1H),7.04(d,J＝8.8Hz,2H),6.91(d,J＝8.8Hz,1H),6.47(s,1H),3.89(s,3H),3.80(S,3H),3.34-3.33(m,4H),1.63-1.53(m,6H).
实施例3：2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮
(3；2-(4-(4-(dimethylamino)piperidin-1-yl)phenyl)-7,8-dihydroxy-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000012
步骤(1)7,8-二(苄氧基)-2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-4H-色烯-4-酮(3a)的合成
在手套箱中，向7,8-二(苄氧基)-2-(4-溴苯基)-4H-色烯-4-酮(1d,41.0g,79.9mmol)、Xphos(7.4g,15.6mmol),Pd 2(dba) 3(7.15g,7.8mmol)和碳酸铯(50.7g,15.6mmol)的混合物中加入1,4-二氧六环(400mL)和4-二甲氨基哌啶(15.0g,117mmol)。所得混合物升温至110℃搅拌2.5小时。反应结束后，混合物冷却至室温，倾入水(300mL)中，加入EtOAc(200mL×3)萃取，分出有机相，用无水硫酸钠干燥。滤液减压浓缩，所得残留物经硅胶柱层析(洗脱剂：DCM/MeOH(v/v)＝40/1)到10:1)纯化，得到黄色固体，即为产物7,8-二(苄氧基)-2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-4H-色烯-4-酮(产物3a,26.88g,产率60％)。LC-MS(ESI):m/z 561.68[M+H] +.
步骤(2)2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮(3)的合成
向化合物7,8-二(苄氧基)-2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-4H-色烯-4-酮(3a,26.88g,47.94mmol)、THF(200mL)和甲醇(50mL)的混合物中，加入Pd/C(2.6g,10％含量)，所得混合物抽除空气用氢气填充，重复三次。所得混合物在室温下搅拌过夜。反应结束后，硅藻土过滤，除去催化剂，滤液减压浓缩得到棕色残留物。向残留物中加入MeOH(30mL)和Et 2O(50mL)，升温至50℃搅拌15分钟，有固体析出。减压过滤，滤饼用乙醚洗涤，收集滤饼，减压干燥得到黄色固体，即为产物2-(4-(4-(二甲氨基)哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮(产物3,15.50g,产率85％)。LC-MS:381.44[M+H] +,96.92％(纯度,UV 214nm)。
1H NMR(400MHz,DMSO-d 6)δ10.18(s,1H),9.37(s,1H),7.96(d,J＝9.2Hz,2H),7.36(d,J＝8.4Hz,1H),7.04(d,J＝8.8Hz,2H),6.91(d,J＝8.8Hz,1H),6.67(s,1H),3.34-3.33(m,4H),2.63(m,1H),2.26(s,6H),1.83-1.93(m,4H).
实施例4：2-(4-(4-(二甲基)哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮
(4；2-(4-(4,4-dimethylpiperidin-1-yl)phenyl)-7,8-dihydroxy-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000013
步骤(1)7,8-二(苄氧基)-2-(4-(4,4-二甲基哌啶-1-基)苯基)-4H-色烯-4-酮(4a)的合成
在手套箱中，向7,8-二(苄氧基)-2-(4-溴苯基)-4H-色烯-4-酮(1d,41.0g,79.9mmol)、Xphos(7.4g,15.6mmol),Pd 2(dba) 3(7.15g,7.8mmol)和碳酸铯(50.7g，15.6mmol)的混合物中加入1,4-二氧六环(400mL)和4,4-二甲基哌啶(13.24g，117mmol)。所得混合物升温至110℃搅拌2.5小时。反应结束后，混合物冷却至室温，倾入水(300mL)中，加入EtOAc(200mL×3)萃取，分出有机相，用无水硫酸钠干燥。滤液减压浓缩，所得残留物经硅胶柱层析(洗脱剂：DCM/MeOH(v/v)＝40/1到10:1)纯化，得到黄色固体，即为产物(1e，26.11g,产率60％)。LC-MS(ESI):m/z 546.6[M+H] +.
步骤(2)2-(4-(4,4-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮(4)的合成
向化合物7,8-二(苄氧基)-2-(4-(4,4-二甲基哌啶-1-基)苯基)-4H-色烯-4-酮(4a,26.88g,47.94mmol)、THF(200mL)和甲醇(50mL)的混合物中，加入Pd/C(2.6g,10％含量)，所得混合物抽除空气用氢气填充，重复三次。所得混合物在室温下搅拌过夜。反应结束后，硅藻土过滤，除去催化剂，滤液减压浓缩得到棕色残留物。向残留物中加入MeOH(30mL)和Et 2O(50mL)，升温至50℃搅拌15分钟，有固体析出。减压过滤，滤饼用乙醚洗涤，收集滤饼，减压干燥得到黄色固体，即为产物2-(4-(4,4-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮(4,共14.01g,产率80％)。
LC-MS:366.41[M+H] +,97.02％(纯度,UV 214nm)；
1H NMR(400MHz,DMSO-d 6)δ10.18(s,1H),9.37(s,1H),7.96(d,J＝9.2Hz,2H),7.36(d,J＝8.4Hz,1H),7.04(d,J＝8.8Hz,2H),6.91(d,J＝8.8Hz,1H),6.67(s,1H),3.34-3.33(m,4H),1.63-1.53(m,4H)1.23-1.33(s,6H).
实施例5：2-(4-(3,5-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮
(5；2-(4-(3,5-dimethylpiperidin-1-yl)phenyl)-7,8-dihydroxy-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000014
参照实施例4的合成方案合成上述结构化合物，即2-(4-(3,5-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮；LC-MS:366.41[M+H] +。
实施例6：3-氟-7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(6；3-fluoro-7,8-dihydroxy-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000015
参照实施方案二的合成方案合成上述结构化合物，即3-氟-7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:356.11[M+H] +。
实施例7：3-氘代-7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000016
参照实施方案二的合成方案合成上述结构化合物，即3-氘代-7,8-二羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:339.14[M+H] +。
实施例8：7-羟基-8-甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(8；7-hydroxy-8-methoxy-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000017
参照实施方案一的合成方案合成上述结构化合物，即7-羟基-8-甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:352.15[M+H] +。
实施例9：7,8-二羟基-2-(4-(4-甲氧基哌啶-1-基)苯基)-4H-色烯-4-酮
(9；7,8-dihydroxy-2-(4-(4-methoxypiperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000018
参照实施方案二的合成方案合成上述结构化合物，即7,8-二羟基-2-(4-(4-甲氧基哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:368.15[M+H] +。
实施例10：7,8-二羟基-2-(4-(4,4-二氘代-哌啶-1-基)苯基)-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000019
参照实施方案二的合成方案合成上述结构化合物，即7,8-二羟基-2-(4-(4,4-二氘代-哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:340.51[M+H] +。
实施例11：7,8-二甲氧基-3-甲基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(11；7,8-dimethoxy-3-methyl-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000020
参照实施方案一的合成方案合成上述结构化合物，即7,8-二甲氧基-3-甲基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:380.21[M+H] +。
实施例12：3-氟-7-羟基-8-甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
(12；3-fluoro-7-hydroxy-8-methoxy-2-(4-(piperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000021
参照实施方案二的合成方案合成上述结构化合物，即3-氟-7-羟基-8-甲氧基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:370.57[M+H] +。
实施例13：7,8-二甲氧基-3-氘代-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000022
参照实施方案一的合成方案合成上述结构化合物，即7,8-二甲氧基-3-氘代-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:367.43[M+H] +。
实施例14：3-氘代-7-甲氧基-8-羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000023
参照实施方案二的合成方案合成上述结构化合物，即3-氘代-7-甲氧基-8-羟基-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:353.31[M+H] +。
实施例15：7,8-二氨基-3-氯-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000024
参照实施方案二的合成方案合成上述结构化合物，即7,8-二氨基-3-氯-2-(4-(哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:371.12[M+2H] +。
实施例16：(S)-3-氟-7,8-二羟基-2-(4-(3-甲基哌啶-1-基)苯基)-4H-色烯-4-酮(16；e(S)-3-fluoro-7,8-dihydroxy-2-(4-(3-methylpiperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000025
参照实施方案二的合成方案合成上述结构化合物，即(S)-3-氟-7,8-二羟基-2-(4-(3-甲基哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:369.94[M+H] +。
实施例17：3-溴-2-(4-(4,4-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮
(17；3-bromo-2-(4-(4,4-dimethylpiperidin-1-yl)phenyl)-7,8-dihydroxy-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000026
参照实施方案二的合成方案合成上述结构化合物，即3-溴-2-(4-(4,4-二甲基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮；LC-MS:445.07[M+H] +。
实施例18：3-氘代-2-(4-(4-丙基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮
Figure PCTCN2019098578-appb-000027
参照实施方案二的合成方案合成上述结构化合物，即3-氘代-2-(4-(4-丙基哌啶-1-基)苯基)-7,8-二羟基-4H-色烯-4-酮；LC-MS:381.25[M+H] +。
实施例19：(S)-3,8-二氟-7-羟基-2-(4-(3-甲基-1-基)苯基)-4H-色烯-4-酮
(19；(S)-3,8-difluoro-7-hydroxy-2-(4-(3-methylpiperidin-1-yl)phenyl)-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000028
参照实施方案二的合成方案合成上述结构化合物，即(S)-3,8-二氟-7-羟基-2-(4-(3-甲基哌啶-1-基)苯基)-4H-色烯-4-酮；LC-MS:372.21[M+H] +。
实施例20：(S)-3,8-二氟-2-(4-(3-氟哌啶-1-基)苯基)-7-羟基-4H-色烯-4-酮
(20；(S)-3,8-difluoro-2-(4-(3-fluoropiperidin-1-yl)phenyl)-7-hydroxy-4H-chromen-4-one)
Figure PCTCN2019098578-appb-000029
参照实施方案二的合成方案合成上述结构化合物，即(S)-3,8-二氟-2-(4-(3-氟哌啶-1-基)苯基)-7-羟基-4H-色烯-4-酮；LC-MS:376.43[M+H] +。
实施例21：本发明化合物对TrkB受体的激活作用
(1)实验方法：N171-82Q WT雄性小鼠与雌性B6C3F1/J杂种小鼠交配，取18天B6C3F1/J孕鼠，异氟烷麻醉，解剖取出胚胎，放到含有Leibovitz’s buffer的10cm培养皿中。断头，剥离脑壳，取出完整鼠脑，放入新的Leibovitz’s buffer的10cm培养皿中。分离脑中的神经皮层放入含有3mL Leibovitz’s buffer的15mL离心管中。1000rpm离心1分钟，轻柔的移去离心管内Leibovitz’s buffer，加入750μL的0.25％trypsin-EDTA，75μL的0.1％DNase(2000U/mL)，37℃消化15min。加入750μL含有5％FBS的DMEM培养液终止消化，混匀细胞，1000rpm离心5分钟。移去Trypins/DNase混合液，加入750μL的Neurobasal medium(B-27,Glutamax,P/S)，用移液枪轻轻吹吸30下使细胞混匀。用40μm过滤器过滤细胞悬浮液，转移至另一全新50mL离心管中。取10μL细胞悬浮液，加入90μL DMEM中(稀释10倍)，吸取10μL上述液体于血球计数板上，使悬液充满盖片和计数板之间，计数，计算板四大格细胞总数，压线细胞只计算左侧和上侧细胞，按以下公式计算：
细胞数/mL＝四大格细胞总数/4×10 4/mL
细胞总数/mL＝四大格细胞总数/4×10 4/mL×稀释倍数。
计数后，用Neurobasal选择培养基稀释细胞，使1mL Neurobasal培养液含有0.5×10 6个细胞。
将PDL溶液提前在37℃水浴锅加热，6孔板每孔加PDL溶液200μL，37℃过夜，使PDL均匀涂满细胞孔板，吸去PDL溶液，PBS洗2遍。使用前，在超净台里将PBS吸干，开盖晾干。每个孔加入2mL细胞悬液(37℃，5％CO 2)，培养细胞贴壁后，换培养液，培养3d，观察神经元生长状况。每隔3d换全培养液1次，每次换半液。培养至原代皮层神经元细胞成熟(一般培养至第14天即可成熟)。
待原代皮层神经元细胞成熟后，重新加入适量新鲜配制的培养液最终使每孔终体积为2mL。设置空白对照组(Vehicle)，阳性对照组BDNF(10ng/mL)和对照化合物组(500nM)，受试化合物组6组(500nM)。每个剂量设置三组平行组。在37℃培养10min，然后倒掉培养液，用预冷的PBS洗涤细胞，每孔内加40μL预冷好的裂解液(RIPA buffer 10mL和蛋白酶和磷酸酶抑制剂100μL)，冰上裂解30min。裂解完后，超声波细胞粉碎机粉碎细胞，于4℃下14000rpm离心20min，上清分装转移至新的1.5mL离心管中，提取蛋白。Western blotting实验检测细胞内(A)p-TrkB和t-TrkB；(B)p-AKT和t-AKT的蛋白水平。
对照化合物为现有技术中公开的具有TrkB激动活性的化合物：
Figure PCTCN2019098578-appb-000030
(2)数据处理：应用软件Sigmaplot 13.0对资料进行统计学分析，各组数据以均数±标准误
Figure PCTCN2019098578-appb-000031
表示。计量资料多组间比较采用单因素方差分析(One-way ANOVA)及LSD两两事后检验，P＜0.05即认为有统计学差异。
(3)实验结果：实验结果如表1所示，其中受试化合物实施例1的p-TrkB/t-TrkB和p-AKT/t-AKT电泳图和蛋白相对强度图如图1所示。其中，图1A为p-TrkB/t-TrkB蛋白电泳图，图1C为p-AKT/t-AKT的蛋白电泳图；Sigmaplot 13.0软件统计p-TrkB/t-TrkB和p-AKT/t-AKT蛋白水平相对强度，图1B为p-TrkB/t-TrkB蛋白水平统计结果；图1D为p-AKT/t-AKT的蛋白水平统计结果。
表1 p-TrkB/t-TrkB和p-AKT/t-AKT蛋白相对强度
组别 p-TrkB/t-TrkB蛋白相对强度 p-AKT/t-AKT蛋白相对强度
Vehicle 100.000±13.095 100.001±1.856
BDNF 274.260±8.047* 229.306±3.912*
受试化合物/实施例1 216.727±8.642* 219.663±8.085*
受试化合物/实施例2 206.453±7.574* 209.651±3.294*
受试化合物/实施例3 234.685±14.695* 240.213±8.376*
受试化合物/实施例4 216.198±8.865* 215.698±5.487*
受试化合物/实施例5 213.254±12.379* 219.904±6.954*
受试化合物/实施例7 218.156±12.556* 220.514±3.476*
对照化合物组 138.890±15.547* 147.748±4.832*
注：与Vehicle组相比，*P＜0.05，n＝3。
(4)实验结论：原代神经皮层神经元细胞经BDNF(10ng/mL)培养后，p-TrkB的相对强度显著增强，为Vehicle组的2.74倍(P＜0.05)，受试化合物组实施例1～5化合物使蛋白相对强度增强了2.06～2.34倍，也表现出了显著性的差异(P＜0.05)。以上数据证明实施例化合物在500nM剂量下对于BDNF-TrkB信号通路的下游信号AKT增强的效果与BDNF相当；本发明化合物的TrkB激动活性显著优于公开的具有TrkB激动活性的对照化合物。本发明实施例化合物在原代神经皮层神经元细胞中可以激活TrkB受体及其下游AKT信号通路。
脑源性神经营养因子(BDNF)对多种神经元集群具有神经营养性作用，这些神经元集群包括感觉神经元、运动神经元、黑质多巴胺能神经元以及基底前脑胆碱能神经元，这些神经元涉及数种神经和神经精神病症。临床前证据表明BDNF可能适用于多种神经和神经精神病症的治疗；然而，这种肽的体内不稳定性和其无法有效穿过血脑屏障限制了它的有用性。
实施例22：本发明化合物磷酸-Akt S473 ELISA分析
(1)实验方法：实施例21相同方法培养原代皮层神经元细胞，待成熟后，重新 加入适量新鲜配制的培养液最终使每孔终体积为2mL。设置空白对照组(Vehicle)，阳性对照组BDNF(10ng/mL)和对照化合物组(500nM)，受试化合物组6组(500nM)。每个剂量设置三组平行组。在37℃培养10min，然后倒掉培养液，用预冷的PBS洗涤细胞，每孔内加40μL预冷好的裂解液(RIPA buffer 10mL和蛋白酶和磷酸酶抑制剂100μL)，冰上裂解30min。裂解完后，超声波细胞粉碎机粉碎细胞，于4℃下14000rpm离心20min，上清分装转移至新的1.5mL离心管中，提取蛋白。p-Akt ELISA实验定量检测细胞内p-Akt蛋白水平。
配置系列浓度p-Akt标准溶液，测定吸光度值，绘制标准曲线。磷酸-Akt1(Ser473)夹心ELISA试剂盒购自细胞信号传导公司(目录号7160)。将抗体包被到96孔板中4℃过夜，洗涤。包被后的平板使用2％牛血清白蛋白(BSA)封闭，充分洗涤后，用样品稀释液(供应于试剂盒中)100μL添加至每个孔中稀释所提取的蛋白，并且在4℃下孵育过夜。在用200μL洗涤缓冲液(供应于试剂盒中)洗涤4次后，添加100μL/孔检测抗体并且在37℃下孵育1小时。4次洗涤后，添加100μL的HRP-联第二抗体(供应于试剂盒中)并且在37℃下孵育30分钟。最终洗涤后，将100μL的TMB底物添加至每个孔中并且在37℃下孵育10分钟，用TMB作为底物检测信号，因为TMB可被氧化为蓝色溶液。通过添加100μL/孔终止溶液来终止反应，反应终止后一个小时内检测结果可以维持稳定。使用酶标仪记录每个孔在450nm和650nm下的值。通过从450nm处的读数中减去650nm处的读数(溶液本身的干扰)确定光密度(OD)。根据OD值对应标准曲线，得到各孔中的p-Akt浓度。
(2)数据处理：各组数据以均数±标准误
Figure PCTCN2019098578-appb-000032
表示。将各阳性对照组(BNDF)、对照化合物组(同实施例21中的对照化合物)和各受试化合物组的检测结果与空白对照组的比值作为指标，对比阳性对照组、对照化合物组和各受试化合物组的p-Akt检测结果。计量资料多组间比较采用单因素方差分析(One-way ANOVA)及LSD两两事后检验，P＜0.05即认为有统计学差异。
(3)实验结果：实验结果如图2所示，图2中可以清楚的观察到阳性对照组(BNDF)、对照化合物组和各受试化合物组(实施例1～5和实施例7)的p-Akt蛋白相对含量；阳性对照组p-Akt蛋白相对含量231％，实施例1～5和实施例7的p-Akt S473蛋白相对含量分别为：212.00±2.122％，221.67±3.69％，247.33±3.18％，224.33±3.27％，217.32±2.57％，218.33±2.31％；对照化合物的p-Akt蛋白相对含量为138.33±2.08％。
(4)实验结论：
本发明实施例化合物对于BDNF-TrkB信号通路的下游信号AKT的激活效果与BDNF相当；本发明化合物的AKT的激活效果显著优于对照化合物。本实验结果与实施例21 Western blotting实验结果相符。
实施例23：本发明化合物在亨廷顿疾病的转基因小鼠中的药效实验分析
本发明实施例化合物的活性，通过以下实验进行说明。所有使用动物的实验操作 均根据IACUC指南进行。
(1)实验方法：7周龄N171-82Q HD雄性小鼠与雌性B6C3F1/J杂种小鼠配对后得到新生小鼠，21日龄时将雄性小鼠分笼饲养，取0.5cm小鼠尾组织提取DNA，确定小鼠基因型，得到实验所需N171-82Q HD小鼠和WT小鼠。随机分为WT Vehicle组(正常小鼠)、WT受试化合物组(正常鼠给药组,5mg/kg/d,n＝10)、HD Vehicle组(HD鼠空白组,n＝15)、HD受试化合物组(HD鼠给药组1～5,5mg/kg/d,n＝10)。受试化合物混悬于1％CMC-Na溶液中，灌胃给药至研究结束。每周测量一次体重。在小鼠第12周、18周、24周龄时进行行为学测试考察运动功能。20周各取3例组织进行western blotting和免疫组化实验，在24周进行MRI扫描，研究结束时统计受试化合物对小鼠死亡率的影响。
(2)数据处理：应用软件Sigmaplot 13.0对资料进行统计学分析，各组数据以均数±标准误
Figure PCTCN2019098578-appb-000033
表示。行为学测试结果采用双因素(基因型和处理因素)方差分析(Two-way ANOVA)及两两事后比较检验，体重数据的比较体重统计采用双因素(基因型和年龄)重复测量方差分析，死亡率采用Kaplan–Meier方法统计。其它计量资料用单因素方差分析进行统计，P＜0.05即认为有统计学差异。
(3)具体检查方法如下：
①行为学测试考察运动功能
通过转棒实验和三种规格平衡木实验评估小鼠运动功能，穿越平衡木时间越短，掉落潜伏期越长则证明小鼠运用协调能力越强。
斜坡平衡木实验：采用100厘米长和11毫米宽的方形平衡木，与水平地面呈45度角放置。平衡木末端放置以黑箱子(12×12×12厘米)，明亮的灯光照亮起始平台。在平衡木下方放置垫片提供缓冲，以防动物从梁上脱落。在测试前1小时，分别在距黑箱子八分之一、四分之一和全长距离处训练小鼠穿过梁3次。如果小鼠在训练期间停止走动，手持长镊子轻轻按压尾部以鼓励运动。训练实验后，小鼠在不会受到干扰的环境下休息至少一个小时。开始正式实验，每个小鼠横穿平衡梁的最大时间截至60秒，如果移动时间大于60秒，则记录为60秒。
高空行走平衡木(11mm)实验一：采用80厘米长和直径为11毫米的圆形平衡木，放置离地面约50cm高度。以下步骤同斜坡平衡木实验。
高空行走平衡木(5mm)实验二：采用80厘米长和5毫米宽的方形平衡木，放置离地面约50cm高度。以下步骤同斜坡平衡木实验。
转棒实验：小鼠疲劳转棒仪最大时间设置为5分钟，加速度设置由4rpm加至40rpm，将小鼠放置到正在运转的转棒仪上，开始计时，记录小鼠保持在杆上的时间，即为掉落潜伏期，以此表示其运动协调能力。在转棒实验开始的前一天，每只老鼠历经训练5分钟，训练课程之后在笼中休息。第二天开始正式实验，将小鼠放到转盘上进行三轮实验，每次实验完毕休息30分钟，进行下一轮实验。结果统计取三轮实验的平均值。
对WT Vehicle组、WT受试化合物组、HD Vehicle组、HD受试化合物组的受试小鼠在四种运动功能考察实验中的表现进行统计，实验结果如下表2～表5。
表2 斜坡平衡木实验小鼠横穿平衡梁时间实验结果
Figure PCTCN2019098578-appb-000034
注：与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝10～15。
表3 高空行走平衡木(11mm)实验一小鼠横穿平衡梁时间实验结果
Figure PCTCN2019098578-appb-000035
Figure PCTCN2019098578-appb-000036
注：与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝10～15。
表4 高空行走平衡木(5mm)实验二小鼠横穿平衡梁时间实验结果
Figure PCTCN2019098578-appb-000037
注：与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝10～15。
表5 转棒实验掉落潜伏期实验结果
Figure PCTCN2019098578-appb-000038
Figure PCTCN2019098578-appb-000039
注：与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝10～15。
根据以上表2～表4平衡木实验结果可知：与WT Vehicle对照组的小鼠相比，在第12周时，HD Vehicle小鼠即表现出了运动缺失症状，18周与第12周相比，HD小鼠在平衡梁上的时间相当，第24周的HD小鼠运动功能急剧恶化，从第12到第24周，HD小鼠穿越平衡梁的时间逐渐增加，这表明这些N171-82Q HD小鼠表现出了典型的进行性运动障碍。
相比于HD Vehicle组，HD受试化合物组表现出对运动能力极好的改善作用。从三种规格的平衡木实验结果来看，在第12周、18周、24周三个时间点的所有实验，与HD Vehicle组相比，HD受试化合物组穿越平衡梁所用时间均显著缩短(P＜0.05)，与WT Vehicle组用时相当。
根据表5的转棒仪实验结果，与WT Vehicle组相比，HD Vehicle组在第12周尚未表现出显著性的运动障碍，在第18周运动能力缺失，但无显著性差异；但在24周时运动能力显著下降(P＜0.05)。相比于HD Vehicle组，HD受试化合物组表现出对运动能力极好的改善作用。在第12周、18周、24周三个时间点的所有实验，与HD Vehicle组相比，HD受试化合物组小鼠的掉落潜伏期均显著延长(P＜0.05)；在第24周的实验结果中，HD给药组的掉落潜伏期与WT Vehicle组小鼠相当。
综合平衡木实验和转棒仪实验证明本发明实施例所制备的受试化合物对HD模型小鼠表现出了极好的改善运动障碍的作用。
②体重和生存率研究
所有小鼠每周测量一次体重。每天对小鼠的生存情况进行监测，记录死亡情况，统计生存率。若小鼠被翻转放置后无法恢复到正常位置，并在轻轻刺激30秒后才开始运动也可认定为生命结束。
HD Vehicle组小鼠整体开始死亡时间较早，在小鼠约七个月月龄时(210天)，11只中有6只小鼠死亡，死亡率为54.5％；HD给药组小鼠仅死亡了3只，死亡率为30％。由此可以说明本发明化合物可以推迟HD小鼠的发病时间。
③体内MRI扫描及脑体积测量
对WT Vehicle组、WT受试化合物组、HD Vehicle组和HD受试化合物组，给药至24周，每组各取4只小鼠进行体内MRI扫描及脑体积测量。采用9.4T核磁共振成像仪，配备有三轴梯度和动物成像探头。用1％异氟烷麻醉小鼠，监测呼吸，整个扫描过程中保持恒温。用三维的T2加权快速自旋回声序列技术获得图像，该技术成像分辨率和对比度足以自动对小鼠的大脑和子结构进行体积描述。其参数如下：回声时间(TE)/重复时间(TR) 1/440/700ms，分辨率为 1/40.1×0.1×0.1毫米，回声列车长度为 1/44，平均数 1/42和翻转角度为 1/4408。该仪器含有一个强大的linux集群，可以编码运行大型变形扩散度度量映射(LDDMM)，从而得到高强度标准化图像。这些转换编码了图像之间的形态差异，并可以基于变形形态学测量(DBM)来进行分析，以检测脑容量的区域变化。对全脑(Brain)，纹状体(Neocortex)及神经皮层(CaudatePutamen)的体积进行分析。
用MRI扫描技术，通过对实验对象和模板图像之间的形态差异进行分析，并运用LDDMM方法对小鼠脑组织结构进行计算分析统计。实验结果见表6，其中受试化合物实施例1的脑体积、纹状体体积和神经皮层体积统计结果见图3，图3中A为全脑体积统计结果，图3中B为纹状体体积统计结果，图3中C为神经皮层体积统计结果。
表6 MRI检测全脑、纹状体及神经皮层的体积结果(mm 3)
Figure PCTCN2019098578-appb-000040
与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝4。
由上表6可知，与WT Vehicle鼠相比，HD Vehicle小鼠显示出了显著的全脑区域、纹状体和神经皮层的萎缩。HD鼠给予本发明实施例制备的受试化合物1～5(5mg/kg/d)后，整个大脑、神经皮层和纹状体的萎缩程度显著降低(P＜0.05)。本发明受试化合物可阻止HD鼠的脑萎缩，给药24周后，HD受试化合物组的整个大脑、神经皮层和纹状体的体积和正常的WT Vehicle鼠体积相当。
④DARPP 32蛋白水平的检测
多巴胺和cAMP调节的磷蛋白(Dopamine and adenosine 3'5'-monophosphate-regulated phospho-protein,DARPP-32)，分子量为32kDa，是多巴胺信号级联的一个基本组成部分，HD病理学的特征是在纹状体中大量丢失MSNs，而MSNs可以表达高水平的DARPP32，因此，DARPP32可以作为HD模型中神经损伤和神经功能障碍的标志。各实验组小鼠待行为学评价全部完成，给药至25周时，每组各取3只老鼠，分离海马组织、纹状体、神经皮层组织，提取纹状体组织中的总蛋白，Western blotting实验检测DARPP 32的蛋白水平。
纹状体组织中总蛋白的提取方法如下：将纹状体组织块置于100μL裂解液(RIPA buffer10mL，蛋白酶抑制剂100μL，磷酸酶抑制剂100μL)中，置于冰上裂解30min，超声波细胞粉碎机粉碎组织，于4℃下14000rpm离心20min。上清分装转移至新的1.5mL离心管中，提取蛋白，Western blotting实验检测DARPP 32蛋白水平。
Western blotting实验测量DARPP32水平的结果如表7所示，其中受试化合物实施例1的DARPP32电泳图和DARPP 32/β-action蛋白水平相对强度见图4；图4中A为DARPP32电泳图，图4中B为DARPP 32/β-action蛋白水平相对强度统计图。
表7 DARPP32水蛋白相对强度实验结果
Figure PCTCN2019098578-appb-000041
注：与WT Vehicle组相比， #P＜0.05，与HD Vehicle组相比，**P＜0.05，n＝3。
由表7可知，与WT Vehicle组相比，HD Vehicle组的纹状体中DARPP-32的水平显著降低；与HD Vehicle组相比，HD受试化合物组(实施例1～5，5mg/kg/d)鼠的纹状体中DARPP-32的水平均显著升高。
上述实验结果显示：在HD小鼠的纹状体中，DARPP-32的水平显著降低(P＜ 0.05)，说明HD小鼠的纹状体存在神经损伤和神经功能障碍；HD受试化合物组DARPP-32的水平相较于HD Vehicle组显著升高，表明本发明实施例制备的受试化合物有效阻止了DARPP32的这种损失，进而可以有效治疗神经损伤和神经功能障碍。
⑤免疫组化法观察小鼠神经皮层中mHtt蛋白聚集体的情况
各实验组小鼠待行为学评价全部完成，给药至25周时，每组各取3只老鼠，用于检测DARPP 32蛋白水平小鼠分离出的右侧脑于4％多聚甲醛中后固定24h，经后固定的脑组织再放入30％蔗糖溶液中至沉底后，用冰冻切片机沿冠状切取厚度为35μm的脑片置于DPBS中储存。取储存的脑片，PBS漂洗一次，封闭用1mL山羊血清+9mL PBS+1％TritonX-100，常温置于摇床封闭2～3h。封闭完成后，用PBS漂洗3次，每次5min，用小毛笔刷将脑片取出放于添加了一抗(anti-Huntingtin,clone mEM48,1:25,mouse，一抗用封闭液配制)的24孔板中进行孵育，置于–4℃冷室摇床过夜。PBS洗涤后，放于添加了二抗的24孔板中(555，anti-mouse)进行洗涤，置于室温摇床孵育2h。PBS洗涤后，放入含有800μL DAPI(用PBS配制，1:10000)的24孔板中进行染色，室温摇床10min。再将脑片取出放在含有800μL PBS的24孔板中洗1次，10min，室温摇床。将载玻片放平于实验台上，正面朝上，在载玻片上滴加PBS，脑片在PBS上展开。等待晾干后，EM48抗体(anti-Huntingtin)对mHtt蛋白进行免疫荧光染色，拍照观察。
脑组织免疫荧光染色后，对右侧大脑皮层中的六个视野的mHtt蛋白聚集的细胞进行计数，取平均值进行分析统计。大脑皮层中具有mHtt蛋白聚集的平均细胞数量统计结果如表8所示。其中受试化合物实施例1的EM48抗体免疫染色的神经皮层代表性图像如图5所示，Scale bars为50μm，其中浅色点即为具有mHtt蛋白聚集的细胞。
表8 大脑皮层中具有mHtt蛋白聚集的平均细胞数量统计结果
Figure PCTCN2019098578-appb-000042
注：与HD Vehicle组相比，*P＜0.05，n＝3。
由表8的实验结果表明，HD小鼠神经皮层中具有明显的mHtt蛋白聚集，本发明受试化合物可以显著减少HD小鼠大脑皮层中的mHtt蛋白聚集(*P＜0.05)。
(4)实验结论：
①本发明受试化合物在行为学测试中表现出了卓越的挽救HD小鼠运动功能障碍的性能，显著提高HD小鼠的运动协调能力，在第12周、18周、24周三个时间点， 各HD受试化合物组小鼠在三种规格平衡木实验中穿越横梁的时间均与Vehicle处理的HD小鼠相当；24周给药的转棒法实验中，给药组HD小鼠掉落潜伏期明显增长，各HD受试化合物组小鼠的掉落潜伏期与WT Vehicle组小鼠相当。
②本发明受试化合物可以推迟HD小鼠的发病时间，延长HD小鼠的发病潜伏期。
③本发明受试化合物大大的减弱了HD小鼠整个大脑、神经皮层和纹状体的萎缩程度。
④本发明受试化合物可阻止HD小鼠纹状体中DARPP32的损失，保持纹状体内DARPP32水平，改善HD小鼠纹状体中的MSNs的功能。
⑤本发明受试化合物减少HD小鼠神经皮层中mHtt蛋白聚集体从而减弱神经元的损伤。
本项实验证明本发明受试化合物具有显著的神经保护作用，可以用于治疗神经系统疾病。
实施例24：本发明化合物对永久性脑缺血损伤大鼠的保护作用
(1)实验方法：SD大鼠，雄性，220-250g左右，由西安交通大学医学院实验动物中心提供。每14只/组，随机分组。分为假手术(Sham)组，模型(Model)组、阳性对照依达拉奉(Edaravone)组和受试化合物组(1～5)。手术前12小时禁食，自由饮水，运用线栓阻塞中脑动脉的造模方法造模。大鼠麻醉后固定与木板上，近心端结扎颈总动脉，远心端结扎颈外动脉，保留颈内动脉入颅骨主干。在颈内动脉分叉处预留结扎线，使用动脉夹暂时夹闭颈内动脉远端，在颈总动脉分叉处剪一小口，插入5cm长且顶端烧制为直径0.3mm的圆球状3-0尼龙线，松开动脉夹，将线栓缓缓推进18～20mm处，有阻力时停止进线，此时尼龙线栓进入中动脉并阻断血流，缺血期开始，缝合皮肤。假手术组除不插入线栓外，其余步骤同上。
缺血后0h、3h、6h和12h各给一次药，之后分别在每天给药一次，连续给药7天。受试化合物以1％CMC-Na溶液配置成4mg/mL的溶液，口服灌胃给药，依达拉奉组静脉给药剂量0.078mg/kg。
①缺血后密切观察小鼠的活动，记录其死亡情况，对小鼠缺血后6h、1d、2d、3d、4d、5d、6d、7d的死亡率进行统计。
②提鼠尾离开地面约1尺，观察两前肢状况；将大鼠置于水平地面，推动其双肩，观察两侧抵抗力有无差异；大鼠置于地面，观察其行走情况。采用五级四分评分法(0～4分)，分数越高，说明其神经行为损伤越严重。
0分：行为完全正常为；
1分：提起鼠尾离开地面，手术对侧前肢内旋、内收；
2分：大鼠置地面，用手挤压两侧检查其抗力，手术对侧抗力下降；
3分：大鼠置地面，观察其行走，围绕手术对侧转圈；
4分：损伤极其严重，已无法自主活动。
③缺血7天后脑梗死体积百分比：TTC染色观察梗死面积。
各组大鼠手术再灌24h后脊椎脱臼处死，迅速剥离出脑组织，放于冰箱–20℃冻至适当硬度去除嗅球、小脑和低位脑干，以1mm间隔行连续冠状切片，共5片，放入0.5％氯化三苯基四氮唑(TTC)磷酸盐缓冲液中(pH＝7.4)，37℃孵育恒温箱内避光孵育15min，后置于多聚甲醛中固定后观察(固定时间以24h以内为宜)，正常脑组织着玫瑰红色，梗塞脑组织呈苍白色。拍照后将切片扫描入扫描仪中保存图像。运行Image J处理软件，计算脑梗死面积及层面总面积。
脑梗塞体积(％)＝(手术对侧半球的体积－手术侧半球未梗塞部分的体积)/手术对侧半球的体积×100％
(2)数据统计分析：应用SPSS18.0统计软件，进行统计学分析，各组数据以均值±标准误
Figure PCTCN2019098578-appb-000043
表示，计量资料多组均数比较采用单因素方差分析及组间多重比较，行为学评分等级资料采用秩和检验，死亡率采用Kaplan–Meier方法统计。P＜0.05即认为有统计学差异。
(3)实验结果
①对缺血6h、1d、2d、3d、4d、5d、6d、7d各时间点进行神经行为学评分，评估受试化合物对永久性脑缺血大鼠神经行为学的影响，实验结果如下表9所示。
表9 受试化合物对永久性脑缺血大鼠神经行为学的影响
Figure PCTCN2019098578-appb-000044
Figure PCTCN2019098578-appb-000045
Figure PCTCN2019098578-appb-000046
注：与Sham组相比， #P＜0.05；与Model组相比，**P＜0.05。n为实验各组动物起始数量
由表9可知，在所有时间点，Model组与Sham组均表现出了显著性差异，说明造模成功。缺血后第0～3天，模型组行为学症状有进行性加重的趋势，随后有所缓解。阳性对照依达拉奉组仅在第7天表现出了差异。Model组第3天和第7天的神经行为学评分分别为3±0.38和2.5±0.33，受试化合物实施例1～5(20mg/kg)给药后第3天和第7天神经行为学评分分别为1.87±0.33～2.23±0.36和1.01±0.27～1.21±0.22，因此，受试化合物(20mg/kg)组给药后第3天和第7天的神经行为学评分均显著低于模型组(P＜0.05)，本实验证明了本发明受试化合物能够有效减轻大鼠的神经行为学症状。
②大鼠取脑进行TTC染色，正常脑组织着玫瑰红色，梗死区为白色。统计各组的脑梗死体积百分比结果见表10。假手术组(Sham)、模型组(Model)、对照组(Edaravone)和其中1组受试化合物组(实施例1化合物)的脑组织TTC染色实验结果如图6所示。
表10 受试化合物对永久性脑缺血大鼠脑梗死体积的影响
Figure PCTCN2019098578-appb-000047
Figure PCTCN2019098578-appb-000048
由表10可知，Model组梗死体积百分比为44.4％±6.31％，说明造模成功。实施例1～5所制备的受试化合物(20mg/kg)组能够减少梗死体积，梗死体积百分比为19.58％±9.56％～21.33％±7.68％，因此本实验结果证明受试化合物实施例1～5(20mg/kg)可显著降低脑梗死体积(P＜0.05)。
(4)实验结论：
本实验证明受试化合物对永久性脑缺血损伤有保护作用，能减轻大鼠的行为学症状，减少脑梗死体积。
综合上述实验结果，本发明化合物对神经系统退行性病变和中枢性脑损伤具有保护和治疗作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (15)

  1. 一种用于治疗神经系统疾病的化合物，其特征在于，其为式(I)所示化合物，或式(I)所示化合物的立体异构体、几何异构体、互变异构体、溶剂化物、代谢产物、药学上可接受的盐，或式(I)所示化合物的前药：
    Figure PCTCN2019098578-appb-100001
    其中，R 1和R 2独立选自-OH、-NH 2、F、Cl、Br、C 1-C 12烷基、C 1-C 12羟基烷基、C 1-C 12杂烷基、C 1-C 12卤代烷基、C 1-C 12烷氧基、C 1-C 12烷氨基、-OC(＝O)R 6和-NHC(＝O)R 7；R 6和R 7独立选自C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氨基；
    R 3为H、D、F、Cl或Br；
    R 4和R 5独立选自H、D、F、Cl、Br、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基和C 1-C 6烷氨基；m和n各自独立地为0、1、2、3或4。
  2. 根据权利要求1所述化合物，其特征在于，所述R 1和R 2独立选自-OH、-NH 2、F、C 1-C 6羟基烷基、C 1-C 6杂烷基、C 1-C 6烷氧基和C 1-C 6烷氨基。
  3. 根据权利要求1或2所述化合物，其特征在于，所述R 1和R 2独立选自-OH、-NH 2、F和-OCH 3。
  4. 根据权利要求1所述化合物，其特征在于，所述R 4和R 5独立选自H、D、F、Cl、Br、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、-N(CH 3) 2、-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3和-OCH(CH 3) 2。
  5. 根据权利要求1所述化合物，其特征在于，所述m和n各自独立地为0、1或2。
  6. 根据权利要求1所述化合物，其特征在于，式(I)所示化合物具有式(II)所示结构；
    Figure PCTCN2019098578-appb-100002
    其中，R 3为H、D、F、Cl或Br；
    R 4和R 5独立选自H、D、F、Cl、Br、-CH 3、-CH 2CH 3、-CH 2CH 2CH 3、-CH(CH 3) 2、-N(CH 3) 2、-OCH 3、-OCH 2CH 3、-OCH 2CH 2CH 3和-OCH(CH 3) 2；
    m和n各自独立地为0、1或2。
  7. 根据权利要求1所述化合物，其特征在于，式(I)所示化合物为如下之一化合物：
    Figure PCTCN2019098578-appb-100003
    Figure PCTCN2019098578-appb-100004
  8. 一种药物组合物，其特征在于，包含权利要求1～7任一项所述的化合物。
  9. 根据权利要求8所述药物组合物，其特征在于，还包括药学上可接受辅料和/或其他神经系统疾病治疗药物，所述其他神经系统疾病治疗药物包括用于治疗亨廷顿病的药物、用于治疗帕金森病的药物、用于治疗抑郁症的药物、用于治疗运动失调的药物、用于治疗脑卒中的药物、用于治疗中枢神经系统损伤疾病的药物、用于治疗肌萎缩侧索硬化的药物和用于治疗多发性硬化症的药物。
  10. 权利要求1-7中任一项所述的化合物和权利要求8-9任一项所述的药物组合物在制备用于预防或治疗神经系统疾病的药物中的用途。
  11. 根据权利要求10所述的用途，其特征在于，所述神经系统疾病包括神经元损伤、神经元退化、脑萎缩相关疾病或病症、中枢神经系统损伤、脑卒中、抑郁症、焦虑症、肌萎缩侧索硬化、阿尔茨海默氏病、自闭症、亨廷顿病、瑞特综合征、癫痫 症、帕金森氏病、创伤后应激障碍、糖尿病性神经病变、多发性硬化症、周围神经病。
  12. 权利要求1-7中任一项所述的化合物和权利要求8-9任一项所述的药物组合物在制备用于激动TrkB受体的药物中的用途。
  13. 一种用于激动TrkB受体的方法，包括向有需要的个体或样本给予有效量的权利要求1～7任一项所述的化合物或权利要求8～9任一项所述的药物组合物。
  14. 一种用于预防、治疗或减轻神经系统疾病的方法，包括向有需要的个体给予治疗有效量的权利要求1～7任一项所述的化合物或权利要求8～9任一项所述的药物组合物。
  15. 根据权利要求14所述的方法，其中，，所述神经系统疾病包括神经元损伤、神经元退化、脑萎缩相关疾病或病症、中枢神经系统损伤、脑卒中、抑郁症、焦虑症、肌萎缩侧索硬化、阿尔茨海默氏病、自闭症、亨廷顿病、瑞特综合征、癫痫症、帕金森氏病、创伤后应激障碍、糖尿病性神经病变、多发性硬化症、周围神经病。
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