KR101229365B1 - Pharmaceutical composition comprising (z)-n-(3-(2,4-difluorophenylamino)-1-(4-nitrophenyl)-3-oxoprop-1-en-2-yl)-2-methoxybenzamide for preventing and treating of neurodegenerative disease - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing (Z)-N-(3-(2,4-difluorophenylamino)-1-(4-nitrophenyl)-3-oxoprop-1-en-2-yl)-2-methoxybenzamide for preventing and treating neurodegenerative diseases is provided to suppress A beta decomposition and to prevent and treat Alzheimer' disease. CONSTITUTION: A pharmaceutical composition for preventing and treating Alzheimer's disease, Parkinson' disease, amylotrophic lateral scelerosis(ALS), or dementia contains (Z)-N-(3-(2,4-difluorophenylamino)-1-(4-nitrophenyl)-3-oxoprop-1-en-2-yl)-2-methoxybenzamide or a pharmaceutically acceptable salt thereof as an active ingredient. The compound of chemical formula 1 specifically suppresses a SI pocket of GCP II(Glutamate Carboxypeptidase II) and strengthens synapse at an early stage. The compound of chemical formula 1 maintains homeostasis of intracellular amyloid beta(A beta).

Description

[Ｚ] N [3 [2,4-difluorophenylamino] 1 [4nitrophenyl] 3oxoprop 1,2 days] Preventing and treating neurodegenerative diseases containing 2 methoxybenzamide as an active ingredient Pharmaceutical composition comprising (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide for preventing and treating of neurodegenerative disease}

The present invention provides (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2 -Methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide) or its It relates to a pharmaceutical composition for preventing and treating neurodegenerative diseases containing a pharmaceutically acceptable salt as an active ingredient.

GCPII (glutamate carboxypeptidaseII) is a type II intrinsic membrane protein with a clear molecular weight of 94 to 100 kDa present in the brain, mainly in the glia, and NAA (N-acetylaspartylglutamate) to NAA (N Hydrolysis with -acetylaspartate and glutamate affects glutamatergic transmission. Glutamate produced by NAAG hydrolysis is a major excitatory neurotransmitter in the central nervous system and acts as an essential component of the nervous system, but excessive accumulation in the brain causes death and degeneration of brain cells, resulting in Alzheimer's, stroke, stroke, dementia, Huntington's disease, and peak. It is known to cause degenerative brain diseases such as (Pick) disease and Creutzfeid-Jakob disease (Choi, WH, Oh, YS, Ahn, JY, Kim, SR and Ha, TY (2005) Antioxidative and protective effects of Ulmus davidiana there is . japonica extracts on glutamate-induced cytotoxicity in PC12 cells. Korean J. Food Sci . Technol. 37, 479-483; Jeon, HJ, Park, SW and Mun, BS (2004) Effects of Gwibitang on glutamate-induced death in rat neonatal astrocytes. J Korean Oriental Med . 25, 184-193; Lee, J., Kim, MS, Lee, C., Kim, HY, Choi, DH, Kim, TY, Son, Y. and Park, R. (2003) Role of morphine in the glutamate-induced oxidative damage of C6 glial cells. Korean J Anesthesiol . 45, 271-277).

GCPII has various names depending on the site of enzyme activity and expression. Because of the strong expression of GCPII in the prostate (the location of GCPII is unknown), PSMA is called, and since the central nervous system metabolizes the brain neurotransmitter, NAAG, GCPII is called NAALADase, and the proximal Glutamate from glutamate carboxypeptidase II (GCPII) as poly-g-glutamated folate, folate hydrolase FOLH1 and carboxypeptidase in small intestine (gamma-linked glutamate)

In addition, the structure of GCPII provides insight into the catalytic mechanism and substrate specificity of GCPII. GCPⅡ has a binuclear Zn ^{2 +} center (binuclear Zn ^{2 +} centre) in the active site, two zinc atoms in the active site may share a binding ligand carboxylate (carboxylate bridging ligand). α-NAAG binds to a type of glutamate side chain that interacts with the arginine portion of GCPII, and oxygen and C-terminus bind zinc ions from carbonyl attack. The remainder of the substrate (eg, NAA in α-NAAG) is received from the substrate-binding space that specifically interacts with Arg-210. Larger substrates (e.g., poly-γ-glutamated folates with up to four γ-linked glutamates (9)) are present in three-dimensional collisions with pockets. Some structural rearrangements may be required to avoid and have alternating methods of coupling. GCPII also has two active sites, the S1 'pocket and the S1 pocket (Mlcochova P., Plechanovova A., Barinka C., Mahadevan D., Saldanha JW, Rjlisek L., Konvalinka J. (2007) Mapping of the active site of glutamate carboxypeptidase II by site-directed mutagenesis.FEBS J. 274 (18): 4731-41). If the S1 pocket acts as a 'fine-tuning' of GCPII substrate specificity, the S1 'pocket contributes to the major high affinity binding of NAAG and GCPII inhibitors.

Up to now, the main function of GCP II has been reported to have a NAAG hydrolysis action. Thus, a potent and selective GCPII inhibitor, 2-PMPA (2- (Phosphonomethyl) pentanedioic acid), can be used to treat brain glutamate in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. It has been reported to reduce and provide a neuroprotective effect. However, until now, no compound had specific inhibitory activity in the S1 pocket of GCPII.

The present inventors have found a compound that specifically inhibits the S1 pocket without inhibiting the S1 'pocket using virtual screening to identify an inhibitory compound specific for the S1 pocket of GCPII, and specifically inhibits the S1 pocket. Compound to play a key role in maintaining the homeostasis of Aβ in neurodegenerative diseases, has the effect of causing synaptic strengthening has completed the present invention by revealing that it can be useful as an active ingredient in the prevention and treatment of neurodegenerative diseases. .

The object of the present invention is (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide) Or to provide a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases containing a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide a composition for screening a prophylactic and therapeutic agent for neurodegenerative diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to 1) simultaneously or sequentially inhibiting the compound represented by the formula (1) and the test substance in the GCPII expressing cell line; 2) measuring the activity level of the S1 or S1 'pocket of GCPII in the cell line of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. It is.

Still another object of the present invention is to 1) simultaneously or sequentially inhibiting the compound represented by Formula 1 and the test substance in GCPII; 2) measuring the activity level of the S1 or S1 'pocket of GCPII of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. It is.

Another object of the present invention to provide a health food for the prevention and improvement of neurodegenerative diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides (Z) -N- (3- (2,4-difluorophenylamino) -1- (4) represented by the following general formula (1) which specifically inhibits the S1 pocket of GCPII: -Nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4- nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide) or a pharmaceutically acceptable salt thereof as an active ingredient, provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases:

[Formula 1]

.

.

The present invention also provides a composition for screening a prophylactic and therapeutic agent for neurodegenerative diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention 1) the step of simultaneously or sequentially inhibiting the compound represented by the formula (1) and the test substance in the GCPII expression cell line; 2) measuring the activity level of the S1 or S1 'pocket of GCPII in the cell line of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. do.

In addition, the present invention 1) the step of simultaneously or sequentially inhibiting the compound represented by the formula (1) and the test substance in GCPII; 2) measuring the activity level of the S1 or S1 'pocket of GCPII of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. do.

In another aspect, the present invention provides a health food for the prevention and improvement of neurodegenerative diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Compounds represented by the formula (1) of the present invention by inhibiting the degradation of Aβ by specifically inhibiting the S1 pocket of GCPII in neurodegenerative diseases, synaptic strengthening occurs, can play an important role in maintaining homeostasis of Aβ neurons It can be usefully used as a pharmaceutical composition for the prevention and treatment of degenerative diseases.

Figure 1 shows the effect of 2-PMPA on Aβ and NAAG degradation:
(a, b) rhGCPII was incubated with Aβ ₄₀ or NAAG with or without 2-PMPA or EDTA, and Aβ ₄₀ or NAAG levels were detected by ELISA or NAAG cleavage assay; (c) After infecting lentiviral GCPII into mouse primary astrocytes, Aβ ₄₀ of the monomer was treated intracellularly and 2-PMPA at various doses to treat the Aβ ₄₀ in cell growth medium. Concentration was measured by ELISA assay; (d, e) Intraperitoneal injections of 8-month-old APP Swedish / PS1E9 transgenic mice were injected with 10 mg / kg of PPM 2 / wk of 2-PMPA for 1 month (9 times) and the membrane fraction was NAAG. And ELISA analysis.
2 shows a regioselective mutation of GCPII:
(a) the sequence of primers was used for regioselective mutation experiments; (b) Expression of various mutant proteins in transduced HEK293 cells.
Figure 3 shows NAAG degradation activity in the S1 'pocket mutant of GCPII:
(a) Mutant proteins with substitutions in the S1 'pocket (R210A, K699S) did not degrade N-acetyl-aspartyl-glutamate (NAAG), but about 40% of other proteins with mutations in the S1 pocket (R536L, R548P). Has a degree NAAG degradation activity; (b) NAAG degradation activity of GCPII was not affected by Αβ peptide.
Figure 4 shows the effect on Aβ degradation of GCPII mutants:
(a, b) in H4 cells but overexpressed S1 'mutant (K699S) is an exploded amyloid _{-β 40 (amyloid-β 40)} , S1 mutant (G548P) was not decomposed; (c) Remaining Aβ was reduced by K699S except G548P.
5 is a diagram showing the GCPII inhibitor binding mode: (a) is a specific binding to the S1 site (selection), (b) is a specific binding to the S1 'site (non-screening).
Figure 6 is a figure confirmed by Western blot seven compounds that appear to inhibit the degradation of GCPII Aβ.
7 is a diagram confirming the effect of the compound of Formula 1 on NAAG degradation.

Hereinafter, the present invention will be described in detail.

The present invention provides (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl)-represented by the following general formula (1) which specifically inhibits the S1 pocket of GCPII: 3-oxoprop-1-en-2-yl) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop -1-en-2-yl) -2-methoxybenzamide) or a pharmaceutically acceptable salt thereof as an active ingredient, provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases.

[Formula 1]

.

.

The compound according to the present invention may be prepared by methods known to those skilled in the art of organic synthesis, or commercially available, but is not limited thereto.

The neurodegenerative disease may be any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, preferably Alzheimer's disease.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases, characterized by specifically inhibiting the S1 pocket of GCPII.

The present invention also provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases, characterized by causing synaptic strengthening.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of neurodegenerative diseases, characterized by maintaining the homeostasis of Aβ in the cell.

In addition, the compounds of the present invention can specifically inhibit the S1 pocket of GCPII, it can be used to measure the substrate resolution specific to the S1 'pocket using the same.

To identify the effect of 2-PMPA, a specific inhibitor of GCPII, on the Aβ peptide, we incubated recombinant human GCPII (rhGCPII) with Aβ _1-40 and added 2-PMPA at various doses and ELISA. Analyzed. As a result, rhGCPII degraded Aβ _1-40 peptide by about 80% compared to the control group, but 2-PMPA confirmed that there was no effect on Aβ _1-40 degradation of GCPII (see FIG. 1A). It was confirmed that 2-PMPA did not block Aβ _1-40 degradation of lentiviral GCPII in primary mouse astrocytic cells (see FIG. 1C), and hydrolysis of NAAG by GCPII was completely blocked by 2-PMPA. (See FIG. 1B). In addition, 2-PMPA was treated in transgenic AD model mice to determine the effect of 2-PMPA in mouse brain. We treated 2-PMPA (10 mg / kg, intraperitoneally, 2 × / wk for 1 month) in 8 month old APP Swedish / PS1Δ9 transgenic mice. Aβ _1-40 and in the cortical membrane fraction Levels of Aβ _1-42 were analyzed by ELISA and NAAG degradation activity of GCPII was determined by NAAG analysis. As a result, NAAG degradation was reduced about 90% in mice treated with 2-PMPA compared to the PBS control (see FIG. 1D), but Aβ _1-40 and It was confirmed that the total level of Aβ _1-42 was not changed by treatment with 2-PMPA (see FIG. 1E). Therefore, it was confirmed that NAAG or Aβ degradation sites are different in GCPII.

Based on the fact that GCPII consists of two active sites designated S1 and S1 'pockets, we found seven mutations of GCPII (R210A, D387N, P388A, R536L, G548P, Y552I), which are various mutations of GCPII [ref]. , K699S) (see FIG. 2A). Two complementary oligonucleotide primers, with each mutation, were used to convert amino acids to regioselective mutations. To confirm the expression of various GCPII mutants, mutant plasmids were transduced into HEK293T cells and then Western blotting confirmed the expression changes of the protein. As a result, the pattern of the band was similar to rhGCPII and it was confirmed that one band was prominent at 98 kDa, the predicted size of GCPII (see FIG. 2B).

We observed a change in the enzyme activity of the GCPII mutant. To analyze the enzymatic activity of various mutations, mutant proteins with substitutions in the S1 'pockets (R210A, K699S) and S1 pockets (R536L, G548P) were transduced into H4 cells. To measure the hydrolysis of NAAG, cell lysates were incubated with specific substrates of NAAG (20 μΜ) labeled with [H] ³ and GCPII. As a result, the S1 'pocket mutants (R210A, K699S) completely lost the activity of hydrolyzing NAAG, but the S1 pocket mutants (R536L, G548P) was confirmed to maintain about 40% activity compared to wild type GCPII (See FIG. 3A). In addition, rhGCPII was incubated with [H] ³ labeled NAAG (20 μM) and Αβ peptides by concentration to determine the competitive inhibitory effect between NAAG degradation and Αβ degradation of GCPII. As a result, it was confirmed that the NAAG degradation activity of GCPII was not affected by the Αβ peptide (see FIG. 3B). Therefore, it was confirmed that NAAG hydrolysis in the S1 'pocket was not affected by Aβ, confirming that the mechanism of NAAG degradation and Aβ degradation in GCPII was different.

To identify the Αβ clearance activity of mutant GCPII, we transduced various mutant plasmids into PC3 cells known to not express endogenous GCPII. After 48 hours, the cell lysates were mixed with Aβ (2 μM) and incubated overnight. The remaining Aβ peptide was confirmed by dot blotting and western blotting using 6E10 antibody. As a result, it was confirmed that the K699S mutant degraded Aβ more than the wild type, but the G548P mutant lost Aβ degradation activity (see FIGS. 4A and B). In addition, to confirm the degradation activity of GCPII in exogenously treated Aβ peptides in the culture medium, the following day after transduction, ELISA analysis was performed by treating Aβ with 1 ng / ml in the medium and incubating at 37 ° C. for 8 hours. Was performed. As a result, similar to Western blotting, it was confirmed that the remaining Aβ was reduced by K699S except for G548P (see FIG. 4C). Therefore, it was confirmed that the action of NAAG hydrolytic activity and Aβ degradation activity is differently regulated by GCPII enzymes through the fact that S1 pocket is more important than S1 'pocket in degradation of Aβ.

We performed virtual screening for GCPII against 396,047 compounds in the ChemDiv database to identify inhibitor compounds specific for the S1 pocket of GCPII. Among them, compounds that specifically bind to S1 sites were selected, and compounds that bind to S1 'sites that cut NAAG were excluded. As a result, 1972 compounds were selected and 100 structures representing overlapping scaffolds among 1,947 hit compounds were selected. Of these, 58 compounds were cultured together with recombinant GCPII and Aβ to identify compounds that affect the degree of Aβ degradation of GCPII. As a result, the present inventors found that (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1- represented by Chemical Formula 1 above. En-2-yl) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl ) -2-methoxybenzamide) could be identified.

In one embodiment of the present invention, the compound of Formula 1 was mixed with recombinant human GCPII and Aβ, and then analyzed by the blotting Αβ degradation activity of GCPII. As a result, it was confirmed that the compound specifically inhibits Aβ degradation by specifically acting on the S1 pocket of GCPII (see FIG. 6).

In one embodiment of the present invention, to determine the effect of the compound of Formula 1 on NAAG degradation, Western blotting was performed after mixing recombinant human GCP II and NAAG. As a result, it was confirmed that the compound did not decompose NAAG of GCPII and thus did not act on the S1 ′ pocket of GCPII (see FIG. 7).

Therefore, the compound represented by the formula (1) of the present invention is an inhibitory compound specific for the S1 pocket of GCPII that significantly inhibits the degradation of ACP of GCPII without affecting the degradation of NAAG.

A [beta] plays a role in regulating the release of synaptic vesicles of neurons, and a large amount of A [beta] is known to cause synaptic damage. However, a small amount of A [beta] is known to induce synaptic strengthening when a small amount of A [beta] is injected to compensate for synaptic loss in early Alzheimer's disease. In particular, there has been a report that synaptic strengthening occurs when the Aβ breakdown is prevented using a thiorphan, an inhibitor of the Aβ cleavage enzyme called neprilysin (Efrat Abramov, Amyloid-β as a positive endogenous regulator of release probability at hippocampal synapses, Nature neuroscience, vol. 12, num. 12, 2009).

It is also known that synapse loss is a major cause of cognitive and memory loss in neurodegenerative diseases including Alzheimer's disease. Especially in Alzheimer's disease such as senile dementia and vascular dementia, synapse loss occurs before accumulation of toxic protein Aβ and entanglement of tau protein. Thus, the compounds of the present invention can preferably be used in the early stages of Alzheimer's disease.

Observations of dementia model mice genetically engineered to develop pathologies such as senile dementia in humans resulted in significant impairment of cognitive function five months before Aβ accumulation and tau protein entanglement in the brain, resulting in Aβ accumulation and tau protein entanglement. It has been reported that this is not a root cause of dementia but is caused by synaptic loss. A 12-week administration of bryostatin and a synthesized bryostatin-like substance on PKC enzymes that regulate synapse production at the molecular level was reported to promote the production of new synapses and preserve the remaining synapses. It became. In addition, the decrease in PKC enzyme and the increase in water-soluble Aβ also disappeared the characteristic symptoms of dementia such as Aβ accumulation, tau protein entanglement and cognitive loss.

Therefore, the compound of the present invention, in neurodegenerative diseases, can inhibit Aβ degradation according to the stage of the disease to maintain homeostasis of Aβ in cells, and also cause synaptic strengthening, thereby preventing and treating neurodegenerative diseases. It can be usefully used.

The present invention includes not only the compound represented by Chemical Formula 1, but also a pharmaceutically acceptable salt thereof, and possible solvates, hydrates, racemates, or stereoisomers which can be prepared therefrom.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is 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 Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salts according to the invention are dissolved in conventional methods, for example, by dissolving a compound of formula 1 in an excess of aqueous acid solution and using the water miscible organic solvent, such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation.

An equivalent amount of the compound represented by the formula (1) and an aqueous acid solution or alcohol may be heated, and then the mixture is evaporated to dryness or the precipitated salt may be produced by suction filtration.

Bases can also 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 alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.

The composition comprising the compound of the present invention, preferably containing 0.1 to 50% by weight based on the total weight of the composition is not limited thereto.

The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The pharmaceutical compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be used. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

 In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the composition of the present invention, for example, starch, calcium carbonate, sucrose (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally, any parenteral administration method can be used, systemic or topical administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 0.03 g / kg, preferably at 0.001 to 8 mg / kg. Administration may be administered once a day or may be divided several times. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a composition for screening a prophylactic and therapeutic agent for neurodegenerative diseases containing the compound represented by the formula (1) as an active ingredient.

Since the compound represented by the formula (1) of the present invention specifically inhibits only the S1 pocket of GCPII, it may be usefully used to purely see the activity of the S1 'pocket. In addition, since the S1 and S1 'pockets are attached to each other to the extent that the pores (pore) are bisected to each other, there is a high possibility of mutual influence. It can be usefully used for the composition for screening a prophylactic and therapeutic agent for degenerative diseases.

Neurodegenerative diseases may include Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, preferably Alzheimer's disease, but is not limited thereto.

The compound represented by Formula 1 may be a compound that specifically inhibits the S1 pocket of GCP II.

The present invention comprises the steps of: 1) simultaneously or sequentially inhibiting the compound represented by Formula 1 and the test substance in the GCPII expressing cell line; 2) measuring the activity level of the S1 or S1 'pocket of GCPII in the cell line of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. do.

In addition, the present invention 1) the step of simultaneously or sequentially inhibiting the compound represented by the formula (1) and the test substance in GCPII; 2) measuring the activity level of the S1 or S1 'pocket of GCPII of step 1); And 3) selecting a test substance whose activity level of the S1 or S1 ′ pocket of GCPII of step 2) is increased or decreased compared to a control which is not treated with the test substance. do.

Since the compound represented by the formula (1) of the present invention in step 2) specifically inhibits S1, it can be used both to select a test substance related to the activity of the GCPII S1 and S1 'pocket.

The method of viewing the activity of GCPII in step 2) is not limited thereto, and may be the method of viewing the Aβ degrading activity of GCPII or the NAAG degrading activity of GCPII.

Compared to the control group in step 3), both the test substance that inhibits the activity of the S1 or S1 'pocket of the GCP in which the S1 pocket is inhibited or the test substance that increases the activity can be used as a prophylaxis and treatment for neurodegenerative diseases.

The present invention also provides a neurodegenerative disease prevention and improvement health food containing the compound represented by the formula (1) as an active ingredient.

Compound of the present invention represented by the formula (1) specifically inhibits the S1 pocket of GCPII, inhibits the degradation of Aβ to cause synaptic strengthening, maintain the homeostasis of Aβ in the cell health for the prevention and improvement of neurodegenerative diseases It can be usefully used as a food.

The compound according to the present invention can be added to the dietary supplements, such as food, beverages, for the purpose of preventing and improving neurodegenerative diseases.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The compound represented by the formula (1) of the present invention may be added to food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the compound in the health food may be 0.1 to 90 parts by weight of the total food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compounds of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages.

These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples, and Production Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples, Experimental Examples, and Production Examples.

< Example  1> 2- PMPA Is Aβ and NAAG  Determine impact on decomposition

<1-1> Cell Culture and Transduction

HEK293T cells and H4 cells (American Type Culture Collection, ATCC, Edinburg, VA, USA) were treated with 10% fetal bovine serum (FBS) (FBS; GIBCO) and 1% penicillin / streptomycin ( Dulbecco's modified Eagle's Medium (DMEM) (DMEM; GIBCO) added with GIBCO, PC3 cells were added RPMI medium 1640 (GIBCO) with 10% fetal calf serum (FBS; GIBCO) and 1% penicillin / streptomycin (GIBCO) ) Was incubated at 37 ° C. in a humidified incubator containing 5% CO ₂ . Transient transduction was performed using Lipofectamine 2000 reagent or Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions.

<1-2> Aβ Peptide  Ready

Aβ _1-40 or Aβ _1-42 (Invitrogen) peptides were finally prepared with cold hexafluoroisopropanol (HFIP) (HFIP; Sigma) at 1 mM concentration. The mixture was shaken at room temperature for 40 minutes and left at 4 ° C. for 20 minutes, and then HFIP was removed in vacuo. Peptide pellets were stored at -70 ° C. The pellet was dissolved in DMSO to a final 5 mM concentration and distilled water was added to a final 1 mM concentration.

<1-3> ELISA  2- through analysis PMPA The effect of VEH on Aβ degradation

Vectors encoding hGCPII and various mutant hGCPII genes were transduced into PC3 cells in 12-well plates. After about 30 hours, the culture medium was treated with Aβ _1-40 or with or without 2-PMPA ((2- (Phosphonomethyl) pentanedioic acid). Aβ _1-42 peptide was changed to treated media and cells were incubated again for 8 hours. The medium was obtained and remaining Aβ _1-40 or using ELISA kit (Invitrogen) following the manufacturer's protocol. Aβ _1-42 was analyzed. Cells transduced with the empty pcDNA3 empty vector were used as negative controls.

As a result, as shown in Figure 1a rhGCPII is about 80% Aβ _1-40 compared to the control group Although degraded, 2-PMPA was found to have no effect on Aβ _1-40 cleavage of GCPII (FIG. 1A).

<1-4> NAAG  2- through decomposition analysis PMPA end NAAG  Determine impact on decomposition

The endogenous or overexpressed glutamate carboxypeptidase II (GCPII) protein activity was measured in the following manner. Cell lysates (50 μg) were treated with NAAG (with or without 20 nM or EDTA of GCPII specific inhibitor 2-PMPA at a total volume of 100 μl containing 50 mM HEPES and 150 mM NaCl for one hour at 37 ° C.). Incubated with 20 μM of N-acetyl-L-aspartyl-L- [3,4-3H] glutamate (NEN corporation). After the reaction, the sample mixture was used for AG 1-X8 anion-exchange resin (Biorad) prepared in a 96 well column (Havard apparatus) and centrifuged at 2000 rpm for 5 minutes. It was. The resin bound with the sample mixture was eluted with 0.5 mM formate (100 ml) and centrifuged at 2000 rpm for 5 minutes. All eluted samples that were transferred were mixed with 1 ml of flash solution (Optiphase HiSafe; Wallac) and radioactivity was measured by a scintillation counter (Wallac Inc.).

As a result, as shown in Fig. 1b, it was confirmed that 2-PMPA completely blocked NAAG hydrolysis by GCPII (Fig. 1b).

<1-5> Effect of GCP Ⅱ plasmid and lentivirus 2- PMPA with virus produced on the decomposition of Aβ alkylene Tyva virus GCPⅡ OK

hGCPII plasmids were prepared as described below. RNA was isolated from U87-MG cells corresponding to human astrocytes, and cDNA was synthesized using reverse transcriptase. For amplification of hGCPII in the synthesized cDNA, PCR was performed using the following primers. PCR products were cloned into pcDNA3 vectors for plasmid generation:

Forward 5′-GATGTGGAATCTCCTTCACGAAAC-3 ′; And

Reverse 5′-ATCCTCTTAGGCTACTTCACTCAAAG-3 ′.

The lentiviral hGCP II was introduced into Macrogen Inc. Prepared by (Seoul, Korea). hGCPII cDNA was cloned into a lentiviral construct (LentiM1.4-hGCPII) comprising an IRES sequence with human cytomegalovirus (CMV) promoter and fluorescent protein (GFP). For virus production, hGCPII, VSV-G and gag-pol expression vectors were transduced together in 293T cells using Lipofectamine Plus (Invitrogen, Carlsbad, Calif., USA), then treated in AβDMF cells of monomer and 2-PMPA Was treated. The concentration of Aβ _1-40 in the cell growth medium was analyzed by the method of Example <1-3>.

As a result, as shown in Figure 1c 2-PMPA was confirmed that did not block the Aβ _1-40 degradation of lentiviral GCPII (Fig. 1c).

<1-6> transgenic animals

Dual-transformed APP Swedish / PS1ΔE9 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and duplexed by crosslinking with wild-type mice in a C57BL / 6J background strain. ). All animals were bred according to standard animal care protocols and maintained in a pathogen-free facility at the Korean Food and Drug Administration. Mouse genotypes were confirmed by PCR using the following primers:

Mouse prion protein (PrP):

Forward 5'-CCTCTTTGTGACTATGTGGACTGATGTCGG-3 ';

Reverse 5'-GTGGATAACCCCTCCCCCAGCCTAGACC-3 ';

Human APP:

Forward 5'-GACTGACCACTCGACCAGGTTCTG-3 '; And

Reverse 5'-CTTGTAAGTTGGATTCTCATATCCG-3 '.

To measure the effect of GCPII inhibitors at the brain level of Aβs, the treated group was injected with 2-PMPA (10 mg / kg) dissolved in PBS 2X / wk for one month by intraperitoneal injection. The untreated group was injected with the same amount of PBS as a control. Treatment was started when the mice were eight months old.

<1-7> 2- PMPA Effects in the mouse brain

To determine the effect of 2-PMPA in the brain of mice, 10 mg / kg of 2-PMPA for 1 month in the abdominal cavity of 8 month old APP Swedish / PS1Δ9 mice transformed by the method of Example <1-6>. Or PBS 2 / wk nine times. Aβ _1-40 and in the cortical membrane fraction The levels of Aβ _1-42 were analyzed by the method of Example <1-3>, and the NAAG degradation activity of GCPII was measured by the method of Example <1-4>.

As a result, as shown in FIG. 1D, NAAG degradation in mice treated with 2-PMPA was reduced by about 90% compared to the PBS control group (FIG. 1D), and as shown in FIG. 1E, by treatment with 2-PMPA. Aβ _1-40 and It was confirmed that the levels of Aβ _1-42 did not change (FIG. 1E).

< Example  2> GCP Confirmation of Regioselective Mutation Expression of Ⅱ

<2-1> GCP Regioselective mutation of II Site - directed mutagenesis )

The pcDNA-hGCPII plasmid (50 ng) was used as a template and each mutation was introduced into two complementary oligonucleotide primers (Table 1) that produce the desired mutations. Extended with 2.5 U of Pfu Ultra High-Fidelity DNA polymerase (stratagene) and opened circular strand (95 ° C., 5 min → (95 ° C., 50 sec → 55 ° C., 1 min) → 80 ° C., 8 min 20 sec → 72 ° C., 1 min) 22 cycles → 4 ° C., infinity (∞)) resulted in mutant primers (125 ng). Methylated, nonmutated parental DNA templates were digested with 20 U of Dpn I (NEB) at 37 ° C. for one hour. The circular dsDNA was then converted into DH5a competent cells. Each mutation was identified by sequencing (Cosmo corporation).

As a result, as shown in Fig. 2a, seven mutations of GCPII (R210A, D387N, P388A, R536L, G548P, Y552I, K699S) were produced (Fig. 2A).

<2-2> Western Western blotting  through GCP Ⅱ Mutation Expression

To confirm the expression of the GCPII mutant, the mutant plasmid was transduced into HEK293T cells following the method of Example <1-1>, and the cells were then phosphate buffered saline (PBS) (PBS; GIBCO). Washed and dissolved in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris at pH8.0) with a protease inhibitor cocktail (Sigma). Lysates were measured for protein concentration by Bradford assay. The same concentration of protein was boiled for 10 minutes by addition of β-mercaptoethanol and separated by 10% SDS-PAGE. Proteins were electrophoresed into polyvinylidene difluoride (PVDF) membranes (GE Healthcare) and blocked with TBS-T in 5% nonfat dry milk at room temperature for one hour. The membrane was then placed in 5% skim dry milk at 4 ° C. overnight in anti-PSMA (abcam; ab41034, 1: 1000), Y-PSMA (Maine Biotech, 1: 1000) or anti-a-tubulin antibody Incubated with (Sigma-Aldrich; T6199, 1: 50000). After washing three times with TBST-T, the blot was either HRP-conjugated goat anti-mouse IgG (Jackson IR; 115-035-071, 1: 10000) or HRP-conjugated goat anti-rabbit IgG (Jackson) for 2 hours at room temperature. IR; 115-035-046, 1: 10000). After washing three times, proteins were detected using a chemiluminescent substrate (Thermo Scientific).

As a result, as shown in FIG. 2B, the pattern of the band was similar to rhGCPII, and one band was found to be prominent at 98 kDa, which is the expected size of GCPII (FIG. 2B).

< Example  3> GCP II mutation NAAG  Determine impact on decomposition

To determine the effect of GCPII mutations on NAAG degradation For this purpose, mutant proteins with substitutions in the S1 'pockets (R210A, K699S) and S1 pockets (R536L, G548P) were transduced into H4 cells according to the method of Example <1-1>. To measure the hydrolysis of NAAG, the cell lysates were incubated with the specific substrates of NAAG (20 μΜ) labeled with [H] ³ and GCPII according to the method of Examples <1-4> above. According to the method of Example <2-2>, changes in protein expression were confirmed.

As a result, as shown in FIG. 3A, the S1 'pocket mutants (R210A, K699S) completely lost the activity of hydrolyzing NAAG, but the S1 pocket mutants (R536L, G548P) were about 40% more active than the wild type GCPII. It was confirmed that it was maintained (Fig. 3a).

To determine the effect of Aβ on NAAG degradation of GCPII, rhGCPII was incubated simultaneously with the [H] ³ labeled NAAG (20 μM) and Aβ peptides according to the method of Example <1-4>.

As a result, as shown in Figure 3b, it was confirmed that the NAAG degradation activity of GCPII was not affected by the Αβ peptide (Figure 3b).

< Example  4> GCP Identification of the Effect of Ⅱ Mutation on Aβ Degradation

<4-1> GCP II / Aβ Complex modelling

MODE of the combination of Aβ-S1 and S1 'pockets can be set to program O (http://xray.bmc.uu.se/alw). yn / A-Z_frameset.html) (PDB file: 1z0q (Ab), 2oot (GCPII)). Computational modeling (computational docking) was performed using the manual docking (manual docking) method.

To determine the effect of GCPII mutations on Aβ degradation To this end, according to the method of Example <1-5> and the method of Example <4-1>, various mutant plasmids were transduced into PC3 cells known to not express endogenous GCPII. After 48 hours, the cell lysates were mixed with Aβ (2 μM) and incubated overnight.

<4-2> dot Dot blotting  through GCP Identification of the Effect of Ⅱ Mutation on Aβ Degradation

In order to confirm the amount of Aβ peptide remaining without being cut by the expressed GCPII, the cell lysate and the mixture of Aβ (2 μM) were mixed with the following dot blotting method and the method of Example <2-2>. Confirmed. 1 μl of the cell lysate and Aβ (2 μM) and the mixture were dropped onto nitrocellulose membrane and dried completely. The dried membrane was blocked for one hour with TBS-T in 5% nonfat dry milk at room temperature. The membrane was then incubated with anti-Aβ antibody, 6E10 (SIGNET; SIG-39300, 1: 2000) in 5% skim dry milk at 4 ° C. overnight. After washing three times with TBS-T, the membrane was incubated with HRP-confugated goat anti-mouse IgG (Jackson IR; 115-035-071, 1: 10000) for 1 hour at room temperature. After washing three times, the protein was detected using a chemiluminescent substrate.

As a result, as shown in Figure 4a, b K699S mutant was a little more degraded Aβ than wild type, but G548P mutant was confirmed that lost the activity of Aβ degradation (Fig. 4a, b).

In order to confirm the degradation activity of GCPII in Aβ peptide, according to the method of Example <1-3> and Example <4-1>, Aβ was treated at 1 ng / ml in the medium the day after the transduction. And incubated at 37 ° C. for 8 hours.

As a result, it was confirmed that the remaining Aβ was reduced by K699S except for G548P as shown in FIG. 4C (FIG. 4C).

As a result, the present inventors have found that NAAG may bind to the S1 'pocket, but not Aβ, and that Aβ may occur in the S1 pocket. Based on the above results, the present inventors confirmed the structure of the active site of GCPII and identified inhibitory compounds of GCPII by virtual screening based on the structure.

< Example  6> GCP II Identification of Inhibitory Compounds

<6-1> GCP Virtual search for Ⅱ

For screening inhibitors for Aβ resolution to GCPII, 396,047 compounds from the ChemDiv database were identified by binding modeling with the S1 site of GCPII to identify appropriate compounds.

A virtual screening study for GCP II was performed using the UNITY built into Tripos' SYBYL 7.3 program. Virtual screening was carried out by docking a total of 396,047 compounds contained in ChemBridge and ChemDiv's databases in a known three-dimensional crystal structure of GCPII (PDB ID: 2PVW). In particular, the three-dimensional structure 2PVW of GCPII has two ligand binding sites (S1, S1 '), of which the S1' site is a glutamate binding site associated with NAAG degradation, and 2-PMPA is known as a selective inhibitor of GCPII. The S1 site is known to be involved in the degradation of Aβ.

We limited the ligand binding site to the S1 site during docking studies to find ligands that selectively bind to the S1 site. After the virtual screening, the ligands bound to S1 site were selected first with a docking score of -40 or more, and then visually inspected to dock at S1 'site or to S1-S1' site. The materials docked over were filtered out.

In addition, the inventors selected compounds in the case of specifically binding to the S1 site that is expected to cut Aβ (FIG. 5A), and exclude compounds that bind to the S1 ′ site to cut NAAG (FIG. 5B).

As a result, the inventors obtained a total of 1,947 hit compounds. The material to be used for the in vitro assay selected 100 structures representing overlapping scaffolds among 1,947 hit compounds.

<6-2> GCP Identification of compounds affecting the degree of Aβ degradation of Ⅱ

Of the 100 compounds selected in Example <6-1>, 63 compounds commercially available from ChemDiv were used to analyze the effect on the degree of Aβ degradation of GCPII. Of the 63 compounds, 58 compounds dissolved in DMSO were cultured with recombinant GCPII and Aβ to identify compounds that affect the degree of Aβ degradation of GCPII.

As a result, a compound represented by the following general formula (1) that significantly inhibits the degradation of Aβ was identified:

[Formula 1]

.

.

<6-3> Western Western blotting  Of the compound represented by Formula 1 GCP Analysis of Aβ Degradation Activity of Ⅱ

 In order to determine the effect of the compound represented by Formula 1 on the Aβ degradation of GCPII, 30.8 ng / ml of purely isolated recombinant human GCPII and Aβ 1-42 8 mM were mixed, and the compound was added in 10 mM increments. The volume was adjusted to 25 ml and incubated at 37 ° C. for 16 hours. After incubation, the mixture was loaded on a 4-12% gradient gel about 3-5 ml and then separated. The separated protein was transferred to a PVDF membrane and then blocked with 5% non fat dry milk (TBS-T) for 1 hour. 6E10 (Covance, 1: 5000) was stored overnight at 4 ° C. to observe degraded Aβ 1-42. After washing three times with TBS-T and incubated with HPR-conjugated goat anti-mouse IgG (HRP-conjugated goat anti-mouse IgG) (Jackson IR, 1: 10000) at room temperature for 2 hours. After washing three times with TBS-T once again, Aβ was detected with a chemiluminescent substrate (Thermo Scientific) (FIG. 6).

As a result, as shown in Figure 6, it was confirmed that the compound represented by the formula (1) significantly inhibits the Aβ degradation of GCPII.

<6-4> Western Western blotting  Of the compound represented by Formula 1 NAAG  Degradation Activity Assay

Whole was mixed with ^[3 H] compounds of the general formula (I) of NAAG, 10mM of the purely separated order to examine the effects of the NAAG decomposition of GCPⅡ recombinant human GCPⅡ 35 ng / ml and 20 mM compound represented by the formula (1) The volume was adjusted to 100 ml using 50 mM Tris buffer and incubated at 37 ° C. for 1 hour. After 20 minutes of mixing 1: 1 with distilled water (DW) and AG1-X8 resin, the resin was loaded by 100 ml in a 96 well column plate (Havard apparatus). After the incubation, the mixtures were put in a column loaded with resins and centrifuged at 2000 rpm for 5 minutes. 100 ml of 0.5 M formate was added to the column and centrifuged for 5 minutes at 2000 rpm after 5 minutes. After centrifugation, the sample passed through the column was transferred to a 2 ml tube, 1 ml of liquid scintillation solution (Optiphase HiSafe) was added and stirred, and then measured using a radioactive scintillation counter.

As a result, it was confirmed that in the case of formula 1 does not affect the decomposition of NAAG (Fig. 7).

As a result, the present inventors were able to select an inhibitory compound specific for the S1 pocket of GCPII which remarkably suppresses the degradation of ACP of GCPII without affecting the degradation of NAAG.

The following are some examples of formulations containing the compounds of the present invention, but the present invention is not limited thereto.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

1-1. Sanje  Produce

Compound 1 500 mg

Lactose 100 mg

10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

Compound 1 500 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Manufacture of capsules

Compound 1 500 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

Compound 1 500 mg

Sterile sterilized water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Liquid  Produce

Compound 1 100 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, The liquid is prepared by sterilization.

< Manufacturing example  2> Manufacture of health food

Compound 1 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 mg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

< Manufacturing example  3> Manufacture of health drinks

Compound 1 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 liter container, And used for manufacturing.

Although the composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

< Manufacturing example  4> Manufacture of Other Health Foods

4-1. Manufacturing of beverages

Honey 522 mg

5 mg &lt; RTI ID = 0.0 &gt;

Nicotinic acid amide 10 mg

3 mg of sodium riboflavin hydrochloride

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Compound 1 0.48-1.28 mg

200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

4-2. Of chewing gum  Produce

Gum base 20%

Sugar 76.36-76.76%

Compound 1 0.24-0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

4-3. Manufacture of candy

50-60% sugar

Starch syrup 39.26 ~ 49.66%

Compound 1 0.24-0.64%

Orange flavor 0.1%

The composition and the content of the candy were prepared using a conventional method.

4-4. Manufacture of flour food products

0.5 to 5 parts by weight of Compound 1 was added to 100 parts by weight of wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers, and noodles to prepare health promoting food.

4-5. dairy product( dairy products Manufacturing

5 to 10 parts by weight of compound 1 was added to 100 parts by weight of milk, and the milk was used to prepare various dairy products such as butter and ice cream.

4-6. Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were alphanated by a known method to distribute the dried ones, and then prepared into a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla were also steamed and dried in a known manner to prepare a powder having a particle size of 60 mesh using a grinder. The grains and seeds prepared above and Compound 1 of the present invention were combined and prepared in the following ratios.

Brown Rice 30%

15% rate

Barley 20%

Perilla 7%

Black Bean 7%

Black sesame 7%

Compound 1 3%

Ganoderma lucidum 0.5%

Foxglove 0.5%

<110> Korea Center for Disease Control and Prevention <120> Pharmaceutical composition to preventing and treating of          Alzheimer's disease comprising GCPII mutant <130> 11p-08-09 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 2653 <212> DNA <213> Artificial Sequence <220> <223> GCPII mutant (K699S) <400> 1 ctcaaaaggg gccggatttc cttctcctgg aggcagatgt tgcctctctc tctcgctcgg 60 attggttcag tgcactctag aaacactgct gtggtggaga aactggaccc caggtctgga 120 gcgaattcca gcctgcaggg ctgataagcg aggcattagt gagattgaga gagactttac 180 cccgccgtgg tggttggagg gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 240 gccggctctg ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc 300 accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg tggcttcttt 360 ctcctcggct tcctcttcgg gtggtttata aaatcctcca atgaagctac taacattact 420 ccaaagcata atatgaaagc atttttggat gaattgaaag ctgagaacat caagaagttc 480 ttatataatt ttacacagat accacattta gcaggaacag aacaaaactt tcagcttgca 540 aagcaaattc aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat 600 gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat aattaatgaa 660 gatggaaatg agattttcaa cacatcatta tttgaaccac ctcctccagg atatgaaaat 720 gtttcggata ttgtaccacc tttcagtgct ttctctcctc aaggaatgcc agagggcgat 780 ctagtgtatg ttaactatgc acgaactgaa gacttcttta aattggaacg ggacatgaaa 840 atcaattgct ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag 900 gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga ccctgctgac 960 tactttgctc ctggggtgaa gtcctatcca gatggttgga atcttcctgg aggtggtgtc 1020 cagcgtggaa atatcctaaa tctgaatggt gcaggagacc ctctcacacc aggttaccca 1080 gcaaatgaat atgcttatag gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1140 gttcatccaa ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca 1200 ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg acctggcttt 1260 actggaaact tttctacaca aaaagtcaag atgcacatcc actctaccaa tgaagtgaca 1320 agaatttaca atgtgatagg tactctcaga ggagcagtgg aaccagacag atatgtcatt 1380 ctgggaggtc accgggactc atgggtgttt ggtggtattg accctcagag tggagcagct 1440 gttgttcatg aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga 1500 agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg ttctactgag 1560 tgggcagagg agaattcaag actccttcaa gagcgtggcg tggcttatat taatgctgac 1620 tcatctatag aaggaaacta cactctgaga gttgattgta caccgctgat gtacagcttg 1680 gtacacaacc taacaaaaga gctgaaaagc cctgatgaag gctttgaagg caaatctctt 1740 tatgaaagtt ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc 1800 aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat tgcttcaggc 1860 agagcacggt atactaaaaa ttgggaaaca aacaaattca gcggctatcc actgtatcac 1920 agtgtctatg aaacatatga gttggtggaa aagttttatg atccaatgtt taaatatcac 1980 ctcactgtgg cccaggttcg aggagggatg gtgtttgagc tagccaattc catagtgctc 2040 ccttttgatt gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt 2100 atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga ttcacttttt 2160 tctgcagtaa agaattttac agaaattgct tccaagttca gtgagagact ccaggacttt 2220 gacaaaagca acccaatagt attaagaatg atgaatgatc aactcatgtt tctggaaaga 2280 gcatttattg atccattagg gttaccagac aggccttttt ataggcatgt catctatgct 2340 ccaagcagcc acaactcata tgcaggggag tcattcccag gaatttatga tgctctgttt 2400 gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag acagatttat 2460 gttgcagcct tcacagtgca ggcagctgca gagactttga gtgaagtagc ctaagaggat 2520 tctttagaga atccgtattg aatttgtgtg gtatgtcact cagaaagaat cgtaatgggt 2580 atattgataa attttaaaat tggtatattt gaaataaagt tgaatattat atataaaaaa 2640 aaaaaaaaaa aaa 2653 <210> 2 <211> 750 <212> PRT <213> Artificial Sequence <220> <223> GCPII mutant (K699S) <400> 2 Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val Ala Thr Ala Arg   1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Ghe Phe              20 25 30 Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe Ile Lys Ser Ser Asn Glu          35 40 45 Ala Thr Asn Ile Thr Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu      50 55 60 Leu Lys Ala Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile  65 70 75 80 Pro His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile                  85 90 95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His             100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile         115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe     130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly Asp Leu Val Tyr                 165 170 175 Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met             180 185 190 Lys Ile Asn Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val         195 200 205 Phe Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly     210 215 220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys 225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg Gly                 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr             260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala Val Gly         275 280 285 Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr Asp Ala Gln Lys     290 295 300 Leu Leu Glu Lys Met Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly Ser Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn                 325 330 335 Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val             340 345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro         355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly     370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val Arg 385 390 395 400 Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro Arg Arg Thr Ile                 405 410 415 Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr             420 425 430 Glu Trp Ala Glu Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala         435 440 445 Tyr Ile Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val     450 455 460 Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu 465 470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser                 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile             500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln Arg Leu         515 520 525 Gly Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn Trp Glu Thr Asn     530 535 540 Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val Glu Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val                 565 570 575 Ala Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val             580 585 590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala         595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr     610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp Phe Asp Lys Ser                 645 650 655 Asn Pro Ile Val Leu Arg Met Met Asn Asp Gln Leu Met Phe Leu Glu             660 665 670 Arg Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg         675 680 685 His Val Ile Tyr Ala Pro Ser Ser His Asn Ser Tyr Ala Gly Glu Ser     690 695 700 Phe Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705 710 715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala Ala                 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala             740 745 750 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer F of hGCPII cDNA to amplify from whole cDNA via PCR <400> 3 gatgtggaat ctccttcacg aaac 24 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer R of hGCPII cDNA to amplify from whole cDNA via PCR <400> 4 atcctcttag gctacttcac tcaaag 26 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PrP-F <400> 5 cctctttgtg actatgtgga ctgatgtcgg 30 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> PrP-R <400> 6 gtggataacc cctcccccag cctagacc 28 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> human APP-F <400> 7 gactgaccac tcgaccaggt tctg 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> human APP-R <400> 8 cttgtaagtt ggattctcat atccg 25 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R210A-F <400> 9 gggaagtttt cgcgggaaat aaggttaaaa atg 33 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> R210A-R <400> 10 catttttaac cttatttccc gcgaaaactt tccc 34 <210> 11 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> D387N-F <400> 11 gtgtttggtg gtattaatcc tcagagtgga gcag 34 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> D387N-R <400> 12 ctgctccact ctgaggatta ataccaccaa acac 34 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> P388A-F <400> 13 gtgtttggtg gtattgacgc tcagagtgga gcag 34 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> P388A-R <400> 14 ctgctccact ctgagcgtca ataccaccaa acac 34 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R536L-F <400> 15 gcttcaggca gagctctgta tactaaaaat tgg 33 <210> 16 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> R536L-R <400> 16 ccaattttta gtatacagag ctctgcctga agc 33 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> G548P-F <400> 17 gaaacaaaca attcagcccc tatccactgt atcac 35 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> G548P-R <400> 18 gtgatacagt ggataggggc tgaatttgtt tgtttc 36 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Y552I-F <400> 19 cagcggctat ccactgattc acagtgtcta tgaaac 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Y552I-R <400> 20 gtttcataga cactgtgaat cagtggatag ccgctg 36 <210> 21 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> K699S-F <400> 21 caagcagcca caactcatat gcaggggagt c 31 <210> 22 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> K699S-R <400> 22 gactcccctg catatgagtt gtggctgctt g 31

Claims (17)

(Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl represented by the formula (1) ) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide ) Or a pharmaceutically acceptable salt thereof as an active ingredient,
Compound represented by the following formula (1) is Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, characterized by specifically inhibiting the S1 pocket of GCP II (glutamate carboxypeptidase II) to cause synaptic strengthening in the early stage of the disease Any of the prophylactic and therapeutic pharmaceutical compositions selected from the group consisting of:
[Formula 1]

.
delete delete delete The method of claim 1, wherein the compound represented by Formula 1 prevents any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, characterized in that to maintain the homeostasis of amyloid beta (Aβ) in cells Therapeutic pharmaceutical composition.
(Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl represented by the formula (1) ) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide ) Or a pharmaceutically acceptable salt thereof as an active ingredient,
Compound represented by the following formula (1) prevents any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, characterized in that specifically inhibit the S1 pocket of GCPII (glutamate carboxypeptidase II) Compositions for Screening Therapeutics:
[Formula 1]

.
delete delete 1) in vitro, treating the GCPII (glutamate carboxypeptidase II) expressing cell line with the compound represented by Formula 1 and the candidates simultaneously or sequentially;
2) measuring the activity level of the S1 or S1 'pocket of GCPII in the cell line of step 1); And
3) with Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia comprising screening for candidates whose activity levels of the S1 or S1 'pockets of GCPII of step 2) were increased or decreased compared to the control without treatment of the candidates. A method for screening any one prophylactic and therapeutic agent selected from the group consisting of:
[Formula 1]

.
delete 1) in vitro, simultaneously or sequentially treating GCPII (glutamate carboxypeptidase II) with a compound represented by the following formula (1) and a candidate:
2) measuring the activity level of the S1 or S1 'pocket of GCPII of step 1); And
3) with Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia comprising screening for candidates whose activity levels of the S1 or S1 'pockets of GCPII of step 2) were increased or decreased compared to the control without treatment of the candidates. A method for screening any one prophylactic and therapeutic agent selected from the group consisting of:
[Formula 1]

.
delete (Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl represented by the formula (1) ) -2-methoxybenzamide ((Z) -N- (3- (2,4-difluorophenylamino) -1- (4-nitrophenyl) -3-oxoprop-1-en-2-yl) -2-methoxybenzamide ) Or a pharmaceutically acceptable salt thereof as an active ingredient,
Compound represented by the following formula (1) is Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, characterized by specifically inhibiting the S1 pocket of GCP II (glutamate carboxypeptidase II) to cause synaptic strengthening in the early stage of the disease Any one improved health food selected from the group consisting of:
[Formula 1]

.
delete delete delete The method of claim 13, wherein the compound represented by Formula 1 is any one improvement health food selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease and dementia, characterized in that to maintain the homeostasis of Aβ in the cell.

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