KR20150041669A - Heterovalent inhibitors targeting N-end Rule Pathway and its use - Google Patents

Abstract

Disclosed is a compound represented by chemical formula 1 and a composition for inhibiting N-end rule pathway including the same. The compound according to the present invention is a heterovalent inhibitor for targeting simultaneously two active sites of N-end rule E3 ligases of a mammal which has a UBR box and a ClpS box for recognizing each substrate of type 1 and type 2. Accordingly, effectively controlling N-end rule pathway is possible by working mutual collaboratively, and the invention can be usefully used for treating a disease, which is related to proteolysis dysregulation such as a heart disease and a neurodegenerative disease.

Description

Heterovalent inhibitors targeting Heterovalent inhibitors targeting and N-terminal Rule Pathway and its use

The present invention is in the field of therapeutic agents for diseases caused by abnormal N-end rule regulation.

Decrease in protein concentration can occur by transcriptional reduction, translation reduction, and / or increased degradation. The vast majority of cellular proteins are degraded by the ubiquitin-proteasome system (UPS), which consists of substrate mobilization and substrate ε degradation machinery.

The ubiquitin-proteasome system (UPS) is the primary mechanism by which cells regulate the levels of individual proteins. Through UPS, cells maintain physiological function, adapt to environmental and pathological changes, and regulate protein quality. Substrate mobilization is mediated by three classes of enzymes: E1 activates ubiquitin, E2 acts as ubiquitin transporter, and E3 is ubiquitin protein ligase that attaches activated ubiquitin to protein substrates. Ubiquitinated proteins are recognized by the property of binding to ubiquitin and are targeted and degraded to lysosomes (Lys63 binding) or proteasome (Lys11, Lys29 or Lys48 binding) [Dahlmann, BMC Biochem. 22: Suppl 1: S3 (2007) and Miranda et al., Molecular Interventions 7: 157-167 (2007).

In such a UPS, the target protein is covalently bound to the ubiquitin through an isopeptide bond between the C-terminal carboxylic acid of ubiquitin (Ub) and the ε-amino group of the internal Lys of the target protein. This ubiquitin process is first mediated by the Ub-charged E2 Ub conjugating enzyme and the substrate-specific E3 Ub ligase, and this process is also reversed by deubiquitinating enzymes (DUBs). Thus, the selectivity of most ubiquitin-mediated proteolysis is first regulated by the time and spatial activation of the degradation signal (degron) recognized by the specific E3 Ub ligase.

The N-end Rule pathway is a proteolytic pathway that degrades a protein with an N-terminal amino acid, which is related to the in vivo half-life of the protein. N-terminal amino acids have been recognized as being essential for degradation signals (N-degron) that generate the N-end Rule pathway.

The UPS and N-end Rule pathway is a major mechanism for degrading damaged proteins and eliminating dysfunctional proteins produced by transcription, translation and / or folding errors. When the UPS or N-end Rule pathway is functioning abnormally, accumulation of denatured protein followed by agglutination is considered to play an important role in many age-related diseases, including neurodegenerative diseases. For example, it was assumed that beta amyloid accumulation in Alzheimer's disease is partly a result of loss of UPS and / or lysosome processing to remove A [beta] aggregates. Pathological protein accumulation is also involved, for example, in a number of other neurodegenerative diseases such as Parkinson's disease (a leucocyte containing alpha synuclein) and Huntington's disease (Huntingtin protein) [Upadhya et al., BMC Biochem. 22: Suppl 1: S1 (2007)]. BACE degradation is also mediated by the ubiquitin proteasome pathway [Qing, H. et al., FASEB J. 18: 1571-1573 (2004)].

International Patent Application Publication No. WO 2010/132390 relates to a method for reducing the concentration of ubiquitinated protein, which comprises administering a heterocyclic compound to a subject in need of a decrease in the concentration of the ubiquitinated protein, To a method for reducing the level of the < / RTI >

United States Patent Application Publication No. US2002-0233730 discloses a method for controlling autophagy by monitoring the change of autophagosome in a cell after administering a test substance to a cell, .

As mentioned above, inhibition of the N-end Rule pathway can treat a wide variety of diseases, and therefore, there is a need to develop new therapeutic agents and methods targeting substances involved in the inhibition of the N-end Rule pathway.

The present invention provides heterobalant inhibitors targeting the N-end rule pathway and uses thereof.

In one embodiment, the present invention provides a compound of formula 1: < EMI ID =

[Chemical Formula 1]

In Formula 1,

n is an integer from 1 to 15;

Ra and Rb are different

Ra is H, C ₁ -C ₇ alkyl-NHC (= NH) NH 2, C ₁ -C ₇ alkyl-NH 2, C ₁ -C ₅ alkylphenyl or heteroaryl, said phenyl and heteroaryl being optionally substituted by hydrogen, C ₁ -C ₅ alkyl;

R b is C ₁ -C ₈ alkyl, C ₁ -C ₇ alkylphenyl, or heteroaryl, wherein said alkyl and phenyl are optionally substituted with hydrogen, hydroxy, halogen, or C ₁ -C ₅ alkyl;

or

Ra is C_One~ C₈Alkyl, C_One~ C₇Alkyl, phenyl or heteroaryl, said alkyl and phenyl being optionally substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, or C_One~ C₅Alkyl;

R b is H, C ₁ -C ₇ alkyl-NHC (═NH) NH ₂ , C ₁ -C ₇ alkyl-NH ₂ , C ₁ -C ₅ alkylphenyl or heteroaryl, , Or C ₁ to C ₅ alkyl; And

Rc is biotin or a derivative thereof, or C ₁ -C ₅ alkyl, said alkyl being optionally substituted by hydrogen, halogen, or C ₁ -C ₅ alkyl.

In one embodiment according to the present application, in the compound of formula (I), wherein Ra is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, 2-imidazolyl , 3-imidazolyl, 4-imidazolyl, 2-pyrrole, 3-pyrrole, 2-pyridine, 3-pyridine, 4-pyridine, 2-pyrimidine, 3-pyrimidine or 4-pyrimidine; Wherein Rb is methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, benzyl, 2- hydroxyphenylmethyl, 3- hydroxyphenylmethyl, ; Rc is methyl, ethyl, n-propyl, or isopropyl; Or Ra is a group selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, benzyl, 2- hydroxyphenylmethyl, 3-hydroxyphenylmethyl, ego; Rc is methyl, ethyl, n-propyl, or isopropyl; Wherein R b is selected from the group consisting of H, - (n-propyl) -NHC (= NH) NH ₂ , - (n-butyl) NH ₂ , benzyl, 2-imidazolyl, Or a pharmaceutically acceptable salt thereof, wherein the compound is a pyrrole, 3-pyrrole, 2-pyridine, 3-pyridine, 4-pyridine, 2-pyrimidine, 3-pyrimidine or 4-pyrimidine.

In another embodiment, in the compound of formula 1, n is an integer from 1 to 15; Wherein Ra is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, or 4-imidazolyl; Rb is isopropyl, 2-butyl, benzyl, 4-hydroxyphenylmethyl, or 3-indole; Or Ra is isopropyl, 2-butyl, benzyl, 4-hydroxyphenylmethyl, or 3-indole; Wherein Rb is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, or 4-imidazolyl, and; Wherein Rc is methyl, or ethyl. (1), or a pharmaceutically acceptable salt thereof.

In another embodiment, in the compound of formula 1, n is 4; Wherein Ra is - (n- _{propyl) -NHC (= NH) NH 2} ; wherein Rb is benzyl; Or wherein Ra is benzyl; wherein Rb is - (n- propyl) -NHC (= NH) NH _2, and, the Rc is methyl acceptable salts, chemical compounds of formula (I), or a medicament.

The compounds according to the present invention are heterologous inhibitors which simultaneously target two active sites of the mammalian N-end rule E3 ligase with a UBR box and a ClpS box recognizing type 1 and type 2 substrates, respectively, It is possible to effectively control the N-end Rule pathway.

Accordingly, in another aspect, the present invention provides a pharmaceutical composition for inhibiting, or modulating, the N-end Rule pathway, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention. Such a composition may be used for the treatment of various diseases requiring N-end Rule pathway control such as heart diseases such as hypertension, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, atherosclerosis (arteriosclerosis) Or arrhythmia; Or neurodegenerative diseases such as proteinopathies such as Alzheimer's disease, Parkinson's disease, Levy's somatic dementia, muscular atrophy sclerosis (ALS), Huntington's disease, spinal cord cerebral ataxia or spinobulbar musculular atrophy, Can be effectively used for prevention or treatment.

The compounds according to the present invention are heterologous inhibitors which simultaneously target two active sites of the mammalian N-end rule E3 ligase with a UBR box and a ClpS box recognizing type 1 and type 2 substrates, respectively, It is possible to effectively control the N-end Rule pathway. The heterotrophic inhibitors according to the present invention are cytotoxic, cell-passable in mammalian cells and can significantly delay degradation of the degradation substrate of the N-end rule substrate. It is also stable against endopeptidase and has a high inhibition efficiency, and can bind ligands to binding motifs and can act efficiently on both type 1 and type 2 substrates. Therefore, the compounds according to the present invention can be useful for the treatment and prevention of heart diseases, neurodegenerative diseases and the like which are closely related to the abnormal N-end rule pathway.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the HPLC profile of 20% water in acetonitrile solution of the compound of Example 1 (RF-C2) according to one embodiment of the present invention.
Figure 2 shows the HPLC profile of a 20% solution of the compound of Example 2 (RF-C3) in water in an acetonitrile solution according to one embodiment of the present invention.
Figure 3 shows the HPLC profile in a 100% methanol solution of the compound of Example 3 (RF-C3) according to one embodiment herein.
Figure 4 shows the HPLC profile of the compound (RF-C8) of Example 4 in a methanol solution according to one embodiment of the invention.
Figure 5 shows the HPLC profile of a 15% solution of the compound of Example 5 (RF-C9) in water in an acetonitrile solution according to one embodiment of the invention.
Figure 6 shows the HPLC profile in a 100% methanol solution of the compound (RF-C11) according to one embodiment of the present invention.
Figure 7 shows the HPLC profile in a 100% methanol solution of the compound of Example 6 (RF-C15) according to one embodiment of the invention.
Figure 8 shows the HPLC profile in a 100% methanol solution of the compound of Example 7 (GV-C5) according to one embodiment of the invention.
Figure 9 shows the HPLC profile in a 100% methanol solution of the compound of Example 8 (RR-C5) according to one embodiment of the invention.
Figure 10 shows the HPLC profile in a 100% methanol solution of the compound of Example 9 (FF-C5) according to one embodiment of the invention.
11A shows generation of the generation of a model N-end rule substrate, X-nsP4, according to one embodiment of the present invention. Using rabbit reticulocyte fusants, ^f DHFR ^h -Ub ^R48 -X-nsP4 ^f The fusion proteins (superscripts "h" and "f" represent HA and flag epitopes, respectively, and X represents various amino acid residues) were expressed in Invitro by the deubiquitination element on the Ub-X junction The DHFR-Ub reference group (B, lower band) and the short-lived X-nsP4 substrate (X is a destabilizing residue of the N-end rule pathway such as Arg)
FIG. 11B shows in vitro decomposition of a model N-end rule substrate according to one embodiment of the present invention. Arg-nsP4, a type 1 model substrate in the N-end rule pathway, was specifically stabilized by 2mM of Arg-Ala dipeptide but was not specifically stabilized by Ala-Arg (stabilized) dipeptide or Trp-Ala Type 2) did not. The newly synthesized protein was labeled with ³⁵ S-methoin. SDS-PAGE / autoradiography.
FIG. 11C shows the level of Arg-nsP4 in FIG. 11B quantified using a Phosphorimager and normalized to DHFR.
Figure 11d shows immunoprecipitation and anti-flag immunblotting using anti-Ub chains. The ubiquitinated Arg-nsP4 was mainly detected in the absence of the Arg-Ala dipeptide inhibitor. * Denotes a nonspecific signal.
Figure 11e shows the specific effects of Arg-Ala (type 1) and Trp-Ala (type 2) on Met-nsP4, Arg-nsP4, and Tyr-nsP4 ubiquitination in anti- Lt; RTI ID = 0.0 > immune < / RTI > Invitro transcription / translation reactions were performed in the presence of 100 [mu] M vestatin and 5 [mu] M MG132. STV = streptavidin.
Figure 11f quantifies the relative amount of ubiquitinated X-GFP in Figure < RTI ID = 0.0 > 11e. ≪ / RTI > Film images were quantified with densitometry.
Fig. 11g is a visualization of ubiquitinated Arg-nsP4 by immunoprecipitation using an anti-flag antibody and immunoblotting with HA. The type 1 model substrate, Arg-nsP4, was actively ubiquitinated in the cells. The plasmids expressing HA-Ub and Arg-nsP4 were simultaneously transferred to wild-type mice in the presence / absence of MG132.
Figure 12 shows an in silico docking analysis and biochemical analysis to demonstrate unstable moieties.
12A shows a combining mode of UBR2 box (PDB code: 3NY3) with (a) RIFS, (b) KIFS, (c) HIFS, and (d) WIFS.
FIG. 12B shows a combining mode of UBR1 box (PDB code: 3NY1), (a) RIFS, (b) KIFS, (c) HIFS, and (d) WIFS.
FIG. 12C shows the binding affinity (kcal / mol) and dissociation constant (μM) of the type 1 peptide with UBR2 (a) and UBR1 box (b) in comparison with experimental data.
12D shows the coupling mode of the ClpS domain (PDBcode: 3DNJ) with (a) WLFV, (b) FLFV, (c) YLFV, and (d) LLFV. The YLFV peptide structure was extracted from the complex of ClpS with another peptide made from PyMOL.
12E shows the binding affinity (kcal / mol) and dissociation constant ([mu] M) of Type 2 peptide and ClpS box in comparison with experimental data. RLFV type 1 peptides were found to be inadequate to bind to the ClpS domain.
Fig. 12F shows the stable X-nsP4 level as a densitometry. Corresponding DHFR levels were used to standardize. Pro level was derived from DHFR-Ub-Pro-nsP4, but the Un site behind Pro was not cleaved.
13A shows the structures of RF-Cn compounds, control compounds and dipeptide inhibitors. N-terminal Arg and Phe, Type 1 and Type 2 destabilizing residues are shown as orange and green backgrounds, respectively, and Gly and Val are shown as yellow background.
Figure 13b shows the relative amounts of Arg-nsP4 (Type 1) quantified after treatment of various RF-Cn compounds at 10, 100, and 250 μM.
Figure 13c shows the relative amounts of Tyr-nsP4 (Type 2) quantified after treatment of various RF-Cn compounds at 10, 100, and 250 μM.
Figure 13d shows two possible UBR2-ClpS structures and RF-C5 binding obtained using the Gramm-X protein-protein docking web server and the semi-experimental PM6 method of the Gaussian 09 program.
Figure 13E shows the calculated binding affinities (kcal / mol) and dissociation constants (μM) of RF-C5 and the two possible UBR2-ClpS protein stereochemistry (a) and (b) of Figure 3E.
Figure 13f shows that inhibition was significantly increased via heterobalance in both Type 1 (Arg-nsP4) and Type 2 (Tyr-nsP4) model substrates. Results from in vitro transcription / translation studies with SDS-PAGE / immunoblot using Odyssey infrared imaging system.
Figure 14a shows that RF-C11 forms micelles in aqueous solution, while RF-C5 does not. Average particle size was measured by density-course analysis with dynamic light scattering (DLS).
FIG. 14B shows the result of measurement of the cytotoxicity of a heterobalant compound using MTT analysis.
14C is a graph showing the effect of RF-C11 on intracellular primary cardiac myocytes (endocytosis) using biotin-GV-C11 / RF-C11 Red, respectively b and c), and anti-troponin I (green).
Fig. 14D is a result showing that transiently overexpressed RGS4 is stabilized by RF-C11. Fig.
Figure 14E shows the results of pulse chase analysis of RGS4, a physiological N-end rule substrate in MEF.
14F is a result of quantifying the data shown in FIG. 14D using a PhosphorImager.

It should be noted that the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiments of the present invention and are not intended to represent all of the technical ideas of the present invention so that various equivalents And variations are possible.

In this paper, the interaction between Type 1 and UBR boxes and the interaction between ClpS domain and Type 2 are comparable using computer nano-docking analysis and biochemical analysis using model n-end rule path based on nsP4, Terminal < RTI ID = 0.0 > His < / RTI > functions as a destabilizing moiety.

In one embodiment, the present invention relates to a compound of formula 1: < EMI ID = 2.1 >

Wherein n is an integer from 1 to 20, particularly an integer from 1 to 15;

Ra and Rb are different,

Wherein Ra is H, C ₁ -C ₇ alkyl-NHC (= NH) NH ₂ , C ₁ -C ₇ alkyl-NH ₂ , C ₁ -C ₅ alkylphenyl or heteroaryl, , Or C ₁ -C ₅ alkyl, preferably Ra is H, - (n-propyl) -NHC (= NH) NH ₂ , - (n-butyl) NH ₂ , benzyl, Pyridine, 3-pyridine, 4-pyridine, 2-pyrimidine, 3-pyrimidine or 4-pyrimidine, more preferably, Ra is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, or 4-imidazolyl, and;

R b is C ₁ -C ₈ alkyl, C ₁ -C ₇ alkylphenyl, or heteroaryl, said alkyl and phenyl being optionally substituted with hydrogen, hydroxy, halogen, or C ₁ -C ₅ alkyl, preferably Rb Is methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, benzyl, 2- hydroxyphenylmethyl, 3- hydroxyphenylmethyl, Preferably, Rb is isopropyl, 2-butyl, benzyl, 4-hydroxyphenylmethyl, or 3-indole; And

Rc is biotin or a derivative thereof or C ₁ -C ₅ alkyl, said alkyl being optionally substituted by hydrogen, halogen, or C ₁ -C ₅ alkyl, preferably Rc is methyl, ethyl, n- , Or isopropyl, and more preferably methyl or ethyl.

In one embodiment according to the present disclosure, n is 4, Ra is - (n-propyl) -NHC (= NH) NH ₂ , and Rb is benzyl; Wherein Rc is methyl.

Substituents located at each of Ra and Rb in the compounds of formula (I) according to the present invention described above may be used interchangeably. That is, the substituent of Ra and Rb is not limited as defined above, and the substituent of Ra may be located at Rb, and vice versa.

The compounds of formula (I) herein include, in addition to the compounds of formula (I), their pharmaceutically acceptable acid addition or base addition salts, their solvates, optical isomers thereof.

Pharmaceutically acceptable acid addition salts comprise a therapeutically active non-toxic addition salt which may be formed by a compound of formula I wherein such salts are prepared by reacting a compound of formula I in base form with a suitable acid, Lt; RTI ID = 0.0 > free acid. ≪ / RTI > Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propionate, naphthalene-1, < / RTI > < RTI ID = 0.0 & -Sulfonate, naphthalene-2-sulfonate or mandelate.

Conversely, the acid addition salt can be converted to the free base form by treatment with an appropriate base.

Compounds of formula (I) containing an acidic proton can be converted into their therapeutically active non-toxic metal or amine addition salt forms (salt addition salts) by treatment with an appropriate organic or inorganic base. Suitable basic bases include, for example, ammonium salts, alkali and alkaline earth metal salts, especially lithium salts, potassium, magnesium and calcium salts, salts with organic salts such as benzathine, N-methyl-D-glucamine, Salts, and salts with amino acids such as, for example, arginine and lysine.

Also included are quaternary ammonium salts of the compounds of Formula 1 herein. The quaternary ammonium salt can be obtained by reacting a basic nitrogen present in the compound of formula (I) with a suitable quaternizing agent. The quaternizing agents include alkyl halides, aryl halides, or aryl alkyl halides, such as methyl iodide, benzyl iodide, alkylcfluoromethanesulfonate, alkyl methanesulfonate, and alkyl p-toluenesulfonate . Quaternary ammonium salts carry a positively charged nitrogen, and pharmaceutically acceptable counter ions include chloro, bromo, iodo, trifluoroacetate, and acetate ions.

Conversely, the compound in the form of the salt may be converted to the free form by treatment with an appropriate acid.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound of the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, , Or may be prepared by drying, or by distillation of the solvent and excess acid under reduced pressure, followed by drying or crystallization in an organic solvent.

Also included are all possible solvates which may be prepared from the compounds of formula (1) herein. Solvates include hydrates, alcoholates, and the like.

Also included are all optical isomers of the compound of Formula 1 herein. The optical isomers herein include all possible isomers of the compound of formula (I). The stereoisomer of the optical isomer has an R- or S-configuration.

The compound of Formula 1 may be synthesized according to the following Reaction Scheme 1, 2 or 3.

[Reaction Scheme 1]

According to the above Scheme 1, N-carbonylation and N-deprotection reactions, a second N-carbonylation and N-deprotection reaction, a third N-carbonylation N-deprotection reaction, And an N-deprotection reaction, and an N-deprotection reaction in that order.

[Reaction Scheme 2]

According to Reaction Scheme 2, the synthesis of the 12th series compound is carried out in the order of esterification reaction, amination reaction, N-carbonylation reaction and hydrolysis reaction, and N-carbonylation, And then the N-carbonylation reaction and the N-deprotection reaction of the compound 12 and the compound 16 are carried out to obtain the final compound Were synthesized.

[Reaction Scheme 3]

According to Reaction Scheme 3, the reaction is carried out in the order of an esterification reaction, an amination reaction, an N-carbonylation reaction, a hydrolysis reaction, an N-carboronylation reaction, and an N-deprotection reaction.

As used herein, the term " N-carboronylation reaction " refers to the reaction of introducing a carbonyl group (- (C = O) -) into N, resulting in an amide bond.

The term "N-deprotection reaction" as used herein refers to a reaction for removing the protecting group when a protecting group is introduced into the N of the amine. The reaction conditions for the deprotection reaction depend on the type of the protecting group.

The term "ester reaction" as used herein refers to a reaction for forming an ester group, and an ester group can be formed through an O-alkylation reaction or the like.

The term " hydrolysis reaction " as used herein refers to a reaction in which an ester group is decomposed to form a carboxylic acid.

The term " amination reaction " as used herein refers to a reaction in which an amine group (-NH2) is introduced into an alkyl group.

As used herein, the term " Z " refers to a protecting group for N of an amine, such as a carbobenzyloxy (Cbz) group, a p-methoxybenzylcarbonyl (Moz or MeOZ) group, a tert- butyloxycarbonyl (BOC) group, a carbamate group, p-methoxybenzyl (PMB), 3,4-dimethoxy Benzyl (DMPM), p-methoxyphenyl (PMP) group, tosyl (Ts) group, and other sulfonamide (Nosyl & A benzyl group was used in the examples according to one embodiment of the present invention.

In another aspect, the present invention relates to a pharmaceutical composition for inhibiting the N-end Rule pathway comprising the compound of formula (1).

The N-end rule pathway herein refers to a proteolytic pathway that degrades a protein having a specific destabilizing moiety, i.e., N-degron, at its N-terminus. The substrate of the N-end rule pathway is recognized by a protein called N-recognin with an N-degron binding region called the UBR box. This UBR box recognizes the substrate through its interaction with the first two residues of the substrate, which is called the N-end rule. In the N-end rule pathway, protein substrates with specific N-terminal amino acids called destabilizing residues have a short life span. The destabilizing residues in eukaryotes are classified as Type 1, a positively charged amino acid such as Arg, Lys, or His, and Type 2, a bulky hydrophobic amino acid such as Phe, Leu, Tyr, and Ile.

The heterotrophic inhibitor of the present invention targets both the recognition motifs of the UBR 1 and 2 proteins while having a different length of tether. One end of the tetra is an amino acid group of type 1, and the other end is mainly of type 2 amino acid group (or vice versa), so that the UBR box and the ClpS box each have a simultaneous interaction with each other, thereby inhibiting the N-end rule path.

The N-end Rule pathway is a major mechanism for disrupting damaged proteins along with UPS and eliminating dysfunctional proteins produced by transcription, translation and / or folding errors. When the N-end Rule pathway is functioning abnormally, accumulation of denatured protein followed by progressive aggregation appears to play an important role in many age-related diseases, including heart disease and neurodegenerative diseases (MJ Lee, et al (2012), J. Biol. Chem., 287, 24043; Komatsu et al., (2006), Nature, 441, 880-884). Fortunato and Kroemer (2009), Autophagy, 5 (6)) have been reported to be associated with liver disease, heart disease, muscle disease and pancreatic disease (Levine and Kroemer, .

In another aspect thereof, the present invention relates to a composition for preventing and treating heart disease, which comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof. Such heart diseases include, but are not limited to, hypertension, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, atherosclerosis (atherosclerosis), cerebrovascular disease, stroke, and arrhythmia.

In another aspect, the present invention relates to a composition for preventing and treating neurodegenerative diseases, comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof. The neurodegenerative diseases include, but are not limited to, adrenal leukodystrophy, alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-related dementia, Kennedy's disease ), Krabbe's disease, Lewy body dementia, neuroborreliosis, Machado-Joseph disease, multi-system dysfunction (multi ple system atrophy, multiple sclerosis, narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease Pick's disease, primary lateral sclerosis, prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease ), Spontaneous anemia secondary to spinal cord secondary to pernicious anemia of spinal cord, Spielmeyer-Vogt-Sjogren-Batten disease, spinal cord cerebral ataxia but are not limited to, spinocerebellar ataxia, spinal muscular atrophy, Steele-Richardson-Olszewski disease, Tabes dorsalis, toxic encephalopathy, .

In another aspect, the present invention relates to a composition for the prevention and treatment of proteinopathy associated with protein denaturation, comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof. Diseases associated with protein denaturation include, but are not limited to, Alzheimer's disease, Parkinson's disease, Levy's somatic dementia, ALS, Huntington's disease, spinal cord cerebral ataxia and spinobulbar muscular atrophy, .

As used herein, the term "treatment" refers to any action that improves or alters the relevant symptoms by administration of a composition according to the invention. Those skilled in the art will be able to ascertain, by reference to the data provided by the Korean Medical Association, the precise criteria of the disease for which the composition of the present invention is effective, .

The term "prophylactic," as used herein, refers to any act that inhibits or delays the onset of a related disorder upon administration of a composition according to the present disclosure. It will be apparent to those skilled in the art that compositions of the present invention that are effective in the removal of denatured proteins due to autophagy abnormalities can prevent such diseases if they are taken prior to the appearance of the symptoms or symptoms of the disease due to accumulation of denatured proteins.

Accordingly, the pharmaceutical composition of the present invention may be administered simultaneously or sequentially, and may be administered in combination with other pharmaceutically active ingredients for treating the diseases.

The therapeutic agent or pharmaceutical composition according to the present invention may be formulated into a suitable form together with a commonly used pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " refers to compositions which are physiologically acceptable and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. Examples of pharmaceutically acceptable carriers include, for example, water, suitable oils, saline, aqueous carriers for parenteral administration such as aqueous glucose and glycols, etc., and may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the composition according to the present invention may contain various additives such as a suspending agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, an interface activator, a diluent, an excipient, a pH adjuster, An antioxidant, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington ' s Pharmaceutical Sciences, Current Edition.

The composition of the present invention may be prepared in a unit dosage form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Into a capacity container. The formulations may be in the form of solutions, suspensions or emulsions in oil or aqueous media, or in the form of powders, granules, tablets or capsules.

The method of administration of the pharmaceutical composition of the present invention can be easily selected according to the formulation, and can be administered to mammals such as livestock, human, and the like in various routes. For example, it may be formulated in the form of powders, tablets, pills, granules, dragees, hard or soft capsules, liquids, emulsions, suspensions, syrups, elixirs, external preparations, suppositories, sterilized injection solutions, Or parenterally, and parenteral administration may be particularly preferable.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol, gelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease, 60 kg), it is about 1 ng to 10 mg / day, especially about 1 μg to 1 mg / day. It will be apparent to those skilled in the art that doses may be additive or subtracted, as the dosage can vary depending on various conditions, and thus the dose is not intended to limit the scope of the invention in any way.

The number of administrations can be administered once or several times a day within a desired range, and the administration period is not particularly limited.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

The experimental methods and materials used in the Examples are as follows.

Experimental Methods and Materials

One. In bito  Degradation / ubiquitination assay

The degradation analysis with time- and concentration-dependent bits was performed as follows. Briefly, a biotin-lysine-tRNA complex (Transcend tRNA, Promega) was added to the reaction mixture to randomly label the DHFR-Ub-X-nsP4 fusion protein with biotin. The biotinylated protein was detected with streptavidin (Pierce) conjugated horseradish peroxidase (HRP). For quantitative immunoblotting, protein was visualized by infrared fluorescence using an oddity imaging system and IR die-conjugated streptavidin (Li-COR). It was added to ³⁵ S- methionine (Amersham Bioscience), and in vitro transcription to supplement (supplement) / translation reaction to the self-emitting (autoradiographic) analysis. To produce the DHFR-Ub-X-nsP4 plasmid, X-nsP4 was amplified by PCR, digested with SmaI / XbaI and subcloned into the UPR vector pcDNA3 (dEheI) FDHUMCM. The ubiquitination of the test protein was characterized in the presence of 5 [mu] M MG132 followed by anti-Ub immunoprecipitation and anti-biotin Western blotting.

2. Expression of N-end rule model substrate in mammalian cells

Using Lipofectin 2000 (Invitrogen), mammalian cells such as MEFs and cardiac cells were transiently transfected for 4-6 hours until cells were > 95% confluent in 6- And transferred into plasmid DNA. Post transfection Cell lysates were collected in RIPA buffer at approximately 36 hours and used for immunoblotting or immunoprecipitation. Total ubiquitinated Arg-GFP concentration was detected in the presence of 10 μM MG 132. For pulse-chase analysis, cells were labeled after 35 hours of treatment with ³⁵ S-methionine / cysteine ( ³⁵ S-expression protein label mix; Perkin-Elmer), followed by cycloheximide chase . Whole cell extracts were prepared and immunoprecipitated, followed by SDS-PAGE and autoradiography as described above.

3. In Silico  Docking analysis of protein-protein and protein-ligand interactions

The crystal structure of the UBR1, UBR2, and Clps boxes was obtained in Protein Data Bank (PDB ID: 3NY1, 3NY3, and 3DNJ, respectively). Since the "bound" UBR2-ClpS structure is not known, UBR2 and ClpS boxes were docked using a Gramm-X protein-protein docking web server. We have found two possible UBR2-ClpS structures with the most stable binding energy. The RIFS and YLFV peptide structures were extracted from the complex with UBR2 box and ClpS, respectively. Other peptides were made with the Mutagenesis module in the PyMOL package (http://sourceforge.net/projects/pymol/). The RF-C5 structure was optimized with the Gaussian 09 program using the semi-empirical PM6 method.

의 회전 단계 범위, 1의 엘리티즘(elitism), 0.02의 돌연변이 속도, 0.8의 크로스 오버 속도, 0.06의 국소 탐색 속도 및 5천만 에너지 이벨류에이션. 2.0 Å RMSD 의 톨러런스(tolerance)를 사용하여 도킹된 입체구조를 클러스터시켰다. 가장 많은 클러스터의 가장 낮은 결합에너지를 실험 데이타와 비교하였다. 하기 방정식에 근거한 해리 상수(dissociation constant)로부터 실험적 결합 자유 에너지를 계산하였다: △G = RT lnK_d, 상기에서, R은 기체 상수(1.987 cal K^-1 mol^-1)이고 T는 온도(298.15K)이다. PBD2PQR 웹 서버를 사용하여 pH=7에서의 PQR 파일 및 AMBER 포스 필드(force field)를 만들었다. 정전기 전위는 APBS 패키지를 사용하여 계산하였다. 모든 구조 도면은 PyMOL 패키지를 사용하여 제조하였다.
Polarhydrogen was added using AutoDockTools version 1.5.4 and Gasteiger charges assigned to proteins, peptides and RF-C5. For each protein, an AutoGrid version 4.2 was used to create an affinity grid centered on the active site. The grid was large enough to contain all active sites. We simulated ligand-receptor docking using AutoDock version 4.2 with the Lamarckian genetic algorithm. The docking parameters are as follows: 200 docking test, 300 population size, random starting position and steric structure, 2.0 A translational step range, 50

A rotation step range of 1, an elitism of 1, a mutation rate of 0.02, a crossover rate of 0.8, a local search speed of 0.06 and a valence of 50 million. The docked steric structure was clustered using a tolerance of 2.0 A RMSD. The lowest binding energy of the largest number of clusters was compared with experimental data. Experimental binding free energy was calculated from the dissociation constant based on the following equation: ΔG = RT lnK _d , where R is the gas constant (1.987 cal K ^-1 mol ^-1 ) and T is the temperature (298.15K )to be. A PQR file at pH = 7 and an AMBER force field were generated using a PBD2PQR web server. The electrostatic potential was calculated using the APBS package. All structural drawings were prepared using the PyMOL package.

4. Measurement of cell viability

Cell viability was assessed using modified MTT assay. RF-C11 and GV-C11 DMSO concentrations at various concentrations (from 0.1 [mu] M to 1000 [mu] M) were added to the cells. After incubation for 4 hours, 25 μL of a 5 mg / mL MTT solution was added to the sample and the plate was incubated at 37 ° C. for 2 hours. Triazolyl blue tetrazolium bromide (MTT, Sigma Aldrich) was added to each well (final concentration 0.5 mg / mL) and the humidity of 95% atmosphere and 5% CO2 was incubated for 2 hours at 37 ° C under controlled conditions. HCl isopropanol solution (0.08 N) was added to dissolve the blue MTT-fomarzan product and the cells were incubated at room temperature for 30 minutes. The absorbance of the solution was found at 570 nm (test) and 630 nm (reference).

5. Synthesis and Analysis of Hetero-Balanced Inhibitors

 Amino acids with a protecting group were purchased from Novabiochem. Organic solvents were purchased from Ranbaxy Fine Chemicals and other compounds were purchased from Sigma-Aldrich. 1H NMR spectra were recorded using a Varian FT 200 MHz or Bruker FT 300 MHz. Mass spectra were measured by Micro mass QuattroLC (ESI). Analytical TLC was developed on a silica gel coated aluminum plate (Merck). Purity of the final product was determined using a Varian Prostar HPLC with a Prostr 325 UV-Vis detector at a flow rate of 1 ml / min in a Hibar Purospher STAR column (4.6 mm-250 mm, RP-18e) .

Dichloromethane, DIPEA, DMF, DMSO, N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride, HOBt (1-hydroxy-1H-benzotriazolehydrate) rt room temperature, TFA (trifluoroacetic acid), THF (tetrahydrofuran), TMS-OTf (trimethylsilyltrifluoro methanesulfonate), HATU (1- [Bis (dimethylamino) methylene] -1H-1,2,3- b] pyridinium 3-oxid hexafluorophosphate).

Amino acids described herein are also abbreviated as follows: Amino acids used herein are as follows: Ala: A. Arg: R, Asp: D, Asn; N, Cys: C, Gly: G, Glu: E, His: H, Ile: I, Ile: I, Leu: L, Lys: K, Met: Thr: T, Tyr: Y. Trp: W, Val: V.

Example 1. Synthesis of Compound 8a (RF-C2)

1. Synthesis of N (ε) -Z-Lysine (-OMe) C2-NHBoc (1a)

HOBt (0.463 g, 3.02 mmol) and EDCI (0.579 g, 3.02 mmol) were successively added with stirring to ice-cold dry DCM solution of Boc-Gly-OH (0.529 g, 3.02 mmol). After a half hour N (ε) -Z-lysine methyl ester hydrochloride (1 g, 3.02 mmol) dissolved in dry DCM was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly basic. The resulting solution was stirred overnight at room temperature. The solution was then diluted with DCM and washed successively with 1N HCl, water and brine. The organic layer was dried to evaporate the solvent. The residue was purified by column chromatography using a 1% methanol in chloroform solution. The yield of pure N (ε) -Z-Lysine (-OMe) C2-NHBoc was 92.2%, which was a gummy yellow gummy compound. (Rf = 0.55 in 5% methanol in chloroform).

^{1 HNMR (200MHz, CDCl3):} δ = 1.2-1.5 [m, 13H], 1.75-1.9 [m, 2H], 3.1-3.3 [m, 2H], 3.65-3.8 [m, 5H], 4.4-4.6 [ 1H, s, 2H], 5.1-5.3 [m, 1H], 5.35-5.5 [m, 1H], 6.9-7.1 [d, 1H], 7.2-7.5 [s, 5H]. ESI-MS: m / z = 452.7 (calculated value for C22H33O7N3 = 451.5).

2. Synthesis of N (ε) -Z-Lysine (-OMe) C2-NH2 (2a)

To a dry DCM solution of N (ε) -Z-Lysine (-OMe) C2-NHBoc (1.2 g, 2.66 mmol) was slowly added TFA at 0 ° C. and the mixture was stirred at room temperature for 3 hours. The nitrogen gas was then flushed to remove DCM and excess TFA. The resulting gummy residue was purified by chromatography using a 2% methanol in chloroform solution. 0.84 g (90% yield) of N (ε) -Z-Lysine (-OMe) C2-NH2 in yellow solid. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 352.7 (calculated value for C17H25O5N3 = 351.4).

3. Synthesis of N (ε) -Z-Lysine (-OMe) C2F (3a)

HOBt (0.29 g, 1.91 mmol) and EDCI (0.37 g, 1.91 mmol) were added successively with ice cooling to a dry DCM solution of Boc-Phe-OH (0.51 g, 1.91 mmol). After half an hour N (ε) -Z-Lysine (-OMe) C2-NH2 (0.84 g, 2.39 mmol) dissolved in dry DCM was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly basic. The resulting solution was stirred overnight at room temperature. It was then diluted with DCM and washed successively with 1N HCl, water and brine. The organic layer was dried and the organic solvent was evaporated. The residue was chromatographed using a 2% methanol in chloroform solution to give 1.079 g (75.5% yield) of pure N (ε) -Z-Lysine (-OMe) C2F as a pale yellow liquid. ^{1 HNMR (200MHz, CDCl3):} δ = 1.2-1.6 [m, 11H], 1.6-1.9 [m, 4H], 2.8-3.0 [m, 2H], 3.05-3.15 [m, 2H], 3.6 [s, 2H], 5.2-5.3 [m, 1H], 5.4-5.55 [m, 2H] d, 1H], 6.9-7.4 [m, 12H]. ESI-MS: m / z = 622 [M + Na < + >] (calculated value for C31H42O8N4 = 598.7).

4. Synthesis of Lysine (-OMe) C2F (4a)

N (?) - Z-Lysine (-OMe) C2F (1 g, 1.67 mmol) was dissolved in HPLC grade methanol (15 ml). The air in the container was removed by flushing with nitrogen several times. Charcoal 10% Pd (0.4 g) was carefully added to the solution and the inner wall of the flask was rinsed with HPLC grade methanol. The air in the flask was removed by flushing the nitrogen again. Hydrogen gas was then passed through the reaction mixture and left to stir overnight. It was then filtered through celite and washed with HPLC grade grade methanol. The filtrate and the washing solution were combined and the organic solvent was evaporated to obtain 0.816 g (90%) of Lysine (-OMe) C2F on a white semi-solid basis. This was used directly in the next reaction without further purification. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 465.9 (calculated value for C23H36O6N4 = 464.6).

5. Synthesis of Lysine (-OMe) C2FC2-NHZ (5a)

HOBt (0.183 g, 1.2 mmol) and EDCI (3.5 g, 1.2 mmol) were successively added with stirring to ice-cold dry DCM solution of Z-Gly-OH (0.25 g, 1.2 mmol). Lysine (-OMe) C2F (0.695 g, 1.5 mmol) dissolved in dry DCM after half an hour was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly basic. The resulting solution was stirred overnight at room temperature. It was then diluted with DCM and washed successively with 1N HCl, water and brine. The organic layer was dried to evaporate the organic solvent. The residue was chromatographed using a chloroform solution of 1.5% methanol to obtain 0.69 g (70.3% yield) of pure yellow N (竜) -Z-Lysine (-OMe) C2FC2-NHZ in yellow semisolid. (Rf = 0.3 in 5% methanol in chloroform). M, 2H], 3.6-3.8 [m, 2H], 2.8-3.5 [m, 4H], 3.6 [ ], 4.2-4.3 [m, IH], 4.45-4.6 [m, IH], 5.1 [s, 2H], 5.65-5.75 , 1H], 7.1-7.4 [m, 12H]. ESI-MS: m / z = 656.9 [M + H +], 679.1 [M + Na +] (calculated value for C33H45O9N5 = 655.7).

6. Synthesis of Lysine (-OMe) C2FC2-NH2 (6a)

N (ε) -Z-Lysine (-OMe) C2FC2-NHZ (0.69 g, 1.05 mmol) was dissolved in HPLC grade THF (15 ml). Nitrogen was flushed several times to remove air in the container. 10% Pd on charcoal was carefully added to the solution and the inner wall of the flask was rinsed with HPLC grade THF. The air in the flask was again flushed with nitrogen to remove. The hydrogen gas was then passed through the reaction mixture and allowed to stir overnight. It was then filtered through celite and washed with HPLC grade THF. The filtrate and washings were combined and the solvent was evaporated to yield 0.49 g (90%) of Lysine (-OMe) C2FC2-NH2 in white semi- solid. This was used for the next reaction without further purification (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 522.8 (calculated value for C25H39O7N5 = 521.6).

7. Synthesis of PRF-C2 (7a)

HOBt (0.112 g, 0.74 mmol) and EDCI (0.141 g, 0.74 mmol) were successively added to a dry DCM solution of Z-Arg- (Z) 2-OH (0.424 g, 0.74 mmol) . Lysine (-OMe) C2F2-NH2 (0.48 g, 0.92 mmol) dissolved in dry DMF after half an hour was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly basic. The resulting solution was stirred overnight at room temperature. The organic layer was then dried to evaporate the solvent. The residue was purified by chromatography using 5% methanol in chloroform to obtain 0.558 g (56.1% yield) of white rubbery semi-solid pure PRF-C2. ^{1 HNMR (300MHz, CDCl3 + CD3OD} ): δ = 1.4-2.2 [m, 19H], 3.0-3.5 [m, 4H], 3.9 [s, 3H], 4.0-4.4 [m, 7H], 4.4-4.7 [ m, 2H], 5.2-5.5 [m, 6H], 6.8-7.2 [m, 3H], 7.3-7.6 [m, 20H], 9.9 [s, 1H]. ESI-MS: m / z = 1082 [M + H +] & 1104 [M + Na +] (calculated value for C55H69O14N9 = 1080.8).

8. Synthesis of compound RF-C2 (8a)

Under a nitrogen atmosphere, PRF-C2 (0.1 g, 0.093 mmol) was dissolved in TFA (2 ml) and stirring was continued in an ice bath. Thioanisole (0.4 ml, 3.4 mmol) and TMSOTf (0.6 ml, 3.32 mmol) were successively added to the cold solution. The reaction mixture was allowed to come to room temperature and stirring was continued for 24 hours. The reaction was then cleared with HPLC grade methanol. Methanol and excess TFA were removed by flushing with nitrogen. The methanol-acetone system was repeated and the resulting residue was recrystallized. Finally, chloride ion exchange chromatography (Amberlite IRA-400Cl chloride ion exchange resin) was performed to obtain 0.031 g (57% yield) of pure yellowish white semi-solid RF-C2. (Rf = 0.1 in 20% methanol in chloroform). ESI-HRMS: m / z = 578.3391 ([M + H] <+>, calculated value for C26H44O6N9 = 578.3414). RP-HPLC: Rt = 3.9 min (20% water in acetonitrile), purity &gt; 99%.

Example 2. Synthesis of compound 8b (RF-C3)

1. Synthesis of N (ε) -Z-Lysine (-OMe) C3-NHBoc (1b)

Was synthesized according to the method of Example 1, step 1, using 3- (N-Boc) -amino propionic acid in place of Boc-Gly-OH.

Yield: 86.7% (Rf = 0.55 in 5% methanol in chloroform). 2H NMR (300 MHz, CDCl3):? = 1.4-1.7 [m, 13H], 1.75-1.9 [m, 2H], 2.3-2.45 , 2H], 3.7 [s, 3H], 4.45-4.55 [m, 1 H], 5.0-5.1 [s, 2H], 5.2-5.3 [ [m, 6H]. ESI-MS: m / z = 489.0 [M + Na &lt; + &gt;] (calculated value for C23H35O7N3 = 465.5).

2. Synthesis of N (ε) -Z-Lysine (-OMe) C3-NH2 (2b)

The procedure of Example 2 was repeated.

Yield: 90% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 366 (calculated value for C18H27O5N3 = 365.4).

3. Synthesis of N (ε) -Z-Lysine (-OMe) C3F (3b)

The procedure of Example 3 was repeated.

Yield: 64.9% (Rf = 0.5 in 5% methanol in chloroform). ^{1 HNMR (300MHz, CDCl3):} δ = 1.2-1.6 [m, 15H], 1.6-1.9 [m, 2H], 2.15-2.45 [m, 2H], 2.8-3.25 [m, 4H], 3.7-3.8 [ s, 3H], 4.15-4.25 [q, IH], 4.4-4.5 [q, IH], 4.9-5. 0 [ m, 1H], 7.1-7.4 [m, 11H]. ES-MS: m / z = 635 [M + Na +] (calculated value for C32H44O8N4 = 612.7).

4. Synthesis of Lysine (-OMe) C3F (4b)

The procedure of Example 1, Step 4 was repeated.

Yield: 95.3% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 480.0 (calculated value for C24H38O6N4 = 478.6).

5. Synthesis of Lysine (-OMe) C3FC3-NHZ (5b)

(N-Z) -amino propionic acid instead of Z-Gly-OH.

Yield: 56.8% (Rf = 0.3 in 5% methanol in chloroform). ^{1 HNMR (200MHz, CDCl3):} δ = 1.2-1.9 [m, 15H], 2.25-2.55 [m, 4H], 2.8-3.5 [m, 8H], 3.7 [s, 3H], 4.2-4.3 [m, 1H], 4.35-4.5 [m, 1H], 5.0-5.1 [s, 2H], 5.2-5.35 [m, 1H], 7.1-7.4 [m, 10H]. ESI-MS: m / z = 685.3 (calculated value for C35H49O9N5 = 683.8).

6. Synthesis of Lysine (-OMe) C3FC3-NH2 (6b)

The procedure of Example 6 was repeated.

Yield: 90% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 551 (calculated value for C27H43O7N5 = 549.7).

7. Synthesis of PRF-C3 (7b)

The procedure of Example 7 was repeated.

Yield: 64.6% (Rf = 0.4 in 5% methanol in chloroform). ^{1 HNMR (300MHz, CDCl3):} δ = 1.2-1.5 [m, 15H], 1.6-1.8 [m, 4H], 2.3-2.65 [m, 8H], 3.1-3.25 [m, 2H], 3.4-3.6 [ m, 4H], 3.7 [s, 3H], 4.05-4.2 [m, 1H], 4.2-4.4 [m, 2H], 5.0-5.4 [m, 6H], 7.15-7.45 [ MS: m / z = 1108 (calculated value for C 57 H 73 O 14 N 9 = 1108.2).

8. Synthesis of RF-C3 (8b)

The procedure of Example 8 was repeated.

Yield: 61.6% (Rf = 0.1 in 20% methanol in chloroform). ESI-HRMS: m / z = 606.3728 ([M + H] <+>, calculated value for C28H48O6N9 = 606.3727). RP-HPLC: Rt = 2.9 min (20% water in acetonitrile), purity &gt; 99%.

Example 3. Synthesis of compound 18a (RF-C5)

1. Synthesis of Compound 12a

1-1. Synthesis of tert-butyl-5-bromopentanoate (9a)

N, N'-Dimethylaminopyridine (1.68 g, 13.75 mmol) and DCC (6.83 g, 33.15 mmol) were added to a cold DCM solution of 5-bromopentanoic acid (5 g, 27.62 mmol) Were successively added. After half an hour tert-butanol (15.8 ml, 165.75 mmol) was added to the reaction mixture. The resulting solution was stirred overnight at room temperature. It was then diluted with DCM and washed successively with water and brine. The organic layer was dried to evaporate the solvent. The residue was purified by chromatography using a hexane solution of 1% ethyl acetate to give 4.1 g (62.6% yield) of pure tert-butyl-5-bromopentanoate as a pale yellow liquid. (Rf = 0.8 in 20% ethylacetate in hexane). 1H NMR (200MHz, CDCl3):? = 1.44 [s, 9H], 1.5-1.6 [m, 2H], 1.84-1.96 [m, 2H], 2.24 t, 2H], 3.4 t, 2H]. ESI-MS: m / z = 258 (calculated value for C9H17O2Br = 237.1).

1-2. Synthesis of tert-butyl-5-aminopentanoate (10a)

tert-Butyl-5-bromopentanoate (3.6 g, 15.19 mmol) was dissolved in dry DMF and sodium azide (1.48 g, 22.77 mmol) was added thereto. The reaction mixture was heated to 70-80 &lt; 0 &gt; C and stirred for 17-18 h. It was then cooled to room temperature. Diluted with chloroform and washed successively with water and brine. The chloroform layer was dried to evaporate the solvent. Triphenylphosphine (2.29 g, 8.77 mmol) was added to the rubbery crude material. THF was added thereto after a vigorous evolution of nitrogen gas. After nitrogen evolution ceased, a few drops of water were added to the reaction mixture and stirring continued for an additional 4 hours. THF was then evaporated. The residue was purified by chromatography using a chloroform solution of 5% methanol to give 2.79 g (96% yield) of pure tert-butyl-5-aminopentanoate as a yellow liquid. (Rf = 0.1 in 5% methanol in chloroform). ^{1 HNMR (200MHz, CDCl3):} δ = 1.28 [s, 12 H], 1.44 [s, 9H], 1.5-1.6 [m, 4H], 2.1-2.2 [t, 2H], 2.76-2.84 [t, 2H ], 2.92-3.4 [m, 2H].

1-3. Synthesis of RC5 (-CO2But) (11a)

HOBt (0.35 g, 2.31 mmol) and EDCI (0.44 g, 2.30 mmol) were added successively to a cold DCM solution of Z-Arg- (Z) 2-OH (1.54 g, 2.82 mmol) . After half an hour tert-butyl-5-aminopentanoate (0.50 g, 2.89 mmol) dissolved in dry DCM was added to the reaction mixture. DIPEA was added to make the reaction mixture weakly basic. The resulting solution was stirred overnight at room temperature. It was then diluted with chloroform and washed successively with water and brine. The organic layer was dried to evaporate the solvent. The residue was chromatographed using a chloroform solution of 1% methanol to obtain 1.73 g (72% yield) of pure white RC5 (-CO2But). (Rf = 0.8 in 5% methanol in chloroform). [M, 2H], 1.4 [s, 9H], 1.5-1.8 [m, 6H], 2.1 [t, 2H], 2.7-2.9 [m, 2H] [M, 2H], 4.2-4.4 [m, 1H], 4.9-5.1 [m, 6H], 6.2 [d, 1H], 6.5-6.6 ], 9.3-9.5 [m, 2H]. ESI-MS: m / z = 732 (calculated value for C39H49O9N5 = 731.8).

1-4. Synthesis of RC5 (-CO2H) (12a)

To a dry DCM solution of RC5 (-CO2But) (0.95 g, 1.3 mmol) was slowly added TFA at 0 &lt; 0 &gt; C and the mixture was stirred at room temperature for 4 h. The nitrogen was then flushed to remove DCM and excess TFA. The gummy residue was purified by chromatography using a 2% methanol in chloroform solution to give 68 g (77.5% yield) of pure white RC5 (-CO2H). (Rf = 0.5 in 5% methanol in chloroform). ESI-MS: m / z = 676 (calculated value for C35H41O9N5 = 675.7).

2. Synthesis of Compound 16a

2-1. Synthesis of N (?) - Z-Lysine (-OMe) C5-Br (13a)

HOBt (0.74 g, 4.82 mmol) and EDCI (1.15 g, 6.02 mmol) were successively added thereto while stirring with ice-cooled dry DCM solution of 5-bromobaleric acid (1.09 g, 6.03 mmol). N (epsilon) -Z-lysine methyl ester hydrochloride (2 g, 6.04 mmol) dissolved in dry DCM after half an hour was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly basic. The reaction mixture was stirred overnight at room temperature. It was then diluted with chloroform and washed successively with 0.5 N HCl, water and brine. The organic layer was dried to evaporate the solvent. The residue was purified by chromatography using a 1% methanol in chloroform solution to give 1.83 g (66% yield) of pure white N (ε) -Z-Lysine (-OMe) C5-Br. (Rf = 0.6 in 5% methanol in chloroform). 2H NMR (200 MHz, CDCl3):? = 1.25-1.4 [m, 2H], 1.45-1.6 [m, 2H], 1.65-1.9 [m, 6H], 2.25 [ 2H], 7.3 [s, 3H], 4.55 [m, 1H], 4.95 [t, . ESI-MS: m / z = 458 (calculated value for C20H29O5N2Br = 457.4).

2-2. Synthesis of N (?) - Z-Lysine (-OMe) C5-NH2 (14a)

(1.5 g, 3.28 mmol) was dissolved in dry DCM and sodium azide (0.26 g, 3.94 mmol) was added. The reaction mixture was heated and stirred at 70-80 &lt; 0 &gt; C for 17-18 hours. It was then cooled to room temperature, diluted with chloroform, and washed successively with water and brine. The organic layer was dried to evaporate the solvent. Triphenylphosphine (1.29 g, 4.92 mmol) was added to the crude material. When it was observed that the nitrogen gas was generated violently, THF was added. After the evolution of nitrogen gas ceased, a few drops of water were added to the reaction mixture and stirred for an additional 4 hours at room temperature. THF was then evaporated with a rotary evaporator. The residue was purified by chromatography using a chloroform solution of 5% methanol to obtain 1.2 g (94% yield) of pure yellow N (ε) -Z-Lysine (-OMe) C5-NH2 as a yellow rubbery liquid. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 394 (calculated value for C20H31O5N3 = 393.5).

2-3. Synthesis of N (?) - Z-Lysine (-OMe) C5F-NHBoc (15a)

HOBt (0.39 g, 2.53 mmol) and EDCI (0.48 g, 2.53 mmol) were successively added thereto while ice-cooling and stirring with a dry DCM solution of Boc-Phe-OH (0.54 g, 2.04 mmol). N (ε) -Z-Lysine (-OMe) C5-NH2 (1 g, 2.54 mmol) dissolved in dry DCM was added to the reaction mixture. DIPEA was added to make the reaction mixture slightly basic. The resulting solution was stirred overnight at room temperature. It was then diluted with chloroform and washed successively with water and brine. The organic layer was dried to evaporate the solvent. The residue was purified by chromatography using a 2% methanol in chloroform solution to obtain 0.9 g (55.28% yield) of pure white N-ethylsilylacetate N (ε) -Z-Lysine (-OMe) C5F. (Rf = 0.55 in 5% methanol in chloroform). 2H NMR (300 MHz, CDCl3):? = 1.3-1.4 [s, 9H], 1.5-1.7 [m, 6H], 2.15-2.35 [t, 2H], 2.9-3.1 [ 1H], 5.0- [t, 1H], 5.1 [s, 2H], 5.4 [m, 1H], 6.5-6.65 [m , 1H], 7.05 [d, 1H], 7.15-7.4 [m, 10H]. ESI-MS: m / z = 641 (calculated value for C34H48O8N4 = 640.8).

2-4. Synthesis of Lysine (-OMe) C5F (16a)

N (ε) -Z-Lysine (-OMe) C5F-NHBoc (0.8 g, 1.25 mmol) was dissolved in HPLC grade methanol. Nitrogen was flushed several times to remove air from the round bottom flask. Charcoal 10% Pd (0.5 g) was carefully added to the solution and the inner wall of the flask was rinsed with HPLC grade methanol. Nitrogen was again flushed to remove air from the flask. Hydrogen gas was then passed through the reaction mixture and stirred overnight. It was then filtered through celite and washed with HPLC grade methanol. The filtrate and washings were combined and the solvent was evaporated to give 0.54 g (85.1%) of white semisolid Lysine (-OMe) C5F. It was used directly in the next reaction without further purification. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 507 (calculated value for C26H42O6N4 = 506.6).

3. Synthesis of Compound 18a

3-1. Synthesis of PRF-C5 (17a)

HOBt (0.061 g, 0.33 mmol) and HATU (0.15 g, 0.39 mmol) were successively added thereto while stirring under ice cooling with a dry DCM solution of RC5 (-CO2H) (0.27 g, 0.9 mmol). After 30 minutes, Lysine (-OMe) C5F (0.25 g, 0.49 mmol) dissolved in dry DCM was added to the reaction mixture. DIPEA was added dropwise to make the reaction mixture weakly basic. The resulting solution was stirred overnight at room temperature. It was then diluted with chloroform and washed successively with water and brine. The organic layer was dried to evaporate the solvent. Using a chloroform solution of 2% methanol, 0.33 g (57.5% yield) of pure rubber PRF-C5, a semi-solid of yellow rubber, was obtained. (Rf = 0.5 in 5% methanol in chloroform, v / v). 2H NMR (300 MHz, CDCl3 + CD3OD):? = 0.7-0.8 [m, 2H] 1.1-1.2 [s, 9H], 1.2-1.7 [m, 16H], 2.0-2.2 [ m, 4H], 3.3 [t, 4H], 3.6 [s, 3H, (merged with solvent peak)], 3.7 [ , 4.3-4.4 [m, 1H], 4.95-5.25 [m, 6H], 7.15-7.35 [m, 20H]. ESI-MS: m / z = 1164 (calculated value for C61H81O14N9 = 1163.4).

3-2. Synthesis of RF-C5 (18a)

PRF-C5 (0.1 g, 0.086 mmol) was dissolved in TFA (2 ml) under a nitrogen atmosphere at 0 ° C. Thioanisole (0.4 ml, 3.4 mmol) and TMS-OTf (0.6 ml, 3.32 mmol) were successively added to the cold solution. The reaction mixture was allowed to come to room temperature and stirring was continued for 24 hours. The reaction was then cleared with HPLC grade methanol. Nitrogen flush was applied to remove methanol and excess TFA. The residue was dissolved in a minimal amount of HPLC grade methanol and excess diethyl ether was added. A gummy yellow liquid precipitated. These were collected by centrifugation and the methanol-diethyl ether system was repeatedly reprecipitated. Finally, chloride ion exchange chromatography (using Amberlite IRA-400Cl chloride ion exchange resin) yielded 0.037 g (65% yield) of pure yellow RFS 5 as a semi-solid. (Rf = 0.1 in 20% methanol in chloroform). ESI-HRMS: m / z = 662.4375 ([M + H] <+>, calculated value for C32H56O6N9 = 662.4348). RP-HPLC: Rt = 2.3 min (100% methanol), purity &gt; 99%.

Example 4. Synthesis of compound 18b (RF-C8)

1. Synthesis of Compound 12b

1-1. Synthesis of tert-butyl-8-bromooctanoate (9b)

Was carried out in the same manner as 1-1 of Example 3 using 8-bromooctanoic acid instead of 5-bromopentanoic acid.

Yield: 70.5% (Rf = 0.85 in 20% ethylacetate in hexane). 2H NMR (300 MHz, CDCl3):? = 1.28-1.36 [m, 6H], 1.4-1.48 [s, 9H], 1.55-1.64 [m, 2H], 1.8-1.92 , 2H], 3.36 - 3.44 [t, 2H].

1-2. Synthesis of tert-butyl-8-amino octanoate (10b)

Was carried out in the same manner as in 1-2 of Example 3.

Yield: 90.5% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 216.5 (calculated value for C12H25O2N = 215.3).

1-3. Synthesis of RC8 (-CO2But) (11b)

The procedure of Example 1-3 was repeated.

Yield: 65.2% (Rf = 0.85 in 5% methanol in chloroform). 1H NMR (300 MHz, CDCl3 + CD3OD):? = 1.1-1.35 [m, 10H], 1.45 [s, 9H], 1.5-1.7 [m, 4H], 2.1-2.25 [ , 2H], 3.8-4.1 [m, 3H, (merged with solvent peak)], 5.05-5.25 [m, 6H], 7.25-7.4 [m, 15H]. ESI-MS: m / z = 775 (calculated value for C42H55O9N5 = 773.9).

1-4. Synthesis of RC8 (-CO2H) (12b)

The procedure of Example 1-4 was repeated.

Yield: 81.1% (Rf = 0.5 in 5% methanol in chloroform). 1H NMR (200MHz, CDCl3 + CD3OD):? = 1.15-1.4 [m, 10H], 1.5-1.75 [m, 4H], 2.2-2.35 [t, 2H], 2.9-3.1 [ , 2H], 4.1-4.2 [m, 1H], 5.0-5.25 [m, 6H], 7.3-7.5 [m, 15H]. ESI-MS: m / z = 718 (calculated value for C38H47O9N5 = 717.8).

2. Synthesis of Compound 16b

2-1. Synthesis of N (?) - Z-Lysine (-OMe) C8-Br (13b)

Was carried out in the same manner as in 2-1 of Example 3 using 8-bromo octanoic acid instead of 5-bromobaleric acid.

Yield: 64.85% (Rf = 0.7 in 5% methanol in chloroform). 2H NMR (300 MHz, CDCl3):? = 1.3-1.8 [m, 14H], 1.8-1.9 [m, 2H], 2.15-2.25 [t, 2H], 3.15-3.25 [ , 2H], 3.75 [s, 3H], 4.55-4.65 [m, IH], 4.8-4.9 [t, IH], 5.1 [s, 2H], 6.1 [d, ]. ESI-MS: m / z = 501 (calculated value for C23H35O5N2Br = 499.4).

2-2. Synthesis of N (?) - Z-Lysine (-OMe) C8-NH2 (14b)

The procedure of Example 2-2 was repeated.

Yield: 88.5% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 436.8 (calculated value for C23H37O5N3 = 435.6).

2-3. Synthesis of N (?) - Z-Lysine (-OMe) C8F-NHBoc (15b)

The procedure of Example 2-3 was repeated.

Yield: 61% (Rf = 0.6 in 5% methanol in chloroform). [M, 25H], 2.1-2.2 [t, 2H], 2.9-3.2 [m, 6H], 3.7 [s, 3H], 4.15-4.3 [ ], 4.5-4.6 [m, IH], 5.0-5.15 [m, 2H], 5.25-5.4 [m, IH], 6.1-6.2 [ [m, lH]. ESI-MS: m / z = 684 (calculated value for C37H54O8N4 = 682.9).

2-4. Synthesis of Lysine (-OMe) C8F (16b)

Was carried out in the same manner as in 2-4 of Example 3.

Yield: 80% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 549 (calculated value for C29H48O6N4 = 548.7).

3. Synthesis of Compound 18b

3-1. Synthesis of PRF-C8 (17b)

RC5 (-CO2H) and Lysine (-OMe) C5F instead of 12b and 16b.

Yield: 53.6% (Rf = 0.6 in 5% methanol in chloroform). 1H NMR (200MHz, CDCl3):? = 1.1-1.4 [m, 33H], 1.4-1.9 [m, 8H], 2.05-2.15 [t, 2H], 2.15-2.25 [t, 2H], 2.75-3.3 [ , [8H], 3.7 [s, 3H], 3.75-3.9 [m, 2H], 3.95-4. 1 [ [m, 6H], 5.7 [m, IH], 5.95-6.0 [m, IH], 6.3-6.35 [m, IH], 6.6 [m, IH], 7.15-7.45 [m, 20H]. ESI-MS: m / z = 1248 (calculated value for C67H93O14N9 = 1248.1).

3-2. Synthesis of RF-C8 (18b)

The procedure of Example 3-2 was repeated.

Yield: 68.5% (Rf = 0.1 in 20% methanol in chloroform). ESI-HRMS: m / z = 746.5274 ([M + H] <+>, calculated value for C38H68O6N9 = 746.5292). RP-HPLC: Rt = 3.8 min (100% methanol), purity &gt; 99%.

Example 5. Synthesis of compound 18c (RF-C9)

1. Synthesis of Compound 12c

1-1. Synthesis of tert-butyl-15-bromopentadecanoate (9c)

Yield: 75% (Rf = 0.9 in 20% ethyl acetate in hexane). M, 2H], 1.8-1.92 [q, 2H], 2.12-2.2 [t, 1 H] , 2H], 3.32-3. 4 [t, 2H].

1-2. Synthesis of tert-butyl-9-aminononate (10c)

To a solution of tert-butyl-8-bromooctanoate (1.87 g, 6.71 mmol) in dry DMSO (3 ml) was added sodium cyanide (2.63 g, 53.68 mmol). Sodium iodide was added in a catalytic amount. The reaction mixture was heated to 80-90 &lt; 0 &gt; C and stirred for 2-3 hours. It was then cooled to room temperature. Diluted with chloroform and washed successively with cold water and brine. The organic layer was dried to evaporate the solvent. The crude material was dissolved in cold dry MeOH and anhydrous nickel chloride (0.081 g, 0.625 mmol) was added. After some time, sodium borohydride (1.65 g, 43.54 mmol) was added to the reaction mixture for 2-3 hours. The reaction mixture was then filtered through celite and washed with HPLC grade MeOH. MeOH was removed and the crude material was dissolved in chloroform. Worked up with brine water and dried. The residue was purified by chromatography using a 5% methanol in chloroform solution to give 1.26 g (88.6% yield) of pure tert-butyl-9-aminonate in yellow liquid. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 230 (calculated value for C13H27O2N = 229.4).

1-3. Synthesis of RC9 (-CO2But) (11c)

The procedure of Example 1-3 was repeated.

Yield: 60.5% (Rf = 0.85 in 5% methanol in chloroform). [M, 25H], 2.15-2.25 [m, 4H], 2.3-2.4 [t, 2H], 5.05-5.3 [m, 6H], 7.2-7.4 [m , 15H]. ESI-MS: m / z = 789 (calculated value for C43H57O9N5 = 787.9).

1-4. Synthesis of RC9 (-CO2H) (12c)

The procedure of Example 1-4 was repeated.

Yield: 75.5% (Rf = 0.5 in 5% methanol in chloroform). 1H NMR (200MHz, CDCl3 + CD3OD):? = 1.1-1.4 [m, 12H], 1.5-1.7 [m, 4H], 2.2-2.3 [t, 2H], 2.9-3.0 [ [m, 2H], 4.05-4.1 [m 1H] 5.0-5.25 [m, 6H], 7.2-7.4 [m, 15H]. ESI-MS: m / z = 732 (calculated value for C39H49O9N5 = 731.8).

2. Synthesis of Compound 16c

2-1. N (?) - Z-Lysine (-OMe) C15-Br (13c)

Was carried out in the same manner as in 2-1 of Example 3 using 9-bromonano-nanoic acid instead of 5-bromobaleric acid.

Yield: 60.1% (Rf = 0.75 in 5% methanol in chloroform). 1H NMR (300MHz, CDCl3):? = 1.2-1.7 [m, 26H], 1.7-1.9 [m, 2H], 2.1-2.2 [t, 2H], 3.1-3.2 [q, 2H] 2H], 3.7 [s, 3H], 4.5-4.6 [m, 1H], 4.75-4.85 [m, 1H], 5.05 , 5H].

2-2. Synthesis of N (?) - Z-Lysine (-OMe) C9-NH2 (14c)

N (ε) -Z-Lysine (-OMe) C8-Br (1.23 g, 2.46 mmol) was dissolved in dry DMSO and sodium cyanide (0.97 g, 19.72 mmol) was added. Sodium iodide was added in a catalytic amount. The reaction mixture was heated to 80-90 &lt; 0 &gt; C and stirred for 2-3 hours. It was then cooled to room temperature. Diluted with chloroform and washed successively with cold water and brine. The organic layer was dried to evaporate the solvent. A red yellow product was obtained. The crude material was dissolved in ice-cold dry MeOH and anhydrous nickel chloride (0.24 g, 1.88 mmol) was added. After some time sodium borohydride (0.5 g, 13.15 mmol) was added to the reaction mixture and left for 2-3 hours. The reaction mixture was then filtered through celite and washed with HPLC grade MeOH. MeOH was removed with a rotary evaporator and the crude material was dissolved in chloroform. Worked up with brine water and dried. The residue was purified by chromatography using a 5% methanol in chloroform solution to give 0.9 g (81.8% yield) of pure tert-butyl-9-aminononate as a yellow liquid. (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 451 (calculated value for C24H39O5N3 = 449.6).

2-3. Synthesis of N (epsilon) -Z-Lysine (-OMe) C9F-NHBoc (15c)

The procedure of Example 2-3 was repeated.

Yield: 56.5% (Rf = 0.6 in 5% methanol in chloroform). 2H NMR (300 MHz, CDCl3):? = 1.1-1.9 [m, 27H], 2.15-2.25 [m, 2H], 2.95-3.3 [ ], 4.55-4.65 [m, IH], 5.1 [s, 2H], 5.3-5.4 [m, IH], 5.9-6. 0 [ ]. ESI-MS: m / z = 697 (calculated value for C38H56O8N4 = 696.9).

2-4. Synthesis of Lysine (-OMe) C9F (16c)

The procedure of Example 2-4 was repeated.

Yield: 88.5% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 563 (calculated value for C30H50O6N4 = 562.7).

3. Synthesis of Compound 18c

3-1. Synthesis of PRF-C9 (17c)

RC5 (-CO2H) and Lysine (-OMe) C5F instead of 12c and 16c.

Yield: 61.2% (Rf = 0.6 in 5% methanol in chloroform). 2H NMR (200 MHz, CDCl3 + CD3OD):? = 1.2-1.7 [m, 41H], 2.1-2.3 [m, 6H], 3.0-3.2 [m, 4H], 3.3-3.35 [ , 3H], 3.85-4.0 [m, 2H], 5.0-5.3 [m, 6H], 7.15-7.45 [m, 20H]. ESI-MS: m / z = 1277 (calculated value for C69H97O14N9 = 1276.6).

3-2. Synthesis of RF-C9 (18c)

The procedure of Example 3-2 was repeated.

Yield: 72% (Rf = 0.1 in 20% methanol in chloroform). ESI-HRMS: m / z = 774.5579 ([M] &lt; + &gt;, calculated value for C40H71O6N9 = 774.5604). RP-HPLC: Rt = 3.7 min (15% water in acetonitrile), purity &gt; 99%.

Example 6. Synthesis of compound 18d (RF-C15)

1. Synthesis of Compound 12d

1-1. Synthesis of tert-butyl-15-aminopendadecanoate (10d)

was carried out in the same manner as 1-2 of Example 5 using tert-butyl-14-bromotetradecanoate instead of tert-butyl-8-bromooctanoate. Yield: 85% (Rf = 0.1 in 5% methanol in chloroform).

1-2. Synthesis of RC15 (-CO2But) (11d)

The procedure of Example 1-3 was repeated.

Yield: 67.5% (Rf = 0.9 in 5% methanol in chloroform).

1-3. Synthesis of RC15 (-CO2H) (12d)

The procedure of Example 1-4 was repeated.

Yield: 79% (Rf = 0.6 in 5% methanol in chloroform).

2. Synthesis of Compound 16d

2-1. Synthesis of N (?) - Z-Lysine (-OMe) C15-NH2 (14d)

-Z-Lysine (-OMe) N (ε) -Z-Lysine (-OMe) In the same manner as in Example 2-2-2, N (ε) -Z-Lysine (-OMe) C14-Br was used instead of C8-Br. Yield: 90.5% (Rf = 0.1 in 5% methanol in chloroform).

2-2. Synthesis of N (epsilon) -Z-Lysine (-OMe) C15F-NHBoc (15d)

The procedure of Example 2-3 was repeated.

Yield: 61.8% (Rf = 0.7 in 5% methanol in chloroform).

2-3. Synthesis of Lysine (-OMe) C15F (16d)

The procedure of Example 2-4 was repeated.

Yield: 92.5% (Rf = 0.1 in 5% methanol in chloroform).

3. Synthesis of Compound 18d

3-1. Synthesis of PRF-C15 (17d)

RC5 (-CO2H) and Lysine (-OMe) C5F instead of 12d and 16d.

Yield: 65.1% (Rf = 0.7 in 5% methanol in chloroform). 1 H NMR (200 MHz, CDCl 3 + CD 3 OD):? = 1.1-1.4 [m, 20H], 1.5 [s, 9H], 1.5-1.85 [m, 10H], 2.15-2.25 [ M, 4H], 3.75-4.4 [m, 4H], 5.0-5.4 [m, 6H], 6.2-6.3 [m, 2H], 6.5-6.6 [m, 2H], 7.25-7.45 [m, 20H].

3-2. Synthesis of RF-C15 (18d)

The procedure of Example 3-2 was repeated.

Yield: 74.3% (Rf = 0.1 in 20% methanol in chloroform). ESI: m / z = 943 ([M + H] +, calculated value for C52H96O6N9 = 943.3), 472 ([M + 2H] 2+, calculated 472.1). RP-HPLC: Rt = 3.87 min (100% methanol), purity > 99%.

Example 7. Synthesis of Compound 18e (GV-C5)

1. Synthesis of Compound 12e

1-1. Synthesis of GC5 (-CO2But) (11e)

Was carried out in the same manner as in 1-3 of Example 3 using Z-Gly- (Z) 2-OH instead of Z-Arg- (Z) 2-OH.

Yield: 63.1% (Rf = 0.6 in 5% methanol in chloroform). [M, 2H], 3.8 [d, 2H], 5.1 [s, 9H], 1.5 [ 2H], 5.55 [m, IH], 6.35 [m, IH], 7.25-7.35 [m, 5H]. ESI-MS: m / z = 365 (calculated value for C19H28O5N2 = 364.4).

1-2. Synthesis of GC5 (-CO2H) (12e)

The procedure of Example 1-4 was repeated.

Yield: 79% (Rf = 0.4 in 5% methanol in chloroform). ESI-MS: m / z = 309 (calculated value for C15H20O5N2 = 308.3).

2. Synthesis of Compound 16e

2-1. Synthesis of N (?) - Z-Lysine (-OMe) C5V (15e)

Was carried out in the same manner as in 2-3 of Example 3 using Boc-Val-OH instead of Boc-Phe-OH.

Yield: 65% (Rf = 0.6 in 5% methanol in chloroform). 1 H NMR (300 MHz, CDCl 3):? = 0.9-1.0 [d, 6H], 1.4 [s, 9H], 1.5-1.9 [m, 10H], 1.95-2.05 [m, 1H], 2.15-2.35 [ 1H], 5.05 [s, 2H], 5.25 [s, 2H], 3.15-3.2 [m, 4H], 3.4-3.5 [d, 1H], 6.85 [m, 1H], 7.15 [m, 1H], 7.25-7.4 [s, 5H].

2-2. Synthesis of Lysine (-OMe) C5V (16e)

Was carried out in the same manner as in 2-4 of Example 3.

Yield: 78.1% (Rf = 0.1 in 5% methanol in chloroform). ESI-MS: m / z = 459 (calculated value for C22H42O6N4 = 458.6).

3. Synthesis of Compound 18e

3-1. Synthesis of PGV-C5 (17e)

RC5 (-CO2H) and Lysine (-OMe) C5F instead of 12e and 16e.

Yield: 54.5% (Rf = 0.6 in 5% methanol in chloroform). 1 H NMR (300 MHz, CDCl 3 + CD 3 OD):? = 0.8-1.0 [d, 6H], 1.45-1.55 [s, 9H], 1.55-2.0 [m, 15H] [m, 6H], 3.65-3.8 [m, 6H], 4.4 [m, (merged with CD3OD solvent peak), 1H], 5.1 [s, 2H], 7.25-7.4 [m, 5H]. ESI-MS: m / z = 749 (calculated value for C37H60O10N6 = 748.9).

3-2. GV-C5 (18e)

The procedure of Example 3-2 was repeated.

Yield: 60.5% (Rf = 0.1 in 10% methanol in chloroform). ESI-HRMS: m / z = 537.33955 ([M + Na] &lt; + &gt;, calculated value for C24H46O6N6Na = 537.3371). RP-HPLC: Rt = 3.8 min (100% methanol), purity &gt; 99%.

Example 8. Synthesis of Compound 18f (RR-C5)

1. FC5 (-CO2But) (11f)

Was carried out in the same manner as in 1-3 of Example 3 using Z-Phe- (Z) 2-OH instead of Z-Arg- (Z) 2-OH.

Yield: 58.9% (Rf = 0.7 in 5% methanol in chloroform). [M, 1H], 5.05 [s, 2H], 2.9-3.2 [m, 4H] 5.45 [d, 1H], 5.85 [t, 1H], 7.1-7.35 [m, 10H]. ESI-MS: m / z = 455 (calculated value for C26H34O5N2 = 454.6).

2. Synthesis of FC5 (-CO2H) (12f)

The procedure of Example 1-4 was repeated.

Yield: 84.4% (Rf = 0.5 in 5% methanol in chloroform). ESI-MS: m / z = 399 (calculated value for C22H26O5N2 = 398.5).

3. Synthesis of PRR-C5 (17f)

To a dry DMF solution of RC5 (-CO2H) (0.53 g, 0.78 mmol) was added HATU (0.3 g, 0.78 mmol) while cooling with ice and stirring. After 30 minutes, L-lysine methyl ester di-hydrochloride (0.1 g, 0.43 mmol) dissolved in dry DMSO was added to the reaction mixture. The DIPEA was then added dropwise until the reaction mixture became weakly alkaline. The reaction mixture was stirred at room temperature for 48 hours. It was then diluted with chloroform and washed successively with 0.5 N HCl, water and brine. The organic layer was dried to evaporate the solvent. The residue was purified by chromatography using a 1% methanol in chloroform solution to give 0.38 g (60% yield) of pure PRR-C5 as a white solid.

(Rf = 0.7 in 5% methanol in chloroform). ^{1 HNMR (200MHz, CDCl3 + CD3OD} ): δ = 1.3-1.4 [m, 4H], 1.4-1.7 [m, 12H], 2.05-2.25 [m, 4H], 2.9-3.05 [m, 4H], 3.05- 3.2 [m, 2H], 3.7 [s, 3H], 3.75-4.0 [m, 6H], 4.35-4.45 [m, 1H], 5.05-5.25 [m, 12H], 7.15-7.4 [m, 30H]. ESI-MS: m / z = 1472 (calculated value for C77H94O10N6 = 1447.6).

4. Synthesis of RR-C5 (18f)

The procedure of Example 3-2 was repeated.

Yield: 47% (Rf = 0.0, spot resided at baseline in 20% methanol in chloroform). ESI-HRMS: m / z = 336.2387 ([M + 2H] 2+ calculated value for [C29H60O6N12] / 2 = 336.2334). RP-HPLC: Rt = 3.6 min (100% methanol), purity > 99%.

Example 9. Synthesis of compound 18g (FF-C5)

1. Synthesis of PFF-C5 (17 g)

Was carried out in the same manner as in Example 8, 3 using FC5 (-CO2H) instead of RC5 (-CO2H).

Yield: 59.8% (Rf = 0.6 in 5% methanol in chloroform). ¹ H NMR (400 MHz, CDCl 3 + CD 3 OD):? = 1.3-1.4 [m, 4H], 1.45-1.85 [m, 6H], 2.1-2.3 [m, 4H], 2.75-3.15 [ s, 3H], 4.25-4.3 [m, 3H], 4.95-5.1 [m, 4H], 7.15-7.35 [m, 20H]. ESI-MS: m / z = 944 [M + Na &lt; + &gt;] (calculated value for C51H64O10N6 = 921.1).

2. Synthesis of FF-C5 (18 g)

The procedure of Example 3-2 was repeated.

Yield: 53% (Rf = 0.1 in 10% methanol in chloroform). ESI-HRMS m / z = 653.4009 ([M + H] +, calculated value for C35H53O6N6 = 653.4021), 327.2064 ([M + 2H] 2+ calculated 327.2017). RP-HPLC: Rt = 3.9 min (100% methanol), purity > 99%.

3. Synthesis of RF-C11 (18h)

RF-C11 (n = 10) was prepared by a conventional method [MJ Lee, K. Pal, T. Tasaki, S. Roy, Y. Jiang, JYAN, R. Banerjee and YT Kwon, Proc. Natl. Acad. Sci. USA, 105; 100 (2008). Yield: 60.8% (Rf = 0.1 in 20% methanol in chloroform). The final compound was identified using ESI-MS and purified by HPLC. ESI-MS: m / z = 831 ([M + H] +, calculated value for C44H80O6N9 = 831.2), 416 ([M + 2H] +, calculated value for C44H80O6N9 = 416.2). RP-HPLC: Rt = 3.78 min (100% methanol), purity > 98%.

Example

Experimental Example 1. Identification of two independent binding sites binding to destabilizing residues causing proteasome degradation contained in N-recognin

To assess the N-degron of mammals, a novel N-end rule substrate in a rabbit reticulocyte lysate rich in UPS elements was first identified here. The DHFR-Ub-X-nsP4 fusion protein (FIG. 11a) was expressed in the fusant, where the DHFR-Ub reference (DHFR-Ub) And the X-nsP4 model substrate were produced. Arg-nsP4 with the type 1 destabilizing moiety disintegrated quickly and was not detected at steady state (Fig. 11b, lane 1). These results are in contrast to those of the dipeptide inhibitor Arg-Ala in the presence of besatin (Figure 11b, lane 2 compared to lanes 6 and 10), which stabilizes the dipeptide N-end rule inhibitor in reticulocyte lysate . Neither Ala-Arg (stabilized) nor Trp-Ala (type 2) stabilized Arg-nsP4 at 2 mM concentrations (FIGS. 11b and 11c). These results show that despite the relatively simple recognition motif, the N-domain of the UBR box and N-recognin is highly selective and independent.

When treated with the proteasome inhibitor MG132, the ubiquitinated form of Arg-nsP4 became more abundant by immunoprecipitation, which was visualized by immunoblotting against Ub (FIG. 11d). Arg-nsP4 (type 1) was rapidly ubiquitinated in steady state, but this process was significantly reduced by the Arg-Ala dipeptide (Fig. 11d, lanes 3 and 4). We also investigated the ubiquitination of other model substrates such as Met-nsP4 (stabilized) and Phe-nsP4 (type 2) (Fig. 11e and f). Met-nsP4 (stabilized) has a long lifetime and is not ubiquitinated irrespective of dipeptide, whereas Arg-nsP4 (type 1) and Phe-nsP4 (type 2) are rapidly degraded by ubiquitination Lanes 9-12). The type 2 dipeptide inhibitor Trp-Ala specifically blocked Phe-nsP4 ubiquitination when type 1 dipeptides blocked Arg-nsP4 ubiquitination. In order to study the ubiquitination of N-end rule substrates in mammalian cells, plasmids expressing HA-tagged ubiquitin and flag-tagged Arg-nsP4 were co-transfected into mouse embryonic fibroblasts (MEF). After treatment with MG132, substantially ubiquitinated Arg-nsP4 was obtained by immunoprecipitation (Fig. 11G).

Taken together, this result implies that the model N-end rule substrate degrades in the reticulocyte fusion system and mammalian cells through rapid ubiquitination. In the substrate based on nsP4, the destabilizing moiety was sufficient to constitute N-degron and excluded the effect of other degradation crystals. Type 1 and 2 destabilized N-terminal residues are highly selective and indicate that the N-recognin-N-degron interaction is completely independent.

Experimental Example 2. Conservation of eukaryotic N-degron

The eukaryotic N-end rule pathway system was first identified in Saccharomyces cerevisiae (DK Gonda, et al, (1989), J. Biol. Chem., 264, 16700). In mammals, destabilizing residues are classified into three types (Tasaki and YT Kwon, (2007) Trends Biochem. Sci., 32, 520). Type 3 destabilizing residues included Ala, Ser and Thr, which are now considered to be stabilizing residues. The original model substrate was based on β-galactosidase, and no other substrate was tested systematically. Because N-degron requires an unstructured N-terminal extension with an accessible internal Lys and an N-terminal amino acid, the ability of the N-recognin to be able to conjugate with Ub is significantly different from substrate to substrate. Further, the N-terminal His was found to have a weak binding affinity (370 μM dissociation constant) in the UBR box compared to Arg (19 μM) based on isothermal titration calorimetry (ITC) analysis. Matta-Camacho, et al, (2010), Nat. Struct Mol. Biol., 17, 1182). The linkage between the UBR box and type 1 destabilization is 10 ² -10 ³ greater than the bond between ClpS and type 2 residues, which further indicates that the destabilizing moiety should be reevaluated.

The binding affinities between Type 1 peptides in the N-end rule pathway and UBR boxes of UBR 1 or UBR 2 (hereinafter UBR 1 box and UBR 2 box) were determined using the Autodock ligand-receptor with Lamarckian genetic algorithm Docking analysis (see Figures 12a &amp;12b; methods described in the docking process). The crystal structure of the UBR 1 and UBR 2 boxes is derived from the Protein Data Bank (PDB ID: 3NY1 and 3NY3, respectively) and the peptide structure was made in the mutation module of the PyMol package based on the RIFS peptide extracted from the complex structure of UBR 2 . Type 2 WIFS peptides were used to demonstrate the docking method. The lowest binding energy obtained from the docking analysis is 10-10 ² times lower than the binding energy obtained from the ITC data (Fig. 12c, panel a). The RIFS, KIFS, and HIFS peptides had binding affinities of -8.15, -7.71, and -7.29 kcal / mol, respectively, for the UBR 2 box, corresponding to dissociation constants of 1.08, 2.26, and 4.5 μM. Type 1 peptides tended to have binding affinities similar to ITC experiments. However, unlike the experimental data, docking analysis showed that Lys and His were also efficiently bound to the UBR 2 box. Type 2 peptides (WIFS) have ~ 103-fold higher binding affinity and exhibit inadequate interactions with active sites (Figures 12a-12c). Interestingly, one box of UBR has weak binding affinities (-6.91 and -5.60 kcal / mol, respectively) in Lys and His, whereas Arg binding to one box of UBR is comparable to binding to UBR 2 box (-8.07 And -8.15 kcal / mol, respectively) (Figure 12c, panels a & b).

These results also indicate that His and Lys can bind to the UBR box more strongly than previously. UBR 1 and UBR 2 have been considered to be functionally unnecessary sequelogs. However, the UBR 2 box may have evolved to interact with the specific destabilizing residues of UBR 1. This independent role of UBR 1 and UBR 2 can be explained by the difference between the UBR 1 and UBR 2 knockout mouse phenotypes. Furthermore, by docking analysis, type 1 residues interacting with the UBR box were shown to be as strong as type 2-ClpS interactions, further demonstrating the advantages of heterobarate inhibitors targeting both UBR and ClpS boxes .

In yeast, the ubiquitination of the N-end rule substrate is mediated by the E3 ligase alone, UBR 1, whereas in mammals, the seven UBR proteins defined by the UBR box are mediated by this process. Of the seven UBRs, UBR 1 and UBR 2 are the only E 3 containing the N-domain. The ClpS domain, the east ortholog of ClpS, recognizes bacterial N-end rule substrates such as Leu, Phe, Tyr, and Trp, which are type 2 destabilizing residues in cells. The binding affinity between ClpS and type 2 residues is within the submicromolar range when measured by fluorescence anisotropy using a fluorescein-labeled peptide.

However, here we found a weak binding affinity, similar to the type 1 residue-URB box interaction, using docking analysis (Figures 12d and 12e). The N-terminal Trp has an exceptionally high binding affinity of -9.51 kcal / mol, corresponding to a dissociation constant of 0.11 μM. The binding affinities of the N-terminal Phe, Tyr, and Leu were -7.76, -7.41, and? 7.01 kcal / mol, respectively. These affinities are similar to the interaction of Lys and His with the UBR2 box. Type 1 and Type 2 interactions are highly substrate-specific and only N-terminal Arg has a binding affinity of -3.01 kcal / mol for ClpS box (Fig. 12e, data not shown).

The in silico docking results are supported by in vitro biochemical analysis results in FIG. To this end, it includes residues of type 1 (Arg, His), type 2 (Phe, Tyr, Leu), tertiary (Glu, Asp, Cys), and stabilization (Met, Ala, Pro, Gly, Ser) Lt; / RTI &gt; construct with 13 different N-terminal amino acids (Fig. 12F). Consistent with the docking analysis, the His-nsP4 protein was rapidly degraded via UPS (FIG. 12f & FIG. 15). Model substrates known as destabilizing moieties were initially classified using β-galactosidase-based assays, all of which were short-lived in reticulocyte melts. As previously observed in yeast and mammals using the? -galactosidase model, deubiquitination of Ub-Pro-nsP4 was slow and the fusion protein was stable (Fig. 15, lane 3). Interestingly, Ala-nsP4 degraded more slowly than Met-, Gly-, and Ser-nsP4 (Fig. 12f). The N-terminal Ala was originally classified as type 3, a destabilizing residue with a relatively long half-life (> 4.4 hours in Ala-β-galactosidase). Recently, however, the N-terminal Ala is considered to be a stabilizing moiety. It is unclear whether the N-domain box or UBR box interacts with the N-terminal Ala through the penultimate N-terminal residue. The N-terminal Cys is a destabilizing residue in the nsP4 and beta -galactosidase models.

To trigger degradation, Cys-degron requires a certain concentration of active oxygen or nitrogen monoxide in the cell. Here we have found a protein with a very diverse stability with Cys-degron in mammalian cells, which appears to reflect intracellular oxygen status (data not shown). Secondary Cys (Cys-2) oxidation is biochemically and functionally related to cell physiology and homeostasis. Recently, a new regulator of the homeostatic response to hypoxia in plants has been identified as an N-end rule substrate with pro-N-degron Cys-2 (D. J. Gibbs, et al., (2011) Nature 479, 415). Ethylene Response Factor Group VII translational factors, including RESPONSIVE ELEMENTS 1 and 2 (HRE1 and HRE2) and Related to AP2.12 (RAP2.12), are degraded through UPS in the normoxic state, , Which leads to gene expression that promotes survival in anaerobic metabolism and hypoxia. Arabidopsis and the human genome each encode at least 206 and 502 proteins with Cys-2 degron, respectively.

The N-end rule in yeast E3 ligase has only UBR box, whereas mammal has UBR box and ClpS box at the same time, which exist to recognize type 1 and type 2 substrates, respectively. We used this characteristic of mammalian N-end rule E3 ligase to simultaneously target two active sites and cooperatively increase the efficiency of the interaction with this enzyme, and showed successful application of this.

Experimental Example 3: Inhibition of N-end rule substrate degradation by RF-C5 heterobalant molecules

Many protein-protein, enzyme-substrate and receptor-ligand interactions use a variety of interactions to increase affinity and selectivity, such as antigen-antibody, virus-cell, and bacterial toxin-cell interactions . As described above, the UBR protein provides a unique structure of scaffolds capable of heteroblast inhibition through two independent substrate-binding motifs: the UBR box for type 1 residues and the type 2 residue for type 1 residues N-domain.

Furthermore, as revealed by the docking studies of the present invention, unlike previously known, the two interactions have comparable binding affinities and furthermore, heterologous inhibitors that simultaneously target the UBR box and N-domain It can be useful. As a proof-of-concept test of heterobalance inhibition targeting UBR proteins, we have developed RF-C11, a prototype heterobalant small molecule. RF-C11 contains an N-terminal Arg for type 1 interactions and an N-terminal Phe for type 2 interactions, which are linked to the ε- and α-amino groups of the core Lys of the 11 carbon- Lt; / RTI &gt; When interacting with N-recognin, the Arg binding to the UBR domain will partially constrain the unbound Phe ligand, thereby increasing the local Phe concentration in the vicinity of the N-domain. This restriction will increase the probability of Phe binding to the N-domain. Phe bond will also increase the potential for Arg binding to the UBR domain. However, RF-C11 only increased the binding affinity to a moderate degree, due to the non-optimized tether length and ligand affinity for the target. Thermodynamically, a multi-valent inhibitor is most efficient when the flexible tethe is close to or slightly longer than the inter-bond site spacing in the target protein, since this length does not cause steric hindrance and does not reduce the entropy of the coordination, To maximize the most efficient concentration of &lt; / RTI &gt;

Various RF-Cn derivatives (where n is the length of the hydrocarbon chain tetra) were synthesized here to determine the appropriate tetra-length for the bivalent inhibitor and to identify the structure-activity relationship (Fig. 13a). Also, RF-Cn, such as GV-Cn (Gly and Val, both of which have stabilizing residues at their ends), R-Cn (a monovalent inhibitor of Arg with n = 1 to 14), and F-Cn Compounds were synthesized. Arg-Ala (RA, type 1) and Phe-Ala (FA, type 2), the traditional dipeptide inhibitors, were compared to the RF-Cn series compounds. 13 by using the in vitro N-end rule substrates model of Figure 11 the inhibition efficiency of the RF time-Cn as shown in were tested and dose-dependent manner (Fig. 13b & 13c 16). As shown in FIG. 13, the heterobalant RF-Cn compound more strongly inhibited the N-end rule pathway than the mono-balent compound or dipeptide, and the effect was maximized when n = 5. The highest activity was observed at 40 minutes when n = 5 for both type 1 (Arg-nsP4, Figure 13b) and type 2 (Tyr-nsP4, Figure 13c). All bivalent inhibitors with n = 3 to n = 11 are stronger inhibitors than Arg-Ala, but RF-C2 or RF-C15 did not, because the heterobalant interactions cooperate and the distance between the two binding motifs is expected I suggest that it is closer than it is. Since the full length structure of the UBR protein containing both the UBR box and the N-domain has not yet been interpreted, a combined structure was constructed using the Gramm-X protein-protein docking web server. The two structures extracted by the analysis had the lowest binding energies when the UBR 2 box and ClpS domain formed complexes as compared to the independent structure (Fig. 13d). These stereostructures reflected the fact that independent recognition motifs could closely combine RF-C5 with strong binding affinity, reflecting heterobalant interaction (Figure 13e). The binding affinities (-8.70 and -8.83 kcal / mol) of the UBR-ClpS stereochemistry to RF-C5 were stronger than all Type 1 and Type 2 interactions measured except for the N-terminal Trp (Figure 12c &Amp; 12e). At the lowest binding energies with the complex, Arg and Phe of the RF-C5 a-carbon stretched 13.5 ANGSTROM to 15 ANGSTROM apart (FIG. 13D). Thus, RF-C2 in the UBR 1/2 protein is too short to appear across both binding motifs. In a Type 2 model, the effect of heterobalance was not as significant as in Type 1 inhibition. These results are probably due to several factors. It is most simply explained that the interaction between the N-terminal Arg and the UBR box is internally stronger than the interaction between the N-terminal Phe and the ClpS domain (Figures 12e & 12e). However, the most plausible mechanism is that it is possible to adjust between binding pockets, especially considering that the two domains are located close together. Type 2 substrate interactions involve allosteric stereoconstitution changes in the ClpS domain, which can modulate UBR box binding to type 1 residues.

The present inventors have previously demonstrated that multi-balancing of RF-C11 increases binding efficiency by comparing with mono-balances RR-C11 and FF-C11. Furthermore, this finding was confirmed using a combination of a single-titer Arg or Phe (R-C5 + F-C5, Figure 13a) and a mono-valent ligand. The nsP4-based N-end rule model substrate was randomly included with biotinylated lysine, which was detected with streptavidin (STV) -frared conjugate and quantitative odd-numbered infrared imaging system. The heterotrophic ligand (RF-C5) and control were tested in in vitro translation reaction at 100 μM and samples were taken at 40, 60, 80 and 100 min (FIG. 13 e). As expected, the merged mono-valent-tetrahed ligand (R-C5 + F-C5) significantly less inhibits than the single heterobarate ligand (RF-C5). In addition, it was found that RF-C5 has a higher inhibitory effect than RR-C5, FF-C5, or GV-C5. In addition, these results indicate that merging two separate individual interactions does not have the same thermodynamic and kinetic effects as multivalent interaction.

Experimental Example 4. Inhibition of N-end rule pathway in mammalian cells by RF-C11

Fig. 14 shows the result of applying a heterobalance inhibitor to a cell culture system. Due to charge destabilizing moieties and hydrophilic tetra, the RF-Cn series compounds form micelles in aqueous solution. Penta Although Murray hetero balance bit inhibitor with a stator inhibits the N-end rule path in vitro more effectively and, RF-C11 is that, in the inhibited better in mammalian cells probably RF-C11 are stable in aqueous solution Micelles can form and pass through the plasma membrane. In dynamic light scattering, RF-C11 forms stable single lamellar micelles with a narrow size distribution (497 ± 40.2 nm) and a relatively low critical micelle concentration (~ 1 μM or less), while RF-C5 does not form micelles or liposomes (FIG. 14A). RF-C11 and its structural control, GC-C11, did not show cytotoxicity even at millimolar concentrations (RF-C11 is IC ₅₀ > 2 mM and IC ₅₀ = 331.1 μM (GV-C11), FIG. The ATE1 branch of the N-end rule pathway was associated with cell autonomic regulation of myocardial muscle cell proliferation during embryogenesis and hypertrophism.

To determine if there is a possibility of applying RF-C11 to myocardial cells, the present inventors isolated first myocardial cells from the embryonic mouse heart and treated them with a 25 [mu] M biotinylated heterobalance inhibitor. GV-C11 and RF-C11 internalized in myocardial cells, which were visualized by anti-troponin immunostaining, were observed by immunostaining with streptavidin-rhodamine (FIG. 14C). Since RF-C11 is rapidly internalized (intracellular) by mammalian cells, we examined whether the heterobalant inhibitor would affect the physiological substrate of the N-end rule pathway such as RGS4.15. It was found that the steady state level of transiently overexpressed RGS4 is significantly increased by RF-C11 and not by the Arg-Ala dipeptide (Fig. 14d). Cells were cultured with 10, 50, or 100 μM RF-C11 or Arg-Ala dipeptide in serum presence / absence after 24 hours of simultaneous transfer of MEF with RGS4 and LacZ plasmids. RGS4 was stabilized by 10 μM MG132. The effect of RF-C11 is even more important when there is no serum in the culture medium during the treatment period, which is usually the case for positive charge-mediated intracellular delivery. 14E shows pulse chase analysis of RGS4, physiological N-end rule substrate in MEFs. Transfected cells were pre-cultured with 100 μM RF-C11 or GV-C11, followed by anti-RGS4 immunoprecipitation, SDS-PAGE analysis and autoradiography. In the pulse-tracing assay, transiently overexpressed RGS4 was rapidly degraded in mouse embryonic fibroblasts, which was shown to be significantly degraded by RF-C11 (Figs. 14e and 14f). In contrast, GV-C11 had no significant effect on RGS4 stability. These results indicate that the non-cytotoxic and plasma membrane permeable heterobalant RF-C11 strongly inhibits the N-end rule pathway in mammalian cells.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (11)

Claims 1. A compound of the formula (1), or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In Formula 1,
n is an integer from 1 to 15;
Ra and Rb are different
Ra is H, C_One~ C₇Alkyl-NHC (= NH) NH₂, C_One~ C₇Alkyl-NH₂, C_One~ C₅Alkylphenyl or heteroaryl, said phenyl and heteroaryl being optionally substituted by one or more substituents selected from the group consisting of hydrogen, halogen, or C_One~ C₅Alkyl;
Rb is C_One~ C₈Alkyl, C_One~ C₇Alkyl, phenyl or heteroaryl, said alkyl and phenyl being optionally substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, or C_One~ C₅Alkyl;
or
Ra is C_One~ C₈Alkyl, C_One~ C₇Alkyl, phenyl or heteroaryl, said alkyl and phenyl being optionally substituted with one or more substituents selected from the group consisting of hydrogen, hydroxy, halogen, or C_One~ C₅Alkyl;
Rb is H, C_One~ C₇Alkyl-NHC (= NH) NH₂, C_One~ C₇Alkyl-NH₂, C_One~ C₅Alkylphenyl or heteroaryl, said phenyl and heteroaryl being optionally substituted by one or more substituents selected from the group consisting of hydrogen, halogen, or C_One~ C₅Alkyl; And
Rc is biotin or a derivative thereof, or C_One~ C₅Alkyl, said alkyl being optionally substituted by one or more substituents selected from the group consisting of hydrogen, halogen, or C_One~ C₅Alkyl.

2. The compound of claim 1, wherein n is an integer from 1 to 15;
Wherein Ra is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, 2-imidazolyl, 3-imidazolyl, 4-imidazolyl, 2 Pyrrole, 3-pyrrole, 2-pyridine, 3-pyridine, 4-pyridine, 2-pyrimidine, 3-pyrimidine or 4-pyrimidine;
Wherein Rb is methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, benzyl, 2- hydroxyphenylmethyl, 3- hydroxyphenylmethyl, ; Rc is methyl, ethyl, n-propyl, or isopropyl; or
Wherein Ra is methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, benzyl, 2- hydroxyphenylmethyl, 3-hydroxyphenylmethyl, 4-hydroxyphenylmethyl, ; Rc is methyl, ethyl, n-propyl, or isopropyl;
Wherein R b is selected from the group consisting of H, - (n-propyl) -NHC (= NH) NH ₂ , - (n-butyl) NH ₂ , benzyl, 2-imidazolyl, Or a pharmaceutically acceptable salt thereof, wherein the compound is a pyrrole, 3-pyrrole, 2-pyridine, 3-pyridine, 4-pyridine, 2-pyrimidine, 3-pyrimidine or 4-pyrimidine.
3. The compound of claim 2, wherein n is an integer from 1 to 15;
Wherein Ra is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, or 4-imidazolyl;
Rb is isopropyl, 2-butyl, benzyl, 4-hydroxyphenylmethyl, or 3-indole; or
Ra is isopropyl, 2-butyl, benzyl, 4-hydroxyphenylmethyl, or 3-indole;
Wherein Rb is H, - (n- _{propyl) -NHC (= NH) NH 2} , - (n- butyl) NH _2, benzyl, or 4-imidazolyl, and;
Wherein Rc is methyl, or ethyl. A compound of formula (I), or a pharmaceutically acceptable salt thereof. The method of claim 3,
N is 4;
Wherein Ra is - (n-propyl) -NHC (= NH) NH2 _;
Rb is benzyl; or
Ra is benzyl;
Wherein Rb is - (n- propyl) -NHC (= NH) NH _2, and,
Wherein Rc is methyl, or a pharmaceutically acceptable salt thereof.
A composition for inhibiting an N-end Rule pathway, comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.
A composition for preventing and treating heart disease, comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.
The composition according to claim 6, wherein the heart disease is hypertension, ischemic heart disease, coronary artery disease, angina pectoris, myocardial infarction, atherosclerosis (atherosclerosis), cerebrovascular disease, stroke or arrhythmia.
A composition for preventing and treating neurodegenerative diseases, comprising a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.
9. The method of claim 8, wherein the neurodegenerative disease is selected from the group consisting of Adrenal Leukodystrophy, Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy, Canavan disease, cerebral palsy, cockayne syndrome, corticobasal degeneration, Creutzfeldt- Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington ' s disease,
Huntedton's disease, HIV-related dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, neuroborreliosis, Machado-Joseph disease, Multiple system atrophy, multiple sclerosis, seizure sleep &lt; RTI ID = 0.0 &gt;
for example, narcolepsy, Niemann Pick disease, Parkinson ' s disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, Prion disease, progressive supranuclear palsy, Refsum's disease, Sandhoff disease, Schilder's disease, toxic anemia secondary subacute of spinal cord secondary Spiegelmeyer-Vogt-Sjogren-Batten disease, spinocerebellar ataxia, spinal muscular atrophy, stellate myelopathy, spinal cord degeneration, - Steele-Richardson-Olszewski disease, Tabes dorsalis, and toxic encephalopathy. A composition for preventing and treating neurodegenerative diseases.
10. The composition for preventing and treating neurodegenerative diseases according to claim 9, wherein the neurodegenerative disease is a proteinopahty associated with protein denaturation.
11. The method of claim 10, wherein the disease associated with protein degeneration is selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia of the Levies, muscular atrophy sclerosis (ALS), Huntington's disease, spinal cord cerebral ataxia or spinobulbar musculular atrophy A composition for the prevention and treatment of metastatic disease.

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