Compound specifically binding to tau protein aggregate and application thereof

Abstract

The invention provides a micromolecular bifunctional chimera compound specifically binding to tau protein aggregate, the compound has a structure as shown in formula (I): A-linker-B (I), A is a depolymerization group specifically binding to pathological tau protein aggregate and promoting depolymerization of the tau protein aggregate, and B is a difunctional chimera group specifically binding to the tau protein aggregate and promoting depolymerization of the tau protein aggregate. B is a clearing group which is used for recruiting p62, LC3B or ubiquitin linker assemblies MDM2, cIAP, VHL and CRBN required by formation of cell autophagosome so as to promote tau protein aggregate to be cleared through cell autophagy or through a ubiquitin-proteasome pathway, and the linker is a linking group for connecting the group A and the group B. The compound can inhibit formation of tau protein aggregates and promote removal of pathological tau protein aggregates at the same time, and can be applied to prevention and treatment of AD, FTD and other diseases with abnormal aggregation of tau proteins.

Description

Technical Field

[0001] The invention discloses a compound and belongs to the field of pharmacy. Background Art

[0002] Tau is a microtubule-associated protein encoded by the Mapt gene located on human chromosome 17. It is highly expressed in neurons and is particularly enriched in axons. Tau protein exerts its core physiological function by binding to α- and β-tubulins, stabilizing microtubule structure and maintaining cytoskeletal integrity. It also participates in the maintenance of neuronal morphology, axonal transport, and the regulation of synaptic plasticity.

[0003] Under certain pathological conditions, abnormal post-translational modifications of tau protein (such as hyperphosphorylation and acetylation) lead to abnormal tau aggregation in cells, forming neurofibrillary tangles and causing neurodegeneration. For example, the aggregation of hyperphosphorylated tau protein within brain neurons is a core pathological hallmark of Alzheimer's disease (AD). Furthermore, abnormal tau aggregation is a key cause of many other neurodegenerative diseases, including frontotemporal dementia (FTD), aging-related tau astrogliopathy (ARTAG), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and chronic traumatic encephalopathy (CTE). Furthermore, tau protein aggregation is also implicated in the pathogenesis of epilepsy, stroke, retinal degeneration, and even certain tumors. Given these common pathological characteristics, the development of drugs targeting tau protein and its aggregates holds great promise for the treatment of related diseases.

[0004] Currently, the development of targeted drugs for tau protein mainly focuses on the following aspects (Congdon EE, Ji C, Tetlow AM, Jiang Y, Sigurdsson EM. Tau-targeting therapies for Alzheimer disease: current status and future directions. Nat Rev Neurol. 2023. 9(12):715-736): (1) Designing interfering RNA or antisense oligonucleotides against tau protein mRNA to reduce the production of tau protein. (2) Developing vaccines or monoclonal antibodies against tau protein monomers (including tau fragments or those containing abnormal modification sites) to promote tau protein degradation through immune pathways. (3) Proteolysis-targeting chimeras (PROTACs) designed for tau monomers, including small molecules and peptide forms, are used to specifically induce the degradation of tau protein monomers. (4) Short peptides or small molecules that inhibit the aggregation of monomeric tau protein. (5) Dephosphorylation-targeting chimera (DEPTAC) that selectively promotes the dephosphorylation modification of monomeric tau protein, including peptides and small molecules.

[0005] The shortcomings of existing drugs targeting tau protein include: (1) tau interfering RNA, antisense oligonucleotides, vaccines, antibodies, peptide PROTACs, and DEPTACs are all large molecules with low efficiency in crossing the blood-brain barrier when administered peripherally and are easily degraded by nucleases and proteases in the body. (2) All existing drugs are designed for monomeric tau protein, lacking pathological specificity, and can widely affect tau proteins with normal physiological functions, with potential toxic side effects. (3) They are ineffective in clearing tau aggregates, which are the real cause of neurodegeneration in diseases such as AD.

[0006] The purpose of the present invention is to provide a bifunctional chimeric compound that specifically binds to tau protein aggregates and promotes their disaggregation and clearance. Summary of the Invention

[0007] Based on the above objectives, the present invention first provides a bifunctional chimeric compound that specifically binds to tau protein aggregates, wherein the compound has a structure as shown in formula (I): A-linker-B (I), Among them, A is a disaggregating group that specifically binds to pathological tau protein aggregates and promotes the disaggregation of pathological tau protein aggregates, and is also named "TFB (Tau Fibril Binder)" in the present invention.

[0008] B is a scavenging group that recruits p62, LC3B or ubiquitin ligase components MDM2, cIAP, VHL, and CRBN required for the formation of cellular autophagy, and promotes the clearance of tau protein aggregates through cellular autophagy or the ubiquitin-proteasome pathway. It is also named "FB (Functional Binder)" in the present invention.

[0009] The linker connects TFB and FB, enhancing the structural flexibility of the chimera without any specific physiological or pathological effects. Ultimately, the chimera specifically promotes the disaggregation and clearance of tau protein aggregates.

[0010] In a preferred embodiment, the group A is selected from the group having a structure as shown in any one of formulas (II) to (VIIII): (II), The compound of formula (II) is (2Z)-2-{[1-(3,4-dimethylphenyl)-1H-pyrazol-4-yl]methylene}-6,7-dimethyl[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one ((2Z)-2-{[1-(3,4-Dimethylphenyl)-1h-Pyrazol-4-Yl]Methylidene}-6,7-Dimethyl[1,3]Thiazolo[3,2-A]Benzimidazol-3(2h)-One), with PubChem CID: 1516497, which is also named C1 compound in the present invention.

[0011] (III), The compound of formula (III) is: 3,4-dimethyl-N-[4-(5-methyl-1,3-benzoxazol-2-yl)phenyl]benzamide (3,4-Dimethyl-N-[4-(5-Methyl-1,3-Benzoxazol-2-Yl)Phenyl]Benzamide), its PubChem CID: 1365388, which is also named C2 compound in the present invention.

[0012] (IV), The compound of formula (IV) is: 3-[1,3-dioxo-5-(4-oxo-4H-3,1-benzoxazin-2-yl)-1,3-dihydro-2H-isoindol-2-yl]phenyl acetate (3-[1,3-Dioxo-5-(4-Oxo-4h-3,1-Benzoxazin-2-Yl)-1,3-Dihydro-2h-Isoindol-2-Yl]Phenyl Acetate), its PubChem CID: 1035007, which is also named C4 compound in the present invention.

[0013] (V), The compound of formula (V) is: N-(1,3-Benzodioxol-5-yl)-2-(3,4-dimethylphenyl)-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carboxamide (N-(1,3-Benzodioxol-5-Yl)-2-(3,4-Dimethylphenyl)-1,3-Dioxo-2,3-Dihydro-1H-Isoindole-5-Carboxamide), its PubChemCID: 1365998, which is also named C5 compound in the present invention.

[0014] (VI), The compound of formula (VI) is 5,5'-Bis[2-(3-acetylphenyl)-1H-isoindole-1,3(2H)-dione] (5,5'-Bis[2-(3-Acetylphenyl)-1h-Isoindole-1,3(2h)-Dione), with PubChem CID: 1089414. It is also named C6 compound in the present invention.

[0015] (VII) The compound of formula (VII) is: N-(2,4-dimethylphenyl)-4-(3-methyl-4-oxo-3,4-dihydro-1-phthalazinyl)benzamide (N-(2,4-Dimethylphenyl)-4-(3-Methyl-4-Oxo-3,4-Dihydro-1-Phthalazinyl)Benzamide), its PubChem CID: 1292350, which is also named C7 compound in the present invention.

[0016] (VIII), The compound of formula (VIII) is: 4-Nitro-2-[4-(4-Oxo-4h-3,1-Benzoxazin-2-yl)phenyl]-1h-isoindole-1,3(2h)-dione (4-Nitro-2-[4-(4-Oxo-4h-3,1-Benzoxazin-2-Yl)Phenyl]-1h-Isoindole-1,3(2h)-Dione), its PubChem CID: 1366027, which is also named C8 compound in the present invention.

[0017] (VIIII), The compound of formula (VIIII) is: 5-[5-[(Z)-(6,7-Dimethyl-1-Oxo-[1,3]Thiazolo[3,2-A]Benzimidazol-2-Ylidene)methyl]furan-2-yl]-2-hydroxybenzoic acid (5-[5-[(Z)-(6,7-Dimethyl-1-Oxo-[1,3]Thiazolo[3,2-A]Benzimidazol-2-Ylidene)Methyl]Furan-2-Yl]-2-Hydroxybenzoic Acid), its PubChem CID: 2256714, which is also named C10 compound in the present invention.

[0018] In a preferred embodiment, the group B is selected from the group having a structure as shown in any one of formulas (X) to (XXII): (X), Compound name: Cysteine-SC-cGMP (S-guanylationed Cys, CAS number: 2241669-85-0) (Takahashi D, Moriyama J, Nakamura T, Et Al. Autacs:Cargo-Specific Degraders Using Selective Autophagy. Mol Cell. 2019;76(5):797-810.e10.).

[0019] (XI), The compound is a p62 ligand (Takahashi D, Ora T, Sasaki S, Ishii N, Tanaka T, Matsuda T, Ikeda M, Moriyama J, Cho N, Nara H, Maezaki H, Kamaura M, Shimokawa K, Arimoto H. Second-Generation Autacs For Targeted Autophagic Degradation. J Med Chem. 2023 Sep 14;66(17):12342-12372. Doi: 10.1021 / Acs.Jmedchem.3c00861. Epub 2023 Aug 17. PMID: 37589438.), which belongs to the first generation of AUTAC (Autophagy-Targeting Chimera) autophagy ligands, and is coupled to PEG. The intermediate of the linker is HY-150408 (FBnG-(Cys-acetamide)-CH2-PEG3-CH2-CH2-CH2-NH2, CAS number: 2241669-84-9).

[0020] (XII), The compound is a p62 ligand (Takahashi D, Ora T, Sasaki S, Ishii N, Tanaka T, Matsuda T, Ikeda M, Moriyama J, Cho N, Nara H, Maezaki H, Kamaura M, Shimokawa K, Arimoto H. Second-Generation Autacs For Targeted Autophagic Degradation. J Med Chem. 2023 Sep 14;66(17):12342-12372. Doi: 10.1021 / Acs.Jmedchem.3c00861. Epub 2023 Aug 17. PMID: 37589438.), which belongs to the second-generation AUTAC autophagy ligand. The intermediate of its coupling PEG linker is HY-158985 (Amino-PEG3-2G degrader-1, CAS No.: 2267315-06-8).

[0021] (XIII), Recombinant strain(GW5074) of LC3B fragment(Li Z, Wang C, Wang Z, et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature.2019;575(7781):203-209.Fu Y, Chen N, Wang Z, Luo S, Ding Y, Lu B. Degradationof lipid droplets by chimeric autophagy-tethering compounds. Cell Res. 2021;31(9):965-9 Tan S, Wang D, Fu Y, Zheng H, Liu Y, Lu B. Targeted clearance of mitochondria by an autophagy-tethering compound (ATTEC) and its potentialtherapeutic effects. Sci Bull (Beijing). 2023;68(23):3013-3026.),Operator:C 15 H8Br2INO2 ,CASE:220904-83-6

[0022] (XIIII), The compound is an LC3B ligand (Li Z, Wang C, Wang Z, et al. Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds. Nature. 2019;575(7781):203-209. Fu Y, Chen N, Wang Z, Luo S, Ding Y, Lu B. Degradation oflipid droplets by chimeric autophagy-tethering compounds. Cell Res. 2021;31(9):965-979.Tan S, Wang D, Fu Y, Zheng H, Liu Y, Lu B. Targeted clearance ofmitochondria by an autophagy-tethering compound (ATTEC) and its potential therapeutic effects. Sci Bull (Beijing). 2023;68(23):3013-3026.), Name: 5,7-Dihydroxy-4-Phenylcoumarin, CAS No.: 7758-73-8.

[0023] (XV), The compound (YT-8-8) is a p62 ligand (Ji Ch, Kim Hy, Lee Mj, Et Al. TheAutotac Chemical Biology Platform For Targeted Protein Degradation Via TheAutophagy-Lysosome System [Published Correction Appears In Nat Commun. 2022Apr 12;13(1):2108. Doi: 10.1038 / S41467-022-29845-W.]. Nat Commun. 2022;13(1):904). CAS No.: 892572-23-5, PubChem CID: 4720656.

[0024] (XVI), The compound (YOK-1304) is a p62 ligand (Ji Ch, Kim Hy, Lee Mj, Et Al. TheAutotac Chemical Biology Platform For Targeted Protein Degradation Via TheAutophagy-Lysosome System [Published Correction Appears In Nat Commun. 2022Apr 12;13(1):2108. Doi: 10.1038 / S41467-022-29845-W.]. Nat Commun. 2022;13(1):904). CAS No.: 2409960-03-6, PubChem CID: 146309589.

[0025] (XVII), The compound (YOK-2204) is a p62 ligand (Ji Ch, Kim Hy, Lee Mj, Et Al. TheAutotac Chemical Biology Platform For Targeted Protein Degradation Via TheAutophagy-Lysosome System [Published Correction Appears In Nat Commun. 2022Apr 12;13(1):2108. Doi: 10.1038 / S41467-022-29845-W.]. Nat Commun. 2022;13(1):904). CAS No.: 2409959-99-3, PubChem CID: 152195557.

[0026] (XVIII), The compound is a p62 ligand (Ji Ch, Kim Hy, Lee Mj, Et Al. The AutotacChemical Biology Platform For Targeted Protein Degradation Via The Autophagy-Lysosome System [Published Correction Appears In Nat Commun. 2022 Apr 12;13(1):2108. Doi: 10.1038 / S41467-022-29845-W.]. Nat Commun. 2022;13(1):904), CAS No.: 2410081-72-8.

[0027] (XVIIII), The compound (Nutrin-3a) is an MDM2 ligand (Lai Ac, Crews Cm. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nat Rev Drug Discov. 2017;16(2):101-114. Doi:10.1038 / Nrd.2016.211), CAS No.: 675576-98-4 (DMSO).

[0028] (XX), The compound (bestatin) is a cIAP ligand (Lai Ac, Crews Cm. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nat Rev DrugDiscov. 2017;16(2):101-114. Doi:10.1038 / Nrd.2016.211), CAS No.: 58970-76-6.

[0029] (XXI), The compound is a Vhl ligand (VH032) (Lai Ac, Crews Cm. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nat Rev Drug Discov. 2017;16(2):101-114. Doi:10.1038 / Nrd.2016.211). CAS No.: 1448188-62-2.

[0030] (XXII), The compound (pomalidomide) is a CRBN ligand (Lai Ac, Crews Cm. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nat Rev DrugDiscov. 2017;16(2):101-114. Doi:10.1038 / Nrd.2016.211), CAS number: 19171-19-8.

[0031] In a preferred embodiment, the linker is selected from PEG, alkyl, polyol, alkynyl, triazole, piperazine, piperidine or a combination thereof.

[0032] More preferably, the linker is selected from one having the following structural formula: , Wherein, m and n are integers of 0-10.

[0033] Particularly preferably, the linker has a structure as shown in formula (XXIII), (XXIII), The compound is a linker, and the SMILES is CCCOCCOCCOCCCNC(CCC(NC)=O)=O, which plays the role of connecting the compound of formula (III) and the compound of formula (XII). It is an intermediate produced in the process of synthesizing the chimeric compound by the reaction of intermediate 3 (SMILES: O=C(CCC(NCC1=CC(C(NC2=CC=C(C3=NC4=C(C=CC(C)=C4)O3)C=C2)=O)=CC=C1C)=O) with HY-158985 (PubChem CID:142515353). The substrate of the synthesis reaction comes from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.

[0034] In one embodiment of the present invention, the bifunctional chimeric compound has a structure as shown in formula (XXIIII): 52 H 56 FN 11 O8): (XXIIII), The SMILES of the bifunctional chimeric compound is: NC1=NC(C2=C(N(CC3=CC=C(F)C=C3)C(C4=CN(N=C4)CCCOCCOCCOCCCNC(CCC(NCC5=C(C)C=CC(C(NC6=CC=C(C=C6)C7=NC8=C(O7)C=CC(C)=C8)=O)=C5)=O)=O)=O)=N2)N1)=O. The bifunctional chimeric compound (XXIIII) is chimerized by a TFB group having formula (III), a linker group having formula (XXIII) and an FB group having formula (XII).

[0035] Secondly, the present invention provides a method for preparing the above-mentioned bifunctional chimeric compound, which comprises the following steps: (1) 4-(tert-Butoxy)-4-oxobutanoic acid was dissolved in dimethylformamide (DMF), and TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) and N,N-diisopropylethylamine (DIEA) were added under stirring. The resulting mixture was reacted at 20-30°C to obtain tert-butyl succinimidyl succinate; (2) Dissolve tert-butyl diimidosuccinate in DMF, add G74437 and DIEA, and stir the resulting mixture at 20°C to 30°C to obtain intermediate 1; (3) Dissolve intermediate 1 in anhydrous acetonitrile, add F75622, N-methylimidazole (NMI), and TCFH (tetramethylformamidine hexafluorophosphate) in sequence under stirring, and stir the resulting mixture at 20°C-30°C to obtain intermediate 2; (4) Dissolve intermediate 2 in anhydrous dichloromethane (DCM), add trifluoroacetic acid (TFA), and stir at 20°C~30°C to obtain intermediate 3; (5) The intermediate 3 was dissolved in DMF, and HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine hexafluorophosphate), DIEA, and HY-158985 were added under stirring. The mixture was stirred at 20°C to 30°C to obtain the bifunctional chimeric compound.

[0036] Furthermore, given that the bifunctional chimeric compound provided by the present invention has excellent dual functions of binding to and disaggregating tau protein aggregates, the present invention provides the use of the above-mentioned bifunctional chimeric compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing diseases related to tau protein.

[0037] In a preferred embodiment, the diseases associated with tau protein include Alzheimer's disease, frontotemporal dementia, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, argyrophilic grain disease, aging-related tau astrocytopathy, chronic traumatic encephalopathy, spheroidal glial tauopathy, Parkinson's disease, Huntington's disease, stroke, epilepsy, and cancer.

[0038] Finally, the present invention provides the use of a compound having a structure as shown in any one of Formulas (II)-(VIIII), or a pharmaceutically acceptable salt thereof, in the preparation of a drug for treating and / or preventing diseases associated with tau protein. Compounds having a structure as shown in any one of Formulas (II)-(VIIII) are already disclosed in the prior art and are used in various technical fields. The present invention has discovered that these compounds have excellent affinity for tau protein aggregates. Therefore, they can not only be used to construct the bifunctional chimeric compounds provided by the present invention, but can also be used independently to prepare drugs that specifically bind to tau protein aggregates to treat and / or prevent diseases associated with tau protein.

[0039] The above drugs can be administered through nasal inhalation, oral inhalation, sublingual administration, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, transcutaneous injection, intraperitoneal injection, epidural injection, intrathecal injection, intracerebral injection, and local application to other parts of the body.

[0040] The present invention provides a small molecule monomer that can specifically recognize and bind to tau protein aggregates in the human body and a bifunctional small molecule chimera constructed based on the small molecule monomer, which can inhibit the formation of tau protein aggregates and promote the clearance of pathological tau protein aggregates. It is used in the prevention and treatment of a large class of diseases with abnormal tau protein aggregation, such as AD and FTD. Its advantages are: (1) It specifically targets tau protein aggregates without affecting monomeric tau protein, has better pathological specificity, and has lower expected toxic side effects. (2) It is a small molecule with better specificity for passing through the blood-brain barrier. It is easier to enter the brain through peripheral administration to exert therapeutic effects. It is not easily degraded in the body and can exert its effects more continuously and stably. (3) As a small molecule compound, it has better stability in the environment and is easier to store and transport than biological macromolecules such as proteins and peptides, which have strict requirements for environmental cleanliness and low temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1Schematic diagram of the preparation route of bifunctional chimeric compounds; Figure 2 . HNMR detection spectrum of bifunctional chimera compound; Figure 3 SPR binding curve of small molecule monomer (TFB); Figure 4 SPR binding curves of C2 and its chimeric compounds with tau protein monomers; Figure 5 SPR binding curves of the chimeric compound and tau protein aggregates; Figure 6 Electron micrographs showing the chimera inhibiting tau protein aggregation and statistical graphs showing the reduction in tau fiber length; Figure 7 . Electron microscopic images and fluorescence intensity statistics showing that the chimera reduces the level of tau aggregates in neurons. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0043] The TFB group small molecule monomers C1, C2, C4, C5, C6, C7, C8, and C10 disclosed in the present invention that can specifically bind to tau protein aggregates are all commercially available: C1: STK764160, PubChem CID: 1516497, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number 8011-4139.

[0044] C2: STK070666, PubChem CID: 1365388, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number 3878-0618.

[0045] C4: STK087082; PubChem CID: 1035007, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number 8004-9036.

[0046] C5: STK295815; PubChem CID: 1365998, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number STK295815.

[0047] C6: STK044853; PubChem CID: 1089414, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number STK044853.

[0048] C7: STK263772; PubChem CID: 1292350, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number STK263772.

[0049] C8: STK156461; PubChem CID: 1366027, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number STK156461.

[0050] C10: 5-[5-[(Z)-(6,7-dimethyl-1-oxo-[1,3]thiazolo[3,2-a]benzimidazol-2-ylidene)methyl]furan-2-yl]-2-hydroxybenzoic acid; PubChem CID: 2256714, purchased from Shanghai Taosu Biochemical Technology Co., Ltd., catalog number 5670901.

[0051] The FB groups disclosed in the present invention that can recruit p62, LC3B or ubiquitin ligase components MDM2, cIAP, VHL, and CRBN required for cellular autophagy formation and promote the clearance of tau protein aggregates through cellular autophagy or ubiquitin-proteasome pathways can all be commercially obtained.

[0052] The preparation of linker was completed by Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., and the synthetic substrates involved were provided by the company. The synthesis methods and conditions are as follows: Figure 1 shown.

[0053] Example 1. Preparation of chimeras The preparation of the chimera of formula (XXIII) was completed by Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd. The synthetic substrates and conditions involved were provided by the company. The synthetic route is as follows Figure 1 The steps are briefly described as follows: (1) 4-(tert-butoxy)-4-oxobutanoic acid (chemical formula C8H 14 O4) (5.0 g, 28.7 mmol, 1.0 eq.) was dissolved in 80 mL DMF (dimethylformamide, N,N-dimethylformamide, chemical formula C3H7NO), and TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate, chemical formula C9H) was added under stirring. 16BF4N3O3) (9.06 g, 30.1 mmol, 1.05 eq.) and DIEA (N,N-diisopropylethylamine, chemical formula C8H 19 N) (7.42 g, 57.4 mmol, 2.0 eq.), the mixture was reacted at 20-30°C for 1 hour to obtain tert-butyl succinimidylsuccinate (chemical formula C 12 H 17 NO6), yield 42%.

[0054] (2) Tert-butyl diimidosuccinate (673 mg, 2.48 mmol, 1.0 eq.) was dissolved in DMF (30 mL), and G74437 (500 mg, 2.48 mmol, 1.0 eq.) and DIEA (801 mg, 6.20 mmol, 2.5 eq.) were added. The mixture was stirred at 20°C–30°C for 1 hour to obtain intermediate 1 in a yield of 95%.

[0055] (3) Intermediate 1 (300 mg, 0.934 mmol, 1.0 eq.) was dissolved in anhydrous acetonitrile (CH3CN, 150 mL), and F75622 (220 mg, 0.981 mmol, 1.05 eq.), NMI (N-Methylimidazole, chemical formula C4H6N2) (138 mg, 1.68 mmol, 1.8 eq.), TCFH (Chloro-N,N,N',N'-tetramethylformamidinium Hexafluorophosphate, chemical formula C5H 12 C l F6N2P) (471 mg, 1.68 mmol, 1.8 eq.), the mixture was stirred at 20°C-30°C for 2 h to obtain intermediate 2 in a yield of 59%.

[0056] (4) Intermediate 2 (290 mg, 0.550 mmol, 1.0 eq.) was dissolved in anhydrous DCM (dichloromethane, chemical formula CH2Cl2) (6 mL), and TFA (trifluoroacetic acid, chemical formula C2HF3O2) (3 mL) was added. The mixture was stirred at 20℃~30℃ for 2 hours to obtain intermediate 3 with a yield of 77%.

[0057] (5) Intermediate 3 (38.0 mg, 0.0805 mmol, 1.0 eq.) was dissolved in DMF (5 mL) and HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridiniumhexafluorophosphate, chemical formula C) was added under stirring. 10 H 15 F6N6OP) (32.1 mg, 0.0845 mmol, 1.05 eq.), DIEA (31.3 mg, 0.242 mmol, 3.0 eq.), and HY-158985 (50 mg, 0.0885 mmol, 1.1 eq.) were stirred at 20°C–30°C for 1 hour to obtain the final chimera as an off-white solid in a yield of 38%.

[0058] The chimera is an off-white solid with a purity of 98.7469%. HNMR detection shows that the peak shifts of each H in the chimera molecule are as follows: Figure 2 The NMR spectrum data are as follows: 1 H NMR (400 MHz, DMSO- d6) δ 11.02 (s, 1H), 10.53 (s, 1H), 8.34 (t, J =5.7 Hz, 1H), 8.22 – 8.14 (m, 3H), 8.07 – 8.01 (m, 2H), 7.87 – 7.80 (m, 4H),7.66 – 7.60 (m, 1H), 7.57 (s, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.21 (d, J = 8.3Hz, 1H), 7.17 – 7.10 (m, 4H), 6.76 (s, 2H), 5.41 (s, 2H), 4.31 (d, J = 5.5Hz, 2H), 4.16 (t, J = 6.9 Hz, 2H), 3.43 – 3.42 (m, 2H), 3.40 – 3.36 (m, 5H), 3.34 – 3.24 (m, 5H), 3.01 (dd, J = 12.8, 6.6 Hz, 2H), 2.45 – 2.40 (m, 5H), 2.39 – 2.32 (m, 5H), 1.95 (p, J = 6.4 Hz, 2H), 1.54 (p, J = 6.6 Hz, 2H).

[0059] Example 2. SPR detection and analysis method: The Biacore 8K+ biomolecular interaction instrument was used to analyze the interaction between target tau aggregates and screening small molecules using surface plasmon resonance. The main steps were as follows: A protein well on a CM5 sensor chip was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at a rate of 10 mL / min for 10 minutes. Tau aggregates were then mixed with sodium acetate solution (pH 4.0) and immobilized on the surface of the protein well at a rate of 10 mL / min for 3 minutes. The well was then blocked with ethanolamine (10 mL / min for 10 minutes). An adjacent well serving as a reference was similarly activated and blocked. Both wells were then equilibrated with buffer. Different TFB small molecule or chimera stock solutions were diluted to a concentration range of 0.05, 0.1, 0.19, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μM, and flowed at 30 mL / min for 180 s per run. At the end of each flow, the chip's protein reservoir was regenerated with 10 mM acetic acid-hydrochloric acid solution (pH 1.5) at 30 mL / min. Data from the sample reservoir were collected using Biacore 8K+ Control Software (GE Healthcare) and subtracted from the data from the reference reservoir. The data were globally fitted to a 1:1 Langmuir binding model using Biacore 8K+ Evaluation Software (GE Healthcare) to obtain association and dissociation constants.

[0060] In the SPR detection of 8 small molecule monomers (TFBs), different small molecule monomers can bind to tau protein aggregates. Small molecules at varying concentrations were incubated with purified human tau fibrils (a form of tau aggregates) in vitro. Surface plasmon resonance (SPR) experiments revealed that the steady-state affinity (Kd) values of different TFBs for tau fibrils were: C1: 0.98 μM, C2: 10.7 μM, C4: 9.63 μM, C5: 12.2 μM, C6: 10.7 μM, C7: 1.25 μM, C8: 0.2 μM, and C10: 0.58 μM. The absolute values of the relative response amplitudes showed that at a concentration of 50 μM, the response amplitudes were C10>C2>C8 ≈ C7>C4>C5≈C1>C6. The stability of the affinity increase with increasing concentration indicated that C2>C1>other compounds. Therefore, C2, C10, C8, C4, and C1 all showed good affinity for tau, among which C2 showed the most stable trend of affinity increasing with concentration, followed by C6, C4, and C5, which also had a certain affinity with tau aggregates (see Figure 3 ), based on comprehensive judgment, C2 is preferred for constructing chimeric compounds.

[0061] Correspondingly, C2 molecules and chimeras have no significant affinity for tau protein monomers (see Figure 4 ).

[0062] Based on the same detection conditions and methods, the chimera (XXIIII) constructed based on C2 also showed good affinity to tau aggregates, with a steady-state Kd value of 0.21 μM (see Figure 5 ), with affinity better than C2 monomer.

[0063] Example 3. Transmission electron microscopy observation of tau fiber formation and disaggregation Different concentrations of chimeras were incubated with tau aggregates in vitro for 48 hours, and tau fiber formation and disaggregation were observed under transmission electron microscopy.

[0064] Tau-P301S fibrils and chimeras (final reaction concentrations of 0, 10, 20, 40, and 80 μg) were mixed in a pre-prepared 10 mM 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and incubated at 37°C for 48 hours. Samples were then collected for transmission electron microscopy (TEM) preparation. Six microliters of sample were dripped onto glow-discharged carbon-coated TEM grids (CF150-Cu, Electron Microscopy Sciences) and allowed to stand for 5 minutes. The grids were then negatively stained with 2% uranyl acetate for 2 minutes. Observations and images were taken using a FEI Tecnai T12 Quick room-temperature transmission electron microscope equipped with a Gatan 2048×2048 charge-coupled detector camera operating at 120 kV. The length of each tau fibril within the field of view was automatically calculated using ImageJ software. For the tau fiber length between different groups, one-way analysis of variance plus Tukey's multiple comparison test was used to compare the differences between groups. p Values less than 0.05 were considered to be significantly different.

[0065] The results showed that compared with the same dose of DMSO solvent, the chimera at a concentration of 10-80 μM could significantly reduce the length of tau fibers, proving that the chimera can effectively inhibit tau protein aggregation (see Figure 6 The mean (variance) of tau fiber length in each group was: 67.41 (8.65) nm in the DMSO control group, 55.10 (11.86) nm in the 10 μM group, 49.23 (6.83) nm in the 20 μM group, 48.25 (5.70) nm in the 40 μM group, and 46.56 (6.72) nm in the 80 μM group.

[0066] Example 4. Effect of the chimera on intracellular tau aggregate levels in neurons of rats overexpressing human tau-P301S In primary cultured rat hippocampal neurons overexpressing human tau-P301S, preformed human tau fibrils were added to induce the formation of tau aggregates in neurons. 3 μM chimeras were then added to treat the cells for 72 hours. After fixation, tau aggregates were immunofluorescently stained using AT8 antibody (an antibody that specifically recognizes tau aggregates), and the immunostaining fluorescence intensity was calculated.

[0067] Detection and analysis methods: Pregnant SD mice of gestational age of 12 to 15 days were obtained. After anesthesia with 1% sodium pentobarbital, the abdominal wall was disinfected, the peritoneum and uterus were opened, the fetuses were removed and placed on ice, and the mother mice were killed by cervical dislocation. The fetuses were rinsed with normal saline and soaked in 75% ethanol for 1 minute for disinfection. The skull was cut open with sterile instruments in a clean bench to remove the brain tissue and immersed in DMEM / F12 culture medium. The brain tissue was then placed on sterile wet filter paper to remove the cerebellum and separate the cerebral hemispheres. The cortex was turned over to expose the hippocampus and the meninges and blood vessels were removed. The hippocampus tissue was minced and digested with an equal volume of 0.25% trypsin (final concentration 0.125%) at 37°C for 20 minutes, gently shaking three times during the period. After digestion, the digestion was stopped with DMEM / F12 containing 10% FBS. After standing on ice, the cell suspension was obtained by pipetting 30 times with a cut pipette and filtered. The cells were plated on polylysine-coated culture plates and first incubated with 10% The cells were cultured in DMEM / F12 with FBS for 4–6 hours, then fully replaced with neuronal culture medium (97% Neurobasal-A, 2% B27, 1% GlutaMAX, 1% penicillin-streptomycin). After 3–4 days, half of the medium was replaced and the cells were observed daily. Lentivirus overexpressing human tau-P301S was added to the culture system for infection. Five days later, preformed human tau fibrils were added to the cultured neurons for 12 hours to induce intracellular tau aggregates. The cells were then treated with 3 μM chimeras for 72 hours, fixed, and harvested. Tau aggregates were immunofluorescently stained using AT8 antibodies on cell slides. After sealing, the slides were photographed using a two-photon laser confocal microscope (AXR, Nikon). ImageJ software was used to automatically calculate the fluorescence intensity of AT8 immunostaining. One-way analysis of variance with Tukey's multiple comparison test was used to compare differences between groups. p Values less than 0.05 were considered to be significantly different.

[0068] The chimera was found to significantly reduce the level of intracellular tau aggregates in neurons (see Figure 7 The mean (variance) of AT8 immunofluorescence intensity in each group was: 39.42 (8.86) in the DMSO control group and 32.11 (8.96) in the chimera group.

Claims

1. A bifunctional chimeric compound that specifically binds to tau protein aggregates, characterized in that The compound has a structure as shown in formula (I): A-linker-B (I), Among them, A is a disaggregation group that specifically binds to pathological tau protein aggregates and promotes the disaggregation of pathological tau protein aggregates, B is a clearance group that recruits p62, LC3B or ubiquitin ligase components MDM2, cIAP, VHL, CRBN required for cellular autophagy formation, thereby promoting the formation of tau protein aggregates through cellular autophagosomes or clearance through the ubiquitin-proteasome pathway, and the linker is a connecting group that connects group A and group B.

2. The bifunctional chimera compound according to claim 1, characterized in that The group A is selected from a structure as shown in any one of formulas (II) to (VIIII): (II), (III), (IV), (V), (VI), (VII) (VIII), (VIII).

3. The bifunctional chimera compound according to claim 1, characterized in that The group B is selected from a structure as shown in any one of formulas (X) to (XXII): (X), (XI), (12), (XIII), (XIII), (15), (XVI), (XVII), (18), (XVIII), (XX), (XXI), (XXII)。 4. The bifunctional chimera compound according to claim 1, characterized in that The linker is selected from PEG, alkyl, polyol, alkynyl, triazole, piperazine, piperidine or a combination thereof.

5. The bifunctional chimera compound according to claim 4, characterized in that The linker is selected from one of the following structural formulas: , Wherein, m and n are integers of 0-10.

6. The bifunctional chimera compound according to claim 5, characterized in that The linker has a structure as shown in formula (XXIII), (XXIII)。 7. The bifunctional chimera compound according to claim 1, characterized in that The bifunctional chimeric compound has a structure as shown in formula (XXIIII): (XXIII), The SMILES of the bifunctional chimeric compound is: NC1=NC(C2=C(N(CC3=CC=C(F)C=C3)C(C4=CN(N=C4)CCCOCCOCCOCCCNC(CCC(NCC5=C(C)C=CC(C(NC6=CC=C(C=C6)C7=NC8=C(O7)C=CC(C)=C8)=O)=C5)=O)=O)=N2)N1)=O.

8. A method for preparing the bifunctional chimera compound according to claim 7, characterized in that: The method comprises the following steps: (1) 4-(tert-Butoxy)-4-oxobutanoic acid was dissolved in DMF, TSTU and DIEA were added under stirring, and the resulting mixture was reacted at 20-30°C to obtain tert-butyl succinimidyl succinate; (2) Dissolve tert-butyl diimidosuccinate in DMF, add G74437 and DIEA, and stir the resulting mixture at 20°C to 30°C to obtain intermediate 1; (3) Intermediate 1 was dissolved in anhydrous CH3CN, and F75622, NMI, and TCFH were added in sequence under stirring. The resulting mixture was stirred at 20°C-30°C to obtain intermediate 2; (4) Dissolve intermediate 2 in anhydrous DCM, add TFA, and stir at 20°C~30°C to obtain intermediate 3; (5) The intermediate 3 was dissolved in DMF, and HATU, DIEA, and HY-158985 were added under stirring. The mixture was stirred at 20°C to 30°C to obtain the bifunctional chimeric compound.

9. Use of the bifunctional chimeric compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing diseases associated with tau protein.

10. The use according to claim 9, characterized in that The diseases associated with tau protein include Alzheimer's disease, frontotemporal dementia, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary age-related tauopathy, argyrophilic grain disease, aging-related tau astrocytopathy, chronic traumatic encephalopathy, spheroidal glial tauopathy, Parkinson's disease, Huntington's disease, stroke, epilepsy, and cancer.

11. Use of a compound having a structure as shown in any one of formulae (II) to (VIIII) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing diseases associated with tau protein.

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