KR101705226B1 - Pharmaceutical composition comprising CX-4945 for prevention or treatment of Degenerative Brain Diseases - Google Patents

Abstract

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (CX-4945) as an active ingredient. More particularly, the present invention relates to a pharmaceutical composition for preventing and treating degenerative brain diseases, which comprises CX2 and Cdc2-like kinase (Clk) 4945 showed a strong inhibitory effect on Dyrk1A in vitro and inhibited DTPA in a competitive manner with ATP and strongly inhibited Dyrk1A at the cellular level, resulting in nerve fiber entanglement and Tau , Amyloid precursor protein (APP) and PS1 (presenilin 1), inhibit NFATc phosphorylation, increase transcriptional activity of NFATc, and increase the expression of wings, eyes, and nerves when the Drosophila Dyrk1A minibrain is overexpressed in the Drosophila model. It was confirmed that the neural developmental abnormality was significantly improved INDY and proINDY, which are generally used as Dyrk1A inhibitors, by confirming that the phosphorylation of Tau protein is remarkably lowered in Dyrk1A overexpressing mice. Thus, 5 - [(3-chlorophenyl) Amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) can be usefully used as a composition for preventing and treating degenerative brain diseases associated with Dyrk1A.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating degenerative brain diseases containing CX-4945 as an active ingredient,

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient, to a pharmaceutical composition for the prevention and treatment of degenerative brain diseases.

Alzheimer's disease (AD) is a degenerative brain disorder that gradually declines in mental function as brain tissue loses function in the process of aging. The hallmark of this disease is that it causes serious obstacles to memory and emotion. In modern medicine, it is recognized as an 'incurable disease' with no clear treatment. It is one of the main causes of dementia which is mainly present in the elderly. Histopathological features include general atrophy of the brain, enlargement of the ventricles, multiple lesions of the nerve fibers and hyperchromatic spots. Clinical features include gradual decline of intellectual functions such as memory, judgment, and language ability, daily living skills, sight, and disability of behavior patterns. In addition, psychiatric symptoms such as depression, devastation of personality, and aggressive behavior are accompanied. These symptoms progress gradually and eventually lead to death. The period from the onset to the gradual death is about 6 to 8 years, but sometimes it is over 20 years depending on the person. In the United States, about 3 percent of the population aged 65 to 74, about 19 percent of the population aged 75 to 84, and 50 percent of the population aged 85 or older are suffering from the disease.

Dyrk1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) is a serine / threonine kinase that plays various roles in the adult central nervous system. The Dyrk1A gene is located in a human Down Syndrome critical region (DSCR), and the increased activity of Dyrk1A is associated with Down syndrome, Alzheimer's disease, Parkinson's disease, , Huntington ' s disease and pick ' s disease.

Transgenic mouse models overexpressing Dyrk1A in humans or rats exhibit hippocampal-dependent spatial learning and Down's syndrome phenotypes including motor neuron deficiency and developmental delays, which are important functions of Dyrk1A in Down's syndrome related mental retardation Suggesting. In particular, the stability of microtubule assembly, which is important for cytoskeletal maintenance, is abnormal in Alzheimer's disease and Down syndrome. In this process, Tau protein plays a pivotal role in controlling the microtubule stability. Abnormal Tau expression and hyperphosphorylation are common features in Alzheimer's and Down's syndrome. Dyrk1A plays an important role in the phosphorylation of various sites of Tau protein in various cell models. Among them, hyperphosphorylation occurs in the Thr-212 residue of Tau in the brain of patients with Alzheimer's disease, and Thr-212 residue of Tau is hyperphosphorylated in human Dyrk1A transgenic mice. Taken together, these results suggest that Dyrk1A may play an important role in the pathogenesis of hyperphosphorylation of Tau in Alzheimer 's disease and Down syndrome.

In particular, in Alzheimer's patients, in addition to the formation of neurofibrillary tangles composed of neuropathologically hyperphosphorylated Tau aggregates, the accumulation of insoluble precipitates of amyloid beta peptide is observed. Dyrk1A It is known that direct phosphorylation of the related amyloid β precursor protein, presenilin, etc., promotes the production of amyloid plaques, one of the important pathogenic agents of Alzheimer's disease.

Lewy bodies, one of the pathological features of Parkinson's disease, are also commonly found in the brain of Alzheimer's and Down's syndrome patients. Dyrk1A is a major component of the Lewy bodies, alpha-synuclein It is known that phosphorylation increases α-synuclein aggregation and eventually causes apoptosis.

In addition, Dyrk1A has been shown to phosphorylate a major protein HIP-1 associated with the onset of Huntington's disease and eventually participate in apoptosis and differentiation of hippocampal neuronal cells.

In addition, Dyrk1A regulates calcineurin / NFAT signaling and plays an important role in human developmental processes. NFATc transcription factors (transcription factors) are Usually while present in a protein that is phosphorylated in the cytoplasm is Ca ^{2 +} concentration in the cell goes up Ca ^{2 +} by-dependent protein phosphatase calcineurin NFATc the dephosphorylation and NFATc nuclear Lt; / RTI > The nucleated NFATc forms a transcription complex with the partner protein NFATn and binds to the target gene phtomotor to induce target gene expression. Dyrk1A induces phosphorylation of NFATc to migrate back to the cytoplasm, resulting in suppression of the expression of the target gene.

Amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid, 5 - [(3-chlorophenyl) amino] benzo [ naphthyridine-8-carboxylic acid, silmitasertib) is a protein that is frequently found in human cancer cells and is a protein that regulates various cellular activities such as cell growth and auto-apoptosis, such as casein kinase 2 (CK2) ≪ / RTI > Adam Siddiqui-Jain et al. Have shown that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) , And inhibits the proliferation of cancer cells by inhibiting the expression level of CK2a, a catalytic subunit of CK2 (Siddiqui-Jain A , et al. Cancer Res. 70 (24): 10288-10298, 2010 ). Clinical studies are underway to use 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) as a CK2 inhibitor. Safety and pharmacodynamic properties have been identified, and further evaluation of randomized drug combinations through secondary trials is underway (Sean E, Cylene Pharmaceuticals. Accessed 22 Mar 2011).

Recent studies have shown that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) plays an important role in intracellular pre- mRNA splicing as well as CK2 (Clk), which binds to CDC2. Interestingly, according to previous studies, many small molecules known to inhibit Clk, such as TG003, KH-CB19, Leucettine, and L41, are commonly known to inhibit Dyrk. This is because Clk and Dyrk1 have an ATP-binding pocket with a similar structure.

Accordingly, the present inventors have made efforts to investigate the role of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on Dyrk1A. And Ck-2,6-naphthyridine-8-carboxylic acid (CX-4945), known as potent inhibitors of Clk, are also potent against Dyrk1A in vitro Inhibitory effect of ATP and competitive inhibition of Dyrk1A at the cellular level, inhibiting nerve fiber entanglement and phosphorylation of Tau, APP and PS1, which induce senile factors, and inhibition of NFATc It was confirmed that NFATc transcriptional activity was increased by inhibiting phosphorylation, and the development of wings, eyes, and nervous system development that occurred when the Drosophila Dyrk1A minibrain was overexpressed was significantly improved in the Drosophila model. In the Dyrk1A overexpressing mouse, Lower (3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-5-carboxylic acid, which is generally superior to Harmine, INDY and proINDY used as Dyrk1A inhibitors, The inhibitory effect of 8-carboxylic acid (CX-4945) on Dyrk1A provides a useful tool for the study of degenerative brain diseases associated with Dyrk1A and can lead to the invention of new therapeutic strategies for diseases Respectively.

It is an object of the present invention to provide a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-dihydroxybenzo [c] -2,6-naphthyridine-8- -naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a pharmaceutical composition for preventing and treating degenerative brain diseases.

In order to achieve the object of the present invention, the present invention provides a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also relates to a method for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-dihydroxybenzo [c] -2,6-naphthyridine-8- -naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a health functional food for preventing and improving degenerative brain diseases.

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention relates to a pharmaceutical composition for preventing and treating degenerative brain diseases, which comprises a strong inhibition of CK2 and Cdc2-like kinase (CX-4945), which is known as a substance, has a potent inhibitory effect on Dyrk1A in vitro in the presence of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid Inhibits Dyrk1A at a cellular level, inhibits phosphorylation of microtubule-related proteins Tau, APP, PS1, dephosphorylates NFATc, and inhibits NFATc , And that in the Drosophila model, the wings, eyes, and nerves appear when the Drosophila minerbrain Dyrk1A is overexpressed And DYK1A overexpression mice were significantly lower than those of Harmine, INDY, and proINDY, which are generally used as Dyrk1A inhibitors, by confirming that the phosphorylation of Tau protein is significantly lowered in Dyrk1A overexpressing mice , And 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) can be usefully used as a composition for preventing and treating degenerative brain diseases associated with Dyrk1A have.

Figure 1 is a schematic illustration of a method for preparing 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (CX-4945).
FIG. 2 is a graph showing the effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) and Harmine, INDY and proINDY Fig.
Figure 3 shows the inhibitory effect on Dyrk by performing a kinase profiling assay:
(A) shows the inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on Dyrk, Harmine inhibitory effect on Dyrk.
FIG. 4 is a graph showing competitive ATP competitive Dyrk1A inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945).
Figure 5 (A) shows the molecular modeling results of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- Is a diagram showing the bonding structure of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) with DyrklA.
6A shows the effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on the phosphorylation inhibition of Tau protein by western blotting (B) is a graph obtained by quantifying the result of (A), and (C) is a figure confirmed by Western blotting experiment of the inhibitory effect of phosphorylation of Harmine on Tau protein , And (D) are graphs that quantify the results of (C).
FIG. 6A is a graph showing the phosphorylation inhibitory effect of INDY on Tau protein by Western blotting, FIG. 6B is a graph obtained by quantifying the result of (A), and FIG. (D) is a graph showing quantification of the results of (C). FIG.
7A shows the effect of inhibiting the amyloid precursor protein (APP) phosphorylation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- (B) is a graph obtained by quantifying the result of (A), and (C) is a graph showing the results of 5 - [(3-chlorophenyl) amino] benzo [ (CX-4945) inhibited the phosphorylation of PS1 (presenilins 1) by Western blotting, and (D) is a graph showing quantification of the results of (C).
Figure 8 is a diagram showing the intracellular location of the Flag-NFATc1 protein under various conditions:
The upper left shows the position of the Flag-NFATc1 protein in the control (no control) and the upper right shows the position of the Flag-NFATc1 protein after ionomycin (IM) treatment. In the middle, Dyrk1A was overexpressed and Flag-NFATc1 protein was located. On the lower right, IM, Dyrk1A was overexpressed and Flag-NFATc1 protein was located. On the lower left, Dyrk1A was overexpressed after IM treatment NFATc1 protein after treatment with 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945).
9 is a graph showing the Dyrk1A inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) measured through transcriptional activity of NFATc .
10A is a diagram showing a phenotype due to overexpression of a wing-specific minibrain (mnb).
Figure 10b (A) shows the phenotypic effect of minibrain-overexpressing Drosophila individuals on the concentration of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- (B) shows the phenotype inhibitory effect of minibrain overexpressing Drosophila individuals according to the concentration of Harmine.
11A is a graph showing the results of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945 ). ≪ / RTI >
Figure 11b shows the inhibition of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on neurogenic phenotypes induced by Drosophila nervosa system- The effect is confirmed.
Figure 11C shows the inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on adult mortality from over- It is also confirmed.
FIG. 12 shows the inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) in Dyrk1A-
(A) is a graph showing the phosphorylation of Tau protein in the hippocampus of a normal mouse and a Dyrk1A-overexpressing mouse by Western blotting. (B) ] Benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945), the effect of reducing the phosphorylation of the Tau protein in the hippocampus was confirmed by Western blotting.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine -8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a pharmaceutical composition for preventing and treating degenerative brain diseases.

The CX-4945 preferably has a structure represented by the following formula (1);

The CX-4945 is characterized by inhibiting the activity of Dyrk1 (Dual specificity tyrosine-phosphorylation-regulated kinase 1).

The inhibition of Dyrk1A activity is characterized in that CX-4945 binds to ATP binding pocket of Dyrk1A.

CX-4945 is characterized by inhibiting phosphorylation of Tau, amyloid precursor protein (APP) and PS1 (presenilin 1) protein through Dyrk1A inhibition, dephosphorylating NFATc1 and increasing transcription activity of NFATc1.

It is preferable that 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. It is not limited.

In a specific embodiment of the present invention we have found that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945), known as CK2 inhibitor, , It was found that Dyrk1A and Dyrk1B are the most potent and selective inhibitors of various Dyrk proteins and that the inhibitory effect of Dyrk1A is 20 times stronger than that of Harmine which is a known Dyrk1A inhibitor 3) and 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) were competitively inhibited by biochemical methods (See FIG. 4), and molecular modeling studies have also shown that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- (see FIG. 5). The hydrogen bond is formed by forming a hydrogen bond in the pocket.

In addition, the present inventors confirmed the phosphorylation of Tau, which is a representative substrate of Dyrk1A in the cells. As a result, the phosphorylation, which was not observed when the Tau protein alone was overexpressed, was conspicuously observed when Dyrk1A was overexpressed. Phenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was increased to decrease the phosphorylation of Tau and this effect was stronger than that of Harmine (See FIG. 6A), the other Dyrk1A inhibitors INDY and proINDY were found to be about 10 (3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- (See FIG. 6B). As a result, it was confirmed that the inhibition of the phosphorylation of APP (amyloid precursor protein) and PS1 (presenilin 1) ( 7).

The present inventors also confirmed that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) can control DYRK1A-related calcineurin / NFAT signal , And the position of the Flag-NFATc1 fusion protein expressed in the cells is confirmed, it is mainly in the cytoplasm. After ionomycin (IM) treatment, Dyrk1A is overexpressed and then 5 - [(3-chlorophenyl) amino] benzo (CX-4945), it was confirmed that Flag-NFATc1 was rearranged to the nucleus even though DyrklA was present (see FIG. 8), and NFATc reporter The expression of luciferase was dramatically increased (over 20 times) when IM and PMA (phorbol 12-myristate 13-acetate) were treated and overexpressed Dyrk1A, / 3 < / RTI > (5. 4-fold increase), and under this condition, 5 - [(3-chlorophenyl) [C] it was confirmed that the expression of luciferase rapidly increased again when the handle-2,6-naphthyridin-8-carboxylic acid (CX-4945) at different concentrations (see Fig. 9).

Further, the present inventors confirmed the Dyrk1A inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) (Mnb) gene, which is a Drosophila homologue of Dyrk1A, was cloned and analyzed using the UAS / Gal4 system. As a result, it was confirmed that in the F1 generation that crosses the wing promoter MS1096-Gal4 line and UAS-minibrain (mnb) (Fig. 10A), and a short vein phenotype due to over-minibrain overexpression was observed in 100 nM 5 - [(3-chlorophenyl) amino] Benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was inhibited by about 30% c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) Level minibrain phenotype inhibitory effect (Fig. 10B).

In addition, the present inventors confirmed that when Tau was expressed using GMR-gal4, which is an eye-specific promoter, a change occurred in the development of fruit fly, and when the minibrain and Tau were expressed at the same time, these changes were more severe (CX-4945) treated with 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid significantly inhibited changes in minibrain and Tau (See FIG. 11A), and the development of central nervous system and peripheral nervous system was confirmed by overexpression of minibrain, and 5 - [(3-chlorophenyl) amino] benzo [c] -2,6- (Fig. 11B), the nervous system-specific minibrain overexpression was significantly inhibited by the minibrain-induced embryonic development of the embryo of the F1 generation that had been fed with riboflavin-8-carboxylic acid (CX-4945) More than 70% (CX-4945) treated with 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) showed a mortality rate of more than 50% (CX-4945) acts as an inhibitor of the minibrain, a homologue of Drosophila Dyrk1A, in the presence of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (See Fig. 11C).

In addition, the present inventors have also found that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) inhibits Dyrk1A- As a result, it was confirmed that the degree of phosphorylation of the Tau protein located in the hippocampus of the Dyrk1A-overexpressing mouse was significantly higher than that of the normal mouse, 30 minutes after oral administration of [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) at a concentration of 75 mg / kg was administered to the hippocampus of Dyrk1A- Phosphorylation of the existing Tau protein was found to be significantly lower than that of the control vehicle, which was similar to that seen in normal mice (see FIG. 12).

Thus, the 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) of the present invention has a potent inhibitory effect on Dyrk1A in vitro In addition, it suppresses Dyrk1A at cellular level, inhibits nerve fiber entanglement and inhibits phosphorylation of Tau, APP, PS1, which induces senile factors, and inhibits NFATc phosphorylation. , It was confirmed that the transcription activity of NFATc was increased and the development of wings, eyes, and nervous system development that occurred when the Drosophila Dyrk1A minibrain was overexpressed was significantly improved in the Drosophila model and the phosphorylation of Tau protein was significantly lowered in Dyrk1A- (3-chlorophenyl) amino] benzo [c] -2,6-diazabicyclo [2.2.1] heptane of the present invention, as compared with Harmine, INDY and proINDY which are generally used as Dyrk1A inhibitors (CX-4945) or a pharmaceutically acceptable salt thereof may be useful as an active ingredient of a composition for preventing and treating degenerative brain diseases associated with Dyrk1A such as Down syndrome, Alzheimer's disease, Parkinson's disease, and the like.

The 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention can be used in the form of a pharmaceutically acceptable salt, Acid addition salts formed by pharmaceutically acceptable free acids are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the invention can be prepared by a conventional method, for example by reacting 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- In an acid aqueous solution, and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Heating the same amount of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) and an acid or alcohol in water, Or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

The 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention can be obtained not only by a pharmaceutically acceptable salt, Including all salts, hydrates, and solvates that may be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method, for example, by reacting 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- Is dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like and added with an excessive amount of an organic acid or an aqueous acid solution of an inorganic acid, followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and then dried to obtain an additional salt, or the precipitated salt can be produced by suction filtration.

When the composition of the present invention is used as a medicament, it is preferable to use 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- May be formulated and administered in a variety of oral or parenteral administration forms, such as, but not limited to, the following formulations at the time of clinical administration.

Examples of the formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations may contain, in addition to the active ingredient, a diluent (e.g., lactose, dextrose, (E.g., silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycols), such as, for example, water, rosin, sucrose, mannitol, sorbitol, cellulose and / or glycine. The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain additives such as starch, agar, alginic acid or its sodium salt A disintegrating or boiling mixture and / or an absorbent, a colorant, a flavoring agent, and a sweetening agent.

The pharmaceutical composition containing the 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient The composition may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. At this time, 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof is formulated for parenteral administration As an active ingredient may be mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which may be prepared into an ampule or vial unit dosage form. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

The dose of the composition of the present invention to the human body may be varied depending on the age, body weight, sex, dosage form, health condition, and disease severity of the patient. When the patient is 60 kg body weight, And may be 0.01 to 500 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.

The present invention also relates to a method for the preparation of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-dihydroxybenzo [c] -2,6-naphthyridine-8- -naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a health functional food for preventing and improving degenerative brain diseases.

The above-mentioned CX-4945 preferably has a structure represented by the above formula (1).

The CX-4945 is characterized by inhibiting the activity of Dyrk1.

The inhibition of Dyrk1A activity is characterized in that CX-4945 binds to ATP binding pocket of Dyrk1A.

The CX-4945 is characterized by inhibiting phosphorylation of Tau, APP, and PS1 proteins through Dyrkl A inhibition, dephosphorylating NFATc, and increasing the transcriptional activity of NFATc.

The degenerative brain disease is preferably one selected from the group consisting of Down's syndrome, Alzheimer's disease, Parkinson's disease and Huntington's disease, but is not limited thereto.

The 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention shows a strong inhibitory effect on Dyrk1A in vitro In addition, it suppresses Dyrk1A at a cellular level, inhibits nerve fiber entanglement, inhibits phosphorylation of Tau, APP, PS1, which induces senile factors, inhibits phosphorylation of NFATc, It was confirmed that the transcription activity of NFATc was increased and the wings, eyes and nervous system developmental abnormalities observed when the Drosophila Dyrk1A minibrain was overexpressed were significantly improved in the Drosophila model and the phosphorylation of Tau protein was significantly lowered in the Dyrk1A overexpressing mouse INDY, and proINDY, which are generally used as Dyrk1A inhibitors, the present inventors have found that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6- 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.

The use of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof for the prevention of degenerative brain diseases And to be used for improvement, it may be prepared by a variety of methods known in the food or pharmaceutical arts and may be prepared in any food form that can be taken orally by itself or in combination with a pharmaceutically acceptable carrier, excipient, diluent, Can also be produced. Preferably in the form of beverage, ring, granule, tablet or capsule.

The health functional food of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable. For example, in the case of beverage preparation, 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) or its pharmaceutically acceptable salt In addition, one or more components may be further included in citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, and the like.

The amount of the active ingredient of the health functional food according to the present invention may be appropriately selected depending on the age, sex, weight, condition and symptom of a person who desires to prevent or improve the degenerative brain disease, Day to 0.01 g to 10.0 g. By taking the health functional food having such a content, prevention and improvement of degenerative brain diseases can be obtained.

The present invention also relates to the use of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylate in isolated cells, cell-free or in vitro A method for inhibiting Dyrk1 comprising the step of treating (5 - [(3-Chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof to provide.

The CX-4945 preferably has a structure represented by the formula (1), but is not limited thereto.

The CX-4945 is characterized by inhibiting the activity of Dyrk1.

The inhibition of Dyrk1A activity is characterized in that CX-4945 binds to ATP binding pocket of Dyrk1A.

The CX-4945 is characterized by inhibiting phosphorylation of Tau protein through Dyrkl A inhibition, dephosphorylating NFATc, and increasing the transcriptional activity of NFATc.

The 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) of the present invention shows a strong inhibitory effect on Dyrk1A in vitro In addition, it suppresses Dyrk1A at a cellular level, inhibits nerve fiber entanglement, inhibits phosphorylation of Tau, APP, PS1, which induces senile factors, inhibits phosphorylation of NFATc, It was confirmed that the transcription activity of NFATc was increased and the wings, eyes and nervous system developmental abnormalities observed when the Drosophila Dyrk1A minibrain was overexpressed were significantly improved in the Drosophila model and the phosphorylation of Tau protein was significantly lowered in the Dyrk1A overexpressing mouse INDY, and proINDY, which are generally used as Dyrk1A inhibitors, the present inventors have found that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6- 8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof may be useful in inhibiting Dyrk1 methods for biological research related to Dyrk1.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1 > 5 - [(3- Chlorophenyl ) Amino] Benzo  [c] -2,6- Naphthyridine -8-Carboxylic acid (CX-4945) Dyrk1 Inhibitory Effect

Many small molecules known to inhibit Cdc2-like kinase (Clk), such as TG 003, KH-CB19 and Leucettine L41, are known to inhibit Dyrk in common. This is because Clk and Dyrk1 have kinase domains of similar sequence and structure. Thus, the present inventors have found in recent studies that a substance called 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) Inhibitory potency, and confirmed the possibility of similar efficacy against Dyrk1 (dual specificity tyrosine-phosphorylation-regulated kinase 1), which is structurally similar to Clk in the extension line. In addition, Harmine (Harmine), which is known to be the most powerful Dyrk1A inhibitor to date, which has been widely used, and INDY and proINDY, which are known inhibitors of Dyrk1A, have been used in the experiments.

Specifically, the purified purified Dyrk1A, Dyrk1B, Dyrk3, and Dyrk4 with various concentrations of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- And then subjected to a kinase profiling analysis using a Kinase Profiler service (Invitrogen) using fluorescence-based immunoassay The inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on Dyrk1A, Dyrk1B, Dyrk3 and Dyrk4 was evaluated as 0.001, 1 and 10 μM of CX-4945, and ATP concentration (ATP concentration equal to Km for ATP) appropriate for the protein phosphorylase activity, respectively, and IC ₅₀ Values were determined to compare the inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on each phosphorylase. The inhibitory effect of Harmine as a control group was confirmed in the same manner as above.

As a result, 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was found to be more effective than Dyrk1A and Dyrk1B The strongest and most selective (IC ₅₀ 6.4 ~ 6.8 nM). The inhibitory effect of Dyrk1A on 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) (Fig. 3).

When the above results and structure were analyzed, it was found that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- Considering that INDY and proINDY have a slightly lower inhibitory effect on Dyrk1A than Harmine, it is suggested that this difference is due to 5 - [( 3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was the most potent Dyrk1A inhibitor.

< Example  2 > 5 - [(3- Chlorophenyl ) Amino] Benzo  [c] -2,6- Naphthyridine -8-Carboxylic Acid (CX-4945) Competitive Inhibitory Effect of ATP

To confirm that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) inhibits Dyrk1A in a manner that competes with ATP, The activity of Dyrk1A was measured by varying the concentration of ATP under the treatment conditions of [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) Burk graph was drawn.

As a result, as shown in Fig. 4, a typical ATP competitive inhibition pattern appeared (Fig. 4).

In order to confirm whether 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) is well fit in the ATP binding site of Dyrk1A, Molecular modeling studies were carried out.

As a result, as shown in Fig. 5, it was confirmed that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- 4945) binds to the ATP binding pocket of DyrklA (FIG. 5A), and the nitrogen of L241 of DyrklA and the carboxyl group of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- two hydrogen bonds between the carboxyl group and the nitrogen of D307 are predicted and the hydrogen bond of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) One hydrogen bond between the tricyclic nitrogen was predicted (Fig. 5B).

This type of coupling is very similar to the one found in the previous studies through the co-crystal structure between Dyrk1A and harmine, INDY, and proINDY. Based on these results, it is concluded that 5 - [(3-chlorophenyl) amino] benzo [ , And 6-naphthyridine-8-carboxylic acid (CX-4945) competitively bind to the ATP binding site of DyrklA.

< Example  3> 5 - [(3- Chlorophenyl ) Amino] Benzo  [c] -2,6- Naphthyridine -8-carboxylic acid (CX-4945) inhibitory effect on Dyrk1A

&Lt; 3-1 > 5 - [(3- Chlorophenyl ) Amino] Benzo  [c] -2,6- Naphthyridine -8-carboxylic acid ( CX -4945) inhibited Tau phosphorylation

To test whether 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) can inhibit Dyrk1A at the cellular level, 293T The phosphorylation of Tau, a representative substrate of Dyrk1A, was confirmed in the cells. Tau is a microtubule associated protein, Dyrk1A mainly phosphorylates Thr212 of Tau protein, and this phosphorylation has been clearly observed in hippocampal tissues of Down syndrome model mice overexpressing Dyrk1A.

Specifically, 293T cells were cultured in 6-well plates at ⁵ × 10 ⁵ cells for 12 hours and co-transformed with 1 μg each of Tau and Dyrk1A-expressing DNA. After culturing for 24 hours, 0.001, 0.01, 0.1, and 1 μM of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- After incubation, cells were harvested and disrupted to obtain a cell extract containing the entire protein of 293T cells. Then, the whole protein was developed by SDS-PAGE and transferred to a polyvinylidene fluoride transfer membrane (GE Healthcare, USA) of 0.45 μm in size, and the mixture was transferred to 5% skim milk (Anti-Tau antibody) (Thermosa), anti-pTau (T212) antibody (Invitrogen) and anti-Dyrk1a antibody (Santa Cruz) as a primary antibody were mixed with 5% skim milk containing tris buffer Diluted 1: 1000 with tris-buffered saline tween-20 (TBST), and treated with the blocking membrane to react overnight. Then, the cells were washed with TBST four times for 10 minutes each, and the secondary antibody was reacted. After the reaction, the cells were rinsed with TBST four times for 10 minutes each, and the proteins on the moving membrane were washed with WEST-ZOL plus western blotting detection system (Intron Biotechnology Co., USA) and LAS- 4,000 image analyzer (LAS-4000 Image analyzer, Fuji Film, Japan) was used to detect the phosphorylation level of the protein. INDY or proINDY which is another DyrklA inhibitor instead of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) as a control, Respectively. HNRNPA1 and GAPDH were expressed in the same manner as above using anti-hNRNP A1 (anti-hnRNA A1, Gideon Dreyfuss, University of Pennsylvania, USA) antibody and anti-GAPDH antibody as primary antibodies for comparison of expression levels Respectively.

As a result, as shown in FIGS. 6A and 6B, it was confirmed that phosphorylation, which was not observed when the Tau protein was overexpressed in 293T cells, was clearly observed when Dyrk1A was overexpressed, and 5 - [(3-chlorophenyl) amino ] Benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) at the cellular level, Amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) showed strong inhibitory effect of DyrklA. The Tau protein phosphorylation inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) at the cellular level was also confirmed to be stronger than that of Harmine (Fig. 6A). In addition, 5 - [(3-chlorophenyl) amino] and benzo [c] -2,6- Tau Protein Phosphorylation inhibitory effect of the 6-naphthyridin-8-carboxylic acid (CX-4945) has IC ₅₀ of approximately 100-200 nM, Harmine was slightly lower at 300-500 nM and the other Dyrk1A inhibitors INDY and proINDY were found to be about 2,000 nM, indicating that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridin- And about 10-fold lower inhibitory effect than that of carboxylic acid (CX-4945) (Fig. 6B).

These results indicate that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) is the most potent Dyrk1A inhibitor Respectively.

&Lt; 3-2 > 5 - [(3- Chlorophenyl ) Amino] Benzo  [c] -2,6- Naphthyridine -8-carboxylic acid ( CX -4945), amyloid precursor protein (APP), PS1 (presenilin 1) phosphorylation inhibitory effect

In addition to the phosphorylation of Tau by inhibiting Dyrk1A, the phosphorylation of APP and PS1, also known as 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- Respectively.

Specifically, APP and Dyrk1A, PS1 and Dyrk1A were overexpressed in the same manner as in Example <3-1>, and then 5 - [(3-chlorophenyl) amino] benzo [c] (T668) antibody and an anti-Dyrk1a antibody or an anti-PS1 antibody and an anti-Dyrk1a antibody (CX-4945) as a primary antibody after treatment with 8-carboxylic acid Antibodies were used to detect the phosphorylation level of the protein. The expression levels of hnRNPA1 and GAPDH were determined using the anti-hNRNP A1 antibody and the anti-GAPDH antibody as primary antibodies.

As a result, as shown in Fig. 7, it was confirmed that phosphorylation of APP and PS1 was reduced similarly to Tau protein phosphorylation (Fig. 7).

These results clearly demonstrate Dyrk1A inhibition of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945), which is common in degenerative brain diseases Hyperphosphorylation of Tau, APP and PS1, which are essential for the formation of neurofibrillary tangles and neurofibrillary tangles, is studied by using 5 - [(3-chlorophenyl) amino] benzo [c] Naphthyridine-8-carboxylic acid (CX-4945).

< Example  4> Calcineurin / Enchant ( calcineurin / NFAT ) &Lt; / RTI &gt; signal from 5 - [(3- Chlorophenyl ) Amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945)

The present inventors have confirmed that 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) can modulate Dyrk1A-related calcineurin / NFAT signals.

Specifically, 293T cells expressing the Flag-NFATc1 fusion protein were cultured in Slied 8 well (ibidi) at 2 × 10 ⁴ cells for 12 hours, and then cultured in 4% paraformaldehyde solution for 20 minutes After fixing the cells, the plates were washed three times with PBS, reacted with 0.5% Triton X-100 solution for 5 minutes, and washed three times with PBS. The cells were then incubated with 2% BSA for 20 minutes to allow the other antibodies to bind. Anti-Flag antibody (Alexa fluor 488, Invitrogen Inc.) After the reaction, the cells were washed three times with PBS and confirmed by fluorescence microscopy.

As a result, as shown in Fig. 8, when the position of the Flag-NFATc1 fusion protein expressed in 293T cells is confirmed, it is mainly found in the cytoplasm (Fig. 8, top left). At this time, when the cell is treated with ionomycin (IM), which is a calcium ion carrier, Flag-NFATc1 accumulates in the nucleus (upper right of FIG. 8). In addition, overexpression of Dyrk1A blocks nuclear accumulation of Flag-NFATc1 and remains in the cytoplasm (Fig. 8, left center). When IM is treated and Dyrk1A is overexpressed, Flag-NFATc1 still remains in the cytoplasm , Which is consistent with the role of Dyrk1A as a negative regulator of the calcineurin / NFAT pathway. In addition, Dyrk1A was found to be present when 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- , It was confirmed that Flag-NFATc1 was rearranged to the nucleus (lower left of FIG. 8).

The Dyrk1A inhibitory effect of this 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was also confirmed by the transcriptional activity of NFATc. A luciferase reporter containing the NFAT reponse element (NFAT-RE) useful for measuring transcriptional activity was used. NFAT-RE-luciferase reporter was overexpressed in 293T cells, treated with IM and PMA (phorbol 12-myristate 13-acetate) to increase intracellular Ca ²⁺ concentration, overexpress Dyrk1A, Phenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945).

As a result, as shown in FIG. 9, when IM and PMA were treated, the expression of luciferase was abruptly increased (over 20 times), and overexpression of Dyrk1A resulted in a luciferase expression of 1/3 ). &Lt; / RTI &gt; In addition, when the 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was treated at different concentrations under this condition, the expression of luciferase again increased Respectively. Even when treated with 10 uM, its activity was about seven times that of the case where 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- (Fig. 9).

The results showed that Dyrk1A inhibitory activity of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) Lt; RTI ID = 0.0 &gt; NFAT &lt; / RTI &gt; signaling.

< Example  5> Minibrain  In the overexpressed Drosophila model, 5 - [(3- Chlorophenyl ) Amino] Benzo  The inhibitory effect of [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on Dyrk1A

To confirm the Dyrk1A inhibitory effect of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) The minibrain (mnb) gene, which is a Drosophila homologue, was cloned and induced by tissue specific gene overexpression using the UAS / Gal4 system.

Specifically, embryos of the F1 generation were collected by crossing with a Gal4 transgenic flora expressing a tissue-specific expression of UAS-minibrain transformed Drosophila to obtain 5 - [(3-chlorophenyl) amino] benzo [c] (Dextrose 7.0%, yeast 5.0%, corn starch 3.5%, agar agar 0.5%) containing 1, 10, 100, 1000, 10000 and 25000 nM of threonine-8-carboxylic acid (CX- , propionic acid 0.5%, Tegosept 0.7%), and the effects of CX-4945 on the phenotype of adult wing were analyzed.

As a result, as shown in Figs. 10a to 10b, 90% of the F1 generations obtained by crossing the wing promoter MS1096-Gal4 line and UAS-minibrain (mnb) to induce the overexpression of the minibrain specifically in the Drosophila wing, (Fig. 10a). It was confirmed that this short-vein phenotype due to minibrain overexpression caused 100 nM 5 - [(3-chlorophenyl) amino] benzo [c ] -2,6-naphthyridine-8-carboxylic acid (CX-4945) was inhibited by about 30% in the fed florets and in the case of harmine, 5 - [(3-chlorophenyl) amino] benzo [c] It was confirmed that the inhibitory effect of minibrain phenotype was about 40% at 10000 nM or more which is 100 times the optimal concentration of 2,6-naphthyridine-8-carboxylic acid (CX-4945) (FIG.

In addition, the inhibitory effect of Dyrk1A on Tau protein phosphorylation and the Dyrk1A phosphorylation activity of 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX- , We used the UAS / Gal4 system to differentiate either minibrain or Tau alone using GMR-gal4, a Drosophila eye-specific promoter, or overexpressing both minibrain and Tau, using 5 - [(3- Phenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) in the eyes of the Drosophila adults.

As a result, as shown in Fig. 11 (a), when the minibrain or Tau was expressed, the change in the occurrence of the fruit fly was observed, and when the minibrain and the Tau were simultaneously expressed, (CX-4945) was significantly inhibited by minibrain and Tau (Fig. 11 (a)).

In addition, after minibrain overexpression in the nervous system using the elav-Gal4 promoter line specific to the nervous system, the F1 generation embryos within 24 hours before waking up to the first instar larvae were collected and fixed with 4% formalin, Synaptobrevin-GFP, a fluorescent protein, was also expressed and examined under a fluorescence confocal microscope to analyze the structure and morphology of the nervous system. In addition, after overexpression of mininbrain specifically for the nervous system, adult worms were repaired after 10 days or more incubation until adult worms were worn out and adult mortality was confirmed.

As a result, as shown in Fig. 11B to Fig. 11C, abnormalities of the central nervous system and peripheral nervous system were confirmed by overexpression of minibrain, and 5 - [(3-chlorophenyl) amino] benzo [c] It was confirmed that the nervous system abnormalities caused by the minibrain were remarkably inhibited in the F1 generation embryos obtained by feeding 6-naphthyridine-8-carboxylic acid (CX-4945) for 7 days and crossing (FIG. 11b) Overexpression was more than 70% lethal mortality compared to the development of the nervous system, whereas treatment with 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) (CX-4945) was found to inhibit the homology of Drosophila Dyrk1A with 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine- Lt; / RTI &gt; minibrain (Fig. 11C).

< Example  6> Dyrk1A  Overexpression of 5 - [(3- Chlorophenyl ) Amino] Benzo  The inhibitory effect of [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) on Dyrk1A

To confirm whether 5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) exhibits Dyrk1A inhibitory activity at higher animal levels, Dyrk1A Over-expressing mice were prepared by previously known methods (Ahn et al., 2006 Neurobiol Dis).

As a result, as shown in FIG. 12, it was confirmed that the degree of phosphorylation of the Tau protein in the hippocampus of the Dyrk1A overexpressing mouse was significantly higher than that of the normal mouse, Phosphorylation of Tau protein present in the hippocampus of Dyrk1A-overexpressing mouse 30 minutes after the oral administration of 75 mg / kg of the oral dyslipidemic acid (CX-495) Was significantly lower than that of the control vehicle, which was similar to that of normal mice (Fig. 12).

This result indicates that the 5-aminobenzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) inhibits the blood-brain barrier (BBB) Effectively inhibiting Dyrk1A. &Lt; / RTI &gt;

Claims (8)

8-carboxylic acid (5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the CX-4945 inhibits the activity of Dyrk1 (Dual specificity tyrosine-phosphorylation-regulated kinase 1) wherein the composition is selected from the group consisting of Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease and picks disease.
delete The method of claim 1, wherein the CX-4945 inhibits phosphorylation of Tau, amyloid precursor protein (APP) and PS1 (presenilin 1) protein, dephosphorylates NFATc1, and increases the transcriptional activity of NFATc1 Wherein the disease is selected from the group consisting of Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, and picks disease.
delete 8-carboxylic acid (5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8- (CX-4945) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the CX-4945 inhibits the activity of Dyrk1 (Dual specificity tyrosine-phosphorylation-regulated kinase 1) wherein the health functional food is selected from the group consisting of Down syndrome, Alzheimer's disease, Parkinson's disease, Huntington's disease, and picks disease.
delete delete (5 - [(3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid in isolated cells, cell-free or in vitro. 3-chlorophenyl) amino] benzo [c] -2,6-naphthyridine-8-carboxylic acid (CX-4945) or a pharmaceutically acceptable salt thereof.

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