WO2023074937A1 - Compound as ubr box domain ligand - Google Patents

Abstract

The present description pertains to a novel compound as a ligand binding to a UBR box domain that is involved in an intracellular protein degradation pathway, and a use thereof.

Description

Compounds as UBR box domain ligands

The disclosure herein relates to ligand compounds that bind to the UBR box domain.

Intracellular proteins have a range of retention periods. Cells use proteolytic regulatory mechanisms to protect against damage caused by misfolded, aggregated or abnormal proteins. That is, cells regulate the amount and function of proteins in vivo through proteolysis.

Intracellular protein degradation is largely composed of two pathways: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. The UPS consists of two classes of E3-E2 ubiquitin ligases and deubiquitylases (DUBs). E3-E2 ubiquitin ligase recognizes a substrate through a degradation signal called "degron" and conjugates Ub, a 9 kDa protein, to a lysine residue inside the target protein. Mammalian E3 ligases, of which there are more than 800, are likely to target different degrons, and thus the UPS is expected to be involved in almost all physiological processes in eukaryotes.

Disassembly signals, one of the characteristics of proteins, not only cause proteins to be rapidly degraded, but are also involved in the specificity of UPS. The degradation signals present at the N-terminus were collectively referred to as "N-degron", and the half-life of a protein was determined by the amino acid residue present at the N-terminus of the protein. It's called the "N-end rule."

As N-recognin that recognizes the N-terminal degradation signal of a protein, UBR (Ubiquitin protein ligase E3 component n-recognin) is used. At this time, the ubiquitinated substrate produced by the binding of N-lecognin to the N-end rule ligand is delivered to the proteasome and degraded into short peptides. Specifically, the UBR box domain in the UBR recognizes the N-terminal amino acid of the substrate and ubiquitinates the substrate through the RING domain or the HECT domain, thereby degrading the substrate through the proteasome pathway.

The present inventors first discovered or cloned N-lecognines, UBR1, UBR2, UBR3, and UBR5, and found that they had a UBR box domain as a substrate recognition domain (Tasaki et al. 2005).

The present specification relates to a novel compound and its use as a ligand that binds to the UBR box domain associated with an intracellular proteolytic pathway.

The present specification is to provide a small molecule compound that binds to the UBR box domain and a proteolytic use thereof.

In one embodiment, the present specification intends to provide a ligand compound that binds to the UBR box domain. At this time, the UBR box domain includes UBR box domains in UBRs 1 to 7.

In one embodiment, the present specification intends to provide a composition for inhibition of UBR box domain substrate binding comprising a ligand compound binding to the UBR box domain.

In a specific embodiment, the present specification is intended to provide a pharmaceutical composition for treating a UBR-related disease, including a ligand compound that binds to a UBR box domain, and a use thereof.

In a more specific embodiment, the present specification describes muscle loss caused by muscle atrophy (Becker, Congennital, Duchenne, Distal, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, myotonic liposarcoma, caused by excessive protein breakdown, with muscle wasting diseases including ocuophargyngeal (muscular dystrophy), sarcopenia and cancer cachexia; UBR box and UBR protein in cystic fibrosis, Johanson-Blizzard syndrome, urethral obstruction sequence, autoimmune pancreatitis or Usher syndrome. It is intended to provide pharmaceutical compositions and uses thereof for the treatment of diseases known to be related diseases.

In the present specification, a compound having the structure of Formula 1, an isomer thereof, or a salt thereof is provided.

[Formula 1]

X ₁ is any one selected from the following,

-(CH ₂ )nO R,

-(CH ₂ ) _n (C ₆ -C ₁₂ aryl)OR,

-CH ₂ (3-12 membered-heteroaryl or heterocyclic)OR;

-CH ₂ (cycloalkyl)OR,

-NH(CH ₂ )nOR, and

-CH ₂ NH(CH ₂ )nOR,

At this time, in each functional group, n is independently an integer selected from 1, 2, 3, 4, 5, and 6, and R is H or C _1-4 alkyl;

X ₂ is selected from H, halo, cyano, methyl and ethyl; and,

X ₃ is C _1-3 alkyl or amino.

In this case, in one embodiment, in Chemical Formula 1, the X ₂ is H, F, Cl or CH ₃ .

In this case, in one embodiment, in Chemical Formula 1, X ₃ is CH ₃ or NH ₂ .

In a specific embodiment, the X ₁ may be any one selected from the following:

-(CH ₂ ) ₂ OMe

-(CH ₂ ) ₃ OMe

-(CH ₂ ) ₄ OMe

-(CH ₂ ) ₅ OMe

-CH ₂ (C ₆ H ₄ )OMe

-(CH ₂ ) ₂ (C ₆ H ₄ )OMe

-NH(CH ₂ ) ₂ OMe

-NH(CH ₂ ) ₃ OMe

-(CH ₂ ) ₂ OEt

-(CH ₂ ) ₃ OEt

-(CH ₂ ) ₄ OEt

-(CH ₂ ) ₅ OEt

-CH ₂ (C ₆ H ₄ )OEt

-(CH ₂ ) ₂ (C ₆ H ₄ )OEt

-NH(CH ₂ ) ₂ OEt

-NH(CH ₂ ) ₃ OEt

In a more specific embodiment, in Formula 1, X ₁ is -(CH ₂ ) ₄ OMe, -(CH ₂ ) ₅ OMe, -CH ₂ (C ₆ H ₄ )OMe, -(CH ₂ ) ₂ (C ₆ H ₄ )OMe, and -NH(CH ₂ ) ₃ OMe.

In a more specific embodiment, in Chemical Formula 1, X ₃ may be CH ₃ or NH ₂ .

As a preferred embodiment, in the compound of the present application, in Formula 1, X ₁ is -(CH ₂ ) ₄ OMe, -(CH ₂ ) ₅ OMe, -CH ₂ (C ₆ H ₄ )OMe, -(CH ₂ ) ₂ (C ₆ H ₄ )OMe or -NH(CH ₂ ) ₃ OMe, X ₂ is H, F, Cl, CH ₃ , and X ₃ is CH ₃ , NH ₂ .

More preferably X ₁ is -(CH ₂ ) ₄ OMe, -CH ₂ (C ₆ H ₄ )OMe or -NH(CH ₂ ) ₃ OMe, X ₂ is H or Cl and X ₃ is NH ₂ .

In one embodiment of the present application, the compound may be a compound selected from the following or a salt thereof:

3-(2-(3-methoxyphenyl)acetamido)benzamide;

4-chloro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

3-(5-methoxypentanamido)benzamide;

4-chloro-3-(3-(3-methoxypropyl)ureido)benzamide;

4-chloro-3-(3-(4-methoxyphenyl)propanamido)benzamide;

3-(6-methoxyhexanamido)benzamide;

3-(2-(3-methoxyphenyl)acetamido)-4-methylbenzamide;

4-fluoro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

1-(5-acetyl-2-chlorophenyl)-3-(3-methoxypropyl)urea;

1-(3-acetylphenyl)-3-(3-methoxypropyl)urea;

N-(5-acetyl-2-chlorophenyl)-2-(3-methoxyphenyl)acetamide, and

N-(3-acetylphenyl)-2-(3-methoxyphenyl)acetamide.

In another aspect of the present application, a pharmaceutical composition for treating a UBR-related disease comprising the compound or a pharmaceutically acceptable salt thereof and a method for treating a UBR-related disease using the same are provided.

At this time, in one embodiment, The UBR-related diseases are caused by muscle atrophy (Becker, Congennital, Duchenne, Distal, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, myotonic, ocuophargyngeal) (muscular dystrophy), sarcopenia or cancer cachexia ( Liposarcoma caused by excessive protein breakdown, cystic fibrosis, Johanson-Blizzard syndrome, as well as muscle wasting diseases, including cancer cachexia ), urethral obstruction sequence, autoimmune pancreatitis or Usher syndrome.

One invention disclosed herein is to provide a ligand compound having higher binding force to the UBR box domain. The UBR box domain ligand compound of the present application can effectively inhibit the binding of the UBR box domain substrate.

In addition, the present application can provide various uses of the UBR protein degradation system using such a ligand compound. For example, UBR-related diseases (eg, muscle loss, etc.) can be treated through UBR box domain ligand compounds.

1 is an experimental result of confirming whether muscle actin is an Arg/N-degron pathway substrate using an immunoblotting method.

FIG. 2 is an experimental result confirming whether the compounds ( Compounds 1, 2, and 4) inhibit the degradation of ACTC1, a substrate of UBR proteins in muscle cells, in muscle cells using an immunoblotting method.

Figure 3 is an experimental result confirming whether the compound (Compound 3) inhibits the degradation of ACTA2, a substrate for UBR proteins in muscle cells, in muscle cells using immunoblotting.

Hereinafter, with reference to the accompanying drawings, the content of the invention will be described in more detail through specific implementation examples and examples. The content of the invention disclosed by this specification may be implemented in various ways, and is not limited to the specific implementation described herein. Modifications and other implementations thereof should be understood to be included within the scope of the claims.

term definition

Definitions of key terms used in this specification are as follows.

UBR (Ubiquitin protein ligase E3 component n-recognin)

As used herein, the term UBR means an abbreviation for Ubiquitin protein ligase E3 component n-recognin. The UBR is N-lecognin that recognizes the N-terminal residue of a protein, and it is known that at least 7 types of UBRs 1 to 7 exist in mammals. The UBR is N-lecognin, which is involved in the N-terminal law pathway, which is a proteolytic pathway in vivo. Specifically, the UBR recognizes the N-terminal degradation signal (N-degron) of a protein and is involved in the process of degradation of a substrate protein through the ubiquitin proteasome pathway.

UBR box domain

As used herein, the term UBR box domain is a domain present in UBR protein and is a zinc finger motif. The UBR proteins include UBR 1, UBR 2, UBR 3, UBR 4, UBR 5, UBR 6 and UBR 7. The UBR box domain is known as a domain to which a substrate protein binds. A compound as a UBR box domain ligand disclosed herein can bind to the UBR box domain and inhibit binding of a UBR box domain substrate. Thus, the UBR box domain ligand compounds disclosed herein can affect proteolytic pathways in cells.

RING domain

As used herein, the term RING domain exists in UBR 1, 2 and 3 proteins. The RING domain may also be used as the term RING ubiquitination domain. The RING domain is a zinc finger motif present in UBR protein. The RING domain is a domain that plays an important role in the process of transferring ubiquitin from E2 to a substrate protein, and the RING domain serves to cause the process of transferring ubiquitin to a substrate protein in one step.

HECT domain

As used herein, the term HECT domain is known to exist within the UBR 5 protein. The HECT domain may also be used as the term HECT ubiquitination domain. The HECT domain is a domain that plays an important role in the process of transferring ubiquitin from E2 to a substrate protein. Ubiquitin in E2 is transferred to the HECT domain and then transferred to the substrate protein. That is, the HECT domain serves to make the process of transferring the ubiquitin to the substrate protein in two steps.

zinc finger motif

The term zinc finger motif used herein refers to a protein structural motif in which one or more zinc ions are present to stabilize the protein structure. The UBR box domain and RING domain of the present specification are zinc finger motifs.

PROTAC

PROTAC (proteolysis targeting chimera; PROTAC) is a heterobifunctional small molecule composed of two active domains and a linker, and is a technology capable of removing specific unwanted proteins. Instead of acting as an enzyme inhibitor, Protec works by inducing selective intracellular protein degradation. Protek consists of two covalently linked protein-binding ligands. One can bind the E3 ubiquitin ligase and the other binds to the target protein signifying degradation. Recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. Since Protek only needs to bind to the target rather than inhibiting the activity of the target protein with high selectivity, many efforts are currently underway to convert previously ineffective inhibitor molecules of the target protein into Protek for next-generation drugs.

The protein disclosed herein uses a "bifunctional compound" comprising a ligand that binds to UBR, an E3 ligase, particularly a ligand that binds to the UBR box domain in UBR and a target protein (or peptide) binding ligand. to be The UBR box domain binding ligand binds to the UBR box domain. The UBR box domain is known as a domain to which an N-terminal residue sequence or an N-terminal degradation signal binds. This domain is involved in the process of protein degradation by the N-terminal pathway rule. Thus, the UBR box domain binding ligand can affect the proteolytic process through the N-terminal law pathway.

In the N-terminal law pathway, N-degrons are recognized by N-recognin, and UBR (Ubiquitin protein ligase E3 component n-recognin) was discovered as N-recognin. It is known that the UBR recognizes an N-terminal residue sequence or an N-terminal degradation signal through the UBR box domain. That is, UBR recognizes a protein degradation signal through the UBR box domain, and through this, the protein degradation process proceeds.

The protein degradation process by the UBR may include the following. The UBR box domain recognizes a substrate having an N-terminal degradation signal, ubiquitin is bound to the substrate, and the substrate to which ubiquitin is bound can be degraded by the proteasome. That is, a substrate having an N-terminal degradation signal can be degraded by the ubiquitin proteasome system (UPS).

Therefore, the protein disclosed herein uses a bifunctional compound including a UBR box domain binding ligand and a target protein (or peptide) binding ligand to position a protein to be degraded in close proximity to the UBR, particularly the UBR box domain let it At this time, the protein to be degraded located close to the UBR box domain is ubiquitinated by the UBR box domain and degraded through the proteasome pathway.

At this time, in one embodiment, the protector may include a UBR box domain binding ligand and a target protein binding ligand. At this time, the protector may further include a linker, and the linker may serve to connect the two ligands by being present between the UBR box domain binding ligand and the target protein binding ligand.

ligand

The term ligand as used herein refers to a substance that specifically binds to a protein. The protein includes an enzyme or a receptor, and when the protein is an enzyme, the ligand may mean a substrate that binds to the enzyme, and when the protein is a receptor, the ligand may mean a hormone that binds to the receptor.

A UBR box domain ligand compound provided herein refers to a compound that binds to a UBR box domain. In one embodiment, the compound refers to a compound that binds to a UBR box domain in a UBR protein. In a specific embodiment, the compound refers to a compound that binds to a UBR box domain present in one or more proteins of UBR1 to 7. However, it is not limited thereto.

A compound as a UBR box domain ligand provided herein may act competitively with a substrate of a UBR box domain. That is, the compound may inhibit binding of a substrate of the UBR box domain. In addition, the compound may inhibit substrate decomposition by inhibiting binding of the substrate.

aliphatic

An “aliphatic” or “aliphatic group” is a hydrocarbon moiety that can be straight-chain (i.e., unbranched), branched or cyclic, can be fully saturated, or can contain one or more units of unsaturation, but the unsaturation is not aromatic. indicates In some variations, the aliphatic group is unbranched or branched. In another variation, the aliphatic group is cyclic. In some variations, unless otherwise specified, aliphatic groups contain from 1 to 20 carbon atoms. In certain embodiments, aliphatic groups contain 1 to 12 carbon atoms. In certain embodiments, aliphatic groups contain 1 to 8 carbon atoms. In certain embodiments, aliphatic groups contain 1 to 6 carbon atoms. In certain embodiments, aliphatic groups contain 1 to 5 carbon atoms, in certain embodiments, aliphatic groups contain 1 to 4 carbon atoms, in other embodiments aliphatic groups contain 1 to 3 carbon atoms, and In another embodiment, aliphatic groups contain 1 to 2 carbon atoms. Suitable aliphatic groups include, for example, linear or branched alkyl, alkenyl and alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. includes

"heteroaliphatic"

The term "heteroaliphatic" refers to an aliphatic group in which one or more carbon atoms are independently replaced by one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen or phosphorus. In certain embodiments, one or two carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, or phosphorus. Heteroaliphatic groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and may be "heterocycle", "heterocyclyl", "heterocycloaliphatic" or "heterocyclic". "Includes groups. In some variations, the heteroaliphatic group is branched or unbranched. In another variation, the heteroaliphatic group is cyclic. In another variation, the heteroaliphatic group is acyclic.

pharmaceutically acceptable salts

The term “pharmaceutically acceptable salt” may collectively refer to any salt that equally retains the biological effectiveness and properties of the compound of Formula 1 according to one embodiment and is desirable in terms of pharmaceutical, biological or other properties. Non-limiting examples of such a salt include a salt obtained by adding an inorganic base or an organic base to the compound of Formula 1, or an acid addition salt. Examples of organic acids capable of forming such acid addition salts include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, and examples of the inorganic acid include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents and other references mentioned herein are incorporated by reference in their entirety.

Problems with the prior art

The fate and activity of cellular proteins are changed through binding to ubiquitin (Ub) molecules, which occurs in a series of enzymatic reactions. Specifically, the E1 activating protein transfers Ub to the E2 conjugating protein, resulting in a covalently linked intermediate, E2-Ub. And the final step of ubiquitination occurs by the E3 ligase enzyme, and various kinds of proteins (RING, HECT, and RBR) transfer Ub from E2-Ub to the substrate through various mechanisms. Currently, cereblon and VHL-based therapeutics, which are generally performed through this process, are being studied, and in addition, molecules that recognize E3 ligase capable of attaching a ligand to a target protein are being studied.

At this time, proteolytic agents must form an enzymatic complex in the cell that acts in a catalytic manner to deplete the target protein. In other words, for effective target protein degradation, a ternary complex of E3 ligase-ligand (PROTAC)-target protein must be stably formed in cells, but it is not easy to artificially form such a three-dimensional structure, Therefore, there has been a problem in that the target protein degradation efficiency is also low. In particular, it is also very difficult to find a low-molecular compound that has a structure and physicochemical properties suitable for including two separate binding moieties in the same molecule and serves as a linker in creating such a three-dimensional structure.

The present application utilizes a system in which UBR, N-lecognin, recognizes the N-terminal degradation signal of a target protein, and the UBR degrades the target protein by binding ubiquitin to the target protein. Since the UBR system used by the present application uses the N-end rule pathway, compared to the above problems, there is no need to form a complicated ternary complex, so the protein degradation efficiency is remarkably high. there is.

Hereinafter, the UBR system of the present application and the UBR box domain ligand compound for using the same will be described in detail.

I. UBR box domain

One. outline

A compound as a UBR box domain ligand provided herein binds to a UBR box domain. The UBR box domain is known as a domain to which an N-terminal residue sequence or an N-terminal degradation signal binds. This domain is involved in the process of protein degradation by the N-terminal pathway rule. Thus, the compound may affect the proteolytic process through the N-terminal law pathway.

2. N-end rule pathway

Cells regulate the amount of protein through proteolysis. At this time, it is known that the protein degradation process proceeds through a process of recognizing degron, which is a protein degradation signal. Specifically, protein degradation is regulated depending on the N-terminal residue sequence of the protein, and proteolysis signals present at the N-terminus are collectively referred to as N-degron. The N-degrons include those having a positively charged residue (eg, arginine, lysine, histidine) or a large hydrophobic residue (phenylalanine, leucine, tryptophan, isoleucine, tyrosine) at the N-terminus. In this way, the term N-end rule was used based on the relationship that the half-life of a protein is determined by what amino acid residue exists at the N-terminus of the protein. The term N-end rule is also used as the term "N-degron pathway".

3. Proteolytic system by N-lecognin UBR

UBR stands for Ubiquitin protein ligase E3 component n-recognin, and UBR is N-recognin that recognizes the N-terminal degradation signal of proteins. It is known that at least 7 types of UBRs 1 to 7 exist in mammals.

In particular, among the UBRs 1 to 7, UBR1, UBR2, UBR3 and UBR5 act as ubiquitin protein ligase E3 and are known to have a RING domain or a HECT domain. Substrates of the N-terminal law binding to the UBR are degraded by the ubiquitin proteasome pathway. For example, misfolded proteins in cells are degraded through the ubiquitin proteasome pathway, as they can aggregate and block the proteasome or impair other cellular functions if left unattended for long periods of time. (Ji and Kwon, 2017). That is, a substrate having an N-terminal degradation signal can be degraded by the ubiquitin proteasome system (UPS).

4. UBR box domain

It is known that the UBR recognizes an N-terminal residue sequence or an N-terminal degradation signal through the 'UBR box domain'. That is, UBR is N-lecognin associated with the N-terminal pathway law, which is a protein degradation pathway, and the UBR box domain in UBR is a substrate binding domain.

Specifically, the UBR box domain in the UBR recognizes the N-terminal amino acid of the substrate and ubiquitinates the substrate through the RING domain or the HECT domain, thereby degrading the substrate through the proteasome pathway. The UBR box domain common to UBR is a zinc finger motif having a size of about 70 residues, and is known as a highly conserved substrate-binding domain.

As such, the UBR box domain plays an important role in the intracellular protein degradation pathway through the recognition of N-terminal degradation signals. Thus, ligands that bind to the UBR box domain can affect proteolytic pathways in cells. This application relates to compounds as ligands that bind to the UBR box domain, which is involved in intracellular proteolytic pathways.

II. UBR box domain ligand

One. outline

One) The compounds herein reflect the structure of the UBR box domain and the binding properties with N-terminal pathway substrates.

The UBR box domain ligand compound disclosed herein was designed in consideration of the structure of the UBR box domain and the binding form between the UBR box domain and the N-terminal pathway substrate.

Various amino acids present in the UBR box domain interact and bind with amino acids of the N-terminal pathway substrate through ionic interaction, hydrogen bond, and hydrophobic interaction. By analyzing these binding modes, in the present specification, a low-molecular-weight compound capable of forming a suitable binding mode with the UBR box domain is synthesized and provided. Furthermore, in the present specification, compounds represented by Chemical Formulas 1 to 12 are provided.

2) The compounds of the present specification have a structure that binds well to the UBR box domain.

In the present specification, as a ligand compound having high binding to the UBR box domain, a compound having the structure of [Formula 1] below is disclosed:

[Formula 1]

In the present specification, various compounds are designed and provided based on [Formula 1]. At this time, candidates for X ₁ , X ₂ , and X ₃ were derived in consideration of binding ability with the UBR box domain. A more detailed description of the various compounds based on [Formula 1] is described below.

2. Formula 1

[Formula 1]

(1) X ₁

In Formula 1, X ₁ may be a substituted or unsubstituted C _1-6 alkylaliphatic hydrocarbon or a substituted or unsubstituted C _1-6 heteroaliphatic hydrocarbon. In this case, the substitution may be made by at least one selected from NO, S, aryl, heteroaryl, cycloalkyl, alkoxy, arylalkoxy, heteroarylalkoxy, and cycloalkylalkoxy.

In one embodiment, the X ₁ may be any one selected from among the following.

-(CH ₂ )nO R,

-(CH ₂ ) _n (C ₆ -C ₁₂ aryl)OR,

-CH ₂ (3-12 membered-heteroaryl or heterocyclic)OR;

-CH ₂ (cycloalkyl)OR,

-NH(CH ₂ )nOR, and

-CH ₂ NH(CH ₂ )nOR

At this time, n in each functional group is independently an integer of 1 to 6, and R may be H, C _1-4 alkyl.

'Alkyl' means an aliphatic hydrocarbon radical, and includes both straight-chain and branched hydrocarbon radicals. For example, C*?*alkyl is an aliphatic hydrocarbon having 1 to 6 carbon atoms, methyl, ethyl, propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, sec-butyl, tert- Includes all butyl, neopentyl, isopentyl, etc.

'Aryl' is an organic radical derived from an aromatic hydrocarbon by the removal of one hydrogen, and contains single or multiple fused ring systems containing 5 to 14 substituted or unsubstituted ring atoms in each ring, as appropriate; It includes even a form in which a plurality of aryls are connected by a single bond. Specific examples include phenyl, naphthyl, biphenyl, terphenyl, anthryl, indenyl, fluorenyl, phenanthryl, triphenylenyl, pyrenyl, perylenenyl, chrysenyl, naphthacenyl, fluoranthenyl, etc. , but not limited thereto.

'Heteroaryl' means a 5- to 12-membered aromatic radical containing 1 to 3 heteroatoms selected from the group consisting of N, O and S atoms, a 5- to 6-membered monocyclic heteroaryl radical and a benzene ring. or a bicyclic heteroaryl radical formed by fusion with a pyridine ring. In addition, the term 'heterocycle' refers to a 3- to 12-membered mono- or poly-cyclic ring containing one or more heteroatoms O, N or S, preferably 1 to 4 identical or different heteroatoms. means and does not include an aromatic ring. Non-limiting examples of heteroaryl rings or heterocyclic rings include oxetane, pyrrolidine, pyrrole, tetrahydrofuran, furan, tetrahydrothiophene, thiophene, imidazolidine, imidazole, pyrazolidine, pyrazole, Pyrrolidine, oxazolidine, oxazole, isoxazolidine, isoxazole, thiazolidine, thiazole, isothiazolidine, isothiazole, dioxolane, dithiolane, oxadiazole, thiadiazole, dithiazole, tetrazole, oxatetrazole, tiatetrazole, piperidine, pyridine, pyrimidine, tetrahydropyran, pyran, thian, thiopyran, piperazine, diazine, morpholine, oxazine, dioxane, Indole, indoline, benzodioxole, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzisoxazole, benzothiazole, benzothiadiazole, benzotriazole, quinoline, isoquinoline, purine, puropyridine, mono- or di-azabicycles such as quinuclidine, diazabicycloheptane, monoazabicyclooctane, diazaspiroundecane, hexahydropyrrolopyrrole, pyrrolopyrrole, pyrrolopyridine, imidazopyridine chopped, dihydrobenzodioxin, dihydrobenzofuran, and the like, but are not limited thereto.

'Cycloalkyl', unless otherwise defined, means a saturated aliphatic 3 to 10 membered ring, preferably a 3 to 7 membered ring. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

Heterocycloalkyl refers to a ring structure containing at least one heteroatom selected from P, N, O and S in addition to the ring-carbon atom in the above cycloalkyl.

In a more specific embodiment, the X ₁ may be any one selected from among the following.

-(CH ₂ ) ₂ OMe

-(CH ₂ ) ₃ OMe

-(CH ₂ ) ₄ OMe

-(CH ₂ ) ₅ OMe

-CH ₂ (C ₆ H ₄ )OMe

-(CH ₂ ) ₂ (C ₆ H ₄ )OMe

-NH(CH ₂ ) ₂ OMe

-NH(CH ₂ ) ₃ OMe

-(CH ₂ ) ₂ OEt

-(CH ₂ ) ₃ OEt

-(CH ₂ ) ₄ OEt

-(CH ₂ ) ₅ OEt

-CH ₂ (C ₆ H ₄ )OEt

-(CH ₂ ) ₂ (C ₆ H ₄ )OEt

-NH(CH ₂ ) ₂ OEt

-NH(CH ₂ ) ₃ OEt

(2)X ₂

In Formula 1, X ₂ may be selected from H, halo, cyano, methyl, and ethyl. Examples of the "halo" may be fluoro, chloro, bromo or iodo.

In a specific embodiment, X ₂ may be H, Cl, F or CH ₃ .

(3)X ₃

In Formula 1, X ₃ may be C1-3alkyl or amino.

In a specific embodiment, it may be CH ₃ or NH ₂ .

As a specific example, Chemical Formula 1 may have a structure selected from the following:

[Formula 1-1]

[Formula 1-2]

1) X ₁

In one embodiment, the X ₁ may be any one selected from among the following.

-(CH ₂ )nO R,

-(CH ₂ ) _n (C ₆ -C ₁₂ aryl)OR,

-CH ₂ (3-12 membered-heteroaryl or heterocyclic)OR;

-CH ₂ (cycloalkyl)OR,

-NH(CH ₂ ) _n OR, and

-CH ₂ NH(CH ₂ ) _n OR.

At this time, in each functional group, n is independently an integer selected from 1, 2, 3, 4, 5, and 6, and R may be H or C _1-4 alkyl.

In a specific embodiment, the aryl may be phenyl or naphthyl.

In a specific embodiment, the heteroaryl may be a saturated or unsaturated 3-12 membered ring containing 1-3 heteroatoms selected from O, S, and N in the ring.

In one specific embodiment, the cycloalkyl may be cyclobutyl, cyclopentyl, or cyclohexyl.

In specific embodiments, R may be methyl or ethyl. Preferably it may be methyl.

In a more specific embodiment, the X ₁ may be selected from the following structures:

In a more specific embodiment, the X ₁ may be selected from the following structures:

2) X ₂

In Formulas [1-1] and [1-2], X ₂ may be selected from H, halo, cyano, methyl, and ethyl. Examples of the "halo" may be fluoro, chloro, bromo or iodo. In a more specific embodiment, the X ₂ may be H, Cl or F.

The compounds disclosed herein may exist in the form of stereoisomers or salts thereof, and isomers or salts of such compounds are included in the scope of the present specification.

III. Specific examples of compounds as UBR box domain ligands

One. Specific examples of compounds

The following describes specific examples of the compounds disclosed herein. The specific examples below are exemplary structures for easy understanding of the compound disclosed herein, and the scope of the compound disclosed herein is not limited to the examples below.

As a more specific embodiment, a specific exemplary compound for [Formula 1-1] may be selected from the following.

[Formula 1-1]

.

In this case, the X ₁ and X ₂ in the compound are listed in Table of Contents Ⅱ. The same applies as described for X ₁ and X ₂ of the UBR box domain ligand.

Table 1. Specific exemplary compounds for formula [1-1]

At this time, the compound may be considered in the form of one of its possible isomers or a form of a mixture thereof. For example, all stereoisomers, including enantiomers and diastereomers, or mixtures thereof (eg, racemic mixtures) are contemplated. The compounds also include pharmaceutically acceptable salts, solvates and polymorphs of known compounds.

In another specific embodiment, the compound disclosed herein may have a structure of [Formula 1-2]:

[Formula 1-2]

.

In this case, X ₁ and X ₄ in the compound are listed in Table of Contents Ⅱ. The same applies as described for X ₁ and X ₂ of the UBR box domain ligand.

Specific exemplary compounds for [Formula 1-2] may be selected from the following.

Table 2. Specific exemplary compounds for formula [1-2]

At this time, the compound may be considered in the form of one of its possible isomers or a form of a mixture thereof. For example, all stereoisomers, including enantiomers and diastereomers, or mixtures thereof (eg, racemic mixtures) are contemplated. The compounds also include pharmaceutically acceptable salts, solvates and polymorphs of known compounds.

One. salts of the above compounds

A compound disclosed herein may be considered in the form of a salt thereof. At this time, the salt includes a pharmaceutically acceptable salt. Salts disclosed herein include acid addition salts or basic addition salts.

Exemplary acids forming the salt include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, glutaric acid, and the like, and exemplary bases forming the salt include lithium, sodium, potassium, calcium, magnesium , methylamine, trimethylamine, and the like. However, it is not limited thereto and may be appropriately selected by those skilled in the art.

IV. use of the compound

One. Inhibit UBR box domain substrate binding

A composition for inhibition of UBR box domain substrate binding

The compounds disclosed herein can be used to prepare compositions for inhibiting binding of UBR box domain substrates. In one embodiment, a composition comprising the compound disclosed herein can be used to inhibit binding of a UBR box domain substrate by binding to a UBR box domain. In another embodiment, a composition containing the compound may be used for preventing decomposition of a substrate that is degraded by being bound to the UBR box domain. In a specific embodiment, a composition containing the compound may be used to prevent a substrate from being degraded by the ubiquitin-proteasome pathway by binding to the UBR box domain.

In a specific embodiment, a composition including a compound disclosed herein may be used for inhibiting binding of a substrate having an N-terminal residue binding to a UBR box domain. In a specific embodiment, N- It can be used for inhibiting the binding of a substrate having a terminal residue. However, it is not limited thereto, and may be used for inhibiting binding of a substance known in the art as a substrate of a UBR box domain.

2. Treatment of UBR-related diseases

The compound of the present specification or a salt thereof has a property of binding to the UBR box domain. That is, it is a compound that functions as a ligand that binds to the UBR box domain. Therefore, using these compounds, it is possible to inhibit the degradation of proteins that are degraded through binding to the UBR box domain in vivo, and UBR-related diseases can be treated using this mechanism.

One) pharmaceutical composition

The compounds disclosed herein can be used in the manufacture of pharmaceutical compositions for treating a subject in need thereof.

In this case, the treatment includes an effect of improving symptoms of a specific medical condition or delaying the progression of a disease. In this case, the subject includes humans and non-human animals. At this time, the pharmaceutical composition may include a pharmaceutically acceptable carrier, excipients and / or additives together with the compound. Such pharmaceutically acceptable carriers, excipients and/or additives include, but are not limited to, water, saline, glycols, glycerol, animal and vegetable fats, oils, starches, etc. Pharmaceutically acceptable carriers, excipients known in the art and/or additives.

2) treatment method

Provided herein are methods of treatment comprising administering a compound disclosed herein or a pharmaceutically acceptable salt thereof to a subject in need thereof.

“Administering” refers to providing, contacting, and/or delivering a compound or compounds by any suitable route to achieve a desired effect. Administration can be oral, sublingual, parenteral (eg intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, administration via buccal, rectal, vaginal, intranasal, ophthalmic, inhalation and implantation.

At this time, administration of the compound or a pharmaceutically acceptable salt thereof may have an effect of improving symptoms of a specific medical condition or delaying the progression of a disease compared to a subject not receiving the administration. In this case, the subject includes humans and non-human animals.

- UBR-related diseases

In one embodiment, the present specification provides a treatment method comprising administering the compound or a pharmaceutically acceptable salt thereof to a subject having a UBR-related disease. That is, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, can be used to treat a UBR-associated disease. In one specific embodiment, the compound or a pharmaceutically acceptable salt thereof can be used to treat a specific disease that can be treated by inhibiting the degradation of a protein that is degraded through binding to the UBR box domain.

The specific disease is caused by muscle atrophy (Becker, Congennital, Duchenne, Distal, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, myotonic, ocuophargyngeal) (muscular dystrophy), sarcopenia or cancer cachexia. muscle wasting diseases, including cachexia, as well as liposarcoma, cystic fibrosis, and Johanson-Blizzard syndrome caused by excessive protein breakdown. , diseases known to be related to the UBR box and UBR protein, such as urethral obstruction sequence, autoimmune pancreatitis or Usher syndrome.

In one embodiment, the compound or pharmaceutically acceptable salt thereof can be used to treat muscle loss mediated by UBR. For example, it is known that the rapid loss of muscle mass accompanying disease states such as cancer, sepsis, hyperthyroidism, etc. is related to increased protein degradation in muscle, which is associated with activation of the ubiquitin proteasome system. . At this time, it is known that ubiquitin binding is increased through activation of the N-terminal law pathway, resulting in muscle loss [ALFRED L. GOLDBERG et al. 1998, 1999]. Accordingly, a compound disclosed herein, or a pharmaceutically acceptable salt thereof, may be used to treat the above diseases by preventing the muscle loss pathway from being activated through binding to the UBR box domain. However, it is not limited thereto, and the specific disease includes all diseases known as UBR-related diseases in the art.

V. Bifunctional Compounds and Uses Thereof

The present application provides a bifunctional compound or a salt thereof. The bifunctional compound includes a UBR box domain binding ligand (UBL) and a target protein (or peptide) binding ligand (TBL).

As an embodiment, the bifunctional compound may be a compound having a [UBL-TBL] structure or a salt thereof.

As another embodiment, the bifunctional compound may be a compound having a [UBL-L(linker)-TBL] structure or a salt thereof.

The UBR box domain binding ligand (UBL) is a compound having the structure of Chemical Formula 1 described above. Description of Formula 1 is as described above.

The TBL (target protein (or peptide) binding ligand) is a compound that binds to a target protein (or peptide) to be degraded, and in this case, the TBL may be a known compound.

For example, the TBL may be an inhibitor compound of a known target protein (or peptide). For example, the TBLs are Hsp90 inhibitors, kinase inhibitors, androgen receptor inhibitors, HDM2 & MDM2 inhibitors, compounds targeting human BET bromodomain-containing proteins, HDAC inhibitors, human lysine methyltransferase inhibitors, angiogenesis inhibitors, nuclear It may be a hormone receptor compound, an immunosuppressive compound or a compound targeting the aryl hydrocarbon receptor (AHR), but is not limited thereto. The TBLs also include pharmaceutically acceptable salts, enantiomers, solvates and polymorphs of known compounds, as well as other small molecules capable of targeting a protein of interest.

The L (linker) may be a compound that connects the UBL (UBR box domain binding ligand) and the TBL (target protein (or peptide) binding ligand) without affecting each function. In this case, L may be a known linker compound.

As the linker, a known linker well known in the art may be used. For example, the linker may be arbitrarily selected from linkers described in Korean Patent Application No. 10-2020-7032733, US Patent Application No. 17/006193, and US Patent Application No. 17/082839. The linker can be designed in various ways considering the structures of UBL and TBL.

The bifunctional compounds include enantiomers, diastereomers, solvates, or polymorphs.

In addition, the present application provides a composition for protein degradation comprising a bifunctional compound and a use thereof.

The bifunctional compound may use the property that UBL binds to the UBR box domain to position the target protein (or peptide) bound to the TBL of the bifunctional compound to be close to the UBR, particularly the UBR box domain. At this time, the target protein (or peptide) located close to the UBR box domain is ubiquitinated by the UBR box domain and can be degraded through the proteasome pathway. Therefore, the protein to be degraded (ie, the desired target protein (or peptide)) can be more effectively degraded by using the bifunctional compound. Therefore, the composition containing the bifunctional compound can be used for inducing or promoting the degradation of a target protein (or peptide) by the ubiquitin-proteasome pathway by binding to the UBR box domain.

In addition, the present application provides a pharmaceutical composition containing a bifunctional compound and a method for treating a disease using the same.

The pharmaceutical composition may be used for treatment of disease. At this time, the disease is protein (or peptide) degradation, protein mutation, misfolded protein, protein accumulation, aggregated (aggregated) protein (and / or peptide), overexpressed protein, truncated protein, abnormal structure Eggplant may be a disease caused by protein problems, such as protein.

Example

Hereinafter, the invention disclosed in this application will be described in detail through examples.

These examples are solely for explaining the invention disclosed in this application in more detail, and it is clear that the scope of the present invention is not limited by these examples to those skilled in the art. It will be self-explanatory for

Example 1. Compound Synthesis

list of compounds

Compound 1: 3-(2-(3-methoxyphenyl)acetamido)benzamide;

Compound 2: 4-chloro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

Compound 3: 3-(5-methoxypentanamido)benzamide;

Compound 4: 4-chloro-3-(3-(3-methoxypropyl)ureido)benzamide;

Compound 5: 4-chloro-3-(3-(4-methoxyphenyl)propanamido)benzamide;

Compound 6: 3-(6-methoxyhexanamido)benzamide;

Compound 7: 3-(2-(3-methoxyphenyl)acetamido)-4-methylbenzamide;

Compound 8: 4-fluoro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

Compound 9: 1-(5-acetyl-2-chlorophenyl)-3-(3-methoxypropyl)urea;

Compound 10: 1-(3-acetylphenyl)-3-(3-methoxypropyl)urea;

Compound 11: N-(5-acetyl-2-chlorophenyl)-2-(3-methoxyphenyl)acetamide, and

Compound 12: N-(3-acetylphenyl)-2-(3-methoxyphenyl)acetamide.

¹ H NMR spectra were recorded on a Bruker Avance III 400 MHz and Bruker Fourier 300 MHz, with TMS used as an internal standard.

LCMS was measured on a quadrupole mass spectrometer on an Agilent 1260HPLC and 6120MSD. (Column operating in ES (+) or (-) ionization mode: C18 (50 Х 4.6 mm, 5 μm); T = 30 ^o C; Flow rate = 1.5 mL/min; Detected wavelengths: 220 nm, 254 nm)

Experimental Example 1-1. Preparation of compound 1, 3-(2-(3-methoxyphenyl)acetamido)benzamide [3-(2-(3-methoxyphenyl)acetamido)benzamide]

To a mixture of 3-aminobenzamide (164 mg, 1.20 mmol) and TBTU (579 mg, 1.80 mmol) was added anhydrous DMF (8 ml), followed by 2-(3-methoxyphenyl)acetic acid (200 mg, 1.2 mmol) and Et ₃ N (0.5 ml) was added. The reaction mixture was stirred overnight at room temperature. After removing the solvent under reduced pressure, the mixture was diluted with EA and washed with water. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column (DCM/MeOH=20/1) to give compound 1 (3-(2-(3-methoxyphenyl)acetamido)benzamide, 290 mg, Yield 85%) was obtained as an off-white solid.

¹ H-NMR (DMSO-d ₆ , 600 MHz): δ 10.27 (s, 1H), 8.03 (s, 1H), 7.92 (s, 1H), 7.77 (d, 1H), 7.52 (d, 1H), 7.37-7.32 (m, 2H), 7.23 (t, 1H), 6.92-6.90 (m, 2H), 6.82 (d, 1H), 3.74 (s, 3H), 3.62 (s, 2H)

LCMS; Chemical Formula: C ₁₆ H ₁₆ N ₂ O ₃ ; Mass Calcd.:284.3; MS Found: 285.1 [MS+1].

Experimental Example 1-2. Compound 2, Preparation of 4-chloro-3- (2- (3-methoxyphenyl) acetamido) benzamide [4-chloro-3- (2- (3-methoxyphenyl) acetamido) benzamide]

To a mixture of 3-amino-4-chlorobenzamide (205 mg, 1.20 mmol) and TBTU (579 mg, 1.80 mmol) was added anhydrous DMF (8 ml), followed by 2-(3-methoxyphenyl)acetic acid (200 mg, 1.2 mmol) and Et3N (0.5 ml) were added. The reaction mixture was stirred overnight at room temperature. After removing the solvent under reduced pressure, the mixture was diluted with EA and washed with water. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column (DCM/MeOH=20/1) to give compound 2 (4-chloro-3-(2-(3-methoxyphenyl)acetamido)benzamide , 303 mg, yield 79.2%) as a white solid.

¹ H-NMR (DMSO-d ₆ , 600 MHz): δ9.81 (s, 1H), 8.14 (s, 1H), 8.04 (s, 1H), 7.68 (d, 1H), 7.58 (d, 1H) , 7.45 (s, 1H), 7.25 (t, 1H), 6.95–6.93 (m, 2H), 6.83 (d, 1H), 3.75 (s, 3H), 3.71 (s, 2H).

LCMS; Chemical Formula: C ₁₆ H ₁₅ ClN ₂ O ₃ ; Mass Calcd.:318.7; MS Found: 319.5 [MS+1].

Experimental Example 1-3. Preparation of compound 3, 3-(5-methoxypentanamido)benzamide [3-(5-methoxypentanamido)benzamide]

To a mixture of 3-aminobenzamide (154mg, 1.13mmol) and TBTU (547mg, 1.70mmol) anhydrous DMF (6ml) was added followed by 5-methoxypentanoic acid (150mg, 1.13mmol) and Et3N (0.47ml) was added. The reaction mixture was stirred overnight at room temperature. After removing the solvent under reduced pressure, the mixture was diluted with EA and washed with water. The organic layer was dried over MgSO4 and filtered. The filtrate was concentrated in vacuo, and the residue was purified by flash column (DCM/MeOH=20/1) to give compound 3 (3-(5-methoxypentamido)benzamide, 190.5 mg, 67% yield) as beige Obtained as a colored solid.

¹ H-NMR (DMSO-d ₆ , 600 MHz): δ9.99 (s, 1H), 8.02 (s, 1H), 7.90 (s, 1H), 7.76 (d, 1H), 7.51 (s, 1H) , 7.36–7.31 (m, 2H), 3.33 (m, 2H), 3.22 (s, 3H), 2.32 (m, 2H), 1.62 (m, 2H), 1.53 (m, 2H).

LCMS; Chemical Formula: C ₁₃ H ₁₈ N ₂ O ₃ ; Mass Calcd.:250.3; MS Found: 251.1 [MS+1].

Experimental Example 1-4. Preparation of compound 4, 4-chloro-3- (3- (3-methoxypropyl) ureido) benzamide [4-chloro-3- (3- (3-methoxypropyl) ureido) benzamide]

To a solution of 3-amino-4-chlorobenzamide (200 mg, 0.7 mmol) in THF (6 ml) was added Et3N (141 mg, 1.4 mmol) followed by 1-isocyanato-3-methoxypropane (82 mg, 0.84 mmol in THF (2 ml)) was added to the reaction. The reaction mixture was stirred at 80 °C for 8 hours. After cooling to room temperature, the solvent was removed in vacuo. The residue was purified by flash column (100% DCM to 5% MeOH in DCM) to give compound 4 (4-chloro-3-(3-(3-methoxypropyl)ureido)benzamide, 120 mg, yield 60 %) was obtained as a white solid.

¹ H-NMR (DMSO-d ₆ , 600 MHz): δ8.58-8.57 (m, 1H), 8.08 (s, 1H), 7.93 (s, 1H), 7.47-7.45 (m, 1H), 7.41- 7.39 (m, 1H), 7.36 (br s, 1H), 7.01 (t, 1H), 3.38-3.33 (m, 2H), 3.24 (s, 3H), 3.16-3.13 (m, 2H), 1.69-1.64 (m, 2H).

LCMS; Chemical Formula: C ₁₂ H ₁₆ ClN ₃ O ₃ ; Mass Calcd.:285.7; MS Found: 286.1 [MS+1].

Experimental Example 1-5. Preparation of compound 5, 4-chloro-3-(3-(4-methoxyphenyl)propanamido)benzamide [(4-chloro-3-(3-(4-methoxyphenyl)propanamido)benzamide)]

Compound 5 can be obtained by using the same preparation method as Experimental Example 1-2, using 3-(4-methoxyphenyl)propanoic acid instead of 2-(3-methoxyphenyl)acetic acid.

Experimental Example 1-6. Preparation of compound 6, 3-(6-methoxyhexanamido)benzamide [(3-(6-methoxyhexanamido)benzamide)]

Compound 6 can be obtained by the same preparation method as in Experimental Example 1-3 using 6-methoxyhexanoic acid instead of 5-methoxypentanoic acid.

Experimental Example 1-7. Preparation of compound 7, 3-(2-(3-methoxyphenyl)acetamido)-4-methylbenzamide [(3-(2-(3-methoxyphenyl)acetamido)-4-methylbenzamide)]

Compound 7 can be obtained by using the same preparation method as in Experimental Example 1-2, using 3-amino-4-methylbenzamide instead of 3-amino-4-chlorobenzamide.

Experimental Example 1-8. Preparation of compound 8, 4-fluoro-3-(2-(3-methoxyphenyl)acetamido)benzamide [(4-fluoro-3-(2-(3-methoxyphenyl)acetamido)benzamide)]

Compound 8 can be obtained by using the same preparation method as in Experimental Example 1-2, using 3-amino-4-fluorobenzamide instead of 3-amino-4-chlorobenzamide.

Experimental Example 1-9. Compound 9, 1-(5-acetyl-2-chlorophenyl)-3-(3-methoxypropyl)urea [(1-(5-acetyl-2-chlorophenyl)-3-(3-methoxypropyl)urea)] manufacture of

Compound 9 can be obtained by using the same preparation method as in Experimental Example 1-4, using 1-(3-amino-4-chlorophenyl)ethan-1-one instead of 3-amino-4-chlorobenzamide.

Experimental Example 1-10. Preparation of compound 10, 1-(3-acetylphenyl)-3-(3-methoxypropyl)urea [(1-(3-acetylphenyl)-3-(3-methoxypropyl)urea)]

Compound 10 can be obtained by using the same preparation method as in Experimental Example 1-4, using 1-(3-aminophenyl)ethan-1-one instead of 3-amino-4-chlorobenzamide.

Experimental Example 1-11. Compound 11, N-(5-acetyl-2-chlorophenyl)-2-(3-methoxyphenyl)acetamide [(N-(5-acetyl-2-chlorophenyl)-2-(3-methoxyphenyl)acetamide) ] manufacture of

Compound 11 can be obtained by the same preparation method as in Experimental Example 1-2, using 1-(3-amino-4-chlorophenyl)ethan-1-one instead of 3-amino-4-chlorobenzamide.

Experimental Example 1-12. Compound 12, N-(3-acetylphenyl)-2-(3-methoxyphenyl)acetamide [( N Preparation of -(3-acetylphenyl)-2-(3-methoxyphenyl)acetamide)]

Compound 12 can be obtained by the same production method as in Experimental Example 1-1, using 1-(3-aminophenyl)ethan-1-one instead of 3-aminobenzamide.

Example 2. Binding assay experiment

Example 2-1: Confirmation of whether muscle actin is an Arg/N-degron pathway substrate

After culturing the L6 cell line, which is a rat muscle-derived cell, in a DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained with 5% carbon dioxide, each cell was dispensed into a 12-well plate. An additional 24 hours of incubation was performed so that the cells completely attached to the surface of the plate.

To confirm whether MG132 increases UBR1 binding, cells were harvested after treatment with MG132 (10 uM) alone for 24 hours. To extract proteins from the pooled cells, 50 uL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, sample buffer was added to each sample and reacted at 100 °C for 5 minutes. 5 uL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in [Figure 1]. Immunoblotting was plotted representatively from three or more independent experiments.

Referring to Figure 1, it was confirmed that the levels of ACTA1, ACTC1, ACTG2 increased more by MG132 than the control group. In addition, it was confirmed that the levels of ACTA1 and ACTG2 increased when the UBR protein was knocked down. That is, since it was confirmed that muscle actin is an Arg/N-degron pathway substrate, the compounds of the present application were evaluated for inhibiting muscle cell actin degradation using the muscle actin protein. The intramuscular proteins used in subsequent experiments were ACTC1 and ACTA2.

Example 2-2: Evaluation of muscle cell actin ACTC1 degradation inhibition through immunoblotting

In order to evaluate actin degradation in muscle cells by the compounds, L6 cell line, which is a rat muscle-derived cell, was cultured using DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained with 5% carbon dioxide.

In order to measure the binding force of UBR1 according to the treatment of selected compounds 1, 2, and 4 among the present compounds, each cell was seeded in a 12-well plate. Additional culture was performed for 24 hours to allow the cells to adhere completely to the surface of the plate. In order to confirm whether the compound increases UBR1 binding, cells were harvested after treatment with the compound (5 uM) alone for 24 hours.

To extract proteins from the pooled cells, 50 uL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, sample buffer was added to each sample and reacted at 100 °C for 5 minutes. 5 uL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in [Fig. 2]. Immunoblotting was plotted representatively from three or more independent experiments.

Referring to Figure 2, it can be confirmed that the level of ACTC1 is further increased by Compound 1, Compound 2, and Compound 4 compared to the control group (Control). That is, it was confirmed that when the compound according to the present invention was treated, degradation of ACTA1, an intramuscular protein, was inhibited through binding to UBR1.

Example 2-3: Evaluation of muscle cell actin ACTA2 degradation inhibition through immunoblotting

In order to evaluate actin degradation in muscle cells by the compounds, L6 cell line, which is a rat muscle-derived cell, was cultured using DMEM medium containing 10% FBS and 1% streptomycin/penicillin in an incubator maintained with 5% carbon dioxide.

In order to measure the binding force of UBR1 according to the treatment with compound 3 selected from among the present compounds, each cell was seeded in a 12-well plate. Additional culture was performed for 24 hours to allow the cells to adhere completely to the surface of the plate. In order to confirm whether the compound increases UBR1 binding, cells were harvested after treatment with the compound (5 uM) alone for 24 hours.

To extract proteins from the pooled cells, 50 uL of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% triton-X-100, 2 mM NaF, 2 mM EDTA, 2 mM b-glycerophosphate, 5 mM sodium orthovanadate, 1 mM PMSF, leupeptin, and aprotenin) were injected and the cells were lysed. Based on the measured total protein concentration, sample buffer was added to each sample and reacted at 100 °C for 5 minutes. 5 uL was taken from the sample after the reaction was dispensed into each well of an acrylamide gel, and immunoblotting was performed. The results of the experiment are shown in [Fig. 3]. Immunoblotting was plotted representatively from three or more independent experiments.

Referring to Figure 3, it can be confirmed that the level of ACTC1 is further increased by compound 3 compared to the control group (Control). That is, it was confirmed that, when compound 3 according to the present invention was treated, degradation of ACTA1, an intramuscular protein, was inhibited through binding to UBR1.

Through these Binding assay test results, it can be confirmed that the UBR ligand compound of the present application has the property of binding to the UBR box domain. This means that the binding of the UBR box domain substrate can be effectively inhibited through the UBR box domain ligand compound of the present application.

One invention disclosed herein is to provide a ligand compound having higher binding force to the UBR box domain. The UBR box domain ligand compound of the present application can effectively inhibit the binding of the UBR box domain substrate.

In addition, the present application can provide various uses of the UBR protein degradation system using such a ligand compound. For example, UBR-related diseases (eg, muscle loss, etc.) can be treated through UBR box domain ligand compounds.

Claims (11)

1. A compound having the structure of Formula 1, an isomer thereof, or a salt thereof:

   [Formula 1]

   

   X ₁ is any one selected from the following,

   -(CH ₂ )nO R,

   -(CH ₂ ) _n (C ₆ -C ₁₂ aryl)OR,

   -CH ₂ (3-12 membered-heteroaryl or heterocyclic)OR;

   -CH ₂ (cycloalkyl)OR,

   -NH(CH ₂ )nOR, and

   -CH ₂ NH(CH ₂ )nOR,

   At this time, in each functional group, n is independently an integer selected from 1, 2, 3, 4, 5, and 6, and R is H or C _1-4 alkyl;

   X ₂ is selected from H, halo, cyano, methyl and ethyl; and,

   X ₃ is C1-3 alkyl or amino;
2. According to claim 1,

   In Formula 1, X ₂ is H, Cl, F or CH ₃ A compound, an isomer thereof, or a salt thereof.
3. According to claim 1,

   In Formula 1, X ₃ is CH ₃ or NH ₂ A compound, an isomer thereof, or a salt thereof
4. According to claim 1,

   In Formula 1, X ₁ is -(CH ₂ ) ₂ OMe, -(CH ₂ ) ₃ OMe, -(CH ₂ ) ₄ OMe, -(CH ₂ ) ₅ OMe, -CH ₂ (C ₆ H ₄ )OMe , -(CH ₂ ) ₂ (C ₆ H ₄ )OMe, -NH(CH ₂ ) ₂ OMe, -NH(CH ₂ ) ₃ OMe, -(CH ₂ ) ₂ OEt, -(CH ₂ ) ₃ OEt, - (CH ₂ ) ₄ OEt, -(CH ₂ ) ₅ OEt, -CH ₂ (C ₆ H ₄ )OEt, -(CH ₂ ) ₂ (C ₆ H ₄ )OEt, -NH(CH ₂ ) ₂ OEt, and -NH(CH ₂ ) ₃ A compound selected from OEt, an isomer thereof, or a salt thereof
5. According to claim 1,

   In Formula 1, X ₁ is -(CH ₂ ) ₄ OMe, -(CH ₂ ) ₅ OMe, -CH ₂ (C ₆ H ₄ )OMe, -(CH ₂ ) ₂ (C ₆ H ₄ )OMe, and -NH(CH ₂ ) ₃ A compound selected from OMe, an isomer thereof, or a salt thereof
6. According to claim 3,

   In Formula 1, X ₃ is CH ₃ or NH ₂ A compound, an isomer thereof, or a salt thereof.
7. According to claim 1,

   In Formula 1,

   X ₁ is -(CH ₂ ) ₄ OMe, -CH ₂ (C ₆ H ₆ )OMe, -NH(CH ₂ ) ₃ OMe -(CH ₂ ) ₂ (C ₆ H ₄ )OMe, or -(CH ₂ ) ₅ OMe,

   X ₂ is H, Cl, F or CH ₃ ;

   A compound in which X ₃ is CH ₃ or NH ₂ , an isomer thereof, or a salt thereof.
8. The compound of claim 1, wherein the compound of Formula 1 is selected from the following compounds, isomers thereof, or salts thereof:

   3-(2-(3-methoxyphenyl)acetamido)benzamide;

   4-chloro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

   3-(5-methoxypentanamido)benzamide;

   4-chloro-3-(3-(3-methoxypropyl)ureido)benzamide;

   4-chloro-3-(3-(4-methoxyphenyl)propanamido)benzamide;

   3-(6-methoxyhexanamido)benzamide;

   3-(2-(3-methoxyphenyl)acetamido)-4-methylbenzamide;

   4-fluoro-3-(2-(3-methoxyphenyl)acetamido)benzamide;

   1-(5-acetyl-2-chlorophenyl)-3-(3-methoxypropyl)urea;

   1-(3-acetylphenyl)-3-(3-methoxypropyl)urea;

   N-(5-acetyl-2-chlorophenyl)-2-(3-methoxyphenyl)acetamide, and

   N-(3-acetylphenyl)-2-(3-methoxyphenyl)acetamide.
9. According to any one of claims 1 to 8,

   A pharmaceutical composition for treating a UBR-related disease comprising the compound or a pharmaceutically acceptable salt thereof.
10. According to claim 9,

    The UBR-related disease is caused by muscle atrophy (Becker, Congennital, Duchenne, Distal, Emery-Dreifuss, Facioscapulohumeral, Limb-girdle, myotonic, ocuophargyngeal) (muscular dystrophy), sarcopenia or cancer cachexia ( Liposarcoma caused by excessive protein breakdown, cystic fibrosis, Johanson-Blizzard syndrome, as well as muscle wasting diseases, including cancer cachexia ), obstructive urinary tract disease (urethral obstruction sequence), autoimmune pancreatitis (autoimmune pancreatitis) or Usher disease (Usher syndrome), such as UBR box and UBR protein-related diseases known as a pharmaceutical composition for treatment.
11. A bifunctional compound having the following structural formula or a salt thereof:

    UBR box domain binding ligand (UBL)—target protein binding ligand (TBL); or

    UBR box domain binding ligand (UBL) - linker (L) - target protein binding ligand (TBL);

    At this time, the TBL is a compound that binds to a target protein or peptide,

    The L is a linker, chemically connecting or combining UBL and TBL,

    The UBL is an isomer thereof of claim 1, or a salt thereof.

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