WO2019154195A1 - 神经退行性疾病的治疗药物及其应用 - Google Patents

神经退行性疾病的治疗药物及其应用 Download PDF

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WO2019154195A1
WO2019154195A1 PCT/CN2019/073773 CN2019073773W WO2019154195A1 WO 2019154195 A1 WO2019154195 A1 WO 2019154195A1 CN 2019073773 W CN2019073773 W CN 2019073773W WO 2019154195 A1 WO2019154195 A1 WO 2019154195A1
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compound
formula
pharmaceutically acceptable
kit
solvate
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PCT/CN2019/073773
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English (en)
French (fr)
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裴钢
俞飚
黄世超
曹鑫
石富春
周悦
安玉谦
陆婧
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上海东西智荟生物医药有限公司
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Priority to KR1020207025708A priority Critical patent/KR20200118158A/ko
Priority to RU2020128178A priority patent/RU2799454C2/ru
Priority to EP19751440.9A priority patent/EP3750904A4/en
Priority to JP2020564302A priority patent/JP7295145B2/ja
Priority to US16/967,584 priority patent/US11643428B2/en
Priority to AU2019218153A priority patent/AU2019218153B2/en
Publication of WO2019154195A1 publication Critical patent/WO2019154195A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • 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
    • A61P25/14—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
    • A61P25/24—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
    • 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
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars

Definitions

  • the present invention belongs to the field of pharmacy, and more particularly, to a novel therapeutic drug for neurodegenerative diseases and its use.
  • a neurodegenerative disease is a general term for a group of diseases caused by degenerative degeneration of chronic progressive central nervous tissue.
  • the main diseases include Parkinson's Disease (PD), Alzheimer's Disease (AD), Huntington Disease (HD), Amyotrophic Lateral Sclerosis (ALS) and the like.
  • Alzheimer's disease also known as Alzheimer's disease
  • Alzheimer's disease is a chronic progressive neurodegenerative disease characterized by progressive memory loss, cognitive dysfunction, and loss of independent self-care.
  • the main pathological feature of Alzheimer's disease is the formation of amyloid plaques and neurofibrillary filaments in the brain of patients.
  • Amyloid plaques are characteristic pathological changes of Alzheimer's disease, which are mainly formed by extracellular accumulation of amyloid- ⁇ (A ⁇ ) protein produced abnormally in cells.
  • a ⁇ amyloid- ⁇
  • a ⁇ oligomerized A ⁇
  • cascade reactions including free radical reactions, mitochondrial oxidative damage and inflammatory responses acting directly or indirectly on neurons and glial cells), leading to synaptic dysfunction and neuronal damage, and causing activation of microglia and astrocytes , accelerate the formation of nerve fiber tangles, leading to cognitive impairment after long-term effects.
  • a large number of recent studies have provided a variety of evidence to support the "A ⁇ hypothesis", showing the central role of A ⁇ in the pathogenesis of Alzheimer's disease.
  • Parkinson’s disease is a common neurodegenerative disease. It is clinically characterized by slow response, tremors, stiff body, and further loss of balance. Brain tissue studies in PD patients have found that the substantia nigra dopaminergic neurons in the disease patients are lost. Lewy inclusions are one of the hallmarks of degenerative neurons in Parkinson's disease. Studies have shown that many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and DLB (Dementia with Lewy Body) Lewy inclusion bodies are formed in the brain tissue of patients. Some studies have also supported the transplantation of neural stem cells for the treatment of Huntington's disease and amyotrophic lateral sclerosis.
  • neural progenitor cells In the mammalian brain, the proliferation and self-renewal of neural progenitor cells (NPCs) lasts throughout the life process and is an important part of nerve regeneration.
  • NPCs neural progenitor cells
  • AD Alzheimer's Disease
  • Promoting nerve regeneration is considered a potential treatment for anti-aging and aging-related neurodegenerative diseases. Therefore, one possible approach is to transplant embryonic neural stem cells or neural stem cells induced in vitro for cell replacement therapy. However, this new and complex technology is still controversial, especially their safety issues and cell sources.
  • Another approach is to use pharmacological means to activate endogenous neural stem cells for the purpose of treating neurodegenerative diseases.
  • Pharmacological methods are simple to operate and can specifically target specific functions of neural stem cells. Therefore, activation of endogenous neural stem cells is not only a viable treatment, but also a preventive measure. However, those skilled in the art also need to find a suitable drug that can better cross the internal barrier to effectively activate endogenous neural stem cells, thereby achieving effective treatment.
  • Y is independently selected from: O, N;
  • the compound is a mixture of any ratio of the beta configuration, the alpha configuration, and the beta configuration;
  • the compound is a mixture of any ratio of the alpha configuration, the beta configuration, the alpha configuration, and the beta configuration.
  • R1 to R4 are independently selected from the group consisting of hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen, or two adjacent groups of R1 to R4 are bonded to each other and The parent rings together form a ring structure.
  • R1 to R4 are independently selected from the group consisting of hydrogen, Hydroxy, C1-C2 alkyl.
  • the compound comprises
  • the compound comprises
  • the compound comprises
  • the compound does not comprise
  • a compound of formula (I), or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof for the preparation of a prophylactic, palliative Or a drug or kit for the treatment of neurodegenerative diseases, depression or stroke.
  • the neurodegenerative disease is:
  • a neurodegenerative disease characterized by a decrease in the number of substantia nigra dopaminergic neurons
  • a neurodegenerative disease characterized by a decrease in the content of striatal dopaminergic nerve fibers.
  • the intracerebral neuroinflammation is characterized by a marked increase in inflammatory factor expression; such inflammatory factors such as TNF-[alpha] and IL- l[beta].
  • the neurodegenerative diseases include: Alzheimer's disease, Parkinson's disease, Lewy body dementia (DLB), Huntington's disease, amyotrophic lateral sclerosis.
  • DLB Lewy body dementia
  • Huntington's disease amyotrophic lateral sclerosis.
  • the compound comprises:
  • a pharmaceutical composition comprising: the compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically thereof thereof An acceptable salt; and a pharmaceutically acceptable carrier.
  • the compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof is an effective amount in the pharmaceutical composition; preferably
  • the effective amount is 0.01-50% by weight, such as, but not limited to, 0.01-5%, 0.03-3%, 0.05-1%, 20-30%, 40-50%, etc.; more preferably It is 0.03-30%; further more preferably 0.05-10%.
  • the pharmaceutical composition provides a dosage form comprising: a powder, a powder, a tablet, a pill, a capsule, a sustained release, an immediate release, an injection, an infusion, and a suspension.
  • kits comprising: the compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically thereof thereof An acceptable salt; or a pharmaceutical composition as described.
  • a method of preventing, ameliorating or treating a neurodegenerative disease, depression or stroke comprising: administering to a subject in need of treatment an effective amount of the formula (I) a compound or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof.
  • a process for the preparation of a compound of formula III, IV or V comprising the steps of: beta-rhamnoside, alpha-1-aminorhamnoside or beta The 1-aminorhamnose glycoside is reacted with tetrabutylammonium fluoride to obtain a compound having the structure shown in formula III-V;
  • the beta rhamnoside is obtained by reacting 2,3,4-O-triacetyl rhamnose with (4-O-tert-butyldimethylsilyl)-ferulic acid
  • the acid chloride is obtained by a reaction.
  • the ⁇ -1-aminorhamnoside and/or ⁇ -1-aminorhamnoside is obtained by reacting 2,3,4-O-triacetyl-1-amino rhamnose with (4-O-tert-butyldimethylsilyl)-ferulic acid chloride is obtained by reaction.
  • FIG. 1 PL201 promotes neurogenesis in vivo.
  • PL201 reduces the number of A ⁇ plaques in the body.
  • PL201 increases the content of striatal dopaminergic nerve fibers.
  • PL202 reduces the expression of inflammatory factors IL-1 ⁇ and iNOS.
  • FIG. 15 Nuclear magnetic resonance spectrum and 1H NOESY spectrum of PL202; where a is a nuclear magnetic resonance spectrum and b is a NOESY spectrum.
  • FIG. 18 Black matter TH staining map; where a is H 2 O+NS, b is PL201+NS, c is H 2 O+MPTP, and d is PL201+MPTP.
  • the inventors have intensively studied that the compound of formula (I) can significantly ameliorate the symptoms of neurodegenerative diseases.
  • the compounds of formula (I) are effective in enhancing the function of neural stem cells; they can not only prevent, but also serve as a treatment to promote nerve regeneration to combat cognition related to aging and neurodegenerative diseases. The function is down.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group containing from 1 to 4 carbon atoms, preferably from 1 to 2 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl.
  • alkenyl as used herein, includes straight-chain and branched hydrocarbon groups containing at least one carbon-carbon double bond and 2 to 4 carbon atoms, preferably 2 to 3 carbon atoms.
  • alkynyl as used herein includes both straight-chain and branched hydrocarbon groups containing at least one carbon-carbon triple bond and from 2 to 4 carbon atoms, preferably from 2 to 3 carbon atoms.
  • halogen refers to F, Cl, Br, or I.
  • isomer as used herein includes: geometric isomers, enantiomers, diastereomers (eg, cis and trans isomers, conformational isomers).
  • solvate denotes a compound carrying a solvent molecule, for example, the solvate may be a hydrate.
  • the term "containing” means that the various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms “consisting essentially of” and “consisting of” are encompassed by the term “contains.”
  • a "pharmaceutically acceptable" ingredient is a substance which is suitable for use in humans and/or animals without excessive adverse side effects (e.g., toxicity, irritation, and allergy), i.e., has a reasonable benefit/risk ratio.
  • a "pharmaceutically acceptable carrier” is a method for delivering a compound, an isomer, a solvate, a precursor, or a pharmaceutically acceptable salt thereof of the formula (I) of the present invention to an animal or a human.
  • the carrier can be a liquid or a solid.
  • the present invention first provides a compound of formula (I):
  • the position of X is a schematic position, and is not limited to one side of R1 in the figure (R1 and The position between the groups), which may also exist between R1 and R2, between R2 and R3, between R3 and R4, and R4
  • R1 and R2 between R2 and R3, between R3 and R4, and R4
  • R4 between groups, for example, the compound can also be:
  • a six-membered heterocyclic ring X is O; Y is independently selected from: O, N; when Y represents O, the compound is a mixture of any ratio of ⁇ configuration, ⁇ configuration, and ⁇ configuration; when Y represents N
  • the compound is in any ratio mixture of the alpha configuration, the beta configuration, the alpha configuration, and the beta configuration;
  • R1 to R4 are independently selected from the group consisting of: hydrogen, hydroxy, C1-C4 alkyl, C2-C4 alkenyl.
  • the C2-C4 alkynyl group, the halogen, or two adjacent groups of R1 to R4 are bonded to each other and form a ring structure (which may be an O-containing ring structure) together with the parent ring.
  • R1 to R4 are independently selected from the group consisting of hydrogen, hydroxy, and C1-C2 alkyl.
  • the present invention also includes isomers, solvates, precursors, or pharmaceutically acceptable salts thereof of the above compounds of the formula (I), as long as they also have the same or substantially the same functions as the compounds of the formula (I).
  • pharmaceutically acceptable salt means a salt formed by reacting a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal.
  • salts include, but are not limited to, (1) salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid, maleic acid, or arginine.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates, or other conventional "prodrugs".
  • the compound has one or more asymmetric centers. Therefore, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis or trans isomers.
  • precursor of a compound means a compound which is converted into a structural formula (I) by a metabolic or chemical reaction of a precursor of the compound in a patient after administration by an appropriate method, or a chemical structural formula (I). a salt or solution of a compound.
  • the compound includes the following compounds represented by Formula II (PL201), III (PL202), IV (PL171), and V (PL172), and the compounds of Formula III and Formula IV are particularly preferred. .
  • the compound of formula II is ( ⁇ -L-pyran rhamido) ferulic acid ester, including but not limited to,
  • the compound of formula III is ( ⁇ -L-pyran rhamido) ferulic acid ester, including but not limited to,
  • the compound of formula IV is (1-amino- ⁇ -L-pyran rhamnoyl) ferulic acid amide, including but not limited to,
  • the method of preparing a compound of formula III comprises the steps of:
  • the beta glycosidation product of the formula VI is mixed with tetrabutylammonium fluoride to obtain a de-tert-butyldimethylsilyl (TBS) product;
  • the de-tert-butyldimethylsilyl (TBS) product is hydrolyzed to provide a compound of formula III;
  • the first step is carried out by dropping (4-O-tert-butyldimethylsilyl) into 2,3,4-O-triacetyl rhamnose under ice bath. - ferulic acid chloride.
  • the reaction of the first step described above is carried out at room temperature; the room temperature is 10-40 ° C, preferably 15-30 ° C, more preferably 20-25 ° C.
  • the first step further comprises separately obtaining ⁇ and by column chromatography.
  • the mixing in the second step above is to treat tetrabutyl fluoride at room temperature.
  • Ammonium is added dropwise to the beta glycosidation product.
  • the method of preparing a compound of formula IV comprises the steps of:
  • the ⁇ -glycosylation product of the formula VIII is separately mixed with tetrabutylammonium fluoride to obtain an ⁇ -de-tert-butyldimethylsilyl (TBS) product;
  • the ⁇ -de-tert-butyldimethylsilyl (TBS) product is separately hydrolyzed to obtain a compound of the formula IV;
  • the first step is carried out by dropwise addition of (4-O-tert-butyldimethyl) to 1-amino-2,3,4-O-triacetyl rhamnose in an ice bath.
  • Silyl)-ferulic acid chloride is a compound that has a side chain length of 1 methylene glycol.
  • the reaction of the first step described above is carried out at room temperature; the room temperature is 10-40 ° C, preferably 15-30 ° C, more preferably 20-25 ° C.
  • the first step further comprises the step of obtaining an alpha glycosidation product by column chromatography, respectively.
  • the mixing in the second step above is to treat tetrabutyl fluoride at room temperature.
  • the ammonium salt is separately dropped into the ⁇ -glycosylation product.
  • the method of preparing a compound of formula V comprises the steps of:
  • 1-amino-2,3,4-O-triacetyl rhamnose and (4-O-tert-butyldimethylsilyl)-ferulic acid chloride are mixed to obtain a structure such as a beta glycosidation product of formula IX;
  • the beta glycosidation product of the formula IX is mixed with tetrabutylammonium fluoride to obtain a ⁇ -de-tert-butyldimethylsilyl (TBS) product;
  • the ⁇ -de-tert-butyldimethylsilyl (TBS) product is separately hydrolyzed to obtain a compound of formula V;
  • the first step is carried out by dropwise addition of (4-O-tert-butyldimethyl) to 1-amino-2,3,4-O-triacetyl rhamnose in an ice bath.
  • Silyl)-ferulic acid chloride is a compound that has a side chain length of 1 methylene glycol.
  • the reaction of the first step described above is carried out at room temperature; the room temperature is 10-40 ° C, preferably 15-30 ° C, more preferably 20-25 ° C.
  • the first step further comprises the step of separately obtaining a beta glycosidation product by column chromatography.
  • the mixing in the second step described above is carried out by separately dropping tetrabutylammonium fluoride into the beta glycosidation product at room temperature.
  • the compounds of the present invention can be obtained by a variety of methods well known in the art, using known starting materials, such as chemical synthesis or from organisms (such as animals or plants). The method of extraction, which is included in the present invention.
  • the synthesized compound can be further purified by column chromatography, high performance liquid chromatography or the like.
  • the present inventors have found in studies that the compound of the formula (I) of the present invention can significantly improve the symptoms of neurodegenerative diseases.
  • the compounds of the present invention are capable of inhibiting neuroinflammation, reducing A ⁇ production, enhancing neural stem cells, and enhancing the function of dopaminergic neurons.
  • the compounds of the present invention have a significant improvement in the learning and memory ability of animals.
  • the mechanism of action of the compound of the present invention it is capable of enhancing the function of neural stem cells, and thus it is also effective for Huntington's disease and amyotrophic lateral sclerosis.
  • the mechanism of action of the compound according to the present invention is also effective for depression and stroke.
  • the compound of the formula (I) of the invention is capable of enhancing the function of neural stem cells, from which it can be understood that it is also effective for depression and stroke.
  • the present invention provides the use of a compound of the formula (I) or an isomer, a solvate thereof, a precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a prophylactic, A drug or kit that relieves or treats a neurodegenerative disease, depression, or stroke.
  • the present invention also provides the use of a compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a composition, a kit or a kit for inhibiting neuroinflammation Pill box.
  • the present invention also provides the use of a compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a composition, a kit for promoting neural stem cell production Or a pill box.
  • the present invention also provides the use of a compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a composition, a kit or a kit for reducing A ⁇ production. Pill box.
  • the present invention also provides the use of a compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a combination for increasing the number of dopaminergic neurons in the substantia nigra Object, kit or kit.
  • the present invention also provides the use of a compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a striatum dopaminergic nerve fiber.
  • the compound of the formula (II) (PL201), the compound of the formula (III) (PL202), the compound of the formula (IV) (PL171) and the compound of the formula (V) (PL172) provided by the present invention are excellent in the above-mentioned uses.
  • the effect is particularly excellent in the effect of the formula (III) and the compound (IV).
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) an effective amount of the compound of the formula (I), or an isomer, solvate, precursor thereof, or a pharmaceutically acceptable salt thereof And (b) a pharmaceutically acceptable carrier or excipient.
  • the content of the compound of the formula (I) or an isomer, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, of the pharmaceutical composition is an effective amount.
  • the compound of the formula (I) or a pharmaceutically acceptable salt thereof may be contained in an amount of from 0.001 to 50% by weight.
  • the pharmaceutical composition contains 0.01 to 20% by weight of the compound of the formula (I) or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present invention may be in a variety of dosage forms as long as it is a dosage form capable of efficiently reaching the mammalian body.
  • it can be selected from the group consisting of powders, powders, tablets, pills, capsules, sustained release agents, controlled release agents, injections, infusion solutions, and suspensions. Based on the type of disease to which the compounds of the present invention are treated, one skilled in the art can select a dosage form that is convenient to use.
  • preferred pharmaceutical compositions are solid compositions, especially tablets and solid filled or liquid filled capsules. From the standpoint of the granule length which is easy to administer, the preferred pharmaceutical composition is an oral preparation.
  • the compounds of the invention or pharmaceutical compositions thereof may also be stored in a sterilizing device suitable for injection or drip.
  • the effective administration dose of the compound of the formula (I) as an active ingredient may vary depending on the mode of administration and the severity of the disease to be treated. However, usually, when the compound of the present invention is administered at a dose of about 0.01 to 100 mg/kg of animal body weight per day, a satisfactory effect can be obtained, preferably at a dose of 1-3 divided times per day, or in a sustained release form. Dosing. This dosage regimen can be adjusted to provide an optimal therapeutic response. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • Example 1 PL201 improves learning and memory ability of AD mice
  • APP/PS1 transgenic male mice aged 5-6 months were randomly divided into model group and polysaccharide administration group.
  • PL201 was administered by intragastric administration and administered daily (10 mg). /kg mouse body weight), while 10 non-transgenic mice were used as a negative control group (no PL201).
  • the Morris water maze behavioral experiment was used to detect the learning of APP/PS1 mice by PL201. The impact of cognitive function.
  • This experiment uses the classic Morris water maze test procedure, which consists of two parts, the positioning navigation test and the space exploration test. A total of 7 days, 4 days and 7 days were added to the space exploration test.
  • mice in the model group were slow to respond, and there was a circle along the barrel wall after entering the water. There is no escape behavior, after artificially leading it to the platform, it jumps into the water. After many trainings, the platform is finally found, but the latency of finding the platform is obviously prolonged.
  • the mice in the drug-administered group continue to search for the platform as the number of training increases. improve.
  • the above three groups all have certain spatial memory ability.
  • the latency of the model group mice was worse than that of the control group, suggesting that the learning and memory ability of the model group mice decreased, and the model mice better simulated the learning and memory impairment of AD.
  • Example 2 PL201 promotes in vivo neurogenesis
  • the present inventors performed a PL201 administration test on AD mice (selective 5-6 month old AD mice), and each mouse was intragastrically administered with PL201, and 100 l was administered at a concentration of 10 mg/kg of mouse body weight.
  • Another control group was given 100 l of water once a day for 90 days, and 50 mg/kg of mouse body weight of 5-bromo-2'-deoxyuridine (BrdU) was started intraperitoneally at 60 days once a day. After 7 days of injection, the mice were anesthetized and the PFA was perfused for 90 days after administration, and the whole brain was taken for the next experiment.
  • the mouse brain of the mature neuronal marker NeuN:PFA in the mouse hippocampus was continuously fixed in 4% PFA for 24 hours, and then allowed to stand in 30% sucrose fixative for 72 hours.
  • the cerebellum portion was removed, and the brain olfactory bulb was placed upright on the filter paper, and frozen at -80 ° C for at least 24 hours.
  • the longitudinal section was frozen at a thickness of 30 m, and the DG part was taken according to the mouse brain map.
  • the cut brain slices are placed in tissue protection solution (30% sucrose, 30% ethylene glycol, 0.1 M PB) and can be stored at -20 ° C for more than half a year.
  • a group of brain slices were taken during staining, and blocked in blocking solution (10% sputum serum, 0.3% Triton X-100, PBS) for 45 minutes at room temperature, and then added with primary antibody at 4 ° C overnight (the ratio of primary antibody dilution: rat anti-BrdU , 1:2000; rabbit anti-Ki67, 1:1000; goat anti-Dcx, 1:200; mouse anti-NeuN, 1:200; goat anti-Sox2, 1:60), and then incubated with a suitable fluorescent secondary antibody for 1 hour at room temperature.
  • DAPI stains the nucleus.
  • the stained brain slices were then photographed with Olympus FV100i or Leica SP-8. The number of positive or double stained cells was analyzed by Image Pro Plus software.
  • brain slices requiring antigen retrieval were treated in an antigen retrieval solution (10 mM sodium citrate, pH 6.5) at 95 ° C for 20 minutes before serum blocking.
  • BrdU staining brain slices were treated with 2M hydrochloric acid at 37 ° C for 30 minutes before serum blocking, and then washed with 0.1 M borate buffer (pH 8.5).
  • the inventors found that there were more BrdU/NeuN double positive neonatal neurons in the hippocampus of PL201-administered mice compared to the control mice, as shown in FIG.
  • Example 3 PL201 alleviates the inhibition of proliferation of neural stem cells by A ⁇ in vitro
  • a ⁇ As the main pathogenic protein of AD, A ⁇ has an inhibitory effect on the proliferation of neural stem cells.
  • the present inventors examined the proliferative ability of human neural stem cells by the method of EdU incorporation, and investigated whether PL201 can alleviate the inhibitory effect of A ⁇ on neural stem cell proliferation.
  • 3L of human neural stem cells were cultured in DMEM/F12 (containing N2/B27 and 10 ng/ml bFGF), cultured in 96-well plates for 24 hours, pre-treated with A ⁇ (5 ⁇ M) for 30 minutes, and then added with PL201 (30M). After the drug was incubated for 72 hours, EdU staining was used to detect the proliferation of neural stem cells.
  • Example 4 PL201 inhibits neuroinflammation
  • Neuroinflammation in the AD brain is one of the causes of cognitive decline.
  • Microglia are key cells that mediate neuroinflammation.
  • the present inventors investigated whether PL201 has an inhibitory effect on neuroinflammation by detecting the expression of inflammatory factors on mouse glioma cell BV-2.
  • Mouse glioma BV-2 was cultured in DMEM and plated in 24-well plates for 24 hours.
  • PL201 (0, 10, 30, 100 ⁇ M) and LPS (300 ng/ml) were added.
  • Trizol extracts RNA and detects the expression of related inflammatory factors by QPCR.
  • Example 5 PL201 reduced the number of A ⁇ plaques in vivo.
  • a ⁇ plaque deposition in the AD brain is one of the key causes of neurological damage, which in turn leads to cognitive decline. Therefore, in this embodiment, it is further explored whether PL201 also has an effect on the number of A ⁇ plaques in the brain.
  • the inventors of the present invention applied a whole brain sample of AD mice after administration to a vertical longitudinal section of 30 m thickness and subjected to thioflavin S and DAPI staining. The stained brain slices were then photographed with Olympus FV100i or Leica SP-8.
  • Example 6 PL201 reduces A ⁇ generation
  • a ⁇ is the major causative protein of AD.
  • the inventors investigated whether the PL201 has an effect of inhibiting the reduction of A ⁇ production by detecting the level of A ⁇ on the neuroblastoma cell SK-N-SH.
  • SK-N-SH Cultured in DMEM, cultured in 24-well plates for 24 hours, then added PL201 (dosages 0, 30, 100, 300 ⁇ M), plus After the drug was incubated for 24 hours, the culture supernatant was taken out and the protein level of total A ⁇ was measured by ELISA.
  • Mitochondrial membrane potential is the main indicator of mitochondrial function, and mitochondrial function is an important indicator of the pathogenesis of AD.
  • the inventors investigated whether the PL201 has an effect of improving mitochondrial function by detecting the mitochondrial membrane potential level of human neural stem cell cells.
  • Mitochondrial function changes are closely related to the regulation of metabolic signaling pathways such as AMPK, and these signaling pathways are also widely considered to be closely related to the pathogenesis of AD.
  • the present inventors examined the level of AMPK phosphorylation in human neural stem cell cells by immunoblotting assay to investigate whether PL201 has a function of regulating metabolic signaling pathways.
  • 3L of human neural stem cells were cultured in DMEM/F12 (containing N2/B27 and 10 ng/ml bFGF), cultured in 12-well plates for 24 hours, pre-treated with A ⁇ (5 ⁇ M) for 30 minutes, and then added with PL201 (30M). After the drug was incubated for 72 hours, the cells were lysed by Laemmli's sample buffer in PBS, and the relevant protein bands were detected by SDS PAGE electrophoresis, rabbit anti-phospo-AMPK and total AMPK antibodies.
  • Example 9 PL201 improves the detection index of mouse climbing rod method
  • the MPTP-induced PD animal model is the most classical animal model similar to the pathological changes and clinical features of human Parkinson's disease.
  • the main symptoms of PD are static tremor, increased muscle tone, decreased exercise, etc.
  • the U-turn time and climb time of the climbing rod experiment can represent the overall activity coordination ability of the mouse.
  • mice were randomly divided into 4 groups: saline group (NS), MPTP model saline group (NS+MPTP), PL201 compound 50 mg intragastric administration group (PL201), MPTP model gavage PL201 compound group (PL201). +MPTP).
  • the animals were started on the day of the grouping, the saline group and the MPTP model were intragastrically administered with normal saline, and the other two groups were given PL201 compound once a day for 7 days.
  • the model drug MPTP or NS was administered from the 7th day, the saline group (NS) and the PL201 compound 50 mg intragastric administration group (PL201) were intraperitoneally injected with normal saline 5 ml/kg, and the other animals were intraperitoneally injected with MPTP 25 mg/kg, 1 per day. Times, a total of 5 days.
  • mice were subjected to a climbing rod test to evaluate the motor coordination ability of the mice. Place the mouse head gently on the top of the rough pole (1 cm in diameter and 50 cm in height). The time from the head up to the head down to the head is recorded as the time to turn around, and the time from the downward movement of the mouse to the time when the limbs all reach the sole is recorded as Time to Climb down, over 30. The seconds are recorded in 30 seconds. Each mouse was repeatedly tested 5 times for averaging.
  • Example 10 PL201 increased striatal dopaminergic nerve fiber content
  • the substantia nigra dopaminergic neurons and striatal dopaminergic nerve fiber loss are the main pathological features of Parkinson's disease. Immunostaining is used to detect the loss of striatal dopaminergic nerve fibers. To explore whether PL201 has increased striatal dopaminergic nerve fibers. The role of the content.
  • mice were randomly divided into 4 groups: saline group (NS), MPTP model saline group (NS+MPTP), PL201 compound 50 mg intragastric administration group (PL201), MPTP model gavage PL201 compound group (PL201). +MPTP).
  • the animals were started on the day of the grouping, the saline group and the MPTP model were intragastrically administered with normal saline, and the other two groups were given PL201 compound once a day for 7 days.
  • the model drug MPTP or NS was administered from the 7th day, the saline group (NS) and the PL201 compound 50 mg intragastric administration group (PL201) were intraperitoneally injected with normal saline 5 ml/kg, and the other animals were intraperitoneally injected with MPTP 25 mg/kg, 1 per day. Times, a total of 5 days.
  • each group of animals was anesthetized with 10% chloral hydrate. After perfusion, the brain tissue was separated and placed in a 1.5 ml EP tube. 250 ⁇ l of RIPA protein lysate (ThermoFisher) was used to ultrasonically disrupt the tissue and collect. The supernatant was assayed for protein concentration. The protein concentration was adjusted to 2 ⁇ g/ ⁇ l, and 5 ⁇ reduction of Loadingbuffer boiling denaturing protein was added, and the sample was applied to 10% SDS-polyacrylamide gel electrophoresis, and transferred to a methanol-activated PVDF membrane by a 100V wet transfer method.
  • RIPA protein lysate ThermoFisher
  • 5% skim milk TBST was blocked at room temperature for 1 h, and added with TH (1:1000) primary antibody at 4 ° C overnight. After washing the non-specific binding primary antibody with TBST buffer, add Goat Anti-Mouse/Rabbit fluorescent secondary antibody (LI-COR), incubate for 1 hour at room temperature, wash the TBSTbuffer three times, and detect the fluorescence signal by Odyssey near-infrared fluorescence scanner.
  • LI-COR Goat Anti-Mouse/Rabbit fluorescent secondary antibody
  • the PL201 compound has protective effects on the damage of mouse striatum dopaminergic neuron fibers and increases the content of striatal dopaminergic nerve fibers, as shown in Fig. 10.
  • the experimental results show that the PL201 compound has a therapeutic effect on Parkinson's disease.
  • Example 11 PL201 increased the number of dopaminergic neurons in the substantia nigra and striatum dopaminergic nerve fiber content
  • the substantia nigra dopaminergic neurons and striatal dopaminergic nerve fiber loss are the main pathological features of Parkinson's disease.
  • the tyrosine hydroxylase (TH) immunohistochemistry is used to detect the striatum dopaminergic nerve fiber loss.
  • TH tyrosine hydroxylase
  • mice were randomly divided into 4 groups: saline group (NS), MPTP model saline group (NS+MPTP), PL201 compound 50 mg intragastric administration group (PL201), MPTP model gavage PL201 compound group (PL201). +MPTP).
  • the animals were started on the day of the grouping, the saline group and the MPTP model were intragastrically administered with normal saline, and the other two groups were given PL201 compound once a day for 7 days.
  • the model drug MPTP or NS was administered from the 7th day, the saline group (NS) and the PL201 compound 50 mg intragastric administration group (PL201) were intraperitoneally injected with normal saline 5 ml/kg, and the other animals were intraperitoneally injected with MPTP 25 mg/kg, 1 per day. Times, a total of 5 days.
  • each group of animals was anesthetized with 10% chloral hydrate and brain was taken after perfusion of 4% paraformaldehyde. After fixing 4% paraformaldehyde for 24 hours, the sample was transferred to a 30% sucrose solution and dehydrated to the bottom of the sample. Coronal sections of the midbrain and striatum were made in a -20 degree cryostat. 30 microns.
  • the primary antibody was monoclonal mouse anti-TH (1:1000, rabbit anti-mouse antibody), incubated for 2.5 hours at room temperature, TBST solution (8 g sodium chloride, 0.2 g potassium chloride and 3 g Tris-base, distilled water to volume) 1 liter, adjusted to pH 7.4 with hydrochloric acid) After washing three times, the secondary antibody (goat anti-rabbit antibody) was labeled with horseradish oxide enzyme (HPR) and incubated for 1 hour at room temperature. It was developed with the developer diaminobenzidine (DAB), dehydrated with ethanol gradient, transparent with xylene, and sealed with neutral gum. The stained sections were analyzed by Image-proplus software.
  • HPR horseradish oxide enzyme
  • the total density of TH positive staining in the substantia nigra region was used as the measure of the number of dopaminergic neurons in the substantia nigra region.
  • the average optical density of TH positive staining in the striatum was used as the striatal dopaminergic nerve. The measure of fiber density is counted.
  • the PL201 compound has protective effects on the damage of dopaminergic neurons and striatal dopaminergic nerve fibers in mice, and increases the number of dopaminergic neurons in the substantia nigra and the dopaminergic nerve fibers in the striatum.
  • Example 12 PL202 reduces A ⁇ generation
  • a ⁇ is the major causative protein of AD.
  • the inventors examined whether the isoform PL202 of PL201 has an effect of inhibiting the reduction of A ⁇ production by detecting the level of A ⁇ on the neuroblastoma cell SK-N-SH.
  • SK-N-SH was cultured in DMEM, and cultured in 24-well plates for 24 hours, then PL202 (0, 30, 100 ⁇ M) was added. After co-incubation for 24 hours, the culture supernatant was taken out and the protein level of total A ⁇ was measured by ELISA.
  • Neuroinflammation in the AD brain is one of the causes of cognitive decline.
  • Microglia are key cells that mediate neuroinflammation.
  • the present inventors investigated whether PL202 has an inhibitory effect on neuroinflammation by detecting the expression of inflammatory factors on mouse glioma cell BV-2.
  • Mouse glioma cells BV-2 were cultured in DMEM and plated in 24-well plates for 24 hours.
  • PL202 (0, 30, 100, 300 ⁇ M) and LPS (300 ng/ml) were added.
  • Trizol extracts RNA and detects the expression of related inflammatory factors by QPCR.
  • Example 14 PL171 reduces A ⁇ generation
  • a ⁇ is the major causative protein of AD.
  • the inventors examined whether the PL171 has an effect of inhibiting the reduction of A ⁇ production by detecting the level of A ⁇ on the neuroblastoma cell SK-N-SH.
  • SK-N-SH Culture of neuroblastoma cells SK-N-SH and detection of A ⁇ : SK-N-SH was cultured in DMEM, and cultured in 24-well plates for 24 hours. PL171 (300 ⁇ M) was added and incubated for 24 hours. The culture supernatant was taken out and the protein level of total A ⁇ was measured by ELISA.
  • Example 15 PL172 reduces A ⁇ generation
  • a ⁇ is the major causative protein of AD.
  • the inventors examined whether the isoform PL172 of PL171 has an effect of inhibiting the reduction of A ⁇ production by detecting the level of A ⁇ on the neuroblastoma cell SK-N-SH.
  • SK-N-SH Culture of neuroblastoma cells SK-N-SH and detection of A ⁇ : SK-N-SH was cultured in DMEM and plated in 24-well plates for 24 hours. PL172 (300 ⁇ M) was added and incubated for 24 hours. The culture supernatant was taken out and the protein level of total A ⁇ was measured by ELISA.
  • 2,3,4-O-triacetyl rhamnose (5.8 g, 20 mmol) was dissolved in 100 mL of anhydrous dichloromethane, and 50 mL of (4-O-TBS)-ferulic acid chloride was added dropwise in an ice bath. 6.52 g, 20 mmol) (Free Radical Res. 2015, 102) in anhydrous dichloromethane and 1.0 mL anhydrous pyridine. The reaction was stopped by stirring at room temperature for 2 hours. It was diluted with 200 mL of dichloromethane, washed with saturated sodium chloride and dried over MgSO4.

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Abstract

本发明涉及新型的神经退行性疾病的治疗药物及其应用。提供了一种新型的式(I)化合物，在体外和体内实验中，其均能有效促进神经干细胞的增殖；其还能作为一种促进神经再生的治疗手段，对抗衰老和神经退行性疾病相关的认知功能下降。

Description

神经退行性疾病的治疗药物及其应用 技术领域
本发明属于药学领域，更具体地，本发明涉及新型的神经退行性疾病的治疗药物及其应用。
背景技术
神经退行性疾病是指一组由慢性进行性的中枢神经组织退行性变性而产生的疾病的总称。主要疾病包括帕金森病(Parkinson’s Disease，PD)、阿尔茨海默病(Alzheimer’s Disease，AD)、亨廷顿病(Huntington Disease，HD)、肌萎缩侧索硬化症(Amyotrophic Lateral Sclerosis，ALS)等。
阿尔茨海默症亦称为早老性痴呆，是一种慢性进行性的神经退行性疾病，主要表现为渐进性的记忆能力下降，认知功能障碍以及失去生活独立自理能力。随着人口老龄化的不断加剧，AD的发病率也逐年升高，已成为最重要的公众关注健康问题。阿尔兹海默症的主要病理学特征为病人大脑内形成的淀粉样蛋白斑和神经纤维丝缠结。淀粉样蛋白斑是阿尔兹海默症的特征性病理学变化，主要由细胞内异常大量产生的淀粉样蛋白-β(Aβ)蛋白在细胞外积聚形成的。目前，有多个理论试图解释其致病机理。Hardy和Selkoe提出的“Aβ假说”是目前被广为接受的理论。该理论认为，在复杂的遗传和环境因素长期作用下，神经细胞异常地大量产生Aβ，积累形成寡聚体和淀粉样蛋白斑，Aβ(尤其是寡聚化的Aβ)通过一系列级联反应(包括自由基反应、线粒体氧化损伤和炎症反应等直接或间接地作用于神经元和胶质细胞)，导致突触功能异常和神经元损伤，并且引起小胶质细胞和星型胶质细胞激活，加速神经纤维丝缠结的形成，长期作用后导致认知障碍。近期大量研究提供了多方面的证据支持“Aβ假说”，显示了Aβ在阿尔兹海默症致病机理中的核心作用。
帕金森症(Parkinson’s disease)是一种常见的神经退行性疾病，在临床上，它主要表现为反应迟缓，震颤，机体僵硬，进一步失去平衡等症状。对PD病人脑组织研究发现，疾病患者中脑黑质多巴胺能神经元丧失。Lewy包涵体是帕金森病中变性神经元的标志性病变之一，研究表明，许多神经退行性疾病，包括阿尔茨海默症(Alzheimer’s disease)、帕金森症和DLB症 (Dementia with Lewy Body)等病人脑组织中都有Lewy包涵体形成。一些研究结果也支持移植神经干细胞有助于亨廷顿病和肌萎缩侧索硬化症的治疗。
在哺乳动物大脑中，神经干细胞(Neural Progenitor Cells，NPC)的增殖和自我更新持续于整个生命过程，是神经再生(Neurogenesis)的重要环节。在衰老、长期压力以及神经系统疾病，比如阿尔兹海默症(Alzheimer’s Disease，AD)发生的情况下，神经干细胞的增殖和自我更新能力下降，导致认知功能受损。促进神经再生被认为是抵抗衰老和衰老相关神经退行性疾病的潜在治疗手段。因此，一种可能的方案是移植胚胎神经干细胞或者体外诱导的神经干细胞来进行细胞替换疗法。但是，这种新创的复杂技术目前还存在一定争议，尤其是它们的安全性问题和细胞来源等问题。另一种方案是用药理学手段来激活内源的神经干细胞，以达到治疗神经退行性疾病的目的。药理学手段操作简便，并且可以特异性靶向神经干细胞的特定功能，因此，激活内源神经干细胞不仅是一种可行的治疗手段，还能作为预防手段。然而，本领域技术人员还需要找到合适的、能给较好地穿越体内屏障以有效激活内源神经干细胞的药物，从而实现有效的治疗。
发明内容
本发明的目的在于提供新型的神经退行性疾病的治疗药物及其应用。
在本发明的第一方面，提供式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐，
Figure PCTCN2019073773-appb-000001
其中，
Figure PCTCN2019073773-appb-000002
为六元杂环，X为O；
Y独立地选自：O，N；
当Y表示O时，所述化合物为β构型、α构型和β构型的任意比例混合物；
当Y表示N时，所述化合物为α构型、β构型、α构型和β构型的任意比例混合物。
R1～R4独立地选自：氢、羟基、C1-C4烷基、C2-C4链烯基、C2-C4链炔基、卤素，或R1～R4中相邻两个基团相互连接、并与母环共同构成环结构。
在一个优选例中，所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐中，R1～R4独立地选自：氢、羟基、C1-C2烷基。
在另一优选例中，所述化合物包含
Figure PCTCN2019073773-appb-000003
在另一优选例中，所述化合物包含
Figure PCTCN2019073773-appb-000004
在另一优选例中，所述化合物包含
Figure PCTCN2019073773-appb-000005
在另一优选例中，所述的化合物不包含
Figure PCTCN2019073773-appb-000006
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备预防、缓解或治疗神经退行性疾病、抑郁症或中风的药物或药盒。
在一个优选例中，所述的神经退行性疾病是：
以脑内发生神经炎症为特征的神经退行性疾病；或
以Aβ生成显著性增加为特征的神经退行性疾病；或
以学习记忆能力显著性下降为特征的神经退行性疾病；或
以神经干细胞功能下降为特征的神经退行性疾病。；或
以运动协调能力下降为特征的神经退行性疾病；或
以黑质多巴胺能神经元数量下降为特征的神经退行性疾病；或
以纹状体多巴胺能神经纤维含量下降为特征的神经退行性疾病。
在另一优选例中，所述的脑内发生神经炎症以炎症因子表达显著性提高为特征；所述的炎症因子如TNF-α和IL-1β。
在另一优选例中，所述的神经退行性疾病包括：阿尔茨海默症，帕金森症，路易体痴呆(DLB症)，亨廷顿病、肌萎缩侧索硬化症。
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备抑制神经炎症的组合物、试剂盒或药盒。
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备促进神经干细胞功能的组合物、试剂盒或药盒。
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备降低Aβ生成的组合物、试剂盒或药盒。
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高黑质多巴胺能神经元数量的组合物、试剂盒或药盒。
在本发明的另一方面，提供所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高纹状体多巴胺能神经纤维含量的组合物、试剂盒或药盒。
在一个优选例中，所述的化合物包括：
Figure PCTCN2019073773-appb-000007
又称PL201；
Figure PCTCN2019073773-appb-000008
又称PL202；
Figure PCTCN2019073773-appb-000009
又称PL171；和
Figure PCTCN2019073773-appb-000010
又称PL172。
在本发明的另一方面，提供一种药物组合物，所述的药物组合物包含：所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐；和药学上可接受的载体。
在一个优选例中，所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐在药物组合物中是有效量的；较佳地，所述的有效量按照重量含量为0.01-50％，例如但不限于，0.01-5％、0.03-3％、0.05-1％、20-30％、40-50％等；更佳地为0.03-30％；进一步更佳地为0.05-10％。
在本发明的另一方面，提供所述的药物组合物的剂型包括：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂、注射剂、输液剂、混悬剂。
在本发明的另一方面，提供一种药盒，所述的药盒中包括：所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐；或所述的药物组合物。
在本发明的另一方面，提供一种预防、缓解或治疗神经退行性疾病、抑郁症或中风的方法，所述方法包括：给予需要治疗的对象有效量的所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐。
在本发明的另外一方面，提供了一种如式Ⅲ、Ⅳ或Ⅴ所示的化合物的制备方法，所述方法包括步骤：使β-鼠李糖苷、α-1-氨基鼠李糖苷或β-1-氨基鼠李糖苷分别与四丁基氟化铵反应得到结构如式Ⅲ～Ⅴ所示的化合物；
Figure PCTCN2019073773-appb-000011
Figure PCTCN2019073773-appb-000012
在另一优选例中，所述β鼠李糖苷是通过将2,3,4-O-三乙酰基鼠李糖与(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯反应而得到。
在另一优选例中，所述α-1-氨基鼠李糖苷和/或β-1-氨基鼠李糖苷是通过将2,3,4-O-三乙酰基-1-氨基鼠李糖与(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯反应而得到。
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、PL201改善AD小鼠Morris水迷宫指标。
图2、PL201促进体内神经发生。
图3、PL201缓解Aβ体外对神经干细胞增殖的抑制。
图4、PL201抑制神经炎症。
图5、PL201减少体内Aβ斑块数量。
图6、PL201减少体外Aβ生成。
图7、PL201增加线粒体膜电位。
图8、PL201提高AMPK磷酸化水平。
图9、PL201改善小鼠爬杆法检测指标。
图10、PL201增加纹状体多巴胺能神经纤维含量。
图11、PL202减少Aβ生成。
图12、PL202减少炎症因子IL-1β和iNOS的表达。
图13、PL171减少Aβ生成。
图14、PL172减少Aβ生成。
图15、PL202的核磁共振谱图和1H NOESY谱；其中a图是核磁共振图谱，b图是NOESY谱。
图16、PL171的核磁共振谱图、1H NOESY谱和1H-1H COSY谱；其中a图是核磁共振图谱，b和c图是1H NOESY谱，d图是1H-1H COSY谱。
图17、PL172的核磁共振谱图。
图18、黑质TH染色图；其中a图是H 2O+NS，b图是PL201+NS，c图是H 2O+MPTP，d图是PL201+MPTP。
具体实施方式
本发明人经过深入的研究，式(I)化合物能够显著改善神经退行性疾病的症状。在体外和体内实验中，式(I)化合物均能有效增强神经干细胞的功能；其不仅能够预防，还能作为一种促进神经再生的治疗手段，来对抗衰老和神经退行性疾病相关的认知功能下降。
术语
本文所用的术语“烷基”指直链或支链饱和的、含有1-4个碳原子(较佳地1-2个碳原子)的脂族烃类基团。例如，烷基包括但不限于甲基，乙基，正丙基，异丙基，正丁基，异丁基，叔丁基。
本文所用的术语“链烯基”包括含有至少一个碳碳双键和2-4个碳原子(较佳地2-3个碳原子)的直链和支链烃基。
本文所用的术语“链炔基”包括含有至少一个碳碳三键和2-4个碳原子(较佳地2-3个碳原子)的直链和支链烃基。
本文所用的术语“卤素”指F、Cl、Br、或I。
本文所用的术语“异构体”包括：几何异构体、对映异构体、非对映异构体(如顺反异构体，构象异构体)。
本文所用的
Figure PCTCN2019073773-appb-000013
的表示方法是本领域人员熟知的，其表示一个带有X原子的杂环。在本发明的优选方式中，所述的
Figure PCTCN2019073773-appb-000014
为六元杂环。
本文所用的
Figure PCTCN2019073773-appb-000015
的表示方法是本领域人员熟知的，其表示基团可选的R1～R4取代在环上的任意一个或多个可被取代的位置。并且，在不同的取代位置上，基团的选择可以是不同的。
本文所用的术语“溶剂合物”表示携带有溶剂分子的化合物，例如，所述的溶剂合物可以是水合物。
本发明中，术语“含有”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。
本发明中，“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)即有合理的效益/风险比的物质。
本发明中，“药学上可接受的载体”是用于将本发明的式(I)化合物、异构体、溶剂合物、前体，或它们的药学上可接受的盐传送给动物或人的药学上或食品上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。
化合物
本发明首先提供了一种如结构式(I)所示的化合物：
Figure PCTCN2019073773-appb-000016
应理解，式(I)中，X的位置为示意的位置，并不限于图中R1的一侧(R1与
Figure PCTCN2019073773-appb-000017
基团之间的位置)，其也可以存在于R1与R2之间，R2与R3之间，R3与R4之间，R4与
Figure PCTCN2019073773-appb-000018
基团之间，例如，该化合物也可以是：
Figure PCTCN2019073773-appb-000019
其中，
Figure PCTCN2019073773-appb-000020
为六元杂环，X为O；Y独立地选自：O，N；当Y表示O时，所述化合物为β构型、α构型和β构型的任意比例混合物；当Y表示N时，所述化合物为α构型、β构型、α构型和β构型的任意比例混合物；R1～R4独立地选自：氢、羟基、C1-C4烷基、C2-C4链烯基、C2-C4链炔基、卤素，或R1～R4中相邻两个基团相互连接、并与母环共同构成环结构(可以是含O的环结构)。
作为本发明的一种优选方式，R1～R4独立地选自：氢、羟基、C1-C2烷基。
本发明还包括上述式(I)化合物的异构体、溶剂合物、前体，或它们的药学上可接受的盐，只要它们也具有与式(I)化合物具有相同或基本相同的功能。所述的“药学上可接受的盐”是指化合物与无机酸、有机酸、碱金属或碱土金属等反应生成的盐。这些盐包括(但不限于)：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以酯、氨基甲酸酯，或其它常规的“前体药物”的形式。化合物具有一个或多个不对称中心。所以，这些化合物可以作为外消旋的混合物、单独的对映异构体、单独的非对映异构体、非对映异构体混合物、顺式或反式异构体存在。
所述的“化合物的前体”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成结构式(I)的一种化合物，或化学结构式(I)的一个化合物所组成的盐或溶液。
作为本发明的优选方式，所述的化合物包括以下的如式Ⅱ(PL201)、Ⅲ(PL202)、Ⅳ(PL171)和Ⅴ(PL172)所示的化合物，式Ⅲ和式Ⅳ化合物是特别优选的。
Figure PCTCN2019073773-appb-000021
式Ⅱ化合物是(α-L-吡喃鼠李糖基)阿魏酸酯，包括但不限于，
Figure PCTCN2019073773-appb-000022
式Ⅲ化合物是(β-L-吡喃鼠李糖基)阿魏酸酯，包括但不限于，
Figure PCTCN2019073773-appb-000023
式Ⅳ化合物是(1-氨基-α-L-吡喃鼠李糖基)阿魏酸酰胺，包括但不限于，
Figure PCTCN2019073773-appb-000024
式Ⅴ化合物(1-氨基-β-L-吡喃鼠李糖基)阿魏酸酰胺，包括但不限于，
Figure PCTCN2019073773-appb-000025
在本发明的一种实施方式中，式Ⅲ化合物的制备方法包括步骤：
第一步，将2,3,4-O-三乙酰基鼠李糖和(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯混合，反应得到结构如式Ⅵ所示的β糖苷化产物；
第二步，将结构如式Ⅵ所示的β糖苷化产物与四丁基氟化铵混合，得到脱叔丁基二甲基甲硅烷基(TBS)产物；
第三步，将脱叔丁基二甲基甲硅烷基(TBS)产物水解得到式Ⅲ化合物；
Figure PCTCN2019073773-appb-000026
在本发明的一个实施例中，上述第一步在冰浴下的2,3,4-O-三乙酰基鼠李糖中滴入(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯。
在本发明的一个实施例中，上述第一步的反应在室温下进行；所述室温为10-40℃，优选为15-30℃，更优选为20-25℃。
在本发明的一个实施例中，上述第一步还包括通过柱层析分别得到α和
β糖苷化产物的操作。
在本发明的一个实施例中，上述第二步中的混合是在室温下将四丁基氟
化铵滴入β糖苷化产物中。
在本发明的一种实施方式中，式Ⅳ化合物的制备方法包括步骤：
第一步，将1-氨基-2,3,4-O-三乙酰基鼠李糖和(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯混合，反应得到结构如式Ⅷ所示的α糖苷化产物；
第二步，分别将结构如式Ⅷ所示的α糖苷化产物与四丁基氟化铵混合，得到α脱叔丁基二甲基甲硅烷基(TBS)产物；
第三步，分别将α脱叔丁基二甲基甲硅烷基(TBS)产物水解得到式Ⅳ化 合物；
Figure PCTCN2019073773-appb-000027
在本发明的一个实施例中，上述第一步在冰浴下的1-氨基-2,3,4-O-三乙酰基鼠李糖中滴入(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯。
在本发明的一个实施例中，上述第一步的反应在室温下进行；所述室温为10-40℃，优选为15-30℃，更优选为20-25℃。
在本发明的一个实施例中，上述第一步还包括通过柱层析分别得到α糖苷化产物的操作。
在本发明的一个实施例中，上述第二步中的混合是在室温下将四丁基氟
化铵分别滴入α糖苷化产物中。
在本发明的一种实施方式中，式Ⅴ化合物的制备方法包括步骤：
第一步，将1-氨基-2,3,4-O-三乙酰基鼠李糖和(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯混合，反应得到结构如式Ⅸ所示的β糖苷化产物；
第二步，分别将结构如式Ⅸ所示的β糖苷化产物与四丁基氟化铵混合，得到β脱叔丁基二甲基甲硅烷基(TBS)产物；
第三步，分别将β脱叔丁基二甲基甲硅烷基(TBS)产物水解得到式Ⅴ化合物；
Figure PCTCN2019073773-appb-000028
在本发明的一个实施例中，上述第一步在冰浴下的1-氨基-2,3,4-O-三乙酰基鼠李糖中滴入(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯。
在本发明的一个实施例中，上述第一步的反应在室温下进行；所述室温为10-40℃，优选为15-30℃，更优选为20-25℃。
在本发明的一个实施例中，上述第一步还包括通过柱层析分别得到β糖苷化产物的操作。
在本发明的一个实施例中，上述第二步中的混合是在室温下将四丁基氟化铵分别滴入β糖苷化产物中。
本领域人员应理解，在得知了本发明化合物的结构以后，可通过多种本领域熟知的方法、利用公知的原料，来获得本发明的化合物，比如化学合成或从生物(如动物或植物)中提取的方法，这些方法均包含在本发明中。
合成的化合物可以进一步通过柱层析法、高效液相色谱法等方式进一步纯化。
用途
本发明人在研究中发现，本发明的式(I)化合物能够显著改善神经退行性疾病的症状。本发明的化合物能够抑制神经炎症、降低Aβ生成、增强神经干细胞、增强多巴胺能神经元的功能。经过实验论证，本发明的化合物对于动物的学习记忆能力有显著的改善。根据本发明的化合物的作用机制，其能够增强神经干细胞的功能，因此其对于亨廷顿病、肌萎缩侧索硬化症也是有效的。根据本发明的化合物的作用机制，其对于抑郁症和中风也是有效的，抑郁症或中风发病过程中，也涉及脑内发生神经炎症的发生，进而引起神经干细胞的减少和神经功能的改变；本发明的式(I)化合物能够增强神经干细胞的功能，籍此可以理解，其对于抑郁症和中风也是有效的。
基于本发明人的新发现，本发明提供了式(I)所示的化合物或其异构体、溶剂合物、前体，或它们的药学上可接受的盐的用途，用于制备预防、缓解或治疗神经退行性疾病、抑郁症或中风的药物或药盒。
本发明还提供了式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备抑制神经炎症的组合物、试剂盒或药盒。
本发明还提供了式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备促进神经干细胞生成的组合物、试剂盒或药盒。
本发明还提供了式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备降低Aβ生成的组合物、试剂盒或药盒。
本发明还提供了式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高黑质多巴胺能神经元数量的组合物、试剂盒或药盒。
本发明还提供了式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高纹状体多巴胺能神经纤维含量的组合物、试剂盒或药盒。
本发明提供的式(II)的化合物(PL201)、式(Ⅲ)的化合物(PL202)、式(Ⅳ)的化合物(PL171)和式(Ⅴ)的化合物(PL172)在上述用途中有优异的效果，其中式(Ⅲ)和化合物的(Ⅳ)效果特别优异。
药物组合物
本发明还提供了一种药物组合物，含有：(a)有效量的式(I)所述的化合物、或其异构体、溶剂合物、前体，或它们的药学上可接受的盐；和(b)药学上可接受的载体或赋形剂。
在本发明的中，所述的药物组合物中，式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的含量是有效量。例如，可以含有按照重量比例为0.001-50％的式(I)所示的化合物或其药学上可接受的盐。较佳的，所述的药物组合物含有按照重量比例为0.01-20％的式(I)所示的化合物或其药学上可接受的盐。
本发明所述的药物组合物的剂型可以是多种多样的，只要是能够使活性成分有效地到达哺乳动物机体的剂型都是可以的。比如可选自：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂、注射剂、输液剂、混悬剂。根据本发明的化合物所治疗的疾病类型，本领域人员可以选择方便应用的剂型。
从易于制备和储存的立场看，优选的药物组合物是固态组合物，尤其是片剂和固体填充或液体填充的胶囊。从易于给药的粒长看，优选的药物组合物是口服制剂。本发明的化合物或其药物组合物也可储存在适宜于注射或滴注的消毒器具中。
式(I)化合物作为活性成分的有效施用剂量可随给药的模式和待治疗的疾病的严重程度而变化。然而，通常当本发明的化合物每天以约0.01-100mg/kg动物体重的剂量给予时，能得到令人满意的效果，较佳地每天以1-3次分开的剂量给予，或以缓释形式给药。可调节此剂量方案以提供最佳治疗应答。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
数据统计分析
所有实验数据表示为平均值±标准误差。不同处理组之间采用t检验进行比较。多组结果之间采用one-way ANOVA进行分析，并用Fisher’s protected least significant difference test或Bonferroni t test进行事后检验，或者采用two-way ANOVA进行分析，并用Tukey post hoc test进行事后检验。P<0.05时认为组间有显著性差异。
实施例1、PL201改善AD小鼠学习记忆能力
Morris水迷宫实验
选用5-6月龄的APP/PS1转基因雄性小鼠(AD小鼠)20只，按随机数字表法分为模型组及多糖给药组，PL201采用灌胃给药方式，每天给药(10mg/kg小鼠体重)，同时用非转基因小鼠10只作为阴性对照组(不给予PL201)，连续给药90天后，利用Morris水迷宫行为学实验来检测PL201对APP/PS1小鼠的学习，认知功能的影响。
本实验采用的是经典Morris水迷宫测试程序，包括两部分，即定位航行试验和空间探索试验实验，共进行7天，第4天和第7天加入空间探索试验。
从图1实验结果可知，此次定位航行实验，3组动物经过3天训练，其逃避潜伏期均缩短，说明各小鼠均能顺利完成水迷宫的空间学习任务。以 逃避潜伏期作为检测指标。
结果显示：阴性对照组小鼠反应比较迅速，入水后能快速找到平台，随着训练次数增加，寻找平台的潜伏期时间缩短；而模型组小鼠反应迟钝，入水后沿桶壁有画圈行为，无逃避行为，人为将其引领平台后，又跳入水中，经多次训练后，最终找到平台，但寻找平台潜伏期明显延长；给药组小鼠随着训练次数的增加，寻找平台的能力不断提高。以上3组都具有一定的空间记忆能力，模型组小鼠的潜伏期成绩差于对照组，提示模型组小鼠学习记忆能力出现下降，模型鼠较好的模拟了AD的学习记忆障碍。给药组小鼠潜伏期与模型组相比有显著差异具有统计学意义，可见PL201给药后AD小鼠学习和记忆能力有明显改善。
实施例2、PL201促进体内神经发生
神经发生的异常、神经炎症以及Aβ斑块的沉积和AD认知能力的下降有着紧密的联系。因此，本实施例中，进一步探究PL201是否对于神经发生有影响。
本发明人对AD小鼠(选取5-6个月大小AD小鼠)进行PL201给药实验，每只小鼠灌胃给药PL201，给予100l，给药浓度为10mg/kg小鼠体重。另对照组给100l水，每日一次，持续给药90天，而在60天开始腹腔注射50mg/kg小鼠体重的5-溴-2’-脱氧尿苷(BrdU)，每日一次，共注射7天，给药90天后将小鼠麻醉后PFA灌流，取全脑进行下一步的实验。
针对获取的全脑，检测小鼠海马中成熟神经元标志物NeuN：PFA灌流过的小鼠大脑继续在4％PFA中固定24小时，再在30％蔗糖固定液中静置72小时。处理完毕后，去除小脑部分，将大脑嗅球朝上直立置于滤纸上，-80℃冻存至少24小时。以30m厚度垂直纵向冰冻切片，按小鼠大脑图谱取DG部分，共切8X 9X＝72张，共2.16mm，按切片顺序排列，每8片取1张，合并后编为一组脑片，进行染色。切好的脑片置于组织保护液中(30％蔗糖，30％乙二醇，0.1M PB)，可在-20℃保存半年以上。染色时取一组脑片，在封闭液(10％驴血清，0.3％TritonX-100，PBS)中室温封闭45分钟，之后加入一抗4℃孵育过夜(一抗稀释比例为：大鼠抗BrdU，1：2000；兔抗Ki67，1：1000；羊抗Dcx，1：200；鼠抗NeuN，1：200；羊抗Sox2，1：60)，再与合适的荧光二抗室温孵育1小时。DAPI对细胞核染色。之后 用Olympus FV100i或Leica SP-8对染色的脑片进行拍照。阳性或双染细胞的数量通过Image Pro Plus软件分析。对于和BrdU共染，需要进行抗原修复的脑片，于血清封闭之前，在抗原修复液(10mM柠檬酸钠，pH 6.5)中95℃处理20分钟。BrdU染色，脑片在血清封闭之前，用2M的盐酸在37℃处理30分钟，再用0.1M的硼酸缓冲液(pH 8.5)清洗。
本发明人发现，与对照组小鼠相比，PL201给药的小鼠海马中具有更多的BrdU/NeuN双阳性新生神经元，如图2。
上述结果表明，PL201能够极为显著地促进小鼠体内神经发生。
实施例3、PL201缓解Aβ体外对神经干细胞增殖的抑制
Aβ作为AD的主要致病蛋白，对神经干细胞增殖有着抑制作用。本实施例中，本发明人通过EdU掺入的方法来检测人神经干细胞的增殖能力，探究PL201是否能够缓解Aβ对神经干细胞增殖的抑制作用。
人神经干细胞3L培养在DMEM/F12(含N2/B27及10ng/ml bFGF)中，铺至96孔板培养24小时后，加入Aβ(5μM)预处理30分钟，再加入PL201(30M)，加药后共孵育72小时，EdU染色检测神经干细胞增殖水平。
结果显示，Aβ处理抑制神经干细胞增殖能力，而加入PL201能显著缓解Aβ对神经干细胞增殖的抑制作用，如图3。
实施例4、PL201抑制神经炎症
AD大脑中的神经炎症是造成认知能力下降的原因之一。小胶质细胞是介导神经炎症关键细胞。本实施例中，本发明人通过检测小鼠胶质瘤细胞BV-2上炎症因子的表达，探究PL201是否具有抑制神经炎症的作用。
小鼠胶质瘤细胞BV-2培养在DMEM中，铺至24孔板培养24小时后，加入PL201(用量0、10、30、100μM)和LPS(300ng/ml)，加药后共孵育24小时，Trizol抽提RNA，并通过QPCR检测相关炎症因子的表达。
结果显示，PL201处理能显著抑制炎症因子TNFα和IL-1β的表达，在较低剂量时就有明显效果，如图4。
实施例5、PL201减少体内Aβ斑块数量。
AD大脑中的Aβ斑块沉积是造成神经损伤，进而导致认知能力下降的 关键原因之一。因此，本实施例中，进一步探究了PL201是否对脑内Aβ斑块数量也有影响。
本发明人对实施例2中的给药后AD小鼠全脑样品，以30m厚度垂直纵向冰冻切片并进行硫黄素S和DAPI染色。之后用Olympus FV100i或Leica SP-8对染色的脑片进行拍照。
本发明人发现，与对照组小鼠相比，PL201给药的小鼠脑中具有更少的A，如图5。
上述结果表明，PL201能够显著地减少小鼠体内Aβ产生。
实施例6、PL201减少Aβ生成
Aβ是AD的主要致病蛋白。本实施例中，本发明人通过检测神经母细胞瘤细胞SK-N-SH上Aβ的水平，探究PL201是否具有抑制减少Aβ生成的作用。
神经母细胞瘤细胞SK-N-SH培养及Aβ检测：SK-N-SH培养在DMEM中，铺至24孔板培养24小时后，加入PL201(用量为0、30、100、300μM)，加药后共孵育24小时，取出培养上清，并通过ELISA检测总Aβ的蛋白水平。
结果显示，PL201处理能显著抑制Aβ生成，如图6。
实施例7、PL201增加线粒体膜电位
线粒体膜电位是反映线粒体功能的主要指标，而线粒体功能受损是AD发病的重要指标。本实施例中，本发明人通过检测人神经干细胞细胞线粒体膜电位水平，探究PL201是否具有改善线粒体功能的作用。
使用JC-1对细胞线粒体染色20分钟，并用相应缓存液清洗后，进行免疫荧光分析。
结果显示，Aβ处理降低线粒体膜电位，而PL201处理能减少这种下降的程度，如图7。
实施例8、PL201提高AMPK磷酸化水平
线粒体功能改变与AMPK等代谢信号通路的调控有着密切关系，而这些信号通路也被广泛认为与AD发病关系紧密。本实施例中，本发明人通过免疫印迹试验检测人神经干细胞细胞中AMPK磷酸化水平，探究PL201是否具有调控代谢信号通路的作用。
人神经干细胞3L培养在DMEM/F12(含N2/B27及10ng/ml bFGF)中，铺至12孔板培养24小时后，加入Aβ(5μM)预处理30分钟，再加入PL201(30M)，加药后共孵育72小时，PBS清洗Laemmli’s sample buffer裂解细胞后，SDS PAGE电泳，兔抗phospo-AMPK和总AMPK抗体检测相关蛋白条带。
结果显示，Aβ处理降低AMPK磷酸化水平，而PL201处理能减少这种下降的程度，如图8。
实施例9、PL201改善小鼠爬杆法检测指标
MPTP对脑黑质多巴胺能神经元有选择性的破坏作用，MPTP导致的PD动物模型是最为经典的类似人类帕金森病理变化及临床特征的动物模型。PD的主要症状表现为静止性震颤、肌张力增高、运动减少等，爬杆实验的掉头时间和爬下时间可以代表小鼠的整体活动协调能力。
取小鼠随机分为4组：生理盐水组(NS)、MPTP模型灌胃生理盐水组(NS+MPTP)、PL201化合物50mg灌胃给药组(PL201)，MPTP模型灌胃PL201化合物组(PL201+MPTP)。
生理盐水组(NS)11只动物、MPTP模型灌胃生理盐水组(NS+MPTP)14只动物、PL201化合物50mg灌胃给药组(PL201)4只动物、MPTP模型灌胃PL201化合物组(PL201+MPTP)15只动物。
动物分组当天开始给药，生理盐水组和MPTP模型灌胃生理盐水组灌胃生理盐水，其余两组均给予PL201化合物，每天给药1次，连续给药7天。从第7天起给予造模药物MPTP或NS，生理盐水组(NS)和PL201化合物50mg灌胃给药组(PL201)动物腹腔注射生理盐水5ml/kg，其余动物腹腔注射MPTP25mg/kg,每天1次，共5天。
实验第3天，对小鼠进行爬杆实验检测，评价小鼠运动协调能力。将小鼠头向上轻柔的放在粗糙的杆顶(直径1cm，高50cm)。小鼠从头向上调整至头完全向下的时间记录为潜伏期(Time to turn around)，小鼠从向下运动至四肢全部到达杆底的时间记录为爬下时间(Time to Climb down)，超过30秒按照30秒记录。每只小鼠重复检测5次取平均值。
结果显示：MPTP模型灌胃生理盐水组(NS+MPTP)潜伏期(Time to turn around)和爬下时间(Time to Climb down)明显延长，与生理盐水组(NS)相比差异有一定程度的意义。给予PL201化合物组潜伏期(Time to  turn around)和爬下时间(Time to Climb down)较MPTP模型灌胃生理盐水组(NS+MPTP)有一定程度缩短。
实验第3天，给予PL201化合物组潜伏期(Time to turn around)和爬下时间(Time to Climb down)有不同程度缩短，与MPTP模型灌胃生理盐水组(NS+MPTP)相比，差异有显著意义。
结果显示，PL201化合物对给药后3天MPTP PD小鼠模型的运动起始及协调能力有改善作用，如图9。
实施例10、PL201增加纹状体多巴胺能神经纤维含量
黑质多巴胺能神经元和纹状体多巴胺能神经纤维丢失是帕金森病的主要病理特征，用免疫印迹检测纹状体多巴胺能神经纤维丢失情况，探究PL201是否具有增加纹状体多巴胺能神经纤维含量的作用。
取小鼠随机分为4组：生理盐水组(NS)、MPTP模型灌胃生理盐水组(NS+MPTP)、PL201化合物50mg灌胃给药组(PL201)，MPTP模型灌胃PL201化合物组(PL201+MPTP)。
生理盐水组(NS)11只动物、MPTP模型灌胃生理盐水组(NS+MPTP)14只动物、PL201化合物50mg灌胃给药组(PL201)4只动物、MPTP模型灌胃PL201化合物组(PL201+MPTP)15只动物。
动物分组当天开始给药，生理盐水组和MPTP模型灌胃生理盐水组灌胃生理盐水，其余两组均给予PL201化合物，每天给药1次，连续给药7天。从第7天起给予造模药物MPTP或NS，生理盐水组(NS)和PL201化合物50mg灌胃给药组(PL201)动物腹腔注射生理盐水5ml/kg，其余动物腹腔注射MPTP25mg/kg,每天1次，共5天。
各组动物于MPTP造模结束后第7天，10％水合氯醛麻醉，灌流后取脑组织分离纹状体置于1.5mlEP管中，加入250μl RIPA蛋白裂解液(ThermoFisher)超声破碎组织，收集上清液检测蛋白浓度。调整蛋白浓度为2μg/μl加入5×还原Loadingbuffer沸煮变性蛋白，加样至10％SDS-聚丙烯酰胺凝胶电泳，100V湿转法转移至甲醇激活的PVDF膜上。5％脱脂牛奶TBST室温封闭1h，加入TH(1：1000)一抗4℃孵育过夜。TBST buffer清洗非特异结合的一抗后，加入Goat Anti-Mouse/Rabbit荧光二抗(LI-COR)，室温孵育1h后，TBSTbuffer清洗3次，Odyssey近红外荧光扫描仪检测荧光信号并统计。
结果显示，MPTP造模7天后，给予PL201化合物组与MPTP模型灌胃生理盐水组(NS+MPTP)相比纹状体多巴胺能神经纤维含量明显增加。PL201化合物对小鼠纹状体多巴胺能神经元纤维的损伤有保护作用，提高纹状体多巴胺能神经纤维含量，如图10。实验结果表明，PL201化合物具有治疗帕金森症的作用。
实施例11、PL201增加黑质多巴胺能神经元数量和纹状体多巴胺能神经纤维含量
黑质多巴胺能神经元和纹状体多巴胺能神经纤维丢失是帕金森病的主要病理特征，用酪氨酸羟化酶(tyrosine hydroxylase，TH)免疫组化检测纹状体多巴胺能神经纤维丢失情况，探究PL201是否具有增加黑质多巴胺能神经元数量的作用。
取小鼠随机分为4组：生理盐水组(NS)、MPTP模型灌胃生理盐水组(NS+MPTP)、PL201化合物50mg灌胃给药组(PL201)，MPTP模型灌胃PL201化合物组(PL201+MPTP)。
生理盐水组(NS)11只动物、MPTP模型灌胃生理盐水组(NS+MPTP)14只动物、PL201化合物50mg灌胃给药组(PL201)4只动物、MPTP模型灌胃PL201化合物组(PL201+MPTP)15只动物。
动物分组当天开始给药，生理盐水组和MPTP模型灌胃生理盐水组灌胃生理盐水，其余两组均给予PL201化合物，每天给药1次，连续给药7天。从第7天起给予造模药物MPTP或NS，生理盐水组(NS)和PL201化合物50mg灌胃给药组(PL201)动物腹腔注射生理盐水5ml/kg，其余动物腹腔注射MPTP25mg/kg,每天1次，共5天。
各组动物于MPTP造模结束后第7天，10％水合氯醛麻醉，4％多聚甲醛灌流后取脑。4％多聚甲醛后固定24小时，再将样本转入30％蔗糖溶液中脱水至样本沉底，在-20度冰冻切片机中做中脑与纹状体部位冠状切片，小鼠脑切片厚度30微米。一抗为单克隆小鼠抗TH(1：1000，兔抗鼠抗体)，室温孵育 2.5小时，TBST溶液(8克氯化钠，0.2克氯化钾和3克Tris-base，蒸馏水定容到1升，用盐酸调至pH 7.4)洗三遍后用辣根氧化物酶(HPR)标记二抗(羊抗兔抗体)，室温孵育1小时。用显色剂二氨基联苯胺(DAB)显色，乙醇梯度脱水，二甲苯透明，中性树胶封片。用Image-proplus软件对染色切片进行分析，以黑质区TH阳性染色总密度作为黑质区多巴胺能神经元数的量度，纹状体TH阳性染色平均光密度作为纹状体多巴能胺神经纤维密度的量度进行统计。
结果显示，MPTP造模7天后，给予PL201化合物组与MPTP模型灌胃生理盐水组(NS+MPTP)相比黑质多巴胺能神经元数量和纹状体多巴胺能神经纤维含量明显增加。PL201化合物对小鼠黑质多巴胺能神经元和纹状体多巴胺能神经纤维的损伤有保护作用，提高黑质多巴胺能神经元数量和纹状体多巴胺能神经纤维含量。实验结果表明，PL201化合物具有治疗帕金森症的作用。如图18。
实施例12、PL202减少Aβ生成
Aβ是AD的主要致病蛋白。本实施例中，本发明人通过检测神经母细胞瘤细胞SK-N-SH上Aβ的水平，探究PL201的异构体PL202是否具有抑制减少Aβ生成的作用。
神经母细胞瘤细胞SK-N-SH培养及Aβ检测：SK-N-SH培养在DMEM中，铺至24孔板培养24小时后，加入PL202(用量为0、30、100μM)，加药后共孵育24小时，取出培养上清，并通过ELISA检测总Aβ的蛋白水平。
结果显示，PL202处理能显著抑制Aβ生成，如图11。
实施例13、PL202抑制神经炎症
AD大脑中的神经炎症是造成认知能力下降的原因之一。小胶质细胞是介导神经炎症关键细胞。本实施例中，本发明人通过检测小鼠胶质瘤细胞BV-2上炎症因子的表达，探究PL202是否具有抑制神经炎症的作用。
小鼠胶质瘤细胞BV-2培养在DMEM中，铺至24孔板培养24小时后，加入PL202(用量0、30、100、300μM)和LPS(300ng/ml)，加药后共孵育24小时，Trizol抽提RNA，并通过QPCR检测相关炎症因子的表达。
结果显示，PL202处理能显著抑制炎症因子IL-1β和iNOS的表达，如图12。
实施例14、PL171减少Aβ生成
Aβ是AD的主要致病蛋白。本实施例中，本发明人通过检测神经母细胞瘤细胞SK-N-SH上Aβ的水平，探究PL171是否具有抑制减少Aβ生成的作用。
神经母细胞瘤细胞SK-N-SH培养及Aβ检测：SK-N-SH培养在DMEM中，铺至24孔板培养24小时后，加入PL171(用量为300μM)，加药后共孵育24小时，取出培养上清，并通过ELISA检测总Aβ的蛋白水平。
结果显示，PL171处理能显著抑制Aβ生成，如图13。
实施例15、PL172减少Aβ生成
Aβ是AD的主要致病蛋白。本实施例中，本发明人通过检测神经母细胞瘤细胞SK-N-SH上Aβ的水平，探究PL171的异构体PL172是否具有抑制减少Aβ生成的作用。
神经母细胞瘤细胞SK-N-SH培养及Aβ检测：SK-N-SH培养在DMEM中，铺至24孔板培养24小时后，加入PL172(用量为300μM)，加药后共孵育24小时，取出培养上清，并通过ELISA检测总Aβ的蛋白水平。
结果显示，PL172处理能显著抑制Aβ生成，如图14。
实施例16、PL202的合成
Figure PCTCN2019073773-appb-000029
将2,3,4-O-三乙酰基鼠李糖(5.8g,20mmol)溶于100mL无水二氯甲烷中，冰浴下滴入50mL(4-O-TBS)-阿魏酸酰氯(6.52g,20mmol)(Free Radical Res.2015,102)的无水二氯甲烷溶液和1.0mL无水吡啶。室温下搅拌2小时停止反应。加入200mL二氯甲烷稀释，饱和氯化钠洗涤，MgSO4 干燥浓缩。柱层析(石油醚:乙酸乙酯＝3:1)分别得到α和β糖苷化产物9.12g(79％)和1.1g(9％)。将其中β糖苷化产物(490mg,0.85mmol)溶于干燥的THF(8.0mL)，稀释，饱和氯化钠洗涤，MgSO4干燥浓缩，得脱TBS产物275mg。将所得产物270mg(0.58mmol)溶于3.0mL甲醇，0℃下滴入33％CH 3NH 2的甲醇溶液0.5mL，在0℃下反应1小时。迅速减压浓缩柱层析分离(乙酸乙酯:甲醇＝6:1)，得PL202 59mg(0.17mmol).ESI(+)-MS:341.3[M+1]+；H-NMR(CD 3OD,400MHz)δ7.80(d,J＝12Hz,1H),7.23(d,J＝1Hz,1H),7.12(dd,J 1＝8Hz,J 2＝1Hz,1H),6.84(d,J＝6Hz,1H),6.42(d,J＝13Hz,1H),5.75(d,J＝1Hz,1H),3.99(d,J＝2Hz,1H),3.92(s,3H),3.55-3.53(m,1H),3.41-3.37(m,2H),1.34(d,J＝4Hz,3H)。
为了验证其构型，还进行了NOESY(nuclear overhauser effect spectroscopy)实验，得到了其1H-1HNOESY谱图，证明得到的PL202为β构型。如图15。
实施例17、PL171的合成
Figure PCTCN2019073773-appb-000030
将1-氨基-2,3,4-O-三乙酰基鼠李糖(5.86g,19mmol)溶于100mL无水二氯甲烷中，冰浴下滴入(4-O-TBS)-阿魏酸酰氯(6.5g,20mmol)(Free Radical Res.2015,102)的无水二氯甲烷溶液和1.0mL无水吡啶。室温下搅拌2小时停止反应。加入200mL二氯甲烷稀释，饱和氯化钠洗涤，MgSO4干燥浓缩。柱层析(石油醚:乙酸乙酯＝10:1to 2:1)分别得到α和β糖苷化产物8.22g(70％)和1.2g(10％)。将其中α糖苷化产物(500mg,0.86mmol)溶于干燥的THF(8.0mL)，稀释，饱和氯化钠洗涤，MgSO4干燥浓缩，得脱TBS产物300mg。将所得产物300mg(0.65mmol)溶于3.0mL二氯甲烷，0℃下滴入33％CH 3NH 2的甲醇溶液0.5mL，在0℃下反应1小时。迅速减压浓缩柱层析分离(二氯甲烷:甲醇＝20:1to 6:1)，得 PL171 70mg(0.21mmol).ESI(+)-MS:340.3[M+1] +；H-NMR(CD3OD,400MHz)δ7.56(d,J＝12Hz,1H)7.19(d,J＝4Hz,1H),7.07(dd,J 1＝6Hz,J 2＝1Hz,1H),6.82(d,J＝4Hz,1H),6.62(d,J＝12Hz,1H),5.28(d,J＝1Hz,1H),3.92(s,3H),3.85(d,J＝4Hz,1H),3.54-3.52(m,1H),3.38-3.34(m,2H),1.32(d,J＝4Hz,3H)。
为了验证其构型，还进行了NOESY(nuclear overhauser effect spectroscopy)实验和1H-1H COSY(correlated spectroscopy)实验，得到了其1H NOESY和1H-1H COSY谱图，证明得到的PL171为α构型。如图16。
实施例18、PL172的合成
Figure PCTCN2019073773-appb-000031
将1-氨基-2,3,4-O-三乙酰基鼠李糖(5.86g,19mmol)溶于100mL无水二氯甲烷中，冰浴下滴入(4-O-TBS)-阿魏酸酰氯(6.5g,20mmol)(Free Radical Res.2015,102)的无水二氯甲烷溶液和1.0mL无水吡啶。室温下搅拌2小时停止反应。加入200mL二氯甲烷稀释，饱和氯化钠洗涤，MgSO4干燥浓缩。柱层析(石油醚:乙酸乙酯＝10:1to 2:1)分别得到α和β糖苷化产物8.22g(70％)和1.2g(10％)。将其中β糖苷化产物(500mg,0.86mmol)溶于干燥的THF(8.0mL)，稀释，饱和氯化钠洗涤，MgSO4干燥浓缩，得脱TBS产物250mg。将所得产物300mg(0.54mmol)溶于3.0mL二氯甲烷，0℃下滴入33％CH 3NH 2的甲醇溶液0.5mL，在0℃下反应1小时。迅速减压浓缩柱层析分离(二氯甲烷:甲醇＝20:1to 6:1)，得PL172 45mg(0.13mmol).ESI(+)-MS:340.3[M+1] +；H-NMR(CD3OD,400MHz)δ7.53(d,J＝16Hz,1H)7.14(d,J＝41Hz,1H),7.05(dd,J 1＝8Hz,J 2＝1Hz,1H),6.80(d,J＝8Hz,1H),6.56(d,J＝16Hz,1H),5.56(d,J＝4Hz,1H),3.89(s,3H),3.85-3.83(m,1H),3.54-3.52(m,1H),3.44(t,J＝12Hz,1H),3.31-3.3(m,1H)1.27(d,J＝4Hz,3H)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (21)

  1. 式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐，
    Figure PCTCN2019073773-appb-100001
    其中，
    Figure PCTCN2019073773-appb-100002
    为六元杂环，X为O；
    Y选自O或N；
    R1～R4独立地选自：氢、羟基、C1～C4烷基、C2～C4链烯基、C2～C4链炔基、卤素，或R1～R4中相邻两个基团相互连接、并与母环共同构成环结构。
  2. 如权利要求1所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐，其特征在于，
    R1～R4独立地选自：氢、羟基、C1～C2烷基。
  3. 如权利要求1所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐，其特征在于，所述化合物如式Ⅲ、Ⅳ或Ⅴ所示：
    Figure PCTCN2019073773-appb-100003
  4. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备预防、缓解或治疗神经退行性疾病、抑郁症或中风的药物或药盒。
  5. 如权利要求4所述的用途，其特征在于，神经退行性疾病是：
    以脑内发生神经炎症为特征的神经退行性疾病；或
    以Aβ生成显著性增加为特征的神经退行性疾病；或
    以学习记忆能力显著性下降为特征的神经退行性疾病；或
    以神经干细胞功能下降为特征的神经退行性疾病；或
    以运动协调能力下降为特征的神经退行性疾病；或
    以黑质多巴胺能神经元数量下降为特征的神经退行性疾病；或
    以纹状体多巴胺能神经纤维含量下降为特征的神经退行性疾病。
  6. 如权利要求5所述的用途，其特征在于，所述的神经退行性疾病包括：阿尔茨海默症，帕金森症，路易体痴呆，亨廷顿病、肌萎缩侧索硬化症。
  7. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备抑制神经炎症的组合物、试剂盒或药盒。
  8. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备促进神经干细胞功能的组合物、试剂盒或药盒。
  9. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备降低Aβ生成的组合物、试剂盒或药盒。
  10. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高黑质多巴胺能神经元数量的组合物、试剂盒或药盒。
  11. 权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐的用途，用于制备提高纹状体多巴胺能神经纤维含量的组合物、试剂盒或药盒。
  12. 如权利要求4～11任一所述的用途，其特征在于，所述的化合物包括如式Ⅱ、Ⅲ、Ⅳ或Ⅴ所示：
    Figure PCTCN2019073773-appb-100004
  13. 一种药物组合物，其特征在于，所述的药物组合物包含：
    权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐；和
    药学上可接受的载体。
  14. 如权利要求12所述的药物组合物，其特征在于，所述的药物组合物的剂型包括：粉剂、散剂、片剂、丸剂、胶囊剂、缓释剂、控速释剂、注射剂、输液剂、混悬剂。
  15. 一种药盒，其特征在于，所述的药盒中包括：权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐；或
    权利要求13或14任一所述的药物组合物。
  16. 一种预防、缓解或治疗神经退行性疾病、抑郁症或中风的方法，其特征在于，所述方法包括：给予需要治疗的对象有效量的权利要求1～3任一所述的式(I)所示化合物或其异构体、溶剂合物或前体，或它们的药学上可接受的盐。
  17. 一种如式Ⅲ所示的化合物的制备方法，其特征在于，所述方法包括步骤：使β吡喃鼠李糖苷与四丁基氟化铵反应得到结构如式Ⅲ所示的化合物；
    Figure PCTCN2019073773-appb-100005
  18. 如权利要求17所述的制备方法，其特征在于，所述β吡喃鼠李糖苷是通过将2,3,4-O-三乙酰基鼠李糖与(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯反应而得到。
  19. 一种如式Ⅳ所示的化合物的制备方法，其特征在于，所述方法包括步骤：使α-1-氨基鼠李糖苷与四丁基氟化铵反应得到结构如式Ⅳ所示的化合物；
    Figure PCTCN2019073773-appb-100006
  20. 一种如式Ⅴ所示的化合物的制备方法，其特征在于，所述方法包括步骤：使β-1-氨基鼠李糖苷与四丁基氟化铵反应得到结构如式Ⅴ所示的化合物；
    Figure PCTCN2019073773-appb-100007
  21. 如权利要求19或20所述的制备方法，其特征在于，所述α-1-氨基 鼠李糖苷和/或β-1-氨基鼠李糖苷是通过将2,3,4-O-三乙酰基-1-氨基鼠李糖与(4-O-叔丁基二甲基甲硅烷基)-阿魏酸酰氯反应而得到。
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