KR100883132B1 - A cleavage agent selectively acting on soluble assembly of amyloidogenic peptide or protein - Google Patents

Abstract

The present invention relates to synthetic cleavage agents that selectively recognize and cleave soluble associations of amyloid-forming peptides or proteins and methods of selectively cleaving soluble associations of amyloid-forming peptides or proteins using these synthetic cleavage agents.

Amyloid, peptide, protein, association, cleavage

Description

A cleavage agent selectively acting on soluble assembly of amyloidogenic peptide or protein

The present invention relates to synthetic molecules that selectively recognize and cleave soluble assemblies of amyloidogenic peptides or proteins, and methods of selectively cleaving soluble aggregates of amyloidogenic peptides or proteins using the same. . By cleaving the soluble association of amyloid-forming peptides or proteins, the biological activity of the amyloid-forming peptides or proteins can be inhibited.

A series of diseases that accumulate insoluble protein aggregates called amyloids and cause deleterious effects are called amyloidoses (Bittan, G .; Fradinger, EA; Spring, SM; Teplow, DB Amyloid 2005, 12, 88 ). . There are various types of amyloid-forming peptides or proteins that cause amyloidosis (Kelly, JF Curr . Opin . Struct . Biol . 1996 , 6 , 11). Amyloids formed by a variety of amyloid-forming peptides or proteins with unique structures and functions have a common feature of having a fibrous form and a cross-beta-fold structure.

Amyloid forming peptides or proteins form soluble aggregates, including various oligomers and protofibrils, which are then converted into insoluble fibrils. The experimental evidence accumulated so far indicates that soluble oligomers of amyloid-forming peptides or proteins are the main cause of amyloidosis (Bittan, G .; Fradinger, EA; Spring, SM; Teplow, DB Amyloid 2005, 12 , 88). . Soluble oligomer forms of amyloid-forming peptides or proteins, such as amyloid beta (Aβ peptide, amylin, α-synuclein, prion, polyglutamine, and poyglutamine), respectively, are known as Alzheimer's disease ), Type II diabetes, Parkinson's disease, spongiform encepahlopathies, and Huntington's disease are identified as the main toxic substances (Demuro, AG; Mina, E .; Kayed, R .; Milton, S .; Parker, I .; Glabe, CG J. Biol . Chem . 2005, 280 , 17294).

Herein, the chemical and biological properties of soluble oligomers of amyloid-forming peptides or proteins will be described using Alzheimer's disease as an example. Alzheimer's disease is a major cause of senile dementia. Alzheimer's disease is characterized by the loss of nerve cells, plaques composed of Aβ peptides, and multiple lesions of nerve fibers (neurofibrillary tangles) found in the brain (Selkoe, DJ Physiol . Rev. 2001 , 81 , 741 ). Aβ peptides consist mainly of oligopeptides consisting of 40 or 42 amino acids formed by cleaving β-amyloid precursor proteins by β- and γ-secretase (Sambamurti, K; Greig, NH; Lahiri). , DK Neuromol. Med. 2002, 1, 1). Aβ consisting of 40 or 42 amino acids is often abbreviated Aβ ₄₀ or Aβ ₄₂ . The amino acid sequence of Aβ ₄₂ is shown in FIG. 1, where two C-terminal amino acids are removed to form the amino acid sequence of Aβ ₄₀ . The main component of amyloid deposits is Aβ ₄₂ .

The first proposed amyloid cascade hypothesis assumes that as the production of Aβ ₄₂ increases, fibrous aggregates form, moreover, spot-like deposits that cause cognitive decline and neuropathy in Alzheimer's disease. (Hardy, JA; Higgins, GA Science 1992 , 256 , 184). A poor correlation between dementia and amyloid spots has been reported in patients with Alzheimer's disease (Katzman, R .; Terry, R .; De Teresa, R .; Brown, T .; Davies, P .; Fuld). , P .; Renbing, X .; Peck, A. Ann. Neurol . 1988 , 23 , 138: Naslund, J .; Haroutunian, V .; Mohs, R .; Davis, K L .; Davies, P .; Greengard , P .; Buxbaum JD J. Am. Med. Ass. 2000, 283, 1571), though this has been considerable support for this hypothesis until recently. However, soluble Aβ ₄₂ was found in the brain of Alzheimer's disease patients (Kuo, YM; Emmerling, MR; Vigo-Pelfrey, C .; Kasunic, TC; Kirkpatrick, JB; Murdoch, GH; Ball, MJ; Roher, AE J. Biol . Chem . 1996 , 271 , 4077), as reported that symptoms of Alzheimer's disease are more related to the amount of soluble Aβ ₄₂ than the amount of amyloid plaques (Lue, LF; Kuo, YM; Roher, AE; Brachova, L .; Shen, Y .; Sue, L .; Beach, T .; Kurth, JH; Rydel, RE; Rogers, J. Am. J. Pathol . 1999 , 155 , 853: McLean, CA; Cherny, RA; Fraser, FW; Fuller, SJ; Smith, MJ; Beyreuther, K .; Bush, AI; Masters, CL Ann. Neurol . 1999 , 46 , 860), the amyloid stage hypothesis has been modified. According to the modified hypothesis (Hardy, J .; Selkoe, DJ Science 2002 , 297 , 353), the development of synaptic dysfunction in the brain of Alzheimer's disease patients is not due to insoluble amyloid fibril or Aβ monomers. Soluble oligomers of Aβ. Additional evidence has recently been reported to support the fact that soluble oligomers such as dodecamer Aβ ₄₂ play a role of neurotoxic intermediates in the symptoms of Alzheimer's disease (Tanzi, RE Nature Neurosci . 2005 , 8 977: Snyder, EM; Nong, Y .; Almeida, CG; Paul, P .; Moran, T .; Choi, EY; Nairn, AC; Salter, MW; Lombroso, PJ; Gouras, GK; Greengard, P. Nature Neurosci . 2005, 8 , 1051: Barghorn, S .; Nimmrich, V .; Striebinger, A .; Krantz, C .; Keller, P .; Janson, B .; Bahr, M .; Schmidt, M .; Bitner Harlan, J. Barlow, E. Ebert, R. Hillen . H. J. Neurochem . 2005 , 95, 834: Lesne S. Koh, MT Kotilinek, L. Kayed, R .; Glabe, CG; Yang, A .; Gallagher, M .; Ashe, KH Nature 2006 , 440 , 352).

In the process of Aβ ₄₂ soluble associations such as various oligomers and protofibrils are formed reversibly or partially irreversibly, and then insoluble fibrils are irreversibly formed (Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB; Teplow, DB Proc . Natl. Acad . Sci . USA 2003 , 100 , 330: Moss, MA; Nichols, MR; Reed, DK; Hoh, JH; Rosenberry, TL Mol . Pharmacol. 2003, 64, 1160: Lesne S .; Koh, MT; Kotilinek, L .; Kayed, R .; Glabe, CG; Yang, A .; Gallagher, M .; Ashe, KH Nature 2006 , 440 , 352). The various oligomers of Aβ ₄₂ have different structures (Urbanic, B .; Cruz, L .; Yun, S .; Buldyrev, SV; Bitan, G .; Teplow, DB; Stanley . HE Proc . Natl . Acad . Sci . USA 2004 , 101 , 17345).

To develop therapies to relieve the neurotoxic Aβ ₄₂ causes may be seeking to facilitate the removal of the oligomer of Aβ ₄₂ in the brain. One way to achieve this is to inhibit the activity of one of β- and γ-secretases to inhibit the production of Aβ from precursor proteins (Hardy, J .; Selkoe, DJ Science 2002 , 297 , 353). It is also possible to suppress the oligomerization of Aβ by using an immunological agent of Aβ.

(Schenk, D .; Barbour, R .; Dunn, W .; Gordon, G .; Grajeda, H .; Guido, T .; Hu, K .; Huang, J .; Johnson-Wood, K .; Khan , K .; Kholodenko, D .; Lee, M .; Liao, Z .; Lieberburg, I .; Motter, R .; Mutter, L .; Soriano, F .; Shopp, G .; Vasquez, N .; Vandevert , C .; Walker, S .; Wogulis, M .; Yednock, T .; Games, D .; Seubert, P. Nature 1999 , 400 , 173: DeMattos, RB; Bales, KR; Cummins, DJ; Dodart, J.-C .; Paul, SM; Holtzman. DM Proc . Natl . Acad . USA 2001 , 98 , 8850). Small molecule with high affinity for dissolved Aβ (Cohen, T .; Frydman-Marom, A .; Rechter, M .; Gazit, E. Biochemistry 2006 , 45, 4727), inhibits oligomerization of Aβ You may. Endorulin conversion known to be responsible for the degradation of Aβ

Enzymes (endothelin converting enzyme), insulin degrading enzymes and neprilysin

enhances the activity of neprilysin, which reduces the amount of Aβ ₄₂ oligomers in the brain.

(Choi, DS; Wang, D .; Yu, GQ; Zhu, G .; Kharazia, VN; Paredes, JP; Chang, WS; Deitchman, JK; Mucke, L .; Messing, RO Proc . Natl . Acad . Sci . 2006 , 103 , 8215).

As discussed for the soluble oligomer of Aβ ₄₂ associated with Alzheimer's disease, various strategies have been adopted as a treatment for amyloidosis (Dobson, CM Science 2004 , 304 , 1259). Among these strategies are methods to stabilize the original state of amyloid-forming peptides or proteins, to inhibit the activity of enzymes that produce amyloid-forming peptides or proteins from precursor proteins, and to alter the synthesis of amyloid-forming peptides or proteins or their precursor proteins. And a method for promoting the removal of amyloid-forming peptides or proteins, a method for inhibiting the formation of fibrous aggregates, and a method for preventing the accumulation of precursors in the fibrous aggregates.

As a new method of reducing the amount of soluble oligomers of amyloid-forming peptides or proteins, the inventors have devised a way to cleave soluble assemblies using synthetic molecules. Synthetic molecules that selectively cleave soluble aggregates of amyloid-forming peptides or proteins (hereinafter abbreviated as "cutting agents") can be regarded as artificial enzymes capable of selectively removing soluble aggregates of amyloid-forming peptides or proteins. . As a result of continuous research to devise a cleavage agent having such characteristics, a cleavage agent was devised by linking a recognition site that selectively recognizes an soluble assembly of an amyloid-forming peptide or protein and a reaction site that cleaves a peptide bond. The present invention has been completed by confirming that the use of a cleavage agent can effectively achieve the original purpose of reducing the amount of soluble oligomers of amyloid-forming peptides or proteins.

Accordingly, the present invention aims to provide a cleavage agent that removes soluble aggregates of amyloid-forming peptides or proteins.

The present invention also aims to provide a pharmaceutical composition for preventing or treating amyloidosis comprising the cleavage agent and a pharmaceutically acceptable carrier.

The present invention relates to a cleavage agent of Formula 1 that selectively cleaves soluble associations of amyloid-forming peptides or proteins:

[Formula 1]

Where

R is A, A- (Y) _o- (CH ₂ ) _p- (Y) _o -A, A- (CH = CH) -A, A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A and A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (O) _o- (CH ₂ ) _p- (Y) _o -A

A are each independently a C _{6 - 14} aryl group, or an oxygen, sulfur, and a heteroaryl group denotes a 5- to 14-membered, containing one or more hetero atoms selected from the group consisting of nitrogen,

Here, the aryl or heteroaryl are each C _{1 - 15} alkyl, hydroxy, C _{1 - 15} alkoxy, C _{1 - 15} alkyl-carbonyl-oxy, C _{1 - 15} alkyl sulfonyloxy, amino, mono- or di-C _{1 - 15} alkylamino, C _{1 - 15} alkylcarbonyl, amino, C _{1 - 15} alkyl sulfonylamino, C _{3 - 15} cycloalkyl-amino, formyl, C _{1 - 15} alkylcarbonyl, carboxy, C _{1 - 15} alkyloxy carbonyl carbonyl, carbamoyl, mono- or di-C _{1 - 15} alkyl, carbamoyl, C _{1 -15-alkyl} sulfanyl-carbonyl, C _{1 - 15} alkyl sulfanyl thiocarbonyl, C _{1 - 15} alkoxy-carbonyl-oxy, carbamoyl oxy, mono- or di-C _{1 - 15} alkyl, carbamoyl-oxy, C _{1 - 15} alkyl sulfanyl-carbonyl-oxy, C _{1 - 15} alkoxy-carbonyl-amino, ureido, mono or di or tri C _{1 - 15} alkyl ureido, C _{1 - 15} alkyl sulfamic nilka amino, bots, captopril, C _{1 - 15} alkyl sulfanyl, C _{1 - 15} alkyl di-sulfanyl, sulfo, C _{1 - 15} alkoxy-sulfonyl, sulfamoyl, mono- or di-C _{1 - 15} alkyl, Sulfamoyl, tri C _{1- 15} alkyl carbonyl and silanol, or from the group consisting of halogen and substituted by one or more substituents independently selected is unsubstituted;

Y represents O or N-Z ', wherein Z' represents hydrogen or C _1-9 alkyl;

L represents a linker;

Z is a catalyst part and represents a metal ion-ligand complex;

n independently represents an integer of 1 to 6;

m and o independently represent 0 or 1;

p represents the integer of 0-5.

The cleavage agent according to the present invention is composed of a target recognition site for recognizing the soluble assembly of the amyloid-forming peptide or protein and a catalytic moiety having the activity of cleaving the peptide bond, thereby selectively recognizing the soluble assembly of the amyloid-forming peptide or protein. And the ability to rapidly cleave peptide bonds. Therefore, in the presence of various kinds of biomolecules, there is an effect of selectively inhibiting the biological activity of the soluble oligomer of the amyloid-forming peptide or protein.

Hereinafter, a cleavage agent of Chemical Formula 1 according to the present invention will be described in detail.

As described in the oligomerization and fibril formation process following the association of Aβ ₄₂ , various soluble oligomers and protofibrils are formed reversibly or partially irreversibly in the process of assembling amyloid-forming peptides or proteins from which fibrils are irreversibly formed. (Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB; Teplow, DB Proc . Natl . Acad . Sci . USA 2003 , 100 , 330). Protofibrils can be considered soluble or insoluble polymers (Moss, MA; Nichols, MR; Reed, DK; Hoh, JH; Rosenberry, TL Mol . Pharmacol. 2003 , 64 , 1160: Hull, R .; Westermark, GT; Westermark, P .; Kahn, S. J. Clin . Endicrinol. Metab . 204 , 89 , 3629) Therefore, eliminating one or more of the various soluble associations of amyloid-forming peptides or proteins may result in the amyloid-forming peptides or proteins. It is possible to reduce the total amount of the soluble association of and to inhibit fibril formation.

One way to devise effective cleavage agents is to mimic the catalysis of enzymes. The enzymatic action is characterized by the formation of a complex with the substrate using the active site of the enzyme, followed by catalytic conversion of the bound substrate. By forming the enzyme-substrate complex, the effective molar concentration of the catalytic functional groups of the enzyme on the substrate is greatly increased and a large rate increase is achieved. Cleavage agent of formula 1 according to the present invention is an artificial enzyme.

The cleavage agent according to the present invention has a target recognizer capable of recognizing a soluble assembly of an amyloid-forming peptide or protein and thus can selectively bind to one or more soluble associations. The target recognizer binds to the soluble assembly and then the catalytic moiety included in the cleavage agent according to the invention cleaves the peptide bond in the soluble assembly. As a result of peptide cleavage, the chemical and biological properties of amyloid-forming peptides or proteins are lost.

The production of various types of soluble associations and insoluble fibrils of amyloid-forming peptides or proteins was proposed for Aβ ₄₂ (Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB). ; Teplow, DB Proc . Natl . Acad . Sci . USA 2003 , 100 , 330). Included in the rectangle in Figure 2 represents a soluble association. Soluble associations include soluble oligomers and soluble protofibrils, which can be considered very large soluble oligomers. The process of converting large assemblies into smaller ones, such as protofibrils or insoluble fibrils, is slower, so the formation of large assemblies can be considered to be a process that is irreversibly or partially irreversible. Some amyloid-forming peptides or proteins have also been reported to have fibril formation as a reversible process and in equilibrium with monomers. (Wetzel, R. Acc . Chem . Res. 2006 , 39 , 671) As shown in FIG. 2, the cleavage of one assembly to reduce the concentration of the other assembly can easily be converted to the cleaved assembly. The concentration is also expected to be effectively reduced.

Cleavage agents according to the present invention can achieve the desired function by combining with one or more of those in the rectangles shown in FIG. 2 and then cleaving the peptide bonds of the amyloid-forming peptide or protein. When the target recognizing portion of the cleaving agent forms a complex with the soluble assembly, the catalytic portion of the cleaving agent may be located in the complex very close to the peptide bond of the amyloid-forming peptide or protein. Then, the amyloid-forming peptide or protein can be effectively cleaved by the attack of the catalyst portion. The reaction between the cleavage agent and the target can be summarized in Scheme 1 below, similar to the Michaelis-Menten equation applied to the enzymatic reaction.

Scheme 1

As is known for the various derivatives of the Co ^III complex, if the cleavage agent catalyzes to hydrolyze the peptide bond, the cleavage agent will be regenerated after the target is cleaved (Jeon, JW; Son, SJ; Yoo, CE; Hong, IS; Song, JB; Suh, J. Org . Lett . 2002 , 4 , 4155: Chae, PS; Kim, M .; Jeung, C .; Lee, SD; Park, H .; Lee, S .; Suh, J. J. Am. Chem . Soc . 2005 , 127 , 2396).

FIG. 3 shows the process of reducing the soluble and insoluble aggregates of amyloid-forming peptides or proteins by the action of a cleavage agent. In Figure 3 (AP) _ass-m represents the association to be cleaved. (AP) _ass-m is reduced as the cutting by the cutting agent in accordance with the present invention the concentration of the (AP) _{m ass-less,} therefore also the concentration of the other aggregate that is easily converted to (AP) _ass-m. However, the amount of protofibrils or fibrils that are not readily converted to (AP) _ass-m is not effectively reduced. However, such a procedure toffee rate at which the formed fibrils or fibril is decreased from the _{(AP) ass-m (AP} ) ass-m whereby a decrease in the concentration.

Target Recognition

Effective cleavage agents should be able to form complexes at very low cleavage concentrations with soluble associations of target amyloid-forming peptides or proteins. Thus, the cleavage agent according to the present invention comprises a target recognizer capable of selectively and strongly binding to the soluble assembly.

Interactions between the aromatic side chains of amyloid-forming peptides or proteins play an important role in association formation (Cohen, T .; Frydman-Marom, A .; Rechter, M .; Gazit, E. Biochemistry 2006 , 45 , 4727), soluble oligomers of other types of amyloid-forming peptides or proteins have also been proposed to have common structural morphological elements (Kayed, R .; Head, E .; Thompson, JL; McIntire, TM; Milton, SC; Cotman, CW; Glabe, CG Science 2003 , 300 , 486).

Therefore, any organic group having a high affinity for the microregions formed by the aromatic chains can be used as a target recognition unit according to the present invention without limitation.

Preferably, the target recognition unit according to the present invention is A, A- (Y) _o- (CH ₂ ) _p- (Y) _o -A, A- (CH = CH) -A, A- (Y) _o- ( CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A and A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (O) _o- (CH ₂ ) _p- (Y) _o -A is selected from the group consisting of.

Here, A is independently C _{6 - 14} aryl, or an oxygen, sulfur, and a 5- to 14-membered heteroaryl, containing one or more hetero atoms selected from the group consisting of nitrogen.

Preferably, A is selected from the group consisting of

Wherein each X is independently selected from C, N, NH, O and S.

More preferably, A is selected from the group consisting of

Wherein X represents NH, O, or S, respectively.

Here, each of A is C _{1 - 15} alkyl, hydroxy, C _{1 - 15} alkoxy, C _{1 - 15} alkyl-oxy-carbonyl, C _{1 -15-alkyl} sulfonyloxy, amino, mono- or di-C _{1 - 15} alkylamino , C _{1 - 15} alkylcarbonyl, amino, C _{1 -15-alkyl-sulfonylamino,} C _{3 - 15} cycloalkyl-amino, formyl, C _{1 - 15} alkylcarbonyl, carboxy, C _{1 - 15} alkyl-oxy-carbonyl, carbazole together, mono- or di-C _{1 - 15} alkyl, carbamoyl, C _{1 - 15} alkyl sulfanyl-carbonyl, C _{1 - 15} alkyl sulfanyl thiocarbonyl, C _{1 - 15} alkoxy-carbonyl-oxy, carbamoyl oxy, mono- or di C _{1 - 15} alkyl, carbamoyl-oxy, C _{1 -15-alkyl} sulfanyl-carbonyl-oxy, C _{1 - 15} alkoxy-carbonyl-amino, ureido, mono or di or tri C _{1 - 15} alkyl ureido, C _{1 - 15} alkyl, sulfanyl-carbonyl-amino, bots, captopril, C _{1 - 15} alkyl sulfanyl, C _{1 - 15} alkyl di-sulfanyl, sulfo, C _{1 - 15} alkoxy-sulfonyl, sulfamoyl, mono- or di-C _{1 - 15} alkyl sulfamoyl, tree C _{1 - 15} Kill sila carbonyl or halogen, and is substituted by one or more substituents independently selected from the group consisting of is unsubstituted.

Preferably, A is C _{1 - 6} alkyl, C _{1 - 6} alkoxy, amino, mono- or di-C _{1 - 12} alkylamino, C _{1 - 6} alkylcarbonyl, amino, C _{1 - 6} alkyl sulfonyl, amino, C _{6 - 15} cycloalkylamino, C _{1 - 6} alkylcarbonyl. Carbamoyl, mono- or di-C _{1 - 6} alkyl carbamoyl, C _{1 - 6} alkoxycarbonyl, amino, ureido, mono or di or tri C _{1- 6} alkyl ureido, C _{1 - 6} alkyl sulfanyl-carbonyl-amino, C _{1 - 6} alkylsulfanyl, C _{1 - 6} alkyl, di-sulfanyl, sulfamoyl, mono- or di-C _{1 - 6} alkyl-sulfamoyl, tri C _{1 - 6} alkyl silanol independently represent at least one group selected from the group consisting of carbonyl and halogen It is substituted or unsubstituted by a substituent.

More preferably, A is C _{1 - 4} alkyl, C _{1 - 4} alkoxy, amino, mono- or di-C _{1 - 8} alkylamino, C _{6 -12} cycloalkylamino, C _{1 - 4} alkylcarbonyl group with a halogen and Unsubstituted or substituted by one or more substituents independently selected from

Wherein Y represents O or N-Z 'and Z' represents C _1-9 alkyl, preferably C _1-4 alkyl.

Here, p independently represents the integer of 0-5, Preferably it represents the integer of 0-2.

Here, o represents 0 or 1 independently.

The cleavage agent according to the present invention comprises 1 to 6, preferably 1 to 4, more preferably 1 or 2, target recognition units.

Catalyst part

Some of the metal ions exhibit catalytic activity in the reaction of hydrolyzing peptide bonds without the aid of organic functional groups or other metal ions (Suh, J. Acc . Chem . Res. 1992 , 25 , 273: Suh, J. Acc . Chem. Res. 2003, 36 , 562). In view of the catalytic activity of the metal ions for the peptide hydrolysis reaction, the metal ions can be utilized as a key element of the catalytic part according to the present invention.

The catalyst portion according to the present invention consists of a metal ion-ligand complex. Due to the above-described target recognition portion, when the cleavage agent according to the present invention is combined with the amyloid-forming peptide or the soluble assembly of the protein, the effective concentration between the catalytic portion and the cleavage site of the target molecule becomes very high, so that the peptide binding of the target molecule is effectively performed. I can cut it.

Many metal ions with the ability to cleave peptides or proteins are known. Preferably, the metal ion used in the catalyst unit according to the present invention is Co ^III , Cu ^I , Cu ^II , Ce ^IV , Ce ^V , Cr ^III , Fe ^II , Fe ^III , Mo ^IV , Ni ^II , Pd ^II , Pt ^II , V ^V and Zr ^IV . More preferably, the metal ion is Co ^III , Cu ^II Or Pd ^II . Most preferably, the metal ion is Co ^III .

The inventors have also discovered that it is important to limit the ligand of the catalytic moiety to specific structures in order to inhibit their biological activity by selectively cleaving soluble oligomers of amyloid-forming peptides or proteins. Ligands used in the catalyst section according to the invention are selected from the group consisting of:

Wherein each nitrogen atom included in the ligand may be replaced by an atom selected from the group consisting of oxygen, sulfur and phosphorus atoms; Ligands are C _{6 -} may be fused with heteroaryl of ₁₄ aryl or 5-to 14-membered.

Preferably, the ligand used in the catalyst portion is selected from the group consisting of:

More preferably, the ligand is a ring of 12 atoms and is selected from the group consisting of:

Here, the ligand is a C _{1 - 15} alkyl, hydroxy, C _{1 - 15} alkoxy, C _{1 - 15} alkyl-carbonyl-oxy, C _{1 - 15} alkyl sulfonyloxy, amino, mono- or di-C _{1 - 15} alkyl, C _{1 - 15} alkylcarbonyl, amino, C _{1 - 15} alkylsulfonyl, amino, formyl, C _{1 - 15} alkylcarbonyl, carboxy, C _{1 - 15} alkyl-oxy-carbonyl, carbamoyl, mono- or di-C _1-15 alkyl carbamoyl, C _{1 - 15} alkyl sulfanyl-carbonyl, C _{1 - 15} alkyl sulfanyl thiocarbonyl, C _{1 - 15} alkoxy-carbonyl-oxy, carbamoyl oxy, mono- or di-C _{1 - 15} alkyl, carbamoyl-oxy, C _{1 - 15} alkyl sulfanyl-carbonyl-oxy, C _{1 -15} alkoxycarbonyl, amino, ureido, mono or di or tri C _{1 - 15} alkyl ureido, C _{1 - 15} alkyl sulfanyl-carbonyl-amino, bots, captopril, C _{1 - 15} alkyl sulfanyl, C _{1 - 15} alkyl di-sulfanyl, sulfo, C _{1 - 15} alkoxy-sulfonyl, sulfamoyl, mono- or di-C _{1 - 15} alkyl sulfamoyl, tri C _1-15 alkyl carbonyl and Silas Sphere with halogen It substituted by one or more substituents independently selected from the group or is unsubstituted.

Preferably, the ligands are C _{1 -} is a substituted or unsubstituted by a _{6-alkyl-carbonyl-oxy} group, and one or more substituents independently selected from consisting of halogen _{- 6} alkyl, C _{1 - 6} alkoxy, C _1.

More preferably, the ligand is a C _{1 -} is a substituted or unsubstituted by a _4-alkoxy groups and one or more substituents independently selected from consisting of halogen _{- 4} alkyl, C _1.

Linker

The cleavage agent according to the present invention may be directly connected to the target recognition unit (R) and the catalyst unit (Z), or by a linker. In the embodiment of combining the target recognizer and the catalyst by the linker, one target recognizer and the catalyst are each connected by the linker, or two or more target recognizers are respectively connected to the catalyst by the separate linker, as shown below. Parallel type, in which two or more target recognition units are connected to the catalyst unit by one linker having a branch structure, two or more target recognition units are connected to each other by one linker, and one recognition unit is connected to the catalyst unit by a separate linker. It is possible, but not limited to, to be connected in series to form a cleavage agent by connecting several targets to the catalyst using these various connection methods simultaneously:

는 링커를 나타내고, R은 표적 인식부를 나타내며, Z는 촉매부를 나타낸다.Where

Represents a linker, R represents a target recognition portion, and Z represents a catalyst portion.

The linker may comprise a main chain and a substituent optionally attached to the main chain that connects directly between the target recognition site and the catalytic site or between two target recognition sites. When the target recognizer binds to the target protein, the catalyst cleaves one or more of the peptide bonds in the target protein. Increasing the effective concentration between the cleavage site and the catalyst site on the protein can enhance the reactivity of the catalyst site. An effective way to control the effective concentration is to control the relative position between the target recognition site and the catalyst site contained in the cleavage agent. . It is the length and shape of the linker that is used to adjust the relative position.

The linker used in the present invention is composed of a skeleton composed of one or more elements independently selected from the group consisting of carbon, nitrogen, oxygen, silicon, sulfur, and phosphorus, which is used to connect the target recognizer and the catalyst or connect the two target recognizers. And the number of atoms constituting the skeleton is 1 to 30, preferably 1 to 20, and more preferably 1 to 15. Atoms included in the backbone of the linker include alkanes, alkenes, alkynes, carbonyls, thiocarbonyls, amines, ethers, silyls, sulfides, disulfides, sulfonyls, sulfinyls, phosphoryls, phosphinyls, amides, imides, esters and It is in the form of a functional group independently selected from the group consisting of thioesters.

Here, the linker is C _{1 - 9} alkyl, hydroxy, C _{1 - 9} alkoxy, C _{1 - 9} alkyl-carbonyl-oxy, C _{1 - 9} alkyl sulfonyloxy, amino, mono- or di-C _{1 - 9} alkyl, C _{1 - 9} alkyl-carbonyl amino, C _{1 - 9} alkyl sulfonyl amino, formyl, C _{1 - 9} alkyl-carbonyl, carboxy, C _{1 - 9} alkyl-oxy-carbonyl, carbamoyl, mono- or di-C _{1 - 9} alkyl carbamoyl, C _{1 - 9} alkyl sulfanyl-carbonyl, C _{1 - 9} alkyl sulfanyl thiocarbonyl, C _{1 - 9} alkoxyl Brassica carbonyl oxy, carbamoyl oxy, mono- or di-C _{1 - 9} alkyl, carbamoyl-oxy, C _{1 - 9} alkyl sulfanyl-carbonyl-oxy, C _{1 - 9} alkoxycarbonyl, amino, ureido, mono or di or tri C _{1 - 9} alkyl ureido, C _{1 - 9} alkyl sulfanyl-carbonyl-amino, bots, captopril, C _{1 - 9} alkyl sulfanyl, C _{1 - 9} alkyl di-sulfanyl, sulfo, C _{1 - 9} alkoxy sulfonyl, sulfamoyl, mono- or di-C _{1 - 9} alkyl sulfamoyl, tri C _{1 - 9} alkyl silanol carbonyl and In a group consisting of halogen Substituted by one or more substituents selected neutral or is unsubstituted.

Preferably, the linker is a C _{1 - 6} alkyl, C _{1 - 6} alkoxy, mono- or di-C _{1 - 6} alkylamino, C _{1 - 6} alkyl-carbonyl amino, C _{1 - 6} alkyl-sulfonylamino, C _{1 - 6} alkyl carbonyl, carbamoyl, mono- or di-C _{1 - 6} alkyl carbamoyl, C _{1 - 6} alkoxycarbonyl, amino, ureido, mono or di or tri C _{1 - 6} alkyl ureido, C _{1 - 6} alkyl sulfanyl carbonyl carbonyl amino, C _{1 -} independently of the _six groups of the alkyl silanol carbonyl and _halogen-6 alkylsulfanyl, C _{1 - 6} alkyl, di-sulfanyl, sulfamoyl, mono- or di-C _{1 - 6} alkyl-sulfamoyl, tri C ₁ Unsubstituted or substituted by one or more substituents selected.

More preferably, the linker is a C _{1 - 4} alkyl, C _{1 - 4} alkoxy, mono- or di-C _{1 - 4} alkylamino, C _1-4 alkylcarbonyl amino, C _{1 - 4} alkyl-sulfonylamino, C _{1 - 4} alkylcarbonyl, carbamoyl, mono- or di-C _{1 - 4} alkyl, carbamoyl, C _{1 - 4} alkoxycarbonyl, amino, ureido, mono or di or tri C _{1 - 4} alkyl ureido, C _{1 - 4} alkyl sulfanyl independently from ₄ alkyl silanol group consisting of carbonyl and _{halogen-carbonyl} amino, C _{1 - 4} alkyl sulfanyl, C _{1 - 4} alkyl, di-sulfanyl, sulfamoyl, mono- or di-C _{1 - 4} alkyl-sulfamoyl, tri C ₁ It is unsubstituted or substituted by one or more substituents selected from.

Those skilled in the art to which the present invention belongs, a combination chemical experiment method for selecting a structure of a linker suitable for controlling effective concentrations between cleavage sites and catalyst sites for various target proteins and synthetic catalysts within the above definitions. Could be devised.

The cleavage agent according to the present invention utilizes the interaction between the aromatic microstructure of the soluble assembly of the amyloid-forming peptide or protein and the aromatic component included in the target recognition portion of the cleavage agent to recognize the target. Therefore, it does not matter how many kinds of compounds are composed of a soluble association having such an aromatic microstructure. That is, soluble assemblies formed by one or more types of amyloid-forming peptides or proteins as well as soluble assemblies formed by two or more types of amyloid-forming peptides or proteins can be targeted as cleavage agents according to the present invention. In addition, when the biomolecules of other types intervene in the process of forming such a soluble assembly, they can be used as targets of the cleavage agent according to the present invention. The cleavage agent according to the present invention may selectively cleave only soluble assemblies of peptides or proteins related to one type of amyloidosis, or cleave soluble assemblies of peptides or proteins related to two or more types of amyloidosis.

Although not limited thereto, the cleavage agent according to the present invention is particularly effective for cleavage of oligomers as follows.

(1) oligomers of Aβ ₄₀ and Aβ ₄₂ associated with Alzheimer's disease

Aβ ₄₀ and Aβ _{42 form} various oligomers with protofibrils and fibrils by self-association, respectively, as shown in FIG. 2. It is known that the association rate of Aβ ₄₀ and Aβ ₄₂ is so high that it starts to oligomerize in minutes when the concentration of Aβ ₄₂ monomer is several μM or more and turns into protofibrils smaller than 0.1 μm in solution or on the solid surface in a few hours. (Kowalewski, T .; Holtzman, DM Proc . Natl . Acad . Sci . USA. 1999 , 96 , 3688: Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB; Teplow, DB Proc . Natl . Acad . Sci. USA 2003 , 100 , 330). Aβ ₄₀ is known to form dimers (dimers), trimers (trimers), tetramers (tetramers), etc. by equilibrium reactions, and Aβ ₄₂ forms pentamers (penters) and hexamers (hexamers). (Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB; Teplow, DB Proc . Natl . Acad . Sci . USA 2003 , 100 , 330-335). Recent literature has reported that Aβ ₄₂ is a mixture of large oligomers and Aβ ₄₀ is in equilibrium with dimers (Hepler, RW; Grimm, KM; Nahas, DD; Breese, R .; Dodson, EC ; Acton, P .; Keller, PM; Yeager, M .; Wang, H .; Shughrue, P .; Kinney, G .; Joyce, JG Biochemistry 2006 , 45 , 15157-15167).

The association of Aβ ₄₀ and Aβ ₄₂ is sensitively affected by the experimental conditions (Hepler, RW; Grimm, KM; Nahas, DD; Breese, R .; Dodson, EC; Acton, P .; Keller, PM; Yeager, M .; Wang, H .; Shughrue, P .; Kinney, G .; Joyce, JG Biochemistry 2006 , 45 , 15157-15167).

It is not known at this stage whether the cleavage agent of the present invention targets and cleaves oligomers of various types of oligomers formed by amyloid-forming peptides or proteins. However, if the target oligomer is cleaved and the concentration is lowered, the concentration of other oligomers in equilibrium with this target will be reduced accordingly, and the amount of oligomers causing the amyloidosis will be reduced.

Some of the cleavage agents presented in the examples of the present invention have demonstrated the ability to cleave oligomers of various types of amyloid-forming peptides or proteins. In Example 1 of the present invention, as well as Aβ ₄₂ Cleavage agent A is cutting the oligomer of A ( beta) ₄₀ . The β-amyloid precursor protein is cleaved by secretase enzymes to form Ab ₄₀ mainly in life. Aβ ₄₀ is responsible for various physiological functions. Excessive cutting of Ab ₄₀ may inhibit its normal function. However, in Alzheimer's disease, the amount of Aβ ₄₀ is 30-40 times the normal level (Lue, LF; Kuo, YM; Roher, AE; Brachova, L .; Shen, Y .; Sue, L .; Beach, T .; Kurth, JH; Rydel, RE;.. Rogers, J. Am J. Pathol 1999, 155, 853-862) in the process of cutting the Aβ ₄₂ Aβ ₄₀ is even slightly larger cut is not a problem.

The antibody made by the oligomers of Aβ ₄₂ to Aβ ₄₂ oligomers target, as well as Aβ ₄₀ oligomers also recognize and α- healing of other amyloidogenic proteins or peptides Klein, Oh was pressed, poly glutamine, lysozyme, insulin, prion peptide 106-126 Aggregates have also been reported to have the ability to recognize (Kayed, R .; Head, E .; Thompson, JL; McIntire, TM; Milton, SC; Cotman, CW; Glabe, CG Science 2003 , 300 , 486-489 ). These results suggest that various types of water-soluble amyloid oligomers have a common morphology and suggest that there is a possibility of treating various types of amyloidosis with a single drug. Among the cleavage agents presented in the examples of the present invention are the ability to cleave amyloid oligomers that cause two or more amyloidosis, which is in agreement with the results of antibody studies.

(2) Amylin oligomers related to type 2 diabetes

Oligomers of amylin (human islet amyloid polypeptide) have recently been identified as one of the causes of type 2 diabetes (Janson, J .; Ashley, RH; Harrison, D .; McIntyre, S .; Butler, PC). Diabetes 1999 , 48 , 491-498: Kayed, R .; Head, E .; Thompson, JL; McIntire, TM; Milton, S. C; Cotman, CW; Glabe, CG Science 2003 , 300 , 486-489: Kayed , R .; Sokolov, Y .; Edmonds, B .; McIntire, TM; Milton, SC; Hall, JE; Glabe, CG J. Biol . Chem. 2004 , 279 , 46363-46366: Meier, JJ; Kayed, R .; Lin, C.-Y .; Gurlo, T .; Haataja, L .; Jayasinghe, S .; Langen, R .; Glabe, CG;... Butler, PC Am J. Endicrinol Metab 2006, 291, E1317 -E1324: Ritzel, RA; Meier, JJ; Lin, C.-Y .; Veldhuis, JD; Butler, PC Diabetes 2007 , 56 , 65-71: Lin, CY; Gurlo, T .; Kayed, R .; Butler , AE; Haataja, L .; Glabe, CG; Butler, PC Diabetes 2007 , 56 , 1324-1332). Amylin (Am) is a cyclic oligopeptide consisting of 37 amino acids, which form amyloid by self-association.

As shown in the examples of the present invention, the cleavage agent of the present invention is believed to cleave the oligomer of Am. At this stage it is not known which cleavage agent of the present invention targets and cleaves any of the various oligomers of Am. However, if the target oligomer is cleaved and the concentration is lowered, the concentration of other oligomers in equilibrium with this target will be reduced accordingly, and the amount of oligomers that cause type 2 diabetes will be reduced.

(3) alpha-neuclein oligomers involved in Parkinson's disease

One of the causes of Parkinson's disease is alpha-synuclein (Giasson, BI; Murry, IVJ; Trojanowski, JQ; Lee, VM J. Biol . Chem . 2001 , 276 , 2380-2386: Vladimir N .; Nversky, N .; Li, J .; Fink, AL J. Biol. Chem . 2001 , 276 , 10737-10744). Alpha-Synuclein (Syn) is a protein consisting of 140 amino acids that form amyloid by self-assembly.

Pharmaceutical composition

Cleavage agents according to the present invention can be used in the prevention or treatment of amyloidosis by effectively cleaving soluble associations formed by amyloidogenic peptides or proteins and inhibiting their biological activity. Accordingly, the present invention also relates to a pharmaceutical composition for preventing or treating amyloidosis, comprising a cleavage agent of Formula 1 and a pharmaceutically acceptable salt. Preferably, amyloidosis includes, but is not limited to, Alzheimer's disease, type II diabetes, Parkinson's disease, spongy encephalopathy or Huntington's disease.

When the cleavage agent according to the present invention is administered for clinical purposes, the specific dose level for each patient is the weight, sex, health condition, diet, time of administration of the drug, administration method, excretion rate, drug mixture and severity of the disease. And so on.

Cleavage agents according to the invention can be administered by any route as desired. Injection, oral and nasal administration are preferred, but can also be administered through the skin, abdominal cavity, larynx and rectum.

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. Particularly, capsules and tablets are useful. Tablets and pills are preferably prepared with enteric agents. Solid dosage forms can be prepared by mixing the cleavage agent of formula 1 according to the invention with one or more inert diluents such as sucrose, lactose, starch and the like and a carrier such as a lubricant, disintegrant, binder, etc., such as magnesium stearate .

The present invention is explained in more detail based on the following examples. However, these examples are only intended to help the understanding of the present invention, and the scope of the present invention in any sense is not limited to the examples.

Example  One

Cleavage A was synthesized according to the route of FIG. 4.

Chemical formula 1a Synthesis of Compounds

4-Aminomethyl-benzoic acid (1.02 g, 6.8 mmol) and 2-aminophenol (0.69 g, 7.4 mmol) were mixed with polyphosphoric acid (10 g) and heated to 170 ° C. under nitrogen for 1.5 hours. The reaction mixture was cooled to room temperature and then poured into 10% K ₂ CO ₃ aqueous solution. The resulting precipitate was filtered off and the precipitate was recrystallized from acetone-water and bleached with activated charcoal in tetrahydrofuran (THF) -water to give 4-benzooxazol-2-yl-benzylamine ( 1a ). R _f 0.65 (EtOAc / hexanes 1: 2); ¹ H NMR (CDCl ₃ ): δ 8.20 (d, 2 H), 7.76 (dd, 1 H), 7.66 (dd, 1 H), 7.55 (d, 2 H), 7.41 (m, 2 H), 3.90 (s, 2 H); MS (MALDI-TOF) m / z 225.33 (M + H) ⁺ calcd for C ₁₄ H ₁₃ N ₂ O ₁ 225.09.

Chemical formula 1c Synthesis of Compounds

Cyanuric chloride ( 1b ) (0.20 g, 1.1 mmol.), A compound of Formula 1a (0.20 g 0.90 mmol) and diisopropylethylamine (DIEA) (0.38 mL, 2.7 mmol) were dissolved in THF (50 mL), reacted at 0 ° C. for 4 hours, and then purified by column chromatography (4-benzooxa). Zol-2-yl-benzyl)-(4,6-dichloro- [1,3,5] triazin-2-yl) -amine ( 1c ) was obtained. R _f 0.7 (EtOAc / hexanes 1: 4); ¹ H NMR (CDCl ₃ ): δ 8.13 (d, 2 H), 7.70 (d, 1 H), 7.51 (d, 1 H), 7.40 (d, 2 H), 7.27 (d, 2 H), 4.58 (d, 2H); ¹³ C NMR (300 CDCl ₃ ): δ 171.26, 170.17, 166.04, 162.43, 150.72, 141.96, 139.84, 128.161, 128.09, 126.96, 125.34, 124.73, 120.06, 110.65, 76.60, 45.05; MS (MALDI-TOF) m / z 372.28 (M + H) ⁺ , calcd for C ₁₇ H ₁₂ Cl ₂ N ₅ O 372.03.

Chemical formula 1d  To 1f Synthesis of Resin

Compound of Formula 1c (55 mg, 0.15 mmol) was dissolved in THF (1.5 mL) and mixed with PS-thiophenol resin (Argonaut Technologies; 50 mg, 0.074 mmol) and DIEA (0.10 mL, 0.74 mmol) and left at 65 ° C. for one day. After filtration, the resin was washed with N, N-dimethylformamide (DMF), methylene chloride (MC), MeOH and MC and dried with nitrogen gas to obtain a resin of formula 1d .

Resin of formula 1d was mixed with N-methyl-2-pyrrolidinone (NMP; 1 mL), and then n-butanol (1 mL), butylamine (49 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol) were added. Then heated at 120 ° C. for 8 hours. After filtration, the resin was washed with DMF, MC, MeOH and MC and dried with nitrogen gas to obtain a resin of formula 1e .

M -chloroperoxybenzoic acid ( m- CPBA; 130 mg, 0.74 mmol) was dissolved in a solution of 1,4-dioxane (1.8 mL) and 1N NaOH aqueous solution (160 μL), and a resin of Formula 1e was added thereto. Then, stirred at room temperature for 4 hours. After filtration, the resin was washed with 1,4-dioxane and MC and dried with nitrogen gas to obtain a resin of formula 1f .

Chemical formula 1h  Synthesis of Compounds

Resin of formula 1f was added to acetonitrile (1.5 mL) and then PS-DIEA resin (Argonaut Technologies; 30 mg, 0.12 mmol) and a compound of formula 1 g (Chae, PS; Kim, M .; Jeung, C .; Lee) , SD; Park, H .; Lee, S .; Suh, J. J. Am. Chem . Soc. 2005 , 127 , 2396-2397) (39 mg, 0.074 mmol) was added. After reacting at 80 ° C. for 8 hours, the resin was washed with MC (1 mL × 3) after filtration. The product obtained by volatizing the filtrate was purified by column chromatography to obtain 10- {3- [4- (4-benzooxazol-2-yl-benzylamino) -6-butylamino- [1,3,5] triazine 2-ylamino] -propyl] -1,4,7,10 tetra aza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester ( 1h ) was obtained.

R _f 0.5 (EA only); ¹ H NMR (CDCl ₃ ): δ 8.13 (d, 2H), 7.70 (d, 1H), 7.51 (d, 1H), 7.40 (d, 2H), 7.27 (d, 2H), 4.58 (d, 2H) , 3.8-3.2 (br, 14H), 2.5 (br, 6H), 2.1 (br, 2H), 1.63 (br, 2H), 1.5-1.3 (m, 31H), 0.9-0.8 (dd 3H); ¹³ C NMR (CDCl ₃ ): δ 164.89, 161.91, 154.33, 149.67, 142.56, 141.06, 126.72, 124.82, 124.01, 123.53, 118.89, 109.53, 78.51, 78.31, 75.58, 53.92, 52.91, 48.92, 48.08, 43.31 39.35, 37.97, 30.86, 28.67, 27.65, 19.03, 13.10; MS (MALDI-TOF) m / z 902.99 (M + H) ⁺ , calcd for C ₄₇ H ₇₂ N ₁₁ O ₇ 902.55.

Synthesis of triazine derivatives using resins in this and the following examples was carried out according to literature methods (Khersonsky, SM; Chang, Y. T. J. Comb. Chem . 2004 , 6 , 474) unless otherwise noted . will be.

Chemical formula 1i Compound of and Cutting agent A Synthesis of

Diethyl ether (1 mL) was added to a mixture of the compound of Formula 1h (5 mg) in 50 mL MC dissolved with 50% trifluoric acid (TFA) for 5 hours. The precipitate was separated by centrifugation and washed several times with diethyl ether. N- (4-benzooxazol-2-yl-benzyl) -N'butyl-N "[3- (1,4,7,10 tetraaza-cyclodode-1-sil) -propyl]-[1 , 3,5] TFA salt of triazine-2,4,6-triamine ( 1i ) was obtained. NMR and MS data were determined using the TFA salt of the compound of Formula 1i ; ¹ H NMR (CDCl ₃ ): δ 8.17 (d, 2 H), 7.73 (d, 1 H), 7.55 (d, 1H), 7.43 (d, 2H), 7.33 (d, 2H), 4.42 (br, 2H), 3.4-3.0 (br, 14H), 2.85 (br, 6H), 2.62 (br, 2H), 1.68 (br, 2H), 1.36-1.23 (m, 4H), 0.9-0.8 (dd 3H); ¹³ C NMR (CDCl ₃ ): δ 164.89, 162.67, 150.62, 141.76, 127.80, 126.12, 125.30, 124.73, 119.83, 110.65, 76.60, 51, 48.90, 44.87, 42.80, 42.32, 38.07, 30.89, 29.70, 23.71, 19.71, 19. , 14.07; MS (MALDI-TOF) MS (MALDI-TOF) m / z 602.73. (M + H) ⁺ , calcd for C ₃₂ H ₄₈ N ₁₁ O 602.40; HRMS m / z 602.4043. (M + H) ⁺ , calcd for C ₃₂ H ₄₈ N ₁₁ O 602.4038.

5-7 equivalents of LiOH was added to a solution of 1i TFA salt dissolved in methanol at a concentration of about 5 mg / 50 μL, followed by the method reported in the literature (Kim, MG; Kim, M.-s .; Lee, SD; Suh, J. J. Inorg . Biol . Chem . 2006, 11. 867) CoCl _{2 ·} H ₂ O in equivalent weight to bind the ligand Co ^II Given the day and shake out the back made for a complex air Co ^II Complexes to Co ^III Oxidized to complex. As the oxidation progressed, the solution turned dark purple. Co ^III by detection at 545 nm using HPLC By separating the complex, and then, after volatilization to stand at 37 ℃ for 1 hour, the obtained solid is dissolved in 0.1 M NaOH solution, this solution was neutralized with HCl to pH 6-8, and allowed to stand for several days at room temperature, and then cutting the A Used as stock solution. ICP was used to determine the cobalt content to determine the concentration of cleavage agent in this solution.

Cutting agent A Activity test

(1) cleavage of oligomers of Aβ ₄₀ and Aβ ₄₂ associated with Alzheimer's disease

In this example and in each of the examples below, the activity test of each cleavage agent was carried out in an Eppendorf tube at 37 ° C. and pH 7.50 (0.050 M phosphoric acid).

The following filtration experiments were performed to collect quantitative information on the process of decreasing the amount of monomers and small oligomers of Aβ ₄₀ and Aβ ₄₂ under the conditions of this experiment. Aβ ₄₀ or Aβ ₄₂ prior to exposure of the reaction medium of pH 7.50 in order to generate a monomer form the initial reaction was treated with Aβ ₄₀ or Aβ ₄₂ with NaOH (Fezoui, Y .; Hartley, DM; Harper, JD; Khurana, R .; Walsh, DM; Condron, MM; Selkoe, DJ; Lansbury, PT Jr .; Fink, AL; Teplow, DB Amyloid 2000 , 7, 166-178). The results of measuring the amount of Aβ ₄₀ or Aβ ₄₂ filtered through the membrane having a cutoff molecular weight of 10000 at various self-association times are summarized in FIG. 5. The results indicate that Aβ ₄₂ (molecular weight 4514) passes through the membrane almost immediately after exposure (> 80%) to pH 7.50 medium. Since the membrane pass experiment takes about 10 minutes, the oligomer of the size cannot pass through the membrane. It is known that the amount of dimers and trimers of Aβ ₄₂ is much less than that of monomers (Bitan, G .; Kirkitadze, MD; Lomakin, A .; Vollers, SS; Benedek, GB; Teplow, DB Proc . Natl. Acad . Sci . USA 2003 , 100 , 330; Hepler, RW; Grimm, KM; Nahas, DD; Breese, R .; Dodson, EC; Acton, P .; Keller, PM; Yeager, M .; Wang, H .; Shughrue, P .; Kinney, G .; Joyce, JG Biochemistry 2006 , 45 , 15157-15167), and Aβ ₄₂ are almost always present as monomers at the beginning of the reaction. After 3 or 36 hours, about two-thirds or 90% of the original Aβ ₄₂ changes into large aggregates that cannot pass through the membrane, respectively. On the other hand, in the case of Aβ ₄₀ , more than 90% at the beginning of the reaction and 50% pass through the membrane after 24 hours.

The MALDI-TOF mass spectrum measured after reacting Aβ ₄₀ or Aβ ₄₂ (4.0 μM) with cleavage agent A is shown in FIGS. 6 and 7. As shown here, Aβ ₄₀ and Aβ ₄₂ are cleaved by cleavage agent A and Aβ _1-20 and Aβ _1-21 (Aβ cleavage products in this example and in each of the following examples are named according to the amino acid sequence of Aβ ₄₂ and m The cleavage product corresponding to the peak indicated by the / z value was confirmed by MALDI LIFT-TOF / TOF MS). Since the intensity of the peak of the MALDI-TOF MS is independent of the relative concentration, there is a possibility that there is a significant amount of cleavage product present without showing a strong MALDI-TOF MS peak.

Unless otherwise stated in this embodiment and the following examples, Aβ ₄₀ or Aβ ₄₂ The cleavage reaction was initiated by adding Aβ ₄₀ or Aβ ₄₂ to the solution of the cleavage agent and the cleavage yield was measured as follows. The product solution formed as a result of the cleavage reaction was passed through a membrane with a cutoff molecular weight of 10000 to remove the aggregates of Aβ ₄₀ or Aβ ₄₂ and then the cleavage product was separated using HPLC to measure the total amount of cleavage product. After cleavage of the cleavage product to the amino acid state by alkaline hydrolysis, the total amount of cleavage product was determined by measuring the total amount of amino acids using fluorescarmine. The cleavage yield was calculated by comparing the amount of cleavage product to the amount of Aβ ₄₀ or Aβ ₄₂ that was first applied.

The cleavage yields measured after varying the concentration of the cleavage agent A at various concentrations of Aβ ₄₀ or Aβ ₄₂ (4.0 μM) at 37 ° C. and pH 7.50 for 36 hours are summarized in FIG. 8. In this example and the following examples, the cleavage yield of Aβ ₄₀ or Aβ ₄₂ is the average of the values measured using 4-6 reaction solutions and the relative standard deviation of each cleavage yield is 5-15%. 9 was previously summarized in FIG. 9 by measuring the cleavage yield when Aβ ₄₀ or Aβ ₄₂ was allowed to undergo self-integration for a predetermined time and then reacted with cleavage agent A. FIG. In order to obtain information on the progress of the cleavage reaction, Aβ ₄₀ or Aβ ₄₂ was reacted with cleavage agent A at 37 ^° C., pH 7.50 for a predetermined time, and the cleavage yields measured were summarized in FIG. 10.

Fig. According to the results of 9 of Aβ ₄₂ was 24 hours integrated dictionary itself following Applying a cutting agent A does the cutting reaction hardly occurs, which can be seen as they hayeotgi changes in Aβ ₄₂ are polymerized as a pro toffee fibrils or fibril. Aβ was ₄₂ for 3-6 hours and then integrated dictionary itself does not decrease to be Applying a cutting agent A as compared with a reaction, without going through a pre-step integration is significantly cutting the yield, it is greatly reduced even during the 3-6 hours Aβ ₄₂ monomers in the 5 Contrasts with the facts. The result is that Aβ ₄₂ It shows that oligomers, but not monomers, protopibrils, or fibrils were cleaved by cleavage agent A. Aβ ₄₀ cleavage reaction occurs to a significant extent even if the pre-integration proceeds for 24 hours and then reacts with the cleavage agent A, and further prolongation of the pre-integration time reduces the cutting yield. This is compared to the Aβ ₄₂ to Aβ ₄₀ matches that for Pro toffee fibrils or fibril formation rate does not cut the first A cut is much slower, and the fact that even in the case of Aβ ₄₀ Pro toffee fibrils or fibril. Increasing the pre-association time to 3-18 hours does not significantly reduce the yield of Aβ ₄₀ cleavage by the cleavage agent A , indicating that Aβ ₄₀ is cleaved in the oligomer state and not in the monomeric state.

In the case of cleavage agent A , the upper limit of cleavage yield observed at high cleavage concentrations is about 30%. Aβ ₄₂ oligomers exist as short-lived intermediates and the cleavage of oligomers by cleavage agents competes with the polymerization of oligomers. The cleavage reaction is a primary reaction to the oligomer concentration so that if the cleavage agent concentration is constant, the half-life of the oligomer cleavage reaction is independent of the peptide concentration. The polymerization reaction of the oligomer is at least a secondary to the peptide concentration so that the half-life resulting from the polymerization becomes longer as the peptide concentration is lowered. The amount of Aβ ₄₂ in the brain of Alzheimer's disease patients is much less than 1 nM (Lue, LF; Kuo, YM; Roher, AE; Brachova, L .; Shen, Y .; Sue, L .; Beach, T .; Kurth, JH; Rydel, RE; Rogers, J. Am. J. Pathol . 1999 , 155 , 853-862).

In this embodiment, the concentration of the Aβ ₄₂ 4.0 μM work when cutting the concentration was able to observe the cutting reaction in 100 nM, the concentration of the low Aβ _42, such as a living body the cutting reaction in a much lower cutting claim concentration than 100 nM significantly proceed will be.

 (2) cleavage of oligomers of amylin related to type 2 diabetes

The results of measuring the amount of amylin (Am) through the membrane having a cutoff molecular weight of 10000 at various self-association times are summarized in FIG. 11. The membrane can pass through small oligomers, such as Am and its dimers or trimmers, and the amount decreases to less than half within a few hours.

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage agent A and then purified by HPLC is shown in FIG. 12. The cleavage reaction products of Am in this example and the following examples show the MALDI-TOF mass spectrum measured after HPLC purification by the method described below. As shown in FIG. 12, amylin is cleaved by cleavage agent A and Am _{20 -37} and Am _{19 -37} (In this example and in each of the examples below, Am cleavage products are named according to the amino acid sequence of Am and the m / z value The cleavage product corresponding to this indicated peak was confirmed by MALDI LIFT-TOF / TOF MS). Since the intensity of the peak of the MALDI-TOF MS is independent of the relative concentration, there is a possibility that there is a significant amount of cleavage product present without showing a strong MALDI-TOF MS peak.

Unless otherwise stated in this example and the following examples, the cleavage reaction of Am was initiated by adding Am to the solution of the cleaver and the cleavage yield was measured as follows. The product solution formed as a result of the cleavage reaction was passed through a membrane with a cutoff molecular weight of 10000 to remove the aggregates of Am and then the cleavage product was separated using HPLC to measure the total amount of cleavage product. After cleavage of the cleavage product to the amino acid state by alkaline hydrolysis, the total amount of cleavage product was determined by measuring the total amount of amino acids using fluorescarmine. The cleavage yield was calculated by comparing the amount of cleavage product to the amount of Am initially applied.

Am varying the concentration of cutting agent A (4.0 [mu] M) was reacted for 36 hours at 37 [deg.] C., pH 7.50, and then the cleavage yield measured was summarized in FIG. In this example and the following examples, the amputation yield of Am is the average of the values measured using 4-6 reaction solutions, and the relative standard deviation of each amputation yield is 5-15%.

 (3) cleavage of oligomers of alpha-neuclein involved in Parkinson's disease

In this example and the following examples, a slightly modified analog of the structure of alpha-synuclein (Syn) was used as a substrate and obtained by genetic recombination. That is, a histidine tag (LEHHHHHH) was attached to the C-terminus to easily purify Syn by nickel chelate purification, and leucine was introduced instead of methionine as an amino acid at position 5 to avoid confusion during transcription.

In order to collect data on the speed at which Syn forms a very large aggregate by self-assembly, allow Syn to remain in place for a period of time and then associate it with a 0.22 mm Millipore filter. The result of measuring the amount is summarized in FIG. 14. It can be seen that a significant amount of protofibrils or fibrils are formed in a few days that cannot be adsorbed to or passed through the filter.

Unless otherwise stated in this example and the following examples, the cleavage reaction of Syn was initiated by adding Syn to a solution of the cleavage agent and the cleavage yield was measured as follows. The product solution formed as a result of the cleavage reaction was passed through a membrane with a cutoff molecular weight of 10000 to remove Syn and its aggregates, then the cleavage product was separated using HPLC to measure the total amount of cleavage product. After cleavage of the cleavage product to the amino acid state by alkaline hydrolysis, the total amount of cleavage product was determined by measuring the total amount of amino acids using fluorescarmine. The cleavage yield was calculated by comparing the amount of cleavage product to the amount of Syn first applied.

The cleavage yield measured after various reactions with Syn (70 μM) at 37 ° C., pH 7.50 at various concentrations of cleavage A was summarized in FIG. 15.

In this example and the following examples, the cleavage yield is an average of the values obtained by using 4-6 reaction solutions. Since the molecular weight of Syn used in the present embodiment is about 15000, some of the protein fragments formed by the cleavage of Syn may not pass through the membrane having a cutoff molecular weight of 10000. With this in mind, the cut yields presented in Figure 15 are of considerable high value.

(4) reaction with control peptides or proteins

The following control experiment was performed about cutting agent A. Peptides or proteins used in this control experiment are those that do not form amyloid. This control experiment was performed to confirm that cleavage agent A did not show cleavage ability under the experimental conditions used in this example. . Two peptides that contain 42 amino acids that make up Aβ ₄₂ , but a completely different sequence of Aβ ₄₂ No cleavage reaction was observed when (KVKGLIDGAHIGDLVYEFMDSNSAIFREGVGAGHVHVAQVEF, AIAEGDSHVLKEGAYMEIFDVQGHVFGGKIFRVVDLGSHNVA) (4.0 μM) was reacted with cleavage A (3.0 μM) at 37 ^° C., pH 7.50 for 36 hours. Equine cardiac myoglobin, bovine serum g-globulin, bovine serum albumin, human serum albumin, egg white lysozyme, egg white ovalbumin, bovine pancreatic insulin (2-7 μM each) and cutting agent A (5.0 μM) and 37 ° C, No cleavage reaction was observed when reacted at pH 7.50 for 36 hours.

Removing the recognition site contained in the cutting agent A when Aβ _40, Aβ _42, Am, PSY clan to ensure that the oligomer is capable of cutting a loss, such as Syn Co ^III No cleavage reaction was observed when the complex (20 μM) was reacted with Aβ ₄₀ (4.0 μM), Aβ ₄₂ (4.0 μM), Am (4.0 μM), Syn (70 μM) at 37 ^° C, pH 7.50 for 36 hours. .

Example  2

Cleavage agent B was synthesized according to the route of FIG. 16.

Chemical formula 2a Synthesis of Compounds

A mixture of 2-aminothiophenol (1.3 g, 10 mmol) and N- methyl- N- (2-hydroxyethyl) -4-aminobenzaldehyde (1.8 g, 10 mmol) in dimethylsulfoxide (10 mL) was prepared. It heated at 170 degreeC for 1.5 hours. After cooling to room temperature the reaction mixture was poured into water and extracted with ethyl acetate (EA; 50 mL × 2). The organic layer was removed moisture using Na ₂ SO ₄ . The solid obtained by volatilizing the solvent was recrystallized in acetonitrile to give 2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino] -ethanol ( 2a ) as a yellow solid. R _f 0.20 (EA / hexanes 1: 1); ¹ H NMR (CDCl ₃ ): δ 7.97 (d, 1 H), 7.95 (d, 2 H), 7.85 (d, 1 H), 7.45 (t, 1 H), 7.32 (t, 1 H), 6.81 (d, 2H), 3.88 (t, 2H), 3.60 (t, 2H), 1.80 (br s, 3H); ¹³ C NMR (CDCl ₃ ): δ 154.09, 151.61, 134.38, 129.03, 126.10, 124.34, 122.23, 121.67, 111.96, 77.02, 60.19, 54.66, 39.03; MS (MALDI-TOF) 285.35 m / z (M + H) ⁺ calcd for C ₁₆ H ₁₇ NOS 285.10.

Chemical formula 2b Wow  2c Synthesis of Compounds

Stir the solution of compound 1g (2.9 g, 5.5 mmol) in acetonitrile (100 mL) and add N-α-Cbz-L-alanine (1.2 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol). gave. 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU; 2.1 g, 5.5 mmol) was added and the reaction mixture was stirred for 1 hour. gave. The solid obtained by volatilizing the solution was dissolved in EA (100 mL), and then the solution was washed with 5% aqueous citric acid solution (50 mL), 5% aqueous Na ₂ CO ₃ solution (50 mL) and (50 mL) brine, and washed with Na _2. Water was removed using SO ₄ . The solvent was volatilized and then purified by column chromatography using 10-[( S ) -3- (2-benzyloxycarbonylamino-propionylamino) -propyl] -1,4,7,10-tetraaza-cyclododecane -1,4,7-tricarboxylic acid tri-tert-butyl ester ( 2b ) was obtained as a colorless oil. R _f 0.2 (EA / hexane 1: 1). ¹ H NMR (CDCl ₃ ): δ 7.30 (s, 5H), 5.02 (s, 2), 3.50-3.10 (br, 15H) 2.60-2.30 (br, 6H), 1.57-1.49 (br, 2H), 1.39 -1.36 (m, 27H), 1.18 (s, 3H); ¹³ C NMR (CDCl ₃ ): δ 171.58, 154.82, 154.79, 154.22, 135.42, 127.55, 78.49, 65.71, 53.42, 49.56, 48.92, 47.57, 47.18, 46.53, 45.25, 37.68, 29.92, 27.64, 18.32; MS (MALDI-TOF) m / z 735.88 (M + H) ⁺ calcd for C ₃₇ H ₆₃ N ₆ O ₉ 735.46.

Compound of Formula 2b (2.0 g, 2.7 mmol) and 1.0 g 10% Pd / C in a suspension of 80 mL EtOH was stirred under 1 atmosphere of hydrogen for 24 hours. The catalyst was filtered off using celite and then the solvent was evaporated to give 10-[( S ) -3- (2-benzyloxycarbonylamino-propionylamino) -propyl] -1,4,7,10- Tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester ( 2c ) was obtained as a solid; ¹ H NMR (CDCl ₃ ): δ 7.49 (s, 1H), 3.63-3.18 (br, 15H), 2.72-2.52 (br, 6H), 1.88-1.75 (br, 2H), 1.73-1.60 (m, 2H ), 1.50-1.40 (m, 27H), 1.40-1.30 (d, 3H); ¹³ C NMR (CDCl ₃ ): δ 171.98, 155.03, 154.69, 154.25, 78.57, 78.42, 78.28, 53.60, 52.95, 49.72, 49.01, 47.80, 47.07, 46.54, 46.18, 37.56, 29.92, 27.64, 22.99; MS (MALDI-TOF) 601.58 m / z (M + H) ⁺ calcd for C ₂₉ H ₅₇ N ₆ O ₇ 601.42.

Chemical formula 2d Synthesis of Compounds

The compound of formula 1b (0.20 g, 1.1 mmol), the compound of formula 2a (0.20 g, 0.70 mmol) and DIEA (0.38 mL, 2.7 mmol) were mixed with THF (50 mL) and stirred at room temperature for 8 hours. The material obtained by volatilizing the solvent was separated by column chromatography to obtain (4-benzothiazol-2-yl-phenyl)-[2- (4,6-dichloro- [1,3,5] triazin-2-yl jade. C) -ethyl] -methyl-amine ( 2d ) was obtained. R _f 0.7 (EA / hexane 1: 4); ¹ H NMR (CDCl ₃ ): δ 7.97 (t, 3H), 7.85 (d, 1H), 7.45 (t, 1H), 7.36 (t, 1H), 6.78 (d, 2H), 4.67 (t, 2H) , 3.84 (t, 2 H), 3.12 (br, 3 H); ¹³ C NMR (CDCl ₃ ): δ 172.53, 171.84, 168.15, 155.13, 151.39, 135.02, 129.21, 126.27, 124.59, 122.66, 122.46, 121.74, 112.14, 67.78, 50.58, 38.89; MS (MALDI-TOF) m / z 432.29 (M + H) ⁺ calcd for C ₁₉ H ₁₆ Cl ₂ N ₅ OS 432.04.

Chemical formula 2e  To 2 g Synthesis of Resin

Compound of formula 2d (62 mg, 0.15 mmol) was dissolved in THF (1.5 mL) and then PS-thiophenol resin (Argonaut Technologies) (50 mg, 0.074 mmol) and DIEA (0.10 mL, 0.74 mmol) were added. This mixture was heated at 65 ° C. overnight. After filtration, the separated resin ( 2e ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

To the suspension obtained by adding the resin of Formula 2e to NMP (1 mL) was added n-butanol (1 mL), 4-chlorobenzylamine (71 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol). This mixture is 120 It heated at 8 degreeC. The resin ( 2f ) isolated by filtration was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

m-CPBA (130 mg, 0.74 mmol) was dissolved in 1,4-dioxane (1.8 mL) and 1N NaOH aqueous solution (160 μL), and a resin of Formula 2f was added thereto, followed by shaking at room temperature for 4 hours. The resin ( 2g ) separated by filtration was washed with 1,4-dioxane and MC (3 mL x 3 each) and dried under nitrogen gas.

Chemical formula 2h  Wow 2i Compound of and Cutting agent B Synthesis of

Resin of formula 2g was mixed into acetonitrile (1.5 mL) and PS-DIEA resin (from Argonaut Technologies) (30 mg, 0.12 mmol) and compound of formula 2c (39 mg, 0.074 mmol) were added. 80 reaction mixture Heat at 8 ° C. After filtration, the separated resin was washed with MC (1 mL x 3) and the liquid was combined with the filtrate. The solution obtained by filtration of this solution was purified by column chromatography to obtain 10- {3- [4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino-ethoxy} -6- (4-Chloro-benzylamino)-[1,3,5] triazin-2-ylamino]-( S ) -propionylamino} -propyl) -1,4,7,10 tetraza-cyclododecane -1,4,7-tricarboxylic acid tri-tert-butyl ester ( 2h ) was obtained. R _f 0.2 (EA only); ¹ H NMR (CDCl ₃ ): δ 8.13 (d, 2 H), 7.70 (d, 1 H), 7.51 (d, 1 H), 7.40 (d, 2 H), 7.27 (d, 2 H), 4.58 (d, 2H), 3.8-3.2 (br, 12H), 2.5 (br, 6H), 2.1 (br, 2H), 1.63 (br, 4H), 1.5-1.3 (m, 31H), 0.9-0.8 (dd 3H); ¹³ C NMR (CDCl ₃ ): δ 164.89, 161.91, 154.33, 149.67, 142.56, 141.06, 126.72, 124.82, 124.01, 123.53, 118.89, 109.53, 78.51, 78.31, 75.58, 53.92, 52.91, 48.92, 48.08, 43. , 37.97, 30.86, 28.67, 27.65, 19.03, 13.10; MS (MALDI-TOF) m / z 1102.59 (M + H) ⁺ , calcd for C ₅₅ H ₇₈ ClN ₁₂ O ₈ S 1102.54.

The compound of formula 2h was treated with TFA as described for 1 h in Example 1 to give 2- [4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy } -6- (4-chloro-benzylamino)-[1,3,5] triazin-2-ylamino] -N- [3- (1,4,7,10 tetraza-cyclodode-1- Real) -propyl]-(S) -propionamide ( 2i ). NMR and MS data were determined using the TFA salt of the compound of formula 2i . ¹ H NMR (CDCl ₃ ): δ 7.95 (s, 1H), 7.92-7.7.85 (q, 3H), 7.45 (t, 1H), 7.37-7.28 (m, 5H), 6.79 (t, 2H), 4.70-4.40 (br, 2H), 3.78 (br, 2H), 3.13-2.70 (br, 15H), 2.74 (s, 3H), 2.58-2.44 (br, 6H), 1.93 (m, 1H), 1.66- 1.55 (br, 2 H), 1.44-1.19 (m, 5 H); ¹³ C NMR (CDCl ₃ ): δ 173.57, 169.69, 168.84, 163.26, 162.01, 155.42, 152.65, 138.23, 135.63, 132.63, 130.76, 130.52, 130.07, 127.63, 125.90, 123.26, 123.08, 122.51, 113.29, 48. , 52.18, 51.49, 50.44, 45.22, 43.73, 43.18, 39.98, 31.51, 24.91; MS (MALDI-TOF) m / z 801.57 (M + H) ⁺ , calcd for C ₄₀ H ₅₄ ClN ₁₂ O ₂ S 801.39; HRMS m / z 801.3907. (M + H) ⁺ , calcd for C ₄₀ H ₅₄ ClN ₁₂ O ₂ S 801.3896.

A stock solution of cutting agent B was obtained from 2i according to the method described for A in Example 1.

Cutting agent B Activity test

(1) cleavage of oligomers of Aβ ₄₀ and Aβ ₄₂ associated with Alzheimer's disease

The MALDI-TOF mass spectrum measured after reacting Aβ ₄₀ or Aβ ₄₂ (4.0 μM) with cleavage B is shown in FIGS. 17 and 18. As shown here, Aβ ₄₀ and Aβ ₄₂ are cleaved by the cleavage agent B and Aβ _1-20 and Aβ _1-21 are included in the cleavage product. Since the intensity of the peak of the MALDI-TOF MS is independent of the relative concentration, there is a possibility that there is a significant amount of cleavage product present without showing a strong MALDI-TOF MS peak.

The cleavage yields measured after varying the concentration of cleavage agent B at various concentrations of Aβ ₄₀ or Aβ ₄₂ (4.0 μM) at 37 ^° C., pH 7.50 for 36 hours are summarized in FIG. 19. In the case of cleavage agent B , the upper limit of cleavage yield observed at high cleavage concentrations is about 12% and 17% for Aβ ₄₀ and Aβ _42, respectively. As described in Example 1, the cleavage reaction was observed at 30-50 nM cleavage concentration when the concentration of Aβ ₄₂ was 4.0 μM, but much lower than 30-50 nM at low concentrations of Aβ ₄₂ such as living bodies. The cleavage reaction will proceed significantly even at low cleavage concentrations.

(2) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage B and then purified by HPLC is shown in FIG. 20. As shown here, amylin is cleaved by cleavage agent B and Am _{20 -37} , Am _{19 -37} and Am _{17 -37} are included in the cleavage product.

Am varying the concentration of cleavage B (4.0 [mu] M) was reacted for 36 hours at 37 [deg.] C., pH 7.50, and then the cleavage yields measured were summarized in FIG.

Am was previously summarized in FIG. 22 by measuring the cleavage yield when the self-integration was allowed to proceed for a predetermined time and then reacted with the cleavage agent B. FIG. According to the results of FIG. 22, when Am was pre-integrated for 36 hours or more, and then the cutting agent B was added, the cleavage reaction hardly occurred, which may be because Am was polymerized and changed to protofibrils or fibrils. The 6-hour pre-integration of Am followed by addition of the cutting agent B does not significantly reduce the cleavage yield compared to the reaction without undergoing the pre-integration step, in contrast to the fact that Am monomers and small oligomers are significantly reduced for 6 hours in FIG. To achieve. This is the result of Am It shows that oligomers, but not monomers, protofibrils, or fibrils were cleaved by cleavage agent B.

In order to obtain information on the progress of the cleavage reaction, Am was reacted with cleavage agent B at 37 ° C., pH 7.50 for a predetermined time, and the cleavage yields measured were summarized in FIG. 23. The results in FIG. 23 show that the reaction yield does not increase even when the reaction time is extended for more than 36 hours.

(3) cleavage of oligomers of alpha-neuclein involved in Parkinson's disease

The cleavage yield measured after various reactions with Syn (70 μM) at 37 ° C., pH 7.50 with varying concentrations of cleavage B was summarized in FIG. 24. Since the molecular weight of Syn used in the present embodiment is about 15000, some of the protein fragments formed by the cleavage of Syn may not pass through the membrane having a cutoff molecular weight of 10000. In view of this, the cutting yields shown in FIG. 24 are of considerable high value.

 (4) reaction with control peptides or proteins

The same result was obtained when the control experiment performed in Example 1 was carried out with respect to the cutting agent B. FIG.

Example  3

Cleavage C was synthesized according to the route of FIG. 25.

Chemical formula 3a  Wow 3b Synthesis of Resin

Resin (50 mg, 0.046 mmol) of formula 2e was suspended in NMP (1 mL), n-butanol (1 mL) and cyclododecylamine (66 μL, 0.51 mmol) and DIEA (63 μL, 0.36 mmol) were added. It was. This mixture is 120 It was made to react at 8 degreeC. After filtration, the separated resin ( 3a ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

The resin of Formula 3a was added to a mixture of m-CPBA (80 mg, 0.46 mmol) in 1,4-dioxane (1.8 mL) and a 1 N NaOH (93 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 3b ) obtained after filtration was washed with 1,4-dioxane and MC v (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 3d  Wow 3e Compound of and Cutting agent C Synthesis of

The resin of formula 3b was suspended in acetonitrile (1.5 mL) and 3c with PS-DIEA resin (Argonaut Technologies) (19 mg, 0.075 mmol) (Chae, PS; Kim, M .; Jeung, C .; Lee, SD; Park, H .; Lee, S .; Suh, J. Am. Chem . Soc . 2005 , 127 , 2396-2397) (28 mg, 0.046 mmol) was added and reacted at 80 ° C for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and volatilized, and the obtained material was purified by column chromatography to obtain 10- {3- [3- (4- {2-[(4- Benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy} -6-cyclododecylamino- [1,3,5] triazin-2-ylamino) -propionylamino] -propyl} -1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid-tert-butyl ester ( 3d ) was obtained. R _f 0.3 (EA only); ¹ H NMR (CDCl ₃ ): δ 7.95 (t, 3H), 7.83 (d, 1H), 7.41 (t, 1H), 7.33 (br, 1H), 6.78 (m, 2H), 4.67 (br, 2H) , 3.75 (br, 2H), 3.67 (br, 2H), 3.55-3.18 (br, 14H), 3.10 (s, 3H), 2.60 (br, 5H), 2.50 (br, 4H), 1.61-1.58 (br , 6H), 1.45-1.41 (m, 27H), 1.33 (br, 18H); ¹³ C NMR (CDCl ₃ ): δ 184.41, 173.21, 171.70, 169.97, 168.67, 156.28, 155.60, 155.24, 154.34, 150.84, 134.47, 129.75, 128.97, 125.99, 125.01, 124.21, 122.21, 121.46, 111.46. , 62.94, 56.52, 55.11, 51.07, 49.97, 48.36, 47.41, 46.54, 40.92, 39.27, 36.76, 35.63, 29.69, 28.62, 23.50, 21.32; MS (MALDI-TOF) m / z 1144.02 (M + H) ⁺ , calcd for C ₆₀ H ₉₅ N ₁₂ O ₈ S 1143.70,

The compound of formula 3d was treated with TFA in the same manner as described for 1 h in Example 1 to give 3- (4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino]- Ethoxy} -6-cyclododecylamino- [1,3,5] triazin-2-ylamino) -N- [3- (1,4,7,10-tetraaza-dodec-1-yl) TFA salt of -propyl] -propanamide ( 3e ) was obtained. NMR and MS data were measured using the TFA salt of the compound of formula 3e ; ¹ H NMR (CDCl ₃ ): δ 7.94 (br, 3H), 7.83 (d, 1H), 7.45 (br, 1H), 7.35 (br, 1H), 6.75 (d, 2H), 4.57 (br, 2H) , 3.81 (br, 2H), 3.63 (br, 2H), 3.3-2.9 (br, 17H), 2.76 (br, 5H), 2.44 (br, 4H), 1.65- 1.61 (br, 6H), 1.31 (br , 18H); ¹³ C NMR (CDCl ₃ ): δ 188.78, 188.78, 172.02, 169.91, 169.79, 169.44, 157.24, 151.78, 133.60, 129.56, 126.89, 125.41, 125.16, 121.81, 120.57, 112.28, 112.02, 66.26, 65.26, 65. , 49.67, 48.68, 44.63, 42.51, 39.33, 39.16, 37.01, 30.20, 29.94, 23.82, 23.51, 21.48; MS (MALDI-TOF) m / z 843.79 (M + H) ⁺ , calcd for C ₄₅ H ₇₁ N ₁₂ O ₂ S 843.55; HRMS m / z 843.5547. (M + H) ⁺ , calcd for C ₄₅ H ₇₁ N ₁₂ O ₂ S 843.5538.

A stock solution of cutting agent C was obtained from 3e according to the method described for A in Example 1.

Cutting agent C Activity test

(1) cleavage of oligomers of Aβ ₄₀ and Aβ ₄₂ associated with Alzheimer's disease

Even when cleavage C (0.1-10 μM) was reacted with Aβ ₄₀ (4.0 mM) for 36 hours at 37 ° C., pH 7.50, no evidence of cleavage of Aβ ₄₀ in the MALDI-TOF mass spectrum was obtained.

The MALDI-TOF mass spectrum measured after reacting Aβ ₄₂ (4.0 μM) with cleavage C is shown in FIG. 26. As shown here, Aβ ₄₂ is cleaved by cleavage agent C and Aβ _1-20 is included in the cleavage product. Since the intensity of the peak of the MALDI-TOF MS is independent of the relative concentration, there is a possibility that there is a significant amount of cleavage product present without showing a strong MALDI-TOF MS peak.

The cleavage yield measured after varying the concentration of cleavage C with Aβ ₄₀ or Aβ ₄₂ (4.0 μM) at 37 ° C., pH 7.50 for 36 hours is summarized in FIG. 27. For cleavage C , the upper limit of cleavage yield observed at high cleavage concentrations is about 12% for Aβ ₄₂ . As described in Example 1, the cleavage reaction was observed at 100 nM cleavage concentration when the concentration of Aβ ₄₂ was 4.0 μM. The cleavage concentration was much lower than 100 nM at low concentrations of Aβ ₄₂ such as living bodies. The cleavage reaction will also proceed significantly.

(2) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage C and then purified by HPLC is shown in FIG. 28. As shown here, amylin is cleaved by cleavage C and Am _{17 -37} is included in the cleavage product.

Am varying the concentration of cleavage C (4.0 μM) and the cleavage yield measured after reacting at 37 ° C., pH 7.50 for 36 hours is summarized in FIG. 29.

(3) reaction with control peptides or proteins

The same result was obtained when the control experiment performed in Example 1 was performed with respect to the cutting | disconnection agent C.

Example  4

Cleavage agent D was synthesized according to the route of FIG. 30.

Chemical formula 4a  Wow 4b Synthesis of Resin

The resin of formula 2e (50 mg, 0.046 mmol) was suspended in NMP (1 mL) and n-butanol (1 mL) and cyclododecylamine (66 μL, 0.51 mmol) and DIEA (63 μL, 0.36 mmol) were added. do. This mixture was reacted at 120 ° C for 8 hours. After filtration, the separated resin ( 4a ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

The resin of Formula 4a was added to a mixture of m-CPBA (80 mg, 0.46 mmol) in 1,4-dioxane (1.8 mL) and a 1 N NaOH (93 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 4b ) obtained after filtration was washed with 1,4-dioxane and MC (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 4c  Wow 4d Compound of and Cutting agent D Synthesis of

The resin of formula 4b was suspended in acetonitrile (1.5 mL) and 3c with PS-DIEA resin (Argonaut Technologies) (19 mg, 0.075 mmol) (28 mg, 0.046 mmol) was added and reacted at 80 ° C for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and volatilized, and the obtained material was purified by column chromatography to obtain 10- {3- [3- (4- {2-[(4- Benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy} -6- (2-methyl-benzylamino)-[1,3,5] triazin-2-ylamino) -propionylamino ] -Propyl} -1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid-tert-butyl ester ( 4c ) was obtained. R _f 0.4 (EA only); ¹ H NMR (CDCl ₃ ): δ 7.96 (t, 3H), 7.83 (d, 1H), 7.43 (t, 1H), 7.32 (br, 1H), 7.15 (br, 4H), 6.77 (d, 2H) , 4.53 (br, 4H), 3.75 (br, 2H), 3.66 (br, 2H), 3.5-3.1 (br, 14H), 3.09 (s, 3H), 2.58 (br, 4H), 2.49 (br, 4H ), 2.32 (s, 3H), 1.60 (br, 2H), 1.46-1.41 (m, 27H); ¹³ C NMR (CDCl ₃ ): δ 186.87, 179.99, 173.28, 171.18, 168.68, 155.26, 154.35, 150.86, 136.88, 136.10, 134.50, 130.27, 128.99, 127.27, 126.09, 126.01, 125.02, 124.12124, 122.24. , 111.55, 80.31, 79.51, 63.54, 56.56, 55.10, 54.95, 50.98, 49.95, 48.35, 47.65, 40.01, 39.03, 36.93, 29.71, 28.65, 19.10; MS (MALDI-TOF) m / z 1081.89 (M + H) ⁺ , calcd for C ₅₆ H ₈₁ N ₁₂ O ₈ S 1081.59.

The compound of formula 4c was treated with TFA in the same manner as described for 1 h in Example 1 to give 3- (4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino]- Ethoxy} -6- (2-methyl-benzylamino)-[1,3,5] triazin-2-ylamino) -N- [3- (1,4,7,10-tetraaza-dode- TFA salt of 1-sil) -propyl] -propanamide ( 4d ) was obtained. NMR and MS data were determined using the TFA salt of the compound of formula 4d ; ¹ H NMR (CDCl ₃ ): δ 7.96 (t, 3H), 7.85 (d, 1H), 7.49 (br, 1H), 7.37 (br, 1H), 7.15 (br, 4H), 6.80 (d, 2H) , 4.56 (m, 4H), 3.58 (br, 2H), 3.3-2.8 (br, 17H), 2.33 (br, 8H), 2.31 (s, 3H), 1.48 (br, 2H); ¹³ C NMR (CDCl ₃ ): δ 186.11, 180.06, 171.65, 169.85, 169.01, 151.51, 151.17, 136.08, 134.83, 132.77, 130.66, 129.65, 129.42, 127.99, 127.93, 127.82, 126.31, 126.121. , 112.09, 111.90, 66.12, 62.87, 53.89, 50.44, 49.72, 44.38, 44.22, 42.34, 42.09, 39.36, 36.63, 29.71, 19.07; MS (MALDI-TOF) m / z 781.73 (M + H) ⁺ , calcd for C ₄₁ H ₅₇ N ₁₂ O ₂ S 781.44; HRMS m / z 781.4457. (M + H) ⁺ , calcd for C ₄₁ H ₅₇ N ₁₂ O ₂ S 781.4443.

A stock solution of cutting agent D was obtained from 4d according to the method described for A in Example 1.

Cutting agent D Activity test

(1) cleavage of oligomers of Aβ ₄₀ and Aβ ₄₂ associated with Alzheimer's disease

Even when cleavage agent D (0.1-10 μM) was reacted with Aβ ₄₀ (4.0 μM) for 36 hours at 37 ° C., pH 7.50, no evidence of Ab ₄₀ cleavage in the MALDI-TOF mass spectrum was obtained.

The MALDI-TOF mass spectrum measured after reacting Aβ ₄₂ (4.0 μM) with cleavage D is shown in FIG. 31. As shown here, Aβ ₄₂ is cleaved by cleavage agent D and Aβ _1-20 is included in the cleavage product. Since the intensity of the peak of the MALDI-TOF MS is independent of the relative concentration, there is a possibility that there is a significant amount of cleavage product present without showing a strong MALDI-TOF MS peak.

The cleavage yields measured after varying the concentration of cleavage agent D with Aβ ₄₀ or Aβ ₄₂ (4.0 mM) at 37 ° C., pH 7.50 for 36 hours are summarized in FIG. 32. For cleavage C , the upper limit of cleavage yield observed at high cleavage concentrations is about 12% for Aβ ₄₂ . As described in Example 1, the cleavage reaction was observed at 50-100 nM cleavage concentration when the concentration of Aβ ₄₂ was 4.0 μM, but much lower than 50-100 nM at low concentrations of Aβ ₄₂ such as living bodies. The cleavage reaction will proceed significantly even at low cleavage concentrations.

(2) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage agent D and then purified by HPLC is shown in FIG. 33. As shown here, amylin is cleaved by cleavage agent D and Am _{20 -37} and Am _{19 -37} are included in the cleavage product.

Am varying the concentration of the cutting agent D (4.0 μM) and 37 hr, pH 7.50 for 36 hours and then the cut yields measured are summarized in FIG.

 (3) reaction with control peptides or proteins

The same result was obtained when the control experiment performed in Example 1 was carried out with respect to the cutting agent D.

Example  5

Cleavage E was synthesized according to the route of FIG. 35.

Chemical formula 5a Wow 5b  Synthesis of Resin

Resin of formula 2e (55 mg, 0.15 mmol) is suspended in NMP (1 mL) and n-butanol (1 mL) and butylamine (49 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol) are added. This mixture was reacted at 120 ° C for 8 hours. After filtration, the separated resin ( 5a ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

The resin of Formula 5a was added to a mixture of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and 1N NaOH (160 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 5b ) obtained after filtration was washed with 1,4-dioxane and MC (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 5c  Wow 5d Compound of and Cutting agent E Synthesis of

The resin of Formula 5b was suspended in acetonitrile (1.5 mL), and PS-DIEA resin (30 mg, 0.12 mmol) and 1 g (39 mg, 0.074 mmol) were added and reacted at 80 ° C. for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and evaporated under reduced pressure, and the obtained material was purified by column chromatography to obtain 10- [3- (4- {2-[(4-benzothiatia). Zol-2-yl-phenyl-methyl-amino] -ethoxy} -6-butylamino- [1,3,5] triazin-2-ylamino) -propyl] -1,4,7,10-tetra Aza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester ( 5c ) was obtained: R _f 0.2 (EA / hexane 1: 1); ¹ H NMR (CDCl ₃ ): δ 7.99 ( t, 2H), 7.86 (d, 1H), 7.44 (t, 1H), 7.31 (t, 1H), 6.79 (d, 2H), 4.48 (t, 2H), 3.80 (t, 2H), 3.54-3.11 (br, 16H), 3.11 (s, 3H), 2.61 (br, 6H), 1.73 (m, 2H), 1.53 (m, 2H), 1.46-1.38 (br, 27H), 1.29 (m, 2H), 0.90 (t, 3H); ¹³ C NMR (CDCl ₃ ): δ 170.47, 168.58, 167.25, 156.09, 155.69, 155.35, 154.43, 154.40, 150.92, 134.53, 128.97, 125.97, 124.19, 122.29, 121.56, 121.33, 111.57, 111.57 79.55, 79.34, 76.61, 62.54, 55.04, 54.16, 51.13, 50.02, 47.97, 40.52, 39.09, 31.75, 29.68, 28.66, 28.50, 24.93, 20.01, 13.79; MS (MALDI- TOF) m / z 962.33 (M + H) ⁺ , calcd for C ₄₉ H ₇₅ N ₁₁ O ₇ S 962.28.

The compound of formula 5c was treated with TFA in the same manner as described for 1 h in Example 1 to give 6- {2-[(4-benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy} N -butyl- N '-[3- (1,4,7,10-tetraaza-cyclododec-1-yl) -propyl]-[1,3,5] triazine-2,4-diamine ( 5d ) of TFA salt was obtained. NMR and MS data were determined using the TFA salt of the compound of Formula 5d ; ¹ H NMR (MeOD): δ 7.90 (q, 4 H), 7.49 (t, 1 H), 7.37 (t, 1H), 6.85 (d, 2H), 4.73 (t, 2H), 3.89 (t, 2H ), 3.2-3.0 (br, 12H), 2.98-2.86 (br, 4H), 2.84-2.75 (br, 4H), 2.68 (t, 3H), 1.76 (q, 2H), 1.52 (q, 2H), 1.31 (q, 2 H), 0.89 (t, 3 H); ¹³ C NMR (CDCl ₃ ): δ 169.61, 161.13, 160.64, 160.32, 152.72, 151.72, 151.55, 133.49, 128.69, 128.64, 126.35, 124.67, 121.48, 120.93, 120.16, 118.24, 114.36, 111.84, 66.28, 49.98, 49.98, 49.98 , 44.31, 42.01, 41.86, 40.69, 38.95, 38.65, 37.84, 37.52, 30.51, 22.62, 19.55, 12.63; HRMS m / z 662.4073 (M + H) ⁺ , calcd for C ₃₄ H ₅₂ N ₁₁ OS 662.4077.

To a solution of 5d TFA salt dissolved in methanol at a concentration of about 3 mg / 50 μL, 5-7 equivalents of LiOH was added to the ligand and CoCl _{2 ·} H ₂ O was added to the equivalent to bind 5d of Co ^II. Given the day and shake out the back made for a complex air Co ^II Complexes to Co ^III Oxidized to complex. As the oxidation progressed, the solution turned dark purple. Co ^III by detection at 545 nm using HPLC After separating the complex, the volatilized solid was dissolved in water, left at room temperature for several days, and used as a stock solution of the cutting agent E. ICP was used to determine the cobalt content to determine the concentration of cleavage agent in this solution.

Cutting agent E Activity test

(1) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage agent E and then purified by HPLC is shown in FIG. 36. As shown here, amylin is cleaved by the cleavage agent E and Am _{17 -37} , Am _{16 -37} and Am _{13 -37} are included in the cleavage product.

Am varying the concentration of the cutting agent E (4.0 μM) at 37 ^° C., pH 7.50 for 36 hours and then the cleavage yields measured are summarized in FIG. 37.

 (2) reaction with control peptides or proteins

The same result was obtained as a result of the control experiment performed in Example 1 with respect to the cutting agent E. FIG.

Example  6

Cleavage agent F was synthesized according to the route of FIG. 38.

Chemical formula 6a Wow  6b Synthesis of Compounds

Stir the solution of compound 1g (2.9 g, 5.5 mmol) in acetonitrile (100 mL) and add N- α-Cbz-L-leucine (0.80 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol). gave. HBTU (2.1 g, 5.5 mmol) was added and the reaction mixture was stirred for 1 hour. The solid obtained by volatilizing the solution was dissolved in EA (100 mL), and then the solution was washed with 5% aqueous citric acid solution (50 mL), 5% aqueous Na ₂ CO ₃ solution (50 mL) and (50 mL) brine, and washed with Na _2. Water was removed using SO ₄ . The solvent was volatilized and then purified by column chromatography using 10-[(R) -3- (4-methyl-2-phenoxycarbonylamino-pentanoylamino) -propyl] -1,4,7,10-tetraaza - cycloalkyl FIG decane -1,4,7- tree carboxylic acid tri-tert-butyl to give the ester (6a) as a colorless oil. R _f 0.5 (EA / hexane 2: 1); ¹ H NMR (CDCl ₃ ): δ 7.31-7.26 (br, 5H), 4.75- 4.33 (br, 2H), 3.63-3.38 (br, 15H), 2.67-2.29 (br, 6H), 1.71-1.58 (m , 5H), 1.50-1.29 (br, 27H), 0.96-0.92 (t, 6H) MS (MALDI-TOF) m / z MS (MALDI-TOF) m / z 776.63 (M + H) ⁺ , calcd for C ₄₀ H ₆₈ N ₆ 0 ₉ 776.50.

Compound of formula 6a (1.8 g, 2.7 mmol) and 1.0 g 10% Pd / C in a 80 mL EtOH suspension was stirred for 24 hours under 1 atmosphere of hydrogen. The catalyst was filtered off using celite and then the solvent was evaporated to give 10- [3- (2-amino-4-methyl-pentanoylamino) -propyl] -1,4,7,10-tetraaza-cyclo Dodecane-1,4,7-tricarboxylic acid tri- tert -butyl ester ( 6b ) was obtained as a solid; ¹ H NMR (CDCl ₃ ): δ 7.72 (s, 1H), 4.60-4.20 (br, 2H), 3.70-3.13 (br, 15H), 2.75-2.50 (br, 6H), 1.86-1.62 (m, 4H ), 1.54-1.36 (m, 28H), 1.05-0.84 (t, 6H); ¹³ C NMR (MeOD): δ 176.12, 156.31, 156.11, 155.91, 79.70, 54.16, 53.91, 53.18, 49.23, 46.84, 43.98, 37.53, 37.70, 27.70, 27.52, 24.51, 22.84, 22.02, 21.26; MS (MALDI-TOF) m / z 643.57 (M + H) ⁺ , calcd for C ₃₂ H ₆₂ N ₆ O ₇ 643.47.

Chemical formula 6c Wow 6d  Synthesis of Resin

Resin (55 mg, 0.15 mmol) of formula 2e is suspended in NMP (1 mL) and n-butanol (1 mL) and piperidine (57 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol) are added. . This mixture was reacted at 120 ° C for 8 hours. After filtration, the separated resin ( 6c ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

Resin of Chemical Formula 6c was added to a mixture of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and a 1 N NaOH (160 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 6d ) obtained after filtration was washed with 1,4-dioxane and MC (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 6e  Wow 6f Compound of and Cutting agent F Synthesis of

The resin of formula 6d was suspended in acetonitrile (1.5 mL), PS-DIEA resin (30 mg, 0.12 mmol) and 6b (48 mg, 0.074 mmol) were added and reacted at 80 ° C. for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and evaporated under reduced pressure. The obtained material was purified by column chromatography to obtain 10-{(R) -3- [2- (4- {2 -[(4-benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy} -6-piperidin-1-yl- [1,3,5] triazin-2-ylamino) 4-Methyl-pentanoylamino] -propyl} -1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri- tert -butyl ester ( 6e ) was obtained. R _f 0.2 (EA / hexane 2: 1); ¹ H NMR (CDCl ₃ ): δ 7.96 (q, 3H), 7.84 (d, 1H), 7.45 (t, 1H), 7.30 (t, 1H), 6.78 (d, 2H), 4.56-4.55 (br, 1H), 4.45 (t, 2H), 3.78 (t, 2H), 3.69-3.67 (br, 4H), 3.61-3.21 (br, 14H), 3.11 (s, 3H), 2.75-2.31 (br, 6H) , 1.72 (m, 1 H), 1.72-1.50 (br, 10 H), 1.49-1.29 (br, 27 H), 0.93 (m, 6H); ¹³ C NMR (CDCl ₃ ): δ 173.17, 170.46, 168.57, 166.78, 165.42, 156.13, 155.78, 155.30, 154.41, 150.89, 134.53, 129.00, 125.99, 124.23, 122.30, 121.60, 121.36, 111.56, 79.44. , 54.88, 53.95, 51.10, 49.96, 49.15, 48.14, 44.46, 41.68, 39.10, 36.99, 29.69, 28.66, 28.51, 25.77, 24.87, 24.72, 24.46, 23.25, 21.80; MS (MALDI-TOF) m / z 1087.60 (M + H) ⁺ , calcd for C ₅₆ H ₈₆ N ₁₂ O ₈ S 1087.64.

The compound of formula 6e was treated with TFA in the same manner as described for 1 h in Example 1 to give 2-((S) -4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl -Amino] -ethoxy} -6-piperidin-1-yl- [1,3,5] triazin-2-ylamino) -4-methyl-pentanoic acid [3- (1,4,7 A TFA salt of, 10-tetraaza-cyclodode-1-yl) -propyl] -amide ( 6f ) was obtained. NMR and MS data were determined using the TFA salt of the compound of formula 6f ; ¹ H NMR (MeOD): δ 7.90 (q, 4H), 7.48 (t, 1H), 7.37 (t, 1H), 6.88 (d, 2H), 4.70-4.68 (br, 1H), 4.52 (t, 2H ), 3.89 (t, 2H), 3.82-3.68 (br, 4H), 3.20-3.05 (br, 13H), 3.02-2.93 (br, 4H), 2.85-2.80 (br, 4H), 2.59 (t, 2H ), 1.75-1.50 (br, 7H), 0.89 (t, 6H); ¹³ C NMR (MeOD): δ 173.16, 169.35, 161.64, 161.45, 158.40, 157.85, 157.30, 153.47, 151.32, 133.84, 128.59, 126.14, 124.46, 121.35, 120.62, 116.53, 111.61, 65.65, 65.65, 65.65, 65.65. 50.01, 46.77, 45.25, 44.06, 42.06, 41.84, 41.09, 37.83, 36.60, 25.09, 24.54, 23.81, 21.90, 20.64, 14.04; HRMS m / z 787.4913 (M + H) ⁺ , calcd for C ₄₁ H ₆₃ N ₁₂ O ₂ S 787.4918.

A stock solution of cutting agent F was obtained from 6f according to the method described for E in Example 5.

Cutting agent F Activity test

(1) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 mM) with cleavage F and then purified by HPLC is shown in FIG. 39. As shown here, amylin is cleaved by the cleavage agent F and Am _{19 -37} And Am _{17 -37} are included in the cleavage product.

Am varying the concentration of cleavage F (4.0 μM) and 37 ^° C., pH 7.50 for 36 hours, and the cleavage yields measured were then summarized in FIG. 40.

 (2) reaction with control peptides or proteins

The same result was obtained as a result of the control experiment performed in Example 1 with respect to the cutting agent F. FIG.

Example  7

Cleavage agent G was synthesized according to the route of FIG. 41.

Chemical formula 7a Wow 7b  Synthesis of Resin

The resin of formula 2e (55 mg, 0.15 mmol) was suspended in NMP (1 mL) and n-butanol (1 mL) and cyclododecylamine (75 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol) were added. do. This mixture was reacted at 120 ° C for 8 hours. After filtration, the separated resin ( 7a ) was washed with DMF, MC, MeOH and MC (each 3 mL × 3) and dried under nitrogen gas.

The resin of Formula 7a was added to a mixture of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and a 1 N NaOH (160 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 7b ) obtained after filtration was washed with 1,4-dioxane and MC (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 7c  Wow 7d Compound of and Cutting agent F Synthesis of

The resin of formula 7b was suspended in acetonitrile (1.5 mL), and PS-DIEA resin (30 mg, 0.12 mmol) and 6b (48 mg, 0.074 mmol) were added and reacted at 80 ° C. for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and evaporated under reduced pressure. The obtained material was purified by column chromatography to obtain 10-{(R) -3- [2- (4- {2 -[(4-benzothiazol-2-yl-phenyl-methyl-amino] -ethoxy} -6-cyclododecylamino- [1,3,5] triazin-2-ylamino) -4-methyl -Pentanoylamino] -propyl} -1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri- tert -butyl ester ( 7c ) was obtained R _f 0.2 (EA / Hexanes 2: 1); ¹ H NMR (CDCl ₃ ): δ 7.96 (q, 3H), 7.84 (d, 1H), 7.43 (t, 1H), 7.30 (t, 1H), 6.77 (d, 2H), 4.47-4.41 (br, 3H), 4.14-4.04 (br, 1H), 3.77 (t, 2H), 3.68-21 (br, 14H), 3.12 (s, 3H), 2.75-2.34 (br, 6H), 1.72-1.55 (br, 5H), 1.52-1.40 (br, 27H), 1.38-1.28 (br, 22H), 0.87 (m, 6H); ¹³ C NMR (CDCl ₃ ): δ 173.56, 170.42, 168.50, 166.90 , 165.93, 156.07, 155.74, 155.26, 154.39, 150.91, 134.50, 128.97, 125.95, 124.20, 122.26, 121.62, 121.53, 121.32, 111.59, 111.49, 79.49, 79.34, 79.23, 76.73, 63.17 , 63.06, 51.05, 49.87, 47.99, 47.55, 47.32, 39.21, 38.83, 30.58, 29.65, 28.64, 28.49, 25.00, 24.83, 24.10, 23.95, 23.73, 23.51, 23.32, 23.08, 22.15, 21.74, 21.17; MS (MALDI -TOF) m / z 1185.53 (M + H) ⁺ , calcd for C ₆₃ H ₁₀₀ N ₁₂ O ₈ S 1185.75.

Compound ( 7c ) was treated with TFA in the same manner as described for 1 h in Example 1 to give 2-((S) -4- {2-[(4-benzothiazol-2-yl-phenyl) -methyl -Amino] -ethoxy} -6-cyclododecylamino- [1,3,5] triazin-2-ylamino) -4-methyl-pentanoic acid [3- (1,4,7,10- A TFA salt of tetraaza-cyclododec-1-yl) -propyl] -amide ( 7d ) was obtained. NMR and MS data were determined using the TFA salt of the compound of formula 7d ; ¹ H NMR (MeOD): δ 7.90 (q, 4H), 7.47 (t, 1H), 7.35 (t, 1H), 6.81 (d, 2H), 4.80-4.53 (br, 3H), 4.12-4.03 (br , 1H), 3.89 (t, 2H), 3.22-3.12 (br, 10H), 3.10-3.00 (br, 3H), 2.97-2.92 (br, 4H), 2.90-2.73 (br, 4H), 2.63 (t , 2H), 1.76-1.60 (br, 5H), 1.40-1.12 (br, 22H), 0.72 (t, 6H); ¹³ C NMR (MeOD): δ 172.79, 169.50, 161.97, 161.41, 160.99, 159.38, 157.83, 157.28, 151.60, 133.48, 128.80, 126.32, 124.63, 121.45, 120.93, 116.52, 111.63, 66.45, 65.47, 53. 46.79, 44.15, 41.96, 41.92, 37.52, 24.66, 23.61, 23.53, 23.37, 23.52, 23.10, 22.90, 22.10, 20.86, 20.46, 14.06; HRMS m / z 885.5991 (M + H) ⁺ , calcd for C ₄₈ H ₇₇ N ₁₂ O ₂ S 885.6013.

A stock solution of cutting agent G was obtained from 7d according to the method described for E in Example 5.

Cutting agent G Activity test

(1) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage G and then purified by HPLC is shown in FIG. 42. As shown here, amylin is cleaved by the cleavage agent G and Am _{20 -37} , Am _{17 -37} and Am _{14 -37} are included in the cleavage product.

Am varying the concentration of the cutting agent G (4.0 μM) at 37 ^° C., pH 7.50 for 36 hours and then the cleavage yields measured were summarized in FIG. 43.

(2) reaction with control peptides or proteins

The same result was obtained when the control experiment performed in Example 1 was carried out with respect to the cutting agent G.

Example  8

Cleavage agent H was synthesized according to the route of FIG. 44.

Synthesis of Compounds of Formulas 8a and 8b

Stir the solution of 1 g of compound (2.9 g, 5.5 mmol) in acetonitrile (100 mL) and add N- α-Cbz-L-tyrosine (1.1 g, 5.5 mmol) and DIEA (2.9 mL, 17 mmol). gave. HBTU (2.1 g, 5.5 mmol) was added and the reaction mixture was stirred for 1 hour. The solid obtained by volatilizing the solution was dissolved in EA (100 mL), and then the solution was washed with 5% aqueous citric acid solution (50 mL), 5% aqueous Na ₂ CO 3 solution (50 mL) and (50 mL) brine, and Na ₂ SO ₄ was removed. Moisture was removed. The solvent was volatilized and then purified by column chromatography using 10-{(S) -3- [3- (4-hydroxy-phenyl) -2-phenoxycarbonylamino-propylamino] -propyl} -1,4, 7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri- tert -butyl ester (8a) was obtained as a colorless oil. R _f 0.5 (EA / hexane 3: 1). 1 H NMR (CDCl ₃ ): δ 7.42-7.23 (br, 5H), 7.07-6.85 (d, 2H), 6.77-6.62 (d, 2H), 4.60-4.13 (br, 4H), 3.77-2.92 (br , 16H), 2.69-2.54 (br, 4H), 2.50-2.37 (br, 2H), 1.65-1.31 (br, 29H); MS (MALDI-TOF) m / z 827.75 (M + H) ⁺ , calcd for C ₄₃ H ₆₆ N ₆ O ₁₀ 827.04.

Compound of Formula 8a (2.0 g, 2.7 mmol) and 1.0 g 10% Pd / C in a suspension of 80 mL EtOH was stirred under 1 atmosphere of hydrogen for 24 hours. The catalyst was filtered off using celite and then the solvent was evaporated to give 10- {3-[(R) -2-amino-3- (4-hydroxy-phenyl) -propionylamino] -propyl} -1 , 4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri- tert -butyl ester ( 8b ) was obtained as a solid; ¹ H NMR (CDCl ₃ ): δ 7.2 (s, 1H), 7.05-6.97 (d, 2H), 6.81-6.72 (d, 2H), 3.65-3.58 (m, 1H), 3.53-3.20 (br, 14H ), 3.12-3.00 (m, 2H), 2.75-2.48 (br, 8H), 1.76-1.64 (m, 2H), 1.54-1.38 (m, 27H); ¹³ C NMR (CDCl ₃ ): δ 173.51, 155.91, 155.51, 130.40, 127.44, 115.62, 79.91, 79.65, 76.58, 56.12, 54.55, 49.76, 47.73, 39.63, 38.56, 37.13, 29.62, 28.61, 28.45, 24.23; MS (MALDI-TOF) m / z 692.84 (M + H) ⁺ , calcd for C ₃₅ H ₆₀ N ₆ O ₈ 692.90.

Chemical formula 8c Wow 8d  Synthesis of Resin

Resin of formula 2e (55 mg, 0.15 mmol) was suspended in NMP (1 mL) and n-butanol (1 mL) and dicyclohexylamine (115 μL, 0.58 mmol) and DIEA (120 μL, 0.87 mmol) were added. do. This mixture was reacted at 120 ° C for 8 hours. After filtration, the separated resin ( 8c ) was washed with DMF, MC, MeOH and MC (3 mL × 3 each) and dried under nitrogen gas.

The resin of Formula 8c was added to a mixture of m-CPBA (130 mg, 0.74 mmol) in 1,4-dioxane (1.8 mL) and a 1 N NaOH (160 μL) aqueous solution, and shaken at room temperature for 4 hours. . The resin ( 8d ) obtained after filtration was washed with 1,4-dioxane and MC (3 mL × 3 each) and dried under nitrogen gas.

Chemical formula 8e  Wow 8f Compound of and Cutting agent H Synthesis of

The resin of formula 8d was suspended in acetonitrile (1.5 mL), and PS-DIEA resin (30 mg, 0.12 mmol) and 8b (51 mg, 0.074 mmol) were added and reacted at 80 ° C. for 8 hours. After filtration, the separated resin was washed with MC (1 mL × 3), the filtrate and the washing solution were combined and evaporated under reduced pressure. The obtained material was purified by column chromatography to obtain 10-{(S) -3- [2- (4- {2 -[(4-benzothiazol-2-yl-phenyl) -methyl-amino] -ethoxy} -6-dicyclohexylamino- [1,3,5] triazin-2-ylamino) -3- (4-hydroxy-phenyl) -propionylamino] -propyl} -1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester (8e) Got. R _f 0.2 (EA: hexane 1: 4); ¹ H NMR (CDCl ₃ ): δ 7.87 (t, 3H), 7.73 (d, 2H), 7.35 (t, 2H), 7.20 (m, 2H), 6.75-6.48 (br, 4H), 4.45-4.41 ( m, 3H), 3.73-3.68 (m, 2H), 3.62-3.08 (br, 18H), 3.05-2.65 (br, 6H), 2.19-2.17 (m, 2H), 1.93-1.85 (m, 2H), 1.75-1.62 (br, 4H), 1.60-1.42 (br, 31H), 1.58-1.20 (br, 12H); ¹³ C NMR (CDCl ₃ ): δ 169.55, 168.64, 165.09, 154.35, 151.53, 150.73, 135.81, 134.52, 129.04, 128.26, 125.53, 124.28, 122.30, 121.75, 121.38, 111.68, 76.71, 64.43, 64.24, 56.16. , 50.96, 47.21, 39.29, 34.24, 30.35, 29.99, 29.80, 29.72, 28.58, 28.46, 26.12, 25.58, 25.37, 21.22, 20.25, 20.18, 20.09; HRMS m / z 1232.7149 (M + H) ⁺ , calcd for C ₆₆ H ₉₆ N ₁₂ O ₉ S 1232.7144.

Compound ( 8e ) was treated with TFA in the same manner as described for 1 h in Example 1 to give 2- (4-{(S) -2-[(4-benzothiazol-2-yl-phenyl) -methyl -amino] -ethoxy} -6-dicyclohexyl-amino- [1,3,5] triazin-2-yl) -3- (4-hydroxy-phenyl) - N - [3- (1 , TFA salt of 4,7,10-tetraaza-cyclododec-1-yl) -propyl] -propionamide ( 8f ) was obtained. NMR and MS data were determined using the TFA salt of the compound of formula 8f ; ¹ H NMR (CDCl ₃ ): δ 8.04 (t, 3H), 7.75 (d, 2H), 7.53 (t, 2H), 7.42 (m, 2H), 6.78-6.64 (br, 4H), 4.48-4.45 ( m, 3H), 3.81-3.74 (m, 2H), 3.63-2.75 (br, 24H), 2.25-2.15 (m, 3H), 1.85-1.63 (br, 12H), 1.60-1.42 (br, 10H); ¹³ C NMR (CDCl ₃ ): δ 169.55, 168.64, 164.59, 160.23, 153.72, 142.86, 130.91, 128.97, 128.26, 125.52, 122.10, 118.18, 117.51, 113.69, 79.80, 74.60, 73.43, 56.70, 56.53, 22.59, 39.59 , 30.88, 30.47, 30.31, 29.56, 27.98, 25.25, 24.93, 21.19, 19.92; HRMS m / z 932.5573 (M + H) ⁺ , calcd for C ₅₁ H ₇₂ N ₁₂ O ₃ S 932.5571.

A stock solution of cutting agent H was obtained from 8f according to the method described for E in Example 5.

Cutting agent H Activity test

(1) cleavage of oligomers of amylin related to type 2 diabetes

The MALDI-TOF mass spectrum measured for cleavage products obtained by reacting Am (4.0 μM) with cleavage H and then purified by HPLC is shown in FIG. 45. As shown here, amylin is cleaved by the cleavage agent H and Am _{1 -19} is included in the cleavage product.

Am varying the concentration of the cutting agent H (4.0 μM) at 37 ^° C., pH 7.50 for 36 hours and then the cleavage yields measured were summarized in FIG. 46.

(2) reaction with control peptides or proteins

The same result was obtained when the control experiment performed in Example 1 was carried out with respect to the cutting agent H.

1 shows the amino acid sequence of Aβ ₄₂ ,

FIG. 2 is a schematic view showing the production of soluble and insoluble aggregates of amyloid-forming peptides or proteins,

3 is a view schematically showing a process of reducing the amount of soluble and insoluble aggregates of amyloid-forming peptides or proteins by the action of a cleavage agent,

4 is a view schematically showing a synthesis process of the cutting agent A in Example 1 of the present invention,

FIG. 5 shows the amount of portion Aβ ₄₀ (○) and Aβ ₄₂ (●) (initial concentration 4.0 μM) filtered through a membrane having a cutoff molecular weight of 10000 after self-association for a period of time at 37 ° C. in a pH 7.50 solution. Is a graph shown (each data point is the average of at least five experiments),

6 is a MALDI-TOF mass spectrum of a product obtained after reacting the cleavage agent A (3.0 μM) of Example 1 with Aβ ₄₀ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

FIG. 7 is a MALDI-TOF mass spectrum of a product obtained after reacting cleavage agent A (1.0 μM) of Example 1 with Aβ ₄₂ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

8 shows Aβ ₄₀ (○) and varying the concentration ( C _o ) of the cutting agent A of Example 1; Aβ ₄₂ (●) This is a graph showing the cleavage yield measured after reacting (4.0 μM) with pH 7.50 and 37 ° C for 36 hours, depending on log C _o / M.

9 shows Aβ ₄₀ (grey bars) at pH 7.50 and 37 ° C. or Aβ ₄₂ (black bars) (4.0 μM) was pre-self associated beforehand followed by the addition of cleavage agent A (3.0 μM) of Example 1, showing that the yield of the reaction cleaved for 36 hours was dependent on the time of pre-self association It's a graph,

10 shows Aβ ₄₀ (○) at pH 7.50 and 37 ° C. And Aβ ₄₂ (●) (4.0 μM) is a graph showing the tendency for the yield to be cleaved by the cleavage agent A of Example 1 to depend on the cleavage reaction time,

FIG. 11 is a graph showing the amount of portion of Am (initial concentration 4.0 μM) filtered through a membrane with a cutoff molecular weight of 10000 after self-association for a period of time at 37 ^° C. in a pH 7.50 solution,

12 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleavage A (3.2 μM) of Example 1 was reacted with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

FIG. 13 shows the cut yields measured after changing the concentration ( C _o ) of the cutting agent A of Example 1 and reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours, depending on the log C _o / M. Indicate,

FIG. 14 is a graph showing the amount of Syn (initial concentration 70 μM) filtered through a 0.22 μm Millipore filter (Millipore Millex-GV 4MM) after self-association for a period of time at 37 ° C. in a pH 7.50 solution. ,

Figure 15 is an embodiment 1 cut the concentration of agent A in the (C _o) a change gamyeo After reaction for 3 days in Syn (70 μM) and pH 7.50 and 37 ℃ measured cutting yield is dependent on the log C _o / M Is a graph showing the trend,

16 is a view schematically showing a synthesis process of the cutting agent B in Example 2 of the present invention,

FIG. 17 is a MALDI-TOF mass spectrum of a product obtained after the cleavage agent B (3.0 μM) of Example 2 was reacted with Aβ ₄₀ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

FIG. 18 is a MALDI-TOF mass spectrum of a product obtained after reacting a cleavage agent B (0.50 μM) of Example 2 with Aβ ₄₂ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

19 is Aβ ₄₀ (○) while changing the concentration ( C _o ) of the cutting agent B of Example 2 or Aβ ₄₂ (●) This is a graph showing the cleavage yield measured after reacting (4.0 μM) with pH 7.50 and 37 ° C for 36 hours, depending on log C _o / M.

FIG. 20 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleaving B (1.0 μM) of Example 2 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

FIG. 21 is a change in the cutting agent B concentration ( C _o ) of Example 2, and the cut yield measured after reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours depends on log C _o / M. Is a graph showing the incense,

22 shows Am at pH 7.50 and 37 ° C. (4.0 μM) was self-associated in advance and the yield of the reaction cleaved for 36 hours after addition of the cleavage agent B (1.0 μM) of Example 2 is a graph showing a tendency to depend on the time of self-association beforehand

FIG. 23 is a graph showing that the yield at which Am (4.0 μM) is cleaved by the cutting agent B (1.0 μM) of Example 2 at pH 7.50 and 37 ° C. depends on the cleavage reaction time,

24 is a second embodiment cut concentration of the B in the (C _o) a change gamyeo After reaction for 3 days in Syn (70 μM) and pH 7.50 and 37 ℃ measured cutting yield is dependent on the log C _o / M Is a graph showing the trend,

25 is a view schematically showing a synthesis process of the cutting agent C in Example 3 of the present invention,

FIG. 26 is a MALDI-TOF mass spectrum of a product obtained after the cleavage C of Example 3 (1.00 μM) was reacted with Aβ ₄₂ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

27 is a β ₄₀ (○) while changing the concentration ( C _o ) of the cutting agent C of Example 3 or Aβ ₄₂ (●) This is a graph showing the cleavage yield measured after reacting (4.0 μM) with pH 7.50 and 37 ° C for 36 hours, depending on log C _o / M.

28 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleavage of C (3.2 μM) of Example 3 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

29 is a graph showing that cutting yields measured after changing the concentration ( C _o ) of the cutting agent C of Example 3 after 36 hours of reacting Am (4.0 μM) with pH 7.50 and 37 ° C. depend on log C _o / M Is a graph showing

30 is a view schematically showing a synthesis process of the cutting agent D in Example 4 of the present invention,

31 is a MALDI-TOF mass spectrum of a product obtained after reacting the cleavage agent D (1.00 μM) of Example 4 with Aβ ₄₂ (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

32 is Aβ ₄₀ (○) while changing the concentration ( C _o ) of the cutting agent D of Example 4 or Aβ ₄₂ (●) This is a graph showing the cleavage yield measured after reacting (4.0 μM) with pH 7.50 and 37 ° C for 36 hours, depending on log C _o / M.

FIG. 33 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleaving D (0.38 μM) of Example 4 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

34 is a graph showing that the cutting yields measured after changing the concentration ( C _o ) of the cutting agent D of Example 4 and reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours depended on the log C _o / M. This is a graph.

35 is a view schematically showing a synthesis process of the cutting agent E in Example 5 of the present invention,

FIG. 36 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleaving E (0.89 μM) of Example 5 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

37 is a graph showing that the cutting yields measured after changing the concentration ( C _o ) of the cutting agent E of Example 5 and reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours are dependent on log C _o / M. Is a graph showing

38 is a view schematically showing a synthesis process of the cutting agent F in Example 6 of the present invention,

39 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleaving F (1.6 μM) of Example 6 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

40 is a graph showing that the cutting yields measured after reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours while changing the concentration ( C _o ) of the cutting agent F of Example 6 depended on the log C _o / M Is a graph showing

41 is a view schematically showing a synthesis process of a cutting agent G in Example 7 of the present invention,

FIG. 42 is a MALDI-TOF mass spectrum of a product purified by HPLC after digesting G (0.89 μM) of Example 7 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

43 is a graph showing that the cutting yields measured after reacting Am (4.0 μM) with pH 7.50 and 37 ° C. for 36 hours while changing the concentration ( C _o ) of the cutting agent G of Example 7 depended on log C _o / M Is a graph showing

44 is a view schematically showing a synthesis process of the cutting agent H in Example 8 of the present invention,

FIG. 45 is a MALDI-TOF mass spectrum of a product purified by HPLC after cleaving H (7.1 μM) of Example 8 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours,

46 is a graph showing that cutting yields measured after changing the concentration of cutting agent H ( C _o ) of Example 8 with Am (4.0 μM) at pH 7.50 and 37 ° C. for 36 hours depend on log C _o / M. This is a graph.

Claims (15)

A compound of formula [Formula 1]

Where R is A, A- (Y) _o- (CH ₂ ) _p- (Y) _o -A, A- (CH = CH) -A, A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A and A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A- (Y) _o- (CH ₂ ) _p- (Y) _o -A independently selected target recognition unit, A is independently selected from the group consisting of C _1-4 alkyl, C _1-4 alkoxy, amino, mono or diC _1-8 alkylamino, C _6-12 cycloalkylamino, C _1-4 alkylcarbonyl and halogen Formula substituted or unsubstituted by one or more substituents

Is selected from the group consisting of: wherein X represents NH, O, or S, respectively Y represents O or N-Z ', wherein Z' represents hydrogen or C _1-9 alkyl; L represents a linker, Here, the linker is composed of a skeleton composed of 1 to 20 elements independently selected from the group consisting of carbon, nitrogen, oxygen, silicon, sulfur and phosphorus, and the atoms included in the skeleton are alkanes, alkenes, alkynes, carbonyls, thio Exists in the form of a functional group independently selected from the group consisting of carbonyl, amine, ether, silyl, sulfide, disulfide, sulfonyl, sulfinyl, phosphoryl, phosphinyl, amide, imide, ester and thioester, and also is a linker Is C _1-6 alkyl, C _1-6 alkoxy, mono or diC _1-6 alkylamino, C _1-6 alkylcarbonylamino, C _1-6 alkylsulfonylamino, C _1-6 alkylcarbonyl, carba Moyl, mono or diC _1-6 alkylcarbamoyl, C _1-6 alkoxycarbonylamino, ureido, mono or di or triC _1-6 alkylureido, C _1-6 alkylsulfanylcarbonylamino, C _1-6 alkylsulfanyl, C _1-6 alkyl di-sulfanyl, sulfamoyl, mono- or di-C _1-6 Al Sulfamoyl, tri C _1-6 alkyl carbonyl and silanol or halogen is substituted by one or more substituents independently selected from the group consisting of and unsubstituted; Z is a catalyst and represents a metal ion-ligand complex, Wherein the metal ion is Co ^III , Cu ^II or Pd ^II and the ligand is one or more substituents independently selected from the group consisting of C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkylcarbonyloxy and halogen Unsubstituted or substituted by

Selected from the group consisting of; n independently represents an integer of 1 to 6; m and o independently represent 0 or 1; p represents the integer of 0-5. delete delete delete delete delete delete delete delete The compound of claim 1, wherein at least one R, L and Z in the compound of Formula 1 is further substituted _by- (L) _m- (R) _n , wherein R, Z, L, m and n are selected from the Compounds as defined in claim. delete delete delete A pharmaceutical composition for preventing or treating Alzheimer's disease, type II diabetes, Parkinson's disease, cavernous encephalopathy or Huntington's disease, comprising the compound as defined in claim 1 or a pharmaceutically acceptable salt thereof. delete

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