Compounds as UBR box domain ligands

Abstract

The present specification relates to compounds as UBR box domain ligands. The present specification provides small molecule compounds that bind to the UBR box domain. Furthermore, the present specification provides compositions for inhibiting UBR box domain substrate binding, including ligand compounds that bind to the UBR box domain, pharmaceutical compositions for treating UBR-related diseases, and uses thereof.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 015,945, filed April 27, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The subject matter disclosed herein relates to compounds as UBR box domain ligands. The UBR box domain is a domain commonly present in the ubiquitin protein ligase E3 component n-recognin (UBR) protein of the N-end rule pathway. In this case, the UBR box domain is known as the domain to which the substrate binds. The UBR box domain is essential for binding to the N-terminal residue of the substrate to form a multi-ubiquitin chain on the substrate, and it is known that the substrate is degraded through this process.

[0003] The present disclosure relates to compounds that function as ligands that bind to UBR box domains. [Background technology]

[0004] Cells regulate the amount and function of proteins in vivo by degrading them. In this case, proteins in vivo can be degraded depending on their N-terminal residue sequence, and this degradation pathway is known as the N-end rule pathway. That is, the N-end rule pathway is a proteolytic system that uses the N-terminus of a specific protein as a degradation signal. The N-end rule pathway can include the following proteolytic processes:

[0005] In eukaryotes, N-recognin recognizes degradation signals at the N-terminus of proteins, and the N-recognin can bind ubiquitin to the protein to be degraded, thereby degrading the protein. In this case, N-terminal degradation signals can include those having a positively charged residue at the N-terminus (type 1: e.g., arginine, lysine, and histidine) or a large hydrophobic residue (type 2: phenylalanine, leucine, tryptophan, isoleucine, and tyrosine). The present inventors first discovered or cloned N-recognin UBR1, UBR2, UBR3, and UBR5, and revealed that N-recognin has a UBR box domain as a substrate recognition domain (Tasaki et al., 2005). In this case, the ubiquitinated substrate generated by the binding of N-recognin to an N-end rule ligand is delivered to the proteasome and degraded into short peptides. In this process, specific N-terminal residues (Nt-Arg, Nt-His, Nt-Lys, Nt-Trp, Nt-Phe, Nt-Tyr, Nt-Leu, Nt-Leu) provide most of the hydrogen bonds required for N-recognin to target N-end rule substrates and are therefore essential determinants for binding (Sriram and Kwon, 2010).

[0006] UBR is an abbreviation for ubiquitin protein ligase E3 component n-recognin, and UBR is an N-recognin that recognizes the N-terminal degradation signal of proteins. It is known that there are at least seven types of UBR1 to 7 in mammals. Furthermore, the UBR box domain, which is a zinc finger motif with a size of about 70 residues, is known as a highly conserved substrate binding domain [Kwon et al., 1998; Xie and Varshavsky, 1999; Kwak et al., 2004; Varshavsky, 1996; Varshavsky, 1997; Kwon et al., 2011; and Zenker et al., 2014].

[0007] That is, UBR is an N-recognin associated with the N-end rule pathway, a protein degradation pathway, and the UBR box domain of UBR is a substrate-binding domain. In particular, among UBR1-7, UBR1, UBR2, UBR3, and UBR5 are known to act as ubiquitin protein ligase E3 and have a RING domain or HECT domain. N-end rule substrates bound to UBR are degraded via the ubiquitin proteasome pathway. Specifically, the UBR box domain contained in UBR recognizes the N-terminal amino acid of the substrate and ubiquitinates the substrate via the RING domain or HECT domain, thereby degrading the substrate via the proteasome pathway. For example, if a misfolded protein remains in the cell for a long period of time, the protein may aggregate and inhibit the proteasome or reduce other cellular functions, so it is degraded via the ubiquitin proteasome pathway (Ji and Kwon, 2017).

[0008] Thus, the UBR box domain plays an important role in the intracellular protein degradation pathway by recognizing N-terminal degradation signals, and ligands that bind to the UBR box domain can affect the intracellular protein degradation pathway.

[0009] As mentioned above, the present specification relates to compounds as ligands that bind to UBR box domains that are involved in intracellular protein degradation pathways. Summary of the Invention [Problem to be solved by the invention]

[0010] The present specification provides a small molecule compound that binds to a UBR box domain. In this case, the UBR box domain includes a UBR box domain contained in UBR1 to 7. The small molecule compound can function as a ligand suitable for binding to the UBR box domain.

[0011] In one embodiment, the present specification provides a composition for inhibiting UBR box domain substrate binding, comprising a ligand compound that binds to the UBR box domain.

[0012] In a specific embodiment, the present specification provides a pharmaceutical composition, and use thereof, for treating a UBR-associated disease, the composition comprising a ligand compound that binds to a UBR box domain.

[0013] In more specific embodiments, the present specification provides pharmaceutical compositions and uses thereof for treating diseases including muscle wasting caused by muscular dystrophies (Becker, congenital, Duchenne, distal, Emery-Dreifuss, facioscapulohumeral, limb-girdle, myotonic, oculopharyngeal), muscle wasting diseases mediated by muscle wasting or degradation including sarcopenia or cancer cachexia, diseases caused by excessive protein degradation including liposarcoma, cystic fibrosis, Johansson-Blizzard syndrome, obstructive uropathy (secondary to urethral obstruction), autoimmune pancreatitis, or known diseases associated with the UBR box and UBR protein including Usher syndrome, wherein the composition comprises a ligand compound that binds to the UBR box domain. [Means for solving the problem]

[0014] Provided herein are compounds having the structure of Formula 1, or a salt thereof: [Formula 1] [ka] In the formula, A1 is CH3 or NH2.

[0015] In this case, by way of example, the present specification provides a composition for inhibiting UBR box domain substrate binding comprising the present compound, or a salt thereof, wherein A1 is CH3 or NH2.

[0016] In this case, as an example, the present specification provides a pharmaceutical composition for treating a UBR-related disease comprising the present compound or a pharma- ceutically acceptable salt thereof, wherein A1 is CH3 or NH2, and a method for treating a UBR-related disease by using the present compound.

[0017] In this case, by way of example, the UBR-associated disease may be selected from muscle wasting caused by muscular dystrophies (Becker, congenital, Duchenne, distal, Emery-Dreifuss, facioscapulohumeral, limb-girdle, myotonic, oculopharyngeal), muscle wasting diseases mediated by muscle wasting or degradation including sarcopenia or cancer cachexia, diseases caused by excessive protein degradation including liposarcoma, cystic fibrosis, Johansson-Blizzard syndrome, obstructive uropathy (secondary to urethral obstruction), autoimmune pancreatitis, or Usher syndrome.

[0018] ADVANTAGES The invention disclosed herein provides ligand compounds with high binding strength for UBR box domains.

[0019] The UBR box domain ligand compound can inhibit the substrate binding of the UBR box domain, and various uses can be provided by utilizing this property. For example, the UBR box domain ligand compound can treat UBR-related diseases (e.g., sarcopenia, etc.). [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 shows the results of an experiment conducted by immunoblotting to determine whether muscle actin is a substrate for the Arg / N-degron pathway.

[0021] [Diagram 2] FIG. 1 shows the results of an experiment conducted using an in vitro transcription / translation method to confirm whether the expression of R-nsp4, which should be degraded, is suppressed by binding compounds (compounds 1 and 2) to UBR1.

[0022] [Diagram 3] FIG. 1 shows the results of an experiment conducted by immunoblotting to confirm whether compounds (compounds 1 and 2) inhibit the degradation of actin, a substrate of UBR protein, in muscle cells.

[0023] [Figure 4] FIG. 1 shows the results of binding between Compound 1 and Compound 2 by microscale thermophoresis (MST). [Diagram 5] FIG. 1 shows the results of binding between Compound 1 and Compound 2 by microscale thermophoresis (MST). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The contents of the present invention will be described in more detail below through specific exemplary embodiments and examples with reference to the accompanying drawings. Please note that the accompanying drawings include some exemplary embodiments of the present invention, but not all exemplary embodiments. The contents of the invention disclosed herein can be implemented in various ways and are not limited to the specific exemplary embodiments described herein. Those having ordinary skill in the art to which the invention disclosed herein belongs will be able to think of many modifications and other exemplary embodiments of the contents of the invention disclosed herein. Therefore, it should be understood that the contents of the invention disclosed herein are not limited to the specific exemplary embodiments described herein, and modifications and other exemplary embodiments are also included in the scope of the claims.

[0025] [Terminology definition] The following are definitions of key terms used in this specification:

[0026] Ubiquitin Protein Ligase E3 Component n-Recognin (UBR) The term UBR as used herein refers to the abbreviation for ubiquitin protein ligase E3 component n-recognin. UBR is an N-recognin that recognizes the N-terminal residue of a protein, and at least seven types of UBR1 to 7 are known to exist in mammals. UBR is an N-recognin and is related to the N-end rule pathway, which is an in vivo protein degradation pathway. Specifically, UBR recognizes the N-terminal degradation signal (N-degron) of a protein and is involved in the process in which a substrate protein is degraded via the ubiquitin proteasome pathway.

[0027] UBR Box Domain The term UBR box domain as used herein refers to a domain present in UBR proteins and is a zinc finger motif. UBR proteins include UBR1-7 proteins. The UBR box domain is known as a domain to which substrate proteins bind. The compounds disclosed herein as UBR box domain ligands can inhibit substrate binding of the UBR box domain by binding to the UBR box domain. Furthermore, the compounds disclosed herein as UBR box domain ligands can affect intracellular protein degradation pathways.

[0028] RING domain The term RING domain used herein is known to exist in UBR1, 2 and 3 proteins. RING domain can also be replaced with RING ubiquitination domain. RING domain is a domain that exists in protein and is a zinc finger motif. RING domain is a domain that plays an important role in the process that ubiquitin that exists in E2 is transferred to substrate protein, and RING domain plays a role in the process that ubiquitin is transferred to substrate protein in one step.

[0029] HECT Domain The term HECT domain used herein is known to exist in UBR5 protein. HECT domain can also be replaced with HECT ubiquitination domain. HECT domain is a domain that plays an important role in the process in which ubiquitin present in E2 is transferred to substrate protein. Ubiquitin present in E2 is carried to HECT domain and then transferred to substrate protein. That is, HECT domain plays a role in the process in which ubiquitin is transferred to substrate protein in two steps.

[0030] Zinc finger motif As used herein, the term zinc finger motif refers to a protein structural motif in which one or more zinc ions are present to stabilize the structure of the protein. The UBR box domain and RING domain herein are zinc finger motifs.

[0031] As used herein, the term ligand refers to a substance that specifically binds to a protein. Proteins include enzymes and receptors, and when the protein is an enzyme, the ligand may refer to a substrate that binds to the enzyme, etc., and when the protein is a receptor, the ligand may refer to a hormone that binds to the receptor, etc.

[0032] The compound as the UBR box domain ligand provided herein means a compound that binds to the UBR box domain. As an example, the compound refers to a compound that binds to the UBR box domain in the UBR protein. As a specific example, the compound refers to a compound that binds to the UBR box domain present in one or more proteins of UBR1 to 7. However, the compound is not limited thereto.

[0033] The compounds provided herein as UBR box domain ligands can act competitively with the substrate of the UBR box domain. That is, the compounds can inhibit the substrate binding of the UBR box domain. Furthermore, the compounds can inhibit the degradation of the substrate by inhibiting the binding of the substrate.

[0034] Unless otherwise defined, 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.

[0035] Specific details of the present invention will be disclosed below.

[0036] I. UBR box domains 1. Overview The compound provided herein as UBR box domain ligand binds to UBR box domain.UBR box domain is known as the domain that N-terminal residue sequence or N-terminal degradation signal binds.This domain is related to the process that protein is degraded by N-end rule pathway.Therefore, this compound can affect the protein degradation process via N-end rule pathway.

[0037] 2. N-end rule pathway Cells regulate the amount of proteins through protein degradation. In this case, it is known that the process of protein degradation is carried out by the process of recognizing degrons, which are protein degradation signals. Specifically, protein degradation is regulated depending on the N-terminal residue sequence of the protein, and proteolytic signals present at the N-terminus are collectively called N-degrons. N-degrons include those that have positively charged residues (e.g., arginine, lysine, and histidine) or large hydrophobic residues (phenylalanine, leucine, tryptophan, isoleucine, and tyrosine) at the N-terminus. As mentioned above, the term N-end rule has been used based on the association that the half-life of a protein is determined by the amino acid residues present at the N-terminus of the protein.

[0038] 3. UBR Box Domain In the N-end rule pathway, N-degrons are recognized by N-recognins, and the ubiquitin protein ligase E3 component n-recognin (UBR) was discovered as an N-recognin. UBR is known to recognize N-terminal residue sequences or N-terminal degradation signals by the UBR box domain. That is, UBR recognizes protein degradation signals through the UBR box domain, and through recognition of the protein degradation signal, it carries out the degradation process of the protein.

[0039] The protein degradation process by UBR may include the following: The UBR box domain recognizes substrates with N-terminal degradation signals, ubiquitin binds to the substrate, and the ubiquitin-bound substrate can be degraded by the proteasome, i.e., substrates with N-terminal degradation signals can be degraded by the ubiquitin proteasome system (UPS).

[0040] II. UBR box domain ligands 1. Overview II. UBR box domain ligands 1. Overview 1) The compounds of the present specification reflect the structure of the UBR box domain and the properties of binding to the N-terminal pathway substrate. The compounds disclosed in the present specification as UBR box domain ligands are 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.

[0041] Various amino acids present in the UBR box domain interact and bind with amino acids in the N-terminal pathway substrates through ionic interactions, hydrogen bonds, hydrophobic interactions, etc. By analyzing these binding modes, small molecule compounds capable of forming suitable binding modes with the UBR box domain have been synthesized and are provided herein. Additionally, compounds according to formula 1 are provided below.

[0042] 2) The compounds herein have a core structure that enhances binding to UBR box domains.

[0043] 1) Formula 1 [ka] [Formula 1]

[0044] In the above formula 1, A1 is CH3 or NH2.

[0045] In this case, the compounds of formula 1 disclosed herein may exist in the form of their stereoisomers or salts, and such isomers or salt forms of the compounds are also included within the scope of the present specification.

[0046] 2) Specific examples of compounds i) Compound By way of example, compounds disclosed herein as UBR box domain ligands can be selected from those listed below: [Compound 1] [ka] [Compound 2] [ka] In this case, the compound may be considered in any of its possible isomeric forms or in the form of mixtures thereof, for example all stereoisomers, including enantiomers and diastereomers, or mixtures thereof (for example racemic mixtures).

[0047] ii) Salts of the Compounds The compounds disclosed herein may be considered in the form of their salts. In this case, the salts include pharma- ceutically acceptable salts. The salts disclosed herein include acid addition salts or base addition salts. Exemplary acids that form salts include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, and glutaric acid, and exemplary bases that form salts include lithium, sodium, potassium, calcium, magnesium, methylamine, and trimethylamine. However, the acids and bases are not limited thereto, and can be easily selected by those skilled in the art.

[0048] III. Use of the Compounds 1. UBR box domain inhibits substrate binding Compositions for inhibiting UBR box domain substrate binding The compounds disclosed herein can be used to prepare compositions for inhibiting UBR box domain substrate binding. As an example, a composition comprising a compound disclosed herein can be used to inhibit UBR box domain substrate binding by binding to the UBR box domain. As another example, a composition comprising the compound can be used for preventing a substrate that binds to the UBR box domain and is degraded from being degraded. As a specific example, a composition comprising the compound can be used for preventing a substrate that is bound to the UBR box domain from being degraded by the ubiquitin-proteasome pathway.

[0049] As a specific example, a composition comprising a compound disclosed herein can be used for inhibiting the binding of a substrate having an N-terminal residue that binds to a UBR box domain. As a specific example, a composition comprising a compound disclosed herein can be used for inhibiting the binding of a substrate having an N-terminal residue such as arginine (Arg), lysine (Lys), histidine (His), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), leucine (Leu) and isoleucine (Ile). However, the application is not limited thereto, and the composition can be used for inhibiting the binding of a substance known in the art as a substrate of a UBR box domain.

[0050] With reference to the Examples, it can be seen that the compounds disclosed herein inhibit the degradation of substrates by binding to the UBR (see Figures 1, 2 and 3).

[0051] 2. Treatment of UBR-related diseases The compound of the present invention or its salt has the property of binding to UBR box domain.That is, the compound of the present invention is a compound that functions as a ligand that binds to UBR box domain.Therefore, these compounds can be used to inhibit the degradation of proteins that are degraded by binding to UBR box domain in the body, and such a mechanism can be used to treat UBR-related diseases.

[0052] 1) Pharmaceutical Composition The compounds disclosed herein can be used in the preparation of a pharmaceutical composition for treating a subject in need thereof.

[0053] In this case, treatment includes having an effect of alleviating the symptoms of a particular disease state or delaying the progression of the disease. In this case, the subject includes humans and non-human animals. In this case, the pharmaceutical composition may contain a pharmaceutically acceptable carrier, excipient and / or additive together with the above-mentioned compound. The pharmaceutically acceptable carrier, excipient and additive include, but are not limited to, water, saline, glycol, glycerol, animal and vegetable oils and fats, oils, starch, etc., and include all acceptable carriers, excipients and / or additives known in the art that are pharmaceutically acceptable.

[0054] 2) Treatment method The present specification provides a method of treatment, comprising administering the compound disclosed herein or its pharmaceutically acceptable salt to a subject in need of administration.In this case, the administration of the compound or its pharmaceutically acceptable salt can have the effect of alleviating the symptoms of a particular disease or delaying the progression of a disease, compared with the subject who does not administer the compound or its salt.In this case, the subject includes human and non-human animals.

[0055] -UBR-related diseases As an example, 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, the compound or a pharmaceutically acceptable salt thereof disclosed herein can be used to treat a UBR-related disease. As a specific example, the compound or a pharmaceutically acceptable salt thereof can be used to treat a specific disease to be treated by inhibiting the degradation of a protein that is degraded by binding to a UBR box domain.

[0056] Specific diseases include muscle wasting caused by muscular dystrophies (Becker, congenital, Duchenne, distal, Emery-Dreifuss, facioscapulohumeral, limb-girdle, myotonic, oculopharyngeal), muscle wasting diseases mediated by muscle wasting or degradation including sarcopenia or cancer cachexia, diseases caused by excessive protein degradation including liposarcoma, cystic fibrosis, Johansson-Blizzard syndrome, obstructive uropathy (secondary to urethral obstruction), autoimmune pancreatitis, or known diseases associated with UBR boxes and UBR proteins including Usher syndrome. As an example, the compound or a pharma- ceutically acceptable salt thereof can be used to treat muscle wasting mediated by UBR. For example, the early loss of muscle mass associated with pathologies such as cancer, sepsis, and hyperthyroidism is associated with increased degradation of intramuscular proteins, which is known to be associated with activation of the ubiquitin proteasome system. In this case, it is known that activation of the N-end rule pathway in particular increases ubiquitin binding, resulting in muscle wasting [ALFRED L. GOLDBERG et al., 1998, 1999]. Accordingly, the compounds disclosed herein or their pharmacologic acceptable salts can be used to treat diseases by binding to the UBR box domain and preventing activation of the muscle wasting pathway. However, the present invention is not limited thereto, and the specific diseases include all diseases known in the art as diseases related to UBR.

[0057] V. Working Examples Example 1. Synthesis of Compounds [Table 1]

[0058] List of Compounds [Table 1] 1H NMR spectra were recorded on a Bruker Avance III 400 MHz and a Bruker Fourier 300 MHz with TMS as internal standard. LCMS was measured on an Agilent 1260 HPLC and 6120 MSD quadrupole mass spectrometer (column: C18 (50 × 4.6 mm, 5 μm) operated in ES (+) or (-) ionization mode; T = 30 °C; flow rate = 1.5 mL / min; detection wavelengths: 220 nm, 254 nm). Experimental Example 1-1 Preparation of compound 1 (N-(5-acetyl-2-chlorophenyl)-5-methoxypentanamide) [ka] Step 1) Synthesis of B2 To a mixture of B1 (tetrahydro-2H-pyran-2-one, 5.00 g, 50 mmol, 1.0 equiv) and trimethyl orthoformate (10.6 g, 100 mmol, 2.0 equiv) in MeOH (100 mL) was added concentrated H2SO4 (3 mL) dropwise at 25°C. The mixture was stirred at 65°C for 16 h. The solvent was concentrated under reduced pressure. 8 M aqueous sodium hydroxide solution (10 mL) was added and the mixture was stirred at 80°C for 2 h. After cooling to 10°C, the reaction mixture was acidified with 4 N HCl and extracted with EA (50 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give B2 (5-methoxypentanoic acid, 4.0 g, 60% yield) as a colorless oil.

[0059] 1HNMR (CDCl3,400MHz): δ3.41(t,J=6.0Hz,2H),3.34(s,3H),2.39(t,J=7.6Hz,2H),1.62-1.74(m,4H).

[0060] Step 2) Synthesis of compound 1 A mixture of B2 (5-methoxypentanoic acid, 0.2 g, 1.52 mmol, 1.0 equiv) and HATU (0.69 g, 1.82 mmol, 1.2 equiv) in DMF (5 mL) was stirred at 25 °C for 30 min. Then, 1-(3-amino-4-chlorophenyl)ethan-1-one (0.26 g, 1.52 mmol, 1.0 equiv) and triethylamine (0.31 g, 0.30 mmol, 2.0 equiv) were added. The mixture was stirred at 50 °C for 48 h. The mixture was poured into water (50 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give the crude product, which was purified by prep-HPLC. The collected fractions were concentrated to remove most of the CH3CN. The remaining fraction was lyophilized to give compound 1 (N-(5-acetyl-2-chlorophenyl)-5-methoxypentanamide, 38 mg, 8.8% yield) as a white solid.

[0061] 1HNMR(CDCl3,400MHz):δ9.03(s,1H),7.84(s,1H),7.67(dd,J=8.4Hz,2Hz,1H),7.46(d,J=8.4Hz,1H),3.4 5(t,J=6.4Hz,2H),3.35(s,3H),2.61(s,3H),2.52(t,J=7.2Hz,2H),1.85-1.88(m,2H),1.69-1.73(m,2H).

[0062] LCMS;Mass Calcd.:283.7;MS Found:283.9.

[0063] Experimental Example 1-2 Preparation of compound 2 (4-chloro-3-(5-methoxypentanamido)benzamide) [ka] A mixture of B2 (5-methoxypentanoic acid, 0.2 g, 1.52 mmol, 1.0 equiv) and HATU (0.69 g, 1.82 mmol, 1.2 equiv) in DMF (5 mL) was stirred at 25 °C for 30 min. Then, 3-amino-4-chlorobenzamide (0.26 g, 1.52 mmol, 1.0 equiv) and triethylamine (0.31 g, 0.30 mmol, 2.0 equiv) were added. The mixture was stirred at 50 °C for 48 h. The mixture was poured into water (50 mL) and extracted with EA (50 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to give the crude product, which was purified by prep-HPLC. The collected fractions were concentrated to remove most of the CH3CN. The remaining fraction was lyophilized to give compound 2 (4-chloro-3-(5-methoxypentanamido)benzamide, 48 mg, 11.1% yield) as a yellow solid.

[0064] 1HNMR(CDCl3,400MHz):δ8.82(s,1H),7.89(s,1H),7.64(dd,J=8.4Hz,2Hz,1H),7.46(d,J=8.4Hz,1H),5.93- 6.38(m,2H),3.45(t,J=6.0Hz,2H),3.36(s,3H),3.52(t,J=7.2Hz,2H),1.81-1.88(m,2H),1.66-1.73(m,2H).

[0065] LCMS; Mass Calcd.:284.7;MS Found:284.9.

[0066] Example 2. Binding assay experiments Example 2-1 Confirmation of whether muscle actin is a substrate of the Arg / N-degron pathway using immunoblotting

[0067] L6 cell line, a rat muscle-derived cell line, was cultured in a 5% carbon dioxide-maintained incubator using DMEM medium containing 10% FBS and 1% streptomycin / penicillin, and the cultured cells were dispensed into 12-well plates. The cells were further cultured for 24 hours to allow them to completely attach to the plate surface. To confirm whether MG132 increases UBR1 binding, the cells were treated with MG132 (10uM) alone for 24 hours and then harvested. To extract proteins from the harvested cells, 50uL of lysate preparation buffer (20mM Tris, pH7.4, 150mM NaCl, 1% Triton-X-100, 2mM NaF, 2mM EDTA, 2mM b-glycerophosphate, 5mM sodium orthovanadate, 1mM PMSF, leupeptin, aprotinin) was injected into each sample to lyse the cells. Sample buffer was added to each sample based on the measured total protein concentration, and the mixture was reacted at 100°C for 5 minutes. 5uL was taken from the fully reacted sample and dispensed into each well of an acrylamide gel, followed by immunoblotting, and the experimental results are shown in [Figure 1].

[0068] For immunoblotting, a representative experiment from three or more independent experiments is depicted.

[0069] As shown in Figure 1, it was confirmed that the levels of ACTA1, ACTC1, and ACTG2 were increased by MG132 compared to the control. Furthermore, it was confirmed that the levels of ACTA1 and ACTG2 were increased when UBR protein was knocked down. In other words, it was confirmed that muscle actin is a substrate of the Arg / N-degron pathway.

[0070] Example 2-2 Confirmation of R-nsP4 Degradation Inhibition by In Vitro Transcription / Translation Method Using the TnT (registered trademark) Quick Coupled Transcription / Translation System kit, R-nsP4 expression of the compound was confirmed. A premix was prepared using Transcend Biotin-Lysyl-tRNA, methionine, bestatin, TnTquick Master mix, and DHFR-Ub-R-nsP4 plasmid, and then the compound (1 uM) was mixed into the premix. Each sample was reacted at 30°C for 40 minutes, and then 5X SDS loading dye was added thereto. The resulting mixture was reacted at 95°C for 2 minutes, and then 5 uL was taken and dispensed into each well of an acrylamide gel, followed by immunoblotting, and the experimental results are shown in [Figure 2]. In the in vitro transcription / translation method, a representative experiment from three or more independent experiments was diagrammed.

[0071] 2, it can be seen that the R-nsP4 level is increased by compounds 1 and 2 compared to the control. That is, it was confirmed that the R-nsP4 level is increased by binding to UBR1 when treated with the compound according to the present invention.

[0072] Example 2-3 Evaluation of inhibition of actin degradation in muscle cells by immunoblotting To evaluate the actin degradation of the compounds in muscle cells, rat muscle-derived L6 cell line was cultured in DMEM medium containing 10% FBS and 1% streptomycin / penicillin in an incubator maintained at 5% carbon dioxide. To measure the UBR1 binding intensity in response to treatment with representative compounds selected from the present compounds, cells were dispensed into 12-well plates. The cells were cultured for an additional 24 hours to allow the cells to fully attach to the plate surface. After 24 hours of treatment with the compounds (5uM) alone, the cells were harvested to determine whether the compounds increased UBR1 binding. To extract proteins from the harvested cells, 50uL of lysate preparation buffer (20mM Tris, pH7.4, 150mM NaCl, 1% Triton-X-100, 2mM NaF, 2mM EDTA, 2mM b-glycerophosphate, 5mM sodium orthovanadate, 1mM PMSF, leupeptin, aprotinin) was injected into each sample to lyse the cells. Sample buffer was added to each sample based on the measured total protein concentration, and the mixture was reacted at 100°C for 5 minutes. 5uL was taken from the fully reacted sample and dispensed into each well of an acrylamide gel, and then immunoblotting was performed. The experimental results are shown in Figure 3. For immunoblotting, a representative experiment from three or more independent experiments is illustrated.

[0073] 3, it was confirmed that the level of ACTA1 was increased by Compound 1 and Compound 2 compared to the control. That is, it was confirmed that the degradation of intramuscular protein ACTA1 was inhibited by binding to UBR1 when treated with the compound according to the present invention.

[0074] Example 2-4: Measurement of the Presence or Absence of Binding of UBR1 to Compounds Using MST 1) Preparation of UBR1 protein The Gln97-Pro168 portion of human UBR1 (UniProt ID: Q8IWV7) corresponding to the UBR box was cloned into a modified expression vector and then expressed in E. coli. After affinity chromatography, the tag was removed with protease, and then Gly-His-Met was added to the N-terminus. After ion chromatography, the UBR box protein of UBR1 was finally purified by gel filtration chromatography in a buffer composition of 10 mM NaCl, 20 mM Tris-HCl, 2 mM β-mercaptoethanol, and pH 7.5.

[0075] 2) Labeling of UBR1 UBR box protein The dye in the Monolith protein labeling kit RED-NHS 2nd generation (catalog number MO-L011) has an NHS ester group that forms a covalent bond with primary amines (lysine residues). This dye is optimized for Monolith series devices equipped with a RED detector. Purified UBR1 UBR box protein was labeled using this kit according to the protocol provided.

[0076] 3) Measurement of the binding between UBR1 and ligand using MST Thermophoresis is a phenomenon in which particles move due to a temperature gradient. Particles in high temperature regions have more kinetic energy than particles in low temperature regions, and collide more frequently with surrounding particles that have more energy. As a result, particles move from high temperature regions to low temperature regions.

[0077] Thermophoresis of proteins is usually different from that of protein-ligand complexes because the size, charge and solvation energy change with the ligand binding. Furthermore, MST can detect the change in solvent entropy of protein molecules caused by ligand binding, even if the size and charge of the protein do not change significantly upon ligand binding. Therefore, we used MST to measure the binding of UBR1 UBR box protein with ligand compounds, and confirmed that the presented ligand binds to the UBR1 UBR box.

Claims

1. Formula 1: [Formula 1] 【Chemistry 7】 or a salt thereof, In the formula, A １ NH ２ or a salt thereof.

2. 1. A composition for inhibiting UBR box domain substrate binding, said composition comprising a compound of formula 1: [Formula 1] 【Chemistry 7】 or a salt thereof, A composition comprising a compound or a salt thereof, wherein A 1 is CH 3 or NH 2 .

3. A pharmaceutical composition for treating a UBR-related disease, the pharmaceutical composition comprising a compound of formula 1: [Formula 1] 【Chemistry 7】 or a salt thereof, A pharmaceutical composition comprising a compound, or a pharma- ceutically acceptable salt thereof, wherein A1 is CH3 or NH2.

4. 4. The pharmaceutical composition of claim 3, wherein the UBR-related disease comprises muscle wasting caused by muscular dystrophy (Becker, congenital, Duchenne, distal, Emery-Dreifuss, facioscapulohumeral, limb-girdle, myotonic, oculopharyngeal), muscle wasting diseases mediated by muscle wasting or degradation including sarcopenia or cancer cachexia, diseases caused by excessive protein degradation including liposarcoma, cystic fibrosis, Johanson-Blizzard syndrome, obstructive uropathy (secondary to urethral obstruction), autoimmune pancreatitis, or known diseases associated with the UBR box and UBR protein including Usher syndrome.

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