Autophagy-activating compound targeting tau protein

Abstract

The present disclosure relates to a compound that targets tau and induces or activates autophagy, a preparation method therefor, and uses thereof More specifically, the present invention relates to a compound that targets tau protein and promotes the formation of autophagosomes via lysosomal pathways, a preparation method therefor, and a treatment of tau protein-related diseases using same.

Description

TAU protein-targeting autophagy-activating compound

[0001] The present invention relates to compounds that target tau and activate autophagy, methods for preparing the same, and uses thereof. More specifically, the present invention relates to compounds that target tau protein and induce the formation of lysosome-mediated autophagosomes, methods for preparing the same, and the use thereof for the treatment of tau protein-related diseases.

[0002]

[0003] Many diseases of aging are associated with extracellular or intracellular deposits of amyloid or amyloid-like proteins. The best-characterized amyloid protein that forms extracellular aggregates is amyloid beta (Aβ). Other examples of amyloid proteins that form extracellular aggregates include prions, transthyretin (ATTR), or ADan (ADanPP). Meanwhile, amyloid-like proteins that primarily form intracellular aggregates include tau, alpha-synuclein, TAR DNA-binding protein 43 (TDP-43), and huntingtin (htt).

[0004] Amyloid or amyloid-like deposits are formed by protein misfolding and their aggregation, forming β-sheet assemblies of multiple peptides or proteins held together by intermolecular hydrogen bonds. Although amyloid or amyloid-like proteins have different primary amino acid sequences, their deposits often contain many shared molecular components and form a characteristic β-sheet quaternary structure. The link between amyloid deposits and disease remains largely unclear, but a wide range of protein aggregates, both associated with and unassociated with disease pathology, have been shown to be toxic, suggesting that common molecular features of amyloids are involved in or responsible for disease pathogenesis. Various multimers of β-sheet-aggregated peptides or proteins are also associated with toxicity for different peptides or proteins, ranging from dimers to soluble low-molecular-weight oligomers, protofibrils, or insoluble fibrillar deposits.

[0005] Previous studies have attempted to remove specific Abs based on the hypothesis that amyloid plaque accumulation is the underlying mechanism of AD and that removing Ab oligomers or fibrils could halt or reverse the disease. For example, studies have been conducted on Ab antibodies as modalities for removing Ab oligomers or fibrils, and aducanumab and recanemab have been approved by the FDA for their ability to slow cognitive decline. However, their clinical efficacy has primarily been demonstrated in early AD with mild cognitive impairment, and they are associated with adverse effects, including cerebral hemorrhage and edema. Therefore, further research is needed to understand the efficacy and side effects of anti-Ab antibodies.

[0006] In addition to Ab, tau is also known as a key target. Indeed, tau-specific genetic deletion or tau immunization can ameliorate clinical and pathological abnormalities in AD animal models, highlighting the crucial role of tau in AD pathogenesis. Furthermore, recent studies suggest a crucial role for lysosomal dysfunction in the pathogenesis of AD and a crucial mechanism for tau in lysosomal damage associated with AD.

[0007] Tau is a freely soluble, "naturally unfolded" protein that tightly binds to microtubules (MTs), facilitating their assembly and stability. MTs are crucial for the integrity of the neuronal cytoskeleton, and thus for the proper formation and function of neuronal circuits, and thus for learning and memory. Tau dissociates from microtubules due to hyperphosphorylation, and hyperphosphorylated tau can aggregate and cause neurotoxicity. Accumulated hyperphosphorylated tau is termed tau tangle or neurofibrillary tangles (NFTs). Tau and NFTs are toxic inside cells, and the impaired function of microtubules, caused by the dissociation of tau, can also lead to cell death. Diseases involving tau aggregates are generally associated with tauopathies, most notably Alzheimer's disease (AD) or Parkinson's disease (PD). Higher levels of non-transportable fibers (NFTs) are found in the brains of Alzheimer's disease patients compared to healthy individuals. The tau hypothesis, which posits that neuronal cell death due to toxicity of tau protein and NFTs is the primary cause of Alzheimer's disease, is clinically recognized. For this reason, tau protein is considered a diagnostic biomarker for Alzheimer's disease. Therefore, research is being conducted to inhibit tau aggregates by applying lysosome-based targeted protein degradation technology to tau protein to treat and prevent diseases involving tau aggregates.

[0008] Meanwhile, there are two main pathways for protein degradation: ubiquitin-proteasome degradation and autophagy-lysosome degradation. While soluble proteins can be removed by both pathways, aggregated or amyloid proteins are known to be preferentially removed by the autophagy-lysosome pathway. Most pathogenic proteins that accumulate in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, or amyloid-like proteins are aggregated or amyloid-like proteins, and promoting the removal of Ab or Tau oligomers is inevitably required. Therefore, the prevention or treatment of Alzheimer's disease is being carried out through the development of new drugs or medications.

[0009] One object of the present invention is to provide a tau-specific autophagy degradation compound comprising a tau protein binding moiety, a linker, and a lysosomal degradation tag moiety. The compound is characterized by targeting the tau protein and inducing and activating autophagy.

[0010] Another object of the present invention is to provide a method for producing the compound.

[0011] Another object of the present invention is to provide a use of the compound for the prevention or treatment of tauopathy. For example, the tauopathy may be, but is not limited to, Alzheimer's disease or Huntington's disease.

[0012] In addition, another object of the present invention is to provide a use of the compound for preventing or treating cognitive dysfunction or neuroinflammation.

[0013] The problems to be solved by the present invention are not limited to the purposes mentioned above. Other unmentioned objectives and advantages of the present invention can be understood through the following description and will be more clearly understood through the embodiments of the present invention. Furthermore, it will be readily apparent that the problems to be solved and advantages of the present invention can be realized by the means and combinations thereof set forth in the claims.

[0014] To achieve the above purpose, the present inventors have completed the present invention by discovering a novel AUTAC compound that specifically acts on abnormal cells in which tau protein is overexpressed or accumulated, thereby inducing effective degradation of tau protein and minimizing side effects by removing tau protein through appropriate structural combination and optimization of a tau binding site, a lysosomal degradation tag site, and a linker.

[0015]

[0016] Tau-specific degradation-inducing compounds

[0017] The present invention provides compounds that specifically bind to a target protein and activate a lysosomal target protein degradation pathway. The target protein may be an amyloid or amyloid-like protein. As an example, the target protein may be a tau protein. The "tau protein" may include any of a naturally occurring form of the tau protein, a homolog, or a variant that retains protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the native protein). For example, a tau variant or homologue may have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a segment of 50, 100, 150, or 200 contiguous amino acids) compared to a naturally occurring form. For example, the tau protein may be a protein identified by UniProt: P10636 or a functional fragment thereof. The tau protein may be a free soluble tau protein. The tau protein may be a monomeric tau protein. The tau protein may be an oligomeric tau protein. The tau protein may be an aggregated tau protein. The tau protein may be an acetylated tau helix (PHF) or a complex of the tau protein and the tau protein. The tau protein may be directly or indirectly associated with another disease-causing protein. For example, the tau protein may be directly or indirectly associated with amyloid beta protein.

[0018] The above target protein degradation by lysosomes is a method in which the lysosomal degradation pathway is utilized for protein degradation, and may be according to LYTAC (Lysosome-Targeting Chimeras) or AUTAC (Autophagy-Targeting Chimeras).

[0019] In one embodiment, the present invention provides a compound that specifically binds to tau protein and activates a lysosomal target degradation pathway. The compound may comprise a tau binding moiety, a linker moiety, and a lysosomal degradation tag moiety. The compound may have the tau binding moiety and the lysosomal degradation tag moiety chemically linked by a linker moiety.

[0020] [Correction under Rule 91 08.04.2025] [Tau binding site] - [Linker site] - [Lysosomal degradation tag site]

[0021] The above tau binding moiety may include a compound that specifically binds to the tau protein. For example, the tau protein binding moiety may be leucomethylene blue (LMB) of the following [chemical formula i] or a derivative or analog thereof.

[0022] [chemical formula i]

[0023]

[0024] For example, the tau protein binding site may be methylene blue (MB) of the following [chemical formula ii] or a derivative or analog thereof.

[0025] Below [chemical formula ii]

[0026]

[0027] The above linker portion may connect the tau binding portion and the lysosomal degradation tag portion.

[0028] According to one embodiment, the linker portion may be a compound of the following [chemical formula iii] or [chemical formula iv].

[0029] [Chemical formula iii]

[0030] H2N-Y-NH2

[0031] Here Y is -C 1-6 Alkyl-, -C 1-6 Alkyl-OC 1-6It may be alkyl- or cycloalkyl. For example, Y is -C 1-4 Alkyl-, -C 1-4 Alkyl-OC 1-4 Alkyl- or 3-8 membered cycloalkyl. For example, Y can be -(CH2)3-, -(CH2)4-, -(CH2)2-O-(CH2)2- or cyclohexyl.

[0032] [chemical formula iv]

[0033]

[0034] The lysosomal degradation tag moiety may induce degradation of the tau protein through the endosome-lysosomal pathway. The lysosomal degradation tag moiety may induce degradation of the tau protein through the autophagy-lysosomal pathway. The lysosomal degradation tag moiety may be a cGMP-based degradation tag. The lysosomal degradation tag may be an S-guanylation lysosomal degradation tag. For example, the lysosomal degradation tag moiety may be a compound of the following [chemical formula v].

[0035] [chemical formula v]

[0036]

[0037] In this specification, the compound of formula v is also referred to as p-fluorobenzyl guanine tag or FBnG tag.

[0038] According to one embodiment of the present invention, the tau-specific autophagy-inducing degradation compound of the present invention may be a compound of [chemical formula I] having the following structure.

[0039] [Chemical Formula I]

[0040]

[0041] Here, X is -NH-Y-NH- or and,

[0042] Y is -C 1-6 Alkyl-, -C 1-6 Alkyl-OC1-6 It can be alkyl- or cycloalkyl.

[0043] For example, the above Y is -C 1-4 Alkyl-, -C 1-4 Alkyl-OC 1-4 Alkyl- or 3-8 membered cycloalkyl. For example, Y can be -(CH2)3-, -(CH2)4-, -(CH2)2-O-(CH2)2- or cyclohexyl.

[0044] According to one specific example, the compound of formula I may be a compound of the following [Chemical Formula 1]. In the present specification, the compound of [Chemical Formula 1] is also referred to as TA-1.

[0045]

[0046] According to another specific example, the compound of formula I may be a compound of the following [chemical formula 2]. In the present specification, the compound of [chemical formula 2] is also referred to as TA-2.

[0047]

[0048] According to another specific example, the compound of formula I may be a compound of the following [chemical formula 3]. In the present specification, the compound of [chemical formula 3] is also referred to as TA-3.

[0049]

[0050] According to another specific example, the compound of formula I may be a compound of the following [chemical formula 4]. In the present specification, the compound of [chemical formula 4] is also referred to as TA-4.

[0051]

[0052] According to another embodiment of the present invention, the compound of formula I may be a compound of the following [chemical formula 5]. In the present specification, the compound of [chemical formula 5] is also referred to as TA-5.

[0053] [Chemical Formula 5]

[0054]

[0055]

[0056] Method for preparing a tau-specific degradation-inducing compound

[0057] The present invention provides a method for preparing a compound I that specifically binds to a target protein and activates a target protein degradation pathway by lysosomes.

[0058] According to one embodiment,

[0059] A first step of obtaining a compound of the following chemical formula vi by adding triphosgene and sodium carbonate to a compound of the following chemical formula i and performing a carbamoylation reaction;

[0060] [chemical formula i]

[0061]

[0062] [chemical formula vi]

[0063]

[0064] A second step of obtaining a compound of the following chemical formula vii or viii by subjecting the compound of the above chemical formula vi to a nucleophilic substitution reaction with a compound of the following chemical formula iii or iv;

[0065] [Chemical formula iii]

[0066] H2N-Y-NH2

[0067] [chemical formula iv]

[0068]

[0069] [chemical formula vii]

[0070]

[0071] [chemical formula viii]

[0072]

[0073] and

[0074] A step of obtaining a compound of formula I by subjecting the compound of formula vii or formula viii to a peptide coupling reaction with a compound of formula v:

[0075] [chemical formula v]

[0076]

[0077] [Chemical Formula I]

[0078]

[0079] A method for preparing a compound of formula I including is provided.

[0080] In the compound of the above manufacturing method, X and Y are as described above, and R is the same as the definition of Y.

[0081]

[0082] In one embodiment, the method may comprise the steps of: a) reacting a 3,7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl chloride compound (compound 7 of FIG. 1B) with the compound of formula iii; and a) reacting the compound prepared in a) with N-acetyl-S-(2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)-L-cysteine ​​(compound 6 of FIG. 1A).

[0083] In one embodiment, the method may comprise the steps of: a) reacting a 3,7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl chloride compound (compound 7 of FIG. 1B) with the compound of formula iv; and a) reacting the compound prepared in a) with N-acetyl-S-(2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)-L-cysteine ​​(compound 6 of FIG. 1A).

[0084]

[0085] In one embodiment, the present invention provides a compound prepared according to the above preparation method.

[0086]

[0087] Uses of Tau-Specific Degradation-Inducing Compounds

[0088] The compound represented by the above chemical formula I according to the present invention can effectively remove tau protein from a subject.

[0089] The term "subject," as used herein, refers to an individual organism, such as an individual mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a non-human mammal, such as, but not limited to, a sheep, goat, cow, cat, or dog.

[0090] Specifically, the compounds of the present invention remove tau proteins by inducing, activating, or mediating tau protein autophagy. Examples of such tau proteins are described above. The compounds of the present invention selectively bind to tau proteins and mediate tau protein removal by activating the lysosomal degradation pathway. Therefore, the compounds of the present invention improve tau protein-related lysosomal dysfunction. Furthermore, the compounds of the present invention can reduce tau-mediated lysosomal stress by removing tau proteins. The compounds of the present invention prevent tau protein accumulation or remove accumulated tau proteins. Furthermore, the compounds of the present invention improve lysosomal dysfunction or reduce lysosomal stress. Furthermore, the compounds of the present invention improve neurological deficits or disorders by reducing lysosomal stress. For example, the compounds of the present invention reduce amyloid or amyloid beta accumulation by improving lysosomal dysfunction.

[0091] In one embodiment, the compound is provided to maintain or increase cognitive memory capacity in a subject, or to slow memory loss.

[0092] In one embodiment, the compound is provided to reduce the level of tau protein, non-phosphorylated tau protein, phosphorylated tau protein, or hyperphosphorylated tau protein in a subject.

[0093]

[0094] The present invention provides a pharmaceutical composition for preventing, improving or treating tauopathy comprising the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0095] Diseases involving tau aggregates are generally listed as tauopathies, which include Alzheimer's disease (AD), familial AD, PART (primary age-related tauopathy), Creutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease (GSS), inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), amyotrophic lateral sclerosis (ALS), Guam type parkinson-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, arginine particle disease, corticobasal degeneration (CBD), diffuse neurofibrillary tangles with calcification, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Hallervorden-Spatz disease, multiple system atrophy (MSA), Niemann-Pick disease type C, globus-pontino-substantia nigra degeneration, and Pick's disease. (PiD), progressive subcortical gliosis, progressive supranuclear palsy (PSP), subacute sclerosing panencephalitis, tangle-predominant dementia, postencephalitic parkinsonism, myotonic dystrophy, subacute sclerosis panencephalopathy, mutations in LRRK2, chronic traumatic encephalopathy (CTE), familial British dementia, familial Danish dementia, other frontotemporal lobar degenerations, Guadeloupe parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-associated mental retardation, leukoencephalopathy with globular glial inclusions, epilepsy, dementia with Lewy bodies (LBD), mild cognitive impairment (MCI), multiple sclerosis, Parkinson's disease, HIV-associated dementia, adult-onset diabetes, senile cardiac amyloidosis, glaucoma, ischemic stroke, AD psychosis, and Huntington's disease.

[0096] In some embodiments, the tauopathy is a neurodegenerative tauopathy. In some embodiments, the tauopathy is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis / parkinsonism-dementia complex of Guam, non-Guamanian motor neuron disease with neurofibrillary tangles, arginine granulomatous dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotetemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-Ponto-Nigral degeneration, Pick's disease, It may be progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic parkinsonism, or myotonic dystrophy.

[0097] In addition, the present invention provides a pharmaceutical composition for preventing, improving or treating cognitive dysfunction or neuroinflammation, comprising the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0098] As used herein, the terms "treat," "treating," and "treatment" refer to any action that provides benefit to a patient to whom the compound is administered, including treatment of any disease state or condition modulated by a protein to which the compound binds. Disease states or conditions that can be treated using the compounds of the present invention, including neurological and neurodegenerative diseases, are described above.

[0099] As used herein, the term "pharmaceutical composition" means a mixture comprising a compound of the present invention and a pharmaceutically acceptable excipient, such as a diluent or carrier. The pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable for a particular dosage form purpose. Pharmaceutically acceptable excipients used in the preparation of the pharmaceutical composition include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils.

[0100] In some embodiments, methods are provided for administering a pharmaceutical composition comprising a composition of the present invention to a subject in need thereof. In some embodiments, the composition of the present invention can be administered to a human. While the description of pharmaceutical compositions provided herein primarily relates to pharmaceutical compositions for administration to humans, those of ordinary skill in the art will appreciate that such compositions are generally suitable for administration to all types of animals. Modifications of pharmaceutical compositions for administration to various animals are well understood, and a skilled veterinary pharmacologist can design and / or implement such modifications, if necessary, simply by routine experimentation. The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition of the present invention may vary depending on the identity, size, and / or disorder of the subject being treated, and on the route by which the composition is administered.

[0101] The pharmaceutical composition of the present invention may be administered by any route. In some embodiments, the pharmaceutical composition may be administered by various routes, including orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, subcutaneously, intracerebroventricularly, transdermally, intradermally, rectally, intravaginally, intraperitoneally, topically (by powders, ointments, creams, and / or drops), mucosally, nasally, orally, enterally, sublingually; by intratracheal instillation, bronchial instillation, and / or by inhalation; and / or by oral spray, nasal spray, and / or aerosol. Specifically contemplated routes may be intravenous injection, local administration via the blood and / or lymphatic supply, and / or direct administration to the affected area. In general, the most suitable route of administration will depend on various factors, including the properties of the agent (e.g., stability in the gastrointestinal environment) and the impairment of the subject (e.g., whether the subject can tolerate oral administration).

[0102] The pharmaceutical composition may be formulated as an injectable preparation. The injectable preparation may be, for example, a sterile injectable aqueous or oleaginous suspension, prepared according to the known art using dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension, or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, 1,3-butanediol. Acceptable vehicles and solvents include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly employed as a solvent or suspending medium. Any bland fixed oil, including synthetic mono- or diglycerides, may be employed for this purpose. In addition, fatty acids, such as oleic acid, may be used in the preparation of injectables. Injectable preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, or by including a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0103] The compounds of the present invention described herein may typically be prepared in dosage unit form for easy and uniform administration. However, the total daily dosage of the compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject may depend on a variety of factors, including the disease, disorder, or disorder being treated and its severity; the activity of the specific active ingredient employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and excretion rate of the specific active ingredient employed; the duration of treatment; drugs used in combination or concurrently with the specific active ingredient employed; and other factors well known in the medical arts.

[0104] In one embodiment, the pharmaceutical composition may be administered at a dosage level sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg of body weight of the subject once or more times per day to achieve the desired therapeutic effect. The desired dosage may be delivered three times per day, twice per day, daily, every other day, every three days, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered via multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). It will be appreciated that the dosage ranges described herein provide guidance for administration of the pharmaceutical compositions provided to adults. For example, the amount administered to children or adolescents can be determined by a physician or one skilled in the art and may be less than or equal to that administered to adults. The precise amount of a compound according to the invention required to achieve an effective amount will vary from subject to subject depending on, for example, the species, age, and overall disorder, side effects, or severity of the disorder, the identity of the specific compound, the mode of administration, and the like.

[0105] In one embodiment, the compounds or pharmaceutical compositions of the present invention may be used in combination therapy. The specific combination of treatments (therapeutics or procedures) to be used in combination therapy may take into account the desired therapeutic effect to be achieved and the suitability of the desired therapeutic agents and / or procedures.

[0106] The pharmaceutical compositions of the present invention may be administered alone or in combination with one or more therapeutically active agents. The compositions may be administered concurrently with, prior to, or subsequent to, one or more other therapeutic agents or medical procedures. Generally, each agent may be administered at a dosage and / or time schedule established for that agent. The compounds of the present invention and the therapeutically active agents may be administered together in a single composition or separately in different compositions. The particular combination employed in combination therapy may take into account the desired therapeutic effect to be achieved and / or the suitability of the procedure and / or the therapeutically active agents comprising the peptides of the present invention. The combination employed may achieve the desired effect for the same disorder (e.g., the compounds of the present invention may be administered in combination with another therapeutically active agent used to treat the same disorder), and / or they may achieve different effects (e.g., controlling any side effects).

[0107] The term "combination administration" or "combination therapy" means that at least two compounds or compositions are administered simultaneously to a patient, such that an effective amount or effective concentration of each of the two or more compounds can be found in the patient's body at a given time. While the compounds according to the present invention may be co-administered to a patient simultaneously, this term encompasses both simultaneous administration of the two or more agents or administration at different times, provided that an effective concentration of all co-administered compounds or compositions is found in the subject at a given time. In certain preferred embodiments of the present invention, one or more of the compounds of the present invention as described above are co-administered in combination with at least one additional bioactive agent, including an agent for treating a tauopathy, particularly an agent for treating Alzheimer's disease.

[0108] In one embodiment, the pharmaceutical composition of the present invention may be administered in combination with any therapeutically active agent or procedure (e.g., surgery, radiation therapy) useful for treating, alleviating, ameliorating, alleviating, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms or conditions.

[0109] In another embodiment, the present invention provides a method for activating and degrading the lysosomal degradation pathway in a subject, such as, for example, a cell, tissue, mammal, or human patient, comprising administering an effective amount of a compound effective for lysosomal degradation of a protein in the subject, or an AUTAC composition comprising an effective amount of the compound, as described herein. In certain embodiments, the protein is tau protein.

[0110] In one embodiment, the present invention provides a method for modulating protein activity of tau protein by disaggregating tau aggregates in a patient in need thereof, comprising administering to the patient an amount of a compound as described herein.

[0111] In another embodiment, the present invention provides a method for treating a disease or disorder in a patient in which dysregulated protein activation (tau aggregation and accumulation) contributes to the disease or disorder, comprising administering to the patient an effective amount of a compound as described herein to modulate protein activation within the patient. In one embodiment, the protein is tau protein.

[0112] In another aspect, the present invention provides a method of modulating and degrading tau protein in a subject, such as, for example, a cell, tissue, mammal, or human patient, comprising administering an effective amount of a compound as described herein that induces tau protein in the subject to be degraded by the lysosomal degradation pathway, or a composition comprising an effective amount of a compound as described herein.

[0113] In another aspect, the present invention provides a method of treating or alleviating symptoms of a disease associated with tau aggregation and accumulation in a subject, such as, for example, a cell, tissue, mammal, or human patient, comprising administering an effective amount of a compound or a composition comprising an effective amount of the compound, as described herein, effective to treat or alleviate symptoms of a disease associated with tau aggregation in the subject.

[0114] In another aspect, the present invention provides a kit comprising a compound or composition as described herein. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Furthermore, the kit of the present invention may preferably include instructions describing proper use. Such a kit may be conveniently used, for example, in a clinical setting, to treat patients with neurological disorders.

[0115]

[0116] The present invention exhibits the effect of inducing and activating tau-specific degradation. Therefore, the compound of the present invention can be beneficially utilized in the prevention, alleviation, or treatment of diseases related to tauopathy.

[0117] Furthermore, the present invention exhibits a lysosomal stress-reducing effect through the degradation of tau protein. Since the removal of accumulated tau protein reduces lysosomal stress, it is useful for the prevention, alleviation, or treatment of tauopathy-related diseases, such as Alzheimer's disease, cognitive dysfunction, or neuroinflammation, in individuals with accumulated tau protein.

[0118] Furthermore, the present invention exhibits an effect of activating or restoring autophagy activity through the degradation of tau protein. The compound according to the present invention not only activates autophagy activity against tau protein, but also restores autophagy activity by removing accumulated tau protein. Therefore, it is expected to be very beneficial in the prevention, alleviation, or treatment of tauopathy-related diseases, such as Alzheimer's disease, in individuals with accumulated tau protein.

[0119] In addition to the effects described above, specific effects of the present invention are described below while explaining specific details for carrying out the invention.

[0120]

[0121] Figure 1 is a reaction diagram of the synthesis process of TauAutac 1 to 5 (TA-1 to TA-5). Figure 1a is a reaction diagram of the synthesis process of the FBnG tag. Figure 1b is a reaction diagram of the synthesis process of TA-1 to TA-5.

[0122] Figure 2 shows the results of an immunoblot experiment confirming the removal of tau protein by TA. Figure 2a shows the tau protein removal effect of TA-1 to TA-5. Figure 2b compares the tau protein removal effect of TA-3, San152805, LMB, and the combination of San152805 and LMB. Figure 2c shows the dose-response relationship of TA-3. Figure 2d shows the tau protein lysosomal degradation effect of TA-3.

[0123] Figure 3 shows the results of an experiment to investigate the effects of TA-3 on Tau or Aß accumulation and amyloid plaques in human tau-overexpressing 6xTg mice. Figure 3a is an immunoblot and graph showing the levels of human tau accumulated in the cortex and hippocampus of 6xTg mice. Figure 3b is an immunohistochemistry and graph showing phospho-tau accumulation stained with the AT8 antibody in the cortex and hippocampus of 6xTg mice.

[0124] Figure 4 shows the results of an experiment to determine whether TA-3-induced Tau degradation occurs through enhanced autophagy activity in vivo. Figure 4a shows the results of confirming the co-localization of AT8 and LC3 to study the autophagic degradation of phosphorylated tau. Figure 4b shows the results of confirming the co-localization of AT8 and LAMP1 to determine whether tau degradation proceeds to the lysosomal stage of autophagy. Figure 4c shows the results of confirming the increase and decrease of CHMP2B puncta to determine whether there are changes in lysosomal stress induced by Tau in vivo. Figure 4d shows the results showing that TA-3 administration improved the neuroinflammatory signs of GFAP+ glial infiltration. Figure 4e shows the results showing that TA-3 administration improved the neuroinflammatory signs of Iba1+ microglial infiltration.

[0125] Figure 5 shows the results of behavioral test experiments in 6xTg mice with induced cognitive memory impairment following treatment with TA-3. Figure 5a shows that the exploration time ratio and recognition memory were significantly restored by treatment with TA-3 in 6xTg mice. Figure 5b shows that alternation in the Y-maze test was significantly improved by treatment with TA-3 in 6xTg mice, indicating that the willingness to explore a novel environment was restored. Figure 5c shows that memory impairment in the passive avoidance test (PAT) was restored by treatment with TA-3 in 6xTg mice.

[0126] Hereinafter, the present invention will be described in more detail through experimental methods and examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0127]

[0128] <Example 1> Synthesis of TauAUTAC

[0129] 1.1 Synthesis of TauAUTAC

[0130] FBnG Tag Synthesis

[0131] To synthesize tert-Butyl (6-chloro-9H-purin-2-yl) carbamate (compound 2 in Figure 1a), (Boc)2O (1.5 g, 7.08 mmol) was added to a stirred solution of 2-amino-6-chloropurine (1.0 g, 5.9 mmol) in DMSO (12 ml) at 0 °C, and the mixture was slowly heated to RT. An amount of DMAP equivalent to the amount of the catalyst was then added, and the solution was stirred for 8 h. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and concentrated in vacuo to yield compound 1 in Figure 1a, which was used in the next step without further purification. THF (30 ml), 60% NaH (350 mg, 8.8 mmol) was added in portions to a stirred solution of compound 1 in Figure 1a at 0 °C, and the mixture was slowly heated to RT. After the reaction was completed, the mixture was quenched with ice water, extracted with ethyl acetate, and washed with brine (2 ml). The organic layer was then concentrated in vacuo, and the residue was purified by column chromatography (MeOH / CH2Cl2) to give compound 2 of Figure 1a as a white solid (1.1 g, 72% in two steps). 1H NMR (300 MHz, DMSO-d6): δ 13.60 (br, 1H), 10.22 (br, 1H), 8.45 (s, 1H), 1.43 (s, 9H); LC-MS: [M+H] + 270.1.

[0132] To a stirred solution of compound 2 (1 g, 3.7 mmol) of Figure 1a in THF (19 ml) were added tert-butyl(6-chloro-9-(4-fluorobenzyl)-9H-purin-2-yl) carbamate (compound 3 of Figure 1a), 4-fluorobenzyl alcohol (0.6 ml), and triphenylphosphine (2.4 g, 9.2 mmol), followed by diethyl azodicarboxylate (2 ml) at RT. The reaction was completed by stirring for 6 h. The reaction mixture was concentrated under vacuum, and the residue was purified by column chromatography (MeOH / CH2Cl2) to give compound 3 of Figure 1a as a yellow solid (1.0 g, 74%). 1 H NMR (300MHz, CDCl3); LC-MS: [M+H] + 378.1.

[0133] To synthesize 2-amino-9-(4-fluorobenzyl)-1,9-dihydro-6H-purin-6-one (compound 4 in Figure 1a), a solution of compound 3 in Figure 1a (1 g) was added to an 80% formic acid solution (10.9 ml), stirred, and heated at 80°C for 2 h. The reaction mixture was concentrated in vacuo to complete drying, and the residue was repeatedly washed with CH2Cl2 and 2% MeOH / CH2Cl2 to obtain approximately 92% pure 4 (601 mg, 65%). 1 H NMR (400 MHz, DMSO-d6); LC-MS: [M+H] + 260.1.

[0134] To synthesize N-acetyl-S-(2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)-L-cysteine ​​(compound 6 in Figure 1a), compound 4 in Figure 1a (900 mg) was partially dissolved in 100 ml of distilled water, and 30 ml of bromine water was slowly added dropwise to the reaction mixture at room temperature using a dropping funnel. After stirring at RT for 12 h, the resulting precipitate was filtered, washed with water, and dried in an oven to obtain compound 5 in Figure 1a, which was used in the next step without further purification. Compound 5 of Figure 1a, K2CO3 (3.8 g, 27 mmol), and N-acetyl-L-cysteine ​​(NAC) (2.2 g, 14 mmol) were dissolved in DMF (15 ml), and the mixture was heated to 90°C for 5 h. After the mixture was cooled to RT, the crude material was purified by stepwise acid coagulation. For example, DMF was removed in vacuo, the crude residue was dissolved in water, and the undissolved solid was filtered off. The clear filtrate was then slowly acidified to pH ~6, the solid was removed, and further acidified to pH ~4. The solution was maintained for a certain period of time to obtain compound 6 of Figure 1a (600 mg, 2 steps 58%) as a pure white solid. 1 H NMR (400MHz, DMSO-d6): δ 12.88(s, 1H), 10.63(s, 1H), 8.51(d, J=7.1, 2H), 7.19(t, J=7.1, 2H), 7.16(t, J=7, 2H), 6.51(s, 2H), 5.08(s, 1H), 5.07(s, 1H), 4.43-447(m, 1H), 3.56, JD= 16.46, JD), 4.46, 16.

[0135]

[0136] Synthesis of TA-1 to TA-5

[0137] To synthesize 3,7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl chloride (compound 7 in Figure 1b), CH2Cl2 (25 m) and Na2CO3 (6.63 g, 62.52 mmol) were added to a methylene blue solution (5.00 g, 15.63 mmol) and stirred at 40°C under a nitrogen atmosphere. Sodium dithionite (10.89 g, 62.52 mmol) dissolved in 70 ml of water was directly injected into the solution using a syringe apparatus. The mixture was then stirred at 40°C under nitrogen until the solution turned yellow. The reaction mixture was cooled in ice water, and then triphoszine (2.78 g, 9.38 mmol) was added to 20 ml of dichloromethane and stirred for an additional hour. The solution was added to 200 ml of ice water, stirred, and the resulting mixture was extracted with 3 x 100 ml of CH2Cl2. The combined extracts were washed with brine, dried over Na2SO4, evaporated on a rotary evaporator, and purified by column chromatography (ethyl acetate / n-hexane = 1 / 10) to produce compound 7 of Figure 1b as a blue solid. Yield 2.83 g, 55%. 1 H NMR (400MHz, DMSO-d6): LC-MS: [M+H] + 348.8.

[0138] To synthesize compounds 8 (8(1-4) and 8(5)) of Fig. 1b, Et3N (1.5 eq.) was added to a stirred solution of compound 7 of Fig. 1b (1 eq.) in CH2Cl2 at 0 °C, followed by the addition of the corresponding amine (1.2 eq.), and the mixture was stirred at room temperature for 30 min. The reaction mixture was quenched by adding water and extracted several times with IPA / CHCl3 (1 / 4). The combined extracts were washed with brine, dried over Na2SO4, and evaporated on a rotary evaporator to give compounds 8(1-4) of Fig. 1b and 8(5) of Fig. 1b as blue solids, which were used in the next reaction step without further purification.

[0139] To synthesize TA-1 to TA-5, DIPEA was added to a stirred solution of the 6 compounds (1 eq.) in DMF (Fig. 1b), followed by HATU (1.5 eq.), and the mixture was stirred at room temperature for 10 min. Then, the corresponding amine 8(1-4) compound or 8(5) compound (1.2 eq.) in DMF was added to the reaction mixture, and the mixture was stirred at RT for 4 to 6 h. The reaction mixture was quenched by adding water and stirred several times to obtain a blue solid, which was filtered, dried in an oven, and purified by column chromatography (MeOH / CH2Cl2) to obtain pure TA-1 to TA-5.

[0140]

[0141] 1.2 Analysis of TauAUTAC TA-1 to TA-5

[0142] TA-1:(R)-N-(3-(2-Acetamido-3-((2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)propanamido)propyl)-3,7-bis(dimethylamino)-10H-phenothiazine-10-carboxamide. 1 H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H), 10.64 (s, 1H), 8.44 (d,J=8.0 Hz, 1H), 8.03 (t,J=5.5 Hz, 1H), 7.33-7.24 (m, 2H), 7.23-7.11 (m, 4 H), 6.73-6.62 (m, 4H), 6.54 (brs, 2H), 6.10 (t,J=5.7 Hz, 1H), 5.07 (s, 1H), 4.54-4.36 (m, 1 H), 3.18-2.98 (m, 6H), 2.88 (s, 12H), 1.85 (s, 3H), 1.58-1.44 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ 169.8, 163.0, 160.7, 156.1, 155.7, 154.0, 153.2, 148.7, 142.5, 133.5, 132.8, 129.5, 129.4, 128.8, 127.7, 117.0, 116.1, 115.8, 111.6, 110.7, 54.0, 45.1, 42.3, 40.7, 36.5, 30.0, 23.0; LC-MS: [M+H] + 788.9 ; HRMS (ESI): calcd. for C 37 H 42 FN 12 O4S2[M + H] + 788.2847; found 788.2897.

[0143] TA-2: (R)-N-(4-(2-Acetamido-3-((2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)propanamido)butyl)-3,7-bis(dimethylamino)-10H-phenothiazine-10-carboxamide. 1 H NMR (400 MHz, DMSO-d6) δ 10.71 (brs, 1H), 8.40 (d,J=8.1 Hz, 1H), 8.04 (t,J=5.5 Hz, 1H), 7.31-7.09 (m, 6H), 6.73-6.56 (m, 5H), 5.99 (t,J=5.6 Hz, 1H), 5.07 (s, 2H), 4.47 (dd,J= 13.4, 8.0 Hz, 1H), 3.54-3.36 (m, 1H), 3.31-3.20 (m, 1H), 3.11-2.94 (m, 4H), 2.88 (s, 12H), 1.85 (s, 3H), 1.50-1.28 (m, 4H); 13C NMR (75 MHz, DMSO-d6) δ 169.9, 163.6, 160.3, 156.2, 155.7, 154.2, 153.2, 148.9, 142.6, 133.6, 133.0, 129.5, 129.4, 128.7, 127.8, 117.1, 116.1, 115.8, 111.6, 110.7, 52.8, 45.1, 40.7, 35.2, 27.5, 26.8, 23.0; LC-MS: [M+H] + 802.9; HRMS (ESI): calcd. for C 38 H 44 FN 12 O4S2[M + H] + 802.3003; found 788. 802.3061.

[0144] TA-3:(R)-N-(2-(2-(2-Acetamido-3-((2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)propanamido)ethoxy)ethyl)-3,7-bis(dimethylamino)-10H-phenothiazine-10-carboxamide. 1 H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.41 (d,J=8.0 Hz, 1H), 8.08 (t,J=5.5 Hz, 1H), 7.31-7.10 (m, 6H), 6.72-6.61 (m, 4H), 6.53 (brs, 2H), 5.97 (t,J=5.5 Hz, 1H), 5.07 (s, 2H), 4.49 (td,J=8.1, 5.4 Hz, 1H), 3.44-3.35 (m, 5H), 3.23-3.13 (m, 5H), 2.88 (s, 12H), 1.84 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.2, 169.9, 163.2, 160.9, 156.1, 155.6, 154.1, 153.2, 148.9, 142.7, 133.5, 133.0, 129.5, 129.4, 128.7, 127.7, 117.0, 116.0, 115.8, 111.6, 110.7, 69.5, 69.1, 52.8, 44.8, 40.7, 39.1, 35.1, 23.0; LC-MS: [M+H] + 818.9. HRMS (ESI): calcd. for C 38 H 44 FN 12 O5S2[M + H] + 818.2952; found 818.3006.

[0145] TA-4:(R)-N-(4-(2-Acetamido-3-((2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)propanamido)cyclohexyl)-3,7-bis(dimethylamino)-10H-phenothiazine-10-carboxamide. 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.32 (d,J=8.1 Hz, 1H), 7.93-7.82 (m, 1H), 7.34-7.27 (m, 2H), 7.24-7.11 (m, 4H), 6.74-6.60 (m, 4H), 6.55 (brs, 2H), 5.28 (d,J=7.3 Hz, 1H), 5.09 (s, 2H), 4.57-4.39 (m, 1H), 3.78-3.63 (m, 1H), 3.62-3.52 (m, 2H), 2.87 (s, 12H), 1.82 (s, 3H), 1.63-1.41 (m, 8H); 13C NMR (101 MHz, DMSO-d6) δ 169.6, 169.0, 160.6, 155.9, 154.4, 153.9, 152.8, 148.5, 142.3, 133.4, 133.0, 129.4, 129.3, 128.4, 127.0, 117.0, 115.9, 115.8, 111.3, 110.7, 52.3, 44.9, 40.6, 35.3, 28.5, 28.4, 28.0, 22.8; LC-MS: [M+H] + 829.0; HRMS (ESI): calcd. for C 40 H 46 FN 12 O4S2[M + H] + 828.3160; found 828.3212.

[0146] TA-5:(R)-N-(3-((2-amino-9-(4-fluorobenzyl)-6-oxo-6,9-dihydro-1H-purin-8-yl)thio)-1-(4-(3,7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl)piperazin-1-yl)-1-oxopropan-2-yl)acetamide. 1 H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.37 (d,J=8.5 Hz, 1H), 7.51 (d,J=8.9 Hz, 2H), 7.25-7.10 (m, 3H), 6.76-6.60 (m, 4H), 6.53 (brs, 2H), 5.04 (s, 2H), 5.03-4.91 (m, 1H), 3.66-3.54 (m, 2H), 3.46-3.35 (m, 2H), 3.30-3.23 (m, 2H), 3.20-3.05 (m, 4H), 2.87 (s, 12H), 1.76 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 169.3, 168.7, 162.5, 155.9, 155.1, 154.1, 153.2, 151.7, 142.6, 133.0, 132.9, 129.5, 129.4, 124.5, 116.9, 116.0, 115.7, 110.9, 53.9, 48.3, 44.9, 42.2, 29.1, 28.0, 22.5; LC-MS: [M+H] + 801.0; HRMS (ESI): calcd. for C 38 H 42 FN 12 O4S2[M + H] + 799.2847; found 799.2814.

[0147]

[0148] <Example 2> Experimental materials and methods

[0149] 2.1 Animals

[0150] 6xTg mice were generated by crossing 5xFAD (B6SJL) transgenic mice (Jackson Laboratory, Bar Harbor, ME, USA) with JNPL3 Tg mice (Tacon Biosciences Inc., Albany, NY, USA), and genotyped by PCR using genomic DNA from ear biopsy samples. Wild-type (WT) and JNPL3+ / -5xFAD+ / -(6xTg) mice were housed under a 12-h light / dark cycle with free access to food and water in an automatically controlled environment at 22 ± 2°C and 50 ± 10% relative humidity. All animal experiments were performed in accordance with the Animal Care and Use Guidelines of Gachon University, Seoul, Korea (LCDI-2021-0171).

[0151]

[0152] 2.2 In vivo drug therapy

[0153] TA-3 was dissolved in distilled water (DW) containing 5% NMP, 5% polyethylene glycol (PEG), and 5% solute solution, and DW containing 5% NMP, 5% PEG, and 5% solute solution was used as a vehicle. TA-3 was injected intraperitoneally (ip) at 5 mg / kg daily for 2 months into 4-month-old 6xTg or wild-type (WT) mice.

[0154]

[0155] 2.3 In vivo evaluation of microdialysis probe recovery

[0156] A total of six kDa CMA 7 microdialysis probes (Harvard Apparatus, Holliston, MA, USA) were connected to a PHD ULTATM syringe pump (Harvard Apparatus, Holliston, MA, USA) with PE / PVC tubing (0.6 x 1.6 mm). Filtered artificial cerebrospinal fluid (aCSF) buffer containing 122 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgCl2, 3.0 mM KH2PO4, and 25.0 mM NaHCO3 was perfused at a flow rate of 0.5 μl / min through the microdialysis probe inlet to equilibrate the microdialysis probes, and the outlet was stored in an empty EP tube for 1 h. The membranes of the microdialysis probes were soaked in ethanol. Postdialysis was performed with aCSF containing TA-3 to estimate the in vivo recovery. In vivo recovery was calculated equivalent to postdialysis loss using the following equation: In vivo recovery = (C perfusate - C diaysate ) / C perfusate , where C perfusate is the TA-3 concentration of perfusate (inlet of the microdialysis probe), and C diaysate is the TA-3 concentration of the dialysate (outlet of the microdialysis probe). Recovery is the TA-3 concentration of the brain interstitial fluid (ISF) from the measured brain ISF dialysate concentration (C ISF ) was used to calculate.

[0157]

[0158] 2.4 Behavioral Testing

[0159] To investigate changes in memory and cognition after TA-3 treatment, three behavioral tests were performed, including neurological assessments. All tests were automatically recorded and aggregated using Noldus Information Technology (Wageningen, The Netherlands). For the novel object recognition (NOR) test, mice were acclimated to an open-field chamber (38 cm wide × 38 cm high × 40 cm long) for 20 min on the first day. The following day, the mice were placed in the same open-field chamber and exposed to two identical objects for 5 min. To prevent movement of the experimental animals, the objects (10 ± 2 cm high) were colored and filled with stones and placed in opposite corners of the chamber, 5 cm from the wall. After 24 h, one of the familiar objects was replaced with a novel object of a different color and shape, and the mice were allowed to explore the area containing the familiar and novel objects for 5 min. Memory scores were calculated as the percentage of exploration time for each object (see Figure 7a). The Y-maze test was performed using a white polyvinyl plastic maze consisting of three arms measuring 40 cm in length, 6.8 cm in width, and 15.5 cm in depth. Mice were placed in the maze for 8 min, and one point (alternation) was given for entering all three arms consecutively. The voluntary alternation was calculated by the following equation: number of alternations / (total entries - 2) x 100 (%) (see Figure 7b). The passive avoidance test was performed for 3 consecutive days using an avoidance learning box (Gemini Passive Avoidance System, San Diego Instruments, CA, USA) consisting of adjacent light and dark chambers connected by remotely operated gates. The light chamber was illuminated by a fixed W LED light, and an adaptation period was allowed on the first day to allow free exploration of the chamber. On the following day, entering the dark chamber resulted in an electric shock (0.05 ms for 3 s) to the foot.3 mA) was given, and 24 hours later, the delay time before entering the dark room was measured (see Fig. 7c).

[0160]

[0161] 2.5 Organizational Preparation

[0162] Mice were anesthetized with a mixture of Zoletil (8.3 mg / kg) and Rompun (15 mg / kg), and their brains were collected. The cortex and hippocampus were dissected from each hemisphere and immediately frozen in liquid nitrogen for immunoblotting. The other hemisphere of each mouse brain was fixed in 4% paraformaldehyde at 4°C for 24 h, dehydrated in a 30% sucrose solution for 3 days, and then frozen in molds filled with optimal cutting temperature compound (Sakura, Osaka, Japan). The frozen tissues were sectioned at 30 μm thickness using a cryomicrotome (Cryotome, Thermo Electron Corporation, Waltham, MA, USA) and stored in a cryoprotectant solution (30% ethylene and 30% glycerol) at 4°C until use.

[0163]

[0164] 2.6 Tissue staining

[0165] For immunohistochemistry, 30 μm sections were incubated in 3% H2O2 for 20 min and then washed in PBS for 10 min. After blocking with 0.5% BSA-3% normal goat serum in 0.4% PBS-T for 1 h, the slices were incubated with primary antibodies overnight at 4°C. The following day, the sections were incubated with secondary antibodies for 1 h, then incubated using the ABC kit (PK-6102, Newark, CA, USA) and colorized with DAB solution (Abcam, Cambridge, UK). Stained tissues were imaged under a light microscope and quantified using ImageJ software. Immunofluorescence staining was performed after washing with 0.2% Triton X-100 in PBS and blocking with 1% BSA-3% normal goat serum in 0.4% PBS-T for 1 h. The brain sections were reacted with primary antibodies (AT8, 6E10, LAMP1, LC3) in PBS-T overnight at 4°C. The next day, the sections were reacted with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h at RT. For amyloid plaque staining, Thioflavin-S solution was applied to the sections and reacted for 10 min at RT. After DAPI counterstaining, images were captured using a Nikon TS2-S-SM microscope equipped with a Nikon DS-Qi2 camera (Nikon Microscope, Tokyo, Japan). Regions of interest (ROIs) were designated, and fluorescence intensities were quantified using Image J software.

[0166]

[0167] 2.7 Immunoblot analysis

[0168] RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing protease inhibitors (Roche Applied Science, Mannheim, Germany) and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) was added to the cell or brain samples, and the lysates were homogenized on ice for 30 min and centrifuged at 20,000 g for 10 min. Supernatants were electrophoresed on 15% Novex gels (Invitrogen) (for LC3) or 4–12% Bis-Tris gradient gels (NUPAGE, Invitrogen) (for all other proteins) with or without ß-mercaptoethanol (for visualization of total tau or other proteins). After transfer to nitrocellulose or PVDF membranes, immunoblot analysis was performed using the ECL method (Pierce). To obtain the RIPA-insoluble fraction, the pellet remaining after centrifugation was washed once with RIPA buffer at 20,000 g at 4°C, resuspended in insoluble fraction buffer (1 M sucrose, 2% SDS in RIPA buffer) and incubated for 1 h at RT. After centrifugation at 20,000 g at RT, the supernatant was used as the RIPA-insoluble fraction. The prepared samples were stored at -80°C until use. Proteins were quantified using the BCA protein assay (Thermo Fisher Scientific, Matham, Massachusetts) and fractionated on 8% or 15% sodium dodecyl sulfate-polyacrylacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Mercrose, NJ, USA) for immunoblot analysis.

[0169]

[0170] 2.8 Cell Therapy

[0171] To study the effect of TA-1 to TA-5 compounds synthesized in Example 1 on tau clearance, T-REx-Tau40-HEK293 cells were treated with 500 ng / ml doxycycline for 24 hours, then treated with test chemicals without doxycycline for another 24 hours, and immunoblot analysis was performed as described above.

[0172]

[0173] 2.9 Statistical Analysis

[0174] All data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 9.1.0 (221) software (GraphPad Software Inc., San Diego, CA, USA). A two-tailed Student's t-test was used to compare values ​​between two groups. A one-way analysis of variance with Tukey's post hoc test was used to compare values ​​across multiple groups. A two-way analysis of variance with the Bonferroni test was used to analyze the effect of two variables or categories on the results. Statistical significance was set at P < 0.05.

[0175]

[0176] <Example 3> Experimental results

[0177] 3.1 Chemical Synthesis of TauAutac

[0178] As autophagy degraders, previously reported S-guanylated lysosomal degradation tags (Autacs) were selected. Methylene blue (MB), which can bind and block Tau polymerization, was selected as the warhead targeting Tau. Furthermore, after synthesizing linkers consisting of 2–5 atoms, three moieties were combined to design five different novel TauAutacs (TAs). To synthesize the lysosomal degradation tags, the six-step synthetic sequence began with Boc protection of 2-amino-6-chloropurine (denoted as compound 1 in Figure 1a), followed by transfer of the protecting group to the 2-amino group, yielding compound 2 in Figure 1a in 72% yield in the second step. Mitsunobu reaction of compound 2 of Figure 1a with 4-fluorobenzyl alcohol gave compound 3 of Figure 1a in a 74% yield, which was then hydrolyzed with 80% formic acid to afford 2-amino-9-(4-fluorobenzyl)-1H-purin-6(9H)-one (represented as compound 4 of Figure 1a) in a 65% yield. Compound 4 of Figure 1a was then brominated with 5% aqueous bromine (represented as compound 5 of Figure 1a) at room temperature (RT), followed by coupling with N-acetyl cysteine ​​(NAC) to afford compound 6 of Figure 1a in a 58% yield in the acid fragment in two steps (see Figure 1a).

[0179] After the tag synthesis was completed, MB Warhead was synthesized in two steps. Commercially available MB was reduced to leucomethylene blue (LMB) using Na2CO3 / Na2S2O4, which reacted in situ with triphosgene to give the stable acid chloride derivative LMB (represented as compound 7 in Figure 1b) in 55% yield. Optionally, compound 7 in Figure 1b was combined with various linkers (linear, cyclic, or heterolinear) to obtain various MB-attached linker fragments, represented as compounds 8(1-4) and 8(5) in Figure 1b, and finally, TA-1 to TA-5 were synthesized via amide bonds between compounds 8(1-4) and 8(5) in Figure 1b and compound 6 in Figure 1a (see Figure 1b).

[0180]

[0181] 3.2 Removal of tau protein by TauAUTAC

[0182] We investigated whether TAs consisting of LMB (Tau Warhead), a linker, and an Autocrine tag could reduce tau accumulation in inducible tau-expressing HEK293 (T-REx-Tau40-HEK293) cells treated with doxycycline for 24 hours. All TAs, TA-1 to TA-5, significantly reduced tau oligomer accumulation, with TA-3 and TA-5 exhibiting the most dramatic effects at concentrations ranging from 0.1 to 10 nM (Fig. 2a). This likely reflects the superior binding of LMB to tau and the productive synergy between the linker and Autoc tag. In addition to its effect on tau oligomerization, TA-3 significantly reduced the accumulation of tau monomers, which can induce aggregation (Figs. 2a and 2f). Therefore, we conducted additional experiments using TA-3.

[0183] The effect of TA-3 showed a significant lysosomal degradation effect of the recognized target Warhead (tau binding site or target binding site), surpassing the simple binding effect of San152085 (degradation tag; compound 6 in Figure 1a) and LMB at equilibrium molar concentrations (Figure 2b). The dose-response relationship showed the maximum clearance of accumulated tau oligomers at a concentration of approximately 10 nM TA-3, and the EC 50 was calculated to be 1.25 nM (Fig. 2c). The clearance of accumulated tau by TA-3 was reduced by bafilomycin A1 but not by lactacystin, suggesting lysosomal degradation of tau (see Fig. 2d).

[0184]

[0185] 3.3 Improvement of AD phenotype by TauAutac in vivo

[0186] Next, the in vivo effects of TA-3 were studied using 6xTg mice. 6xTg mice express a mutant form of Tau (P301L) in addition to APPSwe / Ind / fl (K670N / M671L / V717F / I716V) and mutant PS1 (M146L / L286V). Compared with other AD animal models, 6xTg mice exhibit younger onset of amyloid plaques and Tau NFTs and accelerated neurological deficits, making them a model that allows for the simultaneous study of the effects of TA on Tau or Ab clearance and AD phenotype. Pharmacokinetic studies after single intraperitoneal (ip) administration of TA-3 at different doses (1, 5, 25 mg / kg) revealed that TA-3 concentrations in the brain interstitial fluid at 24 h (C last,ISF) was 10.08 ± 0.67 ng / g (12.33 ± 0.82 nM) and the whole brain TA-3 concentration at 24 h was 13.38 ± 0.09 ng / g (16.37 ± 0.11 nM). Since this concentration was similar to the TA-3 concentration that showed maximal Tau clearance in vitro (see Table 1), a 5 mg / kg TA-3 ip administration dose was selected for further study.

[0187] [Table 1]

[0188]

[0189]

[0190] Furthermore, we investigated the effect of TA-3 on tau accumulation in the brain of 6xTg mice. When 6xTg mice were treated with TA-3 for 2 months starting at 4 months of age, the levels of human tau accumulated in the cortex and hippocampus of 6xTg mice were significantly reduced compared to vehicle-treated 6xTg mice (Fig. 3a). In wild-type mice, human tau recognized by the Tau-13 antibody was absent (Fig. 3a). The total tau levels recognized by the Tau-5 antibody (specific for 218-225 of human tau) in the cortex and hippocampus of 6xTg mice treated with TA-3 for 2 months were also significantly reduced (Fig. 3a).

[0191] Next, we investigated phosphorylated tau, a key feature of AD. Accumulation of phosphorylated tau detected by several antibodies recognizing phosphorylated Ser and / or Thr [AT8, AT180, AT270, and 44-752G (P396)] was significantly reduced in RIPA-soluble fractions of the cortex and hippocampus of 6xTg mice treated with TA-3 for two months. This reduction in phosphorylated tau by TA-3 was also observed in the RIPA-insoluble fraction, with both fractions demonstrating TA-3-induced phosphorylated tau removal. Furthermore, immunohistochemical studies demonstrated that TA-3 treatment significantly reduced phosphorylated tau accumulation stained with AT8 antibody in the cortex and hippocampus of 6xTg mice compared to vehicle treatment (Fig. 3b). Additionally, co-localization of thioflavin-S staining with Ab was significantly reduced by TA-3 administration, suggesting that TA-3 may reduce Ab and amyloid plaque deposition through improvement of lysosomal dysfunction in vivo.

[0192] Next, we investigated whether TA-3-mediated Tau degradation occurs via enhanced autophagy activity in vivo. Co-localization between phosphorylated tau, detected by AT8 and LC3, was increased by 2 months of TA-3 treatment (Fig. 4a), suggesting autophagic degradation of phosphorylated tau. Furthermore, TA-3 treatment increased co-localization between phosphorylated tau and the lysosomal marker LAMP1 (Fig. 4b), suggesting that accelerated autophagic Tau degradation proceeds to the lysosomal stage of autophagy. When we investigated whether Tau-induced lysosomal stress in vivo was altered by TA-3 treatment (Fig. 4c), we confirmed that neuroinflammation, evidenced by infiltration of GFAP+ astrocytes and Iba1+ microglia, which play a key role in neuroinflammation in AD, was significantly reduced by TA-3 treatment due to TA-3-mediated downregulation of Abs capable of inducing Tau or inflammasome activation (see Figs. 4d and 4e).

[0193] After observing that TA-3 reduced tau accumulation in the brain of 6xTg mice, we performed a series of behavioral tests to investigate whether the neurological deficits of 6xTg mice could be improved by reducing tau accumulation in the brain. In the novel object recognition (NOR) test to assess cognition and memory, vehicle-treated 6xTg mice spent more time near familiar objects compared to novel objects, whereas wild-type mice spent more time exploring the novel object compared to the familiar object (Fig. 5a). This suggests a recognition memory impairment in 6xTg mice. Surprisingly, TA-3-treated 6xTg mice spent more time exploring the novel object compared to the familiar object, similar to wild-type mice (Fig. 5b), showing that TA-3 treatment significantly restored the exploration time ratio and recognition memory. This is thought to be due to the TA-3-mediated reduction in tau and Ab accumulation in the brain. As control parameters, there were no significant differences in total distance or speed between groups. In the Y-maze test, vehicle-treated 6xTg mice showed a significant decrease in spontaneous alternation compared to wild-type mice, indicating a decreased willingness to explore a novel environment (Fig. 5b). In contrast, TA-3-treated 6xTg mice showed a significant improvement in alternation compared to vehicle-treated 6xTg mice, suggesting a restored willingness to explore a novel environment (Fig. 5b). As control parameters, there were no significant differences in total number of entries between groups. In the passive avoidance test (PAT), vehicle-treated 6xTg mice showed significant memory impairment as evidenced by a decrease in latency (Fig. 5c). However, TA-3-treated 6xTg mice showed a significantly increased latency compared to vehicle-treated 6xTg mice (Fig. 5c), suggesting a protective effect of TA-3 on memory impairment in AD.These results collectively suggest that TA-3 can improve neurological deficits in 6xTg mice by reducing lysosomal stress and autophagy impairment through target-specific tau clearance.

[0194] Reduced Ab accumulation due to reduced lysosomal stress or improved autophagy activity, and reduced neuroinflammation due to reduced TA or Ab accumulation may also contribute to the improvement of neurological deficits in 6xTg mice treated with the tau-specific degrader TA-3.

[0195]

[0196] Although the embodiments of this specification have been described in more detail above, this specification is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of this specification. Therefore, the embodiments disclosed in this specification are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The protection scope of this specification and the present invention should be interpreted by the claims, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of rights of this specification and the present invention.

Claims

1. A compound represented by the following chemical formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula I] In the above chemical formula I, X is -NH-Y-NH- or and; Y is -C 1-6 Alkyl-, -C 1-6 Alkyl-OC 1-6 Alkyl- or cycloalkyl.

2. In paragraph 1, The above Y is -C 1-4 Alkyl-, -C 1-4 Alkyl-OC 1-4 A compound which is an alkyl- or 3-8 membered cycloalkyl, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

3. In paragraph 1, The above Y is a compound which is -(CH2)3-, -(CH2)4-, -(CH2)2-O-(CH2)2- or cyclohexyl, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

4. In the first paragraph, the compound represented by the chemical formula I is selected from the group consisting of the following compounds, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: .

5. A pharmaceutical composition for preventing or treating tauopathy, comprising a compound according to any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition according to claim 5, wherein the tauopathy is a neurodegenerative tauopathy.

7. In the fifth paragraph, the tauopathy is Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis / parkinsonism-dementia complex of Guam, non-Guamanian motor neuron disease with neurofibrillary tangles, dementia with arginine particles, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pallido-Ponto-Nigral degeneration, A pharmaceutical composition selected from Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle only dementia, postencephalitic parkinsonism, and myotonic dystrophy.

8. A pharmaceutical composition for maintaining or improving cognitive function, comprising a compound according to any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition for preventing or treating cognitive dysfunction or neuroinflammation, comprising a compound according to any one of claims 1 to 4, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

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