WO2009038411A2

WO2009038411A2 - Beta-secretase inhibiting compounds having oxo-dihydro-pyrazole moiety

Info

Publication number: WO2009038411A2
Application number: PCT/KR2008/005592
Authority: WO
Inventors: Hyo-Shin Kwak; Hyejin Jung; Kyeongsik Min; Heuisul Park; Deog-Young Choi; Dongchul Lim; Jae-Ung Choi; Mijeong Park; Hyunjung Park
Original assignee: Lg Life Sciences, Ltd.
Priority date: 2007-09-21
Filing date: 2008-09-22
Publication date: 2009-03-26
Also published as: KR20090031331A; WO2009038411A3

Abstract

Disclosed are compounds represented by Formula (I) as defined in the specification, or pharmaceutically acceptable salts or isomers thereof, and a pharmaceutical composition for inhibiting beta-secretase activity comprising a therapeutically effective amount of the same.

Description

BETA-SECRETASE INHIBITING COMPOUNDS HAVING OXO-

DIHYDRO-PYRAZOLE MOIETY

FIELD OF THE INVENTION

The present invention relates to a novel compound for inhibiting beta-secretase activity or a pharmaceutically acceptable salt or isomer thereof, preparation thereof, and a pharmaceutical composition comprising a therapeutically effective amount of the same.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease which gradually progresses with age and accounts for 50 to 70% of dementia patients. The major symptoms of AD include memory loss, decline in cognitive-reasoning abilities and the like. Most Alzheimer's cases occur around age 65 and progress for about 9 years to result in the death of Ihe patients. The number of AD patients is increasing as society develops and ages, so that it is estimated that there will be approximately 6 million patients within 10 years in the USA and this number will further increase beyond that.

Likewise, the proportion of the elderly population is also increasing in Korea, and thus social problems associated with increased prevalence of senile dementia are becoming more serious. Unfortunately, no therapeutic agent has yet been developed which can treat the underlying pathogenic causes of Alzheimer's disease. Until now, acetylcholine esterase inhibitors have been exclusively used as a general therapeutic agent. Aricept™ (Pfizer), Exelon™ (Novartis), and Reminyl™ (Janssen) are known as representative examples of the acetylcholine esterase inhibitors. Strictly speaking, these drugs cannot be defined as therapeutic agents of Alzheimer's disease, because they do not address the root cause of the disease and result in only partial recovery from the disease in some patients (about 40-50%), and their therapeutic effects are seen for a limited time period only. In addition, the intrinsic nature of the disease requires long-term administration of the drugs, but administration of these therapeutic drugs is accompanied by various adverse side effects including liver toxicity.

Therefore, there is an urgent need for development of a novel therapeutic agent that can halt and reverse the progression of Alzheimer's and related diseases. As an approach to find a pathogenic cause responsible for Alzheimer's disease, long-term studies have been undertaken on familial early-onset Alzheimer's disease patients. As a result, it was revealed that mutations in some genes are primarily responsible for the pathogenesis of Alzheimer's disease.

As the common physiological results of the above-mentioned genetic pathogenic factors, the production of beta amyloid consisting of 42 amino acid residues (hereinafter, sometimes referred to as "Ab42") was found to increase. Based on this finding, it is assumed that the production of Ab42 is a main causative agent of Alzheimer's disease pathogenesis. Therefore, if a method capable of decreasing the production of Ab42 is developed, it will be a direct and effective therapeutic measure capable of blocking a pathogenic mechanism of Alzheimer's disease.

Beta-amyloid proteins are produced from a high-molecular weight amyloid precursor protein (APP) found in neuronal cells through serial cleavage events by 3 types of proteases (secretases). This process takes place at the Golgj apparatus of neuronal cells, wherein APP and secretases are anchored in the Golgi membrane. The N-terminus of beta-amyloid (Ab) corresponds to the 99th amino acid from the C-terminus of APP, and this site is cleaved by beta-secretase (hereinafter, sometimes referred to as "beta-site APP cleaving enzyme" or "BACE"). The C- terminus of the membrane-bound beta-amyloid (Ab) is cleaved by gamma-secretase to generate a beta-amyloid (Ab) protein which is then secreted extracellυlaiy from neuronal cells. Alternatively, APP may also be cleaved at different sites via an alternative pathway; for example, where the middle site of Ab (between 16^th and 17^th amino acid residues from the N-terminus) is cleaved by alpha- secretase, sAPP alpha having a high molecular weight is produced and secreted. This alternate pathway precludes the formation of beta-amyloid.

Cleavage of APP by the action of BACE and gamma-secretase may result in a variety of beta amyloid (Ab) proteins having different lengths, usually a 40-amino acid fragment (AMO) and a 42-amino acid fragment (Ab42). In comparison with Ab40, Ab42 tends to easily aggregate and accelerates the formation of amyloid plaques in the brains of diseased patients, thereby resulting in gradual necrosis of the surrounding neuronal cells. This is assumed to be a major pathogenic mechanism of Alzheimer's disease.

Ab40 and Ab42 are produced in a ratio of about 9: 1 under normal conditions. However, it is known that where levels of two amyloid proteins AMO and AM2 are increased or where levels of AM2 are selectively increased by mutations of Presenilin 1 and 2 genes, the onset of Alzheimer's disease is further accelerated and the symptoms of the disease are more severe. Therefore, it can be said that lowering of AM2 production is the most important factor for development of anti- Alzheimer drugs. For this purpose, there is a need for development of beta- or gamma-secretase inhibitors.

Therefore, many multinational pharmaceutical companies have been investing heavily in research and development in the secretase inhibitor field, especially in the area of beta- or gamma- secretase inhibitors capable of suppressing the production of 42-amino acid beta-amyloid fragments, which form plaques assumed to be an underlying pathological cause of Alzheimer's disease. Unfortunately, there are no noteworthy results of studies in terms of the development of a therapeutic agent that is capable of arresting a mechanism of beta-amyloid production to directly prevent the progression of the concerned disease.

The beta- and gamma-secretases are known as aspartic proteases and found in the membrane-bound form. However, no gene coding for gamma-secretase has yet been identified. Further, it is known that substrates for gamma-secretase are not limited to APP, but that the enzyme also participates in the cleavage of Notch proteins, which are known to play crucial roles in regulating cell fate decision during differentiation processes. In particular, gene-knockout animals from which a gamma-secretase gene has been deleted died in utero, and recent clinical tests on gamma-secretase inhibitors showed significant drug toxicity. For these reasons, gamma-secretase inhibitors are not likely to be promising drug candidates. As a result, it has not yet been confirmed whether gamma-secretase inhibitors can be developed as safe anti-Alzheimer medications.

On the other hand, in case of BACE, the gene thereof was identified through various methods by many pharmaceutical companies and the in vivo activity of beta-secretase was reported in 1999. Furthermore, the X-ray crystal structure of beta-secretase was determined and a peptide inhibitor having high affinity for beta-secretase was also known in the art. As well, it was reported that a gene-knockout animal with deletion of the BACE gene exhibits a normal phenorype, thus suggesting that a BACE-specific inhibitor can be developed as a safer and efficient anti-dementia drug. Taken altogether, these findings indicate that BACE is a more feasible target for the treatment of Alzheimer's disease, as compared to gamma-secretase. As discussed above, upon considering that conventional commercially available drugs merely exhibit palliative effects of disease symptoms, thus providing substantially no effects on the progression of the concerned disease, it is certain that development of the BACE-specific inhibitors will lead to the development of a novel and remarkable drug against Alzheimer's disease. In recent years, many pharmaceutical companies have published study results on BACE inhibitors, for example by Merck (WO 2006/078577, WO 2006/060109 and WO 2006/057983), Elan (WO 2004/022523 and WO 2005/095326), Schering-Plough (WO 2006/014762 and WO 2006/014944), BMS (WO 2005/182105 and WO 2005/030758), and Eli-Lilly (WO 2005/108358 and WO 2006/034093). In Hie beginning, Elan has reported BACE inhibitor compounds, which were compounds having a small molecular weight and low blood-brain barrier (BBB) permeability. However, these compounds exhibited various limitations due to selectivity for cathepsin D (Cat. D) or in vivo cytotoxicity. A great deal of attention has been recently focused on Merck compounds. Through early introduction of dicarbonyl compounds and subsequently sulfonamide compounds from fellow pharma giant Sunesis, Merck reported excellent inhibitory effects and selectivity of the drug compounds. Since then, numerous drug companies issued various compounds in the form of Merck compounds.

Due to excellent BACE inhibitory effects and high Cat. D selectivity, these compounds, particularly sulfonamide compounds were expected to provide the possibility that they may be developed as an anti-Alzheimer drug. However, their low oral applicability and poor BBB permeability are obstacles to development of the drug. For these reasons, there are very little reports on compounds that lower the production of Ab42 in practical animal models. Apart from sulfonamide compounds, Lilly reported efficacy of drug compounds (lowering of Ab42 levels) in a practical animal model, through S.C administration of peptidomimetic compounds having a low molecular weight. However, these peptidomimetic compounds still suffer from disadvantages such as low oral applicability and poor BBB permeability. Therefore, there is no report yet on compounds exerting excellent effects. Schering-Plough also reported peptidic compounds, but these compounds failed to show desired efficacy in the brain of a practical animal model as they were known to be a Pgp substrate. That is, peptidic beta-secretase inhibitors exhibit limited applicability as oral preparations, due to their intrinsic molecular specificities. In particular, sulfonamide type compounds also have poor BBB permeability which makes it difficult to observe desired efficacy of the drug in an animal model. To this end, there is a strong need for development of a compound having excellent beta- secretase inhibitory activity, distinctively different from sulfonamide compounds.

SUMMARY OF THE INVENTION

Therefore, the present invention has been made to solve the above problems and other technical problems that have yet to be resolved.

More specifically, the present invention is intended to improve blood-brain barrier (BBB) permeability of a drug compound which is a technical problem suffered by conventional sulfonamide compounds.

Therefore, an object of the present invention is to provide a novel compound having beta- secretase inhibitory activity through five-membered heterocyclic compounds apart from sulfonamide in terms of a chemical structure, and pharmaceutically acceptable salts and isomers thereof.

It is another object of the present invention to provide a method for preparing the said compound. It is a further object of the present invention to provide a composition for inhibiting beta- secretase activity, comprising a therapeutically effective amount of the said compound as an active ingredient. It is yet another object of the present invention to provide a use of the said novel compound for improvement of cognitive functions or treatment or prevention of neurodegenerative diseases such as Alzheimer's disease.

In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a compound represented by Formula (T):

ris l to 3; R¹ is selected from the group consisting of hydrogen, alkyl, alkene, and -(CH₂)P-A-R⁷ wherein p is 0 to 2, A is aryl or heteroaryl, and R⁷ is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, -NHC(O)-alkyl and aryloxy;

R² is selected from the group consisting of hydrogen, alkyl, alkoxy and -(CR⁸R^)P-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently selected from the group consisting of hydrogen, alkyl and alkoxy, and R¹⁰ is selected from the group consisting of cycloalkyl, aryl, heterocycle and heteroaryl; and

B is any one of substituents represented by Formulae (i) to (iii):

(ϋi) wherein m and n are each independently 0 or 1 , X is C or N,

R³ and R⁴ are each independently selected from the group consisting of hydrogen, alkyl and -(O)Q-(CH₂)P-R¹¹ wherein p is 0 to 2, q is 0 or 1 , and R¹¹ is aryl or heteroaryl,

R⁵ is selected from the group consisting of hydrogen, alkyl, alkoxy and -(0^-(CH₂)P-R¹ ' wherein p is 0 to 2, q is 0 or 1 , and R¹ ' is aryl or heteroaryl, and R⁶ is selected from the group consisting of hydrogen, alkyl, alkoxy, and -(CH₂)p-A'-R¹² wherein p is 0 to 2, A' is phenyl, heteroaryl, or a bicyclic compound containing phenyl, R is selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, NHR¹³ and NR¹³R¹⁴ wherein R^I3and R¹⁴ are each independently hydrogen or alkyl, or R¹³and R¹⁴ are cyclized to form alkylene; or a pharmaceutically acceptable salt or isomer thereof. The alkyl, alkoxy, aryl, cycloalkyl, heterocycle, and heteroaryl may be substituted or unsubstituted. When substituted, the substituent may be at least one selected from the group consisting of halogen, amino, alkylamino, dialkylamino, alkylacylamino, Ci-C₄ alkyl, hydroxy, Q- C₄ alkyl alkoxy, aryl alkoxy and oxo. Where appropriate, these substituents may also be substituted. When substituted, the substituents are as exemplified above and the substituents may be taken together to form a cyclic structure.

Further, the heteroaryl and heterocycle are each independently a 4 to 8-membered ring containing 1 to 3 hetero atoms selected from the group consisting of O, N and S, and having 0 to 3 double bonds, and preferably a 5 or 6-membered ring having 1 to 2 double bonds.

Compounds of Formula (I) in accordance with the present invention have a chemical structure that is distinctly different from that of conventional known sulfonamide- or peptide-based beta-secretase inhibitor compounds. As will be illustrated in Experimental Examples which will follow, the compounds of the present invention exhibit improved BBB permeability to thereby exert excellent inhibitory effects on human beta-secretase which is correlated with improvement of cognitive functions or prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

As used herein, the term "isomer" means a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is optically or sterically different therefrom.

Unless otherwise indicated, the term "compound of Formula (T)" is intended to encompass compounds of Formula (1) per se as well as pharmaceutically acceptable salts and isomers thereof.

The term "alkyl" means an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, which means that it does not contain any alkene or alkyne moiety. The alkyl moiety may also be an "unsaturated alkyl" group, which means that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic. The alkyl group may have 1 to 20 carbon atoms. The alkyl group may also be a medium- sized alkyl having 1 to 10 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. By way of example only, "Ci-C₄ alkyl" indicates that there are one to four carbon atoms in the alkyl chain, Ie., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. When the alkyl group is used alone or in combination with alkyloxy, it may be a linear or branched hydrocarbon radical.

Typical examples of the alkyl group may include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cycloburyl, cyclopentyl, and cyclohexyl. The term "alkoxy" refers to oxo alkyl having 1 to 10 carbon atoms.

The term "cycloalkyl" refers to an unsaturated aliphatic 4 to 10-membered ring, including cyclohexyl.

The term "aryl" refers to an aromatic group which has at least one ring having a conjugated pi (π) electron system and includes both carbocyclic aryl (for example, phenyl) and heterocyclic aryl (for example, pyridine) groups. This term is intended to include monocyclic or fused-ring polycyclic (Ie., rings which share adjacent pairs of carbon atoms) groups. Specifically, aryl means a 4 to 10-membered, preferably 6 to 10-membered aromatic monocyclic or multicyclic group, including phenyl, naphthyl, and the like.

The term "heteroaryl" refers to a 4 to 8-membered ring having 1 to 3 hetero atoms selected from the group consisting of N, O and S and 0 to 3 double bonds. The heteroaryl may be preferably an aromatic 4 to 8-membered ring and more preferably a 5 or 6-membered ring, which may be fused with benzo or C₃-C₈ cycloalkyl. Examples of monocyclic heteroaryl may include, but are not limited to, thiazole, oxazole, thiophene, furan, pyrrole, imidazole, isόxazole, pyrazole, triazole, thiadiazole, tetrazole, oxadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and the like. Examples of tricyclic heteroaryl may include, but are not limited to, indole, indoline, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzisoxazole, benzothiazole, benzothiadiazole, benzotriazole, quinoline, isoquinoline, purine, furopyridine and the like.

The term "heterocycle" refers to a 3 to 10-membered ring which has 1 to 3 hetero atoms selected from the group consisting of N, O and S, may be fused with benzo or C₃-Cg cycloalkyl, and contains 1 or 2 double bonds or may be saturated. The heterocycle may preferably be a 4 to 8- membered ring, and more preferably a 5 or 6-membered ring, which may include, but are not limited to, piperidine, morpholine, thiomorpholine, pyrrolidine, imidazolidine, tetrahydrofuran, piperazine, and the like. Other terms used herein can be interpreted as having their usual meanings in the art to which the present invention pertains.

In one preferred embodiment of the present invention, in the compound of Formula (T), ris 1 to 3;

R¹ is selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₂-C₆ alkene and - (CH₂)P-A-R⁷ wherein p is 0 to 2, A is 6 to 10-membered aryl or 5 or 6-membered heteroaryl, and R⁷ is selected from the group consisting of hydrogen, halogen, C₁-C₃ alkyl, C]-C₃ alkoxy, NHC(O)- (C)-C₆ alkyl) and 5 or 6-membered aryloxy;

R² is selected from the group consisting OfCi-C₆ alkyl, Ci-C₆alkoxy and -(CR⁸RV-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently hydrogen or Ci-C₄ alkyl, and R¹⁰ is selected from the group consisting of 5 or 6-membered cycloalkyl, 6 to 10-membered aryl, and 5 or 6- membered heteroaryl; and

B is any one of substituents represented by Formulae (i) to (iii):

(iii) wherein m and n are each independently 0 or 1 ,

X is C or N,

R³ and R⁴ are each independently selected from the group consisting of hydrogen, C₁-C₆ alkyl and -(O)q-(CH₂)p-R^u wherein p is 0 to 2, q is 0 or 1, and R¹¹ is phenyl or 5 or 6-membered heteroaryl, R⁵ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆alkoxy and -(O)q-

(CH₂)P-R¹ ' wherein p is 0 to 2, q is 0 or 1 , and R¹ ' is phenyl or 5 or 6-membered heteroaryl, and

R⁶ is selected from the group consisting of hydrogen, Cj-C₆ alkyl and -(QHyp-A'-R¹² wherein p is 0 to 2, A' is phenyl, 5 or 6-membered heteroaryl, or a bicyclic compound containing phenyl, and R¹² is selected from the group consisting of hydrogen, halogen, hydroxy, C₁-C₆ alkyl, Q-C_όalkoxy, NHR¹³ and NR¹³R¹⁴ wherein R¹³and R¹⁴ are each independently hydrogen or Q-C₄ alkyl, or R¹³and R¹⁴ may be cyclized to form alkylene. In Formula (I), the number of R¹ may be in a range of 1 to 3, and R¹ may be positioned at carbon and/or nitrogen of the pyrazole ring.

Specifically as in Formula (I'), R¹ in the compound of Formula (T) may consist of R\ substituted at N on position 2 and R^ substituted at C on position 4 of the pyrazole ring.

In one preferred embodiment of the present invention, R¹ is selected from the group consisting of hydrogen, Cj-C₄ alkyl, C₂-C₄ alkene and -(CH₂)p-A-R⁷ wherein p is 0 to 2, A is phenyl or pyridine, and R⁷ is selected from the group consisting of hydrogen, halogen, trifluoromethyl, trifluoromethoxy, acetylamino and 5 or 6-membered aryloxy, particularly preferably hydrogen, methyl, hydroxyethyl, vinyl, phenyl, 4-chlorophenyl, 4-trifluoromethyloxyphenyl, 3- trffluoromethylphenyl, 3-trifluorophenyl, 4-trifluoromethylphenyl, 2-trifluoromethylphenyl, benzyl, pyridin-2-yl, 3-phenoxyphenyl and 2-acetylaminophenyl. Preferably, R² is Q-Qalkoxy or -(CR⁸RV-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently hydrogen or Ci-C₄ alkyl, and R¹⁰ is selected from the group consisting of 5 or 6- membered cycloalkyl, 6 to 10-membered aryl and 5 or 6-membered heteroaryl. More preferably, R² is selected from the group consisting of cyclohexyl, phenyl, 3,5-difluorophenyl and naphthyl.

R³ is preferably selected from the group consisting of hydrogen, Ci-C₆ alkyl and [-(O)q- (CH₂)P-R¹¹] wherein R¹¹ may be phenyl or 5 or 6-membered heteroaryl. R³ is more preferably hydrogen, benzyloxy or benzyl. R⁴ is preferably selected from the group consisting of hydrogen, C₁-C₄ alkyl and O- (CH₂)P-R¹¹ wherein R¹¹ may be phenyl or 5 or 6-membered heteroaryl. R⁴ is more preferably selected from the group consisting of hydrogen, methyl, isobutyl and benzyloxy.

R⁵ is preferably selected from the group consisting of hydrogen, C₁-C₆ alkyl and -O-R¹¹ wherein R¹¹ may be phenyl or 5 or 6-membered heteroaryl. R⁵ is more preferably hydrogen or phenoxy.

R⁶ is preferably selected from the group consisting of hydrogen, C₁-C₄ alkyl and -A' -R¹² wherein A' is phenyl, 5 or 6-membered heteroaryl, or a bicyclic compound containing phenyl, and R¹² is selected from the group consisting of hydrogen, halogen, hydroxy, Ci-C₆ alkyl, Ci-C₆ alkoxy, NHR¹³ and NR¹³R¹⁴ wherein R¹³and R¹⁴ are each independently hydrogen or Ci-C₄ alkyl, or R¹³and R¹⁴ are cyclized to form alkylene. More preferably, R⁶ is preferably selected from the group consisting of hydrogen, methyl, 3-dimethylaminophenyl, 3-(t-butyl)-phenyl, (3-isopropyl-[l,2,4]- oxadiazol-5-yl), 3-(pyrrolidin-l-yl)phenyl, and (l-hydroxy-indan-5-yl).

The compound in accordance with the present invention may form a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" means acid addition salts of the compound with acids capable of forming a non-toxic acid addition salt containing pharmaceutically acceptable anions, for example, inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid and hydroiodic acid; organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fiimaric acid, and maleic acid; or sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid. These acid addition salts may be prepared by conventional techniques known in the art, based on the chemical structure of Formula (I). As used herein, the term "isomer" means a compound of the present invention or a salt thereof, that has the same chemical formula or molecular formula but is optically or stereochemically different therefrom. For examples, the compounds of Formula (I) in accordance with the present invention may have an asymmetric carbon center, and therefore can be present in the form of optical isomers (R or S isomeric forms), racemates, diastereomeric mixtures, and individual diasteromers. When the compound of the present invention has a double bond, there may be present geometrical isomers (trans and cis isomeric forms). The present invention encompasses all these isomeric forms and mixtures.

Unless otherwise specified, it should be understood that the compound of Formula (I) in the context of the present invention is intended to encompass pharmaceutically acceptable salts and isomers thereof, all of which are included within the scope of the present invention. For convenience of illustration, the compound of the present invention is simply expressed as a compound of Formula

O)-

Representative examples of the compound of Formula (T) in accordance with the present invention include the following compounds:

N-[(2S,3R)4-[(3-dime%laminophenyl)me%lamino]-3-hydroxy-l-phenyl butan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(2S,3R)-l-(3,5-difluorophenyl)-4-[(3-dime%laminophenyl)me%lamino]-3- hydroxybutan-2-yl]-5-oxo- 1 -phenyl4H-pyrazole-3-carboxamide; l-(4-chlorophenyl)-N-[(2S,3R-l-(3,5-difluorophenyl)-4-[(3- dimethylaminophenyl)methylamino]-3-hydroxybutan-2-yl]-5-oxo-4H-pyrazole-3-carboxami

N-[(lR,2S)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2S)-pyrrolidin-2-yl]propan-2-yl]-5- oxo- 1 -phenyMH-pyrazole-S-carboxamide; N-[(2S,3R)4-[(3-dime%laminophenyl)me%lamino]-3-hydτoxy-l-phenylbutan-2-yl]-5- oxo-l-[4-(trifluoromethoxy)phenyl]-4H-pyrazx)le-3-carboxamide;

N-[(2S,3R)-4-[(3-dimethylaminophenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo-l-[3-(trifluoromethyl)phenyl]-4H-pyrazole-3-carboxamide; N-[(lS,2R)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2S)-pyrrolidin-2-yl]propan-2-yl]-5- oxo- 1 -phenyl4H-pyrazole-3-cark)xarnide;

N-[(2S,3R)-l-(3,5-dMuorophenyl)4-[(3-dimethylaminophenyl)methylaniino]-3- hydroxybutan-2-yl]-5-oxo- 1 -[3-(1iifluorOmethyl)phenyl]-4H-pyrazole-3-carboxamide;

N-[(lR,2R)-l-[(3R,8aS)-U,3,4,6,7,8,8a-octahydropyrrolo[l,2-d]pyrazb-3-yl]-3-(3,5- difluorophenyl)- 1 -hydroxypropan-2-yl]-5-oxo- 1 -phenyMH-pyrazole-S-carboxamide;

N-[(lS,2S)-l-[(3R,8aS)-l,23,4,6,7,8,8a-()ctahydropyπOlo[l,2-d]pyrazin-3-yl]-3-(3,5- dffluorophenyl)-l-hydroxypropan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(2S,3R)-4-[(3-dimethylaminophenyl)methylamino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo- 1 -[2-(trifluoromethyl)phenyl]-4H-pyrazole-3-carboxamide; l-b)enzyl-N-[(2S,3R)4-[(3-dime%lammophenyl)methylamino]-3-hydroxy-l- phenylbutan-2-yl]-5-oxo-4H-pyrazole-3-carboxamide;

N-[(lR,2S)-3-(3,5-Muorophenyl)-l-hydroxy-l-[(2R,4R)-4-phenylmethoxypyrrolidin-2- yl]propan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(lR,2S)-3-(3,5-dMuorophenyl)-l-hydroxy-l-[(2R)-pyrroHdin-2-yl]propan-2-yl]-5- oxo-l-phenyMH-pyrazole-S-carboxamide;

N-[(2S,3R)-4-[(3-dime%laminophenyl)me1hylamino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo- 1 -pyridin^-yMH-pyrazole-S-carboxamide;

N-[(2S,3R)-4-[(3-dime1hylarainophenyl)methylamino]-3-hydroxy-l-phenylbι^ oxo-l-(3-phenoxyphenyl)4H-pyra2Dle-3-carboxamide; N-[(lS,2S)-l-[(3S,8aS)-l-methyl-4-oxo-2,3,6,7,8,8a-hexahydro-lH-pyirolo[l,2- d]pyrazin-3-yl]-3-(3,5-difluorøphenyl)- 1 -hydroxypropan-2-yl]-5-oxo- 1 -phenyl-4H-pyrazole-3- carboxamide; l-benzyl-N-[(2S,3R)-4-[(3-t-butylphenyl)me%lan±io]-3-hyctoxy-l-phenylbutan-2-yl]- 5-oxo4H-pyrazole-3-carboxamide;

N-[(lS^S)-3-(3,5-difluorophenyl)-l-hydrøxy-l-[(3R,7S>7-phenoxy-l,2,3Λ6,7,8,8a- octahydropyrrolo[l,2-d]pyrazm-3-yl]pro^

N-[(lS,2R)-l-hydroxy-3-mphiMen-2-yl-l-[(2R)-pyrrølidin-2-yl]piOpan-2-yl]-5-oxo-l- phenyl-4H-pyrazole-3-carboxamide; N-[(lR,2S)-l-hydroxy-3-mphthden-2-yl-l-[(2R>pyπOlidin-2-yl]piOpan-2-yl]-5-oxo-l- phenyl4H-pyrazole-3-carboxamide;

N-[(2S,3R)4-[(3-dime1hylaniinophenyl)methylarnino]-3-hydroxy- 1 -phenylbutan-2-yl]- 1 - methyl-5-oxo-4H-pyrazole-3-carboxamide;

N-[(2S,3RH-[(3-t-butylphenyl)me%lamino]-l-(3,5-difluorophenyl)-3-hydroxybutan-2- yl]-5-oxo-l-[3-(trifluorømethyl)phenyl]4H-pyrazole-3-carboxamide; l-benzyl-N-[(2S,3R)-4-[(3-t-butylphenyl)me%lamino]-l-(3,5-difluorophenyl)-3- hydroxybutan-2-yl]-5-oxo4H-pyrazole-3-carboxamide;

N-[(2S,3R)4-[(3Klimethylaminophenyl)methylamino]-3-hydroxy-l-phenylbuto^ ethenyl-5-oxo4-methyl-pyrazole-3-carboxaniide; N-[(2S,3R)4-[(3-dime%lammophenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]-l-

(2-hydroxyethyl)-5-oxo4-methyl-pyrazole-3-carboxamide;

N-[(lR,2S)-3-cyclohexyl-l-hydroxy-l-[(2R)-pyrrolidin-2-yl]propan-2-yl]-5-oxo-l- phenyl4H-pyrazole-3-carboxamide; N-[(2S,3R)-3-hydroxy-l-phenyl-4-[(3-piOpan-2-yl-lA4-oxadiazol-5- yl)methylamino]butan-2-yl]-5-oxo-l-phenyl4H-pyra2Ole-3-carboxamide;

N-[(2S,3R)-3-hy(k)xy-l-phenyl-4-[(3-^ oxo- 1 -phenyMH-pyrazole-S-carboxarnide; 1 -(2-acetylaminophenyl)-N-[(2S,3R)-4-[(3-dime%laminophmyl)me1hylaniino]-3- hydroxy- 1 -phenylbutan-2-yl]-5-oxopyrrolidine-3-carboxamide;

N-[(2S,3R)-3-hydroxy-4-[(l -hydroxy-2,3-dihydrø-lH-inden-5-yl)methylamino]-l - phenylbutan-2-yl]-5-oxo-l-phenyl4H-pyrazole-3-carboxamide;

N-[(2S,3R)-l-(3,5-difluoroρhenyl)-3-hydroxy-4-[(3-pyrroUdin-l- ylphenyl)methylamino]butan-2-yl]-5-oxo-l -phenyMH-pyrazole-S-carboxamide;

N-[(lR,2R)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2R,5S)-5-(2-methylpropyl)piperazin-2- yl]propan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(lS,2S)-3-(3,5-dmuorophenyl)-l-hydroxy-l-[(2R,5S)-5-(2-me%lpropyl)piperazin-2- yl]propan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide; N-[(lR^S)-3-(3,5-cMuorophenyl)-l-hydroxy-l-[(2R)-pyiτolidin-2-yl]propan-2-yl]-5- oxo-1 -phenyMH-pyrazole-S-carboxamide;

N-[(lS,2S)-l-[(2S)4-benzyl-3-oxopiperazin-2-yl]-l-hydrøxy-3-phenylpropan-2-yl]-5- oxo-l-phenyl-4H-pyrazole-3-carboxamide; and

N-[(lR,2S)-3-(3,5-Muorophenyl)-l-hydroxy-l-[(2R,4RH-phenylmethoxypyrrolidin-2- ylJpropan^-yy-S-oxo-l-CS-trifluoromethy^phenyMH-pyrazole-S-carboxamide.

In accordance with yet another aspect of the present invention there is provided a method for preparing a compound of Formula (I). For a better understanding of the present invention, preparation of the compound of Formula (T) will be illustrated with reference to exemplary synthetic Reaction Schemes. However, skilled persons having knowledge about synthesis of compounds in the art to which the present invention pertains could readily prepare the compound of Formula (I) according to various synthetic methods, based on the chemical structure of Formula (I). Therefore, these methods also fall within the scope of the present invention. That is, the compound of Formula (I) may be prepared by any combination of various synthetic methods disclosed herein or in prior art. Therefore, the below description associated with preparation processes is provided only to disclose exemplary ones, and Hie scope of the present invention is not limited to only such preparation processes as described herein.

As shown in Reaction Scheme 1, the compound of Formula (I) can be synthesized by amide-coupling reaction of carboxylic acid of Formula 2 with an amine compound of Formula 3.

[Reaction Scheme 1]

In Reaction Scheme 1 , R¹, R² and B are as defined above. Examples of known coupling agents usable in the amide coupling may include, but are not limited to, carbodiimides such as dicyclohexylcarbodiimide (DCC), 1 -(3-dimethylaminopropyl)- 3-ethylcarbodiimide (EDC), 1 ,1 '-dicarbonyldiimidazolc (CDl), etc. which are used in a mixture with 1-hydroxybenzDtriazole (IIOBT) or l-hydroxy-7-azabenzotriazo]e (HOAT); bis-(2-oxo-3- oxazolidinyl)-phosphinic acid chloride (BOP-Cl), diphenylphosphorylazide (DPPA), N- [dimethylainino-lH-l,2,3-triazole[4,5-b]pyridin-l-ylnie%lene]-N-me%lmetriaiieaniiniLm (HATU)₅ CtC. In addition, examples of solvents that can be used in amide coupling may include DCM, DMF, and DCE. Examples of bases for activation of the reaction may include triethylamine, diisopropylethylamine, etc.

The carboxylic acid of Formula 2 is commercially available or can be synthesized by cyclization and hydrolysis from a compound of Formula 4 or 5 and a hydrazine compound of Formula 6, according to the synthetic procedure of Reaction Scheme 2 below.

[Reaction Scheme 2] RO₂C-^-E — CO₂R

o o + R^I-N-NH₂ ^{l) Cyclizati}?ⁿ lOSf ^OH

._oJUl _o H IO Hydrolysis _Ri/ ^N-N J

O ²

5 Cyclization may be carried out by heating reactants in the presence of a base, for example triethyl amine, N-methylmorpholine, diisopropyl ethyl amine, or K₂CO₃, in a solvent such as DMF,

THF, dioxane or DMSO. The reaction is conventionally carried out at a temperature of 60 to 120 ^°C for 4 to 24 hours.

Hydrolysis to obtain the compound of Formula 2 is carried out in a mixed solution of water and an organic solvent, using a base. Examples of the organic solvent usable in hydrolysis may include THF, methanol, and dioxane. Examples of the base may include LiOH, KOH, and

NaOH.

The hydrazine compound of Formula 6 is commercially available or may be obtained by conversion of an amine group of an amine compound of Formula 7 into a hydrazine group, according to a known method disclosed in Journal of the American Chemical Society, 198(48),

15374-75, 2006. [Reaction Scheme 3]

Ri-NH₂ ^{l) Nitr0Sati0n} , Rⁱ-N -NH₂ ii) [H] H

7 6

Nitrosation may be carried out in the presence of hydrochloric acid, using NaNO₂. Reduction of the nitroso compound may be carried out in the presence of hydrochloric acid, using SnCl₂.

Among the amine compounds of Formula 3, a compound where B is in the form of a chain can be synthesized from a commercially available epoxide, according to a synthetic procedure as disclosed in Reaction Scheme 4 below.

[Reaction Scheme 4]

In Reaction Scheme 4, R⁶ is as defined above, an azide compound of Formula 9 can be obtained by ring-opening of an epoxide compound using NaN₃, and an amine compound of Formula 10 can be obtained by reduction of the azide compound through hydrogenation using a

Pd/C catalyst. The reaction is carried out in a solvent such as methanol, ethanol, and dioxane, at 1 to

3 atm. Reductive animation to obtain a compound of Formula 3-1 is carried out using a compound (ketone or aldehyde) containing a carbonyl group. Examples of an available reducing agent may include sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, etc. In order to facilitate the reaction, acid may be used as a catalyst. Examples of an available acid catalyst may include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid; organic carbonic acids such as acetic acid, and trifluoroacetic acid; and amine salts such as ammonium chloride. Particularly preferred is hydrochloric acid or acetic acid.

Epoxide that is not readily commercially available can be prepared by stereoselective synthesis as shown in Reaction Scheme 5 below.

[Reaction Scheme 5]

11 1122 13

14 15 16 17

In Reaction Scheme 5, R² is -CH₂-R², and X is a leaving group, such as bromine, iodine, or methanesulfonyl. Stereoselective alkylation is carried out using a chiral auxiliary of Formula 11. Then, hydrolysis of the compound of Formula 12 is carried out to prepare an amino acid derivative of Formula 13. The hydrolysis reaction to obtain the compound of Formula 13 was previously described. The reaction is carried out using iodomethane in the presence of a base to obtain an iodocarbonyl compound of Formula 15. Reduction of the compound of Formula 15 is then carried out to obtain an iodohydrin compound of Formula 16. Reduction is carried out using NaBH₄, NaB(CN)H₃, or the like. Further, an epoxide compound of Formula 17 can be obtained by cyclization of the iodohydrin compound in the presence of a base.

A heterocyclic compound such as oxadiazole can be prepared according to a synthetic procedure in Reaction Scheme 6 below:

[Reaction Scheme 6]

First, an amidoxime compound of Formula 18 is reacted with oxalate to synthesize an oxadiazole compound of Formula 20, and reduction of the reaction product is then carried out using diisobutylaluminum (DIBAL) to obtain an aldehyde compound of Formula 21.

In Formula (I) in accordance with the present invention, a compound where B was cyclized can be synthesized using a known method disclosed in WO 2006/034093, according to a synthetic procedure in Reaction Scheme 7 below.

[Reaction Scheme 7] H

21 22 23 24

In Reaction Scheme 7, R² is as defined above, and n is 0 or 1. In Reaction Scheme 7, a nitro compound of Formula 21 and an aldehyde of Formula 22 are reacted under basic conditions to prepare a compound of Formula 23, and a nitro group of Formula 23 is reduced in the presence of hydrogen to obtain a compound of Formula 24.

The nitro compound of Formula 21 can also be synthesized by a known method disclosed in WO2006/034093, according to a synthetic procedure of Reaction Scheme 8.

[Reaction Scheme 8]

25 26 27

D

21

In Reaction Scheme 8, R is as defined above. Aldehyde of Formula 25 and nitromethane are subjected to coupling reaction under basic conditions to obtain a compound of Formula 26 which is then acetylated to prepare a compound of Formula 27. Following dehydroxylation, a compound of Formula 28 is reduced to synthesize a compound of Formula 21.

Aldehyde of Formula 22 can be synthesized from an amino acid.

[Reaction Scheme 9]

)n ^RedUCtiOn , )n ^OxidatiOrU ⁾n

29 30 22

An alcohol compound of Formula 30 can be obtained from a compound of Formula 29 using a reducing agent, for example NaBH₄ or LAH, in methanol or THF. Then, the alcohol compound is subjected to oxidation to thereby obtain a compound of Formula 22. An oxidant may be SO₃P_y, and a solvent may be DCM.

Analogously to the procedure described in Reaction Scheme 7, a piperazine compound can be synthesized according to a synthetic procedure of Reaction Scheme 10 below.

[Reaction Scheme 10]

31 32 33 34

Aldehyde of Formula 31 and piperazine of Formula 32 are reacted in the presence of a base to obtain a compound of Formula 33. The deprotection of the Boc group from the compound of Formula 33 is then carried out to obtain a compound of Formula 34.

Meanwhile, a bicyclic compound can be synthesized according to a synthetic procedure of Reaction Scheme 11.

[Reaction Scheme 11]

BnO

I, -Reduction =^ A ii) Oxidation

38 39

In Reaction Scheme 11, R i5 is as defined above, and [RA] means reductive amination. Analogously to the procedure described in Reaction Scheme 9, aldehyde of Formula 36 is synthesized from a serine derivative of Formula 35. Then, a compound of Formula 37 is synthesized through reductive amination of Formula 36. By repeating the same procedure, aldehyde of Formula

38 is obtained, followed by deprotection of the Boc group and reductive amination to obtain a compound of Formula 39.

Separation of a conventional mixture is carried out by column chromatography, whereas a final compound may be separated by recrystallization or normal-phase or reverse-phase HPLC

(Waters, Delta Pack, 300 x 50 mmlD., C18 5 μso, 100A). When recrystallization or HPLC is employed, a desired compound can be obtained in the form of a trifluoroacetate. When it is desired to obtain hydrochloride, an ion exchange resin may be employed.

As described above, after the reaction according to the method of the present invention is complete, the reaction product can be separated and purified by conventional post-treatments, for example, chromatography, recrystallization, and the like.

In accordance with yet another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting beta-secretase, comprising a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt or isomer thereof as an active ingredient and a pharmaceutically acceptable carrier. If desired, the composition may further comprise one or more diluents or excipients.

The compound of Formula (I) exhibits excellent inhibitory effects on beta-secretase, so the present invention provides a beta-secretase inhibitor composition comprising a compound of Formula (J) in conjunction with a pharmaceutically acceptable carrier. In particular, the composition in accordance with the present invention exhibits excellent effects on improvement of cognitive functions or treatment or prevention of neurodegenerative diseases, particularly Alzheimer's disease without being limited thereto. The term "pharmaceutical composition" as used herein 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 to an organism. Multiple techniques of administering a compound exist in the art including, but are not limited to oral, injection, aerosol, parenteral, and topical administrations. Pharmaceutical compositions can also be obtained by reacting compounds with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

The term "therapeutically effective amount" means that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease being treated. Thus, a therapeutically effective amount refers to that amount which has the effect of (i) reversing the rate of progress of a disease, (ii) inhibiting or slowing to some extent further progress of the disease, and/or, (iii) relieving to some extent (or, preferably, eliminating) one or more symptoms associated with the disease.

The term "carrier" means a chemical compound that facilitates the incorporation of a compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) is a commonly utilized carrier as it facilitates the uptake of many organic compounds into the cells or tissues of an organism.

The term "diluent" defines chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form of the compound. Salts dissolved in buffered solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of human body fluid. Since buffer salts can control the pH of a solution at low concentrations, a buffered diluent rarely modifies the biological activity of a compound.

The term "physiologically acceptable" defines a carrier or diluent that does not abrogate the biological activity and properties of the compound. The compounds described herein can be administered to a human patient per se, or in pharmaceutical compositions in which they are mixed with other active ingredients, as in combination therapy, or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990.

a) Routes of Administration

Compounds of the present invention may be administered via any conventional routes, depending upon desired applications. Preferred routes of administration may, for example, include injection, oral and intranasal administrations. Alternatively, the active compounds may be administered by dermal, intraperitoneal, retroperitoneal and rectal routes.

b) Composition/Formulation

The pharmaceutical composition of the present invention may be manufactured in a manner that is itself known, e.g. , by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g. , in Remington's Pharmaceutical Sciences, supra.

Injectable preparations, for example aqueous or oily suspensions for sterile injection may be prepared by a conventional method known in the art, using suitable dispersants, wetting agents and/or suspending agents. Examples of solvents that can be used in the formulation of injectable preparations may include water, Ringer's solution, and isotonic NaCl solution. In addition, sterile fixing oil is also conventionally used as a solvent or suspending medium. Any non-irritable fixing oil including monoglyceride and diglyceride may be used for this purpose. In addition, fatty acids such as oleic acid may also be used for injectable preparations.

Examples of solid dosage forms for oral administration may include capsules, tablets, pills, powders and granules. Particularly preferred are capsules and tablets. Tablets and pills may be preferably provided with enteric coatings. The solid dosage form may be prepared by mixing an active compound of Formula (I) in accordance with the present invention with one or more inert diluents (such as sucrose, lactose, and starch) and carriers such as lubricants (such as magnesium stearate), disintegrants, binders, and the like.

c) Effective Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. When the compound of the present invention is administered for clinical purposes, a daily dosage of the compound, which may be administered to a subject in a single or divided doses, is preferably in a range of 10 to 100 mg/kg. As will be apparent to those skilled in the art, a specific dose of the active ingredient for individual patients may vary depending on various factors such as kinds of compounds to be used, weight, sex, health conditions, and dietary habits of patients, treatment duration, administration manners, excretion rates, drug mixing and severity of disease.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Now, the present invention will be described in more detail with reference to the following

Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present inventioa

Abbreviations ased in the preceding Reaction Schemes and the following Preparations and Examples are as follows:

Bu: butyl i-Bu: isobutyl

Nap: naphthyl

BOC (boc): t-butoxycarbonyl c-Hex: cyclohexyl

Bn: benzyl

DCM: dichloromethane

DMF: N,N-dimethyrformamide

EDC: l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride EtOAc: ethyl acetate

Hex: normal hexane

HOBT: hydroxybenzotriazole

LDA: lithium dϋsopropyl amide Ph: phenyl

TFA: trifluoroaectic acid

Preparation 1: 3-phenoxy-phenyl-l -hydrazine 3-phenoxy-phenylamine (3.7 g, 20 mmol) was dissolved in HCl (6 mL) at 0°C, and a solution of NaNO₂(13.8 g, 20 mmol) in H₂O (3 mL) was added thereto. The mixture was stirred for 10 min, and a solution of SnCl₂ in HCl was added dropwise thereto, followed by stirring for 3 hours. After the reaction was complete, a solid material was filtered, washed with diethyl ester, and dried to afford the title compound (3.36 g, 84 %). Mass[M+l] =201

Preparation 2: S-oxo-l-rS-phenoxyphenylM^-dihydro-l-H-pyrazole-S-carboxylic acid Step A: S-oxo-l-CS-phenoxyphenyllAS-dihydro-l-H-pyrazole^-carboxylic acid methyl ester

3-phenoxy-phenyl-l -hydrazine (4.2 g, 20 mmol) synthesized in Preparation 1 was added to diethyl acetylene diester (3.2 g, 20 mmol) in THF, to which Et₃N (2.8 mL, 20 mL) was then added, followed by stirring at 80 ^"C for 15 hours. After the reaction was complete, the reaction mixture was cooled and neutralized with addition of IN HCl, followed by EtOAc extraction. The organic extract was dried over MgSO₄ and distilled under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/5) to afford the title compound (4.91 g, 89%).

Step B: S-oxo-l-G-phenoxyphenvlM^-dihvdiO-l-H-pyrazole-S-carboxvlic acid S-oxo-l-CS-phenoxypheny^^S-dihydro-l-H-pyrazole-S-carboxylic acid methyl ester (4.0 g) obtained in Step A was dissolved in methanol, to which water and LiOH were then added, followed by stirring at room temperature for 3 hours. After the reaction was complete, the reaction mixture was adjusted to pH of 2 to 3 with addition of IN HCl, followed by EtOAc extraction. The organic extract was dried over MgSO₄ and distilled under reduced pressure to afford the title compound.

Mass[M+l] =297

Preparations 3 to 10 Analogously to the procedure described in Preparation 2, compounds of Preparations 3 to

10 as listed in Table 3 below and represented by the following general formula were synthesized using commercially available aniline or hydrazine.

[Table 3]

Preparation 11: 4-me1fayl-5-oxo-l-vinyl-4,5-dihvdro-lH-pyra2iole-3-carboxylic acid Step A: 4-metfayl-5-oxo-l-vinyl4,5-dihvdro-lH-pyrazole-3-carboxylic acid methyl ester

The title compound was obtained as a by-product during preparation of the compound of Preparation 10.

Step B: 4-methyl-5-oxo-l-vinyl4,5-dihvdiO-l-H-pyraziole-3-carboxylic acid

Analogously to the procedure described in Step B of Preparation 2, the title compound was synthesized using 4-methyl-5-oxo-l-vinyl-4,5-dihydro-lH-pyrazole-3-carboxylic acid methyl ester obtained in Step A. Mass[M+l] =155

Preparation 12: (nS,2RV3-amino-l-benzyl-2-hydroxypropylVcarbamic acidt-butyl ester

Step A: ((lS2RV3-azido-l-benzyl-2-hvdiOxypropyl)-carbamic acid t-butyl ester

Commercially available epoxide (2.6 g, 10 mmol) was dissolved in a sat'd NH₄Cl aqueous solution, and the mixture was stirred in ethanol at 80 ^"C for 12 hours. After the reaction was complete, the reaction mixture was concentrated under reduced pressure, diluted with EtOAc, washed with IN HCl, and extracted with EtOAc. The organic extract was purified by column chromatography (EtOAc/n-Hex = 1/4) to afford the title compound (2.9 g, 95%). Step B: ((I S,2R>3-amino-l -benzyl^-hydroxypropylVcarbarnic acid t-butyl ester

((lS,2R)-3-azido-l-benzyl-2-hydroxypropyl)-carbaniic acid t-butyl ester (2.0 g, 0.65 mmol) obtained in Step A was placed in a hydrogen reaction vessel, to which 1 g of Pd/C was then added. The reaction mixture was reacted at 50 psi for 4 hours and filtered through celite to afford the title compound (1.10 g, 94%) which was then directly used without further purification. Mass[M+l] =281

Preparation 13: [(lS2RV3-arnino-l-(3₁5-Muoro-bemyl)-2-hydroxy-propyl]-carbarnic acid t-butyl ester Step A: (2S,5RV2,5-dihvdro-3,6-dimethoxy-5-isopropyl-2-(3,5-dMuoro)benzylpyrazine

(TR.)-2,5-dihydro-3,6-dirnethoxy-2-isopropylpyrazine (9.2 g, 50 mmol) was dissolved in THF (100 mL), to which n-BuLi (4M in THF, 15 mL) was then added at -78 ^°C, followed by addition of difluorobenzyl chloride (9.72 g, 60 mmol). After the reaction was complete, NH₄Cl was added to the reaction mixture which was then extracted with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/1) to afford the title compound (12.4 g, 80%).

Step B: (Sy2-amino-3-(3,5-difluorophenyl)propionic acid methyl ester

(2S,5R)-2,5-dihydro-3,6-dimethoxy-5-isopropyl-2-(3,5-dMuoro)ber^lpyrazine (12 g, 38.7 mmol) obtained in Step A was dissolved in acetonitrile (50 mL) and 2N HCl (20 mL) was added thereto, followed by stirring at room temperature for 4 hours. After the reaction was complete, NaHCO₃ was added, followed by EtOAc extraction. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was recrystallized from EtOAc/n-Hex to afford the title compound (7.07 g, 85%). Step C: (SV2-t-butoxycarbonylamino-3-G.5-difluorophenyl^')piOpionic acid methyl ester

(S)-2-amino-3-(3,5-difluorøphenyl)propionic acid methyl ester (7 g, 32.5 mmol) obtained in Step B was dissolved in water, to which NaOH was then added, followed by addition of (BOC)₂O (7.80 g, 35. 7 mmol). 12 hours later, the reaction was terminated with addition of IN HCl, followed by EtOAc extraction. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/8) to afford the title compound (9.89 g, 88%).

Step D: [(SVl-fB^-dMuorobenzylVS-cMoro^-oxo-propyl^'lcarbamic acid t-butyl ester

Chloroiodomethane (9.14 g, 31.4 mmol) was dissolved in THF, to which LDA (synthesized from nBuLi and diisopropyl amine, 33 mmol) was then added, followed by addition of (S)-2-t-butoxycarbonylamino-3-(3,5-difluorophenyl)propionic acid meihyl ester (9 g, 28.5 mmol) obtained in Step C. The mixture was stirred at room temperature for 12 hours. After the reaction was complete, NH₄Cl was added, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/5) to afford the title compound (9.56 g, 79%).

Step E: [(I S,2SV1 -(S^-difluorobenzyπ^-hydroxy-S-chloro-propyllcarbamic acid t-butyl ester [(S^l^jS-difluorobenzyl^-chloro^-oxo-propyycarbamic acid t-butyl ester (9 g, 21 mmol) obtained in Step D was dissolved in MeOH (60 mL), to which NaBH₄ (1.55 g, 42 mmol) was then added, followed by stirring at room temperature for 12 hours. After the reaction was complete, water was added thereto with subsequent addition of IN HCl and stirring for 30 min, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/3) to afford the title compound (8.0 g, 89%).

Step F: [(S)-2-(3,5-difluorophenylVl-(S)-oxiranyl-ethyll-carbamic acid t-butyl ester [(lS^S^l^S^-difluorober^l^-hydroxy-S-chloro-propylJcarbarnic acid t-butyl ester

(6.5 g, 15.2 mmol) obtained in Step E was dissolved in THF (45 mL), to which NaOH (0.6 g, 15.2 mmol) was then added, followed by stirring at room temperature for 12 hours. After the reaction was complete, IN HCl was added thereto, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/5) to afford the title compound (3.40 g, 75%).

Step G: [flS,2R)-3-amino-l-(3,5-α4fluoro-rjeri2^1)-2-hyαVoxy-propyl]-carbarnic acid t-butyl ester

Analogously to the procedure described in Steps A and B of Preparation 12, the title compound was obtained using [(S)-2-(3,5κMuorophenyl)-l-(S)-oxiranyl-ethyl]-carbarnic acid t- butyl ester obtained in Step F. MS[MH] =217 (M+l)

Preparation 14: 3-fdimethylairiino)berizaldehyde

Commercially available 3-(dimethylamino)benzyl alcohol (750 mg, 5 mmol) was dissolved in DCM and DMSO, to which Et₃N (2.8 mL, 20 mmol) and SO₃Py (1.59 g, 10 mmol) were then added. The reaction solution was stirred at room temperature for 2 hours. After the reaction was complete, DCM was distilled under reduced pressure and a saturated NH₄Cl aqueous solution was added, followed by extraction with ethyl ester. The organic extract was washed with water, dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/2) to afford the title compound (670 mg, 90%). MS[MH-I] = 150 (M+l)

Preparation 15: 3-(t-butyl)benzaldehyde

3-(t-butyl)phenylbromide (1.07 g, 5 mmol) was dissolved in THF, to which n-Buli (4M in

THF, 1.5 mL) was then added, followed by addition of DMF and stirring at 0^°C for 3 hours. After the reaction was complete, NH₄Cl was added thereto, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 1/3) to afford the title compound (680 mg,

H O).

MS[MH-I] = 163 (M+l)

Preparation 16: 3-isopropyl-5-formyl-oxadiazole Step A: Methyl 3-isopropyl-5-oxadiazole carboxylate

Isopropyl amidoxime (1.03 g, 10 mmol) was dissolved in DMF (30 mL), and Et₃N (2. 8 mL, 20 mmol) and HOBT (2.30 g, 15 mmol) were added thereto. Pyruvic acid (1.04 g, 10 mmol) and EDC (2. 30 g, 13 mmol) were added to the mixture which was then stirred at room temperature for 15 hours. After the reaction was complete, DMF was removed by distillation under reduced pressure, followed by dilution with a saturated aqueous solution of NaHCO₃ and extraction with EtOAc. The organic extract was washed with brine, dried over MgSO₄ and distilled under reduced pressure. The residue was dissolved in DMF and pyridine was added thereto, followed by stirring at 80 ^°C for 15 hours. After the reaction was complete, a saturated aqueous solution OfNH₄Cl was added thereto, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and distilled under reduced pressure. The residue was purified by column chromatography (EtOAc/n- Hex = 1/5) to afford the title compound (12.8 g, 75%).

Step B: 3-isopropyl-5-(hydroxymethyl^')oxadia2ple Methyl 3-isopropyl-5-oxadiazole carboxylate (1.0 g, 4.67 mmol) obtained in Step A was dissolved in THF (10 mL) and LiBH₄ (186 mg, 9.3 mmol) was added thereto, followed by stirring at room temperature for 15 hours. After the reaction was complete, water was gradually added, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and distilled under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/5) to afford the title compound (431 mg, 65%).

Step C: 3-isopropyl-5-formyl-oxadiazole

Analogously to the procedure described in Preparation 14, the title compound was obtained using 3-isopropyl-5-(hydroxymethyl)oxadiazole obtained in Step B. Mass[M+l] = 140

Preparation 17: 5-formylindan-l-ol Step A: 5-bromoindan-l-ol

5-bromo-l-indanone (300 mg, 1.42 mmol) was dissolved in 50 mL of methanol, and the mixture was cooled to 0^°C, followed by addition of sodium borohydride (65 mg, 1.7 mmol). After stirring for 30 min, an ice bath was removed and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was extracted with ethyl acetate and a saturated ammonium chloride aqueous solution. The organic layer was taken, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to afford the title compound (260 mg, 86%).

Step B: [(5-bromo-2,3-dihvdro-lH-inden-yl^')oxy]ft-butyl)dimethylsilane 5-bromoindan-l-ol (260 mg, 1.22 mmol) obtained in Step A was dissolved in methylene chloride (50 mL) and the mixture was cooled to 0⁰C, followed by addition of trimethyldimethylsilyl trifluoromeihane sulfonate (355 mg, 1.34 mmol). After stirring for 30 min, the reaction temperature was elevated to room temperature. The reaction was terminated with addition of a saturated aqueous solution of ammonium chloride, followed by extraction with a 0.5N HCl aqueous solution. The organic layer was taken, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was directly used in subsequent reactions without further purification.

Step C: l-([t-butyl(dimethyl)silylloxy}indane-5-carbaldehyde

Analogously to the procedure described in Preparation 15, the title compound (326 mg, 97%) was obtained using [(5-bromo-2,3-dihydro-lH-inden-yl)oxy](t-butyl)dimethylsilane (399 mg, 1.22 mmol) obtained in Step B. The compound was directly used in subsequent reactions without further purification. Mass[M+l] =163

Preparation 18: 3-(pyrtOlidin-l-yl)ben2aldehvde Step A: N-[3-(hvdroxymethyl)phenyl1pyrrolidine

3-hydroxymethyl aniline (246 mg, 2 mmol) was dissolved in acetonitrile, and Et₃N (560 fd, 4 mmol) was added thereto, followed by addition of dibromobutane (430 mg, 2 mmol). The reaction mixture was reacted in a microwave oven for 5 min, and EtOAc was added thereto, followed by washing with water and drying over MgSO₄. The residue was purified by column chromatography (EtOAc/n-Hex = 1/4) to afford the title compound.

Step B: 3-(pyrrolidin-l-yl)ben2aldehyde Analogously to the procedure described in Preparation 14, the title compound was obtained using N-[3-(hydroxymethyl)phenyl]pyrrolidine obtained in Step A. Mass[M+l] =176

Preparation 19: [πS,2RH-benzyl-3-G-dimethylamino-benzylan^ acid t-butyl ester

[(lS,2R)-3-arnino-l-(3,5-difluoro-ben2yl)-2-hydroxy-propyl]-carbamic acid t-butyl ester (360 mg, 2 mmol) obtained in Preparation 13 and 3-(dimetiiylamino)benzaldehyde (300 mg, 2 mmol) obtained in Preparation 14 were placed in dichloroethane (15 mL), to which NaBH(OAc)₃ (880 mg, 4 mmol) was then added. The reaction mixture was stirred at room temperature for 4 hours, followed by addition of DMC and extraction with sat'd NaHCO₃ (aqueous). EtOAc was added to the residual aqueous solution, followed by dilution and extraction. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/1) to afford the title compound (590 mg, 94%). Mass[M+l] =314

Preparations 20 to 27

Analogously to the procedure described in Preparation 19, compounds of Preparations 20 to 27 as listed in Table 4 below were obtained using aldehydes commercially available or synthesized in Preparations 14 to 18 and amines synthesized in Preparations 12 and 13.

(R^= CH₂-R⁶)

[Table 4]

Preparation 28: fSVN-BOC-prolinal

Analogously to the procedure described in Steps C and D of Preparation 16, the title compound was obtained using commercially available (S)-N-BOC-proline methyl ester. Mass[M+l] =200

Preparation 29: (RVN-BOC-prolinal

Analogously to the procedure described in Steps C and D of Preparation 16, the title compound was obtained using commercially available (R)-N-BOC-proline methyl ester. Mass[M+l] =200

Analogously to the procedure described in Steps C and D of Preparation 16, the title compound was obtained using commercially available (R)-3-benzyloxyproline methyl ester. Mass[M+l] =306

Preparation 31 : (2R,5SV2-formyl-5-isopropyl-piperazine- 1 ,4-dicarboxylic acid di-t-butyl ester Step A: r(SV2-benzyloxy-l-formyl-ethyl)-carbamic acid t-butyl ester Analogously to the procedure described in Preparation 14, the title compound was obtained using commercially available ((R)-2-benzyloxy-l-hydroxymethyl-ethyl)-carbamic acid t- butyl ester.

Step B: (S>2-[(R>3-benzyloxy-2-t-butoxycarto^ acid methyl ester

Analogously to the procedure described in Preparation 19, the title compound was obtained using commercially available (S)-isoleucine methyl ester and ((S)-2-benzyloxy-l-formyl- ethyl)-carbamic acid t-butyl ester obtained in Step A.

Step C: rS)-2-(((RV2-amino-3-benzyloxy-piOpylaminoV3-methyl-butyric acid methyl ester

(S)-2-[(R)-3-benzyloxy-2-t-butoxycarbonylamino-propylamino]-3-methyl-butyric acid methyl ester (1.5 g, 3.65 mmol) obtained in Step B was dissolved in DCM, to which TFA was then added, followed by stirring at room temperature for 2 hours. After the reaction was complete, the solvent was evaporated under reduced pressure. The residue was used directly used without further purification.

Step D: (3S,6RV6-benzyloxymethyl-3-isopiOpyl-piperazin-2-one (S)-2-(((R)-2-amino-3-ber^loxy-propylamino)-3-me%l-butyric acid methyl ester (1.0 g,

3.23 mmol) obtained in Step C was dissolved in DMF, to which Et₃N (1.4 mL) was then added, followed by stirring at room temperature for 12 hours. After the reaction was complete, the reaction mixture was diluted with a saturated aqueous solution OfNaHCO₃, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/4) to afford the title compound (748mg, 84%).

Step E: (3S,6R)-6-hvdroxymethyl-3-isopropyl-piperazin-2-one

Analogously to the procedure described in Step B of Preparation 12, the title compound was obtained using (3 S,6R)-6-ben2yloxymethyl-3-isopropyl-piperazin-2-one obtained in Step D.

Step F: ((2R,5S)-5-isopropyl-piperazin-2-yl^*)-methanol

((3S,6R)-6-hydroxymethyl-3-isopropyl-piperazin-2-one (500 mg, 2.67 mmol) obtained in Step E was dissolved in THF (10 mL), to which LAH (180 mg) was then added, followed by stirring at room temperature for 6 hours. After the reaction was complete, H₂O was added thereto, followed by addition of IN HCl and stirring at room temperature for 30 min. When a white emulsion was precipitated, extraction with EtOAc was carried out. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 2/3) to afford the title compound (390 mg, 85%). After removal of the solvent under reduced pressure, the product compound was directly used in subsequent reactions without further purification.

Step G: (2R.5SV2-hvdroxymethyl-5-isopropyl-piperazine-L4-dicarboxylic acid di-t-butyl ester ((2R,5S)-5-isopropyl-piperazm-2-yl)-methanol (340 mg, 2 mmol) obtained in Step F was dissolved in DCM, to which Et₃N (560 μJL, 4 mmol) was then added, followed by addition of (BOC)₂O (571 mg, 2.4 mmol) and stirring at room temperature for 4 hours. After the reaction was complete, IN HCl was added thereto, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/8) to afford the title compound (706 mg, 95%).

Step H: (2R,5S)-2-formyl-5-isopropyl-piperazine-l,4-dicarboxylic acid di-t-butyl ester

Analogously to the procedure described in Preparation 14, the title compound was obtained using (2R,5S)-2-hydroxyrnethyl-5-isopropyl-piperazine-l,4-dicarboxylic acid di-t-butyl ester obtained in Step G. Mass[M+l] = 370

Analogously to the procedure described in Preparation 31, the title compound was obtained using commercially available ((R)-2-ben2yloxy-l-hydroxymethyl-ethyl)-carbamic acid t- butyl ester and (S)-proline methyl ester. Mass|M+l] = 355 Preparation 33 : GR,5S,8SV7-phenoxy-3-fomiyl-hexahvdropyrrolo[l 2-a]pyrazine-2-carboxylic acid t-butyl ester

Analogously to the procedure described in Preparation 31, the title compound was obtained using commercially available ((R)-2-benzyloxy-l-hydroxymethyl-ethyl)-carbamic acid t- butyl ester and (2S,4S)-4-phenoxy pyrrolidine-2-carboxylic acid methyl ester. Mass[M+l] = 446

Preparation 34: (RV2-[nS2S)-2-amino-3-O,5-difluorophenylVl-hvdroxypropyl]pyrrolidine-l- carboxylic acid t-butyl ester Step A: l-G.5-difluoroV2-nitro-ethanol

3,5-difluorobenzaldehyde (2 g, 14.1 mmol) was dissolved in THF, to which K₂CO₃ (0.3 g, 2.26 mmol) was then added, followed by addition of nitromethane (2.0 rnL, 36.6 mmol) and stirring for 12 hours. After the reaction was complete, a solid material was filtered through celite, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/4) to afford the title compound (2.19 g, 76%).

Step B: 3,5-difluoro-5-[(E)-2-nitrovinyl1ben2ene l-(3,5-difluoro)-2-nitro-ethanol (2.19 g, 10.7 mmol) obtained in Step A was dissolved in DCM, followed by addition of 4-dimethylaminopyridine (139 mg) and then (Ac)₂O (1.11 mL, 11.8 mmol). The reaction solution was stirred at room temperature for 0.5 hours, diluted with DCM, sequentially washed with a 2% HCl aqueous solution, a NaCl saturated aqueous solution and a

NaHCO₃ saturated aqueous solution, dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/10) to afford the title compound (1.13 g, 56%).

Step C: 3,5-difluoro-5-(2-nitro-ethyl)benzene 3,5-difluoro-5-[(E)-2-nitrovinyl]benzene (1.1 g, 6.02 mmol) obtained in Step B was placed in DMSO and acetic acid solution, and NaBH₄ (91 mg, 24.08 mmol) was added thereto. The reaction solution was stirred below 30 ^°C for 1.5 hours. After the reaction was complete, the reaction solution was diluted with EtOAc, washed with H₂O and NaHCO₃, dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (2- propanol/chloroform = 1/4) to afford the title compound (1.08 g, 95%).

Step D: (RV2-[(lS,2SV3-f3,5-difluoro-phenylH-hydroxy-2-r^^ acid t-butyl ester

3,5-difluoro-5-(2-nitro-ethyl)benzene (260 mg, 1.38 mmol) obtained in Step C was dissolved in THF, to which (Bu)₄NF (1.26 mL, 1. 26 mmol) was then added, followed by addition of (R)-I -BOC-prolinal and stirring at room temperature for 30 min. After the reaction was complete, the reaction mixture was diluted with EtOAc, washed with H₂O, dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/5) to afford the title compound (360 mg, 67%).

Step E: (RV2-[(lS,2SV2-amino-3-(3,5-dfluoro-phenylVl-hvdroxy-propyll-pyrrolidine-l- carboxylic acid t-butyl ester

(R)-2-[(lS,2S)-3<3,5-dMuoro-phenyl)-l-hydroxy-2-mtro-propyl]-pyrrolidine-l- carboxylic acid t-butyl ester (560 mg, 1.13 mmol) obtained in Step D was dissolved in THF, to which Raney nickel (Ni) was then added, followed by stirring at room temperature for 4 hours. After the reaction was complete, the reaction mixture was filtered through celite and extracted with EtOAc. The organic extract was washed with H₂O and NaHCO₃, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 10/1) to afford the title compound (250 mg, 48%). Mass[M+l] = 357

Preparation 35: (R)-2-[(lS2R)-2-amino-3-(3,5-difluorophenylVl-hvdroxypropyl]pyrrolidine-l- carboxylic acid t-butyl ester Analogously to the procedure described in Step E of Preparation 34, Ihe title compound was obtained using an isomer obtained during Step D of Preparation 34 (isomer ratio = 4:1). Mass[M+l] = 357

Preparations 36 and 37: (SV2-rriS2SV2-amino-3-(3,5-difluoro-phenylVl-hvdroxy-propyl1- pyrrolidine- 1-carboχylic acid t-butyl ester and (S)-2-(^"(lSj2R)-2-amino-3-(3,5-difluoro-phenyl)-l- hydroxy-propyl]-pyrrolidine-l-carboxylic acid t-butyl ester

Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was obtained using 3,5-difluoro-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34 and (S)- 1 -BOC-proline aldehyde (isomer ratio = 4:1). Mass[M+l] = 357

Preparations 38 and 39: (RV2-rriS,2SV2-amino-3-rnaphthyl-l-ylVl-hvdroxy-propyη-pyrrolidine-l- carboxylic acid t-butyl ester and (R)-2-[(lS,2S)-2-amino-3-(naphthyl-l-yl)-l-hydroxy-propyll- pyrrolidine-1-carboxylic acid t-butyl ester Analogously to the procedure described in Preparation 34, the title compound was obtained using 2-(naphthyl-l-yl)-nitroethane and (R)-l-BOC-proline aldehyde (isomer ratio = 4: 1). Mass[M+l] = 371

Preparation 40: (K)-2-|Yl S2SV2-aniino-3-cvclohexyl-l-hydroxypropyl1pyrrolidine-l-carboxylic acid t-butyl ester

Analogously to the procedure described in Preparation 34, the title compound was obtained using 2-cyclohexyl-nitroethane and (R)-I -BOC-proline aldehyde.

Mass[M+l] = 327

Preparation 41: gR,5SV2-|^"αS2S)-2-amino-3-G.5-difluoro-phenylVl-hvdroxypropyl1-5-isobutyl- piperazine-l,4-dicarboxylic acid di-t-butyl ester

Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was obtained using (2R,5S)-2-formyl-5-isopropyl-piperazine-l,4-dicarboxylic acid di-t- butyl ester obtained in Preparation 31 and 3,5-difluoro-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34.

Mass[M+l] = 528

Preparations 42 and 43: (3R,8aSV3-rπS.2SV2-amino-3-(3,5-diiluoro-phenvn-l-hvdroxypiOPyl1- hexahydro-pyrrolo[l^alpyrazine-2-carboxylic acid t-butyl ester and (3R,8aS)-3-[(lS,2R)-2-amino- 3-(3,5-difluoro-phenyl)- 1 -hydroxypropyli-hexahydro-pyrrolof 12a]pyrazine-2-carboxylic acid t- butyl ester Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was obtained using (3R,5S)-3-fonOyl-hexahydropyrrolo[l,2-a]pyrazine-2-carboxylic acid t-butyl ester obtained in Preparation 32 and 3,5-difluoro-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34 (isomer ratio = 4: 1). Mass[M+l] = 511

Preparation 44: (3R.7S.8aSV3-rπS^SV2-arnino-3-(3.5-difluoro-phenylVl-hvdroxypropyll-7- phenoxy-hexahydro-pyrrolo[l,2a]pyrazine-2-carboxylic acid t-butyl ester

Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was obtained using (3R,5S,8S)-7-phenoxy-3-fomiyl-hexahyαVopyπOlo[l,2-a]ρyrazine- 2-carboxylic acid t-butyl ester obtained in Preparation 33 and 3,5-difluoro-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34. Mass[M+l] = 504

Preparation 45: (2R.4RV2-[π S^SV2-amino-3-r3,5-difluoro-phenylVl -hydroxypropylH- benzyloxy-pyrroliαϋne-1-carboxylic acid t-butyl ester

Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was obtained using (R)-3-benzyloxy prolinal obtained in Preparation 30 and 3,5- difluoro-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34. Mass[M+l] = 463

Preparation 46: (^'S)-2-[πS2S)-2-amino-l-hvdroxy-3-phenylpropyl^"|-4-benzyl-3-oxo-piperazine-l- carboxylic acid t-butyl ester Step A: (S)-4-benzyl-2-|Tl S,2SV2-(dibenzylamino)-l-hvdroxy-3-phenylpropyl]-3-oxo-piperazine- 1-carboxylic acid t-butyl ester

Dϋsopropylamine (1.42 mL, 10.08 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) and the mixture was cooled to -78 ^°C. n-butyl Miium (2.5 M n-hexane solution, 3.9 mL) was added dropwise to the resulting solution. The mixture was stirred for 5 min and then stirred on an ice bath for 30 min. The reaction mixture was cooled to -78 ^°C, and a solution of t-butyl 4-benzyl- 3-oxopiperazine-l-carboxylate (B) (2.44 g, 8.40 mmol) in 15 mL of anhydrous tetrahydrofuran was added dropwise thereto, followed by stirring at that temperature for 1.5 hours. Thereafter, a solution of 2(S)-2-(dibenzylamino)-3-phenylpropanal (A) (2.99 g, 9.07 mmol) in anhydrous tetrahydrofuran (15 mL) was added to the reaction mixture which was gradually warmed to room temperature. After stirring at room temperature for 16 hours, the reaction was terminated with addition of water, followed by extraction with saturated ammonium chloride (aqueous) and diethyl ether. The organic layer was taken, dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc/n-Hex = 1/5) to afford the title compound (1.92 g, 24%).

Step B: (SV2-[(lS,2S)-2-aniino-l-hydroxy-3-phenylpropyl]-4-benzyl-3-oxo-piperazine-l- carboxylic acid t-butyl ester

(S)4-ber^l-2-[(lS,2S)-2-(dibenzylarnino>l-hydroxy-3-phenylpropyl]-3-oxo- piperazine-1-carboxylic acid t-butyl ester (550 mg, 0.887 mmol) obtained in Step A was dissolved in 20 mL of ethanol, to which one drop of acetic acid was then added, followed by addition of 500 mg of palladium hydroxide and stirring under 1 atm hydrogen (balloon) for 18 hours. The reaction mixture was filtered through celite, followed by extraction with IN hydrochloric acid (aqueous) and diethyl ether. The aqueous layer was adjusted to a pH of about 8 with addition of an aqueous solution of 6N sodium hydroxide, followed by extraction with methylene chloride. The organic extract was dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure to afford the title compound (117 mg, 30%). Mass[M+l] = 440

Preparation 47: (SV3-((lS,2SV2-amino-l-hydroxy-3-phenyl-propyl)-l-methyl-hexahydro- pyrrolofl 2-a1pyrazin-4-one

Step A: rSV2-rmethoxy-methyl-carbamoylVpyrroHdine-l-carboxylic acid t-butyl ester Commercially available (S)-l-BOC-proline (2 g, 9.3 mmol) was placed in DCM (30 mL), followed by sequential addition of triethylamine (5. 22 mL, 37.2 mmol), HOBt (1.89 g, 13.95 mmol), EDC (2.14 g, 13. 95 mmol) and meihoxy methyl amine (0.91 g, 9.3 mmol) and stirring at room temperature for 12 hours. After the reaction was complete, a saturated aqueous solution of NH₄Cl was added to the reaction mixture which was then extracted with EtOAc. The organic extract was washed with a saturated aqueous solution OfNaHCO₃ and dried over anhydrous MgSO₄. The solvent was removed under reduced pressure. The residue was purified by column chromatography (eluent: EtOAc/n-Hex = 1/1) to afford the title compound (1.29 g, 54%).

Step B: (Sy2-acetyl-pyrrolidine-l-carboxylic acid t-butyl ester (S^^methoxy-meΛyl-carbamoyO-pyrrolidine-l-carboxylic acid t-butyl ester (1.29 g,

4.99 mmol) obtained in Step A was placed in THF (10 mL). The reaction mixture was cooled to - 78 "C, and methyl magnesium grignard (12 mL, 15 mmol) was added thereto. The reaction mixture was then warmed to room temperature and stirred for 1 hour. The reaction mixture was cooled to - 78 ^°C and a saturated aqueous solution OfNH₄Cl was added to terminate the reaction, followed by extraction with EtOAc. The organic extract was dried over anhydrous MgSO₄ and the solvent was removed under reduced pressure. The residue was purified by column chromatography (eluent: EtOAc/n-Hex = 1/1) to afford the title compound (1.03 g, 97 %).

Step C: (^"SVl-pyrrolidin-2-yl-ethanone

Analogously to the procedure described in Step C of Preparation 31, the title compound was obtained using (S^-acetyl-pyrrolidine-l-carboxylic acid t-butyl ester obtained in Step B.

Step D: r(SV2-((SV2-aceM-pyrroUdm-l-ylVl-benzyloxymethyl-2-oxo-ethyl]-carbamic acid t-butyl ester

Analogously to the procedure described in Step A, the title compound was obtained using (S)-l-pyrrolidin-2-yl-ethanone obtained in Step C and commercially available (S)-3-benzyloxy-2-t- butoxycarbonylamino-propionic acid.

Step E: (SVl-methyM-oxo-octahydro-pyrrolofl^-aipyrazme-S-carboxyaldehyde

Analogously to the procedure described in Step C of Preparation 31, Preparation 19, and Steps E, G and H of Preparation 31, the title compound was obtained using [(S)-2-((S)-2-acetyl- pvrroHdin-l-yl)-l-benzyloxymethyl-2-oxo-ethyl]-carbamic acid t-butyl ester obtained in Step D.

Step F: rSV3-(αS.2SV2-aniino-l-hvdroxy-3-phenyl-proDylVl-methyl-hexahvdro-pyrrolo|^'1.2- a]pyrazin-4-one

Analogously to the procedure described in Steps D and E of Preparation 34, the title compound was synthesized using (S)-l-me%14-oxo-octahydro-pyirolo[l^-a]pyrazine-3- carboxyaldehyde obtained in Step E and 3,5-difluoiO-5-(2-nitro-ethyl)benzene obtained in Step C of Preparation 34. Mass[M+l] = 440

Preparation 48: l^-acetylamino-phenvD-S-oxo^S-dihydro-lH-pyrazole-S-carboxylic acid

Analogously to the procedure described in Preparations 1 and 2, the title compound was synthesized using N-(2-amino-phenyl)-acetamide. Mass[M+l] = 262

Example 1 : N-[f2S3RH-[(3-dimethylarcmophenyl^

5-oxo-l-phenyl-4H-pyrazole-3-carboxamide

Step A: (2R,3S)-3-arnino-l-O-dimethylaniino-ben2ylainino)4-phenyl-butan-2-ol

[(lS^R)-l-benzyl-3-(3-dime1hylatnino-benzylarnmo)-2-hydroxy-propyl]-carbarm acid t- butyl ester (314 mg, 1 mmol) obtained in Preparation 19 was dissolved in DCM (5 mL), and TFA (2 mL) was added thereto, followed by stirring at room temperature for 1 hour. After the reaction was complete, the solvent was removed under reduced pressure. The residue was directly used in subsequent reactions without further purification.

Step B: N-[r2S3RM-[G-dimethylaminophenyl)methylaminol-3-hydroxy-l -phenylbutan-2-yl1-5- oxo- 1 -phenyHH-pyrazole-S-carboxamide

(2R,3S)-3-amino-l-(3-dimethylamino-benzylamino)^l-phenyl-butan-2-ol (100 mg, 0.32 mmol) obtained in Step A was dissolved in DMF, and the reaction solution was adjusted to a pH of 8 to 9 with addition OfEt₃N (280 ≠, 2 mmol), followed by addition of EDC (74.4 mg, 0.41 mmol) and HOBT (973.4 mg, 0.48 mmol) and stirring at room temperature for 12 hours. After the reaction was complete, a saturated aqueous solution Of NaHCO₃ was added, followed by extraction with EtOAc. The organic extract was dried over MgSO₄ and distilled under reduced pressure. The residue was purified by column chromatography (EtOAc: n-Hex = 2: 1) to afford the title compound (133 mg, 84%).

¹H NMR (500 MHz, CDCl₃): δ 7.69(d, IH), 7.33 (t, IH), 7.18 (t, IH), 7.06 (t, IH), 6.70 (d, 2H), 6.63 (s, IH), 6.60-6.52 (m, 3H), 4.10(brs, IH), 3.85 (t, 2H), 3.82-3.77 (m, 2H), 2.99-2.90 (m, 2H), 2.87-2.75 (m, 2H), 2.77 (s, 6H)

Examples 2 to 21

Analogously to the procedure described in Example 1 , compounds of Examples 2 to 21 as listed in Table 6 below and represented by the following general formula were synthesized using acids synthesized in Preparations 2 to 11 and amine compounds obtained in Preparations 19 to 27 and 48.

[Table 6]

(Me: methyl, Pπpropyl, and *: isomeric form)

Example 22: N-r(lR.2SV3-r3,5-difluorophenylVl-hvdroxy-l-rr2RVpyrrolidin-2-vnpropan-2-yll-5- oxo-l-phenvMH-pyrazole^-carboxamide Step A: 0lV2-{riS2SV3-r3,5-difluoro-phenyl)-l-hvdiOxy-2-[(5-oxo-l-phenyl-4,5-dihvdro-lH- pyrazole^-carbonylVarninoJ-propyll-pyrrolidine-l-carboxylic acid t-butyl ester

Analogously to the procedure described in Step B of Example 1, the title compound was obtained using (R)-2-[(l S^S)-2-arnmo-3-(3,5-diiluoro-phenyl)-l-hydroxy-propyl]pym)lidine-l- carboxylic acid t-butyl ester obtained in Preparation 34 and commercially available 5-oxo-l-phenyl- 4,5-dihydro-lH-pyrazole-3-(^boxyric acid. Step B: N-^lR^Sl-S-rB^-dmuorophenvn-l-hvdroxy-l-rQRVpyrrolidin^-ylipiOpan^-yll-S-oxo- 1 -phenyl-4H-pyrazple-3-c^boxamide

Analogously to the procedure described in Step A of Example 1, the title compound was obtained using (R)-2-{(lS,2S)-3-(3,5-difluoro-phenyl)-l-hydroxy-2-[(5-oxo-l-phenyl-4,5-dihydro- lH-pyrazole-S-carbony^-aniinoJ-propylJ-pyπOlidine-l-carboxylic acid t-butyl ester obtained in Step A.

¹H NMR (500MHz, MeOD): δ 8.02-8.00 (m, IH), 7.73 (d, 2H), 7.50 (t, 2H), 7.34 (t, IH), 6.94 (d, 2H), 6.76 (t, IH), 5.96 (s, IH), 4.38-4.35 (m, IH), 3.96-3.94 (m, IH), 3.72-3.70 (m, IH), 3.64- 3.61(m, IH), 3.56-3.52(m,lH), 3.29-3.26 (m, 2H), 3.02(d, 2H), 2.12-1.92 (m, 2H)

Examples 23 to 33

Analogously to the procedure described in Example 22, compounds of Examples 23 to 33 as listed in Table 7 below and represented by the following general formula were synthesized using acid synthesized in Preparation 5, commercially available 5-oxo-l-phenyl4,5-dihydro-lH-pyrazole- 3-carboxylic acid, and amine compounds obtained in Preparations 34 to 41 and 45 to 46.

[Table η

(*: isomeric form)

Examples 34 to 37

Analogously to the procedure described in Example 21, compounds of Examples 34 to 37 as listed in Table 8 below and represented by the following general formula were synthesized using commercially available 5-oxo-l-phenyl-4,5-dihydro-lH-pyrazole-3-carboxylic acid and amine compounds obtained in Preparations 42 to 44 and 47.

[Table 8]

( * and **: isomeric form)

Experimental Example 1 : Enzymatic activity of recombinant beta-secretase 2 Step A: Construction of vector expressing recombinant beta-secretase 2

BACE cDNA (ATCC, Cat. No. 6896840) was purchased which was synthesized based on a human BACE2 gene sequence (Accession No. BC014453) disclosed in the public Genbank data base. Only the ectodomain which corresponds to the region of from the amino acid residues 1 to 466 with exclusion of a transmembrane domain and a cytoplasmic domain in the entire BACE gene was re-cloned, and then the base sequence of Fc region which corresponds to 230 amino acids (from the amino acid residues 1 to 466) of human Immunoglobulin G (hlgG) was linked to the 3' end thereof. The BACE (ectodomain)-IgG Fc (hereinafter, referred to as "BACE-Fc") was ligated between BamHL and Xhol sites of pCDNA3 (Invitrogen) as a mammalian expression vector to construct a BACE2-Fc expression vector, designated as pCDNA3 BACE2-Fc.

Step B: Construction of mammalian cell line expressing BACE2-Fc fusion protein Chinese hamster ovary (CHO) DHFR- cells (ATCC Accession No. CRL9096) were cultured in an alpha-minimum essential medium (α-MEM, GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS, GIBCO-BRL) and then transferred to a 100 mm culture plate. When the cells reached confluence, they were transfected with the BACE2-Fc-expressing vector pCDNA3

BACE2-Fc using Lipofectamine Plus (Life Technologies). Selection of transfectant cells was carried out in a medium containing 10% dialyzed fetal bovine serum (dFBS, JRH) supplemented with 1 mg/mL of Geneticin (G418 sulfate, GIBCO-BRL). The culture medium was replaced with a fresh one every 4 days. Then, 100 clones were isolated and cultured on a 24-well culture plate. Among these isolated clones, 20 clones showing acceptable growth rates were sub-cultured on a 24-well culture plates for 3 days at an equal cell density (2 x 10⁵ cells/mL/24-well). An amount of BACE2- Fc protein secreted into the medium was quantified by ELISA method using goat anti-human IgG

(Pierce). As a result, clone #66 showing the highest growth rate and BACE2-Fc expression (3 mg/1

L of culture) was selected.

Step C: Production and purification of BACE2-Fc fusion protein 2 x 10⁵ cells/mL of the CHO DHFR- BACE2-Fc #66 cell line were inoculated into a roller bottle containing 250 mL of α-MEM supplemented with 10% dFBS, and cultured in a Roll-In cell incubator (Bellco) at 37 ^°C and 40 rpm for 4 days. When cells reached confluence, they were washed once with 250 mL of a serum-free medium (SFII, GD3CO-BRL), and 500 mL of a serum- free medium containing insulin (0.5 βg/mL, SIGMA) was added, followed by culture for 3 days. After the culture medium was harvested, 500 mL of a serum-free medium was again added thereto and then the cells were cultured for another 3 days. This procedure was repeated two times. All conditioned media harvested were centrifuged at 7000 rpm for 20 min (Beckman, JA 10 rotor) to isolate only the wash solution. The wash solution was filtered through a 0.45 μm filter and was then passed through a protein A sepharose chromatography column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 7.0), followed by washing with 20 mM sodium phosphate buffer (pH 7.0) to completely remove the non-adsorbed proteins. 100 mM sodium acetate buffer (pH 3.5) was added to elute the adsorbed proteins, so the BACE2-Fc protein (MW 75 kDa) of over 95% purity was obtained.

Step D: Beta-secretase 2 activity assay using fluorescence-labeled specific substrate

To determine enzymatic activity of beta-secretase 2 and inhibition efficiency of synthetic compounds, a Fluorescence Resonance Energy Transfer (FRET) enzyme activity assay was carried out using the purified B ACE2-Fc fusion protein and a fluorescence-labeled beta secretase 2-specific substrate. This will be briefly illustrated in the following.

From the entire amino acid sequence of an amyloid precursor protein (APP) known as an intracellular beta secretase 2-specific substrate, a peptide corresponding to a 10-amino acid region containing a beta-secretase cleavage site was synthesized with inclusion of EDANS as a fluorophore and DABCYL as a quenching group linked thereto. When this fluorescence-labeled substrate is reacted with BACE2-Fc, the BACE2 functional region is cleaved and the quenching group is detached, and EDANS emits fluorescence at 510 nm upon excitation with 350 nm light, whereby the level of fluorescence is measured to accurately and conveniently determine the extent of the cleavage reaction. Each of synthetic compounds at a concentration of 10 mM was dissolved in DMSO and then stored at 20 ^°C. For determination of activity, a 10 mM DMSO solution was first added to the right row of a 96-well plate, and then nine successive doubling dilutions were carried out with an equal volume of DMSO. 10 μi of the diluted compound solution was added to a 96-well assay plate containing 600 μM of a fluorescence-labeled BACE substrate dissolved in 15 μJL of reaction buffer (50 mM sodium acetate, pH 4.5, 0.05% CHAPS) and 10 βi of 50% DMSO, so that DMSO was adjusted to a final concentration of 10%, and the inhibitor was treated through nine successive doubling dilutions from 500 uM. 65 μl of the purified BACE2-Fc fusion protein solution was added to a final concentration of 0.4 μg/mL and reacted at room temperature for 1 hour. An amount of the reaction product was measured by the magnitude of fluorescence at a 350 nm excitation wavelength and a 510 nm emission wavelength, using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). The concentration of a synthetic compound inhibiting 50% of beta- secretase activity, Le., IC₅₀ and Ki were determined by comparing the measurement value with that of a control group with no addition of the synthetic compound.

Experimental Example 2: Enzymatic activity of recombinant cathepsin D

To determine enzymatic activity of cathepsin D and inhibition efficiency of synthetic compounds, a Fluorescence Resonance Energy Transfer (FRET) enzyme activity assay was carried out using cathepsin D (Calbiochem, #219401) and fluorescence-labeled cathepsin D-specific substrate (Bachem #M-2455). This will be briefly illustrated in the following. As a cathepsin D-specific substrate, a 10-aa peptide was synthesized with inclusion of Mca as a fluorophore and Dnp as a quenching group linked thereto. When this fluorescence-labeled substrate is reacted with cathepsin D, the cafhepsin D functional region is cleaved and the quenching group is detached, and Mca emits fluorescence at 393 nm upon excitation with 328 nm light, whereby the level of fluorescence is measured to accurately and conveniently determine the extent of the cleavage reaction.

Each of synthetic compounds at a concentration of 10 mM was dissolved in DMSO and then stored at 20 ^°C. For determination of activity, a 10 mM DMSO solution was first added to the right row of a 96-well plate, and then nine successive doubling dilutions were carried out with an equal volume of DMSO. 10 μl of the diluted compound solution was added to a 96-well assay plate containing 7 μM of a fluorescence-labeled cathepsin D substrate dissolved in 50 μl of reaction buffer (50 mM sodium acetate, pH 4.0) and 10 μl of 25% DMSO, so that DMSO was adjusted to a final concentration of 5%, and the inhibitor was treated through nine successive doubling dilutions from 250 μM. 30 μl of the purified cathepsin D fusion protein solution was added to a final concentration of 0.75 ng/mL, followed by reaction at 37 ^°C for 1 hour. An amount of the reaction product was measured by the magnitude of fluorescence at a 328 nm excitation wavelength and a 393 nm emission wavelength, using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). The concentration of a synthetic compound inhibiting 50% of cathepsin D activity, Ie., IC₅₀ and Ki were determined by comparing the measurement value with that of a control group with no addition of the synthetic compound.

Experimental Example 3: Secreted alkaline phosphatase (SEAP) activity assay Step A: Establishment of permanent cell line expressing SEAP-APPsw-KK A gene which expresses SEAP and Swedish mutant form of APP (CRE-SEAP- APP695sweKK) under the control of cAMP response element (CRE) was cloned into pcDNA3.1(+)Neo (Invitrogen) which is a mammalian expression vector. Neuro-2a cells (ATCC Accession No. CCL-131) were cultured in a Dulbecco's minimum essential medium (DMEM, GIBCO-BRL) supplemented with 10% FBS, and then transferred to a 6-well culture plate. When the cells reached confluence, they were transfected with the CRE-SEAP- APP695sweKK expression vector using Lipofectamine 2000 (Life Technologies). Individual clones were isolated and then cultured again on a 6-well culture plate. Following selection of clones, 100 clones showing acceptable growth rates were cultured on a 24-well culture plate for 3 days, followed by culture in DMSO/10 uM Forskolin medium for 6 hours. 50 /^/well of the culture was aliquoted and reacted with 50 μl of AttoPhos (Promega). The magnitude of fluorescence was measured at room temperature for 30 min, at a 450 nm excitation wavelength and a 580 nm emission wavelength, using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). Among the selected clones, 4 clones showing high SEAP activity in forskolin/DMSO and exhibiting the greatest SEAP activity by forskolin were selected. The selected clones were sub-cultured on a 96- well culture plate for one day at an equal cell density (2 X 10⁴ cells/96-well), followed by treatment with DMSO/10 μM Forskolin. After 6 hours, activity of SEAP secreted into the medium was assayed. Clone #159 showing the highest activity of SEAP was selected.

Step B: SEAP activity assay

The clone N2A SEAP-APPsw-KK #159 expressing CRE-SEAP-APP695sweKK was seeded at a density of 2 X 10⁴ cells/80 μJL to each well of a 96-well culture plate. 1OmM Forskolin and DMSO were diluted 100 times with the culture medium and then 10 μt/weil of the dilution was added to the 96-well plate. Each of synthetic compounds at a concentration of 10 mM was dissolved in DMSO and then stored at 20 ^°C. For determination of activity, a 10 mM DMSO solution was first added to the right row of a 96-well plate, and then seven successive 3-fold dilutions were carried out with an equal volume of DMSO. 10 μi of the serial compound dilution was diluted 10 times with 90 μi of the culture. DMSO was adjusted to a final concentration of 1.1% and the inhibitor was treated through seven successive doubling dilutions from 100 μM. After treatments were complete, the cells were cultured in a 6% CO₂ incubator at 37 ^°C for 5 hours. In order to measure an amount of SEAP secreted into the medium, the culture was first subjected to heat inactivation at 65 ^°C for 30 min to thereby abolish activity of other alkaline phosphatases. 50 μi of the heat-inactivated culture and 50 μi of AttoPhos (Promega) were reacted at room temperature for 30 min. The magnitude of fluorescence was measured at a 450 ran excitation wavelength and a 580 nm emission wavelength, using a fluorescent plate reader (SpectraMax Gemini XS, Molecular Device). The concentration of a synthetic compound inhibiting 50% of SEAP activity, Ie., IC₅₀ was determined by comparing the measurement value with that of a control group with no addition of the synthetic compound.

Experimental Example 4: Assay of amyloid beta in neurons of transgenic mice Step A: Culture of primary neurons of transgenic mice (APP/PSldE9)

Offspring mice from interbreeding of transgenic (APP/PSldE9) male and female mice were included in experiments. The brains of 3 or 4-day-old mice were dissected, and the hippocampus tissue and cerebral membrane were removed at 4^°C and minced, followed by treatment with DNase (Sigma, D5025) and protease (Sigma, P5147) and placement in an incubator at 37 ^°C for 20 to 25 min. After being separated into cells, 4 X 10⁵ cells/well were seeded onto a 24- well plate coated with poly-L-lysine and containing a medium (27.6 mL Neurobasal + 1.5 mL FBS + 600 μi B27 + 300 μi of 200 mM L-glutamine). The cells were cultured in an incubator at 37 "C for 7 days, and then used for subsequent experiments.

Step B: AMO activity assay Each of synthetic compounds at a concentration of 10 mM was dissolved in DMSO and then stored at 20 ^°C . For determination of activity, a 10 mM DMSO solution was first added to the right row of a 96-well plate, and then six successive 3-fold dilutions were carried out with an equal volume of DMSO. The serial compound dilution was diluted 250 times with a medium (29.1 mL Neurobasal + 600 μi of B27 + 300 μi of 200 mM L-glutamine + 7.5 μi of 100 mM L- glutamate). 350 μi of the dilution was added to the cultured primary neurons and the cells were then cultured in an incubator at 37 ^°C for 8 hours. An expression level of the beta-amyloid peptide secreted into the medium was measured by sandwich ELISA (Biosource, #KHB3482) using two types of antibodies specific for the beta-amyloid peptide, which will be briefly illustrated below.

50 μi of the water-soluble beta-amyloid or cell culture diluted to different concentrations and 50 μi of reporter antibodies were mixed and added to an antibody-coated plate, followed by reaction at room temperature for 3 hours (or one or more days at 4 ^°C ). The culture plate was washed five times with a 1-fold volume of a wash solution (Biosource, #KHB3482), and 100 μi of horseradish peroxidase (HRP)-coηjugated antibodies diluted 100 times with an antibody dilution solution (Biosource, #KHB3482) was added thereto, followed by reaction at room temperature for 30 min. Then, HRP-conjugated antibodies were removed and the plate was washed five times with a 1-fold volume of a wash solution, to which 100 μi of a chromogene solution (Biosource, #KHB3482) was added, followed by reaction at room temperature for 30 min. Then, 100 μi of a stop solution (Biosource, #KHB3482) was added thereto, followed by reaction at room temperature for 30 min. The optical density (absorbance) was measured at 450 nm using a microplate reader (SpectraMax 340, Molecular Device). The concentration of a synthetic compound inhibiting 50% of intracellular beta-secretase activity, Ie., IC₅₀ was determined by comparing the measurement value with that of a control group treated with 0.4% DMSO and with no addition of the synthetic compound.

Experimental Example 5: In vivo assay of beta-secretase activity

The inhibition degree of intracellular beta-secretase activity by synthetic compounds can be determined by using a cell line producing a beta-amyloid peptide from an amyloid precursor protein (APP).

Step A: Establishment of permanent cell line expressing amyloid precursor protein (APP)

A mutant form of APP gene (APP75 INFEV), expression of which is under the control of Tet-response element (TRE), was cloned into a PBI-L vector (ClonTech) as a mammalian expression vector that expresses a luciferase gene under the control of Tet-response element (TRE). Neuro-2a cells (ATCC Accession No. CCL- 131) were cultured in a Dulbecco's minimum essential medium (DMEM, GIBCO-BRL) supplemented with 10% FBS, and then transferred to a 6-well culture plate. When the cells reached confluence, they were transfected with the pBI-L APP751 NFEV expression vector using Lipofectamine 2000 (Life Technologies). Individual clones were isolated and then cultured again on a 6-well culture plate. Following selection of clones, 100 clones showing acceptable growth rates were cultured on a 96-well culture plate for 1 day. The culture medium was replaced with a medium containing 1 μg/mL of doxycycline, followed by culture for 24 hours. 50 ≠ of Bright-Glo luciferase reagent (Promega) was added to each well which was then left at room temperature for 15 min. Then, luminescence of each well was measured using a luminometer (Victor). Following selection of clones, 4 clones showing the highest expression level of luciferase were sub-cultured on a 24-well culture plate for one day at an equal cell density (3 X 10⁵ cells/mL/24-well). The culture medium was replaced with Opti-MEM (GIBCO-BRL) containing 1 μg/mL of doxycycline, followed by culture for 24 hours. An amount of the beta- amyloid peptide secreted into the medium was measured using an ELISA method using antibodies specific for the beta-amyloid peptide. As a result, clone #79 showing the highest growth rate and beta-amyloid peptide expression level was selected.

Step B: ELISA assay of water-soluble beta-amyloid precursor protein (sAPP)

3 X 10⁵ cells/well of Neuro-2a APP751 NFEV # 79 with permanent expression of a mutant form of APP were seeded onto a 24-well culture plate. The cells were cultured in a 6% CO₂ incubator at 37 ^°C for 24 hours. When the cells reached confluence, the culture medium was replaced with Opti-MEM (GIBCO-BRL) containing 300 μi of 1 μg/mL of doxycycline and the beta-secretase inhibitor (compounds of Examples) diluted to concentrations of individual steps, followed by culture for 24 hours. An expression level of water-soluble beta-amyloid precursor secreted into the medium was measured using a human sAPP assay kit (TBL) according to the manufacturer's instructions. This will be briefly illustrated in the following.

100 μi of the water-soluble beta-amyloid precursor or cell culture diluted to different concentrations was added to an antibody-coated plate, followed by reaction at room temperature for 4 hours (or one or more days at 4 "C). The culture plate was washed seven times with a wash solution (PBS + 0.05% Tween 20), and 100 μi of HRP-conjugated antibodies diluted 30 times with an antibody dilution solution (PBS + 1% BSA + 0.05% Tween 20) was added thereto, followed by reaction at room temperature for 30 min. Then, the plate was washed nine times with a wash solution, to which 100 μi of a TMB solution was added, followed by reaction at room temperature for 30 min. Then, 100 μi of a IN sulfuric acid (H₂SO₄) solution was added thereto, followed by reaction at room temperature for 30 min. The optical density (absorbance) was measured at 450 nm using a microplate reader (SpectraMax 340, Molecular Device). The concentration of a synthetic compound inhibiting 50% of intracellular beta-secretase activity, i.e., IC₅₀ was determined by comparing the measurement value with that of a control group treated with 1 % DMSO and with no addition of the synthetic compound.

Experimental Example 6: Beta-amyloid assay

Quantitative analysis of beta-amyloid was carried out by ELISA using two types of antibodies (Human beta amyloid 1-40 colorimetric immunoassay kit, Biosource, California, USA). Two antibodies used in ELISA were an antibody that specifically recognizes the N-terminus of beta- secretase-cleaved beta amyloid and an antibody that binds to the C-terminus thereof. Two different antibodies and the beta amyloid protein were reacted at room temperature for 3 hours (or one or more days at 4°C). The plate was washed four times with a wash solution, and reacted with HRP- conjugated anti-rabbit IgG antibodies for 30 min. The plate was washed four times with a wash solution, and tetramethylbenzidine as the HRP substrate was added, followed by reaction at room temperature for 30 min. The optical density (absorbance) was measured at 450 nm using a microplate reader (SpectraMax 340, Molecular Device). Reduction of beta amyloid was determined by comparing the measurement value with that of a control group.

Assay of sAPPbeta (secreted amyloid precursor protein beta) was also carried out analogously to Hie procedure described in ELISA of beta-amyloid.

Experimental Example 7: Animal tests (in vivo assay)

In order to investigate whether the activity of beta-secretase was inhibited, the inhibition degree of production of beta amyloid which is a beta-secretase cleavage product was examined in animals. The animals used in the experiments were transgenic mice harboring both a Swedish mutant form of beta-APP (chimeric mouse/human amyloid precursor protein 695swe) and a mutant form of presenilin 1 (presenilin l-dE9) (Jankosky JL et al., Biomolecular engineering, 17(6), 157- 165, 2001). The beta-secretase inhibitor was administered at a dose that is expected to result in a decrease of the amyloid protein via an intraperitoneal or subcutaneous route but does not cause toxicity. Following administration of the drug, the animals were anesthetized at a given time point, and blood and cerebral tissues were isolated. The blood was collected in a heparinized tube by cardiac puncture and centriiuged at 13,000 rpm for 10 min (Eppendorf) to separate the plasma The plasma was stored together with the excised brain tissues (cerebral cortex and hippocampus) at 80 ^°C until use. For analysis, the plasma was diluted 5 times, and inhibitory effects on amyloid formation were examined by ELISA as described above. A 4-fold volume of PBS was added to the excised brain tissues which were then homogenized with a sonicator. The homogenate was reacted with guanidine at room temperature for 4 hours. For extraction of the amyloid protein with guanidine- buffer, guanidine was adjusted to a final concentration of 5M using 8.2 M guanidine/82 mM Tris HCl (pH 8.0). The beta amyloid protein was extracted and diluted 1:500 in BSAT-DPBS (Dulbecco's phosphate buffered saline with 5% BSA and 0.03% Tween-20), followed by analysis.

Experimental Example 8: Dosing and formulation

A drug compound was dissolved in 10% hydroxypropyl-beta-cyclodextrin (HPCD) and was then administered to a subject, typically at a dose of 15 to 100 mg/kg/day, once or three to five times.

The compounds of the present invention had a Ki value of about 10 to 100000 nM, preferably 10 to about 1000 nM, and more preferably 10 to 100 nM. In addition, IC₅₀ for SEAP was in the range of 100 to 100000 nM, and preferably 100 to 10000 nM. The compound of Example 15 had a Ki value of 24 nM, IC₅₀ of 1500 nM for SEAP, and 83-fold higher selectivity for cat. D.

INDUSTRIAL APPLICABILITY As apparent from the foregoing, compounds of Formula (I) in accordance with the present invention exhibit excellent inhibitory effects on human beta-secretase. Therefore, these compounds can be used as drugs for improvement of cognitive functions or for prevention and treatment of neurodegenerative diseases such as Alzheimer's disease.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

WHAT IS CLAIMED IS

1. A compound represented by Formula (T):

wherein ris l to 3;

R¹ is selected from the group consisting of hydrogen, alkyl, alkene, and -(CH₂)p-A-R⁷ wherein p is 0 to 2, A is aryl or heteroaryl, and R⁷ is selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, -NHC(O)-alkyl and aryloxy;

R² is selected from the group consisting of hydrogen, alkyl, alkoxy and -(CR⁸R⁹^-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently selected from the group consisting of hydrogen, alkyl and alkoxy, and R¹⁰ is selected from the group consisting of cycloalkyl, aryl, heterocycle and heteroaryl; and B is any one of substituents represented by Formulae (i) to (ϋi):

(iii) wherein m and n are each independently 0 or 1 , X is C or N,

R³ and R⁴ are each independently selected from the group consisting of hydrogen, alkyl and -(OXi-(CH₂)P-R¹ ' wherein p is O to 2, q is O or 1 , and R¹ ' is aryl or heteroaryl,

R⁵ is selected from the group consisting of hydrogen, alkyl, alkoxy and -(O)q-(CH₂)p-Rⁿ wherein p is 0 to 2, q is 0 or 1 , and R¹¹ is aryl or heteroaryl, and R⁶ is selected from the group consisting of hydrogen, alkyl, alkoxy, and -(CH₂)P-A' -R¹² wherein p is 0 to 2, A' is phenyl, heteroaryl, or a bicyclic compound containing phenyl, R¹² is selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, NHR¹³ and NR¹³R¹⁴ wherein R¹³and R¹⁴ are each independently hydrogen or alkyl, or R¹³and R¹⁴ are cyclized to formalkylene, wherein the alkyl, alkoxy, aryl, cycloalkyl, heterocycle, and heteroaryl may be unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, amino, alkylamino, dialkylamino, alkylacylamino, Cj-C₄ alkyl, hydroxy, C₁-C₄ alkyl alkoxy, aryl alkoxy and oxo, and the heteroaiyl and heterocycle are each independently a 4 to 8-membered ring containing 1 to 3 hetero atoms selected from the group consisting of O, N and S, and having 0 to 3 double bonds; or a pharmaceutically acceptable salt or isomer thereof.

2. The compound according to claim 1 , wherein ris 1 to 3;

R¹ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkene and - (CH₂)P-A-R⁷ wherein p is 0 to 2, A is 6 to 10-membered aryl or 5 or 6-membered heteroaiyl, and R⁷ is selected from the group consisting of hydrogen, halogen, Ci-C₃ alkyl, Ci-C₃ alkoxy, NHC(O)- (C₁-C₆ alkyl) and 5 or 6-membered aryloxy;

R² is selected from the group consisting Of Ci-C₆ alkyl, Q-C_όalkoxy and -(CR⁸R^p-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently hydrogen or C₁-C₄ alkyl, and R¹⁰ is selected from the group consisting of 5 or 6-membered cycloalkyl, 6 to 10-membered aryl, and 5 or 6- membered heteroaryl; and B is any one of substituents represented by Formulae (i) to (iii):

(Hi) wherein m and n are each independently 0 or 1 , X is C or N, R³ and R⁴ are each independently selected from the group consisting of hydrogen, Ci-C₆ alkyl and -(0^-(CH₂)P-R¹ ' wherein p is 0 to 2, q is 0 or 1, and R¹¹ is phenyl or 5 or 6-membered heteroaryl,

R⁵ is selected from the group consisting of hydrogen, Cj-C₆ alkyl, Ci-C₆alkoxy and -(O)q- (CH₂)P-R¹ ' wherein p is 0 to 2, q is 0 or 1 , and R^π is phenyl or 5 or 6-membered heteroaryl, and R⁶ is selected from the group consisting of hydrogen, Ci-C₆ alkyl and -(CH₂)p-A'-R¹² wherein p is 0 to 2, A' is phenyl, 5 or 6-membered heteroaryl, or a bicyclic compound containing phenyl, and R¹² is selected from the group consisting of hydrogen, halogen, hydroxy, Ci-C₆ alkyl, Ci-C₆ alkoxy, NHR¹³ and NR¹³R¹⁴ wherein R^I3and R¹⁴ are each independently hydrogen or Ci-C₄ alkyl, or R^I3and R¹⁴ are cyclized to form alkylene.

3. The compound according to claim 2, wherein R¹ is selected from the group consisting of hydrogen, C₁-C₄ alkyl, C₂-C₄ alkene and -(CH₂)p-A-R⁷ wherein p is 0 to 2, A is phenyl or pyridine, and R⁷ is selected from the group consisting of hydrogen, halogen, trifluoromethyl, trifluoromethoxy, acerylamino and 5 or 6-membered aryloxy.

4. The compound according to claim 3, wherein R¹ is selected from the group consisting of hydrogen, methyl, hydroxyethyl, vinyl, phenyl, 4-chlorophenyl, 4-trifluoromethyloxyphenyl, 3- trifluoromethylphenyl, 4-trifluoromethylphenyl, 2-trifluoromethylphenyl, benzyl, pyridin-2-yl, 3- phenoxyphenyl and 2-acetylaminophenyl.

5. The compound according to claim 2, wherein R² is C₁-C₆ alkoxy or -(CR^^p-R¹⁰ wherein p is 0 to 2, R⁸ and R⁹ are each independently hydrogen or Ci-C₄ alkyl, and R¹⁰ is selected from the group consisting of 5 or 6-membered cycloalkyl, 6 to 10-membered aryl and 5 or 6- membered heteroaryl.

6. The compound according to claim 5, wherein R² is selected from the group consisting of cyclohexyl, phenyl, 3,5-difluorophenyl and naphthyl.

7. The compound according to claim 2, wherein R³ is selected from the group consisting of hydrogen, C₁-C₆ alkyl and [-(COq-(CH₂)P-R¹¹] wherein R¹¹ is phenyl or 5 or 6-membered heteroaryl.

8. The compound according to claim 7, wherein R³ is hydrogen, benzyloxy or benzyl.

9. The compound according to claim 2, wherein R⁴ is selected from the group consisting of hydrogen, Ci-C₄ alkyl and 0-(CH₂)P-R¹ ' wherein R¹ ' is phenyl or 5 or 6-membered heteroaryl.

10. The compound according to claim 9, wherein R⁴ is selected from the group consisting of hydrogen, methyl, isobutyl and benzyloxy.

11. The compound according to claim 2, wherein R⁵ is selected from the group consisting of hydrogen, C₁-C₆ alkyl and -O-R¹¹ wherein R¹ ' may be phenyl or 5 or 6-membered heteroaryl.

12. The compound according to claim 11 , wherein R⁵ is hydrogen or phenoxy .

13. The compound according to claim 2, wherein R⁶ is selected from the group consisting of hydrogen, C₁-C₄ alkyl and -A'-R¹² wherein A' is phenyl, 5 or 6-membered heteroaryl, or a bicyclic compound containing phenyl, and R¹² is selected from the group consisting of hydrogen, halogen, hydroxy, C₁-C₆ alkyl, Ci-C₆ alkoxy, NHR¹³ and NR¹³R¹⁴ wherein R¹³and R¹⁴ are each independently hydrogen or Ci-C₄ alkyl, or R^l3and R¹⁴ are cyclized to form alkylene.

14. The compound according to claim 13, wherein R is selected from the group consisting of hydrogen, methyl, 3-dimethylaminophenyl, 3-(t-butyl)-phenyl, (3-isopropyl-[l,2,4]-oxadiazol-5- yl), 3-(pyrrolidin-l-yl)phenyl, and (l-hydroxy-indan-5-yl).

15. The compound according to claim 1, wherein the compound of Formula (I) is any one selected from:

N-[(2S,3R)4-[(3-dimethylaminophenyl)methylamino]-3-hydroxy-l-phenyl butan-2-yl]-5-oxo- 1 -phenyMH-pyrazole-S-carboxamide; N-[(2S,3R)-l-(3,5-dMuorophenylH-[(3-dime%lammophenyl)me%lamino]-3- hydroxybutan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide; l-(4-chlorophenyl)-N-[(2S,3R-l-(3,5-difluorophenyl)-4-[(3- dimethylaminophenyl)me1hylamino]-3-hydroxybu1an-2-yl]-5-oxo-4H-pyrazole-3-carto^ N-[(lR,2S)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2S)-pyrrolidin-2-yl]propan-2-yl]-5- oxo-l-phenyl4H-pyraz»le-3-carboxarnide;

N-[(2S,3R)4-[(3-dimelhylaminophenyl)methylamino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo- 1 -[4-(trifluoromethoxy)phenyl]-4H-pyrazole-3-carboxamide; N-[(2S3R)-4-[(3-dimethylaminophenyl)me%lamko]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo-l-[3-(tiifluoiOmethyl)phenyl]4H-pyrazDle-3-carboxamide;

N-[(lS^R)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2S)-pyrroHdin-2-yl]propan-2-yl]-5- oxo-l-phenyl4H-pyrazole-3-carboxamide;

N-[(2S,3R)-l-(3,5-difluorophenyl)4-[(3-dimethylaminophenyl)methylamino]-3- hydroxybutan-2-yl]-5-oxo-l-[3-(1rifluoromethyl)phenyl]4H-pyrazole-3-carboxamide;

N-[(lR^R)-l-[(3R,8aS)-l,2,3,4,6,7,8,8a-octahydropyrrolo[l,2-d]pyrazin-3-yl]-3-(3,5- difluorophenyl)-l-hydroxypropan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(lS,2S)-l-[(3R,8aS)-l,2,3,4,6,7,8,8a-octahydropyiτolo[l,2-d]pyrazin-3-yl]-3-(3,5- difluorophenyl)- 1 -hydroxypropan-2-yl]-5-oxo- 1 -phenyl^H-pyrazole-S-carboxamide; N-[(2S,3R)4-[(3-dmethylammophe^^^ oxo- 1 -[2-(trifluorømethyl)phenyl]-4H-pyrazole-3-carboxamide; l-benzyl-N-[(2S,3R)4-[(3-dimethylaminophenyl)methylamino]-3-hydroxy-l- phenylbutan-2-yl]-5-oxo4H-pyrazole-3-carboxamide;

N-[(lR,2S)-3-(3,5-Muorophenyl)-l-hydroxy-l-[(2R,4R)4-ρhenylmethoxypyrrolidin-2- yl]propan-2-yl]-5-oxo-l -phenyMH-pyrazole-S-carboxamide;

N-[(lR^S)-3-(3,5-dffluorophenyl)-l-hydroxy-l-[(2R)-pyrrolidin-2-yl]prøpan-2-yl]-5- oxo-l-phenyl4H-pyrazole-3-carboxamide;

N-[(2S,3R)4-[(3-dime%laminophenyl)me%lainino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo-1 -pyridin^-yMH-pyrazole-S-carboxamide; N-[(2S,3R)-4-[(3-dime%laminophenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]-5- oxo-l-(3-phenoxyphenyl)-4H-pyrazole-3-carboxamide;

N-[(lS,2S)-l-[(3S,8aS)-l-methyl4-oxo-2,3,6,7,8,8a-hexahydro-lH-pyrrolo[l,2- d]pyrazm-3-yl]-3-(3,5-dMuorophenyl)-l-hydroxypropan-2-yl]-5-oxo-l-phenyl-4H-pyra^ carboxamide; l-benzyl-N-[(2S,3R)4-[(3-t-bιitylphenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]- 5-oxo4H-pyrazole-3-carboxamide;

N-[(lS,2S)-3-(3,5-difluoiOphenyl)-l-hydroxy-l-[(3R,7S)-7-phenoxy-l,2,3,4,6,7,8,8a- octahydropyiTolo[12-d]pyra-dn-3-yl]propan-2-yl]-5-oxo-l-phenyl4H-pyrazole-3-carboxamide; N-[(l S,2R)- 1 -hydroxy-S-naphthalen^-yl- 1 -[(2R)-pyrrolidin-2-yl]propan-2-yl]-5-oxo- 1 - phenyMH-pyrazole-S-carboxamide;

N-[(lR,2S)-l-hydroxy-3-naphthden-2-yl-l-[(2R)-pyrrolidin-2-yl]propan-2-yl]-5-oxo-l- phenyl-4H-pyrazole-3-carboxamide;

N-[(2S3R)-4-[(3-dime1hylaminophenyl)me1hylamino]-3-hydroxy-l-phenylbutan-2-yl]-l- methyl-5-oxo-4H-pyrazole-3-carboxamide;

N-[(2S,3RH-[(3-t-butylphenyl)me%lamino]-l-(3,5-difluoiOphenyl)-3-hydroxybutan-2- yl]-5-oxo-l-[3-(1rifluoromethyl)phenyl]4H-pyrazole-3-carboxarnide; l-benzyl-N-[(2S,3R)-4-[(3-t-butylphenyl)me%lamino]-l-(3,5-difluoroρhenyl)-3- hydroxybutan-2-yl]-5-oxo-4H-pyrazole-3-carboxamide; N-[(2S,3R)4-[(3-dimethylamhophenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]-l- ethenyl-5-oxo-4-methyl-pyrazDle-3-carboxamide;

N-[(2S,3R)4-[(3-dime%laminophenyl)me%lamino]-3-hydroxy-l-phenylbutan-2-yl]-l- (2-hydroxye1hyl)-5-oxo-4-methyl-pyraziole-3-carboxamide; N-KlR^SyS-cyclohexyl-l-hydroxy-l-P^-pyrroHdin^-ylJpropan^-yy-S-oxo-l- phenyMH-pyrazole-3-carboxamide;

N-[(2S,3R)-3-hydrøxy- 1 -phenyl-4-[(3-propan-2-yl- 1 ,2,4-oxadiazol-5- yl)methylamino]butan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide; N-[(2S,3R)-3-hydroxy-l-phenyl4-[(3-pyrrolidin-l-ylphenyl)methylamino]butan-2-yl]-5- oxo-l-phenyl-4H-pyrazole-3-carboxamide; l-(2-acetylaminophenyl)-N-[(2S3RH-[(3-dimethylaminophenyl)melhylatnino]-3- hydroxy- 1 -phenylbutan^-ylJ-S-oxopyrrolidine-S-carboxamide;

N-[(2S3R)-3-hydroxy4-[(l-hydroxy-2,3-dihydro-lH-inden-5-yl)methylamino]-l- phenylbutan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxaniide;

N-[(2S,3R)-l-(3,5-difluorophenyl)-3-hydroxy-4-[(3-pyrroUdin-l- ylphenyl)methylamino]butan-2-yl]-5-oxo- 1 -phenyMH-pyrazole-S-carboxamide;

N-[(lR,2R)-3-(3,5-difluoroρhenyl)-l-hyckoxy-l-[(2R,5S)-5-(2-me%lpropyl)piperazin-2- yl]propan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide; N-[(l S,2S)-3-(3,5-difluoiOphenyl)-l -hydroxy- 1 -[(2R,5S)-5-(2-methylpropyl)piperazin-2- yl]propan-2-yl]-5-oxo-l-phenyl-4H-pyrazole-3-carboxamide;

N-[(lR^S)-3-(3,5-difluorophenyl)-l-hydroxy-l-[(2R)-pyrrolidin-2-yl]propan-2-yl]-5- oxo-l-phenyl4H-pyrazole-3-carboxamide;

N-[(lS,2S)-l-[(2S)4-benzyl-3-oxopiperazin-2-yl]-l-hydroxy-3-phenylpropan-2-yl]-5- oxo- 1 -phenyMH-pyrazole-S-carboxamide; and

N-[(lR^S)-3-(3,5-dmuorophenyl)-l-hydroxy-l-[(2R,4RH-phenylmethoxypyrrolidin-2- yl]prøpan-2-yl]-5-oxo-l-(3-trifluoromethyl)phenyMH-pyrazole-3-carboxamide.

16. A pharmaceutical composition for inhibiting beta-secretase, comprising a therapeutically effective amount of a compound of Formula (T) of claim 1 or a pharmaceutically acceptable salt or isomer thereof as an active ingredient and a pharmaceutically acceptable carrier.

17. The composition according to claim 16, wherein the composition is used for the improvement of cognitive functions or treatment or prevention of neurodegenerative diseases.

18. The composition according to claim 17, wherein the composition is used for the treatment of Alzheimer's disease.