WO2015199503A1 - Pharmaceutical composition for preventing and treating degenerative brain diseases, containing cx-4945 as active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating degenerative brain diseases, containing 5-[(3-chlorophenyl)amino]benzo [c]-2,6-naphthyridine-8-carboxylic acid (CX-4945) as an active ingredient. Specifically, the CX-4945, which is known as a strong inhibitor of CK and Cdc2-like kinase (Clk), was also verified to show a strong inhibitory effect on Dyrk1A in vitro; performs an inhibitory action in a competitive manner with ATP; strongly inhibits Dyrk1A even at the cellular level, thereby inhibiting the phosphorylation of Tau, amyloid precursor protein (APP), and presenilin 1 (PS1) as causative factors of neurofibrillary tangles and senile plaques; inhibits the phosphorylation of NFATc, thereby increasing transcriptional activity of NFATc; was also verified to distinctively remedy abnormalities in wings, eyes, and the nervous system, occurring at the time of over-expression of minibrain, which is drosophila Dyrk1A, in drosophila models; and was also verified to significantly lower the phosphorylation of Tau protein in Dyrk1A-overexpressed mice. Generally, the 5-[(3-chlorophenyl)amino]benzo [c]-2,6-naphthyridine-8-carboxylic acid (CX-4945) shows a higher inhibitory efficacy compared with Harmine, INDY, and proINDY, used as Dyrk1A inhibitors, and thus can be favorably used as a composition for preventing and treating degenerative brain diseases associated with Dyrk1A.

Description

 【Specification】

 [Name of invention]

 Pharmaceutical composition for the prevention and treatment of degenerative brain diseases containing CX-4945 as an active ingredient

Technical Field

 The present invention relates to 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-car ^ carboxylic acid (5 一 [(3 一 Chlorophenyl) amino] benzo [c] -2, 6-napht hyr idi ne ~ 8-car boxy 1 io acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient relates to a pharmaceutical composition for the prevention and treatment of degenerative brain diseases.

[Background]

Alzheimer's disease (AD) is a degenerative brain disease in which mental function declines gradually as brain tissues lose their function during the aging process. The disease is characterized by serious impairments in memory and emotion. In modern medicine, it is recognized as an incurable disease with no obvious cure. It is one of the main causes of dementia in the elderly. Histopathologic features include general atrophy of the brain, enlargement of the ventricles, multiple lesions of neurofibers, and stroma. Clinical features include gradual decline in intellectual functions such as memory, judgment, and language skills, and everyday life skills, perceptions, and behavioral disorders. Along with this, psychiatric symptoms such as depression, desolation of personality and violent behavior are accompanied. These symptoms gradually progress and eventually lead to death. The onset of death after the onset is about 6-8 years, but some people have more than 20 years. Currently, about 3% of people aged 65-74, about 19% of people aged 75-84, and 50% of people aged 85 or older are said to have this disease. DyrklA (Dual Specificity Tyrosine Phosphorylation Control Kinase-1A, Dual specificity t yr os i ne-phosphory 1 at i on-r egu 1 at ed kinase 1A) is a serine / threonine kinase It plays various roles within the nervous system. The gene of DyrklA is located in the Down syndrome critical region (DSCR) of human 21, and the increased activity of DyrklA is due to down syndrome, Alzheimer's disease and Parkinson's disease. It has been reported to be associated with neurodegenerative diseases such as' s disease, Huntington's disease and pick's disease.

 Transgenic mouse models that overexpress human or rat DyrklA show a phenotype of Down's syndrome, including hippocampal spatial learning, motor neuron deficiency, and developmental delay, which is an important function of DyrklA in mental retardation associated with Down's syndrome. Suggests. In particular, microtubule assembly stability, which is important for maintaining cytoskeleton, is abnormal in Alzheimer's disease and Down's syndrome. In this process, Tau protein plays a pivotal role in controlling microtubule stability. Abnormal Tau expression and hyperphosphorylation are common features in Alzheimer's and Down syndrome. DyrklA plays an important role in the phosphorylation of various positions of the Tau protein in several cell models. In particular, the phosphorylation of Τέιι Thr-212 residue (residue) in the brains of Alzheimer's disease patients, and the hyperphosphorylation of Tau Thr-212 residues in mice transformed with human DyrklA. Taken together, these findings suggest that DyrklA may make a significant contribution to the pathogenesis of Tau hyperphosphorylation in Alzheimer's disease and Down's syndrome.

In particular, in Alzheimer's patients, in addition to the formation of neurofibrillary tangles composed of neuropathologically superphosphorylated aggregates of Tau, extracellular accumulation of insoluble precipitates of amyloid β peptides appears. DyrklA is the amyloid β precursor protein associated with it. protein and presenilin directly phosphorylate to promote the production of amyloid plaques, which is one of the major pathogens of Alzheimer's disease.

 In addition, Lewy bodies, one of Parkinson's disease and pathological features, are common in the brains of Alzheimer's and Down's syndrome. DyrklA is a major component of Lewy bodies, alpha-synuclein (a—synuc). Phosphorylation of lein increases the formation of α-Synuclein and eventually leads to cell death. In addition, DyrklA has been found to phosphorylate the important protein HIP-1 associated with the development of Huntington's disease, resulting in apoptosis and differentiation of hippocampal neuronal cells.

In addition, DyrklA is known to regulate cal cineur in / NFAT signaling and play an important role in human developmental stages. NFATc transcription factors (transcr ipt ion factors) are normally while when present in a protein that is phosphorylated in the ^cytoplasm, the Ca ²⁺ concentration in the cell goes up Ca ²⁺ dependent protein phosphatase calcineurin NFATc the ride by nyurin. Phosphorylation NFATc moves into the nucleus. NFATc entering the nucleus forms a transcription complex with the partner protein NFATn and binds to the promoter of the target gene to induce target gene expression. DyrklA phosphorylates NFATc, causing it to migrate back into the cytoplasm, resulting in suppressed expression of the target gene. CX-4945 (5- [(3_chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8—carboxylic acid,

5- [(3-Ch lor opheny 1) ami no] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid, Si lmi tasert ib) It is frequently found in cancer cells and is known as an inhibitor of casein kinase 2 (CK2), a protein that regulates various cellular activities such as cell growth and self-killing. Adam Siddiqui-Jain et al. Describe the oral administration of 5 — [(3-chlorophenyl) amino] benzo [(：]-2,6-naphthyridine-8-carboxylic acid (0 (-4945). It has been reported to inhibit the proliferation of cancer cells by inhibiting the expression level of CK2 α, a catalytic subunit of CK2, which shows activity as a selective inhibitor bioavailable by administration (Siddiqui-Jain A, et al. Cancer Res. 70 (24): 10288-10298, 2010). Clinical studies are currently underway to use 5-[(3-chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8-carboxylic acid (CX-4945) as a CK2 inhibitor and is a primary clinical trial. Safety and pharmacodynamic properties have been identified in the study, and further studies are being conducted on randomized drug combinat ions in secondary clinical trials (Sean E, Cylene Pharmaceuticals.Accessed 22 Mar 2011 ).

Recent studies have shown that 5-[(3-chlorophenyl) amino] benzo [(：]-2,6-naphthyridine-8-carboxylic acid (CX-4945) plays an important role in regulating intracellular pre_mRNA splicing in addition to CK2. It has been shown to be a potent inhibitory effect on cdc2-like kinase (Clk) Interestingly, previous studies have found that many small molecule substances known to inhibit Clk, such as TG-003, KH-CB19, and Leucettine L ₄ i, are commonly known to inhibit Dyrk This is because Clk and Dyrkl have ATP-binding pockets of similar structure, CK2, Clk, and Dyrk all belong to CMGC super family kinase. Efforts to study the role of (3_chlorophenyl) amino] benzo [c] -2,6-naphthyradine-8-carboxylic acid (CX-4945) resulted in 5- being known as potent inhibitors of CK2 and Clk. [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid ( CX-4945) showed potent inhibitory effects on DyrklA in vitro, inhibits it in a competitive way with ATP, and strongly inhibits DyrklA at the cellular level, neurofibrillary tangles and senile plaques. Inhibition of phosphorylation of Tau, APP and PS1, the inhibitory factor, inhibition of NFATc phosphorylation, increased transcriptional activity of NFATc, and overexpression of Drosophila DyrklA minibrain in Drosophila model Has significantly improved wing, eye, and nervous system development.

By confirming that the phosphorylation of Tau protein was significantly lowered in DyrklA overexpressing mice, it showed higher inhibitory effect than Harmine, INDY, and proINDY, which are generally used as DyrklA inhibitors, and thus, 5-[(3-chlorophenyl) amino] The inhibitory effects of benzo [c] -2, 6-naphthyridine-8—carboxylic acid (CX-4945) on DyrklA provide a useful tool for the study of degenerative brain brain disease associated with DyrklA and new treatments for the disease. The present invention has been completed by confirming that it can lead to the invention of the strategy.

[Detailed Description of the Invention]

 [Technical problem]

The object of the present invention is 5-[(3-chlorophenyl) amino] benzo [c] _2 ′ 6-naphthyridine-8-ca Ξ. Λ]> (5- [(3-Ch 1 or opheny 1) am i no] benzo [c] -2, 6-napht hyr idi ne-8 ^to car boxy 1 ic acid; CX-4945) or a pharmaceutical thereof To provide a pharmaceutical composition for preventing and treating degenerative planetary brain disease containing an acceptable salt as an active ingredient.

 'Technical Solution

 In order to achieve the object of the present invention, the present invention provides 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (5-[(3-Chl or opheny 1) ami no] benzo [c] -2, 6-naphthyr idine-8-carboxyl ic acid; CX-4945) or a pharmaceutically acceptable salt thereof. It provides a pharmaceutical composition for preventing and treating degenerative brain disease containing as an active ingredient.

The invention also relates to 5-K3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (5 _ [(3— Ch 1 or opheny 1) am i no] benzo [c ] —2, 6-napht hyr idi ne—8— c ar boxy 1 ic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. to provide. The invention also relates to 5 — [(3-chlorophenyl) amino] benzo [(] — 2,6-naphthyridine-8-carboxylic acid in isolated cells, cell-free or in vitro. (5-[(3- Ch lor opheny 1) ami no] benzo [c] -2, 6-napht hyr idi ne-8 ^to c ar boxy 1 ic acid; CX-4945) Also provided are methods of inhibiting Dyrkl comprising treating an acceptable salt, The present invention also provides a pharmaceutically effective amount of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (5— [(3-Ch 1 or opheny Damino] benzo [c] -2, 6- napht hyr idi ne-8-car boxy 1 ic acid; CX-4945) In another aspect, the present invention provides a method for treating degenerative brain disease, comprising administering to a pharmaceutically effective amount of 5-[(3-chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8. —Carboxylic acids (5-[(3-Chlor is 3henyl) amino] benzo [c] -2,6-nap It provides a method for preventing degenerative brain disease, comprising administering hthyridine-8-carboxylic acid (CX-4945) to a subject, and the present invention also provides a method for use as a pharmaceutical composition for the prevention and treatment of degenerative brain disease. [(3-Chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8-carboxylic acid (5-[(3— Chlorophenyl) ami no] benzo [c] -2, 6-napht hyr idi ne-8 -car boxy 1 ic acid; CX—4945) The present invention also provides 5-[(3-chlorophenyl) amino] benzo [c] _2 for use in the prevention and treatment of degenerative brain diseases. , 6-naphthyridine— 8-carboxylic acid (5-[(3-Chlorophenyl) ami no] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid; CX 一 4945) Serves the purpose. Hereinafter, the present invention will be described in detail. The present invention relates to 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (5— [(3-Ch 1 oropheny 1) am i no] benzo [c] 2, 6 napht hyr idine-8-car boxy lie acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient provides a pharmaceutical composition for preventing and treating degenerative brain disease.

 The CX-4945 preferably has a structure represented by the following [Formula 1];

 [Formula 1]

 The CX 4945 is characterized by inhibiting the activity of DyrkKDual specificity tyrosine phosphorylat ion regulated kinase 1).

 Inhibition of DyrklA activity is characterized in that CX-4945 occurs by binding to the ATP binding pocket of DyrklA. . The CX-4945 inhibits phosphorylation of Tau, APP (amyloid precursor protein) and PSKpreseniHn 1) protein through DyrklA inhibition, dephosphorylation of NFATcl, and is characterized by increasing the transcriptional activity of NFATcl.

The degenerative brain diseases include down syndrome, Alzheimer's disease (alzheimer's disease) ， _. It is preferably, but not limited to, any one selected from the group consisting of parkinson's disease, hunt ington's disease and pick's disease. In a specific embodiment of the present invention, the inventors have found that 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8—carboxylic acid (CX-4945), known as a CK2 inhibitor, As a result of confirming the inhibitory effect against Dyrk, among the various Dyfk proteins, DyrklA and DyrklB are the most potent and high selectivity inhibitors, and DyrklA inhibitor is 20 times stronger than the known DyrklA inhibitor Harmine. (See FIG. 3) and the inhibition of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxic acid (CX-4945) in an ATP competitive manner. Confirmed by biochemical methods (see FIG. 4), and in molecular modeling studies, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) -A-DyrklA To form hydrogen bonds in the ATP binding pocket Was (see Fig. 5) it confirmed. In addition, the present inventors confirmed that the phosphorylation of Tau, a representative substrate of DyrklA in the cells, the phosphorylation that did not appear when overexpressing only Tau protein is clearly seen when overexpressing DyrklA, and here 5-[(3-chloro Phenyl) amino] benzo [c] -2,6—naphthyridine-8-carboxylic acid (CX-4945) was found to decrease the phosphorylation of Tau with treatment, which was stronger than Harmine. (See FIG. 6A), the other DyrklA inhibitors INDY and proINDY were more effective than 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945). It was confirmed that the inhibitory effect was about 10 times lower (see FIG. 6b), and that it also inhibited the phosphorylation of the well-known APP myloid precursor protein and PSKpreseni I in 1). As with the Tau protein phosphorylation, the phosphorylation of APP and PS1 was Decreasing It was confirmed (Fig. 7, see) a. In addition, the inventors have identified whether 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridane-8-carboxylic acid (CX-4945) can modulate DyrklA related calcineur in / NFAT signals. As a result, the flag-NFATcl fusion protein, which is transferred to the cytoplasm by DyrklA overexpression under ionomycin (IM) treatment conditions, becomes 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphti. It was confirmed to be rearranged back to the nucleus by treatment with lidine-8-carboxylic acid (CX-4945) (see FIG. 8), and as a result of confirming the transcriptional activity through the reporter including the NFATc-responsive element, IM And PMA (phorbol 12-myri state 13-acetate) significantly increased luciferase expression (more than 20-fold) and overexpressed DyrklA, resulting in a 1/3 (5.4-fold increase in luciferase) expression. Inhibited with 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine- When 8-carboxylic acid (CX-4945) was treated by concentration, it was confirmed that luciferase expression increased rapidly again (see FIG. 9).

In addition, the present inventors have identified at the individual level the inhibitory effect of DyrklA on 5-[(3-chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8-carboxylic acid (CX-4945) identified in cell experiments. DyrklA cloned the Drosophila homolog, the minibrain (mnb) gene, was cloned and identified using the UAS / Gal4 system. In the F1 generation, which crossed the wing promoter MS1096-Gal4 line with UAS-mimbrain (nmb), 90 It was confirmed that the incidence of L5 vein was abnormal in 5% or more individuals (see FIG. 10A), and the short vein phenotype due to such minibrain overexpression was 100 nM 5-[(3-chlorophenyl) amino. Benzo [c] _2,6-naphthyridine— was found to inhibit about 30% in Drosophila fed 8-carboxylic acid (CX-4945), 5 ^: [(3-chlorophenyl) amino] benzo for harmine. 10,00 100 times the optimal concentration of [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) It was confirmed that the 40% level of minibrain phenotype inhibition at 0 nM or more (Fig. 10b). In addition, the present inventors have a change in Drosophila eye development when Tau is expressed using an eye specific promoter, GMR-gal4. When simultaneously expressing minibrain and Tau This change was exacerbated when killing, and in each case treated with 5- [(3-chlorophenyl) amino] benzo [c] _2, 6-naphthyridane-8-carboxylic acid (CX-4945). In the case of minibrain and Tau changes were significantly inhibited (see Fig. 11a), the overexpression of the mini brain was confirmed the development of the central nervous system and peripheral nervous system, and 5- [(3— Chlorophenyl) amino] Benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) fed after 7 days of breeding embryos of the F1 generation of neuronal dysfunction by minibrain markedly (FIG. Lib), the neurological specific minibrain overexpression showed adult mortality more than 70% above the neurogenesis, 5- [(3-chlorophenyl) amino] benzo [c] _2, 6 Subjects treated with -naphthyridine-8-carboxylic acid (CX-4945) Since mortality was reduced by more than 65%, 5-[(3-chlorophenyl) amino] benzo [c] _2 and 6-naphthyridine-8-carboxylic acid (CX-4945) acted as inhibitors of minibrain, Drosophila DyrklA. (See FIG. 11C):

 In addition, the inventors of the present inventors have determined that 5- [(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945) exhibit DyrklA inhibitory efficacy even at higher animal levels. Experiments were performed in DyrklA overexpressing mice that mimic the syndrome, and as previously known, the phosphorylation of the T212 position of the Tau protein in the hippocampus of DyrklA overexpressing mice was markedly higher than in normal mice. 5 Hippocampus of DyrklA overexpressing mice 30 minutes after oral administration of [[3-chlorophenyl) amino] benzo [(;]-2,6-naphthyridine-8-carboxylic acid (CX-4945) at a concentration of 75 mg / kg Phosphorylation of the Tau protein present in the rat was found to be significantly lower than that of the control group vehi c le, which was similar to that seen in normal mice (FIG. 12). Reference) .

Accordingly, the 5- [(3—chlorophenyl) amino] benzo [(：]-2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention is potent inhibitory to DyrklA in vitro. Effect Inhibition of ATP in a competitive manner, potent inhibition of DyrklA at the cellular level, inhibition of Tau, APP, PS1 phosphorylation and neuronal entanglement, and inhibition of NFATc phosphorylation In addition, it was confirmed that the NFATc transcriptional activity was increased, and the Drosophila model significantly improved the development of wing, eye, and nervous system abnormalities in the overexpression of Drosophila DyrklA minibrain, and markedly decreased Tau protein phosphorylation in DyrklA overexpressed mice. As a result, it showed higher inhibitory efficacy than Harmine, INDY, and proINDY, which are generally used as DyrklA inhibitors, and thus, 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine of the present invention. -8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof is associated with DyrklA such as Down syndrome, Alzheimer's disease and Parkinson's disease The neurodegenerative disease can be useful in the prevention and the active ingredient of the therapeutic composition coming. 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the invention may be used in the form of pharmaceutically acceptable salts, salts Acid addition salts formed with pharmaceutically acceptable free acids are useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and Obtained from non-toxic organic acids such as al 3 -dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrile, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, Iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate , Suverate, Sebacate, Fumarate, Maliate, Butin- 1, 4-dioate, nucleic acid-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate terephthalate, benzenesulfonate , Toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartuite, methane Sulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates or mandelate.

 Acid addition salts according to the invention may be prepared by conventional methods, for example, by using an excess of 5-[(3-chlorophenyl) amino] benzo [c] _2, 6-naphthyridine-8-carboxylic acid (CX-4945). It can be prepared by dissolving in an aqueous acid solution and precipitating the salt with a water-soluble organic solvent such as methane using ethanol acetone or acetonitrile. Equivalent amount of 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) and acid or alcohol in water, and then the mixture is evaporated. It can also be prepared by drying by drying, or by suction filtration of the precipitated salt.

 Bases can also be used to make pharmaceutically acceptable metal salts. Alkali or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt at no cost, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the silver salts obtained are reacted with alkali metal or alkaline earth metal salts with suitable negative salts (eg silver nitrate).

 In addition, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention is not only a pharmaceutically acceptable salt, but also a conventional It includes all salts, hydrates, and solvates that can be prepared by the process.

Addition salts according to the invention can be prepared by conventional methods, for example 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) Narcissus Aqueous organic solvents such as acetone, methane, ethane, or acetonitrile may be dissolved and added to an excess of an organic acid or an acidic aqueous solution of an inorganic acid, followed by precipitation or crystallization. This mixture can then be evaporated from solvent or excess acid to evaporate and dried to yield additional salts or prepared by suction filtration of the precipitated salts.

 When the composition of the present invention is used as a medicament, 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable thereof Pharmaceutical compositions containing salts as active ingredients may be formulated and administered in various oral or parenteral dosage forms as described below, but are not limited thereto.

 Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, liquids, suspensions, emulsifiers, syrups, granules, elixirs, etc. Contains dextrose, sucrose, manny, sorbitan, cellulose and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols) . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellose, sodium carboxymethylcellose and / or polyvinylpyridine, optionally starch, agar, alginic acid or Disintegrating or boiling mixtures such as sodium salts thereof and / or absorbents, colorants, flavoring agents, and sweetening agents.

Pharmaceutical preparation containing 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient of the present invention. The topical composition may be administered parenterally, and the parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. Wherein 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable thereof, for formulation into a parenteral formulation Pharmaceutical compositions containing possible salts as active ingredients are mixed in water with stabilizers or buffers to form solutions or suspensions. And can be prepared in ampule or vial unit dosage forms. The composition may be sterile and / or contain preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and is common practice. It may be formulated according to the granulation or coating method. In addition, the dosage of the composition of the present invention to the human body may vary depending on the age, weight, sex dosage form, health condition and degree of disease of the patient, generally based on an adult patient weighing 60 kg, 0.001 to 1,000 mg / day, preferably 0.01 to 500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

In addition, the present invention is 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-ka- ^ " ^Λ 1 ^: (5- [(3-Ch 1 or opheny Γ ) am i no] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid; CX-4945) or pharmaceutically acceptable salts thereof And it provides a dietary supplement for improvement.

The CX-4945 preferably has a structure represented by the above [Formula 1] ^'

 The CX-4945 is characterized by inhibiting the activity of Dyrkl.

 Inhibition of DyrklA activity is characterized in that CX-4945 occurs by binding to the ATP binding pocket of DyrklA.

 The CX-4945 is characterized by inhibiting phosphorylation of Tau, APP and PS1 proteins through DyrklA inhibition, dephosphorylation of NFATc, and increasing transcriptional activity of NFATc.

The degenerative brain disease is preferably any one selected from the group consisting of Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Peek's disease, but is not limited thereto. 5-[(3-chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention exhibits a strong inhibitory effect on DyrklA in vitro. Inhibition of ATP in a competitive and competitive manner, strongly inhibiting DyrklA at the cellular level, inhibiting the phosphorylation of neurofibrillation and senile plaques, Tau, APP, PS1, and phosphorylation of NFATc Inhibition increased the transcriptional activity of NFATc and markedly improved the development of wing, eye, and nervous system developments in the Drosophila model of Drosophila DyrklA minibrain, and markedly enhanced phosphorylation of Tau protein in DyrklA overexpressed mice. By confirming the lowering, showing a higher inhibitory effect compared to Harmine, INDY, and proINDY generally used as a DyrklA inhibitor, 5-[(3-chlorophenyl) amino] Division [c] -2, 6- naphthyridin-8-carboxylic acid (CX- 4945) or a pharmaceutically acceptable salt thereof may be useful as an active ingredient of a dietary supplement for the prevention and improvement of degenerative brain diseases.

5-[(3-chlorophenyl) amino] benzo [c] _2, 6-naphthyridane-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof of the present invention prevents such degenerative brain diseases. and in order to be used for the improvement, it is subject to production by the ^"well-known various methods in sikpumhak or pharmaceutical field embracing itself or food chemical acceptable carrier, excipient, any food combined common as a diluent which can be taken orally It may also be prepared in form. Preferably in the form of beverages, pills, granules, tablets or capsules. The health functional food of the present invention may further include ingredients conventionally added and food-acceptable at the time of food production. For example, when prepared in a beverage, 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof of the present invention In addition, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc. may further include one or more components.

The amount that can be included as an active ingredient of the dietary supplement according to the present invention is degenerated It may be appropriately selected depending on the age, sex, weight status, and symptoms of the disease of a person who wants to prevent and improve sexual brain disease, and preferably, it may be included in an amount of 0.01 g to 10.0 g per day for an adult. By ingesting a dietary supplement that has the effect of preventing and improving degenerative brain disease can be obtained. The invention also relates to 5-[(3-chlorophenyl) amino] benzo [c] —2,6-naphthyridine-8 in isolated cells, in cel l-free or in vitro. -Carboxylic acid (5- [(3—Chlor phenyl) amino] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid; CX-4945) or a pharmaceutically acceptable salt thereof It provides a Dyrkl inhibition method comprising the step of treating.

But not always limited thereto and wherein: CX-4945 are preferable ^and, having a structure represented by the [formula 1].

 The CX-4945 is characterized by inhibiting the activity of Dyrkl.

 Inhibition of DyrklA activity is characterized in that CX-4945 occurs by binding to the ATP binding pocket of DyrklA.

The CX-4945 is characterized by inhibiting phosphorylation of the Tau protein through DyrklA inhibition, dephosphorylation of NFATc, and increasing transcriptional activity of NFATc. 5 — [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention has a potent inhibitory effect on DyrklA in vitro. It inhibits Ayr in a competitive manner with ATP, and strongly inhibits DyrklA at the cellular level, inhibits phosphorylation of Tau, APP and PS1, which are neurofibrillation and senile plaques, and inhibits NFATc phosphorylation. In addition, it was confirmed that the NFATc transcriptional activity was increased, and that Drosophila DyrklA minibrain significantly improved wing, eye and nervous system development in Drosophila model, and Tau protein phosphorylation was markedly low in DyrklA overexpressed mice. In general, DyrklA 5 — [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention by showing higher inhibitory efficacy compared to Harmine, INDY and proINDY used as inhibitors. ) E is a pharmaceutically acceptable salt thereof can be usefully used as a Dyrkl inhibition method for biological research involving Dyrkl.

In addition, the present invention provides a pharmaceutically effective amount of 5-[(3-chlorophenyl) amino] benzo

[c] -2, 6-naphthyridine-8-carboxylic acid (5- [(3-Ch 1 or opheny 1) ami no] benzo [c] -2,6- naphthyridine-8-carboxylic acid; CX—4945) It provides a method for treating degenerative lethal disease comprising the step of administering to a subject suffering from degenerative brain disease.

 In addition, the present invention provides a pharmaceutically effective amount of 5-[(3-chlorophenyl) amino] benzo

[c] -2,6-naphthymodine-8-carboxylic acid (5-[(3-Chlorophenyl) amino] benzo [c; 卜 2,6-naphthyridine-8-carboxylic acid; CX—4945) administered to a subject Provided is a method for preventing degenerative brain disease, comprising the steps of: 유효한 The pharmaceutically effective amount implies an amount sufficient to treat the disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type of disease of the individual. , Severity, drug activity, drug sensitivity, time of administration, route of administration and rate of administration, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention exhibits a strong inhibitory effect on DyrklA in vitro. Inhibition in a competitive manner with ATP, and strongly inhibits DyrklA at the cellular level, inhibits the phosphorylation of Tau, APP, PS1, which causes neurofibrillation and senile plaques, and inhibits phosphorylation of NFATc. ， Increasing NFATc transcriptional activity, and in Drosophila model, Drosophila DyrklA minibrain overexpressed wing, eye and nervous system development. By confirming that the phosphorylation of the Tau protein is significantly lowered, it shows higher potency than Harmine, INDY, and proINDY, which are generally used as DyrklA inhibitors, and thus the 5-[(3-chlorophenyl) amino] benzo of the present invention. [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof has a potent effect on degenerative brain disease, and thus may be useful for the prevention and treatment of degenerative brain disease. Can be.

Advantageous Effects

The present invention relates to 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxy-¾: (5- [(3-Ch 1 or opheny 1) am i no] benzo [c] -2, 6 ^ napht hyr idi ne-8-c ar boxy 1 ic acid; CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention and treatment of degenerative brain diseases 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945), known as a potent inhibitor of CK2 and Cdc2-like kinase (Clk) It has been shown to exhibit potent inhibitory effects on DyrklA in vitro and to inhibit ATP in a competitive manner, and to inhibit DyrklA at the cellular level. Tau, APP, a microtubule-related protein , Inhibition of PS1 phosphorylation, dephosphorylation of NFATc, increased NFATc transcriptional activity, and Drosophila DyrklA minibrain in overdose It was confirmed that significantly improved the development of wing ^' , eyes, nervous system abnormality, and markedly lower phosphorylation of Tau protein even in DyrklA overexpressing mice, Harmine, commonly used as a DyrklA inhibitor INDY, showing a higher inhibitory effect compared to proINDY, the 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8—carboxylic acid (CX-4945) is associated with DyrklA. It can be usefully used as a composition for preventing and treating degenerative brain diseases. [Brief Description of Drawings] 1 is 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (5-[(3-Chlorophenyl) amino] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid; CX # 4945).

 Figure 2 shows 5-[(3-chlorophenyl) amino] benzo [c] —2,6-naphthyridine-8-carboxylic acid (CX-4945) and Harmine, INDY and proINDY, previously known as the most potent inhibitors of DyrklA. Figure shows the structure of.

 3 is a diagram showing the inhibitory effect on Dyrk by performing kinase profiling assay:

 (A) is a diagram showing the inhibitory effect of 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphthymodine-8-carboxylic acid (CX-4945) on Dyrk, (B) Figure shows the inhibitory effect of Harmine on Dyrk.

 4 is a diagram showing the ATP competitive DyrklA inhibitory effect of 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945).

 5 (A) shows molecular modeling results of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) and DyrklA. B) is a diagram showing a binding structure of 5-[(3_chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) and DyrklA.

 FIG. 6A (A) is a Western blot showing the phosphorylation inhibitory effect of Tau protein of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) (B) is a diagram confirmed by performing a western blotting experiment, (B) is a graph showing the quantification of the results of (A), (C) is a Western blot experiment to confirm the inhibitory effect of phosphorylation of Tau protein of Harmine (D) is a graph which quantifies the result of (C) and shows it graphically.

Figure 6b (A) is a diagram confirming the effect of inhibition of phosphorylation of Tau protein of INDY by performing a Western blot experiment, (B) is a diagram showing a graph quantifying the results of (A) (C) shows the effect of proINDY on inhibition of phosphorylation of Tau protein by Western blot experiment. (D) shows the graph quantifying the results with (C).

 FIG. 7A shows the effect of inhibiting APP myloid precursor protein phosphorylation of 5- [(3-chlorophenyl) amino] benzo [c] -2,6—naphthyridine-8-carboxylic acid (CX-4945). (B) is a graph quantifying the results of (A), (C) is 5- [(3-chlorophenyl) amino] benzo [c] _2, 6 PSKpreseni l in 1) phosphorylation inhibitory effect of -naphthyradine-8-carboxylic acid (CX-4945) was confirmed by Western blot experiment, and (D) is a graph quantifying the results of (C). .

 Figure 8 shows the location of Flag-NFATcl protein and its cell under various conditions: the upper left shows the location of the Flag-NFATcl protein without any treatment with the control (control), and the upper right shows the ionomycin. (Ionomycin, IM) shows the location of Flag-NFATcl protein, the left center shows the position of Flag-NFATcl protein after overexpressing DyrklA, and the bottom right shows the overexpression of DyrklA after IM treatment. NFATcl protein position, lower left shows 5-[(3_chlorophenyl) amino] benzo [c] -2, 6_naphthyridine-8-carboxylic acid (CX-4945) after overexpressing DyrklA following IM treatment ) And the location of the Flag-NFATcl protein.

 9 is a diagram showing the DyrklA inhibitory effect of 5- [(3-chlorophenyl) amino] benzo [c] _2 and 6-naphthyridine-8-carboxylic acid (CX-4945) measured through the transcriptional activity of NFATc. Figure 10a is a diagram showing the phenotype by wing-specific minibrain (mnb) overexpression.

Figure 10b (A) is a mini brain overexpressing Drosophila individuals according to the concentration of 5- [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) (B) is a diagram confirming the phenotype inhibitory effect of minibrain overexpressing Drosophila individuals according to the concentration of Ha ine. FIG. 11A shows 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid for phenotype with Drosophila eye specific minibrain overexpression and phenotype with Tau overexpression. CX-4945) confirmed the inhibitory effect.

 FIG. Lib shows inhibition of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on the neurogenic aberrant phenotype by Drosophila nervous system specific minibrain overexpression The effect is confirmed.

 11C shows the inhibitory effect of 5-[(3-chlorophenyl) amino] benzo [c] —2,6-naphthyridine-8-carboxylic acid (0 (-4945) on adult mortality by Drosophila nervous system specific minibrain overexpression. Figure is confirmed.

 Figure 12 shows 5 _ [(3-chlorophenyl) amino] benzo [c]-in DyrklA overexpressing mice.

Figure 2 shows the inhibitory effect of 6-naphthyridine-8-carboxylic acid (CX-4945):

(A) is a diagram confirming the phosphorylation of Tau protein in the hippocampus of normal and DyrklA overexpressing mice by Western blot experiment, (B) is a 5- [(3-chlorophenyl) amino in DyrklA overexpressing mice The effect of Tau protein phosphorylation on hippocampus after administration of benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) ol was confirmed by Western blot experiment.

[Best form for implementation of the invention]

 Hereinafter, the present invention will be described in detail by way of examples.

 However, the following examples are merely to illustrate the present invention, the contents of the present invention is not limited by the following examples.

Example 1 Dyrkl Inhibitory Effect of 5- [(3-Chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945)

Inhibits Cdc2-l ike kinase (Clk) such as TG-003, KH-CB19 and Leucett ine L ₄₁ Many known low molecular weight substances are known to inhibit Dyrk in most cases. This is because Clk and Dyrkl have kinase domains of similar sequence and structure. Accordingly, the present inventors have recently described the substance 5-C (3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) disclosed in FIG. It has been shown to have a strong inhibitory effect and similar to that of Dyrkl (Dual speci f ic i ty tyr os i ne-phosphory 1 at i on-r egu 1 at ed kinase 1), which is structurally similar to Clk in extension lines. The possibility of having efficacy was identified. In addition, Harmine, which is the most potent DyrklA inhibitor to date, which has been widely used in the structure shown in FIG. 2, and INDY and proINDY, which are known as inhibitors of another DyrklA, were used in the experiment.

Specifically, various concentrations of 5- [(3-chlorophenal) amino] benzo [d] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) were isolated and purified from DyrklA, DyrklB, Dyrk3 and Dyrk4. And kinase profiling (kinase prof il) using a kinase profiler service (provided by Invitrogen) using fluorescence-based immunoassay. ing) assays to determine the inhibitory effects of 5 _ [(3 ᅳ chlorophenyl) amino] benzo [c] -2,6_naphthyridine-8-carboxylic acid (CX-4945) on DyrklA, DyrklB, Dyrk3 and Dyrk4. CX-4945 concentrations of 0.001, 0.01, 0.1, 1, and 10 μΜ and ATP concentrations (ATP concentration equal to Km value for ATP), respectively, suitable for phosphatase activity of the protein, and measured using an IC ₅₀ Determine the value to determine 5- [(3-chlorophenyl) amino] benzo [(：]-2 for each kinase. 6-naphthyridin compared the inhibitory effects of naphthyridin-8-carboxylic acid (CX-4945). It confirmed the inhibitory effect of Hantiine as a control in the same manner as described above.

As a result, as shown in Fig. 3, 5-[(3-chlorophenyl) amino] benzo [c] _2,6-naphthyridine-8-carboxylic acid (0 (-4945) was found to be found in DyrklA and DyrklB, among other Dyrk proteins. Most potent and highly selective (IC ₅₀ 6.4-6.8 nM) Confirmed. In addition, when compared to the Harmine 5 _ [(3_-chlorophenyl) amino] benzo [c] 2,6- ^~ is 20 times the inhibitory effect of the DyrklA naphthyridin-8-carboxylic acid (0-4945) that the much stronger It was confirmed (FIG. 3).

 When analyzing the results and structure, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (0 (-4945) has a very similar structure to Harmine. Although it has a benzyl ring (Fig. 2), this difference is believed to be an important factor in the difference between DyrklA inhibitory effect, INDY and proINDY showed a slightly lower DyrklA inhibitory effect than Harmine Considering having, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) identified the most potent DyrklA inhibitor cancer.

Example 2 ATP Competitive Inhibitory Effect of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945)

 5- [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) inhibits DyrklA in a manner competing with ATP to determine whether [(3-Chlorophenyl) amino] benzo [c] —2,6-naphthyradine-8-carboxylic acid (CX-4945) in the treatment conditions, DyrklA activity was measured at different concentrations of ATP and This was confirmed by drawing a Lineweaver-Burk graph.

 As a result, as shown in FIG. 4, a typical ATP competitive mode suppression pattern was shown (FIG. 4). In addition, to confirm that 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) actually fits the ATP binding site of DyrklA. Molecular modeling studies through computer simulations were performed.

As a result, as shown in Fig. 5, 5-[(3 chlorophenyl) amino] benzo [c]- It was confirmed that 2,6-naphthyridine_8-carboxylic acid (0 (-4945) fits well in the ATP binding pocket of DyrklA (FIG. 5A), and the nitrogen and 5-[(3-chloro of L241 of DyrklA Phenyl) amino] two hydrogen bonds are expected between the carboxyl group of benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945), nitrogen of D307 and 5-[( 3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine—one hydrogen bond was predicted between the tricyclic nitrogen of 8-carboxylic acid (CX-4945) (FIG. 5B).

 This binding form is very similar to that found in the co-crystal structure between DyrklA, harmine, INDY, and proINDY in previous studies, and based on these results, 5-[(3-chlorophenyl) amino] benzo [c] -2. It was confirmed that, 6-naphthyridine-8-carboxylic acid (CX-4945) competitively binds to the ATP binding site of DyrklA, thereby showing inhibitory effect.

Example 3 DyrklA Inhibitory Effect of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) at the cellular level

 <3-1> 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid

(CX-4945) Inhibitory Effect of Tau Phosphorylation

 Mammalian cells to test whether 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridane-8-carboxylic acid (CX-4945) can inhibit DyrklA at the cellular level Phosphorylation of Tau, a representative substrate of DyrklA, was confirmed in 293T cells. Tau is a microtubule-related protein, and DyrklA phosphorylates mainly Thr212 of Tau protein, and this phosphorylation has been clearly observed in the hippocampal tissues of DyrklA-overexpressed Down syndrome model mice.

Specifically, 293T cells were incubated for 12 hours at 5xl0 ⁵ cell number in 6 well plates, and co-transformation of Tau and DyrklA expressing DNA 1 was performed. After incubation for 24 hours, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was added. After 6 hours of incubation with 0.001, 0.01, 0.1 and 1 μM treatment, cells were obtained and disrupted to obtain cell extracts containing the total protein of 293Τ cells. The entire protein was then developed by SDS-PAGE and transferred to a 0.45 polyvinyl idene fluoride transfer membrane (GE Healthcare, USA) to 5% skim milk. After blocking with anti-Tau antibody (Thermo), anti-pTau (T212) antibody (Invitrogen) and anti-Dyrkla antibody (Santa Cruz). Diluted 1: 1000 in tris buffered saline tween-20 (TBST) and treated the blocked transfer membrane to react overnight. Then, washed four times for 10 minutes each with TBST, and the second hanger was reacted. After reaction, wash each time 4 times with TBST for 10 minutes each time, the protein on the transfer membrane was washed with WEST-ZOL plus western blotting detection system (Intron Biotechnology, USA) and LAS — Protein phosphorylation level was detected using a LAS-4000 Image analyzer (FUJIFILM, Japan). Same method as above using another DyrklA inhibitor, Harmine, INDY or proINDY instead of 5-[(3-chlorophenyl) amino] benzo [c] -2,6—naphthyridine-8—carboxylic acid (CX-4945) as a control Was performed. As a control for comparing the amount of expression, the expression of hnRNPAl and GAPDH in the same manner using anti-hnRNP AKanti-hnRNA Al, Gideon Dreyfuss, University of Pennsylvania, USA) antibody and anti-GAPDH antibody as the primary antibody Confirmed.

As a result, as shown in Figures 6a to 6b, it was confirmed that the phosphorylation that did not appear when overexpressing only Tau protein in 293T cells was apparent when overexpressing DyrklA, where 5-[(3-chlorophenyl Phosphorylation of Tau decreased with increasing concentrations of amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945). (3_chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) shows a potent inhibitory effect of DyrklA. In addition, 5- [(3-chlorophenyl) amino] benzo at the cellular level It was confirmed that the Tau protein phosphorylation inhibitory effect of [c] _2,6-naphthyridine-8-carboxylic acid (CX-4945) was stronger than that of Harmine (FIG. 6A). In addition, Tau protein ^' phosphorylation inhibitory effect of 5 — [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was approximately 100-200 nM for IC _50. Harmine was slightly lower, 300-500 nM, and other DyrklA inhibitors, INDY and proINDY, were found to be about 2,000 nM. 5-[(3-chlorophenyl) amino] benzo [c] _2, 6-naphthy An inhibitory effect was confirmed about 10-fold lower than that of lidine-8-carboxylic acid (CX-4945) (FIG. 6B).

 As in <Example 1>, these results indicate that 5-[(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945) are the most potent DyrklA inhibitors. Confirmed.

<3-2> Amyloid precursor protein (PSKpreseni 1 in 1) phosphorylation of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) Inhibitory effect

 5-[(3-chlorophenyl) americo] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) inhibits DyrklA, in addition to Tau's phosphorylation, the well-known phosphorus of APP and PS1 It was confirmed whether oxidation was also inhibited.

 Specifically, after overexpressing APP and DyrklA, PS1 and DyrklA in the same manner as in Example <3-1>, 5 _ [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8 Carboxylic acid (CX-4945) was treated by concentration and cell extracts were obtained as anti-APP antibodies, anti-pAPP (T668) antibodies and anti-Dyrkla antibodies or anti-PS1 antibodies and anti- Dyrkla antibodies were used to detect phosphorylation levels of proteins. As a control group for comparing the expression amount, the expression of hnRNPAl and GAPDH was confirmed by the same method using anti-hnRNP A1 antibody and anti-GAPDH antibody as the primary antibody.

As a result, as shown in Fig. 7, it was confirmed that the phosphorylation of APP and PS1 was reduced as in the Tau protein phosphorylation (Fig. 7). These results clearly show the inhibition of 5- [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyradine ᅳ 8-carboxylic acid (CX-4945) and DyrklA, and in degenerative brain diseases Hyperphosphorylation of Tau, APP, and PS1, which is critical for the formation of common neuropathology, Amyloid plaque and Neurofibrillary tangles, is described as 5-[(3-chlorophenyl) amino] benzo [c] -2. It is an important result showing that, 6_naphthyridine-8-carboxylic acid (CX-4945) can be inhibited.

Example 4 Inhibition of DyrklA of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) in calcineurin / NFAT signal In the present study, we found that 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) is associated with DyrklA for the development of Down syndrome, Alzheimer's disease. We examined whether calcineurin / NFAT signals play an important role in.

Specifically, 293T cells expressing the Flag-NFATcl fusion protein were incubated with 2X10 ⁴ cell numbers in Slied 8 wells (ibidi) for 12 hours, followed by 20 minutes with 4% paraformaldehyde solution. After fixing the cells, the plate was washed three times with PBS, and then reacted for 5 minutes with 0.5% 'Triton X-100 solution and washed three times with PBS. Afterwards, reaction was performed with 2¾ BSA for 20 minutes to prevent other antibodies from binding. After reacting the primary antibody (anti-Flag: Sigma), the secondary antibody (ant i mouse-Alexa fluor 488: Invitrogen) was added again. After reaction, the resultant was washed three times with PBS and confirmed by fluorescence microscopy.

As a result, as shown in Figure 8, when confirming the position of the Flag-NFATcl fusion protein expressed in 293T cells, it can be seen that mainly stay in the cytoplasm (top left of Figure 8). At this time, when cells are treated with ionomycin (IM), a calcium ion carrier, Flag-NFATcl accumulates in the nucleus (Fig ^. 8, upper right). In addition, overexpression of DyrklA blocks nuclear accumulation of Flag-NFATcl and remains in the cytoplasm (middle of FIG. 8 left), — When IM was treated after overexpressing DyrklA, Flag-NFATcl remained in the cytoplasm (bottom right of FIG. 8), consistent with its role as a negative regulator of the calcineur in / NFAT pathway of DyrklA. The result is. In addition, after overexpressing DyrklA and treating with IM, 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) In spite of the presence of DyrklA, it was confirmed that Flag-NFATcl was rearranged back to the nucleus (bottom left of FIG. 8). The inhibitory effect of DyrklA on 5-[(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine—8-carboxylic acid (CX-4945) was also confirmed through the transcriptional activity of NFATc. Luciferase reporters containing NFAT-reponsive element (NFAT-RE) useful for measuring transcriptional activity were used. NFAT-RE-luciferase reporter and DyrklA were overexpressed in 293T cells and then treated with IM and PMA (phorbol 12-myr i state 13-acetate) to increase intracellular Ca ²⁺ concentration and 5- [(3-chlorophenyl ) Amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was treated by concentration.

 As a result, as shown in FIG. 9, the expression of luciferase was rapidly increased (more than 20 times) in the treatment of IM and PMA, and the expression of luciferase was increased to 1/3 (5.4 times increase) by overexpressing DyrklA. It was confirmed that it is suppressed). In addition, the expression of luciferase was returned when 5-[(3-chloropronyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was treated under these conditions by concentration. It was confirmed that the increase sharply. Even when 10 uM was treated, it was about 7 times more active than 5-[(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945). It was confirmed that this increased (Fig. 9).

These results confirmed the DyrklA inhibitory effect of 5- [(3-chlorophenyl) amino] benzo [c] — 2,6-naphthyridine-8-carboxylic acid (CX-4945) once again, Down syndrome. It demonstrates that NFAT signaling is associated with the development of Alzheimer's disease. Example 5 Validation of DyrklA Pumice Efficacy of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) in Minibrain Overexpressing Drosophila Model

 In order to confirm the inhibitory effect of DyrklA on the individual level of 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945) identified in the above cell experiment, Drosophila homologue, minibrain (mnb) gene, was cloned and tissue-specific gene overexpression was induced using the UAS / Gal4 system.

 Specifically, embryos of the F1 generation were collected by crossing with a Gal4 transgenic Drosophila that expresses a UAS-minibrain transgenic Drosophila and specifically, 5-[(3-chlorophenyl) amino] benzo [c] -2, 6-naphti. Drosophila standard medium containing 1, 10, 100, 1000, 10000 and 25000 nM of Lidan-8-carboxylic acid (CX-4945), respectively (dextrose 7.0%, yeast 5.0%, corn starch 3.5%, agar 0.5% , Propionic acid 0.5%, Tegosept 0.7%), and then analyzed the effect of CX-4945 based on the phenotype of the stratospheric wing.

As a result, as shown in Figures 10a to 10b, 90% of the F1 generation by crossing the wing promoter MS1096-Gal4 line and UAS-minibrain (mnb) to induce overexpression of minibrain specifically in the fruit fly wings In the above subjects, it was confirmed that the incidence of L5 vein was abnormal (FIG. 10a), and the short vein phenotype due to the minibrain overexpression was 100 nM 5-[(3-chlorophenyl) amino] benzo [ c] -2 and 6-naphthyridane-8-carboxylic acid (CX-4945) were fed about 30% inhibition in fruit flies, harmine 5- [(3-chlorophenyl) amino] benzo [c] _2, 6_ naphthyridine -8-carboxylic acid (CX-4945) It was confirmed that the inhibitory effect of the 40% level of minibrain phenotype at 10000 nM or more 100 times the optimal concentration (Fig. 10b). In addition, the Tau protein phosphorylation of DyrklA and the effect of inhibiting DyrklA phosphorylation activity of 5 ′ [(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945) To identify at the individual level, Drosophila overexpressed minibrain or Tau alone using GMR-gal4, a Drosophila eye specific promoter using the UAS / Gal4 system, or overexpressed minibrain and Tau together. [(3-Chlorophenyl) amino] Benzo [c] -2,6-naphthyridine-8-carboxylic acid (0 -4945) was grown in a medium containing the fruit fly's eyes were analyzed phenotype under the dissecting microscope.

 As a result, as shown in Figure 11a, when the expression of minibrain or Tau Drosophila eye development is changed, when the expression of minibrain and Tau at the same time it was confirmed that this change is more severe, 5- [( 3-chlorophenyl) amino] benzone [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945) were treated with the changes that minibrain and Tau significantly inhibited (Fig. 11a) ). In addition, after overexpressing minibrain in the nervous system using the nervous system-specific elav-Gal4 promoter line, the F1 generation embryos collected within 24 hours before waking into the first larva were collected and «fixed in formalin. Synaptobrevin-GFP, a fluorescent protein, was expressed together and observed under fluorescence confocal microscopy to analyze the nervous system structure and morphology. In addition, after overexpressing the mininbrain specifically in the nervous system, the adult mortality was confirmed by the number of waking layers after incubation for more than 10 days until the stratification.

As a result, as shown in lib to FIG. 11C, abnormal development of the central nervous system and peripheral nervous system was confirmed by overexpression of minibrain, and 5-[(3-chlorophenyl) amino] benzo [c]-was observed in the parent generation. 2, 6-naphthyridine-8-carboxylic acid (CX-4945) ols were fed for 7 days and then crossed to confirm that neuronal developmental abnormalities caused by minibrain were significantly suppressed in the better F1 generation embryos (FIG. Lib). Specific minibrain overexpression shows adult mortality of at least 70% above neuronal development, whereas 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxylic acid (CX-4945 ) Processed 65% Abnormal mortality was found to be reduced, so 5-[(3-chlorophenyl) amino] benzo [c] -2 and 6-naphthyridine-8-carboxylic acid (CX-4945) act as inhibitors of the minibrain, Drosophila DyrklA. It was confirmed (FIG. 11C). Example 6 5- [(3-chlorophenyl) amino] benzo [c]-in DyrklA overexpressing mice

Validation of DyrklA Inhibitory Effect of 2, 6-naphthyridine-8-carboxylic Acid (CX-4945)

 5- [(3-chlorophenyl) amino] benzo [c] — mimics Down's syndrome to determine whether 2,6-naphthyridine-8-carboxylic acid (0 (-4945) exhibits DyrklA inhibitory efficacy at higher animal levels Experiments were performed by preparing DyrklA overexpressing mice by a method known to Kichon (Ahn et al., 2006 Neurobiol Di s).

 As a result, as shown in FIG. 12, it was confirmed that the degree of phosphorylation of the T212 position of the Tau protein present in the hippocampus of DyrklA overexpressing mice was significantly higher than that of the normal mouse, and 5- [(3 -Chlorophenyl) amino] Tau protein present in the hippocampus of DyrklA overexpressing mice 30 minutes after oral administration of benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) at a concentration of 75 mg / kg Phosphorylation was confirmed to be significantly lower than that of the control vehicle, which is similar to the level seen in normal mice (Fig. 12). These results indicate that 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine-8-carboxic acid (CX-4945) is a blood-brain barr ier; BBB) effectively inhibits DyrklA.

Claims

[Range of request]  [Claim 1]  5-[(3-chlorophenyl) amino] benzo [(：]-2,6-naphthyridine-8-carboxylic acid (5-[(3- Ch 1 or opheny 1) am i no] benzo [c] -2 , 6-napht hyr idi ne-8-car boxy 1 ic acid; CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. [Claim 2]  The pharmaceutical composition for preventing and treating degenerative brain disease according to claim 1, wherein the CX-4945 inhibits the activity of DyrkKDual specificity tyros ine-phosphorylat ion-regulated kinase 1). [Claim 3]  The degenerative brain disease of claim 1, wherein the CX-4945 inhibits phosphorylation of Tau, APP (amyloid precursor protein) and PSKpresenilin 1) proteins, dephosphorylates NFATcl, and increases transcription activity of NFATcl. Pharmaceutical compositions for the prevention and treatment. [Claim 4]  The method of claim 1, wherein the degenerative brain disease is Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease. (huntington's disease) and pick's disease (pick's disease) is a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, characterized in that any one selected from the group consisting of. [Claim 5] 5-[(3-chlorophenyl) amino] benzo [<:]-2,6-naphthyridine-8-carboxylic acid (5-[(3- Chlorophenyl) ami no] benzo [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. [Claim 6]  [Claim 6] The health functional food for preventing and improving degenerative brain disease according to claim 5, wherein the CX-4945 inhibits the soaring of Dyrkl. [Claim 7]  The degenerative brain disease of claim 5, wherein the degenerative brain disease is any one selected from the group consisting of Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease and Pick's disease. [Claim 8]  5-[(3_chlorophenyl) amino] benzo [c] -2,6—naphthyridine-8-carboxylic acid (5- [in isolated cells, cell-free or in vitro) (3— Chlorophenyl) ami no] benzo [c] -2, 6-napht hyr i d i ne-8-car boxy 1 i c acid; CX-4945) or a pharmaceutically acceptable salt thereof. [Claim 9] Pharmaceutically effective amount of 5-[(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine 一 8 一 ^！ "Lboxic acid (5 一 [(3-Ch 1 or opheny 1) ami no] benzo [c] _2, 6-napht hyr idi ne— 8— carboxylic acid (CX-4945) to a subject with degenerative brain disease. [Claim 10]  Pharmaceutically effective amount of 5- [(3-chlorophenyl) amino] benzo [c] -2, 6-naphthyridine 一 8—carboxylic acid (5— [(3-Ch 1 oropheny 1) am i no] benzo [c] — 2, 6-napht hyr idine-8-carboxyl ic acid (CX-4945) to the subject comprising the step of preventing degenerative brain disease [Claim 11]  5-[(3-chlorophenyl) amino] benzo [c] -2,6phennaphthyridine-8-carboxylic acid (5-[(3-) for use as a pharmaceutical composition for the prevention and treatment of degenerative brain diseases. Chlorophenyl) amino] benzo [c] -2, 6-napht hyr i d i ne-8-car boxy 1 i c acid; CX—4945). [Claim 12]  5- [(3-chlorophenyl) amino] benzo [c] -2,6—naphthyridine-8-carboxylic acid (5-[(3-ailorophenyl) amino] benzo for preventing degenerative brain disease and as a dietary supplement [c] -2, 6-napht hyr idi ne-8-car boxy 1 ic acid; CX-4945).