KR20090033583A - Compounds having glysin aryl amide moiety as inhibitors of beta-secretase - Google Patents

Abstract

A compound is provided to inhibit human beta-secretase containing glysin aryl amide moiety and to be useful for neurodegenerative diseases such as Alzheimer disease. A compound for inhibiting beta-secretases is a compound of the following chemical formula I, its pharmaceutically allowable salts or isomers. In the chemical formula I, m and n are independently 0 or 1; R^1 and R^2 are selected from the group consisting of hydrogen, halogen, cyano, hydroxy, -(CH2)p-R, -O-(CH2)p-R, -N[-(CH2)p-R][-(CH2)p-R], -S-(CH2)p-R, -C(O)N(R')(R"), -N(R')-C(O)R", -S(O)-(CH2)p-R, -S(O)2-(CH2)p-R, and -S(O)N(H)-(CH2)p-R; R^3 is selected from the group consisting of hydrogen, halogen and -(CH2)p-R; R^4 and R^5 are independently selected from the group consisting of -(CH2)q-R, -Y-(CH2)q-R, -N(-(CH2)q-R)(-(CH2)q-R), -C(O)NH-(CH2)q-R, -C(O)-(CH2)q-R, -C(O)-Y-(CH2)q-R, and -C(O)-Y-N[(CH2)q-R][(CH2)q-R].

Description

Compounds for inhibiting beta-secretase including glycine arylamides {Compounds Having Glysin Aryl Amide Moiety as Inhibitors of Beta-Secretase}

The present invention relates to novel compounds that inhibit the activity of beta-secretase, pharmaceutically acceptable salts or isomers thereof, methods for their preparation, and pharmaceutical compositions containing them in pharmacologically effective amounts.

Alzheimer's disease (senile dementia) is a neurological disease that develops gradually with age, accounting for about 50-70% of all dementia patients. The main symptoms are memory loss and loss of cognitive function, and statistically, most of them develop before and after 65 years and die after about 9 years of progression. As society develops and ages, the number of patients suffering from Alzheimer's disease is increasing. It is estimated that there will be about 6 million patients in the US within the next decade, which will increase over time.

Korean society is also on the rise of the elderly, especially the social problems caused by senile dementia patients. However, to date, no therapeutic agent has been developed to treat the underlying cause of Alzheimer's disease, and acetylcholine esterase inhibitor is the only one that can be used as a general therapeutic agent. For example, Aricept ^{™ of} Pfizer, Exelon ^{™ of} Norvatis and Reminyl ^™ of Janssen are commonly used as the representative therapeutic agents.

These acetylcholine esterase inhibitors do not cure the underlying cause of Alzheimer's disease, and only provide temporary relief of symptoms in some patients (approximately 40-50%) and do not last long. Therefore, it is difficult to define it as a cure in a strict sense. In addition, the nature of the disease is required to take long-term, in the case of the drug, accompanied by various side effects, including liver toxicity has also been revealed as a problem.

Therefore, the development of a therapeutic agent that can prevent the progress of the disease is an urgent problem. As a way to find the cause of Alzheimer's disease, studies on patients with hereditary early on-set Alzheimer's disease have been underway for a long time, and as a result, mutations in several genes have been found to be the main cause. .

As a common physiological result of these genetic pathogens, it was found that the production of beta amyloid consisting of 42 amino acids (hereinafter sometimes abbreviated as 'Ab 42') is increased, so that the production of Ab 42 is a major factor in the development of Alzheimer's disease. Presumed to be a causative agent. Therefore, when a method for reducing the production of Ab 42 is developed, it is expected to be the most direct and effective therapeutic agent that can block the pathogenesis of Alzheimer's disease itself.

Beta amyloid proteins are produced by a series of cleavage by three secretases from a large molecular weight precursor called Amyloid Precursor Protein (APP), which is made in neurons. This process is carried out in a membrane vesicle called Golgi (or Endosome) in the cytoplasm of neurons, and APP and its protease secretases are located and act on the membrane. The N-terminus of beta amyloid (Ab) corresponds to the 99th amino acid from the C-terminus of APP, which is abbreviated as beta-secretase (hereinafter sometimes referred to as 'BACE' (beta-site APP Cleaving Enzyme)). Beta amyloid (Ab) protein is cleaved by a gamma-secretase (beta) amyloid (Ab) protein is produced, and then secreted out of neurons. The APP precursor may be cleaved at a completely different position via a separate pathway, with alpha-secretase cutting off the intermediate region of the Ab (between the 16th and 17th amino acids from the N-terminus). Molecular weight sAPPa is produced and secreted, and no further beta amyloid is produced.

The beta amyloid (Ab) protein of various lengths is produced by the BACE and gamma secretase, 40 amino acids (Ab 40) and 42 amino acids (Ab 42) predominantly. Ab 42, unlike Ab 40, tends to aggregate more easily and, when accumulated in the patient's brain, promotes the formation of amyloid plaque, which causes the necrotic cells in the surrounding area to slowly necrosis. It is estimated as the main pathogenesis of Alzheimer's disease.

Ab 40 and Ab 42 are produced in a ratio of about 9: 1 under normal circumstances. However, the overall increase in the amount of synthesis of the two types of amyloid protein or the selective increase of Ab 42 by the aforementioned mutation of the Presenilin 1 & 2 gene further promotes the onset of Alzheimer's disease, and the symptoms also appear to be serious. Known. Therefore, reducing the synthesis of Ab 42 can be said to be the most important part of the development of Alzheimer's treatment, which requires the development of beta or gamma secretase inhibitors.

Many multinational pharmaceutical companies are investing heavily in R & D in this area, and beta or gamma secretase inhibitors that reduce the production of beta amyloid consisting of 42 amino acids, which are believed to be the primary cause of Alzheimer's disease. Development is the dominant. However, in view of the development of a therapeutic agent that can prevent the progression of the disease by inhibiting the production mechanism of beta amyloid itself, there is no remarkable research result yet.

On the other hand, both beta and gamma secretase are known as aspartic protease and are known to be attached to the membrane. However, in the case of gamma secretase inhibitors, the gene has not yet been identified. In addition, the substrate is not limited to the APP precursor protein, and is known to be involved in the cleavage of Notch protein, which is known to play an important role in cell-fate decision during differentiation. In particular, transgenic animals from which the gamma secretase gene itself has been removed have not only been shown to die in the fetus, but the prospects are unclear because of the recent clinical trials of the gamma secretase inhibitors. Thus, much remains to be seen whether gamma secretase inhibitors can be developed as safe treatments for Alzheimer's disease.

On the other hand, in the case of BACE, several pharmaceutical companies have identified the gene in a variety of ways, and it was published in 1999 showing its activity as a beta secretase in vivo. In addition, the X-ray crystal structure has been found, and a high affinity peptide inhibitor for this is also well known. In particular, genetically deficient transgenic animals showed normal traits with no abnormalities, suggesting that BACE specific inhibitors could be developed as a safer and more effective treatment for dementia. Thus, compared to gamma secretase, BACE is a more promising target.

As discussed above, considering that existing commercially available drugs are only effective in alleviating symptoms and have no effect on the progress of the disease itself, the development of BACE specific inhibitors is a new concept for Alzheimer's disease. There is no doubt that this will be a breakthrough in drug development. In addition, since BACE inhibitors have been relatively recently studied, a full small molecule chemical compound has not yet been announced in addition to peptide variants, which may be a good opportunity for the development of new global drugs.

Recently, as BACE inhibitors, Merck (WO2006078577, WO2006060109, Wo2006057983), Ellan (WO2004022523, WO2005095326), Schering-Plaugh (WO2006014762, Wo2006014944), BMS (WO2005182105, WO2005030758), Eli-Lilly (WO200534093358), WO20034093358, etc. Has published active research results. However, many of the compounds are peptidic compounds and have low oral potential and low cerebral permeability. Recently, Eli-Lilly has reported an efficacy in animal experiments through a compound of low molecular weight peptide form, but the route of administration is not oral, but S.C. Considering the selectivity and oral potential for Cat.D, there seems to be considerable limitation.

On the other hand, recently published data by Johnson & Johnson (WO2007050612, WO2007058583, WO2006024932) and Wyeth (WO2007072925, WO2007016012, WO2006076284) have reported non-peptidic compounds beyond the existing peptide form, Cerebral permeability is known to be good.

Therefore, researches on non-peptide compounds, which are outside of the existing peptidic compounds, have been actively conducted, and this study also relates to compounds having excellent activity against BACE enzyme as non-peptide compounds. In particular, the present compounds have excellent cerebral permeability through oral administration.

The present invention aims to solve the problems of the prior art and the technical problems that have been requested from the past.

In particular, it is an object of the present invention to provide novel compounds, pharmaceutically acceptable salts or isomers thereof which have the inhibitory efficacy of beta secretase.

Another object of the present invention is to provide a method for preparing such novel compounds.

Still another object of the present invention is to provide a composition for inhibiting activity of beta-secretase, which comprises a pharmacologically effective amount of the compound as an active ingredient.

Another object of the present invention is to use such a novel compound as an active ingredient, to provide a use for the treatment or prevention of neurodegenerative diseases such as cognitive function improvement or Alzheimer's disease.

In order to achieve this object, the present invention provides a compound represented by the following formula (I), a pharmaceutically acceptable salt or isomer thereof.

(I)

(I)

Where

m and n are each independently 0 or 1,

R ¹ and R ² are each independently hydrogen, halogen, cyano, hydroxy,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, -N [-(CH ₂ ) _p -R ] [-(CH ₂ ) _p -R], -S- (CH ₂ ) _p -R, -C (O) N (R ') (R "), -N (R')-C (O) R ", -S (O)-(CH ₂ ) _p -R, -S (O) _2- (CH ₂ ) _p -R And -S (O) N (H)-(CH ₂ ) _p -R, or R ¹ and R ² together may form a ring;

R ³ is selected from the group consisting of hydrogen, halogen and-(CH ₂ ) _p -R, and R ⁴ And / or may form a ring together with R ⁵ ;

R ⁴ and R ⁵ are each independently-(CH ₂ ) _q -R, -Y- (CH ₂ ) _q -R, -N (-(CH ₂ ) _q -R) (-(CH ₂ ) _q -R ), -C (O) NH- (CH ₂ ) _q -R, -C (O)-(CH ₂ ) _q -R, -C (O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R];

here,

p and q are each independently selected from 0 to 4,

R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heterocycle and heteroaryl,

R 'and R "are each independently hydrogen, alkyl,-(CH ₂ ) _p -cycloalkyl,-(CH ₂ ) _p -aryl,-(CH ₂ ) _p -heterocycle and-(CH ₂ ) _p -hetero Aryl selected from the group consisting of aryl and R " May form a ring together,

Y is Cycloalkyl, aryl, aryl-heteroaryl, aryloxy, heterocycle and heteroaryl;

In the above,

The alkyl, alkoxy, aryl, cycloalkyl, heterocycle and heteroaryl may have a substituted or unsubstituted structure, where the substituents are halogen, cyano, amino, alkylamino, dialkylamino, alkylacylamino, alkyl , Hydroxy, C ₁ -C ₄ alkyl alkoxy, aryl alkoxy and oxo can be at least one selected from the group consisting of, halogen, cyano, amino, alkylamino, dialkylamino, hydroxy, C _1- C ₄ alkyl, alkoxy, phenyl, aryloxy and oxo. In some cases, these substituents may also be substituted, in which case the substituents are as described above. In addition, the substituents may be bonded to each other to form a ring structure.

In addition, the heteroaryl and heterocycle are each independently a 4-8 membered ring containing 1 to 3 heteroatoms selected from the group consisting of O, N and S, including 0 to 3 double bonds, preferably Preferably, it is a 5-6 membered ring containing 1 or 2 double bonds.

The compound of formula (I) according to the present invention has a structure different from that of beta secretase inhibitors, such as sulfonamide-based or peptide-based compounds, which are known in the art, and as shown in the following experimental examples, cognitive function is improved. Or an excellent inhibitory effect on human beta secretase related to the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

In the definitions for the substituents of the compounds of formula (I) according to the invention, the term 'alkyl' means an aliphatic hydrocarbon group. The alkyl moiety can be a "saturated alkyl" group, meaning that it does not contain any alkenes or alkyne moieties. The alkyl moiety may be an "unsaturated alkyl" moiety, meaning that it contains at least one alkene or alkyne moiety. "Alkene" moiety means a group of at least two carbon atoms consisting of at least one carbon-carbon double bond, and an "alkyne" moiety means at least two carbon atoms having at least one carbon-carbon triple bond Means a group. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain or cyclic.

Alkyl groups may have from 1 to 20 carbon atoms. The alkyl group may be a medium sized alkyl having 1 to 10 carbon atoms. The alkyl group may be lower alkyl having 1 to 6 carbon atoms. For example, C ₁ -C ₄ alkyl is one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t -Selected from the group consisting of butyl. In addition, when used alone or in combination with alkyloxy, respectively, it means a straight or branched chain hydrocarbon radical.

Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. It is not limited only to these.

The term 'alkoxy' means oxo alkyl having 1 to 10 carbon atoms.

The term 'cycloalkyl' means an unsaturated aliphatic 3-10 membered ring including cyclohexyl.

The term 'aryl' refers to an aryl group having at least one ring having a shared pi electron field and comprising a carbocyclic aryl (eg phenyl) and a heterocyclic aryl group (eg pyridine) Means. The term includes monocyclic or fused ring polycyclic (ie rings that divide adjacent pairs of carbon atoms) groups. 4 to 10 membered, preferably 6 to 10 membered aromatic monocyclic or multicyclic ring groups including phenyl, naphthyl and the like.

The term 'heteroaryl' comprises an aromatic 4-8 membered ring, preferably 1 to 3 heteroatoms selected from the group consisting of N, O and S, which can be fused with benzo or C ₃ -C ₈ cycloalkyl It means a 5-6 membered ring. Examples of monocyclic heteroaryl include thiazole, oxazole, thiophene, furan, pyrrole, imidazole, isoxazole, pyrazole, triazole, thiadiazole, tetrazole, oxadiazole, pyridine, pyridazine, Pyrimidine, pyrazine and similar groups, but is not limited to these. Examples of bicyclic heteroaryl include indole, indolin, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzisoxazole, benzthiazole, benzthiadiazole, benztriazole, quinoline, isoquinoline, purine , Furypyridine and similar groups, but are not limited to these.

The term 'heterocycle' includes one or two hetero atoms selected from the group consisting of N, O and S, and is selected from benzo or C ₃ -C ₈ 3 to 10 membered ring, preferably 4 to 8 membered ring, more preferably 5 to 6 membered ring, which may be fused with cycloalkyl and are saturated or include 1 or 2 double bonds. Furan, thiophene, pyrrole, pyrroline, pyrrolidine, oxazole, thiazole, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, isothiazole, triazole, thia Diazole, pyran, pyridine, piperidine, morpholine, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, hydrofuran and the like, but are not limited thereto.

Other terms may be interpreted as meanings commonly understood in the field to which the present invention belongs.

In the compound of formula 1 according to the invention, R ¹ and R ² are preferably hydrogen, halogen, cyano,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, -N ( H)-(CH ₂ ) _p -R, -N [-(CH ₂ ) _p -R] [-(CH ₂ ) _p -R], -S- (CH ₂ ) _p -R, -C (O) N (R ') (R ") and N (R')-C (O) R", or may form a ring together, and R 'and R "are each independently hydrogen, alkyl ,-(CH ₂ ) _p -aryl,-(CH ₂ ) _p -cycloalkyl,-(CH ₂ ) _p -heterocycle, or together may form a ring.

In one preferred embodiment, R ¹ is hydrogen, halogen, cyano, hydroxy,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R and -N [-(CH ₂ ) _p- R] [-(CH ₂ ) _p -R]. Wherein p is selected from 0 to 4, preferably 0 or 1, R may be selected from the group consisting of hydrogen, alkyl, heterocycle, aryl, cycloalkyl and heterocycle, preferably hydrogen, C ₁ -C ₄ alkyl, pyrrolidine, piperidine, phenyl, benzyl, cyclopentyl, and cyclopropyl.

R ¹ is particularly preferably hydrogen, halogen, cyano, anilino, (3-cyano) anilino, (4-fluoro) anilino, pyrrolidinemethyl, piperidinemethyl, (3- Trifluoro) anilino, (3-trifluoromethyl) anilino, isobutylamino, isopentylamino, cyclopentylamino, benzylamino, phenoxy and benzyloxy.

R ² may be more preferably selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, cycloalkyl-alkoxy, heteroaryl-alkoxy and alkyl-aryl, and particularly preferably hydrogen, chloro, methyl, Can be selected from the group consisting of isopentyloxy, cyclopentyloxy, (thiophen-2-yl) propyloxy and (thiophen-2-yl) ethyloxy.

R ³ is preferably-(CH ₂ ) _p -R, or may form a ring together with R ⁴ , more preferably selected from the group consisting of hydrogen, phenyl, cyclohexyl, benzyl and piperidine Or a ring structure together with R ^4, and particularly preferably hydrogen or piperidine.

R ⁴ is preferably-(CH ₂ ) _q -R, -N (-(CH ₂ ) _q -R) (-(CH ₂ ) _q -R), -C (O) NH-R,- C (O)-(CH ₂ ) _q -R, -C (O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R], and more preferably-(CH ₂ ) _q -R, -C (O) NH-R, -C (O)-(CH ₂ ) _q -R, -C (O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R] , Where Y is Cycloalkyl, aryl, heterocycle, and heteroaryl. Especially preferably,-(CH ₂ ) _q -aryl, -C (O)-(CH ₂ ) _q -aryl, -C (O) NH-aryl, -C (O) NH-cycloalkyl and -C ( O) -aryl- (CH ₂ ) _q -R.

In one preferred embodiment, R ⁴ is benzyl, phenethyl, acetyl, aminobenzyl, (3-dimethyl) aminobenzyl, -C (O) cyclohexyl, -C (O) benzyl, -C (O) phenethyl , -C (O) (diphenylmethyl), -C (O) [(3-dimethyl) amino] benzyl, -C (O) NH (3,5-dichlorophenyl), -C (O) NH (phenyl ), -C (O) NH (cyclohexyl), -C (O) (3-benzyloxy-pyrrolidin-2-yl), -C (O) {[3- (4-phenylimidazole) Methyl] phenyl}, -C (O) {3- (piperidine) methylphenyl}, -C (O) {3- (piperidine) ethylphenyl}, -C (O) {3- (diisobutyl Amino) methylphenyl}, -C (O) {3-pyrrolidinephenyl}, -C (O) {3- (piperidine) methylthiophene}, -C (O) [3- (pyrrolidinemethyl ) Thiophen-3-yl], -C (O) [5- (piperidinmethyl) thiophen-2-yl], -C (O) [4- (benzyloxy) pyrrolidin-2-yl ], -C (O) {[7- (tetrahydrofuran-4-yl) amino] indol-2-yl}, -C (O) {[7- (phenoxy-3-yl) amino] indole- 2-yl} and -C (O) [3-N (cyclopropylmethyl, cyclopropyl-piperidine, methyl) phenyl].

R ⁵ may preferably be hydrogen or —W— (CH ₂ ) _r —R, wherein W may be directly bonded or piperidine, pyrrolidine, aminopyrrolidine, azetidine and amino have.

In one preferred example, R ⁵ is hydrogen, (3-dimethyl) aminoethyl, benzyl, phenoxy, phenethyl, pyrrolidine, piperidine, cyclopropyl-piperidine, cyclopropyl-methyl-piperidine , Cyclopentyl-piperidine and C (O) -methyl-dimethylamino, and may be selected from the group consisting of hydrogen, benzyl, piperidine, cyclopropylpiperidine and cyclopentylpiperidine It may be selected from the group consisting of.

The compounds according to the invention can also form pharmaceutically acceptable salts. The 'pharmaceutically acceptable salts' are salts with acids which form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, and the like. Organic carbonic acid, such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or naphthalene Acid addition salts formed by sulfonic acids such as sulfonic acids and the like. These acid addition salts can be prepared by known techniques based on the structure of formula (I).

The compounds of the present invention or pharmaceutically acceptable salts thereof may also exist in the form of hydrates or solvates.

The 'isomer' refers to a compound of the present invention or a salt thereof having the same chemical formula or molecular formula but different optically or stericly. The compounds of formula (I) according to the invention may have an asymmetric carbon center and therefore exist as optical isomers (R or S isomers), racemates, diastereomeric mixtures and individual diastereomers, when having double bonds, Geometric isomers (trans, cis isomers) may also be present. All these isomers and mixtures thereof are also within the scope of the present invention.

Unless stated otherwise below, compounds of formula (I) include pharmaceutically acceptable salts and isomers thereof, all of which are to be construed as being within the scope of the present invention. For convenience of explanation, it is briefly referred to herein as a compound of formula (I).

Representative compounds of Formula 1 according to the present invention include the compounds shown in Table 1 below.

 TABLE 1

More preferred compounds of the formula (1) according to the present invention include the following compounds.

N ^2- (piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenethyl) glycinamide

N ^2- (piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenethyl) glycinamide

N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (4-phenoxy-2-isopentyloxyphenyl) -N ^2- (phenethyl) glycinamide

N ^2- (piperidin-4-yl) -N- (4-phenoxy-2- (3-thiophen-3-yl) -phenyl) -N ^2- (phenethyl) glycinamide

N ^2- (piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide

N ^2- (3-dimethylamino-benzyl) -N- (4-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide

N ^2- (piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzyl) glycineamide

N ^2- (1-cyclopentyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (3,5-dichlorophenylaminocarbonyl) glycinamide

N ^2- (1-cyclopentyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide

N ^2- (1-cyclopropyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide

N ^2- (piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide

N ^2- (piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide

N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide

N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (phenethylcarbonyl) glycinamide

N- {2-oxo-2-[(4-phenoxyphenyl) amino] ethyl} -3- (pyrrolidin-1-ylmethyl) phenylamide

N- {2-oxo-2-[(3-phenoxyphenyl) amino] ethyl} -3- (piperidin-1-ylmethyl) phenylamide

(4R) N [(4-phenoxy) phenyl]]-4-benzyloxy-1- (2-dimethylaminoethyl) -pyrrolidine-2-carboxamide

The present invention also relates to a method for preparing a compound of Formula (I), wherein in Formula (I), wherein m and n are both 0, the compound of Formula 1 is a carboxylic acid such as It can synthesize | combine by the amide coupling reaction of the same amine compound.

Scheme 1

R ¹ , R ² , R ³ , R ⁴ and R ⁵ in the scheme are as defined above.

An amine such as Chemical Formula 2 is commercially available, but some compounds may be synthesized by the same method as in Scheme 2 below.

Scheme 2

That is, a commercially available compound such as Chemical Formula 4 and a phenolic compound such as Chemical Formula 5 are reacted with each other in basicization to synthesize a compound such as Chemical Formula 6, and then subjected to an alkoxylation reaction and to reduce the nitro group. The same amine compound can be obtained.

Compounds containing nitrogen instead of oxygen can be synthesized from nitroaniline in the same manner as in Scheme 3 below.

Scheme 3

That is, by performing a reducing amino reaction from a nitro aniline, such as the formula (8), and reducing the nitro group can be synthesized a compound of the formula (9). On the other hand, in the case of the anilino-type compound, the compound of Formula 11 may be synthesized through synthesis using CuCl.

The acid compound represented by Chemical Formula 3 may be synthesized from glycine methyl ester by the same method as in Scheme 4 below.

Scheme 4

Compounds such as Formula 13 can be synthesized from glycine methyl esters via reductive amination reactions with BOC-piperidinone. By acylating the compound of Formula 13 with acid chloride, an amide compound as shown in Formula 14 can be obtained, and a carbamate type compound as shown in Formula 15 can be obtained by performing a linkage reaction using isocyanate. In addition, by performing the reductive coupling reaction once more, an amine compound such as Chemical Formula 16 may be synthesized.

On the other hand, for some compounds in which R ⁵ is hydrogen can be synthesized through two amidation reactions and reductive amination reaction in the same manner as in Scheme 5.

Scheme 5

The reaction may preferably be carried out in conventional solvents which do not adversely affect the reaction, particularly preferably in the group consisting of DMF, dimethylacetamide, tetrahydrofuran, methylene chloride, dichloroethane, methanol and water One or more solvents selected may be used, but are not limited thereto.

In addition, as a coupling agent which can be used for the coupling reaction, a known coupling agent can be used, for example, dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-ethyl Carbodiimides, such as carbodiimide (EDC) and 1,1'-dicarbonyldiimidazole (CDI), are referred to as 1-hydroxybenzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT). ), Or bis- (2-oxo-3-oxazolidinyl) -phosphinic chloride (BOP-Cl), diphenylphosphoryl azide (DPPA), N- [dimethylamino-1H- 1,2,3-triazole [4,5-b] pyridin-1-ylmethylene] -N-methylmethanealuminum (HATU) and the like, but are not limited thereto.

Separation of common mixtures can be carried out using column chromatography, and for final compounds can be separated by recrystallization or normal or reversed HPLC (Waters, Delta Pack, 300 × 50 mmI.D., C18 5 μm, 100 A). In the case of recrystallization or purification through HPLC, a compound can be obtained in the form of trifluoroacetic acid salt, and an ion exchange resin can be used in case of obtaining hydrochloride.

As mentioned above, after the reaction according to the method of the present invention is completed, the product can be separated and purified by conventional post-treatment methods such as chromatography, recrystallization and the like.

The present invention also relates to a pharmaceutical composition for inhibiting beta secretase, comprising a pharmaceutically effective amount of the compound of formula (I), a pharmaceutically acceptable salt or isomer thereof, and a pharmaceutically acceptable carrier as an active ingredient. In addition, the composition may additionally include a diluent or an excipient, as the case may be.

Since the compound of Formula (I) has an excellent inhibitory effect on beta secretase, it provides a beta secretase inhibitor composition comprising the same with a pharmaceutically acceptable carrier. In particular, the composition according to the present invention exhibits an excellent effect on improving cognitive function or treating or preventing neurodegenerative diseases, and in particular, on the prevention and treatment of Alzheimer's disease, but is not limited thereto.

The term 'pharmaceutical composition' means a mixture of a compound of the invention with other chemical components such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound into the organism. Various techniques for administering a compound exist, including, but not limited to, oral, injection, aerosol, parenteral and topical administration. Pharmaceutical compositions may also be obtained by reacting acid compounds such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

The term 'therapeutically effective amount' means that the amount of the compound administered relieves to some extent one or more symptoms of the disorder being treated. Thus, the pharmacologically effective amount may be selected from (1) the effect of reversing the rate of progression of the disease; (2) the effect of inhibiting or somewhat slowing further progression of the disease; And / or (3) an amount that has some effect of alleviating (preferably eliminating) one or more symptoms associated with the disease.

The term 'carrier' is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into organisms' cells or tissues.

The term 'diluent' is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

The term 'physiologically acceptable' is defined as a carrier or diluent that does not impair the biological activity and the properties of the compound.

The compounds used herein may be administered to human patients as such or as pharmaceutical compositions in combination with other active ingredients or with a suitable carrier or excipient, such as in a combination therapy. Techniques for formulation and administration of compounds in this application can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990.

a) Route of administration

The compounds of the present invention can be administered by any route as desired. Injection, oral and nasal administration are preferred, but can also be administered through the skin, abdominal cavity, larynx and rectum.

b) Composition / Formulation

Pharmaceutical compositions of the invention can be prepared in a known manner, for example, by means of conventional mixing, dissolving, granulating, sugar-making, powdering, emulsifying, encapsulating, trapping and or lyophilizing processes. . Thus, pharmaceutical compositions for use according to the present invention comprise one or more pharmacologically acceptable compositions comprising excipients or auxiliaries which facilitate the treatment of the active compounds into formulations which can be used pharmaceutically. It may also be prepared by conventional methods using a carrier. Proper formulation is dependent upon the route of administration chosen. Any of the known techniques, carriers and excipients can be used suitably and as understood in the art, for example in Remingston's Pharmaceutical Sciences described above.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be prepared using suitable dispersing, wetting, or suspending agents according to known techniques. Solvents that can be used for this are water, Ringer's solution and isotonic NaCl solution, and sterile fixed oils are also commonly used as solvents or suspending media. Any non-irritating fixed oil may be used for this purpose, including mono- and diglycerides, and fatty acids such as oleic acid may also be used in the preparation of injectables.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Particularly, capsules and tablets are useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the active compounds of formula (I) according to the invention with one or more inert diluents such as sucrose, lactose, starch and the like and carriers such as lubricants such as magnesium stearate, disintegrants, binders and the like. .

c) Effective amount

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prolong the survival of the subject to be treated or to prevent, alleviate or alleviate the symptoms of a disease. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, in particular in terms of the detailed disclosure provided herein.

The total daily dose to be administered to the host in a single dose or in separate doses when administering a compound of the present invention for clinical purposes is preferably in the range of 10 to 100 mg / kg body weight, but specific dose levels for individual patients may be used. It may vary depending on the specific compound, the weight of the patient, sex, health condition, diet, the time of administration of the drug, the method of administration, the rate of excretion, the mixing of the drug and the severity of the disease.

The invention is described in more detail by the following preparations and examples, although the scope of the invention is not limited in any way by them.

Explanation of abbreviations and terms used in the names of the compounds in the above reaction schemes, preparation examples and examples are as follows.

The following preparations more specifically illustrate the preparation of intermediates required for the synthesis of the example compounds according to the invention.

Preparation Example 1 3- (3-cyano-phenyl) aminoaniline

Step A: 3- (3-cyanoanilino) nitrobenzene

Dissolve 3-cyanobenzene bromide (1.8 g, 10 mmol) in THF (30 mL) and add CuI (79 mg, 1 mmol) and K ₂ CO ₃ (1.3 g, 12 mmol) for 48 hours at 80 ° C. Heated during. After the reaction was completed, the solid was removed by using a celite filter, 1N HCl was added to the filtrate, and the organic material was extracted with EtOA. The residual organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc / n-Hex = 1/10) to give the title compound (1. 3 g, 54%). Obtained.

Mass [M + H] = 240 (M + 1)

Step B: 3- (3-cyano-phenyl) aminoaniline

3- (3-cyanoanilino) nitrobenzene (1.0 g, 4. 18 mmol) obtained in step A was dissolved in methanol (10 mL), and then NH ₄ Cl (1.12 g, 20. 9 mmol) was added. The reaction temperature was raised to 60 ° C., and iron (1.17 g, 20.9 mmol) was added under heating. After heating at 60 ° C. for 30 min, the reaction was removed solids through a celite filter and the organics extracted with EtOAc. The residual organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc / n-Hex = 1/5) to give the title compound (550 mg, 63%). .

Mass [M + H] = 210 (M + 1)

Preparation Example 2 3- (3-Fluoro-phenyl) aminoaniline

The title compound was obtained in the same manner as in Preparation Example 1 using 3-fluorobenzyl bromide and 3-nitro aniline.

Mass [M + H] = 203 (M + 1)

Preparation Example 3: 3- (3-trifluoromethylphenyl) aminoaniline

The title compound was obtained in the same manner as in Preparation Example 1 using 3-trifluoromethyl phenyl bromide and 3-nitro aniline.

Mass [M + H] = 253 (M + 1)

Preparation Example 3 3-isobutylamino aniline

Step A: 3- (isobutylamino) -1-nitro-benzene

3-nitroaniline (6.9 g, 50 mmol) and isopropyl aldehyde (3.6 g, 50 mmol) were added to dichloroethane (100 mL) and NaBH (OAc) ₃ (21.3 g, 100 mmol) was added, followed by 12 at room temperature. Stir for hours. After the reaction was completed, the mixture was diluted with saturated NaHCO ₃ aqueous solution and the organics were extracted using dichloromethane and EtOA. The extracted organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc / n-Hex = 1/10) to obtain the title compound (7.37 g, 76%). .

Mass [M + H] = 195 (M + 1)

Step B: (3-isobutylamino) aniline

The title compound was obtained in the same manner as in Step B of Preparation Example 1 using 3- (isobutylamino) -1-nitro-benzene obtained in Step A.

Mass [M + H] = 165 (M + 1)

Preparation Example 5 3-cyclopentylamino-aniline

The title compound was obtained in the same manner as in Preparation Example 4 using 3-nitroaniline and isobutyl aldehyde.

Mass [M + H] = 176 (M + 1)

Preparation Example 6 4- (piperidin-1yl) methylaniline

The title compound was obtained in the same manner as in Preparation Example 4 using 4-nitro benzaldehyde and piperidine.

Mass [M + H] = 191 (M + 1)

Preparation Example 7 3- (benzylamino) aniline

The title compound was obtained in the same manner as in Preparation Example 4 using 3-nitroaniline and benzaldehyde.

Mass [M + H] = 198 (M + 1)

Preparation Example 8 4-phenoxy-2-cyclopentyloxyaniline

Step A: 4-fluoro-2-cyclopentyloxy-nitrobenzene

4-Fluoro-2-hydroxy-nitrobenzene (1.57 g, 10 mmol) was dissolved in THF (30 mL) and PPh ₃ (2.62 g, 10 mmol) and cyclopentanol (860 mg, 10 mmol) were added. After lowering the temperature of the reaction solution to 0 ℃, DEAD (40% toluene solution, 2.2 M, 5 mL, 11 mmol) was added slowly and stirred at room temperature for 12 hours. The reaction was terminated with aqueous NH ₄ Cl solution, extracted with EtOAc and washed with 1N HCl. The organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 1/15) to give the title compound (1.48 g, 65%).

Mass [M + H] = 226 (M + 1)

Step B: 4-phenoxy-2-cyclopentyloxy-nitrobenzene

4-fluoro-2-cyclopentyloxy-nitrobenzene (1.0 g, 4.44 mmol) obtained in step A was dissolved in DMF (15 mL), and phenol (941 mg, 10 mmol) and K ₂ CO ₃ (4.05 g) , 30 mmol) was added and stirred at 60 ° C. for 15 hours. When the reaction was terminated, DMF was removed under reduced pressure, the organics were extracted with EtOAc diluted with NH ₄ Cl aqueous solution, and washed with 1N NaHCO ₃ aqueous solution. The organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 1/15) to afford the title compound.

Mass [M + H] = 300 (M + 1)

Step C: 4-phenoxy-2-cyclopentyloxyaniline

The title compound was obtained in the same manner as in Step B of Preparation Example 1 using 4-phenoxy-2-cyclopentyloxy-nitrobenzene obtained in step B.

Mass [M + H] = 270 (M + 1)

Preparation Example 9 4-phenoxy-2-isopentyloxyaniline

The title compound was obtained by the same method as Steps B and C of Preparation Example 8 using commercially available 4-fluoro-2-hydroxy-nitrobenzene and isopentyl bromide.

Mass [M + H] = 272 (M + 1)

Preparation Example 10 4-phenoxy-2- (3-thiophen-3-yl) propyloxy aniline

The title compound was obtained by the same method as Steps B and C of Preparation Example 8 using commercially available 4-fluoro-2-hydroxy-nitrobenzene and 3- (chloropropyl) thiophene.

Mass [M + H] = 312 (M + 1)

Preparation Example 11 N- (1-BOC-piperidin-4-yl) -N- (2-phenylethyl) glycine

Step A: N- (1-BOC-piperidin-4-yl) glycine methylester

The title compound was obtained in the same manner as in Step A of Preparation Example 4 using glycine methyl ester and 1-BOC-piperidine.

Mass [M + H] = 273 (M + 1)

Step B: N- (1-BOC-piperidin-4-yl) -N- (2-phenylethyl) glycine methylester

The title compound was obtained in the same manner as in Step A of Preparation Example 4 using N- (1-BOC-piperidin-4-yl) glycine methylester and phenethyl aldehyde obtained in Step A.

Mass [M + H] = 377 (M + 1)

Step C: N- (1-BOC-piperidin-4-yl) -N- (2-phenylethyl) glycine

N- (1-BOC-piperidin-4-yl) -N- (2-phenylethyl) glycine methylester (1.0 g, 2.65 mmol) obtained in step B was dissolved in methanol (5 mL) and water (4 mL) was added and LiOH (463 mg, 13.25 mmol) was added and stirred at room temperature for 3 hours. After the reaction was completed, methanol was removed under reduced pressure, diluted with 1N HCl, and the organics were extracted with EtOAC. The extracted organic solution was dried over anhydrous MgSO ₄ and the solvent was removed under reduced pressure, and then used in the next reaction without further purification.

Mass [M + H] = 363 (M + 1)

Preparation Example 12 N- (1-BOC-piperidin-4-yl) -N-[(3,5-dichloro) anilinocarbonyl] glycine

Step A: N- (1-BOC-piperidin-4-yl) -N-[(3,5-dichloro) anilinocarbonyl] glycine methylester

N- (1-BOC-piperidin-4-yl) glycine methyl ester (1.0 g, 3.67 mmol) obtained in step A of Preparation Example 11 was dissolved in dichloromethane (15 mL), and Et ₃ N (1.03 mL, 7.4 mmol) was added, followed by 3,5-dichloro isocyanate (690 mg, 3.67 mmol) and stirred at room temperature for 4 hours. The reaction was terminated with aqueous NH ₄ Cl solution, extracted with EtOAc and washed with 1N HCl. The organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 3/7) to give the title compound (1.19 g, 90%).

Mass [M + H] = 361 (M + 1)

Step B: N- (1-BOC-piperidin-4-yl) -N-[(3,5-dichloro) anilinocarbonyl] glycine

N- (1-BOC-piperidin-4-yl) -N-[(3,5-dichloro) anilinocarbonyl] glycine methylester obtained in step A was used as in step B of Preparation Example 11 Performed by the method to give the title compound.

Mass [M + H] = 347 (M + 1)

Preparation Example 13: N- (1-BOC-piperidin-4-yl) -N- (phenylacetyl) glycine

Step A: N- (1-BOC-piperidin-4-yl) -N- (phenylacetyl) glycine methylester

Commercially available phenylacetic acid (499 mg, 3.67 mmol) and N- (1-BOC-piperidin-4-yl) glycine methylester (1 g, 3.67 mmol) obtained in step A of Preparation Example 11 were converted to DMF. It was dissolved in (15 mL), and Et ₃ N (1.02 mL, 7.3 mmol) was added, followed by addition of EDC (825 mg, 4.77 mmol) and HOBT (842 mg, 5.5 mmol), followed by stirring at room temperature for 12 hours. Upon completion of the reaction, the mixture was diluted with NaHCO ₃ , organics were extracted with EtOA, and washed with NaHCO ₃ and 1N HCl. The remaining organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 1/5) to obtain the title compound (1.21 g, 85%). It was.

Mass [M + H] = 391 (M + 1)

Step B: N- (1-BOC-piperidin-4-yl) -N- (phenylacetyl) glycine

The title compound was obtained in the same manner as in Preparation Step B using N- (1-BOC-piperidin-4-yl) -N- (phenylacetyl) glycine methylester obtained in step A.

Mass [M + H] = 377 (M + 1)

Preparation 14-24

Using the commercially available phenyl glycine methyl ester, glycine methyl ester acid and the carbonyl compound was selectively carried out in the same manner as in Preparation Examples 11 to 13 to synthesize the compounds of Preparation Examples 14 to 24.

Preparation Example 24 (S) -N-phenyl-2-piperidin-1-yl succinamic acid

Step A: (S) -2-benzyloxycarbonylamino-N-phenyl-succinamic acid t-butylester

The title compound was obtained by the same method as Step A of Preparation Example 13 using commercially available (S) -2-benzyloxycarbonylamino-succinic acid t-butyl ester and aniline.

Mass [M + H] = 365 (M + 1)

Step B: (S) -2-Amino-N-phenyl-succinamic acid t-butylester

(S) -2-benzyloxycarbonylamino-N-phenyl-succinamic acid t-butylester (1.0 g, 2.73 mmol) obtained in step A was dissolved in methanol (10 mL) and Pd / C (100 mg) After the addition was stirred for 12 hours using a hydrogen reactor (hydrogen = 45 psi). When the reaction was completed, the methanol was removed after the celite filter and used for the next reaction without further purification.

Mass [M + H] = 265 (M + 1)

Step C: (S) -N-phenyl-2-piperidin-1-yl-succinamic acid t-butylester

The title compound was obtained in the same manner as Step A of Preparation Example 4 using N-ASP (Ot-Bu) -N-Ph and glutaaldehyde obtained in Step B.

Mass [M + H] = 333 (M + 1)

Step D: (S) -N-phenyl-2-piperidin-1-yl succinamic acid

Piperidine-N-ASP (Ot-Bu) -N-Ph obtained in step C was dissolved in DCM, TFA was added, stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure. Used for the next reaction without further purification.

Mass [M + H] = 233 (M + 1)

Preparation Example 25 3- (pyrrolidin-1-ylmethyl) benzoic acid

Step A: Methyl 3- (pyrrolidin-1-ylmethyl) benzoate

Commercially available methyl 3-bromomethyl benzoate (2.29 g, 10 mmol) was dissolved in dichloromethane (30 mL), Et ₃ N (2.8 ml, 20 mmol) was added, followed by pyrrolidine (1.42 g , 20 mmol) was added and stirred at room temperature for 12 hours. Upon completion of the reaction, the mixture was diluted with aqueous NH ₄ Cl solution and the organics were extracted with EtOAc. The extracted organics were dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 2/1) to give the title compound (1.32 g, 60%).

Mass [M + H] = 220 (M + 1)

Step B: 3- (Pyrrolidin-1-ylmethyl) benzoic acid

The title compound was obtained by the same method as Step C of Preparation Example 11 using methyl 3- (pyrrolidin-1-ylmethyl) benzoate obtained in Step A.

Mass [M + H] = 206 (M + 1)

Example 1: N- {2-oxo-2-[(4-phenoxyphenyl) amino] ethyl} -3- (pyrrolidin-1-ylmethyl) phenylamide

Step A: N ² -BOC-N- (4-phenoxyphenyl) glycineamide

Commercially available 4-phenoxyaniline (555 mg, 3 mmol) and BOC-N-Gly-OH (525 mg, 3 mmol) were dissolved in DMF (10 mL) and Et ₃ N (840 uL, 6 mmol) EDC (698 mg, 3.9 mmol) and HOBt (688 mg, 4.5 mmol) were added, followed by stirring at room temperature for 12 hours. When the reaction was terminated, diluted with NaHCO ₃ , the organics were extracted with EtOAc, and washed with NaHCO ₃ and 1N HCl. The remaining organic solution was dried over anhydrous MgSO ₄ , the solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent EtOAc: n-Hex = 1/9) to obtain the title compound (843 mg, 82%). .

Mass [M + H] = 343 (M + 1)

Step B: N- (4-phenoxyphenyl) glycineamide

N ² -BOC-N- (4-phenoxyphenyl) glycineamide (500 mg, 1.45 mmol) obtained in step A was dissolved in dichloromethane (2 mL) and TFA (2 mL) was added. Was stirred and the solvent was removed under reduced pressure. Used for the next reaction without further purification.

Mass [M + H] = 243 (M + 1)

Step C: N- {2-oxo-2-[(4-phenoxyphenyl) amino] ethyl} -3- (pyrrolidin-1-ylmethyl) phenylamide

The title compound was carried out in the same manner as in Step A using N- (4-phenoxyphenyl) glycinamide obtained in Step B and 3- (pyrrolidin-1-ylmethyl) benzoic acid obtained in Preparation Example 25. Obtained.

¹ H NMR (500 MHz, CDCl ₃ ): δ 8.57 (s, 1H), 7.85 (s, 1H), 7.76 (d, 1H), 7.52 (d, 3H), 7.42 t, 1H), 7.31 (t, 2H), 7.18 (m 1H), 7.07 (t, 1H), 6.67 (m, 4H), 4.32 (d, 2H), 3.69 (s, 2H) 2.55 (s, 4H), 1.80 (s, 4H)

Mass [M + H] = 443 (M + 1)

Example  2 to 18

Using a commercially available aniline and aniline obtained in Preparation Examples 1 to 7 in the same manner as in Example 1, m is 0, R ⁴ is -C (O) R ^{4 '} , R ⁵ is hydrogen Phosphorus Examples 2 to 15 represented by Formula II were synthesized.

(II)

(II)

In the table, c-hex represents cyclohexyl.

Example 19: N ² -(Piperidin-4-yl) -N- (4-phenoxyphenyl) glycineamide

Step A: N ² -(1-BOC-piperidin-4-yl) -N- (4-phenoxyphenyl) glycinamide

The title compound was obtained in the same manner as in Step A of Preparation Example 4 using N- (4-phenoxyphenyl) glycineamide and 1-BOC-piperidine obtained in Step B of Example 1.

Mass [M + H] = 426 (M + 1)

Step B: N ² -(Piperidin-4-yl) -N- (4-phenoxyphenyl) glycineamide

The title compound was carried out in the same manner as in Step B of Example 1 using N ^2- (1-BOC-piperidin-4-yl) -N- (4-phenoxyphenyl) glycinamide obtained in Step A. Obtained.

Mass [M + H] = 326 (M + 1)

Example 20 N ² -(Piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ² -(Phenethyl) glycineamide

Step A: N ² -(1-BOC-piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ² -(Phenylethyl) glycineamide

4-phenoxy-2-cyclopentyloxyaniline obtained in Preparation Example 8 and N- (1-BOC-piperidin-4-yl) -N- (2-phenylethyl) glycine obtained in Preparation Example 11 were used. Was carried out in the same manner as in Steps A and B of Example 1 to obtain the title compound.

¹ H NMR (CDCl ₃ ) δ 8.34 (d, 1H), 7.40-7.00 (m, 11H), 6.60-6.56 (m, 2H), 4.78-4.74 (m, 1H), 4.23-4.10 (m, 3H) , 3.31 (s, 2H), 2.84-2.64 (m, 8H), 1.90-1.80 (m, 8H), 1.77-1.65 (m, 2H), 1.44-1.40 (m, 9H)

Mass [M + H] = 613 (M + 1)

Step B: N ² -(Piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ² -(Phenylethyl) glycineamide

Using N ^2- (1-BOC-piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenylethyl) glycinamide obtained in step A The procedure was carried out in the same manner as in Example 1, to obtain the title compound.

¹ H NMR (MeOD): δ 7.83 (d, 1H), 7.36-7.10 (m, 9H), 6.98 (d, 2H), 6.67 (s, 1H), 6.50 (d, 1H), 4.79-4.72 (m , 1H), 3.64 (s, 2H), 3.60-3.55 (m, 1H), 3.17-3.05 (m, 8H), 2.36-2.33 (m, 2H), 2.08-2.05 (m, 2H), 1.95-1.74 (m, 4H), 1.60-1.58 (m, 4H)

Mass [M + H] = 513 (M + 1)

Example 21: N ² -[1- (cyclopropylmethyl) piperidin-4-yl] -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ² -(Phenethyl) glycineamide

Using N ^2- (piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenylethyl) glycinamide and cyclopropyl aldehyde obtained in Example 20 Was carried out in the same manner as in Step A of Preparation Example 4 to obtain the title compound.

¹ H NMR (CDCl ₃ ): δ 8.35 (d, 1H), 7.31-6.95 (m, 11H), 6.59-6.54 (m, 2H), 4.73-4.72 (m, 1H), 3.31 (s, 2H), 3.28-3.20 (m, 2H), 2.88-2.75 (m, 4H), 2.61-2.57 (m, 1H), 2.32-2.25 (m, 2H), 1.90-1.63 (m, 14H), 0.95-0.86 (m , 1H), 0.55-0.52 (m, 2H), 0.15-0.09 (m, 2H)

Mass [M + H] = 567 (M + 1)

Example  22 to 51

Commercially available aniline or aniline synthesized in Preparation Examples 8 to 10 and the acid synthesized in Preparation Examples 11 to 25 were selectively carried out in the same manner as in Examples 20 and 21, wherein n in Formula I was 0. The compounds of Examples 22 to 51 represented by the following Chemical Formula III were obtained.

(III)

(III)

In the above table, Ph is phenyl, c-Pro is cyclopropyl, c-Pen is cyclopentyl c-Hex is cyclohexyl.

Example  52: (2S, 4R) -N [(4- Phenoxy ) Phenyl ]]-4- Benzyloxy - Pyrrolidine -2- Carboxamide

Step A: (2S, 4R) -N [(4- Phenoxy ) Phenyl ]]-4- Benzyloxy -One- BOC - Pyrrolidine -2- Carboxamide

The title compound was obtained in the same manner as in Example 1 using commercially available (2S, 4R) -4-benzyloxy-1-BOC-pyrrolidine-2-carboxylic acid and 4-phenoxyaniline.

¹ H NMR (CDCl ₃ ): δ 8.36 (d, 1H), 7.41-7.30 (m, 7H), 7.15-7.08 (m, 2H), 7.03-7.00 (m, 2H), 6.64-6.59 (m, 2H ), 4.85-4.83 (m, 1H), 4.77-4.58 (m, 3H), 4.45-4.23 (m, 1H), 4.01-3.95 (m, 1H), 3.75-3.55 (m, 1H), 2.65-2.55 (m, 1H), 2.21-2.08 (m, 1H), 1.93-1.30 (m, 17H)

Mass [M + H] = 573 (M + 1)

Step B: (2S, 4R) -N [(4- Phenoxy ) Phenyl ]]-4- Benzyloxy - Pyrrolidine -2- Carboxamide

Example B step 1 using the (2S, 4R) -N [(4-phenoxy) phenyl]]-4-benzyloxy-1-BOC-pyrrolidine-2-carboxyamide obtained in step A The same procedure was followed to yield the title compound.

¹ H NMR (MeOD): δ 7.66 (d, 1H), 7.41-7.29 (m, 7H), 7.14-7.10 (m, 2H), 7.01-6.99 (m, 2H), 6.68 (d, 1H), 6.51 (dd, 1H), 4.78-4.75 (m, 1H), 4.72-4.69 (m, 1H), 4.67-4.60 (m, 2H), 4.52-4.48 (m, 1H), 3.61-3.58 (m, 1H) , 3.52-3.48 (m, 1H), 2.84-2.78 (m, 1H), 2.22-2.15 (m, 1H), 1.99-1.77 (m, 6H), 1.65-1.60 (m, 2H)

Mass [M + H] = 473 (M + 1)

Example  53: (2S, 4R) -N [(4- Phenoxy ) Phenyl ]]-4- Benzyloxy -1- (2- Dimethylaminoethyl )- Pyrrolidine -2- Carboxamide

Step A of Preparation Example 4 using (2S, 4R) -N [(4-phenoxy) phenyl]]-4-benzyloxy-pyrrolidine-2-carboxyamide and dimethylglycine egg obtained in Example 52. The same procedure was followed to obtain the title compound.

¹ H NMR (CDCl ₃ ): δ 9.14 (s, 1H), 8.12 (d, 1H), 7.37-7.29 (m, 7H), 7.09-7.06 (m, 1H), 6.98-6.96 (m, 2H), 6.61 (d, 1H), 6.55 (dd, 1H), 4.76-4.73 (m, 1H), 4.54-4.49 (m, 2H), 4.20-4.16 (m, 1H), 3.62-3.59 (m, 1H), 3.54-3.51 (m, 1H), 3.36-3.30 (m, 1H), 3.23-3.14 (m, 2H), 2.94-2.92 (m, 1H), 2.52-2.46 (m, 1H), 2.18-2.04 (m , 6H), 1.98-1.60 (m, 12H)

Mass [M + H] = 570 (M + 1)

Experimental Example 1 Determination of Recombinant Beta Secretase 2 Enzyme Activity

Step A: Preparation of the Recombinant Beta Secretase2 Expression Vector

CDNA (ATCC, Cat #: 6896840) based on the human BACE2 gene sequence (accession: BC014453) known from Genebank, an existing public data base, was purchased. Cloning only the portion corresponding to amino acid sequence 1-466 except for the trnsmembrane domain and the cytoplasmic domain, ectodomain, of the entire BACE gene, and then human immunoglobulin at its 3 'end. Fc partial nucleotide sequences corresponding to 230 amino acids of G (hIgG) (amino acid sequences 1-466) were attached. BACE2-Fc protein expression vector pCDNA3 BACE2 Fc was prepared by conjugating the BACE (ectodomain) -IgG Fc (hereinafter referred to as BACE-Fc) between BamHI and XhoI of a mammalian expression vector pCDNA3 (Invitrogen).

Step B: Preparation of a BACE2-Fc Fusion Protein Expressing Mammalian Cell Line

A-MEM (a-minimum essential medium) (GIBCO) containing 10% fetal bovine serum (FBS) (GIBCO-BRL) containing Chinese hamster ovary (CHO) DHFR-cells (ATCC Accession # CRL9096) -BRL) culture was then transferred to a 100 mm culture plate, cells were grown to cover the bottom of the culture vessel, and then transformed into the BACE2-Fc protein expression vector pCDNA3 BACE2-Fc using Lipofectamine Plus (Life Technologies). Transformed cells were replaced every 4 days with 10% dialyzed fetal bovine serum (dFBS) (JRH) culture containing 1 mg / ml Geneticin (G418 sulfate) (GIBCO-BRL). Selective culture was carried out, and about 100 clones were isolated and cultured in 24-well culture plates. Twenty clones with good cell growth rate among the isolated clones were incubated for 3 days in a 24-well culture plate with the same cell number (2 × 10 ⁵ cells / ml / 24-well). The amount of BACE2-Fc protein secreted into the culture was quantified by ELISA method using goat anti-human IgG (Pierce), and the fastest growth rate and the amount of BACE2-Fc expression (1 liter culture reagent) Clone # 66, representing 3 mg).

Step C: BACE2-Fc Fusion Protein Production and Purification

The CHO DHFR-BACE2-Fc # 66 cell line was placed in a roller bottle containing 250 ml of a-MEM medium containing 10% dFBS at 2 X 10 ⁵ cells / ml, and at 40 rpm in a Roll-In cell incubator (Bellco). Incubated at 37 ° C. for 4 days while rotating at speed. Once the cells have grown to completely cover the culture vessel, wash them once with 250 ml serum-free medium (SFII; GIBCO-BRL) and then again with serum-free serum containing 500 ml of insulin (0.5 ug / ml) (SIGMA). The medium was added and incubated for 3 days. After the culture was collected, the process was repeated twice with 500 ml of serum-free medium and incubated for 3 days. Then, the collected cell culture medium was centrifuged at 7000 rpm for 20 minutes (Beckman, JA 10). rotor) and then only the washing liquid was separated. After passing through the 0.45 um filter, the solution was passed through a Protein A sepharose chromatography column (Pharmacia) equilibrated with 20 mM sodium phosphata buffer (pH 7.0) and washed again with 20 mM sodium phosphata buffer (pH 7.0). All proteins that were not removed. By adding 100 mM sodium acetate buffer (pH 3.5) to drop the adsorbed protein, BACE2-Fc protein (MW 75 KDa.) With a purity of 95% or more was obtained.

Step D: Beta Secrita2 Activity Assay Using Fluorescent Label Specific Substrate

In order to measure the enzyme activity of the beta secretase 2 and the inhibitory effect of the synthetic compounds, Fluorescence Resonance Energy Transfer (FRET) enzyme activity assay using the purified BACE2-Fc fusion protein and the fluorescently labeled beta secrita2 specific substrate was established. . This is briefly described as follows.

Peptide-specific substrates corresponding to 10 amino acid sites including the beta secretase ablation portion of the total amino acid sequence of Amyloid precursor protien (APP), which is known as the intracellular beta secretase 2 specific substrate, were combined with EDANS, a fluorophore. It was synthesized by attaching the quenching group DABCYL. When the fluorescent label substrate is reacted with BACE2-Fc, the quenching group is separated while the BACE2 action site is cleaved, and EDANS exhibits 510 nm wavelength fluorescence by 350 nm excitation light. The accuracy can be measured sensitively and simply.

Each synthetic compound was dissolved in DMSO at a concentration of 10 mM and stored at 20 ° C. In order to measure the activity, the 10 mM DMSO solution was first placed in the rightmost column of the 96 well plate, and then diluted twice with the same amount of DMSO to 9 steps. 10 ul of the diluted compound solution was added to a 96-well assay plate containing 600 uM fluorescent labeled BACE substrate dissolved in 15 ul of reaction buffer (50 mM sodiumacetate, pH 4.5, 0.05% CHAPS) and 10 ul of 50% DMSO. The addition resulted in a final DMSO concentration of 10% and an inhibitor treatment concentration ranging from 500 uM to 9 fold dilutions. After adding 65 ul of the purified BACE2-Fc fusion protein solution to a final concentration of 0.4 ug / ml, the reaction was performed at room temperature for 1 hour. The reaction product was measured using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device) as fluorescence at 350 nM excitation and 510 nm emission wavelength. By comparing this measurement with that in the control group without addition of the synthetic compound, the concentrations of the synthetic compound, namely IC ₅₀ and K i , which inhibited 50% of the beta secretase activity were determined.

Experimental Example 2: Determination of Recombinant Casceptin enzyme activity

In order to measure the enzyme activity of the casceptindi and the inhibitory effect of the synthetic compounds, Fluorescence Resonance Energy Transfer (FRET) enzyme assay using the casceptindi (Calbiochem, # 219401) and the fluorescently labeled casceptin specific substrate (Bachem # M-2455) Was established. This is briefly described as follows.

Known as a casceptin specific substrate, a peptide specific substrate corresponding to 10 amino acid sites is attached to Mca, a fluorophore, and Dnp, a quenching group. When the fluorescently labeled substrate reacts with CASE, the quenching group is cleaved as the CASE synthesizing site is cleaved and Mca emits 393 nm of fluorescence by excitation light of 328 nm. And can be measured easily.

Each synthetic compound was dissolved in DMSO at a concentration of 10 mM and stored at 20 ° C. In order to measure the activity, the 10 mM DMSO solution was first placed in the rightmost column of the 96 well plate, and then diluted twice with the same amount of DMSO to 9 steps. The final DMSO was added to a 96-well assay plate containing 7 uM fluorescently labeled Casceptin substrate dissolved in 50 ul of reaction buffer (50 mM sodiumacetate, pH 4.0) and 10 ul of 25% DMSO. Concentrations were 5% and inhibitor treatment concentrations were from 250 uM to 2-fold dilutions of 9 steps. 30 ul of the purified Casceptindi fusion protein solution was added to a final concentration of 0.75 ng / ml and the reaction was performed at 37 ° C. for 1 hour. The amount of reaction product was measured by fluorescence at 328 nM excitation and 393 nm emission wavelength using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). By comparing this measurement with that in the control group without addition of the synthetic compound, the concentrations of the synthetic compound, i.e., IC ₅₀ and Ki, which inhibited 50% of the Casceptin activity was determined.

Experimental Example 3: Measurement of SEAP (Secreted alkaline phosphatase secreted alkaline phosphatase) activity

Step A: Establish Permanent Cell Line Expressing SEAP-APPsw-KK

Genes expressing SEAP and swedish mutant amyloid precursors (CRE-SEAP-APP695sweKK) under the control of cAMP response elements (CRE) were cloned into mammalian expression vector pcDNA3.1 (+) Neo (Invitrogen). Neuro-2a cells (ATCC Accession # CCL-131) were incubated with Dulbecco's minimum essential medium (DMEM) culture medium containing 10% FBS (GIBCO-BRL), then transferred to a 6-well culture plate and the cells were After overgrowth was transformed with the CRE-SEAP-APP695sweKK expression vector using Lipofectamine 2000 (Life Technologies). Each clone was isolated and incubated again on a 6-well culture plate. 100 clones of the selected clones with good cell growth rate were incubated for 3 days in a 24-well culture plate, then incubated for 6 hours in DMSO / 10uM Forskolin media, and then 50 μl of each well was removed and 50 ul Reaction with Attophos (Promega). Fluorescence at 450 nM excitation and 580 nm emission wavelength was measured with a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device) at room temperature for 30 minutes. Among the selected clones, four clones with the highest SEAP activity in forskolin / DMSO with the highest SEAP activity were selected. Selected clones were incubated for 1 day with the same number of cells (2 X 10 ⁴ cells / 96-well), then treated with DMSO / Forskolin10uM to measure secreted SEAP activity after 6 hours to determine the best clone # 159. Screened.

Step B: SEAP Activity Assay

N2A SEAP-APPsw-KK # 159 expressing the CRE-SEAP-APP695sweKK was placed in a 96-well culture vessel with 2 × 10 ⁴ cells / 80 ul of cells per well. Forskolin 10mM and DMSO were prepared and diluted 100-fold with culture medium, and 10 ul was added to 96-well each.

Each synthetic compound was dissolved in DMSO at a concentration of 10 mM and stored at 20 ° C. In order to measure the activity, the 10 mM DMSO solution was first placed in the rightmost column of the 96 well plate, and then diluted three times with the same amount of DMSO was performed sequentially to step 7. 10 ul of serially diluted compounds are diluted 10-fold with 90 ul of culture. The final DMSO concentration was 1.1% and the inhibitor treatment concentration was from 2 u dilutions of 100 uM. After all treatments were incubated for 5 hours in a 37 ℃ incubator in the presence of 6% CO ₂ . In order to measure the amount of SEAP secreted into the culture medium, the reaction was first performed at 65 ° C. for 30 minutes by heat to remove other alkaline phosphatase activity. 50 ul of the thermally inactivated culture and 50 ul of Attophos (Promega) were reacted at room temperature for 30 minutes, and fluorescence was measured at 450 nM excitation and 580 nm emission wavelength with a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). By comparing this measured value with the measured value in the control group to which the synthetic compound was not added, the concentration of the synthetic compound that inhibits 50% of the SEAP activity, namely IC ₅₀ , was determined.

Experimental Example  4: Genotyping In mice  Amyloid Beta Measurement in Neurons

Stage A: Transgenic Mice (APP / PS1dE9) Primary Neuronal Cell Culture

Experiments were performed with mice born from cross- genotyped (APP / PS1dE9) male and female mice. Take out the brains of rats 3 ~ 4 days old, remove the hippocampus tissue and membranes at 4 ℃, and then chop them and treat them with DNA enzymes (Sigma, D5025) and protease (Sigma, P5147). Leave for 25 minutes. After separation into cells, use a culture medium (27.6 ml Neurobasal + 1.5 ml FBS + 600 ul B27 + 300 ul of 200 mM of L-Glutamine) in a 24-well plate covered with Poly-L-Lysine. ⁵ cells. After incubation for 7 days at 37 ℃ incubator.

Step B: Determination of Ab40 Activity

Each synthetic compound was dissolved in DMSO at a concentration of 10 mM and stored at 20 ° C. In order to measure activity, the 10 mM DMSO solution was first placed in the rightmost column of a 96 well plate, and then diluted three times with the same amount of DMSO up to six steps in sequence. The diluted compounds were diluted 250-fold with culture solution (neurobasal 29.1 ml + B27 600 ul + 200 mM L-Glutamine 300 ul + 100 mM L-Glutamate 7.5 ul) and 350 ul was added to the cultured primary neurons. The cells were incubated in a 37 ° C. incubator for hours. The expression level of beta amyloid peptide secreted into the culture solution was measured by sandwich ELISA method (Biosource, # KHB3482) using two antibodies specific for beta amyloid peptide.

Aqueous beta-amyloid or cell culture diluted to 50 ul of each concentration was added to the plate coated with the antibody while mixing with 50 ul of the identified antibody and reacted at room temperature for 3 hours (or at least 1 day at 4 ° C). After washing 5 times with 1-fold wash solution (Biosource, # KHB3482), 100 ul of HRP (horse radish peroxidase) -attached antibody diluted 100-fold with antibody dilution solution (Biosource, # KHB3482) was added, and the mixture was kept at room temperature for 30 minutes. Reacted. Then, the HRP antibody was removed, washed five times with 1-fold wash solution, and then 100 ul of chromogen solution (Biosource, # KHB3482) was added and reacted at room temperature for 30 minutes. Then, 100 ul of a stop solution (Biosource, # KHB3482) was added and reacted at room temperature for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader (SpectraMax 340, Molecular Device). This measurement was compared with that in the control treated with 0.4% DMSO without the addition of the synthetic compound to determine the concentration of the synthetic compound, i.e., IC ₅₀ , which inhibited 50% of intracellular beta secretase activity.

Experimental Example 5: Measurement of Intracellular Beta Secretase Activity

Inhibition of intracellular beta secretase activity of synthetic compounds can be measured using a cell line making beta amyloid peptides from amyloid precursors (APP).

Step A: Establish Permanent Cell Line Producing Amyloid Precursor

Mutant amyloid precursor gene (APP751NFEV) under the control of TRE (Tet-response element) was cloned into PBI-L vector (CloneTech), a mammalian expression vector expressing the luciferase gene under the control of TRE (Tet-response element). Neuro-2a cells (ATCC Accession # CCL-131) were incubated with Dulbecco's minimum essential medium (DMEM) culture medium containing 10% FBS (GIBCO-BRL), then transferred to a 6-well culture plate and the cells were Overgrowth was then transformed with the pBI-L APP751 NFEV expression vector using Lipofectamine 2000 (Life Technologies). Each clone was isolated and incubated again on a 6-well culture plate. One hundred clones with good cell growth rate among the selected clones were incubated for one day in a 96-well culture plate, exchanged with a culture solution containing 1 ug / ml Doxicycline, and then incubated for 24 hours. After treatment with 50 μl of Bright-Glo Luciferase Reagent (Promega), the solution was left at room temperature for 15 minutes, and then the luminescence of each well was measured using a luminometer (Luminometer, Victor). Four clones with the highest luciferase expression among the selected clones were incubated for 1 day in a 24-well culture plate at the same cell count (3 X 10 ⁵ cells / ml / 24-well), then 1 ug / After exchange with OPTI-MEM (GIBCO-BRL) culture containing ml Doxicycline and incubated for 24 hours, the amount of beta amyloid peptide secreted into the culture was quantified by ELISA method using an antibody selective to beta amyloid peptide. As a result, clone # 79 was selected that exhibited the fastest growth rate and the highest amount of beta amyloid peptide expression.

Step B: Water Soluble Beta-amyloid Precursor (sAPP) ELISA Assay

Neuro-2a APP751 NFEV # 79 cells, a cell line expressing the mutant amyloid precursors, were placed in a 24-well incubator at 3 × 10 ⁵ cells per well in a 37 ° C. incubator for 24 hours in the presence of 6% CO ₂ . Incubated for Once the cells were grown to completely cover the culture vessel, exchange them with Opti-MEM (GIBCO-BRL) containing 300 ul of 1 ug / ml Doxicycline and a beta secretase inhibitor (Example compound) diluted at each concentration. Incubate again for 24 hours. Aqueous beta-amyloid precursor expression amount secreted into the culture was measured according to the manufacturer's experimental plan using the Human sAPP assay kit (IBL), briefly described as follows.

Aqueous beta-amyloid precursors or cell cultures diluted to 100 ul of each concentration were added to the plate coated with the antibody and reacted at room temperature for 4 hours (or at least 1 day at 4 ° C). After washing 7 times with washing solution (PBS with 0.05% Tween20), 100 ul of HRP (horse radish peroxidase) antibody diluted 30 times with antibody dilution solution (1% BSA, PBS with 0.05% Tween20) was added. After the addition, the mixture was reacted at room temperature for 30 minutes. After washing 9 times with the washing solution, 100 ul of TMB solution was added and reacted at room temperature for 30 minutes. After 100 ul of 1N sulfuric acid (H ₂ SO ₄ ) solution was added and reacted at room temperature for 30 minutes, the absorbance at 450 nm was measured using a microplate reader (SpectraMax 340, Molecular Device). This measurement was compared with the measurement in the control treated with only 1% DMSO without addition of the synthetic compound to determine the concentration of the synthetic compound, i.e., IC ₅₀ , which inhibits 50% of intracellular beta secretase activity.

Experimental Example 6: Beta Amyloid Protein Assay

Beta amyloid protein (beta-amyloid) was quantitated by enzyme-linked immune antibody assay (ELISA) using two antibodies (Human beta amyloid 1-40 colorimetric immunoassay kit, Biosource, California, USA). The two antibodies used in the enzyme-linked immune antibody assay consist of an antibody that specifically recognizes the N-terminus of the beta amyloid protein truncated by beta secretase and an antibody that binds to the C-terminus. The two antibodies and the beta amyloid protein were reacted at room temperature for 3 hours (or at least 1 day at 4 ° C), then washed 4 times with a washing solution and then treated with a horseradish peroixdase (HRP) attached anti-rabbit IgG's peroxidase antibody. The reaction was carried out for 30 minutes. After washing four times with the wash solution, tetramethylbenzidine, a substrate of HRP, was added and reacted at room temperature for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader (SpectraMax 340, Molecular Device). The degree of reduction of beta amyloid protein was determined by comparing this measurement with that in the control.

The secreted amyloid precursor protein beta (sAPPbeta) protein is also carried out in the same manner as the enzyme-linked immuno antibody assay used to quantify beta amyloid protein.

Experimental Example 7: Animal Testing in vivo  assay)

In order to confirm that the activity of beta secretase was inhibited, the degree of inhibition of production of beta amyloid protein, a product of beta secretase, was tested in animals. The animals used in the test were transgenic mice (Jankosky JL et al) containing a Swedish mutant (chimeric mouse / human amyloid precursor protein 695swe) of the beta amyloid protein precursor and a mutant gene of presenilin 1 (presenilin 1-dE9). , Biomolecular engineering, 17 (6), 157-165, 2001). Beta-secretase inhibitors are administered at concentrations that are expected to reduce amyloid protein but not toxicity through intraperitoneal or subcutaneous administration. Animals were anesthetized at a defined time after drug administration and blood and brain tissue separated. Blood was collected in a heparin-coated tube via cardiac blood collection, centrifuged at 13,000 rpm for 10 minutes (Eppendorf), and then only plasma was separated and stored at 80 ° C until analyzed with the extracted brain tissue (cerebral cortex and hippocampus). . For analysis, the plasma was diluted five-fold and the inhibitory effect of amyloid protein in plasma was confirmed by the enzyme-linked immune antibody assay mentioned above. After the brain tissues were extracted, the brain tissues were pulverized in a 4 times volume of PBS, and finally, after reacting at room temperature for 4 hours at 5M Guanidine concentration using 8.2 M guanidine / 82 mM Tris HCl (pH 8.0), beta Amyloid protein was extracted and analyzed by diluting 1: 500 beta amyloid protein using BSAT-DPBS (Dulbecco's phosphate buffered saline with 5% BSA and 0.03% Tween-20).

Experimental Example 8: Dosing and formulation

The drug was dissolved in 10% HPCD (hydroxypropyl-beta-Cyclodextrin) and generally administered at a concentration of 15 mg / kg to 100 mg / kg once or three to five times daily.

The compounds of the present invention have a range of Ki from about 0.1 uM to 500 uM, preferably from about 0.1 uM to 100 uM, more preferably from about 0.1 uM to 10 uM, and Ki of Example 50 is 3.5 uM.

As described above, the compounds of formula (I) according to the present invention exert an excellent inhibitory effect on human beta secretase. Therefore, the compound according to the present invention can be used as a medicament for improving cognitive function or preventing and treating neurodegenerative diseases such as Alzheimer's disease.

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

Claims (15)

The present invention provides compounds of Formula I, pharmaceutically acceptable salts or isomers thereof:

(I) In the above formula m and n are each independently 0 or 1; R ¹ and R ² are each independently hydrogen, halogen, cyano, hydroxy,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, -N [-(CH ₂ ) _p -R ] [-(CH ₂ ) _p -R], -S- (CH ₂ ) _p -R, -C (O) N (R ') (R "), -N (R')-C (O) R ", -S (O)-(CH ₂ ) _p -R, -S (O) _2- (CH ₂ ) _p -R And -S (O) N (H)-(CH ₂ ) _p -R, or R ¹ and R ² together may form a ring; R ³ is selected from the group consisting of hydrogen, halogen and — (CH ₂ ) _p —R, or R ⁴ And / or may form a ring together with R ⁵ ; R ⁴ and R ⁵ are each independently-(CH ₂ ) _q -R, -Y- (CH ₂ ) _q -R, -N (-(CH ₂ ) _q -R) (-(CH ₂ ) _q -R ), -C (O) NH- (CH ₂ ) _q -R, -C (O)-(CH ₂ ) _q -R, -C (O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R]; here, p and q are each independently selected from 0 to 4, R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heterocycle and heteroaryl, R 'and R "are each independently hydrogen, alkyl,-(CH ₂ ) _p -cycloalkyl,-(CH ₂ ) _p -aryl,-(CH ₂ ) _p -heterocycle and-(CH ₂ ) _p -hetero Aryl selected from the group consisting of aryl and R " May form a ring together, Y is Cycloalkyl, aryl, aryl-heteroaryl, aryloxy, heterocycle and heteroaryl; In the above, Alkyl, alkoxy, aryl, cycloalkyl, heterocycle and heteroallyl may be of substituted or unsubstituted structure, where the substituents are halogen, cyano, amino, alkylamino, dialkylamino, alkylacylamino, hydroxy At least one selected from the group consisting of alkyl, alkoxy, aryloxy and oxo, Heteroaryl and heterocycle are each independently a 4-8 membered ring containing 1 to 3 heteroatoms selected from the group consisting of O, N and S, and containing 0 to 3 double bonds. The method of claim 1, R ¹ and R ² are each independently hydrogen, halogen, cyano,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, -N (H)-(CH ₂ ) _p -R, -N [-(CH ₂ ) _p -R] [-(CH ₂ ) _p -R], -S- (CH ₂ ) _p -R, -C (O) N (R ') (R ") and N Or (R ')-C (O) R ", or together form a ring; R ³ may be — (CH ₂ ) _p —R or may form a ring with R ⁴ ; R ⁴ is-(CH ₂ ) _q -R, -N (-(CH ₂ ) _q -R) (-(CH ₂ ) _q -R), -C (O) NH-R, -C (O)- (CH ₂ ) _q -R, -C (O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R] It is selected from the group consisting of; R ⁵ is hydrogen or —W— (CH ₂ ) _r —R; Where p and q are each independently selected from 0 to 4, r is selected from 0 to 2, and Y is Is selected from the group consisting of aryl, heterocycle and heteroaryl, W is directly bonded or is selected from the group consisting of piperidine, pyrrolidine, aminopyrrolidine, azetidine and amino, Compounds represented by the formula, pharmaceutically acceptable salts or isomers thereof. The method of claim 1, R ¹ is hydrogen, halogen, cyano, hydroxy,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, and -N [-(CH ₂ ) _p -R] [-(CH ₂ ) _p -R], Wherein p is selected from 0 to 4 and R is selected from the group consisting of hydrogen, alkyl, heterocycle, aryl, cycloalkyl and heterocycle, a compound represented by formula (1), pharmaceutically acceptable Salts or isomers of. The method of claim 3, wherein R ¹ is hydrogen, halogen, cyano, hydroxy,-(CH ₂ ) _p -R, -O- (CH ₂ ) _p -R, and -N (-(CH ₂ ) _p -R) (-(CH ₂ ) _p -R), Wherein p is 0 or 1 and R is selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl, pyrrolidine, piperidine, phenyl, benzyl, cyclopentyl, and cyclopropyl I, pharmaceutically acceptable salts or isomers of I The compound of claim 4, wherein R ¹ is hydrogen, halogen, cyano, anilino, (3-cyano) anilino, (4-fluoro) anilino, pyrrolidinemethyl, piperidinemethyl, (3- Trifluoro) anilino, (3-trifluoromethyl) anilino, isobutylamino, isopentylamino, cyclopentyl amino, benzylamino, phenoxy and benzyloxy Compound of I, pharmaceutically acceptable salts or isomers thereof. The compound of formula (I) according to claim 1, wherein R ² is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, cycloalkyl-alkoxy, heteroaryl-alkoxy and alkyl-aryl. Its salts or isomers. 7. The compound of claim 6 wherein R ² is selected from the group consisting of hydrogen, chloro, methyl, isopentyloxy, cyclopentyloxy, (thiophen-2-yl) propyloxy and (thiophen-2-yl) ethyloxy A compound of formula (I), pharmaceutically acceptable salts or isomers thereof, characterized in that: The compound of claim 1, wherein R ³ is selected from the group consisting of hydrogen, phenyl, cyclohexyl, benzyl, and piperidine, or forms a ring structure with R ^4. Salts or isomers thereof, which are scientifically acceptable. The compound of claim 1, wherein R ⁴ and R ⁵ are each independently — (CH ₂ ) _q —R, —C (O) NH—R, —C (O) — (CH ₂ ) _q —R, —C ( O) -Y- (CH ₂ ) _q -R, and -C (O) -YN [(CH ₂ ) _q -R] [(CH ₂ ) _q -R], A compound represented by formula (1), a pharmaceutically acceptable salt or isomer thereof. 10. The compound of claim 9, wherein R ⁴ is each independently (3-dimethyl) aminoethyl, benzyl, phenethyl, acetyl, aminobenzyl, (3-dimethyl) aminobenzyl, -C (O) cyclohexyl, -C (O ) Benzyl, -C (O) phenethyl, -C (O) (diphenylmethyl), -C (O) [(3-dimethyl) amino] benzyl, -C (O) NH (3,5-dichlorophenyl ), -C (O) NH (phenyl), -C (O) NH (cyclohexyl), -C (O) (3-benzyloxy-pyrrolidin-2-yl), -C (O) {[ 3- (4-phenylimidazole) methyl] phenyl}, -C (O) {3- (piperidine) methylphenyl}, -C (O) {3- (piperidine) ethylphenyl}, -C (O) {3- (Diisobutylamino) methylphenyl}, -C (O) {3-pyrrolidinephenyl}, -C (O) {3- (piperidine) methylthiophene}, -C ( O) [3- (pyrrolidinmethyl) thiophen-3-yl], -C (O) [5- (piperidinmethyl) thiophen-2-yl], -C (O) [4- ( Benzyloxy) pyrrolidin-2-yl], -C (O) {[7- (tetrahydrofuran-4-yl) amino] indol-2-yl}, -C (O) {[7- (phenoxy Cy-3-yl) amino] indol-2-yl} and -C (O) [3-N (cyclopropylmethyl, cyclopropyl-piperidine, methyl) phenyl] A compound represented by formula (1), a pharmaceutically acceptable salt or isomer thereof, which is selected. The compound of claim 1, wherein R ⁵ is hydrogen, (3-dimethyl) aminoethyl, benzyl, phenoxy, phenethyl, pyrrolidine, piperidine, cyclopropyl-piperidine, cyclopropyl-methyl-piperidine , Cyclopentyl-piperidine, C (O) -methyl-dimethylamino, a compound represented by the formula (1), a pharmaceutically acceptable salt or isomer thereof. The compound of formula (I), a pharmaceutically acceptable salt or isomer thereof, according to claim 1, wherein the compound of formula (I) is any one selected from the group consisting of: N ^2- (piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenethyl) glycinamide N ^2- (piperidin-4-yl) -N- (4-phenoxy-2-cyclopentyloxyphenyl) -N ^2- (phenethyl) glycinamide N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (4-phenoxy-2-isopentyloxyphenyl) -N ^2- (phenethyl) glycinamide N ^2- (piperidin-4-yl) -N- (4-phenoxy-2- (3-thiophen-3-yl) -phenyl) -N ^2- (phenethyl) glycinamide N ^2- (piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide N ^2- (3-dimethylamino-benzyl) -N- (4-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide N ^2- (piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzyl) glycineamide N ^2- (1-cyclopentyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (3,5-dichlorophenylaminocarbonyl) glycinamide N ^2- (1-cyclopentyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide N ^2- (1-cyclopropyl-piperidin-4-yl) -N- (4-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide N ^2- (piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (phenethyl) glycineamide N ^2- (piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (benzylcarbonyl) glycinamide N ^2- (1-cyclopropylmethyl-piperidin-4-yl) -N- (3-phenoxy-phenyl) -N ^2- (phenethylcarbonyl) glycinamide N- {2-oxo-2-[(4-phenoxyphenyl) amino] ethyl} -3- (pyrrolidin-1-ylmethyl) phenylamide N- {2-oxo-2-[(3-phenoxyphenyl) amino] ethyl} -3- (piperidin-1-ylmethyl) phenylamide (4R) N [(4-phenoxy) phenyl]]-4-benzyloxy-1- (2-dimethylaminoethyl) -pyrrolidine-2-carboxamide A pharmaceutical composition for inhibiting beta secretase, comprising as an active ingredient a pharmaceutically effective amount of a compound of formula (I) according to claim 1, a pharmaceutically acceptable salt or isomer thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is used to improve or prevent cognitive function or to treat or prevent neurodegenerative diseases. The pharmaceutical composition of claim 14, wherein the composition is used to treat Alzheimer's disease.