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WO2020093370A1 - 一种特异性降解tau蛋白的小分子化合物及其应用 - Google Patents

一种特异性降解tau蛋白的小分子化合物及其应用 Download PDF

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WO2020093370A1
WO2020093370A1 PCT/CN2018/114838 CN2018114838W WO2020093370A1 WO 2020093370 A1 WO2020093370 A1 WO 2020093370A1 CN 2018114838 W CN2018114838 W CN 2018114838W WO 2020093370 A1 WO2020093370 A1 WO 2020093370A1
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small molecule
tau protein
disease
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汪义朋
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汪义朋
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    • 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
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • 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
    • 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
    • A61P25/28—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the invention relates to the technical field of bifunctional molecular compounds, in particular to a small molecule compound (named "JT01”) which specifically degrades tau protein and its application.
  • JT01 small molecule compound
  • AD Alzheimer ’s disease
  • Symptomatic drugs which can only temporarily relieve symptoms but not delay the progression of the disease.
  • Disease-modifying drugs drugs that can truly change the progress of AD.
  • AD senile plaques
  • NFTs neurofibrillary tangles
  • tau lesions rather than A ⁇ lesions are positively correlated with the degree of dementia in AD.
  • tau mediates A ⁇ -induced neurotoxicity, which is necessary for the neurotoxicity of A ⁇ ; and tau is a prion-like protein (Prion-like protein) that can spread between neurons and cause the spread of tau lesions. It may be a necessary drug target for AD.
  • tau aggregation is also seen in frontotemporal dementia (frontotemporal dementia linked to chromosome 17) parkinsonism (FTDP-17), Pick's disease (Pick's disease, PiD), linked to chromosome 17 with Parkinson's disease.
  • frontotemporal dementia frontotemporal dementia linked to chromosome 17
  • parkinsonism FTDP-17
  • Pick's disease Pick's disease, PiD
  • Parkinson's disease Parkinson's disease
  • PSP Progressive supranuclear palsy
  • CBD corticobasal degeneration
  • PART primary age-related tauopathy
  • ARTAG aging-related tau astrogliopathy
  • CTE chronic traumatic encephalopathy
  • GGT spherical glial tauopathy
  • Parkinson ’s disease Disease Parkinson's disease, PD
  • Huntington's disease Huntington's disease
  • tauopathies tau diseases
  • Tau protein is an important cause of such diseases, so it is also an important therapeutic target for such diseases.
  • siRNA, miRNA or antisense oligonucleotide to reduce the expression of target protein. Due to the poor distribution of these oligonucleotides in tissues, poor pharmacokinetics, and the possibility of off-target, their clinical application is currently limited and needs further improvement.
  • Enhance the degradation of target protein A common method is to enhance the activity of protein degradation systems including protease systems and autophagy systems. However, non-specific enhancement of the activity of the protein degradation system is likely to cause the degradation of other non-target proteins and serious side effects. Therefore, no drugs that activate the protein degradation system have been approved for clinical application. The ideal method is to only selectively enhance the degradation of the target protein and avoid the degradation of the non-target protein caused by enhancing the activity of the protein degradation system.
  • the purpose of the present invention is to construct a small molecule compound specific for tau protein.
  • the inventors of the present invention have found through in-depth research that the use of protein degradation targeting chimeras (PROteolysis, TArgeting, Chimeras, PROTAC) technology can construct a bifunctional molecular compound, one end of which can specifically bind the target protein and the other end of the specific Combined with a specific ubiquitin ligase, the two are connected via a linker.
  • protein degradation targeting chimeras PROteolysis, TArgeting, Chimeras, PROTAC
  • the compound thus constructed can simultaneously bind the target protein and ubiquitin ligase, so that the target protein is close to the ubiquitin ligase, so it can enhance the ubiquitination of the target protein and finally be degraded by the proteasome.
  • the PROTAC technology has the following advantages: (1) It can act on many traditionally difficult targets. Many traditional small molecule drugs must act on specific binding pockets of the target protein in order to exert their inhibitory effect. The PROTAC technology does not have this limitation. As long as it can interact with any segment of the target protein and does not require a high affinity, it can cause rapid degradation of the target protein and thereby inhibit the function of the target protein, so it can act on many traditional difficulties. The target of the medicine. (2) The PROTAC technology can be used repeatedly in the cell and can play a similar catalytic effect, so the therapeutic effect can be achieved without high concentration.
  • the present invention provides a small molecule compound (ie JT01) that specifically degrades tau protein, wherein the chemical structure of the compound is TBM-L-ULM or its pharmaceutically acceptable salt, enantiomer, stereo Isomers, solvates, polymorphs, or N-oxides, where TBM is a tau protein binding moiety, L is a linker group, and ULM is a ubiquitin ligase binding moiety, and the tau protein binding moiety is The ubiquitin ligase binding portion is connected through a linker group.
  • TBM is a tau protein binding moiety
  • L is a linker group
  • ULM is a ubiquitin ligase binding moiety
  • tau protein binding moiety is The ubiquitin ligase binding portion is connected through a linker group.
  • ULM is capable of binding E3 ubiquitin ligase.
  • the E3 ubiquitin ligase is VHL E3 ubiquitin ligase or CRBN E3 ubiquitin ligase.
  • ULM is a group having a structure represented by formula (1)
  • R 1 is a hydroxyl group or a group metabolized into a hydroxyl group in a patient or a subject;
  • R 2 is -NH-R 4 -Ar-HET, wherein R 4 is a C1-C3 alkylene group, Ar is a C6-C12 arylene group, and HET is an optionally substituted thiazole or isothiazole;
  • R 3 is -CHR 5 -M-, ULM is connected to the linker group L through M, where R 5 is a C1-C4 alkyl group, M is a bond, a C1-C4 alkylene group, -NH- or- NH-C (O) -R 6- , wherein R 6 is a C1-C4 alkylene group.
  • L is a group -XYZ-, X is connected to TBM, and Z is connected to ULM, wherein X is a bond, a C1-C4 alkylene group, -NH- or -NH-C (O) -R 7- , wherein R 7 is a bond or a C1-C4 alkylene group;
  • Y is -R 8- (R 10 -ER 11 ) n -R 9- , wherein R 8 and R 9 are each a bond or a C1-C8 alkylene group, and R 10 and R 11 are each a C1-C4 alkylene group Group, n is an integer of 0-10, E is O, S, amido, piperazinyl, NR 12 , S (O), S (O) 2 , -S (O) 2 O, -OS (O) 2 , OS (O) 2 O, Where E 1 is O, S, CHR 12 or NR 12 , R 12 is H or C1-C3 alkyl substituted with one or two hydroxyl groups;
  • Z is -AB-, where A is a bond, O or S, and B is a bond, a C1-C4 alkylene group or -R 13 -C (O)-, where R 13 is a C1-C4 alkylene group.
  • TBM is a group having a structure represented by formula (2), or a group having a structure represented by formula (2) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 position is further modified by a substituted group, wherein, TBM through formula (2) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 position with the linker Group L is connected,
  • R 14 is a C1-C4 alkylene group
  • R 15 and R 16 are each a C1-C4 alkyl group
  • R 17 is a bond, H, C1-C4 alkyl group or -R 18 -C (O)-
  • R 18 is a C1-C4 alkylene group.
  • the structural formula of the small molecule compound that specifically degrades tau protein is:
  • the present invention also provides a method for degrading tau protein in a patient in need thereof, comprising administering an effective amount of the above-mentioned small molecule compound to the patient.
  • the small molecule compound is administered to the patient by at least one method selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, abdominal cavity, epidural, and sheath Internal and intravenous routes.
  • the invention also provides the application of the above-mentioned small molecule compound in the preparation of a medicament for treating or preventing diseases related to tau protein.
  • the disease is Alzheimer's disease (AD), frontotemporal dementia linked to chromosome 17 with Parkinson's disease (Frontotemporal dementia linked to chromosome 17), Parkinsonism (FTDP-17), Pick's disease (Pick's disease, PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), primary age-related tauopathy (PART), addiction Argyrophilic grain disease (AGD), aging-related tau astrogliopathy (ARTAG), chronic traumatic encephalopathy (chronic traumatic encephalopathy, CTE), spherical glial cell tauopathy (Globular At least one of glial tauopathy (GGT), Parkinson's disease (PD), Huntington's disease (HD), stroke and epilepsy.
  • AD Alzheimer's disease
  • FTDP-17 Parkinsonism
  • Pick's disease PiD
  • PPP progressive supranuclear palsy
  • CBD corticobasal degeneration
  • PART primary age-related tauopathy
  • the immunoblot test confirmed that the small molecule compound specifically degrading tau protein of the present invention can enhance the degradation of tau protein in cells, thereby reducing the content of tau protein. This indicates that the small molecule compound specifically degrading tau protein according to the present invention can play a role in preventing and treating a series of tau diseases including Alzheimer's disease.
  • FIG. 1 is a nuclear magnetic resonance spectrum of a small molecule compound prepared in Example 1 of the present invention.
  • FIG. 2 is a comparative photograph of the degradation of tau protein using different concentrations of the small molecule compounds of the present invention
  • Fig. 3 is a quantitative analysis chart of the degradation of tau eggs using different concentrations of the small molecule compounds of the present invention
  • Figure 4 shows a fluorescence photograph of the small molecule compound of the present invention reducing the total tau protein content in HEK293 tau cells.
  • the chemical structure of the small molecule compound specifically degrading tau protein according to the present invention is TBM-L-ULM or a pharmaceutically acceptable salt, enantiomer, stereoisomer, solvate, polymorph or N -An oxide, wherein TBM is a tau protein binding portion, L is a linker group, and ULM is a ubiquitin ligase binding portion, the tau protein binding portion and the ubiquitin ligase binding portion are connected by a linker group .
  • ULM is capable of binding E3 ubiquitin ligase.
  • the E3 ubiquitin ligase is VHL E3 ubiquitin ligase or CRBN E3 ubiquitin ligase.
  • ULM is a group having a structure represented by formula (1),
  • R 1 is a hydroxyl group or a group metabolized into a hydroxyl group in a patient or a subject;
  • R 2 is -NH-R 4 -Ar-HET, where R 4 is a C1-C3 alkylene group (such as methylene, ethylene, or propylene), and Ar is a C6-C12 arylene group ( (Such as phenyl or naphthyl), HET is optionally substituted thiazole or isothiazole;
  • R 3 is -CHR 5 -M-
  • ULM is connected to the linker group L through M, where R 5 is a C1-C4 alkyl group (such as methyl, ethyl, propyl or butyl) and M is a bond , C1-C4 alkylene (such as methylene, ethylene, propylene or butylene), -NH- or -NH-C (O) -R 6- , wherein R 6 is C1-C4 Alkylene (such as methylene, ethylene, propylene or butylene).
  • ULM is connected to the linker group L through "-M-".
  • "-M-" is -NH-C (O) -R 6 - time
  • "- R 6 -" and the linker group L is directly connected.
  • R 1 is a hydroxyl group
  • R 2 is -NH-R 4 -Ar-HET, wherein R 4 is a methylene group, Ar is a phenyl group
  • R 3 is- CHR 5 -NH- or -CHR 5 -NH-C (O) -R 6- , wherein R 5 is -C (CH 3 ) 3 and R 6 is methylene.
  • L is a group -X-Y-Z-, X is connected to TBM, and Z is connected to ULM,
  • X is a bond, C1-C4 alkylene (such as methylene, ethylene, propylene or butylene), -NH- or -NH-C (O) -R 7- , where R 7 Is a bond or a C1-C4 alkylene group (such as methylene, ethylene, propylene or butylene);
  • Y is -R 8- (R 10 -ER 11 ) n -R 9- , wherein R 8 and R 9 are each a bond or a C1-C8 alkylene group (such as methylene, ethylene, propylene, (Butylene, pentylene, hexylene, heptylene or octylene), R 10 and R 11 are each C1-C4 alkylene (such as methylene, ethylene, propylene or butylene) , N is an integer of 0-10 (such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), E is O, S, amido, piperazinyl, NR 12 , S (O), S (O) 2 , -S (O) 2 O, -OS (O) 2 , OS (O) 2 O, Where E 1 is O, S, CHR 12 or NR 12 , R 12 is H or C1-C3 alkyl substituted with one or two hydroxyl groups;
  • Z is -AB-, where A is a bond, O or S, B is a bond, a C1-C4 alkylene group (such as methylene, ethylene, propylene or butylene) or -R 13 -C ( O)-, wherein R 13 is a C1-C4 alkylene group (such as methylene, ethylene, propylene or butylene).
  • TBM is a group having a structure represented by formula (2), or a group having a structure represented by formula (2) 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 position is further modified by a substituted group, wherein, TBM through formula (2) 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 position and the linker The group L is connected,
  • R 14 is a C1-C4 alkylene group (such as methylene, ethylene, propylene, or butylene)
  • R 15 and R 16 are each a C1-C4 alkyl group (such as methyl, ethyl , Propyl or butyl)
  • R 17 is a bond, H, C1-C4 alkyl (such as methyl, ethyl, propyl or butyl) or -R 18 -C (O)-, wherein, R 18 It is a C1-C4 alkylene group (such as methylene, ethylene, propylene or butylene).
  • the substitution groups at the positions 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 may be halogen (such as fluorine or chlorine) C4 alkyl (such as methyl, ethyl, propyl or butyl), C1-C4 alkoxy (such as methoxy, ethoxy, propoxy or butoxy), carboxyl, amino, amine Group, C6-C18 aryl group (such as phenyl) or benzyl.
  • C4 alkyl such as methyl, ethyl, propyl or butyl
  • C1-C4 alkoxy such as methoxy, ethoxy, propoxy or butoxy
  • carboxyl amino, amine Group
  • C6-C18 aryl group such as phenyl
  • benzyl such as phenyl
  • TBM is connected to the linker group L through the 5 position in formula (2)
  • R 14 is ethylene
  • R 15 and R 16 are each methyl
  • R 17 is a bond, methylene or -CH 2 -C (O)-.
  • the structural formula of the small molecule compound that specifically degrades tau protein is:
  • the specific preparation process includes the following steps:
  • Dissolve compound 4-8 in DMF add DIPEA and 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane under N 2 protection- 16-oleic acid. Then, HATU was added after the reaction solution was cooled to 0-5 ° C. The reaction solution was then stirred at room temperature under N 2 protection overnight. After the reaction was completed, the reaction mixture was poured into ice water, extracted with EA, and the organic phase was washed with brine. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified with a silica gel column to obtain compound 4-9.
  • the present invention also provides a method for degrading tau protein in a patient in need thereof, comprising administering to the patient an effective amount of the above compound provided by the present invention.
  • the compound may be administered to the patient by at least one method selected from the group consisting of: nasal, inhalation, topical, oral, intramuscular, subcutaneous, transdermal, intraperitoneal, epidural, intrathecal, and intravenous Internal route.
  • the present invention also provides the use of the above compound in the preparation of a medicament for treating or preventing diseases related to tau protein.
  • the disease may be Alzheimer's disease, frontotemporal dementia linked to chromosome 17 with Parkinson's disease, Pick's disease, progressive supranuclear palsy, cortical basal ganglia degeneration, primary age-related tau disease At least one of argyrophilic granulopathy, aging-related tau astrocyte disease, chronic traumatic encephalopathy, spherical glial tauopathy, Parkinson's disease, Huntington's disease, stroke, and epilepsy.
  • reaction mixture was poured into ice water, extracted with EA, and the organic phase was washed with brine.
  • the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified with a silica gel column to obtain 75 mg of compound 4-9.
  • HEK293 tau cells stable expression of wild-type full-length tau protein
  • DMEM medium for culturing in DMEM medium for 24h, add different concentrations of small molecule compounds (0 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M , 10 ⁇ M, 20 ⁇ M), continue to culture for 24h.
  • the cells were lysed with RIPA solution and the cell extracts were collected.
  • the proteins in the cell extracts were separated by 10% polyacrylamide gel electrophoresis (PAGE), and then immunoblotted with anti-total tau protein antibody (tau5) to quantitatively analyze tau protein content.
  • PAGE polyacrylamide gel electrophoresis
  • tau5 anti-total tau protein antibody
  • the working fluid is prepared from the A liquid (self-leading) and the B liquid (public) in the kit at a ratio of 50: 1.
  • Add the working solution to the 96-well plate add 95 ⁇ l to each well in a rapid suspension, cover the lid and shake it quickly in the same direction after addition, do not touch the bottom of the 96-well plate with your hands, incubate with a plastic box at 37 ° C for 30min;
  • bromophenol blue and ⁇ -mercaptoethanol (reducing agent) are added.
  • Bromophenol blue: ⁇ -mercaptoethanol 1: 3
  • mixed solution: sample 1:10, 100 ° C boiling water bath 10min, shake on a shaker for 20 seconds and then aliquot. If the result is not good, sample the sample in a boiling water bath at 100 °C for 10min before loading each sample.
  • sample needle, sample, plug, Marker, electrophoresis solution, electrophoresis tank, distilled water bottle sample needle, sample, plug, Marker, electrophoresis solution, electrophoresis tank, distilled water bottle
  • Transfer membrane (labeled NC membrane, transfer solution, filter paper, ice box, basin, plate, transfer tank, plastic plate, cleaning tweezers)
  • the transfer current is constant current 276mA
  • the voltage is general At 140V (can be supplemented with methanol to increase the voltage)
  • the specific transfer time is determined according to the molecular weight of the protein to be transferred.
  • the time for the molecular weight of the transferred protein is ⁇ 100kDa for 1h, and the time for> 100kDa is 1.5h.
  • Immunoblotting color development cleaning tweezers, box with double distilled water, milk, plastic bag, toilet paper, primary antibody, ice box, tablet, transparent plastic, TBST, black plastic bag, secondary antibody
  • FIG. 2 is a comparison photograph of the degradation of tau protein using different concentrations of the small molecule compounds in the above test example.
  • FIG. 3 is a quantitative analysis chart of the degradation of tau eggs using the different concentrations of the small molecule compounds in the above test example. The results showed that 1 ⁇ M, 10 ⁇ M, and 20 ⁇ M compounds can dose-dependently reduce the content of tau protein in cells.
  • Figure 4 shows a fluorescent picture of the use of different concentrations of the small molecule compounds of the present invention to reduce the total tau protein content in HEK293 tau cells.
  • the small molecule compound specifically degrading tau protein according to the present invention can degrade the content of tau protein in cells.
  • the abnormal accumulation of tau protein in the cell is involved in more than 20 neurodegenerative diseases, and its accumulation is positively related to the neurodegeneration and memory impairment of these degenerative diseases.
  • tau protein can achieve prevention or / and treatment of tau-related Of neurodegenerative degenerative diseases such as Alzheimer's disease, frontotemporal dementia linked to chromosome 17 with Parkinson's disease, Pick's disease, progressive supranuclear palsy, cortical basal ganglia degeneration, primary age-related sexual tau disease, argyrophilic granulopathy, aging-related tau astrocyte disease, chronic traumatic encephalopathy, spherical glial tauopathy, Parkinson's disease, Huntington's disease, stroke and epilepsy.
  • neurodegenerative degenerative diseases such as Alzheimer's disease, frontotemporal dementia linked to chromosome 17 with Parkinson's disease, Pick's disease, progressive supranuclear palsy, cortical basal ganglia degeneration, primary age-related Sexual tau disease, argyrophilic granulopathy, aging-related tau astrocyte disease, chronic traumatic encephalopathy, spherical glial tauopathy, Parkinson's disease, Huntington'

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Abstract

一种特异性降解tau蛋白的小分子化合物及其应用，该特异性降解tau蛋白的小分子化合物的化学结构为TBM-L-ULM或其药学上可接受的盐、对映体、立体异构体、溶剂化物、多晶型物或N-氧化物，其中，TBM为tau蛋白结合部分，L为连接体基团，ULM为泛素连接酶结合部分，所述tau蛋白结合部分与所述泛素连接酶结合部分通过连接体基团连接。所述的特异性降解tau蛋白的小分子化合物能增强细胞中tau蛋白的降解，从而降低tau蛋白的含量。

Description

一种特异性降解tau蛋白的小分子化合物及其应用 技术领域
本发明涉及双功能分子化合物技术领域，具体涉及一种特异性降解tau蛋白的小分子化合物(命名为“JT01”)及其应用。
背景技术
阿尔茨海默病(Alzheimer’s disease,AD)是最常见的一种痴呆症，约占痴呆症的50-70％。据统计，2016年中国有约1000万，全球则有4400万AD患者。随着人口的老龄化，AD的发病率将进一步上升，预计到2050年中国将有约4000万AD患者。目前所有治疗AD的药物都是表征药物(Symptomatic drugs)，只能短暂缓解症状而不能延缓病情的进展。全球迫切期待有能真正改变AD进展的新药(Disease-modifying drugs)。
AD的两个特征性的病理变化是老年斑(Senile plaques，SPs)及神经原纤维缠结(Neurofibrillary tangles,NFTs)，分别是由β-淀粉样蛋白(Aβ)及高度磷酸化的tau蛋白形成的聚合物，其中tau病变而不是Aβ病变与AD的痴呆程度呈正相关。最新的研究显示tau介导Aβ诱导的神经毒性，为Aβ的神经毒性所必需；并且tau是朊脘病毒样蛋白(Prion-like protein)，能在神经元间传播从而导致tau病变的扩散，提示其可能是AD必需的药物靶点。朊
除了AD外，tau的聚集也见于连锁于17号染色体伴帕金森病的额颞叶痴呆(frontotemporal dementia linked to chromosome-17 parkinsonism,FTDP-17)、皮克氏病(Pick’s disease,PiD)、进行性核上麻痹(progressive supranuclear palsy,PSP)、皮质基底节变性(corticobasal degeneration,CBD)、原发性年龄相关性tau病(primary age-related tauopathy,PART)、嗜银颗粒病(argyrophilic grain disease,AGD)、老化相关tau星形胶质细胞病(aging-related tau astrogliopathy,ARTAG)、慢性创伤性脑病(chronic traumatic encephalopathy,CTE)、球形胶质细胞tau病变(Globular glial tauopathy,GGT)、帕金森病(Parkinson’s disease,PD)、亨廷顿病(Huntington’s Disease,HD)等一系列神经退行性疾病。这类疾病包括AD被统称为tau疾病(tauopathies)。Tau蛋白是引起这类疾病的重要原因，故也是这类疾病的重要治疗靶点。
目前，虽然已有多种基于tau蛋白的治疗提案，但其中最有吸引力的方案之一是降低细胞内tau蛋白的含量。该方案之所以受青睐，主要基于下述原因：(1)大量的证据表明，降低细胞内tau蛋白的含量，在动物模型中甚少引起副作用；(2)降低tau蛋白的含量可抑制tau蛋白的聚集，而tau蛋白的聚集则是引起神经元退行性变的重要原因；(3)降低tau蛋白的含量可降低多种因素(比如Aβ)引起的神经元兴奋性神经毒性作用。因此，降低tau蛋白也被认为是一种新的潜在的治疗癫痫及中风的方案。
降低细胞内靶蛋白常用的技术方法有两种。(1)用siRNA、miRNA或反义寡核苷酸降低靶蛋白的表达。由于这些寡核苷酸在组织中的分布不好，药物代谢动力学较差，加之有脱靶的可能性，目前其在临床的应用受到限制，还有待进一步改进。(2)增强靶蛋白的降解。常见的方法是增强蛋白降解系统包括蛋白酶系统及自噬系统的活性。但非特异性地增强蛋白降解系统的活性易引起其它非靶蛋白的降解而严重副作用，故目前尚未有激活蛋白降解系统的药物获批在临床应用。理想的方法是只选择性增强靶蛋白的降解而避免因增强蛋白降解系统的活性而导致的非靶蛋白的降解。
发明内容
本发明的目的是构建一种特异性针对tau蛋白的小分子化合物。为了实现此目的，本发明的发明人通过深入研究发现，利用蛋白降解靶向嵌合体(PROteolysis TArgeting Chimeras,PROTAC)技术可以构建一种双功能分子化合物，其一端能特异结合靶蛋白，另一端特异结合特定的泛素连接酶，两者之间经由连接体基团(linker)连接。如此构建的化合物因能同时结合靶蛋白及泛素连接酶，使靶蛋白临近泛素连接酶，故能增强靶蛋白的泛素化，最终经由蛋白酶体降解。而且，所述PROTAC技术除了对靶蛋白的选择性，还有下面的优点：(1)能作用于许多传统难以成药的靶点。许多传统的小分子药物必须作用于靶蛋白的特定的结合口袋(binding pockets)，才能发挥抑制作用。PROTAC技术则没有这种限制，其只要能与靶蛋白的任何区段相互作用，并且无需很高的亲和力，即可导致靶蛋白的快速降解从而抑制靶蛋白的功能，故能作用于许多传统难以成药的靶点。(2)PROTAC技术在细胞内能被重复使用，能起到类似催化的效应，故无需很高浓度即可达到治疗效果。
为此，本发明提供了一种特异性降解tau蛋白的小分子化合物(即JT01)，其中，该化合物的化学结构为TBM-L-ULM或其药学上可接受的盐、对映体、立体异构体、溶剂化物、多晶型物或N-氧化物，其中，TBM为tau蛋白结合部分，L为连接体基团，ULM为泛素连接酶结合部分，所述tau蛋白结合部分与所述泛素连接酶结合部分通过连接体基团连接。
在优选的实施方式中，ULM能够结合E3泛素连接酶。
进一步优选地，所述E3泛素连接酶为VHL E3泛素连接酶或CRBN E3泛素连接酶。
在优选的实施方式中，ULM为具有式(1)所示结构的基团
Figure PCTCN2018114838-appb-000001
其中，R 1为羟基或者在患者或对象体内代谢成羟基的基团；
R 2为-NH-R 4-Ar-HET，其中，R 4为C1-C3的亚烷基，Ar为C6-C12的亚芳基，HET为任选取代的噻唑或异噻唑；
R 3为-CHR 5-M-，ULM通过M与连接体基团L相连，其中，R 5为C1-C4的烷基，M为键、C1-C4的亚烷基、-NH-或-NH-C(O)-R 6-，其中R 6为C1-C4的亚烷基。
在优选的实施方式中，L为基团-X-Y-Z-，X与TBM相连，Z与ULM相连，其中，X为键、C1-C4的亚烷基、-NH-或-NH-C(O)-R 7-，其中R 7为键或C1-C4的亚烷基；
Y为-R 8-(R 10-E-R 11) n-R 9-，其中R 8和R 9各自为键或C1-C8的亚烷基，R 10和R 11各自为C1-C4的亚烷基，n为0-10的整数，E为O、S、酰胺基、哌嗪基、NR 12、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2、OS(O) 2O、
Figure PCTCN2018114838-appb-000002
其中E 1为O、S、CHR 12或NR 12，R 12为H或者任选一个或两个羟基取代的C1-C3的烷基；
Z为-A-B-，其中A为键、O或S，B为键、C1-C4的亚烷基或-R 13-C(O)-，其中R 13为C1-C4的亚烷基。
在优选的实施方式中，TBM为具有式(2)所示结构的基团，或者为式(2)所示结构的基团中①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位被取代基团进一步修饰的基团，其中，TBM通过式(2)中的①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位与所述连接体基团L相连，
Figure PCTCN2018114838-appb-000003
其中，R 14为C1-C4的亚烷基，R 15和R 16各自为C1-C4的烷基，R 17为键、H、C1-C4的烷基或-R 18-C(O)-，其中，R 18为C1-C4的亚烷基。
在优选的实施方式中，所述特异性降解tau蛋白的小分子化合物的结构式为：
Figure PCTCN2018114838-appb-000004
本发明还提供了降解有需要的患者体内的tau蛋白的方法，包括给予所述患者有效量的上述小分子化合物。
在优选的实施方式中，所述小分子化合物通过选自以下至少一种方式被给予所述患者：鼻服、吸入、局部、口服、肌内、皮下、经皮、腹腔、硬膜外、鞘内和静脉内途径。
本发明还提供了上述小分子化合物在制备用于治疗或预防与tau蛋白有关的疾病的药物中的应用。
可选地，所述疾病为阿尔茨海默病(AD)、连锁于17号染色体伴帕金森病的额颞叶痴呆(Frontotemporal dementia linked to chromosome-17 parkinsonism,FTDP-17)、皮克氏病(Pick’s disease,PiD)、进行性核上麻痹(progressive supranuclear palsy,PSP)、皮质基底节变性(corticobasal degeneration,CBD)、原发性年龄相关性tau病(primary age-related tauopathy,PART)、嗜银颗粒病(argyrophilic grain disease,AGD)、老化相关tau星形胶质细胞病(aging-related tau astrogliopathy,ARTAG)、慢性创伤性脑病(chronic traumatic encephalopathy,CTE)、球形胶质细胞tau病变(Globular glial tauopathy,GGT)、帕金森病(Parkinson’s disease,PD)、亨廷顿病(Huntington’s Disease,HD)、脑卒中和癫痫中的至少一种。
通过免疫印迹试验证实本发明所述的特异性降解tau蛋白的小分子化合物能增强细胞中tau蛋白的降解，从而降低tau蛋白的含量。由此表明，本发明所述的特异性降解tau蛋白的小分子化合物可在预防和治疗包括阿尔茨海默病在内的一系列tau疾病中发挥作用。
附图说明
图1是本发明实施例1制备的小分子化合物的核磁共振谱图；
图2是使用不同浓度的本发明所述的小分子化合物对tau蛋白进行降解的对比照片；
图3是使用不同浓度的本发明所述的小分子化合物对tau蛋进行降解的定量分析图表；
图4显示了本发明所述的小分子化合物可减少HEK293 tau细胞中的总tau蛋白含量 的荧光照片。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
本发明所述的特异性降解tau蛋白的小分子化合物的化学结构为TBM-L-ULM或其药学上可接受的盐、对映体、立体异构体、溶剂化物、多晶型物或N-氧化物，其中，TBM为tau蛋白结合部分，L为连接体基团，ULM为泛素连接酶结合部分，所述tau蛋白结合部分与所述泛素连接酶结合部分通过连接体基团连接。
在优选情况下，ULM能够结合E3泛素连接酶。进步一步优选地，所述E3泛素连接酶为VHL E3泛素连接酶或CRBN E3泛素连接酶。
在较优选的实施方式中，ULM为具有式(1)所示结构的基团，
Figure PCTCN2018114838-appb-000005
其中，R 1为羟基或者在患者或对象体内代谢成羟基的基团；
R 2为-NH-R 4-Ar-HET，其中，R 4为C1-C3的亚烷基(如亚甲基、亚乙基或亚丙基)，Ar为C6-C12的亚芳基(如苯基或萘基)，HET为任选取代的噻唑或异噻唑；
R 3为-CHR 5-M-，ULM通过M与连接体基团L相连，其中，R 5为C1-C4的烷基(如甲基、乙基、丙基或丁基)，M为键、C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)、-NH-或-NH-C(O)-R 6-，其中R 6为C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)。其中，ULM通过“-M-”与连接体基团L相连。当“-M-”为-NH-C(O)-R 6-时，“-R 6-”与连接体基团L直接相连。
进一步优选地，在式(1)所示结构中，R 1为羟基；R 2为-NH-R 4-Ar-HET，其中，R 4为亚甲基，Ar为苯基；R 3为-CHR 5-NH-或-CHR 5-NH-C(O)-R 6-，其中，R 5为-C(CH 3) 3，R 6为亚甲基。
在较优选的实施方式中，L为基团-X-Y-Z-，X与TBM相连，Z与ULM相连，
其中，X为键、C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)、-NH-或-NH-C(O)-R 7-，其中R 7为键或C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)；
Y为-R 8-(R 10-E-R 11) n-R 9-，其中R 8和R 9各自为键或C1-C8的亚烷基(如亚甲基、亚 乙基、亚丙基、亚丁基、亚戊基、亚己基、亚庚基或亚辛基)，R 10和R 11各自为C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)，n为0-10的整数(如0、1、2、3、4、5、6、7、8、9或10)，E为O、S、酰胺基、哌嗪基、NR 12、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2、OS(O) 2O、
Figure PCTCN2018114838-appb-000006
其中E 1为O、S、CHR 12或NR 12，R 12为H或者任选一个或两个羟基取代的C1-C3的烷基；
Z为-A-B-，其中A为键、O或S，B为键、C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)或-R 13-C(O)-，其中R 13为C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)。
在较优选的实施方式中，TBM为具有式(2)所示结构的基团，或者为式(2)所示结构的基团中①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位被取代基团进一步修饰的基团，其中，TBM通过式(2)中的①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位与所述连接体基团L相连，
Figure PCTCN2018114838-appb-000007
其中，R 14为C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)，R 15和R 16各自为C1-C4的烷基(如甲基、乙基、丙基或丁基)，R 17为键、H、C1-C4的烷基(如甲基、乙基、丙基或丁基)或-R 18-C(O)-，其中，R 18为C1-C4的亚烷基(如亚甲基、亚乙基、亚丙基或亚丁基)。
其中，修饰式(2)所示结构的基团中①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位的取代基团可以为卤素(如氟或氯)、C1-C4的烷基(如甲基、乙基、丙基或丁基)、C1-C4的烷氧基(如甲氧基、乙氧基、丙氧基或丁氧基)、羧基、氨基、胺基、C6-C18的芳基(如苯基)或苄基。
进一步优选地，在式(2)所示结构中，TBM通过式(2)中的⑤位与所述连接体基团L相连，R 14为亚乙基，R 15和R 16各自为甲基，R 17为键、亚甲基或-CH 2-C(O)-。 在最优选的实施方式中，所述特异性降解tau蛋白的小分子化合物的结构式为：
Figure PCTCN2018114838-appb-000008
上述特异性降解tau蛋白的小分子化合物可以按照以下工艺路线制备：
Figure PCTCN2018114838-appb-000009
Figure PCTCN2018114838-appb-000010
具体制备过程包括如下步骤：
(1)制备化合物B
将上述化合物A溶于N,N-二甲基乙酰胺(DMA)中，然后在N 2保护下加入醋酸钾(KOAc)、4-甲基噻唑和醋酸铅(Pd(AcO) 2)。将悬浮液在真空下脱气并用氢气置换。然后搅拌反应悬浮液并在N 2下加热回流过夜。反应完全后，过滤反应溶液，将滤液浓缩至干，然后通过硅胶柱纯化，得到化合物B(黄色油状物)。
(2)制备化合物C
将化合物B溶于甲醇(MeOH)和NH 4OH中，然后在N 2保护下加入雷尼镍(Reney Ni)。将悬浮液在真空下脱气并用氢气置换。然后将反应混悬液在H 2下在室温下搅拌过夜。反应完成后，过滤反应溶液，将滤液浓缩至干，然后通过硅胶柱纯化，得到化合物 C(浅黄色油状物)。
(3)制备化合物D
将化合物C溶于N,N-二甲基甲酰胺(DMF)中，在N 2保护下加入N,N-二异丙基乙胺(DIPEA)和Boc-(2S，4R)-4-羟基-2-吡咯烷羧酸。然后在反应溶液冷却至0～5℃后加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(HATU)。然后将反应溶液在N 2保护下在室温下搅拌过夜。反应完全后，将反应混合物倒入冰水中，用乙酸乙酯(EA)萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩，然后通过硅胶柱纯化，获得化合物D。
(4)制备化合物E
将化合物D溶于二氯甲烷(DCM)中，并加入三氟乙酸(TFA)。将反应溶液在室温下搅拌反应。反应完全后进行浓缩，获得化合物E。
(5)制备化合物4-7
将化合物E溶于DMF中，在N 2保护下加入DIPEA、N-Boc-L-叔亮氨酸。然后在反应溶液冷却至0～5℃后加入HATU。然后将反应溶液在N 2保护下在室温下搅拌过夜。反应完全后，将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩，然后通过硅胶柱纯化，获得化合物4-7。
(6)制备化合物4-8
将化合物4-7溶于DCM中，并加入TFA。将反应溶液在室温下搅拌反应。反应完全后进行浓缩，获得化合物4-8。
(7)制备化合物4-9
将化合物4-8溶于DMF，在N 2保护下加入DIPEA和2,2-二甲基-4-氧代-3,8,11,14-四氧杂-5-氮杂十六烷-16-油酸。然后在反应溶液冷却至0～5℃后加入HATU。然后将反应溶液在N 2保护下在室温下搅拌过夜。反应完全后，将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥，浓缩，用硅胶柱纯化，获得化合物4-9。
(8)制备化合物4-10
将化合物4-9溶于DCM中，并加入EA/HCl。将反应溶液在室温下搅拌反应。反应完全后进行浓缩，获得化合物4-10。
(9)制备化合物4-12
将化合物3-3和肌氨酸溶于DMF中，并加入三乙胺(TEA)。然后将反应溶液在N 2保护下搅拌过夜。反应完成后，将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩。通过硅胶色谱法纯化粗产物，得到化合物4-12。
(10)制备化合物4(目标化合物)
将化合物4-12溶于DMF中，并在N 2保护下加入DIPEA和化合物4-10。然后在反应溶液冷却至0～5℃后加入HATU。然后将反应溶液在N 2保护下在室温下搅拌过夜。反应完全后，将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥，浓缩，用硅胶柱纯化，得到化合物4(吸湿性白色泡沫状固体)。
本发明还提供了降解有需要的患者体内的tau蛋白的方法，包括给予所述患者有效量的本发明提供的上述化合物。
在上述方法中，所述化合物可以通过选自以下至少一种方式被给予所述患者：鼻服、吸入、局部、口服、肌内、皮下、经皮、腹腔、硬膜外、鞘内和静脉内途径。
本发明还提供了上述化合物在制备用于治疗或预防与tau蛋白有关的疾病的药物中的应用。所述疾病可以为阿尔茨海默病、连锁于17号染色体伴帕金森病的额颞叶痴呆、皮克氏病、进行性核上麻痹、皮质基底节变性、原发性年龄相关性tau病、嗜银颗粒病、老化相关tau星形胶质细胞病、慢性创伤性脑病、球形胶质细胞tau病变、帕金森病、亨廷顿病、脑卒中和癫痫中的至少一种。
以下将通过实施例对本发明进行详细描述。
实施例
(1)制备化合物B
将化合物A(1.82g，10mmol)溶于25mL DMA中，然后在N 2保护下加入KOAc(1.98g，20mmol)、4-甲基噻唑(1.5g，15mmol)和Pd(AcO) 2(220mg，1mmol)。将悬浮液在真空下脱气并用氢气置换三次。然后搅拌反应悬浮液并在N 2下加热回流过夜。通过薄层层析法(Thin Layer Chromatography，TLC)确认反应完全。过滤反应溶液，将滤液浓缩至干，然后通过硅胶柱纯化，得到1.5g化合物B，为黄色油状物。
(2)制备化合物C
将化合物B(300mg，1.498mmol)溶于10mL MeOH和1mL NH 4OH中，然后在N 2保护下加入200mg雷尼镍(Reney Ni)。将悬浮液在真空下脱气并用氢气置换三次。然后将反应混悬液在H 2下在室温下搅拌过夜。通过TLC(DCM/EA＝1：1)确认反应完成。过滤反应溶液，将滤液浓缩至干，然后通过硅胶柱纯化，得到110mg化合物C，为浅黄色油状物。
(3)制备化合物D
将化合物C(110mg，0.538mmol)溶于3mL DMF中，在N 2保护下加入DIPEA(200mg，1.547mmol)和Boc-(2S，4R)-4-羟基-2-吡咯烷羧酸(131mg，0.566mmol)。然后在反应溶液冷却至0～5℃后加入HATU(245mg，0.644mmol)。然后将反应溶液在N 2下在室温下搅拌过夜。通过TLC(DCM/EA＝1：1)确认反应完全。将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩，然后通 过硅胶柱纯化，获得100mg化合物D。
(4)制备化合物E
将化合物D(100mg，0.239mmol)溶于3mL DCM中，并加入三氟乙酸(TFA)(0.5mL，6.73mmol)。将反应溶液在室温下搅拌1小时。通过TLC(EA/MeOH＝9：1)确认反应完全，并通过高真空泵浓缩，获得220mg粗化合物E。
(5)制备化合物4-7
将化合物E(220mg，约0.239mmol)溶于3mL DMF中，在N 2保护下加入DIPEA(300mg，2.320mmol)、N-Boc-L-叔亮氨酸(58mg，0.251mmol)。然后在反应溶液冷却至0～5℃后加入HATU(110mg，0.289mmol)。然后将反应溶液在N 2保护下在室温下搅拌过夜。通过TLC(PE/EA＝1：1)确认反应完全。将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩，然后通过硅胶柱纯化，获得103mg化合物4-7。
(6)制备化合物4-8
将化合物4-7(103mg，0.194mmol)溶于3mL DCM中，并加入TFA(0.5mL，mmol)。将反应溶液在室温下搅拌1小时。通过TLC(PE/EA＝1：1)确认反应完全，并通过高真空泵浓缩，获得85mg化合物4-8。
(7)制备化合物4-9
将化合物4-8(85mg，0.194mmol)溶于3mL DMF，在N 2保护下加入DIPEA(300mg，2.320mmol)和2,2-二甲基-4-氧代-3,8,11,14-四氧杂-5-氮杂十六烷-16-油酸(63mg，0.205mmol)。然后在反应溶液冷却至0～5℃后加入HATU(89mg，0.234mmol)。然后将反应溶液在N 2保护下在室温下搅拌过夜。通过TLC(EA/MeOH＝9：1)确认反应完全。将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥，浓缩，用硅胶柱纯化，获得75mg化合物4-9。
(8)制备化合物4-10
将化合物4-9(75mg，0.104mmol)溶于3mL DCM中，并加入EA/HCl(0.5mL，2N)。将反应溶液在室温下搅拌1小时。通过TLC(EA/MeOH＝9：1)确认反应完全，并通过高真空泵浓缩，获得103mg(0.104mmol)化合物4-10。
(9)制备化合物4-12
将化合物3-3(150mg，0.394mmol)和肌氨酸(174mg，1.953mmol)溶于15mL DMF中，并加入TEA(400mg，3.943mmol)。然后将反应溶液在N 2保护下在90℃下搅拌过夜。通过TLC(EA/DCM＝1：1和EA/MeOH＝9：1)确认反应完成。将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥并浓缩。通过硅胶色谱法纯化粗产物，得到化合物4-12(63mg，0.145mmol)。
(10)制备化合物4(目标化合物)
将化合物4-12(63mg，0.145mmol)溶于5mL DMF中，并在N 2保护下加入DIPEA(200mg，1.547mmol)和化合物4-10(103mg，0.104mmol)。然后在反应溶液冷却至0～5℃后加入HATU(48mg，0.236mmol)。然后将反应溶液在N 2保护下在室温下搅拌过夜。通过TLC(EA/MeOH＝9：1)确认反应完全。将反应混合物倒入冰水中，用EA萃取，并将有机相用盐水洗涤。合并有机相，用无水硫酸钠干燥，浓缩，用硅胶柱纯化，得到69mg化合物4，为吸湿性白色泡沫状固体。该化合物的核磁共振谱图如图1所示。HPLC纯度：97.8％(254nm)；Mass：m/z 1036[M+1] +。
测试例
细胞培养和药物处理HEK293 tau细胞(稳定表达野生型全长tau蛋白)，在DMEM培养基中培养24h后，加入不同浓度的上述实施例制备的小分子化合物(0μM，0.01μM，0.1μM，1μM，10μM，20μM)，继续培养24h。再用RIPA液裂解细胞并收集细胞提取液，用10％聚丙烯酰胺凝胶电泳(PAGE)分离细胞提取液中的蛋白质，再用抗总tau蛋白抗体(tau5)进行免疫印迹，定量分析tau蛋白含量。其中，样品的制备、样品蛋白质含量的测定以及免疫印迹法的详细操作过程参照以下内容。
一、样品的制备
(1)在显微镜下观察细胞培养板内细胞成长状况和密度；
(2)将1×PBS和细胞培养板放在冰上预冷，按照6孔板每孔加入80μl/6cm培养皿加入150μl配制1×缓冲液+PMSF(1:100)+蛋白水解酶混合抑制剂(1:1000)混合液；
(3)用1ml加样枪贴着细胞培养板底壁吸出培养基，贴壁加入1ml 1×PBS，根据细胞密度轻柔冲洗1-3遍，用1ml和200μl加样枪贴着细胞培养板底壁吸出PBS，加入混合液后用超纯水清洗细胞刮子，用细胞刮子稍用力刮孔板底部，吸出细胞悬液至1.5ml EP管中，不同样品间需清洗细胞刮子；
(4)煮沸10min(先以最高温度煮沸再以150℃维持沸腾，打开抗氧表位)；
(5)离心后20kHz超声20次(为了打开DNA链，超声机按I打开O关闭，每超完一个样品后先在双蒸水中超三次再用卫生纸擦干，超声枪头不能接触气泡和EP管底部)；
(6)振荡混匀置于-20℃冰箱。
二、样品蛋白质含量的测定(BCA法)
(1)将蛋白样品震荡后进行适当稀释(样品各取5μl与45μl双蒸水混合稀释，各设2个平行样)，离心震荡；
(2)设六个标准管，分别取20mg/ml BSA(100mg BSA溶于5ml双蒸水中)0μl、 10μl、20μl、30μl、40μl、50μl，分别加1000μl、990μl、980μl、970μl、960μl、950μl双蒸水配制成0μg/μl、0.2μg/μl、0.4μg/μl、0.6μg/μl、0.8μg/μl、1.0μg/μl的标准蛋白；
(3)将稀释过的蛋白样品和稀释过的标准蛋白分别加入96孔板内(5μl/孔，用PCR枪贴壁和底的交界处，每加一个孔换一个枪头)，各设3个平行孔。
(4)工作液由试剂盒中A液(自领)和B液(公用)按50:1比例配制而成。将工作液加入96孔板中，每孔快速悬空加入95μl，加完后盖上盖子迅速贴底沿同方向震荡，手不要触碰96孔板底，用塑料盒垫着37℃孵育30min；
(5)用1ml注射器针头去气泡，打开BioTek开关，打开Gen5，点左箭头图标，点击OK，导出Excel；
(6)复制标准蛋白OD值，框选OD值和标准蛋白浓度插入散点图。选择数据点添加趋势线，显示公式，显示R平方值(小数点后应至少有2个9)，去掉异常值。复制样品蛋白OD值，上方输入相应样品组号，去掉异常值。
三、tau蛋白含量测定(免疫印迹法)
1、搭架子(两种玻璃板，三个瓶子，五个试剂，滤纸，卫生纸，垃圾桶，枪，枪头，梳齿)
(1)擦净桌面和底架，洗净梳齿、玻璃板、蒸馏水瓶和上、下部胶瓶，并烘干上、下部胶瓶，拿出配制电泳胶的试剂恢复至室温；
(2)将较高的玻璃板朝内叠在一起，按住上部使下部紧贴桌面使其平齐，将夹子向外翻夹紧，放在底架上用夹子扣住。
(3)注入双蒸水检验是否漏液，若漏液则重装后再检漏。
2、制备电泳胶(见表1，天冷时AP和TEMED可加1.5倍)
表1
Figure PCTCN2018114838-appb-000011
(1)依次加入20％Arc/Bis，Tris缓冲液，TEMED和10％AP，用移液器吹打混匀，整个过程防止混合液中混有气泡；
(2)沿两个角分别将分离胶缓缓注入到胶膜内(吸取时深入液面下轻柔吹打混匀，每次枪头留少量液体以防产生气泡)，每块胶用量为3×900μl，观察胶不漏后沿两个角分别用双蒸水将胶膜的空隙处填满(防止氧气抑制聚合并保持下部胶水平，可多放一段时间)；
(3)等待凝胶30min左右待分离胶凝固后倾去双蒸水，并用滤纸将剩余水吸尽， 并用记号笔标出下部胶的上沿；
(4)沿两个角分别将浓缩胶缓缓注入到胶膜内，斜着从左往右插入所需规格梳齿(上样量＜20μl用小梳齿，上样量＞20μl用大梳齿)，在泳道间补胶避免缩胶，等待凝胶50min。
3、样品的处理
BCA法测定蛋白浓度后，加入现配的溴酚蓝和β-巯基乙醇(还原剂)，溴酚蓝：β-巯基乙醇＝1:3，混合液：样品＝1：10，100℃沸水浴10min，在振荡器上振荡20秒钟后分装，若结果不佳每次上样前将样品100℃沸水浴10min。
4、上样和蛋白的电泳分离(上样针，样品，排插，Marker，电泳液，电泳槽，蒸馏水瓶)
清洗电泳架下面的导电丝，将转移到电泳架上，用记号笔标出泳道及编号，缓慢垂直拔出梳齿，用电泳液加满凝胶槽，用微量加样器取样品加入各个泳道(Marker加1μl于第1和8泳道，加入溴酚蓝和1×Buffer混合液加于第15泳道平衡)。上完样后将电泳架转移至电泳槽，加电泳液后盖上盖子使红色对红色，黑色对黑色，加样后先用恒流10mA/块胶电泳约30min(按两次启动)，待溴酚蓝指示剂电泳至浓缩胶与分离胶交界处成以线状时，改为恒压100V(若无法恒压可调高电流)电泳约60min至溴酚蓝到凝胶底部且Marker完全跑开。
5、转膜(标记NC膜，转膜液，滤纸，冰盒，盆子，盘子，转膜槽，塑料板，清洗镊子)
(1)将NC膜用记号笔标记后浸于回收的转膜液10min-20min(有利于蛋白质的固定，能平衡凝胶且去除SDS)，按住两旁的卡口取下凝胶槽，用小板撬起玻璃板和白瓷板右侧中间部分，期间保持剩余胶的电泳。
(2)依欲显分子量范围用玻璃板垂直略倾斜并轻微地左右来回一次切胶，用镊子将浸过转膜液的三层滤纸贴在胶上，用小板小心地将胶撬起放在的海绵上(滤纸朝下)，另一面贴上倒置的NC膜，将胶和NC膜浸没至转膜液中(胶在上)用玻璃棒赶气泡，用镊子夹起小心放在手上(胶在上)，用镊子将浸过转膜液的三层滤纸贴在胶上，倒置放在海绵上，再贴上三层滤纸。由下至上放置黑塑料板→一层海绵→三层滤纸→胶→NC膜→三层滤纸→一层海绵→透明塑料板，若不紧可用橡皮筋固定。
(3)红对红，白对黑，将转膜槽放入冰浴中(通电前不要将胶长时间泡在转膜液中，以免蛋白质扩散分解)，转移电流为恒流276mA，电压一般在140V(可以适当补充甲醇提高电压)，具体转移时间根据所需要转移的蛋白质的分子量的大小决定，转移的蛋白质的分子量＜100kDa时时间为1h，＞100kDa时时间为1.5h。
6、免疫印迹显色(清洗镊子，装有双蒸水的盒子，牛奶，保鲜袋，卫生纸，一抗，冰盒，平板，透明胶，TBST，黑色塑料袋，二抗)
(1)封闭：转膜结束后小心地将NC膜用含5％脱脂奶粉的TBS封闭液于室温振荡封闭1h或4℃过夜，回收未与胶接触的滤纸。
(2)一抗孵育：取出NC膜，用1×TBS漂去膜表面残留的奶渍，用镊子夹着NC膜竖在卫生纸上除去多余的水，将NC膜Marker一侧朝外置于保鲜袋中用卫生纸排水排气。加入一抗(可加入0.1％的吐温20降低背景)封口贴在平板上(有Marker和蛋白的一面朝上)，透明胶带不要压在目标条带上，于4℃孵育过夜。
(3)二抗孵育：次日从孵育袋中取出NC膜并回收一抗，用TBST缓冲液漂洗3×5min，用1×TBS漂去膜表面残留的盐离子，用镊子夹着NC膜竖在卫生纸上除去多余的水，将NC膜置于保鲜袋中用卫生纸排水排气。避光加入荧光素标记的羊抗兔或羊抗鼠的Odyssey二抗(可加入0.1％的吐温20降低背景)，封口贴在平板上(有Marker和蛋白的一面朝上)，透明胶带不要压在目标条带上，于室温慢摇孵育1h(超过1h可能会增加背景，天冷可延长至2h)后，从孵育袋中取出NC膜并回收二抗，用TBST缓冲液漂洗3×5min。漂洗完毕后，用1×TBS漂洗去掉膜表面残留的盐离子。
(4)显色：先用蘸有无水乙醇的擦镜纸将玻璃板擦干净。将Marker侧朝下，按分子量从小到大在扫描仪由上至下方向放置，覆盖塑料薄膜并排气。打开Odyssey软件，点击File→New→Browse→输入日期，点击Obtain image选择扫膜长、宽，扫描成像并保存原图，点击Alter image调整图像，用文本框标记样品和抗体类型，点击Export image file→Save，收好膜后再用卫生纸将玻璃板擦干净。
图2是上述测试例中使用不同浓度的所述小分子化合物对tau蛋白进行降解的对比照片。图3是上述测试例中使用不同浓度的所述小分子化合物对tau蛋进行降解的定量分析图表。结果显示，1μM、10μM、20μM化合物可剂量依赖性降低细胞中tau蛋白的含量。图4显示使用不同浓度本发明所述的小分子化合物可减少HEK293 tau细胞中的总tau蛋白含量的荧光图片。
由此可见，本发明所述的特异性降解tau蛋白的小分子化合物可降解细胞中的tau蛋白含量。由于tau蛋白在细胞内异常聚集参与20多种神经退行性疾病，其聚积量与这些退行性疾病的神经变性和记忆障碍正相关，因此，降解tau蛋白即可达到预防或/和治疗tau-相关的神经退行性变性病，如阿尔茨海默病、连锁于17号染色体伴帕金森病的额颞叶痴呆、皮克氏病、进行性核上麻痹、皮质基底节变性、原发性年龄相关性tau病、嗜银颗粒病、老化相关tau星形胶质细胞病、慢性创伤性脑病、球形胶质细胞tau病变、帕金森病、亨廷顿病、脑卒中和癫痫。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (10)

  1. 一种特异性降解tau蛋白的小分子化合物，其特征在于，该化合物的化学结构为TBM-L-ULM或其药学上可接受的盐、对映体、立体异构体、溶剂化物、多晶型物或N-氧化物，其中，TBM为tau蛋白结合部分，L为连接体基团，ULM为泛素连接酶结合部分，所述tau蛋白结合部分与所述泛素连接酶结合部分通过连接体基团连接。
  2. 根据权利要求1所述的小分子化合物，其特征在于，ULM能够结合E3泛素连接酶。
  3. 根据权利要求2所述的小分子化合物，其特征在于，所述E3泛素连接酶为VHL E3泛素连接酶或CRBN E3泛素连接酶。
  4. 根据权利要求1-3中任意一项所述的小分子化合物，其特征在于，ULM为具有式(1)所示结构的基团，
    Figure PCTCN2018114838-appb-100001
    其中，R 1为羟基或者在患者或对象体内代谢成羟基的基团；
    R 2为-NH-R 4-Ar-HET，其中，R 4为C1-C3的亚烷基，Ar为C6-C12的亚芳基，HET为任选取代的噻唑或异噻唑；
    R 3为-CHR 5-M-，ULM通过M与连接体基团L相连，其中，R 5为C1-C4的烷基，M为键、C1-C4的亚烷基、-NH-或-NH-C(O)-R 6-，其中R 6为C1-C4的亚烷基。
  5. 根据权利要求1所述的小分子化合物，其特征在于，L为基团-X-Y-Z-，X与TBM相连，Z与ULM相连，
    其中，X为键、C1-C4的亚烷基、-NH-或-NH-C(O)-R 7-，其中R 7为键或C1-C4的亚烷基；
    Y为-R 8-(R 10-E-R 11) n-R 9-，其中R 8和R 9各自为键或C1-C8的亚烷基，R 10和R 11各 自为C1-C4的亚烷基，n为0-10的整数，E为O、S、酰胺基、哌嗪基、NR 12、S(O)、S(O) 2、-S(O) 2O、-OS(O) 2、OS(O) 2O、
    Figure PCTCN2018114838-appb-100002
    其中E 1为O、S、CHR 12或NR 12，R 12为H或者任选一个或两个羟基取代的C1-C3的烷基；
    Z为-A-B-，其中A为键、O或S，B为键、C1-C4的亚烷基或-R 13-C(O)-，其中R 13为C1-C4的亚烷基。
  6. 根据权利要求1所述的小分子化合物，其特征在于，TBM为具有式(2)所示结构的基团，或者为式(2)所示结构的基团中①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位被取代基团进一步修饰的基团，其中，TBM通过式(2)中的①、②、③、④、⑤、⑥、⑦、⑧、⑨或⑩位与所述连接体基团L相连，
    Figure PCTCN2018114838-appb-100003
    其中，R 14为C1-C4的亚烷基，R 15和R 16各自为C1-C4的烷基，R 17为键、H、C1-C4的烷基或-R 18-C(O)-，其中，R 18为C1-C4的亚烷基。
  7. 根据权利要求1所述的小分子化合物，其特征在于，所述小分子化合物的结构式为：
    Figure PCTCN2018114838-appb-100004
  8. 降解有需要的患者体内的tau蛋白的方法，包括给予所述患者有效量的权利要求1-7中任意一项所述的小分子化合物；
    优选地，所述小分子化合物通过选自以下至少一种方式被给予所述患者：鼻服、吸入、局部、口服、肌内、皮下、经皮、腹腔、硬膜外、鞘内和静脉内途径。
  9. 权利要求1-7中任意一项所述的小分子化合物在制备用于治疗或预防与tau蛋白有关的疾病的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述疾病为阿尔茨海默病、连锁于17号染色体伴帕金森病的额颞叶痴呆、皮克氏病、进行性核上麻痹、皮质基底节变性、原发性年龄相关性tau病、嗜银颗粒病、老化相关tau星形胶质细胞病、慢性创伤性脑病、球形胶质细胞tau病变、帕金森病、亨廷顿病、脑卒中和癫痫中的至少一种。
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